Isolation and Identification of Compounds from the Leaf Extract of Dillenia indica Linn Md. Abdul Muhit*, Syed Mohammed Tareq, Apurba Sarker Apu, Debasish Basak and Mohammad S. Islam Department of Clinical Pharmacy and Pharmacology, University of Dhaka, Dhaka, Bangladesh, Abstract: Crude extract of the leaf of Dillenia indica linn. was taken under phytochemical investigation. The crude methanolic extract was partitioned with different solvent system by increasing their polarities (h-hexane, carbon tetrachloride and chloroform). The compounds were fractionated and isolated from n-hexane and chloroform partitionates by using vacuum liquid chromatography, column chromatography and preparative thin layer chromatographic technique. Detection was accomplished with UV detection at 254 nm. The structures of the isolated compounds were established by extensive spectroscopic studies (IH NMR spectroscopy) and chemical shifts are reported in ppm with respect to TMS or residual non-deutarated solvent signal. The n-hexane and chloroform fractionate yielded total of four compounds namely 3,5,7-trihydroxy-2"4°-dimethoxy flavone Keywords: Dillnia indica, phytochemical investigation, Dilleniaceae Introduction The chemical constituents of the medicinal plants, particularly the secondary metabolites have pronounced pharmacological actions on animal system and organs. Several bioactive compounds were isolated from the plant sources such as digoxin, digitoxin, morphine, reserpine, taxol, vinblastine, vincristine, quercetin (Ghani A, 2003) etc. which have different pharmacological and nutritional properties. The discovery of a novel chemical component from a medicinal plant may form the basis of development of various therapeutic agents with better activity. More than 500 medicinal plants have been reported to possess medicinal properties in Bangladesh and Dillenia indica linn. (Family: Dilleniaceae) is one of the most common edible species among them (Ghani A, 2003). Dillenia indica is an evergreen tree widely grows in tropical forests in the India, Bangladesh, Sri Lanka and China, The common local names are Chalta or Chalita (Bengali and Hindi), Karambel or thor for Carespondence Karmal in Marathi, and Ramphal in Nepal (Kirtikar & Basu, 1981). The leaves, bark, stem and fruits of the plat are used in traditional medicine for a long time. ‘The fruit juice is mixed up with sugar and water and used as cooling beverage in treatment of fever and also cardiotonic (Shome et.al, 1980). The leaves and bark are used as laxative and astringent. Alcoholic extract of the D. indica showed central nervous system depressant activity (Bhakuni et al. 1968). Phyltochemical studies showed that D. indica species contains the lupeol group of triterpene such as betulinic acid, betulin, betulinaldehyde and flavonol such as myricetin (Parvin et al. 2009). Stem bark contains myricetin, isorrhamnetin, dillenetin and glucosides etc (Pavanasasivam and Sultanbawa, 1975). Here we report the chemical constituents present in the leaf extract by chromatographic and spectrophotometric analysis and established the presence of triterpenoids as well as flavonoids, Bangladesh Pharmaceutical Joural. Vol. 13, No 1, January 2010 49 ISSN no.: 0301-4606Materials and methods: General experimental procedure The 'H NMR spectra was recorded using an Ultra Shield Bruker DPX-400 (400 MHz) instrument. For NMR spectrum studies deutarated chloroform was used and the 6 values for tH spectra were referred to the residual non-deutarated solvent signals. Collection of plant materials Plant sample of Dillenia indica was collected from Dhaka in August 2007. The plant sample was identified by taxonomist and a voucher specimen was given from National herbarium, Dhaka, Bangladesh (Voucher specimen no; DACB 34359) which was kept for further references. Extraction and fractionation of Plant material The fresh leaves were collected, cut into small pieces and air dried for several days. The plant materials were then ground into coarse powder. The dried and ground plant powder (500g) was extracted with methanol (25 liters) in an air tight, clean flat bottomed container for 7 days at room temperature with occasional stirring and shaking, The extract was then filtered first through a fresh cotton plug and finally with a Whatman filters paper. taolc Gre oe 8 am Extacton with hear (100 x3) ————, Uarer ler a retin with Caton acl (103) The filtrate was concentrated using a rotary evaporator (Heidolph, UK) at low temperature (90C) and pressure, The weight of the crude extract was 78 gm. Solvent-solvent partitioning (Figure: 1) was done by Using the protocol designed by Kupchan and Tsou (1873) and modified version of Wagenen et al(1993) The crude extract (6 gm) was triturated with 90% methanol. The prepared solution was then fractionated successively using solvents of, increasing polarity, such as n-hexane (HX: 820 mg), carbon tetrachloride (CT: 850 mg) and chloroform (CF: 665 mg). All the three fractions were evaporated to dryness by using rotary evaporator at low temperature of 390C and kept in air tight containers for further analysis. Isolation and Identification of compounds The chloroform (CF: 665 mg) soluble materials of methanolic extract were fractionated by gel permeation column chromatography (Red and Sarker, 1988). The column was packed with Sephadex (LH-20) and soaked with a ratio of n-hexane:dichloromethane:methanol (25:1) for at least 12 hours. The column was then eluted with the same solvent mixture and finally the column was washed with dichloromethane and methanol mixtures of increasing polarity. Total 100 ml volume was collected in the mentioned solvent system (Fraction no. 1-28), 50 ml was collected in dichloromethane: methanol (9:1) (fraction no, 29-42), 50 ml was collected in dichloromethane (1): methanol (1) (fraction no, 43-53), 100 ml was collected in 100% methanol (fraction no, 54-56). The Compound 1 (6.7 mg), white amorphous materials were collected from the column fractions 52-55. The R-value of the compound 1 was determined as 051 in chloroform-methanol (97:3) and the yield value was 0.073%. Compound 2 (46 mg) was ‘arb eaclarieaabe ction gee tation collected from the column fractions 23-28 by T sme (6 7 etn wih eto (00 3) preparative thin layer chromatography (Liu and Kindetlerer, 1999) (Stationary phase-silca gel Fase, mobile phase- 5% methanol in chloroform, thickness of plates - 0.5%). The R-value of the compound 2 was determined as 0.60 in chloroform-methanol (95:5) and the yield value was 0.061%. Bangladesh Pharmaceutical Journal. Vol. 13, No 1, January 2010 ISSN no.: 0301-4606 50‘The n-hexane (HX: 820 mg) partitioned compounds were then fractionated by vacuum liquid chromatography (Coll and Bowden, 1986). The column was packed with silica gel (Keisel gel 60H, mesh 70-230) under vacuum and washed with petroleum ether. The n-hexane partitionates were adsorbed onto silica gel, allowed to dry and subsequently applied on top of the adsorbent layer. Then the column was eluted with petroleum ether, ethyl acetate and methanol mixtures of increasing polarities to provide 31 fractions (60 ml each), Evaporation of the solvents, colorless crystalline compound 3 (3.8 mg) was isolated from the fraction No, 14-18 eluted with 50% ethyl acetate in petroleum ether and colorless needle shaped crystal compound 4 (4.3 mg) were isolated from the fraction no. 24-27 eluted with 10% ethyl acetate in petroleum ether. The Ri-value of the compound 3 was determined as 0.55 in ethyl acetate-toluene (10:90) and the yield value ‘was 0.049%, The Ry-value of the compound 4 was determined as 0.33 in ethyl acetate-toluene (6:95) and the yield value was 0.055%, Result Repeated chromatographic separation and purification of the cold methanolic extracts of the leaf of Dillenia indica provided a total of four compounds (compound 1, 2, 3 and 4), the structure of which were determined by extensive 'H NMR spectral analysis as well as by comparison of their spectral data with previously reported values. Dillenetin (3,5,7-trihydroxy-3°,4’-dimethoxy flavone) (1): White amorphous powder; 'H NMR spectrum (400 MHz, CDCb): 8 12.65 (1H, br. s, HO-5), 760 (1H, dd, J=76, 2.0 Hz, H-6'), 758 (1H, d, 2.0 Hz, H-2'), 6.95 (1H, d, O76 Hz, H-5), 6.93 (IH, s, H-8),3.95 (3H, s, OMe), 2.97 (3H, s, OMe). Betulinic acid (2): White mass; 'H NMR spectrum (#00 MHz, CDCl): 6 4.75 (1H, br. s, Hy-29), 4.62 (1H, br. s, Hy-29), 3.20 (1H, dd, J=11.2, 4.9 Hz, H-3), 3.00 (1H, m, H-19), 1.70 GH, s, Me-30), 093 GH, s, Me-26), 0.98 (3H, s, Me-23), 0.95 (3H, s, Me-27), 0.83 (3H, s, Me-25), 0.76 (SH, s, Me-24) B-Sitosterol (3): Colorless needles;1H NMR (400 MHz, CDC): 6 5.39 (1H, m, H-6), 3.51 (1H, m, H-3), 1.05 (BH, s, Me-19), 0.96 (3H, d, J+6.5 Hz, Me-21), 0.89 (BH, t, JA74 Hz, Me-29), 0.87 (3H, d, 6.7 Hz, Me-26), 0.85 (3H, d, +67 Hz, Me-27), 0.72 (3H, s, Me-18), Stigmastero! (4): Colorless needle shaped crystal; 1H NMR (400 MHz, CDCI): 8 6.35 (1H, m, H-6), 5.13 (IH, dd, $14.4, 8.4 Hz, H- 22), 5.03 (1H, dd, J=14.4, 84 Hz, H- 23), 3.51 (1H, m, H-3), 1.0 GH, s, CHs-10), 0.91 (BH, d, J+6.4 Hz, CHs-20), 0.85 (3H, d, JH74 Hz, CHb-27), 0.81 GH, d, 74 Hz, CH»-26), 0.67 (3H, , CHs-13). Discussion The 'H NMR spectrum of compound 1 (Figure: 2) showed one broad singlet value at 6 12.65 which revealed hydroxyl groups at C-5 positions. The double doublet 6 760 with coupling constants 26 Hz and 2.0 Hz centered at could be assigned with H-6" The doublet 6 758 and & 6.95 with coupling constants 2.0 Hz and 76 Hz indicates H-2° and H-5" The splitting pattern and coupling constants revealed the presence of a trisubstituted benzene ring. The spectrum displayed two singlet at 6 3.95 and 6 3.97 indicating the presence of two methoxy groups. The spectrum also revealed on proton singlet value at 6 6.93 indicated H-8, The spectra were compared with published data of Parvin et al. (2009) works. The 'H NMR spectrum of compound 2 (Figure: 3) revealed the presence of a lupene type carbon skeleton. It displayed signals attributable to an exomethylene group at 6 4.62 and 475 (1H, each, brs) which together with an allylic methyl at 6 1.70 which indicated an isopropeny! function, The double doublet 6 3.20 with couplings of 11.2 and 4.9 Hz centered at could be assigned to H-3. The large coupling of this proton (H-3) with the vicinyl methylene protons suggested a B (beta) orientation of the hydroxyl group at C-3. In addition, the spectrum also showed a multiplet at 6 3.00 for the methine proton at C-19 and five methyl group resonances at 0.76, 0.83, 0.95, 0.97 and 0.98, On the basis of the above spectral features, compound 2 was identified as betulinic acid. The identity of 2 as betulinic acid was confirmed by comparison of these Bangladesh Pharmaceutical Journal. Vol. 13, No 1, January 2010 51 ISSN no.: 0301-4606Fig2: Compound 1Diertin) Fig Compound 3(p-stostro data with published values of Ikuta and kokawa (1988) as well as by co-TLC with an authentic sample The 'H NMR spectrum (400 MHz, DCL) of compound 3 (Figure: 4) has revealed a one proton multiplet at 6 3.51, the position and multiplicity of which was indicative of H-3 of the steroid nucleus. The typical H-6 of the steroidal skeleton was evident as a multiplet at 6 5.39 that integrated for one proton. The spectrum further revealed signals at 6 0.72 and & 1.05 (BH each) assignable to two tertiary methyl groups at C-13 and C-10, respectively. The 1HINMR spectrum showed two doublets centered at 8 0.87 (6.7 Hz) and 0.85 (6.7 Hz) which could be attributed to two methyl groups at C-25. The doublet at § 0.96 U= 6.5 Hz) was demonstrative of a methyl group at C-20. On the other hand, the triplet of three-proton intensity at 6 0.89 could be assigned to Fig S: Compound 4(Sigmasterst the primary methyl group attached to C-28. The above spectral features are in close agreement to those observed for -sitosterol in literature (Manoharan ef al, 2005) (Escudero et al, 1985). The *H NMR spectra of compound 4 (Figure: 5) revealed a one-proton multiplet at & 3.51, the position and multiplicity of which was indicative of H-3 of the steroidal nucleus. The typical signal for the olefinic H-6 of the steroidal skeleton was evident from a multiplet at 6 5.35 integrating for one-proton, The olefinic protons (H-22 and H-23) appeared as characteristics downfield signals at 6 5.13 and & 5.03 respectively in the 'H NMR spectrum. Each of the signal was observed as double doublets (4=14.4, 84 Hz) which indicated coupling with the neighboring olefinic and methine protons. The spectrum further revealed signals at 6 0.67 and & Bangladesh Pharmaceutical Journal. Vol. 13, No 1, January 2010 52 ISSN no.: 0301-46061.00 (three-proton each) assignable to two tertiary methyl groups at C-13 and C-10, respectively. The 'H NMR spectrum also showed two doublets centered at 6 0.81 (74 Hz) and 0.85 U=74 Hz) which could be attributed to the two methyl groups at C-25, The doublet at 6 0.91 U=6.4 Hz) was demonstrative of a methyl group at C-20. These spectral features are in close agreement to those observed for stigmasterol by Khan (1991). On this basis, the identity of compound 4 was confirmed as stigmasterol (Greca et al, 1980) Conclusion Medicinal plants used in the folk medicine may be an interesting and largely unexplored source for the development of potential _new compounds Cindequist et al. 2005). But it is necessary to isolate the active principles and characterized their constituents for the beneficial of human being. It was our attempt to identify the new compounds in this plant that revealed four compounds and all of them are previously established Acknowledgement The authors would like to thank Prof. Dr. Mohammad ‘Abdur Rashid, Dean, Faculty of Pharmacy, University of Dhaka, Dhaka for the interpretation of 'H NMR spectra of the compound and BCSIR, Dhaka, for their technical assistance. References: Bhakuni DS, Dhar ML, Dhar MM, Dhawan BN, Mehrotra BN (1968). Screening of Indian plants for biological activity Pt. I. Indian J Exp Biol; 7 250-262. Coll JC, Bowden BF (1986). The Application of Vacuum Liquid Chromatography to the Separation of Terpene Mixtures. J. Nat. Prod.; 49 (5): 934-936. Escudero J, Lopez C, Rabanal RM, Valverde S (1985), ‘Secondary metabolites from Satureja species. New triterpenoid from Satureja acinos. Journal of Natural Products; 48: 128-131 Ghani A, (2003), “Medicinal plants of Bangladesh with chemical constituents and uses". 2nd edn, Asiatic society of Bangladesh, Dhaka, Ramna., p.42 Greca MD, Monaco M, Previtra L (1990), Stigmasterols from Typha latifolia. Journal of Natural Products; 53:1430-1435, Ikuta A, Itokawa H (1988). Trterpenoids of Paeonia japonica callus tissue. Phytochemistry; 27: 2813-2815. Khan RIL Natural product: A laboratory guide, 2nd Edn. Academic press, N.Y. USA,, 1991 Kirtikar KR, Basu BD. (1981). Indian medicinal plants. 2nd edn India, Allahabad: Lalit M. and Basu, p.53. Kupchan SM, Tsou. G. (1973). Tumor inhibitors. A, new antileukemic simaroubolide from Brucea antidysenterica. J. Org. Chem. 38: 178-179 Lindequist U, Niedermeyer TH, Julich WD (2008). The pharmacological potential of mushrooms. Evid Based Complement Alternat Med; 2: 285-299, Liv QT, Kinderlerer JL (1999). Preparative thin-layer chromatographic separation and subsequent gas chromatographic-mass. spectrometric analysis. of monoacylglycerols derived from butter oil by fungal degradation. Journal of Chromatography A. 855 (2) 617-626 Manoharan KP, Huat Benny TK, Yang D (2008) Cycloartane type triterpenoids from the rhizomes of Polygonum bistorta. Phytochemistry; 66: 1168-1173, Parvin MN, Rahman MS, Islam MS, Rashid MA\ (2008). Chemical and biological investigations of Dillenia indica Linn. Bangladesh J Pharmacology; 4 122-125 Pavanasasivam G, Sultanbawa MUS (1975) Chemical investigation of ceylonese plants. Part XI (4)-3,4" 5,7-Tetrahydroxy-3"-methoxyflavanone [C+ dihydroisorhamnetin] and _3,5,7-trihydroxy-3", 4-dimethoxyflavone (dillenetin): two new natural products from Dillenia indica L. J. Chem. Soc., Perkin Trans. 1;6: 612-613. Reid RG, Sarker SD (1998). Isolation of Natural Products by Low-Pressure Column Chromatography. Natural Products Isolation; 4: 111-140. Shome U, Khanna RK, Sharma HP (1980) Pharmacognostic studies on Dillenia indica Linn. I+ Fruit and Seed. Proc. Indian Acad Sci (Plant Sci); 89:91-104 Wagenen BCV,, Larsen R, Cardellina JH, Ran Dazzo D, Lidert ZC, Swithenbank C (1983). Ulosantoin, a potent insecticide from the Sponge Ulosa ruetzler J. Org. Chem,, 58: 335-337, Bangladesh Pharmaceutical Journal. Vol. 13, No 1, January 2010 53 ISSN no.; 0301-4605