| Ganoderma lucidum [(Leyss.ex.Fr.) Karst] is a precious medicinal fungi, and has over 2000 years of history as folk medicine. The G.lucidum with high triterpenoids production was investigated in this thesis, and we primarily analyzed the identification of ganoderic acids, the biotransformation of soybean isoflavone by G.lucidum, and the anticancer activity and mechanism of converted products of isoflavone.The American G. lucidum was screened in five tested strains for the following experiment, and the production of its mycelia biomass, intracellular triterpenoids, intracellular polysaccharides, extracellular polysaccharides, extracellular triterpenoids could reach 1.22±0.03g/100ml,39.29±0.25mg/100ml,23.44±0.76mg/100ml,11.50± 0.74mg/ml,14.24±0.55mg/ml, respectively. Based on the analysis of ITS sequences, the American G. lucidum was identified as G. lucidum, which could be used in health food with edible safety. According to optimized the fermentation medium and conditions of G. lucidum, the results showed that when the G. lucidum was grown in seed culture for 8 days, and then was inoculated with 10%(v/v) into the optimized fermentation medium (wort 4.10%, yeast extract 1.8%, KH2PO4 0.3%, MgSO4 0.15%, VBi 0.005%, pH 5.40) and fermented for 7 days at 28? with shaking at 180rpm, the production mycelia biomass and intracellular triterpenoids could reach maximum. In this paper, the wort was prepared with European Brewery Cenvention (EBC) method, and it is the first time to use wort into fermentation medium of G. lucidum for increasing the production of intracellular triterpenoids.Ten ganoderic acids of G. lucidum were preliminarily identified by HPLC-ESI-MS, and their probable ingredients were as following:Ganolucidic acid A, Ganoderic acid A, Ganoderenic acid B, Elfvingic acid A, Ganoderic acid F, 7,15-dihydroxy-4,4,14-trimethyl-3,11-dioxochol-8-en-24-oic acid, Lucidenic acid C, 3?-hydroxy-4,4,14-trimethyl-7,11,15-trioxochol-8-en-24-oic acid, Ganoderic acid H, 3,7,15-trihydroxy-4,4,14-trimethyl-11-oxo-chol-8-en-24-oic acid. In addition, G. lucidum could enrich the amino acids, especially essential amino acids in the liquid fermentation, and which could also enrich trace elements, but the enrichment capabilities for different elements were different.After fermentation, the mycelium and fermentation broth were mixed and mashed. To make the slurry of G. lucidum with ?-glucosidase activity (1.0U/ml) as transformation reaction solution (pH=5), soybean isoflavone (5g) was added, and then was transformed at 60? for 48h, the results showed that the convert rate of daidzein and genistein could reach 96.63%,87.82%, respectively. More importantly, this is the first time to transformed soybean isoflavone by the slurry of G. lucidum which enriched bioactive substances. For the determination of antioxidant capacity of converted production, each antioxidant index of convert products (TSI) and soybean isoflavone (SI) was significantly increased with the increasing of substrate concentration (0.5-10mg/ml), and at the same substrate concentration, the inhibition of OH radical capacity (only substrate concentration<6mg/ml), the DPPH scavenging capacity, SOD activity, and T-AOC activity were all significantly high than SI.The convert products were extracted by 30%,50%,75%(v/v) ethanol, respecyively, and then named as TSI-1, TSI-2 and TSI-3. For MTT assay, TSI-1 (120?g/ml), TSI-2 (100?g/ml) and TSI-3 (60?g/ml) could significantly inhibit the proliferation and promote the apoptosis of HTL9, MCF-7 and HepG2. The effect of TSI-2 (100?g/ml) on cell apoptosis of HTL9 was the most significant, its late apoptotic cells increased from 8.27% to 40.13%, and early apoptotic cells increased from 0.95% to 9.05%. In addition, after treatment by TSI-2 (100?g/ml), HTL9, MCF-7 cells were arrested in G1 phase, while HepG2 was arrested in S phase, which were unable to carry on the division and then induced apoptosis.After treatment for HTL9 cell by TSI (100?g/ml), the relative content of Bax was significantly increased, while the ratio of Bcl-2/Bax was decreased, simultaneously, the significant increasing of Cyto-C protein content could activate the Caspase-3, Caspase-8, and make their relative content increase. Thus it could indicate that TSI (100?g/ml) could induce apotosis through the mitochondrial pathway. In addition, the relative expressions of anti-apototic factors (Survivin, NF-?B) were significantly reduced, while the relative expression of pro-apoptosis factor factor (p53) was increased. All these results showed that TSI (100?g/ml) could induce apoptosis of HTL9 by regulating multiple genes associated with cell apoptosis. |
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