湘西杜仲有效成分提制分析及其指纹图谱研究
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摘要
随着人们对化学药品毒副作用的认识及对健康要求的不断提高,天然药物的用途和需求不断扩大,中草药的优势和特色越来越被世界重视。如何充分提取中草药的有效成分,提高中草药的利用率和临床疗效,是中药现代化面临的主要课题和重要任务。本文以湘西重要中药资源—杜仲为研究对象,以现代新兴样品预处理技术(微波提取技术等)为工具,从杜仲中提取和分离以绿原酸为代表的多酚酸类化合物,研究了各种因素对提取效率的影响。另外,本文还建立了杜仲药材指纹图谱质量控制标准,并对杜仲药材的微量元素及其抗菌活性进行了相关研究,为合理利用杜仲药材提供了相关参照。具体内容如下:
1.杜仲药材有效成分绿原酸的提制分析研究
研究比较采用不同的提取溶剂、提取方法、提取条件来优化提取工艺,得到绿原酸的最佳提取条件。实验结果表明:微波助提取比常规提取方法大大缩短了提取时间,提高了提取效率;采用正交实验因子设计法对微波提取条件进行了优化(最佳提取工艺为:乙醇浓度60%、固液比1:50、辐射时间2min、微波压力2atm);在最佳液相色谱操作条件下,测定了杜仲叶、未烘烤的杜仲皮、已烘烤的杜仲皮和中成药杜仲平压片中绿原酸的含量。实验可知杜仲叶中绿原酸明显高于杜仲皮,且杜仲叶资源丰富、易得,为开发绿原酸提供了丰富的资源。本文研究得出:微波提取技术具有高效、便捷、快速的优点;杜仲药材有效成分绿原酸的分析方法简便、准确、线性范围宽、重现性好,适合对含有绿原酸的原料和产品进行检测和质量控制。
2.杜仲专属性对照物质及其指纹图谱的创建
利用高效液相色谱(HPLC)和液-质联用技术(LC-MS)对杜仲药材进行指纹图谱专属性研究。色谱分离过程研究采用梯度洗脱程序,采用“计算机相似度评价”软件来对不同杜仲药材的相似度进行评价。实验结果发现:地理位置越靠近的杜仲药材的相似度越接近,不同地理位置(不同气候条件)的杜仲药材质量稍有差异。同时结合质谱数据,确定了杜仲药材的主要活性成分,适用于杜仲药材的基源鉴定和质量评价。
3.杜仲药材中微量元素的测定
杜仲被称为神奇之树,它的叶、皮和花中都含有人体不可缺少的微量元素,具有很高的药用价值。本文研究采用火焰原子吸收光谱法对杜仲药材中的多种元素进行了含量测定。实验研究发现:杜仲药材中含有丰富的Mg、Cu、Fe、Zn、Mn等元素,在人体的新陈代谢过程中发挥着重要的作用,且杜仲叶中各元素的含量较杜仲皮要高。杜仲药材中Pb、Cr含量较低,是一种安全无毒副作用的中药,可以放心大胆的用药。杜仲药材中微量元素的研究,为进一步开发利用杜仲药材提供了科学依据。同时本文还对几种植物样品前处理方法进行了对比研究,发现微波消解法具有溶样能力强、速度快、污染少、简单、方便等优点,是目前植物中元素含量测定的最佳样品处理方法。
4.杜仲叶提取物的抑菌效果研究
以乙醇和水组成的混合溶液作提取剂,从杜仲叶中微波提取主要活性成分绿原酸,并研究了在最佳微波提取条件下提取溶液的抗菌活性。结果表明:杜仲绿原酸具有抗大肠杆菌的生物活性,提取液最低抑菌浓度为100mg/mL。
As people have realized the toxic effects of chemical medicine and have improved their health consumption conceptions, the use and requirements of natural medicines have been expanded constantly. More and more people have emphasized the characters and advantages of Chinese herds. How to completely extract active components and improve the utilization and clinical effects of traditional Chinese herbs, have become the main subject and task of traditional Chinese herbs in its modernization. The noval types of sample pretreatment techniques (microwave-assisted extraction approaches) have been used for extraction and isolation of polyphenolic compounds, such as chlorogenic acid, caffeic acid, syringin, et al, from Eucommia ulmodies Oliv. E.ulmodies is one of the precious Traditional Chinese Medicine which distributes in the west of Hunan province. In this work, the parameters which influence the extraction efficiencies in each technique had been investigated. Additional, the quality control standard of Eucommia ulmodies Oliv was established, which was used to control the quality of E.ulmodies. Then the research about trace elements and antibiotic functions had been done, which can provide reference informations for the rational use of E.ulmodies. The concrete contents are as follows:
1. The research of extraction of Chlorogenic Acid from E. ulmodies
The optimum technics of extracting chlorogenic acid from E.ulmodies was studied. During the extracting process, extracting solvents, methods and conditions were studied. The result showed that: Microwave-assisted extraction was fast and efficient. The optimum conditions were: the volume fraction of ethanol, 0.6; the pressure, 2atm; time, 2min. The material/solution ratio was 1:50. Under the same HPLC condition, we detected the content of chlorogenic acid in leaves, unroasted bark, roasted bark and Du Zhong Ping Ya Pian. The content of chlorogenic acid in leaves was higher than that in bark. Meanwhile the leaves of E.ulmodies were easy to get, it can provide abundance chlorogenic acid resource. Microwave-assisted extraction was a kind of newfashioned, high efficient, convenience and fast extraction mean, meanwhile the HPLC method had good precision, repeatability. The result was dependable. It can be adopt to determinate the content and control the quality of material and products that contain chlorogenic acid.
2. Selection and fingerprints of the control substances for plant drug Eucommia ulmodies Oliv by HPLC and LC-MS
The selection of the control substances and their fingerprints of this plant medicine were investigated by using HPLC and LC-MS. The gradient elution mode was applied in chromatographic separation, and the data were analyzed by“Computer Aided Similarity Evaluation”software to compare the similarity of the E. ulmodies from different habitats. Low similarity was found in the samples from nonadjacent provinces, while high similarity was obtained in those from the adjacent provinces. The LC-MS fingerprints of E. ulmodies showed the main active constituents and could be used for its original identification and quality evaluation.
3. Determination of elements in the E.ulmodies
The E.ulmodies is a miraculous tree. Its leaves bark and flowers contain trace elements in human. The elements of the E.ulmodies were determined by FAAS. The results showed that: There were abundance elements, such as Mg Cu Fe, et al. The contents of them were higher in leaves than in bark, and they played a very important part in the metabolish of the flesh. While the contents of the element, such as Pb Cr, were very low. So E.ulmodies is a very safe Chinese traditional medicine. The experiment results provided the scientific basis for further research. Meanwhile by comparing different methods, the microwave digestion had the merits of efficiency, rapidity, less pollution and convenience. It is the best sample preparation method for determination plant’s trace elements contents.
4. Study on antibiotic ability of leaves extracts of E.ulmodies
Chlorogenic acid was extracted from E.ulmodies by Microwave-Extraction method. The solvent was a mixed solution of ethanol and water. The antibiotic ability of chlorogenic acid under optimum extraction condition was studied. The result showed that, the extract had the antibiotic ability towards Escherichia coli, and the MIC was 100 mg/mL.
1.杜仲药材有效成分绿原酸的提制分析研究
研究比较采用不同的提取溶剂、提取方法、提取条件来优化提取工艺,得到绿原酸的最佳提取条件。实验结果表明:微波助提取比常规提取方法大大缩短了提取时间,提高了提取效率;采用正交实验因子设计法对微波提取条件进行了优化(最佳提取工艺为:乙醇浓度60%、固液比1:50、辐射时间2min、微波压力2atm);在最佳液相色谱操作条件下,测定了杜仲叶、未烘烤的杜仲皮、已烘烤的杜仲皮和中成药杜仲平压片中绿原酸的含量。实验可知杜仲叶中绿原酸明显高于杜仲皮,且杜仲叶资源丰富、易得,为开发绿原酸提供了丰富的资源。本文研究得出:微波提取技术具有高效、便捷、快速的优点;杜仲药材有效成分绿原酸的分析方法简便、准确、线性范围宽、重现性好,适合对含有绿原酸的原料和产品进行检测和质量控制。
2.杜仲专属性对照物质及其指纹图谱的创建
利用高效液相色谱(HPLC)和液-质联用技术(LC-MS)对杜仲药材进行指纹图谱专属性研究。色谱分离过程研究采用梯度洗脱程序,采用“计算机相似度评价”软件来对不同杜仲药材的相似度进行评价。实验结果发现:地理位置越靠近的杜仲药材的相似度越接近,不同地理位置(不同气候条件)的杜仲药材质量稍有差异。同时结合质谱数据,确定了杜仲药材的主要活性成分,适用于杜仲药材的基源鉴定和质量评价。
3.杜仲药材中微量元素的测定
杜仲被称为神奇之树,它的叶、皮和花中都含有人体不可缺少的微量元素,具有很高的药用价值。本文研究采用火焰原子吸收光谱法对杜仲药材中的多种元素进行了含量测定。实验研究发现:杜仲药材中含有丰富的Mg、Cu、Fe、Zn、Mn等元素,在人体的新陈代谢过程中发挥着重要的作用,且杜仲叶中各元素的含量较杜仲皮要高。杜仲药材中Pb、Cr含量较低,是一种安全无毒副作用的中药,可以放心大胆的用药。杜仲药材中微量元素的研究,为进一步开发利用杜仲药材提供了科学依据。同时本文还对几种植物样品前处理方法进行了对比研究,发现微波消解法具有溶样能力强、速度快、污染少、简单、方便等优点,是目前植物中元素含量测定的最佳样品处理方法。
4.杜仲叶提取物的抑菌效果研究
以乙醇和水组成的混合溶液作提取剂,从杜仲叶中微波提取主要活性成分绿原酸,并研究了在最佳微波提取条件下提取溶液的抗菌活性。结果表明:杜仲绿原酸具有抗大肠杆菌的生物活性,提取液最低抑菌浓度为100mg/mL。
As people have realized the toxic effects of chemical medicine and have improved their health consumption conceptions, the use and requirements of natural medicines have been expanded constantly. More and more people have emphasized the characters and advantages of Chinese herds. How to completely extract active components and improve the utilization and clinical effects of traditional Chinese herbs, have become the main subject and task of traditional Chinese herbs in its modernization. The noval types of sample pretreatment techniques (microwave-assisted extraction approaches) have been used for extraction and isolation of polyphenolic compounds, such as chlorogenic acid, caffeic acid, syringin, et al, from Eucommia ulmodies Oliv. E.ulmodies is one of the precious Traditional Chinese Medicine which distributes in the west of Hunan province. In this work, the parameters which influence the extraction efficiencies in each technique had been investigated. Additional, the quality control standard of Eucommia ulmodies Oliv was established, which was used to control the quality of E.ulmodies. Then the research about trace elements and antibiotic functions had been done, which can provide reference informations for the rational use of E.ulmodies. The concrete contents are as follows:
1. The research of extraction of Chlorogenic Acid from E. ulmodies
The optimum technics of extracting chlorogenic acid from E.ulmodies was studied. During the extracting process, extracting solvents, methods and conditions were studied. The result showed that: Microwave-assisted extraction was fast and efficient. The optimum conditions were: the volume fraction of ethanol, 0.6; the pressure, 2atm; time, 2min. The material/solution ratio was 1:50. Under the same HPLC condition, we detected the content of chlorogenic acid in leaves, unroasted bark, roasted bark and Du Zhong Ping Ya Pian. The content of chlorogenic acid in leaves was higher than that in bark. Meanwhile the leaves of E.ulmodies were easy to get, it can provide abundance chlorogenic acid resource. Microwave-assisted extraction was a kind of newfashioned, high efficient, convenience and fast extraction mean, meanwhile the HPLC method had good precision, repeatability. The result was dependable. It can be adopt to determinate the content and control the quality of material and products that contain chlorogenic acid.
2. Selection and fingerprints of the control substances for plant drug Eucommia ulmodies Oliv by HPLC and LC-MS
The selection of the control substances and their fingerprints of this plant medicine were investigated by using HPLC and LC-MS. The gradient elution mode was applied in chromatographic separation, and the data were analyzed by“Computer Aided Similarity Evaluation”software to compare the similarity of the E. ulmodies from different habitats. Low similarity was found in the samples from nonadjacent provinces, while high similarity was obtained in those from the adjacent provinces. The LC-MS fingerprints of E. ulmodies showed the main active constituents and could be used for its original identification and quality evaluation.
3. Determination of elements in the E.ulmodies
The E.ulmodies is a miraculous tree. Its leaves bark and flowers contain trace elements in human. The elements of the E.ulmodies were determined by FAAS. The results showed that: There were abundance elements, such as Mg Cu Fe, et al. The contents of them were higher in leaves than in bark, and they played a very important part in the metabolish of the flesh. While the contents of the element, such as Pb Cr, were very low. So E.ulmodies is a very safe Chinese traditional medicine. The experiment results provided the scientific basis for further research. Meanwhile by comparing different methods, the microwave digestion had the merits of efficiency, rapidity, less pollution and convenience. It is the best sample preparation method for determination plant’s trace elements contents.
4. Study on antibiotic ability of leaves extracts of E.ulmodies
Chlorogenic acid was extracted from E.ulmodies by Microwave-Extraction method. The solvent was a mixed solution of ethanol and water. The antibiotic ability of chlorogenic acid under optimum extraction condition was studied. The result showed that, the extract had the antibiotic ability towards Escherichia coli, and the MIC was 100 mg/mL.
引文
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