Isolation, Purification, Structure Characterization And Bioactivity Of Tea Flower Polysaccharide

Tea (Camellia sinensis) flower is rich in nutritional components and active substances, from this point, tea flower as a kind of potential resources which has important application values. The physicochemical properties and the chemical structure of tea flower polysaccharide (TFP) and its fractions (TFP-1 and TFP-2) were studied in this doctoral dissertation. The solution behavior and three-dimensional conformation of TFP were also investigated here, with the advanced methods of isolation and purification technique, chemical and instrument analysis technique of biomacromolecular. The correlation between bioactivities and the conformation of polysaccharides was discussed.Response surface methodology was applied to optimize the hot-water extraction parameters for TFP with a Box-Behnken design. The optimal conditions to obtain the highest yield of TFP were temperature,90℃; time,1.89 h and liquid-solid ratio,21.75. The protein of TFP are removed by using one of the four following methods (sevag reagent, trichloroacetic acid, HC1 or CaCl2), respectively. Considering the deproteinization rate and inventory of polysaccharide, sevag method was chosen for the deproteinization. The decoloration by active carbon, organic solvent and H2O2 were also studied, the results showed that active carbon was more effective in this case. TFP-1 and TFP-2, which were two homogeneous polysaccharide fractions, were separated from TFP by Sephadex G-100 column chromatography. After freeze-dried, it was found that TFP-1 and TFP-2 were grayish powder and the contents of uronic acid, protein and sugar were 2.7 and 7.1%; 1.5 and 2.9%; 83.6 and 87.9%, respectively.The high performance gel filtration chromatography analysis showed that TFP-1 was composed of Glc:Xyl:Rha:Gal=1.0:1.2:0,81:0.98 with a molecular weight of 167.5 kDa, while TFP-2 comprised Glc:Xyl:Rha:Ara=1.0:0.76:2.3:2.3 with a molecular weight of 10.1 kDa. TFP-1 and TFP-2 were water soluble and have trace protein conjugate with polysaccharide. In addition, no starch, phenol, reducing sugar and nucleic acid has been found in them. The results ofβ-elimination reaction indicated that the chain of polysaccharides and protein was connected by 0-linked chemical bond. The IR analysis showed that TFP-1 containedαandβtype glycosidic linkages, while onlyαtype glycosidic linkage in TFP-2. The'H NMR revealed that TFP-1 containedα-L-Rhap,α-D-Galp, a-D-GalpNAc,α-D-Xylp,α-D-Glcp andβ-D-Glcp residues, while. TFP-2 was illustrated to haveα-L-Rhap,α-L-Arap,α-D-Xylp,α-D-Glcp andα-D-GlcpNAc residues.By using technique of atomic force microscope, we found that with the concentrations of 5 and 20μg/mL, the diameters of spherical lumps of TFP-1 and TFP-2 were about 250 nm and 150 nm,500 nm and 200 nm, respectively. The polysaccharide solution showed significant shear thinning behavior, and under a same shear rate, the viscosity showed TFP>TFP-1> TFP-2; The three dimensional conformation and solution behavior were studied by using the technique of laser light scattering and viscometer, the results confirmed that TFP-1 and TFP-2 existed almost as a branched spherical shape in 0.1 M NaCl aqueous. The conformation could be changed by the factors of pH, temperature, ion intensity etc. The results of SEM and X-ray indicated that the state structure of tea flower polysaccharides was noncrystal.The antioxidant effects of TFP and its fractions against DPPH radical, superoxide radical and hydroxyl radical were investigated in vitro assay systems compared with ascorbic acid, the antioxidant activity showed TFP-2>TFP-1>TFP, which was attributed to its relatively low molecular weight, low apparent viscosity and particularity of chemical composition. Administration of TFP (75,150 and 300 mg/kg) for 28 d continuously was found not only to be protective on liver lipid peroxidation induced by bromobenzene in mice through increasing the activity of superoxidase dismutase, total antioxidant capacity, but also markedly to attenuate the enhancement of malondialdehyde content in a dose dependent manner.After administration of TFP (75,150 and 300 mg/kg) for 14 d, there were a enhancement for macrophage phagocytic activity and delayed type hypersensitivity response induced by dinitrofluorobenzene in normal mice. Using S180 tumor bearing mice as model, research showed that continuous administration of TFP (75,150 and 300 mg/kg) for 7 d was found to inhibited the growth of transplanted S180 and prolonged the mice survival days, promoted the plasma interleukin-2, interferon-γlevels and improved the T-lymphocyte subsets CD4+ and CD4+/CD8+ ratio.Furthermore, TFP-2 was presented dose dependent inhibitory effects against a-glucosidase andα-amylase in vitro assay systems. Moreover, continuous administration of TFP-2 (75,150 and 300 mg/kg) for 21 d improved the life quality of alloxan-induced diabetic mice, and also caused a significant (p<0.01) decrease in blood glucose levels.