| Cyclocarya paliurus(Batal.).Iljinskajais native to China and the sole species in its genus belongs to the genus Cyclocarya Iljinskaja(Juglangdaceae).It is a precious tree species with various values,such as medicinal,health care,timber use and landscaping.Polysaccharide is one of the important bioactive substances in the leaves of C.paliurus,which has extensive biological activities,such as hypoglycemic,hypolipidemic,enhancing immunity,antioxidant,antifatigue,antimicrobial and anticancer.In this study,the leaves of C.paliurus at different developmental stages(from the smallest fully expanded leaves to the leaves with the largest width and thickness,it is divided into four developmental stages: F1,F2,F3 and F4)were used as materials,the polysaccharides were extracted by hot water extraction,the content of polysaccharides was determined by phenol-sulfuric acid method and the polysaccharides were refined by deproteinization,decolorization and other impurity removal methods.The differences of the content,physical and chemical properties of polysaccharide were explored in the leaves of C.paliurus at different developmental stages.Transcriptome sequencing was performed to analyze genes related to polysaccharide biosynthesis and accumulationin the leaves of C.paliurus at different developmental stages to elucidate the biosynthetic pathway of polysaccharides in the leaves development of C.paliurus.The antioxidant properties of polysaccharide were comparatively investigated by DPPH radical assay,hydroxyl radical assay,superoxide radical assay and iron ion reducing ability to explore the differences of the antioxidant activity of the polysaccharides in the leaves of C.paliurus at different developmental stages.The inhibition ability of lipase activity,binding ability of sodium glycocholate and sodium taurocholate of polysaccharides were determined to explore the differences of the hypolipidemic ability in vitro of the polysaccharides in the leaves of C.paliurus at different developmental stages.The main results of this study are shown as follows:(1)During different developmental stages of C.paliurus,the polysaccharide content increased at first and then decreased.The content of polysaccharide in the leaves was the highest at F3 stage,16.373 mg/g(1.637%),while that at F1 stage was the lowest(10.572 mg/g).There was no significant difference in the content of uronic acid in polysaccharides in the leaves of C.paliurus during different developmental stages.The content of conjugated protein showed a trend of first decline and then increase.Among them,the content of conjugated protein in polysaccharide at F1 stage was the highest(2.944%)and the lowest at F2 stage(0.715%).Ultraviolet and infrared spectra of polysaccharides in leaves during different developmental stages confirmed that the polysaccharides contained conjugated proteins.Infrared spectrum analysis confirmed that the polysaccharides contained uronic acid,and also containedĪ²-glycosidic bond configuration.The infrared spectra of polysaccharides in the leaves during four developmental stages are similar,indicating that the skeleton of the carbon chain is the same,but the molecular weight,monosaccharide composition ratio and some sugar chain connection modes of polysaccharides might be different.(2)A total of 70386240613 sequencing data were obtained by transcriptome sequencing,which were assembled into 296593 unigenes.The number of unigene annotated to Swissprot,NR,NT,PFAM,GO,KEGG,KOG,CDD and Tr EMBL databases was 118930,173809,104770,57649,145508,10104,64021,89954 and 118930,respectively.And 191829 sequences were annotated to at least one database.A total of 880 DEGs were identified in the leaves between F1 and F2 stages.A total of 690 DEGs were identified in the leaves between F2 and F3 stages.A total of 3649 DEGs were identified in the leaves between F3 and F4 stages.The results showed that there were differences in gene expression between different leaf development stages.(3)A total of 470 glycosyltransferase genes,556 glycoside hydrolases genes,173 carbohydrate esterases genes,99 carbohydrate-binding modules genes,and 35 polysaccharide lyases genes were identified by CAZy database based on gene sequence similarity.Among them,470 glycosyltransferases genes were divided into 57 glycosyltransferase families,including 40 differentially expressed genes,which belong to 13 glycosyltransferase families.The biosynthesis pathway of C.paliurus polysaccharides showed that each enzyme was encoded by multiple genes and had different expression trends during four developmental stages.Correlation analysis found that a total of 150 differentially expressed transcription factors were correlated with the content of polysaccharides and grouped into 38 transcription factor families.The expression of a gene encoding UDP-glucose 4-epimerasewas significantly positively correlated with the content of polysaccharides,while the expression of six genes related with the biosynthesis of polysaccharides were significantly negatively correlated with the content of polysaccharides.(4)In a specific range of polysaccharide concentration,the clearance rate of DPPH radicals,superoxide anion radicals,hydroxyl radicals,and the iron ion reducing ability of polysaccharides in the leaves of C.paliurus during different developmental stages have a dose-dependent relationship with the concentration of polysaccharides.The clearance rate of radicals and the iron ions reduction ability of these polysaccharides increased with the increasing concentration,but it did not show a linear relationship.Compared with the other three stages,the polysaccharides in the leaves at F1 stage had higher antioxidant activity.The scavenging rate of DPPH radicals at F1 stage was similar to that of Vitamin C(Vc)and higher than those at the other three stages,while the scavenging rate of DPPH radicals of polysaccharides in the leaves at F4 stage was less than 50%.With the increasing of concentration,the superoxide anion radicals scavenging rates of polysaccharides in the leaves at F1,F2 and F3 stages were similar and lower than Vc,while that of polysaccharides in the leaves at F4 stage was lower than those at the other three stages and less than 50%.The hydroxyl radicals scavenging rate of polysaccharides in leaves at F1 stage was higher than those at the other three stages,but all of the four stages were lower than Vc and less than 50%.The iron ion reduction ability of polysaccharides at four stages was F1>F3>F2>F4.Considering the factors such as polysaccharide content and leaf morphology,this result suggested that leaves at F3 stage was the best choice for products to develop C.paliurus polysaccharide antioxidant products.(5)There were differences in the hypolipidemic activities in vitro of polysaccharides in the leaves of C.paliurus during different developmental stages.The inhibition ability of lipase,and the binding ability of sodium glycocholate and sodium taurocholate of polysaccharides in the leaves of C.paliurus during different developmental stages showed a dose-dependent relationship with the concentration of polysaccharides.The inhibition and binding ability of these polysaccharides increased with the increasing of concentration,but it did not show a good linear relationship.The lipases activity inhibition rates of polysaccharides in the leaves at F1 and F2 stages were lower than those at the other two stages,while at high concentration(6 mg/m L),the inhibition rates of polysaccharides in the leaves at F3 and F4 stages were twice higher than those at the other two stages.The binding rate of sodium glycocholate and sodium taurocholate from polysaccharide during four development stages was different with different concentrations,and the polysaccharides in the leaves at the F3 stage had higher binding ability of sodium glycinate.The polysaccharides at F4 stage had higher sodium taurocholate binding ability.These results showed that the polysaccharides in the leaves at F3 and F4 stages had higher hypolipidemic activity in vitro,indicating that the leaves at F3 and F4 stages were the best to develop polysaccharide hypolipidemic products. |
PDF Full Text Request