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International Journal of Biological Macromolecules 61 (2013) 89– 96

Contents lists available at SciVerse ScienceDirect

International Journal of Biological Macromolecules

jo ur nal homep age: www.elsev ier .com/ locate / i jb iomac

tructural characterization of an immunoenhancing glucan isolatedrom a mushroom Macrolepiota dolichaula

urajit Samantaa, Ashis K. Nandia, Ipsita K. Sena, Praloy K. Majia, K. Sanjana P. Devib,apas K. Maitib, Syed S. Islama,∗

Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore 721102, West Bengal, IndiaDepartment of Biotechnology, Indian Institute of Technology (IIT) Kharagpur, Kharagpur 721302, West Bengal, India

a r t i c l e i n f o

rticle history:eceived 18 April 2013eceived in revised form 22 May 2013ccepted 2 June 2013vailable online xxx

a b s t r a c t

A water soluble branched glucan (PS-I) was isolated from aqueous extract of the fruit bodies of anedible mushroom Macrolepiota dolichaula, having average molecular weight ∼2.02 × 105 Da. The struc-ture of this PS-I was determined using total hydrolysis, methylation analysis, Smith degradation, partialhydrolysis, and 1D/2D NMR experiments. Total hydrolysis and methylation analysis results showed thepresence of (1 → 3, 6)-, (1 → 6)-, (1 → 4)-, (1 → 3)-linked and terminal �-d-glucopyranosyl residues in a

eywords:acrolepiota dolichaula

ranched glucanMR studies

mmunoactivation

relative proportion of nearly 1:2:1:1:1. All the chemical and NMR results indicated that the PS-I was abranched glucan, and the repeating unit of this glucan consisted of a backbone chain of three (1 → 6)-linked-�-d-glucopyranosyl residues where one of the backbone residues is branched at O-3 with (1 → 3)-moiety which is further attached to another (1 → 4)- residue and terminated with a non-reducing �-d-glucopyranosyl residue. The PS-I exhibited in vitro macrophage activation in RAW 264.7 cell line as wellas splenocyte and thymocyte activation in mouse cell culture medium.

. Introduction

Mushrooms are well known for their sweet taste and food fla-oring materials. It also used as an important source of biologicallyctive molecules. Now days, mushrooms have drawn the attentionf immunobiologist and pharmacologist for their immunostimu-atory, antitumor, and antioxidant properties [1–3]. The glucans in

ushroom are present mostly as linear �-(1 → 3)- [4–6], �-(1 → 6)-7–9] and non linear with �-(1 → 3) backbone branched at O-610,11], and �-(1 → 6) backbone branched at O-3 [12,13].

The genus Macrolepiota, family Agaricaceae was first establishedy Singer [14]. Few years back, 30 species of the genus Macrolepi-ta were recognized all over the world [15]. In past, this genus wasifferentiated into two clades by DNA studies [16]. Recently, onhe basis of molecular phylogenetic analysis, the genus Macrole-iota has been differentiated into three clades, namely/volvalae,macrosporae and/macrolepiota [17]. Macrolepiota dolichaula is onef the edible species [18] belongs to the clade/macrolepiota [17] and

istributed through out the world. It was reported that this mush-oom contained vitamin B1, vitamin C, vitamin B2, and vitamin A19]. This mushroom showed laccase enzymatic activity in wheat

∗ Corresponding author. Tel.: +91 03222 276558x437;ax: +91 03222 275329/9932629971.

E-mail address: sirajul 1999@yahoo.com (S.S. Islam).

141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijbiomac.2013.06.010

© 2013 Elsevier B.V. All rights reserved.

straw and czapek medium [20]. Two water soluble polysaccharides(PS-I & PS-II) have been isolated from the hot aqueous extract of thismushroom. Detailed structural elucidation and some preliminarystudy of immunostimulating properties of the PS-I were carried outand reported herein first time.

2. Materials and methods

2.1. Isolation and purification of the polysaccharide

The edible mushroom M. dolichaula (500 g) was collected fromVidyasagar University garden, West Bengal, and fruit bodies werewashed with distilled water which were then crushed and boiledfor 10 h. The whole mixture was kept overnight at 4 ◦C and then fil-tered through linen cloth. The filtrate was centrifuged at 8400 rpm(using a Heraeus Biofuge Stratos Centrifuge) for 40 min at 4 ◦C.The supernatant was collected and precipitated in 1:5 (v/v) EtOH.It was kept overnight at 4 ◦C and again centrifuged as abovefor 30 min. The precipitated polysaccharide was dissolved in aminimum volume of distilled water and dialyzed through dialy-sis tubing of cellulose membrane (Sigma–Aldrich, retaining > m.w.12,400) against distilled water to remove low molecular weight

materials. The material was then collected from the dialysis bagand a water-soluble part was obtained after centrifugation andfreeze dried to yield water-soluble crude polysaccharide (600 mg).This crude polysaccharide (30 mg) was purified by gel-permeation

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hromatography on a Sepharose-6B column (65 × 2 cm). Ninety fiveest tubes were collected and monitored by the phenol–sulfuriccid method [21] at 490 nm using Shimadzu UV-vis spectropho-ometer, model-1601. Two homogeneous fractions, PS-I (test tubeo. 18–30, yield 5 mg) and PS-II (test tube no. 40–52, yield 12 mg)ere collected through freeze-drying to get pure polysaccharides.

he present work deals with the PS-I only. The same procedureas repeated several times to get the PS-I, yield 90 mg and used for

hemical and NMR analysis.

.2. General methods

Optical rotation was measured on a Jasco polarimeter (Model-1020) at 31.8 ◦C. The average molecular weight was measured byel chromatography as reported earlier [22–24]. For monosaccha-ide analysis, the PS-I (3 mg) was hydrolyzed with 2 M CF3COOH2 mL) in a round-bottom flask at 100 ◦C for 18 h in a boiling waterath and the analysis was carried out as described in previousapers [22–24] by Gas–liquid-chromatography (GLC). The abso-

ute configuration of the monosaccharide units was determinedy the method of Gerwig et al. [25]. Methylation and periodatexidation experiments were carried out as described in the ear-ier reports [22–24]. The GLC instrument Hewlett-Packard model730 A was used, with flame ionization detector and glass columns1.8 m × 6 mm) packed with 3% ECNSS-M (A) on Gas Chrom Q100–120 mesh) and 1% OV-225 (B) on Gas Chrom Q (100–120

esh) for monosaccharide analysis. All GLC analyses were per-ormed at 170 ◦C. GLC–MS analysis was performed on ShimadzuLC–MS Model QP-2010 plus automatic system, using ZB-5MS cap-

llary column (30 m × 0.25 mm). The program was isothermal at50 ◦C; hold time 5 min, with a temperature gradient of 2 ◦C min−1

p to a final temperature of 200 ◦C. Finally NMR experiments26,27] were carried out by a Bruker Avance DPX-500 instrumentt 30 ◦C as reported earlier [22–24].

.3. Smith degradation [13,28,29]

The polysaccharide (30 mg) was oxidized with 0.1 M NaIO45 mL) at 25 ◦C, 72 hrs and kept in dark. 1,2-ethanediol was added totop oxidation and the solution was dialyzed against distilled H2O.he dialyzed material was reduced with NaBH4 and kept overnightt room temperature. The mixture was neutralized with 50% AcOH,nd again dialyzed with distilled water, and finally freeze dried. Thisaterial was subjected to mild hydrolysis with 0.5 M CF3COOH for

5 h at 25 ◦C to eliminate the residues of oxidized part attachedo the polysaccharide chain. The excess of acid was removed byepeated evaporation of water at 37 ◦C and finally, it was purifiedy passing through a Sephadex G-25 column and freeze dried. Aart of this dried material was used for methylation analysis andhe remaining part was used for 13C NMR studies.

.4. Partial hydrolysis [13,30]

The polysaccharide (35 mg) was partially hydrolyzed with 6 mL.1 M CF3COOH at 100 ◦C for 1 h and the excess acid was removed byepeated evaporation of water at 37 ◦C. The residue was dissolvedn water, to which three volumes of ethanol was added. The pre-ipitate was washed with ethanol, freeze-dried and the fraction F2as obtained which was used for methylation analysis as well as

3C NMR analysis. The supernatant was dried by evaporation, and

he residue was dissolved in a minimum volume of water for reduc-ion. After reduction with NaBH4 at 25 ◦C for 2 h, the product waseutralized with 1 M AcOH and it was desalted by passing through

Sephadex G-25 column. The carbohydrate containing eluate (F1)

logical Macromolecules 61 (2013) 89– 96

was collected, freeze-dried and subjected to methylation analysis[22–24].

2.5. Test for macrophage activity by nitric oxide assay

RAW 264.7, a murine macrophage cell line obtained fromNational Centre for Cell Sciences (NCCS), Pune, India, was grow-ing in Dulbecco’s modified Eagle’s medium (DMEM) and seededin 96 well flat bottom tissue culture plates at 5 × 105 cells/mLconcentrations (180 �L) [31]. Cells were left overnight for attach-ment and treated with different concentrations (12.5, 25, 50,100 and 200 �g/mL) of the PS-I. After 48 h of treatment cul-ture supernatant of each well were collected and NO contentwas estimated using Griess Reagent [32] at 540 nm (1:1 of 0.1%1-napthylethylenediamine in 5% phosphoric acid and 1% sulfanil-amide in 5% phosphoric acid). Lipopolysaccharide (LPS), L6511 ofSalmonella enterica serovar Typhimurium (Sigma, St. Louis, USA)was used as positive control.

2.6. Splenocyte and thymocyte proliferation assay

A single cell suspension of spleen and thymus was preparedfrom Swiss Albino mice under aseptic conditions by homoge-nization in Hank’s balanced salt solution (HBSS). This study wasapproved by the ethics committee. The suspension was centrifugedto obtain cell pellet. The contaminating RBC was removed byhemolytic Gey’s solution. After two washes in HBSS the cellswere resuspended in complete RPMI (Roswell Park Memorial Insti-tute) with serum and antibiotics added. RPMI and fetal bovineserum (FBS) has been obtained from Gibco whereas antibioticswere obtained from Himedia. Cell concentration was adjusted to1 × 106 cells/mL and viability of splenocytes and thymocytes (astested by trypan blue dye exclusion) was always over 90%. Thecells (180 �L) were plated in 96 well flat bottom tissue cultureplates and incubated with 20 �L of various concentrations (12.5,25, 50, 100 and 200 �g/mL) of PS-I. PBS (10 mM, Phosphate BufferSaline, pH 7.4) was taken as negative control whereas LPS (4 �g/mL,Sigma) and Concanavalin A (Con A, 10 �g/mL) served as positivecontrols. All cultures were set up in triplicate for 72 h at 37 ◦C ina humidified atmosphere of 5% CO2. Proliferation of splenocytesindicated as Splenocyte Proliferation Index (SPI) and thymocytesas Thymocyte Proliferation Index (TPI) were checked by standardMTT assay method. The data are reported as the mean ± standarddeviation of six different observations and compared against PBScontrol [32,33].

3. Results and discussion

3.1. Isolation, purification, and chemical analysis

Fresh fruit bodies of the mushroom M. dolichaula (500 g) werewashed thoroughly with water, then distilled water and boiledfor 10 h followed by centrifugation and alcohol precipitation.The precipitated material was dissolved in water, dialyzed, cen-trifuged, and freeze dried to yield 600 mg of crude polysaccharide.The crude polysaccharide (30 mg), on fractionation througha Sepharose 6B column yielded two polysaccharide fractions(Fig. 1), which were collected and freeze dried, yielding purifiedpolysaccharide PS-I (5 mg) and PS-II (12 mg). The PS-I showedspecific rotation [�]D

31.8–20.46 (c 0.052, H2O). The molecularweight [34] of PS-I was determined as ∼2.02 × 105 Da from a

calibration curve prepared with standard dextrans. The GLCanalysis of the hydrolyzed alditol acetates of PS-I showed thepresence of glucose only. Absolute configuration determinationby the method of Gerwig et al. [25] showed that glucose was

S. Samanta et al. / International Journal of Bio

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ig. 1. Gel permeation chromatogram of crude polysaccharide, isolated from andible mushroom Macrolepiota dolichaula using Sepharose 6B column.

resent in D-configuration. The PS-I was methylated using Ciu-anu and Kerek method [35], followed by hydrolysis and alditolcetate preparation for the determination of the mode of linkagesresent in the PS-I. The GLC–MS analysis of these alditol acetatesevealed 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-d-glucitol,,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-d-glucitol,,3,5-tri-O-acetyl-2,4,6-tri-O-methyl-d-glucitol and 1,5-di-O-cetyl-2,3,4,6-tetra-O-methyl-d-glucitol in a molar ratio ofearly 1:2:1:1:1, respectively. Thus, the PS-I was found toontain (1 → 3,6)-, (1 → 6)-, (1 → 4)-, (1 → 3)-linked and ter-inal glucopyranosyl residues in the same ratio. The GLC-MS

nalysis of alditol acetates of the periodate oxidized [36,37],aBH4-reduced, methylated [38] PS-I was found to contain,3,5,6-tetra-O-acetyl-2,4-di-O-methyl-d-glucitol and 1,3,5-tri--acetyl-2,4,6-tri-O-methyl-d-glucitol in a molar ratio of nearly:1 which further confirmed the presence of (1 → 3,6)- and1 → 3)-linked glucopyranosyl residues while the other residues

ere consumed during oxidation. All these results suggested

hat the PS-I to be a branched glucan which may have severalossible repeating unit. Therefore, Smith degradation [13,28,29]nd partial hydrolysis [13,30] analysis were carried out to

ig. 2. (a) 1H NMR spectrum (500 MHz, D2O, 30 ◦C) of the PS-I isolated from an edible musS-I isolated from an edible mushroom Macrolepiota dolichaula.

logical Macromolecules 61 (2013) 89– 96 91

determine the most probable structure of the repeating unitpresent in the PS-I. GLC-MS analysis was done with the alditolacetates of methylated, reduced Smith degraded product (SDPS).The GLC–MS data of this methylated SDPS revealed the presenceof 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl-d-glucitol and 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-d-glucitol in a molar ratio of nearly1:1. From the partial hydrolysis of the PS-I, two fractions wereobtained; partially hydrolyzed oligosaccharide (F1) and partiallyhydrolyzed polysaccharide (F2). GLC-MS analysis of the methylatedproduct of F1 revealed the presence of (1 → 4)-, (1 → 3)-linked,and terminal glucopyranosyl moieties and methylation analysis ofF2 revealed the presence of 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-d-glucitol only. This result clearly indicated that F2 polysaccharide(backbone chain of the PS-I) consists of (1 → 6)-linked glucopyra-nosyl residues and F1 oligosaccharide branched from O-3 of thepolysaccharide backbone. Now, there were two possibilities ofbranching; one where (1 → 4)-linked glucopyranosyl and anotherone where (1 → 3)-linked glucopyranosyl of F1 oligosaccharideattached with O-3 of the backbone. But the result of methylationanalysis of SDPS suggested that the (1 → 3)-linked glucopyranosylof F1 attached with O-3 of the backbone.

3.2. NMR and structural analysis of the PS-I

The NMR experiments (1D/2D NMR) were carried out along withthe chemical analysis for determining the structure of the repeat-ing unit of PS-I. In the 1H NMR spectrum (500 MHz, Fig. 2a) at 30 ◦Cof PS-I, the anomeric signals appeared at ı 4.76 (A), 4.74 (B), 4.51 (Cand D) and 4.49 (E) for six anomeric protons where ı 4.51 and 4.49signals indicated the presence of two residues. In 13C NMR spec-trum (125 MHz, Fig. 2b) four signals were observed in the anomericregion at ı 102.4, 102.5, 102.8 and 103.0 in a molar ratio of nearly1:1:2:2. The anomeric carbon chemical shift values of residues A–Ewere assigned from the HSQC spectrum (Fig. 3). All the 1H and 13Csignals (Table 1) were determined from the DQF-COSY, TOCSY andHSQC experiments. Again, from DQF-COSY experiment the protoncoupling constants and from proton coupled 13C experiments theC-1, H-1 coupling constants were measured.

The configurations of all the residues from A–E were confirmedas �-anomer from the coupling constant values JH-1,H-2 ∼8.5 Hz andJC-1,H-1 ∼160 Hz along with their range of anomeric proton chem-ical shifts (ı 4.76–4.49) and anomeric carbon chemical shifts (ı

hroom Macrolepiota dolichaula. (b) 13C NMR spectrum (125 MHz, D2O, 30 ◦C) of the

92 S. Samanta et al. / International Journal of Biological Macromolecules 61 (2013) 89– 96

Fig. 3. Part of HSQC spectrum of the PS-I isolated from an edible mushroom Macrolepiota dolichaula.

Fig. 4. DEPT-135 spectrum (D2O, 30 ◦C) of the PS-I isolated from an edible mushroom Macrolepiota dolichaula.

Table 1The 1H NMRa and 13C NMRb chemical shifts for the PS-I isolated from the mushroom Macrolepiota dolichaula in D2O 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

→4)-�-d-Glcp-(1→ 4.76 3.39 3.46 3.68 3.62 3.71c, 3.88d

A 102.4 73.0 75.6 81.0 75.3 60.8→3)-�-d-Glcp-(1→ 4.74 3.50 3.72 3.42 3.46 3.71c, 3.88d

B 102.5 72.8 84.3 69.5 75.7 60.8�-d-Glcp-(1→ 4.51 3.30 3.49 3.39 3.46 3.70c, 3.86d

C 102.8 73.1 76.0 69.7 75.8 60.8→3,6)-�-d-Glcp-(1→ 4.51 3.50 3.72 3.44 3.62 3.83c, 4.18d

D 102.8 72.8 84.3 69.5 75.0 68.3→6)-�-d-Glcp-(1→ 4.49 3.30 3.48 3.37 3.60 3.86c, 4.19d

E 103.0 73.1 76.0 69.7 75.6 68.9e, 68.7f

a The values of chemical shifts were recorded keeping HOD signal fixed at ı 4.70 ppm at 30 ◦C.b The values of chemical shifts were recorded with reference to acetone as internal standard and fixed at ı 31.05 ppm at 30 ◦C.c,d Interchangeable.e For residue EI .f For residue EII .

S. Samanta et al. / International Journal of Biological Macromolecules 61 (2013) 89– 96 93

Table 2NOESY data for the PS-I isolated from the mushroom Macrolepiota dolichaula in D2O at 30 ◦C.

Glycosyl residue Anomeric proton NOE contact proton

ı ı Residue Atom

→4)-�-d-Glcp-(1→ A 4.76 3.623.72 AB H-5H-3→3)-�-d-Glcp-(1→ B 4.74 3.503.723.72 BBD H-2H-3H-3�-d-Glcp-(1→ C 4.51 3.493.463.68 CCA H-3H-5H-4→3,6)-�-d-Glcp-(1→D 4.51 3.503.723.623.864.19 DDDEIEI H-2H-3H-5H-6aH-6b

3.86a4.19a3.83b4.18b EEEIIEIIDD H-3H-5H-6aH-6bH-6aH-6b

1cecvlatsaarlE(itkECDr

Nmac6sl(c3DEicg

FM

Table 3The significant 3JH,C connectivities observed in an HMBC spectrum for the anomericprotons/carbons of the sugar residues of the PS-I isolated from the mushroomMacrolepiota dolichaula in D2O at 30 ◦C.

Residue Sugar linkage H-1/C-1 ıH/ıC Observed connectivities

ıH/ıC Residue Atom

A →4)-�-d-Glcp-(1→ 4.76 84.3 B C-3102.4 3.72 B H-3

3.39 A H-2B →3)-�-d-Glcp-(1→ 4.74 84.3 D C-3

102.5 3.72 D H-33.50 B H-2

C �-d-Glcp-(1→ 4.51 81.0 A C-4102.8 3.68 A H-4

3.30 C H-23.49 C H-3

D →3,6)-�-d-Glcp-(1→ 4.51 68.9 EI C-6102.8 3.86 EI H-6a

4.19 EI H-6b3.50 D H-2

E →6)-�-d-Glcp-(1→ 4.49 68.7a EII C-6103.0 68.3b D C-6

3.86c EII H-6a4.19d EII H-6b3.83e D H-6a4.18f D H-6b3.30 E H-23.48 E H-3

a For cross peak between EI H-1 and EII C-6.b For cross peak between EII H-1 and D C-6.c For cross peak between EI C-1 and EII H-6a.d For cross peak between EI C-1 and EII H-6b.

→6)-�-d-Glcp-(1→ E 4.49 3.483.60

a For EI H-1 to both EII H-6a and EII H-6b contacts.b For EII H-1 to both D H-6a and D H-6b contacts.

03.0–102.4). The large JH-2,H-3 (∼10.0 Hz) and JH-3,H-4 (∼9.5 Hz)oupling constant values of the residues A–E indicated the pres-nce of glucopyranosyl moiety (Glcp) in the PS-I. The downfieldhemical shift of C-4 (ı 81.0) of residue A with respect to standardalue of methyl glycosides [39,40] indicated that it was a (1 → 4)-inked �-d-Glcp. Similarly, residue B was (1 → 3)-linked �-d-Glcps the C-3 signal (ı 84.3) of residue B showed downfield shift. Allhe chemical shift values of residue C were resembled nearly to thetandard values of methyl glycosides [39,40] indicating that it was

non-reducing end �-d-Glcp. The carbon signals of C-3 (ı 84.3)nd C-6 (ı 68.3) of residue D, appeared at downfield region withespect to standard values, indicating the presence of (1 → 3,6)-inked �-d-Glcp. C-6 of two E residues assigned as EI (ı 68.9) andII (ı 68.7), showed down field shift, indicative of the presence of1 → 6)-linked �-d-Glcp. The C-6 of EI resonated slightly downfieldn comparison to C-6 of EII due to the neighboring effect [41,42] ofhe rigid part D. The residues EI and EII differed only of C-6 valueseeping other values unaltered. The presence of (1 → 6)-linking inI and EII was further confirmed by downward displacement of the-6 signals in DEPT-135 spectrum (Fig. 4). The C-6 signal of residue

did not show any reverse displacement in DEPT-135 spectrum aseported earlier [13,30] (Fig. 4).

The sequence of glycosyl residues was determined from theOESY (Fig. 5 and Table 2) as well as ROESY (not shown) experi-ents followed by confirmation with the HMBC experiments (Fig. 6

nd Table 3). The NOESY experiment showed the inter-residualontacts: AH-1/BH-3; BH-1/DH-3; CH-1/AH-4; DH-1/EIH6a, EIH-b; EIH-1/EIIH-6a, EIIH-6b; and EIIH-1/DH-6a, DH-6b along withome intra-residual contacts. Thus, the NOESY connectivities estab-ished the sequences as, A (1 → 3) B; B (1 → 3) D; C (1 → 4) A; D1 → 6) EI; EI (1 → 6) EII; and EII (1 → 6) D. The long range 13C–1Horrelation of the HMBC experiment, the cross-peaks AH-1/BC-; AC-1/BH-3; BH-1/DC-3; BC-1/DH-3; CH-1/AC-4; CC-1/AH-4;H-1/EIC-6; DC-1/EIH-6a, EIH-6b; EIH-1/EIIC-6; EIC-1/EIIH-6a,

IIH-6b; EIIH-1/DC-6; and EIIC-1/DH-6a, DH-6b along with other

ntra-residual peaks were observed (Fig. 6). Thus, it can be con-luded that the PS-I was a branched glucan with (1 → 6)-linkedlucopyranosyl backbone and branched occurred at O-3 of D

ig. 5. Part of NOESY spectrum of the PS-I isolated from an edible mushroomacrolepiota dolichaula. The NOESY mixing time was 300 ms.

e For cross peak between EII C-1 and D H-6a.f For cross peak between EII C-1 and D H-6b.

residue with (1 → 3)-linked B residue followed by (1 → 4)-linkedA residue and non-reducing end C residue. Hence the hexasaccha-ride repeating unit present in the PS-I was established as:

EII D E I→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→

3↑1

β-D-Glc p-(1 →4)-β-D-Glc p-(1 →3)-β-D-Glc pC A B

For authentication of the linkages, again NMR experimentswere carried out with Smith degradation product (SDPS) andpartially hydrolyzed polysaccharide (F2) of the PS-I. The 13CNMR (125 MHz, 30 ◦C) spectrum (Fig. 7 and Table 4) of SDPSexhibited two anomeric signals at ı 102.7 and 103.2 correspond-ing to �-d-Glcp-(1→ (X) and →3)-�-d-Glcp-(1→ (Y) residuesrespectively. The non-reducing �-d-Glcp-(1→ unit (X) was pro-duced from →3)-�-d-Glcp-(1→ (B) due to complete oxidationof A and C residues during Smith degradation. Again, →3)-�-d-

Glcp-(1→ (Y) was produced from the →3,6)-�-d-Glcp-(1→ (D)residue due to periodate oxidation followed by Smith degrada-tion. The carbon signals C-1, C-2, and C-3 of the glycerol moiety

94 S. Samanta et al. / International Journal of Biological Macromolecules 61 (2013) 89– 96

Fig. 6. Part of HMBC spectrum of the PS-I isolated from an edible mushroom Ma

Table 4The 13C NMRa chemical shifts of Smith-degraded glycerol-containing disaccharideof the PS-I isolated from the mushroom Macrolepiota dolichaula in D2O at 30 ◦C.

Sugar residue C-1 C-2 C-3 C-4 C-5 C-6

�-d-Glcp-(1→ X 102.7 73.2 76.7 70.6 75.9 61.1→3)-�-d-Glcp-(1→Y 103.2 73.0 84.4 69.7 75.6 60.7Gro-(3→ Z 66.4 72.1 62.5

s

(gatHP

a

o

iawp

a The values of chemical shifts were recorded with reference to acetone as internaltandard and fixed at ı 31.05 ppm at 30 ◦C.

Gro) were assigned as ı 66.4, 72.1, and 62.5 respectively. Thelycerol (Z) moiety generated from →6)-�-d-Glcp-(1→ (EI)fter periodate oxidation followed by Smith degradation andhis moiety was attached to →3)-�-d-Glcp-(1→ moiety (Y).ence, a glycerol containing disaccharide was obtained from theS-I after Smith degradation and the structure was established

s: β-D-Glc p-(1 →3)-β-D-Glcp-(1 →3)-Gro Therefore, the above result further confirmed that the branching

ccurred at O-3 of D with B residue.The 13C NMR (125 MHz, 30 ◦C) spectrum of F2 indicated that

t was a polymeric chain of simple (1 → 6)-linked-�-d-Glcp unitss characteristic signal for C-6 at ı 68.7 and C-1 at ı 103.0ere observed. Thus, 13C NMR analysis of partially hydrolyzedolysaccharide (F2) along with Smith degraded product (SDPS),

Fig. 7. 13C NMR spectrum (125 MHz, D2O, 30 ◦C) of the Smith-degraded

crolepiota dolichaula. The delay time in the HMBC experiment was 80 ms.

clearly indicated that it was a branched glucan with (1 → 6)-linkedbackbone where branching occurred at O-3 of one unit of the back-bone followed by (1 → 3)-, (1 → 4)- and non-reducing end �-d-Glcp.

Several types of water soluble �-glucans are reported fromthe edible mushrooms. Non-linear glucans consisting of (1 → 6)-�- [22,43,44] and (1 → 3); (1 → 6)-�-linked [45,46] backbone withbranching at O-3 or O-6 have been reported. A glucan with differ-ent molecular weight from an edible mushroom Sarcodon aspratus(Berk.) S. Ito. have been reported [43] which resembles close tothe structure of the present glucan differing only in the numberof (1 → 6)- and (1 → 3)-�-d-Glcp residues in the backbone andbranched chain respectively.

3.3. Immunological studies of the PS-I

Some immunological studies were also carried out with thePS-I. Macrophage activation of the PS-I was observed in vitro. Ontreatment with different concentrations of the PS-I, an enhancedproduction of NO was observed in a dose-dependent manner withoptimum production of 21 �M NO per 5 × 105 macrophages at

100 �g/mL concentration (Fig. 8a). Hence, the effective dose of thePS-I for macrophage activation was 100 �g/mL.

Proliferation of splenocytes and thymocytes is an indica-tor of immunoactivation [47]. The splenocyte and thymocyte

glucan isolated from an edible mushroom Macrolepiota dolichaula.

S. Samanta et al. / International Journal of Bio

Fig. 8. (a) In vitro activation of macrophage stimulated with different concentra-tions of the PS-I isolated from an edible mushroom Macrolepiota dolichaula in termsoet

am2poPiicw1s

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f NO production. Effect of different concentrations of the PS-I isolated from andible mushroom Macrolepiota dolichaula on proliferation of splenocytes (b) andhymocytes (c) (*Significant compared to PBS control with P < 0.05).

ctivation tests were carried out in Swiss Albino mice cell cultureedium with the PS-I by the MTT [3-(4,5-dimethylthiazol-2-yl)-

,5-diphenyltetrazolium bromide] method [48,49]. The splenocyteroliferation index (SPI) as compared to PBS control if closer to 1r below indicates low stimulatory effect on immune system. TheS-I induced splenocytes and thymocytes activations were shownn Fig. 8b and c, respectively and the asterisks on the columnsndicate the statistically significant differences compared to PBSontrol. Maximum proliferation index of splenocyte and thymocyte

as observed at 100 and 25 �g/mL of the PS-I, respectively. Hence,

00 �g/mL of the PS-I can be considered as efficient splenocytetimulator whereas 25 �g/mL as efficient thymocyte stimulator.

[

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logical Macromolecules 61 (2013) 89– 96 95

4. Conclusions

A water-soluble branched glucan (PS-I) was isolated from theaqueous extract of an edible mushroom M. dolichaula. The puri-fied branched glucan obtained by gel-filtration chromatographyshowed significant splenocyte, thymocyte and macrophage activa-tion. Architectural details of the repeating unit of the M. dolichaulawere determined by the chemical and 1D/2D NMR experiments,and the structure of the hexasaccharide repeating unit in the PS-Iwas established as:

→6) -β-D-Glc p-(1→6) -β-D-Glc p-(1 →6)-β-D-Glc p-(1 →3↑1

β-D-Glcp-(1 →4)-β-D-Glc p-(1 →3) -β-D-Glc p

Acknowledgements

The authors are grateful to Dr. S. Roy, Director, IICB, Kolkatafor providing instrumental facilities. Mr. Barun Majumdar of BoseInstitute, Kolkata is acknowledged for preparing NMR spectra. Dr.Krishnendu Acharya, Mycologist, Department of Botany, Univer-sity of Calcutta is acknowledged for identifying the mushroom. Oneof the authors (S.S.) is thankful to CSIR, India for providing juniorresearch fellowship (CSIR-09/599(0048)/2012-EMR-I).

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