Rev. bras. farmacogn. vol.26 número4

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w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Isolation

and

characterization

of

flavanols

from

Anthocephalus

cadamba

and

evaluation

of

their

antioxidant,

antigenotoxic,

cytotoxic

and

COX-2

inhibitory

activities

Madhu

Chandel

a

_,

_Manish

_Kumar

a

_,

_Upendra

_Sharma

b

_,

_Neeraj

_Kumar

b

_,

_Bikram

_Singh

b,∗

_,

Satwinderjeet

Kaur

a,∗

a_Department_of_Botanical_and_{Environmental}_Sciences,_Guru_Nanak_Dev_University,_Amritsar,_India

b_Natural_Product_Chemistry_and_Process_Development_Division,_{CSIR-Institute}_of_Himalayan_Bioresource_Technology,_Palampur,_India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received4November2015 Accepted25February2016 Availableonline2May2016

Keywords:

Anthocephaluscadamba Antioxidant

Antigenotoxic Cytotoxic

Cyclooxygenase-2inhibitoryactivity

a

b

s

t

r

a

c

t

Insearchofleadmoleculesforuseindiseasepreventionandasfoodadditivefromnaturalsources, two flavanols wereisolated from leaves ofAnthocephalus cadamba (Roxb.)Miq., Rubiaceae.Their structureswereestablishedas6-hydroxycoumarin-(4′′_→_8)-(₋_{)-epicatechin}_and

6-hydroxycoumarin-(4′′_→_8)-(₋_{)-epicatechin-(4}_→₆′′′_)-(₋_{)-epicatechin}_on_the_basis_of_{spectroscopic}_data._Both_the_compounds

exhibitedpotentantioxidantandantigenotoxicactivity.6-Hydroxycoumarin-(4′′_→_8)-(₋_{)-epicatechin}

scavenged DPPH, ABTS+._and _superoxide _anion _radicals_with _IC

50 values of 6.09␮g/ml, 5.95␮g/ml

and42.70␮g/mlrespectivelywhereastheIC50valuesfor6-hydroxycoumarin-(4′′→8)-(−

)-epicatechin-(4→6′′′_)-(₋_{)-epicatechin}_were_6.62_␮_g/ml_for_DPPH_free_radicals,_6.93_␮_g/ml_for_ABTS_radical_cations_and

49.08␮g/mlforsuperoxideanionradicals.Boththecompoundsalsoexhibitedpotentreducing poten-tialinreducingpowerassayandprotectedtheplasmidDNA(pBR322)againsttheattackofhydroxyl radicalsgeneratedbyFenton’sreagentinDNAprotectionassay.InSOSchromotest, 6-hydroxycoumarin-(4′′_→_8)-(₋_{)-epicatechin}_decreased_the_induction_factor_induced_by_4NQO₍₂₀_␮_g/ml)_and_aflatoxin_B1

(20␮g/ml)by31.78%and65.04%respectivelyataconcentrationof1000␮g/ml.Ontheotherhand, 6-hydroxycoumarin-(4′′_→_8)-(₋_{)-epicatechin-(4}_→₆′′′_)-(₋_{)-epicatechin}_decreased_the_genotoxicity_of_these

mutagensby37.11%and47.05%respectively.ItalsoshowedcytotoxicityinCOLO-205cancercellline withGI50of435.71␮g/ml.Boththecompoundsshowedmoderatecyclooxygenase-2inhibitoryactivity.

Introduction

Cancerisamajorpublichealthprobleminallpartsoftheworld. Diversemolecular,biochemicalandcellularmechanismsateach stageof carcinogenesisareaccountable foraltering normal cell cycleprocess and converting a normal cell toa cancerous one (Seifriedetal.,2007).Ithasbeenobservedthatabout20%ormore ofcancercasescouldbepreventedbyincreasedintakeoffruitsand vegetablesindailydiet(RuhulAminetal.,2009).Natural prod-ucts/phytochemicalsarebeingexploredfortheirchemopreventive propertiesduetotheirnon-toxicnature,protectiveeffectsagainst oxidants and theirrecognition as dietary additives (Suh et al., 2009;Linetal.,2011;Liuetal.,2011;Wangetal.,2011;Kundu

∗ _{Corresponding}_authors.

E-mails:bikramsingh@ihbt.res.in(B.Singh),satwinderjeet.botenv@gndu.ac.in (S.Kaur).

etal.,2014;Bohnetal.,2014).Oxidativestressisthemajor deter-minantinthedevelopmentofdiversediseases(Halliwell,1997; Flora,2007; Halliwelland Gutteridge, 2007; Ziech etal., 2010; Ayala-Pena,2013).Plantderivedconstituentsprovideprotection fromROS-inducedDNAdamageandconsequentlyfrom carcino-genesis(Lamsonetal.,2010).Numerousreportshaveshownthe capabilityofphytoconstituentstoprovideprotectionagainstfree radicalinducedailments(Sahinetal.,2010;Ungvarietal.,2010; Negietal.,2011;Scapagninietal.,2011;Xiongetal.,2012;Ziech etal.,2012;Carmona-Ramirezetal.,2013).Phytochemicalsisolated fromdifferentpartsoftheplantsbelongingtodiverseclassesof plantsecondarymetabolitesareaccountableforantioxidant prop-ertiesofmedicinalplants(Chungetal.,1998;Pietta,2000).Toxic effectsofantioxidantsofsyntheticoriginhavelimitedtheir uti-lizationinfoodproducts(Itoetal.,1986;Peters etal.,1996;Li etal.,2002).Naturalplantproductsarefrequentlyreportedas effi-cientchemopreventiveagents(SurhandFerguson,2003).Immense researchcarriedoutinplantscienceshasleadtotheidentification

http://dx.doi.org/10.1016/j.bjp.2016.02.007

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of various plants used in ancient times for their medicinal potential.

Anthocephaluscadamba(Roxb.)Miq.,Rubiaceae,isanAyurvedic medicinalplant,usedintreatingvariousailments.Itisusedasa folkmedicineinthetreatmentoffeverandanemia,asantidiuretic andfor improvementof semenquality. Earlier,in astudyfrom thesamelaboratory,wehavereportedthatamongallfractions, EAAC fractionof A. cadamba leavesexhibited highest potential tocounteroxidativestressinvariousinvitroantioxidantassays (Chandeletal.,2012).Therefore,thepresentstudywasundertaken towardanefforttoisolatetheactivecompoundsresponsibleforthe potentialantioxidantactivityofEAACfractionandtoevaluatethe isolated molecules for theirantioxidant/antigenotoxic/cytotoxic properties.

Materialsandmethods

Bacterialstrain/celllinesandchemicals

EscherichiacoliPQ37 strainwaspurchasedfromInstitut Pas-teur,France.HeLaandCOLO-205cancercelllineswereobtained fromNationalCentreforCellSciences(NCCS),Pune. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), ferric chloride, l_-ascorbic _acid, _NADH (nicotinamideadeninedinucleotide),PMS(phenazine methosul-phate),NBT(nitrobluetetrazoliumchloride),ortho-nitrophenyl␤ -d_{-galactopyranoside}_(ONPG),_para_{-nitrophenylphosphate}_(PNPP), fetal bovineserum(FBS), DMEMculturemedium, RPMIculture medium, MTT, dimethyl sulfoxide (DMSO), penicillin, trypsin, antibiotic/antimycotic solution, HEPES, NaHCO3, streptomycin

wereobtainedfromHiMediaPvt.LimitedMumbai,India.Potassium persulfate, ABTS [2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)diammoniumsalt]andrutinfromSigma(St.Louis,MO,USA). PlasmidpBR322waspurchasedfromGeneiPvt.Ltd.,Banglore.All otherreagentsusedwereofanalyticalgrade(AR).

Collectionofplantmaterial

The plant material used (leaves of Anthocephalus cadamba (Roxb.) Miq., Rubiaceae) was procured during July 2010 from campusofGuruNanakDevUniversity(G.N.D.U.),Amritsar, Pun-jab(India).ThespecimenwasidentifiedandkeptatHerbarium, Department of Botanical and Environmental Sciences, G.N.D.U. withVoucherspecimenno.6557/2011.

ColumnchromatographyofEAACfraction

EAACfraction(15g)waspreparedasmentionedinthe fraction-ationprocedure(Fig.1)anddissolvedin10mlofMeOH,mixedwith silicagelandtheslurrywasmade.Thecolumnwaselutedusinga gradientofhexane/EtOAc(100/0),(98/2),(95/5),(85/15),(80/20), (75/25),(70/30),(60/40),(50/50),(40/60),(30/70),(20/80),(10/90), (0/100)andfinallythecolumnwaselutedwithMeOH.Three frac-tionswerecollectedwhenelutingwithhexane/EtOAc((10/90)viz. EALAC1,EALAC2andEALAC3.Agradientofhexane/EtOAc(100/0), (75/25),(70/30),(65/35),(60/40),(55/45),(50/50),(40/60),(20/80), (0/100)wasusedtofurtherfractionateEALAC2(500mg)andthe columnwaselutedwithMeOH.Compound1(20mg)wasobtained fromfractionselutingin(55:45)n-hexane/EtOAc.

EALAC3 (7.91g) was dissolved in EtOAc and washed with H2O (3×300ml). The EtOAc layerwas dried over sodium

sul-fate and concentrated to obtain EALAC3-S fraction. A gradient ofCHCl3/MeOH(100/0),(95/5),(92/8),(90/10),(85/15),(80/20),

(75/25),(70/30),(60/40),(50/50),(30/70),(0/100)wasusedto col-umnchromatograph EALAC3-Sfraction and finally elutionwas donewithMeOH.Thinlayerchromatographyoffractionscollected inCHCl3/MeOH(90/10)gave singlespotandthefractionswere

concentratedandlyophilizedtoobtaindarkbrowncolored com-pound2(50mg).Compound1wasagainobtainedfromprecipitates obtainedatgradientCHCl3/MeOH(95/5)asmentionedinFig.1.

Antioxidantactivity

DPPH-radicalscavengingassay

ScavengingofDPPHradicalswasassayedusingtheprotocolof Blois(1958)withminormodifications.

ABTSradicalscavengingassay

ThespectrophotometricanalysisofABTS+•_scavenging_activity wasdeterminedaccordingtotheprotocolgivenbyReetal.(1999) withslightmodifications.

Reducingpowerassay

Reducing potential of both the compounds wasdetermined usingthemethodofOyaizu(1986).

Superoxideanionradicalscavengingassay

Themeasurementofsuperoxideanionscavengingactivityof theisolatedcompoundswasperformedaccordingtothemethod ofNishikimietal.(1972)withslightmodifications.

Rutinwasused asstandard antioxidantcompound inDPPH, ABTS.+_,_reducing_power_and_superoxide_anion_radical_scavenging

assays.

DNAprotectionassay

Tomeasurethehydroxylradicalscavengingeffectoftheisolated compounds,DNAnickingexperimentwasperformedaccordingto theprotocolofLeeetal.(2002).

Antigenotoxicactivity

SOSchromotest

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Anthocephalus cadamba (Leaves powder, 1.4 g)

Refluxed with 95% ethanol

Ethanol extract (ETAC) after solvent evaporation (325.4 g)

Dissolved in distilled water

Aqueous ethanol extract Ethyl acetate

EAAC (15 g) Marc

Subjected to column chromatography eluting with a gradient of Hex:EtoAc

Hex:EtoAc::10:90

EALAC1 EALAC2 (500 mg)

Hex:EtoAc::55:45

Compound 1

Upper EtoAc layer Lower aqueous layer

Filteration through sodium-sulfate

Concentrated and dried (EALAC3-S, 1.92 g)

Subjected to column chromatography eluting with a gradient of CHCI3:MeOH

CHCI₃:MeOH::95:5 CHCI₃:MeOH::90:10

Compound 2

Compound 1 Precipitates (200 mg)

CHCI3:MeOH::95:5 Subjected to column chromatography

eluting with a gradient of CHCI3:MeOH

Subjected to column chromatography eluting with a gradient of Hex:EtoAc

EALAC3 (7.91 g)

Dissolved in EtoAc

EtoAc soluble part

Fig.1. Isolationofcompounds1and2fromleavesofAnthocephaluscadamba.

(hydroxymethyl)amino-methane.Theabsorptionwasmeasuredat 420nmusingareferencesolutioninwhichcultureisreplacedbyL medium.

Theenzymeactivitieswerecalculatedaccordingtothe simpli-fiedmethod:

Enzymeunits (U)=A420×1000

t

A420: opticaldensityat 420nm; t: substrateconversiontime in

minutes.

Inductionfactor (IF)= Rc Ro

Rc:␤-galactosidase activity/alkalinephosphatase activity deter-minedforthetestcompoundatconcentrationc,

Ro:␤-galactosidaseactivity/alkalinephosphataseactivityinthe absenceofthetestcompound.

Anti-genotoxicitywas expressedas percentage inhibition of genotoxicityaccordingtotheformula:

Inhibition (%)=100−IF1−IF0 IF2−IF0

×100

where:

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IF2istheinductionfactorofpositivecontrol(4NQOandaflatoxin

B1)

IF0 theinductionfactor oftheblank(without anytest

com-pound).

Cytotoxicity

MTTassay

The cytotoxicityof compounds 1 and 2 on Helaand COLO-205 cancer cell lines was assessed by MTT assay. Cell plating (1×104_/well)_was_done_in_96-well_plates._After₂₄_h_incubation,

cells were incubated with different concentrations of the test sampleinDMSO(0.1%)for24h.Additionof10␮lofMTT(5mg/ml, phosphate-buffered saline solution) was done followed by 2h incubation.The mediumwasdiscardedandDMSOwasusedto dissolvethecrystalsofreducedMTTformed.Opticaldensitywas noticedat570nm.The%ofcytotoxicitywasdeterminedusingthe formulagivebelow:

%cytotoxicity=A0−A1

A0 ×100 where,

A0=absorbanceofcontrol A1=absorbanceoftestsample

InvitroCOX-2inhibitoryactivity

InhibitionofCOX-2activityofisolatedcompoundsfromEAAC fractionfromleavesof A. cadamba wasassessed withthehelp of ‘COX (ovine/human)inhibitor screening assay’ kit (Item No. 560131,CaymanChemicalsCompany,USA).

Statisticalanalysis

The results were presented as the mean±standard error. Regressionanalysiswascarriedoutbybestfit methodandIC50

valueswerecalculatedusingregressionequation.Thedatawere analyzedforstatisticalsignificanceusinganalysisofvariance (one-wayANOVA).Thedifferenceamongaveragevalueswascompared byhonestlysignificantdifference(HSD)usingTukey’stest.The sig-nificancewascheckedat*p≤0.05.

Resultsanddiscussion

Analysisofcompounds1_and2

Compound1

HRMSofcompound1displayedamolecularionpeakatm/z 452.7750correspondingtothemolecularformulaC24H20O9(see

supplementarydataassociatedwiththisarticle).Totaltwentyfour carbonsignalswereobservedin13_C_NMR_spectra_of_compound₁

includingtenmethines,twomethylenesandtwelvequaternaryas evidentfromDEPTspectra.SignalformethyleneatıC28.2(C-4)

correlatingwithıH2.86(1H,m)andıH 2.94(1H,m),inHMQC

spectrumwasfoundtobeadjacenttotwooxygenatedmethines atıH 4.20 (1H,m), ıC66.0 (C-3)and ıH 4.82(1H,overlapped), ıC79.1(C-2)byHMBCanalysis,hence,suggestedthepresenceof

flavan-3-oltypeofunitincompound1.Threearomaticsignalat [ıH6.84(1H,s);ıC114.9(C-2′)],[ıH6.72(1H,m),ıC115.5(C-5′)]

and[ıH6.69(1H,m),ıC118.4(C-6′)]showedlongrangecorrelation

withquaternaryaromaticcarbonsatıC130.7(C-1′),ıC144.9(C-3′)

andıC145.3(C-4′)inHMBCspectraofcompound1.Other

oxy-genatedaromaticcarbonsC-5,C-7,C-9,C3′_and_C4′_were_observed atı156.2,152.5,151.0,144.9and145.3respectively.Onthebasis

oftheseNMRchemicalshiftsandcomparisonwithliterature val-ues(Agrawal andBansal,1989)partialskeleton ofcompound1 appearedtobe(−)-epicatechin.

In1_H_NMR_spectrum,_only_one_singlet _for_H-6_was_observed

insteadofmeta-coupledprotonsforH-6andH-8incaseof(− )-epicatechinthatindicatedthepresenceofsubstitutionatC-8.13_C

NMRspectrumofcompound1showedadditionalsignalsforester typecarbonylatıC169.8,methyleneat[ıH2.83–2.88(2H,m),ıC

37.2(C-3′′_)],_aliphatic_methine_at_[_ı

H4.45(12H,m),ıC34.3(C-3′′)],

aromaticmethines[ıH6.78(1H,m),ıC114.3(C-5′′)],[ıH6.63(1H,

m),ıC114.0(C-7′′)],[ıH6.99(1H,m),ıC118.3(C-8′′)]andthree

qua-ternarycarbonsatıC152.5(C-6′′),144.1(C-9′′)and134.3(C-10′′).

TheanalysisoftheseNMRvaluessuggested6-hydroxycoumarin skeleton.ThesubstitutionatC-8wasconfirmedby13_C_NMR

spec-trumwhichrevealeddownfieldquaternarycarbonforC-8atıC

105.1.Thisquaternarycarbonatı105.1(C-8of(−)epicatechin) showedlongrangecorrelations(HMBC)withmethineatı34.3 (C-4′′_of_{6-hydroxycoumarin)}_suggested_that_both_moieties_were_C-C linkedwhichwasfurtherconfirmedfromspectraldata.Thus,on thebasisofNMRspectraldata(seesupplementarydataassociated withthisarticle)thestructureofcompound1waselucidatedas 6-hydroxycoumarin-(4′′_→_8)-(₋_{)-epicatechin.}_The_structure_is ten-tativelyassignedasforcoumarinunitlinkageatC-6insteadofC-8 similarNMRvaluesandcorrelationareexpected.

Compound2

HRMSofcompound2displayeda molecularionpeakatm/z 741.8964(seesupplementarydataassociatedwiththisarticle) cor-respondingtothemolecularformulaC39H32O15.1Hand13CNMR

signals arevery similartotheNMR of compound 1with addi-tionalsignalscorrespondingtoonemoreepicatechinmoiety.The linkagebetweentwoepicatechinsunitswereestablishedbylong rangecorrelationbetween[ıH2.97–3.07(1H,m),ıC36.7(C-4)]

C-4andıC107.3(C-6′′′)inHMBCspectrum(seesupplementarydata

associatedwiththisarticle).Thus,onthebasisofNMRspectraldata, ‘compound2’wascharacterizedas6-hydroxycoumarin-(4′′_→ 8)-(−)epicatechin-(4→6′′′_)-(₋_{)-epicatechin.}

O

O HO

O

HO

OH

OH OH

OH

1

O

O HO

O

HO

OH

OH OH

OH

HO OH

O HO

OH OH

2

6"

2"

4"

8

5 4

2 1' 3'

6"

2"

4"

8

5 4

2 1' 3'

6"' 5"'

3"'

2"' 1""

3""

4""

Antioxidantactivity

Compounds1and2exhibitedpotentradicalscavenging activ-ityinDPPHassay.Thecompounds1and2scavengedtheDPPH radicalsby77.08%and76.91%respectivelywithIC50of6.09␮g/ml

(compound 1) and 6.62␮g/ml (compound 2) which waslower thanthestandardantioxidantcompoundrutin(IC5054.05␮g/ml)

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y=11.34ln(x)+29.51 r∗∗∗_{= 0.966}

0 20 40 60 80 100

100 80 60 40 20 0

Concentration (µg/ml)

y=19.12ln(x)+15.91 r∗∗=0.943

0 20 40 60 80 100

100 80 60 40 20 0

Inhibition, %

DPPH assay ABTS+. assay

y=0.737x+6.489 r∗∗∗_=0.992

0 20 40 60 80 100

100 80 60 40 20 0

y=0.618x+23.61 r∗∗∗=0.957

0 20 40 60 80 100

100 80 60 40 20 0

Inhibition, %

radical anion Superoxide

scavenging assay Reducing power assay

Fig.2.Antioxidantactivityofcompound1fromAnthocephaluscadambaleavesindifferentassays.

scavengingeffectexhibitedbycompounds1and2was28.35%and 24.99%whichdosedependentlyincreasedto99.76%and99.59% respectivelyatthehighesttestedconcentrationof100␮g/ml.The IC50valueforcompound1wascalculatedas5.95␮g/ml,whereas

compound2showedIC50 valueof6.93␮g/ml whichwaslower

thanthestandardantioxidantcompoundrutin(Figs.2and3).In reducingpowerassayboththecompoundsviz.1and2showed potentreductionpotentialof76.00%and55.57%athighesttested concentration(100␮g/ml)respectively.Standardantioxidant com-poundrutinshowedhigherIC50(IC50ofrutin160.77␮g/ml)than

theisolatedcompounds(IC50ofcompound1:70.27␮g/ml;IC50

ofcompound2:85.48␮g/mlwithrespecttorutin)(Figs.2and3). Bothcompoundsviz.compounds1and 2exhibitedpronounced superoxideanionradicalscavengingwithIC50of42.70␮g/mland

49.08␮g/mlrespectivelywhichwaslessthanthatofstandardrutin (IC5058.75␮g/ml)(Figs.2and3).Boththecompoundsshowedthe

abilitytoprotectthedamagedpBR322plasmidDNAfromtheattack ofhydroxylradicalsgeneratedbyFenton’sreaction(Fig.4).

Thehigher antioxidantactivityofcompounds1and 2might beattributedtothepresence ofmore hydroxyl groups present intheAandBringofthemolecules.Thenumberand configura-tionofhydroxylgroupsandthearrangementoffunctionalgroups aboutthenuclearstructuremayberesponsiblefortheantioxidant capacityofphenolics(Caoetal.,1997;ShekherPannalaetal.,2001). Theresultsofthepresentstudyareinaccordancewiththe sev-eralreportsshowingthepotentantioxidantactivityofflavonoids (Horvathovaetal.,2003;Gulcinetal.,2005;Rahmanetal.,2006; Kalpanaetal.,2009;Emametal.,2010;Hoetal.,2012;Jayasinghe et al.,2012; Tatsimo etal., 2012).Thereis a clearrelationship betweentheantioxidantpowerandthestructuralcharacteristics offlavonoids.Thepotentantioxidantactivityofcompounds1and 2mightbeduetothepresenceofO-dihydroxygroupsinthe B-ring,themeta5,7-dihydroxyarrangementsintheAringanda-OH

groupatposition3.Variousstudiesreportedthepresenceofabove mentionedstructuralcharacteristicsasthemajordeterminantsof antioxidantactivityofflavonoids(RattyandDas,1988;Borsetal., 1990;VanAckeretal.,1996;Rice-Evansetal.,1997;Pietta,2000; Lopez-Velezetal.,2003;Villanoetal.,2005;WolfeandLiu,2008). Moreover,flavonoidscanterminateradicalchainreactionsby servingaselectronandhydrogendonorsandtherebyconverting freeradicalstomorestableproducts(Kellyetal.,2002;Yenand Chen,1995).Thesuperoxideanionsscavengingactivityand antiox-idationof flavonols(quercetin,rutin,morin), flavones(acacetin, hispidulin)andflavanones(hesperidin,naringin)wasstudiedby Yuting et al. (1990). Rutin was found to be the most potent scavengerfollowedbyquercetinandnaringin, whilemorinand hispidulinwereveryweak.Caietal.(1997)attributedthe anti-carcinogeniceffectsofdifferentclassesofflavonoidsi.e.flavonol (quercetin),flavones(luteolin),andisoflavone(genistein)totheir antioxidantactivity.Quercetinandluteolinwerefoundtobepotent scavengerofH2O2andsuperoxideanionradicalsandalsoinhibited

thelipidperoxidationefficiently,whilegenisteinshoweda moder-ateeffectinradicalscavengingandexhibitedweakinhibitoryeffect inlipidperoxidationassay.

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y=11.62ln(x)+28.02 r∗∗∗=0.955

0 20 40 60 80 100

100 80 60 40 20 0

Inhibition, %

y=20.21ln(x)+10.89 r∗∗=0.932

0 20 40 60 80 100

100 80 60 40 20 0

Inhibition, %

DPPH assay ABTS+. assay

y=0.533x+4.434 r∗∗∗=0.996

0 20 40 60 80 100

100 80 60 40 20 0

Inhibition, %

y=0.697x+15.79 r∗∗∗=0.950

0 20 40 60 80 100

100 80 60 40 20 0

Inhibition, %

radical anion Superoxide

scavenging assay Reducing power assay

Fig.3. Antioxidantactivityofcompound2fromAnthocephaluscadambaleavesindifferentassays.

wogonoside isolated from radixof Scutellaria baicalensis. Dose-dependentscavengingofhydroxylradicals,DPPHradicalsandalkyl radicalswasobservedbybaicaleinandbaicalinwhilewogoninand wogonosideshowedveryweakinhibitoryeffectsontheseradicals. Amongallthetestedcompoundsbaicaleinwasthemosteffective antioxidantanditspotentantioxidantactivitywasattributedto thepresenceofo-tri-hydroxylstructureintheAring.Hiranoetal. (2001)evaluatedtheDPPH•_scavenging_activity_of_flavanols (cate-chin,epicatechin[EC],epigallocatechin[EGC],epicatechingallate [ECG], epigallocatechin gallate [EGCG]), flavonols (myricetin, quercetin,kaempferol)andflavones(apigeninandluteolin).EGCG wasthemostpotentDPPHradicalscavenger,whileluteolinwas theleastactive.Theyhavealsoevaluatedtheeffectofflavonoids onLDL oxidation and the inhibitory effectwas in theorder of

luteolin>ECG>EC>quercetin>catechin>EGCG>EGC>myricetin> kaempferol>apigenin.

Variousotherworkersgaveacomparativeaccountof antioxi-dantactivityofflavonoidsindifferentinvitroantioxidantassays andattributedthedifferenceintheiractivitytothepresenceor absence ofsubstituents onring A,ring Bor ring C (Gao etal., 1999;Pietta,2000;Miraetal.,2002;KhandujaandBhardwaj,2003; Emametal.,2010).

Antigenotoxicactivity

Identification of natural products with specific molecular and cellular targets can provide an effective approach to can-cer chemoprevention. Such an approach can be accomplished

1 2 3 4 5 6 ₁ ₂ ₃ ₄ ₅ ₆

100 70 50

10 10 50 70 100

Compound 2

Nicked circular form Linear form Supercoiled form

Compound 1

Figure4.Effectofcompounds1and2fromAnthocephaluscadambaleavesinplasmidDNAnickingassay. Lane1:NegativeControl(DW+pBR322plasmidDNA)

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Table1

Antigenotoxiceffectofcompound1fromleavesofAnthocephaluscadambaagainst4NQOandAFB1inSOSchromotestusingE.coliPQ37testerstrain.

Treatment Dose(␮g/ml) ␤-Galactosidaseunits Alkalinephosphataseunits R Inductionfactor(IF) Mean±SE Mean±SE

4NQO(direct-actingmutagen)

Control(nontreatedcells) 6.23±0.55 29.17±1.99 0.21 1.00 Positivecontrol(4NQO) 20␮g/ml 46.74±4.19 16.57±0.66 2.82 13.43 Negativecontrol(compound1only) 10 4.52±0.24 23.72±2.30 0.19 0.90a

30 4.38±0.29 24.34±2.08 0.18 0.86a

100 4.44±0.28 26.43±2.01 0.17 0.81a

300 4.92±0.19 24.06±1.22 0.20 0.95a

1000 5.10±0.18 23.76±1.67 0.21 1.00a

4NQO+compound1 10 30.58±3.83 12.65±0.45 2.42 11.52

30 34.34±2.95 12.60±0.49 2.73 13.00

100 33.86±2.92 14.32±1.62 2.36 11.24a

300 33.89±2.84 16.09±1.44 2.11 10.04a

1000 31.95±6.21 16.03±0.93 1.99 9.48a

AflatoxinB1(S9-dependentmutagen)

Control(nontreatedcells) 10.42±0.75 61.92±5.04 0.17 1.00

Positivecontrol(aflatoxinB1) 20␮g/ml 97.42±2.99 60.87±5.19 1.60 9.41

Negativecontrol(compound1only) 10 10.53±0.85 62.57±3.16 0.17 1.00a

30 11.42±0.19 61.13±3.98 0.19 1.12a

100 8.93±1.29 60.73±2.62 0.15 0.88a

300 9.03±1.03 61.00±5.48 0.15 0.88a

1000 9.48±0.51 67.18±5.52 0.14 0.82a

AflatoxinB1+compound1 10 61.10±9.04 59.18±6.71 1.04 6.12a

30 61.27±6.48 57.55±5.18 1.06 6.24a

100 57.13±8.15 56.98±5.02 1.00 5.88a

300 51.65±4.71 69.77±10.6 0.74 4.35a

1000 45.68±4.06 68.45±9.59 0.67 3.94a

R,␤-galactosidaseunits/alkalinephosphataseunits;IF,Rc/Ro.

a_Level_of_statistical_{significance:}_p_≤_0.05_with_respect_to_4NQO_and_Aflatoxin_B1.

throughisolation,characterizationandpreclinicalevaluationfor their development as chemopreventive agents (Lippman and Hong,2002;Gupta,2007).Evaluationofantimutagenicactivities of natural products becomes necessary, as there is concor-dancebetweenantimutagenicityandanticarcinogenicity(Maron and Ames, 1983; El-Sayed et al., 2007). Antimutagenic stud-ies of botanical extracts form the foundation for selecting the leadextractsfor longtermand costlyinvivo chemoprevention investigationstogetherwiththeseparation and chemical struc-tural elucidation ofpossible active compounds(EI-Sayedet al., 2013).

IntheSOSchromotest,itwasascertainedthatdifferent concen-trationsofcompounds1and2addedtotheindicatorbacteriawere notgenotoxicastheinductionfactorinducedbythetesteddoses wasbelow1.5.Table1andFig.5showedthatataconcentrationof 10␮g/ml,compound1inhibitedthegenotoxicityofAFB1(IF=9.41)

by39.12%whichdosedependentlyincreasedandata concentra-tionof1000␮g/ml,itshowedaninhibitionof65.04%.Similarly, compound1inhibitedtheinductionfactorof4NQO(IF=13.43)by 15.37%ataconcentrationof10␮g/mland31.78%at1000␮g/ml.As seenfromTable2andFig.5,compound2didnoteffectively mod-ulatedthegenotoxicityof4NQOandAFB1.Compound2reduced theinductionfactor of4NQO(IF=10.62)and AFB1(IF=8.80)by 37.11%and47.05%athighesttestedconcentrationof1000␮g/ml respectively.

Epicatechinsareoneofthecomponentsofteapolyphenolsand in a report by Itoet al. (1989),protective effects of hot water greenteaextractswereevaluatedagainsttheAFB1-induced chro-mosomal aberrations in bone marrow cells. Mutagenic effects mightbeinhibitedbycatechinsduetoflavanol–mutagenadduct formation(Stich,1991).Kuroda(1996)reportedthe bioantimu-tagenic activity of catechins against the direct-actingmutagen

100

80

60

40

20

0 100

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20

0

10 30 100 300 1000 10 30 100

Compound 1 Compound 2

Concentration (

µ

g/ml)

% Inhibition

4NQO Aflatoxin B1 300 1000

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Table2

Antigenotoxiceffectofcompound2fromleavesofAnthocephaluscadambaagainst4NQOandAFB1inSOSchromotestusingE.coliPQ37testerstrain.

Treatment (␮g/ml) ␤-Galactosidaseunits Alkalinephosphataseunits R Inductionfactor(IF) Mean±SE Mean±SE

4NQO(direct-actingmutagen)

Control(nontreatedcells) 5.71±1.14 26.72±1.19 0.21 1.00 Positivecontrol(4NQO) 20␮g/ml 56.16±6.19 25.17±1.55 2.23 10.62 Negativecontrol(compound2only) 10 4.16±0.74 19.20±3.74 0.22 1.04a

30 3.92±0.72 19.62±3.54 0.20 0.95a

100 5.01±0.53 19.31±3.25 0.26 1.24a

300 5.38±0.39 20.05±3.59 0.27 1.29a

1000 6.20±0.62 19.96±3.67 0.31 1.48a

4NQO+compound2 10 43.24±5.13 22.59±2.35 1.91 9.10

30 41.01±6.15 22.67±2.17 1.81 8.62

100 35.26±4.24 22.32±2.21 1.58 7.52

300 40.67±5.80 23.15±2.34 1.78 8.48

1000 37.73±7.02 25.46±2.67 1.48 7.05

AflatoxinB1(S9-dependentmutagen)

Control(nontreatedcells) 11.92±0.21 78.13±3.18 0.15 1.00

Positivecontrol(aflatoxinB1) 20␮g/ml 95.80±1.34 72.63±2.69 1.32 8.80

Negativecontrol(compound2only) 10 11.08±0.99 74.63±2.13 0.15 1.00a

30 11.62±0.35 76.77±1.81 0.15 1.00a

100 12.92±0.43 76.10±4.41 0.17 1.13a

300 11.15±0.18 76.57±1.82 0.15 1.00a

1000 10.58±0.32 79.52±0.67 0.13 0.87a

AflatoxinB+compound2 10 56.12±2.51 79.83±3.26 0.70 4.67a

30 62.13±1.37 75.40±1.99 0.82 5.47a

100 60.30±6.34 75.40±1.98 0.80 5.33a

300 66.38±3.91 76.35±2.24 0.87 5.80a

1000 59.45±3.22 77.43±1.83 0.77 5.13a

R,␤-galactosidaseunits/alkalinephosphataseunits;IF,Rc/Ro.

a_Level_of_statistical_{significance:}_p_≤_0.05_with_respect_to_4NQO_and_Aflatoxin_B1.

i.e.4NQO.Matsumotoetal.(1996)andWeisburgeretal.(1997) reportedthat epicatechins modulatethe activityof detoxifying enzymesi.e.reductionofcytochromeP450andincreaseinphase II enzymes. Bhouri et al. (2011) reported the antigenotoxicity of two flavonoids, kaempferol 3-O-␤-isorhamninoside (K3O-ir) andrhamnocitrinand3-O-␤-isorhamninoside(R3O-ir)againstthe genotoxicityinducedbynitrofurantoineandaflatoxinB1.K3O-ir andR3O-irreducedthegenotoxicityofaflatoxinB1significantly by96.64%and90.26%,respectivelyatthehighesttested concentra-tionof10␮g/ml.Flavonoidscanalsoactasdesmutagensbydirectly interactingwithmutagensandinactivatingthem(Heoetal.,1994). Antigenotoxicpotentialofapigenin(flavone)againstmitomycinC inducedgenotoxicdamageinmousebonemarrowcellswas stud-iedbySiddiqueandAfzal(2009).Resultsofthestudydemonstrated thatapigenineffectivelydiminishedthegenotoxictyofmitomycin Casreflectedfromdecreaseofsisterchromatidexchanges(SCEs) andchromosomalaberrationsinmousebonemarrowcells.Hayder etal.(2004)reportedtheantigenotoxicactivityofextractsfrom Myrtuscommunisandalltheextractswerefoundeffectiveagainst thegenotoxicityofAFB1andnifuroxazideandtheyattributedthe potentialofthetestedextractstowardantigenotoxicitytothe pres-enceofflavonoids,coumarinsandtannins.

Antiproliferativeandinvitro_COX-2_inhibitory_activity

Only compound 2 exhibited cytotoxicity against COLO 205 celllinewithpercentinhibitionof 52.06%at500␮g/ml(Fig.6). Howeverboththecompoundswerefoundtobeineffectiveagainst HeLa cellline.Procyanidins(flavonoids)-richgrape seedextract showedgrowthinhibitoryeffectand inductionofapoptoticcell deathina humanprostate carcinomaDU145cell line(Agarwal et al., 2002). Wang et al. (1999) investigated the mechanism of inductionof apoptosisby apigenin,myricetin, quercetinand kaempferol in HL-60 leukemiacells. Apigenin was foundtobe mostpotentinreducingthecellviabilitywithIC50of50␮M.Park

and Min(2011) reported that quercetininhibited invasion and

proliferationofgliomacellsbydownregulationofphospholipase D1,aregulatorofcellproliferationandtumorigenesis.Compound 1fromA.cadambaleavesinhibitedtheCOX-2by17.79%at concen-trationof1␮Mwhereas compound2showed25.64%inhibition atsameconcentration.Compound2wasfoundeffectiveagainst COLO-205 and this antiproliferative activity of the compound may partly be due to the inhibition of COX-2. Overexpression of COX-2hasbeen observedin colon tumors (Kawamori etal., 1998; Crofford, 1997; Roelofs et al., 2014). Therefore specific COX-2 inhibitors could potentially serve as chemopreventive agents. Ye et al. (2004) investigated the anticancer activity of genistein on human oral squamous carcinoma line (SCC-25). Genisteininhibitedthegrowthoforalsquamouscarcinomacells withIC50ofapproximately200␮MviaG2/Marrest.Genisteinat

aconcentrationof0.1␮Meffectivelydecreasedtheexpressionof COX-2.

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Colo-205 cell line

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Conclusions

The isolated phytochemicals from A. cadamba have the potential to alleviate the genotoxicity of environmental muta-gens/carcinogens.Thesehavealsobeenfoundtopossesssignificant antioxidantactivitygreaterthanthatofstandardcompoundrutin. The results clearly show the chemopreventive potential of A. cadambaphytochemicalsand canbeapromisingnaturalsource inhealthandmedicine.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethattheyhave fol-lowed theprotocolsof theirworkcenter onthepublication of patientdata.

Right to privacy and informed consent. The authors have obtainedthewritteninformedconsentofthepatientsorsubjects mentionedinthearticle.Thecorrespondingauthorisinpossession ofthisdocument.

Authors’contributions

MCcarriedoutextraction,isolationandmajorbioactivitypart ofpresentwork.MKandMCcollectivelyconductedcytotoxicand COX-2inhibitoryactivitiesalongwithstatisticallyanalysisofthe data.US,NKandBScarriedoutstructuralelucidationand char-acterizationofisolatedcompounds.SKdesigned,supervisedand criticallycheckedthemanuscript.Allauthorsreadandapproved thefinalversionofmanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

The authors are thankful to UGC (DRS-SAP), New Delhi for providingfinancialassistanceandDirector,IHBT,Palampurfor pro-vidingthenecessaryfacilitiestocarryoutisolationpart.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2016.02.007.

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