| Zhuling is a kind of precious medicinal herbs which derived from sclerotia of grifola umbellate. At present, wildlife resources of grifola umbellate are almost exhausted day by day, and artificial cultivation are limited by the natural conditions. All of these lead to the market shortages.It has become the research hotspot , to obtain mycelium and active substance in mycelium of grifola umbellate by liquid fermentation culture. For the future development of the medicinal value of mycelium,and to explore the chemical background for pharmaco- logical activity of mycelium and sclerotia of grifola umbellate, we did researches as follows:Study on the steroids production which cultured by liquid fermentation; Doing liquid fermentation test of 70L fermentor and Comparing the chemical constituents between sclerotia and liquid fermentation mycelium of grifola umbellate; Analysis of the chemical constituents of sclerotia of grifola umbellate. The main results are as follows:1. To research on the culture condition of grifola steroidal by liquid , using steroids content as the key indicator and mycelia growth & polysaccharide content as the auxiliary indicators. The result shows the optimum medium composition is: C6H12O6 28.0g/L, YE 4.00g/L, KH2PO41.00g/L, MgSO4 1.00g/L, VB1 0.1g/L, 1%CMC10mL/L. Under this culture condition, the dry weight of grifola umbellateus mycelium was 2.050g, and the content of steroids in this fermentation liquid and dry mycelium of grifola umbellate reach to 3.768mg/100mL and 3.864mg/g. Besides, the content of polysaccharides in this fermentation liquid and the mycelium of grifola umbellateus have reached to11.542 mg/g, 5.885mg /100mL.2. In liquid fermentation test of 70L fermentor, when the inoculation amount to 3%, 4% and 5% , stable breading period of mycelium of grifola umbellate respectively is 8, 6, 5 days. But the time between stationary phase and decline phase of mycelium of grifola umbellate isn't affected by the inoculation amount. In all the five times fermentations the stationary phase last about 5 days, after that mycelium cell become autolysis, and the dry weight of mycelium reduced. Besides, the content of steroids of mycelium of grifola umbellate low in logarithmic phase, high in the late stationary phase. So we can conclude that steroids mainly accumulated in the stationary phase of mycelium of grifola umbellate. 3. The two methanol-soluble, extracted by petroleum ether, were subjected to the chemical constitution analysis by GC/MSD. 66 compounds were identified from the little polar and polar fractions of the extraction of sclerotia of grifola umbellate. The compounds in the little polar fraction were mainly alkanes, while the compounds in polar fraction were mainly fatty acids, fatty acid esters and dimethyl phthalates. 50 compounds were identified from the extraction of the fermented mycelium of grifola umbellate, and in the both little-polar fraction and polar fraction of the extraction, fatty acids, fatty acid esters and its derivatives were in majority.4. Isolation and purification of the methanol extracts of sclerotia of grifola umbellate was carried out on the basis of preparative thin layer chromatography, column chromatography and recrystallization. Based on their physical and chemical properties and spectral analysis (IR, 1H NMR, 13C NMR), Eight of them were identified as:L1, Ergosta-5,7,22-trien-3-one and Ergosta-7,22 -dien-3-one; L2, docosanoi cacid; L3, 12-hentriacontanol; L4, peroxy-ergosterol; L5, N-(2'- hydroxy-tetracosanoyl)-1,3,4-trihydroxyl-2-octodecanine; L6, Polyporusterone A and B. Specially, it is the first time for 12-hentriacontanol to be isolated from sclerotia of grifola umbellate.In this thesis, eight compound have been isolated from the methanol soluble extract of sclerotia of grifola umbellate, and 12-hentriacontanol is firstly isolated. Obtained the optimum medium for liquid fermentation culture accumulate the steroids in mycelium of grifola umbellate. Using this medium for liquid fermentation test of 70L fermentor, and the content of steroids in late stationary phase of mycelium became high. At last, the difference of chemical constituent between sclerotia and mycelium of grifola umbellate of has been analyzed by GC-MS. All the above results, firstly enriched the chemical constituent on sclerotia grifola umbellate, and then provided some base for the production of grifola umbellateus steroids in industry scale in the future, lastly illuminated the chemical background for pharmacological activity of mycelium and sclerotia of grifola umbellate. |
