Isolation And Purification Of Mulberry Hypoglycemic Active Substance DNJ And The Study About Its Pharmacological Activity

Objects:1. To establish the methods of the determination of alkaoid and to optimize the extraction process of mulberry alkaloids based on single factor experiment and Response Surface Methodology with DNJ content index;2. To optimize the best separation effect cation exchange resin and to determine the best separation and purification scheme including the column flow rate, eluent concentration and eluent flow rate3. To research vitro inhibitory activity using mulberry alkaloid for amylase, invertase and maltase, and to optimize reation conditions and mulberry alkaloid’s α-glucosidase inhibition activity.Methods:1. Determine the contents of alkaloids of mulberry through HPLC-ELSD method and to study the four factors (concentration of ethanol, pH value, the solid-liquid ratio and extraction time) which influence on the extraction rate and the best separation conditions using the single factor experiment and response surface analysis method;2. Base on the previous section of HPLC-ELSD detect conditions using target alkaloid content, to filter out the best resin type and examine the column flow rate, eluent concentration and eluent flow rates and other factors which is the best separation and purification of alkaloids;3. The vitro α-glucosidase reaction system using three α-glucosidase enzymes, amylase, sucrase and maltase kits, respectively to test mulberry alkaloids inhibition rate of enzyme activity experiments and using Lineweaver-Burk double reciprocal diagram method to do further the best type of enzyme reaction system and determine inhibition kinetics. Results:1. Using HPLC-ELSD method with DIKMA Platisil NH2(4.6mm X250mm,5μm), the mobile phase of acetonitrile-water (90:10),at a flow rate of1.0ml·min-1, ELSD drift tube temperature of40℃, the carrier gas flow rate of3.5X105Pa and other conditions as established the determination method of alkaloids; methodological study showed that0.356~2.848μg· mL-1within the range of the mass concentration and the peak area is linear, while precision RSD=0.54%, stability RSD=0.98%, reproducibility RSD=1.46%, recoveries RSD=2.75%and other indicators have shown this method not only high precision, excellent stability and reproducibility, but also shows feasible; Using HPLC-ELSD method is used to determine the content of mulberry leaf alkaloids, by single factor and response surface analysis experiment and determine the optimum process parameters of mulberry leaf alkaloid extraction which result is66.53%ethanol concentration, pH value of6.662.73h leaching time; under this condition, mulberry leaf alkaloids actual amount extraction was0.1967%, compared with extraction amount in other papers research, previous literature has improved significantly;2. By the process of investigation, select001×7cation exchange resin as the types of resin separation of alkaloids; on its process conditions, the optimum separation process is:the sample liquid on alkaloids concentration of1.75mg·mL-1, the concentration of ammonia in ethanol at a concentration of eluant (80:20), flow rate1.ml·min-1eluant effect best, after001×7type cation exchange resin treatment of mulberry alkaloids can reach more than29%;3. Activity test in vitro shows that mulberry DNJ inhibitory effect of three kinds of α-glycosidase enzymes, its only have inhibitory effect on sucrase, and with the increase of concentration of DNJ inhibition rate is positively related to increasing; optimum reaction system in experiment show that enzyme dosage20uL, reaction time11min and the substrate concentration55mg·mL-1which is the best reaction conditions; in addition, Lineweaver-Burk double bottom figure methods shows DNJ extracts of sucrose enzyme inhibition is noncompetitive inhibition reaction type.Conclusion:1. HPLC-ELSD determination method of alkaloids in mulberry has high repetition rate and is stable and feasible; 2. Quadratic mathematical model of leaching mulberry alkaloids with dilute alcohol and acid medium, which explains that DNJ extraction process using Box-Behnken model and response surface methodology is accurate and reliable;3.001×7cation exchange resin can be used for separation and purification of mulberry leaf alkaloids, and is high yield and good effect;4. Mulberry alkaloid DNJ on invertase activity is significantly inhibited and the type of noncompetitive inhibition.