【作者】 金青哲；

【导师】 裘爱泳；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2008， 博士

【摘要】 苯丙烯酰-5-羟色胺化合物（PAHA）具有清除自由基、抗氧化、抗肿瘤和强紫外吸收等活性，正日益受到关注，已有研究主要集中于各PAHA物质的分离、鉴定及其药理方面，而适合于工业化的提取纯化研究却很少，为此，本文以红花籽中二个主要PAHA，即香豆酰-5-羟色胺（CS）和阿魏酰-5-羟色胺（FS）为研究重点，在建立PAHA定量分析方法基础上，采用醇法浸取、生物转化、大孔树脂和硅胶吸附组合技术，进行了从红花籽粕中提取纯化PAHA工艺的研究，旨在得到回收率和纯度高且易于产业化的工艺条件。论文建立了快速检测PAHA的反相高效液相色谱分析方法，线性范围50-2000 ng，平均加标回收率99．0％，重复测定的变异系数小于3．2％；建立了艾氏试剂测定PAHA总量的分光光度法，线性范围0．05-0．25 mmol／L，平均加标回收率99．5％，重复测定的变异系数小于2．0％。两种方法的测定结果令人满意。由正交法确定了60％乙醇回流浸提红花籽PAHA的优化工艺条件，提取得率和纯度分别为9．96％和7．87％，PAHA得率为0．784％。浸出过程符合缩芯模型，受孔内扩散所控制。当物料粒度为35-80目时，模型可较好描述PAHA浸出动力学过程，误差20％以内。通过响应面法确定了β-D-葡萄糖苷酶转化香豆酰-5-羟色胺单葡萄糖苷（CSG）、阿魏酰-5-羟色胺单葡萄糖苷（FSG）为CS、FS的优化工艺条件，CSG、FSG的转化率均达90％以上，酶转化物中CS和FS总量由转化前的6．40％提高1．22倍，至7．76％。2-羟基牛蒡子苷等杂质含量下降，提取物品质提高。研究建立了XDA-1大孔吸附树脂柱层析法纯化PAHA的方法，确定了恒组成溶剂洗脱和分步洗脱条件。恒组成溶剂洗脱时，产物中CS和FS总量由7．76％提高到58．1％，较粗提物提高7．5倍，收率84．4％。分步洗脱时，产物中CS和FS总量由3．68％提高到47．0％，较粗提物提高12．8倍，收率为72．9％。在此基础上，建立了硅胶柱层析进一步精制PAHA的方法，产品纯度由58．1％提高至90．4％，收率87．2％。采用熔点测定、紫外和红外光谱、高压液相色谱／质谱、核磁共振谱对C₁₈层析柱分离得到的二个PAHA单体进行了结构鉴定，经与文献对照，分别确认为香豆酰-5-羟色胺（CS）和阿魏酰-5-羟色胺（FS），结构为反式。表明提取纯化和生物转化过程对CS、FS的化学结构无明显影响。采用DPPH·法、邻二氮菲法、邻苯三酚自氧化法和Rancimat法研究了四种PAHA化合物（FS、CS、CSG、FSG）的清除自由基活性，结果表明，清除自由基能力依次为CS≈FS>FSG>CSG，并存在浓度依赖关系。采用噻唑蓝法（MTT）初步研究了四种PAHA化合物的体外抗肿瘤活性。结果表明，CS、FS对肺癌细胞H446、肝癌细胞HepG-2、黑素瘤细胞B16具有一定的抑制癌细胞增殖活性，并存在浓度依赖关系，而对乳腺癌细胞MCF-7、MB-231无抑制活性。更多还原

【Abstract】 Phenylpropanoid amides of 5-hydroxytryptamine （PAHA） from safflower （carthamus tinctorius L.） seeds have a number of biological effects including free radical scavenging, anti-oxidant, anti-tumor, and UV absorption activities, and thus have attracted gaining attention globally in academia. However, previous researches have been concentrated primarily on the separation, characterization and pharmacological aspects, yet have paid scarce attention to larger scale extraction and purification of PAHA. This work is mainly concerned with N-（p-coumaroyl）serotonin （CS） and N-feruloylserotonin （FS）, two principal components of PAHA from safflower （carthamus tinctorius L.） seeds. Methods for quantitative determination of PAHA were established, followed consecutively by extraction of crude PAHA with ethanol aqueous solution, bioconversion, purification of PAHA, macroporous resin chromatography and silicon gel chromatography, and the process conditions was established for recovering PAHA at high yields and purity.A reversed phase high performance liquid chromatographic （RP-HPLC） method was established for rapid analysis of PAHA in safflower seed, which shows an average added standard recovery of 99.0% and a constant of variation of <3.2% in a linear range of 50-2000 ng. And a novel spectrophotometric method using p-dimethylaminobenzaldehyde （Ehrlich’s reagent）, was also developed for the determination of total PAHA in safflower （carthamus tinctorius L.） seeds, which shows an average added standard recovery of 99.5% and a constant of variation of <2.0% in a linear range of 0.05-0.25 mmol/L. Both methods exhibit good accuracy and precision.The process for crude PAHA extraction by 60% aqueous ethanol under reflux was established through orthogonal experiments, which gave a PAHA yield, purity, and extraction ratio of 9.96%, 7.87%, and 0.784%, respectively. The extraction process fitted well to the shrinking unreacted core model, with the pertaining kinetics governed by intra-particle diffusion. When the particle sizes of safflower cake meal is ranged between 35-80 meshes, this model can define the extraction kinetics of PAHA with an error below 20%.Deglycosidation of PAHA extracts from safflower cake meal withβ-D-glucosidase was optimized using response surface design to convert N-（p-coumaroyl）serotonin glucoside （CSG） and N-feruloylserotonin glucoside （FSG） to CS and FS, which gave conversion rates of >90% for both CSG and FSG, and remarkable reduction of impurities such as 2-hydroxyarctrin.A process of XDA-1 macroporous resin column chromatography for purifying PAHA was established. Conditions for both isocratic and stepwise elution were determined, which provided 7.5- and 12.8-fold concentration of PAHA with a recovery of 84.4% and 72.9%, respectively. A silica gel chromatographic process was then developed, using petroleum ether/acetone system as the eluent, to further purify PAHA to a purity and recovery of 90.4% and 87.2%, respectively.The two isoforms of PAHA as obtained were characterized with melting point determination, UV and IR spectroscopy, HPLC/MS, and NMR spectroscopy and, with reference to relevant literature, identified as N-（p-coumaroyl）serotonin （CS） and N-feruloylserotonin （FS）, both in trans configuration. It was suggested that the chemical structure of CS and FS were little changed in the extraction, purification, and biotransformation procedures.The free radical scavenging activities of FS, CS, CSG, and FSG were evaluated using DPPH·system, Fe²⁺/phen system, and pyrogallol auto-oxidation system, and by Rancimat assay. The free radical scavenging activities exhibited are dose-dependent and follow the order of CS≈FS>FSG>CSG.The cytotoxic effects of four isoforms of PAHA, i.e., FS, CS, CSG, and FSG, on various human malignant tumor cell lines were tested in vitro by MTT assay. FS and CS showed dose-dependent anti-proliferative activities to the lung tumor cell line H446, liver tumor cell line HepG-2, and the melanoma cell line B16, while had little such activities towards the breast tumor cell lines MCF-7 and MB-231.更多还原