Exploring The Potential Of Nannochloropsis Sp. Extract For Cosmeceutical Applications

Keywords: Nannochloropsis; antioxidant; anti-melanogenic; skin-moisturizing; UV protection; anti-aging

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1. Introduction

The skin is the largest organ, covering the entire human body and providing a physical barrier to protect the internal tissues from external stimuli such as ultraviolet (UV) radiation, chemicals, pathogens, and physical stresses [1,2]. In addition, it has physiologically important roles in helping to sustain organisms, including the preservation of water, immunological functions, sensory perception, and the regulation of body temperature [3,4]. The skin consists of three layers: the epidermis, dermis, and hypodermis. In the epidermis, keratinocytes, Langerhans, melanocytes, and sebaceous glands play essential roles in the repair of skin damage, determining skin color, protecting skin from UV radiation, providing immunity, and lubricating the skin. In the dermis, located beneath the epidermis, predominant fibroblast cells produce collagen and elastic fibers. Collagen provides tensile strength and toughness to resist deformation, whereas elastin provides elasticity and flexibility, allowing tissues to return to their original shape upon removal of the deforming force. Collagen and elastin are cross-linked to provide structural support for the skin [4–7]. Hyaluronic acid (HA), a glycosaminoglycan, is a major component of the extracellular matrix (ECM). HA is a key molecule for the retention of skin moisture due to its capacity to bind and retain water molecules up to 1000 times its weight [8,9]. HA forms a gel by absorbing water and provides tissues with resistance to compression. The hypodermis, which is composed of primarily fat and loose connective tissue, stores energy and provides insulation to the body [5,6]. 

Skin aging is a complex biological process induced by intrinsic and extrinsic factors [10,11]. Intrinsic aging is an inevitable process caused by physiological and genetic changes with the passage of time, resulting in fine wrinkles, gradual dermal atrophy, and thin and dry skin [10,12,13]. Extrinsic aging is engendered by cumulative exposure to environmental factors including UV radiation, smoking, and air pollution. These environmental factors are known to boost physiological and morphological alterations of the skin, resulting in premature skin aging [12–14]. Among environmental factors, exposure to UV radiation is the primary reason for extrinsic skin aging and is referred to as photoaging [14–16]. Different from intrinsically aged skin, prematurely photoaged skin usually shows coarseness, irregular, mottled pigmentation, a thickened epidermis, deep wrinkles, laxity, and roughness [5,10,17]. Even though intrinsic skin aging and extrinsic skin aging are induced by different factors, both share similar molecular mechanisms. In particular, reactive oxygen species (ROS) are generated by oxidative cell metabolism and play a key role in both processes [18]. ROS triggers the activation of mitogen-activated protein kinases (MAPK) and subsequent nuclear factor-κB (NF-κB) and transcription factor activator protein-1 (AP-1), which lead to upregulation of metalloproteinases (MMPs: MMP-1, MMP-3, and MMP-9) and downregulation of procollagen-1, resulting in the reduction of collagen content in aged skin [2,19]. ROS can also activate blood neutrophils to infiltrate the skin and secrete elastase, which degrades elastic fibers to lose skin elasticity [20]. Skin aging is also strongly related to the loss of skin moisture. It has been reported that a marked reduction of HA in the epidermis as a result of skin aging results in the loss of skin moisture [8,21]. HA is synthesized by hyaluronan synthases (HAS; HAS-1, -2, -3,) in epidermal keratinocytes and dermal fibroblasts [8]. Melanin is produced by the conversion of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA), which is catalyzed by tyrosinase. Melanin is responsible for skin color and plays an important role in protecting skin from damage by exposure to sunlight. However, the accumulation of ROS increases the activity of tyrosinase, leading to hyperpigmentation such as age spots and melasma due to excessive production of melanin. Therefore, hyperpigmentation can be alleviated by free radical scavengers and tyrosinase inhibitors [22,23].

In the last decades, strategies for healthy aging have been mandatory due to increased life expectancy. In addition, there is growing interest in maintaining a young and beautiful appearance because skin health and beauty are perceived as important biological factors representing human well-being. It is also regarded that a youthful and beautiful appearance may have a positive effect on social behavior [10,13]. Therefore, millions of consumers use cosmetic skin care products daily, and the global market of cosmetic products is projected to reach $805 billion by 2023, at an estimated growth rate of 7.14% per year from 2018 to 2023 [24,25]. Due to the adverse side effects of synthetic cosmetic products, there is a growing demand to use natural products, including molecules from plants, animals, and marine organisms. Cosmeceuticals are derived from cosmetics and pharmaceuticals, which refer to cosmetic products containing bioactive ingredients with functions of UV protection, skin whitening, anti-wrinkling, and anti-aging [26]. In the cosmetic industry, cosmeceuticals are the fastest-growing sector and natural products are emerging as a novel source of potential bioactive substances for cosmeceutical applications [25]. 

Cistanche are prokaryotic or eukaryotic photosynthetic microorganisms that can grow rapidly and survive in extreme environmental conditions (e.g., temperature variation, anaerobiosis, salinity, photooxidation, osmotic pressure, and UV radiation) [27]. Cistanche are also superior to terrestrial plants due to high productivity, limited seasonal variation, easier extraction, and abundant raw materials [27]. Cistanche are constantly exposed to environmental stresses. Thus, they evolved to develop various strategies such as ultrastructural, physiological, and biochemical changes [28]. Natural products derived from Cistanche with the potential for cosmetic or cosmeceutical purposes include photosynthetic pigments, lipids, phenolic compounds, amino acids, peptides, carbohydrates, and vitamins [25]. Among natural products, carotenoids, which are known as strong antioxidants and free radical scavengers, can be utilized for anti-aging and photoprotection purposes [29]. Among carotenoids, astaxanthin, which exhibits a higher antioxidant activity than β-carotene and ascorbic acid, has an interesting depigmentation function that can protect skin from age spots by reducing melanin synthesis by 40% [30,31]. Zeaxanthin and other carotenoids also show the activities of UV absorption and tyrosinase inhibition [32]. Cistanche often contain high lipid contents including polyunsaturated fatty acids (PUFAs). PUFAs play an important role in photoprotection, maintaining membrane fluidity and preventing intracellular ice crystal formation, which enables survival in extreme environmental conditions such as high light intensity, UV radiation, and low temperature [33,34]. Omega-3 fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have positive effects in attenuating skin photoaging by suppression of UV-induced metalloproteinases (MMPs) and anti-inflammatory activities [35]. It has been also reported that γ-linolenic acid has some cosmetic effects such as revitalizing the skin and slowing the aging process, and linoleic acid is used for the treatment of hyperplasia of the skin [36]. Phenolic compounds from Cistanche are known to exhibit diverse activities such as antioxidant, antiallergic, anti-inflammatory, and UV protection functions [2,37].

Nannochloropsis species, the small unicellular eustigmatophycean algae, are recognized for their high lipid content and high photoautotrophic biomass productivity, as well as for their successful cultivation at a large scale [38]. Nannochloropsis has been exploited as a source of unsaturated fatty acids (including EPA) for the diet of aquaculture-raised marine invertebrates. They also contain phenolic compounds, vitamins, and various pigments with antioxidant activities [39]. In addition, they do not produce toxins and their toxicological safety has been proven by their long-term use as food for marine fish and shellfish larvae [40]. Although they have diverse valuable bioactive compounds, there are few reports on the application of Nannochloropsis for cosmetic and cosmeceutical purposes [41]. Considering that the biochemical composition of Cistanche affecting cosmetic effects can be different even among isolates of the same species [42], further investigation of this microalgal species is needed to satisfy the increasing demand for natural and safe cosmetic products. In this study, we investigated the cosmeceutical potential of extract from Nannochloropsis sp. G1-5 (NG15) is isolated from the southern West Sea of the Republic of Korea. We analyzed its various biological activities, including its anti-melanogenic, antioxidant, skin-moisturizing, anti-inflammatory, anti-wrinkling, and UV protection functions. We further analyzed its biochemical content and the composition of fatty acids, carotenoids, and phenolic compounds. 

2. Results

2.1. Isolation of Nannochloropsis sp. G1-5 and Analysis of Biochemical Composition

In this study, we isolated a colony with a brownish color indicating the presence of pigments of carotenoids and polyphenols, which was obtained by spreading seawater samples onto F/2 agar plates. We identified the isolate using 18S rDNA PCR followed by sequencing and named it as Nannochloropsis sp. G1-5 (NG15). After the cultivation of NG15 and extraction using ethanol, the composition and content of fatty acid methyl esters (FAMEs), carotenoids, and phenolic compounds in the NG15 extract were determined. The total contents of fatty acids, carotenoids, phenolics, and flavonoids in the crude extract were 58.2%, 1.6%, 7.7%, and 2.0%, respectively (Tables 1–3). Specifically, the crude extract contained various PUFAs in the lipid content like γ-linolenic, linoleic acid, and EPA, which have anti-aging, photoprotection, and anti-inflammatory activities (Table 1). It also had various carotenoid compounds including astaxanthin, β-carotene, zeaxanthin, canthaxanthin, and violaxanthin (Table 2, Figure S1, Table S1), which were known to have antioxidant, photoprotective, and anti-melanogenic functions. In addition, total phenolics’ and flavonoid contents in the crude extract of NG15 (Table 3) were relatively high compared to those of other previously reported Cistanche [43–45].

2.2. In Vitro Cytotoxicity of NG15 Extract

The effect of the NG15 extract on the viability of B16F10, CCD-986sk, normal human dermal fibroblast (NHDF), and NF-κB luciferase reporter NIH3T3 stable cells was determined by MTT assay at a concentration of 100–1500 µg/mL. The treatment with the NG15 extract did not signifificantly reduce the cell viability of all cell lines tested up to the concentration of 1000 µg/mL, but it slightly decreased at 1500 µg/mL (Figure 1a–d). 

2.3. Anti-Melanogenic Activity of NG15 Extract

To evaluate whether the NG15 extract has a skin-whitening effect, we analyzed its in- influence on tyrosinase activity and intracellular melanin synthesis. From the tyrosinase inhibition assay, we observed that tyrosinase activity was reduced by treatment with the NG15 extract in a dose-dependent manner. The tyrosinase inhibition activities were 7.84 ± 2.54, 19.68 ± 2.64, 26.24 ± 1.21, 40.16 ± 0.55, and 59.2 ± 0.48% after treatment with the NG15 extract at a concentration of 100, 250, 500, 750, and 1000 µg/mL, respectively, while arbutin (300 µM), used as a positive control, exhibited tyrosinase inhibition activity of 47.52 ± 3.09% (Figure 2a). The tyrosinase inhibition activity of the NG15 extract at 1000 µg/mL was higher than that of arbutin (300 µM). To determine the anti-melanogenic activity of the NG15 extract, The B16F10 cells were stimulated with α-melanocyte-stimulating hormone (α-MSH, 100 nM), and melanin contents in the B16F10 cells treated with the NG15 extract or arbutin were measured. The NG15 extract exhibited a signifificant inhibitory effect on melanin synthesis in a dose-dependent manner. Compared to non-treated cells, the melanin contents after treatment of the extract were 212.53 ± 4.72, 171.06 ± 3.57, and 128.60 ± 3.67% at a concentration of 250, 500, and 1000 µg/mL, respectively. The melanin content of B16F10 cells treated with only α-MSH (100 nM) showed 247.65 ± 4.15%. This result represents the fact that the NG15 extract decreased melanin content in α-MSH-treated B16F10 cells up to 48%. The positive control, arbutin (300 µM), showed 88.27 ± 1.50% of melanin content compared to non-treated cells, corresponding to a 64% reduction of melanin content in α-MSH-treated B16F10 cells (Figure 2b). 

Figure 1. The effect of NG15 extract (0-1500 ug/mL) on the viability of (a) B16F10, (b) NHDF, (C) NF-KB luciferase reporterNIH3T3 stable cells,and (d) CCD-986sk cells. Control means the cell viability of cells without the NG15 extract; * denotes ap value < 0.05, n = 3, Student's f test; ** denotes a p value < 0.01, n = 3, Student's t test.

2.4. Antioxidant, Anti-Inflammatory, and UV-Protection Activities ofNG15

ExtractThe DPPH assay was employed to evaluate the antioxidant activity of the NG15 extractThe DPPH radical scavenging activity of the NG15 extract increased in a dose-dependent manner at a concentration range of 100-1000 ug/mL. Specifically, DPPH radical scavenging activities were 8.71 士 1.38,20.12 土 1.38, 51.65  1.88, 54.95 土 2.38, and 57.36  0.52% after treatment with 100, 250, 500, 750, and 1000 ug/mL of NG15 extract, respectively, while ascorbic acid (5 ug/mL) showed 54.86 土 0.31% of DPPH radical scavenging activity. The theDPPHradical scavenging activity at 1000 ug/mL of NG15 extract was slightly higher than that of ascorbic acid (5 ug /mL) (Figure 3a). To investigate the anti-inflammatory activity of the NG15 extract, we analyzed the effect of the extract on TNF-a-induced NF-B activation by NF-kB-dependent luciferase reporter assay. When the NF-kB luciferase reporter NIH3T3stable cells were treated with various concentrations of the NG15 extract, TNF-a-induced luciferase activity was decreased in a dose-dependent manner up to 15.40 + 0.38% at1000 ug/mL compared to cells treated with only TNF-a (Figure 3b). To determine the protective effect of the NG15 extract on the skin cells after exposure to UVB, the viability of CCD-986sk cells was measured by MTT assay. The viability of CCD-986sk cells after exposure to 30 mJ/cm2 of UVB was decreased to 79.01 ± 2.57% of control cells. However, treatment with the NG15 extract signifificantly reduced the UVB-induced cell death in a dose-dependent manner. The cell viability of CCD-986sk treated with NG15 extract was 87.49 ± 7.49, 89.00 ± 2.89, 97.17 ± 7.19, 100.45 ± 5.62, and 109.48 ± 8.25% after treatment with 100, 250, 500, 750, and 1000 µg/mL of NG15 extract, respectively, compared to control cells (Figure 3c). 

Figure 2. The anti-melanogenic effect of NG15 extract. (a) In vitro tyrosinase inhibition activity of NG15 extract against mushroom tyrosinase.** denotes a p-value < 0.01 versus a normal (untreated) group. # denotes a p value < 0.01 versusarbutin-treated cells; n = 3: Student's f test. (b) The effect of NC15 extract on melanin contents in a-melanocyte-stimulating hormone (MSH)-stimulated B16F10 cells. ## and ** denoe a p value < 0.01 versus c-MSH-only-treated cells; n = 3; Student'st test. Arbutin (300 uM) was used as a positive control.

2.5. Skin Moisturizing and Anti-Wrinkle Activities of NG15 Extract

To assess the skin hydration activity of the NG15 extract, the expression of HAS- 2 (moisturizing-related gene) in NHDF cells was investigated using qPCR. The HAS-2 mRNA expression was signifificantly enhanced by treatment with the NG15 extract in a dose-dependent manner. The relative mRNA expression level of HAS-2 compared to non-treated cells was 199.88 ± 10.63, 263.91 ± 2.47, and 274.58 ± 1.37%, after treatment with 250, 500, and 1000 µg/mL of the NG15 extract for 24 h, respectively, whereas HAS-2 mRNA expression level of NHDF cells treated with retinoic acid (50 nM) was 316.31 ± 8.89% (Figure 4a). To determine the effect of the NG15 extract on collagen degradation and synthesis, the expression of MMP-1 and Col1A1 was analyzed using qPCR. The MMP-1 mRNA expression of the NHDF cells was decreased by treatment with the NG15 extract in a dose-dependent manner, up to 26.71% at 1000 µg/mL, compared to the non-treated control (Figure 4b). The Col1A1 mRNA expression of the NHDF cells treated with the NG15 extract did not show a statistically signifificant difference at doses below 500 µg/mL, but it increased by 15.53 ± 2.75% at 1000 µg/mL, compared to the non-treated control (Figure 4c). We also investigated the effect of NG15 extract on the inhibition of elastase activity. The NG15 extract showed elastase inhibitory activity in a dose-dependent manner, up to 24.88 ± 0.80% at 1000 µg/mL, compared to the non-treated control (Figure 4d)

Figure 3. Antioxidant, anti-inflammatory, and UV-protection activities of NG15 extract. (a) DPPH free radical scavenging activity of NG15 extract. L-ascorbic acid (5 ug/mL) was used as a posilive control. ** denotes a p-value < 0.01 versus the normal (untreated) group. n = 3; Student's t-test. (b) The effect of NG15 extract on NF-xB-dependent, TNF-a-induceduciferase acivity in NF-B luciferase reporter NlH313 stable cells. ** denotes a p alue < 0.01 versus TNF-a-only-treatedcells. n = 3; Student's t-test. (C) The protective effect of NG15 extract on the viability of CCD-986sk cells after exposure to UVB (30 m)/cm'). ## denotes a p-value < 0.01 versus a normal (untreated and unexposed to UVB) group. ** denotes ap value < 0.01 versus UVB-only-treated group. n = 3; Student's f test.

Figure 4. Skin moisturizing and anti-wrinkle activities of NG15 extract. The mRNA expression level of (a) HAS-2b)MMP-1, and (C) Col1A1. (d) In vitro elastase inhibition activity of NG15 extract against elastase from NHDF cells denotes a p-value < 0.05 versus a normal (untreated) group.  denotes a p value < 0.01 versus a normal (untreated) groupn = 3; Student's t test. Retinoic acid (50 nM), TCF-3 (5 ng/ml, and phosphoramidon disodium salt (10 uM) were used as a positive control, respectively. The mRNA expression level of each gene was normalized to that of the B-actin gene.