| 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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