【作者】 秦昆明；

【导师】 蔡宝昌；

【作者基本信息】 南京中医药大学 ， 中药学， 2010， 硕士

【摘要】 百合知母汤始见于张仲景《金匮要略》,书中记载百合知母汤由百合七枚,知母三两组成,主治“百合病”。百合病以精神恍惚不定,伴有口苦、小便赤、脉洪数为临床表现特点,病因病机为热病以后余热未清,或情志不遂郁而化火,导致阴虚内热。百合知母汤为中医临床治疗百合病的主要方剂,百合宁心安神,润肺止咳；知母清热泻火,滋阴润燥,百合甘寒清润而不腻,知母苦寒降火而不燥,百合偏于补,知母偏于泻,二药伍用,一润一清,一补一泻,共奏润肺清热,宁心安神之效。百合知母汤主治阴虚或温热病后余热末清,以致头昏、心烦不安、失眠等症,以及情志不遂、精神恍惚、不能自制等症。本室前期研究表明,百合知母汤对绝经综合征具有较好的疗效,为了阐明百合知母汤治疗绝经综合征的药效物质基础,本文对百合知母汤的质量控制、配伍变化及体内药效物质进行了研究,通过研究,初步建立百合知母汤的质量控制方法；研究了百合知母汤配伍前后的成分变化；初步明确了百合知母汤的体内药效物质基础。本论文的实验研究从以下五个方面进行,通过本研究,可以为百合知母汤的制剂开发提供相关的科学依据：一、百合知母汤的指纹图谱研究及其与单味药的相关性分析研究和建立百合知母汤的高效液相色谱(HPLC)指纹图谱,为研究百合知母汤的药效物质基础及配伍变化提供手段。采用Agela Venusil XBP-C18 (4.6 mm×250 mm,5μm)色谱柱；乙腈和0.1%甲酸为流动相二元梯度洗脱；检测波长为315 nm；柱温25℃；流速：1 mL／min。以芒果苷为参照物,在相同色谱条件下测定了10批不同产地的百合与知母制备的百合知母汤的指纹图谱,获得共有16个共有指纹峰,通过与对照品的保留时间及紫外光谱比较,标定了5-羟甲基糠醛(5-HMF)、新芒果苷、芒果苷、异芒果苷、王百合苷B的位置。该方法建立的百合知母汤的指纹图谱特征性、重现性较好,方法稳定、可靠,可以为百合知母汤的质量控制提供参考；本实验归属了百合知母汤指纹图谱中的主要色谱峰,并确定了煎煮过程中的主要变化成分为5-HMF。二、百合知母汤及其组方药味的红外光谱研究采用傅里叶变换红外光谱(FTIR)技术对百合知母汤及其组方药味进行了研究。实验测试了单味药百合、知母及百合知母汤剂的红外光谱,结果显示：在百合知母汤剂中保留了百合、知母单味药汤剂中的某些吸收峰,如百合知母汤光谱2933和1059 cm-1处等；百合、知母单味药光谱中的某些吸收峰并未在百合知母汤光谱中出现,见百合光谱1724 cm-1和知母光谱1453 cm-1等；在百合知母汤剂光谱中出现了新的吸收峰,见百合知母汤光谱1871和1522 cm-1处等。同时,因吸收峰位置的改变,说明各基团在百合、知母、百合知母汤中所处的化学环境不同或汤剂中化学物质的结构发生了变化。实验结果表明,百合知母汤及其组方药味的红外谱图特征具有一定的变化规律,根据对特征峰的指认和归属,可初步揭示百合知母汤及其组方药味的红外光谱差异；单味药的特征峰在百合知母汤中得到较好的显示,该方法可以用于百合知母汤的宏观质量控制,也为探索方剂的配伍规律提供了新思路。三、百合知母汤及其组方药味的高效液相色谱-电喷雾质谱研究建立了百合知母汤的高效液相色谱-电喷雾质谱(HPLC-ESI-MS)分析方法,对百合知母汤及其组方药味中的主要成分进行分析鉴定。实验采用反相C18色谱柱,以乙腈-0.05%甲酸水溶液为流动相,二元线性梯度洗脱进行色谱分离,并与电喷雾质谱联用,根据正离子模式和负离子模式下的分子离子峰获得化合物分子量信息,通过与文献数据或部分对照品对照,确定化合物的可能结构。实验结果显示：百合知母汤中各主要化学成分在正离子模式中响应较好。在相同的条件下,通过对百合知母汤及其组方药味进行比较分析,归属并鉴定了百合知母汤中的38个成分,包括3种黄酮,4种酚酸糖苷和31种皂苷类成分。本实验表明高效液相色谱-电喷雾质谱联用技术可以为鉴别百合知母汤中的化学成分提供简便、快速的方法。四、百合知母汤在大鼠体内的代谢产物分析采用高效液相色谱-电喷雾质谱联用技术研究了百合知母汤在大鼠体内的代谢成分,从而初步阐明百合知母汤在大鼠体内的药效物质基础。实验采用Shim-Pack VP-ODS C18色谱柱,柱温35℃,流动相为0.05%甲酸水溶液和乙腈,流速为0.2 mL/min,检测器为电喷雾四级杆质谱。通过对大鼠灌胃百合知母汤前后的血液样品、尿液样品和粪便样品进行分析检测,确定百合知母汤进入体内的成分,从而为阐明百合知母汤的药效物质奠定基础。结果发现,在大鼠给药后的血液样品、尿液样品和粪便样品中共检测到37个代谢成分,其中包括23个原型成分和14个转化成分。五、百合知母汤在家兔体内的代谢产物分析采用高效液相色谱-电喷雾质谱联用技术研究了百合知母汤在家兔体内的代谢成分,从而初步阐明百合知母汤在家兔体内的药效物质基础。实验采用Shim-Pack VP-ODS C18色谱柱,柱温35℃,流动相为0.05%甲酸水溶液和乙腈,流速为0.2 mL／min,检测器为电喷雾四级杆质谱。通过对家兔灌胃百合知母汤前后的血液样品、尿液样品和粪便样品进行比较分析,确定百合知母汤进入体内的成分,从而为阐明百合知母汤的药效物质奠定基础。结果发现,在家兔给药后的血液样品、尿液样品和粪便样品中共检测到25个代谢成分,其中包括19个原型成分和6个转化成分。更多还原

【Abstract】 Baihe Zhimu Tang (BZT) comes from JinKuiYaoLue-a study on traditional Chinese medicine written by Zhang Zhongjing of Han Dynasty. This empirical formula is used as a treatment of Lily Disease and consists of Bulbus Lilii and Rhizoma Anemarrhenae. Lily Disease is a disease of functional disorder manifested by abnormality in diet habit, sleep and behavior, which is similar to menopause syndrome.Former research in our laboratory have revealed that Baihe Zhimu Tang is effective in rat model and can significantly adjust some important indexes. In this research, we want to explore the effective substances of BZT and set up methods to control the quality of BZT, and we also want to investigate the compatibility mechanism of BZT.Our studies were performed in five aspects as following, the results could provide scientific evidence for development of the new drug.1. High performance liquid chromatographic fingerprints of Baihe Zhimu Tang and its correlation to single herbsThe high performance liquid chromatographic (HPLC) fingerprints of Baihe Zhimu Tang were established for evaluating effective substance basis and compatibility regulation of Baihe Zhimu Tang. An Agela Venusil XBP-C18 column (4.6 mm×250 mm,5μm) was employed, and the gradient elution of acetonitrile and 0.1%formic acid was used as mobile phase, the detection wavelength was set at 315 nm, the column temperature was 25℃and the flow rate was 1 mL/min. By taking mangiferin as reference substance, the fingerprints of ten batches of Baihe Zhimu Tang, prepared by Bulbus Lilium and Rhizoma Anemarrhena from different producing areas, were analyzed under the same chromatographic condition, the results showed that there were sixteen common peaks contained in the tested samples. Five constituents were identified as 5-hydroxymethyl-2-furaldehyde (5-HMF), neomagiferin, mangiferin, isomangiferin and regaloside B by comparing with the peak retention time of reference substance and ultraviolet spectra. The HPLC fingerprints of Baihe Zhimu Tang established with above method show good characteristic and repeatability, and the method is stable and reliable, it can be used for the quality control of Baihe Zhimu Tang. The main chromatographic peaks of the HPLC fingerprints of Baihe Zhimu Tang are identified in the experiment and also 5-HMF is identified as the main constituent which changed significantly in decocting process.2. Infrared spectrum analysis of Baihe Zhimu Tang and its single herbsThe fourier transform infrared spectra (FTIR) of decoction of Bulbus Lilii, Rhizoma Anemarrhenae and Baihe Zhimu Tang (BZT) were tested. The change in the mixed decoction was discussed to study the relationship between Bulbus Lilii and Rhizoma Anemarrhenae after decoction. The results showed that some components of Bulbus Lilii and Rhizoma Anemarrhenae were retained in the mixed decoction of BZT, such as 2933 cm-1 and 1059 cm-1, but some components that never appeared in the two component spectra increased, such as 1724 cm-1 in Bulbus Lilii and 1453 cm-1 in Rhizoma Anemarrhenae. New absorption peaks were generated in the mixed decoction of BZT, such as 1871 cm-1 and 1522 cm-1. It can be showed that there are some differences in the chemical environment of the various chemical groups in BZT, and with the variation in absorption peak position, possibly some new chemical constituents were created. Pharmaceutical effective substances in BZT are not simply the addition of Bulbus Lilii and Rhizoma Anemarrhenae. This method can be used in the quiality control of BZT and proposed a new method in the compatibility research of traditional Chinese medicinal formulas.3. Analysis of chemical components in Baihe Zhimu Tang and its single herbs by high performance liquid chromatography-electrospray ionization-mass spectrometryA method was established for identifying the chemical components of a traditional Chinese medicinal formula Baihe Zhimu Tang and its single herbs by combining high performance liquid chromatography and electrospray ionization mass spectrometry (HPLC-ESI-MS). Samples were analyzed by a reversed-phase C18 column using a binary eluent of acetonitrile and water (with 0.05% formic acid) under gradient conditions, and analyzed by HPLC-MS techniques with an ESI source. The molecular ions of compounds in both negative and positive modes were observed for molecular mass information, and the potential structures were identified by attentive studying on the mass spectra of compounds and comparing with reference data and some of standards. The results showed that in MS detection, saponins in Baihe Zhimu Tang and its single herbs are easily to become positive ions in the electrospray ionization procedure, and they have strong responses, but the mass spectrometry signals of flavonoids and phenolic glucosides are week.38 compounds in Baihe Zhimu Tang including 3 flavonoids,4 phenolic glucosides and 31 saponins were identified through analyzing and comparing the total ion chromatograms (TIC) and mass spectra of Baihe Zhimu Tang and its single herbs. This method has the advantages of simple operation, rapid measurement and it is a powerful tool for identification of chemical components in Baihe Zhimu Tang.4. Screening and analysis of metabolic components in rat plasma, urine and feces after oral administration of Baihe Zhimu TangIn this part, high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) methods were developed for the analysis of metabolic components of BZT. A reversed-phase Shim-Pack VP-ODS C18 column with the column temperature at 35℃was used in the HPLC experiments, and the mobile phase system consists of aqueous formic acid (0.05%, v/v) and acetonitrile using a gradient elution at the flow rate of 0.2 mL/min. The ESI-MS was operated with a single-quadrupole mass spectrometer in both negative and positive ion modes. After oral administration of extracts of BZT, the rat’s plasma, urine and feces were also analyzed; 37 metabolic components including 23 original components and 14 transformative components of BZT were detected. The analysis of metabolic components in BZT by HPLC-ESI-MS methods could be a useful means of identifying the multi-components of BZT and to hint at their possible metabolic mechanism of action in the body. The results also narrow the range of active compounds to be found in BZT, and pave the way for further investigations of pharmacology and active mechanism.5. Screening and analysis of metabolic components in rabbit plasma, urine and feces after oral administration of Baihe Zhimu TangIn this part, the same high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) methods were used for the analysis of metabolic components of BZT in rabbit. After oral administration of extracts of BZT, the rabbit’s plasma, urine and feces were also analyzed; 25 metabolic components including 19 original components and 6 transformative components of BZT were detected. The analysis of metabolic components in BZT narrow the range of active compounds to be found in BZT, and pave the way for further investigations of pharmacology and active mechanism.更多还原