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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma
Short communication
Universidade Federal do Rio de Janeiro, Faculdade de Farmcia, CCS, Bl. A 2o andar, Ilha do Fundo 21941-590, RJ, Brazil
Institute of Food Chemistry, Technische Universitt Braunschweig, Schleinitzstrasse 20, 38106 Braunschweig, Germany
c
Curso de Farmcia/Campus UFRJ-Maca, Rua Aluisio da Silva Gomes, 50, Granja dos Cavaleiros, Maca 27930-560, RJ, Brazil
d
Laboratrio de Cincias Qumicas, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Av. Alberto Lamego, 2000, Campos dos Goytacazes
28013-602, RJ, Brazil
e
Universidade Federal do Rio de Janeiro, Ncleo de Pesquisas de Produtos Naturais, CCS, Bl. H, Ilha do Fundo 21941-590, RJ, Brazil
b
a r t i c l e
i n f o
Article history:
Received 22 May 2013
Received in revised form 10 October 2013
Accepted 12 October 2013
Available online 22 October 2013
Keywords:
Indole alkaloids
Aspidosperma
pH-zone-rening countercurrent
chromatography
a b s t r a c t
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 rening 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 identied as 3aricine (circa 17 mg), isoreserpiline (ca. 22 mg) and 3-reserpiline (ca. 40 mg), with relative purity of 79%,
89% and 82% respectively, in a one-step separation of 2 h. Two of them 3-aricine and isoreserpiline
were isolated and identied for the rst time in this species.
2013 Elsevier B.V. All rights reserved.
1. Introduction
High-speed countercurrent chromatography (HSCCC) is an
hydrodynamic preparative technique based on the distribution
coefcient (K) of substances between the two phases of a biphasic
solvent system, where one of them is the stationary phase and the
other acts as mobile phase [1,2]. Specially for the case of ionizable
molecules like organic acids and bases, a method proposed by Ito
[3,4] allows the separation process also according to pKa values
and hydrophobicity of the substances. For the separation of alkaline organic compounds, this method consists on the addition of a
basic retainer to the stationary phase and an acidic eluter to the
mobile phase [4].
pH-Zone rening CCC shows some advantages when compared
to conventional CCC, for example the increase of the sample loading
capacity, the high concentration of the fractions and the possibility of monitoring the analysis by measuring the pH value of each
2. Experimental
167
Table 1
pH-Zone rening experiments A to D, solvent system MtBE-H2 O.
Experiment
Retainer (TEA)
concentration (mM)
Eluter concentration
A
B
C
D
15
10
10
5
10 mM formic acid
10 mM formic acid
15 mM formic acid
5 mM hydrochloric acid
168
Fig. 1. TLC plates with the results of the pH-zone rening CCC. Experimental conditions: TLCsilica-gel plates developed with: ethyl-acetate:acetone:water (25:8:2) and a
drop of a concentrated NH4 OH solution, detection: Dragendorffs reagent; CCCcoil volume: 80 ml, ow-rate: 2 ml/min, rotation speed: 850 rpm, sample loading: 200 mg,
fraction size: 4 ml, solvent system: MtBEwater; (A) 15 mM TEA in the OSP and 10 mM formic acid in the AMP, SPR: 67%; (B) 10 mM TEA in the OSP and 10 mM formic acid
in the AMP, SPR: 40%; (C) 10 mM TEA in the OSP and 15 mM formic acid in the AMP, SPR: 67%; (D) 5 mM TEA in the OSP and 5 mM hydrochloric acid in the AMP, SPR: 55%.
OSPorganic stationary phase, AMPaqueous mobile phase, SPRretention of stationary phase.
Fig. 2. Chemical structures of the alkaloids isolated from A. rigidum Rusby (1, 2 and 4).
169
Fig. 3. HPLC analyses of the dichloromethane extract from the barks of A. rigidum (A) and the isolated alkaloids (B, C, and D). Experimental: Rexchrom Regis reversed phase
ODS reversible analytical column (25 cm 4.6 mm, 5 m), temperature of 25 C, mobile phase: H2 O/TFA (pH 3; 0.025%) and CH3 CN/TFA (0.025%) stepwise gradient from
65:35 to 55:45 (0 to 10 min); from 55:45 to 40:60 (10.1 to 15 min); from 40:60 to 25:75 (15.1 to 28 min) and 0:100 from 28.1 to 30 min, ow rate 1.0 ml/min, detection:
250 nm, injection volume: 20 l.
170
171
offering the resources to run the LCMS analysis. This work was
partially supported by CNPq (scholarship and grant).
4. Conclusions
References
Acknowledgments
The authors are thankful for Dr. Mas help with the pH-zone
rening practical application and Dr. Itos explanations about some
theoretical doubts. In addition, we would like to thank Prof. Dr.
R. Braz-Filho and Prof. Dr. B. Gilbert, for the help with the identication of the alkaloids. We are also deeply indebted to the
Centro Nacional de Ressonncia Magntica Nuclear Jiri Jonas and
LAMAR NPPN-UFRJ, Rio de Janeiro, for the NMR experiments
and to ARQMO, Associaco de Comunidades Remanescentes de
Quilombos do Municpio de Oriximin, Oriximin-PA, Brazil, for
supervising the plant collection. Furthermore, we wish to acknowledge the help provided by Prof. Dr. Peter Winterhalter and Dr.
Gerold Jerz (Technische Universitt Braunschweig, Germany) for