液质联用技术应用于小柴胡汤药效物质基础研究
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摘要
中药复方的药效物质基础是指中药复方中针对某一病症发挥药效作用的全部活性药效物质总和。这些活性物质来源复杂,包括复方原单味药材固有成分、制备过程形成的产物(如炮制加工、煎煮过程)等。复方的药效物质基础不是全方成分机械地组合,而是多种活性物质相互作用的结果。
小柴胡汤方出自东汉张仲景的《伤寒论》,由柴胡、黄芩、人参、半夏、炙甘草、生姜、大枣等7味药材组成,用于治疗伤寒少阳证。小柴胡汤汤组方精良,配伍严谨。在长期的医疗实践中,本方广泛用于肝胆、消化、内分泌、血液、免疫等系统疾病的治疗。
指纹图谱技术是控制中药质量的主要方法之一,具有整体性、宏观性和模糊性的特点,在一定程度上确实反映了中药材的内在质量,但作为一种科学客观的质量控制方法,目前还不够完善,尤其表现在制作中药指纹图谱时,其中色谱峰所代表化学成分还不够明确,很多化学成分的药理作用机制尚不清楚。我们认为中药指纹图谱至少应该对其色谱图上的各色谱峰进行明确化归属,才能科学客观地控制与评价中药质量,从而为中药体内成分分析与代谢研究奠定基础。本文采用高效液相色谱飞行时间质谱联用技术,建立了快速鉴别小柴胡汤中化学成分的分析方法。同时建立了中药复方小柴胡汤的化学成分数据库,其中收纳了小柴胡汤中七味药材已知的化学成分400多个,并计算出其精确分子量,其中包括皂苷类成分,如柴胡皂苷和甘草皂苷;黄酮类成分如黄芩苷、汉黄芩苷和甘草甘;生物碱类成分,如麻黄碱;以及二苯庚烷类和姜辣素类等成分。在小柴胡汤总离子流图中,共鉴别出38个化合物,其中鉴别出甘草中化学成分17个、黄芩中化学成分10个、人参中化学成分4个、柴胡中化学成分4个、生姜中化学成分3个,半夏和大枣未检出。从小柴胡汤的定性结果可以看出,小柴胡汤中含有多种化学成分,其中含量最高的为黄酮类化学成分,主要来自于黄芩和甘草,在液相色谱上较早出峰。皂苷类成分也是小柴胡汤中重要的化学成分,可能由于其极性较小的缘故,在液相色谱中较后出峰。这些成分在小柴胡汤中的含量较高,且为生物活性物质,可为小柴胡汤药效物质基础研究奠定基础。
2005年版中国药典中以黄芩苷的含量作为小柴胡颗粒质量控制的标准。由于有较好的紫外吸收,黄酮类化合物的含量测定多采用紫外检测器。小柴胡汤中黄芩苷、汉黄芩苷含量较高,其含量的多少对小柴胡汤质量影响显著。本文用回流提取的方法,按照原方比例制备小柴胡汤。采用高效液相色谱紫外检测器,Agilent Zorbax XDB-C18(150mm×4.6mm, 5μm)色谱柱,流动相为乙腈-水系统,对小柴胡汤中黄芩苷、汉黄芩苷进行含量测定。与药典相比,增加了对汉黄芩苷的测定。本方法可以用于不同制剂小柴胡汤的质量控制研究,也为药物代谢及药物动力学研究奠定基础。
多数中药以汤剂的形式口服给药。实际上,这种传统复方在发挥药效作用之前,除了在炮制、煎煮过程中会产生动态变化外,进入机体后亦要发生动态变化。本文采用HPLC-MS方法测定口服给药小柴胡汤后,大鼠血浆中黄芩苷和汉黄芩苷的含量,所建立含量测定方法简便可靠,血浆中内源性物质均不干扰样品峰,方法学验证结果表明该含量测定方法适合于生物样品中黄芩苷和汉黄芩苷的含量测定。该方法成功应用于SD大鼠单剂量口服给药小柴胡汤和黄芩提取物后的药物动力学研究。分别口服给药小柴胡汤和黄芩提取物后,从药时曲线中可以看出,黄芩苷和汉黄芩苷均有两个吸收峰,提示可能存在肝肠循环,且给药小柴胡汤组大鼠的半衰期较给药黄芩提取物组明显缩短,说明小柴胡汤中的某些化学成分加快了黄芩苷和汉黄芩苷在体内的消除。
The effective substances of Chinese multiherb remedy is refers to the total compounds which have effective function to some disease. These effective substances’sources are complex, including coming from raw material of herb medicines, the products in preparation process (for example to processing, decoction process) and so on. The effective substance of Chinese multiherb remedy is not to combine the substances in herb medicines mechanically, but is the result of many kinds of effective substances’interaction.
The Chinese multiherb medicine Xiaochaihu-tang is composed of seven herb medicines and was first described in Shang Han Lun by Zhang Zhongjing (150 to 219 A. D. in the Chinese Eastern Han Dynasty). Xiaochaihu-tang (or Shosaiko-to in Japanese) has attracted a great deal of attention for its possible healing effects on chronic hepatitis and its beneficial effect to prevent the development of hepatocellular carcinoma in patients with cirrhosis of the liver. In addition, many studies in animal models or on cell lines have demonstrated that Xiaochaihu-tang or its ingredients possess cytoprotective effects on experimental liver injuries, an antiinflammatory effect on chronic hepatitis, preventive and therapeutic effects on experimental hepatic fibrosis via the inhibition of hepatic stellate cells, and lipid peroxidation in hepatocytes.
Because of the complexity of Chinese herb medicine, it is necessary to fully understanding the compounds in Chinese herb remedy. A data base of compounds in Xiaochaihu-tang has been established, which including about 500 compounds from seven herb medicines. The exact molecular weight is also displayed. To quickly identify the compounds, a analysis method apply HPLC-TOF-MS has been established and the total analysis time was 42min. In the total ion current chromatogram of Xiaochaihu-tang, 38 compounds was identified and 17 compounds were from Gancao, 10 compounds were from Huangqin, 4 compounds were from Renshen, 4 compounds were from Chaihu, 3 compounds were from Shengjiang, no compound was identified in Banxia and Dazao. As the result, it is conclude that flavone compounds have a large amount in Xiaochaihu-tang and they mainly from Huangqin and Gancao positioned earlily in the chromatogram. Saikosaponin are important compounds in Xiaochaihu-tang, maybe as their small polarity, they appeared late in chromatogram.
China pharmacopoeia in 2005 version set baicalin as the qulity control of Xiaochaihu-partical. Because of well ultraviolet absorption, flavone were usually detected with UV detection. Baicalin and wogonoside are two important compounds in Xiaochaihu-tang, their amount will remarkable influence the qulity of it. Reflux extraction was used to produce Xiaochaihu-tang. It is used Agilent Zorbax XDB-C1(8150mm×4.6mm, 5μm)column and ACE/H2O systerm. Compared to China pharmacopoeia, the method is apply to the determination of baicalin and wogonoside meanwhile which will raise the qulity control to Chinese multiherb medicine Xiaochaihu-tang and set foundation to drug metablism and pharmacokinetic research.
Most of herb medicine is oral administration by decotion. Actually, the Chinese herb medition will interact during processing and decotong and also in vivo. The method of determination of baicalin and wogonoside in vivo was established with HPLC-MS. The method was apple to the baicalin and wogonoside pharmacokinetic reaserch in rats after oral administration of Xiaochaihu-tang and Huangqin extraction respective. Baicalin and wogonoside both have two absorption peaks. It is supposed that maybe exist enterohepatic cycle. Compared to oral administration Huangqin extraction, the half life of baicalin and wogonoside is more shorter of Xiaochaihu-tang, which suggest the other compounds in Xiaochaihu-tang accelerate the elimination of baicalin and wogonoside.
小柴胡汤方出自东汉张仲景的《伤寒论》,由柴胡、黄芩、人参、半夏、炙甘草、生姜、大枣等7味药材组成,用于治疗伤寒少阳证。小柴胡汤汤组方精良,配伍严谨。在长期的医疗实践中,本方广泛用于肝胆、消化、内分泌、血液、免疫等系统疾病的治疗。
指纹图谱技术是控制中药质量的主要方法之一,具有整体性、宏观性和模糊性的特点,在一定程度上确实反映了中药材的内在质量,但作为一种科学客观的质量控制方法,目前还不够完善,尤其表现在制作中药指纹图谱时,其中色谱峰所代表化学成分还不够明确,很多化学成分的药理作用机制尚不清楚。我们认为中药指纹图谱至少应该对其色谱图上的各色谱峰进行明确化归属,才能科学客观地控制与评价中药质量,从而为中药体内成分分析与代谢研究奠定基础。本文采用高效液相色谱飞行时间质谱联用技术,建立了快速鉴别小柴胡汤中化学成分的分析方法。同时建立了中药复方小柴胡汤的化学成分数据库,其中收纳了小柴胡汤中七味药材已知的化学成分400多个,并计算出其精确分子量,其中包括皂苷类成分,如柴胡皂苷和甘草皂苷;黄酮类成分如黄芩苷、汉黄芩苷和甘草甘;生物碱类成分,如麻黄碱;以及二苯庚烷类和姜辣素类等成分。在小柴胡汤总离子流图中,共鉴别出38个化合物,其中鉴别出甘草中化学成分17个、黄芩中化学成分10个、人参中化学成分4个、柴胡中化学成分4个、生姜中化学成分3个,半夏和大枣未检出。从小柴胡汤的定性结果可以看出,小柴胡汤中含有多种化学成分,其中含量最高的为黄酮类化学成分,主要来自于黄芩和甘草,在液相色谱上较早出峰。皂苷类成分也是小柴胡汤中重要的化学成分,可能由于其极性较小的缘故,在液相色谱中较后出峰。这些成分在小柴胡汤中的含量较高,且为生物活性物质,可为小柴胡汤药效物质基础研究奠定基础。
2005年版中国药典中以黄芩苷的含量作为小柴胡颗粒质量控制的标准。由于有较好的紫外吸收,黄酮类化合物的含量测定多采用紫外检测器。小柴胡汤中黄芩苷、汉黄芩苷含量较高,其含量的多少对小柴胡汤质量影响显著。本文用回流提取的方法,按照原方比例制备小柴胡汤。采用高效液相色谱紫外检测器,Agilent Zorbax XDB-C18(150mm×4.6mm, 5μm)色谱柱,流动相为乙腈-水系统,对小柴胡汤中黄芩苷、汉黄芩苷进行含量测定。与药典相比,增加了对汉黄芩苷的测定。本方法可以用于不同制剂小柴胡汤的质量控制研究,也为药物代谢及药物动力学研究奠定基础。
多数中药以汤剂的形式口服给药。实际上,这种传统复方在发挥药效作用之前,除了在炮制、煎煮过程中会产生动态变化外,进入机体后亦要发生动态变化。本文采用HPLC-MS方法测定口服给药小柴胡汤后,大鼠血浆中黄芩苷和汉黄芩苷的含量,所建立含量测定方法简便可靠,血浆中内源性物质均不干扰样品峰,方法学验证结果表明该含量测定方法适合于生物样品中黄芩苷和汉黄芩苷的含量测定。该方法成功应用于SD大鼠单剂量口服给药小柴胡汤和黄芩提取物后的药物动力学研究。分别口服给药小柴胡汤和黄芩提取物后,从药时曲线中可以看出,黄芩苷和汉黄芩苷均有两个吸收峰,提示可能存在肝肠循环,且给药小柴胡汤组大鼠的半衰期较给药黄芩提取物组明显缩短,说明小柴胡汤中的某些化学成分加快了黄芩苷和汉黄芩苷在体内的消除。
The effective substances of Chinese multiherb remedy is refers to the total compounds which have effective function to some disease. These effective substances’sources are complex, including coming from raw material of herb medicines, the products in preparation process (for example to processing, decoction process) and so on. The effective substance of Chinese multiherb remedy is not to combine the substances in herb medicines mechanically, but is the result of many kinds of effective substances’interaction.
The Chinese multiherb medicine Xiaochaihu-tang is composed of seven herb medicines and was first described in Shang Han Lun by Zhang Zhongjing (150 to 219 A. D. in the Chinese Eastern Han Dynasty). Xiaochaihu-tang (or Shosaiko-to in Japanese) has attracted a great deal of attention for its possible healing effects on chronic hepatitis and its beneficial effect to prevent the development of hepatocellular carcinoma in patients with cirrhosis of the liver. In addition, many studies in animal models or on cell lines have demonstrated that Xiaochaihu-tang or its ingredients possess cytoprotective effects on experimental liver injuries, an antiinflammatory effect on chronic hepatitis, preventive and therapeutic effects on experimental hepatic fibrosis via the inhibition of hepatic stellate cells, and lipid peroxidation in hepatocytes.
Because of the complexity of Chinese herb medicine, it is necessary to fully understanding the compounds in Chinese herb remedy. A data base of compounds in Xiaochaihu-tang has been established, which including about 500 compounds from seven herb medicines. The exact molecular weight is also displayed. To quickly identify the compounds, a analysis method apply HPLC-TOF-MS has been established and the total analysis time was 42min. In the total ion current chromatogram of Xiaochaihu-tang, 38 compounds was identified and 17 compounds were from Gancao, 10 compounds were from Huangqin, 4 compounds were from Renshen, 4 compounds were from Chaihu, 3 compounds were from Shengjiang, no compound was identified in Banxia and Dazao. As the result, it is conclude that flavone compounds have a large amount in Xiaochaihu-tang and they mainly from Huangqin and Gancao positioned earlily in the chromatogram. Saikosaponin are important compounds in Xiaochaihu-tang, maybe as their small polarity, they appeared late in chromatogram.
China pharmacopoeia in 2005 version set baicalin as the qulity control of Xiaochaihu-partical. Because of well ultraviolet absorption, flavone were usually detected with UV detection. Baicalin and wogonoside are two important compounds in Xiaochaihu-tang, their amount will remarkable influence the qulity of it. Reflux extraction was used to produce Xiaochaihu-tang. It is used Agilent Zorbax XDB-C1(8150mm×4.6mm, 5μm)column and ACE/H2O systerm. Compared to China pharmacopoeia, the method is apply to the determination of baicalin and wogonoside meanwhile which will raise the qulity control to Chinese multiherb medicine Xiaochaihu-tang and set foundation to drug metablism and pharmacokinetic research.
Most of herb medicine is oral administration by decotion. Actually, the Chinese herb medition will interact during processing and decotong and also in vivo. The method of determination of baicalin and wogonoside in vivo was established with HPLC-MS. The method was apple to the baicalin and wogonoside pharmacokinetic reaserch in rats after oral administration of Xiaochaihu-tang and Huangqin extraction respective. Baicalin and wogonoside both have two absorption peaks. It is supposed that maybe exist enterohepatic cycle. Compared to oral administration Huangqin extraction, the half life of baicalin and wogonoside is more shorter of Xiaochaihu-tang, which suggest the other compounds in Xiaochaihu-tang accelerate the elimination of baicalin and wogonoside.
引文
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