Isolation And Evaluation Of Anti-Neuritis Active Ingredients In Two Strains Of Medicinal Fungi

Hericium erinaceus and Cyathus striatus,as medicinal and food-derived valuable herbs in China,are widely spread among the people for their medicinal effects of stomachic and pain relief and their delicious taste.However,the specific functional active molecules for their efficacy have not been clarified,so it is important for pharmaceutical research to search for biofunctional active molecules from their fermented extracts and to characterize the structures and screen the activities.In this thesis,the fermentation of two rare medicinal fungi was carried out on a large scale,and the fermentation extracts of Hericium erinaceum and Cyathus striatus were separated more systematically using a combination of normal-phase silica gel column chromatography,semi-preparative liquid chromatography,and gel column chromatography,followed by comprehensive analysis of multi-wavelength spectroscopy（NMR,MS,UV,IR,etc.）data combined with computational ECD（circular dichroism）,X ray single crystal diffraction chemistry and other means to determine their absolute conformation.A total of 25monomeric compounds were isolated and their structures were determined,of which 8 were new compounds.Further,a variety of anti-inflammatory models were used to screen their activities,and the molecules with prominent activities were investigated in more depth for the mechanism of activity.The main findings are as follows:（1）A total of 12 compounds（1-12）were identified after repeated column chromatographic separation and purification of Hericium erinaceum in 200 vials of rice solid scale culture using the one strain-many compounds（OSMAC）strategy,including 10 cyathane diterpenes（1-10）,1 lupinane type triterpene（11）,and 1 ergosterone（12）.Among them,1was a new compound and the absolute configuration was determined by ECD calculations.Compound 2 was a new natural product.（2）A total of 13 compounds（13-25）were isolated and identified from the fermentor scale fermentation using modified molasses medium after adopting the OSMAC strategy for the screening medium of Cyathus striatus,including 11 cyathane diterpenes（13-23）,1ergosterone sterols（24）,and 1 lupane-type triterpene（25）.Among them,13-18 were new compounds and 23 were found for the first time from Cyathus striatus.（3）Evaluation of anti-neuritis activity showed that compounds 1,2,13,16,17,20 and23 exhibited some anti-neuritis activity with IC₅₀ values of 5.82±1.80,31.61±0.27,9.92±0.75,9.88±0.63,15.48±0.51,13.70±0.57,10.37±2.02μM,where compounds 1 and 16showed extremely strong activities,all of which were superior to the positive control drug indomethacin.Thus,we confirmed that 1 and 16 significantly down-regulated the expression of inflammatory markers i NOS and COX-2 at the protein level by Western Blotting experiments;and by in-depth cellular pathway studies,we found that 1 and 16 inhibited MAPK phosphorylation and nuclear translocation of NF-κB subunits.It is hypothesized that compounds 1 and 16 may exert anti-neuroinflammatory effects through MAPK and NF-κB pathways.（4）.Neurotrophic activity evaluation showed that compounds 13-18 and 23 exhibited neurotrophic activity at concentrations of 5μM,and 13,23 and NGF synergistically induced up to 34.4%and 35.1%of PC-12 cell differentiation,which was approximately three times higher than that of NGF control,in a concentration-dependent relationship.In combination with Western Blotting technique,it was confirmed at the protein level that 13 enhanced the expression of growth-related proteins GAP-43 andβ3-tubulin in NGF-induced PC-12 cells.In conclusion,the present study was conducted to systematically isolate and evaluate the activity of the secondary metabolites of two medicinal fungi with stomachic and analgesic properties,and further found that the active molecules cyathane diterpenes derived from Cyathus striatus and Hericium erinaceum have the potential to treat neuroinflammation in Alzheimer’s disease（AD）,providing a theoretical basis for the development of anti-AD drugs.It is worthy of further development and research.