Anti-skin Aging Potential of Sargassum thunbergii Ethanolic Extract: Antioxidant, Anti-inflammatory, and Antiwrinkle Effects on L929 Fibroblast Cells

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Introduction
As the average life expectancy of humans has been increasing, skin aging is gaining attention, and consequently, the skin aging-related nutraceutical market has been established as one of the largest markets in modern times. Skin aging is a consequence of the natural aging process and the presence of excessive amounts of reactive oxygen species (ROS) in skin exposed to xenobiotics or solar ultraviolet (UV) radiation.
inducible nitric oxide synthase (iNOS), and cyclooxygenase-(COX-) 2 [2]. Enzymes such as hyaluronidase, collagenase, elastase, and matrix metalloproteinases (MMPs) play important roles in the degradation of the extracellular matrix (ECM), which is closely associated with skin aging and wrinkling. To counteract the aging process, the nutraceutical industry is focused on identifying compounds with antioxidant, anti-inflammatory, and MMP/elastase/collagenase inhibitory activities or compounds that can enhance collagen expression [3].
A number of secondary metabolites isolated from marine macroalgae have exhibited potential dermatological benefits. Phenolic compounds have emerged as one of the most extensively researched classes of active substances present in algae. For example, phenolic-enriched extracts of algae may show tyrosinase [4], hyaluronidase [5], and MMP [6] inhibitory activities, as well as antioxidant, antiinflammatory [7], and antiallergenic [8] effects. Consequently, these extracts are desirable ingredients for use in functional foods and cosmetic products. The extraction of marine algae phenolic compounds is most commonly achieved by solvent extraction. In these extraction procedures, polar solvents such as methanol, ethanol, and acetone or their aqueous mixtures are used to obtain polar phenolic compounds such as phlorotannins [9]. Extracts destined for the food, pharmaceutical, and cosmetic industries are standardly prepared by an ethanol-based process [10].
Marine algae phenolic compounds may be purified by a number of techniques, such as chromatography [11], ultrafiltration [12], and liquid-liquid fractionation with ethyl acetate. However, the application of these processes for purification of phenolics for human consumption is limited. The main drawbacks of these methods include low yields, high costs, and the use of non-food-grade solvents. The macroporous resin separation method overcomes the majority of the limitations of these techniques and is approved by the Food and Drug Administration for food product purification [13]. Polyphenols are adsorbed on the resin via hydrophobic binding and aromatic stacking, and they are subsequently desorbed with a mixture of water and organic solvent (e.g., ethanol). Macroporous resins including HP-20 [14], XAD-16N [13], and HPD-300 [15] have been used for purification of marine algae polyphenols in the previous studies.
Sargassum thunbergii is a common brown algae species that is distributed along the marine coast of China and has great economic and ecological value for sea cucumber culturing and seaweed beds. S. thunbergii extracts have been reported to exhibit various beneficial health effects. For example, enzymatic-digested sulfated polysaccharide from S. thunbergii had protective effect against H 2 O 2 -induced oxidative stress in cells and zebrafish model [16]. Methanolextracted polyunsaturated fatty acid components and indole derivatives of S. thunbergii attenuated ROS-mediated oxidative stress in cellular systems [17] and inhibited lipid accumulation and adipogenesis [18]. The genus Sargassum is known to produce interesting phenolic compounds of high molecular weight [19]. However, there are no reports on the anti-skin aging effects of the phenolic-enriched ethanol extract from S. thunbergii. Fibroblasts are a major cell component of the dermis and produce ECM proteins as well as structural proteins (collagen and elastin), adhesive proteins (laminins and fibronectin), glycosaminoglycans, and proteoglycans. L929 mouse fibroblasts are widely used as an in vitro system for skin toxicity assessment or skin exposure experiments [20]. In this study, we optimized the extraction process of ethanol extract to maximize the extraction efficiency of phenolic compounds from S. thunbergii; the resulting crude ethanol extract was enriched by application of the macroporous adsorption resin SP700. Furthermore, the antiskin aging potential of the ethanolic extract from S. thunbergii (STEE) was evaluated using L929 cells.

Materials and Preparation
Procedure of S. thunbergii Extract. S. thunbergii was collected from Dongtou Islands, located in the southeast of Zhejiang Province in China, and its geographical location are 120°59 ′ 45 ″ -121°15 ′ 58 ″ east longitude and 27°41 ′ 19 ″ -28°01 ′ 10 ″ north latitude. It dried under the shade before storage at −20°C until use. S. thunbergii extract was prepared according to the method described by Kim et al. [14]. Samples were powdered using a grinder and extracted with 40% ethanol. The ethanolic extract was centrifuged and concentrated using a vacuum rotary evaporator (BUCHI, Switzerland). The concentrated solution was purified with macroporous adsorption resin (Mitsubishi Chemical Corporation, Japan) by adsorptiondesorption. The purified solution was freeze-dried and stored at −20°C.
2.2. Response Surface Methodology (RSM). Optimization of extraction was performed using RSM. Based on preliminary single-factor screening (data not shown), solid/lipid ratio (X 1 ), ethanol concentration (X 2 ), and extraction time (X 3 ) were considered to be the most important variables affecting the STEE extraction process. The independent variables and their code variable levels are shown in Table 1.
Design-Expert (v8.0.6) software was used to develop the statistical design, RSM modeling, and data analysis. To verify the interactions between the major operating variables and to determine their effect on the extraction rate, we optimized the experimental protocols using the Box-Behnken statistical design method. According to this, as shown in Table 2, 17 experimental runs were required to investigate three parameters at three levels. This experimental design generates a second-degree polynomial model (Y) of the following form: where Y represents the response variable, which in our case is the extraction rate; X 1 , X 2 , and X 3 are the levels of the independent variables; a, b, and c are the linear terms; e, f , and h are the interaction terms; d, g, and i are the quadratic terms; and l is a constant.

Journal of Food Processing and Preservation
The extraction rate (response of an experiment) is determined by the total polyphenol content of the sample using the Folin-Ciocalteu method.

Determination of Total Phenolic Content (TPC).
The total phenolic content of the extracts was evaluated using the modified Folin-Ciocalteu reagent method [21]. Gallic acid was used as a standard phenolic compound at concentrations of 10, 20, 30, 40, and 50 μg/mL, respectively. 1 mL of extract solution containing 1 mg of extract in distilled water was added to 5 mL of the Folin-Ciocalteu reagent (10%) and mixed thoroughly. After 3-8 min, 4 mL of 7.5% Na 2 CO 3 was added, and the mixture was allowed to stand for 1 h with occasional shaking. The resulting absorbance was measured at 765 nm using a microplate reader (Tecan, Morrisville, NC, USA).

Purification of STEE by Macroporous Adsorption Resin.
Crude extracts were further purified by SP700 resin according to the method developed by the Liu Zhiyu Laboratory. Briefly, crude extracts were centrifuged at 12000 g for 30 min, and the supernatants were filtered using 0.45 μM filters. Prior to loading the extract, the chromatographic column (ND8/DB08 Ø: 10 mm; h: 300 mm) with SP700 resin (Mitsubishi Chemical, Japan) was equilibrated with 3 bed volumes (BV) of deionized water. Thereafter, the crude extracts (4 mg/mL) were loaded onto the resin at a flow rate of 1 BV/h. Following sample adsorption, the loaded resin was washed with 1 BV of deionized water. To desorb the STEE, the loaded SP700 macroporous resin was eluted with 40% (v/v) ethanol solution at a rate of 2 BV/h. The eluent was concentrated with a rotary evaporator (BUCHI, Switzerland) to remove ethanol. Ultimately, the purified concentrates of STEE were freeze-dried under vacuum and stored at -80°C.
2.5. Measurement of Antioxidant Activity In Vitro 2.5.1. DPPH Radical Scavenging Assay. The DPPH radical scavenging activity of STEE was determined by the modified previous method [22,23]. L-Ascorbic acid was used as a positive control with antioxidant activity. In brief, STEE at various concentrations of 1.25, 2.5, 5, 10, 20, 40, and 80 μg/mL was mixed with ethanol solution of DPPH radical (final concentration was 150 μM) in equal amounts. L-Ascorbic acid is at the same concentration as STEE. After incubating this mixture for 30 min, the absorbance was measured at 517 nm using a spectrophotometer (Tecan, Morrisville, NC, USA).

Hydroxyl Radical Scavenging
Assay. Hydroxyl radical scavenging activity was performed according to the

Cellular Antioxidant Enzyme Activity and Lipid
Peroxidation. Cellular antioxidant enzyme activity and intracellular lipid peroxidation were evaluated according to the method described by Chen et al. [22,23]. Briefly, L929 cells were cultured in 60 mm tissue culture dishes at 7 × 10 5 cells/ dish. After treatment with different concentrations of STEE (0, 31.25, 62.5, and 125 μg/mL) and 200 μM H 2 O 2 for 24 h and 6 h, respectively, the cells were collected and resuspended in 400 μL phosphate-buffered saline (PBS). The cells were disrupted using TissueLyser II (Qiagen, Hilden, Germany), and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, as well as total antioxidant capacity (T-AOC) of the lysates, were measured with appropriate assay kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. Intracellular lipid peroxidation was evaluated by measuring malondialdehyde (MDA) production using the manufacturer's instructions in commercial kit (Jiancheng Bioengineering Institute, Nanjing, China).
2.9. Measurement of Intracellular ROS Production. The free radical scavenging activity of STEE was evaluated using 1,1-diphenyl-2-picryl-hydrazyl. Briefly, L929 cells were seeded in a 24-well microplate at 1 × 10 5 cells/well. After 12 h of serum starvation and 24 h of STEE treatment, 25 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) prepared in phenol red-free RPMI1640 medium was added to the cells for 30 min at 37°C in the dark. After two washes with prewarmed PBS, 200 μM H 2 O 2 prepared in phenol red-free RPMI1640 with 10% FBS was added for 30-45 min at 37°C in the dark. The cells were washed and maintained in phenol red-free RPMI1640 containing 10% FBS for fluorescence microscopy observation (excitation/ emission: 485/535 nm). For flow cytometry analysis, cells were treated with trypsin (without EDTA) for 4 min at 37°C until most of the cells were detached. The cells were centrifuged and washed twice with PBS, resuspended in phenol red-free RPMI1640 medium containing 10% FBS, and then measured using a flow cytometer within 1 h.
2.10. RNA Isolation, Reverse Transcription, and Quantitative PCR (qPCR) Analysis. L929 cells were pretreated with different concentrations of STEE for 24 h. This study was evaluated according to the method described by Chen et al. [22,23]. Briefly, the total RNA sample was collected and extracted using an RNAprep Pure kit (Tiangen Biotech, Beijing, China) at 1 h and 3 h after the end of exposure to 200 μM H 2 O 2 . Reverse transcription was performed with 1 μg total RNA using a PrimeScript RT Master Mix (Takara Bio, Otsu, Japan), and real-time qPCR was performed using a FastStart Universal SYBR Green Master kit. The primers used for amplification of the target genes (inflammationrelated genes, such as COX-2, iNOS, TNF-α, and IL-6) and reference gene (glyceraldehyde 3-phosphate dehydrogenase, GAPDH) are listed in Table 3. Relative quantification of target gene expression levels was performed with the 2 −ΔΔCt method [24].

Evaluation of MMP Inhibitory Activity by Gelatin
Zymography. MMP-2 and MMP-9 activities in L929 cells treated with STEE were assessed by gelatin zymography. Cells were seeded in 6-well plates and cultured overnight and then treated with STEE in serum-free medium for 24 h. Conditioned medium was collected and centrifuged to remove dead cells, and the total protein content of 10x concentrated conditioned medium was determined with the Bradford assay [27]. The concentrated medium was resolved on a 10% polyacrylamide gel containing 1% gelatin. After electrophoresis, the gel was washed twice with 50 mM Tris-HCl (pH 7.5) containing 2.5% Triton X-100 at room temperature to remove SDS, rinsed, and incubated at 37°C in buffer containing 5 mM CaCl 2 , 1μM ZnCl 2 , 50mM Tris-HCl, and 1% Triton X-100 for 24 h to allow the digestion of gelatin by MMP. The gel was stained with Coomassie blue R250 staining solution for 30 min. Areas of gelatin hydrolyzed by MMP were visualized as white bands by a three-step destaining.

Evaluation of Elastase/Collagenase Inhibitory Activity.
The inhibition of elastase and collagenase activities by STEE was evaluated as previously described [28]. The assay for elastase inhibition was performed in HEPES buffer (0.1 M The collagenase inhibition assay was performed in 50 mM tris(hydroxymethyl)-methyl-2-aminoethane sulfonate (TES) buffer (Sigma-Aldrich; T1375) with 0.36 mM CaCl 2 (pH 7.4). The collagenase substrate N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA) (Sigma-Aldrich; F5135) was dissolved in sterile water to achieve a final concentration of 1 mM. Collagenase from Clostridium histolyticum (Sigma-Aldrich; C0130) was dissolved in TES buffer at a concentration of 1 mg/mL. Sodium citrate (200 mM (pH 5.0)) and ninhydrin solution (4%, w/v) were added to sterile water. Each experimental group consisted of 25 μL collagenase, 25 μL TES buffer, and 25 μL of the test sample. The blank groups contained 75 μL TES buffer, the negative control groups contained 25 μL collagenase and 50 μL TES buffer, and the positive control groups contained 25 μL collagenase, 25 μL TES buffer, and 25 μL EGCG (0.114 mg/ mL). The tubes were incubated in a water bath at 37°C for 20 min. A 100 μL volume of FALGPA was added to each tube, followed by incubation for 60 min at 37°C. Equal volumes of sodium citrate and ninhydrin solution were combined, and 200 μL of the mixture was added to each tube; the tubes were placed in a boiling water bath for 5 min, and when they had cooled, 200 μL of 50% isopropanol was added. The content of each tube was transferred to a 96well plate, and the absorbance at 540 nm was read on a microplate reader.
2.13. In Vitro Skin Irritation Test. The in vitro skin irritation test was conducted based on the test guidelines of the Organization for Economic Co-operation and Development (OECD TG439) [29] and standard operating procedures for the EpiSkin™ skin irritation test method. The epidermis units were transferred to wells of 12-well plates containing 2 mL maintenance media and preincubated in a 37°C, 5% CO 2 incubator for at 24 h. After preincubation, the epidermis units were exposed to 10 μL STEE (1, 10, 100 mg/mL), 5% SDS (positive control), or H 2 O (negative control) for 15 min at room temperature. After the 15 min exposure, treated units were rinsed thoroughly with 25 mL of sterile PBS to remove all residual test material from the epidermal surface. The remaining PBS on the surface was wiped with cotton bud without damaging the epidermis. The tissue units were transferred into wells prefilled with the new maintenance medium and incubated at 37°C, 5% CO 2 for 42 h. The EpiSkin™ units were transferred to the MTT-filled wells (0.3 mg/mL) for 3 h at 37°C, 5% CO 2 . The tissue units were removed with a punch, and the epidermis was gently separated from the collagen matrix; both parts were placed into the microtubes, and 500 μL of acidic isopropanol was added to each tube. The tubes were stored for 4 h at room temperature and protected from light. Each tube was vortexed at the middle of the incubation period to help extraction. Two wells per tissue were transferred from each tube into a 96-well plate, and the absorbance at 570 nm was read on a microplate reader.
2.14. Hemolysis Assay. The horse erythrocyte hemolysis experiment was carried out as previously described [30]. A 4% horse erythrocyte suspension was prepared in PBS solution. Different concentrations of STEE were incubated with an equal volume of horse erythrocytes at 37°C for 2 h. PBS and Triton X-100 were used as negative and positive controls, respectively. Centrifuge the solution at 900 × g for 5 minutes, followed by the transfer of 200 μL of the resulting supernatant to a 96-well plate. Subsequently, the OD value of each well was measured at a wavelength of 500 nm.
2.15. Statistical Analysis. All assays were replicated three times, and the experimental results were expressed as means ± standard deviations. The statistical analysis was carried 5 Journal of Food Processing and Preservation out by one-way analysis of variance (ANOVA) using Graph-Pad Prism. Prism reports results as nonsignificant (ns) at p > 0:05, significant (symbolized by " * ") at 0:01 < p ≤ 0:05, very significant (" * * ") at 0:001 < p ≤ 0:01, and extremely significant (" * * * ") at p < 0:001.  Table 2. By applying multiple regression analysis to the experimental data, a second-order polynomial equation, which describes the correlation between STEE extraction rate and the three variables, was obtained, as below:

Results
Y % ð Þ = 1:59 + 0:068X 1 + 0:031X 2 + 0:081X 3 − 0:025X 1 X 2 + 0: where Y represents the STEE extraction rate and X 1 , X 2 , and X 3 represent the solid/lipid ratio, concentration of ethanol, and extraction time, respectively. As shown in Table 4, the statistical significance of the model was determined by p value (p < 0:0001) and indicated that the model was highly significant. In addition, the F value (lack of fit) of 3.51 and the associated p value of 0.1283 indicated excellent correlation between the predicated and experimental values. The determination coefficient (R 2 = 0:9744) and the adjusted determination coefficient (R 2 adj = 0:9414) were used to test the applicability of the model, indicating a satisfactory correlation of rate values determined by experiment and those predicted by the equation. Furthermore, a low coefficient of variation (C:V:% = 1:74) clearly indicated high reliability of the experimental values. These results suggested that the model could work well for the prediction of polyphenol extraction rate from S. thunbergii.

Analysis of Response Surface Plot and Contour Plot.
Response surfaces were plotted by Design-Expert software to assess the interactions between variables and to determine the optimal level of each independent variable required to obtain the maximum response. 3D response surfaces are shown in Figure 1. Figures 1(a) and 1(c) illustrate the interaction surfaces of the ratio of solid/liquid with ethanol concentration and extraction time, respectively. The steep slopes and elliptical contours observed in these figures indicate a substantial effect magnitude and strong interaction between the variables. Figure 1(b) displays the interaction surface between the ratio of solid/liquid and extraction time, revealing a flat surface. This observation suggests a weaker influence and a less significant interaction between these variables.

Verification of Predictive Model.
To determine the accuracy of the model equation, a verification experiment was carried out based on the optimal conditions: ratio of liquid to solid, 25 : 1 mL/g; concentration of ethanol, 40.2%; and extraction time, 40 min. The mean extraction rate (n = 3) of polyphenol was 1.703%, which correlates closely with the predicted value of 1.704%. This demonstrates and verifies the accuracy of the response model and indicates that the model designed in the study is both fit for the purpose and accurate.

Characterization of STEE Desorbed Using SP700
Macroporous Resin. In the primary extract, that is, prior to static adsorption and desorption, the TPC of STEE was  Journal of Food Processing and Preservation 6.7%. The phenolic concentration of STEE desorbed from SP700 was 43.4%. Therefore, the use of SP700 facilitated enrichment (6.5 times) of polyphenol in the fraction desorbed with respect to the primary extract. DPPH, hydroxyl, and superoxide anion radical scavenging activities were used to determine in vitro antioxidant activity, and the results are shown in Figure 2. STEE and L-ascorbic acid exhibited significantly antioxidant activities to DPPH, hydroxyl, and superoxide anion radicals in vitro. The half maximal inhibitory concentration (IC 50 ) values of STEE and L-ascorbic acid to DPPH radical scavenging activities were 2.713 μg/mL and 6.237 μg/mL, respectively (Figure 2(a)). The IC 50 values of STEE and L-ascorbic acid to hydroxyl radical scavenging activities were 81.32 μg/mL and 235.4 μg/mL, respectively (Figure 2(b)). The IC 50 values of STEE and L-ascorbic acid to superoxide anion radical scavenging activities were 9.718 mg/mL and 0.2552 mg/mL, respectively (Figure 2(c)). STEE exhibited substantially higher DPPH radical and hydroxyl radical scavenging activities compared to Lascorbic acid. However, the superoxide anion radical scavenging activity of STEE was considerably weaker than that of L-ascorbic acid.

STEE Reversed the H 2 O 2 -Induced Decrease in Cell
Viability. L929 cells were first treated with various concentrations of STEE (0-1000 μg/mL) for 24 h to determine whether STEE is cytotoxic. STEE at concentrations of 31.25-250 μg/mL had no effect on cell viability relative to the control group (p > 0:05; Figure 3(a)), although it was toxic at 500 μg/mL (p < 0:05). Therefore, 31.25-250 μg/mL STEE was used in subsequent assays.
Cells were treated with various concentrations of H 2 O 2 (0-500 μM) for 3 h to determine the H 2 O 2 concentration that would induce oxidative damage. H 2 O 2 reduced cell viability in a concentration-dependent manner (Figure 3(b)); 200 μM H 2 O 2 reduced viability by approximately 50% and was selected as the concentration used in subsequent experiments. Cell viability was reduced to 75.5% of the control value upon exposure to H 2 O 2 (Figure 3(c)). However, pretreating the cells with STEE (31-125 μg/mL) reversed this effect in a concentration-dependent manner.

Intracellular ROS Scavenging
Effects of STEE. DCFH-DA, a nonfluorescent compound, is hydrolyzed by intracellular esterase upon crossing the plasma membrane and entering the cytoplasm. This conversion yields nonfluores-cent DCFH, which is subsequently oxidized to fluorescent DCF by intracellular ROS, resulting in green fluorescence. Therefore, the fluorescence intensity serves as an indicator of cellular ROS levels [31]. This study is aimed at investigating the effects of different concentrations of STEE (0-125 μg/mL) and H 2 O 2 (200 μM) on intracellular ROS levels. The scavenging effect of STEE was assessed using fluorescence microscopy and flow cytometry. The fluorescence microscopy results demonstrated a notable elevation in green fluorescence following exposure to H 2 O 2 . However, the preincubation of STEE exhibited a marked reduction in the green fluorescence levels (Figure 4(a)). The flow cytometry analysis revealed a 9.4-fold increase in ROS levels in H 2 O 2 -exposed cells compared to control cells. Additionally, the gradual decrease in peak displacement (Figure 4(b)), representing the ROS level, was observed after the addition of STEE to the cells at 24 h.

STEE Enhanced Antioxidant Activity and Inhibited Lipid
Peroxidation. SOD and GSH-Px are two important antioxidant enzymes that protect tissue from oxidative damage. We evaluated the protective mechanisms of STEE by measuring the levels of SOD, GSH-Px, and T-AOC. Compared to the control group, SOD, T-AOC, and GSH-Px were decreased by H 2 O 2 exposure, but this was abrogated by STEE pretreatment. We also found that the increase in MDA content induced by H 2 O 2 (p < 0:05)-reflecting the oxidation of cellular lipids due to oxidative stress-was reversed by pretreatment with 62.5 or 125 μg/mL STEE (p < 0:05) ( Figure 5).

STEE Alleviated H 2 O 2 -Induced Inflammation in L929
Cells. H 2 O 2 exposure for 1 or 3 h stimulated the mRNA expression of proinflammatory genes, including COX-2, iNOS, TNF-α, and IL-6, as compared to control cells ( Figure 6); treatment with STEE decreased the levels of these transcripts in a dose-dependent manner.

STEE Suppressed MMP-2, Elastase, and Collagenase
Activities. We evaluated whether STEE alters the gelatinolytic activities of the MMP-2 and MMP-9 secreted by L929 cells by gelatin zymography. In gelatin zymography experiments, the band intensity corresponds to the activity levels of MMP-2 and MMP-9. Our findings revealed that the exposure of L929 cells to STEE resulted in a dose-dependent weakening of MMP-2 bands, while the MMP-9 bands did not exhibit significant changes (Figure 7(a)). Thus, STEE concentrations ranging from 0 μg/mL to 125 μg/mL demon-strated a significant reduction in MMP-2 enzymatic activity, while leaving MMP-9 activity largely unaffected. STEE at concentrations from 10 μg/mL to 100 μg/mL showed significant (p < 0:05) increases in inhibitory activities against collagenase and elastase. EGCG, which was used as a standard reference material, showed potent inhibitory effects against collagenase and elastase (Figure 7(b)).
To evaluate the cytotoxicity of STEE, horse erythrocytes were treated with 0-4000 μg/mL STEE. The results showed that as the concentration of STEE increased, hemolysis of horse erythrocytes was induced. It caused less than 10% hemolysis at concentrations up to 500 μg/mL. The EC50 of hemolysis effect is 1275 μg/mL which was a higher concentration than the observed maximum effective concentration (125 μg/mL) against H 2 O 2 exposure (Figure 8(b)).

Discussion
Distinct Sargassum species have demonstrated efficacy in mitigating various dermal conditions including UVB photodamage, acne-induced inflammation, and hyperpigmenta-tion [32,33] as well as inflammation induced by fine particulate matter and compromised skin barrier functionality [34]. Phenolics are secondary metabolites that allow algae to adapt to environmental stressors such as UV radiation, heat, salinity, or cold. Hence, the extraction efficiency of

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Journal of Food Processing and Preservation phenolic compounds serves as a crucial parameter in this study for obtaining the desired extracts. Aqueous ethanol is a safe and effective solvent and was our solvent choice for extraction of the polar phenolic compounds from brown algae. In the current study, we identified that 40% ethanol facilitated the optimal recovery of phenolic extracts from STEE. Otero et al. [35] reported higher recovery of phenolics in 50% ethanol than in pure ethanol. Gam et al. revealed that the free radical scavenging activity, tyrosinase inhibitory activity, and collagenase inhibitory activity of S. thunbergii extracts were optimized when the ethanol concentration was 53% [32]. In previous RSM studies, ethanol was identified as the single most important contributor in extraction of brown algae phenolics [36]. However, in this study, extraction time was identified as the most critical variable, that is, the variable that most affected the response process. The utilization of SP700 macroporous resin led to a remarkable 6.5-fold increase in the content of polyphenols in STEE. However, it remains the direction of our future research to continue to improve the purity of phenolic compounds and to clarify the structural characteristics of the major phenolic compounds that exert skin-protective effects in STEE.
Controlling radical levels seems to be a major mechanism through which we cope with skin aging. The present investigation delineates the potent antioxidant capabilities of S. thunbergii extract in vitro, as evidenced by its hydroxyl radical, DPPH radical, and intracellular ROS scavenging activities. Key players in cellular antioxidant defense such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and total antioxidant capacity (T-AOC) as well as malondialdehyde (MDA), a lipid peroxidation and cell injury indicator, were examined [37]. The escalation in SOD, T-AOC, and GSH-Px and the decrement in MDA level observed by STEE treatment indicated that STEE protects cells against oxidative damage by enhancing the antioxidant systems of fibroblasts. Extensive research has been conducted to employ a diverse array of extraction techniques for the extraction of bioactive natural antioxidants from S. thunbergii. In a study by Park et al., water-soluble extracts from S. thunbergii were prepared using carbohydrate hydrolysis which also showed efficient scavenging activity against hydroxyl, DPPH, and alkyl radicals [38]. The subcritical water extraction process yielded a highly effective extract of S. thunbergii, demonstrating notable scavenging activities against ABTS, DPPH, and FRAP radicals [39]. The methanolic extract of S. thunbergii significantly reduced intracellular ROS-mediated cell damage and upregulated the expression of SOD-1 and glutathione reductase at the mRNA and protein levels [17].
The link between ROS and skin inflammation is well known. The interaction between ROS and cytokines such as TNF-α and IL-6 leads to a vicious cycle that increases immune activation and oxidative damage and exacerbates skin inflammation. iNOS and COX-2 play an important role in immunity against infectious agents by stimulating the production of NO and prostaglandin E2, respectively [40], and were reportedly associated with 12-O-tetradecanoylphorbol-13-acetate-(TPA-) induced inflammation in mouse skin [41]. Additionally, the expression of both enzymes was enhanced in the skin of SKH-1 mice following benzanthrone and UVB exposure [42]. In the present study, COX-2, iNOS, TNF-α, and IL-6 levels were upregulated upon H 2    inflammatory effects of phenolic compounds extracted from diverse species of brown algae on skin cells. Dieckol from E. cava suppressed skin inflammation including atopic dermatitis in HaCaT human keratinocytes [7], and eckol from Eisenia bicyclis reduced the production of NO as well as iNOS, COX-2, and TNF-α expression in Propionibacterium acnes-induced HaCaT cells [43]. Phlorotannins extracted from brown seaweed Ascophyllum nodosum blocked lipopolysaccharide-induced TNF-α and IL-6 release by macrophages [44], and the anti-inflammatory effects of 6,6′ -bieckol and 6,8′-bieckol were demonstrated in a mouse ear model induced by arachidonic acid, 12-O-tetradecanoylphorbol-13-acetate (TPA), and oxazolone [45]. Brown algae phenolics can potentially be used as an active ingredient in antiwrinkle cosmetic products owing to their MMP, hyaluronidase, collagenase, and elastase inhibitory activities [46]. In skin cosmetics, MMPs accelerate skin collagen and elastin fiber degradation. A total of 18 MMPs are expressed in the skin [47]. Collagen fibers are primarily degraded by MMP-1 and MMP-2, whereas elastin fibers are mainly degraded by elastases (i.e., MMP-2) and MMP-9 [48]. Gelatin zymography is used to evaluate the gelatindegrading activities of proteolytic enzymes, such as MMP-2 (gelatinase A) and MMP-9 (gelatinase B) [49]. In this study, we found that STEE inhibited the gelatinolytic activity of MMP-2 in a dose-dependent manner without affecting L929 cell viability. Since the first report of the inhibitory effects of E. cava phlorotannins on MMP-2 and MMP-9 [50], 6,6 ′ -bieckol from E. cava and eckol and dieckol from Ecklonia stolonifera have been shown to suppress MMP activity in human dermal fibroblasts [51,52]. Our study indicated that MMP-9 activity was not reduced following treatment with STEE. The diversity and specificity of the MMP inhibitory property of natural products were proved in many studies. Qu et al. found that the level of MMP-2 mRNA expression and MMP-9 activity is significantly inhibited by a polysaccharide from S. thunbergii; however, there were no changes in MMP-9 mRNA expression and MMP-9 activity following the treatment [53]. In another study, the MMP inhibitory activities of caffeic acid and chlorogenic acid were investigated. The results indicated that caffeic acid inhibits MMP-1 and MMP-9, but not MMP-3, whereas chlorogenic acid inhibits MMP-3, but not MMP-1 and MMP-9 [54]. AE-941, an orally bioavailable standardized extract of cartilage, inhibits the gelatinolytic activity of MMP-2 and exerts minor inhibitory effects on MMP-1, MMP-7, MMP-9, and MMP-13 [55]. The catalytic domain of MMPs includes a notable S1′ "pocket" located to the right of the zinc atom. The depth, length, and amino acid sequence of the peptide around the S1′ pocket determine, to a large extent, the specificity of substrates and inhibitors [56]. The specificity of the MMP inhibitory property of STEE might be responsible for its structure and its binding modes with catalytic domains. Given that elastase and collagenase cause wrinkles and skin aging by degrading elastin and collagen, evaluating the antielastase and anticollagenase activities of STEE can provide useful information on its potential application as a cosmetic agent [57]. STEE was found to suppress the activities of human leukocyte elastase and C. histolyticum collagenase type 1; similar inhibition has also been shown for dieckol from E. cava [58] and S. plagyophyllum ethanol extract [59].
Tests of skin corrosion and irritation have long been performed using rabbits. However, the growing ethical recognition of animal welfare has prompted the replacement of in vivo testing of skin corrosion and irritation using animals by alternative methods using human skin models which closely mimics the biochemical and physiological properties of the upper parts of the human skin. EpiSkin™, EpiDerm™, and SkinEthic™ are the most widely used for the assessment of skin irritation. According to OECD TG439, EpiSkin™ viability is judged to be noncorrosive when viability is above 50% after 15 min exposure and 42 h incubation. Thus, STEE is noncorrosive to human skin based on the in vitro assessment.

Conclusion
In this study, the purity of up to 43.4% for phenolic compounds in STEE was achieved by the use of response surface methodology and purification with SP700 macroporous  Figure 8: Toxicity assessment of STEE: (a) Cell viabilities of the 3D human skin model EpiSkin™ in the skin irritation test. An MTT assay was performed on EpiSkin™ samples treated with STEE for 15 min and postincubated for 42 h. NC and PC were treated with H 2 O or 5% SDS, respectively; (b) the hemolytic activity of STEE on horse erythrocytes. The 100% hemolysis was conducted using the treatment of the positive control, 0.1% Triton X-100. The erythrocytes that was resuspended in PBS was used as negative control.