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Decapeptide-12 Effect On The Production Of Melanin

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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 \[[@B24-molecules-24-03653]\]. 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 ([Figure 1](#molecules-24-03653-f001){ref-type=”fig”}). This finding means that the peptide penetrated more easily in the epidermis than in the dermis.

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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 ([Figure 1](#molecules-24-03653-f001){ref-type=”fig”}). 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 Octastatin^®^ and the change in melanin production on the skin surface was analyzed by melanin contents using a Sysmex^®^-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% ([Figure 2](#molecules-24-03653-f002){ref-type=”fig”}). 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. [Figure 3](#molecules-24-03653-f003){ref-type=”fig”} shows that 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 (*p* \< 0.001) 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 Octastatin^®^ or 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.

Octastatin Explained

It was reported that Octastatin^®^ has a weak effect on the mRNA expression of *TYR*, *TYR2*, *TRP1*, and *TYRP1* \[[@B27-molecules-24-03653]\]. 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 \[[@B28-molecules-24-03653]\]. 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. 4. Materials and Methods {#sec4-molecules-24-03653} ======================== 4.1. Materials {#sec4dot1-molecules-24-03653} ————– 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 {#sec4dot2-molecules-24-03653} ———————————— The peptide sequences of the synthesized decapeptide  were S-(3-(3-Acetyl-1-oxo-1,4-diazepane-2-carbonyl)-2-oxopropyl)-[l]{.smallcaps}-valine-2-naphthylamine (C~52~H~74~N~6~O~10~S). The peptide 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 {#sec4dot3-molecules-24-03653} ——————————————– 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 {#sec4dot4-molecules-24-03653} —————— 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.