RevistaBrasileiradeFarmacognosia27(2017)50–53
w ww.e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
Validated
high
performance
thin
layer
chromatography
method
for
simultaneous
determination
of
quercetin
and
gallic
acid
in
Leea
indica
Ankita
A.
Patel,
Aeshna
A.
Amin,
Arpit
H.
Patwari,
Mamta
B.
Shah
∗DepartmentofPharmacognosy,L.M.CollegeofPharmacy,Ahmedabad,Gujarat,India
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received14October2015 Accepted24May2016
Availableonline19September2016
Keywords: Gallicacid HPTLC Leeaindica Quercetin
Simultaneousestimation Validation
a
b
s
t
r
a
c
t
Asensitiveandreliablehighperformancethinlayerchromatographymethodhasbeendevelopedfor
thesimultaneousestimationofquercetinandgallicacidinLeeaindica,Vitaceae.Ethylacetateextract
preparedfromhydrolysedaqueousalcoholicextract(70%)wasappliedonsilicagelG60F254 plate.
Theplatewasdevelopedusingtoluene-ethylacetate-formicacid,5:4:1(v/v/v)asamobilephaseand
detectionandquantificationwereperformedbydensitometricscanningat254nm.Thesystemwas
foundtogivewellresolvedbandsforquercetin(Rf0.63)andgallicacid(Rf0.45)fromotherconstituents
presentintheextractofL.indica.Thecorrelationcoefficientwasfoundtobe0.991and0.999withrelative
standarddeviation,0.97–1.23%and0.1–1.13%forquercetinandgallicacidrespectivelyinthedeveloped
method.Theaccuracyofthemethodwasconfirmedbyconductingrecoverystudiesatdifferentlevels
usingthestandardadditionmethod.Theaveragerecoveryofquercetinandgallicacidwasfoundclose
to99%suggestingtheaccuratenessofthemethod.Theproposedvalidatedhighperformancethinlayer
chromatographicmethodoffersanew,sensitive,specificandprecisegaugeforquantificationofquercetin
andgallicacidinL.indica.
©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen
accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Leeaindica(Burm.f.)Merr.,(Syn.:L.sambucinaWilld.,L. gigan-tea Griff., L. umbraculifera Cl., Stephylea indica Burm. f.) Merr.,
Vitaceae, a shrub foundto be growing in the forests of
tropi-calandsubtropicalcountriesincludingIndia,SriLanka,Malaysia,
Thailand,AndamanandPhilippines(Stewartand Brandis,1874;
Gamble, 1956). In traditional systems of medicine the leaf is
valuedin vertigoand diabetes, whilethe rootasantidysentric,
anthelmintic,spasmolyticandsudorificandtotreatcardiacand
skindiseases(ChatterjeeandPrakashi,1997;Khare,2007;Rahman et al., 2007).The leaf is reported to possess antioxidant (Saha et al., 2004, Reddy et al., 2012), analgesic (Saifuzzaman et al., 2013), antimicrobial(Srinivasanetal.,2010),phosphodiesterase inhibitory(Temkitthawonetal.,2008),sedative,anxiolytic(Raihan etal.,2011)andcytotoxic(Nurhananetal.,2008;Emranetal.,2012; Raihanetal.,2012)activities.Theplantisreportedtocontain˛
-tocopherol,-amyrin(Sahaetal.,2005),lupeol,-sitosterol,ursolic acid,farnesol,phloridzin,gallicacid(Srinivasanetal.,2008),and quercitrin(Joshietal.,2013).Flavanoids,kaempferol,quercetin,
∗ Correspondingauthor.
E-mail:[email protected](M.B.Shah).
quercitrin,andmearnsitrinhavebeenreportedfromotherspecies
ofgenusLeea(L.guineense)(Becketal.,2003).
HPTLCisasanacquiescentandcommonlyusedtechniquefor
qualitativeandquantitativeanalysisofchemicalmarkersinherbal
raw materials. HPTLC has advantages of simplicity, sensitivity,
accuracy,andisonethemostapproachedtechniquefordeveloping
fingerprintandmarker-basedstandardizationofherbaldrugsand
iscommonlyappliednotonlyfortheidentification,assayand test-ingforpuritybutalsoforstability,dissolutionorcontentuniformity ofrawmaterialsandformulatedproducts(Mukherjeeetal.,2010).
Taking in to consideration the non-existence of a routine
methodofanalysis,asimpleHPTLCmethodforquantitative
esti-mationofquercetinandgallicacidsimultaneouslyisproposedfor
L.indica.Theproposedmethodwasvalidatedbyspecificity,range, linearity, accuracy,precision,detection limit, quantitativelimit, androbustnessaccordingtotheICHguidelines(ICH,1996,2005).
Materialsandmethods
Chemicalsandsolvents
Ethylacetate,methanol,toluene,HCl,NaOH,distilledwaterused wereofanalyticalgrade.Referencestandardsquercetinandgallic
acid(purity99.9%)werepurchasedfromSigma–Aldrich.
http://dx.doi.org/10.1016/j.bjp.2016.05.017
A.A.Pateletal./RevistaBrasileiradeFarmacognosia27(2017)50–53 51
Plantmaterial
ThefreshshrubofLeeaindica(Burm.f.)Merr.,Vitaceae,was
pro-curedfromWaghaiBotanicalGarden,Dang,Gujaratinthemonth
ofSeptember2012andauthenticatedbythetaxonomistofSouth
GujaratUniversity,Surat,India.Voucherspecimenof plant(LM
5412)hasbeendepositedinDepartmentofPharmacognosyand
Phytochemistry,L.M.CollegeofPharmacy,Ahmedabad.Thefresh
plantwascut,driedproperly,powdered,andpassedthrough60#
sieve.Thepowderedsamplewasstoredinanairtightcontainerat
roomtemperature.
HPTLCanalysis
Quercetinand gallic acidstandard stocksolutions were
pre-paredbydissolving2mgofthecompoundinmethanolin10ml
volumetricflaskandvolumewasadjusteduptothemarkwith
methanol,fromthissolution1–5lsolutionswasappliedon pre-coatedTLCplatesforquercetinwhile1–6lwasspottedforgallic acidforcalibrationpurpose.
Accuratelyweighed10gofplantpowderwasextracted
exhaus-tivelywithethanol(100ml×3)underrefluxfor2h/cycle.Ethanolic
extractwasevaporatedtodrynessundervacuumandresiduewas
dissolvedin80mlaqueousmethanol(70%).Theaqueousmethanol
solutionwashydrolysedbyusing40ml2NHCland40mltoluene
andrefluxingfor2h.Afterneutralizingthesolutionby5%NaOH,
itwasagainrefluxedfor1h.Theaqueousphasewassuccessively
extractedwithtoluene (2× 30ml)andethylacetate(4×50ml).
Ethyl acetate extract wasevaporated to dryness and 10mg of
extractwasdissolvedin5mlmethanoland15lofmethanolic
solutionwasappliedtoaTLCplate.Co-TLCstudywasconducted
usingsilicagelG60F254plateandtoluene-ethylacetate-formic
acid,5:4:1 (v/v/v)asa mobilephase.The plateswereobserved
underUVafterderivatizingusingNaturalproducts-polyethylene
glycolreagent(NP/PEG)forquercetinand5%FeCl3forgallicacid.
HPTLC analysis was performed on 10cm×10cm aluminum
backed HPTLCplatescoatedwitha 0.2mm layerof silicagelG
60F254 (Merck, India). The plates were washed with methanol
and activated in an oven at 110◦C for 5minprior toanalysis.
Thestandardandsamplesolutionswereappliedtotheplatesin
formofbandsof5mmlong,1mmfrombottomoftheplateand
1mmfromtheedgebymeansofautomaticTLCsampler(CAMAG
semiautomaticIV).Theplatesweredevelopedusingtoluene-ethyl
acetate-formicacid5:4:1(v/v/v)asmobilephaseinCAMAGtwin
troughchamber,presaturatedwithmobilephasevaporfor30min
beforedevelopment.Afterdevelopment,theplatewasdriedinair
for5minandscannedimmediatelyat254nmusingTLCscanner
(CAMAGscanner-III).ATLCvisualizerwasusedforthe
photodoc-umentationoftheplates.
Validationofmethod
ThemethodwasvalidatedasperICHguidelines.Calibration
curveforquercetinandgallicacidwereobtainedbyplottingthe
peak areaagainst theiramountappliedtotheplate.Thelimits
ofdetection(LOD)andquantification(LOQ)weredeterminedby
equationasperICHguidelines.Instrumentalprecisionwas
deter-minedbyrepeatedanalysisofstandardquercetinandgallicacid.
Repeatabilitywasassessedforeachreferencestandardbyapplying threeconcentrationsofthesestandardsintriplicateontheplate,
which wereexpressedinterms ofpercentage relative standard
deviation(%RSD)andstandarderror(SE).Theintra-dayprecision
wasdeterminedatthreedifferentconcentrationlevelsof
differ-entmarkers,200,400and600ng/spot,sixtimesonthesameday,
andtheinter-dayprecisionwasdeterminedatthreedifferent con-centrationsofmarkers,100,300and500ng/spot,sixtimesonfive
Table1
Validationparametersforquercetinandgallicacidestimation.
Parameters Results
Quercetin Gallicacid
Correlationcoefficient 0.991 0.999
Linearityrange(ng) 200–1000 200–1200
Precision(C.V.)
Repeatabilityofmeasurement 0.89 0.359
Repeatabilityofapplication 0.53 0.42
Intraday 0.23–0.43 0.2–0.6
Interday 0.97–1.23 0.1–1.13
Limitofdetection(ng/spot) 21.31 14.8
Limitofquantification(ng/spot) 64.57 55.04
Accuracy(%) 98.02–99.09 99.28–100.26
Specificity Specific Specific
differentinterval days overa period ofone week.System
suit-abilitywastestedbyapplying600ng/spotofquercetinandgallic
acidstandardsolutiontotheplateseventimesandmeasuringthe
relativestandarddeviation(RSD%)forthepeakareasandRF.To
studytheaccuracyandprecisionofthemethod,recoverystudies
wereperformedbythemethodofstandardaddition.The
recov-ery ofaddedstandard wasstudiedattwodifferentlevels,each
beinganalyzedinamannersimilartothatdescribedfortheassay
(Biringanineetal.,2006;Wagneretal.,2008).
Resultsanddiscussion
QuercetinandgallicacidwerequantifiedbyHPTLCforthefirst timeinL.indica.Theidentityofquercetinwasconfirmedby
co-chromatographyand overlainabsorptionspectrawithreference
standard,whenscannedat254nm.Alsothebathochromicshiftin
UVspectrawithAlCl3/HClrelativetomethanolspectraasserted
presenceofquercetinintheplant.
ThedensitometricchromatogramofHPTLCfingerprintofthe
ethylacetateextractisshowninFig.1.ThepeaksresolvingatRf0.63
and0.45intestsolutionwerefoundtobesuperimposingwiththose ofrespectivestandardsofquercetinandgallicacid.Peakpuritywas
studiedbyUVoverlayspectraobtainedfromthestandardsand
fromthecorrespondingpeaksfromtest(Fig.2).
Thecontentofquercetinandgallicacidinplantwascalculated
onthebasisofpeakareaandwasfoundtobe0.13%and0.041%
(w/w)respectively.Thecalibrationplotsindicatethatthepeak-area
responsewasapolynomialfunctionoftheamountofstandards
quercetinandgallicacidintherange200–1000ngand200–1200ng respectively.Thecorrelationcoefficientwasfoundtobe0.991and
0.999withRSD,0.97–1.23%and0.1–1.13%forquercetinandgallic
acidrespectivelyinthedevelopedmethod.Intradayandinterday
precisionwascalculatedforthedevelopedmethodbyperforming
theanalysisatthreedifferentlevelsonsamedayaswellason
dif-ferentdaysrespectively.RSDforrepeatabilityofmeasurementof
peakareabasedon7timesmeasurementof600ng/spotof
stan-dardsquercetinandgallicacidwerefoundtobe0.89and0.359%
respectively.RSDforrepeatabilityofmeasurement ofpeakarea
basedona7timemeasurementof1l/spotofsampleextractwas
foundtobe0.53and0.42%quercetinandgallicacidrespectively. Thelimitsofdetection(LOD)andlimitofquantification(LOQ)were
determinedtobe21.31and64.57ng/spotforquercetinand14.86
and 55.04ng/spot for gallicacidrespectively. Percentage
recov-eryofquercetinandgallicacidwerefoundtobeintherangeof
98.02–99.09and99.28–100.26respectively,whichindicatedthat
thedeveloped methodwasaccurateand satisfactory.Summary
ofallvalidationparametersforthedevelopedHPTLCmethodfor
simultaneousestimationofquercetinandgallicacidarelistedin
52 A.A.Pateletal./RevistaBrasileiradeFarmacognosia27(2017)50–53
0
–0.10 0.10 0.30 0.50
Track 9. ID: Ext 2
0.70 0.90 –0.10
0 50 100 150 200 250 300 350 400
AU
0.10 0.30 0.50 0.70 0.90
–0.10 0 100 200 300 400 500 600
Gallic acid Quercetin
0.10 0.30
1
2
0.50 0.70 0.90 Rf
50 100 150 200 250 300 350 400 450
AU
AU
500
A
B
C
Fig.1. DensitometricchromatogramofhydrolysedmethanolextractofLeeaindica(A),referencestandardsgallicacid(B)andquercetin(C)scannedat254nm.
0.0
200.0 250.0 300.0 350.0 400.0 450.0
Spectra comparison Spectra comparison
500.0 550.0 600.0 [nm] 700.0 10.0
20.0 30.0 40.0 50.0 60.0 70.0 80.0 [AU] 100.0
A
B
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 [AU] 100.0
0.0
200.0 250.0 300.0 350.0 400.0 450.0 500.0 550.0 600.0 [nm] 700.0 10.0
20.0 30.0 40.0 50.0 60.0 70.0 80.0 [AU] 100.0
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 [AU] 100.0
Fig.2. UVOverlainspectraofgallicacid(A)andquercetin(B)standardsandintestsolutiontrackscannedat254nm.
The method wasfound to be precise, reliable, and suitable
because the recovery in each case was more than 98%. Thus
thepresent HPTLC method enablessimple, rapid and accurate
quantificationofquercetinandgallicacidinplantofL.indica simul-taneously.
Authors’contributions
AAP(M.Pharm.student)contributedincollectingplantsample
andidentification,confectionof herbarium,runningthe
A.A.Pateletal./RevistaBrasileiradeFarmacognosia27(2017)50–53 53
AHPcontributedtochromatographicanalysis.AAAcontributedto
criticalreadingofthemanuscript.MBSdesignedthestudy,
super-visedthelaboratoryworkandcontributed tocriticalreading of
themanuscript.Alltheauthorshavereadthefinalmanuscriptand
approvedthesubmission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
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