【作者】 章海锋；

【导师】 陈启和；

【作者基本信息】 浙江大学 ， 食品科学， 2010， 硕士

【摘要】 天麻素(gastrodin)是一种酚型糖苷类化合物,它具有显著的生物学活性和药理活性。本论文主要研究黄绿密环菌生物转化合成天麻素的转化条件优化以及产物的分离纯化,对分离得到的产物进行结构鉴定,并对合成的天麻素进行动物试验评价,研究其抗炎以及免疫调节功能,并初步研究生物转化体系中葡萄糖基转移酶的酶学性质,为后续深入研究和调控天麻素生物合成提供试验依据。首先研究建立转化体系中天麻素和对羟基苯甲醇的检测方法,确定转化体系中天麻素和对羟基苯甲醇的反相高效液相色谱法(RP-HPLC),色谱柱为Waterssymmetry C18反相柱(4.6 mm×150 mm,5μm),流动相:0.02%磷酸溶液-甲醇(体积比为5:95),检测波长:220 nm,流速:0.5 mL/min,柱温35:℃。通过单因素实验和响应面实验设计对生物转化中的条件进行优化,确定相应的转化条件为初始pH 4.5,在30 mL转化液中底物浓度为3 mg/mL,加入静息细胞量为6.5 g,添加质量分数为3%的葡萄糖和1.5%的吐温80,并对转化动力学进行相应的研究,初步构建了转化合成动力学模型。初步研究转化体系中糖基转移酶的酶活测定方法和酶学性质,测得糖基转移酶的最适反应温度为50℃,在30℃稳定性最好;糖基转移酶的最适作用pH为6,在pH为6-7范围内稳定性较好。采用硅胶柱色谱、葡聚糖凝胶Sephadex LH-20等色谱分离方法相结合对转化产物进行分离纯化,分离得到的两个组分经RP-HPLC、核磁共振分析等分析证实是天麻素和对羟基苯甲醇。对合成的天麻素进行动物实验,研究其抗炎及对免疫功能的影响。研究证实合成的天麻素具有抗炎的作用,并且对正常小鼠的非特异性免疫和细胞免疫功能具有增强作用。更多还原

【Abstract】 Gastrodin is the important ingredients of Gastrodia elata Blume owing to its important pharmacological properties. In this thesis, the major research is focused on the optimization of biotransformation conditions for gastrodin from HBA, separation and purification of the products, and structural identification, and then carrying out the animal experiments to study its anti-inflammatory effection and immune function using the synthetic gastrodin, and finally investigating the characterisitcs of glucosyltransferase from the biotransformation system. This study will provide the basis for understanding the biotransformation mechanism and regulating the gastrodin production.The HPLC system used throughout this study consisted of two Waters 510 pumps, a sample injector （Rheodyne, Cotati, CA, USA） with a 20μL loop, and a Waters 996 photodiode array detector. The column used was a reversed-phase Symmetry C18 column （150 mm×3.9 mm i.d., 5 urn; Waters）. The mobile phase was acetonitrile （solvent A）-0.02% phosphoric acid solution （solvent B） in the aso-gradient mode as follows: 95% A and 5% B. The flow rate was 0.5 mL/min, and the effluent was monitored at 220 nm. Routine sample calculations were made by comparison of the peak area with that of the standard curve.Single factor experiments and response surface methodology were applied to optimize the bioconversion conditions for gastrodin production. The optimized results showed that the optimum transformation conditions composed of 3 mg/mL HBA, 6.5 g/30 mL inoculums level, 1.5% Tween 80, pH 4.5, and transformation temperature at 28℃. The bioconversion kinetics model was simulated and constructed in this study.The glucosyltransferase enzyme for biosynthesis of gastrodin from HBA was preliminary studied. The optimum temperature of the enzyme reaction was 50℃, and the pH was 6.0. The enzyme was stable at less than 50℃, and pH 6.0～7.0. The transformed products were obtained and identified by extraction, chromatography and its analysis from ¹H, ¹³C-NMR. The bioactive functions of the synthetic gastrodin were examined in vivo design, which include anti-inflammatory and immune function. It was confirmed that the synthetic gastrodin had anti-inflammatory effection and immune stimulatory function.更多还原