Structural Characterization And Regulatory Mechanism Of Glycolipid Metabolism Of Gracilaria Lemaneiformis Polysaccharide And Its Degradation Products

Gracilaria lemaneiformis is economical red algae whose main active ingredient is polysaccharide with hypoglycaemic,hypotensive,hypolipidemic,and anti-tumor effects,demonstrating high health and nutritional value.However,the high molecular weight,viscosity and complex structure of polysaccharide is not conducive to its further application.Gracilaria lemaneiformis polysaccharide was degraded by hydrogen peroxide combined with vitamin C（H₂O₂-Vc）.This research aimed to explore the effects of degradation on the structure and analyze the configuration and connectivity patterns of glyosidic bonds in the polysaccharide of Gracilaria lemaneiformis.In this study,enzyme inhibition rate in vitro,cell and animal experiments were combined to explore the activity and mechanism of its regulation of glycolipid metabolism.The main findings and results of this topic were as follows:1.Polysaccharide of Gracilaria lemaneiformis was extracted by hot water and alcohol precipitation,and degraded by H₂O₂-Vc（molar ratio 1:1）.According to single factor and response surface optimization experiments,the optimal degradation scheme was obtained as follows:H₂O₂-Vc concentration was 18.7 m M,reaction time was 0.5 h,and reaction temperature was 56℃.2.The degradation product of Gracilaria lemaneiformis polysaccharide（GLP-HV）was obtained by H₂O₂-Vc with the optimal scheme.The purpose of this study was to explore the changes of structural properties of GLP-HV under the controls of GLP（Gracilaria lemaneiformis polysaccharide without degradation）,GLP-H（Gracilaria lemaneiformis polysaccharide degraded by H₂O₂ alone）and GLP-V（Gracilaria lemaneiformis polysaccharide degraded by Vc alone）.The monosaccharides of these four polysaccharides were mainly glucose and galactose by HPLC.The average molecular weight of GLP,GLP-HV,GLP-H and GLP-V were 1478,16,1330 and 1001 k Da,respectively,detected by gel permeation chromatography.In addition,the average particle sizes of GLP,GLP-HV,GLP-H and GLP-V were 258.05±18.82,85.14±1.42,118.64±5.41 and 133.53±5.55μm,respectively,obtained through laser particle analyzer.The effects of H₂O₂-Vc on the color and viscosity of these polysaccharides were analyzed by chromatic difference meter and viscometer,and it was found that GLP-HV became brighter and less viscous.Moreover,the functional groups of these four polysaccharides were explored using a Fourier transform infrared spectrometer,which indicated that the degradation of H₂O₂-Vc did not change the functional groups of the polysaccharides.The microstructure of these four polysaccharides was analyzed by scanning electron microscope and atomic force microscope,and it was found that the structure of polysaccharides was broken and the particles became smaller after degradation.Furthermore,the configuration and connectivity patterns of glyosidic bonds of GLP-HV were explored by gas chromatograph-mass spectrometerheci and nuclear magnetic resonance spectrometer.The results illustrated that the main chain of GLP-HV mainly formed by→4)-α-D-Glcp-（1→、→3）-β-D-Galp-（1→and a small quantity of→4,6）-α-D-（2-O-Me）-Glcp-(1→,and the branched chain was connectedβ-D-Glcp-（1→and the O-4 from sugar residues→4,6）-α-D-（2-O-Me）-Glcp-(1→.3.The free radical scavenging ability in vitro of the four polysaccharides was GLP-HV>GLP-H,GLP-V>GLP,and the semi-inhibitory concentrations of GLP-HV on ABTS⁺,Fe³⁺,DPPH and superoxide free radicals were 9.62±0.35,4.97±0.18,23.85±1.78 and 3.56±0.0028 mg/m L,respectively.It was observed that GLP-HV significantly enhanced the activities of antioxidant enzymes,decreased the strength of active oxygen radicals and calcium ions,and alleviated the oxidative damage of cells（p<0.05）.RT-PCR experiment demonstrated that GLP-HV observably up-regulated Nrf-2,HO-1,NQO-1 and ZO-1 genes,and down-regulated Keap-1gene（p<0.05）.Correlation analysis revealed that GLP-HV ameliorated oxidative stress in cells through the Nrf-2 signaling pathway,thereby improving the glycolipid metabolism in cells.4.The semi-inhibitory concentrations of GLP-HV onα-glucosidase,pancreatic lipase and cholesterol esterase were 0.265±0.03,2.92±0.28 and 5.81±0.46 mg/m L,respectively.The kinetic experiments of enzymes explained that GLP-HV presented mixed competitive and non-competitive inhibition types onα-glucosidase and pancreatic lipase,and non-competitive inhibition type on cholesterol esterase.Moreover,the fluorescence spectra analysis showed that GLP-HV represented a dynamic quenching effect onα-glucosidase and cholesterol esterase fluorescence,and a static quenching effect on pancreatic lipase fluorescence.These results indicated that GLP-HV expressed an obvious effect on regulating glycolipid metabolism in vitro.5.The cells experiment exhibited that GLP-HV observably decreased the contents of TC,TG,LDL-C,FFA,LPL,LPO,leptin,MDA and LDH,and increased the contents of HDL-C,T-AOC,SOD,CAT,GSH-PX and glycogen in high glucose and high lipid cells（p<0.05）.In addition,GLP-HV significantly fortified the glucose consumption,production and absorption,reduced the contents of reactive oxygen species and calcium ions（p<0.05）,and then improved the glucose and lipid metabolism and oxidative damage of cells.RT-PCR experiment demonstrated that GLP-HV up-regulated genes levels of related factors in IR/IRS-2/PI3k/Akt/Glut4signaling pathway,dramatically up-regulated AMPK and HSL gene levels,and down-regulated SREBP-1c,ACC and FAS genes levels（p<0.05）.Western blotting results demonstrated that GLP-HV up-regulated or down-regulated the protein levels of related factors in the IR/IRS-2/PI3k/Akt/Glut4 and PPAR-γ/AMPK signaling pathways.Correlation analysis showed that the regulatory activity of Gracilaria lemaneiformis polysaccharides on glycolipid metabolism was positively correlated with reducing sugar,3,6-dehydrated galactose,sulfate group and carbonyl group,but negatively correlated with molecular weight and relative viscosity.Moreover,the regulatory activity of Gracilaria lemaneiformis polysaccharides on glycolipid metabolism in cells was highly correlated with IR/IRS-2/PI3K/Akt/Glut4 and PPAR-γ/AMPK signaling pathways,suggesting that Gracilaria lemaneiformis polysaccharides had a regulatory effect on glycolipid metabolism in cells.6.Animal experiment displayed that the body weight and fasting blood glucose of mice with glycolipid metabolism disorder were significantly decreased after 8weeks of GLP-HV feeding（p<0.05）.The serum contents of LPO,LPL,TBA,FFA,leptin,insulin and glycated serum protein were also memorably reduced,while the contents of adiponectin and LDH were significantly increased（p<0.05）.Moreover,GLP-HV markedly ameliorated glucose tolerance,insulin tolerance,lipid levels and oxidative stress of mice,reduced ALT,AST,BUN and Cr contents,and restored physiological functions of liver and kidney（p<0.05）.The liver was stained with HE,PAS and oil red O,respectively,and the results implied that GLP-HV had repair effect on the liver and improved glycogen and fat accumulation in mice.The results of RT-PCR and western blotting manifested that GLP-HV regulated glycolipid metabolism in mice through IR/IRS-2/PI3k/Akt/Glut4 and PPAR-γ/AMPK signaling pathways.The results of 16SrDNA high-throughput sequencing showed that a high-sugar and high-fat diet had an effect on the composition and structure of the gut microbiome in mice,suppressing the growth of Firmicutes,promoting the growth of Bacteroidetes and Proteobacteria,and altering the abundance and diversity of the gut microbiome.GLP-HV improved gut microbiome in mice with glycolipid metabolism disorders,with species composition and structure favoring the normal group.