【作者】 徐金龙；

【导师】 秦路平； 张巧艳； 郑承剑；

【作者基本信息】 第二军医大学 ， 生药学， 2012， 硕士

【摘要】 牡荆子为马鞭草科植物牡荆Vitex negundo var. cannabifolia的干燥成熟果实，味苦、辛，性温；归肺、大肠经。具有除湿祛痰，止咳平喘，理气止痛的功效，现代药理研究表明牡荆子具有镇痛、平喘、抗炎、抗菌、抗氧化等作用。该植物生于低山向阳的山坡路边或灌木丛中，产于全国大部分地区，主要分布于华东及河北、湖南、湖北、广东、广西、四川、贵州等。近些年来对于牡荆属植物化学成分研究表明，牡荆属植物富含木脂素类、黄酮类、二萜类等成分，这些成分也显示出了一系列活性如：抗氧化、抗炎、抗菌、镇痛等。牡荆子在民间与传统用药上作为抗炎平喘的药物来使用，但是由于对牡荆子的系统研究较为缺乏，导致其物质基础不明确，因此，我们对牡荆子进行系统的化学成分研究，阐明其物质基础，为其进一步开发打下研究基础。本课题主要涵盖以下两个方面：一、牡荆子化学成分研究牡荆子药材经低温干燥及打粉后，用80%乙醇反复回流提取3次，每次2小时。减压回流后得到的总浸膏分别用二氯甲烷、石油醚、正丁醇、乙酸乙酯和水萃取，采用柱层析、薄层色谱、半制备液相等方法从二氯甲烷性部位中分离出30个化合物，并应用理化常数测定和光谱(IR, UV, MS,¹³C-NMR,¹H-NMR,2D-NMR)分析技术鉴定了其中的23个化合物。分别为牡荆内酯、前牡荆内酯、白桦酸、紫花牡荆素、北美草素-7-甲醚、对羟基苯甲醛、β-谷甾醇、香草醛、6-羟基-4β-（4-羟基-3-甲氧基苯基）-3α-羟甲基-5-甲氧基-3,4-二氢-2-萘醛、6-羟基-4β-（4-羟基-3-甲氧基苯基）-3α-羟甲基-7-甲氧基-3,4-二氢-2-萘醛、泡桐素、（3S,5S,8R,9R,10S）-3-乙酰氧基-14,15,16-三降-13,9-半日花烷交酯、异落叶松脂素、芝麻素、对羟基肉桂酸、熊果酸、金合欢素、2,5-二羟基-1-甲氧基吡酮、15-羟基脱氢松香酸、5-羟基-6,7,3’,4’-四甲氧基黄酮、8-羟基-5,7,3’,4’-四甲氧基黄酮、对羟基苯甲酸与对甲氧基肉桂醛。我们通过同属植物（黄荆、牡荆、蔓荆、单叶蔓荆以及穗花牡荆）化学成分的比较，发现该属植物大多含有紫花牡荆素，说明紫花牡荆素为该属植物的特征性成分，并且主要成分为紫花牡荆素的植物提取物在欧洲广泛的用于治疗经前期综合征。但由于其药代动力学特征不明确，影响其进一步应用，所以对其进行药代动力学研究，明确其药代学特征，对紫花牡荆素开发成新药显得尤为重要。二、药代动力学研究药物代谢动力学是定量描述药物进入体内以后的吸收、分布、代谢、排泄过程，通过测定生物样本中的药物或者代谢产物的浓度，可以更好的阐明药物的药效或者毒性，为新药研究的代谢筛选和临床用药的安全有效提供重要依据。随着组合化学技术和天然药物分离制备技术的发展，大量的候选药物需要进行药代动力学评价，这就要求建立并优化相关分析方法对生物样品中的目标成分进行高灵敏度、快速准确、低消耗的定量分析。紫花牡荆素（Casticin，3’,5-dihydroxy-3,4’,6,7-tetramethoxyflavone）又名蔓荆子黄素（vitexicarpin），作为一个重要的黄酮类成分，广泛的存在于牡荆属植物中，例如蔓荆、穗花牡荆、单叶蔓荆。据报道紫花牡荆素具有抗肿瘤、抗炎、抗高催乳素血症、抗氧化、镇痛等活性。近年来，紫花牡荆素被认为是抗经前期综合征的主要活性物质，与传统抗经前期综合征的药物（镇痛药、雌激素）相比，从天然产物中得到的紫花牡荆素具有更少的副作用，并在欧洲广泛的用于治疗经前期综合征。鉴于紫花牡荆素具有抗炎、抗高催乳素血症、抗氧化、镇痛等作用，其发展成为抗经前期综合征药物的潜力很大，所以建立一种选择性、灵敏性高的定性、定量检测方法，对紫花牡荆素而言是十分必要的。虽然在以往的研究中，建立了几种通过HPLC-DAD定性、定量检测紫花牡荆素的方法，但这些方法主要着重于原材料的检测，而针对于生物样品的检测报道很少。本文主要的目的在于建立一个快速准确、简单可信的LC-MS检测方法，并将其应用于紫花牡荆素的临床前药代动力学研究。更多还原

【Abstract】 Mu-Jing-Zi is the ripe fruits of Vitex negundo var. cannabifolia （Verbenaceae） and isused to dispel the moisture, relieve cough and asthma, regulate Qi and relieve pains inTraditional Chinese Medicine. Modern pharmacological studies show that Mu-Jing-Zi hasthe effects of analgesic, antiasthmatic, anti-inflammatory, antibacterial and antioxidant.Vitex negundo var. cannabifolia is a shrub growing in the most area of China. It is mainlydistributed in Hebei, Hubei, Hunan, Guangdong, Guangxi, Sichuan and Guizhou province.Recently, the research results show that the plant has been proved to be a rich sources oflignans, as well as flavonoids and diterpenoid, which exhibit a wide variety of biologicalactivities including antioxidant, anti-inflammatory, antisepticise and analgesic effects.Mu-Jing-Zi is commonly used in clinical practice for curing cough and anti-inflammatory infolk medicine. However, the insufficient research of Mu-Jing-Zi resulted in unknownactive chemical constituents. Therefore, figuring out the constituents of Mu-Jing-Zi isbeneficial to its deeper development. The main contents of our study are as following twoaspects:1Study on chemical components of Vitex negundo var. cannabifolia fruits.The air-dried and powdered seeds of Vitex negundo var. cannabifolia were extracted with80%EtOH （×3）, each extraction period lasting2h. After removal of the solvent underreduced pressure, the residue was suspended in H2O and partitioned sequentially withpetroleum ether, CH₂Cl₂, EtOAc and n-butanol, respectively. The CH2Cl2-soluble part wassubjected to column chromatography, TLC, and semi-preparative high performance liquid.A total of30with known compounds were isolated from dichlormethane fraction and23structures were elucidated by detailed spectroscopic analyses on the basis of NMR, IR, andMS data. The substance are vitexilactone, previtexilactone, betulinic acid, casticin,3’,4’,5-hydroxy-7-methoxy–flavanone, p-hydroxy benzaldehyde, β-sitosterol, vanillin,vitedoin A,6-hydroxy-4β-（4-hydroxy-3-methoxyphenyl）-3α-hydroxymethyl-7-methoxy-3,4-dihydro-2-naphthaldehyde, paulownin, vitedoin B, isolariciresinol, sesamin,cis-p-coumaric acid, ursolic acid, acacetin,2,5-Dihydroxy-l-methoxyxanthone,15-Hydroxydehydroabietic acid,5-hydroxy-6,7,3’,4’-tetramethoxyflavone,8-hydroxy-5,7,3’,4’-tetramethoxyflavone, p-hydoxybenzoic acid and 4-Methoxybenzaldehyde respectively. By comparing with the chemical compositions ofVitex negundo L., Vitex negundo var. cannabifolia, Vitex trifolia L., Vitex trifolia L. var.simplicifolia Cham. and Vitex agnus-castus L., we found that casticin is the characteristiccomponents of the Vitex species. In the process of earlier study, casticin is considered asthe major active chemical constituent for the treatment of premenstrual syndrome （PMS） inthe medicinal plants of Vitex species. However, due to its pharmacokinetic properties is notclear, casticin is hard to further study. So, to clarify its pharmacokinetic characteristics isparticularly important to develop casticin into new drug.2Study on pharmacokinetics of casticin in Rat Plasma.Pharmacokinetics is the study of the time course of a drug within the body andincorporates the processes of absorption, distribution, metabolism and excretion. To betterinterpret the effect and toxicity of the drug, an analytical method should be established todetermine the drug and its metabolites quantitatively, which help us to know about theirmechanisms of action and their pharmacokinetic properties during the initial state of drugdevelopment and in clinical therapy. With the improvement of combinatorial chemistry andseparation and purification of natural products, a number of candidate compoundsappeared and required pharmacokinetic analysis. Investigators have shown increasinginterest in developing and optimizing high-throughput analytical methods with moresensitivity，accuracy and lower consumption for quantitating small molecule drugs,metabolites, and other xenobiotic biomolecules in biological matrices. Casticin（3’,5-dihydroxy-3,4’,6,7-tetramethoxyflavone）, also named Vitexicarpin, as a major flavonecomponent has been isolated from a number of medicinal plants of Vitex species, such asVitex trifolia L., Vitex agnus-castus L. and Vitex rotundifolia L. It has been reported thatcasticin has antitumor, anti-inflammation, anti-hyperprolactinemia, antioxidant, andanti-nociceptive properties. In recent years, this compound is considered as the majoractive chemical constituent for the treatment of premenstrual syndrome （PMS） in themedicinal plants of Vitex species. In comparison with conventional drugs such asantiinflammatory agents （ibuprofen）, psychotropic drugs （selective serotonin reuptakeinhibitors）, estrogen and progesterone, naturally occurring products containing casticinhave been extensively used to treat PMS in European for little adverse effects. Consideringthe anti-inflammation, anti-nociceptive, anti-hyperprolactinemia and estrogenic activities of casticin, it has a good potential to be developed as a new therapeutic reagent for PMS.For the development of casticin as a new drug, it is necessary to establish a selective andsensitive analytical method for the quantitative estimation of casticin in preclinicalbiopharmaceutical and clinical pharmacology studies. Several methods for the separationand quantification of casticin have been described using high performance liquidchromatography （HPLC） with ultra-violet detection. However, these methods mainlyfocused on the quantification of casticin in raw materials or pharmaceutical preparations.Few studies can be found in biological specimen. The purpose of this study was to developa simple, rapid and reliable LC-MS assay for the determination of casticin in rat plasmaand applied the assay to the pharmacokinetic study in rats after oral and intravenousadministrations of casticin.更多还原