Depigmenting potential of lichen extracts evaluated by in vitro and in vivo tests

Abstract

Melanin is the main pigment of human skin, playing the primary role of protection from ultraviolet radiation. Alteration of the melanin production may lead to hyperpigmentation diseases, with both aesthetic and health consequences. Thus, suppressors of melanogenesis are considered useful tools for medical and cosmetic treatments. A great interest is focused on natural sources, aimed at finding safe and quantitatively available depigmenting substances. Lichens are thought to be possible sources of this kind of compounds, as the occurrence of many phenolic molecules suggests possible effects on phenolase enzymes involved in melanin synthesis, like tyrosinase. In this work, we used four lichen species, Cetraria islandica Ach., Flavoparmelia caperata Hale, Letharia vulpina (L.) Hue, and Parmotrema perlatum (Hudson) M. Choisy, to obtain extracts in solvents of increasing polarity, viz. chloroform, chloroform-methanol, methanol, and water. Cell-free, tyrosinase inhibition experiments showed highest inhibition for L. vulpina methanol extract, followed by C. islandica chloroform-methanol one. Comparable results for depigmenting activities were observed by means of in vitro and in vivo systems, such as MeWo melanoma cells and zebrafish larvae. Our study provides first evidence of depigmenting effects of lichen extracts, from tyrosinase inhibition to cell and in vivo models, suggesting that L. vulpina and C. islandica extracts deserve to be further studied for developing skin-whitening products.

Keywords: Cetraria islandica; Letharia vulpina; Lichen secondary metabolites; Melanogenesis; Tyrosinase; Zebrafish.

Figure 1. Percent inhibition of mushroom tyrosinase activity induced by lichen extracts.

Percent inhibition of mushroom tyrosinase activity, induced by chloroform (A), chloroform-methanol (B), methanol (C) and water (D) extracts, obtained from Letharia vulpina, Cetraria islandica, Parmotrema perlatum, and Flavoparmelia caperata.

Figure 2. Bioautography assay of the lichen extracts along with TLC profile visualized at 350 nm.

Representative lanes of TLC plates subjected to bioautography assay of the chloroform-methanol extract of C. islandica (A) and of the methanol extract of L. vulpina (B). For each lichen species, TLC plates have been observed under fluorescence at 350 nm (right lane), and under visible light after bioautography (left lane), showing white spots corresponding to compounds with tyrosinase inhibition (see Materials and Methods for technical details).

Figure 3. Melanin assay performed on MeWo melanoma cells exposed to lichen extracts.

Melanin assay performed on MeWo melanoma cells exposed for 72 h to different concentrations of C. islandica chloroform-methanol (above), or L. vulpina methanol (below) extracts. Arbutin (8 mM) was used as positive control. Data are means ± s.d. of 505 nm absorbances derived from three independent samples read in duplicate.

Figure 4. Effects of melanogenic inhibition on the pigmentation of zebrafish treated with C. islandica extracts.

Effects of melanogenic inhibition on the pigmentation of zebrafish treated from 8 hpf to 56 hpf with C. islandica extract. All images are oriented to have rostral to the right and dorsal at the top. Bright field images of embryos treated with (A) arbutin (10 mM) used as a positive control; untreated zebrafish used as negative control (B); C. islandica chloroform-methanol extract: 5 µg/ml (C); 25 µg/ml (D); 50 µg/ml (E); 65 µg/ml (F). Scale bar: 100 µm.

Figure 5. Effects of melanogenic inhibition on the pigmentation of zebrafish treated with L. vulpina extracts.

Effects of melanogenic inhibition on the pigmentation of zebrafish treated from 8 hpf to 56 hpf with L. vulpina extract. All images are oriented to have rostral to the right and dorsal at the top. Bright field images of embryos treated with (A) arbutin (10 mM) used as a positive control; untreated zebrafish used as negative control (B); L. vulpina methanol extract: 6 µg/ml (C); 12 µg/ml (D), 22.5 µg/ml (E), 45 µg/ml (F). Scale bar: 100 µm.

Figure 6. Dose response curves obtained with image analysis data of zebrafish pigmentation after exposure to different lichen extracts.

Dose response curves obtained with image analysis data of zebrafish pigmentation after exposure to different lichen extracts (see Figs. 4–5): (A) C. islandica chloroform-methanol extract, (B) L. vulpina methanol extract. Dots indicate means ± s.d., n = 12 − 60; continuous lines indicate logistic regression curves for determination of IC₅₀ values. * =p < 0.01 according to Dunnett’s test.