琼脂糖凝胶分离纯化大黄等中药有效成分的研究
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摘要
凝胶色谱技术是六十年代初发展起来的一种快速而又简单的分离分析技术,设备简单、操作方便,不需要污染性有机溶剂,对高分子物质有很好的分离效果。目前已经广泛应用于生物化学、分子生物学、生物工程学、分子免疫学以及医学等有关领域,该技术不但应用于科学实验研究,而且已经大规模地用于工业生产。琼脂糖凝胶是由球状琼脂糖经两次交联而制得的凝胶,其高交联结构显著提高了颗粒刚性和物理化学稳定性,粒度小而粒径分布范围窄,属于高效凝胶,可在高流速下使用,并具有分离范围宽和分辨率高等特点。已用于天然中草药中有效成分的分离,成功的分离了黄酮类和多酚类化合物,引起了国内外分离工作者的广泛关注。
中药有效成分的提取分离与纯化技术研究是中药现代化的核心,也是中药企业发展的必经之路。开展中药化学成分提取分离新技术的研究,对于提高企业的科技实力与竞争力,促进中药现代化的发展具有重要意义。目前,应用琼脂糖凝胶作为固定相在分离纯化天然药物中的蒽醌、黄酮和多酚等有效成分方面已获得令人满意结果。本文对琼脂糖凝胶分离纯化大黄、大豆、虎杖、知母、甘草和连翘叶中的有效成分进行了研究,建立了分离纯化这几种中草药中有效成分的新方法,取得了相关核心技术,并利用紫外光谱(UV)、高效液相色谱(HPLC)、核磁共振波谱(NMR)等对目标产物的纯度和化学结构进行了分析和鉴定,结合各组分的化学结构式及凝胶色谱保留时间,对琼脂糖凝胶作为固定相分离纯化蒽醌类化合物、黄酮类化合物、异黄酮类化合物和木脂素类化合物的机理进行了探讨。
1.大黄中有效成分的分离纯化
以琼脂糖凝胶为固定相,建立了分离纯化大黄蒽醌类化合物和苯乙烯酸的新方法,以55%的甲醇(0.01%的HAC)水溶液作为流动相进行洗脱,可以得到苯乙烯酸、大黄酸、芦荟大黄素、大黄酚、大黄素甲醚、大黄素等六部分,分别收集,得到化合物的纯度均在95%以上。经一步分离从大黄粗提物中分离得到苯乙烯酸、大黄酸、芦荟大黄素、大黄酚、大黄素甲醚和大黄素六种高纯度化合物。
根据大黄药材粗提物中各组分的化学结构式以及在琼脂糖凝胶上的保留时间,对琼脂糖凝胶(Superose 12)色谱分离纯化大黄有效成分的机理进行了探讨。研究结果表明琼脂糖凝胶分离纯化中草药中蒽醌类化合物的机理是目标化合物结构式中的羟基与Superose 12合成残留基团的氢键作用和疏水作用二者的协同作用,建立了分离纯化中草药中蒽醌类化合物的新方法。
2.大豆中异黄酮类化合物的分离纯化
应用琼脂糖凝胶作为固定相分离纯化大豆中的异黄酮类化合物,以40%的甲醇和60%的甲醇溶液作为流动相梯度洗脱,经一步分离从大豆提取物中得到大豆苷、染料木苷、黄豆黄苷、大豆苷元、染料木素和黄豆黄素六种高纯度化合物,纯度分别为99.0%, 99.0%, 90.5%, 99.0%, 94.5%和99.0%。
根据大豆粗提物中各组分的化学结构式以及在琼脂糖凝胶上的保留时间,对琼脂糖凝胶色谱分离纯化异黄酮类化合物的机理进行了探讨。研究结果表明琼脂糖凝胶分离纯化中草药中异黄酮类成分的机理是目标化合物结构式中的羟基与Superose 12合成残留基团的氢键作用和疏水作用二者的协同作用,建立了分离纯化中草药中异黄酮类化合物的新方法。
3.虎杖中有效成分的分离纯化
以琼脂糖凝胶为填料,建立了分离纯化虎杖中有效成分的新方法。以65%的甲醇溶液作为流动相,一步分离可以得到白藜芦醇苷、大黄素-8-O-β-D-葡萄吡喃糖甙、白藜芦醇和大黄素四种高纯度化合物。根据虎杖药材粗提物中各组分的化学结构式以及在琼脂糖凝胶上的保留时间,对琼脂糖凝胶(Superose 12)色谱分离纯化虎杖有效成分的机理进行了探讨。研究结果表明琼脂糖凝胶分离纯化虎杖有效成分的机理是目标化合物结构式中的羟基与Superose 12合成残留基团的氢键作用和疏水作用二者的协同作用,建立了分离纯化虎杖有效成分的新方法。
4.知母中有效成分的分离纯化
建立了琼脂糖凝胶作为固定相分离纯化知母中有效成分的新方法,以60%的甲醇溶液作为洗脱流动相,从知母粗提物中一步分离得到单甲基-顺-扁柏树脂酚、顺-扁柏树脂酚两种高纯度木脂素类化合物和新芒果苷、芒果苷两种黄酮类化合物,纯度分别为96.1%, 98.0%, 97.2%和98.1%。
根据中药知母粗提物中各组分的化学结构式以及在琼脂糖凝胶上的保留时间,对琼脂糖凝胶色谱分离纯化木脂素、黄酮类化合物的机理进行了研究。最终得出琼脂糖凝胶分离纯化知母中有效成分的机理是目标化合物结构式中的羟基与Superose 12合成残留基团间的氢键作用和疏水作用二者的协同作用,建立了分离纯化知母中有效成分的新方法。
5.甘草中有效成分的分离纯化
以琼脂糖凝胶作为固定相,建立了分离纯化甘草中有效成分的新方法,即以60%的甲醇溶液作为流动相进行洗脱,经一步分离从甘草中分离得到异甘草素葡萄糖芹菜苷、甘草糖苷A、异甘草素和甘草香豆素四种高纯度化合物,纯度分别为91.3%, 95.1%, 91.8%和96.7%。
根据甘草粗提物中各组分的化学结构式以及在琼脂糖凝胶上的保留时间,对琼脂糖凝胶色谱分离纯化甘草中有效成分的机理进行了探讨。研究结果表明琼脂糖凝胶分离纯化甘草中有效成分的机理是目标化合物结构式中的羟基与Superose 12合成残留基团的氢键作用和疏水作用二者的协同作用,开创了分离纯化甘草中有效成分的新方法。
6.连翘叶中有效成分的分离纯化
建立了琼脂糖凝胶作为固定相分离纯化连翘叶中有效成分的新方法,即以30%的甲醇溶液作为流动相进行洗脱,从连翘叶中一步分离得到连翘苷、连翘酯苷和芦丁三种高纯度化合物,纯度分别为99.2%,96.3%和99.2%。
根据连翘叶粗提物中各组分的化学结构式以及在琼脂糖凝胶上的保留时间,对琼脂糖凝胶色谱分离纯化连翘叶中有效成分的机理进行了探讨。研究结果表明琼脂糖凝胶分离纯化连翘叶中有效成分的机理是目标化合物结构式中的羟基与Superose 12合成残留基团的氢键作用和疏水作用二者的协同作用,开创了分离纯化连翘叶中有效成分的新方法。
Gel media based on cross-linked dextran and cross-linked agarose are widely used for size exclusion chromatography of large biomolecules, which was developed in the 1960’s. Superose 12, the gel that is prepared by spherical agarose with cross-linking two times. The high cross-linked structure increased its rigidity and stability observably. So it can be used in high velocity. And now Superose 12 has been widely used in the separation and purification of high molecular weight compounds. It was also used in the separation and purification of active components from natural products.
It has become an urgent task to investigate Chinese traditional medicinal herbs with modern technological instrument and establish integrate quality appraisal system. And it is also the road that must be taken in the development of the Chinese native medicine enterprise. So carrying on the research of the new method of traditional Chinese medicine plays an important role in promoting the development of modern traditional Chinese pharmacology and the Chinese medicine modernization. Successful application of adsorption chromatography on cross-linked 12% agarose has been reported for the purification of flavones and polyphenols.
In the present paper, under the optimized experiment conditions, active components from common traditional Chinese herbs (Rheum officinale Baill., Glycine max (L.)Merr Polygonum cuspidatum Sieb.et Zucc, etc.) were separated and purified. The purity of the obtained compounds after Superose 12 separation was above 95% determined by high performance liquid chromatography (HPLC) analysis and their chemical structures were identified by 1H-NMR, 13C-NMR and standard samples. And also discussed the separated mechanism of hydroxyanthraquinone, flavonoid and lignans compounds on cross-linked 12% agarose.
1. Isolation and purification of hydroxyanthraquinone compounds from the roots of Rheum officinale Baill. by Superose 12
A chromatographic method for isolation and purification of chemical constituents from the well-known traditional Chinese drugDa-huang (roots of Rheum officinale Baill.) was established by using 12% cross-linked agarose gel, Superose 12, as the separation media. 55% methanol (including 0.01% HAC) was used as the eluent for separation of cinnamic acid, rhein, aloe-emodin, chrysophanol, physcion and emodin from Da-huang crude extract. As a result, cinnamic acid and five kinds of hydroxyanthraquinones including rhein, aloe-emodin, chrysophanol, physcion and emodin were obtained in one-stepp seperation with high purity.
The retention behavior of hydroxyanthraquinones on Superose 12 was studied according to the chemical structure and the retention time. The results of the present study clearly demonstrate that Superose 12 can be used for the separation of hydroxyanthraquinones and that the retention of hydroxyanthraquinones on Superose 12 is based on a mixture of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of hydroxyanthraquinones and the residues of the cross-linking reagents used in the manufacturing process of Superose 12.
2. Isolation and purification of Sobyean isoflavones from sobyean by Superose 12 Six soy isoflavones including daidzin, genistin, glycitin, daidzein, glycitein and genistein with the purity of 99.0%, 99.0%, 90.5%, 99.0%, 94.5% and 99.0%, were isolated and purified by adsorption chromatography Superose 12 from soybean in one-step separation. When 40% and 60% methanol were used as the mobile phase in gradient mode at a flow rate of 0.5 ml/min, good separation results could be obtained.
The retention behavior of soy isoflavones on Superose 12 was studied according to the chemical structure and the retention time. The results of the present study clearly demonstrate that Superose 12 can be used for the separation of soy isoflavones and that the retention of soy isoflavones on Superose 12 is based on a mixture of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of soy isoflavones and the residues of the cross-linking reagents used in the manufacturing process of Superose 12.
3. Isolation and purification of four compounds from the Chinese medicinal herb Polygonum cuspidatum Sieb.et Zucc by adsorption chromatography on cross-linked 12% agarose
The four compounds of the well-known traditional Chinese drug Polygonum cuspidatum Sieb. et Zucc can be separated by adsorption chromatography on the cross-linked 12% agarose gel Superose 12. Sixty five percent methanol was used as the eluent for separation of piceid, resveratrol, anthraglycoside B and emodin from Hu-zhang crude extract. The purity of each compound is over 95% as determined by HPLC.
The retention behavior of the compounds from Polygonum cuspidatum Sieb.et Zucc on Superose 12 was studied according to the chemical structure and the retention time. The results of the present study clearly demonstrate that Superose 12 can be used for the separation of the compounds from Polygonum cuspidatum Sieb.et Zucc and that the retention of these compounds on Superose 12 is based on a mixture of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of the compounds and the residues of the cross-linking reagents used in the manufacturing process of Superose 12.
4. Isolation and purification of four compounds from the Chinese medicinal herb Rhizoma Anemarrhenae by adsorption chromatography on 12% cross-linked agarose Four bioactive compounds including two lignans compounds cishinkiresinol, (-)-4′-O-methylnyasol and two flavonoid compounds mangiferin, neomangiferin were separated from the methanol extract of Rhizoma Anemarrhenae by adsorption chromatography. Sixty percent methanol was used as the mobile phase. The purities of the four compounds reached 96.1%, 98.0%, 97.2% and 98.1%.
The retention behavior of the compounds from Rhizoma Anemarrhenae on Superose 12 was studied according to the chemical structure and the retention time. The results of the present study clearly demonstrate that Superose 12 can be used for the separation of the compounds from Rhizoma Anemarrhenae and that the retention of these compounds on Superose 12 is based on a mixture of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of the compounds and the residues of the cross-linking reagents used in the manufacturing process of Superose 12.
5. One step isolation and purification of four compounds from the Chinese medicinal Radix Glycyrrhiza by adsorption chromatography on 12% cross-linked agarose Adsorption chromatography (Superose 12) was successfully used for the isolation and purification of four compounds from Radix Glycyrrhiza, the Chinese herbal drug. Sixty percent methanol was used as the mobile phase. Four kinds of compounds including licuraside, licorice-glycoside A, isoliquiritigenin and glycycoumarin were obtained after one step separation, with the purity of 91.3%, 95.1%, 91.8% and 96.7%, respectively, as determined by high performance liquid chromatography (HPLC).
The retention behavior of the compounds from Radix Glycyrrhiza on Superose 12 was studied according to the chemical structure and the retention time. The results of the present study clearly demonstrate that Superose 12 can be used for the separation of the compounds from Radix Glycyrrhiza and that the retention of these compounds on Superose 12 is based on a mixture of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of the compounds and the residues of the cross-linking reagents used in the manufacturing process of Superose 12.
6. Isolation and purification of three compounds from the leaves of Forsythia suspensa by adsorption chromatography on 12% cross-linked agarose Superose 12, adsorption chromatography was successfully applied to the separation and purification of forsythiaside, phillyrin and rutin from the leaves of Forsythia suspensa.
The optimum solvent system composed of methanol-water (30:70, v/v) led to the successful preparation of forsythiaside, phillyrin and rutin with high purity (99.2%, 96.7% and 99.2%).
The retention behavior of the compounds from the leaves of Forsythia suspensa on Superose 12 was studied according to the chemical structure and the retention time. The results of the present study clearly demonstrate that Superose 12 can be used for the separation of the compounds from the leaves of Forsythia suspensa and that the retention of these compounds on Superose 12 is based on a mixture of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of the compounds and the residues of the cross-linking reagents used in the manufacturing process of Superose 12.
中药有效成分的提取分离与纯化技术研究是中药现代化的核心,也是中药企业发展的必经之路。开展中药化学成分提取分离新技术的研究,对于提高企业的科技实力与竞争力,促进中药现代化的发展具有重要意义。目前,应用琼脂糖凝胶作为固定相在分离纯化天然药物中的蒽醌、黄酮和多酚等有效成分方面已获得令人满意结果。本文对琼脂糖凝胶分离纯化大黄、大豆、虎杖、知母、甘草和连翘叶中的有效成分进行了研究,建立了分离纯化这几种中草药中有效成分的新方法,取得了相关核心技术,并利用紫外光谱(UV)、高效液相色谱(HPLC)、核磁共振波谱(NMR)等对目标产物的纯度和化学结构进行了分析和鉴定,结合各组分的化学结构式及凝胶色谱保留时间,对琼脂糖凝胶作为固定相分离纯化蒽醌类化合物、黄酮类化合物、异黄酮类化合物和木脂素类化合物的机理进行了探讨。
1.大黄中有效成分的分离纯化
以琼脂糖凝胶为固定相,建立了分离纯化大黄蒽醌类化合物和苯乙烯酸的新方法,以55%的甲醇(0.01%的HAC)水溶液作为流动相进行洗脱,可以得到苯乙烯酸、大黄酸、芦荟大黄素、大黄酚、大黄素甲醚、大黄素等六部分,分别收集,得到化合物的纯度均在95%以上。经一步分离从大黄粗提物中分离得到苯乙烯酸、大黄酸、芦荟大黄素、大黄酚、大黄素甲醚和大黄素六种高纯度化合物。
根据大黄药材粗提物中各组分的化学结构式以及在琼脂糖凝胶上的保留时间,对琼脂糖凝胶(Superose 12)色谱分离纯化大黄有效成分的机理进行了探讨。研究结果表明琼脂糖凝胶分离纯化中草药中蒽醌类化合物的机理是目标化合物结构式中的羟基与Superose 12合成残留基团的氢键作用和疏水作用二者的协同作用,建立了分离纯化中草药中蒽醌类化合物的新方法。
2.大豆中异黄酮类化合物的分离纯化
应用琼脂糖凝胶作为固定相分离纯化大豆中的异黄酮类化合物,以40%的甲醇和60%的甲醇溶液作为流动相梯度洗脱,经一步分离从大豆提取物中得到大豆苷、染料木苷、黄豆黄苷、大豆苷元、染料木素和黄豆黄素六种高纯度化合物,纯度分别为99.0%, 99.0%, 90.5%, 99.0%, 94.5%和99.0%。
根据大豆粗提物中各组分的化学结构式以及在琼脂糖凝胶上的保留时间,对琼脂糖凝胶色谱分离纯化异黄酮类化合物的机理进行了探讨。研究结果表明琼脂糖凝胶分离纯化中草药中异黄酮类成分的机理是目标化合物结构式中的羟基与Superose 12合成残留基团的氢键作用和疏水作用二者的协同作用,建立了分离纯化中草药中异黄酮类化合物的新方法。
3.虎杖中有效成分的分离纯化
以琼脂糖凝胶为填料,建立了分离纯化虎杖中有效成分的新方法。以65%的甲醇溶液作为流动相,一步分离可以得到白藜芦醇苷、大黄素-8-O-β-D-葡萄吡喃糖甙、白藜芦醇和大黄素四种高纯度化合物。根据虎杖药材粗提物中各组分的化学结构式以及在琼脂糖凝胶上的保留时间,对琼脂糖凝胶(Superose 12)色谱分离纯化虎杖有效成分的机理进行了探讨。研究结果表明琼脂糖凝胶分离纯化虎杖有效成分的机理是目标化合物结构式中的羟基与Superose 12合成残留基团的氢键作用和疏水作用二者的协同作用,建立了分离纯化虎杖有效成分的新方法。
4.知母中有效成分的分离纯化
建立了琼脂糖凝胶作为固定相分离纯化知母中有效成分的新方法,以60%的甲醇溶液作为洗脱流动相,从知母粗提物中一步分离得到单甲基-顺-扁柏树脂酚、顺-扁柏树脂酚两种高纯度木脂素类化合物和新芒果苷、芒果苷两种黄酮类化合物,纯度分别为96.1%, 98.0%, 97.2%和98.1%。
根据中药知母粗提物中各组分的化学结构式以及在琼脂糖凝胶上的保留时间,对琼脂糖凝胶色谱分离纯化木脂素、黄酮类化合物的机理进行了研究。最终得出琼脂糖凝胶分离纯化知母中有效成分的机理是目标化合物结构式中的羟基与Superose 12合成残留基团间的氢键作用和疏水作用二者的协同作用,建立了分离纯化知母中有效成分的新方法。
5.甘草中有效成分的分离纯化
以琼脂糖凝胶作为固定相,建立了分离纯化甘草中有效成分的新方法,即以60%的甲醇溶液作为流动相进行洗脱,经一步分离从甘草中分离得到异甘草素葡萄糖芹菜苷、甘草糖苷A、异甘草素和甘草香豆素四种高纯度化合物,纯度分别为91.3%, 95.1%, 91.8%和96.7%。
根据甘草粗提物中各组分的化学结构式以及在琼脂糖凝胶上的保留时间,对琼脂糖凝胶色谱分离纯化甘草中有效成分的机理进行了探讨。研究结果表明琼脂糖凝胶分离纯化甘草中有效成分的机理是目标化合物结构式中的羟基与Superose 12合成残留基团的氢键作用和疏水作用二者的协同作用,开创了分离纯化甘草中有效成分的新方法。
6.连翘叶中有效成分的分离纯化
建立了琼脂糖凝胶作为固定相分离纯化连翘叶中有效成分的新方法,即以30%的甲醇溶液作为流动相进行洗脱,从连翘叶中一步分离得到连翘苷、连翘酯苷和芦丁三种高纯度化合物,纯度分别为99.2%,96.3%和99.2%。
根据连翘叶粗提物中各组分的化学结构式以及在琼脂糖凝胶上的保留时间,对琼脂糖凝胶色谱分离纯化连翘叶中有效成分的机理进行了探讨。研究结果表明琼脂糖凝胶分离纯化连翘叶中有效成分的机理是目标化合物结构式中的羟基与Superose 12合成残留基团的氢键作用和疏水作用二者的协同作用,开创了分离纯化连翘叶中有效成分的新方法。
Gel media based on cross-linked dextran and cross-linked agarose are widely used for size exclusion chromatography of large biomolecules, which was developed in the 1960’s. Superose 12, the gel that is prepared by spherical agarose with cross-linking two times. The high cross-linked structure increased its rigidity and stability observably. So it can be used in high velocity. And now Superose 12 has been widely used in the separation and purification of high molecular weight compounds. It was also used in the separation and purification of active components from natural products.
It has become an urgent task to investigate Chinese traditional medicinal herbs with modern technological instrument and establish integrate quality appraisal system. And it is also the road that must be taken in the development of the Chinese native medicine enterprise. So carrying on the research of the new method of traditional Chinese medicine plays an important role in promoting the development of modern traditional Chinese pharmacology and the Chinese medicine modernization. Successful application of adsorption chromatography on cross-linked 12% agarose has been reported for the purification of flavones and polyphenols.
In the present paper, under the optimized experiment conditions, active components from common traditional Chinese herbs (Rheum officinale Baill., Glycine max (L.)Merr Polygonum cuspidatum Sieb.et Zucc, etc.) were separated and purified. The purity of the obtained compounds after Superose 12 separation was above 95% determined by high performance liquid chromatography (HPLC) analysis and their chemical structures were identified by 1H-NMR, 13C-NMR and standard samples. And also discussed the separated mechanism of hydroxyanthraquinone, flavonoid and lignans compounds on cross-linked 12% agarose.
1. Isolation and purification of hydroxyanthraquinone compounds from the roots of Rheum officinale Baill. by Superose 12
A chromatographic method for isolation and purification of chemical constituents from the well-known traditional Chinese drugDa-huang (roots of Rheum officinale Baill.) was established by using 12% cross-linked agarose gel, Superose 12, as the separation media. 55% methanol (including 0.01% HAC) was used as the eluent for separation of cinnamic acid, rhein, aloe-emodin, chrysophanol, physcion and emodin from Da-huang crude extract. As a result, cinnamic acid and five kinds of hydroxyanthraquinones including rhein, aloe-emodin, chrysophanol, physcion and emodin were obtained in one-stepp seperation with high purity.
The retention behavior of hydroxyanthraquinones on Superose 12 was studied according to the chemical structure and the retention time. The results of the present study clearly demonstrate that Superose 12 can be used for the separation of hydroxyanthraquinones and that the retention of hydroxyanthraquinones on Superose 12 is based on a mixture of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of hydroxyanthraquinones and the residues of the cross-linking reagents used in the manufacturing process of Superose 12.
2. Isolation and purification of Sobyean isoflavones from sobyean by Superose 12 Six soy isoflavones including daidzin, genistin, glycitin, daidzein, glycitein and genistein with the purity of 99.0%, 99.0%, 90.5%, 99.0%, 94.5% and 99.0%, were isolated and purified by adsorption chromatography Superose 12 from soybean in one-step separation. When 40% and 60% methanol were used as the mobile phase in gradient mode at a flow rate of 0.5 ml/min, good separation results could be obtained.
The retention behavior of soy isoflavones on Superose 12 was studied according to the chemical structure and the retention time. The results of the present study clearly demonstrate that Superose 12 can be used for the separation of soy isoflavones and that the retention of soy isoflavones on Superose 12 is based on a mixture of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of soy isoflavones and the residues of the cross-linking reagents used in the manufacturing process of Superose 12.
3. Isolation and purification of four compounds from the Chinese medicinal herb Polygonum cuspidatum Sieb.et Zucc by adsorption chromatography on cross-linked 12% agarose
The four compounds of the well-known traditional Chinese drug Polygonum cuspidatum Sieb. et Zucc can be separated by adsorption chromatography on the cross-linked 12% agarose gel Superose 12. Sixty five percent methanol was used as the eluent for separation of piceid, resveratrol, anthraglycoside B and emodin from Hu-zhang crude extract. The purity of each compound is over 95% as determined by HPLC.
The retention behavior of the compounds from Polygonum cuspidatum Sieb.et Zucc on Superose 12 was studied according to the chemical structure and the retention time. The results of the present study clearly demonstrate that Superose 12 can be used for the separation of the compounds from Polygonum cuspidatum Sieb.et Zucc and that the retention of these compounds on Superose 12 is based on a mixture of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of the compounds and the residues of the cross-linking reagents used in the manufacturing process of Superose 12.
4. Isolation and purification of four compounds from the Chinese medicinal herb Rhizoma Anemarrhenae by adsorption chromatography on 12% cross-linked agarose Four bioactive compounds including two lignans compounds cishinkiresinol, (-)-4′-O-methylnyasol and two flavonoid compounds mangiferin, neomangiferin were separated from the methanol extract of Rhizoma Anemarrhenae by adsorption chromatography. Sixty percent methanol was used as the mobile phase. The purities of the four compounds reached 96.1%, 98.0%, 97.2% and 98.1%.
The retention behavior of the compounds from Rhizoma Anemarrhenae on Superose 12 was studied according to the chemical structure and the retention time. The results of the present study clearly demonstrate that Superose 12 can be used for the separation of the compounds from Rhizoma Anemarrhenae and that the retention of these compounds on Superose 12 is based on a mixture of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of the compounds and the residues of the cross-linking reagents used in the manufacturing process of Superose 12.
5. One step isolation and purification of four compounds from the Chinese medicinal Radix Glycyrrhiza by adsorption chromatography on 12% cross-linked agarose Adsorption chromatography (Superose 12) was successfully used for the isolation and purification of four compounds from Radix Glycyrrhiza, the Chinese herbal drug. Sixty percent methanol was used as the mobile phase. Four kinds of compounds including licuraside, licorice-glycoside A, isoliquiritigenin and glycycoumarin were obtained after one step separation, with the purity of 91.3%, 95.1%, 91.8% and 96.7%, respectively, as determined by high performance liquid chromatography (HPLC).
The retention behavior of the compounds from Radix Glycyrrhiza on Superose 12 was studied according to the chemical structure and the retention time. The results of the present study clearly demonstrate that Superose 12 can be used for the separation of the compounds from Radix Glycyrrhiza and that the retention of these compounds on Superose 12 is based on a mixture of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of the compounds and the residues of the cross-linking reagents used in the manufacturing process of Superose 12.
6. Isolation and purification of three compounds from the leaves of Forsythia suspensa by adsorption chromatography on 12% cross-linked agarose Superose 12, adsorption chromatography was successfully applied to the separation and purification of forsythiaside, phillyrin and rutin from the leaves of Forsythia suspensa.
The optimum solvent system composed of methanol-water (30:70, v/v) led to the successful preparation of forsythiaside, phillyrin and rutin with high purity (99.2%, 96.7% and 99.2%).
The retention behavior of the compounds from the leaves of Forsythia suspensa on Superose 12 was studied according to the chemical structure and the retention time. The results of the present study clearly demonstrate that Superose 12 can be used for the separation of the compounds from the leaves of Forsythia suspensa and that the retention of these compounds on Superose 12 is based on a mixture of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of the compounds and the residues of the cross-linking reagents used in the manufacturing process of Superose 12.
引文
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