Phellinus linteus Grown on Germinated Brown Rice Inhibits IgE-Mediated Allergic Activity through the Suppression of FcεRI-Dependent Signaling Pathway In Vitro and In Vivo

Phellinus linteus (PL) has been used as a traditional herbal medicine owing to its immune regulatory activity. Previous studies reported that PL grown on germinated brown rice (PBR) exerted immunomodulatory, anticancer, and anti-inflammatory activities. However, role of PBR on type I hypersensitive reactions has not been studied yet. We found that PBR contained more polyphenolic compounds than PL extract. Among fractions, PBR butanol fraction (PBR-BuOH) significantly contained the most amounts of total polyphenolic contents compared with all extracts or fractions. In this study, anti-allergic activity of PBR-BuOH was examined using in vitro and in vivo models of immunoglobulin E/antigen- (IgE/Ag-) stimulated allergy. The inhibitory activity of degranulation was higher in PBR-BuOH (IC50 41.31 ± 0.14 μg/mL) than in PL-BuOH (IC50 108.07 ± 8.98 μg/mL). We observed that PBR-BuOH suppressed calcium influx and the level of TNF-α and IL-4 mRNA expression in a dose-dependent manner. The phosphorylation of Fyn, Gab2, PI3K, Syk, and IκB protein is reduced by PBR-BuOH. Oral administration of PBR-BuOH inhibited allergic reactions including the extravasation of Evans blue dye, ear swelling, and infiltration of immune cells in mice with passive cutaneous anaphylaxis (PCA). These findings suggest that PBR-BuOH might be used as a functional food, a health supplement, or a drug for preventing type I hypersensitive allergic disease.


Introduction
Type I hypersensitivity reaction is mediated by immunoglobulin E (IgE) antibodies binding to high-affinity IgE receptors (FcεRI) [1][2][3] and includes allergic rhinitis, atopic dermatitis, and asthma [4,5]. e prevalence rate of type I hypersensitivity is increased by triggers such as food, medications, insects, and unspecified causes over the last 10 years worldwide [6][7][8][9][10][11]. e current treatments for IgEmediated allergic diseases are merely confined to the avoidance of allergens, anti-histamine treatment, and corticosteroid therapy with less efficacy and more side effects. However, it is reported that immediate hypersensitivity reactions could be induced by the unidentified food allergens [11][12][13][14], corticosteroids [11,15], aspirin [16], progestogens [17], and antibiotics [18]. Current therapeutic agents for reducing the allergic reaction such as corticosteroids downregulated the expression of inflammatory mediators such as histamine and cytokines, but they can cause side effects that induce anaphylaxis involving urticaria, bronchospasm, angioedema, and cardiovascular collapse [11,19]. Many reports are interested in improving the quality of care for those who suffer from allergic diseases [11] and examining whether the various complementary and alternative medicine therapies such as acupuncture, diet therapy, herbal medicines, and physical techniques are used for regulating diverse allergic disorders [7].
Phellinus linteus (PL) has been traditionally used as a natural medicine in Asian countries for immune regulatory activities [20][21][22]. Wild PL cannot be obtained in large quantities because they are difficult to grow on mulberry tree and are expensive [23]. In this study, we used Phellinus linteus grown on germinated brown rice (PBR), which is inoculated and cultured the mycelium of PL on germinated brown rice, that is, different from regular PL. In the previous studies, PBR exhibited physiological functions such as antiinflammatory [24,25], anticancer [26,27], and anti-oxidant activities [23]. Unlike PL, PBR reduces IgE production through downregulating 2 responses and has the immunemodulating of the balance of 1 and 2 cytokines in murine mesenteric lymph node lymphocytes [28]. e reducing IgE production of 2 cell helps to modulate the hypersensitivity via suppressing IL-4 secretion and B cell activation in IgE-FcεRI-mediated allergic responses. In addition, previously they reported that ergosterol peroxide and atractylenolide III were found in PBR but not in wild PL [29,30]. e contents of β-glucan and c-aminobutyric acid in PBR was higher than that in regular PL and brown rice [25,31]. Ergosterol peroxide possesses immunosuppressive [32], anti-inflammatory [33], and antibacterial effects [34]. β-glucan and c-aminobutyric acid inhibited the allergic activity in both IgE-mediated mast cells and type I hypersensitivity animal models [35][36][37]. However, the effect of PBR and its underlying mechanisms against type I hypersensitivity reaction has not been studied yet. In this study, we investigated the inhibitory activity of PBR against IgE/Agstimulated RBL-2H3 cell activation and PCA reaction in mice.

Preparation of PBR Extract and Fractionation.
Mycelia of PL (voucher number Kucari 0904) produced by solid-state culture and PBR (voucher number Kucari 0905) were kindly provided by the Cell Activation Research Institute (Seoul, Republic of Korea) and deposited in the Herbarium at the College of Bioscience and Biotechnology, Konkuk University (Seoul) [25,26]. Each PBR or PL (1 kg) was powdered using a grinder and then was extracted with sterilized water (dry sample : water, 1 : 2) for 3 h at 95-100°C (Figure 1(a)). After filtration, the hot water extract was concentrated with a vacuum evaporator (Figure 1(a), yield of PBR and PL, 31.3 ± 1.1 and 24.9 ± 2.2%). Concentrated PBR was successively partitioned with hexane (yield 7.5 ± 0.9%), ethyl acetate (EA, 9.6 ± 0.5%), and water saturated n-butanol (BuOH, 9.9 ± 0.5%) (Figure 1(a)). PL also was successively partitioned with butanol using the same procedure as PBR. Each layer was concentrated by a rotary evaporator using vacuum (EYELA, Tokyo, Japan) at 40°C.

Determination of Total Polyphenol Contents (TPCs).
TPC in each extract was determined using a modified Folin-Ciocalteu method [38]. In each tube, 20 μL of each extract and fractions of PBR or PL (50 mg/mL) and gallic acid (0-500 μg/mL) was incubated with 20 μL of Folin-Ciocalteu reagent for 5 min.
en, 120 μL of 7% sodium carbonate (Na 2 CO 3 ) was added and incubated at room temperature (RT) for 30 min. Absorbance was measured at 720 nm using a UV-Visible spectrophotometer (Epoch, BioTek Instruments, VT, USA). TPC was expressed as mg of gallic acid equivalents (GAEs)/g of dry mass.

Cell
Culture. RBL-2H3 cells (rat basophilic leukemia cells, catalog number CRL-2256) were obtained from the American Type Culture Collection (ATCC) Biological Resource Center (Manassas, VA, USA). Cells were cultured in minimum essential medium (MEM; Invitrogen Co., Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 100 U/mL penicillin-streptomycin (Gibco) and were grown in a 75T cell culture flask at 37°C under humidified air containing 5% CO 2 [1]. en, 200 μL of 0.1 M Na 2 CO 3 /NaHCO 3 solution (pH 10.0) was added to stop the reaction. Degranulation was calculated with the measurement of the released β-hexosaminidase as previously described [30,40].

Reverse Transcription-Polymerase Chain Reaction (RT-PCR).
Reverse transcription PCR was performed as described previously [1,40]. Total RNA was isolated from RBL-2H3 cells, using TRIzol reagent, according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). PCR was performed according to the manufacturer's protocol (Qiagen, Hilden, Germany). PCR program for TNF-α was done as follows: initial denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 62.2°C for 10 s, and extension at 72°C for 45 s, with a final extension at 72°C for 5 min. PCR program for IL-4 was done as follows: initial denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 10 s, and extension at 72°C for 25 s, with a final extension at 72°C for 5 min. PCR program for GAPDH was done as follows: initial denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 62°C for 10 s, and extension at 72°C for 25 s, with a final extension at 72°C for 5 min [42]. PCR program for Fcε receptor was performed as follows: initial denaturation at 95°C for 15 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 49°C for 90 s (for FcεRI α-subunit) or annealing at 51.9°C for 90 s (for FcεRI β-subunit), and extension at 72°C for 90 s, with a final extension step at 72°C for 5 min. PCR program for IFN-c was done as follows: initial denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 57.3°C for 10 s, and extension at 72°C for 25 s, with a final extension at 72°C for 5 min.  Figure 1: e procedure for sample extraction and fractionation (a) and the measurement of total polyphenol content in Phellinus linteus grown on germinated brown rice (PBR) and Phellinus linteus (PL) (b) ( ### p < 0.001 vs. total hot water extract of PL and * * * p < 0.001 vs. total hot water extract of PBR). Data are shown as mean ± SD values (n � 3). GAEs, gallic acid equivalents.

Passive Cutaneous Anaphylaxis (PCA).
Six-week-old female BALB/c mice, which are housed in under specific pathogen free conditions, were obtained from Orient Bio (Orient Bio Inc., Gyeonggi-do, Seongnam, Republic of Korea). ey were maintained in cages at 22 ± 2°C and humidity 55 ± 5% and were exposed to 12 h light/12 h darkness cycles each day. Animals were randomly divided into four groups of six mice and were acclimated to laboratory conditions for 5-7 days. ey were fed with standard diet and allowed free access to drinking water. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Gachon University (Orient Bio Inc., Gyeonggi-do, Seongnam, Republic of Korea) (GIACUC-R2017014).
DNP-specific IgE (1 μg/mL) was injected into the ear of the mice. After 24 h, mice were intravenously injected with 1 mg/mL DNP-BSA (Ag) containing 1% Evans blue dye. To measure the activity of PBR-BuOH (25 mg/kg) or cetirizine (20 mg/kg, CZ), mice were orally administered the abovementioned dosages 1 h before DNP-BSA administration. e dye was extracted from each excised ear tissue with 700 μL formamide at 63°C overnight as previously described [1,30]. e absorbance was measured with a microplate spectrophotometer at 620 nm (Epoch, BioTek Instruments, VT, USA).

Histopathologic Assessment.
Ear samples were fixed using 10% formalin and were embedded in paraffin as previously described [1,43]. Paraffin-embedded sections were stained with hematoxylin and eosin (H&E); in general, the nuclei are stained blue-purple, whereas the cytoplasm and extracellular matrix are stained pink [1,44]. Images of stained samples were taken by Nikon eclipse Ti (Nikon Instruments Incorporated, Melville, NY) using CCD camera (Point Grey Research Inc., Richmond, BC, Canada).

Statistical Analysis.
Results are presented as means ± standard deviation (SD). Statistical differences between the control and the treatment groups were determined by Student's t-test and one-way ANOVA followed by Dunnett's t-test or Duncan's t-test (p < 0.05). e experimental data were analyzed using the Statistical Package for the Social Sciences-12 (SPSS Inc., Chicago, IL, USA).
ese data indicate PBR-BuOH might exert biological activity due to the presence of active molecules.

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In addition, PBR-hot water extracts significantly increased the level of IFN-c mRNA expression in a dose-dependent manner in IgE/Ag-induced RBL-2H3 cells (Figure 3).

Effect of PBR on Calcium Influx of IgE/Ag-Stimulated RBL-2H3 Cells.
IgE-mediated degranulation requires increased calcium mobilization [40,41]. To evaluate whether PBR-BuOH affects the calcium influx induced by IgE/antigen, RBL-2H3 cells were stained with calcium-sensitive dye Fluo-4. We observed that fluorescence intensity decreased in PBR-BuOH-treated RBL-2H3 cells, compared with that in IgE/Ag-stimulated RBL-2H3 cells (Figure 4(a)). e quantitative analysis revealed that the levels of intracellular calcium were decreased in PBR-BuOH-treated RBL-2H3 cells (Figure 4(b)). is result indicated that PBR-BuOH can inhibit IgE/Ag-stimulated degranulation by suppressing the intracellular calcium mobilization.

Effect of PBR-BuOH on FcεRI mRNA Expression and the Activation of FcεRI-Dependent Signaling Molecules in IgE/Ag-Stimulated RBL-2H3 Cells.
To evaluate the level of IgE/Aginduced FcεRI mRNA expression, we used by RT-PCR. PBR-BuOH (25 and 50 μg/mL) significantly suppressed the level of IgE/Ag-induced FcεRI α-subunit and FcεRI β-subunit mRNA expression in a dose-dependent manner ( Figure 5). ese results indicated that PBR-BuOH might reduce the rate of FcεRI α-subunit cross linking with IgE and inhibit the activation of FcεRI β-subunit.

Effect of PBR-BuOH on the Activation of FcεRI-Dependent and NF-κB Signaling Molecules in IgE/Ag-Stimulated RBL-2H3
Cells. Activation of FcεRI signaling events in mast cells results in the degranulation and the production of proinflammatory cytokines. e Src family kinase (e.g., Fyn and Syk) and downstream molecules (e.g., Gab2, PI3K, and ERK1/2) are involved in FcεRI signaling pathways and NF-κB signaling pathway. First, to elucidate the mechanism of its action, we investigated whether PBR-BuOH inhibited phosphorylation of Fyn, Gab2, and PI3K. PBR-BuOH decreased the expression of phosphorylated Fyn, Gab2, and PI3K in a dose-dependent manner compared with the IgE/Ag-stimulated control ( Figure 6(a)). Here, we determined whether PBR-BuOH could suppress the activation of Syk, ERK1/2, NF-κB, and IκBα. PBR-BuOH decreased the levels of p-Syk, p-ERK1/ 2, NF-κB, and p-IκBα (Figure 6(b)). ese results indicate that PBR-BuOH may exert its antiallergic activity through downregulation of the FcεRI-dependent and NF-κB signaling pathway in IgE/Ag-stimulated RBL-2H3 cells (Figure 7).

Inhibitory Effect of PBR-BuOH on IgE/Ag-Mediated Passive Cutaneous Anaphylaxis (PCA) in BALB/C Mice.
To measure the inhibitory effect of PBR-BuOH on IgE/Agmediated type I allergic response, we used an IgE-dependent PCA that is immediate hypersensitivity reaction in the dermis. After IgE sensitization (injected into the ear), DNP-BSA (Ag) containing 1% Evans blue dye was injected through the tail vein (Figure 8(a)). PBR-BuOH, administered orally, at a dose of 25 mg/kg suppressed the amount of extravasated Evans blue dye in the ear of mice with IgE/Agmediated PCA, compared with IgE/Ag-stimulated control mice (Figures 8(b) and 8(c)).  Evidence-Based Complementary and Alternative Medicine

Effect of PBR-BuOH on Histopathological Changes of Ear Tissues on IgE/Ag-Mediated PCA in BALB/C Mice.
To identify inflammatory cell infiltration and ear swelling, the tissue sections were stained with H&E. e ear thickness in PBR-BuOH (25 mg/kg/day) or CZ (20 mg/kg/day)-treated mice was decreased, compared with that in the mice with IgE/ Ag-mediated PCA (Figure 9(a)). e number of infiltrated immune cells was also decreased after the oral administration of PBR-BuOH (30.1 ± 16.0; p < 0.001) or CZ (30.3 ± 11.5; p < 0.05), compared with that in the IgE/Ag-stimulated control group (42.4 ± 14.9) (Figure 9(b)). ese results suggest that PBR-BuOH suppressed the infiltration of inflammatory cells and reduced dermal and epidermal thickening, compared with that in the IgE/Ag-stimulated control group.

Discussion
In the present study, we demonstrated that Phellinus linteus grown on germinated brown rice (PBR) alleviated IgE/Agstimulated hypersensitivity by suppressing FcεRI-mediated pathway along with improvements in antiallergic activity.
Recently, several studies reported that PBR showed enhanced biological activities, including immune regulation [28], anti-inflammatory [24,25], anticancer [26,27], and antioxidant activities, compared with PL [23]. e levels of ergosterol peroxide and atractylenolide III was only found in PBR, not in PL [25,29]. It is reported that ergosterol peroxide has anticancer activity against colon cancer cells [33] and atractylenolide III has antiallergic activity by inhibiting

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IgE/Ag-mediated hypersensitivity response [30]. Other groups reported that both PL and germinated brown rice exert anti-inflammatory and antiallergic activities [24,25]. In addition, PL or germinated brown rice contains the polyphenolic compounds such as protocatechuic acid and gallic acid, which inhibited the expression of cytokines (TNF-α and IL-4) exerted by IgE-mediated allergy-induced basophils [45][46][47][48]. In this study, we found that the total polyphenolic content (TPC) in hot water extract of PBR was significantly higher than that in hot water extract of PL. Particularly, PBR-BuOH contained the highest amount of TPC compared with the other fractions ( Figure 1). However, the antiallergic activity and its mechanism of PBR have not been reported yet.
us, in this study, we investigated whether PBR-BuOH showed the antiallergic activity and its mechanisms in IgE/Ag-mediated allergy responses.
To identify the antiallergic effect of PBR-BuOH, we observed the released inflammatory mediators of degranulation in IgE/Ag-stimulated RBL-2H3 cells. e granules of basophils and mast cells are degranulated by IgEmediated reactions, cross linking with FcεRI, and IgE/antigen (Ag)-complex [1,40,49]. Activated basophil initiates the degranulation of secretory granules including histamine, serotonin, and β-hexosaminidase [50]. Among them, β-hexosaminidase is one of the lysosomal enzymes, which may be fused with secretory lysosome and endosome in RBL-2H3 cells [51]. It is used as an indicator of degranulation [52][53][54] and has defense against bacterial infiltration by degrading the cell wall peptidoglycan [55]. In the present study, PBR-BuOH markedly reduced degranulation, suppressing the activity of released β-hexosaminidase in IgE/Ag-stimulated RBL-2H3 cells (Figure 2). PBR-BuOH also exhibits enhanced degranulation-suppressing activity compared with PL-BuOH ( Figure 2). As the immediate hypersensitivity continues, a large amount of cytokines (IL-3, IL-4, IL-5, IL-6, TNF-α, and GM-CSF) and chemokines (IL-8, and MIP1α) are secreted by IgE/Agstimulated basophils and mast cells within a few hours [50,51]. Some cytokines such as TNF-α and IL-4 can be used as fascinating therapeutic target molecules for IgE-mediated allergic responses [51,54,56] and proinflammatory 2 cell responses in allergic diseases [57]. TNF-α is found in secretory granules and induces endothelial activation, adhesion molecule expression, and inflammatory cell recruitment [52]. IL-4 acts upon naive T-cells, induces them to differentiate into allergen-specific 2 cells, and stimulates B-cell differentiation to produce IgE [50,[58][59][60]. Some studies conducted on allergic rhinitis and asthma showed that there is an association of IL-4 with significant increase in TNF-α [56]. Previous reports have shown that PBR inhibits the production of IgE by modulating the 1/ 2 balance, decreasing the concentration of IL-4, a 2 cytokine, in murine lymphocytes [28]. erefore, we examined whether PBR-BuOH suppressed IgE/Ag-induced gene expression of TNF-α and IL-4. We found that PBR-BuOH significantly reduced TNF-α and IL-4 mRNA expression (p < 0.05, Figure 3). In addition, we found that PBR-hot water extract increased the level of IFN-c mRNA expression in RBL-2H3 cells (Figure 3), which enhances 1-mediated responses and inhibits 2 differentiation and mast cell activation [61][62][63][64]. is result suggests that the antiallergic effect of PBR-BuOH is a result of its reduction of TNF-α and IL-4 expression in basophils.
Basophils and mast cells have FcεRI for IgE on their cell membranes [65,66], which is divided into the IgE-binding portion (α-subunit) and the signaling portion (β-and c-subunits). Some asthmatic patients have more FcεRIexpressed immune cells, compared with non-asthmatic control patients [67,68]. Increased numbers of FcεRIexpressed immune cells can rapidly respond to small amounts of IgE/antigen (Ag) complex and lead to the allergic reaction by releasing cytokines, chemokines, and other mediators, leading to activation of recruited other immune cells [69]. In brief, reducing the number of immune cells that can bind the IgE/Ag complex is targeted for treating hypersensitivity reaction. Some studies have also reported that FcεRI α-subunit-deficient mice do not induce degranulation because IgE cannot bind to the cell membrane receptor of basophils and mast cells [1,70]. Since IgE-binding FcεRI α-subunit activates the antigen-binding site of IgE molecule, induction of FcεRI α-subunit increases IgE-mediated allergy [59,[71][72][73]. Our data showed that PBR-BuOH decreased the expression of FcεRI α-subunit mRNA in IgE/antigenstimulated RBL-2H3 cells ( Figure 5). erefore, PBR-BuOH mitigates the allergic response by inhibiting the expression of FcεRI α-subunit, which it induces the degranulation of granules.
e IgE/Ag-mediated degranulation process can be divided into two phases: (1) the translocation of granules to cell membrane [74] and (2) the fusion between granules and cell membrane [53,74,75]. First, Fyn/Gab2/PI3K signaling pathway was activated by the phosphorylation of FcεRI [52,65,[76][77][78][79]. Fyn and Gab2 can regulate not only the formation of microtubule but also the translocation of granules to the cell membrane [52,74,78,80]. In this study, e expression of FcεRI subunit mRNA were determined by quantitative RT-PCR. Representative images from three independent experiments are shown. Data were analyzed by one-way ANOVA/Duncan's t-test (p < 0.05). Different letters indicate significant differences between groups. 8 Evidence-Based Complementary and Alternative Medicine we found that PBR-BuOH significantly decreased the expression of FcεRI β-subunit mRNA in IgE/Ag-stimulated RBL-2H3 cells ( Figure 5) and significantly inhibited the phosphorylation of Fyn, Gab2, and PI3K ( Figure 6(a)). Our observations suggest that PBR-BuOH might inhibit the translocation of granules for degranulation by negatively regulating FcεRI-mediated signaling pathway. Second, we postulate that PBR-BuOH would suppress the calcium-dependent exocytosis of cytoplasmic granules. e increased intracellular calcium constitutes calcium-calmodulin complex that leads to the exocytosis of granules by inducing the SNARE complex activation [53,74,75]. Consistent with previously reported studies, we found that stimulation of IgE/Ag increases the levels of intracellular calcium in RBL-2H3 cells (Figure 4) [40,41]. We observed that PBR-BuOH inhibited calcium influx assay compared with that in IgE/ Ag-stimulated RBL-2H3 cells (Figure 4). We have deduced the reason for blocking degranulation by PBR its suppressing activity on the activation of FcεRI-mediated signaling molecules and calcium influx. Additionally, NFκB signaling pathway induces the production of inflammatory cytokines in IgE-mediated allergic reaction [81][82][83]. Some studies demonstrated that phosphorylated Syk is known to induce NFκB and NFAT activation [84]. As shown Figure 6, PBR-BuOH reduced total NFκB protein expression and inhibited the phosphorylation of IκBα, Syk, and ERK1/2 MAP kinase protein in IgE/Ag-stimulated RBL-2H3 cells ( Figure 6(b)). ese data suggest that PBR-BuOH possesses antiallergic activity by suppressing the level of phosphorylated Syk, ERK1/2 kinase protein, and IκBα protein.
PCA is a representative local type I allergy that is induced in mice by injecting IgE into the ears and intravenously injecting antigen into the tails [30,85,86]. e antigen-antibody interactions increase the production of vasoactive substances such as histamine in immune cells of subcutaneous tissue [87] and induce the permeability and extravasation of the Evans blue dye injected into the circulation along with the antigen [88]. We observed that PBR-BuOH decreased the amount of extravasated Evans blue dye in the ear of mice, compared with that in the IgE/Ag-mediated allergic group (Figure 8). IgE-mediated PCA induces the expression of endothelial selectins and trafficking molecules that can bind with immune cells [89], and then the infiltration of immune cells occurs in the ear tissues [90][91][92][93][94]. e results showed that PBR-BuOH significantly inhibits IgE-mediated PCA by reducing the number of infiltrating inflammatory cells and the ear swelling ( Figure 9). In this study, these findings implied that PBR-BuOH decreased the vascular expansibility and the activated immune cells caused by the degranulation and released cytokines of IgE/Ag-stimulated basophils and mast cells in dermis, which were in accordance with our in vitro results. In addition, Hong et al. conducted a clinical trial in which PBR powder was orally administered for 12 weeks to an atopic dermatitis patient at an average age of   Evidence-Based Complementary and Alternative Medicine 7.3 years [95]. e symptomatic effect of atopic dermatitis, a type of hypersensitive reaction, was verified based on previous research that has significantly decreased the itching and objective SCORAD indices of the patient [95].

Conclusion
Our study demonstrated that PBR-BuOH has more potent antiallergy activity than PL-BuOH in IgE/Ag-mediated allergic response through suppressing degranulation and decreasing the level of TNF-α and IL-4 mRNA expression. PBR inhibited the phosphorylation of Fyn, Gab2, and PI3K in FcεRI-mediated signaling pathway and calcium mobilization. In addition, PBR-BuOH suppressed IgE-mediated type I hypersensitivity in the PCA murine model. Our findings suggest that PBR could be developed as functional food, health supplement, and drug for treating or preventing type I allergic diseases.

Data Availability
Phellinus linteus grown on germinated brown rice (PBR, Kucari 0905, Patent: 1280949) and the mycelium of Phellinus linteus (PL, Kucari 0904, PDK4708) used to support the findings of this study have been deposited in the Cell Activation Research Institute Co., Ltd. e data used to support the findings of this study are currently under embargo while the research findings are commercialized. Requests for data, 6 months after publication of this article, will be considered by the corresponding author.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.