Antibacterial Fractions from Erodium cicutarium Exposed-Clinical Strains of Staphylococcus aureus in Focus

Abstract

Followed by a buildup of its phytochemical profile, Erodium cicutarium is being subjected to antimicrobial investigation guided with its ethnobotanical use. The results of performed in vitro screening on Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans strains, show that E. cicutarium has antimicrobial activity, with a particular emphasis on clinical S. aureus strains-both the methicillin sensitive (MSSA) and the methicillin resistant (MRSA) S. aureus. Experimental design consisted of general methods (the serial microdilution broth assay and the agar well diffusion assay), as well as observing bactericidal/bacteriostatic activity through time (the "time-kill" assay), investigating the effect on cell wall integrity and biofilm formation, and modulation of bacterial hemolysis. Observed antibacterial activity from above-described methods led to further activity-guided fractionation of water and methanol extracts using bioautography coupled with UHPLC-LTQ OrbiTrap MS⁴. It was determined that active fractions are predominantly formed by gallic acid derivatives and flavonol glycosides. Among the most active phytochemicals, galloyl-shikimic acid was identified as the most abundant compound. These results point to a direct connection between galloyl-shikimic acid and the observed E. cicutarium antibacterial activity, and open several new research approaches for future investigation.

Keywords: Erodium cicutarium; MRSA; anti-hemolytic; bioautography; biofilm; fractionation; galloyl-shikimic acid; phenolic composition.

Figure 1

“Time-kill” assay of E. cicutarium water (Podvinje-W) and methanolic (Podvinje-M) extracts from the Podvinje locality on MSSA and MRSA strains (CFU, colony forming unit).

Figure 2

Bacterial viability reduction (%) in the “time-kill” assay for E. cicutarium water (Podvinje-W) and methanolic (Podvinje-M) extracts from the Podvinje locality on MSSA and MRSA strains at predefined time points t₀, t₁, t₃, t₆, t₉ i t₂₄ (one-way ANOVA and Tukey’s post hoc test; *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001).

Figure 3

S. aureus ATCC 6538 protein leakage after cell integrity loss when treated with E. cicutarium methanolic extract from the Podvinje locality (Podvinje-M) and Triton-X100, compared to a negative control (N = 3; mean ± S.D; one-way ANOVA and Tukey’s post hoc test; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.000).

Figure 4

Anti-hemolytic activity of E. cicutarium water (Podvinje-W) and methanolic (Podvinje-M) extracts from the Podvinje locality on MSSA and MRSA strains (*, statistically significant difference among c₁ and c₂; c₁ = MIC/2; c₂ = MIC/4; p ≤ 0.01; t-test).

Figure 5

(A) Bioautography in situ—chromatograms of water (W) and methanolic (M) E. cicutarium extracts from the Podvinje locality (applied in triplicate) covered with inoculated MHA with S. aureus ATCC 6538 (1.5 × 10⁶ CFU/mL) and stained with bacterial viability indicator TTC (1%, m/V). Zones of bacterial growth inhibition are yellow, while the parts with viable bacteria are in red. (B) Chromatograms of water (W) and methanolic (M) E. cicutarium extracts of from the Podvinje locality (applied in triplicate) on TLC silica gel F₂₅₄ plates, developed with mobile phase acetonitrile/water/formic acid = 30:8:2 (v/v/v), and visualized with NSR (1%, m/V) at 366 nm. (C) Same chromatograms at 254 nm.