Germacrene D, A Common Sesquiterpene in the Genus Bursera (Burseraceae)

The volatile components of the leaves of five Bursera species, B. copallifera, B. exselsa, B. mirandae, B. ruticola and B. fagaroides var. purpusii were determined by gas chromatography–mass spectrometry (GC–MS). Germacrene D was one of the predominant components (15.1–56.2%) of all of these species. Germacrene D has also been found in other Bursera species and some species of Commiphora, the sister group of Bursera, suggesting that the production of germacrene D might be an ancient trait in the genus Bursera.


Introduction
The genus Bursera (Burseraceae) comprises approximately 100 species with a geographical distribution extending from the southwestern United States to Peru. The genus predominates in the tropical dry forests of Mexico where about 85 species coexist and about 75 of them are endemic [1][2][3]. The genus Bursera is divided into two sections, Bullockia and Bursera. Bursera release their resins when they are attacked by their herbivore, Blepharida beetles (Chrysomelidae: Alticinae) [4,5]. The

OPEN ACCESS
resin is reported to decrease the survival and growth of Blepharida beetles [5,6]. The chemical analysis of the leaf components of B. biflora and B. schlechtendalii revealed that their resins consisted mostly of mono-and sesquiterpenes, and alkanes [7,8].
The volatile chemistry of five species, B. copallifera, B. excelsa, B. mirandae, B. ruticola (belonging to section Bullockia) and B. fagaroides var. purpusii (from section Bursera), were analyzed in this study. Bursera copallifera is native to the dry forests from the state of Nayarit to Oaxaca and Puebla at altitudes of between 1,000 and 1,900 m. Bursera excelsa is mostly found along the Pacific coast at low altitudes, while B. mirandae has a narrow distribution in the north of the states of Guerrero and Oaxaca. Bursera ruticola is a narrow endemic form the tip of Baja California. Bursera fagaroides var. purpusii is an abundant species also of relative wide distribution in the South of Mexico at altitudes between 300 and 900 m. All of these species are abundant either locally or regionally. Yet, little information is known about their chemistry.

Results and Discussion
The components found in the five Bursera species analyzed are summarized in Table 1. Since the chemical profiles of the field samples were very similar to those of the greenhouse plants, only the latter ones are presented. The similarity of the chemical profiles between field and greenhouse samples suggests that the production of these compounds is quite stable even in the face of environmental changes. The chemical profiles of each one of these species were species-specific. For example, some compounds such as α-thujene, α-phellandrene, β-ocimene, and others were detected in only one of the species. Some compounds such as α-pinene, β-caryophyllene, germacrene D and bicyclogermacrene, however, were detected in all of the species examined as either major (higher than 10% of total) or minor (less than 10% of total) components. The ratio of monoterpenes to sesquiterpenes was also different among the species (Table 1). The composition pattern could be classified into three types; (1) monoterpene-rich group (B. fagaroides var. purpusii), (2) sesquiterpene-rich group (B. copallifera and B. excelsa), and (3) the intermediate group (both monoterpene-and sesquiterpene-rich; B. mirandae and B. ruticola). It seems that terpene production in the species of the section Bursera tends to be biased toward monoterpenes, and a high percentage of these compounds have been found in the leaves of other species belonging to this section such as B. lancifolia, B. rezedowskii, B. schlechtendalii and B. morelensis (75-95%) [6].
Germacrene D was one of the predominant components found in the leaves of the five species analyzed in this study (15.1-56.2%; Table 1). The concentration of germacrene D was 0.14 ± 0.02 mg per gram of fresh leaves (mg/g.l., means ± SD, N = 5) in B. copallifera, 0.14 ± 0.03 mg/g.l. in B. excelsa, 0.12 ± 0.03 mg/g.l. in B. mirandae, 1.06 ± 0.33 mg/g.l. in B. ruticola and 0.13 ± 0.06 mg/g.l. in B. fagaroides var. purpusii, respectively. A preliminary chemical screening of the leaves of 26 other Bursera species also revealed the presence of germacrene D in all of them (2.6-53.8%, data not shown), suggesting that this compound is common in the genus. Germacrene D has been recently reported from the leaves of B. simaruba [9] and the stem of B. graveolens [10], respectively. Germacrene D has also been found in some species of Commiphora, the sister group of Bursera, such as C. africana (Noge and Becerra, unpublished), C. holtziana [11] and C. myrrha [12]. Thus, producing germacrene D might be an ancient trait in the genus Bursera.   Germacrene D has been found not only in angiosperms and gymnosperms but also in bryophites (Table 2), yet despite its wide distribution, its biological function in plants is still not well understood. It has been proposed that germacrene D plays a role as a precursor of various sesquiterpenes such as cadinenes and selinenes [13,14]. Plant terpenes have often been reported as anti-herbivore defenses [15]. It has also been suggested that germacrene D, by itself may have deterrent effects against herbivores and it has been reported to have insecticidal activity against mosquitoes [16], as well as repellent activity against aphids [17] and ticks [11].
Bursera is attacked by a group of specialized chrysomelid beetles, the genus Blepharida. These beetles show a preference for colonizing chemically similar plants that are not necessarily phylogenetically close [18]. This preference for chemically similar plants might impose pressures on plants to develop divergent chemical components. Thus, the common presence of germacrene D in Bursera may be advantageous as a source of other terpenes that could make the plants chemically different and thus help them escape from herbivory by unadapted Blepharida beetles. We are currently investigating its potential role against Bursera's herbivores. Being such an abundant and frequent compound in this genus, it is likely to play an interesting role in the functioning of these plants.  The genus Bursera is one of the dominant members of the tropical dry forests of Mexico in terms of both diversity and abundance. This study is a contribution to the chemical knowledge of this important group of plants.

Plant materials
Samples of leaves of one to five mature individuals from each species were collected from natural field populations and immediately extracted in dichloromethane. We also collected cuttings of each species and rooted them in a mixture of 70% pumice and 30% topsoil. All cuttings took and were kept in a greenhouse at the University of Arizona under similar environmental conditions to those in the field. When these cuttings developed leaves, they were also extracted for chemical analysis. Voucher specimens of all except for B. ruticola are deposited at the University of Arizona Herbarium (ARIZ) and the Mexican National Herbarium at the Autonomous National University of Mexico (MEXU) [2]. Voucher specimen of B. ruticola is deposited at ARIZ [3].

Chemical analysis
One microliter of each extract was analyzed by GC-MS (an Agilent 6890N gas chromatograph linked to an Agilent 5975B mass spectrometer, operated at 70 eV, with a HP-5MS capillary column, 30 m × 0.25 mm i.d., 0.25 μm in film thickness) and GC (the same gas chromatograph as GC-MS with a flame ionization detector, with a DB-5MS capillary column, 25 m × 0.32 mm i.d., 0.52 μm in film thickness). The oven temperature was programmed from 50 ºC (3 min holding) to 290 ºC at a rate of 10 ºC/min and then held for 5 min. The injector temperature was maintained at 200 ºC. When the authentic standard of germacrene D was analyzed with the injector temperature at 250 ºC, some artificial peaks were observed. However, none of these peaks were detected when the injector was maintained at 200 ºC. The analyses were replicated five times using different leaves for each species. The same analyses were performed on the leaf extractions collected in the field.

Conclusions
The GC-MS analysis of the leaf extract of five Bursera revealed that they produce species-specific chemical components in their leaves. The study also shows the presence of germacrene D as a common sesquiterpene in the genus.