【作者】 汪洋；

【导师】 秦路平； 韩婷；

【作者基本信息】 第二军医大学 ， 生药学， 2010， 博士

【摘要】 苍耳子(Fructus Xanthii),为菊科植物苍耳Xanthium sibirium Patr.的干燥成熟带总苞的果实,收载于历版中国药典,在我国资源丰富,为历代治疗鼻病及头痛的要药。然而,古代文献记载和现代临床应用都发现苍耳对人体具有一定的毒性作用,毒性一直制约着其临床应用。关于苍耳子毒性的隐患始终未能排除,毒性成分及其作用机理未能阐明。因此,本课题首先采用小鼠急性毒性实验来明确苍耳子毒性损伤的靶器官主要为肝脏,采用体外肝细胞实验筛选毒性成分,并对目标成分进行整体动物毒性评价,在此基础上利用代谢组学技术,探讨了苍耳子的毒理学作用机制,为苍耳子的合理应用提供科学依据。本课题主要涵盖以下几方面内容:1.苍耳子毒性物质基础研究通过小鼠急性毒性跟踪筛选,以半数致死量LD50为指标,确定了苍耳子的毒性部位为水提取物经AB-8大孔树脂柱层析的水洗脱部位(D1),并对此部位的化学成分进行了系统的研究,从中分离出7个化合物,分别为贝壳杉烯苷类、酚酸类和含硫杂原子类化合物。2.苍耳子毒性部位的毒性评价采用小鼠急性毒性实验,通过病理组织学观察明确苍耳子毒性损伤的靶器官主要为肝脏,小鼠血清生化指标和肝组织匀浆生化指标检测结果表明,小鼠发生了严重的脂质过氧化损伤。本实验结果提示苍耳子的中毒原因可能是自由基攻击细胞膜中的不饱和脂肪酸,发生了脂质过氧化反应,从而导致肝细胞膜通透性的改变。在此基础上评价了苍耳子的慢性肝毒性,结果表明在长期连续重复多次给药的情况下,小鼠出现了较为严重的肝毒性反应,动物的小叶周围脂肪变性及溶解性坏死,有较多量的淋巴、浆细胞等炎细胞浸润,并伴有较明显的纤维组织增生。由于慢性鼻炎、鼻窦炎等疾病需长期用药,剂量使用不当可引起慢性蓄积中毒,导致肝功能损害。所以长期、大剂量服用苍耳子对机体的不良影响应引起足够的重视。3.毒性成分的筛选和毒性评价采用人正常肝细胞株L-02和正常大鼠肝细胞株BRL作为体外细胞毒性的试验模型,比较研究苍耳子主要成分的细胞毒性。结果表明苍术苷、羧基苍术苷和4’-去磺基苍术苷随着浓度逐渐升高,抑制作用逐渐增强,浓度为10-4 mol/L时能够明显抑制对两种细胞的增殖作用。苍术苷等造成了不同程度的细胞皱缩现象,细胞间隙扩大,随着给药浓度增加,细胞出现空泡化,死细胞明显增多。苍术苷等引起LDH漏出增加,细胞通透性增加,具有一定的细胞毒作用。并采用整体动物实验对目标成分苍术苷进行了毒性评价,结果表明苍术苷高剂量组对小鼠的肝脏造成了较严重的损伤,且中毒机理可能与苍耳子毒性部位相似,证明了苍耳子中的主要毒性成分为贝壳杉烯苷类化合物,与体外实验结果相符。4.利用代谢组学技术探讨苍耳子的毒性作用机制利用1H NMR技术研究苍耳子处理后大鼠尿液和血浆中内源性代谢产物谱的变化,研究发现尿液中乳酸盐、葡萄糖、马尿酸盐、醋酸盐、三甲胺N氧化物、色氨酸、二甲胺、肌醇、柠檬酸盐、N甲基烟酸盐和酪氨酸明显升高,而酮戊二酸、肌酐、异戊酸盐、琥珀酸盐、组氨酸、丁酸盐、二甲基甘氨酸、N-乙酰基-半胱氨酸、牛磺酸和赖氨酸含量下降。血浆中异戊酸盐、乳酸盐、缬氨酸、谷氨酸盐、高密度脂蛋白、乙酸盐、肌酐、丙酮和尿素升高,而甘氨胆碱和葡萄糖明显下降。柠檬酸盐是三羧酸循环的中间产物,主要在肝细胞线粒体中进行代谢;琥珀酸盐的含量的降低是由于降低脂肪酸分解代谢。这些成分的变化影响肝细胞线粒体的能量代谢;烟酸盐及甲基烟酰胺均为色氨酸NAD+通路的产物,色氨酸代谢产物的升高伴随肝脏中脂质过氧化酶的生成减少,影响局部氧化还原平衡。肌醇,乳酸的浓度升高,是体内缺氧代谢的结果。所以苍耳子的毒性主要与其影响了肝细胞能量代谢有关。尿液中的柠檬酸盐、马尿酸盐、色氨酸等可以作为苍耳子的主要生物标志物研究。综上,苍耳子毒性作用靶器官主要是肝脏,贝壳杉烯苷类化合物是主要的毒性成分,其毒性作用与苍耳子引起动物肝脏的脂质过氧化损伤和影响肝细胞能量代谢机理有关。本课题将代谢组学与传统毒理学研究方法相结合,初步揭示了中药苍耳子毒性作用的肝损伤机制。我们的研究显示,用代谢组学方法揭示的生物化学变化很容易与传统手段的测定结果相联系,更容易发现药物作用的生物化学物质基础和作用机制,因此,药物毒性作用机制的代谢组学研究必将在成分复杂的中药体系中发挥更大的作用。苍耳子的临床应用十分广泛,正常情况下使用苍耳子是安全的,但长期、大剂量使用会出现毒性反应,因此在临床上长期用药不要超过3-9 g,肝损伤患者应用时需要调整剂量,长期应用时要注意监测其肝功能。其他含贝壳杉烯苷类中药可能也有类似的毒性毒理机制,因此,应该建立质量标准,控制毒性成份的含量,加强对此类中药的不良反应监测。随着代谢组学技术的发展,此项技术将会越来越多的用于其他中草药毒性毒理的研究,对于实现中药现代化具有重要的意义。更多还原

【Abstract】 Fructus Xanthii named Cang-Er-Zi is the ripe fruits with involucre of Xanthium sibirium Patr. It is a traditional Chinese medicine that is used in curing nasal diseases and headache according to the Chinese Pharmacopoeia. Fructus Xanthii is in great need in clinic. However, utilization of Fructus Xanthii is limited by its toxicity. Fructus Xanthii is toxicant,and it maybe result in poisoning when used excessively,without preparation or in inappropriate preparation.Recently studies have demonstrated that Fructus Xanthii may have the potential of hepatotoxicity. However, so far there is little studies concening the toxic mechanisms and the toxicologic assessment of its essential components. So, there are no identified conclusions about its toxicity. In this study, in vivo and in vitro experiments have been applied to explore the toxicity and the target organs of Fructus Xanthii, and compared the toxicity of its essential components. After then, the toxic mechanisms are elucidated based on metabonomic technologies in order to offer some toxicological information for its safely clinical usage. The main contents of our study are as following three aspects:1 Study on toxic components of Fructus XanthiiToxicity response of different extractions from Fructus Xanthii was compared using acute toxicity experiment LD50 and MTD in mice. Toxic fraction(D1) was determined. Bioactivity guided isolation of toxic fractions and compounds were undertaken by various chromatographic methods. 7 known compounds were isolated from the toxic fraction. The major types of the isolated compounds were phenolic acids, kaurene glucosides and heterocycles.2 Evaluation of toxic fractions of Fructus XanthiiThe results of level of serum transaminase, biochemical parameters of liver tissue and histopathlogy showed that the main toxic target organ of Fructus Xanthii is liver using acute toxicity experiment. It demonstrated that Fructus Xanthii induce hepatotoxicity in mice by way of its induction of oxidative stress as lipid peroxidation in liver, which merited further studies. Then, the chronic liver toxicity experiment was also determined. It deserves attention that long-term and high-dosage administration of Fructus Xanthii can damage the liver to varied degrees.3 Evaluation of toxic components of Fructus XanthiiComparative toxicity studies of its essential components indicated that atractyloside, carbxyatractyloside and 4’-desulphate-atractyloside are main toxic constituents using cell test. It was found that the three kaurene glycosides can strongly inhibite human normal liver L-02 and rat normal liver BRL cells growth. Then, the toxicity of atractyloside was determined in toxicity experiment in mice. The results demonstrated that the toxic constituents of Fructus Xanthii are kaurene glucosides.4 Investigation of toxic methanism of Fructus Xanthii using integrated metabonomic technologyA integrated metabonomic study with high-resolution 1H NMR spectroscopy has been applied to investigate the biochemical composition of urine and plasma obtained from Fructus Xanthii treated rats. It was found that the level ofβ-HB, lactate, valine, Glutamate, Creatinine, acetone, lactate and allantoin was elevated,and the levels of GPC and glucose were decreased in blood plasma. The level of lactate, hippurate, acetate, TMAO, tryptophane, MA, myoinositol, citrate, N-methy-nicotinamide and tyrosine increased in urine,whileα-ketoglutarate, Creatinine, 2-hydroxy-isovalerate, succinate, histidine, 3-hydroxy butyrate, DMG, NAC, taurine and lysine decreased significantly. The increased level of citrate, N-methy-nicotinamide and tryptophane is the typical biomarker of induction of oxidative stress as lipid peroxidation in liver of Fructus Xanthii.Above all, the main toxic target organ of Fructus Xanthii is liver. Kaurene glycosides are main toxic constituents. From these results, we can infer that Fructus Xanthii induce hepatotoxicity in mice by way of its lipid peroxidation and energy metabolismin. Combination with biochemistry and histological research,we used metabonomic analysis of urines and serum samples to visualize significant alterations in metabolite expression patterns as a result of induced metabolic responses. The study of these metabolite alterations also allowed several major metabolic pathways.Fructus Xanthii is widely and safely used in Chinese medicines usually, while the toxic effects will appear when it was used in large dose and for a long time. There for, when it is used for a long time, the dose will not exceed 3-9 g. When the patients have liver injury, the dose must be modified, and functions of kidney and liver should be monitored. Other herb containing kaurene glycosides may have similar toxic effects. Quality criteria should be established to control the content of toxic substance acordingly. With the development of metabonomics,this novel technology might provide additional discrimination in the toxicological effects of other herb in the future, which will contributes to the modemlization of traditional Chinese medicines.更多还原