Isolation, Identification And Analysis Of Hepatoprotective Constituents From Chrysanthemum Indicum L

Chrysanthemum indicum L belongs to the family Compositae. The ethanolic extractand its fractions partitioned by different polar solvents were prepared, then tested on anacute liver damage model in Con A-induced mice. Eight compounds from higherhepatoprotective fraction were obtained and structurally elucidated. Then thecomponents from Chrysanthemum indicum L were screened in vitro forhepatoprotective activity. Finally, the extracts and its fractions were analyzed in order toexplore a material basis of hepatoprotective activity for wild chrysanthemum. Thisthesis is divided into four parts, of which the specific contents are as follows:1. Hepatoprotective capacity test of extracts from Chrysanthemum indicum LChrysanthemum indicum L was extracted3times, each time2h, by80%ethanolwith meterial to solvent ratio of1:8. The ethanol extract was combined, concentratedand further patitioned by petroleum ether, ethyl acetate, n-butyl alcohol sequently to getpetroleum ether, ethyl acetate, n-butyl alcohol and water soluable fractions. Then theenthanol extract and its fractions were tested on ConA-induced acute liver injury miceto explore the protective effect of extracts from Chrysanthemum indicum L.The resultsshowed that the ethyl acetate layer exhibited better hepatoprotective effect than then-butanol layer. 2. Separation of compounds in the hepatoprotective part from Chrysanthemum indicumL.Different chromatographic methods including positive silicagel, reverse silicagel C18and Sephadex LH-20dextrangel, were applied to isolate the components in hepatoprot-ective parts. Through HPLC, LC-MS, NMR analysis,8compounds were identified as6-methoxy-5,7,4′-trihydroxyflavone (hispidulin, Ⅰ),5,6,7,4’-tetrahydroxyflavone(scutellarein, Ⅱ),5,7,3’,4’-trihydroxy-3,6,8-trimethoxyflavone, Ⅲ, kaempf-erol-7-O-β-D-glucosyl (1→6)-α-L-rhamnoside (Isorhoifolin, Ⅳ),5,7,4’-trihydroxy-3,6,3’–trimethoxyflavone (jaceidin,Ⅴ), acacetin-7-O-β-D-glucosyl (1→6)-α-L–rham-noside (linarin, Ⅵ),5,7-dihydroxy-4’-methoxyflavone (acacetin, Ⅶ),3,5,7,3’,4’–pentahydroxyflavone (quercetin, Ⅷ), among which compound Ⅰ, Ⅱ, Ⅳ, Ⅴ werefirstly reported from Chrysanthemum indicum L.3. Screening test on TGF β1-induced hepatic stellate cell (HSC) proliferation of10isolated components form Chrysanthemum indicum LTen isolated compounds, including compounds Ⅰ, Ⅲ~Ⅷ,3,5-caffeoylquinic acid,1,3-caffeoylquinic acid and3,4-caffeoylquinic acid, were tested and evaluated forability to inhibit TGF β1-induced HSC proliferation. The results showed that activecompounds, including quercetin (Ⅷ),3,5-caffeoylquinic acid,1,3-caffeoylquinic acidand3,4-caffeoylquinic acid had significant antiproliferation capacity with IC50less than100μM. Compound Ⅰ, Ⅲ, Ⅳ also exhibited good antiproliferation capacity with IC50ranged in150~300μM, whereas the obundant compound linarin showed little inhibitoryeffect on HSC proliferation with IC50more than600μM.4. Preliminary analysis of hepatoprotective components from Chrysanthemum indicumLThe content of isolated8compounds were indentified in the extracts ofChrysanthemum indicum L by retention time (RT) and measured by peak areanormalization method in order to preliminarily explore the hepatoprotective basis ofChrysanthemum indicum L. Among the isolated compounds, the contents of quercetinand linarin were highest while that of compound Ⅴ is the lowest.The effectivecompounds Ⅳ, Ⅷ arehigher in the n-butanol layer. The most active compounds,3, 5-caffeoylquinic acid and3,4-caffeoylquinic acid, are highest in the ethyl acetate layerwhich caused obvious hepatoprotective activity in this part.