Extraction And Separation Of Phenolic Components From Hawthorn By Column Chromatography

Hawthorn is an important medicinal and edible plant resource.Studies by many scholars at home and abroad have shown that it contains a variety of physiologically active ingredients and thus has a variety of pharmacological effects.In the previous research,our laboratory found that the main phenolic active ingredients in hawthorn fruit are flavanols,including epicatechin and its polymer oligomeric proanthocyanidins;followed by phenolic acid,especially the one contained Eucomic acid(EA)with the characteristic aroma of hawthorn.There have been some reports on the extraction and separation of hawthorn proanthocyanidins,but in view of the complexity of hawthorn fruit components,from the perspective of the comprehensive utilization of its biological resources,column chromatography separation method is still the key to the extraction and separation of hawthorn phenolic components.Based on the previous research,this paper optimizes the separation and purification process of hawthorn proanthocyanidins macroporous adsorption resin;compares the preparation of EA by three column chromatography methods;evaluates the inhibitory activity of EA on tyrosinase,the main results as follows:1.Compare the adsorption-desorption performance of two macroporous adsorption resins HPD826 and D101 to proanthocyanidins in hawthorn fruit through static experiments.The adsorption capacity,resolution and adsorption rate constant of HPD826 type are better than those of D101 type.The adsorption and separation procedure of hawthorn proanthocyanidins was optimized by dynamic tests with HPD826 resin:the loading concentration was 25 mg/mL,the loading flow rate was 1.5 mL/min,and the desorption procedure was:washing with 1.5 BV;45%ethanol desorption 1.5 BV;90%1 BV was eluted by ethanol activation;2 BV was finally washed with water,and the elution flow rate was 1.5 mL/min.With this procedure,the recovery rate of proanthocyanidins in the desorbed part of 45%ethanol was 81.48±3.4%.The HPLC determination showed that the prepared extracts were mainly epicatechin and proanthocyanidin B2,and the total content of 10 proanthocyanidins reached dry weight 42%.2.Extraction and purification of EA by means of column chromatography using three different types of packing materials,the establishment of EA separation and purification process conditions.Using hawthorn as raw material,the process of separation and purification of EA by dextran gel LH-20 was developed,and the influence of various process single factors on the separation and purification of EA was investigated.The best separation conditions obtained through the experiment are:sample flow rate 0.25 mL/min,sample volume 2.5 BV,eluent water,elution flow rate 0.25 mL/min,elution volume 4 BV.With this procedure,the EA recovery rate of the hydrolyzed and adsorbed part is 88.26 ± 2.9%,and the column chromatography method is used to separate high-efficiency and high purity.Preliminary proof can be used as a technical means to purify EA.The adsorption effect of 10 kinds of macroporous adsorption resins was compared,and D101 macroporous adsorption resin was selected to investigate the influence of multiple single factors on the separation and purification effect of EA.The best separation conditions were:sample flow rate 1 mL/min,sample volume 12.5 BV,eluent 40%ethanol solution,elution flow rate 1 mL/min,elution volume 5 BV.With this procedure,the EA recovery of the desorption portion of the 40%ethanol solution was 71.48±1.8%.Preliminary proof that the macroporous adsorption resin can be used as a preliminary separation method.The process of separation and purification of EA by D301 ion exchange resin was studied,and the effects of various process factors on the separation and purification of EA were investigated.The separation conditions were determined as follows:sample flow rate 1 mL/min,sample volume 4 BV,eluent 2 mol sodium chloride solution,elution flow rate 1 mL/min,elution volume 10 BV.With this procedure,the EA recovery of 2 mol of sodium chloride desorption fraction was 30.11±2.2%.The results show that the effect of ion exchange resin separation of EA needs to be improved.3.The in vitro test method was used for the first time to evaluate the inhibitory activity of tyrosine oxidase on purified EA.Its IC50 value is 0.165 ± 0.78 mmol.It provides theoretical support for the development of new whitening cosmetic additives.