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

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: 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 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 Keywords: 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 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 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. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 6 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. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 7 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 8 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 9 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). References 1. Yu, Z.; Ming, G.; Zhixiang, C.; Liquan, D.; Jingyu, L.; Fang, Z. Fitoterapia. 2010, 81, 1163-1170. 2. Moradali, M. F.; Mostafavi, H.; Ghods, S.; Hedjaroude, G. A. Int. s Immunopharmacol. 2007, 7, 701-724. 3. Chen, Y. , Xie, M. Y., Nie, S. P.; Li, C.; Wang, Y. X. Food Chemistry. 2008, 107, 231–241. [...]... H-2 Atom Structural 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 b Department of. .. 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... between Pleurotus florida and Lentinula edodes) Figure 2 (a) 13C NMR spectrum (125 MHz, D2O, 30 ºC) of the polysaccharide isolated 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... 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... 4.19IId 68.8II a Values of the 1H chemical shifts were recorded with respect to the HOD signal fixed at  4.70 ppm at 30 C b Values of the 13C 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 CI II For residue CII 21 Table 2 ROE data for the polysaccharide isolated from a hybrid mushroom (protoplast fusion... 9 Bhunia, S K.; Dey, B.; Maity, K K.; Patra, S.; Mandal, S.; Maiti, S.; Maiti, T K.; Sikdar, S R.; Islam, S S Carbohydr Res 2010, 345, 2542-2549 10 Maity, K.; Kar (Mandal), E.; Maity, S.; Gantait, S K.; Das, D.; Maiti, S.; Maiti, T K.; Sikdar, S R.; Islam, S S Int J Biol Macromol 2011, 48, 304–310 11 Das, D.; Mondal, S.; Roy, S K.; Maiti, D.; Bhunia, B.; Maiti, T K.; Sikdar, S R.; Islam, S S Carbohydr... H- 6a/ b contact 22 Table 3 The significant 3 JH,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 →3,6)-β-D-Glcp-(1→ B β-D-Glcp-(1→ 4.51 102.9 C →6)-β-D-Glcp-(1→ 4.49 103.0 p For cross peak between CII H-1 and. .. V E C.; Chang, S T.; Life Science 1997, 60, 763-771 5 Franz, G Planta Med 1989, 55, 493-497 6 Kishida, E.; Sone, Y.; Misaki, A Carbohydr Polym 1992, 17, 89-95 7 Mandal, S.; Maity, K K.; Bhunia, S K.; Dey, B.; Patra, S.; Sikdar, S R.; Islam, S S Carbohydr Res 2010, 345, 2657-2663 8 Dey, B.; Bhunia, S K.; Maity, K K.; Patra, S.; Mandal, S.; Maiti, S.; Maiti, T K.; Sikdar, S R.; Islam, S S Carbohydr Res... Vincendon, M Carbohydr Polym 1982, 2, 135-144 31 Yoshioka, Y.; Tabita, R.; Saito, H.; Uehara, N.; Fukuoka, F Carbohydr Res 1985, 140, 93-100 32 Ohno, N.; Saito, K.; Nemoto, J.; Kaneko, S.; Adachi, Y.; Nishijima, M.; Miyazaki, T.; Yadomae, T Biol Pharm Bull 1993, 16, 414–419 11 Figure captions Figure 1 1H NMR spectrum (500 MHz, D2O, 30ºC) of the polysaccharide isolated from a hybrid mushroom (protoplast fusion... 974-978 12 Patra, S.; Maity, K K.; Bhunia, S K.; Dey, B.; Mandal, S.; Maiti, T K.; Sikdar, S R.; Islam, S S Carbohydr Res 2011, 346, 1967-1972 13 Rout, D.; Mondal, S.; Chakraborty, I.; Pramanik, M.; Islam, S S Med.Chem Res 2004, 13, 509-517 14 Rout, D.; Mondal, S.; Chakraborty, I.; Pramanik, M.; Islam, S S Carbohydr Res 2005, 340, 2533-2539 15 Rout, D.; Mondal, S.; Chakraborty, I.; Islam, S S Carbohydr . 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 ,. 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, . 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,