Extraction of Solasodine from Dry Fruits and Leaves of Solanum laciniatum Ait. and the Synthesis of 16-Dehydropregnenolone Acetate from Solasodine by Phase-Transfer Catalysis

A simple extraction and isolation process of solasodine, a natural precursor to synthesize steroidal drugs, from fruits and leaves of Solanum laciniatum Ait. was developed. The optimum concentration of 2-propanol for the extraction of crude glycosides was 70%. The suitable hydrolysis condition of solasodine from crude glycosides was by 1 N hydrochloric acid in 2-propanol. Pure solasodine from both fruits and leaves of Solanum laciniatum Ait. was obtained without any requirement of column chromatography. The yield of pure solasodine were 0.34±0.04% and 0.44±0.16% of the dry weight of fruits and leaves, respectively. The maximum yield of 37.0% of pure 16-DPA was obtained by using tetrabutylammonium hydrogen sulfate as a phase-transfer catalyst and potassium dichromate as an oxidizing agent. The results indicated the novel economic with environmental friendly method of solasodine extraction and synthesis of 16-dehydropregnenolone acetate from solasodine by phase-transfer catalysis. INTRODUCTION Solasodine (1, Fig. 1), the nitrogen analogue of diosgenin (2, Fig. 1), has been reported as a valuable steroidal precursor for the supplementary source of the commercial synthesis of several steroidal drugs (Rodriguez et al., 1979; Sree et al., 1982). It could be primarily obtained from various plants of genus Solanum (Crabbe and Fryer, 1982). Solasodine exists in many forms of glycoalkaloids, which occur mainly as triosides such as, solasonine (3, Fig. 1) and solamargine (4, Fig. 1) (Crabbe and Fryer, 1982). Several isolation procedures of solasodine have been reported from berries, leaves or stems of S. khasianum (Sree et al., 1982), S. marginatum L. (Guerrero, 1976), S. asperum Vahl. (Bhattacharyya, 1984), S. paludosum Moric (Bhattacharyya, 1984), S. eleagnifolium Cav. (Rodriguez et al., 1979), S. aculestisimum Jacq., S. sisymbriifolium Lam. and S. laciniatum Ait. (Kangaroo apple), an indigenous vegetable plant of Australia and New Zealand (Conner, 1987). S. laciniatum gives high yield of solasodine and can be grown well in other parts of the world such as Thailand. The isolation and purification methods of solasodine from Solanum spp. were different in detail techniques. In general, isolation of solasodine from either dry or fresh plants can be performed by extraction and followed by chemical hydrolysis or by extraction in concomitant with chemical hydrolysis as a one-step production (Guerrero, 1976; Bhattacharyya, 1984, 1989). Hydrolysis has also been performed by microbial cells (Rodriguez et al., 1979). To obtain solasodine in pure form is complicate since this plant contains considerable amount of other materials, which can also be co-extracted (Guerrero, 1976). Several methods have been reported to remove these impurities. However, most of them were complicated and require toxic organic solvents. Column chromatography is sometimes used but can cause high cost as well as product loss and time consuming. Proc. WOCMAP III, Vol. 5: Quality, Efficacy, Safety, Processing & Trade in MAPs Eds. E. Brovelli, S. Chansakaow, D. Farias, T. Hongratanaworakit, M. Botero Omary, S. Vejabhikul, L.E. Craker and Z.E. Gardner Acta Hort. 679, ISHS 2005 106 Solasodine can be readily converted to 16-dehydropregnenolone acetate (16-DPA, 5, Fig. 1), an important intermediate and convenient precursor for the production of various steroidal drugs such as sex hormones, corticosteroids, contraceptive drugs and other steroid-related drugs. Several synthesis methods of solasodine to 16-DPA have been developed. The general steps involved acetylation of solasodine to O,Ndiacetylsolasodine; transformation by isomerisation to O,N-diacetylpseudosolasodine; oxidation to give 16-(acetylamino-α-methyl-valeryloxy)-pregnenolone acetate and hydrolysis of the compound to 16-DPA. Intermediates in each step may be either isolated or continued to the next step. Most of these methods are expensive and environmental harmful from the chemical used, especially the conventional chromium catalysis in the oxidation step. In this study, a novel simple isolation and purification process of solasodine from fruits and leaves of S. laciniatum, cultivated in Chiang Mai, Thailand, and a novel method for 16-DPA synthesis from solasodine by phase-transfer catalysis (PTC) will be developed. MATERIALS AND METHODS Materials Fruits (code No. 160695-3P) and leaves (code No. G1090395) of S. laciniatum at the age of 3.5 months grown in Bann Huay Sai District, Chiang Mai, Thailand were collected, dried in an oven at 60°C and ground into powder by a simple grooved-disc mill. The reference standards of solasodine, 16-DPA, and other chemicals were purchased from Sigma Co. (St. Louis, MO, USA). All solvents for chromatographic purposes were of HPLC grade. Other solvents were reagent grade. Isolation of Solasodine 1. Effects of 2-Propanol Concentrations on Crude Glycoside Extraction. The dried leaf powder (500 g) was extracted 4-5 times by 0 to 100% v/v of 2-propanol (1.5 dm each) at 70°C in an Erlenmeyer flask. The filtrates were pooled and vacuum evaporated using the rotary evaporator (Buchi, Switzerland). The filtrate was heated to 80°C and stirred while adding 30% aqueous ammonia solution until the pH reached 9-10. The formed precipitate was collected and the crude glycosides were hydrolyzed by 1 N hydrochloric acid in 2-propanol in a round-bottom flask for 3 h. The 20% sodium hydroxide solution was added to precipitate the crude solasodine. The pure solasodine was obtained by crystallization in methanol. 2. Comparison of Various Methods for Hydrolysis of Glycosides. The dried leaf powder (1 kg) was extracted by the same procedure as in 1., but using distilled water instead of 2-propanol. The crude glycosides were divided into four portions and hydrolyzed by four methods which were electrolysis, 1 N hydrochloric acid in aqueous, ethanol and 2-propanol. For electrolysis method, the aqueous extract (1.5 dm) in 0.02 N hydrochloric acid was applied with a DC current (30 A) for 2 h using 2 pairs of aluminum plate electrodes (10×10 cm) in a 3 dm-tank. 3. Comparison of Solasodine