Structural Characterization, Physicochemical Properties And Hepatoprotective Activity Of Polysaccharides From Toona Sinensis Leaves

In this paper,Toona sinensis leaves was used as raw material,and the structural characteristics,physicochemical properties and hepatoprotective activity of Toona sinensis leaves polysaccharide were studied.T.sinensis leaves was dried and pulverized to powder,and the powder was subjected to hot water extraction,80%ethanol precipitation,decoloration with macroporous resin AB-8 and deproteinization with Sevag reagent to obtain crude polysaccharide fraction?CTSP?.Then CTSP was further separated and purified by DEAE-Cellulose 52 ion exchange column and Sephacryl S-400 HR gel column,and two homogeneous polysaccharide fractions?named as TSP-1 and TSP-2?were obtained,their molecular weights were 833.6 and 81.6 KDa,respectively.Chemical composition analysis indicated that the total carbohydrate contents of TSP-1 and TSP-2 were 89.62%and 86.95%,respectively,their uronic acid contents were 4.94%and 4.72%,respectively,and they contained no protein.Besides,both TSP-1 and TSP-2 were composed of Manp,Glcp A,Glcp,Galp,Xylp and Araf with the molar ratios of 17.16:7.46:25.37:21.64:14.92:13.43and 11.42:7.14:21.42:14.28:12.14:33.57,respectively.According to the results of monosaccharide compositions,FT-IR,periodate oxidation,Smith degradation,methylation analysis and NMR analysis,the backbone of TSP-1 might consist of?6)-?-D-Glcp-?1?,?3,6?-?-D-Manp-?1?and?6?-?-D-Galp-?1?,and the branches were substituted at C-6 of?3,6?-?-D-Manp-?1?.Four branches might consist of?3?-?-D-Glcp A-?1?,?4?-?-D-Xylp-?1?,?6?-?-D-Galp-(1?,?-L-Araf-(1?and?-D-Glcp-?1?.While the backbone of TSP-2 might be composed of?6?-?-D-Galp-?1?,?3,5?-?-L-Araf-?1?,?4?-?-D-Xylp-?1?and?6?-?-D-Glcp-?1?,and the branches were substituted at C-3 of?3,5?-?-L-Araf-(1?.Two branches might consist of?-D-Galp-(1?,?-D-Glcp-?1?,?3,6?-?-D-Manp-?1?and?3?-?-D-Glcp A-(1?.The results of Cango red assay and atomic force microscope?AFM?showed that TSP-1 and TSP-2 had no triple helix structure.Physicochemical properties showed that TSP-1 and TSP-2 had lamellar morphology,and TSP-1 and TSP-2 had thermal stability.The intrinsic viscosities of TSP-1 and TSP-2in NaNO₃ solution were 11.42 mL/g and 6.13 mL/g,respectively.Rheological analysis indicated that the viscosities of TSP-1 and TSP-2 were affected by polysaccharide concentration,Ca²⁺and extreme pH.Furthermore,TSP-1 exhibited a weak gel behavior at the concentrations of 0.5%-2.0%,while TSP-2 showed a weak gel behavior at the concentration of 2%.High performance size-exclusion chromatography coupled multiple angle laser light scattering?HPSEC-MALLS?and transmission electron microscopy?TEM?analysis showed that TSP-1 behaved as a sphere in solution,while TSP-2 behaved like a rigid rod.In vivo hepatoprotective activity showed that TSP-1 and TSP-2 could improve CCl₄-induced mice acute liver injury.Pretreatment with TSP-1 and TSP-2 significantly decreased the activities of aspartate aminotransferase?AST?and alanine aminotransferase?ALT?in mice serum,increased the activities of superoxide dismutase?SOD?,glutathione peroxidase?GSH-Px?,catalase?CAT?and the level of glutathione reductase?GSH?in mice liver.Meanwhile,the level of malondialdehyde?MDA?in mice liver was notably reduced after pretreatment with TSP-1 and TSP-2.Furthermore,administration with TSP-1 and TSP-2 could obviously decrease the concentrations and relative expression levels of tumor necrosis factor-??TNF-??,interleukin IL-6?IL-6?in mice liver.Histopathological observation showed that TSP-1 and TSP-2 could attenuate CCl₄-induced hepatocyte injury to some extent and reduced cell necrosis area.