Extraction, Purification, Structural Characterization And Biological Activity Of Polysaccharides From Lindernia Crustacea (L.)F.Muell

Lindernia crustacea（L.）F.Muell is a genus of plants in the genus Lindernia crustaceae.In China,it is mainly distributed in the south,and foreign countries are mainly widely distributed in the tropics and subtropics.Modern pharmacological studies have shown that Lindernia crustacea have anti-inflammatory,analgesic,anticancer,antiviral,antioxidant and hypoglycemic biological activities.In this project,Lindernia crustacea from Yanshan District,Guilin City,Guangxi Province was selected as the material,extracted Lindernia crustacea polysaccharides,isolated,purified and characterized their structure,and carried out in vitro antioxidant and hypoglycemic activity research,to lay a foundation for the in-depth development and application of Lindernia crustacea.The main research contents are as follows:1.Extraction of Lindernia crustacea polysaccharidesIn this study,the polysaccharide yield of Lindernia crustacea was extracted by hot water extraction method and ultrasonic assisted extraction method,and the polysaccharide yield of the two methods was compared by a combination of univariate test and orthogonal test,and the best extraction process was selected.The total sugar content of polysaccharide samples was determined by the anthrone sulfuric acid method,and the reducing sugar content was determined by DNS method,and the difference between the two was recorded as polysaccharide content.The experimental results show that the best process for extracting Lindernia crustacea polysaccharides in hot water is as follows:feed-liquid ratio of 1:20,extraction time of 2.5h,extraction temperature of 75°C,extraction twice of extraction The polysaccharide yield was 13.42±0.33%,the total sugar content was 24.77±1.54%,and the reducing sugar content was 7.17±0.12%;The optimal process of ultrasonic assisted extraction method is:ultrasonic time 50 min,material-liquid ratio 1:25,ultrasonic power 400 W After 2extractions,the polysaccharide yield was 17.32±0.14%,the total sugar content was32.53±1.35%,and the reducing sugar content was 4.54±0.03%.Ultrasound-assisted extraction is more suitable for the extraction of Lindernia crustacea polysaccharides.2.Separation and purification of Lindernia crustacea polysaccharides（1）The CLPS was reconstituted with ultrapure water,and the alcohol precipitation was graded with absolute ethanol to obtain LPS-1（40%alcohol precipitation part）,LPS-2（60%alcohol precipitation part）and LPS-3（80%alcohol precipitation part）three polysaccharide purification components,the yields of which were 54.37±1.01%,30.12±0.63%and 29.54±0.37%,respectively.The total sugar,reducing sugar and protein content of the three components were determined,and the total sugar content of LPS-3 was59.76±0.24%,the content of reducing sugar was 4.28±0.03%,and the protein content was7.17±0.14%.The total sugar content of LPS-2 was 56.27±0.55%,the reducing sugar content was 4.26±0.04%,and the protein content was 7.39±0.22%.The total sugar content of LPS-1 was 45.58±0.83%,the reducing sugar content was 4.76±0.02%,and the protein content was 8.22±0.03%.（2）With protein removal rate,decolorization rate and polysaccharide retention rate as indicators,LPS-3 was studied on protein removal and decolorization treatment.The results showed that the protein removal effect of TCA method was better than that of Sevag method,when the TCA content reached 6%,the removal rate of protein was 75.44%,and the retention rate of polysaccharides was 64.54%;The decolorization effect of macroporous adsorption resin AB-8 was better than that of D101,and the decolorization rate of AB-8 was 80.34%,and the polysaccharide retention rate was 80.34%.74.53%。（3）Purification of LPS-3 with DEAE-52 and Sephadex G-100 to obtain Lindernia crustacea purified polysaccharide LPS-A.3.Structural characterization of Lindernia crustacea polysaccharides（1）The monosaccharide composition and molar ratio of LPS-A were determined by ion chromatography,and the monosaccharide composition and molar ratio of LPS-A were rhamnose:arabinose:glucosamine hydrochloride:galactose:glucose:xylose:mannose:glucuronic acid=0.92:0.64:0.84:2.69:2.81:0.37:1.20:0.53。（2）The relative molecular mass of LPS-A determined by HPGPC was 27049 Da,Mw/Mn=1.408,the ratio was close to 1,indicating that the molecular mass distribution of LPS-A was relatively concentrated,and the analysis of the results of dextran gel column chromatography showed that the purity of LPS-A was high.（3）Through ultraviolet spectrum analysis,it was found that LPS-A had a strong absorption peak near the wavelength of 200 nm,which conformed to the ultraviolet absorption characteristics of polysaccharides,260 and 280 nm There is no obvious absorption,indicating that the purification effect of polysaccharides is good,and there are no or very few impurities such as proteins and nucleic acids.（4）The results of infrared spectroscopy showed that the purified polysaccharide LPS-A had a wide band in the region of 3000-3750 cm^-1 and a strong absorption peak at 3247cm^-1,indicating that the polysaccharide hydroxyl group was used O-H telescopic vibration;The weak absorption peak at 2920 cm^-1 was caused by the C-H telescopic vibration of the polysaccharide alkyl,which also indicates that LPS-A is a polysaccharide.（5）The polysaccharide morphology was magnified by scanning electron microscopy,and LPS-A showed that its surface was a honeycomb at 500,1000,2000 times,and the surface of the connected part was smooth.After further magnification to 3000,5000,8000,the visible hollow part is of various shapes,round or irregular strips.（6）The thermogravimetric analysis results show that the thermal stability of L PS-A is relatively good below 267°C,and a certain degree of heat treatment will not cause thermal decomposition of polysaccharides.4.Exploration of Lindernia crustacea polysaccharide activityThe results of in vitro antioxidant activity experiments showed that the antioxidant activity of Lindernia crustacea polysaccharide samples showed a dose effect,and with the increase of polysaccharide purity,the antioxidant activity of the samples also increased,but they were weaker than the positive control VC at the same concentration.The results of in vitro hypoglycemic activity of Lindernia crustacea polysaccharides showed that Lindernia crustacea polysaccharides had inhibitory effects on bothα-amylase andα-glucosidase,and showed a significant dose-response relationship,and the inhibitory activity of Lindernia crustacea polysaccharides on both enzymes was lower than that of the positive control acarbose under the same concentration conditions.