Isolation and chemical characterization of a glucogalactomannan of the medicinal mushroom Cordyceps militaris

Article abstract of DOI:10.1016/j.carbpol.2013.04.049

Cordyceps militaris dried fruiting bodies were extracted with 5% KOH solution. The extract was purified by freeze-thawing treatment, and dialysis (100 kDa), giving rise to a homogeneous polysaccharide (M_w 23,000 Da). Its monosaccharide composition was mannose (56.7%), galactose (34.5%), and glucose (8.8%). The anomeric configurations were determined by their coupling constants. A complex polysaccharide was identified by NMR and methylation analysis. The HSQC spectrum showed signals at δ 107.7/5.06 and 106.1/5.14; 105.9/5.12 relative to β-D-Galf, and O-2-substituted β-D-Galf units, respectively. The sign at δ 104.4/5.21 corresponded to α-D-Galf. Other signals corresponded to α-D-Manp O-6- and O-2-substituted (δ 100.2/4.94; 100.5/5.27; 100.6/5.23; 100.7/5.16), and δ-D-Manp 2,6-di-O-substituted (from δ 99.3 to 99.9). The main linkages, confirmed by methylation analysis, showed the derivatives: 2,3,4-Me₃-Manp (11.9%) and 3,4,6-Me₃-Manp (28.6%). The branches were (1 → 6)-linked-α-D-Manp or (1 → 2)-linked-β-D-Galf, terminating with β-D-Galf, α-D-Galf, α-D-Galp, or α-D-Manp. 42.7% of the partially hydrolyzed product consisted of 3,4,6-Me₃-Manp, suggesting a (1 → 2)-linked backbone.

Full text of DOI:10.1016/j.carbpol.2013.04.049

Carbohydrate Polymers 97 (2013) 74–80
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Isolation and chemical characterization of a glucogalactomannan of
the medicinal mushroom Cordyceps militaris
Fhernanda R. Smiderlea, Guilherme L. Sassakia, Leo J.L.D. Van Griensvenb,
Marcello Iacominia,∗
a Department of Biochemistry and Molecular Biology, Federal University of Parana, CP 19046 Curitiba, PR, Brazil
b Plant Research International, Wageningen University and Research Centre, Bornsesteeg 1, 6708 PD Wageningen, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Cordyceps militaris dried fruiting bodies were extracted with 5% KOH solution. The extract was purified
by freeze-thawing treatment, and dialysis (100 kDa), giving rise to a homogeneous polysaccharide (Mw
23,000 Da). Its monosaccharide composition was mannose (56.7%), galactose (34.5%), and glucose (8.8%).
The anomeric configurations were determined by their coupling constants. A complex polysaccharide
was identified by NMR and methylation analysis. The HSQC spectrum showed signals at ı 107.7/5.06 and
106.1/5.14; 105.9/5.12 relative to ␤-d-Galf, and O-2-substituted ␤-d-Galf units, respectively. The sign at ı
104.4/5.21 corresponded to ␣-d-Galf. Other signals corresponded to ␣-d-Manp O-6- and O-2-substituted
(ı 100.2/4.94; 100.5/5.27; 100.6/5.23; 100.7/5.16), and ␣-d-Manp 2,6-di-O-substituted (from ı 99.3 to
99.9). The main linkages, confirmed by methylation analysis, showed the derivatives: 2,3,4-Me3-Manp
(11.9%) and 3,4,6-Me3-Manp (28.6%). The branches were (1 → 6)-linked-␣-d-Manp or (1 → 2)-linked-␤-
d-Galf, terminating with ␤-d-Galf, ␣-d-Galf, ␣-d-Galp, or ␣-d-Manp. 42.7% of the partially hydrolyzed
product consisted of 3,4,6-Me3-Manp, suggesting a (1 → 2)-linked backbone.
Received 14 March 2013
Received in revised form 11 April 2013
Accepted 12 April 2013
Available online 24 April 2013
Keywords:
Cordyceps militaris
Glucogalactomannan
Ascomycete
NMR spectrometry
© 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Fujita, 2007; Wang et al., 2012; Yu et al., 2009). It contains many
active components such as cordycepin, ergosterol, mannitol, and
The scientific community have provided plenty of data show-
ing that mushroom extracts demonstrate interesting biological
properties such as antitumor (Daba & Ezeronye, 2003), antiinflam-
matory (Komura, Carbonero, et al., 2010), antiviral (Lindequist,
Niedermeyer, & Jülich, 2005), and immunomodulatory effects
(Chanput et al., 2012; Chen, Zhang, Shen, & Wang, 2010; Lin et al.,
2012; Smiderle et al., 2011). These extracts may contain differ-
ent molecules as steroids, polyphenols, hydroquinones, triterpenes,
ubiquitin-like proteases, anti-protozoal compounds, proteins, gly-
coproteins, and polysaccharides that are involved in such biological
effects (Lindequist et al., 2005).
Several mushrooms have been studied for their pharmacological
potentials. Among them, Cordyceps militaris, an entomopathogenic
fungus belonging to the class Ascomycetes, is the one of the most
important traditional Chinese medicines, being the second most
commercialized species in China, Korea, and Japan (Das, Masuda,
Sakurai, & Sakakibara, 2010; Wang et al., 2012). C. militaris is used as
a folk tonic in East Asia and the studies related to its pharmacolog-
ical properties suggest that this mushroom can exert antioxidant,
antiviral, and immuno-protective activities (Ohta, Lee, Hayashi, &
polysaccharides, as ␤-glucans (Holliday & Cleaver, 2008).
Most of the benefits provided by mushroom extracts can be
attributed to their polysaccharides, which have attracted much
attention in the past 50 years (Ren, Perera, & Hemar, 2012). These
polymers, also known as “biological response modifiers (BRMs),”
are able to stimulate the innate immune system and to promote
the stimulation of the host’s defence mechanism, exerting antitu-
moral, antiviral, and antimicrobial activity (Lindequist et al., 2005;
Ramberg, Nelson, & Sinnott, 2010; Schepetkin & Quinn, 2006).
The most studied mushroom polysaccharides are ␤-glucans,
which are vastly isolated from Basidiomycetes (Ren et al., 2012;
Smiderle et al., 2006), and present plenty of biological activi-
ties (Baggio et al., 2012; Chen & Seviour, 2007; Smiderle et al.,
2013). However also heteropolysaccharides are encountered in
mushrooms, showing interesting therapeutic importance (Zhang,
Cui, Cheung, & Wang, 2007). The most common heteropolysac-
charides isolated from Basidiomycetes are heterogalactans, as
mannogalactans (Smiderle et al., 2008), fucogalactans (Komura,
Carbonero, et al., 2010), and mannofucogalactans (Zhang et al.,
2007). The Ascomycetes present mainly heteropolysaccharides
based on d-mannan main chains as glucomannans and galactoman-
nans (Barreto-Bergter & Gorin, 1983). The chemical structure of
these heteropolymers is more complex than the ␤-glucans. There
are considerable studies on these molecules because they can
∗
Corresponding author. Tel.: +55 41 3361 1655; fax: +55 41 3266 2042.
E-mail address: iacomini@ufpr.br (M. Iacomini).
0144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2013.04.049

F.R. Smiderle et al. / Carbohydrate Polymers 97 (2013) 74–80
75
be used for different purposes as taxonomic classification of the
species (Carbonero, Mellinger, Eliasaro, Gorin, & Iacomini, 2005)
and for treatment of various diseases (Ren et al., 2012).
Cordyceps militaris
Dried mushroom 28.7 g
Elucidation of the chemical structure of polysaccharides has
shown to be challenging, considering that these molecules can
present different monosaccharide composition, linkages, ␣/␤-
configuration, and molecular weight, and that this chemical
characteristic can influence their bioactivity (Lehtovaara & Gu,
2011). Generally, the greater the molecular weight and the higher
the water solubility of the polysaccharides, the higher the anti-
tumor activity (Daba & Ezeronye, 2003). A study based on seven
potent antitumor polysaccharide–protein complexes from Gan-
oderma tsugae, has found that heteropolysaccharides, with Mw
of about 10,000 Da, containing galactose, glucose, mannose, and
fucose, showed the highest antitumoral activity (Ren et al., 2012).
Besides, the polysaccharides can assume distinct quaternary
structures, such as single or multiple-helices. For instance, lenti-
nan, the ␤-d-glucan isolated from Shiitake (Lentinus edodes), in
its triple-helical conformation, was found to inhibit the growth of
solid tumours (sarcoma-180) in mouse. This inhibitory effect was
not observed when the mice were treated with the single-helical
polysaccharide (Zhang, Li, Xu, & Zeng, 2005).
Polysaccharides belong to a structurally diverse class of macro-
molecules. The monosaccharide units of these polymers can
interconnect at several points to produce various branched or linear
structures. This vast potential variability in carbohydrate structures
could offer possibilities to the precise regulatory mechanisms of
various cell–cell interactions in higher organisms. All this informa-
tion shows how important it is to define the chemical structure of
the biologically active polysaccharides. Chemical characterization
of these molecules is required to provide enough data for the elu-
cidation of their biological effects. Considering this, the aim of the
present study was to isolate, purify and chemically characterize a
complex heteropolysaccharide from C. militaris.
CHCl3-MeOH 1:1
Apolar extract
Residue I
H2O at 25 ºC, for 6 h, 3x
Aq. extract I
Residue II
H2O at 100 ºC, for 6 h, 3x
Aq. extract II
Residue III
5% KOH at 100 ºC, for 6 h
Alkaline extract
(K5)
Residue IV
Freeze-thawing process
Precipitate
(PK5)
Supernatant
(SK5)
Me2SO treatment, at 50 ºC, 30 min
Soluble Fraction
(SD-SK5)
Insoluble Fraction
(PD-SK5)
Dialysis 100 kDa Mr cut-off membranes
Eluted Fraction
Retained Fraction
(R100-PD) 0.9%
Glucogalactomannan
Fig. 1. Extraction and purification steps of the glucogalactomannan (R100-PD) of C.
militaris.
2. Experimental
2.1. Fungal material
for 24 h, to remove the solvent. This fraction was dialysed (100 kDa,
cut-off) against distilled water, giving rise to a purified polysaccha-
ride, which was retained by the dialysis membrane (R100-PD).
Fruiting bodies of C. militaris (L.) Link (strain: MCI 10304,
Mushtech Cordyceps Institute) were a kind gift from Dr. J. M. Sung
of Kangwon National University (Chuncheon, Korea).
2.3. Alditol acetates preparation for monosaccharide composition
analysis
2.2. Extraction and purification of the heteropolysaccharide
Each polysaccharide fraction (1 mg) was hydrolyzed with 2 M
TFA at 100 ◦C for 8 h, followed by evaporation to dryness. The dried
carbohydrate samples were dissolved in 0.5 N NH4OH (100 ␮L),
held at room temperature for 10–15 min in reinforced 4 ml Pyrex
tubes with Teflon lined screw caps. NaBH4 (1 mg) was added, and
the solution was kept at 100 ◦C for 10 min, in order to reduce aldoses
to alditols (Sassaki et al., 2008). The excess of NaBH4 was neutral-
ized by the addition of acetic acid (30 ␮L), and removed by the
addition of methanol (×2) under a N2 stream in a fume hood. The
reduced product was dried and acetylation of the Me-alditols was
performed in pyridine–Ac2O (200 ␮L; 1:1, v/v), for 30 min at 100 ◦C.
The resulting alditol acetates were analyzed by GC–MS, and the
sugars were identified by their typical retention times and elec-
tron impact profiles (Sassaki, Gorin, Souza, Czelusniak, & Iacomini,
2005a).
The dried mushroom (28.7 g) was submitted to several extrac-
tion steps as shown in Fig. 1. Briefly, the material was firstly
treated with CHCl3:MeOH (1:1, v/v), using a Soxhlet, under heat-
ing (50 ◦C), for 3 days. After removing the apolar compounds and
the excess of solvents, the residue I was successively submitted
to cold (25 ◦C) and hot (100 ◦C) aqueous extractions (for 6 h, 3×
for each extraction). The aqueous extracts were used for other
studies. The remaining residue III was extracted twice with 5%
KOH solution at 100 ◦C, for 6 h, giving rise to an alkaline extract
(K5), which was neutralized with glacial acetic acid and dialysed
(12–14 kDa), for 24 h. The extract (K5) was solubilized in water
and submitted to freezing followed by mild thawing at 4 ◦C (Gorin
& Iacomini, 1984). This process was repeated 5× to guarantee a
complete separation of the water-soluble (SK5) of the non-soluble
(PK5)polysaccharides. Bothfractionswereseparatedby centrifuga-
tion (12,000 rpm, at 4 ◦C, for 20 min), and freeze-dried. The soluble
fraction (SK5) was the focus of this study, after a treatment
with dimethylsulfoxide (50 mL), for 30 min, at 50 ◦C. The Me2SO-
insoluble material (PD-SK5) was recovered by centrifugation
(10,000 rpm, at 20 ◦C, for 15 min) and dialysed against tap water
2.4. Methylation analysis
Per-O-methylation of the polysaccharides was carried out using
the modified method of Ciucanu and Kerek (1984). An aliquot
of each dried polysaccharide (10 mg) was completely solubilized

76
F.R. Smiderle et al. / Carbohydrate Polymers 97 (2013) 74–80
in dimethylsulfoxide (Me2SO, 1 mL), followed by the addition of
iodomethane (MeI, 1 mL) and powdered NaOH (20 mg) (Ciucanu
& Kerek, 1984). The mixture was stirred for 30 min or until it
turned to solid phase, which was left to react for 14–18 h. The
material was re-solubilized in water, on ice, and neutralized with
glacial acetic acid. After dialysis (8 kDa) against tap water to remove
the excess of salts, the partially O-methylated polysaccharides
were freeze-dried and the methylation procedure was repeated
to guarantee a complete methylation of the free hydroxyls. After
this step, the sample was partitioned between chloroform (3 mL)
and distilled water (3 mL). The chloroform phase, containing the
per-O-methylated derivatives, was evaporated and submitted to
hydrolysis. The aqueous phase, containing salts and residues of the
reaction was discarded. The hydrolysis was proceeded as follows:
an aliquot of the dried per-O-methylated derivatives was treated
with 3% MeOH-HCl (1 mL), for 2 h, at 80 ◦C, followed by evapora-
tion under N2. The methanolized material was then submitted to
hydrolysis with 1 M H2SO4 (1 mL), for 9 h or 12 h, at 100 ◦C.
Fig. 2. Elution profiles of fractions SK5, PD-SK5, and R100-PD determined by HPSEC
using refractive index detectors, and 0.1 M NaNO3 as eluent.
3. Results and discussion
The steps of the polysaccharide extraction are shown in Fig. 1.
The yield of each fraction was calculated on the basis of initial
weight of dried mushroom (28.7 g). The material was firstly treated
with CHCl3:MeOH (1:1, v/v), as described in Section 2.2, and con-
tained 3% apolar compounds and 97% residue I. The latter was used
for the following extractions. The cold (3.5%) and hot (1.1%) water
extracts were not used in the present study. The remaining residue
III, after being extracted with 5% KOH solution, presented an extract
of 9.8% yield (K5), which was submitted to freeze-thawing for
several times, and centrifugation to separate the soluble polysac-
charides from the non-soluble ones. This procedure gave rise to a
fraction (SK5, 4.9%), that consisted of mannose (37.0%), galactose
(14.8%), and glucose (48.2%); its HPSEC analysis showed an het-
erogeneous profile (Fig. 2). Considering that ␣- and/or ␤-glucans
are present in mushrooms in great amounts (Schepetkin & Quinn,
2006), this fraction was treated with Me2SO, which is able to
solubilize mainly ␤-glucans, and after centrifugation, the Me2SO-
insoluble fraction (PD-SK5) was recovered showing a considerable
reduction in the amount of glucose (9.5%), compared to mannose
and galactose (Table 1). Its HPSEC profile confirmed the reduction
of glucose by the absence of peaks at ∼45 min and after 60 min
(Fig. 2). This fraction still presented a small peak at ∼58 min, which
was removed after a dialysis on membrane of 100 kDa cut-off. The
retained polysaccharide on this membrane (R100-PD) showed 0.9%
yield and had a homogeneous profile (Fig. 2). The monosaccha-
ride composition of the purified R100-PD showed mannose (56.7%),
galactose (34.5%), and glucose (8.8%), suggesting the presence of
a heteropolysaccharide (Table 1). The molecular weight of this
molecule was estimated at 23,000 Da.
After this step the samples were reduced by NaB2H4 and acety-
lated as above (alditol acetates preparation), to give a mixture of
partially O-methylated alditol acetates, which was analyzed by
GC–MS using a DB-225 capillary column as described above. The
derivatives were identified from m/z of their positive ions, by com-
parison with standards, and the results were expressed as relative
percentage of each component (Sassaki, Gorin, et al., 2005).
2.5. Gas chromatography–mass spectrometry (GC–MS) analysis
The analyses were performed using a Varian (model 3300)
gas chromatograph linked to a Finnigan Ion-Trap model 810 R-
12 mass spectrometer, with He as carrier gas. A capillary column
(30 m × 0.25 mm i.d.) of DB-225, held at 50 ◦C during injection and
then programmed at 40 ◦C/min to 220 ◦C or 210 ◦C (constant tem-
perature) was used for qualitative and quantitative analysis of
alditol acetates and partially O-methylated alditol acetates, respec-
tively (Sassaki, Gorin, et al., 2005).
2.6. Nuclear magnetic resonance spectroscopy
NMR spectra (1H-, 13C-, and DEPT-13C-NMR, COSY, TOCSY,
NOESY, HSQC, coupled HSQC) were obtained using a 400 MHz
Bruker Avance III spectrometer with a 5 mm inverse probe. The
analyses were performed at 50 or 70 ◦C in D2O, and the chemi-
cal shifts are expressed in ı ppm relative to external standard of
acetone at ı 30.2 (13C) and 2.22 (1H).
In order to characterize the glycosidic linkages of the isolated
heteropolysaccharide, methylation analysis was performed and
the results are shown in Table 2. The analysis showed a complex
and branched polysaccharide by the presence of non-reducing end
units, as 2,3,4,6-Me4-Manp (1.3%), 2,3,5,6-Me4-Galf (11.5%), and
2,3,4,6-Me4-Galp (10.9%), and the derivatives 2,3-Me2-Glcp (9.1%),
and 3,4-Me2-Manp (14.9%). The main chain is probably composed
of (1 → 2)-linked-mannopyranose, which can be concluded from
2.7. Determination of homogeneity and molar mass
The homogeneity and molar mass of the purified glucogalac-
tomannan was determined by high performance steric exclusion
chromatography (HPSEC), using a refractive index (RI) detector.
The eluent was 0.1 M NaNO3, containing 0.5 g/L NaN3. The solutions
were filtered through a membrane of 0.22 ␮m pore size (Millipore).
The molar mass of the polymer was estimated using Astra software
4.70.
Table 1
Monosaccharide composition of the fractions obtained from C. militaris.
2.8. Partial hydrolysis
Fractions
Yield%
Monosaccharides (mol%)a
An aliquot of the fraction R100-PD (100 mg) was partially
hydrolyzed with aqueous trifluoroacetic acid (TFA), adjusted to pH
2.0 (1 ml), at 100 ◦C, for 12 h. A polymeric product (PH) was obtained
by precipitation with EtOH (3:1; v/v) from a small volume of water,
and then retained on a dialysis membrane (2 kDa cut-off). The PH
fraction was lyophilized and analyzed by NMR spectrometry and
GC–MS.
Man
Gal
Glc
SK5
4.9
1.2
0.9
–
37.0
58.4
56.7
84.7
14.8
32.1
34.5
10.4
48.2
9.5
8.8
PD-SK5
R100-PD
PH
4.9
a
Alditol acetates obtained on successive hydrolysis, NaBH4 reduction, and acety-
lation, followed by GC–MS analysis.

F.R. Smiderle et al. / Carbohydrate Polymers 97 (2013) 74–80
77
Fig. 3. 13C-NMR spectra of native (R100-PD) (A) and partially degraded (PH) (B) glucogalactomannan, in D2O at 50 ◦C (chemical shifts are expressed in ppm).
the presence of high amounts of 3,4,6-Me3-Manp (28.6%). Accord-
ing to the methylation data, the branching points may be situated
mainly at O-6 position of mannose residues and there is also the
presence of (1 → 6)-linked-Manp fragments, which was shown by
the presence of the derivative 2,3,4-Me3-Manp (11.9%).
(JC1/H1) of each signal, being ␣ for ∼170–175 Hz and ␤ for values
∼160 Hz (Perlin & Casu, 1969), except for the furanoside units that
do not follow this pattern (Chambat, Joseleau, Lapeyre, & Lefebvre,
1978; Sassaki, Iacomini, & Gorin, 2005b).
The signs of C1/H1 at low field at ı 105.9/5.12; 106.1/5.14;
and 107.7/5.06 confirmed the presence of ␤-galactofunarose
2-O-substituted and as non-reducing end units, respectively
(Barreto-Bergter & Gorin, 1983; Cordeiro et al., 2005; Giménez-
Abián et al., 2007). The sign (C1/H1) at ı 104.4/5.21, showed the
presence of terminal ␣-galactofunarose, and its ␣-configuration
was confirmed by the JC1/H1 at 174 Hz (Chambat et al., 1978; Prieto,
Leal, Bernabé, & Hawksworth, 2008; Sassaki, Iacomini, et al., 2005).
The high field signals of C1/H1 at ı 98.3/5.05 could be attributed
to the 2-O-substituted and non-reducing end ␣-galactopyranosyl
units (Gorin & Iacomini, 1985; Perry & MacLean, 2004; Smiderle
et al., 2008).
Galactomannans have been isolated from fungus (Barreto-
Bergter & Gorin, 1983; Giménez-Abián, Bernabé, Leal, Jiménez-
Barbero, & Prieto, 2007) and lichens (Carbonero, Tischer, Cosentino,
Gorin, & Iacomini, 2003; Gorin & Iacomini, 1985) by alkaline
extractions and such polymers have shown a great variety of side-
chain structures. These heteropolysaccharides presented mainly
(1 → 6)-linked-␣-d-Manp as main chain O-2-substituted by ␣-d-
Galp-(1 → 2)-␣-d-Manp fragments (Barreto-Bergter & Gorin, 1983;
Carbonero et al., 2003). The side-chains may also be substituted
at O-4 by ␣-d-Galp, ␣-d-Manp, and ␤-d-Galf (Gorin & Iacomini,
1985). A different structure was proposed for the galactomannan
of the fungus Lineolata rhizophorae, that presented a main chain
of (1 → 6)-linked-␣-d-Manp, substituted at O-3 by non-reducing
ends of ␤-d-Galf or by ␤-d-Galf-(1 → 5)-␤-d-Galf disaccharides
(Giménez-Abián et al., 2007).
The C2/H2 of the 2-O-substituted ␤-Galf units was shown by a
shift to 87.1/4.21 ppm, while the C2/H2 of the non-reducing end
␤-Galf was at ı 81.4/3.83 (Ahrazem et al., 2006; Cordeiro et al.,
2005).
NMR analysis [1H, 13C (Fig. 3), HSQC (Fig. 4), coupled HSQC,
COSY, TOCSY, and NOESY] contributed to elucidate the structure of
the heteropolysaccharide. The main signals, which are summarized
in Table 3, were assigned according to the correlation among the
NMR spectra and to literature values of similar polysaccharides. The
␣ or ␤-configurations were determined by the coupling constants
Signals (C1/H1) relative to the main chain of (1 → 2)-linked-␣-
d-Manp were observed at ı 100.5/5.27, 100.6/5.23, and 100.7/5.06,
while the sign of (1 → 6)-linked-␣-d-Manp fragments was at ı
Table 2
Partially O-methylalditol acetates formed on methylation analysis of native (R100-
PD) and partially degraded (PH) glucogalactomannan isolated from C. militaris.
Partially O-methylated Mol%
Mol%
PHb
Linkage typec
alditol acetatesa
R100-PD
2,3,4,6-Me4-Manp
2,3,4,6-Me4-Glcp
2,3,5,6-Me4-Galf
2,3,4,6-Me4-Galp
3,4,6-Me3-Manp
3,5,6-Me3-Galf
2,3,4-Me3-Manp
3,4,6-Me3-Galp
2,3-Me2-Glcp
1.3
–
11.5
10.9
28.6
3.8
11.9
8.0
9.1
3.5
2.4
–
7.9
42.7
–
30.7
–
5.9
6.1
Manp-(1→
Glcp-(1→
Galf-(1→
Galp-(1→
2→)-Manp-(1→
2→)-Galf-(1→
6→)-Manp-(1→
2→)-Galp-(1→
4,6→)-Glcp-(1→
2,6→)-Manp-(1→
3,4-Me2-Manp
14.9
a
Analyzed by GC–MS, after methylation, total acid hydrolysis, reduction with
NaBD4 and acetylation.
b
R100-PD after the partial hydrolysis.
Based on derived O-methylalditol acetates.
Fig. 4. Anomeric region of HSQC spectrum of glucogalactomannan (R100-PD), in
c
D2O at 50 ◦C (chemical shifts are expressed in ppm).

78
F.R. Smiderle et al. / Carbohydrate Polymers 97 (2013) 74–80
Table 3
1H and 13C NMR chemical shifts of the C. militaris glucogalactomannan, and coupling constants (JC1/H1) of C1/H1. Assignments are based on 13C, 1H, DEPT, COSY, HSQC, and
coupled HSQC analysis.
Units
13C signa
1H signa
JC1/H1 (Hz)
Referencesb
C1/H1 of ␤-d-Galf-(1→
107.7
106.1
105.9
104.4
103.1
102.5
102.3
102.1
100.7
100.6
100.5
100.2
99.9
99.7
99.5
99.3
98.3
87.1
81.9
81.4
78.4
5.06
5.14
5.12
5.21
4.43
4.75
4.54
5.06
5.16
5.23
5.27
4.94
5.11
5.37
5.20
4.92
5.05
4.21
4.03
3.83
4.03
4.12
3.89/3.99
3.78/4.00
3.71/3.85
3.64/3.79
3.79/3.88
174
173
173
174
161
165
159
173
176
171
172
170
175
173
172
171
173
–
–
–
–
–
–
–
–
–
a, b, c
b
b
d, e, f
g, h
g, h
g, h
C-1/H-1 of →2)-␤-d-Galf-(1→
C-1/H-1 of →2)-␤-d-Galf-(1→
C-1/H-1 of ␣-d-Galf-(1→
C-1/H-1 of →4,6)-␤-d-Glcp-(1→
C-1/H-1 of →4,6)-␤-d-Glcp-(1→
C-1/H-1 of →4,6)-␤-d-Glcp-(1→
C1/H1 of ␣-d-Manp-(1→
i
C1/H1 of →2)-␣-d-Manp-(1→
C1/H1 of →2)-␣-d-Manp-(1→
C1/H1 of →2)-␣-d-Manp-(1→
C1/H1 of →6)-␣-d-Manp-(1→
C1/H1 of →2,6)-␣-d-Manp-(1→
C1/H1 of →2,6)-␣-d-Manp-(1→
C1/H1 of →2,6)-␣-d-Manp-(1→
C1/H1 of →2,6)-␣-d-Manp-(1→
C1/H1 of ␣-d-Galp-(1→
j, k
j, k
j, k
j, k, l
m
j, k, n
j, k, n
j, k, n
n, o, p
C-2/H-2 of →2)-␤-d-Galf-(1→
C-4/H-4 of ␤-d-Galf-(1→
q
b
b
c
c
g
i
r
r
i
C-2/H-2 of ␤-d-Galf-(1→
C-2 of →2)-␣-d-Manp-(1→ and →2,6)-␣-d-Manp-(1→
C-2 of →2)-␣-d-Manp-(1→ and →2,6)-␣-d-Manp-(1→
C-6/H-6 of →4,6)-␤-d-Glcp-(1→
C-6/H-6 of →6)-␣-d-Manp-(1→ and →2,6)-␣-d-Manp-(1→
C-6/H-6 of ␤-d-Galf-(1→
77.9
66.1
65.7
62.8
62.5
61.2
C-6/H-6 of ␤-d-Galf-(1→
C-6/H-6 of ␣-d-Manp-(1→ and →2)-␣-d-Manp-(1→
–
a
The chemical shifts are expressed as ppm (ı).
b
In accordance with references: a, Giménez-Abián et al., 2007; b, Cordeiro et al., 2005; c, Barreto-Bergter & Gorin, 1983; d, Chambat et al., 1978; e, Sassaki, Iacomini, et al.,
2005; f, Prieto et al., 2008; g, Mandal et al., 2012; h, Cui et al., 2000; i, Komura, Ruthes, et al., 2010; j, Viccini et al., 2009; k, Carbonero et al., 2003; l, Rosado et al., 2002; m,
Omarsdottir et al., 2006; n, Gorin & Iacomini, 1985; o, Smiderle et al., 2008; p, Perry & MacLean, 2004; q, Ahrazem et al., 2006; r, Gorin & Mazurek, 1975.
100.2/4.94. The 2,6-di-O-substitution of the ␣-d-Manp residues
was confirmed by the presence of signals from ı 99.3 to 99.9 ppm.
The O-2 and O-6 substitutions of ␣-d-Manp units were con-
firmed by the presence of signals from ı 77.8 to 78.4 (C2)
(Barreto-Bergter & Gorin, 1983), and 65.7 (C6) (Komura, Ruthes,
et al., 2010).
Different from lichen and yeast galactomannans, the isolated
heteropolysaccharide of C. militaris showed a low amount of
glucose (8.8%) in its structure. This was observed from the meth-
ylation analysis, by the presence of the derivative 2,3-Me2-Glcp
(9.1%), that was confirmed by the HSQC signals (C1/H1) at ı
102.3/4.54; 102.5/4.75; and 103.1/4.43 ppm (Cui, Wood, Blackwell,
& Nikiforuk, 2000; Mandal et al., 2012). The JC1/H1 of these units
was ∼160–165 Hz, which confirmed the ␤-configuration. The sub-
stituted C6 of these units was observed at ı 66.1 ppm.
These data suggested that C. militaris alkaline extract contains
a glucogalactomannan, with a main chain of (1 → 2)-linked-␣-d-
Manp, that can be substituted at O-6 by (1 → 6)-linked-␣-d-Manp
or (1 → 2)-linked-␤-d-Galf fragments, terminating with ␣-d-Manp,
␣-d-Galp, ␣-d-Galf, or ␤-d-Galf units. COSY and NOESY NMR cor-
relations have also indicated that 4,6-di-O-substituted-␤-d-Glcp
is present in the structure, probably linked to the main chain
as a fragment similar to ␣-d-Manp-(1 → 2)-␣-d-Manp-(1 → 6)-
␤-d-Glcp-(1 → . The ␤-d-Glcp of this trisaccharide may be also
substituted at O-4 by non-reducing end units as ␣-d-Galp, ␣-d-
Galf or ␤-d-Galf. The other correlations were not possible because
of the large amount of signals at the region from 70 to 80 ppm.
Similar polysaccharides were isolated from C. militaris mycelia
(Lee, Kwon, Yun, et al., 2010) and from the liquid culture broth
(Lee, Kwon, Won, et al., 2010). The first authors have shown a
molecule with a backbone of (1 → 6)-linked d-mannopyranosyl and
(1 → 6)-linked d-glucopyranosyl residues, with d-Manp branches
at O-4. The latter authors isolated a glucogalactomannan with a
backbone of (1 → 2)-linked d-mannopyranosyl and (1 → 6)-linked
d-mannopyranosyl residues, which occasionally branches at O-6.
Yu et al. (2007) have also isolated a glucogalactomannan
from the fruit bodies of C. militaris, although its structure was
quite different. The backbone of this heteropolysaccharide was
composed of (1 → 6)-linked ␣-d-mannopyranosyl residues, being
occasionally substituted at O-3 by ␣-d-glucopyranosyl and ␤-d-
galactopyranosyl residues. An acid water-soluble polysaccharide,
isolated from C. sinensis mycelium, had also showed mannose, glu-
cose, and galactose at a ratio of 3.5:1:1.5 (Chen et al., 2010). This
polymer promoted phagocytosis of RAW264.7 cells and stimulated
NO production (Chen et al., 2010). The fungus of genus Cordy-
ceps have been appreciated because of its therapeutic benefits,
therefore, the study of their polysaccharide structures is neces-
sary for providing new insights about mechanism of action of such
molecules.
In contrast with the above mentioned authors, the backbone of
the glucogalactomannan isolated from C. militaris, in the present
study, was composed of (1 → 2)-linked ␣-d-mannopyranosyl
residues instead of (1 → 6)-linkages. The branches occurred at O-
6, mainly by (1 → 6)-linked-␣-d-Manp or (1 → 2)-linked-␤-d-Galf
fragments, that can be terminated with non-reducing end units of
␣-d-Manp, ␣-d-Galp, ␣-d-Galf or ␤-d-Galf.
The galactomannan of the Ascomycete fungus Sporonthrix
schenckii consists of a similar backbone of a core of 2-O- and 2,6-
di-O-substituted ␣-d-mannopyranosyl units, with three or four
consecutive (1 → 2) linkages occurring between them (Barreto-
Bergter & Gorin, 1983).
Considering the complexity of analyzing these heteropolysac-
charides, and that they may present a variety of main chains and
side chains, the fraction R100-PD was submitted to a partial hydrol-
ysis, with TFA, pH 2.0, at 100 ◦C, for 12 h. The resulting polymeric
product was recovered by EtOH precipitation, as described in item

F.R. Smiderle et al. / Carbohydrate Polymers 97 (2013) 74–80
79
heteropolysaccharides from the genera Parmotrema and Rimelia. FEMS Microbi-
ology Letters, 246(2), 273–278.
Carbonero, E. R., Tischer, C. A., Cosentino, C., Gorin, P. A. J., & Iacomini, M. (2003).
Structural characterization of a galactomannan from the cyanolichen Leptogium
azureum. Carbohydrate Polymers, 53, 469–473.
Chambat, G., Joseleau, J. P., Lapeyre, M., & Lefebvre, A. (1978). Presence of d-
galactofuranose in the capsular polysaccharide of Klebsiella serotype K-41:
Synthesis of 5,6-di-O-methyl-d-galactofuranose. Carbohydrate Research, 63,
323–326.
Chanput, W., Reitsma, M., Kleinjans, L., Mes, J. J., Savelkoul, H. F., & Wichers,
H. J. (2012). ␤-Glucans are involved in immune-modulation of THP-
2.8, giving rise to the PH fraction. Its monosaccharide composition
showed a reduction of the galactose and glucose amounts (Table 1),
which was expected considering that the galactofuranose residues
are more liable to mild hydrolysis than the pyranosyl monosaccha-
rides. This reduction was confirmed by the 13C-NMR experiment
(Fig. 3) that presented higher resolution signals in the anomeric
region as well as the signals relative to the (1 → 2) and (1 → 6)
linkages, at ı 78.1; 78.6; and 65.7 ppm, respectively. No signals
of ␣- or ␤-d-galactofuranose were observed in the PH spectrum,
confirming its complete removal from the structure, showing that
these residues were localized at the branching chains. The meth-
ylation analysis of PH confirmed these results, by the absence of
the derivatives 2,3,5,6-Me4-Galf and 3,5,6-Me3-Galf. The increase
observed in the amounts of 3,4,6-Me3-Manp (42.7%) and 2,3,4-Me3-
Manp (30.7%) strongly indicates that this heteropolymer consists of
a backbone of (1 → 2) linkages, and branches of (1 → 6) linkages.
Considering the diversity of structures presented in this article,
other possibilities of side-chains formations are not discarded.
The galactomannans are usually found in Lichens and
pathogenic Ascomycetes (Ahrazem et al., 2006; Barreto-Bergter &
Gorin, 1983; Cordeiro et al., 2005). These heteropolysaccharides,
which contain high amounts of mannose and galactofuranose,
were not observed for Basidiomycetes. The medicinal mushroom
C. militaris is an entomopathogenic fungus that also belongs to the
phylum Ascomycetes, and the present study showed that the chem-
ical structure of its glucogalactomannan is in accordance with the
other members of this taxonomic group.
1
macrophages. Molecular Nutrition and Food Research, 56(5), 822–833.
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Chen, W., Zhang, W., Shen, W., & Wang, K. (2010). Effects of the acid polysaccharide
fraction isolated from a cultivated Cordyceps sinensis on macrophages in vitro.
Cellular Immunology, 262, 69–74.
Ciucanu, I., & Kerek, F. (1984). A simple and rapid method for the permethylation
of carbohydrates. Carbohydrate Research, 131, 209–217.
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Cui, W., Wood, P. J., Blackwell, B., & Nikiforuk, J. (2000). Physicochemical properties
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from higher basidiomycetes mushrooms. African Journal of Biotechnology, 2(12),
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Das, S. K., Masuda, M., Sakurai, A., & Sakakibara, M. (2010). Medicinal uses of
the mushroom Cordyceps militaris: Current state and prospects. Fitoterapia, 81,
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Giménez-Abián, M. I., Bernabé, M., Leal, J. A., Jiménez-Barbero, J., & Prieto, A. (2007).
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4. Conclusions
The present study has shown the isolation and purification
of a heteropolysaccharide from the alkaline extract of the fun-
gus C. militaris. The spectroscopy and spectrometry analyses
suggested that this polymer is a glucogalactomannan, with a
backbone of (1 → 2)-linked-␣-d-Manp, that can be substituted
by (1 → 6)-linked-␣-d-Manp or (1 → 2)-linked-␤-d-Galf fragments,
terminating with ␣-d-Manp, ␣-d-Galp, ␣-d-Galf or ␤-d-Galf units.
The NMR data have also indicated side chains of ␣-d-Manp-
(1 → 2)-␣-d-Manp-(1 → 6)-␤-d-Glcp-(1 → . The ␤-d-Glcp of this
trisaccharide may be also substituted at O-4 by non-reducing end
units as ␣-d-Galp, ␣-d-Galf or ␤-d-Galf. The similarity of the gluco-
galactomannan structure with other heteropolysaccharides from
Ascomycetes fungi just shows their taxonomic proximity.
Gorin, P. A. J.,
&
Mazurek, M. (1975).
Further studies on the assign-
ments of signals in 13C magnetic resonance spectra of aldoses and
derived methyl glycosides. Canadian Journal of Chemistry, 53, 1212–
1222.
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the genus Cordyceps (Fr.) Link (Ascomycetes). A review. International Journal of
Medicinal Mushrooms, 10(3), 134–219.
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Marcon, R., et al. (2010). Structure of Agaricus spp. fucogalactans and their
anti-inflammatory and antinociceptive properties. Bioresource Technology, 101,
6192–6199.
Komura, D. L., Ruthes, A. C., Carbonero, E. R., Alquini, G., Rosa, M. C. C., Sassaki, G.
L., et al. (2010). The origin of mannans found in submerged culture of basid-
iomycetes. Carbohydrate Polymers, 79, 1052–1056.
Lee, J. S., Kwon, J. S., Won, D. P., Lee, K. E., Shin, W. C., & Hong, E. K. (2010). Study on
macrophage activation and structural characteristics of purified polysaccharide
from the liquid culture broth of Cordyceps militaris. Carbohydrate Polymers, 82,
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Acknowledgements
Lee, J. S., Kwon, J. S., Yun, J. S., Pahk, J. W., Shin, W. C., Lee, S. Y., et al. (2010). Structural
characterization of immunostimulating polysaccharide from cultured mycelia
of Cordyceps militaris. Carbohydrate Polymers, 80, 1011–1017.
Lehtovaara, B. C., & Gu, F. X. (2011). Pharmacological structural, and drug delivery
properties and applications of 1,3-␤-glucans. Journal of Agricultural and Food
Chemistry, 59, 6813–6828.
Lin, J. G., Fan, M. J., Tang, N. Y., Yang, J. S., Hsia, T. C., Lin, J. J., et al. (2012). An extract
of Agaricus blazei Murill administered orally promotes immune responses in
murine leukemia BALB/c mice in vivo. Integrative Cancer Therapies, 11(29)
http://dx.doi.org/10.1177/1534735411400314
The authors would like to thank Dr. J. M. Sung of Kangwon
National University (Chuncheon, Korea), for the fungal material,
and the Brazilian funding agencies Conselho Nacional de Desen-
volvimento Científico e Tecnológico (CNPq), and the Fundac¸ ão
Araucária.
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