European Journal of Pharmacology 597 (2008) 86–91

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European Journal of Pharmacology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Immunopharmacolgy and Inflammation

Anti-inflammatory and analgesic properties in a rodent model of a (1→3),(1→6)-linked

β-glucan isolated from Pleurotus pulmonarius
Fhernanda R. Smiderle a, Lorena M. Olsen a, Elaine R. Carbonero a, Cristiane H. Baggio b, Cristina S. Freitas b,
Rodrigo Marcon c, Adair R.S. Santos c, Philip A.J. Gorin a, Marcello Iacomini a,d,⁎
a
Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, CP 19046, 81531-980, Curitiba, PR, Brazil
b
Departamento de Farmacologia, Universidade Federal do Paraná, CP 19031, 81531-980, Curitiba, PR, Brazil
c
Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, 88040-900, Florianópolis, SC, Brazil
d
Departamento de Pedagogia, Faculdade Doutor Leocádio José Correia, 82640-070, Curitiba, PR, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: A glucan was extracted with hot water from the basidiomycete Pleurotus pulmonarius and shown to have a (1→3)-
Received 18 April 2008 linked β-D-glucopyranosyl main-chain substituted at O-6 of every third unit by single β-D-glucopyranosyl non-
Received in revised form 18 August 2008 reducing end units. This was shown by mono- and bidimensional nuclear magnetic resonance (NMR)
Accepted 21 August 2008
spectroscopy, methylation analysis, and a controlled Smith degradation. The glucan was tested for its effects on
Available online 29 August 2008
the acetic acid-induced writhing reaction in mice, a typical model for quantifying inflammatory pain. It caused a
Keywords:
marked and dose-dependent anti-inflammatory response, demonstrated by the inhibition of leukocyte migration
Pleurotus pulmonarius to injured tissues (82 ± 6%) with an ID50 of 1.19 (0.74–1.92) mg/kg. Furthermore, animals previously treated with
Mushroom the glucan (3 mg/kg i.p.), showed a reduction of 85± 5% of writhes, after receiving the acetic acid injection.
β-glucan Furthermore, in the formalin test, the glucan (3–30 mg/kg, i.p.) also caused significant inhibition of both the early
Chemical analysis (neurogenic pain) and the late phases (inflammatory pain) of formalin-induced licking. However, it was more
Anti-inflammatory and analgesic effects potent and effective in relation to the late phase of the formalin test, with mean ID50 values for the neurogenic and
the inflammatory phases of N 30 and 12.9 (6.7–24.6) mg/kg and the inhibitions observed were 43± 5% and 96± 4%,
respectively. These data showed that the glucan had potent anti-inflammatory and analgesic (antinociceptive)
activities, possibly by the inhibition of pro-inflammatory cytokines.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction addition, they can stimulate the production of proinflammatory

A wide variety of bioactive compounds, isolated from many species of
mushrooms, have been identified (Wasser, 2002). Among these, most of
them were terpenoids, steroids, fatty acids, proteins, lectins, proteoglycans,
and especially polysaccharides (Liu et al., 2007; Moradali et al., 2007).
Basidiomycetes have been widely studied over the past thirty years in
terms of their polysaccharide composition and therapeutic application
(Smith et al., 2003; Zhang et al., 2007). The main polysaccharides present
in mushrooms are structurally different β-glucans (Rowan et al., 2003;
Carbonero et al., 2006), fucomannogalactans (Alquini et al., 2004), xylo-
mannans (Smiderle et al., 2006), and mannogalactans (Rosado et al., 2003).
β-Glucans are a basis of fungal cell wall structure. They are not found
in animals, so that as carbohydrates they can be recognized by the innate
immune system of vertebrates (Brown and Gordon, 2003). In vitro
experiments showed that β-glucans can directly activate leukocytes,
stimulating their phagocytic, cytotoxic, and antimicrobial activity. In
⁎ Corresponding author. Departamento de Bioquímica e Biologia Molecular, Universi-
dade Federal do Paraná, CP 19046, 81531-980, Curitiba, PR, Brazil. Tel.: +55 41 33611655;
fax: +55 41 3266 2042.
E-mail address: iacomini@ufpr.br (M. Iacomini).
mediators, such as cytokines and chemokines (Schepetkin and Quinn,
2006), and have anti-tumor (Mizuno et al., 1990), anti-oxidative (Toklu
et al., 2006), anti-inflammatory (Dore et al., 2007), and immunomodu-
lating (Zhang et al., 2007) activities.
Studies on anti-inflammatory properties of carbohydrates have led to
positive results. Experiments on mice showed that acetic acid injection
induces an inflammatory process by causing tissue injury. Some conse-
quences of inflammation are increased capillary permeability and leu-
kocyte migration to damaged areas. The administration of drugs, such as
dexamethasone and indometacin, before such damage, diminish these
effects due to their anti-inflammatory activity (Vinegar et al.,1979). Anti-
nociception can also be demonstrated in this experimental model, since
the pro-inflammatory mediators cause hyperalgesia (Quinn, 2002).
Extracts of mushrooms have been tested for these activities, although
these contained mainly terpenoids and lipids and not polysaccharides
(Park et al., 2005; Kim et al., 2007; Diyabalanage et al., 2008).
Since there have been few investigations on their therapeutic
properties, we have now isolated and determined the detailed chemical
structure of a β-glucan from the edible mushroom Pleurotus pulmonar-
ius. Its analgesic (antinociceptive) and potential anti-inflammatory
effects were measured using a rodent model.

0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2008.08.028
 F.R. Smiderle et al. / European Journal of Pharmacology 597 (2008) 86–91
2. Materials and methods
2.1. General experimental procedures
Gas liquid chromatography-mass spectrometry (GC-MS) was
performed using a Varian (model 3300) gas chromatograph linked
to a Finnigan Ion-Trap model 810 R-12 mass spectrometer, with He as
carrier gas. A capillary column (30 m × 0.25 mm i.d.) of DB-225, held at
50 °C during injection and then programmed at 40 °C/min to 220 °C or
210 °C (constant temperature) was used for qualitative and quanti-
tative analysis of alditol acetates and partially O-methylated alditol
acetates, respectively (Sassaki et al., 2005). Nuclear magnetic
resonance (NMR) (13C and coupled 1H (obs.),13C heteronuclear multi-
ple quantum correlation (HMQC) spectra were obtained using a
400 MHz Bruker model DRX Avance spectrometer incorporating
Fourier transform. Samples were dissolved in dimethyl sulfoxide-d6
and examined at 70 °C. Chemical shifts are expressed in ppm (δ)
relative to resonances of acetone at δ 30.20 (13C) and 2.22 (1H).
2.2. Fungal material
The mushroom Pleurotus pulmonarius (Fr.) Quel. was furnished by
Renato A. Yamasita, Fazenda Bom Jesus das Araucárias, located in the
town of Reserva (24°39′05″ S; 50°50′40″ W), State of Paraná (PR),
Brazil. A voucher specimen was deposited in the Museu Botânico
Municipal (n° 332755), located in Curitiba-PR, Brazil.
2.3. Polysaccharide extraction and purification
The dried mushroom (200 g) was milled and the powder was
extracted with chloroform-methanol (4:1 v/v) at 60°C for 3 h (×3, 350 ml
each), to remove apolar compounds. The residue was submitted to
successive cold and hot aqueous extraction for 6 h (×7, 3000 ml each).
Each aqueous extract was evaporated to a small volume and the
polysaccharide was precipitated by addition to excess ethanol (3:1 v/v)
and centrifuged at 13,600 g at 10 °C for 20 min. The sediment was
dialyzed against tap water for 24 h, concentrated under reduced
pressure and freeze-dried. The polysaccharides obtained from cold
aqueous extraction were analyzed in a previous investigation
(Smiderle et al., in press)and the hot aqueous extract was processed.
It was dissolved in water and the solution was submitted to a freeze-
thawing process (Gorin and Iacomini, 1984). The precipitate, obtained
after centrifugation (13,600 g at 4 °C for 20 min), was treated with 2%
aqueous sodium hydroxide (100 ml), for 3 h, at 80 °C. After neutrali-
zation (acetic acid), the solution was dialyzed against tap water for
24 h and then resubmitted to the freeze-thawing process, giving
rise to a soluble fraction of glucan after centrifugation (13,600 g at 4 °C
for 20 min). This purified polysaccharide was selected and then ana-
lyzed for its chemical structure and anti-inflammatory and analgesic
(antinociceptive properties).
2.4. Monosaccharide composition
Each polysaccharide fraction (1 mg) was hydrolyzed with 2 M
trifluoroacetic acid at 100 °C for 8 h, followed by evaporation to
dryness. The residue was successively reduced with excess of sodium
borohydride and acetylated with acetic anhydride-pyridine (1:1 v/v)
at room temperature for 12 h (Wolfrom and Thompson, 1963a,b). The
resulting alditol acetates were analyzed by GC-MS (see item 2.1), and
identified by their typical retention times and electron impact profiles.
2.5. Methylation analysis
Per-O-methylation of the glucan (10 mg) was carried out by
dissolution in dimethyl sulfoxide (1 ml), followed by iodomethane
(1 ml), and powdered sodium hydroxide (20 mg) (Ciucanu and Kerek,
87
1984). Each sample was submitted to vigorous stirring for 30 min, at
25 °C and maintained overnight, water then being added, the mixture
neutralized (acetic acid), and the solution dialyzed against distilled
water, and freeze-dried. The product was submitted to one more cycle
of methylation, and then partitioned between chloroform and water,
and the lower chloroform layer evaporated to dryness. The resulting
per-O-methylated mixture was treated with 45% (v/v) formic acid at
100 °C for 10 h, followed by evaporation to dryness (Stortz et al., 1997).
The residues were converted into partially O-methylated alditol
acetates, and analyzed by GC-MS (see item 2.1).
2.6. Controlled Smith degradation
The purified glucan (150 mg) was submitted to oxidation with
0.05 M aqueous sodium periodate (20 ml) for 72 h at 25 °C in the dark
(Goldstein et al., 1965; Gorin et al., 1965; Delgobo et al., 1998). The
solution was then dialyzed against tap water for 48 h and treated
with sodium borohydride (pH 9–10) for 20 h, and after dialysis, the
sample was freeze-dried. An aliquot (10 mg) was methylated and ana-
lyzed by GC-MS, and another one (40 mg) was submitted to 13C NMR
spectroscopy.
2.7. Determination of homogeneity of glucan
The determination of homogeneity of the purified glucan was
performed on a Waters high-performance size-exclusion chromato-
graphy (HPSEC) apparatus coupled to a differential refractometer (RI)
and a Wyatt Technology Dawn-F Multi-Angle Laser Light Scattering
detector (MALLS). Waters Ultrahydrogel columns (2000, 500, 250 and
120) were connected in series and coupled to multidetection
equipment, using a 0.1 M sodium nitrite solution as eluent, containing
0.5 g/l sodium azide, at 25 °C. The polysaccharide solution (1 mg/ml)
was dissolved in the same solvent and filtered through a nitrocellulose
Millipore® membrane, with pores of 0.22 μm. HPSEC data were
collected and analyzed by the Wyatt Technology ASTRA program.
2.8. Experimental animals
Male Swiss mice (25–35 g) were kept in an automatically
controlled temperature room (23 ± 2 °C) in 12 h light–dark cycles,
with freely available water and food. Animals were acclimatized to the
laboratory for at least 2 h before testing and were used only once for
experiments. These were performed after following the protocol by
the Institutional Ethics Committee of the Federal University of Santa
Catarina (n: 23080.0011700/2005-03/UFSC) and were carried out in
accordance with current guidelines for the care of laboratory animals
and ethical guidelines for investigation of experimental pain in
conscious animals (Zimmermann, 1983). The number of animals and
intensity of noxious stimuli used were the minimum necessary to
demonstrate consistent effects of the drug treatments.
2.9. Abdominal constriction, peritoneal capillary permeability and
leukocyte infiltration caused by intraperitoneal injection of 0.6% aqueous
acetic acid
The abdominal constrictions were induced according to previously
described procedures (Santos et al., 1999; Lucena et al., 2007), This
resulted in contraction of the abdominal muscle together with a
stretching of the hind limbs in response to an intra-peritoneal (i.p.)
injection of 0.6% aqueous acetic acid (0.45 ml/mouse, made up in
saline), at the time of the test. At the beginning of the experiment,
mice were pre-treated intravenously with 2.5% Evans blue dye
solution (10 ml/kg), used as a peritoneal capillary permeability
marker. One hour later, mice were pre-treated with the glucan by i.
p. (0.3–3 mg/kg). In a separate series of experiments, was investigated
the effect of indomethacin (10 mg/kg, i.p.) or dexamethasone (2 mg/kg
88 F.R. Smiderle et al. / European Journal of Pharmacology 597 (2008) 86–91

ments using linear regression GraphPad software (Graph Pad software,

San Diego, CA, USA). The statistical significance of differences between
groups was detected by ANOVA followed by Newman-Keuls test.
P-values less than 0.05 were considered to be significant.

3. Results

A β-glucan was isolated from the mushroom Pleurotus pulmonarius

and its anti-inflammatory and analgesic (antinociceptive) activities
were tested in a rodent model. Its chemical structure was determined
in detail, in order to compare it and other glucans, in future studies,
with their therapeutic properties.

3.1. Chemical structure of the purified β-glucan

A polysaccharide fraction was obtained from milled mushroom via

hot aqueous extraction, and was submitted to hot aqueous extraction
followed by a freeze-thawing process (Gorin and Iacomini, 1984).
Partial precipitation took place and centrifugation gave rise to soluble
(2.5% yield) and insoluble (6% yield) fractions (Fig. 1). GC-MS of alditol
Fig. 1. Extraction and purification of β-glucan. acetates, formed on successive acid hydrolysis, sodium borohydride
reduction, and acetylation, showed that the insoluble fraction
s.c.), used as positive controls 30 min and 4 h before the acetic acid consisted of xylose (2%), mannose (5%), galactose (3%), and glucose
injection, respectively. Negative control animals received a similar (90%). Since this fraction was gel-like when in contact with water, and
volume of vehicle (10 ml/kg). After the challenge, the mice were unsui table for biological tests, it was treated with aqueous sodium
placed individually into glass cylinders of 20 cm diameter, and the hydroxide at 80 °C and resubmitted to the freeze-thawing process. The
abdominal constrictions were counted over a period of 20 min. soluble fraction (4% yield), then obtained, was homogeneous when
Antinociceptive activity is expressed as the reduction in the number of analyzed by HPSEC, and was composed exclusively of glucose.
abdominal constrictions, the difference between negative control
animals and pre-treated mice (with glucan, indomethacin or dex-
amethasone). Immediately after the test, the animals were sacrificed
by cervical dislocation and their peritoneal cavity was washed with
1 ml cold sterile saline plus heparin (25 IU/ml) and after 30 s of gentle
manual massage, the exudate was retrieved, and the volume was
measured. Total cell counts were performed with a Neubauer chamber
via optical microscopy, after diluting a sample of the peritoneal fluid
with Türk solution (1:20). A sample of the collected fluid (700 μl) was
centrifuged at 320 g for 10 min and the absorbance of the supernatant
was read at 610 nm with an ELISA analyzer. The peritoneal capillary
permeability induced by acetic acid is expressed in terms of dye
(μg/ml), which leaked into the peritoneal cavity according to the stan-
dard curve of Evans blue dye (Lucena et al., 2007).

2.10. Formalin-induced nociception

The procedure used was essentially the same as that described

previously (Santos et al., 1999). Animals received 20 μl of a 2.5%
formalin solution (0.92% formaldehyde) made up in saline, injected via
intraplantar (i.pl.) administration in the ventral surface of the right
hindpaw. They were observed from 0 to 5 min (neurogenic phase)
and 15 to 30 min (inflammatory phase) and the time spent on licking
the injected paw was recorded with a chronometer and considered as
indication of nociception. Animals received an intraperitoneal injec-
tion of saline solution (10 ml/kg; control group) or glucan (3–30 mg/kg,
i.p.) 30 min before formalin injection. Each animal was placed in
the chamber for 5 min before irritant injection in order to allow
acclimatization to the new environment.

2.11. Statistical analysis

The results are expressed as means ± S.E.M., except for the ID50 values
(i.e. the dose of glucan reducing the abdominal constriction, peritoneal
capillary permeability and leukocyte infiltration responses or licking by
50%, relative to the control value), which are expressed as geometric
means accompanied by their respective 95% confidence limits. The ID50 Fig. 2. 13C NMR (A) and HMQC (B) spectra of native β-glucan in dimethyl sulfoxide-d6 at
values were determined by linear regression from individual experi- 70 °C: chemical shifts are expressed in δ ppm.
 F.R. Smiderle et al. / European Journal of Pharmacology 597 (2008) 86–91 89

Fig. 3. Chemical structure of β-glucan isolated from Pleurotus pulmonarius.

Methylation analysis of the glucan gave rise to partially O-methylated

alditol acetates corresponding to non-reducing end- (20%), 3-O- (58%)
and 3,6-di-O-substituted glucopyranosyl (22%) units. Fig. 4. 13C NMR spectrum of controlled Smith degraded β-glucan in dimethyl sulfoxide-
13
C NMR (Fig. 2A) and coupled 1H (obs.)13C HMQC spectroscopy d6 at 70 °C: chemical shifts are expressed in δ ppm.
(Fig. 2B) of the purified glucan showed signals characteristic of a fungal
β-glucan (Gorin, 1981; Barreto-Bergter and Gorin, 1983). The HMQC 2.63) mg/kg and inhibition of 85± 5% at a dose of 3 mg/kg (Fig. 5A). The
spectrum contained C1/H1 signals at δ 103.0/4.15 from non-reducing positive control, with indomethacin (10 mg/kg, i.p.), a non-steroidal anti-
end-units, and others at δ 103.0/4.44 corresponding to 3-O- (A) and inflammatory drug, gave rise to a nociception inhibition of 92 ± 5%, while
3,6-di-O-substituted Glcp (B) residues (Fig. 3). A β-glycosidic config- the glucocorticoid dexamethasone (2 mg/kg, s.c.), reduced partially but
uration was shown by C-1 signals at high (Hall and Johnson, 1969) and significantly the nociception, with an inhibition of 30 ± 8% (Fig. 5A).
H-1 signals at low frequency. Signals at δ 86.5 and 86.2 arose from To confirm the analgesic activity of the glucan, the formalin test
substitution at O-3 in units A, while those at δ 86.0 and 76.6 are from (Tjølsen et al., 1992) was performed. This is different from most pain
similar substitutions in units B and free O-3 by non-reducing end of
β-Glcp units (C), respectively. Non-substituted and O- substituted –CH2
signals are at δ 61.2; 60.9; 60.8 and 68.3, respectively. All the signals
were assigned (Table 1), based on literature data (Yoshioka et al., 1985;
Carbonero et al., 2006; Santos-Neves et al., 2008).
A controlled Smith degradation was carried out on the glucan frac-
tion and the product was analyzed by 13C NMR spectroscopy (Fig. 4).
Six signals at δ 102.9; 86.2; 76.4; 72.9; 68.5 and 61.0 were present,
which arose from C-1, C-3, C-5, C-2, C-4 and C-6, respectively,
consistent with a linear (1→3)-linked β-D-Glcp chain (Gorin, 1981).
This and above methylation and NMR data show that the polysacchar-
ide isolated was a β-D-glucan with such a main chain, substituted at O-
6 at every third unit, by single-unit β-D-Glcp side-chains (Fig. 3).

3.2. Analgesic properties of the purified β-glucan

In order to evaluate the glucan for its therapeutic properties, its

effect on the acetic acid-induced writhing reaction in mice was tested.
This test, described as a typical model for measuring inflammatory
pain, has long been used as a screening tool for assessment of analgesic
or anti-inflammatory properties of new agents (Collier et al., 1968).
It consists of administration of a 0.6% aqueous acetic acid injection
(0.45 ml/mouse, i.p.), which causes common effects of inflammation,
such as pain, leukocyte infiltration, and increase in capillary perme-
ability. The glucan was administrated (0.3–3 mg/kg, i.p.), 30 min prior
to irritant injection, and the results were noted (for more details, see
Materials and methods).
Painful sensations were quantified by the number of abdominal
writhes, counted after the acetic acid injection. The results indicated that
systemically administered glucan induced a dose-related inhibition of
the acetic acid-induced nociceptive response, with an ID50 of 1.26 (0.60–

Table 1
1
H and 13C NMR chemical shifts of native β-glucan

Units/assignmentsa 1 2 3 4 5 6
6a 6b
13
→3)-β-Glcp-(1→ C 103.0 72.6 86.5/86.2 68.5 76.4 60.9
1
H 4.44 3.24 3.41 3.20 3.19 3.62 3.40
13
→3,6)-β-Glcp-(1→ C 103.0 72.6 86.0 68.5 74.8 68.3
1 Fig. 5. Effect of i.p. administration of the glucan (0.3–3 mg/kg) on acetic acid-induced
H 4.44 3.24 3.41 3.20 3.42 3.98 3.48
13 abdominal constriction (A), leukocyte infiltration (B), and Evans blue leakage (C) in
β-Glcp-(1→ C 103.0 73.6 76.6 70.3 76.4 60.9
1 mice. Legend (Fig. 5). Data are expressed as means ± S.E.M.; n = 6–8 mice per group.
H 4.15 3.00 3.05 3.04 3.19 3.62 3.40
⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 vs. control (C), and #P b 0.001 vs. saline (V), ANOVA
a
The chemical shifts are expressed as ppm, δ. followed by Newman-Keuls test.
90 F.R. Smiderle et al. / European Journal of Pharmacology 597 (2008) 86–91
measurement models, since it is possible to evaluate the way an animal
responds to moderate, continuous pain generated by injured tissue.
This model is constituted by two distinct phases: the neurogenic pain
and the inflammatory pain. The early phase (neurogenic pain) results
from the direct irritating effect on nociceptors activating primary
afferent fiber, and the late phase (inflammatory pain) is mediated
by a combination of peripheral input and spinal cord sensitization
(Hunskaar and Hole, 1987; Tjølsen et al., 1992).
The nociceptive responses were quantified by the time of paw licking
after the formalin injection. The results depicted in Fig. 6A and B show
that the glucan caused significant inhibition of both neurogenic (0 to
5 min) and inflammatory phases (15 to 30 min) of formalin-induced
licking. However, its antinociceptive effects were significantly more
pronounced (P b 0.05) against the second phase of this model of pain. The
calculated mean ID50 values for these effects were N 30 mg/kg and 12.9
(6.7–24.6) mg/kg and the inhibitions observed were 43 ± 5% and 96± 4%
at a dose of 30 mg/kg, for the first and second phases, respectively.
3.3. Anti-inflammatory effect of the β-glucan
Neutrophils are the main leukocyte subtype participating in organism
defense, and their migration from blood vessels into the tissue is a crucial
process in the host-response against microorganism infections (Malech
and Gallin, 1987). Although they have a protective role in inflammation,
tissue damage is a deleterious consequence of the intense migration of
neutrophils, as observed in immune inflammatory diseases (Jones et al.,
1991). Thus, the results of Fig. 5B show that systemically administered
glucan (0.3–3 mg/kg) also caused a marked reduction of leukocyte infil-
tration (total cell migration) induced by acetic acid. The calculated mean
ID50 for this effect was 1.19 (0.74–1.92) mg/kg and the inhibition was
82 ±6%, at a dose of 3 mg/kg. Furthermore, dexamethasone (2 mg/kg, s.c.)
also caused a marked inhibition (92 ±2%) of this effect while indometha-
cin (10 mg/kg i.p.) showed a lower activity (39 ±12%) (Fig. 5B).
In addition, increased vascular permeability is one of the essential
features of the acute inflammatory response (Zhou et al., 2007). Its
development, when induced by acetic acid, is known to correspond to
Fig. 6. Effect of i.p. administration of the glucan (3–30 mg/kg) on formalin-induced licking
(neurogenic phase, panel A, and inflammatory phase, panel B) in mice. Legend (Fig. 6). Data
are expressed as means ± S.E.M.; n =6–8 mice per group. ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 vs.
control (C), and #P b 0.001 vs. saline (V), ANOVA followed by Newman-Keuls test.
the early exudative stage of inflammation (Whittle, 1964). Our results
indicated that systemically administered glucan only reduced by 37 ± 6%
(3 mg/kg) abdominal capillary permeability (Evans Blue dye exudation),
induced by the injurious injection, while indomethacin (10 mg/kg)
inhibited it almost totally, whereas dexamethasone (2 mg/kg) had no
effect (Fig. 5C).
Furthermore, the potent and concentration-dependent antinoci-
ception elicited by the glucan, given by the i.p. route, against the
inflammatory phase of formalin-induced pain, is particularly relevant,
as most of the non-steroidal anti-inflammatory drugs tested have
been reported to be largely effective in preventing the inflammatory
pain response induced by formalin (Shibata et al., 1989; Malmberg and
Yaksh, 1992; Corrêa and Calixto, 1993; Vaz et al., 1996).
4. Discussion
Fungi are well known to contain β-glucans with (1→3)-linked
β-D-glucopyranosyl main-chains partially substituted at O-6 with
single-unit β-D-glucopyranosyl side-chains, one of these being the
presently examined example (Fig. 3). However, they have a great
number of different chemical structures depending on the degree and/
or position of substitution with side chains, which can be either regular
or irregular (Barreto-Bergter and Gorin, 1983).
Fungal β-glucans give rise to a number of biological activities and our
objective is now to correlate anti-inflammatory and antinociceptive
activities with their chemical structure in the present and future
investigations. Various research groups have tested mushroom extracts
for these effects. Methanol extracts from Taiwanofungus camphoratus
(Liu et al., 2007), Inonotus obliquus (Park et al., 2005), and Agrocybe
aegerita (Diyabalanage et al., 2008) presented anti-inflammatory and
antinociceptive effects, but these extracts were only composed of apolar
compounds. However, polysaccharide-containing aqueous extracts
from Geastrum saccatum (Dore et al., 2007) and Ganoderma tsugae
(Lin et al., 2006) have been investigated. A crude extract with high
carbohydrate content from G. saccatum (Dore et al., 2007) gave rise to a
significant inhibition of polymorphonuclear cell migration (57.6%), but
only at a high dose of 30 mg/kg. Furthermore, the extract contained a
pool of structurally different glucans, so that the bioactive molecule(s)
could not be pinpointed. A supplementation with Ganoderma tsugae
extracts (Lin et al., 2006) significantly decreased, in mouse models, the
number of infiltrating leukocytes and lymphocytes, and also reduced
inflammatory mediators. However, the supplemented extract was a
mixture of carbohydrates, triterpenes, proteins, and others.
The most important transmission pathways for inflammatory
pain are ion-sensitive peripheral polymodal nociceptors (acid sensi-
tive ion channel, valinoid receptor 1, and glutamate receptors) and
algogenic substances (bradykinin, prostaglandin and cytokines as
Tumor Necrosis Factor-α, Interleukin-1β, Interleukin-8) (Ribeiro et al.,
2000; Ikeda et al., 2001). Our data indicate that the antinociceptive
and anti-inflammatory action of the glucan in the chemical pain
models (i.e. acetic acid-induced writhing and formalin-induced
licking) could be due to inhibition of cytokine release or antagonism
of acid sensitive ion channels, valinoid and/or glutamate receptors. It
follows that decreased levels of cytokines and lipid mediators in the
peritoneal cavity may be the reason for the reduction of cell migration.
Furthermore, reduction of vascular permeability could be associated
with reduced levels of prostaglandins and leukotrienes, as both are
inflammatory mediators related to vasodilatation (Doherty et al., 1985;
Kolaczkowska et al., 2002).
Our results demonstrate that the β-glucan has an activity similar to
those of non-steroidal anti-inflammatory and glucocorticoid drugs.
Hence, it can be suggested that the glucan inhibits pro-inflammatory
cytokines (such as Interleukin-1β and Tumor Necrosis Factor-α) released
by the injurious injection (Baggiolini et al., 1994; Ribeiro et al., 2000).
However, more experiments are necessary to identify the specific mech-
anism behind this inhibition of inflammation.
 F.R. Smiderle et al. / European Journal of Pharmacology 597 (2008) 86–91
Acknowledgements
The authors would like to thank the Brazilian funding agencies
CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior),
CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico),
FAPESC (Fundação de Apoio à Pesquisa Científica Tecnológica do Estado
de Santa Catarina), FINEP [Financiadora de Estudos e Projetos, Rede
Instituto Brasileiro de Neurociência (IBN-Net)] and Fundação Araucária
for financial support, and the Mr Renato A. Yamasita for furnishing fungi.
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