Pigmentation issues are common conditions associated with excessive or insufficient production of melanin. Recently peptides are investigated to discover novel melanogenesis regulators as low molecular weight compounds to regulate skin pigmentation. In this study, an internal library of peptides obtained through in silico enzymatic digestion of phycocyanin from microalgae S. platensis was tested to apprehend their anti-melanogenic effects. Seven peptides were investigated for their inhibitory potential against mushroom and B16-F10 murine tyrosinase enzymes. According to the results, P5 (SPSWY) and P7 (AADQRGKDKCARDIGY) were effective in lowering the activity of mushroom and B16-F10 tyrosinases. P5 was the most potent (IC 50 value, 12.1 µM) in mushroom which was followed by P2 (MAACLR, 86.9 µM). Although the peptides were particularly powerful in inhibiting monophenolase activity, only moderate inhibition was observed for diphenolase activity in mushroom tyrosinase assay. Apart from tyrosinase inhibition, P2 and P3 (RCLNGRL) were efficient DPPH radical scavengers at low concentrations (IC 50< 200 µM). In the mammalian assay system, P5 and P7 were noticeably effective to decrease tyrosinase enzyme activity with IC 50 values of 48.9 and 34.2 µM, respectively. However, although P4 (RYVTYAVF) was a potent mushroom tyrosinase inhibitor, it increased melanin synthesis up to 3-fold in B16-F10 cells. The results indicate that C-terminal tyrosine residue is important for tyrosinase inhibition. This study shows, for the first time, that microalgae proteins can be regarded as sources for melanogenesis regulation.
The melanogenesis pathway is responsible for the production of melanin pigment which gives color to skin, eyes, and hair. Melanogenesis is a complex path mediated by transcription factors, central signaling pathways, and also enzymatic reactions. Upon synthesis, melanin is transported in melanosome granules from melanocytes to keratinocytes through the epidermis to contribute to color formation. In addition, melanin is an essential molecule in skin homeostasis. It protects the skin from detrimental UV radiations and other environmental hazards. Excessive melanin production may cause a variety of skin pigmentation disorders such as freckles, age spots, café au lait marks, melasma, and post-inflammatory hyperpigmentation. These problems are generally not harmful to the individuals as they are phenotypically observed as uneven dark skin patches on the body. Abnormal melanin accumulation can also cause debilitating health issues such as skin cancers. Recently, melanin is found to be related to the pathology of common neurodegenerative diseases (e.g. Parkinson’s and Alzheimer’s) which is linked to the neuromelanin deposits in the substancia nigra in the brain. On the other hand, downregulation of melanin production can cause hypopigmentation issues mainly known as vitiligo. Apart from the clinical perspective, pigmentation disorders may affect the individual’s psychology along with physical health.
Tyrosinase is a copper containing-enzyme which initiates the melanin synthesis in melanocytes. It catalyzes two distinct reactions; hydroxylation of monophenols (monophenolase activity) or conversion of diphenols into quinone derivatives (diphenolase activity). The first step in melanin synthesis is the hydroxylation of L-tyrosine to subsequent intermediates which is also the rate-limiting step. A multitude of compounds has been discovered either to decrease or increase melanin synthesis through interfering tyrosinase enzyme activity. The severity of compounds targeting hyperpigmentation is greater than hypopigmentation due to the prevalence of hyperpigmentation issues. There are numerous compounds which have been proven to inhibit tyrosinase enzyme; such as kojic acid, hydroquinone, azelaic acid, arbutin, and vitamin C. Although they are recognized as fine inhibitors with in vitro and in vivo studies, they are associated with cytotoxicity (kojic acid, hydroquinone), stability (kojic acid, arbutin, and vitamin C), and carcinogenesis with prolonged use. Hydroquinone is banned by the European Committee (24. Dir. 2000/6/EG) because of the risk of toxicity and carcinogenesis, so there is a significant search for new compounds to treat skin pigmentation problems with high stability, negligible side effects, and high skin penetration. Natural molecules have remarkable potential, as they are regarded to be safe and largely free of side effects. Therefore, the inhibition of tyrosinase by proteins, protein hydrolysates, peptides, and amino acids have emerged as a novel focus in the search of natural tyrosinase inhibitors. Some previous studies have shown the effectiveness of natural, synthetic, and kojic acid/ caffeic acid coupled peptides as tyrosinase inhibitors. Schurink et al. have gone through SPOT synthesis and concluded that peptides with certain combinations of amino acids such as arginine, valine, alanine, leucine, phenylalanine, and C-terminal tyrosine residues efficiently inhibit tyrosinase. Later, Ochiai et al. confirmed the importance of the position of tyrosine residue with experimental and computational studies.
Microalgae are considered sustainable and environmentally friendly feedstocks for cosmetics, pharmaceuticals, and nutraceuticals. Its prosperous content in lipids, pigments, polysaccharides, and other fine chemicals is effective for cosmetic purposes. Microalgae are also rich in protein (up to 80%). Recently, microalgae proteins have been evaluated as protein hydrolysates that include peptides with antioxidant, antifungal, anticancer, antibacterial, and many other bioactive potentials. However, the contribution of microalgae as tyrosinase inhibitors is limited. Recently, cyanobacterial phycocyanin has been shown to be a remarkable tyrosinase inhibitor by downregulating p38/ MAPK and GRB2/ ERK signaling pathways. Although phycocyanin is an outstanding tyrosinase inhibitor as well as antioxidant protein; high molecular weight and hydrophilicity limit transdermal administration. Nevertheless, the discovery of phycocyanin as melanogenesis regulator is encouraging to position microalgae proteins as novel sources for tyrosinase inhibitors. However, there is not enough evaluation of tyrosinase inhibitory compounds based on microalgae peptides in the literature.
In this study, we created a novel approach to decipher tyrosinase inhibitory peptides from S. platensis phycocyanin with an in silico peptide prediction tool. With this method, we avoided a lengthy trial and error strategy to discover bioactive peptides and we created a straightforward approach to evaluate peptides for tyrosinase inhibition activities. To the best of our knowledge, this is the first study about the use of in silico enzymatic hydrolysis to screen low molecular weight tyrosinase inhibitory peptides from microalgal proteins. With this method, we discovered several peptides, such as SPSWY, AADQRGKDKCARDIGY, GRF, and MAACLR successfully inhibiting tyrosinase enzyme either in mushroom or murine assay systems. We additionally found one particular peptide, RYVTYAVF, which increases tyrosinase enzyme activity up to 3-fold in B16-F10 cells and could be a candidate for hypopigmentation disorders. These peptides are novel, pioneering melanogenesis regulatory peptides from microalgal sources which have been deciphered via a combination of in silico and in vitro experimental organization.
3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide (MTT), TritonX-100, phenylmethylsulphonyl fluoride (PMSF), butylated hydroxytoluene (BHT), trypsin/EDTA, L-DOPA, L-tyrosine, mushroom tyrosinase enzyme, kojic acid, synthetic melanin, dimethyl sulfoxide (DMSO) and bovine serum albumin (BSA) were purchased from Sigma, Germany. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin were purchased from Gibco. Gentamycin sulphate was purchased from In silico hydrolysis of phycocyanin Phycocyanin from S. platensis has two subunits (α-chain, P72509; β-chain, P7250, Fig. 2b) having 162 and 172 amino acids and 17.601 and 18.093 kDa molecular weights, respectively. Both subunits begin with methionine and end with serine. The highest amino acid is alanine (14.8% in α-chain; 16.9% in β-chain) followed by serine, leucine, and glycine (Fig. 2a). Histidine and tryptophan amino acids are 0.6% of α-chain and not present in β-chain). The combination of amino acids (alanine, valine, Discussion In this study, we have screened an internal oligopeptide library acquired from proteolytic digestion of phycocyanin sequence with proteases in the BIOPEP web server. This is a novel approach to discover anti-melanogenic peptides from natural protein sources, as an alternative to in vitro enzymatic hydrolysis, spot synthesis, or fermentation-based p
