Triterpenoid Saponins from Stauntonia chinensis

Experimental

Plant Materials. The stems of Stauntonia chinensis were collected from NanNing, Guangxi Zhuang Autonomous Region, P. R. China and identified by associate chief pharmacist Jin-Wei Huang at Guangxi Institute of Minority Medicine. The voucher specimen (20090801) was deposited with the Herbarium of College of Pharmacy, South Central University for Nationalities.

Extraction and Isolation. The stems of Stauntonia chinensis (16 kg) were extracted with 60% EtOH three times and then successively partitioned with EtOAc and n-BuOH. The extract of n-BuOH (300 g) was chromatographed on silica gel with CHCl3-MeOH (100:0, 98:2, 95:5, 9:1, 8:2, 7:3, 1:1, 3:7, 0:100 v/v) to give 15 fractions (fr.1-fr.15). Fr. 10 (9.6 g) was subjected to CC on silica gel with CHCl3-MeOH (1:0, 99:1, 95:5, 9:1, 0:1) and further purified by octadecylsilane CC with H2O/MeOH (95:5→0:1) to obtain 3 (10.3 mg). Fr.12 (30 g) was subjected to CC on silica gel with EtOAc-EtOH (1:0, 98:2, 95:5, 9:1, 8:2, 7:3, 0:1) to afford compounds 2 (13.0 mg), 6 (8.1 mg), 7 (15 mg), 8 (15.0 mg). Fr. 13 (45 g) was subjected to CC on silica gel with EtOAc-EtOH (1:0, 98:2, 95:5, 9:1, 8:2, 7:3, 0:1) to give five fractions (Frs. 13.1-13.5). Fr. 13.2 (3.6 g) was successively separated by CC (ODS; H2O/MeOH; (95:5→0:1)) to give compound 1 (29 mg). Fr. 13.2.3 (314.8 mg) was successively separated by CC (ODS; H2O/MeOH; (9:1→0:1)) and further purified by semi-prep. HPLC (CH3CN/H2O 36:74, 2.5 mL/min) to afford compound 5 (9.6 mg; tR 15.7 min) and 10 (18 mg; tR 24.4 min). Fr. 13.5 (14.1 g) was subjected to CC on silica gel with CHCl3-MeOH (99:1, 98:2, 95:5, 9:1, 8:2, 7:3, 6:4, 0:1) and further purified by semi-prep. HPLC (CH3CN/H2O 30:70, 2.5 mL/min) to afford compound 4 (14.3 mg; tR 11.8 min) and 9 (14.0 mg; tR 19.2 min).

Yemuoside YM36 (1): white amorphous powder; [α]D = +42.5 (c 0.48, MeOH); 1H NMR and 13C NMR (in C5D5N) spectroscopic data, see supporting information; HRESIMS m/z 803.4186 [M+Na]+ (calcd. for C41H64O14Na, 803.4194).

Yemuoside YM37 (2):white amorphous powder; [α]D = +40.8 (c 0.50, MeOH); 1H NMR and 13C NMR (in C5D5N) spectroscopic data, see supporting information; HRESIMS m/z 889.4548 [M+Na]+ (calcd. for C45H70O16Na, 889.4562).

Acidic Hydrolysis. Compounds 1-2 (3 mg) were respectively added to trifluoro-acetic acid (TFA, 4 N) solution (6 mL), then heated for 3 h under 90 ºC. After cooled to room temperature, the hydrolytical solution was extracted with chloroform (3 × 6.0 mL). The water layer was concentrated to 0.5 mL. The completely concentrated water layer and reference substances of β-D-glucose, α-ʟ-arabinose and β-Dxylose and α-ʟ-rhamnose were derived by reacting with 1.5 mg hydroxylamine hydrochloride and 0.5 mL pyridine for 1 hourat 90 ºC. After cooling, 0.9 mL Ac2O was added and the mixture was heated at 90 ºC for 1 h. The reaction mixtures were evaporated under reduced pressure, and the resulting aldononitrile peracetates were analyzed by GC-MS. The samples (10 μL) were injected into a HP-1 30 m × 0.2 mm chromatographic column (Agilent, Santa Clara, CA, USA). The tR values of β-D-glucose, β-D-xylose, α-ʟ-arabinose and and α-ʟ-rhamnose derivatives were 11.04, 9.08, 8.98 and 8.86 min, respectively.

Cytotoxic Activity. Cell viability was measured using the MTT assay. The HepG2 cells were cultured in DMEM medium containing heat inactivated 10% (v/v) fetal bovine serum (FBS) and 100 U/mL penicillin, 100 μg/mL streptomycin. The cells were incubated in a humidified atmosphere of 5% CO2 at 37 °C and pass-aged every 3 days by trypsinization (0.25%). For experiments, HepG2 cells were in-cubated in complete medium with 10% fetal bovine serum in 96-well plates (1000 cells/well). Cells were treated with or without different concentrations of saponins (dissolved in DMSO) when the cells reached 70-80% confluence. Test compounds and blank group had four repetitions. After 24 h, the medium was removed and the serum free DMEM containing 10% MTT (5 mg/mL) were added to each wells. 4 hours later, the culture medium containing MTT was removed and 100 μL DMSO was add to each well to dissolve the formazan and placed on a shaker for 10s. The absorbance values was measured at a wavelength of 492 nm using a microplate reader, and the absorbance values were expressed as a percentage of un-treated control cells (control = 100%).