棉籽油中主要游离棉酚降解产物化学结构鉴定及生殖毒性研究

发布时间：2018-04-14 20:07

本文选题：棉籽油 + 游离棉酚降解产物　；参考：《新疆医科大学》2017年硕士论文

【摘要】：目的:棉籽油中游离棉酚含量检测方法及限量已经有国家标准,但由于棉籽油中游离棉酚在常温下极易降解(30天内降解80%以上),因此该方法很难反映棉籽油的脱毒情况。本实验通过自制未脱毒棉籽油样品,考察游离棉酚在棉籽油中的降解情况,确定主要游离棉酚降解产物的存在并鉴定其化学结构,通过体内、外毒性实验考察主要游离棉酚降解产物的生殖毒性并建立棉籽油中主要游离棉酚降解产物的含量测定方法。方法:1.根据棉籽油生产工艺,本实验采用正己烷溶剂浸提法提取未脱毒棉籽油样品,室温放置。采用HPLC测定不同放置时间的棉籽油样品,建立游离棉酚与降解产物之间的线性关系。2.采用C18层析柱和半制备型高效液相色谱仪制备主要游离棉酚降解产物,用于质谱结构鉴定。3.采用液相色谱-傅立叶变换离子回旋共振质谱仪测定主要游离棉酚降解产物的准确分子量、推测分子式并鉴定其结构。4.采用GC-2小鼠精母细胞毒性实验和小鼠精子畸变实验考察主要游离棉酚降解产物的体内、外毒性。5.建立主要游离棉酚降解产物含量测定方法,测定24批市售棉籽油中主要游离棉酚降解产物含量。结果:1.棉籽油中游离棉酚与降解产物之间的线性关系为Y=-3.00674395X+2058258982,r=0.9900。2.经质谱仪检测,主要游离棉酚降解产物准分子离子为573.25033,推测其分子式为C34H3708,鉴定其结构为四甲氧基棉酚。3.细胞毒性实验结果为:应用SPSS Statistics 17.0软件计算,主要游离棉酚降解产物四甲氧基棉酚对小鼠GC-2精母细胞的半数抑制浓度IC50为6.554μg/mL。小鼠精子畸变实验结果为:空白组小鼠精子畸形率为1.1 ±0.42%,损伤对照组小鼠精子畸形率为8.2±0.31%,高剂量组小鼠精子畸形率为4.4±0.51%,中剂量组小鼠精子畸形率为3.0±0.33%,低剂量组小鼠精子畸形率为1.8±0.44%,采用秩和检验法处理实验数据,阳性组与对照组精子畸形率相比P0.05,有显著性差异,高、中、低试验组与对照组精子畸形率相比P0.05,有显著性差异。4.对24批市售棉籽油样品进行检测,其中有20批棉籽油样品检测到主要游离棉酚降解产物,含量为0.139～0.912μg/mL。结论:通过测定棉籽油中主要游离棉酚降解产物的含量反映游离棉酚的脱毒情况,这种方法切实可行,为新疆地区棉籽油的生产和监管提供科学有效的检测方法。
[Abstract]:Objective: the determination method and limit of free gossypol in cottonseed oil have already had the national standard, but because the free gossypol in cottonseed oil can easily degrade more than 80% in 30 days at room temperature, it is difficult to reflect the detoxification of cottonseed oil by this method.In this experiment, the degradation of free gossypol in cottonseed oil was investigated by self-made samples of undetoxified cottonseed oil, the existence of main degradation products of free gossypol was determined and its chemical structure was identified.The reproductive toxicity of the main free gossypol degradation products was investigated and a method for determining the content of the main free gossypol degradation products in cottonseed oil was established.Method 1: 1.According to the production process of cottonseed oil, the samples of undetoxified cottonseed oil were extracted by n-hexane solvent extraction and placed at room temperature.The linear relationship between free gossypol and degradation product was established by HPLC.The main free gossypol degradation products were prepared by C18 column and semi-preparation high performance liquid chromatograph, which were used to identify the structure of gossypol by mass spectrometry.The molecular weight of the main degradation products of free gossypol was determined by liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer. The molecular formula was deduced and its structure was identified.GC-2 mouse spermatocyte toxicity test and mouse sperm aberration test were used to investigate the in vivo and in vitro toxicity of the main free gossypol degradation products.A method was established to determine the content of major free gossypol degradation products in 24 batches of cottonseed oil.The result is 1: 1.The linear relationship between free gossypol and degradation products in cottonseed oil is YANG-3.00674395X 2058258982 ru 0.9900.2.The main free gossypol degradation product excimer ion is 573.25033, and its molecular formula is C34H3708, and its structure is identified as tetramethoxygossypol. 3.The results of cytotoxicity test showed that the median inhibitory concentration (IC50) of tetramethoxygossyl, the main degradation product of free gossypol, on mouse GC-2 spermatocytes was 6.554 渭 g 路mL ~ (-1), calculated by SPSS Statistics 17.0 software.The results of sperm aberration test in mice were as follows: sperm abnormality rate of blank group was 1.1 卤0.42, that of injured control group was 8.2 卤0.31, that of high dose group was 4.4 卤0.51, that of middle dose group was 3.0 卤0.33 and that of low dose group was small.The deformity rate of rat sperm was 1.8 卤0.44. The experimental data were processed by rank sum test.There was significant difference in sperm malformation rate between positive group and control group (P 0.05). There was significant difference in sperm malformation rate between test group and control group (P 0.05).Twenty-four batches of cottonseed oil samples were tested, of which 20 batches of cottonseed oil samples detected the main free gossypol degradation products, the content of which was 0.139 ~ 0.912 渭 g 路mL ~ (-1).Conclusion: the detoxification of free gossypol is reflected by the determination of the main free gossypol degradation products in cottonseed oil. This method is feasible and provides a scientific and effective method for the production and supervision of cottonseed oil in Xinjiang.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS227;R114

【参考文献】