| Akebia trifoliate?Thunb.?Koidz,a kind of woody lianas,belongs to Akebia of lardizabalaceae,which is one of the basic plants of the “Chinese pharmacopoeia” 2015.Its dry stems have the activities of diuretic,antioxidant and beneficial to some kinds of women diseases.Because of its good diuretic effect,it was widely used for the gonorrhea,upset,urine red,edema,dampness,et al.However,its active fraction and constituents of diuretic effect are not clear.In this study,natural chemical and pharmacological experiments were used to elucidate the active fraction and its antioxidant active was studied.The solution was acquired by extracting the stems of A.Trifoliate coarse powders?4.9 Kg?with 95% ethanol at room temperature,soaking for six times,and 48 hours every time,the extracts?280.6 g?was obtained after vacuum concentration.The ethanol extract was suspended in the water,then successively extracted with petroleum ether,ethyl acetate and n-butanol.Based on the above operation,we obtained the petroleum ether fraction 36.8 g?13.1%?,ethyl acetate fraction 29.7 g?10.6%?,n-butanol fraction 137.7 g?49.1%?and residual water soluble fraction 70.6 g?25.2%?.The ethanol extract of A.trifoliate were extracted with petroleum ether,ethyl acetate,n-butanol and water dissolving fractions,and their diuretic effect was investigated through metabolic cages experiments.The compounds from activity part of n-butanol fraction?P<0.01?were isolated and purified by silica column and Sephadex LH-20,eight compounds were isolated from this part.Their structures were identified by NMR,HRMS and comparison spectral data with literatures.Eight compounds are hederagenin?1?,syringicacid-4-O-?-Lrhamnopyranoside?2?,?-sitosterol-3-O-?-D-glycoside?3?,vanillic acid-?-glucoside?4?,glucosyringic acid?5?,mannitol?6?,hederagenin-3-O-?-L-arabinopyranoside?7?,inositol?8?.Compounds 2,4,5 were isolated from the Akebia for the first time.All compounds were tested for their diuretic activity,compared with the saline control group mannitol?P<0.05?,hederagenin-3-O-?-L-arabinopyranoside?P<0.05?and hederagenin?P<0.05?showed significant diuretic effect.DPPH radical scavenging assay was used for screening the active fractions of ethanol extract for the first time.The compounds of active fraction of ethyl acetate part were isolated and purified by various chromatographic methods,compounds ?-sitosterol?9?,vanillic acid?10?,6,7,10-trihydroxy-8-octadecenoicacid?11?,oleanolic acid?12?,2,6-dimethoxy-1,4-benzoquinone?13?,tetratriacontane?14?,aesculetin?15?,caffeic acid?16?,calceolarioside B?17?were isolated from this fraction.Their structures were identified by NMR,physical and chemical properties.Compounds 10,13,14 were isolated from the Akebia for the first time.The antioxidant activity results showed that caffeic acid(IC50=14.939 ?g.mL-1)and aesculetin(IC50=15.631 ?g.mL-1)possessed significant scavenging activity which were comparable with the Vc(IC50 =17.269 ?g.mL-1),they are the basis of antioxidant activity of A.trifoliate. |
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