Isolation,Purification,Structural Identification Of A Novel Small Molecule Garlic Polysaccharide And Its Anti-inflammatory Activity

Plant polysaccharides have been used more and more in the development of functional foods,clinical drugs and special cosmetics.This is due to its anti-oxidation,anti-inflammatory,improvement of immunity and many other physiological activities,meanwhile,it has a small side effects and high safety characteristics.Therefore,the systematic exploration of the purification,structure identification and biological activity of new natural plant functional polysaccharides has become one of the research hotspots of contemporary scholars.China is the largest garlic exporter in the world and has abundant garlic resources.At the same time,garlic has been designated as one of the medicinal edible foods by the Ministry of Health of China because of its medicinal functions for thousands of years.Jinxiang County is one of the most important garlic producing areas in China.Because of the excellent characteristics of its garlic varieties,it is well known at home and abroad,and is known as"the hometown of garlic in China".Garlic polysaccharide is the most abundant in garlic dry kind of chemical composition and it has significant anti-inflammatory,anti-oxidation,anti-tumor,enhance immunity,and protecting the liver function.At the same time,the research of biological activity of garlic polysaccharide in vitro cumulative research and the studies I have published have shown that garlic polysaccharide can be adjusted through the intestinal flora and its metabolites,and protect the intestinal tract and improve the intestinal inflammation.However,there are few reports on the extraction,purification,structural identification and analysis of Jinxiang small molecule garlic polysaccharide and its anti-inflammatory activity.Therefore,this study to Jinxiang garlic as raw material by hot water extraction method and through ion chromatography column and rubber coagulation chromatography column extraction purification get a new kind of small molecular weight of pure garlic polysaccharide components,through the chemical and instrument analysis method(IC,FT-IR,GC-MS,1D NMR,2D NMR)to further analyses the structure of the purified component of garlic polysaccharide.The anti-inflammatory effect and mechanism of garlic polysaccharides in cellular and animal inflammatory models were thoroughly explored by using a variety of advanced molecular biological techniques such as enzyme-linked immunity,RT-q PCR and Western blot.At the same time,from the perspective of interaction between UC and intestinal microorganisms,the regulation of intestinal flora and metabolites of DSS-induced mouse UC by garlic polysaccharide were systematically studied by using advanced 16S r DNA diversity high-throughput sequencing technology and LC-MS untargeted metabolomics technology.To provide theoretical basis and practical guidance for the development and utilization of clinical drugs and health food of garlic polysaccharide.The main results and conclusions are as follows.(1)Extraction,separation and purification of garlic polysaccharides,their physicochemical properties and antioxidant activity in vitroBoth the crude garlic polysaccharide sample(CWSGP)and the garlic polysaccharide purified component(WSGP)were light yellow loose powder with yield of 4.57%and 3.71%,respectively.According to the corresponding standard curve and the formula of total sugar content,reducing sugar content and protein content,the contents of total sugar,reducing sugar and protein in CWSGP were 91.03%,0.57%and 1.36%,respectively.CWSGP was purified by DEAE cellulose-52 anion exchange column,and two fractions,CGP-1 and CGP-2,were obtained with yields of 4.23%and 23.46%,respectively.The CGP-2 was further purified by Sephadex G-100 dextran gel chromatography column to obtain a single homogeneous component WSGP,the yield was 13.86%.The HPGPC spectrogram showed a single uniform symmetrical peak,and the UV spectrogram showed no absorption peak at 220-280 nm,indicating that the purified polysaccharide group was pure.The peak molecular weight(MP),mass average molecular weight(MW)and number average molecular weight(MN)of WSGP were measured as 1914 Da,1853 Da and 1807 Da,respectively.WSGP has obvious scavenging ability of DPPH free radical,hydroxyl free radical,superoxide anion free radical,ABTS free radical,Fe²⁺chelating force and reducing power and other antioxidant activities in vitro.(2)Structure identification and analysis of the extracted and purified small molecule polysaccharide WSGP from Jinxiang garlicThe WSGP was composed of fucose(2.60%),glucose(32.50%)and fructose(64.90%)based on the chemical and instrument analysis method(IC,FT-IR,GC-MS,1D NMR,2D NMR).The characteristic absorption peaks of O-H,C-H,C=O,C=C,-COOH,C-OH,C-O-C,and-CH₂ functional groups exist in WSGP.WSGP mainly contains 5 methylated sugar residues,which are 1,3,4,5-Me4-Manf,1,3,4,5-Me4-Glcf,2,3,4,6-Me4-Glcp,3,4,5-Me3-Manf and 3,4,5-Me3-Glcf,and the corresponding 5 glycosidic bonds are Manf-(2?,Glcf-(2?,Glcp-(1?,?1)-Manf-(2?and?1)-Glcf-(2?.The corresponding molar percentage of the above glycosidic bonds are 1.20%,1.11%,15.13%,40.26%and 42.30%.WSGP was detected to be composed of two monosaccharides in NMR,and the monosaccharide residues were?-D-Fruf-2,1 and?-D-Glcp-1,respectively.At the same time,?-D-Glcp-1?2-?-D-Fruf-1?and2-?-D-Fruf-1?2-?-D-Fruf-1?bonding modes existed.In summary,WSGP is a fructose containing both?and?-configurational glycosidic bonds and mainly?-type glycosidic bonds.The main chain linkage mode is?1)-Fruf-(2?,and the structural formula is?-D-Glcp-1(2-?-D-Fruf-1)n(n=9-10).(3)Effect of WSGP on LPS-induced macrophage(RAW264.7)inflammatory response and its mechanismIn the range of 62.5?1000?g/m L WSGP concentration,the cell survival rate was more than 95.18%,indicating that WSGP had no toxicity to macrophage RAW264.7.WSGP showed dose-dependent inhibition of RAW264.7NO,IL-1?,IL-6 and TNF-?in LPS-induced macrophages at different concentrations(62.5?1000 g/m L),and dose-dependent inhibition of IL-1?,IL-6 and TNF-?at m RNA expression levels.WSGP at different concentrations(100 and200 g/m L)could significantly inhibit LPS-induced the phosphorylation of p65 and I?B-?in NF-?B signaling pathway of RAW264.7 and the phosphorylation of STAT3 in STAT3 signaling pathway.(4)The effect of WSGP on DSS-induced ulcerative colitis in mice and its mechanismGarlic polysaccharide WSGP intervention of DSS induced acute ulcerative colitis in mice have significantly inhibiting and improving effect,embodied in its apparent index improved significantly colitis mice reduced food intake,weight loss and waste of properties,and the degree of occult blood,colon shorten,spleen symptoms such as edema,and significantly reduce the DAI score.The results of HE staining showed that the intervention of WSGP effectively improved the colon tissue injury induced by DSS in mice,significantly reduced the histopathological score,and the crypt(Cr),high surface colonic cells(Co)and peripheral muscular layer(M)were more intact,and the submucosa(Sm)edema was reduced.The results of AB-PAS staining showed that WSGP significantly inhibited the decrease of goblet cell(Gc)and mucin secretion in DSS induced colitis.Immunofluorescence results showed that WSGP significantly inhibited the reduction of ZO-1,Occludin and Caludin-1 tight junction proteins in DSS-induced colitis mice.Meanwhile,WSGP can significantly inhibit NO secretion in serum of DSS-induced colitis mice.It significantly inhibited the secretion of inflammatory cytokines(IL-6,TNF-?and IL-1?)and their m RNA expression levels.WSGP inhibits the phosphorylation of p65,I?B-?and STAT3 proteins in the NF-?B and STAT3 signaling pathways in the colitis mice,and then inhibits the synthesis and secretion of inflammatory cytokines,and finally improves the inflammation of colitis mice.All of the above results were dose-dependent.(5)WSGP regulation of intestinal flora and its metabolites in mice with ulcerative colitis induced by DSSGarlic polysaccharide WSGP alleviates and improves DSS-induced ulcerative colitis in mice through intestinal flora regulation and its metabolites regulation mainly through the following aspects:significantly inhibit the reduction of the contents of acetic acid,propionic acid,isobutyric acid,n-valerate and total SCFAs.It can effectively improve the number of OUT,the decrease of?-diversity index(Chao1,ACE,Shannon and Simpson)and the disorder of?-diversity flora structure in mice with colitis,thus inhibiting the development and occurrence of inflammation in mice with colitis.Significantly inhibited or enriched the abundance of Muribaculaceae?Lachnospiraceae?Lachnospiraceae?NK4A136?group?Mucispirillum?Helicobacter?Ruminococcus?1 and Ruminiclostridium?5,which were identified as key microorganisms closely related to inflammatory intestinal disease in this study.At the same time,significantly regulated the balance of Cholic Acid,Phosphatidylcholine,Ginsenoside Mc,2,4,6-Triacetylglycitin,N,N-(2,2-dihydroxy-ethyl)arachidonoyl amine,N-Acetyl-D-glucosamine and L-Serine which were identified as key metabolites closely related to inflammatory bowel disease in this study.Dietary intervention treatment of WSGP can play an anti-inflammatory role in reversing the changes of intestinal microbiota and its metabolites caused by DSS-induced colitis in mice.WSGP prevents DSS-induced disruption of cell processing and microbial signaling pathways and restores dysfunction in colitis mice by regulating metabolic pathways.