Isolation, Purification and Hypoglycemic Activity of Polysaccharides from Rosa roxburghii Tratt Fruit and Their Effect on Gut Microflora

Author:Wang Lei

Supervisor:fu xiong


Degree Year:2019





Rosa roxburghii Tratt(R.roxburghii),which belongs to the family of Rosaceae,is largely planted in mountainous areas in Southwest China,especially in Guizhou province.The fruit is commonly used as an edible food and folk medicine in traditional Chinese medicine for the treatment of various diseases such as diabetes,cancer,cacochylia,diarrhea,hypertension,hyperlipemia,and scurvy.On account of the beneficial effects,it has attracted much attention on various active substances of R.roxburghii fruits,including flavonoids,ascorbate,polysaccharides,superoxide dismutase and organic acid.However,to date,the hypoglycemical activity,underlying mechanism and structure-activity relationship of the polysaccharides from the fruits of R.roxburghiiare still unclear.Therefore,in the present study,R.roxburghii fruits were chosen as the experimental material.The extraction,purification,physicochemical properties,structural characteristics of polysaccharides(designated as RTFP)from R.roxburghii fruits were investigated.Furthermore,the in vitro hypoglycemic activity and their effects on gut microbiota of RTFP were evaluated by the combination of chemical,cell,and animal experiments.Meanwhile,the RTFP-3 was functional modificated by preparations of RTFP-3-functionalized selenium nanoparticles(RP3-SeNPs)complexes.The protective effects and molecular mechanisms of RP3-SeNPs on INS-1 cell apoptosis were investigated,with the aim of providing evidence for the application of RP3-SeNPs in the treatment of diabetes mellitus.The main results were listed as follows:(1)Water-soluble polysaccharides(RTFP)were extracted from R.roxburghii fruit by hot water extraction method.Four polysaccharide fractions(RTFP-1,RTFP-2,RTFP-3 and RTFP-4)were isolated from R.roxburghii fruits by purification using DEAE-sepharose fast flow column.RTFP-1 and RTFP-3 had the highest yield,accounting for 12.30%and 23.84%of RTFP.High performance gel permeation chromatography(HPGPC)revealed that RTFP-1and RTFP-3 were homogeneous with the average molecular weights of 128.7 and 66.7 kDa,respectively.Methylation and 1D/2D nuclear magnetic resonance(NMR)analyses indicated that RTFP-1 consisted of→5)-α-L-Araf-(1→,→6)-α-D-Galp-(1→,→3)-α-D-GalpNAc-(1→,→4)-β-D-GalpA-(1→,β-D-Xylp-(1→andα-D-Glcp-(1→.However,RTFP-3 was mainly composed of→6)-α-D-Galp-(1→,→4)-α-D-GalpA-(1→,→5)-α-L-Araf-(1→,→3,4)-β-L-Fucp-(1→,→4)-β-D-Glcp-(1→andβ-D-Glcp-(1→.(2)Bioassay results showed that RTFP-1 and RTFP-3 exerted inhibitory activities onα-glucosidase in a mixed inhibition type,and their half inhibitory concentration(IC50)values were 2.04 and 4.15 mg/mL,respectively.The enzyme-inhibitor dissociation constant Ki for RTFP-1 and RTFP-3 were 5.104 and 19.889 mg/mL,respectively,and the inhibitor-enzyme-substrate dissociation constant Kiss were 0.421 and 7.007 mg/mL,respectively.The quenching ofα-glucosidase by RTFP-1 and RTFP-3 was initiated by static quenching,and the number of binding sites was approximately equal to 1.04 and 1.44,suggesting that a moderate affinity was formed.Moreover,RTFP-3 had hypoglycemic effects on insulin resistance of HepG2 cells through stimulating the bioactivity of hexokinase(HK)and pyruvate kinase(PK).RT-PCR results showed that RTFP-3 could down-regulate the expression of G6Pase mRNA,thus promoting the glucose uptake of IR-HepG2 cells.(3)Selenium nanoparticles(SeNPs)functionalized with RTFP-3(2 mg/mL)was prepared via a facile,single-step and green in-situ synthesis method.The in vitro protective effects of RP3-SeNPs on INS-1 cells against H2O2-induced cell apoptosis were investigated.Structural characterization indicated that RTFP-3 functionalized SeNPs(RP3-SeNPs)with an average diameter size of 104.5 nm was of highly uniform and extremely stable compared to bare SeNPs.Bioassay results revealed that RP3-SeNPs possessed much better protective and suppressed activities against H2O2-induced INS-1 cell apoptosis as compared to its single components.Mechanistic studies exhibited that RP3-SeNPs effectively blocked the overproduction of intracellular ROS,mitochondria damage,activation of caspase-3,-8,and-9in INS-1 cells,indicating that RP3-SeNPs functioned through attenuating oxidative stress and down-regulating expression of uncoupling protein-2(UCP-2).(4)The digestion properties of RTFP-3 under saliva,simulated gastric and small intestinal conditions were studied.The results indicated that human saliva had no effect on RTFP-3.RTFP-3 was slightly degraded in simulated gastric and small intestinal juices.The molecular weight(Mw)of RTFP-3 was decreased from 67.59 to 44.22 kDa.Meanwhile,the reducing sugars were increased from 0.503 to 1.744 mM.However,there was no monosaccharide released after gastrointestinal digestion.Then,the prebiotic effects of RTFP-3 were examined by in vitro fermentation model.RTFP-3 could significantly increase the production of total short-chain fatty acids from 23.49 to 44.29 mM.Furthermore,RTFP-3could significantly modulate the microbial structure by lowering the ratio of Firmicutes to Bacteroidetes from 14.89 to 4.68 after 48 h fermentation,and improving the relative abundances of some beneficial gut microbiota.(5)Supplementation of RTFP(300,600 and 900 mg/kg)in the diet to male db/db mice for 8 weeks significantly casued the decrease of body weight,fasting blood glucose,insulin and blood lipid levels.RTFP could also down-regulate the expression levels of key genes for glycogenesis and lipogenesis(G6Pase、FAS、ACC-1、SREBP-1c and PPAR-γ)in the liver of model mice,and reduce the accumulation of fat in the liver tissue.The 16S rRNA gene sequence data indicated that RTFP not only reversed gut dysbiosis,as indicated by the decreased Firmicutes-to-Bacteroidetes ratios,but also bacteria beneficial to the intestine(Bacteroidaceae,Bacteroidaceae S24-7 group and Lactobacillaceae)were induced to grow,and harmful bacteria(Enterocaccaceae and Desulfovibrionaceae)were inhibited.