Structural Analysis,in Vitro Digestion And Fermentation,and Hepatoprotective Activity Of Polysaccharides From Ginger Stems And Leaves

Polysaccharides are high-molecular carbohydrate polymers consisting of monosaccharides units linked by glycosidic bonds.At present,a variety of polysaccharides with different activities have been isolated from plant tissues of roots,stems,leaves,flowers and fruit,which not only have low side effects but also have significant effects on disease treatment and health improvement.Numerous reports have presented that plant polysaccharides had many bioactivities including hepatoprotective,hypoglycaemic,anti-inflammatory,hypoglycemic lipid and blood pressure,immunological enhancement,anti-tumour and probiotic viability.Therefore,the research for finding active polysaccharides from new plant resources has been becoming a popular focus for researchers in recent years.Ginger(Zingiber officinaie Roscoe),also known as ground pungent,ginger root and spicy clouds,is a perennial and persistent herb of the Zingiber genus in the ginger family.China is rich in ginger resources,the annual production and export of ginger are steady in the forefront.In the process of ginger harvesting and processing,a large number of ginger by-products are produced,such as harvesting by-products of above-ground ginger stems and leaves as well as processing by-products involving ginger peel and ginger residue.However,those by-products are mostly discarded,which results in environmental pollution and waste of ginger resources.Some studies about active polysaccharides from ginger have been reported at home and abroad.Besides,many studies about polysaccharides from ginger peel and ginger residue that are by-products of ginger processing also have been published.However,there are few studies related to polysaccharides from ginger stems and leaves.Therefore,in order to improve the comprehensive utilization value of ginger stems and leaves,the active polysaccharides can be extracted from the waste of ginger stems and leaves,whose specific bioactivities can be systematically studied to provide academic support for the development of pharmaceuticals or cosmetic additives.It is worth looking forward to the great significance for ginger farmers to increase their income,enterprises to profit and transform high value-added resources.In this thesis,the polysaccharide fractions from ginger stems and leaves(GSLP)were extracted using two different extraction methods.More specifically,the physicochemical properties,radical scavenging activities,isolation and purification processes,structural analysis,in vitro digestion and fermentation characteristics of GSLP were systematically investigated.In addition,according to the principle of gut-liver axis,the protective effects and mechanisms of purified GSLP-3 on alcoholic liver injury were investigated.The specific results of this thesis were summarized as follows:(1)Effects of extraction methods on the physico-chemical properties and antioxidant activity of crude polysaccharides from ginger stems and leavesThe crude GSLP were prepared by hot water extraction(HWE)and ultrasonicassisted extraction(UAE),and the effects of HWE and UAE on the physicochemical properties,structural characteristics and antioxidant activity of the crude GSLP were investigated.The results showed that the crude GSLP prepared by UAE(UAE-GSLP)had higher yield,meanwhile,UAE-GSLP contained higher total sugar,uronic acid and sulfate contents than those prepared by HWE(HWE-GSLP).Besides,the average particle size of UAE-GSLP was smaller,the absolute value of zeta potential was larger,the apparent viscosity was smaller,and the molecular weight distribution was relatively smaller as compared with HWE-GSLP.In addition,UAE-GSLP exhibited stronger antioxidant activity than that of HWE-GSLP.Thus,it was advisable to select the UAE method for the preparation of GSLP for subsequent experiments.(2)Isolation,purification and structural analysis of polysaccharides from ginger stems and leavesThe crude UAE-GSLPs were isolated and purified using the method of anion column chromatography and dextran gel.Follewed by the purified fractions were structurally analysed by the combination of chemical and instrumental analysis.The results showed that the crude UAE-GSLPs were purified to yield five polysaccharide fractions,of which GSLP-3 and GSLP-4 were the two major homogeneous polysaccharide fractions as the subsequent research materials.Both GSLP-3 and GSLP-4 were composed of the same eight monosaccharides,while the molar percentages of the corresponding monosaccharides were significantly different.The results of periodate oxidation,Smith degradation,methylation and NMR analysis showed that the glycosidic bonds of GSLP-3 were mainly composed of α-L-Araf-(1→,→5)-α-L-Araf-(1→,6)-β-D-Galp-(1→,→3,6)-β-Galp-(1→,→6)-α-D-Galp-(1→,β-D-Galp-(1→,→3)-β-D-Galp-(1→,β-D-Xylp-(1→ and →4)-α-Galp A-(1→2,whereas the glycosidic bonds of GSLP-4 were mainly composed of α-L-Araf-(1→,→5)-α-L-Araf-(1→,→2)-α-D-Araf-(1→,→6)-α-Galp-(1→,→3)-β-Galp-(1→,→3,6)-β-Galp-(1→,→2,4-α-LRhap-(1→,α-Xylp-(1→,→3)-β-D-Galp A-(1→ and →3,4)-α-D-Galp A-6-OMe-(1→.The results of circular dichroism(CD)and atomic force microscopy(AFM)displayed that both GSLP-3 and GSLP-4 presented a polymeric form with larger particles in aqueous solution.In addition,the result of Congo red experiment showed that GSLP-3had a three-stranded helical structure while GSLP-4 did not present.Besides,the result of XRD diffraction exihited that GSLP-3 and GSLP-4 mainly existed in an amorphous form.(3)In vitro simulated digestion and fermentation of polysaccharides fractions from ginger stems and leavesThe digestive characteristics of GSLP-3 and GSLP-4 were studied by in vitro simulated oral,gastric and small intestinal fluids digestion,also the fermentation characteristics of GSLP-3 and GSLP-4 were studied by in vitro human fecal microflora fermentation.The results displayed that GSLP-3 and GSLP-4 had good stability in the simulated oral,gastric and small intestinal digestion systems.Concretely,the average molecular weight as well as the reducing sugars of GSLP-3 and GSLP-4 did not change significantly,meanwhile no free monosaccharides were produced.In the fermentation system,it was been found that 43.30% and 58.27% of carbohydrates were consumed after 48 h fermentation by GSLP-3 and GSLP-4,respectively.At the same time,GSLP-3 and GSLP-4 notably increased the content of total short-chain fatty acids(SCFAs)during the in vitro fermentation process.GSLP-3 and GSLP-4 were able to interfere the fermentation process and alter the compositions of the gut microbiota at the phylum and genus levels,thereby promoting host gut health.Detailly,both GSLP-3 and GSLP-4significantly increased Firmicutes abundance and inhibited the growth of Clostridium at the phylum level.In addition,Both GSLP-3 and GSLP-4 significantly increased the abundance of Megasphaera and Bifidobacterium at the genus level.Meanwhile,both GSLP-3 and GSLP-4 significantly reduced the abundance of the pathogenic bacterium of Fusobacterium.These results suggested that GSLP-3 and GSLP-4 as potential prebiotics could regulate gut homeostasis and thus reduce diseases induced by gut microbiota disorders.(4)Protective effects and mechanisms of GSLP-3 on alcohol-induced liver injury in C57BL/6J miceWe established the liver injury model in male mice of the C57BL/6J strain on a Lieber-De Carli liquid alcohol diet,and the protective effects of GSLP-3 on alcoholic liver injury in mice were investigated by feeding GSLP-3.Subsequently,the physiological and biochemical evaluation indexes related to liver injury were measured,besides,the transcriptomics and gut microbiomics were explored.The results showed that GSLP-3 had significant protective effects on constructed model mice.Specifically,GSLP-3 significantly increased the activities of ethanol dehydrogenase(ADH)and acetaldehyde dehydrogenase(ALDH)in the liver,also decreased the levels of glutamic oxaloacetic transaminase(AST)and glutamic alanine transaminase(ALT)in serum of mice.In addition,GSLP-3 could increase the activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)as well as reduce the activity of malondialdehyde(MDA).At the same time,GSLP-3 could alleviate the abnormal changes of lipid levels in the liver,as well as reduce fat accumulation and degeneration.In addition,GSLP-3could significantly decrease the gene expression and secretion of pro-inflammatory factors including IL-6,IL-1β and TNF-α in the serum of mice,and reduce the content of endotoxic lipopolysaccharide(LPS),thus alleviating the inflammatory response and improving the condition of liver damage.Based on the theory of gut-liver axis,the protective mechanisms of GSLP-3 on alcohol-induced liver injury were further analyzed as follows: five signaling pathways related to immune and inflammation response were screened by GO and KEGG enrichment analysis,namely NOD-like receptor signaling pathway,Toll-like receptor signaling pathway,chemokine signaling pathway,PI3K/AKT signaling pathway and NF-κB signaling pathway.Based on the validation results of the expression levels of relevant genes,we drawed the conclusions that GSLP-3 could alleviate the immune and inflammation response in C57BL/6J mice and play a protective role against alcohol-induced liver injury.We further analyzed the protective effects of GSLP-3 on alcoholic liver injury from the perspective of gut microbiota,and the outcomes exhibited that GSLP-3 could markedly reduce the relative abundance of Proteobacteria and increase the relative abundance of Verrucomicrobia.Besides,the relative abundances of Akkermansia and Lactobacillus were notably increased,while the abundance of Escherichia-Shigella was decreased.