Fitoterapia 76 (2005) 562 – 566 www.elsevier.com/locate/fitote
Licanolide, a new triterpene lactone from Licania tomentosa Rachel O. Castilho*, Rodrigo R. de Oliveira, Maria A.C. Kaplan Nu´cleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Centro de Cieˆncias da Sau´de, Bloco H, Cidade Universita´ria, CEP 21941-590, Rio de Janeiro, R.J., Brazil Received 15 April 2004; accepted in revised form 27 April 2005 Available online 20 June 2005
Abstract Licanolide (3h-hydroxylupane-20,28-olide), a novel triterpene lactone with new stereochemical pattern, was isolated from fresh fruits of Licania tomentosa in addition to betulinic and palmitoleic acid. The structure elucidation was based on spectroscopic methods including two-dimensional NMR experiments (HSQC, HMBC and NOESY). D 2005 Elsevier B.V. All rights reserved. Keywords: Licania tomentosa; Licanolide (3h-hydroxylupane-20,28-olide); Triterpene lactone
1. Introduction The Chrysobalanaceae encompasses 17 genera and about 450 species represented by trees and shrubs growing in tropical and subtropical lowlands [1]. Some of Chrysobalanaceae spp. have been used in the tropics as fruit trees and many of them are used popularly as medicine in several countries, mainly for the treatment of dysentery, diarrhea, and leucorrhea [2–5]. Licania tomentosa has not been investigated for chemical constituents or pharmacological action. However phytochemical studies on many Licania spp. such as L. densiflora [6], L. heteromorfa [7], L. pittieri [8] and L. pyrifolia [9] as well
* Corresponding author. Tel.: +55 67 363 8477. E-mail address:
[email protected] (R.O. Castilho). 0367-326X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2005.04.018
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as other Chrysobalanaceae resulted in the isolation and characterization of two major classes of compounds, triterpenes and flavonoids metabolites [10–13]. The present paper reports the isolation of a new triterpene lactone, licanolide (1) and two known compounds, betulinic acid (2) and palmitoleic (3) from fruits of the title plant.
2. Experimental 2.1. General Melting points were measured on a Thomas-Hoover or a Koffler Micro Melting apparatus and are uncorrected. Optical rotations were measured on a JASCO DIP-360 polarimeter. CG/MS were determined on HP5890SII chromatograph and VG Autospec spectrometer. Samples were run on DB-1 bonded phase fused silica capillary column (30 m 0.20 mm). Helium was carrier gas. UV and IR were determined on Shimadzu. 1H- and 13C-NMR (one- and twodimensional) spectra were measured on a Bruker (Rheinstetten, Germany) model DRX 400 at 400.13 and 100.61 MHz, respectively, with TMS as internal standard. Thin-layer chromatography (TLC) was performed on Merck F254 silica plates (0.25 mm) and spots were detected by spraying with ceric sulfate followed by heating. 2.2. Plant material L. tomentosa Benth. (Chrysobalanaceae), collected in the State of Rio de Janeiro, Brazil and identified by Dr Rosa Fuks, Rio de Janeiro Botanical Garden was deposited in the Museu Nacional Herbarium (R195.942), Universidade Federal do Rio de Janeiro, UFRJ. 2.3. Extraction and isolation L. tomentosa fresh fruits (1300 g) were extracted at r.t. successively with hexane and MeOH. The concentrated hexane extract (8 g) was Si-gel CC eluting with a binary mixtures of hexane, EtOAc and MeOH to afford fractions 1–100. The hexane: EtOAc 20% eluted fractions (49–54) were purified by crystallization with pentane and dichloromethane, giving two crystalline compounds (1, 13 mg and 2, 57 mg) and (3, 30 mg). 3h-Hydroxylupane-20,28-olide (1), needle crystals (CHCl3), mp 277–278 8C with decomposition; [a]D25 + 38 (c 0.2, CHCl3);. UV max (EtOH) 385, 228 nm; IR bands (nujol): 3755; 3524; 2933; 2864; 2368; 1762; 1463; 1386; 1250; 1144; 1068; 1006; 935; 872, 736 and 584 cm 1; 1H-NMR (400 MHz, CDCl3): y 0.87 (3H, s, CH3-26), 0.94 (3H, s, CH3-24), 0.97 (3H, s, CH3-23), 1.02 (3H, s, CH3-25), 1.05 (3H, s, CH3-27), 1.09 (3H, s, CH3-29), 1.15 (3H, s, CH3-30), 3.0 (1H, m, H-9), 3.1 (1H, m, H-19), 3.45 (1H, m, H-3); 13 C-NMR (100 MHz, CDCl3): y 37.5 (C-1), 27.8 (C-2), 75.8 (C-3), 37.2 (C-4), 49.4 (C-5), 20.9 (C-6), 26.4 (C-7), 40.4 (C-8), 52.6 (C-9), 36.4 (C-10), 21.6 (C-11), 34.1 (C-12), 48.0 (C-13), 41.3 (C-14), 29.5 (C-15), 30.3 (C-16), 43.7 (C-17), 50.2 (C-18), 56.9 (C-19), 87.5 (C-20), 24.8 (C-21), 32.5 (C-22), 26.8 (C-23), 17.0 (C-24), 23.4 (C-25), 21.1 (C-26), 17.3 (C-27), 179.3 (C-28), 33.0 (C-29), 188 (C-30). CG-MS m/z: 470 [M]+ (2.5), 411 (2.5), 262
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O
O
HO Fig. 1. Structure of lincanolide (1).
(27.5), 249 (8.75), 233 (10), 220 (22.5), 207 (42.5), 189 (96.25), 175 (43.75), 133 (41.25), 119 (65), 95 (76.25), 81 (90), 69 (81.25), 55 (100).
3. Results and discussion The hexane extract from fresh fruits of L. tomentosa was chromatographed on silica gel column eluting with solvents of increasing polarities to obtain compounds 1–3. Physical and spectral data for 2 and 3 agreed well with those published for betulinic and palmitoleic acid [14,15]. Licanolide, identified as 3h-hydroxylupane-20, 28-olide, (1) was obtained as needle shape crystals (Fig. 1). Molecular formula after methylation was determined as C31H50O3 on the basis of its CG/MS. Furthermore, the mass fragmentation of the compound (1) derivative exhibited characteristic pattern of the lupane peaks at m/z 411, 262, 233, 175, 207 and 189. The IR spectrum showed absorption band at 1763 cm 1 and 3524 cm 1, suggesting the presence of y-lactone and hydroxyl group. The 1H-NMR Table 1 1 H and 13C-NMR data (400 and 100 MHz, respectively, CDCl3) of 1 C
Ma
yC
yH
HMBC
C
M
1 2 3 4 5 5 7 8
CH2 CH2 CH C CH CH2 CH2 C
37.5 27.8 75.8 37.2 49.4 20.9 26.4 40.4
1.25, 1.35 1.60, 1.63 3.45 – 2.35 1.20 1.63, 1.67 –
16 17 18 19 20 21 22 23
CH2 C CH CH C CH2 CH2 CH3
30.3 43.7 50.2 56.9 87.5 24.8 32.5 26.8
9 10 11 12 13 14 15
CH C CH2 CH2 CH C CH2
52.6 36.4 21.6 34.1 48.0 41.3 29.5
3.0 – 1.63, 1.71 1.45, 1.50 1.71 – 1.30, 1.20
H2, H5, H25 H1, H3 H1, H2, H23, H24 H2, H6, H23, H24 H6, H7, H23, H24 H5, H7 H5, H6, H9, H25 H6, H9, H11, H13, H26, H27 H1, H11, H12, H26 H1, H2, H5, H25 H9 H11 H13
24 25 26 27 28 29 30
CH3 CH3 CH3 CH3 C CH3 CH3
17.0 23.4 21.1 17.3 179.3 33.0 18.9
a
M = Multiplicity.
yC
yH 1.10, 1.30 – 2.30 3.10 – 1.45, 1.50 1.20, 1.30 0.97 0.94 1.02 0.87 1.05 – 1.09 1.15
R.O. Castilho et al. / Fitoterapia 76 (2005) 562–566
29
565
30
O H H 19 O 21
20
H HH 25
HO
27
3 24
12 13 26
28 17
H
16
H
23
Fig. 2. NOESY correlations of lincanolide (1).
spectrum (Table 1) showed resonances for seven methyl singlets, the lack of the double bond between C20 and C29 characteristic of betulinic acid [14]. The multiplet at y 3.45 established the presence of a hydroxyl in position 3. The 13C-NMR (Table 1) of (1) exhibits a signal at y 75.8 (h-OH at C-3 ) [15], at y 87.5 and 179.3 (lactone between C-20 and C-28), and lack of double bound signals corresponding to C-20 and C29 of the betulinic acid. The HMBC spectrum confirmed the presence of y-lactone group between C-20 and C-28 by correlations of the carbon at y 87.5, and the two methyl groups at C-29 and C-30, the signals in the 1H-NMR spectrum appearing at y 1.09 and 1.15, respectively. Comparison of 13C-NMR data with the literature [16–20] confirmed that triterpene belongs to the lupane series with the C-3 OH in h orientation, lack of C20–C29 double bond and the presence of C28–C20 lactone group, confirmed by H,H-NOESY experiment and observed Overhouser effects (Fig. 2). In conclusion, detailed analysis of spectra led to the assignment of structure (1) to the novel compound.
Acknowledgements The authors thank CAPES and CNPq for financial support.
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