高含量磷脂酰胆碱制备方法研究
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摘要
磷脂是生命活动的基础物质,磷脂酰胆碱(PC)是磷脂中的主要有效成分,主要来源于大豆和蛋黄。PC有防止肝炎及脂肪肝,降低胆固醇,预防心血管疾病及抗癌等功能,被世界卫生组织列为“九大长寿食品之一”。随着人民生活水平的提高,药用级高纯度PC的需求趋旺。由于我国制备技术研究开发滞后,高纯度PC主要依赖于国外进口,价格昂贵。因此,研究如何从大豆和蛋黄中提纯PC非常必要。
本文主要围绕以下三部分内容展开工作:
首先,全面深入地研究了以蛋黄粉或大豆浓缩磷脂为原料,粗提精制蛋黄磷脂酰胆碱(ePC)与大豆磷脂酰胆碱(sPC)的工艺过程,ePC和sPC质量指标符合医药级标准,为产业化生产奠定了基础。
ePC的制备分为溶剂浸取和吸附精制两部分。研究了溶剂浸取蛋黄的工艺,采用单因素法和正交设计法考察浸取时间、温度、乙醇浓度等对PC含量和收率的影响,提出了优化操作条件。蛋黄粉中的中性油脂等用丙酮去除,得到含量为68.0%的粗磷脂,达到国家对粉末磷脂含量的要求,溶剂浸取总收率高达95.5%。在吸附精制阶段,比较了不同孔径分布的200～300目粗孔硅胶与80～120目、100～200目粗孔2号硅胶分离效果,确定后者为最佳吸附剂。基于吸附分离的共性,用TLC法初步选定流动相体系为正己烷-异丙醇-水系统;在制备色谱柱上,以PE、PC的保留时间及分离度为指标,调整得到流动相最适配比为46:46:8。优化温度、流速、进样浓度和进样量等操作条件,得到纯度高达94%的产品,收率为60.9%。
本文提出了直接以大豆浓缩磷脂为原料经层析纯化一步得到符合药品级规格的PC的分离方法,与报道的溶剂浸提或超临界CO_2除杂后再经层析分离的工艺相比,工艺路线简单,消耗低,更具工业应用价值。通过比较层析产品中PC含量、收率和流动相的消耗,选用100～200目粗孔2号硅胶为固定相,流动相为正己烷:异丙醇:水(46:46:7.4),最适上载量为0.086 gPC.(g硅胶)~(-1),产品纯度高达85.5%,收率接近100%。
Natural phospholipids, main constituents of biological membranes, have a lot of important biological functions to all living organisms. High-content of phosphatidyl-choline (PC) is the fraction products of phospholipids extracted mainly from soybean or egg yolk. It not only can prevent the occurrence of hepatitis, fatty liver and diseases in heart and blood vessel, but also has functions of lowering cholesterol amount and anticancer. Therefore, World Health Organization (WHO) lists it as one of the nine longevity food. With the improvement of people's life, the demand of high-content PC is bigger and bigger. However, our country lags in manufacture technology and spends much on importing expensive high-content PC from foreign countries each year. How to refine PC from soybean or egg yolk becomes an international attracting project in recent years. And it is significant, necessary and imperative in our country.The project focuses on the three parts as following.First, the purification techniques of high-content ePC and sPC were investigated systematically and roundly. The raw materials are egg yolk powder or soybean concentrated lecithin. The quality indexes of ePC and sPC products fit for the national medicine standard. This is a new point of this paper.The process of ePC includes two parts such as solvent extraction and adsorption separation. We studied the influence of extraction time, temperature, ethanol concentration, solvent volume and times on the purity and recovery of PC with single factor design and orthogonal design. Neutral lipids (NL) and glycolipids (GL) in powder was removed by acetone. The purity of product is 68.0%, which reaches the national requirement of powder lecithin. The total recovery is 95.5%.In the adsorption process, 100~200 mesh coarse silica gel-Ⅱ was selected as the adsorbent by comparing the separation results of three different adsorbents (200~300 mesh coarse silica gel, 80~120 mesh coarse silica gel-Ⅱ and 100~200 mesh coarse silica gel-Ⅱ). Based on the adsorption separation commonness, TLC was utilized to
screen out mobile phase system. Hexane-isopropanol-water was determined as mobile phase. The optimal proportion of mobile phase (46:46:8) was obtained by considering retention time and resolution of PE and PC. After optimizing temperature, flow rate, injection concentration and sample weight, we obtained ePC with the content as high as 94%, which exceeds the requirement of medicine on PC. The recovery of PC in this process is 60.9%.The adsorbent, mobile phase and optimal loadability had been paid more attention in the investigation of sPC preparation from soybean concentrated phospholipids. We put forward a separation method that soybean concentrated phospholipids were directly used as raw material in column chromatography (CC). High-content PC reaching the medicine standard was obtained. Compared with the traditional method that PC was refined with CC after solvent extraction or superficial critical CO2 to remove impurities, the method is simple, low-consumption and valuable in industrial application. 100-200 mesh coarse silica gel-II was selected as adsorbent by comparing purity, recovery and solvent consumption. Hexane-isopropanol-water (46:46:7.4) was chosen as mobile phase. The optimal loadability of CC is 0.086 gPC/ (g silica gel). The purity of refine product is as high as 85.5% at a recovery of near 100 % on the optimal condition hereinbefore.Second, Adsorption equilibrium amount of sPC and ePC on 100-200 mesh coarse silica gel- II were obtained by using static adsorption method in different temperatures and concentrations. The isotherm was fitted by Freundlich and Langmuir equations respectively. And Langmuir equation matches the experiment results well. The general mass transfer model, including axial dispersion, external-film mass transfer and pore diffusion, was further applied to describe the adsorption process of for ePC and sPC on fixed bed with silica gel- II. This is a new point of the work. The pore diffusion coefficient and mass transfer coefficent were obtained by optimization the adsorption breakthrough of ePC and sPC. And these coefficients' values were used to calculate the elution curves of ePC at different flow rates and temperatures. The results show the model can predict the adsorption process of sPC and ePC in the fixed bed of silica gel. The effects of flow rate, initial inset concentration and column
temperature on adsorption breakthrough curves were studied dynamically. All these results are helpful to industrial design and scale-up.Third, a new method of analyzing phospholipids contents is brought forward. Comparison on phospholipids analysis was made between three detectors, i.e., ELSD, UV, and RI. The results show that ELSD is the optimum detector in phospholipids analysis. The gradient ternary solvent procedure of hexane-isopropanol-water was determined by carefully adjusting the ratio of three solvents and flow rate, and switch time of intermediate gradients. It is the first time to baseline resolve ten phospholipids and regress the calibration curves of PE, PI, PC, SM and LPC. Six kinds of soybean lecithin and egg yolk lecithin with these phospholipids were quantified. The coefficient of variation (C.V.) of retention time for five phospholipids are all less than 0.5% and the C.V. of peak areas are less than 6%. Study results prove the method has advantages such as fast, good resolution, high sensitivity in detection, good repeatability and accuracy.
本文主要围绕以下三部分内容展开工作:
首先,全面深入地研究了以蛋黄粉或大豆浓缩磷脂为原料,粗提精制蛋黄磷脂酰胆碱(ePC)与大豆磷脂酰胆碱(sPC)的工艺过程,ePC和sPC质量指标符合医药级标准,为产业化生产奠定了基础。
ePC的制备分为溶剂浸取和吸附精制两部分。研究了溶剂浸取蛋黄的工艺,采用单因素法和正交设计法考察浸取时间、温度、乙醇浓度等对PC含量和收率的影响,提出了优化操作条件。蛋黄粉中的中性油脂等用丙酮去除,得到含量为68.0%的粗磷脂,达到国家对粉末磷脂含量的要求,溶剂浸取总收率高达95.5%。在吸附精制阶段,比较了不同孔径分布的200～300目粗孔硅胶与80～120目、100～200目粗孔2号硅胶分离效果,确定后者为最佳吸附剂。基于吸附分离的共性,用TLC法初步选定流动相体系为正己烷-异丙醇-水系统;在制备色谱柱上,以PE、PC的保留时间及分离度为指标,调整得到流动相最适配比为46:46:8。优化温度、流速、进样浓度和进样量等操作条件,得到纯度高达94%的产品,收率为60.9%。
本文提出了直接以大豆浓缩磷脂为原料经层析纯化一步得到符合药品级规格的PC的分离方法,与报道的溶剂浸提或超临界CO_2除杂后再经层析分离的工艺相比,工艺路线简单,消耗低,更具工业应用价值。通过比较层析产品中PC含量、收率和流动相的消耗,选用100～200目粗孔2号硅胶为固定相,流动相为正己烷:异丙醇:水(46:46:7.4),最适上载量为0.086 gPC.(g硅胶)~(-1),产品纯度高达85.5%,收率接近100%。
Natural phospholipids, main constituents of biological membranes, have a lot of important biological functions to all living organisms. High-content of phosphatidyl-choline (PC) is the fraction products of phospholipids extracted mainly from soybean or egg yolk. It not only can prevent the occurrence of hepatitis, fatty liver and diseases in heart and blood vessel, but also has functions of lowering cholesterol amount and anticancer. Therefore, World Health Organization (WHO) lists it as one of the nine longevity food. With the improvement of people's life, the demand of high-content PC is bigger and bigger. However, our country lags in manufacture technology and spends much on importing expensive high-content PC from foreign countries each year. How to refine PC from soybean or egg yolk becomes an international attracting project in recent years. And it is significant, necessary and imperative in our country.The project focuses on the three parts as following.First, the purification techniques of high-content ePC and sPC were investigated systematically and roundly. The raw materials are egg yolk powder or soybean concentrated lecithin. The quality indexes of ePC and sPC products fit for the national medicine standard. This is a new point of this paper.The process of ePC includes two parts such as solvent extraction and adsorption separation. We studied the influence of extraction time, temperature, ethanol concentration, solvent volume and times on the purity and recovery of PC with single factor design and orthogonal design. Neutral lipids (NL) and glycolipids (GL) in powder was removed by acetone. The purity of product is 68.0%, which reaches the national requirement of powder lecithin. The total recovery is 95.5%.In the adsorption process, 100~200 mesh coarse silica gel-Ⅱ was selected as the adsorbent by comparing the separation results of three different adsorbents (200~300 mesh coarse silica gel, 80~120 mesh coarse silica gel-Ⅱ and 100~200 mesh coarse silica gel-Ⅱ). Based on the adsorption separation commonness, TLC was utilized to
screen out mobile phase system. Hexane-isopropanol-water was determined as mobile phase. The optimal proportion of mobile phase (46:46:8) was obtained by considering retention time and resolution of PE and PC. After optimizing temperature, flow rate, injection concentration and sample weight, we obtained ePC with the content as high as 94%, which exceeds the requirement of medicine on PC. The recovery of PC in this process is 60.9%.The adsorbent, mobile phase and optimal loadability had been paid more attention in the investigation of sPC preparation from soybean concentrated phospholipids. We put forward a separation method that soybean concentrated phospholipids were directly used as raw material in column chromatography (CC). High-content PC reaching the medicine standard was obtained. Compared with the traditional method that PC was refined with CC after solvent extraction or superficial critical CO2 to remove impurities, the method is simple, low-consumption and valuable in industrial application. 100-200 mesh coarse silica gel-II was selected as adsorbent by comparing purity, recovery and solvent consumption. Hexane-isopropanol-water (46:46:7.4) was chosen as mobile phase. The optimal loadability of CC is 0.086 gPC/ (g silica gel). The purity of refine product is as high as 85.5% at a recovery of near 100 % on the optimal condition hereinbefore.Second, Adsorption equilibrium amount of sPC and ePC on 100-200 mesh coarse silica gel- II were obtained by using static adsorption method in different temperatures and concentrations. The isotherm was fitted by Freundlich and Langmuir equations respectively. And Langmuir equation matches the experiment results well. The general mass transfer model, including axial dispersion, external-film mass transfer and pore diffusion, was further applied to describe the adsorption process of for ePC and sPC on fixed bed with silica gel- II. This is a new point of the work. The pore diffusion coefficient and mass transfer coefficent were obtained by optimization the adsorption breakthrough of ePC and sPC. And these coefficients' values were used to calculate the elution curves of ePC at different flow rates and temperatures. The results show the model can predict the adsorption process of sPC and ePC in the fixed bed of silica gel. The effects of flow rate, initial inset concentration and column
temperature on adsorption breakthrough curves were studied dynamically. All these results are helpful to industrial design and scale-up.Third, a new method of analyzing phospholipids contents is brought forward. Comparison on phospholipids analysis was made between three detectors, i.e., ELSD, UV, and RI. The results show that ELSD is the optimum detector in phospholipids analysis. The gradient ternary solvent procedure of hexane-isopropanol-water was determined by carefully adjusting the ratio of three solvents and flow rate, and switch time of intermediate gradients. It is the first time to baseline resolve ten phospholipids and regress the calibration curves of PE, PI, PC, SM and LPC. Six kinds of soybean lecithin and egg yolk lecithin with these phospholipids were quantified. The coefficient of variation (C.V.) of retention time for five phospholipids are all less than 0.5% and the C.V. of peak areas are less than 6%. Study results prove the method has advantages such as fast, good resolution, high sensitivity in detection, good repeatability and accuracy.
引文
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