rebaudiana ( Jackson et al. 2009), where preliminary results were obtained using desorption electrospray ionisation mass spectrometry (DESI-MS). We now describe an isolation procedure and structural analysis of fructooligosaccharides (FOS) from aqueous extracts of roots and leaves of S. rebaudiana, including determination of their degree of polymerisation (DP), using matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF) and electrospray ionisation mass spectrometry (ESI-MS). Dried, powdered leaves of Stevia rebaudiana (Bert.) Bertoni were purchased from SteviaFarma, Maringá, Paraná State, Brazil. Roots were collected in the nearby Medicinal
Plant Garden. Voucher specimens of the plant material are deposited in the herbarium of the Department
of Biology at the Maringá State University (identification number 14301-HUEM). All reagents were of analytical XL184 price grade. Dried roots (100.0 g) were powdered and successively extracted with refluxing hexane (1000 mL) for 4 h and methanol (1000 mL) for 4 h. Insoluble material was separated and extracted with boiling water for 4 h (1000 mL). The extract was cooled, stirred for 3 h, and centrifuged buy ABT-199 (8000g, 30 min). The supernatant was evaporated to a small volume, added to EtOH (3×, v/v), left at 4 °C overnight; insoluble fructooligosaccharides were formed as a precipitate on addition to three volumes of EtOH (v/v). Centrifugation (8000g; 20 min) provided sediment, which was collected, washed twice with EtOH at the same 3:1 concentration and dried to give a product (15.7 g). This was dissolved in water (200 mL),
and the solution then submitted to freezing followed by gentle thawing at 4 °C ( Iacomini, Gorin, & Baron, 1988) until no more precipitate appeared. Centrifugation gave, following freeze-drying, the soluble component containing root fructooligosaccharides (RFOS, 4.6 g). Dried leaves of S. rebaudiana (100 g) were extracted with refluxing acetone (1000 mL) for 2 h (3×). The residue was extracted with refluxing water (1000 mL) for 2 h (3×), which was evaporated to a small volume, and added to EtOH (3×, v/v). The resulting precipitate (4.75 g) was dissolved in water (100 mL), and the solution was treated with 10% aqueous Fenbendazole TCA (100 mL) to precipitate protein. After centrifugation, the supernatant was neutralised with aq. NaOH, dialysed and freeze-dried. The residue was dissolved in H2O (100 mL), and the solution was submitted to freeze–thawing until no more precipitate appeared. Centrifugation gave, following freeze-drying, the soluble component (2.68 g). Afterwards, 1.0 g was submitted to treatment using Sartorius ultrafiltration equipment (Model 16249). Commercial membranes with a molecular mass cut-off (MMCO) of 100 kDa and 30 kDa (Millipore) were used. Each one was cut to size and soaked overnight in deionised H2O, prior to use. Filtration experiments were carried out at a constant pressure of 4 bar.