brasiliensis mycelia. Since this compound presented a potential anti-HSV activity, its mechanism of action was also evaluated. The fruiting bodies of Agaricus brasiliensis Wasser strain UFSC 51 (syn A. subrufescens, A. blazei) were collected in Biguaçu, Santa Catarina State, Southern Brazil. The characterization BTK inhibitor of the species

was performed by Dr. Maria Alice Neves, and a voucher specimen (FLOR 11 797) was deposited in the FLOR Herbarium (Universidade Federal de Santa Catarina). The mycelium of A. brasiliensis was isolated and cultivated on potato dextrose agar (PDA) (Oxoid, UK) at 25 °C during 7 days. The liquid inoculum was produced by transference of mycelial disks to flasks containing Melin-Norkrans Modified medium (MNM) ( Marx, 1969) and cultivated at 25 °C during 10 days. Mycelia were filtered and fragmented in 300 mL of NaCl 0.8%. The inoculum was then added to MNM in an airlift bioreactor (5 L) and cultivated during 7 days at 26 °C. The liquid culture was centrifuged and the mycelial biomass was dehydrated at 55 °C until constant weight. Agaricus brasiliensis

polysaccharide was this website isolated as previously described ( Camelini et al., 2005), with minor modifications. Fifty grams of dried mycelial biomass were blended twice with five volumes of distilled water and refluxed at 100 °C for 3 h. The material was filtered under vacuum through a Whatman n°42 filter paper. Three volumes of ethanol were added to the filtrate. The mixture was maintained PDK4 at 4 °C for 24 h and centrifuged (1100 g, 10 min). The mycelial polysaccharide was freeze-dried and designated as MI. To produce the sulfated derivative, MI was sulfated using the pyridine-chlorosulfonic acid reagent as described by Zhang et al. (2003). After sulfation, resulting polysaccharides were dialyzed through

a 5 kDa molecular weight cut-off membrane (Spectrum Laboratories, Rancho Dominguez, CA) against distilled water and freeze-dried yielding the sulfated derivative (MI-S). MI and MI-S were characterized by spectroscopic methods [Fourier transform infrared (FTIR) and 13C Nuclear magnetic resonance (13C NMR)] and elemental analyses (C, H, O, S). Determination of homogeneity and molecular weight (Mw) was carried out by high-performance gel filtration chromatography (HPGFC) using a Perkin Elmer series 200 equipment coupled with a RI detector, using a gel filtration column (TSK-Gel 5000 PW 7.8 × 300 mm connected to a TSK PWH 5 × 7 mm guard column; Tosoh, Japan). Samples were eluted with 0.2 M NaCl mobile phase at a flow rate of 1 mL/min. Mean Mw was estimated by comparison with retention times of standard dextrans.