Journal of Chromatography A, 1319 (2013) 166171

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Journal of Chromatography A
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Short communication

Application of pH-zone-rening countercurrent chromatography for

the separation of indole alkaloids from Aspidosperma rigidum Rusby
Mariana N. Vieira a,b, , Suzana G. Leito a , Paula C.C. Porto a , Danilo R. Oliveira a ,
Shaft Corra Pinto a,c , Raimundo Braz-Filho d , Gilda G. Leito e
a

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

Corresponding author. Tel.: +49 17656523379; fax: +49 55 21 2562 6413.

E-mail addresses: mnvieira87@gmail.com, m.neves-vieira@tu-bs.de
(M.N. Vieira).
0021-9673/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.chroma.2013.10.044

fraction collected. On the other hand, one obvious disadvantage

is that the analyte must be ionic (or ionizable) [4]. Furthermore,
such approach in does not allow continuous operation, due to the
fact that the system has to be re-established again after each run.
Nevertheless, this method was successfully applied to the separation of several types of alkaloids [58], including those of the indole
type, which are also the major components of Aspidosperma rigidum
Rusby [9,10].
Species of Aspidosperma genus (Apocynaceae) are generally
trees found in Central and South America, commonly known in
the North of Brazil as Carapanaba, which means mosquitos tree
[11]. In this region, the teas made from its barks are popularly
used to treat several diseases [1214]. To date, only nine alkaloids
were described for A. rigidum. extracts: 3-reserpiline, burnamine,
picraline, caboxine A, caboxine B, isocaboxine, (-) carapanaubine,
isocarapanaubine and haplocidine [10,15].
Based on the ethnopharmacological background and on the
lack of recent studies about this plant, the aim of the present
work was to apply the modern technique of CCC in the pHzone rening mode for the separation of the components present
in the dichloromethane extract of Carapanaba (A. rigidum
Rusby).

M.N. Vieira et al. / J. Chromatogr. A 1319 (2013) 166171

2. Experimental

167

Table 1
pH-Zone rening experiments A to D, solvent system MtBE-H2 O.

2.1. Plant material

Experiment

Retainer (TEA)
concentration (mM)

Eluter concentration

Samples of A. rigidum Rusby were collected in August 2008, in

the Brazilian Amazon region of Oriximin (Par state), at Jauari
community (S 01 15.326 , W 056 .02437 ). Plants were collected as
part of a bioprospecting project in quilombola communities from
Oriximin that received authorization by the Directing Council of
Genetic Heritage (Conselho de Gesto do Patrimnio Gentico),
through the Resolution no. 213 (6.12.2007), published in the Federal Ofcial Gazette of Brazil on December 27, 2007. Samples were
identied by Dr. Washington Marcondes-Ferreira, from Universidade Estadual de Campinas, Campinas, Brazil. A voucher specimen
is deposited at the Instituto Nacional de Pesquisas da Amaznia, INPA, herbarium (Manaus, AM), under the registration INPA
233366.
Dried and ground barks (506 g) of A. rigidum were submitted
to extraction by maceration in percolator rst with hexane, than
dichloromethane, followed by ethyl acetate and methanol, in this
order.

A
B
C
D

15
10
10
5

10 mM formic acid
10 mM formic acid
15 mM formic acid
5 mM hydrochloric acid

2.2. Choice of the solvent systems

The solvent systems tested by test tubes assay were composed
of MtBE-CH3 CNwater in different proportions, as proposed by
Ito [4]. First, two test tubes containing the solvent system to be
tested were prepared. Then, the acid to be used as eluter was added
to one of them and the base (retainer) was added to the other.
Different acids were tested, such as acetic, formic, sulfuric, triuoroacetic and hydrochloric acids; the base used was triethylamine.
Later, small amounts of the extract were dissolved in both tubes
and equal amounts of each phase from the two tubes were spotted separately in a silica gel 60 F254 TLC plate (Merck, Darmstadt,
Germany, Art. 5554) developed with ethyl acetate:acetone:water
(25:8:2) and a drop of a concentrated ammonium hydroxide solution. The result was visualized under UV light (264 and 366 nm) in
a Spectronline Model CC-80 (Spectronics Corporation, USA) spectrometer and then the TLC plate was stained with Dragendorffs
[16] reagent to detect the alkaloids. The K values were estimated
using the equation formulated by Conway [1].
2.3. CCC apparatus
Separations were performed on a P.C. Inc. (Potomac, MD, USA)
countercurrent chromatograph equipped with a triple polytetrauoroethylene multi-layer coil (15 ml + 80 ml +280 ml, 1.6 mm i.d.)
equilibrated by a counterweight. The rotation speed is adjustable
from 0 to 1000 rpm. The 80 ml coil was used in all experiments.
The solvents were pumped with a HPLC Solvent Delivery System
Model M-45 Waters (USA) and the fractions were collected in a
Super Fraction Collector SF-2120 Advantec (Japan).
2.4. pH-Zone rening experiments A to D
Separations were performed by pH-zone rening CCC in the
reverse-displacement mode. The two-phase solvent system utilized was composed of MtBE and water and prepared as described
by Ito and Ma [5]. Different concentrations of the retainer (TEA)
in the organic stationary phase and of the eluter formic acid in
the aqueous mobile phase were tested, as follows: experiment
(A)15 mM TEA and 10 mM formic acid, experiment (B)10 mM
TEA and 10 mM formic acid, and experiment (C)10 mM TEA and
15 mM formic acid. Also, an experiment using 5 mM TEA and 5 mM
hydrochloric acid was performed (experiment D). To prepare the
sample solution approximately 200 mg of the dichloromethane

extract of the barks of A. rigidum were dissolved in a mixture of

3 ml of the organic stationary phase with the retainer (TEA) and
3 ml of the aqueous mobile phase free of the acidic eluter.
2.5. Separation procedures
First, the column was lled with the organic stationary phase,
saturated with the mobile phase and containing the retainer (TEA),
in a ow rate of 5 ml/min. Then, the rotation was turned on at the
speed of 850 rpm and the sample solution was injected through
the sample port, using a 5 ml loop. After this, the aqueous mobile
phase containing the acidic eluter was pumped at a ow rate of
2 ml/min. About 60 fractions of 4 ml were collected (rotation was
turned off at tube 40). Fractions were analyzed by TLC using the
same conditions as specied in Section 2.3 and pooled together
by means of chromatographic similarity. Retention of the stationary phase was determined by measuring the volume of stationary
phase displaced until the solvent front; i.e. the rst tube that presented two phases, then subtracting it from the total coil volume
and calculating the percentage of stationary phase that remained
inside the coil [17]. The pH values of each fraction were manually
measured with a portable pH meter model Q400BC, from Quimis
(Brazil).
2.6. Identication of the isolated substances
The fractions containing the isolated alkaloids were concentrated in rotary evaporator (under reduced pressure, at 40 C) and
then identied by the combination of 1D-/2D-NMR and MS spectral
data. NMR experiments were performed on a Varian 500 MHz spectrometer. All samples were dissolved in deuterated chloroform, and
tetramethylsilane (TMS) was used as internal standard. Chemical
shifts () are reported in ppm. The MS experiments were made by
direct injection on a GCMS QP 5000 Shimadzu equipment, using
electron impact ionization at 70 eV. UV data were obtained from
HPLC-DAD injections.
2.7. HPLC analyses
The relative purity of the fractions was obtained by HPLC
in a Merk-HITACHI LaChrom (Germany) apparatus L-7000, with
UV/DAD detector L-7450, using a reversed phase ODS reversible
analytical column (25 cm 4.6 mm, 5 m) from Rexchrom Regis
(USA) at room temperature of 25 C. The mobile phase solvent
system consisted of a mixture of H2 O/TFA (pH 3; 0.025%) and
CH3 CN/TFA (0.025%) in a stepwise gradient as follows: 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. The
injection volume was 20 l, the ow rate 1.0 ml/min and detection
was performed at  250 nm. The dichloromethane extract, as well
as the fractions containing the separated alkaloids, were dissolved
in a mixture (1:1) of the mobile phase components.
LCMS experiments were performed, in order to determinate
the molecular weight of the isolated alkaloids. Samples ran in a
HCT-Ultra ETD II (Bruker Daltonics) equipment, with a Prontosil
and pre-column
C18-Aq column of 250 2.0 mm, 5 micron (100 A)

168

M.N. Vieira et al. / J. Chromatogr. A 1319 (2013) 166171

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).

M.N. Vieira et al. / J. Chromatogr. A 1319 (2013) 166171

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.

of the same material, at room temperature of 20 C. The mobile

phase solvent system consisted of a mixture of H2 O/TFA (pH 3;
0.025%) and CH3 CN/TFA (0.025%) in a linear gradient from 80:20
to 10:90 (0 to 50 min). The injection volume was 10 l, the ow

rate 0.25 ml/min and detection was performed by DAD ( between

200 and 700 nm) and MS. The MS measurements were made in the
range of m/z 100 to 2000 with electrospray ionization in the positive
mode. The fragments obtained represented m/z [M+ + 1].

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M.N. Vieira et al. / J. Chromatogr. A 1319 (2013) 166171

3. Results and discussion

Preliminary thin layer chromatography (TLC) analysis of the
extracts obtained from the bark of A. rigidum showed the presence of four major alkaloids in the dichloromethane extract when
stained with the Dragendorffs reagent and, therefore, this was chosen for the separation experiments. As alkaloids can be regarded as
weak organic bases, the reverse mode displacement pH-zone CCC
technique was applied for the purication of these compounds.
According to Ito [4] the suitable two-phase solvent system for
pH-zone rening CCC is the one in which Kacid  1 in the experiments from the acidic condition, what was observed when adding
formic acid to the test tubes, and Kbase  1 in the experiments from
the basic condition. The solvent systems tested were composed
of MtBE-CH3 CNwater in different ratios, as follows: 1:0:1 (S1),
4:1:5 (S2), 6:3:8 (S3) and 2:2:3 (S4) (v/v/v). Three of these (S2 to
S4) showed an ideal Kacid value, but Kbase 1. The best condition
was achieved when no CH3 CN was added to the system, making
the system MtBE and water the most suitable choice (data not
shown).
3.1. Fractionation of the A. rigidum dichloromethane extract
Four experiments were performed in order to optimize the concentrations of the retainer base (TEA) and eluter acid (Table 1).
Each of the three rst experiments (A to C) provided, from
circa 200 mg of the dichloromethane extract of A. rigidum, around
17 mg of 3-aricine, 1, 22 mg of isoreserpiline, 2, and 40 mg of 3reserpiline, 4, in a one-step separation of 2 h.
Fig. 1 shows the results of the four pH-zone rening experiments (A to D) as analyzed by both TLC and pH measurements of
each fraction tube. In experiment A (Fig. 1A) the elution of the alkaloids begins after around 54 min, coinciding with the decrease of the
mobile phases pH value. First, 3-reserpiline, 4, elutes as a mixture
with a small amount of 3 (non identied alkaloid) until isoreserpiline, 2, starts to elute. After another pH decrease, 2 elutes, followed
by 3-aricine, 1. The stationary phase retention was of 67% and the
pH at zones are observed at pH 6.5, 5.0, 3.7 and 2.7.
In experiment B (Fig. 1B), elution of the alkaloids starts around
10 min earlier than in A. First, a small decrease in the mobile phase
pH was observed, representing the beginning of the elution of
the mixture of 3-reserpiline, 4, and 3. This lasts for 6 min, then
the pH decreases again and elution of isoreserpiline, 2, starts. In
this experiment, there is an increase in resolution between 2 and
1, in comparison with A. The stationary phase retention was of
40% and the pH at zones are observed at pH 9.0, 5.5, 3.5 and
2.4.
Fig. 1C shows the result of experiment C. Here, elution begins
even faster than in B, with the decrease of the pH values observed
at around 38 min after the run started. In this experiment, although
3-reserpiline, 4, eluted in a mixture with 3, the proportion of the
latter was signicantly smaller. It is worth noting that also cleaner
fractions of 2 were obtained, as well as those containing isoreserpiline, as the increased resolution was maintained. This fact was
corroborated by the observation of an additional pH at zone, compared to experiments A and B. These were at pH 7.3, 5.9, 4.7, 3.6 and
2.7. The stationary phase retention was of 67%.
All the experiments showed a reasonable separation of the components from the alkaloidic dichloromethane extract of A. rigidum
and allowed the isolation of three alkaloids 1, 2 and 4 with
relative purity of 79%, 89% and 82% respectively. In all experiments, alkaloid 3 eluted in mixture either with 2 or 4. Comparing
the experiments, it was possible to observe that increasing the
eluter acid concentration in the aqueous mobile phase resulted in
a shorter retention time of the analytes. Reducing the concentration of the retainer base in the upper organic phase changed the

stationary phase properties, increasing the resolution between the

alkaloids.
The pH-zone rening CCC method is characterized by the production of pH-zones according to pKa and hydrophobicity of the
analytes [17], but in experiments A to C the major components were
eluted with an abrupt variation from basic to acid in a few minutes
in all experiments. In terms of chromatogram, it could represent a
peak sharpening, that occurs when the K of the retainer (Kr ) falls
between the analytes K under acidic (Kacid ) and basic (Kbase ) conditions [4]. This fact can be due to the utilization of an organic acid,
which is relatively weak when compared to a mineral acid, in the
aqueous mobile phase. Although it is not usual to add an organic
modier to the inorganic (aqueous) phase, this work showed that
in some cases it can provide the most suitable condition for the
separation of the compounds.
On the other hand, 3-aricine, 1, did not seem to be affected
by the change in the retainers and/or eluters concentrations. It
eluted at the latest plateau during circa 18 min, after the retainer
be entirely removed from the system, which means that its Kacid
and Kbase values are greater than Kr [4].
In order to analyze the inuence of the chemical nature of
the acid in the separation, an experiment using the inorganic
hydrochloric acid, instead of the organic formic acid was also made.
As shown in Fig. 1D the pH at zones were better dened. Elution
of the alkaloids started after around 40 min, yielding fractions containing only 3-reserpiline, 4, followed, however, by mixtures of
3 + 2 and 3 + 1. The stationary phase retention was of 55% and the
pH at zones are observed at pH 5.6, 5.0 and 2.6.
The four pH-zone rening experiments (A to D) afforded the
separation of indole alkaloids from the dichloromethane extract of
the barks of A. rigidum to different extents. By using hydrochloric
acid it was possible to separate 3-reserpiline, 4, from the other
alkaloids, but not to resolve 2 from 1. With the organic formic acid;
however, this separation was possible in experiment C, although a
major overlapping of the other components was observed.

3.2. Identication of the alkaloids

Identication of the alkaloids 1, 2 and 4 was carried out by
the combination of data from HPLC-UV, LCESIMS and 1D-/2DNMR analyses. Therefore, 1 was identied as 3-aricine (MW 382
u) [1821], 2 as isoreserpiline (MW 412) and 4 as 3-reserpiline
(MW 412) [18,19,21], as shown in Fig. 2. Compound 3 remains
unidentied.
3-aricine (1): UV and 1 H-NMR data [1821]; 13 C NMR
(125 MHz, CD3 OD) 168.16 (C-22), 155.48 (CH-17), 153.60 (C10), 134.95 (C-2), 131.85 (C 13), 127.17 (C-8), 111.17 (CH-12),
110.25 (CH-11), 109.49 (C-16), 106.42 (C-7), 99.49 (CH-9), 72.15
(CH-19), 60.43 (CH-3), 55.66 (CH2 -21, 54.83 (MeO-10), 53.48 (CH2 5), 50.21 (MeO-22), 38.44 (CH-20), 33.32 (CH2 -14, 31.24 (CH-15),
21.04 (CH2 -6), 17.43 (CH3 -18). MS-EI (70 eV): m/z 281 (29%), 253
(50%), 199 (37%), 186 (100%).isoreserpiline (2): UV, 1 H and 13 C-NMR
data [18,19,21]. MS-EI (70 eV): m/z 311 (40%), 283 (74%), 229 (46%),
216 (100%).
3-reserpiline (4): UV, 1 H and 13 C-NMR data [18,19,21]. MS-EI
(70 eV): m/z 311 (59%), 283 (100%), 216 (50%).

3.3. Analysis of the fractions by HPLC

The extracts, as well as the separated fractions, were analyzed
by HPLC and three intense peaks could be detected (Fig. 3A), which
correspond, respectively, to compounds 2 and 4 (Rt 16.27 min, coeluted), 1 (Rt 17.84 min) and 3 (Rt 21.28 min). Although alkaloids
2 and 4 showed the same retention time in this HPLC conditions,

M.N. Vieira et al. / J. Chromatogr. A 1319 (2013) 166171

171

they were well separated in the pH zone rening CCC experiments,

as shown by their NMR data.

offering the resources to run the LCMS analysis. This work was
partially supported by CNPq (scholarship and grant).

4. Conclusions

References

The overall results of our work demonstrate that the pH-zone

rening counter-current chromatography was successfully applied
to the fractionation of indole alkaloids from the dichloromethane
extract of Aspidosperma rigidum Rusby. Two of them 3-aricine,
1, and isoreserpiline, 2 were isolated and identied for the rst
time in this species. We also demonstrated that both organic and
inorganic acids can be used as eluters in the aqueous mobile phase
of the pH-zone rening countercurrent fractionation of these indole
alkaloids. Although the prole of the experiments was different, all
of them produced efcient separations. The present method may
be applied to various other indole alkaloids from natural products.

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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