Isolation,purification And Activity Of Anti-lung Cancer Protein And Polysaccharide From Ophiocordyceps Sinensis

Ophiocordyceps sinensis is a traditional and valuable biological resource with food and medicine value.It is considered to have a long cognitive history in China.It is a kind of precious Chinese medicinal material with edible and medicinal value,and can exert the effect of "tonifying the kidney and lungs,stopping bleeding and resolving phlegm".A large number of studies have shown that Ophiocordyceps sinensis and its related products can have a good effect on lung diseases,including lung cancer.In this study,we extracted 16.06 g of crude active protein extract(Protein A)from 100 g Ophiocordyceps sinensis.Protein C,D and E were obtained from anion exchange column after ammonium sulfate precipitation.Used a cation exchange column to further separate stronger activity Protein C to obtain Protein F,G and H.Using SDS-PSGE electrophoresis,it was founded that the Protein H at approximately25 k D and 70 k D can be divided into Protein I and J.Finally,considering the requirements of protein activity and protein content,we choosed to recycle gel to recover Protein I and J.The IC50 of Protein I with stronger anti-lung cancer activity was 139.33,107.92 and 83.91 μg/m L at 24 h,48 h and 72 h,respectively.Using mass spectrometry to analyze the Protein I and BLAST the NCBI gene library,it was found that it can mainly match three types of protein components: serine protease,superoxide dismutase and translation elongation factor 1 alpha.The Protein I was further analyzed and tested.The lactate dehydrogenase(LDH)test found that the Protein I can increase the LDH leakage rate of lung cancer cells.The results of the cell scratch experiment showed that this protein may have an impact on the migration of A549 cells.Apoptosis detection founded that with the increase of the concentration of Protein I,the apoptosis rate increased significantly.Cell cycle testing founded that with the increase of the concentration of Protein I,there were obvious dissimilarity in cells cycle,mainly in the G0/G1 phase.Western Blot detection founded that after treatment of Protein I,it can increase the expression of Bax in A549 cells,inhibit the expression of Bcl-2,and inhibit the expression of P-Rb and CDK4/CDK6.Extraction of polysaccharides from the residue after extracting active protein from Ophiocordyceps sinensis,and the extraction rate was 19.6 mg/g of crude polysaccharide(PS1).The crude polysaccharide after deproteinization can be divided into two parts by using anion exchange chromatography,the passing part PS3 a and the binding part PS3 b.SDS-PAGE analyzed the homogeneity of polysaccharides,and the results showed that both PS3 a and PS3 b contained a small amount of contaminated protein.Further using ethanol proteinal precipitation separation to four parts: PS3a1,PS3a2,PS3b1 and PS3b2.Infrared scanning of the four-part polysaccharides,it can be inferred that PS3a1 and PS3a2 contain α-D-glucopyranose;PS3b1 may contain both α-pyranose and β-pyranose;all four-part polysaccharides contain glycoproteins.Using A549 cells as the test subject by MTT method,it was detected that PS3b1 had the best effect,reaching 67.68% inhibition rate.Further antioxidant activity test found that the antioxidant activity of PS3b1 increased with the increase in concentration after reaching a certain concent ration.The study of Ophiocordyceps sinensis anti-lung cancer active protein and subsequent active polysaccharides can contribute a small part of the future lung cancer treatment and research,and provide a reference direction for future research.