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.elsevier.com/locate/ijbiomac Structural characterization of an immunoenhancing glucan isolated from a mushroom Macrolepiota dolichaula Surajit Samanta a , Ashis K. Nandi a , Ipsita K. Sen a , Praloy K. Maji a , K. Sanjana P. Devi b , Tapas K. Maiti b , 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 a r t i c l e i n f o Article history: Received 18 April 2013 Received in revised form 22 May 2013 Accepted 2 June 2013 Available online xxx Keywords: Macrolepiota dolichaula Branched glucan NMR studies Immunoactivation 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 an edible mushroom Macrolepiota dolichaula, having average molecular weight ∼2.02 × 10 5 Da. The struc- ture of this PS-I was determined using total hydrolysis, methylation analysis, Smith degradation, partial hydrolysis, and 1D/2D NMR experiments. Total hydrolysis and methylation analysis results showed the presence of (1 → 3, 6)-, (1 → 6)-, (1 → 4)-, (1 → 3)-linked and terminal -d-glucopyranosyl residues in a relative proportion of nearly 1:2:1:1:1. All the chemical and NMR results indicated that the PS-I was a branched 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 well as splenocyte and thymocyte activation in mouse cell culture medium. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Mushrooms are well known for their sweet taste and food fla- voring materials. It also used as an important source of biologically active molecules. Now days, mushrooms have drawn the attention of immunobiologist and pharmacologist for their immunostimu- latory, antitumor, and antioxidant properties [1–3]. The glucans in mushroom are present mostly as linear -(1 → 3)- [4–6], -(1 → 6)- [7–9] and non linear with -(1 → 3) backbone branched at O-6 [10,11], and -(1 → 6) backbone branched at O-3 [12,13]. The genus Macrolepiota, family Agaricaceae was first established by Singer [14]. Few years back, 30 species of the genus Macrolepi- ota were recognized all over the world [15]. In past, this genus was differentiated into two clades by DNA studies [16]. Recently, on the basis of molecular phylogenetic analysis, the genus Macrole- piota has been differentiated into three clades, namely/volvalae, /macrosporae and/macrolepiota [17]. Macrolepiota dolichaula is one of the edible species [18] belongs to the clade/macrolepiota [17] and distributed through out the world. It was reported that this mush- room contained vitamin B 1 , vitamin C, vitamin B 2 , and vitamin A [19]. This mushroom showed laccase enzymatic activity in wheat ∗ Corresponding author. Tel.: +91 03222 276558x437; fax: +91 03222 275329/9932629971. E-mail address: sirajul 1999@yahoo.com (S.S. Islam). straw and czapek medium [20]. Two water soluble polysaccharides (PS-I & PS-II) have been isolated from the hot aqueous extract of this mushroom. Detailed structural elucidation and some preliminary study of immunostimulating properties of the PS-I were carried out and 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 from Vidyasagar University garden, West Bengal, and fruit bodies were washed with distilled water which were then crushed and boiled for 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 above for 30 min. The precipitated polysaccharide was dissolved in a minimum 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 bag and a water-soluble part was obtained after centrifugation and freeze dried to yield water-soluble crude polysaccharide (600 mg). This crude polysaccharide (30 mg) was purified by gel-permeation 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.06.010