Cynanchum otophyllum Schneid., Qingyangshen in Chinese, is a species of Cynanchum L.(Asclepiadaceae), and a Chinese herbal medicine(CHM)distributed extensively over Southwest China. Pharmacodynamic and clinical experiments have established that the chloroform extract and ethyl acetate extract from the rhizome of C. otophyllum were especially effective against epilepsy and chronic hepatitis(Pei et al, 1981; 1987; Zhou, 1991). Since 1984, Qingyangshen Tablets(containing the total glycosides of C. otophyllum)have been manufactured by Lijiang Pharmaceutical Co., Yunnan Baiyao Group, China. The steroidal constituents from the plants of Cynanchum L. have been reported(Hayashi and Mitsuhashi, 1972). From the rhizome of C. otophyllum, researchers isolated nine constituents including two C₂₁ steroidal glycosides(Mu and Zhou, 1983a; 1983b; Mu et al, 1986). Consequently, Mu et al(1986)developed C. otophyllum into three novel medicines(Patents of China: ZL 98 1 18938.5, ZL 98 1 18173.2, and ZL 96 1 11270.0). The authors carried out further important investigations. However, most compounds in total glycosides were difficult to separate. To study these compounds, the authors used the acidic hydrolysis reaction universal in the research on glycosides to obtain secondary glycosides that are easy to separate. Moreover, some glycosides were easy to separate after other glycosides changed to secondary glycosides. From the ethyl acetate extract(total glycosides) and its acidic hydrolysis part of the rhizome of C. otophyllum, the authors isolated seven new carbohydrates(Zhao et al, 2007; 2008a) and three new C₂₁ steroidal glycosides(Zhao et al, 2005; 2008b). Seven compounds were isolated from the rhizome(Zhao et al, 2009). Furthermore, this article reports three new C₂₁ steroidal glycosides obtained from the acidic hydrolysis part, such as deacetylmetaplexigenin 3-O-β-D-ole and ropyranosyl-(1→4)- α-D-ole and ropyranosyl-(1→4)-α-D-ole and ropyranoside(1), deacetylmetaplexigenin 3-O-α-D-ole and ropyranosyl-(1→4)- β-D-thevetopyranosyl-(1→4)-α-D-ole and ropyranoside(2), and deacetylmetaplexigenin 3-O-β-D-cymaropyranosyl-(1→4)-α- D-ole and ropyranoside(3). Considering their structures, compounds 1 and 2 might be native glycosides, and compound 3 might be a native glycoside or artificial product as a fragment of corresponding native glycoside.

Melting points were determined on a WC-1 Micromelting Point Apparatus(uncorrected, Instrument Plant of Sichuan University, China). Optical rotations were measured on a Horiba Sepa-300 Digital Polarimeter(Japan). The IR spectra were measured on a Perkin-Elmer 577 Spectrophotometer(USA). The UV spectra were measured on a Shimadzu Double-beam 210A Spectrometer(Japan). FAB-MS was performed on a VG AutoSpec-3000 Spectrometer(UK). Bruker Am-400 and DRX-500 instruments(USA)were used to record ¹H-NMR, 2D NMR(400 MHz), and ^13C-NMR. C₅D₅N was the solvent and the internal st and ard was at room temperature. Column chromatography(CC)was carried out on silica gel. Silica gel(200-300 mesh)for CC and silica gel plate(GF-254)for thin-layer chromatography(TLC)were the products of Qingdao Haiyang Chemical Group Co., China.

The rhizome of Cynanchum otophyllum Schneid. was bought from a drug market in Kunming. It was identified by Dr. Yue-mao Shen and a voucher specimen(KUN No. 0776933)was deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences.

The dried powder of the rhizome of C. otophyllum(40 kg)was refluxed with 95% EtOH(120 L × 3). The extract was evaporated, extracted with EtOAc(6 L), and defatted with petroleum ether(1.4 L). The extract was the total glycosides(700 g). A part of the total glycosides(500 g)were dissolved in 2.25 L MeOH-0.025 mol/L H₂SO₄(1:2)in water bath at 70 ^oC. After 2 h, Ba(OH)₂ solution was added to adjust the pH value to 7, and then filtered to remove BaSO₄. The solution was dried up to give a crude aglycones(100 g).

The crude aglycones(100 g)were separated into 21 fractions(Frs. 1-21)through CC over silica gel(300 g)by elution with CHCl₃(1874 mL), and then with a mixture of CHCl₃-MeOH(100:1, 1000 mL), CHCl₃-MeOH(100:3, 1000 mL), and finally CHCl₃-MeOH(100:8.5, 1582 mL). Fr. 8(between 2597 and 2716 mL, 12 g)produced three fractions by CC(76 g)eluted with CHCl₃-MeOH(100:1.5, 100:2, 100:3, 1000 mL each), and then the second fraction(Fr. 8-2)yielded three subfractions(Frs. 8-2-a, 8-2-b, and 8-2-c)by CC(54.5 g)eluted with petroleum ether-acetone(10:4.5, 300 mL; 10:5, 600 mL; 10:7, 227 mL), and finally the third subfraction(Fr. 8-2-c)further produced four fractions(Frs. 8-2-c-A, 8-2-c-B, 8-2-c-C, and 8-2-c-D)by CC(70.5 g)eluted with petroleum ether-ethyl acetate(35:65, 1500 mL; 0:100, 150 mL). Fr. 8-2-c-D(between 900 and 1650 mL, 0.4 g)produced two fractions by CC(40 g)eluted with petroleum ether-ethyl acetate(25:75, 500 mL). The first fraction(Fr. 8-2-c-D-1)was subjected to CC(15 g)eluted with CHCl₃-MeOH(100:5, 200 mL), and one fraction(Fr. 8-2-c-D-1-1)was obtained, which was further subjected to CC(35 g)eluted with CHCl₃-MeOH(100:3, 250 mL; 100:4, 10 mL; 100:10, 130 mL)to yield compound 1(between 99 and 161 mL, 89 mg, 0.089%). Fr. 14(between 4478 and 4507 mL, 0.4 g)produced two fractions by CC(74 g)eluted with CHCl₃-MeOH(100:8, 1000 mL), and the first fraction(Fr. 14-1)yielded three subfractions(Frs. 14-1-1, 14-1-2, and 14-1-3)by CC(53 g)eluted with petroleum ether-acetone(10:7.5, 500 mL; 10:8, 300 mL). Subfraction 14-1-2 yielded one fraction by CC(5.5 g)eluted with CHCl₃-acetone(6:4, 100 mL; 1:1, 50 mL), and this fraction(Fr. 14-1-2-1)was subjected t o CC(6 g)eluted with CHCl₃-MeOH(100:8, 100 mL)to afford compound 2(between 33 and 53 mL, 46 mg, 0.046%). Fr. 8-2-c-C(between 800 and 850 mL, 30 mg)was subjected to CC(5.5 g)eluted with CHCl₃-MeOH(100:4, 100 mL)(one fraction Fr. 8-2-c-C-1 was obtained), which was then subjected to CC(5.5 g)eluted with CHCl₃-MeOH(100:2, 70 mL; 100:3, 20 mL), to afford compound 3(between 24 and 48 mL, 21 mg, 0.021%).

Compound 1 was obtained as a colorless gum, mp 123-126 ^oC, [α]20.0 D +24.6(c 0.29, EtOH). UV(EtOH)λ_max(log ε): 255.6(3.21)nm; IR(KBr)υ_max: 3442, 2932, 2366, 2339, 1699, 1634, 1456, 1368, 1317, 1195, 1165, 1098, 1060, 1003, 911, 537, 419 cm^-1. ¹H-NMR, ¹³C-NMR, and 2D NMR data are shown in Table 1. FAB^--MS m/z: 811 [M-H]^-(100), 773(4), 699(2.5), 667(13.5), 255(9.5), 85(4.5); HR-FAB^--MS m/z: 811.4508 [M-H]^-(calcd for C₄₂H₆₇O₁₅, 811.4480). The molecular formula of compound 1 was determined as C₄₂H₆₈O₁₅ by HRFAB-MS. The ¹³C-NMR and DEPT spectra showed one carbonyl, one pair of double bonds, nine methyls, and numerous methylenes, methines, and quaternary carbons. The spectra were compared with the ¹³C-NMR and DEPT data(Zhang et al, 2000)of known C₂₁ steroidal aglycones, and the aglycone was determined to be deacetylmetaplexigenin. In compound 1, the anomeric carbon at δ 96.4 corresponded to the proton at δ 5.25 d in the HMQC spectrum, which had a long-range correlation with C-3 of the aglycone in the HMBC spectrum, and the signal for C-3 was at δ 77.9, so compound 1 was a 3-O-glycoside of deacetylmetaplexigenin. The anomeric carbon resonances at δ_C 96.4, 100.6, and 102.2 revealed the presence of three sugar residues. In Table 1, the proton at δ 5.25 d correlated with the signal at δ 96.4 in the HMQC spectrum, and had a correlation with H-2′ in the ¹H-¹H COSY spectrum. The assignment for C-2′(δ 37.0)was obtained from the correlation with H-2′(δ 1.87 m an d 2.51 m)in the HMQC spectrum. These protons had correlations with H-1′ and H-3′ in the ¹H-¹H COSY spectrum, from which C-3′(δ 77.8)was obtained. In this case, the carbons at δ 96.4, 37.0, 77.8, 83.2, 69.0, and 18.4 were determined to be the carbons of the sugar by ¹H-¹H COSY and HMQC spectra. The methoxy group(δ 58.9)was located by the correlation of its proton signal at δ 3.54 s, with C-3′ in the HMBC spectrum. The ¹³C-NMR data of the sugar were compared with those in literature(Zhang et al, 2000) and the sugar was determined to be α-D-ole and ropyranose. C-4′ was found at δ 83.2, and in the HMBC spectrum, it showed a long-range correlation with the proton at δ 5.10 d, which was correlated with the carbon at δ 100.6 in the HMQC spectrum. Consequently, the O-C-4′ was linked with the sugar unit whose anomeric carbon(C-1″)was at δ 100.6. On the basis of the correlations between the protons in the ¹H-¹H COSY spectrum and the long-range correlation of MeO- in the HMBC spectrum in Table 1, all the ¹³C-NMR data of the II unit were determined. The data were compared with those in literature(Zhang et al, 2000) and the II moiety was determined to be α-D-ole and ropyranose. Since H-4″(δ 3.48)had a long-range correlation with the resonance at δ 102.2 in HMBC spectrum, and C-4″ resonated at δ 83.5, so O-C-4″ was linked with the sugar unit III with the anomeric carbon at δ 102.2. This sugar was determined to be β-D-ole and ro- pyranose(Table 1). Therefore, compound 1 was elucidated as deacetylmetaplexigenin 3-O-β-D-ole and ropyranosyl-(1→4)-α-D- ole and ropyranosyl-(1→4)-α-D-ole and ropyranoside(Figure 1).

Table 1

Table 1 1H-NMR and 13C-NMR spectral data for compound 1 in C5D5N(400 MHz, coupling constants in Hz) Carbon 13C 1H 1H-1H COSY HMBC Carbon 13C 1H 1H-1H COSY HMBC Deacetylmetaplexigenin 1′ 96.4 d 5.25 d(9.6) H-2′ C-3 1 39.0 t 1.80 m; 1.85 m H-2 C-5 2′ 37.0 t 1.87 m; 2.51 m H-1′; H-3′ C-1′ 2 30.0 t 2.03 m; 2.07 m H-1 - 3′ 77.8 d 3.83 m H-2′ - 3 77.9 d 4.05 m H-4 - 4′ 83.2 d 3.48 m H-5′ C-5′; C-1″ 4 39.4 t 2.40 m; 2.55 m H-3 C-5; C-6; C-9 5′ 69.0 d 4.17 m H-4′; H-6′ C-4′ 5 139.4 s - - - 6′ 18.4 q 1.35 m; 3H H-5′ C-4′; C-5′ 6 119.6 d 5.32 s H-7 - MeO-3′ 58.9 q 3.54 s; 3H - C-3′ 7 35.1 t 2.27 m; 2.48 m H-6 C-5; C-6; C-8 α-D-Ole 8 74.3 s - - - 1″ 100.6 d 5.10 d(9.6) H-2″ C-4′ 9 45.0 d 1.57 m H-11 C-19 2″ 37.2 t 2.29 m; 2.51 m H-1″; H-3″ C-3″; C-4″ 10 37.0 s - - - 3″ 78.1 d 4.05 m H-2″; H-4″ - 11 29.5 t 1.82 m; 1.87 m H-9 C-5; C-18 4″ 83.5 d 3.48 m H-3″; H-5″ C-1″; C-3″; C-5″; 12 69.0 d 4.17 m - - C-6″; C-1″′ 13 60.4 s - - - 5″ 69.1 d 4.17 m H-4″; H-6″ C-4″ 14 89.4 s - - - 6″ 18.7 q 1.35 m; 3H H-5″ C-2″; C-4″; C-5″ 15 34.3 t 2.08 m; 2H H-16 C-7; C-16 MeO-3″ 59.0 q 3.59 s; 3H - C-3″ 16 32.8 t 2.08 m; 3.44 m H-15 C-13; C-15 β-D-Ole 17 92.6 s - - - 1″′ 102.2 d 4.73 t(9.6, 16) - - 18 9.5 q 1.94 s; 3H - - 2″′ 37.4 t 2.29 m; 2.51 m H-3″′ C-3″′; C-4″′ 19 18.4 q 1.35 s; 3H - C-5 3″′ 81.4 d 3.44 m H-2″′ C-1″′; C-6″′ 20 209.7 s - - - 4″′ 76.2 d 3.45 m H-5″′ C-1″′; C-6″′ 21 28.0 q 2.59 s; 3H - - 5″′ 73.0 d 3.57 s H-4″′; H-6″′ - α-D-Ole 6″′ 18.8 q 1.54 d(6.0); 3H H-5″′ C-4″′; C-5″′ MeO-3″′ 57.2 q 3.43 s; 3H - C-3″′

Table 1 1H-NMR and 13C-NMR spectral data for compound 1 in C5D5N(400 MHz, coupling constants in Hz)

Figure 1

Figure 1 Structures of glycosides 1-3

Compound 2 was obtained as a colorless gum, mp 123-126 ^oC, [α]20.0 D +22.3(c 0.21, EtOH). UV(EtOH)λ_max(log ε): 255.6(3.25)nm; IR(KBr)υ_max: 3443, 2968, 2934, 1700, 1634, 1456, 1369, 1317, 1196, 1166, 1151, 1085, 1005, 912, 865, 674, 564 cm^-1. ¹H-NMR, ¹³C-NMR, and 2D NMR data are shown in Table 2. FAB^--MS m/z(rel. int.): 827 [M-H]^-(100), 728(1), 667(2), 515(1.5), 361(2), 283(1.5), 255(5), 99(1); HR-FAB-MS m/z: 827.4446 [M-H]^-(calcd for C₄₂H₆₇O₁₆, 827.4429). The molecular formula of compound 2 was determined as C₄₂H₆₈O₁₆ from HRFAB-MS. The ¹³C-NMR and DEPT spectra were compared with those of known C₂₁ steroidal aglycones(Zhang et al, 2000), and the aglycone unit was determined to be deacetylmetaplexigenin. In compound 2, the anomeric carbon at δ 96.4 corresponded to the proton at δ 5.25 d in HMQC spectrum, which had a long-range correlation with C-3 of the aglycone in the HMBC

spectrum, and C-3 was at δ 77.8, so this compound was also a C-3 glycoside of the deacetylmetaplexigenin. Three anomeric carbons were observed(δ_C 96.4, 106.3, and 100.5), revealing the presence of three sugar residues. The sugar at δ 96.4 was determined to be α-D-ole and ropyranose in the same way as previously described(Table 2). The resonance of C-4′ located at δ 83.1, and its corresponding proton(δ 3.56 m)in the HMQC spectrum, had a long-range correlation with the resonance at δ 106.3 in the HMBC spectrum. Consequently, O-C-4′ was linked with the sugar whose anomeric carbon was at δ 106.3. This sugar was determined to be β-D-theveto- pyranose(Table 2). The signal of C-4″ was at δ 83.4 whose corresponding proton(δ 3.45)in the HMQC spectrum had a long-range correlation with the carbon at δ 100.5 in the HMBC spectrum. Thus, O-C-4″ was linked with the sugar unit III. This sugar was determined to be α-D-ole and ro- pyranose(Table 2). Therefore, compound 2 was elucidated as deacetylmetaplexigenin 3-O-α-D-ole and ropyranosyl-(1→4)-β- D-thevetopyranosyl-(1→4)-α-D-ole and ropyranoside(Figure 1).

Table 2

Table 2 1H-NMR and 13C-NMR spectral data for compound 2 in C5D5N(400 MHz, coupling constants in Hz) Carbon 13C 1H 1H-1H COSY HMBC Carbon 13C 1H 1H-1H COSY HMBC Deacetylmetaplexigenin 1′ 96.4 d 5.25 d(9.2) H-2′ C-3 1 39.0 t 1.08 m; 1.83 m H-2 C-18 2′ 37.3 t 1.85 m; 2H H-1′ - 2 30.0 t 2.04 m; 2H H-1 - 3′ 78.1 d 4.03 m H-4′ - 3 77.8 d 3.84 m H-4 - 4′ 83.1 d 3.56 m H-3′; H-5′ C-6′; C-1″ 4 39.4 t 2.41 m; 2.52 m H-3 C-5; C-6 5′ 69.1 d 3.90 m H-4′ - 5 139.4 s - - - 6′ 18.6 q 1.56 t(5.6); 3H - C-3′; C-4′ 6 119.6 d 5.31 s H-7 C-8; C-10 MeO-3′ 59.0 q 3.59 s; 3H - C-3′ 7 35.1 t 2.27 m; 2.49 m H-6 C-5; C-6; C-9 β-D-The 8 74.3 s - - - 1″ 106.3 d 4.74 m H-2″ C-4′; C-3″ 9 45.0 d 1.58 t(5.6) H-11 C-12 2″ 75.1 d 3.89 s H-1″; H-3″ - 10 37.4 s - - - 3″ 87.9 d 3.60 s H-2″ MeO-3″ 11 29.5 t 1.87 m; 2.39 m H-9; H-12 C-18 4″ 83.4 d 3.45 m - C-1″′ 12 69.0 d 3.93 m H-11 C-14 5″ 72.8 d 3.71 m H-6″ - 13 60.4 s - - - 6″ 18.7 q 1.56 t(5.6); 3H H-5″ C-2″; C-4″ 14 89.4 s - - - MeO-3″ 61.1 q 3.88 s; 3H - C-3″ 15 34.3 t 2.09 m; 2H H-16 - α-D-Ole 16 32.8 t 2.09 m; 3.34 m H-15 C-14 1″′ 100.5 d 5.08 d(9.6) H-2″′ C-4″ 17 92.6 s - - - 2″′ 36.9 t 1.83 m; 2H H-1″′ - 18 9.5 q 1.96 s; 3H - - 3″′ 78.2 d 4.03 m - - 19 18.4 q 1.35 m; 3H - C-12 4″′ 75.9 d 3.60 s - C-3″′; C-5″′; C-6″′ 20 209.7 s - - - 5″′ 69.3 d 4.18 m H-6″′ - 21 28.0 q 2.60 s; 3H - - 6″′ 18.4 q 1.34 m; 3H H-5″′ C-2″′; C-5″′ α-D-Ole MeO-3″′ 58.9 q 3.53 s; 3H - C-3″′

Table 2 1H-NMR and 13C-NMR spectral data for compound 2 in C5D5N(400 MHz, coupling constants in Hz)

Compound 3 was obtained as a colorless gum, mp 101-104 ^oC, [α]20.0 D +42.6(c 0.20, EtOH); UV(EtOH)λ_max(log ε): 255.6(2.66)nm; IR(KBr)υ_max: 3442, 2932, 1700, 1634, 1456, 1367, 1318, 1274, 1194, 1165, 1147, 1089, 1062, 1004, 912, 865, 577 cm^-1; ¹H-NMR, ¹³C-NMR, and 2D NMR data are shown in Table 3; FAB^--MS m/z: 667 [M - H]^-(100), 559(3), 523(39.5), 280(6.5), 255(7), 194(6), 97(6.5); HR-FAB^--MS m/z: 667.3668 [M - H]^-(calcd for C₃₅H₅₅O₁₂, 667.3694). The molecular formula of compound 3 was C₃₅H₅₆O₁₂, deduced from HRFAB-MS. The ¹³C-NMR and DEPT spectra were compared with those of known C₂₁ steroidal aglycones(Zhang et al, 2000), and the aglycone unit was determined to be deacetylmetaplexigenin. In compound 3, the anomeric carbon at δ 96.5 corresponded to the proton at δ 4.83 d in the HMQC spectrum, which had a long-range correlation with C-3 at δ 77.9 of the aglycone in the HMBC spectrum, and compound 3 was a 3-O-glycoside of de- acetylmetaplexigenin. The anomeric carbon resonances at δ_C 96.5 and 100.6 revealed the presence of two sugar residues. The sugar at δ 96.5 was determined to be α-D-ole and ropyranose in the same way as that in compound 2(Table 3). C-4′ was found to be at δ 83.7, and in the HMBC spectrum, it displayed a long-range correlation with the proton at δ 4.73, showing an HMQC correlation with the carbon at δ 100.6. This sugar was determined to be β-D-cymaropyranose(Table 3). Therefore, compound 3 was elucidated as deacetylmetaplexigenin 3-O-β-D-cymaropyranosyl-(1→4)-α- D-ole and ropyranoside(Figure 1).

Table 3

Table 3 1H-NMR and 13C-NMR spectral data for compound 3 in C5D5N(400 MHz, coupling constants in Hz) Carbon 13C 1H 1H-1H COSY HMBC Carbon 13C 1H 1H-1H COSY HMBC Deacetylmetaplexigenin 19 18.7 q 1.15 s; 3H - C-12 1 39.3 t 1.08 m; 1.82 m H-2 C-4;C-5 20 209.5 s - - - 2 30.0 t 1.56 m; 2.05 m H-1; H-3 - 21 27.6 q 3.08 s; 3H - - 3 77.9 d 3.49 m H-2 C-1′ α-D-Ole 4 39.5 t 2.14 m; 2.34 m - C-3; C-5; C-6 1′ 96.5 d 4.83 d(1.6) H-2′ C-3 5 139.8 s - - - 2′ 37.3 t 1.50 m; 1.93 m H-1′; H-3′ C-5′; C-6′ 6 119.4 d 5.30 m H-7 - 3′ 78.6 d 3.54 m H-2′ C-5′ 7 35.1 t 2.06 m; 2.11 m H-6 - 4′ 83.7 d 3.19 dd(6.8, 2.8) H-5′ C-6′ 8 74.4 s - - - 5′ 69.0 d 3.77 m H-4′; H-6′ C-1′ 9 44.8 d 1.48 m - C-18 6′ 18.5 q 1.13 s; 3H H-5′ C-4′; C-5′ 10 37.6 s - - - MeO-3′ 57.9 q 3.39 s; 3H - C-3′ 11 29.6 t 1.83 m; 2H - C-5; C-17 β-D-Cym 12 69.3 d 3.74 m - - 1″ 100.6 d 4.73 m H-2″ C-4′ 13 60.5 s - - - 2″ 37.3 t 1.51 m; 1.94 m H-1″; H-3″ C-6″ 14 89.1 s - - - 3″ 78.1 d 3.80 d(3.2) H-2″ - 15 34.3 t 1.84 m; 2.04 m H-16 C-13; C-17 4″ 73.9 d 3.11 m H-5″ C-5″; C-6″ 16 32.4 t 2.87 dd(2.0, 2.4); 2H H-15 C-14; C-20 5″ 71.0 d 3.63 m H-4″; H-6″ - 17 92.5 s - - - 6″ 18.7 q 1.18 s; 3H H-5″ C-4″; C-5″ 18 8.6 q 1.33 s; 3H - C-12; C-13 MeO-3″ 58.8 q 3.40 s; 3H - C-3″

Table 3 1H-NMR and 13C-NMR spectral data for compound 3 in C5D5N(400 MHz, coupling constants in Hz)

In conclusion, C₂₁ steroidal glycosides in Qingyangshen Tablets are composed of a C₂₁ steroidal aglycone moiety of the 3-OH substitution and a sugar chain consisted of several sugar residues which are 2-deoxy-hexose. All of the linkages among them are 1→4 linkages. Since Qingyangshen Tablets are particularly effective against epilepsy and chronic hepatitis, this structure class is particularly effective against epilepsy and chronic hepatitis, which is the structure-activity relationship of Qingyangshen Tablets.