International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 2, Suppl 4, 2010

ResearchArticle

HPTLCMETHODFORTHEDETERMINATIONOFPLUMBAGINFROMPLUMBAGOZEYLANICA LINN.(ROOT)
PAWARR.K.1,SHARMASHIVANI2,SINGHK.C.2ANDSHARMARAJEEVK.3
1,3

PharmacopoeialLaboratoryforIndianMedicine,Ghaziabad201002(U.P.)2Departmentofchemistry,R.S.S.(P.G.)College,Pilkhuwa, Ghaziabad(U.P.)Email:[email protected] Received:01September2010,RevisedandAccepted:30September2010

ABSTRACT A simple, rapid, selective and quantitative HPTLC method has been developed for determination of Plumbagin in different samples of Plumbago zeylanica Linn.root.Thechloroformextractof Plumbago zeylanica Linn.rootsampleswereappliedonTLCAluminiumplateprecoatedwithSilica gel60GF254 anddevelopedusingToluene:Ethyl acetate(3:1)v/vasa mobilephase.Theplatewassprayed(derivatized)withAnisaldehyde SulphuricAcidreagentfollowedbyheatingat110 0Cfor10minutesanddetectionandquantificationwerecarriedoutdensitometricallyusingan UVdetectoratwavelengthof270nm.Contentofmarkercompoundinthesampleswerefoundsimilar. Keywords:Plumbagin,PlumbagozeylanicaLinn.Root.,Chitrakroot,Chitrakmul,HPTLC. INTRODUCTION Plumbago zeylanica Linn Syn. Plumbago rosea Linn (Family Plumbaginaceae) known vernacularly as Chitrak, Chitra, Chitraka, Chitrakmul, Agni, Pathi, Ushana, Chita, Chitramulam, Ceylong LeadwortorwhiteLeadwortisfoundwildinthetropics,subtropics andthroughoutIndiaincludingWestBengal,Biharand peninsular India.Itisalsowidelycultivatedasanornamentalplant.Itisamuch branchedshrubwithlongtuberousrootandastriatestem14. Therootandrootbarkarebitter,stomachiccarminative,astringent to bowels, anthelmintic, piles bronchitis, itching, diseases of lever, consumption,ascetics.Therootisbitter,laxative,expectorant,tonic, abortifacient, alexipharmic, good appetizer, useful in laryngitis, rheumatism, diseases of spleen, ringworm, scabies. Paste of root withmilk,vinegarorsaltandwaterisappliedinleprosyand other skin diseases externally. Tincture of root bark is used as an antipatriotic . it acts as a powerful sudorific. Leaves are caustic, vesicantaphrodisiacandgoodforscabies5. Plants contains number of naphthaquinone derivatives viz. plumbagin, 3chloroplumbagin, 3,3biplumbagin, elliptinone, chitranone, zeylinone, isozeylinone, droserone, plumbagic acid, plumbazeylanone, naphthelenone and isoshinanolone5. Fructose, glucose, invertase and protease isolated from root bark. 3,3 bisplumbagin, chitranone (binaphthaquinone), droserone, elliptinone, isozeylinone, catechol tannin 8]. Amino acids; (2, 3 dihydroxybenzoyl)butyric acid (plumbagic acid), vanillic acid, 1,2(3)tetra hydro3,3bisplumbagin, isoshinanolone, dihydrosterone and sitosterol also islated from plant [3,8]. Plumbagin shows as anticancer and antitumor activity5,9. Aspartic acid, tryptophan, tyrosine, threonine, alanine, histidine, glycine, methionine, hydroxyproline, were isolatedfrom the aerial parts 9, 15.Lupeolandlupenylacetatehavebeenisolatedfromtheroot. Literature survey reveals that the TLC, HPLC and HPTLC methods arereportedbutnomethodasyetisreportedforthedetermination of Plumbagin in Plumbago zeylanica Linn., root. A simple, rapid, economical, precise and accurate HPTLC method has been established for the determination of plumbagin in Plumbago zeylanica Linn., root powder. This method can be used for phytochemical profiling of Plumbago zeylanica Linn., root and quantificationofPlumbagin. MATERIALANDMETHOD Plantmaterial TheChitrakrootwasprocuredfromtheLocalMarket,Ghaziabad.It wasidentifiedandauthenticatedbytheBotanistsofPharmacopoeial LaboratoryforIndianMedicine,Ghaziabad.Onegenuinesamplealso taken from the Museum of Pharmacopoeial Laboratory for Indian Medicine,Ghaziabad. EquipmentsCammagLinomatVapplicator,CammagTwinTrough Chamber (size 20x10 cm) with SS lid, Cammag Dipping Chamber, TLCAluminiumprecoatedplatewithSilicagel60GF254(size10X10 cm;0.2mmthick)E.Merck. ChemicalsAnalyticalgrade;Alcohol,Toluene,ethylacetate,Formic acid, Chloroform, Methanol, Anisaldehyde, Sulphuric acid and n Hexane were used; obtained from S.D. Fine Chem. Ltd. (Mumbai, India). TLC Aluminium pre coated plate with Silica gel 60 GF 254 (10X10 cm2; 0.2 mm thick) used were obtained from E. Merck Ltd. (Mumbai, India). Reference standard Plumbagin procured from AldrichChem.Co.Milw,WI33201(4142733850/19,064048142 5). Experimental Sample preparation 1g of coarselypowdereddrug samples were extracted with 10 ml Chloroform for 24 hours by cold extraction method.TheextractswerefilteredbyWhatmannno.42filterpaper andmakeupto10mlinavolumetricflask. Standard Preparation 5mg of standard Plumbagin dissolved in 5mlofChloroformandmadeupto5mlinstandardvolumetricflask. Chromatography TLCAluminumprecoatedplatewithSilicagel60GF254 (20x10cm2; 0.2 mm thick) was used with Toluene : Ethyl acetate (3:1) V/V as mobile phase. Chloroform extract of samples and Plumbagin standardsolutionappliedonplatebyusingLinomatVapplicator. Cammag Twin Trough Glass Chamber (20x10 cm2) with SS lid was usedfordevelopmentofTLCplate.TheTwinTroughGlassChamber was saturated with mobile phase for 30 minutes. TLC plate was developed to 8 cm distance above the position of the sample application.Theplatewasremovedfromthechamberandairdried at room temperature. This plate was sprayed (derivatized) with AnisaldehydeSulphuricAcidreagentfollowedbyheatingat1100C for 10 minutes and HPTLC finger print profile was snapped by CammagReprostarIII,beforedeivatizationunderUV254nm,366 nm and after derivatization (Fig.1). The plate was scanned before derivatization using Camag TLC Scanner III at wavelength 270nm. WincatsanintegratedSoftware4.02wasusedforthedetectionas wellasfortheevaluationofdata. Methodvalidationandrecoverystudy To study the accuracy and precision of the proposed method, recovery experiment was carried out. To a fixed amount of chloroformextractofsamples,thestandardsolutionof Plumbagin

Pawaretal. IntJPharmPharmSci,Vol2,Suppl4,219223 was added (ratio 9:1 v/v)andtotal amountofstandard Plumbagin weredetermined.Percentrecoverywascalculatedfromtheamount ofPlumbaginfoundviagraph(TableNo.3). Linearityofdetectorresponse,assayandrecovery In order to establish linearity, standard solution of Plumbagin (1mg/ml) applied on TLC Aluminium pre coated plate with Silica gel60GF254(20X10cm2;0.2mmthick),2l,4l,6lonTrackNo.S1, S2&S3respectivelyandforassay,9lofChloroformextractofboth samplesappliedonTrackNo.T1&T2andforrecoverystudy,then hexane extract of both samples were spiked with standard Plumbaginsolution(ratio9:1v/v)andapplied10lonTrackNo.T3 &T4onthesameplate.TLCplateswasdevelopedto8cmdistance abovethepositionofthesampleapplicationandremovedfromthe chamberandairdriedatroomtemperature.ThisHPTLCfingerprint profilewassnappedbyCammagReprostarIII,beforederivatization underUVLight254nm,366nmandafterderivatization(Fig.1).The plate was derivatized with Anisaldehyde Sulphuric Acid reagent followed by heating at 1100C for 10 minutes and scanned immediately using Camag TLC Scanner III at wavelength 270nm. WincatsanintegratedSoftware4.02wasusedforthedetectionas well as for the evaluation of data. It was observed that Plumbagin appeared at Rf. 0.84 (dark grey colour). The peaks, graph and spectraobtainedweregiveninFig.2and3andRf.values,colourof bands (Table No.1), quantity of Plumbagin, linearity, standard deviation&regressioncoefficientfoundviagraph(TableNo.2)and calculatedquantityofPlumbagin&%recoveryweregiveninTable No.3. Table1:TLCDetailsofCHCl3ExtractofPlumbagozeylanicaLinn.(Root) Sr.No. 1. Detection/ visualization UnderUV 254nm UnderUV 366nm CitrakRoot (TrackNo.T1,T2,T3andT4) Rf.values Colourofband 0.05 grey 0.10 grey 0.46 grey 0.84 darkgrey 0.05 lightgreen 0.10 brown 0.25 brightgreen 0.46 brightskyblue 0.70 skyblue 0.84 red 0.96 blue 0.05 violet 0.10 violet 0.15 violet 0.46 darkviolet 0.57 lightviolet 0.65 lightviolet 0.73 lightviolet 0.84 yello StandardPlumbagin (TrackNo.S1,S2andS3) Rf.values Colourofband 0.84 darkgrey 0.84 red 0.84 yellow RESULTSANDDISCUSSION Of the various mobile phases tried, the mobile phase containing Toluene:Ethylacetate(3:1)v/vandtheactiveprinciplePlumbagin resolvedasadarkgreycolourbandatRf.0.84veryefficientlyfrom theothercomponentsinChloroformextractof Plumbago zeylanica Linn. (root) (Fig.1). Sharp peaks of Plumbagin (Standard and samples)wereobtainedwhentheplatewasscannedatwavelength 270nm (Fig.2). Quantity of Plumbagin found in samples were obtained automatically (Table No. 2) via graph (Fig.3) and % Plumbaginfoundinsamplesand%recoverywerecalculated(Table No.3). Quantity of Plumbagin found in Local Market Sample, Ghaziabad(U.P.)is4.9589mgin1gdrugsample(0.49589%w/w) and quantity of Plumbagin found in Museum Sample of PLIM, Ghaziabadis4.7922mgin1gdrugsample(0.47922%w/w).The% recovery of Plumbagin in Local Market Sample, Ghaziabad (U.P.) is 99.84% w/w and 99.67%w/w in Museum Sample of PLIM, Ghaziabad(U.P.).Themean%recoverywas99.76%. Theaccuracyandreproducibilityofthemethodwasestablished by means of recovery experiment. The mean recovery was close to 100%whichindicatestheaccuracyofthemethod. The robustness of the method was studied, during method development,bydeterminingtheeffectofsmallvariation,ofmobile phasecomposition(2%),chambersaturationperiod,development distance, derivatization time, and scanning time (10% variation of each). No significant change of Rf. or response to Plumbagin was observed,indicatingtherobustnessofthemethod.

2.

3.

Afterderivatization

Table2:Quantityappliedonplateandvaluesfoundviagraph Sr. No. 1. 2. 3. 4. 5. 6. 7. TrackNo. TrackT1 TrackT2 TrackS1 TrackS2 TrackS3 TrackT3 TrackT4 Volumeofextractandstandard solutionappliedonplate 9l 9l 2l 4l 6l 10l 10l QuantityofBoswellic acidviagraph 4.466g 4.318g 2.000g 4.000g 6.000g 5.459g1g =4.459g 5.307g1g =4.307g Linearity&RegressionCoefficientandStandard deviationviagraph

T1ChloroformextractofLocalMarketsample,Ghaziabad,T2ChloroformextractofMuseumSampleofPLIM,Ghaziabad,S1Plumbaginstandard solution (1mg/ml),S2 Plumbagin standardsolution (1mg/ml),S3 Plumbagin standard solution(1mg/ml), T3Chloroform extract (spiked with std.solution)ofLocalMarketSample,Ghaziabad,T4Chloroformextract(spikedwithstd.solution)ofMuseumSampleofPLIM,Ghaziabad 220

Pawaretal. IntJPharmPharmSci,Vol2,Suppl4,219223 Table3:Summaryofresults Sr.No. 1. 2. 3. Samplefrom Quantityof Plumbaginin1g %Plumbagin %Recovery LocalMarketSample,Ghaziabad 4.9589mg 0.4959%w/w 99.84%w/w MuseumSampleofPLIM,Ghaziabad 4.7922mg 0.4792%w/w 99.67%w/w

T1T2S1 S2 S3T3T4 UV254nm

T1 T2S1S2 S3 T3T4 UV366nm T1T2S1 Afterderivatization Fig.1:H.P.T.L.C.FingerprintofPlumbagozeylanicaLinn.(Root) 221 S2 S3T3T4

Pawaretal. IntJPharmPharmSci,Vol2,Suppl4,219223

(TrackS1)

(TrackS2) PeaksofPlumbagin@270nm

(TrackS3)

(TrackT1)

(TrackT2) (TrackT3) PeaksofPlumbagozeylanicaCHCl3Extract@270nm Fig.2:PeaksofPlumbagozeylanicaLinn.(Root)inallTracks

(TrackT4)

GraphConc.vsAU

SpectraofPlumbagin@270nm Fig.3:GraphandspectrraofPlumbagozeylanicaLinn.(Root) SpectraofPlumbagininalltrcks@270nm

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Pawaretal. IntJPharmPharmSci,Vol2,Suppl4,219223 CONCLUSION The proposed HPTLC method is simple, rapid, accurate, reproducible, selective and economic and can be used for routine qualitycontrolanalysisof Plumbago zeylanicaLinn.(root)powder andquantitativedeterminationofPlumbagininrootpowder. REFERNCES 1. 2. 3. 4. 5. 6. Dhiman Anil Kumar, Ayurvedic Drug Plants, Daya Publishing House,Delhi,2006,p105. Husain Akhtar & et. al., Dictionary of Indian Medicinal Plant, CIMAP,Lucknow,1992,p358. Phondke G.P., The Wealth of India, Raw Materials, Revised Edition,Vol.VIII,NISCAIR,CSIR,NewDelhi,1992,p162. Prajapati/Purohit and et. al., A Handbook of Medicinal Plants, ACompleteSourceBook,Agrobio,India,2004,p410. Handa,S.S.,IndianHerbalPharmacopoeia,Vol.2,1999,p103. ThakurR.S,PUriH.S.,HusainAkhtar,MajorMedicinalPlantof India,CIMAP,Lucknow,1989,p415. 7. 8. 9. 10. 11. 12. 13. 14. 15. Singh V.K., Govil J.N., et. al., Recent progress in Medicinal plants,Vol.11,DrugDevelopmentfromMolecules,p267. Chaterjee Asima, Pakrashi S. C., The Treatise on Indian MedicinalPlants,Vol.4,p55. ElizabethM.WilliamsonMajorHerbsofAyurveda,2002,p239. 10. Rastogi Ram P., Mehrotra B.N., Compandium of Indian MedicinalPlants,CDRI,PID,NewDelhi,1993,Vol.2,p547. Rastogi Ram P., Mehrotra B.N., Compandium of Indian MedicinalPlants,CDRI,PID,NewDelhi,Vol.3,p512. Rastogi Ram P., Mehrotra B.N., Compandium of Indian MedicinalPlants,CDRI,PID,NewDelhiVol.4,p579. Rastogi Ram P., Mehrotra B.N., Compandium of Indian MedicinalPlants,CDRI,PID,NewDelhi,Vol.5,p673. ChettyK.Madhavaandet.al.EthanobotanicalLeaflets10:294 304,2006. DinaB.,SahaS.JournalofIndianChemicalSociety,64,4:261, 1987. DinaB.,SahaS.JournalofIndianChemicalSociety,67:88,1990.

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