Study On Chemical Constituents Of Anemone Reflexa

The plants of genus Anemone (family Ranunculaceae) are widely distributed in the world and mostly of them are in Europe and Asia. The species of genus Anemone is diverse. There are about 150 in the world and about 52 species exsited in China. Rhizome is the main medicinal parts of the genus, for example, the rhizome of A. raddeana was contained in the Chinese Pharmacopeia. From the accumulation of experience in private medicine to modern pharmacological studies confirmed that the genus was used to curing rheumatism, eliminate carbuncle swollen, detoxification, and so on. The plants of genus Anemone contain many kinds of chemical constituents and some compounds have performanced significant pharmacological activity. So far, the investigation of chemical constituents and pharmacological activity of Anemone reflexa have not been reported.In this study, the systematic investigation on the chemical consitituents and biological activities of Anemone reflexa were carried out for the first time. To investigate the chemical constituents of Anemone reflexa, the roots and stems of this plant were extracted with 75% EtOH under reflux. The crude extract was suspended in water and then extracted with petroleum ether and n-BuOH, sequentially. The n-BuOH extract was purified by Sephadex LH-20, silica gel and ODS column chromatographies, as well as semi-preparative HPLC, repeatedly. 20 compounds were isolated and were identified according to their physical and chemical properties and spectroscopic data, They were ferulic acid (1),?-sitosterol (2), ?-daucosterol (3),3?-O-?-L-arabinopyranosyl oleanolic acid (4),3?-O-?-D-glucuronopyranosyl oleanolic acid (5),3?-O-?-L-rhamnopyranosyl-(1?2)-[?-D-glucopyranosyl-(1?4)]-a-L-arabinopyranosyl oleanolic acid (6), pulsatillosi-de D (7),3?-O-?-D-xylopyranosyl-(1?3)-?-L-rhamnopyranosyl-(1?2)-[?-D-glucopyran-osyl-(1?4)]-?-L-arabinopyranosyl oleanolic acid (8), 3?-O-?-L-rhamnopyranosyl-(1?2)-[?-D-glucopyranosyl-(1?4)-?-D-glucopyranosyl-(1?4)]-?-L-arabinopyranosyl olean-olic acid (9), 3?-O-?-D-glucopyranosyl-(1?4)-?-D-glucopyranosyl-(1?3)-?-L-rhamno-pyranosyl-(1?2)-?-L-arabinopyranosyl oleanolic acid (10),3?-O-?-D-glucopyranosyl-(1 ?3)-?-L-rhamnopyranosyl-( 1 ?2)-[?-D-glucopyranosyl-( 1 ?4)-?-D-glucopyranosyl-(1?4)]-?-L-arabinopyranosyl oleanolic acid (11),3?-O-?-D-ribopyranosyl-(1?3)-?-L-rhamnopyranosyl-(1?2)-[?-D-glucopyranosyl-( 1 ?4)-?-D-glucopyranosyl-( 1 ?4)]-?-L-arabinopyranosyl oleanolic acid (12), anhuienoside D (13), 3?-O-6-O-methyl-glucurono-pyranosyl oleanolic acid 28-O-?-D-glucopyranosyl-(1?3)-?-L-rhamnopyranosyl-(1?4)-?-D-glucopyranosyl-(1?6)-?-D-glucopyranoside (14),3?-O-6-O-butyl-glucuronopyran-osyl oleanolic acid 28-O-?-D-glucopyranosyl-(1?3)-?-L-rhamnopyranosyl-(1?4)-?-D-glucopyranosyl-(1?6)-?-D-glucopyranoside (15),anhuienoside E (16),hederasaponin B(17), hederacolchiside F (18), sieboldianoside B (19), hederacolchiside E (20). All of the compounds were obtained from Anemone reflexa for the first time. Compounds 9, 11, 12,14 and 15 were identified as new compounds and compound 10 was firstly isolated from genus Anmone.The cytotoxicity assay showed that compounds 6-12 displayed significant cytotoxicity against human hepatocellular carcinoma HepG2 cells and human leukemia HL-60 cells. And compound 6 showed the most potent cytotoxicity to these two cancer cell lines with IC50 at 6.98 ?mol/L and 4.83 ?mol/L, respectively. The experimental results further validated the structure-activity relationship. The results showed that the structure-activity relationship of the saponins which was summarized by us is right. It is that the compounds with well activity must have the following structures, a free carboxylic acid at C-28 of the oleanane skeleton and ?-L-rhamnopyranosyl-(1?2)-?-L-arabinopyranosyl group at C-3.Innovations and significance:1. The studies on the chemical constituents of Anemone reflexa were carried out for the first time and 20 compounds were isolated and identified. Seventeen of them are oleanane saponins, including five new compounds and a compound which was discovered from genus of Anemone for the first time.2. New compounds 14 and 15 possess glucuronic acid methyl and butyl ester respectively at C-3, which were rare found in this genus.3. The aglycones of most saponins which were isolated from this genus and had showed cytotoxicity were oleanolic acid. Through the preliminary screening of anti-tumor activity, we confirmed the structure-activity relationship of saponins. Those indicated that a large number of anti-tumor components exsited in this plant.