| Artemisia sphaerocephala Krasch seeds powder is a traditional food additive which is added in noodles to improve its elasticity and chewing quality.So far,many researchers focused on investigation of the emulsifying and physicochemical properties of A.sphaerocephala Krasch polysaccharide(ASKP)which is the main active ingredient of A.sphaerocephala Krasch seeds.However,there are a few studies regarding the biological activity of ASKP as well as its potential application in functional food delivery system.In this dissertation,we have evaluated the liver protective and immunomodulatory activity of ASKP using high-fructose induced hepatic steatosis mice and macrophages,respectively.Besides,the evaluation of delivery efficiency of Ly/ASKP-3 nanocomplexes for curcumin was investigated.The main research contents and results are as follows:1.Polysaccharide ASKP was obtained from A.sphaerocephala Krasch seeds by means of water boiling and ethanol sedimentation.ASKP was identified as the acidic heteropolysaccharides by HPLC which was mainly composed of D-glucose(26.7%),D-xylose(19.4%),D-galactose(17.2%),D-galacturonic acid(13.6%),D-mannose(9.4%),L-arabinose(8.0%)and D-glucuronic acid(5.8%).2.The present study was to investigate whether ASKP alleviated insulin resistance and hepatic steatosis in mice fed HF water.Mice fed with 20%fructose in drinking water for 8 weeks significantly displayed hyperglycemia,insulin resistance,liver oxidative stress and steatosis.Administration of ASKP at 400 and 800 mg/kg·bw significantly reduced the fasting serum glucose,insulin concentrations,and the index of homeostasis model assessment of basal insulin resistance(HOMA-IR)of the mice fed 20%HF water.In the oral glucose tolerance test,the administration of ASKP at 400 and 800 mg/kg bw had a reduced plasma glucose concentration after 15 min of glucose loading in HF-fed mice,indicating that ASKP improved glucose intolerance.Besides,ASKP could inhibit hepatic steatosis and oxidative damage induced by HF by decreasing the hepatic TC,TG,NEFA and MDA levels,and increasing the hepatic GSH-Px and SOD activities in HF-fed mice.Liver H&E and Oil red O staining results were consistent with the biochemical results suggesting that ASKP could ameliorate the high fructose-induced mice liver pathology damage.3.20%high fructose water could cause the intestinal barrier disruption in mice,leading to transferation of endotoxin(LPS)into the blood circulation which might induce inflammation reflected by the elevation of serum TNF-a.IL-1 and IL-6 levels.At the same time,HF could also cause the significant increase of serum uric acid in mice.However,administration of ASKP effectively reduced the serum endotoxin,uric acid,TNF-α、IL-1 and IL-6 levels in mice fed 20%HF water,suggesting that ASKP could alleviate the endotoxemia and inflammation induced by HF.The results of metabonomic profiling analysis based on GC-MS showed that HF-induced metabolic disorders of serum fatty acid C14:1 and C18:1t and hepatic fatty acid C16:1 and C18:1 were effectively attenuated by intervention of ASKP.4.High-throughput sequencing based on 16SrDNA revealed that 20%HF water could cause an abnormal increase in proportion of Firmicute at phylum level,and an increasing proportion of Oscillibacter as well as a decreasing proportion of Akkermansia at genus level.The increase in the ratio of Firmicute-to-Bacteroidetes and the decrease in Akkermansia level were closely related to HF-induced insulin resistance and hepatic steatosis.Interestingly,ASKP intervention reduced the relative abundance of Firmicute and Oscillibacter,and increased the relative abundance of Akkermansia.All these results indicated that ASKP could reverse the HF-induced alteration of intestinal microflora in mice,and adjust the composition of intestinal flora of the mice fed 20%HF water similar to that of the normal mice.5.Cellulose DEAE-52 and Sephadex G-150 were adopted to purify ASKP,and the main elution peak ASKP-1 was obtained.The structure characterization of ASKP-1 was identified by UV,FT-IR,HPLC,ESEM and methylation.High performance size exclusion chromatography(HPSEC)results showed that the molecular weight of ASKP-1 was 9.08×105Da.ASKP-1 had no absorptions at 260 nm and 280 nm in the J UV spectrum,indicating the absence of protein and nucleic acid.FT-IR spectra showed that ASKP-1 had the characteristic absorption peak of carbohydrate.The monosaccharide compositions of ASKP-1 were determined by a validated HPLC-UV technique,and the results showed that ASKP-1 was composed of mannose,glucose and galactose in the mole percentages of 14.1%,59.6%and 19.6%.Meanwhile,methylation analysis showed that the main components of ASKP-1 were 1→4)-Glcp(39.8%),1→6)-Galp(18.8%),1→3,6)-Manp(19.6%),1→)-Glcp(10.8%),2→6)-Manp(4.0%)and 2→3,5)-Araf(7.0%).6.The immunoregulatory activity of ASKP-1 was evaluated using mouse peritoneal macrophages RAW264.7.The results showed that ASKP-1 markedly induced the release of cytotoxic molecules(NO and ROS)and secretion of the cytokines(TNF-a,IL-6 and INF-β)and significantly enhanced the phagocytosis of macrophages.Furthermore,TLR4 was found to be a recognized target of ASKP-1 and its related mitogen-activated protein(MAPK)and phosphoinositide 3-kinase(PI3K)/Akt,including phosphorylated ERK,JNK,p38 and Akt,were rapidly activated by ASKP-1 in RAW264.7 macrophages.Moreover,ASKP-1 was found to cause the degradation of NF-κB subunit p65 inhibitor IκB-α,resulting in the transfer of nuclear translocation factor NF-κB subunit p65 into the nucleus.All these findings suggest that TLR4-mediated MAPK,PI3K/Akt and NF-κB pathways are involved in ASKP-1-induced macrophage activation,and ASKP-1 is a potential immunomodulating function food.7.The acidic heteropolysaccharide ASKP-3,which was negatively charged,was purified from ASKP.HPLC analysis showed that ASKP-3 consisted of 1.2%mannose,15.7%glucuronic acid,10.3%glucose,62.4%xylose,4.8%galactose and 5.6%arabinose with the average molecular weight of 8.41×105 Da.A facile approach to fabricate nanocomplex in moderate condition via self-assembly of positive charged lysozyme(Ly)and negative charged ASKP-3 was investigated for the first time to encapsulate and protect curcumin(Cur),and the potential application of ASKP-3 in Cur delivery system was discussed.The interaction between Ly and ASKP-3,size and morphology were studied by zeta potential,transmission electron microscopy(TEM)and X-ray photoelectron spectroscopy(XPS).The results indicated that Ly/ASKP-3 nanocomplex exhibited sphere with diameters 216.6 ±5.7 nm and narrow particle size distribution.The encapsulation efficiency(EE)and loading capacity(LC)of Cur for the Ly/ASKP-3 nanocomplex prepared with Ly/ASKP-3 ratio 1:1 were 79.52 ± 4.14%and 15.47 ± 2.85%,respectively.Cur-loaded Ly/ASKP-3 nanocomplex dramatically increased Cur stability in phosphate buffer at physiological pH.The MTT assay indicated that encapsulated Cur exhibited higher anticancer activity than free Cur.Meanwhile,Cur-loaded nanocomplex could be effectively endocytosed by HepG2 cells,resulting in enhanced cancer-cell apoptosis when compared to free curcumin.Furthermore,the curcumin-loaded nanocomplex significantly increased serum Cur in animal models,and antagonised the Cur’s low oral bioavailability and poor absorption.The worthwhile endeavor elucidated that proteins/polysaccharides nanocomplex was feasible to solubilize and protect sensitive amphiphilic bioactive compounds and possessed extensive potential in food application with various purposes. |
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