Antioxidative, Anti-Inflammatory, And Anti-Aging Properties Of Mycosporine-Like Amino Acids: Molecular And Cellular Mechanisms in The Protection Of Skin-Aging

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Keywords: mycosporine-like amino acids; mycosporine-2-glycine; UV-absorbing compound; sunscreen; anti-aging; anti-oxidation; anti-inflflammation; anti-protein-glycation activity

1. Introduction

The skin, the largest human organ, is constantly exposed to the external environment. Exposure to a variety of environmental stress factors, particularly ultraviolet (UV) radiation in sunlight, can damage the skin. Sunlight can be broken down into three types of nonionizing electromagnetic radiation—infrared (IR) (780–3000 nm), visible (400–780 nm), and UV (100–400 nm). The percentages of energy radiated to the Earth, in the total energy emitted by the Sun, are 53% IR, 39% visible, and 8% UV [1]. On the basis of its physiological and biological effects, UV radiation can be further divided into three main bands—the 315–400 nm band (designated as UV-A), the 280–315 nm band (designated as UV-B), and the 100–280 nm band (designated as UV-C) [2]. Solar UV radiation is drastically diminished as it passes through the ozone layer and the atmosphere; as a result, the proportion of UV rays in the sunlight reaching the Earth’s surface is made up of 95% UV-A and 5% UV-B [1]. 

Although it comprises only a small portion of the total UV radiation, UV-B is thought to be more harmful than UV-A, since UV-B is most active in damaging the skin and eyes [3]. UV-A and UV-B are also known to be genotoxic, meaning they can induce photochemical damage in cellular DNA and proteins [4,5]. Consequently, exposure to UV-A and UV-B, stimulates skin photoaging and can be responsible for the induction of skin cancer [6]. Skin photoaging is characterized by the development of pigmentary disorders, such as solar lentigines, fine and coarse wrinkles, and benign, premalignant, and malignant skin tumors on sun-exposed skin [7]. Highly energetic UV-C radiation has no biological significance because it does not reach the Earth’s surface, due to its complete absorption by the ozone layer and the atmosphere [1,3]. The depletion of the ozone layer, over the past few decades, has increased the amount of solar UV radiation reaching the Earth’s surface [8], in particular UV-B levels, since UV-A is not absorbed by the ozone layer [9]. 

Many marine organisms that are exposed to UV radiation have developed photoprotective mechanisms [10]. For example, in cyanobacteria, which dominate the marine environment, UV protection mechanisms have evolved at the molecular, cellular, and behavioral levels [11]. Cyanobacteria can synthesize various types of “sunscreen” compounds, which confer protection against UV radiation. Mycosporine-like amino acids (MAAs), Stoneman, and carotenoids are known to be key compounds in cyanobacteria that can absorb wavelengths in the UV range. These natural products are promising candidate molecules in the field of cosmeceutical compounds discovery [12]. In fact, MAAs have already been commercialized as Helioguard®365. This cosmetic reagent contains the liposomal MAAs, shinorine (SHI), and Porphyra-334 (P334), that was originally extracted from the red alga Porphyra umbilicalis, and has been successfully commercialized as a natural and safe sunscreen compound [12]. Additionally, MAAs are thought to be multifunctional secondary metabolites, in the cells of producers [13]. Many MAAs are known to act as antioxidants [14], while several recent reports have suggested that MAAs have potential therapeutic applications for reducing skin-aging processes. From this point of view, recently, several review reports with special emphasis on the potential use of MAAs in cosmetic products, have been published [15–17]. In this paper, we review MAAs and their potential as anti-skin-aging ingredients, by describing a basic overview of their structure, before moving on to a detailed account of the most recent experimental observations, accumulated thus far. The mechanisms by which MAAs might act to protect the skin from aging is discussed at, both, the cellular and the molecular level. In particular, the prominent potential anti-aging activity of the MAA mycosporine-2-glycine (M2G), which is biosynthesized by the halotolerant cyanobacterium Aphanothece halophytic, is highlighted. 

2. Mechanisms of the UV-Induced Skin Aging
UV radiation is important for our health as UV-B exposure can induce the production of a crucial nutrient, vitamin D, in our skin [18]. However, long-term and repeated exposure to UV can promote the skin photoaging process, including skin cancer formation [19]. The mechanisms by which UV-mediated cellular damage is induced are briefly described in this section.

2.1. Cellular DNA Damage

Direct and indirect toxic effects of UV radiation on the DNA molecule, mediate photoaging. Direct absorption of UV-B photons by DNA, can result in the generation of pyrimidine dimers, leading to defects in the DNA strand [20]. UV-B radiation leads mostly to the formation of cis-syn cyclobutadipyrimidines (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). 6-4PPs can be converted into related Dewar valence isomers (DewPPs), upon UV-excitation at 314 nm. Such DNA damage interferes with DNA replication and transcription, and brings various harmful effects to the cell, such as mutation, instability of the chromosome, and cell death. UV-A does not directly alter the structure of DNA as DNA does not strongly absorb radiation in the UV-A range [21]. However, UV-A can damage DNA indirectly, via a photosensitized reaction mediated by generating the radical singlet oxygen (1O2), resulting in purine base modifications [20]. The singlet oxygen anion 

oxidizes the guanine moiety, followed by the generation of 8-oxo-7,8-dihydro guanine (8-oxo-G) and 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxo-dG). As 8-oxo-G and 8-oxo-dG can associate with adenine instead of cytosine, transition mutations might occur.

2.2. Reactive Oxygen Species Generation

Reactive oxygen species (ROS), initiators of oxidative stress, are oxygen-containing reactive chemical species that include hydrogen peroxide (H2O2), hydroxyl radicals (·OH), superoxide anion radicals (·O2 −), and 1O2. In our skin, exposure to UV radiation is known to be associated with the generation of ROS. These ROS can activate skin-aging cascades, such as matrix metalloproteinase (MMP)-1-mediated aging and NF-κB-TNF-α-mediated, inflammation-induced aging [22]. The variety of ROS generation mechanism by UV, depending on the UV radiation wavelength range. In addition to 1O2 generation, as mentioned above, it has been reported that UV-A radiation can induce the generation of ·O2 − by the activation of intracellular nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, NOX [23], and association with advanced glycation end-products (AGEs) [24]. Sakurai et al. elucidated that both 1O2 and ·O2 - were generated in the skin of mice exposed to UV-A [25]. H2O2, ·O2 −, and ·OH species might be generated in AGEs, during exposure to UV-A [24]. UV-B is also known to lead to the production of H2O2, ·O2 −, and ·OH [26]. Although the source of these UV-B-induced ROS remains unclear, recently it was reported that NADPH oxidase, NOX1, is associated with UV-B-induced p38/MAPK activation and cytotoxicity, via ROS generation in keratinocytes [26]. 

To prevent skin damage induced by excess UV-induced ROS and regulate epidermal homeostasis, skin cells possess an antioxidative function that acts as an endogenous defense system [20]. This system mainly consists of six enzymes—superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), thioredoxin oxidase (TRXR), and peroxiredoxin (PRDX) (Figure 1). SOD and CAT eliminate ·O2 − and H2O2, respectively, and ultimately convert ·O2 − to H2O, whereas GPX, GR, TRXR, and PRDX eliminate H2O2, by regulation of the redox conditions of glutathione and thioredoxin. In addition to this enzymatic system, non-enzymatic molecules, such as vitamin C (ascorbic acid), vitamin E (α-tocopherol), glutathione, and ureic acid, play a major role as antioxidants in the skin [27]. These small molecules scavenge and neutralize free radicals, by providing an extra electron to make an electron pair.

Figure 1. Removal of reactive oxygen species (ROS) by an antioxidant defense system consisting of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), thioredoxin oxidase (TRXR), and peroxiredoxin (PRDX). GSHred and GSSGox indicate reduced glutathione and oxidized glutathione, respectively. TRXred and TRXox indicate reduced thioredoxin and oxidized thioredoxin, respectively

Exposure to UV radiation induces inflammation by triggering chemical reactions in the skin. Distinct patterns of inflammation are caused by exposure to specific wavelengths of light. The three groups, UV-A, UV-B, and UV-C, have been classifified, based on these different patterns of inflflammation [28]. Erythema induced in the skin, following exposure to UV-B radiation is characterized as sunburn. Inflflammatory responses induced by UV-B are mostly achieved through a variety of mediators, including nitric oxide (NO), inducible NO synthase (iNOS), prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and other cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6) (Figure 2). These molecules are predominantly regulated by nuclear factor-kappa B (NF-κB), and are mostly produced in keratinocytes, which are the predominant cell type in the epidermis [29]. It has been reported that the expression of the COX-2 protein, which is responsible for PGE2 production, is upregulated, following exposure to UV-B, in both human skin and in cultured human keratinocytes [30]. ROS are also known to be associated with the inflflammatory response, as it has been observed that COX-2 expression was induced by ROS in different cell types [31]

Figure 2. UV-B-induced inflflammatory response. Nitric oxide (NO), inducible NO synthase (iNOS), prostaglandin E2 (PGE 2), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and other cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6), are shown as mediators

2.4. Induction of Matrix Metalloproteinases

UV radiation upregulates the expression of matrix metalloproteinases (MMPs) in the skin. MMPswhich is known to be responsible for the destruction of extracellular matrix (ECM) proteins, such as collagen, which play an important role in maintaining skin homeostasis and skin aging (32). MMPs are secreted by keratinocytes and dermal fibroblasts, in response to multiple stimuli, including oxidative stress and cytokines, in addition to UV radiation. The repeated induction of these collagen-degrading enzymes, over the long term, is thought to cause collagen damage, which is one of the reasons for photoaging. Although several MMPs are expressed in mammalian skin, it has been suggested that MMP-1 is the major collagen-degrading enzyme responsible for collagen destruction, in several photo-damaged skins (33). The upregulation of MMP expression is stimulated by the activator protein-1(AP-1), which is known to be a UV-inducible transcription factor (34 In fact, the AP-1 regulatory element exists in the 5' flanking region of MMP genes. Transforming growth factor-beta (ICF-P) and NF-KBare also known to be involved in the induction of MMPs in the skin (34).

2.5. Induction of Protein Glycation

Protein glycation (non-enzymatic glycosylation), also known as the first step of the Maillard reaction, involves the formation of covalent bonds between proteins and reducing sugars. A condensation reaction between the free amino groups of proteins and the carbonyl groups of sugars, results in the formation of a Schiff base, followed by an Amadori product. Excessive products are oxidized and dehydrated to form stable, molecular, cross-linking products, called advanced glycation end-products (AGEs) [35]. Protein glycation influences the physical and functional properties of a protein, as it causes conformational changes in the protein structure [36]. In skin, it has been reported that glycation of collagen type I is associated with the development of skin dullness and decreased skin elasticity [37]. AGEs are also involved in the generation of ROS. Masaki et al. reported that exposure of AGEs to UV-A irradiation in vitro, resulted in the generation of ROS, such as ·O2 −, H2O2, and ·OH, as mentioned above [24]. In humans, skin autofluorescence, a biomarker for AGEs, can function as an endogenous photosensitizer that induces ROS generation, following exposure to UV-A radiation [35]. Thus, a glycation reaction followed by AGE formation is thought to be one of the fundamental mechanisms associated with skin aging, under environmental conditions, especially UV radiation [24]. 

3. Natural Compounds that Prevent UV radiation (UVR)-Induced Photo-Damage

Wavelength dependency is crucial in determining photobiological effects. Short-wavelength UV-C is the most damaging type of UV, but because it is filtered out by the ozone layer, only UV-B and UV-A are considered to be of biological significance. Considering their impacts, only UV-A causes indirect DNA damage, via the generation of ·O2 −, an initiator of important ROS (see Section 2.2). UV-A penetrates deep into the dermis of the skin. Prolonged UV-A exposure can lead to photoaging and suppression of the immune system. Direct DNA damage, however, is caused by UV-B, via the formation of CPDs and 6-4PPs. UV-B also causes skin damage and transformation, for example, by affecting skin structures and causing wrinkling (a sign of photoaging), roughness, and premature aging, and even leading to skin malignancies and fatal diseases, such as skin cancers [38]. To prevent or ameliorate these adverse effects, the blockage of excessive UV or alleviating the cascade of UV-induced photo-damage is crucial. Sunscreen agents (either physical blockers or chemical agents) are often applied to the skin to block excessive UV. Recently, there has been a growing demand for artificial chemical sunscreens to be replaced with natural ones. This is because of the adverse or side effects of artificial chemical sunscreens, and because of environmental concerns. Natural sunscreens and natural compounds that possess properties that resist UV-induced photodamage are, therefore, of great interest. Some natural compounds can lead to signifificant improvements in skin health and performance. Natural compounds that can provide protection against UVR-induced photo-damage can be categorized on the basis of these mechanisms—(1) blockage of UV photons; (2) involvement in the DNA repair system; (3) antioxidant activity; (4) anti-immunomodulatory activity; (5) anti-inflflammatory activity; and (6) having an inhibitory action on the cellular matrix [39]. In this section, we describe the natural compounds from plant extracts and a class of secondary metabolites which have pharmacological relevance as superior sunscreen compounds, called mycosporine-like amino acids (MAAs). 

3.1. Plant Extracts

The database of natural compounds (NCs) contains more than 320,000 compounds, reported in Super Natural II. Of these, more than 50,000 compounds obtained from plant extracts have been submitted to the database. The largest class of natural plant extracts which have been shown to have the most relevant activity and the most beneficial effects against UV-B-induced photo-damage reported to date are those which work via inhibitory effects on the signaling pathways (for example, NF-κB, MARK, and AP-1), and biomarker proteins of the target signaling pathways (for example, COX-2 in inflflammatory signaling). Inhibitory effects on these signaling pathways further modulate cellular signaling targets and antioxidant properties, to deplete ROS generation. For example, pomegranate fruit extract has been demonstrated to inhibit UV-B-mediated phosphorylation of the NF-κB and MARK pathways, in normal human epidermal keratinocytes (NHEK) [40]. Inhibition of UV-B-induced activation of MARK and NF-κB by proanthocyanidins, a class of polyphenols isolated from grape seeds, polyphenols from green tea, and quercetin from onion, have been reported [41]. Resveratrol, a class of natural phenols obtained from grape skin, cranberries, and peanuts, possesses the capacity to reduce NF-κB activation [42]. COX-2, a biomarker protein of the inflflammatory cascade, is inhibited by phenolic compounds, such as caffeine acid from white grapes [43], kaempferol from grapes [44], and curcumin from turmeric [45]. Comprehensive studies into natural plant agents that can protect against UVB-induced photodamage have been summarized in previous review papers [46–48].

3.2. Mycosporine-Like Amino Acids

Mycosporine-like amino acids (MAAs) were first discovered in fungi, and associated with a physiological role in light-stimulated sporulation. In 1993, a direct effect as a photo-protectant was made evident by the compound’s ability to block photons. It was revealed that MAAs could prevent 3 out of 10 photons from hitting a cytoplasmic target in cyanobacteria [49]. This ability has led MAAs to be known as primary sunscreen. Accumulating evidence has shown that some MAAs possess additional beneficial functions. Their pronounced photo-protective potential was first noted when they were proven to be ROS scavengers, an ability which arose via their antioxidant ability [14,50]. Further, distinct biological functions have since been proved, including DNA damage protection, anti-inflflammatory activity, and inhibitory action toward AGEs. These multiple beneficial functions make MAAs very interesting biomolecules. More details of these natural compounds will be further reviewed in the next section. 

4. Molecular Properties of MAAs

In this section, the current basic understanding of MAAs is summarized in three parts—a general description of MAAs, their chemical structure, and their biosynthetic pathways. 

4.1. General Description

‘Mycosporine’ is a common term used for fungal UV-absorbing metabolites that have been substituted with amino acid residues [51]. Since the 1960s, mycosporine derivatives, grouped as MAAs, have been found and identified from a wide range of organisms, including marine organisms such as red algae, sea stars, corals, dinoflagellates, cyanobacteria, and lichens [12,52]. MAAs comprise a cyclohexenone or cycloheximide ring, as their core chromophore ring structure is substituted with amino acid residues or imino alcohols, or some further modifications (Figure 3). For example, in the structure of P334, threonine, and glycine bind to the C1 and C3 positions of the core chromophore structure, respectively. MAAs are considered to be the most effective UV-A-absorbing compounds in nature [53]. MAAs exhibit a maximum absorbance within the UV-A and UV-B range (from 310 to 362 nm), with high molar extinction coefficients (ε = 28,100–50,000 M−1 cm−1 ). In organisms that accumulate MAAs, these compounds are believed to contribute to the suppression of UV-induced stress, by dissipating excess energy in the form of heat, without generating ROS, after absorbing UV radiation. MAAs are highly water-soluble compounds, due to their zwitterionic properties, derived from their amino acid substitutions, and therefore, they generally accumulate in the cytosolic space. It has been reported that MAAs might act as multifunctional compounds within cells. Besides their UV-protective role, another important biological role of MAAs is their antioxidant properties [14]. Other properties of MAAs, proposed so far, include DNA-protective activity [54], anti-inflflammatory activity [55,56], activity to promote osmotic equilibrium [57,58], and involvement in cell–cell interactions [13].

Figure 3. Chemical structures of the selected mycosporine-like amino acids (MAAs)—mycosporineglycine, shinorine, porphyra-334, mycosporine-2-glycine, palythine, and euhalothece-362

4.2. Chemical Structure

MAAs can be roughly divided into two groups-mono-substituted MAAs and di-substituted MAAs. In mono-substituted MAAs, the C3 position on the cyclohexenone structure is substituted with an amino compound. One of the mono-substituted MAAs, mycosporine-glycine (MG), whichis a common intermediate for the bioproduction of di-substituted MAAs, has been suggested to be an important protectant against sunlight damage in marine organisms, via the elimination of'O, (591. The absorption maximum of MC is reported to be 310 nm. On the other hand, the absorption maxima of di-substituted MAAs, vary from 320 to 362 nm, depending on the type of substituent example, the absorption maxima of P334 (C1: Thr; C3: Gly), palythine (C1: -NH; C3: Glyand M2G (C1: Gly; C3: Gly) are 334, 320, and 331 nm, respectively [14]. In di-substituted, MAAsa protonated nitrogen atom on the imine group, results in the formation of a zwitterion followed by conjugation and delocalization of the positive charge on the nitrogen atom, over the core ring structureThis conjugation stimulates UV absorption by MAAs. The extent of resonance delocalization can affect the extinction coefficient and absorption maximum of each MAA (14). Further modification of the substituents of MAAs by condensation, dehydration, decarboxylation, oxidation, reductionsulfonation, or glycosylation might also affect them.To date, more than 30 different MAAs have been identified. To identify and characterize theseMAAs, a variety of experimental techniques have been used, such as high-performance liquid chromatography (HPLC) analysis, mass spectrometry (MS) analysis including liquid chromatographyLC)-MS, amino acid analysis, infrared (IR) spectroscopic analysis, nuclear magnetic resonance (NMR analysis, and gas chromatography (GC-MS analysis. An appropriate combination of these analytical techniques, along with technological improvements in analytical instruments, has been effective in helping to characterize the structure of MAAs (12). T prepare MAA materials for analysis, preparative liquid chromatographic techniques have often been used, following the extraction of MAAs from organisms using organic solvents, such as methanol and ethanol (60].