General Experimental Procedures

1H, 13C and 2D NMR spectra were generated using a JEOL JNM-ECA 400 and JEOL JNM-ECA600 instruments (JEOL, Tokyo, Japan) using TMS as an internal standard. High resolution election spray ionization mass spectrometry (HRESIMS) data were acquired using a Waters SYNAPT G2-Si HDMS spectrometer (Waters, Milford, MA, USA). The aforementioned instruments were analyzed at the Center for University-Wide Research Facilities, Jeonbuk National University (Jeonju, Korea). Column Chromatography (C.C) was performed using Silica gel (Kieselgel 60, 200–400 mesh, Merck, Darmstadt, Germany). Each fraction was monitored by TLC profiling using silica gel 60 F254 and RP-18 F254s (Merck, Burlington, USA), and medium-pressure liquid chromatography (MPLC, CombiFlash RF, Teledyne lsco, Lincoln, NE, USA) was used to separate the fractions of the extract. Semipreparative high-speed liquid chromatography (semipreparative HPLC) was conducted using a Shimadzu LC-6AD instrument (Shimadzu Corp., Tokyo, Japan) equipped with an SPD-20A detector and Phenomenex Luna C18 (21.2 mm × 250 mm, 5 µm) column. All fractions and compounds were analyzed using an Agilent 1200 series HPLC system (Agilent, Santa Clara, USA).

Plant Material and Extraction

Ishige okamurae Yendo 1907, Fucaceae, were collected from the coast of Jeju island, Korea, between March and June 2021, from the area known as para Jeju. The samples were washed five times with tap water and dried in a constant-temperature room for 1 week. The dried I. okamurae (3 kg) were extracted with 70% EtOH (20 l) at 70 ℃ (5 h, × 20) and filtered (No. 10, 600 mm, Hyundai Micro Co., Seoul, South Korea). The filtrates were then concentrated under reduced pressure to obtain 100 g of extract. The residue (100 g) was suspended in distilled water (2 l), and the aqueous layer was partitioned with hexane, EtOAc and BuOH. The EtOAc layer (16.05 g) was subjected to silica gel column chromatography eluted with chloroform:methanol (1:0 → 0:1, v/v), to obtain 16 fractions (EA1 ~ EA16). Fraction EA2 (1.8 g) and EA8 (1.02 g) was subjected to MPLC [column: ODS RediSepRf (40 g); mobile phase: water:MeOH (1:0 → 0:1, v/v)] to yield 7 subfractions (EA2-1∼7) and 7 subfractions (EA8-1∼7). Fraction EA8-3 (20.7 mg) was further purified by preparative HPLC (Phenomenex Luna C18 column 250 × 21.2 mm, 5 μm) and isocratic elution with 30% CH3CN in H2O, resulting in the isolation of compound 1 (4.5 mg, Fig S3). For further research, diphlorethohydroxycarmalol was purchased from ChemNorm Biotech Co., Ltd (> = 98% (HPLC), Lot No. TFS20210619, Wuhan, China).

Diphlorethohydroxycarmalol (1): brownish yellow amorphous powder; ESI–MS m/z 512.1 [M + H]+; 1H NMR (DMSO-d6, 500 MHz, Fig. S1) δH 6.06 (1H, s), 5.87 (2H, s), 5.78 (1H, t, J = 2 Hz), 5.69 (1H, s), 5.67 (2H, d, J = 2 Hz); 13C NMR (DMSO-d6, 125 MHz, Fig. S2) δC 160.1, 158.9, 154.9, 151.3, 146.0, 143.0, 139.6, 138.8, 135.1, 133.8, 130.7 126.4, 125.5, 124.1, 122.9, 96.1, 95.0, 94.3, 93.7, 92.4 (Zou et al. 2008).

Cell Culture

The human keratinocyte cell line, HaCaT, was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin–streptomycin. The cells were incubated at 37 °C in a humidified atmosphere of 90–95% and 5% CO2.

Cell Viability

Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The HaCaT cells were seeded at a density of 3 × 104 cells/ well in 200 μL culture medium in 96-well plates and incubated for 24 h at 37 °C. The cells were treated with ethanol extract of I. okamurae or diphlorethohydroxycarmalol (1) at various concentrations: 1, 3, 6, 10, 30, 60, and 100 μg/ml of extract of I. okamurae and, 1, 3, 6, 10, 30, 60, and 100 μM 1. After 24 h of incubation, MTT solution was added to the plates, and the cells were further incubated 3 h at 37 °C. The results formazan crystals were dissolved in DMSO, and the absorbance was read at 540 nm using a microplate reader.

Real-Time Quantitative Polymerase Chain Reaction

Total RNA was isolated from HaCaT cells treated with extract of I. okamurae, diphlorethohydroxycarmalol or cyclosporine and/or tumor necrosis factor (TNF)-α (50 ng/mL)/ interferon (IFN)-γ (50 ng/ml) as well as from ear tissues from each group, using TRIzol reagent. First-strand complementary DNA (cDNA) was synthesized using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara Bio Inc., Shiga, Japan), and quantitative PCR was performed using a Bio-Rad T100 thermal cycler (Bio-Rad, Hercules, CA, USA) following the manufacturer’s protocol. Real-time PCR was carried out with a StepOnePlus Real-Time PCR System using TaqMan probes and TaqMan Real-Time PCR Master mix (Applied Biosystems, Foster City, CA, USA). For normalization, 18 s rRNA or GAPDH was used as an endogenous control. The following TaqMan primers and probes were purchased from Applied Biosystems, Thermo Fisher Scientific: TNF-α (Hs01113624_g1, Mm00443258_m1), interleukin (IL)-1β (Hs01555410_m1, Mm00434228_m1), IL-6 (Hs00174131_m1, Mm00446190_m1), IL-8 (Hs00174103_m1), CCL17 (Hs00171074_m1, Mm01244826_g1), and CCL22 (Hs01574247_m1, Mm00436439_m1).

Enzyme-Linked Immunosorbent Assay

Serum IgE, IgG1, IgG2a, cytokine (IL-1β, IL-6, IL-8, and TNF-α), and chemokine (CCL17/TARC, CCL22/MDC) levels in the cell culture medium were determined using ELISA kits (Thermo Fisher Scientific, Waltham, MA, USA; BD Biosciences, San Diego, CA, USA; R&D Systems, Minneapolis, MN, respectively) according to the manufacturer’s instructions. The absorbance was measured at 450 nm using a microplate reader.

Western Blotting

Total protein was extracted as previously described HaCaT cells (1 × 106 cells/well in 6-well plates) were pretreated with extract of I. okamurae, 1 or cyclosporine for 1 h and then stimulated with TNF-α/IFN-γ for 30 min. The cells were lysed with 100 μL of cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA), and the lysed samples were vortexed, incubated for 30 min on ice and centrifuged at 17,949 × g for 10 min at 4 °C. The supernatants were collected and quantified using a DC protein assay kit (Bio-Rad, Contra Costa County, CA, USA). Equal amounts of protein lysate were subjected to electrophoresis on an 8–12% SDS-PAGE gel and then transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% bovine serum albumin in tris-buffered saline, the membrane was incubated with primary antibody against the target protein, washed, and subsequently incubated with horseradish peroxidase-conjugated IgG secondary antibody. The bands were developed using a West-Queen RTS Western Blot Detection Kit (iNtRON Bio, Seongnam, Korea). The following antibodies were purchased from Cell Signaling Technology: phosphor(p)-p38 (#4511S, rabbit monoclonal, 1:1000), p38 (#8690S, rabbit monoclonal, 1:1000), p-ERK (#9101S, rabbit monoclonal, 1:1000), ERK (#4348S, rabbit monoclonal, 1:1000), actin (#4967S, rabbit monoclonal, 1:1000), and p-NF-κB (#3033S, rabbit polyclonal, 1:1000). The following antibodies were purchased from Santa Cruz: p-JNK (#sc-6254, mouse polyclonal, 1:1000), JNK (#sc-7345, mouse polyclonal, 1:1000), and p-Stat1 (#sc-8394, mouse polyclonal, 1:1000).

Animals

Female BALB/c mice (6 weeks) were purchased from Samtako (Osan, Korea). All animals had ad libitum access to standard rodent chow and filtered water during the study. The animals were housed (5 per cage) in a laminar air flow room maintained at a temperature of 22 ± 2 °C, relative humidity of 55 ± 5%, on a 12-h light/dark cycle throughout the study. Animal care and treatment protocols were conducted in accordance with the guidelines established by the Public Health Service Policy on the Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB-AEC-21103).

Generation of DNCB/HDM-Induced Atopic Dermatitis-Like Lesions

A total of 25 mice were divided into five groups (n = 5): the vehicle phosphate-buffered saline (PBS), DNCB/HDM vehicle (PBS), DNCB/HDM and extract of I. okamurae (50 and 100 mg/kg), and DNCB/HDM and dexamethasone (DX) (1 mg/kg) groups. During the first week of induction DNCB (2%, 20 µl/ear) was applied once to each ear for sensitization. Then, both ears of each BALB/c mouse were challenged with DNCB/HDM (DNCB: 1%, 20 µl/ear, HDM: 10 mg/ml, 20 µµl/ear) for 3 weeks. Ethanol extract of I. okamurae (50 and 100 mg/kg) or DX (1 mg/kg) was orally administered by gavage for 5 consecutive days per week at the time of the DNCB/HDM challenge. Analysis of DPHC (25 mg/kg) was performed in the AD model, and diphlorethohydroxycarmalol was administered instead of extract of I. okamurae.

Histological Assay

Mouse ear tissues were fixed in 4% (w/v) paraformaldehyde in PBS, (pH 7.4). The fixed tissues were embedded in paraffin and cut into 4-μm-thick sections, which were stained with hematoxylin and eosin (H&E) and toluidine blue.

Statistical Analysis

Statistical analysis was performed using Prism 5 software (GraphPad Software, San Diego, CA, USA). The data are presented as the mean ± SD of nine individual experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test.