Heteroglycan of an edible mushroom Termitomyces clypeatus: Structure elucidation and antioxidant properties

Article abstract of DOI:10.1016/j.carres.2015.05.005

A water-soluble heteroglycan (PS) of an average molecular weight ～1.98 ×10⁵ Da was isolated from the aqueous extract of an edible mushroom Termitomyces clypeatus (R. Heim). The structure of the polysaccharide (PS) was established using total hydrolysis, methylation analysis, Smith degradation, and 1D/2D NMR experiments. Total hydrolysis indicated the presence of d-glucose, d-galactose, d-mannose, and l-fucose in a molar ratio of 4.10:1.95:1.0:0.95, respectively. The chemical and NMR analysis indicated the presence of a repeating unit with a backbone consisting of one each of the residues (1→3)-α-d-galactopyranosyl, (1→3)-α-d-mannopyranosyl, (1→3)-α-d-glucopyranosyl, (1→3)-β-d-glucopyranosyl, (1→6)-β-d-glucopyranosyl, and (1→6)-α-d-galactopyranosyl, respectively. The (1→3)-α-d-mannopyranosyl residue was found branched at O-2 with terminal α-l-fucopyranosyl moiety and (1→3)-β-d-glucopyranosyl residue was branched at O-6 with terminal α-d-glucopyranosyl residue. The PS exhibited antioxidant properties.

Full text of DOI:10.1016/j.carres.2015.05.005

Carbohydrate Research 413 (2015) 30e36
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Heteroglycan of an edible mushroom Termitomyces clypeatus:
structure elucidation and antioxidant properties
a
a
a
a
Manabendra Pattanayak , Surajit Samanta , Prasenjit Maity , Ipsita K. Sen ,
a
a
b
b
a, *
Ashis K. Nandi , Dilip K. Manna , Payel Mitra , Krishnendu Acharya , Syed S. Islam
Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore 721102, West Bengal, India
Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata
00019, West Bengal, India
a
b
7
a r t i c l e i n f o
a b s t r a c t
5
Article history:
Received 19 March 2015
Received in revised form
A water-soluble heteroglycan (PS) of an average molecular weight ~1.98 ꢀ10 Da was isolated from the
aqueous extract of an edible mushroom Termitomyces clypeatus (R. Heim). The structure of the poly-
saccharide (PS) was established using total hydrolysis, methylation analysis, Smith degradation, and 1D/
2
L
7
May 2015
D NMR experiments. Total hydrolysis indicated the presence of
-fucose in a molar ratio of 4.10:1.95:1.0:0.95, respectively. The chemical and NMR analysis indicated the
presence of a repeating unit with a backbone consisting of one each of the residues (1/3)- -gal-
actopyranosyl, (1/3)- -mannopyranosyl, (1/3)- -glucopyranosyl, (1/3)- -glucopyranosyl,
1/6)- -glucopyranosyl, and (1/6)- -galactopyranosyl, respectively. The (1/3)- -mannopyr-
anosyl residue was found branched at O-2 with terminal -fucopyranosyl moiety and (1/3)-
-glucopyranosyl residue. The PS exhibited
D-glucose, D-galactose, D-mannose, and
Accepted 9 May 2015
Available online 27 May 2015
a-D
a
-D
a-
D
b-D
Keywords:
Termitomyces clypeatus (R. Heim)
Heteroglycan
NMR analysis
Antioxidant activities
(
b
-
D
a
-D
a-D
a-
L
b-D-
glucopyranosyl residue was branched at O-6 with terminal
antioxidant properties.
a-D
©
2015 Elsevier Ltd. All rights reserved.
1. Introduction
lateritic forest soil of South West Bengal and Odisha, India during
SeptembereOctober every year. Several polysaccharides from Ter-
1
2
13
Mushrooms are consumed as delicious food and considered a
source of biologically active compounds. The polysaccharides of
mitomyces striatus, Termitomyces eurhizus, Termitomyces micro-
carpus,
1
14
15
and Termitomyces robustus
have been isolated,
edible mushrooms have a lot of pharmaceutical applications in
characterized and reported by our group. One water soluble poly-
saccharide (PS) has been isolated from the aqueous extract of the
mushroom through gel permeation chromatography and charac-
terized as heteroglycan. It showed promising antioxidant activities
as evidenced from its ferrous ion chelating ability, superoxide
radical scavenging activity, and high reducing power property. The
detailed structural investigations and antioxidant properties of the
PS was carried out and reported herein.
2
3e8
industries. Several polysaccharides
from the edible mushrooms
have been isolated, purified, and characterized. Reactive oxygen
species (ROS) are produced during biochemical reactions in human
body and uncontrolled production of the oxygen derived free rad-
icals generate various diseases like cancer, coronary heart diseases,
9
Alzheimer, and neurodegenerative disorders. In recent years, the
research work has been focused on the use of mushroom poly-
saccharides as neutralizing agents of the free radicals as well as its
10
protective role for human lymphocytes.
2. Results and discussion
Termitomyces clypeatus commonly called by local people as Bada
bali chattu, Batki chattu and Bihiduni chattu in India belongs to the
genus Termitomyces, family lyophyllaceae. The edible mushroom11
normally grows in association with the termite's guts of the
2.1. Isolation, purification and chemical analysis of polysaccharide
The hot aqueous extract of fresh fruit bodies (500 g) of edible
mushroom T. clypeatus was cooled, filtered, and precipitated in
ethanol. The residue was dialyzed, centrifuged, and freeze dried,
yield; 650 mg. One homogeneous fraction was obtained from the
crude polysaccharide on separation through Sepharose-6B. The
*
Corresponding author. Tel.: þ91 03222 276558x437, þ91 9932629971 (mobile);
fax: þ91 03222 275329.
E-mail address: sirajul_1999@yahoo.com (S.S. Islam).
http://dx.doi.org/10.1016/j.carres.2015.05.005
0
008-6215/© 2015 Elsevier Ltd. All rights reserved.

M. Pattanayak et al. / Carbohydrate Research 413 (2015) 30e36
31
2
5.7
pure polysaccharide (PS) showed specific rotation [
a
]
D
þ12.9 (c
COOH,
confirmed as
D
-galactopyranosyl moieties. The
a
-configuration of
0
.11, H O) and the average molecular weight of the PS was deter-
2
both A and D residues were assigned from the coupling constant
values JH-1,H-2 ~3.1 and JC-1,H-1 ~171 Hz. The downfield shift of C-6 (
66.5) of eCH with respect to the standard values of methyl
indicated that the residue A was substituted at C-6,
which was further confirmed from DEPT-135 spectrum (Fig. 2b).
Hence, residue A was established as (1/6)-linked -galactopyr-
anosyl residue. The downfield shift of C-3 ( 78.3) with respect to
standard value of methyl glycoside indicated that residue D was
5
mined as ~1.98 ꢀ10 Da. The PS on acid hydrolysis by 2 M CF
3
d
followed by alditol acetate preparation and analysis through gas-
liquid chromatography was found to contain glucose, galactose,
mannose, and fucose in a molar ratio of 4.10:1.95:1.0:0.95, respec-
tively. The absolute configuration of the monosaccharide was
2
18,19
glycoside
a-D
16
determined by the method of Gerwig et al. and found that
glucose, galactose, and mannose had the D configuration but fucose
was present in the L configuration. The polysaccharide was meth-
d
2
0
(1/3)-linked
Residue B was assigned as a terminal
which was strongly supported by the appearance of proton signal at
1.22, and a carbon signal at 15.1 for a CH group, and small
coupling constant JH-3,H-4 (<3 Hz). The appearance of the anomeric
proton signal for residue B at 5.16 and the coupling constant value
of JH-1,H-2 ~ 3.75 Hz clearly indicated that -fucose was -linked. This
anomeric configuration was further confirmed by He C coupling
a-D-galactopyranosyl.
1
7
ylated according to the method of Ciucanu and Kerek. The GLC-
MS analysis of the alditol acetates of methylated product showed
the presence of 1,5-di-O-acetyl-2,3,4-tri-O-methylfucitol; 1,5-di-O-
acetyl-2,3,4,6-tetra-O-methylglucitol; 1,3,5-tri-O-acetyl-2,4,6-tri-
O-methylglucitol; 1,3,5-tri-O-acetyl-2,4,6-tri-O-methylgalactitol;
a-L-fucopyranosyl moiety,
d
d
3
d
1,5,6-tri-O-acetyl-2,3,4-tri-O-methylglucitol;
1,5,6-tri-O-acetyl-
,3,4-tri-O-methylgalactitol; 1,2,3,5-tetra-O-acetyl-4,6-di-O-meth-
ylmannitol; 1,3,5,6-tetra-O-acetyl-2,4-di-O-methylglucitol in a ra-
tio of 0.95:1.0:1.05:1.0:1.10:0.95:1.0:0.95. These results indicated
the presence of terminal fucopyranosyl, terminal glucopyranosyl,
L
a
1
13
2
13
constant JC-1,H-1 ~ 171 Hz. The rest of carbon values in C corre-
sponded to the standard values of methyl glycosides indicating
residue B was glycosidically linked terminal
a-L-fucopyranosyl
(
1/3)- glucopyranosyl, (1/3)- galactopyranosyl, (1/6)- gluco-
moiety.
pyranosyl, (1/6)- galactopyranosyl, (1/2,3)- mannopyranosyl,
and (1/3,6)-linked glucopyranosyl moieties in the PS.
Based on the anomeric proton chemical shift and coupling
constant, JH-1,H-2 ~3 Hz and JC-1,H-1 ~171 Hz it was confirmed that
residues C and F were present in
and JH-3,H-4 coupling constant values (~10.0 Hz) confirmed that
residues C and F were -glucopyranosyl moieties. The downfield
shift of C-3 ( 81.2) with respect to standard values of methyl gly-
cosides indicated that residue C was linked at this position. Thus
a-configuration. The large JH-2,H-3
2
.2. NMR and structural analysis of PS
D
d
The proton NMR spectrum (500 MHz; Fig. 1,Table 1) of this PS at
ꢁ
3
5
d
0
C contained seven signals in decreasing order at
d
5.19, 5.16,
21,22
the residue C was (1/3)-linked
a
-D
-glucopyranosyl
moiety.
.09, 5.06, 4.97, 4.50, and 4.49 for anomeric protons. The peak at
5.09 is almost double in comparison to the other peaks; therefore
The carbon signals from C-1 to C-6 of residue F corresponded nearly
to the standard values of methyl glycosides. Thus the residue F was
it consists of two residues. Hence, they were designated as A, B, C,
13
terminal
The anomeric proton signals of residue E at
values of JH-1,H-2 (~1.6 Hz), JH-2,H-3 (~3.5 Hz) and JC-1,H-1 of 170 Hz
clearly indicated that it was present in -configuration. This was
further confirmed from the large coupling values JH-3,H-4 ~7.5 Hz
and JH-4,H-5 ~10 Hz. The downfield shifts of C-2 ( 78.3) and C-3 (
8.6) with respect to standard values of methyl glycoside indicated
that the moiety E was (1/2,3)-linked unit. Hence, these observa-
a-D-glucopyranosyl moiety.
D, E, F, G, and H, respectively (Table 1). In C NMR spectrum
125 MHz; Fig. 2a) at the same temperature, seven signals were
found in the anomeric region at 103.0, 102.7, 102.6, 102.2, 101.8,
d
5.06 with low
(
d
a
100.1, and 97.9 ppm. From HSQC spectrum (Fig. 3a; Table 1), the
anomeric carbon signals at
9
4
d
103.0, 102.7, 102.6, 101.8, 100.1, and
5.06), H (
4.97), respectively and
d 102.2 was correlated to the anomeric proton signals C
d
d
7.9 were correlated to the anomeric proton signals E (
.49), G ( 4.50), B ( 5.16), A ( 5.19) and F (
d
d
7
d
d
d
d
the peak at
5.09) and D (d 5.09). All the H and C NMR signals (Table 1) were
1
13
tions confirmed that residue E was (1/2,3)-a-D-mannopyranosyl
moiety.
(d
assigned from DQF-COSY, TOCSY, and HSQC experiments. From
DQF-COSY experiment the proton coupling constants were
measured and one-bond CeH coupling were measured from proton
coupled 13C spectrum.
Residues A and D has large coupling constant JH-2,H-3 ~9 Hz and
relatively small JH-3,H-4 ~3.5 Hz (Table 2) and thus, they were
Residue G and H were established as
b-configuration from
coupling constant (Table 2) values JH-1,H-2 (~8.0 Hz), JC-1,H-1
(
(
~160 Hz) and the large JH-2,H-3 and JH-3,H-4 coupling constant values
~10.0 Hz) of residues G and H confirmed their -glucopyranosyl
84.3) and C-6 ( 69.0)
D
configuration. The downfield shifts of C-3 (
d
d
Fig. 1. 1H NMR spectrum (500 MHz, D
O, 30 C) of the PS isolated from the edible mushroom T. clypeatus.
ꢁ
2

32
M. Pattanayak et al. / Carbohydrate Research 413 (2015) 30e36
Table 1
The 1H NMRa and13C NMRb chemical shifts for the PS isolated from the mushroom T. clypeatus in D
O at 30
ꢁ
C
2
Glycosyl residue
H-1/C-1
H-2/C-2
H-3/C-3
H-4/C-4
H-5/C-5
H-6a, H-6b/C-6
c
/
6)-
a
-
D
-Galp-(1/
5.19
100.1
5.16
101.8
5.09
102.2
5.09
102.2
5.06
103.0
4.97
97.9
4.50
102.6
4.49
3.82
68.9
3.81
68.3
3.87
69.6
3.80
66.2
3.98
78.3
3.48
71.5
3.47
72.7
3.30
73.1
3.85
69.6
3.87
69.6
4.05
81.2
3.98
78.3
3.92
78.6
3.73
73.5
3.73
84.3
3.38
76.0
3.98
69.3
3.73
72.7
3.47
69.3
3.87
69.6
3.73
66.2
3.63
69.6
3.43
69.6
3.38
70.1
4.19
69.6
4.19
68.9
3.82
72.9
4.05
70.1
3.82
73.5
3.81
71.7
3.46
75.6
3.47
75.6
3.64 , 3.87d
66.5
1.22
A
a
-
L
-Fucp-(1/
B
/
15.1
c
d
d
d
d
d
d
3)-
a
a
-
-
D
D
-Glcp-(1/
-Galp-(1/
3.71 , 3.87
C2
1,22
60.8
c
/
D2
/
E
3)-
3.74 , 3.74
0
60.7
c
2,3)-
a
-
D
-Manp-(1/
3.73 , 3.87
61.1
c
a
-
D
-Glcp-(1/
3.73 , 3.89
F
/
60.8
c
3,6)-
6)-
b
-
D
-Glcp-(1/
3.81 , 4.17
G
/
H
69.0
c
b
-
D
-Glcp-(1/
3.82 , 4.19
102.7
68.8
c,d
Interchangeable.
a
ꢁ
The values of chemical shifts were recorded keeping HOD signal fixed at
The values of chemical shifts were recorded with reference to acetone as internal standard and fixed at
d
4.70 at 30 C.
b
ꢁ
d 31.05 at 30 C.
of residue G with respect to standard values of methyl glycoside
appearance of these cross peaks and NOESY connectivity, the
structure of the repeating unit in the PS was proposed as:
indicated that it was (1/3,6)-linked
H the downfield shift of C-6 ( 68.8) with respect to standard values
of methyl glycoside indicated that it was (1/6)-linked -gluco-
pyranosyl. The chemical shift values of eCH of both the residues of
b-D-glucopyranosyl. In residue
d
b-D
2
G and H indicated that the C-6 positions were substituted and
further confirmed from DEPT-135 spectrum (Fig. 2b). The se-
quences of glycosyl residue were determined from NOESY (Fig. 4,
Table 3) as well as ROESY (not shown) experiments. The NOESY
experiment showed the inter-residual contacts: AH-1/DH-3; DH-1/
EH-3; EH-1/CH-3; CH-1/GH-3; GH-1/HH-6a, 6b; HH-1/AH-6a, 6b;
BH-1/EH-2; FH-1/GH-6a, 6b along with other intra-residual con-
tacts (Fig. 2). Thus, the NOESY connectivity established the se-
quences as, A (1/3) D; D (1/3) E; E (1/3) C; C (1/3) G; G (1/6)
H; H (1/6) A; B (1/2) E; F (1/6) G. On the basis of the
Finally, Smith degraded material (SDPS) of PS was prepared to
13
confirm the linkages of the heteroglycan. The C NMR (125 Hz)
ꢁ
spectrum (Fig. 5, Table 4) of SDPS at 30 C showed three
anomeric carbon signals at
anomeric carbon signals at
1/3)- -Manp (I) and (1/3)-b-D
d
103.2, 102.7, and 102.2. The
103.2 and 102.7 corresponded to
-Glcp (J), respectively,
d
(
a
-
D
Fig. 2. (a) 13C NMR spectrum (125 MHz, D
O, 30 C) (b) with insert of the part of DEPT-135 spectrum (D O, 30 C) of the PS isolated from the edible mushroom T. clypeatus.
ꢁ ꢁ
2 2

M. Pattanayak et al. / Carbohydrate Research 413 (2015) 30e36
33
ꢁ
2
Fig. 3. HSQC spectrum (D O, 30 C) of (a) anomeric part and (b) other than anomeric part of the PS isolated from the edible mushroom T. clypeatus.
Table 2
Coupling constant data for the PS isolated from the mushroom T. clypeatus
whereas the carbon signal at
1/3)- -Glcp (K) and terminal
carbon signals C-1, C-2, and C-3 of the glycerol moiety (Gro) were
assigned as 66.2, 72.1, and 62.5, respectively. The terminal
Galp (L) residue of SDPS was generated from the (1/3)- -Galp
D) and (1/3)- -Manp unit (I) from (1/2,3)- -Manp (E)
residue of parent PS during Smith degradation. The carbon signal
at 78.2 clearly indicated the presence of I moiety. The (1/3)-
-Glcp residue (K) attached to I was retained during oxidation
previously denoted as C of the parent PS. The carbon signal at
81.9 clearly confirmed the presence of K residue. Further, the
(1/3)- -Glcp residue (J) attached to K was formed from
(1/3,6)- -Glcp (G), during oxidation followed by degradation.
The carbon signal at 84.3 clearly confirmed the presence of J
residue in the SDPS. The glycerol moiety M was generated from
1/6)- -Glcp residue (H) after periodate oxidation followed
d
102.2 resembled both to the
(
a
-
D
a-D
-Galp (L) residues. The
Glycosyl residue
H-1/C-1
Coupling constant
values(Hz)
d
a-D-
/
6)-
a
-
D
-Galp-(1/
5.19
100.1
JH-1,H-2 ~3.1
JH-2,H-3 ~9.0
JH-3,H-4 ~3.5
JC-1,H-1 ~171
JH-1,H-2 ~3.75
JH-3,H-4 <3.0
JC-1,H-1 ~171
JH-1,H-2 ~3.0
JH-2,H-3 ~10.0
JH-3,H-4 ~10.0
JC-1,H-1 ~171
JH-1,H-2 ~3.1
JH-2,H-3 ~9.0
JH-3,H-4 ~3.5
JC-1,H-1 ~171
JH-1,H-2 ~1.6
JH-2,H-3 ~3.5
JH-3,H-4 ~7.5
JH-4,H-5 ~10.0
JC-1,H-1 ~170
JH-1,H-2 ~3.0
JH-2,H-3 ~10.0
JH-3,H-4 ~10.0
JC-1,H-1 ~171
JH-1,H-2 ~8.0
JH-2,H-3 ~10.0
JH-3,H-4 ~10.0
JC-1,H-1 ~160
JH-1,H-2 ~8.0
JH-2,H-3 ~10.0
JH-3,H-4 ~10.0
JC-1,H-1 ~160
a-D
A
(
a
-
D
a-D
a
-
L
-Fucp-(1/
5.16
101.8
d
a-
B
D
/
3)-
3)-
a
a
-
-
D
D
-Glcp-(1/
-Galp-(1/
5.09
102.2
d
C
b-D
b-D
/
5.09
102.2
d
D
(
b-D
/
2,3)-
a
-
D
-Manp-(1/
5.06
103.0
by Smith degradation, and this moiety was attached to J. Hence,
Smith degradation results in the formation of a glycerol con-
taining tetrasaccharide obtained from the parent PS and the
structure of which was established as:
E
a
-
D
-Glcp-(1/
4.97
97.9
F
/
3,6)-
b
-
D
-Glcp-(1/
4.50
102.6
These results further confirmed the repeating unit present in the
PS of the edible mushroom T. clypeatus.
G
Similar kind of polysaccharide with same sugar composition but
differing in ratios was isolated from another mushroom, Termito-
/
6)-
b
-
D
-Glcp-(1/
4.49
102.7
12
H
myces striatus belonging to the same genus. The present PS con-
tains -glucose, -galactose, -mannose, and -fucose in a molar
ratio of nearly 4:2:1:1 but in T. striatus the ratio is 4:2:2:2. In the
D
D
D
L

34
M. Pattanayak et al. / Carbohydrate Research 413 (2015) 30e36
Fig. 4. Part of NOESY spectrum of the PS of the edible mushroom T. clypeatus. The NOESY mixing time was 300 ms.
present PS
terminated with
Galp and -Glcp remained in the skeleton chain and terminated
with -Glcp and
signals are also different in both the molecules.
a
-
D
-Manp and
b
-
D
-Glcp remain in the backbone chain
NMR values for both fragments (1/3,6)-
102.6; E.L, 4.49, 102.5] and (1/6)- -Glcp [T.C,
4.48, 102.7] were found closer, but the other linkages and
chemical shifts values are found different.
b
-
D
-Glcp [T.C,
d 4.50,
a
-L-Fucp and
a-
D-Glcp whereas, in T. Striatus
a
-D
-
d
b-
D
d
4.49, 102.7; E.L,
a
-D
d
1
13
a-
D
b
-D-Glcp, respectively. The linkages and H,
C
10
Another PS reported earlier from Entoloma lividoalbum was
found to contain same sugar composition but different ratios
2
2
.3. Antioxidant properties
5
:2:1:1. The common fragments (1/3,6)-b-D-Glcp and (1/6)-b-D-
.3.1. Chelating ability of ferrous ions
Glcp were present in the skeleton chain of both the PS isolated from
T. clypeatus and E. lividoalbum. But only the difference is that the
Dietary nutrients with metal chelators having transition metal
2þ
þ
2þ
2þ
23
ions e.g., Fe , Cu , Pb , Co may act as preventive antioxidant.
branching terminates with
Glcp in E. lividoalbum (E.L), respectively. The anomeric H and
a
-
D
-Glcp in T. clypeatus (T.C) and
b
-
D
-
The chelating ability of ferrous ions measures the ability of sec-
ondary antioxidants. Primary antioxidants prevent oxidative dam-
age directly through scavenging the free radicals; while secondary
antioxidants act indirectly by preventing the formation of free
1
13
C
2
4
2þ
radicals. Ferrozine quantitatively forms complexes with Fe . In
the presence of chelating agent, the complex formation is dis-
rupted, thus resulting in the reduction of red color. Reduction
therefore allows estimation of the chelating ability of the coexisting
Table 3
NOESY data for the PS isolated from the mushroom T. clypeatus
Glycosyl residue
Anomeric proton
NOE contact proton
d
d
residue
atom
25
chelator. Ferrous ion chelating ability of PS was found (Fig. 6a) to
/
6)-
a
-
D
-Galp-(1/
5.19
3.98
3.82
3.98
3.81
D
A
E
B
B
B
G
C
H-3
H-2
H-2
H-2
H-3
H-5
H-3
H-2
H-3
H-2
H-3
H-3
H-2
H-5
H-6a
H6b
H-2
H-6a
H-6b
H-2
H-3
H-5
H-6a
H-6b
H-2
H-3
H-5
be 54.1% at 500 mg/mL concentration.
A
a
-
L
-Fucp-(1/
5.16
B
2.3.2. Reducing power
Reducing power of any compound is recognized as a parameter
that can be considered as an antioxidant. The assay of the reducing
3
4
.87
.19
/
C
3)-
3)-
a
a
-
-
D
D
-Glcp-(1/
-Galp-(1/
5.09
5.09
3.73
3.87
3.92
3.80
3þ
2þ
power of the present PS revealed that it could reduce Fe to Fe
.
This change was monitored at 700 nm by measuring the intensity
of the Perl's Prussian blue color. The reducing power of PS was
compared to that of butylated hydroxyanisole (BHA), a synthetic
antioxidant. From Fig. 6b, the EC50 value of PS was observed 260
mL.
/
E
D
D
D
C
E
E
G
G
F
H
H
G
G
G
A
A
H
H
H
3.98
/
2,3)-
a
-
D
-Manp-(1/
5.06
4.97
4.50
4.05
3.98
mg/
E
3.82
a
-
D
-Glcp-(1/
3.81
4.17
F
2.3.3. Superoxide radical scavenging assay
3.48
Superoxide radical is known to be very harmful for cellular
/
3,6)-
b
-
D
-Glcp-(1/
3.82
4.19
G
components and plays a major role in the formation of other
reactive oxygen species such as hydroxyl radical, hydrogen
peroxide and singlet oxygen in living system. The data presented in
Fig. 6c indicated the scavenging activity of PS increased with
increasing concentration. The EC50 value of the PS was found
180 mg/mL. These results suggested that the PS exhibited scav-
enging effect on the generation of superoxide anion radicals that
could prevent or ameliorate oxidative cell damage.
3
3
3
.47
.73
.46
/
6)-
b
-
D
-Glcp-(1/
4.49
3.64
3.87
H
3
3
3
.30
.38
.47

M. Pattanayak et al. / Carbohydrate Research 413 (2015) 30e36
35
Fig. 5. 13C NMR spectrum (125 MHz, D
O, 30 C) of the Smith-degraded glycerol containing tetrasaccharide isolated from the edible mushroom T. clypeatus.
ꢁ
2
Earlier report established that reactive oxygen species (ROS),
such as hydroxyl radicals, superoxide anions, and hydrogen
peroxide, are frequently generated in the living cell during meta-
freeze-dried, yield; 19 mg. It was further purified by passing
through Sepharose 6B column in several lots to yield 110 mg of pure
polysaccharide (PS).
2
6
bolism and play a vital role in cell signaling. However, excessive
amount of ROS induces oxidative stress causes damage to cellular
macromolecules. Most of the antioxidants used to preserve
adequate function of cells against homeostatic disturbances caused
by oxidative stress, are usually synthetic and have been suspected
3.2. General methods
Optical rotation was measured by Jasco polarimeter (Model P-
ꢁ
34
1
020) at 25.7 C. The average molecular weight of the poly-
27
of causing liver damage and carcinogenesis. Therefore, there is an
alarming need for the researcher to search natural and safe anti-
oxidants. Earlier reports showed that mushroom polysaccharide
saccharide was determined by gel chromatography as reported in
2
8e30
possess the potential to scavenge ROS.
Results showed that the
polysaccharide isolated from T. clypeatus have the potential to
scavenge intracellular ROS.
3
. Materials and methods
3.1. Isolation and purification of the polysaccharide
Fresh fruit bodies of the mushroom T. clypeatus (500 g) was
washed, crushed and boiled in 500 mL of distilled water for 10 h,
ꢁ
kept overnight at 4 C and filtered. The water-soluble crude poly-
saccharide (650 mg) was isolated by the method described previ-
10,31,32
ously.
A portion (25 mg) of this crude polysaccharide was
purified by gel-permeation chromatography on Sepharose-6B col-
3
3
umn and monitored by the phenolesulfuric acid procedure at
90 nm using Shimadzu UVevis spectrophotometer, Model-1601.
4
One fraction was obtained (test tubes 16e34), collected, and
Table 4
13
a
The C NMR chemical shifts of Smith-degraded glycerol-containing tetrasaccharide
of the mushroom T. clypeatus in D
ꢁ
2
O at 30
C
Sugar residue
C-1
C-2
C-3
C-4
C-5
C-6
/
3)-
3)-
3)-
a
b
a
-
D
-Manp-(1/
103.2
70.6
78.2
67.2
73.5
61.8
I
/
J
-D
-Glcp-(1/
102.7
102.2
102.2
66.2
73.2
69.6
68.1
72.1
84.3
81.9
70.6
62.5
69.7
68.1
70.5
75.6
72.9
71.1
60.8
60.8
61.1
/
-D-Glcp-(1/
K
a
-D-Galp-(1/
L
Gro-(3/
M
Fig. 6. (a) Ferrous ion chelating ability, (b) reducing power, (c) superoxide radical
scavenging activity of the PS isolated from the edible mushroom T. clypeatus. All the
results are the mean±SD of three separate experiments, each in triplicate.
a
The values of chemical shifts were recorded with reference to acetone as in-
ꢁ
ternal standard and fixed at
d
31.05 at 30 C.

36
M. Pattanayak et al. / Carbohydrate Research 413 (2015) 30e36
our earlier works.28 For monosaccharide analysis, the PS (3 mg) was
University of Calcutta has identified the mushroom. One of the
authors (M.P.) is thankful to Council of Scientific and Industrial
Research-UGC, India for providing junior research fellowship [UGC:
Sr. No. 2061210344, Ref. No.:17-06/2012(i) EU-V].
hydrolyzed with 2 M CF
3
COOH (2 mL) in a round-bottom flask at
ꢁ
100 C for 18 h in a water bath and the analysis was carried out as
2
0,35,36
described in earlier publications.
Methylation experiment
37
was carried out by the method described previously. The absolute
configuration of monosaccharide was determined by the method of
Gerwig et al.16 Smith degradation was carried out by the method
described previously.10 Gas-liquid-chromatographic (GLC) analysis
was done by using a HewlettePackard Model 5730 A having a flame
ionization detector and glass columns (1.8 mꢀ6 mm) packed with
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Acknowledgements
1
3
The authors are grateful to Professor S. Roy, Director, IICB, Kol-
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Bose Institute, Kolkata, is acknowledged for preparing NMR spectra.
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