Compositional Variation and Bioactivity of the Leaf Essential Oil of Montanoa guatemalensis from Monteverde, Costa Rica: A Preliminary Investigation

Background: Montanoa guatemalensis is a small to medium-sized tree in the Asteraceae that grows in Central America from Mexico south through Costa Rica. There have been no previous investigations on the essential oil of this tree. Methods: The leaf essential oils of M. guatemalensis were obtained from different individual trees growing in Monteverde, Costa Rica, in two different years, and were analyzed by gas chromatography—mass spectrometry. Results: The leaf oils from 2008 were rich in sesquiterpenoids, dominated by α-selinene, β-selinene, and cyclocolorenone, with lesser amounts of the monoterpenes α-pinene and limonene. In contrast, the samples from 2009 showed no α- or β-selinene, but large concentrations of trans-muurola-4(14),5-diene, β-cadinene, and cyclocolorenone, along with greater concentrations of α-pinene and limonene. The leaf oils were screened for cytotoxic and antimicrobial activities and did show selective cytotoxic activity on MDA-MB-231 breast tumor cells. Conclusion: M. guatemalensis leaf oil, rich in cyclocolorenone, α-selinene, and β-selinene, showed selective in vitro cytotoxic activity to MDA-MB-231 cells. The plant may be a good source of cyclocolorenone.


Introduction
Montanoa guatemalensis B.L. Rob. & Greenm. (Asteraceae), "tubú", is a small to medium tree (3-15 m) that is found in Central America from Mexico through Costa Rica. The leaves are around 13 × 17 cm and have 2-4 shallow lobes. The flowers have large heads (5 cm diameter) with white ray flowers (2.5 cm long) and orange inner disk flowers [1]. This tree has been planted as windbreaks in Monteverde [2]. Germacranolide sesquiterpenoids have been isolated from M. guatemalensis [3], but to our knowledge, the volatile composition of this tree has not been previously investigated. In this work, we present the leaf essential oil composition of M. guatemalensis.

Plant Material
Leaves of Montanoa guatemalensis were collected from two different mature trees (tree A and tree B, flowering stage) on 3 May 2008, and two different mature trees (tree C and tree D, flowering stage) on 7 May 2009, from the property of Hotel El Bosque, Monteverde, Costa Rica (10.3059 N, 84.8144 W, 1380 m above sea level). The plant was identified by William Haber, and a voucher specimen (Haber 425) has been deposited in the herbarium of the Missouri Botanical Garden. The fresh leaves were chopped and hydrodistilled using a Likens-Nickerson apparatus with continuous extraction with CHCl3 to give the yellow essential oils (Table 1).

Gas Chromatographic-Mass Spectral Analysis
A gas chromatographic-mass spectral analysis was performed on the essential oils of M. guatemalensis using an Agilent 6890 GC with Agilent 5973 mass selective detector (Santa Clara, CA, USA) (EIMS, electron energy = 70 eV, scan range = 45-400 amu, and scan rate = 3.99 scans/s), and a fused silica capillary column (HP-5ms, 30 m × 0.25 mm) coated with 5% phenyl-polymethylsiloxane (0.25 mm phase thickness). The carrier gas was helium with a flow rate of 1 mL/min, and the injection temperature was 200 °C. The oven temperature was programmed to initially hold for 10 min at 40° C, then ramp to 200 °C at 3 °C/min and finally to 220 °C at 2 °C/min. The interface temperature was 280 °C.
A 1% w/v solution of each sample in CHCl3 was prepared, and 1 μL was injected using a splitless injection technique.
Identification of the oil components was based on their retention indices determined by reference to a homologous series of n-alkanes, and by comparison of their mass spectral fragmentation patterns with those available in commercial libraries [4,5] as well as our own in-house library [6]. The percentages of each component are reported as raw percentages based on total ion current without standardization. The chemical compositions of the M. guatemalensis leaf oils are summarized in Table 2.

Antibacterial Screening
The M. guatemalensis leaf oils were screened for antibacterial activity against Bacillus cereus Solutions were added to wells in four replicates. Medium controls and DMSO controls (10 μL DMSO/mL) were used. Tingenone (100 μg/mL) was used as a positive control [10]. After the addition of compounds, plates were incubated for 48 h at 37 °C; medium was then removed by suction, and 100 μL of fresh medium was added to each well. In order to establish percent kill rates, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA) assay for cell viability was carried out [11]. After colorimetric readings were recorded (using a Molecular Devices (Sunnyvale, CA, USA) SpectraMAX Plus microplate reader, 570 nm), average absorbances, standard deviations, and percent kill ratios (%killoil/%killDMSO) were calculated. Cytotoxic activities of the essential oils are summarized in Table 3.
The leaf oils of M. guatemalensis were screened for antibacterial activity against Staphylococcus aureus, Bacillus cereus, and Escherichia coli, but showed only marginal activity against B. cereus ( Table 3). The oils were also screened for in vitro cytotoxic activity against MDA-MB-231 human mammary adenocarcinoma and Hs578T human mammary ductal carcinoma cells. The oils did show selective cytotoxicity to MDA-MB-231 cells over Hs578T cells (Table 3). A previous report indicated the leaf essential oil of Montanoa ovalifolia from Colombia (composition not reported) had shown marginal cytotoxic activity to Vero and HeLa cells, but no activity against HepG2 or Jurkat cells [31].

Conclusions
This is the first analysis of the volatiles from Montanoa guatemalensis. The leaf oil, rich in cyclocolorenone, α-selinene, and β-selinene, showed selective in vitro cytotoxic activity to MDA-MB-231 cells. It is not clear, however, what components are responsible for the cytotoxicity, but the plant may be a good source of cyclocolorenone. Although this work presents preliminary results, it should serve as a template for further experimentation on the leaf oil compositions of M. guatemalensis, seasonal, individual, and year-to-year variations.