Synergistic Antioxidant Effect Of EGCG,Grape Seed Proanthocyanidins And Dihydromyricetin And Their Synergistic Inhibitons On Mouse Mammary Carcinoma 4T1 Cells

With the rapid development of researches on free radicals and the continuous deepening of the relationship between free radicals and diseases,antioxidants have been applied to many aspects such as food,health food,daily necessities,cosmetics and so on.Natural antioxidants are regarded as safe and low-toxic,with abundant biological activities,such as anti-inflammatory,preventing cardiovascular and cerebrovascular diseases,anti-bacterial,and anti-cancer.Thus natural antioxidants have become one of the global research hotspots.Among them,EGCG,grape seed proanthocyanidins(GSP),dihydromyricetin(DMY)gained a lot of concern.It was found that the combination of multiple antioxidants had better effect than single component,in the other word,synergistic effect exists between different antioxidants.The synergistic effects of tea polyphenols,proanthocyanidins and dihydromyricetin on antioxidant activity,inhibition of diabetes-related enzymes and inhibition of proliferation of mouse mammary carcinoma 4T1 cells were systematically studied in order to provide basis for dietary guidance and relevant product development.The main results are as follows:1.DPPH,ABTS and hydroxyl radicals scavenging assay were used to determine the antioxidant effects of single substances and combination of EGCG,GSP and DMY in vitro,and their synergistic effect were evaluated using isobologram.The antioxidant capacity of the three natural products increased with the increasing of concentration.Their antioxidant capacity were EGCG>DMY>GSP.In the three antioxidant assays,their orders of antioxidant capacity were the same.The groups that showed the best antioxidant synergistic effect were:the combination of EGCG:GSP:DMY=4.8:1.2:4 in DPPH free radical scavenging experiment(synergistic rate was 29.28%),the combination of EGCG:GSP:DMY=4.8:1.2:4 in ABTS free radical scavenging experiment(synergistic rate was 15.42%)and the combination of EGCG:GSP:DMY=4:4:2 in hydroxyl radical scavenging experiment(synergistic rate was 55.69%).2.The inhibition on three diabetes-related enzymes of EGCG,GSP and DMY and their conbinations were determined.The three diabetes-related enzymes were a-amylase,a-glucosidase and DPP-IV enzyme.The inhibition of three natural products on the activity of a-amylase,a-glucosidase and DPP-IV enzyme were EGCG>GSP>DMY,GSP>EGCG>DMY,and EGCG>GSP>DMY,respectively.The natural products showed different inhibition on different enzymes.In the study of the inhibition of conbinations on the three diabetes related enzymes,synergistic effect among EGCG,GSP and DMY was found.In the inhibition experiment ofa-amylase,the best combination was EGCG:GSP:DMY=4:1:5,and the synergistic rate was 25.95%.The best combination to inhibit the synergistic effect of a-glucosidase was EGCG:GSP:DMY=3.2:4.8:2,and the synergistic rate was 27.69%.In DPP-IV inhibition experiment,the combination of EGCG:GSP:DMY=1.6:6.4:2 showed the best synergistic effect,whose synergistic rate was 66.65%.3.Mouse mammary carcinoma 4T1 cells were used to evaluate the anti-cancer activity of the EGCG,GSP,DMY and their combinations,in order to further explore the synergistic effect among the three natural products.It was found that EGCG,GSP and DMY could inhibit the proliferation of 4T1 cells and promote the apoptosis of 4T1 cells.DMY had the strongest inhibitory effect on the proliferation of 4T1 cells(IC50=20.05+1.66 ug/mL).Combinations of three natural products in different proportions were prepared to determine their inhibition on the proliferation of 4T1 cells.The groups that showed the best synergistic effect were as follows:the combination of EGCG:GSP=6:4,whose synergistic rate was 61.10%;EGCG:DMY=2:8,whose synergistic rate was 22.64%;GSP:DMY=6:4,whose synergistic rate was 57.10%.When the three natural products were combined together,they showed good synergistic effects.The group with the highest synergistic rate was EGCG:GSP:DMY=2.4:1.6:6,and the synergistic rate was 63.99%.4.The strongest synergistic inhibitory combinations were screened out,namely,EGCG-GSP(6:4),EGCG-DMY(2:8),GSP-DMY(6:4),EGCG-GSP-DMY(2.4:1.6:6),to explore the synergistic inhibitory effect and mechanism of combinations of EGCG,GSP and DMY on mouse mammary carcinoma 4T1 cells.It was found that the natural product combinations could block cell cycle to a certain extent and increase the apoptotic rate of 4T1 cells,and the effect of the combination of natural products was better than that of several natural products alone.The ROS levels in 4T1 cells were significantly increased by the four natural product combinations(p<0.05),and the effect of EGCG-GSP-DMY(2.4:1.6:6)combination was better than that of three substances alone or combinations of two materials.The activities of caspase 9 and caspase 3 in mouse mammary carcinoma 4T1 cells increased with the application of various combinations of natural products,suggesting that inducing apoptosis is an important way to inhibit the growth and proliferation of 4T1 cells.Combinations of natural products in each group could affect NO release level in 4T1 cells,and the effect on NO release level may promote 4T1 cell apoptosis and inhibit 4T1 cell growth.