| Glycoside isoflavones are rich in Fructus sophorae. There are no reports on biotransformation of glycoside isoflavones in Fructus sophorae, especially on biotransformation by edible and medical fungi. Thereforth, in this dissertation, Fructus sophorae biotransformation study by edible and medical fungi and anti-breast-cancer cell activities of its products were explored systematically, which including screening of biotransformation strains for glycoside isoflavones in Fructus sophorae, identification of the main biotransformation products, biotransformation technic of the main biotransformation products, genistein-solubility determination and fitting in different solvents, and anti-breast-cancer cell proliferation and apoptosis of biotransformation products. Completion of these studies, we can access to deepen the application of bio-resources theoretically and technically and to find new anti-breast cancer drugs and the biotransformation in Fructus sophorae. The main research results were as follows.61 edible and medical fungi were used to screen by method of plate transparent circle, then 18 stains were obtained after strain map analysis, in which strains DS1 (Schizophyllum commune) and N3 were further selected by determinationβ-glucosidase production in solid and liquid screening medium. Strain DS1 was finally selected as Fructus sophorae biotransformation strain after further analysis of the biotransformation products in Fructus sophorae medium.Two products with high purity (up to 95%) were purificated from 2d and 16d's biotransformation medium respectively. The purified products were identified as genistein and isoprunetin after LC-MS and NMR spectrum analysis. These two products were first reported to produce by Schizophyllum commune DS1 transformation, which is a new era for the research and application of isoprunetin biotransformation.For genistein biotransformation preparation in Fructus sophorae,β-glucosidase was firstly separated and purified with 43.4 kDa molecular weight and good thermal stability in optimum temperature 50℃, pH 5-6. Its enzymatic reaction kinetic parameters were Km =9.43 mmol/L, and Vmax=3.39 mmol/(L.min) using pNPG as its substrate. Secondly, the optimum enzymatic hydrolysis technics were determined and the biotransformation rate reached to 8.68±0.65%(g/g) under the conditions of 0.3U of enzyme dosage,4 hours of enzymatic hydrolysis time,50℃, pH 5.0,30 mmol/L Ca2+ and 80 g/L of Fructus sophorae extraction. Finally, the optimum Fructus sophorae fermentation technics by Schizophyllum commune DS1 were also determinated that the age of inoculum was 3 days, and inoculum volume was 9%, and Fructus sophorae concentration was 5%, and biotransformation time was 2 days, and pH was natural. Genistein biotransformation rate reached to 3.5mmol/L under these conditions. Genistein biotransformation kinetic model was summerized as follows: y(t)=(?) The main genistein biotransformation pathway from Fructus sophorae fermented by Schizophyllum commune was further clarified:4'-glucoside of sophoricoside in Fructus sophorae was hydrolyzed to produce genistein, and percent 79 of genistein in biotransformation system came from the hydrolysis of sophoricoside.For isoprunetin biotransformation preparation in Fructus sophorae, the main isoprunetin biotransformation pathway in Fructus sophorae fermented by Schizophyllum commune was firstly clarified that sophoricoside was hydrolyzed to produce genistein firstly, and 5-OH of genistein was methoxy to produce isoprunetin. Secondly, preliminary fermentation conditions were obtained, and the yield of isoprunetin reached to 3.4mmol/L by biotransformation for 12 days under the conditiones. finally, the fermentation kinetic model of isoprunetin was obtained as:y(t)=0.628+0.286 x (t-2.5). (t≥2.5).The genistein-solubilities determination and fitting in different solvents were studied. The genistein-solubilities in water, methanol, ethanol, isopropanol, butanol and ethyl acetate were determinated and fitted by Apelblat model. It was found that Apelblat model could fit the experiment data of solubilities, and methanol could be a good solvent to extract genistein. The genistein-solubilities in different methanol-water solvents were determinated and fitted by empirical model. It was found that the empirical model could fit the experiment data of the solubilities. Based on the concentration of genistein in biotransformation system, a good suggestion to extract genistein from biotansformation system was that 75% methanol-water solvent was suitable to extract genistein.Finally, anti-breast-cancer MCF-7 cell proliferation and apoptosis of biotransformation products were studied. Fructus sophorae biotransformated by Schizophyllum commune had a higher activity of anti-proliferation to MCF-7 cell line than the control sample, and the activity products were mainly existed in the alcohol extracting solution. There were a good dose and time effect of genistein and isoprunetin which were mainly biotransformation products on the proliferation of MCF-7 cells. Under lower or higher dose, the anti-proliferation activity of isoprunetin was preferable to that of genistein, but there was no distinguish of inhibition rate in 10μmol/L dosage of the two products. Isoprunetin also had a strong activity of anti-proliferation to HuVEC cells. Isoprunetin promoted the apoptosis of MCF-7 cells by morphologic observation, and the apoptosis rate was 12.1% after isoprunetin affected MCF-7 cells for 72 hours by flow cytometer FCM method, and the MCF-7 cells mainly stopped at G1-S stage. The promote-apoptosis function of isoprunetin to MCF-7 cells was further confirmed by protein determination of caspase-3 and PARP, then apoptosis pathway of MCF-7 cells affected by isoprunetin was interposed by caspase-3.
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