| Daphne genkwa Sieb.et Zucc., indigenous to the provinces along both the Yangtze River and the Yellow River of China, is a member of the family Thymelaeaceae (daphne). It is used as a folk medicine in China mainly for diuretic, pesticide, antitussive, expectorant, antifertility and antitumours purpose. The chemical constituents of the 95% EtOH extracts obtained from the flower buds of Daphne genkwa were investigated. Herein, we report the biological examination of compounds’cytotoxic activities against four tumor cell lines, as well as the neuroprotective properties of the isolated compounds which were tested by their inhibitory effects on LPS-induced NO production in microglia cells. We also described the quantitative determination of daphne-type diterpenes from both crude drug and rice vinegar processed drug of Daphne genkwa.By means of various chromatographic methods such as silica gel, sephadex LH-20, ODS column chromatography and semi-preparative HPLC,52 compounds were isolated from the 95% EtOH extracts of the flower buds of Daphne genkwa, and their structures were elucidated on the basis of spectroscopic data and physico-chemical methods. These compounds include 25 diterpenes, 11 flavones,5 lignans,7 sterols and 1 amide.14 new compounds were named as genkwanine M (1), genkwanine N (2), genkwanine P (3), genkwanine Q (4), genkwanine R (5), genkwanine O (6), genkwanine I (7), genkwanine S (8), genkwanine T (9), genkwanine U (10), genkwanine V (11), 12-O-(2’E,4’E-decadienoyl)-4-hydroxyphorbol-13-acetyl (12), isoyuanhuadine (13) and (-)-9’-hydroxysesamin (27).The other 37 known compounds were identified as yuanhuadine (14), daphnane-type diterpene ester-7 (15), orthobenzoate 2 (16), genkwanine A (17), genkwanine H (18), genkwanine D (19), genkwanine F (20), genkwadaphnine (21), yuanhuatine (22), yuanhuafine (23), yuanhuapine (24), yuanhuacine (25), genkdaphin (26), (-)-pinoresinol (28), (-)-lariciresinol (29), pinoresinol (30), genkwanin (31), apigenin (32), luteolin (33), kampherol (34),7,3’-di-O-methylluteolin (35),8-methoxykaempferol (36), naringenin (37), tiliroside (38), genkwanin-5-O-β-D-glucoside (39), genkwanin-5-O-β-D-primeveroside (40), kaempferol-3-O-β-D-glucoside (41), isodaphnoretin B (42), umbelliferone (43), aurantiamide acetate (44), stigmasta-5-en-3β,7α-diol (45), stigmasta-5-en-3β,7β-diol (46), stigmasta-4-en-3β,6β-diol (47),7-ketositosterol (3β-hydroxysitost-5-en-7-one 48), ergo sterol peroxide (49),β-sitosteryl-3β-glucopyranoside-6’-O-palmitate(50),β-sitosterol (51), palmitate-glyceride (52).Among them, compound 16 is a novel natural product, compounds 36,46-50 were obtained from genus daphne for the first time, and compounds 15,28,37 and 45 were obtained from Daphne genkwa for the first time. An overview on the spectral, structural characteristics and biosynthesis of daphnane-type diterpenes was taken.The antitumor activities of 22 daphne-type diterpenes and 4 sterols against four tumor cell lines HL-60, A-549, Hep3B and U87 were tested. Most of daphne-type diterpenes were found to possess potent antitumor activities. The structure-activity relationship of daphne-type diterpenes was discussed. The results showed that the presence of acyl groups at C-3, C-20 or C-14 in the molecular might have an important role in cytotoxic activity. Sterols have no effects on cytotoxic activity, except for compound 45 was found to possess cytotoxic activity against HL-60 cell line.We have, therefore, tested the 18 compounds for their inhibitory effects on NO production by LPS-activated N9 microglial. The results showed that 13 daphne-type diterpenes and 1 sterols had inhibitory effects on LPS-induced NO production. In order to avoid the possible effects of reduced viability on NO release, the cytotoxic activity of the compounds on microglial cells in the presence or abrsence of LPS was assessed using MTT assays. A lot of compounds possess cytotoxic activity against N9 microglial. There was no obvious structure-activity relationship between daphne-type diterpenes and NO release activity.As a Chinese medicinal plant, Daphne genkwa was always used after vinegar processing for decreasing irritants. The quantitative determination of sixteen daphne-type diterpenes from both crude drug and rice vinegar processed drug of Daphne genkwa was conducted by reversed phase high performance liquid chromatography with mass spectrometry (LC-MS). The differences between pre-processing and processed Daphne genkwa provided the evidence for the study on the medicinal processing. |
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