| Nowadays,with a prevalent tendency,diabetes following cardio-cerebrovascular disease and cancer has become another chronic one that threatens human health.The prevalence,mortality and disability rate of diabetes have been increasing significantly.So looking for new hypoglycemic functional factors in natural products has become a research hotspot.α-glucosidase inhibitor and α-amylase inhibitor are two new types of oral drugs,widely used in clinical treatment of diabetes.With the breakthrough point of digestion,absorption and metabolic pathway of sugar,they can be applied to treating different types of diabetes and reduce the complications of diabetes effectively.Therefore,developing natural hypoglycemic drugs from natural products with high security and effective components has a very broad space and prospect.With α-glucosidase activity potential as the guidance and α-glucosidase inhibitor in vitro screening model,this experiment used Luoping small ginger in Yunnan as the raw material to screen α-glucosidase inhibitor and determine the type of the enzyme inhibition.Hyperglycemia mice models were constructed by alloxan to research the effects of ginger extract with α-glucosidase inhibition on the postprandial blood glucose and general condition of hyperglycemic mice.Macroporous resin was used to further purify ginger extract with α-glucosidase inhibition so that the experiment basis could be provided on understanding the hypoglycemic effect of ginger.The main results were as follows:(1)The screening of in vitro screening models and ginger cultivarsThe optimum in vitro screening model was: the overall reaction volume 1.0 mL,successively adding enzyme fluid(10 mg/mL)0.2 mL,phosphate buffer solution(pH6.8)0.5 mL,ginger extract 0.2 mL under 37℃ water bath heating for 10 min,then maltose as substrates(0.5 mol/L)0.1 mL and 25 min later using Na2CO3 solution(1 mol/L)1.0 mL to terminate the reaction.By comparison with α-glucosidase inhibition of 34 kinds of ginger ethanol extract,the result showed that Luoping small ginger in Yunnan had the highest inhibitory activity,inhibition rate of which was 88.58% and its enzyme inhibition activity was positively correlated with its concentration(2)The study on optimum extraction conditions of α-glucosidase and α-amylase inhibitors from ginger and their enzyme kinetics researchThe influencing factors of extracting α-glucosidase inhibitor from Luoping small ginger by ethanol,from big to small,were the extraction time,the solid-liquid ratio and the ethanol volume fraction.And the optimum extraction process was: the solid-liquid ratio 1:3,the extraction time 1.0 h and the ethanol volume fraction 90%.Under these conditions,the inhibition rate was 91.77%;the influencing factors of extracting α-amylase inhibitor from Luoping small ginger by ethyl acetate,from big to small,were the solid-liquid ratio,the extraction time and the extraction temperature.And the optimum extraction process was: the solid-liquid ratio 1:3,the extraction time 2.0 h and the extraction temperature 40 ℃.Under these conditions,the inhibition rate was 69.71%.The α-glucosidase inhibition of ginger ethanol extract was noncompetitive and reversible,the Michaelis constant(Km)being 3.14×10-3 mol/L.In addition,the α-amylase inhibition of ginger ethyl acatate extract was irreversible,the Michaelis constant(Km)being-0.205×10-3 mol/L.(3)The verification on the treatment effects of ginger α-glucosidase inhibitor in miceThe α-glucosidase inhibitor extracted from ginger had significant hypoglycemic effect after the normal mice loaded with starch for 0.5 h.And compared with the blank group,the blood glucose value of mice decreased by 5 mmol/L,closing to that of acarbose with no obvious difference(P>0.05).But when the normal mice loaded with starch for 1.0-1.5 h,the hypoglycemic effect compared with that of acarbose had significant difference(P<0.01),suggesting that the persistent effect of α-glucosidase inhibitors in ginger on decreasing postprandial blood glucose values was worse than that of acarbose.The α-glucosidase inhibitor extracted from ginger had certain effect of normal mice on postprandial serum insulin level,better than that of acarbose.There were no significant effects of normal mice given α-glucosidase inhibitor by gavage on body weight,drinking amount and food intake.The α-glucosidase inhibitor extracted from ginger had very significant hypoglycemic effect after the hyperglycemia mice loaded with starch for 0.5 h.And compared with the blank group,the mice’ blood glucose value decreased by 6.41 mmol/L with statistical significance(p<0.01).But its hypoglycemic effect was worse than that of acarbose with significant difference(P<0.05).There was a certain therapeutic effect of hyperglycemia mice on symptoms of drinking more,eating more,urinating more and losing weight.(4)The verification on the prevention effects of ginger α-glucosidase inhibitor in hyperglycemia miceAfter the mice being given α-glucosidase inhibitor extracted from ginger by gavage for prevention,there was certain protective effect of hyperglycemia mice caused by alloxan.When the hyperglycemia mice loaded with starch were given α-glucosidase inhibitor for 0.5 h,the blood sugar value of them was significantly decreased by 5.57 mmol/L with statistical significance(p<0.05).Besides,after the mice being given α-glucosidase inhibitor extracted from ginger by gavage for prevention,the symptoms of hyperglycemia mice caused by alloxan could be relieved to some extent.(5)The study on process conditions of α-glucosidase inhibitor extracted from ginger by macroporous resinSelected from AB-8,S-8,AL-1-8,HPD-400 and D4020 macroporous resins,the AB-8 macroporous resin was the one with best adsorption and desorption performance on α-glucosidase inhibitor extracted from ginger.And the optimum purification process of α-glucosidase inhibitor in ginger was: the sample liquid pH value 6,adsorption time 3.0 h,the volume-mass ratio of ginger extract and resin 12.5:1,and 80% ethanol as elution with 4 times column volume.By AB-8 macroporous resin,the enzyme activity inhibition rate of α-glucosidase inhibitor in ginger was 93%,the purification effect of which was good. |
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