| Polysaccharide is an important bioactive macromolecule of plant chemical components.It has many biological activities,such as immune regulation,anti-tumor,anti-inflammatory,anti-oxidation,hypoglycemic and so on.Solanum muric atum is herbaceous plant of Solanaceae family.It has many biological activities,such as anti-oxidation,anti-tumor and anti-diabetes.The main research contents are as follows:?1?Response surface method was used to optimize the extraction of SMP.The results of Box-Behnken experiment showed the best extraction conditions of the polysaccharide were:the ratio of solid to liquid was 1:61,the extraction temperature was 90?,and the extraction time was 214 min.The extraction rate was 10.12±0.36%,which was in agreement with the prediction value of response surface method?10.29%?.Antioxidant experiments in vitro showed that the crude polysaccharide?SMP?has strong scavenging activities of hydroxyl radical,DPPH radical and ABTS radical.The EC50were 2.194 mg/m L,0.94 mg/m L and 3.68 mg/m L,respectively.?2?The crude polysaccharide?SMP?from Solanum muricatum was isolated and purified.Firstly,The protein in the crude polysaccharides from Solanum muricatum was removed by trifluoroacetic acid.Then,the neutral polysaccharide SMP-0 and acid polysaccharide SMP-3 were purified by DEAE-52 anion exchange chromatography.The neutral polysaccharide SMP-0b and acidic polysaccharide SMP-3a were obtained after purified by Sephadexgel column chromatography.Scanning electron microscopy showed that the SMP-0b mainly presented irr egular lamellar structure,while the SMP-3a mainly presented lamellar structure and occasionally fragmentary.The results of AFM showed that SMP-0b mainly showed random linear chain,while SMP-3a mainly showed many spherical and uneven blocks.Thermogravimetric analysis showed that SMP-0b and acid polysaccharide SMP-3a had good thermal stability,and the pyrolysis temperature was 258?and 230?,respectively.?3?SMP-0b and SMP-3a are plant polysaccharides with unique molecular structure.The homogeneity and high purity of these two purified polysaccharides were determined by high performance gel permeation chromatography.The aver age molecular weight of neutral polysaccharide SMP-0b was 13.51 k Da,and the average number was 9.91 k Da,and the polydispersity index was 1.36.The weight average molecular weight of SMP-3a was 227 k Da,the number average molecular weight was 125.1 k Da,and the polydispersity index was 1.81.FT-IR analysis showed that both SMP-0b and SMP-3a had typical characteristic ab sorption peaks of polysaccharide.The analysis of monosaccharide compositions howed that SMP-0b was composed of arabinose,mannose,glucose and galactose,with a molar ratio of 5.31:2.92:42.23:25.38;SMP-3a was mainly composed of rhamnose,arabinose,galactose and galacturonic acid,with a molar ratio of 1.09:2.64:1.54:1.The results of methylation analysis revealed that SMP-0b was consisted of T-linked-Araf,1,3,6-linked-Manp,1,4-linked-Galp,1,4-linked-Glcp,and 1,6-linked-Glcp and the relative molar percentages were 6.30%,6.71%,17.56%,16.59%and 52.84%,respectively.SMP-3a was consisted of T-lin ked-Araf,1,5-linked-Araf,1,2-linked-Rhap,1,4-linked-Galp A and 1,4,6-linked-Ga lp and the relative molar percentages were15.7%,32.7%,18.2%,17.7% and 15.7%.The results of NMR analysis showed that the main chain of SMP-0b was consisted of?4)-?-D-Galp-?1?,?3,6?-?-D-Manp-?1?and?6?-?-D-Glcp-(1?,and the branch chain was composed of ?-L-Araf-?1?and?4?-?-D-Glcp-?1?.The side chain was substituted at O-3 of?3,6?-?-D-Manp-?1?.The main chain of SMP-3a was detected to be?2?-?-L-Rhap-?1?4?-?-D-Galp A-?1?4?-?-D-Galp-?1?and the side chain was substituted at O-6 of?4,6?-?-D-Galp-(1?.The branch of SMP-3a was composed of ?-L-Araf-?1?and?5?-?-L-Ara f-(1?.?4?SMP-0b and SMP-3a could activate macrophages.CCK-8 assay and morphological observation indicated that SMP-0b and SMP-3a can obviously promote the proliferation of RAW264.7 macrophages and stimulate the differentiation of macrophages;Neutral red test showed that SMP-0b and SMP-3a can enhance the phagocytic ability of macrophages;Meanwhile,SMP-0b and SMP-3a can significantly promote the production of nitric oxide in macrophages and promote the secretion of TNF,IL-6 and IL-1?in RAW264.7 macrophages.These results indicated that SMP-0b and SMP-3a can exert their immunoregulatory activities by activating macrophages.?5?SMP-0b and SMP-3a could regulate the immune related genes of RAW264.7 macrophages.Compared with the negative control group,869 differentially expressed genes were screened out after the treatment of SMP-0b,including 353 up-regulated genes and 516 down-regulated genes.Three significant GO terms screened out according to the biological process classification by GO enrichment analysis were immune related.There are 204 differentially expressed genes enriched in GO:0002376,including 92 up-regulated genes and 112 down-regulated genes;139 differentially expressed genes enriched in GO:0006955,inc luding 69 up-regulated genes and 70 down-regulated genes;128 differentially expressed genes in GO:0002682,including 61 up-regulated genes and 67 down regulated genes.Through KEGG pathway analysis,according to the classification of environmental information processing,six pathways with significant enrichment of differentially expressed genes were screened out.Then,the position of the differentially expressed genes in the cyclokine-cyclokine receptor interaction pathway was determined by KO analysis,and the related genes upstream and downstream were determined.Compared with the negative control group,1117 differentially expressed genes were screened out after the treatment of SMP-3a,including 523 up-regulated genes and 594 down-regulated genes.The first three most significant GO terms screened by GO enrichment analysis are related to immunity.There are 271 differentially expressed genes enriched in GO:0002376,including 148 up-regulated genes and 123 down-regulated genes;185differentially expressed genes enriched in GO:0006955,including 108 up-regulated genes and 77 down-regulated genes;and 178 differentially expressed genes enriched in GO:0002682,including 103 up-regulated genes and 75 down-regulated genes.According to the classification of environmental information proce ssing by KEGG pathway analysis,four pathways with significant enrichment of differentially expressed genes were screened out.Then,the position of the dif ferentially expressed genes in the cyclokine-cyclokine receptor interaction pathway was determined by KO analysis,and the related genes upstream and downst ream were determined.Finally,the relative expression levels of TNF,IL1?,C CL2,CCR1,CXCR4 and CX3CR1 genes were detected by RT-qPCR.The results were consistent with RNA-seq,which proved the reliability of the results of RNA-seq.The above results showed that the neutral polysaccharide SMP-0b and acid polysaccharide SMP-3a,which were isolated from Solanum muricatum were two new polysaccharides with novel structures,and they could exert immunomodulatory activity by activating macrophages. |
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