【作者】 李少春；

【导师】 李培锋；

【作者基本信息】 内蒙古农业大学 ， 基础兽医学， 2009， 博士

【摘要】 本文在实验室提取纯化牛羊混合胆汁中有效成分工艺的基础上,通过改进和完善工艺、扩大试验规模,建立了一套稳定、可靠的从牛羊混合胆汁中提取分离天然牛磺酸、胆酸、甘氨胆酸及牛磺胆酸的小试工艺技术路线,并对提取过程中废弃的的原料及试剂进行了回收套用研究,同时开展了有效成分甘氨胆酸在大鼠体内的药代动力学研究。旨在为下一步开展从牛羊混合胆汁中提取有效成分的中试实验研究奠定试验基础,为牛羊胆汁的综合开发利用提供可靠的理论依据和可能性,同时为阐明甘氨胆酸在大鼠血清及组织中的分布和变化规律,开发脏器生化药物新资源提供线索。提取牛磺酸的小试工艺研究采用脱蛋白、除色素、碱解酸化等方法开展了从牛羊混合胆汁中提取天然牛磺酸的小试工艺研究。采用纸层析法和红外光谱扫描法对提取物进行定性检测,试验结果证明提取样品与牛磺酸标准品显色斑点Rf值相同,红外光谱特征吸收峰一致,表明提取出的样品为牛磺酸;采用薄层扫描法测定其含量,结果证明其纯度平均达98.6%;测定了方法回收率,结果表明牛磺酸提取量平均达13.1g/L,回收率平均达86.7﹪,这与实验室工艺提取牛磺酸的结果一致,表明该提取工艺稳定、可靠。提取胆酸、甘氨胆酸、牛磺胆酸的小试工艺研究采用脱蛋白、除色素、萃取、柱层析分离等方法开展了从牛羊混合胆汁中分离、提取和纯化胆酸、甘氨胆酸及牛磺胆酸三种有效成分的小试提取工艺研究。采用正交试验筛选了柱层析最佳条件,以容积为10L的层析柱进行分离纯化,固定相为硅胶,流动相为氯仿和正丁醇混合溶剂,一次装样量3000mL胆汁,流动相采用梯度洗脱,先按4:1比例分离胆酸和甘氨胆酸,然后按3:2比例分离甘氨胆酸和牛磺胆酸,流动相洗脱速度为3～5mL/s。采用薄层层析法和红外光谱扫描法对提取物进行定性检测,结果证明三种提取样品分别与胆酸、甘氨胆酸及牛磺胆酸标准品显色斑点Rf值相同,红外光谱特征吸收峰均一致,表明提取出的三种样品分别为胆酸、甘氨胆酸、牛磺胆酸结晶体。采用薄层扫描法测定其含量,结果证明三种成分平均纯度分别达98.0%、98.4%、98.6%。首次测定了方法回收率,结果显示三种成分提取量平均分别达11.0g/L、9.1g/L、9.3g/L,回收率平均达64.4﹪、63.7﹪、67.8﹪,这与实验室工艺提取三种有效成分结果一致,表明小试提取工艺稳定、可行。采用留样观察法研究了不同批次的三种有效成分在室温环境和4℃封闭避光环境下的稳定性,结果表明,三种白色粉末样品在室温环境中均不稳定,易受潮变质,在4℃封闭避光环境下均具有较高的稳定性,不易变质。废旧原料、试剂的回收套用研究采用精馏法回收了提取工艺中的废旧乙醇,试验结果表明回收乙醇纯度平均达95%以上,符合提取工艺纯度要求;采用碱洗、酸洗及去离子水洗涤等方法对废旧硅胶进行了回收,结果表明回收硅胶物理性状与新硅胶无差异,pH值相同;将回收乙醇和硅胶重新套用到提取工艺中,结果证明提取效果与新乙醇、硅胶提取效果无显著差异,表明使用回收乙醇和硅胶不影响对四种有效成分的提取效果,可以重复利用。甘氨胆酸在大鼠体内的药代动力学研究采用固相萃取-反相高效液相色谱法测定了大鼠血清及肝、肾、脑组织中甘氨胆酸的含量。结果表明,在0.15625～10.0μg/ml范围内血清中甘氨胆酸浓度与峰面积呈良好的线性关系,回归方程Y= 39579X + 57823,R2=0.9984;以信噪比S/N=3为标准,最低检测浓度为0.02μg/ml;平均回收率为98.49%;日内RSD为0.52%～2.00%,日间RSD为0.74%～4.98%;稳定性试验第一组的平均含量为99.35%,RSD为2.24%,第二组的平均含量为100.87%,RSD为4.73%;测定了肝、肾、脑组织中甘氨胆酸含量,采用体外甘氨胆酸标准品制备标准曲线的方法计算了相应的组织浓度,其结果仅反映组织中甘氨胆酸浓度的相对变化规律,不能体现真正的组织浓度值,但对组织样品的稳定性进行了较细致的考察,其结果和血样结果基本一致。该方法具有简便、稳定、准确、实用的特点,适用于甘氨胆酸含量的分析。采用固相萃取-反相高效液相色谱法测定了给药后不同时间血清及肝、肾、脑组织中甘氨胆酸的浓度。结果显示:⑴在血清中符合一级吸收二室模型,药动学方程:C= 41.17e-1.256t+84.7 e-0.491t-125.87e-0.667t。⑵在肝脏中符合一级吸收一室模型,药动学方程:C= 18.734(e-0.042t-e-0.733t)。⑶在肾脏中符合一级吸收一室模型,药动学方程:C= 105.88(e-0.076t-e-0.081t)。⑷在脑组织中符合一级吸收一室模型,药动学方程:C= 5.312(e-0.082t-e-0.098t)。结果表明,甘氨胆酸口服给药后,在大鼠体内吸收快、分布广、消除较缓慢,在体内能保持较长时间的药物浓度,有利于其临床药理作用的发挥。更多还原

【Abstract】 The paper mainly include four parts of reseaching in extraction and purification of effective components from the mixed bovine and ovine bile : (1) Established a reliable suitable small-experiment technology route, which is used to be extracted and separated Taurine, Cholic acid, Glycocholic acid and Taurocholic acid, by improving and perfecting the original technology and magnifying the experiment scale at the base of the previous research in our laboratory. (2) Stduied on the recycling and reusing technology of the raw material and reagent at the extracting process. (3) Stduied on the pharmacokinetics of glycocholic acid in rat. Depending on the above research, it can be said that it has been provided experimental basic for the further research in the middling experiment of extracting the active component from the mixed bovine and ovine bile, supplied the reliable theory and possibility for comprehensive delevoping utilizing the bovine and ovine bile, and illustrated the distribution variation law of glycocholic acid in the blood-serum and texture of rat which is regarded to helping developing the biocherical drug from animal organs.The study on the extracting small experiment of TaurineThe extracting small experiment of Taurine was studied by adopting deproteinizatio, depigmentation, alkaline hydrolysis and acidolysis. Bile extraction was qualitatively detected by using the paper chromatography and infrared spectral scanning method, and the results showed that the extraction has the same Rf value and infrared spectra’s characteristic absorption peak as the Taurine standard preparation which can prove that this extraction is Taurine. The tlc-scanning experiment showed that the purity coefficient of extraction is up to 98.6% averagely. The recovery rate detected experiment showed that the extraction amount of taurine is 13.1g/L and the recovery rate is 86.7﹪which is consisted with the results of laboratory technology and showed that this technology is reliable suitable.The study on the extracting small experiment of Choleic acid, Glycocholic acid and Taurocholic acid.The extracting small experiments of Choleic acid, Glycocholic acid and taurocholic acid were studied by adopting deproteinizatio, depigmentation, extraction and column chromatographic fractionation.The optimum condition was determined via orthogonal experiments, and the result shows that the technology of bile extraction and purification adopts 10L chromato bar with 3L bile, gel silica as fixed phase, the mixed chloroform and isopropanol as mobile phase, gradient elution with mobile phase (the mobile phase ratio in extraction of cholalic acid and glycocholic acid is 4:1 , and then becomes 3:2 in extraction of glycocholic acid and glycocholic acid), and eluting velocity of 3～5mL/s .These three kinds of Bile extraction were qualitatively detected by using the paper chromatography and infrared spectral scanning method, and the results show that These extractions have the same Rf value and infrared spectra’s characteristic absorption peak as choleic acid, glycocholic acid and taurocholic acid standard preparation which can prove that These three kinds of Bile extraction are choleic acid, glycocholic acid and taurocholic acid respectively.The TLC-scanning experiment shows that the purity coefficient of these three kinds of bile extraction are up to 98.0%、98.4%、98.6%averagely, respectively. The recovery rate detected experiment shows that these three kinds of bile extraction amounts are 11.0g/L、9.1g/L、9.3g/L and the recovery rates are 64.4﹪、63.7﹪、67.8﹪respectively which are consisted with the results of laboratory technology and shows that this technology is reliable suitable.The stability of these three kinds of bile extraction is detected with the remaining sample and observing methods at room temperature and 4℃closed lucifuge environment ,and the results show that they have high stability.The study on the recycling and reusing technology of the raw material and reagent The waste ethanol was recovered by rectification method and the result shows that the purity coefficient of recovered ethanol is up to 95% according to the process purity requirement. The waste gel silica was recovered by alkali washing, acid cleaning and deionized water washing, and the results shows that the physical properties and PH of recovered gel silica are not remarkably different from the new gel silica. Recovered ethanol and gel silica were used in the extraction process, and the abtained result is not remarkably different from the previous result of using the new ethanol and gel silica in the process, which indicats that recovered ethanol and gel silica have no significant effect on the extraction of four active components and can be reutilizated.The study on pharmacokinetics of Glycocholic acid in rats Solid extraction -reversed-phase high performance liquid was used to detect the concentration of glycocholic acid in blood serum,liver, kidney and brain tissue in rats.The results manifest that the concentration of glycocholic acid has satisfactory linear correlation whith peak area, regression equation: Y= 39579X + 57823,R2=0.9984;The lowest detected concentration is 0.02μg/mL with S/N=3 ; Average recovery rate is 98.49%; RSD为0.52%～2.00%in day, RSD为0.74%～4.98%between days;The average recovery rate is 99.35%, RSD is 2.24% in the first team, and it is 100.87%, RSD is 4.73% in the second team at the stability test. When the concentration of glycocholic acid was detected in the liver, kidney and brain tissue, corresponding tissue concentration was calculated by preparing the standard curve with the glycocholic acid standard preparation in vitro, but the result can only reflect the relative chang law of the concentration of glycocholic acid in tissue but not in real tissue. The result of tissue stability experiment is consistent with that of the serum experiment. This method has the convenient,stable, accurate and pragmatic characteristics and is suitable to analyze the glycocholic acid content.After the rats was administrated with glycocholic acid on the does of 0.14g/kg·b·w, the concentration of glycocholic acid was detected in the blood serum, liver, kidney and brain tissue and the obtained data was analysed using the pharmacokinetics software of DAS ver1.0.The results show that Serum drug-time curve is consistent with first-order absorptiontwo-compartment model, its kinetic equation is C= 41.17e-1.256t+84.7 e-0.491t-125.87e-0.667t.Liver drug-time curve is consistent with first-order single compartment model, its kinetic equation is C= 18.734(e-0.042t-e-0.733t).Kidney drug-time curve is consistent with first-order single compartment model, its kinetic equation is C= 105.88(e-0.076t-e-0.081t).Brain tissue drug-time curve is consistent with first-order single compartment model, its kinetic equation is C= 5.312(e-0.082t-e-0.098t).The above data demonstrates that glycocholic acid has the high exhaustion, wide distribution and slow elimination characteristcs in rat which is beneficial to exert the clinical pharmacologic action by keeping the drug concentration for a long time.更多还原