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,daPharmacognosyDepartment,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±2.0g/ml
(35.4M),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://
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 C18silica10mparticlesize,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.
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±0.5g/ml),followedbyEtOAcandn-BuOHextracts
(IC50=83.59±2.1and127.37±0.8g/ml,respectively)compared
toascorbicacid(IC50=44.16±0.9g/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′/6′andH-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±2.0g/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
766 M.I.Abdelhady,A.A.Motaal/RevistaBrasileiradeFarmacognosia26(2016)763–766
35.4M,respectively,Table1b)comparedtoDoxorubicin®(IC
50
value3.7±0.21g/mlcorrespondingto6.4M).
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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