A furostanol saponin mixture and a known phytoecdysteroid were isolated from the roots of Helleborus orientalis Lam. Their structures were established as 1) and of 2 gave 20-hydroxy-β-ecdyson (aglycone 2). Their structures were elucidated by spectral analysis.Helleborus extracts exhibit high biological activity with antialgic, myorelaxant, antirheumatic, and blood vessel regulating actions [1]. Previously isolated classes of constituents, furostanol saponin [2][3][4][5], phytoecdysteroids [6-9], and bufadienolides [10], were characterized from Helleborus species.This study describes the isolation and the exact structure determination of two glycosides, a furostanol saponin 1a,b and a phytoecdysteroid 2, from Helleborus orientalis Lam roots.Compounds 1a,b was obtained as a mixture of the C-22 hydroxyl and C-22 methoxyl forms. It gave two spots at TLC due to the existence of 22-hydroxy and 22-methoxy derivatives of the glycoside. Acid hydrolysis of the mixture gave aglycone 1. The 1 HMR spectral data of aglycone 1 contained signals of olefinic protons at 5.62, 4.78, and 4.83 ppm, two methoxyl protons at 3.21 and 3.63 ppm, and methyl groups at 0.96 (21-CH 3 ), 0.79 (18-CH 3 ), and 1.15 ppm (19-CH 3 ). Furthermore, the presence of a semiketal carbon signal at 109.4 ppm suggested the furostanol nature of the aglycone 1. The EI-MS spectrum showed an ion peak at m/z: 491 [M+H] + for C 29 O 6 H 46 . The above 1 H NMR spectral data and a comparison of the 13 C NMR signals (Table 1) and of -85.92 of aglycone 1 with those described in the literature [1,2] showed the structure of the aglycone1 to be (1β,3β,11α,22α)-22,26-dimethoxyfurosta-5,25(27)-dien-1,3,11-triol.The furostanol glycosidic nature of compounds 1a,b was evidenced by the furostan steroidal structure (916 and 833 cm -1 ) in the IR spectra. The 1 H NMR spectral data of the mixture of compounds 1a,b contained a signal for one anomeric proton at 4.24 ppm (d, J = 7.3 Hz, H-1′). The 13 C NMR spectral data (Table 1) contained an anomeric carbon signal at 104.0 ppm. Furthermore, the presence of a semiketal carbon signal at 110.4 and 110.7 ppm suggested the furostanol nature of compounds 1a,b. The peak intensities of these semiketal carbon signals were 0.775 and 0.742, respectively. The ratio of these components (1a and 1b) in the mixture of was 1:1. The negative FAB-MS showed an ion peak at m/z: 571.4 [M-OCH 3 -2H 2 O] -, suggesting the molecular formula as C 34 O 11 H 54 . The structure of 1a was established as 26-[(β-D-glucopyranosyl)oxy]-22α-hydroxyfurosta-5,25(27)-dien-1β,3β,11α-triol and 1b as 26-[(β-D-glucopyranosyl)oxy]-22α-methoxyfurosta-5,25 (27)-dien-1β,3β,11α-triol. In this paper, the NMR data of 1a and 1b are reported for the first time. Compounds 1a,b are presumed to exist as C-22 hydroxyl and C-22 methoxyl derivatives. It is thought that furostanol saponin exists in the form of just 1a in the roots of Helleborus orientalis Lam and that compound 1b may be artifacts of the extraction and isolation procedure. Such interconversion between hydroxyl and methox...