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RevistaBrasileiradeFarmacognosia26(2016)763–766

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

Short

communication

A

cytotoxic

C

-glycosylated

derivative

of

apigenin

from

the

leaves

of

Ocimum

basilicum

var.

thyrsiflorum

Mohamed

I.S.

Abdelhady

a,b,∗

,

Amira

Abdel

Motaal

c,d

aPharmacognosyDepartment,FacultyofPharmacy,HelwanUniversity,Cairo,Egypt

bPharmacognosyDepartment,FacultyofPharmacy,UmmAl-QuraUniversity,Makkah,SaudiArabia cPharmacognosyDepartment,FacultyofPharmacy,CairoUniversity,Cairo,Egypt

dPharmaceuticalBiologyDepartment,FacultyofPharmacyandBiotechnology,GermanUniversityinCairo(GUC),Cairo,Egypt

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received12February2016

Accepted7June2016

Availableonline20July2016

Keywords: Ocimumbasilicum

HCT116

Cytotoxic Antioxidant C-glycosylation

Apigeninderivative

a

b

s

t

r

a

c

t

Thestandardized80%ethanolicextractoftheleavesofOcimumbasilicumvar.thyrsiflorum(L.)Benth., Lamiaceae,growinginKSA,exhibitedasignificantantioxidantactivitycomparedtotheethylacetateand butanolextracts,whichwascorrelatedtoitshigherphenolicandflavonoidcontents.Chromatographic separationofthe80%ethanolextractresultedintheisolationoftenknowncompounds;cinnamicacid, gallicacid,methylgallate,ellagicacid,methylellagicacid,apigenin,luteolin,vitexin,isovitexin,and3′′-O -acetylvitexin.Compound3′′-O-acetylvitexin,aC-glycosylatedderivativeofapigenin,wasisolatedforthe firsttimefromgenusOcimum.The80%ethanolicextractand3′′-O-acetylvitexinshowedsignificant cyto-toxicactivitiesagainsttheHCT116humancoloncancercellline[IC50values22.3±1.1and16.8±2.0␮g/ml

(35.4␮M),respectively].

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Herbsprovideduswithsomeoftheveryimportantlifesaving drugsusedinthearmamentariumofmodernmedicine(Goyaletal., 2007).Someworld’spopulationdependsontraditionalmedicine becauseofscarcity,highcostoforthodoxmedicineand unpleas-antsideeffects(Agrawaletal.,2011).Amongtheplantsknownfor theirmedicinalvaluearetheplantsofgenusOcimum,family Lami-aceae,whicharerichinphenolicconstituentsandareveryuseful fortheirtherapeuticpotentials(Nahaketal.,2011).Severalstudies haveshownvariousactivitiesofOcimumspeciesincluding bac-tericidal,antiulcer,antidiarrheal,antiinflammatory,antioxidative, anticancer,forcoughandkidneymalfunction,hypoglycemic, ner-voussystemstimulationandprotectionfromradiation(Elansary

and Mahmoud, 2015; Kadan et al., 2016). The pharmaceutical

potentialityof Ocimum species may beattributed totheir pro-foundbiologicaleffectsduetothepresenceofactivepolyphenols, ashydroxycinnamicacids(caffeic acidandrosmarinicacid)and flavonoids,mainlyintheformofderivativessuchasestersand gly-cosides(Wangetal.,2004).Aninterestingplantwhichbelongsto

∗ Correspondingauthor.

E-mail:miabdelhady@uqu.edu.sa(M.I.Abdelhady).

genusOcimumisO.basilicumL.whichisnativetoIndiaandis culti-vatedinotherregionsofAsia,Africa,theMediterraneanregionand KSA.

ChemicalandbiologicalinvestigationofO.basilicumvar. thyr-siflorumgrowinginKSAwascarriedout.Thecytotoxicactivityof aC-glycosylatedderivativeofapigeninwasstudiedagainsthuman coloncancercelllines,alongwiththeparentstandardizedethanolic extract.

Materialsandmethods

General

NMR(1Hand13CNMR)spectrawererecordedat300MHzfor 1Hand125MHzfor13ConaVarianMercury300.ESI-MS

spec-traweremeasuredusingmassspectrometerconnectedtoanESI-II ion source (Finnigan, Lc-MSLCQdeca. Advantage MAX, Finnigan SurveyorLCpump). TheUVanalysesforpurecompoundswere recordedonaShimadzuUV240spectrophotometer.UV-VS spec-trophotometer(MiltonRoy601) wasusedfor determinationof totalphenoliccontent.Stationaryphasesusedwerepolyamide6S (SeelzeHannover,Germany),sephadexLH-20(Fluka,Switzerland) andcellulose(Pharmacia,Uppsala,Sweden).Purityoftheisolated compoundswas testedby HPLC/DAD (HewlettPackard,Agilent

http://dx.doi.org/10.1016/j.bjp.2016.06.004

0102-695X/©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

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764 M.I.Abdelhady,A.A.Motaal/RevistaBrasileiradeFarmacognosia26(2016)763–766

O.basilicum

leaves, 1100 g

80% ethanolic extract, 50 g

Defatting, reflux 60ºC

80% ethanolic extract, 30 g

Fraction I

(0-10%)

Compounds 1,

2, 3, and 5

Unidentified

compounds

Compounds 4,

8, 9 and 10

Compounds 6

and 7

Fraction II

(10-30%)

Fraction III

(30-50%)

Fraction IV

(50-90%)

Fraction V

(90-100%)

Sephadex 30%-70% MeOH Sephadex, BIW Cellulose C,

10%-70% MeOH, Sephadex, MeOH:H2O Cellulose C,

10%-50% MeOH, Sephadex, MeOH:H2O Cellulose C,

BIW, Sephadex, MeOH:H2O Phenolic free

(Free sugars)

Polyamide 6S, H2O:MeOH

Fig.1. Schemedemonstratingthefractionationofthe80%ethanolicextractofOcimumbasilicum.BIW,butanolisopropanolwater.

1100,quaternarypumpG1311A,vacuumdegasserG1322A, col-umnovenG1316A,photodiodearraydetectorG1315A,column C18silica10␮mparticlesize,Lichrocart,WaterIreland).

DPPH (1.1-diphenyl-2-picrylhydrazyl) was purchased from Sigma-AldrichCo.(StLouis,MO).Sodiumphosphate,ammonium molybdate,Folin-Ciocalteu’sreagent,ascorbicacid,gallicacidwere purchasedfromMerckChemicalCo(Darmstadt,Germany).

LeavesofOcimumbasilicumvar.thyrsiflorum(L.)Benth., Lami-aceae,werefreshly collectedfromTaif,KSA. Thesampleswere collectedinMay2012.AsampleoftheplantwasidentifiedbyProf. Dr.MohamedM.Milad,DepartmentofBiology,FacultyofApplied Sciences,UmmAlquraUniversity,KSA.Avoucherspecimen(no. 402)wasdepositedattheherbariumofFacultyofPharmacy, Hel-wanUniversity.

Threeseparateportions(50geach)oftheairdriedgroundleaves wereextractedwith80%aqueousethanol(80%EtOH),ethylacetate (EtOAc)andn-butanolsaturatedwithwater(n-BuOH)(500ml×3) underreflux (70◦C), yielding three extractsof 2, 1.2 and 1.5g,

respectively.Fortheisolationofpurecompounds,thedriedleaves (1100g) wereextracted with80% EtOH (2.5l×5) underreflux (70◦C).Fractionationandisolationofcompoundswascarriedout

asshowninFig.1.

8-C-ˇ-d-(3′′-O-acetyl)glucopyranosylapigenin(1)

Paleyellowamorphouspowder(21mg);Rfvalues, 0.29(S1),

0.56 (S2), dark purple spot under UV light which turned into

green with FeCl3 and greenish yellow with Naturstoff spray

reagents;UV(MeOH):maxnm: 269,301, 335,(+NaOMe):287,

336,401,(+NaOAC):282,305, 386,(+AlCl3):275,304(sh),350,

390, (+AlCl3/HCl): 277, 302 (sh), 346, 391; Negative ESI-MS:

m/z 473.3743 [M−H]− 431.1153 [M-COCH3](calculated for

C23H22O11,474);1HNMR(300MHz,DMSO-d6):ıppm13.25(1H,

s,H-bondedOH-5),8.03(2H,d,H-2′/6),6.90(2H,d,H-3/5),6.63

(1H,s, H-3),6.27(1H,s,H-6),5.17(1H,m, H-3′′),4.97(1H,brs,

H-1′′),4.37(1H,brt,H-2′′),3.94(1H,m,H-4′′),3.83(1H,m,H-5′′),

3.53(2H,d,H-6′′),1.96(3H,s,methylgpoftheacetyl);13CNMR

(125MHz,DMSO-d6):ı ppm182.54 (C-4),164.86(C-2),163.04

(C-7),161.59(C-5),160.83(C-4′),156.44(C-9),129.41(C-2/6),

122.05(C-1′),116.26(C-3/5),104.47(C-8),105.06(C-10),102.88

(C-3),98.59(C-6),82.29(C-5′′), 79.11(C-3′′), 73.84(C-1′′),71.29

(C-2′′),71.26(C-4′′),61.74(C-6′′),24.14(CH3ofacetyl),169.47(CO

ofacetyl).

1

O O O O O

OH

OH OH

HO HO

HO

Thetotalphenolic contentsof 80%EtOH,EtOAcand n-BuOH extracts of O. basilicum were determined using Folin-Ciocalteu reagentand gallicacidasa referencestandardaccordingtothe methoddescribedbyKumaretal.(2008).Thetotalphenoliccontent wasexpressedasmggallicacidequivalent(GAE)/gextract.

Thetotalflavonoidcontentsofthethreeextractswere deter-minedusingtheproceduredescribedbyKumaranandKarunakaran

(2006)usingquercetinasastandard.Thetotalflavonoidcontentin

eachextractwasdeterminedasmgquercetinequivalent(QE)/g extract.

Theabilityof80%EtOH,EtOAcandBuOHextractsofO.basilicum toscavengeDPPHradicalswasevaluatedaccordingtothe proce-duredescribedbyMensoretal.(2001).Ascorbicacidwasusedas areferencestandard.

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M.I.Abdelhady,A.A.Motaal/RevistaBrasileiradeFarmacognosia26(2016)763–766 765

Table1a

DPPHradicalscavengingactivityofOcimumbasilicumextracts.

Concentration(␮g/ml) Ascorbicacid 80%EtOH EtOAc n-BuOH

12.5 28.59±0.25 15.23±1.10 13.33±0.54 9.43±1.46

25 37.03±0.40 27.62±1.76 19.56±1.30 14.64±1.88

50 54.26±0.21 59.35±2.67 39.47±1.22 23.43±2.72

100 87.43±0.35 76.48±2.36 54.41±1.87 40.21±3.12

IC50(␮g/ml) 44.16±0.9 53.85±0.5 83.59±2.1 127.37±0.8

thehumancoloncancercelllinesHCT116,usingDoxorubicin®asa positivecontrol.Datawereanalyzedbyone-wayanalysisof vari-ance (ANOVA). Differenceswere considered significantwhen p valueswere<0.05.

Resultsanddiscussion

The 80% EtOH, EtOAcand n-BuOH extracts from the leaves of O. basilicum were standardized to their total phenolic and flavonoid contents. The 80% EtOH extract was stan-dardized to contain the highest content of phenolics and flavonoids (77.3±3.0GAE/g, 43.6±1.5QE/g, respectively), fol-lowed by EtOAc (48.3±2.1GAE/g, 32.6±2.2QE/g) and n-BuOH extracts(29.2±1.4GAE/g,15.4±2.6QE/g).

The standardized extracts were assessed for their capac-ity to scavenge DPPH free radical along with ascorbic as a positive control (Table 1a). The 80% EtOH extract of the leavesof O.basilicumexhibitedpronouncedantioxidantactivity (IC50=53.85±0.5␮g/ml),followedbyEtOAcandn-BuOHextracts

(IC50=83.59±2.1and127.37±0.8␮g/ml,respectively)compared

toascorbicacid(IC50=44.16±0.9␮g/ml),owingtoitshigher

phe-nolicandflavonoidalcontents.Itiswellknownthatthereisastrong relationshipbetweentotalphenolcontentandantioxidantactivity, asphenolspossessstrongscavengingabilityforfreeradicalsdue totheirhydroxylgroups.Thus,thephenoliccontentofplantsmay directlycontributetotheirantioxidantaction(AbdelMotaaland

Shaker,2011).

Tencompoundswereisolatedfromtheactive80%EtOHextract wherenineofthemwerechemicallyidentifiedusingUV,1HNMR, 13CNMR,ornegativeESI-MS,andbycomparisonwithpreviously

publisheddata(Meyeretal.,2006;López-Lázaro,2009;Lietal.,

2011;Choo et al.,2012; Kubacey etal., 2012).Thecompounds

werecinnamicacid,gallicacid,methylgallate,ellagicacid,methyl ellagicacid,apigenin,luteolin,vitexin,isovitexin,wherevitexinand isovitexinarethe8-and6-C-glucosidesofapigenin,respectively. Compound1wasisolatedasa paleyellowamorphouspowder. AccordingtothechromatographicpropertiesandUVspectraldata, compound1wasexpectedtobeC-glycosylapigenin.TheUV spec-truminMeOHexhibitedthetwocharacteristicabsorbtionbands atmax269nm(bandII)and335nm(bandI)ofapigeninnucleus.

UponadditionofNaOAc,abathochromicshiftofbandII(≈+8)was diagnosticforafree7-OH group.Theremainingdiagnosticshift reagentswereincompleteaccordance with5,7,4′

-trihydroxy-C-glycosylflavonestructure (Mabryet al.,1970).NegativeESI-MS spectrumexhibited themolecularion peak atm/z473[M−H]−

(calculatedforC23H22O11,474)andfragmentionpeakatm/z431

afterlossofanacetylmoietyindicatinganapigeninacetylhexoside structure.1HNMRspectrumshowedanAXcouplingsystemoftwo

orthodoublets,eachintegratedfortwoprotonsatıH8.03and6.90

assignedtoH-2′/6andH-3/5,respectively,of1,4-disubstituted

ring-B.InadditionthetwosingletsignalresonancesatıH6.63and

6.27assignabletoH-3andH-6,respectively,showed characteris-ticsofanapigeninmoietymissinganH-8resonancesignal.The anomericprotonappearedasbroadsingletatıH4.97givingthe

suggestionofthepresenceofaC-glucosidemoiety.Theabsenceof H-8gavetheexpectationofC-glucosidationonC-8.Thisevidence

O

O OH HO

OH O

OH HO

O

HO O

H H

H

H

H H

H

H H

H

H

Fig.2. StructureandkeyHMBC(H→C)ofcompound10.

wasconfirmedfromthedownfieldshiftof13C-resonanceofC-8to

ıC104.47andtheupfieldofC-7andC-9toıC163.04and156.44,

respectively.Moreover,theC-glucosidemoietywasconfirmedas ␤-glucopyranosidedependingonthecharacteristicupfieldlocation ofC-1′′atıC73.84anddownfieldlocationsofC-5′′andC-3′′atıC

82.29and79.11,respectively.ThedownfieldshiftofC-3′′atıC79.11

indicatedthepositionofacetylmoietywithrespecttothoseof C-glucoside.HMBCapprovedthisstructureandthelinkagebetween theacetylmoietyandC-3′′(Fig.2).All1Hand13Cresonanceswere

assignedbycomparisonwiththecorrespondingvaluesof struc-turallyrelatedcompoundsofpreviouslypublisheddata(Kimetal.,

2005;Zhouetal.,2013).

Thuscompound1wasidentifiedas8-C-␤-d-(3′′-O-acetyl)

glu-copyranosylapigeninor3′′-O-acetylvitexinandwasisolatedforthe

firsttimefromgenusOcimum.

Recentlyapigeninbecameinterestingasabeneficialhealth pro-motingagentbecauseofitslowintrinsictoxicity.ItsC-glycosylated derivatives,vitexinandisovitexin,werereportedtopossess anti-diabetic,anti-Alzheimer’sdisease,andanti-inflammatoryactivities (Choietal.,2014).Vitexin-2-O-xylosidepreviouslyshowedahigher activityagainstcoloncancercelllinesCaCo-2(IC50120±9)than

againstbreastcancercellsMCF-7(IC50350±48)(Papietal.,2013).

The 80%EtOH extract and compound 1 weretested against HCT116coloncarcinomaandshowedasignificantcytotoxic

activ-ity (IC50 value 22.3±1.1 and 16.8±2.0␮g/ml corresponding to

Table1b

Cytotoxicactivitiesofthe80%ethanolextractandcompound10ofOcimumbasilicum

againstHCT116coloncelllines.

Concentration(␮g/ml) Survivalfraction(mean±SD)

80%EtOHextract Compound10

5 0.824±0.021 0.761±0.034

10 0.657±0.013 0.582±0.007

25 0.467±0.032 0.401±0.003

50 0.346±0.016 0.317±0.031

100 0.303±0.042 0.234±0.026

IC50(␮g/ml) 22.3±1.1 16.8±2.0

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766 M.I.Abdelhady,A.A.Motaal/RevistaBrasileiradeFarmacognosia26(2016)763–766

35.4␮M,respectively,Table1b)comparedtoDoxorubicin®(IC

50

value3.7±0.21␮g/mlcorrespondingto6.4␮M).

Conclusion

TheethanolicextractoftheleavesofO.basilicumvar. thyrsiflo-rumshowedpotentantioxidantactivityowingtoitsrelativelyhigh phenolicand flavonoidcontents, comparedtotheethyl acetate andn-butanolextracts.Fourteencompoundswereisolatedfrom theactiveethanolicextract,wherecompound1,aC-glycosylated derivativeofapigenin,wasisolatedforthefirsttimefromOcimum genusandidentifiedas8-C-␤-d-(3′′-O-acetyl)

glucopyranosylapi-genin(or) 3′′-O-acetyl-vitexin.Bothcompound 1and itsparent

extractshowedsignificantcytotoxicactivitiesagainsttheHCT116

humancoloncancercellline.

Authorcontributions

MIandAAparticipatedinstudyconceptanddesign, acquisi-tionofdata,analysisandinterpretationofdata,criticalrevision ofthemanuscriptforimportantintellectualcontent,anddrafted themanuscript. MIcarriedouttheextraction, fractionationand isolationofpurecompounds.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

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Imagem

Fig. 1. Scheme demonstrating the fractionation of the 80% ethanolic extract of Ocimum basilicum
Fig. 2. Structure and key HMBC (H → C) of compound 10.

Referências

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