【作者】 领小；

【导师】 博·格日勒图；

【作者基本信息】 内蒙古大学 ， 植物学， 2008， 博士

【摘要】 胡椒碱是荜茇中提取的有效成分之一。胡椒碱衍生物是以胡椒碱作为原料，用化学方法合成的一系列胡椒碱衍生物。荜茇是胡椒科植物荜茇（Piper longumL．）的未成熟果穗，是常用于中、蒙、藏药。临床研究证明荜茇具有明显的降血脂作用。内蒙古大学蒙药研究所从荜茇中得到胡椒碱及与胡椒碱具有类似化学结构的胡椒碱衍生物，发现其中的胡椒碱和荜茇宁具有显著降血脂活性。另外，根据胡椒碱和荜茇宁的分子结构，以胡椒碱为原料合成了一系列胡椒碱衍生物。胡椒碱衍生物中胡椒酸乙酯（简称为GBA）的降血脂活性好，毒性非常小，具有开发成降血脂创新一类药的研究价值。以开发新药为目的研究胡椒碱衍生物时，寻找和确定一种能够快速、简便、精确、高效地检测胡椒碱衍生物的分析方法是十分必要。另外在研究胡椒碱衍生物的药物代谢和作用机理方面有必要研究生物体内血液中胡椒碱衍生物与血清白蛋白的结合情况等基础研究工作。首次详细研究了金属离子存在下，胡椒碱在胶束体系中的荧光特性，以及实验条件对荧光强度的影响，建立了灵敏度较高、稳定性较好的荧光分析新方法。实验结果表明：阳离子表面活性剂溴化十六烷基三甲基铵对锰（Ⅱ）-胡椒碱的荧光具有增敏作用。胡椒碱的质量浓度在2．02～10．1μ·mL^-1范围内，与其荧光强度呈良好的线性关系，检出限为0．0602μ·mL^-1，相对标准偏差为1．10％。该方法应用于蒙药荜茇中胡椒碱的测定，结果满足分析要求。研究发现非离子表面活性剂OP-10对荜茇宁的荧光有明显的增敏作用，据此首次建立了测定荜茇宁的荧光分析新方法。实验结果表明：荜茇宁的质量浓度在2．0-16．0μg·mL^-1范围内，与其荧光强度呈良好的线性关系，检出限为0．065 1μg·mL^-1，方法的相对标准偏差为1．40％。该方法应用于蒙药荜茇中荜茇宁的测定，结果令人满意。采用荧光光谱及紫外光谱法研究了胡椒碱、荜茇宁和GBA与牛血清白蛋白（BSA）的相互作用。确定胡椒碱、荜茇宁和GBA分别都与BSA形成复合物，从而猝灭BSA的内源性荧光。猝灭的主要原因是静态猝灭和非辐射能量转移作用。计算得到胡椒碱、荜茇宁和GBA与BSA的结合常数K_A，分别为1．02×10⁷L·mol^-1（25℃）和1．11×10⁷L·mol^-1（37℃）；2．40×10⁵ L·mol^-1（25℃）和1．01×10⁶ L·mol^-1（37℃）；9．48×10⁵ L·mol^-1（25℃）和1．36×10⁶ L·mol^-1（37℃）。得到胡椒碱、荜茇宁和GBA与BSA结合位点数n，分别为1．45（25℃）和1．46（37℃）；1．10（25℃）和1．24（37℃）；1．18（25℃）和1．24（37℃）。根据Forster偶极-偶极非辐射能量转移理论得到胡椒碱、荜茇宁和GBA与BSA的结合距离r，分别为3．28 nm（25℃）和3．30nm（25℃）；3．12 nm（25℃）和3．15 nm（37℃）；2．68 nm（25℃）和2．81 nm（37℃）。通过热力学参数的计算，确定胡椒碱、荜茇宁和GBA与牛血清白蛋白的相互作用均是一个熵增加和吉布斯自由能降低的自发过程。根据热力学参数与作用力之间的关系，确定主要作用力为疏水作用力。通过同步荧光光谱研究了BSA构象的变化。并进一步讨论了常见金属离子对胡椒碱衍生物与BSA的相互作用的影响。采用荧光光谱及紫外光谱法研究了胡椒碱、荜茇宁和GBA及胰蛋白酶（trypsin）的相互作用。确定胡椒碱、荜茇宁和GBA与胰蛋白酶形成复合物从而猝灭胰蛋白酶的内源性荧光。猝灭原因主要为静态猝灭和非辐射能量转移。计算得到胡椒碱、荜茇宁和GBA与胰蛋白酶的结合常数K_A，分别为9．29×10⁵L·mol^-1（25℃）和1．44×10⁶ L·mol^-1（37℃）；1．19×10⁶ L·mol^-1（25℃）和9．57×10⁵L·mol^-1（37℃）；1．66×10⁶ L·mol^-1（25℃）和5．05×10⁵L·mol^-1（37℃）。根据Forster非辐射能量转移理论得到胡椒碱、荜茇宁和GBA与胰蛋白酶的结合距离r，分别为2．79nm（25℃）、3．02 nm（37℃）；3．08 nm（25℃）、2．93 nm（37℃）；3．04 nm（25℃）、2．99nm（37℃）。根据热力学参数与作用力之间的关系，确定了胡椒碱衍生物与胰蛋白酶的主要作用力，胡椒碱与胰蛋白酶的主要作用力为疏水作用力；荜茇宁与胰蛋白酶的主要相互作用力为静电相互力；GBA与胰蛋白酶的主要相互作用力为范德华力。并确定胡椒碱、荜茇宁与胰蛋白酶的相互作用是一个熵增加和吉布斯自由能降低的自发过程。更多还原

【Abstract】 Pipeline was one of the active components that extracted from Long Pepper. Piperine derivatives were synthesized from Piperine. Long Pepper was the grown ear-fruits of Piper Longum L., which frequently used as Chinese medicine and Mongolian medicine. Clinical practice indicated that Long Pepper had significant antilipidic effect. Piperine and its derivatives with similar structure were extracted from Long Pepper. Our research group found that Piperine and piperlonguminine had significant antilipidic effect. According to the structures of Piperine and its derivatives, a series of derivatives such as ethyl piperate （GBA） were synthesized using piperine as starting materials. In these derivatives, GBA was the most effective one with low toxicity. GBA was very valuable to do study for developing it into new kind of anti-hyperlipidemic medicine. Therefore, it is important to study and get a quick and convenient method for detecting and analyzing these kinds of compounds for our study on developing the compounds into new drugs in the future. The coalescence of these compounds and bovine serum albumin （BSA） should be studied.The fluorescence characteristics of piperine in micellar system with cation were studied. The influences of experimental condition on fluorescence intensity also were studied. New sensitive and stable fluorescence methods for analyzing the derivatives of GBA and GBB were developed. The experiments indicated that cationic surfactant-cetyltrimethylammonium bromide was able to enhance the fluorescence intensity of Mn（II）-piperine. There was a linear relationship between the enhancement of fluorescence intensity and the concentration of cationic surfactant-cetyltrimethylammonium bromide. The linear range of concentration of piperine was 2.02-10.1μg·mL^-1with the detection limit of 0.0602μg·mL^-1. The relative standard deviation （RSD） was 1.10%. The proposed method has been successfully applied to the quantitative determination of piperine in Long Pepper used as Mongolian medicine. The results were very satisfactory.The experiments indicated that non-ionic surfactant OP-10 enhanced the fluorescence intensity of piperlonguminine. There was a linear relationship between the enhancement of fluorescence intensity and the concentration of non-ionic surfactant OP-10. Based on this result, a sensitive method was developed for the quantitative determination of piperlonguminine. The linear range of concentration of piperlonguminine was 2.0-16.0μg·mL^-1with the detection limit of 0.0651μg·mL^-1. The relative standard deviation （RSD） was 1.40%. The proposed method was successfully applied to the quantitative determination of piperine, of Long Pepper. The results were good.The interaction between piperine, piperlonguminine, GBA and bovine serum albumin （BSA） was studied by fluoroscopy and UV spectroscopy. The experimental results showed that the molecules of piperine, piperlonguminine, GBA quenched the intrinsic fluorescence of BSA by forming complex. The mechanism of fluorescence quenching was confirmed combining by both static quenching and non-radiation energy transferring. The apparent binding constants （K_A） were calculated, were it 1.02×10⁷ L·mol^-1（25℃） and 1.11×10⁷L·mol^-1（37℃）; 2.40×10⁵ L·mol^-1（25℃） and 1.01×10⁶ L·mol^-1 （37℃）; 9.48×10⁵ L·mol^-1’ （25℃） and 1.36×10⁶ L·mol^-1 （37℃）. The value of binding sites （n） were calculated, were it 1.45（25℃） and 1.46（37℃）; 1.10（25℃） and 1.24（37℃）; 1.18（25℃） and 1.24（37℃）. According to the Forster theory of non-radiation energy transfer, the binding distances （r） were obtained, were it 3.28 nm （25℃） and 3.30 nm （25℃）; 3.12 ran （25℃） and 3.15 nm （37℃）; 2.68 nm （25℃） and 2.81 nm （37℃）. The process of binding was a spontaneous molecular interaction in which entropy increased and Gibbs free energy decreased, indicating that the interaction of between piperine, piperlonguminine, GBA and BSA was driven mainly by hydrophobic force. The effect of piperine on the conformation of BSA was also analyzed by synchronous fluoroscopy. The influences of ordinary ions on the interaction of piperine derivatives with BSA were discussed. The relations of structure, pharmacology and combination characteristics of piperine derivatives were also discussed.The interactions between piperine, piperlonguminine, GBA and trypsin were investigated by fluoroscopy and UV spectroscopy. The experimental results showed that the piperine, piperlonguminine and GBA quenched the intrinsic fluorescence of trypsin by forming complex. The result of fluorescence quenching might be caused by static quenching and non radiative energy transferring. The apparent binding constants （K_A） were calculated, were it 9.29×10⁵ L·mol^-1 （25℃） and 1.44×10⁶ L·mol^-1 （37℃）; 1.19×10⁶ L·mol^-1 （25℃） and 9.57×10⁵ L·mol^-1 （37℃）; 1.66×10⁶ L·mol^-1 （25℃） and 5.05×10⁵ L·mol^-1 （37℃）. According to the Forster theory of non-radiation energy transfer, the binding distances （r） were obtained, were it 2.79 run （25℃） and 3.02 nm （37℃）; 3.08 nm（25℃） and 2.93 nm（37℃）; 3.04 nm（25℃） and 2.99 nm（37℃）. According to the relationship of parameters of thermodynamics and the interactions force, determined the inter force between the pipeline derivatives and trypsin. The relationships of the structure, pharmacology and the combination of piperine derivatives were also discussed.更多还原