Bioactivity Analysis of Ingredients and Flavonoids Compounds from Cirsium Japonicum DC
Supervisor:jiang jian guo
Cirsium japonicum DC,belonging to the family of Compositae,is a wild perennial plant widely used for Cool blood and hemostasis.In China,C.japonicum is listed as a health food,and has been reported to possess many bioactivities.The main components were extracted,and their bioactivities were investigated.The flavonoids were further isolated and purified to obtain 13 compounds and their antioxidant,anti-inflammation and anti-atherosclerosis activities were evaluated and the mechanisms were also investigated.(1)To analyze the health effects of C.japonicum,flavonoids,saponins,essential oil,coumarin and alkaloids were extracted from C.japonicum,and antioxidative effect,hepatoprotective effect,cytotoxicity,anti-inflammatory effect against RAW 264.7 macrophages induced by lipopolysaccharide(LPS),and antiproliferative effects against human lung adenocarcinoma cell A549 were investigated.Results showed that flavonoids exhibited best antioxidative capacity,followed by saponoin and cumarin.Coumarin exhibited greatest cell toxicity(IC50=162.7 μg/mL),and alkaloids also showed slightly cytotoxicity at high concentration.Flavonoids could significantly reverse CCl4-induced hepatocyte L02 cell viability.Saponin could significantly inhibit cancer cell proliferation,especially for A549 cell and the inhibition effect reached 47.0% at concentration of 0.2 mg/mL,which might result from the promotion of reactive oxygen species generation in cancer cell.The saponin,essential oil and flavonoids could dose-dependently inhibit nitric oxide production in LPS-induced RAW 264.7 cells,whose inhibition rates were 65.4%,73.0% and 80.4% at concentration of 50 μg/ml,respectively.These results indicated that different components from C.japonicum exhibited different bioactivities,and flavonoids were the main effective ingredients in C.japonicum,which deserved further investigation.(2)C.japonicum was extracted with 70% ethanol,and then loaded on a macroporous resin column chromatography to get five fractions.Results showed that fraction 50% ethanol fraction(CJ-50)exhibited the best antioxidant and anti-inflammation effects.Six compounds were isolated and purified from CJ-50 fraction through column chromatography.The total flavonoids were also further isolated and purified to get nine compounds in which two compounds were also obtained from CJ-50.So total 13 compounds were obtained,which were luteolin-7-O-β-D-glucuronide(1),luteolin-7-O-β-D-glucuronide methyl ester(2),apigenin-7-O-β-D-glucuronide(3),linarin(4),apigenin-7-O-β-D-glucuronide methyl ester(5),pectolinarin(6),kaempferol(7),quercetin(8),astragalin(9),quercetin-3-O-glucopyranoside(10),kaempferol-3,7-di-O-α-L-rhamnoside(11),kaempferol-3-O-rutinonoside(12),rutin(13),and compounds 1-3,5,9-12 were isolated and identified from C.japonicum for the first time.(3)Cell viability results showed that different compounds exhibited different inhibition effects against HepG2 cells,in which the effects of astragalin and kaempferol-3-O-rutinonoside were better.Luteolin-7-O-β-D-glucuronide and luteolin-7-O-β-D-glucuronide methyl ester showed better antioxidant activity.Further investigation indicated that these two luteolin derivatives could reduce intracelluer ROS level and increase the content of GSH in cells.(4)The isolated compounds displayed different inhibitory effects on LPS-induced macrophage NO production,among which luteolin-7-O-β-D-glucuronide exhibited strong inhibition effect.Further investigation showed that luteolin-7-O-β-D-glucuronide could inhibit the production of inflammatory cytokines,which might be through inhibiting the activation of mitogen-activated protein kinases(MAPKs).(5)Oil red O staining and Dil-oxidized low-density lipoprotein(Dil-ox-LDL)uptake results showed that apigenin-7-O-β-D-glucuronide could inhibit macrophage uptake ox-LDL and the accumulation of lipids,decrease the content of LDL-Cholesterol and trigryceride.Further investigation showed that apigenin-7-O-β-D-glucuronide could inhibit the expression of CD36 and upregulate the expression of Scavenger Receptor Class B Member 1 in ox-LDL induced RAW 264.7 cell,which might be through the MAPK pathway.