【作者】 王祝伟；

【导师】 孙毓庆；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2005， 博士

【摘要】 红毛五加（Acanthopanax giraldii Harms）是五加科五加属植物，因其外表密被棕红色长刺毛，故以“红毛五加”为正名。其药用部位为茎皮，有别于其他类五加皮。本论文以红毛五加为研究对象，分别从LC-DAD-ESI-MS／MS成分分析、单体的制备和结构鉴定、含量测定、多维指纹图谱和抗炎药理研究五方面进行实验，现分述如下：一、红毛五加化学成分的LC-DAD-ESI-MS／MS分析采用LC-DAD-ESI-MS／MS联用法，对红毛五加茎皮、根皮、叶子和果实，以及五加科植物剌五加、细柱五加和短梗五加的茎皮、根皮、叶子和果实的化学成分进行定性分析和比较。通过供试品与对照品的色谱峰的保留时间和紫外光谱对比、以及一级和二级质谱图解析，鉴定了10种化合物：尿苷、腺苷、鸟苷、紫丁香树脂苷、原儿茶酸、紫丁香苷、绿原酸、咖啡酸、金丝桃苷和芦丁，其中后6种为首次在红毛五加中发现。根据未知物的紫外光谱图、一级和二级质谱的碎片解析和它们的色谱行为，推断出红毛五加中还含有咖啡酰奎宁酸、二咖啡酰奎宁酸、阿魏酰奎宁酸等具有多种异构体的奎宁酸衍生物。同时比较了红毛五加与五加科其他植物化学成分的异同，为红毛五加药材的质量控制研究奠定了基础。二、红毛五加中紫丁香树脂苷和原儿茶酸的制备与结构鉴定采用大孔吸附树脂对红毛五加水提液进行粗分离后，用C₁₈固相萃取小柱再进一步富集有效成分，用半制备液相色谱进行组分的分离收集，制备了两种单体化合物。通过对它们的UV、IR、¹H-NMR、¹³C-NMR和MS图谱的综合解析，确证两化合物为紫丁香树脂苷和原儿茶酸。本法操作简便、快速、产率高、纯度高，适于推广。三、红毛五加中9种成分的含量测定在红毛五加成分鉴定的基础上，采用高效液相色谱法，分别对红毛五加中紫丁香苷、紫丁香树脂苷、绿原酸、咖啡酸、芦丁、金丝桃苷、腺苷、鸟苷和原儿茶酸这9种成分进行了含量测定，并对于刺五加、细柱五加和短梗五加中这些成分的含量进行了比较。由于尿苷在红毛五加中含量较小而且在各产地药材中分布不均，所以未对其进行含量测定。由含量测定结果可看出，红毛五加茎皮中紫丁香苷的含量低于紫丁香树脂苷，有些产地的红毛五加中几乎不含有紫丁香苷，而紫丁香苷在刺五加茎皮中含量却很高。红毛五加根皮中紫丁香树脂苷含量高于茎皮。通过将红毛五加的茎皮、木质部、髓部剥离，分别测定，发现这三部分中紫丁香树脂苷的含量有很大差异。木质部中紫丁香树脂苷含量约是茎皮的3倍，髓部含量最低。传统上，红毛五加以茎皮入药，而弃去其木质部，本实验结果表明这将大大损失了紫丁香树脂苷的含量。由绿原酸和咖啡酸的含量测定结果表明，不同批次之间的红毛五加药材中绿原酸和咖啡酸的含量有很大的差别，但能从中看出随着放置时间的增长，绿原酸的含量降低，咖啡酸的含量有增高的趋势。刺五加中咖啡酸的含量低于红毛五加。红毛五加根皮中绿原酸在所测定的药材中含量最高，达14.2 mg·g^-1。通过LC-DAD-MS／MS分析，发现这些五加科植物的叶子和果实含有很多黄酮类成分，芦丁和金丝桃苷便是红毛五加中含量较高的两种黄酮类化合物。由含量测定结果表明，红毛五加叶子和果实中芦丁含量高于金丝桃苷，而细柱五加、刺五加和短梗五加果实中的芦丁含量低于金丝桃苷，有的甚至几乎不含有芦丁。本实验全面对红毛五加茎皮、根皮、叶子和果实中的主要化学成分进行了含量测定，为更好的控制药材质量以及与五加皮的其它药材进行对比研究提供了依据。四、红毛五加LC-DAD-ESI-MS／MS多维指纹图谱研究对红毛五加茎皮药材进行多维指纹图谱研究，建立了红毛五加茎皮药材的LC-UV指纹图谱和LC-MS指纹图谱，两者可相互补充，为该药材的质量评价提供了有效的鉴定方法。用高效液相色谱紫外检测法建立了红毛五加茎皮的LC-UV指纹图谱，对22批样品测定后进行聚类分析，并分别采用中南大学和国家药典委的计算机辅助相似度评价系统，建立了共有模式，计算各个样品的相似度。比较了相关系数和夹角余弦两种不同相似度测度计算相似度的差异，以及不同指纹图谱相似度评价系统得出的相似度的异同。计算样品的定量系数以衡量样品指纹图谱总体含量的差异，确定需通过相关系数和定量系数双指标体系来综合评定药材的质量的方法。综合聚类分析和相似度软件计算结果，红毛五加茎皮指纹图谱的合格标准初步定为相似度大于0.9，定量系数大于0.9。本实验还建立了红毛五加茎皮药材的LC-MS指纹图谱。选择10批红毛五加茎皮样品，以绿原酸的萃取离子色谱图为内参比峰。比较了各样品质谱总离子流色谱图相似度和紫外色谱图相似度，可以看出两种方法对于多数样品的评价趋势基本一致。五、红毛五加茎皮的抗炎药理实验研究本实验采用用小鼠耳肿胀法，大鼠足跖肿胀法两个急性非特异性炎症模型和大鼠佐剂关节炎特异性炎症模型，观察红毛五加茎皮总提物的三个不同剂量灌胃给药对炎症的预防和治疗作用。并考察了从红毛五加中分离出的酚酸类化合物粗品对非特异性急性炎症的影响。实验结果初步显示酚酸类成分可能是红毛五加抗炎作用的活性部位。为配合红毛五加茎皮多维指纹图谱研究，先后选用了两批药材进行药理实验。实验结果显示，使用后一批药材得到的药理实验结果与病理模型对照组无显著性差异。比较它们的药材指纹图谱可以看出，后一批药材化学成分的总体含量远远低于前一批药材，而且酚酸类化合物中的绿原酸含量的差异更为明显。药理实验结果验证了我们将聚类分析中的第一类为合格品、第二类为劣质品的判断的正确性。这同时也提示我们，中药指纹图谱的研究在中药现代化进程中的重要性。本研究的创新之处在于：（1）首次对红毛五加茎皮、根皮、叶子和果实采用LC-DAD-ESI-MS／MS联用法进行化学成分分析，共鉴定出10种化合物，其中6种为首次在红毛五加中发现。并对其中的9种成分进行了含量测定，只有尿苷因含量较低而未测定含量。（2）根据未知物的紫外光谱图、一级和二级质谱的碎片解析和它们的色谱行为，推断出红毛五加中还含有咖啡酰奎宁酸、二咖啡酰奎宁酸、阿魏酰奎宁酸等具有多种异构体的奎宁酸衍生物。（3）比较了红毛五加茎皮、根皮、叶子和果实的成分差异，又比较了红毛五加与刺五加、细柱五加和短梗五加化学成分的异同，提出了以紫丁香苷的含量作为判别红毛血加与刺五加的指标成分。（4）首次对红毛五加茎皮药材进行LC-DAD-ESI-MS／MS多维指纹图谱研究，分别建立了红毛五加茎皮药材的LC-UV指纹图谱和LC-MS指纹图谱，两者可相互补充，为该药材的质量评价提供了有效的鉴定手段。更多还原

【Abstract】 Acanthopanax giraldii Harms is a kind of herbal plant that belongs to the family of Araliaceae, and it is fully covered with the reddish brown thorn, so it is given the name of "Hongmaowujia". In this thesis, the analysis of the components of A. giraldii Harms by LC-DAD-ESI-MS/MS technique, compounds preparation and identification, contents determination, multidimensional fingerprint analysis of A. giraldii Harms and anti-inflammation pharmacological experiments were investigated.1. Analysis of the components of A. giraldii Harms by LC-DAD-ESI-MS/MS techniqueThe LC-DAD-ESI-MS/MS technique was used for analysis of the components in the stem bark, root bark, leaf and fruit of A. giraldii Harms, and those of the stem bark, root bark, leaf and fruit of the of A. senticosus, A. gracilistylus W.W. Smith and A. sessiliflorus were also investigated and compared. 10 compounds were identified in the A. giraldii Harms by the comparison of the LC retention time, UV spectra and the mass spectra with corresponding authentic samples. There were uridine, adenosine, guanosine, liriodendrin, protocatechuic acid, syringin, chlorogenic acid, caffeic acid, hyperoside and rutin, and the last 6 compounds in the A. giraldii Harms have not been reported to date. Caffeoylquinic acid, dicaffeoylquinic acid and feruloylquinic acid were also inferred by their UV spectra, mass spectra and fragments of their MS/MS spectra. The components analysis provided scientific basis for the quality control study of A. giraldii Harms.2. Preparation and identification of liriodendrin and protocatechuic acid from A. giraldiiHarmsThe water extract of A. giraldii Harms was pre-isolated by the macroporous adsorption resin （D-101） and C₁₈ solid phase extraction （SPE） cartridge, and the enriched extract was purified by semi-preparative RP-HPLC to give two compounds. Structure identification was performed by the analysis of UV, IR, ¹H-NMR, ¹³C-NMR and MS data, and they were confirmed as liriodendrin and protocatechuic acid. The developed method was simple, fast, reproducible and easy to operate.3. Determination of 9 compounds in the A. giraldii HarmsBased on the LC-DAD-ESI-MS/MS analysis, syringin, liriodendrin, chlorogenic acid, caffeic acid, rutin, hyperoside, adenosine, ganosine and protocatechuic acid in the A. giraldii Harms were determined, and the corresponding components in the A. senticosus, A. gracilistylus W. W. Smith and A. sessiliflorus were also detected and compared. Only uridine was not determined because of its low content in the A. giraldii Harms.As was shown in the determination results, the content of syringin in the stem bark of A. giraldii Harms was lower than that of liriodendrin, while the content of syringin in the A.senticosus was very high. The content of liriodendrin in the root bark of A. giraldii Harms was higher than that in the stem bark. Cutting the stem of A. giraldii Harms into stem bark, xylem and center three parts and determining, it was found that there was a great difference in the contents of liriodendrin in the three parts. The content of liriodendrin in the xylem part was 3 times of that in the stem bark, and that in the center part was the lowest. Traditionally the stem bark of A. giraldii Harms was peeled to be used for the treatment, and the xylem was then discarded. While the determination results suggested that the content of liriodendrin was greatly decreased.It was found in the determination results of chlorogenic acid and caffeic acid that the contents of chlorogenic acid and caffeic acid in the various batches of A. giraldii Harms were significantly different. However, there was a trend that as the storage time increasing, the content of chlorogenic acid decreased, and the content of caffeic acid rose. The content of caffeic acid in the A. senticosus was lower than that in the A. giraldii Harms. The content of chlorogenic acid in the root bark of A. giraldii Harms was 14.2 mg.g^-1 which was the highest in the all determined samples.By the LC-DAD-MS/MS analysis, it was found that the leaves and fruits of Araliaceae contained some flavone components, and rutin and hyperoside were the main flavone compounds in the leaf and fruit of A. giraldii Harms. By the determination results, the content of rutin in the leaf and fruit of A. giraldii Harms was higher than that of hyperoside, while the content of rutin in the fruit of A. senticosus, A. gracilistylus W. W. Smith and A. sessiliflorus was lower than that of hyperoside.In this experiment, the main components in the stem bark, root bark, leaf and fruit of A. giraldii Harms were determined, which provided a foundation for the quality control and comparison with other Wujia Pie.4. LC-DAD-ESI-MS/MS multidimensional fingerprint analysis of A. giraldii HarmsA study on the multidimensional fingerprint of the stem bark of A. giraldii Harms was performed. Both the LC-UV and LC-MS fingerprints were developed, and it was found that they were in a complementary manner for the fingerprint analysis of A. giraldii Harms, and the combination of them could reflect the quality of A. giraldii Harms overall.In the LC-UV fingerprint analysis, 22 samples were classified by Hierarchical Cluster and Step Discrimination analysis, and two computer aided similarity evaluation system were used to appraise the Similarity and set up the Common Mode. The differences between Pearson correlation and Cosine of the angle, and differences of similarity calculated by two evaluation software were compared. Quantification coefficient was used to judge the whole amount of the fingerprint, and both the correlation and quantification coefficient were needed to appraise the quality of the material. Based on the results of Hierarchical Cluster and computer aided evaluation system, the samples with similarities more than 0.9 and quantification coefficient more than 0.9 were defined as the valid samples.10 samples were involved to set up the LC-MS fingerprint of the stem bark of A. giraldii Harms, and the extracted ion chromatogram of chlorogenic acid was chosen as the reference peak. The similarities of the LC-UV and LC-MS fingerprints of the stem bark of A. giraldii Harms were compared, and the evaluation results of the two method were identical for most of the samples.5. Pharmacological experiment of anti-inflammation of A. giraldii HarmsMice ear swelling model, rats hind paw edema model and the model of adjuvant arthritis in rats by injection of Freund’s complete adjuvant were used to observe the protective and therapeutic action of three different doses of the extract of the stem bark of A. giraldii Harms. Moreover, the effect of the isolated phenolic acids from the A. giraldii Harms on the acute inflammation was also investigated. As was shown in the results, it was possible that the phenolic acid compounds would be the active part of anti-inflammation action of the A. giraldii Harms.Two batches of the stem bark of A. giraldii Harms were used in the pharmacological experiment. It was found that there was no significant difference between the test group and the model control group while using the No.2 batch of A. giraldii Harms. As was shown in the fingerprints of the two batches material, the total contents of the components in the No.2 batch were significantly lower than that in the No.1 batch, and the content of chlorogenic acid was especially low in the No.2 batch. The pharmacological results validated our conclusion that No. 1 layer samples in the Hierarchical Cluster analysis were in good quality, and those of in the No. 2 layer were bad. It also suggested that the fingerprint analysis of the traditional Chinese medicine（TCM） played a very important role in the process of the modernization of TCM.The innovation points of this study（1） The LC-DAD-ESI-MS/MS technique was used for analysis of the components in the stem bark, root bark, leaf and fruit of A. giraldii Harms. 10 compounds were identified from the A. giraldii Harms, and 6 in the l0 compounds in the A. giraldii Harms have not been reported to date. 9 compounds in the A. giraldii Harms were determined except uridine because of its low content.（2） Based on the UV spectra, mass spectra and fragments of the MS/MS spectra, caffeoylquinic acid, dicaffeoylquinic acid and feruloylquinic acid in the A. giraldii Harms were also inferred.（3） The components in the stem bark, root bark, leaf and fruit of A. giraldii Harms were compared, and the components in the A. giraldii Harms, A. senticosus, A. gracilistylus W. W. Smith and A. sessiliflorus were also investigated and compared. Moreover, a deduction was put forward that the content of syringin should be as the standard to distinguish A. giraldii Harms from A. senticosus.（4） A study on the multidimensional fingerprint of the stem bark of A. giraldii Harms was performed. Both the LC-UV and LC-MS fingerprints were developed, and it was found that they were in a complementary manner for the fingerprint analysis of A. giraldii Harms, and the combination of them could reflect the quality of A. giraldii Harms overall.更多还原