A Study On Correlation Between Lectin From Araceae And Toxity

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.