The effects of fucoidan‐rich polysaccharides extracted from Sargassum horneri on enhancing collagen‐related skin barrier function as a potential cosmetic product

Sargassum horneri came ashore after flowing from the South China Sea to Jeju Island a few years ago. This caused a significant environmental impact on coastal areas where S. horneri has accumulated because of decomposition and the release of toxic substances, such as hydrogen sulfide.

Skin comes into direct contact with the external environment and protects the body from harmful factors, such as temperature, humidity, and ultraviolet light.Because skin cells do not multiply smoothly, the skin can suffer from wrinkling, loss of elasticity, and dryness, which may be classified into endogenous and extrinsic aging processes. 1Endogenous aging occurs naturally and is characterized by decreased skin elasticity, the development of a rough skin texture, and pronounced wrinkles.Extrinsic aging is caused by external elements, including ultraviolet light, reactive oxygen species (ROS), and stress. 2e stratum corneum is the primary barrier between the skin and the external environment.It is an important factor for determining the condition of the skin.When defects occur from irritants or endogenous factors, such as aging, hormones, and stress, resistance decreases, and a decrease in the function of the epidermal bilayer lipid membrane occurs.In addition, transepidermal water loss (TEWL) and increased sensitivity to irritants result in sensitive and dry skin. 3Because of these factors, skin wrinkling increases and elasticity decreases over time. 4Collagen can undergo structural changes in response to external stimuli that contribute to accelerated skin aging, which is accompanied by decreased levels of collagen and elastin in dermal cells. 5S can increase the activity of collagenases, such as matrix metalloproteinases (MMP), and suppress collagen synthesis.
Collagen contains glycosaminoglycans and elastin fibers and is closely associated with skin elasticity in dermal tissue. 6,7Ultraviolet B (UVB) radiation increases the expression of MMP in dermal fibroblasts in response to extracellular matrix damage.MMP activation significantly contributes to connective tissue damage caused by photoaging. 8Ultraviolet radiation is known to alter skin collagen homeostasis by at least two mechanisms: direct degradation of collagen and decreased collagen biosynthesis. 4This process occurs because of increased activity of interstitial collagenases, including (MMP-1), stromelysin (MMP-3), and gelatinase (MMP-9), in response to oxidative stress. 9,10Skin wrinkling studies have shown that increased intracellular concentrations of ROS and increased collagenase activity play important roles in winkle generation following exposure to ultraviolet light. 11own algae are key components of marine ecosystems and a rich source of chemical and biological diversity.Sargassum horneri is a brown seaweed found on the coasts of East Asia that grows abundantly while attached to rocks in the upper and middle intertidal zones. 12S. horneri exhibits various pharmacological and cosmeceutical properties.It is a rich source of nutrients, including dietary fiber, vitamins, amino acids, and polysaccharides. 13Fucoidan extracted from S. horneri exerts therapeutic benefits based on its antioxidant, anti-inflammatory, and skin barrier functions. 14Fucoidan is a sulfated polysaccharide consisting of fucose, galactose, mannose, uronic acid, and xylose. 15Previous studies have examined the ingredients of functional foods and cosmetics that exhibit antiphotoaging effects. 16The production of fucoidan from brown algae under various conditions has been reported for several seaweed species. 17 the present study, we extracted fucoidan using S. horneri obtained from the coast of Jeju Island.Fucoidan demonstrated antioxidant and antiaging effects in human skin cells.

| Preparation of fucoidan from S. horneri
Fucoidan samples from S. horneri were prepared based on the process described by Sinurat et al. with modifications (Figure 1). 18,19ied S. horneri samples collected off the Jeju coast were provided by the Para Jeju Company.S. horneri was pulverized into powder using a grinder.The dried samples were decolored by stirring with 80% ethanol for 5 h at 200 rpm and then extracted with distilled water containing 0.2 mol HCl for 6 h at 90°C.Next, 2% calcium chloride (w/v) was added to the samples.Precipitated alginic acid was removed from the filtrate using a 0.5 μm filter (HU-005).Extracts were then dried using a vacuum concentrator (Eyela, Japan) and mixed with ethyl alcohol (EtOH) at a 1:2 ratio (v/v).Supernatants were heated at 40°C for 4 h using distilled water.Hydrolyzed extracts were filtered from the supernatant and dried.The resulting precipitates were designated S. horneri fucoidan extract (SHFE).

| Total sulfate content
The activity of SHFE was determined using the BaCl 2 -Gelatin method previously described by Dodgson. 20Briefly, SHFE was incubated with 2 M Tris-HCl buffer for 1 h.The supernatants were mixed with 4% TCA and 0.5% BaCl 2 -Gelatin.After incubation for 15 min at room temperature, the amount of sulfate was measured at an absorbance of 360 nm.

| Total carbohydrate content
Total carbohydrate content was determined using the Phenol-sulfuric acid method. 21A 5% phenol solution was added to SHFE samples and mixed with distilled water.A standard solution, L-(-)-Fucose (Sigma, USA), was mixed with distilled water.Subsequently, 1 mL of concentrated sulfuric acid was added rapidly to the mixture.After incubating for 10 min, the absorbance was measured at 490 nm using a spectrophotometer.

| DPPH assay
DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging was performed based on the method described by Blois et al. with some modifications. 22DPPH with ethanol was added to each sample.
After incubating for 15 min, the absorbance was measured at 517 nm.

| Cell culture
Human fibroblast cells (CCD 986sk, ATCC CRL-1947™) were cultured in high glucose Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin.The cells were subcultured once every 2 days and were incubated at 37°C in an atmosphere containing 5% CO 2 .

| Measurement of procollagen type-1 synthesis
Type 1 procollagen was measured using procollagen type 1 c-peptide enzyme immunoassay kits (Takara Bio, Japan) based on the manufacturer's instructions.The absorbance of the treated samples was measured at 450 nm using a plate reader.

| Western blot analysis
CCD 986sk cells were pretreated with SHFE (50, 100, and 500 μg/ mL) following induction with UVB irradiation (20 mJ/cm 2 ).Cells were harvested and lysed using ripa buffer.After analyzing the lysed protein, standardization was performed using a concentration gradient of bicinchoninic acid (BCA).After separation on 10% SDSpolyacrylamide gels under denaturing conditions, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes.After blocking with skim milk for 1 h, the membranes were incubated with primary antibodies (MMP-1, MMP-3, and β-actin) overnight.After washing with Tris-buffered saline with 0.1% tween 20 (TBST), the membranes were incubated with HRP-conjugated secondary antibodies.Protein bands were visualized using enhanced chemiluminescence reagents and quantified using the Chemi Doc Imaging System.

| Clinical trial
A clinical test was performed at the Korea Institute of Dermatological Sciences in Korea by Good Clinical Practice guidelines and approved by the Institutional Ethics Committee (approval number: KIDS-BTG011-DRA).A lotion containing 1% SHFE and a placebo lotion without SHFE were compared using a formulation containing various ingredients (Table 1).A total of 23 subjects (37-to 60-year-old females in good general health) were recruited for the trial to evaluate the effects of the formulation on the skin barrier.Inclusion criteria were as follows: healthy females ranging in age from 20 to 65 without skin diseases.After being briefed on all of the relevant information concerning the study, the subjects voluntarily signed an informed consent form.Also, all of the subjects were healthy without any clinical complaints regarding acute or chronic physical diseases, including skin diseases.The exclusion criteria were as follows: (1) pregnant or lactating female volunteers, (2) females who were potentially pregnant, (3) individuals with a photo-allergic or photosensitive disease history, (4) individuals who used steroid-containing skin care products for more than 1 month for the treatment of skin diseases, (5) individuals who had been rejected for similar testing procedures within the previous 6 months, (6) individuals who had sensitive or hypersensitive skin, (7) individuals who presented with any abnormal skin findings such as scarring, moles, pimples, erythema, and telangiectasia symptoms at the skin test sites, (8) individuals who used the same or similar cosmetics or medications on the test sites within 6 months after starting the test, and (9) individuals who were inappropriate for inclusion in the study through the discretionary judgment of the researcher.
In this study, skin irritation was induced by sodium lauryl sulfate (SLS) treatment to both forearms of the subjects using a Finn Chamber (SmartPractice, USA) before instrumental measurement.A filter paper disk was placed inside a 12 mm diameter Finn Chamber, 60 μL of a 2% SLS solution was added to the disk, and it was placed in the test area for patch testing.The patch was applied for 24 h and after it was removed, skin irritation was visually evaluated.
Instrumental measurements were also performed on the test substance in the applied and nonapplied areas.

| Skin barrier test by vapometer and video microscope
A vapometer (Delfin Technologies Ltd., Finland) and a video microscope (KONG PC Camera, Bomtech, Korea) were used to evaluate the improvement in the skin barrier after treatment with the test substance.The vapometer was used to measure TEWL by the same tester by applying the same pressure to the probe on both forearms of the subjects (test substance-applied and unapplied areas).The vapometer measures the amount of evaporation caused by an increase in relative humidity inside the chamber through a humidity sensor and expressed as g/m 2 /h.A significant (p < 0.05) decrease in the change of the measured value of the test substance site compared with the unapplied site was considered improved skin barrier enhancement.A video microscope was used to capture images by the same tester in the same position with uniform lighting for consistent imaging at 80× magnification on both forearms (test substance-applied and unapplied areas).Instrumental measurements were taken before the use of the test substance (after SLS stimulation) and after 3 weeks of use.

| Statistical analysis
Experiments were performed in triplicate.Data are presented as the means ± standard deviation.p < 0.05 were considered statistically significant.Statistical analyses were performed using the SPSS ver.

| Radical scavenging activity of SHFE
DPPH is a chemically stable, water-soluble free radical that turns from purple to yellow upon receiving hydrogen radicals from a TA B L E 1 Formulation of the test and placebo lotion.substance with antioxidant capacity. 23We measured the scavenging activity of SHFE in a dose-dependent manner, up to 1000 μg/ mL.The DPPH radical elimination rate for SHFE at a concentration of 1000 μg/mL was 66.35% ± 3.14% (Figure 2).Although SHFE had lower antioxidant activity compared with vitamin C (IC 50 , 4.89 μg/ mL).These findings demonstrate that fucoidan extracted from S.

Phase
horneri has antioxidant properties.

| Effect of SHFE on procollagen synthesis
To determine the effect of SHFE on UVB-induced skin wrinkling, the procollagen synthesis activity was assessed in UVB-irradiated CCD 986sk fibroblast cells.Adenosine was used as a positive control for the determination of procollagen production.As shown in Figure 4, UVB-treated cells resulted in a downregulation of procollagen activity; however, the presence of SHFE significantly increased the procollagen synthesis in a dose-dependent manner (p < 0.05).The inhibitory effect of SHFE at a concentration of 500 μg/mL was greater compared with that of adenosine treatment (160.43% ± 0.99% vs. 201.35%± 3.47%).
These results indicate that SHFE is effective at inducing procollagen synthesis activity in UVB-irradiated fibroblast cells.

| Expression of MMP-1 and MMP-3 of SHFE
To determine the effect of SHFE on MMP-1 and MMP-3 expression, we measured MMP-1 and MMP-3 activity in UVB-exposed CCD 986sk fibroblast cells following treatment with SHFE.SHFE treatment of UVB-exposed cells decreased MMP-1 and MMP-3 levels by 59.53% and 14.51%, respectively, compared with untreated UVB-exposed cells (Figure 5A).SHFE treatment resulted in a significantly greater reduction in MMP-1 expression compared with adenosine treatment (Figure 5B).These results indicate that activation of MMP-1 and MMP-3 in response to SHFE treatment involves increased procollagen synthesis.

| Effect of SHFE on skin barrier in clinical trials
The skin barrier effects of SHFE were further evaluated in a clinical study on healthy women.conducted with volunteers who satisfied all the inclusion criteria.A vapometer and a video microscope were used to evaluate the improvement in skin barrier enhancement before and after 3 weeks of use of the test substance.The improvement of skin barrier effects was assessed in both forearms and showed that TEWL was reduced by 74.14% in the SHFE lotion versus 57.28% in the placebo after 3 weeks of use (Figure 6).The results suggest that the use of the SHFE lotion demonstrates skin barrier effects in clinical trials.

| DISCUSS ION
In the present study, we focused on the biological effects of fucoidan extracts from S. horneri seaweed and demonstrated the antiwrinkle effects on UVB-induced fibroblast cells.A large amount of S.
horneri came ashore after flowing from the South China Sea to Jeju Island a few years ago.This resulted in a significant environmental impact on coastal areas where S. horneri accumulated because of decomposition and the release of toxic substances, such as hydrogen sulfide. 13,14Several studies have reported that S. horneri has antiinflammatory activities and is a rich source of bioactive compounds, including polysaccharides. 24,25Therefore, we developed and evaluated a biological ingredient from fucoidan-rich S. horneri.
Fucoidan is a complex sulfated polysaccharide found predominantly in marine brown algae.It has various pharmacological and cosmeceutical properties, including antioxidant, anti-inflammatory, and anticancer effects. 26We prepared fucoidan extracts using S.
horneri according to the process shown in Figure 1.Based on an SHFE yield of 5.16% ± 0.59%, the main monosaccharide components of SHFE were fucose and glucose.Furthermore, the total sugar and sulfate content was 59.00% ± 0.34% and 30.81% ± 0.47%, respectively (Table 2).Wu et al. reported similar compositional properties for low molecular weight fucoidan extracted from Laminaria japonica, which predominantly consists of fucose and galactose. 27S. horneri extracts containing high-molecular-weight, sulfated polysaccharides also contain substantial amounts of fucose, galactose, mannose, xylose, and rhamnose, which have been demonstrated to exhibit anti-inflammatory effects both in vitro and in vivo. 17In the present study, SHFE had a similar composition to fucoidan, including the presence of sulfated polysaccharides.
We demonstrated that SHFE has DPPH radical scavenging activity of approximately 66.35% ± 3.14% at a concentration of 1000 μg/mL (Figure 2).Furthermore, the total polyphenol content was 26.54 ± 0.237 μg/mL for the same concentration (data not shown).In previous studies, extracts from brown seaweeds have shown promise as valuable sources of antioxidants because of their high phenolic compound content. 28,29In general, most antioxidants are at higher concentrations in the epidermal tissue compared with the dermal tissue. 30The antioxidant effects of SHFE may be mediated by radical scavenging activity because of its high total polyphenolic content.
UV radiation contributes to skin aging through cellular modifications, including type I procollagen production. 31 of UVB-induced fibroblasts resulted in the increased synthesis of procollagen compared with adenosine treatment (Figure 4).Varani et al. reported extrinsic skin aging resulting from UV exposure serves as a regulator of type I collagen synthesis. 32Increased collagen expression may enhance the production of MMP-1 and MMP-3.Our data indicate that the expression of MMP-1 was decreased to 59.53% compared with controls in UVB-irradiated fibroblasts following SHFE treatment (Figure 5A).Furthermore, the expression of MMP-3 was decreased by approximately 14.51% compared with UVB-irradiated fibroblasts (Figure 5B).In previous studies, fucoidan powder was shown to inhibit UVB-induced MMP-1 expression and down-regulate type I procollagen in HS68 cells. 33Furthermore, L-fucose and fucose-rich polysaccharides were shown to increase the expression of elastin and collagen by MMPs. 34,35These results indicate that SHFE contributes to antiaging by increase procollagen synthesis and inhibiting MMP-1 and MMP-3 expression.
The epidermal barrier is essential for preserving the body's homeostasis and shielding it from various external elements, such as environmental factors, chemical exposure, and ultraviolet radiation. 36A TEWL test is typically conducted to assess the integrity of the epidermal barrier, which is linked to a deficiency in the skin barrier. 37The present study demonstrated that SHFE lotion significantly reduced transdermal water loss after 3 weeks compared with the placebo (Figure 6A).We observed a reduction in skin irritation caused by SLS exposure using a video microscope (Figure 6B).Thinning of the epidermis and dermis is the most prominent feature of aging human skin. 38The results of the present study demonstrate that fucoidan-rich polysaccharides extracted from S. horneri exhibit antiaging effects on UVB-induced human fibroblast cells.SHFE may be used as an additive with functional antiaging effects in a range of cosmetic products to restore skin hydration in the epidermal barrier.

| CON CLUS ION
We demonstrated that S. horneri extracts are composed of fucoidan with similarities to sulfated polysaccharides.Fucoidan extracted from S. horneri exhibits antiaging effects on UVB-induced human fibroblast cells.Specifically, SHFE reduces the expression of MMP-1 and MMP-3 in a dose-dependent manner.Thus, SHFE may serve as a beneficial ingredient with functional antiaging properties in various cosmetic products to restore skin hydration within the epidermal barrier.

Cells ( 1 ×
104 cells/well) were seeded into 96-well plates and incubated for 24 h.After incubation, the wells were treated with various concentrations of sample and incubated overnight.Cytotoxicity was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.Cells were incubated with MTT for 4 h, dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 540 nm using a microplate reader.

4 F I G U R E 3 F I G U R E 5
Effects of SHFE on type I collagen production by human dermal fibroblasts.Human dermal fibroblasts were treated with SHFE or UVB radiation at various concentrations and intensities, respectively, for 24 h at 37°C.Supernatants were measured by ELISA.Adenosine was used as a positive control.*p < 0.05 compared with the UVB-irradiated treatment group.SHFE, S. horneri fucoidan extracts; ELISA, enzymelinked immunosorbent assay.Effect of SHFE on fibroblast viability.Cells were treated with fucoidan extracted from Sargassum horneri at concentrations ranging from 5 to 1000 μg/mL.Data are presented as the mean ± standard deviation of at least three independent experiments.*p < 0.05 versus control.SHFE, S. horneri fucoidan extracts.Effects of SHFE and adenosine on MMP-1 and MMP-3 expression in UVB-irradiated human fibroblast cells.Western blot analysis of β-Actin, MMP-1, and MMP-3 for the SHFE group (A) and adenosine (positive control) group (B).Data are presented as the mean ± standard deviation.*p < 0.05 compared with the UVB-irradiated treatment group.F I G U R E 6 Effects of Sargassum horneri fucoidan extract (SHFE)-treated lotion on transepidermal water loss (TEWL) during the treatment period.(A) TEWL values (n = 23; mean age 48 ± 8 yrs) as measured before and after application of SHFE are expressed as the means ± standard deviation.(B) Change in skin images of placebo and SHFE treatment for 3 weeks (N = 13).*p < 0.05, versus values in the placebo group.