Structure characterization of the mannofucogalactan isolated from fruit bodies of Quinine conk Fomitopsis officinalis

Article abstract of DOI:10.1016/j.carbpol.2018.06.103

The mannofucogalactan as a major component of water extract was obtained from fruit bodies of Fomitopsis officinalis by extraction with boiling water followed by deproteination, decoloration, and purification using anion-exchange chromatography and size exclusion chromatography. Its structure was characterized using the data of monosaccharide composition, methylation analysis, one- and two-dimensional NMR spectroscopy. The studied polysaccharide was a branched mannofucogalactan with a backbone composed of partially 3-O-methylated 1,6-O-linked α-D-galactopyranosyl residues. Almost every second residue in the backbone was substituted at O-2 by 3-O-α-D-mannopyranosyl-α-L-fucopyranosyl and β-D-galactopyranosyl residues. The non-reducing terminal α-L-fucopyranosyl units, which were identified by GC–MS analyses, appeared to be the part of mannofucogalactan side chains also.

Full text of DOI:10.1016/j.carbpol.2018.06.103

Accepted Manuscript
Title: Structure characterization of the mannofucogalactan
isolated from fruit bodies of Quinine conk Fomitopsis
officinalis
Authors: Victoria V. Golovchenko, Daria S. Khramova,
Shinen Naranmandakh, Jamsranjav Ganbaatar, Alexander O.
Chizhov, Alexander S. Shashkov
PII:
S0144-8617(18)30758-6
DOI:
Reference:
https://doi.org/10.1016/j.carbpol.2018.06.103
CARP 13777
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Received date:
Revised date:
Accepted date:
12-4-2018
22-6-2018
22-6-2018
Please cite this article as: Golovchenko VV, Khramova DS, Naranmandakh S, Ganbaatar
J, Chizhov AO, Shashkov AS, Structure characterization of the mannofucogalactan
isolated from fruit bodies of Quinine conk Fomitopsis officinalis, Carbohydrate
Polymers (2018), https://doi.org/10.1016/j.carbpol.2018.06.103
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Structure characterization of the mannofucogalactan isolated from fruit bodies of Quinine conk
Fomitopsis officinalis
Victoria V. Golovchenko a*, Daria S. Khramova a, Shinen Naranmandakh b, Jamsranjav
Ganbaatarc, Alexander O. Chizhov d, Alexander S. Shashkov d
a Institute of Physiology, Komi Science Centre, The Urals Branch of the Russian Academy of Sciences, 50,
Pervomaiskaya str., Syktyvkar, 167982, Russia
b National University of Mongolia, School of Life Sciences, Ulaanbaatar-51, Mongolia
c Institute of Chemistry and Chemical Technology, the Mongolian Academy of Sciences, Ulaanbaatar-51,
Mongolia
Graphical abstract
Highlights




The mannofucogalactan was extracted from Fomitopsis officinalis by hot water.
Backbone of mannofucogalactan composed from 1,6-linked D-Galp residues.
3-O-D-Manp-L-Fucp/-D-Galp/L-Fucp units substitute Galp residues of backbone at O-2.
Part of the Galp residues of backbone is methylated at 3-O-position.
d N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 47, Leninsky prospect,
Moscow, 119991, Russia
Abstract
The mannofucogalactan as a major component of water extract was obtained from fruit bodies
of Fomitopsis officinalis by extraction with boiling water followed by deproteination, decoloration, and
purification using anion-exchange chromatography and size exclusion chromatography. Its structure was
characterized using the data of monosaccharide composition, methylation analysis, one- and two-
dimensional NMR spectroscopy. The studied polysaccharide was a branched mannofucogalactan with a
backbone composed of partially 3-O-methylated 1,6-O-linked -D-galactopyranosyl residues. Almost

every second residue in the backbone was substituted at O-2 by 3-O--D-mannopyranosyl--L-
fucopyranosyl and -D-galactopyranosyl residues. The non-reducing terminal -L-fucopyranosyl units,
which were identified by GC–MS analyses, appeared to be the part of mannofucogalactan side chains also.
* Corresponding author: tel/fax: +7 (8212) 241001, e-mail: lemnan@mail.ru
Keywords: medicinal fungi; Fomitopsis officinalis; mannofucogalactan; spectroscopy NMR; structure.
1. Introduction
Macrofungi (mushrooms) have been used as food component and medicine from ancient
times. The mushrooms are extensively applied in traditional Oriental medicine for many centuries, in
which knowledge and practice of use of mushroom and of their water-soluble fractions (decoctions and
essences) were primarily originated. However, mushrooms were used in folk medicine as curatives in
other countries also (Chang, 1999; Hobbs, 2000; Wasser, 2014). Ancient traditions revealed the
importance of several mushroom species, namely, Shiitake mushrooms [Lentinus edodes (Berk.) Sing],
Lingzhi (in China)/Reishi (in Japan) [Ganoderma lucidum (W.Curt.:Fr.) P. Karst.], Split gill mushroom
[Schizophyllum commune (Fr.:Fr.)], Chaga/birch fungus [Inonotus obliquus (Pers.:Fr.) Pilát], Turkey tail
[Trametes versicolor (L.: F) Pilát], Quinine conk [Fomitopsis officinalis (Vill.:Fr.) Bond. et Sing.], Tinder
bracket mushrooms [Fomes fomentarius (L.: Fr.) Fr.], Birch bracket fungus/Razor strop fungus [Piptoporus
betulinus (Bull.:Fr.) P. Karst.] (Chang & Wasser, 2012; Grienke, Zöll, Peintner & Rollinger, 2014; Shikov et
al., 2014; Wasser, 2002). Nowadays mushrooms are promoted as natural product-based pharmaceuticals
with lower toxic effects and providing high potential for the treatment of different pathological processes.
The polysaccharides and polysaccharide complexes have been shown to be bioactive
components of mushrooms. Primarily they attracted considerable attention due to their efficient
immunomodulatory, anti-cancer (Meng, Liang, & Luo, 2016; Moradali, Mostafavi, Ghods, & Hedjaroude,
2007; Ren, Perera, & Hemar, 2012; Zhang, Cui, Cheung, & Wang, 2007) and anti-inflammatory effects (Li,
Zhang, & Ma, 2014; Muszyńska, Grzywacz-Kisielewska, Kała, & Gdula-Argasińska, 2018; Smiderle et al.,
2008a; Taofiq, Martins, Barreiro, & Ferreira, 2016). The structure of a vast amount of glucans and different
heteropolysaccharides have been precisely defined recently. The immunomodulating and anti-tumor
2

activity of fungi were associated mainly with β-D-glucans, whereas heteropolysaccharides also presented
in mushrooms revealed the pronounced biological activities (Ruthes, Smiderle, & Iacomini, 2016; Villares,
Mateo-Vivaracho, & Guillamón, 2012).
The investigation of structure of fungi polysaccharides is highly desirable, due to their potential
biological activity. Although, studies including clinical trials need to be carried out to ascertain the safety
of these compounds as adequate alternatives to conventional drugs (Corrêa, Brugnari, Bracht, Peralta, &
Ferreira, 2016), component composition of many fungi used in folk medicine has not been studied yet.
This study is a part of our complex elucidation of structure and biological activity of polysaccharides of
fruit bodies of the Quinine conk/Agarikon [Fomitopsis officinalis (Vill.) Bondartsev & Singer], which belong
to a group of xylotroph basidiomycetes growing on stems of larch trees. These fungi attracted interest
due to extensive use of its basidiocarps for medical purposes, which even resulted in a decrease of fungi
population (Chlebicki, Mukhin, & Ushakova, 2003). Nevertheless, the structural characteristics of its
polysaccharides have not been investigated yet. Here we report the isolation and structure analysis of a
mannofucogalactan as major component of water extract of these fungi. Further our experiments aimed
to investigate structure of polysaccharides obtained by alkaline extraction, and to study the cytotoxic
activity of all polysaccharides isolated from these fungi in order to reveal the relations between chemical
structures with biological properties.
2. Materials and methods
2.1. Materials and chemical reagents
Chemical reagents: ethyl alcohol, ethanol, C2H5OH (96%, JSC Kirov Pharmaceutical Factory,
Russia); methyl alcohol, methanol, CH3OH (99.9%, Reakhim, Russia); sodium hydroxide, NaOH (98%, Fluka,
Germany); sodium chloride, NaCl (99%, Sigma-Aldrich, USA); hydrogen peroxide solution, H2O2 (29.0-
32.0% %, Sigma-Aldrich, USA); lead (II) acetate trihydrate, Pb(CH3COO)2  3H2O (99.999%, Sigma-Aldrich,
USA); (R)-(−)-2-butanol (Aldrich, USA); dichloromethane, CH2Cl2 (≥99.8%, Sigma-Aldrich, USA), chloroform,
CHCl3 (99.9%, Ekos-1, Russia); ammonium hydroxide solution, NH4OH (≥ 25 NH3 in H2O, 99.9%, Ekos-1,
Russia); 1-methylimidazole, C4H6N2 (99%, Sigma-Aldrich, USA); acetyl chloride (≥98%, Merck, Germany);
3

iodmethane, CH3I (99%, Merck, Germany); trifluoroacetic acid, CF3COOH (99%, Acros organics, USA);
pyridine, C5H5N (99%, Ekos-1, Russia); acetic acid, CH3COOH (99.9%, Khimreactive, Russia); sodium
borohydride, NaBH4 (98.5%, Sigma-Aldrich, USA); dimethylsulfoxide, DMSO (≥99.9 %, PanReac Quimica,
Spain); D2O (99.9 atom % D, Sigma-Aldrich, USA); sodium sulphate, Na2SO4 (99%, Ekros, Russia); toluene,
C6H5-CH3 (99 %, Ekos-1, Russia); sulfuric acid, H2SO4 (99.9%, Vekton, Russia); phenol, C6H5OH (99%,
Reakhim, Russia).
The Folin & Ciocalteu’s phenol reagent (Sigma-Aldrich, USA) was used in Lowry assay.
For Ion Exchange chromatography we used DEAE-cellulose (Sigma-Aldrich, USA), for gel
permeation chromatography – Sephacryl S-300 high resolution (Albersham Bioscientes, Sweden).
The pullulans: 1.3, 6, 12, 22, 50, 110, 200, 400, and 800 kDa) (Fluka, Germany); bovine serum
albumin (≥96%, Sigma-Aldrich, USA); L-(–)-fucopyranose (≥99%, Sigma-Aldrich, USA), D-(+)-
mannopyranose (≥99%, Sigma-Aldrich, USA) and D-(+)-galactopyranose (≥99%, Sigma-Aldrich, USA); myo-
inositol (≥99%, Sigma-Aldrich, USA) were used as standards.
Biological material: the fruit bodies of Fomitopsis officinalis (Vill.: Fr.) Bond. et Sing. 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.
2.2. Isolation of polysaccharide FoHWE, it’s separation and purification
Dry fruit bodies of F. officinalis (200 g) were milled in a blender, and the powder was defatted
with 96% ethanol at 60°C for 5 h (2 × 350 mL) to remove soluble lipids, sugars or phenolic compounds.
The supernatant was removed by filtration. The polysaccharides were extracted twice with boiling water
under reflux for 4 h (3  1000 mL). The residue was separated by centrifugation and the extracts were
collected, combined, concentrated by rotary evaporator to 100 – 200 mL. For the deproteinization 2%
lead acetate was added to the extract to its final concentration of 0.4 – 0.5 % (Chen, 2012). The extract
4

was decolorized by addition of 30 % H2O2 to its final concentration of 5 %, and then 10 % aqueous NH4OH
was added to achieve pH 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 during 2 days at 10 °C and concentrated on a rotary evaporator. Then
polysaccharides were precipitated with 95 % ethanol, dissolved in water after centrifugation and
lyophilized, the traces of ethanol was removed on a rotary evaporator. A fraction FoHWE was obtained as
a result of the extraction. The yield of the fraction FoHWE and its monosaccharide composition are
presented in Table 1.
The yield of polysaccharide FoHWE was calculated as follows:
m(FoHWE)
ω(FoHWE) =
× 100%,
m(dry biological material)
where m (FoHWE) (g) – weight of polysaccharide FoHWE obtained after extraction, m (dry biological
material) (g) – weight of dried fruit bodies of F. officinalis used for the extraction.
Anion-exchange chromatography and gel permeation chromatography were used for separation and
purification of polysaccharide FoHWE. Polysaccharide FoHWE (50 mg) was dissolved in 5 mL of distilled
water and solution was applied to a column (2.5 cm × 40 cm) of DEAE-cellulose (OH--form). The column
was stepwise eluted with distilled water, 0.01, 0.1, 0.2, 0.3 and 0.5M NaCl solutions (400 mL of each
eluent) at a flow rate of 1 mL/min. The fractions were collected at 10 min intervals using low-pressure
system Pharmacia Biotech (Sweden) with a fraction collector F RAC-100, pump P-50, Uvicord SII, Recorder
REC 101. The carbohydrates content of each tube was determined by phenol-sulfuric acid method (Dubois
et al., 1956). One major fraction, eluted with water, was collected, concentrated, dialyzed, and further
purified by gel permeation chromatography on a column of Sephacryl S-300 (1.6 cm×100 cm, V0=27 mL),
which was eluted with 0.15M NaCl solution included NaN3 (0.02%) at a flow rate of 0.3 mL/min. The eluate
(2.5 mL/tube) was collected and detected as described above. As a result, one major purified fraction
FoHWE-1 (kav 0.43) was collected, dialyzed and lyophilized with a yield 37.35 mg (Table 1). The elution
curves for anion-exchange column and gel filtration column are presented in Supplementary Data (Fig.
1S).
5

The yield polysaccharide FoHWE-1 was calculated as follows:
m(FoHWE
̵
ω(FoHWE
̵
1) × 100%,
m(FoHWE)
where m (FoHWE-1) – weight of purified polysaccharide FoHWE-1 obtained after anion-exchange and gel
permeation chromatography, m (FoHWE) – weight of crude polysaccharide FoHWE.
2.3. General analytical methods
Specific optical rotation was measured in 1 mL cell on a Polatronic MHZ polarimeter (Germany) at
20°C. The absorbance was measured using Ultrospec 3000 spectrophotometer (Pharmacia Biotech,
England). The solutions were concentrated with a Laborota 4002 rotary evaporator (Heidolph, Germany)
under reduced pressure at 40 °C. The samples were centrifuged with a 6 K 15 centrifuge (Sigma, Germany)
at 11000 rpm at 4 °C for 20 min, lyophilized using an Edwards lyophilizer (England) under a constant
vacuum of <10 mTorr at – 65 °C.
The monosaccharide composition of polysaccharides was determined as described earlier in
details (Popov et al., 2014). The content of monosaccharides was detected by gas–liquid chromatography
(GLC) after the polysaccharides hydrolysis and transformation into their alditol acetates. The calculation
of monosaccharides content (weight %) was made as follows:
S(i)×m(In)
ω(i) =
× 100%,
(
)
( )
S In ×k i ×m
where S(i) – area of each monosaccharide peak on GLC chromatogram; S(In) – area of myo-inositol (as
internal standard) peak on GLC chromatogram; m(mg) – weight of polysaccharide used for hydrolysis; k(i)
– considering weight response-factors for each monosaccharide. k(i) was detected as S(i) (standard
monosaccharide)/S(In) (myo-inositol), which were used in equal weight ratios.
Table 1 presented data calculated as a relative percentage of sum of all monosaccharides and
protein, which were determined in simples, as follows:
ω(i)
a(i) =
× 100%,
n
∑
( )
ω i +ω(protein)
i=1
6

where a(i) – calculated content of monosaccharide (weight %),  (i) – content of each monosaccharide
(weight %),  (protein) – content of protein detected by Lowry method (weight %).
Protein concentration was determined using Lowry’s procedure (Lowry, Roserbourgh, Farr, &
Randall, 1951) with bovine serum albumin as a standard.
The monosaccharides were determined at 490 nm using the phenol-sulphuric acid assay
(Dubois, Gilles, Hamilton, Rebers, & Smith, 1956).
The homogeneity and molecular weight of the polysaccharide samples were determined by high
performance gel-permeation chromatography (HPGPC). Pullulans (1.3, 6, 12, 22, 50, 110, 200, 400, and
800 kDa) were used as standards. The details of procedure were described earlier (Golovchenko,
Khramova, Ovodova, Shashkov, & Ovodov, 2012).
The enantiomeric configuration of monosaccharides was determined by the method described
earlier (Gerwig, Kamerling, & Vliegenthart, 1978) with minor modification. The polysaccharide (3.0 mg)
was hydrolyzed with CF3COOH at 100°C for 8 h, and then the acid was removed by co-distillation with
water. Mixture of monosaccharides was dissolved in 0.1mL (R)-(−)-2-butanol and added 15 µL of acetyl
chloride; the mixture was heated at 80 C for 16 h. Then the solution was evaporated; butylglycosides was
dissolved in 0.2 mL pyridine and 0.2 mL acetic anhydride was added; the mixture was heated at 100C for
1 h. The solution was evaporated, O-acetylated butylglycosides were dissolved in 0.1 mL in CHCl3 and
analyzed by GLC using a capillary column VF-5 ms (Varian, USA; 0.25 mm, 30 m), a temperature program
(3 °C/min) raised from 150 (1 min) to 210 °C (3 min). The (R)-(−)-2-butyl 2,3,4,6-tetra-O-acetyl-glycosides
were identified by the comparison with those prepared from the D-mannopyranose, D-galactopyranose
and L-fucopyranose.
2.4. Methylation analysis
The polysaccharide fraction FoHWE-1 was activated with powdered NaOH and methylated with
CH3I (Ciucanu & Kerek, 1984) as described earlier (Cerqueira et al., 2011) with minor modifications. The
sample (4 mg) was dispersed in dried DMSO (2mL) and sonicated occasionally until it was fully dispersed.
7

NaOH pellets (80 mg) powdered under argon were added to the solution. The mixture was stirring using
a magnetic stirrer at 250 rpm (Heidolph MR 3001, Germany) for 2 h at 23 °C. The mixture was cooled and
1 mL of methyl iodide was added under continuous stirring at 700 rpm in an ice bath for 1 hours. The
reaction was stopped with water (2 mL) and the resulting partially methylated derivatives were extracted
with chloroform (3 mL). The organic layer was collected and washed with water (4  2mL), dried with
Na2SO4 and evaporated to dryness and remethylated. The methylation process was repeated three times.
The fully methylated polysaccharides were then hydrolyzed with 2 M CF3COOH (1 mL) at 121 °C for 1 h
using thermostat (Jouan, France), cooled and rotary evaporated at 35 °C. The partially methylated sugars
were dissolved in 2M NH4OH (0.3 mL), reduced with NaBH4 (20 mg) (1 h at 23°C) and with conc. CH3COOH
(0.2 mL) to decompose the excess of the reducing agent and finally rotary evaporated to dryness. The
acetylation of the partially methylated alditols was performed by adding of 1-methylimidazole (0.5 mL)
and acetic anhydride (2 mL) for 30 min at 23 °C. Water (2 mL) was added to the solution to decompose
the excess of acetic anhydride. The partially methylated alditol acetates were extracted with
dichloromethane (3 mL). The dichloromethane layer was washed with water (2 × 3 mL), dried with Na2SO4,
and evaporated to dryness. The partially methylated alditol acetates were dissolved in dichloromethane
(100 L) and analyzed by GC–MS.
Partially acetylated, partially methylated alditols were analyzed by Agilent 7890A/5977A GC-MS
mass spectrometer (Agilent, Germany) equipped with a HP-5ms capillary column (Agilent Technologies).
Helium was used as a carrier gas (flow rate 1.8 mL/min). Injector temperature 280 C, injection volume 1
L (autosampler injection mode), split ratio 1 : 10. Temperature program started at 150 C (hold time
1 min), linear increase at 10 C/min to 280 C then linear increase at 35 C/min to 325 C. EI energy was
fixed at 70 eV, scanning range from m/z 50 to m/z 400, ion source temperature 230 C.
The content of methylated monosaccharides (molar %) was detected as follows:
S(i)×Rf(i)
C(i) =
S(i)×Rf(i) × 100%,
n
∑
i=1
where S(i) – area of each methylated monosaccharide peak on GC–MS chromatogram; Rf(i) – considering
molar response-factors previously determined (Sweet et al., 1975).
8

2.5. Nuclear magnetic resonance (NMR) spectroscopy and NMR spectra analyses
The samples (25 mg) were deuterium-exchanged by freeze-drying from D2O for three times and
then dissolved in 500 L D2O with 0.5 L of acetone (99.96 atom % D, Sigma Aldrich) as a reference for
the calibration of the chemical shift (H 2.225 ppm, C 31.45 ppm). All homo- and heteronuclear NMR
experiments of the samples were carried out on a Bruker DRX Avance II 600 MHz spectrometer (Germany)
at a probe temperature that providing a minimum overlap of the signal of deuterated water with the
polymer signals (313 K). 2D NMR spectra were obtained using a standard Bruker software.
3. Results and discussion
3.1. Isolation, purification and composition of mannofucogalactan
Using boiling water the fraction FoHWE (yield 3.68 % of the air-dried material) was isolated from
defatted with hot 96 % ethanol fruit bodies of F. officinalis. The extract was centrifuged, deproteinizated
by lead acetate (Chen et al., 2012), and decolorized with H2O2. After removing of insoluble precipitate by
centrifugation, the extract was dialyzed against distilled water, and polysaccharides were precipitated
from the extract with an excess of ethanol and the solution was freeze-dried. The monosaccharide
composition of polysaccharide fractions (Table 1) was identified by GLC after the complete acid hydrolysis
of polysaccharides with 2 M aqueous CF3COOH and transformation of monosaccharides to corresponding
alditol acetates. The polysaccharides of fraction FoHWE contained fucose, mannose, glucose, galactose
residues as major components of carbohydrate chains, but had a heterogeneous HPGPC elution profile
(Fig.1a). Therefore, the fraction FoHWE was successively separated by anion-exchange chromatography
-
using a DEAE-cellulose (OH -form) column and gel permeation chromatography on a column of Sephacryl
S-300 by water elution. As a result, polysaccharide fraction FoHWE-1 was obtained (yield 74.7 %) as a
dominant component.
9

The fraction FoHWE-1 exhibited an asymmetrical single peak on HPGPC with Mw/Mn 1.4,
however, homogeneity FoHWE-1 was significantly higher than FoHWE (Mw/Mn 13.6). Its molecular
weight (MW) was equal to 17.4 kDa (Fig. 1b), and its specific rotation was equal to []D20 + 77.4 (c 0.155,
H2O). The monosaccharide composition of the fraction FoHWE-1 was typical for mannofucogalactans
(Table 1).
There was no absorption peak at 280 nm in the UV spectrum of the fraction FoHWE-1 and the
Lowry assay confirmed that it was free of protein.
3.2. Structural characterization of mannofucogalactan
3.2.1. Interpretation of NMR data
In order to confirm the types of linkages in the mannofucogalactan of fraction FoHWE-1, NMR
spectroscopy was used. The sugar residues were assigned applying 1H/1H correlation spectroscopy (COSY),
1
total correlation spectroscopy (TOCSY), rotating frame Overhauser effect spectroscopy (ROESY), H/13C
heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlation (HMBC)
experiments. The assignments of the protons and carbons (by HSQC measurements) of the residues are
listed in Table 2.
1
The five anomeric protons were observed in H NMR spectrum of the fraction FoHWE-1 (Fig.
2a), but 1H/13C HSQC spectrum (Fig. 2b) contained an anomeric region with signals for six spin systems C-
1/H-1 at  103.64/5.14, 102.56/5.13, 102.56/5.09, 99.23/5.05, 99.23/5.00, 104.63/4.47. The sugar
residues were designated (A–F) according to the decreasing chemical shifts of the anomeric protons
(Table 2). As judged by the absence from the 13C NMR spectrum of signals within  82–88 ppm, all sugar
residues were in the pyranose form (Buchmarinov et al., 2004). The integrated ratio of the H-1 signals in
1
the H NMR spectra showed that the peak area ratio of A+B:C:D:E:F of the fraction FoHWE-1 was
approximately 1.5:0.3:1.2:1.0:0.4.
10

Determination of protons position of residue (A) revealed stepwise connectivities from H-1 (H
5.14) to H-2 (H 4.09), H-3 (H 3.91), H-4 (H 3.69), H-5 (H 3.81) in the COSY spectrum, and the TOCSY
spectrum showed the correlation of H-1(H 5.14) with H-2 (H 4.09), and H-5 (H 3.81) with H-6-s (H 3.79
and 3.90). The HMBC spectrum demonstrated the correlation of C-5 (C 74.67) with the H-4 (H 3.69) and
H-6 (H 3.79).
The single correlation peak of H-1/H-2 (H/H 5.13/3.95) was detected in the COSY spectrum for
residue (B), and the TOCSY spectrum showed connectivities from H-1 (H 5.13) to H-3 (H 3.98). The
positions H-4 and H-5 were detected from cross peaks between methyl group protons (H 1.25) and H-4
(H 3.99) and H-5 (C 4.17) in ROESY spectrum (Fig.3a). Additionally the cross peaks between methyl group
protons (H 1.25) and C-4 (C 72.72) and C-5 (C 68.17) were identified in HMBC spectrum, and confirmed
in HSQC spectrum (Fig. 2c).
The positions of H-2 (H 3.87) and H-3 (H 4.09) in the COSY spectrum for residue (D) were
detected, in TOCSY spectrum the positions of H-2 (H 3.87), H-3 or/and of H-4 (H 4.09), and H-5 (H 4.16)
were detected from correlations with H-1 (H 5.05). In addition, the ROESY spectrum included correlation
peaks of H-1/H-2 at H/H 5.05/3.87; H-1/H-5 at H/H 5.05/4.16; H-1/H-6 at H/H 5.05/3.70 and two
correlation peaks of low intensity of H-1/H-4 or H-1/H-3 at H/H 5.05/4.09 and H-1/H-6’ at H/H 5.05/3.98
(Fig. 3b). The positions of H-6; H-6’ were detected in TOCSY spectrum from correlations with H-5 (H/H
4.16/3.70; 3.98) (Fig. 4).
The positions of H-2 (H 3.87) and H-3 (H 3.89) from correlation with H-1 (H 5.00) were
detected in the COSY and TOCSY spectrum for residue (E). Additionally position of H-4 (H 4.04) was
detected from correlation with H-1 in the TOCSY spectrum too. In the ROESY spectrum correlation peaks
11

of H-1/H-2 at H/H 5.00/3.87 and H-1/H-6 at H/H 5.00/3.72 and two correlation peaks of low intensity of
H-1/H-4 at H/H 5.00/4.04 and H-1/H-5 at H/H 5.00/4.20 were observed (Fig. 3b). The positions of H-6-s
were detected in TOCSY spectrum from correlations of H-5/H-6; H-6’ (H/H 4.20/3.72; 3.92) (Fig. 4).
The COSY spectrum showed the connectivities from H-1 (H 4.47) to H-5 (H 3.71) in residues
(F). The TOCSY spectrum showed the connectivities from H-1 (H 4.47) to H-4 (H 3.95), and from H-5 (H
3.71) to H-6-s (H 3.80 and 3.91). The HMBC spectrum demonstrated correlation of C-5 (C 76.42) with the
H-6 (H 3.80). The ROESY spectrum included the correlation signals of H-1 (H 4.47) with H-2 (H 3.58), H-
3 (H 3.67), H-5 (H 3.71) and H-6-s (H 3.80 and at H 3.91) (Fig. 3c).
On basis of the proton assignments, data of HMBC experiments, computerized approach to the
structural analysis of polysaccharides (Lipkind, Shashkov, Knirel, Vinogradov, & Kochenkov, 1988), and
comparison with data reported earlier (Carbonero, Mellinger, Sassaki, Gorin, & Iacomini, 2008b; Cho, Yun,
Yoo, & Koshino, 2011; Smiderle, Alquini et al., 2004) the correlations of carbons and protons were
detected in the HSQC spectrum (Fig. 2c), and chemical shifts from C-1 to C-6 are listed in Table 2 and Fig.
5.
The -configuration for sugar residues (A–E) and -configuration for sugar residue (F) were
detected from chemical shifts of C-5-s and H-5-s. Chemical shift of H-5 at  3.81 ppm and C-5 at  74.67
ppm for Manp residues are characteristic of -configuration of Manp residues (Jansson, Kenne &
Widmalm, 1989; Lipkind et al., 1988). Chemical shift of C-5 at  68.17 ppm is typical for -configuration
of Fucp residues (Jansson et al., 1989). Chemical shifts in the range  70-72 ppm are characteristic of C-5
of Galp with -configuration of Galp residues, but chemical shift at  76.42 ppm is typical for -
configuration of Galp residues (Lipkind et al., 1988).
Positions of signals of residues (A) and (F) were identified as corresponding to the α-
mannopyranose and β-galactopyranose residues located at non-reducing terminal positions, respectively.
The positions of signals of C-6-s atoms at C 62.42 ppm evidently point to the absence of substitutes at
these carbons (Lipkind et al., 1988).
12

1
The residue (B) was identified as the 3-O-substituted fucopyranosyl residue. The H/13C HSQC
spectrum contained signals at  16.95/1.25 ppm, both carbon and proton chemical shifts were typical for
methyl group signals of 6-deoxyhexopyranose; here fucopyranose is the only monosaccharide of this type
identified in carbohydrate chains of polysaccharide. The down-field shift of C-3 (C 78.88) and highfield
shift of C-2 (C 68.83) with respect to standard values for glycopyranoses (Lipkind et al., 1988) indicated
the presence of 3-O-substitution.
Positions of signals of residues (D) and (E) and downfield shift of C-6-s (C 68.44 and 67.86,
respectively) and additionally of C-2 (C 78.75) of residue (D) with respect to standard values for
glycopyranoses (Lipkind et al., 1988) indicated these residues as the 2,6-di-O-substituted and 1,6-linked
α-galactopyranose residues, respectively.
The residue (C) was not identified and the assignment of each signal was not detected, due to
the presence of only one cross peak of H-1/H-2 with low intensity at H/H 5.09/3.86 ppm in the COSY
spectrum, which overlapping with intensive signals of H-2 of residues (D) and (E). The TOCSY spectrum
also failed to be useful because of overlapping and/or no proton correlation. The residue (C) appeared to
be the non-reducing end -L-Fucp residues. The anomeric region of HSQC spectrum presented in Fig. 2a
was similar to anomeric region of HSQC spectrum of mannofucogalactan from edible mushroom Grifola
frondosa (Oliveira et al., 2018), where the authors identified it as non-reducing end -L-Fucp residues.
Moreover, the positions of H-1 and H-2 of residue (C) conformed to positions of H-1 and H-2 of non-
reducing end -L-Fucp residues of mannofucogalactan G. frondosa (Oliveira et al., 2018).
In addition, the 3-O-methylated 1,6-linked α-Galp residues (namely (G) residue) were
determined. The 1H/13C HSQC spectrum in high-field contained the signal at C/H 57.6/3.49 ppm distinctive
1
for methyl group. The H/13C HMBC indicated on the correlation between proton at H 3.49 ppm and
carbon at C 80.4 ppm and carbon of methyl group at C 57.6 ppm with proton at H 3.58 ppm. The ROESY
spectrum revealed the correlation between the proton of methyl group at H 3.49 ppm and proton at H
3.58 ppm. The presence and position of cross peak at C/H 80.4/3.58 ppm in HSQC spectrum (Fig. 2c)
confirmed the presence of 3-O-Me--Galp residues as noted previously (Carbonero et al., 2008; Oliveira
13

et al., 2018, Ruthes, Rattmann, Carbonero, Gorin, & Iacomini, 2012; Smiderle et al., 2008c). In the TOCSY
spectrum (Fig. 4) in the region of correlation of signals of H-5-s with H-6-s presented three spin systems
for three different galactose residues substituted at 6-O position, two of which belonged to (D) and (E)
residues, the third apparently – to (G) residue. In the 1H/13C HSQC spectrum three spin systems of C-6/ H-
6; 6’ at C/H 68.44/3.70; 3.98 and 68.20/3.72; 3.92 and 67.86/3.72; 3.92 characterizing three different
galactose residues substituted at 6-O position were detected also. Position of H-5 for (G) residue was
detected from cross peak at  H/H 4.24/3.92; 3.72 in the TOCSY spectrum (Fig. 4). The correlation of C-4/H-
4 at C/H 70.15/4.24 ppm for (G) residue was detected in HSQC spectrum (Fig. 2c). The assignments of C-6
for (D), (E) and (G) residues were assigned as C 68.44, 68.20 and 67.86 ppm, respectively, similar to the
positions of C-5 signals of these residues (Fig. 2c) and data presented for analogical mannofucogalactan
of Grifola frondosa (Oliveira et al., 2018). The presence of cross peaks of C-6 (D) with H-3 (G) at C/H
68.44/3.58 and with H-6 (E) and (G) at C/H 68.44/3.92 and with H-3 (D) at C/H 68.44/4.09 in the HMBC
spectum indicated the neighborhood of these residues.
The sequences of glycosyl residues of the polysaccharide along with some intra-residual
contacts were determined from ROESY experiments (Fig. 3). As a result, the following inter-residual
correlations were detected:
H-1(A)/H-3(B) at  H/H 5.14/3.98 demonstrated the presence of
α-D-Manp-(1→3)-α-L-Fucp-(1→;
6)- -D-Galp-(1

...
...
2)
...
3)- -L-Fucp-(1

H-1(B)/H-2(D) at  H/H 5.13/3.87 and proton of methyl group (B)/H-1(D) at  H/H 1.25/5.05 demonstrated
the presence of
6)- -D-Galp-(1

...
...
2)
-D-Galp-(1

14

H-1(F)/H-2(D) at  H/H 4.47/3.87 demonstrated the presence of
The substitution of fucopyranose residues by non-reducing terminal mannose residues and the
substitution of galactose residues by fucose residues were confirmed by the presence of correlation peaks
at  C/H 103.5/3.98 and 102.56/3.87 in HMBC spectrum, respectively.
Absolute D-configuration of Manp residues was adopted from genetic considerations since
natural L-Manp has not been found so far. Absolute L-configuration of -Fucp was found in -D-Manp-
(1→3)--Fucp fragment based on the regularities reported previously (Lipkind et al., 1988; Shashkov,
Lipkind, Knirel, & Kochetkov, 1988). Correspondingly, absolute D-configuration of -Galp was determined
in -L-Fucp-(1→2)--Galp fragment. The absolute configuration of the sugars was confirmed by the
method described earlier (Gerwig et al., 1978). These are in agreement with previously published data
(Carbonero et al., 2008a; Usui, Iwasaki, & Mizuno, 1981).
3.2.2. Interpretation of methylation analysis
The NMR data were supported by the methylation analysis (the original GC/MS spectra are
presented in Supplementary Data (Fig. 2S)). The polysaccharide FoHWE-1 was methylated by the Ciucanu
& Kerek method (Ciucanu & Kerek, 1984) and converted into the mixtures of partially O-methylated alditol
acetates, which were analyzed by GC–MS with use of characteristic retention times (T-values) (Bjorndal,
Hellerqvist, Lindberg, & Svensson, 1970; Sims, Carnachan, Bell, & Hinkley, 2018) and molar response
factors (Rf) previously determined for the partially methylated alditol acetates individual constituents
(Sweet, Shapiro, & Albersheim, 1975). The methylation analysis showed that all sugar components were
pyranoses.
According to the peak areas, five major components were identified by their retention times and
typical fragmentation patterns: non-reducing terminal units: Manp-(1→; Galp-(1→, linear units: →6)-
15

Galp-(1→; →3)-Fucp-(1→; and 2,6-di-O-substituted galactose residues as points of branching (Table 3),
which were detected in NMR spectra (Table 2). Results of methylation and NMR analysis indicated that
1,6-linked and 2,6-di-O-substituted galactose residues presented in almost equal amounts (Table 3). Other
residues were identified as structural units of side chains.
The presence of non-reducing terminal Fucp units was confirmed by GC–MS analyses. As shown
earlier for mannofucogalactans from Flammulina velutipes (Mukumoto & Yamaguchi, 1977; Smiderle et
al., 2008b) and edible mushroom Grifola frondosa (Oliveira et al., 2018), and fucomannogalactan
(Carbonero et al., 2008a) and heterogalactan (Shida, Haryu, & Matsuda, 1975) from Lentinus edodes, the
-L-Fucp residues appeared to form side chains of F. officinalis mannofucogalactan.
In addition, the following unusual substituents for mannofucogalactan were identified by GC–MS
analyses as minor components: 3,6-substituted residues of Manp and Galp, 1,2- and 1,6-linked residues
of Hexp (Glcp and/or Manp), 1,4-linked residues of Hexp (Manp and/or Galp), 1,2- and 1,3-linked residues
of Glcp and Galp, respectively. Accordingly to polydespersity of polysaccharide fraction FoHWE-1 (Mw/Mn
1.4), we can assume presence of minor amount of glucomannan and/or glucan and/or mannan in FoHWE-
1. The attachment of these substituents to the side chains to the mannofucogalactan appeared to be
unlikely, but can not be completely excluded.
Thus, the three types of side chains were identified in obtained mannofucogalactan: 3-O-D-
mannopyranosyl-L-fucopyranosyl, -D-Galp, -L-Fucp residues. The structural elements of two types of
side chains were uniquely identified by NMR spectroscopy: 3-O-D-mannopyranosyl-L-fucopyranosyl and
-D-Galp, therefore, the approximate ratio of side chains was calculated from data of methylation
analysis.
The NMR and methylation analysis data revealed that the major component in water soluble
polysaccharide of fruit bodies of Quinine conk F. officinalis was partially 3-O-methylated branched
mannofucogalactan, in which almost every second residue was substituted at O-2 by 3-O-D-
16

mannopyranosyl-L-fucopyranosyl or -D-Galp or -L-Fucp residues, the mentioned units presented in an
approximate ratio of 5 : 5 : 2. We propose the following repetitive unit of mannofucogalactan:
6)- -D-Galp-(1
6)- -D-Galp-(1
6)-3-OMe- -D-Galp-(1
...



...
2)
n
Side chain
Side chains - a : b : c ~ 5 : 5 : 2
a) -D-Manp-(1
3)- -L-Fucp-(1




-(1
-D-Galp
-L-Fucp-(1
b)
c)
This structure was similar to those previously proposed for the mannofucogalactans obtained
from other fungi of the Basidiomycetes group (Ruthes et al., 2016), and contained 1,6-O-galactan as
backbone, in which almost every second residue was substituted at O-2 by side chains. 3,6-substituted
residues of Galp detected by GC–MS analyses in mannofucogalactans of F. officinalis also might be
branching points of backbone mannofucogalactans of F. officinalis. 3-O-Me--D-Galp residues were
detected in mannofucogalactans as structural units of backbone. 3-O-Me--D-Galp residues have been
early determined in the backbone of fucogalactans (Ruthes et al., 2012, 2013b; Samanta et al., 2015) and
mannogalactans (Carbonero et al., 2008b; Maity et al., 2014; Rosado et al., 2003; Smiderle et al., 2008;
Silveira et al., 2015; Zhang, Xu, & Sun, 2013). Recently, 3-O-Me--D-Galp residues were also determined
in the structure of mannofucogalactan obtained from edible mushroom Grifola frondosa (Oliveira et al.,
2018).
The side chains of mannofucogalactan of F. officinalis included single L-fucopyranosyl and/or 3-
O--D-mannopyranosyl-L-fucopyranosyl residues as it was found for mannofucogalactans of other
Basidiomycetes (Alquini et al., 2004; Cho, Koshino, Yu, & Yoo, 1998; Cho et al., 2011; Mukumoto &
Yamaguchi, 1977; Ruthes et al., 2013; Shida et al., 1975; Smiderle et al., 2008b; Usui et al., 1981; Wang et
al., 2014; Zhang, Xiao, Deng, He, & Sun, 2012). However some peculiar features in the structure of the
side chains of mannofucogalactan of F. officinalis were found, specifically non-reducing terminal -D-Galp
17

residues were detected as side chains of this mannofucogalactan. They were found to substitute of Galp
residues of backbone at O-2 position. Earlier these units were detected only in fucogalactan isolated from
Agaricus bisporus, and authors suggested that -D-Galp residues substituted of 1,6-linked -D-Galp
residues of backbone at O-2 position (Komura et al., 2010; Ruthes et al., 2012, 2013b).
4. Conclusions
This study presents structure of branched partially 3-O-methylated mannofucogalactan as a major
component of water extract. The extract was obtained by boiling water from fruit bodies of medicine
fungi Quinine conk or Agarikon Fomitopsis officinalis (Vill.) Bondartsev & Singer, which belong to a group
of xylotroph basidiomycetes, growing on stems of larch trees. Generally the structure of backbone of
mannofucogalactan from F. officinalis was composed of 1,6-O-linked D-galactopyranosyl residues, in
which almost every second residue was substituted at O-2 by 3-O-D-mannopyranosyl-L-fucopyranosyl,
non-reducing terminal -D-galactopyranosyl and fucopyranosyl units (approximate ratio of 5 : 5 : 2.).
Acknowledgements
This work was supported by the Russian Foundation for Basic Research (Grant No. 18-54-91005).
References
Alquini, G., Carbonero, E. R., Claudino, R. F., Tischer, C. A., Kemmelmeier, C., & Iacomini, M. (2004).
Polysaccharides from the fruit bodies of the basidiomycete Laetiporus sulphureus (Bull.: Fr.) Murr.
FEMS Microbiology Letters, 230, 47–52.
Bjorndal, H., Hellerqvist, C. G., Lindberg, В., & Svensson, S. (1970). Gas-liquid chromatography and mass
spectrometry in methylation analysis of polysaccharides. Angewandte Chemie International
Edition (English), 9, 610-619.
18

Bushmarinov I. S., Ovchinnikova O. G., Kocharova N. A., Blaszczyk A., Toukach F. V., Torzewska A.,
Shashkov A. S., Knirel Yu. A., & Rozalski A. (2004). Structure of the O-polysaccharide of Providencia
stuartii O49. Carbohydrate Research, 339, 1557–1560.
Carbonero, E. R., Gracher, A. H. P., Komura, D. L., Marcon, R., Freitas, C. S., Baggio, C. H., Torri, G., &
Iacomini, M. (2008a). Lentinus edodes heterogalactan: Antinociceptive and anti-inflammatory
effects. Food Chemistry, 111, 531–537.
Carbonero, E. R., Gracher, A. H. P., Rosa, M. C.C., Torri, G., Sassaki, G.L., Gorin, P.A.J., & Iacomini, M.
(2008b). Unusual partially 3-O-methylated -galactan from mushrooms of the genus Pleurotus.
Phytochemistry, 69, 252–257.
Cerqueira, M. A., Souza, B. W. S., Simões, J., Teixeira, J. A., Domingues, M. R. M., Coimbra, M. A., & Vicente,
A. A. (2011). Structural and thermal characterization of galactomannans from non-conventional
sources. Carbohydrate Polymers, 83, 179–185.
Chang, S.-T. (1999). Global impact of edible and medicinal mushrooms on human welfare in the 21st
century: nongreen revolution. International Journal Medicinal Mushrooms, 1, 1–8.
Chang S.T. & Wasser S.P. (2012). The role of culinary-medicinal mushrooms on human welfare with a
pyramid model for human health. International Journal Medicinal Mushrooms, 1, 195-134.
Chen, Y., Xie, M., Li, W., Zhang, H., Nie Sh., Wang, Y., & Li C. (2012). An effective method for
deproteinization of bioactive polysaccharides extracted from Lingzhi (Ganoderma atrum), Food
Science and Biotechnology, 21, 191-198.
Chlebicki, A., Mukhin, V., & Ushakova, N. (2003). Fomitopsis officinalis on Siberian Larch in the Urals.
Mycologist, 17, 116-120.
Cho, S.-M., Yun, B.-S., Yoo, I.-D., & Koshino, H. (2011). Structure of fomitellan A, a mannofucogalactan
from the fruiting bodies of Fomitella fraxinea. Bioorganics and Medicinal Chemistry Letters, 21,
204–206.
19

Cho, S.-M., Koshino, H., Yu, S.-H., & Yoo, I.-D. (1998). A mannofucogalactan, fomitellan A, with mitogenic
effect from fruit bodies of Fomitella fraxinea (Imaz.). Carbohydrate Polymers, 37, 13–18.
Ciucanu, I. & Kerek, F. (1984). A simple and rapid method for the permethylation of carbohydrates.
Carbohydrate Research, 131, 209-217.
Corrêa, R. C. G., Brugnari, T., Bracht, A., Peralta, R. M., & Ferreira, I. C. F. R. (2016). Biotechnological,
nutritional and therapeutic uses of Pleurotus spp. (Oyster mushroom) related with its chemical
composition: A review on the past decade findings. Trends in Food Science & Technology, 50, 103-
117.
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P., & Smith, F. (1956). Colorimetric method for
determination of sugars and related substances. Analytical chemistry, 28, 350-356.
Gerwig, G. J., Kamerling, J. P., & Vliegenthart, J. F. G. (1978). Determination of the D and L configuration
of neutral monosaccharides by high-resolution capillary g.l.c. Carbohydrate Research, 62, 349-
357.
Golovchenko, V.V., Khramova, D. S., Ovodova, R. G., Shashkov, A. S., & Ovodov, Y. S. (2012). Structure of
pectic polysaccharides isolated from onion Allium cepa L. using a simulated gastric medium and
their effect on intestinal absorption. Food Chemistry, 134, 1813–1822.
Grienke, U., Zöll, M., Peintner, U., & Rollinger, J. M. (2014). European medicinal polypores – A modern
view on traditional uses. Journal of Ethnopharmacology, 154, 564–583.
Hobbs, C. (2000). Medicinal value of Lentinus edodes (Berk.) Sing. (Agaricomycetideae). A literature
review. International Journal Medicinal Mushrooms, 2, 287–302.
Komura, D. L, Carbonero, E. R., Gracher, A. H., Baggio, C. H., Freitas, C. S., Marcon, R., Santos A. R.S., Gorin
Ph. A. J., & Iacomini M. (2010). Structure of Agaricus spp. fucogalactans and their anti-
inflammatory and antinociceptive properties. Bioresource Technology, 101, 6192–6199.
20

Jansson, P.E., Kenne, L., & Widmalm, G. (1989). Computer-assisted structural analysis of polysaccharides
with an extended version of CASPER using 1H- and 13C-NMR data. Carbohydrate research, 188,
169-191.
Li, Y., Zhang, J., & Ma, H. (2014). Chronic inflammation and gallbladder cancer. Cancer Letters, 345, 242–
248.
Lipkind, G. M., Shashkov, A. S., Knirel, Y. A., Vinogradov, E. V., & Kochenkov, N. K. (1988). A computer-
assisted structural analysis of regular polysaccharides on the basis of 13C N.M.R. data.
Carbohydrate research, 175, 59-75.
Lowry, B. O., Roserbourgh, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin
phenol reagent. Journal of Biological Chemistry, 193, 265–275.
Maity, P., Pattanayak, M., Maity, S., Nandi, A. K., Sen, I. K., Behera, B., Maiti, T. K., Mallick, P., Sikdar, S. R.,
& Islam, S. S. (2014). A partially methylated mannogalactan from hybrid mushroom pfle 1p:
purification, structural characterization, and study of immunoactivation. Carbohydrate Research,
395, 1–8.
Meng, X., Liang, H., & Luo, L. (2016). Antitumor polysaccharides from mushrooms: a review on the
structural characteristics, antitumor mechanisms and immunomodulating activities.
Carbohydrate Research, 424, 30-41.
Moradali, M.-F., Mostafavi, H., Ghods, S., & Hedjaroude, G.-A. (2007). Immunomodulating and anticancer
agents in the realm of macromycetes fungi (macrofungi). International Immunopharmacology, 7,
701–724.
Mukumoto, T. & Yamaguchi, H. (1977). The chemicall structure of a mannofucogalactan from the fruit
bodies of Flammulina velutipes (Fr.) Sing. Carbohydrate Research, 59, 614–621.
Muszyńska, B., Grzywacz-Kisielewska, A., Kała, K., & Gdula-Argasińska, J. (2018). Anti-inflammatory
properties of edible mushrooms: A review. Food Chemistry, 243, 373–381.
21

Oliveira, G. K. F, Viano da Silva, E., Ruthes, A. C., Lião, L. M., Iacomini M., Carbonero E. R. (2018). Chemical
structure of a partially 3-O-methylated mannofucogalactan from edible mushroom Grifola
frondosa. Carbohydrate Polymers, 187, 110–117.
Popov, S., Ovodova, R., Golovchenko, V., Khramova, D., Markov, P., Smirnov, V., Shashkov, A., & Ovodov,
Yu. (2014). Pectic polysaccharides of the fresh plum Prunus domestica L. isolated with a simulated
gastric fluid and their anti-inflammatory and antioxidant activities. Food Chemistry, 143, 106-113.
Ren, L., Perera, C., & Hemar, Y. (2012). Antitumor activity of mushroom polysaccharides: A review. Food
& Function, 3, 1118-1130.
Rosado, F. R., Carbonero, E. R., Claudino, R. F., Tischer, C. A., Kemmelmeier, C., & Iacomini, M. (2003). The
presence of partially 3-O-methylated mannogalactan from the fruit bodies of edible
basidiomycetes Pleurotus ostreatus ‘forida’ Berk. and Pleurotus ostreatoroseus Sing. FEMS
Microbiology Letters, 221, 119-124.
Ruthes, A. C., Carbonero, E. R., Córdova, M. M., Baggio, C. H., Sassaki, G. L., Philip, A. J. G., Santos, A. R. S.,
& Iacomini, M. (2013a). Fucomannogalactan and glucan from mushroom Amanita muscaria:
Structure and inflammatory pain inhibition. Carbohydrate Polymers, 98, 761–769.
Ruthes, A. C., Rattmann, Y. D., Carbonero, E. R., Gorin, P. A. J., & Iacomini, M. (2012). Structural
characterization and protective effect against murine sepsis of fucogalactans from Agaricus
bisporus and Lactarius rufus. Carbohydrate Polymers, 87, 1620–1627.
Ruthes A. C., Rattmann Y. D., Malquevicz-Paiva S. M., Carbonero E. R., Córdova M. M., Baggio C. H., Santos
A. R. S., Gorin Ph. A. J., & Iacomini M. (2013b). Agaricus bisporus fucogalactan: Structural
characterization and pharmacological Approaches. Carbohydrate Polymers, 92, 184–191.
Ruthes, A.C., Smiderle, F.R., & Iacomini, M. (2016). Mushroom heteropolysaccharides: A review on their
sources, structure and biological effects. Carbohydrate Polymers, 136, 358-375.
Taofiq, O., Martins, A., Barreiro, M. F., & Ferreira, I. C. F. R. (2016). Anti-inflammatory potential of
mushroom extracts and isolated metabolites. Trends in Food Science & Technology, 50, 193-210.
22

Samanta, S., Nandi, A. K., Sen, I. K., Maity, P., Pattanayak, M., Devi, K. S. P., Khatua, S., Maiti, T. K., Acharya
K., & Islam S. S. (2015). Studies on antioxidative and immunostimulating fucogalactan of the edible
mushroom Macrolepiota dolichaula. Carbohydrate Research, 413, 22-29.
Shashkov, A. S., Lipkind, G. M., Knirel, Y. A., & Kochetkov, N. K. (1988). Stereochemical factors
13
determining the effects of glycosylation on the C chemical shifts in carbohydrates. Magnetic
resonance in chemistry, 26, 735-747.
Shida, M., Haryu, K., & Matsuda, K. (1975). On the water-soluble heterogalactan from the fruit bodies of
Lentinus edodes. Carbohydrate Research, 41, 211–218.
Shikov, A. N., Pozharitskaya, O. N., Makarov, V. G., Wagner, H, Verpoorte, R., & Heinrich, M. (2014).
Medicinal Plants of the Russian Pharmacopoeia; their history and applications. Journal of
Ethnopharmacology, 154, 481–536.
Silveira, M. L. L., Smiderle, F. R., Agostini, F., Pereira, E. M., Bonatti-Chaves, M., Wisbeck, E., Ruthes, A. C.,
Sassaki, G. L., Cipriani, T. R., Furlana, S. A., & Iacomini, M. (2015). Exopolysaccharide produced by
Pleurotus sajor-caju: Its chemicalstructure and anti-inflammatory activity. International Journal of
Biological Macromolecules, 75, 90–96.
Sims, I. M., Carnachan, S. M., Bell, T. J., & Hinkley, S.F.R. (2018). Methylation analysis of polysaccharides:
Technical advice. Carbohydrate Polymers, 188, 1–7.
Smiderle, F. R., Carbonero, E. R., Mellinger, C. G., Sassaki, G. L., Gorin, P. A. J., & Iacomini, M. (2008b).
Characterization of a heterogalactan: Some nutritional values of the edible mushroom
Flammulina velutipes. Food Chemistry, 108, 329–333.
Smiderle, F. R., Olsen, L. M., Carbonero, E. R., Baggio, C. H., Freitas, C. S., Marcon, R., Santos A.R.S., Gorin
P.A.J., & Iacomini M. (2008a). Anti-inflammatory and analgesic properties in a rodent model of a
(1→3),(1→6)-linked -glucan isolated from Pleurotus pulmonarius. European Journal of
Pharmacology, 597, 86–91.
23

Smiderle, F. R., Olsen, L. M., Carbonero, E. R., Marcon, R., Baggio, C. H., Freitas, C. S., Santos, A. R. S., Torri,
G., Gorin, P. A. J., & Iacomini, M. (2008c). A 3-O-methylated mannogalactan from Pleurotus
pulmonarius: Structure and antinociceptive effect. Phytochemistry, 69, 2731–2736.
Sweet, D. P., Shapiro, R. H., & Albersheim, P. (1975). Quantitative analisis by various G.L.C. response-factor
theories for partially methylated and partially ethylated alditol acetates. Carbohydrate Research,
40, 2217-2254.
Usui, T., Iwasaki, Y., & Mizuno, T. (1981). Isolation and characterization of two kinds of heterogalactan
from the fruit bodies of Ganoderma applanatum by employing a column of concanavalin A-
sepharose 4B. Carbohydrate Research, 92, 103–114.
Villares, A., Mateo-Vivaracho, L., & Guillamón, E. (2012). Structural Features and Healthy Properties of
Polysaccharides Occurring in Mushrooms. Agriculture, 2, 452-471.
Wang, Y., Shen, X., Liao, W., Fanga, J., Chen, X, Dong, Q., & Ding, K. (2014). A heteropolysaccharide, L-
fuco-D-manno-1,6--D-galactan extracted from Grifola frondosa and antiangiogenic activity of its
sulfated derivative. Carbohydrate Polymers, 101, 631–641.
Wasser S. P. (2002). Medicinal mushrooms as a source of antitumor and immunomodulating
polysaccharides. Applied Microbiology and Biotechnology, 60, 258–274.
Wasser S. P. (2014). Medicinal Mushroom Science: Current Perspectives, Advances, Evidences, and
Challenges. Biomedical Journal, 37, 345-356.
Zhang, M., Cui, S. W., Cheung, P. C. K., & Wang, Q. (2007). Antitumor polysaccharides from mushrooms:
A review on their isolation process, structural characteristics and antitumor activity. Trends in
Food Science & Technology, 18, 4–19.
Zhang, A., Xiao, N., Deng, Y., He, P., & Sun P. (2012). Purification and structural investigation of a water-
soluble polysaccharide from Flammulina velutipes. Carbohydrate Polymers, 87, 2279–2283.
24

Zhang, A., Xu, M., Fu, L., & Sun, P. (2013). Structural elucidation of a novel mannogalactan isolated from
the fruiting bodies of Pleurotus geesteranus. Carbohydrate Polymers, 92, 236–240.
Fig. 1. HPGPC profile of fractions FoHWE (a) and FoHWE-1 (b) with calibration curve. The pullulans with
Mw 800, 400, 200, 110, 50, 22, 12, 6 and 1.3 kDa were used as standards, which were designated I-IX,
respectively.
25

Fig. 2. The anomeric region of 1H NMR spectrum (a), anomeric region (b) and C-2 – C-6 region (c) of 1H/13C
HSQC spectrum of mannofucogalactan FoHWE-1. The cross-peaks were labeled by letters A–F as
explained in the text.
26

Fig. 3. The parts of anomeric region of1H/1H ROESY spectrum of mannofucogalactan FoHWE-1 differently
enlarged for better visibility of the cross-peaks showing relevant connections.
Fig. 4. 1H/1H TOCSY spectrum of cross-connections of H-5-s/H-6-s -Gal residues region in
mannofucogalactan FoHWE-1.
27

Fig. 5. 13C NMR spectrum of mannofucogalactan FoHWE-1.
Table 1
Chemical characterization of the polysaccharide fractions.
Content, % c
Fuc Xyl Man Glc Gal
Total
sugar
Mw,
kDa
Mn,
kDa
Mw/Mn
Yield,
%
PC
content,
Protein
d
%
FoHWE
3.68 a
4.2
0.0
13.7 2.0 20.2 26.3 33.5 85.3
13.1 1.4 18.6 4.1 62.8 89.7
151.7
17.4
11.2
12.1
13.6
1.4
FoHWE- 74.7 b
1
a – yield of the dried material.
b – yield calculated from the amount of FoHWE applied to the column.
c – calculated as a relative percentage of sum of all monosaccharides and protein, which were detected
in simples.
d - data were calculated as weight %.
28

Table 2
Chemical shifts of the signals in the 1H and 13С NMR spectra of the polysaccharide FoHWE-1.
13С NMR chemical shifts (δС acetone 31.45) and
Residue
1Н (italic, δН acetone 2.225 ppm)
C-1
C-2
C-3
C-4
C-5
C-6
O-CH3
H-1
H-2
H-3
H-4
H-5
H-6; H-6’
103.64; 103.55 71.38 71.72 68.17 74.67 62.42
-Man-(1
5.14
4.09 3.91 3.69 3.81 3.79;3.90
68.83 78.88 72.72 68.17 16.95 *
3.95 3.98 3.99 4.17 1.25
78.75 69.73 70.88 70.59 68.44
3.87 4.09 4.09 4.16 3.70; 3.98
69.60 70.88 70.88 70.42 68.20
3.87 3.89 4.04 4.20 3.72; 3.92
72.08 74.07 69.97 76.42 62.42
3.58 3.67 3.95 3.71 3.80;3.91
(A)
102.56
5.13
3)--Fucp-(1
(B)
99.23
5.05
2,6)--Galp-(1
(D)
99.23
5.00
6)--Galp-(1
(E)
104.63
4.47
-D-Gal-(1
(F)
→6)-α-D-Galp-3-OMe(1→ n.d.
(G)
n.d.
80.4 n.d.
3.58
70.15 67.86
57.6
4.24 3.72; 3.92 3.49
n.d. – not determined;
* – for methyl group.
Table 3
Retention times and Molar % of partially O-methylated sugars, in the form of their alditol acetates from
the hydrolyzate of permethylated mannofucogalactan FoHWE-1
29