Hydroethanolic Extract of Punica granatum Inhibits Cryptococcus by Depolarising Mitochondrial Membranes

Cryptococcal infections are distributed worldwide and mainly caused by Cryptococcus neoformans and Cryptococcus gattii. The reduced number of antifungals and increasing number of cases of resistance require the search for new therapeutic options, such as natural products. Among these, Punica granatum L. has demonstrated antifungal activity. The present study aimed to evaluate the in vitro activity of the hydroethanolic extract of the leaf of P. granatum (HEPg) alone or in antifungal combination against C. neoformans and C. gattii and the interference of P. granatum in the mitochondrial membrane of Cryptococcus using flow cytometry. The minimum inhibitory concentration was determined, which showed inhibitory activity against Cryptococcus isolates. The fractional inhibitory concentration resulted in an indifferent interaction between the combination of amphotericin B + HEPg, whereas the combination of fluconazole + HEPg was synergistic against C. gattii. The depolarisation of mitochondrial membranes was more pronounced when C. gattii was previously treated with P. granatum, either individually or in combination with antifungal agents. In contrast, prior treatment of C. gattii with fluconazole promoted the hyperpolarisation of mitochondrial membranes. Considering the growing search for alternative forms of treatment for cryptococcosis, this study highlights the antifungal potential of P. granatum.