Study On The Extraction, Separation Of Flavonoids In Lotus Leaf And Its Enzymatic Activity Of Glucokinase

The leaf of Nelumbo nucifera Gaertn is widespread in the world.Lotus leaf is a kind of natural plant material used both in food and medicine field,involving many active materials.It has recently been found to display significant antiobesit, anti-hyperlipidemia,antioxidative,free radical scavenging activities,anti-aging, bacteriostasis,antiviral and hypoglycemic action.The flavonoid is one of the main active materials.In this paper,the extraction,separation of flavonoids in lotus leaf and its enzymatic activity of glucokinase have been studied profoundly,which will provide theoretic foundation for exploitation of lotus leaf.The main results were as follow:1.With the extraction ratio of flavonoids as studying index,based on the signal factor experiment,the optimum parameters and scopes are ensured by the method of Plackett-Burman design and response surface methodology.The results showed that, the ratio of solvent to material,ethanol concentration and extraction times were the main factors to affect the extraction rate of flavonoids.The optimum parameters are as follow:the ethanol concentration was 67%,the ratio of solvent to material was 34:1,the times of extraction was 3,and the ultrasonic extraction time was 40min.The total flavoniods extraction ratio was 3.17%at the optimum conditions.2.Polyamide resin was chosen by experiment of static adsorption-desorption of four kinds of resin HPD-100,HPD-100 A,HPD-400 A and polyamide,and the factor of affecting the rate of adsorption and desorption of polyamide resin are researched, such as ratio of diameter to height,effluent concentration,volume of eluting water,elution concentration and volume of eluting agents.The optimum absorption conditions were that the ratio of diameter to height was 1:6,the effluent concentration was 10.668 mg·mL^-1;the optimum desorption conditions were that the eluting solvent was 70%ethanol,and its volume was 5 BV.3.Fraction A,B,C and D were separated from water and ethanol extracts of lotus leaf by column chromatography.Fraction D was identified of quercetin by the thin layer chromatography.The method reproducibility expressed as relative standard deviation(RSD) was 7.9%for quercetin.The method recovery was 88.6%.Quercetin was qualitative and quantitative determination of 0.17 mg·g^-1 in lotus leave by HPLC-UV.4.The quercetin of lotus leaf was purified with polyamide solid-phase extraction (SPE) by HPLC-UV.The volume of eluting solvent was 14 BV.The method reproducibility expressed as relative standard deviation(RSD) was 9.08%,and the average recovery of quercetin was 94.99%for quercetin by SPE.5.The microextraction in a packed capillary(MEPC) was used to separate and purify flavonoids in lotus leaf as a new method for sample preparation.The volume of eluting solvent was 14 BV.The method reproducibility expressed as relative standard deviation(RSD) was 4.61%,and the average recovery of quercetin was 89.55%for quercetin by MEPC6.The enzymatic activity of glucokinase was studied in vitro models with the extract and fraction A,B,C,D from lotus leaf.The results showed that only fraction C increased the enzymatical activity of glucokinase,while the extract of lotus leaf and other fractions had decreased the enzymatical activity of glucokinase.EC₅₀ of fraction C was 0.0803 mg·mL^-1.