Preliminary Study On The Preparation And Anti-tumor And Anti-hepatic Effects Of The Crude Polysaccharides CP50 And CP80 Of Golden Cicada Flower

Cordyceps cicadae,originally recorded in the Southern and Northern Dynasties,is a dry complex formed by the ergot family fungus cicada and its host mountain cicada nymph.Cordyceps cicadae contain a variety of active ingredients,of which polysaccharide is the main active ingredients.It has pharmacological activities such as anti-tumor,anti-aging,immune regulation,antioxidant,hypolipidemic,and kidney protection.In this study,activated carbon decolorization,Sevage deproteinization,and 50%and 80%ethanol precipitation were used to prepare crude polysaccharide CP50 and CP80.The antitumor activity of the two crude polysaccharides was studied with human cervical cancer cell Hela,and then,their anti-tumor mechanism was preliminaryly studied.In addition,this study initially evaluated these two crude polysaccharides on liver injury of mice induced by the toxicity of the chemotherapy drug cyclophosphamide.This study is expected to provide reference for the rational development and clinical application of Cordyceps cicadae.The main results of the experimental study are as follows:1.Study on the preparation process of the crude polysaccharides of Cordyceps cicadae.Polysaccharide of Cordyceps cicadae was prepared by water extract-alcohol precipitation method.The single-factor test and the orthogonal test were used to select the best conditions of Sevage meethod for the deproteinization of the polysaccharides.The best conditions of deproteinization were chloroform and n-butanol to 4:1 ratio,the amount added to the polysaccharide was 1:3,deproteinized for 4 times.Under this condition,the protein removal rate of the polysaccharide was46.35%.According to this method,the yields of CP50 and CP80 were 2.69%and18.79%,and the polysaccharide contents were 2.11%and 6.23%,respectively.2.Comparison of antitumor activity and mechanism of crude polysaccharide CP50 and CP80.Using human cervical cancer cells Hela as model cells,and comparison of the two crude polysaccharides CP50 and CP80 by CCK-8methodwere conducted.The IC₅₀ of the two polysaccharides were 1203?g/mL and778.9?g/mL,respectively.When the concentration of CP80 were 200,400,and 800?g/mL,the proliferation of Hela cells were significantly inhibited,and the survival rates were 84.85%,71.58%and 58.60%?P<0.01 or P<0.001?,respectively;while the concentration of CP50 was up to 400?g/mL,the proliferation inhibition of Hela cells was only statistically significant,which was 82.98%?P<0.05?,and as the concentration of CP80 increases,the cell proliferation inhibition trend was gentler than that of CP80,therefore we concluded that CP80 had better antitumor activity.Further results observed that CP80 has an inhibitory effect on HeLa cell morphology and migration;ROS activity was measured by DCFH-DA method,Western blot and qPCR method were used to determine the expression of Bax,Bcl-2,and p53 protein and gene levels.Compared with the Control group,CP80 administration concentration significantly increased ROS activity in Hela cells?P<0.05?,Bax,p53protein and gene levels were significantly up-regulated?P<0.05?,Bcl-2 protein and gene levels were significantly down-regulated?P<0.05?.Therefore,it is inferred that the crude polysaccharide CP80 of Cordyceps cicadae may cause apoptosis by increasing the ROS activity and mediating the p53 signaling pathway in Hela cells.3.Study on the pretect effect of the liver injury caused by cyclophosphamidethe with crude polysaccharide CP50 and CP80.Liver injury models were constructed by giving different CPA doses in C57BL/6 mice.the liver index and spleen index of CP50 and CP80 high and low dose groups were reduced compared with the model group;the activities of ALT,AST and MDA were increased,and high dose group?200 mg/kg?of CP80 was significantly increased the activity of MDA?P<0.05?.The results showed that the polysaccharides had a certain protective effect on liver injury induced by CPA,and the effect of CP80 was slightly better than CP50.