Isolation And Purification Of Tartary Buckwheat Flavonoids And Its Regulatory Effects In Hepatocytes And Vascular Injury

Tartary buckwheat (Fagopyrum tataricum L., Gaench), a very important edible and medicinal plant, has been used as an effective food in the treatment of many diseases in the folk, including diabetes, hypertension, hyperglycemia, and dyslipidemia. Studies have shown that these beneficial health effects are bound up with its high content of flavonoids. However, the mechanisms of tartary buckwheat flavonoids on liver protection and vascular protection are unclear.Therefore, this study selected tartary buckwheat as raw material, tartary buckwheat flavonoids crude extracts was obtained by methanol. After purification with macroporous adsorption resin, the purified tartary buckwheat flavonoids (TBF) was obtained. The method of HPLC analysis was used to identify tartary buckwheat flavonoids extracts. Then, the study explored the protective effects of TBF in different models through animal and cell experiments.The main researches were as follows:(1) The dried tartary buckwheat flour was extracted by 25 L of methanol-water (75: 25, v/v) with reflux for 2 h, and repeated three times. The combined extracts were centrifuged at 3,000 g for 10 min, and concentrated with a rotary evaporator under vacuum, and then freeze-dried. The enrichment of flavonoids was carried out in the glass column with AB-8 resin under the optimized conditions. The purified tartary buckwheat flavonoid fraction was named TBF in this study. The quantification of rutin and quercetin as major flavonoids in TBF was carried out using a reversed-phase HPLC column. As a result, the content of rutin and quercetin presented in TBF reached 536.2 mg/g and 371.6 mg/g, accounting for 90.8% of TBF.(2) This study was performed to investigate the liver and vascular changes in high trimethylamine-N-oxide (TMAO) diet-fed mice, and the possible vasoprotective and hepatoprotective effects of purified tartary buckwheat flavonoid fraction (TBF). Mice fed 1.5% TMAO in drinking water for 8 weeks significantly displayed vascular dysfunction and liver damage (p<0.01). The administration of TBF at 400 and 800 mng/kg.bw significantly elevated plasma NO and eNOS concentrations, and serum HDL-C and PGI2 levels, and lowered serum TC, TG, LDL-C, ET-1 and TX-A2 levels of TMAO-fed mice. TBF also reduced serum AST and ALT activities, and hepatic NEFA and MDA levels, and increased the hepatic GSH-Px and SOD activities in TMAO-fed mice, which were consistent with the observations of the histological alterations of the liver. This report firstly showed that dietary TMAO might cause liver damage and TBF prevented TMAO-induced vascular dysfunction and hepatic injury.(3) The protective effect of purified tartary buckwheat flavonoids fraction (TBF) against oxidative stress induced by a high-glucose challenge, which causes insulin resistance, was investigated on hepatic HepG2 cells in this study. Results suggest that treatment of HepG2 cells with TBF alone improved glucose uptake and antioxidant enzymes, and activated Nrf2, and attenuated the IRS-1 Ser307 phosphorylation, and enhanced total levels of IRS-1. Furthermore, the high glucose-induced changes in antioxidant defences, Nrf2, p-MAPKs, p-IRS1 Ser307, and IRS-1 levels, and glucose uptake were also significantly inhibited by pre-treatment with TBF. Interestingly, the selective MAPK inhibitors significantly enhanced the TBF-mediated protection by inducing changes in redox status, glucose uptake, p-(Ser307) and total IRS-1 levels. This report firstly showed that TBF could recover redox status of insulin-resistant HepG2 cells, suggesting that TBF significantly protected the cells against high glucose-induced oxidative insult, and these beneficial effects of TBF on redox balance and insulin resistance were mediated by targeting MAPKs.