Preliminary Study On The Screening Of Anti-aging Active Sites And Their Effects Of Cordyceps Cicadae

Cordyceps cicadae is a kind of bug fungus complex formed by the fungus of ergot family parasitized on cicada larvae,which is the same genus as the asexual type of Cordyceps.C.cicadae contains polysaccharides,nucleosides,cordycepic acid and other chemical components,and is often used in folklore as a dual-use medicinal herb,with a cold nature and sweet taste,and was mainly used in ancient times for the treatment of children’s frightened wind and night cries,external wind fever,red and swollen eyes,opacity covering the eyes and other symptoms.Modern research has found that C.cicadae has immunomodulatory,antioxidant and anti-aging,neuroprotective,antitumor,hepatoprotective and other pharmacological effects.Previously,our research group found that the n-butanol extract site of C.cicadae had a protective effect against oxidative damage in PC12 cells;CPA-1 and CPB-1,polysaccharides of C.cicadae,induced dendritic cell maturation;C.cicadae polysaccharides increased the immunomodulatory activity of RAW264.7 cells and significantly prolonged the lifespan of Drosophila under hydrogenperoxide（H₂O₂）stress conditions.In order to further investigate the anti-aging effect of C.cicadae,this thesis utilized the D-galactose（D-gal）aging rat model to screen the anti-aging active site of C.cicadae,and then performed the composition analysis and content determination of the active site,and preliminarily investigated its anti-aging mechanism of action,in order to provide reference for the rational development and utilization of C.cicadae herbs and the development of new anti-aging drugs.The main study contents and results are as follows:1.Screening of the active site for anti-aging in C.cicadae using D-gal senescence rat modelThe D-gal aging rat model was used to screen the anti-aging active sites of C.cicadae based on the effects of its aqueous,polysaccharide,n-butanol and mixed polysaccharide and n-butanol sites on rat behavior,organ indices,antioxidant indexes and pathological tissues.The results showed that the desire for locomotion and organ indices were reduced in the model group rats;superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）activity was reduced and malondialdehyde（MDA）levels were increased;Pathological damage was observed in liver cells and brain hippocampal cells;indicating that the rats showed aging characteristics after D-gal induction.High dose of aqueous extract of C.cicadae（400 mg/kg）significantly increased the total distance of exercise and central compartment dwell time in aging rats（P<0.01）;elevated thymus index and liver index in aging rats（P<0.01 or P<0.05）;increased SOD and GSH-Px contents and decreased MDA levels in serum and brain tissues of aging rats（P<0.01）;improved the pathological damage of D-gal pathological damage to liver and brain tissues of rats.As a whole,all the administered parts of C.cicadae showed some degree of anti-aging activity,among which C.cicadae aqueous extract and C.cicadae crude polysaccharide showed better overall effect,but there was no significant difference in anti-aging activity between them.The aqueous extract of C.cicadae contains the components in the subsequent isolated parts,and the most widely used method of Chinese herbal medicine extraction is the water decoction method,and finally C.cicadae aqueous extract was selected as the anti-aging active site.2.The chemical composition of aqueous extracts of C.cicadae was analyzed and the content of nucleoside components was determinedLiquid chromatography-tandem mass spectrometry（LC-MS）was used to analyze the chemical composition of the aqueous extract of C.cicadae.23 compounds were initially resolved,including 8 amino acids,8 nucleosides,4 unsaturated fatty acids and 3 other compounds.High performance liquid chromatography（HPLC）method was developed for the simultaneous determination of the six nucleosides in the aqueous extract powder of C.cicadae,and the contents of the six major nucleosides in three batches of C.cicadae aqueous extract powder were determined.The results showed that the precision,reproducibility,stability and spiked recovery of the method were good,and the total contents of the six nucleosides in the three batches of aqueous extracts were 7106.87μg/g,7171.69μg/g and 6874.93μg/g,respectively.3.A preliminary study on the anti-aging effect of aqueous extracts of C.cicadaeThe expression levels of messenger ribonucleic acid（m RNA）of NAD-dependent deacetylase Sirtuin-1（Sirt1）,adenylate-activated protein kinase（Ampk）and P53 and the expression of AMPK/SIRT1 signaling pathway-related proteins in rat hippocampus of the cerebral cortex by Real-Time quantitative polymerase chain reaction（q RT-PCR）and protein immunoblotting（Western bolt）,and a preliminary investigation of the anti-aging mechanism of aqueous extract of C.cicadae.The results of q RT-PCR showed that high doses of aqueous extract of C.cicadae（400 mg/kg）significantly increased the m RNA expression levels of Sirt1 and Ampk（P<0.01）and significantly decreased the m RNA expression levels of P53（P<0.01）.The results of WB showed that high dose of aqueous extract of C.cicadae（400 mg/kg）reversed the effect of D-gal on the protein expression of AMPK/SIRT1pathway in rats,as shown by significantly increasing SIRT1 and phosphorylated adenylate-activated protein kinase（p-AMPK）/AMPK protein expression（P<0.01）,while significantly decreasing phosphorylated P53（p-P53）/P53 protein expression（P<0.01）.The above experimental results suggest that the anti-aging effect of aqueous extracts of C.cicadae may be related to the regulation of AMPK/SIRT1 signaling pathway.