Study On Extraction,Separation, Enrichment And Purification Of Anthocyanins From Blue Honeysuckle

Blue honeysuckle(Lonicera caerulea L.)is a perennial deciduous shrub belonging to Lonicera Linn,genus of Caprifoliaceae family.It is riched in anthocyanins and other components,which possess antioxidant,anti-inflammatory,anti-tumor and other activities.At present,domestic and foreign researches on anthocyanins monomer preparation from blue honeysuckle are still in the laboratory groping stage.There is little literature reports of fast separation and purification of anthocyanins monomer components from blue honeysuckle.In the present study,the new and green deep eutectic solvent-based negative pressure cavitation extraction(DES-NPCE)was used for the rapid and efficient extraction of anthocyanins from blue honeysuckle.The extraction process conditions of DES-NPCE were optimized.The process of separation and purification of anthocyanins from blue honeysuckle using macroporous resin followed by flash chromatography were developed.The optimum parameters of extraction,separation and purification of anthocyanins were established,which provides scientific basis for comprehensive development and efficient utilization of blue honeysuckle resource.The results were as follows:1.The process of deep eutectic solvent-based negative pressure cavitation extraction was optimized by a Box-Behnken Design(BBD)and response surface methodology(RSM).The optimum parameters were as follows:Deep eutectic solvents(DESs):ChCl/lactic acid(1/2 mole ratio,20%water content)Extraction time:30 min Extraction pressure:-0.08 MPa Liquid/solid ratio:16 mL/g Extraction temperature:53 ℃Based on the above optimized conditions,the average extraction yield of anthocyanins was 6.601 mg/g(n=3),which was 1.44 and 1.25 folds to those of 70%ethanol-based negative pressure cavitation-assisted extraction and deep eutectic solvents-based ultrasound-assisted extraction.2.The process conditions for enrichment and separation of anthocyanins in blue honeysuckle from DESs by macroporous resins was studied.The optimum parameters were as follows:Resin type:D101 resin Sample concentration:anthocyanins 0.501 mg/mL Processing volume:4 BV Adsorption flow rate:1 BV/h Desorption solution and volume:3 BV distilled water and 6 BV 30%ethanol Desorption flow rate:3 BV/hAfter one treated by D101 resin under the above optimum conditions,the purity and recovery yield of anthocyanins reached 24.8%and 81.3%,respectively.The antioxidant activity of the extracts enriched by macroporous resin was assessed and compared.The results showed that the DPPH assay with IC50 value of 0.189 mg/mL,ABTS assay with IC50 value of 0.055 mg/mL,and reducing power test with IC50 value of 0.045 mg/mL,respectively.3.Separation and purification of anthocyanins main monomer cyanidin-3-glucoside by flash chromatography was studied.The operating parameters of flash chromatography were optimized.Stationary phase type:ODS-C18 reverse phase silica gel Gradient elution:5%formic acid aqueous solution(A)-methanol(B):0-60 min,20%(B)Sample/silica gel ratio:1:100(g/g)Elution flow rate:30 mL/minAfter one flash chromatography run under the optimal parameters,the purity and recovery yield of cyanidin-3-glucoside reached 96.6%and 90.3%,respectively.The antioxidant activity of cyanidin-3-glucoside purified by flash chromatography was assessed and compared.The results showed that the DPPH assay with IC50 value of 0.158 mg/mL,ABTS assay with IC50 value of 0.040 mg/mL,and reducing power test with IC50 value of 0.035 mg/mL,respectively.In this study,it was completed that the optimization of deep eutectic solvent-based negative pressure cavitation extraction of anthocyanins from blue honeysuckle.The enrichment and separation of anthocyanins from DESs using macroporous resin was performed and the purification of anthocyanins monomer by flash chromatography was studied.The yield of anthocyanins and cyanidin-3-glucoside reached 4.95 mg/g and 3.93 mg/g,the purity of anthocyanins and cyanidin-3-glucoside reached 24.8%and 96.6%,respectively.At the same time,the antioxidant activities of the extracts enriched by macroporous resin and cyanidin-3-glucoside were evaluated with excellent results.This study provides scientific basis for reasonable development and utilization of blue honeysuckle resource.Meanwhile,it provides important technical support for green and efficient extraction,separation and purification of other natural products.