| Longan is a characteristic fruit in southern China and a traditional Chinese medicine in Chinese Pharmacopoeia.Studies have shown that longan polysaccharide has a variety of biological activities such as anti-tumor,immune regulation,but longan polysaccharide is difficult to be digested or directly absorbed by human body.Intestinal flora may participate in the immune regulation of longan polysaccharide.In order to clarify the metabolites of longan polysaccharide in intestinal flora and its possible immunomodulatory activity,and the effect of longan polysaccharide on the composition of human intestinal flora.In this paper,longan polysaccharide and human intestinal microflora were co cultured in vitro.The metabolites of longan polysaccharide in human intestinal microflora were analyzed by LCTo F-MS/MS and GC-TOF-MS/MS.The effect of longan polysaccharide on human intestinal microflora was analyzed by metagenomic sequencing.The activation of mesenteric lymph node cells by longan polysaccharide and its metabolites was compared.The main results are as follows1.Analysis of intestinal flora metabolism of longan polysaccharide in vitro:(1)Analysis of intestinal flora metabolism of longan polysaccharide in vitroThe changes of total sugar,reducing sugar,short chain fatty acid and antioxidant activity of longan polysaccharide(LP)and human intestinal microflora during anaerobic co culture in vitro were analyzed.The results showed that the contents of total sugar and reducing sugar in LP group were 53.67% ～ 30.16% and 126% ～ 19.72% of those in 0 h after4 ～ 48 h culture,respectively.At 4 h of culture,FRAP value,ABTS+·scavenging capacity and copper ion reduction capacity of LP group reached the maximum,which were 75.65 ±0.28 μg TE/m L,69.25% ± 1.25% and 235.45 ± 3.40 μg TE/m L,respectively.At 8 ～ 24 h,FRAP,ABTS+·scavenging capacity and copper ion reduction capacity of LP group were significantly higher than those of control group(CON).At 16 ～ 48 h,the NH3-N value of LP group was significantly lower than that of control group.Short chain fatty acids(SCFAs)in LP group increased first and then decreased.In LP group,acetic acid,propionic acid,isobutyric acid,butyric acid,2-Methylbutyric acid,isovaleric acid,valeric acid and caproic acid reached the highest at 16 h,which were 82.51 ± 8.76 mg/L,87.01 ± 10.14 mg/L,17.91± 3.22 mg/L,66.60 ± 9.23 mg/L,11.65 ± 2.22 mg/L,40.12 ± 7.81 mg/L,25.94 ± 4.01 mg/L and 1.95 ± 0.30 mg/L,respectively.Propionic acid,isobutyric acid and 2-Methylbutyric acid were the newly generated SCFAs.Acetic acid,butyric acid,isovaleric acid,valeric acid and hexanoic acid were 2.26,87.97,36.71,55.97 and 5.13 times higher than those at 0 h,respectively.Propionic acid,acetic acid,butyric acid and isovaleric acid were the most abundant SCFAs.In CON group,propionic acid,isobutyric acid,butyric acid,2-Methylbutyric acid and isovaleric acid were the highest at 24 h,which were 10.67 ± 0.99mg/L,4.68 ± 0.67 mg/L,14.33 ± 0.96 mg/L,2.78 ± 0.61 mg/L and 10.50 ± 1.42 mg/L,respectively.Butyric acid and isovaleric acid were 18.93 and 9.60 times of 0 h,respectively;the highest concentration of valeric acid was 8.56 ± 4.30 mg/L at 48 h,which was 16.19 times of 0 h.The four SCFAs with higher production were butyric acid,acetic acid,propionic acid and isovaleric acid,which were significantly different from LP group.(2)Changes of microflora and functional genome in the process of longan polysaccharide metabolism by intestinal floraThe results of metagenome sequencing showed that there was little difference in intestinal flora between LP and CON groups at phylum,family and genus levels.At 48 h,the relative abundance of Proteobacteria in LP and CON groups decreased from 10.59% at0 h to 7.90% and 7.86% respectively,and the relative abundance of Firmicutes increased from 39.98% at 0 h to 56.23% and 54.73% respectively.At the family level,longan polysaccharide could promote the proliferation of Lactobacillus salivarius,its relative abundance increased from 4.85% at 0 h to 8.60%.KEGG pathway analysis showed that there were significant changes in 10 pathways of LP metabolism,including carbohydrate metabolism,global and overview maps,amino acid metabolism and nucleotide metabolism.There were significant differences in glutathione metabolism,sulfur relay system,NF kappa B signaling pathway and PPAR signaling pathway between LP group and con group.Longan polysaccharide may affect the immune response process through the above four pathways.(3)Metabolite analysis of longan polysaccharide intestinal floraMetabonomics analysis showed that at 8 h,10 specific metabolites in LP group were saccharic acid、dodecanoic acid、phenylethylamine、trigonelline、(r)-mandelic acid、harman、4-pyridoxic acid、alpha-hydroxyisobutyric acid、stigmasterol、5-alpha-cholestan-3-one1.The 16 h specific metabolites were propionic acid、betaine、histamine、byssochlamic acid、l-homoserine、glycerol、d-arabitol and so on.The 24 h specific metabolites were lalanine、creatinine、pipecolic acid、ethenyl acetate and so on.The common metabolites at8 h and 16 h were maleic acid、phytosphingosine、2-oxovaleric acid、fluvoxamino acid、gamma-linolenic acid、beta-sitosterol、beta-glutamic acid 1、palatinose.Combined with the above results and literature analysis,the metabolite histamine、gamma-linolenic acid、trigonelline、betaine、SCFAs、l-homoserine、beta-sitosterol、phytosphingosine may be involved in the regulation of host immunity by metabolites of longan polysaccharide intestinal flora.(4)The effect of longan polysaccharide and its metabolites on the activation of mesenteric lymph node cellsThe results showed that longan polysaccharide and its metabolites could significantly increase the secretion of IL-10 and decrease the secretion of TNF-α and IL-12 in mouse mesenteric lymph node(MLN)cells.In the 4～12 h,the IL-10 secretion of LP group was257.33 pg/m L,212.75 pg/m L and 172.75 pg/m L respectively,which were significantly higher than that of con group,which were 1.80 times,1.23 times and 1.46 times respectively;The secretion of TNF-α was 96.69 pg/m L,126.69 pg/m L and 142.08 pg/m L,which were 71.54%,86.82% and 86.83% of con group,respectively;The secretion of IL-12 was 180.44 pg/m L,171.56 pg/m L and 188.22 pg/m L,which were significantly lower than that of con group(87.12%,85.11% and 88.04% respectively).These results indicate that longan polysaccharide can regulate the secretion of mln cytokines and inhibit the inflammatory response in the early stage of intestinal flora metabolism(4～12 h). |
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