Sacran was extracted from cyanobacterium Aphanothece sacrum, and was gifted by Daito Kasei Kogyo Co., Ltd. (Osaka, Japan). RPMI-1640 medium was purchased from Nissui Pharmaceuticals (Tokyo, Japan). TRIzol^® Reagent and fatal bovine serum (FBS) were purchased from Invitrogen (Tokyo, Japan). 2,4-Dinitrofluoroben- zene (DNFB) was purchased from Wako Pure Chemical Industries (Osaka, Japan). Monoclonal anti-dinitro- phenyl IgE (Anti-DNP-IgE) and dinitrophenyl-bovine serum albumin (DNP-BSA) protein were purchased from Cosmo Bio (Tokyo, Japan). Phorbol-12-myristate-13-acetate (PMA) was purchased from Sigma Aldrich (Tokyo, Japan). All other chemicals and solvents were of analytical reagent grade.

Sacran was extracted from cyanobacterium Aphanothece sacrum followed by previous report [15] . Briefly, the Aphanothece sacrum (1 kg) was freeze-thawed, washed by distilled water to remove aqueous pigment (phycobilin), immersed and stirred in a water bath filled with a large amount of dilute hydrochloric acid (pH 3) at 60˚C for 24 h. After washed with 50% - 70% (v/v) isopropanol overnight, a polysaccharide was collected by filtration and air dried at 60˚C. The polysaccharide was extracted using 0.1 M sodium hydroxide solution at 70˚C with agitation for 3 h. Then, the polysaccharides solution was adjusted to a pH 7 to 8 by 0.1 M hydrochloride and filtered by sterile cotton gauge. The filtrate was slowly poured into absolute isopropanol to precipitate white fibrous materials, which were then redissolved in hot water and reprecipitated 3 times. The polysaccharide fibers were dried (yield, 0.65 wt% - 0.75 wt% to wet biomaterials and 60 wt% - 70 wt% to dried ones). The aqueous solution showed no specific absorption in the wavelength range of 220 to 600 nm in UV-visible spectroscopy, which confirmed that there was no contamination by proteins, nucleic acid, chromospheres, and/or other chemicals with UV-visible absorption.

This case descriptive, clinical and biochemical study was conducted in the Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University. Twenty five patients (5 males and 20 females) were enrolled in this study. The nature and aim of this study were explained for each patient. Formal consent was taken from each patient before this study. The clinical studies were approved by the Ethics Committee of Kumamoto University (Approval No.745). The patients used the sacran solution (0.05% (w/v)) for every morning and night after taking shower for 4 weeks. The patients enrolled in this study were well-controlled by standard therapy. During this clinical study, the standard therapy for patients did not changed at all. About 13 questionnaire items as indicated in Table 1, we obtained an answer from the patients with atopic dermatitis after treatment with sacran solution for 2 weeks and 4 weeks. Also, the dermatologists evaluated an area of disorder on face, total score of rash.

Trans-epidermal water loss (TEWL) was determined by a VapoMeter Wireless SWL4 (Delfin technology, Finland). Before each measurement time, the apparatus will be calibrated according to the recommendations of the manufacturer. Measurements will be undertaken after a 20 min rest period, according to the guidelines set out by Standardization Group appointed by the European Society of Contact Dermatitis [16] . Room temperature will be kept between 20˚C and 24˚C and the relative humidity will be comprised between 40% (w/v) and 60% (w/v). Three acquisitions will be performed on the bony areas of the upper cheek of left and right sides.

TESTSKIN™ LSE-high, a three-dimensional cultured skin, was purchased from TOYOBO Life Science (Osaka, Japan). RAW264.7 cells, a murine macrophage-like cell line, and RBL-2H3 cells, a rat basophilic leukemia cell

line, were obtained from Riken Bioresource Center (Tsukuba, Japan) and ATCC (Summit Pharmaceuticals International Corporation, Tokyo, Japan), respectively. RAW264.7 cells and RBL-2H3 cells were grown in RPMI- 1640 medium, containing 1 × 10⁵ mU/mL of penicillin, 0.1 mg/mL of streptomycin supplemented with 10% FBS at 37˚C in a humidified 5% CO₂ and 95% air atmosphere.

Total RNA was isolated from TESTSKIN™ or RAW264.7 cells after stimulation with DNFB or PMA, respectively, using TRIzol^® Reagent according to the manufacturer’s protocol. The RNA (0.5 μg) was subsequently reverse-transcribed using ReverTra Ace^® qPCR RT Master Mix (TOYOBO, Osaka, Japan). The expression levels of MCP-1, TNF-α, IL-6, IL-1β and COX-2 were determined using real time PCR assay. Real-time PCR was performed on a CFX96™ Real-Time PCR (BIO RAD, Tokyo, Japan) using 1 μL of cDNA for each sample. THUNDERBIRD™ SYBR^® qPCR Mix (TOYOBO, Osaka, Japan) was used to detect products, and 10 μM concentrations of the following primers were used: mouse GAPDH forward: 5’-GGTGAAGGTCGGTGTGAA CGGATT-3’, mouse GAPDH reverse: 5’-AATGCCAAAGTTGTCATGGATGACC-3’, MCP-1 forward: 5’-ATGCAATCAATGCCCCAGTC-3’, MCP-1 reverse: 5’-TGCAGATTCTTGGGTTGTGG-3’, mouse TNF-α forward: 5’-AACATCCAACCTTCCCAAACG-3’, mouse TNF-α reverse: 5’-CTCTTAACCCCCGAACTCCC AG-3’, mouse IL-6 forward:5’-TGGAGTCACAGAAGGAGTGGCTAAG-3’, mouse IL-6 reverse: 5’-TCTGAC CACAGTGAGGAATGTCCAC-3’, mouse IL-1β forward: 5’-AAACCTCTTCGAGGCACAAG-3’, mouse IL-1β reverse: 5’-GTTTAGGGCCATCAGCTTCA-3’, mouse COX-2 forward: 5’-AGCACTGCATCCTGC CAGCTC-3’, mouse COX-2 reverse: 5’-AGAGGACTGTGGCTCTGACC-3’. The relative amount of cDNA in each sample was normalized using GAPDH, and the melting curve was used to verify specificity. PCR was set at 95˚C initially for 30 s, followed by 40 cycles of 95˚C × 15 s, 55˚C × 15 s, 72˚C × 45 s. The relative amount of cDNA in each sample was normalized using GAPDH, and the melting curve was used to verify specificity.

The release of β-hexosaminidase from RBL-2H3 cells were evaluated by the following modified method [17] . RBL-2H3 cells in 24-wellplate (5 × 10⁵ cells/mL) were sensitized with anti-DNP IgE (0.45 μg/mL), and incubated for 24 h. Then, the cells were washed with 400 μL of Siraganian buffer (buffer A) [119 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl₂, 1 mM CaCl₂, 25 mM piperazine-N,N’-bis (2-ethanesulfonic acid) (PIPES), 0.1% BSA, and 40 mM NaOH, pH 7.2]. An aliquot of buffer A (160 μL) was added and incubated for 10 min at 37˚C. The 20 μL of test sample solution was added to each well and incubated for 10 min, followed by the addition of 20 μL of antigen (DNP-BSA, final concentration 10 μg/mL) at 37˚C for 20 min to stimulate cell degranulation. The supernatants (50 μL) were transferred into 96 well microplate and incubated with 50 μL of substrate PNAG (1 mM p-nitrophenyl-N-acetylb-D-glucosaminide) in 0.1 M citrate buffer (pH 4.5) at 37˚C for 3 h. The reaction was stopped by adding 200 μL of stop solution (0.1 M Na₂CO₃/NaHCO₃, pH 10.0). The absorbance was measured with a microplate reader at 405 nm. The percentage of β-hexosaminidase release into supernatant was calculated according to the following formula: β-Hexosaminidase release ratio (%) = 100 × S/(S + CL), where CL and S are the absorbance of cell lysate and the absorbance of supernatant, respectively.

In statistical analysis of grades in clinical studies evaluated by dermatologists, the p-value was determined employing the Wilcoxon signed-rank test, followed by the Bonferroni correction. In statistical analysis for in vitro data are presented as the mean ± S.E. Significant differences between experimental values were determined using Scheffe’s test, with p < 0.05 considered significance.

We analyzed the effects of sacran solution on the symptom of atopic dermatitis based on the 13 questionnaire results. The average scores more than 2 or less than 2 indicates the improvement or exacerbation of the symptoms of atopic dermatitis, respectively. As shown in Figure 1, almost all of the average scores of each patient treated with sacran solutions were more than 2. In addition, the scores of sleep disorder and itching tended to

decrease during the sacran treatment for 4 weeks with significant differences compared those of initial states (0 week) (Figure 2). These results suggest that the patients judged amelioration of their atopic dermatitis by the treatment with sacran solution.

Based on the dermatologist analysis, the area of rash on face in atopic dermatitis patients were significantly decreased (Figure 3(a)). In addition, the total scores of rash were also lowered by the treatment with sacran solution for 4 weeks. Taken together, the sacran solution may have the potential to ameliorate the symptom of atopic dermatitis.

The dry itchy skin worsens the severity of atopic dermatitis and associated with the increase in scratching behavior [18] . Next, we therefore investigated the effects of topical application of sacran on TEWL in 10 humans with dry skin (Figure 4). As the results, sacran solution markedly suppressed TEWL, compared to hyaluronan, a well-known polysaccharide that is reported to improve skin moisture and provide skin barrier repair activity [19] . Thereby, these results suggest that sacran can ameliorate the skin barrier protection and repair in atopic dermatitis patients.

The itchy skin elicits scratching behavior, leading to mechanical skin injury and the release of various inflammatory cytokines or chemokines [20] [21] . Therefore, we examined the effect of sacran on inflammatory cytokine and chemokine mRNA levels in immatured dermal skin model stimulated with DNFB. Here, we used immatured dermal skin model (Toyobo, Co, Ltd, Osaka, Japan) simulated the skin of atopic dermatitis patients having low skin barrier function. DNFB was chemical antigen to evoke contact hypersensitivity reaction and was widely used as an inducer of atopic dermatitis. After treatment with DNFB and sacran for 24 h, inflammatory cytokine and chemokine mRNA levels were determined by real-time PCR. As shown in Figure 5, MCP-1, TNF-α, IL-1β and IL-6 mRNAs were significantly down-regulated by the treatment with sacran, compared to those of DNFB alone. These results suggest that sacran may have the potential to reduce inflammatory cytokine and chemokine mRNA levels in skin of atopic dermatitis.

To reveal the anti-inflammatory activity of sacran, it is extremely important to investigate whether a sacran penetrates skin or not. Therefore, we examined the skin penetration of sacran in matured and immatured dermal skin models after treatment for 24 h. As a results, biotinylated sacran (0.05% (w/v)) was penetrate the dermal in immatured dermal skin model, but not in matured dermal skin model, simulated the skin of healthy human having rigid skin barrier function (data not shown). These results suggest that a sacran can permeate into the skin of atopic dermatitis with low skin barrier functions, but not into the healthy human skin.

PMA, a specific activator of protein kinase C (PKC), induces suitable inflammatory responses. Next, to examine

whether a sacran can inhibit inflammatory responses in macrophages, we investigated the effect of sacran on the mRNA expression levels of TNF-α and COX-2 in RAW264.7 cells stimulated with PMA. The results showed that 0.05% (w/v) sacran solution significantly suppressed the mRNA expression of TNF-α and COX-2 in macrophages, compared with PMA alone (Figure 6). These results indicate that sacran can reduce the inflammatory responses in macrophages.

β-Hexosaminidase released from activated mast cells is responsible for the allergic inflammatory responses associated with disease states of atopic dermatitis. Therefore, to examine the anti-allergic activity of sacran, we evaluated the inhibitory effect of sacran on β-hexosaminidase release in RBL-2H3 cells, which is now known to be an analog of rat mucosal mast cells [22] . As shown in Figure 7, a sacran significantly lowered β-hexosami- nidase release to 70%, compared to control. These results suggest that a sacran can inhibit the allergic inflammatory responses.

The anti-inflammatory effects displayed by sacran, a unique cyanobacteria-derived glycosaminoglycanoid, could be related to the similarity of its chemical structure to that of glycosaminoglycans, sulfated polysaccharides, which possess numerous bioactivities including an anti-inflammatory effect [23] - [27] . The high sulfate and carboxyl group content of glycosaminoglycans permits them to interact with a wide range of proteins, enzymes, cytokines, chemokines, lipoproteins, and adhesion molecules [6] - [8] [28] . Also, Ngatu et al. reported that epicutaneous application of sacran to 2,4,6-trinitrochlorobenzene-induced NC/Nga mice significantly inhibited the development of allergic dermatitis skin lesions, and decreased the number of scratching behavior episodes by improving the stability, elasticity, and hydration of skin barrier as well as inhibiting the production of T_H2 cytokines (IL-4, IL-5), T_H1 cytokines (TNF-α, IFN-γ) and inflammatory chemokines such as MCP-1 and

eotaxin; thus, inhibiting IgE and eosinophilic infiltration in mice [14] . These unique properties exhibited by sacran are not popular to most sulfated polysaccharides.