1. Introduction
The aging process is classified into two categories: intrinsic and extrinsic aging. The intrinsic aging is governed by genetic predisposition, whereas extrinsic aging is caused by radiation, stress, smoking, and pollution [
1,
2]. Skin aging is characterized by changes in skin thickness, structural changes in dermal and epidermal layers, and elastic fibers of the dermal layer [
1,
2]. The clinical features of skin aging include coarse, thin, lax skin accompanied by wrinkles, lentigines, irregular hyperpigmentation, telangiectasias, and sallowness [
3]. People become more concerned about their skin condition as they age [
4]. Skin aging is characterized by wrinkle formation accompanied by changes in the generation and breakdown of collagen [
5]. A previous study reported that matrix metalloproteinase (MMP) production is induced by UVB exposure, which leads to increased extracellular matrix (ECM) degradation and wrinkle formation [
6,
7]. The secretion of MMPs from keratinocytes increasingly impairs collagen synthesis and the degradation of collagen and ECM proteins, leading to wrinkling and skin aging [
6,
7].
Drugs are generally prescribed and used according to the medical condition of patients but only for a limited duration [
8]. Since cosmetics are generally used for several years, to inhibit wrinkle formation they should be certified to be safe [
9]. It is also important to determine the safety of individual constituents used on the skin in various cosmetic products [
10]. The safety of cosmetics is determined by experts to evaluate adverse events, including erythema, edema, swelling, and papules after they are applied to normal skin [
11].
The Trebouxiophyceae
Micractinium (
Micractinium sp.) is a genus of microalgae (Chlorophyta) and a polyphyletic group of unicellular photosynthetic eukaryotes that comprises of many species of green algae [
12,
13].
Micractinum sp. used in the study was discovered as a new species on a sea ice on coast of the Baton Peninsula in the Antarctic, making it an excellent candidate for the discovery of new bioactive compounds (
Supplementary Figure S1).
Micractinum sp. can live both sea and fresh water, and recent study has been reported that
Micractinium singularis which is the closest species of
Micractinium sp. was discovered living in sea water Janghang harbor, Korea [
14]. To survive in such harsh environments, these microalgae have been reported to produce special compounds such as antifreeze proteins, polyunsaturated fatty acids, UV radiation-screening compounds, and antioxidants [
15,
16,
17]. Among them, the anti-wrinkly effects of 1-monoeicosapentaenoin (1-MEST) have not yet been reported. To identify whether 1-MEST is a potential anti-wrinkle agent, we evaluated the effects of 1-MEST in human epidermal keratinocytes (HEKs) in vitro. Moreover, the safety and efficacy of 1-MEST were assessed for inhibiting wrinkle formation via clinical trials, and we confirmed the anti-wrinkle effects of 1-MEST and its potential use as an anti-wrinkle agent.
4. Discussion
This study aimed to evaluate the safety and efficacy of 1-MEST as an anti-wrinkle agent in a split-face randomized, double-blind placebo-controlled trial. First, we observed that 1-MEST did not show in vitro cytotoxicity in HEKs, and suppressed the effects of MMPs in the HEKs after irradiation with UVB. Second, we found that our test cream containing 0.1% 1-MEST ameliorated wrinkle roughness and depth, as evaluated by primary outcomes. Lastly, we recognized that our test cream had better efficacy than the placebo cream on skin wrinkles evaluated through secondary outcomes and did not cause any adverse events (
Supplementary Table S1). Thus, the overall results of the present study suggest that 1-MEST is a potential anti-wrinkle agent and can be used to alleviate skin wrinkle formation.
The skin ECM is composed mainly of elastic fibers and collagen, which play an important role in the skin tissue and cells by providing a structural framework. Studies have reported that skin sagging and coarse wrinkle formation occurs through ECM degradation by MMPs [
6,
7,
22]. Notably, the expression of MMPs is induced by prolonged UV light exposure, which causes wrinkle formation due to the breakdown of ECM proteins and collagen [
6,
7,
23]. Therefore, we propose that inhibition of MMP expression is a potential strategy to inhibit wrinkle formation. Based on the above observations, we investigated the secretion and expression of MMPs to determine the anti-wrinkle efficacy of 1-MEST using human primary cells (HEKs). Our results showed that treatment with 1-MEST inhibited the secretion of MMP-1 and the expression of MMP-9 in HEKs. However, we did not elucidate the mechanisms underlying the suppressive effect of 1-MEST on MMP secretion in this study, and therefore, further studies are warranted to clarify these phenomena.
It is well known that UV light is composed UVA, UVB, and UVC and distinguished by wavelength such as UVA having longest (315–400 nm), UVB having mid-range (290–320 nm), and UVC having shortest (100–280 nm). Whereas most UVC is absorbed in atmosphere, UVB can reach epidermis area and UVB can penetrate dermis area [
24]. Among the UV light, UVA is considered to play an important role in skin photoaging and UVB is well known as mutagen and inducer of skin cancer. On the other hand, other groups reported that the UVB are mostly responsible for skin changes such as wrinkle formation, epidermal thickening, degradation of matrix macromolecules, vascularization, and immunosuppression. However, UVA is partly absorbed and has lower efficiency in skin damage such as erythema [
25,
26,
27]. In addition, previous studies reported that high-dose UVB irradiation led to apoptotic (e.g., sunburn) [
28,
29,
30] or cancerous phenotypes [
31,
32] in skin, while low-dose UVB exposure accelerated skin aging or photoaging [
1]. Thus, we suggest that both of UVA and low-dose UVB should be considered to evaluate anti-skin aging efficacy. Moreover, given that a skin wrinkle formation is started by structural changes in both of epidermal and dermal layers through ECM degradation due to MMPs expression, not only keratinocytes but also fibroblasts should be investigated on the MMPs expression after exposure UVA or low-dose UVB. In this study, the aim of in vitro study was to investigate whether 1-MEST has a role in the response of keratinocytes to UVB irradiating. In results, 1-MEST inhibited UBV-induced skin damage. In accordance with the in vitro results, anti-wrinkle activity of 1-MEST was clinically proven.
Skin aging is usually classified as intrinsic (chronological) and extrinsic (photoaging) aging. Extrinsic aging shows photoaged skin mainly due to exposure to ultraviolet B and environmental pollutants, while intrinsic aging is related with aging factors including genes, neuroendocrine, and skin diseases. Intrinsic aging can be accelerated by external factors including ultraviolet radiation, pollutants and microbial insults, resulting in a complex biological factory where structural proteins, lipids, neuroendocrine, melatonin and vitamin D are produced to protect skin damage. UV can regulate neuronal signals but chronic exposure to UV induces oxidative stress which leads to neuroendocrine dysregulation and decreased antioxidant defense systems in the skin. Long term of UV absorption by the skin not only decrease skin function but also induces skin pathology (e.g., cancer, aging, autoimmune diseases). The protective abilities to deal with external stressors are regulated by the cutaneous neuroendocrine systems [
33,
34,
35]. These skin functions have interaction between skin’s endocrine system and central nerve system to maintain and cutaneous homeostasis, with UVB being more efficient than UVA. UV radiation makes crosstalk between skin and brain for neuroendocrine system to control body homeostasis by producing neuropeptides, biogenic amines, serotonin, melatonin, acetylcholine, steroids, cytokines, quinones, indoles, and 7-dehydrocholesterol, precursor of vitamin D. In the neuroendocrine aspects of skin aging, most of biologically relevant molecules have chromophores such as aromatic rings corresponding to UV absorption [
36,
37]. Among them, melatonin is reported as an effective antioxidant hormone to reduce oxidative stress, p53 activation, and NF-κB pathway in radiation-induced premature senescence in terms of indirect way, also it can directly scavenge free radical for skin rejuvenation [
38]. Thus, we suggest that further studies would be necessary to regard homeostatic function of the skin or photoprotective and anti-aging properties of melatonin, considering current strategies against skin aging.
In our clinical trial, only women participated, and we could not obtain these results from men. Thus, our results are not applicable to both sexes, and the study should be performed including male participants. Moreover, a larger study cohort with even sex distribution should be included in future studies. Nevertheless, our study outcomes prove that 1-MEST is a potential anti-wrinkle agent. If our findings are validated in future studies, we believe that 1-MEST would be a potential therapeutic agent for cosmetics and skin treatment use.