| Objective:(1)The method suitable for the extraction of Brassica rapa L.was screened,and the technological parameters of the extraction method were optimized.(2)Spectroscopy and chromatography techniques were used to characterize the primary structure of Brassica rapa L.polysaccharide.in order to lay a foundation for the study of structure-activity relationship of Brassica rapa L.polysaccharide.(3)To evaluate the hypoglycemic activity and hepatoprotective effect of Brassica rapa L.polysaccharide,so as to provide reference basis for the development of hypoglycemic,hepatoprotective drug preparation or health food.Methods and results:(1)The hot water extraction method,ultrasonic assisted extraction method and complex enzyme method were used to extract Brassica rapa L.polysaccharide,and optimization of three extraction methods by response surface,and the effects of different extraction methods on the extraction rate of polysaccharides were compared.Results: the optimal extraction process of hot water extraction were as follows:liquid-material ratio 32:1 m L/g,extraction temperature 92℃,extraction time 2.3 h,the average extraction rate was 7.12% under these conditions.The optimal extraction process of ultrasonic assisted extraction were as follows:liquid-material ratio 33:1m L/g,extraction temperature 62℃,ultrasonic power 250 W,ultrasonic time 43 min,and the average extraction rate was 7.32% under these conditions.The optimum conditions of compound enzymatic hydrolysis were as follows: p H 5.0,temperature52 ℃,enzyme addition 2.6%,reaction 51 min,and the average extraction rate was7.82% under these conditions.The yield of the three extraction methods was from high to low: compound enzyme method > ultrasound-assisted extraction method > hot water extraction method.In addition,the combination of ultrasonic cavitation and enzymatic hydrolysis can greatly improve the extraction efficiency of polysaccharide.Therefore,the combination of ultrasonic and compound enzyme was used to extract Brassica rapa L.polysaccharide in the follow-up experiment.(2)Study on the technology of step-by-step extraction of Brassica rapa L.polysaccharide by ultrasound combined with compound enzymes.On the basis of previous research,taking the extraction rate of polysaccharides as the index,through single factor investigation and Box-Behnken analysis and design,the process of step-by-step extraction of Brassica rapa L.polysaccharide by ultrasound combined with compound enzyme was optimized.Results: the liquid to material ratio 33:1 m L/g,ultrasonic temperature 62 ℃,ultrasonic power 250 W,ultrasonic 43 min,adding2.5% compound enzyme,adjusting p H to 5.4,temperature 50 ℃,and enzymolysis reaction 52 min,and the average extraction rate was 12.62% under these conditions,which was not significantly different from the predicted value of 12.79%.(3)Isolation,purification and primary structure characterization of Brassica rapa L.polysaccharide.Brassica rapa L.polysaccharide were deproteinized by enzymatic method and sevage method,oligosaccharides and other small molecular impurities were dialyzed,and further purified by DEAE-52 cellulose column chromatography and Sephacryl S - 200 HR gel chromatography.the structure of Brassica rapa L.polysaccharide was characterized by UV,FT -IR,CG-MS and Congo red experiment.Results: The crude polysaccharide of Brassica rapa L was separated and purified by column chromatography to obtain the component BP-1-1.UV analysis showed that the polysaccharide component BP-1-1 contained no nucleic acid and protein,infrared scanning analysis showed that it contained α-glycosidic bond and uronic acid,and Congo red test showed that there might be no triple helix structure in BP-1-1.According to GC-MS analysis,BP-1-1 contains are composed of Rha,Ara,Man,Glu and Gal,the molar ratio of five monosaccharides obtained by area normalizationmethod was 1.00 : 0.76 : 1.69 : 2.11 : 2.07.(4)Study on hypoglycemic activity of Brassica rapa L.polysaccharide.To study the hypoglycemic activity of Brassica rapa L.polysaccharide on diabetic mice induced by high glucose and high fat combined with streptozotocin in vivo and the hypoglycemic effect of polysaccharides on α-glucosidase inhibition test in vitro.In vivo hypoglycemic activity examined the effects of polysaccharides on drinking water intake,body weight,blood sugar and sugar tolerance in mice,and the in vitro hypoglycemic activity were mainly measured by enzyme marker to investigate the inhibitory effect of polysaccharides on α-glucosidase.Results: Brassica rapa L.polysaccharide had a certain inhibitory effect on α-glucosidase,and the inhibitory effect was enhanced with the increase of polysaccharide concentration,and Brassica rapa L.polysaccharide could improve the mental state and hair color of type Ⅱdiabetic mice.relieving the symptoms of polydipsia,polyuria and polyuria,it can effectively inhibit the weight loss caused by the disorder of glucose metabolism,and significantly reduce the fasting blood glucose value of diseased mice and improve the oral glucose tolerance of diabetic mice.(5)Study on the protective effects of Brassica rapa L.polysaccharide on acute alcoholic liver injury in mice.By establishing the model of acute alcoholic liver injury in mice,the protective effect of Brassica rapa L.polysaccharide on acute alcoholic liver injury in mice was studied from the aspects of serum,biochemical indexes of liver function and pathological sections of liver tissue.Results: Brassica rapa L.polysaccharide could significantly reduce the levels of ALT,AST and TG in serum and the content of MDA and TG in liver and liver index in mice with acute alcoholic liver injury,and significantly increase the activity of SOD in liver tissue.Through the pathomorphological observation of liver tissue,it was found that all doses of polysaccharide could significantly reduce the injury of hepatocytes in mice.Conclusion:(1)the ultrasonic combined with compound enzyme step-by-step extraction method is suitable for the extraction of Brassica rapa L.polysaccharide,and the extraction rate of this method is high,so this extraction method is used in thefollow-up study.(2)The Brassica rapa L.polysaccharide component BP-1-1 contains no nucleic acid and protein,BP-1-1 contains no nucleic acid and protein,but contains α-glucoside bond and uronic acid,which may not have a triple helix structure,but is composed of five monosaccharides.(3)The Brassica rapa L.polysaccharide have hypoglycemic activity and can effectively relieve type Ⅱdiabetes.In addition,Brassica rapa L.polysaccharide have better hepatoprotective effect,its in the treatment of diabetes and liver therapy with the development and utilization prospects.. |
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