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 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
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 (IL-
4, -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 inammatory
inltrate 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.24
Previously, we have investigated the extracts of the
mushroom studied here, Lentinus edodes, and also the Lions
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.58 The activity of a bioprocessed polysaccharide
isolated from Lentinus edodes mushroom mycelium culture
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.1016
There have been reports of bioactivity from the same and
dierent 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-inammatory proper-
ties 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
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
and exhibited low cytotoxicity in a brine shrimp assay.20 In a
dierent 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 caeic
and rosmarinic acids;25 and (d) antioxidative avonoids,
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-inammatory
and antiallergic eects.
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 nd 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 dene 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 eort we also determined the
proximate composition of the test materials by standard
methods and their components by GC/MS.
MATERIALS AND METHODS
Materials. Dulbeccos modied Eagles medium (DMEM),
phosphate-buered 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 tetra-
zolium 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 conrmed by
the Korean Center of Microorganisms (Seoul, Korea). The mycelia
grown on PDA were inoculated with inoculating loop into a 250 mL
Erlenmeyer ask 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)
774
Article
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 rst analyzed for carbohydrate, protein,
lipid, and sodium content according to the ocially 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
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
the volatilities of the polar compounds using 100 L of N-methyl-N-
trimethylsilyltriuoroacetamide (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 m). 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 ow 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 identied 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 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-
inactivated FBS. Penicillin (100 U/mL) and streptomycin (100 mg/
mL) were also added to the medium. Cells were cultured at 37 C in a
humidied 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 g/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
manufacturers instructions. Absorbance of the nal 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 eects of BPUBE and NPUBE.32
Briey, 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 g/mL each)
for 48 h at 37 C humidied 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 (Bio-
Rad) at 570 nm and a reference wavelength of 655 nm.
DOI: 10.1021/acs.jafc.5b04972
J. Agric. Food Chem. 2016, 64, 773784
Journal of Agricultural and Food Chemistry
Mice. Pathogen-free female Balb/c mice, 68 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 2022 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
modication.33 Briey, acclimatized Balb/c mice were arbitrarily
divided into the following four groups (n = 10), avoiding any
intergroup dierence in body weight: vehicle, OVA-, NPUBE-, and
BPUBE-treated groups. Mice were intraperitoneally (ip) sensitized
with 20 g of OVA emulsied with 0.2 mL of 1% aluminum hydroxide
(w/v) in phosphate-buered 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 sacriced by CO2 inhalation 24 h after the last (day 30)
to assess the suppressive eects 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.
Figure 1. Schematic diagram of the experimental protocol. Mice were
sensitized on days 1, 8, and 15 by ip injection of 20 g of ovalbumin
(OVA) emulsied 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 uids 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 (magnica-
tion, 40) for dierential 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 sacriced mice.
Measurement of OVA-Specic IgE Level in BALF. The OVA-
specic IgE release into BALF was measured with a mouse ovalbumin
specic IgE ELISA assay kit (Bio-Rad) according to the manufacturers
instruction. Briey, appropriately diluted BALF was transferred to the
775
Article
96-well microplate precoated with rat anti-mouse IgE monoclonal
antibody, and incubated at room temperature for 1 h. After thorough
washing with wash buer, horseradish peroxidase (HRP)-conjugated
ovalbumin was added to the wells. Then, incubation at room
temperature was continued for 1 h. After nal 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-specic 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, IL-
10, 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 manufacturers instructions.
Absorbance of the nal 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-Specic IgG Subclass Levels in
Serum. IgG in mouse serum was determined using an ELISA kit
(MyBioSource, San Diego, CA, USA) according to the manufacturers
protocol, and IgG1 and IgG2a levels were measured as follows. Briey,
96-well plates were precoated with 100 L of OVA solution (5 g/mL
in PBS) overnight at 4 C. After washing with PBS, the plates were
blocked with 150 L 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 anti-
mouse 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 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
of the nal 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 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
then cut to a thickness of 4 m 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 inammatory cell inltration.
RNA Isolation and Semi-Quantitative RT-PCR. Total cellular
RNA was prepared from homogenized lung tissue according to the
acid phenol guanidinium thiocyanatechloroform extraction meth-
od.34 For reverse transcription, total RNA (1 g) was incubated with
AMV reverse transcriptase (5 U) and oligo (dT18) as primer. DNA
amplication was then primed in a reaction mixture containing dNTP
mix (400 M each), Taq polymerase (2.5 U), and primer sets (20 M
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-ATGTCCGAAGCAA-
ACATCAC-3; iNOS antisense primer, 5-TAATGTCCAGGAA-
GTAGGTG-3; -actin sense primer, 5-GTGGGGCGCCCCA-
GGCACCA-3; -actin antisense primer, 5-GTCCTTAATGTCA-
CGCACGATTTC-3. PCR was conducted using a thermocycler
(model PTC-200, MJ Research, Reno, NV, USA) with one cycle for 5
DOI: 10.1021/acs.jafc.5b04972
J. Agric. Food Chem. 2016, 64, 773784
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. Amplied 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
quantied 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) buer (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 g) were
separated on 12% SDSpolyacrylamide 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 -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 quantied 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. Signicant dierences between means
were determined by the ANOVA test using the Statistical Analysis
Software package SAS (Cary, NC, USA). p < 0.05 is regarded as
signicant.
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
carbohydrate crude protein crude lipid sodium (mg/100 g)
NPUBE 72.5 6.7 1.7 12.64
BPUBE 75.8 8.6 0.0 51.18
contained 116 characterized compounds (Table 2). Some
components remained unidentied, and others were tentatively
identied. Among the identied compounds, 59 compounds
were found only in BPUBE and 56 structurally dierent
compounds in NPUBE. Because the chemical nature of the
identied compounds varied widely, it is dicult to determine
which compound or combination of compounds might be
responsible for the observed bioactivity.
Eects of BPUBE on Total and OVA-Specic IgE
Production. To examine whether BPUBE has capacity to
suppress asthma in vivo, the inhibitory eects of BPUBE on the
secretion of IgE, an allergic immunoglobulin, were rst
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 specic 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
776
Article
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 inammation in vivo.
Next, we examined whether such inhibitory capacity on IgE
production was also active in vivo using BALF from an OVA-
sensitized/challenged asthmatic mouse model. As depicted in
Figure 2, oral administration of BPUBE (10 mg/kg body
weight) markedly suppressed OVA-specic 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).
Eects of BPUBE on OVA-Specic 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-specic 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 OVA-
sensitization/challenge.
Eects of BPUBE on Th1, Th2, and Treg Cytokine
Production. To examine whether BPUBE can regulate the
dierentiation pathway of CD4 T cells for a balanced Th1/Th2
and Treg immune reaction, changes in Th1, Th2, and Treg
cytokine production proles 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 aects 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 vehicle-
treated 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
inammatory responses including asthmatic diseases. This
nding showed a possibility that BPUBE has capacity of
suppressing allergic asthma though activation of Treg.
Eects of BPUBE on Inammatory Leucocyte Inux
into BALF. To evaluate the eects 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 inltrating cells was approximately 3.6-fold higher in
the BALF than in vehicle-treated normal control group (Figure
4). Such inltration of immune cells was suppressed to a great
extent by BPUBE administration (about 62% inhibition) and to
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
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 signicantly dierent between groups at p < 0.05.
a lesser extent by NPUBE (about 31% inhibition). Eosinophil
counting based on their morphological characteristics in OVA-
sensitized and challenged mice administered with BPUBE
showed a signicant decreased ratio of eosinophilia (about 55%
reduction, Figure 4). NPUBE also showed similar eects but
with lower reduction ratios in eosinophilia than those by
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
Lung. As shown in Figure 4, eosinophils were the most
massively inltrated inammatory cell in BALF from OVA-
sensitized and challenged mice. This was conrmed by the
observation that more inammatory cells inltrated between
the trachea and blood vessels in OVA-sensitized mice than in
the vehicle-treated control mice (Figure 5). In BPUBE-
779
Figure 3. OVA-specic 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 sacriced 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 signicantly dierent between groups at p < 0.05.
administered asthmatic mice, inltration of inammatory cells
was markedly decreased in the lung, and the activity of BPUBE
was higher than that of NPUBE.
Next, we examined whether massive inammatory cell
inltration 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
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.

Figure 4. Inhibition of leukocyte inltration into BALF by BPUBE/
NPUBE administrations in OVA-sensitized/challenged mice. (A)
Total leukocyte inltration changes by BPUBE/NPUBE adminis-
tration. (B) Lymphocyte, neutrophil, macrophage, and eosinophil
inltration prole 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 (magni-
cation, 40) for dierential 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 signicantly dierent
between groups at p < 0.05.
secretions in BALF (about 73% and 84% inhibition,
respectively). As already mentioned, NPUBE also suppressed
780
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-
ties in mice.
A comparison of the proximate composition of the not-
bioprocessed 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 signicant 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 right-
hand columns in the table show that the 57 compounds in
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
nature of compounds also diers. For example, compounds
1116 are present in NPUBE but not in BPUBE, whereas
compounds 1721 are absent in NPUBE and present in
BPUBE. The same dierence is apparent for other groups of
compounds. It seems that exposure of the elm bark to the
liquid mycelium culture signicantly changes the composition
of the resulting bioprocessed (fermented) product.
Because of the complexity of the structural features of many
of the compounds, it is dicult to subdivide the listed
compounds into specic 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 uids are
781
one of the genetic biomarkers that are related to allergic
asthma. We therefore hypothesized that the inhibitory eect 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 inammatory 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 Devel-
opment 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 broblast
proliferation and regulate cytokine production.
Biomarkers of antiallergenicity include the inhibition of
antigen-induced release of proinammatory 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
DOI: 10.1021/acs.jafc.5b04972
J. Agric. Food Chem. 2016, 64, 773784
Journal of Agricultural and Food Chemistry
Figure 6. Inhibition of proinammatory proteins by BPUBE/NPUBE
in lung tissue from OVA-sensitized/challenged mice. (A) Semi-
quantitative 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/-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 inammatory
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 IL-
10 production to near normal level might play a critical role in
ameliorating allergic asthma; (e) reduced the number of
lymphocytes in bronchoalveolar lavage uid; and (f) exhibited
potent suppressive eects on lung airway inammation and
protected the morphology of lung tissues against inammatory
cell inltration.
These benecial eects on asthma-associated biomarkers
(cytokine in serum and bronchoalveolar uid, immunological,
histopathological, and other biomarker levels, and inammatory
eosinophil inltration in the trachea and lungs), as well as RT-
782
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
bioactive formulation as an anti-inammatory 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
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.
AUTHOR INFORMATION
Corresponding Authors
*(S.H.N.) Tel: 82-31-219-2619. Fax: 82-31-219-1615. E-mail:
shnam@ajou.ac.kr.
*(M.F.) WRRC/ARS/USDA, 800 Buchanan St., Albany, CA
94710, United States. Tel: 01-510-559-5615. Fax 01-510-559-
6162. E-mail: mendel.friedman@ars.usda.gov.
Funding
We thank the Technological Innovation R&D Program (No. S-
2014-C1477-00002) of the Small and Medium Business
Administration (SMBA, Korea) and the research fund of
Ajou University for nancial support.
Notes
The authors declare no competing nancial 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 uid; BCS, bovine calf serum;
BPUBE, bioprocessed Ulmus parvifolia bark extract; BSA,
bovine serum albumin; CD4+, immune T cells; COX-2,
DOI: 10.1021/acs.jafc.5b04972
J. Agric. Food Chem. 2016, 64, 773784
Journal of Agricultural and Food Chemistry
cyclooxygenase-2; ELISA, enzyme-linked immunosorbent
assay; FBS, fetal bovine serum; HEPES, a tertiary amine used
to buer 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-buered saline; PDA,
potato dextrose agar medium; PGD2, prostaglandin D2; RT-
PCR, reverse transcriptase polymerase chain reaction; Th, T-
helper 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, 10811091.
(3) Palomares, O.; Martn-Fontecha, M.; Lauener, R.; Traidl-
Hoffmann, C.; Cavkaytar, O.; Akdis, M.; Akdis, C. A. Regulatory T
cells and immune regulation of allergic diseases: Roles of IL-10 and
TGF-. Genes Immun. 2014, 15, 511520.
(4) Bohm, 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, 887897.
(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, 348356.
(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, 98619869.
(7) Kim, S. P.; Nam, S. H.; Friedman, M. Hericium erinaceus (Lions
Mane) mushroom extracts inhibit metastasis of cancer cells to the lung
in CT-26 colon cancer-transplanted mice. J. Agric. Food Chem. 2013,
61, 48984904.
(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, 55905596.
(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, 23842391.
(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, 5257.
(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 C913 mycelia. J. Agric. Food Chem. 2014, 62, 53165320.
(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, 17791781.
(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
783
Article
activity in B16 melanoma-bearing mice. Cancer Immunol. Immunother.
2012, 61, 21432152.
(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, 16711674.
(17) Moon, Y. H.; Rim, G. R. Studies on the constituents of Ulmus
parvifolia. Korean J. Pharmacogn. 1995, 26, 17.
(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, 274281.
(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, 907917.
(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, 507509.
(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,
118125.
(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, 482487.
(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, 256265.
(24) Hamidpour, R.; Hamidpour, S.; Hamidpour, M.; Shahlari, M.
Frankincense (Ru 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,
221226.
(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, 124130.
(26) Castell, M.; Perez-Cano, F. J.; Abril-Gil, M.; Franch, A.
Flavonoids on allergy. Curr. Pharm. Des. 2014, 20, 973987.
(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, 1098710994.
(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. Ocial 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 anti-
inflammatory 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, 5563.
(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 respon-
siveness following chronic exposure to aerosolised allergen. Thorax
1998, 53, 849856.
(34) Chomczynski, P.; Sacchi, N. Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform extrac-
tion. Anal. Biochem. 1987, 162, 156159.
DOI: 10.1021/acs.jafc.5b04972
J. Agric. Food Chem. 2016, 64, 773784
Journal of Agricultural and Food Chemistry Article

(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, 162168.
(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, 12651269.
(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, 10851091.
(38) Turlo, J.; Lubinski, O.; Gutkowska, B. Isolation of lentinan, an
immunomodulating (13)--D-glucan from submerged cultivated
mycelium of Lentinus edodes and culture medium. Acta Polym.
Pharm. 2004, 61 (Suppl), 4042.
(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, 12111219.
(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, S719S726.
(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, 5968.
(43) Kim, M. A.; Shin, Y. S.; Pham, L. D.; Park, H. S. Adult asthma
biomarkers. Curr. Opin. Allergy Clin. Immunol. 2014, 14, 4954.
(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.

784 DOI: 10.1021/acs.jafc.5b04972

J. Agric. Food Chem. 2016, 64, 773784