Three New Steroidal Glycosides from the Roots of Cynanchum auriculatum

Three new steroidal glycosides, cyanoauriculosides F, G and H (1-3), were isolated from the roots of Cynanchum auriculatum (Asclepiadaceae) along with two known steroidal derivatives. On the basis of spectroscopic analysis and chemical methods, their structures were identified as 20-O-acetyl-8,14-seco-penupogenin-8-one 3-O-α-L-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranoside (1), 2′,3′-Z-gagaminine 3-O-α-L-cymaropyranosyl-(1→4)-β-D-cymaro-pyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranoside (2), 17-O-acetyl-kidjoranin 3-O-α-L-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-cymaro-pyranosyl-(1→4)-β-D-digitoxopyranosyl-(1→4)-β-D-digitoxopyranoside (3), gagaminine 3-O-α-L-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-digino-pyranosyl-(1→4)-β-D-cymaropyranoside (4) and wilfoside D1N (5).


Introduction
Cynanchum auriculatum is a famous traditional medicine widely used in south China for the prevention of hair graying, strengthening sinews and bones, and enhancing immunity [1]. In previous papers, we reported the isolation of five new C-21 steroidal glycosides, named cyanoauriculosides A-E, OPEN ACCESS from the roots of C. auriculatum [2]. Many C-21 steroidal glycosides isolated from C. auriculatum species have shown certain antitumor activities in vitro [3,4]. In a further phytochemical investigation of traditional Chinese medicinal plants to search for novel biologically active compounds, three new steroidal glycosides named cyanoauriculosides F-H (1-3, Figure 1) were obtained from the roots of Cynanchum auriculatum (Asclepiadaceae), along with two known steroidal derivatives. All the structures were established on the basis of spectroscopic analysis and chemical methods.

Results and Discussion
Compound 1, obtained as a white amorphous powder, showed a positive reaction in the Libermann-Buchard and Keller-Killiani tests, indicating the presence of a steroidal skeleton with a 2-deoxysugar moiety [5] ] + arising from the aglycone moiety. From 1 H-NMR, 13 C-NMR, HSQC and HMBC data, one acetyl group was also identified by the observation of a proton signal at δ H 1.96 (3H, s) and two carbon signals at δ C 170.5, 21.6. Comparison of the 13 C-NMR data of the aglycone portion of 1 with that of penupogenin [6,7], showed that the major difference between the two substances was the presence of an additional acetyl group in 1, and the fact that the chemical shift of C-20 in 1 was deshielded by ca. 6 ppm. This observation suggested that the extra acetyl group was located at C-20, which was supported by HMBC correlation between H-20 (δ H 4.03, m) and C-10′ (δ C 170.5). Additionally, the chemical shifts of 1 are different from those of penupogenin at C-7 (+8.1 ppm), C-9 (+13.2 ppm), C-14 (-7.4 ppm) and C-18 (+8.9 ppm), and the chemical shift of C-8 appears at δ 209.8. It could be further speculated that the hydroxy group at C-8 was oxidized into a carbonyl, which was supported by HMBC correlations between H-14 (δ H 4.95, m) and C-18 (δ C 20.3), and between H-9 (δ H 2.21, m) and C-8 (δ C 209.8). Thus, the aglycone of compound 1 was determined to be 20-O-acetyl-8,14-seco-penupogenin-8-one. In the NOESY spectrum, NOE correlations between H-9 (δ H 2.21, m) and H-12 (δ H 5.17, m) provided evidence for a β-linked 12-O-cinnamoyl group. Based on the literature [8], the stereochemistry of the C-14 hydroxyl group was assigned as β.
With respect to the glycosidic portion, it contained four anomeric C-atoms with signals at δ (C) 96.5 (C1 I ), 101.0 (C1 II ), 99.5 (C1 III ) and 99.1 (C1 IV ), corresponding to anomeric H-atom signals at δ (H) 5.10 (overlap), 5.10 (overlap), 4.95 (overlap) and 5.05 (overlap), which indicated that there were four sugar moieties in 1. Acidic hydrolysis of 1 afforded a sugar mixture of cymarose and diginose, identified by TLC comparison with authentic samples. Comparing the 13 C-NMR spectrum with that of penupogenin showed that the chemical shift of 1 are different from those of penupogenin at C-2 (-2.3 ppm), C-3 (+5.4 ppm) and C-4 (-4.4 ppm), due to glycosidation, therefore the sugar moiety was linked to the C (3)-O of the aglycone. Furthermore, HMBC correlations between H-C (1 I ) at δ (H) 5.10 and C (3) at δ (C) 76.9 were observed. Signals of each sugar unit ( Table 2)  Compound 2, obtained as a white amorphous powder, showed positive reactions in the Libermann-Buchard and Keller-Killiani tests, indicating again the presence of a steroidal skeleton with a 2deoxysugar moiety [5]. Its molecular formula C 64 H 91 NO 20 was deduced from the HRESIMS spectrum (m/z 1,216.6033 [M+Na] + , calcd 1,216.6032). The 1 H-NMR and 13 C-NMR data (Tables 1-2) suggested that 2 was a C-21 steroidal glycoside.    was also found in the aglycone moiety. The 1 H-NMR data of the aglycone portion of 2 was compared with the data of gagaminine [6], showing that the major difference was the coupling constant of H-2′ and H-3′ was 12.0 Hz, so it could be deduced that the relative configuration at C-2′ and C-3′ was cis. Thus, the aglycone of compound 2 was proposed to be 2′,3′-Z-gagaminine. In the NOESY spectrum, NOE correlations between H-9 (δ H 1.71, m) and H-12 (δ H 5.18, m) gave evidence for a 12-O-cinnamoyl group that was β-linked. According to the literature [8], the stereochemistry of the C-14 hydroxyl group was assigned as β. The chemical shifts of C (13) and C (14) appear at δ 56.9 and 88.8, respectively. It could be deduced that the C/D ring junction was cis compared with the same carbons at δ 41.6-42.7 and 58.7-59.2 for the trans form [8]. By comparing the spectroscopic data of the sugar moiety in 2 with those of 1, compound 2 was seen to possess the same sugar substitution pattern as that of 1. Thus, compound 2 was determined to be 2′,3′-Z-gagaminine 3-O-α-L-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranoside, and named cyanoauriculoside G.

General
Column chromatography was carried using silica gel (200-300 mesh), and Thin-Layer Chromatography (TLC) was performed on silica gel GF 254 from the Qingdao Haiyang Chemical Group Co., P. R. China. RP-18 silica gel was purchased from YMC CO., LTD., Japan. NMR spectra were run on a Bruker DRX-500 MHz spectrometer with TMS as internal standard. HRESIMS were measured on Micromass Q-Tof-Ultima mass spectrometer. The optical rotation was measured on a Jasco P-1020 polarimeter. HPLC was performed on an Ultimate 3000 apparatus using 5C18-MS-II column (ODS, 250 × 10 mm, 5 μm) and monitored by an UV detector.

Plant material
The roots of the C. auriculatum were collected from Jishou, Hunan Province, P. R. China, in September 2007, and identified by Prof. Ding-Rong Wan. The voucher specimen (07091201) was deposited in the Herbarium of College of Pharmacy, South Central University for Nationalities.

Acid hydrolysis
A soln. of 1, 2, and 3 (each 5 mg) in MeOH was treated with 0.05 mol/L HCl, 4-dioxane 1:1 (1 mL) at 60 °C for 1.5 h, respectively. After removing dioxane, the soln. was extracted with EtOAc (3 × 2 mL). The aq. layer was neutralized by NaOH and concentrated under reduced pressure to give the sugar fraction. The presence of the monosaccharides in the hydrolysates of each compound was confirmed by TLC comparison with authentic samples. Cymarose was detected from compounds 1-3; diginose was detected from compounds 1 and 2; digitoxose was detected from compound 3. The R f values of di gitoxose, diginose and cymarose were 0.51, 0.66 and 0.76, respectively with CHCl 3 : MeOH (95:5), 0.07, 0.18 and 0.23, respectively with P.E.: Me 2 CO (3:1).     ; For 1 H-NMR and 13 C-NMR spectroscopic data (in C 5 D 5 N), see Table 1

Conclusions
Twenty compounds were isolated from the dry roots C.auriculatum Royle ex Wight, including thirteen C-21 steroidal glycosides. The anti-tumour activity of these C-21 steroidal glycosides compounds has been studied. Further research on isolation and identification of more bioactive compounds will be helpful to understand this traditional medicine.