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Asian Journal of Pharmaceutical and Clinical Research

Vol. 4, Issue 2, 2011

ISSN - 0974-2441

ResearchArticle

DEVELOPMENTANDVALIDATIONOFHPTLCMETHODFORTHEDETERMINATIONOF BOSWELLICACIDFROMBOSWELLIASERRATAROXB.(EXUDATE)

PAWARR.K.1,SHARMASHIVANI2,SINGHK.C.2ANDSHARMARAJEEVKR.3
1,3

PharmacopoeialLaboratoryforIndianMedicine,Ghaziabad201002(U.P.),2Departmentofchemistry,R.S.S.(P.G.)College,Pilkhuwa, Ghaziabad(U.P.),IndiaEmail:pawarplim@gmail.com

ABSTRACT A simple, rapid, selective and quantitative HPTLC method has been developed for determination of Boswellic acid in different samples of Boswellia serrata Roxb. exudate. The nhexane extract of Boswellia serrata Roxb.( exudate) samples were applied on TLC Aluminium plate pre coatedwithSilicagel60GF254 anddevelopedusingToluene:Ethylacetate:Formicacid(5:4.5:0.5)v/vasamobilephase.Theplatewassprayed (derivatized) with Anisaldehyde Sulphuric Acid reagent followed by heating at 1100C for 10 minutes and detection and quantification were carriedoutdensitometricallyusinganUVdetectoratwavelengthof530nm.Contentofmarkercompoundinthesampleswerefoundsimilar. Keywords:Boswellicacid,BoswelliaserrataRoxb.,Kundruexudate,Shallaki,HPTLC. INTRODUCTION BoswelliaserrataRoxb.ExColebr.Syn.Boswelliaserratavar.glabra (Roxb)Bennett,BoswelliaglabraRoxb.(FamilyBurseraceae)iswell known as Kundru or Shallaki and distributed in dry forests from PunjabtoWestBengalandinPeninsularIndia.Commonatthefoot of Western Himalaya , in Rajasthan, Gujrat, Maharastra, Madhya Pradesh, Bihar Orissa Andhra Pradesh and further south in the Peninsula. Reported to be threatened in North Eastern Region of India.Itisamediumsizetolargesized,deciduoustree,upto18mm in height, an evergreen and spiny tree. Leaves are alternate, imparipinnate and crowded towards the end of branches; leaflets 1731, opposite, sessile, ovate or ovate lanceolate, crenate and pubscent. Flowersaresmallandwhite,inaxillaryracemesorpenicles.Drupes thefruitsareabout1.2cmlongandtrigonous,splittinginto3valves and subtended by the woody disk. Seeds are compressed and pendulous. Flowering is during MarchApril and fruiting in winter season. The oleoresin exudates out during winter and gets deposited on the various parts of tree trunk. The oleoresin is secreted in the schizogenous duct in the bark which are scattered justbelowthebastfibres. The oleoresin exudes as colourless semifluid liquid which gradually becomes whitish to golden yellow and solidifies slowly with time. Sometimes, it is reddish brown, greenish yellow or dull yellow to orange in colour. Oleogum resin exudates and bark are used in medicine. The bark is sweet, acrid, cooling and tonic. It is goodforasthma,dysentery,ulcers,haemorrhoidsandskindiseases. Gumresin exudates obtained from the plant is sweet, bitter, astringent and commercially known as Indian oilbanum or Indian frankincenseorSallaiguggul. It is used as antipyretic, expectorant, diuretic, ecbolic, antiseptic, antidysenteric, diaphoretic, stomachic and uretherorrhea, orchiopathy, bronchitis, asthma, cough, skin diseases, ulcers, tumours, cystic breast, chronic laryngitis, jaundice and arthritis. It shows antiinflammatory and antiarthritic activity have been mainlyattributedtoacomponentintheresincontainingBoswellic acidandisusedforrheumaticpatients[19]. Inadditiontouseforarthritisthisgummyresinisalsomentionedin traditional Ayurvedic texts as a remedy for diarrhea, dysentery, ringworm, boils, fevers (antipyretic) skin and blood diseases, cardiovascular diseases, mouth sores, vaginal discharges, hair loss, jaundice, hemorrhoids, syphilitic diseases, irregular menses and to stimulate the liver. Modern medicine and pharmacology point to Boswellia serratas use as a anti arthritic, antiinflammatory, anti hyperlipidemic (control blood lipids), antiatheroscleotic (anti coronary plaque), analgesic (painreliver) and hepoprotective (protectstheliver).[1014] Structureofboswellicacid Theresearchhasimplicated[16]abeneficialrolefortheresininthe treatment osteoartharities, soft tissue rheumatism, low back pain, goutandrheumatoidarthritisisacreepingcrippingdiseasecausing great physical suffering, it is possible to alleviate physical pain, increase movement (mobility) and prevent further tissue injury throughpropertreatment.TreatmentwithBoswelliaserrata,onthe otherhandBoswellicacid[17]significantlyreducedtheinfiltrationof leucocytes into the knee joint in turn significantly reducing inflammationcausingimmunewhitebloodcellresponse. SothatBoswellicacidisanactiveconstituentsandusedasamarker. Literature survey reveals that the TLC and HPTLC methods are reportedbutnomethodasyetisreportedforthedeterminationof BoswellicacidinBoswelliaserrataRoxb.exudates. With increasing demand for herbal products in medicines and cosmeticsthereisanurgentneedforstandardization.Sotheaimof The gum resin contains a mixture of triterpene acids known as Boswellic acid (,, boswellic acid) acetyl boswellic acid, 11 keto boswellic acid, acetyl11keto boswellic acid and their derivatives [10]. Volatile oil contains thujene, phellandrene, phellandrene, terpineol, dlimonene, myrcene, terpene, p cymeme [11]; a diterpene alcohol serratol and four tetracyclic triterpene acids 3 acetoxytirucall 8,24dien21oic acid, 3 ketotirucall8,24dien21oicacid,3hydroxytirucall8,24dien oicacid,3hydroxytirucall8,24dien21oicacid.[114,18]. It is also fond to contain arabinose, rhamnose, glucose, galactose, fructose,idose,galacturonicacidandsitosterolisolatedfromgum [1217,]. Essential oil from gum gave phenolocresol, mcresol, p cresol, thymol, and carvacrol and carboxylic acid campholenic acid,2,2,4trimethylcyclopent3en1ylaceticacidandcampholytic acid[2].

Pawaretal. AsianJPharmClinRes,Vol4,Issue2,2011,7276 the work is to develop a simple, rapid, selective and cost effective HPTLC method for the determination of Boswellic acid in Boswelliaserrataexudate. MATERIALANDMETHOD Plantmaterial The Kundru exudate was procured from the Local Market, Ghaziabad. It was identified and authenticated by the Botanists of Pharmacopoeial Laboratory for Indian Medicine, Ghaziabad. One genuine sample also taken from the Museum of Pharmacopoeial LaboratoryforIndianMedicine,Ghaziabad. Equipment A Cammag (Switzerland) HPTLC system equipped with a sample applicatorLinomatV,TwintroughglassChamber(20x10cm2)with SS lid, TLC Scanner III, Reprostar III and Wincats an integrated Software4.02(Switzerland),Rotavapour. Chemical&reagents Analytical grade; Alcohol, Toluene, ethyl acetate, Formic acid, Chloroform, Methanol, Anisaldehyde, Sulphuric acid and nHexane wereused;obtainedfromS.D.FineChem.Ltd.(Mumbai,India).TLC AluminiumprecoatedplatewithSilicagel60GF254 (20x10cm2;0.2 mmthick)usedwereobtainedfromE.MerckLtd.(Mumbai,India). Reference standard Boswellic acid procured from Natural RemediesPvt.Ltd.,Bangalore,India. Sample&standardpreparation Samplepreparation 1gofcoarselypowdereddrugsampleswereextractedwith10mln Hexane for 24 hours by cold extraction method. The extracts were filtered by Whatmann filter paper and make up to 10 ml in a volumetricflask. Standardpreparation 5mgofstandardBoswellicaciddissolvedin3mlofnHexaneand madeupto5mlinstandardvolumetricflask. H.P.T.L.C(HighPerformanceThinLayerChromatography) TLCAluminiumprecoatedplatewithSilicagel60GF254 (20x10cm2; 0.2 mm thick) was used with Toluene : Ethyl acetate: Formic acid (5:4.5:0.5)V/Vasmobilephase.Nhexaneextractofsamplesand BoswellicacidstandardsolutionappliedonplatebyusingLinomat Vapplicator.CammagTwinTroughGlassChamber(20x10cm2)with SSlidwasusedfordevelopmentofTLCplate. TheTwinTroughGlassChamberwassaturatedwithmobilephase for30minutes.TLCplatewasdevelopedto8cmdistanceabovethe positionofthesampleapplication.Theplatewasremovedfromthe chamberandairdriedatroomtemperature. Thisplatewassprayed(derivatized)withAnisaldehydeSulphuric Acid reagent followed by heating at 1100C for 10 minutes and HPTLC finger print profile was snapped by Cammag Reprostar III, before deivatization under UV 254 nm, 366 nm and after derivatization(Fig.1). The derivatized plate was scanned immediately using Camag TLC Scanner III at wavelength 530nm. Wincats an integrated Software 4.02wasusedforthedetectionaswellasfortheevaluationofdata. MethodValidationandRecoverystudy To study the accuracy and precision of the proposed method, recoveryexperimentwascarriedout.Toafixedamountofnhexane

extract of samples, the standard solution of Boswellic acid was added(ratio9:1v/v)andtotalamountofstandardBoswellicacid weredetermined.Percentrecoverywascalculatedfromtheamount ofBoswellicacidfoundviagraph(TableNo.3). Linearityofdetectorresponse,assayandrecovery Inordertoestablishlinearity,standardsolutionofBoswellicacid (1mg/ml) applied on TLC Aluminium pre coated plate with Silica gel60GF254 (20X10cm2;0.2mmthick),10l,5l,1lonTrackNo. S1, S2 & S3 respectively and for assay, 9l of nhexane extract of bothsamplesappliedonTrackNo.T1&T2andforrecoverystudy, thenhexaneextractofbothsampleswerespikedwithstandard Boswellicacidsolution(ratio9:1v/v)andapplied10lonTrackNo. T3&T4onthesameplate. TLC plates were developed to 8 cm distance above the position of thesampleapplicationandremovedfromthechamberandairdried atroomtemperature. ThisHPTLCfingerprintprofilewassnappedbyCammagReprostar III,beforederivatizationunderUVLight254nm,366nmandafter derivatization(Fig.1).TheplatewasderivatizedwithAnisaldehyde SulphuricAcidreagentfollowedbyheatingat1100Cfor10minutes and scanned immediately using Camag TLC Scanner III at wavelength530nm. WincatsanintegratedSoftware4.02wasusedforthedetectionas wellasfortheevaluationofdata.ItwasobservedthatBoswellic acidappearedatRf.0.84(darkvioletcolour).Thepeaks,graphand spectraobtainedweregiveninFig.2and3andRf.values,colourof bands(TableNo.1),quantityofBoswellicacid,linearity,standard deviation&regressioncoefficientfoundviagraph(TableNo.2)and calculatedquantityofBoswellicacid&%recoveryweregivenin TableNo.3. RESULTSANDDISCUSSION Of the various mobile phases tried, the mobile phase containing Toluene : Ethyl acetate: Formic acid (5:4.5:0.5) v/v and the active principleBoswellicacidresolvedasadarkvioletcolourbandatRf. 0.84veryefficientlyfromtheothercomponentsinnhexaneextract ofBoswelliaserraraRoxb.(exudates)(Fig.1). Sharp peaks of Boswellic acid (Standard and samples) were obtainedwhentheplatewasscannedatwavelength530nm(Fig.2). Quantity of Boswellic acid found in samples were obtained automatically(TableNo.2)viagraph(Fig.3)and%Boswellicacid foundinsamplesand%recoverywerecalculated(TableNo.3). Quantity of Boswellic acid found in Local Market Sample, Ghaziabad(U.P.)is1.5678mgin1gdrugsample(0.15678%w/w) andquantityofBoswellicacidfoundinMuseumSampleofPLIM, Ghaziabadis1.7100mgin1gdrugsample(0.17100%w/w).The% recovery of Boswellic acid in Local Market Sample, Ghaziabad (U.P.)is98.10%w/wand97.05%w/winMuseumSampleofPLIM, Ghaziabad(U.P.).Themean%recoverywas97.58%. 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 Boswellic acid wasobserved,indicatingtherobustnessofthemethod. Table3

Sr.No. 1. 2. 3.

Samplefrom Quantityof Boswellicacidin1g %Boswellicacid %Recovery

Localmarketsample,ghaziabad 1.5678mg 0.15678%w/w 98.10%w/w

Museumsampleofplim,ghaziabad 1.7100mg 0.1710%w/w 97.05%w/w 73

Pawaretal. AsianJPharmClinRes,Vol4,Issue2,2011,7276 Table1 Sr.No. Detection/visualization Kundruexudate (TrackNo.T1,T2,T3andT4) Rf.values Colourofband 0.52 darkgrey 0.56 darkgrey 0.69 darkgrey 0.76 darkgrey 0.89 darkgrey 0.52 blue 0.69 blue 0.89 brightskyblue 0.13 lightviolet 0.20 lightviolet 0.27 lightviolet 0.34 darkviolet 0.40 violet 0.52 violet 0.62 darkviolet 0.69 darkviolet 0.76 darkbrown 0.81 darkbrown 0.89 darkbrown Table2 Sr.No. 1. 2. 3. 4. 5. 6. TrackNo. T1 T2 S1 S2 S3 T3 Volume appliedon plate 9l 9l 10l 5l 1l (9+1)l Quantityappliedon plate 900g 900g 10g 5g 1g 900g+1g QuantityofBoswellic acidviagraph 1.424g 1.562g 10.000g 5.000g 1.000g 2.397g1g=1.397g Linearity&RegressionCoefficientand Standarddeviationviagraph StandardBoswellicacid(TrackNo.S1,S2andS3) Rf.values 0.62 Colourofband Nosignificantban Nosignificantban darkviolet

1.

UnderUV254nm

2. 3.

UnderUV366nm Afterderivatization

7. T4 900g+1g 2.516g1g=1.516g (9+1)l T1nHexaneextractofLocalMarketsample,Ghaziabad,T2nHexaneextractofMuseumSampleofPLIM,Ghaziabad S1Boswellicacidstandardsolution(1mg/ml),S2Boswellicacidstandardsolution(1mg/ml) S3Boswellicacidstandardsolution(1mg/ml),T3nHexaneextract(spikedwithstd.solution)ofLocalMarketSample,Ghaziabad T4nHexaneextract(spikedwithstd.solution)ofMuseumSampleofPLIM,Ghaziabad

T1T2S1S2S3T3T4 UV254nm

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Pawaretal. AsianJPharmClinRes,Vol4,Issue2,2011,7276

T1T2S1S2

S3

T3T4

T1T2S1

S2S3 AfterDerivatization

T3T4

UV366nm

Fig.1:T.L.C.FingerprintofBoswelliaserraraRoxb.(Exudate)

(TrackS1)

(TrackS2) PeakofBoswellicAcid(@530nm

(TrackS3)

(TrackT1)

(TrackT2)

(TrackT3)

(TrackT4)

PeaksofBoswelliaserrataExtract(@530nm Fig.2:PeaksofBoswelliaserraraRoxb.(Exudate)inallTracks 75

Pawaretal. AsianJPharmClinRes,Vol4,Issue2,2011,7276

Conc.(g)GraphConc.vsAU SpectraofBoswellicacidinalltracks@530nm

Fig.3:GraphandSpectraofBoswelliaserraraRoxb.(Exudate) REFERNCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Dhiman A. K., Ayurvedic Drug Plants, Daya Publishing House,Delhi,2006,p326327. HusainA.&et.al.,DictionaryofIndianMedicinalPlant,CIMAP, Lucknow,1992,p82. The Wealth of India, Raw Materials, Revised Edition, Vol I, NISCAIR,CSIR,NewDelhi,1988,p148. Khare C.P., Encyclopedia of India, Rational Western Therapy, AyurvedicandotherTraditionalUsage,2004. Prajapati/Purohitandet.al.,AHandbookofMedicinalPlants,A CompleteSourceBook,Agrobio,India,2004,p96. SharmaR.K.,GovilJ.N.,SinghV.K.,RecentprogressinMedicinal plants,Vol.7,EthanomedicineandPharmacognosyII,p404. ThakurR.S,PuriH.S.,AkhtarHusain,MajorMedicinalPlantof India,CIMAP,Lucknow,1989,p124. IndianPharmacopoeia,2007,p2045. Chaterjee Asima, Pakrashi S. C., The Treatise on Indian MedicinalPlants,Vol.3,p63. ElizabethM.WilliamsonMajorHerbsofAyurveda,2002,p79. QualityStandardsofIndianMedicinalPlantsVol.2,p19. Rastogi Ram P., B.N. Mehrotra, Compandium of Indian MedicinalPlants,CDRI,PID,NewDelhi,1993,Vol.2,p105. Rastogi Ram P., B.N. Mehrotra, Compandium of Indian MedicinalPlants,CDRI,PID,NewDelhi,Vol.3,p101. Rastogi Ram P., B.N. Mehrotra, Compandium of Indian MedicinalPlants,CDRI,PID,NewDelhiVol.4,p115. PharmacognosyReviews,Vol.1,JanMay,2007,p137. Sharma M.L.& et.al., Indian Journal of Pharmacology, 11 (6): 647652,1989. KulkarniR.R.,etal.,IndianJournalofPharmacology,24(1):98 101,1992.

SpectraofBoswellicacid@530nm CONCLUSION The proposed HPTLC method is simple, rapid, accurate, reproducible, selective and economic and can be used for routine qualitycontrolanalysisofBoswelliaserraraRoxb.(exudate)powder and quantitative determination of Boswellic acid in exudate powder.

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