EFFECT OF VEGETATION CYCLE ON CHEMICAL CONTENT AND ANTIBACTERIAL ACTIVITY OF SATUREJA MONTANA L

Effect of vegetation cycle on phytochemical composition of the essential oil obtained from the aerial parts of wild-growing winter savory (Satureja montana L.) from Montenegro was analysed by GC-MS and its antibacterial activity tested at different oil concentrations. A total of 36 and 34 constituents were identified in the hydrodistilled oil obtained from herb before flowering and during flowering stage, with major components: thymol (37,36% and 27,68%), carvacrol (15,47% and 4,40%), γ-terpinene (11,75% and 8.66%) and p-cymene (7,86% and 31, 37%), respectively. The gained results revealed that essential oil of S. montana has rather significant antibacterial activity against chosen bacteria Staphylococcus aureus, Escherichia coli and Bacillus subtilis. Also, it was found that vegetation cycle affects the chemical composition and antibacterial activity of savory essential oil. Essential oil gained prior to herb flowering period showed stronger antibacterial activity in comparison with the oil gained during herb flowering.


INTRODUCTION
Plants produce an enormous array of secondary metabolites, and it is commonly accepted that a significant part of its chemical diversity serves to protect plants against microbial pathogens (Dixon, 2001).Researchers have been interested in biologically active compounds isolated from plant species for the elimination of pathogenic microorganisms because of the resistance that they have developed to antibiotics (Hunter & Reeves, 2002).
Winter savory (Satureja montana) is a perennial herb in the family Lamiaceae, native to warm temperate regions of southern Europe.It is a semi-evergreen, semi-woody subshrub growing to over 20-30 cm tall.The whole herb is mildly antiseptic, aromatic, carminative, digestive, mildly expectorant and stomachic.It is well known as aromatic and medicinal herb while its essential oil is used in the food industry as a flavoring agent, in the making liqueurs and in perfumery.The positive effects of savory on human health are attributed to its active constituents such as essential oil, triterpenes, flavonoids and rosmarinic acid (Bezic et al., 2005).Essential oils are highly concentrated volatile, aromatic essences of aromatic plants and they have been known since ancient times to possess biological activity.They have a complex composition, containing from a few dozen to several hundred constituents, especially hydrocarbons and oxygenated compounds which are highly odoriferous (Rios & Recio, 2005).The use of essential oils has a potential interest in the pharmacological field and in food preservation technology for its antimicrobial effects (Ciani et al., 2000).
The objectives of this study were: (i) the extraction of the essential oil from herba of wild-growing winter savory from Montenegro during different vegetation stage, (ii) the identification and quantification of the components of the obtained essential oils and (iii) the determination and comparison of their antibacterial activity against chosen Gram-positive and Gram-negative bacteria by disk diffusion method.

Plant Material
Many factors can influence the amount of essential oil in aromatic herbs, such as climate and environmental conditions and age of plants.To avoid this influence in the present work fresh leaves of S. montana were collected manually from the same collection site in the Podgorica region (central part of Montenegro), in summer 2008, prior the flowering (in May) and during flowering (in August).After flowering (in October), the plant material was not collected, because, in this stage of development, the plant has very small leaves.Voucher specimens were deposited in Herbarium, Department of Biology, Faculty of Natural Sciences and Mathematics, University of Montenegro, voucher numbers: S809/08 and S905/08.
The initial water inherent in the herb leaves found to be 10.7 % (w/w) using a Dean and Stark apparatus with n-heptane as the reflux solvent.Herb material was milled in a coffee mill and, after sieving, sample with a mean particle diameter size of 0.8 mm was obtained.A prepared batch was kept in an airtight resalable polypropylene bag and stored at 8ºC for maximum 2 days before use, in order to avoid losses of volatile compounds.
Herb material (40g) was submitted to hydrodistillation in a Clevenger-type apparatus for 2 hours according to Yugoslav Pharmacopoeia IV.The obtained oil was dried over anhydrous sodium sulphate, measured, poured in hermetically sealed dark-glass containers and stored in a freezer at -4°C until analyzed by GC-MS.

Gas Chromatography-Mass Spectrometry
The GC-MS analyses were carried out using a Shimadzu 2010+ gas chromatograph-mass spectrometer equipped with a ZB-5ms (30m x 0,25mm x 0,25μm) capillary column.The column temperature was programmed from 35°C (5 min) to 300°C at 5°C/min.The injection port temperature was 260°C while the interface temperature was 305°C.The samples of oil were injected by splitting and the split ratio was adjusted to 1:100.Helium was used as the carrier gas at a flow rate of 1.2 ml/min and 61.8 kPa inlet pressure.The MS conditions were: the ionisation voltage 70 eV, scanning interval 1.5 s, detector voltage 1.0 kV and m/z range 40 -500.The components were identified by comparing their mass spectral data with those in the WILEY229 and the NIST107 mass spectra libraries, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature and whenever possible, by co-injection with authentic standards (Fluka, Great Britain).

Disc diffusion method
The antimicrobial activity of the oil was evaluated by the disc diffusion method, using Mueller-Hinton agar for bacteria, by determination of inhibition zones.Two Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria were used.Test microorganisms were obtained from Clinical Department of National Institute in Montenegro.Ceftalexine, erythromycin and nalidixine acid were used as positive standards in order to control the sensitivity of the microorganisms.A 100 µl suspension of any tested bacteria containing about 10 8 cells/ml spreaded on Mueller Hinton Agar (MHA) mediums using sterile swabs.Sterile blank disk 6.0 mm in diameter were impregnated with 1, 3 and 5 µl essential oil/disk and finally placed on the agar surface.Plates were incubated at 37 o C for 24 hours and then the inhibition zones were measured in diameters.Disks soaked in the solvent (5% DMSO) were used as a negative control.
All tests of inhibitory activity were carried out in duplicate and the developing inhibition zones were compared with those of reference disks.
The essential oil of winter savory before and during flowering stage consisted chiefly of oxygenated monoterpenes (60.93% and 45.36%), monoterpene  hydrocarbons (31.00% and 50.8%), while sesquiterpene hydrocarbons and oxygenated sesquiterpenes were only present in a relatively small percentage (8.07%and 3.76%), respectively.
Characteristic chromatographs of the hydrodistillated S. montana essential oil at isolated from the herb at different vegetation stage, prior to flowering and during flowering, are shown in Figure 1.
The content of p-cymene increased four times with the maturation of the plant (August) as well as terpinen-4-ol and borneol.The content of thymol methyl ether and carvacrol methyl ether increase with maturation of the plant, while content of eugenol-methyl-ether identified only in young plant.The content of thymol and carvacrol in savory is variable and depends on the origin and vegetative stage of the plant (Slavkovska et al., 2001).With reference to previous studies, thymol and carvacrol, in particular, were found to be principal constituents of the oils isolated from several Croatian Satureja species (Skocibusic & Bezic, 2004).
Similar results of essential oil analysis for this plant was reported by (Mastelic & Jerkovic, 2003) for winter-savory from Croatia (near Sinj) with 30,88% of thymol and 3,81 of carvacrol in August while (Bezic et al., 2005) in contrary, reported that essential oil from Croatia (from Biokovo) has high content of carvacrol (45,7%) and small content of thymol (3,9%).Similar results of essential oil analysis for this plant, but from different locality (also in Croatia) were reported by (Milos et al., 2001) who identified carvacrol as main components of the oil.It is well known that environmental conditions, stage of development and especially chemotype of plant can affect composition and content of essential oil.

Antibacterial activity
The antibacterial activity assay for S. montana essential oil, as summarized in Table 2, showed that different microorganisms tested had different susceptivity to the same essential oil.Savory oil, isolated from the herb before flowering period, was very potent against all chosen microorganisms at all concentration while essential oil isolated from the herb during flowering period showed activity at all concentration only against medically important pathogen Staphylococcus aureus.This oil had significant effect on another medically important pathogen E. coli at lower oil concentration (1µL and 3µL) while the activity was rather high at concentration of 5µL (29.5 mm).Additionally this oil showed no effect on B. subtilis at lowest oil concentration (1µL) while the activity was rather high at concentration of 3 µL and 5µL (27 mm and 39 mm).The essential oil activities against tested microorganisms were increased with increased amount of investigated essential oil.
High antimicrobial activity of savory essential oil is due to the presence of phenolic components, such as thymol and its isomer carvacrol as well as its precursors, γ-terpinene and p-cymene which activities has been confirmed (Slavkovska et al., 2001).Thymol disintegrates the outer membrane and increase the permeability of the cytoplasmic membrane to ATP of E. coli (Helander et al., 1998).Carvacrol is an isomer of thymol and has been shown to cause damage in B. subtilis cells (Ultee & Smid, 2001).The presence of the hydroxyl group seems to be more important for the antimicrobial activities of these compounds than the ability to expand and consequently to destabilize the bacterial membrane.Thus, thymol and p-cymene have almost same structure, although cymene lacks the hydroxyl group present in thymol that results in an increase of the antibacterial activity.
The antimicrobial activity of the savory essential oil could, in part, be associated with borneol which antimicrobial activity was previously reported (Tabanca et al., 2001;Knoblach et al., 1989).Also, a number of researchers had shown the components present in lower amount in savory essential oil such as α-terpineol, terpinen-4-ol and p-cymene could also contribute to the antimicrobial activity of the oil.Terpinen-4-ol was concluded to be responsible for bacteriostatic activity against several microorganisms, especially against bacteria that infect the urinary tract (Barel et al., 1991); p-cymene and α-terpineol had been also reported to be antibacterial (Cosentino et al., 1999).In fact, it was also possible that the components in lower percentage might be involved in some type of synergism with the other active compounds.
Although the antibacterial activity of essential oils from many herb species has been extensively surveyed (Rios & Recio, 2005) their antimicrobial mechanism has not been reported in great details.Since the active antimicrobial compounds of essential oils are terpenes and phenolics in nature, it seems reasonable that their mode of action might be similar to that of other phenolic compounds (Shunying et al., 2005).Individual essential oil contains complex mixtures of such compounds; however, little is known about the effect of interaction between individual constituents on antimicrobial activity.Interactions between constituents may lead to additive, synergistic or antagonistic effects (Delaquis et al., 2002).

CONCLUSIONS
The gained results revealed that essential oil of S. montana has very significant antibacterial activity against chosen bacteria.These results confirm the Table 2. Antimicrobial activity of the Satureja montana L. essential oil and some standard antibiotics potential use of S. montana essential oil in the pharmaceutical industry, and may be useful as an alternative antibacterial agent in the folk medicine.Also, it was found that herb vegetation cycle notably affects the yield, chemical composition and antibacterial activity of S. montana essential oil.Essential oil gained before flowering period showed stronger antibacterial activity, probably due to the higher content of phenolic monoterpenes, thymol and carvacrol.

Fig. 2 .
Fig. 2. Comparison of the main compounds identified in essential oil of Satureja montana from different vegetation stage not active; NK: nalidixine acid; CFK: ceftalexine; ERM: erythromycin.

Table 1 .
Relative percentage composition of Satureja montana L. essential oil depending on the stage of plant development