Tao Xing-Bao, Wu Hao
School of Pharmacy, Nanjing University of Chinese Medicine Nanjing 210023, China.
School of Pharmacy, Nanjing University of Chinese Medicine Nanjing 210023, China Key Laboratory of Traditional Chinese Medicine Processing in Jiangsu Province Nanjing 210023, China Engineering Research Center for Standardization of Traditional Chinese Medicine Processing, Ministry of Education Nanjing 210023, China.
Zhongguo Zhong Yao Za Zhi. 2023 Feb;48(4):951-957. doi: 10.19540/j.cnki.cjcmm.20220718.302.
The present study investigated the effect of immersion in the excipient lime water on the toxic component lectin protein and explained the scientific connotation of lime water detoxication during the processing of Pinelliae Rhizoma Praeparatum. Western blot was used to investigate the effects of immersion in lime water with different pH(pH 10, 11, and 12.4), saturated sodium hydroxide, and sodium bicarbonate solution on the content of lectin protein. The protein compositions of the supernatant and the precipitate after immersing lectin protein in lime water of different pH were determined by the SDS-PAGE method combined with the silver staining technique. The MALDI-TOF-MS/MS technique was used to detect the molecular weight distribution of peptide fragments in the supernatant and precipitate after immersing lectin protein in lime water of different pH, and circular dichroism spectroscopy was used to detect the ratio changes in the secondary structure of lectin protein during the immersion. The results showed that immersion in lime water at pH>12 and saturated sodium hydroxide solution could significantly reduce the content of lectin protein, while immersion in lime water at pH<12 and sodium bicarbonate solution had no significant effect on lectin protein content. The corresponding lectin protein bands and molecular ion peaks were not detected at the 12 kDa position in the supernatant and precipitate after immersing the lectin protein in lime water at pH>12, which was attributed to the fact that lime water immersion at pH>12 could significantly change the ratio of the secondary structure of lectin protein, resulting in irreversible denaturation, while lime water immersion at pH<12 did not change the ratio of the secondary structure of lectin protein. Therefore, pH>12 was the key condition for the detoxication of lime water during the processing of Pinelliae Rhizoma Praeparatum. Lime water immersion at pH>12 could cause irreversible denaturation of lectin protein, resulting in a significant decrease in the inflammatory toxicity of Pinelliae Rhizoma Praeparatum, which played a key role in detoxification.
本研究考察了浸泡于辅料石灰水中对毒性成分凝集素蛋白的影响，并阐释了法半夏炮制过程中石灰水解毒的科学内涵。采用蛋白质免疫印迹法研究不同pH值（pH 10、11和12.4）的石灰水、饱和氢氧化钠溶液及碳酸氢钠溶液浸泡对凝集素蛋白含量的影响。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）法结合银染技术测定不同pH值石灰水浸泡凝集素蛋白后上清液和沉淀的蛋白质组成。采用基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF-MS/MS）技术检测不同pH值石灰水浸泡凝集素蛋白后上清液和沉淀中肽段的分子量分布，并采用圆二色光谱法检测浸泡过程中凝集素蛋白二级结构的比例变化。结果表明，pH>12的石灰水和饱和氢氧化钠溶液浸泡可显著降低凝集素蛋白含量，而pH<12的石灰水和碳酸氢钠溶液浸泡对凝集素蛋白含量无显著影响。凝集素蛋白经pH>12的石灰水浸泡后，上清液和沉淀在12 kDa位置未检测到相应的凝集素蛋白条带和分子离子峰，这是由于pH>12的石灰水浸泡可显著改变凝集素蛋白二级结构比例，导致不可逆变性，而pH<12的石灰水浸泡未改变凝集素蛋白二级结构比例。因此，pH>12是法半夏炮制过程中石灰水解毒的关键条件。pH>12的石灰水浸泡可使凝集素蛋白发生不可逆变性，导致法半夏的炎性毒性显著降低，在解毒过程中起关键作用。
