Antimicrobial Activity of Glycyrrhizinic Acid Is pH-Dependent

In recent years,
antimicrobial hydrogels have attracted
much attention
in biomedical applications due to their biocompatibility and high
water content. Glycyrrhizin (GA) is an antimicrobial that can form
pH-dependent hydrogels due to the three carboxyl groups of GA that
differ in pKa value. The influence of
GA protonation on the antimicrobial activity, however, has never been
studied before. Therefore, we investigated the effect of the pH on
the antimicrobial activity of GA against Pseudomonas
aeruginosa, Staphylococcus aureus, MRSA, Staphylococcus epidermidis, Acinetobacter baumannii, Klebsiella pneumoniae, Klebsiella
aerogenes, and two strains of Escherichia
coli. In general, the antimicrobial activity of GA
increases as a function of decreasing pH (and thus increasing protonation
of GA). More specifically, fully protonated GA hydrogels (pH = 3)
are required for growth inhibition and killing of E.
coli UTI89 and Klebsiella in the
suspension above the hydrogel, while the staphylococci strains and A. baumannii are already inhibited by fully deprotonated
GA (pH = 6.8). P. aeruginosa and E. coli DH5α showed moderate susceptibility,
as they are completely inhibited by a hydrogel at pH 3.8, containing
partly protonated GA, but not by fully deprotonated GA (pH = 6.8).
The antimicrobial activity of the hydrogel cannot solely be attributed
to the resulting pH decrease of the suspension, as the presence of
GA significantly increases the activity. Instead, this increased activity
is due to the release of GA from the hydrogel into the suspension,
where it directly interacts with the bacteria. Moreover, we provide
evidence indicating that the pH dependency of the antimicrobial activity
is due to differences in GA protonation state by treating the pathogens
with GA solutions differing in their GA protonation distribution.
Finally, we show by LC–MS that there is no chemical or enzymatic
breakdown of GA. Overall, our results demonstrate that the pH influences
not only the physical but also the antimicrobial properties of the
GA hydrogels.