Study On Structures And Bioactivities Of Polysaccharides From Fructus Mori

Objective:As a kind of traditional Chinese medicinal material,Herbal sangshen is dry ear of Moras alba L.belonging to Moraceae family,which has high content of polysaccharide.In recent years,a large number of studies have proved the pharmacological effects of macromolecular polysaccharides.The aim of this topic is to study the polysaccharide structure of Fructus Mori and its effects on intestinal flora,anti-A?42aggregation,and anti-angiogenesis of its sulfated derivatives.We attempt to study the structures and bioactivities of polysaccharides,which are from ear of Moras alba L.,and we also hope to lay the foundation for the research on polysaccharide material basis and quality control of this herb,and its related new drugs development or health products.Methods:In this study,crude Fructus Mori polysaccharide?FMP?was extracted from dried Fructus Mori by using enzymes assisted extraction method?cellulase,amylase,papain?.The crude polysaccharide FMP was separated and purified by DEAE-Fast Flow and Sephacryl S-300 HR and Sephacryl S-100 HR,respectively.The purified polysaccharide was analyzed for purity and molecular weight by high performance gel permeation chromatography?HPGPC?.Monosaccharide composition analysis was carried out by sugar alcohol acetate derivatization and 3-methyl-1-phenyl-2-pyridinone?PMP?derivatization.The methylation method was used to analyze the sugar residue linkage types,and the Conrad method was employed for uronic acid reduction.According to partial acid hydrolysis with trifluoroacetic acid?TFA?,infrared spectroscopy?IR?,nuclear magnetic resonance?NMR?and combined with other methods were employed to determine the polysaccharide structure.In this study,Trypticase Soy Broth?TSB?was used to culture Bacteroides gingivalis?B.thetaiotaomicron,B.ovatus and B.fragilis?to study the effects of Fructus Mori polysaccharides on the growth of Bacteroides gut strain.Stably transfect APP and?-secretory proteins of amyloid A?₄₂,which are from the Chinese hamster ovary cell of enzyme 1?CHO/APPBACE1?,and the Switish mutant HEK293-APPsw cell stably transfected with the A?₄₂2 precursor protein APP,they all are the main cell models for the experiment of inhibiting the secretion of A?₄₂2 by the polysaccharide.Thioflavin T method was employed to determine the effect of Fructus Mori polysaccharide on aggregation of A?₄₂.The toxicity experiments of Fructus Mori polysaccharide on human normal liver cell LO2 were detected by MTT method.Sulfated derivatives of Fructus Mori polysaccharide were prepared by chlorosulfonic acid-pyridine method?1:3?.The tube formation assay of human microvascular endothelial cells?HMEC-1?was used to investigate the anti-angiogenic ability of Fructus Mori polysaccharides and their sulfated derivatives.Result:The crude Fructus Mori polysaccharide FMP was extracted?152 g,yield:15.2%?.six kinds homogeneous polysaccharides FMP-2?500 mg,yield 0.05%of 1.00 kg dried medicinal material?,FMP-5?4.67g,yield:0.47%?,FMP-6-H?15.56 g,yield:1.56%?,FMP-6-S1?600 mg,yield:0.06%?,FMP-6-S2?2.5 g,yield:0.25%?and FMP-6-S4?1.71 g,yield:0.17%?were obtained.HPGPC analysis showed that all six polysaccharides were homogeneous polysaccharides with molecular weights of 12.6 kDa,17.6 kDa,17.5 kDa,327.1 kDa,86.83 kDa and 11.23 kDa,respectively.Detailed structures of four homogeneous polysaccharides?FMP-6-S2,FMP-6-S4,FMP-6-S1 and FMP-6-H?were characterized.The results of sugar composition of FMP-6-S2 showed that it was composed of rhamnose?Rha?,galacturonic acid?GalA?,galactose?Glc?and arabinose?Ara?in a molar ratio of 30.86:24.78:28.70:15.61.According to the results of methylation and nuclear magnetic resonance analysis,the polysaccharide FMP-6-S2 was RG-I type polysaccharide and was composed of 1,4-?-D-GalpA and 1,2-?-L-Rhap with the branches was substituted at the C-4 position of 1,2-?-L-Rhap.,the chains were composed of terminal?T?-?-D-Galp,1,4-?-D-Galp,1,3,6-?-D-Galp,T-?-L-Araf and 1,5-?-L-Araf.Proteoglycan FMP-6-S4 is mainly composed of 80%polysaccharides and 20%proteins.The polysaccharide of proteoglycan FMP-6-S4 is mainly composed of galacturonic acid,arabinose,galactose,glucose and rhamnose in a molar ratio of 62.13:4.50:3.70:3.87:5.80.The protein of proteoglycan FMP-6-S4 mainly contains aspartic acid,glutamic acid,glycine,arginine,leucine and proline by the automatic amino acid analyzer,the molar ratio is 4.33:7.27:1.77:1.99:1.61:3.11.The main chain of the repeating of the polysaccharide in proteoglycan FMP-6-S4 is 1,4-?-D-GalpA alternately attached by 1,2-?-L-Rhap,while the C-3 position of?-D-GalpA were replaced by?-L-HexpA and?-D-GalpA.T-?-L-Araf,1,5-?-L-Araf and?-D-Galp or?-D-Glcp,1,6-?-D-Glcp residues were replaced by at the C-4 position of?-L-Rhap.Analysis of sugar composition showed that FMP-6-S1 consisted of rhamnose,galacturonic acid,galactose,arabinose and glucuronic acid,the molar ratio is19.02:17.86:38.05:20.54:3.03:1.5.The results of methylation and NMR showed that the main chain of polysaccharide FMP-6-S1 was similar to that of FMP-6-S2,but its branches were different from that of RG-I polysaccharide with a main chain of 1,4-?-D-GalpA and1,2-?-L-Rhap and branches substituted at C-4 on rhamnose.The branches of FMP-6-S1were composed of T-?-D-Galp?Glcp?,T-?-D-GlcpA,1,4,6-?-D-Galp,1,6-?-D-Galp,1,3-?-D-Galp,1,4-?-D-Galp,T-?-L-Araf and 1,5-?-L-Araf residues.The results of sugar composition showed that FMP-6-H mainly consisted of galacturonic acid?93.52%?and trace of arabinose,glucose,galactose and mannose.The methylation analysis showed that all galacturonic acid residues were 1,4-linkage type,it can be directly speculated that FMP-6-H is a straight galacturonan.Intestinal flora bioactivity test results showed that FMP-6-S2 and its degraded product FMP-6-S2-01a can promote the growth of the beneficial bacteria B.thetaiotaomicron.The further study shows that B.thetaiotaomicron can produce short-chain fatty acid including acetic acid and butyric acid when it was treated by FMP-6-S2 or FMP-6-S2-01a.The short-chain fatty acids were not only the energy source for intestinal bacteria but also for the intestinal mucosal cells to maintain intestinal homeostasis and immune regulation.The results of anti-A?₄₂2 production and agglutination activities showed that proteoglycan FMP-6-S4 could not only inhibit A?₄₂2 aggregation,but also significantly inhibited the secretion of A?₄₂2 in CHO/APPBACE1 and HEK293-APPsw cells.In addition,polysaccharide FMP-6-S2 also has a lower inhibition effect on the A?₄₂2 aggregation.The results of anti-angiogenic activity assays indicated that sulfated derivatives of FMP-6-S2and FMP-6-H could inhibit the tube formation of HMEC-1 cells in vitro.Conclusion:Six kinds of homogeneous polysaccharides?FMP-2,FMP-5,FMP-6-H,FMP-6-S1,FMP-6-S2 and FMP-6-S4?were extracted and isolated from Fructus Mori.FMP-2 is a neutral polysaccharide and others are pectic polysaccharides.FMP-6-S1 and FMP-6-S2 belong to RG-I type polysaccharides,FMP-6-S4 is RG-II type proteoglycan,but their molecular weight and monosaccharide composition are different.These made them have different activities.Polysaccharides FMP-6-S2 and FMP-6-S2-01a could promote bacterial growth of Bacteroides spp.,which is a dominant group of gut bacteria.It seemed that they could play an important role in improving gut flora,and also could lay the theoretical foundation based on Fructus Mori polysaccharide drug development to regulate the intestinal flora.Sulfated derivatives of FMP-6-H and FMP-6-S2 could inhibit the tube formation of HMEC-1 cells,but its inhibition rate was not very strong.In vitro experiments showed that proteoglycan FMP-6-S4 dose-dependently inhibited A?₄₂production in CHO/APP BACE1 cells stably transfected with APP and BACE1 and HEK293-APPsw cells stably transfected with APP Switish mutation.Besides,they could also inhibit A?₄₂2 aggregation.Thus,proteoglycan FMP-6-S4 can be a lead compound for the treatment of Alzheimer's disease and has the potential to be developed as a new drug for the treatment of Alzheimer's disease.In summary,Fructus Mori polysaccharides might be one of the active substances for treating constipation and anti-aging in herbal Sangshen.