2014 Volume 62 Issue 1 Pages 35-44
Eight new bisdesmosidic triterpenoid saponins, clematiunicinosides A–H (1–8), along with eleven known ones (9–19), were isolated from the roots of Clematis uncinata. Their structures were elucidated on the basis of spectroscopic analysis and chemical evidence. All the isolated saponins were tested for their cytotoxic activities on human caski cervical cancer (Caski) cells, and compounds 13, 17 and 19 exhibited inhibitory effect on Caski cells.
The genus Clematis, belonging to the family Ranunculaceae, comprises of more than 300 species distributed throughout the world.1) Three plants of this genus (Clematis chinensis OSBECK, C. hexapetala PALL., and C. mandshurica RUPR.) are officially used as “Weilingxian,” a traditional Chinese herbal drug, for the treatment of inflammation and cancer in China.2) Previous phytochemical investigations had demonstrated that triterpenoid saponins are the dominant constituents of this genus.3) Some of them showed significant anti-inflammatory and antitumor activities.4–7) During the course of our ongoing program to search for new saponins from traditional Chinese medicine,8–12) Clematis uncinata was selected as a subject for this study. The plant Clematis uncinata is mainly distributed in southern China, such as Guizhou, Zhejiang, Fujian, Guangdong and Guangxi provinces.13) The blank in chemical constituent, the potential medicinal importance and our interest in the chemistry of saponins prompted us to carry out a phytochemical investigation on this plant, which led to the isolation of eight new bisdesmosidic triterpenoid saponins, clematiunicinosides A–H (1–8), together with eleven known ones (9–19). In addition, the cytotoxic effects of the isolated triterpenoid saponins on Caski cells were evaluated. The present paper reports the isolation, structural elucidation and antiproliferative activities of these compounds.
The 70% (V/V) EtOH extract of the roots of C. uncinata was subjected to D101 macroporous resin column eluted successively with H2O, 10% EtOH, 30% EtOH, 50% EtOH and 70% EtOH. The 50% EtOH and 70% EtOH eluates were further purified by repeated column chromatography over middle chromatogram isolated (MCI) gel, octadecyl silica (ODS), silica gel and preparative HPLC to afford eight new bisdesmosidic triterpenoid saponins (1–8), named as clematiunicinosides A–H, as well as eleven known ones (9–19).
Clematiunicinoside A (1) was obtained as an amorphous powder. It showed positive reactions to the Liebermann-Burchard and Molish tests, which suggested that 1 might be a triterpenoid glycoside. The molecular formula of 1 was established as C70H114O34 on the basis of the quasi-molecular ion at m/z 1497.7111 [M−H]− (Calcd for C70H113O34, 1497.7118) by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The IR spectrum showed the presence of hydroxyl (3412 cm−1) and carboxyl (1734 cm−1) groups. The 1H-NMR spectrum of 1 exhibited signals for seven angular methyl groups (δH 0.86, 0.88, 0.88, 1.07, 1.16, 1.24, 1.30, each 3H, s), one oxygenated methine [δH 3.40 (1H, dd, J=2.7, 11.5 Hz)], and one olefinic proton [δH 5.40 (1H, br s)]. In the 13C-NMR spectrum of 1, seventy carbon signals were observed including seven methyl carbons (δC 15.7, 17.1, 17.5, 23.7, 26.1, 28.2, 33.1), one oxygenated methine carbon (δC 88.7), two olefinic carbons (δC 122.9, 144.1), and one carbonyl carbon (δC 176.5). With the assistance of 1H–1H correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), HSQC-total correlation spectroscopy (TOCSY), TOCSY and nuclear Overhauser effect spectroscopy (NOESY) experiments, all the 1H- and 13C-NMR signals of 1 were assigned and showed in Tables 1 and 2. The aglycone of 1 was identified as oleanolic acid by comparison of NMR data with those published in the literature.14)
| Position | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
|---|---|---|---|---|---|---|---|---|
| Ara | ||||||||
| 1 | 4.70 d (7.2) | 4.92 d (6.0) | 4.85 d (6.0) | 4.82 d (5.5) | 4.84 d (6.0) | 4.84 d (6.0) | 4.82 d (6.0) | |
| 2 | 4.53 dd (8.0, 6.0) | 4.50 dd (8.0, 6.0) | 4.56 dd (8.0, 6.0) | 4.54 dd (8.0, 5.5) | 4.57 dd (8.0, 6.0) | 4.57 dd (8.0, 6.0) | 4.53 dd (8.0, 6.0) | |
| 3 | 4.24 | 3.92 | 4.28 | 4.24 | 4.25 | 4.25 | 4.24 | |
| 4 | 4.23 | 4.11 | 4.26 | 4.23 | 4.24 | 4.24 | 4.21 | |
| 5a | 4.36 br d (9.0) | 4.33 br d (9.0) | 4.30 | 4.29 br d (9.0) | 4.30 br d (9.0) | 4.30 br d (9.0) | 4.28 br d (9.0) | |
| 5b | 3.75 br d (9.0) | 3.57 br d (9.0) | 3.81 br d (10.4) | 3.80 br d (9.0) | 3.81 br d (9.0) | 3.81 br d (9.0) | 3.80 br d (9.0) | |
| Xyl | ||||||||
| 1 | 4.81 d (7.5) | |||||||
| 2 | 4.24 | |||||||
| 3 | 4.19 | |||||||
| 4 | 4.15 | |||||||
| 5a | 4.32 | |||||||
| 5b | 3.70 t (10.5) | |||||||
| Rib | ||||||||
| 1 | 5.95 d (4.0) | 5.95 d (4.0) | 5.95 d (4.4) | 5.84 d (5.5) | 5.80 d (5.5) | 5.82 d (5.5) | 5.82 d (6.0) | 5.80 d (5.5) |
| 2 | 4.33 | 4.31 | 4.31 | 4.12 | 4.07 | 4.09 | 4.10 | 4.08 |
| 3 | 4.52 | 4.51 | 4.55 | 4.71 | 4.60 | 4.62 | 4.66 | 4.65 |
| 4 | 4.17 | 4.17 | 4.18 | 4.38 | 4.30 | 4.15 | 4.31 | 4.32 |
| 5a | 4.36 | 4.31 | 4.34 | 4.36 | 4.39 | 4.31 | 4.43 | 4.27 |
| 5b | 4.18 | 4.15 | 4.17 | 4.31 | 4.27 | 4.29 | 4.30 | 4.08 |
| Rha1 | ||||||||
| 1 | 6.30 br s | 6.33 br s | 6.28 br s | 6.54 br s | 6.23 br s | 6.25 br s | 6.25 br s | 6.22 br s |
| 2 | 4.90 br s | 4.86 br s | 4.91 br s | 4.95 | 4.85 br s | 4.87 br s | 4.88 br s | 4.85 br s |
| 3 | 4.73 | 4.73 | 4.74 | 4.74 | 4.67 | 4.68 | 4.68 | 4.66 |
| 4 | 4.45 | 4.43 | 4.43 | 4.46 | 4.41 | 4.43 | 4.43 | 4.41 |
| 5 | 4.67 | 4.71 | 4.28 | 4.72 | 4.60 | 4.62 | 4.66 | 4.58 |
| 6 | 1.56 d (6.0) | 1.56 d (6.0) | 1.54 d (6.0) | 1.52 d (6.0) | 1.52 d (6.0) | 1.53 d (6.0) | 1.53 d (6.0) | 1.52 d (6.0) |
| Rha2 | ||||||||
| 1 | 5.84 br s | 5.82 br s | 5.83 br s | 5.84 br s | 5.82 br s | 5.85 br s | 5.85 br s | 5.83 br s |
| 2 | 4.67 br s | 4.66 br s | 4.65 br s | 4.66 br s | 4.65 br s | 4.67 br s | 4.67 br s | 4.65 br s |
| 3 | 4.54 dd (9.5, 3.5) | 4.53 dd (9.5, 3.5) | 4.54 | 4.54 dd (9.5, 3.5) | 4.53 dd (9.5, 3.5) | 4.54 dd (9.5, 3.5) | 4.55 dd (9.5, 3.5) | 4.54 dd (9.5, 3.5) |
| 4 | 4.32 | 4.31 | 4.32 | 4.33 | 4.32 | 4.33 | 4.33 | 4.33 |
| 5 | 4.95 | 4.94 | 4.94 | 4.93 | 4.93 | 4.95 | 4.95 | 4.94 |
| 6 | 1.69 d (6.0) | 1.67 d (6.0) | 1.70 d (6.0) | 1.68 d (6.0) | 1.68 d (6.0) | 1.68 d (6.0) | 1.68 d (6.0) | 1.68 d (6.0) |
| Rha3 | ||||||||
| 1 | 5.45 br s | |||||||
| 2 | 4.65 br s | |||||||
| 3 | 4.55 | |||||||
| 4 | 4.33 | |||||||
| 5 | 4.25 | |||||||
| 6 | 1.59 d (6.0) | |||||||
| Glc1 | ||||||||
| 1 | 6.23 d (8.0) | 6.21 d (8.0) | 6.27 d (8.0) | 5.00 d (8.0) | 4.96 d (8.0) | 4.96 d (7.5) | 4.95 d (7.5) | 4.89 d (7.5) |
| 2 | 4.13 | 4.10 | 4.10 | 3.93 t (8.0) | 3.88 t (8.0) | 3.92 t (7.5) | 3.91 t (7.5) | 3.89 t (7.5) |
| 3 | 4.21 t (9.0) | 4.20 t (9.0) | 4.20 t (9.0) | 4.20 | 4.23 | 4.23 | 4.15 | 4.17 |
| 4 | 4.28 | 4.30 | 4.30 | 4.17 | 4.25 | 4.26 | 4.21 | 4.29 |
| 5 | 4.10 | 4.08 | 4.07 | 3.90 t (8.5) | 3.91 t (8.5) | 3.92 t (8.5) | 3.86 t (8.5) | 3.65 t (8.5) |
| 6a | 4.66 br d (10.0) | 4.64 br d (10.0) | 4.64 br d (10.0) | 4.47 | 4.47 | 4.41 | 4.38 | 4.21 |
| 6b | 4.31 | 4.30 | 4.33 | 4.31 | 4.27 | 4.29 | 4.30 | 4.16 |
| Glc2 | ||||||||
| 1 | 4.99 d (8.0) | 4.97 d (8.0) | 4.98 d (7.6) | 6.22 d (8.0) | 5.15 d (7.4) | 5.13 d (7.5) | 5.11 d (8.0) | 5.35 d (8.5) |
| 2 | 3.93 t (8.0) | 3.92 t (8.0) | 3.92 t (7.6) | 4.12 | 4.05 t (8.0) | 4.08 t (7.5) | 4.06 t (8.0) | 5.77 t (8.5) |
| 3 | 4.13 | 4.12 | 4.14 | 4.19 | 4.19 | 4.17 | 4.13 | 4.25 |
| 4 | 4.40 t (9.0) | 4.38 t (9.0) | 4.39 | 4.30 | 4.15 | 4.05 | 4.07 | 4.01 |
| 5 | 3.65 d (9.5) | 3.63 d (9.5) | 3.63 d (9.5) | 4.09 | 3.99 t (8.5) | 3.93 t (8.5) | 3.92 t (8.5) | 4.00 |
| 6a | 4.18 | 4.18 t (9.0) | 4.20 | 4.66 br d (10.0) | 4.50 | 4.42 | 3.99 | 4.42 |
| 6b | 4.09 | 4.06 | 4.08 | 4.31 | 4.26 | 4.18 | 4.65 | 4.50 |
| Glc3 | ||||||||
| 1 | 5.12 d (8.0) | 5.07 d (8.0) | 4.98 d (8.0) | 6.21 d (8.0) | 6.23 d (8.0) | 6.23 d (8.0) | 6.22 d (8.0) | |
| 2 | 4.04 t (8.0) | 4.01 t (8.0) | 3.92 dd (9.0, 8.0) | 4.11 | 4.08 | 4.13 | 4.13 | |
| 3 | 4.20 | 4.16 | 4.13 | 4.22 t (9.0) | 4.22 t (9.0) | 4.21 t (9.0) | 4.20 t (9.0) | |
| 4 | 4.22 | 4.23 | 4.38 t (9.0) | 4.28 | 4.31 | 4.32 | 4.29 | |
| 5 | 3.90 t (8.5) | 3.87 t (8.5) | 3.62 d (9.0) | 4.08 | 4.10 | 4.10 | 4.07 | |
| 6a | 4.51 | 4.47 | 4.18 | 4.66 br d (10.5) | 4.67 br d (10.5) | 4.65 br d (10.5) | 4.66 br d (10.5) | |
| 6b | 4.37 | 4.34 | 4.07 | 4.31 | 4.32 | 4.24 | 4.31 | |
| Glc4 | ||||||||
| 1 | 4.98 d (8.0) | 4.99 d (8.0) | 4.99 d (8.0) | 4.98 d (8.0) | ||||
| 2 | 3.92 t (8.0) | 3.94 dd (9.0, 8.0) | 3.93 dd (9.0, 8.0) | 3.92 dd (9.0, 8.0) | ||||
| 3 | 4.13 | 4.15 | 4.15 | 4.14 | ||||
| 4 | 4.38 t (9.0) | 4.41 t (9.0) | 4.41 t (9.0) | 4.40 t (9.0) | ||||
| 5 | 3.64 d (9.0) | 3.65 d (9.0) | 3.65 d (9.0) | 3.66 d (9.0) | ||||
| 6a | 4.18 | 4.19 | 4.20 | 4.19 | ||||
| 6b | 4.07 | 4.09 | 4.09 | 4.05 | ||||
| Glc5 | ||||||||
| 1 | 5.27 d (7.5) | 5.19 d (8.0) | 4.98 d (8.0) | |||||
| 2 | 4.06 t (7.5) | 4.03 t (8.0) | 3.92 | |||||
| 3 | 4.24 | 4.21 | 4.39 | |||||
| 4 | 4.18 | 4.10 | 4.07 | |||||
| 5 | 4.02 | 3.95 | 4.19 t (9.5) | |||||
| 6a | 4.52 | 4.42 | 4.55 | |||||
| 6b | 4.29 | 4.15 | 4.23 | |||||
a) Assignments were established by interpretation of the 1H–1H COSY, TOCSY, NOESY, HSQC, HMBC, and HSQC–TOCSY experiments. b) Overlapped signals are reported whitout designating multiplicity.
| Position | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
|---|---|---|---|---|---|---|---|---|
| 1 | 38.9 | 39.0 | 39.0 | 39.0 | 38.9 | 39.0 | 39.0 | 38.9 |
| 2 | 26.7 | 26.4 | 26.7 | 26.9 | 26.6 | 26.6 | 26.6 | 26.6 |
| 3 | 88.7 | 81.0 | 88.8 | 88.6 | 88.7 | 88.7 | 88.7 | 88.7 |
| 4 | 39.5 | 43.5 | 39.6 | 39.6 | 39.5 | 39.6 | 39.6 | 39.5 |
| 5 | 56.1 | 47.7 | 56.1 | 56.1 | 56.0 | 56.0 | 56.0 | 55.8 |
| 6 | 18.6 | 18.1 | 18.5 | 18.6 | 18.5 | 18.5 | 18.5 | 18.5 |
| 7 | 33.1 | 32.7 | 33.2 | 33.0 | 33.0 | 33.1 | 32.9 | 33.0 |
| 8 | 39.9 | 39.8 | 39.8 | 39.9 | 39.9 | 39.9 | 39.9 | 39.8 |
| 9 | 48.1 | 48.1 | 48.1 | 48.0 | 48.0 | 48.1 | 48.1 | 48.0 |
| 10 | 37.0 | 36.8 | 37.1 | 37.0 | 37.0 | 37.0 | 37.0 | 37.0 |
| 11 | 23.8 | 23.8 | 23.8 | 23.7 | 23.8 | 23.8 | 23.8 | 23.7 |
| 12 | 122.9 | 122.9 | 122.8 | 122.8 | 122.8 | 122.8 | 122.8 | 122.8 |
| 13 | 144.1 | 144.0 | 144.4 | 144.1 | 144.1 | 144.1 | 144.1 | 144.1 |
| 14 | 42.1 | 42.1 | 42.5 | 42.1 | 42.1 | 42.1 | 42.1 | 42.1 |
| 15 | 28.3 | 28.2 | 28.8 | 28.2 | 28.2 | 28.3 | 28.3 | 28.2 |
| 16 | 23.4 | 23.3 | 27.0 | 23.3 | 23.3 | 23.4 | 23.4 | 23.3 |
| 17 | 47.0 | 47.0 | 47.5 | 47.0 | 47.0 | 47.0 | 47.0 | 47.0 |
| 18 | 41.7 | 41.6 | 41.8 | 41.6 | 41.6 | 41.6 | 41.6 | 41.6 |
| 19 | 46.2 | 46.1 | 41.4 | 46.2 | 46.2 | 46.2 | 46.2 | 46.2 |
| 20 | 30.7 | 30.7 | 35.7 | 30.7 | 30.7 | 30.7 | 30.7 | 30.7 |
| 21 | 34.0 | 33.9 | 73.4 | 34.0 | 34.0 | 34.0 | 34.0 | 34.0 |
| 22 | 32.5 | 32.5 | 39.7 | 32.5 | 32.5 | 32.6 | 32.5 | 32.5 |
| 23 | 28.2 | 63.8 | 28.2 | 28.1 | 28.2 | 28.2 | 28.2 | 28.1 |
| 24 | 17.1 | 14.1 | 17.2 | 17.2 | 17.1 | 17.1 | 17.1 | 17.1 |
| 25 | 15.7 | 16.1 | 15.7 | 15.7 | 15.6 | 15.6 | 15.6 | 15.6 |
| 26 | 17.5 | 17.5 | 17.6 | 17.4 | 17.4 | 17.5 | 17.5 | 17.4 |
| 27 | 26.1 | 26.0 | 25.7 | 26.1 | 26.0 | 26.1 | 26.1 | 26.0 |
| 28 | 176.5 | 176.5 | 176.5 | 176.5 | 176.5 | 176.5 | 176.5 | 176.5 |
| 29 | 33.1 | 33.0 | 28.4 | 33.1 | 33.1 | 33.1 | 33.1 | 33.1 |
| 30 | 23.7 | 23.6 | 25.0 | 23.6 | 23.7 | 23.7 | 23.7 | 23.6 |
| Ara | ||||||||
| 1 | 105.2 | 104.6 | 105.3 | 105.1 | 105.2 | 105.2 | 105.2 | |
| 2 | 75.8 | 75.6 | 75.3 | 75.4 | 75.4 | 75.3 | 75.4 | |
| 3 | 74.9 | 75.5 | 74.8 | 74.5 | 74.6 | 74.6 | 74.6 | |
| 4 | 79.8 | 80.8 | 69.4 | 69.2 | 69.3 | 69.5 | 69.3 | |
| 5 | 65.4 | 65.9 | 65.7 | 65.5 | 65.5 | 65.5 | 65.6 | |
| Xyl | ||||||||
| 1 | 106.1 | |||||||
| 2 | 77.1 | |||||||
| 3 | 79.8 | |||||||
| 4 | 71.5 | |||||||
| 5 | 67.0 | |||||||
| Rib | ||||||||
| 1 | 104.6 | 104.8 | 104.7 | 104.7 | 104.6 | 104.8 | 104.7 | 104.6 |
| 2 | 72.8 | 72.7 | 72.9 | 72.5 | 72.5 | 72.5 | 72.5 | 72.7 |
| 3 | 68.9 | 68.7 | 69.0 | 69.6 | 69.4 | 69.8 | 69.8 | 69.3 |
| 4 | 70.3 | 70.2 | 70.2 | 76.4 | 76.4 | 76.4 | 76.4 | 76.4 |
| 5 | 65.3 | 65.2 | 65.3 | 61.8 | 61.7 | 61.7 | 61.7 | 61.6 |
| Rha1 | ||||||||
| 1 | 101.3 | 101.2 | 101.4 | 101.4 | 101.4 | 101.4 | 101.4 | 101.4 |
| 2 | 71.9 | 71.9 | 72.1 | 71.9 | 71.9 | 71.9 | 71.9 | 71.9 |
| 3 | 81.1 | 81.0 | 81.3 | 82.0 | 82.0 | 82.1 | 82.1 | 82.1 |
| 4 | 72.8 | 72.8 | 72.8 | 72.7 | 72.7 | 72.8 | 72.7 | 72.5 |
| 5 | 69.9 | 69.7 | 69.9 | 69.7 | 69.7 | 69.7 | 69.8 | 69.7 |
| 6 | 18.5 | 18.5 | 18.4 | 18.5 | 18.5 | 18.4 | 18.4 | 18.4 |
| Rha2 | ||||||||
| 1 | 102.7 | 102.6 | 102.7 | 102.7 | 102.7 | 102.7 | 102.7 | 102.7 |
| 2 | 72.5 | 72.5 | 72.5 | 72.5 | 72.5 | 72.5 | 72.5 | 72.5 |
| 3 | 72.7 | 72.7 | 72.8 | 72.8 | 72.7 | 72.7 | 72.6 | 72.7 |
| 4 | 73.9 | 73.9 | 74.0 | 74.0 | 73.9 | 74.0 | 74.0 | 73.9 |
| 5 | 70.3 | 70.2 | 70.3 | 70.3 | 70.2 | 70.3 | 70.3 | 70.3 |
| 6 | 18.5 | 18.5 | 18.5 | 18.6 | 18.5 | 18.5 | 18.5 | 18.5 |
| Rha3 | ||||||||
| 1 | 102.8 | |||||||
| 2 | 72.0 | |||||||
| 3 | 72.6 | |||||||
| 4 | 73.9 | |||||||
| 5 | 69.4 | |||||||
| 6 | 18.6 | |||||||
| Glc1 | ||||||||
| 1 | 95.6 | 95.6 | 95.7 | 103.5 | 103.1 | 103.1 | 103.1 | 102.8 |
| 2 | 74.0 | 73.8 | 73.9 | 74.7 | 74.2 | 74.2 | 74.2 | 74.2 |
| 3 | 78.7 | 78.7 | 78.7 | 78.3 | 76.6 | 76.6 | 76.6 | 76.3 |
| 4 | 70.9 | 70.8 | 70.9 | 71.5 | 80.9 | 80.9 | 80.9 | 81.1 |
| 5 | 78.0 | 78.5 | 78.0 | 78.6 | 76.5 | 76.4 | 76.4 | 76.5 |
| 6 | 69.2 | 69.1 | 69.3 | 62.5 | 61.8 | 62.1 | 62.0 | 60.7 |
| Glc2 | ||||||||
| 1 | 104.8 | 104.7 | 104.9 | 95.6 | 104.8 | 104.4 | 104.4 | 102.2 |
| 2 | 75.3 | 75.3 | 75.4 | 73.9 | 74.7 | 73.6 | 73.5 | 73.0 |
| 3 | 76.5 | 76.4 | 76.5 | 78.7 | 78.3 | 88.2 | 88.1 | 86.3 |
| 4 | 78.3 | 78.1 | 78.3 | 70.8 | 71.5 | 69.8 | 69.3 | 70.2 |
| 5 | 77.1 | 77.1 | 77.1 | 78.0 | 78.4 | 77.9 | 77.7 | 77.9 |
| 6 | 61.3 | 61.2 | 61.3 | 69.2 | 62.4 | 61.5 | 68.8 | 62.2 |
| Glc3 | ||||||||
| 1 | 106.8 | 106.9 | 104.8 | 95.6 | 95.6 | 95.6 | 95.6 | |
| 2 | 75.5 | 75.4 | 75.3 | 73.8 | 73.9 | 73.5 | 73.8 | |
| 3 | 78.7 | 78.6 | 76.5 | 78.7 | 78.7 | 78.7 | 78.6 | |
| 4 | 71.3 | 71.1 | 78.2 | 70.8 | 70.9 | 70.8 | 70.8 | |
| 5 | 78.5 | 78.0 | 77.1 | 78.0 | 78.0 | 78.0 | 78.2 | |
| 6 | 62.5 | 62.4 | 61.2 | 69.2 | 69.2 | 69.2 | 69.1 | |
| Glc4 | ||||||||
| 1 | 104.9 | 104.7 | 104.8 | 104.8 | ||||
| 2 | 75.3 | 75.5 | 75.4 | 75.3 | ||||
| 3 | 76.3 | 76.5 | 76.5 | 76.3 | ||||
| 4 | 78.2 | 78.2 | 78.2 | 78.0 | ||||
| 5 | 77.1 | 77.1 | 77.1 | 77.1 | ||||
| 6 | 61.3 | 61.3 | 61.3 | 61.2 | ||||
| Glc5 | ||||||||
| 1 | 105.8 | 105.5 | 105.4 | |||||
| 2 | 75.3 | 75.2 | 74.6 | |||||
| 3 | 78.3 | 78.3 | 78.2 | |||||
| 4 | 71.6 | 71.9 | 71.5 | |||||
| 5 | 78.6 | 77.8 | 78.7 | |||||
| 6 | 62.5 | 61.5 | 62.5 | |||||
Furthermore, the 1H- and 13C-NMR spectra of 1 exhibited seven sugar anomeric proton signals at δH 4.70 (1H, d, J=7.2 Hz), 4.99 (1H, d, J=8.0 Hz), 5.12 (1H, d, J=8.0 Hz), 5.84 (1H, br s), 5.95 (1H, d, J=4.0 Hz), 6.23 (1H, d, J=8.0 Hz) and 6.30 (1H, br s), corresponding to seven anomeric carbon signals at δC 105.2, 104.8, 106.8, 102.7, 104.6, 95.6 and 101.3, respectively (Tables 1, 2). Acid hydrolysis of 1 afforded D-glucose, D-ribose, L-arabinose and L-rhamnose, which were identified by HPLC analysis.15) The relative anomeric configurations of the sugar moieties were determined to be β for the D-glucose (J=8.0 Hz) and α for the L-arabinose (J=7.2 Hz) based on the 3JH1-H2 coupling constants of their anomeric protons. The β configuration for D-ribose could be established by comparison of the NMR data with those reported in the literatures.16,17) The sequences and linkage positions of sugar moieties were subsequently deduced from an HMBC experiment. In the HMBC spectrum of 1, the correlations between H-1 (δH 5.95) of D-ribose and C-3 (δC 81.1) of L-rhamnose1, between H-1 (δH 6.30) of L-rhamnose1 and C-2 (δC 75.8) of L-arabinose, between H-1 (δH 5.12) of D-glucose3 and C-4 (δC 79.8) of L-arabinose, between H-1 (δH 4.70) of L-arabinose and C-3 (δC 88.7) of aglycone, between H-1 (δH 5.84) of L-rhamnose2 and C-4 (δC 78.3) of D-glucose2, between H-1 (δH 4.99) of D-glucose2 and C-6 (δC 69.2) of D-glucose1, as well as between H-1 (δH 6.23) of D-glucose1 and C-28 (δC 176.5) of aglycone were observed. The above findings led to assignment of the sugar sequences and linkage positions of the two oligosaccharide chains were as shown in Fig. 1. Therefore, the structure of 1 was established as 3-O-β-D-ribopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosyl olean-12-en-28-oic acid 28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester.
Clematiunicinoside B (2) was isolated as an amorphous powder. The HR-ESI-MS spectrum of 2 showed a quasi-molecular ion at m/z 1513.7051 [M−H]− (Calcd for C70H113O35, 1513.7062), corresponding to the molecular formula C70H114O35. Similar to 1, the IR spectrum of compound 2 also showed the characteristic absorptions of hydroxyl (3410 cm−1) and carboxyl (1733 cm−1) groups. The 1H-NMR data of 2 displayed the signals for six tertiary methyls (δH 0.85, 0.86, 0.94, 1.09, 1.14, 1.18, each 3H, s), one oxygenated methene [δH 3.91 (1H, t, J=8.5 Hz) and 4.27 (1H, overlapped)], one oxygenated methine [δH 4.23 (1H, overlapped)], one olefinic proton [δH 5.39 (1H, br s)], as well as seven anomeric protons [(δH 4.92 (1H, d, J=6.0 Hz), 4.97 (1H, d, J=8.0 Hz), 5.07 (1H, d, J=8.0 Hz), 5.82 (1H, br s), 5.95 (1H, d, J=4.0 Hz), 6.21 (1H, d, J=8.0 Hz) and 6.33 (1H, br s)]. In the 13C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra of 2, seventy carbon signals were observed, which suggested that 2 was also a triterpenoid saponin with seven sugar units. A comprehensive analysis of the 1H–1H COSY, HSQC, HMBC, HSQC-TOCSY, TOCSY and NOESY spectra allowed the assignment of NMR data of 2 as shown in Tables 1 and 2. The NMR data of 2 assigned to the aglycone part were the same as those of hederagenin.18) Acid hydrolysis of 2 also afforded D-glucose, D-ribose, L-arabinose and L-rhamnose. Comparison of the NMR data of the sugar units in 2 with those of 1 revealed that they were very similar, suggesting that 2 possessed the identical oligosaccharide chains to 1. The linkage sequences and positions of these sugar units were confirmed by the HMBC experiment. Hence, the structure of 2 was established to be 3-O-β-D-ribopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosyl hederagenin 28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester.
Clematiunicinoside C (3) was obtained as an amorphous powder. The molecular formula of 3 was established as C64H104O30 by its HR-ESI-MS (Found m/z 1351.6510 [M−H]−; Calcd for C64H103O30, 1351.6534). The 1H- and 13C-NMR spectra of 3 showed the signals corresponding to a triterpenoid saponin with six sugar units. The 1H–1H COSY, HSQC, HMBC, HSQC-TOCSY, TOCSY and NOESY spectra of 3 allowed the assignment of all proton and carbon signals (Tables 1, 2). The aglycone of 3 was identified to be 21α-hydroxyoleanolic acid by comparison of its NMR data with the literature values.5) Acid hydrolysis of 3 also afforded D-glucose, D-ribose, L-arabinose and L-rhamnose. Comparison of the NMR data assigned to the sugar units of 3 with those of 2 indicated that 3 also possessed two oligosaccharide chains attached at C-3 and C-28 positions of the aglycone, respectively. The difference between them was the absence of the proton and carbon signals of the glucose3 moiety linked to the C-4 position of arabinose in 3. The sequences and linkages of the remained six sugar units could be deduced by an HMBC experiment. In the HMBC spectrum of 3, correlation signals were observed between H-1 (δH 5.95) of D-ribose and C-3 (δC 81.3) of L-rhamnose1, between H-1 (δH 6.28) of L-rhamnose1 and C-2 (δC 75.3) of L-arabinose, between H-1 (δH 4.85) of L-arabinose and C-3 (δC 88.8) of aglycone, between H-1 (δH 5.83) of L-rhamnose2 and C-4 (δC 78.3) of D-glucose2, between H-1 (δH 4.98) of D-glucose2 and C-6 (δC 69.3) of D-glucose1, as well as between H-1 (δH 6.27) of D-glucose1 and C-28 (δC 176.5) of aglycone. Thus, the structure of 3 was identified as 3-O-β-D-ribopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl 21α-hydroxyolean-12-en-28-oic acid 28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester.
Clematiunicinoside D (4) was obtained as an amorphous powder. The HR-ESI-MS spectrum of 4 exhibited a quasi-molecular ion at m/z 1497.7109 [M−H]− (Calcd for C70H113O34, 1497.7113), consistent with the molecular formula C70H114O34. The 1H- and 13C-NMR data of the aglycone part of 4 were closely similar to those of 1, indicating that they possessed the same aglycone (oleanolic acid). Besides the signals assigned to the aglycone part, the NMR data of 4 showed the presence of seven anomeric protons [δH 4.81 (1H, d, J=7.5 Hz), 4.98 (1H, d, J=8.0 Hz), 5.00 (1H, d, J=8.0 Hz), 5.84 (1H, br s), 5.84 (1H, d, J=5.5 Hz), 6.22 (1H, d, J=8.0 Hz) and 6.54 (1H, br s)] as well as seven anomeric carbons (δC 95.6, 101.4, 102.7, 103.5, 104.7, 104.8, 106.1), which indicated that 4 also possessed seven sugar moieties. Acid hydrolysis of 4 afforded D-xylose, D-glucose, L-rhamnose and D-ribose by HPLC analysis.15) The relative anomeric configurations of the sugar moieties were established based on the 3JH1-H2 coupling constants of their anomeric protons as well as by comparison of the NMR data with those reported in the literatures.4,16) In the HMBC spectrum of 4, the correlations between H-1 (δH 5.00) of D-glucose1 and C-4 (δC 76.4) of D-ribose, between H-1 (δH 5.84) of D-ribose and C-3 (δC 82.0) of L-rhamnose1, between H-1 (δH 6.54) of L-rhamnose1 and C-2 (δC 77.1) of D-xylose, between H-1 (δH 4.81) of D-xylose and C-3 (δC 88.6) of aglycone, between H-1 (δH 5.84) of L-rhamnose2 and C-4 (δC 78.2) of D-glucose3, between H-1 (δH 4.98) of D-glucose3 and C-6 (δC 69.2) of D-glucose2, and between H-1 (δH 6.22) of D-glucose2 and C-28 (δC 176.5) of aglycone were observed. Based on the above results, the structure of 4 was elucidated as 3-O-β-D-glucopyranosyl(1→4)-α-D-ribopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-β-D-xylopyranosyl olean-12-en-28-oic acid 28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester.
Clematiunicinoside E (5) was isolated as an amorphous powder. The molecular formula of 5 was determined as C76H124O39 by HR-ESI-MS spectrum (m/z 1659.7601 [M−H]−; Calcd for C76H123O39, 1659.7641). Interpretation of the 2D-NMR spectra (1H–1H COSY, HSQC, HMBC, HSQC-TOCSY, TOCSY and NOESY) of 5 allowed the assignment of all proton and carbon signals as shown in Tables 1 and 2. The aglycone of 5 was also identified as oleanolic acid by comparison of the 1H- and 13C-NMR data with those of 1. In addition, the NMR spectra of 5 exhibited eight anomeric proton and carbon resonances, which indicated the existence of eight sugar moieties in 5. Acid hydrolysis of 5 also gave L-arabinose, D-glucose, D-ribose and L-rhamnose. The relative anomeric configurations of the sugar moieties were further determined based on the 3JH1-H2 coupling constants of their anomeric protons as well as by comparison of the NMR data with those reported in the literatures.4,16) Comparison of the NMR data assigned to the sugar units of 5 with those of 3 indicated that 5 possessed the same sugar chain at C-28 position of the aglycone. Different from 3, the NMR spectra of 5 showed the presence of two additional glucose units [(δH 4.96 (1H, d, J=8.0 Hz) and 5.15 (1H, d, J=7.4 Hz), as well as δC 103.1 and 104.8]. The HMBC correlations between H-1 (δH 5.15) of D-glucose2 and C-4 (δC 80.9) of D-glucose1, and between H-1 (δH 4.96) of D-glucose1 and C-4 (δC 76.4) of D-ribose indicated that the additional two glucose units were attached to C-4 position of glucose1 and ribose, respectively. Based on the above evidence, 5 was identified as 3-O-β-D-glucopyranosyl(1→4)-β-D-glucopyranosyl(1→4)-β-D-ribopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl olean-12-en-28-oic acid 28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester.
The HR-ESI-MS spectrum of 6 showed a quasi-molecular ion at m/z 1821.8112 [M−H]− (Calcd for C82H133O44, 1821.8170), corresponding to the molecular formula C82H134O44, which was 162 mass units more than that of 5. Acid hydrolysis of 6 afforded L-arabinose, D-glucose, D-ribose and L-rhamnose. Comparison of 1H- and 13C-NMR spectra of 6 with those of 5 suggested that they possessed the same oleanolic acid as aglycone and two oligosaccharide chains attached at C-3 and C-28 positions of the aglycone, respectively. The difference between them was the appearance of signals due to an additional glucose unit [δH 5.27 (1H, d, J=7.5 Hz); δC 105.8] in 6. The location of the additional glucose moiety was deduced by the HMBC correlation between H-1 (δH 5.27) of the additional glucose moiety (D-glucose5) and C-3 (δC 88.2) of D-glucose2. Thus, the structure of 6 was established as 3-O-β-D-glucopyranosyl(1→3)-β-D-glucopyranosyl(1→4)-β-D-glucopyranosyl(1→4)-β-D-ribopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl olean-12-en-28-oic acid 28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester.
The molecular formula of 7 was determined as C88H144O48 on the basis of the quasi-molecular ion at m/z 1967.8811 [M−H]− (Calcd for C88H143O48, 1967.8749) by its HR-ESI-MS spectrum. Similar to 6, acid hydrolysis of 7 afforded L-arabinose, D-glucose, D-ribose and L-rhamnose. The NMR data of 7 were very similar to those of 6, except for the presence of signals corresponding to an additional rhamnose unit. In the HMBC spectrum of 7, the correlation between H-1 (δH 5.45) of the additional rhamnose moiety and C-6 (δC 68.8) of D-glucose2 was observed, which indicated that the additional rhamnose unit (L-rhamnose3) was attached to the C-6 position of D-glucose2. Therefore, the structure of 7 was determined as 3-O-β-D-glucopyranosyl(1→3)-[α-L-rhamnopyranosyl(1→6)]-β-D-glucopyranosyl(1→4)-β-D-glucopyranosyl(1→4)-β-D-ribopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl olean-12-en-28-oic acid 28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester.
Clematiunicinoside H (8) showed a molecular formula C92H142O47 by its HR-ESI-MS spectrum at m/z 1997.8654 [M−H]− (Calcd for C92H141O47, 1997.8649). The UV spectrum of 8 showed the absorptions maxima at 244, 296 and 326 nm. The IR spectrum implied the presence of hydroxyl (3413 cm−1) and carboxyl (1715 cm−1) groups as well as aromatic ring (1633, 1450 cm−1). Acid hydrolysis of 8 afforded D-glucose, L-rhamnose, L-arabinose and D-ribose, which were identified by HPLC analysis.15) With the aid of 2D-NMR experiments (1H–1H COSY, HSQC, HMBC, HSQC-TOCSY, TOCSY and NOESY), all proton and carbon resonances in 8 were assigned as shown in Tables 1 and 2. Comparison of the 1H- and 13C-NMR data of 8 with those of 6 revealed that they were similar except that 8 displayed proton and carbon signals due to an additional isoferuloyl moiety [δH 3.75 (3H, s), 6.89 (1H, d, J=8.0 Hz), 6.90 (1H, d, J=16.0 Hz), 7.09 (1H, dd, J=2.0, 8.0 Hz), 7.54 (1H, d, J=2.0 Hz) and 8.10 (1H, d, J=16.0 Hz); δC 55.9, 112.1, 115.4, 116.4, 121.5, 128.6, 145.8, 148.4, 150.9 and 166.8].4) The location of the isoferuloyl moiety could be deduced by an HMBC experiment. Hence, in the HMBC spectrum, the correlation between H-2 (δH 5.77) of D-glucose2 and the carbonyl carbon (δC 166.8) of isoferuloyl moiety was observed, suggesting that the isoferuloyl moiety was attached at the C-2 position of D-glucose2. Based on the above evidence, the structure of 8 was established as 3-O-β-D-glucopyranosyl(1→3)-[(2-O-isoferuloyl)-β-D-glucopyranosyl]-(1→4)-β-D-glucopyranosyl(1→4)-β-D-ribopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl olean-12-en-28-oic acid 28-O-α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester.
Compounds 9–19 were identified as clematernoside E (9), huzhangoside D (10), clematiganoside A (11), huzhangoside B (12), huzhangoside C (13), clematichinenoside C (14), clemastanoside D (15), hederasaponin B (16), CP7 (17), CP6 (18) and CP4 (19), respectively, by comparison of their spectral data with literature values.16,17,19–23)
The cytotoxic activities of all the saponins, as well as their aglycones oleanolic acid and hederagenin against Caski cells were evaluated in this study. Aglycones and most of the saponins did not show obvious effects under the higher concentration of 80 µM with the exception of compounds 13, 17 and 19 (Table 3). Compounds 13, 17 and 19 displayed potent effect with IC50 values of 9.05±3.61 µM, 26.07±1.47 µM and 2.97±1.07 µM, respectively. Herein, cisplatin was used as a positive control with IC50 value of 3.37±0.77 µM. These results suggested that some saponins isolated from Clematis uncinata might show better antitumor activities on Caski cells than their aglycones. Considering the insufficient samples size of our current investigation, the structure–activity relationship of these compounds was still an open question, which need to be solved in the further study.
| Compounds | IC50 (µM) | Cell survival rate (% of blank, at concentration of 80 µM) | Compounds | IC50 (µM) | Cell survival rate (% of blank, at concentration of 80 µM) |
|---|---|---|---|---|---|
| 1 | >80 | 100.62±1.01 | 12 | >80 | 107.33±3.21 |
| 2 | >80 | 93.38±1.76 | 13 | 9.05±3.61 | |
| 3 | >80 | 93.45±2.45 | 14 | >80 | 97.27±3.70 |
| 4 | >80 | 95.38±1.72 | 15 | >80 | 72.84±4.13 |
| 5 | >80 | 96.01±3.19 | 16 | >80 | 85.71±5.05 |
| 6 | >80 | 95.58±2.22 | 17 | 26.07±1.47 | |
| 7 | >80 | 77.76±1.49 | 18 | >80 | 75.51±5.50 |
| 8 | >80 | 101.24±1.46 | 19 | 2.97±1.07 | |
| 9 | >80 | 77.24±2.34 | Oleanolic acid | >80 | 90.73±2.39 |
| 10 | >80 | 98.31±2.91 | Hederagenin | >80 | 83.57±1.82 |
| 11 | >80 | 95.63±0.98 | Cisplatin | 3.37±0.77 |
a) Data was expressed as means±S.E. (n=3).
Optical rotations were measured on a JASCO P-1020 digital polarimeter in a 0.1 dm length cell at room temperature. UV spectra were obtained on a JASCO-V-550 UV/VIS spectrophotometer. IR spectra were recorded on a JASCO FT/IR-480 plus infrared spectrometer. HR-ESI-MS data were obtained on an Agilent 6210 ESI/time-of-flight (TOF) or a Waters Xevo-G2 QTOF mass spectrometer. 1H-, 13C-, and 2D-NMR experiments were performed on Bruker AV-400 or Bruker AV-500 spectrometer using pyridine-d5 as solvent with tetramethylsilane (TMS) as internal standard. Analytical HPLC was performed on an Agilent 1260 chromatography equipped with a G1311C pump, a G1315D photodiode array detector and a Cosmosil 5C18-MS-II Waters column (4.6×250 mm, 5 µm, Nacalai Tesque Inc., Kyoto, Japan). Preparative HPLC was carried on an Agilent 1260 chromatography equipped with a G1310B pump, a G1365D detector and a Cosmosil 5C18-MS-II Waters column (20×250 mm, 5 µm, Nacalai Tesque Inc., Kyoto, Japan). Column chromatographies were performed on silica gel (300–400 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China), D101 macroporous resin (Tianjin Chemical Co., Ltd., Tianjin, China), MCI gel (75–150 µm, Mitsubishi Chemical Co., Tokyo, Japan), and ODS (40–64 µm, Merck, Darmstadt, Germany) columns. TLC was performed using precoated silica gel GF254 plates (Yantai Chemical Industry Research Institute, Yantai, China).
The roots of C. uncinata were collected in Tianmu Mountain, Zhejiang province of P. R. China, in August of 2008 and authenticated by Prof. Guang-xiong Zhou (Institute of Traditional Chinese Medicine & Natural Products, Jinan University). A voucher specimen (No. 2008082301) was deposited in the Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou, P. R. China.
The air-dried roots of C. uncinata (2.0 kg) were pulverized and extracted with 70% (v/v) EtOH under reflux (2×4 L) twice (1.5 h each). The solution was concentrated under vacuum to yield a crude extract (258 g). The EtOH extract was redissolved in water and then subjected to D101 macroporous resin column eluted successively with H2O, 10% EtOH, 30% EtOH, 50% EtOH, and 70% EtOH.
The 50% EtOH eluate (74 g) was subjected to a MCI gel column eluted with gradient mixtures of MeOH–H2O (3 : 7, 5 : 5, 7 : 3, v/v) to give three fractions (Fractions 1–3). Fraction 1 (5 g) was separated by ODS column (5×40 cm) with MeOH–H2O (15 : 85, 20 : 80, 25 : 75, 30 : 70, 35 : 65, 40 : 60, 50 : 50) as eluent to afford three subfractions (1A–C). Subfraction 1A (0.4 g) was isolated by preparative HPLC (CH3CN–H2O, 30 : 70, 6.0 mL/min) to yield compounds 2 (19 mg, tR=17.8 min) and 3 (34 mg, tR=20.1 min). Subfraction 1B (0.8 g) was also isolated by preparative HPLC (CH3CN–H2O, 30 : 70, 6.0 mL/min) to yield compounds 10 (35 mg, tR=25.5 min) and 11 (42 mg, tR=33.3 min). Fraction 2 (12 g) was separated by ODS column eluted with gradient mixtures of MeOH–H2O (25 : 75, 30 : 70, 35 : 65, 40 : 60, 50 : 50, 60 : 40) to afford six subfractions (2A–F). Subfraction 2A (1.4 g) was separated by preparative HPLC on a reversed-phase C18 column using CH3CN–H2O (30 : 70, 6.0 mL/min) as eluent to yield compounds 7 (40 mg, tR=52.1 min), 8 (36 mg, tR=57.2 min), and 9 (32 mg, tR=63.6 min). Subfraction 2C (2.9 g) was also separated by preparative HPLC (CH3CN–H2O, 32 : 68, 6.0 mL/min) to yield compounds 4 (27 mg, tR=42.3 min), 5 (31 mg, tR=38.4 min), and 6 (36 mg, tR=45.2 min), respectively. Subfraction 2E (2.7 g) was isolated successively by preparative HPLC (CH3OH–H2O, 68 : 32, 6.0 mL/min) to yield compounds 1 (34 mg, tR=32.5 min) and 14 (58 mg, tR=27.2 min). Subfraction 2F (1.6 g) was isolated by preparative HPLC (CH3OH–H2O, 68 : 32, 6.0 mL/min) to yield compounds 12 (160 mg, tR=35.5 min) and 13 (65 mg, tR=38.2 min). Fraction 3 (7 g) was isolated by ODS column using MeOH–H2O (30 : 70, 35 : 65, 40 : 60, 45 : 55, 50 : 50, 60 : 40) as eluent to give three subfractions (3A–C). Subfractions 3A (1.2 g) and 3B (4.3 g) were respectively isolated by preparative HPLC (CH3CN–H2O, 35 : 65, 6.0 mL/min) to yield compounds 15 (24 mg, tR=52.3 min) and 16 (33 mg, tR=45.6 min).
The 70% EtOH eluate (23 g) was separated by silica gel column (15×60 cm) using gradient mixtures of CHCl3–CH3OH–H2O (9 : 1 : 0.1, 8 : 2 : 0.2, 7 : 3 : 0.5) as eluent to give five fractions (Fractions A–E). Fraction A (0.7 g) was isolated by preparative HPLC (CH3CN–0.05% TFA, 45 : 55, 6.0 mL/min) to yield compound 19 (42 mg, tR=25.4 min). Fraction C (1.5 g) was also isolated by preparative HPLC (CH3CN–0.05% TFA, 45 : 55, 6.0 mL/min) to yield compounds 17 (18 mg, tR=25.1 min) and 18 (16 mg, tR=28.1 min), respectively.
Clematiunicinoside A (1): Amorphous powder; [α]D20 −46.6 (c=0.10, MeOH); IR νmax (KBr) cm−1: 3412, 2936, 1734, 1642, 1065; 1H-NMR (pyridine-d5, 400 MHz) δ: 0.86 (3H, s, 25-CH3), 0.88 (3H, s, 30-CH3), 0.88 (3H, s, 29-CH3), 1.07 (3H, s, 26-CH3), 1.16 (3H, s, 24-CH3), 1.24 (3H, s, 27-CH3), 1.30 (3H, s, 23-CH3), 3.40 (1H, dd, J=2.7, 11.5 Hz, 3-H), 5.40 (1H, br s, 12-H). 1H-NMR data of sugar moieties and 13C-NMR data, see Tables 1 and 2; HR-ESI-MS m/z: 1497.7111 [M−H]− (Calcd for C70H113O34, 1497.7118).
Clematiunicinoside B (2): Amorphous powder; [α]D20 −35.6 (c=0.13, MeOH); IR νmax (KBr) cm−1: 3410, 2936, 1733, 1642, 1064; 1H-NMR (pyridine-d5, 400 MHz) δ: 0.85 (3H, s, 29-CH3), 0.86 (3H, s, 30-CH3), 0.94 (3H, s, 25-CH3), 1.09 (3H, s, 26-CH3), 1.14 (3H, s, 24-CH3), 1.18 (3H, s, 27-CH3), 4.23 (1H, overlapped, 3-H), 3.91 (1H, t, J=8.5 Hz, 23-Hb), 4.27 (1H, overlapped, 23-Ha), 5.39 (1H, br s, 12-H); 1H-NMR data of sugar moieties and 13C-NMR data, see Tables 1 and 2; HR-ESI-MS m/z: 1513.7051 [M−H]− (Calcd for C70H113O35, 1513.7062).
Clematiunicinoside C (3): Amorphous powder; [α]D20 −40.5 (c=0.10, MeOH); IR νmax (KBr) cm−1: 3411, 2936, 1737, 1643, 1385, 1063; 1H-NMR (pyridine-d5, 400 MHz) δ: 0.90 (3H, s, 25-CH3), 1.04 (3H, s, 30-CH3), 1.12 (3H, s, 26-CH3), 1.15 (3H, s, 24-CH3), 1.21 (3H, s, 29-CH3), 1.29 (3H, s, 23-CH3), 1.37 (3H, s, 27-CH3), 3.30 (1H, dd, J=4.0, 11.6 Hz, 3-H), 3.68 (1H, br s, 21-H), 5.50 (1H, br s, 12-H); 1H-NMR data of sugar moieties and 13C-NMR data, see Tables 1 and 2; HR-ESI-MS m/z: 1351.6510 [M−H]− (Calcd for C64H103O30, 1351.6534).
Clematiunicinoside D (4): Amorphous powder; [α]D20 −54.6 (c=0.10, MeOH); IR νmax (KBr) cm−1: IR (KBr) νmax 3412, 2937, 1644, 1067; 1H-NMR (pyridine-d5, 400 MHz) δ: 0.86 (3H, s, 25-CH3), 0.88 (3H, s, 30-CH3), 0.88 (3H, s, 29-CH3), 1.05 (3H, s, 26-CH3), 1.20 (3H, s, 24-CH3), 1.23 (3H, s, 27-CH3), 1.34 (3H, s, 23-CH3), 3.30 (1H, dd, J=4.0, 11.5 Hz, 3-H), 5.38 (1H, br s, 12-H); 1H-NMR data of sugar moieties and 13C-NMR data, see Tables 1 and 2; HR-ESI-MS m/z: 1497.7109 [M−H]− (Calcd for C70H113O34, 1497.7113).
Clematiunicinoside E (5): Amorphous powder; [α]D20 −47.0 (c=0.10, MeOH); IR νmax (KBr) cm−1: 3413, 2936, 1643, 1065; 1H-NMR (pyridine-d5, 400 MHz) δ: 0.84 (3H, s, 25-CH3), 0.87 (3H, s, 30-CH3), 0.88 (3H, s, 29-CH3), 1.06 (3H, s, 26-CH3), 1.12 (3H, s, 24-CH3), 1.23 (3H, s, 27-CH3), 1.26 (3H, s, 23-CH3), 3.26 (1H, dd, J=3.5, 11.5 Hz, 3-H), 5.39 (1H, br s, 12-H); 1H-NMR data of sugar moieties and 13C-NMR data, see Tables 1 and 2; HR-ESI-MS m/z: 1659.7601 [M−H]− (Calcd for C76H123O39, 1659.7641).
Clematiunicinoside F (6): Amorphous powder; [α]D20 −37.5 (c=0.10, MeOH); IR νmax (KBr) cm−1: 3400, 2935, 1644, 1067; 1H-NMR (pyridine-d5, 400 MHz) δ: 0.86 (3H, s, 25-CH3), 0.88 (3H, s, 30-CH3), 0.88 (3H, s, 29-CH3), 1.07 (3H, s, 26-CH3), 1.13 (3H, s, 24-CH3), 1.24 (3H, s, 27-CH3), 1.28 (3H, s, 23-CH3), 3.27 (1H, dd, J=3.5, 11.0 Hz, 3-H), 5.39 (1H, br s, 12-H); 1H-NMR data of sugar moieties and 13C-NMR data see, Tables 1 and 2; HR-ESI-MS m/z: 1821.8112 [M−H]− (Calcd for C82H133O44, 1821.8170).
Clematiunicinoside G (7): Amorphous powder; [α]D20 −41.75 (c=0.10, MeOH); IR νmax (KBr) cm−1: 3414, 2935, 1735, 1643, 1064; 1H-NMR (pyridine-d5, 400 MHz) δ: 0.86 (3H, s, 25-CH3), 0.89 (3H, s, 30-CH3), 0.89 (3H, s, 29-CH3), 1.07 (3H, s, 26-CH3), 1.13 (3H, s, 24-CH3), 1.24 (3H, s, 27-CH3), 1.28 (3H, s, 23-CH3), 3.27 (1H, dd, J=3.5, 11.5 Hz, 3-H), 5.40 (1H, br s, 12-H); 1H-NMR data of sugar moieties and 13C-NMR data, see Tables 1 and 2; HR-ESI-MS m/z: 1967.8811 [M−H]− (Calcd for C88H143O48, 1967.8749).
Clematiunicinoside H (8): Amorphous powder; [α]D20 −52.8 (c=0.16, MeOH); UV λmax (MeOH) nm (log ε): 244 (3.59), 296 (3.92), 326 (4.05); IR νmax (KBr) cm−1: 3413, 2936, 1715, 1633, 1450, 1264, 1064; 1H-NMR (pyridine-d5, 400 MHz) δ: 0.85 (3H, s, 25-CH3), 0.88 (3H, s, 30-CH3), 0.88 (3H, s, 29-CH3), 1.06 (3H, s, 26-CH3), 1.12 (3H, s, 24-CH3), 1.24 (3H, s, 27-CH3), 1.26 (3H, s, 23-CH3), 3.26 (1H, dd, J=3.0, 11.0 Hz, 3-H), 3.75 (3H, s, OCH3), 5.39 (1H, br s, 12-H), 6.89 (1H, d, J=8.0 Hz, 5′-H), 6.90 (1H, d, J=16.0 Hz, 8′-H), 7.09 (1H, dd, J=2.0, 8.0 Hz, 6′-H), 7.54 (1H, d, J=2.0 Hz, 2′-H), 8.10 (1H, d, J=16.0 Hz, 7′-H); 13C-NMR data of isoferuloyl moiety (pyridine-d5, 100 MHz) δ: 55.9 (OCH3), 112.1 (C-5′), 115.4 (C-2′), 116.4 (C-8′), 121.5 (C-6′), 128.6 (C-1′), 145.8 (C-7′), 148.4 (C-4′), 150.9 (C-3′), 166.8 (C-9′); 1H-NMR data of sugar moieties, and 13C-NMR data of aglycone and sugar moieties, see Tables 1 and 2; HR-ESI-MS m/z: 1997.8654 [M−H]− (Calcd for C92H141O47, 1997.8649).
Each solution of compounds 1–8 (each 10 mg) in 2 mol/L HCl (10 mL) was heated in water bath (80°C) for 4 h. The solution was evaporated with a stream of N2. Each residue was dissolved in pyridine (2.0 mL), and stirred with L-cysteine methyl ester hydrochloride (10 mg) for 1 h at 60°C, and then O-tolyl isothiocyanate (15 µL) was added to the mixture and heated at 60°C for another 1 h. The reaction mixtures were analyzed by HPLC and detected at 250 nm. Analytical HPLC was performed on a Cosmosil 5C18-MS-II column (4.6×250 mm, 5 µm) at 20°C using CH3CN–0.05% CH3COOH (25 : 75, 0.8 mL/min) as the solvent. Peaks were detected with a G1315D photodiode array detector. The absolute configurations of the monosaccharides were confirmed to be D-glucose, D-xylose, D-ribose, L-arabinose and L-rhamnose by comparison of the retention times of monosaccharide derivatives with those of standard samples: D-glucose (tR 19.98 min), L-glucose (tR 18.29 min), D-xylose (tR 23.41 min), L-xylose (tR 21.81 min), D-ribose (tR 24.12 min), L-ribose (tR 16.49 min), D-arabinose (tR 24.31 min), L-arabinose (tR 22.43 min), L-rhamnose (tR 33.82 min), and D-rhamnose (using D-cysteine methyl ester and L-rhamnose, tR 17.82 min), respectively.
The cytotoxicity activities of the compounds on Caski cells were tested with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described in the previous report.24) Briefly, compounds 1–19, oleanolic acid and hederagenin (Guangzhou Qiyun Biotechnology Co., Ltd., Guangzhou, China) were dissolved in dimethyl sulfoxide (DMSO) and diluted in the culture medium to the concentrations ranging from 0.3125–80 µM. Caski cells (6×103 cells/well) were cultured in 96-well plates for 24 h. The medium was replaced by the medium with the different concentrations of compounds and cultured for another 48 h. MTT solution was then added in the medium for coloration. The absorption value was recorded by the Microplate reader (Thermo, U.S.A.) at a wavelength of 570 nm. Cell viability was calculated according to the following formula: cell viability (%)=OD (compound)/OD (control)×100. IC50 values were obtained from cell viability plots fitted with a sigmoidal dose-response function with variable slope using Origin 8 software. Cisplatin was used as a positive control.
This work was supported financially by the Program for Changjiang Scholars and Innovative Research Team in the University (No. IRT0965), the Joint Fund of NSFC-Guangdong Province (No. U0932004), the Science and Technology Planning Project of Guangdong Province (No. 2012A080204005), the National Natural Science Foundation of China (No. 81202427), and the Program for New Century Excellent Talents in University (No. NCET-11-0857).
