Screening And Mechanism Analysis Of Anti-Cancer Active Components From Mycelia Of Phellinus Linteus And Antrodia Cinnamomea

As a valuable medicinal fungus,Phellinus linteus and Antrodia cinnamomea have received much attention in the world.At present,more researches have been done on the fruit body of Phellinus linteus and Antrodia cinnamomea,but these fungi grow slowly in the wild,and are scarce,which is not conducive to large-scale research and application.In this study,the mycelium of Phellinus linteus and Antrodia cinnamomea were cultured in solid-state fermentation or liquid fermentation.The polysaccharide composition and triterpenoids were extracted.In vitro antitumor activity,drug action time,safety,anti-cell migration and other activities were studied.The inhibitory effect of each active fraction on the DNA damage pathway mediated by TOP1/TDP1 in tumor cells was found.The main results are as follows:The following four active substances are obtained by separation and purification: The crude polysaccharide fractionss of Phellinus linteus PS were obtained from the solid-state fermentation;The purified polysaccharide fractions ACPS-1 was obtained from the liquid fermentation.Triterpenoid mixtures ACT-I and ACT-N were obtained from the mycelial mycelium of the solid-state fermentation.Antitumor experiments show that PS can inhibit the proliferation of Hep G2,HeLa,Lovo,S180 and H22 cells in vitro,maximum inhibition rate was 65.00%,92.38%,36.30%,61.83%,94.83%,respectively.ACPS-1 can inhibit the proliferation of A431,HeLa,S180 and H22 cells in vitro,maximum inhibition rate was 36.89%,74.09%,70.61%,69.07%,respectively.ACT-I can inhibit the proliferation of HeLa and S180 cells in vitro,maximum inhibition rate was 73.54%,78.81%,respectively.ACT-N inhibits the proliferation of HeLa and S180 cells in vitro,maximum inhibition rate was 59.87%,71.49%,respectively.The safety analysis of these four active fractions found that PS,ACT-I and ACT-N had no inhibitory effect on RAW264.7 cells.In addition,ACPS-1 had a significant inhibitory effect on RAW264.7 cells at a concentration of more than 100 mg /mL,and the inhibition rate was less than 30%.At the same time,we found that PS,ACPS-1,ACT-I and ACT-N had no inhibitory effect on splenic hybrid cells,and could promote the proliferation of spleen cells in vitro,especially ACT-I and ACT-N,with a proliferation rate of over 120%.The experimental results show that the four active fractions of PS,ACPS-1,ACT-I and ACT-N have high safety,and there is no obvious inhibitory effect on normal cells.Annexin V-FITC /PI cell cycle and apoptosis effect were studied using HeLa cells.Under the action of PS,the proportion of early apoptotic cells in HeLa cells was relatively high,and the DNA replication process remained in the G2/M phase;ACPS-1 mainly induces the late apoptosis in HeLa cells,causing the cell DNA replication process to remain in the G2/M phase.ACT-I and ACT-N induce apoptosis of S180 cells and increase the proportion of apoptosis cells,which increased the proportion of cells that remained in the G2/M phase and remained in the G0/G1 phase.Two key proteins in the repair pathway of DNA damage mediated by topoisomerase I and tyramyl-DNA-phosphodiesterase I were detected by topoisomerase I and tyrolamino-DNA-phosphodiesterase I.Using ELISA method to detect in each of the active fractions under the action of the two kinds of protein content,found in PS,ACPS-1,under the action of ACT-I and ACT-N,topoisomerase I and tyramyl-DNA-phosphodiesterase I content decreased significantly,and in a certain concentration range present a dose dependent.The expression levels of topoisomerase I and tyramyl-DNA-phosphodiesterase I were detected by real-time fluorescence quantitative PCR,and the expression levels of these two proteins were significantly decreased.Using HeLa cells to carry out the wournd closure assays,it was found that PS,ACPS-1,ACT-I and ACT-N could inhibit the migration of HeLa cells,and found that these active fractions inhibit tumor cell proliferation by inhibiting tumor cell migration.In conclusion,the research proves that PS,ACPS-1,ACT-I and ACT-N have a better effect on anti-tumor,and their mechanism of tumor suppressive action is also being further explored.These active fractions,both as health food and medicine,have extremely high application value and huge market.