In Vitro Human Skin Penetration, Antioxidant and Antimicrobial Activity of Ethanol-Water Extract of Fireweed (Epilobium angustifolium L.)

Abstract

Epilobium angustifolium L. is applied as an antiseptic agent in the treatment of skin diseases. However, there is a lack of information on human skin penetration of active ingredients with antioxidative potential. It seems crucial because bacterial infections of skin and subcutaneous tissue are common and partly depend on oxidative stress. Therefore, we evaluated in vitro human skin penetration of fireweed ethanol-water extracts (FEEs) by determining antioxidant activity of these extracts before and after penetration study using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and Folin-Ciocalteu methods. Microbiological tests of extracts were done. The qualitative and quantitative evaluation was performed using gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC-UV) methods. The in vitro human skin penetration using the Franz diffusion chamber was assessed. The high antioxidant activity of FEEs was found. Gallic acid (GA), chlorogenic acid (ChA), 3,4-dihydroxybenzoic acid (3,4-DHB), 4-hydroxybenzoic acid (4-HB), and caffeic acid (CA) were identified in the extracts. The antibacterial activities were found against Serratia lutea, S. marcescens, Bacillus subtilis, B. pseudomycoides, and B. thuringiensis and next Enterococcus faecalis, E. faecium, Streptococcus pneumoniae, Pseudomonas aeruginosa, and P. fluorescens strains. In vitro penetration studies showed the penetration of some phenolic acids and their accumulation in the skin. Our results confirm the importance of skin penetration studies to guarantee the efficacy of formulations containing E. angustifolium extracts.

Keywords: Franz cell; antibacterial activity; antioxidants; herbal extract; phenolic acids; skin.

Figure 1

GC-MS chromatogram of the FEE.

Figure 2

The IR spectrum of the FEE.

Figure 3

Chromatogram of phenolic acid identified in the FEE: gallic acid (1), 3,4-dihydroxybenzoic acid (2), 4-hydroxybenzoic acid (3), chlorogenic acid (4), and caffeic acid (5).

Figure 4

Photographs depicting the FEE reaction to limiting the growth of bacteria from genus Serratia sp. (A) and Bacillus sp. (B).

Figure 5

Mean effect of the different dose of the FEE on tested strains (A), mean susceptibility of the tested strains on the FEE (B). S.l: Serratia lutea; S.m: Serratia marcescens; E.f: Enterococcus faecalis; E.f: Enterococcus faecium; S.p: Streptococcus pneumoniae; P.a: Pseudomonas aeruginosa; P.f: Pseudomonas fluorescens; B.s: Bacillus subtilis; B.p: Bacillus pseudomycoides; B.t: Bacillus thuringiensis. Different letters: values differ significantly between analyzed samples.

Figure 6

Cluster analysis graph for mean antimicrobial activity of the FEE.

Figure 7

The HPLC chromatogram of acceptor fluid (A) and fluid after skin extraction (B), after 24-h penetration of the FEE.

Figure 8

Cumulative mass of phenolic acids in the acceptor fluid during the 24-h penetration (A) and the penetration rate (B) of phenolic acids through the skin during the 24-h experiment, n = 6.

Figure 9

Correlations between the cumulative mass of phenolic acids ChA, GA, 3,4-DHB and the antioxidant activity (DPPH, ABTS) of the acceptor fluid collected during the 24-h permeation study: (A) DPPH vs. ChA mass, (B) DPPH vs. GA mass, (C) DPPH vs. 3,4-DHB mass, (D) ABTS vs. ChA mass, (E) ABTS vs. GA mass and (F) ABTS vs. 3,4-DHB mass. The correlations are presented only for phenolic acids penetrated to the highest degree.