Structural characterization and study of immunoenhancing properties of a glucan isolated from a hybrid mushroom of Pleurotus florida and Lentinula edodes Praloy K.. Islama,* a Departme
Trang 1Structural 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: http://dx.doi.org/10.1016/j.carres.2012.06.017
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: http://dx.doi.org/10.1016/j.carres 2012.06.017
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Trang 2Structural characterization and study of immunoenhancing properties
of a glucan isolated from a hybrid mushroom of Pleurotus florida and
Lentinula edodes
Praloy K Majia, Ipsita K Sena, Birendra Beherab, Tapas K Maitib, Pijush Mallickc, Samir R Sikdarc, Syed S Islama,*
a
Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore
721102, West Bengal, India
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 (1H, 13C, 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-(16)--D-Glcp-(16)--D-Glcp-(16)--D-Glcp-(1
3 ↑
1 -D-Glcp
Trang 3Keywords: Hybrid mushroom; Glucan; NMR spectroscopy; Immunostimulation
Corresponding auther Tel.: +91 03222 276558 x 437; +91 9932629971 (M); fax: +91
03222 275329; e mail: sirajul_1999@yahoo.com
Mushrooms are important for its medicinal value.1 Mushroom polysaccharides have gained importance because of their immunomodulatory2, free radical scavenging3,4, and antitumor5,6 activity Various immunoenhancing polysaccharides from edible mushrooms7-9 and hybrid mushrooms10-12 were reported by our group Four different
polysaccharides isolated from Pleurotus florida13-16 were also reported by our group
Lentinan, a biologically active water insoluble polysaccharide from Lentinula edodes
containing (1→3), (1→6)-β-D-glucan17 has been reported and widely used for cancer therapy Water soluble polysaccharides18-20 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 earlier21 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 []D30 –12 (c 0.8, water)
Molecular weight22 of PS-I was estimated as ~1.80 105 Da from a calibration curve
Trang 4al.24 This PS-I was methylated according to Ciucanu and Kerek25 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 NaBH4-reduced PS-I was found to contain glucose only and periodate-oxidized, reduced, methylated28 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 1H NMR spectrum (500 MHz; Fig 1) at δ 4.51 and 4.49 ppm at 30 C 13C 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
Trang 5to 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 1H and 13C signals (Table 1) were assigned from DQF-COSY, TOCSY, and HSQC experiments The proton coupling constants were measured from DQF-COSY experiment
The large JH-2,H-3 and JH-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 JH-1,H-2 (~8 Hz) and JC-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 glycosides29,30
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 effect7,31 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
Trang 6
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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/CIH-6a, CIH-6b; CIIH-1/AH-6a, AH-6b; CIH-1/CIIH-6a, CIIH-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 IIC-6, C IC-1/CIIH-6a, CIIH-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-(16)--D-Glcp-(16)--D-Glcp-(16)--D-Glcp-(1 3
↑
1 -D-Glcp
Trang 7100 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 bromidemethod.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
Trang 8PEG, 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 CF3COOH (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
Trang 91.4 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 105 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
Trang 10200 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% CO2 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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Trang 13
Figure captions
a hybrid mushroom (protoplast fusion between Pleurotus florida and Lentinula edodes)
from a hybrid mushroom (protoplast fusion between Pleurotus florida and Lentinula
edodes)
.(b) DEPT-135 spectrum (D2O, 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)