Effect Of Submerged Culture Conditions On Active Ingredients, Immune Modulation And Antioxidative Activities Of Cordyceps Sinensis Mycelial Extracts

This study used orthogonal design to investigate submerged liquid fermentention conditions for Cordyceps sinensis CS-1 strain. Mycelial biomass was recorded and extracted with hot water. Phenol-sulfuric acid method and 3,5- dinitro naphthol method were used to detect total saccharide and reducing sugar contents in the extract. Contents of nucleosides, including uridine ,adenosine and cordycepin, were detected by reverse phase high performance liquid chromatography. Immunomodulation, antioxidantive activities and oxidative injury protective effects of the extracts were also studied using mouse splenocyte proliferation, FRAP assay and PC12 cell H2O2 injury model, respectively.The optimal conditions for mycelium biomass were determined as 18℃, pH 6.5 and 9 days fermentation course, which the biomass reached20.395±0.178g/L. Under different orthogonal experimental conditions, average total saccharide content of mycelium extracts was 279.37±43.3mg/g, which was lower than that from natural C. sinensis (374.08±7.45mg/g). The reducing sugar contents (average 40.96±8.85mg/g) were also lower than that from natural C. sinensis (163.81±1.1mg/g). However, all the cultured mycelial extracts showed higher intra-mycelial polysaccharide contents, which ranged from 192.43～312.90 mg/g compared to 238.39±37.75 mg/g in natural C. sinensis extract. The optimal conditions for Intra-mycelial polysaccharide yields were 22℃,pH 6.5,6-days fermentation. Temperature at 25℃,pH 7.5,3-days fermentation were ideal for nucleosides production, which was 2.448 mg/g, 5.9 times higher than that from natural C. sinensis and exceeded the minimum level in Chinese pharmacopoeia quality standard (0.010% or 0.1mg/g).FRAP detection results showed that average antioxidant activity of mycelial extracts was 84.6% equivalent to aqueous extracts from natural C. sinensis. The best culture conditions were 25℃,pH 4.5,6-days fermentation. PC12 cell H2O2-injured model revealed that the extracts had similar oxidative injury protective effects as that from natural C. sinensis. Among of which, the optimal culture conditions were 22℃,pH 4.5,fermentation time of 6 days. Neutral red phagocytosis assay results showed that different concentrations of hot-water extracts could notablely improve Raw264.7 cell phagocytosis and showed a dose-response. The highest phagocytic activity was the extracts from 28°C, pH4.5, 3 days culture.MTT assay showed that mycelium extracts in the concentration range of 0-100μg/ml could significantly stimulate the proliferation of splenocytes of BALB/c mice, but it inhibited cell proliferation when concentration reached 200μg/ml. With the presence of Con A, mycelium extracts restrained splenocytes proliferation at all concentrations ranged from 50-200μg/ml. These results suggested that the immunomodulation effects of C. sinensis mycelium extracts was a bidirectional regulation.Objective analysis showed that the best fermentation conditions was 28°C, pH4.5, fermentation period of 6 days for splenocyte proliferative activity while for inhibition of splenocyte over proliferative activity were 22°C and pH6.5, fermentation time to three days.