| Polysaccharide is a kind of widely existed biological macromolecule,which has many pharmacological activities such as anti-oxidation,anti-diabetes,immunomodulatory and anti-tumor.Lotus leaf,as a traditional Chinese medicine resource with the same origin of medicine and food,has a long history of application in health care and treatment.Polysaccharide is the main bioactive component of Lotus leaf,but there are few relevant studies.In this paper,polysaccharide extraction from lotus leaves was assisted by compound enzymes,and the extraction process was optimized by Box Behnken Design response surface design,providing theoretical basis for the development and utilization of lotus leaf resources.The main research contents are as follows:1.In this study,the optimal values of the five factors including pH,extraction temperature,compound enzyme addition amount,extraction time and liquid-solid ratio were determined by single factor experiment,and the Box-Behnken Design response surface was designed with five factors and three levels.The degree of influence of various factors on the yield of Lotus leaf polysaccharide was as follows: adding amount of complex enzyme > liquid-solid ratio > pH > extraction time > extraction temperature.The optimal extraction conditions were pH 7.0,extraction temperature 52℃,liquid-solid ratio 39:1 m L/g,addition of compound enzyme 0.7%,extraction time 116 min,and the yield of Lotus leaf polysaccharide was 3.26%.2.The optimum extraction of lotus leaf polysaccharide by Sevage method static protein and macroporous resin adsorption decolorizing,then through the DEAE-52 cellulose for separation and purification,collect the highest levels of component named LLP-1.The structure of LLP-1 was characterized,and the composition and molar ratio of LLP-1 monosaccharides were determined by ion chromatography.The structure of LLP-1 was characterized,and the monosaccharide composition and molar ratio of LLP-1 were determined by ion chromatography.The results showed that the monosaccharide composition of LLP-1 was rhamnotang,arabinose,glucosamine hydrochloride,galactose,glucose,xylose and mannose,and the molar ratio was0.49:1.7:0.08:4.23:2.48:0.57:0.44.The molecular weight was determined as 13.592 k Da by high performance gel permeation chromatography.Uv spectrum scanning showed that LLP-1 did not exist nucleic acid,protein.The results of IR spectra show that LLP-1has a pyranose ring structure and has two configurations,α and β.The Congo red experiment determined that LLP-1 had a triple-helical conformation.The microscopic morphology was also determined by SEM scanning electron microscopy.3.In this paper,the anti-inflammatory and anti-oxidative activities of Lotus leaf polysaccharide obtained by the best extraction process were studied in vitro.In vitro cell anti-inflammatory experiments showed that Lotus leaf polysaccharide could play an anti-inflammatory role by down-regulating the expression of inflammatory factors.In vitro free radical scavenging experiments showed that lotus leaf polysaccharide has strong scavenging ability for hydroxyl radical and superoxide anion,and can better scavengers DPPH radical and ABTS cation radical.In addition,it has certain iron reduction ability.In vitro cell antioxidant experiments showed that lotus leaf polysaccharide could increase the proliferation rate of cells with oxidative damage,and improve the antioxidant capacity of cells by activating Nrf2 signaling pathway and up-regulating the gene expression of antioxidant enzymes.All in all,this topic studies the new lotus leaf polysaccharide extraction technology and its optimization,preliminary characterization of the structure of the LLP-1,anti-inflammatory antioxidant activity of lotus leaf polysaccharide was studied and its mechanism,to provide theoretical basis for the development and utilization of lotus leaf resources. |
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