Study On Extraction, Purification And Biological Activity Of Total Flavonoids From Ribes Odoratum Wendl. Leaves

In the present study,ionic liquid were used for the efficient and rapid extraction of total flavonoids from Ribes odoratum Wendl.leaves.The extraction process conditions of ionic liquid-based microwave-assisted extraction were optimized.The process of separation and purification of total flavonoids by macroporous resin of crude extracts,antioxidant activity and anti-inflammation activity of purification material were studied,respectively.An efficient and fast method was for the extraction and purification of total flavonoids and analysis of active constituents Ribes odoratum Wendl.leaves.Our study provided theoretical guidance for efficient utilization and reasonable development of Ribes odoratum Wendl.leaves.The results were as follows:1.The process of ionic liquid-based microwave-assisted extraction was optimized by a Box-Behnken Design(BBD)and response surface methodology(RSM).The optimum parameters were as follows:Ionic liquid:1-butyl-3-methyl imidazole bromine saltIonic liquid concentration:1.6 mol/LLiquid/solid ratio:33:1 mL/gExtraction temperature:60?Extraction time:15 minMicrowave power:600 WUnder the optimal parameters,the average extraction yields of total flavonoids were 44.82mg/g.The ionic liquid-based microwave-assisted extraction process is better than 60%ethanol-based microwave-assisted extraction and ionic liquid-based ultrasound-assisted extraction.2.The macroporous resin was selected in separation and purification of Ribes odoratum Wendl.leaves flavonoids by ionic liquid-based microwave-assisted extraction.6 different types of macroporous resin were screened,and 2 kinds of macroporous resin were selected for further static kinetic curve.Finally,the type AB-8 macroporous resin was selected for dynamic experiment.The results showed that the optimal process for the separation and purification of Ribes odoratum Wendl.leaves flavonoids were as follows:Concentration of adsorption solution:1.10 mg/mLAdsorption rate:2 BV/hEthanol concentration:70%Elution rate:3 BV/hAccording to the optimum conditions of purification,the total content of flavonoids of Ribes odoratum Wendl.leaves increased from 8.61%to 36.68%,and the purity increased by4.26 times,recovery rate was 78.12%.A HPLC method was developed for the simultaneous analysis and determination of rutin,isoquercitrin,astragalin,quercetin and kaempferol of total flavonoids by purification in Ribes odoratum Wendl.leaves,the chromatographic conditions as follows:Chromatographic column:Diamonsil C18 column(250 mm×4.6 mm,5?m),mobile phase consisted of 0.4%phosphoric acid aqueous solution(A)and acetonitrile(B),column temperature 30?,detection wavelength 360 nm,flow rate 1.0 mL/min,injection volume 20?L,gradient elution was performed as follows:0-15 min,10-18%(B);15-30 min,18-18%(B);30-35 min,18-20%(B);35-40 min,20-30%(B);40-45 min,30-60%(B);45-50 min,60-10%(B);50-55 min,10%-10%(B).3.DPPH assay,FRAP assay,ABTS assay were applied to evaluate the antioxidant activity of purification in Ribes odoratum Wendl.leaves by different extraction methods.The result of DPPH assay was V_C>The purification by IL-MAE>The purification by IL-UAE>The purification by 60%ethanol-MAE.The result of FRAP assay was V_C>The purification by IL-MAE>The purification by IL-UAE>The purification by 60%ethanol-MAE.ABTS assay also showed that the purification by IL-MAE had better antioxidant activity.The effects of purification by different extraction methods on the activity of RAW264.7macrophages were investigated by MTT assay.And non-toxic concentration ranges of purification by IL-MAE,purification by IL-UAE and purification by 60%ethanol-MAE were0~200?g/mL.Under the same concentration,inhibition of ionic liquid-based microwave-assisted extraction was purified by the macroporous resin sample on LPS-induced RAW264.7macrophages secretion of NO is better than the other 2 kinds of extraction methods,and with the increase of the concentration of the sample,the content of NO secreted by RAW264.7 cells slightly decreased,when the drug concentration was 200?g/mL,nitrite content of purification by IL-MAE,purification by IL-UAE and purification by 60%ethanol-MAE were 22.35,24.44and 27.25?mol/L.At the same time,the purification by IL-MAE showed a better effect than that of positive control.In the present study,the IL-MAE was chosen to extract the total flavonoids from Ribes odoratum Wendl.leaves.The developed procedures based on IL-MAE and macroporous resin enrichment processes provide an alternative and environmental method for the extraction and purification of natural products.