Chemical composition and antimicrobial activity of essential oils of Ocimum canum Sims . and Ocimum selloi Benth .

This work describes the chemical composition and antimicrobial activity of the volatile oils of Ocimum canum and Ocimum selloi, both occurring in Jequié/BA, northeastern Brazil. The plants were collected in the winter/2005 and summer/2006, the oils extracted by steam distillation and further analyzed by GC-MS. A total of 30 and 31 compounds was identified from the oils of O. selloi and O. canum, respectively. It was observed that the oil content of O. canum showed variation during the seasons, while the oils of O. selloi did not. Methylchavicol and linalool were the main chemical components found in the aerial parts and leaves of O. canum. This finding permitted to characterize this specimen as a new chemotype of O. canum. Regarding the aerial parts of O. selloi, eugenol, 1,8-cineole, transcaryophyllene and linalool were identified as their major components. All extracted oils from the aerial parts showed biological activity against gram-positive cocci – Staphylococcus aureus ATCC 25923 – but only the O. canum one showed activity against gram-negative bacilli – Escherichia coli ATCC 25922.


INTRODUCTION
The world trade for essential oils has been growing approximately 11% a year (Bizzo et al. 2009).In Brazil, the exports of essential oils have shown a significant increase in recent years, reaching U$93,000,000 in 2008 (ALICE-Web System 2009).Among the species producing essential oils, those of the genus Ocimum have a small, but important contribution on the total exports.
The genus Ocimum L. (Lamiaceae) comprises about 160 species distributed in tropical and subtropi-cal Africa, Asia and South America (Gupta 1994), many of which are used in folk medicine, as spices and also to control insects (Grayer et al. 1996).Moreover, there is an economic exploitation by large-scale extraction of essential oils.Ocimum selloi is popularly known as "elixir paregórico" 1 in the states of Rio de Janeiro and Espírito Santo, and as "anis" or "alfavaquinha" in Minas Gerais.These popular terms are related to its pharmacological properties and also to its chemical similarity to other species of Ocimum.
The volatile oils of O. selloi present antidiarrheal, antispasmodic and anti-inflammatory activities confirmed in preclinical testing (Vanderlinde et al. 1994).
The essential oils of Ocimum are composed by compounds such as estragole, eugenol, methyleugenol, citral, linalool, geraniol and thymol.These compounds are required as raw materials for the pharmaceutical, comestics and food industries, and as insecticides (Craveiro and Queiroz 1993, Gupta 1994, Bizzo et al. 2009).
As a continuation of our studies on the composition of aromatic and medicinal plants cultivated in different parts of Brazil (Barbosa et al. 2005, Fonseca et al. 2006, Silva et al. 2007a), we describe in this paper the results of our investigation on the chemical composition and antimicrobial activities of the volatile oils produced by O. canum Sims and O. selloi Benth.

PLANT MATERIAL
The species were collected in August 2005 and January 2006 at 7:00 a.m. in the Garden of Medicinal Plants of the Universidade Estadual do Sudoeste da Bahia (UESB).Specimens were prepared, identified and deposited in the Herbarium of the Universidade Estadual de Santa Cruz (Ilhéus-BA, Brazil) under 4385 and 4387 numbers for O. canum and O. selloi, respectively.

EXTRACTION AND CHEMICAL ANALYSES OF THE VOLATILE OILS
For each species, 100 g of fresh plant material from the aerial parts (leaves and fine stems) and from leaves, separately, were chopped and then subjected to a hydrodistillation in a Clevenger (1.5 h).The resulting oils were dried over anhydrous sodium sulphate (Merck), weighed and the reported yields calculated with respect to the fresh material weight.All distillations were repeated three times and the obtained oils were stored under nitrogen atmosphere and maintained at approximately 0 • C, until their analysis.
The compounds were identified using a GC-MS unit (model GCMS-QP5050A, from Shimadzu, Japan) equipped with a DB-5 fused silica column (30 m × 0.22 mm i.d., film thickness 0.25 µm) and interfaced with an ion trap detector.Oven and injector temperatures were as described above; transfer line temperature, 240 • C; ion trap temp., 220 • C; carrier gas, He at a flow rate of 1.8 mL/min; split ratio 1:10; column pressure of 100 kPa; ionization energy, 70 eV; scan range, 29-450 u; scan time, 1s.The components were characterized by the comparison of their retention indexes (RI) relative to a standard alkane series (C 9 -C 24 ), and also by the comparison of their mass spectrum with reference data from either the equipment database (Wiley 7 library) or literature (Adams 1995).

ANTIBACTERIAL ACTIVITY
The antimicrobial activity tests on the oils were performed by the method of diffusion in agar previously described (Bauer et al. 1966, Casteels et al. 1993, Fontana et al. 2004).The oils were tested against grampositive and gram-negative species, Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922), respectively.A suspension of the tested microorganism (0.5 of MacFarland Scale) was spread on Petri plates with Mueller Hinton agar (Difco Laboratories).A 2 mm hole diameter was opened on the surface of inoculated plates to which were added 10µL of an essential oil.The plates were incubated at 37 • C for 24 hours.After this period of time, the diameters of the inhibition zones were measured using a caliper rule and expressed in millimeters.

RESULTS AND DISCUSSION
The essential oils of aerial parts (leaves and stems) of
The yield of volatile oils extracted from the aerial parts and leaves of O. canum in the summer is doubled compared to the winter, in agreement with the results obtained for O. basilicum L. (Silva et al. 2005).These results suggest that the seasonality may be one of the factors that should be considered when dealing with a possible economic exploitation.Other possible factors related to this variation can be rainfall, climate and leaf development (Gobbo-Neto and Lopes 2007).For the content of the essential oil of O. selloi, a significant variation on the yielding values for leaves and aerial parts in the two samples was not observed.Similar results were previously described by Moraes et al. (2002).Thus, these findings can suggest that, for this species, the seasonality is not a factor that could influence in the amount of essential oils produced.
The chemical composition of the essential oils of the aerial parts and leaves obtained in the winter of 2005 and in the summer of 2006 for O. canum is formed by methylchavicol and linalool, respectively, as its major constituents (Table I, Figures 2, 3, 6 and 10 canum is known to present different chemotypes with the volatile oils constituted mainly of linalool and limonene (Ngassoum et al. 2004, Ravid et al. 1997), methylcinnamate and caryophyllene (Murillo andViña 2003, Martins et al. 1999), fenchone (Lawrence et al. 1980), eugenol (Ekundayo et al. 1989), 1,8-cineole (Bassole et al. 2005), citral (Choudhary et al. 1989), terpinen-4-ol (Sanda et al. 1998) and camphor (Chagonda et al. 2000).Therefore, according to the present study, it can be suggested that the plant under investigation may constitute a new chemotype methylchavicol and linalool.However, proposing the existence of a new chemotype has to be taken carefully since the chemical composition of the volatile oils can vary with the sea-2 Mean of three replications ± SD. son, plant origin, plant age and soil composition, among other factors (Castro et al. 2004, Martins et al. 2006, 2007, Barbosa et al. 2007, 2008).In the present investigation, the influence of the harvest season on the composition of volatile oils is also observed.Specifically in the case of linalool content, a significant increase in aerial parts was observed in the winter compared to the summer.
Eugenol, 1,8-cineole, trans-caryophyllene and linalool were found to be major chemical constituents of essential oils of aerial parts from O. selloi (Table II, Figures 1, 4, 5, 6, 7, 8 and 9) in the two seasons.Therefore, it suggests that the access of O. selloi belongs to the eugenol chemotype (Farago et al. 2004, Paula et al. 2004).There are other chemotypes reported for this plant, such as estragole and methyleugenol (Martins et al. 1997, De Paula et al. 2003, Franca et al. 2008), trans-anethole (Moraes et al. 2002) and elemicine (David et al. 2006).It is noteworthy that, despite the literature describes the existence of an eugenol chemotype, as far as we know there are no GC nor GC-MS analyses reported for this variety.The results reported in the present investigation show small differences in the eugenol, 1,8-cineole, trans-caryophyllene and linalool contents present within the volatile oils from the aerial parts extracted during the winter 2005 and the summer 2006.Chromatograms of essential oils of Ocimum selloi and O. canum analyzed by GC/MS and mass spectra of major compounds identified in these oils, with the Retention time (Rt) of these compounds in the respective oils are presented in the Figures 1-10.
The results presented above should be analyzed carefully because, as previously discussed, individuals of the same species may contain many different volatile compounds.These variations are related to environmental factors such as temperature, soil type, moisture, climate, height and factors intrinsic to the plant, as its pathological condition and age (Barbosa et al. 2007, Martins et al. 2006, Silva et al. 2007b).The content of metabolites may also vary depending on the extraction method used (Charles and Simon 1990, Silva et al. 2004).
The essential oil of the aerial parts of O. selloi showed an 8 mm diameter of inhibition zone against However, it did not show activity against E. coli (ATCC 25922).These results are in agreement with that previously observed (Farago et al. 2004), which reported a small antimicrobial activity of the essential oils of eugenol variety for both microorganims.The observed activity against S. aureus can be attributed to the large amount of eugenol present in the oil, as reported by Nakamura et al. (1999).

791 TABLE II Chemical constituents of the essential oil of Ocimum selloi Benth.
*All values reported as an average of three replicates ± SD; tr -trace compound (less than 0.10%); RI Cal.-retention indices calculated; RI Lit.= Adams 1995.