Study On Effects Of Active Components From Tartary Buckwheat In Hep G2Cells

Buckwheat is of high nutritional values and powerful healthy functions. It is known as the most popular green food. In recent years, some researchs had shown that the main active ingredients of buckwheat, Such as trypsin inhibitor and flavonoids, can cut down blood cholesterol, suppress lipopexia, restrain cancer of colon and gallstones, improve constipation, anti-aging, et al. In order to further reveal the molecular mechanism of anticancer drugs which inhibit cell proliferation and induce cells apoptosis, we used trypsin inhibitor BTI, quercetin and rutin from buckwheat seeds as experimental materials and deeply researched their antitumor effect.The experiment maked use of the gene of Buckwheat trypsin inhibitor (BTI) to transfect the human hepatocellular carcinoma cells Hep G2, then studied the influence of BTI to cells growth and migration preliminarily. MTT assay results indicated that BTI could inhibit the growth of Hep G2in a time dependent manner. The inhibition rate reached35%when Hep G2cells were treated for48h. Through DAPI nuclear staining and flow cytometry analysis, we observed that Hep G2cells showed death signals, and produced apoptotic bodies after they were induced by BTI. The cell cycle regulation factor test showed that the mRNA of CyclinD1, CyclinE1, CDK2, CDK7was decreased, but the mRNA of P21, P53was increased in transfected pEGFP-N1-BT1cells. Western Blot analysis showed that BTI may inhibit cells proliferation and induce cells apoptosis through decreasing the expression of CyclinD1and increasing the expression of P21. Then it can achieve the purpose of cancer therapy. Besides, the adhesion and migration capacities of Hep G2cells were decreased when cells were transfected with BTI.In another experiment, we compared and studied the antitumor effects of quercetin and rutin from tartary buckwheat seeds. MTT assay showed that quercetin and rutin could inhibit the growth of Hep G2cells in time and concentration dependent manners. DAPI nuclear staining showed that nuclear condensed and apoptotic bodies produced. Cell injury healing experiment indicated that quercetin and rutin could suppress cells migration in certain concentration range. And the inhibiting migration capacity of quercetin was bigger than that of rutin. Through flow cytometry analysis, we can test the intracellular reactive oxygen species (ROS) level. We found that quercetin and rutin could induce the increase of ROS levels significantly, and the ROS level of quercetin was higher than that of rutin. We used the total antioxidant competency (T-AOC) detection Kit to detect the T-AOC of quercetin and rutin, and found that the T-AOC of rutin solution was higher than that of quercetin. Two kinds of flavonoids treate Hep G2cells, the T-AOC of the cells and culture medium of quercetin was higher than that of rutin. The results indicate that quercetin and rutin both have good antioxidant competency, and the antioxidant competency is different in different systems.The signal transduction pathways of eukaryotic cells apoptosis are very complex and extremely subtle, it needs many signal molecules to participate. The molecular mechanism of trypsin inhibitors and flavonoids inducing cells apoptosis still need to be further studied, and the thesis laid a experimental basis for this complex mechanism and the exploitation of the value of buckwheat as drugs, the study is originality and meaningful.