【作者】 史闰均；

【导师】 吴皓；

【作者基本信息】 南京中医药大学 ， 中药学， 2011， 硕士

【摘要】 本学位论文得到国家自然基金项目——半夏刺激性毒性的物质基础及炮制解毒机理研究(NO.30772785)、国家自然基金项目——天南星科有毒中药矾制解毒共性规律研究(NO.30973939)、国家“十一五”科技部支撑计划课题——复制法炮制解毒共性技术和相关设备研究(NO.2006BAI09806-10)的资助。半夏为天南星科植物半夏Pinellia ternata(Thunb.)Breit的块茎。自古以来,因其味辛辣、麻舌而刺喉,具有“戟人咽”的刺激性,被列为有毒中药。临床误服或使用不当,可致强烈的皮肤、粘膜接触性的刺激性毒性。本课题组前期研究表明半夏刺激性毒性成分为其含有的具有特殊晶型的“毒针晶”。半夏生品及毒针晶均可引起家兔眼结膜强烈水肿,使小鼠毛细管通透性增强,腹腔渗出液中炎症介质PGE2含量增加,其毒性表现与炎症效应极为相似。课题组进一步研究发现半夏毒针晶是主要由草酸钙、蛋白组成的复合物,其中蛋白含量为6.084%；毒针晶上带有凝集素蛋白,该蛋白能显著引起大鼠腹腔中性粒细胞向腹腔迁移和大鼠足趾肿胀。毒针晶中加入凝集素后,可显著加重炎症反应。这些结果表明半夏的刺激性毒性作用是由毒针晶的机械损伤加上凝集素蛋白的化学损伤双重作用产生。中医临床上半夏中毒用生姜解毒。传统中医理论认为：“半夏性畏生姜,用之以制其毒,功益彰”。梁·陶弘景《本草经集注》中记载“半夏毒,用生姜汁,煮干姜汁并解之”。但生姜解半夏毒的机理不明。现代研究发现生姜具有抗炎镇痛、调节免疫等药理活性。而半夏毒针晶可引起强烈的炎症反应。故本学位论文提出假说“半夏的刺激性毒性在机体可表现为刺激性炎症毒性,生姜对半夏的解毒作用与其拮抗半夏所致的刺激性炎症毒性相关”。本学位论文据此设计了研究路线。本学位论文采用多种炎症模型,以半夏毒针晶为致炎剂,发现半夏毒针晶可引起强烈的炎症毒性反应。研究表明生姜汁确实能显著拮抗半夏毒针晶所致的炎性作用,并筛选得出姜辣素类成分是生姜拮抗半夏毒针晶所致炎性反应的有效部位,其拮抗半夏所致炎症反应的机制不是通过兴奋肾上腺分泌肾上腺皮质激素而发挥作用,可能与抑制半夏毒针晶诱导的炎症介质的合成与释放有关。同时发现姜辣素类成分可拮抗半夏毒针晶中的主要化学刺激物(凝集素蛋白)的致炎效应。主要工作内容和研究结果包括以下几方面：1半夏毒针晶的致炎作用研究采用小鼠毛细管通透性试验、小鼠腹腔炎症试验及大鼠足趾肿胀试验,考察不同浓度半夏毒针晶所致的炎症毒性效应及其量-毒关系。研究结果表明：腹腔注射半夏毒针晶混悬液可使小鼠腹腔毛细血管通透性增加,腹腔渗出液中炎症介质PGE2含量增加,也可引起大鼠足趾肿胀,并在一定剂量范围内表现为典型的量-毒关系。此结果说明半夏毒针晶可诱导炎症,毒针晶的刺激性毒性在机体内主要表现为炎症反应。2生姜汁对半夏毒针晶所致炎性反应的影响采用半夏毒针晶所致小鼠腹腔炎症模型,观察生姜汁对小鼠毛细管通透性、腹腔渗出液中蛋白质含量、白细胞数量的影响；采用半夏毒针晶诱导大鼠足趾肿胀模型,观察生姜汁对半夏毒针晶所致大鼠足趾肿胀的影响。研究结果表明：生姜汁能显著抑制半夏毒针晶腹腔注射所致的小鼠毛细管通透性增加,减少腹腔渗出和白细胞数目,降低渗出液中蛋白质的含量,亦可减轻半夏毒针晶所致的大鼠足趾肿胀,说明生姜汁确实可拮抗半夏毒针晶所致的炎症反应,且其中高剂量组的拮抗效应与阳性药阿司匹林组相当。3生姜各部位的提取及相关成分含量测定生姜挥发油部位采用水蒸气蒸馏的方法提取,并采用气质联用法对其组分进行分析；生姜姜辣素部位为生姜经乙酸乙酯提取后去除挥发油后精制所得,并采用紫外分光光度法对姜辣素部位中总姜辣素含量进行测定,采用HPLC法对姜辣素部位中6-姜酚(6-gingerol)、6-姜烯酚(6-shogaol)进行含量测定；生姜乙酸乙酯提取后的药渣经水提取得到药渣水提物部位。结果获得挥发油、姜辣素、药渣水提物三个部位；生姜挥发油中共鉴定了64个化合物,主要为萜类物质；生姜姜辣素部位中总姜辣素含量为88.15%,6-姜酚和6-姜烯酚的含量分别为19.40%、8.11%。此结果说明姜辣素部位所含的总姜辣素含量较高,且生姜其他部位的提取方法亦可行,可作为生姜拮抗半夏毒针晶所致炎症的筛选部位。4生姜各部位对半夏毒针晶所致炎性反应的影响为筛选生姜中拮抗半夏毒针晶炎性反应的有效部位,采用腹腔注射半夏毒针晶所致小鼠腹腔炎症模型,观察生姜各提取部位对小鼠毛细管通透性、腹腔渗出液中蛋白质含量、白细胞数量的影响；采用半夏毒针晶诱导大鼠足趾肿胀模型,观察生姜各提取部位对大鼠足趾肿胀的作用。研究结果表明：姜辣素部位能显著抑制半夏毒针晶腹腔注射所致的小鼠毛细管通透性增加,降低蛋白质含量和白细胞数目,也可减轻半夏毒针晶所致的大鼠足趾肿胀,而挥发油及药渣水提物未见显著性影响。此结果表明生姜中的姜辣素类成分是拮抗半夏毒针晶所致炎症反应的有效部位。5姜辣素类成分拮抗半夏毒针晶所致炎性反应机制的初步研究为探讨姜辣素类成分的拮抗效应是否依赖于下丘脑-垂体-肾上腺皮质系统,采用半夏毒针晶诱导的正常大鼠和去肾上腺大鼠足趾肿胀试验,观察姜辣素类成分对正常大鼠和去肾上腺大鼠足趾肿胀的影响。对正常大鼠两侧肾上腺中胆固醇与VitC含量进行测定,观察姜辣素类成分对肾上腺中胆固醇与VitC含量的影响。同时采用半夏毒针晶诱导的小鼠腹腔炎症模型,观察姜辣素类成分对小鼠腹腔渗出液中PGE2、NO、MDA的影响;采用半夏毒针晶诱导的正常大鼠足趾肿胀模型,观察姜辣素类成分对大鼠炎足中COX-2、PGE2、TNF-α的影响。研究结果表明：姜辣素类成分可显著抑制半夏毒针晶所致的正常大鼠足趾肿胀和去肾上腺大鼠足趾肿胀,且对正常大鼠肾上腺中胆固醇和VitC的合成释放无显著影响,表明姜辣素类成分拮抗半夏毒针晶所致炎性反应不是通过兴奋肾上腺分泌肾上腺皮质激素而发挥作用的。同时,姜辣素类成分可抑制半夏毒针晶所致的小鼠腹腔渗出液中PGE2、NO、MDA的生成,还能显著抑制半夏毒针晶所致大鼠炎足中COX-2、PGE2、TNF-α的生成,提示可能与抑制COX-2、PGE2、TNF-α、NO、MDA的生成有关。6姜辣素类成分对半夏凝集素所致炎性反应的影响课题组前期研究表明凝集素蛋白是半夏毒针晶中的主要蛋白,可以引起大鼠腹腔白细胞迁移、渗出液中PGE2含量增加和大鼠足趾肿胀,是半夏毒针晶中引起炎症刺激的主要化学物质。故将提取所得的凝集素作为致炎剂,采用小鼠腹腔炎症模型和大鼠足趾肿胀模型,考察姜辣素类成分对半夏凝集素所致炎症反应的影响。结果显示半夏凝集素可显著引起小鼠腹腔渗出液中PGE2含量增高及大鼠足趾肿胀,并可加重毒针晶的致炎作用；姜辣素类成分可显著抑制半夏凝集素所致的炎症反应,亦可拮抗半夏凝集素与毒针晶协同所致的炎症反应。通过上述研究表明,半夏毒针晶可引起机体强烈的刺激性炎症毒性,而生姜汁及生姜中的姜辣素类成分能有效拮抗半夏毒针晶所致的炎性反应,并初步阐明姜辣素类成分拮抗半夏毒针晶炎性反应不依赖于肾上腺分泌肾上腺皮质激素系统,其机制可能与抑制毒针晶诱导的炎症因子有关。同时研究进一步证明半夏凝集素是半夏毒针晶中的主要化学刺激物,姜辣素类成分可拮抗半夏凝集素所致的炎性反应。说明生姜确实可以起到解半夏毒的作用,其作用机制是生姜中的姜辣素类成分可直接拮抗半夏毒针晶刺激后产生的炎症反应,减轻毒针晶上的凝集素蛋白所产生的炎症反应。本学位论文对“生姜解半夏毒”的传统中医理论进行了探索性研究,结果揭示了何为半夏毒,生姜中解半夏毒的成分,并初步揭示了生姜的解毒机制,证实此传统中医理论的科学性,为临床半夏中毒的解毒提供理论基础。本学位论文的研究可为中医理论的现代科学内涵研究提供了较好的借鉴作用。更多还原

【Abstract】 This project was supported by national natural science foundation (No.30772785, NO. 30973939), "Eleven-Five" national projects of technological supporting (No.2006BAI09B06-10).Pinellia ternata (Thunb.) Breit. whose tuberous stems are important Chinese medicines, is taxonomically belonged to Araceae family, and are well known for their irritant properties. If chewed and touched raw, the corms of the plants could result in swelling and burning pain in the mucous membranes, and an unbearable itching in skin. The needle-like raphides of calcium oxalate were proved to be their common poisonous components, named as toxic raphides. The raphides caused severe conjunctival edema in rabbit, increased capillary permeability and content of PGE2 in the exudates in mice. Toxic raphide were a complex compsed by mainly calcium oxalate crystals and protein. The content of protein was 6.084%. The study found that the agglutinin on the toxic raphide can significantly induce rat peritoneal neutrophil migration to peritoneal and rat paw edema. These results indicated that the reason of the toxic effects of raphides were the dual role of mechanical damage by calcium oxalate and chemical inflammation induced by agglutinin.Ginger was used to detoxificate to P. ternate in clinic. Traditional Chinese medicine theory held that "P. ternata feared ginger, and cooked with ginger for detoxification". Tao Hongjing held that "P. ternata was toxic and was detoxificated by ginger juice and dry ginger" However, the mechanism of ginger detoxification to P. ternata was unknown. Modern research showed that ginger had anti-inflammatory, analgesic, immune and other pharmacological activity. This paper was put forward the hypothesis "the irritating toxic of P. ternata in the body can be expressed as an inflammation. Ginger detoxification of P. ternata was related to the antagonistic effect of inflammation caused by P. ternata". This paper was designed according to this line of the research hypothesis.Inflammation models were used to reseach the raphides. The result found that raphides can cause irritant toxicity, and ginger juice can really antagonize to inflammation induced by the raphides. Gingerols were an effective part of ginger which did not depend on the anti-inflammatory mechanisms of the release of adrenal cortex hormone by activating adrenocorticotropic hormone, and its anti-inflammatory mechanism may be an inhibition of inflammatory mediators induced by the raphides in P. ternate. The result also found that gingerols can inhibit the inflammatory effects caused by P. ternate agglutinin. Main contents and findings were the following:1. The inflammatory effect induced by raphides in P. ternateThe mouse capillary permeability test, mouse peritonitis and toe swelling of rat induced by the raphides were applied to investigate the inflammatory effects of different concentrations of raphides. The result showed that raphides can increase the capillary permeability, caused the release of inflammatory mediator (PGE2) and toe swelling of rat.2 Anti-inflammatory effect of ginger juice on inflammation induced by raphides in P. ternateThe mouse peritonitis model induced by the raphides was applied to investigate the anti-inflammatory effects of ginger juice by detecting the capillary permeability, quantity of leukocytes, and the protein content in the exudates. Toe swelling of rat induced by the raphides was used to determine the swelling extent. The result showed that ginger juice exhibited comparable anti-inflammatory effects in terms of the inhibitions on the exudation and leukocytes efflux, and the production of protein. Ginger juice inhibited penetration of capillary vessel in mouse and toe swelling of rat induced by the raphides.3 Three extracts of ginger and component analysisGinger volatile oils, extracted by steam distillation, were analyzed by GC/MS. Gingerols were obtained from ginger which was extracted by ethyl acetate and was removed of volatile oils. The content of total gingerols was determined by UV spectrophotometry. The content of 6-gingeroland 6-shogaol was determined by HPLC. After extracted by the ethyl acetate, ginger dregs were extracted again by water, called as aqueous extract. The result showed that ginger volatile oil, gingerols, and aqueous extract of ginger were obtained. GC/MS analysis showed that there were 64 compounds in volatile oil which were identified mainly terpenoids. The content of total gingerols,6-gingerol, and 6-shogaol were 88.15%,19.40%and 8.11%, respectively. The results were ready to screen the active part from three extracts further.4 Anti-inflammatory effect of three parts of ginger extract on inflammation induced by raphides in P. ternate The mouse peritonitis model induced by the raphides induced was applied to investigate the anti-inflammatory effects of three ginger extracts by detecting the capillary permeability, quantity of leukocytes, and the protein content in the exudates. Toe swelling of rat induced by the raphides was used to determine the swelling extent. The result showed that gingerols exhibited comparable anti-inflammatory effects in terms of the inhibitions on the exudation and leukocytes efflux, and the production of protein. Gingerols inhibited penetration of capillary vessel in mouse and toe swelling of rat induced by the raphides. The volatile oils and aqueous extract did not have the anti-inflammatory effects.5 Anti-inflammatory mechanisms of gingerols related to inhibiting inflammatory by raphides in P. ternateAccording to the results of previous studies, toe swelling induced by the raphides of normal rat and toe swelling induced by the raphides of bilaterally adrenalectomized rats were used to determine the swelling extent. The contents of VitC and cholesterol were measured in normal adrenal glands. The result showed that gingerols had a significantly suppressive effect on paw edema of normal rat and adrenalectomized rat induced by raphides and had no effect on the contents of VitC and cholesterol in normal adrenal glands. The result showed gingerols had an anti-inflammatory effect, which were independent on pituitary-adrenal axis.The content of PGE2, NO and MDA in the mouse exudate were respectively measured in rats with acute peritonitis induced by raphides. The activity of COX-2 and the content such as PGE2, TNF-a were examined in the toe of rats induced by raphides. The result showed that gingerols significantly inhibited the production of NO, PGE2, and MDA in exudate of peritonitis rats. Furthermore, gingerols inhibited the activity of COX-2, and reduced the production of PGE2 and TNF-a in the toe. The anti-inflammatory mechanisms of gingerols may be related to inhibiting the production of inflammatory mediators and free radicals.6 Anti-inflammatory effect of gingerols on inflammation induced by P. ternate agglutininThe mouse peritonitis model induced by the agglutinin was applied to investigate the anti-inflammatory effects of gingerols by detecting the releases of inflammatory mediators PGE2 in the exudates. Toe swelling of rat induced by the agglutinin was used to determine the swelling extent. The result showed that gingerols exhibited comparable anti-inflammatory effects in terms of the inhibitions on the releases of inflammatory mediator(PGE2) and the swelling extent of rat The research showed that raphides of P. ternate can cause intense inflammatory toxicity. Ginger juice and gingerols effectively antagonized to inflammatory induced by raphides of P. ternate. The anti-inflammatory mechanism of gingerols is not related to the release of adrenal cortex hormone by activating adrenocorticotropic hormone, and it may be an inhibition of COX-2, PGE2, TNF-a, NO, and MDA. The result also found that gingerols can inhibit the inflammatory effects induced by agglutinin. This paper clarified the scientific content on the theory of Chinese medicine "P. ternate can be detoxification by Ginger". It provided a basis to the clinical application of ginger detoxification to P. ternate.更多还原