A heteropolysaccharide from aqueous extract of an edible mushroom, Pleurotus ostreatus cultivar: structural and biological studies.

Abstract

A water soluble polysaccharide isolated from the hot aqueous extract of Pleurotusostreatus cultivar was found to contain d-glucose and d-galactose in a molar ratio of nearly 7:1. Structural investigation was carried out using acid hydrolysis, methylation analysis, periodate oxidation, Smith degradation, and NMR studies ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC). On the basis of the above mentioned experiments the structure of the repeating unit of the polysaccharide was established as: This heteroglycan stimulates macrophages, splenocytes, and thymocytes.

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Graphical abstract
pp xxx–xxx A heteropolysaccharide from aqueous extract of an edible mushroom, Pleurotus ostreatus cultivar: structural
and biological studies
Kankan K. Maity, Sukesh Patra, Biswajit Dey, Sanjoy K. Bhunia, Soumitra Mandal, Debsankar Das,
Dibyendu K. Majumdar, Swatilekha Maiti, Tapas K. Maiti, Syed S. Islam*
CAR 5578No. of Pages 1, Model 5G
10 November 2010
1

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Note
A heteropolysaccharide from aqueous extract of an edible mushroom,
Pleurotus ostreatus cultivar: structural and biological studies
Kankan K. Maitya, Sukesh Patraa, Biswajit Deya, Sanjoy K. Bhuniaa, Soumitra Mandala, Debsankar Dasa,
Dibyendu K. Majumdarb, Swatilekha Maitic, Tapas K. Maitic, Syed S. Islama,⇑
aDepartment of Chemistry and Chemical Technology, Vidyasagar University, Midnapore 721102, West Bengal, India
bSangri-la Mushroom, Mohitnagar, Jalpaiguri, West Bengal, India
cDepartment of Biotechnology, Indian Institute of Technology (IIT) Kharagpur, Kharagpur 721302, West Bengal, India
a r t i c l ei n f o
Article history:
Received 14 September 2010
Received in revised form 28 October 2010
Accepted 29 October 2010
Available online xxxx
Keywords:
Pleurotus ostreatus cultivar
Polysaccharide
Structure
NMR spectroscopy
Immunoactivation
20
a b s t r a c t
A water soluble polysaccharide isolated from the hot aqueous extract of Pleurotus ostreatus cultivar was
foundtocontain D-glucoseand D-galactoseinamolarratioofnearly7:1.Structuralinvestigationwascarried
out using acid hydrolysis, methylation analysis, periodate oxidation, Smith degradation, and NMR studies
(1H,13C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC). On the basis of the above men-
tioned experiments the structure of the repeating unit of the polysaccharide was established as:
This heteroglycan stimulates the macrophages, splenocytes, and thymocytes.
? 2010 Published by Elsevier Ltd.
In Asian countries like China, Korea, and Japan mushrooms have
been collected, cultivated, and used for food since ancient times.
Currently mushroom-derived substances with anti-tumor and
immunomodulating properties are used as dietary supplements1,2
or drugs. Several linear3,4and branched glucans5,6and heterogly-
cans7,8isolated from higher basidiomycetes exert strong immuno-
stimulating and anti-tumor activity.
Fungi of the genus Pleurotus, known as oyster mushrooms have
40 species of which Pleurotus florida,9–12Pleurotus sajor-caju,13,14P.
sajor-caju cv Black Japan,15P. florida cv Assam Florida16,17are com-
monlyavailableediblemushroomsandthedetailedstructuralchar-
acterization of the polysaccharides isolated from them have been
reported by our group. Another mushroom of this genus, Pleurotus
ostreatus, famous for its delicious taste and high quantities of pro-
40
50
teins, carbohydrates, minerals, vitamins, and low fat, is also com-
mercially important edible mushroom.18,19It has anti-oxidant,
immunomodulatoryeffects, andcholesterolloweringactivities.20,21
A water soluble heteroglycan22and insoluble b-glucan23with anti-
tumoractivitywereisolatedfromthismushroomandcharacterized.
The present mushroom, P. ostreatus cultivar is a temperature
tolerance species. The main objective of this work is to investigate
any difference that arises in the constituent of the water soluble
polysaccharide of this variety from the original species reported
earlier.22The polysaccharide from P. ostreatus contains glucose,
galactose, and mannose in a molar ratio of 8:2:1 where the main
chain is composed of (1?3)-linked glucose moieties with branch-
ing at C-6 positions. But the present polysaccharide isolated from
this cultivar variety contains only glucose and galactose in a molar
ratio of nearly 7:1 where the main chain consists of mixture of b-
(1?3)-, (1?6)-linked glucose residues with branching at C-3 posi-
tions. We are reporting herein the detailed structural characteriza-
tion of this polysaccharide as well as immunomodulatory studies
including macrophages, splenocyte, and thymocyte activations.
60
70
0008-6215/$ - see front matter ? 2010 Published by Elsevier Ltd.
doi:10.1016/j.carres.2010.10.026
⇑Corresponding author. Tel.: +91 03222 276558x437; mobile: +91 9932629971;
fax: +91 03222 275329.
E-mail address: sirajul_1999@yahoo.com (S.S. Islam).
Carbohydrate Research xxx (2010) xxx–xxx
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
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The fresh fruit bodies of the edible mushroom, P. ostreatus cuti-
var (1 kg) were washed, boiled with distilled water, filtered, centri-
fuged, and the supernatant was precipitated in alcohol. The
precipitated materials on dialysis followed by freeze drying yielded
1.2 g of crude polysaccharide. The water soluble crude polysaccha-
ride on fractionation through Sepharose S-6B column yielded one
homogeneous fraction (POPS) which was identified as a heteropol-
ysaccharide composed of D-galactose and D-glucose. The molecular
weight24of POPS was estimated from a calibration curve prepared
with standard dextrans as ?1.87 ? 105Da. It showed a specific
rotation of ½a?25
Paper chromatographic analysis25of the hydrolyzed product
showed the presence of glucose and galactose. The GLC analysis
of the alditol acetates of POPS showed the presence of glucose
and galactose in the molar ratio of nearly 7:1. The absolute config-
uration of the monosaccharides was determined by the method of
Gerwig et al.26Both the sugar residues, glucose and galactose had D
configuration. The mode of linkages of POPS was determined by
methylation analysis using Ciucanu and Kerek method.27The GLC
and GLC–MS of alditol acetates of the methylated product showed
the presence of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucitol;
1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl-D-glucitol; 1,5,6-tri-O-acetyl-
2,3,4-tri-O-methyl-D-glucitol; 1,3,5,6-tetra-O-acetyl-2,4-di-O-meth
yl-D-glucitol, and 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-galacti-
tol in a molar ratio of nearly 1:2:2:2:1. This result indicated the
presence of terminal D-glucopyranosyl, (1?3)-linked D-glucopyr-
anosyl, (1?6)-linked D-glucopyranosyl, (1?3,6)-linked D-gluco-
pyranosyl, and terminal D-galactopyranosyl moieties in a molar
ratio of nearly 1:2:2:2:1. Thereafter, a periodate oxidation experi-
ment was carried out with the polysaccharide. The GLC analysis
of the alditol acetates of the periodate-oxidized,28,29reduced PS
showed the presence of D-glucose only. The GLC and GLC–MS anal-
80
D+18.6 (c 0.094, water).
90
100
ysis of periodate-oxidized, reduced, methylated PS showed the
presence of 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl-D-glucitol and
1,3,5,6-tetra-O-acetyl-2,4-di-O-methyl-D-glucitol in a molar ratio
of nearly 1:1. This result indicated that (1?6)-linked D-glucopyr-
anosyl, terminal D-glucopyranosyl, and terminal D-galactopyrano-
syl moieties were destroyed during oxidation. These results
confirmed the mode of linkages of these sugar moieties present
in the PS.
The
27 ?C showed the presence of five signals in the anomeric region
at 5.11, 4.98, 4.51, 4.49, and 4.47 ppm in a ratio of nearly
1:1:2:2:2. The sugar residues were designated as A, B, C, D, and
E according to their decreasing anomeric proton chemical shift val-
ues. The13C NMR (125 MHz) spectrum (Fig. 2) at 27 ?C showed that
five anomeric signals appeared at 99.7, 98.6, 103.3, 103.1, and
103.2 ppm in a molar ratio of nearly 1:1:2:2:2. All1H and13C sig-
nals (Table 1) were assigned from DQF-COSY, TOCSY, and HMQC
NMR experiments.
Residue A was assigned to terminal D-galactosyl unit. The galac-
to configuration was assigned from the large coupling constant
JH-2,H-3 (?9 Hz) and a relatively small coupling constant JH-3,H-4
(?3.5 Hz). The anomeric proton chemical shift for residue A at
5.11 ppm and a carbon chemical shift at 99.7 ppm (JC-1,H-
1? 170 Hz) indicated that the galactose was a-linked anomer.
The carbon signals from C-1 to C-6 of residue A corresponded
nearly to the standard values of methyl glycosides.30,31Thus the
residue A was an a-linked terminal D-galactopyranosyl moiety. In
case of residues B, C, D, and E the large JH-2,H-3and JH-3,H-4(9–
10 Hz) indicate their gluco configuration. Residue B had an ano-
meric proton signal at 4.98 ppm and the coupling constants JH-
1,H-2(?3.2 Hz) and (JC-1,H-1? 171 Hz) indicating its a-configura-
tion. The signals from C-1 to C-6 indicated that residue B was a ter-
110
1H NMR spectrum (500 MHz, Fig. 1) recorded in D2O at
120
130
Figure 1.
1H NMR spectrum (500 MHz, D2O, 27 ?C) of the polysaccharide, isolated from Pleurotus ostreatus cultivar.
Figure 2.
13C NMR spectrum (125 MHz, D2O, 27 ?C) of the polysaccharide, isolated from Pleurotus ostreatus cultivar.
2
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minal a-D-glucopyranosyl moiety. The coupling constants JH-1,H-2
(?8 to 9 Hz) and JC-1,H-1(?160 to 162 Hz) of residues C, D, and E
and in addition to their anomeric proton signals at 4.51, 4.49,
and 4.47 ppm, respectively, indicate their b-configuration. The
anomeric carbon signals of residue C, D, and E appeared at 103.3,
103.1, and 103.2 ppm, respectively. The downfield shift of C-3
(84.8 ppm) of residue C indicated that it was (1?3)-linked b-D-glu-
140
copyranosyl moiety. The residue D was assigned as (1?6)-b-D-glu-
copyranosyl moiety as the presence of the downfield shift of C-6
(69.2 ppm). The downfield shifts of C-3 (84.9 ppm) and C-6
(69.3 ppm) of residue E indicated that E was (1?3,6)-linked b-D-
glucopyranosyl moiety. The C-6 linking of residue D and E was con-
firmed from DEPT-135 spectrum (Fig. 3).
The sequences of glycosyl residues of the polysaccharide were
determined on the basis of the phase sensitive ROESY (Fig. 4,
Table 2) as well as NOESY (not shown) experiments. The inter-
residual contacts from AH-1 to EH-3, BH-1 to EH-3, CH-1 to EH-
6a and EH-6b, DH-1 to CH-3, and EH-1 to DH-6a and DH-6b indi-
cated the following sequences:A (1?3) E; B (1?3) E; C (1?6) E; D
(1?3) C; E (1?6) D
Long range13C–1H correlation obtained from the HMBC exper-
iment (Fig. 5, Table 3) corroborated the assigned repeating unit
established from the ROESY experiment. The cross peaks of both
anomeric protons and carbons of each of the sugar residues were
examined and the connectivities were observed from the HMBC
experiment. Inter residual cross peaks AH-1/EC-3; AC-1/EH-3;
BH-1/EC-3; BC-1/EH-3; CH-1/EC-6; CC-1/EH-6a; CC-1/EH-6b;
DH-1/CC-3; DC-1/CH-3; EH-1/DC-6; EC-1/DH-6a; EC-1/DH-6b
with other intra residual peaks were observed (Fig. 5). On the basis
of the appearance of these cross peaks and ROESY connectivities,
the structure of the repeating unit present in the polysaccharide
(POPS) isolated from P. ostreatus cutivar was established as:
150
160
Table 1
The1H NMRaand13C NMRbchemical shifts for the polysaccharide isolated from Pleurotus ostreatus cutivar in D2O at 27 ?C
Glycosyl residue H-1/C-1 H-2/C-2H-3/C-3 H-4/C-4H-5/C-5H-6a, H-6b/C-6
a-D-Galp-(1?
A
5.11
99.7
3.91
67.2
3.99
69.9
3.88
70.5
4.08
70.8
3.69c, 3.71d
61.4
a-D-Glcp-(1?
B
4.98
98.6
3.85
69.2
3.65
73.2
3.48
69.9
3.84
70.8
3.88c, 3.91d
61.1
?3)-b-D-Glcp-(1?
C
4.51 3.51
73.3
3.71
84.8
3.47
68.9
3.58
75.2
3.71c, 3.90d
61.1103.3
?6)-b-D-Glcp-(1?
D
4.493.30
73.5
3.43
76.7
3.38
70.9
3.46
75.3
3.91c, 4.20d
69.2103.1
?3,6)-b-D-Glcp-(1?
E
4.473.31
73.8
3.72
84.9
3.58
68.6
3.62
75.3
3.85c, 4.19d
69.3 103.2
aThe values of chemical shifts were recorded keeping HOD signal fixed at d 4.76 ppm at 27 ?C.
bThe values of chemical shifts were recorded with reference to acetone as internal standard and fixed at d 31.05 ppm at 27 ?C.
c,dInterchangeable.
Figure 3. DEPT-135 spectrum (D2O, 27 ?C) of the polysaccharide, isolated from Pleurotus ostreatus cultivar.
Figure 4. Part of ROESY spectrum of polysaccharide, isolated from Pleurotus
ostreatus cultivar. The ROESY mixing time was 300 ms.
K. K. Maity et al./Carbohydrate Research xxx (2010) xxx–xxx
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Table 2
ROE data for the polysaccharide isolated from Pleurotus ostreatus cutivar
Glycosyl residueAnomeic proton
d
ROE contact proton
d
ResidueAtom
a-D-Galp-(1?
A
5.113.72
3.91
3.99
E
A
A
H-3
H-2
H-3
a-D-Glcp-(1?
B
4.983.72
3.85
3.88
E
B
B
H-3
H-2
H-6a
?3)-b-D-Glcp-(1?
C
4.513.85
4.19
3.71
3.47
3.58
E
E
C
C
C
H-6a
H-6b
H-3
H-4
H-5
?6)-b-D-Glcp-(1?
D
4.493.71
3.30
3.43
3.38
C
D
D
D
H-3
H-2
H-3
H-4
?3,6)-b-D-Glcp-(1?
E
4.473.91
4.20
3.31
3.72
D
D
E
E
H-6a
H-6b
H-2
H-3
Figure 5. HMBC spectrum of polysaccharide isolated from Pleurotus ostreatus cultivar. The delay time in the HMBC experiment was 80 ms.
Table 3
The significant3JH,Cconnectivities observed in an HMBC spectrum for the anomeric protons/carbons of the sugar residues of the polysaccharide of Pleurotus ostreatus cutivar
ResidueSugar linkageH-1/C-1
dH/dC
Observed connectivities
dH/dC
ResidueAtom
A
a-D-Galp-(1?
5.1184.9
67.2
69.9
3.72
E
A
A
E
C-3
C-2
C-3
H-399.7
B
a-D-Glcp-(1?
4.98 84.9
73.2
3.72
E
B
E
C-3
C-3
H-398.6
C
?3)-b-D-Glcp-(1?
4.51 69.3
73.3
3.85
4.19
3.51
3.47
E
C
E
E
C
C
C-6
C-2
H-6a
H-6b
H-2
H-4
103.3
D
?6)-b-D-Glcp-(1?
4.49
103.1
84.8
3.71
3.30
C
C
D
C-3
H-3
H-2
E
?3,6)-b-D-Glcp-(1?
4.47 69.2
73.8
3.91
4.20
3.31
D
E
D
D
E
C-6
C-2
H-6a
H-6b
H-2
103.2
4
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170
Solution) control either closer to 1 or below indicates low stimula-
tory effect on the immune system. The molecule was found to acti-
vate splenocytes and thymocytes as shown in Figure 7A and B,
respectively. At 25 lg/mL of the polysaccharide, both splenocyte
and thymocyte proliferation indexes were maximum as compared
to other concentrations. Hence, 25 lg/mL of the polysaccharide can
be considered as efficient splenocyte and thymocyte proliferators.
Some mushroom and plant polysaccharides showed similar
splenocyte as well as macrophage activation as reported ear-
lier9,16,36by our group.
In order to obtain information on the sequence of the sugar res-
idues in the repeating unit, the polysaccharide was subjected to
Smith degradation32,33studies, and the products were separated
on a Sephadex G-25 column using water as the eluant, resulting
in one fraction (SDPS). GLC analysis of the alditol acetates of the
acid-hydrolyzed product from SDPS showed the presence of D-glu-
cose and glycerol in a molar ratio of nearly 2:1. The alditol acetates
of the methylated product from SDPS were analyzed by GLC and
these methylated sugars were also identified by GLC–MS analysis
using ZB-5MS capillary column which showed the presence of
1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucitol and 1,5,6-tri-O-
acetyl-2,3,4-tri-O-methyl-D-glucitol in a molar ratio of nearly 1:1.
The
two anomeric signals at 4.51 and 4.47 ppm in a molar ratio of
nearly 1:1. The anomeric signals at 4.51 and 4.47 ppm corre-
sponded to b-D-Glcp-(1? (Residue F), and ?6)-b-D-Glcp-(1? (Res-
idue G), respectively. The13C NMR (125 MHz) experiment (Table 4)
of SDPS showed two anomeric carbon signals at 103.1 and
102.9 ppm corresponding to b-D-Glcp-(1? (F) and another ?6)-
b-D-Glcp-(1? (G) residues, respectively. The carbon signals C-1,
C-2, and C-3 of the glycerol moiety were assigned as 66.8, 72.4,
and 62.9 ppm, respectively. The terminal b-D-Glcp unit (F) was
generated during Smith degradation of (1?3)-b-D-Glcp moiety
(C) of the main chain. The (1?6)-b-D-Glcp (G) was produced from
the (1?3,6)-b-D-Glcp (E) residue due to oxidation, followed by
Smith degradation of the terminal a-D-Galp moiety (A) and of the
terminal a-D-Glcp moiety (B) on another side. The glycerol moiety
(H) was generated from (1?6)-b-D-Glcp (D) after periodate oxida-
tion, followed by Smith degradation. Hence, the structure of the
Smith degraded product was established as:
180
1H NMR (500 MHz) experiment (Table 4) of SDPS showed
190
200
210
Therefore, these results supported the above mentioned struc-
ture of the repeating unit of the polysaccharide.
The PS was found to activate the macrophages. Macrophageacti-
vationwasstudiedbynitricoxide(NO) productionin culturesuper-
natantinvitro.Upontreatmentwithdifferentconcentrationsofthis
PS, enhanced production of NO was observed maximum value of
9.1 lM per 5 ? 105macrophages at 25 lg/mL but decreased at con-
centration 50 lg/mL and further increased at 100 lg/mL (Fig. 6).
Proliferation of splenocytes and thymocytes is an indicator of
immunoactivation. The splenocyte and thymocyte activation tests
were carried out in mouse cell culture medium with the polysac-
charide by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide] method.34,35The splenocyte or thymocyte
proliferation index (SPI) compared to PBS (Phosphate Buffer
220
230
1. Experimental
1.1. Isolation and purification of the polysaccharide
The present mushroom P. ostreatus cutivar is a temperature tol-
erance species, adapted at the temperature of West Bengal, India
and can be cultivated within 30–35 ?C. The fresh fruiting bodies
of P. ostreatus cutivar (1 kg) were cultivated and collected from
Sangri-la mushroom farm, Mohitnagar, Jalpaiguri, gently washed
with distilled water, and then boiled for 6 h. The crude polysaccha-
ride (1.2 g) was isolated by the procedures as applied in our previ-
ous papers.14,15The polysaccharide (30 mg) was purified by gel
permeation chromatography and one fraction (test tubes 20–35)
was collected and freeze dried, yielding 14 mg of material. The
purification process was carried out in seven lots.
240
1.2. General methods
Optical rotation was measured on a Jasco polarimeter, model P-
1020 at 26.8 ?C. For monosaccharide analysis, the polysaccharide
sample (3.0 mg) was hydrolyzed with 2 M CF3COOH (2 mL), and
the analysis was carried out as described earlier.10,15,17The molecu-
lar weight of the polysaccharide was determined as reported ear-
lier.15,17
The absolute configuration of
constituent was assigned according to Gerwig et al.26The polysac-
charide was methylated according to Ciucanu and Kerek method.27
Gas-liquidchromatography–massspectrometric(GLC–MS)analysis
wasalsoperformedonShimadzuGLC–MSModelQP-2010Plusauto-
matic system, using ZB-5MS capillary column (30 m ? 0.25 mm).
Theprogramwasisothermalat150 ?C;holdtime5 min,withatem-
perature gradient of 2 ?C min?1up to a final temperature of 200 ?C.
Gas Liquid chromatographic (GLC) analysis was done by using a
Hewlett–Packard Model 5730 A, having a flame ionization detector
and glass columns (1.8 m ? 6 mm) packed with 3% ECNSS-M (A)
on Gas Chrom Q (100–120 mesh) and 1% OV-225 (B) on Gas Chrom
Q (100–120 mesh). All GC analyses were performed at 170 ?C.
250
themonosaccharide
260
Table 4
The1H NMRaand13C NMRbfor Smith degraded polysaccharide (SDPS)
Sugar residueH-1/C-1H-2/C-2H-3/C-3 H-4/C-4 H-5/C-5H-6ac, H6bd/C-6
b-D-Glcp-(1?
F
4.513.49
73.8
3.71
76.3
3.53
69.7
3.65
77.0
3.73, 3.88
61.4 103.1
?6)-b-D-Glcp-(1?
G
4.47 3.41
73.2
3.68
75.2
3.53
70.2
3.65
76.6
3.82, 4.17
69.0102.9
Gro-(3?
H
4.23
3.90
66.8
3.74 3.67
3.52
62.972.4
aValues of the1H chemical shifts were recorded with respect to the HOD signal fixed at d 4.74 at 27 ?C.
bValues of the13C chemical shifts were recorded with reference to acetone as the internal standard and fixed at d 31.05 at 27 ?C.
c,dInterchangeable.
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NMR experiments of polysaccharide and Smith degraded polysac-
charide were carried out applying the methods as reported in our
previous papers.10,15,17All NMR and DEPT-135 NMR experiments
were carried out at 27 ?C.
270
Figure 6. In vitro activation of peritoneal macrophage stimulated with different concentrations of the polysaccharide in terms on NO production.
Figure 7. Effect of different concentrations of the polysaccharide on splenocyte (A) and thymocyte (B) proliferation.
6
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1.3. Periodate oxidation and Smith degradation32,33
Periodate oxidation was performed with this polysaccharide as
described in our earlier reports.10,17The polysaccharide (25 mg)
was oxidized with 0.1 M sodium metaperiodate (2 mL) at 25 ?C
in the dark during 48 h and the Smith degraded material was pre-
pared as described earlier.10
1.4. Test for macrophage activity by Nitric oxide assay
Peritoneal macrophages (5 ? 105cells mL?1) after harvesting
were cultured in complete RPMI (Roswell Park Memorial Institute)
media in 96-well plates.34,35The purity of macrophages was tested
by adherence to tissue culture plates. The polysaccharide was
added to the wells in different concentrations. The cells were cul-
tured for 24 h at 37 ?C in a humidified 5% CO2incubator. Produc-
tion of nitric oxide was estimated by measuring nitrite levels in
cell supernatant with Greiss reaction.37Equal volumes of Greiss re-
agent (1:1 of 0.1% in 1-napthylethylenediamine in 5% phosphoric
acid and 1% sulfanilamide in 5% phosphoric acid) and sample cell
supernatant were incubated together at room temperature for
10 min. Absorbance was observed at 550 nm. Lipopolysaccharide
(LPS), L6511 of Salmonella enterica serotype typhimurium (sigma)
was used as positive control.
280
290
1.5. Splenocyte and thymocyte proliferation assay35,38
A single cell suspension of spleen and thymus was prepared
from the normal mice under aseptic conditions by frosted slides
in Phosphate Buffer Solution (PBS). The suspension was centri-
fuged to obtain cell pellet. The contaminating RBC was removed
by hemolytic Gey’s solution. After washing two times in PBS the
cells were resuspended in complete RPMI medium. Cell concentra-
tion was adjusted to 1 ? 105cells/mL and viability of the sus-
pended cells (as tested by trypan blue dye exclusion) was always
over 90%. The cells (180 lL) were plated in 96-well flat-bottomed
plates and incubated with 20 lL of various concentrations (10–
100 lg/mL) of the polysaccharide. Similar lipopolysaccharide
(LPS) as used in macrophage activation was also used as positive
control. The cultures were set-up for 72 h at 37 ?C in a humidified
atmosphere of 5% CO2. Proliferation was checked by MTT assay
method. The data are reported as the mean ± standard deviation
of five different observations and compared against PBS control.
300
Acknowledgments
310
The authors are grateful to Professor S. Roy, Director, IICB, Kolk-
ata, for providing instrumental facilities. Mr. Barun Majumder of
Bose Institute, Kolkata, is acknowledged for preparing NMR spec-
tra. DST, Govt of India is acknowledged for sanctioning a project
(Ref. No: SR/S1/OC-52/2006 dated 19/02/2007) and also for offer-
ing junior research fellowship.
References
1. Brochers, A. T.; Stern, J. S.; Hackman, R. M.; Keen, C. L.; Gershwin, M. E. Proc. Soc.
Exp. Biol. Med. 1999, 221, 281–293.
2. Wasser, S. P.; Weis, A. L. Int. J. Med. Mush. 1999, 1, 31–62.
3. Kiho, T.; Nagai, Y. S.; Sakushima, M.; Ukai, S. Chem. Pharm. Bull. 1992, 40, 2212–
2214.
4. Yoshida, I.; Kiho, T.; Usui, S.; Sakushima, M.; Ukai, S. Biol. Pharm. Bull. 1996, 19,
114–121.
5. Fujimiya, Y.; Suzuki, Y.; Oshiman, K. I.; Kobori, H.; Moriguchi, K.; Nakashima,
H.; Matumota, Y.; Takahara, S.; Bina, T.; Kakakura, R. Cancer Immunol. Immun.
1998, 46, 147–159.
6. Ukawa, Y.; Ito, H.; Hisamatsu, M. J. Biosci. Bioeng. 2000, 90, 98–104.
7. Mizuno, T.; Minto, K.; Ito, H.; Kawade, M.; Terai, H.; Tsuchida, H. Biochem. Mol.
Biol. Int. 1999, 47, 707–714.
8. Gutierrez, A.; Prito, A.; Martinez, A. T. Carbohydr. Res. 1996, 281, 143–154.
9. Rout, D.; Mondal, S.; Chakraborty, I.; Pramanik, M.; Islam, S. S. Med. Chem. Res.
2004, 13, 509–517.
10. Rout, D.; Mondal, S.; Chakraborty, I.; Pramanik, M.; Islam, S. S. Carbohydr. Res.
2005, 340, 2533–2539.
11. Rout, D.; Mondal, S.; Chakraborty, I.; Islam, S. S. Carbohydr. Res. 2006, 341, 995–
1002.
12. Rout, D.; Mondal, S.; Chakraborty, I.; Islam, S. S. Carbohydr. Res. 2008, 343, 982–
987.
13. Pramanik, M.; Chakraborty, I.; Mondal, S.; Islam, S. S. Carbohydr. Res. 2007, 342,
2670–2675.
14. Pramanik, M.; Chakraborty, I.; Mondal, S.; Islam, S. S. Carbohydr. Res. 2005, 340,
629–636.
15. Roy, S. K.; Maiti, D.; Mondal, S.; Das, D.; Islam, S. S. Carbohydr. Res. 2008, 343,
1108–1113.
16. Roy, S. K.; Das, D.; Mondal, S.; Maiti, D.; Bhunia, B.; Maiti, T. K.; Islam, S. S.
Carbohydr. Res. 2009, 344, 2596–2601.
17. Ojha, A. K.; Chandra, K.; Ghosh, K.; Islam, S. S. Carbohydr. Res. 2010, 345, 2157–
2163.
18. Hernández, D.; Sánchez, J. E.; Yamasaki, K. Bioresour. Technol. 2003, 90, 145–
150.
19. Kalmis, E.; Nuri, A.; Hasan, Y.; Fatih, K. Bioresour. Technol. 2008, 99, 164–169.
20. Jayakumar, T.; Thomas, P. A.; Geraldine, P. Exp. Gerontol. 2007, 42, 183–191.
21. Regina, M. M. G.; Elisabeth, W.; Jamile, R. R.; Jorge, L. N.; Sandra, A. F. Bioresour.
Technol. 2008, 99, 76–82.
22. Tong, H.; Xia, F.; Feng, K.; Sun, G.; Gao, X.; Sun, L.; Jiang, R.; Sun, X. Bioresour.
Technol. 2009, 100, 1682–1686.
23. Yoshioka, Y.; Tabeta, R.; Saito, H.; Uehara, N.; Fukuoka, F. Carbohydr. Res. 1985,
140, 93–100.
24. Hara, C.; Kiho, T.; Tanaka, Y.; Ukai, S. Carbohydr. Res. 1982, 110, 77–87.
25. Hoffman, J.; Lindberg, B.; Svensson, S. Acta Chem. Scand. 1972, 26, 661–666.
26. Gerwig, G. J.; Kamerling, J. P.; Vliegenthart, J. F. G. Carbohydr. Res. 1978, 62,
349–357.
27. Ciucanu, I.; Kerek, F. Carbohydr. Res. 1984, 131, 209–217.
28. Hay, G. W.; Lewis, B. A.; Smith, F. Methods Carbohydr. Chem. 1965, 5, 357–361.
29. Goldstein, I. J.; Hay, G. W.; Lewis, B. A.; Smith, F. Methods Carbohydr. Chem.
1965, 5, 361–370.
30. Agarwal, P. K. Phytochemistry 1992, 31, 3307–3330.
31. Rinuado, R.; Vincendon, M. Carbohydr. Polym. 1982, 2, 135–144.
32. Abdel-Akher, M.; Hamilton, J. K.; Montgomery, R.; Smith, F. J. Am. Chem. Soc.
1952, 74, 4970–4971.
33. Datta, A. K.; Basu, S.; Roy, N. Carbohydr. Res. 1999, 322, 219–227.
34. Ohno, N.; Hasimato, T.; Adachi, Y.; Yadomae, T. Immunol. Lett. 1992, 52, 1–7.
35. Sarangi, I.; Ghosh, D.; Bhutia, S. K.; Mallick, S. K.; Maiti, T. K. Int.
Immunopharmacol. 2006, 6, 1287–1297.
36. Das, D.; Mondal, S.; Roy, S. K.; Maiti, D.; Bhunia, B.; Maiti, T. K.; Islam, S. S.
Carbohydr. Res. 2009, 344, 2581–2585.
37. Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.; Wishnok, J. S.;
Tannenbaum, S. R. Anal. Biochem. 1982, 126, 131–138.
38. Maiti, S.; Bhutia, S. K.; Mallick, S. K.; Kumar, A.; Khadgi, N.; Maiti, T. K. Environ.
Toxicol. Pharmacol. 2008, 26, 187–191.
320
330
340
350
360
370
380
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Please cite this article in press as: Maity, K. K.; et al. Carbohydr. Res. (2010), doi:10.1016/j.carres.2010.10.026