| Rosae Laevigatae Michx.,a perennial shrub belonging to the Rosaceae family,is a kind of Chinese dietetic herb with medicinal functions including anti-oxidation,anti-apoptosis,anti-inflammatory,inhibiting arteriosclerosis,and protecting the liver.R.Laevigatae polysaccharides(RLP)is the major effective component of R.Laevigatae.Nevertheless,the biological activity of polysaccharides is closely related to their chemical structure,molecular weight,and monosaccharide compositions.High molecular weight polysaccharides(over 10k Da)are limited in the applications due to their high apparent viscosity,poor water-solubility,and complex structures and conformations.Low molecular weight polysaccharides have been reported to own higher activity and have greater significance for human health.The separation of low molecular weight polysaccharides has become a hot topic in recent years.At present,few systematical reports on the separation,purification,bioactivity,and molecular mechanism of low molecular weight polysaccharides from RLPs have been documented.In this study,the R.Laevigatae fruits were used as materials,the extraction,purification,structure identification,and antioxidant activity of RLPs were studied systemically.Besides,through the experimental technologies including histology,transcriptomics,metagenomics,physiology and biochemistry,and molecular biology in high-fat diet(HFD)rats,this paper discussed whether the low molecular weight polysaccharides of R.Laevigatae fruits could regulate lipid metabolism and obesity development,and further analyzed the correlation between the potential molecular mechanism and intestinal bacteria changes.The main contents and results of this paper are as follows:(1)Extraction,separation,and purification of polysaccharides from R.Laevigatae fruitsCentral composite design(CCD)was applied to optimize the hot-water extraction of RLP.The optimal conditions were extraction temperature 93~oC,extraction time 2.8 h,water to raw material ratio 22,and extraction frequency 3.The RLP yield was 9.61±0.08%,in accordance with the predicted value of 9.56%.It was proved that the method of CCD was accurate and effective for the optimization of hot water extraction conditions of RLP.Moreover,five RLP fractions including RLP-1a,RLP-2a,RLP-3a,RLP-4a,and RLP-5a were obtained.Among them,the molecular weights of RLP-1a,RLP-2a,and RLP-3a were9004,8761,and 7571 Da,respectively.The above three polysaccharides were homogeneous.RLP-4 consisted of two components with the molecular weights of 87679 Da(92.52%)and3696 Da(7.48%),and RLP-5 consisted of two components with the molecular of 59664 Da(13.75%)and 4463 Da(86.25%).(2)Structure analysis and antioxidant activity of RLPThree fractions of low molecular weights polysaccharides(<10 k Da)from R.Laevigatae fruits,i.e.RLP-1a,RLP-2a,and RLP-3a,were further analyzed in structure.UV spectra showed that RLP-1a,RLP-2a,and RLP-3a did not contain the impurities such as proteins,polypeptides,and nucleic acids.Infrared spectra indicated that the characteristic absorption peaks of polysaccharides were observed in the three fractions.Structural characterization showed that RLP-1a consisted of rhamnose,arabinose,xylose,glucose,and galactose with the ratio of 3.14:8.21:1:1.37:4.90,whereas RLP-2a was composed of rhamnose,mannose,glucose,and galactose with the ratio of 1.70:1:93.59:2.73,and RLP-3a was composed of rhamnose,arabinose,xylose,mannose,glucose,and galactose with ratio of 6.04:26.51:2.05:1:3.17:31.77.Besides,the fractions of RLP-1a and RLP-3a showed higher antioxidant abilities than that of RLP-2a in vitro.(3)Effects of low molecular weight RPLs on lipid metabolism of SD rats with high-fat diet.After intervention with RLPs(the mixture of RLP-1a,RLP-2a,and RLP-3a)for 12weeks,the oral glucose tolerance of rats fed with HFD plus RLPs in HRLP and LRLP groups was significantly increased,and the fasting blood glucose level and insulin resistance index were decreased.Moreover,the lower Lee's index,the body mass index,and the liver index was decreased by 4.32%,15.21%,and 20.97%,respectively.The high-density lipoprotein(HDL-c)was increased by 35%,and the serum triglycerides(TG),total cholesterol(TC),free fatty acids(FFA),and low-density lipoprotein(LDL-c)levels were decreased by 4.32%,15.21%,20.97%,16.3%,47%,27.8%,and 14.8%,respectively.These subsequently inhibited body weight gain in the HFD-fed SD rats.RLPs decreased serum aspartate aminotransferase(AST),alanine aminotransferase(ALT),and?-glutamyltranspeptidase(GGT)levels,and increased the activities of superoxide dismutase(SOD)by 34.7%,glutathione peroxidase(GSH-Px)by 39.9%,and catalase(CAT)by 31.2%.RLPs also significantly reduced the levels of proinflammatory cytokines TNF-?,IL-1?,IL-6,and MCP-1 induced by HFD,improved the oxidative stress environment and inflammation in bodies,and reduced the damages caused by the lipid peroxides and oxidative stress.Besides,RLPs could maintain the normal form of liver tissue and prevent steatosis.(4)Effects of low molecular weight RLPs on expression of lipid metabolism-related genes in liver of HFD ratsIn this study,a total of 1109 differentially expressed genes had been enriched in HRLP vs HFD through RNA-Seq.RLPs could significantly regulate the biological processes related to the glycometabolism and lipid metabolism after being analyzed by Gene Ontology(GO).Then with the analysis by Kyoto Encyclopedia of Genes and Genomes(KEGG),the differential genes were mainly in the peroxisome proliferactor-activated receptors(PPARs)signaling pathway,steroid biosynthesis pathway,and non-alcoholic fatty liver disease(NAFLD)pathway which were related to glycometabolism and lipid metabolism.RLPs activated the PPARs signaling pathway,up-regulated the expression of unsaturated fatty acid synthesis genes FADS2 and scd-1,and up-regulated the expression of the rate-limiting enzyme of fatty acid?-oxidation(Acox3).Besides,RLPs significantly down-regulated the gene expression of a mixed-function oxidase(Cyp2e1)in NAFLD to reduce ROS production.The RNA-seq results were consistent with those of the q RT-PCR analyses.(5)Effects of low molecular weight RLPs on gut microbiota and its metabolites of SD rats with HFD.After 12 weeks of intervention with RLPs,the imbalance of intestinal flora in rats fed with HFD was repaired,and the abundance of intestinal microorganisms in rats with HFD was increased.On the phylum level,the ratio of Firmicutes to Bacteroidetes was decreased.On the genus level,the relative abundances of Clostridium,Blautia,Roseburia,and Dorea were decreased,and the relative abundances of Alistipe,Butyricimonas,and Prevotella were increased.And the relative abundances of Akkermansia and Bacteroides were also increased.Spearman correlation analysis indicated that the abundances of Firmicutes bacteria including Lactobacillus murinus,Treponema,and Bacteroides bacteria were significantly negatively correlated with the main indexes including obesity phenotype,liver function,and inflammatory factor levels,and significantly positively correlated with liver antioxidant enzyme activities.The abundances of Clostridium,Dorea,Gemmiger,and Flavonifractor were significantly negatively correlated with most of the indicators including obesity phenotype,liver function,and inflammatory factor level,and significantly positively correlated with liver antioxidant enzyme activities.The analyses on the contents of SCFAs in fecal samples showed that after RLPs intervention 12 weeks(P<0.05),the contents of propionic acid,butyric acid,isovaleric acid,and valeric acid in the HRLP group were significantly increased 131.82%,222.22%,130.77%,and 190.32%as compared to HFD,respectively.The above results indicated that the low molecular weight RLPs could regulate the gut microbiota of rats fed with HFD,and increase the contents of SCFAs and other gut microbiota metabolites.This study could provide a theoretical basis for further clarifying the low molecular weight RLPs to regulate inflammatory factors,intestinal permeability,glycometabolism,fatty acid oxidation,synthesis,and energy metabolism,and to clarify the coordinated regulation mechanisms with bacteria in regulating the related signaling pathway of the host lipid metabolism,inhibiting the growth of pathogenic microorganisms,and slowing down the development of obesity in the HFD diet-fed rats. |
PDF Full Text Request