V.V. Golovchenko, et al.
Phytochemistry 175 (2020) 112313
by the inclusion 1,6-β-D-glucan and 1,3-α-D-mannan in the former.
Notably, 1,3-α-mannan, having a backbone conformation similar to
that of 1,3-β-glucan, has shown antitumour action comparable to that
of 1,3-β-glucan (Zhang et al., 2007). High molecular weight FoCA
of 30% H
NH
2
O
2
to a final concentration of 5%, and 10% (w/v), aqueous
4
OH was added to increase the pH to 7, and the mixture was kept in
the dark for 15 h at room temperature. The insoluble precipitate was
removed by centrifugation. The aqueous solution of polysaccharide was
dialyzed against distilled water for 48 h at 10 °C and concentrated on a
Heidolph 4002 rotary evaporator (Germany) under reduced pressure at
40 °C. Then, the polysaccharides were precipitated with 95% ethanol,
dissolved in water after centrifugation and lyophilized on a VirTis
freeze-drier (USA) under a constant vacuum of <10 mTorr at −65 °C.
All precipitates were isolated/obtained by centrifugation at 7000 rpm
and 4 °C for 10–20 min on a Sigma 6 K 15 centrifuge with rotor N
12,256 (USA). Finally, three water-soluble crude polysaccharide frac-
tions were obtained by hot water extraction, cold alkali extraction and
hot alkali extraction. The yields of the polysaccharide fractions ob-
tained by cold alkali extraction and hot alkali extraction were 5.60 g
and 2.42 g, or 2.8% and 1.21%, from the dry fruit bodies, respectively.
The structure of mannofucogalactan as the main component of the hot
water extract was reported in our previous paper (Golovchenko et al.,
2018).
(
Mw > 800 kDa) (in comparison with FoHA3, Mw 411 kDa) appeared
to play an important role in the anticancer activity. The activity of β-
glucans strongly depends on their molecular weight. Generally, high
molecular weight glucans (ranging from 500 to 2000 kDa (Mizuno
et al., 1996)) are more effective in suppressing tumour cell growth of
low molecular weight glucans (Meng et al., 2016; Zong et al., 2012;
Zhang et al., 2007).
The mechanisms of action of fungal polysaccharides on cancer cells
is pleiotropic (Khan et al., 2019) and includes cell cycle arrest, depo-
larization of the mitochondrial membrane, nitric oxide biosynthetic
pathways, and the activation of immune processes (production of TNF-
α, cytokines, and other immunomodulators) (Meng et al., 2016). For
example, a Pleurotus nebrodensis polysaccharide acts via cell cycle arrest
at the G2/M phase and the produces cytokines (Cui et al., 2014). In our
experiments, exposure of HeLa cells to 200 μg/mL FoCA 1,3-β-glucan
gave rise to apoptotic bodies around the cells (Fig. 7f). We suppose that
this effect is related to the ability of 1,3-β-glucan to arrest the cell cycle.
The formation of apoptotic bodies at concentrations below the IC50 has
been described earlier for three Cordyceps militaris polysaccharides and
SEC on Sepharose CL-4B was performed for the separation and
purification of polysaccharides obtained by alkali extraction.
Polysaccharide (20 mg) was dissolved in 1 mL of 0.15 M NaCl, and the
solution was applied to
a
column (1.5 cm × 72 cm,
0
V = 23 mL,
explained by cell cycle arrest at the G
0
/G
1
phase (Chen et al., 2015).
V = 73 mL) with Sepharose CL-4B. The gel bed was equilibrated with
t
Our results suggested that the fruit bodies of F. officinalis are a
source of a high molecular weight, water-soluble 1,3-β-glucan posses-
sing cytotoxic activity. However, extraction with boiling water (used
for the extract and decoction preparation) appeared to be in-
appropriate, and β-glucan can be obtained in its pure form by cold al-
kali extraction after the removal of components extractable by boiling
water. In our opinion, β-glucan from F. officinalis fruit bodies is a pro-
mising substance for use in anticancer therapy. However, further in vitro
and in vivo studies are needed to determine of the biochemical and
physiological mechanisms of its anticancer effects and its ability to
stimulate the immune system.
degassed elution of 0.15 M NaCl used as an eluent. Elution was per-
formed at a flow rate of 0.25 mL/min at room temperature. The eluate
was collected in 3.5 mL tubes and the carbohydrate content of each
tube was determined by the phenol-sulfuric acid method (Dubois et al.,
1956). Fractions corresponding to the separated peaks obtained on the
elution curve were combined, concentrated, dialyzed, and lyophilized.
Finally, one main polysaccharide fraction FoCA (Kav 0.02, yield
15.48 mg) was obtained after purification of the crude polysaccharide
from the cold alkali extract, and four polysaccharide fractions were
obtained after purification of crude polysaccharide of hot alkali extract
as follows: FoHA1 polysaccharide with Kav 0.02 (yield 1.8 mg), poly-
saccharide FoHA2 with Kav 0.18 (yield 2.3 mg), polysaccharide FoHA3
(Kav 0.42, yield 6.7 mg) further designated FoHA and polysaccharide
FoHA4 with Kav 0.66 (yield 4.5 mg). The procedure was repeated sev-
eral times affording substantial amounts of fractions FoCA and FoHA3,
which were used for further investigations.
3
. Experimental
3.1. Biological material
The fruit bodies of F. officinalis were collected in November 2012
from trees grown in Eruu soum, Selenge province, Mongolia. They were
identified by Prof. Ch. Sanchir (Botany Institute of Mongolian Academy
of Sceinces, Mongolia). The fruit bodies were washed with distilled
water, cut into small pieces, and dried in a thermostat (Jouan, France)
at 45 °C for 24 h.
3.3. General analytical methods
The monosaccharide composition was determined after hydrolysis
of the polysaccharides, where 2 M aqueous CF COOH (1 mL) containing
3
myo-inositol (0.5 or 1 mg/mL) was added to a weighed portion of
polysaccharide (2–3 mg). The mixture was incubated for 5 h at 100 °C.
The excess acid was removed by repeated evaporation to dryness of the
hydrolysate with methanol. The mixture of monosaccharides was
3.2. Isolation and purification of polysaccharides
Dry fruit bodies of F. officinalis (200 g) were milled in a blender, and
transformed into alditol acetates by using (CH
3
CO)
2
O followed by re-
the powder was defatted with 96% ethanol at 60 °C for 5 h
duction with NaBH
4
in 1 M aqueous NH
4
OH and monosaccharides were
(
2 × 350 mL) to remove the soluble lipids, low molar. weight sugars,
identified by gas–liquid chromatography (GLC) on a Varian 450-GC
chromatograph (Varian, USA) equipped with a flame-ionization de-
tector. GLC was run on a VF-5 ms capillary column (Varian, USA;
0.25 mm, 30 m) using the following temperature program: 175 °C
(isotherm, 1 min), ramp to 250 °C (isotherm, 2 min), at a rate of 3 °C/
min.
and phenolic compounds. The supernatant was removed by filtration
and the remaining residue was air-dried. The polysaccharides were
extracted successively with boiling water under reflux for 4 h, cold (at
4
0
°C) and hot (at 80 °C) 5% (w/v) aqueous solution NaOH containing
.5% (w/v) NaBH for 24 h and 6 h, respectively. Treatment with each
4
extractant was performed twice. The residue after each extraction was
separated by centrifugation and the corresponding extracts were col-
lected and combined. Finally, the aqueous extract (3 L), cold alkali
extract (1 L) and hot alkali extract (1 L) were obtained. The alkali ex-
Protein concentration was determined using Lowry's procedure
(Lowry et al., 1951) with bovine serum albumin as a standard. The
sugar concentration was determined at 490 nm using the phenol-sul-
furic acid assay (Dubois et al., 1956). Absorbance was measured using
an Ultrospec 3000 spectrophotometer (Pharmacia Biotech, England).
The Mw, Mn and MMD of the polysaccharide samples were de-
termined by an SEC separation of polysaccharides with high-perfor-
mance liquid chromatography (HPLC). The chromatographic system
consisted of an LC-20AD pump, a DGU-20A3 degasser, a CTO-10AS
tracts were neutralized with 36% (v/v) aqueous CH COOH and the
3
precipitate was removed by centrifugation as described previously
(
Surenjav et al., 2006). Aqueous lead acetate (2%, w/v) was added to
the extracts to a final concentration of 0.4–0.5% (w/v) for deproteini-
zation (Chen et al., 2012). The extract was decolourized by the addition
8
Golovchenko, Victoria V.
