Abundant Extractable Metabolites from Temperate Tree Barks: The Specific Antimicrobial Activity of Prunus Avium Extracts

Abstract

Tree barks are mainly considered as wood wastes from forestry activities, but represent valuable resources as they may contain antimicrobial compounds. Here, we aimed to evaluate the possible antimicrobial activities of bark extracts and to characterize the chemical composition of the most active extract. Ten methanol bark extracts were tested in vitro against 17 bacterial strains and 5 yeast strains, through minimum inhibitory concentration (MIC) and minimum bactericidal (or fungicidal) concentration (MBC/MFC) assays. The extract from Prunus avium (E2-4) displayed the largest bactericidal activity against Gram-positive bacteria, with a lethal effect on 6 out of 8 strains. Antibiofilm assays of E2-4 were performed by crystal violet staining and enumeration of adhered bacteria. Assays demonstrated a biofilm inhibitory effect of E2-4 against Staphylococcus aureus CIP 53.154 at concentrations equal to or higher than 250 µg/mL. Chemical profiling of E2-4 by ¹³C nuclear magnetic resonance (NMR) revealed the presence of dihydrowogonin as a major constituent of the extract. E2-4 was fractionated by centrifugal partition chromatography and the three fractions containing dihydrowogonin were tested for their antibacterial and antibiofilm activities, revealing similar activities to those of E2-4. Dihydrowogonin was positively assessed as an interesting antimicrobial compound, which could be valued from wastes of Prunus avium barks.

Keywords: 13C nuclear magnetic resonance chemical profiling; Prunus avium; antibiofilm activity; antimicrobial activity; barks; centrifugal partition chromatography; dihydrowogonin; natural products.

Figure 1

Antibiofilm activity of Prunus avium bark extract (E2-4) against S. aureus and P. aeruginosa, after 24h of contact. (a) Evaluation of the planktonic growth of bacteria through optical density measurement at 600 nm. (b) Evaluation of the total biofilm biomass through crystal violet coloration and absorbance measurement at 595 nm. (c) Concentration of bacteria adhered on coverslips, after ultrasonication and culture on agar plates. * indicates a significant difference (P < 0.05) between the condition and the control without E2-4 (0 µg/mL). a: 11 replicates did not present any adhered bacteria. b: 6 replicates did not present any adhered bacteria.

Figure 2

Hierarchical clustering analysis of ¹³C NMR signals detected in all centrifugal partition chromatography (CPC) fractions obtained from the MeOH bark extract of Prunus avium (E2-4).

Figure 3

Antibacterial and antibiofilm activity E2-4 fractions containing dihydrowogonin as the major compound (F1, F2 and F3) against S. aureus after 24 h of contact. Hatched bars: no fraction. (a) Evaluation of the planktonic growth of bacteria through optical density measurement at 600 nm. (b) Evaluation of the total biofilm biomass through crystal violet coloration and absorbance measurement at 595 nm. (c) Concentration of bacteria adhered on coverslips, after ultrasonication and culture on agar plates.