Seasonal Variation and Antimicrobial Activity of Myrcia myrtifolia Essential Oils

Martins D. de Cerqueira, Lourdes C. Souza-Neta, Maria das Graças V. M. Passos, Edeltrudes de O. Lima, Nídia F. Roque, Dirceu Martins, Maria L. S. Guedes and Frederico G. Cruz* Instituto de Química, Departamento do Medicamento and Instituto de Biologia, Faculdade de Farmácia, Universidade Federal da Bahia, Campus de Ondina, 40.170-290 Salvador-BA, Brazil Laboratório de Micologia, Centro de Ciências da Saúde, Universidade Federal da Paraíba, 58.059-900 João Pessoa-PI, Brazil


Introduction
Aromatic plants have been used since ancient times as antiseptics and anti-infectious agents, as aroma in perfumes and cosmetics as well as preservative and flavour ingredients in food and beverages.Their biological properties are directly related with their chemical compositions, which can be affected by environmental, geographical, seasonal and circadian variations.
The family Myrtaceae is constituted by 140 genera with about 3000 species, which are widely distributed in America and Australia. 1 The genus Myrcia comprises more than 300 species that grow in all Brazilian territory.Indigenous tribes and traditional Brazilian communities have used several species of this genus as astringent, against diabetes and diarrhea, as diuretic, to stanch hemorrhages, against the hypertension and ulcers of the mouth. 2 Some works were published regarding the chemistry and the biological properties of Myrcia species.From M. citriofolia, the occurrence of a C-methylated flavone, eucalyptin, was related, 3 from the methanolic extract of M. multiflora were isolated flavonol, flavanone and acetophenone glucosides along with myricitrin, mearnssitin, quercitrin, desmanthin-1 and guaijaverin, this extract were found to show antidiabetic properties and potent inhibitory activities on aldose reductase and α-glucosidase. 4,57][8] In a study with volatile oils of some Myrtaceae species the lack of activity against Escherichia coli and good activity against Staphylococcus aureus and S. epidermidis in the majority of species was related. 9The antifungal activity of essential oils from Myrtaceae species against dermatophytes was attributed to its terpenes constituents. 10he aim of this work was the study of seasonal variation of composition of the leaf volatile oils and the composition of volatile oils from flowers and fruits of M. myrtifolia that was harvested in the sand dunes of Salvador, Bahia, northeastern region of Brazil between 2002 and 2003, along with the study of the antimicrobial properties of the leaf oil collected in October 2002 against six bacteria, two yeasts and five filamentous fungi.Additionally, a preliminary toxicity evaluation of the leaf oil with brine shrimp (Artemia salina) bioassay was performed.

Plant material
Two specimens of M. myrtifolia DC, apart from each other by approximately 100 m, were selected to work.Leaves of the two specimens were collected every month from May 2002 to December 2003 always between 8:00 and 10:00 pm.Flowers and fruits were collected when they were present.The leaves, immature flowers and immature fruits were carefully separated, cleaned and frozen at -20 °C until the extraction.Voucher specimens were deposited in the herbarium Alexandre Leal Costa of the Instituto de Biologia, UFBA, under the number 52168.

Essential oil analysis
The volatile oils were obtained from leaves, fruits and flowers after 3 h hydro-distillation in a Clevenger modified apparatus.The identification of compounds was performed by comparison of their retention indices and mass spectra with those reported in the literature 11,12 and stored in the NIST libraries (Mass Spectral Library, 1998).The retention indices were calculated by co-injection with a standard saturated n-alkanes homologous series.GC analyses were performed using a gas chromatograph HP 5890 equipped with capillary DB-5 column (30 m × 0.25 mm, film thickness 0.25 μm) and a FID detector.The oven temperature was programmed from 60 °C to 240 °C at rate of 3 °C min -1 , and isothermal at 240 °C for 10 min, using H 2 as the carrier gas (1.0 mL min -1 ).Injector and detector temperatures were 220 °C and 300 °C, respectively.GC-MS analyses were performed using a gas chromatograph HP 6890 interfaced with a HP 5873 Mass Selective Detector (ionisation voltage 70 eV) equipped with capillary HP-5MS column (30 m × 0.25 mm, film thickness 0.25 μm), using He as the carrier gas (1.0 mL min -1 ).Oven and injector temperatures were as above.

Microbial strains
Thirteen microbial strains were used to access the antimicrobial properties of the test sample: four Gram Trichophyton rubrum ATCC 28189).Microorganisms were obtained from the culture collections of the Instituto Nacional de Controle de Qualidade em Saúde-INCQS/ Brazil.Bacteria and fungi were grown on Mueller Hinton Agar (MHA) and Sabouraud Dextrose Agar (SDA) (Difco Laboratoires).Inocula were prepared in sterile saline from 24 h, 48 h and 7-14 days cultures of bacteria, yeasts and filamentous fungi respectively, and standardized against 0.5 McFarland Standard to obtain suspensions containing approximately 10 8 cfu mL -1 of bacteria and 10 6 cfu mL -1 of fungi.

Essential oils dilution
The essential oils were tested pure and at dilutions ranging from 8% to 0.125% (v/v), corresponding, respectively, to concentrations of 69.50 mg mL -1 to 1.08 mg mL -1 .The dilutions were prepared in sterile distilled water with addition of Tween 80 (Merck) to the first dilution in a concentration of 10% (v/v), each dilution being mixed on Vortex. 14Due to the similarity of the oils J. Braz.Chem.Soc.composition, only the oil of the leaves collected in October 2002 had its antimicrobial properties and toxicity tested.

Preparation of microorganism culture
To evaluate antimicrobial activity, assays were performed using the agar diffusion method. 14,15For each microorganism, 1 mL of standardized inoculum was transferred to sterile Petri dishes and mixed with 20 mL of molten agar at 45 °C (MHA for bacteria and SDA for fungi).After solidification, 6 mm wells were made on agar (six per plate) to which 50 μL of each oil dilution was transferred.After 30 min at room temperature, plates with bacteria were incubated at 35-37 °C for 24 h, 28-30 °C for 48 h with yeasts and 10-14 days with filamentous fungi.Growth control of test strains and diluent control were simultaneously performed.Susceptibility tests using standard substances obtained from Cecon/SP were performed by applying the diffusion method with filter paper disks of 6 mm diameter Chloranphenicol (30 μg) for Gram-negative bacteria, Vancomycin (30 μg) for Staphylococcus species and Ketoconazol (50 μg) for fungi (NCCLS, 2000).The results were showed on Table 3.All the tests were performed in duplicate.The bioassay results were expressed in terms of mean of inhibition zone diameters: < 9 mm, inactive; 9-12 mm, partially active; 12-18 mm, active; > 18 mm, very active.

Minimum Inhibitory Concentration (MIC)
MIC assays were performed by Broth Microdilution Method as described in National Committee for Clinical Laboratory Standards with 100 mL aliquots of diluted leaf oil and standards antimicrobial agents as controls. 16,17acterial suspensions were standardized with 0.5 McFarland standard and diluted to give final concentrations of 5 × 10 5 cfu mL -1 for bacteria and 0.5-2.5 × 10 3 cfu mL -1 for yeasts.The minimum bactericidal concentrations were visualized by inoculating 20 μL of sterile 0.01% sodium resazurin solution to each plate well and after 3 h incubation.The minimal inhibitory concentration, defined as the lowest concentration that inhibited bacterial growth, was determined by the emergence of a blue colour at the wells indicating absence of growth. 18The minimum fungicidal concentrations were determined by absence of fungal growth when compared with positive and negative controls.The MIC assays were performed with microorganisms for which the oil was considered active in the agar diffusion assay (inhibition zone diameters larger than 12 mm) except to filamentous fungi.

Toxicity against Artemia salina
The brine shrimp lethality assay was performed by the method of McLaughlin. 19Brine shrimp eggs (Artemia salina) were hatched in saline solution of NaCl (38 g L -1 ).
The essential oil was tested at concentrations of 10, 100 and 1000 mg L -1 .Survival was measured after 24 h incubation at 10 °C.The collected data were computerized and LC 50 values determined by Probit analysis.

Results and Discussion
7][8] To verify if this result it would have been caused by a seasonal condition, the essential oils were collected and analyzed monthly during the years 2002 and 2003.To minimize the effects of the variation of other such factors as soil composition, humidity, light intensity, and others, the leaves, flower and fruits were always collected from two specimens very close each other.Sixteen samples of leaves, six from flowers and two from fruits were analyzed between May 2002 and December 2003.In total, twenty-eight compounds were identified in the oils (Tables 1 and 2).The monoterpene fraction was present in higher amount in all samples, 94.1% in average, being hydrocarbons the main compounds.The most abundant component in all samples analyzed was α-pinene.In the leaves, its concentrations varied from 62.0% (May 2002) to 87.3% (October 2002).In the flowers α-pinene concentrations varied from 61.5% (November 2002) to 85.1% (December, 2002).In the immature fruits, its concentrations were 88.1% (May 2002) and 90.9% (June 2003).Although in low concentrations, limonene and β-pinene were the only compounds besides α-pinene, present in all leaves, flowers and fruits samples.trans-Pinocarveol which was present in all leaves samples, it was not present in flowers while in the fruits it was present in May 2002 and not in June 2003.β-Cariophyllene was present in larger concentrations in the flowers.In the fruits and leaves either it was not present or it was present in concentrations smaller than 1.0%.
In a general way, the composition of the leaf oils did not show meaningful seasonal variation, however, the leaf oils yield was found to vary during the year reaching its maximum between August and December.
The evaluation of antimicrobial activity by the agar diffusion method showed that the leaf volatile oil presented activity to some specific microorganisms.Table 3 summarizes the results listing only the microorganisms that had their growth inhibited.The essential oil evaluated in this screening did not present inhibition against Gramnegative bacteria (E.coli, S. choleraesuis, P. aeruginosa, P. mirabilis) and against the fungi C. herbarum and P. notatum.The pure oil was active against S. aureus, methicilin resistant S. aureus (MRSA), C. albicans, C. neoformans and A. fumigatus and was very active against M. canis and T. rubrum.At 8%, the oil was partially active against S. aureus and C. albicans, active against MRSA, C. neoformans and A. fumigatus, and very active against M. canis and T. rubrum.At 1% it just showed partial activity against M. canis.
The minimum inhibitory concentrations of leaf essential oil were determined against four microbial species (Table 4).These results indicated that the oil presented strong activity against S. aureus, methicilin resistant S. aureus and C. albicans with MIC of 0.25%, 0.25% and 0.125%, respectively and weaker activity against C. neoformans.The results obtained by both agar diffusion and broth microdilution methods were different.Many factors vary between assays which include differences in microbial growth, exposure of microorganisms to plant oils, the solubility of oils or oil components, the use and quantity of emulsifier and others elements. 14,20,21In the agar diffusion method, the hydrophobic nature of the majority of essential oil components hamper the uniform diffusion of these substances through the agar medium that may account for differences on the results obtained.
The antimicrobial properties of the oil are supposed to be associated to the high hydrocarbon monoterpene content, especially to its majority component, α-pinene.The antimicrobial activity of this compound was related to its capacity of destruction of cellular integrity, inhibition  of respiration and ion transportation process and to increase membrane permeability of yeast cells. 22,23ermatophytoses (onychomycosis, tinea or ringworm) are infections that occur in the hair, skin, nails and are caused by dermatophyte fungi, mainly T. rubrum and M. canis. 24Some species of Candida have also been related as onychomicosis-causing fungi.The high activity of the essential oil of M. myrtifolia against T. rubrum, M. Canis and C. albicans and the moderate toxicity obtained in a preliminary evaluation with Artemia salina bioassay, LC 50 0.12 0.20 0.18 0.16 0.25 mass of fresh leaves / g 41.0 295.0 407.0 210.0 164.0 79.0 89.0 126.0 155.0 242.0 218.0 312.0 201.0 280.0 280.0 396.0 t = amounts < 0.1%; nm = not measured; -= not detected.Seasonal Variation and Antimicrobial Activity of Myrcia myrtifolia Essential Oils J. Braz.Chem.Soc.

Table 1 .
Seasonal composition of essential oils from leaves of Myrcia myrtifolia collected from May 2002 to December 2003

Table 3 .
Antimicrobial activity of leaf essential oil from M. myrtifolia collected in October 2002, showed as mean of duplicate of zones of growth inhibition (mm)

Table 2 .
Composition of essential oils from flowers and fruits of Myrcia myrtifolia collected in different months and years nm = not measured; -= not detected; Fl = flowers; Fr = fruits.