Nystatin

Abstract

The composition and the antimicrobial activity of the essential oil of Luma chequen from Peru were investigated. Essential oil was analyzed by GC and GC/MS. The oil was characterized by the presence of small amounts of sesquiterpenes (3.1%), and large amounts of monoterpenes (90.1%), of which α-pinene (57.1%), 1,8-cineole (12.1%) and linalool (5.5%) were the major compounds. The oil showed significant antimicrobial activity against Proteus vulgaris, Cryptococcus neoformans and Cladosporium cladosporioides, with MIC values ranging from 0.45 to 1.67 µL/mL,.

Key Word Index

Luma chequen, "arrayan," Myrtaceae, essential oil composition, α-pinene, 1,8-cineole, antimicrobial activity.

Introduction

Luma chequen (Molina) A. Gray (Myrtaceae) is a native tree from Peru, growing sparsely on fresh and moistly soils from 2500 to 3200 m high in Charcana, Cotahuasi, Huaynacotas, Pampamarca, Quechualla, Sayla, Tauria and Toro. Leaves and twigs of L. chequen are widely used in Peru mainly for the treatment of gastrointestinal disorders, post-parturition infections and toothaches. For the treatment of post-parturition infections a bath is prepared with the twigs; to treat gastrointestinal disorders the leaves are crushed and squeezed; for toothaches the leaves are chewed (1).

In continuation with our work on the characterization of aromatic and medicinal plants widely used in the traditional medicines (2-7), the authors now report the chemical composition and antimicrobial activity of the essential oil from aerial parts of Luma chequen from Peru. To our knowledge, this is the first time the composition and antimicrobial activity of this species has been reported.

Experimental

Commercially available L. chequen oil (Biolatina) obtained from leaves of the plant was investigated.

Oil analysis: Analysis of the oil was carried out by GC-FID and GC/MS. Analytical GC was carried out in a Hewlett-Packard 6890 (Agilent Technologies, Palo Alto, CA, USA) gas chromatograph with an HP GC ChemStation Rev. A.05.04 data handling system, equipped with a single injector and two flame ionization detection (FID) systems. A graphpak divider (Agilent Technologies, part no. 5021-7148) was used for simultaneous sampling to two Supelco fused silica capillary columns with two different stationary phases (SPB-I and SupelcoWax 10, 30 m x 0.2 mm, 0.20 µm). Analytical conditions for GC were as follow: oven temperature program: 70°-220°C (3°C/min), 220°C (15 min); injector temperature: 250°C; carrier gas: helium, adjusted to a linear velocity of 30 m/s; splitting ratio 1:40; detectors temperature: 250°C.

GC/MS was performed with a computerized system coupled to a Hewlett-Packard mass selective detector 5973 (Agilent Technologies) operated by HP Enhanced ChemStation software, version A.03.00, using GC parameters as above; interface temperature: 250°C; MS source temperature: 230°C; MS quadrupole temperature: 150°C; ionization energy: 70 eV; ionization current: 60 µA; scan range: 35-350 u; scans/sec: 4.51. The constituents of the oil were identified on the basis of their GC retention indices (RI), calculated by linear interpolation relative to retention times of a series of n-alkanes, and by matching their 70 eV mass spectra with those from a homemade library and /or from literature data (8,9). The relative amounts of individual components were calculated based on GC peak areas without using correction factors.

Antimicrobial activity: Antibacterial and antifungal activities of the oil and the three major constituents (α-pinene, 1,8-cineole and linalool) were evaluated against three Gram-positive and two Gram-negative bacteria, two yeasts and three filamentous fungi by the disk diffusion method (10) as previously reported (6). Chloramphenicol (30 µg), Ampicillin (10 µg) and Nystatin (100 units) disks were used to control the sensitivity of the tested organisms. The microorganisms used were Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Streptococcus faecalis CECT 795, Escherlchia coli ATCC 25922, Proteus vulgaris CECT 484, Candida albicans CECT 1394, Cryptococcus neoformans CECT 1078, Cladosporium cladosponoides CECT 2111, Aspergillus niger CECT 2574 and Aspergillus fumigatus CECT 2071. All the experiments were carried out in triplicate and average and standard deviation (SD) were calculated for the inhibition zone diameters.

Minimal inhibitory concentration (MIC) was evaluated by agar dilution technique modified and adapted to a 96-well microtiter plates. Mueller-Hinton agar and Sabouraud Dextrose agar were used for bacteria and fungi, respectively. Different concentrations of the oils were obtained in dimethyl sulfoxide (DMSO) to give serial two-fold dilutions that were added to each well, resulting in concentrations ranging from 0.33 to 10 µL/mL. Final concentration of DMSO never exceeded 2%. Wells were inoculated with test microorganisms suspensions at a final inoculum of 104 cells/mL for bacteria and yeast and 10^sup 4^ a 10^sup 5^ spores/mL for filamentous fungi.

For each strain tested, the growth conditions and the sterility of the medium were checked in two control columns. The inoquity of the DMSO were also checked at the highest tested concentration. The microtiter plates were then incubated for 24 h at 37°C for the bacteria and for 48 h at 27°C for yeast and filamentous fungi. All experiments were performed in triplicate and repeated if the results differed.

Results and Discussion

The qualitative and quantitative composition of the oil, analyzed by GC and GC/MS, is presented in Table I, where compounds are listed in order of their elution on a polydimethylsiloxane column.

The oil was characterized by high percentage of monoterpenes (90.1%) with oc-pinene (57.1%), 1,8-cineole (12.1%) and linalool (5.5%) as the major compounds. Sesquiterpenic compounds accounted for only 3.1%.

The disk diffusion test, used in the preliminary screening of the antimicrobial activity, showed that the oil of L. chequen was active against all the tested microorganisms, except Staphylococcus epidermidis, Streptococcus faecalis and Aspergillus niger. Nevertheless, the oil proved to be significantly more active against Proteus vulgaris, Staphylococcus aureus, Cryptococcus neoformans and Cladosporium cladosporioides (Table II). Antimicrobial activity of the three major constituents of the oil were also assayed against the same strains. The activity of the oil can be associated with the significant contribution of the α-pinene, 1,8-cineole and linalool.

The antimicrobial activity of the oil was also determined using the dilution technique, by measuring the minimal inhibitory concentration (MIC) against Escherichia coli, Proteus vulgaris, Staphylococcus aureus, Candida albicans, Cryptococcus neoformans, Cladosporium cladosporioides anaAspergillus fumigatus (Table II). The oil showed significant activity against Proteus vulgaris, Cryptococcus neoformans and Cladosporium cladosporioides, with MIC values ranging from 0.45 to 1.67 µL/mL. These results may partially justify the use of this plant in the traditional medicine of Peru.

References

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2. AP. Martins, L.R. Salgueiro, R. Vila, F. Tomi, S. Canigueral, J. Casanova, A. Proença da Cunha and T. Adzet, Essential oil composition of four Piper species from S. Tomé e Príncipe. Phytochemistry, 49, 2019-2023 (1998).

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6. A.P. Martins, L.R. Salgueiro, MJ. Gonçalves, A. Proença da Cunha, R.Vila and S. Cañigueral, Essential oil composition and antimicrobial activity of Santirla trimera bark. Planta Medica, 69, 77-79 (2003).

7. L.R. Salgueiro, C. Cavaleiro, MJ. Gonçalves and A. Proença da Cunha, Antimicrobial activity and chemical composition of the essential oil of Lippia graveolens from Guatemala. Planta Med., 69, 80-83 (2003).

8. R.P. Adams, Identification of Essential Oil Components by Gas Chromatography / Mass Spectroscopy. Allured Publishing Corp., Carol Stream, IL (1995).

9. D. Joulain and W.A. König, The atlas of spectral data of sesquiterpene hydrocarbons. E. B.- Verlag Hamburg, Hamburg (1998).

10. R. Cruickshank, I.P. Duguid, B.P. Marmion and R.H.A. Swain, Microbiologia Médica. Vol II, 4th ed. Fundação Calouste Gulbenkian, Lisbon (1975).

Maria José Gonçalves, Carlos Cavaleiro, António Proença da Cunha and Lígia R. Salgueiro*

Lab. de Fannacognosia, Foc. de Farmácia/CEF, Universidade de Coimbra. R. do Norte, 3000 Coimbra, Portugal

* Address for correspondence

Received: April 2004

Revised: August 2004

Accepted: November 2004

Copyright Allured Publishing Corporation Jan/Feb 2006
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