| G. lucidum, a famous folk medicine in East Asia, has been used as tonic and prescribed for hundreds of years. Ganoderic acid is one of the major bioactive constituents in G. lucidum. It has various bioactives against several serious human diseases, including anticancer and antimetastasis effect. With the development on G. lucidum mycelia fermentation like submerged and two-stage static process, the limitation on raw material supply was relieved, however, lack of efficient preparation technique still heavily hinders the further clinical research and development on such compound. Currently, owing to the poor content and resolution of GAs in G. lucidum extracts, no successful trial on certain specific GA large-quantity preparation has been reported. This thesis probed into the high-performance preparation of antitumor ganoderic acid T (GA-T) from crude G. lucidum mycelia by two-stage static culture process, and a low-cost GA-T preparation method with high product purity and recovery was established in this work.Firstly, a new GA was isolated and discovered from G. lucidum through metabolites analysis, and this compound was named 7-O-ethyl ganoderic acid O (7-O-ethyl GA-O) by common nomenclature. The content of 7-O-ethyl GA-O in G. lucidum mycelia was as high as 1.2-1.5g/100g dried mycelia, which was about 5-10 times higher than that of the others. As this compound could be converted into GA-T by acid treatment, thus, 7-O-ethyl GA-O could be treated as a leading compound in GA-T preparation.Stability of 7-O-ethyl GA-O was a key problem in the design of its preparation process, as this compound was quite unstable against pH, heat, and water. It could be concluded from assays that 7-O-ethyl GA-O possesses the best stability in neutral and non-polar environment. Its degradation kinetics and thermodynamics could be effectively described by first-order reaction model and Arrhenius equation. The activation energy was estimated to be 38.2±3.6kJ/mol in acidic methanol (0.01mM HCl, pH=5.1), 103.2±2.4kJ/mol in aqueous methanol (20% water), and 105.8±3.3kJ/mol in methanol. Based on such data, a process consisting of 7-O-ethyl GA-O normal-phase isolation, crystallization and hydrolysis was designed to prepare GA-T.To overcome the disadvantage of huge usage of non-polar solvent and low total recovery, GAs constituents in the crude extract of G. lucidum mycelia was further analyzed, which inspire a novel solution to GA-T preparation. It was found that the combination of acid hydrolysis and acetylation would remove the main difference on the carbon skeleton and kind of substituted groups of GAs in the crude extract. After treatment, the content and key resolution of GA-T in the crude extract was increased by about 4 and 7 times from 0.44g/100g and 1.05 to 1.69g/100g and 7.90, respectively, which significantly facilitate GA-T preparation. The recovery of the total pretreatment process was about 90%. Coupling this pretreatment process with reversed-phase preparative HPLC, GA-T product purity and recovery could reach about 85% and over 95%, separately. In laboratory scale of 78.5ml column volume, the total process only need 1 day with GA-T output of 2.9g. The solvent consumption of this method was about 3.3L/g. With great performance on production cost, purity and recovery, this preliminarily-established craftwork would be treated as suitable model for further optimization and scale-up in future GA-T production.This work demonstrated low-cost, high-purity, good-recovery GA-T preparation from chemical complicated fermentation mixture by overcoming the initial poor content and resolution of this compound. It also provided a well-performed solution to GA-T large-quantity production to speed up its clinical development. Results from this research would also provide information on the preparation of the other bioactives from fermentation broth and herbal extract. |
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