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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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Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. 23, 575–578 (2009)
DOI: 10.1002/ptr
Copyright © 2008 John Wiley & Sons, Ltd.
Phytother. Res. 23, 575–578 (2009)
Published online 9 December 2008 in Wiley InterScience
( DOI: 10.1002/ptr.2698
Cyclooxygenase-2 Inhibitory and Antioxidant
Compounds from the Truffle Elaphomyces
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
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
while syringic acid strongly inhibited COX-2 activity with an IC50 of 0.4 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
μg/mL. Of the pure compounds, syringic acid
displayed a strong antioxidant activity, with an IC50 of 0.7 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.
Contract/grant sponsor: USDA Agricultural Research Service Specific
Cooperative Agreement; contract/grant number: 58-6408-2-0009.
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
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
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
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
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
(CDCl3, 400 MHz):
9.81 (1H, s, CHO), 7.14 (2H, s,
H-2, H-6), 3.95 (6H, s, 2OCH3); 13C NMR (CDCl3,
100 MHz):
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):
7.21 (2H, s, H-2, H-6), 3.79 (6H, s,
2OCH3); 13C NMR (DMSO-d6, 100 MHz):
(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
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
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).
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
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
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
Figure 3. Effect of syringic acid on DCF production in HL-60
cells. Each data point represents the mean ± SD of duplicate
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.
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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To better understand the pharmacological characters of syringaldehyde (SA), which is a key-odorant compound of whisky and brandy, this review article is the first to compile the published literature for molecular docking that were subsequently validated by in vitro and in vivo assays to predict and develop insights into the medicinal properties of SA in terms of anti-oxidation, anti-inflammation, and anti-diabetes. The molecular docking displayed significantly binding affinity for SA towards tumor necrosis factor-α, interleukin-6, and antioxidant enzymes when inflammation from myocardial infarction and spinal cord ischemia. Moreover, SA nicely docked with dipeptidyl peptidase-IV, glucagon-like peptide 1 receptor, peroxisome proliferator-activated receptor, acetylcholine M2 receptor, and acetylcholinesterase in anti-diabetes investigations. These are associated with (1) an increase glucose utilization and insulin sensitivity to an anti-hyperglycemic effect; and (2) to potentiate intestinal contractility to abolish the α-amylase reaction when concurrently reducing retention time and glucose absorption of the intestinal tract to achieve a glucose-lowering effect. In silico screening of multi-targets concomitantly with preclinical tests could provide a potential exploration for new indications for drug discovery and development.
... 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). ...
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.
... Hexane extracts from basidiomes of G. frondosa resulted in COX-2 inhibition due to three ergosterol-like terpenoids (Gregori et al. 2016). Analogously, ethanolic extract from Elaphomyces granulatus Fr. ("deer's truffle") were reported to inhibit COX-2 in macrophages of RAW 264.7 mice by two novel bioactive derivatives of syringaldehyde and syringic acid (Stanikunaite et al. 2009). ...
Medicinal mushrooms are a rich source of bioactive molecules, nutraceutic products, as well as natural co-formulants and excipients. The fungal cell wall is rich in branched polysaccharides (mainly β-glucans), peptidic residues, and chitin or chitosan. A plethora of secondary metabolites with current or potential application includes e.g. terpenoids, polyketides, ceramides, polyphenolics, l-ergothioneine and others.Mushroom application in cosmetics has been explored for a few decades only, but the increasing demand for bio-based products has stimulated this research field. Strategies against skin aging are the main target of cosmetic and cosmeceutic applications. Based on an integrated approach that pursues psycho-physical wellness, Systemic Aesthetic Medicine focuses on both external (creams, lotions) and internal (food supplements) uses of fungal molecules and extracts, besides the introduction in the diet of bulk fungal biomass (nutritional strategy). Far beyond the pure cosmetology, Systemic Aesthetic Medicine in fact focuses on prevention and treatment of skin diseases and cutaneous symptoms. Main properties reported from mushrooms at this concern include: hydration, anti-oxidant, acceleration of skin cells turnover, stimulation of skin bio-reparation, antiseptic, and immunomodulant.KeywordsMedicinal mushroomsSkinSystemic aesthetic medicineCosmeticCosmeceutic
... Consistent with our results, 21 bioactive compounds were reported to participate in the COX-2 inhibitory pathway in different forms. It is well known that LPS-induced reactive oxygen species production leads to COX-2 induction, and compounds 1, 3,9,15,16,19, and 20 were found to effectively inhibit the production of LPS-induced COX-2 metabolites (20)(21)(22)(23). It has been reported that compound 8 suppressed UVBinduced COX-2 expression by blocking Fyn kinase activity (24). ...
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Inhibition of cyclooxygenase-2 (COX-2) activity is an effective way for treatment of coronary heart disease. And as an important source of COX-2 inhibitors, bioactive compounds of Choerospondias axillaris and pharmacological mechanisms remained lacking in prospective researches. Therefore, for the purpose of accelerating the discovery of natural products targeting designed inhibitors, the COX-2 microreactor composed of functionalized microspheres and magnetic ligand fishing was developed and applied in Choerospondias axillaris , and the physicochemical properties of the COX-2 functionalized microspheres were characterized using Fourier transform infrared spectroscopy (FT-IR), vibrating sample magnetometer (VSM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Furthermore, the bioactive compounds singled out from ethanol decoction without prepurification were dissociated and identified by ultraperformance liquid chromatography plus Q-Exactive Orbitrap tandem mass spectrometry (UPLC-Q-Exactive Orbitrap-MS/MS). Consequently, 21 bioactive compounds consisting of 6 organic acids, 8 flavonoids, and 7 others were separated and characterized from Choerospondias axillaris , which were reported to participate in the COX-2 inhibitory pathway to varying degrees. Therefore, this method could provide a prospective solution for the extraction and identification of active pharmaceutical ingredients and the rapid screening of some enzyme inhibitors in the complex mixtures.
... The antioxidant activities of some truffles species such as Tuber indicum, Tuber magnatum, and Picoa juniperi, among others, were evaluated in previous studies but usually using in vitro colorimetric tests (DPPH, ABTS, etc.) [34,[46][47][48]. These assays might be interesting as preliminary screening through many samples, but their biological significance is limited since antioxidants must pass through the cell membrane and reach their targets. ...
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A PLE (pressurized liquid extraction) method was adjusted following a full-factorial experimental design to obtain bioactive-enriched fractions from Tuber aestivum and Terfezia claveryi. Temperature, time and solvent (water, ethanol and ethanol–water 1:1) parameters were investigated. The response variables investigated were: obtained yield and the levels of total carbohydrate (compounds, β-glucans, chitin, proteins, phenolic compounds and sterols). Principal component analysis indicated water solvent and high temperatures as more adequate parameters to extract polysaccharide-rich fractions (up to 68% of content), whereas ethanol was more suitable to extract fungal sterols (up to 12.5% of content). The fractions obtained at optimal conditions (16.7 MPa, 180 °C, 30 min) were able to protect Caco2 cells from free radical exposure, acting as antioxidants, and were able to reduce secretion of pro-inflammatory cytokines in vitro: IL-6 (50%), and TNFα (80% only T. claveryi ethanol extract), as well as reduce high inhibitory activity (T. aestivum IC50: 9.44 mG/mL).
... Compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) were isolated chromatographically, their structures were estab-62 lished using NMR and MS techniques and compared to related compounds in the litera-63 ture ( Figure 1). This includes 2-deethoxy-2β-hydroxyphantomolin (1) [5], 2β-methoxy-2-deethoxyphantomolin (2) [13], 2-deethoxy-8-O-deacylphantomolin-8-O-tiglinate (3) [14], 8-O-methacryloylelephantane (4) [5], 2,4-bis-O-methyl-8-O-methacryloy lelephantane (5), molephantin (6), molephantinin (7) [15], isochlorogenic acid (8) [16], 1,5-dicaffeoylquinic acid (9) [17], 4,5-dicaffeoylquinic acid (10) [17], apigenin (11) [18], luteolin (12) [19], tricin (13) [20], 2,6-dimethoxy-4-hydroxybenzoic acid (14) [21], 4-formylsyringol (15) [22], cryptomeridiol (16) [23], β-sitosterol-3-O-β-D-glucopyranoside-6 -O-palmitate (17) [24], β-sitosterol-3-O-β-D-glucopyranoside (18) [24] and glycerine monopalmitate (19) [24]. ...
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A total of nine sesquiterpenoid lactones together with phenolic compounds and other terpenes were identified from the crude methanol extract of Elephantopus mollis Kunth. Compounds were isolated using different chromatographic techniques and their structures were determined by NMR and IR spectroscopy as well as mass spectrometry. The structures of some detected compounds were assigned based on LC-ToF-ESI-MS screening of main fractions/subfractions from flash chromatography and comparison with isolated analogues as standards. The findings revealed not only the in-source loss of water as the base peak in hirsutinolides but also the in-source loss of corresponding alcohol when the oxygen at position 1 is alkylated. The present study also draws up a complement of data with respect to hirsutinolide-like sesquiterpene lactones whose LC-MS characteristics are not available in the literature. The chemophenetic significance is also discussed. Some of the isolated compounds were reported for the first time to be found in the species, the genus as well as the plant family. The medium-polar fractions of the crude extract, also containing the larger amount of sesquiterpenoid lactones, exhibited activity both against a cancer cell line and bacterial strains. Isolated lactones were also active against the cancer cell line, while the chlorogenic derivatives also valuable in Elephantopus genus showed potent radical scavenging activity. This is the first report of cytotoxic and antibacterial activities of our samples against the tested strains and cell line. The present study follows the ongoing research project dealing with the characterization of taxa with antibacterial and antiparasitic activities from Cameroonian pharmacopeia
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Background Terfezia claveryi truffles are known for their nutritional value and have been considered among traditional treatments for ophthalmic infections and ailments. Objectives We sought to investigate the in vitro antimicrobial efficacy of several T. claveryi extracts from Saudi Arabia. Certain pathogenic fungi and gram-negative and gram-positive bacteria were included. Methods Dry extracts were prepared using methanol, ethyl acetate, and distilled water, while the latter was used for preparing fresh extracts. The extracts were microbiologically evaluated through the disc-diffusion agar method; the zones of inhibition of microbial growth were measured post-incubation. The minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) were determined in Müller-Hinton Broth through the microdilution susceptibility method. Anti-biofilm activity was assessed for potent extracts. Results Dry extracts showed potent activity (> 16-mm inhibition zones) against gram-positive (Bacillus subtilis IFO3007 and Staphylococcus aureus IFO3060) and gram-negative (Pseudomonas aeruginosa IFO3448 and Escherichia coli IFO3301) bacteria. The activity against fungi was moderate (12–16-mm inhibition zones) for both Aspergillus oryzae IFO4177 and Candida albicans IFO0583; there was no activity against Aspergillus niger IFO4414 growth. Methanolic extract had the lowest MIC and MBC, exhibiting remarkable activity against B. subtilis growth. Fresh extract showed moderate activity against bacterial growth and inactivity against fungal growth. Methanolic extract showed potent anti-biofilm activity (IC50, 2.0 ± 0.18 mg/mL) against S. aureus. Conclusions T. claveryi extracts showed antibacterial effects potentially suitable for clinical application, which warrants further in-depth analysis of their individual isolated compounds.
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Mushrooms fortified with iron (Fe) can offer a promising alternative to counter the worldwide deficiency problem. However, the factors that may influence the efficiency of fortification have not yet been fully investigated. The aim of this study was to compare the effects of three Fe forms (FeCl3 6H2O, FeSO4 7H2O, or FeHBED) in three concentrations (5, 10, or 50 mM) for three mushroom species (Pleurotus eryngii, P. ostreatus, or Pholiota nameko) on their chemical composition, phenolic compounds, and organic acid production. The most effective metal accumulation of all the investigated species was for the 50 mM addition. FeCl3 6H2O was the most favorable additive for P. eryngii and P. nameko (up to 145 and 185% Fe more than in the control, respectively) and FeHBED for P. ostreatus (up to 108% Fe more than in control). Additionally, P. nameko showed the highest Fe accumulation among studied species (89.2 ± 7.51 mg kg−1 DW). The creation of phenolic acids was generally inhibited by Fe salt supplementation. However, an increasing effect on phenolic acid concentration was observed for P. ostreatus cultivated at 5 mM FeCl3 6H2O and for P. eryngii cultivated at 5 mM FeCl3 6H2O and 5 mM FeSO4 7H2O. In the case of organic acids, a similar situation was observed. For P. ostreatus, FeSO4 7H2O and FeHBED salts increased the formation of the determined organic acids in fruiting bodies. P. eryngii and P. nameko were characterized by a much lower content of organic acids in the systems supplemented with Fe. Based on the obtained results, we recommend starting fortification by preliminarily indicating which form of the element is preferred for the species of interest for supplementation. It also seems that using an additive concentration of 50 mM or higher is most effective.
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This database was created and is administered as a cooperative effort between the US Forest Products Laboratory and the US Dairy Forage Research Center. It was designed to provide a coherent, single source of NMR data of lignin model compounds as well as compounds modeling similar structures in grasses and other forage plants. The database exists in four different formats: an interactive HyperCard© stack for the Macintosh® computer, a FileMaker Pro© database for cross-platform use, an Adobe© pdf cross-platform file for viewing and printing, and a hardcopy version derived from the FileMaker Pro database. The first three versions are available for downloading over the internet from the Dairy Forage Research Center web site: The hardcopy is available by request from the authors at the Forest Products Laboratory. The use of trade or firm names in this publication is for reader information and does not imply endorsement by the US Dept. of Agriculture of any product or service. In general 13 C NMR data was collected in three common deuterated solvents (acetone, chloroform and dimethyl sulfoxide) for each compound. The 1 H NMR data was reported for one solvent. A standard set of acquisition parameters was used to acquire and process the spectra to keep the data as uniform and constant as possible. Those compounds with an index number less than 1000 were run on a Bruker 250 MHz spectrometer at FPL and those compounds with an index number between 1000 and 10,000 were run at the DFRC on a Bruker 360 MHz instrument. The order of the compounds in the database reflects their arrival at the spectrometer rather than a preordained plan. Search routines for the software versions allow grouping the compounds with similar traits, whereas the structure index is most useful for the hardcopy version. The inclusion of many analogous series of structures with small structural differences allows calculation of substituent effects that are invaluable for chemical shift predictions of structures not included in the database. The chemical shift assignments for most of the compounds were made by comparison with other compounds, literature values and in some cases other NMR experiments such as long and short range C-H correlations, COSY and DEPT. Every effort was made to correctly assign the chemical shifts; however, limited time and resources precluded confirming the shifts for many of the compounds. The shifts are reported to the second decimal place only to distinguish very close shifts however comparisons between spectra are practical only within ± 0.1 ppm. The authors would greatly appreciate any corrections on misassignments.
Effects of 3, 5 - dimethoxy - 4 - hydroxybenzoic acid and 2, 3, 4 - trihydroxyacetophenone were studied on haemoglobin S (Hb S) polymerisation, analgesia and inflammation using Hb S solution, rats and mice. UV spectrophotometric procedure was used to monitor the polymerization of the Hb S. Acetic acid induced writhing in mice and egg albumin induced rat paw edema procedures were used to evaluate analgesic and anti-inflammatory activities of the compounds respectively. The results indicate that both drugs inhibit the process of polymerization significantly, possibly by direct action on the Hb S molecules. The drugs inhibited acetic acid induced pain and decreased egg albumin induced oedema. It is concluded that 3, 5 - dimethoxy - 4 - hydroxybenzoic acid and 2, 3, 4 - trihydroxyacetophenone may have some value in the management of sickle cell disease.
We previously reported that oroxylin A, a polyphenolic compound, was a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In the present study, three oroxylin A structurally related polyphenols isolated from the Chinese herb Huang Qui, namely baicalin, baicalein, and wogonin, were examined for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. The results indicated that these three polyphenolic compounds inhibited LPS-induced NO production in a concentration-dependent manner without a notable cytotoxic effect on these cells. The decrease in NO production was in parallel with the inhibition by these polyphenolic compounds of LPS-induced iNOS gene expression. However, these three compounds did not directly affect iNOS enzyme activity. In addition, wogonin, but not baicalin or baicalein, inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity. Furthermore, N-nitro-l-arginine (NLA) and N-nitro-l-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS (but not COX-2) protein expression, which was inhibited by these three polyphenolic compounds. Wogonin, but not baicalin or baicalein, similarly inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cells. These results indicated that co-treatment with NOS inhibitors and polyphenolic compounds such as wogonin effectively blocks acute production of NO and, at the same time, inhibits expression of iNOS and COX-2 genes.
Four new iridoid glucosides 1–4, named blumeosides A–D, were isolated from the methanolic stem-bark extract of Fagraea blumei G. DON. (Loganiaceae). They were accompanied by the benzyl-alcohol derivative di-O-methylcrenatin (5) and the flavone C-glucoside swertisin (6). The structures of 1–4 were established by spectroscopic methods, including FAB-MS, and 1H- and 13C-NMR, and by alkaline hydrolysis. Blumeosides A (1) and C (3) are 10-O-(2,5-dihydroxytercphthalo) adoxosidic acid and 10-O-(2-hydroxyterephthalo)adoxosidic acid, respectively. In blumeosides B (4) and D (2), both carboxylic groups of the terephthalic-acid moiety are esterified by adoxosidic-acid units, Blumeosides A–D (1–4) inhibited bleaching of crocin induced by alkoxyl radicals. Blumeosides A (1) and D (2) also demonstrated scavenging properties towards the 2,2-diphenyl-1-picryl-hvdrazvl (CDPPH) radical in TLC autographic and spectrophotometric assays.