高效液相色谱技术在中草药研究中的应用
摘要
中草药不仅具有很好的防病治病作用,而且也为开发研究新的药用成分提供了广阔的资源,因此受到科学界的广泛关注。为了指导临床上安全有效的使用中草药,必须建立快速、灵敏、准确的分析方法,以便强化和规范中草药的质量标准,深化它们在药理学、毒理学及药物代谢动力学等方面的研究。因为中草药不像化学药品那样单纯和规范,它们不仅成分复杂,并且成分之间相互影响和作用,同一成分化合物的分析结果可能会因母体药材或制剂配方的改变而受到不同程度的影响。所以根据分析的样品、目的和要求,对中草药同一成分化合物建立不同的分析方法和定量标准也是十分必要的。现代高效液相色谱(HPLC)技术具有高效、准确、灵敏度高、操作方便等优点,非常适合活性成分复杂、含量低的中草药研究。本论文是在前人的工作基础上,主要开展了以下几方面的创新性研究工作:
(1) 建立了测定银黄制剂和大鼠血浆中绿原酸及黄芩甙的HPLC新方法。
(2) 首次采用HPLC开展了银黄制剂在大鼠体内的药代动力学研究,计算并分析了药代动力学参数。
(3) 首次研究探讨了不同给药方式和剂量对大鼠服用银黄制剂后血液中绿原酸及黄芩甙“药-时曲线”的影响。
(4) 首次建立了同时测定绿原酸、咖啡酸、阿魏酸、原儿茶酸和原儿茶醛的HPLC法,并用于兽用制剂“复方蒲公英合剂”及其原料药材中相关成分的分析研究。
(5) 首次研究探讨了制剂工艺对“复方蒲公英合剂”有效成分含量的影响。
(6) 在国内首次开展了中草药有效成分松萝酸的透皮实验研究。
本论文共分为六章。
第一章:从HPLC的发展、联用技术的应用、定量分析方法以及在中草药研究中的应用几个方面详细介绍了HPLC分析技术的研究进展与应用现状。
第二章:建立了测定大鼠血浆中绿原酸的RP-HPLC-UV法(反相高效液相
Chinese herbal preparations are not only to prevent and treat disease but also to provide wide resources for exploitation of new drugs. Therefore they have become increasingly popular. In order to using Chinese herbal preparations rationally and safely, it is necessary to build sensitive and accurate analytical methods for deepening the study of pharmacokinetic, pharmacology, toxicology and qualitative standard. Chinese herbal preparations are not as simple and standard as chemical drugs, they have complex components which affect and act each other, in which the analytical result of a compound is probable affected by change of parent herb or prescription. It is necessary to build different analytical methods for a compound in Chinese herbal preparations according to analyzed sample, purpose and requirement. The HPLC is convenient to operate with high effectiveness, sensitivity and accuracy, so it is very suitable for the analysis of Chinese herbal preparations that have complex component and low content.On the basis of the previous literatures, the following major innovative works were carried out in this dissertation:(1) The novel HPLC methods were developed for the determination of chlorogenic acid and baicalin in both Yin-Huang preparations and rat plasma.(2) The pharmacokinetic study of Yin-Huang preparations in rat was carried outby HPLC, and pharmacokinetic parameters were calculated and discussed.(3) It was first to research the influence of administration dose and way to trendchange of chlorogenic acid and baicalin in rat dosed with Yin-Huang preparations.(4) A novel HPLC method was developed for simultaneous determination ofchlorogenic acid, caffeic acid, ferulic acid, protocatechuic acid and
protocatechuic aldehyde The developed method can be used for analysis and study of Fufang-Pugongying-Mixture and its compositional herbs.(5) The influence of preparation craft to the component content of Fufang-Pugongying-Mixture was studied by the developed HPLC method for the first time.(6) The study of the in vitro permeability of usnic acid, an effective component of Chinese herbal preparation was carried out through rabbit skin.This dissertation consists of six chapters.Chapter 1: In this chapter, the current status of HPLC technique is given by complete description at development of HPLC, method of qualitative analysis, use of union technique and the application of HPLC in study of Chinese herbal preparations.Chapter 2: A novel RP-HPLC-UV method for the quantification of CGA (chlorogenic acid) in rat plasma was developed. A good linear relationship between CGA concentration in plasma and corresponding peak-area ratio (CGA / internal standard) was obtained in the concentration range of 0.4—40 |ag/ml. The recovery of CGA was in the range of 85.29—89.12%, and LOQ (the limit of quantification) was 0.4 (xg/ml. The analytical method has high precision (RSD < 10.78%) and accuracy (-2.20% < RE < 5.00%). The method was successfully applied to the pharmacokinetic study of CGA in rat following administration of Yin- Huang granule, and the influence of administration dose and way to trend change of CGA in rat blood was researched.Chapter 3: A novel RP-HPLC-UV method for the quantification of BCL (baicalin) in rat plasma was developed. A good linear relationship between BCL concentration in plasma and corresponding peak-area ratio (BCL / internal standard) was obtained in the concentration range of 0.3—30 ng/ml. The recovery of BCL was within 96.31— 107.22%, and the LOQ was 0.3 ug/ml. The analytical method has high precision (RSD<8.08%) and accuracy (-3.00% < RE < 6.67%). The method was successfully applied to the pharmacokinetic study of BCL in rat following administration of Yin-Huang granule or Yin-Huang oral liquid, and the influence of administration way to
trend change of BCL in rat was researched.Chapter 4: A novel RP-HPLC-UV method was developed for simultaneous determination of chlorogenic acid, caffeic acid, ferulic acid, protocatechuic acid and protocatechuic aldehyde. The good linear relationships between concentrations and corresponding peak-areas were obtained in the concentration range of 2—200 ng/ml for the 5 compounds mentioned above. The recoveries of(the 5 compounds were in range of 96.72—104.07%. Their LOD (the limit of determination) values were in range of 0.05—0.78 {ig/ml, and the CV (the coefficient of variation) values reflecting analytical precision in range of 0.89—4.5%. The method can be used as qualitative method of Fufang-Pugongying-Mixture (FFPM), and used for the study of preparation craft of FFPM and analysis of Taraxacum, Lonicera and Angelica.Chapter 5: Through evaluation and comparison of analytical method, the RP-HPLC-UV methods for determining respectively CGA and BCL were selected and developed. The good linear relationships between concentrations and corresponding peak-areas were obtained in the concentration range of 1—50 fxg/ml for CGA and BCL. The LOQ of CGA was 0.2 (j.g/ml and BCL was 0.3 ug/ml, and their recoveries were between 97% and 104%. The analytical methods have high precision and accuracy: RSD<5%, -3%
(1) 建立了测定银黄制剂和大鼠血浆中绿原酸及黄芩甙的HPLC新方法。
(2) 首次采用HPLC开展了银黄制剂在大鼠体内的药代动力学研究,计算并分析了药代动力学参数。
(3) 首次研究探讨了不同给药方式和剂量对大鼠服用银黄制剂后血液中绿原酸及黄芩甙“药-时曲线”的影响。
(4) 首次建立了同时测定绿原酸、咖啡酸、阿魏酸、原儿茶酸和原儿茶醛的HPLC法,并用于兽用制剂“复方蒲公英合剂”及其原料药材中相关成分的分析研究。
(5) 首次研究探讨了制剂工艺对“复方蒲公英合剂”有效成分含量的影响。
(6) 在国内首次开展了中草药有效成分松萝酸的透皮实验研究。
本论文共分为六章。
第一章:从HPLC的发展、联用技术的应用、定量分析方法以及在中草药研究中的应用几个方面详细介绍了HPLC分析技术的研究进展与应用现状。
第二章:建立了测定大鼠血浆中绿原酸的RP-HPLC-UV法(反相高效液相
Chinese herbal preparations are not only to prevent and treat disease but also to provide wide resources for exploitation of new drugs. Therefore they have become increasingly popular. In order to using Chinese herbal preparations rationally and safely, it is necessary to build sensitive and accurate analytical methods for deepening the study of pharmacokinetic, pharmacology, toxicology and qualitative standard. Chinese herbal preparations are not as simple and standard as chemical drugs, they have complex components which affect and act each other, in which the analytical result of a compound is probable affected by change of parent herb or prescription. It is necessary to build different analytical methods for a compound in Chinese herbal preparations according to analyzed sample, purpose and requirement. The HPLC is convenient to operate with high effectiveness, sensitivity and accuracy, so it is very suitable for the analysis of Chinese herbal preparations that have complex component and low content.On the basis of the previous literatures, the following major innovative works were carried out in this dissertation:(1) The novel HPLC methods were developed for the determination of chlorogenic acid and baicalin in both Yin-Huang preparations and rat plasma.(2) The pharmacokinetic study of Yin-Huang preparations in rat was carried outby HPLC, and pharmacokinetic parameters were calculated and discussed.(3) It was first to research the influence of administration dose and way to trendchange of chlorogenic acid and baicalin in rat dosed with Yin-Huang preparations.(4) A novel HPLC method was developed for simultaneous determination ofchlorogenic acid, caffeic acid, ferulic acid, protocatechuic acid and
protocatechuic aldehyde The developed method can be used for analysis and study of Fufang-Pugongying-Mixture and its compositional herbs.(5) The influence of preparation craft to the component content of Fufang-Pugongying-Mixture was studied by the developed HPLC method for the first time.(6) The study of the in vitro permeability of usnic acid, an effective component of Chinese herbal preparation was carried out through rabbit skin.This dissertation consists of six chapters.Chapter 1: In this chapter, the current status of HPLC technique is given by complete description at development of HPLC, method of qualitative analysis, use of union technique and the application of HPLC in study of Chinese herbal preparations.Chapter 2: A novel RP-HPLC-UV method for the quantification of CGA (chlorogenic acid) in rat plasma was developed. A good linear relationship between CGA concentration in plasma and corresponding peak-area ratio (CGA / internal standard) was obtained in the concentration range of 0.4—40 |ag/ml. The recovery of CGA was in the range of 85.29—89.12%, and LOQ (the limit of quantification) was 0.4 (xg/ml. The analytical method has high precision (RSD < 10.78%) and accuracy (-2.20% < RE < 5.00%). The method was successfully applied to the pharmacokinetic study of CGA in rat following administration of Yin- Huang granule, and the influence of administration dose and way to trend change of CGA in rat blood was researched.Chapter 3: A novel RP-HPLC-UV method for the quantification of BCL (baicalin) in rat plasma was developed. A good linear relationship between BCL concentration in plasma and corresponding peak-area ratio (BCL / internal standard) was obtained in the concentration range of 0.3—30 ng/ml. The recovery of BCL was within 96.31— 107.22%, and the LOQ was 0.3 ug/ml. The analytical method has high precision (RSD<8.08%) and accuracy (-3.00% < RE < 6.67%). The method was successfully applied to the pharmacokinetic study of BCL in rat following administration of Yin-Huang granule or Yin-Huang oral liquid, and the influence of administration way to
trend change of BCL in rat was researched.Chapter 4: A novel RP-HPLC-UV method was developed for simultaneous determination of chlorogenic acid, caffeic acid, ferulic acid, protocatechuic acid and protocatechuic aldehyde. The good linear relationships between concentrations and corresponding peak-areas were obtained in the concentration range of 2—200 ng/ml for the 5 compounds mentioned above. The recoveries of(the 5 compounds were in range of 96.72—104.07%. Their LOD (the limit of determination) values were in range of 0.05—0.78 {ig/ml, and the CV (the coefficient of variation) values reflecting analytical precision in range of 0.89—4.5%. The method can be used as qualitative method of Fufang-Pugongying-Mixture (FFPM), and used for the study of preparation craft of FFPM and analysis of Taraxacum, Lonicera and Angelica.Chapter 5: Through evaluation and comparison of analytical method, the RP-HPLC-UV methods for determining respectively CGA and BCL were selected and developed. The good linear relationships between concentrations and corresponding peak-areas were obtained in the concentration range of 1—50 fxg/ml for CGA and BCL. The LOQ of CGA was 0.2 (j.g/ml and BCL was 0.3 ug/ml, and their recoveries were between 97% and 104%. The analytical methods have high precision and accuracy: RSD<5%, -3%
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