TWO NEW CYCLOARTANES FROM Combretum leprosum MART.

(COMBRETACEAE)

VALDIR ALVES FACUNDO*, KATIÚSCIA ALEXANDRE RIOSa, LEANDRO SOARES MOREIRAa, JÚLIO SANCHO L.
TEIXEIRA MILITÃOa, RODRIGUS GUERINO STABELLIa, RAIMUNDO BRAZ-FILHOb AND EDILBERTO ROCHA SILVEIRAc
(Received October 2008; Accepted November 2008)

ABSTRACT

Two new cycloartanes, (1) and (2) and seven known metabolites, three flavonoids
[5,7,3’,4’-tetrahydroxy-3-methoxyflavone (3), 5,3’,4’-trihydroxy-3,7-dimethoxy-
flavone (4) and 5,3’-dihydroxy-3,7,4’-trimethoxyflavone (5)], three triterpenes
[3β,6β,16β-trihydroxy-lup-20(29)-ene (6), mollic acid (7) and arjunolic acid (8)],
and ? -D-glucopiranoside-(3β)-stigmast-5-en-3-yl (9), were isolated from the flo-
wers of Combretum leprosum Mart. The chemical structures of the isolated products
were established on the basis of their spectral data and by comparing with those
reported in the literature.

Key Word Index: Combretum leprosum; Combretaceae; cycloartanes; flavonoids.

RESUMEN

Dos nuevos cicloartanos, (1) and (2), y siete metabolitos conocidos, tres flavo-
noides [5,7,3’,4’-tetrahydroxy-3-methoxyflavona (3), 5,3’,4’-trihydroxy-3,7-dime-
thoxyflavona (4) and 5,3’-dihydroxy-3,7,4’-trimethoxyflavona (5)], tres triterpenos
[3β,6β,16β-trihydroxy-lup-20(29)-eno (6), ácido mollico (7) y ácido arjunólico (8)],
y el a -D-glucopiranósido de (3β)-stigmast-5-en-3-ilo (9), se aislaron de las flores
de Combretum leprosum Mart. Las estructuras químicas de los productos aislados
se establecieron con base a sus datos espectroscópicos y por comparación con los
datos reportados en la literatura.

INTRODUCTION and anthelmintic activities (McGaw et al.,

2001; Katererea et al., 2003) and are used
Plants belonging to the family Combre- in the folk medicine of Africa and India.
taceae are sources of metabolites with These include the treatment of abdominal
interesting biological activities, including pains, backache, bilharzia, chest coughs,
antifungal, antibacterial, anti-inflamma- colds, conjunctivitis, diarrhoea, dysmenor-
tory, DNA-damaging, antischistosomal rhea, earache, fever, headache, hookworm,
a
Departamento de Química, Universidade Federal de Rondônia, 78.900-500 Porto Velho-RO, Brazil
b
Setor de Química de Produtos Naturais, Universidade Estadual do Norte Fluminense, 28013-600 Campos-
RJ, Brazil
c
Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, CP 12200,60021-970
Fortaleza-CE, Brazil
*
Author to whom correspondence should be addressed: Fax: (55) 69-2182-2207. E-mail: [email protected]

76
Two new cycloartanes from Combretum leprosum Mart. (Combretaceae) Rev. Latinoamer. Quím. 36/3 (2008) 77

infertility in women, leprosy, pneumonia, TLC. Silica gel 60 (Merck, 230-240 mesh)
scorpion and snake bite, swelling caused was used for column chromatography.
by mumps, syphilis, toothache and general All compounds were visualized on TLC by
weakness (Adnyana et al., 2000; McGaw spraying with vanillin/perchloric acid/
et al., 2001; Katererea et al., 2003). The EtOH followed by heating.
genus Combretum occur widely in tropi-
cal and subtropical areas and includes
around 370 species (McGaw et al., 2001). Plant material
The presence of a Combretum sp, together
with Combretum leprosum and Combre- The flowers of Combretum leprosum Mart,
tum lanceolato has been reported in Ceará Combretaceae family, were collected in
State. C. leprosum is the major species of May 2001 in Viçosa, Ceará State, Brazil.
tree reported in the natural habitat of Ceará The plant was identified by a botanist of
State (Braga, 1960) and popular informa- the Herbário Prisco Bezerra, Universidade
tion reveals that its bark is used as an anti- Federal do Ceará, Ceará, Brazil, where
dote against snake bites (Mors et al., 2000). a voucher specimen (number 1244) was
A phytochemical investigation carried out deposited.
by Pegel & Rogeres (1976) and Rogeres &
Coombes (2001) on the genus Combretum Extraction and isolation
from Africa reported the isolation of several
glycosilated triterpenes. These triterpe- Dried and powdered flowers (2.7 kg) from
noids isolated from the Combretum belong C. leprosum were extracted with EtOH at
almost exclusively to two distinct groups; room temperature for one week. The extract
viz. 30-carboxy-1a-hydroxycyloartanes was concentrated in vacuo to yield 58.3 g
(Pegel & Rogers, 1998) and 29-carboxy-1a- of crude extract. A portion (30.0 g) of the
hydroxyoleanes (Rogers, 1989). crude extract was subjected to column
This paper describes the isolation and chromatography on silica gel, eluting with
structural elucidation of two new cycloar- n-hexane, EtOAc and MeOH. The EtOAC
tanes, 1 and 2, and the identification of fraction was further purified by column
eight known metabolites, from the flowers chromatography over silica gel, using a
of a C. leprosum specimen. gradient elution with n-hexane-EtOAc
mixtures, to obtain metabolites 3 (32 mg;
80:20), 4 (19 mg; 75:25), 5 (12.1 mg; 60:40)
MATERIALS AND METHODS and 6 (120 mg; 55:45). Silica gel column
chromatography purification of the MeOH
General experimental procedures fraction, using a gradient elution with mix-
tures of EtOAc-MeOH, yielded metabolites
1
H and 13C NMR spectra were obtained 1 (15.1 mg; 80:20), 2 (13.1 mg; 75:25), 7
using a JEOL Eclipse spectrometer opera- (33.2 mg; 85:15), 8 (14.0 mg; 70:30) and 9
ting at 400 MHz and 100 MHz, respec- (10.0 mg; 65:35).
tively; chemical shift values are reported
in d (ppm) and coupling constant (J) in Hz.
Mass spectra were obtained on a Shimadzu RESULTS AND DISCUSSION
QP-2000 mass spectrometer by electron
impact ionization (70 eV). The IR spectra The two new cycloartanes 1 and 2, together
were recorded on a Perkin-Elmer FT-1500 with the seven known metabolites 3-9, were
spectrometer. Sílica gel 60 (Merck, kiesegel isolated from the EtOH extract of the flowers
60 F254, 0.20 mm) were used for analytical of Combretum leprosum. Metabolites 3-9
78 V. A. Facundo, K. A. Rios, L. S. Moreira et al.
Two new cycloartanes from Combretum leprosum Mart. (Combretaceae) Rev. Latinoamer. Quím. 36/3 (2008) 79

Table 1. 1H (400 MHz) and 13C (100 MHz) NMR for triterpenes 1, 2 and 7 in pyridine-d5. Multiplicity and
coupling constants (J), in Hz, are shown in parenthesis.

1 2 7
C dC dH dC dH dC dH
1 32.7 1.68; 1.16 73.1 3.94 (s) 73.1 3.95 (s)
2 30.8 2.20-1.90 39.4 2.53 (d, 13.2) 39.3 2.53 (td, 12.8);
2.33 (t, 13.2) 2.33 (dd, 12.8, 2.6)
3 75.9 4.79 (dd, 11.7, 4.8) 71.3 5.61 (dd, 12.1, 4.0) 71.3 5.60 (dd, 12.1, 4.4)
4 56.0 - 56.3 - 56.3 -
5 45.4 2.79 (m) 38.3 3.46 (dl, 10.6) 38.3 3.46 (dd, 12.1, 4.4)
6 26.8 1.66; 1.14 23.5 1.80; 1.20 23.9 1.75; 1.25
7 26.4 1.54; 1.22 26.4 1.70; 1.40 26.4 2.15; 1.52
8 48.6 1.64 (m) 48.7 1.76 (m) 48.7 1.60
9 20.0 - 21.4 - 21.4 -
10 24.1 - 30.9 - 30.9 -
11 27.2 - 26.8 2.78; 1.25 26.8 2.78; 1.25
12 33.7 - 33.8 1.67; 1.45 33.8 1.67; 1.45
13 47.6 - 46.1 - 46.1 -
14 49.1 - 49.7 - 49.7 -
15 48.7 - 36.4 - 36.4 -
16 77.3 4.30 (dd, 7.7, 6.2) 28.8 - 28.9 -
17 62.6 1.94 (m) 53.1 1.60 53.1 1.60
18 19.8 1.12 (s) 20.0 0.98 (s) 19.0 1.07 (s)
19 31.4 0.70 (d, 4.0) 30.3 0.85 (d) 30.51 0.85 (d, 4.0)
0.43 (d, 4.0) 0.57 (d) 0.57 (d, 4.0)
20 35.7 1.65 37.3 - 36.7 1.28
21 19.6 1.09 (d, 6.3) 18.9 1.04 (d) 18.9 0.97 (d, 6.2)
22 36.8 - 33.2 2.25; 1.80 37.2 1.75; 1.48
23 26.3 2.35; 2.05 129.1 5.86 (m) 25.8 2.10; 1.92
24 126.8 5.32 (t, 7.3) 137.7 5.99 (d, 16.1) 126.4 5.24 (t, 7.0)
25 131.1 - 81.7 - 131.3 -
26 18.3 1.67 (s) 25.9 1.60 (s) 18.2 1.65 (s)
27 26.4 1.68 (s) 25.7 1.59 (s) 26.3 1.71 (s)
28 180.4 - 180.6 - 180.7 -
29 11.0 1.72 (s) 10.3 1.77 (s) 10.3 1.77 (s)
30 20.9 1.37 (s) 19.9 1.09 (s) 20.0 1.01 (s)

were identified by comparing their spectros- sp2 carbons at dH 1.67 and 1.68), 10 methy-
copic data with those reported in the litera- lenes, seven methines [including one sp2 at
ture (Macari et al., 1990; Facundo et al., dC 126.8 and two bound to oxygen atoms
1993; Hui et al., 1999; Marin et al.,2004). at dC 77.3 and 75.9], and seven quaternary
The EIMS of metabolite 1 showed a molecu- carbons [including two sp2 at dC 180.4 and
lar ion peak at m/z 472 [M], corresponding 131.1]. This data was consistent with that
to a molecular formula C30H48O4, suggesting of a cycloartane triterpene (Kasai et al.,
a structure with seven degrees of unsatura- 1999) and was confirmed by the characte-
tion. The IR spectrum of 1 showed strong ristic high-field chemical shift of the H-19
absorption bands at nmax 3400 (hydroxyl) protons [(dH 0.70 and 0.43] in the 1H NMR
and 1700 cm-1 (carboxyl). The combined spectrum of 1 (Banskota et al. 2000). The
analysis of the 1H-NMR and the APT experi- coupling pattern of the H-3 signal (dH 4.79,
ment of 1 (Table 1) indicated the presence dd, J=11.7 and 4.8 Hz) indicated an axial
of 30 carbon signals, corresponding to six orientation for the hydroxyl group. The
methyl groups (including two attached to location of the carboxyl group at C-4 was
80 V. A. Facundo, K. A. Rios, L. S. Moreira et al.

revealed by the HMBC experiment which periment of 1, between H-16 (dH 4.30) and
showed a clear correlation between the C- the carbon atoms at C-13 (dC 47.6), C-14
28 signal at dC 180.4, and the H-3 and H-29 (dC 49.1), CH-20 (dC 35.7) and C-15 (dC 48.7)
protons at dH 4.79 and dH 1.72, respectively (Table 2). It analyzes of the specter of 2D
(Table 2). The g-effect (shielding) of oxygen (NOESY) allowed to consider the orienta-
atoms of the carboxyl group at the 4a-posi- tion 16a for hydroxyl group C-16. Finally,
tion may explain the chemical shifts of the the complete 1H and 13C NMR assignments
methine carbon atoms at C-3 (dC 75.9) and for the structure of 1 were unambiguously
C-5 (dC 45.4), when compared with those made by analyzing 2D experiments such as
described for other triterpenes without a 1
H-1H-COSY, HMQC (Table 1) and HMBC
carboxyl group in this position, dC 78 and (Table 2). Thus, metabolite 1 was identified
56 for C-3 and C-5, respectively (De Pascual as a new triterpenoid, 4a-carboxy-3b,16a-
et al., 1989). The presence of a hydroxyl dihydroxy-24-cycloartene, isolated from
group at C-16 (dC 77.4) was suggested by flowers of Combretum leprosum.
the correlations observed in the HMBC ex-

Table 2. Heteronuclear long-range correlations observed in the HMBC experiments of triterpenes 1, 2 and 7.

1 2 7
2
JCH 3
JCH 2
JCH 3
JCH 2
JCH 3
JCH
C
4 H-5; 3H-29 H-5; 3H-29 H-2a H-3; H-5; 3H-29 2H-2
9 H-8 2H-19 H-11a
10 H-5 H-1; H-5 H-8 H-19b H-2a; 2H-11
13 3H-18 H-16; 3H-30 3H-18 3H-30 3H-18 3H-30
14 3H-30 H-16; 3H-18 3H-30 3H-18 3H-30 3H-18
25 3H-26; 3H-27 3H-26; 3H-27 H-23 3H-26; 3H-27
28 H-3; 3H-29 H-3; 3H-29 3H-26; 3H-27
CH
1 2H-29 2H-19
3 3H-29 2H-2 H-1; H-5; 3H-29
5 H-19a; 3H-29 2H-19; 3H-29
8 2H-29; 3H-30 2H-19 2H-19; 3H-30
16 H-17 - -
17 3H-18; 3H-21 3H-18; 3H-21 3H-18; 3H-21
20 3H-21 H-16 3H-21
23 H-24
24 3H-26; 3H-27 3H-26; 3H-27 3H-26; 3H-27
CH2
1 H-19b - -
2 H-1
6 H-5 H-5
11 H-19b H-19 b H-19b
12 3H-18 3H-18
15 H-16 H-17; 3H-30 H-8; 3H-30 3H-30
19 H-5 H-5 H-1; H-5
22 3H-21 3H-21; H-24 3H-21
CH3
18 H-17
21 H-17
26 H-24; 3H-27 H-24 H-24; 3H-27
27 H-24; 3H-26 H-24 H-24; 3H-26
29 H-5 H-3; H-5
Two new cycloartanes from Combretum leprosum Mart. (Combretaceae) Rev. Latinoamer. Quím. 36/3 (2008) 81

Metabolite 2 gave a positive reaction to the the HMBC experiment of 2 between the
Liebermann–Burchard test, indicating a C-26 and C-27 protons and the C-24 and
triterpene skeleton. Its IR spectrum exhi- C-25 carbons (Tables 1 and 2) and 2 was
bited hydroxyl (nmax 3400 cm-1) and carboxyl identified as, a new triterpene, 4a-carboxy-
(nma 1700 cm-1) absorption bands, while 1a,3b-dihydroxy-25-hydroperoxy-trans-
the EIMS showed ion peaks at m/z 471 23(24)-cycloartene, isolated from flowers
[M+-OOH], 470 [M+-HOOH] and 453 [M+- of Combretum leprosum.
OOH-H2O], suggesting a molecular formula These results also provide further evidence
C30H48O6 and seven degrees of unsatura- of the bifurcation in triterpenoid biosynthe-
tion. A comparative analysis of the 1H and sis within the genus Combretum in which
13
C NMR chemical shifts of triterpenes 1, species produce triterpenoids with either
2 and 7 indicated the presence of hydroxyl a 30-carboxy-1a-hydroxycycloartane or
groups at C-1 and C-3, and a carboxyl func- 29-carboxy-1a-hydroxyoleanes skeleton
tion at C-4, in the structure of 2 (Table 1). (Rogeres & Coombes, 2001).
The 1H and 13C NMR spectra, incuding 2D
experiments, of 2 indicated that the side
chain in this metabolite was different to Acknowledgements
that in 1 and 7; two trans-olefin hydrogens
at dH 5.99 (d, J=16.1 Hz) and 5.86 (m), and The authors are grateful to Conselho Na-
an oxygenated carbon at dC 81.7, confirmed cional de Desenvolvimento Científico e Tec-
a 1,2-disubstituted E-double bond between nológico (CNPq) and FAPERJ for grants and
C-23 (dC: 129.1) and C-24 (dC: 137.7), and research fellowship. The Herbário Prisco
the presence of a hydroperoxide group Bezerra, Universidade Federal do Ceará,
at C-25 (Banskota et al., 2000) (Tables 1 Ceará, Brazil, is gratefully acknowledged
and 2). This was confirmed by the hetero- for botanical identification.
nuclear long-range couplings observed in

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