黄芪、党参质量评价及其提取物活性研究
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摘要
由于中药材是中药饮片和成方制剂的原料,它的质量直接关系到临床的用药安全和疗效,因此中药材的质量控制显得尤为重要。同时,随着中药材在世界范围的应用,中药材质量控制是保证中药“安全、稳定、有效、可控”,实现中药现代化、产业化及国际化的核心问题。要做到中药材质量的稳定可控,就必须要有客观规范的质量评价方式全面科学的评价和控制中药材的质量。近几十年,在药材质量控制方面已有大量的研究,为中药材的安全和质量控制打下了良好基础,但是,在这方面仍然还存在一些问题,需要进一步研究和完善。由于中药材的疗效是多种成分协同作用的结果,测定中药材个别“有效成分或部位”的含量,作为评价中药材质量的指标不够充分、客观,也不符合中医药理论,因而关键的是要建立符合现代医学和中医学理论的多指标质量评价体系。
对不同产地来源的中药材,它的质量受到种源、产地气候、土壤、采收期、用药部位和炮制方法等因素的综合影响。黄芪和党参作为我国大宗中药材,由于多因素的影响,黄芪和党参药材存在品种混杂,质量不稳定的问题。为了保证药品安全有效和稳定可控、筛选优良品种、促进资源合理开发和利用,黄芪和党参的药材质量评价成为现在研究中的热点和重点。本文在《中华人民共和国药典》(2005、2010版)对黄芪、党参内在质量检测的指标的基础上,采用多指标结合的方法建立黄芪、党参综合评价体系,用此体系对不同来源的黄芪、党参药材进行质量评价以及对影响黄芪和党参药材质量的主要因素进行研究,确保药材的质量。另外,本研究对黄芪、党参醇提物的抗氧化活性和抗菌活性进行研究,为探索黄芪、党参治病、保健机理及进一步评价、利用黄芪、党参资源提供参考依据。本研究得出如下主要结论:
1.建立了黄芪药材HPLC-ELSD指纹图谱,检出了15个峰作为定性鉴别的指标峰,结合《中华人民共和国药典》(2005,2010)版中规定黄芪检测项下对内在质量检测的总灰分、酸不溶灰分、水溶性浸出物、黄芪甲苷含量测定,建立黄芪药材质量评价体系,并用此体系对不同来源的黄芪进行质量评价,可知不同产地黄芪药材的质量存在差异,多个指标综合评价能够更加准确的反映黄芪药材的质量。
2.不同产地黄芪样品中黄芪甲苷的含量及HPLC图谱存在一定的差异。为研究不同产地不同气候因子对黄芪药材质量的影响,通过黄芪甲苷含量测定、不同产地黄芪HPLC图谱与气候因子逐步回归分析的方法,研究发现年最高气温、年日照时数和年积温突出影响黄芪甲苷含量,其中较高的年最高气温和年积温利于黄芪甲苷的积累。
3.黄芪、党参样品中均含有17种氨基酸和微量元素。在黄芪、党参所含氨基酸中的7种为人体必需氨基酸,其种类齐全,含量较丰富,以WHO/FAO提出的E/ E + N40 % ,E/ N 0. 6的参考蛋白模式为标准,所测黄芪、党参样品基本接近参考蛋白模式,黄芪、党参可作为重要的氨基酸资源。不同产地黄芪、党参药材中氨基酸总量、必需氨基酸含量都存在一定的差异。在不同产地黄芪、党参样品中均含有钒(V)、铁(Fe)、钴(Co)、镍(Ni)、铜(Cu)、锌(Zn)、硒(Se)、钼(Mo)等微量元素,并且不同产地样品中所含微量元素具有一定的差异。氨基酸和微量元素的含量研究有助于了解它们与黄芪、党参药理与保健价值之间的关系,以及进一步对黄芪、党参药材的质量评价。
4.建立了党参药材HPLC-DAD指纹图谱,结合《中华人民共和国药典》(2005,2010)中对党参内在质量醇溶性浸出物的测定与党参样品的总灰分、酸不溶灰分、党参炔苷、苍术内酯Ⅲ含量测定,建立党参药材质量评价体系,并用此体系对不同来源的党参进行质量评价可知不同产地党参药材的质量存在差异,多个指标综合评价能够更加准确的反映党参药材的质量。
5.对不同产地黄芪、党参醇提取物DPPH自由基清除率的研究表明不同产地的黄芪、党参醇提物的抗氧化活性均存在一定的差异。黄芪、党参醇提物浓度与清除DPPH自由基能力的呈现量效关系,并且黄芪醇提物清除自由基能力大于党参提取物。
6.通过对黄芪、党参醇提物抗菌活性进行研究,黄芪醇提物对金黄色葡萄球菌、表皮葡萄球菌、卡他布朗汉姆氏菌、甲型溶血性链球菌、乙型溶血性链球菌、肺炎克雷伯氏菌、大肠埃希氏菌、枯草芽胞杆菌有抑制作用。尤其是对乙型溶血性链球菌、炭疽杆菌、金黄色葡萄球菌和枯草杆菌的作用较明显。党参醇提物对除去黄芪醇提物有作用的9种菌以外的伤寒沙门氏菌和变形杆菌也有抑制作用。
Quality of medical materials is one of the most decisive factors to the quality and potency of drugs, and quality control to be the core solution to safeguarding the security, effectiveness, stability and controllability of traditional Chinese medicines(TCM)and realizing the modernization, industrialization and internationalization of the industry, also the procedure with most challenge to resolve in the production. The past decades witnessed not only series of promising research on quality control conducted to form fine foundations for Chinese herbs security and quality control, but also disadvantages and shortcoming existed in the present research. Due to the fact that curative effects of the TCM are comprehensive interactions between different materials, the accepted quality assessment system by judging the content of active ingredient of the medical materials proved inadequate on objectivity and hard to be accepted by theories on TCM. Thus, the basic step is to set up a normalized quality assessment criterion to fit the principles in TCM and the acceptance of modern medicine. Quality of the medical materials from diversified regions is determined by its species, place, climate, soil, harvest period, drug use part and processing methods. Astragalus membranaceus and Codonopsis pilosula, as infinitive crude materials in China, show a mixed species and quality diversity for historical and geographical reasons. Quality assessment of materials in A. membranaceus and C. pilosula is the heated topic for research to select fine variety, widen and promote rational exploitation and use of medical resources, and guarantee secured effectiveness and controlled stability of drugs. The paper guided by the quality assessment index on A. membranaceus and C. pilosula in Chinese Pharmcopoeia (2005, 2010), set up a comprehensive assessment system integrating multiple indexes, then applied the system into the quality assessment to A. membranaceus and C. pilosula with diversified origins, analyzed the main factors affecting their quality, safeguarding the security, effectiveness, stability and controllability of the quality. Besides, owning to the clinical use of A. membranaceus and C. pilosula on therapy to chronic intractable disease and the raw antioxidant and antimicrobial extract of them, the paper also implemented study on the antioxidant and antimicrobial activity of their ethanol extracts, and concluded the referential results to the exploration of curative and healthcare mechanism of A. membranaceus and C. pilosula and the assessment and use of the resources. Major research findings concluded include:
1、set up the fingerprint of HPLC-ELSD for A. membranaceus, there are 15 common peaks in the fingerptint; tested the total ash, acid insoluble ash, watery extract and contents of astragaloside IV referring to the assessing indexes of A. membranaceus in Chinese Pharmcopoeia (Edition 2005, 2010), set up the quality assessment system of A. membranaceus; applied the system into the quality assessment of A. membranaceus from various origins and concluded that quality of A. membranaceus from different habitats was different. Multi-indices to assess the quality of A. membranaceus could make assessment result more accurate.
2、contents of astragaloside IV in A. membranaceus samples from different places and the HPLC chromatogram proved certain diversities; illustrated on the effects of annual mean temperature, minimum temperature, maximum temperature, annual accumulative temperature, annual precipitation, annual sunshine duration and frost-free period to contents of astragaloside IV in different places; SAS stepwise regression over the above elements demonstrated that annual maximum temperature, annual sunshine duration and average accumulative temperature evidently affected contents of astragaloside IV and high maximum temperature and average temperature proved favorable to the content accumulation.
3、Both A. membranaceus and C. pilosula samples have 17 types of amino acid and trace element, among them 7 are fertile and complete amino acid essential for human. Samples acid ration approximated the number of reference protein model proposed byWHO/FAO (E/E+N40%, E/N0.6), which proved that A. membranaceus and C. pilosula are important resources of amino acid and the amino acid total content and necessary content varies from places. Samples from different places contain various trace elements of V, Fe, Co, Ni, Cu, Zn, Se and Mo. Research on amino acid and trace elements of their samples helped to construct the relations between their pharmacology and health care capacity, as well furthered the quality assessment to A. membranaceus and C. pilosula.
4、set up the fingerprint of HPLC-ELSD for C. pilosula; tested the total ash, acid insoluble ash, lobetyolin and atractylenolide III referring to the assessing indexes of C. pilosula in Chinese Pharmcopoeia, set up the quality assessment system of C.pilosula; applied the system into the quality assessment of C. pilosula from various origins and concluded that quality of C.pilosula from different habitats was different. Multi-indices to assess the quality of C. pilosula could make assessment result more accurate.
5、research on DPPH radical scavenging activity of A. membranaceus and C. pilosula ethanol extract from different places concluded the difference in their activity. A dose-response relationship existed between the concentration of their ethanol extract and DPPH free radical scavenging capacity, and the former surpassed the latter in the capacity.
6、Research on A. membranaceus and C. pilosula ethanol extracts antimicrobial activity concluded that A. membranaceus extract attained inhibition to Staphylococcus aureus, Staphylococcus epidermdis, Branhamella catarrhalis, Streptococcus hemolysis-α, Streptococcus hemolysis-β, Bacillus anthracis, Klebsiella pneumoniae, Escherichia coli and Bacillus subtilis, more evident to Streptococcus hemolysis-β, Bacillus anthracis, Staphylococcus aureus and Bacillus subtilis., and ethanol extract of C. pilosula attained inhibition to Salmonella typhi and Proteus sp. besides the nine fungi mentioned above.
对不同产地来源的中药材,它的质量受到种源、产地气候、土壤、采收期、用药部位和炮制方法等因素的综合影响。黄芪和党参作为我国大宗中药材,由于多因素的影响,黄芪和党参药材存在品种混杂,质量不稳定的问题。为了保证药品安全有效和稳定可控、筛选优良品种、促进资源合理开发和利用,黄芪和党参的药材质量评价成为现在研究中的热点和重点。本文在《中华人民共和国药典》(2005、2010版)对黄芪、党参内在质量检测的指标的基础上,采用多指标结合的方法建立黄芪、党参综合评价体系,用此体系对不同来源的黄芪、党参药材进行质量评价以及对影响黄芪和党参药材质量的主要因素进行研究,确保药材的质量。另外,本研究对黄芪、党参醇提物的抗氧化活性和抗菌活性进行研究,为探索黄芪、党参治病、保健机理及进一步评价、利用黄芪、党参资源提供参考依据。本研究得出如下主要结论:
1.建立了黄芪药材HPLC-ELSD指纹图谱,检出了15个峰作为定性鉴别的指标峰,结合《中华人民共和国药典》(2005,2010)版中规定黄芪检测项下对内在质量检测的总灰分、酸不溶灰分、水溶性浸出物、黄芪甲苷含量测定,建立黄芪药材质量评价体系,并用此体系对不同来源的黄芪进行质量评价,可知不同产地黄芪药材的质量存在差异,多个指标综合评价能够更加准确的反映黄芪药材的质量。
2.不同产地黄芪样品中黄芪甲苷的含量及HPLC图谱存在一定的差异。为研究不同产地不同气候因子对黄芪药材质量的影响,通过黄芪甲苷含量测定、不同产地黄芪HPLC图谱与气候因子逐步回归分析的方法,研究发现年最高气温、年日照时数和年积温突出影响黄芪甲苷含量,其中较高的年最高气温和年积温利于黄芪甲苷的积累。
3.黄芪、党参样品中均含有17种氨基酸和微量元素。在黄芪、党参所含氨基酸中的7种为人体必需氨基酸,其种类齐全,含量较丰富,以WHO/FAO提出的E/ E + N40 % ,E/ N 0. 6的参考蛋白模式为标准,所测黄芪、党参样品基本接近参考蛋白模式,黄芪、党参可作为重要的氨基酸资源。不同产地黄芪、党参药材中氨基酸总量、必需氨基酸含量都存在一定的差异。在不同产地黄芪、党参样品中均含有钒(V)、铁(Fe)、钴(Co)、镍(Ni)、铜(Cu)、锌(Zn)、硒(Se)、钼(Mo)等微量元素,并且不同产地样品中所含微量元素具有一定的差异。氨基酸和微量元素的含量研究有助于了解它们与黄芪、党参药理与保健价值之间的关系,以及进一步对黄芪、党参药材的质量评价。
4.建立了党参药材HPLC-DAD指纹图谱,结合《中华人民共和国药典》(2005,2010)中对党参内在质量醇溶性浸出物的测定与党参样品的总灰分、酸不溶灰分、党参炔苷、苍术内酯Ⅲ含量测定,建立党参药材质量评价体系,并用此体系对不同来源的党参进行质量评价可知不同产地党参药材的质量存在差异,多个指标综合评价能够更加准确的反映党参药材的质量。
5.对不同产地黄芪、党参醇提取物DPPH自由基清除率的研究表明不同产地的黄芪、党参醇提物的抗氧化活性均存在一定的差异。黄芪、党参醇提物浓度与清除DPPH自由基能力的呈现量效关系,并且黄芪醇提物清除自由基能力大于党参提取物。
6.通过对黄芪、党参醇提物抗菌活性进行研究,黄芪醇提物对金黄色葡萄球菌、表皮葡萄球菌、卡他布朗汉姆氏菌、甲型溶血性链球菌、乙型溶血性链球菌、肺炎克雷伯氏菌、大肠埃希氏菌、枯草芽胞杆菌有抑制作用。尤其是对乙型溶血性链球菌、炭疽杆菌、金黄色葡萄球菌和枯草杆菌的作用较明显。党参醇提物对除去黄芪醇提物有作用的9种菌以外的伤寒沙门氏菌和变形杆菌也有抑制作用。
Quality of medical materials is one of the most decisive factors to the quality and potency of drugs, and quality control to be the core solution to safeguarding the security, effectiveness, stability and controllability of traditional Chinese medicines(TCM)and realizing the modernization, industrialization and internationalization of the industry, also the procedure with most challenge to resolve in the production. The past decades witnessed not only series of promising research on quality control conducted to form fine foundations for Chinese herbs security and quality control, but also disadvantages and shortcoming existed in the present research. Due to the fact that curative effects of the TCM are comprehensive interactions between different materials, the accepted quality assessment system by judging the content of active ingredient of the medical materials proved inadequate on objectivity and hard to be accepted by theories on TCM. Thus, the basic step is to set up a normalized quality assessment criterion to fit the principles in TCM and the acceptance of modern medicine. Quality of the medical materials from diversified regions is determined by its species, place, climate, soil, harvest period, drug use part and processing methods. Astragalus membranaceus and Codonopsis pilosula, as infinitive crude materials in China, show a mixed species and quality diversity for historical and geographical reasons. Quality assessment of materials in A. membranaceus and C. pilosula is the heated topic for research to select fine variety, widen and promote rational exploitation and use of medical resources, and guarantee secured effectiveness and controlled stability of drugs. The paper guided by the quality assessment index on A. membranaceus and C. pilosula in Chinese Pharmcopoeia (2005, 2010), set up a comprehensive assessment system integrating multiple indexes, then applied the system into the quality assessment to A. membranaceus and C. pilosula with diversified origins, analyzed the main factors affecting their quality, safeguarding the security, effectiveness, stability and controllability of the quality. Besides, owning to the clinical use of A. membranaceus and C. pilosula on therapy to chronic intractable disease and the raw antioxidant and antimicrobial extract of them, the paper also implemented study on the antioxidant and antimicrobial activity of their ethanol extracts, and concluded the referential results to the exploration of curative and healthcare mechanism of A. membranaceus and C. pilosula and the assessment and use of the resources. Major research findings concluded include:
1、set up the fingerprint of HPLC-ELSD for A. membranaceus, there are 15 common peaks in the fingerptint; tested the total ash, acid insoluble ash, watery extract and contents of astragaloside IV referring to the assessing indexes of A. membranaceus in Chinese Pharmcopoeia (Edition 2005, 2010), set up the quality assessment system of A. membranaceus; applied the system into the quality assessment of A. membranaceus from various origins and concluded that quality of A. membranaceus from different habitats was different. Multi-indices to assess the quality of A. membranaceus could make assessment result more accurate.
2、contents of astragaloside IV in A. membranaceus samples from different places and the HPLC chromatogram proved certain diversities; illustrated on the effects of annual mean temperature, minimum temperature, maximum temperature, annual accumulative temperature, annual precipitation, annual sunshine duration and frost-free period to contents of astragaloside IV in different places; SAS stepwise regression over the above elements demonstrated that annual maximum temperature, annual sunshine duration and average accumulative temperature evidently affected contents of astragaloside IV and high maximum temperature and average temperature proved favorable to the content accumulation.
3、Both A. membranaceus and C. pilosula samples have 17 types of amino acid and trace element, among them 7 are fertile and complete amino acid essential for human. Samples acid ration approximated the number of reference protein model proposed byWHO/FAO (E/E+N40%, E/N0.6), which proved that A. membranaceus and C. pilosula are important resources of amino acid and the amino acid total content and necessary content varies from places. Samples from different places contain various trace elements of V, Fe, Co, Ni, Cu, Zn, Se and Mo. Research on amino acid and trace elements of their samples helped to construct the relations between their pharmacology and health care capacity, as well furthered the quality assessment to A. membranaceus and C. pilosula.
4、set up the fingerprint of HPLC-ELSD for C. pilosula; tested the total ash, acid insoluble ash, lobetyolin and atractylenolide III referring to the assessing indexes of C. pilosula in Chinese Pharmcopoeia, set up the quality assessment system of C.pilosula; applied the system into the quality assessment of C. pilosula from various origins and concluded that quality of C.pilosula from different habitats was different. Multi-indices to assess the quality of C. pilosula could make assessment result more accurate.
5、research on DPPH radical scavenging activity of A. membranaceus and C. pilosula ethanol extract from different places concluded the difference in their activity. A dose-response relationship existed between the concentration of their ethanol extract and DPPH free radical scavenging capacity, and the former surpassed the latter in the capacity.
6、Research on A. membranaceus and C. pilosula ethanol extracts antimicrobial activity concluded that A. membranaceus extract attained inhibition to Staphylococcus aureus, Staphylococcus epidermdis, Branhamella catarrhalis, Streptococcus hemolysis-α, Streptococcus hemolysis-β, Bacillus anthracis, Klebsiella pneumoniae, Escherichia coli and Bacillus subtilis, more evident to Streptococcus hemolysis-β, Bacillus anthracis, Staphylococcus aureus and Bacillus subtilis., and ethanol extract of C. pilosula attained inhibition to Salmonella typhi and Proteus sp. besides the nine fungi mentioned above.
引文
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