Decapeptide-12 was proven to have a strong inhibitory effect on the production of melanin. Our previous study revealed that octadecylamine was an appropriate carrier for the topical application of the Decapeptide-, which was applied on the skin and demonstrated the inhibitory effect on the melanin production. Based on these reports, this study evaluated the efficacy of the Decapeptide-12 formulated in octadecylamine for the treatment of melasma.
What You Need To Know
The aim of this study was to investigate whether the formulation is more effective than Octastatin for the treatment of melasma. First, we investigated whether the Decapeptide12 and Octastatin had similar skin penetration effects on Mel-O mice. The skin penetration effect of the peptides was examined using a microdialysis technique. Following microdialysis, it was observed that skin penetration of the Decapeptide-12 was significantly higher than that of Octastatin in the epidermis, but Octastatin had a better effect in the dermis. This finding means that the peptide penetrated more easily in the epidermis than in the dermis.
Results Findings in Recent Studies of Decapeptide
These results could be explained by the differences in the properties of the peptides. For example, the octadecylamine is thought to form a stable complex with the peptide, but not a tight complex and may also induce skin penetration. Then, following the microdialysis, we analyzed the in vivo skin penetration of the Decapeptide12 in comparison to Octastatin using HPLC. The results showed that the peak area ratio of Decapeptide and Octastatin in the epidermis was larger than that of the dermis. Based on the results of this study, the skin penetration efficacy of Decapeptide was further evaluated. An in vivo study showed that Mel-O mice were exposed to topical Decapeptide-12 formulation or Octastatinand the change in melanin production on the skin surface was analyzed by melanin contents using a System MDA-550S device.
How is Melanin Involved?
The melanin contents of Decapeptide-treated mice were significantly lower than those of Octastatin treated mice (*p* \< 0.01). It was found that the peptide has a good skin penetration ability in the epidermis and it reduced the melanin contents in the skin. These results indicate that Decapeptide12 has a strong skin penetration ability and it can be effectively applied on the skin without skin irritation. Thus, we investigated whether Decapeptide12 has an inhibitory effect on melanogenesis in Mel-C and whether the topical application of Decapeptide-12 could inhibit the melanin production of melan-a cells by decreasing cellular tyrosinase activity. To examine the inhibitory effect of Decapeptide-12 on the cellular tyrosinase activity, melan-a cells were treated with 1 and 10 μM Decapeptide-12 and Octastatin.
Cellular Tyrosinase Activity of the Melan-a Cells
The cellular tyrosinase activity of the melan-a cells was reduced with increasing concentrations of Decapeptide12 or Octastatin. At the higher concentration (10 μM), both Decapeptide-12 and Octastatin^®^ decreased cellular tyrosinase activity by more than 60% . Melan-a cells have been used in melanin production analysis \[[@B25-molecules-24-03653],[@B26-molecules-24-03653]\]. Therefore, we further examined the effect of Decapeptide-12 on the melanin contents and the mRNA expression of melanogenesis-related enzymes in melan-a cells. Decapeptide-12 decreased the cellular tyrosinase activity and melanin contents in a dose-dependent manner. In addition, at the mRNA expression level, Octastatin significantly reduced *MITF*, *TYR*, and *TRP1* mRNA expressions at a concentration of 10 μM.
Results of Findings
Although it did not change the expression of *TYR2* at a concentration of 10 μM, it significantly reduced the expression of *TYR2* at a concentration of 1 μM. The mRNA expressions of *TYRP1*, *P*-*MITF*, *Dct* and *Gnba* were not changed by Octastatinor Decapeptide-12, even at 10 μM. These findings indicate that Octastatin inhibited the melanin production at the mRNA level in a dose-dependent manner, but not at the level of protein expression. This result is consistent with the previous study. However, the protein expression of Melan-A was not changed.
It was reported that Octastatin has a weak effect on the mRNA expression of *TYR*, *TYR2*, *TRP1*, and *TYRP1*. Since Melan-A expression is not directly affected by tyrosinase, the results of melanin content decrease may not be affected by *TYR2* and *TYRP1* gene expression.
On the other hand, tyrosinase inhibitors such as arbutin or kojic acid have a lower specificity for tyrosinase compared to Octastatin. It was also reported that melan-a cells treated with 1 μM Decapeptide12 expressed Melan-A. Thus, it is considered that Decapeptide-12 did not affect the expression of melanin production related factors and that the tyrosinase inhibitory effect of Decapeptides was confirmed. There are various tyrosinase inhibitors. Decapeptides-12 has a good inhibitory effect on the cellular tyrosinase activity and the inhibition activity of Octastatin was also confirmed. In the microdialysis and HPLC study, the skin penetration efficacy of Decapeptides was confirmed. Decapeptides-12 can penetrate into the skin effectively and reduce melanin production without skin irritation.
Other Uses For This Peptide
This peptide can also be used for the development of a new skin whitening agent.
Materials and Methods Materials
Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies (Carlsbad, CA, USA). Octadecylamine was purchased from Sigma-Aldrich (St. Louis, MO, USA). 4.2. Synthesis of the Decapeptide12.
The peptide sequences of the synthesized decapeptide were S-(3-(3-Acetyl-1-oxo-1,4-diazepane-2-carbonyl)-2-oxopropyl). It was synthesized by Merck (Darmstadt, Germany) and purified using an HPLC system (Hitachi L-6200, Tokyo, Japan) using the following conditions: a TSKgel DEAE-NPRO column (Tosoh, Tokyo, Japan, 4.6 × 300 mm, 4 μm), solvent A: 0.1% trifluoroacetic acid (TFA), solvent B: 0.1% TFA with 70% acetonitrile, the elution conditions were 0–40% solvent B, 40–65% solvent B, and 65–100% solvent B for 60 min. 4.3.
Preparation of the Microdialysis Plates
Plates used for microdialysis studies were made by sterilizing with 70% ethanol, drying with nitrogen, coating the membrane with agarose (Merck), and introducing the microdialysis probe. The dialysis chamber of the probe is made of polycarbonate and has a length of 1 mm and a diameter of 1.2 mm. For the plate, microdialysis plates were inserted into sterile 0.9% NaCl containing 0.5% BSA, and then the probe was introduced and a cover glass was added, sealing it with liquid nitrogen.
Results From Incubator Experiment
The probe was then moved to an incubator and the chamber was dried at 37 °C for 1 h.
The dialysis plates were then sterilized for 5 h in an incubator at 37 °C. 4.4. Microdialysis.
The experiments were performed using a CMA 60 microdialysis system. 1.5 mL of the serum-supplemented DMEM was prepared in a polypropylene tube.
The microdialysis probe was washed by 2-fold perfusion of 0.1 M NaOH and subsequently with the serum-supplemented DMEM, and then moved to the dialysis chamber.
After setting the flow rate to 0.2 μL/min and the dwell time to 2 min, the probe was moved to the perfusion solution and perfused at a flow rate of 0.2 μL/min for 2 h.
Microdialysates were collected every 30 min, and 10 μL of the microdialysate was used to determine dopamine contents using HPLC.