Characterization of Sunflower Oil Extracts from the Lichen Usnea barbata

Abstract

The increasing global emergence of multidrug resistant (MDR) pathogens is categorized as one of the most important health problems. Therefore, the discovery of novel antimicrobials is of the utmost importance. Lichens provide a rich source of natural products including unique polyketides and polyphenols. Many of them display pharmaceutical benefits. The aim of this study was directed towards the characterization of sunflower oil extracts from the fruticose lichen, Usnea barbata. The concentration of the major polyketide, usnic acid, was 1.6 mg/mL extract as determined by NMR analysis of the crude mixture corresponding to 80 mg per g of the dried lichen. The total phenolics and flavonoids were determined by photometric assays as 4.4 mg/mL (gallic acid equivalent) and 0.27 mg/mL (rutin equivalent) corresponding to 220 mg/g and 13.7 mg/g lichen, respectively. Gram-positive (e.g., Enterococcus faecalis) and Gram-negative bacteria, as well as clinical isolates of infected chickens were sensitive against these extracts as determined by agar diffusion tests. Most of these activities increased in the presence of zinc salts. The data suggest the potential usage of U. barbata extracts as natural additives and mild antibiotics in animal husbandry, especially against enterococcosis in poultry.

Keywords: Enterococcus faecalis; Usnea barbata; cytotoxicity; enterococcosis; flavonoids; multidrug resistant bacteria; natural antimicrobial; phenolics; usnic acid.

Figure 1

Chemical structures of (+)-usnic acid (A) and vulpinic acid (B).

Figure 2

(A) ¹H-NMR spectrum of dimethyl fumarate (DMFum). The singlet signal (6.89 ppm) of the alkene moiety is indicated by the red circle; (B) ¹H-NMR spectrum of usnic acid (UA). The well separated singlet signal of the OH proton at C-8 (13.33 ppm) is indicated by the blue circle; (C) ¹H-NMR spectrum of a 1:1 molar mixture (DMFum/UA), showing the expected integral ratio of the DMFum and UA signals of approximately 2:1; (D) ¹H-NMR spectrum of U. barbata extract using sunflower oil as the extractant; (E) ¹H-NMR spectrum of a commercial U. barbata CO₂ extract (Flavex^®). All spectra (except for (B)) are scaled to the same height of the DMFum signals.

Figure 3

Antimicrobial activity of the zinc salt of extract (1) and the total sunflower-oil extract (2) of U. barbata. (A) Staphylococcus aureus (RCMB010010); (B) Micrococcus sp. (RCMB 028); (C) Enterobacter cloacae (RCMB 001, ATCC 23355); (D) Bacillus subtilis (RCMB 015); (E) Staphylococcus epidermidis (RCMB 009); (F) Micrococcus spp (RCMB 028); (G) Proteus vulgaris (RCMB 004, ATCC 13315); (H) Bacillus cereus (RCMB 027).

Figure 4

Antimicrobial activity of U. barbata preparations. (A) Size of the inhibition zone of the sunflower-oil U. barbata extract and its zinc salt on Gram-positive and Gram-negative reference strains; (B) In vitro effect of the salt on S. aureus (n = 10), Salmonella spp (n = 5), E. coli (n = 10), and E. faecalis (n = 10) strains isolated from chickens. Not effective (very small inhibition zone) ≤8 mm, low effective (small inhibition zone) = 8–13, moderately effective (medium inhibition zone) = 14–19 mm, and highly effective (large inhibition zone) ≥20 mm. Sunflower oil was used as negative control and did not cause a zone of inhibition.

Figure 5

The lichen U. barbata used in this study. Whole (left); Macerated (middle); Powder (right).