Isolation, Identification, Fermentation And Determination Of Adenosine In Cordyceps Sinensis

Cordyceps sinensis (Berk.)Sacc as one of the famous traditional tonic medicine in China, has been used to treat deficiency syndrome of the lung, physically and mentally fatigued and short of breath, also has a great effect on reinforcing kidney and lung. The modern researches have proven that the main active ingredients such as polysaccharides, nucleosides, sterol composition in C. sinensis have functions about anti-tumor, anti-inflammatory, resisting free radical formation and so on. One antibiotic ingredents extracted from fermentation broth of C. sinensis have proved to be the good immune inhibitor, which can help organ transplantation and chemotherapy patients to improve the immune function.Cordyceps sinensis and its related fungus refer to the Cordyceps fungus including its anamorph Hirsutella sinensis Liu, Guo, Yu et Zeng, and some other uncertained entomogenous fungus extracted from C. sinensis. There are more than300reports released about C. sinensis, but mostly reported about its anamorph and its active ingredients. Seldom to study on the other fungus isolated from Cordyceps, too. However, the C. sinensis and its relatives are pointed to be the best potential fungi group to find out new bioactive compounds and the precursors to synthesis new drugs. To study the diversity of C. sinensis not only can help to develop the high active biological drugs, but can help people to seek how the Cordyceps grow up.This research is aim to screen the ingredients of fungi medium, the culture temperature and liquid fermentation conditions, in order to compare withe the adenosine among these fungus. Firstly, through the repeated experiment and orthogonal method screen the additions of medium, temperature and selected the best one to isolate and culture the Cordyceps digged from the Tibet naqu area; And identified the species in morphological and moleculer levels; Then optimized the liquid fermentation medium to test the adenosine content in fungus.The result of this research shows:(1) There are total43strains of fungi isolated from wild C. sinensis, including Ascomycetes that have33strains accounted almost for77%of the number of the separations, Basidiomyete and Uncultured Fungus have5strians seperatly. The dominational strains are Cordyceps, Trichoderma and Penicillium. (2) Through observation the colony characteristics and microscopic characteristics of separation of fungus, and blast whole strains in DNA sequence to identifty the stains. Using Cs-1(Tolypocladium sinensis C. L. Li.) Cordyceps fungus and two strains sequences of Trichoderma and Penicilliun to make an evolution tree, analysis the relationship between them.(3) Screening better liquid fermentation culture medium for these species. Cultivation in dark for five days, found that dark culture could improve some stains target biomass by fermented. Cs-1showed the average mycelial dry weight under dark training is1.7g/L, the light training weight1.25g/L; Cs-4in dark training weight1.5g/L, the light training weight1.35g/L; Cs-5in dark training is average1.9g/L, in light training is1.2g/L; Cs-6in dark training is1.7g/L, in lighting training is1.45g/L; Their biomass increased average1.39g/L by dark culture.(4) Choose the best way to extract the adenosine from dry mycelia:use80%methyl to ultrasomic extract for twice,40mins per time. The adenosine content can reach to59.63ug/mL; then use HPLC to test the adenosine fermentated by medium Ⅱ (glucose25g/L, peptone10g/L, yeast powder5g/L, VB20.8mg/L, KH2PO40.8g/L, MgSO4·7H2O0.8g/L). Result:the content of adenosine are similar between Cs-1and Cs-6, the content Cs-1is0.347%a little bit higher than Cs-60.314%, but still lower than0.596%of Cs-4; through the adenosine content have’t report before, its dramtically higher content is worth to study further.(5) Add NH4Cl into medium Ⅲ, to observe the changes of adenosine content of fermentated mycelia. Finally find out that the adenosine content in four strains is all in creased. Proven that NH4Cl can be a precursor to induce the adenosine form.This research described systematically the various species in Cordyceps sinensis, and proved that there are many fungus genus living in Cordyceps sinensis. And extracted some fungus (Pseudogymnoascus roseus Raillo)、(Tolypocladium sinense C.Lan Li)和(Hirsutella sinensis (Berk) Sacc), which reported before. Nevertheless, there is the first time to find Cordyceps nitrolii extracted from Cordyceps sinensis. To compare the adenosine in fermentation mycelium can prove that there is some fungus like Cs-5owes such high active indigents extracted from Cordyceps sinensis. All of these results can help to find new fungus replaces wild Cordyceps sinensis and new medicine make of fungus.