Extraction Of Mulberry Leaf Flavonoids By Ultrasound-assisted Enzymatic Method And Its Antioxidant Activity

Mulberry[Morus alba L.]is a perennial woody plant that is distributed worldwide.Mulberry Leaves is the dried leaf of the mulberry tree of the family Moraceae.As a traditional Chinese medicinal herb with the same origin,it has the effect of dispersing wind-heat,clearing the liver and brightening the eyes.Mulberry leaves are rich in flavonoids,alkaloids,polysaccharides and other active substances,which have significant antioxidant,anti-aging and other biological activities,and are widely used in health food and cosmetics industry.At present,most of the mulberry leaves in China are not fully utilized except for silkworm feed,resulting in environmental problems and great waste of resources.In recent years,the extraction of flavonoids from mulberry leaves as the main raw material for pharmaceutical manufacturing has become a hot spot,but there are still unsolved problems in terms of condition optimization,extraction rate and purity,etc.The effective utilization of mulberry leaf resources and the development of their edible and medicinal values will promote the economic maximization of mulberry resources.In this study,we used ultrasonic-assisted enzymatic extraction of Mulberry Leaf Flavonoids（MLF）and determined the optimal extraction process conditions to investigate the antioxidant activity of MLF through the extraction and purification of Mulberry leaf flavonoids.The specific studies were as follows.1.On the basis of single factor,the optimal extraction process conditions for the extraction of mulberry leaf flavonoids by ultrasonic-assisted enzymatic method were obtained by response surface optimization test:ethanol concentration of 70%,feed-to-liquid ratio of 1:50 g/m L,enzymatic digestion temperature of 60℃,enzyme content of 5%,enzyme ratio（cellulose:pectinase）of 1:3 g/g,enzymatic digestion time of 45min,ultrasonic time of 15 min,and ultrasonic power of 420 W.2.The crude extract of mulberry leaves was purified by using macroporous resin D101,and finally the MLF contained 74.74%of total flavonoids,which was 7.4 times higher than that before purification.The MLF was subjected to compositional analysis,and the UV absorption spectrum and IR absorption spectrum indicated the presence of flavonoids in the extract.The MLF was analyzed by liquid chromatography mass spectrometer and its main components were obtained as:hesperidin,rutin and chrysin,etc.3.By examining the scavenging ability of MLF on DPPH·radical,-OH·radical,ABTS·radical and O₂^-·radical and its protective effect on oxidative damage of sheep erythrocytes,the results showed that MLF had the best effect on the scavenging rate of DPPH·radical and ABTS·radical,and the IC₅₀values of the four free were 0.0452mg/m L for DPPH,0.5242 mg/m L for-OH,3.1768 mg/m L for O₂^-·and 0.0319 mg/m L for ABTS,-OH was 0.5242 mg/m L,O₂^-·was 3.1768 mg/m L,and ABTS was 0.0319mg/m L.The results of AAPH-induced oxidative damage model in sheep erythrocytes showed that MLF was able to reduce cellular MDA levels and restore enzymatic activity stress to near normal levels,thus protecting the basic cell membrane structure.4.Using different concentrations of MLF to treat Hep G2 cells,we found that flavonoids have the ability to significantly reduce the level of ROS in Hep G2 cells,reduce the stimulation of mitochondria by H₂O₂,significantly reduce the rate of apoptosis,reduce the expression of apoptosis-related proteins,MLF can significantly reduce the abnormal leakage of LDH and ALT caused by H₂O₂,regulate the activity of antioxidant enzymes,thus reducing the MDA content to protect cells from damage.In conclusion,MLF showed good antioxidant activity and pharmacological activity by scavenging a variety of free radicals to maintain the integrity of cells.The results of this thesis can provide some theoretical basis for the comprehensive and effective utilization of mulberry leaf resources,especially for the in-depth development of the medicinal value of flavonoids.