Research Article The Protective Effects of Buzui on Acute...

Research ArticleThe Protective Effects of Buzui on Acute Alcoholism in Mice

Chen Chen1 Da-Chao Wen2 Shu-di Gao3 Xiao-yu Hu3 and Cheng Yi1

1Division of Abdominal Cancer West China Hospital Sichuan University Chengdu Sichuan 610041 China2School of Clinical Medicine Chengdu University of Traditional Chinese Medicine Chengdu Sichuan 610072 China3Department of Infectious Diseases Affiliated Hospital of Chengdu University of Traditional Chinese MedicineChengdu Sichuan 610072 China

Correspondence should be addressed to Cheng Yi yicheng6834163com

Received 14 September 2015 Revised 30 November 2015 Accepted 27 December 2015

Academic Editor Yibin Feng

Copyright copy 2016 Chen Chen 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

This study was designed to investigate the role of a traditional buzui recipe in anti-inebriation treatment Buzui consists of FructusSchisandrae Chinensis Fructus Chebulae Fructus Mume Fructus Crataegi Endothelium Corneum Gigeriae Galli and ExcrementumBombycis The buzui mixture was delivered by gavage and ethanol was delivered subsequent to the final treatment The effectsof buzui on the righting reflex inebriation rates and the survival curve are depicted Blood alcohol concentrations alanineaminotransferase (ALT) levels aspartate aminotransferase (AST) levels and alkaline phosphatase (ALP) levels were recordedThe activities of alcohol dehydrogenase (ADH) aldehyde dehydrogenase (ALDH) and superoxide dismutase (SOD) as well asmalonaldehyde (MDA) levels were also measured Our results demonstrated that a traditional buzui recipe showed significanteffects on promoting wakefulness and the prevention of acute alcohol intoxication accelerating the metabolism of alcohol in theliver and reducing the oxidative damage caused by acute alcoholism

1 Introduction

Acute alcohol intoxication (AAI) is the result of a singleepisode of excessive drinking over the oxidative metabolismof the liver Ethanol accumulates in the body and thebrain causing central nervous system suppression after initialexcitement Clinical manifestations are heterogeneous andinvolve different organs and apparatuses with behavioralcardiac gastrointestinal pulmonary neurologicalmetabolicand especially hepatic effects [1] AAI is a clinically commondisease typically to the detriment of the drinkerrsquos healthpersonal relationships and social standing [2ndash4]

The liver is one of the major target organs of ethanolactions [5] Free radical mechanisms contribute to ethanol-induced liver injury [6] In general the production and elim-ination of oxygen free radicals in the body exist in homeosta-sis Hepatocytes have antioxidation enzymes such as super-oxide dismutase (SOD) to mitigate the harm caused by reac-tive oxygen species and oxidative processes When thisbalance is destroyed by alcohol excessive oxygen free radicalsperoxide the biomembrane causing cell damage and disease[7]

Because of social contact with Western countries andthe growing alcohol culture in China people recognize thedangers of alcohol but alcoholism remains a persistent socialconcern How to prevent and avoid the harm of alcohol whendrinking is an outstanding research question Unfortunatelyno effective and safe reagents have yet emerged as AAI pre-ventive drugs Compared with relatively new synthetic drugsfor alcoholic intoxication traditional Chinese medicine hasbeen used in the prevention and treatment of alcohol-induced liver disease for more than two millennia and it hassignificant effects [8] Moreover natural medicines presentwide therapeutic spectra to various targets and promote thegradual recovery of physical conditions without toxicity Thebuzui recipe used in this study derives from the hangoverprescription of Professor Xiao-yu Hu and includes FructusSchisandrae Chinensis Fructus Chebulae Fructus MumeFructus Crataegi Endothelium Corneum Gigeriae Galli andExcrementum Bombycis

In this study we investigated the role of this buzuirecipe (BZ) in inebriation prevention RU-21 a classical drugfor alcoholic intoxication was used as the positive control

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 3539748 8 pageshttpdxdoiorg10115520163539748

2 Evidence-Based Complementary and Alternative Medicine

Table 1 The formula for the buzui recipe (BZ one dose)

Chinese medicinename

Medicinalparts Origin

Amount inpreparation

(g)Schisandra chinensis(Turcz)Baill(Beiwuweizi)

Fruit Heilongjiangprovince 40

Terminalia chebulaRetz(Hezi)

Fruit Yunnanprovince 40

Dark plum fruit(Wumei) Fruit Sichuan

province 50

Crataegus pinnatifidaBunge(shanzha)

Fruit Shandongprovince 40

Chickenrsquos gizzardmembrane(Jineijin)

Gizzard Sichuanprovince 60

Silkworm excrement(cansha) Excrement Sichuan

province 30

We present evidence that AAI may cause uncoordinatedmovements and alterations in hepatocyte structure and func-tioning and that BZ plays a key role in protecting the liverthrough antioxidant effects

2 Materials and Methods

21 Materials and Chemicals Alcohol metabolism supple-ment description (RU-21) (lot 108671) a commonly usedhangover drug was purchased from American Spirit ScienceLtd Detection kits for alanine aminotransferase (ALT) (lot120272) aspartate aminotransferase (AST) (lot 120272) andalkaline phosphatase (ALP) (lot 120166) were provided byBiosino Biotechnology Co Ltd (Beijing China) Detectionkits for superoxide dismutase (SOD) (lot 20120216) mal-ondialdehyde (MDA) (lot 20120212) and aldehyde dehy-drogenase (ALDH) (lot 20120214) were obtained from theNanjing Jiancheng Institute of Biological Engineering (Nan-jing China) The formula for the buzui recipe (BZ onedose) is presented in Table 1 All ingredients were purchasedfrom the Affiliated Hospital of the Chengdu University ofTraditional ChineseMedicine (Chengdu China) and authen-ticated by Professor Zhu-yun Yan (IdentificationDepartmentof the Chengdu University of TCM) according to the Phar-macopoeia of the Peoplersquos Republic of China (2010) Theirvoucher specimens were deposited at Teaching Hospital ofthe Chengdu University of Traditional Chinese Medicine(Chengdu Sichuan China)

22 Animals Two hundred male pathogen-free (SPF) Kun-ming mice weighing 20 plusmn 2 g were purchased from theChengdu Dashuo Experimental Animal Research Center(Chengdu China) They were housed 3 per cage and main-tained at a controlled ambient temperature (23 plusmn 2∘C) underdiurnal conditions (lightndashdark 0700ndash1900) for one weekAll mice were allowed access to laboratory chow and tap

water ad libitum The animals were cared for in accordancewith the Guiding Principles for the Care and Use of Animals

23 Dosage of Reagents After identification by Profes-sor Zhu-yun Yan of the Identification Department of theChengdu University of TCM of genuine medicinal materi-als the herbs were decocted filtered and concentrated to35 gsdotmLminus1 A gastric lavage dose of 1 kgmouse = mg60 kgtimes 1233 (M refers to the dose of Chinese medicine and 60 kgfor adult standard weight) was prepared

Similarly an RU-21 adult clinical dose (12 g times 6 = 72 gkgd equivalent dose mice 72 g60 kg times 1233 = 148 gkgd)was also prepared Before use both treatments were groundand then dissolved in NS to make a suspension with aconcentration of 106mgmL

24 Experimental Design A total of 48 mice were randomlydivided into four groups with 12 mice in each group asfollows A negative control group (Control) B model group(Model) C alcohol metabolism supplement description(RU-21) group (RU-21) and D buzui recipe group (BZ)All mice were fasted on water only for 12 h before theexperiment The RU-21 and BZ groups were dosed with RU-21 148 gkg or BZ 49 gkg by gavage respectivelyThe controland model groups received equal volumes of normal salineinstead Thirty minutes later apart from the control groupgiven an equal volume of saline the remainder was dosedwith 627 gkg ethanol to establish a mouse model of acutealcoholism

25 Estimation of Liver Function Serum was collected 6 hafter modeling Then the serum was centrifuged at3000 rmin for 5min at 4∘C ALT AST and ALP activitiesin the serum were measured using commercially availabledetection kits according to the manufacturerrsquos instructions

26 Histopathology of the Liver and Pathological ScoresLiver tissues from the right liver were fixed in 10 for-malin and embedded with paraffin Then 5120583m thick sec-tions were stained with hematoxylin and eosin (HampE) Thehistopathological changes of the liver were observed withlight microscopy

All slides were read by three investigators who wereblinded to the allocation arm of the animalThe investigatorswere asked to grade the fat variable score and inflammationscore in the liver using a semiquantitative scoring systemwith zero indicating no discernable injury and 4 indicatingthe presence of severe injury [9]

27 Assay of the Metabolism of Ethanol in the Liver Liver tis-sue samples from the left liver were prepared by homogeniza-tion with ice-cold isotonic saline to yield a 10 (wv) tissuehomogenate The homogenate was centrifuged at 3000 rminfor 20min and the resulting supernatant was stored at minus80∘Cfor SOD andMDA estimation SOD activity wasmeasured byxanthine oxidase and theMDA content was measured by thepresence of thiobarbituric acid reactive substances (TBARS)according to Buege (Buege and Aust 1978 [10]) ADH and

Evidence-Based Complementary and Alternative Medicine 3

Table 2 Blood alcohol concentrations following acute alcoholismmice (unit mgsdotmLminus1)

Hour GroupsControl Model RU-21 BZ

0 h 0 0 0 005 h 0 3603 plusmn 079 3033 plusmn 067lowast 2816 plusmn 109lowast998771

1 h 0 3722 plusmn 027 3228 plusmn 07lowast 3112 plusmn 095lowast998771




6 h 0 3313 plusmn 046 2836 plusmn 136lowast 2698 plusmn 117lowastlowast

119875 lt 001 compared with model group 119875 lt 005 compared with RU-21group and 998771119875 lt 001 compared with RU-21 group

ALDH activities in liver were determined by kits accordingto the manufacturerrsquos instructions

28 Blood Ethanol Concentrations Oxidized forms of nic-otinamide-adenine dinucleotide (NAD) were added to theblood standard solution with different concentrations ofalcoholThe absorbance of the reactionmixture was recordedat 340 nm and the regression equation of the ethanol concen-tration was achieved as C = 12376A minus 11862 (119903 = 0967) bythe standard curveThe ethanol concentration in the blood ofmice was calculated by the formula

29 Measures of Drunken Behaviors in Mice and SurvivalCurves An additional 120 mice were divided into the abovefour groups with 30 animals per group for observations ofdrunken behaviors and survival The treatments were thesame as before The time to loss of righting reflex (alcoholtolerance time) and the times to recovery of the righting reflex(sober-up time) were recorded and the numbers of deathsover 24 hours were also recorded

210 Statistical Analyses All statistical analyses were per-formed using SPSS software package version 170 Data wereexpressed as the means plusmn standard deviation (SD) The sig-nificance of the differences between groups was determinedusing analyses of variance (ANOVA) A value of 119875 lt 005(2-sided) was considered statistically significant

3 Results

31 Blood Alcohol Concentrations after Acute AlcoholismMiceAs shown in Table 2 blood alcohol concentrations reachedtheir peak at 2 hours after alcohol intake Compared with themodel group blood alcohol concentrations were significantlyreduced at all times after alcohol administration in both theRU-21 and BZ groups (119875 lt 001) Furthermore blood alcoholconcentrations were significantly reduced in the animalstreated with BZ compared with RU-21 (119875 lt 001 or119875 lt 005)

32 Protective Effects of BZ in Liver Function Serum ALTAST and ALP levels are presented in Table 3 and Figure 1From these results it was apparent that the ALT AST and

Table 3 Comparisons of acute alcohol intoxication in mice ALTAST and ALP levels in the groups (119909 plusmn 119878 119899 = 12)

Groups ALT (UL) AST (UL) ALP (UL)Control 7658 plusmn 1034 10517 plusmn 1234 15958 plusmn 1643Model 12556 plusmn 1489lowast 33111 plusmn 2543lowast 21944 plusmn 2138lowast

RU-21 10240 plusmn 1650lowast 19450 plusmn 2815lowast 14730 plusmn 2020lowast

BZ 8309 plusmn 812lowast998771 18073 plusmn 2035lowast 12491 plusmn 1878lowastlowast

119875 lt 001 compared with control group 119875 lt 005 compared with RU-21group and 998771119875 lt 001 compared with RU-21 group

Table 4 Comparisons of Acute alcohol intoxication in mice SODand MDA levels in the groups (119909 plusmn 119878 119899 = 12)

Groups SOD (Umg prot) MDA (nmolmL)Control 13943 plusmn 485 463 plusmn 035Model 10567 plusmn 1141lowast 982 plusmn 086lowast

RU-21 15745 plusmn 582lowast 432 plusmn 043

BZ 15326 plusmn 733lowast 375 plusmn 043lowast998771lowast

119875 lt 001 compared with Control group 119875 lt 001 compared with Modelgroup 998771119875 lt 005 compared with RU-21 group

ALP levels in the RU-21 and BZ groups were decreasedcompared with those in the model group (119875 lt 001)Compared with the RU-21 group the ALT (119875 lt 001)and ALP levels (119875 lt 005) of mice in the BZ group weresignificantly decreased However no significant differenceswere noted between the RU-21 group and the BZ group inAST (119875 gt 005)

33 The MDA Content and SOD Activity of Each Group inLiver Tissue As presented in Table 4 and Figure 2 comparedwith the control group SOD activity in the model group wassignificantly decreased (119875 lt 001) and MDA levels were sig-nificantly elevated (119875 lt 001) RU-21 and BZ treatment re-sulted in SOD activity increases and MDA level decreases

34 Liver Pathology and Pathological Scores General obser-vations of samples of the livers in the control group includedthe presence of a pale red color smooth surface no spots orbruising and softness with no adhesion to the surroundingtissue The model group was described as dark red smoothslightly tough partial liver swelling tense and dull livercapsule and partial surrounding tissue adhesion The RU-21and BZ groups were slightly darker than normalmouse liversexhibiting surfaces without petechiae with liver capsules thatwere smooth and shiny and without adhesion phenomena

In histological examinations using light microscopy thecontrol group exhibited normal lobular architecture withhepatic cords which are neat and sinusoidal as well as noexpansion gap fatty degeneration necrosis or inflamma-tory cell infiltration The lobule structures of the livers inthe model group were disordered with swelling obviousaround the central vein of the liver cells The cytoplasm wastranslucent exhibiting ballooning degeneration necrosisand visible inflammatory cell infiltration The RU-21 and BZgroups were more comparable to the normal group with


150

100

50

0

ALT

(UL

)

Control Model RU-21 BZ

ALT

998771

(a)

AST400

300

200

100

0

AST

(UL

)

Control Model RU-21 BZ(b)

ALP300

200

100

0

ALP

(UL

)


(c)

Figure 1 Acute alcohol intoxication in mice liver enzyme changes (a) ALT (b) AST (c) ALP Control mice without alcohol or any drugsModel the model group treated with alcohol only RU-21 the positive control group treated with RU-21 before alcohol BZ the experimentgroup treated with buzui recipe before alcohol 119875 lt 005 compared with RU-21 group 998771119875 lt 001 compared with RU-21 group

SOD

200

150

100

50

0

SOD

(Um

g pr

ot)


lowast

lowast

lowast

(a)

MDA

15

10

5

0

MD

A (n

mol

mL)


998771

lowast

lowast

(b)

Figure 2 Comparison of acute alcohol intoxication inmice SOD andMDA among groups (a) SOD (b)MDA Control mice without alcoholor any drugs Model the model group treated with alcohol only RU-21 the positive control group treated with RU-21 before alcohol BZ theexperiment group treated with buzui recipe before alcohol For control model RU-21 and Buzui groups the SOD were 13943 plusmn 485Umgprot 10567plusmn1141Umg prot 15745plusmn582Umg prot and 15326plusmn733Umg prot respectively and theMDAwere 463plusmn035 nmolmL982 plusmn 086 nmolmL 432 plusmn 043 nmolmL and 375 plusmn 043 nmolmL respectively lowast119875 lt 001 compared with control group 119875 lt 001compared with model group and 998771119875 lt 005 compared with RU-21 group


Control Model

RU-21 BZ

Figure 3 Livers of each group with HampE staining

Table 5 Pathological scores (119909 plusmn 119878 119899 = 12)

Groups Fat variable score Inflammation scoreControl 0 0Model 317 plusmn 103lowastlowast 267 plusmn 089lowastlowast

RU-21 217 plusmn 127 233 plusmn 089BZ 133 plusmn 151 158 plusmn 079998771lowastlowast

119875 lt 001 compared with control group 119875 lt 005 compared with modelgroup 119875 lt 001 compared with model group 119875 lt 005 compared withRU-21 group and 998771119875 lt 005 compared with RU-21 group

part of the liver cells swollen In addition the boundariesbetween liver cells were clear and the cytoplasm lightlystained Individual portal areas for a few inflammatory cellswere visible (Figure 3 and Table 5)

Table 6 Comparisons of acute alcohol intoxication in mice ADHand ALDH levels in the groups (119909 plusmn 119878 119899 = 12)

Groups ADH (nmol(gsdotmin)) ALDH (UL)Normal 0175 plusmn 0016 2127 plusmn 371Model 0191 plusmn 0016 4604 plusmn 803lowast

RU-21 0213 plusmn 0015lowast 12446 plusmn 2768lowast

BZ 0295 plusmn 0037lowast998771998771 9373 plusmn 1537lowast998771lowast

119875 lt 005 compared with control group 119875 lt 005 119875 lt 001 comparedwith model group and 998771119875 lt 005 998771998771119875 lt 001 compared with RU-21 group

35 ADH and ALDH Levels in Liver Tissue As shown inTable 6 acute ethanol administered to mice induced incre-mented ADH activity in the liver and this elevation in ADHactivity in BZ-pretreated animals was considerably increasedcompared with the model group or the RU-21 group (119875 lt


Alcohol tolerance time400

300

200

100

0

Tim

e (m

in)

Control Model RU-21 BZ(a)

Sober-up time800

600

400

200

0

Tim

e (m

in)


Figure 4 Comparison of alcohol tolerance time and sober-up time among groups (a) Alcohol tolerance time (b) sober-up time

Table 7 Comparisons of the sober-up times and death rates amonggroups (119909 plusmn 119878 119899 = 30)

Groups 119873Sober-up time(119909 plusmn 119878 min)

Death numbers orrate (119899 )

Normal 30 mdash mdashModel 19 6662 plusmn 4431 11 (37)RU-21 23 3952 plusmn 3195lowast 7 (23)BZ 28 28440 plusmn 270lowast 2 (6)lowast

119875 lt 001 compared with model group and 119875 lt 001 compared with RU-21 group

001) A similar tendency was also shown for ALDH activityamong the model RU-21 and BZ groups but the BZ groupwas lower than the RU-21 group Pretreatment with BZfurther enhanced ADH and ALDH activities in the liver afteralcohol administration

36 Effects on the Behavior of Mice Compared with themodel group the alcohol tolerance times of mice in theRU-21 and BZ groups were significantly extended Similarlythe sober-up time was shortened Significant differenceswere noted between the RU-21 and BZ groups indicatingthat BZ had stronger effects (Figure 4 Table 7) We alsoobserved changes in mouse appetite behavior and varioussymptoms such as urinary incontinence lethargy irritabilityand convulsion These behaviors were common in the modelgroup but rare in the BZ group

37 Survival Curves We excluded the control group fromoursurvival curves because there were no deaths at the endpointBZ significantly improved survival rates after acute alcoholintoxication (Figure 5)

4 Discussion

In this study we observed increased serum ALT AST andALP levels and reductions in the uncoordinated movementsof mice and damage to their livers after acute alcohol

10

09

08

07

06

05

Cum

surv

ival

00 50 100 150 200 250

Time (h)

GroupModelRU-21BZ

Figure 5 The survival curve Model the model group treated withalcohol only RU-21 the positive control group treated with RU-21before alcohol BZ the experiment group treated with buzui recipebefore alcohol

intoxication models RU-21 and BZ both ameliorated liverfunction and prevented AAI however the effects of BZ werestronger

Currently oral gavage and intraperitoneal injection arethe main two methods to dose ethanol for acute alcoholintoxication in mice We choose gavage because it simu-lates normal human drinking and the alcohol is absorbedthrough the digestive tracts of themice Furthermore alcoholadministered intragastrically results in obvious abnormalliver functioning even compared with similar pathologic


changes in the human liver following acute intoxication[11]

We performed prior experiments (data not shown) inaccordance with the related literature using ethanol at dosesof 448 gKg 538Kg 627 gKg 717 gKg and 806 gKgWe observed drunken mice and computed intoxication ratesand mortality at each of these doses After repeated tests wefound that administration in the amount of 627 gkg resultsin inebriation ratesge90andmortality ratesle40 reflectingmodeling success We determined that the mouse becameinebriated when the righting reflex disappeared

The efficiency of traditional Chinese medicine (TCM) isbased on reinforcing an organismrsquos natural healing powerand ability to restore energy homoeostasis Previous studieshave demonstrated that a likely mechanism for at least someof these activities is interaction with redox balance and theprevention of oxidative stress [12] Some traditional Chinesemedicinal seeds and fruits are well known for their antiox-idant properties Regarding the Chinese medicine includedin this recipe Fructus Chebulae might be the mechanismfor the inhibition of oxidative damage [13] Fructus Schisan-drae Chinensis exhibits pharmacological effects includingliver protection antihypoxia antifatigue immunity enhance-ment antioxidation and antitumor [14 15] Fructus Mumeenhances intestinal propulsive motion and motility [16] andit might play a therapeutic role by enhancing SOD activityand lowering MDA content [17] Fructus Crataegimight reg-ulate lipidmetabolism enhancing antioxidant properties andreducing the release of inflammatory factors [18] Endothe-lium Corneum Gigeriae Galli possesses strong hydroxylradical scavenging properties as well as Fe2+ chelating andlipid peroxidation inhibitory and antioxidant activities invitro [19] Extracts from Excrementum Bombycis (BE) whichconsists of various bioactive constituents andmulberry leaves(the preferred food of silkworms) possess anti-inflammatoryantidiabetic and antioxidative effects [20] BZ combinesthese Chinese herb medicines following the principle ofclearing heat and resolving stasis BZ exhibited a positiveeffect in this study

Over recent years studies have extensively searched foreffective herbs for the treatment of alcohol-related diseasesBZ is derived from some uncommon herbs that exhibit anti-inebriation effects including Flos Puerariae and Radix Puer-ariae Its formulation is based on clinically reliable alcoholmetabolism pathways and the recipe refers to TCM theorymatching herbs to improve their natural hepatoprotectiveeffects BZ is innovative andmore importantly no treatment-related side effects were discovered

In the present study we found that buzui has positiveeffects and we believe it may even have more comprehensiveeffects in humanbeings including postponing the inebriationtime reducing sleep duration reducing the degree of inebri-ation and exhibiting prominent effects against liver oxidativedamage

5 Conclusion

Our buzui recipe demonstrated significant effects on promot-ing wakefulness and preventing acute alcohol intoxication

This mixture accelerated the metabolism of alcohol in theliver and reduced the oxidative damage caused by acutealcoholismThese effects are thought to bemediated by inter-actions with redox balance and the prevention of oxidativestress

Ethical Approval

All animal procedures were approved by theAnimal Care andScientific Committee of Chengdu University of TraditionalChinese Medicine

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Chen Chen and Da-chao Wen equally contributed to thepaper

References

[1] L Vonghia L Leggio A Ferrulli M Bertini G Gasbarrini andGAddolorato ldquoAcute alcohol intoxicationrdquoEuropean Journal ofInternal Medicine vol 19 no 8 pp 561ndash567 2008

[2] R D Brewer and M H Swahn ldquoBinge drinking and violencerdquoThe Journal of the American Medical Association vol 294 no 5pp 616ndash618 2005

[3] American Psychiatric Association Diagnostic and StatisticalManual of Mental Disorders American Psychiatric AssociationWashington DC USA 4th edition 2000

[4] H A Sindelar N P Barnett and A Spirito ldquoAdolescent alcoholuse and injuryrdquo Minerva Pediatrica vol 56 no 3 pp 291ndash3092004

[5] C S Lieber ldquoHepatic metabolic and toxic effects of ethanol1991 updaterdquo Alcoholism Clinical and Experimental Researchvol 15 no 4 pp 573ndash592 1991

[6] C S Lieber ldquoAlcoholic liver injury pathogenesis and therapy in2001rdquo Pathologie Biologie vol 49 no 9 pp 738ndash752 2001

[7] J I Beier and C J McClain ldquoMechanisms and cell signaling inalcoholic liver diseaserdquo Biological Chemistry vol 391 no 11 pp1249ndash1264 2010

[8] B J Xu Y N Zheng and C K Sung ldquoNatural medicines foralcoholism treatment a reviewrdquo Drug and Alcohol Review vol24 no 6 pp 525ndash536 2005

[9] Fatty Liver and Alcoholic Liver Disease Group of the ChineseMedical Association ldquoThe diagnosis and treatment of alcoholicliver diseaserdquoChinese Journal of Liver Diseases vol 4 pp 49ndash532010

[10] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquoMethods in Enzymology vol 52 pp 302ndash310 1978

[11] Z Jinbo W Tailing Z Jing et al ldquoRat model of acute alcoholicliver injuryrdquo Journal of China-Japan Friendship Hospital vol 10no 1 article 17 1996

[12] A Matkowski W Jamiołkowska-Kozlowska and I NawrotldquoChinese medicinal herbs as source of antioxidant com-poundsmdashwhere tradition meets the futurerdquo Current MedicinalChemistry vol 20 no 8 pp 984ndash1004 2013


[13] B P Gaire and H Kim ldquoNeuroprotective effects of Fruc-tus Chebulae extracts on experimental models of cerebralischemiardquo Journal of Traditional Chinese Medicine vol 34 no1 pp 69ndash75 2014

[14] W Chen and Y Ji ldquoProgress on pharmacological action offructus schisandrae polysacchariderdquo Food and Drug vol 12 p22 2007

[15] HWagner R Bauer DMelchart P-G Xiao andA StaudingerldquoFructus schisandraemdashWuweizirdquo in Chromatographic Finger-print Analysis of Herbal Medicines pp 37ndash44 Springer ViennaAustria 2011

[16] L Wang and H Y Zhang ldquoComparison of pharmacologicaleffects of FructusMume and its processed productsrdquo Zhong YaoCai vol 33 no 3 pp 353ndash356 2010

[17] A He Y Wang and S Lin ldquoThe effect of Fructus Mume waterextract on experimental ulcerative colitis in micerdquo Journal ofPharmaceutical Practice vol 30 no 5 pp 357ndash360 2012

[18] W Wang B Yang L Wang et al ldquoAntiatherogenic effect ofradix Salvia miltiorrhiza and Fructus Crataegi on experimentalatherosclerosis in ratsrdquo China Journal of Chinese Materia Med-ica vol 36 no 6 pp 784ndash789 2011

[19] Q Xiong X Li R Zhou et al ldquoExtraction characterizationand antioxidant activities of polysaccharides from E corneumgigeriae gallirdquo Carbohydrate Polymers vol 108 no 1 pp 247ndash256 2014

[20] M Moon J G Choi S Y Kim et al ldquoBombycis excrementumreduces amyloid-120573 oligomer-induced memory impairmentsneurodegeneration and neuroinflammation inmicerdquo Journal ofAlzheimerrsquos Disease vol 41 no 2 pp 599ndash613 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 The formula for the buzui recipe (BZ one dose)

Chinese medicinename

Medicinalparts Origin

Amount inpreparation

(g)Schisandra chinensis(Turcz)Baill(Beiwuweizi)

Fruit Heilongjiangprovince 40

Terminalia chebulaRetz(Hezi)

Fruit Yunnanprovince 40

Dark plum fruit(Wumei) Fruit Sichuan

province 50

Crataegus pinnatifidaBunge(shanzha)

Fruit Shandongprovince 40

Chickenrsquos gizzardmembrane(Jineijin)

Gizzard Sichuanprovince 60

Silkworm excrement(cansha) Excrement Sichuan

province 30

We present evidence that AAI may cause uncoordinatedmovements and alterations in hepatocyte structure and func-tioning and that BZ plays a key role in protecting the liverthrough antioxidant effects

2 Materials and Methods

21 Materials and Chemicals Alcohol metabolism supple-ment description (RU-21) (lot 108671) a commonly usedhangover drug was purchased from American Spirit ScienceLtd Detection kits for alanine aminotransferase (ALT) (lot120272) aspartate aminotransferase (AST) (lot 120272) andalkaline phosphatase (ALP) (lot 120166) were provided byBiosino Biotechnology Co Ltd (Beijing China) Detectionkits for superoxide dismutase (SOD) (lot 20120216) mal-ondialdehyde (MDA) (lot 20120212) and aldehyde dehy-drogenase (ALDH) (lot 20120214) were obtained from theNanjing Jiancheng Institute of Biological Engineering (Nan-jing China) The formula for the buzui recipe (BZ onedose) is presented in Table 1 All ingredients were purchasedfrom the Affiliated Hospital of the Chengdu University ofTraditional ChineseMedicine (Chengdu China) and authen-ticated by Professor Zhu-yun Yan (IdentificationDepartmentof the Chengdu University of TCM) according to the Phar-macopoeia of the Peoplersquos Republic of China (2010) Theirvoucher specimens were deposited at Teaching Hospital ofthe Chengdu University of Traditional Chinese Medicine(Chengdu Sichuan China)

22 Animals Two hundred male pathogen-free (SPF) Kun-ming mice weighing 20 plusmn 2 g were purchased from theChengdu Dashuo Experimental Animal Research Center(Chengdu China) They were housed 3 per cage and main-tained at a controlled ambient temperature (23 plusmn 2∘C) underdiurnal conditions (lightndashdark 0700ndash1900) for one weekAll mice were allowed access to laboratory chow and tap

water ad libitum The animals were cared for in accordancewith the Guiding Principles for the Care and Use of Animals

23 Dosage of Reagents After identification by Profes-sor Zhu-yun Yan of the Identification Department of theChengdu University of TCM of genuine medicinal materi-als the herbs were decocted filtered and concentrated to35 gsdotmLminus1 A gastric lavage dose of 1 kgmouse = mg60 kgtimes 1233 (M refers to the dose of Chinese medicine and 60 kgfor adult standard weight) was prepared

Similarly an RU-21 adult clinical dose (12 g times 6 = 72 gkgd equivalent dose mice 72 g60 kg times 1233 = 148 gkgd)was also prepared Before use both treatments were groundand then dissolved in NS to make a suspension with aconcentration of 106mgmL

24 Experimental Design A total of 48 mice were randomlydivided into four groups with 12 mice in each group asfollows A negative control group (Control) B model group(Model) C alcohol metabolism supplement description(RU-21) group (RU-21) and D buzui recipe group (BZ)All mice were fasted on water only for 12 h before theexperiment The RU-21 and BZ groups were dosed with RU-21 148 gkg or BZ 49 gkg by gavage respectivelyThe controland model groups received equal volumes of normal salineinstead Thirty minutes later apart from the control groupgiven an equal volume of saline the remainder was dosedwith 627 gkg ethanol to establish a mouse model of acutealcoholism

25 Estimation of Liver Function Serum was collected 6 hafter modeling Then the serum was centrifuged at3000 rmin for 5min at 4∘C ALT AST and ALP activitiesin the serum were measured using commercially availabledetection kits according to the manufacturerrsquos instructions

26 Histopathology of the Liver and Pathological ScoresLiver tissues from the right liver were fixed in 10 for-malin and embedded with paraffin Then 5120583m thick sec-tions were stained with hematoxylin and eosin (HampE) Thehistopathological changes of the liver were observed withlight microscopy

All slides were read by three investigators who wereblinded to the allocation arm of the animalThe investigatorswere asked to grade the fat variable score and inflammationscore in the liver using a semiquantitative scoring systemwith zero indicating no discernable injury and 4 indicatingthe presence of severe injury [9]

27 Assay of the Metabolism of Ethanol in the Liver Liver tis-sue samples from the left liver were prepared by homogeniza-tion with ice-cold isotonic saline to yield a 10 (wv) tissuehomogenate The homogenate was centrifuged at 3000 rminfor 20min and the resulting supernatant was stored at minus80∘Cfor SOD andMDA estimation SOD activity wasmeasured byxanthine oxidase and theMDA content was measured by thepresence of thiobarbituric acid reactive substances (TBARS)according to Buege (Buege and Aust 1978 [10]) ADH and















3 Results


















150

100

50

0

ALT

(UL

)


ALT

998771

(a)

AST400

300

200

100

0

AST

(UL

)


ALP300

200

100

0

ALP

(UL

)


(c)


SOD

200

150

100

50

0

SOD

(Um

g pr

ot)


lowast

lowast

lowast

(a)

MDA

15

10

5

0

MD

A (n

mol

mL)


998771

lowast

lowast

(b)



Control Model

RU-21 BZ















300

200

100

0

Tim

e (m

in)


Sober-up time800

600

400

200

0

Tim

e (m

in)











4 Discussion


10

09

08

07

06

05

Cum

surv

ival

00 50 100 150 200 250

Time (h)

GroupModelRU-21BZ










5 Conclusion



Ethical Approval






References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























3 Results


















150

100

50

0

ALT

(UL

)


ALT

998771

(a)

AST400

300

200

100

0

AST

(UL

)


ALP300

200

100

0

ALP

(UL

)


(c)


SOD

200

150

100

50

0

SOD

(Um

g pr

ot)


lowast

lowast

lowast

(a)

MDA

15

10

5

0

MD

A (n

mol

mL)


998771

lowast

lowast

(b)



Control Model

RU-21 BZ















300

200

100

0

Tim

e (m

in)


Sober-up time800

600

400

200

0

Tim

e (m

in)











4 Discussion


10

09

08

07

06

05

Cum

surv

ival

00 50 100 150 200 250

Time (h)

GroupModelRU-21BZ










5 Conclusion



Ethical Approval






References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















150

100

50

0

ALT

(UL

)


ALT

998771

(a)

AST400

300

200

100

0

AST

(UL

)


ALP300

200

100

0

ALP

(UL

)


(c)


SOD

200

150

100

50

0

SOD

(Um

g pr

ot)


lowast

lowast

lowast

(a)

MDA

15

10

5

0

MD

A (n

mol

mL)


998771

lowast

lowast

(b)



Control Model

RU-21 BZ















300

200

100

0

Tim

e (m

in)


Sober-up time800

600

400

200

0

Tim

e (m

in)











4 Discussion


10

09

08

07

06

05

Cum

surv

ival

00 50 100 150 200 250

Time (h)

GroupModelRU-21BZ










5 Conclusion



Ethical Approval






References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control Model

RU-21 BZ















300

200

100

0

Tim

e (m

in)


Sober-up time800

600

400

200

0

Tim

e (m

in)











4 Discussion


10

09

08

07

06

05

Cum

surv

ival

00 50 100 150 200 250

Time (h)

GroupModelRU-21BZ










5 Conclusion



Ethical Approval






References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















300

200

100

0

Tim

e (m

in)


Sober-up time800

600

400

200

0

Tim

e (m

in)











4 Discussion


10

09

08

07

06

05

Cum

surv

ival

00 50 100 150 200 250

Time (h)

GroupModelRU-21BZ










5 Conclusion



Ethical Approval






References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















5 Conclusion



Ethical Approval






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 The Protective Effects of Buzui on Acute...

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