| Objective: Extraction and purification of Polygonatum cyrtonema Hua polysaccharide(PCP)to study its structural characterization and its effects and mechanism on non-alcoholic fatty liver disease(NAFLD).Methods:(1)Isolation,purification and structural elucidation of PCP: The total PCP were extracted from the rhizome of Polygonatum cyrtonema Hua by hot water extraction and ethanol precipitation,and the preliminary purified PCP were obtained by decolorization,deproteinization and dialysis.The preliminary purified PCP were graded by DEAE-52 cellulose anion exchange chromatography column to obtain the neutral polysaccharide fraction,PCP1,and the acidic polysaccharide fraction,PCP2.In this study,we focused on the structure of PCP1 and the protective effect of NAFLD.After further purification of PCP1 using a Sephadex G-75 dextran gel column,PCP1 purity by high-performance gel permeation chromatography(HPGPC),PCP1 molecular weight by multi-angle laser light scattering system,and the PCP1 monosaccharide composition by ion chromatography.The glycan structure of PCP1 was analyzed by a combination of Fourier transform infrared(FT-IR),methylation and nuclear magnetic resonance(NMR).(2)Study on the protective effect of PCP on NAFLD: A high-fat diet(HFD)induced C57BL/6J mice for 8 weeks was used to construct the NAFLD model,and the experiment was divided into normal group(ND),HFD,PCP1 low-dose group(PCP1-L,200 mg/kg/day),PCP1 high-dose group(PCP1-H,400 mg/kg /day).After 6weeks of continuous administration,oral glucose tolerance test(OGTT)was performed and feces,serum and liver were collected.Among them,stool was used to analyze intestinal flora and short-chain fatty acids(SCFAs),serum was used to determine lipid levels,and liver was used to determine biochemical indicators of liver injury and oxidative stress indicators as well as H&E staining and oil red O staining.Results:(1)Isolation,purification and structural elucidation of PCP: The yield of crude polysaccharide from Polygonatum cyrtonema Hua was 8.37% after hydroextraction and alcoholic precipitation,decolorization,deproteinization and dialysis,of which the content of PCP1 was 96.2%,PCP2 was 3.8%,the total sugar content of PCP1 was78.9% and the protein content was 0.87%.The results of HPGPC showed that PCP1 is a homogeneous polysaccharide with narrow molecular weight distribution,and its heavy average molecular weight(Mw)and number average molecular weight(Mn)were 4650 and 4420 Da,respectively,with a polydispersity index of 1.05.The results of monosaccharide composition showed that PCP1 is mainly composed of four monosaccharides,and the molar ratios were: fructose : glucose : mannose : galactose =88.1 : 9.7 : 1.6 : 0.3.The comprehensive analysis of PCP1 structure by FT-IR spectroscopy,methylation and NMR spectroscopy showed that the main chain of PCP1 was mainly composed of → 1)-β-D-Fruf-(2 →,→ 1,6)-β-D-Fruf-(2 → and a small amount of →6)-α-D-Glcp-(1→,→4)-β-D-Manp-(1→,β-D-Glcp-(1→,the side chain mainly consists of β-D-Fruf-(2→ attached to the C-6 position of →1,6)-β-D-Fruf-(2→.(2)Protective effect of PCP on NAFLD: PCP1 can alleviate liver injury and dyslipidemia in NAFLD mice.After PCP1 intervention,liver alanine transaminase(ALT),aspartate transaminase(AST)and serum triglycerides(TG)、total cholesterol(TC)and low-density lipoprotein cholesterol levels were significantly reduced,and serum high-density lipoprotein cholesterol(HDL-C)levels were significantly increased in HFD mice.PCP1 could reduce oxidative stress injury and glucose metabolism function.Compared with the HFD group,PCP1 significantly reduced malondialdehyde(MDA)levels and increased superoxide dismutase(SOD)and glutathione(GSH)levels in NAFLD mice.OGTT showed that PCP1 could alleviate the reduced glucose regulation and glucose metabolism disorders in NAFLD mice caused by long-term HFD.Alpha analysis showed that compared with the ND group,the HFD group had significantly lower Chao1,observed species Simpson and Shannon indices were significantly reduced in the HFD group compared to the ND group,however,all of these indices were significantly increased in the PCP1 group compared to the HFD group.In addition,PCo A and Venn analyses also showed that PCP1 ameliorated HFD-induced changes in intestinal community composition.PCP1 improved intestinal flora composition at both the phylum and genus levels.The results of the system-level analysis showed that the relative abundance of Firmicutes to Bacteroidetes was significantly larger in NAFLD mice relative to the ND group,and PCP1 significantly reduced this ratio,suggesting that PCP1 has a good anti-obesity effect.Meanwhile,analysis of the top 40 bacterial genera showed that there was a significant difference in the distribution of bacterial genera between the HFD and ND groups.Compared with the HFD group,PCP1 significantly increased the relative abundance of Bifidobacterium,Allobaculum,Ruminococcus,and Adlercreutzia and decreased the relative abundance of Lactobacillus,Bacteroides,Helicobacter and other genera.This suggests that PCP1 can affect specific beneficial bacteria and thus regulate the structure of intestinal flora.PCP1 can improve the levels of SCFAs in NAFLD mice;the results of measuring the levels of seven SCFAs in mouse feces showed that PCP1 intervention significantly increased the levels of SCFAs,especially isobutyrate and isovalerate,in NAFLD mice.Conclusion: Polysaccharide of Polygonatum cyrtonema Hua,PCP1,is a neutral polysaccharide with molecular weight of 4650 Da,mainly containing fructose,glucose,mannose and galactose,and its repeating sugar chain structural unit mainly consists of→1)-β-D-Fruf-(2→,→1,6)-β-D-Fruf-(2→ and a small amount of →6)-α-D-Glcp-(1→,→ 4)-β-D-Manp-(1 →,β-D-Glcp-(1 →,and the side chain mainly consists ofβ-D-Fruf-(2 → attached at position C-6 of → 1,6)-β-D-Fruf-(2 →.PCP1 has good therapeutic effects on NAFLD mice and can reduce blood lipid levels,reduce liver damage and fat accumulation,alleviate liver oxidative stress,repair intestinal flora structural disorders as well as promoting SCFAs production. |
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