Studies On Anti-tumor Effect In Vitro And Chemical Constituents From Diaphragma Juglandis Fructus Of Xinjiang

Objective:1. The study aims to research anti-tumor activity of different extracts of Diaphragma Juglandis Fructus.2. The effective anti-tumor extracts is separated and purified in the study.3. To optimize extraction and purification of total saponins from Diaphragma Juglandis Fructus.4. To research antioxidant activity of total saponins,total flaconoids and total polysaccharides in vitro.Methods:1. The extracts was extracted from Diaphragma Juglandis Fructus with 95% ethanol and then the ethanolic extract was separated with petroleum ether,chloroform,ethyl acetate,and n-butyl alcohol in turn.The effects of different extracts of Diaphragma Juglandis Fructus on proliferation of human hepatoma bele 7404,human non-small lung cancer cell A549,human colon cancer cell line Hct116,Caco-2 were detected by MTT assay.The cell apoptosis rate of the effective parts on 4 human tumor lines was detected with flow cytometry.2. The effective extracs of Diaphragma Juglandis Fructus was separated and purified by the solvent method,macroporous resin method,silica gel column chromatography with gradient elution,and recrystal method.The structures of crystals were identified by physiochemical properties,spectrum analysis and literatures contrast.3. Taking Oleanolic acid as a reference substance,the content of total saponins was determined by UV.The single factor test and orthogonal test was designed for investigating optimum extraction technology.The total saponins was purified by macroreticular resin.4. using VC as a positive control,the antioxidant activity in vitro of total saponins,total flaconoids and total polysaccharides from Diaphragma Juglandis Fructus was evaluated by scavening rates against DPPH,superoxide anion,hydroxyl radicals and ferric reduing antioxidant power.Result:1.The different extracts of Diaphragma Juglandis Fructus inhibited the proliferation of 4cancer cells,but the inhibitory effects of ethyl acetate and n-butyl alcohol on the proliferation of 4 tumor cells were best.Then, the apoptosis of 4 tumor cells is measured by flow cytometry with ethyl acetate and n-butyl alcohol.The experimental results showthat its apoptosis was 50% at the concentration of 400 μg/ml.2. Three compounds were isolated from n-butyl alcohol extract of Diaphragma Juglandis Fructus,they were dimethyl terephthalate(I),quercetin(II),Jugnaphthalenoside A(III).3. The optimized extraction technology of the total saponins was as follows:the powder of Diaphragma Juglandis Fructus was subjected to reflux extraction for 2 times with 80% ethanol at 80 ℃,each extraction lasting 2 h.For each extraction,the solid-liquid was 25.The optimized purification technology was as follows:selected D101 macroreticular resin, removed impurition with 8BV of water,and eluted with 7BV of 50% ethanol.In this way the purity rose from 27.4% to 50.8%.The elution rate was 82.4%.4. The results showed that the total saponins,total flaconoids and total polysaccharides had savening effects on DPPH,superoxide anion,hydroxyl radicals and ferric reduing antioxidant power.Over the studied concentration range,the radical scavenging activity had a dose-effect relationship.Conclusion:1. ethyl acetate and n-butyl alcohol extracts of Diaphragma Juglandis Fructus are the main anticancer active part in vitro.2. This is the first distraction from Diaphragma Juglandis Fructus isolated compound I and III.3. It is shown by an approval test that the optimized process is stable,feasible and suitable for the extraction and purification of total saponin of Diaphragma Juglandis Fructus.4. Total saponins,total flaconoids and total polysaccharides from Diaphragma Juglandis Fructus have antioxidant activity.