The Composition Analysis Of Golden Cicada Flower And The Research On The Antioxidant Activity Of Polysaccharides

Cordyceps cicadae,a drying complex which composed with the parasitic fungus Cordyceps cicadae Shing and the larvae of Cicada flammata Dist.It is considered to have the similar medical effects to Cordyceps sinensis,and used to be a kind of edible and medically fungus in the folk.It is sweet in taste and cold in nature,relieves heat,arrests convulsion and calms the frightened.C.cicadae are mainly grown in Sichuan,Jiangsu,Zhejiang,Fujian,Anhui and Yunnan provinces.In recent years,large-scale harvesting of C.sinensis has led to the depletion of resources.C.cicadae,contributed to the similar composition and efficacy with C.sinensis,may offer promise as the substitute for it.This present study systematically analyzed the nutrients in C.cicadae,and established a method for simultaneous determination of six nucleosides in C.cicadae by HPLC,and measured its main active components（mannitol,ergosterol and nucleosides）.The in vitro antioxidant activity experiments were carried out to screen the ethanol polysaccharides of different concentrations of ethanol,and the best antioxidant polysaccharides were obtained.The effects on the lifespan and antioxidant activities of Drosophila fed by the polysaccharides were investigated,and the mechanism of action was studied.It provides bases for the further development and utilization of C.cicadae.The main contents are as follows:1.Study on the nutritional composition of C.cicadae.According to the National Food Safety Standards,the main nutrients,crude polysaccharides and metal components in the C.cicadae samples were determined.The results showed that the contents of cellulose,crude protein,crude fat,water and ash were 36.98%～42.84%,33.9%～37.62%,4.46%～8.87%,4.37%～6.06%and 5.77%～9.59%,respectively.The carbohydrate content is calculated from 3.08%to 8.40%.The crude polysaccharide content of C.cicadae is 2.27%～3.58%.The content of metal components Cd,Pb,Hg and As in C.cicadae is lower than the limit standards of heavy metals in edible fungi in national standards（1.0 mg/kg,0.2 mg/kg,0.1 mg/kg and 0.5 mg/kg）.2.LC-MS analysis and content determination of nucleoside components of C.cicadae.The chemical constituents of the ultrasonic extract of C.cicadae were qualitatively analyzed by HPLC-MS method.Seven kinds of nucleosides were identified,which were adenine,N6-（2-hydroxyethyl）adenine and uridine,inosine,guanosine,adenosine and N6-（2-hydroxyethyl）adenosine.A method for simultaneous determination of components（adenine,uridine,inosine,guanosine,adenosine,and HEA）ulteriorly by HPLC was established.The results show that the method has good resolution and peak shape,and the precision,accuracy,stability and repeatability are satisfying,which indicates that the method is suitable for the detection and analysis of nucleoside components.The contents of adenine,uridine,inosine,guanosine,adenosine and HEA were 49.12μg/g,1293.53μg/g,214.36μg/g,503.25μg/g,614.33μg/g,692.12μg/g and 3366.71μg/g,respectively.3.Study on the determination of ergosterol and mannitol of C.cicadae.According to Chinese Pharmacopoeia The contents of ergosterol and mannitol in the sclerotium,coremium and the entire C.cicadae from 12 different habitats were determined by HPLC method UV method,respectively.The results showed that the content of the ergosterol in the entire C.cicadae was 281.88-683.1μg/g.The content of the ergosterol in the coremium was apparently higher than that in the sclerotium,which were 540.57～1800.39μg/g and 129.83～229.55μg/g,respectively.The content of the mannitol in the entire C.cicadae was 82.12～136.24mg/g.The content of the ergosterol in the sclerotium was slightly higher than that in the coremium,which were112.53～154.29mg/g and 68.71～105.62mg/g,respectively.4.Effect of rating polysaccharides of C.cicadae on the In vitro antioxidant activity.Six different C.cicadae rating polysaccharides CP30～CP80 were extracted with different concentration levels of ethanol alcohol.The antioxidant activity of different graded polysaccharides of C.cicadae was conducted by DPPH free radical and·OH radical scavenging capacity determination,total reducing ability and total antioxidant capacity.The results showed that the antioxidant activity in different system had the same tendency:CP70>CP60>CP80>CP50>CP30>CP40,which indicated the polysaccharide component CP70 has the best antioxidant activity in vitro.5.Effect of rating polysaccharides CP70 of C.cicadae on the In vivo antioxidant activity.The effects of C.cicadae polysaccharides of different doses on the lifespan of Drosophila Melanogaster were studied by using the hydrogen peroxide stress model and the normal physiological aging model.Through the determination of antioxidant index（GSH-Px,MDA,CAT）and antioxidant gene（CAT,SOD1,MTH,SIRT2）expression in Drosophila in the middle dose group（5 mg/mL）,the mechanism of anti-aging effect of C.cicadae polysaccharide CP70 on Drosophila in vivo was further explored.The results showed that the C.cicadae polysaccharide CP70 had longevity effect on the physiological aging model and the H₂O₂ acute oxidative damage model,and the middle dose group（5 mg/mL）had the most significant effect.The activity of antioxidant enzymes CAT and GSH-PX was increased,and the content of lipid peroxidation product MDA was decreased.In addition,CP70 significantly increase the expression levels of CAT,SOD1 and MTH mRNA in Drosophila.However,there was no significant difference in SIRT2 mRNA expression.It is speculated that the antioxidant mechanism of C.cicadae polysaccharide CP70 may be achieved by up-regulating the expression of the antioxidant enzymes CAT,SOD1 and the migratory gene MTH gene.