Chemical Constituents From The Whole Plants Of Clematis Heracleifolia

The plants of genus Clematis (family Ranunculaceae) are widely used as folk medicine in China for curing rheumatism, relieving pain and swelling. The pharmacological studies have showed that they exhibited anti-tumor, anti-inflammatory activities and so on. Clematis heracleifolia mainly distributes in Shannxi, Jiangsu, Shandong, Hebei, Hunan and Hubei Province of China. It is rich in natural resources and lives in the hillside ravine, woodside and roadside thickets. So far, its chemical composition has not been reported. In order to clarify the main chemical composition and discover new compounds with anti-tumor activities, the systematic studies on the chemical consitituents and preliminary biological activities of the whole plants were carried out for the first time by the combination of modern chromatographies and spectra analysis techniques, as well as MTT assay. In this way, the thesis was expected to provide new theoretical basis and application prospect for Clematis heracleifolia.To investigate the chemical constituents of the whole plants of Clematis heracleifolia, the plant materials were extracted with 75% EtOH, and the crude extract was suspended in petroleum ether and n-BuOH successively. The n-BuOH extract was purified by silica gel, ODS and Sephadex LH-20 column chromatographies, as well as semi-preparative HPLC. Several isolated saponins were dealed with by alkaline hydrolysis to yield prosapogenins. Then 27 compounds were identified in all according to their physical and chemical properties and spectroscopic techniques.Thereinto,22 compounds were isolated from the n-BuOH extract. They were identified as friedelin (1), epifriedeland (2),?-sitosterol (3), quercetin-3-O-?-D-galactoside (4), isoorientin (5), kalopanax saponin G (6), cauloside D (7), yemuoside YM32 (8), huzhangoside B (9), clematiganoside A (10), huzhangoside C (11), flaccidoside ? (12), oleanolic acid 3-O-?-L-arabinopyranosyl-28-O-?-L-rhamnopyr-anoyl-(1?4)-?-D-glucopyranosyl-(1?6)-?-D-glucopyranosyl ester (13), kalopanax saponin B (14), clemargenoside B (15), ciwujianoside A1 (16),3-O-a-L-rhamnopyranosyl-(1?2)-a-L-arabinopyranosyl oleanolic acid 28-O-?-L-rhamn-opyranosyl-(1?4)-?-D-glucopyranosyl-(1?6)-?-D-glucopyranoide (17),3-O-?-D-xylopyranosyl-(1?3)-?-L-rhamnopyranosyl-(1?2)-a-L-arabinopyranosyl oleanolic acid 28-O-?-L-rhamnopyranosyl-(1?4)-?-D-glucopyranosyl-(1?6)-?-D-glucopyranosyl ester (18),3?-O-a-L-rhamnopyranosyl-(1?4)-?-D-glucopyranosyl moronic acid 28-O-?-D-glucopyranosyl-(1?6)-?-D-glucopyranosyl ester (19), 3?-O-?-L-arabinopyranosyl moronic acid 28-O-a-L-rhamnopyranosyl-(1?4)-?-D-glucopyranosyl-(1?6)-?-D-glucopyranoside (20),3?-O-a-L-arabinopyranosyl moronic acid 28-O-a-L-rhamnopyranosyl-(1?4)-?-D-glucopyranosyl-(1?-6)-?-D-glucopyranoside (21) and 3?,23-dihydroxyolean-18-en-28-oic acid-O-a-L-rhamnopyranosyl-(1?4)-?-D-glucopyranosyl-(1?6)-?-D-glucopyranoside (22) respectively. Five prosapogenins were obtained through alkaline hydrolysis and identified as hederagenin 3-O-a-L-arabinopyranoside (7a), saponin CP4(9a), moronic acid 3?-O-a-L-rhamnopyranosyl-(1?4)-?-D-glucopyranosyl ester (19a), moronic acid 3?-O-?-L-arabinopyranosyl ester (20a) and moronic acid 3?-O-?-D-glucopyranosyl-(1?3)-a-L-arabinopyranosyl (21a). All of the compounds were obtained from Clematis heracleifolia for the first time. Compounds 19-22 were new compounds and named heracleifolianosides A, B, C and D. The structures of the artifacts 19a,20a and 21a were also new. Compound 8 was firstly isolated from genus Clematis.Nine native saponins and five prosapogenins were screened for their anti-tumor activity by MTT assay. Adriamycin was chosen as positive control. The results showed that the monodesmosic saponins 7a,9a,19a,20a and 21a with free carboxyl group at 28 position exhibited different levels of activity against the human tumor MDA-MB-231 cell lines,and 9a displayed significant cytotoxicity against MDA-MB-231 cells and SGC-7901 cells, while the nine bisdesmosides and the monodesmoside with the sugar chain linked at C-28 were inactive. It suggested that the sugar chain linked at C-3 and the free carboxyl group was necessary for the oleanane-type saponins to possess cytotoxic activity against tumor cells. The studies indicated that it might be a feasible way to obtain the preparations with anti-tumor effects by dealing with the Chinese herb extracts with bisdesmosic tritepenoid saponins as the main constituents through a simple procedure of alkaline hydrolysis.Innovative topics and research significance:1. The systematic studies on the chemical constituents of Clematis heracleifolia were carried out for the first time and 27 compounds were identified for the first time, including twenty-one oleanane saponins (17 native ones and 5 artifacts), two friedelanes, two flavonoid glycosides and one sterols.2. Chemical novelty:Four saponins were identified as new compounds. The structural feature of a double bond at 18 (19) position of the aglycone was rarely encountered in the genus. Moreover, the disaccharide chain at C-3 of the aglycone of saponin 21 was found in the genus for the first time. All of the compounds were isolated from the plant for the first time, including one saponin firstly obtained from the genus. Meanwhile, three new prosapogenins were also identified.3. The cytotoxicity assay of several isolated saponins and the five prosapogenins was carried out and the preliminary structure-activity relationship was discussed. The way was proposed that the Chinese herb extracts with bisdesmosic tritepenoid saponins as the main constituents might be developed as anti-tumor preparations by alkaline hydrolysis.4. The studies focused on triterpenoid saponins of Clematis heracleifolia have laid the scientific foundation of its further development, and provided valuable leads for pursuing new antitumor drugs.