Chemical analysis of an immunoenhancing water-soluble polysaccharide of an edible mushroom, Pleurotus florida blue variant

Article abstract of DOI:10.1016/j.carres.2010.09.032

An immunoenhancing polysaccharide isolated from the aqueous extract of the fruit bodies of the mushroom, Pleurotus florida blue variant, was found to consist of d-glucose and d-galactose in a molar ratio of nearly 5:1. On the basis of sugar analysis, methylation analysis, periodate oxidation, Smith degradation, and NMR studies (¹H, ¹³C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of the polysaccharide was established as: The molecule activated macrophages, splenocytes, and thymocytes.

Full text of DOI:10.1016/j.carres.2010.09.032

Carbohydrate Research 345 (2010) 2736–2741
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Chemical analysis of an immunoenhancing water-soluble polysaccharide
of an edible mushroom, Pleurotus florida blue variant
Biswajit Dey a, Sanjoy K. Bhunia a, Kankan K. Maity a, Sukesh Patra a, Soumitra Mandal a, Swatilekha Maiti b,
Tapas K. Maiti b, Samir R. Sikdar c, Syed S. Islam a,
⇑
a Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore 721 102, West Bengal, India
b Department of Biotechnology, Indian Institute of Technology (IIT) Kharagpur, Kharagpur 721 302, West Bengal, India
c Plant Molecular and Cellular Genetics Section, Bose Institute, Kolkata 700 054, West Bengal, India
a r t i c l e i n f o
a b s t r a c t
Article history:
An immunoenhancing polysaccharide isolated from the aqueous extract of the fruit bodies of the mush-
room, Pleurotus florida blue variant, was found to consist of D-glucose and D-galactose in a molar ratio of
nearly 5:1. On the basis of sugar analysis, methylation analysis, periodate oxidation, Smith degradation,
and NMR studies (1H, 13C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC), the structure
of the repeating unit of the polysaccharide was established as:
Received 12 August 2010
Received in revised form 22 September
2010
Accepted 28 September 2010
Keywords:
Pleurotus florida blue variant
Mushroom polysaccharide
Structure
NMR spectroscopy
Immunoactivation
The molecule activated macrophages, splenocytes, and thymocytes.
Ó 2010 Elsevier Ltd. All rights reserved.
Mushrooms are used as food materials with unique flavor and
texture, and are recognized as an important source of biologically
active compounds,1 especially polysaccharides for their immuno-
modulatory and anticancer properties.2 Among different types
of oyster mushrooms of the genus Pleurotus, mainly Pleurotus
sajor-caju,3–5 Pleurotus ostreatus,6 Pleurotus citrinopileatus,7 and
Pleurotus florida,8 are reported as commonly available edible mush-
rooms that contain immunomodulating and antitumor materials.
The hot water extract of the edible mushroom P. florida was found
to consist of different water-soluble and water-insoluble polysac-
charides, which were isolated by our group and reported.9–12 Water
soluble and insoluble polysaccharides were also isolated fromP. flor-
ida, cultivar (Assam Florida) and reported recently.13,14 We report
herein a new type of water-soluble polysaccharide isolated from
the hot water extract of P. florida, blue variant. The main objective
of this study was to investigate any differences that arise in the con-
stituents of polysaccharide of this variant from its original mush-
room P. florida. The present polysaccharide is a galactosyl-glucan
consisting of D-glucose and D-galactose in a molar ratio of nearly
5:1. We report the detailed structural characterization of the mole-
cule as well as some of its immunoenhancing properties, such as
macrophage, splenocyte, and thymocyte activation.
The polysaccharide (PS) on acid hydrolysis by 2 M CF3COOH, fol-
lowed by GLC analysis showed the presence of glucose and galactose
in a molar ratio of nearly 5:1. The absolute configuration15 of the su-
gar residues was determined as D. Its molecular mass,16 determined
from a calibration curve prepared with standard dextrans, was
found to be ꢀ1.74 Â 105 Da. The pure polysaccharide showed a spe-
cific rotation of [
a
]
D À4.11 (c 0.11, H2O, 26.8 °C). The linkages in the
polysaccharide were determined by methylation analysis using the
method of Ciucanu and Kerek,17 followed by hydrolysis and alditol
acetate preparation. The GLC and GLC–MS analyses of partially methyl-
ated alditol acetates revealed the presence of 1,5-di-O-acetyl-2,3,4,
6-tetra-O-methyl-
acetyl-2,4,6-tri-O-methyl-
1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-
129); and 1,3,5,6-tetra-O-acetyl-2,4-di-O-methyl-
43, 87, 117, 129, 189) in a molar ratio of nearly 1:1:3:1. These results
indicated that terminal -galactopyranosyl, (1?3)-linked -gluco-
pyranosyl, (1?6)-linked -glucopyranosyl, and (1?3, 6)-linked
D
-galactitol (m/z 101, 117, 129, 161); 1,3,5-tri-O-
-glucitol (m/z 43, 101, 117, 129, 161);
-glucitol (m/z 99, 101, 117,
-glucitol (m/z
D
D
D
⇑
Corresponding author. Tel.: +91 03222 276558x437, mobile: +91 9932629971;
fax: +91 03222 275329.
D
D
D
E-mail address: sirajul_1999@yahoo.com (S.S. Islam).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.09.032

B. Dey et al. / Carbohydrate Research 345 (2010) 2736–2741
2737
D
-glucopyranosyl moieties were present in the polysaccharide in a
molar ratio of nearly 1:1:3:1. Further, GLC analysis of the alditol ace-
tates of the periodate-oxidized,18,19 NaBH4-reduced PS showed the
presence of
methylated PS exhibited the presence of 1,3,5,6-tetra-O-acetyl-2,4-
di-O-methyl- -glucitol and 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl-
glucitol in a molar ratio of nearly 1:1. These results indicated that
(1?6)-linked -glucopyranosyl and terminal -galactopyranosyl
moieties were destroyed during oxidation. Hence, these observa-
tions confirmed the mode of linkages of these sugar moieties present
in the PS.
D-glucose only. The periodate-oxidized, NaBH4-reduced,
D
D-
D
D
Figure 2. 13C NMR spectrum (125 MHz, D2O, 27 °C) of the polysaccharide, isolated
from Pleurotus florida blue variant.
The proton NMR spectrum (500 MHz, Fig. 1) of the PS at 27 °C
showed four signals in the anomeric region at d 5.12, 4.98, 4.51,
and 4.50 ppm in a molar ratio of nearly 1:1:1:3. The anomeric sig-
nals at d 5.12, 4.98, 4.51, and 4.50 ppm were assigned as A, B, C,
and D, respectively. In the 13C NMR spectrum (125 MHz, Fig. 2)
at 27 °C four anomeric signals appeared at d 103.4, 103.2, 98.6,
and 98.3 ppm in a molar ratio of nearly 3:1:1:1, corresponded to
anomeric carbons of D, C, A, and B residues, respectively. The re-
sponse of the signal at d 103.4 ppm was almost thrice to those of
other signals, which indicated the presence of three units of resi-
due D. All the 1H and 13C signals (Table 1) were assigned using
DQF-COSY, TOCSY, and HMQC experiments. The proton coupling
constants were measured from DQF-COSY experiment.
the standard values indicated that it was (1?3, 6)-linked moiety.
The downfield shift of C-6 (d 69.2 ppm) of residue D indicated that
it was (1?6)-linked moiety. The linking of residues, C and D at C-6
was also confirmed from DEPT-135 spectrum (Fig. 3). These
observations indicated that residue C was a (1?3, 6)-linked-b-
glucopyranosyl moiety and residue D was a (1?6)-linked-b-
glucopyranosyl moiety.
D
-
-
D
The sequence of glycosyl residues of the PS was determined from
NOESY as well as ROESY experiments followed by confirmation with
an HMBC experiment. In NOESY experiment (Fig. 4, Table 2) the
inter-residual contacts from AH-1 to CH-3, BH-1 to DH-6, CH-1 to
BH-3, and DH-1 to CH-6 and DH-6 established the following
sequences;
Residue A was assigned as terminal D-galactopyranosyl unit.
The galacto configuration was assigned from the large JH-2,H-3 cou-
pling constant (ꢀ8 Hz) and relatively small JH-3,H-4 coupling con-
stant (ꢀ3 Hz). The
a
-configuration of residue A (d 5.12 ppm) was
assigned from J
coupling constant (ꢀ3 Hz) and J
H-1,H-2
C-1,H-1
coupling constant (ꢀ171 Hz). The carbon signal at d 98.6 ppm
was assigned to C-1 of residue A. The signals from C-1 to C-6
corresponded nearly to the standard values of methyl glyco-
sides.20,21 These results indicated that residue A was an
a-linked,
terminal
The large JH-2,H-3 and JH-3,H-4 coupling constant values (ꢀ10 Hz)
of residues B, C, and D confirmed their -glucopyranosyl configura-
D-galactopyranosyl moiety.
D
tion. Residue B had an anomeric proton signal at d 4.98 ppm, and
the coupling constants JH-1,H-2 (ꢀ3 Hz) and JC-1,H-1 (ꢀ170 Hz), indi-
In the HMBC spectrum (Fig. 5, Table 3) the cross-peaks of both
anomeric protons and carbons of each of the glycosyl residues
were examined, and both intra and inter-residue connectivities
were observed. Cross-peaks were found between H-1 of residue
A and C-3 of residue C (AH-1, CC-3); C-1 of residue A and H-3 of
residue C (AC-1, CH-3) with other intra-residue coupling between
H-1 of residue A with its own C-2 atom (AH-1, AC-2). Similarly,
cross-peaks between H-1 of residue B and C-6 of residue D (BH-
1, DC-6); C-1 of residue B and H-6a, H-6b of residue D (BC-1,
DH-6a; BC-1, DH-6b) were observed. Cross-peaks were also ob-
served between H-1 of residue D and C-6 of residue C (DH-1, CC-
6); C-1 of residue D and H-6a, H-6b of residue C (DC-1, CH-6a;
DC-1, CH-6b) with other intra-residue coupling between H-1 and
C-1 of residue D with its own C-2, C-6 atoms and H-2, H-3, H-6a
cating its a-configuration. The carbon signal at d 98.3 ppm was as-
signed to C-1 of residue B. The downfield shift of C-3 (d 80.4 ppm)
with respect to standard value indicated that it was (1?3)-linked
residue. The other values of carbons corresponded nearly to the
standard values. Thus, residue B was a (1?3)-linked-a-D-glucopyr-
anosyl moiety.
The coupling constants JH-1,H-2 (ꢀ8 Hz) and JC-1,H-1 (ꢀ160 Hz) of
residues C and D and in addition to their anomeric proton signals
at d 4.51 and 4.50 ppm, respectively, indicated their b-configura-
tion. The anomeric carbon signals of residues C and D appeared
at d 103.2 and 103.4 ppm, respectively. The downfield shifts of C-
3 (d 85.0 ppm) and C-6 (d 68.9 ppm) of residue C with respect to
Figure 1. 1H NMR spectrum (500 MHz, D2O, 27 °C) of the polysaccharide, isolated from Pleurotus florida blue variant.

2738
B. Dey et al. / Carbohydrate Research 345 (2010) 2736–2741
Table 1
The 1H NMRa and 13C NMRb chemical shifts for the polysaccharide isolated from Pleurotus florida blue variant in D2O at 27 °C
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
5.12
98.6
3.89
67.2
3.92
68.5
4.00
69.9
4.08
69.9
3.73c, 3.92d
61.4
4.98
98.3
3.83
69.7
3.86
80.4
3.82
69.2
3.89
73.4
3.69c, 3.89d
61.1
4.51
103.2
3.50
73.3
3.72
85.0
3.44
68.6
3.62
75.3
3.84c, 4.22d
68.9
4.50
103.4
3.31
73.5
3.48
76.6
3.46
69.9
3.61
75.9
3.83c, 4.19d
69.2
a
The values of chemical shifts were recorded keeping HOD signal fixed at d 4.75 ppm at 27 °C.
The values of chemical shifts were recorded with reference to acetone as internal standard and fixed at d 31.05 ppm at 27 °C.
Interchangeable.
b
c,d
To prove information on the sequence of the sugar residues in
the repeating unit, the polysaccharide was subjected to Smith deg-
radation22–24 studies, and the products were separated on a Sepha-
dex 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-glucose
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,
which showed the presence of 1,5-di-O-acetyl-2,3,4,6-tetra-O-
Figure 3. DEPT-135 spectrum (D2O, 27 °C) of the polysaccharide, isolated from
Pleurotus florida blue variant.
methyl-D-glucitol and 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl-D-gluc-
itol in a molar ratio nearly 1:1. The 125-MHz 13C NMR experiment
(Fig. 6, Table 4) of SDPS showed two anomeric carbon signals at d
102.8 and 98.7 ppm corresponding to b-
-Glcp-(1? (B) residues, respectively. The carbon signals C-1, C-2,
and C-3 of the glycerol moiety were assigned as 66.2, 72.4, and
62.8 ppm, respectively. The nonreducing end b- -Glcp unit (F)
was generated from (1?3, 6)-b- -Glcp (residue C) during Smith
degradation of the terminal -Galp moiety (A). The (1?3)-
Glcp residue (B) was retained and found attached to a glycerol moi-
ety (G). The glycerol moiety was generated from (1?6)-b- -Glcp
D-Glcp-(1? (F) and ?3)-a-
D
D
D
a-
D
a-D-
D
(D) after periodate oxidation, followed by Smith degradation.
Hence, Smith degradation results in the formation of an
Table 2
NOESY data for the polysaccharide isolated from Pleurotus florida blue variant
Glycosyl residue
Anomeric proton
NOE contact proton
d
d
Residue
Atom
Figure 4. Part of NOESY spectrum of polysaccharide, isolated from Pleurotus florida
blue variant. The NOESY mixing time was 300 ms.
5.12
4.98
3.72
3.89
3.92
C
A
A
H-3
H-2
H-3
atoms (DH-1, DC-2; DH-1, DC-6; DC-1, DH-2; DC-1, DH-3; DC-1,
DH-6a), respectively. Cross-peaks between H-1 of residue C and
C-3 of residue B (CH-1, BC-3); C-1 of residue C and H-3 of residue
B (CC-1, BH-3) with other intra-residue coupling between H-1 and
C-1 of residue C with its own C-2 atom and H-2, H-3 atoms (CH-1,
CC-2; CC-1, CH-2; CC-1, CH-3), respectively, were also found. Thus,
the HMBC and NOESY connectivities clearly supported the pres-
ence of the following repeating unit in the PS isolated from the
mushroom, P. florida blue variant as:
3.83
3.86
3.89
3.69
3.83
4.19
B
B
B
B
D
D
H-2
H-3
H-6b
H-6a
H-6a
H-6b
4.51
4.50
3.62
3.50
3.72
3.86
C
C
C
B
H-5
H-2
H-3
H-3
3.31
3.48
D
D
H-2
H-3
3.61
3.84
4.22
3.83
4.19
D
C
C
D
D
H-5
H-6a
H-6b
H-6a
H-6b

B. Dey et al. / Carbohydrate Research 345 (2010) 2736–2741
2739
Figure 5. HMBC spectrum of polysaccharide isolated from Pleurotus florida blue variant. The delay time in the HMBC experiment was 80 ms.
Table 3
3
The significant JH,C connectivities observed in an HMBC spectrum for the anomeric
protons/carbons of the sugar residues of the polysaccharide isolated from Pleurotus
florida blue variant
Residue
Sugar linkage
H-1/C-1
dH/dC
Observed connectivities
dH/dC
Residue
Atom
A
a
-
D
-Galp-(1?
5.12
85.0
67.2
C
A
C
C-3
C-2
H-3
Figure 6. 13C NMR spectrum (125 MHz, D2O, 27 °C) of the Smith-degraded
Polysaccharide isolated from Pleurotus florida blue variant.
98.6
3.72
69.2
3.83
4.19
80.4
73.3
3.86
B
C
?3)-
a
-
D-Glcp-(1?
4.98
98.3
D
D
D
C-6
H-6a
H-6b
Table 4
?3,6)-b-
D-Glcp-(1?
4.51
B
C
B
C
C
C-3
C-2
H-3
H-2
H-3
The 13C NMRa chemical shifts of Smith-degraded polysaccharide isolated from
Pleurotus florida blue variant in D2O at 27 °C
103.2
3.50
3.72
Glycosyl residue
C-1
C-2
C-3
C-4
C-5
C-6
D
?6)-b-
D-Glcp-(1?
4.50
68.9
C
C-6
102.8
73.5
76.5
70.9
76.5
61.5
73.5
69.2
3.84
4.22
3.31
3.48
3.83
D
D
C
C-2
C-6
98.7
66.2
70.9
72.4
79.1
62.8
68.6
72.4
61.1
103.4
H-6a
H-6b
H-2
H-3
H-6a
C
D
D
D
a
The values of chemical shifts were recorded with reference to acetone as
internal standard and fixed at d 31.05 ppm at 27 °C.
oligosaccharide (SDPS) from the parent polysaccharide and the
structure was established as:
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.25 The polysaccharide was tested to
proliferate splenocytes and thymocytes and results are shown in
Figure 8A and B, respectively, and the asterisks on the columns
indicate the statistically significant differences compared to PBS
control. The splenocyte proliferation index (SPI) as compared to
PBS (Phosphate Buffered Saline) control closer to 1 or below indi-
Therefore on the basis of above results, the structure of the
repeating unit of the polysaccharide was confirmed.
Macrophage activation by the polysaccharide was observed
in vitro. On treatment with different concentrations of this poly-
saccharide, an enhanced production of NO was observed in a dose
dependent manner with optimum production of 8.1
lg/mL, and subsequently decreased
with further increase in concentration (Fig. 7). Hence, the effective
l
M NO per
cates low stimulatory effect on immune system. At 50
the polysaccharide, splenocyte proliferation index was found max-
imum as compared to other concentrations. Hence, 50 g/mL of
the polysaccharide can be considered as efficient splenocyte
lg/mL of
5 Â 105 macrophages at 10
l
dose of this polysaccharide was observed at 10 lg/mL.

2740
B. Dey et al. / Carbohydrate Research 345 (2010) 2736–2741
pharose 6B column. A single fraction (test tubes 18–45) was ob-
served and collected through lypholyzation, yielding 15 mg of
material. The purification process was carried out in seven lots
(yield; 105 mg).
1.2. General methods
The 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.26 The molecularweight
of the polysaccharide was determined as reported earlier.16,26 The
absolute configuration of the monosaccharide constituent was as-
signed according to Gerwig et al.15 The polysaccharide was methyl-
ated according to Ciucanu and Kerek method.17 Gas-liquid
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  0.25 mm). The pro-
gram was isothermal at 150 °C; hold time 5 min, with a temperature
gradient of 2 °C/min up 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. Perio-
date oxidation was performed with this polysaccharide as described
in the earlier report.18,19,26 NMR experiments were carried out as re-
ported in our previous papers.26,28,29 DEPT-135 NMR experiment
was carried out at 27 °C.
Figure 7. In vitro activation of peritoneal macrophage stimulated with different
concentrations of the polysaccharides in terms of NO production.
1.3. Test for macrophage activity by nitric oxide assay
Peritoneal macrophages (5 Â 105 cells/mL) after harvesting
were cultured in complete RPMI (Roswell Park Memorial Institute)
media in 96-well plates.30,31 The 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% CO2 incubator. Produc-
tion of nitric oxide was estimated by measuring nitrite levels in
cell supernatant with Greiss reaction.32 Equal 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 serovar Typhimurium was used
as positive control.
Figure 8. Effect of different concentrations of the polysaccharide on splenocytes (A)
and thymocytes (B) proliferation. (* Significant compared to the PBS control.)
proliferator. Again, 25 lg/mL of the same sample showed maximum
1.4. Splenocyte and thymocyte proliferation assay31,33
effect on thymocyte proliferation. It is noteworthy to mention that
similar kind of activations was observed with mushroom polysac-
charides9,10,13 reported previously by our group.
A single cell suspension of spleen and thymus was prepared
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
concentration was adjusted to 1 Â 105 cells/mL and viability of
the suspended cells (as tested by trypan blue dye exclusion) was al-
1. Experimental
1.1. Isolation and purification of the polysaccharide
The fresh fruiting bodies of the mushroom, P. florida blue variant
(500 g) were cultivated and collected from Falta experimental
farm, Bose Institute. The material was first washed with water,
then cut into pieces, and boiled with 250 mL distilled water for
6 h. The whole mixture was then kept overnight at 4 °C and filtered
through a linen cloth. The filtrate was centrifuged at 8000 rpm
(using a Heraeus Biofuge stratos centrifuge) for 30 min at 4 °C
and the crude polysaccharide (wt 180 mg) was isolated as de-
scribed in our previous papers.26,27 The crude polysaccharide
(25 mg) was purified by gel permeation chromatography on Se-
ways over 90%. The cells (180
plates and incubated with 20
100 g/mL) of the polysaccharide. The same lipopolysaccharide
lL) were plated in 96-well flat-bottomed
lL of various concentrations (1–
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
atmosphere of 5% CO2. Proliferation was checked by MTT assay
method.25 The data are reported as the mean standard deviation
of six different observations and compared against PBS control.

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2741
12. Rout, D.; Mondal, S.; Chakraborty, I.; Islam, S. S. Carbohydr. Res. 2008, 343, 982–
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Acknowledgments
13. Roy, S. K.; Das, D.; Mondal, S.; Maiti, D.; Bhunia, B.; Maiti, T. K.; Islam, S. S.
Carbohydr. Res. 2009, 344, 2596–2601.
14. Ojha, A. K.; Chandra, K.; Ghosh, K.; Islam, S. S. Carbohydr. Res. 2010, 345, 2157–
2163.
15. Gerwig, G. J.; Kamerling, J. P.; Vliegenthart, J. F. G. Carbohydr. Res. 1978, 62, 349–357.
16. Hara, C.; Kiho, T.; Tanaka, Y.; Ukai, S. Carbohydr. Res. 1982, 110, 77–87.
17. Ciucanu, I.; Kerek, F. Carbohydr. Res. 1984, 131, 209–217.
18. Hay, G. W.; Lewis, B. A.; Smith, F. Methods Carbohydr. Chem. 1965, 5, 357–361.
19. Goldstein, I. J.; Hay, G. W.; Lewis, B. A.; Smith, F. Methods Carbohydr. Chem.
1965, 5, 361–370.
The authors are grateful to Dr. S. Roy, Director, and Dr. A. K. Sen
(Jr.), IICB, Kolkata, for providing instrumental facilities. Mr. Barun
Majumdar of Bose Institute, Kolkata, is acknowledged for preparing
NMR spectra. DST, Government of India is acknowledged for sanc-
tioning a project (Ref No: SR/S1/OC-52/2006 dated 19/02/2007).
The author (B.D.) acknowledges CSIR for offering junior research
fellowship.
20. Agrawal, P. K. Phytochemistry 1992, 31, 3307–3330.
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