Research Article Sea Buckthorn Leaf Extract Protects Jejunum...

Research ArticleSea Buckthorn Leaf Extract Protects Jejunum andBone Marrow of 60Cobalt-Gamma-Irradiated Mice byRegulating Apoptosis and Tissue Regeneration

Madhu Bala1 Manish Gupta1 Manu Saini12 M Z Abdin2 and Jagdish Prasad1

1Department of Radiation Biology Institute of Nuclear Medicine and Allied Sciences Brig SK Mazumdar Marg Delhi 110054 India2Centre for Transgenic Plant Development Department of Biotechnology Jamia Hamdard New Delhi 110062 India

Correspondence should be addressed to Madhu Bala bala44gmailcom

Received 19 March 2015 Accepted 27 July 2015

Academic Editor Jae Youl Cho

Copyright copy 2015 Madhu Bala et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A single dose (30mgkg body weight) of standardized sea buckthorn leaf extract (SBL-1) administered 30min before whole body60Co-gamma-irradiation (lethal dose 10Gy) protected gt90 of mice population The purpose of this study was to investigate themechanism of action of SBL-1 on jejunum and bonemarrow quantify key bioactive compounds and analyze chemical compositionof SBL-1 Study with 9-week-old inbred male Swiss albino Strain lsquoArsquo mice demonstrated that SBL-1 treatment before 60Co-gamma-irradiation (10Gy) significantly (119901 lt 005) countered radiation induced decreases in jejunum crypts (127-fold) villi number (141-fold) villus height (125-fold) villus cellularity (227-fold) cryptal Paneth cells (189-fold) and Bcl2 level (154-fold) It counteredradiation induced increases in cryptal apoptotic cells (164-fold) and Bax levels (188-fold) It also countered radiation (2Gy and3Gy) induced bone marrow apoptosis (159-fold and 185-fold) and micronuclei frequency (172-fold and 26-fold) SBL-1 renderedradiation protection by promoting cryptal stem cells proliferation by regulating apoptosis and by countering radiation inducedchromosomal damage Quercetin Ellagic acid Gallic acid high contents polyphenols tannins and thiols detected in SBL-1 mayhave contributed to radiation protection by neutralization of radiation induced oxidative species supporting stem cell proliferationand tissue regeneration

1 Introduction

The increasing use of ionizing radiation in diagnosis therapyindustry and warfare has increased the threat of unwantedexposures of human race to harmful effects of radiation andtherefore the search for medical radiation countermeasuresis an area of intense research [1 2] Till this date no singlechemical compound has been approved for human use as asafe nontoxic and efficacious radioprotective agent againstthe lethal dose of radiation More recently herbal drugs aregaining better acceptability due to lower toxicity but mostof them are efficacious at sublethal doses of radiation only[3] and are rather ineffective in countering toxicity at lethaldoses The lethal doses of low Linear Energy Transfer (LET)ionizing radiation produce flux of multiple intracellularreactive species and free radicals which severely disturb theredox homeostasis produce oxidative stress and damage

multiple tissues simultaneously resulting in multiple organdysfunctionfailure ultimately leading to death

Nonrecoverable multiple damage to jejunal cells is animportant cause of death after radiation exposure at lethaldoses In gastrointestinal (GI) tract jejunum has sizablenumber of stem cells as well as proliferating cells and istherefore a highly radiation sensitive tissue The radiationexposure causes depletion of crypts of Lieberkuhn resultingin loss of villi cellularity their denudation and shorteningdecreased absorption of nutrients leakage of fluids into thelumen and increased systemic bacterial infections Althoughsome herbs were reported to render protection to GI tractfrom radiation damage those were found effective at low orsublethal doses of ionizing radiation only [3ndash5]

Bone marrow is another highly sensitive tissue to the lowLET radiation and hematopoietic syndrome occurs gt1 Gyleading to the death of irradiated animals within 3-4 weeks

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 765705 10 pageshttpdxdoiorg1011552015765705

2 Evidence-Based Complementary and Alternative Medicine

Bone marrow cells undergo mitotic death or apoptosis aftertotal body exposure to sublethal doses of low LET ionizingradiation (up to 3Gy) Assessment of bone marrow DNAdamage by scoring of micronuclei (MN assay) is one ofthe established methods for evaluating chromosomal DNAdamage caused by ionizing radiations [6] Micronuclei aresmall bodies in the cytoplasm resembling the nuclearmaterialin morphology and staining pattern They are formed whena broken chromosome or chromosome fragment does nottravel to the spindle during mitosis and therefore are notincluded in either of the daughter nuclei [6] Apoptosis isa physiological cell death which is under genetic control Itis characterized morphologically by increased cytoplasmicgranularity cell shrinkage and nuclear and chromosomalcondensation membrane blebbing and the formation ofdistinctive nuclear bodies Cells such as thymocytes lympho-cytes lymphoblasts and stem cells undergo apoptosis shortlyafter irradiation (peaking usually 3-4 h after irradiation) [7]Theprocess of apoptosis is controlled by a diverse range of cellsignals which may originate either extracellularly (extrinsicinducers) or intracellularly (intrinsic inducers)These signalsmay positively (ie trigger) or negatively (ie repress inhibitor dampen) affect apoptosis

The plant Hippophae rhamnoides L (common name seabuckthorn family Eleagnaceae) is a wonder plant loadedwith multitude of antioxidants and nutrients Sea buckthornis deciduous and dioecious shrub with silvery leaves andextensive root system which are very effective in controllingsoil erosionThis plant has been adapted to tolerate high radi-ation stress as well as extreme temperature (minus25∘C to +40∘C)prevalent at high altitude regions in northwest Himalayas(8000ndash11000 feet) for millions of years It was argued thatby virtue of adaptation such a plant may have accumulatedunique secondary metabolites which could be of help incountering radiation toxicity [8] A large number of bioactivecompounds known from this plant such as carotenoids a-tocopherols c-tocopherol b-tocotrienol steroids flavonoidshigh amount of unsaturated fatty acids vitamins A C EandKminerals tannins and polyphenols contributed to thehigh antioxidant hepatoprotective cardioprotective woundhealing properties [9ndash11] Traditionally sea buckthorn isrecommended for treatment of gastric ailments circulatorydisorders hepatic injuries and neoplasia [12] The leaves ofsea buckthorn are extremely popular for their nutraceuti-cal as well as medicinal values Okanagan sea buckthorntea of Canada has health promoting ingredients such ascalcium magnesium potassium beta-carotene flavonoidslycopene polyphenols vitamin E and protein Earlier studiesdemonstrated that sea buckthorn leaves prevented radiationtoxicity to experimental mice Only one time intraperitoneal(ip) treatment with 30mgkg body weight (bw) of SBL-1(extract from H rhamnoides leaves) 30min before irradia-tion with lethal dose of 60Co-gamma-rays (10Gy) rendered94 survivors in mice population for 30 days and beyondwhile all non-SBL-1 treated irradiated (10Gy) animals diedwithin 12 days after irradiation [8] SBL-1 treatment beforeirradiation countered the radiation induced inflammation inmice liver [13] and taste aversion (akin to early nausea andvomiting) in rats [14] The SBL-1 interacted in vitro with

DNA and displayed antioxidant properties in a concentrationdependent manner [15] It is proposed that presence ofmultiple bioactive constituents in a specific proportion wasresponsible for strong radioprotective effects of SBL-1 Thepurpose of this study was to provisionally quantify thekey bioactive compounds from groups of polyphenols andflavonoids in SBL-1 by using reverse phase high performanceliquid chromatography (RP-HPLC) analyze the chemicalcomposition by colorimetric and gravimetric methods andinvestigate the mechanism of radioprotective action of SBL-1by monitoring the bone marrow damage and early (at 48 h)as well as late (up to Day 30) changes in the jejunal histologyand levels of key proteins associated with apoptosis usingexperimental mice as model system

2 Materials and Methods

21 Reagents and Chemicals HPLC grade reference stan-dards 345-trihydroxybenzoic acid (gallic acid ethyl esterpurity 98) was purchased from Acros Organics FischerScientific and 3310158404101584056-pentahydroxy flavones (quercetindihydrate purity 98) was purchased from Fluka Bio-chemika Urea and thiourea were purchased from Hi-MediaLaboratories India 2378-Tetrahydroxy-chromeno[543-cde]chromene-510-dione (ellagic acid) dithiothreitol(DTT) acrylamide bis-acrylamide ammonium persulfatetetramethylethylenediamine (TEMED) sodium dodecylsulphate (SDS) tris-HCL phenylmethanesulfonylfluoride(PMSF) 3-[(3-cholamidopropyl) dimethylammonio]-1-pro-panesulfonate (CHAPS) tween 20 and 331015840551015840-tetra-methylbenzidine (TMB) substrate were purchased fromSigma-Aldrich USA Polyvinylidene fluoride (PVDF) mem-branes were purchased from Millipore (India) Pvt LtdIndia Mouse monoclonal anti-Bcl-2 primary antibodyanti-beta-actin primary antibody rabbit polyclonal anti-Bax primary antibody HRP conjugated anti-mouse IgGsecondary antibody HRP conjugated anti-rabbit IgGsecondary antibody and nonfat dry milk were purchasedfrom Santa Cruz Biotechnology CA Methanol glacialacetic acid formaldehyde hematoxylin and eosin stains andother analytical chemicals and reagents used for HPLC andphytochemical analyses were purchased from Merck PvtLtd India

22 Preparation of Herbal Extract SBL-1 The SBL-1 wasprepared as per procedure described earlier [8] Brieflyfresh green leaves of Hippophae rhamnoides L (family Eleag-naceae) common name sea buckthorn (identified and con-firmed by ethnobotanist the specimen records are preservedat museum Defence Institute of High Altitude ResearchLeh India (voucher specimen number SBTL-2006)) werecollected in the month of September from a specific naturalhabitat from western Himalayas India The leaves werecleaned washed thoroughly with distilled water dried andpowdered The extract was prepared by soaking the driedleaves powder in distilled water (1 1 wv) The water extractwas lyophilized to yield 0125 gram lyophilized extract pergram of dried leaves and was coded as SBL-1 (drug)

Evidence-Based Complementary and Alternative Medicine 3

23 Identification and Quantification of Marker CompoundsThe HPLC fingerprint of SBL-1 was developed using AgilentHPLC system with quaternary pump attached with PDAdetector and Autosampler EZ Chrome Elite software wasused for data computation A reverse phase C-18 columnand mobile phase 01 glacial acetic acid methanol (90 10)were used The detection wavelength was 272 nm Theidentification and quantification of marker compounds wereperformed on the basis of coinjections and matching theretention time with standards The calibration curves wereprepared with standard stocks of 345-trihydroxybenzoicacid (gallic acid ethyl ester purity 98) 3310158404101584056-pentahydroxy flavones (quercetin dihydrate purity 98) and2378-tetrahydroxy-chromeno[543-cde]chromene-510-dione (ellagic acid)The stock solutions of the standards wereprepared in methanol filtered through 022-micron filters(Millipore) and diluted to obtain a suitable concentrationfor unambiguous identification

The total phenolic content was determined by themethoddescribed [16] Total flavonoid content was determined bymethod of [17] Total tannin content was determined byprotein precipitation method [18] The total thiols weremeasured as per method of [19]

24 Experimental Animals The 8-9-week-old male inbredSwiss albino Strain lsquoArsquo mice weighing 28 plusmn 2 g were usedafter the approval of Animal Experimentation Ethics Com-mittee of the Institute The animals were maintained undercontrolled environment at 26 plusmn 2∘C and 12 h lightdark cycleand offered standard animal food (Golden Feed Delhi) aswell as tap water ad libitum All the animal experimentswere conducted according to the guidelines of the Committeefor Protection and Care of Small Experimental Animals(CPCSEA) Delhi India

25 Experimental Procedure for Studies on Jejunum Theanimals were divided into four groups Group I was untreatedcontrol and had three animals which were administeredsterile water (vehicle) only The other three groups wereldquotreatment groupsrdquo that is group II animals were whole body60Co-120574-irradiated (10Gy) group III animals were treatedwith 30mgkg body weight (bw) SBL-1 (drug) only andgroup IV animals were treated with 30mgkg bw drug30min prior to irradiation (10Gy) Groups II III and IV had18 animals per group Forwhole body irradiation eachmousewas placed in a separate wire mesh container and was givenone-time exposure to 10Gy radiation dose using 60Co-120574-raysource (GC-220 Atomic Energy of Canada Ltd Canadadose rate of 031 radsec) The fresh air was continuouslycirculated in the irradiation chamber with the help of pumpto avoid hypoxia For all drug treatments SBL-1 was dissolvedin sterile water filtered and administered intraperitoneally(ip) A set of three animals were sacrificed from eachtreatment group at Days 2 5 10 15 and 30 after treatmentFrom each animal the jejunum portion of intestine (2-3 cm)was taken out after leaving approximately 4 cm segment fromthe pyloric sphincter side of stomach The lumen of jejunumwas flushed with normal ice cold saline to remove particulate

debris One half of the jejunum tissue was preserved at minus20∘Cfor biochemical analyses while the other half was placed in10 neutral buffered formalin for histological studies

26 Experimental Procedure for Studies on Bone MarrowThe animals were divided into six groups (3 mice in eachgroup) and the experiment was repeated 3 times Group Iwas untreated control in which the animals were adminis-tered sterile water only group II animals were treated with30mgkg bw drug only group III animals were whole body60Co-120574-irradiated (2Gy) group IV animals were whole body60Co-120574-irradiated (3Gy) group V animals were treated with30mgkg bw drug 30 minutes prior to irradiation (2Gy)and group VI animals were treated with 30mgkg bw drug30 minutes prior to irradiation (3Gy)

27 Protein Extraction and Western Blotting Jejunum washomogenized in lysis buffer containing urea 7M thiourea2M DTT 65mM CHAPS 032M and PMSF 2mM celldebris was removed by centrifugation supernatant wascollected and the protein concentration was determinedby using Bradford assay [20] Proteins separation was byelectrophoresis on 10 SDS-polyacrylamide gel in 1X SDSPAGE running buffer (25mM tris base 192mM glycine and01 SDS) at 70V for 4 h at 26 plusmn 2∘C For western blottingthe gels were fixed (acetic acid methanol and Milli Qwater 10 50 40) washed and placed in mini Trans blot cell(Bio-RAD USA) together with PVDF membrane (pore size045 120583m)TheTrans blot cell was filledwith the transfer buffer(25mM tris base 192mM glycine and 10 methanol) andcurrent was applied at 25V overnight at 4∘CThemembraneswere dried and dipped in blocking buffer (5 nonfat skimmilk powder) at 4∘C for 18 h treated with primary antibodyand then incubatedwithHRP conjugated secondary antibod-ies TMB solution was used to develop protein bands on theblot Images were quantified by using image analysis softwareNIH image J 146 software (a free to use application for imageanalysis available at httprsbinfonihgovij)

28 Tissue Fixation Staining and Morphometry The forma-lin fixed tissue was dehydrated sequentially with differentconcentration of alcohol and embedded in paraffinwaxNon-serial transverse sections (approximately 5120583m) of jejunumwere cut and stained with hematoxylin and eosin for analysisEach section was separated from the previous one by aminimum of 50 120583mof tissue Microscopic examinations weredone by using light microscope (Axio Scope Observer D1from Carl Zeiss Germany)

29 Surviving Crypt Number Villi Number Villi Height andVillus and Crypt Cellularity Only complete sections whichincluded the opening of crypt and full length of villi frombase to the tip were considered for analyses Villus heightwas determined bymeasuring the distance from the tip of thevillus up to the crypt in pixels A surviving crypt was definedas containing 10 or more adjacent healthy-looking non-Paneth cells somePaneth cells and a lumen [21]Thenumberof crypts was counted in each circumference Only those


crypts which were seen directly against the inner musclelayer were counted Cryptal Paneth cells were identified onthe basis of their position within the hemispheric bases ofthe crypts their truncated shape basal nuclei and deepred staining secretion granules and were counted only ifthey were contained within longitudinally sectioned cryptsresting on the muscle layer All counts and measurementsfrom each tissue specimen were obtained ldquoblindrdquo from aminimum of 4 coded sections Apoptotic cells were scoredwithin the crypts of jejunum Apoptosis was assessed on thebasis of morphological characteristics such as cell shrinkagechromatin condensation and nuclear fragmentation

210 Micronuclei Assay The animals were euthanized bycervical dislocation 30 h after irradiation The bone marrowsfrom both femurs were flushed in the form of a fine suspen-sion and the assay was done according to Schmid method[22]

211 Statistical Analysis The data was presented as mean plusmnstandard deviation (SD) Multivariate analysis of variance(ANOVA) was applied for analyzing statistically impor-tant changes in more than one parameter in histologicalstudy Studentrsquos 119905-test was applied for evaluating significancebetween any two treatments and value at 119901 lt 005 wasconsidered significantly different Statistical analysis was per-formed using SPSS software (SPSS Inc Chicago IL USA)

3 Results

31 Total Polyphenols Flavonoids Tannins and Thiols Con-tents in SBL-1 The SBL-1 (per gram of dried leaves) wasfound to contain 22 plusmn 02 polyphenolics equivalent to gallicacid 093 + 001 flavonoids equivalent to quercetin 20 plusmn18 tannins equivalent to tannic acid and 0827M totalthiols

32 Identification and Quantification of Marker Compoundsby RP-HPLC in SBL-1 The structures of marker com-pounds 345-trihydroxybenzoic acid (gallic acid ethyl ester)3310158404101584056-pentahydroxy flavones (quercetin) and ellagicacid are shown in Figure 1(a) The RP-HPLC retentiontime for standard compound gallic acid was 293min forellagic acid was 580min and for quercetin was 795min(Figure 1(b)) The plant extract showed prominent peaks at293min 580min and 795min (Figure 1(c)) The quan-titative analysis showed that SBL-1 contained gallic acid098ww ellagic acid 020ww and quercetin 007ww

33 Effects of Radiation on Tissue Histology Apoptosis andLevels of Bcl2 and Bax Proteins The villi of jejunum fromhealthy untreated controls were tall and cylindrical Inirradiated animals there were decreases in the villi heightnumber and cellularity as well as in crypts number andcrypt cellularity with time At 48 h after irradiation persection the crypts number was decreased to 73 and villinumber to 63 (significant at 119901 lt 005 in comparison tothe untreated control (Table 1)) Also there were other gross

Table 1 Effects of SBL-1 (30mgkg bw minus30min) before irradiation(10Gy) on villi and crypts number of mouse intestine at 48 h afterirradiation (10Gy)

Treatment Villi numbersection Crypts numbersectionUntreated control 46 plusmn 091 105 plusmn 3SBL-1 alone 45 plusmn 087 102 plusmn 410Gy 29 plusmn 04lowast 77 plusmn 2lowast

SBL-1 + 10Gy 41 plusmn 05 98 plusmn 3lowastSignificantly reduced at 119901 lt 005 in comparison to untreated control

histological changes such as villi fusion and nonrecoverabledecreases in villi height villus cellularity and number ofcrypts (Figures 2(a) and 2(b)) At Day 5 more sterile cryptsand further decreases were observed in villi and cryptscellularity Significant decreases were also observed in cryptalPaneth cells and Bcl2 protein level while significant increaseswere observed in lumen enlargement cryptal apoptotic cellsand levels of Bax proteins (Figures 2(c) and 2(d)) At Day10 the jejunal lumen was highly enlarged villi were nearlyabsent or severely stunted andmost of the crypts were sterile(Figure 2(a))

34 Effects of SBL-1 Treatment on Jejunal Tissue HistologyApoptosis and Levels of Bcl2 and Bax Treatment with SBL-130min before irradiation (10Gy) countered significantly (119901 lt005) the early (within 48 h) radiation induced histologicalchanges The radiation induced decrease in crypts numberwas countered by 127-fold villi number by 141-fold (Table 1)decreases in villus height by 125-fold villus cellularity by227-fold and cryptal Paneth cell number by 189-fold (Fig-ures 2(a) and 2(b)) At Day 5 the increase in cryptal apoptoticcells was countered by 164-fold and levels of Bcl2 and Baxproteins were normalized (Figure 2(d))The villus and cryptscellularity villi height and Paneth cells numbers were furtherimproved (Figure 2(c)) From Day 15 onwards till the end ofthe study there were no significant differences between theSBL-1 treated irradiated animals and untreated controls interms of villi and crypts number and villus height Animalstreated with SBL-1 alone did not show any difference fromthe untreated animals in the jejunum histology as well as inthe levels of Bcl2 and Bax

35 Percentage Micronuclei Frequency in Bone Marrow Incomparison to untreated control animals in the irradiatedanimals significant increase in micronuclei frequency wasobserved at 3Gy (119901 lt 001 26-fold) and 2Gy (119901 lt 001172-fold) respectively In drug alone treated animals and drugtreated irradiated animals the micronuclei frequency wascomparable to untreated control animals (Figure 3(a))

36 Percentage Apoptosis in Bone Marrow In comparison tountreated control animals in the irradiated animals signif-icant increase in apoptosis was observed at 3Gy (119901 lt 001185 folds) and 2Gy (119901 lt 001 159 folds) respectively In drugalone treated animals and drug treated irradiated animals themicronuclei frequency was comparable to untreated controlanimals (Figure 3(b))


O

O

OH

O

O

HH

H

O

O

O

O

OH

OH

HO

HO

Gallic acid Quercetin dihydrateEllagic acid

O

O

OH

HO

OH

OH

OH middot 2H2O

(a)

293

795

580

Retention time

0

1000

2000

3000

(mAU

)0

1000

2000

3000

(mAU

)

2 4 6 8 10 12 140

Retention time (min)

(b)

Retention time

225

261

311

323

351

399

487 5

886

15 795

850

2810

100

200

300

(mAU

)

0

100

200

300

(mAU

)

2 4 6 8 10 12 140


(c)

Figure 1 (a) Chemical structure of gallic acid ellagic acid and quercetin dihydrate (b) The HPLC profile of the standards and (c) plantmaterial (SBL-1) using reverse phase C-18 column and mobile phase 01 glacial acetic acid methanol (90 10) The detection wavelengthwas 272 nm

4 Discussion

Radiation causes toxicity and multiple damages to vitalbiomolecules such as nucleic acids proteins and lipids eitherby direct deposition of energy or indirectly by generatingfree radicals and ROSThe resultant effects are radiation dosedependent and may range from cellular losses to the failureof organs To counter the toxicity caused by lethal doses ofradiation there is a need to identify a pharmacological agentor a group of agents which have the potential to counter theoxidative stress inmultiplemanners such as by quenching thehigh flux of free radicals breaking the chain reaction of ROSand other reactive species and preventing lipid peroxidationBesides these the agents should essentially have the tissueregenerative properties (such as the potential to recoupcellular loss regulate apoptosis and prevent inflammationcarry out DNA replication etc) The sea buckthorn leaveshave nutraceutical antioxidant medicinal and adaptogenicproperties [23] and form common constituent of health pro-moting teas and other beverages The specific standardizedextract SBL-1 from sea buckthorn leaves was successfulin protecting the mice population from radiation toxicityand injuries in many ways such as increasing the survivalof animal population from zero to gt90 restoration ofbody weight loss increasing the in vivo reducing powerof blood plasma [8] and prevention of inflammation byregulating the levels of high mobility group box-1 (HMGB-1)protein interleukin (IL-10) tumor necrosis factor (TNF-120572)and immunoglobulin (IgG) [13]The SBL-1 promoted in vitrocell proliferation had antimutagenic antirecombinogenicand antioxidant effects and demonstrated quenching of

radiation induced superoxide radicals reduction of hydroxylradicals promoted metal chelation and decreased the lipidperoxidation [24]

The polyphenols and flavonoids are important plantmetabolites which impart antioxidant properties The highconcentration of polyphenolics (22 plusmn 02) flavonoids (093+ 001) tannins (20plusmn 18) and thiols (0827M)was detectedin SBl-1 and explained the strong antioxidant potential ofSBL-1 The antioxidant properties of plant polyphenols andflavonoids as well as their beneficial effects have been recentlyreviewed [25] Polyphenols are ubiquitous to the plantextracts and may be present as glycosides and esters Theyact as free radical scavengers specifically for peroxyl radicalssuperoxide anions and hydroxyl radicals Flavonoids are agroup of compounds (flavonols anthocyanins isoflavonoidsflavones etc) which have antioxidant properties due to thepresence of phenolic hydroxyl groups attached to the ringstructure They act as reducing agents hydrogen donors andsuperoxide radical scavengers One of the most studied andpromising compounds of polyphenols (specifically hydroxy-benzoic group) is the gallic acid which also is the precursor ofmany tannins [26] Ellagic acid is a natural phenol antioxidantand is produced in plants by hydrolysis of tannins Quercetin-3-O-glucoside and Quercetin-3-O-galactoside are commonwell-investigated and important flavonoids present in seabuckthorn leaves [27] It was therefore considered importantto quantify the gallic acid ellagic acid and quercetin inSBL-1 by HPLC technique The mobile phase 01 glacialacetic acid methanol (90 10) was optimized after manytrials to obtain separation of standards as well as of plantconstituents (Figures 1(a)ndash1(c)) The concentrations of gallic


SBL-1+10

Gy

SBL-1

alon

eU

ntre

ated

Day 2 Day 5 Day 10 Day 15 Day 30

cont

rol

Radi

atio

n(10

Gy)

(a)

lowast lowast lowast

lowastlowast lowast

lowast

lowast lowast

ControlRadiation

DrugDrug + radiation

0

40

80

120

160

Cel

lsvi

llus

0

100

200

300

Villi

hei

ght (

pixe

ls)

0

5

10

15

20

Pane

th ce

llss

ectio

n

5 10 15 302

Posttreatment time (days)

(b)

Size of lumen Apoptotic (AP) andVilli and crypts Villi height paneth (P) cells

SBL-1+10

Gy

SBL-1

alon

eRa

diat

ion

Unt

reat

ed

(pixels) cellularity

cont

rol

(10

Gy)

(c)

Apop

totic

cells

C R D

Bcl2Actin

Treatments

BaxActin

RadiationDrug

Drug + radiation

0306090

120

15 cr

ypts

5 10 15 302Posttreatment time (days)

D + R

C R D D + R

C R D D + R

050

100150

Abso

rban

ce

0

20

40

Abso

rban

ce

(d)

Figure 2 (a) Effects of one-time administration of SBL-1 (30mgkg bw) 30min before whole body irradiation (10Gy) on gross histology ofmouse jejunum on Day 2 Day 5 Day 10 Day 15 and Day 30 All the irradiated (10Gy) animals died by Day 12 in SBL-1 + 10Gy group 94 ofthe animals survived while no death was observed in mice treated with SBl-1 alone [8] (b)The quantitative changes in the villi height villuscellularity and cryptal Paneth cell count from Day 2 till Day 30 (c) The typical radioprotective effects of SBL-1 on Day 5 in terms of jejunallumen size villi height villus and crypt cellularity and cryptal apoptotic and Paneth cells (d) Quantitative changes in cryptal apoptotic cellsand levels of Bcl2 and Bax proteins on Day 5


controlR2 + drugR1+ drug R2R1Untreated drug

Treatment

lowast

0

05

1

15

2

25

3M

icro

nucle

i (

)

(a)

lowast

lowast

R2 + drugR1+ drug R2R1

TreatmentcontrolUntreated drug

0

05

1

15

2

25

Apop

tosis

()

(b)

Figure 3 (a) Frequency of micronuclei and (b) apoptosis in bone marrow of mice exposed to 2Gy (R1) and 3Gy (R2) 60Co-gamma rayswith or without SBL-1 (Drug) administration (30mgkgbwt)

acid (098ww) ellagic acid (020ww) and quercetin(007ww) were detected in SBL-1 and explained its mul-tiple effects against radiation toxicity and damage

In healthy unirradiated animals the epithelium of jejunalvilli is renewed at regular intervals by newly proliferated cellswhich transit from the zone of proliferation to the zone ofextrusion at the tip of the villi The crypts of Lieberkuhnwhich are reservoirs of stem cells are located at the base ofvilliThe putativemultipotent intestinal stem cells proliferateto increase the production of the clonogenic self-renewingprogenitor cells The daughter cells migrate either towardthe villus and differentiate into enterocytes goblet cells andenteroendocrine cells or inwards to the crypt base giving riseto Paneth cells The Paneth cells are important for innateintestinal defense because they act as regulators of microbialdensity in the small intestine and protect stem cells frominfections [28] Following exposure to ionizing radiation thecells located at the base of the crypts undergo rapid apoptosisor stop dividing temporarily or permanently dependingupon the absorbed radiation dose If all crypt cells diethe crypt is ldquosterilizedrdquo and ultimately disappears The lossof regenerating population of clonogenic cells in irradiatedanimals impairs the normal regeneration of epithelial layerof intestinal villi leading to varying degrees of bluntingfusion attenuation and denudation of the villi This causesmalabsorption electrolyte imbalance diarrhea inflamma-tion infections weight loss and ultimately mortality [29]At biochemical level complex interplay of apoptotic proteinssuch as Bax and p53 as well as antiapoptotic factors such asBcl2 regulates tissue damage and toxicity [30] In this studyper section significant decreases in crypts number to 73and villi number to 63 (Table 1) together with villi fusionand decreases in villi height villi cellularity and number ofcrypts (Figures 2(a) and 2(b)) within 48 h after irradiationindicated themassive cellular damage to the jejunumby lethaldose of 60Co-gamma-irradiation (10Gy) The appearance ofincreased number of sterile crypts and further decreasesin villus and crypts cellularity at Day 5ndashDay 10 indicatedthat most of the stem cells were lost and regeneration ofepithelium was not taking place in the irradiated animals

Significant decreases observed in cryptal Paneth cells andBcl2 protein level and significant increases in cryptal apop-totic cells and levels of Bax proteins suggested that massiveapoptosis was progressing with the passage of time (Figures2(c) and 2(d)) These biochemical as well as histologicalchanges were the typical features of radiation induced GIsyndrome and were corroborated with presentations suchas diarrheas weight loss in irradiated animals from Day 1onwards and death of all animals by Day 12 reported earlier[8]

The early protection from radiation toxicity by seabuckthorn leaves (SBL-1) was evident because there wassignificant countering of radiation induced decreases incrypts number villi number (Table 1) villus height villuscellularity and cryptal Paneth cell number (Figures 2(a) and2(b)) within 48 h The continuation of protective effects ofSBL-1 for subsequent time was observed because at Day 5the radiation induced increase in cryptal apoptotic cells wascountered levels of Bcl2 and Bax proteins were normalized(Figure 2(d)) and villi and crypts cellularity villi heightand Paneth cells numbers were improved (Figure 2(c)) Thecrypts of Lieberkuhn of GI tract are highly radiosensitive andprone to radiation damage The early protection (at 48 h) tocrypt cellularity against radiation damage could be attributedto effective countering of radiation induced multiple freeradicals and reactive species due to the presence of multipleantioxidants (polyphenols tannins flavonoids and thiols)and more specifically gallic acid ellagic acid and quercetinin SBL-1 The flavonoids alone were reported to protect fromradiation induced oxidative stress but were effective at low orsublethal doses of radiation only [5 31] Quercetin alone wasalso reported to protect the human lymphocytes from geneticdamage and oxidative stress caused by low doses of radiationonly [32]

The irradiated cells which undergoDNAdamage beyondrepair ultimately undergo apoptosis and show decrease inlevel of Bcl2 protein and increase in level of Bax protein [33]Postirradiation increase in apoptosis as well as micronucleifrequency in bone marrow is reported In jejunum theradiation induced apoptosis was shown to be accompanied


with increased accumulation of Bax and p53 and simul-taneous decrease in Bcl2 [30] Decrease in the frequencyof micronuclei as well as apoptotic cells at sublethal dosesof radiation (2Gy and 3Gy) suggested that the SBL-1 wasefficient in countering the radiation damage to hematopoieticsystem at sublethal doses also In this study SBL-1 treatedirradiated mice showed the countering of radiation inducedapoptosis with concomitant normalization of Bcl2 and Baxproteins levels This indicated that SBL-1 acted by counteringradiation induced apoptosis as well

There was no significant difference between the SBL-1treated irradiated animals and untreated controls in termsof villi and crypts number and villus height by 15 days andthereafter suggesting that SBL-1 also acted by promoting theproliferation of surviving cells The presence of quercetin inSBL-1 may have contributed to this property The quercetinalone was reported to promote the proliferation of spleno-cytes in the irradiated mice although the concentrationsrequired were much higher and the doses were in multiplesfor 10ndash30 days [34] In SBL-1 treated irradiated animals upto Day 15 significantly greater apoptosis was observed incomparison to untreated controls though the values weresignificantly lower than the irradiated controls (Figure 2(d))This suggested that SBL-1 treatment prior to irradiationpermitted controlled and slow apoptosis of cells till Day15 (Figure 2(d)) and simultaneously promoted proliferationof healthy intestinal stem cells and transition of amplifiedcells which ultimately resulted in crypt and villi regen-eration (Figures 2(a) and 2(b)) The regulated apoptosisof radiation damaged cells by SBl-1 was expected to beadvantageous because increased apoptosis during early timepoints reduced the burden of damaged nonrepairable cellsand hence checked the resultant inflammation and othertoxic effects The presence of flavonoids in SBL-1 could beresponsible for altering the regulatory mechanisms becauseflavonoids have been shown to act by altering the signalingpathways besides acting as antioxidants [35] Our in silicodocking studies had revealed that gallic acid quercetin andgenistein could block the TWEAK binding site on Fn14receptors and therefore contributed to protection againstcarcinogenic effects of radiation [36]

The continuous antimicrobial protection of stem cells isof paramount importance and the Paneth cells importantlycontribute to the innate intestinal defense [28] The propertyof SBL-1 to counter radiation induced decrease in Panethcell number suggested that SBL-1 was helpful in maintainingthe innate immune response of jejunum in the irradiatedmice Treatment with SBL-1 alone did not alter the jejunalhistology or levels of Bcl2 and Bax proteins significantly incomparison to the untreated controlmiceThis demonstratedthe nontoxic nature of SBL-1 at the doses used in thisstudy Other studies have also shown the nontoxic nature ofHippophae leaf extract [37]

This study clearly demonstrated that sea buckthorn leavesextract (SBL-1) was effective in preventing radiation toxicityto jejunum The SBL-1 acted by more than one mechanismsuch as by reducing oxidative stress by regulating apoptosisand by promoting the proliferation of the cryptal stem cellsThe radioprotective effects of SBL-1 on jejunum were higher

than those observed with most investigated single moleculardrugs WR 2721 and MPG alone or in combination [38]The SBL-1 treatment demonstrated the dose reduction factorof 141 on the basis of whole body survival and 197 onthe basis of endogenous spleen CFUs counts the effectiveSBL-1 concentration was 4 times lower than the maximumtolerated dose (MTD) [8] In thismanner SBL-1met with twoessential criteria of an ideal radioprotector in being nontoxicand effective at lethal doses of whole body irradiation Theradioprotective survival effect of SBL-1 against lethal dosesof ionizing radiation was better than the other preparationsfrom sea buckthorn such as SBL-2 from leaves [39] andRH-3 from berries [40] which caused only 82 survivalat 34 of MTD [41] The sea buckthorn leaf extract SBL-1 displayed a number of other distinct advantages over thesea buckthorn berry extract RH-3 such as longer shelflife lower storage and transportation cost higher stabilityof active constituents better ecofriendly collection of plantmaterial and zero destruction of plant germplasm To thebest of our knowledge this is the first report presentingthe protective effects of sea buckthorn leaves on jejunum inlethally irradiated experimental animals

5 Conclusion

The manifestation of gastrointestinal syndrome at dosesabove 6Gy is the common cause of death of irradiatedanimals The study showed that single prophylactic doseof SBL-1 (30mgkg body weight) before lethal irradiation(10Gy) countered the radiation induced atrophy of mucosallayer decrease in jejunum villi number and cellularity cryptsnumber and caused normalization of apoptosis and levelsof Bcl2 and Bax expression These observations suggesteda strong radioprotective action of SBL-1 on the jejunum oflethally irradiated experimental mice The damage sufferedby bone marrow above 1 Gy is another important cause ofdeath of irradiated animals This study showed that singleprophylactic dose of SBL-1 at 30mgkg body weight beforesublethal irradiation (2Gy and 3Gy) significantly counteredthe apoptosis as well as formation of micronuclei in bonemarrow therefore suggesting the radioprotective action ofSBL-1 on hematopoietic system Sea buckthorn leaves forma part of dietary constituents in many parts of the worldbecause of their nutraceutical propertiesThis study togetherwith earlier published reports which demonstrated theradioprotective action of sea buckthorn on immune systemthrough HMGB1 modification in irradiated (10Gy) miceaction of SBL-1 to counter taste aversion (akin to early nauseaand vomiting) in irradiated (2Gy) rats by normalizing jeju-nal serotonin levels antimutagenic and antirecombinogenicaction of SBL-1 in Saccharomyces cerevisiae and significant(gt90) whole body radioprotection at nontoxic doses withdose reduction factor gt 14 suggested that SBL-1 could bedeveloped as a promising medical radiation countermeasurefor human use

Conflict of Interests

No conflict of interests is declared by the authors


Authorsrsquo Contribution

Madhu Bala and Manish Gupta contributed equally to thiswork

Acknowledgments

This research work was supported by Defence Research andDevelopment Organization (DRDO) India Vide Project noINM31112 The authors wish to acknowledge the facilitiesprovided by the director of the Institute of Nuclear Medicineand Allied Sciences and the director of the Defence Instituteof High Altitude Research Leh and efforts of Dr O PChaurasia and Dr S K Dwivedi in procurementcollectionof plant material The author Manish Gupta acknowledgesthe research fellowship grant provided by DRDO

References

[1] L G Burdelya V I Krivokrysenko T C Tallant et al ldquoAn ago-nist of toll-like receptor 5 has radioprotective activity in mouseand primate modelsrdquo Science vol 320 no 5873 pp 226ndash2302008

[2] J P Williams and W H McBride ldquoAfter the bomb dropsa new look at radiation-induced multiple organ dysfunctionsyndrome (MODS)rdquo International Journal of Radiation Biologyvol 87 no 8 pp 851ndash868 2011

[3] S J Hosseinimehr ldquoTrends in the development of radioprotec-tive agentsrdquo Drug Discovery Today vol 12 no 19-20 pp 794ndash805 2007

[4] J Ni A L Romero-Weaver and A R Kennedy ldquoPotential ben-eficial effects of Si-Wu-Tang on white blood cell numbers andthe gastrointestinal tract of 120574-ray irradiated micerdquo InternationalJournal of Biomedical Science vol 10 no 3 pp 182ndash190 2014

[5] P Uma Devi A Ganasoundari B Vrinda K K SrinivasanandM K Unnikrishnan ldquoRadiation protection by the ocimumflavonoids orientin and vicenin mechanisms of actionrdquo Radia-tion Research vol 154 no 4 pp 455ndash460 2000

[6] R J Albertini D Anderson G R Douglas et al ldquoIPCSguidelines for themonitoring of genotoxic effects of carcinogensin humansrdquo Mutation ResearchReviews in Mutation Researchvol 463 no 2 pp 111ndash172 2000

[7] P LOlive G Frazer and J P Banath ldquoRadiation-induced apop-tosis measured in TK6 human B lymphoblast cells using thecomet assayrdquo Radiation Research vol 136 no 1 pp 130ndash1361993

[8] M Bala J Prasad S Singh S Tiwari and R C SawhneyldquoWhole-body radioprotective effects of SBL-1 a preparationfrom leaves of Hippophae rhamnoidesrdquo Journal of Herbs Spicesand Medicinal Plants vol 15 no 2 pp 203ndash215 2009

[9] M S Y Kumar R Dutta D Prasad and K Misra ldquoSubcriticalwater extraction of antioxidant compounds from Seabuckthorn(Hippophae rhamnoides) leaves for the comparative evaluationof antioxidant activityrdquo Food Chemistry vol 127 no 3 pp 1309ndash1316 2011

[10] D T Maheshwari M S Yogendra Kumar S K Verma VK Singh and S N Singh ldquoAntioxidant and hepatoprotectiveactivities of phenolic rich fraction of Seabuckthorn (Hippophaerhamnoides L) leavesrdquo Food and Chemical Toxicology vol 49no 9 pp 2422ndash2428 2011

[11] A Gupta N K Upadhyay R C Sawhney and R KumarldquoA poly-herbal formulation accelerates normal and impaireddiabetic wound healingrdquo Wound Repair and Regeneration vol16 no 6 pp 784ndash790 2008

[12] A Zeb ldquoImportant therapeutic uses of Sea buckthorn (Hip-pophae) a reviewrdquo Journal of Biological Sciences vol 4 no 5pp 687ndash693 2004

[13] S Tiwari and M Bala ldquoHippophae leaves prevent immuno-suppression and inflammation in 60Co-120574-irradiated micerdquo Phy-topharmacology vol 1 no 3 pp 36ndash48 2011

[14] V Gupta M Bala J Prasad S Singh and M Gupta ldquoLeavesof Hippophae rhamnoides prevent taste aversion in gamma-irradiated ratsrdquo Journal of Dietary Supplements vol 8 no 4 pp355ndash368 2011

[15] M Saini S Tiwari J Prasad S Singh M S Y Kumarand M Bala ldquoHippophae leaf extract concentration regulatesantioxidant and prooxidant effects on DNArdquo Journal of DietarySupplements vol 7 no 1 pp 60ndash70 2010

[16] M Senevirathne S-H Kim N Siriwardhana J-H Ha K-WLee and Y-J Jeon ldquoAntioxidant potential of Ecklonia cavaonreactive oxygen species scavenging metal chelating reducingpower and lipid peroxidation inhibitionrdquo Food Science andTechnology International vol 12 no 1 pp 27ndash38 2006

[17] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[18] A E Hagerman and L G Butler ldquoProtein precipitationmethodfor the quantitative determination of tanninsrdquo Journal of Agri-cultural and Food Chemistry vol 26 no 4 pp 809ndash812 1978

[19] J Sedlak and R H Lindsay ldquoEstimation of total protein-bound andnonprotein sulfhydryl groups in tissuewith Ellmanrsquosreagentrdquo Analytical Biochemistry vol 25 no 1 pp 192ndash2051968

[20] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[21] H R Withers and M M Elkind ldquoMicrocolony survival assayfor cells of mouse intestinal mucosa exposed to radiationrdquoInternational Journal of Radiation Biology vol 17 no 3 pp 261ndash267 1970

[22] W Schmid ldquoThe micronucleus testrdquoMutation Research vol 31no 1 pp 9ndash15 1975

[23] T Stobdan G Korekar and R B Srivastava ldquoNutritionalattributes and health application of seabuckthorn (Hippophaerhamnoides L)mdasha reviewrdquo Current Nutrition and Food Sciencevol 9 no 2 pp 151ndash165 2013

[24] S Tiwari S Tyagi J Prasad et al ldquoAnti-oxidant anti-mutagenicand radioprotective properties of sea buckthorn leaf (Hippophaerhamnoides L)rdquo Zeitschrift Arznei Gewurzpfla vol 14 no 2 pp83ndash89 2009

[25] M Carocho and I C F R Ferreira ldquoA review on antioxidantsprooxidants and related controversy natural and syntheticcompounds screening and analysis methodologies and futureperspectivesrdquo Food and Chemical Toxicology vol 51 no 1 pp15ndash25 2013

[26] B Krimmel F Swoboda S Solar and G Reznicek ldquoOH-radicalinduced degradation of hydroxybenzoic- and hydroxycinnamicacids and formation of aromatic productsmdasha gamma radiolysisstudyrdquoRadiation Physics and Chemistry vol 79 no 12 pp 1247ndash1254 2010


[27] N K Upadhyay M S Yogendra Kumar and A Gupta ldquoAntiox-idant cytoprotective and antibacterial effects of Sea buckthorn(Hippophae rhamnoides L) leavesrdquo Food and Chemical Toxicol-ogy vol 48 no 12 pp 3443ndash3448 2010

[28] H Tanabe T Ayabe B Bainbridge et al ldquoMouse paneth cellsecretory responses to cell surface glycolipids of virulent andattenuated pathogenic bacteriardquo Infection and Immunity vol 73no 4 pp 2312ndash2320 2005

[29] C S Potten ldquoA comprehensive study of the radiobiologicalresponse of the murine (BDF1) small intestinerdquo InternationalJournal of Radiation Biology vol 58 no 6 pp 925ndash973 1990

[30] A Takahashi K Ohnishi I Asakawa et al ldquoRadiation responseof apoptosis in C57BL6N mouse spleen after whole-bodyirradiationrdquo International Journal of Radiation Biology vol 77no 9 pp 939ndash945 2001

[31] V Benkovic A H Knezevic D Dikic et al ldquoRadioprotectiveeffects of propolis and quercetin in 120574-irradiated mice evaluatedby the alkaline comet assayrdquo Phytomedicine vol 15 no 10 pp851ndash858 2008

[32] N Devipriya A R Sudheer M Srinivasan and V P MenonldquoQuercetin ameliorates gamma radiation-induced DNA dam-age and biochemical changes in human peripheral bloodlymphocytesrdquoMutation Research vol 654 no 1 pp 1ndash7 2008

[33] R Ciccocioppo A di Sabatino G Gasbarrini and G RCorazza ldquoApoptosis and gastrointestinal tractrdquo Italian Journalof Gastroenterology and Hepatology vol 31 no 2 pp 162ndash1721999

[34] J-H Jung J-I Kang and H-S Kim ldquoEffect of quercetin onimpaired immune function in mice exposed to irradiationrdquoNutrition Research and Practice vol 6 no 4 pp 301ndash307 2012

[35] R J Williams J P E Spencer and C Rice-Evans ldquoFlavonoidsantioxidants or signalling moleculesrdquo Free Radical Biology andMedicine vol 36 no 7 pp 838ndash849 2004

[36] C S Prasad and M Bala ldquoExploring in silico affinity offlavonoids and tannins to human fibroblast growth factor-inducible14 (Fn14) a member of TNF receptor super familyrdquoBioinformation vol 9 no 12 pp 633ndash638 2013

[37] S Saggu HMDivekar V Gupta R C Sawhney P K Banerjeeand R Kumar ldquoAdaptogenic and safety evaluation of seabuck-thorn (Hippophae rhamnoides) leaf extract a dose dependentstudyrdquo Food and Chemical Toxicology vol 45 no 4 pp609ndash617 2007

[38] P G S Prasanna and P U Devi ldquoModification of WR-2721radiation protection from gastrointestinal injury and death inmice by 2-mercaptopropionylglycinerdquo Radiation Research vol133 no 1 pp 111ndash115 1993

[39] M Bala S Tiwari J Prasad and S Singh ldquoHerbal Preparationfrom Hippophae leaves (SBL-2) render survival benefit protecthaemopoietic stem cells and liver stem cells in whole body irra-diated micerdquo in Seabuckthorn (Hippophae L)mdashA MultipurposeWonder Plant Emerging Trends in Research and Technologies VSingh Ed vol 4 Daya Publishing House Astral InternationalNew Delhi India 2014

[40] H C Goel J Prasad S Singh R K Sagar I P Kumar and A KSinha ldquoRadioprotection by a herbal preparation of Hippophaerhamnoides RH-3 against whole body lethal irradiation inmicerdquo Phytomedicine vol 9 no 1 pp 15ndash25 2002

[41] H C Goel and M Bala ldquoSeabuckthorn (Hippophae rham-noides) as a radioprotectorrdquo in Seabuckthorn A MultipurposeWonder Plant V Singh Ed vol 2 pp 419ndash455Daya PublishingHouse New Delhi India 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Bone marrow cells undergo mitotic death or apoptosis aftertotal body exposure to sublethal doses of low LET ionizingradiation (up to 3Gy) Assessment of bone marrow DNAdamage by scoring of micronuclei (MN assay) is one ofthe established methods for evaluating chromosomal DNAdamage caused by ionizing radiations [6] Micronuclei aresmall bodies in the cytoplasm resembling the nuclearmaterialin morphology and staining pattern They are formed whena broken chromosome or chromosome fragment does nottravel to the spindle during mitosis and therefore are notincluded in either of the daughter nuclei [6] Apoptosis isa physiological cell death which is under genetic control Itis characterized morphologically by increased cytoplasmicgranularity cell shrinkage and nuclear and chromosomalcondensation membrane blebbing and the formation ofdistinctive nuclear bodies Cells such as thymocytes lympho-cytes lymphoblasts and stem cells undergo apoptosis shortlyafter irradiation (peaking usually 3-4 h after irradiation) [7]Theprocess of apoptosis is controlled by a diverse range of cellsignals which may originate either extracellularly (extrinsicinducers) or intracellularly (intrinsic inducers)These signalsmay positively (ie trigger) or negatively (ie repress inhibitor dampen) affect apoptosis

The plant Hippophae rhamnoides L (common name seabuckthorn family Eleagnaceae) is a wonder plant loadedwith multitude of antioxidants and nutrients Sea buckthornis deciduous and dioecious shrub with silvery leaves andextensive root system which are very effective in controllingsoil erosionThis plant has been adapted to tolerate high radi-ation stress as well as extreme temperature (minus25∘C to +40∘C)prevalent at high altitude regions in northwest Himalayas(8000ndash11000 feet) for millions of years It was argued thatby virtue of adaptation such a plant may have accumulatedunique secondary metabolites which could be of help incountering radiation toxicity [8] A large number of bioactivecompounds known from this plant such as carotenoids a-tocopherols c-tocopherol b-tocotrienol steroids flavonoidshigh amount of unsaturated fatty acids vitamins A C EandKminerals tannins and polyphenols contributed to thehigh antioxidant hepatoprotective cardioprotective woundhealing properties [9ndash11] Traditionally sea buckthorn isrecommended for treatment of gastric ailments circulatorydisorders hepatic injuries and neoplasia [12] The leaves ofsea buckthorn are extremely popular for their nutraceuti-cal as well as medicinal values Okanagan sea buckthorntea of Canada has health promoting ingredients such ascalcium magnesium potassium beta-carotene flavonoidslycopene polyphenols vitamin E and protein Earlier studiesdemonstrated that sea buckthorn leaves prevented radiationtoxicity to experimental mice Only one time intraperitoneal(ip) treatment with 30mgkg body weight (bw) of SBL-1(extract from H rhamnoides leaves) 30min before irradia-tion with lethal dose of 60Co-gamma-rays (10Gy) rendered94 survivors in mice population for 30 days and beyondwhile all non-SBL-1 treated irradiated (10Gy) animals diedwithin 12 days after irradiation [8] SBL-1 treatment beforeirradiation countered the radiation induced inflammation inmice liver [13] and taste aversion (akin to early nausea andvomiting) in rats [14] The SBL-1 interacted in vitro with

DNA and displayed antioxidant properties in a concentrationdependent manner [15] It is proposed that presence ofmultiple bioactive constituents in a specific proportion wasresponsible for strong radioprotective effects of SBL-1 Thepurpose of this study was to provisionally quantify thekey bioactive compounds from groups of polyphenols andflavonoids in SBL-1 by using reverse phase high performanceliquid chromatography (RP-HPLC) analyze the chemicalcomposition by colorimetric and gravimetric methods andinvestigate the mechanism of radioprotective action of SBL-1by monitoring the bone marrow damage and early (at 48 h)as well as late (up to Day 30) changes in the jejunal histologyand levels of key proteins associated with apoptosis usingexperimental mice as model system

2 Materials and Methods

21 Reagents and Chemicals HPLC grade reference stan-dards 345-trihydroxybenzoic acid (gallic acid ethyl esterpurity 98) was purchased from Acros Organics FischerScientific and 3310158404101584056-pentahydroxy flavones (quercetindihydrate purity 98) was purchased from Fluka Bio-chemika Urea and thiourea were purchased from Hi-MediaLaboratories India 2378-Tetrahydroxy-chromeno[543-cde]chromene-510-dione (ellagic acid) dithiothreitol(DTT) acrylamide bis-acrylamide ammonium persulfatetetramethylethylenediamine (TEMED) sodium dodecylsulphate (SDS) tris-HCL phenylmethanesulfonylfluoride(PMSF) 3-[(3-cholamidopropyl) dimethylammonio]-1-pro-panesulfonate (CHAPS) tween 20 and 331015840551015840-tetra-methylbenzidine (TMB) substrate were purchased fromSigma-Aldrich USA Polyvinylidene fluoride (PVDF) mem-branes were purchased from Millipore (India) Pvt LtdIndia Mouse monoclonal anti-Bcl-2 primary antibodyanti-beta-actin primary antibody rabbit polyclonal anti-Bax primary antibody HRP conjugated anti-mouse IgGsecondary antibody HRP conjugated anti-rabbit IgGsecondary antibody and nonfat dry milk were purchasedfrom Santa Cruz Biotechnology CA Methanol glacialacetic acid formaldehyde hematoxylin and eosin stains andother analytical chemicals and reagents used for HPLC andphytochemical analyses were purchased from Merck PvtLtd India

22 Preparation of Herbal Extract SBL-1 The SBL-1 wasprepared as per procedure described earlier [8] Brieflyfresh green leaves of Hippophae rhamnoides L (family Eleag-naceae) common name sea buckthorn (identified and con-firmed by ethnobotanist the specimen records are preservedat museum Defence Institute of High Altitude ResearchLeh India (voucher specimen number SBTL-2006)) werecollected in the month of September from a specific naturalhabitat from western Himalayas India The leaves werecleaned washed thoroughly with distilled water dried andpowdered The extract was prepared by soaking the driedleaves powder in distilled water (1 1 wv) The water extractwas lyophilized to yield 0125 gram lyophilized extract pergram of dried leaves and was coded as SBL-1 (drug)















3 Results












O

O

OH

O

O

HH

H

O

O

O

O

OH

OH

HO

HO


O

O

OH

HO

OH

OH

OH middot 2H2O

(a)

293

795

580

Retention time

0

1000

2000

3000

(mAU

)0

1000

2000

3000

(mAU

)

2 4 6 8 10 12 140


(b)

Retention time

225

261

311

323

351

399

487 5

886

15 795

850

2810

100

200

300

(mAU

)

0

100

200

300

(mAU

)

2 4 6 8 10 12 140


(c)


4 Discussion





SBL-1+10

Gy

SBL-1

alon

eU

ntre

ated


cont

rol

Radi

atio

n(10

Gy)

(a)


lowastlowast lowast

lowast

lowast lowast

ControlRadiation


0

40

80

120

160

Cel

lsvi

llus

0

100

200

300

Villi

hei

ght (

pixe

ls)

0

5

10

15

20

Pane

th ce

llss

ectio

n

5 10 15 302


(b)


SBL-1+10

Gy

SBL-1

alon

eRa

diat

ion

Unt

reat

ed


cont

rol

(10

Gy)

(c)

Apop

totic

cells

C R D

Bcl2Actin

Treatments

BaxActin

RadiationDrug

Drug + radiation

0306090

120

15 cr

ypts


D + R

C R D D + R

C R D D + R

050

100150

Abso

rban

ce

0

20

40

Abso

rban

ce

(d)




Treatment

lowast

0

05

1

15

2

25

3M

icro

nucle

i (

)

(a)

lowast

lowast



0

05

1

15

2

25

Apop

tosis

()

(b)













5 Conclusion







Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























3 Results












O

O

OH

O

O

HH

H

O

O

O

O

OH

OH

HO

HO


O

O

OH

HO

OH

OH

OH middot 2H2O

(a)

293

795

580

Retention time

0

1000

2000

3000

(mAU

)0

1000

2000

3000

(mAU

)

2 4 6 8 10 12 140


(b)

Retention time

225

261

311

323

351

399

487 5

886

15 795

850

2810

100

200

300

(mAU

)

0

100

200

300

(mAU

)

2 4 6 8 10 12 140


(c)


4 Discussion





SBL-1+10

Gy

SBL-1

alon

eU

ntre

ated


cont

rol

Radi

atio

n(10

Gy)

(a)


lowastlowast lowast

lowast

lowast lowast

ControlRadiation


0

40

80

120

160

Cel

lsvi

llus

0

100

200

300

Villi

hei

ght (

pixe

ls)

0

5

10

15

20

Pane

th ce

llss

ectio

n

5 10 15 302


(b)


SBL-1+10

Gy

SBL-1

alon

eRa

diat

ion

Unt

reat

ed


cont

rol

(10

Gy)

(c)

Apop

totic

cells

C R D

Bcl2Actin

Treatments

BaxActin

RadiationDrug

Drug + radiation

0306090

120

15 cr

ypts


D + R

C R D D + R

C R D D + R

050

100150

Abso

rban

ce

0

20

40

Abso

rban

ce

(d)




Treatment

lowast

0

05

1

15

2

25

3M

icro

nucle

i (

)

(a)

lowast

lowast



0

05

1

15

2

25

Apop

tosis

()

(b)













5 Conclusion







Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















O

O

OH

O

O

HH

H

O

O

O

O

OH

OH

HO

HO


O

O

OH

HO

OH

OH

OH middot 2H2O

(a)

293

795

580

Retention time

0

1000

2000

3000

(mAU

)0

1000

2000

3000

(mAU

)

2 4 6 8 10 12 140


(b)

Retention time

225

261

311

323

351

399

487 5

886

15 795

850

2810

100

200

300

(mAU

)

0

100

200

300

(mAU

)

2 4 6 8 10 12 140


(c)


4 Discussion





SBL-1+10

Gy

SBL-1

alon

eU

ntre

ated


cont

rol

Radi

atio

n(10

Gy)

(a)


lowastlowast lowast

lowast

lowast lowast

ControlRadiation


0

40

80

120

160

Cel

lsvi

llus

0

100

200

300

Villi

hei

ght (

pixe

ls)

0

5

10

15

20

Pane

th ce

llss

ectio

n

5 10 15 302


(b)


SBL-1+10

Gy

SBL-1

alon

eRa

diat

ion

Unt

reat

ed


cont

rol

(10

Gy)

(c)

Apop

totic

cells

C R D

Bcl2Actin

Treatments

BaxActin

RadiationDrug

Drug + radiation

0306090

120

15 cr

ypts


D + R

C R D D + R

C R D D + R

050

100150

Abso

rban

ce

0

20

40

Abso

rban

ce

(d)




Treatment

lowast

0

05

1

15

2

25

3M

icro

nucle

i (

)

(a)

lowast

lowast



0

05

1

15

2

25

Apop

tosis

()

(b)













5 Conclusion







Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SBL-1+10

Gy

SBL-1

alon

eU

ntre

ated


cont

rol

Radi

atio

n(10

Gy)

(a)


lowastlowast lowast

lowast

lowast lowast

ControlRadiation


0

40

80

120

160

Cel

lsvi

llus

0

100

200

300

Villi

hei

ght (

pixe

ls)

0

5

10

15

20

Pane

th ce

llss

ectio

n

5 10 15 302


(b)


SBL-1+10

Gy

SBL-1

alon

eRa

diat

ion

Unt

reat

ed


cont

rol

(10

Gy)

(c)

Apop

totic

cells

C R D

Bcl2Actin

Treatments

BaxActin

RadiationDrug

Drug + radiation

0306090

120

15 cr

ypts


D + R

C R D D + R

C R D D + R

050

100150

Abso

rban

ce

0

20

40

Abso

rban

ce

(d)




Treatment

lowast

0

05

1

15

2

25

3M

icro

nucle

i (

)

(a)

lowast

lowast



0

05

1

15

2

25

Apop

tosis

()

(b)













5 Conclusion







Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Treatment

lowast

0

05

1

15

2

25

3M

icro

nucle

i (

)

(a)

lowast

lowast



0

05

1

15

2

25

Apop

tosis

()

(b)













5 Conclusion







Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















5 Conclusion







Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Sea Buckthorn Leaf Extract Protects Jejunum...

Documents

Transcript of Research Article Sea Buckthorn Leaf Extract Protects Jejunum...