The Anti-tumor Effect And Its Mechanism Of Total Flavonoids In Leaves Of Phyllanthus Emblica L.

Objective:To research the anti-tumor effect and its mechanism of total flavonoids in leaves of Phyllanthus emblica L..Methods:Firstly,total flavonoidsin in leaves of Phyllanthus emblica L,which derived from leaves of Phyllanthus emblica L,was extracted in ethanol by soaking diacolation,and isolated and purified by D101 macroporous resin column chromatography and ethyl acetate.Then,seven kinds of tumor cells were used to analyze cell proliferation by the method of SRB,that was human hepatocellular carcinoma cell（Bel-7404）,hepatocellular carcinoma cell（HepG2）,cervical carcinoma cell（Hela）,gastric cancer cell（SGC7901）,nasopharyngeal carcinoma cell（CNE-2）,lung cancer cell（H460）and breast cancer cell（A2780）;The DNA damage of Bel-7404 cells was detected by IF FM HCS,Caspase-3 activity,cell apoptosis and cell cycle was detected by enzyme linked immunosorbent assay,kit Hoechst 33258 and by flow cytometry,respectively.Tumor model of Bel-7404 nude mice was evaluated the effect of total flavonoids on inhibition rate of tumor.Proteins expression of Bax and Bcl-2 in tumor tissue were measured by immunohistochemistry and Western-blot.Results:Total flavonoids was prepared through soaking diacolation,separation and purification,and the yield of 4.90%.SRB results showed that compared with normal group,total flavonoids had significant inhibitory effect on 7 kinds of tumor cells,especially Bel-7404 cell,H460 cell and CNE-2 cell,IC₅₀0 were 453.46±40.06μg/mL、471.36±50.66μg/mLand 463.26±40.75μg/mL,respectively.The inhibitory effect of high dose was with similar to the positive medicine 5-Fu.Besides,Bel-7404 cells were incubated total flavonoids for 24h,36h,and 48h,IC₅₀0 were453.46±40.06μg/m L、253.75±41.25μg/mL and 152.73±40.55μg/mL,respectively,the rate of inhibition showed the relationship between dose effect and time effect.IF FM HCS detection showed that total flavonoids significantly damaged nuclear DNA of Bel-7404（**p<0.01）in middle and high dose,while the low dose group had no significant effect on DNA damage.Hoechst 33258 showed nuclear pyknosis of Bel-7404 cells and cell thin membrane were observed induced by total flavonoids under a fluorescence microscope,as expected,effect of high dose group was similar to 5-Fu group.FITC-AnnexinV/PI double labeling assay showed that cell apoptosis was increasing along with increasing of total flavonoids of dose.Eventually,results in vivo showed that tumor transplantation inhibition of Bel-7404 were 66.77%,29.03%,20.56%in high（495.55 mg/kg）,middle（247.78 mg/kg）and low（123.89 mg/kg）dose group of total flavonoids,and can reduce the animal’s liver index and spleen index.Immunohistochemistry results the positive area of Bax was significantly increased in the high dose group,and the positive rate of Bcl-2immunoreactivity in the high dose group was significantly lower than that in the control group.Western blotting showed the protein expression of Bax significantly increased with total flavonoids vs model group,while the protein expression of Bcl-2 were decreased in all dose group,with significant difference in high dose.Conclusion:1、Total flavonoids in leaves of Phyllanthus emblica L.2、The total flavonoids of Phyllanthus emblica L.induced Bax expression,inhibited Bcl-2 expression and induced apoptosis,and inhibited the proliferation of Bel-7404 cells.3、Total flavonoids in leaves of Phyllanthus emblica L.can significantly inhibit the growth of Bel-7404 xenografts in nude mice.