The Influence of Elicitors and Two-Stage Culture on Triterpenoids Production and Antitumor activity in Submerged Cultivation of Antrodia cinnamomea

Antrodia cinnamomea was a popular folk medicine that has attracted great attention due to its antitumor activity. In the last decade, the triterpenoids were isolated from A. cinnamomea and their biological activities such as anticancer and hepatoprotection have been reported. Elicitors have been effective in inducing the synthesis of secondary metabolites (e.g., triterpenoids) in submerged cultivation of A. cinnamomea. A defense response was induced in the culture when elicitors were added. Elicitors can enhance the secondary metabolites in fungal cells. The objective of this study was to investigate the effects of different elicitors (chitosan, CaCl2 and combination (chitosan+CaCl2)) on the triterpenoids production in submerged cultivation system of A. cinnamomea. It was found that the maximum biomass reached 15.8 g/L and 12.9 g/L in airlift bioreactor with dual-net draft tube and fed-batch (day 8) cultures, respectively. The elicitors study showed that treatment of chitosan (100 mg/L) not only obtained more biomass than combination treatment, but also achieved the highest total triterpenoids production. However, combination seriously affected the permeability of cell membrane and resulted in the loss of cell viability. Although the use of combination was not appropriate for long-term cultivation, it did enhance the fungus to accumulate the intracellular triterpenoids in a short-term cultivation. In addition, feeding CaCl2 had little effect on accumulation of triterpenoids for short time cultivation, so it was suitable for long-term fermentation. For two-stage culture strategies, the results proved that triterpenoids production could be highly enhanced by means of the control of oxygen limitation and temperature-shift strategy. For instance, the maximum triterpenoids production (1615 mg/L) was observed at static culture with temperature fluctuation (TF-S group), which was significantly higher than the control. In vitro anticancer test, the results indicated that the crude triterpenoids from TF-S group at concentration of 28 μg/ml inhibited 50% cell viability of HeLa cells, while exhibiting under 100 μg/ml no significant cytotoxicity to WS1 (normal cells).