Study On The Acetylation Modification And Effects Of INS-1 Cell Activity Of Polysaccharide From Natural Portulaca Oleracea L.

Insulin is a hormone formed and secreted by pancreaticβ-cells,which is synthesized under the stimulation of endogenous or exogenous substances.Insulin has many functions,not only assisting in the production of fat and liver glycogen,but also lowering blood sugar levels in the body and promoting the formation of protein.Growth factors,nutrients and ion channels can affect the secretion mechanism of insulin.When the content of extracellular Glucose increases(Glucose transporters（GLUTs）will take glucose into the cell by means of transmembrane transport.The glucose in the cell will be accelerated by a series of reactions,such as fermentation decomposition,citric acid cycle,oxidative phosphorylation of ATP generation,etc.In this process,ATP(ATP-sensitive K⁺(K_ATP)channels)shut down,,causing membrane depolarization,ion channels open,and insulin release.Polysaccharides from Portulaca oleracea L.（POPs）are extracted from Portulace oleracea L.after isolation and purification.So far,it has been proved by many scholars that it has many functions,such as resistance to germs,improvement of body immunity,nerve protection,resistance to viruses,reduction of blood sugar level,antioxidant,anti-cancer activity and so on.Domestic and foreign research from purslane polysaccharide composition analysis,a variety of purification methods,structure research,biological antioxidant activity,the modification of various chemical physics method was reported,but the acetylated modification and their antioxidant activity and research on the influence of the biological function of islet beta cells,had never seen the related research.Therefore,after a large number of experiments,the research group obtained relatively pure Portulaca oleracea polysaccharide.The modification process of acetylated Portulaca oleracea polysaccharide was optimized by single factor experiment.MTT assay,ELISA,fluorescence imaging assay and Western blot were used to study the extracellular and extracellular antioxidant activities of Ac-POP and their effects on the biological function ofβcells.1、Polysaccharides from Portulaca oleracea powder were prepared by water extraction,alcohol precipitation,deproteinization,dialysis,percellulose column and other processes,which were acetylated and modified.The acetylated modification process of Portulaca oleracea polysaccharides was optimized by single factor experiment and response surface method.The results showed that the optimal modification process was as follows:reaction temperature of51.90℃,reaction time of 2.78 h,solid-liquid ratio of 1:28.15（g/m L）.At this time,the Degree of substitutin（DS）of Ac-POP can be substitutin（0.60）.Infrared spectra further confirmed the success of acetylation.2、Determination of extracellular antioxidant activity of Ac-POP and POP.The scavenging effects of Ac-POP and POP on DPPH free radical,·OH and O₂^-·were determined using vitamin C（Vc）as positive control.The results showed that both Ac-POP and POP had certain scavenging activity in the concentration range of 0.05-5.0 mg/m L.However,the scavenging ability of POP on DPPH free radical and·OH was significantly higher than that of Ac-POP in this concentration range.When the concentration of polysaccharide was greater than 1.0 mg/m L,the scavenging ability of Ac-POP on O₂^-·was significantly higher than that of POP.3、Antioxidant activity of Ac-POP and POP in INS-1 cells was determined.Using Vc as positive control,the oxidative damage of Ac-POP and POP on INS-1 cells induced by H₂O₂was studied.The results showed that the protective effect of Ac-POP on INS-1 cells was stronger than that of POP in certain concentration range.4、The survival rate of INS-1 cells was dose-dependent on Ac-POP concentration,which was promoted at low concentration and inhibited at high concentration.The effect of Ac-POP on insulin synthesis and secretion was studied by enzyme-linked immunosorbent assay.The results showed that Ac-POP promoted the formation and release of insulin.The effect of Ac-POP on the intracellular ATP content of INS-1 cells was detected by ATP kit.The results showed that Ac-POP could significantly increase the intracellular ATP synthesis.The effect of Ac-POP on mitochondrial membrane potential and cell membrane potential was studied by fluorescence imaging method.The results showed that Ac-POP significantly increased the mitochondrial membrane potential and caused the depolarization of cell membrane.5、Western blot was used to study the changes in the expression levels of insulin secretion-related proteins PDX-1 and GLUT-1 in cells treated with Ac-POP and POP.The results showed that both polysaccharides promoted the expression of PDX-1 protein and GLUT-1protein,and Ac-POP promoted the expression of PDX-1 protein more obviously.