中药活性成分在线色谱筛选新模型研究
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摘要
中药活性物质是指中药及其复方中发挥作用的化学成分,它是阐明中药作用奥秘的关键。由于中药作用的整体性、中药成分和作用机制的复杂性,使得中药效应物质基础的阐明一直进展缓慢。寻找适于中药复杂体系效应物质基础的研究方法,进而建立科学的质量控制方法,一直是中药研究的关键科学问题。长期以来,国内中药活性物质基础研究的主流模式是在化学成分提取、分离和结构鉴定的基础上利用药理模型对所得的纯化合物进行生物活性测试。多年来,这种研究方法阐明了一些中药的效应物质基础。但是,常规的提取分离手段只能分析中药的主要特征性成分,而不能评价这些成分是否具有活性。
本论文的研究目的是构建不同层次的中药药效物质基础的在线筛选色谱模型,并以各类提取物的抗氧化或细胞毒性指标的测定结果进行验证,使效应成分的分离与筛选相结合,克服以往先从中药中分离单体或部位,再分析其药效,致使成分分离与效应筛选脱节的弊端。主要研究工作如下：
1.构建抗肿瘤药物的脂筏色谱在线筛选模型并研究其热力学机理,将由U251细胞提取的脂筏固化于硅胶载体上制备酪氨酸蛋白激酶受体(TrkA)-脂筏色谱柱,进行色谱保留特性研究。由于构建的脂筏-硅胶固定相富含TrkA,固定相能与以TrkA为靶点的药物特异性结合产生保留,而非TrkA靶点的药物在该脂筏色谱柱中无保留行为。经不同时间和不同流动相的使用,证明TrkA-脂筏固定相色谱系统稳定,使用寿命长。首次进行脂筏色谱的机理研究,以TrkA为靶点的药物来他替尼为例,考察了脂筏色谱柱填料粒径、温度、流速对色谱行为的影响,探讨了溶质在固定相和流动相之间的迁移焓变、迁移熵变等热力学参数的变化。结果发现适宜的填料粒径有利于来他替尼在脂筏色谱上的保留及分离；在柱温37℃,流速0.2ml/min的色谱条件下能得到较适宜的保留效果。对于来他替尼而言,与脂筏固定相作用过程的AH0、△S0均为负值,说明这种脂筏固定相的分离机理是氢键或范德华力等协同作用的结果。
2.将构建的脂筏色谱系统应用于中药五倍子的抗肿瘤活性成分筛选,得到的在色谱柱上有保留行为的乙醚部分显示出良好的抑制U251肿瘤细胞毒性。为进一步证明这种柱上保留行为是TrkA与药物间结合力的结果,而非脂筏中其他蛋白的作用,我们在细胞试验的对照组中加入了低浓度的TrkA信号阻断剂,观察在TrkA脂筏柱上有保留的物质在细胞试验中饱和TrkA后,是否还能继续抑制U251细胞的增值。实验结果可见,脂筏色谱筛选出的具有明显保留行为的中药提取物与特异性TrkA抑制剂来他替尼一样,在TrkA信号阻断前体现出良好的抑制细胞增值的效果,信号阻断后则几乎无细胞抑制作用。而氟尿嘧啶并非以TrkA作为靶点,所以在TrkA信号阻断前后均体现出良好的肿瘤细胞抑制效果,证明脂筏色谱是一种针对TrkA的高选择性、高特异性的生物亲和色谱新技术。本实验为TrkA抑制剂的高效筛选提供了范例,证实了脂筏色谱在中药活性成分高效筛选中的应用可能。说明富含TrkA受体的脂筏-硅胶色谱是一种具有生物亲和特性、能够选择性识别与TrkA受体作用的药物的新型先导化合物筛选模型。
3.1,1-二苯基-2-苦肼基自由基(DPPH·)-检测器后衍生化高效液相色谱(HPLC)法在线筛选抗氧化活性成分技术的构建及在水飞蓟素及其脱氢衍生物中的应用。首次合成水飞蓟素的脱氢衍生物,超高效液相色谱(UPLC)-质谱(MS)联用鉴别水飞蓟素脱氢化物的组成；对水飞蓟素及其脱氢衍生物进行DPPH·清除能力、还原力、超氧自由基清除能力、Fe3+还原实验、羟基自由基清除能力等体外抗氧化能力的评价；同时研制DPPH-检测器后衍生化HPLC技术进行抗氧化活性成分的快速在线筛选,并通过动物体内抗氧化实验进行验证。结果发现,体内外抗氧化能力的评价试验中,几乎所有脱氢化物的抗氧化活性指标均明显大于脱氢前的药物,表明水飞蓟素的脱氢化衍生物具有更强的抗氧化活性。DPPH-HPLC法在线筛选出脱氢水飞蓟素的主要抗氧化活性成分为2,3-去氢水飞蓟宾和2,3-去氢水飞蓟亭。说明所构建的中药抗氧化药效物质基础的活性筛选-化学在线分析方法具有切实的可行性。
4. DPPH-薄层色谱(TLC)生物自显影法在线筛选抗氧化活性成分技术的构建及其在绵茵陈中的应用。采用HPLC及TLC法测定绵茵陈提取物中绿原酸的含量,紫外法离线测定提取液清除DPPH·的能力。另建立DPPH-TLC生物自显影法在线分析绵茵陈提取物中的抗氧化成分。结果表明,由生物自显影扫描法测得薄层色谱的色谱峰总积分值明显大于绿原酸单峰面积,表明绵茵陈中除了绿原酸外,还存在其它抗氧化能力较强的活性成分。说明以DPPH·作显色剂,双波长扫描,可利用TLC-生物自显影技术对天然提取物中具有自由基清除及抗氧化活性的活性成分进行快速筛选与评价。
With the development and modernization of traditional Chinese medicines (TCMs), screening active components in complex systes have introduced a key resolution to explore new drugs in TCMs. However, one TCM herb may contain up to hundreds or even thousands of different constituents, but only a few compounds are responsible for the pharmaceutical or toxic effects. With the interference of other components in TCMs, it is extremely difficult to screen the bioactive ingredients rapidly and efficiently.
By far, a variety of strategies have been developed to separate and analyze the constituents of TCM for identification of bioactive compounds and quality control, including screening with animal models, cell bioassays, enzyme/receptor models and traditional chromatographies. The establishment and application of these models play a positive role for screening of active components. However, these methods suffer from some shortcomings such as weak selectivity and lack of the information of screened components of TCMs. So it is often time-consuming and inefficient to separate lead compound from TCMs following with the bioassay guidance, resulting from the poor affinity and selectivity of conventional procedures.
This paper mainly focuses on the research of construction and application of some biochromatographic models, and aims to establish some new methods for screening bioactive components. The paper contains up to six chapters. The author's main contributions are as follows:
1. In this paper we have, for the first time, established a lipid raft stationary phase chromatography (LRSC) to develop a high throughput screening of potential therapeutic compounds for cancer treatment. The stable lipid rafts abundant with one receptor tyrosine kinase, tropomyosin-related kinase A (TrkA) obtained from U251cells were constructed to prepare the stationary phase. An unrelated TrkA inhibitor (gemcitabine) and TrkA targeted anti-tumor drugs (lestaurtinib and gefitinib) were analyzed on LRSC to evaluate the specific affinity of the column. Using lestaurtinib as a model, the retention behavior was investigated and the thermodynamic parameters of transfer from mobile phase to stationary phase were discussed. The results showed that the ideal resolution of lestaurtinib on lipid raft chromatography could be achieved with eligible partical size (10～50μm) of stationary phase at a flow rate of0.2mL/min at37℃. The standard enthalpy change (AH0) and entropy change (ΔS) in chromatographic system were determined by Van't Hoff relationship (lnk-1/T). The binding constants, changes of enthalpy, entropy and Gibbs free energy were obtained as negative value by using lestaurtinib as a model. The retention mechanism of solutes on the prepared stationary phase involves synergistic interaction of hydrogen bonding and van der Waals force.
2. The established LRSC was further applied for the screening of potential therapeutic compounds in cancer treatment using Chinese gallnut as a model. The chromatographic column manifested desirable affinity when the ether fraction of Chinese gallnut was injected into LRSC. The established LRSC system can recognize target components through affinity interactions between the components and TrkA signaling pathway on the lipid rafts. The methyl thiazolyl tetrazolium assay confirmed the anti-tumor effect of the screened ether fraction of Chinese gallnut and further proved the selectivity of LRSC on TrkA-targeted drugs. The mode of the newly constructed LRSC was identified as an efficient approach to screen anti-tumor components in TCMs.
3. In this study,2,3-dehydrosilymarin, a compound exhibiting remarkable antiradical/antioxidant activity, was synthesized from silymarin for the first time. The structures of its main components were verified by ultra-performance liquid chromatography/mass spectrometry (UPLC-MS) and other spectral analysis. The solubility, radical scavenging capacity and liver protecting activity of2,3-dehydrosilymarin were studied with silybin, dehydrosilybin and silymarin employed as controls. The antioxidant activities of silymarin and its derivatives were measured by determining their reducing activity, scavenging DPPH-free radical, superoxide radical, hydroxyl radical and the Fe2+concentration by reduction experiment. In addition, a rapid screening method, online high-performance liquid chromatography/1,1-dipheny1-2-picrylhydrazyl (DPPH-HPLC) system, was developed for identifying individual antioxidants. Both in-vitro and in-vivo results markedly proved that dehydrosilymarin has decent aqueous solubility and remarkable antiradical/antioxidation capacity. Moreover,2,3-dehydrosilybin and2,3-dehydrosilychristin were identified as the two major active compounds comprising2,3-dehydrosilymarin. Our results suggest that2,3-dehydrosilymarin may be a promising and potent alternative for inhibition of free radical and prevention of oxidation. The established on-line HPLC radical scavenging activity measurement makes it possible to directly identify active constituents in complex matrices.
4. An HPLC method and a single wavelength TLC-scanning method were used to determinate the concertration of chlorogenic acid of Herba Artemisiae scopariae extracts. Their free radical scavenging activities were measured with offline UV-2,2-diphenyl-l-picrylhydrazyl (DPPH·) method. The chromatograms were obtained after spraying with DPPH-solution and the peak areas of the spots on TLC were calculated to evaluate the antioxidant capacity of Herba Artemisiae scopariae extracts. The contents of chlorogenic acid determined by TLC-bioautography were consistent with the routine method, and the total integral value measured by bioautography was significantly higher than the single peak area of chlorogenic acid. The results indicated that Herba Artemisiae scopariae had many antioxidant active ingredients including chlorogenic acid. With DPPH as color developing reagent and dual wavelength scanning, TLC-bioautography can be used for fast selecting and evaluating the antioxidant capacity of traditional Chinese medicines.
本论文的研究目的是构建不同层次的中药药效物质基础的在线筛选色谱模型,并以各类提取物的抗氧化或细胞毒性指标的测定结果进行验证,使效应成分的分离与筛选相结合,克服以往先从中药中分离单体或部位,再分析其药效,致使成分分离与效应筛选脱节的弊端。主要研究工作如下：
1.构建抗肿瘤药物的脂筏色谱在线筛选模型并研究其热力学机理,将由U251细胞提取的脂筏固化于硅胶载体上制备酪氨酸蛋白激酶受体(TrkA)-脂筏色谱柱,进行色谱保留特性研究。由于构建的脂筏-硅胶固定相富含TrkA,固定相能与以TrkA为靶点的药物特异性结合产生保留,而非TrkA靶点的药物在该脂筏色谱柱中无保留行为。经不同时间和不同流动相的使用,证明TrkA-脂筏固定相色谱系统稳定,使用寿命长。首次进行脂筏色谱的机理研究,以TrkA为靶点的药物来他替尼为例,考察了脂筏色谱柱填料粒径、温度、流速对色谱行为的影响,探讨了溶质在固定相和流动相之间的迁移焓变、迁移熵变等热力学参数的变化。结果发现适宜的填料粒径有利于来他替尼在脂筏色谱上的保留及分离；在柱温37℃,流速0.2ml/min的色谱条件下能得到较适宜的保留效果。对于来他替尼而言,与脂筏固定相作用过程的AH0、△S0均为负值,说明这种脂筏固定相的分离机理是氢键或范德华力等协同作用的结果。
2.将构建的脂筏色谱系统应用于中药五倍子的抗肿瘤活性成分筛选,得到的在色谱柱上有保留行为的乙醚部分显示出良好的抑制U251肿瘤细胞毒性。为进一步证明这种柱上保留行为是TrkA与药物间结合力的结果,而非脂筏中其他蛋白的作用,我们在细胞试验的对照组中加入了低浓度的TrkA信号阻断剂,观察在TrkA脂筏柱上有保留的物质在细胞试验中饱和TrkA后,是否还能继续抑制U251细胞的增值。实验结果可见,脂筏色谱筛选出的具有明显保留行为的中药提取物与特异性TrkA抑制剂来他替尼一样,在TrkA信号阻断前体现出良好的抑制细胞增值的效果,信号阻断后则几乎无细胞抑制作用。而氟尿嘧啶并非以TrkA作为靶点,所以在TrkA信号阻断前后均体现出良好的肿瘤细胞抑制效果,证明脂筏色谱是一种针对TrkA的高选择性、高特异性的生物亲和色谱新技术。本实验为TrkA抑制剂的高效筛选提供了范例,证实了脂筏色谱在中药活性成分高效筛选中的应用可能。说明富含TrkA受体的脂筏-硅胶色谱是一种具有生物亲和特性、能够选择性识别与TrkA受体作用的药物的新型先导化合物筛选模型。
3.1,1-二苯基-2-苦肼基自由基(DPPH·)-检测器后衍生化高效液相色谱(HPLC)法在线筛选抗氧化活性成分技术的构建及在水飞蓟素及其脱氢衍生物中的应用。首次合成水飞蓟素的脱氢衍生物,超高效液相色谱(UPLC)-质谱(MS)联用鉴别水飞蓟素脱氢化物的组成；对水飞蓟素及其脱氢衍生物进行DPPH·清除能力、还原力、超氧自由基清除能力、Fe3+还原实验、羟基自由基清除能力等体外抗氧化能力的评价；同时研制DPPH-检测器后衍生化HPLC技术进行抗氧化活性成分的快速在线筛选,并通过动物体内抗氧化实验进行验证。结果发现,体内外抗氧化能力的评价试验中,几乎所有脱氢化物的抗氧化活性指标均明显大于脱氢前的药物,表明水飞蓟素的脱氢化衍生物具有更强的抗氧化活性。DPPH-HPLC法在线筛选出脱氢水飞蓟素的主要抗氧化活性成分为2,3-去氢水飞蓟宾和2,3-去氢水飞蓟亭。说明所构建的中药抗氧化药效物质基础的活性筛选-化学在线分析方法具有切实的可行性。
4. DPPH-薄层色谱(TLC)生物自显影法在线筛选抗氧化活性成分技术的构建及其在绵茵陈中的应用。采用HPLC及TLC法测定绵茵陈提取物中绿原酸的含量,紫外法离线测定提取液清除DPPH·的能力。另建立DPPH-TLC生物自显影法在线分析绵茵陈提取物中的抗氧化成分。结果表明,由生物自显影扫描法测得薄层色谱的色谱峰总积分值明显大于绿原酸单峰面积,表明绵茵陈中除了绿原酸外,还存在其它抗氧化能力较强的活性成分。说明以DPPH·作显色剂,双波长扫描,可利用TLC-生物自显影技术对天然提取物中具有自由基清除及抗氧化活性的活性成分进行快速筛选与评价。
With the development and modernization of traditional Chinese medicines (TCMs), screening active components in complex systes have introduced a key resolution to explore new drugs in TCMs. However, one TCM herb may contain up to hundreds or even thousands of different constituents, but only a few compounds are responsible for the pharmaceutical or toxic effects. With the interference of other components in TCMs, it is extremely difficult to screen the bioactive ingredients rapidly and efficiently.
By far, a variety of strategies have been developed to separate and analyze the constituents of TCM for identification of bioactive compounds and quality control, including screening with animal models, cell bioassays, enzyme/receptor models and traditional chromatographies. The establishment and application of these models play a positive role for screening of active components. However, these methods suffer from some shortcomings such as weak selectivity and lack of the information of screened components of TCMs. So it is often time-consuming and inefficient to separate lead compound from TCMs following with the bioassay guidance, resulting from the poor affinity and selectivity of conventional procedures.
This paper mainly focuses on the research of construction and application of some biochromatographic models, and aims to establish some new methods for screening bioactive components. The paper contains up to six chapters. The author's main contributions are as follows:
1. In this paper we have, for the first time, established a lipid raft stationary phase chromatography (LRSC) to develop a high throughput screening of potential therapeutic compounds for cancer treatment. The stable lipid rafts abundant with one receptor tyrosine kinase, tropomyosin-related kinase A (TrkA) obtained from U251cells were constructed to prepare the stationary phase. An unrelated TrkA inhibitor (gemcitabine) and TrkA targeted anti-tumor drugs (lestaurtinib and gefitinib) were analyzed on LRSC to evaluate the specific affinity of the column. Using lestaurtinib as a model, the retention behavior was investigated and the thermodynamic parameters of transfer from mobile phase to stationary phase were discussed. The results showed that the ideal resolution of lestaurtinib on lipid raft chromatography could be achieved with eligible partical size (10～50μm) of stationary phase at a flow rate of0.2mL/min at37℃. The standard enthalpy change (AH0) and entropy change (ΔS) in chromatographic system were determined by Van't Hoff relationship (lnk-1/T). The binding constants, changes of enthalpy, entropy and Gibbs free energy were obtained as negative value by using lestaurtinib as a model. The retention mechanism of solutes on the prepared stationary phase involves synergistic interaction of hydrogen bonding and van der Waals force.
2. The established LRSC was further applied for the screening of potential therapeutic compounds in cancer treatment using Chinese gallnut as a model. The chromatographic column manifested desirable affinity when the ether fraction of Chinese gallnut was injected into LRSC. The established LRSC system can recognize target components through affinity interactions between the components and TrkA signaling pathway on the lipid rafts. The methyl thiazolyl tetrazolium assay confirmed the anti-tumor effect of the screened ether fraction of Chinese gallnut and further proved the selectivity of LRSC on TrkA-targeted drugs. The mode of the newly constructed LRSC was identified as an efficient approach to screen anti-tumor components in TCMs.
3. In this study,2,3-dehydrosilymarin, a compound exhibiting remarkable antiradical/antioxidant activity, was synthesized from silymarin for the first time. The structures of its main components were verified by ultra-performance liquid chromatography/mass spectrometry (UPLC-MS) and other spectral analysis. The solubility, radical scavenging capacity and liver protecting activity of2,3-dehydrosilymarin were studied with silybin, dehydrosilybin and silymarin employed as controls. The antioxidant activities of silymarin and its derivatives were measured by determining their reducing activity, scavenging DPPH-free radical, superoxide radical, hydroxyl radical and the Fe2+concentration by reduction experiment. In addition, a rapid screening method, online high-performance liquid chromatography/1,1-dipheny1-2-picrylhydrazyl (DPPH-HPLC) system, was developed for identifying individual antioxidants. Both in-vitro and in-vivo results markedly proved that dehydrosilymarin has decent aqueous solubility and remarkable antiradical/antioxidation capacity. Moreover,2,3-dehydrosilybin and2,3-dehydrosilychristin were identified as the two major active compounds comprising2,3-dehydrosilymarin. Our results suggest that2,3-dehydrosilymarin may be a promising and potent alternative for inhibition of free radical and prevention of oxidation. The established on-line HPLC radical scavenging activity measurement makes it possible to directly identify active constituents in complex matrices.
4. An HPLC method and a single wavelength TLC-scanning method were used to determinate the concertration of chlorogenic acid of Herba Artemisiae scopariae extracts. Their free radical scavenging activities were measured with offline UV-2,2-diphenyl-l-picrylhydrazyl (DPPH·) method. The chromatograms were obtained after spraying with DPPH-solution and the peak areas of the spots on TLC were calculated to evaluate the antioxidant capacity of Herba Artemisiae scopariae extracts. The contents of chlorogenic acid determined by TLC-bioautography were consistent with the routine method, and the total integral value measured by bioautography was significantly higher than the single peak area of chlorogenic acid. The results indicated that Herba Artemisiae scopariae had many antioxidant active ingredients including chlorogenic acid. With DPPH as color developing reagent and dual wavelength scanning, TLC-bioautography can be used for fast selecting and evaluating the antioxidant capacity of traditional Chinese medicines.
引文
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