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its pronounced anti-tumor activity, which is probably related to the
2.4. Monosaccharide analysis
activation of T-cells, natural-killer cells, and macrophages (Zhang,
Li, Wang, Zhang, & Cheung, 2011).
ness, and the hydrolysis product was submitted to reduction with
NaBH4 (Wolfrom & Thompson, 1963b) and acetylation with Ac2O-
pyridine (1:1, v/v; 300 L) for 12 h at room temperature (Wolfrom
& Thompson, 1963a). The resulting alditol acetates were analyzed
by gas chromatography–mass spectrometry (GC–MS) using a Var-
DB-225 capillary column (30 m × 0.25 mm i.d.), which was main-
tained at 50 ◦C during injection and then programmed to increase
to 220 ◦C at a rate of 40 ◦C min−1, was used for the quantitative
analysis of the alditol acetates (Ruthes et al., 2013). The products
were identified by their typical retention times and electron impact
profiles.
ostreatus and Pleurotus eryngii), or (1 → 4)-linked (from Agaricus
blazei and P. ostreatus) (Synytsya & Novák, 2013; Palacios et al.,
2012), and (1 → 6)--d-glucan (from Agaricus bisporus and Agaricus
brasiliensis) (Smiderle et al., 2013).
In this study, a linear (1 → 3)--d-glucan was isolated, for the
first time, from Pleurotus sajor-caju. We have not found any stud-
ies showing the presence of this polysaccharide in other Pleurotus
species. It was chemically characterized, and its biological proper-
ties were evaluated in vitro, using THP-1 macrophages, and in vivo,
through formalin and peritonitis tests in mice.
2. Materials and methods
2.5. Methylation analysis of the polysaccharide
2.1. Microorganisms and maintenance
The isolated polysaccharide was per-O-methylated according
to the method described by Ciucanu and Kerek (1984), with
slight modifications. The sample (10 mg) was dissolved in Me2SO
(0.5 mL), followed by addition of iodomethane (0.5 mL), and pow-
dered NaOH (200 mg). After vigorous stirring for 30 min, the
mixture was maintained overnight at room temperature. The reac-
tion was interrupted by the addition of water, neutralized with
HOAc, dialyzed against distilled water (2 kDa MWCO membrane)
and freeze-dried. The product was submitted to one more cycle of
methylation, however in the second cycle, after neutralization with
HOAc, the per-O-methylated polysaccharide was recovered by par-
tition between CHCl3 and water. The per-O-methylated derivatives
were hydrolyzed with 45% (v/v) formic acid (HCO2H; 1 mL) at 100 ◦C
for 15 h, followed by evaporation to dryness. The resulting mixture
of O-methylated monosaccharides was reduced with NaBH4 and
acetylated with Ac2O-pyridine (1:1, v/v; 300 L) for 12 h at room
described for alditol acetates (Section2.4), with the exception that
the final temperature was 215 ◦C. The derivatives were identified by
their typical retention times and electron impact spectra (Sassaki,
Gorin, Souza, Czelusniak, & Iacomini, 2005).
P. sajor-caju CCB019 was obtained from the Center for Basid-
iomycete Cultivation of the São Paulo University, Brazil, and
maintained in Petri dishes containing WDA (1 L of wheat extract,
20 g of dextrose, and 15 g of agar) at 4 ◦C (Furlan et al., 1997).
2.2. Cultivation conditions of the fruiting bodies
P. sajor-caju fruiting bodies cultivation was conducted at the
Biotechnology Laboratory of UNIVILLE University, Joinville (SC)
Brazil, using banana straw.
Banana straw was packed in polypropylene bags, supplemented
with rice bran, sterilized, and inoculated using 10% solid inoculum.
The first step of the process, the mycelial growth, was carried out at
25 ◦C, with 60% relative air humidity, artificial light, for 20 days. The
induction of the fruiting body formation (second step) was achieved
by perforating the plastic bags to increase air exchange, and by
exposing them to light for a period of 12 h a day while increasing
relative air humidity to 90%. After 20 days, the fruiting bodies were
harvested, frozen, and freeze-dried.
2.3. Extraction and purification of the polysaccharide
2.6. Nuclear magnetic resonance spectroscopy
Fruiting bodies were milled in a blender and the powder was
defatted by addition of a chloroform–methanol (2:1 v/v) mixture
at 50 ◦C for 24 h. Then, the residue was submitted to successive
aqueous extraction at 100 ◦C for 24 h (10×, 800 mL each). The
hot-water extract (HW) was centrifuged (6000 rpm, 30 min, 10 ◦C)
and reduced to a small volume, by concentration under reduced
pressure in a rotary-evaporator, and the polysaccharides were
recovered from this extract by precipitation with excess of ethanol
(4 volume). The precipitated polysaccharides were then dialyzed
against tap water for 24 h (2 kDa MWCO membrane), concentrated
under reduced pressure, and submitted to freeze-thawing process.
The insoluble fraction (PHW) obtained from freeze-thawing was
recovered by centrifugation (12,000 rpm, 30 min, 5 ◦C), and sub-
mitted to one extraction with 50 mL dimethyl sulfoxide (Me2SO;
80 ◦C for 5 h). The Me2SO-extract (M-PHW) was dialyzed against
tap water for 24 h (2 kDa MWCO membrane) and then resubmit-
ted to the freeze-thawing process, giving rise to a water-insoluble
fraction (G-PHW) recovered by centrifugation (12,000 rpm, 30 min,
5 ◦C). The chemical structure of this fraction was evaluated as well
as its antinociceptive and anti-inflammatory activities.
The NMR spectra mono-(13C) and bidimensional (HSQC) were
obtained using a 400-MHz Bruker Avance III spectrometer with
a 5 mm inverse probe. The 13C-NMR (100.6 MHz) and 1H-NMR
(400.13 MHz) analyses were performed at 70 ◦C, and the sam-
ples were dissolved in D2O or Me2SO-d6. The chemical shifts are
expressed in ppm (␦) relative acetone (for samples in D2O) at ␦
30.2 and ␦ 2.22 or to Me2SO-d6 at ␦ 39.7 and ␦ 2.40 for 13C and 1
H
signals, respectively.
2.7. Homogeneity and average molecular mass (Mw) analysis
The homogeneity and average molecular mass (Mw) of the -
d-glucan were determined by high-performance size-exclusion
chromatography (HPSEC) coupled to refractive index and multi-
angle laser light-scattering detectors (MALLS). Four gel-permeation
Ultrahydrogel columns, with exclusion sizes of 7 × 106, 4 × 105,
8 × 104, and 5 × 103 Da, were used in series. The eluent was 0.1 M aq.
NaNO2 containing 200 ppm aq. NaN3 at 0.6 mL min−1. The sample
was dissolved in the same solution used as eluent at a concentration
of 1 mg mL−1, filtered through a membrane (0.22 m), and injected