Studies On The Chemical Constituents Of Total Flavonoids Of Bidens Bipinnata L. And Their Potential Anti-liver Fibrosis Activities

Bidens bipinnata L. is a annual herb belongs to the Bidens genus(Asteraceae), which is one of the conventional compositae medicinal plants. It has been reported that Bidens bipinnata L. includes no less than 240 species worldwide and mainly distributed in tropical and subtropical areas in Asia. The whole herb was used as folk medicine for the treatment of upper respiratory infection, acute appendicitis, acute icteric hepatitis, gastroenteritis. In recent years, more biological activities of Bidens bipinnata L., have been demonstrated including anti-inflammatory, anti-liver fibrosis, and anti-oxidation. Our team has long focus on the exploration and development of medicinal plants including Bidens bipinnata L., and the results of our previous studies have demonstrated the anti-liver fibrosis effect of total flavonoids of Bidens bipinnata L.(TFB). In the present study, to explore the activity-structure relationship of total flavonoids of Bidens bipinnata L.(TFB), the chemical components were separated and purified, and their function on the proliferation of HSC-T6 cells and the lipopolysaccharide(LPS) induced IL-6 and TNF-α production of macrophage(RAW264.7) cells were investigated.Objective: To isolate, identify of the chemical constituents of total flavonoids of Bidens bipinnata L.(TFB) and screen their anti-liver fibrosis activities.Methods: Compounds from total flavonoids of Bidens bipinnata L.(TFB) were isolated using macroporous resin, reverse-phase silica gel column chromatography, Sephadex LH-20, MCI and ODS chromatography. The structures of the isolated compounds wereelucidated on the basis of their spectroscopic data(1H-NMR and 13C-NMR), and by comparison of their spectroscopic data with those reported in the literature. The proliferation of HSC-T6 cells was evaluated by MTT. The inhibitory activity of the compounds against lipopolysaccharide(LPS) induced IL-6 and TNF-α production in macrophage(RAW264.7) cells were investigated using Elisa method.Results: 15 components were isolated from total flavonoids of Bidens bipinnata L.(TFB) including 3-(hydroxyacetyl)indole(1), kaempferol(2), 6,7,3’,4’-tetrahydroxy-aurone(3), quercetin(4), okanin(5), pinoresinol(6), 3,5,7,4￠-tetra hydroxyl-8-isopentene group flavonoids-3-O-a-L-rhamnopyranoside(7), isookanin(8), bis(2-ethylheptyl) phythalate(9),(2S/2R) isookanin-7-O-β-D-(2’’,4’’,6’’-triacetyl)glucopyranoside(10/11),(3E,11E)-tetradecadiene-5,7,9-tetrayn-1,2,13-triol(12), 2-O-β-D-glucopyranosyloxy-3,11E-en-5,7,9-tetrayn-1-hydroxytrideca(13),(6E, 12E)-tridecadiene-8,10-diyn-1,14-diol-3-O-β-D-glucopyranoside(14), 5-(2-Phenyleth ynyl)-2-β-glucosylmethyl-thiophene(15).6 Effects of the components on the proliferation of HSC-T6 cells assay were performed via MTT method. The result indicated that compounds 2(kaempferol), 4(quercetin), 6(pinoresinol) and 8(isookanin) could inhibit the proliferation of HSC-T6 cells with the concentration ranging from 200 μmol×L-1 to 12.5 μmol×L-1. Among them, compounds 2(kaempferol), 4(quercetin) showed potent inhibitory activities and the IC50 values were 38.41 and 40.44 μmol×L-1, respectively. The inhibitory ability of the components against lipopolysaccharide(LPS) induced IL-6 and TNF-α production in macrophage(RAW264.7) cells were also tested. The result suggested that compounds 2(kaempferol), 3(6,7,3’,4’-tetrahydroxy-aurone), 4(quercetin), and 5(okanin) could decrease the production of IL-6 induced by LPS in RAW264.7 cells with the concentration ranging from 100 μmol×L-1 to 12.5μmol×L-1. Moreover, compounds 2(kaempferol), 3(6,7,3’,4’-tetrahydroxy-aurone), and 4(quercetin) could reduce the concentration of TNF-α in the supernatant of RAW264.7 cells induced by LPS, with theconcentration ranging from 100 μmol×L-1 to 25 μmol×L-1.Conclusion: 15 components were isolated from total flavonoids of Bidens bipinnata L.(TFB), among which compounds 6(pinoresinol) and 9(bis(2-ethylheptyl) phythalate) were isolated from this genus for the first time. Compounds 2(kaempferol), 4(quercetin), 6(pinoresinol) and 8(isookanin) showed inhibitory activities of proliferation of HSC-T6 cells with the concentration ranging from 100 μmol?L-1 to 6.25 μmol?L-1. Among them, compounds 2(kaempferol), 4(quercetin) showed potent inhibitory activities. Compounds 2(kaempferol), 3(6,7,3’,4’-tetrahydroxy-aurone), 4(quercetin), 5(okanin) inhibit the production of IL-6 induced by LPS in RAW264.7 cells with the concentration ranging from 100 μmol?L-1 to 12.5μmol?L-1 and compounds 2(kaempferol), 3(6,7,3’,4’-tetrahydroxy-aurone), 4(quercetin) inhibit the production of IL-6 induced by LPS in RAW264.7 cells with the concentration ranging from 100 μmol?L-1 to 25 μmol?L-1.