Received: 21 Oct 2016 Revised and Accepted: 07 Dec 2016

Transcription

1 Interntionl Journl of Phrmcy nd Phrmceuticl Sciences ISSN Vol 9, Issue 2, 2017 Originl Article DEVELOPMENT OF A DENSITOMETRIC HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHIC METHOD FOR THE QUANTITATIVE ANALYSIS OF URSOLIC ACID IN THE LEAVES OF SPECIES OF GENUS TECOMA AND TABEBUIA OF BIGNONIACEAE FAMILY KALYANI A. KEDAR,b, *, SANJAY. R. CHAUDHARI c, AVANAPU S. RAO d P. E. Society s Modern College of Phrmcy, Nigdi, Pune Mhrshtr , b Jwhrll Nehru Technologicl University (JNTU), Hyderbd, Andr Prdesh, , Indi, c Amrutvhini College of Phrmcy Amrutngr, Sngmner SK, Tl-Sngmner, Dist Ahmedngr, Mhrshtr , Indi, d Bhskr Phrmcy College, Yeknplly, Moinbd (Mndl), R. R. (Dt), Hyderbd , Indi Emil: kk_phrm20@rediffmil.com ABSTRACT Received: 21 Oct 2016 Revised nd Accepted: 07 Dec 2016 Objective: A simple nd sensitive high-performnce thin lyer chromtogrphic (HPTLC) method ws developed for the quntifiction of ursolic cid in the leves of three species of genus Tecom (Tecom gudichudi DC, Tecom cpensis (Thunb.) Lindl, Tecom stns (L.) Juss. ex Kunth) nd genus Tbebui (Tbebui rose Bertol) belong to fmily Bignonicee. Methods: Chromtogrphy ws performed on Silic gel 60 F254 precoted HPTLC Pltes with optimised mobile phse pet ether: ethyl cette: formic cid (7:3:0.5, v/v/v). The plte ws derivtized with p-nisldehyde regent nd scnned t 540 nm. The developed method ws found to give compct spot for ursolic cid t R f vlue 0.43±0.01. The method ws vlidted using Interntionl Council for Hrmoniztion (ICH) guidelines, including linerity, precision, ccurcy, nd robustness. Results: Ursolic cid ws found to be present in four species, i.e., Tecom gudichudi DC (1.48%w/w), Tecom cpensis (Thunb.) Lindl. (0.79%w/w), Tecom stns (L.) Juss. Ex Kunth (1.11%w/w), Tbebui rose (Bertol.) (1.13%w/w). A good linerity reltionship ws found to be ( ng bnd 1) with correltion coefficient (r2) vlue of with ursolic cid. Limit of detection nd limit of quntifiction ws considered to be 40.66, ng per bnd respectively. The developed method ws found to be ccurte nd precise with 1.32%, 1.19% (%RSD) for interdy nd intrdy precision. The ccurcy of the method ws performed by recovery studies t three different concentrtion levels, nd the verge percentge recovery ws found to be 98.05% for ursolic cid. Conclusion: The proposed method for the quntittion of ursolic cid ws found to be reproducible nd simple. Keywords: Bignonicee, Tecom, Tbebui, Ursolic cid, High-performnce thin lyer chromtogrphy 2017 The Authors. Published by Innovre Acdemic Sciences Pvt Ltd. This is n open ccess rticle under the CC BY license ( 0/) DOI: INTRODUCTION Bignoni Linn (Bignonicee) is monotypic genus of woody climbers, ntive to North Americ nd mostly grown for ornment in the tropics of the old world [1]. Bignonicee fmily ws hving 100 gener nd more thn 750 plnt species observed in vrious tropicl regions of Indi. Known numbers of this fmily re Bignoni, Tecom, Ctlp, Tbebui nd Jcrnd. These re succulent herbs, shrubs, stem sometimes reduced to rhizome or tuber. Numerous species of this fmily re observed s poisonous to leeches [2]. In Chrk, Sushrut, the root, brk, stem nd lef of some species of Bignonicee fmily is useful for snke bite, the stem nd wood for scorpion sting. In Bngldesh whole plnt of Tecom gudichudi DC use of remedy for dibetes nd infertility problems [3]. From ll secondry metbolite's pentcyclic triterpenes, re n importnt group of it considered s lupenyl, ursnyl, betulenyl or olenyl. They re presented in plnt species s the form of glycone's sponin triterpenoids [4-5]. Previous reports stte tht species of Bignonicee fmily show the presence of promising ctive constituents such s tnnins, flvonoids, triterpenes, lkloids, crbohydrtes, etc. [6]. Ursolic cid ws pentcyclic triterpene reported in severl plnt species such s Alstoni scholrs R. Br., Diospyros melnoxlon, Holoptele integrifoli [7-9]. Ursolic cid ws reported to for its vrious biologicl ctivities such s ntiinflmtory, ntidibetic nd cn trget severl steps of cncer development. Thus, being promising tool for the tretment nd chemoprevention of cncer [10-12]. The phytochemicl nlysis of vrious species of Bignonicee fmily ws not studied so fr hence; the following reserch dels with to crry n out nlysis of ursolic cid by high-performnce thin lyer chromtogrphy in four species of Bignonicee fmily (Tecom gudichudi DC, Tecom cpensis (Thunb.) Lindl, Tecom stns (L.) Juss. Ex Kunth, Tbebui rose (Bertol.) Berto). MATERIALS AND METHODS Plnt mterils All four species of Bignonicee fmily, i.e. Tecom gudichudi DC (Smple 1), Tecom cpensis (Thunb.) Lindl. (Smple 2), Tecom stns (L.) Juss. Ex Kunth (Smple 3), Tbebui rose (Bertol.) (Smple 4) were collected from different re of Pune district (Mhrshtr), nd the plnts were uthenticted t Botnicl Survey of Indi, Pune with reference no. BSI/WRC/Iden./2015/576 on dted The specimen voucher number is KALKTEG1, KKA-2, KKA-3, KKA-1 specimens of plnts were deposited t the Botnicl Survey of Indi, Pune nd deprtment of Phrmcognosy, Modern College of Phrmcy, Nigdi, Pune. Chemicls nd regents Ursolic cid ws purchsed from Sigm-Adrich (USA), nlyticl grde solvents, regents, silic gel 60 F254 precoted HPTLC Pltes (20 20 cm) were purchsed from Merck (Germny). HPTLC instrumenttion nd experimentl conditions Chromtogrphic nlysis ws done on cm HPTLC Silic gel F254 pltes (Merck, Germny). Smples of extrcts nd stndrds were pplied s bnd length 8 mm wide nd 8 mm prt by the Cmg Linomt V smple pplictor. The ppliction rte of the smple on the plte ws 150nl/s. The pltes were developed in

2 Kedr et l. Int J Phrm Phrm Sci, Vol 9, Issue 2, previously sturted (20 10 cm) twin trough glss chmber t room temperture. Anlysis crried out t 540 nm in bsorbnce remission mode by TLC scnner III (Cmg, Muttenz, Switzerlnd) nd win CATS version softwre (Cmg, Muttenz, Switzerlnd) ws used in this study. Microsoft excel ws lso used to tret dt sttisticlly. Plnt mteril nd Preprtion of extrcts Ethyl cette extrcts of four species of Bignonicee fmily, i.e. Tecom gudichudi DC (Smple 1), Tecom cpensis (Thunb.) Lindl. (Smple 2), Tecom stns (L.) Juss. Ex Kunth (Smple 3), Tbebui rose (Bertol.) (Smple 4) were prepred by weighing 500 gm of ir dried powdered mteril, nd extrction ws crried out by soxhlet extrction ssembly for 6 h. The solution ws filtered, concentrted nd use for HPTLC nlysis of ech smple ws subjected to soxhlet extrction for 6 h. Preprtion of clibrtion curve A stock solution of ursolic cid (stndrd) ws prepred by dissolving 10 mg in 100 ml methnol. For clibrtion 2, 4, 6, 8, 10, 12, 14 µl stndrd solutions were pplied to HPTLC plte in the rnge ng per bnd. Method development Initilly, different mobile phses were used for chromtogrm development from this best resolution ws observed in the selected pet ether: Ethyl cette: Formic cid (7:3:0.5 v/v/v) solvent system, gives compct pek of stndrd ursolic cid nd stndrd in ethyl cette lef extrct of smples Tecom gudichudi DC (Smple 1), Tecom cpensis (Thunb.) Lindl. (Smple 2), Tecom stns (L.) Juss. Ex Kunth (Smple 3), Tbebui rose (Bertol.) (Smple 4). Dry TLC plte derivtive with p-nisldehyde regent, het plte on 105 C nd observed seprtion of bnds it helps in nlysis of ursolic cid in Extrcts [13]. The nlysis ws done t 540 nm. Method vlidtion The nlyticl method ws vlidted for linerity, precision, ccurcy, specificity, limit of detection (LOD) nd limit of quntittion (LOQ) ccording to Interntionl Council for Hrmoniztion [14]. Linerity rnge For determining the linerity rnge of stndrd ursolic cid, series of spots of different volumes (2, 4, 6, 8, 10, 12, 14 µl) ws pplied to get the ng quntity of ursolic cid per bnd, respectively. The plte ws scnned t 540 nm, nd the curve ws prepred with respect to re vs. mount per bnd. Precision nd ccurcy Precision studies were crried out to show the reproducibility of the proposed developing method. Repetbility ws determined by prepring eight replictes of the sme concentrtion (500ng bnd -1 ) of the smple. The intrdy precision study ws crried out by prepring the sme concentrtion of stndrd compound, nd it cn be nlyzing t three different times in dy. The sme procedure ws followed for three different dys to determine interdy precision. The results were reported s % RSD. Specificity The specificity of the method ws determined by nlyzing the stndrd drug nd smple. The presence of ursolic cid in Tecom gudichudi DC (Smple 1), Tecom cpensis (Thunb.) Lindl. (Smple 2), Tecom stns (L.) Juss. Ex Kunth (Smple 3), Tbebui rose (Bertol.) (Smple 4) were confirmed by compring R f of the smple with stndrd. (fig. 4 9) Limit of detection nd quntittion In order to determine the limit of detection (LOD) nd limit of quntittion (LOQ) ws clculted bsed on stndrd devition (SD) nd slope (S) of the clibrtion curve t levels pproching the LOD ccording to formul.{ LOD= 3.3(SD/S) nd LOQ= 10 (SD/S) }. Robustness Robustness ws studied in triplicte t 500ng bnd -1 by mking smll vrition in optimised method prmeters. Vrition in composition of the mobile phse, chmber sturtion time nd ctivtion of HPTLC pltes. The results were exmined in terms of reltive stndrd devition (%RSD) nd stndrd error of the pek re. Mobile phse prepred by the solvent system such s pet ether: ethyl cette: formic Acid in different concentrtion (7: 3: 0.5v/v/v), (7.5: 2.5: 0.5 v/v/v), (6.5:3.5:0.5 v/v/v) ws used for chromtogrphic nlysis. Durtion of sturtion time chnge during chromtogrph development 20±5 min (15, 20 nd 25 min) respectively. The plte ws ctivted t 110 C for 10±5 min (5, 10, 15 min) nd nlyzed t 540 nm. By introducing smll chnges into chromtogrphic method nd % RSD ws obtined less thn 2%, proved the robustness of the proposed method. Assy of ursolic cid Stndrd ursolic cid nd the test smple were spotted on HPTLC plte. The percentge of ursolic cid present in test smples (1, 2, 3 nd 4) ws determined by using clibrtion curve of the stndrd. Thereby, the content of ursolic cid ws clculted for species of Tecom nd Tbebui genus. RESULTS AND DISCUSSION Plnt mteril nd preprtion of extrcts Ethyl cette extrcts of four species of Bignonicee fmily, i.e. Tecom gudichudi DC (Smple 1), Tecom cpensis (Thunb.) Lindl. (Smple 2), Tecom stns (L.) Juss. Ex Kunth (Smple 3), Tbebui rose (Bertol.) (Smple 4) were prepred by soxhlet extrction ssembly nd the yield ws 8.5%, 4.0%, 5.75, 6.2% w/w respectively nd used for HPTLC nlysis. Method development The developed mobile phse ws found to be effective in the seprtion of components in smples Tecom gudichudi DC (Smple 1), Tecom cpensis (Thunb.) Lindl. (Smple 2), Tecom stns (L.) Juss. Ex Kunth (Smple 3), Tbebui rose (Bertol.) (Smple 4) nd shows compct nd shrp peks of stndrd (ursolic cid) nd stndrd in the four smples from selected mobile phse under specific sturtion time (fig. 3 nd 4). The identity of the stndrd in the smple extrcts ws confirmed by the sme R f vlue (0.43±0.01) (fig. 5, 6, 7, 8 nd 9) for ursolic cid in stndrd nd smples. Method vlidtion The linerity of stndrd ursolic cid ws vlidted by the liner regression eqution nd correltion coefficient. The clibrtion curve of ursolic cid ws found to be liner in the rnge of ng bnd- 1 (tble 1 nd fig. 2). Regression eqution nd vlue of r 2 for the reference compound were observed s Y= X nd respectively; improved method shows good linerity nd resolution (tble 1). The men vlues with SD of the slope were nd intercept vlue of SD ws Inter-dy nd intr-dy precision (n= 8) for ursolic cid were crried out t 500 ng bnd -1 nd found to be 1.32% nd 1.19% (%RSD) respectively. Good precision ws observed from developed method for ursolic cid. Limit of detection (LOD) nd limit of quntittion (LOQ) ws considered to be 40 ng nd ng bnd -1 respectively (tble 1). This indicted the sensitivity of the instrument for the quntittion of the compound. The ccurcy of the method ws studied by performing recovery studies t 3 levels of ursolic cid. Pre-nlyzed smples were spiked with 50%, 100% nd 150% of the stndrd ursolic cid nd nlysed by the proposed HPTLC method. The experiment ws conducted three times the percentge recovery t three different levels of ursolic cids ws found to be 98.13, 98.23, 98.46% respectively (tble 3). Robustness ws studied in triplicte t 500ng bnd -1 by mking minor vrition in optimised method prmeters such s the vrition in the composition of the mobile phse, chmber sturtion time, plte ctivtion time. The miniml vlue of SD nd reltive stndrd devition (% RSD) ws obtined indicte tht method ws robust (tble 4). 110

3 Kedr et l. Int J Phrm Phrm Sci, Vol 9, Issue 2, Assy of ursolic cid The developed mobile phse is useful for the quntittion nd identifiction of ursolic cid in smples Tecom gudichudi DC (Smple 1), Tecom cpensis (Thunb.) Lindl. (Smple 2), Tecom stns (L.) Juss. Ex Kunth (Smple 3), Tbebui rose (Bertol.) (Smple 4). Ursolic cid stndrd ws quntified by using developed mobile phse pet ether: ethyl cette: formic cid (7: 3: 0.5 v/v/v). The ethyl cette extrcts of ll four smples 1, 2, 3 nd 4 observed some common R f vlue 0.43, 0.5, 0.6, 0.9 from this 0.43 noted for the presence of ursolic cid compre with stndrd (fig. 3 A-F). From the HPTLC chromtogrm, ll the four smples of the Bignonicee fmily i.e. smple 1 (Tecom gudichudi DC), smple 2 (Tecom cpensis Thunb. Lindl). Smple 3 (Tecom stns (L.) Juss. Ex Kunth), smple 4 (Tbebui rose (Bertol.) were found to contin ursolic cid by compring with R f vlue of the stndrd (fig. 5, 6, 7, 8 nd 9). As per literture, ursolic cid hs been estimted by using HPTLC in severl plnts from this some plnt species re Alstoni scholrs R. Br., Diospyros melnoxlon [7-8]. The ursolic cid content ws observed s 0.302% nd 0.122% w/w in Holoptele integrifoli methnolic nd hexne frction respectively [9]. In some studies, the series of imine nd phthlic cid derivtives of ursolic cid hve been synthesised for ntiinflmmtory nd nti-cncer potentil. So need to find out the rich source of ursolic cid contining medicinl plnts [15]. The outcome of study includes quntittive nlysis of ursolic cid by densitometric high-performnce thin lyer chromtogrphy, from observtion the content of ursolic cid in the Tecom gudichudi DC (1.48%w/w), Tecom cpensis (Thunb.) Lindl. (0.79%w/w), Tecom stns (L.) Juss. Ex Kunth (1.11%w/w), Tbebui rose (Bertol) (1.13%w/w) respectively from the clibrtion curve. Tble 1: Method vlidtion prmeters for the quntittion of ursolic cid by HPTLC Prmeters Results Rnge of linerity (µg/ml) 2-14 Regression of eqution Y= *X Correltion coefficient (r 2 ) LOD (ng/bnd) LOQ (ng/bnd) Tble 2: Interdy nd intrdy precision of HPTLC (n=8) Amount (ng/bnd) Interdy precision Intrdy precision Men re SD %RSD Men re SD %RSD Ursolic cid Men of eight determintions. Tble 3: Accurcy (recovery study) determined for the TLC-densitometric method % of stndrd spiked to the smple Theoreticl content (µg/bnd) Experimentl content (µg/bnd) % men recovery RSD (%) Men of three determintions CH 3 H O H H CH 3 HO CH 3 Fig. 1: Structure of ursolic cid Fig. 2: Clibrtion curve of stndrd ursolic cid t 540 nm 111

4 Kedr et l. Int J Phrm Phrm Sci, Vol 9, Issue 2, Tble 4: Robustness study for the HPTLC method Fctors Chromtogrphic chnges Level Pek re SD %RSD Mobile phse composition (±5) 7.5:2.5: : 3: :3.5: Sturtion time (±5) Activtion of TLC plte (±5) Men of three determintions Fig. 3: Picture of developed TLC plte derivtized with p- nisldehyde regent t 540 nm; mobile phse: pet ether: ethyl cette: formic cid (7: 3: 0.5, v/v/v) Fig. 6: Chromtogrm of Ethyl cette extrct of lef of Tecom gudichudi DC (Smple 1) t 540 nm Fig. 4: 3D disply of ll trcks t 540 nm; mobile phse: pet ether: ethyl cette: formic cid (7: 3: 0.5, v/v/v) Fig. 7: Chromtogrm of Ethyl cette extrct of lef of Tecom cpensis (Thunb.) Lindl. (Smple 2) t 540 nm Fig. 5: Chromtogrm of stndrd ursolic cid (500ng spot -1 ), pek 1(Rf =0.43) t 540 nm Fig. 8: Chromtogrm of Ethyl cette extrct of lef of Tecom stns (L.) Juss. Ex Kunth (Smple 3) t 540 nm 112

5 Kedr et l. Int J Phrm Phrm Sci, Vol 9, Issue 2, Fig. 9: Chromtogrm of Ethyl cette extrct of lef of Tbebui rose (Bertol.) (Smple 4) t 540 nm CONCLUSION In present study HPTLC method ws developed nd vlidted for the determintion of ursolic cid in Tecom gudichudi DC (Smple 1), Tecom cpensis (Thunb.) Lindl. (Smple 2), Tecom stns (L.) Juss. Ex Kunth (Smple 3), Tbebui rose (Bertol.) (Smple 4) lef extrcts, which shows 1.48%w/w, 0.79%w/w, 1.11%w/w nd 1.13%w/w respectively. The proposed method ws found to be simple, ccurte, specific nd robust for the nlysis of ursolic cid in crude drug smple. So these studies shde on chromtogrphic nlysis of four species of Bignonicee fmily. Bsed on these results leves of Tecom gudichudi DC (Smple 1), contin higher ursolic cid s compred to other species. So concentrted frctions or extrct of leves of Tecom gudichudi DC is rich source of ursolic cid nd my be more useful for formultions. ACKNOWLEDGMENT Authors re thnkful to Botnicl Survey of Indi (BSI), Pune, BCUD Svitribi Phule Pune University, Jwhrll Nehru Technologicl University, Hyderbd (JNTUH) nd Modern college of Phrmcy for providing necessry ssistnce. CONFLICTS OF INTERESTS There re no conflicts of interest REFERENCES 1. Anonymous. The welth of Indi: rw mterils, New Delhi Publictions nd Informtion Directorte, C.S.I.R; Kirtikr KR, Bsu BD. Indin Medicinl plnts, Interntionl Book distributors Dehrdun; Rhmtullh M, Smrri W, Rownk J, Shhnz RSZUM, Nsim. An ethnomedicinl, phrmcologicl nd phytochemicl review of some Bignonicee fmily plnts nd description of bignonicee plnts in folk medicinl uses in Bngldesh. Adv Ntr Appl Sci 2010;4: Connolly JD, Hill RA. Triterpenoids. Ntu Prod Rep 2000;17: Allouche Y, Beltrán G, Gforio JJ, Uced M, Mes MD. Antioxidnt nd ntitherogenic ctivities of pentcyclic triterpenic diols nd cids. Food Chem Toxi 2010;48: Choudhry S, Dtt S, Tlukdr AD. Phytochemistry of the fmily bignonicee- review. Assm Univ J Sci Technol Biol Environ Sci 2011;7: Shetty P, Mngonkr K, Sne RT. HPTLC determintion of ursolic cid in Alstoni scholris R. Br J Plner Chromto- Modern TLC 2007;20: Rut K, Dsh B, Singh R. HPTLC quntifiction nd ntimicrobil ctivity of ursolic cid from Diospyros melnoxylon. J Plner Chromto-Modern TLC 2012;25: Dixit P, Pl M, Upreti D. Comprtive studies on the nlyticl nd ntioxidnt ctivities of the medicinlly importnt stem brk of Holoptele integrifoli. J Plner Chromto-Modern TLC 2014;27: Liu J. Phrmcology of olenolic cid nd ursolic cid. J Ethnoprmcol 1995;49: Slvdor JAR, Lel ASM, Sntos RC. In: Pentcyclic Triterpenes s Promising Agents in Cncer. Slvdor JAR. editor. Nov Science Publishers, Inc; New York; Novotny L, Vchlkov A, Biggs D. Ursolic cid: n ntitumorigenic nd chemopreventive ctivity. Neoplsm 2001;48: Wgner H, Bldt S. Plnt drug nlysis. 2nd ed. Springer-Verlg Berlin nd Heidelberg; Interntionl Council for Hrmoniztion (ICH) HARMONISED TRIPARTITE GUIDELINE. Vlidtion of nlyticl procedures: Methodology; p Shriknt B, kirti L. Synthesis nd chrcteriztion of new imine nd pthlic cid derivtives of ursolic cid. Int J Phrm Phrm Sci 2014;10: How to cite this rticle Klyni A Kedr, Snjy R Chudhri, Avnpu S Ro. Development of densitometric high-performnce thin-lyer chromtogrphic method for the quntittive nlysis of ursolic cid in the leves of species of genus Tecom nd tbebui of the bignonicee fmily. Int J Phrm Phrm Sci 2017;9(2):