Abbreviations
DCF-DA
2′,7′-dichlorofluorescein diacetate
DMEM
Dulbecco’s modified Eagle’s medium
DMSO
dimethyl sulfoxide
DPPH
2,2-diphenyl-1-picrylhydrazyl
EGCG
(−)-epigallocatechin gallate
HaCaT
human immortalized keratinocyte cell line
LAS X
Leica Application Suite X
LPS
lipopolysaccharide
MAPKs
mitogen-activated protein kinases
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NAC
N-acetyl-L-cysteine
NRF2
nuclear factor erythroid 2-related factor 2
UEF
Undaria pinnatifida sporophyll extract fraction
UPE
Undaria pinnatifida sporophyll extract
INTRODUCTION
Marine organisms have garnered attention for their diverse applications in human health, including their physiological benefits (El Gamal 2010). Marine algae such as Undaria pinnatifida, an edible brown marine macroalgae mainly found in Korea, Japan, and China, are an abundant source of nutrition that is particularly important in Asian cultures (Xu et al. 2017, Hakim and Patel 2020). U. pinnatifida is known for the excellent health benefits derived from its constituents, such as polysaccharides, dietary fiber, and phytochemicals (Eom et al. 2020). Sporophyll, a reproductive organ of U. pinnatifida, is a typical by-product of post-food processing. It is also a promising source of bioactive compounds for the functional food industry because it contains abundant indigestible polysaccharides (e.g., alginate and fucoidan) (Kim et al. 2016a, Qi et al. 2017). However, extracting sporophyll requires a special processing technique because of its dense, rigid structure (Dong et al. 2019). Since extraction methods can substantially impact the usability of raw materials, and the techniques differ in their energy and organic solvent requirements, improved processing methods are necessary to make extracting sporophyll viable.
Ultrasonic-assisted extraction requires less energy input than other extraction methods (Chemat et al. 2012) and is recognized as a green extraction technology. The principle behind ultrasonic extraction is acoustic cavitation, which collapses bubbles and impacts cell wall surfaces (Tiwari 2015) to facilitate the release of bioactive substances and reduce polysaccharide weight (Kim and Zayas 1991, Kardos and Luche 2001). Ethanol precipitation can be used to refine polysaccharides in aqueous extracts (Jin et al. 2012). Since the optimal ethanol concentration for polysaccharide reactivity is affected by the structural characteristics of the polysaccharides (Xu et al. 2014), it is essential to optimize ethanol concentrations to precipitate the target substances.
Acute skin damage is a common phenomenon that is mainly induced by external factors, such as ultraviolet B (UVB) light (Clydesdale et al. 2001), pathogens (Shah and Shah 2011), and environmental pollutants (Bocheva et al. 2023). Exposure to UVB light damages the cutaneous layer and increases the risk of skin inflammation (Clydesdale et al. 2001, Portugal-Cohen et al. 2017). The effect of UVB on skin can be amplified by interactions with other factors, such as airborne pollutants (Araviiskaia et al. 2019). UVB radiation activates cell signaling molecules, such as mitogen-activated protein kinases (MAPKs); increases the level of inflammation-related enzymes, such as cyclooxygenase-2 (COX-2) (Cho et al. 2005); and induces the excessive production of reactive oxygen species (ROS) (Ansary et al. 2021). Imbalances between ROS production and the antioxidant system cause oxidative stress in living organisms (Pizzino et al. 2017). A variety of environmental factors (e.g., pathogen infection, air pollutants, and UVB exposure) induce oxidative stress (Xie et al. 2019). Lipopolysaccharide (LPS) derived from gram-negative bacteria triggers ROS formation mediated by nicotinamide adenine dinucleotide phosphate oxidase (Shang-Guan et al. 2018), which results in the outbreak of inflammation and chronic diseases (Castaneda et al. 2017, Sharifi-Rad et al. 2020). Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor associated with anti-inflammatory activity that regulates the antioxidant defense system against ROS (Nishikawa et al. 2020). Since various stimuli can cause oxidative stress and inflammatory responses that result in acute skin damage, it is important to find new candidates that prevent oxidative stress and inflammation by targeting related signaling molecules throughout the body.
In this study, we aim to identify the U. pinnatifida sporophyll extract (UPE) fractions with different molecular structures that demonstrate enhanced functions for skin health. We hypothesize that UPE fraction (UEF) is more effective at preventing acute skin damage than unfractionated UPE and assume that the difference in efficacy is caused by their differing structural and chemical characteristics. To test our hypothesis, we fractionated UPE using gradient ethanol precipitation and analyzed the physicochemical properties of the fractions. We also investigated whether the UEFs have preventive effects against skin inflammation and oxidative stress and examined the associated mechanisms of action. This research provides a new perspective on the viability of UEFs as functional materials and their enhanced functionality after ultrasonic extraction and fractionation.
MATERIALS AND METHODS
Preparation of ultrasonic UPE and its fractions
Dried U. pinnatifida sporophyll (Wando-gun, Korea) was stored at 4°C and crushed into a powder prior to the extraction. A total of 400 g of U. pinnatifida sporophyll powder was extracted with 40 L of water using an ultrasonic extraction system (Mirae Ultrasonic, Bucheon, Korea) set to 20 kHz, 1,080 W, and 30°C for 8 h. After centrifugation at 2,800 ×g for 20 min, the supernatant was lyophilized to obtain UPE. The stepwise ethanol precipitation method used to obtain the crude polysaccharide fractions was described by Fernando et al. (2020). Briefly, 1 L of ethanol was added to a 0.5 L solution containing 30 g of extract, and the mixture was precipitated for 1 day at 4°C and then centrifuged (5,000 ×g, 15 min) to collect the precipitate. This precipitate is referred to as UEF1. The upper layer was precipitated for 1 day after adding ethanol (1 L) and then centrifuged (5,000 ×g, 15 min). UEF2, UEF3, and UEF4 were obtained through repetitions of these steps. The four precipitant fractions and the final supernatant were freeze-dried after removing the ethanol.
Fraction yield and composition analysis
The ratio of the dry weight of each fraction to the dry weight of extract was used to calculate the fraction yield. Total carbohydrate content was quantified using the phenol-sulfuric acid method and a total carbohydrate assay kit (Sigma-Aldrich, St. Louis, MO, USA) with glucose as the standard. The protein was quantified using a detergent compatible protein assay kit (Bio-Rad Inc., Hercules, CA, USA) with bovine serum albumin (Sigma-Aldrich) as the standard. The total polyphenol content was evaluated using a modified version of the methodology described by Zhang et al. (2006). Briefly, the sample solution (20 μL) and the sequential standard solutions were placed into a 96-well microplate. Folin-Ciocalteu reagent (100 μL) was introduced into each well, and the samples were thoroughly mixed. After 5 min of rest without disturbance, 7.5% sodium carbonate solution (80 μL) was added to the samples, and all solutions were mixed again. After reacting in a dark place (at 20–25°C) for 2 h, the absorbance was detected using a microplate reader (SpectraMax M2e; Molecular Devices, San Jose, CA, USA) at a wavelength of 750 nm.
Fucoxanthin content analysis
The fucoxanthin content was analyzed following the method described by Eom et al. (2020). A high-performance liquid chromatography (HPLC) system (Dionex, Sunnyvale, CA, USA) equipped with an Inertsil ODS-3 column (4.6 × 250 mm, 5 μm) (GL Science, Tokyo, Japan) was used to quantify the fucoxanthin content. The samples were eluted isocratically with 75% acetonitrile at a mobile phase flow rate of 1 mL min−1. Peaks were detected using a UV detector (450 nm), and the concentration of fucoxanthin was calculated from its peak area.
Analysis of molecular weight and sugar composition
Molecular weight and sugar composition were analyzed using the method described by Lee et al. (2023). The molecular weight of the samples was quantified through size exclusion chromatography using an HPLC system (Agilent, Palo Alto, CA, USA). Samples were dissolved in distilled water and filtered through a 0.45 μm pore-size syringe filter before they were injected into the HPLC system. The analysis was performed using Shodex SB-804HQ and SB-802.5HQ OHPak columns (Showa Denko, Tokyo, Japan) at 55°C with water that was isocratically eluted at a flow rate of 0.6 mL min−1. A refractive index detector was used to detect peaks. The molecular weight of each peak was evaluated using maltooligosaccharides (specifically, glucose to maltohexaose) and the Pullulan series (Shodex Standard Pullulan kit P-82; Showa Denko) as molecular weight markers.
The monosaccharide composition of the samples was analyzed using a high-performance anion-exchange chromatography system equipped with a CarboPac PA10 column (2 × 250 mm; particle size = 10 μm), which was coupled with pulsed amperometric detection (Dionex). Trifluoroacetic acid was used to hydrolyze samples, and the solvent was eluted at a flow rate of 1.0 mL min−1 using an 18 mM NaOH/200 mM NaOH gradient system.
Sulfate content analysis
Sulfate polysaccharide contents were determined following the BaCl2 gelation method described by Dodgson and Price (1962).
Cell culture
Human immortalized keratinocyte cell line HaCaT, mouse macrophage RAW 264.7 cells, and human bronchial epithelial cells BEAS-2B were incubated in Dulbecco’s modified Eagle’s medium (DMEM; high glucose with L-glutamine) cell culture media, which was prepared with a 1% penicillin-streptomycin solution and 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA). Cells were grown in a CO2 incubator (Eppendorf, Hamburg, Germany) with a humidified atmosphere (37°C). DMEM/high glucose with L-glutamine that did not contain phenol red (serum-free media; Hyclone) was used for the starvation of HaCaT cells.
Reagents and materials
LPS, Escherichia coli O111:B4 (Sigma-Aldrich) was diluted in sterilized phosphate-buffered saline (PBS; Hyclone). Fucoidan from U. pinnatifida, N-acetyl-L-cysteine (NAC), and (−)-epigallocatechin gallate (EGCG) (Sigma-Aldrich) were diluted with dimethyl sulfoxide (DMSO). Primary antibodies against HO-1, KEAP1, α/β-tubulin, COX-2, p-ERK1/2, ERK1/2 (Cell Signaling Technology, Beverly, MA, USA), NRF2, Lamin B1 (Abcam, Cambridge, UK), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were diluted according to the manufacturer’s protocols. The secondary antibodies, horseradish peroxidase-conjugated Pierce goat anti-rabbit IgG (H + L) and Alexa Fluor 488 goat anti-rabbit IgG (H + L), were supplied by Thermo Fisher Scientific (Waltham, MA, USA).
Cytotoxicity test
To test cell viability, HaCaT cells (1 × 105 cells mL−1), RAW 264.7 cells (3 × 105 cells mL−1), and BEAS-2B cells (3 × 104 cells mL−1) were seeded into 96-well plates and incubated until they reached 70–80% confluency. Cells were treated with UEFs, UPE, and supernatant (25, 50, and 100 μg mL−1) for 24 h, and then reacted with 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Sigma-Aldrich). After 2 h, 80 μL of media were discarded and 100 μL of DMSO was added. After reacting in a dark room for 30 min, the plate absorbances were detected at a wavelength of 595 nm and evaluated for cell viability.
Western blot
HaCaT cells (1 × 105 cells mL−1) were grown until cells reached 60–70% confluency. After 12 h of alteration of culture media to new serum-free media, cells were pretreated with UEFs, UPE, supernatant (100 μg mL−1), EGCG (10 μM), and fucoidan (12.5, 25, and 50 μg mL−1). After 1 h of treatment, cells were irradiated with UVB (0.03 J cm−2) and harvested with lysis buffer (Cell Signaling Technology) and protease and phosphatase inhibitors (Thermo Fisher Scientific). RAW 264.7 cells (3 × 105 cells mL−1) were pretreated with UEF3 (25, 50, and 100 μg mL−1), stimulated with LPS for 1 h, and harvested following the procedure described for HaCaT cells. Lysate proteins were quantified using a detergent compatible protein assay kit (Bio-Rad Inc.), and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used for different sizes of proteins. Immobilon polyvinylidene fluoride membranes (MilliporeSigma, Burlington, MA, USA) with a pore size of 0.45-μm were used to transfer the proteins, which were then incubated with the primary antibodies overnight. Membranes were reacted with secondary antibodies that were specific to the primary antibodies, and protein expression was detected using a chemiluminescence detection kit (ATTO, Tokyo, Japan). Visual imaging was performed using Gene Gnome XRQ NPC (Syngene, Bengaluru, Karnataka, India), and Image J software was used to quantify protein bands.
Animal study
The animal study was conducted in strict compliance with relevant guidelines, and the Kyungpook National University committee reviewed and approved the study (KNU 2023-0480). The 5-week-old, male, ICR mice were supplied by Saemtako BIO KOREA (Osan, Korea). They were housed in a climate-controlled space at 25°C with 50% humidity and a 12-h light/dark cycle. The mice were allowed to consume sterilized water and a standard diet without restriction. Mouse anesthesia (a 2 : 1 mixture of ketamine and rumpun) was administered by intraperitoneal injection to facilitate hair removal.
Experimental design
Mice were allowed to acclimatize for 2 weeks before the study began. Sterilized water and acetone were used as vehicles; UEF3 was dissolved in sterilized water and EGCG was dissolved in acetone. The 25 mice were randomly assigned to one of five groups (n = 5 per group): (1) control group, (2) UVB-radiated group, (3) UVB + 5 mg kg−1 d−1 UEF3 group, (4) UVB + 25 mg kg−1 d−1 UEF3 group, and (5) UVB + 3 mg EGCG group. Mice were orally administered sterilized water or UEF3 daily for 14 days. On day 12, dorsal hair was removed from each mouse using a clipper and hair removal cream. On day 13, the mice were orally administered sterilized water and UEF3 and subsequently treated with acetone or EGCG applied topically to their dorsal skin. On day 14, 1 h after the oral administration of UEF3 and topical treatment with EGCG, the mice were irradiated with UVB (0.5 J cm−2). The mice were then sacrificed and their dorsal skin was harvested after 4 h of UVB exposure.
Hematoxylin and eosin staining
To observe the epidermal thickness of mice dorsal skin, skin tissue samples were blocked using optimal cutting temperature compound (Leica Biosystems, St. Gallen, Switzerland). The sliced skin tissue blocks (8-μm thickness) were prepared with a histology cryostat (Leica CM1850 Cryostat, Leica Biosystems) and then soaked with 4% formaldehyde (10 min). To stain the cell nucleus, tissues were dipped in hematoxylin reagent (TissuePro Technology, Gainesville, FL, USA) five times. After a water rinse, the tissues were immersed in 95% ethanol 10 times. The cell cytoplasm and extracellular matrix were stained with eosin Y solution (TissuePro Technology) and cleaned with xylene. After the tissue sections were covered with the mounting solution, a fluorescent microscope (Leica Microsystems, Wetzlar, Germany) and Leica Application Suite X (LAS X) software (Leica Microsystems) were used to observe and quantify epidermal thickening.
Immunofluorescence
The process of embedding and sectioning the mouse skin tissue samples followed the steps described in the hematoxylin and eosin staining section. Prepared frozen tissue samples were soaked in 4% formaldehyde for 15 min. After blocking with PBS (mixed with 5% FBS and 0.3% Triton X-100), anti-COX-2 antibody (1 : 400) was added to the samples before incubation overnight at 4°C. The following day, the tissues were incubated with goat anti-rabbit IgG (H&L) conjugated to Alexa Fluor 488 secondary antibody (Abcam) for 1 h. After incubation, the nucleus was counterstained with 4′,6-diamidino-2-phenylindole medium (Abcam). COX-2 expression was observed using fluorescence microscopy (Leica Microsystems).
Evaluation of free radical scavenging capacity
2,2-Diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, and alkyl radical scavenging activities were evaluated using an electron spin resonance (ESR) spectrometer following the methodology described by Ahn et al. (2007).
Intercellular ROS measurements
To quantify ROS production, RAW 264.7 cells (3 × 105 cells mL−1) were grown in 96-well plates and allowed to react with the UEFs, UPE, supernatant (100 μg mL−1), and NAC (25 μM). After 1 h of treatment, LPS (1 μg mL−1) was added to the cells, which were incubated for 24 h. Next, the cells were incubated with 20 μM of 2′,7′-dichlorofluorescein diacetate (DCF-DA) for 30 min before their fluorescence was analyzed using a fluorescent plate reader (Molecular Devices) at an absorbance between 485 and 538 nm. A fluorescence microscope (Leica Microsystems) was used to observe ROS localization. Cell images were captured and edited using Las X (Leica Microsystems).
Cytosol/nucleus fractions
To separate proteins from the cytosol and the nucleus, RAW 264.7 cells (3 × 105 cells mL−1) were grown in 10-cm dishes. After incubation with UEF3 and NAC for 1 h, the cells were treated with LPS. All cell culture media was removed, and the cells were washed twice with PBS. After an additional 1 mL of PBS was added, the cells were collected with scrapers and centrifuged at 500 ×g for 3 min at 4°C. The cell pellets were reacted with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Protein expression was evaluated through western blotting.
Statistical analysis
Data is presented as the mean ± standard deviation for three independent experiments using GraphPad Prism (La Jolla, CA, USA). For statistical analysis, one-way ANOVAs and Dunnett’s post hoc test were carried out in GraphPad Prism (GraphPad). Statistical significance among the groups was defined as p < 0.05.
RESULTS AND DISCUSSION
Changes in yield and structural characteristics via the UPE fractionation system
Interest in the effects of macroalgae on human health is steadily increasing (Magwaza and Islam 2023). However, the processing method of sporophyll has been studied to solve the difficulties caused by its rigid structure (Dong et al. 2019). When we compared water-only and ultrasonic-assisted extraction methods, we found that the ultrasonic-assisted extraction method can remove heavy metals, but water extraction cannot (data not shown). We found that the optimal ultrasonication conditions were 20 kHz, 1,080 W, and 30°C for 8 h to maximize yield, polysaccharide content, and antioxidant activity. Our previous study showed that using an ultrasonication process to extract U. pinnatifida sporophyll altered its carbohydrate molecular size, which resulted in elevated anti-inflammatory effects (Lee et al. 2023). Successful bond formation between precipitated polysaccharides and ethanol depends on the concentration of ethanol; differing concentrations result in the unique physiochemical properties of each fraction (Xu et al. 2014, Tai et al. 2020). To characterize the functional polysaccharides and evaluate their structural properties and physiological activities, we precipitated U. pinnatifida sporophyll extract using a gradient of ethanol concentration and separated fractionated U. pinnatifida sporophyll extract into four fractions (UEF1–4) and supernatant (Fig. 1).
To identify compositional changes induced by the fractionation process, we analyzed the yield and composition of UPE, the UEFs, and the supernatant. The yield decreased as fractionation progressed: UEF1 had a yield of 16.7%, UEF2 was 11.42%, UEF3 was 3.45%, UEF4 was 3.20%, and the supernatant was 53.57%. This decrease is likely due to harvesting the next fraction from the residue of the previous fraction. Although each fraction has different total carbohydrate, protein, and polyphenol contents, they had the highest carbohydrate contents in common. UEF1 had the highest total carbohydrate, protein, and polyphenol contents (63.39, 32.51, and 1.30%, respectively), possibly due to the differences in reactivity between the different polysaccharide components and ethanol. Fucoxanthin content was the highest in UEF1 (10.7%). The other fractions exhibited similar low values (1.1–1.3%), which indicates that most of the fucoxanthin was extracted during the first precipitation step (Table 1). Therefore, we propose that inducing polysaccharide precipitation through an ethanol gradient can subdivide UPE into its UEFs, each of which has unique physicochemical properties.
Changes in polysaccharide molecular weight by UPE fractionation
Physicochemical and biological properties of polysaccharides can vary with the size and weight of the molecule (Gómez-Ordóñez et al. 2012, Yu et al. 2018). Therefore, developing technology to identify and control molecular weight in an industrial setting is essential.
We analyzed the specific molecular weights of UPE, the UEFs, and the supernatant to determine which molecular weights enhance physiological activity (Table 2). Each UEF had a distinct molecular weight range. UPE was composed of two main peaks with peak molecular weights of 1,486 and 0.186 kDa. The ratio of the peak area was 24.62% and 75.38% for each peak, respectively. As fractionation progressed, the molecular weight of the larger peak (Peak 1) tended to decrease, while its peak area ratio gradually increased. UEF3 and UEF4 contained high amounts of large molecular weight polysaccharides that comprised 92.83% of the 1,012 kDa peak and 94.62% of the 826 kDa peak, respectively. This result can likely be explained by the effects of the polysaccharide precipitation process, which can alter the molecular weight of UPE based on the differences in reactivity with varying ethanol concentrations.
Monosaccharide composition and sulfate polysaccharide content of UPE fractions
The monosaccharide composition of the UEFs, UPE, and the supernatant is described in Table 3. Galactose and fucose were the major polysaccharide components that the UEFs and UPE had in common, accounting for more than 80% of the content in all of the samples, with the exception of the supernatant. By contrast, the largest component of the supernatant was glucose (37.35%), followed by xylose (22.56%). Its galactose (8.57%) and fucose (14.58%) contents were relatively low. Galactose and fucose are well known for their high antioxidant activities (He et al. 2012, Yang et al. 2013, Kim et al. 2016b), and sometimes exist as heteropolymers. Since sulfated galactofucan contributes to bioactivity (Zayed et al. 2022), the analysis of the monosaccharide composition and sulfated polysaccharides content suggest that galactofucan is a major polysaccharide with physiological activity present in the UEFs.
Algae extract structure can change with processing to include sulfate in their polysaccharide structure (Silva et al. 2012). We investigated the sulfate polysaccharide content of the UEFs, UPE, and the supernatant. The results are exhibited in Table 4. UEF3 showed the highest sulfate content (33.89 ± 0.05%), followed by UEF4 (26.95 ± 0.10%), UEF2 (25.65 ± 0.15%), and UEF1 (13.02 ± 0.15%). However, UPE and the supernatant had remarkably low sulfate polysaccharide contents compared to the UEFs. These results suggest that sequential precipitation is not linked to the increase in sulfated polysaccharide content. The process of fractionation likely breaks down molecular structure and allows molecules to be aggregated into larger units. This is also relevant to the results of the molecular weight analysis (Table 2), which highlights the need to consider fractionation in extraction techniques to produce the optimal structure and physiological activity.
Biological safety assessment of UPE fractions in vitro
Producing a safe extract without side effects is more important than its biological activity. For this reason, we conducted safety assessments before assessing health benefits. We tested the cellular toxicity of the extracts in three cell lines: HaCaT, RAW 264.7, and BEAS-2B cells. The test concentrations of UPE and its fractions did not cause cytotoxicity in any of the three independent cell lines (Fig. 2), which indicates that fractionated UPE is a safe material for biological use. Therefore, we further investigated the efficacy of UPE and its fractions in the concentration range where cell safety was confirmed.
Preventive efficacy of UPE fractions against acute UVB exposure
Interest in skin health-promoting marine biological materials is increasing (Siahaan et al. 2022). However, most of the research on developing materials for skin health has focused on plant-based products. Therefore, we evaluated the possibility of using UPE and its fractions as a marine nutraceutical for skin inflammation at the cellular and tissue levels.
UVB induces skin inflammation by activating COX-2, an inflammation marker, which mediates inflammatory pathways such as MAPKs (Ansary et al. 2021). Our study assessed whether UPE, the UEFs, and the supernatant could suppress the overexpression of COX-2 mediated by UVB in HaCaT cells. Notably, UEF2, UEF3, UEF4, and the supernatant showed significant inhibitory effects on COX-2 expression, while UEF1 and UPE had no effect (Fig. 3A). We further investigated their impact on MAPK phosphorylation and found that UEF3 repressed the UVB-induced ERK1/2 phosphorylation (Fig. 3B). However, UEF3 had no effect on the JNK1/2 and p38 pathways (data not shown).
Based on the in vitro study, UEF3 is a promising anti-inflammatory material for UVB-induced acute skin damage. Therefore, we examined whether orally administered UEF3 can prevent skin inflammation from UVB exposure in vivo. The process is illustrated in a schematic representation of the overall experiment (Fig. 4A). We analyzed the efficacy of UEF3 using harvested mouse skin tissue and compared its efficacy with EGCG, which is known to alleviate UV-induced skin damage (Katiyar and Mukhtar 2001). Oral administration of UEF3 showed a preventive effect on the abnormal epidermal thickening caused by UVB irradiation. A high dose (25 mg kg−1 d−1) of UEF3 intake resulted in a reduced epidermal thickness (16.98 ± 3.21 μm) that was similar to the results from applying EGCG topically (17.82 ± 3.31 μm) (Fig. 4B). In addition, UEF3 suppressed COX-2 overexpression mediated by UVB in a dose-dependent manner (Fig. 4C), suggesting that oral UEF3 administration exerts anti-skin inflammatory effects similar to those of EGCG, a well-known topical formulation. Oral UEF3 administration did not cause body weight loss, which can indicate toxicity (Supplementary Fig. S1). Therefore, we suggest that UEF3 is a safe food ingredient for skin health without any adverse effects.
Preventive efficacy of UPE fractions against oxidative stress
An imbalance in ROS production and accumulation induces oxidative stress and causes inflammation. Repetitive inflammation may lead to the progression of many diseases, such as cardiovascular diseases, diabetes, and skin cancer (Sharifi-Rad et al. 2020). Since antioxidant efficacy is relevant to skin health, we confirmed the antioxidant properties of UPE, the UEFs, and the supernatant to highlight the preventive efficacy of these ingredients for skin health.
The free radical scavenging capabilities of UPE and its fractions were analyzed using ESR, which revealed their scavenging effects on DPPH, hydroxyl, and alkyl radicals in all samples (Table 5). To further evaluate the biological relevance of their antioxidant potential, we examined the effects of UPE and its fractions on LPS-induced ROS production in RAW 264.7 cells. ROS overproduction was inhibited by UEF3 (76.12 ± 7.85%), UEF4 (78.14 ± 14.04%), UPE (73.72 ± 13.08%), and the supernatant (66.65 ± 11.73%). Their inhibitory effects were comparable to those of NAC (67.13 ± 10.91%), a well-known antioxidant (Zhitkovich 2019) (Fig. 5A & B).
Based on these observed anti-inflammatory and antioxidant activities, we investigated the antioxidant mechanism of UEF3 at the molecular level.
The antioxidant-related gene HO-1 can be activated by NRF2 nuclear translocation and exerts a protective effect against external stimulation (Chiang et al. 2018). We examined whether UEF3 could modulate HO-1 expression via NRF2 activation. UEF3 upregulated HO-1 expression (Fig. 5C) and induced NRF2 nuclear translocation by downregulating KEAP1 expression in the cytoplasm (Fig. 5D). Since the reduction of oxidative stress can alleviate inflammation (Leyane et al. 2022), we demonstrated that UEF3 functions as an anti-inflammatory and antioxidant agent via the upregulation of the HO-1/NRF2 signaling pathway. UEF3 consistently demonstrated the highest efficacy in both anti-inflammatory and antioxidant capacities for skin health. Based on the structural analysis results, increased sulfate polysaccharides seem to be associated with enhanced efficacy, although the answer is likely more complex and related to a variety of components identified in the UPE fractions. Polyphenols and fucoxanthin, which are found in U. pinnatifida, are known to promote human health (Roh et al. 2008, Peng et al. 2011). However, a direct relationship between these compounds and efficacy was not established in our study. Nevertheless, our findings suggest that fractionated UPE can be a safe, functional food material with higher antioxidant and anti-inflammatory effects than conventional extracts. Our study provides a new perspective on the development of safe, edible materials for skin health.
CONCLUSION
Ultrasonic-assisted fractionation of U. pinnatifida sporophyll extract significantly enhanced its preventive effects against acute skin damage and oxidative stress. Structural analysis revealed progressive changes in the components present in each extract after fractionation, which included alternations to carbohydrate molecular weight, monosaccharide composition, and sulfate polysaccharide content. Functional analysis demonstrated that the fractionated extracts exhibited superior skin damage prevention and antioxidant efficacy than the unfractionated extracts. Among the fractions, UEF3 showed the highest efficacy. UEF3 mitigated UV-induced skin damage by regulating the ERK kinase pathway and counteracted oxidative stress by activating the NRF2/HO-1 signaling pathway. These findings suggest that fractionated UPE is a safe, beneficial substance with enhanced physiological benefits compared to conventional extracts. Furthermore, the fractionation process presents a promising strategy to optimize the biological activity of natural products.
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