Method Development for Fractionation of the Butanol Extract from Siparuna glycycarpa by Centrifugal Partition Chromatography (CPC)

Siparuna glycycarpa (Siparunaceae) occurs in the Amazon region. Some species of this genus are used in Brazilian Folk Medicine, being a promising source of bioactive compounds (Leitão et al, 1999). Centrifugal Partition Chromatography (hydrostatic column) is a liquid-liquid separation chromatographic technique that uses a constant-gravity force field produced by a single-axis rotating column. Among the several advantages of the hydrostatic techniques stands the excellent stationary phase retention, especially with solvent systems composed of high-polarity and viscous solvents, making it possible to work at high-flow rates (Costa et al., 2017). In this study, the butanol extract from leaves of S. glycycarpa was fractionated by CPC using BuOH-MeOH-H2O (BuMWat), a polar solvent system which failed stationary phase retention on a hydrodynamic equipment. The leaves were collected at Reserva Florestal Adolpho Ducke, Manaus-AM, Brazil and the crude extract was subjected to liquid-liquid partition between water and the solvents hexane, dichloromethane, ethyl acetate and butanol. The solvent system selection for the fractionation were performed by test tube partition test. The BuOH-MeOH-H2O system was tested at the ratios 10:0:10 (v/v), 9:1:10 (v/v) and 8:2:10 (v/v), with 9:1:10 (v/v) being chosen for fractionation. 500 mg of the extract was dissolved in 10 mL of the upper and lower phases (1:1, v/v) of the system and injected in the 250 mL column of the Gilson CPC System, with rotation of 1000 rpm and 6 mL/min mobile phase flow rate in descending mode. The PLC 2050 Purification System (pump, injector, fraction collector and UV detector) was used. 46 tubes of 12 mL were collected (21 tubes with classic elution and 25 tubes with extrusion). Data were processed using Gilson Glider CPC V5.1e.03 (READER) software. During the fractionation of the BuOH extract in the elution it was possible to monitor (UV detection) the presence of compounds with absorption bands at λ=280 nm, characteristic of phenolic compounds, in fractions 14 to 21. Fractions 36 to 41 collected during the extrusion step showed the presence of two absorption bands (λ=264 nm and λ=352 nm), probably due to the presence of flavonoids. The obtained fractions were analyzed by TLC under UV lamp (254 nm and 365 nm) and pooled according to chemical similarity. 1H NMR (MeOD-d4 and DMSO-d6, 500 MHz) analysis of some of the obtained fractions revealed their hydrolysable tannins nature. The optimization of this method is being developed for the isolation and purification of these compounds and studies continue in order to provide the identification and further information on the chemical structures of these compounds.