Steroidal compounds from Helleborus caucasicus leaves

Abstract

Interest in the genus Helleborus has heightened recently because it is a known source of cardiac glycosides of the bufadienolide group. Several new steroidal glycosides, ecdysteroids, representatives of a new class of natural compounds thionines, macrocyclic suboxides, etc. have been isolated from various Helleborus species in addition to bufadienolides. Total preparations of Helleborus have been used in medicine as analgesic, antirheumatic, and other agents. Mainly the subterrean organs of the plant are studied and used. The leaves have been neglected.Of a total of 25 Helleborus species, 2 grow in Georgia, H. caucasicus A. Br. and H. abchasicus A. Br. The former is endemic to the Caucases; the latter, to Georgia. Roots and rhizomes of H. caucasicus and H. abchasicus accumulate an extraordinarily high amount (14-18%) of neutral lipids for subterrean organs. These differ in their chemical composition from typical plant lipids and exhibit a pronounced specific anticancer effect [1].Analysis (LC/MS) of the butanol extract of the MeOH extract of roots and rhizomes of H. caucasicus (HC) detected 20 steroidal compounds. A total of 12 were isolated and characterized, of these 4 were new glycosides called "caucasicosides" [2,3]. Total steroidal compounds of HC exhibited at very low concentrations (2 × 10 -9 g/μL) cytotoxic activity against several malignant tumor cells [4].HC (like other Helleborus species) is a perennial herbaceous rhizomous evergreen plant with large finger-like leaves. The leaf mass is 23-25% of the total weight of the whole plant. They contain a significant quantity of steroids, which prompted us to study the chemical composition of the HC organs that are reproduced yearly, the leaves.Air-dried leaves of HC were extracted with MeOH (80%). The MeOH was distilled off. The aqueous phase was worked up with CHCl 3 and extracted with n-BuOH to afford total glycosides (8-10% of raw material). LC/MS spectral analysis of these detected over 30 steroids (Table 1). Therefore, the qualitative composition of the leaves was richer than that of the subterrean organs of HC.The BuOH fraction (3 g) was fractionated over a column of silica gel (100 × 2.5 cm, 100/160 μm, Merck) with a mobile phase of CHCl 3 :CH 3 OH:H 2 O (26:14:3) under isocratic conditions. This isolated a nonpolar fraction (630 mg), slightly polar (580), and polar (370).The nonpolar fraction was separated by HPLC (515 HPLC, Waters R 590) over an RP XTerra C 18 column (7.8 × 300 mm, LiChroprep RP18, 10 μm, XTerra) using MeOH of various concentrations. This isolated five pure compounds.Compound 1, amorphous white powder, treatment with H 2 SO 4 gave a blue color, fluoresced blue. The PMR spectrum showed resonances characteristic of methyls with δ 0.92 (3H, s, Me-18), 0.99 (3H, s, Me-19), 1.23 (3H, s, Me-21), 1.22 (3H, s, Me-26), and 1.22 (3H, s, Me-27); one olefinic resonance with δ 5.84 (1H, d, H-6); three methine protons with δ 3.87 (1H, m, H-2), 3.98 (1H, m, H-3), and 3.36 (1H, m, H-21), which confirmed that a secondary alcohol was present. The posi...