Structural characterization and protective effect against murine sepsis of fucogalactans from Agaricus bisporus and Lactarius rufus

Article abstract of DOI:10.1016/j.carbpol.2011.09.071

Fucogalactans from edible Agaricus bisporus (RFP-Ab) and wild Lactarius rufus (RFP-Lr) mushrooms were obtained on aqueous extraction followed by purification. RFP-Ab had M_w 43.8 × 10⁴ g mol ^-1 and RFP-Lr M_w 1.4

Full text of DOI:10.1016/j.carbpol.2011.09.071

Carbohydrate Polymers 87 (2012) 1620–1627
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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Structural characterization and protective effect against murine sepsis of
fucogalactans from Agaricus bisporus and Lactarius rufus
Andrea C. Ruthesa, Yanna D. Rattmanna, Elaine R. Carbonerob, Philip A.J. Gorina, Marcello Iacominia,∗
a Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, CP 19046, CEP 81531-980, Curitiba, PR, Brazil
b Departamento de Química, Universidade Federal de Goiás, Campus Catalão, 75704-020 Catalão, GO, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 August 2011
Received in revised form 2 September 2011
Accepted 23 September 2011
Available online 29 September 2011
Fucogalactans from edible Agaricus bisporus (RFP-Ab) and wild Lactarius rufus (RFP-Lr) mushrooms were
obtained on aqueous extraction followed by purification. RFP-Ab had Mw 43.8 × 104 g mol−1 and RFP-
Lr Mw 1.4 × 104 g mol−1. RFP-Lr had a (1 → 6)-linked ␣-d-Galp main-chain partially substituted at O-2
by nonreducing end-units of ␣-l-Fucp (29%). While RFP-Ab had a similar main chain, it was partially
substituted at O-2 by nonreducing end-units of ␣-l-Fucp (2.8%) and ␤-d-Galp (14.5%), and partially methy-
lated at HO-3. Both RFP-Lr and RFP-Ab were tested in mice against polymicrobial sepsis. Lethality rate,
myeloperoxidase (MPO) activity and cytokine levels were determined. It was observed a reduction in
late mortality rate by 62.5% and 50%, respectively, prevention of neutrophil accumulation in ileum and
decreasing in TNF-␣ and IL-1␤ serum levels.
Keywords:
Edible mushrooms
Agaricus bisporus
Lactarius rufus
Fucogalactans
Sepsis
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Several polysaccharides have been isolated from basid-
iomycetes, such as linear or branched glucans and heterogalactans,
Mushrooms have been used as therapeutic agents and valuable
food sources for centuries in Asia, especially because of their chem-
ical composition and nutritional values. They have high protein
and fiber contents with small amounts of fat (Lakhanpal & Rana,
2005; Manzi, Aguzzi, & Pizzoferrato, 2001), and can be considered
as functional foods, which can provide health benefits beyond the
traditional nutrients they contain.
The practice of using hot-water extracts and decocts of mush-
rooms in oriental medicine, particularly in China, is the basis of
modern studies on the medicinal properties. Approximately 300
species are known to have medicinal or nutraceutical properties
and another 1800 have been identified, which have prospective
medicinal properties (Lakhanpal & Rana, 2005).
which can contain O-methyl groups or a variety of side chains
(Wasser, 2002; Zhang et al., 2007). Most heterogalactans have a
main chain composed of (1 → 6)-linked ␣-d-Galp units with mainly
fucose or mannose as substituents (Carbonero, Gracher, Komura,
et al., 2008; Carbonero, Gracher, Rosa, et al., 2008; Rosado et al.,
2003; Smiderle et al., 2008; Wasser, 2002; Zhang et al., 2007),
although there are few studies relating their detailed structure and
biological activity.
Many of these polysaccharides have been evaluated as bio-
logical response modifiers, especially for their antitumor activity
(Moradali, Mostafavi, Ghods, & Hedjaroude, 2007; Wasser & Weis,
1999; Zhang et al., 2007). Some reports have shown that extracts
from mushrooms can have other effects, such as anti-inflammatory
and antinociceptive activity (Komura et al., 2010; Smiderle
et al., 2008). However, it is not possible to attribute a relation
between structure and activity, because most of the investigations
were carried out using crude polysaccharide extracts (Lindequist,
Niedermeyer, & Julich, 2005; Poucheret, Fons, & Rapior, 2006).
Recent studies concerned anti-inflammatory and antinociceptive
effects of fucogalactans, fucomannogalactans, and mannogalactans
isolated from Agaricus brasiliensis and Agaricus bisporus var. hort-
ensis (Komura et al., 2010), Lentinus edodes (Carbonero, Gracher,
Komura, et al., 2008), and Pleurotus pulmonarius (Smiderle et al.,
2008), respectively.
From these,
a great variety of active molecules, featur-
ing immunomodulatory and antitumoral properties, reduction
of blood pressure, among others (Wasser, 2002; Zhang, Cui,
Cheung, & Wang, 2007), have been identified in many mush-
rooms species. Among these, polysaccharides isolated from fruiting
bodies, mycelia, and culture media (Zhang et al., 2007) deserve
attention.
∗
However, there are very few reports dealing with the ability of
mushroom polysaccharides in reducing mortality caused by sepsis
Corresponding author. Tel.: +55 41 3361 1655; fax: +55 41 3266 2042.
E-mail address: iacomini@ufpr.br (M. Iacomini).
0144-8617/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2011.09.071

A.C. Ruthes et al. / Carbohydrate Polymers 87 (2012) 1620–1627
1621
which was collected by centrifugation (8000 rpm at 5 ◦C for 20 min).
Each sediment was then dissolved in H2O, dialyzed against tap
water for 20 h to remove low-molecular-weight carbohydrates,
giving rise to solutions containing fractions CW-Ab and CW-Lr,
respectively. These were frozen and then allowed to thaw slowly
(Gorin & Iacomini, 1984), resulting in insoluble fractions ICW-Ab
and ICW-Lr, which were separated by centrifugation as described
above. The supernatant fractions SCW-Ab and SCW-Lr, were treated
with Fehling solution (Jones & Stoodley, 1965), giving precipitated
Cu2+ complexes (FP-Ab and FP-Lr, respectively), which were sep-
arated by centrifugation. The precipitates were neutralized with
aceticacid(HOAc), dialyzedagainsttap water(48 h), deionizedwith
mixed ion exchange resins, and then freeze-dried.
Agaricusbisporus
Lactariusrufus
H2O at4º C for 6h (x6, 2l)
ColdAqueousExtract(CW)
ResidueI
EtOH(3:1; v/v); Centrifugation; Dialisys;
Freeze-thawing, Centrifugation
Insolublefraction
Solublefraction
(SCW)
(ICW)
TreatmentwithFehling
solution, Centrifugation
Each fraction was further purified by closed dialysis through a
membrane with a 100 kDa Mr cut-off (Spectra/Por® Cellulose Ester),
giving rise to an eluted (EFP) and a retained (RFP) material (Fig. 1).
Fehlingsupernatant
(FSHW)
Fehlingprecipitate
(FPHW)
2.3. Monosaccharide composition of polysaccharides
Closeddialysisthrougha membrane
with a 100 kDaMrcut-off
Each polysaccharide fraction (1 mg) was hydrolyzed with 2 M
TFA at 100 ◦C for 8 h, followed by evaporation to dryness. The
residues were successively reduced with NaBH4 (1 mg) and acety-
lated with Ac2O-pyridine (1:1, v/v; 200 ␮l) at 100 ◦C for 30 min
following the method of Sassaki et al. (2008). The resulting alditol
acetates were analyzed by gas chromatography–mass spectrom-
etry (GC–MS), using a Varian model 3300 gas chromatograph
linked to a Finnigan Ion-Trap, Model 810-R12 mass spectrome-
ter. Incorporated was a DB-225 capillary column (30 m × 0.25 mm
i.d.) programmed from 50 to 220 ◦C at 40 ◦C min−1, then hold, and
the alditol acetates identified by their typical retention times and
electron impact profiles.
Retainedfraction
(RFP)
Elutedfraction
(EFP)
Fucogalactans
Fig. 1. Scheme of extraction and purification of fucogalactans from A. bisporus (Ab)
and L. rufus (Lr).
in mice. Sepsis is a considerable health problem and a leading cause
of morbidity and mortality in many intensive care units. It rep-
resents a state of overproduction of pro-inflammatory mediators
which frequently occurs after various noxious injuries, especially
bacterial infection, as a consequence of abdominal surgery, appen-
dicitis, perforated ulcers, or an ischemic bowel (Angus et al., 2001).
Thus, in order to compare chemical structure with biological
properties of mushroom polysaccharides, structures of fucogalac-
tans isolated from the worldwide consumed mushroom A. bisporus
(champignon de Paris; RFP-Ab), and from Lactarius rufus (RFP-Lr),
which is used as condiment (Phillips, 2006), were now character-
ized. Their activities against murine sepsis, namely their effects
on lethality, neutrophil migration and cytokine levels were also
investigated.
2.4. Determination of homogeneity of polysaccharides and their
molecular weight (Mw)
The homogeneity and molecular mass (Mw) of the purified
fucogalactans RFP-Ab and RFP-Lr were determined by high perfor-
mance steric exclusion chromatography (HPSEC), using a refractive
index (RI) detector. The eluent was 0.1 M NaNO3, containing
0.5 g l−1 NaN3. The polysaccharide solutions were filtered through
a membrane with 0.22 ␮m diameter pores (Millipore). The spe-
cific refractive index increment (dn/dc) was determined using a
Waters 2410 detector, the samples being dissolved in the elu-
ent, five increasing concentrations, ranging from 0.2 to 1.0 mg ml−1
being used to determine the slope of the increment.
2. Materials and methods
2.1. Biological materials
2.5. Methylation analysis of fucogalactans
Dried fruiting bodies of A. bisporus (champignon de Paris)
were provided by Makoto Yamashita Company (Miriam Harumi
Yamashita), Sao José dos Pinhais, State of Paraná, Brazil. L. rufus
fruit bodies were collected in middle of May, 2005 from soil of a
Pinus sp. reforestation project located in Mafra, State of Santa Cata-
rina, Brazil at latitude: 26◦13ꢀS; longitude: 49◦50ꢀW and altitude
of 826 m above sea level. This mushroom sample was taken to the
laboratory within 12 h of collection, cleaned up from forest debris
(without washing) with a plastic knife, and dried in a freeze-dryer.
Dried samples were then ground.
Per-O-methylation of each isolated polysaccharides (RFP-Lr and
RFP-Ab; 10 mg) was carried out using NaOH–Me2SO–MeI (Ciucanu
& Kerek, 1984). The process, after isolation of the products by
neutralization (HOAc), dialysis, and evaporation was repeated,
and the methylation was found to be complete. The per-O-
methylated derivatives were hydrolyzed with 45% aqueous formic
acid (HCO2H, 1 ml) for 6 h at 100 ◦C, followed by NaB2H4 reduction
and acetylation as above (Section 2.3), to give a mixture of partially
O-methylated alditol acetates, which was analyzed by GC–MS using
a Varian model 4000 gas chromatograph, equipped with fused sil-
ica capillary columns of CP-Sil-43CB. The injector temperature was
maintained at 220 ◦C, with the oven starting at 50 ◦C (hold 2 min)
to 90 ◦C (20 ◦C min−1, then held for 1 min), 180 ◦C (5 ◦C min−1, then
held for 2 min) and to 220 ◦C (3 ◦C min−1, then held for 5 min).
Helium was used as carrier gas at a flow rate of 1.0 ml min−1. Par-
tially O-methylated alditol acetates were identified from m/z of
their positive ions, by comparison with standards, the results being
2.2. Extraction and purification of fucogalactans
Extraction of crude polysaccharides and their purification was
carried out as in Fig. 1. Wiley-milled powder fruiting bodies from
A. bisporus (100 g) and L. rufus (42 g) were extracted with H2O at
4 ◦C for 6 h (6×; 2000 ml). The combined aq. extracts were added
to excess ethanol (EtOH, 3:1; v/v) to precipitate polysaccharide,

1622
A.C. Ruthes et al. / Carbohydrate Polymers 87 (2012) 1620–1627
expressed as a relative percentage of each component (Sassaki,
Gorin, Souza, Czelusniak, & Iacomini, 2005).
were then assayed at a 1:20 dilution in reaction buffer (9.6 mM
3,3,5,5-tetramethylbenzine, 150 nmol l−1 H2O2 in 50 mM potas-
sium phosphate buffer), and read at 620 nm. Results are expressed
as change in optical density per milligram of protein (measured by
Bradford assay).
2.6. Nuclear magnetic resonance (NMR) spectroscopy
Mono- (13C, 1H and DEPT) and bidimensional NMR spectra
(HMQC, COSY, and coupled HMQC) were prepared using a 400 MHz
Bruker model DRX Avance spectrometer incorporating Fourier
transform. Analyses were carried out at 70 ◦C on samples dis-
solved in D2O. Chemical shifts are expressed in ı relative to acetone
at ı 32.77 (13C) and 2.21 (1H), based on DSS (2,2-dimethyl-2-
silapentane-5-sulfonate-d6 sodium salt; ı = 0.0 for 13C and 1H).
2.10. Determination of cytokines levels
Tumor necrosis factor alpha (TNF-␣) and interleukin (IL)-1␤
concentrations were determined in mice serum using enzyme
linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapo-
lis, MN) according to the manufacturer’s instructions.
2.11. Statistical analysis
2.7. Experimental animals
Data were expressed as means SE of five or ten mice exam-
ined in each group. Statistical error was determined by one-way
ANOVA; the post hoc test was Bonferroni’s. Calculations were per-
formed with Graphpad Prism 5.0. p values < 0.05 were considered
significant.
Male albino Swiss mice (3 months old, weighing 25–30 g), from
the Universidade Federal do Paraná colony were used for biological
tests. They were maintained under standard laboratory conditions,
with a constant 12 h light/dark cycle and controlled temperature
(22 2 ◦C), Standard pellet food (Nuvital®, Curitiba/PR, Brazil) and
water were available ad libitum. All experimental procedures were
previously approved by the Institutional Ethics Committee of the
University (authorization number 529).
3. Results and discussion
3.1. Fucogalactans structural characterization
2.8. Procedure to induce sepsis by cecal ligation and puncture
(CLP)
Agaricus is an important fungal genus, considering that some of
its species contain polysaccharides that have been presenting sev-
eral biological activities, especially antitumor. The specie A. bisporus
is one of the most extensively cultivated and consumed mushrooms
in the world. In the other hand, Lactarius spp., such as L. rufus,
which consumption is limited to its use as a condiment in a few
countries, have not been studied in this context. In order to obtain
pure heteropolysaccharides from the fruiting bodies of A. bisporus
and L. rufus, each was submitted to aqueous extraction at 4 ◦C. The
extracted polysaccharides were precipitated with excess ethanol,
obtained as sediments on ultracentrifugation, dialyzed against tap
water 4, and the solution freeze-dried to give CW-Ab and CW-Lr,
respectively (Fig. 1).
Fractionation and purification of CW-Ab and CW-Lr was carried
out by a freeze–thawing procedure (Gorin & Iacomini, 1984), result-
ing in a respective cold water-soluble SCW-Ab (4.2 g) and SCW-Lr
(2.4 g) and discarded insoluble fractions.
Fraction SCW-Lr contained fucose, xylose, galactose and glucose,
while SCW-Ab had in addition, 3-O-methyl-galactose (confirmed
by the presence of the fragments with m/z 130 and 190 from derived
mono-deuterated alditol acetates).
Fractions SCW-Ab and SCW-Lr gave heterogeneous HPSEC elu-
tion profiles, so they were then treated with Fehling solution.
Their respective insoluble Cu2+ complexes FP-Ab (2.1 g) and FP-Lr
(1.9 g) were further purified by closed dialysis through a 100 kDa
Mr cut-off membrane. This procedure gave rise to eluted EFP-Ab
and EFP-Lr and retained RFP-Ab and RFP-Lr fractions, respectively
(Fig. 1). The RFP fractions (RPF-Ab: 205 mg and RPF-Lr: 301 mg)
were homogeneous on HPSEC, and had Mw 43.8 × 104 g mol−1
(dn/dc 0.145 ml g−1) for A. bisporus and Mw 1.4 × 104 g mol−1 (dn/dc
0.151 ml g−1) for L. rufus (Fig. 2A and B, respectively).
Mice were randomly divided into five groups with 10
mice/group: sham-operation, CLP plus saline (10 ml kg−1 s.c.), CLP
plus RFP-Lr (30 mg kg−1 s.c.), CLP plus RFP-Ab (30 mg kg−1 s.c.)
and CLP plus dexamethasone (0.5 mg kg−1 s.c.). Saline was used
as vehicle for dissolving both polysaccharides; and dexametha-
sone was commercially purchased. It was administered 60 ␮l of
each treatment solution, regarding the corporeal weigh of the ani-
mals (∼30 g). Ketamine (80 mg kg−1) and xylazine (20 mg kg−1
)
were injected intraperitoneally to anesthetize the mice before the
surgical procedures. Polymicrobial sepsis was induced by CLP as
previously described (Rittirsch, Huber-Lang, Flierl, & Ward, 2009).
A midline incision about ∼1.5 cm was performed on the abdomen.
The cecum was carefully isolated and the distal 50% was ligated. The
cecum was then punctured twice with a sterile 20-gauge needle and
squeezed to extrude the fecal material from the wounds. The cecum
was replaced and the abdomen was closed in two layers. Sham-
control animals were treated identically, but no cecal ligation or
puncture was carried out. Each mouse received subcutaneous ster-
ile saline injection (1 ml) for fluid resuscitation after surgery. The
mice were then placed on a heating pad until they recovered from
the anesthesia. Food and water ad libitum were provided through-
out the experiment. Survival was monitored for 7 days, each 12 h.
During this period, saline and drugs were subcutaneously admin-
istered daily.
Inotherexperiments, duringthesurgery, miceweretreatedsub-
cutaneously with saline, RFP-Lr, RFP-Ab or dexamethasone. After
18 h post-operation, mice were sacrificed. Their tissues from small
intestines (ileum) and blood were collected and frozen for later use
to determine the myeloperoxidase (MPO) activity and investigate
cytokine production in serum.
Fraction RFP-Ab contained fucose (6%), galactose (84%) and 3-
O-methyl-galactose (10%), whereas RFP-Lr, only contained fucose
(29%) and galactose (71%).
2.9. Ileum MPO activity
Both fractions, RFP-Ab and RFP-Lr were submitted to methy-
MPO activity, assessment measure of neutrophil influx, was
determined according to established protocols (Bradley, Priebat,
Christensen, & Rothstein, 1982). Briefly, ileum tissue was homog-
enized in 0.5 ml of 50 mM potassium buffer pH 6.0 with 0.5%
hexadecyltrimethylammonium bromide, sonicated on ice, and
then centrifuged at 14,000 rpm at 4 ◦C for 15 min. Supernatants
lation (Ciucanu & Kerek, 1984), where GC–MS of resulting
O-methylalditol acetates showed the presence of branched
structures, containing mainly, nonreducing end-units of Fucp
(2,3,4-Me3Fuc), besides the 6-O-(2,3,4-Me3Gal) and 2,6-di-O-
substituted units (3,4-Me2Gal) of galactopyranose. Nonreducing
end-units of Galp (2,3,4,6-Me4Gal) were only detected in the

A.C. Ruthes et al. / Carbohydrate Polymers 87 (2012) 1620–1627
1623
Table 1
indicating the presence of nonreducing end-units of ␤-Galp
(Table 2). The glycosidic configurations of RFP-Lr and RFP-Ab fuco-
galactans units were confirmed by the values of the coupling
constants JC-1/H-1 found in 1H/13C-coupled HMQC spectra. The
nonreducing end- of Fucp and the main-chain units (galactose or
3-O-Me-galactose or both) had the ␣-configuration due to respec-
tive JC-1/H-1 162.6 Hz and 172.6 Hz, respectively, while those of
nonreducing end-units of Galp from RFP-Ab had ␤-configuration,
consistent with JC-1/H-1 161.3 Hz (Perlin & Casu, 1969).
Partially O-methylated acetates formed on methylation analysis of the fucogalactans
obtained from L. rufus (Lr) and A. bisporus (Ab) fruiting bodies: linkage types.
Partially O-methylated
Rtb
% Area of
Linkage typed
alditol acetatesa
fragmentsc
RFP-Ab
RFP-Lr
2,3,4-Me3-Fuc
2,3,4,6-Me4-Gal
2,3,4-Me3-Gal
3,4-Me2-Gal
20.03
21.19
23.67
25.76
2.8
14.5
64.8
17.9
26.9
–
46.0
27.1
Fucp-(1→
Galp-(1→
6→)-Galp-(1→
2,6→)-Galp-(1→
The above methylation analysis indicated the presence of 6-
O- and 2-O-subst. Galp units (Table 2), which was confirmed by
NMR spectroscopy. O-subst. C-2 signals were at ı 82.8 and 81.6
for RFP-Lr and RFP-Ab, respectively (Figs. 3 and 4), and substituted
–CH2 groups of the 6-O-(Galp e 3-O-Me-Galp) and 2,6-di-O-subst.
(Galp) units of the main chain were at ı 69.4/69.7 and 70.0, respec-
tively, giving rise to inverted signals in the DEPT spectra (Fig. 3Aꢀ
and Bꢀ). The presence and position of O-methyl groups of the het-
eropolysaccharide from A. bisporus were confirmed by ı 58.9/3.43
and ı 81.6/3.58 (C/H) signals corresponding to –OCH3 and O-subst.
C-3 substituted/H-3, respectively (Figs. 3B and 4B and Table 2).
The signals of C-2/H-2 to C-6/H-6 at ı 72.0/3.90, 71.2/3.85,
74.5/73.0/3.95, 69.9/4.11 and ı 18.2/1.27 corresponded to Fucp
units, while those at ı 70.1/3.88, 81.6/3.58, 68.2/4.30, 71.7/4.21 and
ı 69.4/69.7/3.72/3.93 were from 3-O-Me-Galp units from RFP-Ab
fucogalactan (Figs. 3 and 4 and Table 2). Due to the presence of ␤-
Galp (14.5%) of the polymer isolated from A. bisporus, the signals of
C-2 (ı 73.6), C-3 (ı 77.6), C-4 (ı 71.1), C-5 (ı 78.4) and C-6 (ı 63.6)
were enhanced in its DEPT spectrum (Fig. 2Bꢀ).
Analysis of the fucogalactan RFP-Lr from L. rufus showed it to
consist of a (1 → 6)-linked ␣-d-galactopyranosyl main-chain, par-
tially substituted at O-2 by nonreducing end-units of ␣-l-Fucp. That
of the RFP-Ab from A. bisporus was related, although the main chain
was partially O-methylated and had some ␤-d-Galp nonreducing
end-units as substituents.
Fucogalactans similar to those now described have been iso-
lated from cultivated mycelium of Coprinus comatus (Fan et al.,
2006), fruiting bodies of Hericium erinaceus (Zhang et al., 2006),
A. brasiliensis and A. bisporus var. hortensis (Komura et al., 2010).
However, RFP-Lr had greater fucose content among the fucogalac-
tans already characterized, while RFP-Ab had more nonreducing
end-units of ␤-Galp than the fucogalactan from the related species
A. bisporus var. hortensis (Komura et al., 2010).
a
GC–MS analysis on a CP-Sil-43CB capillary column.
Retention time (min).
Based on derived O-methyalditol acetates.
Trace.
b
c
d
RFP-Ab isolated from A. bisporus (Table 1). The ratio of units of
Fucp:2,6-di-O-Galp:6-O-Galp was ∼1:0.9:1.5 and 0.1:1:5 for RFP-Lr
from L. rufus and RFP-Ab from A. bisporus, respectively, show-
ing the major structural difference existent when comparing the
two isolated fucogalactanas. These values were confirmed by the
integration of H-1 signals (5.10:5.08:5.01) present in the 1H NMR
spectrum (data not shown).
Further NMR spectroscopy [1H, 13C and DEPT (Fig. 3), HMQC
(Fig. 4), COSY and coupled HMQC (data not shown)] was also helpful
in elucidating the fucogalactan structures (Table 2).
RFP-Lr 13C NMR (Fig. 3A) and HMQC spectra (Fig. 4A), obtained
using D2O as solvent, had signals (C-1/H-1) at ı 103.9/5.10,
101.2/5.08, 100.9/5.08 and 100.7/4.98 corresponding to Fucp units,
2,6-di-O- and 6-O-subst. Galp units, respectively. While those
from RFP-Ab (Figs. 3B and 4B, respectively), had C-1/H-1 sig-
nals at ı 103.9/5.10, 100.7/5.05 and 100.6/5.01 corresponding
to Fucp units, 2,6-di-O- and 6-O-subst. Galp units, respectively.
Anomeric signals corresponding to non-reducing end groups of ␤-
Galp (ı 105.5/4.64) and units of 3-Me-␤-Galp (ı 100.6/5.01) were
present (Figs. 3B and 4B), in agreement with the methylation data
3.2. Biological experiments
It is generally accepted that polysaccharides isolated from
mushrooms and plants enhance various immune responses
in vivo and in vitro. There are also many reports of mushroom
polysaccharide-induced nonspecific resistance against diverse
microbial pathogens.
In order to determine effects on murine sepsis of the fuco-
galactans RFP-Ab and RFP-Lr, isolated from A. bisporus and L. rufus,
respectively, they were tested at doses of 30 mg kg−1. Thus their
effects on the survival rate of infected mice, neutrophil influx, and
cytokine levels were determined.
The results of the survival experiments are shown in Fig. 5A.
Mice treated with saline started to die between 12 h and 24 h after
CLP, with a death rate reaching 37.5% and 87.5% at 24 h and 72 h
post-CLP, respectively. The overall mortality at the end of the obser-
vation period was 100%. The lethality was markedly delayed in mice
treated with both fucogalactans, RFP-Ab and RFP-Lr. At 24 h and
72 h after CLP, the mortality rate in RFP-Lr group was zero and 25%,
respectively. At the end of the study, the overall survival in this
group was 62.5%. As for RFP-Ab, at 24 h and 72 h after CLP, their
mortality rate was zero and 37.5%, respectively, with an overall
survival of 50.0% (Fig. 5A). Dexamethasone-treated mice showed a
Fig. 2. Elution profiles of fractions RFP-Ab (B) and RFP-Lr (C) determined by HPSEC
using light scattering (- - -) and refractive index detectors (—).

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A.C. Ruthes et al. / Carbohydrate Polymers 87 (2012) 1620–1627
Table 2
1H and 13C NMR chemical shifts [expressed as ı (ppm)] of the fucogalactans from A. bisporus (RFP-Ab) and L. rufus (RFP-Lr).a
Units
1
2
3
4
5
6
-O-CH3
a
b
13C
1H
103.9
5.10
72.0
3.85
71.2
3.89
74.5/73.0
3.85
69.9
4.20
18.2
1.27
–
–
–
–
␣-l-Fucp-(1→
13C
1H
105.5
4.64
73.6
3.85
77.6
71.1
4.08
78.4
63.6
–
–
–
–
␤-d-Galp-(1→
3.84
71.1
3.58
71.7
3.80
69.7
13C
1H
101.2/100.7
5.08
82.8/79.4
3.61/3.84
72.2
4.09
69.7
4.01
–
–
2,6→)-␣-d-Galp-(1→
6→)-␣-d-Galp-(1→
4.08
4.21
3.70
13C
1H
100.7
5.01
71.1
3.84
72.4
72.4
4.03
71.7
69.3
69.3
3.93
–
–
3.89
81.6
4.21
71.7
3.72
69.4
13C
1H
100.6
5.01
70.1
3.88
68.2
4.3
69.4
3.93
58.9
3.43
6→)-3-O-Me-␣-d-Galp-(1→
3.58
4.21
3.89
a
Assignments are based on 13C, 1H, DEPT, COSY and HMQC examination.
significant improvement in survival, with an overall survival rate
of 16.7% at the end of the observation period. However, this anti-
inflammatory medicine significantly delayed the onset of death
after CLP (∼36 h versus saline group). No death was observed in
the sham-operated mice.
In terms of neutrophil influx, it was inhibited by polysaccha-
ride compounds. CLP surgery markedly increased ileum tissue MPO
levels compared with sham group (42.3%) (Fig. 5B). This increase
in tissue MPO was significantly prevented by both polysaccha-
rides RFP-Lr and RFP-Ab, with an inhibition of 43.0% and 51.9%,
respectively, versus saline group (Fig. 5B). Dexamethasone, the
anti-inflammatory control, strongly inhibited the MPO activity in
ileum (61.2%).
TNF-␣ and IL-1␤ cytokines levels were lower in sham surgery
control mice. In contrast, 18 h after CLP surgery, both showed
an increase of 49% and 48%, respectively, in comparison to the
sham group (Fig. 6A and B). The treatment with both fucogalactans
showed significant reduction in cytokine production in relation to
saline group. RFP-Lr reduced the levels of TNF-␣ in 33.6% and 38.8%
of IL-1␤, while RFP-Ab reduced 36.5% and 32.3%, respectively. The
increased levels of TNF-␣ and IL-1␤ were significantly prevented
by dexamethasone (0.5 mg kg−1), which promoted an inhibition of
Fig. 3. 13C NMR spectra of fucogalactan fractions RFP-Lr (A) and RFP-Ab (B) with inserts of DEPT –CH2 inversion (Aꢀ and Bꢀ, respectively).

A.C. Ruthes et al. / Carbohydrate Polymers 87 (2012) 1620–1627
1625
Fig. 5. Effects of the polysaccharides RFP-Lr and RFP-Ab on survival rate and
on myeloperoxidase activity in sepsis. (A) Mice were randomly divided into five
groups. All groups except the sham group received cecal ligation and puncture
(CLP) surgery. Saline, RFP-Lr (30 mg kg−1 s.c.), RFP-Ab (30 mg kg−1 s.c.) and dex-
amethasone (0.5 mg kg−1 s.c.) were administered immediately after surgery. The
treatment continued over the next days, each 24 h. The survival rate of mice was
recorded every 12 h over 7 days after CLP. (B) Mice subjected to sepsis by CLP were
treated with saline, RFP-Lr, RFP-Ab or dexamethasone immediately after surgery.
The myeloperoxidase activity (MPO, indicator of neutrophil accumulation) was mea-
sured spectrophotometrically in ileum samples harvested 18 h after the induction
of CLP. Results were expressed as change in optical density per milligram of protein.
Values represent means SEM. ***p < 0.001 and **p < 0.01, indicated value versus
CLP plus saline group; ##p < 0.01, CLP plus saline versus sham. ANOVA followed by
Bonferroni’s test.
Fig. 4. 1H (obs.)/13C HMQC spectra of RFP-Lr (A) and RFP-Ab (B) of fucogalactans
fractions (chemical shifts are expressed in ı ppm).
approximately 42%, in both cases, in comparison with saline group
(Fig. 6, panels A and B).
It is believed that sepsis may lead to aberrant host inflamma-
tory responses, causing cell injury and organ dysfunction. During
sepsis, the intestine is considered not only to be a source of bac-
teremia but also an important target of bacterial products with
major functional consequences for intestinal motility and the gen-
eration of inflammatory mediators and recruitment of immune
cells. Neutrophil infiltration is an important pathophysiologic alter-
ation associated with sepsis. These cells directly damage tissue by
releasing pro-inflammatory mediators, as cytokines, superoxide-
derived free radicals and lysosomal enzymes, such as MPO, which
amplify the systemic inflammatory response and cause multiple
organ failure (Landry & Oliver, 2001; Mainous, Ertel, Chaudry, &
Deitch, 1995).
In this study, a polymicrobial sepsis was induced by cecal lig-
ation and puncture (CLP) in mice to investigate the effects of
fucogalactans RFP-Lr and RFL-Ab. This model mimics the sepsis
in human, caused by pathogens derived from the intestinal tract,
and is considered to closely simulate clinical situation (Otero-
Anton et al., 2001). Lethality was markedly delayed in mice
receiving both polysaccharides (see Fig. 5A). This result could be
attributable to an anti-inflammatory activity, which is consistent
with previous literature findings (Ahn et al., 2006; Kim et al.,
2003; Komura et al., 2010). There are many reports of mushroom
polysaccharides inducing nonspecific resistance against diverse
microbial pathogens in animals (Cisneros, Gibson, & Tzianabos,
1996; Kernodle, Gates, & Kaiser, 1998; Tzianabos & Cisneros, 1996),
as well as in human (Babineau, Hackford, et al., 1994; Babineau,
Marcello, et al., 1994) models. Although the mechanism that mush-
room polysaccharides induced effects is not well understood, a
key host immune response modulation caused by these mush-
room polymers appears to be central (Wasser, 2010). In addition, a
study carried out on experimental sepsis showed that a pretreat-
ment with an acidic polysaccharide isolated from the medicinal
mushroom Phellinus linteus could be able to inhibit bacterial septic
shock by suppressing TNF-␣ production in areas of inflammation,
suggesting that the mushroom polysaccharide may contribute to
prolong the survival of septic shock mice (Kim et al., 2003). A
similar result was observed at the present work, where both fuco-
galactans tested were able to decrease significantly TNF-␣ and also
IL-1␤ serum levels. Several previous studies have demonstrated
that some cytokines, especially TNF-␣, IL-1␤ and IL-6 are strongly
associated with sepsis syndrome, therefore inhibiting the pro-
inflammatory cytokine overproduction during early sepsis may
reduce sepsis outcome (Cohen, 2002; Ebong et al., 1999; Takala,
Nupponen, Kylanpaa-Back, & Repo, 2002). Consequently, the reduc-
tion in mice lethality caused by both fucogalactans may be also
related to the decreasing in TNF-␣ and IL-1␤ serum levels.

1626
A.C. Ruthes et al. / Carbohydrate Polymers 87 (2012) 1620–1627
protection against organ damage, although further investigation
is required to clarify the detailed mechanism.
4. Conclusions
The fucogalactans from A. bisporus and L. rufus have an anti-
inflammatory action, such as those from A. brasiliensis and A.
bisporus var. hortensis (Komura et al., 2010), related to their struc-
tures. The influence of structure on the biological response was
found with similar polymers, having small differences in the main
chain, such as the presence of O-methyl groups, or more signifi-
cant ones in the side chains. Consequently, determination of the
fine structure of the polysaccharides is significant in understand-
ing the relation between their structure and biological activity. To
determine their mechanism of action, other biological assays are
necessary.
Acknowledgements
The authors would like to thank the Brazilian funding agen-
cies: CAPES (Coordenac¸ ão de Aperfeic¸ oamento de Pessoal de Nível
Superior), CNPq (Conselho Nacional de Desenvolvimento Cientí-
fico e Tecnológico), and Fundac¸ ão Araucária for financial support,
and the mycologist André A.R. de Meijer for identification of the
mushrooms.
Fig. 6. Effect of fucogalactans RFP-Lr and RFP-Ab upon TNF-␣ (A) and IL-1␤ (B)
production in infected mice serum. The mice, except the sham group, received
sterile saline, RFP-Lr (30 mg kg−1 s.c.), RFP-Ab (30 mg kg−1 s.c.) or dexametha-
sone (0.5 mg kg−1 s.c.), which were administered during the CLP-surgery, and
the cytokines levels were evaluated 18 h after onset. Each group represents the
mean SEM of four to five animals. *p < 0.05, indicated value versus CLP plus saline
group; ##p < 0.01, CLP plus saline versus sham. ANOVA followed by Bonferroni’s test.
A false-operated group (sham) was also provided for this test.
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