Glucans from alkaline extract of a hybrid mushroom (backcross mating between PfloVv12 and Volvariella volvacea): Structural characterization and study of immunoenhancing and antioxidant properties

Article abstract of DOI:10.1016/j.carres.2011.10.043

Two different glucans (water-soluble PS-I, water-insoluble PS-II) were isolated from the alkaline extract of the fruit bodies of hybrid mushroom. PS-I was found to consist of only (1→6)-linked β-d-glucopyranose. PS-II was composed of terminal, (1→3,4)-linked, and (1→3)-linked β-d-glucopyranosyl moieties in a molar ratio of nearly 1:1:1. PS-I showed macrophages, splenocytes, and thymocytes activation as well as antioxidant property. On the basis of sugar analysis, methylation analysis, and NMR studies (¹H, ¹³C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of these glucans were established as: PS-I →6)-β-D-Glcp-(1→(Water-soluble glucan) PS-II →3)-Β-D-Glcp-(1→3)-β-D-Glcp-(1→4↑1 β-D-Glcp (Water-Insoluble glucan)

Full text of DOI:10.1016/j.carres.2011.10.043

Carbohydrate Research 347 (2012) 107–113
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Glucans from alkaline extract of a hybrid mushroom (backcross mating
between PfloVv12 and Volvariella volvacea): structural characterization
and study of immunoenhancing and antioxidant properties
a
a
a
b
b
Ramsankar Sarkar , Chanchal K. Nandan , Sanjoy K. Bhunia , Swatilekha Maiti , Tapas K. Maiti ,
c
a,
⇑
Samir R. Sikdar , Syed S. Islam
Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore 721102, West Bengal, India
Department of Biotechnology, Indian Institute of Technology (IIT) Kharagpur, Kharagpur 721302, West Bengal, India
Division of Plant Biology, Bose Institute, Centenary Building, P-1/12, C.I.T. Scheme VII M, Kolkata 700054, West Bengal, India
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2011
Received in revised form 26 October 2011
Accepted 28 October 2011
Available online 6 November 2011
Two different glucans (water-soluble PS-I, water-insoluble PS-II) were isolated from the alkaline extract
of the fruit bodies of hybrid mushroom. PS-I was found to consist of only (1?6)-linked b-
D-glucopyra-
nose. PS-II was composed of terminal, (1?3,4)-linked, and (1?3)-linked b- -glucopyranosyl moieties
D
in a molar ratio of nearly 1:1:1. PS-I showed macrophages, splenocytes, and thymocytes activation as
well as antioxidant property. On the basis of sugar analysis, methylation analysis, and NMR studies
1
13
(
H, C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating
Keywords:
Hybrid mushroom
Polysaccharide
unit of these glucans were established as:
NMR spectroscopy
Immunostimulation
Antioxidant property
Ó 2011 Elsevier Ltd. All rights reserved.
1
. Introduction
within the gene pool of any one particular edible mushroom spe-
cies is limited and therefore for further improvement of quality
of edible mushrooms, introgression of characters from different
mushroom strains are needed and that can be done through pro-
Mushrooms have been used as a food or food flavoring materi-
1
als because of their unique and subtle flavor for a long time. Edible
mushrooms are highly nutritious and used as a source of physio-
logically beneficial and nontoxic medicines. Currently mushroom-
2
5
duction of hybrid mushrooms. In our laboratory, water-soluble
3
polysaccharides have already been isolated from the aqueous ex-
6
derived substances having anti-tumor and immunomodulating
tract of fruit bodies of somatic hybrid mushrooms PfloVv5FB and
properties are used as dietary supplements or drugs.4 Characters
PfloVv1aFB and characterized. Water-soluble branched
7
a,b-
(
1?3)(1?6) and insoluble b-(1?3)(1?6)-glucans from Pleurotus
8
,9
10
florida and another glucan from V. volvacia were reported from
parent mushrooms. The aqueous extract of this hybrid mushroom
showed the presence of b-(1?6) glucan and manno galactosyl
⇑
Corresponding author. Tel.: +91 03222 276558x437, mobile: +91 9932629971;
fax: +91 03222 275329.
E-mail address: sirajul_1999@yahoo.com (S.S. Islam).
0
008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.10.043

1
08
R. Sarkar et al. / Carbohydrate Research 347 (2012) 107–113
glucose (communicated) but its alkaline extract showed the pres-
ence of linear soluble b-(1?6) and also insoluble branched
b-(1?3)(1?4) glucans. Here, we report the structural character-
ization of both the glucans and the study of immunoenhancing
and antioxidant properties of the b-(1?6) glucan isolated from
alkaline extract of a novel hybrid mushroom (backcross mating be-
tween PfloVv12 and Volvariella volvacea).5 PfloVv12, was initially
obtained through intergeneric protoplast fusion between P. florida
and V. volvacea.
2
2
. Results and discussion
.1. Structural investigation of water-soluble glucan (PS-I)
The alkaline extract of fruit bodies of hybrid mushroom was
cooled, centrifuged, and precipitated in ethanol. The residue was
dialyzed until alkali free, centrifuged, and freeze–dried to yield
3
00 mg of water-soluble and 900 mg of water-insoluble crude
polysaccharides. The water-soluble crude material was purified,
2
D
5
yielding a single fraction (PS-I). PS-I had a specific rotation ½ ꢀ
a
11
ꢁ
26.6 (c 0.6, water). Molecular weight of PS-I was estimated as
5
12
ꢂ2.2 ꢃ 10 Da. Paper chromatography analysis of the hydrolyzed
PS-I showed the presence of only one spot of glucose. This was also
confirmed by GLC analysis of alditol acetate of hydrolyzed product
of PS-I. The absolute configuration of the glucose residue was
determined as D by the method of Gerwig et al.13 The mode of link-
ages of the sugar moieties present in PS-I was determined by using
1
4
Ciucanu and Kerek method followed by hydrolysis and alditol
acetate preparation. The GLC–MS analysis of the partially methyl-
ated alditol acetates of PS-I revealed the presence of only one peak
of 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-glucitol. This result indi-
cated the presence of only (1?6)-linked glucopyranosyl residue
in the glucan (PS-I). Further GLC analysis of alditol acetates of per-
Figure 1. 1H NMR spectrum (500 MHz, D
bodies of hybrid mushroom.
2
O, 27 °C) of PS-I, isolated from fruit
1
5,16
iodate-oxidized,
4
NaBH reduced, methylated polysaccharide
showed the disappearance of the sugar units. This result is in
agreement with the presence of (1?6)-linked sugar residue in
PS-I.
Table 1
1
H NMR and 13C NMR chemical shifts (ppm) of PS-I isolated from the fruit bodies of
a,b
hybrid mushroom recorded D
2
O at 27 °C
The 500 MHz 1H NMR spectrum (Fig. 1, Table 1) and 13C NMR
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
(
125 MHz) spectrum (Fig. 2, Table 1) showed the anomeric sig-
nals at 4.54 ppm and 103.4 ppm, respectively. From the above
anomeric signals it was confirmed that PS-I consist of only one
sugar unit in a repeating manner, designated as A. The coupling
constant JH-1,H-2 value (ꢂ8.5 Hz) and JC-1,H-1 value (ꢂ160 Hz) sug-
gested that it was b-linked residue. The large JH-2,H-3 and JH-3,H-4
values (ꢂ10.0 Hz) confirmed its glucopyranosyl configuration.
The proton chemical shifts from H-1 to H-6 were assigned from
the DQF-COSY and TOCSY spectrum. On the basis of the proton
assignments, the chemical shifts of C-1 to C-6 were obtained
?6)-b-
A
D
-Glcp-(1?
4.54
103.4
3.34
73.5
3.64
76.0
3.50
70.0
3.52 4.24, 3.87
75.4 69.3
a
Values of the 13C chemical shifts were recorded with reference to acetone as the
internal standard and fixed at d 31.05 at 27 °C.
b
Values of the 1H chemical shifts were recorded with respect to the HOD signal
fixed at d 4.74 at 27 °C.
1
13
from the H– C HMQC spectrum. The carbon signals at 73.5,
6.0, 70.0, 75.4, and 69.3 ppm correspond to C-2, C-3, C-4, C-5
7
and C-6, respectively, of the glucopyranoside residue. The C-6
signal of the glucopyranoside residue at 69.3 ppm was shifted
to 7.5 ppm downfield compared to the standard methyl glyco-
7,18
sides due to the
cated that residue
residue. The (1?6)-linking was also confirmed from the DEPT-
a
-glycosilation1
A
effect. These results indi-
was (1?6)-linked b-D-glucopyranosyl
1
35 NMR spectrum (Fig. 3).
The sequence of glycosyl residues of PS-I was determined from
the NOESY (Fig. 4, Table 2) as well as ROESY experiments followed
by confirmation with a HMBC (Fig. 5, Table 3) experiment. For
explanation of NOESY and HMBC experiments two units of glucose
0
(
A and A ) were considered. NOESY experiment showed inter-
0
0
residual contact from A H-1 to A H-6a and A H-6b along with
other intra-residual contacts. Hence, the following sequence in
PS-I was established as:
Figure 2. 13C NMR spectrum (125 MHz, D
2
O, 27 °C) of PS-I, isolated from fruit
bodies of hybrid mushroom.

R. Sarkar et al. / Carbohydrate Research 347 (2012) 107–113
109
Table 2
NOESY data for PS-I isolated from the fruit bodies of hybrid mushroom
Anomeric proton
Glycosyl residue
NOE contact protons
d (ppm) Residue, atom
d (ppm)
0
0
?
A
6)-b-
D-Glcp-(1?
4.54
4.24
3.87
A H6a
A H6b
3
3
3
.64
.52
.34
A H-3
A H-5
A H-2
ence of only glucose was further confirmed by the GLC analysis of
alditol acetates of PS-II. Thus, above experiments confirmed that
PS-II was a glucan. The absolute configuration of the sugar units
was determined as configuration
D by the method of Gerwig
1
3
et al. The mode of linkages of the sugar moieties were determined
by methylation analysis using the method of Ciucanu and Kerek,14
followed by hydrolysis and alditol acetates preparation. GLC–MS
analysis of the partially methylated alditol acetates revealed the
presence of three peaks of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-
2
Figure 3. DEPT-135 NMR spectrum (D O, 27 °C) of PS-I, isolated from fruit bodies of
hybrid mushroom.
glucitol,
,3,5-tri-O-acetyl-2,4,6-tri-O-methyl-glucitol in a molar ratio of
nearly 1:1:1. Above results indicated the presence of terminal,
1?3,4)-linked, and (1?3)-linked glucopyranosyl moieties in the
water-insoluble glucan. Further GLC–MS analysis of alditol acetates
1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl-glucitol,
and
1
(
1
5,16
of periodate-oxidized,
4
NaBH reduced, methylated polysaccha-
ride showed the peaks of 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl-
glucitol and 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl-glucitol in a mo-
lar ratio of nearly 1:1. This result indicated that terminal D-glucosyl
Long range 13C– H HMBC experiment confirmed the above se-
quences deduced from NOESY experiment. The cross-peaks of both
anomeric proton and carbon of the sugar moiety were examined
and both inter- and intra-residual connectivities were observed
from the HMBC experiment (Fig. 5, Table 3). Cross-peaks were
found between H-1 (4.54 ppm) of residue A and C-6 (69.3 ppm)
1
moiety was consumed during periodate oxidation, while (1?3,4)-
linked, (1?3)-linked residues were retained.
13
The C NMR spectrum (Fig. 6, Table 4) of PS-II was carried out
at 27 °C. The anomeric carbon signal at 103.6 ppm confirmed the b-
0
0
of residue A (A H-1, A C-6) and C-1 (d 103.4 ppm) of residue A
and H-6a (4.24 ppm) and H-6b (3.87 ppm) of residue A (A C-1,
A H-6a and A C-1, A H-6b).
Thus, from all these observations the structure of the repeating
unit of the glucan (PS-I) was established as:
conformation of the
D-glucopyranosyl moieties. The anomeric
0
carbon signal of terminal, (1?3,4)-linked, and (1?3)-linked
D-glu-
0
0
copyranosyl moieties appeared at the same position (103.6 ppm)
and the moieties were designated as X, Y, and Z, respectively. C-
2
to C-6 values of residue X corresponds nearly to the standard
1
7,18
methyl glycosides.
anosyl moiety.
It was assigned as a terminal b-D-glucopyr-
In case of residue Y, the C-3 signal at 87.0 ppm shifted to
0.2 ppm downfield, the C-4 signal at 77.6 ppm shifted to
.0 ppm downfield and the slightly upfield shift of C-2 and C-5 sig-
1
6
nal compared to the standard methyl glycosides confirmed that it
was (1?3,4)-linked b- -glucopyranosyl moiety.
D
1
3
2
.2. Structural investigation of water insoluble glucan (PS-II)
In C spectrum, the C-3 value of residue Z at 87.3 ppm shifted
1
0.5 ppm downfield compared to the standard methyl glycosides
The molecular weight of the glucan (PS-II) was determined from
indicated that it was (1?3)-linked b-D-glucopyranosyl moiety.
1
1
a
calibration curve prepared with standard dextrans
ꢂ2.0 ꢃ 10 Da. Paper chromatographic analysis of the hydrolyzed
as
Since, residue Y is the most rigid part of the backbone of the glucan,
its C-3 value (d 87.0 ppm) appeared at the upfield compared to the
C-3 value (87.3 ppm) of residue Z. Therefore, based on all the
5
12
PS-II was performed and showed only one spot of glucose. The pres-
Figure 4. The NOESY spectrum of PS-I isolated from fruit bodies of hybrid mushroom. The NOESY mixing time was 300 ms.

1
10
R. Sarkar et al. / Carbohydrate Research 347 (2012) 107–113
Figure 5. The HMBC spectrum of PS-I isolated from fruit bodies of hybrid mushroom The delay time in the HMBC experiment was 80 ms.
Table 3
The significant 3
JH,C connectivities observed in a HMBC spectrum for the anomeric protons/carbons of the sugar residues of PS-I of the fruit bodies of hybrid mushroom
Residue
A
Sugar linkage
H-1/C-1 d
H
/d
C
(ppm)
Observed connectivities
Residue
d
H
/d
C
(ppm)
Atom
0
?6)-b-
D
-Glcp-(1?
4.54
69.3
A
C-6
7
5.4
4.24
A
A
A
A
A
C-5
0
0
1
03.4
H-6a
H-6b
H-5
H-2
3
3
3
.87
.52
.34
Figure 6. 13C NMR spectrum (125 MHz, D
Figure 7. In vitro activation of peritoneal macrophage stimulated with different
2
O, 27 °C) of PS-II, isolated from fruit
bodies of hybrid mushroom.
concentrations of the polysaccharide (PS-I) in terms of NO production.
Table 4
The chemical shifts in the 13C NMRa spectrum of PS-II, a water-insoluble glucan
isolated from the fruit bodies of hybrid mushroom in Me
2
6
SO-d at 27 °C
Residue Sugar linkage C-1 C-2 C-3
C-4
C-5
C-6
b-
b-
D
D
-Glcp-(1? (lit.14
-Glcp-(1?
)
104.0 74.1 76.8 70.6 76.8 61.8
103.6 74.6 77.2 70.8 76.5 61.8
X
Y
Z
?3,4)-b-
?3)-b- -Glcp-(1?
D
-Glcp-(1? 103.6 73.4 87.0 77.6 75.4 61.8
103.6 73.7 87.3 69.3 76.9 61.8
D
a
Values of chemical shifts were recorded with reference to acetone as internal
standard and fixed at d 31.05 ppm at 27 °C.
2
.3. Biological characterization
chemical and spectroscopic evidence, the structure of trisaccha-
Some biological studies were carried out with PS-I. Macrophage
rides repeating unit of water-insoluble b-glucan was established
as:
activation of the polysaccharide was observed in vitro. Upon treat-
ment with different concentrations of this PS-I, enhanced produc-

R. Sarkar et al. / Carbohydrate Research 347 (2012) 107–113
111
Figure 9. Ability of PS-I against DPPH radical. Results were presented as
Means ± SD (n = 3).
Figure 8. Effect of different concentrations of the polysaccharide (PS-I) on
splenocyte (A) and thymocyte (B) proliferation.
binding of polysaccharides to cells activate nuclear transcription
2
7,28
factors such as NF-
jB
which translocates to nucleus and en-
hances transcription as well as translation of genes required for
proliferation or NO production. Biological processes like this prolif-
eration and NO production needs one sufficient strength of signal
at the cell surface level for optimum down stream effect and the
strength of signal depends on various factors like cell type, avail-
ability and abundance of specific receptors on the cell surface, nat-
ure of stimulant (polysaccharide). Again strength of stimuli
generated by polysaccharides depends on their molecular mass,
the degree of branching, conformation and chemical modifica-
tion of NO was observed in a dose-dependent manner with opti-
5
mum production of 8.2
l
M NO per 5 ꢃ 10 macrophages at
5
0
l
g/mL (Fig. 7). As depicted from the Figure 7, there is no in-
crease of NO production at concentrations 1 and 10 g/mL. The
NO production increases at 25 g/mL by 10% which was further in-
creased by 50% at 50 g/mL and decreased thereafter at 100 g/mL.
Hence, the effective dose of PS-I was observed at 50 g/mL. From
l
l
l
l
l
2
9,30
tion.
These factors may be responsible for the observed effects
our previous experiences, it is evident that biological phenomenon
like NO production needs an optimum concentration for optimum
activity below which it is incapable of producing sufficient
strength of stimulus and above which it may produce some inhib-
of PS-I in present study that induce maximum NO production at
higher concentrations but stimulates splenocyte and thymocyte
proliferation at lower concentrations. Further studies on mecha-
nism of action of PS-I are needed to get more insight on its action.
It is note worthy to mention that several mushroom polysaccha-
rides also showed similar type of immunostimulation as reported
itory signals as observed in our previous reports.1
9–22
In this pres-
ent case of PS-I similar trend of effect is observed in a dose
dependent manner.
3
1,32
in our previous works.
Splenocyte and thymocyte activation tests were carried out in
mouse cell culture medium with PS-I by the MTT [3-(4,5-dimethyl-
Figure 9 demonstrated DPPH scavenging activity33 caused by
different concentrations of PS-I. A maximum of 66.5% DPPH radical
scavenging activity was observed at 8 mg/mL of PS-I. The EC50 va-
lue of the PS-I for DPPH radicals was 2.5 mg/mL. The scavenging
activity of the PS-I increased steadily from 0.5 to 6 mg/mL, while
it reached a maximum plateau from 0.5 to 2 mg/mL for ascorbic
acid, which indicates the scavenging activity of PS-I against DPPH
radical was less than that of ascorbic acid.
2
3
thiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. Prolif-
eration of splenocytes and thymocytes is an indicator of
immunostimulation. PS-I was found to stimulate splenocytes and
thymocytes as shown in Figure 8A and B, respectively. The spleno-
cyte and thymocyte proliferation index as compared to phosphate
buffer saline (PBS) control either closer to 1 or below indicated low
stimulatory effect on the immune system. At 10
splenocyte and thymocyte proliferation index were found maxi-
mum as compared to other concentrations. Hence, 10 g/mL of
lg/mL of PS-I,
l
3. Conclusion
PS-I can be considered as an efficient splenocyte and thymocyte
stimulator.
Water-soluble (PS-I) and insoluble (PS-II) glucans were isolated
from alkaline extract of fruit bodies of a hybrid mushroom. The
water-soluble polysaccharide was purified by gel-filtration chro-
matography which showed high splenocyte, thymocyte and mac-
rophage activation as well as antioxidant property in a particular
concentration. The water insoluble polysaccharide was purified
by repeated precipitation in alcohol. On the basis of chemical and
NMR analysis, the structure of the repeating unit of the polysac-
charides was established as:
From these experiments it was observed that NO production
occured at higher concentration of PS-I than that of splenocytes
and thymocytes. The possible reasons of differences in effective
concentration of NO production to that of splenocytes and thymo-
cytes proliferations may be due to the following reasons. Dectin-1,
CR3 and TLRs are the receptors at the cell surface to which polysac-
charides like glucans bind for producing a down-stream signal like
proliferation and NO production.2
4–26
Further there are reports that

1
12
R. Sarkar et al. / Carbohydrate Research 347 (2012) 107–113
4
.3. Test for macrophage activity by nitric oxide assay
5
ꢁ1
Peritoneal macrophages (5 ꢃ 10 cells mL ) after harvesting
were cultured in complete RPMI (Rose well Park Memorial Insti-
3
8,39
tute) media in 96-well plates.
tested by adherence to tissue culture plates. The polysaccharide
was added to the wells in different concentrations (1–100 g/
mL). The cells were cultured for 24 h at 37 °C in a humidified 5%
CO incubator. Production of nitric oxide was estimated by mea-
suring nitrite levels in cell supernatant with Greiss reaction.
The purity of macrophages was
l
2
40
Equal volumes of Greiss reagent (1:1 of 0.1% in 1-napthylethylen-
ediamine in 5% phosphoric acid and 1% sulfanilamide in 5% phos-
phoric acid) and sample cell supernatant were incubated
together at room temperature for 10 min. Absorbance was ob-
served at 550 nm. Lipopolysaccharide (LPS), L6511 of Salmonella
enterica serotype typhimurium (sigma, St. Louis, USA) was used
as positive control.
4
4
. Experimental
.1. Isolation and purification of the polysaccharide
Hybrid mushroom was cultivated and collected from Falta
4
.4. Splenocyte and thymocyte proliferation assay39,41
A single cell suspension of spleen and thymus was prepared
campus, Bose Institute, Kolkata 700054. Fresh fruit bodies of hy-
brid mushroom (600 g) were washed with distilled water and
then boiled with 6% NaOH for 45 min. The crude polysaccharide
from the normal mice under aseptic conditions by frosted slides
in PBS (Phosphate Buffered Saline). The suspension was centri-
fuged to obtain cell pellet. The contaminating red blood cells were
removed by hemolytic Gey’s solution. After washing two times in
PBS the cells were resuspended in complete RPMI medium. Cell
34
was isolated by applying the methods reported earlier, yield
00 mg (water-soluble) and 900 mg (water-insoluble). Purifica-
tion of the water-soluble polysaccharide was carried out by
gel-permeation chromatography on Sepharose 6B column
3
5
concentration was adjusted to 1 ꢃ 10 cells/mL and viability of
a
the suspended cells (as tested by trypan blue dye exclusion) was
(
90 ꢃ 2.1 cm) using a Redifrac fraction collector loading 30 mg
always over 90%. The cells (180
tomed plates and incubated with 20
(1–100 g/mL) of the polysaccharide. The same lipopolysaccharide
l
L) were plated in 96-well flat-bot-
crude polysaccharide for each run. Ninety five Test tubes (each
lL of various concentrations
contain 2 mL eluant) were collected and monitored by the phe-
nol–sulfuric acid procedure35 at 490 nm using Shimadzu UV–
l
as used in macrophage activation was also used here as positive
control. The cultures were set-up for 72 h at 37 °C in a humidified
vis spectrophotometer, model-1601. A single fraction (test tube
nos. 16–28) was collected and freeze–dried, yielding 14 mg of
material. The purification process was carried out for several lots
and polysaccharide fraction was again purified and collected. The
water insoluble polysaccharide (PS-II) was purified dissolving in
2
atmosphere of 5% CO . Proliferation was checked by MTT assay
2
3
method. Data were reported as the mean ± standard deviation
of five different observations and compared against PBS control.
6
% NaOH and repeated precipitation in alcohol (1:5 v/v) followed
4
.5. DPPH radical scavenging activity of PS-I
by centrifugation.
The antioxidant activity of the PS-I was measured on the basis
4
.2. General methods
of the scavenging activity of the stable 1,1-diphenyl-2-picryl
hydrazyl (DPPH) free radical test according to the method de-
The optical rotation was measured on a Jasco polarimeter,
3
3
scribed by Yen et al. with some modification. Samples were dis-
solved in distilled water at 0.2, 0.5, 1, 2, 4, and 8 mg/mL. One
milliliter test sample was mixed with two milliliter of freshly pre-
pared DPPH (0.1 mM) in 50% ethanol. After shaking vigorously, the
mixture was incubated at 25 °C for 30 min in the dark, and then the
absorbance was measured at 517 nm. Lower absorbance of the
reaction mixture indicated higher free radical scavenging activity.
Ascorbic acid was used as a standard antioxidant material. The
scavenging activity of the DPPH radicals was calculated as:
model P-1020 at 25 °C. For monosaccharide analysis, the poly-
saccharide sample (3.0 mg) was hydrolyzed with 2 M CF COOH
2 mL), and the analysis was carried out as described earlier.
The molecular weight of the polysaccharide was determined as
3
3
4
(
1
1,34
12
reported earlier.
Paper chromatographic was carried out
as described in our previous papers.8 The absolute configuration
of the monosaccharide constituent was assigned according to
1
3
Gerwig et al. The polysaccharide was methylated according
14
to Ciucanu and Kerek method.
GLC) analysis was performed by using a Hewlett-Packard Model
730 A, having a flame ionization detector and glass columns
Gas liquid chromatographic
(
5
Q ¼ ðA
0
ꢁ A
c
Þ=A
0
ꢃ 100%
Q = percentage reduction of the DPPH. Where A
0
= initial absor-
(
(
1.8 m ꢃ 6 mm) packed with 3% ECNSS-M (A) on Gas Chrom Q
bance. A = absorbance after added sample concentration c.
c
100–120 mesh) and 1% OV-225 (B) on Gas Chrom Q (100–
1
20 mesh). All GLC analyses were performed at 170 °C. Gas li-
Acknowledgments
quid chromatography–mass spectrometric (GLC–MS) analysis
was also performed on Shimadzu GLC–MS Model QP-2010 Plus
automatic system, using ZB-5MS capillary column (30 m ꢃ
The authors are grateful to Prof. S. Roy, Director, IICB, Dr. A.K.
Sen (Jr.), IICB Kolkata, for providing instrumental facilities. Mr. Bar-
un Majumder of Bose Institute, Kolkata, is acknowledged for
preparing NMR spectra. DST, Govt of India is acknowledged for
sanctioning a project (Ref. No: SR/S1/OC- 52/2006 dated 19/02/
2007). One of the authors (R.S.) is grateful to UGC, New Delhi for
offering junior research fellowship.
0
5
.25 mm). The program was isothermal at 150 °C; hold time
min, with a temperature gradient of 2 °C/min up to a final
temperature of 200 °C. Periodate oxidation was performed as de-
scribed in an earlier report.34 NMR experiments were carried out
34,36,37
as reported in our previous papers.
iment was carried out at 27 °C.
DEPT-135 NMR exper-

R. Sarkar et al. / Carbohydrate Research 347 (2012) 107–113
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