Separation And Purification Of Total Flavonoids From Milk Thistle Fruit Receptalcle And Their Bioactivity Evaluation

Milk thistle(Silybum marianum L.)is a year or biennial plant of compositae.The plant was introduced and planted in our country since 1970 s.At present,the research and utilization of milk thistle are mainly concentrated in the fruit part,the utilization value of plant is low.Flavonoids are widely found in plants and have a variety of physiological activities.Flavonoids in milk thistle fruit-silymarin has been widely used as a major component of hepatoprotective drugs.There are few studies on the milk thistle plant,which mainly concentrated in the roots,stems and leaves.No report is found on the part of fruit receptacle.In this reasearch,the active ingredients and alcohol extract antioxidant activity for each part of the milk thistle plant were studied firstly,then the extraction and purification of flavonoids from fruit receptacle of the milk thistle was researched and the main composition of flavonoids were identified.The antioxidant activity and hypoglycemic activity of flavonoids from fruit receptacle were discussed at last.Main conclusions are as follows:1,The content of active ingredients and alcohol extracts antioxidant activity of milk thistle plants(root,stem,leaf and fruit receptacle)were determined,the results showed that flavonoids content in fruit receptacle was most among four parts(9.98±0.58 mg/g),total flavonoids content order was fruit receptacle>root>leaf>stem.Polyphenol content in fruit receptacle was most among four parts(12.19±0.38 mg/g),polyphenol content order was fruit receptacle>root>leaf>stem.Content of polysaccharide in milk thistle root was most(8.02±0.15 mg/g),polysaccharide content order was root>fruit receptacle>leaf>stem.The content of proteins in each part of the milk thistle plant were little,the comparative content order was stem>root>leaf>fruit receptacle.the DPPH scavenging activity,hydroxyl and total antioxidant capacity test results of alcohol extracts from four parts showed that the order of antioxidant activity was fruit receptacle>root>leaf>stem.2,The optimal method was used to optimize the ultrasonic extraction conditions of the total flavonoids.The results showed that the optimum extraction conditions were as follows:the volume fraction of ethanol was 40%,the extraction time was 80 min,the ratio of liquid to material was 40:1,and the extraction temperature was 60?.Under these conditions,the extraction amount of total flavonoids from fruit receptacle was up to 10.18mg/g,and the order of the four factors on the extraction of the total flavonoids from the fruit receptacle was liquid to material>ethanol>Extraction temperature>extraction time.3,The total flavonoids from fruit receptacle were isolated and purified by macroporous resinand gel column chromatography.The structure of the isolated monomer compounds were characterized.It was found that AB-8 macroporous resin had better purification effect.The extraction process was as follows:The concentration of total flavonoids was 3.98mg/ml,the loading volume was 80 mL,pH5.5,ethanol volume fraction 70%,elution volume of 100 mL,adsorption temperature 50?.analytical temperature 60?.Under these conditions,the purity of flavonoids was 73.45 ± 0.26%.,the recovery was 84.04±1,83%.In column chromatography elution test,compound ?(45 mg)was obtained from ethyl acetate:acetone in a 3:1 eluent,and the compound ?(55 mg)was obtained from ethyl acetate:acetone in a 1:1 eluent,compound ?(80 mg)was obtained frome thyl acetat:acetone in a 1:2 eluent.The results of structural identification showed that compound ? has the formula C15H12O7,which is dihydroxapyrein;Molecular formula of compound ? is C25H22O10,which is silychristin;compound ?(molecular formula C25H22O10)is silybin.4,The antioxidant capacity of the total flavonoids from fruit receptacle was evaluated by the determination of DPPH activity,hydroxyl radical scavenging activity,total antioxidant capacity and antioxidant activity of oil.Then,Kunming mice were used as experimental animals,CAT,GSH-Px,T-SOD and MDAcontent in serum and liver were measured to evaluate the antioxidant capacity of total flavonoidsin vivo.Antioxidant test in vitro showed that DPPH removal rate,hydroxyl clearance rate and total antioxidant capacity of the order of the total flavonoids>ascorbic acid>fruit receptacle alcohol extract;lard and soybean oil antioxidant test results show that the total flavonoids fruit receptacle had good antioxidant effect on lard and soybean oil.The results of antioxidant testin vivo showed that the total flavonoids from fruit receptacle could increase the CAT activity and decrease the MDA content in the serum,liver and kidney tissues of the mice.The high dose could increase the T-SOD activity and GSH-PX activity in the serum,liver and kidney tissues of the mice.Therefore,the total flavonoids have good in vitro and in vivo antioxidant capacity.5,The blood glucose,blood lipid,serum MDA,SOD activity and glucose tolerance were measured by establishing type 2 diabetes mellitus model to study the hypoglycemic activity of total flavonoids from fruit receptacle.The results showed that total flavonoids from fruit receptacle had the following effects:It could decrease the blood glucose,TC and TG,TG can be restored to normal levels,the reduction of TC is a certain dose-related.It could increase the concentration of HDL-C anddecrease low density lipoprotein LDL-Cconcentration,HDL-C can be restored to normal levels.Total flavonoids from fruit receptacl could increase the serum SOD activity and decrease MDA content,it also could improve the oral glucose tolerance of rats.The results of liver tissue sections showed that the total flavonoids had a certain inhibitory effect on the liver tissue fattening of diabetic rats.