Major Components from the Epicuticular Wax of Cocos nucifera
Fabiola Escalante Erosa,1 M. Rubí Gamboa-León,1 Jana G. Lecher,1 Gabriela A. Arroyo-Serralta,1 Daniel Zizumbo-Villareal,2 Carlos Oropeza-Salín1 and Luis M. Peña-Rodríguez1*
1 Grupo de Química Orgánica, Unidad de Biotecnología, Centro de Investigación Científica de Yucatán. Calle 43, No. 130, Col. Chuburná de Hidalgo, Mérida, Yucatán, México 97200. *Telephone: 52(9) 981-3923. E-mail: Imanuel@cicy.mx
2 Unidad de Recursos Naturales, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, México 97200.
Recibido el 29 de abril del 2002. ]]> Aceptado el 28 de agosto del 2002.
Abstract
The three major components present in the epicuticular wax from leaves of Cocos nucifera L. were identified as lupeolmethylether (1), skimmiwallin (2) [3β-methoxy-25-ethyl-9,19-cyclolanost-24(241)-ene] and isoskimmiwallin(3) [3β-methoxy-24-ethyl-9,19-cyclolanost-25(251)-ene]. Structural elucidation of the metabolites was carried out by analysis of their spectroscopic data and/or by comparison with those reported in the literature.
Keywords: Cocos nucifera, epicuticular wax, chemotaxonomy, triterpenes, lupeolmethyl ether, skimmiwallin, isoskimiwallin.
Resumen
Los tres componentes principales presentes en la cera epicuticular de Cocos nucifera L. fueron identificados como el éter metílico de lupeol (1), skimmiwallina (2) [3β-metoxi-25-etil-9,19-ciclolanost-24(241)-eno] e isoskimmiwallina (3) [3β-metoxi-24-etil-9,19-ciclolanost-25(251)-eno]. La elucidación estructural de estos metabolitos se llevó a cabo mediante la interpretación de sus datos espectroscópicos y/o por comparación de los mismos con los reportados en la literatura.
Palabras clave: Cocos nucifera, cera epicuticular, quimiotaxonomía, triterpenos, eter metílico de lupeol, esquimiwallina, isoesquimiwallina.
]]> Dedicated to Dr. Barbarín Arreguín Lozano
Introduction
All aerial organs of higher plants are covered by a continuous wax layer on the surface of the cuticle [1]. This layer protects plant cells from various enviromental factors such as drought and UV damage [2], and acts as a first line of defense against insects, bacteria and fungal pathogens [2, 3]. In some higher plants, morphological and chemical studies carried out on epicuticular waxes have been used to correlate the nature and the chemical composition of the wax, with the susceptibility of the plant to insect attack or to chemical agents [4, 5].
The main components of the wax of Brassica oleracea, identified as amyrin-type triterpenes, have been recognized as the metabolites responsible for the repellent effect against Plutella xilostella aphids [6]. These results have been confirmed by similar studies which have shown that amyrins and other triterpens have repellent or toxic activity against insects [3, 7].
Studies carried out on the chemical composition of the epicuticular waxes from various palm species have resulted in the isolation and identification of a number of triterpenes, including lupeol methylether from Orbignya speciosa, Butia capitata and Orbignya phalerata, 3-β-methoxylupane from Orbignya phalerata, and cylindrin from Orbignya cohune [4]. To date no reports on the chemical composition of the epicuticular wax of C. nucifera have been found.
Recently, 18 populations of Cocos nucifera L. growing in different regions of Mexico were grouped into five ecotypes according to their similarities in phenotypic characters and isoenzymatic profiles [8]. Since the chemical composition of the epicuticular wax of a number of plant species has been used as a chemotaxonomic marker for classification [4, 5], the main objective of this investigation was to isolate and identify the major components present in the epicuticular wax of C. nucifera for future use in chemotaxonomical studies. We wish to report herein the identification of lupeolmethylether (1), skimmiwallin (2) and isoskimmiwallin (3) as the major components of the epicuticular wax of C. nucifera.
Results and discussion
The hexane extract from pines of leaves of C. nucifera showed two main components when analyzed by conventional TLC. Silica gel column chromatography purification of the extract yielded two major fractions, each containing one of the main components in apparent pure form. The fraction containing the more polar component showed a single peak by GC and the pure metabolite was identified as lupeol methylether (1) by direct comparison with an authentic sample and by comparing its spectroscopic data with those reported in the literature.
]]> Even though the least polar component appeared as a single spot on TLC, its GC analysis showed that it was in fact a mixture of two metabolites that could only be separated by using AgNO3-impregnated silica gel TLC plates. Successive AgNO3-impregnated silica gel column chromatography and preparative TLC yielded both components in pure form. The EIMS of the less polar metabolite showed a molecular ion peak at m/z 482 indicating a molecular formula of C34H58O and suggesting a triterpenoid structure. While the two signals at 0.32 and 0.56 ppm in its 1H NMR indicated the presence of a cyclopropane ring in the structure and strongly suggested a cycloartane skeleton, the presence of a sharp singlet at 3.36 ppm clearly indicated that the single oxygen in the molecular formula was part of a methoxyl group. Confirmation of the cycloartane skeleton for this metabolite came from its EIMS where the characteristic fragment ion peak at m/z 328, originated by loss of the A ring in cycloartanes [9, 10], was observed.
The same fragment ion peak could be explained as resulting from the loss of a C11H21 side chain in the molecule; this data, together with four methyl signals at 0.90 (d, 6.5 Hz), 0.95 (s), 0.97 (s), and 1.05 ppm (t, 7.5 Hz), and two vinylic proton signals at 4.77 (bd, 1 Hz) and 4.79 (bs) ppm, was in agreement with an eight-carbon side chain having a gem-dimethyl group and a 1,1 disubstituted double bond as substituents. This data proved to be identical to those reported for skimmiwallin (2) [3β-methoxy-25-ethyl-9,19-cyclolanost-24(241)-ene], a cycloartane isolated from the petrol ether extract of Skimmia wallichii [11].
The second most polar component showed identical spectral data to those of 2, suggesting an isomeric structure. Significant differences could only be observed on comparing the HMBC experiment results for each component; while the HMBC experiment of 2 showed a clear 3J correlation between the C27-methyl signal (1.05 ppm) and the sp3-quaternary carbon at 39.51 ppm (C25), the same experiment in the new metabolite showed a definite 3J correlation between the same methyl group (C27, 1.05 ppm) and an sp2-quaternary carbon (C25, 157.92 ppm). This data is in agreement with an isomeric structure 3 for the new metabolite, where the only structural difference between 2 and 3 is located at the side chain of the molecule. Because of the isomeric relationship with 2, we have designated the new metabolite as isoskimmiwallin (3).
The purified metabolites 1-3 were used as standards in GC analyses to show that the epicuticular wax profiles of the five main ecotypes of C. nucifera are qualitatively similar, but quantitatively different [12]. These results also showed that there exists a correlation between the concentrations of 1, 2, and 3 in the epicuticular wax and the resistance or susceptibility of a given ecotype to the lethal yellowing disease of coconut palms. Presently, studies are underway in order to establish if the components, pure or combined, show biological activity against the insect vector that transmits the disease. The results of these studies will be published in due course.
Experimental
General
]]> Samples for IR were dissolved in CHCl3 (Merck, uvasol) and spectra were recorded using a Nicolet Magna Protégé 460 FT-IR instrument. 1H NMR and 13C NMR spectra were recorded on Varian Unity-300, Bruker AMX-400, and Varian Unity Plus-500 spectrometers using CDCl3 as solvent and residual solvent signals for reference. EIMS were recorded at 70 eV on a JEOL-JMSAX505HA and JEOL-JMS-SX102A mass spectrometer for low and high resolution, respectively; while GC-MS analyses were carried out in a Hewlett Packard 5890 gas chromatographer coupled to a 5971 mass selective detector (GC conditions: column Hp Ultra 1; flow rate 1mL/min; oven temperature 280 to 300 °C; gradient 5 C/min; injector 290 °C; detector 300°). Analytical TLC was performed on aluminum-backed Silica gel 60 F254 plates (E. M. Merck, 0.20 mm thickness), both normal and impregnated with a 5% AgNO3 solution, and preparative TLC (PTLC) was performed on glass-coated (0.25 mm thickness) Silica gel 60 F254 (E. M. Merck) plates (20 × 20 cm) impregnated with a 5% AgNO3 solution. Flash column chromatography purifications were run using Silica gel G (200-400 mesh, Aldrich Chemical Co.).Plant material
Pines of Cocos nucifera were collected in September 1999 from plants (Alto del Pacífico and Enano Malayo) growing in the San Crisanto plantation located in Sinanché, Yucatán, México.
Extraction and isolation
Six pines were cut at the base and immersed for 40 seconds in a liter of hexane contained in a measuring cylinder. The solvent was evaporated to dryness under reduced pressure and the crude wax extract was purified by flash column chromatography (Hx/ CH2Cl2 7:3 as the eluting solvent) to produce two major fractions, each showing a single component on TLC. GC-MS analysis of fraction A showed the presence of a single component at Rt10.73 having a fragmentation pattern very similar to that of lupeolmethylether (1). The identity of 1 (60.3 mg) was confirmed by direct comparison with an authentic sample and by comparing its spectroscopic data with those reported in the literature [11].
Although fraction B showed a single component on normal TLC, its GC analysis clearly indicated the presence of two metabolites (Rt14.45 and 15.14 min) that could only be separated using AgNO3-impregnated Silica gel TLC plates (CH2Cl2). Successive purifications using AgNO3-impregnated Silica gel column chromatography (Hx / CH2Cl2 7:3 as the eluting solvent) and PTLC (CH2Cl2 as the eluting solvent) resulted in the isolation of 2 (16.9 mg) and 3 (10.9 mg) in pure form. These components were identified as skimmiwallin (2) and isoskimmiwallin (3), respectively.
Skimmiwallin (2): mp. 156-159°. IR (CHCl3 cm-1) 1097, 1468; HREIMS m/z 482.42881 (calcd. for C34H58O, 482.448767); LREIMS m/z (rel. int.); 482 [M]+ (7), 467 [M-Me]+ (25), 450 [M-MeOH]+ (71), 435 [M-Me-MeOH]+ (100), 407 [M-MeOH-43]+ (46), 381 [M-MeOH-69]+ (30), 328 [M-C11H21]+ (16), 297 [M-C11H21-MeOH]+ (20), 175 [M-C11H21-side chain]+ (74); 1H NMR (400 MHz, CDCl3) see Table 1, 13C NMR (100 MHz, CDCl3) δ 31.79 (C-1), 25.42 (C-2), 40.46 (C-4), 47.63 (C-5), 20.93 (C-6), 25.94 (C-7), 47.97 (C-8), 19.97 (C-9), 26.29 (C-10), 25.50 (C-11), 45.29 (C-13), 48.83 (C-14), 35.54 (C-15), 28.13 (C-16), 52.27 (C-17). For additional 13C NMR data see Table 1.
Isoskimmiwallin (3): IR (CHCl3 cm-1) 1098, 1470; HREIMS m/z 482.35862 (calcd. for C34H58O, 482.448767); LREIMS m/z (rel. int.); 482 [M]+ (7), 467 [M-Me]+ (22), 450 [M-MeOH]+ (60), 435 [M-Me-MeOH]+ (100), 407 [M-MeOH-43]+ (47), 381 [M-MeOH-69]+ (38), 328 [M-C11H21]+ (22), 297 [M-C11H21-MeOH]+ (24), 175 [M-C11H21-side chain]+ (78); 1H NMR (400 MHz, CDCl3) see Table 1, 13C NMR (100 MHz, CDCl3) 31.81 (C-1), 30.78 (C-2), 40.48 (C-4), 47.95 (C-5), 20.94 (C-6), 25.43 (C-7), 47.66 (C-8), 19.98 (C-9), 26.29 (C-10), 25.96 (C-11), 32.86 (C-12), 45.2 (C-13), 45.80 (C-14), 35.54 (C-15), 28.87 (C-16), 52.14 (C-17). For additional 13C NMR data see Table 1.
Acknowledgments
]]> We thank Miguel Fernández-Barrera, Centro de Investigación Científica de Yucatán, for collecting palm leaves; Mariela Padrón-Hernández and Gabriela Arana López for technical assistance; Guillermo Delgado-Lamas, Luis Velasco, Javier Pérez, Isabel Chávez, Héctor Ríos, Beatriz Quiroz (Universidad Nacional Autónoma de México), for LREIMS and NMR spectroscopy; and Solomon Habtemariam, University of Greenwich UK, for HREIMS.
References
1. Gülz, P.-G.; Müeller E.; Prasad, R.B.N. Phytochemistry 1991, 30, 769-773. [ Links ]
2. Taíz, L.; Zeiger, E. Plant Physiology The Benjamin/Cummings Publishing Company, Inc., 1991. [ Links ]
3. Yoon, R.; Alenka, H-R.; Jayakumar P.; Dehua, L; Dusty, P-B., Plant Physiology 1998, 24, 901-911. [ Links ]
4. García, S.; Heinzen, H.; Hubbuch, C.; Martínez, R.; De Vries X.; Moyna, P. Phytochemistry 1995, 39, 1381-1382. [ Links ]
5. Rashotte, A.; Jenks, M.; Nguyen, T.; Feldmann, K., Phytochemistry 1997, 45, 251-255. [ Links ]
6. Sanford, D.; Satish, K., J. Chem. Ecol. 1998, 24, 1611-1627. [ Links ]
7. Manners, G.; Davis, D. Phytochemistry 1984, 23, 1059-1062. [ Links ]
8. Zizumbo, D., Bol. Soc. Bot. México 1998, 62, 157-170. [ Links ]
9. Aplin, R.T.; Hornby G. M. J., Journal of Chemical Society 1966, (B), 1078-1079. [ Links ]
10. Audier, H. E.; Beugelmans, R.; Das, B. C., Tetrahedron Lett. 1966, 36, 4341-4347. [ Links ]
11. Kostova, I.; Simeonov, M.; Iossifova, T.; Tappe, R.; Pardeshi, N. ; Budzikiewicz, H. Phytochemistry 1996, 43, 643-648. [ Links ]
12. Arroyo-Serralta, G.; Escalante-Erosa, F.; Peña-Rodríguez, L.M.; Zizumbo-Villarreal, D., Biochemical Systematics and Ecology 2001, Submitted. [ Links ]
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