Study On The Extraction And Purification Of Flavonoids Crude Extract From Coreopsis Tinctoria Nutt.and Its Anti-aging Effect

Objective:To optimize the extraction of flavonoids crude extract from Coreopsis tinctoria Nutt.Based on the theory of oxidative stress,the anti-aging effect of flavonoids crude extract in Coreopsis tinctoria Nutt.was studied.And further optimize the purification process of the crude extract.Methods:The flavonoids content and extract yield were used as evaluation indexes to investigate the extraction solvent and extraction method.Based on the single factor test results,Box-Behnken response surface method was used to investigate the extraction time,extraction temperature,ethanol concentration and solid-liquid ratio,and to optimize the extraction process;And antioxidant activities of flavonoids crude extract from Coreopsis tinctoria Nutt.was determined by using DPPH free radical scavenging activity,ferric-reducing antioxidant power and inhibiting spontaneous lipid peroxidation;Morris water maze experiment was used to study the effect of flavonoids crude extract on learning and memory in mice induced by D-galactose,SOD activity and MDA content in brain tissue of mice were measured,and the anti-aging mechanism of flavonoids crude extract of Coreopsis tinctoria Nutt.was preliminarily explored;The crude flavonoids were purified by macroporous adsorption resin,and the concentration,loading volume,loading velocity,removal solvent and volume,elution velocity and elution volume of the sample solution were evaluated,and the purification process was optimized.Results:The optimized extraction condition was as follows:the ethanol concentration was60%,the solid-liquid ratio was 1∶67,extraction temperature was 80℃and the extraction time was 21 min.The IC₅₀ value of DPPH free radical-scavenging was 7.16μg·m L^-1.The absorbance was 1.75,when the flavonoid content of Coreopsis tinctoria Nutt.was 100μg·m L^-1.The inhibition rate of lipid peroxidation was 25.13%,when the flavonoid content of Coreopsis tinctoria Nutt.was 200μg·m L^-1.The flavonoids crude extract of Coreopsis tinctoria Nutt.significantly increased the incubation period,the percentage of time in the platform quadrant,the number of times of crossing the platform,and the percentage of time in the original platform quadrant of aging mice（P<0.05）.The flavonoids crude extract of Coreopsis tinctoria Nutt.significantly increased SOD activity and decreased MDA content in aging mice（P<0.05）.The purification process was as follows:the sample concentration was 1.0 mg·m L^-1,the loading flow rate was 1 m L·min^-1,the loading volume was 7 BV,the water elution dosage was 6 BV,the elution flow rate was 1.0 m L·min^-1,the impurity was removed by 1 BV 10%ethanol,and 5 BV was eluted by 50%ethanol.Conclusion:The optimized extraction was stable and feasible,which can be used for extraction of flavonoids crude extract from Coreopsis tinctoria Nutt.The antioxidation test in vitro proves that flavonoids crude extract from Coreopsis tinctoria Nutt.have good antioxidant capacity.The flavonoids crude extract of Coreopsis tinctoria Nutt.can improve the learning and memory ability of D-galactose-induced aging mice,increase SOD activity and decrease MDA content in brain tissue,and inhibit oxidative stress.The optimized purification process was stable and feasible,which can be used for and purification of flavonoids crude extract from Coreopsis tinctoria Nutt.