Study On The Extraction And Purification Of Four Main Compositions Of Anthocyanins From Ribes Nigrum L.

In the present study,the new and green extraction method deep eutectic solvent-based microwave-assisted extraction were used for the efficient and rapid extraction of delphinidin 3-O-glucoside,delphinidin 3-O-rutinoside,cyanidin 3-O-glucoside,cyanidin 3-O-rutinoside from Ribes nigrum L.,and macroporous resins combining with flash chromatographic were applied for enrichment and purification of blackcurrant anthocyanins.The extraction process conditions of deep eutectic solvent-based microwave-assisted extraction were optimized.An efficient,simple and fast method was for the analysis and detection of anthocyanins.Our study provided scientific guidance for reasonable development and efficient utilization of blackcurrant resources.The results were as follows:1.Using quick and easy pH-differential method for determination the amount of anthocyanins from blackcurrant.An efficient HPLC method for simultaneous determination of delphinidin 3-O-glucoside,delphinidin 3-O-rutinoside,cyanidin 3-O-glucoside,cyanidin 3-O-rutinoside from Ribes nigrum L was developed as follow:Chromatographic column:Luna C₁₈（5μm,250 mm×4.6 mm i.d.）,flow rate 1.0m L/min,column temperature 30 ^oC,injection volume 5μL,detection wavelength 520 nm.Mobile phase consisted of 10%formic acid aqueous solution（A）and methanol-acetonitrile（15:85,B）,gradient elution was performed as follows:0-4 min,15-20%（B）;4-6 min,20-22%（B）;6-14 min,22-24%（B）;14-24 min,24-26%（B）;24-35 min,30-35%（B）.2.The process of microwave-assisted deep eutectic solvent 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（mol/mol,20%water content）Extraction temperature:45 ℃Liquid/solid ratio:13:1 m L/gExtraction time:14 minUnder the optimal parameters,the average extraction yields of anthocyanins were 2.03mg/g（n=3）.The deep eutectic solvent-based microwave-assisted extraction process is better than deep eutectic solvent-based ultrasound-assisted extraction and 80%ethanol microwave-assisted extraction.3.A highly efficient,high-yield and high-purity purification process for anthocyanins was developed by flash chromatography combined with macroporous resin.（1）The process parameters for enrichment of anthocyanins by macroporous resin were optimized:Resin type:D101 resinProcessing volume:6 BVDesorption solution and volume:3 BV deionized water and 5 BV 40%ethanol（2）The optimum process parameters for separation and purification of the D101 enriched product including delphinidin 3-O-glucoside,delphinidin 3-O-rutinoside,cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside by flash chromatography were optimized:Stationary phase types:ODS-C₁₈ reverse phase silica gelGradient elution:10%formic acid aqueous solution（A）-methanol-acetonitrile（15:85,B）,isocratic elutionSample/ODS-C₁₈ silica gel:1:150（g/g）Flow rate:25 m L/minAfter purified by one run ODS-C₁₈ silica gel flash chromatography and macroporous resins under the above optimum conditions and recrystallization,the yields of delphinidin 3-O-glucoside,delphinidin 3-O-rutinoside,cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside reached 22.31 mg,35.47 mg,11.83 mg and 31.36 mg with the purities of more than 95%,respectively.The final yields were 0.32 mg/g,0.47 mg/g,0.18 mg/g and 0.43 mg/g,respectively.The developed procedure boasts simple and easy to use,production of high-purity and allows an easy scale-up.This study provides a scientific basis for the comprehensive development and utilization of Ribes nigrum L.Meanwhile,it also provides important data and theoretical basis for large-scale production of anthocyanins from Ribes nigrum L.