Isolation And Structure Elucidation Of Cytotoxic Constituents From Pleurotus Cystidiosus And Optimization Of Liquid Culture Of Wild Medicinal Clavicorona Pyxidata

The techniques of developing active chemical constituents from Pleurotus cystidiosus and Clavicorona pyxidata were explored in this thesis.Pleurotus cystidiosus, which has high nutritional and medicinal value, belongs to Basidio mycotina, Hymenomycetes, Agaricales, Pleurotaceae, Pleurotus. P. cystidiosus was cultured in dishes laid with potato dextrose agar (PDA) medium and the crude organic extract was prepared from this fermented substrate. MTT cytotoxity test showed that three prostate cancer cells LNCaP、DU-145 and PC-3 could be inhibited at the rate of 39.9%、41.2% and 36.6%, while normal human prostate epithelial cells RWPE-1 could only be inhibited at the rate of 23.5%, both by 50 μg/mL of crude organic extract. Subsequently, P. cystidiosus was cultured in dishes laid with ca.20 mL PDA medium with the total volume of 15 L. The mycelium together with culture medium was extracted with the solvent and afforded 4.4 g of a crude organic extract.5 compounds were obtained from the crude extract with the techniques of using different column chromatographic such as RP-18, Sephadex LH-20, and silica gel. Their structures were elucidated through spectroscopic analyses, including the data of NMR, HRMS-EI, IR, UV, CD, and [a]. The five compounds were identified as new sesquiterpenoids after database retrieval. Two compounds were new bisabolane type sesquiterpenoids and named pleuroton A (1) and B (2). Three were clitocybulol derivatives, named clitocybulols D (3), E (4), and F (5). The MTT cytotoxity test showed that all the five compounds could inhibit prostate cancer cells LNCaP、C42B and DU-145, and compound 2 showed the strongest inhibition among these sesquiterpenoids. The median inhibitory concentration (IC50) of compound 2 were 28、52 and 51 nM respectively against LNCaP、C42B and DU-145. The cytotoxity of pleuroton B (2) was confirmed by colony formation assay. Furthermore, pleuroton B (2) could induced the apoptosis of Du-145 cells through the detection of apoptosis cells using annexin V-FITC staining by flow cytometry, the observation of condensed nuclei in the apoptosis cells, and the western blot analysis for the expression of apoptosis-related proteins Bcl-2, Bak, and Bax. Analysis of structure-activity relationships revealed the unusual functional moiety of α,β-unsaturated doubled bond and the hydroxyl at C8 in pleuroton B should contribute to its significant bioactivity.Clavicorona pyxidata belongs Aphyllophorales, Clavariaceae, Clavicorona. C. pyxidata possessed abundant amino acids and terpenoids and high nutritional and medicinal value. At present, the fruiting body of C. pyxidata was harvested from the wild and few was from the artificial cultivate. The utilization of C. pyxidata with the techniques of liquid fermentation was explored in this experiment. Firstly, the chemical compositions were compared between the fruiting body and mycelium of C. pyxidata with the aid of NMR data (1H and 13C) analysis of crude extract from both materials. The analysis of NMR data revealed the chemical compositions in the fruiting body and mycelium of C. pyxidata were similar at high level and indicated the mycelium could replace the fruiting body on a certain degree. Then, the liquid fermentation conditions were optimized. The optimized medium was using glucose as carbon source, yeast powder as nitrogen source, and the initial pH value was 7.0. The optimal working volume was 100 mL in the shake-flask (250 mL). The optimal fermentation time was 20 days. The biomass of C. pyxidata could reached the highest production at 9.45 g/L under the optional conditions by the Plackett-Burman and the steepest ascent experiment.This research would lay the foundation for the further development of chemical resources off. cystidiosus and C. pyxidata by the industrial fermentation.