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Year : 2010  |  Volume : 2  |  Issue : 1  |  Page : 19-21 Table of Contents     

The chemical composition and antimicrobial activity of the leaf oil of Cupressus lusitanica from Monteverde, Costa Rica

Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA

Date of Submission16-Oct-2009
Date of Decision19-Nov-2009
Date of Web Publication13-Mar-2010

Correspondence Address:
William N Setzer
Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0974-8490.60585

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The essential oils from the leaves of three different individuals of Cupressus lusitanica were obtained by hydrodistillation and analyzed by gas chromatography - mass spectrometry. A total of 49 compounds were identified in the leaf oils. The major components of C. lusitanica leaf oil were α-pinene (40%-82%), limonene (4%-18%), isobornyl acetate (up to 10%) and cis-muurola-4(14),5-diene (up to 7%). The essential oil was screened for antimicrobial activity, and it showed antibacterial activity against Bacillus cereus and antifungal activity against Aspergillus niger.

Keywords: cis -muurola-4(14), 5-diene, α-pinene, antimicrobial, composition, Cupressus lusitanica, essential oil, isobornyl acetate, limonene

How to cite this article:
Hassanzadeh SL, Tuten JA, Vogler B, Setzer WN. The chemical composition and antimicrobial activity of the leaf oil of Cupressus lusitanica from Monteverde, Costa Rica. Phcog Res 2010;2:19-21

How to cite this URL:
Hassanzadeh SL, Tuten JA, Vogler B, Setzer WN. The chemical composition and antimicrobial activity of the leaf oil of Cupressus lusitanica from Monteverde, Costa Rica. Phcog Res [serial online] 2010 [cited 2021 Feb 28];2:19-21. Available from: http://www.phcogres.com/text.asp?2010/2/1/19/60585

   Introduction Top

There are 13 species of Cupressus (Cupressaceae) distributed throughout North America. [1] Cupressus lusitanica Mill, known in Costa Rica as ciprés, normally ranges from central Mexico to Honduras but has been cultivated in other parts of the world. In Monteverde, it has been planted as a windbreak to protect dairy cows from harsh winds. [2] The leaves of this plant are used to cure some skin diseases caused by dermatophytes and have also been used to ward off insects from stored grain. [3] In Costa Rica, a drink made by steeping a branch in alcohol is taken to alleviate coughs and cold symptoms. [4] Essential oil compositions of C. lusitanica from Portugal [5,6] and from Cameroon [3] have been reported, but these show wide variation. In this report, we present the leaf essential oil composition and antimicrobial activity of C. lusitanica from Monteverde, Costa Rica.

   Materials And Methods Top

Plant material

Leaves of C. lusitanica were collected from three different mature trees growing in Monteverde, Costa Rica (10.3059 N, 84.8144W, 1380 m above sea level), on May 9, 2009. The plant was identified by William Setzer. [2] The fresh leaves were chopped and hydrodistilled for 4 hours using a Likens- Nickerson hydrodistillation apparatus [7] with continuous extraction with CHCl 3 (50 mL). The chloroform extract was then evaporated to yield yellow essential oils [Table 1].

Gas chromatographic-mass spectral analysis

A gas chromatographic-mass spectral analysis was performed on the essential oils of C. lusitanica using an Agilent 6890 GC with Agilent 5973 mass selective detector (EIMS, electron energy = 70 eV, scan range = 45-400 amu, and scan rate = 3.99 scans/s), and a fused silica capillary column (HP 5 ms, 30 m x 0.25 mm) coated with 5% phenyl-polymethylsiloxane (0.25 µm 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 minutes 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 CHCl 3 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 reported in the literature, [8] and stored on the MS library [NIST database (G1036A revision D.01.00)/ChemStation data system (G1701CA, version C.00.01.080)]. The percentages of each component are reported as raw percentages based on total ion current without standardization. The chemical compositions of the C. lusitanica leaf oils are summarized in [Table 1].

Antimicrobial screening

The essential oil was screened for antimicrobial activity against Gram-positive bacteria, Bacillus cereus (ATCC No. 14579) and Staphylococcus aureus (ATCC No. 29213); Gram-negative bacteria, Pseudomonas aeruginosa (ATCC No. 27853) and Escherichia coli (ATCC No. 10798). Minimum inhibitory concentrations (MICs) were determined using the microbroth dilution technique. [9] Dilutions of the crude extracts were prepared in cation-adjusted Mueller Hinton broth (CAMHB) beginning with 50 µL of 1% w/w solutions of crude extracts in DMSO plus 50 µL CAMHB. The extract solutions were serially diluted (1:1) in CAMHB in 96-well plates. Organisms at a concentration of approximately 1.5 x 10 8 colony-forming units (CFU)/ mL were added to each well. Plates were incubated at 37°C for 24 hours; the final minimum inhibitory concentration (MIC) was determined as the lowest concentration without turbidity. Geneticin was used as a positive antibiotic control; DMSO was used as a negative control. Antifungal activity was determined as described above using Candida albicans (ATCC No. 90028) in yeast-mold (YM) broth with concentration of approximately 7.5 x 10 7 CFU/mL. Antifungal activity against Aspergillus niger (ATCC No. 16888) was determined as above using YM broth inoculated with A. niger hyphal culture diluted to a McFarland turbidity of 1.0. Amphotericin B was the positive control.

   Results And Discussion Top

The hydrodistillation of the fresh leaves of C. lusitanica produced pale yellow essential oils in yields ranging from 0.28% to 0.58%. The main components in all three essential oils were monoterpene hydrocarbons, which included α-pinene (82.3%, 39.9% and 60.0%) and limonene (4.2%, 17.6% and 8.4%) as the major components. This is in contrast to essential oil compositions growing in Portugal (dominated by the diterpene abietadiene, 11%-24%) [6] or Cameroon (composed principally of umbellulone, 17%-18%). [3] It is however qualitatively similar to the oil reported by Carmo and Frazγo [5] (18.0%, α-pinene; 13.2%, β-pinene 1 sabinene), but these workers had only identified 79% of the composition. C. lusitanica leaf oil (combined samples) was screened for antimicrobial activity against Bacillus cereus (MIC = 78 µg/mL), Staphylococcus aureus (MIC = 625 µg/mL), Escherichia coli (MIC = 1250 µg/ mL), Pseudomonas aeruginosa (MIC = 1250 µg/mL), Candida albicans (MIC = 625 µg/mL) and Aspergillus niger (MIC = 78 µg/mL). Thus, C. lusitanica leaf oil showed appreciable activity against the Gram-positive bacterium B. cereus and the mold A. niger only.

   References Top

1.Mabberly DJ. The Plant Book, 2 nd Ed. Cambridge, UK: Cambridge University Press; 1997. p. 200-1.  Back to cited text no. 1      
2.2. Zuchowski W. A Guide to Tropical Plants of Costa Rica. Miami, Florida: Zona Tropical; 2005. p. 223-4.  Back to cited text no. 2      
3.3. Kuiate JR, Bessiére JM, Vilarem G, Amvam Zollo PH. Chemical composition and antidermatophytic properties of the essential oils from leaves, flowers and fruits of Cupressus lusitanica Mill. from Cameroon. Flavour Fragr J 2006;21:693-7.  Back to cited text no. 3      
4.4. Morton JF. Atlas of Medicinal Plants of Middle America, Vol. I. Springfield, Illinois: Charles C. Thomas Publisher; 1981. p. 21.  Back to cited text no. 4      
5.5. Carmo MM, Frazão S. The essential oil of Cupressus lusitanicus Mill. Flavour Fragr J 1989;4:185-6.  Back to cited text no. 5      
6.6. Adams RP, Zanoni TA, Lara A, Barrero AF, Cool LG. Comparisons among Cupressus arizonica Greene, C. benthamii Endl, C. lindleyi Klotz. ex Endl. and C. lusitanica Mill. using essential oils and DNA fingerprinting. J Essent Oil Res 1997;9:303-9.  Back to cited text no. 6      
7.7. Likens ST, Nickerson GB. Determination of certain hop oil constituents in brewing products. Proc Am Soc Brew Chem 1964;5-13.  Back to cited text no. 7      
8.8. Adams RP. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4 th Ed. Carol Stream, Illinois: Allured Publishing; 2007.  Back to cited text no. 8      
9.9. Sahm DH, Washington JA. Antibacterial susceptibility tests: Dilution methods. In: Balows A, Hausler WJ, Herrmann KL, Isenberg HD, Shamody HJ, editors. Manual of Clinical Microbiology, 5 th Ed. Washington DC: American Society for Microbiology; 1991  Back to cited text no. 9      


  [Table 1]

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