Study On The Extraction?purification And Separation Of Flavonoids From Hawthorn Leaves

Hawthorn leaves were used as traditional Chinese medicinal materials in long medicinal history,which have the functions of Anti-oxidation,anti-aging,antibacterial,anti-inflammatory,anticancer,fall blood sugar and blood pressure,prevent cardiovascular disease and so on.There are amounts of flavonoids and polysaccharide compounds in hawthorn leaves,which are the main pharmacological active ingredients of hawthorn lea ves.Extracting the active ingredients from the hawthorn leaves and development health functional foods can realize the high value utilization and increase the use value of the hawthorn leaves.Therefore,there are important social significance and practical significance of investigating extraction and purification of active ingredients from hawthorn leaves to develop new health products,cosmetics and increase farmers' income.The study chose the hawthorn leaves as the material in the experiment,the extraction and purification of ingredients in hawthorn leaves were investigated.The main research results were as follows:1.It can be determined that the optimal harvest time of hawthorn leaves was in early November according to the content of flavonoids,polysaccharide,protein,and water extract in different growing period of hawthorn leaves.2.Flavonoids were extracted by direct water extraction method.In the extraction process,the single factor experiment and orthogonal experiment were used to determine the optimal extraction conditions.The results indicated that the solid-liquid rate,extraction time,temperature,pH and extraction times was 1:35,60 min,60 ?,3.6,3,respectively.The extraction rate of flavonoids was 69.17±0.586 % under the optimal conditions.3.Microwave-ultrasonic assisted extraction technology was applied to extract flavonoids with water as solvent from hawthorn leaves.On the basis of single-factor test and PB test,the response surface methodology was used to determine the optimal conditions with the extraction rate of flavonoids as response value.The best extraction technology conditions were microwave time 63 s,microwave power 264 w,the ratio of solid to liquid 1:32 and ultrasonic power 540 w,the extraction rate of flavonoids was 90.68±0.53 %.4.The effects of aqueous two-phase system(ATPS)combined with ultrasonic technology on the extraction of flavonoids and polysaccharide from hawthorn leaves was investigated.Based on the yield of flavonoids and polysaccharide and partition coefficient(K),ATP of PEG11.5 % and(NH4)2SO414 % was selected from different ATPS as the optimum solvent.The optimal conditions was gained uising a Box-Behnken Design(BBD)of response surface methodology(RSM).The optimum conditions of extracting flavonoids and polysaccharide: the ratio of material to solvent 1:37,ultrasonic time 35 min,ultrasonic power 360 w and ultrasonic temperature 65 ? were the optimum conditions.The yield of flavonoids was 2.869 % and the yield of polysaccharide was 3.005 % under the optimal conditions.5.The D101 resin was selected as the suitable resin by comparing the effect of seven kinds of macroporous resin on the adsorption and desorption of flavonoids.The main purification parameters were determined by the static and dynamic adsorption and desorption experiment of D101 resin as follows: pH of sample was 5.0,injection speed was 1.0 mL/min,sample volume was 70 m L,ethanol concentration was 40 %,elution rates was 1.0 mL/min,the ethanol volume was 100 mL.The purity of flavonoids gained using D101 resin was 42.4 % and it increased 2.8 times than the without purify.It shows that D101 resin has a good effect on the enrichment and purification of flavonoids.6.Sephadex LH-20 column was used to further separate flavonoids in hawthorn leaves by D101 macroporous resin,the suitable purification condition as follows: the elution speed 0.5 mL/min,0 %,20 %,40 %,60 % and ethanol gradient elution.One compound and one single component(compound 3)were obtained from the part of water and 40 % ethanol,which the purity was 83.27 % and 94.27 %.The results of the hydroxyl free radical scavenging ability and reducing power of unpurified and purified flavonoid were as follows: purified flavonoids had better antioxidant activity than unpurified flavonoids.After the analysis of LC-MS,the compound 3 may be inferred as vitexin-2''-O-rhamnoside.