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

Chemicals

All reagents were purchased from Sigma-Aldrich (Milan, Italy), unless otherwise indicated.

Lichen species and extract preparation

Thalli of F. caperata and of P. perlatum were collected in a woodland area of eastern Liguria (NW Italy), L. vulpina in a forest area of Valtournenche (NE Valle d’Aosta, Italy), and C. islandica was purchased from Kubja Ürditalu (Tallinn, Estonia). No permissions for lichen collection are required according to Italian legislation. Lichen materials were identified by one of us (PG) using microscope analysis with the help of identification keys and spot tests. Thereafter lichen material was cleaned from debris, left to dry at room temperature overnight, and stored in paper bags at room temperature until use.

Dried lichen thalli were extracted at room temperature (about 23 °C) with four solvent polarities from chloroform to water: chloroform, chloroform–methanol (9:1), methanol, and water (14.4 g of F. caperata in 70 mL of each solvent, 10.4 g of P. perlatum in 60 mL, 13.3 g of L. vulpina in 75 mL, and 100.6 g of C. islandica in 500 mL). Extractions were carried out for 5 days and 3 times for each solvent, with frequent agitation. The supernatant liquid was then filtered and evaporated to dryness under reduced pressure in a rotary system (Rotavapor Heidolph, Schwabach, Germany) to obtain dried extracts (Souza et al., 2016). Lichen extract yields are reported in Table 1.

Tyrosinase inhibition assay

Lichen extracts were dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 mg/ml. Extract stock solutions were then diluted in water in order to obtain a series of test solutions with final concentrations of 10, 50, 100, 250, 350 and 500 µg/ml. Components of the reaction mix were added to each well of 96-well plates in the following order: 70 µL of phosphate buffer, 60 µL of extract solutions (water for controls), 10 µL of mushroom tyrosinase (Sigma-Aldrich, T3824, 25 kU, 125 U/ml in phosphate buffer, pH 6.8) and 70 µL L-tyrosine (0.3 mg/ml in water). Kojic acid was used instead of lichen extracts as a positive control, at increasing concentrations from 0.5 to 500 µg/ml. Blank samples without enzyme were also included for all conditions. Plates were then incubated at 30 °C for 60 min and absorbance was read at 505 nm in a microplate reader (Spectra Max 340PC). Percent inhibitory activity (I%) was calculated according to the formula ${\displaystyle \mathrm{I}\text{\%}=\left[1-\frac{\left({\mathrm{A}}_{\mathrm{ex}∕\mathrm{en}}-{\mathrm{A}}_{\mathrm{ex}}\right)}{\left({\mathrm{A}}_{\mathrm{en}}-{\mathrm{A}}_{\mathrm{bk}}\right)}\right]\times 100}$

where A_ex∕en = absorbance of sample mixture with extract and enzyme; A_ex = absorbance of sample mixture with extract and without enzyme; A_en = absorbance of sample mixture with enzyme and without extract; A_bk = absorbance of sample mixture without enzyme and extract (blank).

TLC bioautography analysis

Conventional TLC chromatographic profile was conducted on 20 × 20 cm plates (Merck silica gel 60 F₂₅₄) following literature protocols (Culberson & Kristinsson, 1970; White & James, 1985). Briefly, samples of L. vulpina methanolic extract and of C. islandica chloroform-methanol extract were resolubilized in their extraction solvents and then spotted on a TLC plate using a capillary tube. TLC profile was carried out using toluene:acetic acid (200:30 ml) as a mobile phase (solvent C). After the solvent front was reached, the plate was left to dry at room temperature. Dried plates were examined and photographed initially in visible light (daylight) for detecting pigments as colored spots, and then in fluorescence light, using 254 and 350 nm excitations.

To visualize the presence of tyrosinase inhibition activity in each spot, TLC plates were sprayed with L-tyrosine solution (about 2.5 × 10⁻⁵ mmol/cm²) and then with tyrosinase solution (about 3.6 U/cm²). Spots with tyrosinase inhibitory activity appeared white on a dark background (Wangthong et al., 2007).

Cell culture, cell viability, and melanin assays

The MeWo human melanoma cell line (cat. HTB-65, ATCC, Manassas, VA, USA, https://www.lgcstandards-atcc.org/products/all/HTB-65.aspx) was used for cell viability assay, as reported by Pastorino et al. (2017), and for melanin assay, as described by Cornara et al. (2018).

Briefly, for cell viability assay, cells were settled in 96-well plates, exposed for 48 h to a logarithmic series of lichen extract concentrations, probed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reaction, and read at 550 nm in a microplate reader (Spectra Max 340 PC). For melanin assay, cells were settled in 24-well plates, exposed in triplicate to lichen extracts, or arbutin (8 mM) as positive control, washed with PBS, trypsinized, centrifuged, freeze-thawed, dissolved in 1 N NaOH and read at 505 nm in the microplate reader. In particular, non-cytotoxic concentrations were tested: 10 and 50 µg/ml for L. vulpina, and 25 and 50 µg/ml for C. islandica.

Zebrafish depigmentation assay

Adult zebrafish (Danio rerio) were obtained from a commercial dealer and kept in a circulating system with water conductivity of 500–530 Ω/cm at 27 °C, pH 7.0–7.5 under a constant 14/10 light/dark photoperiod. Veterinary care of the animals was carried out according to the Italian law (D.to L.Vo 26/2014) and the experiments were approved by the Institutional Ethics Review Body (University of Genoa) and the Italian ministry of Health (authorization 720/2015-PR). Spawning of adult zebrafish was performed according to standard methods. In particular, synchronized embryos were obtained from natural spawning induced in the morning by turning on light. Embryos were arrayed in 6-well plates containing 2 ml of embryo medium and 15 embryos per well. Extract stock solutions were diluted to the desired concentrations with embryo medium just before use (L. vulpina methanol extract: 6–45 µg/ml; C. islandica chloroform-methanol extract: 5–65 µg/ml). Diluted extracts were added to each well and incubated from 8 to 56 hpf (hours post-fertilization), resulting in 48 h exposure. Arbutin 10 mM was used as positive control. Replacement of the medium was done every 24 h to ensure even distribution of the test compounds. Embryos at 56 hpf were dechorionated by forceps, anesthetized in tricaine methanesulfonate solution (Sigma-Aldrich) and then photographed under a stereomicroscope (Leica M205C). The effects on pigmentation were evaluated using the ImageJ software v. 1.74. The pixel measurement analyzer function was used to evaluate the area of zebrafish pigmentation.

Statistical analysis

Dose–response curves obtained by tyrosinase inhibition, MTT, and zebrafish pigmentation data, were analyzed by a logistic regression model, yielding IC₅₀ and IC₀₅ values that were assumed as median and threshold levels, respectively. Statistic comparisons were done with R 3.0.1 (R Core Team, 2013) environment, using ANOVA test, t Student with Bonferroni’s correction and Dunnett’s tests for multiple comparisons.

Effects on tyrosinase activity

Lichen extracts showed a complex of modulatory effects on mushroom tyrosinase activity, evaluated in vitro in a cell-free assay. Some extracts induced tyrosinase activation, notably F. caperata methanol and chloroform, and L. vulpina water extracts, others showed a biphasic behavior, while some were definitely inhibitory (Fig. 1 and Table 2). In particular, chloroform-methanol (Fig. 1B) and methanol extracts (Fig. 1C) showed on the whole stronger tyrosinase inhibition, with dose-dependent effects. The strongest inhibition activity was recorded for the methanol extract of L. vulpina (Fig. 1C), followed by the chloroform-methanol extract of C. islandica (Fig. 1B). The inhibitory activities of these extracts were the only ones that allowed to estimate IC₅₀ values with 95% CI (Table 2). As a positive control, in these experiments kojic acid induced tyrosinase inhibition with an IC50 of 13.9 µg/ml (95% confidence interval: 12.4–15.7).

Based on the results of cell-free experiments, we selected the L. vulpina methanol and C. islandica chloroform-methanol extracts for testing their depigmenting effects on melanoma cells as an in vitro model, as well as on zebrafish as an in vivo model.

Detection of tyrosinase inhibition by TLC bioautography

The TLC profile made it possible to show the main substances contained in each extract. An identification of these substances is not an aim of this work, but, as an example, the spot of vulpinic acid (yellow under visible light) in L. vulpina methanolic extract is clearly evident (Huneck & Yoshimura, 1996) (Fig. 2). Bioautography data revealed that several compounds exert inhibitory effects on tyrosinase activity. Most notably, the inhibitory activity of C. islandica chloroform-methanol extract was distributed among different bands covering a wide range of polarity (Fig. 2A). Conversely, the tyrosinase inhibitory activity of L. vulpina methanol extract is concentrated in one band that migrates close to the large yellow band corresponding to vulpinic acid (Fig. 2B).

Lichen extract depigmenting effects on MeWO cells

The MeWo human melanoma cell line was used as an in vitro model to explore lichen depigmenting effects. As a first step, cells were subjected to cell viability assay upon exposure for 48 h to increasing concentrations of L. vulpina methanol and C. islandica chloroform-methanol extracts. Dose–response curves of cell viability allowed deriving IC₅₀ values of 88 µg/ml (95% CI [68–113 µg/ml]) for L. vulpina, and of 264 µg/ml (95% CI [213–328 µg/ml]) for C. islandica. Threshold IC₀₅ values were 19 µg/ml (95% CI [9–40 µg/ml]) and 51 µg/ml (95% CI [31–85 µg/ml]), respectively.

Thereafter, the assay of melanin conducted on MeWo cells, after 72 h exposure to different lichen extract concentrations, showed a sharp reduction with respect to controls induced by both extracts. In these experiments, arbutin (8 mM) was used as a positive control, reducing the melanin content of cells to about 50% of controls. The methanol extract of L. vulpina induced a melanin reduction similar to that of arbutin, occurring already at a concentration as low as 10 µg/ml (Fig. 3). This concentration is lower than the threshold for cytotoxic effects measured for this extract, allowing to rule out the possibility of aspecific injurious effects on cells. A similar effect on the melanin content of cells was also observed for the C. islandica chloroform-methanol extract, but only at a concentration of 50 µg/ml (Fig. 3). However, also the effective concentration of this extract was lower than the threshold for cytotoxic effects.

Phenotype-based evaluation of depigmenting effects of lichen extracts using zebrafish

Zebrafish models were used to further substantiate in vivo the effects of the inhibition of melanogenesis by C. islandica and L. vulpina. In order to define the optimal concentration to use, first embryos were subjected to toxicity assay upon exposure for 48 h to increasing concentrations of L. vulpina methanol and C. islandica chloroform-methanol extracts.

Thereafter, we observed that, when treated with subtoxic doses of C. islandica and L. vulpina, zebrafish larvae had a reduction in the pigmentation (Figs. 4 and 5). The extract of L. vulpina showed higher inhibitory activity than C. islandica, as indicated by data from image analysis. Logistic regression curves yielded IC₅₀ values of 44 µg/ml (42–47 µg/ml) for the chloroform-methanol extract of C. islandica, and of 30 µg/ml (25–36 µg/ml) for the methanol extract of L. vulpina (Fig. 6). Finally, depigmenting activity of C. islandica and L. vulpina extracts was also evaluated in the zebrafish embryos by melanin assay (Supplemental Information).