【作者】 颜彦；

【导师】 杨祥良；

【作者基本信息】 华中科技大学 ， 生物制药工程， 2011， 博士

【摘要】 缺血性心脏病是当今世界重大疾病之一,由于目前尚无有效的治疗手段,其发病率和死亡率一直居高不下,严重危害着人类健康。寻找和开发抗心肌缺血药物一直是药物研究中的热点。我国传统中医药和民族药物有着悠久的应用历史,其中有很多物质具有抗心肌缺血的活性,蕴藏着丰富的资源。从这些已知临床应用效果的天然产物中寻找活性药物,是开发新药非常有效同时也是非常重要的途径之一,将达到事半功倍的效果。蒙医药是我国具有鲜明特色的民族医药之一,在内蒙古地区至今仍有广泛应用。但其现代研究起步晚,其中有很多已知确切疗效的药物,不明其物质基础和药理作用。山沉香就是其中之一。山沉香(Syringa pinnatifolia Hemsl.var.alashanensis Ma et S. Q. Zhou)别名贺兰山丁香,主要分布于内蒙古贺兰山地区,为内蒙古地区特有品种,在阿拉善地区民间医药中有着长期的应用。蒙药中山沉香可镇“赫依”病,多用于胸闷气短、心绞痛等心肺疾病的治疗,可治“热邪侵于心脏的一切疾病”,在缺血性心脏病的防治中有着较好的疗效。但是,目前山沉香的化学成分的研究十分薄弱,药理作用的研究更是一片空白,严重制约了该药的进一步应用。本文以山沉香挥发油为主要研究对象,研究了其抗心肌缺血的药效学作用,结合多种动物模型和细胞模型,从不同层次水平上研究了其心肌保护作用。采用顶空液相微萃取法(HS-LPME),结合气质联用技术(GC-MS),对山沉香挥发油的快速分析和药材中花姜酮的含量测定进行了研究。同时开展了山沉香乙酸乙酯提取物化学成分的初步研究。主要结果如下:(1)建立了一种对山沉香挥发油进行快速测定的顶空液相微萃取气质联用法(HS-LPME-GC-MS),对液相微萃取条件进行优化得到的最佳处理条件为:山沉香粉末1.0 g(80目),1.0μL苯甲醇于70℃萃取20 min后进样。采用所建立的方法对山沉香挥发性成分进行了分析,鉴定出52种组分,水蒸汽蒸馏法(SD)提取的挥发油鉴定出50种组分。两种方法所得成分中36种相同。相同组分的总百分含量占水蒸汽蒸馏法所得挥发油的95 %。由于HS-LPME简便、快速、能耗低,所用药材、溶剂均极少,并可达到与SD法相似的结果,可作为传统水蒸气蒸馏的替代方法,用于快速分析山沉香挥发油及其他药材挥发性成分。(2)采用超声法和顶空液相微萃取法结合,研究了UA-HS-LPME-GC测定山沉香药材中花姜酮含量的方法。对UA-HS-LPME条件如超声溶剂、超声时间、萃取溶剂、萃取温度、萃取时间进行了考察。对所建立的方法进行了方法学论证,研究结果表明,方法特异性强,准确率高,重现性好。(3)研究了山沉香挥发油对实验性心肌缺血的保护作用。首先考察了山沉香挥发油对小鼠常压耐缺氧能力的影响。以小鼠在常压密闭环境下耐缺氧的存活时间为指标,考察了其对机体耐缺氧能力的影响。结果显示,山沉香挥发油8 mg/kg和32 mg/kg均能显著延长小鼠常压耐缺氧的存活时间,其中32 mg/kg挥发油组的存活时间与阳性药物普萘洛尔组十分接近,无显著性差异,说明山沉香挥发油能够在整体上增强机体对缺氧状态的耐受程度。这种作用的发挥可能与其增加心肌供血、降低心肌耗氧量有关,对于保护心肌缺血具有重要的意义。采用冠状动脉左前降支结扎法复制心肌缺血模型,对心肌缺血程度、缺血后血清酶谱和损伤标志物进行了检测,考察了山沉香挥发油对大鼠急性心肌缺血的保护作用。并对病理学切片和心肌细胞超微结构进行了观察。研究结果表明,山沉香挥发油可减少大鼠心电图ST段的偏移量,降低心肌缺血的程度。同时山沉香挥发油还可降低冠脉结扎后肌酸激酶(creatine kinase,CK)和乳酸脱氢酶(lactate dehydrogenase,LDH)的水平,降低心肌损伤标志物心肌肌钙蛋白T(cardiac Troponin T,cTn T)的含量,升高超氧化物歧化酶(superoxide dismutase,SOD)的活性。组织病理学切片和心肌细胞的电镜照片的观察发现,山沉香挥发油能够降低模型组心肌细胞的坏死程度和炎症细胞的浸润范围,并且能够保护心肌细胞线粒体、肌质网等重要结构。证明山沉香挥发油具有显著的抗急性心肌缺血的活性,能够提高心肌细胞抗脂质过氧化的能力,稳定心肌细胞膜,保护心肌细胞内部结构和生理功能。考察了山沉香挥发油对大鼠体外血小板聚集的影响。采用电阻法在全血中测定血小板聚集度,以电阻值表征聚集度的大小,以ADP为诱导剂。结果表明,山沉香挥发油能够显著抑制ADP诱导的血小板聚集,高剂量组(5μg/mL)最大抑制率达到47.4%,显示出良好的抗血小板聚集的活性。(4)考察了山沉香挥发油对心肌细胞氧化损伤和缺氧-复氧损伤的影响。研究结果显示,山沉香挥发油对两种模型中损伤所致心肌细胞存活率下降都有显著的保护作用。同时还能降低心肌细胞氧化损伤后LDH的漏出,提高SOD的活性,表明山沉香挥发油对心肌细胞具有直接而显著的保护作用。(5)研究了山沉香不同极性部位对心肌细胞H2O2氧化损伤的保护作用,并根据活性筛选结果,对山沉香乙酸乙酯提取物的化学成分进行了研究。综合采用多种分离手段,从中分离出4个化合物,经结构解析和对比,分别为:落叶松脂素、开环异落叶松树脂酚、3’,4’-缩醛-落叶松脂素和(+)-二氢荜澄茄素。四种化合物均为首次从山沉香中分离得到。本文首次对山沉香挥发油抗心肌缺血的活性进行了研究,明确了其心肌保护的作用,并分析了山沉香挥发油及乙酸乙酯提取物的化学成分,将为山沉香的后续研发提供依据和基础。更多还原

【Abstract】 Ischemic heart disease (IHD) is one of the world’s major diseases, and its incidence and mortality has been high worldwide. It is serious harmful to human health, since no sufficient therapy for this obstinate illness is available. Research and development of anti-ischemic drugs has been the focus of drug research. Attempts are made globally to get complementary and alternative medicines for IHD. In China, the application of Traditional Chinese Medicine (TCM) and folk medicines has a long history. Many of the durgs have anti-ischemic activity, rich in resources. Searching new active drug in these natural products and materials which have definite clinical effect is very effective and important in development of new drugs.Mongolian medicine is one of the most important ethnic drugs in China with its own distinctive feature, which has a long application history and is still widely used in Inner Mongolia. However, because of the late starting of modern research, many of Mongolian medicines have known exact effect but unknown material basis and pharmacological effects. Syringa pinnatifolia Hemsl.var.alashanensis Ma et S. Q. Zhou (Shanchenxiang) is one among these. The stem of Shanchenxiang is one of the best-known traditional herbal medicines frequently used to treat cardiovascular symptoms in Mongolian medicine. It is widely used in Mongolian in treatment of chest tightness, shortness of breath, myocardial ischemia and other cardiovascular diseases, basicly“all the diseases induced by hot evil invasion”, and has a broad application in the prevention and treatment of IHD. However, there is few reported scientific study to support these claimed therapeutic and medicinal effects.In this paper, Syringa pinnatifolia Hemsl.var.alashanensis Ma et S. Q. Zhou essential oil (SPEO) was chosen as the major research object, and its anti-ischemic effect was studied with a variety of animal models and cell models, to confirm different levels of its cardioprotective effect. At the same time, headspace liquid phase microextraction (HS-LPME) was used in the rapid analysis of chemical components of SPEO and determination of zerumbone in the herbal material. At last, chemical constituents of ethyl acetate extract of Shanchenxiang was studied. The main results are as follows:(1) A headspace liquid phase microextraction coupled with gas chromatography- mass spectrum (HS-LPME-GC-MS) method was described for the analysis of volatile compounds from Syringa pinnatifolia Hemsl.var.alashanensis Ma et S. Q. Zhou. Parameters of optimized conditions were: sample powder 1.0 g (80 mesh), 1.0μL of benzyl alcohol extracted at 70℃for 20 min. Analysis of volatile compounds in Shanchenxiang by HS-LPME-GC-MS method was performed under the described conditions and 52 compounds were identified,and the number of the identified compounds was 50 for steam distillation (SD)-GC-MS. HS-LPME-GC-MS shared 36 same compounds with the SD method while the total content of the same components accounted for 95% of the essential oil derived by SD method. HS-LPME is simple, fast, with low energy consumption, few use of drugs and solvents, and can achieve similar results with SD method, indicating that it can be used as an alternative method to SD for the rapid analysis of volatile compounds in Shanchenxiang and other TCMs.(2) Ultrasound assisted HS-LPME gas chromatography (UA-HS-LPME-GC) was applied in determination of zerumbone in Shanchenxiang. UA-HS-LPME parameters, such as ultrasonic solvent, ultrasound time, extraction solvent, extraction temperature and extraction time were optimized. The method was comprehensively validated. The results showed that the method for determination of zerumbone was specific, accurate and reproducible.(3) Effect of SPEO on myocardial ischemia was investigated. In hypoxia tolerance test of mice, the survival time of mice under hypoxia was chosen as an index. The results showed that the SPEO 8 mg/kg and 32 mg/kg could significantly prolong the survival time of mice under hypoxia, the survival time of 32 mg/kg SPEO is close to positive drug propranolol, indicating that SPEO can enhance the body’s overall tolerance of hypoxia. This effect may be related to increasing of myocardial blood flow and reduction of myocardial oxygen consumption which are critical in myocardial ischemia.The acute myocardial ischemia (AMI) model in rats was induced by ligation of left anterior descending artery, and the extent of myocardial ischemia, ischemia and damage markers in serum enzymes were tested to study the effect of SPEO on acute myocardial ischemia. Pathology slides and myocardial ultrastructure were also observed. The results showed that SPEO can reduce the deviation of ST segment in ECG, reducing the extent of myocardial ischemia. SPEO can also decreased the levels of lactate dehydrogenase (LDH), creatine kinase (CK) and cardiac Troponin T (cTn T), while increased the activity of superoxide dismutase (SOD). The protective role of SPEO was further confirmed by histopathological examination. Pretreatment with SPEO exhibited decreased degree of necrosis and less infiltration of inflammatory cells, and can protect the mitochondria, sarcoplasmic reticulum and other important structures in cardiac myocytes. The results suggest that SPEO has significant activity against acute myocardial ischemia. It can increase the ability of myocardial cells against lipid peroxidation, stabilize the cell membrane to protect the myocytes.The in vitro effect of SPEO against platelet aggregation was investigated using adenosine 5’-diphosphate (ADP) as agonist. Electrical resistance method was used in the test and aggregation was induced in the whole blood. The degree of platelet aggregation was represented by resistance quantity. The results presented that SPEO can significantly inhibit the platelet aggregation induced by ADP and high dose group (5μg/mL) exhibited the maximum inhibition rate of 47.4%, indicating good anti-platelet aggregation activity of SPEO.(4) The protective effect of SPEO on H2O2-induced oxidative damage and hypoxia/reoxygenation injury was evaluated. The results show that SPEO can significantly protect the cell injury in these models. Meanwhile, SPEO can reduced the leakage of LDH and increased SOD activity after oxidative injury, indicating a direct and significant protective effect on cardiac myocytes.(5) Effects of different polar fractions of Shanchenxiang on H2O2-induced cardiac myocytes injury were studied. The chemical composition of ethyl acetate extract was studied according to activity screening results. Using a combination of various separation methods, four compounds were finally isolated from the extract. After structural analysis and comparison, the purified compounds are identified as lariciresinol, secoisolariciresinol, 3’,4’-acetal-lariciresinol and (+)-dihydrocubebin, respectively.The results of this study are the first report on effect of SPEO on myocardial ischemia in multiple levels. The role of SPEO in cardioprotection was defined. Phytochemical study and quality control index of the Materia Medica will provide basis and foundation for follow-up research of Syringa pinnatifolia Hems1. var. alashanensis Ma et S. Q. Zhou.更多还原