| Botrychium ternatum?Thunb.?Sw.,which belongs to the family Botrychiaceae,is resource-rich and widely distributed.Botrychium ternatum?Thunb.?Sw.has many clinical efficacies such as clearing heat,calming liver wind,relieve a cough,Stopping bleeding,and removing nebula for improving eyesight and it is a traditional Chinese folk herb with a wide range of clinical applications.In 1977"The Chinese pharmacopoeia"has contained the Botrychium ternatum?Thunb.?Sw.,only identification and microscopic identification,without content determination and other inspection item,and the drug is not included in the current 2015 pharmacopeia.In order to be full and accurate control of the inner quality of the herb medicine,and to lay a foundation for the production and quality control standards specification,it is necessary to carry out in-depth systematic research on its quality standard.In addition,in the early stage,Chinese and foreign plant chemical workers have isolated the flavonoids,polysaccharides and other chemical components.And the flavonoids mainly include ternafin,luteolin,quercetin,kaempferol and so on.They all have different degrees of anti-oxidation,expectorant,anti-inflammatory or anti-tumor activity,is the common active index component,In the early stage,the pharmacological research on the flavonoids in the Botrychium ternatum?Thunb.?Sw.was mainly focused on the expectoration,anti-inflammatory and anti-tumor.For scientific,make full use of the resources of flavonoids in the Botrychium ternatum?Thunb.?Sw.,this study extracted and purified the total flavonoids and to inspect the antioxidation activity of total flavonoids in the shade in order to provide the basis for the development and treatment of the disease based on the anti-oxidation mechanism and the preparation of the agent.Objective:to establish the quality standard of Botrychium ternatum?Thunb.?Sw.,optimize of the extraction process of total flavonoids,study on the separation and purification of its total flavonoids,and use the purified flavonoids to determine the antioxidant activity in vitro and in vivo.Methods:first of all,the source of the herb medicinal materials was determined by the literature research and the identification of the system,and the characters and microscopic characteristics of the herb were observed.The TLC chromatographic study was conducted with luteolin as reference.The method of determination of Botrychium ternatum?Thunb.?Sw.was established by UV-Vis and HPLC.and the water content,total ash,acid-insoluble ash,ethanol soluble extraction were checked.Then,on the basis of single factor experiment,we selected three factors which has significant effect of volumetric fraction of ethanol,extraction time and liquid-solid ratio as influence factor,Using the box-behnken center combination design to build a mathematical model,the extraction rate of total flavonoids as the response value to analytical the response surface,the optimum extraction technology of total flavonoids from the Botrychium ternatum?Thunb.?Sw.was obtained,and the optimum extraction process was determined under this condition.Second,with 80%ethanol reflux extraction and petroleum ether degreasing,macroporous resin adsorption,different concentrations of ethanol elution,get the purification of total flavonoids and use it as the research object,with vitamin C?Vc?and rutin as comparison,discuss the total antioxidant capacity and on?DPPH,?ABTS+free radical,hydroxyl free radical??OH?removal ability,so as to determine the in vitro antioxidant activity of total flavonoids.Finally,vitamin E?VE?as contrast,using carbon tetrachloride?CCl4?determination of liver damage in mice model flavonoids on carbon tetrachloride liver injury in mice liver and kidney tissues of superoxide dismutase?SOD?,catalase?CAT?activity and reduced glutathione?GSH?and the influence of the malondialdehyde?MDA?content,so as to determine the in vivo antioxidant activity of total flavonoids.Results:?1?The TLC method used G taking cyclohexane-aceticether-methyl alcohol-formic acid?20?10?3?1.5?as the developer and ethanol solution of 1%ferric chloride as the chromogenic reagent with the results that it appeared clear spots.The UV method took rutinum as the reference substance,and the content of total flavonoids as index,The contents of samples from 8 different batches were determined,and the lowest content of total flavonoids was 12.55 mg·g-1.The HPLC method took quercetin and kaempferol as the reference substance,and methyl alcohol-water?containing 0.4%phosphoric acid?as the mobile phase;Detection wavelength was 366 nm.The contents of samples from8 different batches were determined,and the lowest content of quercetin was 1.30 mg·g-1,and kaempferol was 1.25 mg·g-1.?2?the optimal extraction process of the total flavonoids in the Botrychium ternatum?Thunb.?Sw.was 80%ethanol leaching time,56 min,and the material liquid ratio 12.5:1.The predicted value of extraction rate of total flavonoid is 1.628%,and its experimental value is 1.609%,and the relative error was 0.32%.The purity of flavonoids was 36.73%.?3?when the mass concentration of the total flavonoids in the Botrychium ternatum?Thunb.?Sw.reached 0.8 mg·ml-1,the removal capacity of the?DPPH radical was 92.9%,which is about to reach the level of Vc.When the mass concentration was 0.6mg·ml-1,the clearance rate of the Vc to·ABTS+was 42.7%,and the clearance rate of the total flavonoids was 41.8%,while the clearance rate of rutin to·ABTS+was only 12.0%.When the concentration of the total flavonoids was 1.0 mg·ml-1,the scavenging capacity of the?OH radical was 68.2%,which was close to the level of Vc.The reduction ability of total flavonoids in the same concentration was significantly lower than that of Vc,but it was significantly higher than that of rutin.?4?Compared with liver damage in the control group,the total flavonoids in each dose group of MDA in mice all have varying degrees of decline,the SOD activity,CAT activity and GSH content all have different degree of improvement,and the total flavonoid dose group is statistically significant?P<0.01?.Conclusion:?1?the microscopy,TLC identification,total flavonoids,quercetin and mountain neophenol content of the herb are accurate and reliable,which can be used for the quality control of Botrychium ternatum?Thunb.?Sw.?2?the optimum process of extracting flavonoids from the Botrychium ternatum?Thunb.?Sw.is stable and easy to operate.?3?the total flavonoids in the Botrychium ternatum?Thunb.?Sw.has obvious antioxidant activity. |
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