Cordyceps Spp. Fermentation, Its Metabolites Separation And Their Bioactivity Study

Cordyceps, an entomopathogenic macrofungus, has been used for centuries as a rare traditional Chinese medicinal herb. It contains various known and untapped bioactive compounds, and is recognized as an important natural resource for the development of health-promoting functional food and for new drug discovery related to immunomodulatory, antitumor, hypoglycemic and hypocholesterolemic activities, etc. The fungi of Cordyceps therefore receive increasing attention around the world as an interesting natural drug resource. Two new and folk medicinal species of Cordyceps, namely Cordyceps taii Z.Q. Liang & A.Y. Liu (1991) (=Metacordyceps taii) and Cordyceps jiangxiensis Z.Q. Liang, A.Y. Liu & Y. C. Jiang (2001), which were discovered in China, were used in the present study. Fermentation for production of mycelial biomass and polysaccharide, evaluation of polysaccharide bioactivity, analysis of Cordyceps mycelia chemical composition, and separation and purification of metabolites from the fermented Cordyceps were performed on the basis of previous work.The production of Cordyceps spp. and its metabolites using fermentation technology is seen as an important project on research yield of Cordyceps. Desirability function, a nonlinear mathematical model, was applied to optimize the fermentation conditions for simultaneous hyper-production of biomass and intracellular polysaccharide by C. jiangxiensis in submerged cultivation for the first time. Under optimal fermentation conditions obtained by desirability function method, the production titer of mycelia and intracellular polysaccharide reached 24.45 and 8.93 g/1, respectively, after a 9-day submerged cultivation, which was 2 folds higher than that of un-optimized conditions. The use of desirability function was proved effective in optimizing multi-response fermentation processes and it is readily applicable to other bioprocesses.A combinatorial experimental design strategy involving both the one-variable-at-a-time method and response surface methodology (Plackett-Burman, steepest ascent, and central combination designs) was employed for the optimization of mycelial growth and exopolysaccharides (EPS) production by C. taii. A quadratic multivariate regression model accurately predicting EPS yield was established through the above optimization process. This optimization gave a surprising increase in EPS yield (>40g/L) compared to the level (0.77g/L) obtained in basal medium in shake-flask culture. The improved production condition will be helpful for the development of EPS in the fields of health foods and drug discovery.Further works such as bioactivity of mycelial polysaccharide from both C. taii and C. jiangxiensis and their mechanism of action were also performed. The antioxidant potency of polysaccharides from C. taii and C. jiangxiensis were evaluated using five different in vitro assays after the fermentation conditions of biomass and polysaccharide production by both C. jiangxiensis and C. taii were established. Among these assays, polysaccharide from C. taii and C. jiangxiensis showed a strong capability of scavenging free radicals and the ferrous ion chelating activity, however, there was a certain variation among different fractions of polysaccharide from the same Cordyceps species. Especially most polysaccharide fractions from two cultured Cordyceps species possessed more potent scavenging superoxide anion radical activity than positive control, and two polysaccharide fractions of C. taii exhibited more significant chelating capability on ferrous ion than positive control. The antioxidant effect of C. taii polysaccharide fraction on D-galactose induced aging mice is associated with enhancing markedly the activities of endogenous antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, and decreasing evidently maleic dialdehyde level. The results suggest that C. taii and C. jiangxiensis are promising sources for development of natural antioxidant. In additional, MPCJ4 and MPCJ5, two enriched-polysaccharide fractions of cultured C. jiangxiensis, exerted antitumor effects at different dose range from 5 to 500μg/mL while had no cytotoxicity. Further investigations indicated that MPCJ4 and MPCJ5 markedly induced cell cycle arrest in the S phase, and resulted in tumor cell death of G1 phase via apoptotic biochemical pathway. These findings thus suggest that MPCJ4 and MPCJ5 might be a promising candidate for application in cancer therapy, and Cordyceps-derivated polysaccharides possess a new antitumor mechanism of action with non-immunologic response.Besides polysaccharide, the mycelial biomass of Cordceps spp. including both C. jiangxiensis and C. taii possess many high-value nutritional ingredient and bioactive compounds. To evaluate the nutritional and health-promoting value of three edible and medicinal mushrooms C. jiangxiensis, C. taii, and Cordyceps gunnii (Berk.) Berk.(1859) and simultaneously get a considerable data for establishing the quality control standard of Cordyceps preparations, their proximate compositions, bioactive ingredients and potential toxic constituents were investigated by using various chromatographic and spectrographic assays such as HPLC, GC/MS, amino acid high speed chromatography, ICP atomic emission spectrometer etc. and routine physical and chemical analysis. The contents of polysaccharide, cordycepic acid, and nucleosides were C. jiangxiensis>C. taii>C. gunnii. The three fungi contained high levels of unsaturated fatty acids (UFA) and essential amino acids. The percent of UFA relative to total fatty acids was 78.7% for C. gunnii,66.9% for C. taii, and 79.0% for C. jiangxiensis. The Cordyceps species displayed similar amino acid profiles and a high level of total amino acids ranging from 146.0 to 168.3 mg/g, where glutamic acid and aspartic acid were the two principal amino acids with over 10% of the total amino acid contents. Other essential amino acids made up 37.05%,39.39%,39.37% of the total amino acids of C. gunnii, C. taii, and C. jiangxiensis, respectively. Abundant amounts of Fe,Zn,Se,Cr and Co were found in these species, while toxic microelements such as Al,Sn,Pb,Cd,As and Hg were presented at very low amounts and could reach the related quality standard of SFDA in China. Taken together, our findings suggest that the three medicinal Cordyceps species can be regarded as a promising source of nutriceuticals and drugs without any health risk.During the above process of detecting nutritional ingredients and bioactive compounds of Cordyceps spp, a new universal and high-throughput method was also developed for quantitative determination of cordycepic acid in Cordyceps species using microplate reader, detected at 412 nm. The linearity of the method was found within 6.3-60μg/ml concentration ranges of cordycepic acid, and the linear correlation coefficient was good (R2> 0.9997) within the test ranges. The limits of detection (LOD) and quantification (LOQ) were 1.8μg/mL and 6.3μg/ml, respectively. The recoveries were in the range of 96.71 to 104.61%. Taken together, compared to conventional method, the new method showed good accuracy, less error, and high-throughput ability, and it is applicable in Cordyceps bioproduct. Besides, a simple high-performance liquid chromatography with photo diode array (HPLC-PDA) method for the separation and quantitative determination of nucleosides and nucleobases in cultured C. jiangxiensis has also been developed. The use of mobile phase with ultrapure water and methanol enabled the efficient separation and determination of seven nucleosides and nucleobases within a 12 min.Validation of the method was done with its linearity, precision, and stability, where a good linear correlation (R2>0.9900) for all analytes was obtaied within the test range, the corresponding LOD and LOQ were 0.01-0.30μg/mL and 0.04-0.96μg/mL, respectively, and the relative standard deviations (RSD) for precision and stability were all within 3%. Under the optimized conditions, six nucleosides and nucleobases were detected in cultured C. jiangxiensis, of which uridnine (7802.14μg/g) and adenosine (2705.92μg/g) were principal nucleosides compounds. In short, the developed HPLC-PDA method could be efficiently applied for the simultaneous analysis of nucleosides and nucleobases during fermentation of C. jiangxiensis.Polysaccharides, cordycepic acid and nucleosides are some major bioactive compounds of Cordyceps spp., but the most valuable compounds should be attributed to some secondary metabolites possessing different pharmcological effects, which are true sources of new drug discovery. Therefore it is necessary to develop the separation and purification of metabolites from the fermented Cordyceps and their bioactivity evaluation, which is useful pathway to discover new compounds and new functions for the development of high-value-added products. With the bioactivity-oriented separation, over 30 compounds were separated from cultured C. jiangxiensis by repeated liquid column chromatography, preparative thin layer chromatography and recrystallzation. The chemical structures of thirteen compounds were elucidated on the basis of spectroscopic methods including UV, IR, ESIMS, HRESIMS, 1D-NMR (1H-NMR,13C-NMR, and DEPT) and 2D-NMR ('H-'H COSY, HMQC, HMBC, and NOESY). Eighteen compounds were identified as tetracosanoic acid (2),1-monostearin (5), jiangxienone (7), docosanoic acid campesterol-ester (8), hexadecanoic acid ergosterol-ester (9), uridine (14), ergosterol (21),5a-Ergosta-7,22-dien-3β-ol (22), succinic acid (23), uracil(24), adenine (25), adenosine (26), nicotinic acid (27), and 3'-methoxyuridine (28),2'-methoxyadenosine (31),2'-deoxyuridine (32), methyl L-pyroglutamate (33), and dipterocarpol (34), of which compounds 7-9 were of new chemical structure belonging to steroides. Ten known compounds (2,5,22-23,27-28,31-34) were obtained from Cordyceps species for the first time. Jiangxienone, a high oxidation degree compound, showed more significant inhibition capability versus first-line antitumor drug cisplatin for inhibiting tumor cell proliferation. Jiangxienone therefore possesses a great potential as a potential new antitumor drug candidate. These findings contributed to a compound source for new drug discovery in Cordyceps-derived natural compound library.