| Schisandra chinensis(Turcz.)Baill(Magnoliaceae schisandra plantsis) is a perennial lignifying liana coming from Heilongjiang, Jilin, Liaoning and other places in China.The fruits and seeds of Schisandra chinensis(Turcz.)Baill are similar to ginseng,which are used in traditional Chinese medicine as stimulants and adaptogenic drugs. Schisandra chinensis(Turcz.)Baill is one of five varieties of national modernization of traditional Chinese medicine industry base. The main compositions of Schisandra chinensis(Turcz.)Baill are lignan, triterpenes,that have been widely applied to functional food and medicine field. In recent years, much research for Schisandra chinensis(Turcz,)Baill has been concentrated on it’s fruit and seeds. At the same time there are some research to analyze the content of the lignans, polysaccharide, volatile oil etc.However few works was done on the chemical compositionsin caculis of Schisandra chinensis(Turcz.)Baill. The caculis is used as raw materials in this study, the triterpenes extracting technology, purification technology, physical and chemical properties, structure, antioxidant activity and inhibiting liver-carcinoma-cells activity of the triterpenes were studied.It will lay the foundation developping the industrialization of Schisandra chinensis(Turcz.)Baill and the main results are as follow:1. The triterpenes from caculis of Schisandra Chinensis(Turcz.) baill were extracted by ultrasonic and microwave,the optimual technologys were found through single test and central composite response surface methodology,respectively.In the extraction process by ultrasonic,the optimual result:the extraction time is 48min, the alcohol content was 64%, the frequency of ultrasound was 68W, the ratio of solid and liquid is 1:60, and the extraction percent is 2.53±0.24%. In the extraction process by microwave, the optimual result:the alcohol content was 53%,the multiple of pretreating is 4.6, the pretreating time is 2 min, the extraction percent is 2.02±0.12%. The response surface methodology (RSM) was used to optimize the process parameters of the triterpenes from caculis of Schisandra Chinensis(Turcz.) Baill.This method not only scientific reasonable and efficient, and has a strong guidance to practical production.2. The purification of triterpenes with macroporous resin from caculis of Schisandra Chinensis(Turcz.) baill were researched.The triterpenes from caculis of Schisandra Chinensis(Turcz.) baill were purification using different types of resin adsorption. AB-8 resin was found to be the best for the purification of triterpenes components.The results showed that AB-8 was the most appropriate resin for the purification total triterpenes,with working solution pH6, flow velocity 2BV/h,Injection concentration 1.5 mg/mL.After purification desorption ratio and product purity reached 90.52% and 34.26%% respectively.This separation and purification methods avoid a toxic organic solvent, only use ethanol, and macroporous adsorption resin can be recycled. 3.The triterpenoids are further purified by silica column chromatography, and qualitative colour-display was analysised by TLC method. the best eluant solvent system is chloroform: methanol=5:1 through with different gradient elution from chloroform:methanol=20:1 to 3:1.The results not only confirm the existence of triterpenoids.but also be used to preparate monomer by high speed countercurrent chromatography.4.Four compounds were isolated from caculis of Schisandra Chinensis(Turcz.) baill by various chromatographic methods such as macroporous resin chromatography,silicagel column chromatography, high speed countercurrent chromatography(HSCCC), assistant with traditional methods like ultrasonic extraction.The structures of the four compounds had been identified on the basis of UV, IR, HPLC-MS, NMR, HMBC, HSQC spectrum means and chemical evidence.They are corosolic acid,lancifodilactone C,nigranoic acid,oleanolic acid。The compounds were first isolated from the caculis of Schisandra Chinensis(Turcz.) baill, corosolic acid,lancifodilactone C by using SciFinder scholar database online retrieval5. The anti-oxidative activities of ethanol extracts were evaluated using various established in vitro systems,including total reducing power,fenton method, adjacent pyrogallol since oxidation, diphenyl picryl-hydrazyl, resistance to oxidation and the POV value. The experimental results showed:triterpenes had better reducing power, stronger antioxidation effect on the·OH and DPPH·, and the quality concentration 50%(IC50) was 0.6mg/mL and 0.077mg/mL to inhibit. It had less effect on inhibiting O2-·. Triterpenes could inhibit peroxidation of lard, and have less effect of lard peroxidation than Vc and TBHQ. Triterpenes exhibit remarkable synergistic antioxidation activity with VC in lard.6. The inhibiting activities of the four monomer compounds isolated from the caculis of Schisandra Chinensis(Turcz.) baill against HepG2,SMMC7721,Bel-7402, HSC-T6 in vitro were studied by cell culture methods.The cell toxicities of the samples were tested by3-(4,5-dimethylthiazol-2-yl)-2-5-diphe-nyltetrazolium bromide (MTT) assaying.To investigate the effect of corosolic acid on proliferation andapoptosis of HSC in vitro and explore the mechanisms of corosolic acid inducing apoptosis of HSC by studying the expressions of apoptosis-regulating proteins Caspase3 and Bcl-2 in HSC were studied. The results showed that the corosolic acid had better inhibitory effects against Bel-7402,SMMC7721,HepG2,HSC-T6 cells with the IC50 value of 12.5～25μg/mL,12.5～25μg/mL,12.5～25μg/mL,5～25μg/mL. The oleanolic acid had certain inhibitory effects against HepG2 cell with the IC50 value of 52μg/mL.The lancifodilactone C had certain inhibitory effects against HepG2 and Bel-7402 cells with the IC50 value of 30μg/mL,10μg/mL. The nigranoic acid had certain inhibitory effects against HepG2,Bel-7402 and HSC-T6 cells with the IC50 value of 21μg/mL、16μg/mL、25μg/mL.7.The rate of HSC-T6 apoptosis was identified by flow cytometry (FCM) and the morphological change of apoptosis was observed by light microscopy.And then the expression of apoptosis-regulating protein, Caspase3, Bcl-2 in HSC-T6 after apoptosis induced by corosolic acid were examined by immunocytochemical staining assay. HE staining:Treatment with 25μg/mL concentrations of corosolic acid for 48h resulted in morphologic changes of HSC-T6,including karyorrhexis and cytoplasm vacuolization. Flow cytometry quantitation:After treating HSC-T6 with corosolic acid at concentration of 5 u g/mL,15μg/mL,25μg/mL for 48h,the apoptosis ratio of HSC-T6 were(11.86±2.75)%,(24.62±3.42)%,(48.24±4.75)%, respectively,which were significantly higher than that of control group(2.44±1.95)%%(P<0.01). Immunocytochemistry:the expressions of caspase3 protein in HSC-T6 cells were up-regulated in dose-dependent manner,but expressions of bcl-2 protein were not significantly different from that of the control group. The results suggested that HSC-T6 cells apoptosis induced by corosolic acid occurs through mechanisms involving mitochondrial pathways and Bcl-2 family proteins.The study also indicated that corosolic acid might be a potential Chinese medical component for inhibiting liver fibrosis. |
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