天南星科有毒中药刺激性毒性成分及矾制解毒共性机制研究
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摘要
本课题来源于“十一五”国家科技支撑计划项目:“复制法矾制解毒共性技术和相关设备研究“(编号:2006BAI09B06-10);国家自然科学基金项目:半夏刺激性毒性的物质基础和炮制解毒机理研究“(编号:30772785)。
2005年版中国药典收载的半夏、天南星和禹白附均属于天南星科有毒中药,另外,天南星科的有毒中药虎掌南星在市场上作为天南星代用品广泛使用。天南星科的这些中药生品具有强烈的刺激性毒性,主要表现为对黏膜的刺激性,可致口唇肿痛、失音、流涎、痉挛、呼吸困难,甚至窒息死亡。因此,中医临床上均使用其炮制品,在炮制工艺中共同的炮制解毒辅料是白矾。根据天南星科有毒中药均采用白矾进行炮制解毒的现象,本课题提出,天南星科的有毒中药可能含有类似的毒性成分;白矾炮制解毒的机理可能存在共性的规律的假说。本文在本课题组前期对半夏的刺激性毒性成分和炮制减毒机理研究的基础上,针对天南星科四种有毒中药半夏、虎掌南星、东北天南星和禹白附是否具有共性刺激性毒性成分及矾制解毒作用是否具有共性机制进行系统深入的研究。
在天南星科四种有毒中药刺激性毒性成分及矾制解毒机制研究工作中,主要工作内容和研究结果包括以下几方面:
1天南星科四种有毒中药具有共性的刺激性毒性成分研究
对天南星科有毒中药半夏、虎掌南星、东北天南星、禹白附的刺激性毒性成分,采用不同极性的溶剂进行提取分离,分别得到四种有毒中药不同极性溶剂提取物;同时采用低速逐级离心技术从四种有毒中药中分离出针晶,以草酸钙化学品为对照,以家兔眼结膜刺激性模型进行筛选,研究表明四种有毒中药中具有特殊晶型的针晶为共性的刺激性毒性成分,命名为“毒针晶”。
2天南星科四种有毒中药刺激性毒性的强度比较及毒针晶量-毒关系研究
在明确天南星科四种有毒中药刺激性毒性成分的基础上,以小鼠急性毒性实验比较生品和毒针晶毒性的强弱,以四种有毒中药生品粉末进行家兔眼结膜刺激性毒性实验,研究表明,四种有毒中药毒性的强弱顺序为:虎掌南星＞半夏＞东北天南星＞禹白附;小鼠腹腔注射四种有毒中药的粉末和毒针晶考察毒针晶对肠系膜粘膜的刺激性作用方式,研究发现毒针晶可直接刺入小鼠肠系膜组织细胞内部。进一步对四种有毒中药毒针晶刺激性毒性强度和浓度的量-毒关系进行研究,结果表明各有毒中药所提取的毒针晶其刺激性毒性均随浓度的增加而增强,在一定的浓度范围内,呈现出线性的量-毒关系,进一步证实毒针晶为该四种有毒中药的共性毒性成分。
3天南星科四种有毒中药毒针晶的研究
天南星科四种有毒中药生品中毒针晶的含量测定结果表明,各中药中毒针晶含量高低依次为:虎掌南星＞半夏＞东北天南星＞禹白附,该结果与四种有毒中药刺激性毒性强度顺序一致,进一步证实毒针晶是天南星科有毒中药共性的刺激性毒性成分。对四种有毒中药的毒针晶采用光学显微、电镜扫描、X-衍射、光电能谱、红外光谱、组成鉴定及含量分析、凝胶电泳实验进行研究。研究结果表明,四种毒针晶的结构晶型和组成成分具有共同的特点。在晶型上,扫描电镜观察毒针晶的细微结构显示,四种中药的毒针晶:均具有针尖样的末端,尖锐锋利,晶体表面具凹槽、布满倒刺的特殊结构;采用X-衍射、光电能谱、红外光谱对四种毒针晶进行组成分析,结果表明针晶中主要含有草酸钙以及-NH_2和-COOH、-S-基团;定性鉴别和组成分析结果表明四种毒针晶均由草酸钙和蛋白及微量的糖类成分组成;以SDS-凝胶电泳分离四种毒针晶蛋白结果表明,四种毒针晶中的主要蛋白条带基本一致,均含有分子量约26KD和51KD的蛋白;四种有毒中药毒针晶中分离获得的针晶蛋白均具有强烈的刺激性和致敏性。测定四种毒针晶中蛋白的含量结果表明;毒针晶蛋白的含量顺序为:虎掌南星＞半夏＞东北天南星＞禹白附,该顺序也与四种中药的毒性强度顺序一致,说明毒针晶毒性的产生不仅与针晶的晶型有关,也与针晶的蛋白有关。
4天南星科四种有毒中药矾制解毒共性机制研究
在确定四种有毒中药刺激性毒性成分的基础上,以毒性成分的含量变化为指标,以针晶中的成分与矾水体系中离子的相互作用和反应过程进行理论分析和实验,对天南星科有毒中药矾制解毒共性机理进行了研究。研究表明天南星科有毒中药矾制解毒机理主要是:白矾溶液中的Al~(3+)能够结合毒针晶中的C_2O_4~(2-),使得毒针晶草酸钙分解破坏,同时白矾溶液呈酸性,在酸性水溶液中毒针晶蛋白溶解或水解,从而彻底破坏了毒针晶的结构和晶型和成分组成,导致刺激性毒性下降或解除。
研究结果表明:天南星科四种常用的有毒中药的刺激性毒性成分同属一类,同为由草酸钙、蛋白为主要成分复合而成具有特殊晶型结构的毒蛋白草酸钙针晶,毒性的产生主要由于毒针晶特殊晶型的机械刺激和针晶蛋白的致敏化学刺激共同作用导致。四种有毒中药的毒性的大小与毒针晶含量高低和毒针晶中蛋白含量高低呈现出一致的规律,以半夏属的虎掌南星为最强,依次为半夏、东北天南星、禹白附。
以白矾溶液炮制四种有毒中药共性的解毒机制是由于矾水体系中的Al~(3+)与毒针晶中草酸钙分子中的草酸根离子结合促使草酸钙分解;针晶的刚性支撑被破坏,同时酸性的矾水体系又使得毒针晶中蛋白溶解或水解,整个晶体被破坏,毒性消失。
综上所述,天南星科有毒中药半夏、虎掌南星、东北天南星及禹白附具有共性刺激性毒性成分,在矾制解毒的机理上也具有共性。
This project was supported "Eleven-Five" national projects of technological supporting (No.2006BAI09B06-10) and national natural science foundation(No.30772785).
Pinellia ternate(Thunb.)Breit.,Arisaema amurense Maxim.and Typhonium giganteum Engl., included in Chinese Pharmacopoeia(2005),are all belonged to Araceae family.And Pinellia pedatisecta Schott is widely used in china herbal market as a substitute for arisaematis.The property and flavor of the four Araceae plants is all documented to be 'acrid,warm and toxic'. That means these crude drugs have intensive acridity and toxicity,causing swelling of the lips, mouth and throat,aphonia,salivation,convulsion,convulsion,and even death from suffocation. So clinical medication is that they are all processed with Potash Alum through duplicating method.Whether the four plants have the same acridity component? Whether the same detoxification processing mechanism present in the common processing method? No report.At the base of our group work on the acridity factor and the processing detoxification mechanism of Pinellia ternate(Thunb.)Breit.,in this paper,we attempt to prove the conjecture.
The work as follows:
1 Common poisonous component in the four types of Araceae herbal medicine.
Using different polarity solvent extraction and separation,different polar solvent extracts were obtained respectively from the four plants.At the same time,the seperation of the calcium oxalate raphides was achieved by centrifugalization.The common poisonous component of the four plants(Araceae) were identified through toxicity screening,rabbits' conjunctival irritation experiment as the animal model.The needle-like raphides of calcium oxalate were proved to be their common poisonous components,which were named as 'poisonous raphides'.
2 The comparison of the irritant and toxic intensity of the four crude drugs and the dose-toxic response relationships of the poisonous raphides.
The irritant and toxic intensity of the four crude drugs(Araceae) and the raphides inside of them was detected through mice acute toxicity experiment and the rabbits' eye irritation experiments,and the result showed the level as follows:Pinellia pedatisecta＞Pinellia ternate＞ Arisaema amurense＞Typhonium giganteum.The action mode of acrid raphides on mucous membranes was investigated through mice mesentery experiment,after intraperitoneal injection with the power pf the four crude drugs.The rusults showed that the poisonous raphids punctured into the cell of the mesenteric tissue directly.Futhur study about the toxic and irritant intensity and the dose-toxic response relationships of poisonous raphides indicated that toxicity enhanced with the increase in concentration of poisonous raphides.The toxicity presented linear doseresponse relationships during a range in concentration.The result further indicated that the raphides were their common poisonous factor.
3 Analysis of poisonous raphides in the four types of Araceae herbal medicine.
Through determination of raphides content in the four crude drugs,the content level showed as follows:Pinellia pedatisecta＞Pinellia ternate＞Arisaema amurense＞Typhonium giganteum. The raphides content level was in line with level of the toxic intensity of the four crude drugs. This consistency further indicated that the poisonous raphides were their common poisonous components.Their morphology was studied by light microscope and Scanning electron microscopy(SEM).The raphides of the four plants all have two types of raphides(long and short).SEM of the raphids showed pointed and blunt ends of a raphide,and a long groove and barbs on it.The result of X-ray diffraction,photoelectric spectra and infrared spectra showed the raphides consist of calcium oxalate monohydrate and-NH,-COOH and -S- functional groups. Results of identification and composition analysis showed that the four plants poisonous raphides all consist of calcium oxalate,proteins and small amount of Polysaccharide (Glycoprotein).SDS-gel electrophoresis separation of raphides proteins results indicated that the major protein bands are basically the same,showing the same bands of 26KD and 51KD The proteins isolated from raphides in the four plants had strong acridity,and allergenicity.Through determination,the content level of the protein in the raphides is as follows:Pinellia pedatisecta＞Pinellia ternate＞Arisaema amurense＞Typhonium giganteum.The content level was also in line with the level of intensity of the toxicity,which indicated that the toxicity derived from not only the blunt structure of the raphides but also from protein.
4 Common mechanism of detoxification processing of the four types of Araceae herbal medicine.
The common mechanism detoxification processing with Potash Alum was studied by analyzing the constituents of the raphides in alaum water solution system.Al~(3+) in alaum water solution have the combination ability with C_2O_4~(2-) which comes from calcium oxalate raphides, which made the raphides decompose.At the same time,the alaum water solution is acidic,and the raphides proteins were hydrolyzed,which completely destroyed the raphides structure and crystal form.Irritation and toxicity,decreased.
The results as above showed that:the toxic components of the four types of herbal medicine (Araceae) belong to one class of toxic components.That is piosonous raphides,having a special crystal structure,needle-like crystal,with calcium oxalate,proteins as the main conponents.The toxicity mainly due to the dual effects of mechanical stimulation of special crystal form and chemical stimulation of protein allergens.The level of toxic and irritant intensity was in line with the level of the raphides concentration of the four crude drugs:Pinellia pedatisecta＞Pinellia ternate＞Arisaema amurense＞Typhonium giganteum.The common mechanism of detoxification processing with Potash Alum was that Al~(3+) in alaum water solution combined with C_2O_4~(2-),which cause the decomposition of calcium oxalate,and the rigid support of crystal is damaged,while the alaum acidic water system also made proteins hydrolyzed,and the entire crystal structure was damaged.The toxicity disappeared
To sum up,the piosonous raphides were the common poisonous factor in Pinellia ternate(Thunb.)Breit,Pinellia pedatisecta Schott,Arisaema amurense Maxim and Typhonium giganteum Engl.The common detoxification mechanism present in the processing methods.
2005年版中国药典收载的半夏、天南星和禹白附均属于天南星科有毒中药,另外,天南星科的有毒中药虎掌南星在市场上作为天南星代用品广泛使用。天南星科的这些中药生品具有强烈的刺激性毒性,主要表现为对黏膜的刺激性,可致口唇肿痛、失音、流涎、痉挛、呼吸困难,甚至窒息死亡。因此,中医临床上均使用其炮制品,在炮制工艺中共同的炮制解毒辅料是白矾。根据天南星科有毒中药均采用白矾进行炮制解毒的现象,本课题提出,天南星科的有毒中药可能含有类似的毒性成分;白矾炮制解毒的机理可能存在共性的规律的假说。本文在本课题组前期对半夏的刺激性毒性成分和炮制减毒机理研究的基础上,针对天南星科四种有毒中药半夏、虎掌南星、东北天南星和禹白附是否具有共性刺激性毒性成分及矾制解毒作用是否具有共性机制进行系统深入的研究。
在天南星科四种有毒中药刺激性毒性成分及矾制解毒机制研究工作中,主要工作内容和研究结果包括以下几方面:
1天南星科四种有毒中药具有共性的刺激性毒性成分研究
对天南星科有毒中药半夏、虎掌南星、东北天南星、禹白附的刺激性毒性成分,采用不同极性的溶剂进行提取分离,分别得到四种有毒中药不同极性溶剂提取物;同时采用低速逐级离心技术从四种有毒中药中分离出针晶,以草酸钙化学品为对照,以家兔眼结膜刺激性模型进行筛选,研究表明四种有毒中药中具有特殊晶型的针晶为共性的刺激性毒性成分,命名为“毒针晶”。
2天南星科四种有毒中药刺激性毒性的强度比较及毒针晶量-毒关系研究
在明确天南星科四种有毒中药刺激性毒性成分的基础上,以小鼠急性毒性实验比较生品和毒针晶毒性的强弱,以四种有毒中药生品粉末进行家兔眼结膜刺激性毒性实验,研究表明,四种有毒中药毒性的强弱顺序为:虎掌南星＞半夏＞东北天南星＞禹白附;小鼠腹腔注射四种有毒中药的粉末和毒针晶考察毒针晶对肠系膜粘膜的刺激性作用方式,研究发现毒针晶可直接刺入小鼠肠系膜组织细胞内部。进一步对四种有毒中药毒针晶刺激性毒性强度和浓度的量-毒关系进行研究,结果表明各有毒中药所提取的毒针晶其刺激性毒性均随浓度的增加而增强,在一定的浓度范围内,呈现出线性的量-毒关系,进一步证实毒针晶为该四种有毒中药的共性毒性成分。
3天南星科四种有毒中药毒针晶的研究
天南星科四种有毒中药生品中毒针晶的含量测定结果表明,各中药中毒针晶含量高低依次为:虎掌南星＞半夏＞东北天南星＞禹白附,该结果与四种有毒中药刺激性毒性强度顺序一致,进一步证实毒针晶是天南星科有毒中药共性的刺激性毒性成分。对四种有毒中药的毒针晶采用光学显微、电镜扫描、X-衍射、光电能谱、红外光谱、组成鉴定及含量分析、凝胶电泳实验进行研究。研究结果表明,四种毒针晶的结构晶型和组成成分具有共同的特点。在晶型上,扫描电镜观察毒针晶的细微结构显示,四种中药的毒针晶:均具有针尖样的末端,尖锐锋利,晶体表面具凹槽、布满倒刺的特殊结构;采用X-衍射、光电能谱、红外光谱对四种毒针晶进行组成分析,结果表明针晶中主要含有草酸钙以及-NH_2和-COOH、-S-基团;定性鉴别和组成分析结果表明四种毒针晶均由草酸钙和蛋白及微量的糖类成分组成;以SDS-凝胶电泳分离四种毒针晶蛋白结果表明,四种毒针晶中的主要蛋白条带基本一致,均含有分子量约26KD和51KD的蛋白;四种有毒中药毒针晶中分离获得的针晶蛋白均具有强烈的刺激性和致敏性。测定四种毒针晶中蛋白的含量结果表明;毒针晶蛋白的含量顺序为:虎掌南星＞半夏＞东北天南星＞禹白附,该顺序也与四种中药的毒性强度顺序一致,说明毒针晶毒性的产生不仅与针晶的晶型有关,也与针晶的蛋白有关。
4天南星科四种有毒中药矾制解毒共性机制研究
在确定四种有毒中药刺激性毒性成分的基础上,以毒性成分的含量变化为指标,以针晶中的成分与矾水体系中离子的相互作用和反应过程进行理论分析和实验,对天南星科有毒中药矾制解毒共性机理进行了研究。研究表明天南星科有毒中药矾制解毒机理主要是:白矾溶液中的Al~(3+)能够结合毒针晶中的C_2O_4~(2-),使得毒针晶草酸钙分解破坏,同时白矾溶液呈酸性,在酸性水溶液中毒针晶蛋白溶解或水解,从而彻底破坏了毒针晶的结构和晶型和成分组成,导致刺激性毒性下降或解除。
研究结果表明:天南星科四种常用的有毒中药的刺激性毒性成分同属一类,同为由草酸钙、蛋白为主要成分复合而成具有特殊晶型结构的毒蛋白草酸钙针晶,毒性的产生主要由于毒针晶特殊晶型的机械刺激和针晶蛋白的致敏化学刺激共同作用导致。四种有毒中药的毒性的大小与毒针晶含量高低和毒针晶中蛋白含量高低呈现出一致的规律,以半夏属的虎掌南星为最强,依次为半夏、东北天南星、禹白附。
以白矾溶液炮制四种有毒中药共性的解毒机制是由于矾水体系中的Al~(3+)与毒针晶中草酸钙分子中的草酸根离子结合促使草酸钙分解;针晶的刚性支撑被破坏,同时酸性的矾水体系又使得毒针晶中蛋白溶解或水解,整个晶体被破坏,毒性消失。
综上所述,天南星科有毒中药半夏、虎掌南星、东北天南星及禹白附具有共性刺激性毒性成分,在矾制解毒的机理上也具有共性。
This project was supported "Eleven-Five" national projects of technological supporting (No.2006BAI09B06-10) and national natural science foundation(No.30772785).
Pinellia ternate(Thunb.)Breit.,Arisaema amurense Maxim.and Typhonium giganteum Engl., included in Chinese Pharmacopoeia(2005),are all belonged to Araceae family.And Pinellia pedatisecta Schott is widely used in china herbal market as a substitute for arisaematis.The property and flavor of the four Araceae plants is all documented to be 'acrid,warm and toxic'. That means these crude drugs have intensive acridity and toxicity,causing swelling of the lips, mouth and throat,aphonia,salivation,convulsion,convulsion,and even death from suffocation. So clinical medication is that they are all processed with Potash Alum through duplicating method.Whether the four plants have the same acridity component? Whether the same detoxification processing mechanism present in the common processing method? No report.At the base of our group work on the acridity factor and the processing detoxification mechanism of Pinellia ternate(Thunb.)Breit.,in this paper,we attempt to prove the conjecture.
The work as follows:
1 Common poisonous component in the four types of Araceae herbal medicine.
Using different polarity solvent extraction and separation,different polar solvent extracts were obtained respectively from the four plants.At the same time,the seperation of the calcium oxalate raphides was achieved by centrifugalization.The common poisonous component of the four plants(Araceae) were identified through toxicity screening,rabbits' conjunctival irritation experiment as the animal model.The needle-like raphides of calcium oxalate were proved to be their common poisonous components,which were named as 'poisonous raphides'.
2 The comparison of the irritant and toxic intensity of the four crude drugs and the dose-toxic response relationships of the poisonous raphides.
The irritant and toxic intensity of the four crude drugs(Araceae) and the raphides inside of them was detected through mice acute toxicity experiment and the rabbits' eye irritation experiments,and the result showed the level as follows:Pinellia pedatisecta＞Pinellia ternate＞ Arisaema amurense＞Typhonium giganteum.The action mode of acrid raphides on mucous membranes was investigated through mice mesentery experiment,after intraperitoneal injection with the power pf the four crude drugs.The rusults showed that the poisonous raphids punctured into the cell of the mesenteric tissue directly.Futhur study about the toxic and irritant intensity and the dose-toxic response relationships of poisonous raphides indicated that toxicity enhanced with the increase in concentration of poisonous raphides.The toxicity presented linear doseresponse relationships during a range in concentration.The result further indicated that the raphides were their common poisonous factor.
3 Analysis of poisonous raphides in the four types of Araceae herbal medicine.
Through determination of raphides content in the four crude drugs,the content level showed as follows:Pinellia pedatisecta＞Pinellia ternate＞Arisaema amurense＞Typhonium giganteum. The raphides content level was in line with level of the toxic intensity of the four crude drugs. This consistency further indicated that the poisonous raphides were their common poisonous components.Their morphology was studied by light microscope and Scanning electron microscopy(SEM).The raphides of the four plants all have two types of raphides(long and short).SEM of the raphids showed pointed and blunt ends of a raphide,and a long groove and barbs on it.The result of X-ray diffraction,photoelectric spectra and infrared spectra showed the raphides consist of calcium oxalate monohydrate and-NH,-COOH and -S- functional groups. Results of identification and composition analysis showed that the four plants poisonous raphides all consist of calcium oxalate,proteins and small amount of Polysaccharide (Glycoprotein).SDS-gel electrophoresis separation of raphides proteins results indicated that the major protein bands are basically the same,showing the same bands of 26KD and 51KD The proteins isolated from raphides in the four plants had strong acridity,and allergenicity.Through determination,the content level of the protein in the raphides is as follows:Pinellia pedatisecta＞Pinellia ternate＞Arisaema amurense＞Typhonium giganteum.The content level was also in line with the level of intensity of the toxicity,which indicated that the toxicity derived from not only the blunt structure of the raphides but also from protein.
4 Common mechanism of detoxification processing of the four types of Araceae herbal medicine.
The common mechanism detoxification processing with Potash Alum was studied by analyzing the constituents of the raphides in alaum water solution system.Al~(3+) in alaum water solution have the combination ability with C_2O_4~(2-) which comes from calcium oxalate raphides, which made the raphides decompose.At the same time,the alaum water solution is acidic,and the raphides proteins were hydrolyzed,which completely destroyed the raphides structure and crystal form.Irritation and toxicity,decreased.
The results as above showed that:the toxic components of the four types of herbal medicine (Araceae) belong to one class of toxic components.That is piosonous raphides,having a special crystal structure,needle-like crystal,with calcium oxalate,proteins as the main conponents.The toxicity mainly due to the dual effects of mechanical stimulation of special crystal form and chemical stimulation of protein allergens.The level of toxic and irritant intensity was in line with the level of the raphides concentration of the four crude drugs:Pinellia pedatisecta＞Pinellia ternate＞Arisaema amurense＞Typhonium giganteum.The common mechanism of detoxification processing with Potash Alum was that Al~(3+) in alaum water solution combined with C_2O_4~(2-),which cause the decomposition of calcium oxalate,and the rigid support of crystal is damaged,while the alaum acidic water system also made proteins hydrolyzed,and the entire crystal structure was damaged.The toxicity disappeared
To sum up,the piosonous raphides were the common poisonous factor in Pinellia ternate(Thunb.)Breit,Pinellia pedatisecta Schott,Arisaema amurense Maxim and Typhonium giganteum Engl.The common detoxification mechanism present in the processing methods.
引文
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