毛穗藜芦乙酸乙酯部位化学成分研究-药物分析学

摘 要毛穗藜芦（Veratrum maaekii Regel）是百合科（ Liliaceae）藜芦属（Veratrum Linn.）的植物，据中国植物志记载：毛穗藜芦主要分布在东北、山东、内蒙等地。藜芦属（Veratrum Linn.）植物是多年生草本植物，大约有 40 种，主要分布在欧洲、亚洲以及北美洲，我国有 13 种和 1 个变种，主要分布在华北、东北、西南、华东、华南。中医通常用藜芦属植物的根和根茎入药，可以治疗中风失语、头痛、癫痫、痢疾、黄疸、疥癣等疾病。主要药用品种有藜芦（Veratrum. nigrum L.）、黑紫藜芦（Veratrum. japonicum（Baker）Loes. f.）、毛穗藜芦（Veratrum. maackii Regel）、兴安藜芦（Veratrum. dahuricum（Turcz.）Loes. f.）、天目藜芦（Veratrum. Schindleri Loes. f.）、大理藜芦（Veratrum. taliense Loes. f. ）等。现在新药研究的一个热点是从天然产物中提取具有药理活性的化学成分，目前，我国学者对藜芦属中草药的化学成分进行了大量的药理学研究，发现藜芦属植物中的甾体生物碱成分有降低血压、抗血栓、抗肿瘤、增强心肌收缩力的作用。虽然已经有很多学者对藜芦属植物进行了一些化学成分、药理和毒理方面的研究，但是总体上研究的还不够深入、不够广泛。藜芦是有毒中药，在发挥其药理作用的同时还有一定的毒性作用，现代研究证明藜芦生物碱既是藜芦的有效成分又是毒性成分。因此，藜芦属中草药的毒性大大限制了藜芦属中草药在临床上的应用和新药开发。由于我们前期研究发现，藜芦生物碱会引起小鼠脑细胞 DNA 损伤，在现有的基础上，有必要对藜芦属中草药的生物碱成分进行更深入、系统的研究，从藜芦属中草药中提取分离制备单体化合物，寻找出高效低毒并且具有药理活性的化学成分，进一步明确藜芦属植物的化学成分，为天然药物化学研究积累资料，为藜芦属植物的进一步药理学研究、毒理学研究和构毒关系研究奠定物质基础。经查阅大量文献发现，迄今为止，关于藜芦、毛叶藜芦、天目藜芦、乌苏里藜芦、狭叶藜芦、兴安藜芦、大理藜芦、黑紫藜芦等品种的化学成分研究的比较多，而关于毛穗藜芦的化学成分研究较为薄弱，鉴于此，本论文对毛穗藜芦乙酸乙酯部位的生物碱成分进行研究，以期提取、分离出新的甾体生物碱，为以后藜芦属中草药的毒性研II究及其构毒关系研究提供单体化合物，为藜芦属中草药的开发和利用提供化学成分方面的实验依据。本论文进一步对毛穗藜芦根及根茎乙酸乙酯部位的化学成分进行了系统的研究，药材经 95%乙醇加热回流提取，氯仿超声提取以及氯仿、乙酸乙酯萃取，得到乙酸乙酯部位，采用硅胶柱层析、大孔吸附树脂(HPD100) 柱层析和葡聚糖凝胶（Sephadex LH-20）柱等柱层析方法以及制备型 TLC，制备型 HPLC 等色谱技术对乙酸乙酯部位的化学成分进行了系统的分离，同时用 5%硫酸乙醇试剂和碘化铋钾试剂显色，着重分离毛穗藜芦乙酸乙酯部位的生物碱成分，并应用核磁共振波谱技术和质谱技术（ 1H-NMR， 13C-NMR，DETP ， 1H-1HCOSY，HMQC ，HSQC ，HMBC ，NOESY ，ESI-MS）对分离的化合物进行结构鉴定，共分离得到 11 个化合物，且都是生物碱，鉴定了其中 9 个化合物的结构分别为 L1*：3-O-D-葡萄吡喃糖基-23- 甲氧基环杷明（3-O-D-glucoside-23-methoxylcyclopamine）；L2：7-乙酰基-15-异戊酰基-3-藜芦酰基计明碱（verussurine）；L3： 3-藜芦酰基-15-异戊酰基计明碱（verabenzoamine）；L4 ：计莫林碱（germerine）；L5：藜芦嗪（verazine ）；L6 ：异藜芦嗪（isoverazine）；L7：veramitaline；L8 ：番茄立啶碱（Tomatillidine）；L9 ：介芬胺（jervine ）。其中L1*是新化合物，L8 是首次从藜芦属植物中分离得到，L2、L3 、L4、L7 、L9 是首次从毛穗藜芦中分离得到。关键词：毛穗藜芦，化学成分，生物碱ABSTRACTVeratrum.maackii Regel is one of species of the genus Veratrum (Liliaceae), according to the “flora Chinese “ records: Veratrum maackii Regel is mainly distributed in the northeast, Shandong, Inner Mongolia etc. Veratrum is a perennial herb, there are about 40 species, mainly distributed in Europe, Asia and North America, there are thirteen species and one varieties in china, mainly distributed in North China, northeast, southwest, East China, South China. The roots and rhizomes of several Veratrum species, including Veratrum. nigrum L. ; Veratrum. japonicum(Baker)Loes. f .; Veratrum. dahuricum(Turcz.)Loes. f. ; Veratrum. maackii Regel ; Veratrum. Schindleri Loes. f. ; Veratrum. taliense Loes. f. have been used to treat stroke, headache, epilepsy, dysentery, jaundice, scabies and other diseases as Traditional Chinese medicine. Nowadays, the hot research on new drug is to extract the chemical constituents with pharmacological activity from natural products , Chinese scholars make a lot of pharmacological studies on the chemical constituents of Veratrum plants, they have found that the steroidal alkaloids of Veratrum plants have the effects of lower blood pressure, antithrombosis, antitumor, enhance myocardial contractility. Although many researchers have been did a lot studies on chemical constituents, pharmacological and toxicological of Veratrum plants, the researches are not thoroughly and not wide enough on the whole.Veratrum plants is a kind of poisonous Traditional Chinese Medicine, to play its pharmacological effects as well as certain toxic effects , Modern research proves that ,Veratrum alkaloids are the effective ingredients and toxic constituents of Veratrum plants , Therefore due to the toxicity of Veratrum plants has greatly limits the application of Veratrum plants in the clinical and the development of new drugs , our previous study found that, Veratrum alkaloids can cause DNA damage of brain cells in mice, So on the existing basis, it is necessary to make a futher and more system study on the active constituents of Veratrum plants , continuing to extract monomer compounds from Veratrum plants, finding out the chemical composition of pharmacological activity with highly efficient and low toxicity, to further clarify the chemical composition of Veratrum plants , accumulation data for futher study of natural medicine chemistry and laid the material IVfoundation for further study on pharmacology research, toxicological studies and Structure toxicity relationships studies of Veratrum plants .Having reviewed the extensive literature found that, so far ,more studies on the chemical constituents of Veratrum. nigrum L; Veratrum. japonicum(Baker)Loes. f. ; Veratrum. stenophyllum Diels ; Veratrum. dahuricum(Turcz.)Loes. f. ; Veratrum. Schindleri Loes. f. ; Veratrum. nigrum L. var.ussuriense Nakai; Veratrum. grandiflorum (Maxim.) Loes. f. ; Veratrum. taliense Loes.f. , while the studies on the chemical constituents of Veratrum. maackii Regel relatively weak. In view of this, my thesis did a lot of research on the alkaloid constituents of Veratrum maackii Regel ethyl acetate parts, so as to extract the new steroidal alkaloids , provide monomer compounds for the study of toxicity and the toxic mechanism of Veratrum plants, and make a basis of chemical composition for the develop and utilize of Veratrum plants.The chemical constituents of ethyl acetate fraction of Veratrum maackii Regel were studied in this article, First, herbs refluxing extraction by 95% ethanol, then,ultrasonic extraction with chloroform and then extraction by chloroform and ethyl acetate , The ethyl acetate fraction is obtained.By using column chromatography with silica gel, macroporous adsorption resin (HPD100) and dextran gel column (Sephadex LH-20) and preparative TLC, preparative HPLC chromatographic technique to separate compounds , At the same time with 5% sulfuric acid ethanol reagent and bismuth potassium iodide reagent, Mainly to extract the alkaloid constituents of Veratrum maackii Regel from ethyl acetate extract.By using nuclear magnetic resonance spectroscopy and mass spectrometry (1H-NMR, 13C-NMR, 1H-1HCOSY, HMBC, HMQC, NOESY, ESI-MS) to identify the structure of compounds which are isolated, eleven compounds are isolated and all of them are alkaloids, Among them,identification the structure of nine compounds ,they are 3-O-D-glucoside-23-methoxylcyclopamine(L1); verussurine(L2); verabenzoamine (L3); germerine(L4); verazine(L5); isoverazine(L6); veramitaline(L7); Tomatillidine(L8); jervine(L9) . L1 is a new compound . L8 is isolated from the Veratrum plants for the first time . L2, L3, L4, L7, L9 are isolated from Veratrum. maackii Regel for the first time .Keywords: Veratrum maackii, chemical constituents, alkaloid缩略语说明LIST OF ABBREVIATIONS2D NMR two -dimensional nuclear magnetic resonanceNMR nuclear magnetic resonance spectroscopyDEPT Distortionless enhancement by polarization transferCOSY Correlation spectroscopyHMBC H-detected heteronuclear multiple-bond correlationHMQC H-detected heteronuclear multiple-quantum correlationNOESY Nuclear overhauser enhancement spectroscopyHRESI-MS High resolution ESI-MSESI-MS Electrospray ionization-mass spectrumTLC thin-layer chromatographyHPLC high-performance liquid chromatographyHz hertzMHz megahertz Chemical shiftg gramL litre(s)mg milligrammin minute(s)VII目 录摘 要 .IABSTRACT.III缩略语说明 .V第一章 藜芦属植物的研究进展 .11 藜芦属植物的化学成分研究 .21.1 甾体生物碱.21.2 黄酮类化合物 .61.3 甾醇类化合物 .71.4 茋类化合物 .71.5 其他成分 .72 藜芦属植物的药理活性研究 .72.1 藜芦生物碱的降血压作用 .72.2 抗血栓和降低血液粘度的作用 .82.3 藜芦生物碱抗肿瘤作用 .92.4 抑菌及杀虫作用 .102.5 藜芦对神经系统的影响 .112.6 对 5-HT 表达的影响和对心室肌细胞的影响 .113 藜芦的毒性和毒理学研究 .123.1 藜芦的毒性.123.2 中药十八反中藜芦的配伍禁忌机制.133.3 藜芦的中毒及解救.144 藜芦的临床应用 .145 本章小结 .156 本论文研究的目的意义 .157 研究方法 .15VIII第二章 毛穗藜芦乙酸乙酯部位的化学成分研究 .191 仪器与材料 .191.1 仪器 .191.2 材料 .192 实验部分 .202.1 提取 .202.2 分离 .202.3 核磁数据及质谱测定 .223 化合物的结构鉴定 .243.1 化合物的结构、编号和名称 .243.2 新化合物的结构鉴定 .263.3 已知化合物的结构鉴定 .294 本章小结 .41参考文献 .45附 录 .45致 谢 .61第一章 藜芦属植物的研究进展1第一章 藜芦属植物的研究进展前言藜芦属（Veratrum）是百合科（Liliaceae）植物，世界上大概有 40 种，我国有 13种 1，藜芦辛、苦、寒，有毒，归肺、胃、肝经。主要药用品种有藜芦（Veratrum. nigrum L.）、毛穗藜芦（ Veratrum. maackii Regel）、黑紫藜芦（Veratrum. japonicum（Baker）Loes.f. ）、狭叶藜芦（Veratrum. stenophyllum Diels）、兴安藜芦（Veratrum. dahuricum（Turcz.）Loes. f.）、天目藜芦（Veratrum. Schindleri Loes. f.）、乌苏里藜芦（Veratrum. nigrum L. var.ussuriense Nakai）、毛叶藜芦（Veratrum. grandiflorum （Maxim.） Loes. f.）、大理藜芦（Veratrum. taliense Loes. f.）等，藜芦属植物在传统医药中有较为广泛的应用，中医通常用藜芦属植物的根和根茎入药，有催吐，祛痰和杀虫的作用。用来治疗中