Depigmenting Potential Of Lichen Extracts Evaluated By in Vitro And in Vivo Tests Part 2
Apr 11, 2023
According to relevant studies,cistanche is a common herb that is known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides. Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase, and the oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.
In addition, cistanche also has the function of promoting collagen production, which can increase the elasticity and luster of the skin and help repair damaged skin cells. Cistanche Phenylethanol Glycosides have a significant down-regulating effect on tyrosinase activity, and the effect on tyrosinase is shown to be competitive and reversible inhibition, which can provide a scientific basis for developing and utilizing the whitening ingredients in Cistanche. Therefore, cistanche has a key role in skin whitening. It can inhibit melanin production to reduce discoloration and dullness; and promote collagen production to improve skin elasticity and radiance. Due to the widespread recognition of these effects of cistanche, many skin whitening products have begun to infuse herbal ingredients such as Cistanche to meet consumer demand, thus increasing the commercial value of Cistanche in skin whitening products. In summary, the role of cistanche in skin whitening is crucial. Its antioxidant effect and collagen-producing effect can reduce discoloration and dullness, improve skin elasticity and luster, and thus achieve a whitening effect. Also, the wide application of Cistanche in skin whitening products demonstrates that its role in commercial value cannot be underestimated.
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Detection of tyrosinase inhibition by TLC bioautography
The TLC profile made it possible to show the main substances contained in each extract. 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 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 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 concerning 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 specific 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. To define the optimal concentration to use, the 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 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 30 µg/ml (25–36 µg/ml) for the methanol extract of L. vulpina (Fig. 6). Finally, the depigmenting activity of C. islandica and L. vulpina extracts was also evaluated in the zebrafish embryos by melanin assay (Supplemental Information).
DISCUSSION
Our study highlighted a complex of in vitro and in vivo depigmenting effects due to specific lichens and extraction solvents. The strategy to choose separate extractions with a variety of solvent polarities, instead of a successive extraction with solvents of increasing polarities, was dictated by the pioneering aspect of the investigation. The study was aimed at disclosing widely available lichen species that could be exploited for their depigmenting effects, with a piece of very limited knowledge about the possible presence of active principles and their interactions. Therefore, we adopted an extract fractionation method that can involve some composition overlap among fractions but maximizes their depigmenting performance, possibly also due to synergistic effects.
As for tyrosinase inhibition in cell-free experiments, our results confirm data from Higuchi et al. (1993), showing tyrosinase inhibition rates of 40.4% for L. vulpina and 13.8% for C. islandica, concerning our 86.2% and 42.6%, respectively, possibly due to the use of cultured lichens and different extraction solvent. We showed the strongest activity for the methanol extract of L. vulpina, followed by the chloroform-methanol extracts of C. islandica. Thus, these extracts were used to explore the anti-melanogenic activity of melanoma cells and zebrafish larvae. Data obtained from these tests confirmed those from cell-free experiments, and in all cases, the methanol extract of L. vulpina induced the strongest effect.
In addition, bioautography assay indicates that different substances contained in these lichens exert tyrosinase inhibition. Although we did not perform a complete characterization of the extracts, we know from the literature the main lichen substances characterizing these lichens: L. vulpina contains atranorin and vulpinic acid, while C. islandica contains lichesterinic, protolichesterinic and fumarprotocetraric acid (Culberson, 1969). However, information regarding the antityrosinase activities of lichen substances in the literature is relatively poor, while only in a few cases it was possible to clarify the mechanisms of inhibition. Recently, Brandão et al. (2017) isolated fumarprotocetraric acid from the lichen Cladonia verticillate and showed uncompetitive, mixed-type inhibition on tyrosinase activity which rose with increasing concentration, at 0.6 mM the acid inhibited tyrosinase activity by 39.8%.
An element that makes comparisons among different lichen species a difficult task is the high variability of chemical composition, which is also subjected to the variation of environmental parameters, habitat, and microclimatic characteristics (e.g., availability of water and light) (Matteucci et al., 2017). These differences may underlie considerable differences in the biological activity of lichen phytocomplexes in which the composition has not been quantitatively characterized. Therefore, further work is necessary to isolate and quantify active compounds from the extracts to better define the components with antityrosinase activity. So far, several works have investigated the possible antityrosinase activity of lichen compounds (e.g., Kwong et al., 2020; Honda et al., 2016; Lopes, Coelho & Honda, 2018). For example, Kim & Cho (2007) determined that methanolic extracts of Usnea longissima and Usnea esculent affected melanin formation independently from their antioxidant action. About their phenolic structure, different constituents are likely to be strong tyrosinase inhibitors, with a much lower IC50 concerning that of the whole extract.
In conclusion, our study provides evidence of depigmenting effects of specific lichen extracts, going from tyrosinase inhibition in cell-free experiments to depigmenting effects in vitro on cultured cells and in vivo on zebrafish larvae. These data indicate that L. vulpina and C. islandica lichen extracts are potential candidates for developing pharmaceutical and cosmetic products for skin whitening. Moreover, data also suggest that L. vulpina could be a good source for the isolation of compounds with strong depigmenting properties. Future objectives in this direction will be the chemical characterization of the lichen extracts and the evaluation of the activity of their most promising constituents.
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
This work was supported by the University of Genova (FRA2018). The funders had no role in the study design, data collection, analysis, the decision to publish, or the preparation of the manuscript.
Grant Disclosures
The following grant information was disclosed by the authors: University of Genova: FRA2018.
Competing Interests
Paolo Giordani is an Academic Editor for PeerJ.
Author Contributions
Animal Ethics
The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):
Data Availability
The following information was supplied regarding data availability:
The raw measurements of tyrosinase inhibition are available in a Supplemental File.
Supplemental Information
Supplemental information for this article can be found online.
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