【作者】 张林；

【导师】 何强；

【作者基本信息】 四川大学 ， 轻工技术与工程， 2023， 硕士

【摘要】 黄花菜作为一种药食同源的经济型农产品在我国已有数千年的种植历史,既具备丰富的营养价值,又兼具清热止血、明目安神等保健功能。黄花菜因含大量糖类、蛋白类营养物质,采摘后极易发生腐败变质,多年以来,黄花菜以晒干干制为主要采后加工方式,便于长期储存。与干制黄花菜相比,鲜黄花菜具备更高的营养价值和保健价值,近年来以黄花菜为原料的黄花产业链发展迅猛,这对黄花类食品的食品食用安全性提出了更高的要求。众所周知,鲜黄花菜处理不当会引起食物中毒,普通成年人一次性摄入处理不当的新鲜黄花菜超过200 g就可能产生头晕、腹泻、呕吐、胃绞痛等急性中毒症状。最早从1978年的黄花食用中毒事件报道开始至2020年三十多年间,大量研究及报道明确指出,黄花菜中的主要致毒成分为秋水仙碱。近两年,有学者指出过去对黄花中秋水仙碱的定性、定量研究存在不足,对黄花中是否存在秋水仙碱产生了质疑。在第一部分研究工作中,对四川渠县黄花进行了更深入和完善的秋水仙碱定性、定量研究,采用高效液相色谱-质谱仪,选取分离效果更好的长色谱柱,验证了黄花中秋水仙碱的存在,并精确检测出与秋水仙碱标准物质相同出峰时间处物质的相对分子量。结果表明,相同洗脱条件下,黄花提取物在与秋水仙碱标准物质几乎同一保留时间处出现明显吸收峰,且该吸收峰处的化合物检测出与秋水仙碱具有相同的相对分子量。进一步通过标准曲线绘制法,测算出黄花菜中秋水仙碱含量约为31.81μg/g,这与过往文献报道的黄花菜导致中毒的食用量保持一致,同时也与我国官方规定秋水仙碱的致毒剂量吻合。在第二部分的研究工作中,探究了秋水仙碱的光降解过程的机理并对光解产物作了定性分析。过往研究中,鲜黄花菜中毒事件屡见报道,而传统晒干黄花未见中毒的报道,因此考虑到黄花中的秋水仙碱是否在干制工艺过程中发生了变化。以秋水仙碱标准物质为研究对象,将秋水仙碱溶液曝露于光照下发现,光照前后,秋水仙碱溶液的紫外吸收扫描光谱图发生了明显变化,进一步将经过充足光照后的秋水仙碱溶液进行了高效液相色谱-质谱分析,确定了光解产物主要成分的分子量,结合紫外扫描波谱分析及以往文献报道,初步推断秋水仙碱的光解产物为去乙酰氨基光秋水仙碱。第三部分研究工作对秋水仙碱的光解进行了动力学分析,并探究了食品加工过程中各种环境因素（温度、p H值、溶剂和光源）对秋水仙碱光解的影响。依据朗伯比尔定律,对秋水仙碱溶液在光照过程中的浓度进行定量分析。结果表明,秋水仙碱的光解过程满足一级动力学方程。对比不同条件下秋水仙碱的光解动力学常数发现,溶剂和光源波长对光解速率影响最大,当光源为波长100-290 nm的紫外光且溶剂为乙醇时光解动力学常数达到最大为0.5862 h^-1,溶液中的离子强度大小对光解转化率影响较大,在低离子强度的无水乙醇溶剂条件下,秋水仙碱的光解转化率最高达到99.8%,而在p H为4的酸性条件下光解转化率仅为55.8%。第四部分研究工作中,按梯度稀释的方法分别配制了不同浓度的秋水仙碱及光照处理后的秋水仙碱溶液,并将二者分别作用于正常条件培养下的大鼠肝细胞（BRL 3A）,探究了两种药物在不同浓度下对大鼠肝细胞细胞活力的影响,并对细胞进行染色,通过倒置显微镜和荧光显微镜观察对比了加药前后大鼠肝细胞形态变化,同时选择了其中一组实验组进一步进行了细胞流式分析,检测药物对大鼠肝细胞细胞周期的影响,在该实验组的药物浓度下,大鼠肝细胞的细胞活力差异最明显。结果表明,秋水仙碱的光解产物对细胞活力的抑制能力显著降低,其半抑制浓度为1.35μg/m L。秋水仙碱的半抑制浓度为0.23μg/m L。此外,秋水仙碱光解产物对大鼠肝细胞的G2/M期阻滞能力显著降低,G2/M期细胞占比约为28.8%,秋水仙碱处理后的细胞G2/M期占比为43.0%,且经过秋水仙碱处理后的细胞,在流式分析结果图中显示细胞碎片更多,秋水仙碱光解产物组处理的细胞碎片更少,说明光照后的秋水仙碱对细胞凋亡的诱导能力显著降低。同时,还进一步探究了秋水仙碱及其光解产物对人肾上皮细胞（293T）和人肺癌细胞（A549）活力的影响,对秋水仙碱光解产物的抗癌活性进行了初步分析,为秋水仙碱衍生物抗癌药物的研发提供理论依据和参考。更多还原

【Abstract】 Daylily flower has been planted for thousands of years in China as an economic agricultural product with homology to medicine and food.It has abundant nutritional value and health care functions such as clearing heat,stanching bleeding,improving eyesight,and calming the nerves.Harvested daylilies rot very fast due to a large amount of polysaccharide and protein.Over the years,the main processing methods of daylily flowers have been hot air drying or natural drying with sunlight for longterm storage.Fresh daylilies have a much higher value of health care and nutrition than dried ones.The development of the daylily industry has been growing fast in recent years,and higher standards of food safety related to daylily foods have been proposed.It is generally known that daylily flowers can cause poisoning if not treated well.An average adult may suffer from dizziness,diarrhea,vomiting,gastralgia and etc.,when taking over 200 g of not-well-treated daylily flowers at a time.From the earliest report of a daylily poisoning event in 1979 to 2020,numerous researches indicate that the main toxic composition of daylily flower is colchicine.In the last two years,some scholars have pointed out that the previous qualitative and quantitative researche of colchicine in daylily flowers was insufficient and incomplete.As a result,they have questioned whether colchicine is present in daylily flowers.In the first part of this research,deeper and more complete qualitative and quantitative research was carried out using LC/MS and a longer chromatographic column with better separation efficiency to detect the accurate peak time and relative molecular weight.The purpose was to prove the existence of colchicine in daylily flowers.The results showed that one big absorption peak whose retention time was extremely close to the colchicine standard was detected,and the relative molecular weight of the main peak substance was also the same as the colchicine standard.Next,the concentration of colchicine in daylily flowers was calculated through standard curve,and it was deduced by calculation that the concentration of colchicine was 31.8075 μg/g.The result matched previous reports and the national standard.In the second part of the research,the photolysis mechanism of colchicine was analyzed and the photolysis product was investigated.Fresh daylily poisoning events were frequently reported previously,while the dried daylily was not.Accordingly,the drying process appeared to cause colchicine change.Therefore,colchicine standard was used as research object and exposed under the light.After exposure,the UV absorbance scanning spectrum of colchicine changed significantly.Irradiated samples were analyzed by LC/MS,and the relative molecular weight of the photolysis product was confirmed.According to the results and previous researches,the structure of photolysis product was preliminarily deduced as deacetamido-lumicolchicine.In the third part of the research,the kinetics of photolysis under varied conditions(p H,temperature,solvent,source of light)was investigated.The quantitative analysis was carried out during the photolysis process based on Lambert-Beer law.The results showed the photolysis of colchicine conformed to first-order kinetics equation.A high degradation rate(K=0.5862 h-1)was observed when colchicine was dissolved in ethanol and irradiated by UV light.The photolysis conversion rate was deeply affected by ionic strength,a high conversion rate was detected(α=99.8%)when colchicine was dissolved in ethanol and a low conversion rate was detected(α=55.8%)when colchicine was dissolved in water with p H4.In the fourth part of the research,colchicine and its photolysis product with serial dilutions were added into BRL 3A cells in order to investigate the cytotoxicity of irradiated colchicine to liver cells.The BRL 3A cells were stained and observed through inverted microscope and fluorescence microscope to compare the impact of colchicine before and after irradiation on cell viability.One of the experimental groups was selected to be performed with flow analysis,detecting the impact of drugs on BRL 3A cell cycle.The cell viability of the selected experimental group showed significant differences.The result showed the cytotoxicity of photolysis product reduced remarkably with IC50 of 1.3478 μg/m L compared with colchicine with IC50 of 0.2283 μg/m L.The cell cycle inhibition of colchicine photolysis product reduced significantly,with a G2/M phase cell proportion of approximately 43.0% while the colchicine group with a G2/M phase cell proportion of approximately 28.8%.Meanwhile,the effects of colchicine and its photolysis products on the viability of human renal epithelial cells(293T)and human lung cancer cells(A549)were further investigated,and the anticancer activity of colchicine photolysis products was preliminarily analyzed,providing theoretical basis and reference for the development of colchicine derivative anticancer drugs derived from colchicine.更多还原