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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 eect 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-specic IgE secretion in bronchoalveolar lavage uid (BALF) was observed in OVA-sensitized/challenged asthmatic
mice. BPUBE also inhibited OVA-specic 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 prole changes caused by BPUBE in serum or BALF. Inammatory 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 inltration near the trachea was associated with reduced eotaxin and vascular cell adhesion molecule-1
(VCAM-1) levels. Changes in proinammatory mediator levels in BALF suggest that BPUBE decreased OVA-sensitization-
induced elevation of leukotriene C4 (LTC4) and prostaglandin D2 (PGD2). The nding 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 inammatory disease of lung airways caused
supplemented with black rice bran protected mice against
salmonellosis through the activation of macrophage-mediated
by triggering stimuli (dust, food, pollen) that results in partially immune response resulting from augmented Th1 immunity.9
or completely reversible constriction of the bronchi. Treatment These results indicate that the mushroom extracts can have an
involves controlling triggering factors and drug therapy. It is impact on the immune system. Some other studies describe
well-known that such susceptibility is attributed to immune several other health-promoting properties of Lentinus edodes
factors including T-helper (Th2) cells and their cytokines (IL- mushroom mycelia.1016
4, -5, and -13). Genetic and environmental components may There have been reports of bioactivity from the same and
interact in the determination of T-helper (Th1) and Th2 dierent elm tree species, which may also be relevant to the
immune responses. The Th2 and other cell types, including present study. The bark, which contains phenolic compounds
eosinophils, mast cells, and neutrophils, form an inammatory and steroidal glucosides, has been used for the treatment of
inltrate in the lung airways leading to adverse symptoms.1 eczema, edema, gonorrhea, and scabies.17 Water extracts of the
Regulatory T cells (Treg) and their cytokine (IL-10) seem to root bark of the same species, Ulmus parvifolia, decreased the
play a key role in the causes and prevention of allergic size of carbuncles induced by Staphylococcus aureus pathogenic
asthma.24 bacteria and exhibited analgesic and anti-inammatory proper-
Previously, we have investigated the extracts of the ties in mice by stimulation of the immune system.18 Extracts
mushroom studied here, Lentinus edodes, and also the Lions also inhibited lipid peroxidation in the linoleic acid system19
Mane mushroom, Hericium erinaceus, to determine their
bioactivities. Cell and mouse studies suggest that mushroom Received: October 13, 2015
extracts may have the potential to modulate the immune Revised: January 13, 2016
system.58 The activity of a bioprocessed polysaccharide Accepted: January 14, 2016
isolated from Lentinus edodes mushroom mycelium culture Published: January 25, 2016

2016 American Chemical Society 773 DOI: 10.1021/acs.jafc.5b04972


J. Agric. Food Chem. 2016, 64, 773784
Journal of Agricultural and Food Chemistry Article

and exhibited low cytotoxicity in a brine shrimp assay.20 In a and treated with amylase (40 U) and cellulase (100 U) at 60 C for 60
dierent elm species, Ulmus davidiana, bark extracts have been min for enzymatic digestion of carbohydrate-based particulate
shown to enhance the immunocompetent properties of mice materials. This preparation was then adjusted to pH 7.0, followed by
(splenocyte proliferation and cytokine production) by activated sterilization in an autoclave. The U. parvifolia bark preparation was
added to a 5 L fermenter (working volume, 3 L), inoculated with the
macrophages,21 and they exhibited free radical scavenging seed culture, and cultured at 28 C and 150 rpm for 5 days. This main
properties.22 culture was treated with an enzyme mixture containing glucanase
There have been several reported studies on the prevention (3,300 U), cellulose (6,000 U), hemicellulose (3,000 U), and pectinase
of asthma in rodents by various plant materials and their (3,000 U) at 60 C for 60 min for cell wall lysis. The enzyme-treated
bioactive compounds. These include (a) Sideritis scardica, an culture mass was then heated at 90 C for 1 h for extraction, and the
endemic herbal species of the Balkan peninsula;23 (b) resultant aqueous phase was recovered and freeze-dried to a solid
frankincense, the resinous extract from the trees of the genus material. The resultant material was termed BPUBE (bioprocessed
Boswellia, and bioactive boswellic acids;24 (c) rosemary Ulmus parvifolia bark extract). U. parvifolia bark extract not subjected
(Rosmarinus off icinalis), a common household plant grown in to bioprocessing by mycelia was also prepared under the same
enzymatic hydrolysis, extraction (90 C/1 h), and freeze-drying
many parts of the world, and its bioactive constituents caeic
conditions, and termed NPUBE (not-bioprocessed Ulmus parvifolia
and rosmarinic acids;25 and (d) antioxidative avonoids, bark extract).
reviewed by Castell et al.26 These observations suggest that Component Analysis by Standard Methods and by GC/MS.
the antioxidative properties of the plant materials and their BPUBE and NPUBE were rst analyzed for carbohydrate, protein,
bioactive constituents might largely govern anti-inammatory lipid, and sodium content according to the ocially published
and antiallergic eects. method.29
Mushrooms obtain their sustenance from decomposing For GC/MS analysis, the two extracts were derivatized in two steps
plants. They secrete enzymes into their host that break down to protect carbonyl function following the method of Kim et al.30
complex carbohydrates and lignins into simpler compounds. Dried samples were dissolved in methoxyamine hydrochloride (100
New, bioactive compounds can therefore be produced from the L; 20 mg/mL) in pyridine, and reacted at 60 C for 1 h. The acidic
protons were exchanged against the trimethylsilyl group to increase
elm bark substrate exposed to mushroom mycelia during the volatilities of the polar compounds using 100 L of N-methyl-N-
bioprocessing. trimethylsilyltriuoroacetamide (MSTFA) at 70 C for 1 h. Each
Because we previously found that bioprocessed Lentinus extract was analyzed by GC/MS using a gas chromatograph, model
edodes mycelia and black rice bran protected mice against 6890GC (Agilent Technologies, Santa Clara, CA), equipped with a
infection by Salmonella via upregulation of the Th1 immune mass spectrometer detector 5975 and DB-1 column (Agilent
reaction,9 it was of interest to nd out whether bioprocessing of Technologies, stationary phase; polyethylene glycol, 30 m 0.25
elm bark in a liquid mycelium culture produced bioactive mm; i.d. 0.25 m). The temperature was programmed at 70 C (4
compounds that would protect mice against allergic asthma via min) with an increase of 10 C/min until 300 C (6 min) was reached.
a similar mechanism. The main objective of this study was Helium gas was used as the carrier with a ow rate of 1 mL/min. Both
injector and detector temperatures were set at 250 C. The injection
therefore to dene the mechanism of protection against allergic was a split ratio of 25:1 in all cases. The injection volume was 1 L.
asthma in mice by the bark of the elm tree Ulmus parvifolia Mass spectra were recorded in electron ionization mode with
bioprocessed with Lentinus edodes mushroom mycelia in liquid ionization energy of 70 eV. Components were identied by retention
culture in terms of Th1/Th2 cells and other biomarkers time in the mass spectra and by comparing the mass spectra with those
associated with the immune system in relation to allergic in a commercial library.31
manifestations. As part of this eort we also determined the Cell Culture. The U266B1 human multiple myeloma B
proximate composition of the test materials by standard lymphocyte cells from American Type Tissue Culture Collection
methods and their components by GC/MS. (ATCC, Manassas, VA, USA) were cultured in a modied 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% heat-
MATERIALS AND METHODS inactivated FBS. Penicillin (100 U/mL) and streptomycin (100 mg/
Materials. Dulbeccos modied Eagles medium (DMEM), mL) were also added to the medium. Cells were cultured at 37 C in a
phosphate-buered saline (PBS), fetal bovine serum (FBS), bovine humidied atmosphere with 5% CO2. The medium was replaced every
calf serum (BCS), and other miscellaneous cell culture reagents were 3 days until the cell reached maximal cell density.
purchased from Hyclone Laboratories (Logan, UT, USA). Potato Measurement of Total IgE Secretion and Cell Viability. For
dextrose agar medium (PDA) was from Difco Laboratory (Detroit, assessment of changes in IgE production levels, U266B1 cells were
MI, USA). Chicken egg ovalbumin (OVA, grade V), human IL-4, stimulated with 10 g/mL lipopolysaccharide (LPS), 5 ng/mL human
aluminum hydroxide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra- IL-4 (Sigma-Aldrich), and either BPUBE or NPUBE (1, 10, and 100g/
zolium bromide (MTT), and analytical grade reagents were obtained mL) for 72 h. The culture supernatants were recovered for IgE assay
from Sigma-Aldrich (St. Louis, MO, USA). U. parvifolia bark, air-dried by an assay kit (Biorbyt, San Francisco, CA, USA) according to the
and minced, was purchased at the Kyung-Dong traditional manufacturers instructions. Absorbance of the nal reaction mixture
pharmaceutical market in Seoul, Korea. was read at 450 nm using a microplate reader (model 550, Bio-Rad,
Preparation of Bioprocessed Ulmus parvifolia Bark Extract. Hercules, CA, USA).
L. edodes fungal mycelia were isolated from the fruitbody and were Cell Viability Assay. MTT staining reported by Mosmann was
cultured on PDA. The genetic identity of the fungus was conrmed by employed to evaluate the cytotoxic eects of BPUBE and NPUBE.32
the Korean Center of Microorganisms (Seoul, Korea). The mycelia Briey, the U266B1 cells were seeded into 96-well plate at a density of
grown on PDA were inoculated with inoculating loop into a 250 mL (1 104) cells/well, and then the cells were treated with appropriate
Erlenmeyer ask containing 50 mL of the liquid medium (2% glucose, concentration of BPUBE and NPUBE (1, 10, and 100 g/mL each)
0.5% yeast extract, 0.5% soy peptone, 0.2% KH2PO4, 0.05% MgSO4, for 48 h at 37 C humidied air containing 5% CO2. The controls,
and 0.002% FeSO4, w/v), and was cultured at 28 C for 5 days in a vehicle and LPS/IL-4-stimulated, were also tested. After treatments,
rotary shaker (120 rpm). This culture was used to seed the main the cells were stained by adding MTT. The resultant intracellular
culture for bioprocessing. chromogen formazan products were solubilized with DMSO.
Bioprocessing was carried out according to the method previously Absorbance of the chromogen was read in a microplate reader (Bio-
reported.27,28 Minced U. parvifolia bark was added to DMEM (30 g/L) Rad) at 570 nm and a reference wavelength of 655 nm.

774 DOI: 10.1021/acs.jafc.5b04972


J. Agric. Food Chem. 2016, 64, 773784
Journal of Agricultural and Food Chemistry Article

Mice. Pathogen-free female Balb/c mice, 68 weeks old, were 96-well microplate precoated with rat anti-mouse IgE monoclonal
purchased from Orient Bio Inc. (Seoul, Republic of Korea). The mice antibody, and incubated at room temperature for 1 h. After thorough
were housed in a stainless steel cage under a 12 h light/dark cycle with washing with wash buer, horseradish peroxidase (HRP)-conjugated
a temperature range of 2022 C and relative humidity of 50 10%. ovalbumin was added to the wells. Then, incubation at room
Mice were fed the pelletized normal commercial chow diet (Cat. No. temperature was continued for 1 h. After nal washing, the substrate,
5L79, Orient Bio.) and tap water ad libitum for 1 week after arrival for tetramethylbenzidine (TMB), was added and the wells were incubated
acclimation. for another 30 min in the dark. The absorbance of each well at 450 nm
Antigen Sensitization, Challenge, and Treatment. The (a reference wavelength of 655 nm) was measured using a microplate
protocol for sensitization and inhalational challenge was carried out reader (Bio-Rad). OVA-specic IgE concentration was calculated
according to the method of Temelkovski et al. with slight based on the results from serial dilution of standard mouse IgE
modication.33 Briey, acclimatized Balb/c mice were arbitrarily included in the ELISA kit.
divided into the following four groups (n = 10), avoiding any Measurement of Cytokine Levels in Serum or BALF. IL-2, IL-
intergroup dierence in body weight: vehicle, OVA-, NPUBE-, and 10, and IL-12 levels in serum, together with IL-4, IL-5, and IL-13 levels
BPUBE-treated groups. Mice were intraperitoneally (ip) sensitized in BALF, were determined using an ELISA kit (R&D Systems,
with 20 g of OVA emulsied with 0.2 mL of 1% aluminum hydroxide Minneapolis, MN, USA) following the manufacturers instructions.
(w/v) in phosphate-buered saline (PBS, pH 7.4). Injections were Absorbance of the nal reaction mixture was read in a microplate
performed three times on days 1, 8, and 15. The sensitized mice were reader (Bio-Rad) at 450 nm and a reference wavelength of 540 nm.
subjected to OVA challenge by placing each mouse individually in a Standard curves for each target substance were created from the
Plexiglas box (29 22 18 cm). Challenge was continued with averages of the absorbance at the same wavelengths and triplicate
repeated exposure to an aerosol of 1% OVA using an ultrasonic readings of the standards in the assay kits.
nebulizer (NE-U12, Omron Co., Kyoto, Japan). Challenge was carried Measurements of OVA-Specic IgG Subclass Levels in
out for 30 min once a day for 5 consecutive days (day 25 to 29). Serum. IgG in mouse serum was determined using an ELISA kit
Vehicle-treated group was subjected to ip injection of 0.2 mL of PBS (MyBioSource, San Diego, CA, USA) according to the manufacturers
plus 0.15 mL of aluminum hydroxide gel, and then challenged with protocol, and IgG1 and IgG2a levels were measured as follows. Briey,
PBS. For NPUBE and BPUBE groups, mice sensitized/challenged 96-well plates were precoated with 100 L of OVA solution (5 g/mL
with OVA as described were orally administered with either of the in PBS) overnight at 4 C. After washing with PBS, the plates were
extracts (10 mg/kg each) for 14 consecutive days (day 16 to 29). PBS blocked with 150 L of 1% bovine serum albumin (BSA) for 30 min at
was used as vehicle, and both extracts were administered once a day. room temperature. Then, the serum samples (50-fold dilution for
All mice were sacriced by CO2 inhalation 24 h after the last (day 30) IgG2 and 5,000-fold dilutions for IgG1) were added to the wells and
to assess the suppressive eects of BPUBE and NPUBE. The described incubated for 2 h at room temperature. HRP-conjugated rat anti-
experimental design is schematically shown in Figure 1. The protocol mouse IgG1 and IgG2a (Life Technologies, Carlsbad, CA, USA) was
for the mouse studies was approved by the Ethics Committee for added, and the samples were incubated for 2 h at room temperature.
Animal Care and Use, Ajou University, Republic of Korea. The plates were washed, and 100 L 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 manufacturers instructions (Biorbyt, San
Francisco, CA, USA). The absorbance of the nal 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
Figure 1. Schematic diagram of the experimental protocol. Mice were of the nal reaction mixtures at 420 nm was measured using a
sensitized on days 1, 8, and 15 by ip injection of 20 g of ovalbumin microplate reader (Bio-Rad). The standard curves for each target
(OVA) emulsied with 0.2 mL of 1% aluminum hydroxide (w/v). substance were created from the averages of triplicate reading
After sensitization/challenge, the mice were exposed to aerosolized 1% (absorbance at 450 nm) of the standards in the assay kits.
OVA from day 25 to 29 using an ultrasonic nebulizer for 30 min/day. Histological Analysis of the Lung. The exsanguinated left lung
BPUBE/NPUBE were orally administered from day 16 to 29. was removed from the chest cavity and xed with 4% paraformalde-
hyde in phosphate buer (0.5 M, pH 7.4). Lobes were isolated,
dehydrated with ethanol, and embedded in paran. The tissues were
Collection of Bronchoalveolar Fluid (BALF) and Blood. For then cut to a thickness of 4 m and mounted onto glass slides. The
collection of BALF, tracheotomy was performed as follows. The sections were dewaxed using xylene and ethanol, stained with
tracheas exposed by cannulating upper tracheas and BALF were hematoxylin and eosin (H&E), and examined by light microscopy
carefully collected by twice lavaging with 1 mL of ice-cold PBS (Olympus) to observe inammatory cell inltration.
containing 0.05 mM EDTA. RNA Isolation and Semi-Quantitative RT-PCR. Total cellular
Collected lavage uids were then centrifuged at 400g for 5 min at 4 RNA was prepared from homogenized lung tissue according to the
C. The recovered supernatants were set aside and kept at 70 C acid phenol guanidinium thiocyanatechloroform extraction meth-
until further analysis. Cell pellets were resuspended in ice-cold PBS, od.34 For reverse transcription, total RNA (1 g) was incubated with
followed by slide preparation by centrifuge using Cytospin (Hanil AMV reverse transcriptase (5 U) and oligo (dT18) as primer. DNA
Science, Incheon, Republic of Korea) and staining with Wright amplication was then primed in a reaction mixture containing dNTP
Giemsa stain. The slides were microscopically observed (magnica- mix (400 M each), Taq polymerase (2.5 U), and primer sets (20 M
tion, 40) for dierential cell counts by counting a total 300 cells per each) representing the target genes as follows: cyclooxygenase-2
slide under light microscopy (model D50, Olympus, Tokyo, Japan). (COX-2) sense primer, 5-TCTCAACCTCTCCTACTAC-3; COX-2
The total cell number in BALF was also measured by microscopic cell antisense primer, 5-GCACGTAGTCTTCGATCACT-3; inducible
counting using a hemocytometer. Blood was collected by cardiac nitric oxide synthase (iNOS) sense primer, 5-ATGTCCGAAGCAA-
puncture from the sacriced mice. ACATCAC-3; iNOS antisense primer, 5-TAATGTCCAGGAA-
Measurement of OVA-Specic IgE Level in BALF. The OVA- GTAGGTG-3; -actin sense primer, 5-GTGGGGCGCCCCA-
specic IgE release into BALF was measured with a mouse ovalbumin GGCACCA-3; -actin antisense primer, 5-GTCCTTAATGTCA-
specic IgE ELISA assay kit (Bio-Rad) according to the manufacturers CGCACGATTTC-3. PCR was conducted using a thermocycler
instruction. Briey, appropriately diluted BALF was transferred to the (model PTC-200, MJ Research, Reno, NV, USA) with one cycle for 5

775 DOI: 10.1021/acs.jafc.5b04972


J. Agric. Food Chem. 2016, 64, 773784
Journal of Agricultural and Food Chemistry Article

min at 94 C, followed by 30 cycles for 30 s at 94 C, 45 s at 72 C, mycelia. Table 3 also demonstrates that both BPUBE and
and one cycle for 5 min at 72 C. Amplied PCR products were NPUBE were not cytotoxic to the U266B1 cells at the
subjected to 1.5% agarose gel electrophoresis and visualized with an examined three doses. The results suggest the potential of
UV illuminator. The intensity of the separated bands of DNA was BPUBE to suppress airway inammation in vivo.
quantied using a gel documentation system (model LAS-100CH, Fuji
Photo Film Co., Tokyo, Japan). Next, we examined whether such inhibitory capacity on IgE
Western Blot Analysis of Lung Tissues. Lung tissue was production was also active in vivo using BALF from an OVA-
extracted with RIPA (radioimmunoprecipitation assay) buer (50 mM sensitized/challenged asthmatic mouse model. As depicted in
Tris Cl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% Figure 2, oral administration of BPUBE (10 mg/kg body
SDS, and 1 mM EDTA, pH 7.4) for preparation of whole cell proteins. weight) markedly suppressed OVA-specic IgE secretion into
Protein concentrations were determined according to the Bradford BALF by about 83%, whereas administration of NPUBE
method using a Bio-Rad Protein kit. Bovine serum albumin (BSA) was showed much lower inhibition of IgE secretion at the same
used as standard. The cell extract containing proteins (30 g) were dose (about 44% inhibition).
separated on 12% SDSpolyacrylamide gels and electrophoretically
Eects of BPUBE on OVA-Specic IgG, IgG1, and
transferred onto a nitrocellulose membrane (Millipore, Billerica, MA,
USA). The membrane was blocked in 5% skim milk at 4 C overnight IgG2a Levels in Serum. We also examined whether BPUBE
and probed with the primary antibodies as follows; anti-mouse modulated additional immunoglobulins involved in allergic
cyclooxygenae-2 (COX-2) goat polyclonal antibody (Santa Cruz, responses, including IgG, IgG1, and IgG2a. As shown in Figure
Dallas, TX, USA), anti-mouse inducible nitric oxide synthase (iNOS) 3, OVA-sensitization/challenge induced marked upregulation
rabbit polyclonal antibody (Cell Signaling Tech., Danvers, MA, USA), of OVA-specic IgG levels (about 105-, 38-, and 4-fold
and anti-mouse -actin monoclonal antibody (Millipore, Billerica, MA, increases in IgG, IgG1, and IgG2a levels, respectively). By
USA). After the primary antibody reaction for at least 3 h, the contrast, mice treated with BPUBE showed a strong reduction
secondary antibody reaction with HRP-conjugated anti-IgG antibody of total IgG and IgG1 serum levels (about 75% and 82%
was performed under the same conditions. Blots were developed using
the ECL detection kit (Pierce, Rockford, IL, USA). The intensity of
reduction, respectively). Serum IgG2a level was also measured
separated protein bands was quantied using a gel documentation in OVA-sensitized/challenged mice administered with BPUBE.
system (model LAS-1000CH, Fuji Photo Film Co., Tokyo, Japan). At The result showed a reduction of IgG2a in response to the
least three separate replicates were determined for each experiment. BPUBE treatment; however, the extent was much lower than
Statistical Analysis. Results are expressed as the mean SD of that of IgG and IgG1 (about 33% reductions). The observed
three independent experiments. Signicant dierences between means drastic reduction of IgG and IgG1 and the moderate reduction
were determined by the ANOVA test using the Statistical Analysis of IgG2a suggest that BPUBE modulates the Th1/Th2 balance
Software package SAS (Cary, NC, USA). p < 0.05 is regarded as by suppressing Th2 immune response elevated by the OVA-
signicant.

sensitization/challenge.
RESULTS Eects of BPUBE on Th1, Th2, and Treg Cytokine
Production. To examine whether BPUBE can regulate the
Composition of BPUBE and NPUBE. Crude lipid and dierentiation pathway of CD4 T cells for a balanced Th1/Th2
sodium compositions markedly changed during bioprocessing and Treg immune reaction, changes in Th1, Th2, and Treg
(Table 1). GC/MS analysis revealed that the two extracts cytokine production proles were determined by ELISA assay
in BALF or serum. As shown in Table 4, oral administration of
Table 1. Proximate Composition of NPUBE and BPUBE BPUBE markedly suppressed the production of Th2 cytokines
% dry wt 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
carbohydrate crude protein crude lipid sodium (mg/100 g)
production, respectively). Compared to BPUBE, NPUBE
NPUBE 72.5 6.7 1.7 12.64 showed much lower inhibitions. Next, we examined whether
BPUBE 75.8 8.6 0.0 51.18 inhibition of Th2 cytokine production adversely aects the
restoration of suppressed Th1 reaction to normal status in
contained 116 characterized compounds (Table 2). Some serum. Table 4 also shows that oral administration of BPUBE
components remained unidentied, and others were tentatively upregulated the OVA-induced downregulated Th1 cytokines
identied. Among the identied compounds, 59 compounds including IL-2 and IL-12 to near normal levels observed in the
were found only in BPUBE and 56 structurally dierent vehicle-treated control mice (about 85% level of the vehicle-
compounds in NPUBE. Because the chemical nature of the treated control). In addition, BPUBE also exerted upregulation
identied compounds varied widely, it is dicult to determine of OVA-induced downregulated IL-10 production (about 87%
which compound or combination of compounds might be level of the vehicle-treated control), the cytokine secreted
responsible for the observed bioactivity. mainly from Treg cells and involved in suppressive functions in
Eects of BPUBE on Total and OVA-Specic IgE inammatory responses including asthmatic diseases. This
Production. To examine whether BPUBE has capacity to nding showed a possibility that BPUBE has capacity of
suppress asthma in vivo, the inhibitory eects of BPUBE on the suppressing allergic asthma though activation of Treg.
secretion of IgE, an allergic immunoglobulin, were rst Eects of BPUBE on Inammatory Leucocyte Inux
determined in vitro in human multiple myeloma U266B1 into BALF. To evaluate the eects of BPUBE on the
cells. Compared to the LPS and IL-4-treated control, 1, 10, and recruitment of immune cells to the airway, we counted total
100 g/mL BPUBE treatments suppressed specic IgE secretion leukocyte cell numbers and the number of lymphocytes,
levels in cell supernatants by about 30, 55, and 61%, neutrophils, macrophages, and eosinophils in BALF. The total
respectively (Table 3). By contrast, NPUBE was found to number of inltrating cells was approximately 3.6-fold higher in
have lower inhibitory activities at the same concentrations (6, the BALF than in vehicle-treated normal control group (Figure
17, and 22% inhibitions, respectively), clearly showing the 4). Such inltration of immune cells was suppressed to a great
elevation of bioactivity through bioprocessing with mushroom extent by BPUBE administration (about 62% inhibition) and to
776 DOI: 10.1021/acs.jafc.5b04972
J. Agric. Food Chem. 2016, 64, 773784
Journal of Agricultural and Food Chemistry Article

Table 2. Compounds Identied by GC/MS Analysis of TMS-Derivatized Elm Bark Extracts


peak area (%)
a
peak tR (min) compd NPUBE BPUBE
1 8.537 decane, 3,6-dimethyl- 0.109
2 8.537 dodecane 0.099
3 8.741 2-undecen-4-ol 0.001
4 8.964 propanoic acid 0.427
5 8.983 isophthalic acid, 2-fluorophenyl pentyl ester 0.007
6 10.014 undecane, 5-methyl- 0.017
7 10.019 heptane, 2,4-dimethyl- 0.019
8 10.102 quinoline, 2-phenyl- 0.004
9 10.104 1-(3-methylbutyl)-2,3,4,6-tetramethylbenzene 0.048
10 10.189 succinic acid, diamide, N,N-diethyl-N,N-diphenyl- 0.003
11 11.405 noscapine 0.022
12 11.412 thioquinox 0.002
13 11.425 1,2,3,4-tetrahydroisoquinoline, 6,7,8-trimethoxy-1,2-dimethyl- 0.001
14 11.439 1H-indene, 1,1,4,5,6,7-hexafluoro- 0.002
15 11.487 2-pentamethyldisilanyloxybutane 0.036
16 11.502 1-(anilinomethylene)-2-indanone 0.005
17 12.691 dibenzo[c,f ]1,7-naphthyridin-9(7H)-one, 8,10,11,12-tetrahydro-8-(3-cyclohexenyl)- 0.003
18 13.894 isophthalic acid, 2,6-dichlorophenyl propyl ester 0.006
19 13.959 phenol, 2-amino-4,6-bis(1,1-dimethylethyl)- 0.002
20 13.988 oxo(6-phenyl-imidazo[1,2-a]pyridin-2-yl)acetic acid, ethyl ester 0.001
21 15.021 cadaverine 0.007
22 15.035 2-(phenylpiperidin-1-yl-methyl)cyclohexanol 0.006
23 15.359 sulfurous acid, 2-ethylhexyl octadecyl ester 0.026
24 15.369 hexadecane 0.089
25 15.394 glycerol 0.014 3.670
26 15.445 cobalt, acetylacetonato(pentamethylcyclopentadienyl)- 0.002
27 15.537 4-methoxyphenyl 0.006
28 15.566 2-(4-methoxyphenyl)-2-propane 0.001
29 19.507 naphtho[2,3-b]furan-9(4H)-one, 4-(acetyloxy)-4a,5,6,7-tetrahydro-3,4a,5-trimethyl-, (4s,-4ar,5s)- 0.008
30 19.518 2H-1,3-thiazine-6-carboxylic acid, 3-aminotetrahydro-2-(methylimino)-4-oxo-, methyl ester 0.002
31 19.537 9,10-anthracenedicarbonitrile 0.001
32 19.538 1-(1,1-bis(ethoxycarbonyl)ethyl)-7-methoxynaphthalene 0.001
33 19.563 1,3,5-triazine-2,4-diamine, 6-(4-tert-butylphenyl)- 0.001
34 20.651 fumaric monoamide, N-(2-bromophenyl)-, butyl ester 0.001
35 20.675 ethanamine, N-chloro-N,1,1-trifluoro- 0.008
36 21.090 dodecane, 4,6-dimethyl- 0.067
37 21.106 sulfurous acid, hexyl pentyl ester 0.012
38 21.574 threitol 0.006
39 21.596 5-bromothiophene-2-carboxamide 0.006
40 22.029 butanoic acid 0.065 0.078
41 22.043 N,N-diethyl-N-[4-[[4-[trifluoromethyl]phenyl]amino]-2-quinazolinyl]-ethane-1,2-diamine 0.002
42 22.060 3-methoxybenzylamine, N,N-diundecyl- 0.001
43 22.106 lysine, 4-hydroxy- 0.001
44 22.209 2-oxo-6-phenyl-4-(4-hydroxyphenyl)-1,2-dihydropyrimidine 0.003
45 22.239 3-ethyl-3-methylheptane 0.023
46 24.448 ursane-3,16-diol, (3b,16b,18a,19a,20b)- 0.002
47 24.462 butanal 0.022
48 24.832 uracil 0.002
49 25.624 phthalic acid, di(3-methylphenyl) ester 0.002
50 25.925 ethylamine, N-heptyl-N-octyl-2-(2-thiophenyl)- 0.038
51 25.944 5,6-dimethyl-4-phenyl-3-cyanopyridine-2(1H)-thione 0.284
52 25.974 heptadecane 0.073
53 25.979 1-iodo-2-methylundecane 0.073
54 26.016 phenylephrine, N,O,O-tris(heptafluorobutyryl) deriv 0.004
55 26.059 phthalic acid, di(3-methylphenyl) ester 0.005
56 26.072 ribitol 0.037
57 26.084 propane(dithioic) acid, ethyl ester 0.005
58 26.389 D-fructose 0.027
59 26.479 1,3-propanedione, 1-(1,3-benzodioxol-5-yl)-3-(4-methoxy-5-benzofuranyl)- 0.003

777 DOI: 10.1021/acs.jafc.5b04972


J. Agric. Food Chem. 2016, 64, 773784
Journal of Agricultural and Food Chemistry Article

Table 2. continued
peak area (%)
a
peak tR (min) compd NPUBE BPUBE
60 26.925 dichloro tetra(cyclopentadienyl)diytterbium 0.002
61 27.292 benzenepropanoic acid 0.008
62 27.418 2,4,6(3H)-pteridinetrione, 1,5-dihydro- 0.002
63 27.424 5-chloro-2-([4-(2-naphthyloxy)butyl]sulfanyl)-4(3H)-pyrimidinone 0.003
64 27.608 phenylpropanolamine 0.114
65 27.611 adrenaline 0.117
66 28.883 2H-1-benzopyran 0.031
67 29.233 octanedioic acid 0.195
68 29.525 D-fructose 6.000 0.182
69 29.724 4,5-dimethylbenzene-1,2-bis[2-(2-pyridyl)ethynyl] 0.381
70 29.780 2H-1-benzopyran 0.074
71 29.798 mannonic acid 0.142
72 29.812 5a-6b-cholest-2-en-6-yl 0.006
73 29.813 1H-phenanthro[9,10-d]imidazole, 1,2-diphenyl- 0.002
74 29.861 3-methoxy-2,4,5-trifluorobenzoic acid, tridecyl ester 0.021
75 29.876 galactose oxime 0.124
76 30.009 D-glucose 3.860 0.095
77 30.347 bicyclo[2.2.2]octa-2,5-diene, 1,4,5,7,7,8,8-heptafluoro-2,3-dimethyl- 0.002
78 30.363 tefluthrine 0.003
79 31.733 benzo[1,2-c:3,4-c:5,6-c]tris[1,2,5]oxadiazole 0.002
80 31.899 2-keto-L-gluconic acid 0.030
81 31.968 D-gluconic acid 0.651
82 32.655 1,3-diphenyl-2-azafluorene 0.001
83 32.994 hexadecanoic acid 0.314 0.524
84 33.049 androst-2-en-17-amine, 4,4-dimethyl-N-(2-phenylethyl)- 0.002
85 33.641 myo-inositol 0.823 0.307
86 34.297 tridecanol, 2-ethyl-2-methyl- 0.035
87 34.316 1,4-dioxaspiro[4.5]decane 0.002
88 36.491 naphthalene, 1-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydro-6,7-dimethoxy-2,3-dimethyl-, (1s,2s,3r)- 0.002
89 36.523 10-(acenaphthylen-1-yl)phenanthren-9-ol 0.002
90 36.561 2-propynoic acid 0.052
91 37.364 2-O-glycerol-D-galactopyranoside 0.004
92 37.899 5-iodopentan-2-one 0.007
93 38.647 D-glucopyranuronic acid 0.010
94 42.587 korupensamin b 0.027
95 42.600 D-glucopyranoside 13.200 38.400
96 42.641 4-norlanosta-17(20),24-diene-11,16-diol-21-oic acid, 3-oxo-16,21-lactone 0.451
97 42.682 5-isoxazolidinecarboxylic acid 0.008
98 42.701 D-glucopyranoside 0.535
99 42.720 4-pyrenamine 0.004
100 42.733 ethyl 3,8-dimethylimidazo[1,5-a]pyrimidine-6-carboxylate 0.012
101 45.583 4-hydroxyanthraquinone-2-carboxylic acid 0.008
102 45.598 cis-13-docosenoic acid 0.111
103 45.729 14-cholest-8-en-11-one 0.001
104 45.761 4,4-(4,4-biphenylylenedioxy)dianiline 0.002
105 46.000 1-dimethyl(pentafluorophenyl)silyloxy-4-methoxybenzene 0.006
106 46.155 salmeterol, N-trifluoroacetyl 0.006
107 47.384 benzo[1,2-c:3,4-c:5,6-c]tris[1,2,5]oxadiazole 0.002 0.003
108 52.307 D-glucopyranoside 0.089 0.134
109 52.326 9-(2-p-tolylethyl)-3,4,5,6,7,9-hexahydro-2H-xanthene-1,8-dione 0.015
110 52.397 benzeneacetonitrile, 2-(hydroxymethyl)- 0.004
111 52.495 16,19-secostrychnidine-10,16-dione, 21,22-epoxy-21,22-dihydro-4,14-dihydroxy-3-methoxy-19-methyl-, (21a,22a)- 0.009
112 52.747 1,3-dipentylheptabarbital 0.018
113 53.757 anthraquinone, 1-(p-bromophenyl)- 0.002
114 54.501 1H-furo[3,4-c][1]benzazepin-1-one, 3,3a,4,5-tetrahydro-4-(1H-indol-3-yl)- 0.002
115 55.266 4-hydroxymandelic acid 0.011
116 55.386 benzene, 1,3,5-tris(2,2-dimethylpropyl)-2-methyl- 0.008
a
Based on mass spectral data.

778 DOI: 10.1021/acs.jafc.5b04972


J. Agric. Food Chem. 2016, 64, 773784
Journal of Agricultural and Food Chemistry Article

Table 3. Eects of BPUBE and NPUBE on Total IgE


Production and Cell Viability in U266B1 Human Multiple
Myeloma Cellsa
IgE concn (ng/
mL) cell survival (%)
vehicle 11.42 0.81 f 100.0 5.4 b
positive control: (10 g/mL LPS + 5 527 32 a 135.4 9.6 a
ng/mL IL-4)
NPUBE 1 g/mL 494 19 a 132 12 a
NPUBE 10 g/mL 439 17 b 129 11 a
NPUBE 100 g/mL 413 33 bc 124.8 9.6 a
BPUBE 1 g/mL 372 19 c 120 10 a
BPUBE 10 g/mL 243 17 d 115.4 9.7 ab
BPUBE 100 g/mL 203 12 e 108.7 5.6 b
a
Data are expressed as the mean SD (n = 10). PBS was used as
vehicle. Values in each column with the same letter are not
signicantly dierent between groups at p < 0.05.

Figure 2. OVA-specic IgE level in bronchoalveolar lavage uid


(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/chal-
lenged Balb/c mice by lavaging tracheas with PBS. After
centrifugation, supernatant was recovered from each mouse group
for quantitation of OVA-specic 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 Figure 3. OVA-specic IgG, IgG1, and IgG2a levels in serum from
NPUBE and BPUBE (10 mg/kg body weight), respectively. Data are OVA-sensitized/challenged asthma model mice administered with
expressed as mean SD (n = 10). Bars not sharing a common letter BPUBE/NPUBE. The sensitized/challenged Balb/c mice were orally
are not signicantly dierent between groups at p < 0.05. administered with BPUBE/NPUBE, and were sacriced to bleed by
cardiac puncture. Serum was collected after blood clotting reaction,
and the resultant supernatant was quantitatively assayed for IgG, IgG1,
a lesser extent by NPUBE (about 31% inhibition). Eosinophil and IgG2a levels of each mouse group using the ELISA method.
counting based on their morphological characteristics in OVA- Representation: vehicle (), negative control not sensitized/
sensitized and challenged mice administered with BPUBE challenged with OVA; vehicle (+), OVA-sensitized/challenged positive
control; NPUBE and BPUBE, mouse groups orally administered with
showed a signicant decreased ratio of eosinophilia (about 55%
NPUBE and BPUBE (10 mg/kg body weight), respectively. Data are
reduction, Figure 4). NPUBE also showed similar eects but expressed as mean SD (n = 10). Bars not sharing a common letter
with lower reduction ratios in eosinophilia than those by are not signicantly dierent between groups at p < 0.05.
BPUBE (about 33% reduction). The number of lymphocytes in
BALF was also signicantly reduced by BPUBE administration.
Eects of BPUBE on Eosinophil Inltration in the administered asthmatic mice, inltration of inammatory cells
Lung. As shown in Figure 4, eosinophils were the most was markedly decreased in the lung, and the activity of BPUBE
massively inltrated inammatory cell in BALF from OVA- was higher than that of NPUBE.
sensitized and challenged mice. This was conrmed by the Next, we examined whether massive inammatory cell
observation that more inammatory cells inltrated between inltration was associated with the expression of potent
the trachea and blood vessels in OVA-sensitized mice than in chemoattraction-related proteins. The results summarized in
the vehicle-treated control mice (Figure 5). In BPUBE- Table 5 show that BPUBE could inhibit eotaxin and VCAM-1
779 DOI: 10.1021/acs.jafc.5b04972
J. Agric. Food Chem. 2016, 64, 773784
Journal of Agricultural and Food Chemistry Article

Table 4. Eects 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 in serum
IL-4 IL-5 IL-13 IL-2 IL-12 IL-10
vehicle 13.3 1.0 d 17.7 1.2 d 16.5 1.3 d 23.6 1.7 a 295 18 a 105.5 8.3 a
OVA only 96.4 5.6 a 138.5 8.6 a 148 10 a 14.39 0.90 d 204 11 d 67.2 4.3 c
OVA + NPUBE 64.6 3.5 b 99.9 5.6 b 103.4 8.2 b 17.7 1.2 c 226 10 c 83.3 5.6 b
OVA + BPUBE 34.2 2.4 c 51.1 2.4 c 51.7 3.1 c 20.0 1.2 b 251 13 b 92.2 8.0 a
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 signicantly dierent between groups at p < 0.05.

OVA-sensitized eotaxin and VCAM-1 secretions but with lower


levels than that by BPUBE (about 42% and 47% suppression,
respectively).
Eects of BPUBE on Proinammatory Mediator Levels
in the Lung. Inammatory mediators such as LTC4 and PGD2
levels were also determined as biomarkers of airway
inammation. 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 inammatory
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 OVA-
sensitized/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, avonoids, lignin monomers
(dilignols), lipids, and fatty acids.35,36 Turlo et al.3739 reported
that the L. edodes mycelia contained an immunomodulating -
glucan, 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 proper-
Figure 4. Inhibition of leukocyte inltration into BALF by BPUBE/ ties in mice.
NPUBE administrations in OVA-sensitized/challenged mice. (A)
A comparison of the proximate composition of the not-
Total leukocyte inltration changes by BPUBE/NPUBE adminis-
tration. (B) Lymphocyte, neutrophil, macrophage, and eosinophil bioprocessed NPUBE and bioprocessed BPUBE shows that
inltration prole changes by BPUBE/NPUBE. BALF was centrifuged bioprocessing increased the carbohydrate, crude protein, and
to obtain cell pellets, which were resuspended in PBS, followed by sodium contents and reduced the crude lipid content (Table 1),
centrifuging onto slide glass and subsequent staining with Wright suggesting that the treatment might also alter the proportions
Giemsa staining. The slides were microscopically observed (magni- of bioactive compounds that might be responsible for the
cation, 40) for dierential cell count by counting a total of 300 cells enhanced activity. The increase in carbohydrate content of the
per slide. Total cell number in BALF was measured by cell counting biofermented product is signicant because it might represent
using a hemocytometer. Data are expressed as mean SD (n = 10). the release into the supernatant mycelium culture of
Bars not sharing a common letter are not signicantly dierent
immunostimulating polysaccharides embedded in the elm tree
between groups at p < 0.05.
bark.
The data on the content of characterized compound shown
secretions in BALF (about 73% and 84% inhibition, in Table 2 seem to reinforce this suggestion. The two right-
respectively). As already mentioned, NPUBE also suppressed hand columns in the table show that the 57 compounds in
780 DOI: 10.1021/acs.jafc.5b04972
J. Agric. Food Chem. 2016, 64, 773784
Journal of Agricultural and Food Chemistry 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 xed with 10% (v/v) paraformaldehyde. The xed tissues were sectioned to 4 m, followed by
staining with hematoxylin and eosin (H&E) and light microscopy (magnication, 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 inammatory cell inltration in lung tissue from each mouse
group. Figures represent at least three individual experiments.

Table 5. Eects of BPUBE and NPUBE on Chemoattractant and Eicosanoid Releases into BALF from OVA-Sensitized/
Challenged Micea
chemoattractants (pg/mL) eicosanoids (ng/mL)
eotaxin VCAM-1 LTC4 PGD2
vehicle 42.7 3.6 d 4.22 0.23 d 44.0 2.3 d 40.6 2.3 d
OVA only 154 11 a 31.4 2.1 a 83.6 4.6 a 78.6 5.5 a
OVA + NPUBE 106.4 8.5 b 18.6 1.4 b 62.9 3.0 b 59.3 3.5 b
OVA + BPUBE 72.2 4.0 c 8.68 0.52 c 51.7 4.1 c 48.4 3.0 c
a
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 signicantly dierent between groups at p < 0.05.

NPUBE dier signicantly from 67 compounds in BPUBE. The one of the genetic biomarkers that are related to allergic
nature of compounds also diers. For example, compounds asthma. We therefore hypothesized that the inhibitory eect on
1116 are present in NPUBE but not in BPUBE, whereas IgE production via blockade of the Ig class switch in vitro might
compounds 1721 are absent in NPUBE and present in indicate whether the bioprocessed formulation has the potential
BPUBE. The same dierence is apparent for other groups of to suppress allergic asthma in vivo. This was found to be the
compounds. It seems that exposure of the elm bark to the case.
liquid mycelium culture signicantly changes the composition Allergic asthma is a multifaceted inammatory disease of the
of the resulting bioprocessed (fermented) product. lung airways. An allergic reaction takes place when the immune
Because of the complexity of the structural features of many system of an animal or human reacts to exposure to an allergen
of the compounds, it is dicult to subdivide the listed (antigen), resulting in bronchial hyperresponsiveness.1 Devel-
compounds into specic categories. We do not know which of opment of allergic asthma depends on the interactions between
the individual compounds or combination of compounds in the multiple susceptibility genes and environmental factors that
mixture that could exhibit additive or synergistic bioactivities determine the balance among Th1, Th2, and Treg cells. The
might be responsible for the exceptional antiallergic properties responsible genes stimulate smooth muscle and broblast
of the bioprocessed product, an aspect that merits further study. proliferation and regulate cytokine production.
Mechanism of Allergic Asthma Formation and Biomarkers of antiallergenicity include the inhibition of
Inhibition. Both the in vitro cell studies and in vivo mouse antigen-induced release of proinammatory cytokines that are
studies contribute to our understanding of the mechanism of involved in mediating and signaling of the immune response at
allergic asthma. Allergic asthma is known to be caused by the genetic level.40 Protein biomarkers can serve as a powerful
IgE:antigen mediated activation of submucosal mast cells and detection tool in both clinical and basic research applications.
subsequent recruitment of eosinophils and Th2 cells from It is also relevant to mention the major role of Treg in
blood in the respiratory tract. High IgE levels in body uids are allergic asthma. Because both Th1 and Th2 are capable of
781 DOI: 10.1021/acs.jafc.5b04972
J. Agric. Food Chem. 2016, 64, 773784
Journal of Agricultural and Food Chemistry 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 (biofunc-
tional) components produced by the fungal mycelium culture
suppressed IgE production in a dose-dependent manner and
that histological examination of the lung tissue from BPUBE-
treated mice, performed at least three times using randomly
selected samples, showed consistently reduced patterns of cell
inltration. 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 eects of
asthma drugs.4144
In vitro studies showed that the bioactive formulation was
not toxic to human multiple myeloma U266B1 cells and elicited
analogous benecial 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 eective.
Signicance for Human Health. The in vitro cell and in
vivo mouse assays demonstrate the potential value of the
Figure 6. Inhibition of proinammatory proteins by BPUBE/NPUBE
bioactive formulation as an anti-inammatory and antiallergic
in lung tissue from OVA-sensitized/challenged mice. (A) Semi-
quantitative analysis of transcription of COX-2 and iNOS genes. (B) combination of natural compounds and possibly also as a
Western blot analysis of COX-2 and iNOS protein expressions. Each therapeutic agent for the treatment and prevention of allergic
gene transcription and protein expression level was expressed as an diseases in humans such as hayfever and asthma. Finally,
R.E. (relative expression) value calculated from target gene/-actin because in the present study the described formulation was not
gene expression. Representation: vehicle (), negative control not cytotoxic to leukemia cells and the elm bark is used in Korea,17
sensitized/challenged with OVA; vehicle (+), OVA-sensitized/ and possibly elsewhere as a natural traditional human medicinal
challenged positive control; NPUBE and BPUBE, mouse groups agent, this novel bioprocessed elm bark might be safe for
orally administered with NPUBE and BPUBE (10 mg/kg each), allergic asthmatic patients. The potent antiallergic asthma
respectively. Data are expressed as mean SD (n = 3). Figures properties observed in the present study suggest the need to
represent at least three individual experiments.
conrm the results in mice by clinical studies with human
patients to determine the value of the formulation to prevent
and/or treat allergic asthma.


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 AUTHOR INFORMATION
incidence and severity of allergic asthma Corresponding Authors
The results of the present study show that oral *(S.H.N.) Tel: 82-31-219-2619. Fax: 82-31-219-1615. E-mail:
administration of BPUBE ameliorated the following adverse shnam@ajou.ac.kr.
aspects associated with allergic asthma in the OVA-sensitized/ *(M.F.) WRRC/ARS/USDA, 800 Buchanan St., Albany, CA
challenged mice: (a) elevated Th1 cytokine production to near 94710, United States. Tel: 01-510-559-5615. Fax 01-510-559-
normal levels; (b) reduced the expression of inammatory 6162. E-mail: mendel.friedman@ars.usda.gov.
mediators due to the apparent reduction in gene expression at Funding
the transcription level; (c) reduced elevated immunoglobulin We thank the Technological Innovation R&D Program (No. S-
subclass productions directly related to Th1 or Th2 immune 2014-C1477-00002) of the Small and Medium Business
reaction, suggesting that the formulation modulates Th1/Th2 Administration (SMBA, Korea) and the research fund of
balance by suppressing Th2 immune response; (d) elevated Ajou University for nancial support.
Treg IL-10 cytokine production to near normal level,
Notes
suggesting that the normalized OVA-induced suppressed IL-
The authors declare no competing nancial interest.


10 production to near normal level might play a critical role in
ameliorating allergic asthma; (e) reduced the number of ACKNOWLEDGMENTS
lymphocytes in bronchoalveolar lavage uid; and (f) exhibited
potent suppressive eects on lung airway inammation and We thank Carol E. Levin for assistance with the preparation of
the manuscript and for her constructive comments.


protected the morphology of lung tissues against inammatory
cell inltration.
These benecial eects on asthma-associated biomarkers ABBREVIATIONS USED
(cytokine in serum and bronchoalveolar uid, immunological, BALF, bronchoalveolar lavage uid; BCS, bovine calf serum;
histopathological, and other biomarker levels, and inammatory BPUBE, bioprocessed Ulmus parvifolia bark extract; BSA,
eosinophil inltration in the trachea and lungs), as well as RT- bovine serum albumin; CD4+, immune T cells; COX-2,
782 DOI: 10.1021/acs.jafc.5b04972
J. Agric. Food Chem. 2016, 64, 773784
Journal of Agricultural and Food Chemistry Article

cyclooxygenase-2; ELISA, enzyme-linked immunosorbent activity in B16 melanoma-bearing mice. Cancer Immunol. Immunother.
assay; FBS, fetal bovine serum; HEPES, a tertiary amine used 2012, 61, 21432152.
to buer the pH of biological experiments; HRP, horseradish (16) Okuno, K.; Uno, K. Efficacy of orally administered Lentinula
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inducible nitric oxide synthase; LPS, lipopolysaccharide; LT, undergoing cancer chemotherapy: a pilot study. Asian Pac. J. Cancer
Prev. 2011, 12, 16711674.
leukotriene; MTT, tetrazolium dye used in cell viability assays;
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NPUBE, not-bioprocessed Ulmus parvifolia bark extract; OVA, parvifolia. Korean J. Pharmacogn. 1995, 26, 17.
chicken egg ovalbumin; PBS, phosphate-buered saline; PDA, (18) Cho, S. K.; Lee, S. G.; Kim, C. J. Anti-inflammatory and
potato dextrose agar medium; PGD2, prostaglandin D2; RT- analgesic activities of water extract of root bark of Ulmus parvifolia.
PCR, reverse transcriptase polymerase chain reaction; Th, T- Korean J. Pharmacogn. 1996, 27, 274281.
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vascular cell adhesion molecule-1

Park, J. C. The inhibitory effects of 12 medicinal plants and their


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