[Molecular dynamics simulation and experimental verification reveal processing detoxification mechanism of Pinelliae...

This study aims to investigate the effects of glycyrrhizic acid and calcium ions from adjuvant solutions on the toxic lectin in Pinelliae Rhizoma Praeparatum and clarify the processing detoxification mechanism. Molecular docking and molecular dynamics simulations were employed to construct a 3D model of lectin and evaluate the interactions of glycyrrhizic acid and/or calcium ions with lectin. Wild-type and mutant lectin proteins were produced via a prokaryotic expression system in Escherichia coli. The interactions of glycyrrhizic acid and/or calcium ions with the lectin proteins were verified by Bio-Layer Interferometry(BLI), and the irritant toxicity was assessed via a rabbit model of conjunctival irritation. The findings revealed that the ternary complex formed by glycyrrhizic acid, calcium ions, and lectin exhibited greater stability. This complex showed significantly reduced root mean square deviation(RMSD), root mean square fluctuation(RMSF), solvent-accessible surface area(SASA), radius of gyration(Rg), Gibbs free energy of solvation(ΔGSOLV), and total binding free energy(ΔTOTAL), but significantly increased number of hydrogen bonds and gas-phase free energy(ΔGGAS). The combined action of glycyrrhizic acid and calcium ions led to a significant increase in the number of interactions in the amino acid residue region 51-56, 170-175, and 232-237 of lectin, with enhanced Coulomb interaction-short range(Coul-SR) and Lennard-Jones-short range(LJ-SR). When the wild-type lectin protein co-bound with glycyrrhizic acid and calcium ions, the affinity constant was 8.10×10~(-6) mol·L~(-1), which was significantly lower than that of the binary complex of wild-type lectin with glycyrrhizic acid or calcium ions and the ternary complex of mutant lectin with glycyrrhizic acid and calcium ions. Additionally, lectin soaked in a mixed solution of glycyrrhizic acid and calcium ions did not significantly exacerbate the irritation of the rabbit conjunctiva when being combined with toxic needle crystals. In conclusion, glycyrrhizic acid and calcium ions demonstrate a synergistic effect in forming a structurally stable and non-toxic supramolecular complex with lectin, thereby achieving the processing detoxification of Pinelliae Rhizoma Praeparatum.