Application of pH-zone-refining countercurrent chromatography for the separation of indole alkaloids from Aspidosperma rigidum Rusby

• pH-zone refining CCC successfully isolated indole alkaloids from A. rigidum.
• Three pure alkaloids were obtained in a one step separation of 2 h.
• Inorganic and organic acids acted as efficient eluters in the aqueous mobile phase.
• The stereoisomers 3β-reserpiline and isoreserpiline were separated.
• 3α-Aricine and isoreserpiline were identified for the first time in this species.

Species of Aspidosperma (Apocynaceae) are characterized by the occurrence of indole alkaloids, but few recent reports on Aspidosperma rigidum Rusby chemical constituents were found. The present work shows the application of pH-zone refining countercurrent chromatography on the separation of alkaloids from the barks of A. rigidum. In this study, the dichloromethane extract was fractionated with the solvent system composed of methyl-tert-butyl ether and water with different concentrations of the retainer triethylamine in the organic stationary phase and formic or hydrochloric acids as eluters in the aqueous mobile phase, in order to evaluate the most suitable condition. In each experiment, from circa 200 mg of the dichloromethane extract of A. rigidum, three major alkaloids were isolated and identified as 3α-aricine (circa 17 mg), isoreserpiline (ca. 22 mg) and 3β-reserpiline (ca. 40 mg), with relative purity of 79%, 89% and 82% respectively, in a one-step separation of 2 h. Two of them – 3α-aricine and isoreserpiline – were isolated and identified for the first time in this species.