Study On The Extraction,purification And Biological Activity Of Flavonoids From Mulberry Seeds

Mulberry is the fruit of Morus alba L.It is rich in nutrients,such as active protein,multi-vitamins,organic acids,free amino acids,minerals and trace elements.It also contains anthocyanins,active polysaccharides,flavonoids and other functional factors,and has a strong biological health function.Because the mulberry has the characteristics of fast maturity,short harvesting time,short shelf life of fresh fruits and perishable,in addition to a small amount for fresh food,some of them have been processed into mulberry juice,vinegar,fruit wine,jam,preserved fruit and other products.However,there is little research on mulberry seed,which is the by-product of mulberry processing.At present,it is only used for feed.It contains rich protein,oil,polyphenols,flavonoids and other components,which have high development and utilization value.It is also used as feed together with other skin residues,resulting in extreme waste of resources.In this paper,the extraction,separation and purification technology of flavonoids from mulberry residue was systematically studied.The anti-oxidation activity and antibacterial activity of flavonoids in vitro were analyzed.Furthermore,the effects of mulberry flavonoids on the growth inhibition of Hep G2 cells and the promotion of oxidative damage and apoptosis were studied.Its main research contents and results are as follows:(1)According to the single factor test and response surface analysis,the optimum extraction conditions of flavonoids by ultrasonic complex enzymatic method were as follows: enzyme dosage 0.32mg/m L,enzymatic hydrolysis temperature 55 ?,enzymatic hydrolysis time 75 minutes,and ultrasonic time 22 minutes.Under these conditions,the yield of flavonoids was 5.32 mg/g.(2)By comparing the adsorption and analytical effects of five macroporous resins on mulberry flavonoids,it was determined that HPD300 macroporous resin is the best purification resin.The optimal purification process were as follows: the concentration of the flavonoid extract was 0.5 mg/m L,the p H was5.0,the loading flow rate was 1.0 m L/min,and the eluent was 70 % ethanol,the elution rate was 1.0m L/min.When the elution volume was 90 m L,the desorption rate was the largest.After purification by HPD300 macroporous resin,the flavonoid content and total antioxidant capacity were 2.28 and 2.80 times higher then before purification,respectively.(3)The antioxidant activity of flavonoids from mulberry seeds showed that flavonoids had strongantioxidant activity.When the concentration was 1.50 mg/m L,the scavenging rate of flavonoids to DPPH free radicals was 89.58 %.When the sample concentration was 1.20 mg/m L,the inhibition rate of flavonoids on hydroxyl radicals was 88.75 %,and the activity of flavonoids against superoxide anion could reach 258.88 U/L,which is close to the clearance rate of free radicals by vitamin C at the same concentration.rate.Oxford cup method was used to test its bacteriostasis effect.It was found that the order of inhibition ability was Salmonella > Escherichia coli > Staphylococcus aureus > yeast.(4)MTT assay was used to detect the growth inhibition of mulberry seed flavonoids on Hep G2 cells.The results showed that mulberry seed flavonoids could inhibit the growth of Hep G2 cells in a certain dose and time effect.When the concentration of flavonoids was 500 ?g/m L,the inhibition rates were 57.05 %,68.39 % and 71.54 % at 24 h,48 h and 72 h respectively.(5)Analysis of the cell cycle by flow cytometry revealed that mulberry flavonoids can block cells in the G0/G1 phase,effectively reducing protein production,inhibiting cell division,and reducing new cell production.According to the analysis of apoptotic cells,early apoptotic cells increased in a dose-dependent manner.When the concentration of flavonoids was lower(c?200 ?g/m L),there was little difference between early apoptotic cells and late apoptotic cells.When the concentration was higher,the proportion of early apoptotic cells was much larger than that of late apoptotic cells.The total apoptosis rate of cells increased significantly.The results of LDH,SOD and MDA showed that the content of LDH and MDA in Hep G2 cells increased significantly after treatment with mulberry flavonoids,while the level of SOD decreased obviously due to the decrease of ROS.Therefore,mulberry flavonoids can significantly inhibit the growth of Hep G2 cells,resulting in oxidative damage of cells.