半夏刺激性毒性成分、炮制减毒机理及工艺研究
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摘要
本项目研究来源于江苏省自然科学基金项目(项目编号：BK2004153)。论文主要针对具有强烈刺激性毒性的半夏，对其刺激性毒性成分进行研究，在阐明半夏的刺激性毒性成分的基础上，进行了半夏的炮制减毒机理和工艺研究，并由此对半夏所属天南星科部分植物的刺激性成分及作用进行了初步研究。
在确定半夏刺激性毒性成分的研究工作中，主要工作内容和研究结果包括：
半夏刺激性毒性作用研究，通过对半夏生品的急性毒性实验研究，测得半夏生品的LD_(50)为3359mg／kg，而水煎液未能测出LD_(50)；半夏生品混悬液能引起动物毛细血管通透性显著增强，引起小鼠腹腔渗出液中的PGE_2含量显著增加，组胺含量显著增加：生品混悬液能引起家兔眼部的强烈水肿和充血，具有极强烈的刺激性作用。研究表明半夏的毒性表现为对接触部位的刺激性炎症反应，当刺激性反应达到一定程度时，可引起动物死亡的毒性反应，因此可以说明半夏的毒性主要表现为刺激性毒性。同时研究还确定了以操作简单方便，可重复，并有客观明确的刺激性强度分值的家兔眼刺激模型和眼刺激性强度作为本课题刺激性研究的主要药理指标。
在明确半夏的毒性即为刺激性毒性后，进行了半夏刺激性毒性成分的初步筛选。通过对半夏的水及各种有机溶剂提取物的家兔眼刺激性实验表明，上述提取物均未表现出半夏所特有的刺激性毒性作用，但提取后的残渣均对家兔眼表现出强烈的刺激性，显微观察表明，残渣中含有大量的草酸钙针晶，而不具备刺激性作用的各提取物中则不合草酸钙针晶；进一步加热和酸碱处理，发现半夏经过高温加热后，仍然保留着刺激性活性，不同pH值酸碱溶液对半夏浸泡后，仅强酸强碱浸泡的残渣基本无刺激感，其余均有较强的刺激性，经显微观察，加热后的半夏针晶形状无明显变化，强酸强碱溶液浸泡的半夏中草酸钙针晶的数量大大减少，针晶形状也被不同程度破坏，而其余具有刺激性的残渣中仍然含有数量不等的较完整的草酸钙针晶。因此，认为半夏的刺激性成分可能就是其中大量的形态特殊的草酸钙针晶。
刺激性毒性研究结果表明，半夏中含有的草酸钙针晶可能为半夏主要的刺激性成分，进一步的研究通过低速逐级离心从生半夏中分离纯化得到草酸钙针晶，经过显微，理化鉴别和X衍射分析，鉴定提取物为由草酸钙形成的针晶；对草酸钙针晶的急性毒性试验表明，半夏纯草酸钙针晶的半数致死量(LD_(50))为15.94mg／kg，而半夏生品混悬液的LD_(50)为3395mg／kg，半夏中草酸钙针晶的毒性是半夏生品的212倍。说明当草酸钙针晶对机体的刺激性达到一定强度时，能够引起机体的死亡，进一步表明半夏的毒性即通过刺激性表现；与不含草酸钙针晶的半夏粉末刺激性比较作用表明，去除了草酸钙针晶的半夏没有刺激性作用，说明半夏中的草酸钙针晶正是引起半夏产生刺激性作用的主要成分；等效性和量效分析表明，相同含量的半夏纯针晶和药材中针晶的刺激性作用具有等效关系，随着针晶浓度的增加，其刺激性强度也在不断增强，呈现出典型的量效关系；显微观察发现针晶能够深入刺入小鼠肠系膜表面并引起组织肿胀，提示针晶刺入后，引起组织的肿胀，而产生炎症反应，引起对机体的刺激性作用，进一步验证刺激性作用即由草酸钙针晶引起。此外考察了加热前后的草酸钙针晶的刺激性作用，两者刺激性作用没有显著性差异。
进一步的研究表明半夏的草酸钙针晶是由草酸钙和蛋白结合而成。将提取得到的草酸钙针晶进行含量测定，按草酸量进行折算后，其中含有草酸钙的含量为53.3961％，说明半夏中的草酸钙针晶并不仅仅由草酸钙形成。根据国际植物生理学杂志文章中关于草酸钙结晶的研究结果和方法，从草酸钙针晶中提取分离得到半夏针晶结合蛋白，刺激性实验表明半夏针晶结合蛋白具有刺激性作用，而半夏的水溶性蛋白没有刺激性作用。将两者进行了SDS-聚丙烯酰胺凝胶电泳分析，经过比对标准蛋白质分子量，发现有刺激性作用的半夏针晶结合蛋白在20KD和31KD之间有两条清晰的谱带，而半夏的水溶性蛋白则没有，推测具有刺激性的半夏针晶结合蛋白的分子量可能为25KD和27KD。
上述研究证实半夏中草酸钙针晶及其结合蛋白具有刺激性作用。实验进一步从草酸钙针晶本身的显微结构，机械压力和黏液细胞对针晶刺激性作用的影响，以及草酸钙针晶结合蛋白刺激性作用研究等方面探讨了半夏草酸钙针晶产生刺激性的作用机制。研究结果表明半夏草酸钙针晶极细长的具针尖末端、倒刺及凹槽，这种结构为针晶刺破粘膜细胞提供基础条件，而草酸钙成分本身并不具备毒性和刺激性；机械压力和黏液细胞通过作用力推动草酸钙针晶刺入组织；针晶结合蛋白对组织具有一定的刺激作用，这种刺激作用与整体针晶作用相比较小，但能加重对破损皮肤的炎症反应。研究表明半夏中草酸钙针晶的刺激性作用是由草酸钙针晶特殊的晶形引起的机械刺激和针晶结合蛋白的化学刺激综合作用的结果。
研究同时比较了数种无毒药材，如茜草、山药、麦冬中草酸钙针晶的异同。研究表明，半夏中含有大量细长的草酸钙针晶，其余药材中不含或只有少量的细长针晶，表明细长针晶的大量存在可能是产生刺激性作用的主要原因；半夏针晶具有特殊的针尖状末端、针晶表面有凹槽和倒刺，其他植物针晶则不具备类似的细微结构；显微观察残留在小鼠肠系膜中的针晶，发现半夏针晶大量残留在肠系膜表面，并多以竖直刺入肠系膜的状态存在，而茜草针晶仅有少量残留，并且仅仅停留在肠系膜表面，没有刺入肠系膜当中，进一步解释了为何同样含有草酸钙针晶，半夏能够产生刺激性作用，而茜草则没有刺激性作用；X-衍射分析的结果则表明半夏和茜草针晶在晶粒大小上存在较大差异，这可能导致了两者针晶在柔韧性和强度上的差别，从而导致针晶刺激性的有无。
通过上述研究，可确定半夏中的刺激性毒性成分主要是由蛋白结合草酸钙形成的特殊晶形的草酸钙针晶。在此基础上，围绕着草酸钙针晶这一刺激性成分经过炮制后所发生的变化，初步探讨了半夏炮制的减毒机理。主要的工作内容和结果包括：
以HPLC法，对经过pH1～pH14浸泡的半夏，市场流通的半夏炮制品种姜半夏和法半夏，以及按照药典法炮制明矾及石灰水浸泡的半夏，分别测定各炮制样品中草酸钙针晶的含量，结果表明，强酸强碱液及明矾、石灰水浸泡的样品中草酸钙针晶含量明显下降，姜半夏和法半夏中的草酸钙针晶含量也明显减少；通过光学显微观察和扫描电镜研究，发现在强酸强碱或明矾、石灰水浸泡条件下，半夏的特殊草酸钙针晶被锈蚀或消解，而使针晶数量减少，即使残留的针晶，也以断碎针晶为主，针晶细微结构均已被破坏，没有针尖状末端和凹槽、倒刺等特殊结构，不再具有刺激性作用；同时在强碱(pH13～14)条件下，有部分针晶成束状被凝固在黏液细胞当中，不能被从黏液细胞中释放出来，针晶内部被锈蚀成半透明状(即针晶的蛋白母核)，使针晶完全丧失了应有的柔韧性和强度，从而不能发挥刺激性作用。而以明矾(pH3.34)和石灰水(pH12以上)炮制半夏，除可用酸碱理论解释外，根据沉淀平衡理论，由于明矾中含有硫酸根离子(SO_4~(2-))，在一定浓度下，可将草酸根离子(C_2O_4~-)从不溶性草酸钙中置换出来，从而达到破坏针晶晶形的目的；而石灰水的强碱性亦可使针晶蛋白变性，同时[OH~-]在一定的浓度条件下也可以使得草酸钙溶解，不再具有刺激性作用；另外，两者成分本身也能使部分针晶凝固，不易被释放出来，而使刺激性降低。
在初步明确了半夏炮制减毒机理的基础上，同样以草酸钙针晶的含量变化为线索，结合药效试验，进行了半夏炮制减毒工艺的研究，主要工作内容和研究结果包括：
采用正交设计，选取辅料用量，浸泡时间和温度三因素，以半夏中所含草酸钙针晶的含量以及对家兔眼刺激性的强度分值为指标，优选最佳的炮制减毒工艺。由综合加权评分结果和方差分析结果表明，炮制因素辅料用量，浸泡时间和温度对明矾和石灰水减毒炮制品种的质量均没有显著影响，明矾炮制减毒的最佳工艺为8％明矾，在30℃下浸泡48小时，石灰水炮制减毒的最佳工艺为15％石灰水，在室温下浸泡24小时。得出结论认为明矾的最佳用量符合药典规定要求，石灰水的用量，由于在实验过程中，随着时间的延长，pH值在不断下降，因此，要以较高浓度较短时间浸泡，才能维持pH12以上，并达到最佳减毒目的。
此外，还开展了对天南星科植物刺激性毒性作用的初步研究，主要工作内容和研究结果包括：
通过光学显微和扫描电镜分析天南星科其它刺激性植物，如白附子，天南星和水半夏中的草酸钙针晶，并进行X-衍射分析，结果白附子、天南星和水半夏中的草酸钙针晶与半夏中草酸钙针晶在形态上相似，均具有针尖状末端，倒刺和凹槽的特殊细微结构，认为这可能提示了这些植物中的草酸钙针晶同样可能是其中主要的刺激性成分；对水半夏、白附子、天南星药材提取的草酸钙针晶的刺激性作用及其量效关系试验表明，生水半夏、生白附子、生天南星中的草酸钙针晶具有强烈刺激性；各药材提取的针晶与各药材生品混悬液在针晶含量相同的条件下，对家兔的眼刺激性作用基本相同，具有等效性；针晶浓度与家兔眼刺激程度呈现确切的量效关系。认为天南星科部分具有刺激性作用的植物中的草酸钙针晶为其主要刺激性成分。
This project came from Natural Science Found of Jiangsu province(No. BIC2004153). Irritant component of pinelliae tuber were mainly studied in this paper. After illuminating the stimulating component, the attenuation mechanism and technology of processing were studied and discussed. Based on the results mentioned above, we studied the irritant effect of some plants in Araceae family.
In order to determining the stimulating component in pinelliae tuber, The main works were done as follows.
Determinantion of irritation or toxicity effect of Pinelliae tuber. In this part, the acute toxicity testing was first performed, LD50 of raw Pinelliae tuber suspl was 3359mg/kg, but couldn't measured in Pinelliae tuber water decoct. Pinelliae tuber suspl also leaded capillary permeability of Jimpy mice to strengthen notably, the content of Jimpy mice's peritoneal exudates increaseed notably, and the content of histamine increased either. The rabbits' eyes showed intensive dropsy and congestion by Pinelliae tuber suspl, indicating the fierce irritation of Pinelliae tuber. From the results, we came to the conclusion that Pinelliae tuber, could induced irritation through inflammatory reaction and inflammatory factor. When irritative response got to some degree, animals would die. At the same time, we made sure the stimulus intensity of rabbits' eyes as the main pharmaco-index in our study because of its simplicity, convenience, reproducibility and definite parameter.
After determining the toxicity was showed as irritation, we started the preliminary screening of irritant component in Pinelliae tuber. After irritation test with the model of rabbits' eyes, no irritation was found in extraction of water and various solvents, but the remain of them showed strong irritation which were observed by light microscope, lots of calcium oxalate crystals could be seen, while there's no calcium oxalate crystals in those non-irritant extraction. After being heated and soaked in mild acid base solution, strong irritation remained, but no irritation found in strong acid base solution. Observed under light microscope, the crystals remained no visible change after being heated and soaked in mild acid base, but the number and form of crystals of those soaked in strong acid base changed largely. So the special calcium oxalate crystals were considered as the main stimulating component in Pinelliae tuber.
Since the calcium oxalate crystal may be the main stimulating component in Pinelliae tuber, the pure crystals was isolated from raw Pinelliae tuber by centrifugalization with low speed, the extract was determined through observation by light microscope, chemi-detecting and X-diffraction analysis as the calcium oxalate monohydrate. The results of acute test of calcium oxalate crystals indicated that a little pure crystals could lead to death(LD_(50) was 15.94mg/kg), explaining that the organism would dead when irritation got to some degree, which confirmed the toxicity of Pinellia tuber showed as irritation all the more. Comparing the irritation of Pinelliae tuber with and without calcium oxalate crystals, we found the latter had no irritation which indicated the crystals were just the main component leading to irritation all the better. The results of isoeffect and quantity-effect relationship analysis confirmed our propose. That was, with the same content of crystals, the pure crystal and Pinelliae tuber had almost the same irritation effect, and with the density of crystals increased gradually, the irritation intensity increased gradually either, showed the typical quantity-effect relationship. Furthmore, crystals could prick into mice's mesenterium deeply, leading to swell of tissue and cell, producing inflammatory reaction and the irritation then. In addition, with being heated or not, there's no significant different on irritation effect, supposing that the attachment protein on crystals mentioned in some foreign papers may have a little irritation but be not the key factor.
To ensure the irritation of attachment protein(binding protein) on crystals, the content of canlcium oxalate and composition of crystals were determined, the water protein in Pinelliae tuber without calcium oxalate crystals and crystals binding protein were compared, the former had no irritation and latter showed just opposite. The SDS-PAGE was employed in determining the difference between the two protein. The results showed the protein with irritation had two clear band between 20KD and 31KD which couldn't be seen in the protein without irritation. Contrasting to the standard protein, we presumed that the molecular weight of stimulating binding protein may be 25KD and 27KD.
From results above, the irritation of calcium oxalate crystals and the binding protein were comfirmed. The irritation mechanism of crystals was studied then from the microstructure of calcium oxalate crystals, the contribution to crystals' irritation effect of mechanical pressure outsid and idioblast in plants, and the irritation of crystals binding protein as well. The results showed that it's the needle tip, barb and groove on the surface of crystals that provided convenience condition for crystals pricing into the mucous cell, not the calcium oxalate component itself, the latter had no toxity and irritation. Mechanical pressure and idioblast also made crystals pricking into tissue easier through driving force. Binding protein could aggravate the inflammatory reaction of damaged skin. We concluded that the irritation mechanism of raphides(needle-like calcium oxalate crystals) could be combination of mechanical release of the raphides with special sharp end disrupting the barrier, which results in traumatic injury, and penetration of irritation protein carried on the crystals' surfaces and grooves to make further chemical injury.
The calcium oxalate crystals in non-irritatant in other plants, such as Indian Madder Root, Common Yan Rhizome and Dwarf Lilyturf Tuber were also compared with those in Pinellia Tuber. Through observation with light microscope and scanning electron microscope, we found there were lots of slender and flexible calcium oxalate crystals in Pinelliae tuber and almost no such crystals in other non-irritation plants, indicating that the flexible calcium oxalate crystals may be the key factors to cause irritation. Moreover, the needle-like tip, barb and groove were the special feature of crystals just in Pinellia Tuber but not those in other non-irritant plants. Furthermore, it's Pinelliae tuber crystals that could be found a lot on the surface of mice' mesenterum and pricked into it deeply, but not the indian madder root crystals. The latter remained little on the mesenterium surface and didn't prick into it, which explaining the Pinelliae tuber crystals could leading to irritation and indian madder root couldn't. The results of X-diffraction analysis also showd that there was very different in crystals size of the two kinds of crystals, which might result in the difference of flexible and strength of crystals and then the difference in irritation.
On the base of the conclusion that the calcium oxalate crystals were the main stimulating component, the attenuation mechanism of Pinelliae tuber precessing were then studied according to the qualitative and quantitative alternation of calcium oxalate crystals after being processed.
The contents of calcium oxalate crystals in Pinelliae tuber samples soaked in acid base solution, the prepared Rhizoma pinelliae with juice of rhizima zingiberis recens and with alumen radix glyrrhigae calcaren bought from market, and Pinelliae tuber samples soaked in alum and limewater were determined by HPLC. The results made clear that the contents of calcium oxalate crystals in samples soaked in strong acid base solution decreased sharply, as the ones soaked in alum and limewater. Meanwhile, the pH of alum and limewater were about 3 and 12 respectively, indicating under the circumstance of strong acid base, the crystals' contents reduced and irritation lowered. Through the results of observation by light microscope and scanning electron microscope, we thought that the attenuation mechanism of processing be the special calcium oxalate crystals in Pinelliae tuber destroyed in strong acid base, which not only reduced the content but destroyed the shape of crystals, the needle-like ends, the barb and groove were all disappeared and no irritation remained. On the condition of strong base(pH13～14), some crystals were corroded to be semitransparent and coagulated in the idioblast, thus couldn't be released and lose their flexible and strenth, leading to non-irritant. The mechanism of processing with alum and limewater could explained by the acid base theory, except that, the two adjuvant themselves could flocculate some crystals and made them not easy to release, which also reduced the irritation.
At the same time, the attenuation technology of Pinelliae tuber was studied on the basis of content change of calcium oxalate crystals, together with pharmacodynamic test. The main research works were as follows.
The orthogonal test was employed to determine the best attenuation technology of the three factors affiliated with three levels including the quantity of adjuvant、soaking temperature and time accounting on the indexes of the contents of calcium oxalate crystals and irritation intension value with the model of rabbits' eyes. Through combination weighing score and analysis of variance, the three factors had no significant influence on the processed products. The best attenuation technology of alum solution was temperature at 600C, time at 48 hours and adding 8% alum per 100kg drugs, such quantities consisting with Chinese Pharmacopeia(Edition 2005). The best attenuation technology of limewater was temperature at ordinary temperature, time at 24 hours and adding 15% limewater per 100kg drugs which was more than that in Chinese Pharmacopeia. Because as time went by, the pH value reduced continuously, and the higher density meant pH value could keep 12 or more, the time was shortened and the density of limewater increased.
Additionaly, we studied the irritation of some plants in Araceae family, such as giant typhonium rhizome, jackinthepulpit tuber and Rhizoma typhonii flagelliformis.
The crystals in Giant Typhonium Rhizome, Jackinthepulpit tuber and Rhizoma Typhonii Flagelliformis were studied through light microscope, scanning microscope andⅩdiffraction analysis, which all had the same shape with those in Pinelliae tuber, such as the same microstructure of needle-like ends, barb and groove, which may indicating the crystals in these plants be the stimulating components either. The test with the model of rabbits' eyes were also employed to determine the irritation of calcium oxalate crystals extracted from these plants. The results indicated that crystals isolated from Giant Typhonium Rhizome, Jackinthepulpit tuber and Rhizoma Typhonii Flagelliformis showed strong irritation. With the same content of calcium oxalate crystals, the irritation of raw medicinal materials and pure calcium oxalate crystals had no difference. The density of calcium oxalate crystal and irritation strength on rabbits' eyes showed undoubted quantity-effect relationship, which meant the calcium oxalate crystal was also the main acridity principle in some Araceae plants.
在确定半夏刺激性毒性成分的研究工作中，主要工作内容和研究结果包括：
半夏刺激性毒性作用研究，通过对半夏生品的急性毒性实验研究，测得半夏生品的LD_(50)为3359mg／kg，而水煎液未能测出LD_(50)；半夏生品混悬液能引起动物毛细血管通透性显著增强，引起小鼠腹腔渗出液中的PGE_2含量显著增加，组胺含量显著增加：生品混悬液能引起家兔眼部的强烈水肿和充血，具有极强烈的刺激性作用。研究表明半夏的毒性表现为对接触部位的刺激性炎症反应，当刺激性反应达到一定程度时，可引起动物死亡的毒性反应，因此可以说明半夏的毒性主要表现为刺激性毒性。同时研究还确定了以操作简单方便，可重复，并有客观明确的刺激性强度分值的家兔眼刺激模型和眼刺激性强度作为本课题刺激性研究的主要药理指标。
在明确半夏的毒性即为刺激性毒性后，进行了半夏刺激性毒性成分的初步筛选。通过对半夏的水及各种有机溶剂提取物的家兔眼刺激性实验表明，上述提取物均未表现出半夏所特有的刺激性毒性作用，但提取后的残渣均对家兔眼表现出强烈的刺激性，显微观察表明，残渣中含有大量的草酸钙针晶，而不具备刺激性作用的各提取物中则不合草酸钙针晶；进一步加热和酸碱处理，发现半夏经过高温加热后，仍然保留着刺激性活性，不同pH值酸碱溶液对半夏浸泡后，仅强酸强碱浸泡的残渣基本无刺激感，其余均有较强的刺激性，经显微观察，加热后的半夏针晶形状无明显变化，强酸强碱溶液浸泡的半夏中草酸钙针晶的数量大大减少，针晶形状也被不同程度破坏，而其余具有刺激性的残渣中仍然含有数量不等的较完整的草酸钙针晶。因此，认为半夏的刺激性成分可能就是其中大量的形态特殊的草酸钙针晶。
刺激性毒性研究结果表明，半夏中含有的草酸钙针晶可能为半夏主要的刺激性成分，进一步的研究通过低速逐级离心从生半夏中分离纯化得到草酸钙针晶，经过显微，理化鉴别和X衍射分析，鉴定提取物为由草酸钙形成的针晶；对草酸钙针晶的急性毒性试验表明，半夏纯草酸钙针晶的半数致死量(LD_(50))为15.94mg／kg，而半夏生品混悬液的LD_(50)为3395mg／kg，半夏中草酸钙针晶的毒性是半夏生品的212倍。说明当草酸钙针晶对机体的刺激性达到一定强度时，能够引起机体的死亡，进一步表明半夏的毒性即通过刺激性表现；与不含草酸钙针晶的半夏粉末刺激性比较作用表明，去除了草酸钙针晶的半夏没有刺激性作用，说明半夏中的草酸钙针晶正是引起半夏产生刺激性作用的主要成分；等效性和量效分析表明，相同含量的半夏纯针晶和药材中针晶的刺激性作用具有等效关系，随着针晶浓度的增加，其刺激性强度也在不断增强，呈现出典型的量效关系；显微观察发现针晶能够深入刺入小鼠肠系膜表面并引起组织肿胀，提示针晶刺入后，引起组织的肿胀，而产生炎症反应，引起对机体的刺激性作用，进一步验证刺激性作用即由草酸钙针晶引起。此外考察了加热前后的草酸钙针晶的刺激性作用，两者刺激性作用没有显著性差异。
进一步的研究表明半夏的草酸钙针晶是由草酸钙和蛋白结合而成。将提取得到的草酸钙针晶进行含量测定，按草酸量进行折算后，其中含有草酸钙的含量为53.3961％，说明半夏中的草酸钙针晶并不仅仅由草酸钙形成。根据国际植物生理学杂志文章中关于草酸钙结晶的研究结果和方法，从草酸钙针晶中提取分离得到半夏针晶结合蛋白，刺激性实验表明半夏针晶结合蛋白具有刺激性作用，而半夏的水溶性蛋白没有刺激性作用。将两者进行了SDS-聚丙烯酰胺凝胶电泳分析，经过比对标准蛋白质分子量，发现有刺激性作用的半夏针晶结合蛋白在20KD和31KD之间有两条清晰的谱带，而半夏的水溶性蛋白则没有，推测具有刺激性的半夏针晶结合蛋白的分子量可能为25KD和27KD。
上述研究证实半夏中草酸钙针晶及其结合蛋白具有刺激性作用。实验进一步从草酸钙针晶本身的显微结构，机械压力和黏液细胞对针晶刺激性作用的影响，以及草酸钙针晶结合蛋白刺激性作用研究等方面探讨了半夏草酸钙针晶产生刺激性的作用机制。研究结果表明半夏草酸钙针晶极细长的具针尖末端、倒刺及凹槽，这种结构为针晶刺破粘膜细胞提供基础条件，而草酸钙成分本身并不具备毒性和刺激性；机械压力和黏液细胞通过作用力推动草酸钙针晶刺入组织；针晶结合蛋白对组织具有一定的刺激作用，这种刺激作用与整体针晶作用相比较小，但能加重对破损皮肤的炎症反应。研究表明半夏中草酸钙针晶的刺激性作用是由草酸钙针晶特殊的晶形引起的机械刺激和针晶结合蛋白的化学刺激综合作用的结果。
研究同时比较了数种无毒药材，如茜草、山药、麦冬中草酸钙针晶的异同。研究表明，半夏中含有大量细长的草酸钙针晶，其余药材中不含或只有少量的细长针晶，表明细长针晶的大量存在可能是产生刺激性作用的主要原因；半夏针晶具有特殊的针尖状末端、针晶表面有凹槽和倒刺，其他植物针晶则不具备类似的细微结构；显微观察残留在小鼠肠系膜中的针晶，发现半夏针晶大量残留在肠系膜表面，并多以竖直刺入肠系膜的状态存在，而茜草针晶仅有少量残留，并且仅仅停留在肠系膜表面，没有刺入肠系膜当中，进一步解释了为何同样含有草酸钙针晶，半夏能够产生刺激性作用，而茜草则没有刺激性作用；X-衍射分析的结果则表明半夏和茜草针晶在晶粒大小上存在较大差异，这可能导致了两者针晶在柔韧性和强度上的差别，从而导致针晶刺激性的有无。
通过上述研究，可确定半夏中的刺激性毒性成分主要是由蛋白结合草酸钙形成的特殊晶形的草酸钙针晶。在此基础上，围绕着草酸钙针晶这一刺激性成分经过炮制后所发生的变化，初步探讨了半夏炮制的减毒机理。主要的工作内容和结果包括：
以HPLC法，对经过pH1～pH14浸泡的半夏，市场流通的半夏炮制品种姜半夏和法半夏，以及按照药典法炮制明矾及石灰水浸泡的半夏，分别测定各炮制样品中草酸钙针晶的含量，结果表明，强酸强碱液及明矾、石灰水浸泡的样品中草酸钙针晶含量明显下降，姜半夏和法半夏中的草酸钙针晶含量也明显减少；通过光学显微观察和扫描电镜研究，发现在强酸强碱或明矾、石灰水浸泡条件下，半夏的特殊草酸钙针晶被锈蚀或消解，而使针晶数量减少，即使残留的针晶，也以断碎针晶为主，针晶细微结构均已被破坏，没有针尖状末端和凹槽、倒刺等特殊结构，不再具有刺激性作用；同时在强碱(pH13～14)条件下，有部分针晶成束状被凝固在黏液细胞当中，不能被从黏液细胞中释放出来，针晶内部被锈蚀成半透明状(即针晶的蛋白母核)，使针晶完全丧失了应有的柔韧性和强度，从而不能发挥刺激性作用。而以明矾(pH3.34)和石灰水(pH12以上)炮制半夏，除可用酸碱理论解释外，根据沉淀平衡理论，由于明矾中含有硫酸根离子(SO_4~(2-))，在一定浓度下，可将草酸根离子(C_2O_4~-)从不溶性草酸钙中置换出来，从而达到破坏针晶晶形的目的；而石灰水的强碱性亦可使针晶蛋白变性，同时[OH~-]在一定的浓度条件下也可以使得草酸钙溶解，不再具有刺激性作用；另外，两者成分本身也能使部分针晶凝固，不易被释放出来，而使刺激性降低。
在初步明确了半夏炮制减毒机理的基础上，同样以草酸钙针晶的含量变化为线索，结合药效试验，进行了半夏炮制减毒工艺的研究，主要工作内容和研究结果包括：
采用正交设计，选取辅料用量，浸泡时间和温度三因素，以半夏中所含草酸钙针晶的含量以及对家兔眼刺激性的强度分值为指标，优选最佳的炮制减毒工艺。由综合加权评分结果和方差分析结果表明，炮制因素辅料用量，浸泡时间和温度对明矾和石灰水减毒炮制品种的质量均没有显著影响，明矾炮制减毒的最佳工艺为8％明矾，在30℃下浸泡48小时，石灰水炮制减毒的最佳工艺为15％石灰水，在室温下浸泡24小时。得出结论认为明矾的最佳用量符合药典规定要求，石灰水的用量，由于在实验过程中，随着时间的延长，pH值在不断下降，因此，要以较高浓度较短时间浸泡，才能维持pH12以上，并达到最佳减毒目的。
此外，还开展了对天南星科植物刺激性毒性作用的初步研究，主要工作内容和研究结果包括：
通过光学显微和扫描电镜分析天南星科其它刺激性植物，如白附子，天南星和水半夏中的草酸钙针晶，并进行X-衍射分析，结果白附子、天南星和水半夏中的草酸钙针晶与半夏中草酸钙针晶在形态上相似，均具有针尖状末端，倒刺和凹槽的特殊细微结构，认为这可能提示了这些植物中的草酸钙针晶同样可能是其中主要的刺激性成分；对水半夏、白附子、天南星药材提取的草酸钙针晶的刺激性作用及其量效关系试验表明，生水半夏、生白附子、生天南星中的草酸钙针晶具有强烈刺激性；各药材提取的针晶与各药材生品混悬液在针晶含量相同的条件下，对家兔的眼刺激性作用基本相同，具有等效性；针晶浓度与家兔眼刺激程度呈现确切的量效关系。认为天南星科部分具有刺激性作用的植物中的草酸钙针晶为其主要刺激性成分。
This project came from Natural Science Found of Jiangsu province(No. BIC2004153). Irritant component of pinelliae tuber were mainly studied in this paper. After illuminating the stimulating component, the attenuation mechanism and technology of processing were studied and discussed. Based on the results mentioned above, we studied the irritant effect of some plants in Araceae family.
In order to determining the stimulating component in pinelliae tuber, The main works were done as follows.
Determinantion of irritation or toxicity effect of Pinelliae tuber. In this part, the acute toxicity testing was first performed, LD50 of raw Pinelliae tuber suspl was 3359mg/kg, but couldn't measured in Pinelliae tuber water decoct. Pinelliae tuber suspl also leaded capillary permeability of Jimpy mice to strengthen notably, the content of Jimpy mice's peritoneal exudates increaseed notably, and the content of histamine increased either. The rabbits' eyes showed intensive dropsy and congestion by Pinelliae tuber suspl, indicating the fierce irritation of Pinelliae tuber. From the results, we came to the conclusion that Pinelliae tuber, could induced irritation through inflammatory reaction and inflammatory factor. When irritative response got to some degree, animals would die. At the same time, we made sure the stimulus intensity of rabbits' eyes as the main pharmaco-index in our study because of its simplicity, convenience, reproducibility and definite parameter.
After determining the toxicity was showed as irritation, we started the preliminary screening of irritant component in Pinelliae tuber. After irritation test with the model of rabbits' eyes, no irritation was found in extraction of water and various solvents, but the remain of them showed strong irritation which were observed by light microscope, lots of calcium oxalate crystals could be seen, while there's no calcium oxalate crystals in those non-irritant extraction. After being heated and soaked in mild acid base solution, strong irritation remained, but no irritation found in strong acid base solution. Observed under light microscope, the crystals remained no visible change after being heated and soaked in mild acid base, but the number and form of crystals of those soaked in strong acid base changed largely. So the special calcium oxalate crystals were considered as the main stimulating component in Pinelliae tuber.
Since the calcium oxalate crystal may be the main stimulating component in Pinelliae tuber, the pure crystals was isolated from raw Pinelliae tuber by centrifugalization with low speed, the extract was determined through observation by light microscope, chemi-detecting and X-diffraction analysis as the calcium oxalate monohydrate. The results of acute test of calcium oxalate crystals indicated that a little pure crystals could lead to death(LD_(50) was 15.94mg/kg), explaining that the organism would dead when irritation got to some degree, which confirmed the toxicity of Pinellia tuber showed as irritation all the more. Comparing the irritation of Pinelliae tuber with and without calcium oxalate crystals, we found the latter had no irritation which indicated the crystals were just the main component leading to irritation all the better. The results of isoeffect and quantity-effect relationship analysis confirmed our propose. That was, with the same content of crystals, the pure crystal and Pinelliae tuber had almost the same irritation effect, and with the density of crystals increased gradually, the irritation intensity increased gradually either, showed the typical quantity-effect relationship. Furthmore, crystals could prick into mice's mesenterium deeply, leading to swell of tissue and cell, producing inflammatory reaction and the irritation then. In addition, with being heated or not, there's no significant different on irritation effect, supposing that the attachment protein on crystals mentioned in some foreign papers may have a little irritation but be not the key factor.
To ensure the irritation of attachment protein(binding protein) on crystals, the content of canlcium oxalate and composition of crystals were determined, the water protein in Pinelliae tuber without calcium oxalate crystals and crystals binding protein were compared, the former had no irritation and latter showed just opposite. The SDS-PAGE was employed in determining the difference between the two protein. The results showed the protein with irritation had two clear band between 20KD and 31KD which couldn't be seen in the protein without irritation. Contrasting to the standard protein, we presumed that the molecular weight of stimulating binding protein may be 25KD and 27KD.
From results above, the irritation of calcium oxalate crystals and the binding protein were comfirmed. The irritation mechanism of crystals was studied then from the microstructure of calcium oxalate crystals, the contribution to crystals' irritation effect of mechanical pressure outsid and idioblast in plants, and the irritation of crystals binding protein as well. The results showed that it's the needle tip, barb and groove on the surface of crystals that provided convenience condition for crystals pricing into the mucous cell, not the calcium oxalate component itself, the latter had no toxity and irritation. Mechanical pressure and idioblast also made crystals pricking into tissue easier through driving force. Binding protein could aggravate the inflammatory reaction of damaged skin. We concluded that the irritation mechanism of raphides(needle-like calcium oxalate crystals) could be combination of mechanical release of the raphides with special sharp end disrupting the barrier, which results in traumatic injury, and penetration of irritation protein carried on the crystals' surfaces and grooves to make further chemical injury.
The calcium oxalate crystals in non-irritatant in other plants, such as Indian Madder Root, Common Yan Rhizome and Dwarf Lilyturf Tuber were also compared with those in Pinellia Tuber. Through observation with light microscope and scanning electron microscope, we found there were lots of slender and flexible calcium oxalate crystals in Pinelliae tuber and almost no such crystals in other non-irritation plants, indicating that the flexible calcium oxalate crystals may be the key factors to cause irritation. Moreover, the needle-like tip, barb and groove were the special feature of crystals just in Pinellia Tuber but not those in other non-irritant plants. Furthermore, it's Pinelliae tuber crystals that could be found a lot on the surface of mice' mesenterum and pricked into it deeply, but not the indian madder root crystals. The latter remained little on the mesenterium surface and didn't prick into it, which explaining the Pinelliae tuber crystals could leading to irritation and indian madder root couldn't. The results of X-diffraction analysis also showd that there was very different in crystals size of the two kinds of crystals, which might result in the difference of flexible and strength of crystals and then the difference in irritation.
On the base of the conclusion that the calcium oxalate crystals were the main stimulating component, the attenuation mechanism of Pinelliae tuber precessing were then studied according to the qualitative and quantitative alternation of calcium oxalate crystals after being processed.
The contents of calcium oxalate crystals in Pinelliae tuber samples soaked in acid base solution, the prepared Rhizoma pinelliae with juice of rhizima zingiberis recens and with alumen radix glyrrhigae calcaren bought from market, and Pinelliae tuber samples soaked in alum and limewater were determined by HPLC. The results made clear that the contents of calcium oxalate crystals in samples soaked in strong acid base solution decreased sharply, as the ones soaked in alum and limewater. Meanwhile, the pH of alum and limewater were about 3 and 12 respectively, indicating under the circumstance of strong acid base, the crystals' contents reduced and irritation lowered. Through the results of observation by light microscope and scanning electron microscope, we thought that the attenuation mechanism of processing be the special calcium oxalate crystals in Pinelliae tuber destroyed in strong acid base, which not only reduced the content but destroyed the shape of crystals, the needle-like ends, the barb and groove were all disappeared and no irritation remained. On the condition of strong base(pH13～14), some crystals were corroded to be semitransparent and coagulated in the idioblast, thus couldn't be released and lose their flexible and strenth, leading to non-irritant. The mechanism of processing with alum and limewater could explained by the acid base theory, except that, the two adjuvant themselves could flocculate some crystals and made them not easy to release, which also reduced the irritation.
At the same time, the attenuation technology of Pinelliae tuber was studied on the basis of content change of calcium oxalate crystals, together with pharmacodynamic test. The main research works were as follows.
The orthogonal test was employed to determine the best attenuation technology of the three factors affiliated with three levels including the quantity of adjuvant、soaking temperature and time accounting on the indexes of the contents of calcium oxalate crystals and irritation intension value with the model of rabbits' eyes. Through combination weighing score and analysis of variance, the three factors had no significant influence on the processed products. The best attenuation technology of alum solution was temperature at 600C, time at 48 hours and adding 8% alum per 100kg drugs, such quantities consisting with Chinese Pharmacopeia(Edition 2005). The best attenuation technology of limewater was temperature at ordinary temperature, time at 24 hours and adding 15% limewater per 100kg drugs which was more than that in Chinese Pharmacopeia. Because as time went by, the pH value reduced continuously, and the higher density meant pH value could keep 12 or more, the time was shortened and the density of limewater increased.
Additionaly, we studied the irritation of some plants in Araceae family, such as giant typhonium rhizome, jackinthepulpit tuber and Rhizoma typhonii flagelliformis.
The crystals in Giant Typhonium Rhizome, Jackinthepulpit tuber and Rhizoma Typhonii Flagelliformis were studied through light microscope, scanning microscope andⅩdiffraction analysis, which all had the same shape with those in Pinelliae tuber, such as the same microstructure of needle-like ends, barb and groove, which may indicating the crystals in these plants be the stimulating components either. The test with the model of rabbits' eyes were also employed to determine the irritation of calcium oxalate crystals extracted from these plants. The results indicated that crystals isolated from Giant Typhonium Rhizome, Jackinthepulpit tuber and Rhizoma Typhonii Flagelliformis showed strong irritation. With the same content of calcium oxalate crystals, the irritation of raw medicinal materials and pure calcium oxalate crystals had no difference. The density of calcium oxalate crystal and irritation strength on rabbits' eyes showed undoubted quantity-effect relationship, which meant the calcium oxalate crystal was also the main acridity principle in some Araceae plants.
引文
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[1] 吴皓，李伟，韩洪滔等．半夏刺激性成分的研究，中国中药杂志，1999，24 (12)：725～730
[1] 董子红，史万瑞．半夏炮制中值得探讨的几个问题．新疆中医药，2004，22 (1)：35
[2] 张贵君．中药师考试千题析(下)．哈尔滨：黑龙江科学技术出版社，1997，303．
[3] 赵建英．用辅料炮制半夏解毒机理分析．中国新医药，2004，3 (9)：90～91
[4] 高昌琨，吴琼，张陆军．半夏及炮制品对动物黏膜刺激性比较．安徽中医学院学报，2001，20 (6)：49～50
[5] 刘彦群，刘凯，何静：壳聚糖家兔眼刺激实验，徐州医学院学报，2002，22 (3)：201～202
[1] 钟凌云，吴皓．天南星科植物中黏膜刺激性成分的研究现状与分析，中国中药杂志，2006，31 (18)：1561～1563
[2] 钟凌云，吴皓，张科卫，等．生半夏中草酸钙针晶刺激性作用研究，中国中药杂志，2006，31 (20)：1706～1710
[3] 刘彦群、刘凯、何静．壳聚糖家兔眼刺激实验，徐州医学院学报，2002，22 (3)：201～202
[4] 吴皓，李伟，韩洪涛，等．半夏刺激性成分的研究[J]．中国中药杂志，1999，24 (12)：725～731