Antimicrobial Activity\nof Glycyrrhizinic Acid Is pH-Dependent

In recent years,\nantimicrobial hydrogels have attracted\nmuch attention\nin biomedical applications due to their biocompatibility and high\nwater content. Glycyrrhizin (GA) is an antimicrobial that can form\npH-dependent hydrogels due to the three carboxyl groups of GA that\ndiffer in p<i>K</i><sub>a</sub> value. The influence of\nGA protonation on the antimicrobial activity, however, has never been\nstudied before. Therefore, we investigated the effect of the pH on\nthe antimicrobial activity of GA against Pseudomonas\naeruginosa, Staphylococcus aureus, MRSA, Staphylococcus epidermidis, Acinetobacter baumannii, Klebsiella pneumoniae, Klebsiella\naerogenes, and two strains of Escherichia\ncoli. In general, the antimicrobial activity of GA\nincreases as a function of decreasing pH (and thus increasing protonation\nof GA). More specifically, fully protonated GA hydrogels (pH = 3)\nare required for growth inhibition and killing of E.\ncoli UTI89 and <i>Klebsiella</i> in the\nsuspension above the hydrogel, while the staphylococci strains and A. baumannii are already inhibited by fully deprotonated\nGA (pH = 6.8). P. aeruginosa and E. coli DH5α showed moderate susceptibility,\nas they are completely inhibited by a hydrogel at pH 3.8, containing\npartly protonated GA, but not by fully deprotonated GA (pH = 6.8).\nThe antimicrobial activity of the hydrogel cannot solely be attributed\nto the resulting pH decrease of the suspension, as the presence of\nGA significantly increases the activity. Instead, this increased activity\nis due to the release of GA from the hydrogel into the suspension,\nwhere it directly interacts with the bacteria. Moreover, we provide\nevidence indicating that the pH dependency of the antimicrobial activity\nis due to differences in GA protonation state by treating the pathogens\nwith GA solutions differing in their GA protonation distribution.\nFinally, we show by LC–MS that there is no chemical or enzymatic\nbreakdown of GA. Overall, our results demonstrate that the pH influences\nnot only the physical but also the antimicrobial properties of the\nGA hydrogels.