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Cyclooxygenase-2 inhibitory and antioxidant compounds from the truffle Elaphomyces granulatus

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The ethanol extract of fruiting bodies of Elaphomyces granulatus, a truffle-like fungus, was evaluated for cyclooxygenase-2 (COX-2) enzyme inhibitory and antioxidant activities. Inhibition of COX-2 activity was evaluated in mouse macrophages (RAW 264.7). The extract of E. granulatus caused a 68% inhibition of COX-2 activity at 50 microg/mL. Bioassay-guided investigation led to the isolation and identification of two active compounds, syringaldehyde and syringic acid. Syringaldehyde moderately inhibited COX-2 activity with an IC(50) of 3.5 microg/mL, while syringic acid strongly inhibited COX-2 activity with an IC(50) of 0.4 microg/mL. The antioxidant activity of the extract and isolated compounds was evaluated in HL-60 cells by the DCFH-DA method. The extract of E. granulatus showed a potent antioxidant effect, with an IC(50) of 41 microg/mL. Of the pure compounds, syringic acid displayed a strong antioxidant activity, with an IC(50) of 0.7 microg/mL, while syringaldehyde showed no activity in the assay.
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COMPOUNDS FROM THE TRUFFLE ELAPHOMYCES GRANULATUS 575
Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. 23, 575–578 (2009)
DOI: 10.1002/ptr
Copyright © 2008 John Wiley & Sons, Ltd.
PHYTOTHERAPY RESEARCH
Phytother. Res. 23, 575–578 (2009)
Published online 9 December 2008 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/ptr.2698
Cyclooxygenase-2 Inhibitory and Antioxidant
Compounds from the Truffle Elaphomyces
granulatus
Rita Stanikunaite1, Shabana I. Khan2, James M. Trappe3 and Samir A. Ross1,2*
1Department of Pharmacognosy, The University of Mississippi, University, MS 38677, USA
2National Center for Natural Products Research, The University of Mississippi, University, MS 38677, USA
3Department of Forest Science, Oregon State University, Corvallis, OR 97331-5752, USA
The ethanol extract of fruiting bodies of Elaphomyces granulatus, a truffle-like fungus, was evaluated for
cyclooxygenase-2 (COX-2) enzyme inhibitory and antioxidant activities. Inhibition of COX-2 activity was evaluated
in mouse macrophages (RAW 264.7). The extract of E. granulatus caused a 68% inhibition of COX-2 activity
at 50 μμ
μμ
μg/mL. Bioassay-guided investigation led to the isolation and identification of two active compounds,
syringaldehyde and syringic acid. Syringaldehyde moderately inhibited COX-2 activity with an IC50 of 3.5 μμ
μμ
μg/mL,
while syringic acid strongly inhibited COX-2 activity with an IC50 of 0.4 μμ
μμ
μg/mL. The antioxidant activity of the
extract and isolated compounds was evaluated in HL-60 cells by the DCFH-DA method. The extract of E.
granulatus showed a potent antioxidant effect, with an IC50 of 41 μμ
μμ
μg/mL. Of the pure compounds, syringic acid
displayed a strong antioxidant activity, with an IC50 of 0.7 μμ
μμ
μg/mL, while syringaldehyde showed no activity in
the assay. Copyright © 2008 John Wiley & Sons, Ltd.
Keywords: Elaphomyces granulatus; Ascomycota; hypogeous; truffle; COX-2; RAW 264.7 cells; antiinflammatory; antioxidants;
DCFH oxidation; HL-60 cells.
Received 4 May 2007
Accepted 29 July 2008
* Correspondence to: Samir A. Ross, National Center for Natural Prod-
ucts Research, P.O. Box 1848, University, MS 38677, USA.
E-mail: sross@olemiss.edu
Contract/grant sponsor: USDA Agricultural Research Service Specific
Cooperative Agreement; contract/grant number: 58-6408-2-0009.
INTRODUCTION
Truffles and truffle-like fungi are characterized by a hypo-
geous, i.e. underground, fruiting habit, having evolved
the use of animal mycophagy for the dispersal of their
spores. These fungi produce aromatic compounds by
which the animals locate them, dig them up and eat
them (Trappe and Claridge, 2005). The animals digest
the nutritious tissues of the fruiting bodies, but the spores
pass through the digestive tract and are defecated
unharmed (Claridge and Trappe, 2005). Because of their
underground fruiting habit, truffles are difficult to find,
hence research on their chemistry and medicinal prop-
erties has been limited and generally focused on the
volatile compounds responsible for their unique aromas
(Diaz et al., 2002, 2003).
In the course of our program aimed at the investiga-
tion of biological activity and chemistry of truffles from
North America, a study on Elaphomyces granulatus
Fr. (Elaphomycetaceae, Ascomycota) was initiated. The
most widely distributed and common hypogeous fungus
in the Northern Hemisphere, it occurs as a beneficial,
mycorrhizal symbiont with feeder rootlets of trees from
subarctic and subalpine forests south to the tropics
(Trappe, 1971). In Europe, E. granulatus has been used
as an aphrodisiac and a cheap but illegitimately marketed
substitute for more expensive truffles (Arora, 1986). The
analysis of the chemical composition of the spore mass
and outer layer of E. granulatus revealed the occurrence
of mixtures of higher aliphatic esters, free fatty acids,
hydrocarbons, mannitol, ergosterol, pyrocatechol, proto-
catechuic acid, salicylic acid, resorcinol, 3-hydroxy- and
4-hydroxy benzoic acids (Solberg, 1976). However, no
other studies of its biological activity and/or chemical
constituents have been reported.
Cyclooxygenase-2 (COX-2) enzyme plays an important
role in the inflammatory process. COX-2 is an induc-
ible isoform of cyclooxygenase enzyme responsible for
the production of pro-inflammatory prostaglandins in
neoplastic and inflamed tissues. COX-2 inhibitors have
a well established role in the treatment of inflamma-
tory disorders, as well as potential application for the
prevention and treatment of other diseases, such as
cancer (Flower, 2003; Amir and Agarwal, 2005). Reac-
tive oxygen species (ROS) and oxidative stress also play
an important role in the etiology and progression of
human degenerative diseases. ROS have been impli-
cated in inflammation, aging, cancer, heart disease and
other disorders (Pietta, 2000). Antioxidants act as ROS
scavengers and are important for protecting against
oxidative tissue damage in vital organs. Although,
numerous in vitro solution-based chemical assay systems,
such as 2,2-diphenyl-1-picrylhydrazyl radical (DPPH)
assay, have been used for the evaluation of antioxidants
(Cuendet et al., 1997), the use of 2,7-dichlorodihydro-
fluorescein diacetate (DCFH-DA) as a specific probe
in a cell based assay provides a better system to evalu-
ate an antioxidant effect in live cells. This method is
useful for the direct examination of ROS inhibitory
activity of natural products in live human cells
(Takamatsu et al., 2003; Choi et al., 2006).
As part of our investigation of biologically active
compounds from E. granulatus, the ethanol extract of
Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. 23, 575–578 (2009)
DOI: 10.1002/ptr
576 R. STANIKUNAITE ET AL.
fruiting bodies of E. granulatus was evaluated for COX-
2 enzyme inhibitory and antioxidant activities in cellu-
lar assays. The bioassay-guided isolation and biological
characterization of compounds from E. granulatus with
antioxidant and COX-2 inhibitory activities were also
performed.
MATERIALS AND METHODS
General experimental procedures. The 1H-NMR, 13C-
NMR, HMBC and HMQC spectra were recorded on a
Bruker DRX 400 MHz. The Bruker NMR spectrometer
operated at 400 MHz for 1H and 100 MHz for 13C. The
NMR spectra were recorded in ppm using the residual
solvent peak as an internal standard. The HRESIMS
data were acquired on a Bruker BioAPEX 30es mass
spectrometer.
Fungal material. Elaphomyces granulatus was collected
by Mr Adrian Beyerle of the North American Truffling
Society (NATS) in Oregon, Wasco Co., Mt Hood
National Forest, near 15-Mile Creek on 16 August 2002.
All specimens were identified by Dr James M. Trappe.
A voucher specimen is deposited in the USDA National
Fungus Collections, Beltsville, MD (BPI 864222). Mr
Adrian Beyerle subsequently provided additional speci-
mens from the area to supplement the original collection.
Additional collections were contributed by other NATS
members.
Extraction and isolation. The fruiting bodies of E.
granulatus were dried for 24 h in a forced air dehydrator
at 35 °C. Powdered material (482 g) was extracted
exhaustively by maceration with 95% EtOH at room
temperature, and the combined extracts were concen-
trated under reduced pressure to yield 12.5 g of residue.
The crude extract was divided into EtOH soluble and
insoluble fractions. The EtOH soluble fraction (9 g) was
subjected to a silica gel gravity column (230 g, 457 mm
× 51 mm) and eluted with chloroform, and chloroform–
methanol (2%–100%) to yield 16 fractions. The active
fractions 4–7 were combined (800 mg) and subjected
to another silica gel column (50 g, 450 mm × 17 mm)
with hexane–chloroform–ethyl acetate (3:3:1–1:1:1–0:1:1)
to give 108 fractions. Fractions 52–74 were combined
(61 mg) and separated on preparative TLC (Si gel
CF254, 250 μm, Uniplate) with hexane–chloroform–ethyl
acetate (1:1:1) to yield syringaldehyde (7 mg). Frac-
tions 98–102 were combined (63 mg) and separated
on preparative TLC (Si gel CF254, 250 μm, Uniplate)
with chloroform–methanol (2%) to yield syringic acid
(10 mg).
Syringaldehyde: pale yellow solid; HRESIMS m/z
183.0638 (calcd for C9H11O4 [M + H]+ 183.0657), 205.0457
(calcd for C9H10O4Na [M + Na]+ 205.0477); 1
H NMR
(CDCl3, 400 MHz):
δ
9.81 (1H, s, CHO), 7.14 (2H, s,
H-2, H-6), 3.95 (6H, s, 2OCH3); 13C NMR (CDCl3,
100 MHz):
δ
190.7 (CHO), 147.4 (C-3, C-5), 141.0 (C-
4), 128.4 (C-1), 106.8 (C-2, C-6), 56.5 (OCH3). Spectra
corresponded with previously reported data (Ralph
et al., 2004).
Syringic acid: pale yellow solid; HRESIMS m/z 199.0580
(calcd for C9H11O5 [M + H]+ 199.0606), 221.0398 (calcd
for C9H10O5Na [M + Na]+ 221.0426); 1H NMR (DMSO-
d6, 400 MHz):
δ
7.21 (2H, s, H-2, H-6), 3.79 (6H, s,
2OCH3); 13C NMR (DMSO-d6, 100 MHz):
δ
167.9
(COOH), 148.1 (C-3, C-5), 140.9 (C-4), 121.1 (C-1),
107.6 (C-2, C-6), 56.6 (OCH3). Spectra corresponded
with previously reported data (Ralph et al., 2004).
Cell-based assay for inhibition of COX-2 activity. Mouse
macrophages (RAW 264.7, ATCC) were cultured in
a 75 cm2 culture flask in RPMI-1640 medium (Gibco)
supplemented with 10% bovine calf serum (Hyclone)
and 60 mg/L amikacin (Sigma), at 37 °C in an environ-
ment of 95% humidity and 5% CO2. For the assay, the
cells were seeded in the wells of 96-well plates (50,000
cells/well) and incubated at 37 °C for 24 h. After wash-
ing with RPMI-1640 medium, supplemented with 3%
bovine calf serum, the cells were incubated with 5 μg/
mL LPS (Escherichia coli 055:B5, Sigma) for 16 h to
induce the production of COX-2. Induced cells were
washed thoroughly with medium to remove LPS com-
pletely, and treated with different concentrations of
test samples for 2 h. Arachidonic acid (300 μM, Sigma)
was added and the cells were further incubated for
30 min. The amount of PGE2 released in the medium
was determined with PGE2 enzyme immunoassay kit
(Cayman Chem. Co.). COX-2 activity was determined
by the conversion of exogenous arachidonic acid to
PGE2 and expressed as the percent of the vehicle con-
trol. The concentration that caused 50% inhibition
of enzyme activity (IC50) was calculated from the dose
curves generated by plotting percent COX-2 activity
against the test concentrations. NS-398 (Cayman Chem.
Co.), a specific inhibitor of COX-2, was included as a
positive control in each assay.
Assay for cytotoxicity to macrophages. RAW 264.7 cells
were cultured as described above. For the assay, cells
were seeded to wells of a 96-well plate at a density of
25 000 cells/well and incubated for 24 h. Different dilu-
tions of test compounds were added to the cells and
incubated for 48 h. Cell viability was determined by the
neutral red assay (Borenfreund et al., 1990). After in-
cubation, the medium was removed and 100 μL of fresh
medium containing 0.2 mg/mL neutral red (Sigma) was
added to each well and incubated for 90 min. The cells
were washed with saline (0.9% NaCl) to remove excess
dye. The solution of acidified isopropanol (0.33% HCl)
was then added to lyse cells. As a result, the incorpo-
rated dye was liberated from viable cells, the absorb-
ance of which was measured at 490 nm using the EL312e
plate reader (Bio-Tek instruments).
Assay for antioxidant activity. Myelomonocytic HL-60
cells (ATCC) were grown in RPMI 1640 medium sup-
plemented with 10% fetal bovine serum (Hyclone) and
60 mg/mL amikacin at 37 °C in an environment of 95%
humidity and 5% CO2. For the assay, 125 μL of the cell
suspension (1 × 106cells/mL) was added to the wells
of a 96-well plate. After treating with different concen-
trations of the test samples for 30 min, the cells were
stimulated with 100 ng/mL phorbol 12-myristate 13-
acetate (PMA, Sigma) for 30 min. DCFH-DA (Mole-
cular Probe, 5 μg/mL) was added and the cells were
incubated for 15 min. The levels of DCF produced were
measured on a PolarStar plate reader with an excita-
tion wavelength at 485 nm and emission at 530 nm as
COMPOUNDS FROM THE TRUFFLE ELAPHOMYCES GRANULATUS 577
Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. 23, 575–578 (2009)
DOI: 10.1002/ptr
described previously (Takamatsu et al., 2003; Choi et al.,
2006). The ability of the test materials to inhibit exo-
genous cytoplasmic ROS-catalysed oxidation of DCFH
to fluorescent DCF in HL-60 cells was measured in
comparison to PMA treated controls without the test
materials. The IC50 values were calculated from dose
curves of the % DCF production versus test concentra-
tions. Vitamin C (Sigma) was included as a positive
control.
Assay for cytotoxicity to HL-60 cells. Cytotoxicity of
the test samples to HL-60 cells was determined by the
XTT method after incubating the cells with test samples
for 48 h as described earlier (Takamatsu et al., 2003).
Briefly, 25 μL of XTT-PMS solution (1 mg/mL XTT
solution supplemented by 25 μM of PMS) was added
to each well. After incubating for 4 h at 37 °C, the
absorbance at 450 nm was measured on a plate reader
(EL312e; Bio-Tek instruments).
RESULTS AND DISCUSSION
The dried fruiting bodies of E. granulatus were exhaus-
tively extracted by maceration with 95% EtOH and
fractionated by various chromatographic techniques.
The bioassay-guided fractionation of E. granulatus led
to the isolation of two active compounds, syringaldehyde
and syringic acid (Fig. 1). Structures of syringaldehyde
and syringic acid were determined by using HRESIMS,
1H-NMR and 13C-NMR experiments.
The extract of E. granulatus and isolated compounds
were evaluated for inhibition of COX-2 activity in a
cell-based assay that utilizes the mouse macrophage cell
line (RAW 264.7). Unstimulated macrophages express
only a small amount of COX-2, while treatment with
bacterial lipopolysaccharide (LPS) leads to the induc-
tion of COX-2, which converts arachidonic acid to PGE2
(Chen et al., 2001). LPS-induced RAW 264.7 macrophages
were incubated in the presence, or absence, of test sam-
ples for 2 h, followed by the addition of arachidonic
acid. The effects of the test samples on COX-2 activity
were determined by measuring the PGE2 produced
in the culture medium. NS-398, a specific inhibitor of
COX-2, was used as a positive control (IC50: 0.2 μg/mL,
0.64 μM).
The extract of E. granulatus showed a potent COX-
2 inhibitory activity with 68% inhibition at 50 μg/mL.
Syringaldehyde inhibited COX-2 activity in a dose-
dependent manner (Fig. 2), with an IC50 of 3.5 μg/mL
(19.23 μM). Syringic acid showed a stronger inhibition
Figure 1. Structures of syringaldehyde and syringic acid.
Figure 2. Inhibition of COX-2 enzyme activity by syringaldehyde
() and syringic acid () in LPS-activated macrophages (RAW
264.7). Each data point represents the mean ± SD of triplicate
determination.
of COX-2 activity in a dose-dependent manner (Fig. 2),
with an IC50 of 0.4 μg/mL (2.02 μM).
The RAW 264.7 cell viability was determined to
exclude the possibility that the observed COX-2 inhibi-
tory effect was due to cytotoxicity. Examination of
cytotoxicity of syringic acid and syringaldehyde in RAW
264.7 macrophages by the neutral red assay indicated
that compounds did not affect the viability of RAW
264.7 cells in concentrations up to 25 μg/mL.
The results of this study demonstrate that syringal-
dehyde has a moderate COX-2 inhibitory activity, while
syringic acid is a strong inhibitor of the COX-2 enzyme.
This study is the first report on the occurrence of
syringaldehyde and syringic acid in E. granulatus, which
could account for the potent COX-2 inhibitory activity
of the mushroom extract. Although in previous studies
syringic acid has been reported to show antiinflam-
matory activity in vivo (Fernandez et al., 1998; Gamaniel
et al., 2000), this is the first report on its activity on the
COX-2 enzyme. The results of this study suggest that
the mechanism responsible for the antiinflammatory
activity of syringic acid might be related to COX-2
inhibition.
The antioxidant activity of the extract and isolated
compounds was evaluated in HL-60 cells using DCFH-
DA. This cell-based method examines directly the abil-
ity of test material to penetrate living cells and inhibit
ROS catalysed oxidation of DCFH to DCF. DCFH-
DA is a non-fluorescent probe that diffuses into cells.
Cytoplasmic esterases hydrolyse DCFH-DA to DCFH
which is oxidized to DCF (2,7-dichlorofluorescin) that
fluoresces. The antioxidant activity of test samples is
determined by measuring the level of DCF produced
in treated cells compared with controls.
The extract of E. granulatus showed a potent anti-
oxidant effect, with an IC50 of 41 μg/mL. The inhibitory
effect of syringic acid on DCF production is shown in
Fig. 3. Syringic acid displayed a strong antioxidant activity
in a dose-dependent manner, with an IC50 of 0.7 μg/mL
(3.54 μM) which is comparable to the effect of vitamin
C, a naturally occurring antioxidant, that showed an
IC50 of 0.5 μg/mL (2.84 μM) in the same assay. Examina-
tion of the cytotoxicity of syringic acid and syringaldehyde
in HL-60 cells indicated that compounds were not
cytotoxic up to a concentration of 31.25 μg/mL.
The results of this study indicate that syringic acid has
a strong antioxidant activity in the cellular-based assay
while syringaldehyde was inactive. Most of the previous
reports on the antioxidant properties of syringaldehyde
Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. 23, 575–578 (2009)
DOI: 10.1002/ptr
578 R. STANIKUNAITE ET AL.
Figure 3. Effect of syringic acid on DCF production in HL-60
cells. Each data point represents the mean ± SD of duplicate
determination.
and syringic acid have utilized solution-based chemical
assay systems that do not evaluate the antioxidant
activity within living cells. One recent study on ROS
production by human neutrophils, induced by opsonized
zymosan or phorbol 12-myristate 13-acetate (PMA),
found ROS inhibitory effects for both syringaldehyde
and syringic acid measured as luminol or lucigenin-
enhanced chemiluminescence (Worm et al., 2001).
This study is the first report on the potent antioxidant
and COX-2 enzyme inhibitory properties of the extract
of E. granulatus. In addition, COX-2 inhibitory activities
of syringaldehyde and syringic acid are reported here
for the first time. E. granulatus seems to have potential
health benefits due to its antioxidant and antiinflam-
matory effects. Consumption of E. granulatus as a dietary
supplement or as a food item could contribute to the
prevention of cancer and inflammatory disorders.
Acknowledgements
We want to thank Ms Shama Moktan and Mr John Trott for their
excellent technical help. We also want to thank Mr Adrian Beyerle
and other members of the North American Truffling Society for pro-
viding specimens of Elaphomyces granulatus. USDA Agricultural
Research Service Specific Cooperative Agreement No. 58-6408-2-0009
is acknowledged for partial support of this work.
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... Currently, mushrooms are part of most beauty products. Several mushroom-based lotions, serums, ointments, creams, and facial packs are used as moisturizers, whitening agents, or antiaging products (Mohorčič et al. 2007;Stanikunaite et al. 2009;Wu et al. 2016a). Mycelium-based leather is also a good substitute for bovine leather having similar features to bovine leather in terms of stiffness, strength, and moisture resistance (Meyer et al. 2021). ...
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The utilization of biological systems has been receiving considerable attention in the past couple of decades in the development of bio-based functional materials. This has been largely inspired by the use of green, biodegradable, and environmentally sustainable materials for the development of new functional biomaterials. The utilization of renewable resources for the production of materials introduces fast-growing and biodegradable fungal mycelium-derived materials for various applications. Mycelium secretes enzymes and decomposes the substrate to take nutrients for growth and make an interwoven three-dimensional network. The elastic, porous, stiff, and dense mycelia are rich in antioxidants, antiviral, and anti-inflammatory compounds. The properties of mycelium-derived materials are greatly dependent upon the feeding substrate, fungus type, and processing conditions. Both pure mycelial materials and their composites secure an important position in the race of utilization of renewable resources for material synthesis. This chapter summarizes the utilization of mycelium-based materials for numerous applications like cosmetics, medicine, textile, construction, packaging, and the food industry. It also describes the potential of mycelial-derived materials as an alternative to the traditional insulators, packaging materials, and bovine leather. It further explains the importance of mycelium-based functional foods, cosmetics, and medicines.
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