Structural characterization and study of immunoenhancing properties of a glucan isolated from a hybrid mushroom of Pleurotus florida and Lentinula edodes pot

Accepted Manuscript
Structural characterization and study of immunoenhancing properties of a glu‐
can isolated from a hybrid mushroom of Pleurotus florida and Lentinula edodes
Praloy K. Maji, Ipsita K. Sen, Birendra Behera, Tapas K. Maiti, Pijush Mallick,
Samir R. Sikdar, Syed S. Islam
PII: S0008-6215(12)00277-7
DOI: />Reference: CAR 6220
To appear in: Carbohydrate Research
Received Date: 7 June 2012
Revised Date: 20 June 2012
Accepted Date: 21 June 2012
Please cite this article as: Maji, P.K., Sen, I.K., Behera, B., Maiti, T.K., Mallick, P., Sikdar, S.R., Islam, S.S.,
Structural characterization and study of immunoenhancing properties of a glucan isolated from a hybrid mushroom
of Pleurotus florida and Lentinula edodes, Carbohydrate Research (2012), doi: />2012.06.017
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Structural characterization and study of immunoenhancing properties
of a glucan isolated from a hybrid mushroom of Pleurotus florida and
Lentinula edodes
Praloy K. Maji
a

, Ipsita K. Sen
a
, Birendra Behera
b
, Tapas K. Maiti
b
, Pijush Mallick
c
,
Samir R. Sikdar
c
, Syed S. Islam
a,
*
a
Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore
721102, West Bengal, India
b
Department of Biotechnology, Indian Institute of Technology (IIT) Kharagpur,
Kharagpur 721302, West Bengal, India
c
Division of Plant Biology, Bose Institute, Centenary Building, P-1/12, C.I.T. Scheme
VII M, Kolkata 700054, West Bengal, India

Abstract A water soluble glucan isolated from hot aqueous extract of fruit bodies of an
edible hybrid mushroom Pfle1r of Pleurotus florida and Lentinula edodes showed
macrophages, splenocytes, and thymocytes activation. The glucan consists of terminal,
(1→3,6)-linked, and (1→6)-linked β-D-glucopyranosyl moieties in a molar ratio of nearly
1:1:3. On the basis of acid hydrolysis, methylation, periodate oxidation study, and NMR
studies (

1
H,
13
C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HSQC, and
HMBC), the structure of the repeating unit of the glucan was established as:

6)-D-Glcp-(16)--D-Glcp-(16)--D-Glcp-(16)--D-Glcp-(1
3
↑
1
-D-Glcp


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Keywords: Hybrid mushroom; Glucan; NMR spectroscopy; Immunostimulation

Corresponding auther. Tel.: +91 03222 276558 x 437; +91 9932629971 (M); fax: +91
03222 275329; e mail:

Mushrooms are important for its medicinal value.
1
Mushroom polysaccharides have
gained importance because of their immunomodulatory
2

, free radical scavenging
3,4
, and
antitumor
5,6
activity. Various immunoenhancing polysaccharides from edible
mushrooms
7-9
and hybrid mushrooms
10-12
were reported by our group. Four different
polysaccharides isolated from Pleurotus florida
13-16
were also reported by our group.
Lentinan, a biologically active water insoluble polysaccharide from Lentinula edodes
containing (1→3), (1→6)-β-D-glucan
17
has been reported and widely used for cancer
therapy. Water soluble polysaccharides
18-20
from L. edodes are also reported. Protoplast
fusion between the strains of Pleurotus florida and Lentinula edodes produced nine new
hybrid strains adopting the procedures as applied earlier
21
out of which six strains pfle 1o,
pfle 1p, pfle 1q, pfle 1r, pfle 1s, and pfle 1v produced fruit bodies. Aqueous extract of the
fruit bodies of one of the hybrid mushroom strains, pfle1r yielded two polysaccharides,
glucan (PS-I) and a heteroglycan (PS-II) consisting of glucose, mannose, and galactose.
Structural investigation of PS-I showed that it is different from the polysaccharides
isolated from either of the aqueous or alkali extract of parent mushrooms Pleurotus

florida and Lentinula edodes. The structural characterization and immunoenhancing
studies of PS-I isolated from the aqueous extract of fruit bodies of hybrid mushroom
strain Pfle1r has been carried out and reporting herein.
This pure polysaccharide (PS-I) had a specific rotation []
D
30
–12

(c 0.8, water).
Molecular weight
22
of PS-I was estimated as ~1.80  10
5
Da from a calibration curve

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prepared with standard dextran. PS-I was hydrolyzed with 2M trifluroacetic acid and then
alditol acetate
23
was prepared for GLC analysis. GLC analysis of alditol acetate of
hydrolyzed product of PS-I confirmed the presence of glucose only. The absolute
configuration of the glucose residue was determined as D by the method of Gerwig et
al.
24

This PS-I was methylated according to Ciucanu and Kerek
25
method followed by
hydrolysis and then alditol acetate was prepared to know the linkages of sugar moieties.
The GLC-MS analysis of the partially methylated alditol acetate of PS-I revealed the
presence of 1,3,5,6-tetra-O-acetyl-2,4-di-O-methyl-glucitol, 1,5,6-tri-O-acetyl-2,3,4-tri-
O-methyl-glucitol, and 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-glucitol in a molar ratio of
nearly 1:3:1. These results indicated the presence of (1→3,6)-, (1→6)-linked, and
terminal glucopyranosyl residues in the glucan (PS-I). These linkages were further
confirmed by periodate oxidation experiment. GLC analysis of alditol acetates of the
periodate-oxidized,
26,27
NaBH
4
-reduced PS-I was found to contain glucose only and
periodate-oxidized, reduced, methylated
28
PS-I exhibited the presence of 1,3,5,6-tetra-O-
acetyl-2,4-di-O-methyl glucitol. These results showed that (1→6)-linked and terminal
glucopyranosyl moieties were consumed during oxidation. Hence, these observations
confirmed the mode of linkages of these sugar moieties present in the PS-I.
Two signals were observed in the anomeric region of the
1
H NMR spectrum (500
MHz; Fig. 1) at δ 4.51 and 4.49 ppm at 30 C.
13
C NMR spectrum (125 MHz; Fig. 2a)
showed three signals in the anomeric region at δ 103, 102.9, and 102.7 ppm at the same
temperature. So, two anomeric proton signals consists of three sugar residues, designated
as A, B and C. On the basis of HSQC spectrum, the anomeric proton signal at δ 4.51 ppm

was correlated to both the carbon signals at δ 102.7 ppm and δ 102.9 ppm, corresponded

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to anomeric carbons of A and B residues respectively. Again, the proton signal at δ 4.49
ppm was correlated to carbon signal at δ 103.0 ppm, corresponded to anomeric carbon of
residue C. The response of the signal at δ 103.0 ppm was almost three times with respect
to other anomeric carbon signals, indicating the presence of three units of residue C. All
the
1
H and
13
C signals (Table 1) were assigned from DQF-COSY, TOCSY, and HSQC
experiments. The proton coupling constants were measured from DQF-COSY
experiment.
The large J
H-2,H-3
and J
H-3,H-4
coupling constant values (~10 Hz) confirmed
glucopyranosyl configuration (Glcp) of all the residues from A-C. In case of all residues
(A-C), the coupling constants J
H-1,H-2
(~8 Hz) and J
C-1,H-1

(~160-161 Hz), anomeric proton
chemical shifts (4.51-4.49 ppm) and anomeric carbon chemical shifts (103.0-102.7 ppm)
confirmed their β-configuration. The downfield shifts of C-3 at δ 84.2 ppm and C-6 at δ
68.7 ppm of residue A with respect to the standard values of methyl glycosides
29,30
indicated that (1→3,6)-β-D-Glcp was present in the PS-I . Since, residue A was the most
rigid part of the backbone of this glucan, it’s C-6 (68.7 ppm) appeared at the upfield
region in comparison to that of the other (1→6)-linked residues (C). Among the three C
residues, one moiety (C
I
) was glycosidically linked to the residue A, hence, its C-6 signal
(69 ppm) showed 0.2 ppm downfield shift with respect to that of another two residues of
C
II
(68.8 ppm) due to neighbouring effect
7,31
of the rigid part ‘A’. The linking at C-6 of
residues A and C was further confirmed from DEPT-135 spectrum (Fig. 2b). The carbon
chemical shifts of residue B from C-1 to C-6 corresponded nearly to the standard values
of methyl glycoside of β-D-glucose. Thus, residue B was established as terminal β-D-
Glcp.

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The sequences of glucosyl moieties were determined from ROESY as well as NOESY

(not shown) experiments. In ROESY experiment (Fig. 3, Table 2), the inter-residual
contacts AH-1/C
I
H-6a, C
I
H-6b; C
II
H-1/AH-6a, AH-6b; C
I
H-1/C
II
H-6a, C
II
H-6b; and
BH-1/AH-3 along with some other intra residual contacts were also observed. The above
ROESY connectivities established the following sequences:
A C
I
C
II
A
→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→ ; →6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→
3 3
↑ ↑



C
I
C

II
A
→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→ ; →6)-β-D-Glcp-(1→
3
↑
1
β-D-Glcp
B

A long range HMBC experiment was carried out to confirm the ROESY
connectivities. In HMBC experiment (Fig. 4, Table 3), inter residual couplings AH-
1/C
I
C6, AC-1/C
I
H-6a, C
I
H-6b; C
II
H-1/AC-6, C
II
C-1/AH-6a, AH-6b; C
I
H-1/C
II
C-6,
C
I
C-1/C
II

H-6a, C
II
H-6b; BH-1/AC-3, BC-1/AH-3 along with some intra residual
couplings were also observed. Thus, the HMBC and ROESY connectivities confirmed
the presence of the following pentasaccharide repeating unit in the glucan isolated from
hybrid mushroom strain Pfle1r of Pleurotus florida and Lentinula edodes as;

C
II
C
II
A C
I

6)-D-Glcp-(16)--D-Glcp-(16)--D-Glcp-(16)--D-Glcp-(1
3
↑
1
-D-Glcp
B

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Some immunological studies were also investigated with the glucan (PS-I).

Macrophage activation of the PS-I was observed in vitro. On treatment with different
concentrations of the PS-I an enhanced production of NO was observed in a dose-
dependent manner with optimum production of 34 µM NO per 5 x 10
5
macrophages at
100 µg/mL of the PS-I (Fig. 5a).
Splenocytes are the cells present in the spleen that include T cells, B cells, dendritic
cells, and macrophages that stimulate the immune response in living organism.
Thymocytes are hematopoietic cells in thymus which generate T cells. The splenocytes
and thymocytes activation tests were carried out in mouse cell culture medium with the
PS-I by the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
method.
32
Proliferation of splenocytes and thymocytes is an indicator of
immunostimulation. The splenocyte and thymocyte proliferation index as compared to
Phosphate Buffer Saline (PBS) control if closer to 1 or below indicates low stimulatory
effect on immune system. The PS-I was found to stimulate splenocytes and thymocytes
as shown in Fig. 5b and c respectively and the asterisks on the columns indicate the
statistically significant differences compared to PBS control. Maximum proliferation
index of splenocyte and thymocyte was observed at 100 µg/mL and 25 µg/mL of the PS-I
respectively as compared to other concentrations. Hence, 100 µg/mL of the PS-I can be
considered as efficient splenocyte stimulator where as 25 µg/mL of the PS-I acts as
thymocyte stimulator.




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1. Experimental
1.1. Preparation of hybrid mushroom strain produced between Pleurotus florida and
Lentinula edodes
The hybrid mushroom strain pfle1r was produced through polyethyleneglycol (30%
PEG, MW-3350)-mediated somatic protoplast fusion between Pleurotus florida and
Lentinula edodes. Hybrid strains were selected based on double selection method and
afterwards maintained in Potato-Dextrose-Agar medium. Spawn of the hybrid strain was
produced on paddy grain and mushroom was produced on paddy straw substrate.
1.2. Isolation and purification of the polysaccharide
The fresh fruit bodies of an edible hybrid mushroom strain Pfle1r were cultivated and
collected from Falta Experimental Farm, Bose Institute, Kolkata. The fruit bodies (450g)
were washed with water and then with distilled water. The mushroom bodies were
crushed and boiled with water for 6 h. The crude polysaccharide (200 mg) was isolated
and purified (20 mg) by gel-permeation chromatography as described in our previous
papers.
10,11
Two fractions, PS-I (test tube 16–31) and PS-II (test tube 37-47) were
obtained, collected, and freeze-dried, yielding 6 mg and 5 mg pure polysaccharide,
respectively. The purification process was repeated several times, obtaining a total of 60
mg of PS-I.
1.3. General methods
The molecular weight was of the PS-I was measured as reported earlier.
10-12
The
optical rotation was measured on a Jasco Polarimeter model P-1020 at 25.5 C. The PS-I
(3.0 mg) was hydrolyzed with 2 M CF

3
COOH (2 mL) in a round-bottom flask at 100 C
for 18 h in a boiling water bath for sugar analysis and the analysis was carried out as

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described in our previous papers.
8,9
The absolute configuration of the monosaccharide
constituents was determined by the method of Gerwig et al.
24
The PS-I was methylated
according to the method of Ciucanu and Kerek
25
. Periodate oxidation experiment was
carried out with the PS-I as described in the earlier report.
9
A gas-liquid chromatographic
analysis (GLC) was done using Hewlett-Packard model 5730 A, having a flame
ionization detector and glass columns (1.8 m x 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
GLC analyses were performed at 170 C. The 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
program was isothermal at 150 C; hold time 5 min, with a temperature gradient of 2 C

min
-1
up to a final temperature of 200 C. The NMR experiments were carried out as
reported in our previous papers.
10-12

1.4. Test for macrophage activity by Nitric oxide assay
RAW 264.7 growing in Dulbecco's modified Eagle's medium (DMEM) was seeded in
96 well flat bottom tissue culture plates at 5 x 10
5
cells/mL concentrations (180 µL).
Cells were kept overnight for attachment and treatment of different concentrations (12.5,
25, 50, 100 or 200 μg/mL) of the PS-I. After 48 hrs of treatment culture supernatant of
each well were collected and NO content was estimated using Griess Reagent.
1.5. Splenocyte and thymocyte proliferation assay
A single cell suspension of spleen and thymus was prepared from normal mice under
aseptic conditions by homogenization in Hank's balanced salt solution (HBSS). The
suspension was centrifuged to obtain cell pellet. The contaminating RBC was removed

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by hemolytic Gey's solution. After two washes in HBSS the cells were resuspended in
complete RPMI. Cell concentration was adjusted to 1×10
6
cells/mL and viability of

splenocytes and thymocytes (as tested by trypan blue dye exclusion) was always over
90%. The cells (180 µL) were plated in 96 well flat bottom tissue culture plates and
incubated with 20 µL of various concentrations of polysaccharide (12.5, 25, 50, 100, or
200 µg/mL). PBS (10 mM, Phosphate Buffer Saline, pH-7.4) is taken as negative control
whereas LPS (4 µg/mL, Sigma) and Concavalin A (Con A, 10 µg/mL) served as positive
controls. All cultures were set up in triplicate for 72 h at 37 °C in a humidified
atmosphere of 5% CO
2
. Proliferation of splenocytes (% Splenocyte Proliferation Index or
% SPI) and Thymocytes (% Thymocyte Proliferation Index or %TPI) were checked by
MTT assay method.
32

Acknowledgements
The authors are grateful to Professor S. Roy, Director, IICB, Kolkata, for providing
instrumental facilities. Mr. Barun Majumdar of Bose Institute, Kolkata is acknowledged
for preparing NMR spectra. P.K.M. (one of the authors) thanks the CSIR for offering
junior research fellowship (CSIR-09/599(0043)/2011-EMR-I).
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17. Sasaki, T.; Takasura, N. Carbohydr. Res. 1976, 47, 99–104.
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Fitoterapia. 2010, 81, 1163-1170.
21. Chakraborty, U.; Sikdar, S.R.; World J. Microbial. Biotechnol. 2010, 26, 213-225.
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23. Lindhall, U. Biochem. J. 1970, 116, 27-34.
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12

Figure captions

Figure 1.
1
H NMR spectrum (500 MHz, D
2
O, 30ºC) of the polysaccharide isolated from
a hybrid mushroom (protoplast fusion between Pleurotus florida and Lentinula edodes).
.
Figure 2. (a)
13
C NMR spectrum (125 MHz, D
2
O, 30 ºC) of the polysaccharide isolated
from a hybrid mushroom (protoplast fusion between Pleurotus florida and Lentinula
edodes).
.(b) DEPT-135 spectrum (D
2
O, 30 ºC) of the polysaccharide isolated from a hybrid
mushroom (protoplast fusion between Pleurotus florida and Lentinula edodes).

Figure 3. The part of ROESY spectrum of the polysaccharide isolated a hybrid
mushroom (protoplast fusion between Pleurotus florida and Lentinula edodes). The
ROESY mixing time was 300ms.

Figure 4. The part of HMBC spectrum of the polysaccharide isolated from a hybrid
mushroom (protoplast fusion between Pleurotus florida and Lentinula edodes). The delay
time in the HMBC experiment was 80 ms.

Figure 5. (a) In vitro activation of raw macrophage stimulated with different
concentrations of the polysaccharide isolated from a hybrid mushroom (protoplast fusion
between Pleurotus florida and Lentinula edodes) in terms of NO production. Effect of
different concentrations of the polysaccharide isolated from a hybrid mushroom
(protoplast fusion between Pleurotus florida and Lentinula edodes) on splenocyte (b) and
thymocyte (c) proliferation (significant compared to the PBS control).










13








Figure 1

14

Figure 2a
























15

Figure 2b

16










Figure 3

17












Figure 4

18

19

20

21
Table 1
The
1
H

a
and
13
C
b
NMR chemical shifts for the polysaccharide isolated from a hybrid mushroom
(protoplast fusion between Pleurotus florida and Lentinula edodes)
in D
2
O at 30

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
→3,6)-β-D-Glcp-(1→
A
4.51
102.7
3.50
72.8
3.71
84.2
3.39
69.5

3.48
75.5
3.85
c
, 4.22
d

68.7

β-D-Glcp-(1→
B


4.51
102.9
3.32
73.03

3.46
75.9
3.44
69.6
3.46
75.9
3.72
c
, 3.88
d

60.7

→6)-β-D-Glcp-(1→
C

4.49
103.0
3.31
73.03
3.60
74.9
3.46
69.6
3.48
75.5
3.84
I
c
, 4.19
I
d

69.0
I







3.82

II
c
, 4.19
II
d

68.8
II









a
Values of the
1
H chemical shifts were recorded with respect to the HOD signal fixed at
 4.70 ppm at 30

C.
b
Values of the
13
C chemical shifts were recorded with reference to acetone as the
internal standard and fixed at  31.05 ppm at 30


C.
c,d
Interchangeable.
I
For residue C
I
.
II
For residue C
II
.













22

Table 2
ROE data for the polysaccharide isolated from a hybrid mushroom (protoplast fusion between
Pleurotus florida and Lentinula edodes)

I
For C
II
H-1 to A H-6a/b contact.
II

For C
I
H-1 to C
II
H-6a/b contact.





Glycosyl residue

Anomeric proton
ROE contact protons


Residue, atom
→3,6)-β-D-Glcp-(1→
A
4.51



3.50
3.48
3.84
4.19
A H-2
A H-5
C

I
H-6a
C
I
H-6b
β-D-Glcp-(1→
B

4.51



3.32
3.46
3.46
3.71

B H-2
B H-3
B H-5
A H-3

→6)-β-D-Glcp-(1→
C

4.49



3.31

3.60
3.48
3.85
I

4.22
I

3.82
II

4.19
II

C H-2
C H-3
C H-5
A H-6a
A H-6b
C
II
H-6a
C
II
H-6b

23
Table 3
The significant
3
J
H,C
connectivities observed in an HMBC spectrum for the anomeric
protons/carbons of the sugar residues of the polysaccharide isolated from a hybrid mushroom
(protoplast fusion between Pleurotus florida and Lentinula edodes)
.
Residues
Sugar linkage
H-1/C-1
Observed connectivities

H
/
C


H
/
C

Residue

Atom
A
→3,6)-β-D-Glcp-(1→

4.51
102.7
69.0
3.84
4.19
3.50
C
I

C
I

C
I

A
C-6
H-6a
H-6b
H-2

B

β-D-Glcp-(1→

4.51

102.9

84.2
73.03
3.71
3.32


A
B
A
B


C-3
C-2
H-3
H-2
C
→6)-β-D-Glcp-(1→


4.49


103.0
68.7

p

68.8
q

73.03
3.85
r

4.22
r

3.82
s

4.19
s

A

C
II

C
A
A
C
II
C
II

C-6
C-6
C-2
H-6a
H-6b
H-6a
H-6b




3.31

C

H-2








p
For cross peak between C
II
H-1 and A C-6.
q

For cross peak between C
I
H-1 and C
II
C-6.
r
For cross peak between C
II
C-1 and A H-6a/b.
s

For cross peak between C
I
C-1 and C
II
H-6a/b.



Structural characterization and study of immunoenhancing properties of a
glucan isolated from a hybrid mushroom of Pleurotus florida and Lentinula
edodes
Praloy K. Maji
a
, Ipsita K. Sen
a
, Birendra Behera
b
, Tapas K. Maiti
b

, Pijush Mallick
c
, Samir R.
Sikdar
c
, Syed S. Islam
a,
*
a
Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore
721102, West Bengal, India
b
Department of Biotechnology, Indian Institute of Technology (IIT) Kharagpur,
Kharagpur 721302, West Bengal, India
c
Division of Plant Biology, Bose Institute, Centenary Building, P-1/12, C.I.T. Scheme
VII M, Kolkata 700054, West Bengal, India




→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→
3
↑
1
β-D-Glcp