The Antimicrobial and Antiviral Activity of Polyphenols from Almond (Prunus dulcis L.) Skin

Abstract

Due to their antimicrobial and antiviral activity potential in vitro, polyphenols are gaining a lot of attention from the pharmaceutical and healthcare industries. A novel antiviral and antimicrobial approach could be based on the use of polyphenols obtained from natural sources. Here, we tested the antibacterial and antiviral effect of a mix of polyphenols present in natural almond skin (NS MIX). The antimicrobial potential was evaluated against the standard American Type Culture Collection (ATCC) and clinical strains of Staphylococcus aureus, including methicillin-resistant (MRSA) strains, by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Herpes simplex virus type I was used for the antiviral assessment of NS MIX by plaque assay. Furthermore, we evaluated the expression of viral cascade antigens. NS MIX exhibited antimicrobial (MIC values of 0.31-1.25 mg/ml) and antiviral activity (decrease in the viral titer ** p < 0.01, and viral DNA accumulation * p < 0.05) against Staphylococcus aureus and HSV-1, respectively. Amongst the isolated compounds, the aglycones epicatechin and catechin showed the greatest activity against S. aureus ATCC 6538P (MIC values of 0.078-0.15 and 0.15 mg/ml, respectively), but were not active against all the other strains. These results could be used to develop novel products for topical use.

Keywords: S. aureus; almonds; antimicrobial; antiviral; herpes simplex virus; polyphenols.

Figure 1

Viability of Vero cells treated with NS MIX. Vero cells were incubated with 1.6, 0.8, 0.6, 0.4, 0.2, and 0.1 mg/mL of NS MIX for 72 h. DMSO was used as a negative control. Cells were then collected and their viability was determined using the ViaLight™ plus cell proliferation and cytotoxicity bioassay kit (Lonza Group Ltd, Basel, Switzerland). The luminescence value was converted into cellular proliferation index as described in Materials and Methods. Results represent the mean of three biological independent experiments ± SD.

Figure 2

Antiviral activity of NS MIX. Vero cells were pre-treated with 0.4, 0.2, and 0.1 mg/mL of NS MIX for 1 h and the cells were then infected with Herpes simplex virus type 1 strain F (HSV-1) for 1 h at 37 °C under gentle shaking. After incubation, the inoculum was removed and the cells were covered with medium containing 0.8% methylcellulose in the presence of NS MIX at 0.4, 0.2, and 0.1 mg/mL, separately. After three days the cells were fixed and stained with crystal violet and the plaques were visualized with an inverted microscope. (A) Plaque reduction assay following the NS MIX treatment. (B) Plaque morphological change due to the NS MIX treatment. Results are the mean of three biological independent experiments ± SD for each dilution. (C) Antiviral activity of acyclovir was tested in Vero cells infected with HSV-1 and incubated with medium containing 0.8% methylcellulose in the presence of acyclovir at 1, 10, and 20 μM. The cells were stained with crystal violet for plaque counting and detection. Data are expressed as a mean (± SD) of at least three experiments and asterisks (**, ***) indicate the significance of p-values less than 0.01, and 0.001, respectively.

Figure 3

NS MIX affected the expression of viral antigens and HSV-1 replication. (A) Normal phase contrast inverted micrographs of Vero cells treated with 0.4 mg/mL of NS MIX. The cells were pre-treated with NS MIX for 1 h at 37 °C, then cells either mock infected or infected with HSV-1 at multiplicity of infection (MOI) of 1 for 1 h and incubated in presence of 0.4 mg/mL of the NS MIX for 24 h. (B) Immunoblot analysis was performed to detect α (ICP0), β (UL42), and γ (US11) viral proteins. GAPDH protein was used as loading control. Band density indicated in the figure was determined with the TINA program (version 2.10, Raytest, Straubenhardt, Germany) and expressed as the fold change over the housekeeping gene GAPDH. (C) Relative quantization of viral DNA was performed using real-time quantitative PCR and analyzed by the comparative Ct method (ΔΔCt). Data are expressed as a mean (± SD) of at least three experiments and asterisk (*) indicate the significance of p-values less than 0.05.