Pressurized Solvent Extraction And Separation Of Monomer Ingredients Red Clover Isolfavones Research

Red clover (Trifolium Pratense.L) for the legume trifolium perennial forage,contains richisoflavones,including relatively high content of formononetin and biochanin. Red cloverisoflavones have many medical functions: anti-inflammatory,anti-cancer,anti-tumor, anti-oxidation,and regulation of human hormone balance, improvement of osteoporosis, alleviate menopausalsyndrome, prevention and treatment of hypercholesterolemia, scavenging free radicals and preventUV, great value for the food and pharmaceutical industry is of great significance in the field ofnatural products. Red clover, the raw material of the test, through the extraction, purification andseparation methods ultimately gets formononetin and biochanin a monomer substances, and finallyresearch antioxidant scavenging free radicals of isoflavones.Use pressurized solvent method and critical water to take the red clover isoflavones, and twomethods are compared and analyzed. Red clover isoflavones, pressurized solvent extraction bysingle factor and orthogonal experiment to determine the optimum conditions: extractiontemperature120°C, extraction time15min, extraction pressure1.4MPa, liquid to solid ratio50ml/g, the final yield is33.61mg g-1. Subcritical water extraction of red clover isoflavones, throughsingle factor and orthogonal experiment to determine the optimum conditions: extractiontemperature120°C, extraction time15min, extraction pressure1.4MPa, liquid to solid ratio50ml/g, the final yield was33.61mg g-1. This paper first adopts two methods for extracting totalisoflavones in red clover, the results showed that the extraction rate is higher than the sub-criticalwater pressurized solvent method. Purification of total isoflavones in red clover by macroporousresin method, first through the analysis and comparison of static adsorption test of5kinds ofmacroporous adsorption resin, the results to determine the best comprehensive effect of AB-8onthe adsorption of isoflavones from red clover, selected70%ethanol as eluent; followed by AB-8macroporous adsorption resin for dynamic test, to get the best purification conditions for: choosethe sample concentration is0.2173mg/mL, volume of the sample3BV, sample flow rate is1mL/min, elution volume is2BV, elution flow rate is2mL/min, under the above conditionsisoflavone recovery rate is96.61%, macroporous resin purification of flavonoids extract purifiedsample purity is5.03%,after purification the purity reaches to65.58%.Again, after the initial purification and separation of purified Red Clover Isoflavones byhigh-speed counter-current chromatography, get formononetin and biochanin xanthein of highpurity. Ultimately determine the optimal solvent system: n-hexane: chloroform: methanol: water =1.5:3:3:2;HSCCC separation operation parameters: host temperature30℃, the engine speed900r/min, the flow phase rate2mL/min, the concentration of sample5.26mg/mL. Separate and purifyred clover isoflavones in the above conditions, we get content of formononetin92.87%andbiochanina83.48%Finally utilizing detection and purification of high performance liquid chromatography getformononetin and biochanina, and drawing the standard curve, calculating its content, liquid phaseconditions: methanol: water (0.2%=60:40phosphate).Antioxidant activity of formononetin and biochanin a: scavenging superoxide free radical,scavenging hydroxyl free radical, scavenging DPPH ability and reducing energy; results show that:the antioxidant capacity of formononetin and biochanin increases with the increaseof concentration; under the same concentration, the antioxidative ability of biochanin a is higherthan that of formononetin.