Screening of Lichen Extracts Using Tyrosinase Inhibition and Toxicity Against Artemia salina

Nine lichen extracts were evaluated for tyrosinase inhibition and toxicity against Artemia salina larvae. Extract compositions were investigated by TLC and NMR analysis. The activity of constituents against tyrosinase was evaluated by bioautography, and the percent of inhibition was calculated based on the dopachrome produced during a set time interval. Cladia aggregata, Cladonia dimorphoclada, Stereocaulon ramulosum and Stereocaulon microcarpum extracts were active for tyrosinase inhibition. Barbatic, usnic, anziaic acids and an unidentified compound present in the extracts, are possibly responsible for tyrosinase inhibition. Cladia aggregata, Cladonia crispatula, Cladonia furcata, Lobaria erosa, Punctelia canaliculata and S. microcarpum proved to be less toxic to A. salina (LC50 > 500 g/mL) than Cladonia confusa and S. ramulosum (LC50 45.0 and 100.3g/mL, respectively), while the extract of C. dimorphoclada was highly toxic (LC50 < 10 g/mL).

Phenolic substances produced by plants or lichens have shown inhibition/activation effects on the activity of enzymes such as phenolases, including tyrosinase [8][9][10]. This enzyme catalyzes a step in the biosynthesis of melanin, which is responsible for skin, eye, and hair pigmentation in a number of animal orders and for cuticle formation in insects [11][12][13]. More recent studies have been conducted on the role of tyrosinase in Parkinson's and other degenerative diseases, and as a tool for the treatment of melanoma [14][15]. Oxidization of o-diphenols to o-quinones catalyzed by phenolases can cause browning in some vegetables and fruits, with consequent losses in visual attractiveness and nutritional quality. Tyrosinase inhibitors have become important in medicine and the cosmetic industry, to prevent hyperpigmentation; in agriculture and public health, in the development of new insecticides; and for applications in the food industry [16][17][18]

General procedures
NMR analysis -1 H, 13 C, and DEPT-135 NMR spectra were obtained in DMSO-d6 and CDCl3. Chemical shifts were calibrated using the solvent signal as reference. All NMR experiments were conducted on a Bruker Advance DPX300 instrument (operating at 300.13 MHz for 1 H and 75.48 MHz for 13 C). The absorbance was measured on a Bioespectro SP220 instrument. TLC -The extracts were chromatographed on aluminum plates coated with GF254 silica gel (0.20 mm, Macherey-Nagel), using the following eluents: (I) toluene : dioxane : acetic acid, 180:45:5 v/v/v; (II) toluene : acetic acid, 85:15 v/v [19]. The spots were visualized under UV (254 nm) and then sprayed with methanol: sulfuric acid (10%) and heated until complete appearance of spots, followed by p-anisaldehyde: sulfuric acid and reheating. Migration of substances was expressed as retention fator (Rf). Tyrosinase enzyme inhibition -Mushroom tyrosinase (Agaricus bisporus) and L-3,4dihydroxyphenylalanine (L-DOPA) were purchased from Sigma-Aldrich and kept below -10 C. Analytical-grade organic reagents were acquired from Synth, Aldrich and Tedia. Fragments of each lichen (240.0-960.0 mg) were cleaned, fragmented, and exhaustively extracted with acetone at room temperature. After solvent evaporation, the extracts were kept in a desiccator. Yields ranged from 2.7% to 8.2%. TLC and NMR methods were employed for analysis of the extracts. Supplementary Material available: NMR data ( 1 H, 13 C and Dept-135) of the extracts.

Tyrosinase enzyme inhibition
All aqueous solutions were prepared with deionized water on the first day of the experiment, then frozen, and used within 3 days. Phosphate buffer (Na2HPO4-NaH2PO4) at 66.7 mmol/L, pH 6.8, was used in all reactions.

Bioautography assay
In order to identify the compounds responsible for tyrosinase inhibition, TLC assays were performed. Extracts (100 μg) of each lichen were solubilized in acetone and spotted on Si-gel plates that were eluted in appropriate solvent mixtures. After development of chromatograms and total elimination of solvents, a tyrosinase solution (100 g/mL in 0.066 M phosphate buffer, pH 6.8) was spread on the plates followed by spray of a L-DOPA solution (0.3 % m/v in distilled water) in order to identify the compounds responsible for tyrosinase inhibition. The positive results (spots with tyrosinase inhibitor appeared white against a brownish-purple background) were observed and photographed until color shifts were no longer perceived.

Determination of enzyme activity
In a glass cuvette, 1.6 mL of tyrosine solution (24 µg/mL in phosphate buffer) and 0.2 mL of substrate (0.6 mM L-DOPA in water) were mixed, and absorbance was measured at 475 nm every 30 s for 5 min, to calculate the rate of dopachrome formation ( = 3.700 mol/L cm) [20]. The enzyme solution was used as the control.

Effect of solvent on enzyme activity
To evaluate the effect of DMSO on enzyme activity, the reaction was prepared with 1.6 mL of a solution containing 24 μg/mL of enzyme and 0.2 mL of DMSO. After 3 min of contact between enzyme and solvent, absorbance readings were taken. The enzyme solution was used as a blank. A 0.2 mL aliquot of L-DOPA solution (0.6 mM) was subsequently added to each cuvette and the mixture was stirred. Only the enzyme solution and solvent were employed in the blank. Readings were taken in triplicate every 30 s for 5 min.

Evaluation of extract effect on enzyme activity
The extracts of Stereocaulon microcarpum, S. ramulosum, Cladia aggregata, and Cladonia dimorphoclada, which showed higher inhibition potential in the bioautography assay, were selected for quantitative evaluation of the inhibition of enzymatic activity. A 0.2 mL aliquot of extract (2 mg/mL in DMSO) was added to 1.6 mL of enzyme solution (24 µg/mL), and the absorbance read after 3 min, followed by addition of 0.2 mL of L-DOPA (0.6 mM). Absorbance was recorded every 30 s for 5 min. A solution of kojic acid in DMSO (2 mg/mL) was used as positive control. A blank experiment without the compounds was also carried out. All the experiments were performed in triplicate at 28-30 C. Percent inhibition (%I) was calculated based on absorbance in the blank system (B) and absorbance in the system (A) after 5 min of reaction, applying the formula [(B -A)/B] × 100. Graphs were constructed using Origin 6.0 software.

Toxicity to Artemia salina
Approximately 100 mg of A. salina eggs were transferred to 1 L of saline solution (38 g/L). After hatching (approximately 48 h), the larvae were transferred to solutions at a ratio of 10 larvae per vial. The extracts (solubilized in saline solution containing 3% DMSO) were evaluated at concentrations of 500, 400, 200, 150, 100, 50, 20, and 10 g/mL. Quinidine sulfate was used as the positive control; the saline solution was the negative control. Survivors were counted after 24 h [21]. The results were employed to determine LC50 (95% confidence interval) using the probit method [22]. Extracts with LC50 < 500 g/mL were considered toxic.

RESULTS AND DISCUSSION
The chemical profiles of the lichens extracts were determined by TLC and NMR analysis. Figure 1 shows the chromatograms of the extracts. The tyrosinase inhibition effects of the lichens extracts were initially assessed by bioautography, and the extracts with promising activities were subjected to quantitative evaluation. All extracts were evaluated for toxicity against A. salina, widely employed for preliminary assessment of the toxicity of extracts and pure substances. Figure 2 shows the structures of the compounds identified in the extracts. Lobaria erosa contains gyrophoric acid, identified by TLC and NMR spectra. The 1 H NMR spectrum exhibits signals at 2.35, 2.43, and 2.50 ppm (methyl groups) and six ArH signals (6.22-6.67 ppm). 13 Narui et al. (1998) [23]. Further to gyrophoric acid, the NMR spectra showed signals indicative of alditols and waxes, as well as low-intensity signals attributed to the depside atranorin.

Protolichesterinic acid
Cladonia confusa contains perlatolic acid and usnic acid, while Stererocaulon ramulosum contains perlatolic acid, atranorin and a compound (RfI = 0.50) that migrates slightly less than perlatolic acid. Perlatolic acid was identified by comparison between its chromatographic behavior and the isolated perlatolic acid obtained in our laboratory. According to Sipman [24], S. ramulosum also contains a compound that migrates slightly less than perlatolic acid and probably corresponds to anziaic acid, which differs from perlatolic acid only by demethylation at C-4 ( Figure 2). Punctelia canaliculata produces atranorin, protolichesterinic acid, and an aliphatic acid possibly similar to caperatic acid [25]. 1 H NMR spectrum of the extract showed an intense signal at 1.22 ppm and others at 0.84 and 1.63 (dublet), in addition to a large signal at 4.39 ppm, suggesting an aliphatic chain structure. Two singlets, at 6.2 and 5.9 ppm, indicated the presence olefinic hydrogens. These signals could be assigned to (+) and ( ̶ ) isomers of the protolichesterinic acid. The spectrum contains no signals attributable to other aliphatic compounds, unless these occur in trace amounts. Stictic acid is the main component of the S. microcarpum extract, in addition to norstictic acid, atranorin, and other unidentified compounds. Its 1 H NMR spectrum exhibited signals corresponding to two aromatic methyl groups (2.17 and 2.49 ppm), one methoxy group (3.90 ppm), one aromatic hydrogen (7.09 ppm), and an aldehyde hydrogen (10.20 ppm). Two signals, at 8.20 and 6.64 ppm, correspond to a hydroxyl and a lactol hydrogen, respectively, indicating a butyrolactone unit. The 13 C and DEPT-135 NMR spectra exhibit signals at 9.59, 21.57, 56.8, and 186.79 ppm that correspond to two methyl, one methoxyl, and one aldehyde group, in addition to other signals indicative of the stictic acid structure [26]. The spectra showed lower-intensity signals indicative of other substances in the S. microcarpum extract. Cladia aggregata contains barbatic acid (figures 1 and S9-S11). Usnic acid is the principal compound in Cladonia dimorphoclada (figures 1 and S16-S17). Fumarprotocetraric acid is present in C. furcata (figures 1 and S6-S8).
Thamnolic acid is present in Cladonia crispatula, whose 13 C and DEPT-135 NMR spectra showed signals corresponding to two ArCH3 groups (14.4 and 21.8 ppm), one ArOCH3 (56.7 ppm), one ArH (106.0 ppm), and one ArCHO group (194.4 ppm). The 13 C spectrum exhibits signals at 170.3 and 171.7 ppm corresponding to two carboxyl groups, in addition to other signals that suggest thamnolic acid as the principal compound in the C. crispatula extract [26].
The preliminary bioautography assay for tyrosinase using 100 g of each extract indicated that the anziaic, usnic and barbatic acids and an unidentified compound (RfI = 0.24), were the active compounds present in S. ramulosum, Cladonia dimorphoclada, Cladia aggregata, and S. microcarpum extracts (Figure 3), respectively. Once it was possible to assess which extract were active and the substances responsible for activity, quantitative assessment of enzyme inhibition were performed. The relationship between enzyme activity (9.10 -3 mmol/min/mg) and inhibition was expressed by the concentration of dopachrome present in the reaction medium as a function of time. Figure 4 shows the effect of enzyme inhibition by the extracts (at a concentration of 200 g/mL) and the concentration of dopachrome formed (mmol/L). This assay showed that the extract of S. microcarpum caused stronger inhibition of the enzyme (32.4%), possibly by action of the unidentified compound (RfI = 0.24). The extracts of S. ramulosum and Cladia aggregata had similar inhibitory effects (23.8% and 21.5%, respectively) and the extract of Cladonia dimorphoclada was less active in inhibiting tyrosinase (16.1%) ( Table 1)   The toxicity of the extracts on A. salina was determined. In this assay, Cladonia crispatula, Cladia aggregata, L. erosa, P. canaliculata, Cladonia furcata, and S. microcarpum extracts exhibited low toxicity against microcrustacean larvae, with LC50 > 500 μg/mL, whereas the extracts of Cladonia confusa and S. ramulosum showed LC50 values of 45.0 and 100.3 μg/mL, respectively. The extract of C. dimorphoclada proved highly toxic, with LC50 < 10 μg/mL (Table 1).
These are promising results, given the inhibitory activity of S. microcarpum and C. aggregata extracts on tyrosinase and their low toxicity against A. salina. Although some of the extracts failed to inhibit tyrosinase, the compounds present in the species investigated are known for other important properties. Gyrophoric acid is a potent protein tyrosine phosphatase 1B (PTP1B) inhibitor, with an IC50 value of 3.6 ± 0.04 M. Selective inhibition of this enzyme is a target for the treatment of type-2 diabetes and obesity [27]. This acid is also a potent antiproliferative agent against growth of human keratinocytes (HaCaT cells), with an IC50 value of 1.7 M, and a topoisomerase I inhibitor (25 M) [28][29]. Pandey et al. [30], reported the anti-Malassezia action of the ethanolic extract of C. aggregata against three fungal species that cause pityriasis versicolor and seborrheic dermatitis. Atranorin and, more effectively, usnic acid inhibit cell proliferation and induce cell death, as revealed by evaluating these compounds against nine human cancer cell lines [31]. The cytotoxicity mechanisms of these compounds have been investigated [32]. Protolichesterinic acid has been reported as active against several tumor cell lines [33][34][35].

CONCLUSION
In conclusion, our results showed that, out of the nine extracts tested, four contained compounds that inhibit tyrosinase (S. ramulosum, Cladonia dimorphoclada, Cladia aggregata and S. microcarpum). The activity of these extracts can be attributed to anziaic, usnic, barbatic, and stictic acids, the principal compounds in these lichen species. Cladia aggregata and S. microcarpum were not toxic to A. salina (LC50 > 500 g/mL), while S. ramulosum and Cladonia dimorphoclada inhibited the enzyme (23.8% and 16.1%, respectively) and were toxic to A. salina (100.32 and <10 g/mL, respectively). The study was novel in revealing the inhibitory effects of the selected lichens extracts against tyrosinase. Further investigations of the effects of the isolated compounds are expected to identify the mechanisms and types of the inhibition processes.

ACKNOWLEDMENTS
K.G. wishes to thank PET (Brazil) for the student research grant. R.G.C. thanks CNPq (Brazil). A.A.S. and L.S.C. acknowledge the support from CNPq and FUNDECT.