Fractionation of Schinus terebinthifolius Extracts by CCC

Schinus terebinthifolius Raddi is popularly known as aroeira, aroeira da praia, aroeira vermelha or pink pepper. It is a native plant of South America, amply distributed in Brazil. It has been used as a medicinal plant for many years due to its anti-inflammatory, wound-healing, antimicrobial and anti-tumor activity; and popularly applied to the treatment of respiratory tract and urinary tract infections, diarrhea, sexually transmitted diseases and ulcers. The purpose of this study was to isolate fractions from the ethanolic extracts of leaf by countercurrent chromatography (CCC). Fresh leaves of S. terebinthifolius were extracted with Ethanol PA (99.9%), in the proportion of 1 gram of plant material to 5 mL of solvent. The extraction was made by ultrasound assisted maceration during 30 minutes. The extracts were filtered and solvent was removed in a rotary evaporator at 50°C and then concentrated under vacuum. This procedure resulted in oily-viscous crude extracts and the yield was 3% (m/m). For the CCC separation a Midi Centrifuge (Berkshire, United Kingdon) equipment coupled to a HPLC UV/Vis, 1200 series, Agilent Technologies detector and a Model 202, Gilson fraction collector was used. A solvent system beginning with Heptane:Ethyl Acetate:Methanol:Water (6/1/6/1) with step gradient to (19/1/19/1) at 40 min in reverse mode was applied. The flow started with 20 mL/min and increased to 40 mL/min at 20 min. The total time of separation was 99 minutes, resulting in 85 fractions in elution mode; and 23 fractions in extrusion mode. The fractions were analyzed by thin-layer chromatography (TLC) using a normal phase silica gel TLC Plates 60 F254 (Merck Art. 05554, Darmstadt, Germany). The mobile phase was CHCl3–EtOAc 2:1 (v/v) and the results were observed under visible and UV light. These fractions were combined, according the TLC results and concentrated in evaporator under vacuum at 40°C. The recovery was 85%. These 26 fractions were analyzed by ultra-high performance liquid chromatography (UPLC Acquity. Waters) coupled to mass spectrometry (TQD Acquity, Waters) with electrospray ionization in the negative and positive ion modes. For the separation a C18 BEH Waters Acquity (2.1 mm × 50 mm × 1.7 μm) column was used. The analytical conditions were: solvent A (0.1% ammonium hydroxide solution in purified water); solvent B chromatographic grade methanol; beginning 30% A and 70% B, ramping to 100% B in 7 min, maintaining these conditions until 9 minutes, returning to the initial conditions at 9.1 minutes andstabilizing until 12 min. Both UHPLC-MS and TLC plates showed that we got enriched fractions of leaf extract and the results confirmed that CCC is a reproducible and fast technique to fractionate a complex matrix such as aroeira leaf extract.