Twelve New Seco-Pregnane Glycosides from Cynanchum taihangense

For our interest in the potential biologically active and structurally unique steroidal glycosides, continued phytochemical investigation of Cynanchum taihangense was carried out; twelve new seco-pregnane glycosides, cynataihosides I–L (1–4), M-T (7–14), and two known glycosides, glaucoside A (5) and atratcynoside F (6), were isolated from the 95% ethanol extract of Cynanchum taihangense. Two new aglycones were found among compounds 10, 11, 13, and 14. The structures of the glycosides were elucidated based on 1D and 2D NMR spectroscopic data, HR-ESI-MS analysis, and chemical evidence. The cytotoxicity of compounds against three human tumor cell lines (HL-60, THP-1, and PC-3) were evaluated by MTT assay. Compound 11 displayed significant cytotoxicity against THP-1 and PC-3 cell line with IC50 values of 5.08 and 22.75 μm, respectively. Compounds 3 and 14 exhibited moderate and selective cytotoxicity on HL-60 and THP-1 with IC50 values of 17.78 and 16.02 μm, respectively.


Results and Discussion
The fresh plants of C. taihangense were extracted with 95% ethanol. After concentration, the water suspension of the extraction was partitioned with petroleum ether, ethyl acetate, and n-butanol, successively. Then, the ethyl acetate and n-butanol extract were subjected to various chromatographic isolation methods, respectively, to give twelve new and two known seco-pregnane glycosides. Their structures and the absolute configurations were elucidated by analysis of 1D/2D NMR spectroscopic data, HR-ESI-MS analysis, and chemical evidence (Supplementary Figures S1-S88).
Cynataihoside Q (11) was white amorphous powder and its positive HR-ESI-MS gave a quasi-molecular ion peak at m/z 655.3096 (calcd, 655.3094) for [M + Na] + ion, based on which the molecular formula was calculated as C 34 H 48 O 11 . A detailed comparison between the 1 H and 13 C NMR data of 11 and those of 10 in Table 2 indicated they had the same aglycone and one β-D-cymaropyranosyl connected with their C-3 hydroxyl group, but different terminal sugar. From the 13 C NMR spectra, the outer deoxysugar units were in good agreement with the terminal β-D-digitoxopyranosyl in sublanceoside B 2 [15]. The HMBC spectrum showed correlations from δ H 5.35 (1H, br d, J = 9.6 Hz, H-1 ) to δ c 82.7 (C-4 ), and from δ H 4.75 (H-3) to δ c 95.5 (C-1 ), which further confirmed the linkage sugar sequence. On acid hydrolysis of 11, cymarose and digitoxose were afforded. Thus, all the above-mentioned evidences with the NOESY spectrum in Figure 3 confirmed 11 as cynataihogenin P 3-O-β-D-cymaropyranosyl-(1→4)-β-D-digitoxopyranoside, and named cynataihoside Q.
The known compounds were identified as glaucoside A [14] (5) and atratcynoside F [6] (6) by comparing their 1 H and 13 C NMR data to those in the literature.

Cytotoxic Activities
The cytotoxic activities of compounds 1-14 against HL-60 (human leukemic promyelocytic cell), THP-1 (human acute monocytic leukemia cell line) and compounds 1, 3, 10-14 against PC-3 (prostate cancer cell line) were evaluated. The cytotoxicity data represented by IC 50 values in µm are shown in Table 3, in which 5-Fluorouracil was used as a positive control and their IC 50 values against HL-60, THP-1, and PC-3 were 9.93, 5.82, and 22.15 µm, respectively. As evident from results, the compounds exhibited varying degrees of cytotoxic activity. Compound 11 was more active than others, especially on THP-1 and PC-3, and showed significant cytotoxicity similar to the positive control. Compounds 3 and 14 moderately and selectively inhibited the proliferation of HL-60 and THP-1 with IC 50 values of 17.78 and 16.02 µm, respectively.

Plant Material
The fresh whole plants (4.7 kg) of C. taihangense were collected in August 2014 at Wangmang Ridge in Shanxi Province, China. A voucher specimen was identified by Prof. Jing-Ming Jia of Shenyang Pharmaceutical University and was deposited in the School of Traditional Chinese Materia Medica of Shenyang Pharmaceutical University (NO. SYPC201408316).

Cell Lines
The human leukemic promyelocytic cell (HL-60), human acute monocytic leukemia cell line (THP-1), and prostate cancer cell line (PC-3) were provided by America Type Culture Collection, ATCC (Rockville, MD, USA).

Extraction and Isolation
The fresh whole plants (4.7 kg) of C. taihangense were extracted with 95% EtOH, and water suspension of the extraction was then partitioned with petroleum ether, ethyl acetate, and n-butanol, successively [11].
The ethyl acetate extract (110 g) was further pre-fractioned by a gel column to give thirteen fractions . Fr. 6 was separated by the same procedures as those in our previous report [11] to afford nine fractions .  was purified via Sephadex LH-20 eluting with MeOH, and then isolated by semi-preparative HPLC eluting with CH 3 OH/H 2 O (67:33 v/v, flowrate 2.5 mL/min) to yield compound