w w w . s b f g n o s i a . o r g . b r / r e v i s t a
Original
Article
Screening
of
flavonoids
rich
fractions
of
three
Indian
medicinal
plants
used
for
the
management
of
liver
diseases
Arti
Gupta
a,∗,
Navin
R.
Sheth
b,
Sonia
Pandey
a,
Jitendra
S.
Yadav
c,
Shrikant
V.
Joshi
aaMalibaPharmacyCollege,UkaTarsadiaUniversityBardoli,Gujarat,India bDepartmentofPharmacognosy,SaurashtraUniversity,Rajkot,Gujarat,India
cDepartmentofPharmaceutics,VidyabhartiTrustCollegeofPharmacy,Bardoli,Gujarat,India
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received15April2015
Accepted7June2015
Availableonline21August2015
Keywords:
Paracetamol
Flavonoid-richfractions
Buteamonosperma Bauhiniavariegata Ocimumgratissimum
Hepatoprotectiveactivity
a
b
s
t
r
a
c
t
ThedecoctionsoftheButeamonosperma(Lam.)Taub.,Fabaceae,BauhiniavariegataL.,Fabaceae,and OcimumgratissimumL.,Lamiaceae,aretraditionallyusedforthetreatmentofvarioustypesofhepatic disorder.Phytochemicalstudieshaveshownthattotalflavonoidsfromtheseplantswerethemajor constituentsofthepickedoutpartofeachplant.Thepresentstudywasplannedtoinvestigatethe hep-atoprotectiveeffectofflavonoidrichfractionsoftheB.monosperma,B.variegataandO.gratissimum againstparacetamolinducedliverdamage.Flavonoidrichfractionswereisolatedbysolvent fraction-ationfromeachplant.Eachfractionwassubjectedtovariousqualitativechemicalteststofindoutthe metabolites.Flavonoidfractionsofeachplantweresubjectedforpharmacologicalscreening.Therats weremonitoredforchangeinlivermorphology,biochemicalparameterslikeserumglutamatepyruvate transaminase,serumglutamateoxaloacetatetransaminase,alkalinephosphataseandtotalbilirubinfor thegroupsreceivingtheflavonoid-richfractions.Allflavonoidrichfractionsshowedsignificant hep-atoprotectiveactivity.Thehistologicalstudiessupportedthebiochemicalparameters.Fromtheresults ofbiochemicalanalysisandhistopathologicalstudies,itcanbeaccomplishedthatintheethylacetate fractionofO.gratissimumshowedhighesthepatoprotectiveactivityascomparedtootherfractions.The presentstudywasthefirstevidenceofflavonoid-richfractionsofeachplanthavearemarkable hepato-protectiveeffect.Allfractionscontainapotenthepatoprotectiveagentsuggestedtobeaflavone,which mayfindclinicalapplicationinameliorationofparacetamol-inducedliverdamage.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
Liver diseases remain one of the serious health problems (Baranisrinivasanetal.,2009).Modernmedicationshavelittlerole toalleviationofhepaticdiseaseandtheplant-basedpreparations whicharechieflyavailablemedicinesemployedforthetreatment ofliverdisorders (Rajuetal.,2008).Thestrengthoftheseplant productsmustbeestablished,thusastoidentifynewer medica-mentsactingagainsthepaticinjury.Intheabsenceofareliableliver protectivedruginthemodernsystemofmedication,anumberof medicinalplantsinAyurvedaarerecommendedforthetreatment ofliverdisorders.Naturaltreatmentsfrommedicinalplantsare thoughttobeefficaciousandsafemedicamentsforhepatotoxicity.
Buteamonosperma(Lam.)Taub.(BM)belongstofamilyFabaceae
is a medium size deciduous tree, found throughout India and
∗ Correspondingauthor.
E-mail:[email protected](A.Gupta).
traditionallyusedforthetreatmentofhepatopathy,ulcers,tumors, and diabetes (Kirtikar et al., 1999).The plant mainly contains flavones(quercetin)(Nadkarni,1994;Guptaetal.,2013a), kino-tannicacidandgallicacid.
BauhiniavariegataL.(BV)belongstothefamilyFabaceae
com-monlyknownasKachnar,isfoundtobebeneficialin Ayurveda as a tonic to the liver and anti-inflammatory, healing activity, antioxidantactivity(BodakheandRam,2007).Ithasbeenreported tocontainquercetin,rutin,apigeninandapigenin7-O-glucoside. Flavonoidsandquercetininparticulararestrongantioxidantsand areknowntoregulatetheactivitiesofvariousenzymesystemsdue totheirinteractionwithvariousbiomolecules(Maldonadoetal., 2003).
OcimumgratissimumL.(OG)belongstofamilyLamiaceaeisan
erect,multi-branchedperennialshrubthatgrowsuptoaheight of two meters with a tap rootand many adventitious rootlets (Ramachandranetal.,1986).EssentialoilsobtainedfromOcimum
speciesshowedvariousmedicinalpotentialsinchemo-preventive, anti-carcinogenic, free radical scavenging, and radio-protective
http://dx.doi.org/10.1016/j.bjp.2015.06.010
uses(Guptaetal.,2002;Onajobi,1986;PrakashandGupta,2000). Additionally,OGleafalsorevealedsignificantchemo-preventive effects on chemical-induced papilloma genesis by modulating metabolizingenzymessuchas cytochromeP450, glutathione-S -transferase,andarylhydrocarbonhydroxylase(Karthikeyanetal., 1999;Prasharetal.,1994).Arecentstudyindicatedthat admin-istered orally aqueous extract of OG leaf could oxidative and toxicantactivityandenhancespecificactivitiesofhepatic antioxi-dantenzymesinrats(IghodaroandEbuehi,2008).Notably,recent studyalsoshowedthattheOGleafaqueousextract(OGAE)maybe importantinprotectingH9c2cellsfromH2O2-inducedcelldeath
byinhibitingthemitochondrialdependentapoptosispathway(Lee etal.,2010).Epicatechin,caffeicacid,rutin,gallicacid,quercetin, epigallocatechingallatewereidentifiedasmajorcomponentsof phenolicacidsandflavonoidsinOGAE(Chiuetal.,2012;Grayer etal.,2000).Ursolicacidwasdeterminedindichloromethaneand ethylacetatefractionsofmethanolicextractofO.gratissimumin previouslypublishedreport(Guptaetal.,2013b).
Theflavonoid,quercetininacetonefractionofB.monosperma, ethyl acetate and n-butanol fractions of B. variegata; dichloromethane and ethyl acetate fractions of O. gratissimum
wereidentifiedinpreviouslypublishedreport(Guptaetal.,2013a). Quercetin(flavonoid)andursolicacid(triterpenicacid)arewell knownforitshepatoprotectiveeffectsinacutechemicallyinduced liverinjuryandchronicliverfibrosisandcirrhosis(Janbazetal., 2004).
Althoughthesestudiesstronglyimplicatedthemedicinaleffects oftheaboveplants,thereisnostudyforthebeneficialeffectsof flavonoidrichfractions oftheseplants onparacetamol-induced hepaticinjury. Therefore, in order tofullydevelop the medical plantresourcesandtojustifytheuseofthispreparation in tra-ditionalmedicineforthetreatmentoflivercomplaint,thepresent studywasdesignedtoinvestigatethehepatoprotectiveeffectof theflavonoid-rich fractions obtained fromdifferent parts of B.
monosperma,B.variegateandO.gratissimumagainst
paracetamol-inducedliverinjuryinvivo.
Materialsandmethods
Animals
Albino Wistarmale rats (125–175g) were used for determi-nation of maximum tolerable dose (MTD) and evaluation of hepatoprotectiveactivity.Theanimalswerehousedin polypropyl-enecagesat25±1◦Cwiththerelativehumidityof55±5%under
12/12hlight/darkcycles.Theywerereceivedstandardchowand waterduringexperimentation.Thefoodwaswithdrawnontheday beforetheexperiment,butfreeaccessofwaterwasallowed.
Aminimumofsixanimalswasusedineachgroup.Throughout theexperiment,animalsweretreatedaccordingtothesuggested internationalethicalguidelinesforthemaintenanceoflaboratory animals. The study protocol wasapproved by theInstitutional AnimalEthicsCommittee,accordingtotheregulationofthe Com-mitteeforthePurposeofControlandSupervisionofExperiments onAnimals(MPC1007:dated:30/01/2010).
Plantmaterials
ThefreshbarkofButeamonosperma(Lam.)Taub.,Fabaceae(BM),
BauhiniavariegateL.,Fabaceae(BV)andfreshleavesof Ocimum
gratissimum L., Lamiaceae (OG) were collected from the
cam-pusofMalibapharmacycollege,Bardoli.Voucherspecimen(No: MPC/13032010/01,2and03)hasbeendepositedintheDepartment ofBioscience,VeerNarmadSouthGujaratUniversity,Surat,India. Ratswereused forhepatoprotective study,withprior approval
fromtheInstitutionalAnimalEthicalCommittee(RegistrationNo. 717/02/a/CPCSEA/30Jan2010) ofMalibaPharmacyCollege,Uka TarsadiaUniversity.
Extractionandfractionationprocedures
Thedried and powdered material of each plant(500g) was extractedwithmethanolatroomtemperatureforthreeweekswith shaking andstirring.Combined methanolicextractswere evap-oratedtodrynessunderreducedpressurebelow40◦Candthen
dissolvedindistilledwaterandsubjectedtosolvent–solvent frac-tionation.
B. monosperma: Methanolic extract obtained was fractionated
withpetroleumether,benzene,chloroform andacetoneinthe orderof theirincreasingpolaritytoobtainrespectivefractions (SharmaandDeshwal,2011).
B.variegata: Methanolicextract wasfractionatedwithhexane,
ethylacetateandn-butanolintheorderoftheirincreasingpolarity toobtainrespectivefractions(Silvaetal.,2008).
O.gratissimum:Methanolicextractwasfractionatedwithhexane,
dichloromethaneandethylacetateintheorderoftheirincreasing polaritytoobtainrespectivefractions(Chattopadhyay,2003).
Eachfractionwasconcentratedtodrynessunderreduced pres-sureandbelow(40–50◦C)onarotaryevaporatortogiveacetone
fractionofB.monosperma[yield9.4%,w/w],ethylacetatefraction [yield2.2%,w/w]and n-butanol fraction[yield5.0%,w/w] ofB.
variegateanddichloromethanefraction[yield4.2%w/w]andethyl
acetatefraction[yield4.8%,w/w]ofO.gratissimum,respectively.
Establishmentofqualitativeandquantitativephytoprofileof
fractionedextracts
Qualitativephytochemicalanalysis
Eachfractionwassubjectedtovariousqualitativechemicaltests usingreportedmethodstodeterminethepresenceorabsenceof metabolitesviz.,alkaloids,tannins,flavonoid,steroid,terpenoids andphenoliccompoundsetc.(Khandelwal,2001).
Chemical test for flavonoids. Chemical testswere performed for
flavonoidsaccordingtoMacdonald(Macdonaldetal.,2010).
Quantitativephytochemicalanalysis
Determinationoftotalphenols. Eachsamplewasmixedwith1ml
Folin-Ciocalteureagentand0.8mlof7.5%Na2CO3.Theresultant
mixturewasevaluatedat765nmafter2hatroomtemperature. Themeanofthreereadingswasusedandthetotalphenoliccontent wasexpressedinmilligramofgallicacidequivalents/1gextract. Thecoefficientofdeterminationwasfoundtober2=0.992(Yuvaraj etal.,2011).
Determinationoftotalflavonoids. Standardquercetinwasusedto
Invivohepatoprotectiveactivityofselectedflavonoidfractionsof
B.monosperma,B.variegateandO.gratissimumagainst
paracetamol-inducedtoxicity
Testanimals
MaleWistarrats(125–175g)wereusedfordeterminationof maximumtolerabledoseandevaluationofhepatoprotective activ-ityofflavonoidrichfractionsofB.monosperma,B.variegateandO.
gratissimum.Theanimalswerehousedinpolypropylenecagesat
25±1◦Cwiththerelativehumidityof55±5%lessthan12/12h
light/darkcycles.Theywerereceivedastandardchowandwater
adlibitumduringexperimentation.Thefoodwaswithdrawnonthe
daybeforetheexperiment,butfreeaccessofwaterwasallowed. Aminimumofsixanimalswasusedineachgroup.Throughout theexperiments,animalsweretreatedaccordingtothesuggested internationalethicalguidelinesforthemaintenanceoflaboratory animals.ThestudyprotocolwasapprovedbytheInstitutional Ani-malEthicsCommittee,accordingtotheregulationofcommitteefor thepurposeofcontrolandsupervisionofexperimentsonanimals (MPC1007:dated:30/01/2010).
Preparationandadministrationoftestsamples
TheapprovedIAECmethodwasadoptedforscreeningof hep-atoprotectiveactivity.Driedfractionsweresuspendedin1%CMC solution before oral administration to animals. Animals were dividedintofivegroupsofsixratseachwereusedforthestudy. Group1and2receivednormal saline(1ml/kgorally)forseven days.Groups 3,4,5,6, 7and8 received100mg/kgbwdoseof silymarin,acetonefractionofB.monosperma,ethylacetateandn -butanolfractionsofB.variegataanddichloromethane andethyl acetatefractionsofO.gratissimumat100mg/kgofeachorally,once a dayforsevendays. On thefifthday,aftertheadministration oftherespectivetreatments,alltheanimalsingroups2,3,4,5, 6,7and8thwereadministeredparacetamol(PCM)2g/kgorally. Ontheseventhday,thebloodsampleswerecollectedviaorbital sinuspuncturefortheestimationofbiochemicalmarkerenzymes. Thentheliverwascarefullyisolatedandcleanedoffextraneous tissueandpreservedin10%neutralformalinandthensubjectedto histopathologicalstudies(Parasuramanetal.,2010;Puratchikody etal.,2006).
Assessmentofbiochemicalparametersofliver
The enzymatic parameters of serum like serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase(SGOT)andserumalkalinephosphatase(ALP)and non-enzymaticparameterliketotal bilirubin(TB)wereassayed accordingtostandardmethods(Varleyetal.,1994).
Statisticalanalysis
Theoutcomesareshownasmean±S.E.M.(n=6).Statistical sig-nificancewasdeterminedbyone-wayanalysisofvariance with
p<0.01and p<0.05 consideredsignificantfollowed byDunnett MultipleComparisonsTest.TheanalysiswasperformedbyGraph PadInStatsoftware.
Results
Phytochemicalscreening
Preliminaryphytochemicalscreeningofalcoholicextractandits fractionsshowedthepresenceofflavonoids,steroids,terpenoids, tannins and phenolic compounds. The chemical test analysis demonstratedthatacetonefractionofB.monosperma,ethylacetate
fractionandn-butanolfractionofB.variegata,dichloromethaneand ethylacetatefractionsofO.gratissimumwererichinphenolic com-pounds.ThephenoliccontentinB.monosperma(acetonefraction),
B.variegate(ethylacetateandn-butanolfractions)andO.
gratissi-mum(dichloromethaneandethylacetatefractions)werefoundto be452±1.6,712.4±2.4,442.5±1.1,735±2.1and1365±1.4mg gallic acid/1g fractionrespectively. The flavonoid contentin B.
monosperma(acetonefraction),B.variegata(ethylacetateandn
-butanolfractions)andO.gratissimum(dichloromethaneandethyl acetatefractions)werefoundtobe251±1.8,417±2.2,227±3.2, 394.5±2.4 and 717±5.2mg quercetin/1g fractionrespectively. Thephenolandflavonoidcontentsareresponsiblefor hepatopro-tectiveactivity;hencethesesolventfractionsweretakenforfurther work.
Evaluationofhepatoprotectiveactivity
Fromtheeffectsofacutetoxicitystudybypreviouslypublished literature,2000mg/kgwasconsideredasmaximumtolerabledose ofalcoholicextractofB.monosperma(Muralidharetal.,2011),B.
variegata(BalamuruganandMuralidharan,2010)andO.
gratissi-mum(Okolietal.,2010).1/20thofthisdosewasconsideredasan experimentaldoseforsubsequenthepatoprotectivestudies.The effectivedoseofeachfractionwasdecidedonthebasisof previ-ouslypublishedreports,i.e.100mg/kgofbodyweight.
ParacetamolhasenhancedthelevelsofSGPT,SGOT,ALPand totalbilirubin.Theresultsindicatedthattheflavonoidrich frac-tionsofB.monosperma,B.variegataandO.gratissimumsignificantly reducedtheelevatedlevelsofSGPT,SGOT,ALPandbilirubinwhen comparedtoparacetamoltreatedgroup.Theresultsareshownin
Figs.1and2.
Histopathologicalexaminationoftheliversectionsconfirmed thatthenormalliverarchitecturewasdamagedwithparacetamol administration. However,pretreatment of fractionsof alcoholic extracts of B. monosperma, B. vareigata and O. gratissimum at 100mg/kgdose,significantlyreducedtheseverityof histopatho-logicalinjury(comparedwiththeparacetamolgroup).Theresults ofthebiochemicaltestsandhistopathologicalobservationssuggest that 100mg/kg of each fractionis effective against liver toxic-ity.Rats treatedwitheach flavonoidfractionof B.monosperma,
B.vareigataandO.gratissimumshowednoticeableimprovement
inhistopathologicalparameters.Thus,eachfractionisconsidered as prominent onparacetamol-induced liver damage. Moreover, at necropsy, livers of rats treated with paracetamol appeared degenerationinhepatocytes,hepaticcellinjury,focalnecrosis, con-gestionincentralvein,vascularswelling,kupffercellproliferation. Furthermore,nogrosspathologicalfindingswerenotedinthe liv-ersoftheothergroupsofrats.TheresultsareshowninTable1and
Fig.3.
Discussion
Because B. monosperma, B. vareigataand O. gratissimum are widely used in folk medicine for the treatment of liver dis-eases, we investigated the hepatoprotective activity of the flavonoid rich fractions of each plant using two paraceta-mol induced liver toxicity models. Several mechanisms may be linked up with the damage done to the liver by different hepatotoxinsin. In the paracetamol model, the drug is sup-posed to be eliminated mainly assulfate and glucuronide. Just a modest quantity (5%) is metabolized via the cytochrome P450 enzyme system to the alkylating metabolite, N-acetyl-p
0 50 100 150 200 250 300 350 400 450 500
PCM treated
Control Silymarin
treated
AcO fraction of
BM treated
EtOAc fraction of BV treated
n-butanol fraction of BV treated
DCM fraction of OG treated
EtOAc fraction of OG treated
SGPT U/l
SGOT U/l
ALP U/l
Level of biochemical parameters
Treatment groups
Fig.1.EffectofallflavonoidfractionsobtainedfromthealcoholicextractofButeamonosperma,BauhiniavariegateandOcimumgratissimumonSGOT,SGPTandALP(U/L)in
paracetamolinducedhepaticinjuryinrats.
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
PCM treated
Control Silymarin
treated
AcO fraction of BM treated
EtOAc fraction of BV treated
n-butanol fraction of BV treated
DCM fraction of OG treated
EtOAc fraction of OG treated Total bilirubin (mg/dl)
Direct bilirubin (mg/dl)
Treatment groups
Level of biochemical parameters
Fig.2.EffectofallflavonoidfractionsobtainedfromthealcoholicextractofButeamonosperma,BauhiniavariegateandOcimumgratissimumontotalbilirubin(mg/dl)in
paracetamolinducedhepaticinjuryinrats.
Table1
Histopathologicalchangesintheliverofrats.
Microscopicobservation Control Paracetamol Silymarin Acetone fr.ofBM
Ethylacetate fractionofBV
n-Butanol fractionofBV
Dichloromethane fractionofOG
Ethylacetate fractionofOG
Degenerationinhepatocytes + +++ ++ ++ ++ ++ ++ ++
Hepaticcellinjury Ø +++ ++ +⊥ +⊥ +⊥ +⊥ +⊥
Focalnecrosis Ø +++ Ø +⊥ +⊥ +⊥ +⊥ +⊥
Congestionincentralvein + +++ + +⊥ +⊥ +⊥ +⊥ +⊥
Vascularswelling + +++ + ++ +⊥ +⊥ +⊥ +⊥
Kupffercellproliferation Ø ++ + ++ +⊥ +⊥ +⊥ ++
Ø,absent;⊥,few;+mild;++,moderate;+++,severe;++++,extremelysevere.
saturated,andthus,higherpartofparacetamolmoleculesare
oxi-dizedtohighlyreactiveNAPQI.Higherdoseofparacetamol and
NAPQIcanalkylateandoxidizeintracellularglutathione(GSH)and proteinthiolgroups,whichresultinthedepletionofliverGSHpool
andsubsequently leadtoincreasedlipid peroxidationand liver
damage(AkahandOdo,2010;Dongetal.,2000).
Inthisstudy,flavonoidfractionofB.monosperma,B.vareigata
andO.gratissimumdemonstratedsignificant(p<0.01andp<0.05)
liverprotectionagainstthehepaticinjuriescausedbythe paraceta-mol.Itisapparentthatseveralphytoconstituentshavethepowerto inducemicrosomalenzymeseitherbyacceleratingtheexcretionof thehepatotoxinorbysuppressionoflipidperoxidationinducedby
Hepatocytes Kupffer cell
Kupffer cell
Sheets of hepatocytes
Sinusoids
A
B
C
D
E
F
G
H
Sinusoid
Sinusoids
Sinusoids Sinusoid
Hepatocytes Hepatocytes
Hepatocytes
Sinusoid Hepatocytes
Degeneration in hepatocytes
Congestion in central vein
Kupffer cell
proliferation CV
CV
CV CV
CV CV
Fig.3. Thephotomicrographsofliversectionfromrats:(A)receivedsaline0.5mlasanormalcontrolgroup(10×10);(B)paracetamol(2g/kg)(10×10);(C)silymarin
(100mg/kgbw.)+paracetamol(10×10);(D)acetonefractionofBM(100mg/kgbw.)+paracetamol;(E)ethylacetatefractionofBV(100mg/kgbw.)+paracetamol;(F)
n-butanolfractionofBV(100mg/kgbw.)+paracetamol;(G)dichloromethanefractionoftheOG(100mg/kgbw.)+paracetamol;and(H)ethylacetatefractionoftheOG
(100mg/kgbw.)+paracetamol.
ofB.monosperma,B.vareigataandO.gratissimumhavepromising
hepatoprotectivepotentials.
Conclusions
The hepatoprotective effect of flavonoid rich fractions of B.
monosperma,B.vareigataandO.gratissimummaybeattributeddue
tothereducedSGPT,SGOT,ALPandbilirubinandimproveddefense ofthehepatocytesagainsttheparacetamol.The histopathologi-cal studiesalsoconfirm theaction of thedrug.Thus thestudy scientificallysupportstheusageofflavonoidrichfractionsofB.
monosperma,B.vareigataandO.gratissimumfortreatmentofliver
disordersandasatonic.Toelucidatetheexactmechanism respon-sibleforthehepatoprotectiveeffectsofflavonoidrichfractionsofB.
monosperma,B.vareigataandO.gratissimum,furtherexperiments
arebeingconductedinourlaboratory.
Authorcontributions
TheAGwasresponsiblefortheplantcollections,performedthe experiments,analyzedthedataandwrotethereport.JSYandSP supervisedtheentirework,analyzedthedataanddidacritical readingofthemanuscript.SVJhelpedinbiologicalstudies.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
TheauthorsexpresstheirsincerethankstoDr.BhavinVyasand JankiDesaifromDepartmentofPharmacologyfortheirhelp.
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