“广枣—降香”君—使对药体内效应物质的研究
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摘要
复方配伍理论是中医理论的重要组成部分。然而,中药自身组成的复杂性和临床用药的辩证灵活性使得复方配伍理论很难用现代科学技术作出清晰表达,因此,如何科学阐明复方配伍理论一直是中医药理论研究中的热点和难点。“君-使”药物是复方中的一组核心对药,符合中药“七情”中的“相使”关系,研究这种重要的配伍关系,发现体内效应物质对于进一步认识中药引经的实质和丰富并发展复方配伍理论有着重要意义,同时也能为高效创新药物的研发提供新的途径。本论文选择“广枣-降香”作为研究对象,系统地研究了降香对君药广枣中的效应物质在正常家兔体内药代动力学过程和组织分布特征的影响。全文共分为四章,主要研究内容如下:
1.建立了兔含药血浆和组织中没食子酸(GA)和原儿茶酸(PA)的高效液相色谱分析新方法,结果显示:没食子酸和原儿茶酸在0.04-2.0 mg·L~(-1)范围内具有良好的线性关系,低、中、高三个浓度的方法回收率、精密度和稳定性等指标均符合美国FDA生物样品的测定要求,可以作为广枣体内效应物质研究的方法,为君.使对药体内动力学和组织分布研究奠定了方法学基础。
2.采用上述方法研究了君药广枣中没食子酸和原儿茶酸在正常家兔体内的药代动力学过程;并研究了使药降香对没食子酸和原儿茶酸药代动力学过程的影响。研究发现与降香配伍后,可促进没食子酸、原儿茶酸由血液向组织中分布,提高了生物利用度,提示降香有可能增强君药广枣在治疗心血管类疾病方面的疗效,为分析降香增效.导向作用,以及科学阐明复方配伍理论提供了依据。
3.研究了广枣与降香配伍前后,没食子酸和原儿茶酸在正常家兔体内的组织分布特征变化,结果表明:没食子酸的分布特征由C_肾>C_脑>C_脾>C_心>C_肝>C_肺转变为C_肾>C_脑>C_肝>C_心>C_脾>C_肺;原儿茶酸的分布特征由C_肾>C_心>C_脾>C_肝>C_肺>C_脑转变为C_肾>C_脾>C_肝>C_心>C_肺>C_脑。结果印证了中医理论在广枣归经与降香引经方面的理论论述,为中药归经、引经等理论的现代研究提供了新思路。
4.结合代谢组学的研究方法,用主成分分析法对获得的色谱数据进行了分析,并结合药时曲线的变化规律,确定了4个未知成分为“广枣-降香”的体内效应物质。进一步通过高效液相色谱-质谱联用技术(HPLC-MS)对其进行了定性分析,推测其中2个成分的分子离子峰分别为:m/z 179.2和m/z 3 18.5,建立了“君-使”对药体内效应物质研究的新模式,为创新药物的开发提供了新途径。
The theory of prescription compatibility is an important part of the theory of traditional Chinese medicine (TCM). But, for the complex composition and clinical application with philosophic thoughts, it's very difficult to express the mechanism of prescription compatibility by modern science and technology. Therefore, how to express the principle of prescription compatibility according to modern science and technology is a hotspot and a tough problem all the time. Monarch-guide compatible herbal medicine is the core of TCM. An important approach to enrich the theories of Traditional Chinese Medicine and develop highly effective new drugs is to find the key compatibility relationship through the studies of efficacious prescriptions and find their bioactive substances. Under the guidance of the theories of Traditional Chinese Medicine, the present research chose "Guangzao - Dalbergia" to systematically study the mechanism of monarch-guide herbal medicine in the body through metabolomics. The influence of guide herb (Dalbergia,JX) was revealed through investigating the pharmacokinetical (PK) changes of bioactive substances in monarch herb(Guangzao,GZ); The main contents are as follows:
1. A new high performance liquid chromatography (HPLC) method for analysis of the gallic acid (GA) and protocatechuic acid (PA) of Guangzao in plasma and tissue is established. The results showed that the linear range of GA and PA is 0.04～2.0 mg·L~(-1). The relative recovery and absolute recovery of low, medium and high concentration, relative standard deviations (RSD) of precision and stability experiments are consistent with the requirements of biological sample analysis according to FDA.
2. After intragastric administration of GZ and GZ-JX, the PK parameters of GA and PA in plasma and tissue of normal rabbits. The results indicated that dalbergia could change the pharmacokinetics of GA, also could increase the biological availability of PA. Further, it might indicate that the the dalbergia enhance curative effect of GZ in the cardiovascular diseases, which will provide evidences of dalbergia's synergism and explaination of the theory of prescription compatibility by modem science and technology.
3. Tissue distribution of GA and PA is investigated to study the influence of synergism between SM and BC. The results show that the concentration sequence of GA in SM group is kidney >brain>spleen>heart>liver>lung. However, it convert to kidney> brain >liver> heart>spleen>lung under the influence of BC. The results show that the concentration sequence of PA in SM group is kidney >heart >spleen >liver>lung>brain, and it convert to kidney>spleen >liver> heart >lung > brain under the influence of BC, verifying the TCM theory on meridian distribution of SM and guidance distribution of BC.
4. The mass data of HPLC is statistically processed by metabonomic ,with SPSS method and considering the change of concentration-time curves, 4 unkown substances were ascertained to be the bioactive substances of GZ-JX. An initial qualitative analysis on them was carried out by HPLC -MS, then the fragment ions corresponded with 2 unknown bioactive substances were supposed to be: m/z179.2 and m/z318.5. A new mode to investigate the bioactive substances of monarch-guide compatible herb was established, which could provide new pathway to develop highly effective new drugs.
1.建立了兔含药血浆和组织中没食子酸(GA)和原儿茶酸(PA)的高效液相色谱分析新方法,结果显示:没食子酸和原儿茶酸在0.04-2.0 mg·L~(-1)范围内具有良好的线性关系,低、中、高三个浓度的方法回收率、精密度和稳定性等指标均符合美国FDA生物样品的测定要求,可以作为广枣体内效应物质研究的方法,为君.使对药体内动力学和组织分布研究奠定了方法学基础。
2.采用上述方法研究了君药广枣中没食子酸和原儿茶酸在正常家兔体内的药代动力学过程;并研究了使药降香对没食子酸和原儿茶酸药代动力学过程的影响。研究发现与降香配伍后,可促进没食子酸、原儿茶酸由血液向组织中分布,提高了生物利用度,提示降香有可能增强君药广枣在治疗心血管类疾病方面的疗效,为分析降香增效.导向作用,以及科学阐明复方配伍理论提供了依据。
3.研究了广枣与降香配伍前后,没食子酸和原儿茶酸在正常家兔体内的组织分布特征变化,结果表明:没食子酸的分布特征由C_肾>C_脑>C_脾>C_心>C_肝>C_肺转变为C_肾>C_脑>C_肝>C_心>C_脾>C_肺;原儿茶酸的分布特征由C_肾>C_心>C_脾>C_肝>C_肺>C_脑转变为C_肾>C_脾>C_肝>C_心>C_肺>C_脑。结果印证了中医理论在广枣归经与降香引经方面的理论论述,为中药归经、引经等理论的现代研究提供了新思路。
4.结合代谢组学的研究方法,用主成分分析法对获得的色谱数据进行了分析,并结合药时曲线的变化规律,确定了4个未知成分为“广枣-降香”的体内效应物质。进一步通过高效液相色谱-质谱联用技术(HPLC-MS)对其进行了定性分析,推测其中2个成分的分子离子峰分别为:m/z 179.2和m/z 3 18.5,建立了“君-使”对药体内效应物质研究的新模式,为创新药物的开发提供了新途径。
The theory of prescription compatibility is an important part of the theory of traditional Chinese medicine (TCM). But, for the complex composition and clinical application with philosophic thoughts, it's very difficult to express the mechanism of prescription compatibility by modern science and technology. Therefore, how to express the principle of prescription compatibility according to modern science and technology is a hotspot and a tough problem all the time. Monarch-guide compatible herbal medicine is the core of TCM. An important approach to enrich the theories of Traditional Chinese Medicine and develop highly effective new drugs is to find the key compatibility relationship through the studies of efficacious prescriptions and find their bioactive substances. Under the guidance of the theories of Traditional Chinese Medicine, the present research chose "Guangzao - Dalbergia" to systematically study the mechanism of monarch-guide herbal medicine in the body through metabolomics. The influence of guide herb (Dalbergia,JX) was revealed through investigating the pharmacokinetical (PK) changes of bioactive substances in monarch herb(Guangzao,GZ); The main contents are as follows:
1. A new high performance liquid chromatography (HPLC) method for analysis of the gallic acid (GA) and protocatechuic acid (PA) of Guangzao in plasma and tissue is established. The results showed that the linear range of GA and PA is 0.04～2.0 mg·L~(-1). The relative recovery and absolute recovery of low, medium and high concentration, relative standard deviations (RSD) of precision and stability experiments are consistent with the requirements of biological sample analysis according to FDA.
2. After intragastric administration of GZ and GZ-JX, the PK parameters of GA and PA in plasma and tissue of normal rabbits. The results indicated that dalbergia could change the pharmacokinetics of GA, also could increase the biological availability of PA. Further, it might indicate that the the dalbergia enhance curative effect of GZ in the cardiovascular diseases, which will provide evidences of dalbergia's synergism and explaination of the theory of prescription compatibility by modem science and technology.
3. Tissue distribution of GA and PA is investigated to study the influence of synergism between SM and BC. The results show that the concentration sequence of GA in SM group is kidney >brain>spleen>heart>liver>lung. However, it convert to kidney> brain >liver> heart>spleen>lung under the influence of BC. The results show that the concentration sequence of PA in SM group is kidney >heart >spleen >liver>lung>brain, and it convert to kidney>spleen >liver> heart >lung > brain under the influence of BC, verifying the TCM theory on meridian distribution of SM and guidance distribution of BC.
4. The mass data of HPLC is statistically processed by metabonomic ,with SPSS method and considering the change of concentration-time curves, 4 unkown substances were ascertained to be the bioactive substances of GZ-JX. An initial qualitative analysis on them was carried out by HPLC -MS, then the fragment ions corresponded with 2 unknown bioactive substances were supposed to be: m/z179.2 and m/z318.5. A new mode to investigate the bioactive substances of monarch-guide compatible herb was established, which could provide new pathway to develop highly effective new drugs.
引文
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