Chem. Pharm. Bull. 54(10) 1440—1442 (2006)

nongjia Forest District, a National Natural Protection Region of China. Most of them were recorded to be used as anti-inflammatory agents in the folk. Kaikoujian, the Chinese name of rhizomes of Tupistra chinensis BAK., is a reputed folk medicine in that district because of its potent to markedly reduce carbuncles and to ameliorate pharyngitis. In the course of our studies on the native folk medicines of Shennongjia Forest District, a pair of diastereoisomeric steroidal saponins were isolated and their structures were elucidated by means of chemical and spectroscopic methods. Although studies on the lipophilic constituents, including the sapogenins of this plant, were reported in several papers, the steroidal saponins, especially those showing anti-tumor activities, were hardly elucidated yet. This paper reported the isolation and structural elucidation of two steroidal saponins from cytotoxic extracts of this plant. Compounds 1 and 2, two distinctly separate peaks appearing on the analytical HPLC, were separated by means of preparative HPLC and purified by repeated semi-preparative HPLC. 1 was obtained as a colorless powder. Its HR-FABMS showed a quasi-molecular ion peak at m/z 1103.5252 [M Na] , which indicated a formula C51H84O24Na (Calcd: 1103.5250). Positive coloration reactions were observed when 1 was subjected to Ehrlich, Molish and Liebermann– Buchard tests, which suggested that 1 have a steroidal saponin skeleton. H-NMR data of 1 contained an olefinic proton at d 5.28 (br s), a methenyl proton at d 4.98 (m), two doublet methyl groups at d 1.01(d, J 7.0 Hz) and 1.31(d, J 7.0 Hz), two singlet methyl groups at d 0.88 and 0.86 (each s), attributable to a steroidal aglycone moiety. Furthermore, the furostanol glycosidic nature of 1 was suggested by the strong absorption bands at 3432 and 1050 cm 1 in the IR spectrum, and a semiketal carbon signal at d 110.65 in the C-NMR spectrum. The above H-NMR spectral data and a comparison of the C-NMR signals of the aglycone moiety of 1 with those described in the literature showed the structure of the aglycone to be (3b ,22a ,25S)-furost-5-en-3,22,26-triol. The aconfiguration of C-22 hydroxyl group of the aglycone moiety was deduced from the semiketal carbon signal at d 110.65, instead of d 115.5 for b-configuration, and it was further confirmed by the ROESY correlation between H-20 (2.21, dq, J 6.5, 7.5 Hz) and H-23 (1.95, m). The 25S stereochemistry was inferred by the resonance of H-27 methyl protons at d 1.01, a few bigger than 1.00; and also by the resonance difference between equatorial proton signal (3.46, dd, J 6.5, 9.5 Hz) and axial proton signal (4.06, m) of H-26: 0.60 ppm, a few bigger than 0.57 ppm. Yamogenin, an isomer of diosgenin, was obtained from the hydrolysate of compound 1, which further confirmed a 25S configuration for compound 1. When 1 was hydrolyzed with 2.0 M HCl, only glucose was detected in the hydrolysate on TLC and PC. Among the 51 carbon signals in the C-NMR spectrum, 27 signals were assignable to the aglycone, the remaining 24 signals were indicative of the presence of four glucose moieties, in good agreement with the four anomeric proton signals appearing at d 5.21 (d, J 7.5 Hz), 5.12 (d, J 8.0 Hz), 4.89 (d, J 7.5 Hz), and 4.78 (d, J 7.5 Hz), and the corresponding anomeric carbon signals at d 106.92, 105.20, 102.68 and 105.13, respectively. The absence of any glycosidation shift for two b-D-glucopyranosyl moieties which contained anomeric protons at d 4.78 and 5.21 as obtained from HMQC and TOCSY data, suggested that there be at most two sugar chains in compound 1. A sugar chain was determined to be attached to C26 by an observation of glycosidation shift of C-26 signal from d 66.00 to 75.37, and this linkage was further indicated by the cross peak between the carbon-13 signal at d 75.37 and the anomeric proton signal at d 4.78 of b-D-glucopyranosyl moiety in the HMBC spectrum of 1. Similarly, a cross peak between the C-3 signal of aglycone at d 78.13 and the anomeric proton signal at d 4.89 in the HMBC spectrum of 1, together with a correlation between H-3 proton at d 3.98 and the anomeric proton at d 4.89 in the NOESY spectrum of 1, indicated that another sugar chain was located at the C-3 position of aglycone. A 1→4 linkage of the middle sugar moiety to the inner one was revealed by a cross peak between the C-4 signal of the inner glucose at d 81.11 and the anomeric proton signal of the middle glucose at d 5.12 in the HMBC spectrum of 1, and further determined by the correlation between the H-4 proton signal of the inner moiety at d 4.57 and the anomeric proton signal at d 5.12 in the NOESY spectrum of 1. Finally, the terminal sugar residue was assigned to be located at the C-2 position of the middle moiety, which was determined by a correlation between the C-2 signal of the middle moiety at d 86.12 and 1440 Vol. 54, No. 10 Chem. Pharm. Bull. 54(10) 1440—1442 (2006)