马兜铃酸类物质的液相色谱联用技术及应用
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摘要
马兜铃酸类物质,包括马兜铃酸(aristolochic acids, AAs)和马兜铃内酰胺(aristolactams, ALs),已经被证实具有肾毒性、致癌和致基因突变作用,含马兜铃酸类物质的药用植物和制剂因此被多数国家禁用。我国也先后取消了关木通、广防己和青木香的药用标准,并对含马兜铃、寻骨风、天仙藤和朱砂莲的中药制剂严格按处方药管理。但是,仍有一些马兜铃酸类物质含量较低的中药或植物药在使用。因此,为保证用药安全,应该加强药品生产、流通和使用各环节的监管。
中医对中药毒性的认识由来已久,在长期的实践中,中医药发展和形成了一系列减毒增效的理论和技术。已经有研究证实,炮制和配伍能够降低关木通和广防己等中药的毒性。我们应该正确认识马兜铃酸类物质的毒性作用,在中医药理论指导下,积极开展含马兜铃酸类物质中草药的实验研究,寻找减毒制毒的规律和途径,发挥其正常的药用疗效,减轻“马兜铃酸事件”所带来的负面影响。本文首先介绍了药用植物及其制剂中马兜铃酸类物质的一系列液相色谱联用检测技术。然后,基于所开发的色谱检测平台,我们对马兜铃酸I的细胞毒性、细胞毒代动力学进行了初步探讨,并建立了关木通配伍过程的成分分析方法,以此从化学角度来研究配伍减毒的物质基础。
具体研究工作如下:
1.为满足药用植物及其制剂中马兜铃酸类物质的筛查需求,我们开发了一个面向最普及的仪器设备和简单的分析环境的高效液相色谱方法。通过对提取、分离和分析条件的优化,我们成功地对12种药用植物和两种制剂中的9个马兜铃酸类物质进行了同时分析。该法以甲醇—0.2% HAc为流动相进行梯度洗脱,各种马兜铃酸类物质获得了良好的色谱分离。分析方法的线性范围约为2个数量级,在所研究的浓度范围内,线性相关系数超过0.9950。日内精密度和日间精密度实验的相对标准偏差小于4.35%。平均回收率落在88.4-98.8%之间。这些结果表明本法准确可靠。相比于先前的文献方法,本实验提出的多组分分析方法在对可疑药品中的马兜铃酸类物质的筛查和定量分析上具有明显的优势,更有效,更实用。
2.马兜铃酸类物质是弱极性的小分子化合物,在电喷雾质谱( electro-spray ionization/mass spectrometry, ESI/MS)中的离子化效率不高,易受离子抑制效应的影响。通过对基质效应、离子化模式和锥孔电压的影响等因素的研究,我们系统探讨了马兜铃酸类物质在ESI质谱中的离子化行为,找到了它们的ESI质谱裂解规律,第一次系统阐述了各种马兜铃酸类物质的离子化机理。在此基础上,通过对实验条件和仪器参数的优化,降低了离子抑制效应,获得了比文献稍高的检测灵敏度。所开发的HPLC-DAD-ESI/MS方法可以同时得到每个色谱峰的保留时间、紫外光谱图、相对分子量和质谱特征碎片,因而拥有良好的定性能力。我们将该方法应用于9种马兜铃科植物和两个制剂中9个马兜铃酸类物质的同时分析,结果令人满意。
3.为进一步提高马兜铃酸类物质的分析灵敏度,我们引入了荧光检测。根据两大类马兜铃酸类物质(AAs和ALs)的紫外、荧光特性和它们在马兜铃科植物中的分布特征(相对高含量的AAs和痕量的ALs),我们将DAD和FLD(fluorescence detection, FLD)串联使用,巧妙地实现了对多数样品中AAs和ALs的直接分析。当分析痕量的AAs时,我们提出了一个简单的衍生方法,即在酸性条件下,将AAs的硝基还原成亚氨基而变成相应的ALs再进行分析,因而也实现了对AAs的高灵敏分析。对不同样品的分析结果表明,DAD和FLD这两种廉价和普及度广的检测器的联用与马兜铃酸类物质在自然界的分布的结合近于完美。我们将这种HPLC-DAD-FLD联用技术应用于各种马兜铃科植物及其制剂的分析,结果表明本法选择性好,灵敏度高。一般情况下,该法的线性范围可达3-4个数量级,且线性相关系数(r2)大于0.9970;检测限可低至0.2 ng/ml;日内精密度和日间精密度良好,其RSDs小于5.74%;马兜铃酸类物质的平均回收率为94.5-99.2%。
4.尽管马兜铃酸类物质的肾毒性已经研究得比较深入,但其毒代动力学方面的数据仍很有限。要获取AA类物质的毒理学或药理学数据,开发一个能对生物样品中AA类物质及其代谢产物进行专一灵敏检测的分析方法,无疑是很有意义的。马兜铃酸(AAs)在生物体内的代谢产物主要是相应的马兜铃内酰胺(ALs)衍生物。本文首次采用HPLC-DAD-FLD联用检测技术对细胞环境中的AA I及其代谢产物同时进行了灵敏专属的检测。本法的线性范围可达3个数量级,且线性相关系数大于0.9994。各种细胞样品的分析结果表明了本法具有合适的精密度和灵敏度,适用于细胞毒代动力学的研究。基于倒置相差显微镜和MTT分析,我们研究了AA I对人正常肝细胞L-02的细胞毒性。同时,对细胞培养液中的AA I及其代谢产物进行了色谱分析。实验检测到了两种代谢产物,一种为AL I,另一种也可归属为马兜铃内酰胺衍生物。依据细胞培养液中的AA I及其代谢产物的浓度—时间数据,进行了AA I毒代动力学参数的初步统计估测。
5.研究人员已经通过动物实验证实了复方配伍能够降低关木通毒性的事实。为了能从化学角度研究关木通配伍过程毒性成分或活性成分的变化情况,我们开发了一个具有良好定性定量能力的HPLC-DAD-ESI/MS分析方法。我们对关木通和黄连中的27个组分进行了追踪分析,并对其中7个主要成分进行了定量分析。关木通配伍黄连后,黄连中的原小蘖碱型生物碱的煎出量明显减少,但关木通中的马兜铃酸类物质仍然不可忽略。关木通配伍大黄的实验得到了类似结论。实验中,我们也发现水煎煮能够明显降低关木通中马兜铃酸类物质的溶出率,同70%乙醇提取方法相比,水回流提取液中AA I和AA II的含量分别降低了58.6%和51.5%,这种现象说明了中药用药的实践存在着合理的科学依据。我们的研究表明,关木通配伍减毒的效应并没有出现在配伍减毒的第一个作用环节即药剂学环节。要弄清关木通配伍减毒的物质基础,进一步的研究工作可以从两方面入手,一是继续对组方药材的有效成分、有效部位进行分离、分析及鉴别;二是进行动物实验、组织器官、细胞亚细胞及分子生物学四个药理水平上的药效和作用机理研究。
Aristolochic acid (AA) analogues, including aristolochic acids (AAs) and aristolactams (ALs), have been found to be nephrotoxic, carcinogenic and mutagenic. So, the herbal plants and their preparations containing aristolochic acid analogues have been prohibited in most of countries. In China, caulis aristolochiae manshureinsis (Guanmutong in Pinyin Name, GMT), radix aristolochiae fangchi (Guangfangji in Pinyin Name, GFJ) and radix aristolochiae (Qingmuxiang in Pinyin Name, QMX) have also been cancelled off, and some Chinese traditional patent medicines (CPMs) containing aristolochia plants have been required to be scrutinized strictly. However, some drugs with lower contents of AA analogues still can be found in markets. For drug safety, it is necessary to strengthen the supervision on the product, sale and use of the drugs.
In fact, curative effect and drug safety have been always the focus of traditional Chinese medicine (TCM). To eliminate or reduce the toxicity, drastic actions and side effects of some drugs, a series of theory and technique have formed through the long practice. Recent studies have verified that drug processing and compatibility can reduce the toxicity of caulis aristolochiae manshureinsis and fructus aristolochiae (Madouling in Pinyin Name, MDL). We should hold on the unbiased viewpoint about“aristolochic acid nephropathy case”(AAN case) and make efforts to reduce the side effects brought by AAN case. The efforts should include fundamental experiment, substance base and pharmacology data of TCM containing AA analogues, and the studies should be carried out under the guideline of TCM theory. In this thesis, a series of hyphenated HPLC techniques were developed to determine simultaneously the AA analogues in herbal plants and their preparations. Then based on the analytical platform, some applications were carried out such as cytotoxicity of AA I, preliminary toxicokinetics, and chemical study of compatability, and some valuable results were obtained. The main points of this thesis are summarized as follows:
1. To meet the requirements of screening for AA analogues in medicinal plants and their preparations, a simple, reliable and effective high-performance liquid chromatography method coupled with photodiode array detector (HPLC-DAD) is presented based on the bench-top instruments. By optimizing the extraction, separation and analytical conditions, the proposed method has been successfully used for the simultaneous determination of nine AA analogues, i.e., AA I, AA II, AA C, AA D, 7-OH AA I, aristolic acid, AL II, AL III and AL IV, in twelve medicinal herbs and two preparations. The separation was completed on a C18 column with aqueous methanol containing 0.2% (V/V) acetic acid as mobile phase. Linearities of around two orders of magnitude were obtained with correlation coefficients exceeding 0.9950. Satisfactory intra-day and inter-day precisions were achieved with RSDs less than 4.35%, and the average recoveries obtained were in the range of 88.4-98.8%. The proposed method appears to be suitable for use as a tool for safety assurance for commercially available suspect samples containing AA analogues. Multicomponent analysis in this work has significant advantages over the previous methods. The proposed method is more effective, more robust.
2. AA analogues are weak or non-polarity small molecules, which are more susceptible to ion suppression. Based on the investigation on the matrix effect, ionization mode and cone voltage, comprehensive fragmentation process of AAs and ALs are discussed in ESI/MS, and systematic ionization mechanism of AA analogues in ESI/MS were elucidated. ALs exhibit single ionization product [M+H]+ in positive ion mode, whereas AAs show multiple ionization products. By optimizing the chromatographic separation and MS parameters, the precursor ions of the derivatives with the best responses were found, and the sensitivities in the determination of the nine derivatives were improved. Based on the investigation of ionization behaviours, a HPLC-DAD-ESI/MS (high-performance liquid chromatography-photodiode array detection-electrospray mass spectrometry) method has been developed for simultaneous analysis of nine derivatives in nine medicinal herbs and two preparations. The proposed method was proven to be a powerful approach for the identification and analysis of AA analogues in aristolochia plants and their preparations with versatility, specificity and sensitivity.
3. To improve the analysis sensitivity, fluorescence detection (FLD) was adopted. According to the UV and FL characteristics and distribution characteristics of AAs and ALs in herbal plants, the hyphenated HPLC-DAD-FLD (high-performance liquid chromatography- diode array detection-fluorescence detection) technique was found to be the optimal tool for the simultaneous analysis of AAs and ALs. Baseline separation was obtained on an ODS C18 analytical column with 0.2% HAc/methanol gradient elution. The hyphenation of DAD and FLD allows the method directly meet the analysis requirements of most herbal plants with high sensitivity and selectivity. For trace analysis, aristolochic acids were reduced to their corresponding aristolactams in acidic solution containing iron powder, and then highly sensitive detection and quantification were carried out. The method was successfully validated in the matrices of various Aristolochiaceae plants and their preparations. Linearities of around 3-4 orders of magnitude were obtained with correlation coefficients exceeding 0.9970. The detection limits were decreased to 0.2 ng/ml. Satisfactory intra-day and inter-day precisions were achieved with RSDs less than 5.74%, and the average recoveries were in the range of 94.5-99.2%.
4. Aristolochic acid I (AA I), a major component of the carcinogenic plant extract, is known to be nephrotoxic, carcinogenic and mutagenic. A simple and sensitive HPLC-DAD-FLD method was developed and validated for the analysis of AA I and its metabolites in cell culture medium. The samples were prepared with ethyl acetate liquid-liquid extraction (LLE). Good separation was obtained on an ODS C18 analytical column with 0.2% HAc/methanol gradient elution. Linearities of about 3 orders of magnitude were gained with correlation coeffiecients exceeding 0.9994. The method appears to be a suitable tool for the cell toxicokinetic study with acceptable precisions and recoveries. In this work, Proliferation and morphology of cells were observed with an inverted phase contrast microscope, and cell viability was analyzed by MTT assay. AA I and its metabolites (AL I and an unknown metabolite) were determined. With this assay, some preliminary cytotoxicity and toxicokinetic experiments were carried out.
5. Recent researches indicate the toxicity of caulis aristolochia manshuriensis can be reduced remarkably when combined with rhizoma coptidis. To trace the chemical ingredients and monitor whether any changes have occurred during the course of the compatibility, a HPLC-DAD-ESI/MS method was developed for the simultaneous analysis of AAs in caulis aristolochia manshuriensis and protoberberine alkaloids in rhizoma coptidis (Huanglian in Pinyin Name, HL) with good separation and suitable sensitivity. Twenty-seven peaks in the chromatograms of the two herbs have been separated and analyzed, and 7 main ingredients were quantified. After decocting with water the contents of AA I and AA II decreased by 58.6 and 51.5%, respectively, which indicates that the decoct method should be a reasonable form of TCM preparations. Although the chemical variations were found during the course of compatibility of GMT with HL, the main toxic ingredients in GMT-HL still cannot be neglected. The similar phenomena were found when GMT was combined with radix et rhizoma rhei (Dahuang in Pinyin Name, DH). No significant reduction of AA analogues in GMT was found at the first stage of compatibility. The more significant de-toxicity effects may occur in organisms. The comprehensive de-toxicity mechanism is still unclear for compatibility of GMT, and need to be investigated further.
中医对中药毒性的认识由来已久,在长期的实践中,中医药发展和形成了一系列减毒增效的理论和技术。已经有研究证实,炮制和配伍能够降低关木通和广防己等中药的毒性。我们应该正确认识马兜铃酸类物质的毒性作用,在中医药理论指导下,积极开展含马兜铃酸类物质中草药的实验研究,寻找减毒制毒的规律和途径,发挥其正常的药用疗效,减轻“马兜铃酸事件”所带来的负面影响。本文首先介绍了药用植物及其制剂中马兜铃酸类物质的一系列液相色谱联用检测技术。然后,基于所开发的色谱检测平台,我们对马兜铃酸I的细胞毒性、细胞毒代动力学进行了初步探讨,并建立了关木通配伍过程的成分分析方法,以此从化学角度来研究配伍减毒的物质基础。
具体研究工作如下:
1.为满足药用植物及其制剂中马兜铃酸类物质的筛查需求,我们开发了一个面向最普及的仪器设备和简单的分析环境的高效液相色谱方法。通过对提取、分离和分析条件的优化,我们成功地对12种药用植物和两种制剂中的9个马兜铃酸类物质进行了同时分析。该法以甲醇—0.2% HAc为流动相进行梯度洗脱,各种马兜铃酸类物质获得了良好的色谱分离。分析方法的线性范围约为2个数量级,在所研究的浓度范围内,线性相关系数超过0.9950。日内精密度和日间精密度实验的相对标准偏差小于4.35%。平均回收率落在88.4-98.8%之间。这些结果表明本法准确可靠。相比于先前的文献方法,本实验提出的多组分分析方法在对可疑药品中的马兜铃酸类物质的筛查和定量分析上具有明显的优势,更有效,更实用。
2.马兜铃酸类物质是弱极性的小分子化合物,在电喷雾质谱( electro-spray ionization/mass spectrometry, ESI/MS)中的离子化效率不高,易受离子抑制效应的影响。通过对基质效应、离子化模式和锥孔电压的影响等因素的研究,我们系统探讨了马兜铃酸类物质在ESI质谱中的离子化行为,找到了它们的ESI质谱裂解规律,第一次系统阐述了各种马兜铃酸类物质的离子化机理。在此基础上,通过对实验条件和仪器参数的优化,降低了离子抑制效应,获得了比文献稍高的检测灵敏度。所开发的HPLC-DAD-ESI/MS方法可以同时得到每个色谱峰的保留时间、紫外光谱图、相对分子量和质谱特征碎片,因而拥有良好的定性能力。我们将该方法应用于9种马兜铃科植物和两个制剂中9个马兜铃酸类物质的同时分析,结果令人满意。
3.为进一步提高马兜铃酸类物质的分析灵敏度,我们引入了荧光检测。根据两大类马兜铃酸类物质(AAs和ALs)的紫外、荧光特性和它们在马兜铃科植物中的分布特征(相对高含量的AAs和痕量的ALs),我们将DAD和FLD(fluorescence detection, FLD)串联使用,巧妙地实现了对多数样品中AAs和ALs的直接分析。当分析痕量的AAs时,我们提出了一个简单的衍生方法,即在酸性条件下,将AAs的硝基还原成亚氨基而变成相应的ALs再进行分析,因而也实现了对AAs的高灵敏分析。对不同样品的分析结果表明,DAD和FLD这两种廉价和普及度广的检测器的联用与马兜铃酸类物质在自然界的分布的结合近于完美。我们将这种HPLC-DAD-FLD联用技术应用于各种马兜铃科植物及其制剂的分析,结果表明本法选择性好,灵敏度高。一般情况下,该法的线性范围可达3-4个数量级,且线性相关系数(r2)大于0.9970;检测限可低至0.2 ng/ml;日内精密度和日间精密度良好,其RSDs小于5.74%;马兜铃酸类物质的平均回收率为94.5-99.2%。
4.尽管马兜铃酸类物质的肾毒性已经研究得比较深入,但其毒代动力学方面的数据仍很有限。要获取AA类物质的毒理学或药理学数据,开发一个能对生物样品中AA类物质及其代谢产物进行专一灵敏检测的分析方法,无疑是很有意义的。马兜铃酸(AAs)在生物体内的代谢产物主要是相应的马兜铃内酰胺(ALs)衍生物。本文首次采用HPLC-DAD-FLD联用检测技术对细胞环境中的AA I及其代谢产物同时进行了灵敏专属的检测。本法的线性范围可达3个数量级,且线性相关系数大于0.9994。各种细胞样品的分析结果表明了本法具有合适的精密度和灵敏度,适用于细胞毒代动力学的研究。基于倒置相差显微镜和MTT分析,我们研究了AA I对人正常肝细胞L-02的细胞毒性。同时,对细胞培养液中的AA I及其代谢产物进行了色谱分析。实验检测到了两种代谢产物,一种为AL I,另一种也可归属为马兜铃内酰胺衍生物。依据细胞培养液中的AA I及其代谢产物的浓度—时间数据,进行了AA I毒代动力学参数的初步统计估测。
5.研究人员已经通过动物实验证实了复方配伍能够降低关木通毒性的事实。为了能从化学角度研究关木通配伍过程毒性成分或活性成分的变化情况,我们开发了一个具有良好定性定量能力的HPLC-DAD-ESI/MS分析方法。我们对关木通和黄连中的27个组分进行了追踪分析,并对其中7个主要成分进行了定量分析。关木通配伍黄连后,黄连中的原小蘖碱型生物碱的煎出量明显减少,但关木通中的马兜铃酸类物质仍然不可忽略。关木通配伍大黄的实验得到了类似结论。实验中,我们也发现水煎煮能够明显降低关木通中马兜铃酸类物质的溶出率,同70%乙醇提取方法相比,水回流提取液中AA I和AA II的含量分别降低了58.6%和51.5%,这种现象说明了中药用药的实践存在着合理的科学依据。我们的研究表明,关木通配伍减毒的效应并没有出现在配伍减毒的第一个作用环节即药剂学环节。要弄清关木通配伍减毒的物质基础,进一步的研究工作可以从两方面入手,一是继续对组方药材的有效成分、有效部位进行分离、分析及鉴别;二是进行动物实验、组织器官、细胞亚细胞及分子生物学四个药理水平上的药效和作用机理研究。
Aristolochic acid (AA) analogues, including aristolochic acids (AAs) and aristolactams (ALs), have been found to be nephrotoxic, carcinogenic and mutagenic. So, the herbal plants and their preparations containing aristolochic acid analogues have been prohibited in most of countries. In China, caulis aristolochiae manshureinsis (Guanmutong in Pinyin Name, GMT), radix aristolochiae fangchi (Guangfangji in Pinyin Name, GFJ) and radix aristolochiae (Qingmuxiang in Pinyin Name, QMX) have also been cancelled off, and some Chinese traditional patent medicines (CPMs) containing aristolochia plants have been required to be scrutinized strictly. However, some drugs with lower contents of AA analogues still can be found in markets. For drug safety, it is necessary to strengthen the supervision on the product, sale and use of the drugs.
In fact, curative effect and drug safety have been always the focus of traditional Chinese medicine (TCM). To eliminate or reduce the toxicity, drastic actions and side effects of some drugs, a series of theory and technique have formed through the long practice. Recent studies have verified that drug processing and compatibility can reduce the toxicity of caulis aristolochiae manshureinsis and fructus aristolochiae (Madouling in Pinyin Name, MDL). We should hold on the unbiased viewpoint about“aristolochic acid nephropathy case”(AAN case) and make efforts to reduce the side effects brought by AAN case. The efforts should include fundamental experiment, substance base and pharmacology data of TCM containing AA analogues, and the studies should be carried out under the guideline of TCM theory. In this thesis, a series of hyphenated HPLC techniques were developed to determine simultaneously the AA analogues in herbal plants and their preparations. Then based on the analytical platform, some applications were carried out such as cytotoxicity of AA I, preliminary toxicokinetics, and chemical study of compatability, and some valuable results were obtained. The main points of this thesis are summarized as follows:
1. To meet the requirements of screening for AA analogues in medicinal plants and their preparations, a simple, reliable and effective high-performance liquid chromatography method coupled with photodiode array detector (HPLC-DAD) is presented based on the bench-top instruments. By optimizing the extraction, separation and analytical conditions, the proposed method has been successfully used for the simultaneous determination of nine AA analogues, i.e., AA I, AA II, AA C, AA D, 7-OH AA I, aristolic acid, AL II, AL III and AL IV, in twelve medicinal herbs and two preparations. The separation was completed on a C18 column with aqueous methanol containing 0.2% (V/V) acetic acid as mobile phase. Linearities of around two orders of magnitude were obtained with correlation coefficients exceeding 0.9950. Satisfactory intra-day and inter-day precisions were achieved with RSDs less than 4.35%, and the average recoveries obtained were in the range of 88.4-98.8%. The proposed method appears to be suitable for use as a tool for safety assurance for commercially available suspect samples containing AA analogues. Multicomponent analysis in this work has significant advantages over the previous methods. The proposed method is more effective, more robust.
2. AA analogues are weak or non-polarity small molecules, which are more susceptible to ion suppression. Based on the investigation on the matrix effect, ionization mode and cone voltage, comprehensive fragmentation process of AAs and ALs are discussed in ESI/MS, and systematic ionization mechanism of AA analogues in ESI/MS were elucidated. ALs exhibit single ionization product [M+H]+ in positive ion mode, whereas AAs show multiple ionization products. By optimizing the chromatographic separation and MS parameters, the precursor ions of the derivatives with the best responses were found, and the sensitivities in the determination of the nine derivatives were improved. Based on the investigation of ionization behaviours, a HPLC-DAD-ESI/MS (high-performance liquid chromatography-photodiode array detection-electrospray mass spectrometry) method has been developed for simultaneous analysis of nine derivatives in nine medicinal herbs and two preparations. The proposed method was proven to be a powerful approach for the identification and analysis of AA analogues in aristolochia plants and their preparations with versatility, specificity and sensitivity.
3. To improve the analysis sensitivity, fluorescence detection (FLD) was adopted. According to the UV and FL characteristics and distribution characteristics of AAs and ALs in herbal plants, the hyphenated HPLC-DAD-FLD (high-performance liquid chromatography- diode array detection-fluorescence detection) technique was found to be the optimal tool for the simultaneous analysis of AAs and ALs. Baseline separation was obtained on an ODS C18 analytical column with 0.2% HAc/methanol gradient elution. The hyphenation of DAD and FLD allows the method directly meet the analysis requirements of most herbal plants with high sensitivity and selectivity. For trace analysis, aristolochic acids were reduced to their corresponding aristolactams in acidic solution containing iron powder, and then highly sensitive detection and quantification were carried out. The method was successfully validated in the matrices of various Aristolochiaceae plants and their preparations. Linearities of around 3-4 orders of magnitude were obtained with correlation coefficients exceeding 0.9970. The detection limits were decreased to 0.2 ng/ml. Satisfactory intra-day and inter-day precisions were achieved with RSDs less than 5.74%, and the average recoveries were in the range of 94.5-99.2%.
4. Aristolochic acid I (AA I), a major component of the carcinogenic plant extract, is known to be nephrotoxic, carcinogenic and mutagenic. A simple and sensitive HPLC-DAD-FLD method was developed and validated for the analysis of AA I and its metabolites in cell culture medium. The samples were prepared with ethyl acetate liquid-liquid extraction (LLE). Good separation was obtained on an ODS C18 analytical column with 0.2% HAc/methanol gradient elution. Linearities of about 3 orders of magnitude were gained with correlation coeffiecients exceeding 0.9994. The method appears to be a suitable tool for the cell toxicokinetic study with acceptable precisions and recoveries. In this work, Proliferation and morphology of cells were observed with an inverted phase contrast microscope, and cell viability was analyzed by MTT assay. AA I and its metabolites (AL I and an unknown metabolite) were determined. With this assay, some preliminary cytotoxicity and toxicokinetic experiments were carried out.
5. Recent researches indicate the toxicity of caulis aristolochia manshuriensis can be reduced remarkably when combined with rhizoma coptidis. To trace the chemical ingredients and monitor whether any changes have occurred during the course of the compatibility, a HPLC-DAD-ESI/MS method was developed for the simultaneous analysis of AAs in caulis aristolochia manshuriensis and protoberberine alkaloids in rhizoma coptidis (Huanglian in Pinyin Name, HL) with good separation and suitable sensitivity. Twenty-seven peaks in the chromatograms of the two herbs have been separated and analyzed, and 7 main ingredients were quantified. After decocting with water the contents of AA I and AA II decreased by 58.6 and 51.5%, respectively, which indicates that the decoct method should be a reasonable form of TCM preparations. Although the chemical variations were found during the course of compatibility of GMT with HL, the main toxic ingredients in GMT-HL still cannot be neglected. The similar phenomena were found when GMT was combined with radix et rhizoma rhei (Dahuang in Pinyin Name, DH). No significant reduction of AA analogues in GMT was found at the first stage of compatibility. The more significant de-toxicity effects may occur in organisms. The comprehensive de-toxicity mechanism is still unclear for compatibility of GMT, and need to be investigated further.
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