Research Article

Received: 26 February 2008, Revised: 26 August 2008, Accepted: 1 September 2007 Published online 11 November 2008 in Wiley Interscience

(www.interscience.wiley.com) DOI 10.1002/pca.1100

Isolation of Limonoids from Seeds of Carapa

John Wiley & Sons, Ltd.

guianensis Aublet (Meliaceae) by High-Speed

Countercurrent Chromatography
Vagner Pereira da Silva,a Rodrigo Rodrigues Oliveirab*
ISOLATION OF LIMONOIDS FROM SEEDS OF CARAPA GUIANENSIS AUBLET (MELIACEAE)

and Maria Raquel Figueiredoa

ABSTRACT:
Introduction – Limonoids are tetranortriterpenoids of considerable interest due to their structural varieties and biological
activities, such as insecticidal, antibacterial, antifungal, antimalarial, anticancer and antiviral. They contain oxygen atoms
that confer a moderate polarity and are responsible for the difficulties in their separation by traditional chromatographic
methods. High-speed countercurrent chromatography (HSCCC) is a versatile liquid–liquid separation technique, in which the
sample is distributed between two non-miscible phases to achieve separation.
Objective – To isolate limonoids from a complex Carapa guianensis seed extract by gradient elution HSCCC and to identify
them by spectrometric and spectroscopic methods.
Methodology – The hexane extract of Carapa guianensis squeezed seeds was prepared by Soxhlet extraction. From this
extract, 800 mg were submitted to gradient mode HSCCC, using the solvent systems hexane:ethyl acetate:methanol:water
1:2:X:1, X = 1.5 (system A) and X = 1.75 (system B). The upper organic phase of the system A was used as stationary phase, and
the lower aqueous phases of both systems as mobile phases. In this procedure, 165 fractions of 4 mL (660 mL) were collected.
Results – Six compounds were isolated. Spectrometric and spectroscopic analysis allowed the identification of the substances,
as follows: methyl angolensate (28.7 mg), 7-descetoxy-7-oxogedunin (17.9 mg), deacetylgedunin (3.7 mg), 6a-acetoxygedunin
(40.1 mg), gedunin (21.0 mg), and andirobin (5.8 mg).
Conclusion – The use of gradient mode in HSCCC was a good alternative, exploiting small variations of partition coefficient
between the substances. Thus it was possible to isolate them in a good relative abundance, compared with classical
chromatographic methods. Copyright © 2008 John Wiley & Sons, Ltd.

Keywords: Limonoids; meliaceae; Carapa guianensis; high-speed countercurrent chromatography

Introduction
Limonoids are highly oxygenated tetranortriterpenoid compounds
that are reported to possess a wide range of biological activities,
such as insecticidal, antifeedant and growth-regulator on insects
(Champagne et al., 1992), antibacterial, antifungal, antimalarial,
and antiviral (Roy and Saraf, 2006). Some limonoids are known
to induce the in vivo production of the detoxifying enzyme
glutathione S-transferase in the liver, and to inhibit the formation
of chemically induced neoplasia in the oral cavity, forestomach,
small intestine, colon, lung and skin of laboratory animals (Manners
et al., 2003; Yu et al., 2005), as well as the proliferation of breast
cancer cells grown in culture (Yu et al., 2005). Recent reports have
also shown the antiprotozoal activity of limonoids on Leishmania
donovani, Trypanossoma brucei rhodesiensis, Trypanossoma
cruzi and Plasmodium falciparum strains (Hay et al., 2007).
Limonoids are found mainly in species of the Rutaceae,
Meliaceae, Cneoraceae and, less frequently, in Simaroubaceae
families (Champagne et al., 1992; Júnior, 2003; Dewick, 2004; Roy
and Saraf, 2006). A rich source of limonoids is the seeds of Carapa
guianensis Aublet, also called andiroba, a tall Meliaceae tree
growing wild throughout South America, West India and South
Africa. In Brazil, it can be found prevalently in flooded areas of
the Amazon rainforest. From the seeds of this plant is extracted
the andiroba oil, which has a long history of traditional use in
South America, e.g. analgesic, anti-inflammatory, insecticide, anti-
bacterial, anti-parasitic and anti-cancer medicines (Ambrozin
et al., 2006). The potential health benefits of the andiroba oil
have led to the search for new methods to separate and to
purify limonoids at satisfactory yield levels.
* Correspondence to: R. R. Oliveira, Laboratório de Ciências Químicas, CCT,
Universidade Estadual do Norte Fluminense Darcy Ribeiro; Avenida Alberto
Lamego 2000, Horto—28013-602, Campos dos Goytacazes—RJ, Brazil. E-
mail: roroliveira@uenf.br
a
Laboratório de Química de Produtos Naturais, Far-Manguinhos, Fundação
Oswaldo Cruz; Rua Sizenando Nabuco 100, Manguinhos 21041-250, Rio de
Janeiro, RJ, Brazil
b
Laboratório de Ciências Químicas, CCT, Universidade Estadual do Norte
Fluminense Darcy Ribeiro; Avenida Alberto Lamego 2000, Horto 28013-602,
Campos dos Goytacazes, RJ, Brazil
Contact/grant Sponsor: Analytical Center and NMR Laboratory of Far-
Manguinhos. Fiocruz (Manguinhos. Rio de Janeiro, Brazil)
Contact/grant Sponsor: PDTIS/Instituto de Tecnologia em Fármacos/FIOCRUZ
77

Phytochem. Anal. 2009; 20: 77–81 Copyright © 2008 John Wiley & Sons, Ltd.

High-speed countercurrent chromatography (HSCCC) is a
separation technique based on the partition of a solute between
two immiscible liquid phases without any solid support matrix.
Therefore it eliminates the irreversible loss of sample observed
in conventional adsorption chromatography. The centrifugal
force provided by rotation retains the stationary phase in the
coil tubes (Marston and Hostettmann, 2006). Recently, gradient
elution in HSCCC has been widely employed in natural product
separations, but rather little attention has been given to the use
of solvent system gradients, in contrast to the usual isocratic
elution (Oliveira et al., 2005). However, gradient elution in HSCCC
may be the choice for the purification of mixtures containing
compounds with similar polarities, where slight differences in
their partition coefficients must be exploited. Some favorable
solvent systems are stable to drastic changes of mobile phase
composition during separation. Therefore, a gradient elution
mode applied to HSCCC appears to simplify separation of crude
plant extracts, whose constituents range over a wide variation of
polarity (Du et al., 2004).
Although there is a risk of disturbing the liquid–liquid equi-
librium and causing elution of the stationary phase, gradients
are indeed possible. Furthermore, in certain circumstances, the
composition of one phase may be systematically varied while
the other remains nearly constant (Marston and Hostettmann,
1994). This method has been successfully applied in HSCCC for
triterpenoid separation (Oliveira et al., 2005).
Experimental
Apparatus. The HSCCC was performed in a Pharma-Tech
Research Corp. (Baltimore, Maryland, USA) model HSCCC-1000
apparatus, equipped with an interchangeable triple coil with
a total capacity of 325 mL, 2.6 i.d. and β value range 0.5–0.75.
The HSCCC system was equipped with an HSCCC Electronic
Controller, Pharma-Tech Research Corp., a solvent pump
ConstaMetric 3500, LDC Analytical, a manual injection valve
with a 10 mL loop and a fraction collector CF1 from Spectrum
Chromatography.
HPLC analysis. The HPLC system consisted of a Shimadzu (Kyoto,
Japan) model Prominence liquid chromatograph, a Shimadzu
LC-20AT pump equipped with a quaternary gradient valve, a
Shimadzu SPD-M20A diode array detector, a Shimadzu SIL-20A
auto-sampler and a Shimadzu CBM 20-A communications bus
module. Separation was achieved on a Merck Lichrospher 100
C18 column (250 × 4.0 mm i.d.; 5 μm particle size). The mobile
phase was acetonitrile:water:methanol (35:35:30 v/v), the injec-
tion volume was 20 μL at 1 mg/mL, the flow rate was 0.9 mL/min
and the effluent was monitored at 210 nm.
GC-MS spectrometry. Analyses were carried out on a Hewlett-
Packard Agilent (Santa Clara, California, USA) model 6890N gas
chromatograph coupled to a 70 eV electron impact ionisation
detector model 5973 mass spectrometer and a model 7683
automatic injector, equipped with an HP-5MS capillary column
(30 m × 0.25 mm i.d.; 0.30 μm film thickness), under the following
conditions: carrier gas, helium at 2.0 mL/min; split ratio, 1:20;
injector temperature, 290°C; ion source temperature 290°C;
and oven temperature programmed from 150 to 290°C at 15°C/min
and then held for 15 min. The volume of injected samples was of
1 μL.
78
www.interscience.wiley.com/journal/pca Copyright © 2008 John Wiley & Sons, Ltd.
V. P. da Silva et al.
NMR spectroscopy. 1H and 13C NMR spectra were acquired on
a Bruker (Rheinstetten, Baden-Württemberg, Germany) equip-
ment model DRX 400 at 400.13 and 100.61 MHz, respectively;
and on a Bruker equipment model Advance 500 at 500.13 and
125.77 MHz, respectively.
Choice of the solvent systems. Test tube experiments and TLC
investigation on the separation of limonoids (Oliveira et al.,
2005) afforded the basis to select and optimise the solvent
systems as hexane:ethyl acetate:methanol:water 1:2:X:1 v/v,
X = 1.5 (system A) and X = 1.75 (system B). Both were mechanically
shaken for 30 min and then degassed using an ultrasonic
cleaner for 15 min before the experiments.
Plant material, extraction and sample preparation. After
extracting the andiroba oil, the squeezed seeds of C. guianensis
were kindly donated by the Company COPALMA, Macapá-AP,
Brazil. This material (937.5 g) was exhaustively submitted to a
Soxhlet extraction with hexane (2.7 L). The extract was then
cooled and the limonoid mixture precipitated. The supernatant
was set apart and the precipitate (5.98 g) was recovered and
labeled as ‘crude hexane extract’. From this extract 800 mg were
dissolved in 5 mL of each upper and lower phases of the solvent
system A. The non-soluble material was discarded, and 10 mL of
the solution containing 749.4 mg of the sample, measured by
differential weight, were injected.
HSCCC separation procedure. For separation with the gradient
solvent system mode, the methanol proportion was modified
during the chromatography run as: 1:2:X:1 v/v, X = 1.5 (A);
X = 1.75 (B). The HSCCC column was filled with the upper
organic phase of solvent system A, and the rotor was started and
set at 900 rpm. The lower aqueous phase of system A was
pumped in the head-to-tail direction at the flow-rate of 1 mL/min.
The organic stationary phase volume (Vs) initially retained in the
HSCCC column was 250 mL (Sf = 76.9%; VM = 75 mL). The solution
(10 mL) containing the limonoid mixture was injected, and the
mobile phase eluted from the chromatographic column was
collected in 4 mL fractions. After collecting 76 fractions (304 mL,
corresponding to Kd = 1), the rotation was stopped and the
lower aqueous phase of system B was then pumped into the coil
at the same operational conditions to afford 89 fractions (356
mL). The rotation was stopped and the coil content (upper and
lower phases) was collected. All fractions were combined
according to their TLC profile (Fig. 1). TLC silicagel plates were
eluted in hexane:ethyl acetate 6:4 and visualised under UV light
254 nm and Godin detection reagent (Godin, 1954). Pure and semi-
pure fractions were also analyzed by GC/MS, by HPLC and by NMR.
The limonoids were identified by elucidation of their 1H- and
13
C-NMR spectra and by comparison with data from the literature.
The HSCCC technique gradient elution mode led to the
separation of six major fractions from the hexane extract of the
seeds of C. guianensis, that yielded as follows: 85–90 (1), 28.7 mg;
96–98 (2), 17.9 mg; 124–129 (4), 40.1 mg; 138–149 (5), 21.0 mg;
and 155–165 (6), 5.8 mg. Additionally, the fraction 99–115, con-
taining a mixture of two substances, was submitted to an open silica
gel column chromatography to yield the pure substance 3 (3.7 mg).
Results and Discussion
The structures of most gedunin family limonoids present oxygen
atoms at C-3, C-7 and C-16, besides an epoxide oxygen bound
Phytochem. Anal. 2009; 20: 77–81
Isolation of limonoids from seeds of Carapa guianensis Aublet (Meliaceae)
Figure 1. TLC monitoring of fractions from the HSCCC separation of a hexane extract of Carapa guianensis seeds: methyl angolensate (1), 7-deacetoxy-
7-oxogedunin (2), deacetylgedunin (3), 6α-acetoxygedunin (4), gedunin (5) and andirobin (6). TLC silicagel plates were eluted in hexane:ethyl acetate
6:4 and the chemical detection was done by spraying Godin reagent.
to C-14 and C-15. All these oxygen atoms confer a moderate
polarity to the compounds and are responsible for the difficulties in
the limonoid separation by traditional chromatographic methods.
Spectrometric and spectroscopic analyses led to the iden-
tification of the limonoids in the six major fractions eluted
from HSCCC, namely methyl angolensate (1), 7-deacetoxy-7-
oxogedunin (2), deacetylgedunin (3), 6α-acetoxygedunin (4),
gedunin (5) and andirobin (6). HPLC chromatograms and the
corresponding limonoids structures are shown in Fig. 2. Sub-
stances 3 and 6 are present in such a small amount that they
could not be reliably indicated in the chromatogram of the
crude extract.
Except for methyl angolensate, the other limonoids isolated
all exhibited similar signals in the 13C NMR spectrum, characteristic
for the gedunin family limonoids, as follows: 203.7–204.7
ppm corresponding to an α,β-unsaturated carbonyl (C-3);
153.5–157.8 and 125.7–127.1 ppm (C-1 and C-2, respectively,
between which the double bond is located); 167.1–170.3 ppm
(C-16, D-ring lactone carbonyl); 66.2–72.5 and 54.3–57.8 ppm
(C-14 and C-15, bound to an epoxide oxygen); and furanyl
ring signals 119.7–121.0 ppm (C-20), 140.9–141.7 ppm (C-21),
109.7–110.4 ppm (C-22) and 143.0–143.8 ppm (C-23).
Methyl angolensate (1): C27H34O7, MW 470
Identification of 1 was supported by the mass spectrum fragment
m/z 470 (M+ and base peak; Baldwin et al., 1967), and by
comparison with 1H and 13C NMR data reported in literature
(Kadota et al., 1990). This compound lacked the signals related
to the epoxide group between C-14 (δC 80.4 ppm) and C-15 (δC
34.0 ppm), as well as those characteristic signals corresponding
to the double bond between C-1 (δC 77.4 ppm) and C-2 (δC 39.6
ppm). The signals 213.7 ppm (carbonyl at C-3) and 111.7 ppm
Phytochem. Anal. 2009; 20: 77–81 Copyright © 2008 John Wiley & Sons, Ltd.
(terminal methylene at C-30) are also typical of the methyl
angolensate molecule.
7-Deacetoxy-7-oxogedunin (2): C26H30O6, MW 438
The mass spectrum of 7-deacetoxy-7-oxogedunin showed the
base peak m/z 315. That is compatible with the profile of other
limonoids (Baldwin et al., 1967). Comparison with 1H and 13C
NMR data from the literature (Kadota et al., 1990) confirmed the
identity of substance 2. The 13C NMR spectrum showed the
signal 208.9 ppm, related to a carbonyl group at C-7.
Although previous analyses have shown that 2 is the major
limonoid in the crude hexane extract, it is not obtained in an
appreciable amount from HSCCC. This happens due to the
sample solubilisation process, in which most of 2 precipitates.
Furthermore, 2 does not separate satisfactorily from substances
1 and 3.
Deacetylgedunin (3): C26H32O6, MW 440
Deacetylgedunin showed the same base peak as 5 (m/z 299) in
the mass spectrum. Nonetheless these substances could be
differentiated by the fragment m/z 317, present in 3 and absent
in 5 (Baldwin et al., 1967). The 1H NMR data were in agreement
with the literature (Ambrozin et al., 2006). The signal δH 3.58 ppm
corresponds to the hydrogen atom at C-7 bound to a hydroxyl
group, which was important for the identification of 3. The 13C
NMR data of 3 (Table 1) have not been published so far.
6a-Acetoxygedunin (4): C30H36O9, MW 540
Mass spectrum fragments m/z 297 as the base peak and m/z 540
were useful for the identification of compound 4. Signals δC 69.5
79
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 V. P. da Silva et al.

Figure 2. HPLC chromatograms of the crude hexane extract (C) and of the six major fractions and structures of the limonoids isolated from C.
guianensis: 1, 68%; 2, 56%; 3, 98%; 4, 93%; 5, 77%; and 6, 91%. Substances 3 and 6 are not shown in the profile C because they are present in a
such small amounts that it is not safe to correlate a peak in the chromatogram to each of them. HPLC conditions: Merck Lichrospher 100 C-18
column (250 × 4.0 mm i.d.; 5 μm); acetonitrile:water:methanol (35:35:30, v/v); injection volume 20 μL at 1 mg/mL; 0.9 mL/min. The effluent was monitored
at 210 nm.

ppm (C-6), δC 170.0 ppm and δC 170.1 ppm (two acetoxy-carbonyl
groups bound to C-6 and C-7, respectively), δH 4.89 ppm (H-7
proton), and the presence of seven methyl groups corroborate
the structure of 4. All complementary 1H and 13C NMR data were
successfully compared with literature information (Kadota et al.,
1990).
Gedunin (5): C28H34O7, MW 482
Fragments m/z 299 (base peak) and m/z 482 (M+) occurring in
the mass spectrum of 5 were assigned as characteristic signals
of the limonoid gedunin (Baldwin et al., 1967). The 13C NMR
signals at 170.1 ppm (acetoxy-carbonyl group at C-7) and 23.5
ppm (methylene group at C-6), and the presence of six methyl
groups, were important for the identification of 5. All 1H and 13C
80
NMR data were successfully compared with those from the
literature (Khalid et al., 1989).
Andirobin (6): C27H32O7, MW 468
The mass spectrum of 6 showed no characteristic base peak.
Confirmation of its molecular structure came from comparing 1H
NMR data from the literature. Important signals are 4.04 ppm for
C-15, and 5.37 and 5.27 ppm for the geminal methylene protons
at C-30 (Powell, 1966). Not much has been reported about this
substance in the literature, so the data acquired by GC/MS could
not be compared. The 13C NMR data of 6 (Table 1) have not been
published so far.
Owing to the high similarity in polarity, previous attempts to
isolate limonoids from the crude hexane extract of the squeezed

www.interscience.wiley.com/journal/pca Copyright © 2008 John Wiley & Sons, Ltd. Phytochem. Anal. 2009; 20: 77–81
Isolation of limonoids from seeds of Carapa guianensis Aublet (Meliaceae)

Acknowledgements
Table 1. The 13C NMR spectral data for deacetylgedunin (3)
and andirobin (6) from C. guianensis The authors wish to thank the Analytical Center and NMR Labora-
tory of Far-Manguinhos, Fiocruz (Manguinhos, Rio de Janeiro,
C δC of 3 δC of 6 Brazil), PDTIS/Instituto de Tecnologia em Fármacos/FIOCRUZ for
financial support and Dr. Maria Auxiliadora Coelho Kaplan, Dr.
1 157.8 153.5 Antônio Carlos Siani, Dr. Benjamin Gilbert and Dr. Diana Negrão
2 125.7 125.7 Cavalcanti for their contributions.
3 204.7 203.7
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