JOURNAL OF MEDICINAL FOOD

J Med Food 00 (0) 2014, 1–6

FULL COMMUNICATION
# Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2014.0012

Anti-Inflammatory Effects of Wild Irish Mushroom Extracts

in RAW264.7 Mouse Macrophage Cells
Yvonne C. O’Callaghan,1 Nora M. O’Brien,1 Owen Kenny,2,3 Tom Harrington,4
Nigel Brunton,3 and Thomas J. Smyth2
1
School of Food and Nutritional Sciences, University College Cork, Cork, Ireland.
2
Teagasc Food Research Centre, Ashtown, Dublin, Ireland.
3
School of Agriculture and Food Science, University College Dublin, Dublin, Ireland.
4
School of Science and Engineering, University of Limerick, Limerick, Ireland.

ABSTRACT Mushrooms and mushroom extracts have traditionally been used as therapies for a wide variety of ailments,
including allergy, arthritis, and other inflammatory disorders. However, more evidence is required on the mechanism by which
mushrooms exert these effects. In the present study, the anti-inflammatory properties of ethanol and hot water extracts
prepared from 27 fungal samples collected between October and November 2011 at various forest locations in the southwest
of Ireland were investigated using the lipopolysaccharide (LPS)–stimulated mouse macrophage (RAW264.7 cells) model of
inflammation. LPS-stimulated cells were incubated in the presence of mushroom extracts at nontoxic concentrations for 24 h
and the production of interleukin-6 (IL-6) was quantified by ELISA. Seven ethanolic and one hot water extract that decreased
IL-6 production were selected for further study. The extracts were then incubated with LPS-stimulated cells for 24 h and the
production of IL-6, tumor necrosis factor-alpha (TNF-a), and nitric oxide (NO) was measured. Ethanolic extracts prepared
from Russula mairei, Lactarius blennius, Craterellus tubaeformis, Russula fellea, and Craterellus cornucopioides demon-
strated selective anti-inflammatory activity by decreasing the production of NO and IL-6 but not TNF-a in LPS-stimulated
RAW264.7 cells. These findings support existing evidence of the anti-inflammatory potential of mushroom extracts.

KEY WORDS:  anti-inflammatory  fungi  RAW264.7  wild mushrooms

INTRODUCTION mor necrosis factor-alpha (TNF-a) and interleukin-6 (IL-6),

is a causative factor in the development of heart disease,
T he medicinal properties of mushrooms have been
exploited for centuries in the preparation of traditional
therapies for a broad range of illnesses, particularly in Asian
rheumatoid arthritis, diabetes, and cancer.11 Wild mush-
rooms and their extracts have been shown to possess both
proinflammatory and anti-inflammatory activities depend-
countries. Recent investigations have demonstrated that
ing on the mushroom species, the method used in the
extracts prepared from wild mushrooms possess an array
preparation of the extracts, and the experimental model se-
of biological activities, including antioxidant,1 anticancer,2
lected for study.12 Both edible and medicinal mushrooms
cardioprotective,3 and immunomodulatory effects.4,5 The
and mushroom extracts, including a glycoprotein extracted
majority (80–85%) of medicinal mushroom products are
from Pleurotus citrinopileatus, a triterpene isolated from
derived from the fruiting body6 and the medicinal properties
Ganoderma lucidum, and crude extracts prepared from ei-
of mushrooms have been attributed to their content of gly-
ther the fruiting body or mycelia of Antrodia cinnamomea,
cosides, alkaloids, tocopherols, phenolics, flavanoids, ter-
Phellinus baumii, Pleurotus ostreatus, or Lentinus poly-
penes, carotenoids, vitamins, and minerals.7 Mushrooms are
chrous, have demonstrated anti-inflammatory effects in the
also a good source of fiber, which accounts for up to 40% of
lipopolysaccharide (LPS)–stimulated RAW264.7 mouse
dry matter,8 and b-glucans that has been demonstrated, from
macrophage model.13–18
several species, to lower serum cholesterol levels9 while also
While the evidence for their anti-inflammatory activity is
having potential antidiabetic effects.10
building, to date only a relatively small number of wild
Chronic inflammation, which is characterized by an ex-
species have been investigated and no studies have been
cess of circulating proinflammatory cytokines, such as tu-
carried out on species collected in Ireland. Therefore, the
objective of the present study was to determine whether
Manuscript received 17 January 2014. Revision accepted 18 June 2014. crude extracts prepared from wild mushrooms harvested in
Address correspondence to: Nora M. O’Brien, MS, PhD, School of Food and Nutritional
Ireland demonstrate anti-inflammatory effects in RAW264.7
Sciences, University College Cork, Cork, Ireland, E-mail: nob@ucc.ie cells by measuring the production of IL-6, TNF-a, and nitric

1
2 O’CALLAGHAN ET AL.
oxide (NO) following exposure of the cells to LPS in the
presence of the mushroom extracts.
MATERIALS AND METHODS
Materials
RAW264.7 cells were purchased from the American Type
Culture Collection (ATCC; LGC Standards, Middlesex,
United Kingdom). Cell culture plastics were purchased from
Cruinn Diagnostics (Dublin, Ireland). Fetal bovine serum
(FBS) was purchased from Invitrogen (Paisley, Scotland). All
other chemicals and reagents were purchased from Sigma
Chemical Co. (Dublin, Ireland) unless otherwise stated.
Sample collection
The 27 fungal samples investigated in this study, which
included both edible and nonedible species, were collected
between October and November 2011 from various forest
locations around the southwest of Ireland, including Cur-
ragh Chase Forest Park, Cratloe Forest Park, and Killarney
National Park.
Each mushroom specimen was collected and identified
by a trained mycologist. These included Russala nigricans,
Clitocybe nebularis, Lactarius deterrimus, Paxilllus in-
volutus, Cortinarius croceus, Russula mairei, Hydnum re-
pandum, Lactarius blennius, Craterellus tubaeformis, Leotia
lubrica, Lycoperdon perlatum, Cortinarius sect. cinnamo-
meobadei, Russula fellea, Craterellus cornucopioides, Suillus
luteus, Hebeloma crustuliniforme, Trichloma ustale, Collybia
confluens, Amanita citrina, Agarius semotus, Inocybe floc-
culosa, Russula ochroleuca, Amanita rubescens, Helvella
crispa, Thelophora terrestris, Laccaria amethystina, and
Mycena pura. Each of the mushroom samples was cleaned
with a soft brush to remove soil debris and frozen. All samples
were freeze dried (A12/60 Freeze Dryer, Frozen In Time Ltd.,
York, United Kingdom) and ground to a fine powder and
stored in vacuum-packed bags at - 80C prior to extraction.
Extraction
Solid-liquid extraction was employed to extract the freeze-
dried mushroom powders with two different solvents, ethanol
(room temperature) and hot water (60C), in a sequential
manner. Ethanol and hot water have previously been used by
Hu et al.19 to prepare bioactive extracts from mushrooms. For
the ethanol extracts, each sample of dried mushroom powder
was mixed in ratio of 1:7.9 w/w with ethanol and for the hot
water extractions (carried out on the material remaining after
ethanol extraction) this ratio was changed to 1:20 w/w due to
the viscosity of the material extracted. Extractions were
performed in an orbital shaker (MaxQ 6000 Shaker, Thermo
Fisher Scientific, Westmeath, Ireland) at 175 rpm. Following
3 h of incubation, ethanol extracts were filtered through a
buchner funnel and the material was re-extracted with fresh
solvent for a further 3 h. The process was repeated with a final
overnight extraction to ensure exhaustive extraction occurred
and the solvents were combined. The residue remaining for
each mushroom sample was dried using nitrogen and then
used for the hot water extract. The hot water extraction was
filtered initially after 6 h and then after 24 h through glass
wool filters and combined. Ethanol was removed from the
ethanol extractions using a rotary evaporator (Heidolph Ro-
tary Evaporator with WB eco bath, Schwabach, Germany)
with the water bath set at 50C. The hot water extracts were
frozen at - 20C and then freeze dried (A12/60 Freeze Dryer,
Frozen In Time Ltd., York, United Kingdom).
The stock solutions of the wild mushrooms (10 mg/mL),
prepared with 10% DMSO in water, were diluted to 100 lg/mL
in Dulbecco’s modified Eagle’s medium (DMEM) and filter
sterilized using 0.22 lm Millex GP, PES membrane filter units
(Millipore, Cork, Ireland) before addition to the cells in culture.
Cell culture
RAW264.7 cells were maintained in DMEM, supple-
mented with 10% FBS, and incubated at 37C and 5% CO2
in a humidified atmosphere. For experiments, the serum
concentration was reduced to 2.5% FBS.
Cell viability
RAW264.7 cells were seeded in 96-well plates at a den-
sity of 0.2 · 104 cells/well and allowed to adhere overnight.
In an initial experiment, the cytotoxicity of each of the ex-
tracts prepared from the 27 mushroom species was investi-
gated at concentrations ranging from 2.5 to 75 lg/mL and
noncytotoxic concentrations (Table 1) were selected for
screening the effect of the extracts on IL-6 production in
LPS-stimulated RAW264.7 cells. The concentration of ex-
tract at which cell viability exceeded 80% was considered as
noncytotoxic as previously reported by López-Garcı́a et
al.20 In the second series of experiments, the eight selected
wild mushroom extracts were added to the cells at concen-
trations ranging from 0 to 50 lg/mL and incubated for 24 h.
Cell viability was determined using the MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] assay
(MTT I proliferation kit, Roche Diagnostics, West Sussex,
United Kingdom) as previously described.21 Cell viability
was quantified by measuring the absorbance at 570 nm
(Varioskan Flash, ThermScientific, Tewksbury, MA, USA)
and was expressed as a percentage of control, untreated cells.
Cytokine production
RAW264.7 cells were seeded in 96-well plates at a density
of 0.2 · 104 cells/well and allowed to adhere overnight. In the
initial screen of the 27 mushroom species, cells were stimu-
lated to produce IL-6 by the addition of 0.2 lg/mL LPS in the
presence of the mushroom extracts at noncytotoxic concen-
trations (outlined in Table 1) and samples were incubated for
24 h. In the second series of experiments, cells were stimu-
lated to produce cytokines by the addition of 0.2 lg/mL LPS
(IL-6) or 0.1 lg/mL LPS (TNF-a) in the presence of the eight
selected mushroom extracts at concentrations of 5 and 10 lg/
mL and samples were incubated for 24 h.
The cell media were harvested and the quantity of IL-6 and
TNF-a in the media was measured by ELISA (eBiosciences,
 ANTI-INFLAMMATORY EFFECTS OF MUSHROOMS IN VITRO 3

Table 1. Production of IL-6 in Lipopolysaccharide-Stimulated RAW264.7 Cells Exposed to Wild Mushroom Extracts

Ethanol extracts Hot water extracts

Concentration (mg/mL) IL-6 (%) Concentration (mg/mL) IL-6 (%)

R. nigricans 40.0 95.2 – 11.7 7.5 90.6 – 5.6
C. nebularis 40.0 85.6 – 7.4 7.5 108.2 – 1.2
L. deterrimus 10.0 87.5 – 3.3 7.5 108.7 – 6.4
P. involutus 50.0 78.4 – 7.1 2.5 121.9 – 8.2
C. croceus 75.0 58.8 – 15.0* 7.5 95.6 – 6.2
R. mairei 75.0 48.5 – 3.1* 7.5 95.1 – 11.0
H. repandum 75.0 72.4 – 0.8 20.0 85.2 – 6.8
L. blennius 75.0 67.6 – 9.6 5.0 105.5 – 2.4
C. tubaeformis 75.0 65.9 – 8.0* 7.5 82.1 – 8.8
L. lubrica 40.0 77.1 – 7.7 7.5 73.7 – 13.8
L. perlatum 40.0 71.8 – 7.4 20.0 88.5 – 6.0
C. cinnamomeobadei 75.0 71.8 – 4.5 7.5 92.1 – 7.7
R. fellea 75.0 59.4 – 5.4* 7.5 96.3 – 8.1
C. cornucopioides 40.0 67.7 – 4.3* 10.0 112.4 – 2.6
S. luteus 75.0 83.6 – 2.1 10.0 106.7 – 3.1
H. crustuliniforme 40.0 85.2 – 4.2 10.0 87.6 – 7.6
T. ustale 50.0 54.5 – 6.1* 50.0 73.7 – 7.0
C. confluens 7.5 90.0 – 4.8 20.0 101.6 – 0.7
A. citrina 7.5 99.4 – 3.1 7.5 102.3 – 6.4
A. semotus 7.5 94.2 – 13.0 5.0 88.9 – 4.0
I. flocculosa ND ND 10.0 125.2 – 14.8
R. ochroleuca 7.5 94.4 – 8.0 5.0 102.9 – 4.2
A. rubescens 75.0 104.4 – 3.5 7.5 111.7 – 9.0
H. crispa ND ND 20.0 68.4 – 6.9*
T. terrestris 2.5 88.2 – 3.5 7.5 78.7 – 5.1
L. amethystina ND ND 50.0 100.9 – 3.8
M. pura 7.5 96.5 – 12.6 50.0 109.2 – 7.8

Data are expressed as a percentage of the control (100%) LPS-stimulated cells, for three independent experiments – standard error.
*Denotes significant difference from control cells (P < .05), ANOVA followed by Dunnett’s.
IL, interleukin; LPS, lipopolysaccharide; ND, not determined.

Hatfield, United Kingdom) as described previously.21 Ab-
sorbance was determined at 450 nm and corrected at 590 nm
(Varioskan Flash; ThermScientific). Results are expressed
as a percentage of the control cells that were exposed to LPS
alone.
NO production
For the measurement of NO production, RAW264.7 cells
were seeded at a density of 8 · 104 cells/well as detailed in
Kenny et al.21 and incubated for 48 h; cells were incubated
with the mushroom extracts at concentrations of 10 and 20
lg/mL in the presence of LPS (0.5 lg/mL) for a further 24 h.
The neutral red uptake assay (NRUA) was used to assess
cell viability under the conditions used to assess NO pro-
duction as this assay is more suitable than the MTT for
measuring viability at higher cell densities. For the NRUA,
media were removed and 200 lL of neutral red dye (40 lg/
mL) was added to the wells. Cells were incubated for 3 h to
allow uptake of the dye. The cells were washed and burst
using 1% glacial acetic acid and the absorbance was mea-
sured at 540 nm (Varioskan Flash; ThermScientific).
For the measurement of NO production, 50 lL of media
from each well was transferred to a 96-well plate and 50 lL
of Greiss reagent (1% sulfanilamide in 5% phosphoric acid:
0.1% N-1-naphtyl-ethylenediamine dichloride in water; 1:1)
was added. Samples were incubated for 20 min and the ab-
sorbance was then measured at 540 nm (Varioskan Flash;
ThermScientific). Results were expressed as a percentage of
the control cells that were exposed to LPS alone.
Statistical analysis
Data represent the mean of at least three independent ex-
periments – standard error. Statistical analysis was evaluated
by Dunnett’s post-test, P < .05 (Graphpad prism 4.0; Graph-
Pad, Inc., San Diego, CA, USA).
RESULTS
The anti-inflammatory activity of the 27 mushroom ex-
tracts, extracted using either ethanol or hot water, was initially
screened by measuring the production of IL-6 in LPS-
stimulated RAW264.7 cells. The concentration of mushroom
extract added to the cells varied from 5 to 75 lg/mL (Table 1)
dependent on the cytotoxicity of the extracts; selected con-
centrations were not cytotoxic (i.e., cell viability exceeded
80%; data not shown). In our study, the extracts prepared
using ethanol were less toxic than those prepared by hot water
4 O’CALLAGHAN ET AL.
extraction and therefore it was possible to add the ethanol
extracts at higher concentrations. Six of the mushroom ex-
tracts (C. croceus, R. mairei, C. tubaeformis, R. fellea, C.
cornucopioides, and T. ustale) prepared by ethanol extraction
caused a significant decrease (P < .05) in IL-6 production
(Table 1) and were selected for further investigation. The
extract of H. crispa prepared using hot water also significantly
decreased (P < .05) IL-6 production and the ethanol extract of
L. blennius nonsignificantly decreased IL-6 production to
67.6% of the LPS-treated control (Table 1) and both these
samples were also included for further investigation.
The viability of RAW264.7 cells exposed to the selected
wild mushroom extracts (0–50 lg/mL) was determined
following a 24-h incubation (Fig. 1) using the MTT assay.
Each of the wild mushroom extracts induced a dose-
dependent decrease in the amount of viable cells. H. crispa
hot water extract caused a significant decrease (P < .05) in
the number of viable cells at the 30 and 50 lg/mL con-
centrations. The initial cytotoxicity screen found that
C. tubaeformis was not cytotoxic (viability > 80%) up to a
concentration of 75 lg/mL following a 24-h incubation;
however, a more thorough investigation found that this ex-
tract induced a significant decrease (P < .05) in cell viability
at the 50 lg/mL concentration to 55.6% of the untreated
control cells (Fig. 1). The cytotoxicity data for the remaining
extracts were consistent between the two series of experi-
ments. Concentrations of 5 and 10 lg/mL were selected for
investigation of the effect of the extracts on cytokine (IL-6
and TNF-a) production as cell viability exceeded 80% at
these concentrations for all of the extracts.
The addition of LPS (0.2 lg/mL) to RAW264.7 cells
caused a 25-fold increase in the level of IL-6 produced by the
cells, from 2.7 to 68.3 pg/mL (data not shown). At the lower
concentration (5 lg/mL) the wild mushroom extracts did not
significantly alter the production of IL-6 in LPS-stimulated
FIG. 1. Cell viability, as determined by MTT, in RAW264.7 cells
following 24-h incubation with extracts of wild mushrooms. Data are
expressed as percentage of untreated control cells and represent three
independent experiments. C. croceus (CC), R. mairei (RM), L.
blennius (LB), C. tubaeformis (CT), R. fellea (RF), C. cornucopioides
(CC2), T. ustale (TU) (all ethanol extracts), and H. crispa (HC) (hot
water extract). *Denotes significant difference from control cells
(P < .05), ANOVA followed by Dunnett’s.
RAW264.7 cells. However, ethanol extracts from five of the
samples (R. mairei, L. blennius, C. tubaeformis, R. fellea, and
C. cornucopioides) significantly (P < .05) decreased IL-6
production at the 10 lg/mL concentration, to between 56.4%
and 72.1% of the LPS-stimulated control cells (Table 2).
Exposure of RAW264.7 cells to LPS (0.1 lg/mL) resulted
in a 15-fold increase in TNF-a production from 12.9 to
191.1 pg/mL (data not shown). The production of TNF-a in
LPS-treated cells incubated with the mushroom extracts was
not significantly different from TNF-a production in cells
exposed to LPS only (Table 2).
Six of the wild mushroom extracts [R. mairei, L. blennius,
C. tubaeformis, R. fellea, C. cornucopioides, and T. ustale
(all ethanol extracts)] demonstrated a dose-dependent de-
crease in NO production in LPS-stimulated RAW264.7 cells
(Table 3). NO production was significantly reduced (P < .05)
to between 44.0% and 66.7% in LPS-stimulated cells in the
presence of these six extracts at the 20 lg/mL concentration
(Table 3). Cell viability was not significantly altered in cells
exposed to LPS and mushroom extracts (10 or 20 lg/mL) in
comparison to untreated control cells, as determined by the
NRUA (data not shown).
DISCUSSION
LPS interacts with Toll-like receptor-4 (TR4) and acti-
vates several pathways involved in the macrophage immune
response, including the NF-jB pathway, the MAPK path-
way, and the JAK-STAT pathway, and results in the pro-
duction of IL-6 and TNF-a. A decrease in the production of
NO, TNF-a, and IL-6 was previously observed in LPS-
stimulated RAW264.7 cells exposed to a triterpene isolated
from G. lucidum,14 a methanolic extract of the mycelia of A.
cinnamomea,17 and a water-soluble lyophilized extract of P.
ostreatus.16
Table 2. Production of IL-6 and TNF-a in LPS-Stimulated
RAW264.7 Cells Exposed to Wild Mushroom Extracts
Interleukin-6 TNF-a
5 mg/mL 10 mg/mL 5 mg/mL 10 mg/mL
Control 100.0 – 5.6 100.0 – 5.6 100.0 – 6.9 100.0 – 6.9
C. croceusa 90.2 – 4.5 97.8 – 6.3 102.6 – 5.1 104.4 – 5.8
R. maireia 71.6 – 3.8 56.4 – 7.9* 105.1 – 4.1 105.8 – 3.1
L. blenniusa 79.6 – 7.3 58.6 – 5.3* 114.1 – 6.4 111.7 – 2.0
C. tubaeformisa 75.3 – 2.9 63.7 – 8.5* 111.0 – 1.8 122.1 – 2.6
R. felleaa 86.9 – 5.6 72.1 – 6.2* 105.1 – 2.7 107.7 – 3.0
C. cornucopioidesa 78.9 – 4.1 69.3 – 9.5* 114.5 – 5.9 119.1 – 9.7
T. ustalea 91.1 – 2.6 88.4 – 5.6 104.2 – 3.5 108.2 – 1.9
H. crispab 96.3 – 1.3 85.1 – 0.7 102.9 – 4.2 111.0 – 8.5
Data are expressed as a percentage of the control LPS-stimulated cells, for
three independent experiments – standard error.
*Denotes significant difference from control cells (P < .05), ANOVA
followed by Dunnett’s.
a
Denotes ethanol extraction.
b
Corresponds to hot water extraction.
 ANTI-INFLAMMATORY EFFECTS OF MUSHROOMS IN VITRO
Table 3. Production of Nitric Oxide in LPS-Stimulated
RAW264.7 Cells Exposed to Wild Mushroom Extracts
10 mg/mL 20 mg/mL
Control 100.0 – 6.1 100.0 – 6.1
C. croceusa 92.2 – 6.8 78.8 – 1.7
R. maireia 74.5 – 2.5* 44.0 – 5.7*
L. blenniusa 70.0 – 3.6* 47.9 – 8.2*
C. tubaeformisa 87.6 – 5.1 66.7 – 10.5*
R. felleaa 79.4 – 7.1 51.9 – 4.5*
C. cornucopioidesa 75.8 – 5.8* 47.1 – 5.9*
T. ustalea 89.8 – 6.6 65.7 – 3.1*
H. crispab 103.5 – 7.1 87.2 – 5.2
Data are expressed as a percentage of the control LPS-stimulated cells, for
three independent experiments – standard error.
*Denotes significant difference from control cells (P < .05), ANOVA
followed by Dunnett’s.
a
Denotes ethanol extraction.
b
Corresponds to hot water extraction.
In an initial screen of IL-6 production, each of the extracts
was added to the cells at the maximum concentration at which
no cytotoxic effects were evident for that extract (Table 1).
Eight of the extracts were selected from this screen for further
investigation; the concentrations selected for the assessment
of cytokine production were 5 and 10 lg/mL as none of the
selected extracts demonstrated cytotoxic effects at these
concentrations (Fig. 1). In the initial screen (Table 1) C.
croceus (ethanol extract) and H. crispa (hot water extract) did
induce a significant decrease in IL-6 production at concen-
trations of 75 and 20 lg/mL, respectively. However, at the
lower concentrations (5 and 10 lg/mL) that were not cyto-
toxic for any of the extracts, C. croceus and H. crispa did not
significantly alter IL-6 production. Ethanol extracts prepared
from the wild mushrooms (R. mairei, L. blennius, C. tubae-
formis, R. fellea, and C. cornucopioides) reduced the pro-
duction of IL-6 and NO in LPS-stimulated RAW264.7 cells
(Tables 2 and 3). T. ustale (ethanol extract) also reduced NO
production (P < .05) relative to the LPS-treated control cells
but IL-6 production was not significantly altered.
To our knowledge, anti-inflammatory activity has not
previously been reported for R. mairei, L. blennius, C. tu-
baeformis, T ustale, or R. fellea. Three of the mushroom
species demonstrating anti-inflammatory activity (R. mairei,
R. fellea, and L. blennius) belong to the family Russulaceae
and may contain a similar profile of secondary metabolites
that could be responsible for their anti-inflammatory effects.
Anti-inflammatory activity has been reported in other spe-
cies of Russula, such as Russula virescens. Hur et al.22
demonstrated in vitro anti-inflammatory activity of a 70%
ethanol extract of Russula virescens in RAW264.7 cell lines
at a concentration of 0.5 mg/mL, which is considerably
higher than the concentrations found to exhibit significant
reduction in NO production in Russula species investigated
in this study (R. mairei (10 lg/mL) and R. fellea (10 lg/mL).
Moro et al.4 recently reported that C. cornucopioides along
with other wild mushroom species exhibited potential anti-
inflammatory activity. The authors speculated that the anti-
5
inflammatory effect of the mushroom extracts including
C. cornucopioides may be, at least in part, related to their
content of pyrogallol. The present study has demonstrated
significant anti-inflammatory activity, in terms of reduced
NO production, at concentrations 50-fold lower (10 lg/mL)
than that reported in Moro et al.4
The mushroom extracts investigated in the present study
did not alter the production of TNF-a in LPS-stimulated
cells. Similarly, Fangkrathok et al.15 observed that TNF-a
was not significantly altered at the mRNA level in LPS-
stimulated RAW264.7 cells incubated with 50 and 100 lg/
mL extract from the mycelia of L. polychrous; a significant
decrease in the expression of IL-6 mRNA, to 80% of the
control, was observed under these conditions. Mollugin, a
bioactive phytochemical extracted from the Rubia cordifolia
plant, was also shown to decrease the production of NO and
IL-6 but not TNF-a in LPS-stimulated RAW264.7 cells.23
Further investigation revealed that mollugin inhibited the
JAK-STAT pathway responsible for the induction of IL-6
but not the NF-jB or MAPK pathways that are involved in
TNF-a production. Therefore, it is possible that the mush-
room extracts (R. mairei, L. blennius, C. tubaeformis, R.
fellea, and C. cornucopioides) may operate by a similar
mechanism to mollugin with the ability to selectively inhibit
NO and IL-6 production. Moro et al.4 also found that the
mushroom species A. bisporus, C. cibarius, and L. delicio-
sus inhibited NO production and IL-6 expression but did not
affect TNF-a expression in LPS-stimulated RAW264.7
cells. Further research is necessary to determine the precise
mechanism involved in the anti-inflammatory effects of
wild Irish mushroom extracts examined in this study.
Several different solvents, including chloroform, hot
water, ethanol, methanol, and hexane, have been used in the
preparation of mushroom extracts24–27 and the composition
of mushroom extracts can vary significantly depending on
the method of extraction. Kim et al.28 found that the b-
glucan content of an extract of Hericium erinaceus was
three- to fourfold higher in hot water (HWE) and micro-
waved ethanolic (MWE) extracts than in either acid (ACE)
or alkaline extracts (AKE); additionally, there were 40 and
27 identified compounds in HWE and MWE, respectively,
in comparison to 16 and 13 in ACE and AKE, respectively.
The ACE and AKE extracts did not demonstrate any bioactive
effects, indicating the importance of the extraction solvent
employed. In our study we found that the ethanolic extracts
demonstrated greater anti-inflammatory potential than the
mushroom extracts prepared using hot water.
In conclusion, a preliminary screen of extracts from 27
different wild Irish mushroom species demonstrated that
seven extracts prepared using ethanol and one hot water
extract significantly decreased the production of IL-6 in
LPS-stimulated RAW264.7 cells. Further investigation re-
vealed that extracts prepared from R. mairei, L. blennius,
C. tubaeformis, R. fellea, and C. cornucopioides caused
selective anti-inflammatory effects by decreasing NO and
IL-6 in LPS-stimulated mouse macrophage cells. To date,
limited information is available on the chemical composition
of these anti-inflammatory species, but based on the activities
6 O’CALLAGHAN ET AL.
demonstrated in this study these species warrant further in-
vestigation to determine the precise chemical identity of anti-
inflammatory compounds responsible for the activities ob-
served. In addition, while more detailed studies would be
required, extracts from the five species outlined just now have
demonstrated anti-inflammatory potential.
ACKNOWLEDGMENT
All authors read and contributed toward the final article.
AUTHOR DISCLOSURE STATEMENT
The authors declare that no competing financial interests
exist.
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