Study On Extraction,Purification And Biological Activities Of Main Flavonoids From Ribes Mandshuricum(Maxim.) Kom.Leaves

In this paper,a green and efficient deep eutectic solvents-based microwave-assisted extraction(DES-MAE)technique was developed to extract the seven major active flavonoids rutin,hyperoside,isoquercetin,trifolin,astragalin,quercetin and kaempferol from Ribes mandshuricum leaves.The extraction process conditions of DES-MAE were optimized.After the completion of the extraction process,macroporous adsorption resin was used for purification seven flavonoids.Then,the antioxidative and anti-inflammatory activities of extracts were evaluated.The main studies and results were as follows:1.A HPLC method was developed for the simultaneous analysis and detection of Rutin,hyperoside,isoquercetin,trifolin,astragalin,quercetin and kaempferol in Ribes mandshuricum leaves,the conditions were as follows:Chromatographic column:Phenomenex Curosil-PFP reverse-phase column(250× 4.6 mm i.d.,5 ?m);Mobile phase:phosphoric acid/water(0.5:99.5,v/v)as solvent A and acetonitrile as B;flow rate:0.8 mL/min;column temperature:40 ?;detection wavelength:360 nm;injection volume:20 ?L.Gradient conditions were as follows:0-7 min,10-15%(B);7-10 min,15-17%(B);10-25 min,17-17%(B);25-27 min,17-18%(B);27-31 min,18-19%(B);31-34 min,19-19%(B);34-38 min,19-26%(B);38-42 min,26-26%(B);42-55 min,26-50%(B);55-60 min,50-10%(B).2.Box-Behnken Design(BBD)method combined with response surface methodology(RSM)was applied to acquire the optimal operating parameters of DES-MAE.The optimal conditions were as follows:Extraction solvent:choline chloride/lactic acid M ratio 1:2 with water content 25%Extraction temperature:54 ?Extraction time:10 minLiquid/solid ratio:27 mL/g.Under the optimal parameters,the yields of rutin,hyperoside,isoquercetin,trifolin,astragalin,quercetin and kaempferol were 1.26 mg/g?0.33 mg/g?2.59 mg/g?4.81 mg/g?1.16 mg/g?0.35 mg/g and 0.10 mg/g DW,respectively.The deep eutectic solvent-based microwave-assisted extraction process is better than deep eutectic solvent-based heat refluxing extraction and deep eutectic solvent-based ultrasound-assisted extraction.3.Macroporous resins for enrichment and separation of rutin,hyperoside,isoquercetin,trifolin,astragalin,quercetin and kaempferol from DES extraction solution:In this work,the direct enrichment of seven main flavonoids from DES solution were performed using macroporous resin D101,NKA-9,AB-8,HPD-400,HP20,HPD-826,X-5 and LSA-10.It was found that among the eight kinds of macroporous resin,X-5 resin was the best in enrichment and separation of rutin,hyperoside,isoquercetin,trifolin,astragalin,quercetin and kaempferol with recoveries of 79.64%,88.08%,81.37%,87.13%,97.36%and 99.39%,respectively.4.The antioxidative and anti-inflammatory activities of extracts wereevaluated:The antioxidant activities of Ribes mandshuricum leaves flavonoids were evaluated by DPPH,ABTS and FRAP assays.We revealed that the Ribes mandshuricum leaves flavonoids had the antioxidant ability.Then,it was discovered that Ribes mandshuricum leaves flavonoids could effectively inhibit the NO production and increase the IL-10 cytokines production in LPS-induced RAW 264.7 cells.It suggested that the Ribes mandshuricum leaves flavonoids had the potential anti-inflammatory effects.To sum up,this study completed that the optimization of DES-MAE of seven main flavonoids from Ribes mandshuricum leaves.The direct enrichment and separation of seven main flavonoids from extracts were performed,and the antioxidative and anti-inflammatory activities of extracts were evaluated.This study provides a theoretical basis and scientific basis for the comprehensive development and efficient utilization of Ribes mandshuricum leaves.