【作者】 刘先琼；

【导师】 吴皓；

【作者基本信息】 南京中医药大学 ， 中药学， 2012， 博士

【摘要】 本学位论文得到两项国家自然科学基金——天南星科有毒中药矾制解毒共性规律研究（NO.30973939）、天南星科有毒中药凝集素蛋白与毒性的相关性和炮制的影响（NO.81173549）以及江苏省普通高校研究生科研创新计划项目（NO.CXZZ11₀786）的资助。2010版《中国药典》收载的半夏（Pinellia ternata （Thunb.） Breit.）、天南星（Arisaema erubescens （Wall.） Schott）、白附子（Typhonium giganteum Engl）及在市场上作为天南星的代用品广泛使用的掌叶半夏（Pinellia pedatisecta Schott.）,均属于天南星科,为临床常用有毒中药,这四种有毒中药临床中毒症状极为相似,误服均出现粘膜及皮肤刺激,口舌肿胀,呼吸缓慢甚至窒息死亡,所以中医临床上均使用炮制品。论文作者所在课题组在前期研究中提出并证实天南星科四种有毒中药半夏、掌叶半夏、天南星及白附子的刺激性毒性成分均为其所含有的具有特殊晶形的针晶复合物,具有强烈的刺激性毒性,可产生严重的刺激性炎症反应,故课题组称为“毒针晶”。毒针晶主要由草酸钙、蛋白组成,前期研究发现组成毒针晶的草酸钙没有刺激性作用,而组成毒针晶的蛋白可显著加重豚鼠破损皮肤的炎症反应。对半夏毒针晶蛋白研究的结果显示,半夏毒针晶上带有的半夏凝集素蛋白是半夏毒针晶进一步诱发炎症反应的刺激物,凝集素蛋白可显著诱导中性粒细胞迁移,引起大鼠足趾肿胀,可显著加重毒针晶引起的家兔眼结膜水肿,具有强烈的促炎作用。天南星科的掌叶半夏、天南星及白附子,与半夏同属天南星科,刺激性毒性反应相似,毒性物质基础都是块茎中含有的毒针晶,故本论文提出,掌叶半夏、天南星、白附子的毒针晶上也存在与半夏类似的化学刺激物。对天南星科有毒中药凝集素蛋白与毒针晶毒性的相关性研究,除本课题组外,未见有研究报道。本学位论文以同属天南星科、毒性表现相似的有毒中药掌叶半夏、天南星及白附子的毒针晶为研究对象,以半夏为参照,考察半夏、掌叶半夏、天南星及白附子的毒针晶中是否存在同类的与刺激性毒性相关的化学成分,研究表明四种有毒中药的毒针晶中带有共性的凝集素（lectin）蛋白成分。进一步的研究结果表明四种毒针晶中的凝集素蛋白均具有显著促炎作用,毒针晶的刺激性毒性是凝集素蛋白随毒针晶刺入组织后诱导炎症反应产生严重的炎症刺激性毒性。论文基本阐明了天南星科有毒中药半夏、掌叶半夏、天南星及白附子凝集素蛋白与毒性的相关性,主要工作内容和研究结果包括以下几个方面：1天南星科有毒中药毒针晶蛋白类成分分析及共性刺激性相关蛋白研究采用SDS-PAGE （Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis,SDS.PAGE）分析,考察半夏、掌叶半夏、天南星及白附子毒针晶的条带,是否存在分子量相同的蛋白条带,结果显示分子量约12kDa处有相同的蛋白条带。继续将毒针晶中约12kDa处的蛋白条带采用混合蛋白胶内酶解后肽段的液相色谱串联质谱联用（1iquid chromatography tandem mass spectrogram,LC-MS-MS）技术分析,结果显示四种毒针晶约12kDa的条带存在共性的凝集素蛋白,分别为半夏凝集素（Pillellia ternata lectin,PTL）,掌叶半夏凝集素（Pillellia pedatisecta lectin,PPL）,天南星凝集素（Arisaema erubescens lectin,AEL）及白附子凝集素（Typhonium giganteum lectin,TGL）。2天南星科有毒中药凝集素的提取分离纯化及性质研究从半夏、掌叶半夏、天南星及白附子新鲜块茎中分离纯化相应的植物凝集素蛋白PTL、PPL、AEL及TGL。提取分离的流程为：半夏、掌叶半夏、天南星及白附子新鲜块茎粗提液经硫酸铵分级沉淀、苯基琼脂糖凝胶（Phenyl FF）疏水柱层析、Tris-HCl（pH=8.0）缓冲体系,收集主峰通过强阴离子（Q FF）交换柱层析,脱盐,冷冻干燥分别得PTL、PPL、AEL及TGL。高效体积排阻色谱（size-exclusion high performance liquid chromatography, SEC-HPLC）及SDS-PAGE电泳分析显示分离所得的PTL、PPL、AEL及TGL均达到色谱纯及电泳纯,测得其分子量约为12kDa。通过不同来源红细胞、温度、pH及糖或糖蛋白考察凝集素蛋白的凝集活性,结果显示：四种凝集素蛋白均能凝集兔、鼠、狗的红细胞,不能凝集鸡及人的红细胞；80℃以下具有凝集活性,50℃以下凝集活性最强,85℃以上凝集活性全部丧失；pH6.0-10.0范围内具有凝集活性,pH6.0-8.0范围内凝集活性保持稳定,pH达到11.0,凝集活性完全丧失；凝集活性不被D-木糖、D-核糖、D-阿拉伯糖、无水葡萄糖、D-半乳糖、D-甘露糖、L-鼠李糖、甲基-α-D-甘露糖苷所抑制,仅被去唾液酸胎球蛋白所抑制。3天南星科四种有毒中药毒针晶中凝集素蛋白与块茎中凝集素蛋白比对鉴定为进一步确定毒针晶上带有的凝集素蛋白是否与相应植物块茎中的凝集素蛋白为同一蛋白,采用SDS-PAGE分析,研究表明从植物块茎中分离获得的半夏、掌叶半夏、天南星及白附子的凝集素蛋白与其毒针晶蛋白具有相同的分子量。进一步对块茎中分离的凝集素蛋白进行LC-MS-MS分析鉴定,与美国国立生物技术信息中心（National Center for Biotechnology Information,NCBI）数据库进行比对,发现从块茎中提取的凝集素蛋白为对应植物的凝集素蛋白。蛋白免疫印迹Westem blotting结果进一步证实四种毒针晶中含有相应的PTL、PPL、AEL及TGL,即从块茎中提取分离的半夏、掌叶半夏、天南星及白附子凝集素蛋白与相应毒针晶上带有的凝集素蛋白为同一蛋白。4天南星科有毒中药毒针晶中凝集素蛋白的含量研究前面的研究结果表明毒针晶上含有凝集素蛋白,采用Western Blotting方法对毒针晶上的凝集素蛋白含量进行半定量分析,结果半夏、掌叶半夏、天南星及白附子毒针晶中的凝集素蛋白占毒针晶蛋白上的含量PTL为19.89%,PPL为24.84%,AEL为15.08%,TGL为11.67%,四种有毒中药凝集素蛋白含量大小顺序为掌叶半夏>半夏>天南星>白附子,该结果与前期课题组研究的四种有毒中药的毒针晶毒性与其蛋白的含量呈相关性的结果一致,表明四种毒针晶的毒性与毒针晶中带有的凝集素蛋白含量呈正相关。5天南星科四种有毒中药凝集素蛋白的促炎作用研究采用大鼠腹腔中性粒细胞迁移模型研究PTL、PPL、AEL及TGL的促炎作用。结果显示腹腔注射PTL、PPL、AEL及TGL后,四种有毒中药的凝集素蛋白出现明显的中性粒细胞向腹腔迁移的现象并呈剂量依赖性,腹腔液中炎症介质一氧化氮（Nitric Oxide,NO）、前列腺素E2（Prostaglandin E2, PGE2）及肿瘤坏死因子α （Tumor necrosis factor-alpha, TNF-α）的含量与空白组相比较均显著升高,表明PTL、PPL、AEL及TGL均可诱导炎症介质释放。进一步考察加热对凝集素蛋白致大鼠腹腔中性粒细胞迁移的影响,结果显示加热组与空白组比较无显著性差异,表明加热可导致凝集素蛋白失活,使其促炎作用显著下降。6天南星科四种有毒中药凝集素蛋白与毒针晶的刺激性协同作用研究采用家兔眼结膜刺激性模型,家兔破损皮肤刺激性模型和大鼠足趾肿胀模型,考察PTL、PPL、AEL及TGL对毒针晶所致家兔眼刺激性,家兔破损皮肤刺激性及大鼠足趾肿胀的影响以及加热处理对凝集素蛋白及毒针晶诱导炎症反应的影响。结果显示单用毒针晶对家兔眼结膜具有强烈的刺激作用,单用PTL、PPL、AEL及TGL无刺激性,毒针晶中加入一定量的PTL、PPL、AEL及TGL后对家兔眼结膜的刺激性毒性显著增强,与单用毒针晶组相比呈显著性差异,表明PTL、PPL、AEL及TGL需要毒针晶刺入黏膜导致黏膜破损才可诱发炎症反应,即PTL、PPL、AEL及TGL具有协同毒针晶引起强烈的家兔眼结膜刺激性毒性；毒针晶经加热处理后与未处理的毒针晶比较,刺激性明显降低,表明加热可破坏毒针晶蛋白的活性,该结果与凝集素蛋白加热后促炎活性显著降低的结果一致。家兔破损皮肤试验结果表明凝集素蛋白可加重毒针晶对家兔破损皮肤的刺激性,加热处理的凝集素蛋白不增强家兔破损皮肤刺激的作用,呈显著性差异。同时毒针晶加热处理后与未处理的毒针晶比较对家兔破损皮肤的刺激性显著降低。大鼠足趾肿胀试验结果显示单用凝集素蛋白,通过皮下注射进入组织的凝集素蛋白即可引起严重的促炎效应,加热处理后凝集素蛋白组与空白组相比无显著性差异。毒针晶中加入凝集素蛋白可显著增强大鼠足趾肿胀,毒针晶组与毒针晶加入加热处理的凝集素蛋白组比较无显著性差异,表明加热可导致凝集素蛋白失活,使其促炎作用显著下降。同时加热处理的毒针晶组与未处理的毒针晶组比较对大鼠足趾肿胀显著降低,进一步表明毒针晶的刺激性毒性与毒针晶带有的凝集素蛋白密切相关。上述实验结果表明凝集素蛋白只有被带入机体与组织接触才可产生促炎效应,即凝集素蛋白需要随毒针晶刺入组织才可产生促炎效应,而且加热可破坏凝集素蛋白的促炎效应。7天南星凝集素蛋白炎症刺激性与炎症相关细胞的研究前述试验证实天南星科四种有毒中药的凝集素蛋白可诱导大鼠中性粒细胞向腹腔迁移,诱发炎症发应,这种作用与机体的炎症相关细胞有何关系?论文采用动物整体试验,腹腔内居留细胞灌洗模型,巯基乙酸盐（Thioglycolate, Tg）刺激大鼠腹腔内巨噬细胞增殖模型,N-甲基-对甲氧基苯乙胺和甲醛的缩合物（Compound48/80N-methyl-p-methoxyphenethylamine with formaldehyde,48/80）致大鼠腹腔内肥大细胞凋亡模型,考察凝集素AEL诱导大鼠中性粒细胞迁移与居留细胞、巨噬细胞及肥大细胞的关系。结果显示AEL诱导大鼠中性粒细胞迁移不依赖于居留细胞,但居留细胞中的巨噬细胞能协同AEL诱导大鼠中性粒细胞迁移,肥大细胞能拮抗AEL诱导大鼠中性粒细胞迁移。进一步采用体外细胞试验考察四种凝集素蛋白对巨噬细胞的影响,测定炎症介质TNF-α、白细胞介素IL-1β（Interleukin-1β, IL-1β）的含量。体内外试验结果均表明凝集素蛋白的炎症刺激性毒性与游离巨噬细胞并刺激巨噬细胞释放炎症介质有关。论文研究表明,半夏、掌叶半夏、天南星及白附子毒针晶中均带有相应植物的凝集素蛋白,且是毒针晶蛋白中的主要毒蛋白,是天南星科四种有毒中药毒针晶引起炎症反应产生强烈刺激性毒性的共性化学刺激物。毒针晶产生刺激性毒性的机制在于毒针晶刺入组织后,产生机械刺激性,同时毒针晶上的凝集素蛋白随毒针晶进入组织诱导中性粒细胞向刺入部位迁移,促使巨噬细胞活化,并刺激巨噬细胞释放炎症介质TNF-a及IL-1β,导致严重的组织水肿和炎症反应。本论文的研究结果揭示了天南星科四种有毒中药半夏、掌叶半夏、天南星及白附子毒针晶产生强烈炎症刺激性的共性机制,基本阐明了天南星科有毒中药凝集素蛋白与其毒性的相关性,为天南星科有毒中药炮制机理的进一步深入研究提供坚实基础,并将为天南星科有毒中药炮制方法的改进与创新提供理论支持。更多还原

【Abstract】 This work was supported by National Nature Science Fund of China （NO.30973939, No.81173549）.2011Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education Institutions （CXZZ11₀786）.Pinellia ternata （Thunb.） Breit, Arisaema erubescens （Wall.） Schott., Typhonium giganteum Engl included in Chinese Pharmacopoeia （2010）, and Pinellia pedatisecta Schott widely used in china herbal market as a substitute for arisaematis are all belonged to Araceae family and are important toxic Chinese medicines. It has been reported that the four Araceae plants possess similar toxic properties, such as causing mucous membrane and skin irritation, mouth and lingua pain, even respiration slowing and suffocation, which have seriously restricted the development of its clinical applications. Our previous research had proved that the four Araceae plants demonstrated toxicity due to the components of raphide including calcium oxalate, protein and trace carbohydrates. For the raphides could aggratate the inflammation toxicity, the raphides were called "toxic raphides". Moreover, investigations showed that the toxicity of the P. ternate raphide might closely relate to the protein components. Among the protein components, a lectin was proven to be the main pro-inflammatory component. The lectin of raphides from P. ternate could induce neutrophil migration and aggratate the inflammation such as severe conjunctival edema in rabbit and footpad edema in rat. P. ternate, P. pedatisecta, A. erubescens and T. giganteum belong to the same Araceae family and have the similar irritation. However, the raphides irritation mechanism has not been well illustrated.The raphides of P. ternata, P. pedatisecta, A. erubescens and T. giganteum were investigated. Analysis of the chemical components of the raphides showed that proteins in the raphides were associated with the irritation. The lectins of the four plants were proved to be the ringleader of inflammation. The irritating effect of the raphides was due to the dual action of the sharp raphides in puncturing soft skin or mucosa and the lectin in bring inflammation. This paper explained the correlation between lectin from Araceae and toxity. Detail researches and findings include the following aspects:1Analysis and identification of the proteins on the raphides of the four plants SDS-PAGE showed the raphides from the four plants of same molecular weight at about12kDa. The12kDa bands were soaked in a mixed protease enzyme solution by liquid chromatography tandem mass spectrogram. The result showed that the lectin is the same proteins of the12kDa bands. The lectin of the four plants is P. ternata lectin（PTL）, P. pedatisecta lectin（PPL）, A. erubescens lectin（AEL） and T. giganteum lectin（TGL）2Purification and character of the lectin from the tuber of P. pedatisecta, A. erubescens and T. giganteum.PPL, AEL and TGL were respectively purified from tuber of Pinellia pedatisecta, Arisaema erubescens and Typhonium giganteum in the sequence of processing steps. Hydrophobic interaction, ion exchange and desalting chromatographic steps were applied to the purification of PPL, AEL and TGL. A single peak on size-exclusion high performance liquid chromatography （SEC-HPLC） and a single band of about12kDa on SDS-PAGE were observed, suggesting that the purity of isolated PPL, AEL and TGL was fairly good. The haemagglutination activities of PTL, PPL, AEL and TGL were investigated by different erythrocytes, temperatures, pH and sugars. PTL, PPL, AEL and TGL could agglutinate rabbit, mice and dog erythrocytes and could not agglutinate chicken erythrocytes and human red blood cells. PTL, PPL, AEL and TGL are stable up to50℃for20min without any loss of activity and is not markedly affected by pH in the range of6.0～10.0. The haemagglutination activity of PTL, PPL, AEL and TGL is inhibited by asialofetuin only, while monosaccharide is not react.3Identification of PPL, AEL and TGL from the tuber and raphides of P. pedatisecta, A. erubescens and T. giganteum.SDS-PAGE analysis showed that the gel of PPL, AEL and TGL from the tuber and raphides of P. pedatisecta, A. erubescens and T. giganteum have the same bands. LC-MS-MS identification showed that12kDa protein bands from PPL, AEL and TGL from the tuber of P. pedatisecta, A. erubescens and T. giganteum was corresponding lectin protein respectively. The result of western blotting also showed that PPL, AEL and TGL were on the raphides of P. pedatisecta, A. erubescens and T. giganteum. All these results demonstrated that the purified PPL, AEL and TGL from the tuber and raphides of P. pedatisecta, A. erubescens and T. giganteum are the same protein respectively.4The content of PTL, PPL, AEL, TGL from the raphide protein It is important that the content of the lectins which are the stimulation of raphide and are decreased by heating posses to the raphide protein. Western Blotting analysis showed that the content of PTL, PPL, AEL and TGL from the raphide protein is19.89%,24.84%,15.08%and11.67%respectively. The results indicated that lectin is the main protein of the raphide protein.5Pro-inflammatory effect of PTL, PPL, AEL and TGLThe in vivo model of neutrophil migration was used to study the inflammatory activity of lectin. PTL, PPL, AEL and TGL （100,200and300μg/mL/cavity） could induce significant and dose-dependent neutrophil migration in the rat peritoneal cavities. Besides, PTL, PPL, AEL and TGL at doses ranging from100to300μg/mL/cavity could significantly increase the concentration of nitric oxide （NO）, prostaglandin E2（PGE2） and tumor necrosis factor alpha （TNF-a） in peritoneal fluid. Compared with control animals, heated PTL, PPL, AEL and TGL did not reduce neutrophil migration. This result indicated that the irritation effects of lectins were decreased by heated.6Synergistic effect of inflammation stimulated by PTL, PPL, AEL and TGL and raphides of P. ternate, P. pedatisecta, A. erubescens and T. giganteum.The models of Draize rabbit eye test, rabbit’s damaged skin test and rat paw edema test were used to study the role of the PTL, PPL, AEL and TGL and raphides of P. ternate, P. pedatisecta, A. erubescens and T. giganteum on inflammatory response. The results showed that the pro-inflammtation effect of PTL, PPL, AEL and TGL could work only when the raphides pierce into the organization, and PTL, PPL, AEL and TGL enhanced the inflammation induced by raphides, and heating could decrease the lectin of the pro-inflammation effect.7Pro-inflammatory effect of PTL, PPL, AEL, TGL and relative study of resident cellsThe role of resident cells on the PTL, PPL, AEL and TGL effect was evaluated using three strategies:（a） reducing the total resident cell population by lavage of rat cavities with saline;（b） increasing macrophage population by treating rats with Tg.;（c） depleting mast cell population by subchronic treatment of rats with compound48/80. This results indicate that PTL, PPL, AEL, TGL-induced neutrophil migration is independent on resident cells. However, macrophages could synergize PTL, PPL, AEL, TGL-induced neutrophil migration, while mast cells could antagonize the process. The pro-inflammation effect of lectin with macrophages was determinating TNF-a, Interleukin-1β （IL-1β） in vitro. These results indicated that PTL, PPL, AEL and TGL-induced neutrophil migration was dependent on macrophages which release TNF-α, IL-1β in vivo and in vitro.Through the researches above, we concluded that the irritation induced by raphides is due to the dual action of the sharp raphides in puncturing and the PTL, PPL, AEL, and TGL in causing inflammation. The pro-inflammation effect of lectin was related to the macrophage which release TNF-α, IL-1β.These findings illustrated the reason why raphides could induce severe inflammation. The researches above could provide the way of studies on the toxic components and detoxification processing skills and mechanism of other poisonous medicines from Araceae.更多还原