Study On Isolation And Extraction Of Polysaccharides From Lycopus Lucidus Turcz.and Their Effects On Proliferation Inhibition And Apoptosis Of Melanoma A375 Cells And Lung Cancer A549 Cells

Objective: To study the extraction method and antitumor activity of Lycopus lucidus turcz.polysaccharide(LLP)from Lycopus lucidus Turcz,Yunnan Province.Methods: The extraction of Lycopus lucidus Turcz polysaccharide was made by water extraction and alcohol precipitation method.The raw materials of Lycopus lucidus Turcz were crushed,and then were recycled with petroleum ether and 95%ethanol respectively.After filtration,distilled water was extracted in water bath at75 ℃,vacuum concentrated,alcohol precipitation,seaveg method was used to deprotein completely alcohol,and freeze-dried.The total polysaccharides(LLP)of Lycopus lucidus Turcz were refined,and the standard curve of glycan was drawn to determine the content of LLP.CCK8 was used to detect the cytotoxic effects of LLP on A375 and A549 cells;the cell scratch and Transwell test were used to detect the migration and invasion ability of LLP to A375 and A549 cells;the apoptosis of A375 and A549 cells induced by LLP was detected by annexin V FITC / PI apoptosis kit;the effect of LLP on the cell cycle of A375 and A549 was detected by cell cycle detection kit;real The expression of 5 genes(Caspase-3,Caspase-8,Caspase-9,CDK-1 and Cyclin B1)in A375 and A549 cells was detected by Real time PCR.Western blotting test detected the effect of LLP on the expression of 10 proteins(Caspase-3,Caspase-8,Caspase-9,Bax,Cyclin B1,Cyclin D1,CDK-1,CDK-4,CDK-6 and Bcl-2).Results: The extraction rate of polysaccharide was 20.77% by the experimental method.LLP at different concentrations can effectively inhibit the proliferation and migration of A375 and A549 cells,and it is concentration dependent.Flow cytometry was used to detect the treatment of A375 and A549 cells with different concentrations of LLP.The results showed that LLP could induce apoptosis of A375 and A549 cells,and the apoptosis rate was also concentration dependent.LLP can up regulate the expression of Caspase-3,Caspase-8,Caspase-9,Bax,and down regulate the expression of Bcl-2.The results of cell cycle study showed that LLP could block A375 cell cycle in G0/G1 and G2/M,and A549 cell cycle in G0/G1.LLP can down regulate the cell cycle related proteins,CDK-1,CDK-4,CDK-6,Cyclin B1 and Cyclin D1.Conclusions: LLP was extracted from Lycopus lucidus Turcz,which proved that LLP has the activity of anti-tumor and non-small cell lung cancer in vitro.The antitumor activity of the tumor cell may be achieved by regulating the apoptosis signal pathway and regulating the cell cycle related proteins.