Propolis Extract for Onychomycosis Topical Treatment: From Bench to Clinic

Abstract

Onychomycosis is a chronic fungal infection of nails, commonly caused by dermatophyte fungi, primarily species of Trichophyton. Because of the limited drug arsenal available to treat general fungal infections and the frequent failure of onychomycosis treatment, the search for new therapeutic sources is essential, and topical treatment with natural products for onychomycosis has been encouraged. Propolis, an adhesive resinous compound produced by honeybees (Apis mellifera), has shown multiple biological properties including significant antifungal and anti-biofilm activities in vitro. In spite of promising in vitro results, in vivo results have not been reported so far. This study assessed an ethanol propolis extract (PE) as a topical therapeutic option for onychomycosis, including its characterization in vitro and its applicability as a treatment for onychomycosis (from bench to clinic). The in vitro evaluation included analysis of the cytotoxicity and the antifungal activity against the planktonic cells and biofilm formed by Trichophyton spp. We also evaluated the capacity of PE to penetrate human nails. Patients with onychomycosis received topical PE treatments, with a 6-month follow-up period. The results of the in vitro assays showed that PE was non-toxic to the cell lines tested, and efficient against both the planktonic cells and the biofilm formed by Trichophyton spp. The results also showed that PE is able to penetrate the human nail. The results for PE applied topically to treat onychomycosis were promising, with complete mycological and clinical cure of onychomycosis in 56.25% of the patients. PE is an inexpensive commercially available option, easy to obtain and monitor. Our results indicated that PE is a promising natural compound for onychomycosis treatment, due to its ability to penetrate the nail without cytotoxicity, and its good antifungal performance against species such as Trichophyton spp. that are resistant to conventional antifungals, both in vitro and in patients.

Keywords: antifungal activity; dermatophytosis; ex vivo nail model; natural products; permeation property; photoacoustic spectroscopy; propolis.

FIGURE 1

Schematic diagram of placement of nail fragment with propolis extract (PE) in the photoacoustic cell. μs = thermal diffusion length (μm); L = initial sample thickness (μm); Ls = field sample thickness (μm).

FIGURE 2

Effect of propolis extract (PE) on HeLa and Vero cell lines. Viability determined by MTT assay. Concentrations of the PE extract ranged from 0.002 to 1.42% of total polyphenols. The results are presented as the average of three independent experiments performed in three replicates, ^∗p < 0.05 compared to control group.

FIGURE 3

In vitro biofilm inhibition by propolis extract (PE) measured through two methods: Crystal violet-staining biomass production (A) and Colony-Forming Units (B) of T. rubrum CMRP2912 and T. interdigitale CMRP2921 biofilms exposed to PE. ^∗p < 0.05 compared to untreated control group.

FIGURE 4

Photoacoustic spectra of: propolis extract (PE); dorsal surface of nail without PE application (Dorsal control); ventral surface of nail without PE application (Ventral control). Arrows indicate bands of optical absorption of PE. In right corner, the band centered at 670 nm.

FIGURE 5

Photoacoustic spectra of nail sample after 24 h of treatment with propolis extract (PE), for different thicknesses. Surface of application of PE (PE dorsal, 419 μm); first measurement without filing the layer on the side opposite to the application site (PE ventral. 419 μm); consecutive measurements after filing the nail layer on the side opposite to the application site until optical absorption bands of PE were detected (PE ventral. 357 μm; PE ventral. 293 μm; PE ventral. 240 μm; PE ventral. 193 μm; PE ventral. 148 μm). Arrows indicate the presence of characteristic bands of PE in 148-μm-thick nail layer, highlighting the band centered at 670 nm.

FIGURE 6

Flow chart of the experimental scheme of the study, from the number (in parentheses) of onychomycosis cases investigated to the successful outcome of treatment with PE. Treated patients, but in other health services where they received other medications and therefore were excluded from this project step. Treated with PE: number of patients who agreed to use PE as the only antifungal drug. Successful outcome: complete recovery of the anatomical appearance of the nail and absence of fungi in the sample.

FIGURE 7

Images showing patients with onychomycosis caused by Trichophyton spp. before and after treatment with PE. Patient with toenail onychomycosis before treatment with PE (A); nail scrapings cleared with KOH plus Parker’s blue–black permanent ink, observed by direct microscopy (B); complete resolution of the nail, fully recovered to its initial appearance (C); and no fungi present in the sample (D).