Extraction Of Total Flavonoids From Tartary Buckwheat And Its Anti-tumor Activity In Vitro

There are many constituents in tartary buckwheat, including flavonoids, highly reactive protein, amino acids, fatty acids, dietary fibre and microelements. Among these components, the content of flavonoids is much higher, which is mainly composed of rutin, quercetin and other essential active ingredient. And flavonoids, as an extremely nutritious substance, exerts a variety of biological activities, such as anti-oxidative, free radical scavenging, anti-allergic, anti-inflammatory, and anti-mutagenic activities. In particular, They have potential effect on chemotherapy treatment. Accumulating evidence suggests that intake of beverages or food products containing flavonoids has been frequently associated with a reduced risk for developing various cancers. In this paper, abundant total flavonoids were extracted from tartary buckwheat and the principal determination of the ingredient, anticancer effects of total flavonoids were studied.Total flavonoids were extracted from tartary buckwheat seeds by using hot water leaching method, alkali water method, ethanol solution leaching method, microwave exeraction method, and ultrasonic wave method. The rate and content of total flavonoids was determined by High Performance Liquid Chromatography. In five extraction methods, the extraction rate of ethanol solution leaching method was highest, microwave extraction method and ultrasonic wave method followed, alkali water method and hot water leaching method were lowest. the microwave extraction method is rapid and efficient, which is the appropriate method for extracting flavonoid substances from tartary buckwheat seeds.To explore the possible influence of the flavonoids from tartary buckwheat on the human solid tumor cells, recently the in vitro anti-cancer effect of flavonoids on esophageal cancer cell line EC9706 and cervical carcinoma cell line HeLa was investigated in our lab. When the two cells were treated with flavonoids for various periods of time and various doses, the MTT assay showed that flavonoids could specifically inhibit the growth of solid tumor cells in a time -and dose -dependent manner. Cell death induced by flavonoids was characterized by nuclear condensation and the production of apoptotic bodies via the light microscopy and DAPI nuclear staining observation, indicating flavonoids can induce cell apoptosis.By flow cytometry analysis, the cell cycle progression and Annexin-Ⅴ-FIFT/PI assay indicated that the cytotoxicity induced by flavonoids was mediated by G2/M cell cycle arrest and the apoptosis rate also increases as compared to controls, which was increased after treatment with flavonoids in range of 0-60μg/ml. Besides, flavonoids-induced apoptosis was shown to involve a concentration-dependent increase in ROS production by flow cytometric analysis. Moreover, western blotting was used for the detection on esophageal cancer cell line EC9706 of the alteration of Bcl-2 and Bax protein ratio.It showed that treatment with flavonoids increased Bax expression but decreased Bcl-2 expression.All above results suggests that flavonoids from tartary buckwheat have anticancer activities, and it appears to be a potent drug against human solid tumor cells (EC9706 and HeLa cells), due to its in vitro potential to not only arrest cell cycle progression at G2/M phase but also induce apoptosis via ROS generation. Therefore it can provid a great deal of referenes for further use and exploitation of flavonoids from tartary buckwheat.