【作者】 刘敏；

【导师】 李坤；

【作者基本信息】 辽宁师范大学 ， 有机化学， 2017， 硕士

【摘要】 目的:优选五谷虫油脂最佳提取工艺;建立柱前衍生化测定五谷虫中脂肪酸含量的高效液相色谱(HPLC)法;探讨五谷虫脂肪酸提取物的相关抗菌活性,为五谷虫的应用奠定理论基础,为新型药物的开发提供新策略。方法:1.以五谷虫油脂得率为指标,筛选出五谷虫油脂提取最佳方法和最佳溶剂,再通过响应面法(RSM)筛选出五谷虫油脂最佳提取时间、提取次数和液料比。2.以亚油酸、软脂酸、油酸、硬脂酸为对照品,采用柱前衍生化高效液相(HPLC)法,选择十七烷酸作为内标物测定五谷虫脂肪酸含量,对五谷虫中脂肪酸进行质量控制。3.以金黄色葡萄球菌(Staphylococcus aureus,SA)和大肠杆菌(Escherichia coli,EC)为研究对象,采用微量稀释肉汤法测定五谷虫脂肪酸提取物的最小抑菌浓度(MIC);通过结晶紫染色法测定五谷虫脂肪酸提取物对细菌生物被膜的影响。结果:1.通过工艺优选,筛选出五谷虫油脂的最佳提取工艺为:采用二氯甲烷超声提取,提取时间为4 h,液料比为15,提取次数为2次。2.运用柱前衍生化HPLC法,测得五谷虫脂肪酸提取物中亚油酸、软脂酸、油酸、硬脂酸的含量分别为0.0955 mg/mL、0.1864 mg/m L、0.1658 mg/m L、0.0550 mg/mL。3.微量稀释肉汤法测得五谷虫脂肪酸提取物对金黄色葡萄球菌的MIC为0.250mg/mL;结晶紫染色法表明,金黄色葡萄球菌的生物被膜形成在1/2 MIC药物浓度下即受到抑制,金黄色葡萄球菌的成熟生物被膜在MIC药物浓度下遭到破坏,且药物浓度显示出剂量依赖性;大肠杆菌基本没有受到五谷虫脂肪酸提取物的明显抑制,其生物被膜的形成和成熟生物被膜均没有受到影响。结论:1.利用响应面法对五谷虫油脂的提取工艺进行优化,与传统正交实验相比,结果更精确,涵盖范围更广,准确度更高。2.柱前衍生化HPLC法测定五谷虫脂肪酸含量,该方法稳定,重现性好,可用于五谷虫脂肪酸的含量测定,并可采用此方法对五谷虫中脂肪酸进行质量控制。3.五谷虫脂肪酸提取物明显抑制金黄色葡萄球菌,该药物对金黄色葡萄球菌的生物被膜形成和成熟生物被膜均产生抑制;对大肠杆菌及其生物被膜没有明显抑制作用。更多还原

【Abstract】 Object:This work is aimed at the optimization of not only the extraction of maggot oils but also the precolumn derivatization HPLC methods for the of the oil content.Through the detail studies of related antibacterial activities of the extraction of fatty acid of maggots,we are looking forward to obtain further fundamental theoretic for the application of maggots and to provide a new sight for the development of new drugs.Methods:1.The best extraction time,extraction times and reasonable liquid-to-material ratio of the extraction of maggot oil were optimized by the response surface method（RSM）on the basis of the screening of the extraction methods and solvents.2.With linoleic acid,palmitic acid,oleic acid,stearic acid as the reference,seventeen alkyl acid as the internal standard,the contents of fatty acids in maggots were determined by the precolumn derivatization of high performance liquid chromatography（HPLC）,and the contents of fatty acids were also determined by the internal standard method3.Taking Staphylococcus aureus（SA）and Escherichia coli（EC）as the research object,the minimum inhibitory concentration（MIC）of fatty acid from maggot is determined by microdilution broth method and the effect was determined by crystal violet staining method.Results:1.The optimized method of maggot oil extraction was ultrasonic extraction,the best solvent was CH₂Cl_2,the best extraction conditions were ultrasonic extraction 4 hours,solvent amount 15 times and 2 times of extraction.2.By the precolumn derivatization of HPLC method for the determination of fatty acid content of maggots,the content of linoleic acid,palmitic acid,oleic acid and stearic acid were,0.0955 mg/mL、0.1864 mg/m L、0.1658 mg/m L、0.0550 mg/m L,respectively.3.Microdilution broth measured fatty acid extract from maggots on Staphylococcus aureus the MIC was 0.250 mg/mL,1/2 MIC showed that the concentration of crystal violet staining method of Staphylococcus aureus biofilm formation was inhibited,the MIC drug concentration damaged Staphylococcus aureus mature biofilm,drug concentration showed a dose dependent,fatty acid extract from maggots on Escherichia coli had no obvious inhibitory effect,the drug also had no obvious effect on Escherichia coli biofilm formation and mature biofilm.Conclusions:1.Compared with the traditional orthogonal experiment,the response surface method is used to optimize the extraction process of Fructus Glutamate oil,and the result is more accurate and higher accuracy.2.The pre-column derivatization of HPLC method is stable and reproducible,and can be used for determination of fatty acid content of maggot.The method was employed to determine the content of fatty acid in maggots.3.Fatty acid extract of Maggot inhibits Staphylococcus aureus obviously,the drug also inhibit the biofilm formation of Staphylococcus aureus and its mature biofilm,but the drug had no obvious effect on Escherichia coli biofilm formation and mature biofilm.更多还原