A Study Of Antrodins And Other Bio-active Metabolites By Antrodia Camphorata Fermentation Production

Antrodia camphorata, also named Niuchangchih, is an edible and medicinal fungus that only grown on the inner cavity of the Cinnamomum kanehirae Hayata (Lauraceae) in Taiwan. The bioactive compounds identified from A. camphorata including tritrpenoids, polysaccharides, steroids, benzenoids and other metabolites, have exhibit hepatoprotective, anti-cancer, immuno-modulatory and anti-inflammatory activities. Recently, both the mycelia and fruiting bodies of A. camphorata were reported to have similar pharmacological activities. Moreover, the maleic/succinic acid derivatives (Antrodins) and ubiquinone derivatives (Antroquinonols) isolated from the mycelia of A. camphorata have shown excellent hepatoprotective and anti-cancer activities. However, most of studies on the artificial culture of A. camphorata aimed at the non-characteristic compounds such as polysaccharides or biomass, resulting in the relativlely low value and weak mark competitiveness of the mycelial products.The aim of this work was to isolate and identify the characteristic compounds from the mycelia of A. camphorata based on HPLC analysise. In order to improve the production of these characteristic compounds, the culture conditions of solid state fermentation (SSF) and submerged fermentation (SmF) were optimized, and methods for product treatment were also developed. Furthermore, a coupling fermentation system was established to overcome the deficiency in submerged fermentation system, in which the characteristic compounds were less than that of solid state fermentation system.The main results of this study are as follows:(1) By the application of column chromatography and preparative HPLC, eight compounds were isolation and purified. The structures of these compounds were elucidated according to the data of MS and NMR and identified to be: Ergostatrien-3β-ol(AC-1); Antrodin C(AC-2 ) ; Antrodin B(AC-3); Antrodin A(AC-4); Antroquinonol(AC-5) ;Antroquinonol B(AC-6) ; 4,7-Dimethoxy-5-methyl-1,3-benzodioxole(AC-7); Ubiquinone-3(AC-8).(2) The HPLC of A. camphorata were established based on the comparative analysis of characteristic compounds produced from A. camphorata by different culture methods. The result shows that there some differences in active compounds between mycelium and fruiting body and mycelial product has its own characteristics. The main bioactive components of A. camphorata in submerged fermentation were Antrodins, especially for Antrodin C, while in solid state fermentation were Antrodins and Antroquinonols.(3) The optimization on culture condition in solid state fermentation was carried out to improve the production of antrodins by A. camphorata. The composition of the medium optimal for Antrodins production were rice 100 g/L, soy bean meal 0.6 g/L, Na2HPO4 0.05 g/L, MgSO4 0.05 g/L with initial moisture content of 52%, inoculum density 35% (v/m) and cultivation for 24 d. The fermentation efficiency was improved by adapting a two-step temperature control strategy, by which the temperature was controlled at 28℃for 16 d and then changed to 25℃for 8 d. Under the optimal conditions, the concentrations of Antrodin A, B and C reached 3096.9 mg/kg, 6876.7 mg/kg and 8095.7 mg/kg, respectively. This is the first report on the Antrodins production by SSF of A. camphorata.(4) The medium for Antrodin C production by SmF of A. camphorata was optimized using the Plackett-Burman design and Response Surface Methodology.A model was constructed and furthermore, the accuracy and reliability of the model were verified by experiments, in which the results were in good agreement with the predicted ones. By solving the quadratic regression model equation, the optimal conditions were determined as: glucose 72 g/L, soybean powder 5.91 g/L, MgSO4 0.614 g/L and the predicted production of Antrodin C was 178.59 mg/L. After culturing under the optimized condition for 7 d, the prodeuction of Antrodin C reached 177.83±0.32 mg/L, increased by 85.8% compared with that of the former 95.72 mg/L.(5) Three approaches for improving dissolved oxygen (DO) concentration, including addition of certain proportion of oxygen, oxygen carrier and H2O2, were applied to study the effect of DO on Antrodin C production in SmF of A. camphorate. The results showed that all the three methods could increase the production of Antrodin C by improving DO, in which the addition of oxygen carrier and H2O2 seems more promising in large scale production.(6) Regulation of cellular permeability exhibited to be another way for improving Antrodin C biosynthesis in SmF of A. camphorate. Results showed that the extracellular Antrodin C content was improved from 41.72% to 74.6% by adding 0.3% surfactant M. Yield of Antrodin C reaeched 322.17 mg/L by adding 0.5% surfactant M in 4 d, according to 35.3% increase than that of control,. In a 5 L fermentor, the yield of Antrodin C and period of biosynthesis were also increased because surfactant M could improve cellular permeability and decrease negative feedback inhibition of high intracellular Antrodin C concentration.(7) The regulation of cellular permeability and in-situ extration were combined to construct a coupling fermentation system for A. camphorata to produce bioactive metabolites including Antrodins and Antroquinonols, which were similar to the production of SSF. The coupling fermentation system can carry out metabolites synthesis and extraction simultaneously. Hence, the yield of Antrodin C and other compounds were significantly increased. After extraction by ethanol, the extract phase can be recovered and reused in the coupling fermentation system. The optimal conditions of coupling fermentation system were determined as follows: adding 0.7% surfactant M and 10% extractant n-dodecane at day 4 and day 5, respectively, and feeding 0.15 mmol/L/h H2O2 to increase DO. Under the optimal conditions, yield of Antrodin C increased to 455.78 mg/L. Meanwhile, the concentration of Antrodin C and Antroquinonol were 2624.42 mg/L and 489.09 mg/L in extractant n-dodecane respectively.(8) Systematic studies were carried out on the basic chemical componentseof A. camphorata. The results showed that mycelium of A. camphorata almost contain all types of amino acid and also many kinds of fatty acids, adenosine and polysaccharides. Changes in chemical components of volatile compounds of A. camphorata during solid state fermentation were studied by GC-MS.