Elm Tree (Ulmus parvifolia) Bark Bioprocessed with Mycelia of

Article pubs.acs.org/JAFC

Elm Tree (Ulmus parvifolia) Bark Bioprocessed with Mycelia of Shiitake (Lentinus edodes) Mushrooms in Liquid Culture: Composition and Mechanism of Protection against Allergic Asthma in Mice Sung Phil Kim,† Sang Jong Lee,‡ Seok Hyun Nam,*,† and Mendel Friedman*,§ †

Department of Biological Science, Ajou University, Suwon 443-749, Republic of Korea STR Biotech Company, Ltd., Chuncheon 200-160, Republic of Korea § Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710, United States ‡

ABSTRACT: Mushrooms can break down complex plant materials into smaller, more digestible and bioactive compounds. The present study investigated the antiasthma effect of an Ulmus parvifolia bark extract bioprocessed in Lentinus edodes liquid mycelium culture (BPUBE) against allergic asthma in chicken egg ovalbumin (OVA)-sensitized/challenged mice. BPUBE suppressed total IgE release from U266B1 cells in a dose-dependent manner without cytotoxicity. Inhibitory activity of BPUBE against OVA-specific IgE secretion in bronchoalveolar lavage fluid (BALF) was observed in OVA-sensitized/challenged asthmatic mice. BPUBE also inhibited OVA-specific IgG and IgG1 secretion into serum from the allergic mice, suggesting the restoration of a Th2-biased immune reaction to a Th1/Th2-balanced status, as indicated by the Th1/Th2 as well as regulatory T cell (Treg) cytokine profile changes caused by BPUBE in serum or BALF. Inflammatory cell counts in BALF and lung histology showed that leukocytosis and eosinophilia induced by OVA-sensitization/challenge were inhibited by the oral administration of BPUBE. Amelioration of eosinophil infiltration near the trachea was associated with reduced eotaxin and vascular cell adhesion molecule-1 (VCAM-1) levels. Changes in proinflammatory mediator levels in BALF suggest that BPUBE decreased OVA-sensitizationinduced elevation of leukotriene C4 (LTC4) and prostaglandin D2 (PGD2). The finding that asthma-associated biomarker levels of OVA-sensitized/challenged mice were much more inhibited with BPUBE treatment than NPUBE (not-bioprocessed Ulmus parvifolia extract) treatment suggested the production of new bioactive compounds by the mushroom mycelia that may be involved in enhancing the observed antiasthmatic properties. The possible relation of the composition determined by proximate analysis and GC/MS to observed bioactivity is discussed. The results suggest that the elm tree (Ulmus parvifolia) bark bioprocessed with mycelia of shiitake (Lentinus edodes) mushrooms has the potential to prevent and/or treat allergic asthma. KEYWORDS: bioprocessing, bioactive, biofunctional, proximate analysis, GC/MS, elm tree bark, Ulmus parvifolia, shiitake mushroom mycelia, Lentinus edodes, mice feeding study, allergic asthma prevention, functional food

■

INTRODUCTION Asthma is a human inflammatory disease of lung airways caused by triggering stimuli (dust, food, pollen) that results in partially or completely reversible constriction of the bronchi. Treatment involves controlling triggering factors and drug therapy. It is well-known that such susceptibility is attributed to immune factors including T-helper (Th2) cells and their cytokines (IL4, -5, and -13). Genetic and environmental components may interact in the determination of T-helper (Th1) and Th2 immune responses. The Th2 and other cell types, including eosinophils, mast cells, and neutrophils, form an inflammatory infiltrate in the lung airways leading to adverse symptoms.1 Regulatory T cells (Treg) and their cytokine (IL-10) seem to play a key role in the causes and prevention of allergic asthma.2−4 Previously, we have investigated the extracts of the mushroom studied here, Lentinus edodes, and also the Lion’s Mane mushroom, Hericium erinaceus, to determine their bioactivities. Cell and mouse studies suggest that mushroom extracts may have the potential to modulate the immune system.5−8 The activity of a bioprocessed polysaccharide isolated from Lentinus edodes mushroom mycelium culture © XXXX American Chemical Society

supplemented with black rice bran protected mice against salmonellosis through the activation of macrophage-mediated immune response resulting from augmented Th1 immunity.9 These results indicate that the mushroom extracts can have an impact on the immune system. Some other studies describe several other health-promoting properties of Lentinus edodes mushroom mycelia.10−16 There have been reports of bioactivity from the same and different elm tree species, which may also be relevant to the present study. The bark, which contains phenolic compounds and steroidal glucosides, has been used for the treatment of eczema, edema, gonorrhea, and scabies.17 Water extracts of the root bark of the same species, Ulmus parvifolia, decreased the size of carbuncles induced by Staphylococcus aureus pathogenic bacteria and exhibited analgesic and anti-inflammatory properties in mice by stimulation of the immune system.18 Extracts also inhibited lipid peroxidation in the linoleic acid system19 Received: October 13, 2015 Revised: January 13, 2016 Accepted: January 14, 2016

A

DOI: 10.1021/acs.jafc.5b04972 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

and treated with amylase (40 U) and cellulase (100 U) at 60 °C for 60 min for enzymatic digestion of carbohydrate-based particulate materials. This preparation was then adjusted to pH 7.0, followed by sterilization in an autoclave. The U. parvifolia bark preparation was added to a 5 L fermenter (working volume, 3 L), inoculated with the seed culture, and cultured at 28 °C and 150 rpm for 5 days. This main culture was treated with an enzyme mixture containing glucanase (3,300 U), cellulose (6,000 U), hemicellulose (3,000 U), and pectinase (3,000 U) at 60 °C for 60 min for cell wall lysis. The enzyme-treated culture mass was then heated at 90 °C for 1 h for extraction, and the resultant aqueous phase was recovered and freeze-dried to a solid material. The resultant material was termed BPUBE (bioprocessed Ulmus parvifolia bark extract). U. parvifolia bark extract not subjected to bioprocessing by mycelia was also prepared under the same enzymatic hydrolysis, extraction (90 °C/1 h), and freeze-drying conditions, and termed NPUBE (not-bioprocessed Ulmus parvifolia bark extract). Component Analysis by Standard Methods and by GC/MS. BPUBE and NPUBE were first analyzed for carbohydrate, protein, lipid, and sodium content according to the officially published method.29 For GC/MS analysis, the two extracts were derivatized in two steps to protect carbonyl function following the method of Kim et al.30 Dried samples were dissolved in methoxyamine hydrochloride (100 μL; 20 mg/mL) in pyridine, and reacted at 60 °C for 1 h. The acidic protons were exchanged against the trimethylsilyl group to increase the volatilities of the polar compounds using 100 μL of N-methyl-Ntrimethylsilyltrifluoroacetamide (MSTFA) at 70 °C for 1 h. Each extract was analyzed by GC/MS using a gas chromatograph, model 6890GC (Agilent Technologies, Santa Clara, CA), equipped with a mass spectrometer detector 5975 and DB-1 column (Agilent Technologies, stationary phase; polyethylene glycol, 30 m × 0.25 mm; i.d. 0.25 μm). The temperature was programmed at 70 °C (4 min) with an increase of 10 °C/min until 300 °C (6 min) was reached. Helium gas was used as the carrier with a flow rate of 1 mL/min. Both injector and detector temperatures were set at 250 °C. The injection was a split ratio of 25:1 in all cases. The injection volume was 1 L. Mass spectra were recorded in electron ionization mode with ionization energy of 70 eV. Components were identified by retention time in the mass spectra and by comparing the mass spectra with those in a commercial library.31 Cell Culture. The U266B1 human multiple myeloma B lymphocyte cells from American Type Tissue Culture Collection (ATCC, Manassas, VA, USA) were cultured in a modified RPMI1640 medium containing 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and 15% heatinactivated FBS. Penicillin (100 U/mL) and streptomycin (100 mg/ mL) were also added to the medium. Cells were cultured at 37 °C in a humidified atmosphere with 5% CO2. The medium was replaced every 3 days until the cell reached maximal cell density. Measurement of Total IgE Secretion and Cell Viability. For assessment of changes in IgE production levels, U266B1 cells were stimulated with 10 μg/mL lipopolysaccharide (LPS), 5 ng/mL human IL-4 (Sigma-Aldrich), and either BPUBE or NPUBE (1, 10, and 100g/ mL) for 72 h. The culture supernatants were recovered for IgE assay by an assay kit (Biorbyt, San Francisco, CA, USA) according to the manufacturer’s instructions. Absorbance of the final reaction mixture was read at 450 nm using a microplate reader (model 550, Bio-Rad, Hercules, CA, USA). Cell Viability Assay. MTT staining reported by Mosmann was employed to evaluate the cytotoxic effects of BPUBE and NPUBE.32 Briefly, the U266B1 cells were seeded into 96-well plate at a density of (1 × 104) cells/well, and then the cells were treated with appropriate concentration of BPUBE and NPUBE (1, 10, and 100 μg/mL each) for 48 h at 37 °C humidified air containing 5% CO2. The controls, vehicle and LPS/IL-4-stimulated, were also tested. After treatments, the cells were stained by adding MTT. The resultant intracellular chromogen formazan products were solubilized with DMSO. Absorbance of the chromogen was read in a microplate reader (BioRad) at 570 nm and a reference wavelength of 655 nm.

and exhibited low cytotoxicity in a brine shrimp assay.20 In a different elm species, Ulmus davidiana, bark extracts have been shown to enhance the immunocompetent properties of mice (splenocyte proliferation and cytokine production) by activated macrophages,21 and they exhibited free radical scavenging properties.22 There have been several reported studies on the prevention of asthma in rodents by various plant materials and their bioactive compounds. These include (a) Sideritis scardica, an endemic herbal species of the Balkan peninsula;23 (b) frankincense, the resinous extract from the trees of the genus Boswellia, and bioactive boswellic acids;24 (c) rosemary (Rosmarinus off icinalis), a common household plant grown in many parts of the world, and its bioactive constituents caffeic and rosmarinic acids;25 and (d) antioxidative flavonoids, reviewed by Castell et al.26 These observations suggest that the antioxidative properties of the plant materials and their bioactive constituents might largely govern anti-inflammatory and antiallergic effects. Mushrooms obtain their sustenance from decomposing plants. They secrete enzymes into their host that break down complex carbohydrates and lignins into simpler compounds. New, bioactive compounds can therefore be produced from the elm bark substrate exposed to mushroom mycelia during bioprocessing. Because we previously found that bioprocessed Lentinus edodes mycelia and black rice bran protected mice against infection by Salmonella via upregulation of the Th1 immune reaction,9 it was of interest to find out whether bioprocessing of elm bark in a liquid mycelium culture produced bioactive compounds that would protect mice against allergic asthma via a similar mechanism. The main objective of this study was therefore to define the mechanism of protection against allergic asthma in mice by the bark of the elm tree Ulmus parvifolia bioprocessed with Lentinus edodes mushroom mycelia in liquid culture in terms of Th1/Th2 cells and other biomarkers associated with the immune system in relation to allergic manifestations. As part of this effort we also determined the proximate composition of the test materials by standard methods and their components by GC/MS.

■

MATERIALS AND METHODS

Materials. Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), fetal bovine serum (FBS), bovine calf serum (BCS), and other miscellaneous cell culture reagents were purchased from Hyclone Laboratories (Logan, UT, USA). Potato dextrose agar medium (PDA) was from Difco Laboratory (Detroit, MI, USA). Chicken egg ovalbumin (OVA, grade V), human IL-4, aluminum hydroxide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and analytical grade reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA). U. parvifolia bark, air-dried and minced, was purchased at the Kyung-Dong traditional pharmaceutical market in Seoul, Korea. Preparation of Bioprocessed Ulmus parvifolia Bark Extract. L. edodes fungal mycelia were isolated from the fruitbody and were cultured on PDA. The genetic identity of the fungus was confirmed by the Korean Center of Microorganisms (Seoul, Korea). The mycelia grown on PDA were inoculated with inoculating loop into a 250 mL Erlenmeyer flask containing 50 mL of the liquid medium (2% glucose, 0.5% yeast extract, 0.5% soy peptone, 0.2% KH2PO4, 0.05% MgSO4, and 0.002% FeSO4, w/v), and was cultured at 28 °C for 5 days in a rotary shaker (120 rpm). This culture was used to seed the main culture for bioprocessing. Bioprocessing was carried out according to the method previously reported.27,28 Minced U. parvifolia bark was added to DMEM (30 g/L) B


Article

Journal of Agricultural and Food Chemistry Mice. Pathogen-free female Balb/c mice, 6−8 weeks old, were purchased from Orient Bio Inc. (Seoul, Republic of Korea). The mice were housed in a stainless steel cage under a 12 h light/dark cycle with a temperature range of 20−22 °C and relative humidity of 50 ± 10%. Mice were fed the pelletized normal commercial chow diet (Cat. No. 5L79, Orient Bio.) and tap water ad libitum for 1 week after arrival for acclimation. Antigen Sensitization, Challenge, and Treatment. The protocol for sensitization and inhalational challenge was carried out according to the method of Temelkovski et al. with slight modification.33 Briefly, acclimatized Balb/c mice were arbitrarily divided into the following four groups (n = 10), avoiding any intergroup difference in body weight: vehicle, OVA-, NPUBE-, and BPUBE-treated groups. Mice were intraperitoneally (ip) sensitized with 20 μg of OVA emulsified with 0.2 mL of 1% aluminum hydroxide (w/v) in phosphate-buffered saline (PBS, pH 7.4). Injections were performed three times on days 1, 8, and 15. The sensitized mice were subjected to OVA challenge by placing each mouse individually in a Plexiglas box (29 × 22 × 18 cm). Challenge was continued with repeated exposure to an aerosol of 1% OVA using an ultrasonic nebulizer (NE-U12, Omron Co., Kyoto, Japan). Challenge was carried out for 30 min once a day for 5 consecutive days (day 25 to 29). Vehicle-treated group was subjected to ip injection of 0.2 mL of PBS plus 0.15 mL of aluminum hydroxide gel, and then challenged with PBS. For NPUBE and BPUBE groups, mice sensitized/challenged with OVA as described were orally administered with either of the extracts (10 mg/kg each) for 14 consecutive days (day 16 to 29). PBS was used as vehicle, and both extracts were administered once a day. All mice were sacrificed by CO2 inhalation 24 h after the last (day 30) to assess the suppressive effects of BPUBE and NPUBE. The described experimental design is schematically shown in Figure 1. The protocol for the mouse studies was approved by the Ethics Committee for Animal Care and Use, Ajou University, Republic of Korea.

96-well microplate precoated with rat anti-mouse IgE monoclonal antibody, and incubated at room temperature for 1 h. After thorough washing with wash buffer, horseradish peroxidase (HRP)-conjugated ovalbumin was added to the wells. Then, incubation at room temperature was continued for 1 h. After final washing, the substrate, tetramethylbenzidine (TMB), was added and the wells were incubated for another 30 min in the dark. The absorbance of each well at 450 nm (a reference wavelength of 655 nm) was measured using a microplate reader (Bio-Rad). OVA-specific IgE concentration was calculated based on the results from serial dilution of standard mouse IgE included in the ELISA kit. Measurement of Cytokine Levels in Serum or BALF. IL-2, IL10, and IL-12 levels in serum, together with IL-4, IL-5, and IL-13 levels in BALF, were determined using an ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions. Absorbance of the final reaction mixture was read in a microplate reader (Bio-Rad) at 450 nm and a reference wavelength of 540 nm. Standard curves for each target substance were created from the averages of the absorbance at the same wavelengths and triplicate readings of the standards in the assay kits. Measurements of OVA-Specific IgG Subclass Levels in Serum. IgG in mouse serum was determined using an ELISA kit (MyBioSource, San Diego, CA, USA) according to the manufacturer’s protocol, and IgG1 and IgG2a levels were measured as follows. Briefly, 96-well plates were precoated with 100 μL of OVA solution (5 μg/mL in PBS) overnight at 4 °C. After washing with PBS, the plates were blocked with 150 μL of 1% bovine serum albumin (BSA) for 30 min at room temperature. Then, the serum samples (50-fold dilution for IgG2 and 5,000-fold dilutions for IgG1) were added to the wells and incubated for 2 h at room temperature. HRP-conjugated rat antimouse IgG1 and IgG2a (Life Technologies, Carlsbad, CA, USA) was added, and the samples were incubated for 2 h at room temperature. The plates were washed, and 100 μL of TMB substrate was added to each well. After incubation for 30 min in the dark, the absorbance was read at 450 nm using a microplate reader (Bio-Rad). Measurement of Eotaxin, VCAM-1, LTC4, and PGD2 Levels in BALF. ELISA analysis was employed to measure eotaxin and VCAM-1 levels following the manufacturer’s instructions (Biorbyt, San Francisco, CA, USA). The absorbance of the final reaction mixture was read in a microplate reader (Bio-Rad) at 450 nm. LTC4 and PGD2 levels in BALF were also determined using the ELISA kit (MyBioSource, San Diego, CA, USA). For both assays, absorbance of the final reaction mixtures at 420 nm was measured using a microplate reader (Bio-Rad). The standard curves for each target substance were created from the averages of triplicate reading (absorbance at 450 nm) of the standards in the assay kits. Histological Analysis of the Lung. The exsanguinated left lung was removed from the chest cavity and fixed with 4% paraformaldehyde in phosphate buffer (0.5 M, pH 7.4). Lobes were isolated, dehydrated with ethanol, and embedded in paraffin. The tissues were then cut to a thickness of 4 μm and mounted onto glass slides. The sections were dewaxed using xylene and ethanol, stained with hematoxylin and eosin (H&E), and examined by light microscopy (Olympus) to observe inflammatory cell infiltration. RNA Isolation and Semi-Quantitative RT-PCR. Total cellular RNA was prepared from homogenized lung tissue according to the acid phenol guanidinium thiocyanate−chloroform extraction method.34 For reverse transcription, total RNA (1 μg) was incubated with AMV reverse transcriptase (5 U) and oligo (dT18) as primer. DNA amplification was then primed in a reaction mixture containing dNTP mix (400 μM each), Taq polymerase (2.5 U), and primer sets (20 μM each) representing the target genes as follows: cyclooxygenase-2 (COX-2) sense primer, 5′-TCTCAACCTCTCCTACTAC-3′; COX-2 antisense primer, 5′-GCACGTAGTCTTCGATCACT-3′; inducible nitric oxide synthase (iNOS) sense primer, 5′-ATGTCCGAAGCAAACATCAC-3′; iNOS antisense primer, 5′-TAATGTCCAGGAAGTAGGTG-3′; β-actin sense primer, 5′-GTGGGGCGCCCCAGGCACCA-3′; β-actin antisense primer, 5′-GTCCTTAATGTCACGCACGATTTC-3′. PCR was conducted using a thermocycler (model PTC-200, MJ Research, Reno, NV, USA) with one cycle for 5

Figure 1. Schematic diagram of the experimental protocol. Mice were sensitized on days 1, 8, and 15 by ip injection of 20 μg of ovalbumin (OVA) emulsified with 0.2 mL of 1% aluminum hydroxide (w/v). After sensitization/challenge, the mice were exposed to aerosolized 1% OVA from day 25 to 29 using an ultrasonic nebulizer for 30 min/day. BPUBE/NPUBE were orally administered from day 16 to 29. Collection of Bronchoalveolar Fluid (BALF) and Blood. For collection of BALF, tracheotomy was performed as follows. The tracheas exposed by cannulating upper tracheas and BALF were carefully collected by twice lavaging with 1 mL of ice-cold PBS containing 0.05 mM EDTA. Collected lavage fluids were then centrifuged at 400g for 5 min at 4 °C. The recovered supernatants were set aside and kept at −70 °C until further analysis. Cell pellets were resuspended in ice-cold PBS, followed by slide preparation by centrifuge using Cytospin (Hanil Science, Incheon, Republic of Korea) and staining with Wright− Giemsa stain. The slides were microscopically observed (magnification, ×40) for differential cell counts by counting a total 300 cells per slide under light microscopy (model D50, Olympus, Tokyo, Japan). The total cell number in BALF was also measured by microscopic cell counting using a hemocytometer. Blood was collected by cardiac puncture from the sacrificed mice. Measurement of OVA-Specific IgE Level in BALF. The OVAspecific IgE release into BALF was measured with a mouse ovalbumin specific IgE ELISA assay kit (Bio-Rad) according to the manufacturer’s instruction. Briefly, appropriately diluted BALF was transferred to the C


Article

Journal of Agricultural and Food Chemistry min at 94 °C, followed by 30 cycles for 30 s at 94 °C, 45 s at 72 °C, and one cycle for 5 min at 72 °C. Amplified PCR products were subjected to 1.5% agarose gel electrophoresis and visualized with an UV illuminator. The intensity of the separated bands of DNA was quantified using a gel documentation system (model LAS-100CH, Fuji Photo Film Co., Tokyo, Japan). Western Blot Analysis of Lung Tissues. Lung tissue was extracted with RIPA (radioimmunoprecipitation assay) buffer (50 mM Tris Cl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA, pH 7.4) for preparation of whole cell proteins. Protein concentrations were determined according to the Bradford method using a Bio-Rad Protein kit. Bovine serum albumin (BSA) was used as standard. The cell extract containing proteins (30 μg) were separated on 12% SDS−polyacrylamide gels and electrophoretically transferred onto a nitrocellulose membrane (Millipore, Billerica, MA, USA). The membrane was blocked in 5% skim milk at 4 °C overnight and probed with the primary antibodies as follows; anti-mouse cyclooxygenae-2 (COX-2) goat polyclonal antibody (Santa Cruz, Dallas, TX, USA), anti-mouse inducible nitric oxide synthase (iNOS) rabbit polyclonal antibody (Cell Signaling Tech., Danvers, MA, USA), and anti-mouse β-actin monoclonal antibody (Millipore, Billerica, MA, USA). After the primary antibody reaction for at least 3 h, the secondary antibody reaction with HRP-conjugated anti-IgG antibody was performed under the same conditions. Blots were developed using the ECL detection kit (Pierce, Rockford, IL, USA). The intensity of separated protein bands was quantified using a gel documentation system (model LAS-1000CH, Fuji Photo Film Co., Tokyo, Japan). At least three separate replicates were determined for each experiment. Statistical Analysis. Results are expressed as the mean ± SD of three independent experiments. Significant differences between means were determined by the ANOVA test using the Statistical Analysis Software package SAS (Cary, NC, USA). p < 0.05 is regarded as significant.

mycelia. Table 3 also demonstrates that both BPUBE and NPUBE were not cytotoxic to the U266B1 cells at the examined three doses. The results suggest the potential of BPUBE to suppress airway inflammation in vivo. Next, we examined whether such inhibitory capacity on IgE production was also active in vivo using BALF from an OVAsensitized/challenged asthmatic mouse model. As depicted in Figure 2, oral administration of BPUBE (10 mg/kg body weight) markedly suppressed OVA-specific IgE secretion into BALF by about 83%, whereas administration of NPUBE showed much lower inhibition of IgE secretion at the same dose (about 44% inhibition). Effects of BPUBE on OVA-Specific IgG, IgG1, and IgG2a Levels in Serum. We also examined whether BPUBE modulated additional immunoglobulins involved in allergic responses, including IgG, IgG1, and IgG2a. As shown in Figure 3, OVA-sensitization/challenge induced marked upregulation of OVA-specific IgG levels (about 105-, 38-, and 4-fold increases in IgG, IgG1, and IgG2a levels, respectively). By contrast, mice treated with BPUBE showed a strong reduction of total IgG and IgG1 serum levels (about 75% and 82% reduction, respectively). Serum IgG2a level was also measured in OVA-sensitized/challenged mice administered with BPUBE. The result showed a reduction of IgG2a in response to the BPUBE treatment; however, the extent was much lower than that of IgG and IgG1 (about 33% reductions). The observed drastic reduction of IgG and IgG1 and the moderate reduction of IgG2a suggest that BPUBE modulates the Th1/Th2 balance by suppressing Th2 immune response elevated by the OVAsensitization/challenge. Effects of BPUBE on Th1, Th2, and Treg Cytokine Production. To examine whether BPUBE can regulate the differentiation pathway of CD4 T cells for a balanced Th1/Th2 and Treg immune reaction, changes in Th1, Th2, and Treg cytokine production profiles were determined by ELISA assay in BALF or serum. As shown in Table 4, oral administration of BPUBE markedly suppressed the production of Th2 cytokines including IL-4, IL-5, and IL-13 when assessed against BALF (about 74, 72, and 73% inhibition for IL-4, IL-5, and IL-13 production, respectively). Compared to BPUBE, NPUBE showed much lower inhibitions. Next, we examined whether inhibition of Th2 cytokine production adversely affects the restoration of suppressed Th1 reaction to normal status in serum. Table 4 also shows that oral administration of BPUBE upregulated the OVA-induced downregulated Th1 cytokines including IL-2 and IL-12 to near normal levels observed in the vehicle-treated control mice (about 85% level of the vehicletreated control). In addition, BPUBE also exerted upregulation of OVA-induced downregulated IL-10 production (about 87% level of the vehicle-treated control), the cytokine secreted mainly from Treg cells and involved in suppressive functions in inflammatory responses including asthmatic diseases. This finding showed a possibility that BPUBE has capacity of suppressing allergic asthma though activation of Treg. Effects of BPUBE on Inflammatory Leucocyte Influx into BALF. To evaluate the effects of BPUBE on the recruitment of immune cells to the airway, we counted total leukocyte cell numbers and the number of lymphocytes, neutrophils, macrophages, and eosinophils in BALF. The total number of infiltrating cells was approximately 3.6-fold higher in the BALF than in vehicle-treated normal control group (Figure 4). Such infiltration of immune cells was suppressed to a great extent by BPUBE administration (about 62% inhibition) and to

■

RESULTS Composition of BPUBE and NPUBE. Crude lipid and sodium compositions markedly changed during bioprocessing (Table 1). GC/MS analysis revealed that the two extracts Table 1. Proximate Composition of NPUBE and BPUBE % dry wt NPUBE BPUBE

carbohydrate

crude protein

crude lipid

sodium (mg/100 g)

72.5 75.8

6.7 8.6

1.7 0.0

12.64 51.18

contained 116 characterized compounds (Table 2). Some components remained unidentified, and others were tentatively identified. Among the identified compounds, 59 compounds were found only in BPUBE and 56 structurally different compounds in NPUBE. Because the chemical nature of the identified compounds varied widely, it is difficult to determine which compound or combination of compounds might be responsible for the observed bioactivity. Effects of BPUBE on Total and OVA-Specific IgE Production. To examine whether BPUBE has capacity to suppress asthma in vivo, the inhibitory effects of BPUBE on the secretion of IgE, an allergic immunoglobulin, were first determined in vitro in human multiple myeloma U266B1 cells. Compared to the LPS and IL-4-treated control, 1, 10, and 100 g/mL BPUBE treatments suppressed specific IgE secretion levels in cell supernatants by about 30, 55, and 61%, respectively (Table 3). By contrast, NPUBE was found to have lower inhibitory activities at the same concentrations (6, 17, and 22% inhibitions, respectively), clearly showing the elevation of bioactivity through bioprocessing with mushroom D


Article

Journal of Agricultural and Food Chemistry Table 2. Compounds Identified by GC/MS Analysis of TMS-Derivatized Elm Bark Extracts

peak area (%) peak

tR (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

8.537 8.537 8.741 8.964 8.983 10.014 10.019 10.102 10.104 10.189 11.405 11.412 11.425 11.439 11.487 11.502 12.691 13.894 13.959 13.988 15.021 15.035 15.359 15.369 15.394 15.445 15.537 15.566 19.507 19.518 19.537 19.538 19.563 20.651 20.675 21.090 21.106 21.574 21.596 22.029 22.043 22.060 22.106 22.209 22.239 24.448 24.462 24.832 25.624 25.925 25.944 25.974 25.979 26.016 26.059 26.072 26.084 26.389 26.479

compda decane, 3,6-dimethyldodecane 2-undecen-4-ol propanoic acid isophthalic acid, 2-fluorophenyl pentyl ester undecane, 5-methylheptane, 2,4-dimethylquinoline, 2-phenyl1-(3-methylbutyl)-2,3,4,6-tetramethylbenzene succinic acid, diamide, N,N′-diethyl-N,N′-diphenylnoscapine thioquinox 1,2,3,4-tetrahydroisoquinoline, 6,7,8-trimethoxy-1,2-dimethyl1H-indene, 1,1,4,5,6,7-hexafluoro2-pentamethyldisilanyloxybutane 1-(anilinomethylene)-2-indanone dibenzo[c,f ]1,7-naphthyridin-9(7H)-one, 8,10,11,12-tetrahydro-8-(3-cyclohexenyl)isophthalic acid, 2,6-dichlorophenyl propyl ester phenol, 2-amino-4,6-bis(1,1-dimethylethyl)oxo(6-phenyl-imidazo[1,2-a]pyridin-2-yl)acetic acid, ethyl ester cadaverine 2-(phenylpiperidin-1-yl-methyl)cyclohexanol sulfurous acid, 2-ethylhexyl octadecyl ester hexadecane glycerol cobalt, acetylacetonato(pentamethylcyclopentadienyl)4-methoxyphenyl 2-(4-methoxyphenyl)-2-propane naphtho[2,3-b]furan-9(4H)-one, 4-(acetyloxy)-4a,5,6,7-tetrahydro-3,4a,5-trimethyl-, (4s,-4ar,5s)2H-1,3-thiazine-6-carboxylic acid, 3-aminotetrahydro-2-(methylimino)-4-oxo-, methyl ester 9,10-anthracenedicarbonitrile 1-(1,1-bis(ethoxycarbonyl)ethyl)-7-methoxynaphthalene 1,3,5-triazine-2,4-diamine, 6-(4-tert-butylphenyl)fumaric monoamide, N-(2-bromophenyl)-, butyl ester ethanamine, N-chloro-N,1,1-trifluorododecane, 4,6-dimethylsulfurous acid, hexyl pentyl ester threitol 5-bromothiophene-2-carboxamide butanoic acid N,N-diethyl-N′-[4-[[4-[trifluoromethyl]phenyl]amino]-2-quinazolinyl]-ethane-1,2-diamine 3-methoxybenzylamine, N,N-diundecyllysine, 4-hydroxy2-oxo-6-phenyl-4-(4-hydroxyphenyl)-1,2-dihydropyrimidine 3-ethyl-3-methylheptane ursane-3,16-diol, (3b,16b,18a,19a,20b)butanal uracil phthalic acid, di(3-methylphenyl) ester ethylamine, N-heptyl-N-octyl-2-(2-thiophenyl)5,6-dimethyl-4-phenyl-3-cyanopyridine-2(1H)-thione heptadecane 1-iodo-2-methylundecane phenylephrine, N,O,O-tris(heptafluorobutyryl) deriv phthalic acid, di(3-methylphenyl) ester ribitol propane(dithioic) acid, ethyl ester D-fructose 1,3-propanedione, 1-(1,3-benzodioxol-5-yl)-3-(4-methoxy-5-benzofuranyl)-

E

NPUBE

BPUBE

0.109 0.099 0.001 0.427 0.007 0.017 0.019 0.004 0.048 0.003 0.022 0.002 0.001 0.002 0.036 0.005 0.003 0.006 0.002 0.001 0.007 0.006 0.026 0.089 0.014

3.670 0.002

0.006 0.001 0.008 0.002 0.001 0.001 0.001 0.001 0.008 0.067

0.065

0.012 0.006 0.006 0.078 0.002 0.001 0.001 0.003 0.023

0.002 0.022 0.002 0.002 0.038 0.284 0.073 0.073 0.004 0.005 0.037 0.005 0.027 0.003 DOI: 10.1021/acs.jafc.5b04972 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 2. continued

peak area (%)

a

peak

tR (min)

compda

60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116

26.925 27.292 27.418 27.424 27.608 27.611 28.883 29.233 29.525 29.724 29.780 29.798 29.812 29.813 29.861 29.876 30.009 30.347 30.363 31.733 31.899 31.968 32.655 32.994 33.049 33.641 34.297 34.316 36.491 36.523 36.561 37.364 37.899 38.647 42.587 42.600 42.641 42.682 42.701 42.720 42.733 45.583 45.598 45.729 45.761 46.000 46.155 47.384 52.307 52.326 52.397 52.495 52.747 53.757 54.501 55.266 55.386

dichloro tetra(cyclopentadienyl)diytterbium benzenepropanoic acid 2,4,6(3H)-pteridinetrione, 1,5-dihydro5-chloro-2-([4-(2-naphthyloxy)butyl]sulfanyl)-4(3H)-pyrimidinone phenylpropanolamine adrenaline 2H-1-benzopyran octanedioic acid D-fructose 4,5-dimethylbenzene-1,2-bis[2-(2-pyridyl)ethynyl] 2H-1-benzopyran mannonic acid 5a-6b-cholest-2-en-6-yl 1H-phenanthro[9,10-d]imidazole, 1,2-diphenyl3-methoxy-2,4,5-trifluorobenzoic acid, tridecyl ester galactose oxime D-glucose bicyclo[2.2.2]octa-2,5-diene, 1,4,5,7,7,8,8-heptafluoro-2,3-dimethyltefluthrine benzo[1,2-c:3,4-c′:5,6-c″]tris[1,2,5]oxadiazole 2-keto-L-gluconic acid D-gluconic acid 1,3-diphenyl-2-azafluorene hexadecanoic acid androst-2-en-17-amine, 4,4-dimethyl-N-(2-phenylethyl)myo-inositol tridecanol, 2-ethyl-2-methyl1,4-dioxaspiro[4.5]decane naphthalene, 1-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydro-6,7-dimethoxy-2,3-dimethyl-, (1s,2s,3r)10-(acenaphthylen-1-yl)phenanthren-9-ol 2-propynoic acid 2-O-glycerol-D-galactopyranoside 5-iodopentan-2-one D-glucopyranuronic acid korupensamin b D-glucopyranoside 4-norlanosta-17(20),24-diene-11,16-diol-21-oic acid, 3-oxo-16,21-lactone 5-isoxazolidinecarboxylic acid D-glucopyranoside 4-pyrenamine ethyl 3,8-dimethylimidazo[1,5-a]pyrimidine-6-carboxylate 4-hydroxyanthraquinone-2-carboxylic acid cis-13-docosenoic acid 14-cholest-8-en-11-one 4,4′-(4,4′-biphenylylenedioxy)dianiline 1-dimethyl(pentafluorophenyl)silyloxy-4-methoxybenzene salmeterol, N-trifluoroacetyl benzo[1,2-c:3,4-c′:5,6-c″]tris[1,2,5]oxadiazole D-glucopyranoside 9-(2-p-tolylethyl)-3,4,5,6,7,9-hexahydro-2H-xanthene-1,8-dione benzeneacetonitrile, 2-(hydroxymethyl)16,19-secostrychnidine-10,16-dione, 21,22-epoxy-21,22-dihydro-4,14-dihydroxy-3-methoxy-19-methyl-, (21a,22a)1,3-dipentylheptabarbital anthraquinone, 1-(p-bromophenyl)1H-furo[3,4-c][1]benzazepin-1-one, 3,3a,4,5-tetrahydro-4-(1H-indol-3-yl)4-hydroxymandelic acid benzene, 1,3,5-tris(2,2-dimethylpropyl)-2-methyl-

NPUBE

BPUBE 0.002 0.008 0.002

0.003 0.114 0.117 0.031 0.195 6.000 0.381 0.074

0.182

0.142 0.006 0.002 0.021 0.124 3.860

0.001 0.314 0.002 0.823

0.095 0.002 0.003 0.002 0.030 0.651 0.524 0.307 0.035 0.002 0.002 0.002

0.052

13.200

0.004 0.007 0.010 0.027 38.400 0.451

0.008 0.535 0.004 0.012 0.008 0.111 0.001 0.002 0.006 0.006 0.002 0.089

0.003 0.134 0.015 0.004 0.009 0.018 0.002 0.002

0.011 0.008

Based on mass spectral data.

F


Article

Journal of Agricultural and Food Chemistry Table 3. Effects of BPUBE and NPUBE on Total IgE Production and Cell Viability in U266B1 Human Multiple Myeloma Cellsa

vehicle positive control: (10 μg/mL LPS + 5 ng/mL IL-4) NPUBE 1 μg/mL NPUBE 10 μg/mL NPUBE 100 μg/mL BPUBE 1 μg/mL BPUBE 10 μg/mL BPUBE 100 μg/mL

IgE concn (ng/ mL)

cell survival (%)

11.42 ± 0.81 f 527 ± 32 a

100.0 ± 5.4 b 135.4 ± 9.6 a

494 439 413 372 243 203

± ± ± ± ± ±

19 17 33 19 17 12

a b bc c d e

132 ± 12 a 129 ± 11 a 124.8 ± 9.6 a 120 ± 10 a 115.4 ± 9.7 ab 108.7 ± 5.6 b

Data are expressed as the mean ± SD (n = 10). PBS was used as vehicle. Values in each column with the same letter are not significantly different between groups at p < 0.05.

a

Figure 2. OVA-specific IgE level in bronchoalveolar lavage fluid (BALF) from OVA-sensitized/challenged allergic model mice orally administered with BPUBE/NPUBE (10 mg/kg body weight). BALF was collected from BPUBE/NPUBE-administered sensitized/challenged Balb/c mice by lavaging tracheas with PBS. After centrifugation, supernatant was recovered from each mouse group for quantitation of OVA-specific IgE level in BALF using the ELISA kit. Representation: vehicle (−), negative control not sensitized/ challenged with OVA; vehicle (+), OVA-sensitized/challenged positive control; NPUBE and BPUBE, mouse groups orally administered with NPUBE and BPUBE (10 mg/kg body weight), respectively. Data are expressed as mean ± SD (n = 10). Bars not sharing a common letter are not significantly different between groups at p < 0.05.

Figure 3. OVA-specific IgG, IgG1, and IgG2a levels in serum from OVA-sensitized/challenged asthma model mice administered with BPUBE/NPUBE. The sensitized/challenged Balb/c mice were orally administered with BPUBE/NPUBE, and were sacrificed to bleed by cardiac puncture. Serum was collected after blood clotting reaction, and the resultant supernatant was quantitatively assayed for IgG, IgG1, and IgG2a levels of each mouse group using the ELISA method. Representation: vehicle (−), negative control not sensitized/ challenged with OVA; vehicle (+), OVA-sensitized/challenged positive control; NPUBE and BPUBE, mouse groups orally administered with NPUBE and BPUBE (10 mg/kg body weight), respectively. Data are expressed as mean ± SD (n = 10). Bars not sharing a common letter are not significantly different between groups at p < 0.05.

a lesser extent by NPUBE (about 31% inhibition). Eosinophil counting based on their morphological characteristics in OVAsensitized and challenged mice administered with BPUBE showed a significant decreased ratio of eosinophilia (about 55% reduction, Figure 4). NPUBE also showed similar effects but with lower reduction ratios in eosinophilia than those by BPUBE (about 33% reduction). The number of lymphocytes in BALF was also significantly reduced by BPUBE administration. Effects of BPUBE on Eosinophil Infiltration in the Lung. As shown in Figure 4, eosinophils were the most massively infiltrated inflammatory cell in BALF from OVAsensitized and challenged mice. This was confirmed by the observation that more inflammatory cells infiltrated between the trachea and blood vessels in OVA-sensitized mice than in the vehicle-treated control mice (Figure 5). In BPUBE-

administered asthmatic mice, infiltration of inflammatory cells was markedly decreased in the lung, and the activity of BPUBE was higher than that of NPUBE. Next, we examined whether massive inflammatory cell infiltration was associated with the expression of potent chemoattraction-related proteins. The results summarized in Table 5 show that BPUBE could inhibit eotaxin and VCAM-1 G


Article


Table 4. Effects of BPUBE and NPUBE on Th1, Th2, and Treg Cytokine Release into Serum and BALF from OVA-Sensitized/ Challenged Micea cytokine production (pg/mL) in BALF IL-4 vehicle OVA only OVA + NPUBE OVA + BPUBE

13.3 96.4 64.6 34.2

± ± ± ±

1.0 5.6 3.5 2.4

d a b c

in serum

IL-5

IL-13

IL-2

17.7 ± 1.2 d 138.5 ± 8.6 a 99.9 ± 5.6 b 51.1 ± 2.4 c

16.5 ± 1.3 d 148 ± 10 a 103.4 ± 8.2 b 51.7 ± 3.1 c

23.6 ± 1.7 a 14.39 ± 0.90 d 17.7 ± 1.2 c 20.0 ± 1.2 b

IL-12 295 204 226 251

± ± ± ±

18 11 10 13

IL-10 a d c b

105.5 ± 8.3 a 67.2 ± 4.3 c 83.3 ± 5.6 b 92.2 ± 8.0 a

Data expressed as the mean ± SD (n = 10). PBS was used as vehicle. NPUBE/BPUBE was orally administered at a dose of 10 mg/kg body weight. Values in each column with the same letter are not significantly different between groups at p < 0.05.

a

OVA-sensitized eotaxin and VCAM-1 secretions but with lower levels than that by BPUBE (about 42% and 47% suppression, respectively). Effects of BPUBE on Proinflammatory Mediator Levels in the Lung. Inflammatory mediators such as LTC4 and PGD2 levels were also determined as biomarkers of airway inflammation. As shown in Table 5, BPUBE administration decreased LTC4 and PGD2 levels to 19% and 21% of those measured in the OVA-sensitized/challenged control group. NPUBE OVA-triggered LTC4 and PGD2 releases were lower than those induced by BPUBE. Nitric oxide (NO) and prostaglandins, synthesized with inducible NO synthase (iNOS) and cyclooxygenase (COX)-2, respectively, are also known to be involved in the inflammatory process. OVA-sensitized/challenged mice displayed marked increases in the expressions of iNOS and COX-2 in the lung tissue compared to the vehicle-treated control. However, BPUBE-administered mice showed a reduction in the expression of iNOS and COX-2 compared to the OVAsensitized/challenged mice (Figure 6), presumably due to a reduction in gene expression at the transcription level.

■

DISCUSSION Composition in Relation to Bioactivities. We did not determine the individual composition of the elm bark or the mycelia used in the present study. Previous investigators reported that the elm bark contained phytosterols, triterpenes, suberins, phenolic compounds, flavonoids, lignin monomers (dilignols), lipids, and fatty acids.35,36 Turlo et al.37−39 reported that the L. edodes mycelia contained an immunomodulating βglucan, chitin, ergosterol, polyphenols, proteins, carbohydrates, and lipids. As noted in the Introduction, Kim et al.9 found that a polysaccharide isolated from the liquid culture of L. edodes mycelia containing black rice bran exhibited antibiotic properties in mice. A comparison of the proximate composition of the notbioprocessed NPUBE and bioprocessed BPUBE shows that bioprocessing increased the carbohydrate, crude protein, and sodium contents and reduced the crude lipid content (Table 1), suggesting that the treatment might also alter the proportions of bioactive compounds that might be responsible for the enhanced activity. The increase in carbohydrate content of the biofermented product is significant because it might represent the release into the supernatant mycelium culture of immunostimulating polysaccharides embedded in the elm tree bark. The data on the content of characterized compound shown in Table 2 seem to reinforce this suggestion. The two righthand columns in the table show that the 57 compounds in

Figure 4. Inhibition of leukocyte infiltration into BALF by BPUBE/ NPUBE administrations in OVA-sensitized/challenged mice. (A) Total leukocyte infiltration changes by BPUBE/NPUBE administration. (B) Lymphocyte, neutrophil, macrophage, and eosinophil infiltration profile changes by BPUBE/NPUBE. BALF was centrifuged to obtain cell pellets, which were resuspended in PBS, followed by centrifuging onto slide glass and subsequent staining with Wright− Giemsa staining. The slides were microscopically observed (magnification, ×40) for differential cell count by counting a total of 300 cells per slide. Total cell number in BALF was measured by cell counting using a hemocytometer. Data are expressed as mean ± SD (n = 10). Bars not sharing a common letter are not significantly different between groups at p < 0.05.

secretions in BALF (about 73% and 84% inhibition, respectively). As already mentioned, NPUBE also suppressed H


Article


Figure 5. Morphological changes in the lung tissues of OVA-sensitized/challenged allergic mice orally administered with BPUBE/NPUBE. Lung tissues from sensitized/challenged Balb/c mice were fixed with 10% (v/v) paraformaldehyde. The fixed tissues were sectioned to 4 μm, followed by staining with hematoxylin and eosin (H&E) and light microscopy (magnification, ×100). Representation: vehicle, negative control not sensitized/ challenged with OVA; OVA-sensitized/challenged positive control; NPUBE and BPUBE, mouse groups orally administered with BPUBE/NPUBE (10 mg/kg each), respectively. Arrows indicate the level of tracheal edema resulted from inflammatory cell infiltration in lung tissue from each mouse group. Figures represent at least three individual experiments.

Table 5. Effects of BPUBE and NPUBE on Chemoattractant and Eicosanoid Releases into BALF from OVA-Sensitized/ Challenged Micea chemoattractants (pg/mL) eotaxin vehicle OVA only OVA + NPUBE OVA + BPUBE

42.7 154 106.4 72.2

± ± ± ±

3.6 d 11 a 8.5 b 4.0 c

eicosanoids (ng/mL)

VCAM-1 4.22 31.4 18.6 8.68

± ± ± ±

0.23 d 2.1 a 1.4 b 0.52 c

LTC4 44.0 83.6 62.9 51.7

± ± ± ±

2.3 4.6 3.0 4.1

PGD2 d a b c

40.6 78.6 59.3 48.4

± ± ± ±

2.3 5.5 3.5 3.0

d a b c

Data are expressed as the mean ± SD (n = 10). PBS was used as vehicle. NPUBE/BPUBE was orally administered at a dose of 10 mg/kg body weight. Values in each column with the same letter are not significantly different between groups at p < 0.05.

a

NPUBE differ significantly from 67 compounds in BPUBE. The nature of compounds also differs. For example, compounds 11−16 are present in NPUBE but not in BPUBE, whereas compounds 17−21 are absent in NPUBE and present in BPUBE. The same difference is apparent for other groups of compounds. It seems that exposure of the elm bark to the liquid mycelium culture significantly changes the composition of the resulting bioprocessed (fermented) product. Because of the complexity of the structural features of many of the compounds, it is difficult to subdivide the listed compounds into specific categories. We do not know which of the individual compounds or combination of compounds in the mixture that could exhibit additive or synergistic bioactivities might be responsible for the exceptional antiallergic properties of the bioprocessed product, an aspect that merits further study. Mechanism of Allergic Asthma Formation and Inhibition. Both the in vitro cell studies and in vivo mouse studies contribute to our understanding of the mechanism of allergic asthma. Allergic asthma is known to be caused by IgE:antigen mediated activation of submucosal mast cells and subsequent recruitment of eosinophils and Th2 cells from blood in the respiratory tract. High IgE levels in body fluids are

one of the genetic biomarkers that are related to allergic asthma. We therefore hypothesized that the inhibitory effect on IgE production via blockade of the Ig class switch in vitro might indicate whether the bioprocessed formulation has the potential to suppress allergic asthma in vivo. This was found to be the case. Allergic asthma is a multifaceted inflammatory disease of the lung airways. An allergic reaction takes place when the immune system of an animal or human reacts to exposure to an allergen (antigen), resulting in bronchial hyperresponsiveness.1 Development of allergic asthma depends on the interactions between multiple susceptibility genes and environmental factors that determine the balance among Th1, Th2, and Treg cells. The responsible genes stimulate smooth muscle and fibroblast proliferation and regulate cytokine production. Biomarkers of antiallergenicity include the inhibition of antigen-induced release of proinflammatory cytokines that are involved in mediating and signaling of the immune response at the genetic level.40 Protein biomarkers can serve as a powerful detection tool in both clinical and basic research applications. It is also relevant to mention the major role of Treg in allergic asthma. Because both Th1 and Th2 are capable of I


Article


PCR and Western blot analysis of associated gene products, all show that the bark of the elm tree bioprocessed with liquid mushroom mycelium culture normalized the antigen-triggered immune imbalance of the two T-helper cells Th1/Th2. They suggest that this treatment overcame the manifestation of the numerous biomarkers associated with the asthmatic syndrome. Noteworthy are the observations that the bioactive (biofunctional) components produced by the fungal mycelium culture suppressed IgE production in a dose-dependent manner and that histological examination of the lung tissue from BPUBEtreated mice, performed at least three times using randomly selected samples, showed consistently reduced patterns of cell infiltration. The results of the present study also show that changes in the mentioned biomarkers are similar to those reported for the mechanism of the antiasthmatic effects of asthma drugs.41−44 In vitro studies showed that the bioactive formulation was not toxic to human multiple myeloma U266B1 cells and elicited analogous beneficial changes in allergic biomarkers, as was found with the asthmatic mouse model. Moreover, although the individual bark and mushroom mycelium extracts exhibited bioactivity in the assays, the elm bark extract processed by the mycelia was more effective. Significance for Human Health. The in vitro cell and in vivo mouse assays demonstrate the potential value of the bioactive formulation as an anti-inflammatory and antiallergic combination of natural compounds and possibly also as a therapeutic agent for the treatment and prevention of allergic diseases in humans such as hayfever and asthma. Finally, because in the present study the described formulation was not cytotoxic to leukemia cells and the elm bark is used in Korea,17 and possibly elsewhere as a natural traditional human medicinal agent, this novel bioprocessed elm bark might be safe for allergic asthmatic patients. The potent antiallergic asthma properties observed in the present study suggest the need to confirm the results in mice by clinical studies with human patients to determine the value of the formulation to prevent and/or treat allergic asthma.

Figure 6. Inhibition of proinflammatory proteins by BPUBE/NPUBE in lung tissue from OVA-sensitized/challenged mice. (A) Semiquantitative analysis of transcription of COX-2 and iNOS genes. (B) Western blot analysis of COX-2 and iNOS protein expressions. Each gene transcription and protein expression level was expressed as an R.E. (relative expression) value calculated from target gene/β-actin gene expression. Representation: vehicle (−), negative control not sensitized/challenged with OVA; vehicle (+), OVA-sensitized/ challenged positive control; NPUBE and BPUBE, mouse groups orally administered with NPUBE and BPUBE (10 mg/kg each), respectively. Data are expressed as mean ± SD (n = 3). Figures represent at least three individual experiments.

■

producing IL-10, IL-10 is not a unique feature of Th2 cells. The main source of T-cell derived IL-10 is the regulatory T cells. It seems that Treg cells play a central role in determining the incidence and severity of allergic asthma The results of the present study show that oral administration of BPUBE ameliorated the following adverse aspects associated with allergic asthma in the OVA-sensitized/ challenged mice: (a) elevated Th1 cytokine production to near normal levels; (b) reduced the expression of inflammatory mediators due to the apparent reduction in gene expression at the transcription level; (c) reduced elevated immunoglobulin subclass productions directly related to Th1 or Th2 immune reaction, suggesting that the formulation modulates Th1/Th2 balance by suppressing Th2 immune response; (d) elevated Treg IL-10 cytokine production to near normal level, suggesting that the normalized OVA-induced suppressed IL10 production to near normal level might play a critical role in ameliorating allergic asthma; (e) reduced the number of lymphocytes in bronchoalveolar lavage fluid; and (f) exhibited potent suppressive effects on lung airway inflammation and protected the morphology of lung tissues against inflammatory cell infiltration. These beneficial effects on asthma-associated biomarkers (cytokine in serum and bronchoalveolar fluid, immunological, histopathological, and other biomarker levels, and inflammatory eosinophil infiltration in the trachea and lungs), as well as RT-

AUTHOR INFORMATION

Corresponding Authors

*(S.H.N.) Tel: 82-31-219-2619. Fax: 82-31-219-1615. E-mail: [email protected]. *(M.F.) WRRC/ARS/USDA, 800 Buchanan St., Albany, CA 94710, United States. Tel: 01-510-559-5615. Fax 01-510-5596162. E-mail: [email protected]. Funding

We thank the Technological Innovation R&D Program (No. S2014-C1477-00002) of the Small and Medium Business Administration (SMBA, Korea) and the research fund of Ajou University for financial support. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We thank Carol E. Levin for assistance with the preparation of the manuscript and for her constructive comments. ABBREVIATIONS USED BALF, bronchoalveolar lavage fluid; BCS, bovine calf serum; BPUBE, bioprocessed Ulmus parvifolia bark extract; BSA, bovine serum albumin; CD4+, immune T cells; COX-2, J


Article


activity in B16 melanoma-bearing mice. Cancer Immunol. Immunother. 2012, 61, 2143−2152. (16) Okuno, K.; Uno, K. Efficacy of orally administered Lentinula edodes mycelia extract for advanced gastrointestinal cancer patients undergoing cancer chemotherapy: a pilot study. Asian Pac. J. Cancer Prev. 2011, 12, 1671−1674. (17) Moon, Y. H.; Rim, G. R. Studies on the constituents of Ulmus parvifolia. Korean J. Pharmacogn. 1995, 26, 1−7. (18) Cho, S. K.; Lee, S. G.; Kim, C. J. Anti-inflammatory and analgesic activities of water extract of root bark of Ulmus parvifolia. Korean J. Pharmacogn. 1996, 27, 274−281. (19) Cho, E. J.; Yokozawa, T.; Rhyu, D. Y.; Kim, H. Y.; Shibahara, N.; Park, J. C. The inhibitory effects of 12 medicinal plants and their component compounds on lipid peroxidation. Am. J. Chin. Med. 2003, 31, 907−917. (20) Ghareeb, M. A.; Refahy, L. A.; Saad, A. M.; El-Shazely, M. A.; Mohamed, A. S.; Osman, N. S. Cytotoxic screening of three Egyptian plants using brine shrimp lethality test. Int. J. Pharm. Pharm. Sci. 2015, 7, 507−509. (21) Lee, Y.; Park, H.; Ryu, H. S.; Chun, M.; Kang, S.; Kim, H. S. Effects of elm bark (Ulmus davidiana var. japonica) extracts on the modulation of immunocompetence in mice. J. Med. Food 2007, 10, 118−125. (22) Jung, M. J.; Heo, S. I.; Wang, M. H. Free radical scavenging and total phenolic contents from methanolic extracts of Ulmus davidiana. Food Chem. 2008, 108, 482−487. (23) Todorova, M.; Trendafilova, A. Sideritis scardica Griseb., an endemic species of Balkan peninsula: traditional uses, cultivation, chemical composition, biological activity. J. Ethnopharmacol. 2014, 152, 256−265. (24) Hamidpour, R.; Hamidpour, S.; Hamidpour, M.; Shahlari, M. Frankincense (Rǔ Xiang; Boswellia species): From the selection of ̅ traditional applications to the novel phytotherapy for the prevention and treatment of serious diseases. J. Tradit. Complement. Med. 2013, 3, 221−226. (25) al-Sereiti, M. R.; Abu-Amer, K. M.; Sen, P. Pharmacology of rosemary (Rosmarinus of f icinalis Linn.) and its therapeutic potentials. Indian J. Exp. Biol. 1999, 37, 124−130. (26) Castell, M.; Perez-Cano, F. J.; Abril-Gil, M.; Franch, A. Flavonoids on allergy. Curr. Pharm. Des. 2014, 20, 973−987. (27) Kim, S. P.; Park, S. O.; Lee, S. J.; Nam, S. H.; Friedman, M. A polysaccharide isolated from the liquid culture of Lentinus edodes (Shiitake) mushroom mycelia containing black rice bran protects mice against a Salmonella lipopolysaccharide-induced endotoxemia. J. Agric. Food Chem. 2013, 61, 10987−10994. (28) Lee, S. J.; Park, S. O. Fermentation of black rice with basidiomycetes mycelia and immune stimulating agent through bioconversion process. Patent Number 10-2013-0077802, Republic of Korea, July 9, 2013. (29) AOAC. Official Methods of Analysis of AOAC International, 17th ed.; AOAC International: Washington, DC, 2000. (30) Kim, S. P.; Yang, J. Y.; Kang, M. Y.; Park, J. C.; Nam, S. H.; Friedman, M. Composition of liquid rice hull smoke and antiinflammatory effects in mice. J. Agric. Food Chem. 2011, 59, 4570− 4581. (31) U.S. Dept. of Commerce. NIST/EPA/NIH (NIST 05) Mass Spectral Library, 7th ed.; Wiley: Washington, DC, 2005. (32) Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55−63. (33) Temelkovski, J.; Hogan, S. P.; Shepherd, D. P.; Foster, P. S.; Kumar, R. K. An improved murine model of asthma: selective airway inflammation, epithelial lesions and increased methacholine responsiveness following chronic exposure to aerosolised allergen. Thorax 1998, 53, 849−856. (34) Chomczynski, P.; Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 1987, 162, 156−159.

cyclooxygenase-2; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; HEPES, a tertiary amine used to buffer the pH of biological experiments; HRP, horseradish peroxidase; IgE, immunoglobulin E; IL, interleukin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; LT, leukotriene; MTT, tetrazolium dye used in cell viability assays; NPUBE, not-bioprocessed Ulmus parvifolia bark extract; OVA, chicken egg ovalbumin; PBS, phosphate-buffered saline; PDA, potato dextrose agar medium; PGD2, prostaglandin D2; RTPCR, reverse transcriptase polymerase chain reaction; Th, Thelper lymphocytes; Treg, regulatory T lymphocyte; VCAM-1, vascular cell adhesion molecule-1

■

REFERENCES

(1) Beers, M. H. The Merck Manual of Diagnosis and Therapy, 18th ed.; Merck Research Laboratories: Whitehouse Station, NJ, 2006. (2) Robinson, D. S. The role of the T cell in asthma. J. Allergy Clin. Immunol. 2010, 126, 1081−1091. (3) Palomares, O.; Martín-Fontecha, M.; Lauener, R.; TraidlHoffmann, C.; Cavkaytar, O.; Akdis, M.; Akdis, C. A. Regulatory T cells and immune regulation of allergic diseases: Roles of IL-10 and TGF-β. Genes Immun. 2014, 15, 511−520. (4) Böhm, L.; Maxeiner, J.; Meyer-Martin, H.; Reuter, S.; Finotto, S.; Klein, M.; Schild, H.; Schmitt, E.; Bopp, T.; Taube, C. IL-10 and regulatory T cells cooperate in allergen-specific immunotherapy to ameliorate allergic asthma. J. Immunol. 2015, 194, 887−897. (5) Kim, S. P.; Kang, M. Y.; Choi, Y. H.; Kim, J. H.; Nam, S. H.; Friedman, M. Mechanism of Hericium erinaceus (Yamabushitake) mushroom-induced apoptosis of U937 human monocytic leukemia cells. Food Funct. 2011, 2, 348−356. (6) Kim, S. P.; Kang, M. Y.; Kim, J. H.; Nam, S. H.; Friedman, M. Composition and mechanism of antitumor effects of Hericium erinaceus mushroom extracts in tumor-bearing mice. J. Agric. Food Chem. 2011, 59, 9861−9869. (7) Kim, S. P.; Nam, S. H.; Friedman, M. Hericium erinaceus (Lion’s Mane) mushroom extracts inhibit metastasis of cancer cells to the lung in CT-26 colon cancer-transplanted mice. J. Agric. Food Chem. 2013, 61, 4898−4904. (8) Kim, S. P.; Moon, E.; Nam, S. H.; Friedman, M. Hericium erinaceus mushroom extracts protect infected mice against Salmonella Typhimurium-induced liver damage and mortality by stimulation of innate immune cells. J. Agric. Food Chem. 2012, 60, 5590−5596. (9) Kim, S. P.; Park, S. O.; Lee, S. J.; Nam, S. H.; Friedman, M. A polysaccharide isolated from the liquid culture of Lentinus edodes (Shiitake) mushroom mycelia containing black rice bran protects mice against salmonellosis through up-regulation of the Th1 immune reaction. J. Agric. Food Chem. 2014, 62, 2384−2391. (10) Matsuhisa, K.; Yamane, S.; Okamoto, T.; Watari, A.; Kondoh, M.; Matsuura, Y.; Yagi, K. Anti-HCV effect of Lentinula edodes mycelia solid culture extracts and low-molecular-weight lignin. Biochem. Biophys. Res. Commun. 2015, 462, 52−57. (11) Wang, L.; Wang, C.; Gao, X.; Xu, N.; Lin, L.; Zhao, H.; Jia, S.; Jia, L. Purification, characterization and anti-aging capacity of mycelia zinc polysaccharide by Lentinus edodes SD-08. BMC Complementary Altern. Med. 2015, 15, 111. (12) Li, X.; Zhong, M.; Liu, B.; Wang, X.; Liu, L.; Zhang, W.; Huang, M. Antiproliferative protein from the culture supernatant of Lentinula edodes C91−3 mycelia. J. Agric. Food Chem. 2014, 62, 5316−5320. (13) Tanigawa, K.; Ito, Y.; Sakai, M.; Kobayashi, Y. [Evaluation of quality of life and immune function in cancer patients receiving combined immunotherapy and oral administration of Lentinula edodes mycelia extract]. Gan To Kagaku Ryoho 2012, 39, 1779−1781. (14) Chen, M. F.; Chung, H. H.; Lu, H. L. Protection of the extracts of Lentinus edodes mycelia against carbon-tetrachloride-induced hepatic injury in rats. Sci. World J. 2012, 2012, 231586. (15) Tanaka, K.; Ishikawa, S.; Matsui, Y.; Kawanishi, T.; Tamesada, M.; Harashima, N.; Harada, M. Combining a peptide vaccine with oral ingestion of Lentinula edodes mycelia extract enhances anti-tumor K


Article

Journal of Agricultural and Food Chemistry (35) Paschke, D.; Abarzua, S.; Schlichting, A.; Richter, D. U.; Leinweber, P.; Briese, V. Inhibitory effects of bark extracts from Ulmus laevis on endometrial carcinoma: an in-vitro study. Eur. J. Cancer Prev. 2009, 18, 162−168. (36) Hartmann, A. M.; Abarzua, S.; Schlichting, A.; Richter, D. U.; Leinweber, P.; Briese, V. Effects of elm bark extracts from Ulmus laevis on human chorion carcinoma cell lines. Arch. Gynecol. Obstet. 2011, 284, 1265−1269. (37) Turlo, J.; Gutkowska, B.; Herold, F. Effect of selenium enrichment on antioxidant activities and chemical composition of Lentinula edodes (Berk.) Pegl. mycelial extracts. Food Chem. Toxicol. 2010, 48, 1085−1091. (38) Turlo, J.; Lubinski, O.; Gutkowska, B. Isolation of lentinan, an immunomodulating (1−3)-β-D-glucan from submerged cultivated mycelium of Lentinus edodes and culture medium. Acta Polym. Pharm. 2004, 61 (Suppl), 40−42. (39) Turlo, J.; Gutkowska, B.; Herold, F.; Dawidowski, M.; Slowinski, T.; Zobel, A. Relationship between selenium accumulation and mycelial cell composition in Lentinula edodes (Berk.) cultures. J. Toxicol. Environ. Health, Part A 2010, 73, 1211−1219. (40) Choi, S. P.; Kang, M. Y.; Koh, H. J.; Nam, S. H.; Friedman, M. Antiallergic activities of pigmented rice bran extracts in cell assays. J. Food Sci. 2007, 72, S719−S726. (41) Kuang, Z.; Wilson, J. J.; Luo, S.; Zhu, S. W.; Huang, R. P. Deciphering asthma biomarkers with protein profiling technology. Int. J. Inflammation 2015, 2015, 630637. (42) Parulekar, A. D.; Diamant, Z.; Hanania, N. A. Role of T2 inflammation biomarkers in severe asthma. Curr. Opin. Pulm. Med. 2016, 22, 59−68. (43) Kim, M. A.; Shin, Y. S.; Pham, L. D.; Park, H. S. Adult asthma biomarkers. Curr. Opin. Allergy Clin. Immunol. 2014, 14, 49−54. (44) Silkoff, P. E.; Strambu, I.; Laviolette, M.; Singh, D.; FitzGerald, J. M.; Lam, S.; Kelsen, S.; Eich, A.; Ludwig-Sengpiel, A.; Hupp, G. C.; Backer, V.; Porsbjerg, C.; Girodet, P. O.; Berger, P.; Leigh, R.; Kline, J. N.; Dransfield, M.; Calhoun, W.; Hussaini, A.; Khatri, S.; Chanez, P.; Susulic, V. S.; Barnathan, E. S.; Curran, M.; Das, A. M.; Brodmerkel, C.; Baribaud, F.; Loza, M. J. Asthma characteristics and biomarkers from the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) longitudinal profiling study. Respir. Res. 2015, 16, 142.

L


Elm Tree (Ulmus parvifolia) Bark Bioprocessed with Mycelia of

Recommend Documents