Extraction And Purification Process Of Total Flavonoids From Loquat Leaf And Antioxidant Research In Vitro.

Background:Loquat leaf is a dry leaf of loquat Eriobotrya japonica(Thunb.)lindl.of the genus loquat in the rosaceae family.It belongs to lung and stomach meridian,and has the functions of clearing lung and relieving cough,reducing anti-nausea and anti-vomiting.As a kind of natural medicine,it has very important social significance for the comprehensive utilization of loquat leaf resources.Therefore,on account of the principles of high yield,low cost and easy operation,the optimal extraction process and purification process of loquat leaf total flavonoids were optimized to prepare the higher purity loquat leaf total flavonoids,and to provides theoretical basis for industrial production of loquat leaf total flavonoids.Objective:(1)To optimize the extraction of total flavonoids from loquat leaf;(2)To optimize the purification of total flavonoids from loquat leaf;(3)To identificate the compound of total flavones in loquat leaf;(4)To explore the antioxidant activity of total flavonoids in loquat leaf.Methods:(1)Four factors influencing the extraction process,such as extraction solvent,extraction time and extraction times,were screened out by single factor test.Using box-behnken response surface analysis,the extraction of total flavonoids from loquat leaves was taken as the index,and the optimal extraction process was optimized.(2)Three factors affecting the purification process,including the concentration of ethanol,the volume of the upper sample,and the pH of the upper sample solution,were screened out by the single-factor test.The box-behnken response surface analysis method was used to evaluate the total flavonoids content of loquat leaves and optimize the best purification process.(3)The total flavonoids of loquat leaf were identified by color identification and LC-MS/MS.(4)The reduction capacity,hydroxyl radical scavenging capacity and DPPH scavenging capacity of total flavonoids in loquat leaves were investigated respectively by Prussian blue method,ortho-diazophil method and DPPH method.Results:(1)The ethanol concentration and the ethanol multiple had a significant impact on the extraction value.The parameters at the maximum value of the model: the ethanol concentration was 71.90%,the ethanol multiple was 10.58,the extraction time was 61.80 min,and the extraction times were 1.92.(2)The influence of alcohol solution elution on the content is very significant.The parameters when the maximum value of the model: the concentration of ethanol was 45.75%,the volume of the sample was 23.93,and the pH of the sample solution was 3.84.(3)Positive reactions were observed in the hydrochloric acid-magnesium powder test,aluminum trichloride reaction,ammonia water reaction and ultraviolet irradiation.The results of LC-MS/MS analysis showed that there were 12 compounds including 2 flavanols,6 flavonoid glycosides,3 flavonols and 1 polyhydroxyl astragalus.(4)DPPH free radical scavenging capacity ranged from strong to weak: loquat leaf total flavone > alcohol extract of total flavonoids from loquat leaf > ascorbate Vc.The reducing power was divided from strong to weak was ascorbic acid Vc > loquat leaf total flavone > loquat leaf total flavone alcohol extract.There was a good linear relationship between the removal of hydroxyl radical and sample concentration of total flavone of loquat leaf.Conclusion:(1)The modified optimal extraction process parameters: the ethanol concentration was set as 72%,the ethanol multiple was 11,the extraction time was 62 min,and the extraction was twice.(2)The modified optimal purification process parameters: the concentration of ethanol was set as 46%,the volume of the sample was set as 24 ml,and the pH of the sample was set as 3.8.(3)The total flavonoids of loquat leaf contain 12 flavonoids such as 2 flavanols,6 flavonoids glycosides,3 flavonols and 1 polyhydroxyl astragalus.(4)The total flavonoids of loquat leaf had better scavenging capacity of DPPH free radical and hydroxyl radical than that of ascorbic acid Vc.The total flavonoids of the purified loquat leaf were significantly improved in the removal of DPPH free radical,hydroxyl radical and reduction.