WO2003011891A1

WO2003011891A1 - A process for the isolation of betulinic acid from nelumbo nucifera gaertn rhizomes

Info

Publication number: WO2003011891A1
Application number: PCT/IN2001/000142
Authority: WO
Inventors: Pulok Kumar Mukherjee; Manjusree Pal; Bishnu Pada Saha
Original assignee: Council Of Scientific And Industrial Research
Priority date: 2001-08-03
Filing date: 2001-08-03
Publication date: 2003-02-13

Abstract

The present invention provides a process for the isolation of betulinic acid by extracting rhizomes of Nelumbo nucifera Gaertn (Family - Nymphaeaceae) with a polar solvent by conventional soxhlet extraction methods, concentrating the extract, adding water to the concentrated residue, extracting the said residue successively with non-polar solvents, extracting the aqueous phase with organic solvent, concentrating the above obtained organic solvent phase and purifying the product by conventional methods such as column chromatography followed by crystallization to give pure betulinic acid.

Description

A PROCESS FOR THE ISOLATION OF BETULINIC ACID FROM NELUMBO NUCIFERA GAERTN RHIZOMES

Field of the invention

The present invention relates to a process for the isolation of betulinic acid. More particularly, the present invention relates to a process for the isolation of betulinic acid from Nelumbo nυcifera Gaertn. (Family Nymphaeaceae). The acid thus obtained has potent therapeutic activity and is useful as anti-HIV, anti-tumoral and melanoma specific cytotoxic agent. Background of the invention

Nelumbo nucifera Gaertn. (Family Nymphaeaceae) is a large aquatic herb with slender elongate, branched creeping stems and stout creeping yellowish white coloured rhizomes. The flower of Nelumbo nucifera Gaertn. is the national flower of India. Almost all parts of this plant are used in Indian Traditional Medicine to cure several diseases [Chopra, R. N., Chopra I. C, and Handa, K. L. (1958), Indigenous Drugs of India, 2^nd ed., U. N. Dhur & Sons Pvt. Ltd., Calcutta, p. 679; Kirtikar, K. R, and Basu, B. D., (1975) in Indian Medicinal Plants ed. by Blatter, E., Caius, J. E., and Mhasker, K. S., Bishen Singh and Mahendra Pal Singh, Dehradun, pp. 116 - 120; Nadkarni, A. K., (1992) Indian Materia Medica, Vol. I, Popular Prakashan, Bombay, pp. 845 - 846]. Evaluation of different therapeutic potentials like the hypoglycaemic [Mukherjee, P. K., Pal,-S. K., Saha, K., and Saha, B. P., Phytotherapy Research, 1995, Vol. 9, pp. 522 - 524], antibacterial rMukherjee, P. K:₅ Balasubramaniam, R., Saha K., Pal, M. and Saha B. P., Indian Drugs, 1995, Vol. 32(6), pρ.274 - 276], antifungal [Mukherjee, P. K., Giri, S. N., Saha, K., Pal, M., and Saha, B. P., Indian J. Microbiology, 1995, Vol. 35(4), pp.327 - 330], diuretic [Mukherjee, P. K., Das, J„ Saha K., Pal, M., and Saha, B. P., Phytotherapy Research, 1996, Vo. 10, pp. 424 - 425], psychopharmacological [Mukherjee, P. K., Balasubramaniam, R., Pal, M. and Saha, B. P., Journal of Ethnopharmacology, 1996,Vol. 54 (2,3), 63 - 67], antipyretic [Mukherjee, P. K., Saha K., Das, J., Giri, S. N., Pal, M. and Saha, B. P., Indian J. Experimental Biology, 1996, Vol. 82(6), pp. 274 - 276], anti-diarrhoeal [Mukherjee, P. K., Das, J., Balasubramaniam, R., Saha K., Pal, M. and Saha, B. P., Indian J. Pharmacology, 1995, Vol. 27, pp. 262 - 264] effects of the rhizome N. nucifera have been investigated.

Betulinic acid has been isolated and identified from several plant species. The hexane extract of Picramnia pentandra showed the presence of 3 - epi-betulinic acid [Herz, W., Santhanam, P. S., and Wahlberg, I., (1972), Phytochemistry, Vol. 11, pp. 3061 - 3063]. The plants Lemaireocereus stellatus, L. pruinosus, Machaereocereus eruca have been found to contain oleanolic acid, betulinic acid and machaeric acid (21 - keto-oleanolic acid) [Djerassi, C, Liu, L. H., Farkar, E., Lippman, A. E., Lennin, A. J., McDonald, R. N., and Taylor, B. J., (1995), J. Am. Chem. Soc, Vol. 77, pp. 1200 - 1203]. 3-epi-betulinic acid along with three other triterpene glycosides were isolated from leaves of Schefflera cotophylla (Lour) Harms (Family - Araliaceae) [Sung, T. V., Steglich, W., and Adam, G., (1991), Phytochemistry, Vol. 30(7), pp. 2349 - 2356]. Oleanolic acid, betulin, betulinic acid, erythrodiol, maniladiol and several other triterpenes have been isolated from cactus species along with some other cactus triterpenes such as longispinogenin, gummosogenin and cochalic acid, all belonging to the Cactaceae family. Oleanolic acid and betulinic acid have also been isolated from several plants of the Lemaireocereus specie such as Lemaireocereus quovedonis, L. griseus, L. hystrix, L. treleasei. The occurrence of betulinic acid or betulin in Cactus ox Lemaireocereus speceis is not surprising in view of their evident structural similarity to the cactus triterpenes thurberogenin and stellatogenin [Djerassi, C, Bowers, A., Burstein, S., Estrada, H., Grossman, J., Herran, J., Lennin, A. J., Manjarraz, A., and Pakrashi, S. C, (1956), J. Am. Chem. Soc. Vol. 78, pp. 2312 - 2315]. Seeds of Diospyros montana Roxb produce betulinic acid from the n-hexane soluble fraction of its ethanolic extract [Misra, G., Nigam, S. K., and Mitra C. R., (1972), Phytochemistry, Vol. 11, pp. 1508 - 1509], Bark of Arbutus menziesii (Family - Ericaceae) produce betulinic acid from the acetic acid insoluble solid of the alkaloidal extractives produced from this plant [Robinson, F. P., and Martel, H., (1970), Phytochemistry, Vol. 9, pp. 907 - 909]. The extractives of the barks and leaves of Alphitonia excelsa, A. petriel and A. whitei and of the wood, bark and leaves of A. macrocarpa are reported to possess triterpenes like betulinic acid, ceanothic acid and alphitolic acid [Branch, G. B., Buragers, D. V., Dunstan, P. J., Foo, L. Y., Green, G. H., Mack, J. P. G., Ritchie, E, and Taylor, W. C, (1972), Aust. J. Chem., Vol. 25, pp. 2209 - 2216]. The bark of Akania luceus (commonly known as 'turnip wood') has been found to contain betulinic acid, betulonic acid and 3— desoxy betulonic acid [Krajniak, E., Ritchie, E., and Taylor, W. C, (1969), Aust. J. Chem., Vol. 22, ppl331 - 1332]. The trunk wood of Caraipa grandifolia Mart (Family - Guttiferae) contains sitosterol, lupeol, lupenone, betulinic acid and vanillin. The trunk wood of C. densiflora Mart contains sitosterol, lupeol, friedelin, betulinic acid and vanillin [Alves De Lima, R., and Gottlieb, O. R., (1972), Phytochemistry, Vol. 11, pp. 2307 - 2309]. Betulinic acid and ursolic acid have been isolated from Hedyotis chrystoricha (Palib) Merr [Fang, Z., Yang, Y., and Zhou, G., (1992), Merr. Chug. Kuo Chung Y o Tsa Chih, Vol. 17(2), pp. 98 - 100].

Betulinic acid a steroidal triterpenoid has been found to possess several pharmacological properties. The cytotoxic activity of betulimc acid and other ellagic acid derivatives from Agrostistachys hookery has been reported [Choi, Y. N., Pezzuto, J. M., Kinghorn, A. D., and Farnsworth, N. R., (1988), Planta Medica, Vol. 54(6), pp. 511 - 513]. Topical application of lμg of 12 - O - tetradecanoyl phorbol -.13 - acetate (TPA) to the ears of mice was shown to induce oedema and this TPA induced inflammation was inhibited by 4 - methyl sterol and triterpene derivatives. The inhibitory effects of sterol and triterpene derivatives including betulinic acid on TPA - induced inflammation roughly paralleled their inhibitory activities against tumor promotion [Yasukawa, K., Takidom, M., Matsu oto, T., Takeuchi, M., Nakagawa, S., (1991), Oncology, Vol. 48(1), pp. 72 - 76].

Betulinic acid and platonic acid isolated from the leaves Syzigium calviforum were found to be inhibitors of HIV replication in H9 lymphocyte cells. Evaluation of anti-HTV activity with eight derivatives of betulinic acid revealed that dihydro-betulinic acid was also a potent inhibitor of HTV replication; the C-3 hydroxy group of C-17 carboxylic acid group, as well as the C-19 substituents, contribute to enhance the anti-HTV activity [Fujioka, T., Kashiwada, Y., Lee, K. H, (1994), J. Natural Product, Vol. 57(2), pp. 243 - 247]. A series of triterpene compounds including betulinic acid characterized by a straight structure activity related relationship were identified as potent and selective inhibitors or human immunodeficiency virus type 1 (HTV - 1) replication. The betulinic acid derivatives showed 50 % inhibitory concentrations (IC 50) against HTV - 1 strain HI B/LAI in the 10 nM range in several cellular infection assayed, but were inactive against HTV - 2. These compounds did not significantly inhibit the in vitro activities of several purified HTV - 1 enzymes; rather they appeared to block virus infection at a post-binding, envelope dependant step involved in the fusion of the virus to the cell membrane [Mayaux, J. F., Bousseall, A., Pauwels, R., Huet, T., and Henin, Y., (1994), Proc. Natl. Acad, Sc , Vol. 91, pp. 3564 - 3568]. Steroidal anti- inflammatory activity of triterpenoids has also been studied. These triterpenes were isolated from Diospyros leucomelas and identified as betulin, betulinic acid and ursoic acid. They showed anti-inflammatory activity in the carrageenan and serotonin paw oedema test and TPA and EPP ear oedema tests [Recio, M. C, Giner, R. M., Manez, S., Gueno, J., Julien, H. R., Hostet mann, K., and Rios, J. L., (1995), Planta Medica, Vol. 61, pp. 9 - 12].

Plant constituents such as taxol, camptothecin, podophyllotoxin, vinblastine and vincristine represent some of the most important drugs currently used for the treatment of human cancers. They are structurally complex and capable of facilitating unique mechanisms of cytotoxicity like necrosis or apoptosis. These lead compounds have been used as models for development of anti-cancer chemotherapeutic drugs. Betulinic acid isolated from the stem bark of Ziziphus mauritiana Lam., (Rhamnaceae) (white birch trees) has been found to be active as a selective inhibitor of human melanoma which acts by induction of apoptosis as a melanoma specific cytotoxic agent [Pisha, E., Chai, H., Lee, I.S., Chagwedera, T. E., Farnsworth, N. R., Cordell, G. A, Beecher, C. W. W., Fong, H. H. S., Kinghorn, A. D., Brown, D. M., Wani, M. C, Wall, M. E., Hieken, T. J., Dasgupta, T. K., and Pezzuto, J. M., (1995), Nature Medicine, Vol. 1(10), pp. 1046 - 1051].

It is therefore important to develop processes for the isolation of triterpenes such as betulinic acid from new and easily available natural sources in a cost effective manner in view of its high utility. Objects of the invention

The main object of the invention is to provide a process for the isolation of betulinic acid useful as anti-HTV, anti-tumoral and melanoma specific cytotoxic agent.

It is another object of the present invention to provide a process for the isolation of betulinic acid from Nelumbo nucifera Gaertn (Family - Nymphaeaceae) in good yield (0.60% w/w with respect to the dry powdered material) and in a cost effective manner. Summary of the invention

It has been found that the rhizome of the plant N. nucifera contains a substantial amount of the pentacyclic triterpenoid, betulinic acid of the general formula

This can be prepared in good yield in an environmentally acceptable manner using a soxhlet extractor and column chromatography and crystallization technique and characterized thereafter. While other parts of the plant were also studied it was observed that the presence of betulinic acid in such parts was minimal or absent.

While different alkaloids and glycosides like nelumbin, nuciferine, nor-nuciferine has been reported from the leaves, flowers and stalks of this plant, there is no report on the chemical constituents of the rhizome so far. The rhizome has been found to contain a good quality of starch, which has potential binding and disintegrating property which can be effectively used in tablet technology [Mukherjee, P. K., Giri, S. N., Saha, K., Pal, M., and Saha, B. P., (1995) Indian Drugs, Vol. 32(8), pp. 392 - 397; Mukherjee, P. K., Giri, S. N., Saha, K., Pal, M., and Saha, B. P., (1996), Indian J. Pharmaceutical Science, Vol. 58(2), pp. 59 - 66]. While proceeding with its chemical identity the methanolic extract of N. nucifera has been found to contain a steroidal triterpenoid, betulinic acid.

The identity of the isolated compound was decided by comparing its spectral data (UV, IR, 1HΝMR, Mass) to values reported earlier [Davy, G. S., Halsall, T. G., and Jones, E. R. H, (1951), J. Chem. Soc, pp. 2696 - 2702; Djerassi, C, Bowers, A, Burstein, S., Estrada, H, Grossman, J., Herran, J., Lennin, A. J., Manjarraz, A., and Pakrashi, S. C, (1956), J. Am. Chem. Soc, Vol. 78, pp. 2312 - 2315; Sung, T. V., Steglich, W., and Adam, G., (1991), Phytochemistry, Vol. 30(7), pp. 2349 - 2356; Misra, G., Νigam, S. K., and Mitra C. R., (1972), Phytochemistry, Vol. 11, pp. 1508 - 1509; Pakrashi, S. C, Bhattacharya, J., Mookherjee, S., and Samanta, T. B., (1968), Phytochemistry, Vol. 7, pp. 461 - 466], TLC comparison with authentic sample, and also by observing the mixed melting point with authentic sample. Other parts of N. nucifera such as leaves, stalks, flowers and seeds were subjected to phyt^'ochemical methods for the isolation of betulinic acid with little success. It was noted that the rhizome showed the presence of substantial amounts of betulinic acid.

Accordingly, the present invention provides a process for the isolation of betulinic acid which comprises extracting rhizomes of Nelumbo nucifera Gaertn (Family - Nymphaeaceae) with a polar solvent at a temperature in the range of 50 - 60°C by conventional soxhlet extraction methods, concentrating the extracts at a pressure in the range of 30 - 50 mm Hg, adding water to the above said concentrate residue, extracting the said residue successively with non-polar solvents, extracting the aqueous phase with organic solvent, concentrating the above obtained organic solvent phase and purifying the product by conventional methods such as column chromatography followed by crystallization to give pure betulinic acid.

In one embodiment of the invention, the polar solvents used for extraction are selected from the group consisting of methanol, ethanol, butanol, propanol, dimethyl formamide and dimethyl sulphoxide.

In another embodiment of the invention, the non-polar solvent used is selected from petroleum ether (b.p. 40 - 60°C), petroleum ether (b.p. 60 - 80°C), carbon tetrachloride, heptane and benzene. In another embodiment of the invention, the organic solvent used for the final extraction are selected from the group consisting of chloroform, methanol and any mixture thereof.

Detailed description of the invention Plant material:

The rhizomes fo the plant N. nucifera Gaertn were collected form Sandhipur, Midnapore district of West Bengal, India. Taxonomic identification of the plant was performed by Dr. D. C. S, Raju of Botanical Survey of India, Shibpur, Howrah. A voucher specimen (N-21) is being maintained in the laboratory for future reference. The rhizomes were sliced into small pieces, dried, pulverized by a mechanical grinder, passed through a 40 mesh sieve and stored in a well closed container for extraction. Extraction and isolation;

The dried powder rhizome of the plant was extracted with methanol in a soxhlet extractor. After evaporation of the solvent from the extract, a brown coloured dry mass was obtained which was taken in a small amount of water and extracted successively with petroleum ether (b.p. 60 - 80°C) and benzene. The aqueous phase of the benzene extract was acidified using 2M sulphuric acid upto pH 5 - 5.5 and extracted with chloroform. This was concentrated under vacuum to dryness and subjected to chromatographic separation of silica gel (60 - 120 mesh) column (20 x 5.5 cm). Elution with the solvent system chloroform: methanol (1:1) afforded a light yellow coloured fraction which when kept under refrigeration showed crystalline precipitate. This was recrystallised from methanol to provide a white needle shaped crystalline material in yields of 0.60 % w/w with respect to the dry powdered rhizomes.

The dried powdered rhizomes was again extracted with ethyl alcohol (95 % v/v) and same procedure was adopted. The amount of betulinic acid obtained was relatively less. The dried powdered rhizomes was also extracted with petroleum ether (b.p. 40 - 60°C and 60 - 80°C), benzene, n-hexane, propanol, butanol, separately concentrated under reduced pressure and the concentrated mass was subjected to column chromatographic separation as described above. Pentacyclic triterpene like betulinic acid was not separated by these methods.

The crystalline compound obtained from the methanolic extract of N. nucifera rhizome has melting point: 276 - 278°C, UV γ_max nm: 223, 420; IR λ_ma_xcm^"1: 1692 (>C = 0), 3452 (OH), 2940, 1644 and 885 (terminal methylene group); 'HΝMR (IOOMHZ, CdCL₃, TMS as international standard): δ 4.77 (IH, d, j = 1.5 Hz, H - 29b), 4.60 (IH, d, j = 1.5 Hz, H - 29a), 3.39 (IH, d, j = 10.8 Hz, H- 27b), 3.00 (H, dd,j = 5.4, 4.3 Hz, H- 3), 1.7 (3H, s, H- 30). The NMR spectrum showed a 15 proton multiplet ranging from τ 8.77 - 9.16 which accounts for five methyl groups. The methyl group of the isopropenyl function resonates as a triplet centered at τ 8.18 and the proton on the hydroxyl bearing carbon resonates as a triplet centered at τ 6.53 suggesting that this carbon is adjacent to a single methylene group. Mass spectrum of the isolated substance showed principal peaks at m/z 456 (M), 438 (m - 18), 423 (M-33), 406 (M-50) and the peaks in the mass range of 190 - 250 were m/z 248, 220, 219, 207, 203 and 1S9. [ ]²³ + 7.9° (c. 0.057, pyridine), The IR, NMR, and mass spectra of the isolated compound were identical with those of an authenticated sample of betulinic acid (found: C, 78.90; H, 10.53% Calc. for C30H48O3: C, 78.90; H, 10.59%).

From the structure of betulinic acid as provided above as formula I, it is clear that betulinic acid, the therapeutically active pentacyclic triterpenoid embodying the invention is shown by way of illustration only and not as a limitation of the invention. The principal features of the invention may be employed in respect of various other plant species for isolation of the pentacyclic triterpene betulinic acid without departing from the scope of the invention.

The steps involved in the isolation of betulinic acid from the rhizome of the N. nucifera are given below:

Rhizomes were collected, dried under shade, pulverized by mechanical grinder and passed through a 40 mesh sieve.

I

Powdered rhizomes were extracted with methanol (b.p. 65°C) in a soxhlet extractor for successive 48 hours with constant supply of heat (temperature 60 - 65°C) by a heating mantle.

I After complete extraction, the extract so obtained was concentrated under reduced pressure (30mm Hg) and at a temperature of 35°C, pH of which was adjusted to 5 by addition of 2M sulphuric acid. i The extract was successively extracted with petroleum ether and benzene.

1

The aqueous phase (pH 5.5 - 6) was further extracted with chloroform.

I The chloroform extract was concentrated under vacuum to 1/10^th of the original extract volume.

I

This extract was subjected to column chromatographic separation over silica gel (60 - 120 mesh) column and eluted with a solvent system comprising chloroform:methanol (1:1).

I

Light yellow coloured eluant separated from column and was refrigerated (4°C) for 24 hours.

I

Crystalline precipitate separated out which was filtered through Whatman filter paper No. 1, and dried in air.

The product was recrystallised from methanol by treating with activated charcoal.

I

White needle shaped crystalline material separated out.

The white needle shaped crystalline material was characterized by studying its different physico-chemical characteristics such as melting point, UV, IR, NMR, mass spectral characteristics and compared with values reported in the art and with an authenticated sample of betulinic acid. The data showed the compound to be betulinic acid.

The following examples are illustrative and should not be construed to limit the scope of the invention. Example 1

200 gm of powdered rhizome of N. nucifera was extracted with methanol for 48 hours in a soxhlet extractor at a temperature of 60°C. The methanol extract was concentrated under reduced pressure, mixed with 10 ml of water and extracted successively with petroleum ether (b. p. 60 - 80°C) and benzene. The aqueous phase after benzene extraction was further extracted with chloroform and this chloroform extract was concentrated under reduced pressure. This extract was passed through a silica gel (60 -120 mesh) column (20 x 5.5 cm) and eluted with a solvent system chloroforrmmethanol (1:1). A yellowish coloured compound eluted out which after collection was concentrated to half its volume and refrigerated for 24 hours at.4°C. A yellow coloured crystalline compound separated out which was filtered through a Whatman filter paper No. 1 and recrystallised from methanol after treatment with activated charcoal. The recrystallised material was found to be a white needle shaped crystalline material which when subjected to study of characteristics such as UV, TR, NMR, mass spectra characteristics and compared with values for an authenticated sample of betulinic acid was identified as betulinic acid (C₃₀H₄8O₃). 200 gm of powdered rhizome yielded 1.125 gm of isolated crystalline betulinic acid. Example 2

400 gm of powdered rhizome of N. nucifera was extracted with methanol for 48 hours in a soxhlet extractor at a temperature of 60°C. The methanol extract was concentrated under reduced pressure, mixed with 10 ml of water and extracted successively with petroleum ether (b. p. 60 - 80°C) and benzene. The aqueous phase after benzene extraction was further extracted with chloroform and this chloroform extract was concentrated under reduced pressure. This extract was passed through a silica gel (60 -120 mesh) column (20 x 5.5 cm) and eluted with a solvent system chloroform:methanol (1:1). A yellowish coloured compound eluted out which after collection was concentrated to half its volume and refrigerated for 24 hours at 4°C. A yellow coloured crystalline compound separated out which was filtered through a Whatman filter paper No. 1 and recrystallised from methanol after treatment with activated charcoal. The recrystallised material was found to be a white needle shaped crystalline material which when subjected to study of characteristics such as UV, R, NMR, mass spectra characteristics and compared with values for an authenticated sample of betulinic acid was identified as betulinic acid (C30H48O3). 400 gm of powdered rhizome yielded 2.90 gm of isolated crystalline betulinic acid. Example 3

1000 gm of powdered rhizome of N. nucifera was extracted with methanol for 48 hours in a soxhlet extractor at a temperature of 60°C. The methanol extract was concentrated under reduced pressure, mixed with 10 ml of water and extracted successively with petroleum ether (b. p. 60 - 80°C) and benzene. The aqueous phase after benzene extraction was further extracted with chloroform and this chloroform extract was concentrated under reduced pressure. This extract was passed through a silica gel (60 -120 mesh) column (20 x 5.5 cm) and eluted with a solvent system chloroform:methanol (1:1). A yellowish coloured compound eluted out which after collection was concentrated to half its volume and refrigerated for 24 hours at 4°C. A yellow coloured crystalline compound separated out which was filtered through a Whatman filter paper No. 1 and recrystallised from methanol after treatment with activated charcoal. The recrystallised material was found to be a white needle shaped crystalline material which when subjected to study of characteristics such as UV, TR, NMR, mass spectra characteristics and compared with values for an authenticated sample of betulinic acid was identified as betulinic acid (C30H48O3). 1000 gm of powdered rhizome yielded 6.25 gm of isolated crystalline betulinic acid.

The process of the present invention provides a therapeutically effective pentacyclic triterpene betulinic acid in good yield (0.53 - 0.60 % w/w with respect to dry powdered rhizomes). This compound is reported to have potent anti-HTV, anti-tumoral and human melanoma specific cytotoxic acitivity. Advantages of the invention

1. A pentacyclic triterpenoid betulinic acid is isolated from a commonly occurring plant N. nucifera Gaertn (Family - Nymphaeaceae).

2. The process of the invention provides a good yield and is economical.

3. The process of the invention is useful for the isolation of compounds similar to betulinic acid from the vast resources of medicinal plants in India.

Claims

We claim:

1. A process for the isolation of betulinic acid comprising extracting rhizomes of Nelumbo nucifera Gaertn (Family - Nymphaeaceae) with a polar solvent at a temperature in the range of 50 - 60°C by conventional soxhlet extraction methods, concentrating the extract so obtained at a pressure in the range of 30 - 50 mm Hg, adding water to the concentrated residue, extracting the said residue successively with non-polar solvents, extracting the aqueous phase with organic solvent, concentrating the above obtained organic solvent phase and purifying the product by conventional methods such as column chromatography followed by crystallization to give pure betulinic acid.

2. A process as claimed in claim 1 wherein the polar solvent used for extraction is selected from the group consisting of methanol, ethanol, butanol, propanol, dimethyl formamide and dimethyl sulphoxide.

3. A process as claimed in claim 1 wherein the non-polar solvent used is selected from petroleum ether (b.p. 40 - 60°C), petroleum ether (b.p. 60 - 80°C), carbon tetrachloride, heptane and benzene.

4. A process as claimed in claim 1 wherein the organic solvent used for the final extraction is selected from the group consisting of chloroform, methanol and any mixture thereof.