【作者】 韩铨；

【导师】 何普明；

【作者基本信息】 浙江大学 ， 茶学， 2011， 博士

【摘要】 茶树花富含营养成分和活性物质,是一类具重要的潜在资源。本文在系统研究茶叶多糖分离纯化、理化性质和一级结构的基础上,利用生物大分子研究的近代最新技术和现代分离、分析手段与药理实验方法,表征茶树花多糖的溶液行为和三维链构象,尝试揭示多糖的高级结构与其重要生物活性的关系。茶树花多糖的提取主要包括茶树花多糖提取条件的优化和茶树花多糖脱蛋白,脱色等方法的选择。采用单因素实验和Box-Behnken中心组合设计实验,确定了热水提取茶树花多糖最佳提取条件：21.75倍量水,90。C提取1.89h。通过综合考量Sevag法、三氯乙酸法、盐酸法和氯化钙法的脱蛋白率和多糖保留量,确定了以Sevag法作为茶树花多糖脱蛋白的方法。实验还比较了活性炭吸附法,H2O2氧化法和溶剂洗涤法的脱色效果,活性炭吸附法能够得到较高的脱色率和多糖保留量。经Sephadex G-100凝胶柱层析分离得到多糖组分TFP-1和TFP-2,冷冻干燥后为灰白色粉末,多糖含量为83.6%和87.9%,蛋白含量为1.5%和2.9%,糖醛酸含量为2.7%和7.1%。高效液相凝胶色谱法分析茶树花多糖组分TFP-1和TFP-2,得到TFP-1的分子量为167.5kDa,单糖组成葡萄糖：木糖：鼠李糖：半乳糖=1.0：1.2：0.81：0.98。TFP-2分子量为10.1kDa,单糖组成葡萄糖：木糖：鼠李糖：阿拉伯糖=1.0：0.76：2.3：2.3。多糖的初步分析显示,TFP-1和TFP-2是水溶性、非淀粉、非酚类物质,不含还原糖和核酸、含有蛋白质的多糖。β-消去反应表明TFP-1和TFP-2中糖链与蛋白质之间通过0-糖肽键相连。通过红外光谱分析得出TFP-1和TFP-2具有典型的糖类化合物的光谱特征,TFP-1同时含有α-吡喃环以及β-吡喃环的特征吸收峰,而TFP-2只含有α-吡喃环的特征吸收峰。1H NMR中的化学位移显示TFP-1含有α-D-Galp、α-D-GalpNAc、α-D-Xylp、α-D-Glcp和β-D-Glcp残基,而TFP-2含有α-L-Arap、α-D-Glcp、α-D-Xylp和α-D-GlcpNAc残基。原子力显微镜(AFM)的图像显示,茶树花多糖链分子间互相缠绕,在浓度为5μg/mL下TFP-1和TFP-2直径为250nm和150nm,在浓度为20μg/mL时直径增加为500nm和200nm。茶树花粗多糖TFP及组分TFP-1和TFP-2在水溶液中表现为剪切变稀,在相同的剪切速率下,茶树花多糖溶液粘度TFP>TFP-1>TFP-2。通过激光光散射、粘度测定和刚果红反应等方法确定了茶树花多糖在0.1MNaCl水溶液中呈现球状构象,且分枝较多。pH值、温度、离子强度等环境因素的改变均影响其立体构象。扫描电镜SEM观察和X-射线衍射分析表明TFP-1和TFP-2为无定形结构,提示多糖的空间结构和溶液行为将明显影响多糖的生物活性。系统的研究了茶树花多糖的体外和体外抗氧化活性。通过比较茶树花粗多糖TFP及组分TFP-1和TFP-2对羟基自由基,超氧阴离子自由基以及DPPH自由基的清除活性,得出抗氧化活性TFP-2>TFP-1>TFP,这可能与组成多糖的成分及水溶性和粘度有关。灌胃茶树花多糖(75、150和300mg/kg) 28d后可抑制溴化苯诱导的小鼠肝脏脂质过氧化,能显著提高小鼠SOD活性和T-AOC水平,并且抑制MDA含量的升高。灌胃茶树花多糖(75、150和300mg/kg)14d后能显著增加正常小鼠巨噬细胞的吞噬活性,对二硝基甲苯诱导的小鼠迟发型超敏反应也有显著的增强。对S180肉瘤小鼠的研究发现,灌胃TFP(75、150和300mg/kg) 7d后能显著改善荷瘤小鼠生活状况,显著增加荷瘤小鼠生存时间和存活率,抑瘤率分别为45.5%,60.9%和64.5%。能显著增加荷瘤小鼠血清中IFN-γ和IL-2含量的影响,并且能增加T细胞亚群CD4+的数量及改善CD4+/CD8-的失调,提示摄入茶树花多糖可明显增强免疫活性。茶树花多糖对四氧嘧啶引起的小鼠实验性糖尿病有明显的防治作用。灌胃茶树花多糖(75、150和300mg/kg)21d后,能明显改善糖尿病小鼠的“三多一少”状况,显著降低四氧嘧啶诱导的糖尿病小鼠的血糖(p<0.01)。茶树花多糖在体外可以抑制α-淀粉酶和α-葡萄糖苷酶活性,在0.2-2.0mg/mL浓度范围内呈量效关系。更多还原

【Abstract】 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.更多还原