Study On Chemical Constituents Of Lonicera Alberti Regel And Their Hepatoprotective Activitis

Objection:To investigate chemical constituents from Lonicera alberti Regel and establish a HPLC method for simultaneously determining the indicative contents,and study preventive effect of extracts of Lonicera alberti leaves on hepatic injury.Methods:1)Chemical constituents were isolated and purified by various chromatographic techniques such as silica gel,macroporous resin,and Sephadex LH-20 column chromatography.Their structures were elucidated by physicochemical properties and spectroscopic data.2)A HPLC method for simultaneously determining the contents of three kinds of caffeoylquinic acids was established.The chromatographic conditions were as follows:Phenomenex C18 column(4.6 mm×250 mm,5 ?m),acetonitrile(A)and 0.2%phosphoric acid solution(B)as the mobile phase for gradient elution(0-13 min.120%A:13-25 min,12%-22%A;25-45 min,22%A),flow rate at 1.0 mL·min-1,detection wavelength at 327 nm.A HPLC method for determining the contents of loganin was established.The chromatographic conditions were as follows:Cosmosil Ph-AR(250 mm× 4.6 mm,5?m),acetonitrile(A)and 0.2%(v/v)phosphoric acid solution(B)as the mobile phase for gradient elution(0-30 min.8%A-9%A).flow rate at 1.0 mL/min,detection wavelength at 236nm.Column temperature was 30 ? and injection volume was 10?L.3)Antioxidant activity of Lonicera alberti Rege extracts were evaluated by determining free radical-scavenging activity on DPPH.Extracts with the strongest antioxidant activity in vitro were used to evaluate the activity of anti-liver injury in vivo.Liver injury model induced by carbon tetrachloride in mice was established.Healthy adult mice were equally randomized to six groups,which include control group,model group,dimethyl diphenyl bicarboxylate(DDB)group,different doses of extracts of Lonicera alberti leaves(500,250,125 mg/kg·BW).Each group was treated with oral administration once a day for successively 7 days.Two hours after the last medication,the mice in all groups(except the control group)received intraperitoneal injection of 0.12%carbon tetrachloride olive oil solution.After 12h,the blood was taken from the neck and the activity of ALT and AST in the serum was measured.Livers and spleens were taken out by laparotomy and weighed to calculate liver index and spleen index.The pathological changes in the livers of mice was observed.Liver homogenate was prepared and the activities of SOD,GSH and the content of MDA were determined Results:1)Ten compounds were separated from L.alberti,and were identified as 3,7-dimethylquercetin(1),3,3'-dim ethyl quercetin(2),quercetin(3),ursolic acid(4),loganin(5),chlorogenic acid(6),quercetin 5-O-?-D-glucoside(7),3,4-O-dicaffeoylquinic acid(8),3,5-O-dicaffeoylquinic acid(9)and 4,5-O-dicaffeoylquinic acid(10).2)Compounds 6,9 and 10 in samples achieved baseline separation and showed good linearity at the HPLC conditions,the average recoveries were 100.15%,99.68%and 99.52%respectively.The contents of three compounds from L.alberti flower(3.99%,1.77%,2.33%)was more than that of leaves(2.62%,1.73%,0.90%)and caulis(0.74%,0.25%,0.15%).Loganin in samples achieved baseline separation and showed good linearity at the HPLC conditions,and the average recovery was 99.23%.The content of loganin from Lonicera alberti leaves was 0.17%and caulis was 0.15%,it was not detected in the flowers.3)30%extract from Lonicera alberti leaves has better DPPH radicals scavenging ability.IC50 of DPPH radicals scavenging ability was 51.50?g·mL-1.30%extract from Lonicera alberti leaves were used to evaluate the activity of anti-liver injury in vivo.Compared with CCl4 model group,the liver indices and the activities of ALT and AST in serum of each treatment group were decreased significantly(P<0.05,or P<0.01),the activities of GSH and SOD in liver homogenate of each treatment group were increased significantly(P<0.05 or P<0.01)and the content of MDA of each treatment group in liver homogenate was reduced significantly(P<0.05 or P<0.01)According to the results of pathological examination,the hepatic injury of each treatment group mice were relieved significantly Conclusion:1)Compound 1,2 and 7 were isolated from Lonicera genus for the first time,and others were found from this plant for the first time.2)The content determination method was accurate,reliable and convenient,so it could be used for content determination of chlorogenic acid,3,5-dicaffeoylquinic acid,4,5-dicaffeoylquinic acid and loganin inL.alberti.3)Extract of Lonicera alberti leaves has better antioxidant ability and preventive effect on acute liver damage in mice induced by CCl4,and its hepatoprotective mechanism might be related to scavenge free radicals and inhibit lipid peroxidation.