【作者】 王瑞；

【导师】 王峥涛； 毕开顺；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2005， 博士

【摘要】 芍药为传统中药，历有赤、白之分。《中国药典》2005年版规定白芍为毛茛科植物芍药Paeonia Lactiflora Pall．的干燥根，置沸水中煮后除去外皮或去皮后再煮，晒干；赤芍为毛茛科植物芍药Paeonia Lactiflora Pall．或川赤芍Paeonia veitchii Lynchde的干燥根，直接晒干。此外，杂芍药Paeonia hybrida Pall．的干燥纺锤状块根直接晒干后称为赤芍或新疆赤芍在新疆地区应用。本文对川赤芍的化学成分与芍药的质量控制方法进行了系统研究。从川赤芍(P．veitchii)中分离并鉴定了28个化合物，其中1个新化合物，为2-O-[α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside]-Benzaldehyde(BH)；18个首次从该种植物中分离得到的化合物，分别为β-谷甾醇亚油酸酯、胡萝卜苷亚油酸酯、亚油酸、棕榈酸、豆甾醇、菠甾醇、stigmast-7-en-3β-ol、木栓酮、表木栓醇、没食子酰芍药苷(GPF)、芍药内酯苷(AF)、水杨醇、异水杨苷、水杨苷、没食子酸甲酯(MG)、1，2，3，4，6-五没食子酰基葡萄糖(PG)、丹皮酚(PN)、熊果苷；其他已知化合物分别为β-谷甾醇、胡萝卜苷、芍药苷(PF)、苯甲酰芍药苷(BPF)、氧化芍药苷、没食子酸(GA)、苯甲酸(BA)、d-儿茶素、蔗糖。采用高效液相色谱法，建立对芍药样品中GA、MG、BH、AF、PF、GPF、BA、PG、BPF、PN等10个化学成分同时进行含量测定的方法，线性关系良好，回归方程的相关系数为0.9995～0.9999；平均回收率为96.9～104.0％，RSD＜3.3％。结果表明(川)赤芍(P．veitchii，具皮)的总成分含量高，其次是赤芍(P．lactiflora，具皮)，总成分含量最低的为新疆赤芍(P．hybrida，具皮)。芍药苷类化合物是芍药的主要成分之一，其中又以芍药苷含量最高。(川)赤芍(P．veitchii，具皮)和新疆赤芍(P．hybrida，具皮)中还含有较多的没食子酸类化合物。BH是(川)赤芍(P．veitchii，具皮)特征性成分，可以作为其专属性指标，具有分类学和鉴别意义。白芍(P．lactiflora，去皮，水煮)和赤芍(P．lactiflora，具皮)来源于同一植物的同一入药部位，但产地加工方法不同。药材经去皮、水煮后得到的白芍，其总成分含量明显下降，芍药内酯苷的含量明显上升。去皮、水煮这种传统加工方法对赤、白芍功效的影响可能大于赤、白芍药材种间差异的影响。所建立的含量测定方法简便，重复性好，可以在同一色谱条件下对白芍和赤芍中化学成分的含量进行比较研究，为评价芍药质量提供了一个定量指标。对芍药样品进行色谱指纹图谱研究。采用薄层色谱法对芍药进行指纹图谱研究，可在同一薄层板上同时对多个样品进行可见光色谱及荧光猝灭色谱图像平行分析比较，扫描轮廓图谱结合薄层色谱图像观察信息更加丰富，操作简便，可辨认性强。同种样品显示高度的相似性，不同种样品在具有各自的共性特征的同时，个体之间有显著差异。采用高效液相色谱法对芍药进行指纹图谱研究，获取反映药材整体特征的化学数据，将所获得的化学数据进行化学模式识别研究，建立统计学识别模型。通过系统聚类分析将芍药样品分为3类：第Ⅰ类为白芍(P．lactiflora，去皮，水煮)，第Ⅱ类为赤芍(P．lactiflora，具皮)，第Ⅲ类为(川)赤芍(P．veitchii，具皮)和新疆赤芍(P．hybrida，具皮)。用逐步判别分析对样品的分类结果建立判别函数，通过判别函数对芍药样品进行区别、鉴定，判别分类与真实分类的判别符合率达100％。采用“中药色谱指纹图谱相似度评价系统”进行相似度计算，对芍药样品进行鉴定、分类和评价。在分析未知样品时，只需计算判别函数或计算未知样品与共有模式间的相似度，即可对未知样品进行鉴定、分类和评价。在此基础上，以13个未知芍药样品为研究对象，进行指纹图谱分析，结果表明采用系统聚类分析法、逐步判别分析法和相似度计算法三种方法分类结果基本一致，得到了相互认证。为鉴别白芍和赤芍提供了新方法采用液相色谱与多级质谱联用(HPLC-PDA-MS~n)技术，研究5个芍药苷类和3个没食子酸类化合物的特征碎片离子和特征紫外吸收，推测其电喷雾质谱裂解规律，并对白芍和赤芍中这8个化合物进行鉴定和色谱峰的归属。根据样品的选择离子(SIM)色谱图，进一步证明BH是(川)赤芍(P．veitchii，具皮)的特征化学成分之一，具有鉴别意义。对(川)赤芍(P．veitchii，具皮)和白芍(P．lactiflora，去皮，水煮)的80％乙醇提取物及其脂溶性成分和水溶性成分进行初步药理活性比较研究，以二甲苯致小鼠耳廓肿胀和醋酸诱发小鼠毛细血管通透性增高观察提取物的抗炎作用；以提取物对小鼠凝血酶原时间(PT)、部分凝血活酶时间(KPTT)和对大鼠血小板聚集的影响，考察提取物的活血化瘀作用。旨在为进一步探讨白芍与赤芍的药理活性奠定基础。本研究在中医药理论和实践的指导下，将中药学、分析化学、药理学、化学统计学和计算机技术相结合，研究了川赤芍的化学成分；初步建立了白芍和赤芍的质量评价方法，对评价中药材质量的方法进行了有益的尝试；探讨了芍药苷类和没食子酸类化合物的电喷雾质谱裂解规律及其在芍药中的分布，并初步比较了白芍和赤芍的药理活性，为白芍和赤芍的更深入研究奠定了基础，为中药现代化做了有意义的探索。更多还原

【Abstract】 Radix Paeoniae Alba （white peony root） and Radix Paeoniae Rubra （red peony root） are commonly used traditional Chinese herbal drug for analgesia, anti-inflammation and activating blood circulation to dissipate blood stasis. According to the Pharmacopoeia of the PRC （edition 2005）, white peony root is the dried root of Paeonia lactiflora Pall.（peeled and boiled before drying） and red peony root is the dried root of Paeonia lactiflora Pall.or P. veitchii Lynch （directly dried）. Furthermore, the dried root of Paeonia hybrida Pall is used as a substitute for red peony root in Xinjiang locally, and called as Xinjiang red peony root or red peony root.In this dissertation, the chemical components of Paeonia veitchii and method of quality assessment of Radix Paeoniae were studied systematically.From the roots of P. veitchii, 28 compounds were isolated and structurally elucidated by chemical and spectroscopic methods (¹H NMR, ¹³C NMR, HMBC, HMQC, NOESY, EI-MS, ESI-MS, HRESI-MS, IR, UV). One of them was a new compound, 2-O-[α-L-arabinopyranosyl-（1→6）-β-D-glucopyranoside]-Benzaldehyde （BH）, and eighteen of them were isolated from this plant for the first time:β-sitosterol linoleate, daucosterol linoleate, linoleic acid, palmitic acid, stigmasterol,α-spinaterol, stigmast-7-en-3β-ol, friedelin, epi-friedelanol, galloylpaeoniflorin （GPF）, albiflorin （AF）, salicyl alcohol, isosalicin, salicin, methyl gallate （MG）, pentagalloylglucose （PG）, paeonol （PN）, arbutin. 9 Known ones,β-sitosterol, daucosterin, paeoniflorin （PF）, benzoylpaeoniflorin （BPF）, oxypaeoniflorin, gallic acid （GA）, benzoic acid （BA）, d-catechin and saccharose, were also isolated.A reversed-phase HPLC method was developed for simultaneous determination of GA, MG, BH, AF, PF, GPF, BA, PG, BPF and PN in white peony root and red peony root, collected from different habitats and different batches. The determination were accomplished by a linear gradient elution system on ODS columns. The linear calibration curves were plotted with satisfied relative coefficients （0.9995～0.9999）. The average recoveries were in the range of 96.9～104.0% with RSD less than 3.3%. The established assay method is simple, rapid, reproducible and provide a quantitative basis for the quality assessment for Radix Paeoniae.The TLC and HPLC chromatographic fingerprints of methanol extracts of white peony root and red peony root were evaluated. Totally samples derived from Paeonia lactiflora, P. veitchii and P. hybrida, respectively, were firstly analyzed by TLC documentation. The results indicated the three species of peony roots could be distinctly authenticated from each other based on their TLC characters, detected by colorimetric or fluorescent spotting, and the samples from the same species displayed higher homogenicity.The methanol extracts of the above mentioned samples were simultaneously analyzed by a newly developed HPLC system and characteristic chromatographic fmgerprints and the chemical data were acquired. The HPLC chromatographic profiles of Radix Paeoniae showing 27 diagnostic peaks from both white and red peony root and studied by chemical pattern recognition methods. The profiling data were randomly treated with hierarchical clustering analysis and divided into three classes, P. lactiflora （peeled）, P. lactiflora （unpeeled）, and P. veitchii plus P. hybrida. Stepwise discriminant analysis （STEPDA） method was also utilized for differentiation of Radix Paeoniae samples and four discriminant functions were produced from the result of classification, in which P. veitchii plus P. hybrida were divided into two classes in stead. When an unknown sample is analyzed by this method, the observed values are imported into the discriminant functions. The analyzed sample is identified to a specified class which give the biggest function value to the discriminant function of the specified class. Based on the results of classification, a similarity analysis method for quality assessment of Radix Paeoniae samples was developed. All the three different methods resulted in the consistent trend and can be used for identifying white peony root and red peony root.High-performance liquid chromatography with on-line photodiode array detection and electrospray ionization tandem mass spectrometry was developed for simultaneous analysis of compounds in the Radix Paeoniae. Combining the separation by HPLC, the characteristic of UV spectra, and the information of molecular structure provided by ESI-MSⁿ, eight compounds in the white peony root and red peony root were identified. Furthermore, the fragmentation pattern of them were proposed. Two inflammatory models of ear edema induced by xylene and elevation of capillary permeability by acetic acid were used for testing the anti-inflammatory effect of extracts of red and white peony roots. The effects on the activating blood and eliminating stasis with prothrombin time （PT）, kaolin partial thromboplastin time （KPTT） and platelet aggregation induced by ADP were studied. The results showed that red peony root and white peony root indicated the similar biological activities in those tests.Conclusively, by using the theory and methodologies of traditional Chinese medicine （TCM）, phytochemistry, analytical chemistry, chemical statistics and pharmacology, red peony root and white peony root were studied in this paper. The chemical components of Paeonia veitchii was elucidated and a new methodology for the quality control of Radix Paeoniae was established. Furthermore, the MSⁿ fragmentation patterns of compounds were proposed and a comparative study on pharmacological effects was carried out, which provided valuable academic evidences for the future research and development of Radix Paeoniae.更多还原