Antimicrobial terpenoids from the oleoresin of the Peruvian medicinal plant Copaifera paupera

Article abstract of DOI:10.1055/s-2002-34399

Twelve known diterpenes 1-11 and 13, and three known sesquiterpenes 14-16, along with a new C₂₀-C₁₅ terpenoid 17, with a structure based on an unprecedented skeleton in which a labdane diterpene is linked to a monocyclic sesquiterpene by an ester bridge, were isolated from the oleoresin of the Peruvian medicinal plant Copaifera paupera (Herzog) Dwyer (Leguminosae). Their structures were elucidated on the basis of spectral analysis, including homo- and heteronuclear correlation NMR experiments (COSY, ROESY, HMQC and HMBC), and by comparison with data in the literature. The leishmanicidal, antimicrobial, cytotoxic, and aldose reductase inhibitory activities were studied. Compounds 1 and 11 showed significant antimicrobial activity (MIC < 10μg/ml) against Gram-positive bacteria, comparable with cephotaxime used as control. Compound 2 exhibited moderate cytotoxic activity against four cancer cell lines.

Full text of DOI:10.1055/s-2002-34399

Benigna M. Tincusi1,²
Ignacio A. JimØnez1
Isabel L. Bazzocchi1
Laila M. Moujir2
Zulma A. Mamani3
JosØP. Barroso 3
Antimicrobial Terpenoids from the Oleoresin of the
Peruvian Medicinal Plant Copaifera paupera
Angel G. Ravelo1
Basilio V. Hernµndez3
Abstract
with data in the literature. The leishmanicidal, antimicrobial, cy-
totoxic, and aldose reductase inhibitory activities were studied.
Twelve known diterpenes 1±11 and 13, and three known sesqui- Compounds 1 and 11 showed significant antimicrobial activity
terpenes 14±16, along with a new C20 ±C15 terpenoid 17, with a (MIC < 10 mg/ml) against Gram-positive bacteria, comparable
structure based on an unprecedented skeleton in which a lab- with cephotaxime used as control. Compound 2 exhibited mod-
dane diterpene is linked to a monocyclic sesquiterpene by an es- erate cytotoxic activity against four cancer cell lines.
ter bridge, were isolated from the oleoresin of the Peruvian med-
icinal plant Copaifera paupera (Herzog) Dwyer (Leguminosae). Key words
Their structures were elucidated on the basis of spectral analysis, Copaifera paupera ´ Leguminosae ´ Copaiba oil ´ terpenoids ´
including homo- and heteronuclear correlation NMR experi- leishmanicidal ´ antimicrobial ´ cytotoxic ´ aldose reductase inhi-
ments (COSY, ROESY, HMQC and HMBC), and by comparison bitory activities
Introduction
leishmanicidal properties of copaiba oils have been known for
centuries, and at the present time they are used as plaster to
808
Copaiba oils, also described as copaiba balsams or oleoresins, threat leishmaniasis (Uta) [4].
are produced by extraction of the trunk of trees belonging to
the genus, Copaifera. These trees are largely distributed in The main constituents of these oils are diterpenes and sesqui-
northern South America, mainly in the Amazonas rain forest. terpenes [2]. Nevertheless, in spite of the existing abundant lit-
Copaiba oils have been used for the treatment of cancer, ulcer, erature on copaiba oils properties, little is known of the rela-
syphilis, bronchitis, and diarrhoea [1], and as bactericidal, tionship between the structures and the activities of the com-
anti-helminthic, analgesic, anti-inflammatory, gastro-protec- ponents.
tive, antitumor, and trypanocidal agents [2]. The oils are used
in the flavour and cosmetics industry, and as diesel-like fuels During the search for new bioactive compounds from South
[3]. In Peru, where resorting to medicinal plants represents a American folk medicinal plants, this work was initiated to inves-
primary health care measure of the native population, the tigate the oil of Copaifera paupera. The present paper describes
the isolation and identification of fifteen known terpenoids, and
a new C20 ±C15 terpenoid. All compounds were identified by ex-
²
Deceased
Affiliation
1 Instituto Universitario de Bio-Orgµnica ªAntonio Gonzµlezº, Universidad de La Laguna, La Laguna, Tenerife,
Spain
2 Departamento de Microbiología y Biología Celular, Facultad de Farmacia, Universidad de La Laguna,
La Laguna, Tenerife, Spain
3 Departamento de Parasitología, Ecología y GenØtica, Facultad de Farmacia, Universidad de La Laguna,
La Laguna, Tenerife, Spain
Correspondence
Isabel López Bazzocchi ´ Instituto Universitario de Bio-Orgµnica ªAntonio Gonzµlezº ´
Universidad de La Laguna ´ Avenida Astrofísico Fco Sµnchez 2 ´ La Laguna ´ 38206 Tenerife ´ Spain ´
E-Mail: ilopez@ull.es ´ Fax: +34-922-318571 ´ Phone: +34-922-318576
Received November 16, 2001 ´ Accepted March 23, 2002
Bibliography
Planta Med 2002; 68: 808±812 ´ ꢀ Georg Thieme Verlag Stuttgart ´ New York ´ ISSN 0032-0943

tensive analysis of spectroscopic data, and comparison with data in May 1997. A voucher specimen (No. CUZ-026527) is deposited
in the literature. The leishmanicidal, antimicrobial, cytotoxic, in the Vargas Herbarium, Facultad of Biology, Universidad Nacio-
and aldose reductase inhibitory activities of some of the com- nal de San Antonio Abad del Cusco, Per‚.
pounds are described.
Copaifera paupera oil (29.4 g) was chromatographed by dry flash
chromatography on silica gel, using n-hexane-EtOAc mixtures of
increasing polarity to afford 67 ” 100 ml frs., which were reduced
to 5 frs. by TLC: A (0±5%, n-hexane-EtOAc), B (5±15%), C (15±
25%), D (25±45%), and E (45±100%). A 40 mg sample of fraction
A (16.2 g) was rechromatographed on semi-preparative HPLC
using n-hexane as eluent to give 14 (tR 22.6 min), and 15 (tR 25.0
min). Fraction B (6.9 g) was rechromatographed on Sephadex LH-
20, using n-hexane-CHCl3-MeOH (2:1:1) as eluent to afford frs.
1±3. Fr. 1 (1.5 g) was then chromatographed on silica gel using
Cl2CH2-acetone (from 10:0 to 5:5) as eluent to give 6 (Rf 0.61,
n-hexane-EtOAc, 6:2). Fr. 2 (2.0 g) was subjected to a silica gel-
column with n-hexane-EtOAc (from 10:0 to 5:5) as eluent to af-
ford 13 and 1 (Rf 0.72, and 0.65, respectively, n-hexane-EtOAc,
6:4). Chromatography of fr. 3 (3.2 g) under the same conditions
yielded 2, 4, and 10 (Rf 0.60, 0.55, and 0.42, respectively, n-hex-
ane-EtOAc, 9:1). Fraction C (2.8 g), after chromatography on Se-
phadex LH-20 (n-hexane-CHCl3-MeOH, 2:1:1), gave rise to
compounds 5 and 11 (Rf 0.53, and 0.40, respectively, n-hexane-
EtOAc, 7:3). Fraction D (2.1 g) was purified by silica gel column,
using Cl2CH2-acetone (from 10:0 to 8:2) as eluent to give 3, 8, 9
and 17 (Rf 0.34, 0.60, 0.51, and 0.58, respectively, Cl2CH2-acetone,
9:1). Chromatography of fraction E (1.4 g) on Sephadex LH-20
(n-hexane-CHCl3-MeOH, 2:1:1) afforded 7 (Rf 0.53, n-hexane-
EtOAc, 6:4), and four additional fractions. Fr. 2 (50 mg) of these
were further purified by silica gel column using n-hexane-EtOAc
(from 10:0 to 0:10) yielded 16 (Rf 0.38, n-hexane-EtOAc, 7:3).
(±)-Copalic acid 1 (856.0 mg), [a]D25: ±15.38 (c 1.5, CHCl3); (±)-me-
thyl copalate 2 (15.4 mg), [a]D25: ±18.58 (c 1.8 , CHCl3); agathic acid
15-methyl ester 3 (17.7 mg), [a]D25: ±18.58 (c 0.4, CHCl3); agathic
acid 15,19-dimethyl ester 4 (2.3 mg), [a]D25: ±15.68 (c 0.2, CHCl3);
(±)-polyalthic acid 5 (145.8 mg), [a]D25: ±34.38 (c 1.8, CHCl3); (±)-
methyl polyalthate 6 (8.8 mg), [a]D25: ±38.98 (c 2.4, CHCl3); (±)-pi-
nifolic acid 7 (51.7 mg), [a]D25: ±27.98 (c 2.1, CHCl3); methyl 3b-hy-
droxy-labda-8(17),13-dien-15-ate 8 (4.3 mg), [a]D25: + 24.88 (c
809
Materials and Methods
General
0.31, CHCl3); (±)-methyl-18-hydroxy-copaiferolate 9 (12.4 mg),
[a]D25: ±21.58 (c 0.2, CHCl3); (+)-14,15-bisnorlabd-8(17)-en-13-
one 10 (3.4 mg), [a]D25: + 31.98 (c 0.4, CHCl3); (±)-kaurenic acid 11
Optical rotations were measured on a Perkin-Elmer 241 auto- (25.5 mg), [a]D25: ±71.78 (c 6.7, CHCl3); 16b-kauran-19-oic acid 13
matic polarimeter and the [a]D are given in 10±1 deg cm2 g±1
.
(65.2 mg), [a]D25: ±51.78 (c 1.2, CHCl3); b-bisabolene 14 (11.5 mg),
IR spectra were recorded in CHCl3 on a Bruker IFS 55 spectro- [a]D25: + 68.18 (c 0.5, CHCl3); caryophyllene oxide 15 (5.5 mg),
photometer and UV spectra were collected in absolute EtOH [a]D25: ±5.98 (c 0.2, CHCl3); 3-methyl-5-(2,2,6-trimethyl-6-hydro-
on a JASCO V-560. 1H- and 13C-NMR spectra were recorded on xy-cyclohexyl)-pentanoic acid 16 (10.0 mg), [a]D25: ±1.98 (c 0.9,
a Bruker Avance 400 NMR spectrometer at 400 MHz and 100 CHCl3); with all other spectroscopic and chemical data identical
MHz, respectively. EIMS and HREIMS were recorded an a Mi- with previously reported data [1], [5], [6], [7], [8], [9], [10], [11],
cromass Autospec spectrometer. TLC 1500/LS 25 Schleicher [12], [13], [14], [15].
and Schüell foils were used for thin-layer chromatography. Si-
lica gel (particle size 40±63 mM, Merk) and Sephadex LH-20 Pauperol (17). Colorless lacquer. [a]D25: ±7.48 (c 0.1, CHCl3). UV
(Pharmacia), were used for column chromatography, and semi- (EtOH): lmax = 220, 211, 202 nm. IR (film): lmax = 3500, 2900,
preparative m-porasil column (Waters, 10 mM, 19 mm ” 25 cm) 2850, 1750, 1680, 1450, 1430, 1370, 1215, 1140, 1080, 880,
for HPLC.
750 cm±1. 1H-NMR (400 MHz): d, see Table 1. 13C-NMR (100
MHz): d, see Table 1. EI-MS: m/z (%) = 572 (M+, 1), 556 (11), 554
(1), 524 (5), 317 (83), 285 (40), 257 (26), 203 (29), 147 (42), 95
Plant material and isolation
Copaifera paupera crude oil obtained from the trunk of the tree (65), 69 (100); HR-EI-MS: calcd. for C36H60O5 572.44408, found
was collected in Madre de Dios, Provincia de Tambopata, Per‚, 572.44621.
Tincussi B M et al. Antimicrobial Terpenoids from¼ Planta Med 2002; 68: 808±812

Table 1 1H-, 13C- and HMBC NMR data for pauperol (17) and 13C-NMR data for 9 and 16
17
16
9
Unit I
Unit II
a
b
a
b
b
b
Position
dH
dC
HMBC
dH
dC
HMBC
dC
dC
1
2
173.7 s
41.4 t
39.8 t
178.4 s
38.5t
2.11, 2.30 dd
(6.2, 14.4)
1c, 1518.4 t
2
41.3 t
18.6 t
3
1.98 m
31.0 d
38.4 t
1.4, 2.19 m
35.9 t
35.4 s
18, 19
23.1 t
8, 17
8
30.8 d
41.4 t
39.7 t
35.3 s
4
51.34 m
23.2 t
1.01 t
1.27 m
7, 13
d
49.5 10, 19
48.4 d
6
57.3 d
74.2 s
43.4 t
20.4 t
41.7 t
35.4 s
21.4 q
32.7 q
23.2 q
2, 4
21.5 t
37.9 t
57.0 d
74.9 s
43.1 t
20.4 t
39.6 t
35.5 s
21.2 q
32.8 q
23.0 q
21.5 t
37.9 t
7
1.60, 2.32 m
1.55 m
8
1.35, 1.63 m
147.7 s
56.1 d
36.8 s
24.2 t
148.0 s
56.0 d
37.9 s
21.1 t
9
10
11
1.35 m
1.89 m
13
12
0.74 s
0.85s
1.10 s
19.9 q
6, 10, 11c, 13
6, 10, 11 c, 13
6, 7c, 8
39.7 t
9, 13c, 14, 16
39.5t
13
161.0 s
114.9 d
161.1 s
114.7 d
14
5.58 s
167.3 s
2.10 s
12, 13c, 15c, 16
150.90 d (6.8)
19.8 q
167.3 s
16
17
18.8 q
12, 13c, 14
7, 8c, 9
18.9 q
106.5t
4.43 s
4.78 s
106.7 t
18
3.56, 3.79
72.8 t
3, 4c, 5, 19, C = O
3, 4 c, 5, 19
72.0 t
dAB (11.0)
19
0.75s
0.65s
3.69 s
17.5q
14.9 q
50.8 q
17.6 q
14.9 q
c
20
1, 5, 9, 10
OCH3
15
a d, CDCl3, J values in Hz.
b Data are based on DEPT and HMQC experiments.
c Two-bond coupling enhancement observed.
810
Leishmanicidal activity
with the medium (105 u.v./ml). The in vitro evaluation of antimi-
The strain of Leishmania used in the presented study was crobial activity was performed following the two-fold dilution
MHOM/PE/95/LQ8 from L. braziliensis. Promastigotes were adapt- method [18]in the 0.1±50 mg/ml range. Results were expressed
ed for growth at 22 8C in RPMI 1640 modified medium (Gibco) as minimal inhibitory concentration (MIC). Each MIC of the differ-
and supplemented with 20% heat inactivated fetal bovine serum ent compounds dissolved in DMSO was determined three times
[17]. Logarithmic phase cultures were used for experimental pur- for all the strain tested. Cephotoxime was used as positive control.
poses. Assays were performed in sterilized, 24-well microtiter
plates (CorningTM). To these wells were added 500,000 parasites, Cytotoxic Assays
in promastigote form obtained from logarithmic phase cultures, Cytotoxic activity was measured against the following cell lines:
and the test drug. The final volume was 500 ml in each well. Differ- P-388 (ATCC CCL-46), suspension culture of a lymphoid neo-
ent concentrations were tested and parasite determination was plasm from a DBA/2 mouse; A-549 (ATCC CCL-185), monolayer
performed after 48 h. by density determination on a Coulter Coun- culture of a human lung carcinoma; HT-29 (ATCC HTB-38),
ter model Z1. Resistance was determined by measuring the IC50. monolayer culture of a human colon carcinoma; MEL-28 (ATCC
Tests were performed at least in triplicate on three different days HTB-72), monolayer culture of a human melanoma. Cells were
in order to verify the results. The final IC50 was calculated as the maintained, in logarithmic growth in EMEM, supplemented
arithmetic mean of the IC50 is obtained. Ketoconazol was used as with 5% fetal calf serum (FCS), 10-2 M sodium bicarbonate, and
positive control (IC50 34.8 mg/ml).
0.1 g/l penicillin G + 0.1 g/l streptomycin sulfate. The compounds
assayed were dissolved in DMSO-MeOH (1:9) and tested follow-
ing the method described previously [19]. Taxol was used as po-
Antimicrobial Assay
The activity was tested against Gram-positive (Staphylococcus sitive control (IC50 0.59 and 0.012 mg/ml for P-388 and A-549, HT-
aureus ATCC 6538, S. epidermidis CECT 232, S. saprophyticus CECT 29, and MEL-28 cell lines, respectively).
235, Bacillus subtilis CECT 39) and Gram-negative (Escherichia coli
CECT 99, Pseudomonas aeruginosa AK 958) bacteria, and yeast Assay of aldose reductase
(Candida albicans UBC 1). Bacterial cultures were developed in Nu- Purification of the recombinant human aldose reductase used in
trient Broth (Oxoid) and the yeast culture in Sabouraud liquid the bioassay is based on the method described by Nishimura et
medium. Inocula were prepared by diluting overnight cultures al. [20]. The aldose reductase inhibitory activity in vitro was de-
Tincussi B M et al. Antimicrobial Terpenoids from¼ Planta Med 2002; 68: 808±812

termined following a modification of the method reported synthetic route with participation of enzymes with acyl transfer-
above. Sorbinil was used as positive control (IC50 0.8 mg/ml) [21]. ase activity could be put forward.
As a continuation of a program oriented towards the discovery of
Results and Discussion
bioactive natural products, the isolated compounds were evalu-
ate for their leishmanicidal, antimicrobial, cytotoxic, and aldose
Repeated chromatography on Sephadex HL-20 and silica gel of reductase inhibitory activities.
Copaifera paupera crude oil yielded twelve known diterpenes,
1±11 and 13, and three known sesquiterpenes 14 and 16, along Compounds 1±3, 5±7, 9, and 11 ± 13 were assayed against
with a new C20 ±C15 terpenoid 17, with a structure based on the Leishmania braziliensis promastigotes in vitro. All of the com-
union of a labdane diterpene and a monocyclic sesquiterpene.
pounds were deemed inactive, with IC50 values up to 100 mg/ml.
The crude oil showed a 20.1% of inhibition at 300 mg/ml.
The known compounds were identified as (±)-copalic acid 1, (±)-
methyl copalate 2 [1], agathic acid 15-methyl ester 3 [5], agathic These compounds were also assayed to determine the minimal
acid 15,19-dimethyl ester 4 [6], (±)-polyalthic acid 5, (±)-methyl inhibitory concentration (MIC) against Gram-positive and
polyalthate 6 [7], (±)-pinifolic acid 7 [8], methyl 3b-hydroxy-lab- Gram-negative bacteria, and the yeast Candida albicans. Com-
da-8(17),13-dien-15-ate 8, (±)-methyl-18-hydroxy-copaiferolate pounds 1 and 11 showed significant activity against B. subtilis,
9[9], (+)-14,15-bisnorlabd-8(17)-en-13-one 10 [10], (±)-kaurenic S. aureus and S. epidermidis, comparable with cephotaxime
acid 11 [11],16b-kauran-19-oic acid 13 [12], b-bisabolene 14 [13], used as control (Table 2). On the other hand, compounds 2, 7,
caryophyllene oxide 15 [14], and 3-methyl-5-(2,2,6-trimethyl-6- 9, 10, 12, and 13 were inactive against all microorganisms as-
hydroxycyclohexyl)-pentanoic acid 16 [15]by comparison of the
sayed at a level of 50 mg/ml. On the basis of their chemical fea-
NMR, IR, and mass spectra with data in the literature. Com- tures, these results suggest that the presence of a carboxylic
pounds 8 and 9, were isolated from a natural source for the first acid moiety could strongly affect the antimicrobial activity, as
time, and compound 12, the methyl ester of (±)-kaurenic acid, the masking of the carboxylic acid as its methyl ester fully elimi-
was obtained by methylation of 11 with diazomethane in the nated the activity (1 versus 2 and 11 versus 12). In addition, the
usual way.
double bonds appear to induce some enhancement of the ac-
tivity (1 versus 7 and 11 versus 13).
Compound 17 has the molecular formula C36H60O5 by HREIMS
and 13C-NMR data. Its H-NMR (Table 1) spectrum revealed sig- Compounds 1, 2, 5, 7 , 11, and 13 were tested for cytotoxic and
nals for seven methyl groups, five of them on quaternary satura- aldose reductase [16]inhibitory (this enzyme appears to initi-
ted carbons (d 0.65, 0.74, 0.75, 0.85, and 1.10), one on a tertiary ate the cataract development process in diabetics) activities.
carbon (d 0.90, d, J = 6.8 Hz), and one on a vinyl carbon (dH The cytotoxic activity was determined against the following tu-
2.10). Two methylene protons of a primary alcohol (d 3.56 and mor cell lines: P-388 (lymphoid neoplasm from a DBA/2
3.79, dAB, J = 11.0 Hz), a methoxy group (d 3.69), and three vinyl mouse), A-549 (human lung carcinoma), HT-29 (human colon
protons (d 4.43, 4.78, and 5.58) were also observed. These data carcinoma) and MEL-28 (human melanoma). Among these
were confirmed by 13C-NMR (Table 1) showing 36 carbons, and compounds, 2 exhibited moderate activity in the four tested
DEPT experiments revealed the presence of eight methyls, fifteen cell lines, with IC50 values of 2.5, 5, 5, and 10 mg/ml against the
methylene, five methine, and five quaternary carbons, two car- P-388, A-549, HT-29 and MEL-28 cell lines, respectively. The re-
bonyl groups (d 167.3 and 173.7) and one quaternary carbon maining tested compounds showed no significant inhibitory
bearing a hydroxyl group (d 74.2). The IR spectrum corroborated activity (IC50 > 10 mg/ml). In the aldose reductase assay inhibi-
the presence of the latter groups through the absorptions at tion was observed only for diterpene 7, which showed weak ac-
1680, 1750 and 3500 cm±1. When 17 was treated with acetic an- tivity with an IC50 value of 25 mg/ml, while quercetrin used as
hydride in pyridine at room temperature it remained unaltered, control showed an IC50 of 0.9 mg/mL.
1
811
which, in conjunction with the IR spectrum, attested to this mol-
ecule having a tertiary alcohol group. The analysis of its COSY, These results partially highlight the antimicrobial potential of
ROESY, HMQC, and HMBC spectra enabled the assignment of the the Copaiba oil as traditional remedy. However, its potential
signals of the two units I and II, which correspond to the mono- use for the treatment of the leishmanisis (Uta), could not be
cyclic sesquiterpene 16 [15]and the labdane diterpene 9 [9], predicted, although the pure metabolites showed no activity
respectively.
The nature of the ester linkage connecting units I and II was con-
firmed through the HMBC (Table 1) experiment, which showed a
three-bond correlation between the carboxyl carbon of the unit I
(C-1, dC 173.7) and H-18 of the unit II (dH 3.56 and 3.79), and by
comparison with the 13C-NMR data of 3-methyl-5-(2,2,6-trime-
thyl-6-hydroxycyclohexyl)-pentanoic acid 16 and the (±)-me-
thyl-18-hydroxy-copaiferolate 9. Thus the structure of 17, for
which the name pauperol is proposed, is that of a monocyclic far-
nesane and a labdane diterpene linked by an ester bridge. These
two types of terpenes are present in the oil (16 and 9), and a bio-
Table 2 Minimal inhibitory concentration (MIC, mg/ml) of selected
terpenoids from Copaifera paupera
Bacteria
1
5
9
11
Control a
B. subtilis
6.3±3.1
10±8
5±4
30±20
50±40
40
30
5±2.5
8±6
8
S. aureus
>50
>50
2.5±1.25
2.5
S. epidermidis
6±4
a Control was cephotaxime.
Tincussi B M et al. Antimicrobial Terpenoids from¼ Planta Med 2002; 68: 808±812

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Acknowledgements
11
We are indebted to the DGES Project BQU2000-0870-CO2-01
and the FEDER Project 1FD1997-0747-CO4-01, for financial
assistance. BIOMAR S.A., Tres Cantos, Madrid, Spain (Dr. D.
Grµvalos and I. Raymundo) is responsible for the cytotoxic and
aldose reductase assays. We thank Prof. Alfedro Tupayachi for
supplying the plant material.
12
13
14
15
16
17
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