2– Feniletanol en las hojas de Brusera velutina Bullock (Burseraceae)
Koji Noge1,4,5, D. Lawrence Venable2 & Judith X. Becerra 3
1 University of Arizona, Department of Entomology, Tucson, AZ 85721, USA.
2 University of Arizona, Department of Ecology and Evolutionary Biology, Tucson, AZ 85721, USA.
3 University of Arizona, Department of Biosphere 2, Tucson, AZ 85721, USA.
4 Present Address: Akita Prefectural University, Department of Biological Production, Akita 010–0195, Japan.
]]> 5 Corresponding author: noge@akita–pu.ac.jp
Recibido en octubre 2010.
Aceptado en junio de 2011.
ABSTRACT
The volatile composition of the leaves of Bursera velutina Bullock (Burseraceae) was determined by a gas chromatograph–mass spectrometer (GC–MS). the major component found was 2–phenylethanol (29.5%). This is the first report of 2–phenylethanol in the leaves of a species of the genus Bursera. in addition, B. velutina also produces monoterpenes, sesquiterpenes, diterpenes and alkanes, making it a species with one of the most complex chemical compositions in the genus. this diverse and unique blend of compounds may play an important role in plant defense against its herbivores.
Key words: Burseraceae, Bursera velutina, 2–phenylethanol, Mexico, terpenes.
RESUMEN
]]> Se identificaron los compuestos químicos volátiles presentes en las hojas de Bursera velutina usando cromatografía de gases y espectrometría de masas. El compuesto de mayor abundancia fue 2–feniletanol (29.5%). Esta es la primera vez que se reporta la presencia de esta substancia en las hojas de plantas del género Bursera. Además de este compuesto aromático, B. velutina produce monoterpenos, sesquiterpenos, diterpenos y alcanos, lo que la convierte en una de las especies de mayor complejidad química en el género. Esta combinación diversa y distinta de compuestos podría jugar un papel importante en la defensa contra sus herbívoros.Palabras clave: Burseraceae, Bursera velutina, 2–feniletanol, México, terpenos.
INTRODUCCIÓN
The genus Bursera Jacq. ex l. (Burseraceae) includes about 100 species of trees and shrubs native to tropical regions of New World, from the southwestern United States to Peru. Bursera's close old World relatives are the source of frankincense (Boswellia) and myrrh (Commiphora). The genus is highly diverse in the tropical dry forests of Mexico where, with about 85 species, it is one of the predominant woody taxa (Rzedowski & Kruse, 1979; Becerra & Venable, 1999; Becerra, 2003). Bursera velutina is a narrow endemic that inhabits the warmest areas at low altitudes on the west side of the Balsas River Basin in Southern Mexico. No previous phytochemical characterization has been reported for the leaves of this species.
Bursera produces an array of terpenes, mostly mono– and sesquiterpenes, and alkanes (Evans et al., 2000; Becerra et al., 2001; Evans & Becerra, 2006; Noge & Becerra, 2009; Noge et al., 2010). These compounds are toxic or repellent to herbivorous insects, and in Bursera they decrease the survival and growth of their specialized herbivores, the chrysomelid genus Blepharida (Becerra, 1994; Becerra et al., 2001). The impact of Blepharida on Bursera often depends on the defensive status of the plants, and individuals with relatively low concentration of terpenes can be completely defoliated by these beetles (Becerra, 1993).
Blepharida beetles show a preference for colonizing chemically similar plants that are not necessarily phylogenetically close (Becerra, 1997). This preference for chemically similar plants might impose selective pressures on plants promoting divergent chemical components (Becerra, 2007). In this study, we investigated the volatile chemical composition of the leaves of B. velutina.
MATERIALS AND METHODS
Plant materials
]]> Samples of leaves of B. velutina were collected from mature individuals in natural populations growing in the vicinity of Altamirano, Guerrero between 100– 102° W and 17–18° N. Voucher specimens are deposited at the University of Arizona Herbarium (ARIZ) under the number Becerra and Venable 377.Sample preparation and chemical analysis
Fresh leaves (28–76 mg) of mature individuals were collected and immediately extracted in 2 ml of dichloromethane at 4 °C for more than 24 h. The extracts were then collected into a new glass vial and kept at 4 °C until chemical analysis. the dichloromethane extract of B. velutina was mixed with the same amount of dichloromethane containing 10 ng/µl 1–dodecene as an internal standard. Then, 1 µl of the mixture was subjected to GC and GC–MS analyses. The volatile components were identified and the yield of essential oil per weight of leaf tissue was determined. The analyses were replicated 15 times using leaf extracts from different leaves from 8 individuals.
GC–MS analysis was carried out by an agilent 6890N gas chromatograph linked to an Agilent 5975B mass spectrometer operated at 70 eV using a HP–5MS capillary column (Agilent Technologies, 30 m X 0.25 mm i.d., 0.25 µm film thickness) with helium carrier gas at 1.2 ml/min in splitless mode. The oven temperature was held at 40 °C for 4 min and programmed to increase at 8 °C/min from 40 °C to 240 °C and finally held at 240 °C for 5 min. The injector temperature was maintained at 200 °C and the detector temperature at 280 °C. all compounds except for sabinene were identified by comparing their GC retention times and mass spectra with those of authentic standards. Sabinene was tentatively identified by comparison of the mass spectrum with that of libraries (Wiley7 and NIST05).
GC analysis was performed on an hewlett–Packard 5890 gas chromatograph with a flame ionization detector, using a DB–5 capillary column (J & W Scientific, 15 m X 0.32 mm i.d., 0.25 µm in film thickness) under the same conditions as those used for GC–MS analysis.
The dichloromethane extract of B. velutina was concentrated by evaporating the solvent, and then the yield of the oil of B. velutina was calculated.
RESULTS AND DISCUSSION
The yield of oil extracted by dichloromethane from fresh leaves was 4.5 ± 0.6% (w/w). The leaf of B.velutina is a rich source of volatile oil similar to the one of Bursera chemapodicta (5.3%, Evans & Becerra, 2006) and the confamilial species, Boswellia sacra (5.5%, Al–Harrasi & Al–Saidi, 2008). Chemical analysis indicated that 2–phenylethanol (29.5%), α–phellandrene (28.8%) and β–phellandrene (11.0%) were the most abundant compounds in the leaves of B . velutina (table 1). Other terpenes and heptane and its derivatives that are already reported from other Bursera species were also found (Evans et al., 2000; Becerra et al., 2001; Evans & Becerra, 2006). This is the first identification of an aromatic compound present in relative large amount in the leaves of a member of the genus Bursera (4.8–6.5 mg/g leaf). Some aromatic compounds such as guaiacol (0.3%), p –cresol (0.2%), vanillin (0.2%) and 2–methoxy–5–methylphenol (0.1%) were detected in the roast aroma extract of B . graveolens as minor components (Yukawa et al., 2006).
Two monoterpenes (limonene, 15.7%; α–terpineol, 10.7%) and two sesquiterpenes (spathulenol, 12.5%; β–eudesmol, 12.9%) have been identified as major components of the bark extract of B . velutina together with 17 other components (Zúñiga et al., 2005). We detected none of these compounds in the leaves of B . velutina. Evans and Becerra (2006) showed that the chemical composition of B . chemapodicta was different between leaves and twigs. Thus, tissue–specific localization or production of resin components may not be unusual in the genus Bursera.
]]> The chemical components found in the Bursera leaves can be classified into three groups based on their biosynthetic pathways (table 2). Terpenes are further divided into three subgroups based on the number of isoprene units and their origin (Davis & Croteau, 2000). 2–phenylethanol is synthesized from phenylalanine in plants (Watanabe et al., 2002; Tieman et al., 2006), and this pathway is fundamentally different from that of terpenes and short–chain aliphatic alkanes. Leaf components of most Bursera species generally include only two or three groups, such as monoterpenes, sesquiterpenes and/or alkanes. Bursera velutina , however, produces more complex blend of compounds that include at least five different basic pathways.2–phenylethanol is known to be a floral fragrance in some plants, having a rose like odor (Knudsen et al., 1993) and attracting various kinds of insects to flowers, such as the cabbage looper moth, Trichoplusia ni (Haynes et al., 1991), the cabbage butterfly, Pieris rapae (Honda et al., 1998), the long–legged chafter, Hoplia communis (Imai et al., 1998) and the lacewing, Chrysopa carnea (Zhu et al., 2005). These flower–visiting insects could act as pollinators. This compound has also been known to inhibit the growth of fungi (Lester, 1965; Terenzi & Storck, 1969) and bacteria by breaking down cell membranes and inhibiting DNA synthesis (Berrah & Konetzka, 1962; Slepecky, 1963; Silver & Wendt, 1967). In terms of anti–herbivore defense, 2–phenylethanol has been identified as a feeding deterrent against pine weevil, Hylobiusabietis , from the non–host plants, Ilex aquifolium and Populus tremula (Eriksson et al., 2008). Thus, this is a multifunctional compound that can play an array of important roles in plants.
Besides having a relatively complex chemical composition, producing a high concentration of 2–phenylethanol makes B . velutina chemically unique, different from other Bursera species. We have analyzed the chemical compositions of 65 of the ~100 species in the genus and only a handful of them produce aromatic compounds and only in trace amounts (Becerra, 2007). Furthermore, all of the other Bursera species that inhabit the distribution area of B . velutina , such as B . trimera, B . kerberii , B . trifoliolata , B . coyucensis and others have very dissimilar chemical compositions to that of B . velutina (Becerra, 2007). Thus, B . velutina is more unlikely to be attacked by their herbivores. For example, neither Blepharida lineata that attacks B . trimera , Blepharida pallida that attacks B . coyucensis , nor Blephari da sparsa that feeds on B . kerberii will accept B . velutina as food (personal observation by J.X.B.). Blepharida flavocostata feeds on B . velutina in the field, but not on the above four sympatric Bursera species (Becerra, 2004). Blepharida beetles prefer chemically similar Bursera species as has been shown for the host recognition of Pieris napi macdunnoughii (Lepidoptera: Pieridae). Both larvae and adults of that crucifer specialist accept two naturalized weeds whose leaf glucosinolate profiles are chemically similar to those of preferred native food plants even the weeds are unfavorable for larval development to pupation (Rodman & Chew, 1980). thus, producing high amounts of 2–phenylethanol may constitute a strong divergence from the sympatric Bursera , making B . velutina chemically distinct and thereby helping avoid herbivory by unadapted Blepharida beetles. Developing unique blends of compounds has also been observed in other burseras. For example, in B . chemapodicta the typical terpene resin composition has been completely replaced with heptane and other hydrocarbon derivatives, which has allowed this species to deter herbivory from Blepharida schlechtendalli, the specialist of the sympatric sister species B . schlechtendalli (Evans & Becerra, 2006).
ACKNOWLEDGMENTS
We thank Leif Abrell and Brenda Jackson (University of Arizona) for the generous gift of chemicals; Phil H. Evans (University of Arizona) for help with chemical analyses. This work was supported by National Science Foundation CAREER grant, a Young Investigator award from the Beckman Foundation, and a grant from the Vice President for Research and the Colleges of Science and Agriculture of the University of Arizona to J.X.B.
LITERATURE CITED
Al–Harrasi, A. & S. Al–Saidi. 2008. Phytochemical analysis of the essential oil from botanically certified oleogum resin of Boswellia sacra Omani Luban). Molecules 13: 2181–2189. [ Links ]
]]>Becerra, J. X. 1993. Adaptations to ecological interactions. PhD thesis. University of Arizona. Tucson. pp. 1–125. [ Links ]
Becerra, J. X. 1994. Squirt–gun defense in Bursera and the Chrysomelid counterploy. Ecology 75: 1991–1996. [ Links ]
Becerra, J. X. 1997. Insects on plants: macroevolutionary chemical trends in host use. science 276: 253–256. [ Links ]
Becerra, J. X. 2003. Evolution of Mexican Bursera (Burseraceae) inferred from ITS, ETS, and 5S nuclear ribosomal DNA sequences. Mol. Phylogenet. Evol. 26: 300–309. [ Links ]
Becerra, J. X. 2004. Molecular systematic of Blepharida beetles (Chrysomelidae: Alticinae) and relatives. Mol. Phylogenet. Evol. 30: 107–117. [ Links ]
]]>Becerra, J. X. 2007. The impact of herbivore–plant coevolution on plant community structure. Proc. Natl. Acad. Sci. 104: 7483–7488. [ Links ]
Becerra, J. X. & D. L. Venable. 1999. Nuclear ribosomal DNA phylogeny and its implications for evolutionary trends in Mexican Bursera (Burseraceae). Am. J. Bot. 86: 1047–1057. [ Links ]
Becerra, J. X., D. L. Venable, P. H. Evans & W. S. Bowers. 2001. Interactions between chemical and mechanical defenses in the plant genus Bursera and their implications for herbivores. Amer. Zool. 41: 865–876. [ Links ]
Berrah, G. & W. A. Konetzka. 1962. Selective and reversible inhibition of the synthesis of bacterial deoxyribonucleic acid by phenethyl alcohol. J. Bacteriol. 83: 738–744. [ Links ]
Davis, E. M. & R. Croteau. 2000. Cyclization enzymes in the biosynthesis of monoterpenes, sesquiterpenes, and diterpenes. Top. Curr. Chem. 209: 53–95. [ Links ]
]]>Eriksson, C., P. E. Månsson, K. Sjödin & F. Schlyter. 2008. Antifeedants and feeding stimulants in bark extracts of ten woody non–host species of the pine weevil, Hylobius abietis . J. Chem. Ecol. 34: 1290–1297. [ Links ]
Evans, P. H. & J. X. Becerra. 2006. Non–terpenoid essential oils from Bursera chemapodicta . Flavour Fragr. J. 21: 616–618. [ Links ]
Evans, P. H., J. X. Becerra, D. L. Venable & W. S. Bowers. 2000. Chemical analysis of squirt–gun defense in Bursera and counterdefense by Chrysomelid beetles. J. Chem. Ecol. 26: 745–754. [ Links ]
Haynes, K. F., J. Z. Zhao & A. Latif. 1991. Identification of floral compounds from Abelia grandiflora that stimulate upwind flight in cabbage looper moths. J. Chem. Ecol. 17: 637–646. [ Links ]
Honda, K., H. Ômura & N. Hayashi. 1998. Identification of floral volatiles from Ligustrum japonicum that stimulate flower–visiting by cabbage butterfly, Pieris rapae. J. Chem. Ecol. 24: 2167–2180. [ Links ]
]]>Imai, T., M. Maekawa, S. Tsuchiya & T. Fujimori. 1998. Field attraction of Hoplia communis to 2–phenylehtanol, a major volatile component from host flowers, Rosa spp. J. Chem. Ecol. 24: 1491–1497. [ Links ]
Knudsen, J. T., L. Tollsten & L. G. Bergström. 1993. Floral scents – a checklist of volatile compounds isolated by head–space techniques. Phytochemistry 33: 253–280. [ Links ]
Lester, G. 1965. Inhibition of growth, synthesis, and permeability in Neurospora crassa by phenethyl alcohol. J. Bacteriol. 90: 29–37. [ Links ]
Noge, K. & J. X. Becerra. 2009. Germacrene D, a common sesquiterpene in the genus Bursera (Burseraceae). Molecules 14: 5289–5297. [ Links ]
Noge, K., N. Shimizu & J. X. Becerra. 2010. (R )–(–)–Linalyl acetate and (S )–(–)–germacrene D from the leaves of Mexican Bursera linanoe. Nat. Prod. Commun. 5: 351–354. [ Links ]
]]>Rodman, J. E. & F. S. Chew. 1980. Phytochemical correlates of herbivory in a community of native and naturalized Cruciferae. Biochem. Syst. Ecol. 8: 43–50. [ Links ]
Rzedowski, J. & H. Kruse. 1979. Algunas tendencias evolutivas en Bursera (Burseraceae). Taxon 28: 103–116. [ Links ]
Silver, S. & L. Wendt. 1967. Mechanism of action of phenethyl alcohol: breakdown of the cellular permeability barrier. J. Bacteriol. 93: 560–566. [ Links ]
Slepecky, R. A. 1963. Inhibition of sporulation and germination of Bacillus magaterium by phenethyl alcohol. Biochem. Biophys. Res. Commun. 12: 369–373. [ Links ]
Terenzi, H. F. & R. Storck. 1969. Stimulation of fermentation and yeast–like morphogenesis in Mucor rouxii by phenethyl alcohol. J. Bacteriol. 97: 1248–1261. [ Links ]
]]>Tieman, D., M. Taylor, N. Schauer, A. R. Fernie, A. D. Hanson & H. J. Klee. 2006. Tomato aromatic amino acid decarboxylases participate in synthesis of the flavor volatiles 2–phenylethanol and 2–phenylacetaldehyde. Proc. Natl. Acad. Sci. 103: 8287–8292. [ Links ]
Watanabe, S., K. Hayashi, K. Yagi, T. Asai, H. MacTavish, J. Picone, C. Turnbull & N. Watanabe. 2002. Biogenesis of 2–phenylethanol in rose flowers: Incorporation of [ 2H 8 ]L–phenylalanine into 2–phenylethanol and its β–D–glucopyranoside during the flower opening of Rosa 'Hoh–Jun' and Rosa damascena Mill. Biosci. Biotechnol. Biochem. 66: 943–947. [ Links ]
Yukawa, C., Y. Imayoshi, H. Iwabuchi, S. Komemushi & A. Sawabe. 2006. Chemical composition of three extracts of Bursera graveolens. Flavour Fragr. J. 21: 234–238. [ Links ]
Zhu, J., J. J. Obrycki, S. A. Ochieng, T. C. Baker, J. A. Pickett & D. Smiley. 2005. Attraction of two lacewing species to volatiles produced by host plants and aphid prey. Naturwissenschaften 92: 277–281. [ Links ]
Zúñiga, B., P. Guevara–Fefer, J. Herrera, J. L. Contreras, L. Velasco, F. J. Pérez & B. Esquivel. 2005. Chemical composition and anti–inflammatory activity of the volatile fractions from the bark of eight Mexican Bursera species. Planta Med. 71: 825–828. [ Links ]
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