Hepatoprotective and Antioxidant Effect of Bauhinia hookeri Extract against Carbon...

Research ArticleHepatoprotective and Antioxidant Effect of BauhiniaHookeri Extract against Carbon Tetrachloride-InducedHepatotoxicity in Mice and Characterization of Its BioactiveCompounds by HPLC-PDA-ESI-MSMS

Eman Al-Sayed1 Olli Martiskainen2 Sayed H Seif el-Din3 Abdel-Nasser A Sabra3

Olfat A Hammam4 Naglaa M El-Lakkany3 and Mohamed M Abdel-Daim56

1 Department of Pharmacognosy Faculty of Pharmacy Ain-Shams University Cairo 11566 Egypt2 Laboratory of Organic Chemistry and Chemical Biology Department of Chemistry University of Turku 20014 Turku Finland3Department of Pharmacology Theodor Bilharz Research Institute Giza 12411 Egypt4Department of Pathology School of Medicine Stanford University Stanford CA 94305 USA5Department of Pharmacology Faculty of Veterinary Medicine Suez Canal University Ismailia 41522 Egypt6Center for Emerging Infectious Diseases Gifu University 1-1 Yanagido Gifu 501-1194 Japan

Correspondence should be addressed to Eman Al-Sayed em alsayedpharmaasueduegand Mohamed M Abdel-Daim abdeldaimmvetsuezedueg

Received 27 February 2014 Revised 26 April 2014 Accepted 26 April 2014 Published 14 May 2014

Academic Editor Sung-Hoon Kim

Copyright copy 2014 Eman Al-Sayed et alThis 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

The hepatoprotective and antioxidant activity of Bauhinia hookeri ethanol extract (BHE) against CCl4-induced liver injury was

investigated in mice BHE was administered (500 and 1000mgkgday) along with CCl4for 6 weeks The hepatic marker enzymes

alanine aminotransferase (ALT) aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were determined in the serumThe antioxidant parameters glutathione (GSH) superoxide dismutase (SOD) glutathione peroxidase (GPx) glutathione reductase(GR) glutathione transferase (GST) and malondialdehyde (MDA) were estimated in the liver homogenate BHE treatmentsignificantly inhibited the CCl

4-induced increase in ALT (44 and 64) AST (36 and 46) ALP (28 and 42) and MDA (39 and

51) levels at the tested doses respectively Moreover BHE treatment markedly increased the activity of antioxidant parametersGSH GPx GR GST and SOD Histological observations confirmed the strong hepatoprotective activity These results suggestthat a dietary supplement of BHE could exert a beneficial effect against oxidative stress and various liver diseases by enhancingthe antioxidant defense status reducing lipid peroxidation and protecting against the pathological changes of the liver Thehepatoprotective activity of BHE is mediated at least in part by the antioxidant effect of its constituents The active constituents ofBHE were identified by HPLC-PDA-ESIMSMS

1 Introduction

The liver is a vital organ that has a crucial role in thedetoxification of various xenobiotics [1] Excessive expo-sure to drugs and environmental pollutants overpowersthe natural protective mechanisms of the liver and leadsto hepatic injury Liver damage is associated with cellularnecrosis plasma membrane damage and depletion in theglutathione level (GSH) The levels of serum markers of liver

damage such as alanine aminotransferase (ALT) aspartateaminotransferase (AST) and alkaline phosphatase (ALP) areelevated [2] Long-standing hepatic injury leads to hepaticsteatosis fibrosis and even to life-threatening conditionssuch as liver cirrhosis and hepatocellular carcinoma [3]Steroids and vaccines have been used for the treatmentof liver diseases however they have serious adverse sideeffects and are of limited therapeutic benefits [3] Silymarina mixture of flavonolignans obtained from milk thistle is

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 245171 9 pageshttpdxdoiorg1011552014245171

2 BioMed Research International

a popular herbal extract used as a hepatoprotective agent [4]However some clinical trials have indicated that the standarddoses of silymarin were ineffective in many patients withchronic liver disease [5] In view of the limited therapeuticoptions available for the treatment of liver diseases thesearch for new and safe hepatoprotective candidates is quiteapparent

Herbal medicines have been used for centuries for thetreatment of several ailments Natural products remain asimportant sources of lead structures for the developmentof many drugs [4] Recently there has been a resurgenceof interest in the use of natural products because of theirreduced side effects when compared to synthetic drugs [3]Plants are considered as potential hepatoprotective agentsbecause they contain a combination of different phytochem-icals that are synergistic in their action [6 7] Dietarypolyphenolic compounds are abundant in many plant foodssuch as fruits vegetables and legumes There is growinginterest in the use of dietary polyphenols because of theirbeneficial health effects [8]Dietary polyphenolic compoundshelp to restore the balance between the natural antioxidantsand free radicals by enhancing the activity of natural antiox-idant defenses such as superoxide dismutase (SOD) glu-tathione peroxidase (GPx) glutathione reductase (GR) andglutathione-S-transferase (GST) and by direct scavenging offree radicals [8 9]

ThegenusBauhinia (Fabaceae) comprises 300 species andare commonly known as ldquocowrsquos pawrdquo tree because of theshape of their leaves [10] Plants of the genus Bauhinia arewidely distributed in Africa Asia and South America Theleaves and stem-bark of these plants have been used in folkmedicine for the treatment of different ailments [10] Severalpharmacological activities have been reported for manyBauhinia species including hepatoprotective antioxidantanti-inflammatory and antihyperlipidemic effects [11 12]B hookeri F Muell is a small ornamental tree native toAustralia [13] Although various pharmacological effects werereported for different Bauhinia species no studies have sofar been conducted on the chemical constituents and thepharmacological activities of B hookeri The first objectiveof this study was to determine the antioxidant and hepato-protective effect of the phenolic-rich ethanol extract of Bhookeri (BHE) against CCl

4-induced hepatotoxicity in mice

with the aim to develop a safe and effective hepatoprotectiveagent Hepatoprotection was determined by assaying theactivities of ALT AST and ALP in the serum of controland treated mice The lipid peroxidation and antioxidantparameters (SOD GSH GPx GR and GST) were estimatedin the liver homogenates to determine the possible mech-anisms of the hepatoprotective activity A histopathologicalexamination of liver sections was conducted to confirmthe hepatoprotective effect The second objective of thepresent study was to identify the bioactive constituentsof B hookeri utilizing the HPLC-PDA-ESIMSMS tech-nique (high-performance liquid chromatography coupledwith diode array detection-electrospray ionization massspectrometry)

2 Materials and Methods

21 Chemicals Carbon tetrachloride (CCl4) ethylenedi-

aminetetraacetic acid (EDTA) hydrogen peroxide (H2O2)

551015840-dithiobis-2-nitrobenzoic acid (DTNB Ellmanrsquos reagent)potassium dihydrogen phosphate (KH

2PO4) reduced

glutathione (GSH) 1-chloro-24-dinitrobenzene (CDNB)nicotinamide adenine dinucleotide phosphate reduced form(NADPH) nicotinamide adenine dinucleotide reducedform (NADH) GR oxidized glutathione (GSSG) nitrobluetetrazolium (NBT) phenazine methosulphate (PMT)trichloroacetic acid (TCA) thiobarbituric acid (TBA) andsilymarin were purchased from Sigma-Aldrich Chemical Co(St Louis MO USA) The assay kits were purchased fromSpectrum MDSS GmbH Hannover Germany

22 Plant Material The leaves of B hookeri were collectedin July 2011 from the botanical garden of the Faculty ofAgriculture Cairo University Cairo Egypt The plant wasbotanically identified by Eng Therese Labib the taxonomyspecialist at the herbarium of El-Orman Botanical GardenGiza Egypt A voucher specimen of B hookeri was depositedat the herbarium of the Department of PharmacognosyFaculty of Pharmacy Ain Shams University Cairo Egypt(ASU BHF2011)

23 Extract Preparation Air-dried powdered leaves of Bhookeri (1700 g) were extracted three times with 80 EtOHThe total extract was concentrated and freeze-dried to obtaina dry powder which was dissolved in absolute EtOH TheEtOH-soluble portion was concentrated and freeze-dried toobtain a dry powder of BHE (140 g)

24 HPLC-PDA-ESIMSMS Method Part of the extractwas dissolved in 20 MeOH (20mgmL) and the solutionwas filtered through 045120583m membranes LC-HRESIMSwas performed on a Bruker micrOTOF-Q Daltonics (API)Time-of-Flight mass spectrometer (Bremen Germany) cou-pled to a 1200 series HPLC system (Agilent TechnologiesWaldbronn Germany) equipped with Starlight diode-arraydetector (PDA) Chromatographic separation was performedon an XBridge C18 (21 times 100mm 35 120583m) column (WatersDublin Ireland) The mobile phase consisted of acetonitrile(A) and 01 formic acid (B) The elution profile was 0ndash3min 100 B (isocratic) 3ndash30min 0ndash30 A in B 30ndash35min 30ndash70 A in B 35ndash37min 70 A in B (isocratic)with constant flow rate of 02mLminTheHPLC system wascontrolled by Hystar software (version 32 Bruker BioSpinGmbH Rheinstetten Germany) The mass spectrometerwas controlled by the Compass 13 for micrOTOF softwarepackage (Bruker Daltonics GmbH Rheinstetten Germany)The ionization technique was a pneumatically assisted elec-trosprayThemass spectrometer was operated in the negativemode and mass detection was performed in the full scanmode in the range ofmz 50ndash2000mzThe following settingswere applied to the instrument capillary voltage 4000Vend plate offset minus500V The drying gas (N

2) flow rate was

84 Lmin and the drying gas temperature was 200∘C For

BioMed Research International 3

collision-induced dissociation (CID) MSMSmeasurementsthe voltage over the collision cell varied from 20 to 70 eVArgonwas used as collision gasThe data were analyzed usingCompass Data Analysis Software (version 40 SP5 BrukerDaltonics GmbH Rheinstetten Germany)

25 Animals Male Swiss albino mice (CD-1 strain) weighing20ndash25 g were purchased from the Schistosome BiologicalSupply Center at the Theodor Bilharz Research InstituteGiza Egypt The animals were housed under standard lab-oratory conditions of controlled temperature (20 plusmn 2∘C)humidity (60 plusmn 5) and under 12 h light and dark cyclesThe mice were fed a standard rodent pellet chow and waterad libitum The animals were acclimatized for at least l weekbefore use All the animal experiments were conducted inaccordancewith theGuide for theCare andUse of LaboratoryAnimals of the National Institutes of Health (NIH 1985) andwere approved by the ethical committee of the Faculty ofPharmacy Ain Shams University Cairo Egypt (ASU 2013-8Research Article 5 approval date August 5th 2013)

26 Acute Toxicity Study A group of 36 normal male miceweighing 20ndash25 g were used to determine the acute oraltoxicity of BHE according to the previous method [14] Themice were divided into six subgroups (119899 = 6 per group) Thesubgroups were treated with graded doses (500 1000 20003000 4000 and 5000mgkg body weight po) of BHE Theanimals were observed for 24 h to record toxicity symptomsand mortality rates

27 Animal Grouping and Experimentation The animalswere divided into five groups (119899 = 9 per group) Group(I) served as the normal control and received vehicleonly (05 wv carboxymethyl cellulose) Group (II) wastreated intraperitoneally with a sublethal dose of CCl

4(20

CCl4olive oil 3 daysweek) for 6 weeks to induce chronic

liver injury The mice in groups (III and IV) were treatedorally with 500 and 1000mgkg body weight of BHE respec-tively together with CCl

4for 6 weeks The extract was given

to the animals 5 daysweek while CCl4was administered 3

daysweek Group (V) was treated with standard silymarinat a daily dose of 500mgkg body weight po 5 daysweekSimilarly CCl

4was administered 3 daysweek The animals

were anesthetized using diethyl ether and then sacrificed bydecapitation 48 h after the last treatment dose and bloodsamples were immediately collectedThe livers were dissectedto two parts The first part was fixed in 10 formalin for thehistopathology The second part was washed with 09 icecold saline A piece of 05 g was homogenized in 25 volumes(wv) ice cold 01M potassium phosphate buffer (pH 74)Thehomogenate was centrifuged at 600 g for 10min to removethe cell debris and the supernatant was centrifuged at 10000 gand the pellet was discarded The supernatant was collectedand stored at minus70∘C for the estimation of liver GSH contentantioxidant enzymes and lipid peroxidation

28 Biochemical Assays The collected blood was allowed toclot the serum was separated at 1800 g and the biochemical

markers of hepatic damage including serum AST (UL)ALT (UL) and ALP (IUL) were estimated according topreviously reported methods [15 16] Liver homogenate wasdeproteinized in 5 (wv) TCA centrifuged at 2000 g for20min and the GSH content was estimated by Ellmanrsquosreagent using a standard curve [17]The SOD assay was basedon the inhibition of NBT reduction to water insoluble blueformazan [18] One unit of enzyme activity was defined asthe amount of enzyme that causes half-maximal inhibitionof NBT reduction in the NADH-PMT-NBT reaction systemThe SOD activity was assayed at 560 nm and expressed in120583moleming liver The hepatic GPx activity was determinedby monitoring GSH oxidation [19] The enzyme reactioncontained NADPH GSH and GR and was initiated by theaddition of H

2O2 The change in absorbance was monitored

at 340 nm One unit of GPx activity was defined as theamount of enzyme that catalyzes the oxidation of 1 120583moleof NADPHming liver The GR activity was assayed bymonitoring the oxidation of NADPH at 340 nm using GSSGas a substrate [20] The GST activity was measured based onthe rate of increase in the conjugate formation between GSHand CDNB and the absorbance was monitored at 340 nmOne unit of GST activity was defined as 1120583mole conjugateformationming liver [21] Malondialdehyde (MDA) is abiomarker of lipid peroxidation and reacts with TBA to forma pink chromogen [22] Briefly 1mL of the liver homogenatewas mixed with 1mL of TCA (10 wv) and was centrifugedat 1850 g for 15min 1mL of TBA solution (067 wv)was added to 1mL of supernatant and boiled for 45minAbsorbance was read after cooling at 530 nm against a blankcontaining all the reagents except the liver homogenate Thecontent of thiobarbituric acid reactive substances (TBARS) inthe samples was calculated using the extinction coefficient ofMDA (156 times 105Mminus1 cmminus1) and the results were expressedas MDA equivalents in nmoleg liver

29 Histopathological Examination Liver sections wereembedded in paraffin and sliced into 5 120583m thick sectionsin a rotary microtome (Leica USA) and then stainedwith hematoxylin-eosin dye (Merck) The histopathologicalexamination of the slides was performed under a micro-scope (Zeiss Germany) with times200 magnification powerThe parameters examined for the assessment of histologicaldamage and their relative score systems were as follows

(1) hepatic architecture preserved partial loss or com-plete loss

(2) hydropic degeneration absent mild moderate ormarked

(3) fatty changes absent or present

(4) central vein congestion present or absent

(5) Kupffer cell hyperplasia absent or present

(6) necrosis absent or present

(7) infiltration of portal tract by lymphocytes absent orpresent


Table 1 LC-PDA-ESIMSMS identification of the major constituents of B hookeri

119873119905119877

(min) UV maximum (nm) (MndashH)minus119898119911 Fragments (MSMS)119898119911 Tentative structural

assignment1 47 222 271 16901 12502 Gallic acid2 130 220 225 (sh) 280 320 31104 17904 14901 13505 Caffeic acid pentose ester3 141 218 230 (sh) 280 325 31104 17904 14901 13505 Caffeic acid pentose ester

4 151 205 225 (sh) 280 57714 42509 40708 2890724508 12503

Procyanidin dimer(epicatechin dimer)

5 164 205 230 (sh) 280 (sh)315 (sh) 29505 16304 14901 11905 Coumaric acid pentose ester

6 174 205 225 (sh) 280 86521 40708 28907 12503 Procyanidin trimer(epicatechin trimer)

7 177 220 280 72916 40708 28907 24508 1690212503 Procyanidin dimer gallate

8 182 200 260 355 6151146309 30104 30003

27103 25503 17900 1510116902 12503

Quercetin 3-O-hexosidegallate

9 185 210 250 356 60916 30104 30003 2710225503 17900 15101

Rutin Quercetin3-O-rhamnosyl (1rarr 6)hexoside

10 194 210 278 44109 28907 24508 16902 12503 Epicatechin gallate

11 196 200 260 355 4630930104 30003 2710225503 17900 15100 Quercetin 3-O-hexoside

12 211 200 265 345 5991144710 28504 28403

25503 22704 16902 12503Kaempferol 3-O-galloylhexoside

13 220 200 265 345 44710 28504 28403 25503 22704 Kaempferol 3-O-hexoside

14 284 220 290 (sh) 318 4410929505 27704 16304 14503

11905 Dicoumaroyl pentose

15 290 220 290 (sh) 315 44109 29505 27704 16304 1450311905 Dicoumaroyl pentose

1

2 3 4

5 76

8

91011

12

13

220

3421

092

181

8016

3644

095

14 15(mAU

)

(min)

120010008006004002000

0 5 10 15 20 25 30 35

Figure 1 HPLC chromatogram of BHE

210 Statistical Analysis All the data were expressed asmeans plusmn SEM Statistical analysis of the data was performedusing the one-way ANOVA test followed by Tukeyrsquos post hoctest to determine the difference between the mean values ofthe different groups All statistical analyses were performedusing the GraphPad InStat software (Version 306 La JollaCA USA) 119875 values lt 005 were considered statisticallysignificant

3 Results

31 Identification of the Constituents of BHE by HPLC-PDA-ESIMSMS In this study 15 compounds were detected inBHE using the HPLC instrument coupled to a PDA detector

and a mass spectrometer The combination of the PDAand mass spectrometry (MSMS) data provided a sensitivemethod for the characterization of the constituents of BHE(Table 1 and Figure 1) Compounds 2 and 3 were identified ascaffeic pentose esters based on their molecular ion [M-H]minus atmz 31104 and the MSMS ion at mz 17904 of caffeic acidwhich results from the loss of a pentose moiety (minus132 amu)A base peak at mz 13505 was detected corresponding to atypical loss of a carboxylic group from the caffeic acid unit[23] Compound 5 showed a molecular ion [M-H]minus at mz29505The loss of a pentose unit resulted in diagnostic peaksof coumaric acid at mz 16304 and 11905 [M-H-pentose-COO]minus Compound 4 produced a [M-H]minus peak atmz 57714and fragment ions at mz 42509 40708 28907 2450812503 which are typical for dimeric procyanidins [24]Fragmentation of dimeric procyanidins through retro-Diels-Alder (RDA) fission results in a fragment ion at mz 42509The fragment ion at mz 40708 arises from subsequentelimination of water from the ion at mz 42509 [24 25]Heterocyclic ring fission (HRF) of the flavan-3-ol structureresults in the loss of a phloroglucinol unit (mz 12503)Direct cleavage of the interflavanoid bonds results in thefragment ion at mz 28907 [24 25] Similarly compound 6was identified as trimeric procyanidins from its molecular


Table 2 Effect of BHE on hepatic marker enzymes after 6 weeks of CCl4 intoxication in mice

Animal groups ALT (UL) AST (UL) ALP (IUL)Control 1323 plusmn 071$ 12240 plusmn 268$ 8106 plusmn 473$

CCl4 4325 plusmn 305lowast 22330 plusmn 532lowast 14365 plusmn 382lowast

BHE (500mgkgday) 2429 plusmn 260(44)

14284 plusmn 545(36)

10414 plusmn 544(28)

BHE (1000mgkgday) 1576 plusmn 272$(64)

12024 plusmn 583$(46)

8376 plusmn 586$(42)

Silymarin (500mgkgday) 2364 plusmn 225(45)

12506 plusmn 343$

(44)8978 plusmn 524$

(38)Data are expressed as the means plusmn SEM (119899 = 9)The numbers in parentheses represent the percentage of reduction from the CCl4-intoxicated groupValues having different superscripts within the same column are significantly different at 119875 lt 005

ion [M-H]minus at mz 86521 and fragment ions at mz 4070828907 12503 [25] Compounds 7 and 10 were identifiedbased on their molecular and fragment ions as previouslydescribed [24] The identification of the flavonoid glycosidesof B hookeri was achieved based on the PDA and MSMSdata as previously described [23 26 27]TheMSMS analysisof compound 9 revealed fragment ion peaks at mz 30104and 30003 which indicated the glycosylation site at the 3-position of quercetin [27] Moreover the MSMS analysisof compound 9 indicated that no distinctive intermediatefragments [M-H-146]minus and [M-H-162]minus were observedconfirming the rutinoside (rhamnosyl (1rarr 6) glucoside)interglycosidic linkage [26] Similarly compounds 8 11ndash13were identified from the [M-H]minus and fragment ion peaks aswell as from the retention time and distinctive PDA data

32 Acute Toxicity No adverse behavioral changes toxicitysymptoms or mortality were observed in mice at doses up to5000mgkg (LD

50gt 5000mgkg) Based on these findings

BHE is considered safe in mice

33 Hepatoprotective Effect A significant increase in theactivity of the serum enzymes ALT AST and ALP (119875 lt0001) was observed in the CCl

4group compared to the

negative control group (Table 2)The treatment of intoxicatedmice with BHE at the tested doses (500 and 1000mgkgday)produced a significant hepatoprotective effect and reducedthe activity of ALT AST and ALP Notably BHE treatmentat a dose of 1000mgkg was more effective in reducing theALT AST and ALP levels compared to silymarin (Table 2)The levels of the hepatic enzymes were comparable and notsignificantly different from the normal control group

34 Antioxidant Activity A marked reduction in hepaticGSH and antioxidant enzymes (GPx GR GST and SOD)wasobserved in CCl

4-intoxicated mice In contrast a significant

increase in the MDA level was evident compared to thenormal control group (Table 3) Administration of BHE at thetwo treatment doses (500 and 1000mgkgday) produced amarked increase in GSH (by 51 and 76 resp) and improvedthe activity of all the antioxidant enzymes relative to theCCl4-intoxicated group (Table 3 and Figure 2) In addition

005

115

225

335

445

Control BHE 1 BHE 2 Silymarin

GSH

(120583m

ole

g liv

er)

$

$$

$

lowast

CCl4

Figure 2 Effect of BHE and silymarin on the hepatic GSHlevel Data are expressed as means plusmn SEM (119899 = 9) BHE 1CCl4+ 500mgkg of BHE BHE 2 CCl

4+ 1000mgkg of BHE

Values having different superscripts within the same column aresignificantly different at 119875 lt 005

the elevated hepatic level of MDA was reduced by 39 and51 at the tested doses respectively compared to the CCl

4-

intoxicated group (Figure 3) The higher dose of BHE wasmore effective in reducing the MDA level compared tosilymarin Notably the GSH GPx GR GST and SOD levelsin the group treated with 1000mgkg of BHE were markedlyhigher compared to the silymarin-treated group MoreoverBHE treatment at a dose of 500mgkg induced a markedincrease in theGR andGST levels compared to the silymarin-treated group These results clearly indicated the strong invivo antioxidant activity provided by BHE

35 Histopathological Observations CCl4induced severe

loss of the hepatic architecture marked hydropic degen-eration fatty changes central vein congestion Kupffercell hyperplasia necrosis and scattered lymphocytes inbetween the hepatocytes and in the sinusoids The patho-logical changes induced by CCl

4were markedly amelio-

rated in the groups treated with BHE at the two treatmentdoses (Figure 4 and Supplementary Data available online athttpdxdoiorg1011552014245171) The liver architectureis preserved in 75 and 875 of animals treated with 500 and1000mgkg of BHE respectively In addition BHE conferred


Table 3 Effect of BHE on lipid peroxidation (MDA) GSH and antioxidant enzymes after 6 weeks of CCl4 intoxication in mice

Animal groups GSH(120583moleg liver)

GPx(120583moleming

liver)

GR(120583moleming

liver)

GST(120583moleming

liver)

SOD(120583moleming

liver)

MDA(nmoleg liver)

Control 374 plusmn 032$ 308 plusmn 029$ 246 plusmn 023$ 4896 plusmn 487$ 35585 plusmn 726$ 3236 plusmn 326$

CCl4 210 plusmn 024lowast 164 plusmn 023lowast 157 plusmn 027$ 2085 plusmn 273lowast 23656 plusmn 408lowast 6437 plusmn 418lowast

BHE (500mgkgday) 318 plusmn 024$(+51)

251 plusmn 024$lowast(+53)

230 plusmn 027$(+46)


29748 plusmn 486(+26)

3903 plusmn 397$(minus39)


300 plusmn 026$(+83)

237 plusmn 033$(+51)

4191 plusmn 431$(+101)

32467 plusmn 628sect

(+37)3132 plusmn 347$

(minus51)

Silymarin (500mgkgday) 338 plusmn 028$(+61)


219 plusmn 036$(+39)



(+33)3610 plusmn 239$

(minus44)Data are expressed as the means plusmn SEM (119899 = 9)The numbers in parentheses represent the percent change from the CCl4-intoxicated groupValues having different superscripts within the same column are significantly different at 119875 lt 005

0

10

20

30

40

50

60

70

80


MD

A (n

mol

eg

liver

)

$ $$$

lowast

CCl4

Figure 3 Effect of BHE and silymarin on lipid peroxidation (hepaticMDA level) Data are expressed as means plusmn SEM (119899 = 9) BHE1 CCl

4+ 500mgkg of BHE BHE 2 CCl

4+ 1000mgkg of BHE


protection against liver damage as evidenced by the markeddecrease in lymphocyte infiltration hydropic degenerationfatty changes central vein congestion Kupffer cell hyperpla-sia and necrotic changes It was clear that the lower dose ofBHE is as effective as silymarin in reducing the Kupffer cellhyperplasia induced by CCl

4intoxication Notably treatment

with BHE at doses of 500 and 1000mgkg reduced the fattychanges central vein congestion and necrosis more than inthe silymarin-treated group Moreover the higher dose ofBHE was more effective in restoring the hepatic architectureand in reducing the lymphocyte infiltration Kupffer cellhyperplasia and hydropic degeneration compared to thesilymarin-treated group Complete amelioration of the fattychanges was evident by the higher dose of BHE

4 Discussion

CCl4intoxication is a widely used experimental model for

liver injury The highly hepatotoxic metabolites namelytrichloromethyl radicals (CCl

3

sdot and CCl3O2

sdot) are generatedduring the metabolic activation of CCl

4by cytochrome P-

450 These radicals have a central role in the initiationof lipid peroxidation inflammation and fatty changes of

the liver [3 28] Moreover CCl4intoxication is associated

with oxidative stress since the CCl3

sdot and CCl3O2

sdot radi-cals alter the antioxidant state of the liver by deactivatingthe hepatic antioxidant enzymes including SOD GPx GRand GST [3] Trichloromethyl radicals also react with thesulfhydryl groups of GSH leading to its deactivation [3] Inthe present study CCl

4treatment markedly increased the

levels of AST ALT and ALP The leakage of the markerenzymes into the blood was associated with marked necrosisloss of hepatic architecture hydropic degeneration fattychanges Kupffer cell hyperplasia central vein congestionand infiltration of the liver by lymphocytes The MDA levelin the liver tissue wasmarkedly increased in response to CCl

4

intoxication indicating oxidative damage of the liver CCl4

administration also reduced the levels of GPx SOD GSTGSH andGR in the liver tissue compared to the normalmiceThe results of the present study demonstrated that treatmentwith BHE returned the increased MDA to its normal levelThe inhibitory effect against lipid peroxidation suggested thatBHE could prevent the liver injury induced by free radicalsalong with the subsequent pathological changes in the liverThe marked reduction in the leakage of liver enzymes intothe serum also confirmed the inhibitory effect of BHE againstlipid peroxidation In contrast the GSH GPx SOD GSTand GR levels were markedly improved compared to thesilymarin-treated group Modulation of these antioxidantdefenses clearly contributed to the antioxidant and hepato-protective activity of BHE In this study the phytochemicalcomposition of B hookeri was identified for the first timeusing the HPLC-PDA-ESIMSMS analysis The identifiedcompounds include gallic acid hydroxycinnamic acid deriva-tives flavonoids epigallocatechin gallate and dimeric andtrimeric procyanidins The remarkable hepatoprotective andantioxidant effect of BHE may be attributed to a synergisticeffect between these compounds [8] Experimental evidenceproved that the whole plant extracts usually possess muchbetter pharmacological activities than single isolated ingre-dients due to synergistic interactions between the individ-ual components [6 7] It is also known that mixtures ofantioxidant compounds are more active than the individualcomponents of these mixtures [29] Flavonoids especiallyflavonols and flavan-3-ols possess various biological effects


200120583m

(a)

200120583m

(b)

200120583m

(c)

200120583m

(d)

200120583m

(e)

Figure 4 Hepatoprotective effect of BHE in CCl4-intoxicated mice (a) Group I (normal control) showing normal hepatic architecture and

normal hepatocytes (b) Group II (CCl4-treated group) showing marked loss of hepatic architecture marked hydropic degeneration central

vein congestion and scattered lymphocytes in between the hepatocytes and in the sinusoids (c and d) Groups III and IV (CCl4+ 500mgkg

or CCl4+ 1000mgkg respectively of BHE) showing preserved hepatic architecture (e) Group V (CCl

4+ 500mgkg of silymarin) showing

normal hepatic architecture and mild hydropic degeneration of hepatocytes (H amp E 200x) Hydropic degeneration (the black arrow) centralvein congestion (the white arrow) and scattered lymphocytes in between the hepatocytes and in the sinusoids (the dotted black arrow)

that contribute to health benefits including antioxidant andhepatoprotective activities [8 29] Flavonols prevent theoxidative stress by direct scavenging of free radicals metalchelation reduction of tocopheryl radicals and induction ofantioxidant enzymes as well as phase II detoxifying enzymessuch as GST Flavonoids also have a membrane-stabilizingeffect [8 9 29] Diverse pharmacological activities havebeen attributed to epigallocatechin gallate including potentantioxidant antifibrogenic and hepatoprotective activity [830] Gallic acid has been reported to exhibit a strong antiox-idant and hepatoprotective activity [31] Proanthocyanidins

are complex polymers of polyhydroxy flavan-3-ol constitutiveunits and are widely present in legumes and fruits [8]Commercial preparations of standardized proanthocyani-dins including grape seed proanthocyanidin extract aremarked as dietary supplements due to their health benefits[32] Proanthocyanidins are known to increase the activityof GST and SOD to elevate the cellular GSH content andto have potent radical-scavenging activity [8] They also havean ameliorative effect on oxidative stress and hepatic fibrosis[32] Grape seed proanthocyanidin extract exhibited a stronghepatoprotective as well as antifibrogenic effects against


dimethylnitrosamine and thioacetamide induced liver injuryin animal models [32 33] Sufficient evidence indicatesthat hepatocellular damage and subsequent infiltration byinflammatory cells have a central role in the activation ofKupffer cells to release several cytokines and free radicalswhich in turn stimulate the transformation of hepatic stellatecells to myofibroblast-like cells ultimately leading to theenhancement of collagen formation and hepatic fibrosis[32 33] The protective effect of BHE against Kupffer cellhyperplasia infiltration by inflammatory cells and otherCCl4-induced pathological changes in liver along with the

protective effect against oxidative stress indicated that thisplant has hepatoprotective and antifibrotic therapeutic poten-tial

5 Conclusion

Based on the results of this study the hepatoprotective effectof BHE is attributed to its ability to reduce the rate of lipidperoxidation to enhance the antioxidant defense status andto guard against the pathological changes of the liver inducedby CCl

4intoxication The strong in vivo antioxidant activity

also suggests that a dietary supplement of BHE may confer abeneficial effect against oxidative stressThe hepatoprotectiveactivity of BHE is concluded to be partly mediated by theantioxidant effect of its constituents This study representsthe first report that employed the HPLC-PDA-ESIMSMStechnique for the identification of the phytoconstituents ofB hookeri The adopted HPLC-PDA-ESIMSMS methodprovided a useful tool to develop a characteristic chromato-graphic fingerprint for the authentication of BHE and for theidentification of its composition

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

Professor Juha-Pekka Salminen Department of ChemistryUniversity of Turku Finland is acknowledged for the use ofthe HPLC-MS instrument during this study

References

[1] M M Abdel-Daim S M M Abuzead and S M Halawa ldquoPro-tective role of Spirulina platensis against acute deltamethrin-induced toxicity in ratsrdquo PLoS ONE vol 8 no 9 Article IDe72991 2013

[2] S R Setty A A Quereshi A H M V Swamy et alldquoHepatoprotective activity of Calotropis procera flowers againstparacetamol-induced hepatic injury in ratsrdquo Fitoterapia vol 78no 7-8 pp 451ndash454 2007

[3] A Srivastava and T Shivanandappa ldquoHepatoprotective effectof the root extract of Decalepis hamiltonii against carbontetrachloride-induced oxidative stress in ratsrdquo Food Chemistryvol 118 no 2 pp 411ndash417 2010

[4] E M Williamson D T Okpako and F J Evans Pharmacologi-calMethods in PhytotherapyResearch Selection Preparation andPharmacological Evaluation of Plant Material vol 1 JohnWileyamp Sons Chichester UK 1996

[5] K R Reddy ldquoSilymarin for the treatment of chronic liverdiseaserdquo Gastroenterology amp Hepatology vol 3 no 11 pp 825ndash826 2007

[6] H Wagner and G Ulrich-Merzenich ldquoSynergy researchapproaching a new generation of phytopharmaceuticalsrdquo Phy-tomedicine vol 16 no 2-3 pp 97ndash110 2009

[7] E M Williamson ldquoSynergy and other interactions in phy-tomedicinesrdquo Phytomedicine vol 8 no 5 pp 401ndash409 2001

[8] X Han T Shen and H Lou ldquoDietary polyphenols andtheir biological significancerdquo International Journal of MolecularSciences vol 8 no 9 pp 950ndash988 2007

[9] S Ramos ldquoCancer chemoprevention and chemotherapydietary polyphenols and signalling pathwaysrdquoMolecular Nutri-tion amp Food Research vol 52 no 5 pp 507ndash526 2008

[10] V C Filho ldquoChemical composition and biological potential ofplants from the genus Bauhiniardquo Phytotherapy Research vol 23no 10 pp 1347ndash1354 2009

[11] S H Bodakhe and A Ram ldquoHepatoprotective properties ofBauhinia variegata bark extractrdquo Yakugaku Zasshi vol 127 no9 pp 1503ndash1507 2007

[12] S Sosa A Braca G Altinier R D Loggia I Morelli andA Tubaro ldquoTopical anti-inflammatory activity of Bauhiniatarapotensis leavesrdquo Phytomedicine vol 9 no 7 pp 646ndash6532002

[13] L Maddigan R Allan and R Reid Coastal Plants of theBurdekinDry Tropics Burdekin Solutions LtdampTownsville andCoastal Dry Tropics Landcare Incorporated South TownsvilleAustralia 2008

[14] R D Bruce ldquoAn up-and-down procedure for acute toxicitytestingrdquo Fundamental and Applied Toxicology vol 5 no 1 pp151ndash157 1985

[15] P R Kind and E J King ldquoEstimation of plasma phosphataseby determination of hydrolysed phenol with amino-antipyrinerdquoJournal of Clinical Pathology vol 7 no 4 pp 322ndash326 1954

[16] S Reitman and S Frankel ldquoA colorimetricmethod for the deter-mination of serum glutamic oxalacetic and glutamic pyruvictransaminasesrdquo American Journal of Clinical Pathology vol 28no 1 pp 56ndash63 1957

[17] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[18] C C Winterbourn R E Hawkins M Brian and R W CarrellldquoThe estimation of red cell superoxide dismutase activityrdquo TheJournal of Laboratory and Clinical Medicine vol 85 no 2 pp337ndash341 1975

[19] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo The Journal of Laboratory and Clinical Medicine vol70 no 1 pp 158ndash169 1967

[20] G Zanetti ldquoRabbit liver glutathione reductase Purification andpropertiesrdquoArchives of Biochemistry and Biophysics vol 198 no1 pp 241ndash246 1979

[21] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS-transferases The first enzymatic step in mercapturic acidformationrdquoThe Journal of Biological Chemistry vol 249 no 22pp 7130ndash7139 1974

[22] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979


[23] K R Maatta A Kamal-Eldin and A R Torronen ldquoHigh-performance liquid chromatography (HPLC) analysis of phe-nolic compounds in berries with diode array and electrosprayionization mass spectrometric (MS) detection Ribes speciesrdquoJournal of Agricultural and Food Chemistry vol 51 no 23 pp6736ndash6744 2003

[24] V Verardo D Arraez-Roman A Segura-Carretero EMarconiA Fernandez-Gutierrez and M F Caboni ldquoIdentification ofbuckwheat phenolic compounds by reverse phase high perfor-mance liquid chromatography-electrospray ionization-time offlight-mass spectrometry (RP-HPLC-ESI-TOF-MS)rdquo Journal ofCereal Science vol 52 no 2 pp 170ndash176 2010

[25] J Hellstrom J Sinkkonen M Karonen and P Mattila ldquoIsola-tion and structure elucidation of procyanidin oligomers fromSaskatoon berries (Amelanchier alnifolia)rdquo Journal of Agricul-tural and Food Chemistry vol 55 no 1 pp 157ndash164 2007

[26] D Del Rio A J Stewart W Mullen et al ldquoHPLC-MSn analysisof phenolic compounds and purine alkaloids in green and blackteardquo Journal of Agricultural and Food Chemistry vol 52 no 10pp 2807ndash2815 2004

[27] C Engels D Grater P Esquivel V M Jimenez M G Ganzleand A Schieber ldquoCharacterization of phenolic compounds injocote (Spondias purpurea L) peels by ultra high-performanceliquid chromatographyelectrospray ionizationmass spectrom-etryrdquo Food Research International vol 46 no 2 pp 557ndash5622012

[28] S-J Lee and K-T Lim ldquoGlycoprotein of Zanthoxylum piperi-tumDC has a hepatoprotective effect via anti-oxidative charac-ter in vivo and in vitrordquo Toxicology in Vitro vol 22 no 2 pp376ndash385 2008

[29] D Prochazkova I Bousova and N Wilhelmova ldquoAntioxidantand prooxidant properties of flavonoidsrdquoFitoterapia vol 82 no4 pp 513ndash523 2011

[30] G L Tipoe T M Leung E C Liong T Y H Lau M L Fungand A A Nanji ldquoEpigallocatechin-3-gallate (EGCG) reducesliver inflammation oxidative stress and fibrosis in carbontetrachloride (CCl

4)-induced liver injury in micerdquo Toxicology

vol 273 no 1ndash3 pp 45ndash52 2010[31] M K Rasool E P Sabina S R Ramya et al ldquoHepatoprotective

and antioxidant effects of gallic acid in paracetamol-inducedliver damage in micerdquo Journal of Pharmacy and Pharmacologyvol 62 no 5 pp 638ndash643 2010

[32] J Li J Li S Li et al ldquoAmeliorative effect of grape seedproanthocyanidin extract on thioacetamide-induced mousehepatic fibrosisrdquo Toxicology Letters vol 213 no 3 pp 353ndash3602012

[33] M-O Shin S Yoon and J-O Moon ldquoThe proanthocyanidinsinhibit dimethylnitrosamine-induced liver damage in ratsrdquoArchives of Pharmacal Research vol 33 no 1 pp 167ndash173 2010

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


a popular herbal extract used as a hepatoprotective agent [4]However some clinical trials have indicated that the standarddoses of silymarin were ineffective in many patients withchronic liver disease [5] In view of the limited therapeuticoptions available for the treatment of liver diseases thesearch for new and safe hepatoprotective candidates is quiteapparent

Herbal medicines have been used for centuries for thetreatment of several ailments Natural products remain asimportant sources of lead structures for the developmentof many drugs [4] Recently there has been a resurgenceof interest in the use of natural products because of theirreduced side effects when compared to synthetic drugs [3]Plants are considered as potential hepatoprotective agentsbecause they contain a combination of different phytochem-icals that are synergistic in their action [6 7] Dietarypolyphenolic compounds are abundant in many plant foodssuch as fruits vegetables and legumes There is growinginterest in the use of dietary polyphenols because of theirbeneficial health effects [8]Dietary polyphenolic compoundshelp to restore the balance between the natural antioxidantsand free radicals by enhancing the activity of natural antiox-idant defenses such as superoxide dismutase (SOD) glu-tathione peroxidase (GPx) glutathione reductase (GR) andglutathione-S-transferase (GST) and by direct scavenging offree radicals [8 9]

ThegenusBauhinia (Fabaceae) comprises 300 species andare commonly known as ldquocowrsquos pawrdquo tree because of theshape of their leaves [10] Plants of the genus Bauhinia arewidely distributed in Africa Asia and South America Theleaves and stem-bark of these plants have been used in folkmedicine for the treatment of different ailments [10] Severalpharmacological activities have been reported for manyBauhinia species including hepatoprotective antioxidantanti-inflammatory and antihyperlipidemic effects [11 12]B hookeri F Muell is a small ornamental tree native toAustralia [13] Although various pharmacological effects werereported for different Bauhinia species no studies have sofar been conducted on the chemical constituents and thepharmacological activities of B hookeri The first objectiveof this study was to determine the antioxidant and hepato-protective effect of the phenolic-rich ethanol extract of Bhookeri (BHE) against CCl

4-induced hepatotoxicity in mice

with the aim to develop a safe and effective hepatoprotectiveagent Hepatoprotection was determined by assaying theactivities of ALT AST and ALP in the serum of controland treated mice The lipid peroxidation and antioxidantparameters (SOD GSH GPx GR and GST) were estimatedin the liver homogenates to determine the possible mech-anisms of the hepatoprotective activity A histopathologicalexamination of liver sections was conducted to confirmthe hepatoprotective effect The second objective of thepresent study was to identify the bioactive constituentsof B hookeri utilizing the HPLC-PDA-ESIMSMS tech-nique (high-performance liquid chromatography coupledwith diode array detection-electrospray ionization massspectrometry)

2 Materials and Methods

21 Chemicals Carbon tetrachloride (CCl4) ethylenedi-

aminetetraacetic acid (EDTA) hydrogen peroxide (H2O2)

551015840-dithiobis-2-nitrobenzoic acid (DTNB Ellmanrsquos reagent)potassium dihydrogen phosphate (KH

2PO4) reduced

glutathione (GSH) 1-chloro-24-dinitrobenzene (CDNB)nicotinamide adenine dinucleotide phosphate reduced form(NADPH) nicotinamide adenine dinucleotide reducedform (NADH) GR oxidized glutathione (GSSG) nitrobluetetrazolium (NBT) phenazine methosulphate (PMT)trichloroacetic acid (TCA) thiobarbituric acid (TBA) andsilymarin were purchased from Sigma-Aldrich Chemical Co(St Louis MO USA) The assay kits were purchased fromSpectrum MDSS GmbH Hannover Germany

22 Plant Material The leaves of B hookeri were collectedin July 2011 from the botanical garden of the Faculty ofAgriculture Cairo University Cairo Egypt The plant wasbotanically identified by Eng Therese Labib the taxonomyspecialist at the herbarium of El-Orman Botanical GardenGiza Egypt A voucher specimen of B hookeri was depositedat the herbarium of the Department of PharmacognosyFaculty of Pharmacy Ain Shams University Cairo Egypt(ASU BHF2011)

23 Extract Preparation Air-dried powdered leaves of Bhookeri (1700 g) were extracted three times with 80 EtOHThe total extract was concentrated and freeze-dried to obtaina dry powder which was dissolved in absolute EtOH TheEtOH-soluble portion was concentrated and freeze-dried toobtain a dry powder of BHE (140 g)

24 HPLC-PDA-ESIMSMS Method Part of the extractwas dissolved in 20 MeOH (20mgmL) and the solutionwas filtered through 045120583m membranes LC-HRESIMSwas performed on a Bruker micrOTOF-Q Daltonics (API)Time-of-Flight mass spectrometer (Bremen Germany) cou-pled to a 1200 series HPLC system (Agilent TechnologiesWaldbronn Germany) equipped with Starlight diode-arraydetector (PDA) Chromatographic separation was performedon an XBridge C18 (21 times 100mm 35 120583m) column (WatersDublin Ireland) The mobile phase consisted of acetonitrile(A) and 01 formic acid (B) The elution profile was 0ndash3min 100 B (isocratic) 3ndash30min 0ndash30 A in B 30ndash35min 30ndash70 A in B 35ndash37min 70 A in B (isocratic)with constant flow rate of 02mLminTheHPLC system wascontrolled by Hystar software (version 32 Bruker BioSpinGmbH Rheinstetten Germany) The mass spectrometerwas controlled by the Compass 13 for micrOTOF softwarepackage (Bruker Daltonics GmbH Rheinstetten Germany)The ionization technique was a pneumatically assisted elec-trosprayThemass spectrometer was operated in the negativemode and mass detection was performed in the full scanmode in the range ofmz 50ndash2000mzThe following settingswere applied to the instrument capillary voltage 4000Vend plate offset minus500V The drying gas (N

2) flow rate was

84 Lmin and the drying gas temperature was 200∘C For






4(20






















119873119905119877



4 151 205 225 (sh) 280 57714 42509 40708 2890724508 12503





8 182 200 260 355 6151146309 30104 30003

27103 25503 17900 1510116902 12503


9 185 210 250 356 60916 30104 30003 2710225503 17900 15101




12 211 200 265 345 5991144710 28504 28403



14 284 220 290 (sh) 318 4410929505 27704 16304 14503



1

2 3 4

5 76

8

91011

12

13

220

3421

092

181

8016

3644

095

14 15(mAU

)

(min)

120010008006004002000

0 5 10 15 20 25 30 35



3 Results








14284 plusmn 545(36)

10414 plusmn 544(28)


12024 plusmn 583$(46)

8376 plusmn 586$(42)


12506 plusmn 343$

(44)8978 plusmn 524$












005

115

225

335

445


GSH

(120583m

ole

g liv

er)

$

$$

$

lowast

CCl4


4+ 1000mgkg of BHE



4-









GPx(120583moleming

liver)

GR(120583moleming

liver)

GST(120583moleming

liver)

SOD(120583moleming

liver)

MDA(nmoleg liver)





230 plusmn 027$(+46)


29748 plusmn 486(+26)



300 plusmn 026$(+83)

237 plusmn 033$(+51)

4191 plusmn 431$(+101)


(+37)3132 plusmn 347$

(minus51)



219 plusmn 036$(+39)



(+33)3610 plusmn 239$


0

10

20

30

40

50

60

70

80


MD

A (n

mol

eg

liver

)

$ $$$

lowast

CCl4



4+ 1000mgkg of BHE





4 Discussion



3

sdot and CCl3O2


4by cytochrome P-




sdot and CCl3O2




4




200120583m

(a)

200120583m

(b)

200120583m

(c)

200120583m

(d)

200120583m

(e)












5 Conclusion






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















4(20






















119873119905119877



4 151 205 225 (sh) 280 57714 42509 40708 2890724508 12503





8 182 200 260 355 6151146309 30104 30003

27103 25503 17900 1510116902 12503


9 185 210 250 356 60916 30104 30003 2710225503 17900 15101




12 211 200 265 345 5991144710 28504 28403



14 284 220 290 (sh) 318 4410929505 27704 16304 14503



1

2 3 4

5 76

8

91011

12

13

220

3421

092

181

8016

3644

095

14 15(mAU

)

(min)

120010008006004002000

0 5 10 15 20 25 30 35



3 Results








14284 plusmn 545(36)

10414 plusmn 544(28)


12024 plusmn 583$(46)

8376 plusmn 586$(42)


12506 plusmn 343$

(44)8978 plusmn 524$












005

115

225

335

445


GSH

(120583m

ole

g liv

er)

$

$$

$

lowast

CCl4


4+ 1000mgkg of BHE



4-









GPx(120583moleming

liver)

GR(120583moleming

liver)

GST(120583moleming

liver)

SOD(120583moleming

liver)

MDA(nmoleg liver)





230 plusmn 027$(+46)


29748 plusmn 486(+26)



300 plusmn 026$(+83)

237 plusmn 033$(+51)

4191 plusmn 431$(+101)


(+37)3132 plusmn 347$

(minus51)



219 plusmn 036$(+39)



(+33)3610 plusmn 239$


0

10

20

30

40

50

60

70

80


MD

A (n

mol

eg

liver

)

$ $$$

lowast

CCl4



4+ 1000mgkg of BHE





4 Discussion



3

sdot and CCl3O2


4by cytochrome P-




sdot and CCl3O2




4




200120583m

(a)

200120583m

(b)

200120583m

(c)

200120583m

(d)

200120583m

(e)












5 Conclusion






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















119873119905119877



4 151 205 225 (sh) 280 57714 42509 40708 2890724508 12503





8 182 200 260 355 6151146309 30104 30003

27103 25503 17900 1510116902 12503


9 185 210 250 356 60916 30104 30003 2710225503 17900 15101




12 211 200 265 345 5991144710 28504 28403



14 284 220 290 (sh) 318 4410929505 27704 16304 14503



1

2 3 4

5 76

8

91011

12

13

220

3421

092

181

8016

3644

095

14 15(mAU

)

(min)

120010008006004002000

0 5 10 15 20 25 30 35



3 Results








14284 plusmn 545(36)

10414 plusmn 544(28)


12024 plusmn 583$(46)

8376 plusmn 586$(42)


12506 plusmn 343$

(44)8978 plusmn 524$












005

115

225

335

445


GSH

(120583m

ole

g liv

er)

$

$$

$

lowast

CCl4


4+ 1000mgkg of BHE



4-









GPx(120583moleming

liver)

GR(120583moleming

liver)

GST(120583moleming

liver)

SOD(120583moleming

liver)

MDA(nmoleg liver)





230 plusmn 027$(+46)


29748 plusmn 486(+26)



300 plusmn 026$(+83)

237 plusmn 033$(+51)

4191 plusmn 431$(+101)


(+37)3132 plusmn 347$

(minus51)



219 plusmn 036$(+39)



(+33)3610 plusmn 239$


0

10

20

30

40

50

60

70

80


MD

A (n

mol

eg

liver

)

$ $$$

lowast

CCl4



4+ 1000mgkg of BHE





4 Discussion



3

sdot and CCl3O2


4by cytochrome P-




sdot and CCl3O2




4




200120583m

(a)

200120583m

(b)

200120583m

(c)

200120583m

(d)

200120583m

(e)












5 Conclusion






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















14284 plusmn 545(36)

10414 plusmn 544(28)


12024 plusmn 583$(46)

8376 plusmn 586$(42)


12506 plusmn 343$

(44)8978 plusmn 524$












005

115

225

335

445


GSH

(120583m

ole

g liv

er)

$

$$

$

lowast

CCl4


4+ 1000mgkg of BHE



4-









GPx(120583moleming

liver)

GR(120583moleming

liver)

GST(120583moleming

liver)

SOD(120583moleming

liver)

MDA(nmoleg liver)





230 plusmn 027$(+46)


29748 plusmn 486(+26)



300 plusmn 026$(+83)

237 plusmn 033$(+51)

4191 plusmn 431$(+101)


(+37)3132 plusmn 347$

(minus51)



219 plusmn 036$(+39)



(+33)3610 plusmn 239$


0

10

20

30

40

50

60

70

80


MD

A (n

mol

eg

liver

)

$ $$$

lowast

CCl4



4+ 1000mgkg of BHE





4 Discussion



3

sdot and CCl3O2


4by cytochrome P-




sdot and CCl3O2




4




200120583m

(a)

200120583m

(b)

200120583m

(c)

200120583m

(d)

200120583m

(e)












5 Conclusion






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















GPx(120583moleming

liver)

GR(120583moleming

liver)

GST(120583moleming

liver)

SOD(120583moleming

liver)

MDA(nmoleg liver)





230 plusmn 027$(+46)


29748 plusmn 486(+26)



300 plusmn 026$(+83)

237 plusmn 033$(+51)

4191 plusmn 431$(+101)


(+37)3132 plusmn 347$

(minus51)



219 plusmn 036$(+39)



(+33)3610 plusmn 239$


0

10

20

30

40

50

60

70

80


MD

A (n

mol

eg

liver

)

$ $$$

lowast

CCl4



4+ 1000mgkg of BHE





4 Discussion



3

sdot and CCl3O2


4by cytochrome P-




sdot and CCl3O2




4




200120583m

(a)

200120583m

(b)

200120583m

(c)

200120583m

(d)

200120583m

(e)












5 Conclusion






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















200120583m

(a)

200120583m

(b)

200120583m

(c)

200120583m

(d)

200120583m

(e)












5 Conclusion






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















5 Conclusion






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Hepatoprotective and Antioxidant Effect of Bauhinia hookeri Extract against Carbon...

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Transcript of Hepatoprotective and Antioxidant Effect of Bauhinia hookeri Extract against Carbon...