Synthesis,Characterization And Biological Activity Of Selenium Nanoparticles Decorated With Polysaccharides Of Gracilaria Lemaneiformis

Selenium（Se）is one of the trace elements needed by the body.Selenium nanoparticles（SeNPs）has become the most promising selenium source in the area of medicine and food science because of its high efficiency and low toxicity properties.However,SeNPs is usually very unstable and tend to easily aggregate into gray or black biologically inert selenium due to its high surface energy.In order to prevent SeNPs aggregation and improve its stability and biological activity,in this paper,SeNPs decorated with polysaccharides of Gracilaria lemaneiformis（GLPs-SeNPs）was prepared with Gracilaria lemaneiformis polysaccharides（GLPs）as modifier.The size,morphology,structure and other characteristics of GLPs-SeNPs were analyzed by a series of characterization methods,its stability was investigated,and the antioxidant and hypoglycemic activities,simulated digestion and safety in vitro,and anti-inflammatory activities of GLPs-SeNPs were also studied,in order to lay a research foundation for the further development and application of GLPs-SeNPs.The main conclusions are as follows:（1）According to the results of single factor test,the ultrasonic temperature of 65℃,ultrasonic time of 20 min,ultrasonic power of 360 W and liquid-material ratio of 120 m L/g were selected as central point experimental factors for response surface analysis,and the optimal extraction conditions of GLPs were optimized as follows:ultrasonic temperature of 70℃,ultrasonic time of 30 min,ultrasonic power of 300 W and liquid-material ratio of 100 m L/g.Under these conditions,the extraction rate of GLPs was 36.24±0.16%.The polysaccharides extracted under the optimized conditions were deproteinized,dialyzed and lyophilized to obtain GLPs.The total sugar content in GLPs was 63.07%,the moisture and ash content were 15.9%and14.6%respectively,and the protein content was only 0.03%.Infrared spectrum analysis showed that GLPs had the characteristic absorption peak of polysaccharides.The average molecular weight（Mw）of GLPs determined by gel permeation chromatography（GPC）was 382.3 k Da.The results of ion chromatography showed that GLPs was composed of fucose,galactose,glucose,xylose,ribose and glucuronic acid,and the molar percentages were0.53,92.85,2.44,2.43,1.02 and 0.73%,respectively.（2）By discussing the effect of GLPs concentration on the particle size of SeNPs,GLPs with a concentration of 2 mg/m L was selected to synthesize GLPs-SeNPs,the average particle size of the prepared GLPs-SeNPs was 92.5nm.SEM and TEM results showed that GLPs-SeNPs were monodisperse and homogeneous spherical nano-particles.UV-Vis and FT-IR spectra analysis showed that GLPs and SeNPs were combined by physical adsorption.The results of EDX analysis showed that Seaccounting for 50.32%of the component elements（C,O,Se）in GLPs-SeNPs.XRD and XPS analysis showed that Sein GLPs-SeNPs was an amorphous and zero-valent elemental selenium.The stability test results showed that GLPs-SeNPs was more stable when stored at 4℃,after stored at 4℃for 42 days,GLPs-SeNPs was still clear and transparent without aggregation and precipitation.GLPs-SeNPs was relatively stable in a high ionic strength and a wide p H range,and also has good thermal stability.（3）The antioxidant and hypoglycemic activities of GLPs-SeNPs in vitro were studied by free radical scavenging assay and enzyme inhibition assay.The results showed that GLPs-SeNPs had stronger scavenging ability to DPPH,ABTS and superoxide anion radical than GLPs,SeNPs and Na₂SeO₃.Its scavenging ability was positively correlated with the concentration in a certain range,and the IC₅₀ was 0.418 mg/m L,0.585 mg/m L and 0.699 mg/m L,respectively.However,SeNPs showed low free radical scavenging activity,and GLPs as a modifier significantly improved the free radical scavenging ability of SeNPs.Compared with GLPs,SeNPs and Na₂SeO₃,GLPs-SeNPs had stronger inhibitory effects onα-amylase andα-glucosidase.Within the range of experimental concentration,the inhibitory effect increased with the increase of concentration,IC₅₀ were 1.55 mg/m L and 2.139 mg/m L,respectively.（4）The results of simulated digestion in vitro showed that in the simulated gastrointestinal digestion stage,due to the low p H in gastric juice,the nanoparticles were unstable and aggregated,and the particle size increased significantly after gastric digestion.However,the GLPs-SeNPs system structure remains basically intact,which shows that GLPs as a modifier can protect the delivery of SeNPs in the gastrointestinal tract,thus improving the bioavailability.The results of cytotoxicity test and blood compatibility test showed that the cytotoxicity and hemolysis rate of GLPs-SeNPs at the same Seconcentration were much lower than that of Na₂SeO₃,indicating that GLPs-SeNPs had good biocompatibility.The median lethal dose(LD₅₀)of GLPs-SeNPs to KM mice（SPF grade）was 137.98 mg Se/kg（95%confidence interval:111.39-205.76 mg Se/kg）calculated by bliss method,indicating that GLPs-SeNPs had less toxicity and had the potential to be developed into a safe and effective oral selenium dietary supplement.（5）In the LPS-induced cell inflammation model,GLPs-SeNPs can inhibit the secretion of IL-1β,IL-6 and TNF-αin LPS-induced RAW264.7 cells.The results of q PCR further showed that each concentration of GLPs-SeNPs could significantly reduce LPS-induced IL-6 m RNA expression in RAW264.7 cells in a concentration-dependent manner.In the experiment of xylene induced ear swelling in mice,the high-dose GLPs-SeNPs group significantly reduced the phenomenon of ear swelling,and alleviated local cell inflammation to a greater extent than the blank group.All experimental groups can effectively reduce the ear swelling degree caused by xylene and the contents of IL-1β,IL-6 and TNF-αin serum of mice.The inhibition rate of swelling increased with the increase of dosage,and the inhibition rate was 25.42%,36.85%and 50.28%respectively.In the experiment of acetic acid-induced peritoneal capillary permeability in mice,with the increase of the dose of GLPs-SeNPs,the increase of acetic acid-induced peritoneal capillary permeability in mice was significantly inhibited,with the inhibition rates reaching 9.03%,28.20%and46.96%,respectively,showing anti-inflammatory effect.