Triterpene glycosides from the tubers of Leontice smirnowii

Abstract

Leontice smirnowii (Trautv.) (Berberidaceae) is an endemic plant of Georgia [1]. Its tubers have been used in folk medicine as an antitubercular remedy [2]. The occurrence of triterpene saponins and alkaloids in the plant as well as the biological activity of the crude extract and monodesmoside fraction has been previously reported [3–7]. The tubers of Leontice smirnowii were collected in Lagodekhi region of Georgia (May, 2005) and dried in the shade. A voucher specimen is deposited in the department of Pharmacobotany, Institute of Pharmacochemistry, Georgia (tubers No. 95343). Dried and powdered tubers of L. smirnowii (1 kg) were extracted with 80% MeOH (7 L). The extract was concentrated under vacuum. After evaporation of solvent the dry extract (298 g) was disolved in MeOH (900 mL) and precipitated in acetone (5 L). The precipitate was filtered and dried to obtain a crude saponin fraction (205 g). Forty grams of crude saponins were subjected to low-pressure liquid chromatography (LPLC) (ChromatoSPAC Prep 100, Lichroprep C-18, 15–25 μm) by using reversed phase RP-18 and eluted with MeOH–H2O (10% to 80% of MeOH) to afford four fractions. The obtained fractions were subjected to column chromatography on silica gel (0.04–0.063 mm, Merck) and eluted with CHCl3–MeOH–H2O (26:14:3) to give the individual compounds: 1 (90 mg), 2 (45 mg), 3 (80 mg), 4 (60 mg). The structures of the glycosides 1–4 were established on the basis of acid hydrolysis and NMR data. Leonticin D (1) was obtained as an amorphous solid with a molecular peak at m/z 865 [M-1]+, indicating a mol. wt. of 866 daltons. Acid hydrolysis of 1 yielded arabinose, glucose, and xylose as the sugar components identified on TLC by comparison with authentic samples. The 1H and 13C NMR data of 1 suggested the presence of α-arabinopyranosyl, β-xylopyranosyl, and β-glucopyranosyl moieties, as shown by three anomeric proton signals at δ 4.37 (d, J = 7.6 Hz), 4.66 (d, J = 7.7 Hz), and 4.58 (d, J = 7.6 Hz), as well as the corresponding anomeric carbons at δ 106.04, 104.86, and 105.05. The 13C NMR spectrum showed that the aglycone of 1 contained 29 carbon atoms out of 63 and consisted of five methyls, eleven methylenes, five methines, and eight quaternary carbons as determined from a DEPT experiment. The 1H NMR spectrum of 1 showed signals for five tertiary methyl groups at δ 1.18, 1.04, 0.95, 0.84, and 0.79, an exomethylene group at δ 4.63 and 4.62 (each br.s), and an olefinic proton at δ 5.32 (br.t). Compound 1 was clearly shown to have two unsaturations: one endocyclic between C-12 (δ 124.32) and C-13 (δ 144.10), while signals at δ 149.28 and δ 107.58 were assigned to an exocyclic double bond involving C-20 and C-29. The 20(29)-exomethylene group was ascertained by long-range correlations from the exomethylene proton signals to the methylene carbons C-19 and C-21, in the heteronuclear multiple-bond connectivity (HMBC) spectrum of 1. The above spectral information allowed to us to establish the structure of aglycone 1 as 3β-hydroxy-30-norolean-12,20(29)-dien-28-oic acid, in agreement with literature data [8]. The aglycone 3β-hydroxy-30-nor-olean-12,20(29)dien-28-oic acid was described for the first time in Leontice species. The HMBC spectrum showed correlations between C-3 (d 91.10) of aglycone and H-1 (δ 4.37) of the arabinose (Ara), and between C-2 (δ 77.53) and C-3 (δ 84.04) of the arabinose and H-1 (δ 4.66) of the xylose (Xyl) and H-1 (δ 4.58) of the glucose (Glc), respectively. Based upon the above observations the structure of 1 was elucidated as 3-O-β-D-glucopyranosyl-(1→3)-[β-Dxylopyranosyl-(1→2)]-α-L-arabinopyranosyl-3-hydroxy-30-nor-olean-12,20(29)-dien-28-oic acid, which was previously reported as trifoside B [9].