毛细管电泳在中药及其煎煮液分析中的应用
摘要
中药成分复杂,分析其有效成分比较困难。如何保证药品质量、提高药物疗效是亟待解决的问题。为了控制药物质量,发现新的药物有效成分,越来越多的分析手段已用于分离和分析中药的活性成分。近年来对中药煎煮过程中有效成分的研究已取得了很大的进步,但距现代化的要求还有很大的差距。毛细管电泳(Capillary electrophoresis,CE)是一种新型的分离分析技术,具有高效、快速、进样体积小、可选择的分离模式多、方法简单等优点;而流动注射和毛细管电泳联用技术(Flow injection-capillary electrophoresis,FI-CE)是近几年研究的热点,在进样和样品预处理方面有强大的优势。鉴于这种情况,本论文从理论和方法上开展了毛细管电泳在中药及其煎煮液分析方面的研究工作。
本论文第二部分充分利用毛细管电泳及流动注射-毛细管电泳联用的优势,对中药浸泡和煎煮过程中活性组分的浓度变化进行了测定,取得了以下几方面创新性的研究成果:
1.建立了同时分离中药甘草根中的甘草次酸和甘草酸的自由溶液毛细管电泳新方法。并且将该方法用于离线测定甘草根浸泡和煎煮过程中不同时间时甘草次酸和甘草酸的含量变化。
2.利用FI高精度进样和强大的样品预处理能力,首次将在线过滤和冷却装置与FI-CE联用,并应用到中药甘草根煎煮过程中有效成分浓度的动态变化研究。
3.将微乳液毛细管电动色谱用于中药蛇床子中欧前胡素和蛇床子素的分离与测定,结果令人满意。
本论文共分为三部分五章:
第一部分:综述
第一章:简单介绍了CE的原理和特点、毛细管电泳在中药及其煎煮液分析中的应用。
第二部分:毛细管电泳在中药活性成分分析中的应用
第二章:建立了同时分离测定甘草根中甘草次酸和甘草酸的自由溶液毛细管电泳新方法。最佳分析条件为:10 mM硼砂,pH 8.80,分离电压20 kV,检测波长254 nm。该方法已用于甘草根离线浸泡和煎煮过程中甘草次酸和甘草酸含量的变化研究,并获得了最佳浸泡和煎煮时间,而且对其它中药煎煮过程中有效成分含量的研究也具有重要的参考价值。
第三章:通过FI强大的样品预处理能力,实现了样品的在线过滤和冷却,建立了一种在线监测甘草根煎煮过程中有效成分动态变化的新方法。最佳分析条件为:10 mM硼砂,pH 8.80,分离电压为10 kV,检测波长为268 nm。通过对甘草根煎煮液的在线监测,得到了甘草根的最佳煎煮时间,而且发现在甘草根煎煮过程中,没有新成分出现,原有的成分也没有消失。将该方法进行改善,可用于其它中药煎煮过程中活性成分的在线测定。
第四章:建立了一种简单、快速、灵敏、准确的微乳液毛细管电动色谱法,同时测定了中药蛇床子中的欧前胡素和蛇床子素。最佳分析条件为:20 mM Tris,20%(v/v)微乳(16.55%(w/v)SDS+40.75%(v/v)正丁醇+5.85%(v/v)正庚烷+36.85%(v/v)水),分离电压15 kV,检测波长254 nm。
第三部分:毛细管电泳在手性分离中的应用
第五章:建立了一种简单、灵敏、准确的自由溶液毛细管电泳新方法,用β-CD为手性选择试剂,分离了盐酸帕洛诺司琼对映体。最佳分析条件为:30 mMNaH_2PO_4,10%甲醇,150 mMβ-CD,pH=3.0,分离电压为15 kV。
Natural medicinal resources provide valuable material for the discovery and development of new drugs of natural origin. The large numbers of other components in traditional Chinese medicines (TCMs) make the screening and analysis of the bioactive components extremely difficult. In the past decades, many effective analysis tools have been used for analyzing the components of TCMs in order to control the quality and discover bioactive compounds. Capillary electrophoresis (CE) is a new analysis technology. It provides advantages in terms of higher efficiency, faster separation time, less injected sample volume and more practicable operating modes. The advantages of the combined flow injection with capillary electrophoresis (FI-CE) system have been demonstrated not only to produce an efficient and reliable mode of sample introduction with improved throughput and precision and outstanding reproducibility of retention time, peak area and peak height, but also to provide on-line sample pretreatment ability. In the second part of this dissertation, the advantages of CE and FI-CE were made use of to separate and determinate active components in the decoction of Glscyrrhiza uralensis Fisch.
On the basis of the previous literatures, in this dissertation, some original studies are carried out as followings:
1. A novel method based on capillary zone electrophoresis (CZE) was developed for determining glycyrrhetinic acid (GTA) and glycyrrhizic acid (GA) in Glycyrrhiza uralensis Fisch root. This method has also been successfully applied to determine the optimum time of the steeping process and decocting process of Glycyrrhiza uralensis Fisch root.
2. The flow injection-capillary electrophoresis (FI-CE) system was used in connection with an on-line decoction system to automatically monitor glycyrrhetinic acid (GTA) and glycyrrhizic acid (GA) in the decoction process of Glycyrrhiza uralensis Fisch root for the first time.
3. A novel microemulsion electrokinetic chromatography (MEEKC) method was established for simultaneous determination of Imporation and Osthol in the medicinal plant Fructus Cnidii.
This dissertation consists of three parts and five chapters.
Part I Review of the development of capillary electrophoresis and application of CE in Traditional Chinese Medicines and their decoctions.
Chapter 1: The principle and characteristics of CE were simply described. The application of CE in study of TCMs and the application of analytical methods in the decoction of TCMs were reviewed and discussed in details.
Part II Study and application of CE in the bioactive components of Traditional Chinese Medicines.
Chapter 2: A fast, sensitive and precise analytical method based on CZE was developed for determining glycyrrhetinic acid (GTA) and glycyrrhizic acid (GA) in Glycyrrhiza uralensis Fisch root. This analysis was carried out with 10 mM borate (adjusted to pH 8.8) as a background electrolyte. The separation was achieved in 5 min. This method has also been successfully applied to determine the optimum time of the steeping process and decocting process of Glycyrrhiza uralensis Fisch root.
Chapter 3: The FI-CE system was used in connection with an on-line decoction system to automatically monitor glycyrrhetinic acid (GTA) and glycyrrhizic acid (GA) in the decoction process of Glycyrrhiza uralensis Fisch root. The separation of GTA and GA took less than 5 min by using a 10 mM borate buffer (adjusted pH to 8.8). The dynamic changes of GTA and GA on the decoction time were obtained in the on-line decoction process of Glycyrrhiza uralensis Fisch root.
Chapter 4: A rapid and effective MEEKC method was established for simultaneous determination of Imporation and Osthol in the medicinal plant Fructus Cnidii. After optimization of separation conditions, baseline separation was obtained for the two analytes within 20 min using a running buffer composed of 20 mMTris +20% (v/v) microemulsion stock solution(16.55%(w/v) SDS + 40.75%(v/v)l-butanol + 5.85%(v/v) n-heptane + 36.85%(v/v) water). The proposed method is a good alternative for simultaneous analysis of coumarins compounds in other medicinal plants.
Part III Study and application of CE in enantioseparation of four stereoisomersof palonosetron.
Chapter 5: The enantioseparation of four stereoisomers of palonosetron hydrochloride by CZE usingβ-CD as chiral selector was described for the first time.β-CD was shown to be effective in separating palonosetron hydrochloride stereoisomers. Baseline separation of palonosetron hydrochloride stereoisomers was achieved within 35 min using a running buffer composed of 30 mM NaH_2PO_4+10% (v/v) methol+150 mMβ-CD(adjusted pH to 3.0)
本论文第二部分充分利用毛细管电泳及流动注射-毛细管电泳联用的优势,对中药浸泡和煎煮过程中活性组分的浓度变化进行了测定,取得了以下几方面创新性的研究成果:
1.建立了同时分离中药甘草根中的甘草次酸和甘草酸的自由溶液毛细管电泳新方法。并且将该方法用于离线测定甘草根浸泡和煎煮过程中不同时间时甘草次酸和甘草酸的含量变化。
2.利用FI高精度进样和强大的样品预处理能力,首次将在线过滤和冷却装置与FI-CE联用,并应用到中药甘草根煎煮过程中有效成分浓度的动态变化研究。
3.将微乳液毛细管电动色谱用于中药蛇床子中欧前胡素和蛇床子素的分离与测定,结果令人满意。
本论文共分为三部分五章:
第一部分:综述
第一章:简单介绍了CE的原理和特点、毛细管电泳在中药及其煎煮液分析中的应用。
第二部分:毛细管电泳在中药活性成分分析中的应用
第二章:建立了同时分离测定甘草根中甘草次酸和甘草酸的自由溶液毛细管电泳新方法。最佳分析条件为:10 mM硼砂,pH 8.80,分离电压20 kV,检测波长254 nm。该方法已用于甘草根离线浸泡和煎煮过程中甘草次酸和甘草酸含量的变化研究,并获得了最佳浸泡和煎煮时间,而且对其它中药煎煮过程中有效成分含量的研究也具有重要的参考价值。
第三章:通过FI强大的样品预处理能力,实现了样品的在线过滤和冷却,建立了一种在线监测甘草根煎煮过程中有效成分动态变化的新方法。最佳分析条件为:10 mM硼砂,pH 8.80,分离电压为10 kV,检测波长为268 nm。通过对甘草根煎煮液的在线监测,得到了甘草根的最佳煎煮时间,而且发现在甘草根煎煮过程中,没有新成分出现,原有的成分也没有消失。将该方法进行改善,可用于其它中药煎煮过程中活性成分的在线测定。
第四章:建立了一种简单、快速、灵敏、准确的微乳液毛细管电动色谱法,同时测定了中药蛇床子中的欧前胡素和蛇床子素。最佳分析条件为:20 mM Tris,20%(v/v)微乳(16.55%(w/v)SDS+40.75%(v/v)正丁醇+5.85%(v/v)正庚烷+36.85%(v/v)水),分离电压15 kV,检测波长254 nm。
第三部分:毛细管电泳在手性分离中的应用
第五章:建立了一种简单、灵敏、准确的自由溶液毛细管电泳新方法,用β-CD为手性选择试剂,分离了盐酸帕洛诺司琼对映体。最佳分析条件为:30 mMNaH_2PO_4,10%甲醇,150 mMβ-CD,pH=3.0,分离电压为15 kV。
Natural medicinal resources provide valuable material for the discovery and development of new drugs of natural origin. The large numbers of other components in traditional Chinese medicines (TCMs) make the screening and analysis of the bioactive components extremely difficult. In the past decades, many effective analysis tools have been used for analyzing the components of TCMs in order to control the quality and discover bioactive compounds. Capillary electrophoresis (CE) is a new analysis technology. It provides advantages in terms of higher efficiency, faster separation time, less injected sample volume and more practicable operating modes. The advantages of the combined flow injection with capillary electrophoresis (FI-CE) system have been demonstrated not only to produce an efficient and reliable mode of sample introduction with improved throughput and precision and outstanding reproducibility of retention time, peak area and peak height, but also to provide on-line sample pretreatment ability. In the second part of this dissertation, the advantages of CE and FI-CE were made use of to separate and determinate active components in the decoction of Glscyrrhiza uralensis Fisch.
On the basis of the previous literatures, in this dissertation, some original studies are carried out as followings:
1. A novel method based on capillary zone electrophoresis (CZE) was developed for determining glycyrrhetinic acid (GTA) and glycyrrhizic acid (GA) in Glycyrrhiza uralensis Fisch root. This method has also been successfully applied to determine the optimum time of the steeping process and decocting process of Glycyrrhiza uralensis Fisch root.
2. The flow injection-capillary electrophoresis (FI-CE) system was used in connection with an on-line decoction system to automatically monitor glycyrrhetinic acid (GTA) and glycyrrhizic acid (GA) in the decoction process of Glycyrrhiza uralensis Fisch root for the first time.
3. A novel microemulsion electrokinetic chromatography (MEEKC) method was established for simultaneous determination of Imporation and Osthol in the medicinal plant Fructus Cnidii.
This dissertation consists of three parts and five chapters.
Part I Review of the development of capillary electrophoresis and application of CE in Traditional Chinese Medicines and their decoctions.
Chapter 1: The principle and characteristics of CE were simply described. The application of CE in study of TCMs and the application of analytical methods in the decoction of TCMs were reviewed and discussed in details.
Part II Study and application of CE in the bioactive components of Traditional Chinese Medicines.
Chapter 2: A fast, sensitive and precise analytical method based on CZE was developed for determining glycyrrhetinic acid (GTA) and glycyrrhizic acid (GA) in Glycyrrhiza uralensis Fisch root. This analysis was carried out with 10 mM borate (adjusted to pH 8.8) as a background electrolyte. The separation was achieved in 5 min. This method has also been successfully applied to determine the optimum time of the steeping process and decocting process of Glycyrrhiza uralensis Fisch root.
Chapter 3: The FI-CE system was used in connection with an on-line decoction system to automatically monitor glycyrrhetinic acid (GTA) and glycyrrhizic acid (GA) in the decoction process of Glycyrrhiza uralensis Fisch root. The separation of GTA and GA took less than 5 min by using a 10 mM borate buffer (adjusted pH to 8.8). The dynamic changes of GTA and GA on the decoction time were obtained in the on-line decoction process of Glycyrrhiza uralensis Fisch root.
Chapter 4: A rapid and effective MEEKC method was established for simultaneous determination of Imporation and Osthol in the medicinal plant Fructus Cnidii. After optimization of separation conditions, baseline separation was obtained for the two analytes within 20 min using a running buffer composed of 20 mMTris +20% (v/v) microemulsion stock solution(16.55%(w/v) SDS + 40.75%(v/v)l-butanol + 5.85%(v/v) n-heptane + 36.85%(v/v) water). The proposed method is a good alternative for simultaneous analysis of coumarins compounds in other medicinal plants.
Part III Study and application of CE in enantioseparation of four stereoisomersof palonosetron.
Chapter 5: The enantioseparation of four stereoisomers of palonosetron hydrochloride by CZE usingβ-CD as chiral selector was described for the first time.β-CD was shown to be effective in separating palonosetron hydrochloride stereoisomers. Baseline separation of palonosetron hydrochloride stereoisomers was achieved within 35 min using a running buffer composed of 30 mM NaH_2PO_4+10% (v/v) methol+150 mMβ-CD(adjusted pH to 3.0)
引文
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