**The Skin-Whitening E** ff**ects of Ectoine via the Suppression of** α**-MSH-Stimulated Melanogenesis and the Activation of Antioxidant Nrf2 Pathways in UVA-Irradiated Keratinocytes**

#### **You-Cheng Hseu 1,2,3,4, Xuan-Zao Chen 1, Yugandhar Vudhya Gowrisankar 1, Hung-Rong Yen 3,4,5,6, Jing-Yuan Chuang 7 and Hsin-Ling Yang 8,\***


Received: 23 December 2019; Accepted: 8 January 2020; Published: 10 January 2020

**Abstract:** Ultraviolet A (UVA)-irradiation induced reactive oxygen species (ROS) production mediates excessive melanogenesis in skin cells leading to pigmentation. We demonstrated the depigmenting and anti-melanogenic e ffects of Ectoine, a natural bacterial osmolyte, in UVA-irradiated human (HaCaT) keratinocytes, and the underlying molecular mechanisms were elucidated. HaCaT cells were pre-treated with low concentrations of Ectoine (0.5–1.5 μM) and assayed for various depigmenting and anti-melanogenic parameters. This pre-treatment significantly downregulated ROS generation, α-melanocyte-stimulating hormone ( α-MSH) production, and proopiomelanocortin (POMC) expression in UVA-irradiated HaCaT cells. Also, antioxidant heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase [quinone 1] (NQO-1), and γ-glutamate-cysteine ligase catalytic subunit (γ-GCLC) protein expressions were mediated via the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) whose knockdown indeed impaired this e ffect signifying the importance of the Nrf2 pathway. Ectoine was mediating the activation of Nrf2 via the p38, protein kinase B (also known as AKT), protein kinase C (PKC), and casein kinase II protein kinase (CKII) pathways. The conditioned medium obtained from the Ectoine pre-treated and UVA-irradiated HaCaT cells downregulated the tyrosinase, tyrosinase-related protein-1 and -2 (TRP-1/-2), cyclic AMP (c-AMP) protein kinase, c-AMP response element-binding protein (CREB), and microphthalmia-associated transcription factor (MITF) expressions leading to melanoma B16F10 cells having inhibited melanin synthesis. Interestingly, this anti-melanogenic e ffect in α-MSH-stimulated B16F10 cells was observable only at 50–400 μM concentrations of Ectoine, signifying the key role played by Ectoine (0.5–1 μM)-treated keratinocytes in skin whitening e ffects. We concluded that Ectoine could be used as an e ffective topical natural cosmetic agen<sup>t</sup> with depigmenting and anti-melanogenic e fficacy.

**Keywords:** Ectoine; keratinocytes; melanogenesis; tyrosinase; α-MSH; Nrf2 # **1. Introduction**

Exposing human skin to UVA radiation triggers ROS generation and also over-producing melanin in the skin cells. The uncontrolled production of ROS could lead to melanoma conditions as well. Most skin whitening agents are targeting and trying to minimize the melanogenesis process through the inhibition of α-MSH and tyrosinase productions [1]. Most skin-tone lightening creams are composed of hydroquinone [2] or hydrocortisone [3], that are known to decrease the formation of melanin, but are also associated with severe side effects. For example, acne, flaky and itchy skin, blue and black discoloration of the skin, ochronosis, burning and stinging, skin irritation, and even inflammation. However, skin whitening agents from the natural sources, for example, Kojic acid (a fungal derivative obtained from *Penicillium* and *Aspergillus* species) is also reported to cause 'contact dermatitis' in individuals who have sensitive skin. In these individuals, more than 1% of kojic acid could cause severe hypersensitive side effects [4,5]. Therefore, only a few naturally derived skin whitening products (oleosin, licorice extract, ascorbic acid, soy protein, and *N*-acetyl glucosamine, etc.) are currently being used in the cosmetic industry [6]. However, the skincare products that are principally targeting the depigmenting properties have at times failed to focus on counteracting the deleterious effects posed by the UVA irradiation-induced ROS production mediated excess melanogenesis in skin cells.

Ectoine is a 'natural extremolyte' produced from several species of microorganisms under stressful conditions [7,8]. This compound was first isolated from the *Ectothiorhodospira* species of bacteria that are living in the Egyptian desert. The cascade of *ect* operon genes (*ect*A, *ect*B, *ect*C, or *ect*D) are involved in the production of this compound. Ectoine is chemically designated as 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid [9]. As a moisture binder, Ectoine helps in, restructuring of the skin cell membrane [10], protection from UV damage and pollution [11,12], moisturizing the skin [13], delaying the premature skin aging [14], etc. In addition to the skin protective roles, Ectoine has been shown to be useful in the treatment of atopic dermatitis [15], Alzheimer's [16], as well as the inhibition of HIV replication [17], radio and chemotherapy [18], and liver cirrhosis [19]. Ectoine is speculated to exhibit its depigmenting and skin whitening properties without causing undesirable side effects [20]. Contrastingly, the molecular mechanisms elicited by Ectoine are not known. Therefore, the objective of this study was to delineate the Ectoine mediated depigmenting and anti-melanogenic mechanisms elicited in UVA-irradiated human (HaCaT) keratinocytes as the cellular model system. The effect of Ectoine induced secretions of skin-protecting agents from the HaCaT cells to the culture medium (conditioned medium) was also tested using a typical melanoma cell (B16F10) line as well.
