Dried pulp powder was defatted with chloroform–methanol (1:1) in order to remove lipids, pigments and other hydrophobic material. Polysaccharides were extracted from the residue with water at 100 °C for 2 h (×7, l L each). The aqueous extracts were obtained by centrifugation Selleck Selumetinib (8000 rpm, 20 min at 25 °C), combined and concentrated under reduced pressure. The polysaccharides were precipitated

with EtOH (3 vol.), collected by centrifugation (3860g, 20 min at 4 °C) and freeze–dried, giving fraction TW. The remaining residue was then extracted four times (1 L each) with aq. 10% KOH, at 100 °C for 2 h and the alkaline extracts were neutralized with acetic acid, dialyzed for 48 h with tap water, concentrated under reduced pressure and freeze–dried, generating fraction TK ( Cordeiro et al., 2012). A freeze–thaw treatment was applied to fractions TW and TK to give cold-water GS-7340 soluble fractions STW and STK, respectively (Fig. 1B). In this procedure, the sample was frozen and then thawed at room temperature. Insoluble polysaccharides (fractions PTW and PTK, respectively) were recovered by centrifugation (3860g, 20 min at 4 °C).

In order to remove starch, these fractions were extensively treated with α-amylase (from Bacillus licheniformis, Sigma A3403) and dialyzed. Fraction STK was submitted to ultrafiltration through membrane (Millipore, PLHK04710-Ultracel) with cut-off of 300 kDa. The retained fraction on the membrane (fraction STK-300R) was further submitted to closed dialysis (in bag with cut-off of 1000 kDa) against distilled water for 48 h. The eluted fraction Arachidonate 15-lipoxygenase through 300 kDa ultrafiltration membrane (STK-300E) was further

treated with Fehling solution and the precipitated material (PF) separated by centrifugation (Fig. 1B). The Cu2+-precipitate was neutralized with AcOH, dialyzed against tap water, deionized with mixed ion exchange resins and then freeze–dried. The yields were expressed as % based on the weight of dried tamarillo pulp that was submitted to extraction (235 g). Neutral monosaccharide components of the polysaccharides and their ratio were determined by hydrolysis with 2 M TFA for 8 h at 100°C, followed by conversion to alditol acetates by successive NaBH4 or NaBD4 reduction and acetylation with Ac2O–pyridine (1:1, v/v, 1 ml) at room temperature for 14 h. The resulting alditol acetates were then extracted with CHCl3. These were analyzed by GC–MS using a Varian model 3300 gas chromatograph linked to a Finnigan Ion-Trap model (ITD 800) mass spectrometer with He as carrier gas. A capillary column (30 m × 0.25 mm i.d.) of DB-225 was used for the quantitative analysis (Cordeiro et al., 2012). During injection, column temperature was held at 50 °C for 1 min, then programmed at 40 °C/min to 220 °C and held at this constant temperature for 19.75 min. Uronic acid contents were determined using the m-hydroxybiphenyl method ( Filisetti-Cozzi & Carpita, 1991).