Amelioration of CCl -Induced Hepatotoxicity in Rabbits by Lepidium sativum...
Transcript of Amelioration of CCl -Induced Hepatotoxicity in Rabbits by Lepidium sativum...
Research ArticleAmelioration of CCl4-Induced Hepatotoxicity in Rabbits byLepidium sativum Seeds
Mazin A Zamzami 1 Othman A S Baothman 12
Fatma Samy3 and Mohamed Kamel Abo-Golayel 14
1Biochemistry Department Faculty of Science King Abdulaziz University Jeddah Saudi Arabia2Microbial Toxicology amp Natural Products Centre Faculty of Science King Abdulaziz University Saudi Arabia3Pathology Department Faculty of Medicine Ain Shams University Cairo Egypt4Medical Research Centre Ain Shams University Hospitals Ain Shams University Cairo Egypt
Correspondence should be addressed to Mohamed Kamel Abo-Golayel magolayelkauedusa
Received 24 December 2018 Revised 19 January 2019 Accepted 30 January 2019 Published 7 March 2019
Academic Editor Vincenzo De Feo
Copyright copy 2019 Mazin A Zamzami et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
The current study aimed to evaluate the probable protective effect of Lepidium sativum seeds (LSS) against CCl4 induced hepaticinjury in New-Zealand rabbits Rabbits were randomly divided into two main groups group-A (noninjured group n=15) wasdivided to subgroups A1 (untreated control) and A2 and A3 which received 200 amp 400 mgkg bw of LSS respectively in theirdiet daily Group-B (injured group n=30) were subcutaneously injected with CCl4 (05 mlkg bw) starting from day one of theexperiment and were equally divided into 3 subgroups B1 received normal standard diet and B2 amp B3 received 200 amp 400mgkg bw of LSS respectively in their diet daily Five rabbits of all subgroups were decapitated 5 and 10 weeks after experimentalrunning Biochemical analysis revealed significant decrease in serum levels of transaminases 120574-GT ALP total bilirubin cholesteroltriglycerides associated with significant increase in the serum levels of T protein and albumin of 200 and 400 mgkg bw ofLSS protected rabbits for 5 and 10 weeks as compared with CCl4 treated rabbits Oxidative stress and depressed antioxidantsystem of the liver tissues were markedly obvious in the CCl4 treated group LSS administration reversed these results towardsnormalization Histopathological examination of LSS protected rabbits (200 mgkg bw of LSS for 10 weeks) showed improvementof the histoarchitectural changes of the liver induced by CCl4 to the normal aspect showing regenerating hepatocytes with nosteatosis discrete chronic venous congestion and discrete inflammatory infiltrateThe current findings provide new evidence thatLSS could reverse the hepatotoxic effects of CCl4 and repair the liver functions
1 Introduction
Carbon tetrachloride (CCl4) is a xenobiotic industrial solventthat is used to induce chemical hepatitis and liver injuriesin experimental animals Carbon tetrachloride-induced liverinjuries are the most common experimental model for moni-toring the hepatoprotective activity of certain drugs A singleexposure to CCl4 as being a strong hepatotoxic xenobioticdirectly leads to severe liver necrosis and steatosis [1 2]Mechanistic studies offer evidence that metabolism of CCl4via CYP2E1 to strongly reactive free radical metabolites playsa crucial role in the proposed mode of action The majormetabolites trichloromethyl (CCl3sdot) and trichloromethyl
peroxy (CCl3O2sdot) free radicals are extremely reactive andare capable of covalent bind to cellular macromoleculespreferring fatty acids of the membrane phospholipids Thefree radicals induce cell membrane lipid peroxidation via dis-rupting polyunsaturated fatty acids within these membranesinitiating a sequential free radical chain reaction [1]
Lepidium sativum is a wild-growing edible annual herbbelongs to Brassicaceae family It is believed to be originatedmainly in Eritrea and Ethiopia Leaves roots and seeds ofLepidium sativum are great profitable crops however thecrop is basically cultivated for seeds In India this herbis known as Asalio and it is used in folk medicine sinceancient eras [3 4] Lepidium sativum seeds (LSS) exert
HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019 Article ID 5947234 17 pageshttpsdoiorg10115520195947234
2 Evidence-Based Complementary and Alternative Medicine
hypoglycaemic activity both in diabetic and normal ratswithout interfering with insulin secretion [5] The seeds arerecognized to improve the symptoms of asthma and recoverlung function in asthmatic patients [6] In folk medicineseveral parts of Lepidium sativum have been utilized forthe treatment of liver problems jaundice spleen diseasesmenstrual problems gastrointestinal disorders bone frac-ture and arthritis [7ndash9] Phytochemical research studies ofLepidium sativum demonstrated the existence of flavonoidsglucosinolates tannins alkaloids triterpenes sterols benzylisothiocyanate [10 11] that were stated to possess anal-gesic anti-inflammatory activities antioxidant capacitiesand hepatoprotective characters [10 12ndash14] Although pre-vious reports revealed that hepatoprotective efficacy of LSSagainst doxorubicin [15] and CCl4 [12] induced intoxicationhas been described up to our knowledge no studies havebeen conducted on the LSS existing in the Arabian regionMoreover the uniqueness of this study is that the active ingre-dients of LSS extract could be responsible for the bioactivityof these seeds Therefore the current study was designed toinvestigate the probable protective effect of Lepidium sativumseeds against CCl4 induced hepatotoxicity in New-Zealandrabbits
2 Materials and Methods
21 Preparation of Lepidium sativum Seeds (LSS) Aque-ous Extract Lepidium sativum seeds were gained fromAlquaseem market of traditional medicine KSA recognizedand identified by a professor of taxonomy and depositedat the Herbarium of Biological Department Faculty ofScience King Abdulaziz University Before extraction LSSwere washed using double deionized distilled water driedand crushed by pestle and mortar The seeds were permittedto dry in the sunlight for 2 days then homogenized to a finepowder and kept in free-moisture impervious container untiluse
22 Chromatographic Analysis of LSS Using GC-MS Chro-matographic analysis using GC-MS was carried out (AgilentTechnologies 7890B GC Systems combined with 5977AMassSelective Detector) Capillary column (HP-5MS Capillary300mtimes025mm IDtimes025120583m film) and helium as carrier gaswere used at a rate of flowof 19mlminwith 1120583l injectionThesample was analyzed with the column held initially for 3 minat 40∘C after injection then the temperature was increasedto 300∘Cwith a 20∘Cmin heating ramp with a 40 min holdInjection was carried out in split-lessmode at 300∘CMS scanrange was (mz) 50ndash550 atomic mass units (AMU) underelectron impact (EI) ionization (70 eV)
23 Preparation of Fatty AcidMethyl Esters Fatty acidmethylesters were prepared fromdifferent sources using commercialaqueous HCl [16] Yields of FAME were similar to thoseobtained with boron trifluoride method Additionally thereagent was very safe and appropriate Shortly the reagentwas made from 97 ml commercial concentrated HCI (35ww) diluted with 415 ml of methanol and was stored in arefrigerator
A lipid sample was dissolved in 020 ml of toluene then150 ml of methanol and 030 ml of the reagent solution wereadded in this order The tube was vortexed and then heatedat 100∘C for 1 h After cooling 1 ml of hexane and 1 ml ofwaterwere added for extraction ofmethyl esters in the hexanephase
24 Silylation Agent BSA N O-Bis (Trimethylsilyl) Acetam-ide The reaction was carried out by adding 100 uL of BSA+ amount of the sample after extraction and heating in waterbath at 70∘C for two hours and after that injected into GCMSunder the above conditions
The constituents were determined by mass fragmenta-tions with the NIST mass spectral search program for theNISTEPANIH mass spectral library Version 22 (Jun 2014)
25 Animal Model Forty-five adult male New-ZealandWhite rabbits (Oryctolagus cuniculus) of 6 months of ageand weighing 3-4 kg were included in this study Rabbitswere purchased from the animal house of King Fahd MedicalResearch Centre (KFMRC) King Abdulaziz University KSARabbits were maintained for Care and Use of LaboratoryAnimals according to the criteria of US National Institutesof Health (NIH Publication No 8523 revised 1985) [17]
26 Treatment of Rabbits Induction of Hepatic Injury andExperimental Design Rabbits were housed in well-ventilatedpolypropylene cages with husk beds Rabbits were left toacclimatize to the laboratory conditions (26-28C∘ 60-80relative humidity 12 h lightdark cycle) for 10 days priorto starting the experimental running during which theyreceived standard diet and tap water ad libitum [18 19]Rabbits were divided into 2 groups normal control group(group -A) and hepatic injured group (group-B) which wassubdivided into the following
Group-A (n15) five rabbits (Subgroup-A1) out of group-A received normal diet without exposure to CCl4 hepaticintoxication to serve as negative control group Five rabbits(Subgroup-A2) received 200mgkg bw of Lepidium sativum Lseeds in their diet on daily basis till the end of the experimentFive rabbits (Subgroup-A3) received 400 mgkg bw of Lepid-ium sativum L seeds in their diet on daily basis till the end ofthe experiment [20] Group-B (n30) all rabbits of this groupwere subcutaneously injected with CCl4 starting from dayone of the experiment at a dose of 05 mlKg bw (20 CCl4in paraffin oil) of previously prepared CCl4 [21 22] Rabbitsof group-B received normal diet and were weighted twice perweek to precisely determine the CCl4 dose Also they weresubdivided as follows
Subgroup-B1 ten rabbits served as a control pollutantgroup (+ve control)Subgroup-B2 ten rabbits received 200 mgkg bw ofLSS mixed with their food dailySubgroup-B3 ten rabbits received 400 mgkg bw ofLSS mixed with their food dailyFive rabbits out of each subgroup of group-B weredecapitated 5 amp 10 weeks after experimental running
Evidence-Based Complementary and Alternative Medicine 3
500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 18000
1e+072e+073e+074e+075e+076e+077e+078e+079e+071e+08
11e+08 12e+08 13e+08 14e+08 15e+08 16e+08 17e+08
11388
11450
12428
13222
1329814028
141 148 15623
Rela
tive A
bund
ance
Time (min)Figure 1 Gas Chromatography Mass Spectrometry (GC-MS) analysis of fatty acids in LSS
Diet hygienic conditions and behaviour were observed andfollowed up on daily basis Rabbits and food were observedensuring that rabbits ate all of their food before addition ofextra seeds in their new meal according to the experimentaldesign At the end of the 5th and 10th weeks of the experimentrabbits were fasted 12-16 hours before sacrificing At theend of 5th and 10th weeks of the study blood sample fromeach target rabbits according to the experimental designwas withdrawn from the ear vein collected in a centrifugetube and kept at room temperature for 20 minutes Serawere separated by a cooling centrifuge at 3000 rpm for15 minutes and stored at -80∘C until recall Then targetrabbits were decapitated and the abdomen of each rabbitwas excised immediately after scarifying The liver of eachrabbit was removed and divided into 3 segments 1st segmentwas immersed directly into 10 formaldehyde solution andthen processed for histopathological preparation and exami-nation the 2nd segment was used forDNAextraction to studythe liver DNA integrity and the 3rd segment was used forpreparation of liver homogenate
Sera of the studied rabbits were used to estimate thelevels of ALT AST 120574-GT ALP T Bilirubin T ProteinAlbumin Cholesterol and Triglycerides which have beenpurchased from (EGY-CHEM for lab technology BaderCity Egypt) The homogenization of pancreas tissues wascarried out according to Gackowski et al [23] Liver tissuehomogenate of each sample was used to estimate the levelsof the antioxidant enzymes Catalase (CAT) glutathione per-oxidase (GPX) Glutathione reductase (GR) Glutathione-S-transferase (GST) and Super-oxide dismutase (SOD) as wellas (MDA) for all the studied rabbits using kits from (BioassayTechnology Laboratory SHANGHAI KORIAN BIOTECHCo)
27 Extraction of DNA of Rabbits Hepatic Tissues and GelElectrophoresis We studied the genomic DNA integrity of
the liver of all the studied rabbits In accordance with thepurification protocol of total DNA from rabbits tissues DNAextractionwas processed using (Spin-ColumnProtocol QIA-GEN Group) Agarose gel (2 agarose gel in 1x TAE buffer)was prepared according to Raj Kumar [24]
28 Histopathological Examination Liver tissues were dis-sected from scarified rabbits removed and fixed in 10formalin solution The fixed specimens were then trimmedwashed and dehydrated in ascending grades of alcoholThesespecimens were cleared in xylene embedded in paraffinsectioned at 4-5 120583 of thickness and stained with Hematoxylinand Eosin then examined microscopically [25]
29 Statistical Analysis Analyzing the data was conducted bystatistical package software (SPSS) The t-test of significancewas tested for identifying the differences between the meansResults were expressed as mean plusmn SEM
3 Results
31 GC-MS Analysis of LSS Extract By comparing the massspectra of the components with the NIST library ten majorfatty acids peaks were obtained out of which thirty-eightphytocomponents in LSS were described and recognized asshown in Figure 1 These ten major fatty acids were E-10-Methyl-11-tetradecen-1-ol acetate Hexadecanoic acid methylester 91215-Octadecatrienoic acid methyl ester cis-Methyl11-eicosenoate Methyl 18-methylnonadecanoate Methyl 11-docosenoate Docosanoic acid methyl ester Octadecanoicacid 910-dihydrox methyl ester bis(trifluoroacetate) Tetra-cosanoic acid methyl ester Benzoic acid 35-dicyclohexyl-4-hydroxy- methyl ester The retention time peak areachemicalformulae andmolecular weight andMZ ratio of allfatty acids found in LSS are depicted in Table 1 Similarly thechemical pattern of LSS extract of polyphenols and carbohy-drates revealed by GC-MS analyses exhibited 15 compounds
4 Evidence-Based Complementary and Alternative Medicine
Table 1 Chemical pattern of LSS extract revealed by GC-MS analyses RT = retention time Area Pct= peak area and Mwt= molecularweight
No RT Area Pct Compound name Mwt(gmol)
Chemicalformula MZ
1 113902 00408 E-10-Methyl-11-tetradecen-1-ol acetate 26824 C17H32O2 5512 114485 59674 Hexadecanoic acid methyl ester 270256 C17H34O2 7413 124277 802415 91215-Octadecatrienoic acid methyl ester 292456 C19H32O2 7924 132204 97366 cis-Methyl 11-eicosenoate 324549 C21H40O2 29245 132962 20426 Methyl 18-methylnonadecanoate 326565 C21H42O2 7416 140306 12517 Methyl 11-docosenoate 35259 C23H44O2 32047 141064 02947 Docosanoic acid methyl ester 354619 C23H46O2 741
8 148058 00701 Octadecanoic acid 910-dihydroxy- methylester bis(trifluoroacetate) 3305026 C19H38O4 741
9 148641 00826 Tetracosanoic acid methyl ester 3826633 C25H50O2 741
10 156218 02718 Benzoic acid 35-dicyclohexyl-4-hydroxy-methyl ester 2643600 C16H24O3 571
821642
116107
126009
134058
134323
142319147773 150368
152751
156087
000E+00
100E+07
200E+07
300E+07
400E+07
500E+07
600E+07
700E+07
8 10 12 14 16 18 20
Rela
tive A
bund
ance
Time (min)
Figure 2 Gas ChromatographyMass Spectrometry (GC-MS) anal-ysis of polyphenols and carbohydrates of LSS
were shown in Figure 2 These active compounds were Glyc-erol 3TMS derivative D-Pinitol pentakis(trimethylsilyl)ether Palmitic Acid TMS derivative 11-Octadecenoic acid(Z)- TMS derivative alpha-Linolenic acid TMS derivativeStearic acid TMS derivative Sinapinic acid 2TMS derivative11-Eicosenoic acid (Z)- TMS derivative Sucrose 8TMSderivative 371115-tetramethylhexadecan-13-diol silylatedSucrose 8TMS derivative Behenic acid TMS derivativeD-(+)-Turanose octakis(trimethylsilyl) ether and beta-Sitosterol TMS derivative The retention time peak areachemicalformulae and molecular weight and MZ ratio ofpolyphenols and carbohydrates found in LSS are depicted inTable 2
32 Assessment of Biochemical Markers As shown in Table 3amp Figure 3(a) serum levels of liver biomarkers ALT AST 120574-GT and ALP activities were significantly elevated (Ple 001)with percentages of 49 128 310 and 421 respectivelyin rabbits injected with CCl4 for 5 weeks and for 10 weekswith percentages of 76 159 468 and 474 respectivelySerum total bilirubin triglycerides and cholesterol levelswere also significantly elevated (Ple 001) in rabbits injected
with CCl4 for 5 weeks with percentages of 139 250and 72 respectively and for 10 weeks with percentages of114 290 and 512 respectively compared to those of thenormal control (Figure 3(b)) In addition serum total proteinand albumin levels were significantly decreased (Ple 001)in rabbits injected with CCl4 for 5 weeks with percentagesof 36 and 30 respectively and for 10 weeks as wellwith percentages of 31 and 26 respectively compared tothose of the normal control Serum ALT AST 120574-GT ALPtotal bilirubin triglycerides and cholesterol activities weresignificantly reduced (Ple 001) in rabbits protected with 200mg kg bw of LSS with percentages of 46 60 31 4850 39 and 42 respectively and for 400 mgkg bw withpercentages of 57 67 49 46 34 29 and 28respectively for 5 weeks compared to the corresponding CCl4treated group (Table 3 and Figures 3(a) and 3(b)) SerumALTAST 120574-GT ALP total bilirubin triglycerides and cholesterollevelswere also significantly reduced in rabbits protectedwith200 and 400mgkg bw of LSS for 10 weeks compared to thoseof the corresponding CCl4 treated group as shown in Table 3and Figures 3(a) and 3(b)
As shown in Table 3 and Figure 3(b) serum total proteinand albumin levels were significantly elevated (Ple 001) withpercentages of 57 37 in 200 mgkg bw of LSS protectedgroup and 39 31 in 400 mgkg bw of LSS protectedgroup for five consecutive weeks compared to those ofthe corresponding CCl4 treated group respectively Samebehaviour was noticed after 10 consecutive weeks of CCL4treatment accompanied with LSS protection the elevationwas 43 29 in 200 mgkg bw of LSS protected group and42 27 and in 400 mgkg bw of LSS protected groupcompared to those of the corresponding CCl4 treated grouprespectively
33 Assessment of Oxidative Stress and Antioxidant ActivityThe present results showed in Table 4 amp Figure 4 exert asignificant decrease (Ple 001) in the enzymatic activities ofCAT SOD GR GST and GPx in the liver homogenateof CCl4 treated rabbits 5 and 10 weeks after experimental
Evidence-Based Complementary and Alternative Medicine 5
Table 2 Chemical pattern of LSS extract of polyphenols and carbohydrates revealed by GC-MS analyses RT = retention time Area Pct=peak area and Mwt= molecular weight
No RT Area Pct Compound name Mwt Chemical formula MZ1 81846 29942 Glycerol 3TMS derivative 3086372 C12H32O3Si3 2052 116107 66731 D-Pinitol pentakis(trimethylsilyl) ether 5550881 C22H54O6Si5 733 126062 42518 Palmitic Acid TMS derivative 3286052 C19H40O2Si 3134 134058 303464 11-Octadecenoic acid (Z)- TMS derivative 3546425 C21H42O2Si 3395 134323 18965 alpha-Linolenic acid TMS derivative 3506107 C21H38O2Si 757 134906 26564 Stearic acid TMS derivative 3566584 C21H44O2Si 1178 136123 10245 Sinapinic acid 2TMS derivative 3685722 C17H28O5Si2 3689 142372 33369 11-Eicosenoic acid (Z)- TMS derivative 3826956 C23H46O2Si 36710 147773 29175 Sucrose 8TMS derivative 9197454 C36H86O11Si8 36111 149521 12532 371115-tetramethylhexadecan-13-diol silylated 298547 C20H42O 23312 150368 32061 Sucrose 8TMS derivative 9197454 C36H86O11Si8 36113 151003 14675 Behenic acid TMS derivative 4127647 C25H52O2Si 11714 15614 15318 D-(+)-Turanose octakis(trimethylsilyl) ether 9487867 C37H89NO11Si8 7315 190612 12082 beta-Sitosterol TMS derivative 4868878 C32H58OSi 129
running as compared to those of the normal control with apercentage of change (53 50 33 32 35amp 46 3737 39 and 47 respectively) Also the present resultsrevealed a significant increase (Ple 001) in the MDA levelsin the liver homogenate of CCl4 treated rabbitrsquos 5 and 10weeks after experimental running as compared to those ofthe normal control with a percentage of change (96amp 120)respectively
Daily oral administration of 200 mgkg bw of LSS withinthe rabbitrsquos diets for 5 weeks starting from day one of theexperiment significantly increased (Ple 001) the enzymaticactivities of CAT SOD GR GST and GPx in the liverhomogenate of injured rabbits with liver cirrhosis for 5 weeksas compared to those of the corresponding CCl4 treatedgroupwith a percentage of change (93 93 39 41 42)and all parameters mentioned were significantly increased(Ple 001) with 70 58 33 25 37 respectively inrabbits administrated 400 mgkg bw of LSS and injuredwith liver cirrhosis for 5 weeks as compared to those ofthe corresponding CCl4 treated group Similarly Daily oraladministration of 200 mgkg bw of LSS within the rabbitrsquosdiets for 10 weeks starting from day one of the experimentsignificantly increased (Ple 001) the enzymatic activities ofCAT SOD GR GST and GPx in the liver homogenateof injured rabbits with liver cirrhosis with a percentageof change (70 58 52 58 74) and all parametersmentioned were reduced significantly (Ple 001) to 73 4747 46 and 65 respectively in rabbits administered 400mgkg bw of LSS and injured with liver cirrhosis for 10 weeksas compared to those of the corresponding CCl4 treatedgroup Also the results revealed that daily oral administrationof 200 and 400 mgkg bw of LSS within the rabbitsrsquo dietsfor 5 and 10 weeks starting from day one of the experimentsignificantly decreased (Ple 001) the MDA levels in the liverhomogenate with a percentage of change (47amp 44 and60amp 56 for 5 and 10 weeks respectively) compared tothose of the corresponding CCl4 treated group (Table 4 ampFigure 4)
34 DNA Results of Untreated LSS Administered CCl4-Treated and LSS Protected Rabbits DNAwas extracted fromliver tissues of all the studied rabbits the banding mannerwas observable in Figure 5 Treatment of rabbits with CCl4increased significantly DNA damage of the hepatic tissuesmore than those of the untreated rabbits An entirely dif-ferent banding manner was detected in CCl4 treated rabbitswhich was absent from the liver tissues of the untreatedrabbits DNA extracted from 200 and 400 mgkg bw ofLSS protected rabbitsrsquo livers administered with CCl4 after5 and 10 weeks of the experimental running showed asignificant repair in hepatic DNA Rabbits treated with only200 and 400 mgkg bw of LSS after 5 and 10 weeks of theexperimental running did not display any kind of DNAfragmentation
35 Histopathological Observations The micrograph shownin Figure 6(a) illustrates the normal aspect of the hepatic liverfor the control group (Subgroup A1) fed with normal dietwithout exposing to CCl4 hepatic intoxication Examinationof liver tissue of the control group in which rabbits receivednormal diet and 200 mgkg bw of LSS in their diet on dailybasis till the end of the experiment without exposing to CCl4hepatic intoxication (Subgroup A2) revealed normal hepaticarchitecture (Figure 6(b)) Examination of the control groupreceived normal diet and 400mgkg bw of LSS in their diet ondaily basis till the end of the experiment without exposing toCCl4 hepatic intoxication (Subgroup A3) revealed only mildinflammation except one case which showed the adverseeffects of the drug showing evident chronic venous conges-tion moderate inflammation and bridging fibrosis (Figures6(c) 6(d) and 6(e))
According to liver tissue in rabbits exposed to CCL4intoxication (Group-B1 control pollutant group) the fol-lowing can be seen hepatocyte feathery degeneration andchronic venous congestion steatosis parenchymal and portalinflammation and fibrosis (Figures 7(a) 7(b) and 7(c)) and(Figures 7(d) 7(e) and 7(f)) respectively
6 Evidence-Based Complementary and Alternative Medicine
Table3Liverp
rofileo
funtreatedLSS
administeredC
Cl4tre
atedand
LSSprotectedrabb
itsEffectofLS
Son
serum
markere
nzym
esALTA
STG
GTALPTBILITP
ALB
TGand
CHOL
inrabb
itstre
ated
with
CCL4indu
cedhepatotoxicityas
indicatedin
thefi
gureA
lldatawe
reexpressedas
meanplusmnSE
MV
aluesw
eres
tatistic
allyteste
dusingtheS
tudent
t-testandsig
nificant
differencesatplt005
andplt001
asindicatedby
(lowast)amp
(lowastlowast)com
paredto
norm
alcontrolby
()amp
()
comparedto
CCL4aft
er5w
eeksand
by(+)amp
(++)
comparedto
CCL4aft
er10
weeks
respectiv
ely
Group
ALT
(UL)
AST
(UL)
GGT(U
L)
ALP
(UL)
TBILI
(mgdl)
TP(gdl)
ALB
(gdl)
CHOL(m
gdl)
TG(m
gdl)
Con
trol
3801plusmn
468
2725plusmn
192
702plusmn12
42202plusmn16
2028plusmn003
650plusmn031
355plusmn017
1632plusmn506
4203plusmn469
LS(200
mgkg
bw)
3148plusmn678
2941plusmn
331
848plusmn12
05702plusmn229lowastlowast
043plusmn003lowast
640plusmn025
324plusmn013
3217plusmn287lowast
5274plusmn279lowast
LS(400
mgkg
bw)
1644plusmn14
6lowastlowast
2076plusmn343lowast
629plusmn229
5308plusmn16
1lowastlowast
046plusmn003lowastlowast
586plusmn047
344plusmn010
3161plusmn
305lowast
2610plusmn304lowast
CCl4(5
wk)
5676plusmn546lowastlowast
6230plusmn391lowastlowast
2872plusmn276lowastlowast
11483plusmn273lowastlowast
067plusmn004lowastlowast
414plusmn014lowastlowast
248plusmn025lowastlowast
8921plusmn
762lowastlowast
14774plusmn74
4lowastof
Change
49128
310
421
139
-36
-30
72250
CCl4(10wk)
6700plusmn476lowastlowast
7069plusmn645lowastlowast
3978plusmn226lowastlowast
12638plusmn371lowastlowast
060plusmn004lowastlowast
445plusmn01lowastlowast
261plusmn022lowastlowast
9996plusmn616lowastlowast
16379plusmn89lowastlowast
of
Change
76159
468
474
114-3
1-2
6512
29
CCl4+LS
(200
mgkg
bw)5
wk
3062plusmn219
2490plusmn302
1962plusmn19
5
5960plusmn237
033plusmn003
649plusmn020
339plusmn012
5135plusmn547
8978plusmn611
of
Change
-46
-60
-31
-48
-50
5737
-42
-39
CCl4+LS
(400
mgkg
bw)5
wk
2423plusmn
291++
2027plusmn309++
1464plusmn17
3++
6146plusmn416++
044plusmn003++
576plusmn062++
326plusmn020++
6306plusmn524++
10539plusmn562++
of
Change
-57
-67
-49
-46
-34
3931
-29
-28
CCl4+LS
(200
mgkg
bw)10wk
3804plusmn371
2390plusmn264
1581plusmn
142
5259plusmn257
038plusmn004
636plusmn038
338plusmn009
5236plusmn447
9870plusmn344
of
Change
-43
-66
-60
-58
-36
4329
-47
-39
CCl4+LS
(400
mgkg
bw)10wk
4018plusmn292++
2516plusmn508++
2292plusmn254++
7954plusmn484++
048plusmn003
++635plusmn038++
334plusmn014++
6557plusmn79
8++
10513plusmn666
++of
Change
-40
-64
-42
-37
-20
4227
-34
-36
Evidence-Based Complementary and Alternative Medicine 7
01020304050607080
ALT
Lev
e (U
L)
ALTlowastlowast
lowastlowast
lowastlowast
++++
0102030405060708090
AST
Lev
e (U
L)
AST
lowastlowast
lowast
lowastlowast
++ ++
05
1015202530354045
GG
T Le
ve (U
L)
GGTlowastlowast
lowastlowast
++
++
020406080
100120140
ALP
Lev
el (U
L)
ALPlowastlowast
lowastlowast
lowastlowastlowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
(a) Effect of LSS aqueous extract on serum marker enzymes ALT AST GGTand ALP in rabbits treated with CCL4 induced hepatotoxicity as indicated inthe figure All data were expressed as mean plusmn SEM Values were statisticallytested using the Student t-test and significant differences at p lt 005 and p lt 001indicated by (lowast) amp (lowastlowast) compared to normal control by () amp () comparedto CCL4 after 5 weeks and by (+) amp (++) compared to CCL4 after 10 weeksrespectively
012345678
TP L
evel
(gL
)
TP
lowastlowastlowastlowast
++ ++
00102030405060708
TBI
LI L
evel
(gL
) TBILI
lowastlowastlowast
lowastlowastlowastlowast
++++
005
115
225
335
4
ALB
Lev
el (g
L)
ALB
lowastlowastlowastlowast
++++
020406080
100120140160180200
TG L
evel
(gL
)
TG
lowast
lowastlowast
lowastlowast
lowastlowast
++++
0
20
40
60
80
100
120
CHO
L Le
vel (
gL)
CHOLlowastlowast
lowastlowast
++++
lowastlowast
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
(b) Effect of with LSS aqueous extract on biochemical parameters TBILI TPALB TG and CHOL in rabbits treated with CCL4 induced hepatotoxicity asindicated in the figure All data were expressed as mean plusmn SEM Values werestatistically tested using the Student t-test and significant differences at p lt 005and p lt 001 indicated by (lowast)amp (lowastlowast) compared to normal control by () amp ()compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4 after 10weeks respectively
Figure 3
8 Evidence-Based Complementary and Alternative Medicine
Table4En
zymaticactiv
itiesofantio
xidant
enzymesandMDAofun
treatedLSS
administeredC
Cl4tre
atedand
LSSprotectedrabb
itsA
lldatawe
reexpressedas
meanplusmnSE
MV
aluesw
ere
statistic
allyteste
dusingtheS
tudent
t-testandsig
nificantd
ifferencesa
tplt005
andplt001
asindicatedby
(lowast)amp
(lowastlowast)com
paredto
norm
alcontrolby
()amp
()
comparedto
CCL4aft
er5
weeksandby
(+)amp
(++)
comparedto
CCL4aft
er10
weeksrespectiv
ely
Group
CAT
(nggtissue)
SOD
(nggtissue)
GR
(nggtissue)
GST
(nggtissue)
GPx
(nggtissue)
MDA(nmolm
l)
Con
trol
3155plusmn
199
2820plusmn10
92029plusmn099
1537plusmn12
12197plusmn17
0647plusmn057
LS(200
mgkg
bw)
3127plusmn18
62631plusmn15
51908plusmn077
1542plusmn067
2129plusmn12
8635plusmn045
LS(400
mgkg
bw)
2843plusmn204
2676plusmn18
81972plusmn12
81555plusmn14
32031plusmn074
635plusmn060
CCl4(5
wk)
1475plusmn258lowastlowast
1406plusmn13
9lowastlowast
1358plusmn061lowastlowast
1041plusmn
108lowastlowast
1409plusmn099lowastlowast
1273plusmn10
6lowastlowast
of
Change
-53
-50
-33
-32
-35
96CCl4(10wk)
1682plusmn15
7lowastlowast
1767plusmn202lowastlowast
1277plusmn13
6lowastlowast
940plusmn073lowastlowast
1152plusmn18
2lowastlowast
1427plusmn10
7lowastlowast
of
Change
-46
-37
-37
-39
-47
120
CCl4+LS
(200
mgkg
bw)5
wk
2846plusmn201
2720plusmn094
1892plusmn093
1472plusmn118
2009plusmn112
667plusmn046
of
Change
9393
3941
42-4
7CCl4+LS
(400
mgkg
bw)5
wk
2519plusmn17
7++
2231plusmn14
0++
1813plusmn094++
1304plusmn13
1++
1941plusmn
192+
+713plusmn072++
of
Change
7058
3325
37-4
4CCl4+LS
(200
mgkg
bw)10wk
2872plusmn201
2796plusmn222
1949plusmn096
1492plusmn15
8
2006plusmn17
9
569plusmn046
of
Change
7058
5258
74-6
0CCl4+LS
(400
mgkg
bw)10wk
2919plusmn17
0++
2610plusmn14
3++
1887plusmn17
3++
1378plusmn086++
1906plusmn14
1++
619plusmn052++
of
Change
7347
4746
65-5
6
Evidence-Based Complementary and Alternative Medicine 9
05
10152025303540
CAT
Leve
(ng
g tis
sue)
CAT
lowastlowastlowastlowast
++++
0
5
10
15
20
25
30
35
SOD
Lev
e (ng
lg ti
ssue
)
SOD
lowastlowast
lowastlowast
++++
0
5
10
15
20
25
GR
Leve
(ng
g tis
sue)
GR
lowastlowastlowastlowast
++++
02468
1012141618
GST
Lev
e (ng
g ti
ssue
)
GST
lowastlowastlowastlowast
++++
0
5
10
15
20
25
GPx
Lev
e (ng
g ti
ssue
)
GPx
lowastlowastlowastlowast
++++
02468
1012141618
MD
A L
eve (
nmol
g ti
ssue
)
MDA
lowastlowast
lowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
Figure 4 Effects of LSS on oxidative stress markers in rabbits treated with CCL4 induced hepatotoxicity as indicated in the figure All datawere expressed as mean plusmn SEM Values were statistically tested using the Student t-test and significant differences at p lt 005 and p lt 001 asindicated by (lowast) amp (lowastlowast) compared to normal control by () amp () compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4after 10 weeks respectively
Examination of liver tissue exposed to CCL4 as well as200 mgkg bw of LSS in their diet on daily basis (GroupB2) reveals that the group which is sacrificed 5 weeksafter experimental running shows evident chronic venouscongestion and feathery degeneration in two cases only mildfeathery degeneration in others no steatosis or isolated vesic-ular steatosis mild parenchymal and portal inflammation
(Figures 8(a) 8(b) and 8(c)) Examination of the groupsacrificed 10 weeks after experimental running shows theimprovement of the histoarchitectural changes of the livertowards the normal aspect showing regenerating hepato-cytes with no steatosis discrete chronic venous congestionand discrete inflammatory infiltrate (Figures 8(d) 8(e) and8(f))
10 Evidence-Based Complementary and Alternative Medicine
M 1 2 3 4 5 6 7 8 9bp
10000
3000
2000
1500
1000
750
500
300
Figure 5 Agarose gel showingDNAdamage byCCl4 and protectiveeffects of various fractions of LSS Lanes from left (M) molecularweight marker (1) negative control (2) LSS (200 mgkg bw) (3) LSS(400mgkg bw) (4) CCl4 for 5weeks (5) CCl4 for 10weeks (6)amp (7)CCl4 + LSS (200 mgkg bw) (8) amp (9) CCl4 + LSS (400 mg kg bw)
Examination of liver tissue of rabbits exposed to CCL4as well as 400 mgkg bw of LSS in their diet on daily basis(Group B3) reveals in the group that scarified 5 weeks afterexperimental running improvement of the histoarchitecturalchanges of the liver as there is no chronic venous congestionor only discrete chronic venous congestion and mild featherydegeneration no steatosis or only isolated vesicular steatosisand mild parenchymal and portal inflammation (Figures9(a) 9(b) and 9(c)) But the group that was sacrificed10 weeks after experimental running shows adverse effectsof the drug in the form of steatosis and chronic venouscongestion which is evident in two cases with mild tomoderate inflammation Bridging fibrosis is seen in one case(Figures 9(d) 9(e) 9(f) and 9(g))
4 Discussion
Hepatic disease became a significant public health concernowing to growing rate of obesity in all age populations [26]The conservative treatments of liver problems such as acuteand chronic liver hepatitis liver cirrhosis and fatty liverare often insufficient due to hazardous effects initiated byhepatotoxic drugs of chemical origin [27] Medicinal plantsowing to their abundant hepatoprotective natural ingredi-ents have been comprehensively investigated to treat liverdisorders In the current study hepatoprotective efficacy ofLSS was investigated via hepatotoxic classical model inducedby carbon tetrachloride
In organ and tissue systems CCl4 is stimulated bycytochrome P-450 dependent mixed oxidase in the hepaticendoplasmic reticulum to procedure free radical trichloro-methyl (CCl3 and Cl3COO) This causes changes in themembrane of the endoplasmic reticulum and other cel-lular membranes resulting in an increase in calcium ionpermeability through the plasma membrane causing severeinstabilities of calcium homeostasis leading to necrotic celldeath [28 29] CCL4 affects many organs causing degenera-tion of liver fatty layer and centrilobular necrosis [29] Thisphenomenon indicates that CCl4 is a powerful hepatotoxic
agent and may be used for the characterization of the hepaticdisease treatment medicines Elevation of liver enzymaticmarkers (ALT AST ALP and bilirubin) and increased lipidperoxidation are further evidences of hepatotoxicity ALTand AST that exist in the hepatocytes can certainly leak intothe peripheral blood as soon as the hepatocytes are injured[29] Lepidium sativum seed analysis by GCMS exhibitedthe presence of high concentrations of mainly 120572-linolenicacid (ALA) which is nminus3 fatty acid an omega-3 fatty acidfound in seeds exerting its bioactivity in ameliorating thehepatic intoxication and oxidative stress including liver injuryinduced by alcohol [30] liver steatosis [31 32] nonalcoholichepatic disease [33] and parenteral nutrition-associated liverdisease [34] Pari and Jalaludeen stated that orientationof the active groups in sinapic acid increases antioxidantefficiency as metal chelators and promotes its protectiveefficacy against liver oxidative stress in arsenic intoxicatedrats [35]The current study showed that sinapic acid as ametalchelator may exert its biological activity in improving liverfunction
Lepidium sativum seed extracts have proved hepatopro-tective effects against CCl4 induced liver damage A researchstudy on male Albino Wistar rats demonstrated significantreduction in hepatotoxicity induced by CCl4 administeredwith 200-400 mgkg of Lepidium sativum seeds extractincluded within the diet Serum level of AST ALT levels andbilirubin levels were significantly elevated in CCl4 treated ratscompared to those of normal control However a significantreduction in the levels of these parameters in Lepidiumsativum seed extracts protected rats was observed The hep-atoprotective effect could be due to the presence of alkaloidcoumarin flavonoids tannin and triterpenes which enhanceantioxidant activity and reduce free radical production fromCCl4 which is the basic triggering factor for hepatotoxicity[36 37]
In the present study we evaluated the hepatoprotectiveefficacy of L sativum seeds in white male New-Zealand rab-bits The present results revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withL sativum seeds significantly repaired their liver injuriousmarker enzymes as well as bilirubin total protein and albu-min hence approving its hepatoprotective effect The resultsare in agreement with those of Afaf et al [12] where theystated the role of LSS as efficient hepatoprotective agent Alsothe current results are consistent with those of Mohamedand his coworkers [38] who reported that preprotectionwith LSEE (Lepidium sativum ethanolic extract) significantlyprohibited the D-GalNLPS induced rise in hepatic enzymes(AST ALT 120574-GGT ALP total bilirubin LDH and total pro-tein) thus significantly alleviated the reduction of lipid per-oxidation and repaired the antioxidant enzymatic status andtotal protein restoring them to normal levels D-GalNLPShas been widely used to examine the mechanisms underlyingacute liver failure [39 40] The hepatoprotective significanceof LSEE could be due to downregulation of cytokines (TNF-120572 IL-6) and stress gene (iNOS and HO-1) mRNA expres-sion and upregulation of IL-10 Lepidium sativum ethanolicextract (LSEE) preprotection also improved the degree ofstructural damage and reduced inflammatory infiltration in
Evidence-Based Complementary and Alternative Medicine 11
(a) (b)
(c) (d)
(e)
Figure 6 Effect of LSS on histopathological changes of liver tissues of normal groups in rabbits (a) Showing normal histological picture ofhepatic lobule that consists of central vein surrounded by normal hepatocytes (HEx100) (b) Showing liver sections of the rabbits treatedwith LSS (200 mgkg bw) which revealed normal histological picture of hepatic lobule that consists of central vein surrounded by normalhepatocytes (HEx100) (c) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealed normal hepatic architecturewith only mild inflammation [arrows] (HEx100) (d) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealedevident chronic venous congestion and moderate inflammation [arrows] (HEx100) (e) Showing liver sections of the rabbits treated with LSS(400 mgkg bw) which revealed portal to portal bridging fibrosis [arrow] (HEx200)
hepatic cells These outcomes established that LSEE alle-viates hepatic impairments and structural injury throughthe decay of oxidative stress inflammation and apopto-sis in the liver [38] Mohamed Sakran and his colleagues[10] suggested that 56-dimethoxy-210158403-methylenedioxy-7-C-120573-D glucopyranosyl isoflavone extracted from Lepidiumsativum seeds could improve the lipid profile and the liverfunctions in serum and reduce the free radicals generationby enhancing the antioxidant defense mechanism Newsuggested isoflavonoid can be used as a probable antioxi-dant against paracetamol hepatotoxicity with its antioxidant
characteristics and could restore the hepatic functionstowards normalization [10]
In the present study we found that improved cholesteroland triglycerides levels in L sativum seeds protected rabbitsas compared to CCl4 treated rabbits also support the hepato-protective effect of LSSThese results can also be correlated tothose of Shukla et al [41] who reported the role of L sativumseeds as hypolipidemic agent Also the results of the presentstudy agreed with those of Abdulrahman et al [20] whoseresults showed that L sativum seeds have hepatoprotectiveactivity that could be due to its antioxidant activity together
12 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
Figure 7 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) In rabbits sacrificed 5 weeks afterexperimental running as shown in (a) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100)(b) steatosis (30) in one case (HEx100) and (c) mild parenchymal inflammation [arrow] (HEx200) In rabbits sacrificed 10 weeks afterexperimental running as shown in (d) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100) (e)steatosis (about 20) and moderate parenchymal and portal inflammation (HEx100) and (f) bridging fibrosis [arrow] (HEx100)
with the presence of anti-inflammatory ingredients in Lsativum seeds extract
The results of the current study revealed that the exposureto CCl4 for 5 and 10 weeks led to a significant decrease inthe enzymatic activities of CAT GPx GR GST and SOD inthe liver homogenate of CCl4 treated rabbits 5 and 10 weeksafter experimental running compared to those of the normalcontrolThe concurrent protection of rabbits exposed toCCl4for 5 and 10weeks with 200amp 400mg kg bw LSS significantlyrepaired their antioxidant status and reduced significantly theMDA levels hence confirming its hepatoprotective efficacy
Although the precise molecular mechanism of action ofLSS is not completely understood some researchers [42]have confirmed the role of LSS in inhibiting oxidative stress
associated liver damage The antioxidant activity of LSScould be due to the presence of phenolic compound andflavonoids in these seeds as have been evidenced through thepreliminary screening study on LSS extract using HPLCOurresults match with those of Lee et al [42] who reported thatphenolic compounds play an essential role against oxidativestress related liver damage
A unique expectation of the current study is to confirmthat CCl4 induces liver DNA injury that was assessed usinggel electrophoretic method The results of the current studyshowed that CCL4 induces DNA injury as presented bya significant progression in liver DNA disintegration ofCCl4 treated rabbits above untreated control in the gelelectrophoresis
Evidence-Based Complementary and Alternative Medicine 13
(a) (b)
(c) (d)
(e) (f)
Figure 8 Effect of LSLS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (200 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) evident chronic venous congestion and feathery degeneration [arrow](HEx100) (b) mild feathery degeneration and isolated vesicular steatosis [arrow] (HEx200) and (c) moderate parenchymal and portalinflammation (HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) regenerating hepatocytes with isolatedvesicular steatosis (HEx100) (e) discrete chronic venous congestion and no steatosis (HEx400) and (f) discrete inflammatory infiltrate(HEx100)
Lipid peroxidation of the nuclear membrane may bedirectly accompanied with nuclear DNA damage [43]Nuclear DNA is concomitant with particular regions of thenuclear membrane [44]The close proximity of nuclear DNAto the nuclear membrane can ease the interaction of genomicDNA with the peroxyl radicals and further reactive inter-mediates formed during membrane lipid peroxidation [43]In the presence of transition metals the hydroxyl radicalsgenerated by CCL4 autoxidation can trigger peroxidationof the nuclear membrane lipids by metal catalyzed Haber-Weiss reaction the lipid peroxides levels and intermediate
free radicals could then be augmented by a chain reactionSubsequently the reactive species oxidize nuclearDNAwhenthe lipid peroxidation reaction occurs in the nuclear mem-brane since the nuclear membrane controls the transportof messenger RNA into the cytoplasm and supports thenuclear division process nuclear membrane peroxidationof lipids and proteins Moreover oxidative nuclear DNAdamage could disrupt these serious cellular functions [43]
DNA findings showed that DNA disintegration is adirect result after CCl4 treatment in rabbitsrsquo liver tissueswhich was reduced by participation of antioxidants through
14 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
(g)
Figure 9 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (400 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) discrete chronic venous congestion (HEx200) (b) centrilobular fibrosis[arrows] regenerating hepatocytes and isolated vesicular steatosis (HEx100) and (c) mild parenchymal and portal inflammation [arrow](HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) mild chronic venous congestion and isolated vesicularsteatosis (HEx100) (e) evident chronic venous congestion [arrows] (HEx100) (f) steatosis (40) with moderate inflammation (HEx100) and(g) portal to portal bridging fibrosis in one case [arrow] (HEx100)
Evidence-Based Complementary and Alternative Medicine 15
administration of 200 mg amp 400 mgkg bw L sativum seedsNearly complete repair of DNA damage was significant aftertreating rabbits with 200 mg amp 400 mgkg bw L sativumseeds The protective effect of L sativum seeds on hepaticDNA injury may be due to the antioxidant activity ofL sativum seeds via direct scavenging of free radicals orinterfering with free radicals generation
Concerning histopathological evaluation in our studyrabbitsrsquo livers exposed to CCL4 show portal and parenchymalinflammatory infiltrate feathery and ballooning degenera-tion fatty change and fibrosis Our data are consistent withRajeswary et al [45] who reported dilatation of sinusoidsfatty change disarrangement of normal hepatocytes and cen-trilobular necrosis Also A S Bernacchi et al [46] reportedthat CCL4 leads to liver necrosis and fat accumulation in therabbit
The present study revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withLSS improved their liver histopathological architecture inthe form of less inflammation regenerating hepatocytes nosteatosis or isolated vesicular steatosis no chronic venouscongestion or just discrete congestion and no fibrosis henceapproving its hepatoprotective effectThe results are in agree-ment with Afaf et al [12] and Abdulrahman et al [20] whoobserved the improvement of the histological structure inextract treated group as the histopathologic findings showedless inflammation well restored hepatocytes and less area ofnecrosis as compared to severe necrosis and inflammationin CCl4 treated group of animals which also supports thehepatoprotective effects of LLS Their findings suggest thepossibility of the presence of anti-inflammatory compoundsin LSS extract which could also be a contributing factortowards the hepatoprotective action of LSS
Althnaian [47] also reported that examination of hep-atocytes showed mild to moderate degree of recovery LSSattenuated the hepatic fat accumulation decreased vacuolardegeneration and improved the architecture of hepatocytes
In our study the reducing effect of LSS on fatty changecaused by CCL4 intoxication is indicating the protective andcurative role against fatty liver Our results are supported byShukla et al [41] who reported that LSS significantly reducedsteatosis Their histopathological observations confirmed thecurative efficacy of L sativum liver damage Microvesicularfatty changes hepatocyte ballooning and steatosis weremarkedly corrected by LSS Also Wadhwa et al [36] foundthat the severe fatty changes in the livers of rats caused byCCl4 were decreased in the treated groups
In the present study chronic venous congestion featherydegeneration and fatty changes show mild improvement inrabbits receiving 200mgkg bw of LSS for 5 weeks and showmoderate improvement in rabbits that received 200mgkg bwof LSS for 10 weeks and rabbits that received 400mgkg bwof LSS for 5 weeks Our data are consistent with Afaf et al[12] who observed that severe centrilobular hepatocellularvacuolation hemorrhages and congestion of the centralveins noticed in CCL4 group showed mild improvement ingroups that received 200 mgkg bw of LSS and moderateimprovement in groups that received 400 mgkg bw ofLSS
On the other hand rabbits that received 400mgkg bw ofLSS for 10 weeks showed evidence of liver toxicity in the formof venous congestion hepatocyte feathery degeneration fattychange and fibrosis These results are compatible with Bafeeland Ali [48] who reported that LSS had minimal effect onliver parenchyma if given in low doses So LSS could be usedas food additive or supplement Mineral content of calciumand phosphate and the presence of essential fatty acids inconsiderable level make LSS of beneficial nutritional valuefor many body organs including the liver However LSS inhigh dose showed some toxic effect on renal parenchyma rep-resented by focal necrosis hepatocyte apoptosis and portalinflammatory cell infiltrate Burow et al [49] reported thatthese hazardous effects of LSS may be due to isothiocyanatesreported to be one of LSS constituents
5 Conclusions and Recommendations
The present study showed that LSS is a key factor ininhibition of the hazardous effects of CCl4 LSS treatmentcould significantly alleviate to a greater extent the degree oflipid peroxidation and restore the antioxidative enzymes andshift liver function to normal levels In addition to antioxi-dants activity LSS attenuated the hepatic fat accumulationdecreased vacuolar degeneration ameliorated the degree ofstructural damage in the hepatic cells and improved thearchitecture of hepatocytes The current findings demon-strated that LSS have marked hepatoprotective activity Infuture work active ingredients of LSS extract should bestudied in various animal models of severe toxicity inducedby CCL4 and other hepatotoxic agents The active ingredientsof this plant are likely to be applied as one of the future drugsagainst acute liver toxicity
Data Availability
Data used to support the findings of this study are availablefrom the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This project was funded by the Deanship of ScientificResearch (DSR) King Abdulaziz University Jeddah undergrant no S-17-314-37 The authors therefore acknowledgewith thanks DSR technical and financial support
References
[1] M K Manibusan M Odin and D A Eastmond ldquoPostu-lated carbon tetrachloride mode of action a reviewrdquo Journalof Environmental Science and Health Part C EnvironmentalCarcinogenesis and Ecotoxicology Reviews vol 25 no 3 pp 185ndash209 2007
[2] N Brautbar and J Williams II ldquoIndustrial solvents andliver toxicity risk assessment risk factors and mechanismsrdquo
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
2 Evidence-Based Complementary and Alternative Medicine
hypoglycaemic activity both in diabetic and normal ratswithout interfering with insulin secretion [5] The seeds arerecognized to improve the symptoms of asthma and recoverlung function in asthmatic patients [6] In folk medicineseveral parts of Lepidium sativum have been utilized forthe treatment of liver problems jaundice spleen diseasesmenstrual problems gastrointestinal disorders bone frac-ture and arthritis [7ndash9] Phytochemical research studies ofLepidium sativum demonstrated the existence of flavonoidsglucosinolates tannins alkaloids triterpenes sterols benzylisothiocyanate [10 11] that were stated to possess anal-gesic anti-inflammatory activities antioxidant capacitiesand hepatoprotective characters [10 12ndash14] Although pre-vious reports revealed that hepatoprotective efficacy of LSSagainst doxorubicin [15] and CCl4 [12] induced intoxicationhas been described up to our knowledge no studies havebeen conducted on the LSS existing in the Arabian regionMoreover the uniqueness of this study is that the active ingre-dients of LSS extract could be responsible for the bioactivityof these seeds Therefore the current study was designed toinvestigate the probable protective effect of Lepidium sativumseeds against CCl4 induced hepatotoxicity in New-Zealandrabbits
2 Materials and Methods
21 Preparation of Lepidium sativum Seeds (LSS) Aque-ous Extract Lepidium sativum seeds were gained fromAlquaseem market of traditional medicine KSA recognizedand identified by a professor of taxonomy and depositedat the Herbarium of Biological Department Faculty ofScience King Abdulaziz University Before extraction LSSwere washed using double deionized distilled water driedand crushed by pestle and mortar The seeds were permittedto dry in the sunlight for 2 days then homogenized to a finepowder and kept in free-moisture impervious container untiluse
22 Chromatographic Analysis of LSS Using GC-MS Chro-matographic analysis using GC-MS was carried out (AgilentTechnologies 7890B GC Systems combined with 5977AMassSelective Detector) Capillary column (HP-5MS Capillary300mtimes025mm IDtimes025120583m film) and helium as carrier gaswere used at a rate of flowof 19mlminwith 1120583l injectionThesample was analyzed with the column held initially for 3 minat 40∘C after injection then the temperature was increasedto 300∘Cwith a 20∘Cmin heating ramp with a 40 min holdInjection was carried out in split-lessmode at 300∘CMS scanrange was (mz) 50ndash550 atomic mass units (AMU) underelectron impact (EI) ionization (70 eV)
23 Preparation of Fatty AcidMethyl Esters Fatty acidmethylesters were prepared fromdifferent sources using commercialaqueous HCl [16] Yields of FAME were similar to thoseobtained with boron trifluoride method Additionally thereagent was very safe and appropriate Shortly the reagentwas made from 97 ml commercial concentrated HCI (35ww) diluted with 415 ml of methanol and was stored in arefrigerator
A lipid sample was dissolved in 020 ml of toluene then150 ml of methanol and 030 ml of the reagent solution wereadded in this order The tube was vortexed and then heatedat 100∘C for 1 h After cooling 1 ml of hexane and 1 ml ofwaterwere added for extraction ofmethyl esters in the hexanephase
24 Silylation Agent BSA N O-Bis (Trimethylsilyl) Acetam-ide The reaction was carried out by adding 100 uL of BSA+ amount of the sample after extraction and heating in waterbath at 70∘C for two hours and after that injected into GCMSunder the above conditions
The constituents were determined by mass fragmenta-tions with the NIST mass spectral search program for theNISTEPANIH mass spectral library Version 22 (Jun 2014)
25 Animal Model Forty-five adult male New-ZealandWhite rabbits (Oryctolagus cuniculus) of 6 months of ageand weighing 3-4 kg were included in this study Rabbitswere purchased from the animal house of King Fahd MedicalResearch Centre (KFMRC) King Abdulaziz University KSARabbits were maintained for Care and Use of LaboratoryAnimals according to the criteria of US National Institutesof Health (NIH Publication No 8523 revised 1985) [17]
26 Treatment of Rabbits Induction of Hepatic Injury andExperimental Design Rabbits were housed in well-ventilatedpolypropylene cages with husk beds Rabbits were left toacclimatize to the laboratory conditions (26-28C∘ 60-80relative humidity 12 h lightdark cycle) for 10 days priorto starting the experimental running during which theyreceived standard diet and tap water ad libitum [18 19]Rabbits were divided into 2 groups normal control group(group -A) and hepatic injured group (group-B) which wassubdivided into the following
Group-A (n15) five rabbits (Subgroup-A1) out of group-A received normal diet without exposure to CCl4 hepaticintoxication to serve as negative control group Five rabbits(Subgroup-A2) received 200mgkg bw of Lepidium sativum Lseeds in their diet on daily basis till the end of the experimentFive rabbits (Subgroup-A3) received 400 mgkg bw of Lepid-ium sativum L seeds in their diet on daily basis till the end ofthe experiment [20] Group-B (n30) all rabbits of this groupwere subcutaneously injected with CCl4 starting from dayone of the experiment at a dose of 05 mlKg bw (20 CCl4in paraffin oil) of previously prepared CCl4 [21 22] Rabbitsof group-B received normal diet and were weighted twice perweek to precisely determine the CCl4 dose Also they weresubdivided as follows
Subgroup-B1 ten rabbits served as a control pollutantgroup (+ve control)Subgroup-B2 ten rabbits received 200 mgkg bw ofLSS mixed with their food dailySubgroup-B3 ten rabbits received 400 mgkg bw ofLSS mixed with their food dailyFive rabbits out of each subgroup of group-B weredecapitated 5 amp 10 weeks after experimental running
Evidence-Based Complementary and Alternative Medicine 3
500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 18000
1e+072e+073e+074e+075e+076e+077e+078e+079e+071e+08
11e+08 12e+08 13e+08 14e+08 15e+08 16e+08 17e+08
11388
11450
12428
13222
1329814028
141 148 15623
Rela
tive A
bund
ance
Time (min)Figure 1 Gas Chromatography Mass Spectrometry (GC-MS) analysis of fatty acids in LSS
Diet hygienic conditions and behaviour were observed andfollowed up on daily basis Rabbits and food were observedensuring that rabbits ate all of their food before addition ofextra seeds in their new meal according to the experimentaldesign At the end of the 5th and 10th weeks of the experimentrabbits were fasted 12-16 hours before sacrificing At theend of 5th and 10th weeks of the study blood sample fromeach target rabbits according to the experimental designwas withdrawn from the ear vein collected in a centrifugetube and kept at room temperature for 20 minutes Serawere separated by a cooling centrifuge at 3000 rpm for15 minutes and stored at -80∘C until recall Then targetrabbits were decapitated and the abdomen of each rabbitwas excised immediately after scarifying The liver of eachrabbit was removed and divided into 3 segments 1st segmentwas immersed directly into 10 formaldehyde solution andthen processed for histopathological preparation and exami-nation the 2nd segment was used forDNAextraction to studythe liver DNA integrity and the 3rd segment was used forpreparation of liver homogenate
Sera of the studied rabbits were used to estimate thelevels of ALT AST 120574-GT ALP T Bilirubin T ProteinAlbumin Cholesterol and Triglycerides which have beenpurchased from (EGY-CHEM for lab technology BaderCity Egypt) The homogenization of pancreas tissues wascarried out according to Gackowski et al [23] Liver tissuehomogenate of each sample was used to estimate the levelsof the antioxidant enzymes Catalase (CAT) glutathione per-oxidase (GPX) Glutathione reductase (GR) Glutathione-S-transferase (GST) and Super-oxide dismutase (SOD) as wellas (MDA) for all the studied rabbits using kits from (BioassayTechnology Laboratory SHANGHAI KORIAN BIOTECHCo)
27 Extraction of DNA of Rabbits Hepatic Tissues and GelElectrophoresis We studied the genomic DNA integrity of
the liver of all the studied rabbits In accordance with thepurification protocol of total DNA from rabbits tissues DNAextractionwas processed using (Spin-ColumnProtocol QIA-GEN Group) Agarose gel (2 agarose gel in 1x TAE buffer)was prepared according to Raj Kumar [24]
28 Histopathological Examination Liver tissues were dis-sected from scarified rabbits removed and fixed in 10formalin solution The fixed specimens were then trimmedwashed and dehydrated in ascending grades of alcoholThesespecimens were cleared in xylene embedded in paraffinsectioned at 4-5 120583 of thickness and stained with Hematoxylinand Eosin then examined microscopically [25]
29 Statistical Analysis Analyzing the data was conducted bystatistical package software (SPSS) The t-test of significancewas tested for identifying the differences between the meansResults were expressed as mean plusmn SEM
3 Results
31 GC-MS Analysis of LSS Extract By comparing the massspectra of the components with the NIST library ten majorfatty acids peaks were obtained out of which thirty-eightphytocomponents in LSS were described and recognized asshown in Figure 1 These ten major fatty acids were E-10-Methyl-11-tetradecen-1-ol acetate Hexadecanoic acid methylester 91215-Octadecatrienoic acid methyl ester cis-Methyl11-eicosenoate Methyl 18-methylnonadecanoate Methyl 11-docosenoate Docosanoic acid methyl ester Octadecanoicacid 910-dihydrox methyl ester bis(trifluoroacetate) Tetra-cosanoic acid methyl ester Benzoic acid 35-dicyclohexyl-4-hydroxy- methyl ester The retention time peak areachemicalformulae andmolecular weight andMZ ratio of allfatty acids found in LSS are depicted in Table 1 Similarly thechemical pattern of LSS extract of polyphenols and carbohy-drates revealed by GC-MS analyses exhibited 15 compounds
4 Evidence-Based Complementary and Alternative Medicine
Table 1 Chemical pattern of LSS extract revealed by GC-MS analyses RT = retention time Area Pct= peak area and Mwt= molecularweight
No RT Area Pct Compound name Mwt(gmol)
Chemicalformula MZ
1 113902 00408 E-10-Methyl-11-tetradecen-1-ol acetate 26824 C17H32O2 5512 114485 59674 Hexadecanoic acid methyl ester 270256 C17H34O2 7413 124277 802415 91215-Octadecatrienoic acid methyl ester 292456 C19H32O2 7924 132204 97366 cis-Methyl 11-eicosenoate 324549 C21H40O2 29245 132962 20426 Methyl 18-methylnonadecanoate 326565 C21H42O2 7416 140306 12517 Methyl 11-docosenoate 35259 C23H44O2 32047 141064 02947 Docosanoic acid methyl ester 354619 C23H46O2 741
8 148058 00701 Octadecanoic acid 910-dihydroxy- methylester bis(trifluoroacetate) 3305026 C19H38O4 741
9 148641 00826 Tetracosanoic acid methyl ester 3826633 C25H50O2 741
10 156218 02718 Benzoic acid 35-dicyclohexyl-4-hydroxy-methyl ester 2643600 C16H24O3 571
821642
116107
126009
134058
134323
142319147773 150368
152751
156087
000E+00
100E+07
200E+07
300E+07
400E+07
500E+07
600E+07
700E+07
8 10 12 14 16 18 20
Rela
tive A
bund
ance
Time (min)
Figure 2 Gas ChromatographyMass Spectrometry (GC-MS) anal-ysis of polyphenols and carbohydrates of LSS
were shown in Figure 2 These active compounds were Glyc-erol 3TMS derivative D-Pinitol pentakis(trimethylsilyl)ether Palmitic Acid TMS derivative 11-Octadecenoic acid(Z)- TMS derivative alpha-Linolenic acid TMS derivativeStearic acid TMS derivative Sinapinic acid 2TMS derivative11-Eicosenoic acid (Z)- TMS derivative Sucrose 8TMSderivative 371115-tetramethylhexadecan-13-diol silylatedSucrose 8TMS derivative Behenic acid TMS derivativeD-(+)-Turanose octakis(trimethylsilyl) ether and beta-Sitosterol TMS derivative The retention time peak areachemicalformulae and molecular weight and MZ ratio ofpolyphenols and carbohydrates found in LSS are depicted inTable 2
32 Assessment of Biochemical Markers As shown in Table 3amp Figure 3(a) serum levels of liver biomarkers ALT AST 120574-GT and ALP activities were significantly elevated (Ple 001)with percentages of 49 128 310 and 421 respectivelyin rabbits injected with CCl4 for 5 weeks and for 10 weekswith percentages of 76 159 468 and 474 respectivelySerum total bilirubin triglycerides and cholesterol levelswere also significantly elevated (Ple 001) in rabbits injected
with CCl4 for 5 weeks with percentages of 139 250and 72 respectively and for 10 weeks with percentages of114 290 and 512 respectively compared to those of thenormal control (Figure 3(b)) In addition serum total proteinand albumin levels were significantly decreased (Ple 001)in rabbits injected with CCl4 for 5 weeks with percentagesof 36 and 30 respectively and for 10 weeks as wellwith percentages of 31 and 26 respectively compared tothose of the normal control Serum ALT AST 120574-GT ALPtotal bilirubin triglycerides and cholesterol activities weresignificantly reduced (Ple 001) in rabbits protected with 200mg kg bw of LSS with percentages of 46 60 31 4850 39 and 42 respectively and for 400 mgkg bw withpercentages of 57 67 49 46 34 29 and 28respectively for 5 weeks compared to the corresponding CCl4treated group (Table 3 and Figures 3(a) and 3(b)) SerumALTAST 120574-GT ALP total bilirubin triglycerides and cholesterollevelswere also significantly reduced in rabbits protectedwith200 and 400mgkg bw of LSS for 10 weeks compared to thoseof the corresponding CCl4 treated group as shown in Table 3and Figures 3(a) and 3(b)
As shown in Table 3 and Figure 3(b) serum total proteinand albumin levels were significantly elevated (Ple 001) withpercentages of 57 37 in 200 mgkg bw of LSS protectedgroup and 39 31 in 400 mgkg bw of LSS protectedgroup for five consecutive weeks compared to those ofthe corresponding CCl4 treated group respectively Samebehaviour was noticed after 10 consecutive weeks of CCL4treatment accompanied with LSS protection the elevationwas 43 29 in 200 mgkg bw of LSS protected group and42 27 and in 400 mgkg bw of LSS protected groupcompared to those of the corresponding CCl4 treated grouprespectively
33 Assessment of Oxidative Stress and Antioxidant ActivityThe present results showed in Table 4 amp Figure 4 exert asignificant decrease (Ple 001) in the enzymatic activities ofCAT SOD GR GST and GPx in the liver homogenateof CCl4 treated rabbits 5 and 10 weeks after experimental
Evidence-Based Complementary and Alternative Medicine 5
Table 2 Chemical pattern of LSS extract of polyphenols and carbohydrates revealed by GC-MS analyses RT = retention time Area Pct=peak area and Mwt= molecular weight
No RT Area Pct Compound name Mwt Chemical formula MZ1 81846 29942 Glycerol 3TMS derivative 3086372 C12H32O3Si3 2052 116107 66731 D-Pinitol pentakis(trimethylsilyl) ether 5550881 C22H54O6Si5 733 126062 42518 Palmitic Acid TMS derivative 3286052 C19H40O2Si 3134 134058 303464 11-Octadecenoic acid (Z)- TMS derivative 3546425 C21H42O2Si 3395 134323 18965 alpha-Linolenic acid TMS derivative 3506107 C21H38O2Si 757 134906 26564 Stearic acid TMS derivative 3566584 C21H44O2Si 1178 136123 10245 Sinapinic acid 2TMS derivative 3685722 C17H28O5Si2 3689 142372 33369 11-Eicosenoic acid (Z)- TMS derivative 3826956 C23H46O2Si 36710 147773 29175 Sucrose 8TMS derivative 9197454 C36H86O11Si8 36111 149521 12532 371115-tetramethylhexadecan-13-diol silylated 298547 C20H42O 23312 150368 32061 Sucrose 8TMS derivative 9197454 C36H86O11Si8 36113 151003 14675 Behenic acid TMS derivative 4127647 C25H52O2Si 11714 15614 15318 D-(+)-Turanose octakis(trimethylsilyl) ether 9487867 C37H89NO11Si8 7315 190612 12082 beta-Sitosterol TMS derivative 4868878 C32H58OSi 129
running as compared to those of the normal control with apercentage of change (53 50 33 32 35amp 46 3737 39 and 47 respectively) Also the present resultsrevealed a significant increase (Ple 001) in the MDA levelsin the liver homogenate of CCl4 treated rabbitrsquos 5 and 10weeks after experimental running as compared to those ofthe normal control with a percentage of change (96amp 120)respectively
Daily oral administration of 200 mgkg bw of LSS withinthe rabbitrsquos diets for 5 weeks starting from day one of theexperiment significantly increased (Ple 001) the enzymaticactivities of CAT SOD GR GST and GPx in the liverhomogenate of injured rabbits with liver cirrhosis for 5 weeksas compared to those of the corresponding CCl4 treatedgroupwith a percentage of change (93 93 39 41 42)and all parameters mentioned were significantly increased(Ple 001) with 70 58 33 25 37 respectively inrabbits administrated 400 mgkg bw of LSS and injuredwith liver cirrhosis for 5 weeks as compared to those ofthe corresponding CCl4 treated group Similarly Daily oraladministration of 200 mgkg bw of LSS within the rabbitrsquosdiets for 10 weeks starting from day one of the experimentsignificantly increased (Ple 001) the enzymatic activities ofCAT SOD GR GST and GPx in the liver homogenateof injured rabbits with liver cirrhosis with a percentageof change (70 58 52 58 74) and all parametersmentioned were reduced significantly (Ple 001) to 73 4747 46 and 65 respectively in rabbits administered 400mgkg bw of LSS and injured with liver cirrhosis for 10 weeksas compared to those of the corresponding CCl4 treatedgroup Also the results revealed that daily oral administrationof 200 and 400 mgkg bw of LSS within the rabbitsrsquo dietsfor 5 and 10 weeks starting from day one of the experimentsignificantly decreased (Ple 001) the MDA levels in the liverhomogenate with a percentage of change (47amp 44 and60amp 56 for 5 and 10 weeks respectively) compared tothose of the corresponding CCl4 treated group (Table 4 ampFigure 4)
34 DNA Results of Untreated LSS Administered CCl4-Treated and LSS Protected Rabbits DNAwas extracted fromliver tissues of all the studied rabbits the banding mannerwas observable in Figure 5 Treatment of rabbits with CCl4increased significantly DNA damage of the hepatic tissuesmore than those of the untreated rabbits An entirely dif-ferent banding manner was detected in CCl4 treated rabbitswhich was absent from the liver tissues of the untreatedrabbits DNA extracted from 200 and 400 mgkg bw ofLSS protected rabbitsrsquo livers administered with CCl4 after5 and 10 weeks of the experimental running showed asignificant repair in hepatic DNA Rabbits treated with only200 and 400 mgkg bw of LSS after 5 and 10 weeks of theexperimental running did not display any kind of DNAfragmentation
35 Histopathological Observations The micrograph shownin Figure 6(a) illustrates the normal aspect of the hepatic liverfor the control group (Subgroup A1) fed with normal dietwithout exposing to CCl4 hepatic intoxication Examinationof liver tissue of the control group in which rabbits receivednormal diet and 200 mgkg bw of LSS in their diet on dailybasis till the end of the experiment without exposing to CCl4hepatic intoxication (Subgroup A2) revealed normal hepaticarchitecture (Figure 6(b)) Examination of the control groupreceived normal diet and 400mgkg bw of LSS in their diet ondaily basis till the end of the experiment without exposing toCCl4 hepatic intoxication (Subgroup A3) revealed only mildinflammation except one case which showed the adverseeffects of the drug showing evident chronic venous conges-tion moderate inflammation and bridging fibrosis (Figures6(c) 6(d) and 6(e))
According to liver tissue in rabbits exposed to CCL4intoxication (Group-B1 control pollutant group) the fol-lowing can be seen hepatocyte feathery degeneration andchronic venous congestion steatosis parenchymal and portalinflammation and fibrosis (Figures 7(a) 7(b) and 7(c)) and(Figures 7(d) 7(e) and 7(f)) respectively
6 Evidence-Based Complementary and Alternative Medicine
Table3Liverp
rofileo
funtreatedLSS
administeredC
Cl4tre
atedand
LSSprotectedrabb
itsEffectofLS
Son
serum
markere
nzym
esALTA
STG
GTALPTBILITP
ALB
TGand
CHOL
inrabb
itstre
ated
with
CCL4indu
cedhepatotoxicityas
indicatedin
thefi
gureA
lldatawe
reexpressedas
meanplusmnSE
MV
aluesw
eres
tatistic
allyteste
dusingtheS
tudent
t-testandsig
nificant
differencesatplt005
andplt001
asindicatedby
(lowast)amp
(lowastlowast)com
paredto
norm
alcontrolby
()amp
()
comparedto
CCL4aft
er5w
eeksand
by(+)amp
(++)
comparedto
CCL4aft
er10
weeks
respectiv
ely
Group
ALT
(UL)
AST
(UL)
GGT(U
L)
ALP
(UL)
TBILI
(mgdl)
TP(gdl)
ALB
(gdl)
CHOL(m
gdl)
TG(m
gdl)
Con
trol
3801plusmn
468
2725plusmn
192
702plusmn12
42202plusmn16
2028plusmn003
650plusmn031
355plusmn017
1632plusmn506
4203plusmn469
LS(200
mgkg
bw)
3148plusmn678
2941plusmn
331
848plusmn12
05702plusmn229lowastlowast
043plusmn003lowast
640plusmn025
324plusmn013
3217plusmn287lowast
5274plusmn279lowast
LS(400
mgkg
bw)
1644plusmn14
6lowastlowast
2076plusmn343lowast
629plusmn229
5308plusmn16
1lowastlowast
046plusmn003lowastlowast
586plusmn047
344plusmn010
3161plusmn
305lowast
2610plusmn304lowast
CCl4(5
wk)
5676plusmn546lowastlowast
6230plusmn391lowastlowast
2872plusmn276lowastlowast
11483plusmn273lowastlowast
067plusmn004lowastlowast
414plusmn014lowastlowast
248plusmn025lowastlowast
8921plusmn
762lowastlowast
14774plusmn74
4lowastof
Change
49128
310
421
139
-36
-30
72250
CCl4(10wk)
6700plusmn476lowastlowast
7069plusmn645lowastlowast
3978plusmn226lowastlowast
12638plusmn371lowastlowast
060plusmn004lowastlowast
445plusmn01lowastlowast
261plusmn022lowastlowast
9996plusmn616lowastlowast
16379plusmn89lowastlowast
of
Change
76159
468
474
114-3
1-2
6512
29
CCl4+LS
(200
mgkg
bw)5
wk
3062plusmn219
2490plusmn302
1962plusmn19
5
5960plusmn237
033plusmn003
649plusmn020
339plusmn012
5135plusmn547
8978plusmn611
of
Change
-46
-60
-31
-48
-50
5737
-42
-39
CCl4+LS
(400
mgkg
bw)5
wk
2423plusmn
291++
2027plusmn309++
1464plusmn17
3++
6146plusmn416++
044plusmn003++
576plusmn062++
326plusmn020++
6306plusmn524++
10539plusmn562++
of
Change
-57
-67
-49
-46
-34
3931
-29
-28
CCl4+LS
(200
mgkg
bw)10wk
3804plusmn371
2390plusmn264
1581plusmn
142
5259plusmn257
038plusmn004
636plusmn038
338plusmn009
5236plusmn447
9870plusmn344
of
Change
-43
-66
-60
-58
-36
4329
-47
-39
CCl4+LS
(400
mgkg
bw)10wk
4018plusmn292++
2516plusmn508++
2292plusmn254++
7954plusmn484++
048plusmn003
++635plusmn038++
334plusmn014++
6557plusmn79
8++
10513plusmn666
++of
Change
-40
-64
-42
-37
-20
4227
-34
-36
Evidence-Based Complementary and Alternative Medicine 7
01020304050607080
ALT
Lev
e (U
L)
ALTlowastlowast
lowastlowast
lowastlowast
++++
0102030405060708090
AST
Lev
e (U
L)
AST
lowastlowast
lowast
lowastlowast
++ ++
05
1015202530354045
GG
T Le
ve (U
L)
GGTlowastlowast
lowastlowast
++
++
020406080
100120140
ALP
Lev
el (U
L)
ALPlowastlowast
lowastlowast
lowastlowastlowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
(a) Effect of LSS aqueous extract on serum marker enzymes ALT AST GGTand ALP in rabbits treated with CCL4 induced hepatotoxicity as indicated inthe figure All data were expressed as mean plusmn SEM Values were statisticallytested using the Student t-test and significant differences at p lt 005 and p lt 001indicated by (lowast) amp (lowastlowast) compared to normal control by () amp () comparedto CCL4 after 5 weeks and by (+) amp (++) compared to CCL4 after 10 weeksrespectively
012345678
TP L
evel
(gL
)
TP
lowastlowastlowastlowast
++ ++
00102030405060708
TBI
LI L
evel
(gL
) TBILI
lowastlowastlowast
lowastlowastlowastlowast
++++
005
115
225
335
4
ALB
Lev
el (g
L)
ALB
lowastlowastlowastlowast
++++
020406080
100120140160180200
TG L
evel
(gL
)
TG
lowast
lowastlowast
lowastlowast
lowastlowast
++++
0
20
40
60
80
100
120
CHO
L Le
vel (
gL)
CHOLlowastlowast
lowastlowast
++++
lowastlowast
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
(b) Effect of with LSS aqueous extract on biochemical parameters TBILI TPALB TG and CHOL in rabbits treated with CCL4 induced hepatotoxicity asindicated in the figure All data were expressed as mean plusmn SEM Values werestatistically tested using the Student t-test and significant differences at p lt 005and p lt 001 indicated by (lowast)amp (lowastlowast) compared to normal control by () amp ()compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4 after 10weeks respectively
Figure 3
8 Evidence-Based Complementary and Alternative Medicine
Table4En
zymaticactiv
itiesofantio
xidant
enzymesandMDAofun
treatedLSS
administeredC
Cl4tre
atedand
LSSprotectedrabb
itsA
lldatawe
reexpressedas
meanplusmnSE
MV
aluesw
ere
statistic
allyteste
dusingtheS
tudent
t-testandsig
nificantd
ifferencesa
tplt005
andplt001
asindicatedby
(lowast)amp
(lowastlowast)com
paredto
norm
alcontrolby
()amp
()
comparedto
CCL4aft
er5
weeksandby
(+)amp
(++)
comparedto
CCL4aft
er10
weeksrespectiv
ely
Group
CAT
(nggtissue)
SOD
(nggtissue)
GR
(nggtissue)
GST
(nggtissue)
GPx
(nggtissue)
MDA(nmolm
l)
Con
trol
3155plusmn
199
2820plusmn10
92029plusmn099
1537plusmn12
12197plusmn17
0647plusmn057
LS(200
mgkg
bw)
3127plusmn18
62631plusmn15
51908plusmn077
1542plusmn067
2129plusmn12
8635plusmn045
LS(400
mgkg
bw)
2843plusmn204
2676plusmn18
81972plusmn12
81555plusmn14
32031plusmn074
635plusmn060
CCl4(5
wk)
1475plusmn258lowastlowast
1406plusmn13
9lowastlowast
1358plusmn061lowastlowast
1041plusmn
108lowastlowast
1409plusmn099lowastlowast
1273plusmn10
6lowastlowast
of
Change
-53
-50
-33
-32
-35
96CCl4(10wk)
1682plusmn15
7lowastlowast
1767plusmn202lowastlowast
1277plusmn13
6lowastlowast
940plusmn073lowastlowast
1152plusmn18
2lowastlowast
1427plusmn10
7lowastlowast
of
Change
-46
-37
-37
-39
-47
120
CCl4+LS
(200
mgkg
bw)5
wk
2846plusmn201
2720plusmn094
1892plusmn093
1472plusmn118
2009plusmn112
667plusmn046
of
Change
9393
3941
42-4
7CCl4+LS
(400
mgkg
bw)5
wk
2519plusmn17
7++
2231plusmn14
0++
1813plusmn094++
1304plusmn13
1++
1941plusmn
192+
+713plusmn072++
of
Change
7058
3325
37-4
4CCl4+LS
(200
mgkg
bw)10wk
2872plusmn201
2796plusmn222
1949plusmn096
1492plusmn15
8
2006plusmn17
9
569plusmn046
of
Change
7058
5258
74-6
0CCl4+LS
(400
mgkg
bw)10wk
2919plusmn17
0++
2610plusmn14
3++
1887plusmn17
3++
1378plusmn086++
1906plusmn14
1++
619plusmn052++
of
Change
7347
4746
65-5
6
Evidence-Based Complementary and Alternative Medicine 9
05
10152025303540
CAT
Leve
(ng
g tis
sue)
CAT
lowastlowastlowastlowast
++++
0
5
10
15
20
25
30
35
SOD
Lev
e (ng
lg ti
ssue
)
SOD
lowastlowast
lowastlowast
++++
0
5
10
15
20
25
GR
Leve
(ng
g tis
sue)
GR
lowastlowastlowastlowast
++++
02468
1012141618
GST
Lev
e (ng
g ti
ssue
)
GST
lowastlowastlowastlowast
++++
0
5
10
15
20
25
GPx
Lev
e (ng
g ti
ssue
)
GPx
lowastlowastlowastlowast
++++
02468
1012141618
MD
A L
eve (
nmol
g ti
ssue
)
MDA
lowastlowast
lowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
Figure 4 Effects of LSS on oxidative stress markers in rabbits treated with CCL4 induced hepatotoxicity as indicated in the figure All datawere expressed as mean plusmn SEM Values were statistically tested using the Student t-test and significant differences at p lt 005 and p lt 001 asindicated by (lowast) amp (lowastlowast) compared to normal control by () amp () compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4after 10 weeks respectively
Examination of liver tissue exposed to CCL4 as well as200 mgkg bw of LSS in their diet on daily basis (GroupB2) reveals that the group which is sacrificed 5 weeksafter experimental running shows evident chronic venouscongestion and feathery degeneration in two cases only mildfeathery degeneration in others no steatosis or isolated vesic-ular steatosis mild parenchymal and portal inflammation
(Figures 8(a) 8(b) and 8(c)) Examination of the groupsacrificed 10 weeks after experimental running shows theimprovement of the histoarchitectural changes of the livertowards the normal aspect showing regenerating hepato-cytes with no steatosis discrete chronic venous congestionand discrete inflammatory infiltrate (Figures 8(d) 8(e) and8(f))
10 Evidence-Based Complementary and Alternative Medicine
M 1 2 3 4 5 6 7 8 9bp
10000
3000
2000
1500
1000
750
500
300
Figure 5 Agarose gel showingDNAdamage byCCl4 and protectiveeffects of various fractions of LSS Lanes from left (M) molecularweight marker (1) negative control (2) LSS (200 mgkg bw) (3) LSS(400mgkg bw) (4) CCl4 for 5weeks (5) CCl4 for 10weeks (6)amp (7)CCl4 + LSS (200 mgkg bw) (8) amp (9) CCl4 + LSS (400 mg kg bw)
Examination of liver tissue of rabbits exposed to CCL4as well as 400 mgkg bw of LSS in their diet on daily basis(Group B3) reveals in the group that scarified 5 weeks afterexperimental running improvement of the histoarchitecturalchanges of the liver as there is no chronic venous congestionor only discrete chronic venous congestion and mild featherydegeneration no steatosis or only isolated vesicular steatosisand mild parenchymal and portal inflammation (Figures9(a) 9(b) and 9(c)) But the group that was sacrificed10 weeks after experimental running shows adverse effectsof the drug in the form of steatosis and chronic venouscongestion which is evident in two cases with mild tomoderate inflammation Bridging fibrosis is seen in one case(Figures 9(d) 9(e) 9(f) and 9(g))
4 Discussion
Hepatic disease became a significant public health concernowing to growing rate of obesity in all age populations [26]The conservative treatments of liver problems such as acuteand chronic liver hepatitis liver cirrhosis and fatty liverare often insufficient due to hazardous effects initiated byhepatotoxic drugs of chemical origin [27] Medicinal plantsowing to their abundant hepatoprotective natural ingredi-ents have been comprehensively investigated to treat liverdisorders In the current study hepatoprotective efficacy ofLSS was investigated via hepatotoxic classical model inducedby carbon tetrachloride
In organ and tissue systems CCl4 is stimulated bycytochrome P-450 dependent mixed oxidase in the hepaticendoplasmic reticulum to procedure free radical trichloro-methyl (CCl3 and Cl3COO) This causes changes in themembrane of the endoplasmic reticulum and other cel-lular membranes resulting in an increase in calcium ionpermeability through the plasma membrane causing severeinstabilities of calcium homeostasis leading to necrotic celldeath [28 29] CCL4 affects many organs causing degenera-tion of liver fatty layer and centrilobular necrosis [29] Thisphenomenon indicates that CCl4 is a powerful hepatotoxic
agent and may be used for the characterization of the hepaticdisease treatment medicines Elevation of liver enzymaticmarkers (ALT AST ALP and bilirubin) and increased lipidperoxidation are further evidences of hepatotoxicity ALTand AST that exist in the hepatocytes can certainly leak intothe peripheral blood as soon as the hepatocytes are injured[29] Lepidium sativum seed analysis by GCMS exhibitedthe presence of high concentrations of mainly 120572-linolenicacid (ALA) which is nminus3 fatty acid an omega-3 fatty acidfound in seeds exerting its bioactivity in ameliorating thehepatic intoxication and oxidative stress including liver injuryinduced by alcohol [30] liver steatosis [31 32] nonalcoholichepatic disease [33] and parenteral nutrition-associated liverdisease [34] Pari and Jalaludeen stated that orientationof the active groups in sinapic acid increases antioxidantefficiency as metal chelators and promotes its protectiveefficacy against liver oxidative stress in arsenic intoxicatedrats [35]The current study showed that sinapic acid as ametalchelator may exert its biological activity in improving liverfunction
Lepidium sativum seed extracts have proved hepatopro-tective effects against CCl4 induced liver damage A researchstudy on male Albino Wistar rats demonstrated significantreduction in hepatotoxicity induced by CCl4 administeredwith 200-400 mgkg of Lepidium sativum seeds extractincluded within the diet Serum level of AST ALT levels andbilirubin levels were significantly elevated in CCl4 treated ratscompared to those of normal control However a significantreduction in the levels of these parameters in Lepidiumsativum seed extracts protected rats was observed The hep-atoprotective effect could be due to the presence of alkaloidcoumarin flavonoids tannin and triterpenes which enhanceantioxidant activity and reduce free radical production fromCCl4 which is the basic triggering factor for hepatotoxicity[36 37]
In the present study we evaluated the hepatoprotectiveefficacy of L sativum seeds in white male New-Zealand rab-bits The present results revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withL sativum seeds significantly repaired their liver injuriousmarker enzymes as well as bilirubin total protein and albu-min hence approving its hepatoprotective effect The resultsare in agreement with those of Afaf et al [12] where theystated the role of LSS as efficient hepatoprotective agent Alsothe current results are consistent with those of Mohamedand his coworkers [38] who reported that preprotectionwith LSEE (Lepidium sativum ethanolic extract) significantlyprohibited the D-GalNLPS induced rise in hepatic enzymes(AST ALT 120574-GGT ALP total bilirubin LDH and total pro-tein) thus significantly alleviated the reduction of lipid per-oxidation and repaired the antioxidant enzymatic status andtotal protein restoring them to normal levels D-GalNLPShas been widely used to examine the mechanisms underlyingacute liver failure [39 40] The hepatoprotective significanceof LSEE could be due to downregulation of cytokines (TNF-120572 IL-6) and stress gene (iNOS and HO-1) mRNA expres-sion and upregulation of IL-10 Lepidium sativum ethanolicextract (LSEE) preprotection also improved the degree ofstructural damage and reduced inflammatory infiltration in
Evidence-Based Complementary and Alternative Medicine 11
(a) (b)
(c) (d)
(e)
Figure 6 Effect of LSS on histopathological changes of liver tissues of normal groups in rabbits (a) Showing normal histological picture ofhepatic lobule that consists of central vein surrounded by normal hepatocytes (HEx100) (b) Showing liver sections of the rabbits treatedwith LSS (200 mgkg bw) which revealed normal histological picture of hepatic lobule that consists of central vein surrounded by normalhepatocytes (HEx100) (c) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealed normal hepatic architecturewith only mild inflammation [arrows] (HEx100) (d) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealedevident chronic venous congestion and moderate inflammation [arrows] (HEx100) (e) Showing liver sections of the rabbits treated with LSS(400 mgkg bw) which revealed portal to portal bridging fibrosis [arrow] (HEx200)
hepatic cells These outcomes established that LSEE alle-viates hepatic impairments and structural injury throughthe decay of oxidative stress inflammation and apopto-sis in the liver [38] Mohamed Sakran and his colleagues[10] suggested that 56-dimethoxy-210158403-methylenedioxy-7-C-120573-D glucopyranosyl isoflavone extracted from Lepidiumsativum seeds could improve the lipid profile and the liverfunctions in serum and reduce the free radicals generationby enhancing the antioxidant defense mechanism Newsuggested isoflavonoid can be used as a probable antioxi-dant against paracetamol hepatotoxicity with its antioxidant
characteristics and could restore the hepatic functionstowards normalization [10]
In the present study we found that improved cholesteroland triglycerides levels in L sativum seeds protected rabbitsas compared to CCl4 treated rabbits also support the hepato-protective effect of LSSThese results can also be correlated tothose of Shukla et al [41] who reported the role of L sativumseeds as hypolipidemic agent Also the results of the presentstudy agreed with those of Abdulrahman et al [20] whoseresults showed that L sativum seeds have hepatoprotectiveactivity that could be due to its antioxidant activity together
12 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
Figure 7 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) In rabbits sacrificed 5 weeks afterexperimental running as shown in (a) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100)(b) steatosis (30) in one case (HEx100) and (c) mild parenchymal inflammation [arrow] (HEx200) In rabbits sacrificed 10 weeks afterexperimental running as shown in (d) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100) (e)steatosis (about 20) and moderate parenchymal and portal inflammation (HEx100) and (f) bridging fibrosis [arrow] (HEx100)
with the presence of anti-inflammatory ingredients in Lsativum seeds extract
The results of the current study revealed that the exposureto CCl4 for 5 and 10 weeks led to a significant decrease inthe enzymatic activities of CAT GPx GR GST and SOD inthe liver homogenate of CCl4 treated rabbits 5 and 10 weeksafter experimental running compared to those of the normalcontrolThe concurrent protection of rabbits exposed toCCl4for 5 and 10weeks with 200amp 400mg kg bw LSS significantlyrepaired their antioxidant status and reduced significantly theMDA levels hence confirming its hepatoprotective efficacy
Although the precise molecular mechanism of action ofLSS is not completely understood some researchers [42]have confirmed the role of LSS in inhibiting oxidative stress
associated liver damage The antioxidant activity of LSScould be due to the presence of phenolic compound andflavonoids in these seeds as have been evidenced through thepreliminary screening study on LSS extract using HPLCOurresults match with those of Lee et al [42] who reported thatphenolic compounds play an essential role against oxidativestress related liver damage
A unique expectation of the current study is to confirmthat CCl4 induces liver DNA injury that was assessed usinggel electrophoretic method The results of the current studyshowed that CCL4 induces DNA injury as presented bya significant progression in liver DNA disintegration ofCCl4 treated rabbits above untreated control in the gelelectrophoresis
Evidence-Based Complementary and Alternative Medicine 13
(a) (b)
(c) (d)
(e) (f)
Figure 8 Effect of LSLS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (200 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) evident chronic venous congestion and feathery degeneration [arrow](HEx100) (b) mild feathery degeneration and isolated vesicular steatosis [arrow] (HEx200) and (c) moderate parenchymal and portalinflammation (HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) regenerating hepatocytes with isolatedvesicular steatosis (HEx100) (e) discrete chronic venous congestion and no steatosis (HEx400) and (f) discrete inflammatory infiltrate(HEx100)
Lipid peroxidation of the nuclear membrane may bedirectly accompanied with nuclear DNA damage [43]Nuclear DNA is concomitant with particular regions of thenuclear membrane [44]The close proximity of nuclear DNAto the nuclear membrane can ease the interaction of genomicDNA with the peroxyl radicals and further reactive inter-mediates formed during membrane lipid peroxidation [43]In the presence of transition metals the hydroxyl radicalsgenerated by CCL4 autoxidation can trigger peroxidationof the nuclear membrane lipids by metal catalyzed Haber-Weiss reaction the lipid peroxides levels and intermediate
free radicals could then be augmented by a chain reactionSubsequently the reactive species oxidize nuclearDNAwhenthe lipid peroxidation reaction occurs in the nuclear mem-brane since the nuclear membrane controls the transportof messenger RNA into the cytoplasm and supports thenuclear division process nuclear membrane peroxidationof lipids and proteins Moreover oxidative nuclear DNAdamage could disrupt these serious cellular functions [43]
DNA findings showed that DNA disintegration is adirect result after CCl4 treatment in rabbitsrsquo liver tissueswhich was reduced by participation of antioxidants through
14 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
(g)
Figure 9 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (400 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) discrete chronic venous congestion (HEx200) (b) centrilobular fibrosis[arrows] regenerating hepatocytes and isolated vesicular steatosis (HEx100) and (c) mild parenchymal and portal inflammation [arrow](HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) mild chronic venous congestion and isolated vesicularsteatosis (HEx100) (e) evident chronic venous congestion [arrows] (HEx100) (f) steatosis (40) with moderate inflammation (HEx100) and(g) portal to portal bridging fibrosis in one case [arrow] (HEx100)
Evidence-Based Complementary and Alternative Medicine 15
administration of 200 mg amp 400 mgkg bw L sativum seedsNearly complete repair of DNA damage was significant aftertreating rabbits with 200 mg amp 400 mgkg bw L sativumseeds The protective effect of L sativum seeds on hepaticDNA injury may be due to the antioxidant activity ofL sativum seeds via direct scavenging of free radicals orinterfering with free radicals generation
Concerning histopathological evaluation in our studyrabbitsrsquo livers exposed to CCL4 show portal and parenchymalinflammatory infiltrate feathery and ballooning degenera-tion fatty change and fibrosis Our data are consistent withRajeswary et al [45] who reported dilatation of sinusoidsfatty change disarrangement of normal hepatocytes and cen-trilobular necrosis Also A S Bernacchi et al [46] reportedthat CCL4 leads to liver necrosis and fat accumulation in therabbit
The present study revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withLSS improved their liver histopathological architecture inthe form of less inflammation regenerating hepatocytes nosteatosis or isolated vesicular steatosis no chronic venouscongestion or just discrete congestion and no fibrosis henceapproving its hepatoprotective effectThe results are in agree-ment with Afaf et al [12] and Abdulrahman et al [20] whoobserved the improvement of the histological structure inextract treated group as the histopathologic findings showedless inflammation well restored hepatocytes and less area ofnecrosis as compared to severe necrosis and inflammationin CCl4 treated group of animals which also supports thehepatoprotective effects of LLS Their findings suggest thepossibility of the presence of anti-inflammatory compoundsin LSS extract which could also be a contributing factortowards the hepatoprotective action of LSS
Althnaian [47] also reported that examination of hep-atocytes showed mild to moderate degree of recovery LSSattenuated the hepatic fat accumulation decreased vacuolardegeneration and improved the architecture of hepatocytes
In our study the reducing effect of LSS on fatty changecaused by CCL4 intoxication is indicating the protective andcurative role against fatty liver Our results are supported byShukla et al [41] who reported that LSS significantly reducedsteatosis Their histopathological observations confirmed thecurative efficacy of L sativum liver damage Microvesicularfatty changes hepatocyte ballooning and steatosis weremarkedly corrected by LSS Also Wadhwa et al [36] foundthat the severe fatty changes in the livers of rats caused byCCl4 were decreased in the treated groups
In the present study chronic venous congestion featherydegeneration and fatty changes show mild improvement inrabbits receiving 200mgkg bw of LSS for 5 weeks and showmoderate improvement in rabbits that received 200mgkg bwof LSS for 10 weeks and rabbits that received 400mgkg bwof LSS for 5 weeks Our data are consistent with Afaf et al[12] who observed that severe centrilobular hepatocellularvacuolation hemorrhages and congestion of the centralveins noticed in CCL4 group showed mild improvement ingroups that received 200 mgkg bw of LSS and moderateimprovement in groups that received 400 mgkg bw ofLSS
On the other hand rabbits that received 400mgkg bw ofLSS for 10 weeks showed evidence of liver toxicity in the formof venous congestion hepatocyte feathery degeneration fattychange and fibrosis These results are compatible with Bafeeland Ali [48] who reported that LSS had minimal effect onliver parenchyma if given in low doses So LSS could be usedas food additive or supplement Mineral content of calciumand phosphate and the presence of essential fatty acids inconsiderable level make LSS of beneficial nutritional valuefor many body organs including the liver However LSS inhigh dose showed some toxic effect on renal parenchyma rep-resented by focal necrosis hepatocyte apoptosis and portalinflammatory cell infiltrate Burow et al [49] reported thatthese hazardous effects of LSS may be due to isothiocyanatesreported to be one of LSS constituents
5 Conclusions and Recommendations
The present study showed that LSS is a key factor ininhibition of the hazardous effects of CCl4 LSS treatmentcould significantly alleviate to a greater extent the degree oflipid peroxidation and restore the antioxidative enzymes andshift liver function to normal levels In addition to antioxi-dants activity LSS attenuated the hepatic fat accumulationdecreased vacuolar degeneration ameliorated the degree ofstructural damage in the hepatic cells and improved thearchitecture of hepatocytes The current findings demon-strated that LSS have marked hepatoprotective activity Infuture work active ingredients of LSS extract should bestudied in various animal models of severe toxicity inducedby CCL4 and other hepatotoxic agents The active ingredientsof this plant are likely to be applied as one of the future drugsagainst acute liver toxicity
Data Availability
Data used to support the findings of this study are availablefrom the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This project was funded by the Deanship of ScientificResearch (DSR) King Abdulaziz University Jeddah undergrant no S-17-314-37 The authors therefore acknowledgewith thanks DSR technical and financial support
References
[1] M K Manibusan M Odin and D A Eastmond ldquoPostu-lated carbon tetrachloride mode of action a reviewrdquo Journalof Environmental Science and Health Part C EnvironmentalCarcinogenesis and Ecotoxicology Reviews vol 25 no 3 pp 185ndash209 2007
[2] N Brautbar and J Williams II ldquoIndustrial solvents andliver toxicity risk assessment risk factors and mechanismsrdquo
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
Stem Cells International
Hindawiwwwhindawicom Volume 2018
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Disease Markers
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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
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Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 3
500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 18000
1e+072e+073e+074e+075e+076e+077e+078e+079e+071e+08
11e+08 12e+08 13e+08 14e+08 15e+08 16e+08 17e+08
11388
11450
12428
13222
1329814028
141 148 15623
Rela
tive A
bund
ance
Time (min)Figure 1 Gas Chromatography Mass Spectrometry (GC-MS) analysis of fatty acids in LSS
Diet hygienic conditions and behaviour were observed andfollowed up on daily basis Rabbits and food were observedensuring that rabbits ate all of their food before addition ofextra seeds in their new meal according to the experimentaldesign At the end of the 5th and 10th weeks of the experimentrabbits were fasted 12-16 hours before sacrificing At theend of 5th and 10th weeks of the study blood sample fromeach target rabbits according to the experimental designwas withdrawn from the ear vein collected in a centrifugetube and kept at room temperature for 20 minutes Serawere separated by a cooling centrifuge at 3000 rpm for15 minutes and stored at -80∘C until recall Then targetrabbits were decapitated and the abdomen of each rabbitwas excised immediately after scarifying The liver of eachrabbit was removed and divided into 3 segments 1st segmentwas immersed directly into 10 formaldehyde solution andthen processed for histopathological preparation and exami-nation the 2nd segment was used forDNAextraction to studythe liver DNA integrity and the 3rd segment was used forpreparation of liver homogenate
Sera of the studied rabbits were used to estimate thelevels of ALT AST 120574-GT ALP T Bilirubin T ProteinAlbumin Cholesterol and Triglycerides which have beenpurchased from (EGY-CHEM for lab technology BaderCity Egypt) The homogenization of pancreas tissues wascarried out according to Gackowski et al [23] Liver tissuehomogenate of each sample was used to estimate the levelsof the antioxidant enzymes Catalase (CAT) glutathione per-oxidase (GPX) Glutathione reductase (GR) Glutathione-S-transferase (GST) and Super-oxide dismutase (SOD) as wellas (MDA) for all the studied rabbits using kits from (BioassayTechnology Laboratory SHANGHAI KORIAN BIOTECHCo)
27 Extraction of DNA of Rabbits Hepatic Tissues and GelElectrophoresis We studied the genomic DNA integrity of
the liver of all the studied rabbits In accordance with thepurification protocol of total DNA from rabbits tissues DNAextractionwas processed using (Spin-ColumnProtocol QIA-GEN Group) Agarose gel (2 agarose gel in 1x TAE buffer)was prepared according to Raj Kumar [24]
28 Histopathological Examination Liver tissues were dis-sected from scarified rabbits removed and fixed in 10formalin solution The fixed specimens were then trimmedwashed and dehydrated in ascending grades of alcoholThesespecimens were cleared in xylene embedded in paraffinsectioned at 4-5 120583 of thickness and stained with Hematoxylinand Eosin then examined microscopically [25]
29 Statistical Analysis Analyzing the data was conducted bystatistical package software (SPSS) The t-test of significancewas tested for identifying the differences between the meansResults were expressed as mean plusmn SEM
3 Results
31 GC-MS Analysis of LSS Extract By comparing the massspectra of the components with the NIST library ten majorfatty acids peaks were obtained out of which thirty-eightphytocomponents in LSS were described and recognized asshown in Figure 1 These ten major fatty acids were E-10-Methyl-11-tetradecen-1-ol acetate Hexadecanoic acid methylester 91215-Octadecatrienoic acid methyl ester cis-Methyl11-eicosenoate Methyl 18-methylnonadecanoate Methyl 11-docosenoate Docosanoic acid methyl ester Octadecanoicacid 910-dihydrox methyl ester bis(trifluoroacetate) Tetra-cosanoic acid methyl ester Benzoic acid 35-dicyclohexyl-4-hydroxy- methyl ester The retention time peak areachemicalformulae andmolecular weight andMZ ratio of allfatty acids found in LSS are depicted in Table 1 Similarly thechemical pattern of LSS extract of polyphenols and carbohy-drates revealed by GC-MS analyses exhibited 15 compounds
4 Evidence-Based Complementary and Alternative Medicine
Table 1 Chemical pattern of LSS extract revealed by GC-MS analyses RT = retention time Area Pct= peak area and Mwt= molecularweight
No RT Area Pct Compound name Mwt(gmol)
Chemicalformula MZ
1 113902 00408 E-10-Methyl-11-tetradecen-1-ol acetate 26824 C17H32O2 5512 114485 59674 Hexadecanoic acid methyl ester 270256 C17H34O2 7413 124277 802415 91215-Octadecatrienoic acid methyl ester 292456 C19H32O2 7924 132204 97366 cis-Methyl 11-eicosenoate 324549 C21H40O2 29245 132962 20426 Methyl 18-methylnonadecanoate 326565 C21H42O2 7416 140306 12517 Methyl 11-docosenoate 35259 C23H44O2 32047 141064 02947 Docosanoic acid methyl ester 354619 C23H46O2 741
8 148058 00701 Octadecanoic acid 910-dihydroxy- methylester bis(trifluoroacetate) 3305026 C19H38O4 741
9 148641 00826 Tetracosanoic acid methyl ester 3826633 C25H50O2 741
10 156218 02718 Benzoic acid 35-dicyclohexyl-4-hydroxy-methyl ester 2643600 C16H24O3 571
821642
116107
126009
134058
134323
142319147773 150368
152751
156087
000E+00
100E+07
200E+07
300E+07
400E+07
500E+07
600E+07
700E+07
8 10 12 14 16 18 20
Rela
tive A
bund
ance
Time (min)
Figure 2 Gas ChromatographyMass Spectrometry (GC-MS) anal-ysis of polyphenols and carbohydrates of LSS
were shown in Figure 2 These active compounds were Glyc-erol 3TMS derivative D-Pinitol pentakis(trimethylsilyl)ether Palmitic Acid TMS derivative 11-Octadecenoic acid(Z)- TMS derivative alpha-Linolenic acid TMS derivativeStearic acid TMS derivative Sinapinic acid 2TMS derivative11-Eicosenoic acid (Z)- TMS derivative Sucrose 8TMSderivative 371115-tetramethylhexadecan-13-diol silylatedSucrose 8TMS derivative Behenic acid TMS derivativeD-(+)-Turanose octakis(trimethylsilyl) ether and beta-Sitosterol TMS derivative The retention time peak areachemicalformulae and molecular weight and MZ ratio ofpolyphenols and carbohydrates found in LSS are depicted inTable 2
32 Assessment of Biochemical Markers As shown in Table 3amp Figure 3(a) serum levels of liver biomarkers ALT AST 120574-GT and ALP activities were significantly elevated (Ple 001)with percentages of 49 128 310 and 421 respectivelyin rabbits injected with CCl4 for 5 weeks and for 10 weekswith percentages of 76 159 468 and 474 respectivelySerum total bilirubin triglycerides and cholesterol levelswere also significantly elevated (Ple 001) in rabbits injected
with CCl4 for 5 weeks with percentages of 139 250and 72 respectively and for 10 weeks with percentages of114 290 and 512 respectively compared to those of thenormal control (Figure 3(b)) In addition serum total proteinand albumin levels were significantly decreased (Ple 001)in rabbits injected with CCl4 for 5 weeks with percentagesof 36 and 30 respectively and for 10 weeks as wellwith percentages of 31 and 26 respectively compared tothose of the normal control Serum ALT AST 120574-GT ALPtotal bilirubin triglycerides and cholesterol activities weresignificantly reduced (Ple 001) in rabbits protected with 200mg kg bw of LSS with percentages of 46 60 31 4850 39 and 42 respectively and for 400 mgkg bw withpercentages of 57 67 49 46 34 29 and 28respectively for 5 weeks compared to the corresponding CCl4treated group (Table 3 and Figures 3(a) and 3(b)) SerumALTAST 120574-GT ALP total bilirubin triglycerides and cholesterollevelswere also significantly reduced in rabbits protectedwith200 and 400mgkg bw of LSS for 10 weeks compared to thoseof the corresponding CCl4 treated group as shown in Table 3and Figures 3(a) and 3(b)
As shown in Table 3 and Figure 3(b) serum total proteinand albumin levels were significantly elevated (Ple 001) withpercentages of 57 37 in 200 mgkg bw of LSS protectedgroup and 39 31 in 400 mgkg bw of LSS protectedgroup for five consecutive weeks compared to those ofthe corresponding CCl4 treated group respectively Samebehaviour was noticed after 10 consecutive weeks of CCL4treatment accompanied with LSS protection the elevationwas 43 29 in 200 mgkg bw of LSS protected group and42 27 and in 400 mgkg bw of LSS protected groupcompared to those of the corresponding CCl4 treated grouprespectively
33 Assessment of Oxidative Stress and Antioxidant ActivityThe present results showed in Table 4 amp Figure 4 exert asignificant decrease (Ple 001) in the enzymatic activities ofCAT SOD GR GST and GPx in the liver homogenateof CCl4 treated rabbits 5 and 10 weeks after experimental
Evidence-Based Complementary and Alternative Medicine 5
Table 2 Chemical pattern of LSS extract of polyphenols and carbohydrates revealed by GC-MS analyses RT = retention time Area Pct=peak area and Mwt= molecular weight
No RT Area Pct Compound name Mwt Chemical formula MZ1 81846 29942 Glycerol 3TMS derivative 3086372 C12H32O3Si3 2052 116107 66731 D-Pinitol pentakis(trimethylsilyl) ether 5550881 C22H54O6Si5 733 126062 42518 Palmitic Acid TMS derivative 3286052 C19H40O2Si 3134 134058 303464 11-Octadecenoic acid (Z)- TMS derivative 3546425 C21H42O2Si 3395 134323 18965 alpha-Linolenic acid TMS derivative 3506107 C21H38O2Si 757 134906 26564 Stearic acid TMS derivative 3566584 C21H44O2Si 1178 136123 10245 Sinapinic acid 2TMS derivative 3685722 C17H28O5Si2 3689 142372 33369 11-Eicosenoic acid (Z)- TMS derivative 3826956 C23H46O2Si 36710 147773 29175 Sucrose 8TMS derivative 9197454 C36H86O11Si8 36111 149521 12532 371115-tetramethylhexadecan-13-diol silylated 298547 C20H42O 23312 150368 32061 Sucrose 8TMS derivative 9197454 C36H86O11Si8 36113 151003 14675 Behenic acid TMS derivative 4127647 C25H52O2Si 11714 15614 15318 D-(+)-Turanose octakis(trimethylsilyl) ether 9487867 C37H89NO11Si8 7315 190612 12082 beta-Sitosterol TMS derivative 4868878 C32H58OSi 129
running as compared to those of the normal control with apercentage of change (53 50 33 32 35amp 46 3737 39 and 47 respectively) Also the present resultsrevealed a significant increase (Ple 001) in the MDA levelsin the liver homogenate of CCl4 treated rabbitrsquos 5 and 10weeks after experimental running as compared to those ofthe normal control with a percentage of change (96amp 120)respectively
Daily oral administration of 200 mgkg bw of LSS withinthe rabbitrsquos diets for 5 weeks starting from day one of theexperiment significantly increased (Ple 001) the enzymaticactivities of CAT SOD GR GST and GPx in the liverhomogenate of injured rabbits with liver cirrhosis for 5 weeksas compared to those of the corresponding CCl4 treatedgroupwith a percentage of change (93 93 39 41 42)and all parameters mentioned were significantly increased(Ple 001) with 70 58 33 25 37 respectively inrabbits administrated 400 mgkg bw of LSS and injuredwith liver cirrhosis for 5 weeks as compared to those ofthe corresponding CCl4 treated group Similarly Daily oraladministration of 200 mgkg bw of LSS within the rabbitrsquosdiets for 10 weeks starting from day one of the experimentsignificantly increased (Ple 001) the enzymatic activities ofCAT SOD GR GST and GPx in the liver homogenateof injured rabbits with liver cirrhosis with a percentageof change (70 58 52 58 74) and all parametersmentioned were reduced significantly (Ple 001) to 73 4747 46 and 65 respectively in rabbits administered 400mgkg bw of LSS and injured with liver cirrhosis for 10 weeksas compared to those of the corresponding CCl4 treatedgroup Also the results revealed that daily oral administrationof 200 and 400 mgkg bw of LSS within the rabbitsrsquo dietsfor 5 and 10 weeks starting from day one of the experimentsignificantly decreased (Ple 001) the MDA levels in the liverhomogenate with a percentage of change (47amp 44 and60amp 56 for 5 and 10 weeks respectively) compared tothose of the corresponding CCl4 treated group (Table 4 ampFigure 4)
34 DNA Results of Untreated LSS Administered CCl4-Treated and LSS Protected Rabbits DNAwas extracted fromliver tissues of all the studied rabbits the banding mannerwas observable in Figure 5 Treatment of rabbits with CCl4increased significantly DNA damage of the hepatic tissuesmore than those of the untreated rabbits An entirely dif-ferent banding manner was detected in CCl4 treated rabbitswhich was absent from the liver tissues of the untreatedrabbits DNA extracted from 200 and 400 mgkg bw ofLSS protected rabbitsrsquo livers administered with CCl4 after5 and 10 weeks of the experimental running showed asignificant repair in hepatic DNA Rabbits treated with only200 and 400 mgkg bw of LSS after 5 and 10 weeks of theexperimental running did not display any kind of DNAfragmentation
35 Histopathological Observations The micrograph shownin Figure 6(a) illustrates the normal aspect of the hepatic liverfor the control group (Subgroup A1) fed with normal dietwithout exposing to CCl4 hepatic intoxication Examinationof liver tissue of the control group in which rabbits receivednormal diet and 200 mgkg bw of LSS in their diet on dailybasis till the end of the experiment without exposing to CCl4hepatic intoxication (Subgroup A2) revealed normal hepaticarchitecture (Figure 6(b)) Examination of the control groupreceived normal diet and 400mgkg bw of LSS in their diet ondaily basis till the end of the experiment without exposing toCCl4 hepatic intoxication (Subgroup A3) revealed only mildinflammation except one case which showed the adverseeffects of the drug showing evident chronic venous conges-tion moderate inflammation and bridging fibrosis (Figures6(c) 6(d) and 6(e))
According to liver tissue in rabbits exposed to CCL4intoxication (Group-B1 control pollutant group) the fol-lowing can be seen hepatocyte feathery degeneration andchronic venous congestion steatosis parenchymal and portalinflammation and fibrosis (Figures 7(a) 7(b) and 7(c)) and(Figures 7(d) 7(e) and 7(f)) respectively
6 Evidence-Based Complementary and Alternative Medicine
Table3Liverp
rofileo
funtreatedLSS
administeredC
Cl4tre
atedand
LSSprotectedrabb
itsEffectofLS
Son
serum
markere
nzym
esALTA
STG
GTALPTBILITP
ALB
TGand
CHOL
inrabb
itstre
ated
with
CCL4indu
cedhepatotoxicityas
indicatedin
thefi
gureA
lldatawe
reexpressedas
meanplusmnSE
MV
aluesw
eres
tatistic
allyteste
dusingtheS
tudent
t-testandsig
nificant
differencesatplt005
andplt001
asindicatedby
(lowast)amp
(lowastlowast)com
paredto
norm
alcontrolby
()amp
()
comparedto
CCL4aft
er5w
eeksand
by(+)amp
(++)
comparedto
CCL4aft
er10
weeks
respectiv
ely
Group
ALT
(UL)
AST
(UL)
GGT(U
L)
ALP
(UL)
TBILI
(mgdl)
TP(gdl)
ALB
(gdl)
CHOL(m
gdl)
TG(m
gdl)
Con
trol
3801plusmn
468
2725plusmn
192
702plusmn12
42202plusmn16
2028plusmn003
650plusmn031
355plusmn017
1632plusmn506
4203plusmn469
LS(200
mgkg
bw)
3148plusmn678
2941plusmn
331
848plusmn12
05702plusmn229lowastlowast
043plusmn003lowast
640plusmn025
324plusmn013
3217plusmn287lowast
5274plusmn279lowast
LS(400
mgkg
bw)
1644plusmn14
6lowastlowast
2076plusmn343lowast
629plusmn229
5308plusmn16
1lowastlowast
046plusmn003lowastlowast
586plusmn047
344plusmn010
3161plusmn
305lowast
2610plusmn304lowast
CCl4(5
wk)
5676plusmn546lowastlowast
6230plusmn391lowastlowast
2872plusmn276lowastlowast
11483plusmn273lowastlowast
067plusmn004lowastlowast
414plusmn014lowastlowast
248plusmn025lowastlowast
8921plusmn
762lowastlowast
14774plusmn74
4lowastof
Change
49128
310
421
139
-36
-30
72250
CCl4(10wk)
6700plusmn476lowastlowast
7069plusmn645lowastlowast
3978plusmn226lowastlowast
12638plusmn371lowastlowast
060plusmn004lowastlowast
445plusmn01lowastlowast
261plusmn022lowastlowast
9996plusmn616lowastlowast
16379plusmn89lowastlowast
of
Change
76159
468
474
114-3
1-2
6512
29
CCl4+LS
(200
mgkg
bw)5
wk
3062plusmn219
2490plusmn302
1962plusmn19
5
5960plusmn237
033plusmn003
649plusmn020
339plusmn012
5135plusmn547
8978plusmn611
of
Change
-46
-60
-31
-48
-50
5737
-42
-39
CCl4+LS
(400
mgkg
bw)5
wk
2423plusmn
291++
2027plusmn309++
1464plusmn17
3++
6146plusmn416++
044plusmn003++
576plusmn062++
326plusmn020++
6306plusmn524++
10539plusmn562++
of
Change
-57
-67
-49
-46
-34
3931
-29
-28
CCl4+LS
(200
mgkg
bw)10wk
3804plusmn371
2390plusmn264
1581plusmn
142
5259plusmn257
038plusmn004
636plusmn038
338plusmn009
5236plusmn447
9870plusmn344
of
Change
-43
-66
-60
-58
-36
4329
-47
-39
CCl4+LS
(400
mgkg
bw)10wk
4018plusmn292++
2516plusmn508++
2292plusmn254++
7954plusmn484++
048plusmn003
++635plusmn038++
334plusmn014++
6557plusmn79
8++
10513plusmn666
++of
Change
-40
-64
-42
-37
-20
4227
-34
-36
Evidence-Based Complementary and Alternative Medicine 7
01020304050607080
ALT
Lev
e (U
L)
ALTlowastlowast
lowastlowast
lowastlowast
++++
0102030405060708090
AST
Lev
e (U
L)
AST
lowastlowast
lowast
lowastlowast
++ ++
05
1015202530354045
GG
T Le
ve (U
L)
GGTlowastlowast
lowastlowast
++
++
020406080
100120140
ALP
Lev
el (U
L)
ALPlowastlowast
lowastlowast
lowastlowastlowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
(a) Effect of LSS aqueous extract on serum marker enzymes ALT AST GGTand ALP in rabbits treated with CCL4 induced hepatotoxicity as indicated inthe figure All data were expressed as mean plusmn SEM Values were statisticallytested using the Student t-test and significant differences at p lt 005 and p lt 001indicated by (lowast) amp (lowastlowast) compared to normal control by () amp () comparedto CCL4 after 5 weeks and by (+) amp (++) compared to CCL4 after 10 weeksrespectively
012345678
TP L
evel
(gL
)
TP
lowastlowastlowastlowast
++ ++
00102030405060708
TBI
LI L
evel
(gL
) TBILI
lowastlowastlowast
lowastlowastlowastlowast
++++
005
115
225
335
4
ALB
Lev
el (g
L)
ALB
lowastlowastlowastlowast
++++
020406080
100120140160180200
TG L
evel
(gL
)
TG
lowast
lowastlowast
lowastlowast
lowastlowast
++++
0
20
40
60
80
100
120
CHO
L Le
vel (
gL)
CHOLlowastlowast
lowastlowast
++++
lowastlowast
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
(b) Effect of with LSS aqueous extract on biochemical parameters TBILI TPALB TG and CHOL in rabbits treated with CCL4 induced hepatotoxicity asindicated in the figure All data were expressed as mean plusmn SEM Values werestatistically tested using the Student t-test and significant differences at p lt 005and p lt 001 indicated by (lowast)amp (lowastlowast) compared to normal control by () amp ()compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4 after 10weeks respectively
Figure 3
8 Evidence-Based Complementary and Alternative Medicine
Table4En
zymaticactiv
itiesofantio
xidant
enzymesandMDAofun
treatedLSS
administeredC
Cl4tre
atedand
LSSprotectedrabb
itsA
lldatawe
reexpressedas
meanplusmnSE
MV
aluesw
ere
statistic
allyteste
dusingtheS
tudent
t-testandsig
nificantd
ifferencesa
tplt005
andplt001
asindicatedby
(lowast)amp
(lowastlowast)com
paredto
norm
alcontrolby
()amp
()
comparedto
CCL4aft
er5
weeksandby
(+)amp
(++)
comparedto
CCL4aft
er10
weeksrespectiv
ely
Group
CAT
(nggtissue)
SOD
(nggtissue)
GR
(nggtissue)
GST
(nggtissue)
GPx
(nggtissue)
MDA(nmolm
l)
Con
trol
3155plusmn
199
2820plusmn10
92029plusmn099
1537plusmn12
12197plusmn17
0647plusmn057
LS(200
mgkg
bw)
3127plusmn18
62631plusmn15
51908plusmn077
1542plusmn067
2129plusmn12
8635plusmn045
LS(400
mgkg
bw)
2843plusmn204
2676plusmn18
81972plusmn12
81555plusmn14
32031plusmn074
635plusmn060
CCl4(5
wk)
1475plusmn258lowastlowast
1406plusmn13
9lowastlowast
1358plusmn061lowastlowast
1041plusmn
108lowastlowast
1409plusmn099lowastlowast
1273plusmn10
6lowastlowast
of
Change
-53
-50
-33
-32
-35
96CCl4(10wk)
1682plusmn15
7lowastlowast
1767plusmn202lowastlowast
1277plusmn13
6lowastlowast
940plusmn073lowastlowast
1152plusmn18
2lowastlowast
1427plusmn10
7lowastlowast
of
Change
-46
-37
-37
-39
-47
120
CCl4+LS
(200
mgkg
bw)5
wk
2846plusmn201
2720plusmn094
1892plusmn093
1472plusmn118
2009plusmn112
667plusmn046
of
Change
9393
3941
42-4
7CCl4+LS
(400
mgkg
bw)5
wk
2519plusmn17
7++
2231plusmn14
0++
1813plusmn094++
1304plusmn13
1++
1941plusmn
192+
+713plusmn072++
of
Change
7058
3325
37-4
4CCl4+LS
(200
mgkg
bw)10wk
2872plusmn201
2796plusmn222
1949plusmn096
1492plusmn15
8
2006plusmn17
9
569plusmn046
of
Change
7058
5258
74-6
0CCl4+LS
(400
mgkg
bw)10wk
2919plusmn17
0++
2610plusmn14
3++
1887plusmn17
3++
1378plusmn086++
1906plusmn14
1++
619plusmn052++
of
Change
7347
4746
65-5
6
Evidence-Based Complementary and Alternative Medicine 9
05
10152025303540
CAT
Leve
(ng
g tis
sue)
CAT
lowastlowastlowastlowast
++++
0
5
10
15
20
25
30
35
SOD
Lev
e (ng
lg ti
ssue
)
SOD
lowastlowast
lowastlowast
++++
0
5
10
15
20
25
GR
Leve
(ng
g tis
sue)
GR
lowastlowastlowastlowast
++++
02468
1012141618
GST
Lev
e (ng
g ti
ssue
)
GST
lowastlowastlowastlowast
++++
0
5
10
15
20
25
GPx
Lev
e (ng
g ti
ssue
)
GPx
lowastlowastlowastlowast
++++
02468
1012141618
MD
A L
eve (
nmol
g ti
ssue
)
MDA
lowastlowast
lowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
Figure 4 Effects of LSS on oxidative stress markers in rabbits treated with CCL4 induced hepatotoxicity as indicated in the figure All datawere expressed as mean plusmn SEM Values were statistically tested using the Student t-test and significant differences at p lt 005 and p lt 001 asindicated by (lowast) amp (lowastlowast) compared to normal control by () amp () compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4after 10 weeks respectively
Examination of liver tissue exposed to CCL4 as well as200 mgkg bw of LSS in their diet on daily basis (GroupB2) reveals that the group which is sacrificed 5 weeksafter experimental running shows evident chronic venouscongestion and feathery degeneration in two cases only mildfeathery degeneration in others no steatosis or isolated vesic-ular steatosis mild parenchymal and portal inflammation
(Figures 8(a) 8(b) and 8(c)) Examination of the groupsacrificed 10 weeks after experimental running shows theimprovement of the histoarchitectural changes of the livertowards the normal aspect showing regenerating hepato-cytes with no steatosis discrete chronic venous congestionand discrete inflammatory infiltrate (Figures 8(d) 8(e) and8(f))
10 Evidence-Based Complementary and Alternative Medicine
M 1 2 3 4 5 6 7 8 9bp
10000
3000
2000
1500
1000
750
500
300
Figure 5 Agarose gel showingDNAdamage byCCl4 and protectiveeffects of various fractions of LSS Lanes from left (M) molecularweight marker (1) negative control (2) LSS (200 mgkg bw) (3) LSS(400mgkg bw) (4) CCl4 for 5weeks (5) CCl4 for 10weeks (6)amp (7)CCl4 + LSS (200 mgkg bw) (8) amp (9) CCl4 + LSS (400 mg kg bw)
Examination of liver tissue of rabbits exposed to CCL4as well as 400 mgkg bw of LSS in their diet on daily basis(Group B3) reveals in the group that scarified 5 weeks afterexperimental running improvement of the histoarchitecturalchanges of the liver as there is no chronic venous congestionor only discrete chronic venous congestion and mild featherydegeneration no steatosis or only isolated vesicular steatosisand mild parenchymal and portal inflammation (Figures9(a) 9(b) and 9(c)) But the group that was sacrificed10 weeks after experimental running shows adverse effectsof the drug in the form of steatosis and chronic venouscongestion which is evident in two cases with mild tomoderate inflammation Bridging fibrosis is seen in one case(Figures 9(d) 9(e) 9(f) and 9(g))
4 Discussion
Hepatic disease became a significant public health concernowing to growing rate of obesity in all age populations [26]The conservative treatments of liver problems such as acuteand chronic liver hepatitis liver cirrhosis and fatty liverare often insufficient due to hazardous effects initiated byhepatotoxic drugs of chemical origin [27] Medicinal plantsowing to their abundant hepatoprotective natural ingredi-ents have been comprehensively investigated to treat liverdisorders In the current study hepatoprotective efficacy ofLSS was investigated via hepatotoxic classical model inducedby carbon tetrachloride
In organ and tissue systems CCl4 is stimulated bycytochrome P-450 dependent mixed oxidase in the hepaticendoplasmic reticulum to procedure free radical trichloro-methyl (CCl3 and Cl3COO) This causes changes in themembrane of the endoplasmic reticulum and other cel-lular membranes resulting in an increase in calcium ionpermeability through the plasma membrane causing severeinstabilities of calcium homeostasis leading to necrotic celldeath [28 29] CCL4 affects many organs causing degenera-tion of liver fatty layer and centrilobular necrosis [29] Thisphenomenon indicates that CCl4 is a powerful hepatotoxic
agent and may be used for the characterization of the hepaticdisease treatment medicines Elevation of liver enzymaticmarkers (ALT AST ALP and bilirubin) and increased lipidperoxidation are further evidences of hepatotoxicity ALTand AST that exist in the hepatocytes can certainly leak intothe peripheral blood as soon as the hepatocytes are injured[29] Lepidium sativum seed analysis by GCMS exhibitedthe presence of high concentrations of mainly 120572-linolenicacid (ALA) which is nminus3 fatty acid an omega-3 fatty acidfound in seeds exerting its bioactivity in ameliorating thehepatic intoxication and oxidative stress including liver injuryinduced by alcohol [30] liver steatosis [31 32] nonalcoholichepatic disease [33] and parenteral nutrition-associated liverdisease [34] Pari and Jalaludeen stated that orientationof the active groups in sinapic acid increases antioxidantefficiency as metal chelators and promotes its protectiveefficacy against liver oxidative stress in arsenic intoxicatedrats [35]The current study showed that sinapic acid as ametalchelator may exert its biological activity in improving liverfunction
Lepidium sativum seed extracts have proved hepatopro-tective effects against CCl4 induced liver damage A researchstudy on male Albino Wistar rats demonstrated significantreduction in hepatotoxicity induced by CCl4 administeredwith 200-400 mgkg of Lepidium sativum seeds extractincluded within the diet Serum level of AST ALT levels andbilirubin levels were significantly elevated in CCl4 treated ratscompared to those of normal control However a significantreduction in the levels of these parameters in Lepidiumsativum seed extracts protected rats was observed The hep-atoprotective effect could be due to the presence of alkaloidcoumarin flavonoids tannin and triterpenes which enhanceantioxidant activity and reduce free radical production fromCCl4 which is the basic triggering factor for hepatotoxicity[36 37]
In the present study we evaluated the hepatoprotectiveefficacy of L sativum seeds in white male New-Zealand rab-bits The present results revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withL sativum seeds significantly repaired their liver injuriousmarker enzymes as well as bilirubin total protein and albu-min hence approving its hepatoprotective effect The resultsare in agreement with those of Afaf et al [12] where theystated the role of LSS as efficient hepatoprotective agent Alsothe current results are consistent with those of Mohamedand his coworkers [38] who reported that preprotectionwith LSEE (Lepidium sativum ethanolic extract) significantlyprohibited the D-GalNLPS induced rise in hepatic enzymes(AST ALT 120574-GGT ALP total bilirubin LDH and total pro-tein) thus significantly alleviated the reduction of lipid per-oxidation and repaired the antioxidant enzymatic status andtotal protein restoring them to normal levels D-GalNLPShas been widely used to examine the mechanisms underlyingacute liver failure [39 40] The hepatoprotective significanceof LSEE could be due to downregulation of cytokines (TNF-120572 IL-6) and stress gene (iNOS and HO-1) mRNA expres-sion and upregulation of IL-10 Lepidium sativum ethanolicextract (LSEE) preprotection also improved the degree ofstructural damage and reduced inflammatory infiltration in
Evidence-Based Complementary and Alternative Medicine 11
(a) (b)
(c) (d)
(e)
Figure 6 Effect of LSS on histopathological changes of liver tissues of normal groups in rabbits (a) Showing normal histological picture ofhepatic lobule that consists of central vein surrounded by normal hepatocytes (HEx100) (b) Showing liver sections of the rabbits treatedwith LSS (200 mgkg bw) which revealed normal histological picture of hepatic lobule that consists of central vein surrounded by normalhepatocytes (HEx100) (c) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealed normal hepatic architecturewith only mild inflammation [arrows] (HEx100) (d) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealedevident chronic venous congestion and moderate inflammation [arrows] (HEx100) (e) Showing liver sections of the rabbits treated with LSS(400 mgkg bw) which revealed portal to portal bridging fibrosis [arrow] (HEx200)
hepatic cells These outcomes established that LSEE alle-viates hepatic impairments and structural injury throughthe decay of oxidative stress inflammation and apopto-sis in the liver [38] Mohamed Sakran and his colleagues[10] suggested that 56-dimethoxy-210158403-methylenedioxy-7-C-120573-D glucopyranosyl isoflavone extracted from Lepidiumsativum seeds could improve the lipid profile and the liverfunctions in serum and reduce the free radicals generationby enhancing the antioxidant defense mechanism Newsuggested isoflavonoid can be used as a probable antioxi-dant against paracetamol hepatotoxicity with its antioxidant
characteristics and could restore the hepatic functionstowards normalization [10]
In the present study we found that improved cholesteroland triglycerides levels in L sativum seeds protected rabbitsas compared to CCl4 treated rabbits also support the hepato-protective effect of LSSThese results can also be correlated tothose of Shukla et al [41] who reported the role of L sativumseeds as hypolipidemic agent Also the results of the presentstudy agreed with those of Abdulrahman et al [20] whoseresults showed that L sativum seeds have hepatoprotectiveactivity that could be due to its antioxidant activity together
12 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
Figure 7 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) In rabbits sacrificed 5 weeks afterexperimental running as shown in (a) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100)(b) steatosis (30) in one case (HEx100) and (c) mild parenchymal inflammation [arrow] (HEx200) In rabbits sacrificed 10 weeks afterexperimental running as shown in (d) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100) (e)steatosis (about 20) and moderate parenchymal and portal inflammation (HEx100) and (f) bridging fibrosis [arrow] (HEx100)
with the presence of anti-inflammatory ingredients in Lsativum seeds extract
The results of the current study revealed that the exposureto CCl4 for 5 and 10 weeks led to a significant decrease inthe enzymatic activities of CAT GPx GR GST and SOD inthe liver homogenate of CCl4 treated rabbits 5 and 10 weeksafter experimental running compared to those of the normalcontrolThe concurrent protection of rabbits exposed toCCl4for 5 and 10weeks with 200amp 400mg kg bw LSS significantlyrepaired their antioxidant status and reduced significantly theMDA levels hence confirming its hepatoprotective efficacy
Although the precise molecular mechanism of action ofLSS is not completely understood some researchers [42]have confirmed the role of LSS in inhibiting oxidative stress
associated liver damage The antioxidant activity of LSScould be due to the presence of phenolic compound andflavonoids in these seeds as have been evidenced through thepreliminary screening study on LSS extract using HPLCOurresults match with those of Lee et al [42] who reported thatphenolic compounds play an essential role against oxidativestress related liver damage
A unique expectation of the current study is to confirmthat CCl4 induces liver DNA injury that was assessed usinggel electrophoretic method The results of the current studyshowed that CCL4 induces DNA injury as presented bya significant progression in liver DNA disintegration ofCCl4 treated rabbits above untreated control in the gelelectrophoresis
Evidence-Based Complementary and Alternative Medicine 13
(a) (b)
(c) (d)
(e) (f)
Figure 8 Effect of LSLS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (200 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) evident chronic venous congestion and feathery degeneration [arrow](HEx100) (b) mild feathery degeneration and isolated vesicular steatosis [arrow] (HEx200) and (c) moderate parenchymal and portalinflammation (HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) regenerating hepatocytes with isolatedvesicular steatosis (HEx100) (e) discrete chronic venous congestion and no steatosis (HEx400) and (f) discrete inflammatory infiltrate(HEx100)
Lipid peroxidation of the nuclear membrane may bedirectly accompanied with nuclear DNA damage [43]Nuclear DNA is concomitant with particular regions of thenuclear membrane [44]The close proximity of nuclear DNAto the nuclear membrane can ease the interaction of genomicDNA with the peroxyl radicals and further reactive inter-mediates formed during membrane lipid peroxidation [43]In the presence of transition metals the hydroxyl radicalsgenerated by CCL4 autoxidation can trigger peroxidationof the nuclear membrane lipids by metal catalyzed Haber-Weiss reaction the lipid peroxides levels and intermediate
free radicals could then be augmented by a chain reactionSubsequently the reactive species oxidize nuclearDNAwhenthe lipid peroxidation reaction occurs in the nuclear mem-brane since the nuclear membrane controls the transportof messenger RNA into the cytoplasm and supports thenuclear division process nuclear membrane peroxidationof lipids and proteins Moreover oxidative nuclear DNAdamage could disrupt these serious cellular functions [43]
DNA findings showed that DNA disintegration is adirect result after CCl4 treatment in rabbitsrsquo liver tissueswhich was reduced by participation of antioxidants through
14 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
(g)
Figure 9 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (400 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) discrete chronic venous congestion (HEx200) (b) centrilobular fibrosis[arrows] regenerating hepatocytes and isolated vesicular steatosis (HEx100) and (c) mild parenchymal and portal inflammation [arrow](HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) mild chronic venous congestion and isolated vesicularsteatosis (HEx100) (e) evident chronic venous congestion [arrows] (HEx100) (f) steatosis (40) with moderate inflammation (HEx100) and(g) portal to portal bridging fibrosis in one case [arrow] (HEx100)
Evidence-Based Complementary and Alternative Medicine 15
administration of 200 mg amp 400 mgkg bw L sativum seedsNearly complete repair of DNA damage was significant aftertreating rabbits with 200 mg amp 400 mgkg bw L sativumseeds The protective effect of L sativum seeds on hepaticDNA injury may be due to the antioxidant activity ofL sativum seeds via direct scavenging of free radicals orinterfering with free radicals generation
Concerning histopathological evaluation in our studyrabbitsrsquo livers exposed to CCL4 show portal and parenchymalinflammatory infiltrate feathery and ballooning degenera-tion fatty change and fibrosis Our data are consistent withRajeswary et al [45] who reported dilatation of sinusoidsfatty change disarrangement of normal hepatocytes and cen-trilobular necrosis Also A S Bernacchi et al [46] reportedthat CCL4 leads to liver necrosis and fat accumulation in therabbit
The present study revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withLSS improved their liver histopathological architecture inthe form of less inflammation regenerating hepatocytes nosteatosis or isolated vesicular steatosis no chronic venouscongestion or just discrete congestion and no fibrosis henceapproving its hepatoprotective effectThe results are in agree-ment with Afaf et al [12] and Abdulrahman et al [20] whoobserved the improvement of the histological structure inextract treated group as the histopathologic findings showedless inflammation well restored hepatocytes and less area ofnecrosis as compared to severe necrosis and inflammationin CCl4 treated group of animals which also supports thehepatoprotective effects of LLS Their findings suggest thepossibility of the presence of anti-inflammatory compoundsin LSS extract which could also be a contributing factortowards the hepatoprotective action of LSS
Althnaian [47] also reported that examination of hep-atocytes showed mild to moderate degree of recovery LSSattenuated the hepatic fat accumulation decreased vacuolardegeneration and improved the architecture of hepatocytes
In our study the reducing effect of LSS on fatty changecaused by CCL4 intoxication is indicating the protective andcurative role against fatty liver Our results are supported byShukla et al [41] who reported that LSS significantly reducedsteatosis Their histopathological observations confirmed thecurative efficacy of L sativum liver damage Microvesicularfatty changes hepatocyte ballooning and steatosis weremarkedly corrected by LSS Also Wadhwa et al [36] foundthat the severe fatty changes in the livers of rats caused byCCl4 were decreased in the treated groups
In the present study chronic venous congestion featherydegeneration and fatty changes show mild improvement inrabbits receiving 200mgkg bw of LSS for 5 weeks and showmoderate improvement in rabbits that received 200mgkg bwof LSS for 10 weeks and rabbits that received 400mgkg bwof LSS for 5 weeks Our data are consistent with Afaf et al[12] who observed that severe centrilobular hepatocellularvacuolation hemorrhages and congestion of the centralveins noticed in CCL4 group showed mild improvement ingroups that received 200 mgkg bw of LSS and moderateimprovement in groups that received 400 mgkg bw ofLSS
On the other hand rabbits that received 400mgkg bw ofLSS for 10 weeks showed evidence of liver toxicity in the formof venous congestion hepatocyte feathery degeneration fattychange and fibrosis These results are compatible with Bafeeland Ali [48] who reported that LSS had minimal effect onliver parenchyma if given in low doses So LSS could be usedas food additive or supplement Mineral content of calciumand phosphate and the presence of essential fatty acids inconsiderable level make LSS of beneficial nutritional valuefor many body organs including the liver However LSS inhigh dose showed some toxic effect on renal parenchyma rep-resented by focal necrosis hepatocyte apoptosis and portalinflammatory cell infiltrate Burow et al [49] reported thatthese hazardous effects of LSS may be due to isothiocyanatesreported to be one of LSS constituents
5 Conclusions and Recommendations
The present study showed that LSS is a key factor ininhibition of the hazardous effects of CCl4 LSS treatmentcould significantly alleviate to a greater extent the degree oflipid peroxidation and restore the antioxidative enzymes andshift liver function to normal levels In addition to antioxi-dants activity LSS attenuated the hepatic fat accumulationdecreased vacuolar degeneration ameliorated the degree ofstructural damage in the hepatic cells and improved thearchitecture of hepatocytes The current findings demon-strated that LSS have marked hepatoprotective activity Infuture work active ingredients of LSS extract should bestudied in various animal models of severe toxicity inducedby CCL4 and other hepatotoxic agents The active ingredientsof this plant are likely to be applied as one of the future drugsagainst acute liver toxicity
Data Availability
Data used to support the findings of this study are availablefrom the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This project was funded by the Deanship of ScientificResearch (DSR) King Abdulaziz University Jeddah undergrant no S-17-314-37 The authors therefore acknowledgewith thanks DSR technical and financial support
References
[1] M K Manibusan M Odin and D A Eastmond ldquoPostu-lated carbon tetrachloride mode of action a reviewrdquo Journalof Environmental Science and Health Part C EnvironmentalCarcinogenesis and Ecotoxicology Reviews vol 25 no 3 pp 185ndash209 2007
[2] N Brautbar and J Williams II ldquoIndustrial solvents andliver toxicity risk assessment risk factors and mechanismsrdquo
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
Stem Cells International
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Hindawiwwwhindawicom Volume 2018
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Disease Markers
Hindawiwwwhindawicom Volume 2018
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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
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Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
4 Evidence-Based Complementary and Alternative Medicine
Table 1 Chemical pattern of LSS extract revealed by GC-MS analyses RT = retention time Area Pct= peak area and Mwt= molecularweight
No RT Area Pct Compound name Mwt(gmol)
Chemicalformula MZ
1 113902 00408 E-10-Methyl-11-tetradecen-1-ol acetate 26824 C17H32O2 5512 114485 59674 Hexadecanoic acid methyl ester 270256 C17H34O2 7413 124277 802415 91215-Octadecatrienoic acid methyl ester 292456 C19H32O2 7924 132204 97366 cis-Methyl 11-eicosenoate 324549 C21H40O2 29245 132962 20426 Methyl 18-methylnonadecanoate 326565 C21H42O2 7416 140306 12517 Methyl 11-docosenoate 35259 C23H44O2 32047 141064 02947 Docosanoic acid methyl ester 354619 C23H46O2 741
8 148058 00701 Octadecanoic acid 910-dihydroxy- methylester bis(trifluoroacetate) 3305026 C19H38O4 741
9 148641 00826 Tetracosanoic acid methyl ester 3826633 C25H50O2 741
10 156218 02718 Benzoic acid 35-dicyclohexyl-4-hydroxy-methyl ester 2643600 C16H24O3 571
821642
116107
126009
134058
134323
142319147773 150368
152751
156087
000E+00
100E+07
200E+07
300E+07
400E+07
500E+07
600E+07
700E+07
8 10 12 14 16 18 20
Rela
tive A
bund
ance
Time (min)
Figure 2 Gas ChromatographyMass Spectrometry (GC-MS) anal-ysis of polyphenols and carbohydrates of LSS
were shown in Figure 2 These active compounds were Glyc-erol 3TMS derivative D-Pinitol pentakis(trimethylsilyl)ether Palmitic Acid TMS derivative 11-Octadecenoic acid(Z)- TMS derivative alpha-Linolenic acid TMS derivativeStearic acid TMS derivative Sinapinic acid 2TMS derivative11-Eicosenoic acid (Z)- TMS derivative Sucrose 8TMSderivative 371115-tetramethylhexadecan-13-diol silylatedSucrose 8TMS derivative Behenic acid TMS derivativeD-(+)-Turanose octakis(trimethylsilyl) ether and beta-Sitosterol TMS derivative The retention time peak areachemicalformulae and molecular weight and MZ ratio ofpolyphenols and carbohydrates found in LSS are depicted inTable 2
32 Assessment of Biochemical Markers As shown in Table 3amp Figure 3(a) serum levels of liver biomarkers ALT AST 120574-GT and ALP activities were significantly elevated (Ple 001)with percentages of 49 128 310 and 421 respectivelyin rabbits injected with CCl4 for 5 weeks and for 10 weekswith percentages of 76 159 468 and 474 respectivelySerum total bilirubin triglycerides and cholesterol levelswere also significantly elevated (Ple 001) in rabbits injected
with CCl4 for 5 weeks with percentages of 139 250and 72 respectively and for 10 weeks with percentages of114 290 and 512 respectively compared to those of thenormal control (Figure 3(b)) In addition serum total proteinand albumin levels were significantly decreased (Ple 001)in rabbits injected with CCl4 for 5 weeks with percentagesof 36 and 30 respectively and for 10 weeks as wellwith percentages of 31 and 26 respectively compared tothose of the normal control Serum ALT AST 120574-GT ALPtotal bilirubin triglycerides and cholesterol activities weresignificantly reduced (Ple 001) in rabbits protected with 200mg kg bw of LSS with percentages of 46 60 31 4850 39 and 42 respectively and for 400 mgkg bw withpercentages of 57 67 49 46 34 29 and 28respectively for 5 weeks compared to the corresponding CCl4treated group (Table 3 and Figures 3(a) and 3(b)) SerumALTAST 120574-GT ALP total bilirubin triglycerides and cholesterollevelswere also significantly reduced in rabbits protectedwith200 and 400mgkg bw of LSS for 10 weeks compared to thoseof the corresponding CCl4 treated group as shown in Table 3and Figures 3(a) and 3(b)
As shown in Table 3 and Figure 3(b) serum total proteinand albumin levels were significantly elevated (Ple 001) withpercentages of 57 37 in 200 mgkg bw of LSS protectedgroup and 39 31 in 400 mgkg bw of LSS protectedgroup for five consecutive weeks compared to those ofthe corresponding CCl4 treated group respectively Samebehaviour was noticed after 10 consecutive weeks of CCL4treatment accompanied with LSS protection the elevationwas 43 29 in 200 mgkg bw of LSS protected group and42 27 and in 400 mgkg bw of LSS protected groupcompared to those of the corresponding CCl4 treated grouprespectively
33 Assessment of Oxidative Stress and Antioxidant ActivityThe present results showed in Table 4 amp Figure 4 exert asignificant decrease (Ple 001) in the enzymatic activities ofCAT SOD GR GST and GPx in the liver homogenateof CCl4 treated rabbits 5 and 10 weeks after experimental
Evidence-Based Complementary and Alternative Medicine 5
Table 2 Chemical pattern of LSS extract of polyphenols and carbohydrates revealed by GC-MS analyses RT = retention time Area Pct=peak area and Mwt= molecular weight
No RT Area Pct Compound name Mwt Chemical formula MZ1 81846 29942 Glycerol 3TMS derivative 3086372 C12H32O3Si3 2052 116107 66731 D-Pinitol pentakis(trimethylsilyl) ether 5550881 C22H54O6Si5 733 126062 42518 Palmitic Acid TMS derivative 3286052 C19H40O2Si 3134 134058 303464 11-Octadecenoic acid (Z)- TMS derivative 3546425 C21H42O2Si 3395 134323 18965 alpha-Linolenic acid TMS derivative 3506107 C21H38O2Si 757 134906 26564 Stearic acid TMS derivative 3566584 C21H44O2Si 1178 136123 10245 Sinapinic acid 2TMS derivative 3685722 C17H28O5Si2 3689 142372 33369 11-Eicosenoic acid (Z)- TMS derivative 3826956 C23H46O2Si 36710 147773 29175 Sucrose 8TMS derivative 9197454 C36H86O11Si8 36111 149521 12532 371115-tetramethylhexadecan-13-diol silylated 298547 C20H42O 23312 150368 32061 Sucrose 8TMS derivative 9197454 C36H86O11Si8 36113 151003 14675 Behenic acid TMS derivative 4127647 C25H52O2Si 11714 15614 15318 D-(+)-Turanose octakis(trimethylsilyl) ether 9487867 C37H89NO11Si8 7315 190612 12082 beta-Sitosterol TMS derivative 4868878 C32H58OSi 129
running as compared to those of the normal control with apercentage of change (53 50 33 32 35amp 46 3737 39 and 47 respectively) Also the present resultsrevealed a significant increase (Ple 001) in the MDA levelsin the liver homogenate of CCl4 treated rabbitrsquos 5 and 10weeks after experimental running as compared to those ofthe normal control with a percentage of change (96amp 120)respectively
Daily oral administration of 200 mgkg bw of LSS withinthe rabbitrsquos diets for 5 weeks starting from day one of theexperiment significantly increased (Ple 001) the enzymaticactivities of CAT SOD GR GST and GPx in the liverhomogenate of injured rabbits with liver cirrhosis for 5 weeksas compared to those of the corresponding CCl4 treatedgroupwith a percentage of change (93 93 39 41 42)and all parameters mentioned were significantly increased(Ple 001) with 70 58 33 25 37 respectively inrabbits administrated 400 mgkg bw of LSS and injuredwith liver cirrhosis for 5 weeks as compared to those ofthe corresponding CCl4 treated group Similarly Daily oraladministration of 200 mgkg bw of LSS within the rabbitrsquosdiets for 10 weeks starting from day one of the experimentsignificantly increased (Ple 001) the enzymatic activities ofCAT SOD GR GST and GPx in the liver homogenateof injured rabbits with liver cirrhosis with a percentageof change (70 58 52 58 74) and all parametersmentioned were reduced significantly (Ple 001) to 73 4747 46 and 65 respectively in rabbits administered 400mgkg bw of LSS and injured with liver cirrhosis for 10 weeksas compared to those of the corresponding CCl4 treatedgroup Also the results revealed that daily oral administrationof 200 and 400 mgkg bw of LSS within the rabbitsrsquo dietsfor 5 and 10 weeks starting from day one of the experimentsignificantly decreased (Ple 001) the MDA levels in the liverhomogenate with a percentage of change (47amp 44 and60amp 56 for 5 and 10 weeks respectively) compared tothose of the corresponding CCl4 treated group (Table 4 ampFigure 4)
34 DNA Results of Untreated LSS Administered CCl4-Treated and LSS Protected Rabbits DNAwas extracted fromliver tissues of all the studied rabbits the banding mannerwas observable in Figure 5 Treatment of rabbits with CCl4increased significantly DNA damage of the hepatic tissuesmore than those of the untreated rabbits An entirely dif-ferent banding manner was detected in CCl4 treated rabbitswhich was absent from the liver tissues of the untreatedrabbits DNA extracted from 200 and 400 mgkg bw ofLSS protected rabbitsrsquo livers administered with CCl4 after5 and 10 weeks of the experimental running showed asignificant repair in hepatic DNA Rabbits treated with only200 and 400 mgkg bw of LSS after 5 and 10 weeks of theexperimental running did not display any kind of DNAfragmentation
35 Histopathological Observations The micrograph shownin Figure 6(a) illustrates the normal aspect of the hepatic liverfor the control group (Subgroup A1) fed with normal dietwithout exposing to CCl4 hepatic intoxication Examinationof liver tissue of the control group in which rabbits receivednormal diet and 200 mgkg bw of LSS in their diet on dailybasis till the end of the experiment without exposing to CCl4hepatic intoxication (Subgroup A2) revealed normal hepaticarchitecture (Figure 6(b)) Examination of the control groupreceived normal diet and 400mgkg bw of LSS in their diet ondaily basis till the end of the experiment without exposing toCCl4 hepatic intoxication (Subgroup A3) revealed only mildinflammation except one case which showed the adverseeffects of the drug showing evident chronic venous conges-tion moderate inflammation and bridging fibrosis (Figures6(c) 6(d) and 6(e))
According to liver tissue in rabbits exposed to CCL4intoxication (Group-B1 control pollutant group) the fol-lowing can be seen hepatocyte feathery degeneration andchronic venous congestion steatosis parenchymal and portalinflammation and fibrosis (Figures 7(a) 7(b) and 7(c)) and(Figures 7(d) 7(e) and 7(f)) respectively
6 Evidence-Based Complementary and Alternative Medicine
Table3Liverp
rofileo
funtreatedLSS
administeredC
Cl4tre
atedand
LSSprotectedrabb
itsEffectofLS
Son
serum
markere
nzym
esALTA
STG
GTALPTBILITP
ALB
TGand
CHOL
inrabb
itstre
ated
with
CCL4indu
cedhepatotoxicityas
indicatedin
thefi
gureA
lldatawe
reexpressedas
meanplusmnSE
MV
aluesw
eres
tatistic
allyteste
dusingtheS
tudent
t-testandsig
nificant
differencesatplt005
andplt001
asindicatedby
(lowast)amp
(lowastlowast)com
paredto
norm
alcontrolby
()amp
()
comparedto
CCL4aft
er5w
eeksand
by(+)amp
(++)
comparedto
CCL4aft
er10
weeks
respectiv
ely
Group
ALT
(UL)
AST
(UL)
GGT(U
L)
ALP
(UL)
TBILI
(mgdl)
TP(gdl)
ALB
(gdl)
CHOL(m
gdl)
TG(m
gdl)
Con
trol
3801plusmn
468
2725plusmn
192
702plusmn12
42202plusmn16
2028plusmn003
650plusmn031
355plusmn017
1632plusmn506
4203plusmn469
LS(200
mgkg
bw)
3148plusmn678
2941plusmn
331
848plusmn12
05702plusmn229lowastlowast
043plusmn003lowast
640plusmn025
324plusmn013
3217plusmn287lowast
5274plusmn279lowast
LS(400
mgkg
bw)
1644plusmn14
6lowastlowast
2076plusmn343lowast
629plusmn229
5308plusmn16
1lowastlowast
046plusmn003lowastlowast
586plusmn047
344plusmn010
3161plusmn
305lowast
2610plusmn304lowast
CCl4(5
wk)
5676plusmn546lowastlowast
6230plusmn391lowastlowast
2872plusmn276lowastlowast
11483plusmn273lowastlowast
067plusmn004lowastlowast
414plusmn014lowastlowast
248plusmn025lowastlowast
8921plusmn
762lowastlowast
14774plusmn74
4lowastof
Change
49128
310
421
139
-36
-30
72250
CCl4(10wk)
6700plusmn476lowastlowast
7069plusmn645lowastlowast
3978plusmn226lowastlowast
12638plusmn371lowastlowast
060plusmn004lowastlowast
445plusmn01lowastlowast
261plusmn022lowastlowast
9996plusmn616lowastlowast
16379plusmn89lowastlowast
of
Change
76159
468
474
114-3
1-2
6512
29
CCl4+LS
(200
mgkg
bw)5
wk
3062plusmn219
2490plusmn302
1962plusmn19
5
5960plusmn237
033plusmn003
649plusmn020
339plusmn012
5135plusmn547
8978plusmn611
of
Change
-46
-60
-31
-48
-50
5737
-42
-39
CCl4+LS
(400
mgkg
bw)5
wk
2423plusmn
291++
2027plusmn309++
1464plusmn17
3++
6146plusmn416++
044plusmn003++
576plusmn062++
326plusmn020++
6306plusmn524++
10539plusmn562++
of
Change
-57
-67
-49
-46
-34
3931
-29
-28
CCl4+LS
(200
mgkg
bw)10wk
3804plusmn371
2390plusmn264
1581plusmn
142
5259plusmn257
038plusmn004
636plusmn038
338plusmn009
5236plusmn447
9870plusmn344
of
Change
-43
-66
-60
-58
-36
4329
-47
-39
CCl4+LS
(400
mgkg
bw)10wk
4018plusmn292++
2516plusmn508++
2292plusmn254++
7954plusmn484++
048plusmn003
++635plusmn038++
334plusmn014++
6557plusmn79
8++
10513plusmn666
++of
Change
-40
-64
-42
-37
-20
4227
-34
-36
Evidence-Based Complementary and Alternative Medicine 7
01020304050607080
ALT
Lev
e (U
L)
ALTlowastlowast
lowastlowast
lowastlowast
++++
0102030405060708090
AST
Lev
e (U
L)
AST
lowastlowast
lowast
lowastlowast
++ ++
05
1015202530354045
GG
T Le
ve (U
L)
GGTlowastlowast
lowastlowast
++
++
020406080
100120140
ALP
Lev
el (U
L)
ALPlowastlowast
lowastlowast
lowastlowastlowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
(a) Effect of LSS aqueous extract on serum marker enzymes ALT AST GGTand ALP in rabbits treated with CCL4 induced hepatotoxicity as indicated inthe figure All data were expressed as mean plusmn SEM Values were statisticallytested using the Student t-test and significant differences at p lt 005 and p lt 001indicated by (lowast) amp (lowastlowast) compared to normal control by () amp () comparedto CCL4 after 5 weeks and by (+) amp (++) compared to CCL4 after 10 weeksrespectively
012345678
TP L
evel
(gL
)
TP
lowastlowastlowastlowast
++ ++
00102030405060708
TBI
LI L
evel
(gL
) TBILI
lowastlowastlowast
lowastlowastlowastlowast
++++
005
115
225
335
4
ALB
Lev
el (g
L)
ALB
lowastlowastlowastlowast
++++
020406080
100120140160180200
TG L
evel
(gL
)
TG
lowast
lowastlowast
lowastlowast
lowastlowast
++++
0
20
40
60
80
100
120
CHO
L Le
vel (
gL)
CHOLlowastlowast
lowastlowast
++++
lowastlowast
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
(b) Effect of with LSS aqueous extract on biochemical parameters TBILI TPALB TG and CHOL in rabbits treated with CCL4 induced hepatotoxicity asindicated in the figure All data were expressed as mean plusmn SEM Values werestatistically tested using the Student t-test and significant differences at p lt 005and p lt 001 indicated by (lowast)amp (lowastlowast) compared to normal control by () amp ()compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4 after 10weeks respectively
Figure 3
8 Evidence-Based Complementary and Alternative Medicine
Table4En
zymaticactiv
itiesofantio
xidant
enzymesandMDAofun
treatedLSS
administeredC
Cl4tre
atedand
LSSprotectedrabb
itsA
lldatawe
reexpressedas
meanplusmnSE
MV
aluesw
ere
statistic
allyteste
dusingtheS
tudent
t-testandsig
nificantd
ifferencesa
tplt005
andplt001
asindicatedby
(lowast)amp
(lowastlowast)com
paredto
norm
alcontrolby
()amp
()
comparedto
CCL4aft
er5
weeksandby
(+)amp
(++)
comparedto
CCL4aft
er10
weeksrespectiv
ely
Group
CAT
(nggtissue)
SOD
(nggtissue)
GR
(nggtissue)
GST
(nggtissue)
GPx
(nggtissue)
MDA(nmolm
l)
Con
trol
3155plusmn
199
2820plusmn10
92029plusmn099
1537plusmn12
12197plusmn17
0647plusmn057
LS(200
mgkg
bw)
3127plusmn18
62631plusmn15
51908plusmn077
1542plusmn067
2129plusmn12
8635plusmn045
LS(400
mgkg
bw)
2843plusmn204
2676plusmn18
81972plusmn12
81555plusmn14
32031plusmn074
635plusmn060
CCl4(5
wk)
1475plusmn258lowastlowast
1406plusmn13
9lowastlowast
1358plusmn061lowastlowast
1041plusmn
108lowastlowast
1409plusmn099lowastlowast
1273plusmn10
6lowastlowast
of
Change
-53
-50
-33
-32
-35
96CCl4(10wk)
1682plusmn15
7lowastlowast
1767plusmn202lowastlowast
1277plusmn13
6lowastlowast
940plusmn073lowastlowast
1152plusmn18
2lowastlowast
1427plusmn10
7lowastlowast
of
Change
-46
-37
-37
-39
-47
120
CCl4+LS
(200
mgkg
bw)5
wk
2846plusmn201
2720plusmn094
1892plusmn093
1472plusmn118
2009plusmn112
667plusmn046
of
Change
9393
3941
42-4
7CCl4+LS
(400
mgkg
bw)5
wk
2519plusmn17
7++
2231plusmn14
0++
1813plusmn094++
1304plusmn13
1++
1941plusmn
192+
+713plusmn072++
of
Change
7058
3325
37-4
4CCl4+LS
(200
mgkg
bw)10wk
2872plusmn201
2796plusmn222
1949plusmn096
1492plusmn15
8
2006plusmn17
9
569plusmn046
of
Change
7058
5258
74-6
0CCl4+LS
(400
mgkg
bw)10wk
2919plusmn17
0++
2610plusmn14
3++
1887plusmn17
3++
1378plusmn086++
1906plusmn14
1++
619plusmn052++
of
Change
7347
4746
65-5
6
Evidence-Based Complementary and Alternative Medicine 9
05
10152025303540
CAT
Leve
(ng
g tis
sue)
CAT
lowastlowastlowastlowast
++++
0
5
10
15
20
25
30
35
SOD
Lev
e (ng
lg ti
ssue
)
SOD
lowastlowast
lowastlowast
++++
0
5
10
15
20
25
GR
Leve
(ng
g tis
sue)
GR
lowastlowastlowastlowast
++++
02468
1012141618
GST
Lev
e (ng
g ti
ssue
)
GST
lowastlowastlowastlowast
++++
0
5
10
15
20
25
GPx
Lev
e (ng
g ti
ssue
)
GPx
lowastlowastlowastlowast
++++
02468
1012141618
MD
A L
eve (
nmol
g ti
ssue
)
MDA
lowastlowast
lowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
Figure 4 Effects of LSS on oxidative stress markers in rabbits treated with CCL4 induced hepatotoxicity as indicated in the figure All datawere expressed as mean plusmn SEM Values were statistically tested using the Student t-test and significant differences at p lt 005 and p lt 001 asindicated by (lowast) amp (lowastlowast) compared to normal control by () amp () compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4after 10 weeks respectively
Examination of liver tissue exposed to CCL4 as well as200 mgkg bw of LSS in their diet on daily basis (GroupB2) reveals that the group which is sacrificed 5 weeksafter experimental running shows evident chronic venouscongestion and feathery degeneration in two cases only mildfeathery degeneration in others no steatosis or isolated vesic-ular steatosis mild parenchymal and portal inflammation
(Figures 8(a) 8(b) and 8(c)) Examination of the groupsacrificed 10 weeks after experimental running shows theimprovement of the histoarchitectural changes of the livertowards the normal aspect showing regenerating hepato-cytes with no steatosis discrete chronic venous congestionand discrete inflammatory infiltrate (Figures 8(d) 8(e) and8(f))
10 Evidence-Based Complementary and Alternative Medicine
M 1 2 3 4 5 6 7 8 9bp
10000
3000
2000
1500
1000
750
500
300
Figure 5 Agarose gel showingDNAdamage byCCl4 and protectiveeffects of various fractions of LSS Lanes from left (M) molecularweight marker (1) negative control (2) LSS (200 mgkg bw) (3) LSS(400mgkg bw) (4) CCl4 for 5weeks (5) CCl4 for 10weeks (6)amp (7)CCl4 + LSS (200 mgkg bw) (8) amp (9) CCl4 + LSS (400 mg kg bw)
Examination of liver tissue of rabbits exposed to CCL4as well as 400 mgkg bw of LSS in their diet on daily basis(Group B3) reveals in the group that scarified 5 weeks afterexperimental running improvement of the histoarchitecturalchanges of the liver as there is no chronic venous congestionor only discrete chronic venous congestion and mild featherydegeneration no steatosis or only isolated vesicular steatosisand mild parenchymal and portal inflammation (Figures9(a) 9(b) and 9(c)) But the group that was sacrificed10 weeks after experimental running shows adverse effectsof the drug in the form of steatosis and chronic venouscongestion which is evident in two cases with mild tomoderate inflammation Bridging fibrosis is seen in one case(Figures 9(d) 9(e) 9(f) and 9(g))
4 Discussion
Hepatic disease became a significant public health concernowing to growing rate of obesity in all age populations [26]The conservative treatments of liver problems such as acuteand chronic liver hepatitis liver cirrhosis and fatty liverare often insufficient due to hazardous effects initiated byhepatotoxic drugs of chemical origin [27] Medicinal plantsowing to their abundant hepatoprotective natural ingredi-ents have been comprehensively investigated to treat liverdisorders In the current study hepatoprotective efficacy ofLSS was investigated via hepatotoxic classical model inducedby carbon tetrachloride
In organ and tissue systems CCl4 is stimulated bycytochrome P-450 dependent mixed oxidase in the hepaticendoplasmic reticulum to procedure free radical trichloro-methyl (CCl3 and Cl3COO) This causes changes in themembrane of the endoplasmic reticulum and other cel-lular membranes resulting in an increase in calcium ionpermeability through the plasma membrane causing severeinstabilities of calcium homeostasis leading to necrotic celldeath [28 29] CCL4 affects many organs causing degenera-tion of liver fatty layer and centrilobular necrosis [29] Thisphenomenon indicates that CCl4 is a powerful hepatotoxic
agent and may be used for the characterization of the hepaticdisease treatment medicines Elevation of liver enzymaticmarkers (ALT AST ALP and bilirubin) and increased lipidperoxidation are further evidences of hepatotoxicity ALTand AST that exist in the hepatocytes can certainly leak intothe peripheral blood as soon as the hepatocytes are injured[29] Lepidium sativum seed analysis by GCMS exhibitedthe presence of high concentrations of mainly 120572-linolenicacid (ALA) which is nminus3 fatty acid an omega-3 fatty acidfound in seeds exerting its bioactivity in ameliorating thehepatic intoxication and oxidative stress including liver injuryinduced by alcohol [30] liver steatosis [31 32] nonalcoholichepatic disease [33] and parenteral nutrition-associated liverdisease [34] Pari and Jalaludeen stated that orientationof the active groups in sinapic acid increases antioxidantefficiency as metal chelators and promotes its protectiveefficacy against liver oxidative stress in arsenic intoxicatedrats [35]The current study showed that sinapic acid as ametalchelator may exert its biological activity in improving liverfunction
Lepidium sativum seed extracts have proved hepatopro-tective effects against CCl4 induced liver damage A researchstudy on male Albino Wistar rats demonstrated significantreduction in hepatotoxicity induced by CCl4 administeredwith 200-400 mgkg of Lepidium sativum seeds extractincluded within the diet Serum level of AST ALT levels andbilirubin levels were significantly elevated in CCl4 treated ratscompared to those of normal control However a significantreduction in the levels of these parameters in Lepidiumsativum seed extracts protected rats was observed The hep-atoprotective effect could be due to the presence of alkaloidcoumarin flavonoids tannin and triterpenes which enhanceantioxidant activity and reduce free radical production fromCCl4 which is the basic triggering factor for hepatotoxicity[36 37]
In the present study we evaluated the hepatoprotectiveefficacy of L sativum seeds in white male New-Zealand rab-bits The present results revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withL sativum seeds significantly repaired their liver injuriousmarker enzymes as well as bilirubin total protein and albu-min hence approving its hepatoprotective effect The resultsare in agreement with those of Afaf et al [12] where theystated the role of LSS as efficient hepatoprotective agent Alsothe current results are consistent with those of Mohamedand his coworkers [38] who reported that preprotectionwith LSEE (Lepidium sativum ethanolic extract) significantlyprohibited the D-GalNLPS induced rise in hepatic enzymes(AST ALT 120574-GGT ALP total bilirubin LDH and total pro-tein) thus significantly alleviated the reduction of lipid per-oxidation and repaired the antioxidant enzymatic status andtotal protein restoring them to normal levels D-GalNLPShas been widely used to examine the mechanisms underlyingacute liver failure [39 40] The hepatoprotective significanceof LSEE could be due to downregulation of cytokines (TNF-120572 IL-6) and stress gene (iNOS and HO-1) mRNA expres-sion and upregulation of IL-10 Lepidium sativum ethanolicextract (LSEE) preprotection also improved the degree ofstructural damage and reduced inflammatory infiltration in
Evidence-Based Complementary and Alternative Medicine 11
(a) (b)
(c) (d)
(e)
Figure 6 Effect of LSS on histopathological changes of liver tissues of normal groups in rabbits (a) Showing normal histological picture ofhepatic lobule that consists of central vein surrounded by normal hepatocytes (HEx100) (b) Showing liver sections of the rabbits treatedwith LSS (200 mgkg bw) which revealed normal histological picture of hepatic lobule that consists of central vein surrounded by normalhepatocytes (HEx100) (c) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealed normal hepatic architecturewith only mild inflammation [arrows] (HEx100) (d) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealedevident chronic venous congestion and moderate inflammation [arrows] (HEx100) (e) Showing liver sections of the rabbits treated with LSS(400 mgkg bw) which revealed portal to portal bridging fibrosis [arrow] (HEx200)
hepatic cells These outcomes established that LSEE alle-viates hepatic impairments and structural injury throughthe decay of oxidative stress inflammation and apopto-sis in the liver [38] Mohamed Sakran and his colleagues[10] suggested that 56-dimethoxy-210158403-methylenedioxy-7-C-120573-D glucopyranosyl isoflavone extracted from Lepidiumsativum seeds could improve the lipid profile and the liverfunctions in serum and reduce the free radicals generationby enhancing the antioxidant defense mechanism Newsuggested isoflavonoid can be used as a probable antioxi-dant against paracetamol hepatotoxicity with its antioxidant
characteristics and could restore the hepatic functionstowards normalization [10]
In the present study we found that improved cholesteroland triglycerides levels in L sativum seeds protected rabbitsas compared to CCl4 treated rabbits also support the hepato-protective effect of LSSThese results can also be correlated tothose of Shukla et al [41] who reported the role of L sativumseeds as hypolipidemic agent Also the results of the presentstudy agreed with those of Abdulrahman et al [20] whoseresults showed that L sativum seeds have hepatoprotectiveactivity that could be due to its antioxidant activity together
12 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
Figure 7 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) In rabbits sacrificed 5 weeks afterexperimental running as shown in (a) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100)(b) steatosis (30) in one case (HEx100) and (c) mild parenchymal inflammation [arrow] (HEx200) In rabbits sacrificed 10 weeks afterexperimental running as shown in (d) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100) (e)steatosis (about 20) and moderate parenchymal and portal inflammation (HEx100) and (f) bridging fibrosis [arrow] (HEx100)
with the presence of anti-inflammatory ingredients in Lsativum seeds extract
The results of the current study revealed that the exposureto CCl4 for 5 and 10 weeks led to a significant decrease inthe enzymatic activities of CAT GPx GR GST and SOD inthe liver homogenate of CCl4 treated rabbits 5 and 10 weeksafter experimental running compared to those of the normalcontrolThe concurrent protection of rabbits exposed toCCl4for 5 and 10weeks with 200amp 400mg kg bw LSS significantlyrepaired their antioxidant status and reduced significantly theMDA levels hence confirming its hepatoprotective efficacy
Although the precise molecular mechanism of action ofLSS is not completely understood some researchers [42]have confirmed the role of LSS in inhibiting oxidative stress
associated liver damage The antioxidant activity of LSScould be due to the presence of phenolic compound andflavonoids in these seeds as have been evidenced through thepreliminary screening study on LSS extract using HPLCOurresults match with those of Lee et al [42] who reported thatphenolic compounds play an essential role against oxidativestress related liver damage
A unique expectation of the current study is to confirmthat CCl4 induces liver DNA injury that was assessed usinggel electrophoretic method The results of the current studyshowed that CCL4 induces DNA injury as presented bya significant progression in liver DNA disintegration ofCCl4 treated rabbits above untreated control in the gelelectrophoresis
Evidence-Based Complementary and Alternative Medicine 13
(a) (b)
(c) (d)
(e) (f)
Figure 8 Effect of LSLS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (200 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) evident chronic venous congestion and feathery degeneration [arrow](HEx100) (b) mild feathery degeneration and isolated vesicular steatosis [arrow] (HEx200) and (c) moderate parenchymal and portalinflammation (HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) regenerating hepatocytes with isolatedvesicular steatosis (HEx100) (e) discrete chronic venous congestion and no steatosis (HEx400) and (f) discrete inflammatory infiltrate(HEx100)
Lipid peroxidation of the nuclear membrane may bedirectly accompanied with nuclear DNA damage [43]Nuclear DNA is concomitant with particular regions of thenuclear membrane [44]The close proximity of nuclear DNAto the nuclear membrane can ease the interaction of genomicDNA with the peroxyl radicals and further reactive inter-mediates formed during membrane lipid peroxidation [43]In the presence of transition metals the hydroxyl radicalsgenerated by CCL4 autoxidation can trigger peroxidationof the nuclear membrane lipids by metal catalyzed Haber-Weiss reaction the lipid peroxides levels and intermediate
free radicals could then be augmented by a chain reactionSubsequently the reactive species oxidize nuclearDNAwhenthe lipid peroxidation reaction occurs in the nuclear mem-brane since the nuclear membrane controls the transportof messenger RNA into the cytoplasm and supports thenuclear division process nuclear membrane peroxidationof lipids and proteins Moreover oxidative nuclear DNAdamage could disrupt these serious cellular functions [43]
DNA findings showed that DNA disintegration is adirect result after CCl4 treatment in rabbitsrsquo liver tissueswhich was reduced by participation of antioxidants through
14 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
(g)
Figure 9 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (400 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) discrete chronic venous congestion (HEx200) (b) centrilobular fibrosis[arrows] regenerating hepatocytes and isolated vesicular steatosis (HEx100) and (c) mild parenchymal and portal inflammation [arrow](HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) mild chronic venous congestion and isolated vesicularsteatosis (HEx100) (e) evident chronic venous congestion [arrows] (HEx100) (f) steatosis (40) with moderate inflammation (HEx100) and(g) portal to portal bridging fibrosis in one case [arrow] (HEx100)
Evidence-Based Complementary and Alternative Medicine 15
administration of 200 mg amp 400 mgkg bw L sativum seedsNearly complete repair of DNA damage was significant aftertreating rabbits with 200 mg amp 400 mgkg bw L sativumseeds The protective effect of L sativum seeds on hepaticDNA injury may be due to the antioxidant activity ofL sativum seeds via direct scavenging of free radicals orinterfering with free radicals generation
Concerning histopathological evaluation in our studyrabbitsrsquo livers exposed to CCL4 show portal and parenchymalinflammatory infiltrate feathery and ballooning degenera-tion fatty change and fibrosis Our data are consistent withRajeswary et al [45] who reported dilatation of sinusoidsfatty change disarrangement of normal hepatocytes and cen-trilobular necrosis Also A S Bernacchi et al [46] reportedthat CCL4 leads to liver necrosis and fat accumulation in therabbit
The present study revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withLSS improved their liver histopathological architecture inthe form of less inflammation regenerating hepatocytes nosteatosis or isolated vesicular steatosis no chronic venouscongestion or just discrete congestion and no fibrosis henceapproving its hepatoprotective effectThe results are in agree-ment with Afaf et al [12] and Abdulrahman et al [20] whoobserved the improvement of the histological structure inextract treated group as the histopathologic findings showedless inflammation well restored hepatocytes and less area ofnecrosis as compared to severe necrosis and inflammationin CCl4 treated group of animals which also supports thehepatoprotective effects of LLS Their findings suggest thepossibility of the presence of anti-inflammatory compoundsin LSS extract which could also be a contributing factortowards the hepatoprotective action of LSS
Althnaian [47] also reported that examination of hep-atocytes showed mild to moderate degree of recovery LSSattenuated the hepatic fat accumulation decreased vacuolardegeneration and improved the architecture of hepatocytes
In our study the reducing effect of LSS on fatty changecaused by CCL4 intoxication is indicating the protective andcurative role against fatty liver Our results are supported byShukla et al [41] who reported that LSS significantly reducedsteatosis Their histopathological observations confirmed thecurative efficacy of L sativum liver damage Microvesicularfatty changes hepatocyte ballooning and steatosis weremarkedly corrected by LSS Also Wadhwa et al [36] foundthat the severe fatty changes in the livers of rats caused byCCl4 were decreased in the treated groups
In the present study chronic venous congestion featherydegeneration and fatty changes show mild improvement inrabbits receiving 200mgkg bw of LSS for 5 weeks and showmoderate improvement in rabbits that received 200mgkg bwof LSS for 10 weeks and rabbits that received 400mgkg bwof LSS for 5 weeks Our data are consistent with Afaf et al[12] who observed that severe centrilobular hepatocellularvacuolation hemorrhages and congestion of the centralveins noticed in CCL4 group showed mild improvement ingroups that received 200 mgkg bw of LSS and moderateimprovement in groups that received 400 mgkg bw ofLSS
On the other hand rabbits that received 400mgkg bw ofLSS for 10 weeks showed evidence of liver toxicity in the formof venous congestion hepatocyte feathery degeneration fattychange and fibrosis These results are compatible with Bafeeland Ali [48] who reported that LSS had minimal effect onliver parenchyma if given in low doses So LSS could be usedas food additive or supplement Mineral content of calciumand phosphate and the presence of essential fatty acids inconsiderable level make LSS of beneficial nutritional valuefor many body organs including the liver However LSS inhigh dose showed some toxic effect on renal parenchyma rep-resented by focal necrosis hepatocyte apoptosis and portalinflammatory cell infiltrate Burow et al [49] reported thatthese hazardous effects of LSS may be due to isothiocyanatesreported to be one of LSS constituents
5 Conclusions and Recommendations
The present study showed that LSS is a key factor ininhibition of the hazardous effects of CCl4 LSS treatmentcould significantly alleviate to a greater extent the degree oflipid peroxidation and restore the antioxidative enzymes andshift liver function to normal levels In addition to antioxi-dants activity LSS attenuated the hepatic fat accumulationdecreased vacuolar degeneration ameliorated the degree ofstructural damage in the hepatic cells and improved thearchitecture of hepatocytes The current findings demon-strated that LSS have marked hepatoprotective activity Infuture work active ingredients of LSS extract should bestudied in various animal models of severe toxicity inducedby CCL4 and other hepatotoxic agents The active ingredientsof this plant are likely to be applied as one of the future drugsagainst acute liver toxicity
Data Availability
Data used to support the findings of this study are availablefrom the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This project was funded by the Deanship of ScientificResearch (DSR) King Abdulaziz University Jeddah undergrant no S-17-314-37 The authors therefore acknowledgewith thanks DSR technical and financial support
References
[1] M K Manibusan M Odin and D A Eastmond ldquoPostu-lated carbon tetrachloride mode of action a reviewrdquo Journalof Environmental Science and Health Part C EnvironmentalCarcinogenesis and Ecotoxicology Reviews vol 25 no 3 pp 185ndash209 2007
[2] N Brautbar and J Williams II ldquoIndustrial solvents andliver toxicity risk assessment risk factors and mechanismsrdquo
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
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Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
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Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
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Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 5
Table 2 Chemical pattern of LSS extract of polyphenols and carbohydrates revealed by GC-MS analyses RT = retention time Area Pct=peak area and Mwt= molecular weight
No RT Area Pct Compound name Mwt Chemical formula MZ1 81846 29942 Glycerol 3TMS derivative 3086372 C12H32O3Si3 2052 116107 66731 D-Pinitol pentakis(trimethylsilyl) ether 5550881 C22H54O6Si5 733 126062 42518 Palmitic Acid TMS derivative 3286052 C19H40O2Si 3134 134058 303464 11-Octadecenoic acid (Z)- TMS derivative 3546425 C21H42O2Si 3395 134323 18965 alpha-Linolenic acid TMS derivative 3506107 C21H38O2Si 757 134906 26564 Stearic acid TMS derivative 3566584 C21H44O2Si 1178 136123 10245 Sinapinic acid 2TMS derivative 3685722 C17H28O5Si2 3689 142372 33369 11-Eicosenoic acid (Z)- TMS derivative 3826956 C23H46O2Si 36710 147773 29175 Sucrose 8TMS derivative 9197454 C36H86O11Si8 36111 149521 12532 371115-tetramethylhexadecan-13-diol silylated 298547 C20H42O 23312 150368 32061 Sucrose 8TMS derivative 9197454 C36H86O11Si8 36113 151003 14675 Behenic acid TMS derivative 4127647 C25H52O2Si 11714 15614 15318 D-(+)-Turanose octakis(trimethylsilyl) ether 9487867 C37H89NO11Si8 7315 190612 12082 beta-Sitosterol TMS derivative 4868878 C32H58OSi 129
running as compared to those of the normal control with apercentage of change (53 50 33 32 35amp 46 3737 39 and 47 respectively) Also the present resultsrevealed a significant increase (Ple 001) in the MDA levelsin the liver homogenate of CCl4 treated rabbitrsquos 5 and 10weeks after experimental running as compared to those ofthe normal control with a percentage of change (96amp 120)respectively
Daily oral administration of 200 mgkg bw of LSS withinthe rabbitrsquos diets for 5 weeks starting from day one of theexperiment significantly increased (Ple 001) the enzymaticactivities of CAT SOD GR GST and GPx in the liverhomogenate of injured rabbits with liver cirrhosis for 5 weeksas compared to those of the corresponding CCl4 treatedgroupwith a percentage of change (93 93 39 41 42)and all parameters mentioned were significantly increased(Ple 001) with 70 58 33 25 37 respectively inrabbits administrated 400 mgkg bw of LSS and injuredwith liver cirrhosis for 5 weeks as compared to those ofthe corresponding CCl4 treated group Similarly Daily oraladministration of 200 mgkg bw of LSS within the rabbitrsquosdiets for 10 weeks starting from day one of the experimentsignificantly increased (Ple 001) the enzymatic activities ofCAT SOD GR GST and GPx in the liver homogenateof injured rabbits with liver cirrhosis with a percentageof change (70 58 52 58 74) and all parametersmentioned were reduced significantly (Ple 001) to 73 4747 46 and 65 respectively in rabbits administered 400mgkg bw of LSS and injured with liver cirrhosis for 10 weeksas compared to those of the corresponding CCl4 treatedgroup Also the results revealed that daily oral administrationof 200 and 400 mgkg bw of LSS within the rabbitsrsquo dietsfor 5 and 10 weeks starting from day one of the experimentsignificantly decreased (Ple 001) the MDA levels in the liverhomogenate with a percentage of change (47amp 44 and60amp 56 for 5 and 10 weeks respectively) compared tothose of the corresponding CCl4 treated group (Table 4 ampFigure 4)
34 DNA Results of Untreated LSS Administered CCl4-Treated and LSS Protected Rabbits DNAwas extracted fromliver tissues of all the studied rabbits the banding mannerwas observable in Figure 5 Treatment of rabbits with CCl4increased significantly DNA damage of the hepatic tissuesmore than those of the untreated rabbits An entirely dif-ferent banding manner was detected in CCl4 treated rabbitswhich was absent from the liver tissues of the untreatedrabbits DNA extracted from 200 and 400 mgkg bw ofLSS protected rabbitsrsquo livers administered with CCl4 after5 and 10 weeks of the experimental running showed asignificant repair in hepatic DNA Rabbits treated with only200 and 400 mgkg bw of LSS after 5 and 10 weeks of theexperimental running did not display any kind of DNAfragmentation
35 Histopathological Observations The micrograph shownin Figure 6(a) illustrates the normal aspect of the hepatic liverfor the control group (Subgroup A1) fed with normal dietwithout exposing to CCl4 hepatic intoxication Examinationof liver tissue of the control group in which rabbits receivednormal diet and 200 mgkg bw of LSS in their diet on dailybasis till the end of the experiment without exposing to CCl4hepatic intoxication (Subgroup A2) revealed normal hepaticarchitecture (Figure 6(b)) Examination of the control groupreceived normal diet and 400mgkg bw of LSS in their diet ondaily basis till the end of the experiment without exposing toCCl4 hepatic intoxication (Subgroup A3) revealed only mildinflammation except one case which showed the adverseeffects of the drug showing evident chronic venous conges-tion moderate inflammation and bridging fibrosis (Figures6(c) 6(d) and 6(e))
According to liver tissue in rabbits exposed to CCL4intoxication (Group-B1 control pollutant group) the fol-lowing can be seen hepatocyte feathery degeneration andchronic venous congestion steatosis parenchymal and portalinflammation and fibrosis (Figures 7(a) 7(b) and 7(c)) and(Figures 7(d) 7(e) and 7(f)) respectively
6 Evidence-Based Complementary and Alternative Medicine
Table3Liverp
rofileo
funtreatedLSS
administeredC
Cl4tre
atedand
LSSprotectedrabb
itsEffectofLS
Son
serum
markere
nzym
esALTA
STG
GTALPTBILITP
ALB
TGand
CHOL
inrabb
itstre
ated
with
CCL4indu
cedhepatotoxicityas
indicatedin
thefi
gureA
lldatawe
reexpressedas
meanplusmnSE
MV
aluesw
eres
tatistic
allyteste
dusingtheS
tudent
t-testandsig
nificant
differencesatplt005
andplt001
asindicatedby
(lowast)amp
(lowastlowast)com
paredto
norm
alcontrolby
()amp
()
comparedto
CCL4aft
er5w
eeksand
by(+)amp
(++)
comparedto
CCL4aft
er10
weeks
respectiv
ely
Group
ALT
(UL)
AST
(UL)
GGT(U
L)
ALP
(UL)
TBILI
(mgdl)
TP(gdl)
ALB
(gdl)
CHOL(m
gdl)
TG(m
gdl)
Con
trol
3801plusmn
468
2725plusmn
192
702plusmn12
42202plusmn16
2028plusmn003
650plusmn031
355plusmn017
1632plusmn506
4203plusmn469
LS(200
mgkg
bw)
3148plusmn678
2941plusmn
331
848plusmn12
05702plusmn229lowastlowast
043plusmn003lowast
640plusmn025
324plusmn013
3217plusmn287lowast
5274plusmn279lowast
LS(400
mgkg
bw)
1644plusmn14
6lowastlowast
2076plusmn343lowast
629plusmn229
5308plusmn16
1lowastlowast
046plusmn003lowastlowast
586plusmn047
344plusmn010
3161plusmn
305lowast
2610plusmn304lowast
CCl4(5
wk)
5676plusmn546lowastlowast
6230plusmn391lowastlowast
2872plusmn276lowastlowast
11483plusmn273lowastlowast
067plusmn004lowastlowast
414plusmn014lowastlowast
248plusmn025lowastlowast
8921plusmn
762lowastlowast
14774plusmn74
4lowastof
Change
49128
310
421
139
-36
-30
72250
CCl4(10wk)
6700plusmn476lowastlowast
7069plusmn645lowastlowast
3978plusmn226lowastlowast
12638plusmn371lowastlowast
060plusmn004lowastlowast
445plusmn01lowastlowast
261plusmn022lowastlowast
9996plusmn616lowastlowast
16379plusmn89lowastlowast
of
Change
76159
468
474
114-3
1-2
6512
29
CCl4+LS
(200
mgkg
bw)5
wk
3062plusmn219
2490plusmn302
1962plusmn19
5
5960plusmn237
033plusmn003
649plusmn020
339plusmn012
5135plusmn547
8978plusmn611
of
Change
-46
-60
-31
-48
-50
5737
-42
-39
CCl4+LS
(400
mgkg
bw)5
wk
2423plusmn
291++
2027plusmn309++
1464plusmn17
3++
6146plusmn416++
044plusmn003++
576plusmn062++
326plusmn020++
6306plusmn524++
10539plusmn562++
of
Change
-57
-67
-49
-46
-34
3931
-29
-28
CCl4+LS
(200
mgkg
bw)10wk
3804plusmn371
2390plusmn264
1581plusmn
142
5259plusmn257
038plusmn004
636plusmn038
338plusmn009
5236plusmn447
9870plusmn344
of
Change
-43
-66
-60
-58
-36
4329
-47
-39
CCl4+LS
(400
mgkg
bw)10wk
4018plusmn292++
2516plusmn508++
2292plusmn254++
7954plusmn484++
048plusmn003
++635plusmn038++
334plusmn014++
6557plusmn79
8++
10513plusmn666
++of
Change
-40
-64
-42
-37
-20
4227
-34
-36
Evidence-Based Complementary and Alternative Medicine 7
01020304050607080
ALT
Lev
e (U
L)
ALTlowastlowast
lowastlowast
lowastlowast
++++
0102030405060708090
AST
Lev
e (U
L)
AST
lowastlowast
lowast
lowastlowast
++ ++
05
1015202530354045
GG
T Le
ve (U
L)
GGTlowastlowast
lowastlowast
++
++
020406080
100120140
ALP
Lev
el (U
L)
ALPlowastlowast
lowastlowast
lowastlowastlowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
(a) Effect of LSS aqueous extract on serum marker enzymes ALT AST GGTand ALP in rabbits treated with CCL4 induced hepatotoxicity as indicated inthe figure All data were expressed as mean plusmn SEM Values were statisticallytested using the Student t-test and significant differences at p lt 005 and p lt 001indicated by (lowast) amp (lowastlowast) compared to normal control by () amp () comparedto CCL4 after 5 weeks and by (+) amp (++) compared to CCL4 after 10 weeksrespectively
012345678
TP L
evel
(gL
)
TP
lowastlowastlowastlowast
++ ++
00102030405060708
TBI
LI L
evel
(gL
) TBILI
lowastlowastlowast
lowastlowastlowastlowast
++++
005
115
225
335
4
ALB
Lev
el (g
L)
ALB
lowastlowastlowastlowast
++++
020406080
100120140160180200
TG L
evel
(gL
)
TG
lowast
lowastlowast
lowastlowast
lowastlowast
++++
0
20
40
60
80
100
120
CHO
L Le
vel (
gL)
CHOLlowastlowast
lowastlowast
++++
lowastlowast
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
(b) Effect of with LSS aqueous extract on biochemical parameters TBILI TPALB TG and CHOL in rabbits treated with CCL4 induced hepatotoxicity asindicated in the figure All data were expressed as mean plusmn SEM Values werestatistically tested using the Student t-test and significant differences at p lt 005and p lt 001 indicated by (lowast)amp (lowastlowast) compared to normal control by () amp ()compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4 after 10weeks respectively
Figure 3
8 Evidence-Based Complementary and Alternative Medicine
Table4En
zymaticactiv
itiesofantio
xidant
enzymesandMDAofun
treatedLSS
administeredC
Cl4tre
atedand
LSSprotectedrabb
itsA
lldatawe
reexpressedas
meanplusmnSE
MV
aluesw
ere
statistic
allyteste
dusingtheS
tudent
t-testandsig
nificantd
ifferencesa
tplt005
andplt001
asindicatedby
(lowast)amp
(lowastlowast)com
paredto
norm
alcontrolby
()amp
()
comparedto
CCL4aft
er5
weeksandby
(+)amp
(++)
comparedto
CCL4aft
er10
weeksrespectiv
ely
Group
CAT
(nggtissue)
SOD
(nggtissue)
GR
(nggtissue)
GST
(nggtissue)
GPx
(nggtissue)
MDA(nmolm
l)
Con
trol
3155plusmn
199
2820plusmn10
92029plusmn099
1537plusmn12
12197plusmn17
0647plusmn057
LS(200
mgkg
bw)
3127plusmn18
62631plusmn15
51908plusmn077
1542plusmn067
2129plusmn12
8635plusmn045
LS(400
mgkg
bw)
2843plusmn204
2676plusmn18
81972plusmn12
81555plusmn14
32031plusmn074
635plusmn060
CCl4(5
wk)
1475plusmn258lowastlowast
1406plusmn13
9lowastlowast
1358plusmn061lowastlowast
1041plusmn
108lowastlowast
1409plusmn099lowastlowast
1273plusmn10
6lowastlowast
of
Change
-53
-50
-33
-32
-35
96CCl4(10wk)
1682plusmn15
7lowastlowast
1767plusmn202lowastlowast
1277plusmn13
6lowastlowast
940plusmn073lowastlowast
1152plusmn18
2lowastlowast
1427plusmn10
7lowastlowast
of
Change
-46
-37
-37
-39
-47
120
CCl4+LS
(200
mgkg
bw)5
wk
2846plusmn201
2720plusmn094
1892plusmn093
1472plusmn118
2009plusmn112
667plusmn046
of
Change
9393
3941
42-4
7CCl4+LS
(400
mgkg
bw)5
wk
2519plusmn17
7++
2231plusmn14
0++
1813plusmn094++
1304plusmn13
1++
1941plusmn
192+
+713plusmn072++
of
Change
7058
3325
37-4
4CCl4+LS
(200
mgkg
bw)10wk
2872plusmn201
2796plusmn222
1949plusmn096
1492plusmn15
8
2006plusmn17
9
569plusmn046
of
Change
7058
5258
74-6
0CCl4+LS
(400
mgkg
bw)10wk
2919plusmn17
0++
2610plusmn14
3++
1887plusmn17
3++
1378plusmn086++
1906plusmn14
1++
619plusmn052++
of
Change
7347
4746
65-5
6
Evidence-Based Complementary and Alternative Medicine 9
05
10152025303540
CAT
Leve
(ng
g tis
sue)
CAT
lowastlowastlowastlowast
++++
0
5
10
15
20
25
30
35
SOD
Lev
e (ng
lg ti
ssue
)
SOD
lowastlowast
lowastlowast
++++
0
5
10
15
20
25
GR
Leve
(ng
g tis
sue)
GR
lowastlowastlowastlowast
++++
02468
1012141618
GST
Lev
e (ng
g ti
ssue
)
GST
lowastlowastlowastlowast
++++
0
5
10
15
20
25
GPx
Lev
e (ng
g ti
ssue
)
GPx
lowastlowastlowastlowast
++++
02468
1012141618
MD
A L
eve (
nmol
g ti
ssue
)
MDA
lowastlowast
lowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
Figure 4 Effects of LSS on oxidative stress markers in rabbits treated with CCL4 induced hepatotoxicity as indicated in the figure All datawere expressed as mean plusmn SEM Values were statistically tested using the Student t-test and significant differences at p lt 005 and p lt 001 asindicated by (lowast) amp (lowastlowast) compared to normal control by () amp () compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4after 10 weeks respectively
Examination of liver tissue exposed to CCL4 as well as200 mgkg bw of LSS in their diet on daily basis (GroupB2) reveals that the group which is sacrificed 5 weeksafter experimental running shows evident chronic venouscongestion and feathery degeneration in two cases only mildfeathery degeneration in others no steatosis or isolated vesic-ular steatosis mild parenchymal and portal inflammation
(Figures 8(a) 8(b) and 8(c)) Examination of the groupsacrificed 10 weeks after experimental running shows theimprovement of the histoarchitectural changes of the livertowards the normal aspect showing regenerating hepato-cytes with no steatosis discrete chronic venous congestionand discrete inflammatory infiltrate (Figures 8(d) 8(e) and8(f))
10 Evidence-Based Complementary and Alternative Medicine
M 1 2 3 4 5 6 7 8 9bp
10000
3000
2000
1500
1000
750
500
300
Figure 5 Agarose gel showingDNAdamage byCCl4 and protectiveeffects of various fractions of LSS Lanes from left (M) molecularweight marker (1) negative control (2) LSS (200 mgkg bw) (3) LSS(400mgkg bw) (4) CCl4 for 5weeks (5) CCl4 for 10weeks (6)amp (7)CCl4 + LSS (200 mgkg bw) (8) amp (9) CCl4 + LSS (400 mg kg bw)
Examination of liver tissue of rabbits exposed to CCL4as well as 400 mgkg bw of LSS in their diet on daily basis(Group B3) reveals in the group that scarified 5 weeks afterexperimental running improvement of the histoarchitecturalchanges of the liver as there is no chronic venous congestionor only discrete chronic venous congestion and mild featherydegeneration no steatosis or only isolated vesicular steatosisand mild parenchymal and portal inflammation (Figures9(a) 9(b) and 9(c)) But the group that was sacrificed10 weeks after experimental running shows adverse effectsof the drug in the form of steatosis and chronic venouscongestion which is evident in two cases with mild tomoderate inflammation Bridging fibrosis is seen in one case(Figures 9(d) 9(e) 9(f) and 9(g))
4 Discussion
Hepatic disease became a significant public health concernowing to growing rate of obesity in all age populations [26]The conservative treatments of liver problems such as acuteand chronic liver hepatitis liver cirrhosis and fatty liverare often insufficient due to hazardous effects initiated byhepatotoxic drugs of chemical origin [27] Medicinal plantsowing to their abundant hepatoprotective natural ingredi-ents have been comprehensively investigated to treat liverdisorders In the current study hepatoprotective efficacy ofLSS was investigated via hepatotoxic classical model inducedby carbon tetrachloride
In organ and tissue systems CCl4 is stimulated bycytochrome P-450 dependent mixed oxidase in the hepaticendoplasmic reticulum to procedure free radical trichloro-methyl (CCl3 and Cl3COO) This causes changes in themembrane of the endoplasmic reticulum and other cel-lular membranes resulting in an increase in calcium ionpermeability through the plasma membrane causing severeinstabilities of calcium homeostasis leading to necrotic celldeath [28 29] CCL4 affects many organs causing degenera-tion of liver fatty layer and centrilobular necrosis [29] Thisphenomenon indicates that CCl4 is a powerful hepatotoxic
agent and may be used for the characterization of the hepaticdisease treatment medicines Elevation of liver enzymaticmarkers (ALT AST ALP and bilirubin) and increased lipidperoxidation are further evidences of hepatotoxicity ALTand AST that exist in the hepatocytes can certainly leak intothe peripheral blood as soon as the hepatocytes are injured[29] Lepidium sativum seed analysis by GCMS exhibitedthe presence of high concentrations of mainly 120572-linolenicacid (ALA) which is nminus3 fatty acid an omega-3 fatty acidfound in seeds exerting its bioactivity in ameliorating thehepatic intoxication and oxidative stress including liver injuryinduced by alcohol [30] liver steatosis [31 32] nonalcoholichepatic disease [33] and parenteral nutrition-associated liverdisease [34] Pari and Jalaludeen stated that orientationof the active groups in sinapic acid increases antioxidantefficiency as metal chelators and promotes its protectiveefficacy against liver oxidative stress in arsenic intoxicatedrats [35]The current study showed that sinapic acid as ametalchelator may exert its biological activity in improving liverfunction
Lepidium sativum seed extracts have proved hepatopro-tective effects against CCl4 induced liver damage A researchstudy on male Albino Wistar rats demonstrated significantreduction in hepatotoxicity induced by CCl4 administeredwith 200-400 mgkg of Lepidium sativum seeds extractincluded within the diet Serum level of AST ALT levels andbilirubin levels were significantly elevated in CCl4 treated ratscompared to those of normal control However a significantreduction in the levels of these parameters in Lepidiumsativum seed extracts protected rats was observed The hep-atoprotective effect could be due to the presence of alkaloidcoumarin flavonoids tannin and triterpenes which enhanceantioxidant activity and reduce free radical production fromCCl4 which is the basic triggering factor for hepatotoxicity[36 37]
In the present study we evaluated the hepatoprotectiveefficacy of L sativum seeds in white male New-Zealand rab-bits The present results revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withL sativum seeds significantly repaired their liver injuriousmarker enzymes as well as bilirubin total protein and albu-min hence approving its hepatoprotective effect The resultsare in agreement with those of Afaf et al [12] where theystated the role of LSS as efficient hepatoprotective agent Alsothe current results are consistent with those of Mohamedand his coworkers [38] who reported that preprotectionwith LSEE (Lepidium sativum ethanolic extract) significantlyprohibited the D-GalNLPS induced rise in hepatic enzymes(AST ALT 120574-GGT ALP total bilirubin LDH and total pro-tein) thus significantly alleviated the reduction of lipid per-oxidation and repaired the antioxidant enzymatic status andtotal protein restoring them to normal levels D-GalNLPShas been widely used to examine the mechanisms underlyingacute liver failure [39 40] The hepatoprotective significanceof LSEE could be due to downregulation of cytokines (TNF-120572 IL-6) and stress gene (iNOS and HO-1) mRNA expres-sion and upregulation of IL-10 Lepidium sativum ethanolicextract (LSEE) preprotection also improved the degree ofstructural damage and reduced inflammatory infiltration in
Evidence-Based Complementary and Alternative Medicine 11
(a) (b)
(c) (d)
(e)
Figure 6 Effect of LSS on histopathological changes of liver tissues of normal groups in rabbits (a) Showing normal histological picture ofhepatic lobule that consists of central vein surrounded by normal hepatocytes (HEx100) (b) Showing liver sections of the rabbits treatedwith LSS (200 mgkg bw) which revealed normal histological picture of hepatic lobule that consists of central vein surrounded by normalhepatocytes (HEx100) (c) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealed normal hepatic architecturewith only mild inflammation [arrows] (HEx100) (d) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealedevident chronic venous congestion and moderate inflammation [arrows] (HEx100) (e) Showing liver sections of the rabbits treated with LSS(400 mgkg bw) which revealed portal to portal bridging fibrosis [arrow] (HEx200)
hepatic cells These outcomes established that LSEE alle-viates hepatic impairments and structural injury throughthe decay of oxidative stress inflammation and apopto-sis in the liver [38] Mohamed Sakran and his colleagues[10] suggested that 56-dimethoxy-210158403-methylenedioxy-7-C-120573-D glucopyranosyl isoflavone extracted from Lepidiumsativum seeds could improve the lipid profile and the liverfunctions in serum and reduce the free radicals generationby enhancing the antioxidant defense mechanism Newsuggested isoflavonoid can be used as a probable antioxi-dant against paracetamol hepatotoxicity with its antioxidant
characteristics and could restore the hepatic functionstowards normalization [10]
In the present study we found that improved cholesteroland triglycerides levels in L sativum seeds protected rabbitsas compared to CCl4 treated rabbits also support the hepato-protective effect of LSSThese results can also be correlated tothose of Shukla et al [41] who reported the role of L sativumseeds as hypolipidemic agent Also the results of the presentstudy agreed with those of Abdulrahman et al [20] whoseresults showed that L sativum seeds have hepatoprotectiveactivity that could be due to its antioxidant activity together
12 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
Figure 7 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) In rabbits sacrificed 5 weeks afterexperimental running as shown in (a) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100)(b) steatosis (30) in one case (HEx100) and (c) mild parenchymal inflammation [arrow] (HEx200) In rabbits sacrificed 10 weeks afterexperimental running as shown in (d) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100) (e)steatosis (about 20) and moderate parenchymal and portal inflammation (HEx100) and (f) bridging fibrosis [arrow] (HEx100)
with the presence of anti-inflammatory ingredients in Lsativum seeds extract
The results of the current study revealed that the exposureto CCl4 for 5 and 10 weeks led to a significant decrease inthe enzymatic activities of CAT GPx GR GST and SOD inthe liver homogenate of CCl4 treated rabbits 5 and 10 weeksafter experimental running compared to those of the normalcontrolThe concurrent protection of rabbits exposed toCCl4for 5 and 10weeks with 200amp 400mg kg bw LSS significantlyrepaired their antioxidant status and reduced significantly theMDA levels hence confirming its hepatoprotective efficacy
Although the precise molecular mechanism of action ofLSS is not completely understood some researchers [42]have confirmed the role of LSS in inhibiting oxidative stress
associated liver damage The antioxidant activity of LSScould be due to the presence of phenolic compound andflavonoids in these seeds as have been evidenced through thepreliminary screening study on LSS extract using HPLCOurresults match with those of Lee et al [42] who reported thatphenolic compounds play an essential role against oxidativestress related liver damage
A unique expectation of the current study is to confirmthat CCl4 induces liver DNA injury that was assessed usinggel electrophoretic method The results of the current studyshowed that CCL4 induces DNA injury as presented bya significant progression in liver DNA disintegration ofCCl4 treated rabbits above untreated control in the gelelectrophoresis
Evidence-Based Complementary and Alternative Medicine 13
(a) (b)
(c) (d)
(e) (f)
Figure 8 Effect of LSLS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (200 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) evident chronic venous congestion and feathery degeneration [arrow](HEx100) (b) mild feathery degeneration and isolated vesicular steatosis [arrow] (HEx200) and (c) moderate parenchymal and portalinflammation (HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) regenerating hepatocytes with isolatedvesicular steatosis (HEx100) (e) discrete chronic venous congestion and no steatosis (HEx400) and (f) discrete inflammatory infiltrate(HEx100)
Lipid peroxidation of the nuclear membrane may bedirectly accompanied with nuclear DNA damage [43]Nuclear DNA is concomitant with particular regions of thenuclear membrane [44]The close proximity of nuclear DNAto the nuclear membrane can ease the interaction of genomicDNA with the peroxyl radicals and further reactive inter-mediates formed during membrane lipid peroxidation [43]In the presence of transition metals the hydroxyl radicalsgenerated by CCL4 autoxidation can trigger peroxidationof the nuclear membrane lipids by metal catalyzed Haber-Weiss reaction the lipid peroxides levels and intermediate
free radicals could then be augmented by a chain reactionSubsequently the reactive species oxidize nuclearDNAwhenthe lipid peroxidation reaction occurs in the nuclear mem-brane since the nuclear membrane controls the transportof messenger RNA into the cytoplasm and supports thenuclear division process nuclear membrane peroxidationof lipids and proteins Moreover oxidative nuclear DNAdamage could disrupt these serious cellular functions [43]
DNA findings showed that DNA disintegration is adirect result after CCl4 treatment in rabbitsrsquo liver tissueswhich was reduced by participation of antioxidants through
14 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
(g)
Figure 9 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (400 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) discrete chronic venous congestion (HEx200) (b) centrilobular fibrosis[arrows] regenerating hepatocytes and isolated vesicular steatosis (HEx100) and (c) mild parenchymal and portal inflammation [arrow](HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) mild chronic venous congestion and isolated vesicularsteatosis (HEx100) (e) evident chronic venous congestion [arrows] (HEx100) (f) steatosis (40) with moderate inflammation (HEx100) and(g) portal to portal bridging fibrosis in one case [arrow] (HEx100)
Evidence-Based Complementary and Alternative Medicine 15
administration of 200 mg amp 400 mgkg bw L sativum seedsNearly complete repair of DNA damage was significant aftertreating rabbits with 200 mg amp 400 mgkg bw L sativumseeds The protective effect of L sativum seeds on hepaticDNA injury may be due to the antioxidant activity ofL sativum seeds via direct scavenging of free radicals orinterfering with free radicals generation
Concerning histopathological evaluation in our studyrabbitsrsquo livers exposed to CCL4 show portal and parenchymalinflammatory infiltrate feathery and ballooning degenera-tion fatty change and fibrosis Our data are consistent withRajeswary et al [45] who reported dilatation of sinusoidsfatty change disarrangement of normal hepatocytes and cen-trilobular necrosis Also A S Bernacchi et al [46] reportedthat CCL4 leads to liver necrosis and fat accumulation in therabbit
The present study revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withLSS improved their liver histopathological architecture inthe form of less inflammation regenerating hepatocytes nosteatosis or isolated vesicular steatosis no chronic venouscongestion or just discrete congestion and no fibrosis henceapproving its hepatoprotective effectThe results are in agree-ment with Afaf et al [12] and Abdulrahman et al [20] whoobserved the improvement of the histological structure inextract treated group as the histopathologic findings showedless inflammation well restored hepatocytes and less area ofnecrosis as compared to severe necrosis and inflammationin CCl4 treated group of animals which also supports thehepatoprotective effects of LLS Their findings suggest thepossibility of the presence of anti-inflammatory compoundsin LSS extract which could also be a contributing factortowards the hepatoprotective action of LSS
Althnaian [47] also reported that examination of hep-atocytes showed mild to moderate degree of recovery LSSattenuated the hepatic fat accumulation decreased vacuolardegeneration and improved the architecture of hepatocytes
In our study the reducing effect of LSS on fatty changecaused by CCL4 intoxication is indicating the protective andcurative role against fatty liver Our results are supported byShukla et al [41] who reported that LSS significantly reducedsteatosis Their histopathological observations confirmed thecurative efficacy of L sativum liver damage Microvesicularfatty changes hepatocyte ballooning and steatosis weremarkedly corrected by LSS Also Wadhwa et al [36] foundthat the severe fatty changes in the livers of rats caused byCCl4 were decreased in the treated groups
In the present study chronic venous congestion featherydegeneration and fatty changes show mild improvement inrabbits receiving 200mgkg bw of LSS for 5 weeks and showmoderate improvement in rabbits that received 200mgkg bwof LSS for 10 weeks and rabbits that received 400mgkg bwof LSS for 5 weeks Our data are consistent with Afaf et al[12] who observed that severe centrilobular hepatocellularvacuolation hemorrhages and congestion of the centralveins noticed in CCL4 group showed mild improvement ingroups that received 200 mgkg bw of LSS and moderateimprovement in groups that received 400 mgkg bw ofLSS
On the other hand rabbits that received 400mgkg bw ofLSS for 10 weeks showed evidence of liver toxicity in the formof venous congestion hepatocyte feathery degeneration fattychange and fibrosis These results are compatible with Bafeeland Ali [48] who reported that LSS had minimal effect onliver parenchyma if given in low doses So LSS could be usedas food additive or supplement Mineral content of calciumand phosphate and the presence of essential fatty acids inconsiderable level make LSS of beneficial nutritional valuefor many body organs including the liver However LSS inhigh dose showed some toxic effect on renal parenchyma rep-resented by focal necrosis hepatocyte apoptosis and portalinflammatory cell infiltrate Burow et al [49] reported thatthese hazardous effects of LSS may be due to isothiocyanatesreported to be one of LSS constituents
5 Conclusions and Recommendations
The present study showed that LSS is a key factor ininhibition of the hazardous effects of CCl4 LSS treatmentcould significantly alleviate to a greater extent the degree oflipid peroxidation and restore the antioxidative enzymes andshift liver function to normal levels In addition to antioxi-dants activity LSS attenuated the hepatic fat accumulationdecreased vacuolar degeneration ameliorated the degree ofstructural damage in the hepatic cells and improved thearchitecture of hepatocytes The current findings demon-strated that LSS have marked hepatoprotective activity Infuture work active ingredients of LSS extract should bestudied in various animal models of severe toxicity inducedby CCL4 and other hepatotoxic agents The active ingredientsof this plant are likely to be applied as one of the future drugsagainst acute liver toxicity
Data Availability
Data used to support the findings of this study are availablefrom the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This project was funded by the Deanship of ScientificResearch (DSR) King Abdulaziz University Jeddah undergrant no S-17-314-37 The authors therefore acknowledgewith thanks DSR technical and financial support
References
[1] M K Manibusan M Odin and D A Eastmond ldquoPostu-lated carbon tetrachloride mode of action a reviewrdquo Journalof Environmental Science and Health Part C EnvironmentalCarcinogenesis and Ecotoxicology Reviews vol 25 no 3 pp 185ndash209 2007
[2] N Brautbar and J Williams II ldquoIndustrial solvents andliver toxicity risk assessment risk factors and mechanismsrdquo
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
6 Evidence-Based Complementary and Alternative Medicine
Table3Liverp
rofileo
funtreatedLSS
administeredC
Cl4tre
atedand
LSSprotectedrabb
itsEffectofLS
Son
serum
markere
nzym
esALTA
STG
GTALPTBILITP
ALB
TGand
CHOL
inrabb
itstre
ated
with
CCL4indu
cedhepatotoxicityas
indicatedin
thefi
gureA
lldatawe
reexpressedas
meanplusmnSE
MV
aluesw
eres
tatistic
allyteste
dusingtheS
tudent
t-testandsig
nificant
differencesatplt005
andplt001
asindicatedby
(lowast)amp
(lowastlowast)com
paredto
norm
alcontrolby
()amp
()
comparedto
CCL4aft
er5w
eeksand
by(+)amp
(++)
comparedto
CCL4aft
er10
weeks
respectiv
ely
Group
ALT
(UL)
AST
(UL)
GGT(U
L)
ALP
(UL)
TBILI
(mgdl)
TP(gdl)
ALB
(gdl)
CHOL(m
gdl)
TG(m
gdl)
Con
trol
3801plusmn
468
2725plusmn
192
702plusmn12
42202plusmn16
2028plusmn003
650plusmn031
355plusmn017
1632plusmn506
4203plusmn469
LS(200
mgkg
bw)
3148plusmn678
2941plusmn
331
848plusmn12
05702plusmn229lowastlowast
043plusmn003lowast
640plusmn025
324plusmn013
3217plusmn287lowast
5274plusmn279lowast
LS(400
mgkg
bw)
1644plusmn14
6lowastlowast
2076plusmn343lowast
629plusmn229
5308plusmn16
1lowastlowast
046plusmn003lowastlowast
586plusmn047
344plusmn010
3161plusmn
305lowast
2610plusmn304lowast
CCl4(5
wk)
5676plusmn546lowastlowast
6230plusmn391lowastlowast
2872plusmn276lowastlowast
11483plusmn273lowastlowast
067plusmn004lowastlowast
414plusmn014lowastlowast
248plusmn025lowastlowast
8921plusmn
762lowastlowast
14774plusmn74
4lowastof
Change
49128
310
421
139
-36
-30
72250
CCl4(10wk)
6700plusmn476lowastlowast
7069plusmn645lowastlowast
3978plusmn226lowastlowast
12638plusmn371lowastlowast
060plusmn004lowastlowast
445plusmn01lowastlowast
261plusmn022lowastlowast
9996plusmn616lowastlowast
16379plusmn89lowastlowast
of
Change
76159
468
474
114-3
1-2
6512
29
CCl4+LS
(200
mgkg
bw)5
wk
3062plusmn219
2490plusmn302
1962plusmn19
5
5960plusmn237
033plusmn003
649plusmn020
339plusmn012
5135plusmn547
8978plusmn611
of
Change
-46
-60
-31
-48
-50
5737
-42
-39
CCl4+LS
(400
mgkg
bw)5
wk
2423plusmn
291++
2027plusmn309++
1464plusmn17
3++
6146plusmn416++
044plusmn003++
576plusmn062++
326plusmn020++
6306plusmn524++
10539plusmn562++
of
Change
-57
-67
-49
-46
-34
3931
-29
-28
CCl4+LS
(200
mgkg
bw)10wk
3804plusmn371
2390plusmn264
1581plusmn
142
5259plusmn257
038plusmn004
636plusmn038
338plusmn009
5236plusmn447
9870plusmn344
of
Change
-43
-66
-60
-58
-36
4329
-47
-39
CCl4+LS
(400
mgkg
bw)10wk
4018plusmn292++
2516plusmn508++
2292plusmn254++
7954plusmn484++
048plusmn003
++635plusmn038++
334plusmn014++
6557plusmn79
8++
10513plusmn666
++of
Change
-40
-64
-42
-37
-20
4227
-34
-36
Evidence-Based Complementary and Alternative Medicine 7
01020304050607080
ALT
Lev
e (U
L)
ALTlowastlowast
lowastlowast
lowastlowast
++++
0102030405060708090
AST
Lev
e (U
L)
AST
lowastlowast
lowast
lowastlowast
++ ++
05
1015202530354045
GG
T Le
ve (U
L)
GGTlowastlowast
lowastlowast
++
++
020406080
100120140
ALP
Lev
el (U
L)
ALPlowastlowast
lowastlowast
lowastlowastlowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
(a) Effect of LSS aqueous extract on serum marker enzymes ALT AST GGTand ALP in rabbits treated with CCL4 induced hepatotoxicity as indicated inthe figure All data were expressed as mean plusmn SEM Values were statisticallytested using the Student t-test and significant differences at p lt 005 and p lt 001indicated by (lowast) amp (lowastlowast) compared to normal control by () amp () comparedto CCL4 after 5 weeks and by (+) amp (++) compared to CCL4 after 10 weeksrespectively
012345678
TP L
evel
(gL
)
TP
lowastlowastlowastlowast
++ ++
00102030405060708
TBI
LI L
evel
(gL
) TBILI
lowastlowastlowast
lowastlowastlowastlowast
++++
005
115
225
335
4
ALB
Lev
el (g
L)
ALB
lowastlowastlowastlowast
++++
020406080
100120140160180200
TG L
evel
(gL
)
TG
lowast
lowastlowast
lowastlowast
lowastlowast
++++
0
20
40
60
80
100
120
CHO
L Le
vel (
gL)
CHOLlowastlowast
lowastlowast
++++
lowastlowast
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
(b) Effect of with LSS aqueous extract on biochemical parameters TBILI TPALB TG and CHOL in rabbits treated with CCL4 induced hepatotoxicity asindicated in the figure All data were expressed as mean plusmn SEM Values werestatistically tested using the Student t-test and significant differences at p lt 005and p lt 001 indicated by (lowast)amp (lowastlowast) compared to normal control by () amp ()compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4 after 10weeks respectively
Figure 3
8 Evidence-Based Complementary and Alternative Medicine
Table4En
zymaticactiv
itiesofantio
xidant
enzymesandMDAofun
treatedLSS
administeredC
Cl4tre
atedand
LSSprotectedrabb
itsA
lldatawe
reexpressedas
meanplusmnSE
MV
aluesw
ere
statistic
allyteste
dusingtheS
tudent
t-testandsig
nificantd
ifferencesa
tplt005
andplt001
asindicatedby
(lowast)amp
(lowastlowast)com
paredto
norm
alcontrolby
()amp
()
comparedto
CCL4aft
er5
weeksandby
(+)amp
(++)
comparedto
CCL4aft
er10
weeksrespectiv
ely
Group
CAT
(nggtissue)
SOD
(nggtissue)
GR
(nggtissue)
GST
(nggtissue)
GPx
(nggtissue)
MDA(nmolm
l)
Con
trol
3155plusmn
199
2820plusmn10
92029plusmn099
1537plusmn12
12197plusmn17
0647plusmn057
LS(200
mgkg
bw)
3127plusmn18
62631plusmn15
51908plusmn077
1542plusmn067
2129plusmn12
8635plusmn045
LS(400
mgkg
bw)
2843plusmn204
2676plusmn18
81972plusmn12
81555plusmn14
32031plusmn074
635plusmn060
CCl4(5
wk)
1475plusmn258lowastlowast
1406plusmn13
9lowastlowast
1358plusmn061lowastlowast
1041plusmn
108lowastlowast
1409plusmn099lowastlowast
1273plusmn10
6lowastlowast
of
Change
-53
-50
-33
-32
-35
96CCl4(10wk)
1682plusmn15
7lowastlowast
1767plusmn202lowastlowast
1277plusmn13
6lowastlowast
940plusmn073lowastlowast
1152plusmn18
2lowastlowast
1427plusmn10
7lowastlowast
of
Change
-46
-37
-37
-39
-47
120
CCl4+LS
(200
mgkg
bw)5
wk
2846plusmn201
2720plusmn094
1892plusmn093
1472plusmn118
2009plusmn112
667plusmn046
of
Change
9393
3941
42-4
7CCl4+LS
(400
mgkg
bw)5
wk
2519plusmn17
7++
2231plusmn14
0++
1813plusmn094++
1304plusmn13
1++
1941plusmn
192+
+713plusmn072++
of
Change
7058
3325
37-4
4CCl4+LS
(200
mgkg
bw)10wk
2872plusmn201
2796plusmn222
1949plusmn096
1492plusmn15
8
2006plusmn17
9
569plusmn046
of
Change
7058
5258
74-6
0CCl4+LS
(400
mgkg
bw)10wk
2919plusmn17
0++
2610plusmn14
3++
1887plusmn17
3++
1378plusmn086++
1906plusmn14
1++
619plusmn052++
of
Change
7347
4746
65-5
6
Evidence-Based Complementary and Alternative Medicine 9
05
10152025303540
CAT
Leve
(ng
g tis
sue)
CAT
lowastlowastlowastlowast
++++
0
5
10
15
20
25
30
35
SOD
Lev
e (ng
lg ti
ssue
)
SOD
lowastlowast
lowastlowast
++++
0
5
10
15
20
25
GR
Leve
(ng
g tis
sue)
GR
lowastlowastlowastlowast
++++
02468
1012141618
GST
Lev
e (ng
g ti
ssue
)
GST
lowastlowastlowastlowast
++++
0
5
10
15
20
25
GPx
Lev
e (ng
g ti
ssue
)
GPx
lowastlowastlowastlowast
++++
02468
1012141618
MD
A L
eve (
nmol
g ti
ssue
)
MDA
lowastlowast
lowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
Figure 4 Effects of LSS on oxidative stress markers in rabbits treated with CCL4 induced hepatotoxicity as indicated in the figure All datawere expressed as mean plusmn SEM Values were statistically tested using the Student t-test and significant differences at p lt 005 and p lt 001 asindicated by (lowast) amp (lowastlowast) compared to normal control by () amp () compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4after 10 weeks respectively
Examination of liver tissue exposed to CCL4 as well as200 mgkg bw of LSS in their diet on daily basis (GroupB2) reveals that the group which is sacrificed 5 weeksafter experimental running shows evident chronic venouscongestion and feathery degeneration in two cases only mildfeathery degeneration in others no steatosis or isolated vesic-ular steatosis mild parenchymal and portal inflammation
(Figures 8(a) 8(b) and 8(c)) Examination of the groupsacrificed 10 weeks after experimental running shows theimprovement of the histoarchitectural changes of the livertowards the normal aspect showing regenerating hepato-cytes with no steatosis discrete chronic venous congestionand discrete inflammatory infiltrate (Figures 8(d) 8(e) and8(f))
10 Evidence-Based Complementary and Alternative Medicine
M 1 2 3 4 5 6 7 8 9bp
10000
3000
2000
1500
1000
750
500
300
Figure 5 Agarose gel showingDNAdamage byCCl4 and protectiveeffects of various fractions of LSS Lanes from left (M) molecularweight marker (1) negative control (2) LSS (200 mgkg bw) (3) LSS(400mgkg bw) (4) CCl4 for 5weeks (5) CCl4 for 10weeks (6)amp (7)CCl4 + LSS (200 mgkg bw) (8) amp (9) CCl4 + LSS (400 mg kg bw)
Examination of liver tissue of rabbits exposed to CCL4as well as 400 mgkg bw of LSS in their diet on daily basis(Group B3) reveals in the group that scarified 5 weeks afterexperimental running improvement of the histoarchitecturalchanges of the liver as there is no chronic venous congestionor only discrete chronic venous congestion and mild featherydegeneration no steatosis or only isolated vesicular steatosisand mild parenchymal and portal inflammation (Figures9(a) 9(b) and 9(c)) But the group that was sacrificed10 weeks after experimental running shows adverse effectsof the drug in the form of steatosis and chronic venouscongestion which is evident in two cases with mild tomoderate inflammation Bridging fibrosis is seen in one case(Figures 9(d) 9(e) 9(f) and 9(g))
4 Discussion
Hepatic disease became a significant public health concernowing to growing rate of obesity in all age populations [26]The conservative treatments of liver problems such as acuteand chronic liver hepatitis liver cirrhosis and fatty liverare often insufficient due to hazardous effects initiated byhepatotoxic drugs of chemical origin [27] Medicinal plantsowing to their abundant hepatoprotective natural ingredi-ents have been comprehensively investigated to treat liverdisorders In the current study hepatoprotective efficacy ofLSS was investigated via hepatotoxic classical model inducedby carbon tetrachloride
In organ and tissue systems CCl4 is stimulated bycytochrome P-450 dependent mixed oxidase in the hepaticendoplasmic reticulum to procedure free radical trichloro-methyl (CCl3 and Cl3COO) This causes changes in themembrane of the endoplasmic reticulum and other cel-lular membranes resulting in an increase in calcium ionpermeability through the plasma membrane causing severeinstabilities of calcium homeostasis leading to necrotic celldeath [28 29] CCL4 affects many organs causing degenera-tion of liver fatty layer and centrilobular necrosis [29] Thisphenomenon indicates that CCl4 is a powerful hepatotoxic
agent and may be used for the characterization of the hepaticdisease treatment medicines Elevation of liver enzymaticmarkers (ALT AST ALP and bilirubin) and increased lipidperoxidation are further evidences of hepatotoxicity ALTand AST that exist in the hepatocytes can certainly leak intothe peripheral blood as soon as the hepatocytes are injured[29] Lepidium sativum seed analysis by GCMS exhibitedthe presence of high concentrations of mainly 120572-linolenicacid (ALA) which is nminus3 fatty acid an omega-3 fatty acidfound in seeds exerting its bioactivity in ameliorating thehepatic intoxication and oxidative stress including liver injuryinduced by alcohol [30] liver steatosis [31 32] nonalcoholichepatic disease [33] and parenteral nutrition-associated liverdisease [34] Pari and Jalaludeen stated that orientationof the active groups in sinapic acid increases antioxidantefficiency as metal chelators and promotes its protectiveefficacy against liver oxidative stress in arsenic intoxicatedrats [35]The current study showed that sinapic acid as ametalchelator may exert its biological activity in improving liverfunction
Lepidium sativum seed extracts have proved hepatopro-tective effects against CCl4 induced liver damage A researchstudy on male Albino Wistar rats demonstrated significantreduction in hepatotoxicity induced by CCl4 administeredwith 200-400 mgkg of Lepidium sativum seeds extractincluded within the diet Serum level of AST ALT levels andbilirubin levels were significantly elevated in CCl4 treated ratscompared to those of normal control However a significantreduction in the levels of these parameters in Lepidiumsativum seed extracts protected rats was observed The hep-atoprotective effect could be due to the presence of alkaloidcoumarin flavonoids tannin and triterpenes which enhanceantioxidant activity and reduce free radical production fromCCl4 which is the basic triggering factor for hepatotoxicity[36 37]
In the present study we evaluated the hepatoprotectiveefficacy of L sativum seeds in white male New-Zealand rab-bits The present results revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withL sativum seeds significantly repaired their liver injuriousmarker enzymes as well as bilirubin total protein and albu-min hence approving its hepatoprotective effect The resultsare in agreement with those of Afaf et al [12] where theystated the role of LSS as efficient hepatoprotective agent Alsothe current results are consistent with those of Mohamedand his coworkers [38] who reported that preprotectionwith LSEE (Lepidium sativum ethanolic extract) significantlyprohibited the D-GalNLPS induced rise in hepatic enzymes(AST ALT 120574-GGT ALP total bilirubin LDH and total pro-tein) thus significantly alleviated the reduction of lipid per-oxidation and repaired the antioxidant enzymatic status andtotal protein restoring them to normal levels D-GalNLPShas been widely used to examine the mechanisms underlyingacute liver failure [39 40] The hepatoprotective significanceof LSEE could be due to downregulation of cytokines (TNF-120572 IL-6) and stress gene (iNOS and HO-1) mRNA expres-sion and upregulation of IL-10 Lepidium sativum ethanolicextract (LSEE) preprotection also improved the degree ofstructural damage and reduced inflammatory infiltration in
Evidence-Based Complementary and Alternative Medicine 11
(a) (b)
(c) (d)
(e)
Figure 6 Effect of LSS on histopathological changes of liver tissues of normal groups in rabbits (a) Showing normal histological picture ofhepatic lobule that consists of central vein surrounded by normal hepatocytes (HEx100) (b) Showing liver sections of the rabbits treatedwith LSS (200 mgkg bw) which revealed normal histological picture of hepatic lobule that consists of central vein surrounded by normalhepatocytes (HEx100) (c) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealed normal hepatic architecturewith only mild inflammation [arrows] (HEx100) (d) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealedevident chronic venous congestion and moderate inflammation [arrows] (HEx100) (e) Showing liver sections of the rabbits treated with LSS(400 mgkg bw) which revealed portal to portal bridging fibrosis [arrow] (HEx200)
hepatic cells These outcomes established that LSEE alle-viates hepatic impairments and structural injury throughthe decay of oxidative stress inflammation and apopto-sis in the liver [38] Mohamed Sakran and his colleagues[10] suggested that 56-dimethoxy-210158403-methylenedioxy-7-C-120573-D glucopyranosyl isoflavone extracted from Lepidiumsativum seeds could improve the lipid profile and the liverfunctions in serum and reduce the free radicals generationby enhancing the antioxidant defense mechanism Newsuggested isoflavonoid can be used as a probable antioxi-dant against paracetamol hepatotoxicity with its antioxidant
characteristics and could restore the hepatic functionstowards normalization [10]
In the present study we found that improved cholesteroland triglycerides levels in L sativum seeds protected rabbitsas compared to CCl4 treated rabbits also support the hepato-protective effect of LSSThese results can also be correlated tothose of Shukla et al [41] who reported the role of L sativumseeds as hypolipidemic agent Also the results of the presentstudy agreed with those of Abdulrahman et al [20] whoseresults showed that L sativum seeds have hepatoprotectiveactivity that could be due to its antioxidant activity together
12 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
Figure 7 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) In rabbits sacrificed 5 weeks afterexperimental running as shown in (a) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100)(b) steatosis (30) in one case (HEx100) and (c) mild parenchymal inflammation [arrow] (HEx200) In rabbits sacrificed 10 weeks afterexperimental running as shown in (d) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100) (e)steatosis (about 20) and moderate parenchymal and portal inflammation (HEx100) and (f) bridging fibrosis [arrow] (HEx100)
with the presence of anti-inflammatory ingredients in Lsativum seeds extract
The results of the current study revealed that the exposureto CCl4 for 5 and 10 weeks led to a significant decrease inthe enzymatic activities of CAT GPx GR GST and SOD inthe liver homogenate of CCl4 treated rabbits 5 and 10 weeksafter experimental running compared to those of the normalcontrolThe concurrent protection of rabbits exposed toCCl4for 5 and 10weeks with 200amp 400mg kg bw LSS significantlyrepaired their antioxidant status and reduced significantly theMDA levels hence confirming its hepatoprotective efficacy
Although the precise molecular mechanism of action ofLSS is not completely understood some researchers [42]have confirmed the role of LSS in inhibiting oxidative stress
associated liver damage The antioxidant activity of LSScould be due to the presence of phenolic compound andflavonoids in these seeds as have been evidenced through thepreliminary screening study on LSS extract using HPLCOurresults match with those of Lee et al [42] who reported thatphenolic compounds play an essential role against oxidativestress related liver damage
A unique expectation of the current study is to confirmthat CCl4 induces liver DNA injury that was assessed usinggel electrophoretic method The results of the current studyshowed that CCL4 induces DNA injury as presented bya significant progression in liver DNA disintegration ofCCl4 treated rabbits above untreated control in the gelelectrophoresis
Evidence-Based Complementary and Alternative Medicine 13
(a) (b)
(c) (d)
(e) (f)
Figure 8 Effect of LSLS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (200 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) evident chronic venous congestion and feathery degeneration [arrow](HEx100) (b) mild feathery degeneration and isolated vesicular steatosis [arrow] (HEx200) and (c) moderate parenchymal and portalinflammation (HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) regenerating hepatocytes with isolatedvesicular steatosis (HEx100) (e) discrete chronic venous congestion and no steatosis (HEx400) and (f) discrete inflammatory infiltrate(HEx100)
Lipid peroxidation of the nuclear membrane may bedirectly accompanied with nuclear DNA damage [43]Nuclear DNA is concomitant with particular regions of thenuclear membrane [44]The close proximity of nuclear DNAto the nuclear membrane can ease the interaction of genomicDNA with the peroxyl radicals and further reactive inter-mediates formed during membrane lipid peroxidation [43]In the presence of transition metals the hydroxyl radicalsgenerated by CCL4 autoxidation can trigger peroxidationof the nuclear membrane lipids by metal catalyzed Haber-Weiss reaction the lipid peroxides levels and intermediate
free radicals could then be augmented by a chain reactionSubsequently the reactive species oxidize nuclearDNAwhenthe lipid peroxidation reaction occurs in the nuclear mem-brane since the nuclear membrane controls the transportof messenger RNA into the cytoplasm and supports thenuclear division process nuclear membrane peroxidationof lipids and proteins Moreover oxidative nuclear DNAdamage could disrupt these serious cellular functions [43]
DNA findings showed that DNA disintegration is adirect result after CCl4 treatment in rabbitsrsquo liver tissueswhich was reduced by participation of antioxidants through
14 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
(g)
Figure 9 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (400 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) discrete chronic venous congestion (HEx200) (b) centrilobular fibrosis[arrows] regenerating hepatocytes and isolated vesicular steatosis (HEx100) and (c) mild parenchymal and portal inflammation [arrow](HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) mild chronic venous congestion and isolated vesicularsteatosis (HEx100) (e) evident chronic venous congestion [arrows] (HEx100) (f) steatosis (40) with moderate inflammation (HEx100) and(g) portal to portal bridging fibrosis in one case [arrow] (HEx100)
Evidence-Based Complementary and Alternative Medicine 15
administration of 200 mg amp 400 mgkg bw L sativum seedsNearly complete repair of DNA damage was significant aftertreating rabbits with 200 mg amp 400 mgkg bw L sativumseeds The protective effect of L sativum seeds on hepaticDNA injury may be due to the antioxidant activity ofL sativum seeds via direct scavenging of free radicals orinterfering with free radicals generation
Concerning histopathological evaluation in our studyrabbitsrsquo livers exposed to CCL4 show portal and parenchymalinflammatory infiltrate feathery and ballooning degenera-tion fatty change and fibrosis Our data are consistent withRajeswary et al [45] who reported dilatation of sinusoidsfatty change disarrangement of normal hepatocytes and cen-trilobular necrosis Also A S Bernacchi et al [46] reportedthat CCL4 leads to liver necrosis and fat accumulation in therabbit
The present study revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withLSS improved their liver histopathological architecture inthe form of less inflammation regenerating hepatocytes nosteatosis or isolated vesicular steatosis no chronic venouscongestion or just discrete congestion and no fibrosis henceapproving its hepatoprotective effectThe results are in agree-ment with Afaf et al [12] and Abdulrahman et al [20] whoobserved the improvement of the histological structure inextract treated group as the histopathologic findings showedless inflammation well restored hepatocytes and less area ofnecrosis as compared to severe necrosis and inflammationin CCl4 treated group of animals which also supports thehepatoprotective effects of LLS Their findings suggest thepossibility of the presence of anti-inflammatory compoundsin LSS extract which could also be a contributing factortowards the hepatoprotective action of LSS
Althnaian [47] also reported that examination of hep-atocytes showed mild to moderate degree of recovery LSSattenuated the hepatic fat accumulation decreased vacuolardegeneration and improved the architecture of hepatocytes
In our study the reducing effect of LSS on fatty changecaused by CCL4 intoxication is indicating the protective andcurative role against fatty liver Our results are supported byShukla et al [41] who reported that LSS significantly reducedsteatosis Their histopathological observations confirmed thecurative efficacy of L sativum liver damage Microvesicularfatty changes hepatocyte ballooning and steatosis weremarkedly corrected by LSS Also Wadhwa et al [36] foundthat the severe fatty changes in the livers of rats caused byCCl4 were decreased in the treated groups
In the present study chronic venous congestion featherydegeneration and fatty changes show mild improvement inrabbits receiving 200mgkg bw of LSS for 5 weeks and showmoderate improvement in rabbits that received 200mgkg bwof LSS for 10 weeks and rabbits that received 400mgkg bwof LSS for 5 weeks Our data are consistent with Afaf et al[12] who observed that severe centrilobular hepatocellularvacuolation hemorrhages and congestion of the centralveins noticed in CCL4 group showed mild improvement ingroups that received 200 mgkg bw of LSS and moderateimprovement in groups that received 400 mgkg bw ofLSS
On the other hand rabbits that received 400mgkg bw ofLSS for 10 weeks showed evidence of liver toxicity in the formof venous congestion hepatocyte feathery degeneration fattychange and fibrosis These results are compatible with Bafeeland Ali [48] who reported that LSS had minimal effect onliver parenchyma if given in low doses So LSS could be usedas food additive or supplement Mineral content of calciumand phosphate and the presence of essential fatty acids inconsiderable level make LSS of beneficial nutritional valuefor many body organs including the liver However LSS inhigh dose showed some toxic effect on renal parenchyma rep-resented by focal necrosis hepatocyte apoptosis and portalinflammatory cell infiltrate Burow et al [49] reported thatthese hazardous effects of LSS may be due to isothiocyanatesreported to be one of LSS constituents
5 Conclusions and Recommendations
The present study showed that LSS is a key factor ininhibition of the hazardous effects of CCl4 LSS treatmentcould significantly alleviate to a greater extent the degree oflipid peroxidation and restore the antioxidative enzymes andshift liver function to normal levels In addition to antioxi-dants activity LSS attenuated the hepatic fat accumulationdecreased vacuolar degeneration ameliorated the degree ofstructural damage in the hepatic cells and improved thearchitecture of hepatocytes The current findings demon-strated that LSS have marked hepatoprotective activity Infuture work active ingredients of LSS extract should bestudied in various animal models of severe toxicity inducedby CCL4 and other hepatotoxic agents The active ingredientsof this plant are likely to be applied as one of the future drugsagainst acute liver toxicity
Data Availability
Data used to support the findings of this study are availablefrom the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This project was funded by the Deanship of ScientificResearch (DSR) King Abdulaziz University Jeddah undergrant no S-17-314-37 The authors therefore acknowledgewith thanks DSR technical and financial support
References
[1] M K Manibusan M Odin and D A Eastmond ldquoPostu-lated carbon tetrachloride mode of action a reviewrdquo Journalof Environmental Science and Health Part C EnvironmentalCarcinogenesis and Ecotoxicology Reviews vol 25 no 3 pp 185ndash209 2007
[2] N Brautbar and J Williams II ldquoIndustrial solvents andliver toxicity risk assessment risk factors and mechanismsrdquo
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 7
01020304050607080
ALT
Lev
e (U
L)
ALTlowastlowast
lowastlowast
lowastlowast
++++
0102030405060708090
AST
Lev
e (U
L)
AST
lowastlowast
lowast
lowastlowast
++ ++
05
1015202530354045
GG
T Le
ve (U
L)
GGTlowastlowast
lowastlowast
++
++
020406080
100120140
ALP
Lev
el (U
L)
ALPlowastlowast
lowastlowast
lowastlowastlowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
(a) Effect of LSS aqueous extract on serum marker enzymes ALT AST GGTand ALP in rabbits treated with CCL4 induced hepatotoxicity as indicated inthe figure All data were expressed as mean plusmn SEM Values were statisticallytested using the Student t-test and significant differences at p lt 005 and p lt 001indicated by (lowast) amp (lowastlowast) compared to normal control by () amp () comparedto CCL4 after 5 weeks and by (+) amp (++) compared to CCL4 after 10 weeksrespectively
012345678
TP L
evel
(gL
)
TP
lowastlowastlowastlowast
++ ++
00102030405060708
TBI
LI L
evel
(gL
) TBILI
lowastlowastlowast
lowastlowastlowastlowast
++++
005
115
225
335
4
ALB
Lev
el (g
L)
ALB
lowastlowastlowastlowast
++++
020406080
100120140160180200
TG L
evel
(gL
)
TG
lowast
lowastlowast
lowastlowast
lowastlowast
++++
0
20
40
60
80
100
120
CHO
L Le
vel (
gL)
CHOLlowastlowast
lowastlowast
++++
lowastlowast
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
(b) Effect of with LSS aqueous extract on biochemical parameters TBILI TPALB TG and CHOL in rabbits treated with CCL4 induced hepatotoxicity asindicated in the figure All data were expressed as mean plusmn SEM Values werestatistically tested using the Student t-test and significant differences at p lt 005and p lt 001 indicated by (lowast)amp (lowastlowast) compared to normal control by () amp ()compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4 after 10weeks respectively
Figure 3
8 Evidence-Based Complementary and Alternative Medicine
Table4En
zymaticactiv
itiesofantio
xidant
enzymesandMDAofun
treatedLSS
administeredC
Cl4tre
atedand
LSSprotectedrabb
itsA
lldatawe
reexpressedas
meanplusmnSE
MV
aluesw
ere
statistic
allyteste
dusingtheS
tudent
t-testandsig
nificantd
ifferencesa
tplt005
andplt001
asindicatedby
(lowast)amp
(lowastlowast)com
paredto
norm
alcontrolby
()amp
()
comparedto
CCL4aft
er5
weeksandby
(+)amp
(++)
comparedto
CCL4aft
er10
weeksrespectiv
ely
Group
CAT
(nggtissue)
SOD
(nggtissue)
GR
(nggtissue)
GST
(nggtissue)
GPx
(nggtissue)
MDA(nmolm
l)
Con
trol
3155plusmn
199
2820plusmn10
92029plusmn099
1537plusmn12
12197plusmn17
0647plusmn057
LS(200
mgkg
bw)
3127plusmn18
62631plusmn15
51908plusmn077
1542plusmn067
2129plusmn12
8635plusmn045
LS(400
mgkg
bw)
2843plusmn204
2676plusmn18
81972plusmn12
81555plusmn14
32031plusmn074
635plusmn060
CCl4(5
wk)
1475plusmn258lowastlowast
1406plusmn13
9lowastlowast
1358plusmn061lowastlowast
1041plusmn
108lowastlowast
1409plusmn099lowastlowast
1273plusmn10
6lowastlowast
of
Change
-53
-50
-33
-32
-35
96CCl4(10wk)
1682plusmn15
7lowastlowast
1767plusmn202lowastlowast
1277plusmn13
6lowastlowast
940plusmn073lowastlowast
1152plusmn18
2lowastlowast
1427plusmn10
7lowastlowast
of
Change
-46
-37
-37
-39
-47
120
CCl4+LS
(200
mgkg
bw)5
wk
2846plusmn201
2720plusmn094
1892plusmn093
1472plusmn118
2009plusmn112
667plusmn046
of
Change
9393
3941
42-4
7CCl4+LS
(400
mgkg
bw)5
wk
2519plusmn17
7++
2231plusmn14
0++
1813plusmn094++
1304plusmn13
1++
1941plusmn
192+
+713plusmn072++
of
Change
7058
3325
37-4
4CCl4+LS
(200
mgkg
bw)10wk
2872plusmn201
2796plusmn222
1949plusmn096
1492plusmn15
8
2006plusmn17
9
569plusmn046
of
Change
7058
5258
74-6
0CCl4+LS
(400
mgkg
bw)10wk
2919plusmn17
0++
2610plusmn14
3++
1887plusmn17
3++
1378plusmn086++
1906plusmn14
1++
619plusmn052++
of
Change
7347
4746
65-5
6
Evidence-Based Complementary and Alternative Medicine 9
05
10152025303540
CAT
Leve
(ng
g tis
sue)
CAT
lowastlowastlowastlowast
++++
0
5
10
15
20
25
30
35
SOD
Lev
e (ng
lg ti
ssue
)
SOD
lowastlowast
lowastlowast
++++
0
5
10
15
20
25
GR
Leve
(ng
g tis
sue)
GR
lowastlowastlowastlowast
++++
02468
1012141618
GST
Lev
e (ng
g ti
ssue
)
GST
lowastlowastlowastlowast
++++
0
5
10
15
20
25
GPx
Lev
e (ng
g ti
ssue
)
GPx
lowastlowastlowastlowast
++++
02468
1012141618
MD
A L
eve (
nmol
g ti
ssue
)
MDA
lowastlowast
lowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
Figure 4 Effects of LSS on oxidative stress markers in rabbits treated with CCL4 induced hepatotoxicity as indicated in the figure All datawere expressed as mean plusmn SEM Values were statistically tested using the Student t-test and significant differences at p lt 005 and p lt 001 asindicated by (lowast) amp (lowastlowast) compared to normal control by () amp () compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4after 10 weeks respectively
Examination of liver tissue exposed to CCL4 as well as200 mgkg bw of LSS in their diet on daily basis (GroupB2) reveals that the group which is sacrificed 5 weeksafter experimental running shows evident chronic venouscongestion and feathery degeneration in two cases only mildfeathery degeneration in others no steatosis or isolated vesic-ular steatosis mild parenchymal and portal inflammation
(Figures 8(a) 8(b) and 8(c)) Examination of the groupsacrificed 10 weeks after experimental running shows theimprovement of the histoarchitectural changes of the livertowards the normal aspect showing regenerating hepato-cytes with no steatosis discrete chronic venous congestionand discrete inflammatory infiltrate (Figures 8(d) 8(e) and8(f))
10 Evidence-Based Complementary and Alternative Medicine
M 1 2 3 4 5 6 7 8 9bp
10000
3000
2000
1500
1000
750
500
300
Figure 5 Agarose gel showingDNAdamage byCCl4 and protectiveeffects of various fractions of LSS Lanes from left (M) molecularweight marker (1) negative control (2) LSS (200 mgkg bw) (3) LSS(400mgkg bw) (4) CCl4 for 5weeks (5) CCl4 for 10weeks (6)amp (7)CCl4 + LSS (200 mgkg bw) (8) amp (9) CCl4 + LSS (400 mg kg bw)
Examination of liver tissue of rabbits exposed to CCL4as well as 400 mgkg bw of LSS in their diet on daily basis(Group B3) reveals in the group that scarified 5 weeks afterexperimental running improvement of the histoarchitecturalchanges of the liver as there is no chronic venous congestionor only discrete chronic venous congestion and mild featherydegeneration no steatosis or only isolated vesicular steatosisand mild parenchymal and portal inflammation (Figures9(a) 9(b) and 9(c)) But the group that was sacrificed10 weeks after experimental running shows adverse effectsof the drug in the form of steatosis and chronic venouscongestion which is evident in two cases with mild tomoderate inflammation Bridging fibrosis is seen in one case(Figures 9(d) 9(e) 9(f) and 9(g))
4 Discussion
Hepatic disease became a significant public health concernowing to growing rate of obesity in all age populations [26]The conservative treatments of liver problems such as acuteand chronic liver hepatitis liver cirrhosis and fatty liverare often insufficient due to hazardous effects initiated byhepatotoxic drugs of chemical origin [27] Medicinal plantsowing to their abundant hepatoprotective natural ingredi-ents have been comprehensively investigated to treat liverdisorders In the current study hepatoprotective efficacy ofLSS was investigated via hepatotoxic classical model inducedby carbon tetrachloride
In organ and tissue systems CCl4 is stimulated bycytochrome P-450 dependent mixed oxidase in the hepaticendoplasmic reticulum to procedure free radical trichloro-methyl (CCl3 and Cl3COO) This causes changes in themembrane of the endoplasmic reticulum and other cel-lular membranes resulting in an increase in calcium ionpermeability through the plasma membrane causing severeinstabilities of calcium homeostasis leading to necrotic celldeath [28 29] CCL4 affects many organs causing degenera-tion of liver fatty layer and centrilobular necrosis [29] Thisphenomenon indicates that CCl4 is a powerful hepatotoxic
agent and may be used for the characterization of the hepaticdisease treatment medicines Elevation of liver enzymaticmarkers (ALT AST ALP and bilirubin) and increased lipidperoxidation are further evidences of hepatotoxicity ALTand AST that exist in the hepatocytes can certainly leak intothe peripheral blood as soon as the hepatocytes are injured[29] Lepidium sativum seed analysis by GCMS exhibitedthe presence of high concentrations of mainly 120572-linolenicacid (ALA) which is nminus3 fatty acid an omega-3 fatty acidfound in seeds exerting its bioactivity in ameliorating thehepatic intoxication and oxidative stress including liver injuryinduced by alcohol [30] liver steatosis [31 32] nonalcoholichepatic disease [33] and parenteral nutrition-associated liverdisease [34] Pari and Jalaludeen stated that orientationof the active groups in sinapic acid increases antioxidantefficiency as metal chelators and promotes its protectiveefficacy against liver oxidative stress in arsenic intoxicatedrats [35]The current study showed that sinapic acid as ametalchelator may exert its biological activity in improving liverfunction
Lepidium sativum seed extracts have proved hepatopro-tective effects against CCl4 induced liver damage A researchstudy on male Albino Wistar rats demonstrated significantreduction in hepatotoxicity induced by CCl4 administeredwith 200-400 mgkg of Lepidium sativum seeds extractincluded within the diet Serum level of AST ALT levels andbilirubin levels were significantly elevated in CCl4 treated ratscompared to those of normal control However a significantreduction in the levels of these parameters in Lepidiumsativum seed extracts protected rats was observed The hep-atoprotective effect could be due to the presence of alkaloidcoumarin flavonoids tannin and triterpenes which enhanceantioxidant activity and reduce free radical production fromCCl4 which is the basic triggering factor for hepatotoxicity[36 37]
In the present study we evaluated the hepatoprotectiveefficacy of L sativum seeds in white male New-Zealand rab-bits The present results revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withL sativum seeds significantly repaired their liver injuriousmarker enzymes as well as bilirubin total protein and albu-min hence approving its hepatoprotective effect The resultsare in agreement with those of Afaf et al [12] where theystated the role of LSS as efficient hepatoprotective agent Alsothe current results are consistent with those of Mohamedand his coworkers [38] who reported that preprotectionwith LSEE (Lepidium sativum ethanolic extract) significantlyprohibited the D-GalNLPS induced rise in hepatic enzymes(AST ALT 120574-GGT ALP total bilirubin LDH and total pro-tein) thus significantly alleviated the reduction of lipid per-oxidation and repaired the antioxidant enzymatic status andtotal protein restoring them to normal levels D-GalNLPShas been widely used to examine the mechanisms underlyingacute liver failure [39 40] The hepatoprotective significanceof LSEE could be due to downregulation of cytokines (TNF-120572 IL-6) and stress gene (iNOS and HO-1) mRNA expres-sion and upregulation of IL-10 Lepidium sativum ethanolicextract (LSEE) preprotection also improved the degree ofstructural damage and reduced inflammatory infiltration in
Evidence-Based Complementary and Alternative Medicine 11
(a) (b)
(c) (d)
(e)
Figure 6 Effect of LSS on histopathological changes of liver tissues of normal groups in rabbits (a) Showing normal histological picture ofhepatic lobule that consists of central vein surrounded by normal hepatocytes (HEx100) (b) Showing liver sections of the rabbits treatedwith LSS (200 mgkg bw) which revealed normal histological picture of hepatic lobule that consists of central vein surrounded by normalhepatocytes (HEx100) (c) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealed normal hepatic architecturewith only mild inflammation [arrows] (HEx100) (d) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealedevident chronic venous congestion and moderate inflammation [arrows] (HEx100) (e) Showing liver sections of the rabbits treated with LSS(400 mgkg bw) which revealed portal to portal bridging fibrosis [arrow] (HEx200)
hepatic cells These outcomes established that LSEE alle-viates hepatic impairments and structural injury throughthe decay of oxidative stress inflammation and apopto-sis in the liver [38] Mohamed Sakran and his colleagues[10] suggested that 56-dimethoxy-210158403-methylenedioxy-7-C-120573-D glucopyranosyl isoflavone extracted from Lepidiumsativum seeds could improve the lipid profile and the liverfunctions in serum and reduce the free radicals generationby enhancing the antioxidant defense mechanism Newsuggested isoflavonoid can be used as a probable antioxi-dant against paracetamol hepatotoxicity with its antioxidant
characteristics and could restore the hepatic functionstowards normalization [10]
In the present study we found that improved cholesteroland triglycerides levels in L sativum seeds protected rabbitsas compared to CCl4 treated rabbits also support the hepato-protective effect of LSSThese results can also be correlated tothose of Shukla et al [41] who reported the role of L sativumseeds as hypolipidemic agent Also the results of the presentstudy agreed with those of Abdulrahman et al [20] whoseresults showed that L sativum seeds have hepatoprotectiveactivity that could be due to its antioxidant activity together
12 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
Figure 7 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) In rabbits sacrificed 5 weeks afterexperimental running as shown in (a) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100)(b) steatosis (30) in one case (HEx100) and (c) mild parenchymal inflammation [arrow] (HEx200) In rabbits sacrificed 10 weeks afterexperimental running as shown in (d) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100) (e)steatosis (about 20) and moderate parenchymal and portal inflammation (HEx100) and (f) bridging fibrosis [arrow] (HEx100)
with the presence of anti-inflammatory ingredients in Lsativum seeds extract
The results of the current study revealed that the exposureto CCl4 for 5 and 10 weeks led to a significant decrease inthe enzymatic activities of CAT GPx GR GST and SOD inthe liver homogenate of CCl4 treated rabbits 5 and 10 weeksafter experimental running compared to those of the normalcontrolThe concurrent protection of rabbits exposed toCCl4for 5 and 10weeks with 200amp 400mg kg bw LSS significantlyrepaired their antioxidant status and reduced significantly theMDA levels hence confirming its hepatoprotective efficacy
Although the precise molecular mechanism of action ofLSS is not completely understood some researchers [42]have confirmed the role of LSS in inhibiting oxidative stress
associated liver damage The antioxidant activity of LSScould be due to the presence of phenolic compound andflavonoids in these seeds as have been evidenced through thepreliminary screening study on LSS extract using HPLCOurresults match with those of Lee et al [42] who reported thatphenolic compounds play an essential role against oxidativestress related liver damage
A unique expectation of the current study is to confirmthat CCl4 induces liver DNA injury that was assessed usinggel electrophoretic method The results of the current studyshowed that CCL4 induces DNA injury as presented bya significant progression in liver DNA disintegration ofCCl4 treated rabbits above untreated control in the gelelectrophoresis
Evidence-Based Complementary and Alternative Medicine 13
(a) (b)
(c) (d)
(e) (f)
Figure 8 Effect of LSLS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (200 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) evident chronic venous congestion and feathery degeneration [arrow](HEx100) (b) mild feathery degeneration and isolated vesicular steatosis [arrow] (HEx200) and (c) moderate parenchymal and portalinflammation (HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) regenerating hepatocytes with isolatedvesicular steatosis (HEx100) (e) discrete chronic venous congestion and no steatosis (HEx400) and (f) discrete inflammatory infiltrate(HEx100)
Lipid peroxidation of the nuclear membrane may bedirectly accompanied with nuclear DNA damage [43]Nuclear DNA is concomitant with particular regions of thenuclear membrane [44]The close proximity of nuclear DNAto the nuclear membrane can ease the interaction of genomicDNA with the peroxyl radicals and further reactive inter-mediates formed during membrane lipid peroxidation [43]In the presence of transition metals the hydroxyl radicalsgenerated by CCL4 autoxidation can trigger peroxidationof the nuclear membrane lipids by metal catalyzed Haber-Weiss reaction the lipid peroxides levels and intermediate
free radicals could then be augmented by a chain reactionSubsequently the reactive species oxidize nuclearDNAwhenthe lipid peroxidation reaction occurs in the nuclear mem-brane since the nuclear membrane controls the transportof messenger RNA into the cytoplasm and supports thenuclear division process nuclear membrane peroxidationof lipids and proteins Moreover oxidative nuclear DNAdamage could disrupt these serious cellular functions [43]
DNA findings showed that DNA disintegration is adirect result after CCl4 treatment in rabbitsrsquo liver tissueswhich was reduced by participation of antioxidants through
14 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
(g)
Figure 9 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (400 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) discrete chronic venous congestion (HEx200) (b) centrilobular fibrosis[arrows] regenerating hepatocytes and isolated vesicular steatosis (HEx100) and (c) mild parenchymal and portal inflammation [arrow](HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) mild chronic venous congestion and isolated vesicularsteatosis (HEx100) (e) evident chronic venous congestion [arrows] (HEx100) (f) steatosis (40) with moderate inflammation (HEx100) and(g) portal to portal bridging fibrosis in one case [arrow] (HEx100)
Evidence-Based Complementary and Alternative Medicine 15
administration of 200 mg amp 400 mgkg bw L sativum seedsNearly complete repair of DNA damage was significant aftertreating rabbits with 200 mg amp 400 mgkg bw L sativumseeds The protective effect of L sativum seeds on hepaticDNA injury may be due to the antioxidant activity ofL sativum seeds via direct scavenging of free radicals orinterfering with free radicals generation
Concerning histopathological evaluation in our studyrabbitsrsquo livers exposed to CCL4 show portal and parenchymalinflammatory infiltrate feathery and ballooning degenera-tion fatty change and fibrosis Our data are consistent withRajeswary et al [45] who reported dilatation of sinusoidsfatty change disarrangement of normal hepatocytes and cen-trilobular necrosis Also A S Bernacchi et al [46] reportedthat CCL4 leads to liver necrosis and fat accumulation in therabbit
The present study revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withLSS improved their liver histopathological architecture inthe form of less inflammation regenerating hepatocytes nosteatosis or isolated vesicular steatosis no chronic venouscongestion or just discrete congestion and no fibrosis henceapproving its hepatoprotective effectThe results are in agree-ment with Afaf et al [12] and Abdulrahman et al [20] whoobserved the improvement of the histological structure inextract treated group as the histopathologic findings showedless inflammation well restored hepatocytes and less area ofnecrosis as compared to severe necrosis and inflammationin CCl4 treated group of animals which also supports thehepatoprotective effects of LLS Their findings suggest thepossibility of the presence of anti-inflammatory compoundsin LSS extract which could also be a contributing factortowards the hepatoprotective action of LSS
Althnaian [47] also reported that examination of hep-atocytes showed mild to moderate degree of recovery LSSattenuated the hepatic fat accumulation decreased vacuolardegeneration and improved the architecture of hepatocytes
In our study the reducing effect of LSS on fatty changecaused by CCL4 intoxication is indicating the protective andcurative role against fatty liver Our results are supported byShukla et al [41] who reported that LSS significantly reducedsteatosis Their histopathological observations confirmed thecurative efficacy of L sativum liver damage Microvesicularfatty changes hepatocyte ballooning and steatosis weremarkedly corrected by LSS Also Wadhwa et al [36] foundthat the severe fatty changes in the livers of rats caused byCCl4 were decreased in the treated groups
In the present study chronic venous congestion featherydegeneration and fatty changes show mild improvement inrabbits receiving 200mgkg bw of LSS for 5 weeks and showmoderate improvement in rabbits that received 200mgkg bwof LSS for 10 weeks and rabbits that received 400mgkg bwof LSS for 5 weeks Our data are consistent with Afaf et al[12] who observed that severe centrilobular hepatocellularvacuolation hemorrhages and congestion of the centralveins noticed in CCL4 group showed mild improvement ingroups that received 200 mgkg bw of LSS and moderateimprovement in groups that received 400 mgkg bw ofLSS
On the other hand rabbits that received 400mgkg bw ofLSS for 10 weeks showed evidence of liver toxicity in the formof venous congestion hepatocyte feathery degeneration fattychange and fibrosis These results are compatible with Bafeeland Ali [48] who reported that LSS had minimal effect onliver parenchyma if given in low doses So LSS could be usedas food additive or supplement Mineral content of calciumand phosphate and the presence of essential fatty acids inconsiderable level make LSS of beneficial nutritional valuefor many body organs including the liver However LSS inhigh dose showed some toxic effect on renal parenchyma rep-resented by focal necrosis hepatocyte apoptosis and portalinflammatory cell infiltrate Burow et al [49] reported thatthese hazardous effects of LSS may be due to isothiocyanatesreported to be one of LSS constituents
5 Conclusions and Recommendations
The present study showed that LSS is a key factor ininhibition of the hazardous effects of CCl4 LSS treatmentcould significantly alleviate to a greater extent the degree oflipid peroxidation and restore the antioxidative enzymes andshift liver function to normal levels In addition to antioxi-dants activity LSS attenuated the hepatic fat accumulationdecreased vacuolar degeneration ameliorated the degree ofstructural damage in the hepatic cells and improved thearchitecture of hepatocytes The current findings demon-strated that LSS have marked hepatoprotective activity Infuture work active ingredients of LSS extract should bestudied in various animal models of severe toxicity inducedby CCL4 and other hepatotoxic agents The active ingredientsof this plant are likely to be applied as one of the future drugsagainst acute liver toxicity
Data Availability
Data used to support the findings of this study are availablefrom the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This project was funded by the Deanship of ScientificResearch (DSR) King Abdulaziz University Jeddah undergrant no S-17-314-37 The authors therefore acknowledgewith thanks DSR technical and financial support
References
[1] M K Manibusan M Odin and D A Eastmond ldquoPostu-lated carbon tetrachloride mode of action a reviewrdquo Journalof Environmental Science and Health Part C EnvironmentalCarcinogenesis and Ecotoxicology Reviews vol 25 no 3 pp 185ndash209 2007
[2] N Brautbar and J Williams II ldquoIndustrial solvents andliver toxicity risk assessment risk factors and mechanismsrdquo
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
8 Evidence-Based Complementary and Alternative Medicine
Table4En
zymaticactiv
itiesofantio
xidant
enzymesandMDAofun
treatedLSS
administeredC
Cl4tre
atedand
LSSprotectedrabb
itsA
lldatawe
reexpressedas
meanplusmnSE
MV
aluesw
ere
statistic
allyteste
dusingtheS
tudent
t-testandsig
nificantd
ifferencesa
tplt005
andplt001
asindicatedby
(lowast)amp
(lowastlowast)com
paredto
norm
alcontrolby
()amp
()
comparedto
CCL4aft
er5
weeksandby
(+)amp
(++)
comparedto
CCL4aft
er10
weeksrespectiv
ely
Group
CAT
(nggtissue)
SOD
(nggtissue)
GR
(nggtissue)
GST
(nggtissue)
GPx
(nggtissue)
MDA(nmolm
l)
Con
trol
3155plusmn
199
2820plusmn10
92029plusmn099
1537plusmn12
12197plusmn17
0647plusmn057
LS(200
mgkg
bw)
3127plusmn18
62631plusmn15
51908plusmn077
1542plusmn067
2129plusmn12
8635plusmn045
LS(400
mgkg
bw)
2843plusmn204
2676plusmn18
81972plusmn12
81555plusmn14
32031plusmn074
635plusmn060
CCl4(5
wk)
1475plusmn258lowastlowast
1406plusmn13
9lowastlowast
1358plusmn061lowastlowast
1041plusmn
108lowastlowast
1409plusmn099lowastlowast
1273plusmn10
6lowastlowast
of
Change
-53
-50
-33
-32
-35
96CCl4(10wk)
1682plusmn15
7lowastlowast
1767plusmn202lowastlowast
1277plusmn13
6lowastlowast
940plusmn073lowastlowast
1152plusmn18
2lowastlowast
1427plusmn10
7lowastlowast
of
Change
-46
-37
-37
-39
-47
120
CCl4+LS
(200
mgkg
bw)5
wk
2846plusmn201
2720plusmn094
1892plusmn093
1472plusmn118
2009plusmn112
667plusmn046
of
Change
9393
3941
42-4
7CCl4+LS
(400
mgkg
bw)5
wk
2519plusmn17
7++
2231plusmn14
0++
1813plusmn094++
1304plusmn13
1++
1941plusmn
192+
+713plusmn072++
of
Change
7058
3325
37-4
4CCl4+LS
(200
mgkg
bw)10wk
2872plusmn201
2796plusmn222
1949plusmn096
1492plusmn15
8
2006plusmn17
9
569plusmn046
of
Change
7058
5258
74-6
0CCl4+LS
(400
mgkg
bw)10wk
2919plusmn17
0++
2610plusmn14
3++
1887plusmn17
3++
1378plusmn086++
1906plusmn14
1++
619plusmn052++
of
Change
7347
4746
65-5
6
Evidence-Based Complementary and Alternative Medicine 9
05
10152025303540
CAT
Leve
(ng
g tis
sue)
CAT
lowastlowastlowastlowast
++++
0
5
10
15
20
25
30
35
SOD
Lev
e (ng
lg ti
ssue
)
SOD
lowastlowast
lowastlowast
++++
0
5
10
15
20
25
GR
Leve
(ng
g tis
sue)
GR
lowastlowastlowastlowast
++++
02468
1012141618
GST
Lev
e (ng
g ti
ssue
)
GST
lowastlowastlowastlowast
++++
0
5
10
15
20
25
GPx
Lev
e (ng
g ti
ssue
)
GPx
lowastlowastlowastlowast
++++
02468
1012141618
MD
A L
eve (
nmol
g ti
ssue
)
MDA
lowastlowast
lowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
Figure 4 Effects of LSS on oxidative stress markers in rabbits treated with CCL4 induced hepatotoxicity as indicated in the figure All datawere expressed as mean plusmn SEM Values were statistically tested using the Student t-test and significant differences at p lt 005 and p lt 001 asindicated by (lowast) amp (lowastlowast) compared to normal control by () amp () compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4after 10 weeks respectively
Examination of liver tissue exposed to CCL4 as well as200 mgkg bw of LSS in their diet on daily basis (GroupB2) reveals that the group which is sacrificed 5 weeksafter experimental running shows evident chronic venouscongestion and feathery degeneration in two cases only mildfeathery degeneration in others no steatosis or isolated vesic-ular steatosis mild parenchymal and portal inflammation
(Figures 8(a) 8(b) and 8(c)) Examination of the groupsacrificed 10 weeks after experimental running shows theimprovement of the histoarchitectural changes of the livertowards the normal aspect showing regenerating hepato-cytes with no steatosis discrete chronic venous congestionand discrete inflammatory infiltrate (Figures 8(d) 8(e) and8(f))
10 Evidence-Based Complementary and Alternative Medicine
M 1 2 3 4 5 6 7 8 9bp
10000
3000
2000
1500
1000
750
500
300
Figure 5 Agarose gel showingDNAdamage byCCl4 and protectiveeffects of various fractions of LSS Lanes from left (M) molecularweight marker (1) negative control (2) LSS (200 mgkg bw) (3) LSS(400mgkg bw) (4) CCl4 for 5weeks (5) CCl4 for 10weeks (6)amp (7)CCl4 + LSS (200 mgkg bw) (8) amp (9) CCl4 + LSS (400 mg kg bw)
Examination of liver tissue of rabbits exposed to CCL4as well as 400 mgkg bw of LSS in their diet on daily basis(Group B3) reveals in the group that scarified 5 weeks afterexperimental running improvement of the histoarchitecturalchanges of the liver as there is no chronic venous congestionor only discrete chronic venous congestion and mild featherydegeneration no steatosis or only isolated vesicular steatosisand mild parenchymal and portal inflammation (Figures9(a) 9(b) and 9(c)) But the group that was sacrificed10 weeks after experimental running shows adverse effectsof the drug in the form of steatosis and chronic venouscongestion which is evident in two cases with mild tomoderate inflammation Bridging fibrosis is seen in one case(Figures 9(d) 9(e) 9(f) and 9(g))
4 Discussion
Hepatic disease became a significant public health concernowing to growing rate of obesity in all age populations [26]The conservative treatments of liver problems such as acuteand chronic liver hepatitis liver cirrhosis and fatty liverare often insufficient due to hazardous effects initiated byhepatotoxic drugs of chemical origin [27] Medicinal plantsowing to their abundant hepatoprotective natural ingredi-ents have been comprehensively investigated to treat liverdisorders In the current study hepatoprotective efficacy ofLSS was investigated via hepatotoxic classical model inducedby carbon tetrachloride
In organ and tissue systems CCl4 is stimulated bycytochrome P-450 dependent mixed oxidase in the hepaticendoplasmic reticulum to procedure free radical trichloro-methyl (CCl3 and Cl3COO) This causes changes in themembrane of the endoplasmic reticulum and other cel-lular membranes resulting in an increase in calcium ionpermeability through the plasma membrane causing severeinstabilities of calcium homeostasis leading to necrotic celldeath [28 29] CCL4 affects many organs causing degenera-tion of liver fatty layer and centrilobular necrosis [29] Thisphenomenon indicates that CCl4 is a powerful hepatotoxic
agent and may be used for the characterization of the hepaticdisease treatment medicines Elevation of liver enzymaticmarkers (ALT AST ALP and bilirubin) and increased lipidperoxidation are further evidences of hepatotoxicity ALTand AST that exist in the hepatocytes can certainly leak intothe peripheral blood as soon as the hepatocytes are injured[29] Lepidium sativum seed analysis by GCMS exhibitedthe presence of high concentrations of mainly 120572-linolenicacid (ALA) which is nminus3 fatty acid an omega-3 fatty acidfound in seeds exerting its bioactivity in ameliorating thehepatic intoxication and oxidative stress including liver injuryinduced by alcohol [30] liver steatosis [31 32] nonalcoholichepatic disease [33] and parenteral nutrition-associated liverdisease [34] Pari and Jalaludeen stated that orientationof the active groups in sinapic acid increases antioxidantefficiency as metal chelators and promotes its protectiveefficacy against liver oxidative stress in arsenic intoxicatedrats [35]The current study showed that sinapic acid as ametalchelator may exert its biological activity in improving liverfunction
Lepidium sativum seed extracts have proved hepatopro-tective effects against CCl4 induced liver damage A researchstudy on male Albino Wistar rats demonstrated significantreduction in hepatotoxicity induced by CCl4 administeredwith 200-400 mgkg of Lepidium sativum seeds extractincluded within the diet Serum level of AST ALT levels andbilirubin levels were significantly elevated in CCl4 treated ratscompared to those of normal control However a significantreduction in the levels of these parameters in Lepidiumsativum seed extracts protected rats was observed The hep-atoprotective effect could be due to the presence of alkaloidcoumarin flavonoids tannin and triterpenes which enhanceantioxidant activity and reduce free radical production fromCCl4 which is the basic triggering factor for hepatotoxicity[36 37]
In the present study we evaluated the hepatoprotectiveefficacy of L sativum seeds in white male New-Zealand rab-bits The present results revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withL sativum seeds significantly repaired their liver injuriousmarker enzymes as well as bilirubin total protein and albu-min hence approving its hepatoprotective effect The resultsare in agreement with those of Afaf et al [12] where theystated the role of LSS as efficient hepatoprotective agent Alsothe current results are consistent with those of Mohamedand his coworkers [38] who reported that preprotectionwith LSEE (Lepidium sativum ethanolic extract) significantlyprohibited the D-GalNLPS induced rise in hepatic enzymes(AST ALT 120574-GGT ALP total bilirubin LDH and total pro-tein) thus significantly alleviated the reduction of lipid per-oxidation and repaired the antioxidant enzymatic status andtotal protein restoring them to normal levels D-GalNLPShas been widely used to examine the mechanisms underlyingacute liver failure [39 40] The hepatoprotective significanceof LSEE could be due to downregulation of cytokines (TNF-120572 IL-6) and stress gene (iNOS and HO-1) mRNA expres-sion and upregulation of IL-10 Lepidium sativum ethanolicextract (LSEE) preprotection also improved the degree ofstructural damage and reduced inflammatory infiltration in
Evidence-Based Complementary and Alternative Medicine 11
(a) (b)
(c) (d)
(e)
Figure 6 Effect of LSS on histopathological changes of liver tissues of normal groups in rabbits (a) Showing normal histological picture ofhepatic lobule that consists of central vein surrounded by normal hepatocytes (HEx100) (b) Showing liver sections of the rabbits treatedwith LSS (200 mgkg bw) which revealed normal histological picture of hepatic lobule that consists of central vein surrounded by normalhepatocytes (HEx100) (c) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealed normal hepatic architecturewith only mild inflammation [arrows] (HEx100) (d) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealedevident chronic venous congestion and moderate inflammation [arrows] (HEx100) (e) Showing liver sections of the rabbits treated with LSS(400 mgkg bw) which revealed portal to portal bridging fibrosis [arrow] (HEx200)
hepatic cells These outcomes established that LSEE alle-viates hepatic impairments and structural injury throughthe decay of oxidative stress inflammation and apopto-sis in the liver [38] Mohamed Sakran and his colleagues[10] suggested that 56-dimethoxy-210158403-methylenedioxy-7-C-120573-D glucopyranosyl isoflavone extracted from Lepidiumsativum seeds could improve the lipid profile and the liverfunctions in serum and reduce the free radicals generationby enhancing the antioxidant defense mechanism Newsuggested isoflavonoid can be used as a probable antioxi-dant against paracetamol hepatotoxicity with its antioxidant
characteristics and could restore the hepatic functionstowards normalization [10]
In the present study we found that improved cholesteroland triglycerides levels in L sativum seeds protected rabbitsas compared to CCl4 treated rabbits also support the hepato-protective effect of LSSThese results can also be correlated tothose of Shukla et al [41] who reported the role of L sativumseeds as hypolipidemic agent Also the results of the presentstudy agreed with those of Abdulrahman et al [20] whoseresults showed that L sativum seeds have hepatoprotectiveactivity that could be due to its antioxidant activity together
12 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
Figure 7 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) In rabbits sacrificed 5 weeks afterexperimental running as shown in (a) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100)(b) steatosis (30) in one case (HEx100) and (c) mild parenchymal inflammation [arrow] (HEx200) In rabbits sacrificed 10 weeks afterexperimental running as shown in (d) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100) (e)steatosis (about 20) and moderate parenchymal and portal inflammation (HEx100) and (f) bridging fibrosis [arrow] (HEx100)
with the presence of anti-inflammatory ingredients in Lsativum seeds extract
The results of the current study revealed that the exposureto CCl4 for 5 and 10 weeks led to a significant decrease inthe enzymatic activities of CAT GPx GR GST and SOD inthe liver homogenate of CCl4 treated rabbits 5 and 10 weeksafter experimental running compared to those of the normalcontrolThe concurrent protection of rabbits exposed toCCl4for 5 and 10weeks with 200amp 400mg kg bw LSS significantlyrepaired their antioxidant status and reduced significantly theMDA levels hence confirming its hepatoprotective efficacy
Although the precise molecular mechanism of action ofLSS is not completely understood some researchers [42]have confirmed the role of LSS in inhibiting oxidative stress
associated liver damage The antioxidant activity of LSScould be due to the presence of phenolic compound andflavonoids in these seeds as have been evidenced through thepreliminary screening study on LSS extract using HPLCOurresults match with those of Lee et al [42] who reported thatphenolic compounds play an essential role against oxidativestress related liver damage
A unique expectation of the current study is to confirmthat CCl4 induces liver DNA injury that was assessed usinggel electrophoretic method The results of the current studyshowed that CCL4 induces DNA injury as presented bya significant progression in liver DNA disintegration ofCCl4 treated rabbits above untreated control in the gelelectrophoresis
Evidence-Based Complementary and Alternative Medicine 13
(a) (b)
(c) (d)
(e) (f)
Figure 8 Effect of LSLS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (200 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) evident chronic venous congestion and feathery degeneration [arrow](HEx100) (b) mild feathery degeneration and isolated vesicular steatosis [arrow] (HEx200) and (c) moderate parenchymal and portalinflammation (HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) regenerating hepatocytes with isolatedvesicular steatosis (HEx100) (e) discrete chronic venous congestion and no steatosis (HEx400) and (f) discrete inflammatory infiltrate(HEx100)
Lipid peroxidation of the nuclear membrane may bedirectly accompanied with nuclear DNA damage [43]Nuclear DNA is concomitant with particular regions of thenuclear membrane [44]The close proximity of nuclear DNAto the nuclear membrane can ease the interaction of genomicDNA with the peroxyl radicals and further reactive inter-mediates formed during membrane lipid peroxidation [43]In the presence of transition metals the hydroxyl radicalsgenerated by CCL4 autoxidation can trigger peroxidationof the nuclear membrane lipids by metal catalyzed Haber-Weiss reaction the lipid peroxides levels and intermediate
free radicals could then be augmented by a chain reactionSubsequently the reactive species oxidize nuclearDNAwhenthe lipid peroxidation reaction occurs in the nuclear mem-brane since the nuclear membrane controls the transportof messenger RNA into the cytoplasm and supports thenuclear division process nuclear membrane peroxidationof lipids and proteins Moreover oxidative nuclear DNAdamage could disrupt these serious cellular functions [43]
DNA findings showed that DNA disintegration is adirect result after CCl4 treatment in rabbitsrsquo liver tissueswhich was reduced by participation of antioxidants through
14 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
(g)
Figure 9 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (400 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) discrete chronic venous congestion (HEx200) (b) centrilobular fibrosis[arrows] regenerating hepatocytes and isolated vesicular steatosis (HEx100) and (c) mild parenchymal and portal inflammation [arrow](HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) mild chronic venous congestion and isolated vesicularsteatosis (HEx100) (e) evident chronic venous congestion [arrows] (HEx100) (f) steatosis (40) with moderate inflammation (HEx100) and(g) portal to portal bridging fibrosis in one case [arrow] (HEx100)
Evidence-Based Complementary and Alternative Medicine 15
administration of 200 mg amp 400 mgkg bw L sativum seedsNearly complete repair of DNA damage was significant aftertreating rabbits with 200 mg amp 400 mgkg bw L sativumseeds The protective effect of L sativum seeds on hepaticDNA injury may be due to the antioxidant activity ofL sativum seeds via direct scavenging of free radicals orinterfering with free radicals generation
Concerning histopathological evaluation in our studyrabbitsrsquo livers exposed to CCL4 show portal and parenchymalinflammatory infiltrate feathery and ballooning degenera-tion fatty change and fibrosis Our data are consistent withRajeswary et al [45] who reported dilatation of sinusoidsfatty change disarrangement of normal hepatocytes and cen-trilobular necrosis Also A S Bernacchi et al [46] reportedthat CCL4 leads to liver necrosis and fat accumulation in therabbit
The present study revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withLSS improved their liver histopathological architecture inthe form of less inflammation regenerating hepatocytes nosteatosis or isolated vesicular steatosis no chronic venouscongestion or just discrete congestion and no fibrosis henceapproving its hepatoprotective effectThe results are in agree-ment with Afaf et al [12] and Abdulrahman et al [20] whoobserved the improvement of the histological structure inextract treated group as the histopathologic findings showedless inflammation well restored hepatocytes and less area ofnecrosis as compared to severe necrosis and inflammationin CCl4 treated group of animals which also supports thehepatoprotective effects of LLS Their findings suggest thepossibility of the presence of anti-inflammatory compoundsin LSS extract which could also be a contributing factortowards the hepatoprotective action of LSS
Althnaian [47] also reported that examination of hep-atocytes showed mild to moderate degree of recovery LSSattenuated the hepatic fat accumulation decreased vacuolardegeneration and improved the architecture of hepatocytes
In our study the reducing effect of LSS on fatty changecaused by CCL4 intoxication is indicating the protective andcurative role against fatty liver Our results are supported byShukla et al [41] who reported that LSS significantly reducedsteatosis Their histopathological observations confirmed thecurative efficacy of L sativum liver damage Microvesicularfatty changes hepatocyte ballooning and steatosis weremarkedly corrected by LSS Also Wadhwa et al [36] foundthat the severe fatty changes in the livers of rats caused byCCl4 were decreased in the treated groups
In the present study chronic venous congestion featherydegeneration and fatty changes show mild improvement inrabbits receiving 200mgkg bw of LSS for 5 weeks and showmoderate improvement in rabbits that received 200mgkg bwof LSS for 10 weeks and rabbits that received 400mgkg bwof LSS for 5 weeks Our data are consistent with Afaf et al[12] who observed that severe centrilobular hepatocellularvacuolation hemorrhages and congestion of the centralveins noticed in CCL4 group showed mild improvement ingroups that received 200 mgkg bw of LSS and moderateimprovement in groups that received 400 mgkg bw ofLSS
On the other hand rabbits that received 400mgkg bw ofLSS for 10 weeks showed evidence of liver toxicity in the formof venous congestion hepatocyte feathery degeneration fattychange and fibrosis These results are compatible with Bafeeland Ali [48] who reported that LSS had minimal effect onliver parenchyma if given in low doses So LSS could be usedas food additive or supplement Mineral content of calciumand phosphate and the presence of essential fatty acids inconsiderable level make LSS of beneficial nutritional valuefor many body organs including the liver However LSS inhigh dose showed some toxic effect on renal parenchyma rep-resented by focal necrosis hepatocyte apoptosis and portalinflammatory cell infiltrate Burow et al [49] reported thatthese hazardous effects of LSS may be due to isothiocyanatesreported to be one of LSS constituents
5 Conclusions and Recommendations
The present study showed that LSS is a key factor ininhibition of the hazardous effects of CCl4 LSS treatmentcould significantly alleviate to a greater extent the degree oflipid peroxidation and restore the antioxidative enzymes andshift liver function to normal levels In addition to antioxi-dants activity LSS attenuated the hepatic fat accumulationdecreased vacuolar degeneration ameliorated the degree ofstructural damage in the hepatic cells and improved thearchitecture of hepatocytes The current findings demon-strated that LSS have marked hepatoprotective activity Infuture work active ingredients of LSS extract should bestudied in various animal models of severe toxicity inducedby CCL4 and other hepatotoxic agents The active ingredientsof this plant are likely to be applied as one of the future drugsagainst acute liver toxicity
Data Availability
Data used to support the findings of this study are availablefrom the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This project was funded by the Deanship of ScientificResearch (DSR) King Abdulaziz University Jeddah undergrant no S-17-314-37 The authors therefore acknowledgewith thanks DSR technical and financial support
References
[1] M K Manibusan M Odin and D A Eastmond ldquoPostu-lated carbon tetrachloride mode of action a reviewrdquo Journalof Environmental Science and Health Part C EnvironmentalCarcinogenesis and Ecotoxicology Reviews vol 25 no 3 pp 185ndash209 2007
[2] N Brautbar and J Williams II ldquoIndustrial solvents andliver toxicity risk assessment risk factors and mechanismsrdquo
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
Stem Cells International
Hindawiwwwhindawicom Volume 2018
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Disease Markers
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Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 9
05
10152025303540
CAT
Leve
(ng
g tis
sue)
CAT
lowastlowastlowastlowast
++++
0
5
10
15
20
25
30
35
SOD
Lev
e (ng
lg ti
ssue
)
SOD
lowastlowast
lowastlowast
++++
0
5
10
15
20
25
GR
Leve
(ng
g tis
sue)
GR
lowastlowastlowastlowast
++++
02468
1012141618
GST
Lev
e (ng
g ti
ssue
)
GST
lowastlowastlowastlowast
++++
0
5
10
15
20
25
GPx
Lev
e (ng
g ti
ssue
)
GPx
lowastlowastlowastlowast
++++
02468
1012141618
MD
A L
eve (
nmol
g ti
ssue
)
MDA
lowastlowast
lowastlowast
++
++
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
ControlCCL
4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
CCL4 + LS (400) 10 wk
CCL4 + LS (400)
5 wk
CCL4 + LS (2
00) 10 wk
CCL4 + LS (2
00)5 wk
CCL4 (10 wk)
CCL4 (5 wk)
LS (400)
LS (200)
Control
Figure 4 Effects of LSS on oxidative stress markers in rabbits treated with CCL4 induced hepatotoxicity as indicated in the figure All datawere expressed as mean plusmn SEM Values were statistically tested using the Student t-test and significant differences at p lt 005 and p lt 001 asindicated by (lowast) amp (lowastlowast) compared to normal control by () amp () compared to CCL4 after 5 weeks and by (+) amp (++) compared to CCL4after 10 weeks respectively
Examination of liver tissue exposed to CCL4 as well as200 mgkg bw of LSS in their diet on daily basis (GroupB2) reveals that the group which is sacrificed 5 weeksafter experimental running shows evident chronic venouscongestion and feathery degeneration in two cases only mildfeathery degeneration in others no steatosis or isolated vesic-ular steatosis mild parenchymal and portal inflammation
(Figures 8(a) 8(b) and 8(c)) Examination of the groupsacrificed 10 weeks after experimental running shows theimprovement of the histoarchitectural changes of the livertowards the normal aspect showing regenerating hepato-cytes with no steatosis discrete chronic venous congestionand discrete inflammatory infiltrate (Figures 8(d) 8(e) and8(f))
10 Evidence-Based Complementary and Alternative Medicine
M 1 2 3 4 5 6 7 8 9bp
10000
3000
2000
1500
1000
750
500
300
Figure 5 Agarose gel showingDNAdamage byCCl4 and protectiveeffects of various fractions of LSS Lanes from left (M) molecularweight marker (1) negative control (2) LSS (200 mgkg bw) (3) LSS(400mgkg bw) (4) CCl4 for 5weeks (5) CCl4 for 10weeks (6)amp (7)CCl4 + LSS (200 mgkg bw) (8) amp (9) CCl4 + LSS (400 mg kg bw)
Examination of liver tissue of rabbits exposed to CCL4as well as 400 mgkg bw of LSS in their diet on daily basis(Group B3) reveals in the group that scarified 5 weeks afterexperimental running improvement of the histoarchitecturalchanges of the liver as there is no chronic venous congestionor only discrete chronic venous congestion and mild featherydegeneration no steatosis or only isolated vesicular steatosisand mild parenchymal and portal inflammation (Figures9(a) 9(b) and 9(c)) But the group that was sacrificed10 weeks after experimental running shows adverse effectsof the drug in the form of steatosis and chronic venouscongestion which is evident in two cases with mild tomoderate inflammation Bridging fibrosis is seen in one case(Figures 9(d) 9(e) 9(f) and 9(g))
4 Discussion
Hepatic disease became a significant public health concernowing to growing rate of obesity in all age populations [26]The conservative treatments of liver problems such as acuteand chronic liver hepatitis liver cirrhosis and fatty liverare often insufficient due to hazardous effects initiated byhepatotoxic drugs of chemical origin [27] Medicinal plantsowing to their abundant hepatoprotective natural ingredi-ents have been comprehensively investigated to treat liverdisorders In the current study hepatoprotective efficacy ofLSS was investigated via hepatotoxic classical model inducedby carbon tetrachloride
In organ and tissue systems CCl4 is stimulated bycytochrome P-450 dependent mixed oxidase in the hepaticendoplasmic reticulum to procedure free radical trichloro-methyl (CCl3 and Cl3COO) This causes changes in themembrane of the endoplasmic reticulum and other cel-lular membranes resulting in an increase in calcium ionpermeability through the plasma membrane causing severeinstabilities of calcium homeostasis leading to necrotic celldeath [28 29] CCL4 affects many organs causing degenera-tion of liver fatty layer and centrilobular necrosis [29] Thisphenomenon indicates that CCl4 is a powerful hepatotoxic
agent and may be used for the characterization of the hepaticdisease treatment medicines Elevation of liver enzymaticmarkers (ALT AST ALP and bilirubin) and increased lipidperoxidation are further evidences of hepatotoxicity ALTand AST that exist in the hepatocytes can certainly leak intothe peripheral blood as soon as the hepatocytes are injured[29] Lepidium sativum seed analysis by GCMS exhibitedthe presence of high concentrations of mainly 120572-linolenicacid (ALA) which is nminus3 fatty acid an omega-3 fatty acidfound in seeds exerting its bioactivity in ameliorating thehepatic intoxication and oxidative stress including liver injuryinduced by alcohol [30] liver steatosis [31 32] nonalcoholichepatic disease [33] and parenteral nutrition-associated liverdisease [34] Pari and Jalaludeen stated that orientationof the active groups in sinapic acid increases antioxidantefficiency as metal chelators and promotes its protectiveefficacy against liver oxidative stress in arsenic intoxicatedrats [35]The current study showed that sinapic acid as ametalchelator may exert its biological activity in improving liverfunction
Lepidium sativum seed extracts have proved hepatopro-tective effects against CCl4 induced liver damage A researchstudy on male Albino Wistar rats demonstrated significantreduction in hepatotoxicity induced by CCl4 administeredwith 200-400 mgkg of Lepidium sativum seeds extractincluded within the diet Serum level of AST ALT levels andbilirubin levels were significantly elevated in CCl4 treated ratscompared to those of normal control However a significantreduction in the levels of these parameters in Lepidiumsativum seed extracts protected rats was observed The hep-atoprotective effect could be due to the presence of alkaloidcoumarin flavonoids tannin and triterpenes which enhanceantioxidant activity and reduce free radical production fromCCl4 which is the basic triggering factor for hepatotoxicity[36 37]
In the present study we evaluated the hepatoprotectiveefficacy of L sativum seeds in white male New-Zealand rab-bits The present results revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withL sativum seeds significantly repaired their liver injuriousmarker enzymes as well as bilirubin total protein and albu-min hence approving its hepatoprotective effect The resultsare in agreement with those of Afaf et al [12] where theystated the role of LSS as efficient hepatoprotective agent Alsothe current results are consistent with those of Mohamedand his coworkers [38] who reported that preprotectionwith LSEE (Lepidium sativum ethanolic extract) significantlyprohibited the D-GalNLPS induced rise in hepatic enzymes(AST ALT 120574-GGT ALP total bilirubin LDH and total pro-tein) thus significantly alleviated the reduction of lipid per-oxidation and repaired the antioxidant enzymatic status andtotal protein restoring them to normal levels D-GalNLPShas been widely used to examine the mechanisms underlyingacute liver failure [39 40] The hepatoprotective significanceof LSEE could be due to downregulation of cytokines (TNF-120572 IL-6) and stress gene (iNOS and HO-1) mRNA expres-sion and upregulation of IL-10 Lepidium sativum ethanolicextract (LSEE) preprotection also improved the degree ofstructural damage and reduced inflammatory infiltration in
Evidence-Based Complementary and Alternative Medicine 11
(a) (b)
(c) (d)
(e)
Figure 6 Effect of LSS on histopathological changes of liver tissues of normal groups in rabbits (a) Showing normal histological picture ofhepatic lobule that consists of central vein surrounded by normal hepatocytes (HEx100) (b) Showing liver sections of the rabbits treatedwith LSS (200 mgkg bw) which revealed normal histological picture of hepatic lobule that consists of central vein surrounded by normalhepatocytes (HEx100) (c) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealed normal hepatic architecturewith only mild inflammation [arrows] (HEx100) (d) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealedevident chronic venous congestion and moderate inflammation [arrows] (HEx100) (e) Showing liver sections of the rabbits treated with LSS(400 mgkg bw) which revealed portal to portal bridging fibrosis [arrow] (HEx200)
hepatic cells These outcomes established that LSEE alle-viates hepatic impairments and structural injury throughthe decay of oxidative stress inflammation and apopto-sis in the liver [38] Mohamed Sakran and his colleagues[10] suggested that 56-dimethoxy-210158403-methylenedioxy-7-C-120573-D glucopyranosyl isoflavone extracted from Lepidiumsativum seeds could improve the lipid profile and the liverfunctions in serum and reduce the free radicals generationby enhancing the antioxidant defense mechanism Newsuggested isoflavonoid can be used as a probable antioxi-dant against paracetamol hepatotoxicity with its antioxidant
characteristics and could restore the hepatic functionstowards normalization [10]
In the present study we found that improved cholesteroland triglycerides levels in L sativum seeds protected rabbitsas compared to CCl4 treated rabbits also support the hepato-protective effect of LSSThese results can also be correlated tothose of Shukla et al [41] who reported the role of L sativumseeds as hypolipidemic agent Also the results of the presentstudy agreed with those of Abdulrahman et al [20] whoseresults showed that L sativum seeds have hepatoprotectiveactivity that could be due to its antioxidant activity together
12 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
Figure 7 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) In rabbits sacrificed 5 weeks afterexperimental running as shown in (a) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100)(b) steatosis (30) in one case (HEx100) and (c) mild parenchymal inflammation [arrow] (HEx200) In rabbits sacrificed 10 weeks afterexperimental running as shown in (d) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100) (e)steatosis (about 20) and moderate parenchymal and portal inflammation (HEx100) and (f) bridging fibrosis [arrow] (HEx100)
with the presence of anti-inflammatory ingredients in Lsativum seeds extract
The results of the current study revealed that the exposureto CCl4 for 5 and 10 weeks led to a significant decrease inthe enzymatic activities of CAT GPx GR GST and SOD inthe liver homogenate of CCl4 treated rabbits 5 and 10 weeksafter experimental running compared to those of the normalcontrolThe concurrent protection of rabbits exposed toCCl4for 5 and 10weeks with 200amp 400mg kg bw LSS significantlyrepaired their antioxidant status and reduced significantly theMDA levels hence confirming its hepatoprotective efficacy
Although the precise molecular mechanism of action ofLSS is not completely understood some researchers [42]have confirmed the role of LSS in inhibiting oxidative stress
associated liver damage The antioxidant activity of LSScould be due to the presence of phenolic compound andflavonoids in these seeds as have been evidenced through thepreliminary screening study on LSS extract using HPLCOurresults match with those of Lee et al [42] who reported thatphenolic compounds play an essential role against oxidativestress related liver damage
A unique expectation of the current study is to confirmthat CCl4 induces liver DNA injury that was assessed usinggel electrophoretic method The results of the current studyshowed that CCL4 induces DNA injury as presented bya significant progression in liver DNA disintegration ofCCl4 treated rabbits above untreated control in the gelelectrophoresis
Evidence-Based Complementary and Alternative Medicine 13
(a) (b)
(c) (d)
(e) (f)
Figure 8 Effect of LSLS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (200 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) evident chronic venous congestion and feathery degeneration [arrow](HEx100) (b) mild feathery degeneration and isolated vesicular steatosis [arrow] (HEx200) and (c) moderate parenchymal and portalinflammation (HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) regenerating hepatocytes with isolatedvesicular steatosis (HEx100) (e) discrete chronic venous congestion and no steatosis (HEx400) and (f) discrete inflammatory infiltrate(HEx100)
Lipid peroxidation of the nuclear membrane may bedirectly accompanied with nuclear DNA damage [43]Nuclear DNA is concomitant with particular regions of thenuclear membrane [44]The close proximity of nuclear DNAto the nuclear membrane can ease the interaction of genomicDNA with the peroxyl radicals and further reactive inter-mediates formed during membrane lipid peroxidation [43]In the presence of transition metals the hydroxyl radicalsgenerated by CCL4 autoxidation can trigger peroxidationof the nuclear membrane lipids by metal catalyzed Haber-Weiss reaction the lipid peroxides levels and intermediate
free radicals could then be augmented by a chain reactionSubsequently the reactive species oxidize nuclearDNAwhenthe lipid peroxidation reaction occurs in the nuclear mem-brane since the nuclear membrane controls the transportof messenger RNA into the cytoplasm and supports thenuclear division process nuclear membrane peroxidationof lipids and proteins Moreover oxidative nuclear DNAdamage could disrupt these serious cellular functions [43]
DNA findings showed that DNA disintegration is adirect result after CCl4 treatment in rabbitsrsquo liver tissueswhich was reduced by participation of antioxidants through
14 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
(g)
Figure 9 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (400 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) discrete chronic venous congestion (HEx200) (b) centrilobular fibrosis[arrows] regenerating hepatocytes and isolated vesicular steatosis (HEx100) and (c) mild parenchymal and portal inflammation [arrow](HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) mild chronic venous congestion and isolated vesicularsteatosis (HEx100) (e) evident chronic venous congestion [arrows] (HEx100) (f) steatosis (40) with moderate inflammation (HEx100) and(g) portal to portal bridging fibrosis in one case [arrow] (HEx100)
Evidence-Based Complementary and Alternative Medicine 15
administration of 200 mg amp 400 mgkg bw L sativum seedsNearly complete repair of DNA damage was significant aftertreating rabbits with 200 mg amp 400 mgkg bw L sativumseeds The protective effect of L sativum seeds on hepaticDNA injury may be due to the antioxidant activity ofL sativum seeds via direct scavenging of free radicals orinterfering with free radicals generation
Concerning histopathological evaluation in our studyrabbitsrsquo livers exposed to CCL4 show portal and parenchymalinflammatory infiltrate feathery and ballooning degenera-tion fatty change and fibrosis Our data are consistent withRajeswary et al [45] who reported dilatation of sinusoidsfatty change disarrangement of normal hepatocytes and cen-trilobular necrosis Also A S Bernacchi et al [46] reportedthat CCL4 leads to liver necrosis and fat accumulation in therabbit
The present study revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withLSS improved their liver histopathological architecture inthe form of less inflammation regenerating hepatocytes nosteatosis or isolated vesicular steatosis no chronic venouscongestion or just discrete congestion and no fibrosis henceapproving its hepatoprotective effectThe results are in agree-ment with Afaf et al [12] and Abdulrahman et al [20] whoobserved the improvement of the histological structure inextract treated group as the histopathologic findings showedless inflammation well restored hepatocytes and less area ofnecrosis as compared to severe necrosis and inflammationin CCl4 treated group of animals which also supports thehepatoprotective effects of LLS Their findings suggest thepossibility of the presence of anti-inflammatory compoundsin LSS extract which could also be a contributing factortowards the hepatoprotective action of LSS
Althnaian [47] also reported that examination of hep-atocytes showed mild to moderate degree of recovery LSSattenuated the hepatic fat accumulation decreased vacuolardegeneration and improved the architecture of hepatocytes
In our study the reducing effect of LSS on fatty changecaused by CCL4 intoxication is indicating the protective andcurative role against fatty liver Our results are supported byShukla et al [41] who reported that LSS significantly reducedsteatosis Their histopathological observations confirmed thecurative efficacy of L sativum liver damage Microvesicularfatty changes hepatocyte ballooning and steatosis weremarkedly corrected by LSS Also Wadhwa et al [36] foundthat the severe fatty changes in the livers of rats caused byCCl4 were decreased in the treated groups
In the present study chronic venous congestion featherydegeneration and fatty changes show mild improvement inrabbits receiving 200mgkg bw of LSS for 5 weeks and showmoderate improvement in rabbits that received 200mgkg bwof LSS for 10 weeks and rabbits that received 400mgkg bwof LSS for 5 weeks Our data are consistent with Afaf et al[12] who observed that severe centrilobular hepatocellularvacuolation hemorrhages and congestion of the centralveins noticed in CCL4 group showed mild improvement ingroups that received 200 mgkg bw of LSS and moderateimprovement in groups that received 400 mgkg bw ofLSS
On the other hand rabbits that received 400mgkg bw ofLSS for 10 weeks showed evidence of liver toxicity in the formof venous congestion hepatocyte feathery degeneration fattychange and fibrosis These results are compatible with Bafeeland Ali [48] who reported that LSS had minimal effect onliver parenchyma if given in low doses So LSS could be usedas food additive or supplement Mineral content of calciumand phosphate and the presence of essential fatty acids inconsiderable level make LSS of beneficial nutritional valuefor many body organs including the liver However LSS inhigh dose showed some toxic effect on renal parenchyma rep-resented by focal necrosis hepatocyte apoptosis and portalinflammatory cell infiltrate Burow et al [49] reported thatthese hazardous effects of LSS may be due to isothiocyanatesreported to be one of LSS constituents
5 Conclusions and Recommendations
The present study showed that LSS is a key factor ininhibition of the hazardous effects of CCl4 LSS treatmentcould significantly alleviate to a greater extent the degree oflipid peroxidation and restore the antioxidative enzymes andshift liver function to normal levels In addition to antioxi-dants activity LSS attenuated the hepatic fat accumulationdecreased vacuolar degeneration ameliorated the degree ofstructural damage in the hepatic cells and improved thearchitecture of hepatocytes The current findings demon-strated that LSS have marked hepatoprotective activity Infuture work active ingredients of LSS extract should bestudied in various animal models of severe toxicity inducedby CCL4 and other hepatotoxic agents The active ingredientsof this plant are likely to be applied as one of the future drugsagainst acute liver toxicity
Data Availability
Data used to support the findings of this study are availablefrom the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This project was funded by the Deanship of ScientificResearch (DSR) King Abdulaziz University Jeddah undergrant no S-17-314-37 The authors therefore acknowledgewith thanks DSR technical and financial support
References
[1] M K Manibusan M Odin and D A Eastmond ldquoPostu-lated carbon tetrachloride mode of action a reviewrdquo Journalof Environmental Science and Health Part C EnvironmentalCarcinogenesis and Ecotoxicology Reviews vol 25 no 3 pp 185ndash209 2007
[2] N Brautbar and J Williams II ldquoIndustrial solvents andliver toxicity risk assessment risk factors and mechanismsrdquo
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
10 Evidence-Based Complementary and Alternative Medicine
M 1 2 3 4 5 6 7 8 9bp
10000
3000
2000
1500
1000
750
500
300
Figure 5 Agarose gel showingDNAdamage byCCl4 and protectiveeffects of various fractions of LSS Lanes from left (M) molecularweight marker (1) negative control (2) LSS (200 mgkg bw) (3) LSS(400mgkg bw) (4) CCl4 for 5weeks (5) CCl4 for 10weeks (6)amp (7)CCl4 + LSS (200 mgkg bw) (8) amp (9) CCl4 + LSS (400 mg kg bw)
Examination of liver tissue of rabbits exposed to CCL4as well as 400 mgkg bw of LSS in their diet on daily basis(Group B3) reveals in the group that scarified 5 weeks afterexperimental running improvement of the histoarchitecturalchanges of the liver as there is no chronic venous congestionor only discrete chronic venous congestion and mild featherydegeneration no steatosis or only isolated vesicular steatosisand mild parenchymal and portal inflammation (Figures9(a) 9(b) and 9(c)) But the group that was sacrificed10 weeks after experimental running shows adverse effectsof the drug in the form of steatosis and chronic venouscongestion which is evident in two cases with mild tomoderate inflammation Bridging fibrosis is seen in one case(Figures 9(d) 9(e) 9(f) and 9(g))
4 Discussion
Hepatic disease became a significant public health concernowing to growing rate of obesity in all age populations [26]The conservative treatments of liver problems such as acuteand chronic liver hepatitis liver cirrhosis and fatty liverare often insufficient due to hazardous effects initiated byhepatotoxic drugs of chemical origin [27] Medicinal plantsowing to their abundant hepatoprotective natural ingredi-ents have been comprehensively investigated to treat liverdisorders In the current study hepatoprotective efficacy ofLSS was investigated via hepatotoxic classical model inducedby carbon tetrachloride
In organ and tissue systems CCl4 is stimulated bycytochrome P-450 dependent mixed oxidase in the hepaticendoplasmic reticulum to procedure free radical trichloro-methyl (CCl3 and Cl3COO) This causes changes in themembrane of the endoplasmic reticulum and other cel-lular membranes resulting in an increase in calcium ionpermeability through the plasma membrane causing severeinstabilities of calcium homeostasis leading to necrotic celldeath [28 29] CCL4 affects many organs causing degenera-tion of liver fatty layer and centrilobular necrosis [29] Thisphenomenon indicates that CCl4 is a powerful hepatotoxic
agent and may be used for the characterization of the hepaticdisease treatment medicines Elevation of liver enzymaticmarkers (ALT AST ALP and bilirubin) and increased lipidperoxidation are further evidences of hepatotoxicity ALTand AST that exist in the hepatocytes can certainly leak intothe peripheral blood as soon as the hepatocytes are injured[29] Lepidium sativum seed analysis by GCMS exhibitedthe presence of high concentrations of mainly 120572-linolenicacid (ALA) which is nminus3 fatty acid an omega-3 fatty acidfound in seeds exerting its bioactivity in ameliorating thehepatic intoxication and oxidative stress including liver injuryinduced by alcohol [30] liver steatosis [31 32] nonalcoholichepatic disease [33] and parenteral nutrition-associated liverdisease [34] Pari and Jalaludeen stated that orientationof the active groups in sinapic acid increases antioxidantefficiency as metal chelators and promotes its protectiveefficacy against liver oxidative stress in arsenic intoxicatedrats [35]The current study showed that sinapic acid as ametalchelator may exert its biological activity in improving liverfunction
Lepidium sativum seed extracts have proved hepatopro-tective effects against CCl4 induced liver damage A researchstudy on male Albino Wistar rats demonstrated significantreduction in hepatotoxicity induced by CCl4 administeredwith 200-400 mgkg of Lepidium sativum seeds extractincluded within the diet Serum level of AST ALT levels andbilirubin levels were significantly elevated in CCl4 treated ratscompared to those of normal control However a significantreduction in the levels of these parameters in Lepidiumsativum seed extracts protected rats was observed The hep-atoprotective effect could be due to the presence of alkaloidcoumarin flavonoids tannin and triterpenes which enhanceantioxidant activity and reduce free radical production fromCCl4 which is the basic triggering factor for hepatotoxicity[36 37]
In the present study we evaluated the hepatoprotectiveefficacy of L sativum seeds in white male New-Zealand rab-bits The present results revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withL sativum seeds significantly repaired their liver injuriousmarker enzymes as well as bilirubin total protein and albu-min hence approving its hepatoprotective effect The resultsare in agreement with those of Afaf et al [12] where theystated the role of LSS as efficient hepatoprotective agent Alsothe current results are consistent with those of Mohamedand his coworkers [38] who reported that preprotectionwith LSEE (Lepidium sativum ethanolic extract) significantlyprohibited the D-GalNLPS induced rise in hepatic enzymes(AST ALT 120574-GGT ALP total bilirubin LDH and total pro-tein) thus significantly alleviated the reduction of lipid per-oxidation and repaired the antioxidant enzymatic status andtotal protein restoring them to normal levels D-GalNLPShas been widely used to examine the mechanisms underlyingacute liver failure [39 40] The hepatoprotective significanceof LSEE could be due to downregulation of cytokines (TNF-120572 IL-6) and stress gene (iNOS and HO-1) mRNA expres-sion and upregulation of IL-10 Lepidium sativum ethanolicextract (LSEE) preprotection also improved the degree ofstructural damage and reduced inflammatory infiltration in
Evidence-Based Complementary and Alternative Medicine 11
(a) (b)
(c) (d)
(e)
Figure 6 Effect of LSS on histopathological changes of liver tissues of normal groups in rabbits (a) Showing normal histological picture ofhepatic lobule that consists of central vein surrounded by normal hepatocytes (HEx100) (b) Showing liver sections of the rabbits treatedwith LSS (200 mgkg bw) which revealed normal histological picture of hepatic lobule that consists of central vein surrounded by normalhepatocytes (HEx100) (c) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealed normal hepatic architecturewith only mild inflammation [arrows] (HEx100) (d) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealedevident chronic venous congestion and moderate inflammation [arrows] (HEx100) (e) Showing liver sections of the rabbits treated with LSS(400 mgkg bw) which revealed portal to portal bridging fibrosis [arrow] (HEx200)
hepatic cells These outcomes established that LSEE alle-viates hepatic impairments and structural injury throughthe decay of oxidative stress inflammation and apopto-sis in the liver [38] Mohamed Sakran and his colleagues[10] suggested that 56-dimethoxy-210158403-methylenedioxy-7-C-120573-D glucopyranosyl isoflavone extracted from Lepidiumsativum seeds could improve the lipid profile and the liverfunctions in serum and reduce the free radicals generationby enhancing the antioxidant defense mechanism Newsuggested isoflavonoid can be used as a probable antioxi-dant against paracetamol hepatotoxicity with its antioxidant
characteristics and could restore the hepatic functionstowards normalization [10]
In the present study we found that improved cholesteroland triglycerides levels in L sativum seeds protected rabbitsas compared to CCl4 treated rabbits also support the hepato-protective effect of LSSThese results can also be correlated tothose of Shukla et al [41] who reported the role of L sativumseeds as hypolipidemic agent Also the results of the presentstudy agreed with those of Abdulrahman et al [20] whoseresults showed that L sativum seeds have hepatoprotectiveactivity that could be due to its antioxidant activity together
12 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
Figure 7 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) In rabbits sacrificed 5 weeks afterexperimental running as shown in (a) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100)(b) steatosis (30) in one case (HEx100) and (c) mild parenchymal inflammation [arrow] (HEx200) In rabbits sacrificed 10 weeks afterexperimental running as shown in (d) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100) (e)steatosis (about 20) and moderate parenchymal and portal inflammation (HEx100) and (f) bridging fibrosis [arrow] (HEx100)
with the presence of anti-inflammatory ingredients in Lsativum seeds extract
The results of the current study revealed that the exposureto CCl4 for 5 and 10 weeks led to a significant decrease inthe enzymatic activities of CAT GPx GR GST and SOD inthe liver homogenate of CCl4 treated rabbits 5 and 10 weeksafter experimental running compared to those of the normalcontrolThe concurrent protection of rabbits exposed toCCl4for 5 and 10weeks with 200amp 400mg kg bw LSS significantlyrepaired their antioxidant status and reduced significantly theMDA levels hence confirming its hepatoprotective efficacy
Although the precise molecular mechanism of action ofLSS is not completely understood some researchers [42]have confirmed the role of LSS in inhibiting oxidative stress
associated liver damage The antioxidant activity of LSScould be due to the presence of phenolic compound andflavonoids in these seeds as have been evidenced through thepreliminary screening study on LSS extract using HPLCOurresults match with those of Lee et al [42] who reported thatphenolic compounds play an essential role against oxidativestress related liver damage
A unique expectation of the current study is to confirmthat CCl4 induces liver DNA injury that was assessed usinggel electrophoretic method The results of the current studyshowed that CCL4 induces DNA injury as presented bya significant progression in liver DNA disintegration ofCCl4 treated rabbits above untreated control in the gelelectrophoresis
Evidence-Based Complementary and Alternative Medicine 13
(a) (b)
(c) (d)
(e) (f)
Figure 8 Effect of LSLS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (200 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) evident chronic venous congestion and feathery degeneration [arrow](HEx100) (b) mild feathery degeneration and isolated vesicular steatosis [arrow] (HEx200) and (c) moderate parenchymal and portalinflammation (HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) regenerating hepatocytes with isolatedvesicular steatosis (HEx100) (e) discrete chronic venous congestion and no steatosis (HEx400) and (f) discrete inflammatory infiltrate(HEx100)
Lipid peroxidation of the nuclear membrane may bedirectly accompanied with nuclear DNA damage [43]Nuclear DNA is concomitant with particular regions of thenuclear membrane [44]The close proximity of nuclear DNAto the nuclear membrane can ease the interaction of genomicDNA with the peroxyl radicals and further reactive inter-mediates formed during membrane lipid peroxidation [43]In the presence of transition metals the hydroxyl radicalsgenerated by CCL4 autoxidation can trigger peroxidationof the nuclear membrane lipids by metal catalyzed Haber-Weiss reaction the lipid peroxides levels and intermediate
free radicals could then be augmented by a chain reactionSubsequently the reactive species oxidize nuclearDNAwhenthe lipid peroxidation reaction occurs in the nuclear mem-brane since the nuclear membrane controls the transportof messenger RNA into the cytoplasm and supports thenuclear division process nuclear membrane peroxidationof lipids and proteins Moreover oxidative nuclear DNAdamage could disrupt these serious cellular functions [43]
DNA findings showed that DNA disintegration is adirect result after CCl4 treatment in rabbitsrsquo liver tissueswhich was reduced by participation of antioxidants through
14 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
(g)
Figure 9 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (400 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) discrete chronic venous congestion (HEx200) (b) centrilobular fibrosis[arrows] regenerating hepatocytes and isolated vesicular steatosis (HEx100) and (c) mild parenchymal and portal inflammation [arrow](HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) mild chronic venous congestion and isolated vesicularsteatosis (HEx100) (e) evident chronic venous congestion [arrows] (HEx100) (f) steatosis (40) with moderate inflammation (HEx100) and(g) portal to portal bridging fibrosis in one case [arrow] (HEx100)
Evidence-Based Complementary and Alternative Medicine 15
administration of 200 mg amp 400 mgkg bw L sativum seedsNearly complete repair of DNA damage was significant aftertreating rabbits with 200 mg amp 400 mgkg bw L sativumseeds The protective effect of L sativum seeds on hepaticDNA injury may be due to the antioxidant activity ofL sativum seeds via direct scavenging of free radicals orinterfering with free radicals generation
Concerning histopathological evaluation in our studyrabbitsrsquo livers exposed to CCL4 show portal and parenchymalinflammatory infiltrate feathery and ballooning degenera-tion fatty change and fibrosis Our data are consistent withRajeswary et al [45] who reported dilatation of sinusoidsfatty change disarrangement of normal hepatocytes and cen-trilobular necrosis Also A S Bernacchi et al [46] reportedthat CCL4 leads to liver necrosis and fat accumulation in therabbit
The present study revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withLSS improved their liver histopathological architecture inthe form of less inflammation regenerating hepatocytes nosteatosis or isolated vesicular steatosis no chronic venouscongestion or just discrete congestion and no fibrosis henceapproving its hepatoprotective effectThe results are in agree-ment with Afaf et al [12] and Abdulrahman et al [20] whoobserved the improvement of the histological structure inextract treated group as the histopathologic findings showedless inflammation well restored hepatocytes and less area ofnecrosis as compared to severe necrosis and inflammationin CCl4 treated group of animals which also supports thehepatoprotective effects of LLS Their findings suggest thepossibility of the presence of anti-inflammatory compoundsin LSS extract which could also be a contributing factortowards the hepatoprotective action of LSS
Althnaian [47] also reported that examination of hep-atocytes showed mild to moderate degree of recovery LSSattenuated the hepatic fat accumulation decreased vacuolardegeneration and improved the architecture of hepatocytes
In our study the reducing effect of LSS on fatty changecaused by CCL4 intoxication is indicating the protective andcurative role against fatty liver Our results are supported byShukla et al [41] who reported that LSS significantly reducedsteatosis Their histopathological observations confirmed thecurative efficacy of L sativum liver damage Microvesicularfatty changes hepatocyte ballooning and steatosis weremarkedly corrected by LSS Also Wadhwa et al [36] foundthat the severe fatty changes in the livers of rats caused byCCl4 were decreased in the treated groups
In the present study chronic venous congestion featherydegeneration and fatty changes show mild improvement inrabbits receiving 200mgkg bw of LSS for 5 weeks and showmoderate improvement in rabbits that received 200mgkg bwof LSS for 10 weeks and rabbits that received 400mgkg bwof LSS for 5 weeks Our data are consistent with Afaf et al[12] who observed that severe centrilobular hepatocellularvacuolation hemorrhages and congestion of the centralveins noticed in CCL4 group showed mild improvement ingroups that received 200 mgkg bw of LSS and moderateimprovement in groups that received 400 mgkg bw ofLSS
On the other hand rabbits that received 400mgkg bw ofLSS for 10 weeks showed evidence of liver toxicity in the formof venous congestion hepatocyte feathery degeneration fattychange and fibrosis These results are compatible with Bafeeland Ali [48] who reported that LSS had minimal effect onliver parenchyma if given in low doses So LSS could be usedas food additive or supplement Mineral content of calciumand phosphate and the presence of essential fatty acids inconsiderable level make LSS of beneficial nutritional valuefor many body organs including the liver However LSS inhigh dose showed some toxic effect on renal parenchyma rep-resented by focal necrosis hepatocyte apoptosis and portalinflammatory cell infiltrate Burow et al [49] reported thatthese hazardous effects of LSS may be due to isothiocyanatesreported to be one of LSS constituents
5 Conclusions and Recommendations
The present study showed that LSS is a key factor ininhibition of the hazardous effects of CCl4 LSS treatmentcould significantly alleviate to a greater extent the degree oflipid peroxidation and restore the antioxidative enzymes andshift liver function to normal levels In addition to antioxi-dants activity LSS attenuated the hepatic fat accumulationdecreased vacuolar degeneration ameliorated the degree ofstructural damage in the hepatic cells and improved thearchitecture of hepatocytes The current findings demon-strated that LSS have marked hepatoprotective activity Infuture work active ingredients of LSS extract should bestudied in various animal models of severe toxicity inducedby CCL4 and other hepatotoxic agents The active ingredientsof this plant are likely to be applied as one of the future drugsagainst acute liver toxicity
Data Availability
Data used to support the findings of this study are availablefrom the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This project was funded by the Deanship of ScientificResearch (DSR) King Abdulaziz University Jeddah undergrant no S-17-314-37 The authors therefore acknowledgewith thanks DSR technical and financial support
References
[1] M K Manibusan M Odin and D A Eastmond ldquoPostu-lated carbon tetrachloride mode of action a reviewrdquo Journalof Environmental Science and Health Part C EnvironmentalCarcinogenesis and Ecotoxicology Reviews vol 25 no 3 pp 185ndash209 2007
[2] N Brautbar and J Williams II ldquoIndustrial solvents andliver toxicity risk assessment risk factors and mechanismsrdquo
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
Stem Cells International
Hindawiwwwhindawicom Volume 2018
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MEDIATORSINFLAMMATION
of
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Hindawiwwwhindawicom Volume 2018
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Disease Markers
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OncologyJournal of
Hindawiwwwhindawicom Volume 2013
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Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
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Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
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Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 11
(a) (b)
(c) (d)
(e)
Figure 6 Effect of LSS on histopathological changes of liver tissues of normal groups in rabbits (a) Showing normal histological picture ofhepatic lobule that consists of central vein surrounded by normal hepatocytes (HEx100) (b) Showing liver sections of the rabbits treatedwith LSS (200 mgkg bw) which revealed normal histological picture of hepatic lobule that consists of central vein surrounded by normalhepatocytes (HEx100) (c) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealed normal hepatic architecturewith only mild inflammation [arrows] (HEx100) (d) Showing liver sections of the rabbits treated with LSS (400 mgkg bw) which revealedevident chronic venous congestion and moderate inflammation [arrows] (HEx100) (e) Showing liver sections of the rabbits treated with LSS(400 mgkg bw) which revealed portal to portal bridging fibrosis [arrow] (HEx200)
hepatic cells These outcomes established that LSEE alle-viates hepatic impairments and structural injury throughthe decay of oxidative stress inflammation and apopto-sis in the liver [38] Mohamed Sakran and his colleagues[10] suggested that 56-dimethoxy-210158403-methylenedioxy-7-C-120573-D glucopyranosyl isoflavone extracted from Lepidiumsativum seeds could improve the lipid profile and the liverfunctions in serum and reduce the free radicals generationby enhancing the antioxidant defense mechanism Newsuggested isoflavonoid can be used as a probable antioxi-dant against paracetamol hepatotoxicity with its antioxidant
characteristics and could restore the hepatic functionstowards normalization [10]
In the present study we found that improved cholesteroland triglycerides levels in L sativum seeds protected rabbitsas compared to CCl4 treated rabbits also support the hepato-protective effect of LSSThese results can also be correlated tothose of Shukla et al [41] who reported the role of L sativumseeds as hypolipidemic agent Also the results of the presentstudy agreed with those of Abdulrahman et al [20] whoseresults showed that L sativum seeds have hepatoprotectiveactivity that could be due to its antioxidant activity together
12 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
Figure 7 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) In rabbits sacrificed 5 weeks afterexperimental running as shown in (a) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100)(b) steatosis (30) in one case (HEx100) and (c) mild parenchymal inflammation [arrow] (HEx200) In rabbits sacrificed 10 weeks afterexperimental running as shown in (d) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100) (e)steatosis (about 20) and moderate parenchymal and portal inflammation (HEx100) and (f) bridging fibrosis [arrow] (HEx100)
with the presence of anti-inflammatory ingredients in Lsativum seeds extract
The results of the current study revealed that the exposureto CCl4 for 5 and 10 weeks led to a significant decrease inthe enzymatic activities of CAT GPx GR GST and SOD inthe liver homogenate of CCl4 treated rabbits 5 and 10 weeksafter experimental running compared to those of the normalcontrolThe concurrent protection of rabbits exposed toCCl4for 5 and 10weeks with 200amp 400mg kg bw LSS significantlyrepaired their antioxidant status and reduced significantly theMDA levels hence confirming its hepatoprotective efficacy
Although the precise molecular mechanism of action ofLSS is not completely understood some researchers [42]have confirmed the role of LSS in inhibiting oxidative stress
associated liver damage The antioxidant activity of LSScould be due to the presence of phenolic compound andflavonoids in these seeds as have been evidenced through thepreliminary screening study on LSS extract using HPLCOurresults match with those of Lee et al [42] who reported thatphenolic compounds play an essential role against oxidativestress related liver damage
A unique expectation of the current study is to confirmthat CCl4 induces liver DNA injury that was assessed usinggel electrophoretic method The results of the current studyshowed that CCL4 induces DNA injury as presented bya significant progression in liver DNA disintegration ofCCl4 treated rabbits above untreated control in the gelelectrophoresis
Evidence-Based Complementary and Alternative Medicine 13
(a) (b)
(c) (d)
(e) (f)
Figure 8 Effect of LSLS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (200 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) evident chronic venous congestion and feathery degeneration [arrow](HEx100) (b) mild feathery degeneration and isolated vesicular steatosis [arrow] (HEx200) and (c) moderate parenchymal and portalinflammation (HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) regenerating hepatocytes with isolatedvesicular steatosis (HEx100) (e) discrete chronic venous congestion and no steatosis (HEx400) and (f) discrete inflammatory infiltrate(HEx100)
Lipid peroxidation of the nuclear membrane may bedirectly accompanied with nuclear DNA damage [43]Nuclear DNA is concomitant with particular regions of thenuclear membrane [44]The close proximity of nuclear DNAto the nuclear membrane can ease the interaction of genomicDNA with the peroxyl radicals and further reactive inter-mediates formed during membrane lipid peroxidation [43]In the presence of transition metals the hydroxyl radicalsgenerated by CCL4 autoxidation can trigger peroxidationof the nuclear membrane lipids by metal catalyzed Haber-Weiss reaction the lipid peroxides levels and intermediate
free radicals could then be augmented by a chain reactionSubsequently the reactive species oxidize nuclearDNAwhenthe lipid peroxidation reaction occurs in the nuclear mem-brane since the nuclear membrane controls the transportof messenger RNA into the cytoplasm and supports thenuclear division process nuclear membrane peroxidationof lipids and proteins Moreover oxidative nuclear DNAdamage could disrupt these serious cellular functions [43]
DNA findings showed that DNA disintegration is adirect result after CCl4 treatment in rabbitsrsquo liver tissueswhich was reduced by participation of antioxidants through
14 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
(g)
Figure 9 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (400 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) discrete chronic venous congestion (HEx200) (b) centrilobular fibrosis[arrows] regenerating hepatocytes and isolated vesicular steatosis (HEx100) and (c) mild parenchymal and portal inflammation [arrow](HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) mild chronic venous congestion and isolated vesicularsteatosis (HEx100) (e) evident chronic venous congestion [arrows] (HEx100) (f) steatosis (40) with moderate inflammation (HEx100) and(g) portal to portal bridging fibrosis in one case [arrow] (HEx100)
Evidence-Based Complementary and Alternative Medicine 15
administration of 200 mg amp 400 mgkg bw L sativum seedsNearly complete repair of DNA damage was significant aftertreating rabbits with 200 mg amp 400 mgkg bw L sativumseeds The protective effect of L sativum seeds on hepaticDNA injury may be due to the antioxidant activity ofL sativum seeds via direct scavenging of free radicals orinterfering with free radicals generation
Concerning histopathological evaluation in our studyrabbitsrsquo livers exposed to CCL4 show portal and parenchymalinflammatory infiltrate feathery and ballooning degenera-tion fatty change and fibrosis Our data are consistent withRajeswary et al [45] who reported dilatation of sinusoidsfatty change disarrangement of normal hepatocytes and cen-trilobular necrosis Also A S Bernacchi et al [46] reportedthat CCL4 leads to liver necrosis and fat accumulation in therabbit
The present study revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withLSS improved their liver histopathological architecture inthe form of less inflammation regenerating hepatocytes nosteatosis or isolated vesicular steatosis no chronic venouscongestion or just discrete congestion and no fibrosis henceapproving its hepatoprotective effectThe results are in agree-ment with Afaf et al [12] and Abdulrahman et al [20] whoobserved the improvement of the histological structure inextract treated group as the histopathologic findings showedless inflammation well restored hepatocytes and less area ofnecrosis as compared to severe necrosis and inflammationin CCl4 treated group of animals which also supports thehepatoprotective effects of LLS Their findings suggest thepossibility of the presence of anti-inflammatory compoundsin LSS extract which could also be a contributing factortowards the hepatoprotective action of LSS
Althnaian [47] also reported that examination of hep-atocytes showed mild to moderate degree of recovery LSSattenuated the hepatic fat accumulation decreased vacuolardegeneration and improved the architecture of hepatocytes
In our study the reducing effect of LSS on fatty changecaused by CCL4 intoxication is indicating the protective andcurative role against fatty liver Our results are supported byShukla et al [41] who reported that LSS significantly reducedsteatosis Their histopathological observations confirmed thecurative efficacy of L sativum liver damage Microvesicularfatty changes hepatocyte ballooning and steatosis weremarkedly corrected by LSS Also Wadhwa et al [36] foundthat the severe fatty changes in the livers of rats caused byCCl4 were decreased in the treated groups
In the present study chronic venous congestion featherydegeneration and fatty changes show mild improvement inrabbits receiving 200mgkg bw of LSS for 5 weeks and showmoderate improvement in rabbits that received 200mgkg bwof LSS for 10 weeks and rabbits that received 400mgkg bwof LSS for 5 weeks Our data are consistent with Afaf et al[12] who observed that severe centrilobular hepatocellularvacuolation hemorrhages and congestion of the centralveins noticed in CCL4 group showed mild improvement ingroups that received 200 mgkg bw of LSS and moderateimprovement in groups that received 400 mgkg bw ofLSS
On the other hand rabbits that received 400mgkg bw ofLSS for 10 weeks showed evidence of liver toxicity in the formof venous congestion hepatocyte feathery degeneration fattychange and fibrosis These results are compatible with Bafeeland Ali [48] who reported that LSS had minimal effect onliver parenchyma if given in low doses So LSS could be usedas food additive or supplement Mineral content of calciumand phosphate and the presence of essential fatty acids inconsiderable level make LSS of beneficial nutritional valuefor many body organs including the liver However LSS inhigh dose showed some toxic effect on renal parenchyma rep-resented by focal necrosis hepatocyte apoptosis and portalinflammatory cell infiltrate Burow et al [49] reported thatthese hazardous effects of LSS may be due to isothiocyanatesreported to be one of LSS constituents
5 Conclusions and Recommendations
The present study showed that LSS is a key factor ininhibition of the hazardous effects of CCl4 LSS treatmentcould significantly alleviate to a greater extent the degree oflipid peroxidation and restore the antioxidative enzymes andshift liver function to normal levels In addition to antioxi-dants activity LSS attenuated the hepatic fat accumulationdecreased vacuolar degeneration ameliorated the degree ofstructural damage in the hepatic cells and improved thearchitecture of hepatocytes The current findings demon-strated that LSS have marked hepatoprotective activity Infuture work active ingredients of LSS extract should bestudied in various animal models of severe toxicity inducedby CCL4 and other hepatotoxic agents The active ingredientsof this plant are likely to be applied as one of the future drugsagainst acute liver toxicity
Data Availability
Data used to support the findings of this study are availablefrom the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This project was funded by the Deanship of ScientificResearch (DSR) King Abdulaziz University Jeddah undergrant no S-17-314-37 The authors therefore acknowledgewith thanks DSR technical and financial support
References
[1] M K Manibusan M Odin and D A Eastmond ldquoPostu-lated carbon tetrachloride mode of action a reviewrdquo Journalof Environmental Science and Health Part C EnvironmentalCarcinogenesis and Ecotoxicology Reviews vol 25 no 3 pp 185ndash209 2007
[2] N Brautbar and J Williams II ldquoIndustrial solvents andliver toxicity risk assessment risk factors and mechanismsrdquo
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
Stem Cells International
Hindawiwwwhindawicom Volume 2018
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Hindawiwwwhindawicom Volume 2018
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Disease Markers
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Oxidative Medicine and Cellular Longevity
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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
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Journal of
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Computational and Mathematical Methods in Medicine
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Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
12 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
Figure 7 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) In rabbits sacrificed 5 weeks afterexperimental running as shown in (a) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100)(b) steatosis (30) in one case (HEx100) and (c) mild parenchymal inflammation [arrow] (HEx200) In rabbits sacrificed 10 weeks afterexperimental running as shown in (d) evident chronic venous congestion and feathery degeneration of hepatocytes [arrows] (HEx100) (e)steatosis (about 20) and moderate parenchymal and portal inflammation (HEx100) and (f) bridging fibrosis [arrow] (HEx100)
with the presence of anti-inflammatory ingredients in Lsativum seeds extract
The results of the current study revealed that the exposureto CCl4 for 5 and 10 weeks led to a significant decrease inthe enzymatic activities of CAT GPx GR GST and SOD inthe liver homogenate of CCl4 treated rabbits 5 and 10 weeksafter experimental running compared to those of the normalcontrolThe concurrent protection of rabbits exposed toCCl4for 5 and 10weeks with 200amp 400mg kg bw LSS significantlyrepaired their antioxidant status and reduced significantly theMDA levels hence confirming its hepatoprotective efficacy
Although the precise molecular mechanism of action ofLSS is not completely understood some researchers [42]have confirmed the role of LSS in inhibiting oxidative stress
associated liver damage The antioxidant activity of LSScould be due to the presence of phenolic compound andflavonoids in these seeds as have been evidenced through thepreliminary screening study on LSS extract using HPLCOurresults match with those of Lee et al [42] who reported thatphenolic compounds play an essential role against oxidativestress related liver damage
A unique expectation of the current study is to confirmthat CCl4 induces liver DNA injury that was assessed usinggel electrophoretic method The results of the current studyshowed that CCL4 induces DNA injury as presented bya significant progression in liver DNA disintegration ofCCl4 treated rabbits above untreated control in the gelelectrophoresis
Evidence-Based Complementary and Alternative Medicine 13
(a) (b)
(c) (d)
(e) (f)
Figure 8 Effect of LSLS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (200 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) evident chronic venous congestion and feathery degeneration [arrow](HEx100) (b) mild feathery degeneration and isolated vesicular steatosis [arrow] (HEx200) and (c) moderate parenchymal and portalinflammation (HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) regenerating hepatocytes with isolatedvesicular steatosis (HEx100) (e) discrete chronic venous congestion and no steatosis (HEx400) and (f) discrete inflammatory infiltrate(HEx100)
Lipid peroxidation of the nuclear membrane may bedirectly accompanied with nuclear DNA damage [43]Nuclear DNA is concomitant with particular regions of thenuclear membrane [44]The close proximity of nuclear DNAto the nuclear membrane can ease the interaction of genomicDNA with the peroxyl radicals and further reactive inter-mediates formed during membrane lipid peroxidation [43]In the presence of transition metals the hydroxyl radicalsgenerated by CCL4 autoxidation can trigger peroxidationof the nuclear membrane lipids by metal catalyzed Haber-Weiss reaction the lipid peroxides levels and intermediate
free radicals could then be augmented by a chain reactionSubsequently the reactive species oxidize nuclearDNAwhenthe lipid peroxidation reaction occurs in the nuclear mem-brane since the nuclear membrane controls the transportof messenger RNA into the cytoplasm and supports thenuclear division process nuclear membrane peroxidationof lipids and proteins Moreover oxidative nuclear DNAdamage could disrupt these serious cellular functions [43]
DNA findings showed that DNA disintegration is adirect result after CCl4 treatment in rabbitsrsquo liver tissueswhich was reduced by participation of antioxidants through
14 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
(g)
Figure 9 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (400 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) discrete chronic venous congestion (HEx200) (b) centrilobular fibrosis[arrows] regenerating hepatocytes and isolated vesicular steatosis (HEx100) and (c) mild parenchymal and portal inflammation [arrow](HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) mild chronic venous congestion and isolated vesicularsteatosis (HEx100) (e) evident chronic venous congestion [arrows] (HEx100) (f) steatosis (40) with moderate inflammation (HEx100) and(g) portal to portal bridging fibrosis in one case [arrow] (HEx100)
Evidence-Based Complementary and Alternative Medicine 15
administration of 200 mg amp 400 mgkg bw L sativum seedsNearly complete repair of DNA damage was significant aftertreating rabbits with 200 mg amp 400 mgkg bw L sativumseeds The protective effect of L sativum seeds on hepaticDNA injury may be due to the antioxidant activity ofL sativum seeds via direct scavenging of free radicals orinterfering with free radicals generation
Concerning histopathological evaluation in our studyrabbitsrsquo livers exposed to CCL4 show portal and parenchymalinflammatory infiltrate feathery and ballooning degenera-tion fatty change and fibrosis Our data are consistent withRajeswary et al [45] who reported dilatation of sinusoidsfatty change disarrangement of normal hepatocytes and cen-trilobular necrosis Also A S Bernacchi et al [46] reportedthat CCL4 leads to liver necrosis and fat accumulation in therabbit
The present study revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withLSS improved their liver histopathological architecture inthe form of less inflammation regenerating hepatocytes nosteatosis or isolated vesicular steatosis no chronic venouscongestion or just discrete congestion and no fibrosis henceapproving its hepatoprotective effectThe results are in agree-ment with Afaf et al [12] and Abdulrahman et al [20] whoobserved the improvement of the histological structure inextract treated group as the histopathologic findings showedless inflammation well restored hepatocytes and less area ofnecrosis as compared to severe necrosis and inflammationin CCl4 treated group of animals which also supports thehepatoprotective effects of LLS Their findings suggest thepossibility of the presence of anti-inflammatory compoundsin LSS extract which could also be a contributing factortowards the hepatoprotective action of LSS
Althnaian [47] also reported that examination of hep-atocytes showed mild to moderate degree of recovery LSSattenuated the hepatic fat accumulation decreased vacuolardegeneration and improved the architecture of hepatocytes
In our study the reducing effect of LSS on fatty changecaused by CCL4 intoxication is indicating the protective andcurative role against fatty liver Our results are supported byShukla et al [41] who reported that LSS significantly reducedsteatosis Their histopathological observations confirmed thecurative efficacy of L sativum liver damage Microvesicularfatty changes hepatocyte ballooning and steatosis weremarkedly corrected by LSS Also Wadhwa et al [36] foundthat the severe fatty changes in the livers of rats caused byCCl4 were decreased in the treated groups
In the present study chronic venous congestion featherydegeneration and fatty changes show mild improvement inrabbits receiving 200mgkg bw of LSS for 5 weeks and showmoderate improvement in rabbits that received 200mgkg bwof LSS for 10 weeks and rabbits that received 400mgkg bwof LSS for 5 weeks Our data are consistent with Afaf et al[12] who observed that severe centrilobular hepatocellularvacuolation hemorrhages and congestion of the centralveins noticed in CCL4 group showed mild improvement ingroups that received 200 mgkg bw of LSS and moderateimprovement in groups that received 400 mgkg bw ofLSS
On the other hand rabbits that received 400mgkg bw ofLSS for 10 weeks showed evidence of liver toxicity in the formof venous congestion hepatocyte feathery degeneration fattychange and fibrosis These results are compatible with Bafeeland Ali [48] who reported that LSS had minimal effect onliver parenchyma if given in low doses So LSS could be usedas food additive or supplement Mineral content of calciumand phosphate and the presence of essential fatty acids inconsiderable level make LSS of beneficial nutritional valuefor many body organs including the liver However LSS inhigh dose showed some toxic effect on renal parenchyma rep-resented by focal necrosis hepatocyte apoptosis and portalinflammatory cell infiltrate Burow et al [49] reported thatthese hazardous effects of LSS may be due to isothiocyanatesreported to be one of LSS constituents
5 Conclusions and Recommendations
The present study showed that LSS is a key factor ininhibition of the hazardous effects of CCl4 LSS treatmentcould significantly alleviate to a greater extent the degree oflipid peroxidation and restore the antioxidative enzymes andshift liver function to normal levels In addition to antioxi-dants activity LSS attenuated the hepatic fat accumulationdecreased vacuolar degeneration ameliorated the degree ofstructural damage in the hepatic cells and improved thearchitecture of hepatocytes The current findings demon-strated that LSS have marked hepatoprotective activity Infuture work active ingredients of LSS extract should bestudied in various animal models of severe toxicity inducedby CCL4 and other hepatotoxic agents The active ingredientsof this plant are likely to be applied as one of the future drugsagainst acute liver toxicity
Data Availability
Data used to support the findings of this study are availablefrom the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This project was funded by the Deanship of ScientificResearch (DSR) King Abdulaziz University Jeddah undergrant no S-17-314-37 The authors therefore acknowledgewith thanks DSR technical and financial support
References
[1] M K Manibusan M Odin and D A Eastmond ldquoPostu-lated carbon tetrachloride mode of action a reviewrdquo Journalof Environmental Science and Health Part C EnvironmentalCarcinogenesis and Ecotoxicology Reviews vol 25 no 3 pp 185ndash209 2007
[2] N Brautbar and J Williams II ldquoIndustrial solvents andliver toxicity risk assessment risk factors and mechanismsrdquo
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 13
(a) (b)
(c) (d)
(e) (f)
Figure 8 Effect of LSLS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (200 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) evident chronic venous congestion and feathery degeneration [arrow](HEx100) (b) mild feathery degeneration and isolated vesicular steatosis [arrow] (HEx200) and (c) moderate parenchymal and portalinflammation (HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) regenerating hepatocytes with isolatedvesicular steatosis (HEx100) (e) discrete chronic venous congestion and no steatosis (HEx400) and (f) discrete inflammatory infiltrate(HEx100)
Lipid peroxidation of the nuclear membrane may bedirectly accompanied with nuclear DNA damage [43]Nuclear DNA is concomitant with particular regions of thenuclear membrane [44]The close proximity of nuclear DNAto the nuclear membrane can ease the interaction of genomicDNA with the peroxyl radicals and further reactive inter-mediates formed during membrane lipid peroxidation [43]In the presence of transition metals the hydroxyl radicalsgenerated by CCL4 autoxidation can trigger peroxidationof the nuclear membrane lipids by metal catalyzed Haber-Weiss reaction the lipid peroxides levels and intermediate
free radicals could then be augmented by a chain reactionSubsequently the reactive species oxidize nuclearDNAwhenthe lipid peroxidation reaction occurs in the nuclear mem-brane since the nuclear membrane controls the transportof messenger RNA into the cytoplasm and supports thenuclear division process nuclear membrane peroxidationof lipids and proteins Moreover oxidative nuclear DNAdamage could disrupt these serious cellular functions [43]
DNA findings showed that DNA disintegration is adirect result after CCl4 treatment in rabbitsrsquo liver tissueswhich was reduced by participation of antioxidants through
14 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
(g)
Figure 9 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (400 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) discrete chronic venous congestion (HEx200) (b) centrilobular fibrosis[arrows] regenerating hepatocytes and isolated vesicular steatosis (HEx100) and (c) mild parenchymal and portal inflammation [arrow](HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) mild chronic venous congestion and isolated vesicularsteatosis (HEx100) (e) evident chronic venous congestion [arrows] (HEx100) (f) steatosis (40) with moderate inflammation (HEx100) and(g) portal to portal bridging fibrosis in one case [arrow] (HEx100)
Evidence-Based Complementary and Alternative Medicine 15
administration of 200 mg amp 400 mgkg bw L sativum seedsNearly complete repair of DNA damage was significant aftertreating rabbits with 200 mg amp 400 mgkg bw L sativumseeds The protective effect of L sativum seeds on hepaticDNA injury may be due to the antioxidant activity ofL sativum seeds via direct scavenging of free radicals orinterfering with free radicals generation
Concerning histopathological evaluation in our studyrabbitsrsquo livers exposed to CCL4 show portal and parenchymalinflammatory infiltrate feathery and ballooning degenera-tion fatty change and fibrosis Our data are consistent withRajeswary et al [45] who reported dilatation of sinusoidsfatty change disarrangement of normal hepatocytes and cen-trilobular necrosis Also A S Bernacchi et al [46] reportedthat CCL4 leads to liver necrosis and fat accumulation in therabbit
The present study revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withLSS improved their liver histopathological architecture inthe form of less inflammation regenerating hepatocytes nosteatosis or isolated vesicular steatosis no chronic venouscongestion or just discrete congestion and no fibrosis henceapproving its hepatoprotective effectThe results are in agree-ment with Afaf et al [12] and Abdulrahman et al [20] whoobserved the improvement of the histological structure inextract treated group as the histopathologic findings showedless inflammation well restored hepatocytes and less area ofnecrosis as compared to severe necrosis and inflammationin CCl4 treated group of animals which also supports thehepatoprotective effects of LLS Their findings suggest thepossibility of the presence of anti-inflammatory compoundsin LSS extract which could also be a contributing factortowards the hepatoprotective action of LSS
Althnaian [47] also reported that examination of hep-atocytes showed mild to moderate degree of recovery LSSattenuated the hepatic fat accumulation decreased vacuolardegeneration and improved the architecture of hepatocytes
In our study the reducing effect of LSS on fatty changecaused by CCL4 intoxication is indicating the protective andcurative role against fatty liver Our results are supported byShukla et al [41] who reported that LSS significantly reducedsteatosis Their histopathological observations confirmed thecurative efficacy of L sativum liver damage Microvesicularfatty changes hepatocyte ballooning and steatosis weremarkedly corrected by LSS Also Wadhwa et al [36] foundthat the severe fatty changes in the livers of rats caused byCCl4 were decreased in the treated groups
In the present study chronic venous congestion featherydegeneration and fatty changes show mild improvement inrabbits receiving 200mgkg bw of LSS for 5 weeks and showmoderate improvement in rabbits that received 200mgkg bwof LSS for 10 weeks and rabbits that received 400mgkg bwof LSS for 5 weeks Our data are consistent with Afaf et al[12] who observed that severe centrilobular hepatocellularvacuolation hemorrhages and congestion of the centralveins noticed in CCL4 group showed mild improvement ingroups that received 200 mgkg bw of LSS and moderateimprovement in groups that received 400 mgkg bw ofLSS
On the other hand rabbits that received 400mgkg bw ofLSS for 10 weeks showed evidence of liver toxicity in the formof venous congestion hepatocyte feathery degeneration fattychange and fibrosis These results are compatible with Bafeeland Ali [48] who reported that LSS had minimal effect onliver parenchyma if given in low doses So LSS could be usedas food additive or supplement Mineral content of calciumand phosphate and the presence of essential fatty acids inconsiderable level make LSS of beneficial nutritional valuefor many body organs including the liver However LSS inhigh dose showed some toxic effect on renal parenchyma rep-resented by focal necrosis hepatocyte apoptosis and portalinflammatory cell infiltrate Burow et al [49] reported thatthese hazardous effects of LSS may be due to isothiocyanatesreported to be one of LSS constituents
5 Conclusions and Recommendations
The present study showed that LSS is a key factor ininhibition of the hazardous effects of CCl4 LSS treatmentcould significantly alleviate to a greater extent the degree oflipid peroxidation and restore the antioxidative enzymes andshift liver function to normal levels In addition to antioxi-dants activity LSS attenuated the hepatic fat accumulationdecreased vacuolar degeneration ameliorated the degree ofstructural damage in the hepatic cells and improved thearchitecture of hepatocytes The current findings demon-strated that LSS have marked hepatoprotective activity Infuture work active ingredients of LSS extract should bestudied in various animal models of severe toxicity inducedby CCL4 and other hepatotoxic agents The active ingredientsof this plant are likely to be applied as one of the future drugsagainst acute liver toxicity
Data Availability
Data used to support the findings of this study are availablefrom the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This project was funded by the Deanship of ScientificResearch (DSR) King Abdulaziz University Jeddah undergrant no S-17-314-37 The authors therefore acknowledgewith thanks DSR technical and financial support
References
[1] M K Manibusan M Odin and D A Eastmond ldquoPostu-lated carbon tetrachloride mode of action a reviewrdquo Journalof Environmental Science and Health Part C EnvironmentalCarcinogenesis and Ecotoxicology Reviews vol 25 no 3 pp 185ndash209 2007
[2] N Brautbar and J Williams II ldquoIndustrial solvents andliver toxicity risk assessment risk factors and mechanismsrdquo
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
14 Evidence-Based Complementary and Alternative Medicine
(a) (b)
(c) (d)
(e) (f)
(g)
Figure 9 Effect of LSS on histopathological changes of liver tissues induced by CCL4 (05 mLkg ip) and treated with LSS (400 mgskg bw) Inrabbits sacrificed 5 weeks after experimental running as shown in (a) discrete chronic venous congestion (HEx200) (b) centrilobular fibrosis[arrows] regenerating hepatocytes and isolated vesicular steatosis (HEx100) and (c) mild parenchymal and portal inflammation [arrow](HEx100) In rabbits sacrificed 10 weeks after experimental running as shown in (d) mild chronic venous congestion and isolated vesicularsteatosis (HEx100) (e) evident chronic venous congestion [arrows] (HEx100) (f) steatosis (40) with moderate inflammation (HEx100) and(g) portal to portal bridging fibrosis in one case [arrow] (HEx100)
Evidence-Based Complementary and Alternative Medicine 15
administration of 200 mg amp 400 mgkg bw L sativum seedsNearly complete repair of DNA damage was significant aftertreating rabbits with 200 mg amp 400 mgkg bw L sativumseeds The protective effect of L sativum seeds on hepaticDNA injury may be due to the antioxidant activity ofL sativum seeds via direct scavenging of free radicals orinterfering with free radicals generation
Concerning histopathological evaluation in our studyrabbitsrsquo livers exposed to CCL4 show portal and parenchymalinflammatory infiltrate feathery and ballooning degenera-tion fatty change and fibrosis Our data are consistent withRajeswary et al [45] who reported dilatation of sinusoidsfatty change disarrangement of normal hepatocytes and cen-trilobular necrosis Also A S Bernacchi et al [46] reportedthat CCL4 leads to liver necrosis and fat accumulation in therabbit
The present study revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withLSS improved their liver histopathological architecture inthe form of less inflammation regenerating hepatocytes nosteatosis or isolated vesicular steatosis no chronic venouscongestion or just discrete congestion and no fibrosis henceapproving its hepatoprotective effectThe results are in agree-ment with Afaf et al [12] and Abdulrahman et al [20] whoobserved the improvement of the histological structure inextract treated group as the histopathologic findings showedless inflammation well restored hepatocytes and less area ofnecrosis as compared to severe necrosis and inflammationin CCl4 treated group of animals which also supports thehepatoprotective effects of LLS Their findings suggest thepossibility of the presence of anti-inflammatory compoundsin LSS extract which could also be a contributing factortowards the hepatoprotective action of LSS
Althnaian [47] also reported that examination of hep-atocytes showed mild to moderate degree of recovery LSSattenuated the hepatic fat accumulation decreased vacuolardegeneration and improved the architecture of hepatocytes
In our study the reducing effect of LSS on fatty changecaused by CCL4 intoxication is indicating the protective andcurative role against fatty liver Our results are supported byShukla et al [41] who reported that LSS significantly reducedsteatosis Their histopathological observations confirmed thecurative efficacy of L sativum liver damage Microvesicularfatty changes hepatocyte ballooning and steatosis weremarkedly corrected by LSS Also Wadhwa et al [36] foundthat the severe fatty changes in the livers of rats caused byCCl4 were decreased in the treated groups
In the present study chronic venous congestion featherydegeneration and fatty changes show mild improvement inrabbits receiving 200mgkg bw of LSS for 5 weeks and showmoderate improvement in rabbits that received 200mgkg bwof LSS for 10 weeks and rabbits that received 400mgkg bwof LSS for 5 weeks Our data are consistent with Afaf et al[12] who observed that severe centrilobular hepatocellularvacuolation hemorrhages and congestion of the centralveins noticed in CCL4 group showed mild improvement ingroups that received 200 mgkg bw of LSS and moderateimprovement in groups that received 400 mgkg bw ofLSS
On the other hand rabbits that received 400mgkg bw ofLSS for 10 weeks showed evidence of liver toxicity in the formof venous congestion hepatocyte feathery degeneration fattychange and fibrosis These results are compatible with Bafeeland Ali [48] who reported that LSS had minimal effect onliver parenchyma if given in low doses So LSS could be usedas food additive or supplement Mineral content of calciumand phosphate and the presence of essential fatty acids inconsiderable level make LSS of beneficial nutritional valuefor many body organs including the liver However LSS inhigh dose showed some toxic effect on renal parenchyma rep-resented by focal necrosis hepatocyte apoptosis and portalinflammatory cell infiltrate Burow et al [49] reported thatthese hazardous effects of LSS may be due to isothiocyanatesreported to be one of LSS constituents
5 Conclusions and Recommendations
The present study showed that LSS is a key factor ininhibition of the hazardous effects of CCl4 LSS treatmentcould significantly alleviate to a greater extent the degree oflipid peroxidation and restore the antioxidative enzymes andshift liver function to normal levels In addition to antioxi-dants activity LSS attenuated the hepatic fat accumulationdecreased vacuolar degeneration ameliorated the degree ofstructural damage in the hepatic cells and improved thearchitecture of hepatocytes The current findings demon-strated that LSS have marked hepatoprotective activity Infuture work active ingredients of LSS extract should bestudied in various animal models of severe toxicity inducedby CCL4 and other hepatotoxic agents The active ingredientsof this plant are likely to be applied as one of the future drugsagainst acute liver toxicity
Data Availability
Data used to support the findings of this study are availablefrom the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This project was funded by the Deanship of ScientificResearch (DSR) King Abdulaziz University Jeddah undergrant no S-17-314-37 The authors therefore acknowledgewith thanks DSR technical and financial support
References
[1] M K Manibusan M Odin and D A Eastmond ldquoPostu-lated carbon tetrachloride mode of action a reviewrdquo Journalof Environmental Science and Health Part C EnvironmentalCarcinogenesis and Ecotoxicology Reviews vol 25 no 3 pp 185ndash209 2007
[2] N Brautbar and J Williams II ldquoIndustrial solvents andliver toxicity risk assessment risk factors and mechanismsrdquo
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 15
administration of 200 mg amp 400 mgkg bw L sativum seedsNearly complete repair of DNA damage was significant aftertreating rabbits with 200 mg amp 400 mgkg bw L sativumseeds The protective effect of L sativum seeds on hepaticDNA injury may be due to the antioxidant activity ofL sativum seeds via direct scavenging of free radicals orinterfering with free radicals generation
Concerning histopathological evaluation in our studyrabbitsrsquo livers exposed to CCL4 show portal and parenchymalinflammatory infiltrate feathery and ballooning degenera-tion fatty change and fibrosis Our data are consistent withRajeswary et al [45] who reported dilatation of sinusoidsfatty change disarrangement of normal hepatocytes and cen-trilobular necrosis Also A S Bernacchi et al [46] reportedthat CCL4 leads to liver necrosis and fat accumulation in therabbit
The present study revealed that concurrent treatmentof rabbits administered with CCl4 for 5 and 10 weeks withLSS improved their liver histopathological architecture inthe form of less inflammation regenerating hepatocytes nosteatosis or isolated vesicular steatosis no chronic venouscongestion or just discrete congestion and no fibrosis henceapproving its hepatoprotective effectThe results are in agree-ment with Afaf et al [12] and Abdulrahman et al [20] whoobserved the improvement of the histological structure inextract treated group as the histopathologic findings showedless inflammation well restored hepatocytes and less area ofnecrosis as compared to severe necrosis and inflammationin CCl4 treated group of animals which also supports thehepatoprotective effects of LLS Their findings suggest thepossibility of the presence of anti-inflammatory compoundsin LSS extract which could also be a contributing factortowards the hepatoprotective action of LSS
Althnaian [47] also reported that examination of hep-atocytes showed mild to moderate degree of recovery LSSattenuated the hepatic fat accumulation decreased vacuolardegeneration and improved the architecture of hepatocytes
In our study the reducing effect of LSS on fatty changecaused by CCL4 intoxication is indicating the protective andcurative role against fatty liver Our results are supported byShukla et al [41] who reported that LSS significantly reducedsteatosis Their histopathological observations confirmed thecurative efficacy of L sativum liver damage Microvesicularfatty changes hepatocyte ballooning and steatosis weremarkedly corrected by LSS Also Wadhwa et al [36] foundthat the severe fatty changes in the livers of rats caused byCCl4 were decreased in the treated groups
In the present study chronic venous congestion featherydegeneration and fatty changes show mild improvement inrabbits receiving 200mgkg bw of LSS for 5 weeks and showmoderate improvement in rabbits that received 200mgkg bwof LSS for 10 weeks and rabbits that received 400mgkg bwof LSS for 5 weeks Our data are consistent with Afaf et al[12] who observed that severe centrilobular hepatocellularvacuolation hemorrhages and congestion of the centralveins noticed in CCL4 group showed mild improvement ingroups that received 200 mgkg bw of LSS and moderateimprovement in groups that received 400 mgkg bw ofLSS
On the other hand rabbits that received 400mgkg bw ofLSS for 10 weeks showed evidence of liver toxicity in the formof venous congestion hepatocyte feathery degeneration fattychange and fibrosis These results are compatible with Bafeeland Ali [48] who reported that LSS had minimal effect onliver parenchyma if given in low doses So LSS could be usedas food additive or supplement Mineral content of calciumand phosphate and the presence of essential fatty acids inconsiderable level make LSS of beneficial nutritional valuefor many body organs including the liver However LSS inhigh dose showed some toxic effect on renal parenchyma rep-resented by focal necrosis hepatocyte apoptosis and portalinflammatory cell infiltrate Burow et al [49] reported thatthese hazardous effects of LSS may be due to isothiocyanatesreported to be one of LSS constituents
5 Conclusions and Recommendations
The present study showed that LSS is a key factor ininhibition of the hazardous effects of CCl4 LSS treatmentcould significantly alleviate to a greater extent the degree oflipid peroxidation and restore the antioxidative enzymes andshift liver function to normal levels In addition to antioxi-dants activity LSS attenuated the hepatic fat accumulationdecreased vacuolar degeneration ameliorated the degree ofstructural damage in the hepatic cells and improved thearchitecture of hepatocytes The current findings demon-strated that LSS have marked hepatoprotective activity Infuture work active ingredients of LSS extract should bestudied in various animal models of severe toxicity inducedby CCL4 and other hepatotoxic agents The active ingredientsof this plant are likely to be applied as one of the future drugsagainst acute liver toxicity
Data Availability
Data used to support the findings of this study are availablefrom the corresponding author upon request
Conflicts of Interest
The authors declare that they have no conflicts of interest
Acknowledgments
This project was funded by the Deanship of ScientificResearch (DSR) King Abdulaziz University Jeddah undergrant no S-17-314-37 The authors therefore acknowledgewith thanks DSR technical and financial support
References
[1] M K Manibusan M Odin and D A Eastmond ldquoPostu-lated carbon tetrachloride mode of action a reviewrdquo Journalof Environmental Science and Health Part C EnvironmentalCarcinogenesis and Ecotoxicology Reviews vol 25 no 3 pp 185ndash209 2007
[2] N Brautbar and J Williams II ldquoIndustrial solvents andliver toxicity risk assessment risk factors and mechanismsrdquo
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
16 Evidence-Based Complementary and Alternative Medicine
International Journal of Hygiene and Environmental Health vol205 no 6 pp 479ndash491 2002
[3] R G Mali S G Mahajan and A A Mehta ldquoLepidiumsativum (Garden cress) a review of contemporary literature andmedicinal propertiesrdquo Oriental Pharmacy and ExperimentalMedicine vol 7 no 4 pp 331ndash335 2007
[4] P N Tiwari and G S Kulmi ldquoPerformance of Chandrasur(Lepidium sativum) under different levels of nitrogen andphosphorusrdquo Vol 26 2004 479-481
[5] M Eddouks M Maghrani N-A Zeggwagh and J B MichelldquoStudy of the hypoglycaemic activity of Lepidium sativumL aqueous extract in normal and diabetic ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 391ndash395 2005
[6] A N Paranjape and A A Mehta ldquoA study on clinical efficacyof Lepidium sativum seeds in treatment of bronchial asthmardquoIranian Journal of Pharmacology amp erapeutics vol 5 no 1pp 55ndash59 2006
[7] A K Al-Asmari A M Al-Elaiwi Md T Athar et al ldquoA reviewof hepatoprotective plants used in saudi traditional medicinerdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 890842 22 pages 2014
[8] M Al-Yahya J Mossa A Ageel and S Rafatullah ldquoPharmaco-logical and safety evaluation studies on Lepidium sativum LSeedsrdquo Phytomedicine vol 1 no 2 pp 155ndash159 1994
[9] S K Ahsan M Tariq M Ageel M A Al-Yahya and A HShah ldquoStudies on some herbal drugs used in fracture healingrdquoInternational Journal of Crude Drug Research vol 27 no 4 pp235ndash239 1989
[10] M Sakran Y Selim and N Zidan ldquoA new isoflavonoid fromseeds of Lepidium sativum L and its protective effect onhepatotoxicity induced by paracetamol in male ratsrdquoMoleculesvol 19 no 10 pp 15440ndash15451 2014
[11] Q V Vo C Trenerry S Rochfort J Wadeson C Leytonand A B Hughes ldquoSynthesis and anti-inflammatory activityof aromatic glucosinolatesrdquo Bioorganic amp Medicinal Chemistryvol 21 no 19 pp 5945ndash5954 2013
[12] I Afaf Abuelgasim H S Nuha and A H MohammedldquoHepatoprotective effect of Lepidium sativum against carbontetrachloride induced damage in ratsrdquo Vol 3 2008 20-23
[13] Y C Yadav D N Srivastav A K Seth V Saini R Balaramanand T K Ghelani ldquoIn vivo antioxidant potential of Lepidiumsativum L seeds in albino rats using cisplatin induced nephro-toxicityrdquo International Journal of Phytomedicine vol 2 no 3 pp292ndash298 2010
[14] N D Raval and B Ravishankar ldquoAnalgesic effect of Lepidiumsativum Linn (Chandrashura) in experimental animalsrdquo AYU(An International Quarterly Journal of Research in Ayurveda)vol 31 no 3 p 371 2010
[15] E Issabeagloo ldquoHepatoprotective effects of Lepidium Sativumon doxorubicin mediated toxicity in ratrdquo Magnt Res Rep vol2 pp 557ndash565 2014
[16] F Behrouzian S M A Razavi and G O Phillips ldquoCressseed (Lepidium sativum) mucilage an overviewrdquo BioactiveCarbohydrates and Dietary Fibre vol 3 no 1 pp 17ndash28 2014
[17] J Hrenak K Arendasova R Rajkovicova et al ldquoProtectiveeffect of captopril olmesartan melatonin and compound 21on doxorubicin-induced nephrotoxicity in ratsrdquo PhysiologicalResearch vol 62 p S181 2013
[18] L Al-Malki Abdulrahman A-G M Kamel A-E Gamal andA-B Hassan ldquoHepatoprotective effect of dandelion (Tarax-acumofficinale) against induced chronic liver cirrhosisrdquo Journalof Medicinal Plants Research vol 7 no 20 pp 1494ndash1505 2013
[19] A Bishayee A Sarkar and M Chatterjee ldquoHepatoprotectiveactivity of carrot (Daucus carota L) against carbon tetrachlo-ride intoxication inmouse liverrdquo Journal of Ethnopharmacologyvol 47 no 2 pp 69ndash74 1995
[20] A K Al-Asmari M T Athar H M Al-Shahrani S I Al-Dakheel and M A Al-Ghamdi ldquoEfficacy of Lepidium sativumagainst carbon tetra chloride induced hepatotoxicity and deter-mination of its bioactive compounds by GC-MSrdquo ToxicologyReports vol 2 pp 1319ndash1326 2015
[21] Z Hesami A Jamshidzadeh M Ayatollahi et al ldquoEffect ofplatelet-rich plasma on CCl4-induced chronic liver injury inmale ratsrdquo International Journal of Hepatology vol 2014 ArticleID 932930 7 pages 2014
[22] A L Al-Malki and A-G M Kamel ldquoHepatoprotective efficacyof chicory alone or combined with dandelion leaves againstinduced liver damagerdquo Life Science Journal vol 10 no 4 pp140ndash157 2013
[23] D Gackowski R Rozalski K Roszkowski A Jawien MFoksinski and R Olinski ldquo8-Oxo-78-dihydroguanine and 8-oxo-78-dihydro-21015840-deoxyguanosine levels in human urine donot depend on dietrdquo Free Radical Research vol 35 no 6 pp825ndash832 2001
[24] R K Gothwal V K Nigam M K Mohan D Sasmal andP Ghosh ldquoExtraction of bulk DNA from thar desert soilsfor optimization of PCR-DGGE based microbial communityanalysisrdquo Electronic Journal of Biotechnology vol 10 no 3 pp400ndash408 2007
[25] N N Than and P N Newsome ldquoA concise review of non-alcoholic fatty liver diseaserdquo Atherosclerosis vol 239 no 1 pp192ndash202 2015
[26] C Pereira L Barros and I C Ferreira ldquoExtraction identifi-cation fractionation and isolation of phenolic compounds inplants with hepatoprotective effectsrdquo Journal of the Science ofFood and Agriculture vol 96 no 4 pp 1068ndash1084 2016
[27] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003
[28] S I Alqasoumi and M S Abdel-Kader ldquoScreening of sometraditionally used plants for their hepatoprotective effectrdquo inPhytochemicals as Nutraceuticals - Global Approaches to eirRole in Nutrition and Health 2011
[29] R O Recknagel E A Glende Jr J A Dolak and R L WallerldquoMechanisms of carbon tetrachloride toxicityrdquoPharmacology amperapeutics vol 43 no 1 pp 139ndash154 1989
[30] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016
[31] L-L Huang J-B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids (PLEFA) vol 88 no 5 pp 347ndash353 2013
[32] MWang X-J Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 17
[33] E Scorletti L Bhatia K G McCormick et al ldquoEffects ofpurified eicosapentaenoic and docosahexaenoic acids in non-alcoholic fatty liver disease results from the welcome studyrdquoHepatology vol 60 no 4 pp 1211ndash1221 2014
[34] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated fatty acids for treatment of parenteral nutritionasso-ciated liver disease a review of the literaturerdquo e Journal ofPediatric Pharmacology and erapeutics vol 16 no 1 pp 31ndash38 2011
[35] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011
[36] S Wadhwa M S Panwar A Agrawal et al ldquoA review onpharmacognostical study of Lepidium sativumrdquo vol 2 2012316-323
[37] S Sharm and N Agarwal ldquoNourishing and healing prowessof garden cress (Lepidium sativum Linn) - a reviewrdquo IndianJournal of Natural Products and Resources (IJNPR) vol 2 no3 pp 292ndash297 2011
[38] M Raish A Ahmad K M Alkharfy et al ldquoHepatoprotectiveactivity of Lepidium sativum seeds against D-galactosaminelipopolysaccharide induced hepatotoxicity in animal modelrdquoBMC Complementary and Alternative Medicine vol 16 no 1p 501 2016
[39] T Nakama S Hirono A Moriuchi et al ldquoEtoposide preventsapoptosis in mouse liver with D-Galactosaminelipopolysac-charide-induced fulminant hepatic failure resulting in reduc-tion of lethalityrdquoHepatology vol 33 no 6 pp 1441ndash1450 2001
[40] A Mignon N Rouquet M Fabre et al ldquoLPS challenge in D-galactosamine-sensitizedmice accounts for caspase- dependentfulminant hepatitis not for septic shockrdquo American Journal ofRespiratory andCritical CareMedicine vol 159 no 4 I pp 1308ndash1315 1999
[41] A K Shukla P Bigoniya and P Srivastava ldquoHypoglycemicactivity of Lepidium Sativum Linn seed total alkaloid onalloxan induced diabetic ratsrdquo Journal of Pharmacy and Biolog-ical Sciences vol 10 no 4 pp 13ndash22 2015
[42] C-C Lee S-R Shen Y-J Lai and S-C Wu ldquoRutin andquercetin bioactive compounds from tartary buckwheat pre-vent liver inflammatory injuryrdquo Food amp Function vol 4 no 5pp 794ndash802 2013
[43] S C Sahu and M C Washington ldquoEffect of ascorbic acid andcurcumin on quercetin-induced nuclear DNA damage lipidperoxidation and protein degradationrdquo Cancer Letters vol 63no 3 pp 237ndash241 1992
[44] W W Franke U Scheer G Krohne and E D Jarasch ldquoThenuclear envelope and the architecture of the nuclear peripheryrdquoe Journal of Cell Biology vol 91 no 3 pp 39sndash50s 1981
[45] H Rajeswary R Vasuki P Samudram andAGeetha ldquoHepato-protective action of ethanolic extracts of Melia azedarach LinnandPiper longumLinn and their combination onCCl4 inducedhepatotoxicity in ratsrdquo Indian Journal of Experimental Biology(IJEB) vol 49 no 4 pp 276ndash281 2011
[46] A S Bernacchi C R de Castro E C de Ferreyra et al ldquoCarbontetrachloride-induced liver injury in the rabbitrdquo British journalof experimental pathology vol 64 no 3 pp 261ndash267 1983
[47] T Althnaian ldquoInfluence of dietary supplementation of gardencress (Lepidium sativum L) on liver histopathology and serumbiochemistry in rats fed high cholesterol dietrdquo Journal ofAdvanced Veterinary and Animal Research vol 1 no 4 pp 216ndash223 2014
[48] S Bafeel and S Ali ldquoThe potential liver toxicity of lepidiumsativum seeds in albino ratsrdquo Research Journal of BiologicalSciences vol 4 no 12 pp 1250ndash1258 2009
[49] M Burow A Bergner J Gershenzon and U Wittstock ldquoGlu-cosinolate hydrolysis in Lepidium sativum - identification of thethiocyanate-forming proteinrdquo Plant Molecular Biology vol 63no 1 pp 49ndash61 2007
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom