Mechanism of therapeutic effects of oral administration of bioprocessed black rice bran and balloon flower root (Platycodon grandiflorum) individually and in combination against atopic dermatitis in mice

Mechanism of therapeutic effects of oral administration of bioprocessed black rice bran and balloon flower root (Platycodon grandiflorum) individually and in combination against atopic dermatitis in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mechanism of therapeutic effects of oral administration of bioprocessed black rice bran and balloon flower root (Platycodon grandiflorum) individually and in combination against atopic dermatitis in mice Kyung Hee Lee, Ki Sun Kwon, Woon Sang Hwang, Alan D Friedman, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8302597/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Atopic dermatitis (AD) is a chronic inflammatory skin disorder frequently associated with dysregulated Th1/Th2 immune balance and impaired regulatory T (Treg) activity. Although conventional treatments can reduce clinical symptoms, concerns regarding long-term adverse effects have led to increased interest in complementary and functional dietary materials with immunomodulatory potential. Bioprocessed black rice bran (BRB-F) and balloon flower root fractions (BFR-F) have individually demonstrated anti-inflammatory activities, but their combined oral efficacy and mechanisms in AD remain insufficiently characterized. Methods Mechanistic in vitro assays were first performed using a human B-cell line to measure IgE secretion, mouse and/or human mast cells to evaluate degranulation and TSLP release, and keratinocyte cultures to quantify TARC, MDC, and IL-6 production. These assays were used to determine whether BRB-F and BFR-F individually or cooperatively modulate immune and epithelial cell activation relevant to AD pathology. Subsequently, an AD-like phenotype was induced in BALB/c mice by topical application of 1-chloro-2,4-dinitrobenzene followed by mite extract. Animals received dietary supplementation with a 3:1 BRB-F:BFR-F mixture (10–80 mg/kg/day), and clinical skin inflammation, ear histopathology, and cytokine profiles were analyzed to assess Th1/Th2 balance and Treg-associated markers. Results Dietary supplementation with the BRB-F:BFR-F binary combination resulted in dose-dependent improvement in AD-like skin lesions and reduced histopathological severity. The mixture suppressed Th2 cytokines (IL-4, IL-5, IL-13) in ear tissue and increased splenic Th1 cytokines (IL-2, IL-12, IFN-γ), accompanied by dose-dependent elevation of galectin-9, suggesting activation of Treg-associated pathways. IL-10 expression was also reduced in vivo. In vitro, BRB-F and BFR-F cooperatively inhibited IgE production by B cells, attenuated mast cell activation and TSLP release, and lowered keratinocyte secretion of TARC, MDC, and IL-6. Conclusions The combined oral administration of bioprocessed BRB-F and BFR-F exerts multi-targeted anti-atopic activity that includes modulation of Th1/Th2 immune balance, activation of Treg-associated signaling, and suppression of B-cell, mast cell, and keratinocyte responses. These findings, confirmed by histopathology of skin tissues before and after treatment, provide preclinical support for the development of BRB-F:BFR-F as a complementary functional material for managing immune dysregulation associated with AD, although validation in human patients will be required. atopic dermatitis bioprocessed black rice bran balloon flower root regulatory T cells immunomodulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Atopic dermatitis (AD), the most common chronic inflammatory skin disease in children and frequently persisting into adulthood, is characterized by intensely pruritic eczematous lesions. The pathology of AD involves multiple interacting contributors, including genetic susceptibility, impaired epidermal barrier function, alterations in skin microbial homeostasis, and dysregulated immune responses [ 1 ]. AD is further associated with a wide spectrum of comorbidities, such as allergic asthma [ 2 ], food allergy [ 3 ], autoimmune disorders [ 4 ], squamous cell carcinoma and melanoma [ 5 ], and additional conditions including rhinitis, ocular complications, psychiatric and infectious diseases, and endocrine and cardiovascular abnormalities [ 6 ]. Patients with AD also appear to have an increased risk of all-cause mortality [ 7 ]. Current therapeutic options—including corticosteroids [ 8 ], IL-4 and IL-13 receptor blockade [ 9 ], triolein [ 10 ], coconut oil [ 11 ], alkaloid and limonoid compounds [ 12 ], probiotics [ 13 ], phototherapy [ 14 ], and other anti-inflammatory approaches [ 15 ]—provide symptomatic improvement but rarely achieve durable disease resolution. Despite substantial progress in both approved and investigational therapies, the long-term management of AD remains challenging [ 16 ], likely reflecting the heterogeneous and chronic nature of the disorder. As part of efforts to identify food-derived functional materials with immunomodulatory potential, we previously reported that black rice bran exhibits anti-inflammatory and anti-allergic properties relevant to chronic inflammatory disorders [ 17 , 18 ], and that bioprocessed black rice bran cultured with shiitake mushroom mycelia protects mice against inflammation and asthma [ 19 ]. These findings motivated the present investigation into the therapeutic potential of an orally administered binary combination of bioprocessed black rice bran and balloon flower root (Platycodon grandiflorum), which contains bioactive compounds reported to be beneficial against asthma and related conditions. Methods Materials An ointment containing house dust mite extract (DFE; Biostir, Hyogo, Japan) and 1-Chloro-2,4-dinitrobenzene (DNCB; Sigma Chemical Co., St. Louis, MO) was used as the antigen and hapten, respectively, for inducing AD-like skin lesions. Unless otherwise specified, all other reagents were obtained from Sigma (St. Louis, MO). DNCB was dissolved in a solution of olive oil and acetone at a ratio of 3:1. The ELISA kit was purchased from R&D Systems (Minneapolis, MN). Preparation of Bioprocessed Black Rice Bran and Bioprocessed Balloon Flower Root Bioprocessed (fermented) black rice bran (BRB-F) and bioprocessed (fermented) balloon flower root (BFR-F) were prepared according to previously reported methods [ 20 ]. Lentinus edodes mycelia grown on potato dextrose agar (PDA) were inoculated into 50 mL of liquid medium and incubated in 250 mL Erlenmeyer flasks at 28°C for 5 days on a rotary shaker (120 rpm). The resulting pre-cultured mycelia were then used to inoculate the main liquid culture, which contained either black rice bran (100 g/L) or balloon flower root (100 g/L). For enzymatic pretreatment, the culture was treated with amylase at 60°C for 60 min. Fermentation proceeded in a 5 L fermenter (working volume 3 L) at 28°C and 150 rpm with a 10% inoculum. After 3 days, the culture mass was subjected to additional enzymatic treatment using amylase and a mixed enzyme preparation to degrade particulate carbohydrate-rich material. The culture was adjusted to pH 6.0 with HCl and subsequently sterilized in an autoclave. The main culture process was initiated by adding a cell-wall–degrading enzyme mixture containing cellulase, hemicellulase, pectinase, glucanase, mannose, and arabinase at 50°C for 60 min. Following enzymatic treatment, the cultures were extracted with hot water at 90°C for 1 h and then lyophilized to obtain the final solid bioprocessed materials. U266.B1 Cell culture and Measurement of IgE production Measurement of IgE production using a B cell line was performed according to previously described methods [ 19 ]. The U266.B1 human multiple myeloma B lymphocyte cell line was obtained from the American Type Tissue Culture Collection (ATCC, Manassas, VA). These cells were cultured in a modified RPMI1640 medium, supplemented with 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and 15% heat-inactivated fetal bovine serum (FBS). Penicillin (100 U/mL) and streptomycin (100 mg/mL) were also included in the medium to prevent bacterial contamination. The cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. The culture medium was replaced every three days, ensuring optimal conditions until the cells reached maximal density. To evaluate changes in IgE production, U266.B1 cells were seeded in 96-well plates at a density of 1×106 cells per well and incubated for 24 hours. The cells were then stimulated with 10 µg/mL lipopolysaccharide (LPS), 5 ng/mL human IL-4, and the respective samples for an additional 72 hours. After incubation, the supernatant was collected and transferred to centrifuge tubes. The samples were centrifuged at 12,000 rpm for 10 minutes to separate the culture supernatants. IgE levels in the supernatants were quantified using an ELISA kit (Cat. No. BMS2097, Invitrogen) following the manufacturer's protocol. The absorbance of the reaction mixture was measured at 450 nm using a microplate reader (VersaMax, Molecular Devices Corp., CA). RBL-2H3 Cell culture and Measurement of β-Hexosaminidase Release Measurement of β-hexosaminidase release using a mast cell line was performed according to previously described methods [ 19 ]. The RBL-2H3 rat basophilic leukemia mast cell line, obtained from ATCC, was cultured in a modified Dulbecco’s Modified Eagle Medium (DMEM). The medium was supplemented with 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and 10% heat-inactivated fetal bovine serum (FBS). To prevent bacterial contamination, penicillin (100 U/mL) and streptomycin (100 mg/mL) were added. The cells were maintained at 37°C in a humidified incubator containing 5% CO2. The culture medium was replaced every 2 to 3 days, allowing the cells to proliferate until maximal density was achieved. To assess β-hexosaminidase activity, RBL-2H3 cells were seeded in 96-well plates at a density of 1×105 cells per well and incubated for 24 hours. Each well received 200 µL of Tyrode buffer (137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1.1 mM MgCl2, 11.9 mM NaHCO3, 0.4 mM NaH2PO4, and 5.6 mM glucose, pH 7.2), along with the test samples, and was incubated for 15 minutes. After incubation, the extracts were removed by washing with Tyrode buffer. To stimulate the cells, calcium ionophore A23187 (10 µM) dissolved in Tyrode buffer was added for 20 minutes. Following stimulation, 50 µL of the supernatant containing released β-hexosaminidase was collected from each well. The collected supernatant was mixed with an equal volume of p-nitrophenyl-N-acetyl-β-glucosaminide solution (1 mM, pH 5.2) and incubated for 1 hour at room temperature. The reaction was stopped by adding sodium carbonate buffer (67 mM, pH 10.2). The absorbance of the final mixture was measured at 405 nm using a microplate reader (VersaMax, Molecular Devices Corp., CA, USA), providing quantitative data on β-hexosaminidase release. HMC-1.2 Cell culture and Measurement of TSLP production Measurement of TSLP production was performed according to the method of Moon and Kim [ 21 ] with slight modifications. The HMC-1.2 human mast cell line, obtained from ATCC, was cultured using a modified Iscove's Modified Dulbecco's Medium (IMDM). The culture medium was supplemented with 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and 10% heat-inactivated fetal bovine serum (FBS). To prevent bacterial contamination, penicillin (100 U/mL) and streptomycin (100 mg/mL) were also included in the medium. The cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. The culture medium was replaced every 2 to 3 days to support cell proliferation until maximal density was reached. To assess changes in thymic stromal lymphopoietin (TSLP) production, HMC-1.2 cells were plated in 96-well plates at a density of 5×105 cells per well and incubated for 24 hours. The samples to be tested were then added to each well, followed by a 2-hour incubation period. Afterward, HMC-1.2 cells were stimulated with 50 µM phorbol 12-myristate 13-acetate (PMA) and 1 µg/mL calcium ionophore A23187 for 7 hours to induce TSLP production. Following stimulation, the supernatant from each well was collected and transferred into centrifuge tubes. The samples were centrifuged at 12,000 rpm for 10 minutes to separate the culture supernatants. The TSLP levels in the supernatants were then measured using a specific ELISA kit (Cat. No. EHTSLP, Invitrogen) according to the manufacturer's instructions. The absorbance of the final reaction mixture was read at 450 nm using a microplate reader (VersaMax, Molecular Devices Corp., CA), providing quantitative data on TSLP production. HaCaT Cell culture and Measurement of TARC, MDC, and IL-6 production Measurement of TARC, MDC, and IL-6 production using a keratinocyte cell line was performed according to the method of Park et al. [ 22 ] with slight modification. The HaCaT human keratinocyte cell line, obtained from the American Type Culture Collection (ATCC), was cultured using a modified Dulbecco’s Modified Eagle Medium (DMEM). The growth medium was supplemented with 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and 10% heat-inactivated fetal bovine serum (FBS). To prevent bacterial contamination, penicillin (100 U/mL) and streptomycin (100 mg/mL) were also added to the medium. The HaCaT cells were maintained at 37°C in a humidified incubator containing 5% CO2. The culture medium was replaced every two to three days, supporting cell proliferation until maximal density was reached. For the assessment of chemokine and cytokine production, HaCaT cells were seeded into 48-well plates at a density of 4×105 cells per well and incubated for 24 hours. After the initial incubation, the cells were further cultured for 24 hours in serum-free DMEM. Test samples were then added to each well, and the cells were incubated for an additional 24 hours. Following this treatment, HaCaT cells were stimulated with 10 ng/mL of TNF-α and IFN-γ for 24 hours to induce the production of the target proteins. After stimulation, the culture supernatants were collected and transferred to centrifuge tubes. The samples were centrifuged at 12,000 rpm for 10 minutes to separate the supernatants. The concentrations of thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), and interleukin-6 (IL-6) in the supernatants were measured using specific ELISA kits (Cat. No. SDN00, DMD00, S6050; R&D Systems) according to the manufacturer’s protocols. The absorbance of the final reaction mixtures was determined at 450 nm using a microplate reader (VersaMax, Molecular Devices Corp., CA), providing quantitative data for TARC, MDC, and IL-6 production. Mice Pathogen-free female Balb/c mice, aged six weeks, were obtained from Koatech (Gyeonggi-do, Korea). Upon arrival, the mice were housed in stainless-steel cages and maintained under a controlled environment featuring a 12-hour light/dark cycle. The room temperature was kept at 23°C with a margin of ± 3°C, and the relative humidity was regulated to 50 ± 10%. For the purpose of acclimation, the mice were provided with unrestricted access to pelletized standard commercial chow diet (Cat. No. 5L79, Orient Bio, USA) and tap water for one week. DFE and DNCB-Induced Allergic Dermatitis (AD) in Ear Skin Allergic dermatitis-like lesions were induced in the ear skin of female BALB/c mice, following a modified protocol based on Choi et al. [ 17 ] and Hwang et al. [ 23 ] The experimental procedure is illustrated in Fig. 1 . The mice were randomly assigned to nine distinct groups (n = 10 per group): vehicle control; positive control (DFE/DNCB plus vehicle); DFE/DNCB plus black rice bran (BRB, 40 mg/kg); DFE/DNCB plus balloon flower root (BFR, 40 mg/kg); DFE/DNCB plus bioprocessed black rice bran (BRB-F, 40 mg/kg); DFE/DNCB plus bioprocessed balloon flower root (BFR-F, 40 mg/kg); and DFE/DNCB plus bioprocessed product mixtures in ratios of 3 BRB-F : 1 BFR-F, 1 BRB-F : 1 BFR-F, and 1 BRB-F : 3 BFR-F (all at 40 mg/kg). To initiate the dermatitis model, each mouse received a topical application of 20 µL of 1% 2,4-dinitrochlorobenzene (DNCB) dissolved in an olive oil/acetone solution (3:1 ratio) to both the front and back of right ear. This was followed three days later by an application of DFE ointment (100 mg/mL). The combined DFE/DNCB exposure was repeated once weekly for five consecutive weeks. One week after the initial challenge with DNCB and mite extract, the mice began receiving a diet containing the respective test samples, administered for a total duration of four weeks. At the end of the study period, all mice were sacrificed by CO2 inhalation, 24 hours after the final treatment (day 33), to evaluate the atopic dermatitis-suppressive effects of bioprocessed black rice bran and bioprocessed balloon flower root. Following blood collection, the ears and spleen of each mouse were excised for subsequent histological analysis and ELISA assessment. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Chuncheon Bioindustry Foundation (CBF IACUC no. 2025-054) and were conducted in accordance with relevant guidelines and regulations. Measurement of ear thickness and cytokines from ear tissue After the mice were sacrificed, ear thickness was determined using a dial thickness gauge (Mitutoyo Corporation, Kanagawa, Japan) to quantitatively assess swelling. Ear tissues were homogenized in a phosphate buffer (pH 7) containing 0.4 M NaCl, 0.05% Tween-20, 0.5% bovine serum albumin (BSA), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 mM ethylenediaminetetraacetic acid (EDTA). The resulting homogenates were then subjected to microcentrifugation at 14,000 × g for 15 minutes at 4 ℃ to recover the supernatant, which contains the soluble cytokines. The concentrations of TSLP, IL-33, IL-31, and IL-10 in the ear tissue supernatants were subsequently measured using ELISA kits (R&D Systems, Minneapolis, MN), following the manufacturer’s instructions. ELISA Measurement of cytokine levels in serum Blood samples were obtained from the sacrificed mice via cardiac puncture. Following collection, the samples were placed upright and allowed to rest for 30 minutes at 4 ℃ to facilitate clot formation of the red blood cells. Subsequently, the blood samples were centrifuged at 2,000 × g for 30 minutes at 4 ℃ using a Micro 17R centrifuge (Hanil Science, Incheon, Republic of Korea). The resulting supernatants were carefully collected and stored at -70 ℃ until further analysis. To quantify the levels of serum IgE and Galectin-9, ELISA kits (R&D Systems, Minneapolis, MN) were used in accordance with the manufacturer’s instructions. RNA isolation Total RNA was extracted from biopsy specimens or mononuclear cells utilizing the protocol established by Chomczynski and Sacchi [ 24 ], with minor modifications tailored for this study. To achieve efficient cell lysis from tissue samples, forty consecutive cryostat sections, each 5 µM thick, were immersed in a buffer containing 4 M guanidinium isothiocyanate. Following the RNA extraction process, the isolated samples underwent treatment with DNase 1 (Promega Corp., Madison, WI) for 30 minutes at 37°C to eliminate any residual DNA. Throughout all enzymatic procedures involving RNA, an RNase inhibitor (Boehringer Mannheim Corp., Indianapolis, IN) was consistently included to protect the integrity of the RNA. Real-Time Polymerase Chain Reaction (qRT-PCR) Quantitative real-time PCR (qRT-PCR) was performed using a Thermal Cycler Dice TP850 (Takarabio Inc., Shiga, Japan) in accordance with the manufacturer’s protocol. The reaction setup involved combining 2 µL of cDNA (containing 100 ng), 1 µL of a primer solution (sense and antisense, 0.4 µM each), 12.5 µL of SYBR Premix Ex Taq (Takarabio Inc.), and 9.5 µL of distilled water (dH2O), resulting in a total reaction volume of 25 µL per tube. The amplification protocol consisted of an initial denaturation step of 10 seconds at 95℃, followed by 40 cycles comprising 5 seconds at 95℃ and 30 seconds at 60℃. This was succeeded by additional steps: 15 seconds at 95℃, 30 seconds at 60℃, and a final 15 seconds at 95℃. Quantification and normalization of mRNA expression levels were conducted using the TP850 software provided by the manufacturer. This ensured accurate assessment of gene expression in the analyzed samples. Statistical analysis Results are expressed as the mean ± SD of three independent experiments. Significant differences between means were determined by the one-way ANOVA test followed by Duncan’s multiple range test using the Statistical Analysis System (SAS, Cary, NC, USA). p < 0.05 was regarded as significant. Percent reductions were calculated using values obtained by subtracting the mean value obtained with vehicle from the mean values obtained using the positive control treatment and the experimental treatment. Results Inhibitory Effects of BRB-F and BFR-F on IgE Production in Human U266.B1 Cells To evaluate the ability of bioprocessed black rice bran (BRB-F) and bioprocessed balloon flower root (BFR-F) extracts to suppress IgE production, assays were conducted using the human B-cell line U266.B1. Both individual extracts and their combinations were examined. Stimulation of U266.B1 cells with lipopolysaccharide (LPS) and interleukin-4 (IL-4) increased IgE secretion by more than 50-fold compared with unstimulated controls. Treatment with the raw extracts, BRB and BFR, led to modest reductions in IgE production, with BRB decreasing IgE levels by 18.1% and BFR by 10.7% relative to the positive control. In contrast, the bioprocessed extracts BRB-F and BFR-F exhibited markedly stronger inhibitory activity, each suppressing IgE production by approximately 60%. Combinations of BRB-F and BFR-F at different ratios (3:1, 1:1, and 1:3) resulted in inhibitory levels comparable to those observed with each extract alone, with no significant differences among mixing ratios. Table 1 Inhibitory effect of IgE production in U266.B1 cells by combined ratios of BRB-F and BFR-F. Treatment IgE concentration (ng/mL) Vehicle 8.9 ± 0.6 e Positive control (10 µg/mL LPS + 5 ng/mL IL-4) 482.7 ± 31.7 a 100 µg/mL BRB 396.8 ± 30.6 bc 100 µg/mL BFR 431.9 ± 25.4 b 100 µg/mL BRB-F 202.7 ± 15.2 d 100 µg/mL BFR-F 205.4 ± 17.9 d 75 µg/mL BRB-F + 25 µg/mL BFR-F (3:1) 204.3 ± 18.1 d 50 µg/mL BRB-F + 50 µg/mL BFR-F (1:1) 198.3 ± 10.3 d 25 µg/mL BRB-F + 75 µg/mL BFR-F (1:3) 209.6 ± 11.9 d Data are shown as the mean ± SD of three independent experiments. Values in each column with the same letter are not significantly different between groups at p < 0.05. Degranulation Inhibitory Effects of BRB-F and BFR-F in RBL-2H3 Mast Cells The inhibitory activity of bioprocessed black rice bran (BRB-F) and bioprocessed balloon flower root (BFR-F) on mast cell degranulation was evaluated using the RBL-2H3 rat basophilic leukemia cell line. Both individual and combined treatments were tested to determine their effects on β-hexosaminidase release, a key marker of mast cell degranulation. Stimulation with A23187 induced a strong degranulation response. Treatment with the raw materials, BRB and BFR, reduced β-hexosaminidase release by 52.7% and 31.5%, respectively, compared with the positive control. BRB-F and BFR-F also inhibited degranulation, decreasing β-hexosaminidase release by 58.4% and 33.9%, respectively. When BRB-F and BFR-F were combined at different mixing ratios, the degree of inhibition was comparable to that produced by BRB-F alone, and no significant differences were detected among the tested combinations (Table 2 ). Table 2 Inhibitory effect of β-hexosaminidase releases in RBL-2H3 cells by combined ratios of BRB-F and BFR-F. Treatment β-hexosaminidase release (%) Vehicle 0.0 ± 7.3 d Positive control (10 µM Calcium ionophore A23187) 100 ± 4.8 a 100 µg/mL BRB 47.3 ± 2.6 c 100 µg/mL BFR 68.5 ± 1.9 b 100 µg/mL BRB-F 41.6 ± 4.5 c 100 µg/mL BFR-F 66.1 ± 3.1 b 75 µg/mL BRB-F + 25 µg/mL BFR-F (3:1) 44.8 ± 3.5 c 50 µg/mL BRB-F + 50 µg/mL BFR-F (1:1) 44.1 ± 4.1 c 25 µg/mL BRB-F + 75 µg/mL BFR-F (1:3) 46.4 ± 2.7 c Data are shown as the mean ± SD of three independent experiments. Values in each column with the same letter are not significantly different between groups at p < 0.05. Evaluation of BRB-F and BFR-F Inhibitory Effects on TSLP Production in HMC-1.2 Cells The inhibitory effects of bioprocessed black rice bran (BRB-F) and bioprocessed balloon flower root (BFR-F) on thymic stromal lymphopoietin (TSLP) production were examined using the human mast cell line HMC-1.2. Both individual and combined treatments were assessed to determine their relative efficacy. Stimulation with PMA and A23187 elevated TSLP production by 2.3-fold compared with baseline. Treatment with the raw materials BRB and BFR reduced TSLP levels by 34.5% and 18.9%, respectively. In contrast, treatment with the bioprocessed forms resulted in greater suppression, with BRB-F reducing TSLP production by 51.9% and BFR-F by 31.3% (Table 3 ). Combination treatments of BRB-F and BFR-F showed that increasing the proportion of BRB-F produced slightly stronger TSLP inhibition, although the degree of suppression was comparable among the tested mixing ratios. Table 3 Inhibitory effect of TSLP production in HMC-1 cells by combined ratios of BRB-F and BFR-F. Treatment TSLP production (pg/mL) Vehicle 36.3 ± 2.4 e Positive control (50 µM PMA + 1 µg/mL Calcium ionophore A23187) 82.9 ± 5.9 a 100 µg/mL BRB 66.8 ± 4.3 c 100 µg/mL BFR 74.1 ± 3.7 b 100 µg/mL BRB-F 58.7 ± 3.6 d 100 µg/mL BFR-F 68.3 ± 2.9 c 75 µg/mL BRB-F + 25 µg/mL BFR-F (3:1) 59.6 ± 4.2 d 50 µg/mL BRB-F + 50 µg/mL BFR-F (1:1) 62.9 ± 5.6 cd 25 µg/mL BRB-F + 75 µg/mL BFR-F (1:3) 66.2 ± 5.1 c Data are shown as the mean ± SD of three independent experiments. Values in each column with the same letter are not significantly different between groups at p < 0.05. Inhibitory Effects of BRB-F and BFR-F on Cytokine and Chemokine Expression in HaCaT Keratinocyte Cells The inhibitory effects of bioprocessed black rice bran (BRB-F) and bioprocessed balloon flower root (BFR-F) on cytokine and chemokine production were evaluated in human HaCaT keratinocyte cells. Both individual and combined treatments were examined to determine their relative efficacy. Stimulation with TNF-α and IFN-γ elevated the expression of TARC, MDC, and IL-6 by 29-fold, 89-fold, and 17-fold, respectively. Treatment with the raw materials BRB and BFR significantly suppressed these induced levels, with BRB showing greater inhibitory activity than BFR by 1.7-fold for TARC, 2.2-fold for MDC, and 1.5-fold for IL-6. Among the bioprocessed materials, BFR-F exhibited higher inhibitory potency than BRB-F, producing 1.5-fold, 1.2-fold, and 0.8-fold stronger suppression of TARC, MDC, and IL-6, respectively. Combination treatments of BRB-F and BFR-F resulted in inhibition levels comparable to those of the bioprocessed extracts alone (Table 4 ). Table 4 Inhibitory effect of TARC, MDC, and IL-6 production in HaCaT cells by combined ratios of BRB-F and BFR-F. Treatment Cytokine and chemokine production (pg/mL) TARC MDC IL-6 Vehicle 12.0 ± 3.7 i 33.5 ± 14.3 h 6.6 ± 1.3 g Positive control (10 ng/mL TNF-α/IFN-γ) 341.7 ± 6.8 a 2999.3 ± 253.3 a 109.7 ± 2.7 a 100 µg/mL BRB 218.0 ± 16.5 c 1703.8 ± 134.7 c 66.1 ± 3.9 c 100 µg/mL BFR 268.1 ± 13.6 b 2421.1 ± 150.7 b 81.4 ± 2.6 b 100 µg/mL BRB-F 130.2 ± 6.4 d 942.2 ± 64.5 d 52.5 ± 3.4 ef 100 µg/mL BFR-F 30.1 ± 6.9 h 600.1 ± 59.6 g 61.4 ± 2.4 d 75 µg/mL BRB-F + 25 µg/mL BFR-F (3:1) 97.4 ± 18.5 e 892.4 ± 80.4 de 50.2 ± 1.0 f 50 µg/mL BRB-F + 50 µg/mL BFR-F (1:1) 55.5 ± 2.7 f 791.7 ± 31.9 e 51.9 ± 1.2 f 25 µg/mL BRB-F + 75 µg/mL BFR-F (1:3) 44.8 ± 4.1 g 717.3 ± 51.2 f 55.0 ± 2.2 e Data are shown as the mean ± SD of three independent experiments. Values in each column with the same letter are not significantly different between groups at p < 0.05. Anti-atopic effects of BRB-F and BFR-F in a mouse model of atopic dermatitis The therapeutic effects of bioprocessed black rice bran (BRB-F) and bioprocessed balloon flower root (BFR-F), administered individually or in combination, were evaluated in a mouse model of atopic dermatitis (AD) induced by repeated exposure to 2,4-dinitrochlorobenzene (DNCB) and mite extract. Disease severity was assessed by measuring ear thickness, serum IgE levels, and the expression of thymic stromal lymphopoietin (TSLP), interleukin-33 (IL-33), and interleukin-31 (IL-31) in ear tissue. In AD-induced mice, ear thickness, serum IgE, TSLP, IL-33, and IL-31 levels were markedly elevated compared with normal controls. Treatment with the bioprocessed materials resulted in significantly greater reductions in these markers than their corresponding raw forms. Among the individual treatments, BRB and BRB-F consistently produced stronger inhibitory effects than BFR and BFR-F. Combination treatments of BRB-F and BFR-F further reduced these biomarkers, with inhibitory efficacy increasing as the proportion of BRB-F increased in the mixture. The greatest therapeutic effect was observed for the 3:1 BRB-F:BFR-F ratio (30 mg/kg BRB-F and 10 mg/kg BFR-F), which reduced ear thickness to 84.5% of the control level, serum IgE to 82.4%, TSLP expression to 85.9%, IL-33 to 92.9%, and IL-31 to 73.7% (Fig. 2 ). Modulation of Th2 and Th1 Cytokine Expression by BRB-F and BFR-F in Atopic Dermatitis Th2 cells are known to predominate in lesioned tissues following the induction of atopic dermatitis (AD) [ 23 ]. To evaluate immune modulation in this model, the mRNA expression levels of Th2 cytokines (IL-4, IL-5, and IL-13) were measured in ear tissue. AD induction resulted in strong upregulation of these cytokines, exceeding a 5-fold increase compared with normal, untreated mice. Administration of the bioprocessed extracts, either alone or in combination, produced greater suppression of Th2 cytokine expression than the corresponding raw materials. The most pronounced inhibitory effect was observed with the 3:1 BRB-F:BFR-F combination, which reduced IL-4 mRNA to 48.8%, IL-5 to 49.1%, and IL-13 to 49.7% of the AD-induced control (Table 5 ). To further examine T-cell–mediated immune responses, expression levels of the Th1 cytokines IL-2, IL-12, and IFN-γ were measured in spleen tissue. AD induction markedly decreased Th1 cytokine expression. Treatment with the bioprocessed materials, particularly the 3:1 BRB-F:BFR-F combination, substantially restored Th1 cytokine levels, with recovery most notable for IL-2 (68.4% of normal), followed by IL-12 (48.3% of normal) and IFN-γ (76% of normal). Table 5 Regulatory effect of Th1 and Th2 cytokines in atopic dermatitis-induced mice by combined ratios of BRB-F and BFR-F. Treatment Cytokines Production Th2 Cytokine mRNA in ear (fold-increase) Th1 Cytokine mRNA in spleen (fold-increase) IL-4 IL-5 IL-13 IL-2 IL-12 IFN-γ Vehicle 1 ± 0.07 f 1 ± 0.06 f 1 ± 0.03 g 1 ± 0.06 a 1 ± 0.07 a 1 ± 0.09 a Positive control 5.41 ± 0.46 a 5.93 ± 0.41 a 5.69 ± 0.43 a 0.75 ± 0.05 d 0.62 ± 0.04 d 0.71 ± 0.05 d 40 mg/kg BRB 5.03 ± 0.37 b 5.13 ± 0.25 b 5.16 ± 0.43 b 0.77 ± 0.07 cd 0.65 ± 0.05 d 0.71 ± 0.06 d 40 mg/kg BFR 5.24 ± 0.3 ab 5.25 ± 0.29 b 5.38 ± 0.39 ab 0.74 ± 0.03 d 0.63 ± 0.02 d 0.72 ± 0.04 d 40 mg/kg BRB-F 3.98 ± 0.31 cd 4.09 ± 0.29 d 4.21 ± 0.33 cd 0.85 ± 0.04 bc 0.82 ± 0.06 bc 0.77 ± 0.03 c 40 mg/kg BFR-F 4.27 ± 0.28 c 4.55 ± 0.28 c 4.49 ± 0.36 c 0.81 ± 0.05 c 0.8 ± 0.07 bc 0.76 ± 0.06 cd 40 mg/kg BRB-F + BFR-F (3:1) 3.26 ± 0.25 e 3.51 ± 0.22 e 3.36 ± 0.16 f 0.94 ± 0.07 ab 0.88 ± 0.08 b 0.85 ± 0.06 b 40 mg/kg BRB-F + BFR-F (1:1) 3.55 ± 0.17 de 3.72 ± 0.41 de 3.7 ± 0.24 de 0.89 ± 0.08 b 0.86 ± 0.05 b 0.82 ± 0.07 bc 40 mg/kg BRB-F + BFR-F (1:3) 3.71 ± 0.29 d 3.86 ± 0.35 de 3.94 ± 0.28 d 0.86 ± 0.05 bc 0.83 ± 0.03 bc 0.8 ± 0.05 bc Data are shown as the mean ± SD of three independent experiments. Values in each column with the same letter are not significantly different between groups at p < 0.05. Effects of BRB-F and BFR-F on IL-10 Cytokine Expression in Atopic Dermatitis Previous studies have shown that excessive secretion of interleukin-10 (IL-10) by Th2 cells is associated with the progression of atopic dermatitis (AD) [ 25 ]. To evaluate this mechanism, IL-10 mRNA and protein expression levels were measured in ear tissue. AD induction resulted in an approximately threefold increase in both IL-10 mRNA and protein expression compared with normal, untreated mice. Administration of the bioprocessed materials produced greater inhibition of IL-10 expression than their raw counterparts. Among the treatment groups, the combination of BRB-F and BFR-F at a 3:1 ratio yielded the strongest effect, reducing IL-10 mRNA expression by 87.2% and protein expression by 70.9% relative to the AD-induced group (Fig. 3 ). Effects of BRB-F and BFR-F on Galectin-9 Expression and Treg Activation in Atopic Dermatitis Galectin-9 plays a key role in immune regulation by promoting the activation of regulatory T (Treg) cells through the induction of transforming growth factor-β1 (TGF-β1), a cytokine associated with suppression of inflammatory responses [ 26 ]. To evaluate whether BRB-F and BFR-F modulate this pathway, serum galectin-9 levels were measured in mice treated with the extracts individually or in combination. Mice with induced atopic dermatitis exhibited a substantial increase in serum galectin-9 levels (78.59 ± 5.58 pg/mL), approximately 3.3-fold higher than those in normal controls (23.67 ± 1.73 pg/mL). Administration of BRB-F and BFR-F further elevated serum galectin-9 levels, and no significant differences were observed between single-agent and combination treatments (Fig. 4 ). Evaluation of Dose-Dependent Effects of BRB-F and BFR-F Mixture on Atopic Dermatitis To identify the optimal dosage for suppressing atopic dermatitis (AD), the bioprocessed black rice bran (BRB-F) and bioprocessed balloon flower root (BFR-F) mixture was evaluated at the previously determined optimal ratio of 3:1 (30 mg/kg BRB-F and 10 mg/kg BFR-F). Mice were administered daily oral doses of 10, 20, 40, and 80 mg/kg of the mixture following AD induction with mite extract and DNCB. Disease severity was evaluated by measuring ear thickness, serum IgE levels, and the expression of TSLP, IL-33, and IL-31 in ear tissue. All biomarkers were significantly elevated in AD-induced mice compared with normal controls. Treatment with the BRB-F:BFR-F mixture produced dose-dependent suppression of AD symptoms. The highest dose (80 mg/kg) reduced ear thickness by 75%, serum IgE by 87.1%, TSLP by 82.9%, IL-33 by 95.4%, and IL-31 by 72.4% relative to the AD control (Fig. 5 ). When compared with a reference group receiving 60 mg/kg γ-linolenic acid (GLA), the 20 mg/kg BRB-F:BFR-F treatment produced comparable or greater inhibition across multiple biomarkers. Regulation of Th1/Th2 Immune Response by BRB-F and BFR-F Mixture To evaluate the effects of the BRB-F and BFR-F mixture on Th1/Th2 immune regulation in atopic dermatitis (AD), mRNA expression levels of key cytokines were measured in mice. Th2 cytokines (IL-4, IL-5, and IL-13) were analyzed in lesional ear tissue, while Th1 cytokines (IL-2, IL-12, and IFN-γ) were quantified in spleen tissue using RT-PCR. AD induction resulted in a marked elevation of Th2 cytokine expression, with IL-4, IL-5, and IL-13 increasing more than fivefold compared with normal mice. Treatment with the BRB-F:BFR-F mixture produced dose-dependent suppression of Th2 cytokine expression, with the strongest effect observed at the highest dose (80 mg/kg), reducing IL-4, IL-5, and IL-13 by 45.3%, 39.2%, and 41.1%, respectively. AD induction also caused significant reductions in the expression of Th1-associated cytokines in the spleen. Administration of the BRB-F:BFR-F mixture resulted in dose-dependent restoration of Th1 cytokine levels, with the highest dose (80 mg/kg) increasing IL-2 to 60.5% of normal, IL-12 to 55.2%, and IFN-γ to 56% (Table 6 ). The 20 mg/kg dose of the BRB-F:BFR-F mixture produced levels of cytokine suppression and recovery comparable to those observed in the reference group treated with 60 mg/kg γ-linolenic acid (GLA). Table 6 Dose-dependent effects of BRB-F and BFR-F (3:1) on Th1 and Th2 cytokines. Treatment Cytokines Production Th2 Cytokine mRNA in ear (fold-increase) Th1 Cytokine mRNA in spleen (fold-increase) IL-4 IL-5 IL-13 IL-2 IL-12 IFN-γ Vehicle 1 ± 0.06 d 1 ± 0.07 d 1 ± 0.06 d 1 ± 0.03 a 1 ± 0.06 a 1 ± 0.05 a Positive 5.59 ± 0.48 a 6.03 ± 0.51 a 5.79 ± 0.52 a 0.62 ± 0.02 e 0.71 ± 0.05 c 0.75 ± 0.05 c 10 mg/kg 5.13 ± 0.38 ab 5.48 ± 0.53 ab 5.29 ± 0.48 b 0.65 ± 0.05 de 0.73 ± 0.06 c 0.77 ± 0.03 c 20 mg/kg 4.83 ± 0.41 b 5.12 ± 0.50 b 5.05 ± 0.41 b 0.72 ± 0.04 d 0.76 ± 0.07 c 0.80 ± 0.05 bc 40 mg/kg 3.77 ± 0.31 c 4.19 ± 0.40 c 4.06 ± 0.32 c 0.81 ± 0.05 bc 0.83 ± 0.06 b 0.86 ± 0.06 b 80 mg/kg 3.51 ± 0.28 c 4.06 ± 0.35 c 3.82 ± 0.31 c 0.85 ± 0.06 b 0.87 ± 0.07 b 0.89 ± 0.05 b GLA 4.93 ± 0.36 b 5.34 ± 0.41 ab 5.16 ± 0.49 b 0.69 ± 0.06 d 0.75 ± 0.08 c 0.78 ± 0.07 c Data are shown as the mean ± SD of three independent experiments. Values in each column with the same letter are not significantly different between groups at p < 0.05. Inhibitory Effects of BRB-F and BFR-F Mixture on IL-10 Expression in Atopic Dermatitis-Induced Mice Measurements of IL-10 mRNA and protein expression in ear tissue revealed significant overexpression of IL-10 in mice with induced atopic dermatitis. Administration of the BRB-F:BFR-F mixture produced dose-dependent suppression of IL-10 expression. The highest dietary dose (80 mg/kg) resulted in the greatest inhibition, reducing IL-10 mRNA by 90.9% and IL-10 protein by 85.2% relative to the AD-induced control (Fig. 6 ). A comparative analysis with γ-linolenic acid (GLA) showed that the 20 mg/kg BRB-F:BFR-F treatment produced stronger IL-10 inhibitory activity than 60 mg/kg GLA. Dose-Dependent Effects of BRB-F and BFR-F Mixture on Galectin-9 Expression To assess the regulatory effects of the BRB-F:BFR-F mixture on galectin-9 expression, serum galectin-9 levels were measured in mice with induced atopic dermatitis (AD). ELISA analysis showed that AD induction resulted in a substantial elevation of serum galectin-9 (61.2 ± 5.4 pg/mL), approximately 3.6-fold higher than that of normal control mice (16.8 ± 2.2 pg/mL) (Fig. 7 ). Administration of the BRB-F:BFR-F mixture produced a dose-dependent increase in galectin-9 expression, yielding 1.4-, 1.5-, 1.8-, and 1.9-fold increases in serum galectin-9 levels at incremental doses. When compared with γ-linolenic acid (GLA), the 10 mg/kg dose of the mixture exhibited comparable stimulatory activity to 60 mg/kg GLA, while higher doses surpassed the effect of GLA. Histopathological Analysis of Ear Skin in Atopic Dermatitis Mice Histological examination of normal mice showed a thin epidermis composed of one to two cell layers, with minimal desquamation of the stratum corneum. In contrast, mice with induced atopic dermatitis (AD) displayed marked epidermal thickening characterized by multiple cell layers, increased numbers of blood vessels, sebaceous glands, hair follicles, and muscle bundles. The granular layer contained abundant keratohyalin granules, and the stratum corneum appeared uneven with extensive desquamation throughout the tissue [ 27 , 28 ]. Dietary administration of the BRB-F:BFR-F mixture at doses of 10, 20, 40, and 80 mg/kg produced dose-dependent improvements in these pathological alterations. Treatment resulted in progressive thinning of the epidermis relative to AD-induced mice, along with reduced granular layer thickness. The stratum corneum became more continuous and uniform, with minor areas of partial desquamation remaining. The most substantial alleviation of epidermal hyperplasia, inflammatory cell infiltration, and stratum corneum exfoliation occurred at the highest dose (80 mg/kg) (Fig. 8 ). The group receiving 60 mg/kg γ-linolenic acid (GLA) exhibited ear tissue morphology comparable to that of the group treated with 20 mg/kg of the BRB-F:BFR-F mixture. Taken together, these findings demonstrate that oral administration of the bioprocessed black rice bran and balloon flower root formulation produces coordinated improvements across cellular, tissue-level, and systemic immune parameters, indicating a consistent therapeutic trajectory that extends beyond single-pathway modulation. Discussion The present study demonstrates that bioprocessing substantially enhances the anti-atopic activities of both black rice bran and balloon flower root. Although the raw materials exhibited inherent but limited suppression of IgE synthesis, fermentation markedly amplified their bioactivity, consistent with previous reports that bioconversion improves the physiological functions and bioavailability of BRB-derived polysaccharides [ 18 , 19 ]. The similar degrees of inhibition produced by different BRB-F:BFR-F mixing ratios suggest that enhanced activity primarily reflects intrinsic properties of each extract rather than synergistic interactions, although the potential for synergy under alternative dosing conditions cannot be excluded. Both raw and bioprocessed extracts were also capable of suppressing mast cell degranulation. The greater inhibition achieved with BRB and BRB-F compared with BFR and BFR-F indicates that mast cell-modulating constituents are more abundant or potent in black rice bran, which is consistent with previous observations that BRB-derived bioactives modulate mast cell–associated inflammatory signaling [ 17 , 21 , 22 ]. These results suggest that anti-degranulation activity relies largely on functional components already present in the raw materials rather than bioactive factors newly generated by fermentation. In keratinocytes, bioprocessing enhanced suppression of pro-inflammatory cytokines and chemokines, mirroring the trends observed in IgE synthesis and mast cell activation. The stronger anti-inflammatory effect of BFR-F relative to BRB-F indicates that balloon flower root contains constituents that more strongly target keratinocyte-mediated inflammation, consistent with the known pharmacological activities of platycodin-containing extracts [ 32 ]. The lack of synergistic or additive effects across BRB-F:BFR-F mixing ratios suggests that the two materials act through complementary but largely independent biochemical pathways in this context. In vivo, bioprocessing significantly enhanced the ability of both materials to attenuate AD pathology. Notably, efficacy increased progressively with the proportion of BRB-F in the formulation, supporting the interpretation that BRB-derived constituents make dominant contributions to moderating AD-associated immune dysregulation. This interpretation aligns with earlier reports demonstrating protective effects of BRB-derived bioactive compounds against allergic and inflammatory disorders [ 17 , 19 ]. The concomitant reductions in IgE, TSLP, IL-33 and IL-31 suggest action across multiple inflammatory axes rather than suppression of a single inflammatory pathway. The BRB-F:BFR-F formulation modulated adaptive immunity in a bidirectional manner by attenuating Th2-dominant inflammation in lesional skin while restoring systemic Th1 cytokine expression. A similar pattern—where normalization of Th1 responses accompanies suppression of Th2 hyperactivation—has been observed in previous successful immunomodulatory interventions for AD [ 23 , 24 ]. These results indicate that the formulation restores immune balance rather than solely inhibiting inflammatory drivers. Bioprocessing also enhanced the ability of both extracts to inhibit IL-10 overexpression in vivo. Since excessive IL-10 secretion is known to suppress Th1-mediated immune responses and promote AD progression [ 25 ], inhibition of IL-10 may contribute to immune homeostasis in this model. Likewise, induction of circulating galectin-9—an immunoregulatory factor associated with Treg activation and attenuation of Th2-dominant inflammation [ 26 ]—may represent an additional component of the therapeutic mechanism. Together, these findings suggest that BRB-F and BFR-F act through coordinated regulation of B-cell, mast cell, keratinocyte, Th2, Th1 and Treg-associated pathways, contributing both to immune rebalancing and improved tissue-level pathology. Importantly, substantial therapeutic activity was observed even at relatively low doses. The 20 mg/kg treatment produced comparable or superior inhibitory activity relative to 60 mg/kg γ-linolenic acid, in agreement with previous findings showing that functional food–derived bioactive ingredients can serve as effective adjuncts in the management of AD [ 15 , 17 ]. Although the highest dose (80 mg/kg) yielded the strongest effects, the robust response at intermediate doses suggests that clinically meaningful benefits may be achievable without maximal exposure. This study has limitations. Because all experiments were conducted in a murine model, translation to clinical applications will require assessment of safety, optimal dosing and efficacy in humans. Moreover, although this work demonstrates improved biological activity following bioprocessing, the specific bioactive molecules and fermentation-driven chemical conversions underlying the therapeutic effects remain incompletely defined. Ongoing studies are aimed at identifying the major immunomodulatory constituents of BFR-F and determining how bioprocessing alters the chemical profiles of both BRB-F and BFR-F. Such work may facilitate further optimization of this binary formulation and provide insight into whether similar immune responses can be achieved in clinical settings. Conclusions This study demonstrates that a novel binary functional food composed of bioprocessed black rice bran (BRB-F) and independently bioprocessed balloon flower root (BFR-F) provides significant therapeutic benefits in a mouse model of atopic dermatitis (AD). Oral administration of the combined extracts at doses ranging from 20 to 80 mg/kg resulted in dose-dependent suppression of multiple AD-associated biomarkers and markedly improved histopathological features of lesional skin. Although each bioprocessed material individually suppressed key immunologic mechanisms underlying AD in vitro and in vivo, the combined formulation at a BRB-dominant 3:1 ratio produced the most pronounced inhibitory effects. These immunological improvements were accompanied by reduced Th2-associated cytokines and enhanced Th1- and Treg-associated immune regulation together with elevated serum galectin-9 levels, suggesting that coordinated modulation of multiple immune pathways underlies the enhanced therapeutic efficacy of the combined treatment. The findings of this investigation support the rationale for conducting clinical trials to determine whether this binary functional food may offer a viable strategy for the treatment or prevention of AD in humans. Furthermore, the present results strengthen the broader hypothesis that this formulation may also influence comorbidities associated with AD, as suggested by previous observations showing that bioprocessed black rice bran in combination with various food ingredients protects mice against asthma [ 19 ], murine colon carcinoma [ 29 ], alcohol-induced fatty liver formation [ 30 ], alcohol-induced hangover symptoms [ 31 ], and reported multiple health benefits of mushroom polysacharides [ 32 , 33 ] and of ballon flower root mentioned below.. Taken together, these outcomes raise the possibility that immunomodulatory effects driven by BRB-F and BFR-F may extend beyond AD and contribute to the improvement of systemic inflammatory conditions linked to immune imbalance. Additional evidence from the literature highlights the potential relevance of balloon flower root bioactive components to immune and inflammatory regulation: Song et al. [ 34 ] reviewed the traditional use of platycodin D in viral illness; Han et al. [ 35 ] demonstrated the influence of Platycodon grandiflorus root on gut microbiota and host metabolism; and Kim et al. [ 36 ] reported anti-inflammatory activity of balloon flower root extracellular vesicles. We previously reported that the bioprocessed black rice bran product is a polysaccharide consisting of glucose and galactose units [ 18 ], whereas the chemical composition of the bioprocessed balloon flower root product remains to be determined. Clarifying the bioactive constituents of BFR-F will be essential for understanding how bioprocessing influences its anti-atopic properties and for optimizing the formulation. Future studies should therefore investigate how bioprocessing alters the chemical composition of balloon flower root, identify the compounds responsible for anti-atopic activity, and determine how these components may be optimized to maximize the therapeutic potential of this binary functional food for AD and its associated comorbidities. Abbreviations AD, atopic dermatitis BRB-F, bioprocessed black rice bran BFR-F, bioprocessed balloon flower root DFE, Dermatophagoides farinae extract DNCB, 1-chloro-2,4-dinitrobenzene IgE, immunoglobulin E IgG2a, immunoglobulin G2a Th, T helper Treg, regulatory T cell qRT-PCR, quantitative real-time polymerase chain reaction ELISA, enzyme-linked immunosorbent assay Declarations Funding This research was financially supported by the Ministry of Trade, Industry & Energy (MOTIE), Korea Institute for Advancement of Technology (KIAT) through the Encouragement Program for The Industries of Economic Cooperation Region(P0004707), and Ministry of Trade, Industry and Energy (MOTIE, Republic of Korea) through the Technology Innovation Program (No. 20008826). Authors’ contributions S.P.K. conceived the idea and, with K.H.L., K.S.K., W.S.H., W.Y.L., J.K., and S.J.L., conducted the experimental studies; M.F., A.D.F., and S.P.K. interpreted the results and prepared a draft of the manuscript. All authors read and approved the final manuscript. Acknowledgements We thank Eun Young Seong for excellent technical assistance. Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate Animal experiments were approved by the Institutional Animal Care and Use Committee of Chuncheon Bioindustry Foundation (CBF IACUC no. 2025-054) and were conducted in accordance with the relevant guidelines and regulations. Availability of data and materials The original source of method description is acknowledged and cited in each case. The datasets used and/or analyzed during the current study and supplementary material are available from the following author on reasonable request: Prof. Sung Phil Kim; E-mails: spkim@ strbiotech. co. kr; ksp11 08@ ajou. ac. kr. References Sroka-Tomaszewska J, Trzeciak M. Molecular mechanisms of atopic dermatitis pathogenesis. Int J Mol Sci. 2021;22:4130. https://doi.org/10.3390/ijms22084130 . 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Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8302597","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":556921146,"identity":"759a2243-b259-4eaa-a52a-fe3c8b608f81","order_by":0,"name":"Kyung Hee Lee","email":"","orcid":"","institution":"STR Biotech Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Kyung","middleName":"Hee","lastName":"Lee","suffix":""},{"id":556921148,"identity":"239661ab-a7b1-4b1e-94b2-3c252d73fa9a","order_by":1,"name":"Ki Sun Kwon","email":"","orcid":"","institution":"STR Biotech Co., 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01:29:43","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":161319,"visible":true,"origin":"","legend":"","description":"","filename":"50c8a53b8b114827b0a57979f1cd0bbe1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8302597/v1/0f07969d2f682c1009f3a685.xml"},{"id":99194138,"identity":"fb15e1d3-8dc3-45e2-9e10-b757f40d5332","added_by":"auto","created_at":"2025-12-30 01:29:43","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":169364,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8302597/v1/0851dc87379277cd43f57e6e.html"},{"id":99317460,"identity":"e5398e6c-5d0d-4afb-b529-25173dfa501b","added_by":"auto","created_at":"2025-12-31 16:30:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":119948,"visible":true,"origin":"","legend":"\u003cp\u003eInduction of Atopic dermatitis. Schematic diagram of the atopic dermatitis induction protocol. 20 μL of 1% DNCB in olive oil/acetone (3:1) was applied to the front/back of the right ear (10 μL to each side) on Day 1, and 3 days later 100 mg/mL of mite extract was applied to the same ear, and these treatments were repeated weekly for five total treatments. One week after the initial challenge, test samples were administered orally for four weeks. Mice were sacrificed on Day 33, 24 hours after the final treatment.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8302597/v1/af9ab9782d18c2bd15f78dc6.png"},{"id":99318002,"identity":"a36260f2-9b64-4dfc-bc06-202873ae0657","added_by":"auto","created_at":"2025-12-31 16:31:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":597421,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of the combined ratio of BRB-F and BFR-F on DNCB and mite extract-induced atopic dermatitis. Atopic dermatitis was induced by DNCB (1%, 20 μL) and DFE ointment (100 mg/mL) applied weekly to the right ear for 5 weeks; One week after the initial challenge, test samples were administered orally for 4 weeks. (A) Ear thickness was assessed using a micrometer. (B) Serum level of IgE was measured using ELISA. (C) TSLP, (D) IL-33, and (E) IL-31 in ear skin were measured by ELISA. Representation: vehicle (-), negative control not stimulated with DNCB and mite extract; vehicle (+), DNCB and mite extract-stimulated positive control; BRB, black rice bran water extracts (40 mg/kg body weight); BFR, Balloon flower root water extract (40 mg/kg body weight); BRB-F, bioprocessed black rice bran extract (40 mg/kg body weight); BFR-F, bioprocessed balloon flower root extract (40 mg/kg body weight); BRB-F : BFR-F (3:1), bioprocessed black rice bran extract (30 mg/kg body weight) and bioprocessed balloon flower root extract (10 mg/kg body weight); BRB-F : BFR-F (1:1), bioprocessed black rice bran extract (20 mg/kg body weight) and bioprocessed balloon flower root extract (20 mg/kg body weight); BRB-F : BFR-F (1:3), bioprocessed black rice bran extract (10 mg/kg body weight) and bioprocessed balloon flower root extract (30 mg/kg body weight) mouse group orally administered with black rice bran and Balloon flower root extracts respectively. Results are presented as mean ± SD (n=10). Bars sharing a common letter are not significantly different between groups at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8302597/v1/0ba9f7fa944c4422ac90501b.png"},{"id":99194119,"identity":"ce19dbc1-b986-4d9b-aa42-d79107515d6a","added_by":"auto","created_at":"2025-12-30 01:29:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":350748,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the combined ratio of BRB-F and BFR-F on the IL-10 expression. The IL-10 in ear was measured by ELISA (A) and RT-PCR intensity (B) are shown for the atopic dermatitis. Results are presented as mean ± SD (n=10). Bars sharing a common letter are not significantly different between groups at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8302597/v1/18126b81b8e4d17a4dd7f56c.png"},{"id":99316608,"identity":"013b9143-7284-4b65-a0a0-64ec49e46b71","added_by":"auto","created_at":"2025-12-31 16:28:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":361377,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the combined ratio of BRB-F and BFR-F on the galectin-9 expression. Galectin-9 in serum was measured by ELISA. Results are presented as mean ± SD (n=10). Bars sharing a common letter are not significantly different between groups at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8302597/v1/65ad00da9d4a1c5c9ac9f0a3.png"},{"id":99316941,"identity":"69174635-3688-4ac9-9183-92ec885e0260","added_by":"auto","created_at":"2025-12-31 16:29:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":586639,"visible":true,"origin":"","legend":"\u003cp\u003eDose-dependent effects of BRB-F and BFR-F (3:1) on DNCB and mite extract-induced atopic dermatitis. (A) Ear thickness was assessed using a micrometer. (B) Serum level of IgE was measured using ELISA. (C) TSLP, (D) IL-33 and (E) IL-31 in ear skin were measured by ELISA. Representation: vehicle (-), negative control not stimulated with DNCB and mite extract; vehicle (+), DNCB and mite extract-stimulated positive control; 10 mg/kg, 3 bioprocessed black rice bran extract + 1 bioprocessed balloon flower root extract (10 mg/kg body weight); 20 mg/kg, 3 bioprocessed black rice bran extract + 1 bioprocessed balloon flower root extract (20 mg/kg body weight); 40 mg/kg, 3 bioprocessed black rice bran extract + 1 bioprocessed balloon flower root extract (40 mg/kg body weight); 80 mg/kg, 3 bioprocessed black rice bran extract + 1 bioprocessed balloon flower root extract (80 mg/kg body weight); GLA 60 mg/kg, γ-linolenic acid (GLA, 60 mg/kg body weight) mouse group orally administered with Black rice bran and Balloon flower root extracts respectively. Results are presented as mean ± SD (n=10). Bars sharing a common letter are not significantly different between groups at p \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8302597/v1/ed143adcaffafc3537fd4b3e.png"},{"id":99317500,"identity":"89b562f2-e405-4e75-aa61-9540a3c0bd0c","added_by":"auto","created_at":"2025-12-31 16:30:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":264602,"visible":true,"origin":"","legend":"\u003cp\u003eDose-dependent effects of BRB-F and BFR-F (3:1) on the IL-10 expression. The IL-10 in ear was measured by ELISA (A) and RT-PCR intensity (B) are shown for the atopic dermatitis. Results are presented as mean ± SD (n=10). Bars sharing a common letter are not significantly different between groups at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8302597/v1/ad3c7161f2a4cef5eb789b44.png"},{"id":99194137,"identity":"b14e5e41-1f06-44ee-8d6e-29f64443bd65","added_by":"auto","created_at":"2025-12-30 01:29:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":302185,"visible":true,"origin":"","legend":"\u003cp\u003eDose-dependent effects of BRB-F and BFR-F (3:1) on the galectin-9 expression. Galectin-9 in serum was measured by ELISA. Results are presented as mean ± SD (n=10). Bars sharing a common letter are not significantly different between groups at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8302597/v1/122afcbb001d75f946b8e1fa.png"},{"id":99194134,"identity":"b43341aa-413d-40bf-9a9e-165307890d33","added_by":"auto","created_at":"2025-12-30 01:29:43","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":914986,"visible":true,"origin":"","legend":"\u003cp\u003eDose-dependent effects of BRB-F and BFR-F (3:1) on histopathological observation of ear skin in atopic dermatitis mouse using H\u0026amp;E Stain (×40). Ear tissues from AD Balb/c mice were fixed with 10% (v/v) paraformaldehyde. The fixed tissues were sectioned to 4μm, followed by staining with hematoxylin and eosin (H\u0026amp;E) and light microscopy (magnification, ×40). Photographs and H\u0026amp;E staining of the ear skin lesions from groups of mice on weeks 5. The black dotted line indicates ear thickness and epidermal thickness (hyperplasia). The red arrow indicates hyperkeratosis. The black arrows indicate exfoliating keratinocyte.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8302597/v1/ee955a3c4417c62e4e7290ac.jpeg"},{"id":102964480,"identity":"acc59129-e6bf-432a-bd8d-559a16643d97","added_by":"auto","created_at":"2026-02-19 04:22:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4715954,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8302597/v1/6fad1a5b-4fba-4903-9835-c8e0a6651b02.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanism of therapeutic effects of oral administration of bioprocessed black rice bran and balloon flower root (Platycodon grandiflorum) individually and in combination against atopic dermatitis in mice","fulltext":[{"header":"Background","content":"\u003cp\u003eAtopic dermatitis (AD), the most common chronic inflammatory skin disease in children and frequently persisting into adulthood, is characterized by intensely pruritic eczematous lesions. The pathology of AD involves multiple interacting contributors, including genetic susceptibility, impaired epidermal barrier function, alterations in skin microbial homeostasis, and dysregulated immune responses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. AD is further associated with a wide spectrum of comorbidities, such as allergic asthma [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], food allergy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], autoimmune disorders [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], squamous cell carcinoma and melanoma [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and additional conditions including rhinitis, ocular complications, psychiatric and infectious diseases, and endocrine and cardiovascular abnormalities [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Patients with AD also appear to have an increased risk of all-cause mortality [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCurrent therapeutic options\u0026mdash;including corticosteroids [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], IL-4 and IL-13 receptor blockade [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], triolein [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], coconut oil [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], alkaloid and limonoid compounds [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], probiotics [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], phototherapy [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and other anti-inflammatory approaches [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u0026mdash;provide symptomatic improvement but rarely achieve durable disease resolution. Despite substantial progress in both approved and investigational therapies, the long-term management of AD remains challenging [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], likely reflecting the heterogeneous and chronic nature of the disorder.\u003c/p\u003e\u003cp\u003eAs part of efforts to identify food-derived functional materials with immunomodulatory potential, we previously reported that black rice bran exhibits anti-inflammatory and anti-allergic properties relevant to chronic inflammatory disorders [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and that bioprocessed black rice bran cultured with shiitake mushroom mycelia protects mice against inflammation and asthma [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These findings motivated the present investigation into the therapeutic potential of an orally administered binary combination of bioprocessed black rice bran and balloon flower root (Platycodon grandiflorum), which contains bioactive compounds reported to be beneficial against asthma and related conditions.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003eAn ointment containing house dust mite extract (DFE; Biostir, Hyogo, Japan) and 1-Chloro-2,4-dinitrobenzene (DNCB; Sigma Chemical Co., St. Louis, MO) was used as the antigen and hapten, respectively, for inducing AD-like skin lesions. Unless otherwise specified, all other reagents were obtained from Sigma (St. Louis, MO). DNCB was dissolved in a solution of olive oil and acetone at a ratio of 3:1. The ELISA kit was purchased from R\u0026amp;D Systems (Minneapolis, MN).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePreparation of Bioprocessed Black Rice Bran and Bioprocessed Balloon Flower Root\u003c/h3\u003e\n\u003cp\u003eBioprocessed (fermented) black rice bran (BRB-F) and bioprocessed (fermented) balloon flower root (BFR-F) were prepared according to previously reported methods [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Lentinus edodes mycelia grown on potato dextrose agar (PDA) were inoculated into 50 mL of liquid medium and incubated in 250 mL Erlenmeyer flasks at 28\u0026deg;C for 5 days on a rotary shaker (120 rpm). The resulting pre-cultured mycelia were then used to inoculate the main liquid culture, which contained either black rice bran (100 g/L) or balloon flower root (100 g/L).\u003c/p\u003e\u003cp\u003eFor enzymatic pretreatment, the culture was treated with amylase at 60\u0026deg;C for 60 min. Fermentation proceeded in a 5 L fermenter (working volume 3 L) at 28\u0026deg;C and 150 rpm with a 10% inoculum. After 3 days, the culture mass was subjected to additional enzymatic treatment using amylase and a mixed enzyme preparation to degrade particulate carbohydrate-rich material.\u003c/p\u003e\u003cp\u003eThe culture was adjusted to pH 6.0 with HCl and subsequently sterilized in an autoclave. The main culture process was initiated by adding a cell-wall\u0026ndash;degrading enzyme mixture containing cellulase, hemicellulase, pectinase, glucanase, mannose, and arabinase at 50\u0026deg;C for 60 min. Following enzymatic treatment, the cultures were extracted with hot water at 90\u0026deg;C for 1 h and then lyophilized to obtain the final solid bioprocessed materials.\u003c/p\u003e\n\u003ch3\u003eU266.B1 Cell culture and Measurement of IgE production\u003c/h3\u003e\n\u003cp\u003eMeasurement of IgE production using a B cell line was performed according to previously described methods [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The U266.B1 human multiple myeloma B lymphocyte cell line was obtained from the American Type Tissue Culture Collection (ATCC, Manassas, VA). These cells were cultured in a modified RPMI1640 medium, supplemented with 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and 15% heat-inactivated fetal bovine serum (FBS). Penicillin (100 U/mL) and streptomycin (100 mg/mL) were also included in the medium to prevent bacterial contamination. The cells were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO2. The culture medium was replaced every three days, ensuring optimal conditions until the cells reached maximal density. To evaluate changes in IgE production, U266.B1 cells were seeded in 96-well plates at a density of 1\u0026times;106 cells per well and incubated for 24 hours. The cells were then stimulated with 10 \u0026micro;g/mL lipopolysaccharide (LPS), 5 ng/mL human IL-4, and the respective samples for an additional 72 hours. After incubation, the supernatant was collected and transferred to centrifuge tubes. The samples were centrifuged at 12,000 rpm for 10 minutes to separate the culture supernatants. IgE levels in the supernatants were quantified using an ELISA kit (Cat. No. BMS2097, Invitrogen) following the manufacturer's protocol. The absorbance of the reaction mixture was measured at 450 nm using a microplate reader (VersaMax, Molecular Devices Corp., CA).\u003c/p\u003e\n\u003ch3\u003eRBL-2H3 Cell culture and Measurement of β-Hexosaminidase Release\u003c/h3\u003e\n\u003cp\u003eMeasurement of β-hexosaminidase release using a mast cell line was performed according to previously described methods [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The RBL-2H3 rat basophilic leukemia mast cell line, obtained from ATCC, was cultured in a modified Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM). The medium was supplemented with 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and 10% heat-inactivated fetal bovine serum (FBS). To prevent bacterial contamination, penicillin (100 U/mL) and streptomycin (100 mg/mL) were added. The cells were maintained at 37\u0026deg;C in a humidified incubator containing 5% CO2. The culture medium was replaced every 2 to 3 days, allowing the cells to proliferate until maximal density was achieved. To assess β-hexosaminidase activity, RBL-2H3 cells were seeded in 96-well plates at a density of 1\u0026times;105 cells per well and incubated for 24 hours. Each well received 200 \u0026micro;L of Tyrode buffer (137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1.1 mM MgCl2, 11.9 mM NaHCO3, 0.4 mM NaH2PO4, and 5.6 mM glucose, pH 7.2), along with the test samples, and was incubated for 15 minutes. After incubation, the extracts were removed by washing with Tyrode buffer. To stimulate the cells, calcium ionophore A23187 (10 \u0026micro;M) dissolved in Tyrode buffer was added for 20 minutes. Following stimulation, 50 \u0026micro;L of the supernatant containing released β-hexosaminidase was collected from each well. The collected supernatant was mixed with an equal volume of p-nitrophenyl-N-acetyl-β-glucosaminide solution (1 mM, pH 5.2) and incubated for 1 hour at room temperature. The reaction was stopped by adding sodium carbonate buffer (67 mM, pH 10.2). The absorbance of the final mixture was measured at 405 nm using a microplate reader (VersaMax, Molecular Devices Corp., CA, USA), providing quantitative data on β-hexosaminidase release.\u003c/p\u003e\n\u003ch3\u003eHMC-1.2 Cell culture and Measurement of TSLP production\u003c/h3\u003e\n\u003cp\u003eMeasurement of TSLP production was performed according to the method of Moon and Kim [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] with slight modifications. The HMC-1.2 human mast cell line, obtained from ATCC, was cultured using a modified Iscove's Modified Dulbecco's Medium (IMDM). The culture medium was supplemented with 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and 10% heat-inactivated fetal bovine serum (FBS). To prevent bacterial contamination, penicillin (100 U/mL) and streptomycin (100 mg/mL) were also included in the medium. The cells were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO2. The culture medium was replaced every 2 to 3 days to support cell proliferation until maximal density was reached. To assess changes in thymic stromal lymphopoietin (TSLP) production, HMC-1.2 cells were plated in 96-well plates at a density of 5\u0026times;105 cells per well and incubated for 24 hours. The samples to be tested were then added to each well, followed by a 2-hour incubation period. Afterward, HMC-1.2 cells were stimulated with 50 \u0026micro;M phorbol 12-myristate 13-acetate (PMA) and 1 \u0026micro;g/mL calcium ionophore A23187 for 7 hours to induce TSLP production. Following stimulation, the supernatant from each well was collected and transferred into centrifuge tubes. The samples were centrifuged at 12,000 rpm for 10 minutes to separate the culture supernatants. The TSLP levels in the supernatants were then measured using a specific ELISA kit (Cat. No. EHTSLP, Invitrogen) according to the manufacturer's instructions. The absorbance of the final reaction mixture was read at 450 nm using a microplate reader (VersaMax, Molecular Devices Corp., CA), providing quantitative data on TSLP production.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eHaCaT Cell culture and Measurement of TARC, MDC, and IL-6 production\u003c/h2\u003e\u003cp\u003eMeasurement of TARC, MDC, and IL-6 production using a keratinocyte cell line was performed according to the method of Park et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] with slight modification. The HaCaT human keratinocyte cell line, obtained from the American Type Culture Collection (ATCC), was cultured using a modified Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM). The growth medium was supplemented with 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and 10% heat-inactivated fetal bovine serum (FBS). To prevent bacterial contamination, penicillin (100 U/mL) and streptomycin (100 mg/mL) were also added to the medium. The HaCaT cells were maintained at 37\u0026deg;C in a humidified incubator containing 5% CO2. The culture medium was replaced every two to three days, supporting cell proliferation until maximal density was reached. For the assessment of chemokine and cytokine production, HaCaT cells were seeded into 48-well plates at a density of 4\u0026times;105 cells per well and incubated for 24 hours. After the initial incubation, the cells were further cultured for 24 hours in serum-free DMEM. Test samples were then added to each well, and the cells were incubated for an additional 24 hours. Following this treatment, HaCaT cells were stimulated with 10 ng/mL of TNF-α and IFN-γ for 24 hours to induce the production of the target proteins. After stimulation, the culture supernatants were collected and transferred to centrifuge tubes. The samples were centrifuged at 12,000 rpm for 10 minutes to separate the supernatants. The concentrations of thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), and interleukin-6 (IL-6) in the supernatants were measured using specific ELISA kits (Cat. No. SDN00, DMD00, S6050; R\u0026amp;D Systems) according to the manufacturer\u0026rsquo;s protocols. The absorbance of the final reaction mixtures was determined at 450 nm using a microplate reader (VersaMax, Molecular Devices Corp., CA), providing quantitative data for TARC, MDC, and IL-6 production.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMice\u003c/h3\u003e\n\u003cp\u003ePathogen-free female Balb/c mice, aged six weeks, were obtained from Koatech (Gyeonggi-do, Korea). Upon arrival, the mice were housed in stainless-steel cages and maintained under a controlled environment featuring a 12-hour light/dark cycle. The room temperature was kept at 23\u0026deg;C with a margin of \u0026plusmn;\u0026thinsp;3\u0026deg;C, and the relative humidity was regulated to 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10%. For the purpose of acclimation, the mice were provided with unrestricted access to pelletized standard commercial chow diet (Cat. No. 5L79, Orient Bio, USA) and tap water for one week.\u003c/p\u003e\n\u003ch3\u003eDFE and DNCB-Induced Allergic Dermatitis (AD) in Ear Skin\u003c/h3\u003e\n\u003cp\u003eAllergic dermatitis-like lesions were induced in the ear skin of female BALB/c mice, following a modified protocol based on Choi et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and Hwang et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] The experimental procedure is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The mice were randomly assigned to nine distinct groups (n\u0026thinsp;=\u0026thinsp;10 per group): vehicle control; positive control (DFE/DNCB plus vehicle); DFE/DNCB plus black rice bran (BRB, 40 mg/kg); DFE/DNCB plus balloon flower root (BFR, 40 mg/kg); DFE/DNCB plus bioprocessed black rice bran (BRB-F, 40 mg/kg); DFE/DNCB plus bioprocessed balloon flower root (BFR-F, 40 mg/kg); and DFE/DNCB plus bioprocessed product mixtures in ratios of 3 BRB-F : 1 BFR-F, 1 BRB-F : 1 BFR-F, and 1 BRB-F : 3 BFR-F (all at 40 mg/kg). To initiate the dermatitis model, each mouse received a topical application of 20 \u0026micro;L of 1% 2,4-dinitrochlorobenzene (DNCB) dissolved in an olive oil/acetone solution (3:1 ratio) to both the front and back of right ear. This was followed three days later by an application of DFE ointment (100 mg/mL). The combined DFE/DNCB exposure was repeated once weekly for five consecutive weeks. One week after the initial challenge with DNCB and mite extract, the mice began receiving a diet containing the respective test samples, administered for a total duration of four weeks. At the end of the study period, all mice were sacrificed by CO2 inhalation, 24 hours after the final treatment (day 33), to evaluate the atopic dermatitis-suppressive effects of bioprocessed black rice bran and bioprocessed balloon flower root. Following blood collection, the ears and spleen of each mouse were excised for subsequent histological analysis and ELISA assessment. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Chuncheon Bioindustry Foundation (CBF IACUC no. 2025-054) and were conducted in accordance with relevant guidelines and regulations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of ear thickness and cytokines from ear tissue\u003c/h2\u003e\u003cp\u003eAfter the mice were sacrificed, ear thickness was determined using a dial thickness gauge (Mitutoyo Corporation, Kanagawa, Japan) to quantitatively assess swelling. Ear tissues were homogenized in a phosphate buffer (pH 7) containing 0.4 M NaCl, 0.05% Tween-20, 0.5% bovine serum albumin (BSA), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 mM ethylenediaminetetraacetic acid (EDTA). The resulting homogenates were then subjected to microcentrifugation at 14,000 \u0026times; g for 15 minutes at 4 ℃ to recover the supernatant, which contains the soluble cytokines. The concentrations of TSLP, IL-33, IL-31, and IL-10 in the ear tissue supernatants were subsequently measured using ELISA kits (R\u0026amp;D Systems, Minneapolis, MN), following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eELISA Measurement of cytokine levels in serum\u003c/h2\u003e\u003cp\u003eBlood samples were obtained from the sacrificed mice via cardiac puncture. Following collection, the samples were placed upright and allowed to rest for 30 minutes at 4 ℃ to facilitate clot formation of the red blood cells. Subsequently, the blood samples were centrifuged at 2,000 \u0026times; g for 30 minutes at 4 ℃ using a Micro 17R centrifuge (Hanil Science, Incheon, Republic of Korea). The resulting supernatants were carefully collected and stored at -70 ℃ until further analysis. To quantify the levels of serum IgE and Galectin-9, ELISA kits (R\u0026amp;D Systems, Minneapolis, MN) were used in accordance with the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eRNA isolation\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from biopsy specimens or mononuclear cells utilizing the protocol established by Chomczynski and Sacchi [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], with minor modifications tailored for this study. To achieve efficient cell lysis from tissue samples, forty consecutive cryostat sections, each 5 \u0026micro;M thick, were immersed in a buffer containing 4 M guanidinium isothiocyanate. Following the RNA extraction process, the isolated samples underwent treatment with DNase 1 (Promega Corp., Madison, WI) for 30 minutes at 37\u0026deg;C to eliminate any residual DNA. Throughout all enzymatic procedures involving RNA, an RNase inhibitor (Boehringer Mannheim Corp., Indianapolis, IN) was consistently included to protect the integrity of the RNA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eReal-Time Polymerase Chain Reaction (qRT-PCR)\u003c/h2\u003e\u003cp\u003eQuantitative real-time PCR (qRT-PCR) was performed using a Thermal Cycler Dice TP850 (Takarabio Inc., Shiga, Japan) in accordance with the manufacturer\u0026rsquo;s protocol. The reaction setup involved combining 2 \u0026micro;L of cDNA (containing 100 ng), 1 \u0026micro;L of a primer solution (sense and antisense, 0.4 \u0026micro;M each), 12.5 \u0026micro;L of SYBR Premix Ex Taq (Takarabio Inc.), and 9.5 \u0026micro;L of distilled water (dH2O), resulting in a total reaction volume of 25 \u0026micro;L per tube. The amplification protocol consisted of an initial denaturation step of 10 seconds at 95℃, followed by 40 cycles comprising 5 seconds at 95℃ and 30 seconds at 60℃. This was succeeded by additional steps: 15 seconds at 95℃, 30 seconds at 60℃, and a final 15 seconds at 95℃. Quantification and normalization of mRNA expression levels were conducted using the TP850 software provided by the manufacturer. This ensured accurate assessment of gene expression in the analyzed samples.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eResults are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three independent experiments. Significant differences between means were determined by the one-way ANOVA test followed by Duncan\u0026rsquo;s multiple range test using the Statistical Analysis System (SAS, Cary, NC, USA). p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was regarded as significant. Percent reductions were calculated using values obtained by subtracting the mean value obtained with vehicle from the mean values obtained using the positive control treatment and the experimental treatment.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eInhibitory Effects of BRB-F and BFR-F on IgE Production in Human U266.B1 Cells\u003c/h2\u003e\u003cp\u003eTo evaluate the ability of bioprocessed black rice bran (BRB-F) and bioprocessed balloon flower root (BFR-F) extracts to suppress IgE production, assays were conducted using the human B-cell line U266.B1. Both individual extracts and their combinations were examined. Stimulation of U266.B1 cells with lipopolysaccharide (LPS) and interleukin-4 (IL-4) increased IgE secretion by more than 50-fold compared with unstimulated controls. Treatment with the raw extracts, BRB and BFR, led to modest reductions in IgE production, with BRB decreasing IgE levels by 18.1% and BFR by 10.7% relative to the positive control. In contrast, the bioprocessed extracts BRB-F and BFR-F exhibited markedly stronger inhibitory activity, each suppressing IgE production by approximately 60%. Combinations of BRB-F and BFR-F at different ratios (3:1, 1:1, and 1:3) resulted in inhibitory levels comparable to those observed with each extract alone, with no significant differences among mixing ratios.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInhibitory effect of IgE production in U266.B1 cells by combined ratios of BRB-F and BFR-F.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIgE concentration (ng/mL)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVehicle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive control\u003c/p\u003e\u003cp\u003e(10 \u0026micro;g/mL LPS\u0026thinsp;+\u0026thinsp;5 ng/mL IL-4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e482.7\u0026thinsp;\u0026plusmn;\u0026thinsp;31.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BRB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e396.8\u0026thinsp;\u0026plusmn;\u0026thinsp;30.6\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BFR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e431.9\u0026thinsp;\u0026plusmn;\u0026thinsp;25.4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BRB-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e202.7\u0026thinsp;\u0026plusmn;\u0026thinsp;15.2\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BFR-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e205.4\u0026thinsp;\u0026plusmn;\u0026thinsp;17.9\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e75 \u0026micro;g/mL BRB-F\u0026thinsp;+\u0026thinsp;25 \u0026micro;g/mL BFR-F (3:1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e204.3\u0026thinsp;\u0026plusmn;\u0026thinsp;18.1\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e50 \u0026micro;g/mL BRB-F\u0026thinsp;+\u0026thinsp;50 \u0026micro;g/mL BFR-F (1:1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e198.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.3\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e25 \u0026micro;g/mL BRB-F\u0026thinsp;+\u0026thinsp;75 \u0026micro;g/mL BFR-F (1:3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e209.6\u0026thinsp;\u0026plusmn;\u0026thinsp;11.9\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eData are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three independent experiments. Values in each column with the same letter are not significantly different between groups at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eDegranulation Inhibitory Effects of BRB-F and BFR-F in RBL-2H3 Mast Cells\u003c/h2\u003e\u003cp\u003eThe inhibitory activity of bioprocessed black rice bran (BRB-F) and bioprocessed balloon flower root (BFR-F) on mast cell degranulation was evaluated using the RBL-2H3 rat basophilic leukemia cell line. Both individual and combined treatments were tested to determine their effects on β-hexosaminidase release, a key marker of mast cell degranulation. Stimulation with A23187 induced a strong degranulation response. Treatment with the raw materials, BRB and BFR, reduced β-hexosaminidase release by 52.7% and 31.5%, respectively, compared with the positive control. BRB-F and BFR-F also inhibited degranulation, decreasing β-hexosaminidase release by 58.4% and 33.9%, respectively. When BRB-F and BFR-F were combined at different mixing ratios, the degree of inhibition was comparable to that produced by BRB-F alone, and no significant differences were detected among the tested combinations (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInhibitory effect of β-hexosaminidase releases in RBL-2H3 cells by combined ratios of BRB-F and BFR-F.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eβ-hexosaminidase release (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVehicle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.3\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive control\u003c/p\u003e\u003cp\u003e(10 \u0026micro;M Calcium ionophore A23187)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BRB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e47.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BFR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e68.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BRB-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e41.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BFR-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e66.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e75 \u0026micro;g/mL BRB-F\u0026thinsp;+\u0026thinsp;25 \u0026micro;g/mL BFR-F (3:1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e44.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e50 \u0026micro;g/mL BRB-F\u0026thinsp;+\u0026thinsp;50 \u0026micro;g/mL BFR-F (1:1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e44.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e25 \u0026micro;g/mL BRB-F\u0026thinsp;+\u0026thinsp;75 \u0026micro;g/mL BFR-F (1:3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e46.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eData are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three independent experiments. Values in each column with the same letter are not significantly different between groups at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of BRB-F and BFR-F Inhibitory Effects on TSLP Production in HMC-1.2 Cells\u003c/h2\u003e\u003cp\u003eThe inhibitory effects of bioprocessed black rice bran (BRB-F) and bioprocessed balloon flower root (BFR-F) on thymic stromal lymphopoietin (TSLP) production were examined using the human mast cell line HMC-1.2. Both individual and combined treatments were assessed to determine their relative efficacy. Stimulation with PMA and A23187 elevated TSLP production by 2.3-fold compared with baseline. Treatment with the raw materials BRB and BFR reduced TSLP levels by 34.5% and 18.9%, respectively. In contrast, treatment with the bioprocessed forms resulted in greater suppression, with BRB-F reducing TSLP production by 51.9% and BFR-F by 31.3% (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Combination treatments of BRB-F and BFR-F showed that increasing the proportion of BRB-F produced slightly stronger TSLP inhibition, although the degree of suppression was comparable among the tested mixing ratios.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInhibitory effect of TSLP production in HMC-1 cells by combined ratios of BRB-F and BFR-F.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTSLP production (pg/mL)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVehicle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e36.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive control\u003c/p\u003e\u003cp\u003e(50 \u0026micro;M PMA\u0026thinsp;+\u0026thinsp;1 \u0026micro;g/mL Calcium ionophore A23187)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e82.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BRB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e66.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BFR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e74.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BRB-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e58.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BFR-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e68.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e75 \u0026micro;g/mL BRB-F\u0026thinsp;+\u0026thinsp;25 \u0026micro;g/mL BFR-F (3:1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e59.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e50 \u0026micro;g/mL BRB-F\u0026thinsp;+\u0026thinsp;50 \u0026micro;g/mL BFR-F (1:1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e62.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e25 \u0026micro;g/mL BRB-F\u0026thinsp;+\u0026thinsp;75 \u0026micro;g/mL BFR-F (1:3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e66.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eData are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three independent experiments. Values in each column with the same letter are not significantly different between groups at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eInhibitory Effects of BRB-F and BFR-F on Cytokine and Chemokine Expression in HaCaT Keratinocyte Cells\u003c/h2\u003e\u003cp\u003eThe inhibitory effects of bioprocessed black rice bran (BRB-F) and bioprocessed balloon flower root (BFR-F) on cytokine and chemokine production were evaluated in human HaCaT keratinocyte cells. Both individual and combined treatments were examined to determine their relative efficacy. Stimulation with TNF-α and IFN-γ elevated the expression of TARC, MDC, and IL-6 by 29-fold, 89-fold, and 17-fold, respectively. Treatment with the raw materials BRB and BFR significantly suppressed these induced levels, with BRB showing greater inhibitory activity than BFR by 1.7-fold for TARC, 2.2-fold for MDC, and 1.5-fold for IL-6. Among the bioprocessed materials, BFR-F exhibited higher inhibitory potency than BRB-F, producing 1.5-fold, 1.2-fold, and 0.8-fold stronger suppression of TARC, MDC, and IL-6, respectively. Combination treatments of BRB-F and BFR-F resulted in inhibition levels comparable to those of the bioprocessed extracts alone (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInhibitory effect of TARC, MDC, and IL-6 production in HaCaT cells by combined ratios of BRB-F and BFR-F.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eCytokine and chemokine production (pg/mL)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTARC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMDC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIL-6\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVehicle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e33.5\u0026thinsp;\u0026plusmn;\u0026thinsp;14.3\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive control\u003c/p\u003e\u003cp\u003e(10 ng/mL TNF-α/IFN-γ)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e341.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2999.3\u0026thinsp;\u0026plusmn;\u0026thinsp;253.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e109.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BRB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e218.0\u0026thinsp;\u0026plusmn;\u0026thinsp;16.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1703.8\u0026thinsp;\u0026plusmn;\u0026thinsp;134.7\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e66.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BFR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e268.1\u0026thinsp;\u0026plusmn;\u0026thinsp;13.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2421.1\u0026thinsp;\u0026plusmn;\u0026thinsp;150.7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e81.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BRB-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e130.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e942.2\u0026thinsp;\u0026plusmn;\u0026thinsp;64.5\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e52.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 \u0026micro;g/mL BFR-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e600.1\u0026thinsp;\u0026plusmn;\u0026thinsp;59.6\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e61.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e75 \u0026micro;g/mL BRB-F +\u003c/p\u003e\u003cp\u003e25 \u0026micro;g/mL BFR-F (3:1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e97.4\u0026thinsp;\u0026plusmn;\u0026thinsp;18.5\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e892.4\u0026thinsp;\u0026plusmn;\u0026thinsp;80.4\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e50 \u0026micro;g/mL BRB-F +\u003c/p\u003e\u003cp\u003e50 \u0026micro;g/mL BFR-F (1:1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e55.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e791.7\u0026thinsp;\u0026plusmn;\u0026thinsp;31.9\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e51.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e25 \u0026micro;g/mL BRB-F +\u003c/p\u003e\u003cp\u003e75 \u0026micro;g/mL BFR-F (1:3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e44.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e717.3\u0026thinsp;\u0026plusmn;\u0026thinsp;51.2\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eData are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three independent experiments. Values in each column with the same letter are not significantly different between groups at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eAnti-atopic effects of BRB-F and BFR-F in a mouse model of atopic dermatitis\u003c/h2\u003e\u003cp\u003eThe therapeutic effects of bioprocessed black rice bran (BRB-F) and bioprocessed balloon flower root (BFR-F), administered individually or in combination, were evaluated in a mouse model of atopic dermatitis (AD) induced by repeated exposure to 2,4-dinitrochlorobenzene (DNCB) and mite extract. Disease severity was assessed by measuring ear thickness, serum IgE levels, and the expression of thymic stromal lymphopoietin (TSLP), interleukin-33 (IL-33), and interleukin-31 (IL-31) in ear tissue. In AD-induced mice, ear thickness, serum IgE, TSLP, IL-33, and IL-31 levels were markedly elevated compared with normal controls. Treatment with the bioprocessed materials resulted in significantly greater reductions in these markers than their corresponding raw forms. Among the individual treatments, BRB and BRB-F consistently produced stronger inhibitory effects than BFR and BFR-F. Combination treatments of BRB-F and BFR-F further reduced these biomarkers, with inhibitory efficacy increasing as the proportion of BRB-F increased in the mixture. The greatest therapeutic effect was observed for the 3:1 BRB-F:BFR-F ratio (30 mg/kg BRB-F and 10 mg/kg BFR-F), which reduced ear thickness to 84.5% of the control level, serum IgE to 82.4%, TSLP expression to 85.9%, IL-33 to 92.9%, and IL-31 to 73.7% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eModulation of Th2 and Th1 Cytokine Expression by BRB-F and BFR-F in Atopic Dermatitis\u003c/h2\u003e\u003cp\u003eTh2 cells are known to predominate in lesioned tissues following the induction of atopic dermatitis (AD) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. To evaluate immune modulation in this model, the mRNA expression levels of Th2 cytokines (IL-4, IL-5, and IL-13) were measured in ear tissue. AD induction resulted in strong upregulation of these cytokines, exceeding a 5-fold increase compared with normal, untreated mice. Administration of the bioprocessed extracts, either alone or in combination, produced greater suppression of Th2 cytokine expression than the corresponding raw materials. The most pronounced inhibitory effect was observed with the 3:1 BRB-F:BFR-F combination, which reduced IL-4 mRNA to 48.8%, IL-5 to 49.1%, and IL-13 to 49.7% of the AD-induced control (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). To further examine T-cell\u0026ndash;mediated immune responses, expression levels of the Th1 cytokines IL-2, IL-12, and IFN-γ were measured in spleen tissue. AD induction markedly decreased Th1 cytokine expression. Treatment with the bioprocessed materials, particularly the 3:1 BRB-F:BFR-F combination, substantially restored Th1 cytokine levels, with recovery most notable for IL-2 (68.4% of normal), followed by IL-12 (48.3% of normal) and IFN-γ (76% of normal).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRegulatory effect of Th1 and Th2 cytokines in atopic dermatitis-induced mice by combined ratios of BRB-F and BFR-F.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e\u003cp\u003eCytokines Production\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eTh2 Cytokine mRNA in ear (fold-increase)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eTh1 Cytokine mRNA in spleen (fold-increase)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIL-4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIL-5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIL-13\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIL-2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIL-12\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eIFN-γ\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVehicle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive control\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40 mg/kg BRB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40 mg/kg BFR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40 mg/kg BRB-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40 mg/kg BFR-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40 mg/kg BRB-F\u0026thinsp;+\u0026thinsp;BFR-F (3:1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40 mg/kg BRB-F\u0026thinsp;+\u0026thinsp;BFR-F (1:1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40 mg/kg BRB-F\u0026thinsp;+\u0026thinsp;BFR-F (1:3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003eData are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three independent experiments. Values in each column with the same letter are not significantly different between groups at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eEffects of BRB-F and BFR-F on IL-10 Cytokine Expression in Atopic Dermatitis\u003c/h2\u003e\u003cp\u003ePrevious studies have shown that excessive secretion of interleukin-10 (IL-10) by Th2 cells is associated with the progression of atopic dermatitis (AD) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To evaluate this mechanism, IL-10 mRNA and protein expression levels were measured in ear tissue. AD induction resulted in an approximately threefold increase in both IL-10 mRNA and protein expression compared with normal, untreated mice. Administration of the bioprocessed materials produced greater inhibition of IL-10 expression than their raw counterparts. Among the treatment groups, the combination of BRB-F and BFR-F at a 3:1 ratio yielded the strongest effect, reducing IL-10 mRNA expression by 87.2% and protein expression by 70.9% relative to the AD-induced group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eEffects of BRB-F and BFR-F on Galectin-9 Expression and Treg Activation in Atopic Dermatitis\u003c/h2\u003e\u003cp\u003eGalectin-9 plays a key role in immune regulation by promoting the activation of regulatory T (Treg) cells through the induction of transforming growth factor-β1 (TGF-β1), a cytokine associated with suppression of inflammatory responses [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To evaluate whether BRB-F and BFR-F modulate this pathway, serum galectin-9 levels were measured in mice treated with the extracts individually or in combination. Mice with induced atopic dermatitis exhibited a substantial increase in serum galectin-9 levels (78.59\u0026thinsp;\u0026plusmn;\u0026thinsp;5.58 pg/mL), approximately 3.3-fold higher than those in normal controls (23.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73 pg/mL). Administration of BRB-F and BFR-F further elevated serum galectin-9 levels, and no significant differences were observed between single-agent and combination treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eEvaluation of Dose-Dependent Effects of BRB-F and BFR-F Mixture on Atopic Dermatitis\u003c/h2\u003e\u003cp\u003eTo identify the optimal dosage for suppressing atopic dermatitis (AD), the bioprocessed black rice bran (BRB-F) and bioprocessed balloon flower root (BFR-F) mixture was evaluated at the previously determined optimal ratio of 3:1 (30 mg/kg BRB-F and 10 mg/kg BFR-F). Mice were administered daily oral doses of 10, 20, 40, and 80 mg/kg of the mixture following AD induction with mite extract and DNCB. Disease severity was evaluated by measuring ear thickness, serum IgE levels, and the expression of TSLP, IL-33, and IL-31 in ear tissue. All biomarkers were significantly elevated in AD-induced mice compared with normal controls. Treatment with the BRB-F:BFR-F mixture produced dose-dependent suppression of AD symptoms. The highest dose (80 mg/kg) reduced ear thickness by 75%, serum IgE by 87.1%, TSLP by 82.9%, IL-33 by 95.4%, and IL-31 by 72.4% relative to the AD control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). When compared with a reference group receiving 60 mg/kg γ-linolenic acid (GLA), the 20 mg/kg BRB-F:BFR-F treatment produced comparable or greater inhibition across multiple biomarkers.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eRegulation of Th1/Th2 Immune Response by BRB-F and BFR-F Mixture\u003c/h2\u003e\u003cp\u003eTo evaluate the effects of the BRB-F and BFR-F mixture on Th1/Th2 immune regulation in atopic dermatitis (AD), mRNA expression levels of key cytokines were measured in mice. Th2 cytokines (IL-4, IL-5, and IL-13) were analyzed in lesional ear tissue, while Th1 cytokines (IL-2, IL-12, and IFN-γ) were quantified in spleen tissue using RT-PCR. AD induction resulted in a marked elevation of Th2 cytokine expression, with IL-4, IL-5, and IL-13 increasing more than fivefold compared with normal mice. Treatment with the BRB-F:BFR-F mixture produced dose-dependent suppression of Th2 cytokine expression, with the strongest effect observed at the highest dose (80 mg/kg), reducing IL-4, IL-5, and IL-13 by 45.3%, 39.2%, and 41.1%, respectively. AD induction also caused significant reductions in the expression of Th1-associated cytokines in the spleen. Administration of the BRB-F:BFR-F mixture resulted in dose-dependent restoration of Th1 cytokine levels, with the highest dose (80 mg/kg) increasing IL-2 to 60.5% of normal, IL-12 to 55.2%, and IFN-γ to 56% (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The 20 mg/kg dose of the BRB-F:BFR-F mixture produced levels of cytokine suppression and recovery comparable to those observed in the reference group treated with 60 mg/kg γ-linolenic acid (GLA).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDose-dependent effects of BRB-F and BFR-F (3:1) on Th1 and Th2 cytokines.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e\u003cp\u003eCytokines Production\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eTh2 Cytokine mRNA in ear (fold-increase)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eTh1 Cytokine mRNA in spleen (fold-increase)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIL-4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIL-5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIL-13\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIL-2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIL-12\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eIFN-γ\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVehicle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10 mg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20 mg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40 mg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e80 mg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGLA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eData are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three independent experiments. Values in each column with the same letter are not significantly different between groups at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eInhibitory Effects of BRB-F and BFR-F Mixture on IL-10 Expression in Atopic Dermatitis-Induced Mice\u003c/h2\u003e\u003cp\u003eMeasurements of IL-10 mRNA and protein expression in ear tissue revealed significant overexpression of IL-10 in mice with induced atopic dermatitis. Administration of the BRB-F:BFR-F mixture produced dose-dependent suppression of IL-10 expression. The highest dietary dose (80 mg/kg) resulted in the greatest inhibition, reducing IL-10 mRNA by 90.9% and IL-10 protein by 85.2% relative to the AD-induced control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). A comparative analysis with γ-linolenic acid (GLA) showed that the 20 mg/kg BRB-F:BFR-F treatment produced stronger IL-10 inhibitory activity than 60 mg/kg GLA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eDose-Dependent Effects of BRB-F and BFR-F Mixture on Galectin-9 Expression\u003c/h2\u003e\u003cp\u003eTo assess the regulatory effects of the BRB-F:BFR-F mixture on galectin-9 expression, serum galectin-9 levels were measured in mice with induced atopic dermatitis (AD). ELISA analysis showed that AD induction resulted in a substantial elevation of serum galectin-9 (61.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4 pg/mL), approximately 3.6-fold higher than that of normal control mice (16.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 pg/mL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Administration of the BRB-F:BFR-F mixture produced a dose-dependent increase in galectin-9 expression, yielding 1.4-, 1.5-, 1.8-, and 1.9-fold increases in serum galectin-9 levels at incremental doses. When compared with γ-linolenic acid (GLA), the 10 mg/kg dose of the mixture exhibited comparable stimulatory activity to 60 mg/kg GLA, while higher doses surpassed the effect of GLA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eHistopathological Analysis of Ear Skin in Atopic Dermatitis Mice\u003c/h2\u003e\u003cp\u003eHistological examination of normal mice showed a thin epidermis composed of one to two cell layers, with minimal desquamation of the stratum corneum. In contrast, mice with induced atopic dermatitis (AD) displayed marked epidermal thickening characterized by multiple cell layers, increased numbers of blood vessels, sebaceous glands, hair follicles, and muscle bundles. The granular layer contained abundant keratohyalin granules, and the stratum corneum appeared uneven with extensive desquamation throughout the tissue [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Dietary administration of the BRB-F:BFR-F mixture at doses of 10, 20, 40, and 80 mg/kg produced dose-dependent improvements in these pathological alterations. Treatment resulted in progressive thinning of the epidermis relative to AD-induced mice, along with reduced granular layer thickness. The stratum corneum became more continuous and uniform, with minor areas of partial desquamation remaining. The most substantial alleviation of epidermal hyperplasia, inflammatory cell infiltration, and stratum corneum exfoliation occurred at the highest dose (80 mg/kg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The group receiving 60 mg/kg γ-linolenic acid (GLA) exhibited ear tissue morphology comparable to that of the group treated with 20 mg/kg of the BRB-F:BFR-F mixture.\u003c/p\u003e\u003cp\u003eTaken together, these findings demonstrate that oral administration of the bioprocessed black rice bran and balloon flower root formulation produces coordinated improvements across cellular, tissue-level, and systemic immune parameters, indicating a consistent therapeutic trajectory that extends beyond single-pathway modulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study demonstrates that bioprocessing substantially enhances the anti-atopic activities of both black rice bran and balloon flower root. Although the raw materials exhibited inherent but limited suppression of IgE synthesis, fermentation markedly amplified their bioactivity, consistent with previous reports that bioconversion improves the physiological functions and bioavailability of BRB-derived polysaccharides [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The similar degrees of inhibition produced by different BRB-F:BFR-F mixing ratios suggest that enhanced activity primarily reflects intrinsic properties of each extract rather than synergistic interactions, although the potential for synergy under alternative dosing conditions cannot be excluded.\u003c/p\u003e\u003cp\u003eBoth raw and bioprocessed extracts were also capable of suppressing mast cell degranulation. The greater inhibition achieved with BRB and BRB-F compared with BFR and BFR-F indicates that mast cell-modulating constituents are more abundant or potent in black rice bran, which is consistent with previous observations that BRB-derived bioactives modulate mast cell\u0026ndash;associated inflammatory signaling [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These results suggest that anti-degranulation activity relies largely on functional components already present in the raw materials rather than bioactive factors newly generated by fermentation.\u003c/p\u003e\u003cp\u003eIn keratinocytes, bioprocessing enhanced suppression of pro-inflammatory cytokines and chemokines, mirroring the trends observed in IgE synthesis and mast cell activation. The stronger anti-inflammatory effect of BFR-F relative to BRB-F indicates that balloon flower root contains constituents that more strongly target keratinocyte-mediated inflammation, consistent with the known pharmacological activities of platycodin-containing extracts [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The lack of synergistic or additive effects across BRB-F:BFR-F mixing ratios suggests that the two materials act through complementary but largely independent biochemical pathways in this context.\u003c/p\u003e\u003cp\u003eIn vivo, bioprocessing significantly enhanced the ability of both materials to attenuate AD pathology. Notably, efficacy increased progressively with the proportion of BRB-F in the formulation, supporting the interpretation that BRB-derived constituents make dominant contributions to moderating AD-associated immune dysregulation. This interpretation aligns with earlier reports demonstrating protective effects of BRB-derived bioactive compounds against allergic and inflammatory disorders [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The concomitant reductions in IgE, TSLP, IL-33 and IL-31 suggest action across multiple inflammatory axes rather than suppression of a single inflammatory pathway.\u003c/p\u003e\u003cp\u003eThe BRB-F:BFR-F formulation modulated adaptive immunity in a bidirectional manner by attenuating Th2-dominant inflammation in lesional skin while restoring systemic Th1 cytokine expression. A similar pattern\u0026mdash;where normalization of Th1 responses accompanies suppression of Th2 hyperactivation\u0026mdash;has been observed in previous successful immunomodulatory interventions for AD [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These results indicate that the formulation restores immune balance rather than solely inhibiting inflammatory drivers.\u003c/p\u003e\u003cp\u003eBioprocessing also enhanced the ability of both extracts to inhibit IL-10 overexpression in vivo. Since excessive IL-10 secretion is known to suppress Th1-mediated immune responses and promote AD progression [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], inhibition of IL-10 may contribute to immune homeostasis in this model. Likewise, induction of circulating galectin-9\u0026mdash;an immunoregulatory factor associated with Treg activation and attenuation of Th2-dominant inflammation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u0026mdash;may represent an additional component of the therapeutic mechanism. Together, these findings suggest that BRB-F and BFR-F act through coordinated regulation of B-cell, mast cell, keratinocyte, Th2, Th1 and Treg-associated pathways, contributing both to immune rebalancing and improved tissue-level pathology.\u003c/p\u003e\u003cp\u003eImportantly, substantial therapeutic activity was observed even at relatively low doses. The 20 mg/kg treatment produced comparable or superior inhibitory activity relative to 60 mg/kg γ-linolenic acid, in agreement with previous findings showing that functional food\u0026ndash;derived bioactive ingredients can serve as effective adjuncts in the management of AD [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although the highest dose (80 mg/kg) yielded the strongest effects, the robust response at intermediate doses suggests that clinically meaningful benefits may be achievable without maximal exposure.\u003c/p\u003e\u003cp\u003eThis study has limitations. Because all experiments were conducted in a murine model, translation to clinical applications will require assessment of safety, optimal dosing and efficacy in humans. Moreover, although this work demonstrates improved biological activity following bioprocessing, the specific bioactive molecules and fermentation-driven chemical conversions underlying the therapeutic effects remain incompletely defined. Ongoing studies are aimed at identifying the major immunomodulatory constituents of BFR-F and determining how bioprocessing alters the chemical profiles of both BRB-F and BFR-F. Such work may facilitate further optimization of this binary formulation and provide insight into whether similar immune responses can be achieved in clinical settings.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates that a novel binary functional food composed of bioprocessed black rice bran (BRB-F) and independently bioprocessed balloon flower root (BFR-F) provides significant therapeutic benefits in a mouse model of atopic dermatitis (AD). Oral administration of the combined extracts at doses ranging from 20 to 80 mg/kg resulted in dose-dependent suppression of multiple AD-associated biomarkers and markedly improved histopathological features of lesional skin. Although each bioprocessed material individually suppressed key immunologic mechanisms underlying AD in vitro and in vivo, the combined formulation at a BRB-dominant 3:1 ratio produced the most pronounced inhibitory effects. These immunological improvements were accompanied by reduced Th2-associated cytokines and enhanced Th1- and Treg-associated immune regulation together with elevated serum galectin-9 levels, suggesting that coordinated modulation of multiple immune pathways underlies the enhanced therapeutic efficacy of the combined treatment.\u003c/p\u003e\u003cp\u003eThe findings of this investigation support the rationale for conducting clinical trials to determine whether this binary functional food may offer a viable strategy for the treatment or prevention of AD in humans. Furthermore, the present results strengthen the broader hypothesis that this formulation may also influence comorbidities associated with AD, as suggested by previous observations showing that bioprocessed black rice bran in combination with various food ingredients protects mice against asthma [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], murine colon carcinoma [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], alcohol-induced fatty liver formation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], alcohol-induced hangover symptoms [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and reported multiple health benefits of mushroom polysacharides [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and of ballon flower root mentioned below.. Taken together, these outcomes raise the possibility that immunomodulatory effects driven by BRB-F and BFR-F may extend beyond AD and contribute to the improvement of systemic inflammatory conditions linked to immune imbalance.\u003c/p\u003e\u003cp\u003eAdditional evidence from the literature highlights the potential relevance of balloon flower root bioactive components to immune and inflammatory regulation: Song et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] reviewed the traditional use of platycodin D in viral illness; Han et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] demonstrated the influence of Platycodon grandiflorus root on gut microbiota and host metabolism; and Kim et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] reported anti-inflammatory activity of balloon flower root extracellular vesicles. We previously reported that the bioprocessed black rice bran product is a polysaccharide consisting of glucose and galactose units [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], whereas the chemical composition of the bioprocessed balloon flower root product remains to be determined. Clarifying the bioactive constituents of BFR-F will be essential for understanding how bioprocessing influences its anti-atopic properties and for optimizing the formulation. Future studies should therefore investigate how bioprocessing alters the chemical composition of balloon flower root, identify the compounds responsible for anti-atopic activity, and determine how these components may be optimized to maximize the therapeutic potential of this binary functional food for AD and its associated comorbidities.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAD, atopic dermatitis\u003c/p\u003e\u003cp\u003eBRB-F, bioprocessed black rice bran\u003c/p\u003e\u003cp\u003eBFR-F, bioprocessed balloon flower root\u003c/p\u003e\u003cp\u003eDFE, Dermatophagoides farinae extract\u003c/p\u003e\u003cp\u003eDNCB, 1-chloro-2,4-dinitrobenzene\u003c/p\u003e\u003cp\u003eIgE, immunoglobulin E\u003c/p\u003e\u003cp\u003eIgG2a, immunoglobulin G2a\u003c/p\u003e\u003cp\u003eTh, T helper\u003c/p\u003e\u003cp\u003eTreg, regulatory T cell\u003c/p\u003e\u003cp\u003eqRT-PCR, quantitative real-time polymerase chain reaction\u003c/p\u003e\u003cp\u003eELISA, enzyme-linked immunosorbent assay\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;This research was financially supported by the Ministry of Trade, Industry \u0026amp; Energy (MOTIE), Korea Institute for Advancement of Technology (KIAT) through the Encouragement Program for The Industries of Economic Cooperation Region(P0004707), and Ministry of Trade, Industry and Energy (MOTIE, Republic of Korea) through the Technology Innovation Program (No. 20008826).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;S.P.K. conceived the idea and, with K.H.L., K.S.K., W.S.H., W.Y.L., J.K., and S.J.L., conducted the experimental studies; M.F., A.D.F., and S.P.K. interpreted the results and prepared a draft of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;We thank Eun Young Seong for excellent technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Animal experiments were approved by the Institutional Animal Care and Use Committee of\u0026nbsp;Chuncheon Bioindustry Foundation\u0026nbsp;(CBF IACUC no. 2025-054) and were conducted in accordance with the relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;The original source of method description is acknowledged and cited in each case. The datasets used and/or analyzed during the current study and supplementary material are available from the following author on reasonable request: Prof. Sung Phil Kim; E-mails: spkim@ strbiotech. co. kr; ksp11 08@ ajou. ac. kr.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSroka-Tomaszewska J, Trzeciak M. Molecular mechanisms of atopic dermatitis pathogenesis. Int J Mol Sci. 2021;22:4130. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms22084130\u003c/span\u003e\u003cspan address=\"10.3390/ijms22084130\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDai L, Liu J, Zhao Q, Li M, Zhou Y, Chen Z, Zhang Y. Investigation of allergic sensitizations in children with allergic rhinitis and/or asthma. 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Antioxidants. 2023;12:1146. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/antiox12061146\u003c/span\u003e\u003cspan address=\"10.3390/antiox12061146\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"atopic dermatitis, bioprocessed black rice bran, balloon flower root, regulatory T cells, immunomodulation","lastPublishedDoi":"10.21203/rs.3.rs-8302597/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8302597/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eAtopic dermatitis (AD) is a chronic inflammatory skin disorder frequently associated with dysregulated Th1/Th2 immune balance and impaired regulatory T (Treg) activity. Although conventional treatments can reduce clinical symptoms, concerns regarding long-term adverse effects have led to increased interest in complementary and functional dietary materials with immunomodulatory potential. Bioprocessed black rice bran (BRB-F) and balloon flower root fractions (BFR-F) have individually demonstrated anti-inflammatory activities, but their combined oral efficacy and mechanisms in AD remain insufficiently characterized.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eMechanistic in vitro assays were first performed using a human B-cell line to measure IgE secretion, mouse and/or human mast cells to evaluate degranulation and TSLP release, and keratinocyte cultures to quantify TARC, MDC, and IL-6 production. These assays were used to determine whether BRB-F and BFR-F individually or cooperatively modulate immune and epithelial cell activation relevant to AD pathology. Subsequently, an AD-like phenotype was induced in BALB/c mice by topical application of 1-chloro-2,4-dinitrobenzene followed by mite extract. Animals received dietary supplementation with a 3:1 BRB-F:BFR-F mixture (10\u0026ndash;80 mg/kg/day), and clinical skin inflammation, ear histopathology, and cytokine profiles were analyzed to assess Th1/Th2 balance and Treg-associated markers.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eDietary supplementation with the BRB-F:BFR-F binary combination resulted in dose-dependent improvement in AD-like skin lesions and reduced histopathological severity. The mixture suppressed Th2 cytokines (IL-4, IL-5, IL-13) in ear tissue and increased splenic Th1 cytokines (IL-2, IL-12, IFN-γ), accompanied by dose-dependent elevation of galectin-9, suggesting activation of Treg-associated pathways. IL-10 expression was also reduced in vivo. In vitro, BRB-F and BFR-F cooperatively inhibited IgE production by B cells, attenuated mast cell activation and TSLP release, and lowered keratinocyte secretion of TARC, MDC, and IL-6.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThe combined oral administration of bioprocessed BRB-F and BFR-F exerts multi-targeted anti-atopic activity that includes modulation of Th1/Th2 immune balance, activation of Treg-associated signaling, and suppression of B-cell, mast cell, and keratinocyte responses. These findings, confirmed by histopathology of skin tissues before and after treatment, provide preclinical support for the development of BRB-F:BFR-F as a complementary functional material for managing immune dysregulation associated with AD, although validation in human patients will be required.\u003c/p\u003e","manuscriptTitle":"Mechanism of therapeutic effects of oral administration of bioprocessed black rice bran and balloon flower root (Platycodon grandiflorum) individually and in combination against atopic dermatitis in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 01:29:38","doi":"10.21203/rs.3.rs-8302597/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a84f3cbd-8e69-4a62-af56-ac70dd17fa2e","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-18T10:25:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-30 01:29:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8302597","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8302597","identity":"rs-8302597","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}