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

Cui, Lulu; Qu, Haidong; Qiao, Kun; Liu, Shuji; Wu, Jingna; Su, Yongchang; Chen, Bei; Liu, Zhiyu

doi:https://doi.org/10.1155/2023/2230456

Journal of Food Processing and Preservation

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Abstract Introduction Methods Results Discussion Conclusion Data Availability Additional Points Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 2230456 | https://doi.org/10.1155/2023/2230456

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

Lulu Cui,¹Haidong Qu,²Kun Qiao,³Shuji Liu,³Jingna Wu,⁴Yongchang Su,³Bei Chen,³and Zhiyu Liu³

Academic Editor: Fabiano A.N. Fernandes

Received05 Dec 2022

Revised02 Jul 2023

Accepted09 Aug 2023

Published06 Sept 2023

Abstract

Increasing life expectancy of the human population has created a demand for functional food with anti-skin aging properties. Skin aging is a degenerative process caused by oxidative stress, inflammation, and matrix destruction. Some secondary metabolites isolated from marine macroalgae have been shown to exhibit potential dermatological benefits. The present study investigated the skin-protective properties of the ethanolic extract from Sargassum thunbergii (STEE) via antioxidant, anti-inflammatory, and antiwrinkle effects on mouse fibroblast L929 cells. The optimal extraction conditions for STEE were determined as follows: ratio of liquid to solid 25 : 1 mL/g, concentration of ethanol 40.2%, and extraction time 40 min. STEE was further purified by adsorption using a macroporous resin, SP700. STEE exhibited high DPPH radical and hydroxyl scavenging activity in vitro, enhanced the viability of H₂O₂-treated L929 cells, and reduced intracellular ROS generation in a dose-dependent manner. Superoxide dismutase and glutathione peroxidase activities and total antioxidant capacity in H₂O₂-treated L929 cells were increased while malondialdehyde content was markedly reduced by pretreatment with STEE. The inhibitory effect of STEE on H₂O₂-induced inflammation was shown by the downregulation of tumor necrosis factor-α, interleukin-6, cyclooxygenase-2, and inducible nitric oxide synthase. Furthermore, STEE reduced the matrix metalloproteinase-2 expression and inhibited collagenase and elastase activities. Moreover, STEE was nonirritating to human skin at 100 mg/mL according to the in vitro skin irritation test using EpiSkin™. Hemolysis assay showed that the hemolysis EC50 (1275 μg/mL) of STEE is much higher than the maximum effective concentration (125 μg/mL) against H₂O₂ exposure. Our results demonstrate that STEE may be a good candidate for the development of anti-skin aging functional food and cosmetic products.

1. 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. ROS are continuously generated during normal metabolism in skin cells and are rapidly removed by nonenzymatic and enzymatic antioxidants; however, uncontrolled release of ROS is implicated in numerous human skin disorders. ROS induce a number of transcription factors such as activator protein- (AP-) 1 and nuclear factor- (NF-) κB [1], thereby contributing to proinflammatory signaling cascades and the activation of inflammation-related factors including interleukin- (IL-) 6, tumor necrosis factor- (TNF-) α, 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, anti-inflammatory [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 H₂O₂-induced oxidative stress in cells and zebrafish model [16]. Methanol-extracted 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 anti-skin aging potential of the ethanolic extract from S. thunbergii (STEE) was evaluated using L929 cells.

2. Material and Methods

2.1. 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 adsorption-desorption. 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 (), ethanol concentration (), and extraction time () 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 () of the following form: where represents the response variable, which in our case is the extraction rate; , , and are the levels of the independent variables; , , and are the linear terms; , , and are the interaction terms; , , and are the quadratic terms; and l is a constant.

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

2.3. 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% Na₂CO₃ 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).

2.4. 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 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% () 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).

2.5.2. Hydroxyl Radical Scavenging Assay

Hydroxyl radical scavenging activity was performed according to the manufacturer’s instructions (Jiancheng Bioengineering Institute, Nanjing, China). L-Ascorbic acid was used as a positive control with antioxidant activity. The concentrations of STEE and L-ascorbic acid were 12.5, 25, 50, 100, 200, 400, and 800 μg/mL, respectively.

2.5.3. Superoxide Anion Radical Scavenging Assay

Superoxide anion radical scavenging activity were performed according to the manufacturer’s instructions (Jiancheng Bioengineering Institute, Nanjing, China). L-Ascorbic acid was used as a positive control with antioxidant activity. The concentrations of STEE and L-ascorbic acid were 0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 mg/mL, respectively.

2.6. Cell Culture

L929 murine fibroblast cell line was supplied by Prof. Yutian Pan (the Engineering Technological Center of Mushroom Industry, Minnan Normal University, China). The cells were cultured in RPMI1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 mg/mL penicillin, and 100 U/mL streptomycin at 37°C and 5% CO₂.

2.7. Cell Viability Assay

Cell viability was evaluated with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, L929 cells were seeded in 96-well plates at 10⁴ cells/well and allowed to attach overnight. After serum starvation by culturing in medium containing 3% FBS for 12 h, the cells were treated with different concentrations of STEE (0-1000 μM) or H₂O₂ (0-500 μM) for 24 h and 3 h, respectively. After adding 20 μL MTS solution for another 45 min, the absorbance at 490 nm was measured with a microplate reader (Tecan, Morrisville, NC, USA). At least three independent experiments were performed.

2.8. 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 cells/dish. After treatment with different concentrations of STEE (0, 31.25, 62.5, and 125 μg/mL) and 200 μM H₂O₂ 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 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 H₂O₂ 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 H₂O₂. 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 (inflammation-related 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].

2.11. 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 CaCl₂, 1 μM ZnCl₂, 50 mM 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.

2.12. 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 HEPES with 0.5 M sodium chloride (pH 7.5)). Human leukocyte elastase (Sigma-Aldrich, St. Louis, MO, USA; E8140) was dissolved in sterile water to obtain a 1 μg/mL stock solution. The substrate N-(methoxysuccinyl)-Ala-Ala-Pro-Val-4-nitroanilide (MAAPVN) (Sigma-Aldrich; M4765) was dissolved in sterile water at 1 mM. A 25 μL volume of HEPES buffer, 25 μL elastase, and 25 μL of test extract were added to a 96-well plate. The blank contained 75 μL HEPES buffer, and the control contained 25 μL elastase and 50 μL HEPES buffer; (−)epigallocatechin-3-gallate (EGCG; 0.114 mg/mL) was used as a positive control. The plate was incubated at 25°C for 20 min. A 100 μL volume of MAAPVN was added to each well, and the plate was incubated for 40 min at 25°C. The optical density (OD) at 410 nm was measured on a microplate reader, and the inhibition rate was calculated as follows: .

The collagenase inhibition assay was performed in 50 mM tris(hydroxymethyl)-methyl-2-aminoethane sulfonate (TES) buffer (Sigma-Aldrich; T1375) with 0.36 mM CaCl₂ (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%, ) 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 96-well 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% CO₂ 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 H₂O (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% CO₂ for 42 h. The EpiSkin™ units were transferred to the MTT-filled wells (0.3 mg/mL) for 3 h at 37°C, 5% CO₂. 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 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 deviations. The statistical analysis was carried out by one-way analysis of variance (ANOVA) using GraphPad Prism. Prism reports results as nonsignificant (ns) at , significant (symbolized by “”) at , very significant (“”) at , and extremely significant (“”) at .

3. Results

3.1. Extraction Optimization

3.1.1. Model Fitting and Statistical Analysis

The design matrix and the corresponding results of the RSM experiments are presented in 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: where represents the STEE extraction rate and , , and 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 value () and indicated that the model was highly significant. In addition, the value (lack of fit) of 3.51 and the associated value of 0.1283 indicated excellent correlation between the predicated and experimental values. The determination coefficient () and the adjusted determination coefficient () 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 () 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.

3.1.2. 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.

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3.1.3. 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 () 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.

3.2. 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 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 (IC₅₀) 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 IC₅₀ 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 IC₅₀ 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 L-ascorbic acid. However, the superoxide anion radical scavenging activity of STEE was considerably weaker than that of L-ascorbic acid.

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3.3. STEE Reversed the H₂O₂-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 (; Figure 3(a)), although it was toxic at 500 μg/mL (). Therefore, 31.25–250 μg/mL STEE was used in subsequent assays.

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Cells were treated with various concentrations of H₂O₂ (0-500 μM) for 3 h to determine the H₂O₂ concentration that would induce oxidative damage. H₂O₂ reduced cell viability in a concentration-dependent manner (Figure 3(b)); 200 μM H₂O₂ 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 H₂O₂ (Figure 3(c)). However, pretreating the cells with STEE (31–125 μg/mL) reversed this effect in a concentration-dependent manner.

3.4. 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 nonfluorescent 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 H₂O₂ (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 H₂O₂. 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 H₂O₂-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.

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3.5. 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 H₂O₂ exposure, but this was abrogated by STEE pretreatment. We also found that the increase in MDA content induced by H₂O₂ ()—reflecting the oxidation of cellular lipids due to oxidative stress—was reversed by pretreatment with 62.5 or 125 μg/mL STEE () (Figure 5).

3.6. STEE Alleviated H₂O₂-Induced Inflammation in L929 Cells

H₂O₂ 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.

Figure 6

STEE inhibits H₂O₂-induced proinflammatory gene expression in L929 cells. Cells were pretreated with different concentrations of STEE (0, 31.25, 62.5, and 125 μg/mL) for 24 h. The mRNA expression of COX-2, iNOS, TNF-α, and IL-6 in L929 cells was measured by qPCR at 1 h and 3 h after the end of exposure to 200 μM H₂O₂. Bars represent of four samples per group. The data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. The means of treatments not sharing a common letter (A-C) are significantly different (comparison between treatments within each time point, ).

3.7. 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 demonstrated 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 () 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)).

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3.8. Toxicity Assessment of STEE

In the in vitro skin irritation test, EpiSkin™ cell viability was in the 5% SDS-treated group. However, the viabilities of the STEE-treated groups were , , or with 1, 10, or 100 mg/mL STEE treatment, respectively (Figure 8(a)). Based on OECD TG439, all the STEE preparations were nonirritants to human skin because the cell viabilities were above 50% after 15 min exposure and 42 h incubation.

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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 H₂O 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.

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 H₂O₂ exposure (Figure 8(b)).

4. Discussion

Distinct Sargassum species have demonstrated efficacy in mitigating various dermal conditions including UVB photodamage, acne-induced inflammation, and hyperpigmentation [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 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 H₂O₂ exposure relative to the control group; however, this was reversed by pretreatment with STEE. This finding parallels that of our groups previous research, which demonstrated that the S. thunbergii polyphenol extract (STPE), purified using XDA-7 macroporous resin, exhibited a significant reduction in the expression of inflammatory factors in UVB-irradiated L929 cells. In addition, a multitude of studies have provided compelling evidence regarding the anti-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 gelatin-degrading 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.

5. 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 resin. STEE exhibits remarkable antioxidant activities against DPPH and hydroxyl radicals in vitro. Moreover, increasing the concentration of STEE leads to a reduction in intracellular ROS accumulation. Additionally, STEE supplementation enhances the levels of T-AOC, SOD, and GSH-Px, while reducing MDA content. Furthermore, STEE significantly downregulates the expression of inflammatory factors, including COX-2, iNOS, TNF-α, and IL-6, in L929 cells. Remarkably, STEE also exhibits inhibitory effects on collagenase and elastase, indicating its potential for safeguarding human skin from aging when employed in cosmetic or functional food applications.

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.

Additional Points

Practical Application. Sargassum thunbergii has significant potential to protect the human skin from aging.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Cui Lulu and Qu Haidong contributed equally to this work.

Acknowledgments

This work was supported by the Research Project of Province-Owned Public Scientific Research Institutes in Fujian Province (no. 2017R1003-13), a grant from the Marine Economy Innovation and Development Project of Fujian (16CZP010SF07), and a grant from Marine Economic Development Subsidy Program of Fujian (FJHJF-L-2019-3). We extend our special thanks to Yutian Pan (the Engineering Technological Center of Mushroom Industry, Minnan Normal University, China) for providing us with L929 cell line.

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Copyright © 2023 Lulu Cui et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Journal of Food Processing and Preservation

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

Abstract

1. Introduction

2. Material and Methods

2.1. Materials and Preparation Procedure of S. thunbergii Extract

2.2. Response Surface Methodology (RSM)

2.3. Determination of Total Phenolic Content (TPC)

2.4. Purification of STEE by Macroporous Adsorption Resin

2.5. Measurement of Antioxidant Activity In Vitro

2.5.1. DPPH Radical Scavenging Assay

2.5.2. Hydroxyl Radical Scavenging Assay

2.5.3. Superoxide Anion Radical Scavenging Assay

2.6. Cell Culture

2.7. Cell Viability Assay

2.8. Cellular Antioxidant Enzyme Activity and Lipid Peroxidation

2.9. Measurement of Intracellular ROS Production

2.10. RNA Isolation, Reverse Transcription, and Quantitative PCR (qPCR) Analysis

2.11. Evaluation of MMP Inhibitory Activity by Gelatin Zymography

2.12. Evaluation of Elastase/Collagenase Inhibitory Activity

2.13. In Vitro Skin Irritation Test

2.14. Hemolysis Assay

2.15. Statistical Analysis

3. Results

3.1. Extraction Optimization

3.1.1. Model Fitting and Statistical Analysis

3.1.2. Analysis of Response Surface Plot and Contour Plot

3.1.3. Verification of Predictive Model

3.2. Characterization of STEE Desorbed Using SP700 Macroporous Resin

3.3. STEE Reversed the H2O2-Induced Decrease in Cell Viability

3.4. Intracellular ROS Scavenging Effects of STEE

3.5. STEE Enhanced Antioxidant Activity and Inhibited Lipid Peroxidation

3.6. STEE Alleviated H2O2-Induced Inflammation in L929 Cells

3.7. STEE Suppressed MMP-2, Elastase, and Collagenase Activities

3.8. Toxicity Assessment of STEE

4. Discussion

5. Conclusion

Data Availability

Additional Points

Conflicts of Interest

Authors’ Contributions

Acknowledgments

References

Copyright

3.3. STEE Reversed the H₂O₂-Induced Decrease in Cell Viability

3.6. STEE Alleviated H₂O₂-Induced Inflammation in L929 Cells