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Steroidal saponins from the fresh tubers of Ophiopogon japonicus
Phytochemistry Letters 15 (2016) 72–80
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Short communication
Steroidal saponins from the fresh tubers of Ophiopogon japonicus Renyi Yana , Yixun Liub , Liping Kangb , Yang Zhaob , Xinguang Sunb , Jie Zhanga , Dexian Jiaa , Baiping Mab,* a b
Ovation Health Science and Technology Co., Ltd., ENN Group, Langfang 065001, PR China Beijing Institute of Radiation Medicine, Beijing 100850, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 September 2015 Received in revised form 15 November 2015 Accepted 17 November 2015 Available online xxx
Eleven novel furostanol saponins, named ophiofurospisides C–E, G–N (1–3, 5–12), one new spirostanol saponin, named ophiopogonin R (13), were isolated from the fresh tubers of Ophiopogon japonicus. Their structures were determined on the basis of spectroscopic techniques (1D and 2D NMR) and HRESIMS. The isolated furostanol saponins possessed two sugar chains located at C-3 and C-26, respectively. Six furostanol saponins (1, 5–9) with disaccharide moiety linked at position C-26 of the aglycone were rare in the plant kingdom. ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Keywords: Ophiopogon japonicus Furostanol saponin Spirostanol saponin
1. Introduction Ophiopogon japonicus (L.f.) Ker-Gawl. (Liliaceae) is an evergreen perennial, widely distributed in China, especially in Sichuan province and Zhejiang province. The tubers of O. japonicus are well known as “Maidong” in China. It has been used in traditional Chinese medicine for thousands of years as a tonic agent. Nowadays, it often combines with Panax ginseng and Schisandra chinensis to treat cardiovascular and cerebrovascular diseases (Jiangsu New Medicinal College, 1986). Previous phytochemical investigations revealed that O. japonicus contain a variety of steroidal saponins, mainly in the spirostanol form (Watanabe et al., 1977; Asano et al., 1993; Chen et al., 2000; Dai et al., 2005; Cheng et al., 2006; Xu et al., 2008; Zhou et al., 2008; Wang et al., 2008a,b; Li et al., 2013; Ye et al., 2013; Liu et al., 2014a,b). However, our former reports about the steroidal saponins of fresh tubers of O. japonicus showed that contained mainly furostanol saponins (Guo et al., 2013; Zhang et al., 2009, 2012). Furostanol saponins can be easily transformed to corresponding spirostanol saponins under the action of the enzyme during drying and storage of plant, and usually regarded as the synthetic precursors of the corresponding spirostanol saponins in plants (Inoue et al., 1996). Thereby, with the aim to ascertain chemical components disparity between dried and fresh tubers, we decided to carry out a continuous phytochemical research on fresh tubers of O. japonicus. As a result, 13 steroidal saponins including 12 new compounds were
* Corresponding author. Fax: +86 10 68214653. E-mail address: [email protected] (B. Ma).
isolated and identified, 12 of which were furostanol saponins. In this paper, the isolation and structural elucidation of the new compounds were described. 2. Results and discussion Using the same solvent extraction method and chromatographic steps as former reports (Guo et al., 2013; Zhang et al., 2009, 2012), 12 furostanol saponins (ophiofurospisides C–N, 1–12) and one spirostanol saponin (ophiopogonin R, 13) were isolated from 60% EtOH extract of fresh tubers of O. japonicus. Among them, only ophiofurospiside F (4) was reported recently from same plant (Liu et al., 2014a); all others are new compounds (Fig. 1). The sugars obtained by aqueous acid hydrolysis of total saponin (Fr.B) (see Section 3.3) were identified as D-xylose, D-glucose, and Lrhamnose by GC (see Section 3.4). The fucose was not detected by GC analysis. It may be the result of low fucose levels in total saponin. In compound 12, the configuration of D-fucose was deduced from the fact that it was the common stereoisomer from natural source. In the 1H NMR spectra, the relatively large 3JH-1,H-2 values (between 6.6 and 9.0 Hz) of the anomeric protons of the glucose, fucose, and xylose moieties indicated b-anomeric orientation for these glycose moieties. The broad singlet of the anomeric proton of rhamnose combined with the carbon signals of C-3 (dc ca. 72.5) and C-5 (dc ca. 69.5) (Zhang et al., 2009, 2012) indicated the a-configuration. The a-configuration of the C-22 hydroxyl group was deduced from the hemiketal carbon signal at dc ca. 110, about 2–4 ppm higher field than that of the b-configuration (dc ca. 114) (Dini et al., 2005). The 25R configuration of furostanol saponins was demonstrated by the chemical shift
http://dx.doi.org/10.1016/j.phytol.2015.11.013 1874-3900/ ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
R. Yan et al. / Phytochemistry Letters 15 (2016) 72–80
Fig. 1. The chemical structures of compounds 1–13.
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R. Yan et al. / Phytochemistry Letters 15 (2016) 72–80
difference between the two protons of H-26 (Dab < 0.48) (Agrawal, 2004, 2005). The configurations of hydroxyl group at C-1, C-12, C13 and C-14 were determined by comparing the 13C NMR spectroscopic data with those reported in literatures (Guo et al., 2013; Zhang et al., 2009, 2012). Ophiofurospiside C (1) was obtained as white amorphous powder. Its molecular formula was determined as C56H92O28 by negative ion HR-ESI-MS with a quasi-molecular ion peak at m/z 1211.5717 [M-H]– (calcd. for C56H91O28, 1211.5697). The major fragment ions at m/z 1061.5 [M-H-18-132]–, 899.4 [M-H-18-132162]–, 753.3 [M-H-18-132-162-146]–, 591.3 [M-H-18-132-162-146162]–, and 429.3 [M-H-18-132-162-146-162-162]– could be attributable to the losses of a pentosyl, a deoxyhexosyl, and three hexosyl moieties from the ion [M-H]–. The 1H NMR spectrum of 1 contained two methyl singlet signals at dH 0.88 (3H, s, Me-18) and 1.06 s (3H, s, Me-19), three methyl doublet signals at dH 1.36 (3H, d, J = 7.2 Hz, Me-21), 0.98 (3H, d, J = 6.6 Hz, Me-27), and 1.71 (3H, d, J = 6.6 Hz, Rha-6), and an olefinic proton signal at dH 5.25 (1H, br s, H-6). In the 13C NMR spectrum, one double bond carbon signals at dC 140.8 (C-5) and 121.9 (C-6); diagnostic quaternary carbon signal at dC 111.4 (C-22) and the oxygenated signals at dC 78.2 (C-3), 90.5 (C-16), 90.9 (C-17), and 75.2 (C-26) were observed. In HMBC spectrum, the correlations between dH 1.36 (Me-21) and 90.9 (C17), and between dH 0.88 (Me-18) and 90.9 (C-17). With above mentioned NMR data, compound 1 was characteristic of a steroidal saponin having (25R)-furosta-5-ene-3b,17a,22a,26-tetraol as the aglycone (Matsuo et al., 2013; Zhao et al., 2009). The presence of five sugar residues was confirmed from the observation of five anomeric proton signals [dH 4.94 (d, J = 7.8 Hz), 6.25 (br s), 5.02 (d, J = 7.8 Hz), 4.72 (d, J = 7.8 Hz) and 5.09 (d, J = 7.8)], giving HSQC correlations with five anomeric 13C NMR signals at dC 100.1, 102.0, 105.8, 104.9, and 105.5, respectively. Complete assignments of each sugar were achieved by extensive 1D and 2D NMR analyses. Evaluation of chemical shifts and spin–spin couplings (Zhang et al., 2009, 2012) allowed the identification of three b-glucopyranosyl, one b-xylopyranosyl and one a-rhamnopyranosyl units in 1. The sequence of the oligosaccharidic chain of 1 was achieved by HMBC experiment. The HMBC correlations between the anomeric 1H NMR signal at dH 4.94 (Glc1 H-1) and dC 78.2 (Agly C-3), between dH 6.25 (br s, Rha H-1) and dC 77.5 (Glc1 C-2), and between dH 5.02 (Xyl H-1) and dC 81.5 (Glc1 C-4) proved the sequence of the sugar chain at C-3 to be a-L-rhamnopyranosyl(1 ! 2)-[b-D-xylopyranosyl-(1 ! 4)]-b-D-glucopyranosyl. Finally, HMBC correlations between dH 5.09 (Glc3 H-1) and dC 70.2 (Glc2 C-6), between dH 4.72 (Glc2 H-1) and dC 75.2 (Agly C-26) allowed the linkage of another sugar chain, b-D-glucopyranosyl(1 ! 6)-b-D-glucopyranosyl, at C-26. Hence, the structure of ophiofurospiside C (1) was concluded to be (25R)-26-O-[b-Dglucopyranosyl-(1 ! 2)-b-D-glucopyranosyl]-5-ene-furosta3b,17a,22a,26-tetraol-3-O-a-L-rhamnopyranosyl-(1 ! 2)-[b-Dxylopyranosyl-(1 ! 4)]-b-D-glucopyranoside. Compound 2 was isolated as a white amorphous powder with a molecular formula of C45H74O21, as determined by HRESIMS with a quasi-molecular ion peak at m/z 949.4667 [M-H]– (calcd. for C45H73O21, 949.4644). HRESIMS showed fragments attributable to the losses of a deoxyhexosyl, two hexosyl residues and three molecules of water. The 1H NMR spectrum of 2 contained three anomeric proton signals at dH 4.82 (1H, d, J = 7.8 Hz, Glc2 H-1), 5.01 (1H, d, J = 7.8 Hz, Glc1 H-1), 6.39 (1H, br s, Rha H-1) and five methyl group signals at dH 0.99 (3H, s, CH3-18), 1.12 (3H, s, CH3-19), 1.41 (3H, d, J = 8.4 Hz, CH3-21), 1.01 (3H, d, J = 7.8 Hz, CH3-27), and 1.79 (3H, d, J = 7.2 Hz, Rha H-6). In 13C NMR spectrum, the signals at dC 91.6 (C-16), 111.2 (C-22), 75.2 (C-26), 139.9 (C-5), and 122.7 (C-6) indicated that 2 is a furostanol saponin possessing unsaturated double bond at C-5 and C-6 (Agrawal et al., 1995). Two oxygenated methine signals at dC 78.3 and 76.4 were attributed to C-3 and C-12,
which showed correlations with proton signals at dH 3.91 (1H, m, H-3) and 4.49 (1H, m, H-12) in the HMQC spectrum, respectively; two oxygenated quaternary carbon signals at dC 88.2 and 94.8 were attributed to C-14 and C-17. The position of hydroxyl groups was confirmed by the long-range correlations between H-21 (dH 1.41, d, J = 8.4 Hz) with C-17, C-22, between H-18 (0.99, s) with C-12, C-14 in the HMBC spectrum. All above data suggested the aglycone of 2 was identical as ophiopogonin M which isolated from the same plant in our lab before (Zhang et al., 2012). In the HMBC spectrum, the long-range correlations between dH 6.39 (H-1 of Rha) and dC 77.9 (C-2 of Glc1), between dH 5.01 (H-1 of Glc1) and dC 78.3 (C-3) indicated the presence of a disaccharide moiety linked at position C-3 of the aglycone. An additional correlation between dH 4.82 (H-1 of Glc2) and dC 75.2 (C-26) revealed the attachment of a single glucose residue at position C-26 of the aglycone. Thus, the structure of ophiofurospiside D (2) was assigned as (25R)-26-Ob-D-glucopyranosyl-5-ene-furosta-3b,12b,14a,17a,22a,26-pentaol-3-O-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranoside. Compound 3 was obtained as a white amorphous powder. The molecular formula was deduced as C50H82O24 based on its negative HRESIMS ion peak at m/z 1065.5144 [M-H]– (calcd. for C50H81O24, 1065.5118). In process to prepare this paper, we found that ophiofurospiside F (4) was reported recently (Liu et al., 2014b). A comparison of NMR and MS data of 3 with those of 4 revealed that they were similar, except for the presence of an additional xylosyl moiety. A detailed comparison of the NMR data of 3 with those of 1 and 4 revealed that 3 and 4 share the same aglycone part, 3 and 1 share the same glycone part. Through analysis the 1D and 2D NMR data of 3, its structure was defined as (25R)-26-O-b-D-glucopyranosyl-5-ene-furosta-3b,14a,17a,22a,26-pentaol-3-O-a-L-rhamnopyranosyl-(1 ! 2)-[b-D-xylopyranosyl-(1 ! 4)]-b-D-glucopyranoside, named ophiofurospiside E. Ophiofurospiside G (5) and ophiofurospiside H (6), white amorphous powder, displayed the same molecular formula of C50H82O23 based on HRESIMS m/z 1049.5162 [M-H]– (calcd. for C50H81O23, 1049.5169) and m/z 1049.5192 [M-H]– (calcd. for C50H81O23, 1049.5169), respectively. HRESIMS of both compounds showed same fragment ion peaks at m/z 887.5 [M-H-162]–, 755.4 [M-H-162-132]–, 737.4 [M-H-162-132-18]–, 575.4 [M-H-162-13218-162]–, and 413.3 [M-H-162-132-18-162-162]– in negative ion mode, suggesting the presence of three hexosyl and a pentosyl moieties. In 1H NMR spectra, there was also four anomeric proton resonances appeared at dH 4.95 (1H, d, J = 7.8 Hz, Glc1 H-1), 5.02 (1H, d, J = 7.8 Hz, Xyl H-1), 4.72 (1H, d, J = 7.8 Hz, Glc2 H-1), and 5.09 (1H, d, J = 7.8 Hz, Glc3 H-1) in 5 and dH 4.95 (1H, d, J = 7.8 Hz, Glc1 H1), 5.02 (1H, d, J = 7.8 Hz, Xyl H-1), 4.84 (1H, d, J = 6.6 Hz, Glc2 H-1), and 5.28 (1H, d, J = 7.8 Hz, Glc3 H-1) in 6. Additionally, diagnostic signals of four methyl, one oxymethine, one oxymethylene, one hemiketal and one olefinic groups were also deduced from NMR spectra of compounds 5 and 6 (Tables 1 and 2). Based on these diagnostic signals, it can easily deduced that the aglycone was (25R)-5-ene-furosta-3b,22a,26-triol (Zhao et al., 2009). By comparing the 13C NMR data with those of 1 indicated that there were one xylose and three glucose moieties in 5 and 6. In HMBC spectrum of 5, correlations of Glc1 H-1 (dH 4.95)/C-3 (dC 78.4), Xyl H-1 (dH 5.02)/Glc1 C-4 (dC 81.0), Glc2 H-1 (dH 4.72)/C-26 (dC 75.4), and Glc3 H-1 (dH 5.09)/Glc2 C-6 (dC 70.2) were observed. Thus, 5 was determined as (25R)-26-O-[b-D-glucopyranosyl-(1 ! 6)]-b-Dglucopyranosyl-5-ene-furosta-3b,22a,26-triol-3-O-b-D-xylopyranosyl-(1 ! 4)-b-D-glucopyranoside. The difference between 5 and 6 in HMBC spectrum was that correlation between Glc3 H-1 and Glc2 C-6 in 5 was replaced by the Glc3 H-1 and Glc2 C-2 in 6 which was supported by the downfield signal of Glc2 C-2 (dC 84.3) and upfield signal of Glc2 C-6 (dC 62.7). Accordingly, 6 was determined as (25R)-26-O-[b-D-glucopyranosyl-(1 ! 2)]-b-D-glucopyranosyl-
R. Yan et al. / Phytochemistry Letters 15 (2016) 72–80
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Table 1 1 H NMR data of compounds 1–3, 5–9, 12 and 13 in pyridine-d5. Pos.
1
2
3
5
6
7
8
9
12
13
1
0.93 (m) 1.74 (m) 1.85 (m) 2.07 (m) 3.83 (m) 2.70 (m) 2.72 (m) 5.25 (br s) 1.48 (m) 1.86 (m) 1.58 (m)
1.02 (m) 1.72 (m) 1.83 (m) 2.06 (m) 3.91 (m) 2.74 (m) 2.78 (m) 5.40 (br s) 1.81 (m) 2.57 (m) 2.03 (m)
1.03 (m) 1.79 (m) 1.86 (m) 2.08 (m) 3.83 (m) 2.71 (m) 2.77 (m) 5.36 (br s) 1.83 (m) 2.47 (m) 2.10 (m)
0.95 (m) 1.70 (m) 1.71 (m) 2.08 (m) 3.86 (m) 2.44 (m) 2.67 (m) 5.27 (br s) 1.51 (m)
1.02 (m) 1.79 (m) 1.90 (m) 2.10 (m) 3.87 (m) 2.76 (m) 2.80 (m) 5.37 (br s) 1.86 (m) 2.48 (m) 2.07 (m)
0.96 (m) 1.71 (m) 1.88 (m) 2.14 (m) 3.89 (m) 2.74 (m) 2.78 (m) 5.28 (br s) 1.47 (m) 1.84 (m) 1.56 (m)
2.67 (m) 2.42 (m) 3.67 (m) 2.67 (m) 2.56 (m) 5.35 (br s) 1.53 (m)
1.05 (m) 1.85 (m) 1.82 (m) 2.08 (m) 3.96 (m) 2.73 (m) 2.75 (m) 5.38 (br s) 1.86 (m) 2.50 (m) 2.09 (m)
0.95 (m) 1.51 (m) 1.57 (m) 1.49 (m) 1.57 (m)
1.98 (m) 1.85 (m) 1.89 (m) 4.49 (m)
1.79 (m) 1.58 (m) 1.63 (m) 1.36 (m) 2.55 (m)
0.86 (m) 1.39 (m) 1.42 (m) 1.10 (m) 1.73 (m)
0.87 (m) 1.37 (m) 1.41(m) 1.10 (m) 1.74 (m)
1.82 (m) 1.58 (m) 1.50 (m) 2.29 (m)
0.88 (m) 1.38 (m) 1.46 (m) 1.12 (m) 1.73 (m)
1.56 (m) 1.46 (m) 1.37 (m) 2.19 (m) 1.65 (m)
1.49 (m) 2.32 (m)
14 15
2.01 (m) 1.59 (m) 2.15 (m)
– 1.85 (m) 2.50 (m)
1.03 (m) 1.83 (m) 1.96 (m)
1.02 (m) 1.40 (m) 1.99 (m)
– 1.93 (m) 2.33 (m)
1.06 (m) 1.84 (m) 2.00 (m)
1.22 (m) 1.88 (m) 2.48 (m)
– 1.93 (m) 2.45 (m)
16
4.74 (d, 7.8)
– 1.66 (m) 2.42 (dd, 14.4, 9.0) 5.31 (t, 9.0)
1.03 (m) 1.78 (m) 1.90 (m) 2.11 (m) 3.92 (m) 2.75 (m) 2.81 (m) 5.35 (br s) 1.84 (m) 2.47 (m) 2.02 (dd, 10.8, 5.4) 1.82 (m) 1.58 (m) 1.62 (m) 1.36 (br d, 12.6) 2.57 (dd, 12.6, 5.4) 1.06 (m) 1.85 (m) 2.49 (m)
3.87 (m)
1.52 (m)
0.96 (m) 1.68 (m) 1.71 (m) 2.08 (m) 3.86 (m) 2.44 (m) 2.67 (m) 5.27 (br s) 1.44 (m) 1.83 (m) 1.54 (m)
5.09 (t, 6.6)
4.92 (m)
4.93 (m)
5.09 (m)
4.94 (m)
5.09 (m)
4.95 (m)
17
–
–
–
1.90 (m)
1.91 (m)
1.92 (m)
1.92 (m)
1.95 (m)
2.76 (m)
18 19 20 21 23
0.88 (s) 1.06 (s) 2.48 (p, 7.2) 1.36 (d, 7.2) 2.07 (m)
5.42 (dd, 14.4, 7.2) 2.90 (dd, 14.4, 7.2) 1.15 (s) 1.13 (s) 2.36 (m) 1.54 (d, 7.2) 2.06 (m)
1.13 (s) 1.13 (s) 2.63 (q, 7.2) 1.41 (d, 7.2) 2.05-2.08 (m)
0.91 (s) 1.05 (s) 2.22 (m) 1.32 (d, 6.6) 2.03 (m)
1.70 (m) 2.07 (m) 2.00 (m) 3.62 (dd, 8.4, 4.8) 3.88 (m) 1.09 (d, 6.6) 5.02 (d, 7.8)
1.72 (m) 2.10 (m) 1.94 (m) 3.57 (dd, 9.6, 6.0) 4.03 (m) 1.01 (d, 7.2) 5.02 (d, 7.8)
4.27 (m) 4.27 (m) 4.15 (m) 3.91 (m) 4.44 (m) 4.51 (m)
4.27 (m) 4.27 (m) 4.17 (m) 3.89 (m) 4.49 (br d, 11.4) 4.35 (m)
2 3 4 6 7 8 9 11 12
0.99 (s) 1.12 (s) 2.56 (m) 1.41 (d, 8.4) 2.03 (m) 2.06 (m) 1.68 (m) 1.66 (m) 2.05 (m) 2.00 (m) 1.92 (m) 1.92 (m) 3.56 (dd, 9.6, 3.62 (dd, 11.4, 5.4) 7.2) 4.02 (m) 3.97 (m) 0.98 (d, 6.6) 1.01 (d, 7.8) 4.94 (d, 7.8) 5.01 (d, 7.8)
1.12 (s) 1.11 (s) 2.62 (m) 1.39 (d, 7.2) 2.07 (m)
0.88 (s) 0.90 (s) 2.22 (m) 1.32 (d, 6.6) 2.02 (m)
0.89 (s) 0.91 (s) 2.23 (m) 1.31 (d, 6.6) 2.01 (m)
1.71 (m) 2.07 (m) 1.93 (m) 3.63 (dd, 9.0, 5.4) 4.01 (m) 1.00 (d, 6.6) 4.94 (d, 7.8)
1.67 (m) 2.00 (m) 1.90 (m) 3.64 (dd, 9.6, 6.0) 3.96 (m) 1.01 (d, 6.6) 4.95 (d, 7.8)
1.61 (m) 2.01 (m) 1.98 (m) 3.86 (m) 4.07 (m)
4.20 (m) 4.22 (m) 4.18 (m) 3.82 (m) 4.43 (m) 4.49 (m)
4.27 (m) 4.26 (m) 4.17 (m) 3.91 (m) 4.35 (m) 4.49 (m)
4.19 (m) 4.20 (m) 4.18 (m) 3.81 (m) 4.41 (m) 4.49 (m)
4.21 (m) 4.23 (m) 4.18 (m) 3.83 (m) 4.43 (br d, 11.4) 4.50 (br d, 11.4)
4.21 (m) 4.20 (m) 4.18 (m) 3.83 (m) 4.45 (br d, 11.4) 4.52 (br d, 11.4)
Rha-1 2 3
6.25 (br s) 4.78 (br s) 4.58 (m)
6.39 (s) 4.82 (br s) 4.65 (m)
4 5 6 Xyl-1 2 3
4.33 (m) 4.92 (m) 1.75 (d, 6.6) 5.02 (d, 7.8) 4.03 (m) 4.09 (m)
4.35 (m) 5.00 (m) 1.79 (d, 7.2)
4 5
4.15 (m) 3.66 (t, 10.8) 4.23 (m)
6.24 (br s) 4.77 (br s) 4.58 (dd, 9.0, 3.6) 4.33 (m) 4.92 (m) 1.76 (d, 6.0) 5.01 (d, 7.8) 3.95 (m) 4.08 (dd, 8.4, 7.8) 4.15 (m) 3.66 (dd, 11.4, 10.8) 4.24 (m)
Glc2-1 2 3 4 5 6
4.72 (d, 7.8) 4.21 (m) 4.21 (m) 4.15 (m) 4.04 (m) 4.32 (m) 4.82 (m)
Glc3-1
5.09 (d, 7.8)
24 25 26
27 Glc1-1/ Fuc-1 2 3 4 5 6
4.82 (d, 7.8) 4.02 (m) 4.24 (m) 4.19 (m) 3.94 (m) 4.53 (m)
4.81 4.01 4.22 4.21 3.93 4.36 4.53
(d, 7.8) (m) (m) (m) (m) (m) (m)
1.05 (d, 6.0) 4.95 (d, 7.8)
6.35 (br s) 4.79 (br s) 4.62 (dd, 9.0, 3.0) 4.34 (m) 4.99 (m) 1.78 (d, 6.6) 5.02 (d, 7.8) 3.97 (m) 4.09 (dd, 9.0, 8.4) 4.16 (m) 3.66 (dd, 11.4, 10.2) 4.26 (dd, 11.4, 4.8) 4.72 (d, 7.8) 4.03 (m) 4.23 (m) 4.14 (m) 4.03 (m) 4.32 (m) 4.81 (d, 9.6)
5.02 (d, 7.8) 3.97 (m) 4.09 (dd, 9.0, 8.4) 4.17 (m) 3.66 (dd, 11.4, 10.2) 4.26 (dd, 11.4, 4.8) 4.84 (d, 6.6) 4.12 (m) 4.11 (m) 4.17 (m) 3.93 (m) 4.38 (m) 4.49 (m)
4.86 4.22 4.30 4.30 3.86 4.34 4.42
5.09 (d, 7.8)
5.28 (d, 7.8)
5.27 (d, 7.8)
(d, 7.8) (m) (m) (m) (m) (m) (m)
4.74 (d, 7.8) 3.96 (m) 4.16 (m) 4.15 (m) 4.05 (m) 4.83 (br d, 10.2) 4.33 (m) 5.10 (d, 7.8)
1.46 (m)
1.90 (m) 1.56 (m)
0.92 (s) 1.41 (s) 2.19 (q, 7.2) 1.48 (d, 7.2) 2.45 (m) 2.20 (m) 1.67 (m) 1.63 (m) 2.02 (m) 1.65 (m) 1.90 (m) 1.96 (m) 3.56 (dd, 9.6, 3.58 (dd, 9.6, 6.0) 6.0) 3.95 (m) 4.02 (m) 0.98 (d, 6.6) 1.26 (d, 7.2) 5.03 (d, 7.2) 4.66 (d, 7.8)
1.01 (s) 1.12 (s) 2.09 (m) 1.11 (d, 7.8) 2.03 (m) 2.75 (m) 4.07 (m)
4.22 (m) 4.18 (m) 4.12 (m) 3.94 (m) 4.34 (m) 4.50 (m)
4.02 (m) 4.06 (m) 4.59 (m) 4.23 (m) 4.49 (br d, 11.4) 4.35 (m)
4.20 (m) 4.22 (m) 4.18 (m) 3.84 (m) 4.39 (m) 4.52 (m)
6.35 (br s) 4.80 (m) 4.61 (m)
6.34 (s) 4.21 (m) 4.79 (m)
4.34 (m) 4.98 (m) 1.76 (d, 6.6)
4.08 (m) 4.86 (m) 1.74 (d, 6.6) 4.98 (d, 7.2) 3.57 (m) 3.93 (m)
6.27 (br s) 4.80 (br s) 4.60 (dd, 11.4, 3.6) 4.43 (m) 4.96 (m) 1.78 (d, 7.2) 5.06 (d, 7.8) 3.98 (m) 4.08 (m)
3.76 (m) 4.26 (m) 3.69 (m)
4.16 (m) 3.68 (m) 4.26 (m)
4.78 (d, 7.8) 4.07 (m) 4.21 (m) 4.67 (m) 3.99 (m) 4.53 (m) 4.36 (m)
4.96 (d, 7.2) 4.06 (m) 4.22 (m) 4.27 (m) 3.98 (m) 4.39 (m) 4.51 (m)
4.72 (d, 7.8) 3.98 (m) 4.22 (m) 4.16 (m) 4.04 (m) 4.32 (m) 4.82 (m) 5.09 (d, 7.2)
1.90 (m) 3.56 (m) 3.63 (m) 1.34 (d, 9.6) 5.03 (d, 9.0)
76
R. Yan et al. / Phytochemistry Letters 15 (2016) 72–80
Table 1 (Continued) Pos.
1
2 3 4 5 6
4.01 (m) 4.16 (m) 4.22 (m) 3.91 (m) 4.36 (m) 4.50 (m)
2
3
5 4.03 4.22 4.21 3.93 4.34 4.50
(m) (m) (m) (m) (m) (m)
6
7
8
9
4.09 (m) 4.21 (m) 4.27 (m) 3.95 (m) 4.30 (m) 4.48 (m)
4.01 (m) 4.22 (m) 4.30 (m) 3.96 (m) 4.41 (m) 4.50 (m)
4.03 (m) 4.21 (m) 4.23 (m) 3.91 (m) 4.35 (m) 4.49 (br d, 11.4)
4.02 (m) 4.16 (m) 4.22 (m) 3.91 (m) 4.34 (m) 4.50 (m)
5-ene-furosta-3b,22a,26-triol-3-O-b-D-xylopyranosyl-(1 ! 4)b-D-glucopyranoside. Ophiofurospiside I (7) was obtained as white amorphous powder. Its molecular formula was determined as C51H84O24 by negative ion HRESIMS with a quasi-molecular ion peak at m/z 1079.5294 [M-H]– (calcd. for C51H83O24, 1079.5274). HRESIMS showed fragment ion peaks at m/z 917.4 [M-H-162]–, 771.4 [M-H162-146]–, 591.4 [M-H-162-146-180]–, and 429.3 [M-H-162-146180-162]– in negative ion mode, suggesting the presence of three hexosyl and one deoxyhexosyl moieties. 1H NMR spectrum of 7 also exhibited four sugar anomeric protons at dH 5.02 (d, J = 7.8 Hz), 6.35 (br s), 4.86 (d, J = 7.8 Hz), and 5.27 (d, J = 7.8 Hz), with their corresponding anomeric carbons at dC 100.3, 102.1, 103.2, and 106.6 in the 13C NMR spectrum, respectively. Two tertiary methyls at dH 1.13 (3H, s) and 1.15 (3H, s), two secondary methyls at dH 1.09 (3H, d, J = 6.6 Hz), 1.54 (3H, d, J = 7.2 Hz), an olefinic proton at dH 5.37 (1H, br s) can be deduced from 1H NMR spectrum. In the 13C NMR spectrum showed the olefinic carbon signals at dC 140.4 (C-5), 122.4 (C-6), hemiketal carbon signal at dC 110.0 (C-22), two oxygenated methines at dC 78.1 (C-3) and 81.9 (C-16), and one oxygenated quaternary carbon signal at dC 86.4 (C-14). All these mentioned NMR data suggested that 7 had an aglycone, (25R)furosta-5-ene-3b,14a,22a,26-tetraol. It is also supported by comparing NMR data with these of ophiopogonins I, J and N, which co-existed in entitled plant (Zhang et al., 2012). A detailed comparison of the NMR data of 7 with those of ophiopogonin J indicated that they had the same aglycone and sugar chain at C-26. The sugar sequence of a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranosyl and its linkage at C-3 were identified by HMBC correlations between H-1 of Rha (dH 6.35) and C-2 of Glc1 (dC 78.0), and between H-1 of Glc1 (dH 5.02) and C-3 (dC 78.1). Thus, ophiofurospiside I (7) was determined to be (25R)-26-O-[b-Dglucopyranosyl-(1 ! 2)]-b-D-glucopyranosyl-5-ene-furosta3b,14a,22a,26-tetraol-3-O-a-L-rhamnopyranosyl-(1 ! 2)-b-Dglucopyranoside. Ophiofurospiside J (8) was obtained as white amorphous powder. Its molecular formula was determined as C45H74O19 by negative ion HRESIMS with a quasi-molecular ion peak at m/z 917.4778 [M-H]– (calcd. for C45H73O19, 917.4746). 1H NMR spectrum of 8 exhibited three sugar anomeric protons at dH 5.02 (d, J = 7.8 Hz), 4.74 (d, J = 7.8 Hz), and 5.10 (d, J = 7.8 Hz), with their corresponding anomeric carbons at dC 102.6, 103.2, and 106.6 in the 13C NMR spectrum, respectively. Two tertiary methyls at dH 1.13 (6H, s), two secondary methyls at dH 1.01 (3H, d, J = 7.2 Hz), 1.41 (3H, d, J = 7.2 Hz), an olefinic proton at dH 5.35 (1H, br s) can be deduced from 1H NMR spectrum. These 1H NMR data, together with olefinic carbon signals at dC 140.9 (C-5), 121.8 (C-6) and the carbon signal at dC 110.7 (C-22) in the 13C NMR spectrum suggested that 8 had a 5,6ene-furostanol skeleton. A detailed comparison of the NMR data of 8 with those of 6 indicated that they had the same aglycone, and only sugar chain at C-3 was different. The glucosyl located at C-3 was deduced from the HMBC correlation between H-1 of Glc1 (dH 5.02, d, J = 7.8 Hz) and C-3 of the aglycone (dC 78.3). Thus, ophiofurospiside J (8) was determined to be (25R)-26-O-[b-D-
12
13
glucopyranosyl-(1 ! 2)]-b-D-glucopyranosyl-5-ene-furosta3b,22a,26-triol-3-O-b-D-glucopyranoside. Ophiofurospiside K (9) was obtained as white amorphous powder. Its molecular formula was determined as C51H84O23 by negative ion HRESIMS with a quasi-molecular ion peak at m/z 1063.5397 [M-H]– (calcd. for C51H83O23, 1063.5325). HRESIMS showed fragment ion peaks at m/z 901.4 [M-H-162]–, 755.4 [M-H162-146]–, 593.4 [M-H-162-146-162]–, and 431.3 [M-H-162-146162-162]– in negative ion mode, suggesting the presence of three hexosyl and one deoxyhexosyl moieties. A detailed comparison of the NMR data of 9 with those of 5 indicated that they had the same aglycone and C-26 disaccharide chain, and the only difference between two compounds was the sugar chain at C-3. Comparing the 13C NMR data with these of compound 2 indicated that they shared the same sugar chain at C-3. It is also supported by the HMBC correlations between H-1 of Glc1 (dH 5.03, d, J = 7.2 Hz) and C-3 of the aglycone (dC 78.3), between H-1 of Rha1 (dH 6.35, br s) and C-2 of the Glc1 (dC 78.0). Thus, ophiofurospiside K (9) was determined to be (25R)-26-O-[b-D-glucopyranosyl-(1 ! 6)]-b-Dglucopyranosyl-5-ene-furosta-3b,22a,26-triol-3-O-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranoside. Ophiofurospiside N (12) was obtained as a white amorphous powder. Its molecular formula was determined to be C50H82O22 by HRESIMS m/z 1033.5271 [M-H]– (calcd. for C50H81O22, 1033.5219). HRESIMS showed fragment ion peaks at m/z 887.4 [M-H-146]–, 775.4 [M-H-146-132]–, 609.3 [M-H-146-132-146]–, and 447.3 [M-H-146-132-146-162]– in negative ion mode, suggesting the sequential losses of two deoxyhexosyl, one pentosyl, and one hexosyl moieties. The combined analysis of 1 H, 13C NMR, and HSQC spectrum of 12, showed the presence of four methyl groups [dH/dC 0.92 s/17.0 (agly 18); 1.41 s/19.2 (agly 19); 1.48 (d, J = 7.2 Hz)/16.4 (agly 21); 1.26 (d, J = 7.2 Hz)/17.5 (agly 27)], an olefinic group [dH/dC 5.35 br s/(139.6, 124.8)], a hydroxymethene [dH/dC 3.58 (dd, J = 9.6, 6.0 Hz), 4.02 m/75.2], two oxygenated methines (dH/dC 3.87 m/84.4; 3.67 m/67.1 and a hemiketal (dC 110.7) suggesting a 5-ene-furosta-1b,3b,22a,26tetraol moiety. Four anomeric proton signals at dH 4.66 (d, J = 7.8 Hz), 6.34 s, 4.98 (d, J = 7.2 Hz), 4.78 (d, J = 7.8 Hz), and 1.74 (d, J = 6.6 Hz) indicated that compound 12 contains four sugar moieties of which one is rhamnose. The NMR spectral data of 12 were almost consistent with those of (25S)-26-O-b-Dglucopyranosyl-furosta-5-ene-1b,3b,22a,26-teraol-1-O-b-Dxylopyranosyl-(1 ! 3)-[a-L-rhamnopyranosyl-(1 ! 2)]-b-D-glucopyranoside, except that the signal of H-26 (dH 3.58, 4.02; Dab = 0.44 < 0.48 demonstrated the 25R configuration of 12 (Agrawal 2004, 2005). Thus, the structure of 12 was determined to be (25R)-26-O-b-D-glucopyranosyl-5-ene-furosta-1b,3b,22a,26-teraol-1-O-b-D-xylopyranosyl-(1 ! 3)-[a-Lrhamnopyranosyl-(1 ! 2)]-b-D-fucopyranoside). The molecular formula of ophiofurospiside L (10) was determined as C50H82O23 by HRESIMS, showing a pseudomolecular ion peak at m/z 1049.5183 [M-H]– (calcd. for C50H81O23, 1049.5169). Further fragment ion peaks were observed at m/z 917.5, 771.4, 591.4, and 429.3 corresponding to the loss of one pentosyl, one
R. Yan et al. / Phytochemistry Letters 15 (2016) 72–80
77
Table 2 13 C NMR data of compounds 1–13 in pyridine-d5. Pos.
1
2
3
5
6
7
8
9
10
11
12
13
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Glc1-1/Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Xyl-1 2 3 4 5 Glc2-1 2 3 4 5 6 Glc3-1 2 3 4 5 6
37.6 30.2 78.2 39.0 140.8 121.9 32.5 32.3 50.3 37.2 21.0 32.2 45.1 53.1 32.0 90.5 90.9 17.3 19.5 43.6 10.5 111.4 36.9 28.1 34.2 75.2 17.5 100.1 77.5 76.3 81.5 77.3 61.7 102.0 72.5 72.8 74.2 69.6 18.7 105.8 75.1 78.4 70.8 67.4 104.9 75.0 78.5 71.6 77.3 70.2 105.5 75.4 78.5 71.6 78.5 62.8
37.6 30.2 78.3 39.0 139.9 122.7 26.6 36.4 39.4 37.1 29.4 76.4 48.6 88.2 39.9 91.6 94.8 21.9 19.3 45.1 10.1 111.2 35.9 28.0 34.2 75.2 17.4 100.3 79.7 77.9 71.9 77.8 62.9 102.1 72.6 72.9 74.2 69.5 18.7
37.8 30.3 78.3 39.0 140.3 122.4 26.2 36.2 43.6 37.4 20.2 26.7 48.5 87.7 40.3 90.8 91.6 20.8 19.4 43.9 10.7 111.2 37.1 28.1 34.3 75.3 17.5 100.0 77.3 76.3 81.6 77.6 61.7 102.0 72.5 72.9 74.2 69.6 18.7 105.8 75.0 78.4 70.8 67.4 105.0 75.3 78.7 71.8 78.5 62.9
37.5 30.2 78.4 39.3 140.9 121.8 32.3 31.7 50.4 37.2 21.2 40.0 40.7 56.6 32.5 81.1 63.9 16.5 19.4 40.7 16.5 110.7 37.1 28.5 34.3 75.4 17.5 102.4 77.6 77.3 81.0 76.3 61.7
37.5 30.3 78.3 39.3 140.9 121.8 32.3 31.7 50.4 37.3 21.2 40.0 40.8 56.7 32.5 81.7 63.8 16.5 19.4 40.9 16.6 110.7 37.1 28.3 34.4 75.2 17.6 102.4 77.6 77.3 84.3 76.3 61.7
37.8 30.3 78.1 39.1 140.4 122.4 26.8 35.6 43.7 37.4 20.5 32.1 45.5 86.4 40.1 81.9 60.6 20.2 19.4 41.0 16.7 111.0 37.5 28.3 34.5 75.2 17.7 100.3 79.7 78.0 71.9 77.8 62.6 102.1 72.6 72.9 74.2 69.5 18.7
37.5 30.3 78.3 39.4 140.9 121.8 32.3 31.7 50.3 37.3 21.2 40.0 40.8 56.7 32.5 81.2 63.8 16.5 19.4 40.9 16.6 110.7 37.1 28.2 34.4 75.2 17.6 102.6 75.2 77.0 81.2 77.0 62.6
37.6 30.3 78.3 39.1 140.9 121.8 32.4 31.7 50.4 37.2 21.1 40.0 40.7 56.7 32.5 81.1 63.9 16.5 19.5 40.8 16.5 110.7 37.2 28.5 34.3 75.2 17.5 100.4 79.7 78.0 71.9 77.9 62.7 102.1 72.6 72.9 74.2 69.5 18.7
77.7 40.8 75.2 39.5 139.2 125.2 32.4 33.0 51.3 43.8 24.2 40.6 40.6 56.8 32.8 81.1 64.2 16.7 13.8 40.9 16.5 110.6 37.3 28.4 34.3 75.3 17.5 100.5 79.6 78.0 71.7 77.7 62.9 102.1 72.6 72.9 74.2 69.5 18.7
105.6 75.0 78.4 70.8 67.4 104.9 75.1 78.5 71.6 77.3 70.2 105.5 75.4 78.6 71.6 78.5 62.8
105.6 75.0 78.4 70.8 67.4 103.2 84.3 78.3 71.4 78.4 62.7 106.6 77.0 78.1 71.5 78.7 62.6
103.2 84.3 78.3 71.4 78.7 62.7 106.6 77.0 78.1 71.5 78.7 62.7
103.2 84.3 78.3 71.5 78.5 62.6 106.6 77.0 78.1 71.5 78.8 62.9
104.9 75.1 78.5 71.7 77.3 70.2 105.5 75.4 78.6 71.9 78.5 62.8
77.4 40.8 75.2 39.5 139.1 125.3 32.3 32.9 51.3 43.8 24.2 40.6 40.6 56.8 32.8 81.1 64.2 16.7 13.7 40.9 16.5 110.7 37.3 28.4 34.3 75.3 17.5 100.2 77.9 76.3 81.4 77.3 61.6 101.9 72.5 72.8 74.2 69.6 18.7 105.8 75.0 78.6 70.8 67.4 105.0 75.2 78.5 71.7 78.4 62.9
84.4 37.2 67.1 43.9 139.6 124.8 32.1 32.7 50.6 42.9 24.1 40.6 40.6 57.2 33.1 81.2 64.0 17.0 15.1 40.8 16.4 110.7 38.1 28.3 34.3 75.2 17.5 100.5 74.8 85.6 72.8 71.7 17.2 101.8 70.9 72.6 74.3 68.3 19.2 106.7 74.8 78.4 71.1 78.5 105.0 75.3 78.5 69.4 78.4 62.9
37.8 30.2 78.1 39.0 140.3 122.4 26.7 35.6 43.6 37.5 20.4 31.9 45.1 86.4 39.8 81.7 62.9 20.0 19.4 42.2 15.2 112.0 41.0 82.3 38.2 65.1 13.6 99.9 77.5 76.3 81.5 77.3 61.7 102.0 72.5 72.8 74.2 69.6 18.7 105.8 75.0 70.8 70.8 67.4 106.5 75.7 78.7 71.7 78.1 62.9
105.0 75.2 78.7 71.7 78.5 62.7
deoxyhexosyl and two hexosyl moieties. The 13C NMR spectrum displayed 50 resonance signals and 27 due to the aglycone part, including one olefinic, one oxygenated methene, three oxygenated methines, and one hemiketal carbons. 13C NMR spectral data of aglycone 10 were close to those of compound 12, except that the signal of C-3 (dC 75.2) was by shielded Dd 8.1, and signal of C-1 was deshielded by Dd + 7.0, suggesting the glycosylation at C-3. The 13 C NMR data of sugar part of 10 were identical with those of 3. On the basis of above evidences, 10 was identified as (25R)-26-O-b-Dglucopyranosyl-5-ene-furosta-1b,3b,22a,26-teraol-3-O-b-Dxylopyranosyl-(1 ! 4)-[a-L-rhamnopyranosyl-(1 ! 2)]-b-D-glucopyranoside. The molecular formula of ophiofurospiside M (11) was deduced to be C45H74O19 on the basis of its quasi-molecular ion peaks at m/z 917.4756 [M-H]– (calcd. for C45H73O19, 917.4746) in its HRESIMS.
105.0 74.8 78.5 71.7 78.3 62.6
Fragment ion peaks at m/z 771.4, 591.4, and 429.3 were corresponded to the loss of one deoxyhexosyl and two hexosyl moieties. The 13C NMR spectrum displayed one olefinic, one oxygenated methene, three oxygenated methines, and one hemiketal carbons. 13C NMR spectral data of aglycone were consistent with those of compound 10. In the 13C NMR spectrum of 11, the data of sugar chain were identical with those of 2. On the basis of above evidences, 11 was identified as (25R)-26-O-b-D-glucopyranosyl-5-ene-furosta-1b,3b,22a,26-teraol-3-O-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranoside. The molecular formula of ophiofurospiside R0 (13) was determined as C50H80O23 by HRESIMS, showing a pseudomolecular ion peak at m/z 1047.5073 [M-H]– (calcd. for C50H79O23, 1047.5012). Further fragment ion peaks were observed at m/z 915.5, 769.3, 607.3, and 445.3 corresponding to the loss of one pentosyl, one
78
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deoxyhexosyl and two hexosyl moieties. Inspection of the 1H NMR spectrum of 12 led to the assignment of two tertiary methyl groups [dH 1.01 (3H, s, Me-18) and 1.12 (3H, s, Me-19)], two secondary methyl groups [dH 1.34 (3H, d, J = 9.6 Hz, CH3-27) and 1.11 (3H, d, J = 7.8 Hz, CH3-21)], and one olefinic proton [dH 5.38 (1H, br s)]. These proton signals together with a distinctive quaternary carbon signal at dC 112.0 showing long-range correlations with one of the methylene protons at dH 3.63 (Agly H-26a) along with the proton at dH 1.11 (Agly Me-21) indicated that the aglycone of 13 was a spirosta-5-ene type. On the other hand, HMBC correlations between the proton signal at dH 4.07 (H-24) and the carbon signal at dC 112.0 (C-22), and between the proton signal at dH 1.34 (Me-27) and the carbon signals at dC 82.3 (C-24), suggested the presence of hydroxyl group at C-24. The NOE correlations between H-23eq (d 2.75)/H-24 (d 4.07), H-27 (d 1.34)/H-24 (d 4.07) and the 13C NMR chemical shifts of C-23–C-27 allowed assignment of the 24S and 25S (Zhang et al., 2012). The aglycone of 13 was identified as (25S)spirosta-5-ene-3b,14a,24b-triol, by comparison of 1H and 13C NMR spectroscopic data obtained from 2D NMR spectra of 13 with those reported in the literature (Zhang et al., 2012). The presence of four sugar residues was confirmed from observation of four anomeric proton signals [dH 5.03 (d, J = 9.0 Hz), 6.27 br s, 5.06 (d, J = 7.8 Hz), and 4.96 (d, J = 7.2 Hz)], giving HSQC correlations with four anomeric carbon signals at dC 99.9, 102.0, 105.8, and 106.5, respectively. Complete assignments of each sugar unit were carried out by extensive 1D and 2D NMR analyses. Two b-glucopyranosyl (Glc1 and Glc2), one b-xylopyranosyl (Xyl), and one a-rhamnopyranosyl (Rha) units were identified in 13. The HMBC correlations between the anomeric 1H NMR signal at dH 5.03 (Glc1 H-1) and dC 78.1 (Agly C-3), between dH 6.27 (Rha-1) and dC 77.5 (Glc1 C-2), between dH 5.06 (Xyl H-1) and dC 81.5 (Glc1 C-4) proved the sequence of the sugar chain at C-3 to be a-Lrhamnopyranosyl-(1 ! 2)-[b-D-xylopyranosyl-(1 ! 4)]-b-D-glucopyranosyl. Finally, HMBC correlation between dH 4.96 (Glc2 H-1) and dC 82.3 (Agly C-24) allowed the linkage of Glc2 at C-24. Thus, 13 was determined to be (25S)-24-O-b-D-glucopyranosyl-spirosta-5ene-3b,14a,24b-triol-3-O-a-L-rhamnopyranosyl-(1 ! 2)-[b-Dxylopyranosyl-(1 ! 4)]-b-D-glucopyranoside. 3. Experimental 3.1. General Optical rotations were measured with a PerkinElmer 343 polarimeter (PerkinElmer, Waltham, MA, USA). The NMR spectra were recorded in pyridine-d5 using a Varian Inova-600 spectrometer (600 MHz for 1H NMR and 150 MHz for 13C NMR) (Palo Alto, CA, USA). The HRESIMS was recorded on a Synapt MS system (Waters Corporation, Milford, MA, USA). Macroporous resin SP825 (Mitsubishi Chemical, Tokyo, Japan) and MCI CHP20 (Mitsubishi Chemical), ODS silica gel (50 mm, YMC, Kyoto, Japan), and silica gel H (Qingdao Marine Chemical, Inc., Qingdao, China) were used for column chromatography. TLC was performed on precoated silica gel 60 plates (Merck, Darmstadt, Germany). HPLC was performed using Agilent 1100 system with the following components: Alltech ELSD 2000 (Alltech, Lexington, KY, USA), and an analytical column, Agela Venusil XBP C18 (2) (5 mm, 4.6 250 mm, Agela, Tianjin, China). Preparative HPLC separations were performed using PrimeLine solvent delivery module (ASI, Salt Lake City, UT, USA) equipped with Shodex refractive index detector (RID) 102 detector (Showa Denko Group, Tokyo, Japan) and a preparative column, Venusil XBP C18 column (5 mm, 10.0 250 mm, Agela, Tianjin, China). GC analysis was carried out on an Agilent 6890 equipped with an H2 flame ionization detector and an HP-5 capillary column (30 m 0.25 mm 0.25 mm) (Agilent Technologies, Milford, MA, USA).
3.2. Plant material The tubers of O. japonicus were collected from the Mianyang region of Sichuan Province, China in April 2007. The plant was identified by Prof. Li-Juan Zhang (Tianjin University of Traditional Chinese Medicine) and a voucher specimen (No. 070403) was deposited at the Herbarium of Beijing Institute of Radiation Medicine, Beijing, China. 3.3. Extraction and isolation The fresh tubers of O. japonicus (dried at 80 C) (56.0 kg) were extracted twice with 60% EtOH (450 L, 1 h each time) under reflux, followed by evaporation of solvent in vacuum. The concentrated extract was separated on SP825 macroporous resin column chromatography eluted successively with EtOH–H2O (1:4, 1:1, and 9:1) to give three fractions (A–C). Fr.B (220 g) was chromatographed on macroporous resin SP825 and eluted with a gradient mixture of EtOH–H2O (2:8, 3:7, 4:6, 11:9, and 8:2) to afford 12 fractions (B1–B12). A part of Fr.B4 (11.3 g) was separated chromatographically over silica gel column and eluted successively with a gradient mixture of CHCl3–MeOH–H2O (65:25:4, 65:35:10, and 60:40:12), and a total of 50 sub-fractions were collected. Sub-fractions 31–43 (3.2 g) were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 17:83 and 20:80), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with MeCN–H2O (23:77) yielded compound 1 (30.0 mg). Sub-fractions 13–17 (600 mg) were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 18:82 and 20:80), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with MeCN–H2O (26:74) yielded compounds 2 (12.0 mg), 3 (17.6 mg), and 4 (9.5 mg). A part of Fr.B5 (20.0 g) was separated chromatographically over silica gel column and eluted successively with a gradient mixture of CHCl3–MeOH–H2O (70:26:6, 65:36:10, and 60:40:12), and a total of 70 sub-fractions were collected. Sub-fractions 42– 48 were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 20:80), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with MeCN–H2O (26:74) yielded compounds 10 (8.2 mg) and 11 (10.3 mg). Sub-fractions 49–51 were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 18:82), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with Me2CO– H2O (18:82) yielded 13 (17.0 mg). Sub-fractions 52–62 were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 20:80), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with Me2CO–H2O (20:80) yielded 7 (8.9 mg). A part of Fr.B6 (27.0 g) was separated chromatographically over silica gel column and eluted successively with a gradient mixture of CHCl3–MeOH–H2O (70:26:6, 65:36:10, and 60:40:12), and a total of 100 sub-fractions were collected. Sub-fractions 38–46 (3.2 g) were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 28:72), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with Me2CO– H2O (35:65) yielded compound 8 (9.3 mg). Sub-fractions 57–62 were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 18:82 and 20:80), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with Me2CO–H2O (20:80) yielded compounds 5 (8.4 mg), 6 (8.1 mg), and 12 (9.3 mg). Sub-fractions 78–80 were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 20:80), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with MeCN–H2O (24:76) yielded compound 9 (46.3 mg).
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3.3.1. Ophiofurospiside C (1) White amorphous powder; a20 D – 79.5 (c 0.110, pyridine); HRESIMS: m/z 1211.5717 [M-H]– (calcd. for C56H91O28, 1211.5697); ESI-MS2: m/z 1193.5725 [M-H-18]–, 1061.5 [M-H-18132]–, 899.4 [M-H-18-132-162]–, 753.3 [M-H-18-132-162-146]–, 591.3 [M-H-18-132-162-146-162]–, and 429.3 [M-H-18-132-162146-162-162]–, and 411.3 [M-H-18-132-162-146-162-162-18]–; 1H NMR data, see Table 1; 13C NMR data, see Table 2. 3.3.2. Ophiofurospiside D (2) White amorphous powder; a20 D – 89.7 (c 0.112, pyridine); HRESIMS: m/z 949.4677 [M-H]– (calcd. for C45H73O21, 949.4644); ESI-MS2: m/z 931.5 [M-H-18]–, 913.4 [M-H-18-18]–, 767.4 [M-H-1818-146]–, 605.4 [M-H-18-18-146-162]–, 443.3 [M-H-18-18-146162-162]–, and 425.3 [M-H-18-18-146-162-162-18]–; 1H NMR data, see Table 1; 13C NMR data, see Table 2. 3.3.3. Ophiofurospiside E (3) White amorphous powder; a20 D – 87.9 (c 0.091, pyridine); HRESIMS: m/z 1065.5144 [M-H]– (calcd. for C50H81O24, 1065.5118); ESI-MS2: m/z 1047.5 [M-H-18]–, 915.4 [M-H-18132]–, 769.3 [M-H-18-132-146]–, 607.3 [M-H-18-132-146-162]–, 445.3 [M-H-18-132-146-162-162]–, and 409.2 [M-H-18-132-146162-162-36]–; 1H NMR data, see Table 1; 13C NMR data, see Table 2. 3.3.4. Ophiofurospiside G (5) White amorphous powder; a20 D – 90.9 (c 0.089, pyridine); HRESIMS: m/z 1049.5162 [M-H]– (calcd. for C50H81O24, 1049.5169); ESI-MS2: m/z 887.5 [M-H-162]–, 755.4 [M-H-162132]–, 737.4 [M-H-162-132-18]–, 575.4 [M-H-162-132-18-162]–, and 413.3 [M-H-162-132-18-162-162]–; 1H NMR data, see Table 1; 13 C NMR data, see Table 2. 3.3.5. Ophiofurospiside H (6) White amorphous powder; a20 D – 59.6 (c 0.079, pyridine); HRESIMS: m/z 1049.5162 [M-H]– (calcd. for C50H81O23, 1049.5169); ESI-MS2: m/z 887.5 [M-H-162]–, 755.4 [M-H-162132]–, 737.4 [M-H-162-132-18]–, 575.4 [M-H-162-132-18-162]–, and 413.3 [M-H-162-132-18-162-162]–; 1H NMR data, see Table 1; 13 C NMR data, see Table 2. 3.3.6. Ophiofurospiside I (7) White amorphous powder; a20 D – 64.5 (c 0.087, pyridine); HRESIMS: m/z 1079.5294 [M-H] – (calcd. for C51H83O24, 1079.5274); ESI-MS2: m/z 917.4 [M-H-162]–, 771.4 [M-H-162146]–, 591.4 [M-H-162-146-180]–, 429.3 [M-H-162-146-180-162]–, and 411.3 [M-H-162-146-180-162-18]–; 1H NMR data, see Table 1; 13 C NMR data, see Table 2. 3.3.7. 7 Ophiofurospiside J (8) White amorphous powder; a20 D – 66.3 (c 0.077, pyridine); HRESIMS: m/z 917.4778 [M-H]– (calcd. for C45H73O19, 917.4746); 1 H NMR data, see Table 1; 13C NMR data, see Table 2.
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3.3.9. Ophiofurospiside L (10) White amorphous powder; a20 D – 70.1 (c 0.110, pyridine); HRESIMS: m/z 1049.5183 [M-H] – (calcd. for C50H81O23, 1063.5169); ESI-MS2: m/z 917.5 [M-H-132]–, 771.4 [M-H-132146]–, 591.4 [M-H-132-146-180]–, 429.3 [M-H-132-146-180-162]–, and 411.3 [M-H-132-162-146-180-162-18]–; 1H NMR data, see Table 1; 13C NMR data, see Table 2. 3.3.10. Ophiofurospiside M (11) White amorphous powder; a20 D – 74.2 (c 0.081, pyridine); HRESIMS: m/z 917.4756 [M-H] – (calcd. for C45H73O19, 917.4746); ESI-MS2: m/z 771.4 [M-H-146]–, 591.4 [M-H-146-180]–, 429.3 [MH-146-180-162]–, and 411.3 [M-H-146-180-162-18]–; 1H NMR data, see Table 1; 13C NMR data, see Table 2. 3.3.11. Ophiofurospiside N (12) White amorphous powder; a20 D – 79.3 (c 0.072, pyridine); HRESIMS: m/z 1033.5271 [M-H]– (calcd. for C50H81O22, 1033.5219); ESI-MS2: m/z 887.4 [M-H-146]–, 775.4 [M-H-146132]–, 609.3 [M-H-146-132-146]–, 447.3 [M-H-146-132-146-162]–, and 429.3 [M-H-146-132-146-162-18]–; 1H NMR data, see Table 1; 13 C NMR data, see Table 2. 3.3.12. Ophiopogonin R0 (13) White amorphous powder; a20 D – 85.4 (c 0.084, pyridine); HRESIMS: m/z 1047.5073 [M-H]– (calcd. for C50H79O23, 1047.5012); ESI-MS2: m/z 915.5 [M-H-132]–, 769.3 [M-H-132146]–, 607.3 [M-H-132-146-162]–, 445.3 [M-H-132-146-162-162]–, and 427.3 [M-H-132-146-162-162-18]–; 1H NMR data, see Table 1; 13 C NMR data, see Table 2. 3.4. Acid hydrolysis and sugar analysis Total saponin (Fr.B) was hydrolyzed in 2 M CF3COOH (5 mL) at 95 C for 5 h. The reaction mixture was extracted with CH2Cl2 (5 mL) three times. The aqueous layer was repeatedly evaporated to dryness with EtOH until neutral. The residue of the sugars was dissolved in anhydrous pyridine (2 mL), 12 mg of L-cysteine methyl ester hydrochloride was added, and the mixture was stirred at 60 C for 1 h. Then, HMDS–TMCS (hexamethyldisilazane–trimethylchlorosilane 2:1) (6.0 mL) was added, and the mixture was kept at 60 C for 30 min. The reference substances, D-glucose, D-xylose, and L-rhamnose (each 1.0 mg) were reacted as above procedure. After centrifugation, the supernatant was analyzed by GC under the following conditions: column temperature: 180 C/250 C; programmed increase, 15 C/min; carrier gas: N2 (1 mL/min); injection and detector temperature: 250 C; injection volume: 1.0 mL, and split ratio: 1/50. The derivatives of D-glucose, D-xylose, and L-rhamnose were detected, with tR values of 17.932 min (Dglucose derivative), 13.005 min (D-xylose derivative) and 14.519 min (L-rhamnose derivative). Acknowledgment This work was financially supported by the Major Program of Municipal Natural Science Foundation of Beijing (No. 7090001). References
3.3.8. Ophiofurospiside K (9) White amorphous powder; a20 D – 68.2 (c 0.091, pyridine); HRESIMS: m/z 1063.5397 [M-H]– (calcd. for C51H83O23, 1063.5325); ESI-MS2: m/z 901.4 [M-H-162]–, 755.4 [M-H-162146]–, 593.4[M-H-162-146-162]–, 431.3 [M-H-162-146-162-162]–, and 413.3 [M-H-162-146-162-162-18]–; 1H NMR data, see Table 1; 13 C NMR data, see Table 2.
Asano, T., Murayama, T., Hirai, Y., Shoji, J., 1993. Comparative studies on the constituents of Ophiopogonis tuber and its congeners-VIII studies on the glycosides of the subterranean part of Ophiopogon japonicus Ker-Gawler cv. Nanus. 2. Chem. Pharm. Bull. 41, 566–570. Agrawal, P.K., 2004. Dependence of 1H NMR chemical shifts of geminal protons of glycosyloxy methylene (H2-26) on the orientation of the 27-methyl group of furostane-type steroidal saponins. Magn. Reson. Chem. 42, 990–993.
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Agrawal, P.K., 2005. Assigning stereodiversity of the 27-Me group of furostane-type steroidal saponins via NMR chemical shifts. Steroids 70, 715–724. Agrawal, P.K., Jain, D.C., Pathak, A.K., 1995. NMR spectroscopy of steroidal sapogenins and steroidal saponins: an update. Magn. Reson. Chem. 33, 923–953. Chen, J.J., Zhu, Z.L., Luo, S.D., 2000. Cixi-ophiopogon A and B, C27 steroidal glycosides from Ophiopogon japonicum. Acta Bot. Yunnanica 22, 97–102. Cheng, Z.H., Wu, T., Yu, B.Y., 2006. Steroidal glycosides from tubers of Ophiopogon japonicus. J. Asian Nat. Prod. Res. 8, 555–559. Dai, H.F., Deng, S.M., Tan, N.H., Zhou, J., 2005. A new steroidal glycoside from Ophiopogon japonicus (Thunb.) Ker-Gawl. J. Integr. Plant Biol. 47, 1148–1152. Dini, I., Tenore, G.C., Trimarco, E., Dini, A., 2005. Furostanol saponins in Allium caepa L. var. tropeana seeds. Food Chem. 93, 205–214. Guo, Y., Liu, Y.X., Kang, L.P., Zhang, T., Yu, H.S., Zhao, Y., Xiong, C.Q., Ma, B.P., 2013. Two novel furostanol saponins from the tubers of Ophiopogon japonicus. J. Asian Nat. Prod. Res. 15, 459–465. Inoue, K., Shimomura, K., Kobayashi, S., Sankawa, U., Ebizuka, Y., 1996. Conversion of furostanol glycoside to spirostanol glycoside by b-glucosidase in Costus speciosus. Phytochemistry 41, 725–727. Jiangsu New Medicinal College, 1986. Zhong Yao Da Ci Dian 3, 2082. Li, N., Zhang, L., Zeng, K.W., Zhou, Y., Zhang, J.Y., Che, Y.Y., Tu, P.F., 2013. Cytotoxic steroidal saponins from Ophiopogon japonicus. Steroids 78, 1–7. Liu, Y., Meng, L.Z., Xie, S.X., Xu, T.H., Sun, L.k., Liu, T.H., Xu, Y.J., Xu, D.M., 2014a. Studies on chemical constituents of Ophiopogon japonicus. J. Asian Nat. Prod. Res. 16, 982–990. Liu, Y., Meng, L.Z., Xie, S.X., Xu, T.H., Sun, L.K., Liu, T.H., Xu, Y.J., Xu, D.M., 2014b. Studies on chemical constituents of Ophiopogon japonicus. J. Asian Nat. Prod. Res. 16, 982–990.
Matsuo, Y., Shinoda, D., Nakamaru, A., Mimaki, Y., 2013. Steroidal glycosides from the bulbs of Fritillaria meleagris and their cytotoxic activities. Steroids 78, 670–682. Watanabe, Y., Sanada, S., Tada, A., Shoji, J., 1977. Studies on the constituents of Ophiopogonis tuber. IV. On the structures of ophiopogonin A, B0 , C, C0 , and D0 . Chem. Pharm. Bull. 25, 3049–3055. Wang, J.Z., Chen, X.B., Wang, F.P., 2008a. Structural elucidation of steroidal glycosides from Ophiopogon japonicus. Chin. J. Org. Chem. 28, 1620–1623. Wang, J.Z., Ye, L.M., Chen, X.B., 2008b. A new C27-steroidal glycoside from Ophiopogon japonicus. Chin. Chem. Lett. 19, 82–84. Xu, Y.J., Xu, T.H., Hao, L.Z., Zhao, H.F., Xie, S.X., Si, Y.S., Han, D., Xu, D.M., 2008. Two new steroidal glucosides from Ophiopogon japonicus (L.f.) Ker-Gawl. Chin. Chem. Lett. 19, 825–828. Ye, Y., Qu, Y., Tang, R.Q., Cao, S.N., Yang, W., Xiang, L., Qi, J.H., 2013. Three new neuritogenic steroidal saponins from Ophiopogon japonicus (Thunb.) Ker-Gawl. Steroids 78, 1171–1176. Zhou, Y.F., Qi, J., Zhu, D.N., Yu, B.Y., 2008. Two new steroidal glycosides from Ophiopogon japonicus. Chin. Chem. Lett. 19, 1086–1088. Zhang, T., Zou, P., Kang, L.P., Yu, H.S., Liu, Y.X., Song, X.B., Ma, B.P., 2009. Two novel furostanol saponins from Ophiopogon japonicus. J. Asian Nat. Prod. Res. 11, 824–831. Zhang, T., Kang, L.P., Yu, H.S., Liu, Y.X., Zhao, Y., Xiong, C.Q., Zhang, J., Zou, P., Song, X. B., Liu, C., Ma, B.P., 2012. Steroidal saponins from the tuber of Ophiopogon japonicus. Steroids 77, 1298–1305. Zhao, Y., Kang, L.P., Liu, Y.X., Liang, Y.G., Tan, D.W., Yu, Z.Y., Cong, Y.W., Ma, B.P., 2009. Steroidal saponins from the rhizome of Paris polyphylla and their cytotoxic activities. Planta Med. 75, 356–363.
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Short communication
Steroidal saponins from the fresh tubers of Ophiopogon japonicus Renyi Yana , Yixun Liub , Liping Kangb , Yang Zhaob , Xinguang Sunb , Jie Zhanga , Dexian Jiaa , Baiping Mab,* a b
Ovation Health Science and Technology Co., Ltd., ENN Group, Langfang 065001, PR China Beijing Institute of Radiation Medicine, Beijing 100850, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 September 2015 Received in revised form 15 November 2015 Accepted 17 November 2015 Available online xxx
Eleven novel furostanol saponins, named ophiofurospisides C–E, G–N (1–3, 5–12), one new spirostanol saponin, named ophiopogonin R (13), were isolated from the fresh tubers of Ophiopogon japonicus. Their structures were determined on the basis of spectroscopic techniques (1D and 2D NMR) and HRESIMS. The isolated furostanol saponins possessed two sugar chains located at C-3 and C-26, respectively. Six furostanol saponins (1, 5–9) with disaccharide moiety linked at position C-26 of the aglycone were rare in the plant kingdom. ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Keywords: Ophiopogon japonicus Furostanol saponin Spirostanol saponin
1. Introduction Ophiopogon japonicus (L.f.) Ker-Gawl. (Liliaceae) is an evergreen perennial, widely distributed in China, especially in Sichuan province and Zhejiang province. The tubers of O. japonicus are well known as “Maidong” in China. It has been used in traditional Chinese medicine for thousands of years as a tonic agent. Nowadays, it often combines with Panax ginseng and Schisandra chinensis to treat cardiovascular and cerebrovascular diseases (Jiangsu New Medicinal College, 1986). Previous phytochemical investigations revealed that O. japonicus contain a variety of steroidal saponins, mainly in the spirostanol form (Watanabe et al., 1977; Asano et al., 1993; Chen et al., 2000; Dai et al., 2005; Cheng et al., 2006; Xu et al., 2008; Zhou et al., 2008; Wang et al., 2008a,b; Li et al., 2013; Ye et al., 2013; Liu et al., 2014a,b). However, our former reports about the steroidal saponins of fresh tubers of O. japonicus showed that contained mainly furostanol saponins (Guo et al., 2013; Zhang et al., 2009, 2012). Furostanol saponins can be easily transformed to corresponding spirostanol saponins under the action of the enzyme during drying and storage of plant, and usually regarded as the synthetic precursors of the corresponding spirostanol saponins in plants (Inoue et al., 1996). Thereby, with the aim to ascertain chemical components disparity between dried and fresh tubers, we decided to carry out a continuous phytochemical research on fresh tubers of O. japonicus. As a result, 13 steroidal saponins including 12 new compounds were
* Corresponding author. Fax: +86 10 68214653. E-mail address: [email protected] (B. Ma).
isolated and identified, 12 of which were furostanol saponins. In this paper, the isolation and structural elucidation of the new compounds were described. 2. Results and discussion Using the same solvent extraction method and chromatographic steps as former reports (Guo et al., 2013; Zhang et al., 2009, 2012), 12 furostanol saponins (ophiofurospisides C–N, 1–12) and one spirostanol saponin (ophiopogonin R, 13) were isolated from 60% EtOH extract of fresh tubers of O. japonicus. Among them, only ophiofurospiside F (4) was reported recently from same plant (Liu et al., 2014a); all others are new compounds (Fig. 1). The sugars obtained by aqueous acid hydrolysis of total saponin (Fr.B) (see Section 3.3) were identified as D-xylose, D-glucose, and Lrhamnose by GC (see Section 3.4). The fucose was not detected by GC analysis. It may be the result of low fucose levels in total saponin. In compound 12, the configuration of D-fucose was deduced from the fact that it was the common stereoisomer from natural source. In the 1H NMR spectra, the relatively large 3JH-1,H-2 values (between 6.6 and 9.0 Hz) of the anomeric protons of the glucose, fucose, and xylose moieties indicated b-anomeric orientation for these glycose moieties. The broad singlet of the anomeric proton of rhamnose combined with the carbon signals of C-3 (dc ca. 72.5) and C-5 (dc ca. 69.5) (Zhang et al., 2009, 2012) indicated the a-configuration. The a-configuration of the C-22 hydroxyl group was deduced from the hemiketal carbon signal at dc ca. 110, about 2–4 ppm higher field than that of the b-configuration (dc ca. 114) (Dini et al., 2005). The 25R configuration of furostanol saponins was demonstrated by the chemical shift
http://dx.doi.org/10.1016/j.phytol.2015.11.013 1874-3900/ ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
R. Yan et al. / Phytochemistry Letters 15 (2016) 72–80
Fig. 1. The chemical structures of compounds 1–13.
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difference between the two protons of H-26 (Dab < 0.48) (Agrawal, 2004, 2005). The configurations of hydroxyl group at C-1, C-12, C13 and C-14 were determined by comparing the 13C NMR spectroscopic data with those reported in literatures (Guo et al., 2013; Zhang et al., 2009, 2012). Ophiofurospiside C (1) was obtained as white amorphous powder. Its molecular formula was determined as C56H92O28 by negative ion HR-ESI-MS with a quasi-molecular ion peak at m/z 1211.5717 [M-H]– (calcd. for C56H91O28, 1211.5697). The major fragment ions at m/z 1061.5 [M-H-18-132]–, 899.4 [M-H-18-132162]–, 753.3 [M-H-18-132-162-146]–, 591.3 [M-H-18-132-162-146162]–, and 429.3 [M-H-18-132-162-146-162-162]– could be attributable to the losses of a pentosyl, a deoxyhexosyl, and three hexosyl moieties from the ion [M-H]–. The 1H NMR spectrum of 1 contained two methyl singlet signals at dH 0.88 (3H, s, Me-18) and 1.06 s (3H, s, Me-19), three methyl doublet signals at dH 1.36 (3H, d, J = 7.2 Hz, Me-21), 0.98 (3H, d, J = 6.6 Hz, Me-27), and 1.71 (3H, d, J = 6.6 Hz, Rha-6), and an olefinic proton signal at dH 5.25 (1H, br s, H-6). In the 13C NMR spectrum, one double bond carbon signals at dC 140.8 (C-5) and 121.9 (C-6); diagnostic quaternary carbon signal at dC 111.4 (C-22) and the oxygenated signals at dC 78.2 (C-3), 90.5 (C-16), 90.9 (C-17), and 75.2 (C-26) were observed. In HMBC spectrum, the correlations between dH 1.36 (Me-21) and 90.9 (C17), and between dH 0.88 (Me-18) and 90.9 (C-17). With above mentioned NMR data, compound 1 was characteristic of a steroidal saponin having (25R)-furosta-5-ene-3b,17a,22a,26-tetraol as the aglycone (Matsuo et al., 2013; Zhao et al., 2009). The presence of five sugar residues was confirmed from the observation of five anomeric proton signals [dH 4.94 (d, J = 7.8 Hz), 6.25 (br s), 5.02 (d, J = 7.8 Hz), 4.72 (d, J = 7.8 Hz) and 5.09 (d, J = 7.8)], giving HSQC correlations with five anomeric 13C NMR signals at dC 100.1, 102.0, 105.8, 104.9, and 105.5, respectively. Complete assignments of each sugar were achieved by extensive 1D and 2D NMR analyses. Evaluation of chemical shifts and spin–spin couplings (Zhang et al., 2009, 2012) allowed the identification of three b-glucopyranosyl, one b-xylopyranosyl and one a-rhamnopyranosyl units in 1. The sequence of the oligosaccharidic chain of 1 was achieved by HMBC experiment. The HMBC correlations between the anomeric 1H NMR signal at dH 4.94 (Glc1 H-1) and dC 78.2 (Agly C-3), between dH 6.25 (br s, Rha H-1) and dC 77.5 (Glc1 C-2), and between dH 5.02 (Xyl H-1) and dC 81.5 (Glc1 C-4) proved the sequence of the sugar chain at C-3 to be a-L-rhamnopyranosyl(1 ! 2)-[b-D-xylopyranosyl-(1 ! 4)]-b-D-glucopyranosyl. Finally, HMBC correlations between dH 5.09 (Glc3 H-1) and dC 70.2 (Glc2 C-6), between dH 4.72 (Glc2 H-1) and dC 75.2 (Agly C-26) allowed the linkage of another sugar chain, b-D-glucopyranosyl(1 ! 6)-b-D-glucopyranosyl, at C-26. Hence, the structure of ophiofurospiside C (1) was concluded to be (25R)-26-O-[b-Dglucopyranosyl-(1 ! 2)-b-D-glucopyranosyl]-5-ene-furosta3b,17a,22a,26-tetraol-3-O-a-L-rhamnopyranosyl-(1 ! 2)-[b-Dxylopyranosyl-(1 ! 4)]-b-D-glucopyranoside. Compound 2 was isolated as a white amorphous powder with a molecular formula of C45H74O21, as determined by HRESIMS with a quasi-molecular ion peak at m/z 949.4667 [M-H]– (calcd. for C45H73O21, 949.4644). HRESIMS showed fragments attributable to the losses of a deoxyhexosyl, two hexosyl residues and three molecules of water. The 1H NMR spectrum of 2 contained three anomeric proton signals at dH 4.82 (1H, d, J = 7.8 Hz, Glc2 H-1), 5.01 (1H, d, J = 7.8 Hz, Glc1 H-1), 6.39 (1H, br s, Rha H-1) and five methyl group signals at dH 0.99 (3H, s, CH3-18), 1.12 (3H, s, CH3-19), 1.41 (3H, d, J = 8.4 Hz, CH3-21), 1.01 (3H, d, J = 7.8 Hz, CH3-27), and 1.79 (3H, d, J = 7.2 Hz, Rha H-6). In 13C NMR spectrum, the signals at dC 91.6 (C-16), 111.2 (C-22), 75.2 (C-26), 139.9 (C-5), and 122.7 (C-6) indicated that 2 is a furostanol saponin possessing unsaturated double bond at C-5 and C-6 (Agrawal et al., 1995). Two oxygenated methine signals at dC 78.3 and 76.4 were attributed to C-3 and C-12,
which showed correlations with proton signals at dH 3.91 (1H, m, H-3) and 4.49 (1H, m, H-12) in the HMQC spectrum, respectively; two oxygenated quaternary carbon signals at dC 88.2 and 94.8 were attributed to C-14 and C-17. The position of hydroxyl groups was confirmed by the long-range correlations between H-21 (dH 1.41, d, J = 8.4 Hz) with C-17, C-22, between H-18 (0.99, s) with C-12, C-14 in the HMBC spectrum. All above data suggested the aglycone of 2 was identical as ophiopogonin M which isolated from the same plant in our lab before (Zhang et al., 2012). In the HMBC spectrum, the long-range correlations between dH 6.39 (H-1 of Rha) and dC 77.9 (C-2 of Glc1), between dH 5.01 (H-1 of Glc1) and dC 78.3 (C-3) indicated the presence of a disaccharide moiety linked at position C-3 of the aglycone. An additional correlation between dH 4.82 (H-1 of Glc2) and dC 75.2 (C-26) revealed the attachment of a single glucose residue at position C-26 of the aglycone. Thus, the structure of ophiofurospiside D (2) was assigned as (25R)-26-Ob-D-glucopyranosyl-5-ene-furosta-3b,12b,14a,17a,22a,26-pentaol-3-O-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranoside. Compound 3 was obtained as a white amorphous powder. The molecular formula was deduced as C50H82O24 based on its negative HRESIMS ion peak at m/z 1065.5144 [M-H]– (calcd. for C50H81O24, 1065.5118). In process to prepare this paper, we found that ophiofurospiside F (4) was reported recently (Liu et al., 2014b). A comparison of NMR and MS data of 3 with those of 4 revealed that they were similar, except for the presence of an additional xylosyl moiety. A detailed comparison of the NMR data of 3 with those of 1 and 4 revealed that 3 and 4 share the same aglycone part, 3 and 1 share the same glycone part. Through analysis the 1D and 2D NMR data of 3, its structure was defined as (25R)-26-O-b-D-glucopyranosyl-5-ene-furosta-3b,14a,17a,22a,26-pentaol-3-O-a-L-rhamnopyranosyl-(1 ! 2)-[b-D-xylopyranosyl-(1 ! 4)]-b-D-glucopyranoside, named ophiofurospiside E. Ophiofurospiside G (5) and ophiofurospiside H (6), white amorphous powder, displayed the same molecular formula of C50H82O23 based on HRESIMS m/z 1049.5162 [M-H]– (calcd. for C50H81O23, 1049.5169) and m/z 1049.5192 [M-H]– (calcd. for C50H81O23, 1049.5169), respectively. HRESIMS of both compounds showed same fragment ion peaks at m/z 887.5 [M-H-162]–, 755.4 [M-H-162-132]–, 737.4 [M-H-162-132-18]–, 575.4 [M-H-162-13218-162]–, and 413.3 [M-H-162-132-18-162-162]– in negative ion mode, suggesting the presence of three hexosyl and a pentosyl moieties. In 1H NMR spectra, there was also four anomeric proton resonances appeared at dH 4.95 (1H, d, J = 7.8 Hz, Glc1 H-1), 5.02 (1H, d, J = 7.8 Hz, Xyl H-1), 4.72 (1H, d, J = 7.8 Hz, Glc2 H-1), and 5.09 (1H, d, J = 7.8 Hz, Glc3 H-1) in 5 and dH 4.95 (1H, d, J = 7.8 Hz, Glc1 H1), 5.02 (1H, d, J = 7.8 Hz, Xyl H-1), 4.84 (1H, d, J = 6.6 Hz, Glc2 H-1), and 5.28 (1H, d, J = 7.8 Hz, Glc3 H-1) in 6. Additionally, diagnostic signals of four methyl, one oxymethine, one oxymethylene, one hemiketal and one olefinic groups were also deduced from NMR spectra of compounds 5 and 6 (Tables 1 and 2). Based on these diagnostic signals, it can easily deduced that the aglycone was (25R)-5-ene-furosta-3b,22a,26-triol (Zhao et al., 2009). By comparing the 13C NMR data with those of 1 indicated that there were one xylose and three glucose moieties in 5 and 6. In HMBC spectrum of 5, correlations of Glc1 H-1 (dH 4.95)/C-3 (dC 78.4), Xyl H-1 (dH 5.02)/Glc1 C-4 (dC 81.0), Glc2 H-1 (dH 4.72)/C-26 (dC 75.4), and Glc3 H-1 (dH 5.09)/Glc2 C-6 (dC 70.2) were observed. Thus, 5 was determined as (25R)-26-O-[b-D-glucopyranosyl-(1 ! 6)]-b-Dglucopyranosyl-5-ene-furosta-3b,22a,26-triol-3-O-b-D-xylopyranosyl-(1 ! 4)-b-D-glucopyranoside. The difference between 5 and 6 in HMBC spectrum was that correlation between Glc3 H-1 and Glc2 C-6 in 5 was replaced by the Glc3 H-1 and Glc2 C-2 in 6 which was supported by the downfield signal of Glc2 C-2 (dC 84.3) and upfield signal of Glc2 C-6 (dC 62.7). Accordingly, 6 was determined as (25R)-26-O-[b-D-glucopyranosyl-(1 ! 2)]-b-D-glucopyranosyl-
R. Yan et al. / Phytochemistry Letters 15 (2016) 72–80
75
Table 1 1 H NMR data of compounds 1–3, 5–9, 12 and 13 in pyridine-d5. Pos.
1
2
3
5
6
7
8
9
12
13
1
0.93 (m) 1.74 (m) 1.85 (m) 2.07 (m) 3.83 (m) 2.70 (m) 2.72 (m) 5.25 (br s) 1.48 (m) 1.86 (m) 1.58 (m)
1.02 (m) 1.72 (m) 1.83 (m) 2.06 (m) 3.91 (m) 2.74 (m) 2.78 (m) 5.40 (br s) 1.81 (m) 2.57 (m) 2.03 (m)
1.03 (m) 1.79 (m) 1.86 (m) 2.08 (m) 3.83 (m) 2.71 (m) 2.77 (m) 5.36 (br s) 1.83 (m) 2.47 (m) 2.10 (m)
0.95 (m) 1.70 (m) 1.71 (m) 2.08 (m) 3.86 (m) 2.44 (m) 2.67 (m) 5.27 (br s) 1.51 (m)
1.02 (m) 1.79 (m) 1.90 (m) 2.10 (m) 3.87 (m) 2.76 (m) 2.80 (m) 5.37 (br s) 1.86 (m) 2.48 (m) 2.07 (m)
0.96 (m) 1.71 (m) 1.88 (m) 2.14 (m) 3.89 (m) 2.74 (m) 2.78 (m) 5.28 (br s) 1.47 (m) 1.84 (m) 1.56 (m)
2.67 (m) 2.42 (m) 3.67 (m) 2.67 (m) 2.56 (m) 5.35 (br s) 1.53 (m)
1.05 (m) 1.85 (m) 1.82 (m) 2.08 (m) 3.96 (m) 2.73 (m) 2.75 (m) 5.38 (br s) 1.86 (m) 2.50 (m) 2.09 (m)
0.95 (m) 1.51 (m) 1.57 (m) 1.49 (m) 1.57 (m)
1.98 (m) 1.85 (m) 1.89 (m) 4.49 (m)
1.79 (m) 1.58 (m) 1.63 (m) 1.36 (m) 2.55 (m)
0.86 (m) 1.39 (m) 1.42 (m) 1.10 (m) 1.73 (m)
0.87 (m) 1.37 (m) 1.41(m) 1.10 (m) 1.74 (m)
1.82 (m) 1.58 (m) 1.50 (m) 2.29 (m)
0.88 (m) 1.38 (m) 1.46 (m) 1.12 (m) 1.73 (m)
1.56 (m) 1.46 (m) 1.37 (m) 2.19 (m) 1.65 (m)
1.49 (m) 2.32 (m)
14 15
2.01 (m) 1.59 (m) 2.15 (m)
– 1.85 (m) 2.50 (m)
1.03 (m) 1.83 (m) 1.96 (m)
1.02 (m) 1.40 (m) 1.99 (m)
– 1.93 (m) 2.33 (m)
1.06 (m) 1.84 (m) 2.00 (m)
1.22 (m) 1.88 (m) 2.48 (m)
– 1.93 (m) 2.45 (m)
16
4.74 (d, 7.8)
– 1.66 (m) 2.42 (dd, 14.4, 9.0) 5.31 (t, 9.0)
1.03 (m) 1.78 (m) 1.90 (m) 2.11 (m) 3.92 (m) 2.75 (m) 2.81 (m) 5.35 (br s) 1.84 (m) 2.47 (m) 2.02 (dd, 10.8, 5.4) 1.82 (m) 1.58 (m) 1.62 (m) 1.36 (br d, 12.6) 2.57 (dd, 12.6, 5.4) 1.06 (m) 1.85 (m) 2.49 (m)
3.87 (m)
1.52 (m)
0.96 (m) 1.68 (m) 1.71 (m) 2.08 (m) 3.86 (m) 2.44 (m) 2.67 (m) 5.27 (br s) 1.44 (m) 1.83 (m) 1.54 (m)
5.09 (t, 6.6)
4.92 (m)
4.93 (m)
5.09 (m)
4.94 (m)
5.09 (m)
4.95 (m)
17
–
–
–
1.90 (m)
1.91 (m)
1.92 (m)
1.92 (m)
1.95 (m)
2.76 (m)
18 19 20 21 23
0.88 (s) 1.06 (s) 2.48 (p, 7.2) 1.36 (d, 7.2) 2.07 (m)
5.42 (dd, 14.4, 7.2) 2.90 (dd, 14.4, 7.2) 1.15 (s) 1.13 (s) 2.36 (m) 1.54 (d, 7.2) 2.06 (m)
1.13 (s) 1.13 (s) 2.63 (q, 7.2) 1.41 (d, 7.2) 2.05-2.08 (m)
0.91 (s) 1.05 (s) 2.22 (m) 1.32 (d, 6.6) 2.03 (m)
1.70 (m) 2.07 (m) 2.00 (m) 3.62 (dd, 8.4, 4.8) 3.88 (m) 1.09 (d, 6.6) 5.02 (d, 7.8)
1.72 (m) 2.10 (m) 1.94 (m) 3.57 (dd, 9.6, 6.0) 4.03 (m) 1.01 (d, 7.2) 5.02 (d, 7.8)
4.27 (m) 4.27 (m) 4.15 (m) 3.91 (m) 4.44 (m) 4.51 (m)
4.27 (m) 4.27 (m) 4.17 (m) 3.89 (m) 4.49 (br d, 11.4) 4.35 (m)
2 3 4 6 7 8 9 11 12
0.99 (s) 1.12 (s) 2.56 (m) 1.41 (d, 8.4) 2.03 (m) 2.06 (m) 1.68 (m) 1.66 (m) 2.05 (m) 2.00 (m) 1.92 (m) 1.92 (m) 3.56 (dd, 9.6, 3.62 (dd, 11.4, 5.4) 7.2) 4.02 (m) 3.97 (m) 0.98 (d, 6.6) 1.01 (d, 7.8) 4.94 (d, 7.8) 5.01 (d, 7.8)
1.12 (s) 1.11 (s) 2.62 (m) 1.39 (d, 7.2) 2.07 (m)
0.88 (s) 0.90 (s) 2.22 (m) 1.32 (d, 6.6) 2.02 (m)
0.89 (s) 0.91 (s) 2.23 (m) 1.31 (d, 6.6) 2.01 (m)
1.71 (m) 2.07 (m) 1.93 (m) 3.63 (dd, 9.0, 5.4) 4.01 (m) 1.00 (d, 6.6) 4.94 (d, 7.8)
1.67 (m) 2.00 (m) 1.90 (m) 3.64 (dd, 9.6, 6.0) 3.96 (m) 1.01 (d, 6.6) 4.95 (d, 7.8)
1.61 (m) 2.01 (m) 1.98 (m) 3.86 (m) 4.07 (m)
4.20 (m) 4.22 (m) 4.18 (m) 3.82 (m) 4.43 (m) 4.49 (m)
4.27 (m) 4.26 (m) 4.17 (m) 3.91 (m) 4.35 (m) 4.49 (m)
4.19 (m) 4.20 (m) 4.18 (m) 3.81 (m) 4.41 (m) 4.49 (m)
4.21 (m) 4.23 (m) 4.18 (m) 3.83 (m) 4.43 (br d, 11.4) 4.50 (br d, 11.4)
4.21 (m) 4.20 (m) 4.18 (m) 3.83 (m) 4.45 (br d, 11.4) 4.52 (br d, 11.4)
Rha-1 2 3
6.25 (br s) 4.78 (br s) 4.58 (m)
6.39 (s) 4.82 (br s) 4.65 (m)
4 5 6 Xyl-1 2 3
4.33 (m) 4.92 (m) 1.75 (d, 6.6) 5.02 (d, 7.8) 4.03 (m) 4.09 (m)
4.35 (m) 5.00 (m) 1.79 (d, 7.2)
4 5
4.15 (m) 3.66 (t, 10.8) 4.23 (m)
6.24 (br s) 4.77 (br s) 4.58 (dd, 9.0, 3.6) 4.33 (m) 4.92 (m) 1.76 (d, 6.0) 5.01 (d, 7.8) 3.95 (m) 4.08 (dd, 8.4, 7.8) 4.15 (m) 3.66 (dd, 11.4, 10.8) 4.24 (m)
Glc2-1 2 3 4 5 6
4.72 (d, 7.8) 4.21 (m) 4.21 (m) 4.15 (m) 4.04 (m) 4.32 (m) 4.82 (m)
Glc3-1
5.09 (d, 7.8)
24 25 26
27 Glc1-1/ Fuc-1 2 3 4 5 6
4.82 (d, 7.8) 4.02 (m) 4.24 (m) 4.19 (m) 3.94 (m) 4.53 (m)
4.81 4.01 4.22 4.21 3.93 4.36 4.53
(d, 7.8) (m) (m) (m) (m) (m) (m)
1.05 (d, 6.0) 4.95 (d, 7.8)
6.35 (br s) 4.79 (br s) 4.62 (dd, 9.0, 3.0) 4.34 (m) 4.99 (m) 1.78 (d, 6.6) 5.02 (d, 7.8) 3.97 (m) 4.09 (dd, 9.0, 8.4) 4.16 (m) 3.66 (dd, 11.4, 10.2) 4.26 (dd, 11.4, 4.8) 4.72 (d, 7.8) 4.03 (m) 4.23 (m) 4.14 (m) 4.03 (m) 4.32 (m) 4.81 (d, 9.6)
5.02 (d, 7.8) 3.97 (m) 4.09 (dd, 9.0, 8.4) 4.17 (m) 3.66 (dd, 11.4, 10.2) 4.26 (dd, 11.4, 4.8) 4.84 (d, 6.6) 4.12 (m) 4.11 (m) 4.17 (m) 3.93 (m) 4.38 (m) 4.49 (m)
4.86 4.22 4.30 4.30 3.86 4.34 4.42
5.09 (d, 7.8)
5.28 (d, 7.8)
5.27 (d, 7.8)
(d, 7.8) (m) (m) (m) (m) (m) (m)
4.74 (d, 7.8) 3.96 (m) 4.16 (m) 4.15 (m) 4.05 (m) 4.83 (br d, 10.2) 4.33 (m) 5.10 (d, 7.8)
1.46 (m)
1.90 (m) 1.56 (m)
0.92 (s) 1.41 (s) 2.19 (q, 7.2) 1.48 (d, 7.2) 2.45 (m) 2.20 (m) 1.67 (m) 1.63 (m) 2.02 (m) 1.65 (m) 1.90 (m) 1.96 (m) 3.56 (dd, 9.6, 3.58 (dd, 9.6, 6.0) 6.0) 3.95 (m) 4.02 (m) 0.98 (d, 6.6) 1.26 (d, 7.2) 5.03 (d, 7.2) 4.66 (d, 7.8)
1.01 (s) 1.12 (s) 2.09 (m) 1.11 (d, 7.8) 2.03 (m) 2.75 (m) 4.07 (m)
4.22 (m) 4.18 (m) 4.12 (m) 3.94 (m) 4.34 (m) 4.50 (m)
4.02 (m) 4.06 (m) 4.59 (m) 4.23 (m) 4.49 (br d, 11.4) 4.35 (m)
4.20 (m) 4.22 (m) 4.18 (m) 3.84 (m) 4.39 (m) 4.52 (m)
6.35 (br s) 4.80 (m) 4.61 (m)
6.34 (s) 4.21 (m) 4.79 (m)
4.34 (m) 4.98 (m) 1.76 (d, 6.6)
4.08 (m) 4.86 (m) 1.74 (d, 6.6) 4.98 (d, 7.2) 3.57 (m) 3.93 (m)
6.27 (br s) 4.80 (br s) 4.60 (dd, 11.4, 3.6) 4.43 (m) 4.96 (m) 1.78 (d, 7.2) 5.06 (d, 7.8) 3.98 (m) 4.08 (m)
3.76 (m) 4.26 (m) 3.69 (m)
4.16 (m) 3.68 (m) 4.26 (m)
4.78 (d, 7.8) 4.07 (m) 4.21 (m) 4.67 (m) 3.99 (m) 4.53 (m) 4.36 (m)
4.96 (d, 7.2) 4.06 (m) 4.22 (m) 4.27 (m) 3.98 (m) 4.39 (m) 4.51 (m)
4.72 (d, 7.8) 3.98 (m) 4.22 (m) 4.16 (m) 4.04 (m) 4.32 (m) 4.82 (m) 5.09 (d, 7.2)
1.90 (m) 3.56 (m) 3.63 (m) 1.34 (d, 9.6) 5.03 (d, 9.0)
76
R. Yan et al. / Phytochemistry Letters 15 (2016) 72–80
Table 1 (Continued) Pos.
1
2 3 4 5 6
4.01 (m) 4.16 (m) 4.22 (m) 3.91 (m) 4.36 (m) 4.50 (m)
2
3
5 4.03 4.22 4.21 3.93 4.34 4.50
(m) (m) (m) (m) (m) (m)
6
7
8
9
4.09 (m) 4.21 (m) 4.27 (m) 3.95 (m) 4.30 (m) 4.48 (m)
4.01 (m) 4.22 (m) 4.30 (m) 3.96 (m) 4.41 (m) 4.50 (m)
4.03 (m) 4.21 (m) 4.23 (m) 3.91 (m) 4.35 (m) 4.49 (br d, 11.4)
4.02 (m) 4.16 (m) 4.22 (m) 3.91 (m) 4.34 (m) 4.50 (m)
5-ene-furosta-3b,22a,26-triol-3-O-b-D-xylopyranosyl-(1 ! 4)b-D-glucopyranoside. Ophiofurospiside I (7) was obtained as white amorphous powder. Its molecular formula was determined as C51H84O24 by negative ion HRESIMS with a quasi-molecular ion peak at m/z 1079.5294 [M-H]– (calcd. for C51H83O24, 1079.5274). HRESIMS showed fragment ion peaks at m/z 917.4 [M-H-162]–, 771.4 [M-H162-146]–, 591.4 [M-H-162-146-180]–, and 429.3 [M-H-162-146180-162]– in negative ion mode, suggesting the presence of three hexosyl and one deoxyhexosyl moieties. 1H NMR spectrum of 7 also exhibited four sugar anomeric protons at dH 5.02 (d, J = 7.8 Hz), 6.35 (br s), 4.86 (d, J = 7.8 Hz), and 5.27 (d, J = 7.8 Hz), with their corresponding anomeric carbons at dC 100.3, 102.1, 103.2, and 106.6 in the 13C NMR spectrum, respectively. Two tertiary methyls at dH 1.13 (3H, s) and 1.15 (3H, s), two secondary methyls at dH 1.09 (3H, d, J = 6.6 Hz), 1.54 (3H, d, J = 7.2 Hz), an olefinic proton at dH 5.37 (1H, br s) can be deduced from 1H NMR spectrum. In the 13C NMR spectrum showed the olefinic carbon signals at dC 140.4 (C-5), 122.4 (C-6), hemiketal carbon signal at dC 110.0 (C-22), two oxygenated methines at dC 78.1 (C-3) and 81.9 (C-16), and one oxygenated quaternary carbon signal at dC 86.4 (C-14). All these mentioned NMR data suggested that 7 had an aglycone, (25R)furosta-5-ene-3b,14a,22a,26-tetraol. It is also supported by comparing NMR data with these of ophiopogonins I, J and N, which co-existed in entitled plant (Zhang et al., 2012). A detailed comparison of the NMR data of 7 with those of ophiopogonin J indicated that they had the same aglycone and sugar chain at C-26. The sugar sequence of a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranosyl and its linkage at C-3 were identified by HMBC correlations between H-1 of Rha (dH 6.35) and C-2 of Glc1 (dC 78.0), and between H-1 of Glc1 (dH 5.02) and C-3 (dC 78.1). Thus, ophiofurospiside I (7) was determined to be (25R)-26-O-[b-Dglucopyranosyl-(1 ! 2)]-b-D-glucopyranosyl-5-ene-furosta3b,14a,22a,26-tetraol-3-O-a-L-rhamnopyranosyl-(1 ! 2)-b-Dglucopyranoside. Ophiofurospiside J (8) was obtained as white amorphous powder. Its molecular formula was determined as C45H74O19 by negative ion HRESIMS with a quasi-molecular ion peak at m/z 917.4778 [M-H]– (calcd. for C45H73O19, 917.4746). 1H NMR spectrum of 8 exhibited three sugar anomeric protons at dH 5.02 (d, J = 7.8 Hz), 4.74 (d, J = 7.8 Hz), and 5.10 (d, J = 7.8 Hz), with their corresponding anomeric carbons at dC 102.6, 103.2, and 106.6 in the 13C NMR spectrum, respectively. Two tertiary methyls at dH 1.13 (6H, s), two secondary methyls at dH 1.01 (3H, d, J = 7.2 Hz), 1.41 (3H, d, J = 7.2 Hz), an olefinic proton at dH 5.35 (1H, br s) can be deduced from 1H NMR spectrum. These 1H NMR data, together with olefinic carbon signals at dC 140.9 (C-5), 121.8 (C-6) and the carbon signal at dC 110.7 (C-22) in the 13C NMR spectrum suggested that 8 had a 5,6ene-furostanol skeleton. A detailed comparison of the NMR data of 8 with those of 6 indicated that they had the same aglycone, and only sugar chain at C-3 was different. The glucosyl located at C-3 was deduced from the HMBC correlation between H-1 of Glc1 (dH 5.02, d, J = 7.8 Hz) and C-3 of the aglycone (dC 78.3). Thus, ophiofurospiside J (8) was determined to be (25R)-26-O-[b-D-
12
13
glucopyranosyl-(1 ! 2)]-b-D-glucopyranosyl-5-ene-furosta3b,22a,26-triol-3-O-b-D-glucopyranoside. Ophiofurospiside K (9) was obtained as white amorphous powder. Its molecular formula was determined as C51H84O23 by negative ion HRESIMS with a quasi-molecular ion peak at m/z 1063.5397 [M-H]– (calcd. for C51H83O23, 1063.5325). HRESIMS showed fragment ion peaks at m/z 901.4 [M-H-162]–, 755.4 [M-H162-146]–, 593.4 [M-H-162-146-162]–, and 431.3 [M-H-162-146162-162]– in negative ion mode, suggesting the presence of three hexosyl and one deoxyhexosyl moieties. A detailed comparison of the NMR data of 9 with those of 5 indicated that they had the same aglycone and C-26 disaccharide chain, and the only difference between two compounds was the sugar chain at C-3. Comparing the 13C NMR data with these of compound 2 indicated that they shared the same sugar chain at C-3. It is also supported by the HMBC correlations between H-1 of Glc1 (dH 5.03, d, J = 7.2 Hz) and C-3 of the aglycone (dC 78.3), between H-1 of Rha1 (dH 6.35, br s) and C-2 of the Glc1 (dC 78.0). Thus, ophiofurospiside K (9) was determined to be (25R)-26-O-[b-D-glucopyranosyl-(1 ! 6)]-b-Dglucopyranosyl-5-ene-furosta-3b,22a,26-triol-3-O-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranoside. Ophiofurospiside N (12) was obtained as a white amorphous powder. Its molecular formula was determined to be C50H82O22 by HRESIMS m/z 1033.5271 [M-H]– (calcd. for C50H81O22, 1033.5219). HRESIMS showed fragment ion peaks at m/z 887.4 [M-H-146]–, 775.4 [M-H-146-132]–, 609.3 [M-H-146-132-146]–, and 447.3 [M-H-146-132-146-162]– in negative ion mode, suggesting the sequential losses of two deoxyhexosyl, one pentosyl, and one hexosyl moieties. The combined analysis of 1 H, 13C NMR, and HSQC spectrum of 12, showed the presence of four methyl groups [dH/dC 0.92 s/17.0 (agly 18); 1.41 s/19.2 (agly 19); 1.48 (d, J = 7.2 Hz)/16.4 (agly 21); 1.26 (d, J = 7.2 Hz)/17.5 (agly 27)], an olefinic group [dH/dC 5.35 br s/(139.6, 124.8)], a hydroxymethene [dH/dC 3.58 (dd, J = 9.6, 6.0 Hz), 4.02 m/75.2], two oxygenated methines (dH/dC 3.87 m/84.4; 3.67 m/67.1 and a hemiketal (dC 110.7) suggesting a 5-ene-furosta-1b,3b,22a,26tetraol moiety. Four anomeric proton signals at dH 4.66 (d, J = 7.8 Hz), 6.34 s, 4.98 (d, J = 7.2 Hz), 4.78 (d, J = 7.8 Hz), and 1.74 (d, J = 6.6 Hz) indicated that compound 12 contains four sugar moieties of which one is rhamnose. The NMR spectral data of 12 were almost consistent with those of (25S)-26-O-b-Dglucopyranosyl-furosta-5-ene-1b,3b,22a,26-teraol-1-O-b-Dxylopyranosyl-(1 ! 3)-[a-L-rhamnopyranosyl-(1 ! 2)]-b-D-glucopyranoside, except that the signal of H-26 (dH 3.58, 4.02; Dab = 0.44 < 0.48 demonstrated the 25R configuration of 12 (Agrawal 2004, 2005). Thus, the structure of 12 was determined to be (25R)-26-O-b-D-glucopyranosyl-5-ene-furosta-1b,3b,22a,26-teraol-1-O-b-D-xylopyranosyl-(1 ! 3)-[a-Lrhamnopyranosyl-(1 ! 2)]-b-D-fucopyranoside). The molecular formula of ophiofurospiside L (10) was determined as C50H82O23 by HRESIMS, showing a pseudomolecular ion peak at m/z 1049.5183 [M-H]– (calcd. for C50H81O23, 1049.5169). Further fragment ion peaks were observed at m/z 917.5, 771.4, 591.4, and 429.3 corresponding to the loss of one pentosyl, one
R. Yan et al. / Phytochemistry Letters 15 (2016) 72–80
77
Table 2 13 C NMR data of compounds 1–13 in pyridine-d5. Pos.
1
2
3
5
6
7
8
9
10
11
12
13
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Glc1-1/Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Xyl-1 2 3 4 5 Glc2-1 2 3 4 5 6 Glc3-1 2 3 4 5 6
37.6 30.2 78.2 39.0 140.8 121.9 32.5 32.3 50.3 37.2 21.0 32.2 45.1 53.1 32.0 90.5 90.9 17.3 19.5 43.6 10.5 111.4 36.9 28.1 34.2 75.2 17.5 100.1 77.5 76.3 81.5 77.3 61.7 102.0 72.5 72.8 74.2 69.6 18.7 105.8 75.1 78.4 70.8 67.4 104.9 75.0 78.5 71.6 77.3 70.2 105.5 75.4 78.5 71.6 78.5 62.8
37.6 30.2 78.3 39.0 139.9 122.7 26.6 36.4 39.4 37.1 29.4 76.4 48.6 88.2 39.9 91.6 94.8 21.9 19.3 45.1 10.1 111.2 35.9 28.0 34.2 75.2 17.4 100.3 79.7 77.9 71.9 77.8 62.9 102.1 72.6 72.9 74.2 69.5 18.7
37.8 30.3 78.3 39.0 140.3 122.4 26.2 36.2 43.6 37.4 20.2 26.7 48.5 87.7 40.3 90.8 91.6 20.8 19.4 43.9 10.7 111.2 37.1 28.1 34.3 75.3 17.5 100.0 77.3 76.3 81.6 77.6 61.7 102.0 72.5 72.9 74.2 69.6 18.7 105.8 75.0 78.4 70.8 67.4 105.0 75.3 78.7 71.8 78.5 62.9
37.5 30.2 78.4 39.3 140.9 121.8 32.3 31.7 50.4 37.2 21.2 40.0 40.7 56.6 32.5 81.1 63.9 16.5 19.4 40.7 16.5 110.7 37.1 28.5 34.3 75.4 17.5 102.4 77.6 77.3 81.0 76.3 61.7
37.5 30.3 78.3 39.3 140.9 121.8 32.3 31.7 50.4 37.3 21.2 40.0 40.8 56.7 32.5 81.7 63.8 16.5 19.4 40.9 16.6 110.7 37.1 28.3 34.4 75.2 17.6 102.4 77.6 77.3 84.3 76.3 61.7
37.8 30.3 78.1 39.1 140.4 122.4 26.8 35.6 43.7 37.4 20.5 32.1 45.5 86.4 40.1 81.9 60.6 20.2 19.4 41.0 16.7 111.0 37.5 28.3 34.5 75.2 17.7 100.3 79.7 78.0 71.9 77.8 62.6 102.1 72.6 72.9 74.2 69.5 18.7
37.5 30.3 78.3 39.4 140.9 121.8 32.3 31.7 50.3 37.3 21.2 40.0 40.8 56.7 32.5 81.2 63.8 16.5 19.4 40.9 16.6 110.7 37.1 28.2 34.4 75.2 17.6 102.6 75.2 77.0 81.2 77.0 62.6
37.6 30.3 78.3 39.1 140.9 121.8 32.4 31.7 50.4 37.2 21.1 40.0 40.7 56.7 32.5 81.1 63.9 16.5 19.5 40.8 16.5 110.7 37.2 28.5 34.3 75.2 17.5 100.4 79.7 78.0 71.9 77.9 62.7 102.1 72.6 72.9 74.2 69.5 18.7
77.7 40.8 75.2 39.5 139.2 125.2 32.4 33.0 51.3 43.8 24.2 40.6 40.6 56.8 32.8 81.1 64.2 16.7 13.8 40.9 16.5 110.6 37.3 28.4 34.3 75.3 17.5 100.5 79.6 78.0 71.7 77.7 62.9 102.1 72.6 72.9 74.2 69.5 18.7
105.6 75.0 78.4 70.8 67.4 104.9 75.1 78.5 71.6 77.3 70.2 105.5 75.4 78.6 71.6 78.5 62.8
105.6 75.0 78.4 70.8 67.4 103.2 84.3 78.3 71.4 78.4 62.7 106.6 77.0 78.1 71.5 78.7 62.6
103.2 84.3 78.3 71.4 78.7 62.7 106.6 77.0 78.1 71.5 78.7 62.7
103.2 84.3 78.3 71.5 78.5 62.6 106.6 77.0 78.1 71.5 78.8 62.9
104.9 75.1 78.5 71.7 77.3 70.2 105.5 75.4 78.6 71.9 78.5 62.8
77.4 40.8 75.2 39.5 139.1 125.3 32.3 32.9 51.3 43.8 24.2 40.6 40.6 56.8 32.8 81.1 64.2 16.7 13.7 40.9 16.5 110.7 37.3 28.4 34.3 75.3 17.5 100.2 77.9 76.3 81.4 77.3 61.6 101.9 72.5 72.8 74.2 69.6 18.7 105.8 75.0 78.6 70.8 67.4 105.0 75.2 78.5 71.7 78.4 62.9
84.4 37.2 67.1 43.9 139.6 124.8 32.1 32.7 50.6 42.9 24.1 40.6 40.6 57.2 33.1 81.2 64.0 17.0 15.1 40.8 16.4 110.7 38.1 28.3 34.3 75.2 17.5 100.5 74.8 85.6 72.8 71.7 17.2 101.8 70.9 72.6 74.3 68.3 19.2 106.7 74.8 78.4 71.1 78.5 105.0 75.3 78.5 69.4 78.4 62.9
37.8 30.2 78.1 39.0 140.3 122.4 26.7 35.6 43.6 37.5 20.4 31.9 45.1 86.4 39.8 81.7 62.9 20.0 19.4 42.2 15.2 112.0 41.0 82.3 38.2 65.1 13.6 99.9 77.5 76.3 81.5 77.3 61.7 102.0 72.5 72.8 74.2 69.6 18.7 105.8 75.0 70.8 70.8 67.4 106.5 75.7 78.7 71.7 78.1 62.9
105.0 75.2 78.7 71.7 78.5 62.7
deoxyhexosyl and two hexosyl moieties. The 13C NMR spectrum displayed 50 resonance signals and 27 due to the aglycone part, including one olefinic, one oxygenated methene, three oxygenated methines, and one hemiketal carbons. 13C NMR spectral data of aglycone 10 were close to those of compound 12, except that the signal of C-3 (dC 75.2) was by shielded Dd 8.1, and signal of C-1 was deshielded by Dd + 7.0, suggesting the glycosylation at C-3. The 13 C NMR data of sugar part of 10 were identical with those of 3. On the basis of above evidences, 10 was identified as (25R)-26-O-b-Dglucopyranosyl-5-ene-furosta-1b,3b,22a,26-teraol-3-O-b-Dxylopyranosyl-(1 ! 4)-[a-L-rhamnopyranosyl-(1 ! 2)]-b-D-glucopyranoside. The molecular formula of ophiofurospiside M (11) was deduced to be C45H74O19 on the basis of its quasi-molecular ion peaks at m/z 917.4756 [M-H]– (calcd. for C45H73O19, 917.4746) in its HRESIMS.
105.0 74.8 78.5 71.7 78.3 62.6
Fragment ion peaks at m/z 771.4, 591.4, and 429.3 were corresponded to the loss of one deoxyhexosyl and two hexosyl moieties. The 13C NMR spectrum displayed one olefinic, one oxygenated methene, three oxygenated methines, and one hemiketal carbons. 13C NMR spectral data of aglycone were consistent with those of compound 10. In the 13C NMR spectrum of 11, the data of sugar chain were identical with those of 2. On the basis of above evidences, 11 was identified as (25R)-26-O-b-D-glucopyranosyl-5-ene-furosta-1b,3b,22a,26-teraol-3-O-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranoside. The molecular formula of ophiofurospiside R0 (13) was determined as C50H80O23 by HRESIMS, showing a pseudomolecular ion peak at m/z 1047.5073 [M-H]– (calcd. for C50H79O23, 1047.5012). Further fragment ion peaks were observed at m/z 915.5, 769.3, 607.3, and 445.3 corresponding to the loss of one pentosyl, one
78
R. Yan et al. / Phytochemistry Letters 15 (2016) 72–80
deoxyhexosyl and two hexosyl moieties. Inspection of the 1H NMR spectrum of 12 led to the assignment of two tertiary methyl groups [dH 1.01 (3H, s, Me-18) and 1.12 (3H, s, Me-19)], two secondary methyl groups [dH 1.34 (3H, d, J = 9.6 Hz, CH3-27) and 1.11 (3H, d, J = 7.8 Hz, CH3-21)], and one olefinic proton [dH 5.38 (1H, br s)]. These proton signals together with a distinctive quaternary carbon signal at dC 112.0 showing long-range correlations with one of the methylene protons at dH 3.63 (Agly H-26a) along with the proton at dH 1.11 (Agly Me-21) indicated that the aglycone of 13 was a spirosta-5-ene type. On the other hand, HMBC correlations between the proton signal at dH 4.07 (H-24) and the carbon signal at dC 112.0 (C-22), and between the proton signal at dH 1.34 (Me-27) and the carbon signals at dC 82.3 (C-24), suggested the presence of hydroxyl group at C-24. The NOE correlations between H-23eq (d 2.75)/H-24 (d 4.07), H-27 (d 1.34)/H-24 (d 4.07) and the 13C NMR chemical shifts of C-23–C-27 allowed assignment of the 24S and 25S (Zhang et al., 2012). The aglycone of 13 was identified as (25S)spirosta-5-ene-3b,14a,24b-triol, by comparison of 1H and 13C NMR spectroscopic data obtained from 2D NMR spectra of 13 with those reported in the literature (Zhang et al., 2012). The presence of four sugar residues was confirmed from observation of four anomeric proton signals [dH 5.03 (d, J = 9.0 Hz), 6.27 br s, 5.06 (d, J = 7.8 Hz), and 4.96 (d, J = 7.2 Hz)], giving HSQC correlations with four anomeric carbon signals at dC 99.9, 102.0, 105.8, and 106.5, respectively. Complete assignments of each sugar unit were carried out by extensive 1D and 2D NMR analyses. Two b-glucopyranosyl (Glc1 and Glc2), one b-xylopyranosyl (Xyl), and one a-rhamnopyranosyl (Rha) units were identified in 13. The HMBC correlations between the anomeric 1H NMR signal at dH 5.03 (Glc1 H-1) and dC 78.1 (Agly C-3), between dH 6.27 (Rha-1) and dC 77.5 (Glc1 C-2), between dH 5.06 (Xyl H-1) and dC 81.5 (Glc1 C-4) proved the sequence of the sugar chain at C-3 to be a-Lrhamnopyranosyl-(1 ! 2)-[b-D-xylopyranosyl-(1 ! 4)]-b-D-glucopyranosyl. Finally, HMBC correlation between dH 4.96 (Glc2 H-1) and dC 82.3 (Agly C-24) allowed the linkage of Glc2 at C-24. Thus, 13 was determined to be (25S)-24-O-b-D-glucopyranosyl-spirosta-5ene-3b,14a,24b-triol-3-O-a-L-rhamnopyranosyl-(1 ! 2)-[b-Dxylopyranosyl-(1 ! 4)]-b-D-glucopyranoside. 3. Experimental 3.1. General Optical rotations were measured with a PerkinElmer 343 polarimeter (PerkinElmer, Waltham, MA, USA). The NMR spectra were recorded in pyridine-d5 using a Varian Inova-600 spectrometer (600 MHz for 1H NMR and 150 MHz for 13C NMR) (Palo Alto, CA, USA). The HRESIMS was recorded on a Synapt MS system (Waters Corporation, Milford, MA, USA). Macroporous resin SP825 (Mitsubishi Chemical, Tokyo, Japan) and MCI CHP20 (Mitsubishi Chemical), ODS silica gel (50 mm, YMC, Kyoto, Japan), and silica gel H (Qingdao Marine Chemical, Inc., Qingdao, China) were used for column chromatography. TLC was performed on precoated silica gel 60 plates (Merck, Darmstadt, Germany). HPLC was performed using Agilent 1100 system with the following components: Alltech ELSD 2000 (Alltech, Lexington, KY, USA), and an analytical column, Agela Venusil XBP C18 (2) (5 mm, 4.6 250 mm, Agela, Tianjin, China). Preparative HPLC separations were performed using PrimeLine solvent delivery module (ASI, Salt Lake City, UT, USA) equipped with Shodex refractive index detector (RID) 102 detector (Showa Denko Group, Tokyo, Japan) and a preparative column, Venusil XBP C18 column (5 mm, 10.0 250 mm, Agela, Tianjin, China). GC analysis was carried out on an Agilent 6890 equipped with an H2 flame ionization detector and an HP-5 capillary column (30 m 0.25 mm 0.25 mm) (Agilent Technologies, Milford, MA, USA).
3.2. Plant material The tubers of O. japonicus were collected from the Mianyang region of Sichuan Province, China in April 2007. The plant was identified by Prof. Li-Juan Zhang (Tianjin University of Traditional Chinese Medicine) and a voucher specimen (No. 070403) was deposited at the Herbarium of Beijing Institute of Radiation Medicine, Beijing, China. 3.3. Extraction and isolation The fresh tubers of O. japonicus (dried at 80 C) (56.0 kg) were extracted twice with 60% EtOH (450 L, 1 h each time) under reflux, followed by evaporation of solvent in vacuum. The concentrated extract was separated on SP825 macroporous resin column chromatography eluted successively with EtOH–H2O (1:4, 1:1, and 9:1) to give three fractions (A–C). Fr.B (220 g) was chromatographed on macroporous resin SP825 and eluted with a gradient mixture of EtOH–H2O (2:8, 3:7, 4:6, 11:9, and 8:2) to afford 12 fractions (B1–B12). A part of Fr.B4 (11.3 g) was separated chromatographically over silica gel column and eluted successively with a gradient mixture of CHCl3–MeOH–H2O (65:25:4, 65:35:10, and 60:40:12), and a total of 50 sub-fractions were collected. Sub-fractions 31–43 (3.2 g) were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 17:83 and 20:80), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with MeCN–H2O (23:77) yielded compound 1 (30.0 mg). Sub-fractions 13–17 (600 mg) were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 18:82 and 20:80), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with MeCN–H2O (26:74) yielded compounds 2 (12.0 mg), 3 (17.6 mg), and 4 (9.5 mg). A part of Fr.B5 (20.0 g) was separated chromatographically over silica gel column and eluted successively with a gradient mixture of CHCl3–MeOH–H2O (70:26:6, 65:36:10, and 60:40:12), and a total of 70 sub-fractions were collected. Sub-fractions 42– 48 were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 20:80), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with MeCN–H2O (26:74) yielded compounds 10 (8.2 mg) and 11 (10.3 mg). Sub-fractions 49–51 were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 18:82), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with Me2CO– H2O (18:82) yielded 13 (17.0 mg). Sub-fractions 52–62 were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 20:80), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with Me2CO–H2O (20:80) yielded 7 (8.9 mg). A part of Fr.B6 (27.0 g) was separated chromatographically over silica gel column and eluted successively with a gradient mixture of CHCl3–MeOH–H2O (70:26:6, 65:36:10, and 60:40:12), and a total of 100 sub-fractions were collected. Sub-fractions 38–46 (3.2 g) were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 28:72), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with Me2CO– H2O (35:65) yielded compound 8 (9.3 mg). Sub-fractions 57–62 were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 18:82 and 20:80), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with Me2CO–H2O (20:80) yielded compounds 5 (8.4 mg), 6 (8.1 mg), and 12 (9.3 mg). Sub-fractions 78–80 were combined and chromatographed using an ODS silica gel column (Me2CO–H2O, 20:80), and final purification by preparative HPLC (flow rate 4.5 mL/min) eluted with MeCN–H2O (24:76) yielded compound 9 (46.3 mg).
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3.3.1. Ophiofurospiside C (1) White amorphous powder; a20 D – 79.5 (c 0.110, pyridine); HRESIMS: m/z 1211.5717 [M-H]– (calcd. for C56H91O28, 1211.5697); ESI-MS2: m/z 1193.5725 [M-H-18]–, 1061.5 [M-H-18132]–, 899.4 [M-H-18-132-162]–, 753.3 [M-H-18-132-162-146]–, 591.3 [M-H-18-132-162-146-162]–, and 429.3 [M-H-18-132-162146-162-162]–, and 411.3 [M-H-18-132-162-146-162-162-18]–; 1H NMR data, see Table 1; 13C NMR data, see Table 2. 3.3.2. Ophiofurospiside D (2) White amorphous powder; a20 D – 89.7 (c 0.112, pyridine); HRESIMS: m/z 949.4677 [M-H]– (calcd. for C45H73O21, 949.4644); ESI-MS2: m/z 931.5 [M-H-18]–, 913.4 [M-H-18-18]–, 767.4 [M-H-1818-146]–, 605.4 [M-H-18-18-146-162]–, 443.3 [M-H-18-18-146162-162]–, and 425.3 [M-H-18-18-146-162-162-18]–; 1H NMR data, see Table 1; 13C NMR data, see Table 2. 3.3.3. Ophiofurospiside E (3) White amorphous powder; a20 D – 87.9 (c 0.091, pyridine); HRESIMS: m/z 1065.5144 [M-H]– (calcd. for C50H81O24, 1065.5118); ESI-MS2: m/z 1047.5 [M-H-18]–, 915.4 [M-H-18132]–, 769.3 [M-H-18-132-146]–, 607.3 [M-H-18-132-146-162]–, 445.3 [M-H-18-132-146-162-162]–, and 409.2 [M-H-18-132-146162-162-36]–; 1H NMR data, see Table 1; 13C NMR data, see Table 2. 3.3.4. Ophiofurospiside G (5) White amorphous powder; a20 D – 90.9 (c 0.089, pyridine); HRESIMS: m/z 1049.5162 [M-H]– (calcd. for C50H81O24, 1049.5169); ESI-MS2: m/z 887.5 [M-H-162]–, 755.4 [M-H-162132]–, 737.4 [M-H-162-132-18]–, 575.4 [M-H-162-132-18-162]–, and 413.3 [M-H-162-132-18-162-162]–; 1H NMR data, see Table 1; 13 C NMR data, see Table 2. 3.3.5. Ophiofurospiside H (6) White amorphous powder; a20 D – 59.6 (c 0.079, pyridine); HRESIMS: m/z 1049.5162 [M-H]– (calcd. for C50H81O23, 1049.5169); ESI-MS2: m/z 887.5 [M-H-162]–, 755.4 [M-H-162132]–, 737.4 [M-H-162-132-18]–, 575.4 [M-H-162-132-18-162]–, and 413.3 [M-H-162-132-18-162-162]–; 1H NMR data, see Table 1; 13 C NMR data, see Table 2. 3.3.6. Ophiofurospiside I (7) White amorphous powder; a20 D – 64.5 (c 0.087, pyridine); HRESIMS: m/z 1079.5294 [M-H] – (calcd. for C51H83O24, 1079.5274); ESI-MS2: m/z 917.4 [M-H-162]–, 771.4 [M-H-162146]–, 591.4 [M-H-162-146-180]–, 429.3 [M-H-162-146-180-162]–, and 411.3 [M-H-162-146-180-162-18]–; 1H NMR data, see Table 1; 13 C NMR data, see Table 2. 3.3.7. 7 Ophiofurospiside J (8) White amorphous powder; a20 D – 66.3 (c 0.077, pyridine); HRESIMS: m/z 917.4778 [M-H]– (calcd. for C45H73O19, 917.4746); 1 H NMR data, see Table 1; 13C NMR data, see Table 2.
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3.3.9. Ophiofurospiside L (10) White amorphous powder; a20 D – 70.1 (c 0.110, pyridine); HRESIMS: m/z 1049.5183 [M-H] – (calcd. for C50H81O23, 1063.5169); ESI-MS2: m/z 917.5 [M-H-132]–, 771.4 [M-H-132146]–, 591.4 [M-H-132-146-180]–, 429.3 [M-H-132-146-180-162]–, and 411.3 [M-H-132-162-146-180-162-18]–; 1H NMR data, see Table 1; 13C NMR data, see Table 2. 3.3.10. Ophiofurospiside M (11) White amorphous powder; a20 D – 74.2 (c 0.081, pyridine); HRESIMS: m/z 917.4756 [M-H] – (calcd. for C45H73O19, 917.4746); ESI-MS2: m/z 771.4 [M-H-146]–, 591.4 [M-H-146-180]–, 429.3 [MH-146-180-162]–, and 411.3 [M-H-146-180-162-18]–; 1H NMR data, see Table 1; 13C NMR data, see Table 2. 3.3.11. Ophiofurospiside N (12) White amorphous powder; a20 D – 79.3 (c 0.072, pyridine); HRESIMS: m/z 1033.5271 [M-H]– (calcd. for C50H81O22, 1033.5219); ESI-MS2: m/z 887.4 [M-H-146]–, 775.4 [M-H-146132]–, 609.3 [M-H-146-132-146]–, 447.3 [M-H-146-132-146-162]–, and 429.3 [M-H-146-132-146-162-18]–; 1H NMR data, see Table 1; 13 C NMR data, see Table 2. 3.3.12. Ophiopogonin R0 (13) White amorphous powder; a20 D – 85.4 (c 0.084, pyridine); HRESIMS: m/z 1047.5073 [M-H]– (calcd. for C50H79O23, 1047.5012); ESI-MS2: m/z 915.5 [M-H-132]–, 769.3 [M-H-132146]–, 607.3 [M-H-132-146-162]–, 445.3 [M-H-132-146-162-162]–, and 427.3 [M-H-132-146-162-162-18]–; 1H NMR data, see Table 1; 13 C NMR data, see Table 2. 3.4. Acid hydrolysis and sugar analysis Total saponin (Fr.B) was hydrolyzed in 2 M CF3COOH (5 mL) at 95 C for 5 h. The reaction mixture was extracted with CH2Cl2 (5 mL) three times. The aqueous layer was repeatedly evaporated to dryness with EtOH until neutral. The residue of the sugars was dissolved in anhydrous pyridine (2 mL), 12 mg of L-cysteine methyl ester hydrochloride was added, and the mixture was stirred at 60 C for 1 h. Then, HMDS–TMCS (hexamethyldisilazane–trimethylchlorosilane 2:1) (6.0 mL) was added, and the mixture was kept at 60 C for 30 min. The reference substances, D-glucose, D-xylose, and L-rhamnose (each 1.0 mg) were reacted as above procedure. After centrifugation, the supernatant was analyzed by GC under the following conditions: column temperature: 180 C/250 C; programmed increase, 15 C/min; carrier gas: N2 (1 mL/min); injection and detector temperature: 250 C; injection volume: 1.0 mL, and split ratio: 1/50. The derivatives of D-glucose, D-xylose, and L-rhamnose were detected, with tR values of 17.932 min (Dglucose derivative), 13.005 min (D-xylose derivative) and 14.519 min (L-rhamnose derivative). Acknowledgment This work was financially supported by the Major Program of Municipal Natural Science Foundation of Beijing (No. 7090001). References
3.3.8. Ophiofurospiside K (9) White amorphous powder; a20 D – 68.2 (c 0.091, pyridine); HRESIMS: m/z 1063.5397 [M-H]– (calcd. for C51H83O23, 1063.5325); ESI-MS2: m/z 901.4 [M-H-162]–, 755.4 [M-H-162146]–, 593.4[M-H-162-146-162]–, 431.3 [M-H-162-146-162-162]–, and 413.3 [M-H-162-146-162-162-18]–; 1H NMR data, see Table 1; 13 C NMR data, see Table 2.
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