The Structure And Immune Activity Of Lotus Root Dregs Polysaccharide And The Preparation Of Polysaccharide Nano-selenium

Lotus root is an aquatic vegetable with a variety of biological functions.The polysaccharide is one of its biologically active ingredients,which has the functions of weight loss,anti-oxidation,immune regulation,blood fat reduction and blood sugar lowering.Se is an indispensable trace element in the human body.Some of the normal physiological functions of the body are inseparable from Se,but its intake is not well controlled.It is easy to cause poisoning due to excessive intake.Nano-selenium is less toxic and has better biological activity.Nano-selenium prepared by using polysaccharide as a template can combine the biological activities of polysaccharide and nano-selenium,and is the development direction of the future food and pharmaceutical industry.In this paper,lotus root slag was prepared by water extraction and alcohol precipitation method,and then to determined the polysaccharide extraction rate,polysaccharide content,sulfate content,uronic acid content and protein content.DEAE Sepharose Fast Flow and Sephadex G-100 was used to separate and purify the polysaccharides from lotus root slag,and analyzed its molecular weight and monosaccharide composition.The binding mode of lotus root slag polysaccharide was investigate by Fourier transform infrared spectroscopy(FT-IR),NMR wave methylation and GC-MS.Stimulate RAW264.7 cells to establish an in vitro inflammation model,study the effects of lotus root slag polysaccharide on macrophage MAPKs and PI3K-AKT signaling pathway,and detect TNF-a,iNOS and IL-6 mRNA in cell fluid by semi-quantitative RT-PCR.Akt,p-Akt,I?B?,p-I?B?,p-ERK,ERK,p-JNK,JNK,p-p38,p38 and intranuclear c-fox and c-Jun levels were detected by Western Blot method.Finally,the isolated and purified lotus root slag polysaccharide was used as a template,and the reduction reaction of sodium selenite and ascorbic acid in aqueous solution was carried out for 4 h at room temperature to prepare nano-selenium of lotus root slag polysaccharide.The molar ratio of different lotus root slag polysaccharides to sodium selenite was explored,and the optimal molar ratio of lotus root slag polysaccharide to sodium selenite was determined by UV full-wavelength scanning method and particle size measurement.The scanning electron microscope,transmission electron microscope,X-raydiffraction,FT-IR and particle size analyzer were used to characterized the nano-selenium of lotus root slagThe main findings are as follows:(1)The yield of polysaccharides prepared from lotus root slag by water extraction and alcohol precipitation method is 7.76±0.79%,the polysaccharide content is 69.19%±0.24%,the uronic acid content is 1.48±0.20%,and the sulfate content is 5.8±0.31%.The protein content was 28.33±1.18 mg/mL.It was found that LRP was acidic polysaccharide by measuring its potential and FT-IR.The molecular weight of LRP was 1.24 kDa by gel permeation chromatography.The monosaccharide component is mainly glucose by GC-MS;FT-IR showed that the lotus root slag polysaccharide had a characteristic absorption peak in the range of 4000?1000 cm-1;the NMR data further supported the LRP linkage through the a-glycosidic bond.(2)The lotus root slag polysaccharide(LRP)was extracted from lotus root slag by water extraction method.In order to research the effects of lotus root polysaccharide on MAPKs and PI3K-Akt signaling pathways in macrophages,we have to stimulating RAW264.7 cells with LRP and established the model of in vitro inflammation.The semi-quantitative RT-PCR was used to detected the expression levels of TNF-a,iNOS and IL-6 mRNA in cell liquid.The levels of Akt,p-Akt,I?B?,p-I?B?,p-ERK,ERK,p-JNK,JNK,p-p38,p38 and intranuclear c-fox and c-Jun were detected by Western Blot.The results showed that LRP was not toxic and could up-regulate the expression level of related inflammatory gene mRNA and increase the phosphorylation level of p38 and I?B?.The morphological changes of control and LRP-treated RAW 264.7 cells were observed by scanning electron microscopy,indicating that LRP can activate RAW.264.7 cells.(3)LRP-SeNPs were prepared by the reduction reaction of ascorbic acid and sodium selenite with lotus root slag polysaccharide as template.The effect of the molar ratio of lotus root slag polysaccharide and sodium selenite solution on the particle size of nano-selenium of lotus root slag polysaccharide was investigated.The ICP was used to determined content of selenium in LRP-SeNPs.The dual-wavelength colorimetry,particle size analyzer,SEM,TEM and XRD were used to characterized the LRP-SeNPs.The experimental results show that the molar ratio of lotus root slag polysaccharide to sodium selenite solution is 30:1(LRP concentration is 6 mg/mL),and the reaction at room temperature for 4 h can obtain stable properties with a particle size of about 40 nm.Left and right lotus root slag polysaccharide nano-selenium particles.It can be seen from ICP that the selenium content of the lotus seed slag polysaccharide nano-selenium is 19.18 mg/L.It can be seen from SEM and TEM that the LRP-SeNPs are relatively uniform and dispersed,and the solution after standing at room temperature for one month is still transparent and clear,and has no coagulation and no precipitation.