| Bamboo leaf flavonoids(BLFs) are a group of natural bioactive substances that exists in the nature widely, it have been proven that flavonoids have the function of antioxidant, antibacterial, scavenging free radicals, inhibiting nitrosation reaction, regulating blood lipids, preventing cardiovascular and cerebrovascular diseases. In order to study the protective effect and antioxidant mechanism of bamboo leaf flavonoids on CCl4induced liver injury in mice, ultrasonic assisted ethanol extraction method was used to optimize the extraction process parameters; AB-8macroporous resin was selected to determine the adsorption performance and optimal purification conditions; then separated the main component of BLFs by high performance liquid chromatography(HPLC). Mice model of liver injury induced by CCl4was established and mice in different BLF groups were fed28d to determine kinds of enzyme activities and make pathology section of liver tissue, so as to take biological overall evaluation on the protective effect of BLFs to acute liver injury. In vitro, cell experiment was used to determine cell activity and intracellular main antioxidant enzymes, so as to study the protective mechanism of BLFs to hepatocytes injured by CCl4at a cellular level. The main results are as follows.(1) The influence of every factor on extraction efficiency of bamboo leaf flavonoids were studied by single factor experiment, and orthogonal test was selected to determine optimal extraction process parameters of BLFs:ethanol concentration60%, solid-to-liquid rat1:15, extraction temperature60℃, ultrasonic time40min.(2) Macroporous resin AB-8was selected as adsorbent, then analyzed the static and dynamic adsorption capacity to BLFs. Results showed that AB-8has high adsorption capacity and good enrichment effect. The optimal conditions for separation and purification:sample concentration1.0mg/mL, pH5.5, adsorption time7h, ethanol concentration60%, adsorption rate1.OmL/min, elution rate1.5mL/min.(3) The flavonoids content of purified solution increased from9.68%to30.21%, the purity increased by3.1times. It is proven that this extraction and purification methods have significant effect on enrichment of BLFs. Method of high performance liquid chromatography analysis was used to verify the main component of BLFs, changed mobile phase and gradient elution to obtain better liquid phase condition to identify BLFs, and four kinds of main flavonoids glycosides:Rutin、Homorrientin、Vitexin、Quercetin were separated from BLFs successfully.(4) By establishing liver injury model induced by CCl4, animal experiment in vivo showed that BLFs can cause significant reduction in ALT,AST activities and MDA content (p<0.05) and significant increase in SOD,GSH-Px activities and liver glycogen content (p<0.05). In addition, the pathological changes degree of liver tissue in BLFs groups were much better than tissue of liver injury group by observing section of liver tissue.(5) cell experiment in vitro showed that:optimal concentration of CCl4is20mmol/L, optimal injury time is6h by establishing NCTC-1469hepatocyte injury model. In this condition, the cell viability is only50%, AST,ALT activities increased significantly, and the degree of cell damage showed a linear correlation with CC14concentration and action time. BLFs showed significant effect to protect NCTC-1469injured by CCl4, increased cell viability and antioxidase activities such as CAT and SOD, inhibited the increase of AST,ALT activities and MDA content caused by cell damage (P<0.05), and showed linear correlation. It is proven that bamboo leaf flavonoids have significant protective effect on liver cell damage caused by CCl4. |
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