高速逆流色谱分离纯化山楂和杏仁皮中主要活性成分的研究
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摘要
本文以山楂、杏仁皮为研究对象,采用高速逆流色谱法对其中的多酚类物质进行研究分析。山楂多酚具有多种重要的生理活性,主要体现在预防和治疗心血管疾病等方面；杏仁皮多酚具有预防心血管疾病、调节免疫、抗氧化、抗菌抗病毒等诸多功效。关于山楂及杏仁皮中的化学成分已有研究报道,但鲜见有关采用高速逆流色谱法对其中的多酚类化合物进行分离纯化的研究报道。本文从山楂、杏仁皮多酚的提取、分离纯化对其进行了深入的研究,并且采用液质联用和核磁技术对分离纯化出的化合物进行分析。
本论文包括五部分：
1.综述
首先从高速逆流色谱的工作原理、特点、仪器类型、两相溶剂体系的选择及目前的研究现状五个方面对高速逆流色谱的研究概况进行综述；然后对山楂和杏仁皮的活性成分及其应用两个方面进行总结；最后对多酚类化合物的提取、分离、功能和应用做了概述。
2.山楂多酚的提取及液质分析
采用热回流提取法提取山楂中的多酚类物质,提取溶剂采用70%的乙醇,料液比按1：15,提取温度为80℃,提取时间为2h,提取次数为3次,在最佳提取条件下,从100 g山楂中得到了62.4g粗提物。将所得粗提物通过高效液相分离分析得到了四种主要酚类化合物,同时利用液质联用技术对四种主要酚类化合物进行具体分析,分别为：绿原酸、原花青素B2、表儿茶素和原花青素C1。
3.高速逆流色谱分离纯化山楂多酚类化合物
采用高速逆流色谱法对山楂粗提物进行研究,将山楂粗提物在液质上分析得出的四种多酚类化合物：绿原酸、原花青素B2、表儿茶素和原花青素C1作为逆流溶剂体系选择的依据,根据四种目标化合物的分配系数及实际分离情况,选择正丁醇：乙酸乙酯：水(1:1:2)作为本实验半制备型高速逆流色谱的两相溶剂系统,本文首次采用响应表面优化法对高速逆流色谱分离纯化条件进行优化,通过响应面法优化试验得到优化条件实际为：流动相的流速为1.5mL/min,仪器转速为850rpm,分离温度为25℃。上相做固定相,下相做流动相。该方法不仅成功地将山楂中的四种多酚类活性物质分离出来,并且从500 mg山楂粗提物中制备得到了-种纯度高达94.8%的物质,质量为9.7 mg,其化学结构通过紫外,液质联用和核磁技术确定为绿原酸。
4.杏仁皮多酚的提取及液质分析
采用微波辅助提取法对杏仁皮多酚类物质进行了提取,通过优化实验,得出最佳提取条件：提取溶剂为70%乙醇,料液比为1：30,微波提取功率为400W,微波辐射时间为10 min以及提取次数为2次。在最佳提取条件下,杏仁皮中多酚的含量达到8.5 mg/g。将所得粗提物通过高效液相分离分析得到了三种主要酚类化合物,同时采用液质联用对三种主要酚类化合物进行具体分析,分别为：绿原酸类二聚体,原花青素类二聚体和原花青素类三聚体。
5.高速逆流色谱分离纯化杏仁皮多酚类化合物
采用高速逆流色谱法对杏仁皮粗提物进行研究,将杏仁皮粗提物在液质上分析得到的三种多酚类化合物：绿原酸类二聚体、原花青素类二聚体和原花青素类三聚体作为逆流溶剂体系选择的依据,根据实际分离情况,选择正丁醇：乙酸乙酯：1%醋酸水溶液(1：1：2)作为本实验半制备型高速逆流色谱仪的两相溶剂系统,通过优化实验,确定流动相流速为1.5 mL/min,仪器转速为1000 rpm,分离温度为25℃,上相做固定相,下相做流动相。在优化条件下,通过高速逆流色谱的一次分离,从300 mg杏仁皮粗提物中可得到一种20.6 mg的物质；经高效液相色谱分析其纯度为97%以上,其化学结构通过紫外,液质联用确定为绿原酸类二聚体。
In this paper, high-speed counter-current chromatography (HSCCC) was developed for the separation and purification of polyphenols from hawthorn and almond skin. Polyphenols of hawthorn had important physiological activities, mainly in the prevention and treatment of cardiovascular diseases; polyphenols of almond skin had the prevention of cardiovascular disease, regulation of the immune, antioxidant, antibacterial and antiviral, and many other effects. Chemical constituents of hawthorn and almond skin had been reported, but the separation and purification of polyphenols from hawthorn and almond skin by HSCCC had not been reported yet. This paper was in-depth research about the extraction, separation and purification of hawthorn and almond skin, meanwhile structure characterization of compounds were by means of liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic. There are five parts in this paper:
1. Review
Firstly, from five aspects, high-speed counter-current chromatography was introduced, which included principles, characteristics, instrument types, how to select two-phase solvent system and the current research status; then, did a summary of the active ingredient and application of hawthorn and almond skin; Finally did an overview of the extraction, separation, function and application of polyphenols.
2. Extraction, separation and LC-MS analysis of polyphenol of hawthorn
The extract obtained under reflux, the optimized extraction conditions were:70% ethanol, a solid-liquid ratio of 1:15, extracting temperature of 80℃and extracting time of 2 h. Under the optimized conditions, a quality of crude extract 62.4 g was obtained from hawthorn(100 g). The crude extract of hawthorn was separated by high performance liquid chromatography, four main phenolic compounds obtained, which were identified as chlorogenic acid, procyanidin B2, epicatechin, and procyanidin C1 by LC-MS.
3. Separation and purification of polyphenol from hawthorn by HSCCC
By LC-MS analysis of four phenolic components from hawthorn, we did a further research about extract of hawthorn by HSCCC; according to the K values of four target components, we choosed solvent systems composed of n-butanol-ethyl acetate-water at a volume ratio of 1:1:2 (v/v/v) for our purification in HSCCC. Optimizing HSCCC conditions with RSM was first time in this paper. The optimal condition obtained using response surface methodology (RSM) as follows:revolution speed, 850 rpm; flow-rate,1.5 mL/min; and separation temperature,25℃. The upper phase was used as the stationary phase while the lower phase was used as the mobile phase.9.7 mg of a product from the crude sample (500 mg)was obtained with purity over 94.8%. The product was identified with UV, LC-MS and H1NMR as chlorogenic acid.
4. Extraction, separation and LC-MS analysis of polyphenol of almond skin
we use microwave assisted extraction of polyphenolic compounds from almond skin, by optimizing the extraction conditions, the results were as follow:70% ethanol, solid to liquid ratio of 1:30, microwave extraction power of 400 W and microwave irradiation time of 10 min. Under the optimized conditions, the content of polyphenol of almond skin was up to 8.5 mg/g. The crude extract of almond skin was separated by high performance liquid chromatography, three main phenolic compounds obtained, which were identified as chlorogenic acid dimer, procyanidin dimers and procyanidin trimers were identified by LC-MS.
5. Separation and purification of polyphenol from almond skin by HSCCC
By LC-MS analysis of three phenolic components from almond skin, we did a further research about extract of almond skin by HSCCC; according to actual separation condition, we choosed solvent systems composed of n-butanol-ethyl acetate-1% acetic acid solution at a volume ratio of 1:1:2 (v/v/v) for our purification in HSCCC. The optimal condition were as follows:revolution speed,1000 rpm; flow-rate,1.5 mL/min; and separation temperature,25℃. The upper phase was used as the stationary phase while the lower phase was used as the mobile phase.300 mg extract could be introduced into the HSCCC system and 20.6 mg of a product was obtained with purity over 97% in one-step. The product was identified with LC-MS as chlorogenic acid dimer.
本论文包括五部分：
1.综述
首先从高速逆流色谱的工作原理、特点、仪器类型、两相溶剂体系的选择及目前的研究现状五个方面对高速逆流色谱的研究概况进行综述；然后对山楂和杏仁皮的活性成分及其应用两个方面进行总结；最后对多酚类化合物的提取、分离、功能和应用做了概述。
2.山楂多酚的提取及液质分析
采用热回流提取法提取山楂中的多酚类物质,提取溶剂采用70%的乙醇,料液比按1：15,提取温度为80℃,提取时间为2h,提取次数为3次,在最佳提取条件下,从100 g山楂中得到了62.4g粗提物。将所得粗提物通过高效液相分离分析得到了四种主要酚类化合物,同时利用液质联用技术对四种主要酚类化合物进行具体分析,分别为：绿原酸、原花青素B2、表儿茶素和原花青素C1。
3.高速逆流色谱分离纯化山楂多酚类化合物
采用高速逆流色谱法对山楂粗提物进行研究,将山楂粗提物在液质上分析得出的四种多酚类化合物：绿原酸、原花青素B2、表儿茶素和原花青素C1作为逆流溶剂体系选择的依据,根据四种目标化合物的分配系数及实际分离情况,选择正丁醇：乙酸乙酯：水(1:1:2)作为本实验半制备型高速逆流色谱的两相溶剂系统,本文首次采用响应表面优化法对高速逆流色谱分离纯化条件进行优化,通过响应面法优化试验得到优化条件实际为：流动相的流速为1.5mL/min,仪器转速为850rpm,分离温度为25℃。上相做固定相,下相做流动相。该方法不仅成功地将山楂中的四种多酚类活性物质分离出来,并且从500 mg山楂粗提物中制备得到了-种纯度高达94.8%的物质,质量为9.7 mg,其化学结构通过紫外,液质联用和核磁技术确定为绿原酸。
4.杏仁皮多酚的提取及液质分析
采用微波辅助提取法对杏仁皮多酚类物质进行了提取,通过优化实验,得出最佳提取条件：提取溶剂为70%乙醇,料液比为1：30,微波提取功率为400W,微波辐射时间为10 min以及提取次数为2次。在最佳提取条件下,杏仁皮中多酚的含量达到8.5 mg/g。将所得粗提物通过高效液相分离分析得到了三种主要酚类化合物,同时采用液质联用对三种主要酚类化合物进行具体分析,分别为：绿原酸类二聚体,原花青素类二聚体和原花青素类三聚体。
5.高速逆流色谱分离纯化杏仁皮多酚类化合物
采用高速逆流色谱法对杏仁皮粗提物进行研究,将杏仁皮粗提物在液质上分析得到的三种多酚类化合物：绿原酸类二聚体、原花青素类二聚体和原花青素类三聚体作为逆流溶剂体系选择的依据,根据实际分离情况,选择正丁醇：乙酸乙酯：1%醋酸水溶液(1：1：2)作为本实验半制备型高速逆流色谱仪的两相溶剂系统,通过优化实验,确定流动相流速为1.5 mL/min,仪器转速为1000 rpm,分离温度为25℃,上相做固定相,下相做流动相。在优化条件下,通过高速逆流色谱的一次分离,从300 mg杏仁皮粗提物中可得到一种20.6 mg的物质；经高效液相色谱分析其纯度为97%以上,其化学结构通过紫外,液质联用确定为绿原酸类二聚体。
In this paper, high-speed counter-current chromatography (HSCCC) was developed for the separation and purification of polyphenols from hawthorn and almond skin. Polyphenols of hawthorn had important physiological activities, mainly in the prevention and treatment of cardiovascular diseases; polyphenols of almond skin had the prevention of cardiovascular disease, regulation of the immune, antioxidant, antibacterial and antiviral, and many other effects. Chemical constituents of hawthorn and almond skin had been reported, but the separation and purification of polyphenols from hawthorn and almond skin by HSCCC had not been reported yet. This paper was in-depth research about the extraction, separation and purification of hawthorn and almond skin, meanwhile structure characterization of compounds were by means of liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic. There are five parts in this paper:
1. Review
Firstly, from five aspects, high-speed counter-current chromatography was introduced, which included principles, characteristics, instrument types, how to select two-phase solvent system and the current research status; then, did a summary of the active ingredient and application of hawthorn and almond skin; Finally did an overview of the extraction, separation, function and application of polyphenols.
2. Extraction, separation and LC-MS analysis of polyphenol of hawthorn
The extract obtained under reflux, the optimized extraction conditions were:70% ethanol, a solid-liquid ratio of 1:15, extracting temperature of 80℃and extracting time of 2 h. Under the optimized conditions, a quality of crude extract 62.4 g was obtained from hawthorn(100 g). The crude extract of hawthorn was separated by high performance liquid chromatography, four main phenolic compounds obtained, which were identified as chlorogenic acid, procyanidin B2, epicatechin, and procyanidin C1 by LC-MS.
3. Separation and purification of polyphenol from hawthorn by HSCCC
By LC-MS analysis of four phenolic components from hawthorn, we did a further research about extract of hawthorn by HSCCC; according to the K values of four target components, we choosed solvent systems composed of n-butanol-ethyl acetate-water at a volume ratio of 1:1:2 (v/v/v) for our purification in HSCCC. Optimizing HSCCC conditions with RSM was first time in this paper. The optimal condition obtained using response surface methodology (RSM) as follows:revolution speed, 850 rpm; flow-rate,1.5 mL/min; and separation temperature,25℃. The upper phase was used as the stationary phase while the lower phase was used as the mobile phase.9.7 mg of a product from the crude sample (500 mg)was obtained with purity over 94.8%. The product was identified with UV, LC-MS and H1NMR as chlorogenic acid.
4. Extraction, separation and LC-MS analysis of polyphenol of almond skin
we use microwave assisted extraction of polyphenolic compounds from almond skin, by optimizing the extraction conditions, the results were as follow:70% ethanol, solid to liquid ratio of 1:30, microwave extraction power of 400 W and microwave irradiation time of 10 min. Under the optimized conditions, the content of polyphenol of almond skin was up to 8.5 mg/g. The crude extract of almond skin was separated by high performance liquid chromatography, three main phenolic compounds obtained, which were identified as chlorogenic acid dimer, procyanidin dimers and procyanidin trimers were identified by LC-MS.
5. Separation and purification of polyphenol from almond skin by HSCCC
By LC-MS analysis of three phenolic components from almond skin, we did a further research about extract of almond skin by HSCCC; according to actual separation condition, we choosed solvent systems composed of n-butanol-ethyl acetate-1% acetic acid solution at a volume ratio of 1:1:2 (v/v/v) for our purification in HSCCC. The optimal condition were as follows:revolution speed,1000 rpm; flow-rate,1.5 mL/min; and separation temperature,25℃. The upper phase was used as the stationary phase while the lower phase was used as the mobile phase.300 mg extract could be introduced into the HSCCC system and 20.6 mg of a product was obtained with purity over 97% in one-step. The product was identified with LC-MS as chlorogenic acid dimer.
引文
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