| 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(LD50 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. |
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