Four columns were used in series, with exclusion

sizes of 7 × 106 Da (Ultrahydrogel 2000, Waters), 4 × 105 Da (Ultrahydrogel 500, Waters), 8 × 104 Da (Ultrahydrogel 250, Waters) and 5 × 103 Da (Ultrahydrogel 120, Waters). The eluent was 0.1 M aqueous NaNO2 containing 200 ppm aqueous NaN3 with a flow rate of 0.6 ml/min, at room temperature. The sample, previously filtered through a membrane (0.22 μm, Millipore), was injected (250 μl loop) at a concentration of 1 mg/ml. The specific refractive index increment (dn/dc) was determined and the results were processed with software ASTRA provided by the manufacturer (Wyatt Technologies). The purified polysaccharides were O-methylated according to the KU-57788 method of ( Ciucanu and Kerek (1984) using powdered NaOH in DMSO–MeI. The per-O-methylated derivatives were hydrolyzed GSK126 with 45% formic acid for 16 h at 100 °C and the resulting mixture of partially O-methylated monosaccharides was

successively reduced with NaBD4 and acetylated with Ac2O–pyridine. The products (partially O-methylated alditol acetates) were examined by capillary GC–MS. A capillary column (30 m × 0.25 mm i.d.) of DB-225, held at 50 °C during injection for 1 min, then programmed at 40 °C/min to 210 °C and held at this temperature for 31 min was used for separation. The partially O-methylated alditol acetates were identified by their typical electron impact breakdown profiles and retention times ( Sassaki, Iacomini, & Gorin, 2005). 13C1H NMR spectra were acquired at 50 °C on a Bruker AVANCE III 400 NMR spectrometer, operating at 9.5 T, observing 13C at 100.61 MHz, equipped with a 5-mm multinuclear inverse detection probe with z-gradient. The water soluble samples were acquired in D2O and the water-insoluble ones in DMSO-d6. All 13C NMR chemical shifts are expressed in ppm relative to CH3 signal from acetone at δ 30.2 or DMSO-d6 at δ 39.7 as internal references. For 1H–13C directed correlation from HSQC (Heteronuclear Single-Quantum Coherence) Decitabine datasheet NMR experiment, the fraction was deuterium-exchanged

three times by freeze-drying with D2O solutions, finally dissolved in DMSO-d6 and transferred into a 5-mm NMR tube. All pulse programs were supplied by Bruker. Experiments were conducted using female Swiss mice (25–35 g) from the Federal University of Paraná colony. Animals were kept under standard laboratory conditions (12 h light/dark cycles, temperature 22 ± 2 °C) with food and water provided ad libitum. Animals were acclimatized to the laboratory for at least 12 h before testing and were used only once for experiments. All the experiments were performed after approval of the respective protocols by the Committee of Animal Experimentation of the Federal University of Paraná (CEUA/BIO – UFPR; approval number 613).