An α-D-galactan and a β-D-glucan from the mushroom Amanita muscaria: Structural characterization and antitumor activity against melanoma

Carbohydrate Polymers 274 (2021) 118647

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

An α-D-galactan and a β-D-glucan from the mushroom Amanita muscaria:
Structural characterization and antitumor activity against melanoma
Matheus Zavadinack a, Daniel de Lima Bellan b, Jessica Loren da Rocha Bertage a,
Shayane da Silva Milhorini a, Edvaldo da Silva Trindade b, Fernanda Fogagnoli Simas b,
Guilherme Lanzi Sassaki a, Lucimara M.C. Cordeiro a, Marcello Iacomini a, *
a
b

Department of Biochemistry and Molecular Biology, Federal University of Paran´
a, Curitiba, PR CEP 81531-980, Brazil
Department of Cell Biology, Federal University of Paran´
a, Curitiba, PR CEP 81531-980, Brazil

A R T I C L E I N F O

A B S T R A C T

Keywords:
A. muscaria
Polysaccharides
α-Galactan
β-Glucan
Chemical structure
Antimelanoma properties

Polysaccharides α-D-galactan (GAL-Am) and β-D-glucan (GLC-Am) were obtained from Amanita muscaria fruiting
bodies. They were purified using different methodologies, such as Fehling precipitation (for both fractions),
freeze-thawing process and ultrafiltration (for GLC-Am). Results showed that the GAL-Am has (1 → 6)-linked
Galp main chain branched at O-2 by terminal Galp units and has not been previously reported. Besides, GLC-Am
has (1 → 3)-linked Glcp in the main chain, substituted at O-6 by (1 → 6)-linked β-Glcp units. Both are watersoluble, with 9.0 × 103 g/moL and 1.3 × 105 g/moL, respectively. GAL-Am and GLC-Am presented a selec­
tive proliferation reduction against B16-F10 melanoma cell line, not affecting non tumoral BALB/3T3 fibroblast
cell line. Furthermore, both fractions reduced clonogenic capacity of melanoma cell line over an extended period
of time. These results were obtained without modulations in B16-F10 cell adhesion, reinforcing the biological
activities towards cell proliferation impairment and eliciting these polysaccharides as promising compounds to
further exploration of their antimelanoma properties.

1. Introduction
The fungal fruiting body, popularly known as mushroom, is an
important source of nutrients and fibers, such as β-glucans (Abreu et al.,
2019), being considered a delicacy food in many cultures and countries
(White et al., 2019).
Commonly known as fly agaric mushroom, A. muscaria (L.:Fr) has
toxic and hallucinogenic molecules in its composition such as muscimol,
muscarina, ibotenic acid and muscazone, besides heterocyclic alkaloids
in small amounts (Kondeva-Burdina et al., 2019; Ruthes et al., 2013a,b).
Despite crude mushroom show toxicity, it is consumed in regions of
Europe, North America and Japan after boiling in salt water and
steeping in vinegar (Coville, 1898; Kiho et al., 1992).
In addition, edible mushrooms present a diverse range of biological
properties, including immunomodulatory, hypolipidemic, antibacterial,
anti-inflammatory, hepatoprotective and antitumor effects (Morales
et al., 2020).
Amongst mushrooms biological activities, their antitumor properties
are especially relevant and explored. Cancer stands out as the second

leading cause of death worldwide (Bray et al., 2018). One of the few
cancer types with a rising incidence rate is melanoma, the most lethal
form of skin cancer given its fast progression to the metastatic stage
(Ward & Farma, 2017). Although recent advances in targeted and im­
munotherapies, cancer and particularly melanoma treatment are mostly
palliative and accompanied of several debilitating adverse effects
(Ramos-Casals et al., 2020), thus engendering the search for new ther­
apeutic approaches. Polysaccharides obtained from mushrooms have
already demonstrated its potential to modulate malignancy related traits
in cancer models, reducing cell proliferation, inducing selective cyto­
toxicity, impairing migration and metastasis and decreasing tumor
growth (Kothari et al., 2018; Ren et al., 2012). Their pharmacological
relevance already reached cancer clinical trials in China and Japan, as
illustrated by PSP - a heteropolysaccharide mainly constituted of Dglucose with a main chain α-(1 → 4) and β-(1 → 3)-linked units - and PSK
– mainly constituted of glucan β-(1 → 4)-linked with (1 → 6)-β-gluco­
pyranosidic side chains – both obtained from the mushroom Coriolus
versicolor (Habtemariam, 2020; Kobayashi et al., 1995).
It is known that polysaccharides biological properties are

* Corresponding author.
E-mail address: (M. Iacomini).
/>Received 31 March 2021; Received in revised form 3 September 2021; Accepted 4 September 2021
Available online 8 September 2021
0144-8617/© 2021 Elsevier Ltd. This article is made available under the Elsevier license ( />

M. Zavadinack et al.

Carbohydrate Polymers 274 (2021) 118647

Fig. 1. Extraction and purification scheme of the β-D-glucan (GLC-Am) and the α-D-galactan (GAL-Am) from A. muscaria (L.:Fr).

Fig. 2. HSQC spectra of the α-galactan (GAL-Am). The sample was analyzed in D2O at 70 ◦ C in a Bruker Avance III 600 MHz (chemical shifts are expressed in δ ppm).

intrinsically linked to their composition and chemical structure (Ruthes
et al., 2013a,b; Vetvicka & Yvin, 2004) and there is limited information
in the literature regarding polysaccharides from A. muscaria. Ruthes
et al. (2013a,b) isolated a fucomannogalactan formed by a (1 → 6)linked α-D-galactopyranosyl main chain partially substituted for nonreducing end units at O-2 mainly by α-L-Fucp and β-D-Manp and a (1
→ 3),(1 → 6) β-D-glucan. Regarding biological effects of these

polysaccharides, the (1 → 3),(1 → 6) β-D-glucan showed potent inhibi­
tion of inflammatory pain (Ruthes et al., 2013a,b) and significant anti­
tumor activity against Sarcoma 180 in mice (Kiho et al., 1992).
Therefore, in the present study we advance in the knowledge of
A. muscaria polysaccharides, describing for the first time the chemical
structure of an unmethylated α-D-galactan, not yet reported for mush­
rooms, as well as the structure of a β-D-glucan. Moreover, their possible
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Carbohydrate Polymers 274 (2021) 118647

Fig. 3. 2D NMR analysis of the GAL-Am fraction. COSY (A),
TOCSY (B), HMBC and 1H (C), and HSQC-TOCSY - in blue
and green - with superimposed HSQC - in red - (D) corre­
lation maps of the α-D-galactan (GAL-Am) from A. muscaria
(L.:Fr). The sample was analyzed in D2O at 70 ◦ C in a Bruker
Avance III 600 MHz (chemical shifts are expressed in δ

ppm). n = carbon number from the sugar ring. The symbols
' and " indicate the units 2,6→)-α-D-Galp-(1 → and α-D-Galp(1→, respectively, while the absence of them represent the
6→)-α-D-Galp-(1 → units.

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Carbohydrate Polymers 274 (2021) 118647

Fig. 3. (continued).

Fig. 4. 2D NOESY analysis of the GAL-Am fraction. The sample was analyzed in D2O at 70 ◦ C in a Bruker Avance III 600 MHz (chemical shifts are expressed in
δ ppm).

antimelanoma activities were further explored against the murine mel­
anoma B16-F10 cell line.

2.2. General extraction and purification processes
General extraction and purification processes performed on the
A. muscaria are represented in the flowchart (Fig. 1). The extraction
processes were carried out using 270.86 g dehydrated fruiting bodies.
The material was defatted with chloroform-methanol (2:1; v/v) at 65 ◦ C
for 3 h using a Soxhlet apparatus. Then, the residue was submitted to
aqueous extraction at room temperature (25 ◦ C) for 6 h (5×, 4 L each)
under stirring. The aqueous extract was concentrated, and the poly­
saccharides precipitated by ethanol addition (3:1, v/v), collected by
centrifugation (20 min, 8000 rpm, 25 ◦ C) and dialyzed (6–8 kDa
membrane) against tap water for 24 h. Insoluble materials that formed

during the dialysis process were removed by centrifugation (20 min,

2. Material and methods
2.1. Biological material
A. muscaria fruiting bodies were collected at the Biological Sciences
Sector gardens (25◦ 26′ 48.4′′ S 49◦ 14′ 16.5′′ W) of the Federal University
of Paran´
a in Curitiba, Paran´
a - Brazil. The respective mushrooms were
cleaned, freeze-dried and then grounded to a powder.

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Carbohydrate Polymers 274 (2021) 118647

Table 1
Chemical shiftsa of the correlation between the 1H/1H and 1H/13C of the adjacent carbons in monosaccharides units of the GAL-Am.
6→)-α-Galp-(1→
1

1

H-1/ H-2
H-2/13C-3
1
H-3/1H-4
1

H-4/1H-5
1
H-5/13C-6a
1
H-5/13C-6b
1

a
b
c
d

Correlations

2,6→)-α-Galp-(1→

b

1

4.98/3.88
3.88/68.4c
3.85/4.03b
4.03/4.17b
3.91/66.7c
3.71/66.7c

1

Correlations

d

b

H-1/ H-2
C-2/1H-3
1
H-3/13C-4
1
H-4/1H-5
13
C-5/1H-6a
13
C-5/1H-6b

5.02/3.97
75.1/3.98c
3.98/66.2c
4.25/4.17b
68.8/3.91c
68.8/3.71c

13

α-Galp-(1→

1

1

5.15/3.88b
3.88/69.5b
3.94/4.03d
4.03/4.18d
4.18/3.76c
–

1

H-1/ H-2
H-2/13C-3
1
H-3/1H-4
1
H-4/1H-5
1
H-5/1H-6c
–
1

H/13C

Assignments are based on 1H and HSQC analysis and are expressed as ppm.
Signals in COSY spectrum.
Signals in HSQC-TOCSY/HSQC spectrum.
Signals in TOCSY spectrum.

10,000 rpm, 25 ◦ C). The soluble polysaccharides were named S-Am and
submitted to Fehling treatment (Brito et al., 2018). Cu2+-complexed

polysaccharides recovered by centrifugation (20 min, 10,000 rpm,
25 ◦ C) gave rise to the fraction (I-1) while the Cu2+-uncomplexed
polysaccharides were named as S-1 fraction. Both were neutralized with
HOAc, dialyzed (2 kDa cut-off membrane) and deionized with mixed ion
exchange resins and then freeze dried. The latter one (S-1) was subjected
to freeze-thawing (Gorin & Iacomini, 1984) cycles until no more coldwater insoluble polysaccharides appear. After each cycle, the precipi­
tated fraction was recovered by centrifugation (10,000 rpm, 20 min,
4 ◦ C) and the soluble portion was named as S-2. This was again treated
with Fehling solution and Cu2+-uncomplexed polymers (fraction S-3)
were further submitted to ultrafiltration (Millipore®; polyethersulfone;
3 kDa membrane) on a filter holder (Sartorius – Model 16,249) with
compressed air at 10 psi carrier gas, generating the retained (GLC-Am)
and the eluted polysaccharide.
Sample I-1, due to the presence of contaminating glucans, suffered a
second precipitation with Fehling reagent, originating a purified gal­
actan polysaccharide (GAL-Am).

2.4. Methylation analysis by GC–MS
GAL-Am and GLC-Am were subjected to methylation process
adapted from Ciucanu and Kerek (1984) by dissolving the sample in
DMSO followed by addition of NaOH powder and methyl iodide, kept
under stirring for 30 min at 25 ◦ C. Samples were maintained overnight;
the reaction was neutralized with acetic acid and lyophilized. The
methylation process was repeated thrice for each sample. The per-Omethylated derivatives were hydrolyzed with 45% formic acid (1 mL) at
100 ◦ C for 15 h (Carbonero et al., 2012). Acid excess was removed by
lyophilization followed by reduction with NaBD4 and acetylation
generating a mixture of partially O-methylated alditol acetates de­
rivatives which were analyzed by GC–MS using a Varian (model 4000)
gas chromatograph equipped with VF5-MS capillary columns. The
injector temperature was maintained at 210 ◦ C and the oven tempera­

ture increased from 50 ◦ C (maintained 2 min) to 90 ◦ C (20 ◦ C/min, then
maintained for 1 min), 180 ◦ C (5 ◦ C/min, then maintained for 2 min)
and to 210 ◦ C (3 ◦ C/min, then maintained for 5 min). Helium was used
as the carrier gas at a flow rate of 1.0 mL/min. Partially O-methylated
alditol acetates were identified by the ion m/z by comparing their pos­
itive ions with standards. The results are expressed as a relative per­
centage of each component (Sassaki et al., 2005).

2.3. Monosaccharide composition
Polysaccharides (5 mg) were hydrolyzed with 2M TFA at 100 ◦ C for
8 h and analyzed by GC–MS as alditol acetate derivatives using a Varian
(model CP-3800) gas chromatograph coupled to an Ion-Trap 4000 mass
spectrometer using a VF5-MS column programmed from 100 to 280 ◦ C
at 10 ◦ C/min, with He as the carrier gas. The monosaccharides obtained
were identified by their typical retention times and electron impact
profiles in comparison with standards (Sassaki et al., 2008).

2.5. Controlled Smith degradation of the β-glucan (GLC-Am)
Fraction GLC-Am (50 mg) was solubilized in 25 mL of distilled water
and oxidized with 25 mL of 0.1 M sodium periodate at room temperature
in the dark under stirring for 72 h (Delgobo et al., 1998). The material
was then dialyzed (2 kDa, cut-off membrane) for 24 h against tap water.
Subsequently it was reduced with NaBH4 to pH ~8 and kept at room

Table 2
1
H and 13C NMR chemical shiftsa of fractions GAL-Amb, GLC-Amc and GLC-Smd from A. muscaria (L.:Fr).
Fractions

Units

GAL-Am

6→)-α-D-Galp-(1→

α-D-Galp-(1→
2,6→)-α-D-Galp-(1→
GLC-Am

β-D-Glcp-(1→
3→)-β-D-Glcp-(1→
3,6→)-β-D-Glcp-(1→

GLC-Sm
a
b
c
d

3→)-β-D-Glcp-(1→

13

C
1
H
13
C
1
H

13
C
1
H
13
C
1
H
13
C
1
H
13
C
1
H
13
C
1
H

1

2

3

4

5

98.1
4.98
95.7
5.15
98.0
5.02
103.2
4.27
103.2
4.55
103.2
4.55
103.0
4.53

69.6
3.88
69.6
3.88
75.1
3.97
73.7
3.09
72.8
3.35
72.8
3.35
72.9
3.31

68.4
3.85
69.5
3.94
66.9
3.98
76.7
3.14
86.3/86.7
3.50
86.1
3.51
86.2
3.49

69.5
4.03
69.5
4.03
66.2
4.25
70.2
3.14
68.7
3.29
68.7
3.29
68.4
3.25

68.8
4.17
71.0
4.18
68.8
4.17
76.4
3.29
76.4
3.29
74.8
3.54
76.4
3.27

Assignments are based on 13C NMR, 1H and HSQC analysis and are expressed as ppm.
GAL-Am: α-D-galactan isolated from A. muscaria.
GLC-Am: β-D-glucan isolated from A. muscaria.
GLC-Sm: GLC-Am that was submitted to a controlled Smith degradation, according to material and methods, Section 2.5.
5

6
a

b

66.7
3.91
61.2

3.76
66.7
3.91
60.9
3.72
60.9
3.72
68.5
4.11
60.9
3.71

66.7
3.71
61.2
3.76
66.7
3.71
60.9
3.50
60.9
3.50
68.5
3.58
60.9
3.48


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Carbohydrate Polymers 274 (2021) 118647

Table 3
Partially O-methylated alditol acetates of the α-D-galactan (GAL-Am) and β-D-glucan (GLC-Am) polysaccharides purified from A. muscaria.
Partially O-methylated alditol acetatesa

Rtb

Relative peak area %c
d

2,3,4,6-Me4-Gal
2,3,4,6-Me4-Glc
2,3,4-Me3-Gal
2,4,6-Me3-Glc
2,3,4-Me3-Glc
2,4-Me2-Glc
3,4-Me2-Gal
a
b
c
d
e

14.678
14.414
16.214
15.423
15.802
17.057

17.418

Linkage types

GAL-Am

GLC-Am

15
–
71
–
–
–
14

–
19
–
45
13
23
–

e

Galp-(1→
Glcp-(1→
6→)-Galp-(1→
3→)-Glcp-(1→

6→)-Glcp-(1→
3,6→)-Glcp-(1→
2,6→)-Galp-(1→

GC–MS analysis on a Varian 4000 capillary column;
Retention time (min).
Based on derived O-methylalditol acetates.
GAL-Am: α-D-galactan isolated from A. muscaria.
GLC-Am: β-D-glucan isolated from A. muscaria.

temperature for 20 h, neutralized with acetic acid and dialyzed (2 kDa)
for 24 h. Then, it was hydrolyzed with TFA pH 2.0 at 100 ◦ C for 30 min,
dialyzed (2 kDa) (Ruthes et al., 2015), subsequently precipitated by
ethanol (3:1, v/v) and lyophilized. The residual polysaccharide was
named GLC-Sm and analyzed by NMR spectroscopy.

during 24 h and transferred to a 5 mm NMR tube. The analyses were
carried out at temperature of 70 ◦ C. The chemical shifts are expressed in
δ (ppm) relative to signals from solvent Me2SO-d6 at δ 39.7 (13C) and 2.5
(1H) or external reference of acetone (at δ 30.2 and 2.22 for 13C and 1H,
respectively).

2.6. Determination of homogeneity and relative molecular weight

2.8. Total sugar and protein content determination

The homogeneity and relative Mw were determined by high perfor­
mance steric exclusion chromatography (HPSEC) using a refractive
index (RI) detector and 4 Waters Ultrahydrogel columns – 120, 250, 500
and 2000 were coupled in series. The eluent was 0.1 M NaNO3, con­

taining 0.5 g/L NaN3. Each sample was dissolved in the solvent (1 mg/
mL) and filtered through a membrane (0.22 μm, Millipore). The relative
Mw was determined using a calibration curve of dextran standards (9.4
kDa, 17.2 kDa, 40.2 kDa, 72.2 kDa, 124 kDa, 266 kDa and 487 kDa, from
Sigma). The data obtained were analyzed by the Wyatt Technology
ASTRA program.

Total sugar content present in GAL-Am and GLC-Am fractions was
determined spectrophotometrically following the method of Dubois
(Dubois et al., 1956). Galactose and glucose were used to construct the
calibration curve for determination of the total sugar content in GAL-Am
and GLC-Am, respectively.
The protein content present in the samples was determined using a
96-well plate by the Bradford method (Bio-Rad, Hercules, CA, USA)
(Bradford, 1976) with bovine serum albumin (BSA) as the standard and
performed according to the manufacturer's instructions.
2.9. In vitro antitumor activity of polysaccharides

2.7. Nuclear magnetic resonance (NMR) spectroscopy

2.9.1. Cell culture and polysaccharide preparation
The B16-F10 murine melanoma (BCRJ, Code 0046) and the nontumorigenic fibroblast BALB/3T3 clone A31 (ATCC, Code CCL-163)
cell lines were used. Cells were cultivated in Dulbecco's Modified Ea­
gle's Medium (DMEM; Gibco, Cat. 12800-017, Waltham, Massachusetts,
USA), supplemented with 10% fetal bovine serum (FBS; Gibco, Cat.

NMR analyses were performed on a Bruker Avance III HD spec­
trometer (Bruker), at base frequencies of 400 MHz (1H), and 100 MHz
(13C), and on a Bruker Avance III 600 MHz spectrometer (Bruker Ger­
many). An aliquot of each sample (20 mg) was dissolved in 500 μL of

solvent (D2O or Me2SO-d6) and left under stirring in a magnetic stirrer

Fig. 5. HSQC spectra of the β-glucan (GLC-Am). The sample was analyzed in Me2SO-d6 at 70 ◦ C, at base frequencies of 400 MHz (1H) (chemical shifts are expressed in
δ ppm).
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Fig. 6. GAL-Am and GLC-Am cell cytotoxicity and proliferation. (A) Neutral red and (B) crystal violet B16-F10 melanoma cell line. (C) Neutral red and (D) crystal
violet – Balb/3T3 non-tumor cell line. These results represent the set of at least three biologically independent experiments. Control represented as a dotted line.

12657029, Waltham, Massachusetts, USA), 0.25 μg/mL penicillin/
streptomycin (Thermo Fisher, Cat. 15140122, Waltham, Massachusetts,
USA), 1.57 g/L sodium bicarbonate (Merck, Cat. 36486, Kenilworth,
New Jersey, USA), and maintained in a humidified incubator at 37 ◦ C
and 5% CO2. For subculture and seeding, cells were always used with
flask confluence not higher than 80%, detached with trypsin/EDTA
(Gibco, Cat. 15400054, Waltham, Massachusetts, USA) and counted in
hemocytometer. GAL-Am and GLC-Am were dissolved at a final con­
centration of 5 mg/mL in DMEM without FBS and sterilized in 0.22 μm
membranes (Millipore, Cat. SLGV033RS, Kenilworth, New Jersey, USA)
following the preparation of serial dilutions in the desired concentra­
tions. DMEM without FBS was used as control in the same volume as the
polysaccharides.

were fixed, stained with CV and imaged, following area measurement
and counting using ImageJ Fiji Software (Schindelin et al., 2012).

2.9.4. Cell adhesion assay
Cell adhesion ability was analyzed on plastic and on pre-coated
Matrigel® (BD Biosciences, Cat. 356234, San Jose, California, USA)
plates. For plate coating, 50 μL of 20 μg/mL Matrigel® diluted in cold
PBS was pipetted per well on 96 well plates and kept overnight at 4 ◦ C.
Wells were washed once with PBS before cell seeding. For cell treatment,
B16-F10 cells (1.2 × 104 cells/well) were seeded in 6 well plates and
then exposed to 10 or 100 μg/mL GAL-Am or GLC-Am for 72 h, following
cell detachment with 2 mM EDTA and counting with a hemocytometer.
3 × 104 cells were seeded per well and kept for 2h30m in a cell incu­
bator. After the adhesion period, wells were washed with PBS (37 ◦ C) for
removal of non-adherent cells. Remaining adherent cells were fixed and
stained with CV, following dye elution and absorbance reading in a
microplate reader at 570 nm.

2.9.2. Cytotoxicity and proliferation analysis
B16-F10 cells (0.5 × 103 cells/well) or BALB/3T3 cells (0.2 × 104
cells/well) were seeded in 96 well plates and exposed or not (control
group) to 10, 100 and 1000 μg/mL of GAL-Am or GLC-Am for 72 h.
Cytotoxicity was assessed by the Neutral Red (NR) assay (Borenfreund &
Puerner, 1985), while cell proliferation was analyzed using Crystal Vi­
olet (CV) dye (Gillies et al., 1986).

2.9.5. Statistical analysis
Significant differences between experimental groups were deter­
mined by unpaired t-test with Welch's correction, using GraphPad Prism
6 software. Data presented as median ± interquartile range unless stated
otherwise.

2.9.3. Clonogenic capacity assay

B16-F10 clonogenic capacity was analyzed in two different ap­
proaches: in the first model, B16-F10 cells (1.2 × 104 cells/well) were
seeded in 6 well plates and then exposed to 10 or 100 μg/mL GAL-Am or
GLC-Am for 72 h, following cell detachment, subsequent seeding in low
density (5.5 × 102 cells/well, 6 well plate) and maintained in culture
without any treatments for more 96 h. In the second approach, non-pretreated B16-F10 cells were seeded in the same low density conditions in
the presence of 10 or 100 μg/mL GAL-Am or GLC-Am for 96 h. Colonies

3. Results and discussion
3.1. Chemical structure of the α-D-galactan (GAL-Am) from A. muscaria
The purification of the galactan (GAL-Am fraction) was developed
sequentially and are depicted in Fig. 1. The crude soluble fraction S-Am
(Suppl. Fig. 1A), obtained after ethanolic precipitation and dialysis, was
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Carbohydrate Polymers 274 (2021) 118647

Fig. 7. GAL-Am and GLC-Am reduce B16-F10 colony formation capacity. (A) Representative images of pre-treated colony formation assay. (a- Control, b- GAL-Am
100 μg/mL, c- GLC-AM 100 μg/mL). (B) Colony counting – pre-treatment. (C) Colony area – pre-treatment. (D) Colony counting – simultaneous treatment. (D) Colony
area – simultaneous treatment. These results represent the set of at least three biologically independent experiments. Data shown as median ± interquartile range for
B and D, and as mean ± SD for C and E.

treated with Fehling solution, generating the Cu2+-complexed fraction I1 (Suppl. Fig. 1B), which after a new treatment with Fehling solution,
gave rise to fraction GAL-Am, containing the purified galactan (Fig. 2).
Results showed that the total sugar content was 95% and protein was
absent in GAL-Am.
GAL-Am showed a homogeneous elution profile in HPSEC analysis,

with Mw 9.08 × 103 g/mol (Suppl. Fig. 2A) and composed of 99% of
galactose (Suppl. Fig. 3A). NMR analyses (1H, 13C, Suppl. Figs. 4 and 5,
HSQC, COSY, TOCSY, HSQC-TOCSY, HMBC and NOESY spectroscopy,
Figs. 2, 3 and 4) contributed to elucidate the α-D-galactan structure
through the signals and intermolecular correlations observed. The
α-configuration was confirmed by the coupling constant JC-1,H-1 = 173

Hz observed in coupled HSQC spectrum (Perlin & Casu, 1969). It is
possible to observe in COSY (Fig. 3A) and TOCSY (Fig. 3B) experiments
signals at δ 4.98/3.85, 5.02/3.97 and 5.15/3.88 indicating the corre­
lation between H-1 and H-2 of the three different monosaccharide
groups present in the HSQC spectra (Fig. 2). HMBC with superimposed
1
H (Fig. 3C) and NOESY (Fig. 4) were performed to confirm the linkage
between the different monosaccharide units. In HMBC experiment, it is
possible to observe signals that correlate the C-2 of the 2,6→)-α-D-Galp(1→ units with the H-1 of the α-D-Galp-(1→ units and between the C-1
and the H-6 of the 6→)-α-D-Galp-(1→ units, which indicates the pres­
ence of the linkage types 1 → 2 and 1 → 6, respectively, which can also
be observed in the result of the Nuclear Overhauser enhancement
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Carbohydrate Polymers 274 (2021) 118647

Fig. 8. B16-F10 cell adhesion. (A) Cell adhesion on plastic. (B) Cell adhesion on Matrigel®. These results represent the set of four biologically independent ex­
periments. Control represented as a dotted line.

experiment, indicating a spatial molecular proximity relationship be­

tween the cited molecules. In HSQC-TOCSY spectrum with super­
imposed HSQC (Fig. 3D) the correlation between H-6 and C-5 (δ 3.91/
68.8, 3.71/68.8, 3.91/68.8, 3.71/68.8, 3.76/71.0 for 5/6a, 5/6b, 5/6a,
5/6b, 5/6c, respectively) is indicated for the three monosaccharides
units. Through the results obtained, we conclude that the anomeric
signals at δ 98.1/4.98 and 98.0/5.02 in the HSQC spectra correspond to
6-O-linked and 2,6-O-linked α-D-Galp units, respectively, and at δ 95.7/
5.15 corresponds to C-1 of terminal α-D-Galp units linked to C-2 of Galp
units in the main chain. Signals at δ 66.7/3.91 and 66.7/3.71 were
assigned to substituted C-6, while those at δ 61.2/3.76 corresponding to
unsubstituted C-6 from terminal units (Brito et al., 2018; Carbonero
et al., 2008). The signals at δ 75.1/3.97 and at δ 69.6/3.88 correspond to
substituted and non-substituted C-2 from Galp units, respectively.
The other observed correlations are indicated in the Table 1 ac­
cording to signals already reported by Brito et al. (2018), Zhang et al.
(2013), Carbonero et al. (2008) and Rosado et al. (2003). Thus, the
assignments of all carbons and hydrogen from the GAL-Am are sum­
marized in Table 2.
The analysis of methylated derivatives of GAL-Am fraction (Table 3
and Suppl. Fig. 6) agrees with NMR analyses. The mainly derivative
observed was the 1,5,6-O-Ac3-2,3,4-Me3-galactitol (71%) from (1 → 6)linked Galp units from main chain. Approximately 14% of the main
chain units were 2-O-substituted by terminal Galp units (15%), which
could be confirmed by the presence of 1,2,5,6-O-Ac4-3,4-Me2-galactitol
and 1,5-O-A2-2,3,4,6-Me4-galactitol derivatives, respectively.
Polysaccharides having (1 → 6)-linked Galp units partially
substituted at O-2 by different side chains, constituting hetero­
polysaccharides, have already been reported for A. muscaria (Ruthes
et al., 2013a,b) and other mushrooms, such as Agaricus bisporus (Komura
et al., 2010), Flammulina velutipes (Zhang et al., 2012), Fomitella fraxinea
(Cho, Yun, et al., 2011) and Ganoderma atrum (Zhang et al., 2014). It is

worth to note that all of them had distinct chemical characteristics when
compared with the α-D-galactan reported in the present work. As far as
we know, α-D-galactan homopolymer has not been described from the
mushroom A. muscaria until now. Besides, α-D-galactans have previ­
ously been observed in mushrooms of the genus Pleurotus, having (1 →
6)-linked and partially 3-O-methylated α-D-Galp in the main chain (Brito
et al., 2018; Carbonero et al., 2008; Rosado et al., 2003). Conversely,
none of them showed 2-O-substitution by α-D-Galp non-reducing end
units. Finally, (1 → 6)-linked α-D-Galp units 2-O-substituted and without
the presence of methyl groups at C-3 have not yet been reported in the
literature.

3.2. Chemical structure of the (1 → 3),(1 → 6)-β-D-glucan (GLC-Am)
from A. muscaria
For the GLC-Am purification, the Cu2+-uncomplexed fraction S-1
(Suppl. Fig. 7A) obtained after the treatment with Fehling solution of SAm fraction has undergone to freezing and thawing process (2×) and a
new treatment with Fehling solution (Fig. 1), generating the Cu2+uncomplexed fraction S-3 (Suppl. Fig. 7B). In order to separate the
mixture of polysaccharides present in the S-3 fraction, an ultrafiltration
(3 kDa membrane) was performed, which gave rise the retained fraction
GLC-Am, containing the purified glucan (Fig. 5). Results showed that the
total sugar content was 96% and the protein was absent in this fraction.
GLC-Am showed a Mw of 1.3 × 105 g/mol with a homogeneous
elution profile in the HPSEC analysis (Suppl. Fig. 2B). It was composed
of 98.8% of glucose (Suppl. Fig. 3B), confirming the presence of a
glucan. In the methylation data (Table 3 and Suppl. Fig. 8) it is possible
to identify and characterize a (1 → 3),(1 → 6)-linked D-glucan repre­
sented by the presence of 1,5-O-Ac2-2,3,4,6-Me4-glucitol (19%), 1,3,5O-Ac3-2,4,6-Me3-glucitol (45%), 1,5,6-O-Ac3-2,3,4-Me3-glucitol (13%)
and 1,3,5,6-O-Ac4-2,4-Me2-glucitol (23%) derivatives, suggesting a β-Dglucan with (1 → 3)-linked main chain, according to the analysis of
residual polysaccharide (GLC-Sm fraction) obtained after controlled
Smith degradation (Suppl. Fig. 9). The six signals observed at δ 103.0/

4.53, 86.2/3.49, 76.4/3.27, 72.9/3.31, 68.4/3.25 and 60.9/3.71;3.48
are relatives to C1/H1, C3/H3, C5/H5, C2/H2, C4/H4, C6/H6a;b,
respectively, also referenced by de Jesus et al. (2018); Morales et al.
(2020) and characteristic of a linear (1 → 3)-β-D-glucan (Table 2).
The HSQC spectra (Fig. 5) of GLC-Am presented signals relative to
the (1 → 3),(1 → 6)-linked D-glucan, with anomeric signals at δ 103.2/
4.55 and 103.2/4.27 (C1/H1), substituted C3/H3 signals at δ 86.3/3.50,
and substituted C6/H6 signals at δ 68.5/4.11 and 68.5/3.58. The H6/C6
signals from terminal β-D-Glcp units can be observed at δ 60.9/3.72 and
60.5/3.50. The NMR chemical shifts are present in Table 2 and were
based on 1H, 13C (Suppl. Figs. 10 and 11), HSQC (Fig. 5) and comparison
with literature data (Bhanja et al., 2014; Kono et al., 2017; Morales
et al., 2020; Ruthes et al., 2015; Zhu et al., 2015).
(1 → 3),(1 → 6) β-D-glucans had already been isolated from the
mushroom A. muscaria and presented different Mw and branching degree
when compared to GLC-Am fraction. Kiho et al. (1992) obtained a
glucan with estimated Mw of 9.5 × 103 g/mol and (1 → 3)-linked main
chain substituted by two (1 → 6)-linked D-Glcp units at every seven
monosaccharide units in the main chain. Besides, Ruthes et al. (2013b)
isolated a (1 → 3),(1 → 6) β-D-glucan with Mw 1.6 × 104 g/mol
substituted at O-6 mostly by terminal β-D-Glcp units. In addition, GLCAm appears to have a Mw similar to the (1 → 3),(1 → 6) β-D-glucan
9


Carbohydrate Polymers 274 (2021) 118647

M. Zavadinack et al.

isolated from Lactarius rufus (Ruthes et al., 2013a), but differs in the
degree of branching and in the composition of the side chains.

characteristics already described as important traits eliciting mushroom
polysaccharides as anticancer compounds (Lemieszek & Rzeski, 2012).
Mushrooms heterogalactans have already shown antitumor activities
(Ruthes et al., 2016). Some of these polysaccharides present a degree of
similarity to GAL-Am, with α-D-galactose (1 → 6)-linked in the backbone
and inducing in vitro cytotoxicity and proliferation reduction against
cancer cells lines (Pires et al., 2017). A fucogalactan obtained from
Macrocybe titans mushroom, which presented a (1 → 6)-linked α-Dgalactose main chain partially substituted at O-2 by α-L-Fucp units with
a Mw of 14.2 kDa did not induce cytotoxicity nor proliferation changes in
B16-F10 cell line, however it was able to reduce cell migration in a
similar concentration to GAL-Am colony formation reduction effects
(100 μg/mL) (Milhorini et al., 2018). A fucomannogalactan with a Mw of
17.1 kDa obtained from the mushroom Hypsizygus marmoreus with (1 →
6)-linked α-D-galactose main chain partially substituted at O-2 by α-LFucp and β-D-Manp was non-cytotoxic to B16-F10 cell line at concen­
trations from 1 to 100 μg/mL, but was able to reduce cell colony area –
similar to GAL-Am - at the highest concentration (Oliveira et al., 2019).
Both previously cited polysaccharides containing a (1 → 6)-linked α-Dgalactose main chain present a relative low Mw close to GAL-Am (9.05
kDa), which could contribute to its biological effects, as already
described for other polysaccharides as a relevant structural feature to­
wards higher biological activity (Cho, Lee, & You, 2011; Choi & Kim,
2013).
Acute and long-term adverse effects related to cancer treatment are
still one of the major concerns and barriers in drug development. Most of
the induced adverse effects revolve around the consequences of target­
ing cell molecules and mechanics common to cancer and normal cells
(Nurgali et al., 2018). Hence, GAL-Am and GLC-Am selective reduction
of B16-F10 proliferation indicates a promising feature of these poly­
saccharides that could potentially be translated to direct antitumor ac­
tivity without compromising non-tumor cells.

3.3. GAL-Am and GLC-Am selectively reduce melanoma cells viability
and proliferation
GAL-Am and GLC-Am significantly reduced murine melanoma B16F10 neutral red uptake in all tested concentrations, as showed in
Fig. 6A (26.2%, 27.9% and 26.8% for GAL-Am; 22.5%, 23.5% and
32.7% for GLC-Am; concentrations of 10, 100 and 1000 μg/mL,
respectively). Similarly, both polysaccharides reduced B16-F10 prolif­
eration at all tested concentrations (Fig. 6B; 21%, 26.3% and 34.4% for
GAL-Am; 27.4%, 34.2% and 37.5% for GLC-Am; concentrations of 10,
100 and 1000 μg/mL, respectively). Strikingly, although GAL-Am and
GLC-Am induced significant reductions in B16-F10 proliferation (and
consequently decrease the neutral red color measure), they did not
impaired murine fibroblast 3T3 cell line in the same manner, with only
GAL-Am at 1000 μg/mL decreasing cell viability. The smaller concen­
trations (10 and 100 μg/mL) that did not induce any effects on 3T3 cell
line were chosen to be used in the next assays.
3.4. GAL-Am and GLC-Am cause B16-F10 colony formation impairment
in extended cultivation periods that persists with treatment withdraw
GAL-Am and GLC-Am treatment were able to affect B16-F10 colony
formation capacity, both when cells were pre-treated and seeded to form
colonies for 96 h without the polysaccharides (Fig. 7A–B; 24.8% and
26.2% reduction in comparison to control group for GAL-Am and GLCAm, respectively, at 100 μg/mL for both compounds) and when cells
were seeded to form colonies in the polysaccharide presence (Fig. 7D;
32.0% and 24.9% reduction in comparison to control group for GAL-Am
and GLC-Am, respectively, at 100 μg/mL for both compounds). In both
conditions and concentrations tested (10 and 100 μg/mL), GAL-Am and
GLC-Am were also able to significantly reduce colonies mean area when
compared to control group (Fig. 7C and E).

CRediT authorship contribution statement

Matheus Zavadinack, Jessica Loren da Rocha Bertage, Shayane da
Silva Milhorini and Guilherme Lanzi Sassaki: Carbohydrate Chemistry
Experiments, Writing - Original Draft; Daniel de Lima Bellan, Edvaldo da
Silva Trindade and Fernanda Fogagnoli Simas: Biological Experiments,
Writing - Original Draft, Lucimara M. C. Cordeiro and Marcello Iaco­
mini: Writing - Review & Editing, Supervision, Funding acquisition.

3.5. B16-F10 cell adhesion is not modulated by GAL-Am nor GLC-Am
B16-F10 treatment with GAL-Am or GLC-AM at 10 or 100 μg/mL did
not alter its capacity to adhere neither in a plastic substrate nor in
Matrigel® coated wells, as showed in Fig. 8. The absence of adhesion
modulation reinforces the previous results, as it shows that both treat­
ments did not reduce the number of adherent cells, but rather reduced
their capacity to proliferate both in normal culture conditions and in low
confluence.
Although not fully understood, it is clearly that polysaccharide
structural features, such as molecular weight, monosaccharide compo­
sition and type of glycosidic linkage, play an important role in poly­
saccharide biological activity (Xu et al., 2017). Several β-glucans
obtained from mushrooms with similar structure to GLC-Am are
described to present direct and indirect cytotoxic and anti-proliferative
activity against tumor cells (Pandya et al., 2019; Ren et al., 2012). One
example is lentinan, a β-glucan (1 → 3)-linked with β-(1 → 6)-glucose
branches, from the mushroom Lentinus edodes with a Mw ranging from
400 to 800 kDa (Pandya et al., 2019). In a similar range of concentra­
tions as used in the present paper, a purified lentinan (605.4 kDa) was
able to significantly reduce the proliferation of murine breast cancer cell
line MCF-7 and murine sarcoma cell line S180 (Zhang et al., 2015).
Another similar β-glucan from L. edodes presented a very similar selec­
tive cytotoxicity as demonstrated by GLC-Am against liver cancer cell

line H22 but not affecting human liver normal cell line HL7702 (Wang
et al., 2017). Interestingly, as GLC-Am presents similar structure to these
two polysaccharides but significantly smaller molecular weight, we
suggest that the β-(1 → 3)-glucose backbone and (1 → 6)-β-glucose
branches play an important role in the biological activity of β-glucans.
Moreover, GLC-Am glucose linkage and its water solubility are chemical

Acknowledgements
The authors would like to thank the Brazilian funding agencies:
CAPES (Funding Code 001) and CNPq (404717/2016-0, 301719/20160, 307314/2018-9) for financial support, the Chemical Technicians
Thiago J. dos Santos and Arquimedes Paix˜
ao de Santana Filho for
GC–MS and NMR analyses, respectively. The authors have declared no
conflicts of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.carbpol.2021.118647.
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