Psidium guajava L. hydroethanolic extract as endodontic irrigant: phytochemical analysis, antioxidant activity, antimicrobial action and biocompatibility

Abstract

Background: The search for novel antimicrobial agents in Endodontics is constant to overcoming persistent infections. <i>Psidium guajava</i> L. is a medicinal plant little explored in Endodontics. The aim of this study was to produce hydroethanolic extract of <i>P. guajava</i> L. and to evaluate its phytochemical composition, antimicrobial and antibiofilm action against standard and clinical strains of <i>Enterococcus faecalis</i> and <i>Candida albicans</i>, and cytotoxicity and genotoxicity on human keratinocyte cultures (HaCaT cells). The findings provide new insights into the potential of <i>P. guajava</i> as an alternative endodontic antimicrobial agent, contributing to the development of more effective and biocompatible therapeutic strategies. Methods: <i>P. guajava</i> hydroethanolic extract was produced using young leaves of guajava and extracted using absolute ethanol and ultrapure water in a ratio (30 g:100 mL). The solid soluble, total flavonoid and total phenols content were determined. The chemical composition was determined <i>via</i> high-performance liquid chromatography (HPLC) analysis, then the free radical suppressive activity was assessed by determining the IC₅₀ value, indicating the concentration required to eliminate 50% of free radicals. Later, the minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) of the extract was evaluated against the strains using clinical and laboratory standards institute (CLSI) guidelines (M27-S4 and M7-A9). Then, the antibiofilm activity was evaluated <i>via</i> MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Finally, the cytotoxicity of the extract was evaluated <i>via</i> Alamar Blue assay, and the genotoxicity <i>via</i> micronucleus assay on human keratinocyte cultures (HaCaT cells). Data were analyzed using ANOVA and Tukey's test or Kruskal-Wallis and Dunn's test. Results: The soluble solids content in the extract was 3.35%. Using the quercetin standard curve, the total flavonoid concentration was 0.130 ± 0.110 mg/mL. In addition, using standard curve for phenolic acids, the total phenolic concentration was 1.770 ± 1.540 mg/mL. HPLC analysis revealed peaks of rutin, quercetin and kaempferol as major flavonoids in the <i>P. guajava</i> L. extract. The extract demonstrated notable antioxidant activity, with an IC₅₀ value of 10.39 µg/mL. The MMC values ranged 1.04-8.37 mg/mL. The extract at 8.37 mg/mL was effective in reducing the biofilms of standard and clinical strains of <i>E. faecalis</i> and <i>C. albicans</i> after 10 min. Cytotoxicity analysis revealed that all tested concentrations exhibited relatively low toxicity toward HaCaT cells. Genotoxicity assessment <i>via</i> the micronucleus assay indicated minimal DNA damage at all tested concentrations. Overall, <i>P. guajava</i> L. hydroethanolic extract at 8.37 mg/mL is the most effective concentration in reducing the biofilm of the standard and clinical strains of <i>E. faecalis</i> and <i>C. albicans</i>, while maintaining biocompatibility with HaCaT cultures.