Eugenol (99%, Reagent plus), β-caryophyllene (98.5%), α-humulene (96%) meta-chloroperoxybenzoic acid (77%), and dichloromethane (99.5%) were obtained from Sigma-Aldrich. Dimethyl sulfoxide (DMSO, 99.8%), sodium hydroxide (98%), diethyl ether (99%) were obtained from Merck. All the chemicals were used without further purification.

The commercial essential oil was obtained from a local pharmacy and was from the Beolab Spa & Wellness Company. The dried flower buds of Eugenia caryophyllata with declared origin from Madagascar were obtained from a local supplier. The plant material was pulverized and was subjected to hydrodistillation for 3 h in Clevenger-type apparatus (for denser than water EO). The essential oil was separated and dried over sodium sulfate for a minimum of 10 minutes. The overall yield of the EO was 14.2% (v/w) established on dry basis. Portion of the hydro distilled oil (2 g) was dissolved in ether (20 mL) and subjected to extraction with 5% (w/w) sodium hydroxide (3x15 mL). The combined NaOH layers were back-extracted with ether (3x10 ml). The combined aqueous layer was transferred to Erlenmayer flask and acidified with concentrated HCl to a pH value of 2. The acidified mixture is transferred to separate funnel and extracted with ether (3 x 15 mL). The combined organic layers were washed with water (3 x 15 mL) and brine (1 x15 mL). The ether layer was dried over sodium sulfate and decanted to a pre-weighed round bottom flask. Removal of solvents via rotary evaporation gave light yellow liquid (1.42 g), which was eugenol with analytical purity, GC-MS 99% pure. The purity was confirmed by comparison with authentic sample (eugenol, 99+% Reagent plus, Sigma-Aldrich). All the samples (isolated eugenol, the commercial EO and hydro distilled EO) were subjected to density, refractive index measurements, and IR and GC-MS analyses prior to the antimicrobial testing. Throughout the study they were stored at 4^oC in dark glass air-tight bottles. Appropriate volumes of the samples were diluted in 100% DMSO, at a maximum concentration of 100 μLmL^-1 (v/v) for each experiment. The FT-IR spectra were recorded on Varian 3100-IR Excalibur series spectrophotometer as liquid films between sodium chloride plates. The refractive index measurements were carried out on a digital Kruss A. Optronic (Germany) refractometer equipped with accurate internal thermostating unit.

The chemical composition of clove bud essential oils was determined using GC and GC-MS. Samples (100 mg) were placed in 10 mL volumetric flask and were filled to the mark with dichloromethane. Gas chromatography (GC) was performed on Agilent 6890 GC system, equipped with flame-ionization detector (FID) and Agilent 5973 GC auto sampler. HP-5 column (30 m x 0.25 mm, 0.25 µm film thickness) was employed and nitrogen was used as a carrier gas at constant flow of 1.0 mLmin^-1. Sample amount injected was 1µL, with applied split ratio of 1:50 at 250°C. The GC conditions were the following: the oven temperature started at 40°C and held for 5 min, then heated at 5°Cmin^-1 to 140 °C, held for 5 min, then heated to 260°C at 10 °Cmin^-1 and held at the final temp for 10 min. The temperature of the detector was at 260°C. The GC-MS analyses were carried out on a Varian 450 GC fitted with a VF-5MS fused silica capillary column (5% diphenyl- 95% dimethyl polysiloxane, 30 m x 0.25mm i.d., film thickness 0.25 μm) interfaced to a Varian 300 single quadrupole mass spectrometer, operated by Varian MS Workstation software. The GC temperature program was identical as in the GC-FID analysis. Helium (purity 99.999%) was used as a carrier gas with a constant flow rate of 1 mLmin^-1. One microliter of the samples was injected using Varian CP 8410 autosampler, with a 50:1 split ratio and injector temperature of 250 °C. The temperatures of the transfer line and ion source were 240°C and 250°C, respectively. All mass spectra were acquired in electron impact (EI) mode at 70 eV in a full scan mode, with a range of 40-350 amu at a rate of 2 scans sec^-1. Data acquisition and processing were carried out with Varian MS Workstation software, with capability of mass spectral matching. Identification of the components was based on comparison of the retention times with those of authentic samples and on computer matching against commercial (NIST 05) and home-made library mass spectra built up from pure substances and components of known oils. The relative amounts of individual components were calculated based on the GC-FID peak areas without response factor corrections. To correctly assign the GC peaks of the isomeric sesquiterpene epoxides (of β-caryophyllene and α-humulene) partial epoxidations with meta-chloroperoxybenzoic acid (mCPBA) were carried using literature procedures [23Bombarda I, Gaydou EM, Smadja J, Faure R. Synthesis of oxygenated compounds derived from caryophyllene. Bull Soc Chim Fr 1995; 132: 836-42., 24Damodaran NP, De S. Studies in sesquiterpenes-XXXVIII. Structure of humulene epoxide-I and humulene epoxide-II. Tetrahedron 1968; 24: 4123-32.
[http://dx.doi.org/10.1016/0040-4020(68)88175-X] ]. β-Caryophyllene was reacted with 0.75 equivalent of mCPBA, at 0 °C for 30 min, in dichloromethane following the procedure of [23Bombarda I, Gaydou EM, Smadja J, Faure R. Synthesis of oxygenated compounds derived from caryophyllene. Bull Soc Chim Fr 1995; 132: 836-42.]. After subsequent work-up, the obtained crude product was analyzed by GC-MS (ca. 3:1 β-caryophyllene epoxide: starting material). α-Humulene was reacted with 1 equivalent of mCPBA in chloroform according to [24Damodaran NP, De S. Studies in sesquiterpenes-XXXVIII. Structure of humulene epoxide-I and humulene epoxide-II. Tetrahedron 1968; 24: 4123-32.
[http://dx.doi.org/10.1016/0040-4020(68)88175-X] ], and the crude product was analyzed by GC-MS (94% humulene epoxide II, 3% humulene epoxide I and ca. 1% starting material).

The antimicrobial activity of the extracts was assessed against six bacterial strains, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Sarcina lutea ATCC 9341, Bacillus pumilus NCTC 8241; two yeasts: Saccharomyces cerevisiae ATCC 9763 and Candida albicans ATCC 10231; and three molds: Penicillium sp., Aspergillus niger ATCC 16404 and Aspergillus sojae.

The 96-well micro-titre assay using resazurin as the indicator of cell growth [25Sarker SD, Nahar L, Kumarasamy Y. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods 2007; 42(4): 321-4.
[http://dx.doi.org/10.1016/j.ymeth.2007.01.006] [PMID: 17560319] , 26Genest S, Kerr C, Shah A, et al. Comparative bioactivity studies on two Mimosa species. BLACPMA 2008; 7: 38-43.] was employed for the determination of the minimum inhibitory concentration (MIC) of the active extracts.

The test organisms were sub-cultured onto fresh plates of Mueller- Hinton agar (Oxoid, UK) for 24 h at 37°C for bacteria and Saboraud dextrose agar (Oxoid, UK) for 5 - 7 days at 25°C for fungi. Colonies from these plates were suspended in Mueller-Hinton broth and Saboraud broth (Oxoid, UK) to a turbidity matching 0.5 McFarland standard (10⁸ colony forming units (cfu) ml^-1).

Into each well of the 96-well micro-titre plate, 50 μL of the medium, 50 μL of the extract, 5 μL of the resazurin and 5 μL of the suspension were dropped. Concentrations of extract used were 100-0.049 μLmL^-1. Dilutions were made using DMSO as solvent. Each plate was incubating for 24 hours at 37°C for bacteria and 5 - 7 days at 25°C for fungi, as described by [27Pretorius JC, Magama S, Zietsman PC. Growth inhibition of plants pathogenic bacteria and fungi by extracts from selected South African plant species. S Afr J Bot 2003; 69(2): 188-92.
[http://dx.doi.org/10.1016/S0254-6299(15)30344-6] ]. A positive control (containing inoculum but no essential oil) and negative control (containing essential oil but no inoculum) were included on each microplate. A colour change from blue to pink was indicative of microbial growth. Solutions of benzylpenicillin, ampicillin and streptomycin (for bacteria) and nystatin (for yeasts and molds) were used for control wells.

In our study, we have used commercial essential oil, hydro distilled one and pure isolated eugenol, 1. The clove buds (declared to be originating from Madagascar) were hydro distilled in Clevenger type of apparatus and the obtained EO was split into two parts. The first one was directly used in the antimicrobial studies, while the second part was used for the isolation of eugenol. The chemical composition of the isolated clove bud essential oil and the commercial clove bud essential oil was determined using GC-FID and GC-MS. Also, the purity of the isolated eugenol (99%) was determined by the GC-MS and refractive index measurement. From the obtained results it can be seen that in both the essential oils, the main component is eugenol 1 (Table 1). It is interesting to point out the absence of eugenyl acetate in the commercial essential oil and the presence of α-humulene. The chemical composition of the isolated essential oil is in agreement with the literature data (references within Table 2) [21Pinto E, Vale-Silva L, Cavaleiro C, Salgueiro L. Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus and dermatophyte species. J Med Microbiol 2009; 58(Pt 11): 1454-62.
[http://dx.doi.org/10.1099/jmm.0.010538-0] [PMID: 19589904] , 22European Pharmacopoeia (VII). 7th ed. Strasbourg: Council of Europe 2011., 28Gaydou EM, Randriamiharisoa R. Multidimensional analysis of gas chromatographic data, application to the differentiation of clove bud and clove stem essential oils from Madagascar. Perfum Flavor 1987; 12: 45-51.-33Chaieb K, Zmantar T, Ksouri R, et al. Antioxidant properties of the essential oil of Eugenia caryophyllata and its antifungal activity against a large number of clinical Candida species. Mycoses 2007; 50(5): 403-6.
[http://dx.doi.org/10.1111/j.1439-0507.2007.01391.x] [PMID: 17714361] ].

Table 1
Key data from the GC-MS analyses of essential oils.

Table 2
Literature survey of origin and chemical composition of clove bud oil.

After the GC-FID/GC-MS analysis we have noted differences in the essential oils. It is interesting to note that both samples, based on the GC-MS analyses, contained, 2.3 and 2.7 wt.% of caryophyllene oxide, 5, which is an oxidation product of β-caryophyllene [34Turek C, Stintzing FC. Impact of different storage conditions on the quality of selected essential oils. Food Res Int 2012a; 46: 341-53.
[http://dx.doi.org/10.1016/j.foodres.2011.12.028] , 35Turek C, Stintzing FC. Stability of essential oils: A review. Compr Rev Food Sci Food Saf 2012b; 12: 40-53.
[http://dx.doi.org/10.1111/1541-4337.12006] ]. Additionally, the commercial essential oil contained significant amounts of β-caryophyllene, 3, (10.41%), and some of α-humulene, 4, (~2%), while the hydro distilled EO contained low amounts of β-caryophyllene, 3, (1.21%). With pure eugenol as a control we had a convenient control probe to test the essential oils with almost the same content of eugenol (84.8 and 86.3 wt.%), but different content of eugenyl acetate and β-caryophyllene. The chemical composition of the isolated essential oil is in agreement with the literature data (references within Table 2).

During our preparation we have thoroughly dried the hydro-distilled clove essential oil over sodium sulfate. We have postulated that the way commercial oils are manufactured they are not additionally dried. Over time and storage, the traces of water can hydrolyze the ester, eugenyl acetate, 2, and if it is originally present in small quantities it may not be detectable by GC analyses. In our case, we wanted to confirm the absence of 2, and to eliminate the possible decomposition and/or hydrolysis at the GC injector. For that purpose we have recorded the FT-IR spectra of all samples (commercial essential oil, the hydro distilled/dried essential oil and pure eugenol). The typical ester absorption band (1743 cm^-1) was absent in the IR spectra of the commercial essential oil, but was present in hydrodistilled and dried essential oil.

A few antimicrobial studies involving clove essential oils with chromatographically determined chemical composition have been done [33Chaieb K, Zmantar T, Ksouri R, et al. Antioxidant properties of the essential oil of Eugenia caryophyllata and its antifungal activity against a large number of clinical Candida species. Mycoses 2007; 50(5): 403-6.
[http://dx.doi.org/10.1111/j.1439-0507.2007.01391.x] [PMID: 17714361] , 21Pinto E, Vale-Silva L, Cavaleiro C, Salgueiro L. Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus and dermatophyte species. J Med Microbiol 2009; 58(Pt 11): 1454-62.
[http://dx.doi.org/10.1099/jmm.0.010538-0] [PMID: 19589904] , 19Chaieb K, Hajlaoui H, Zmantar T, et al. The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): a short review. Phytother Res 2007; 21(6): 501-6.
[http://dx.doi.org/10.1002/ptr.2124] [PMID: 17380552] , 36Leela NK, Sapna VP. Clove. In: Parthasarathy VA, Chempakam B, Zachariah TJ, Eds. Chemistry of Spices. 2008; pp. 146-64.
[http://dx.doi.org/10.1079/9781845934057.0146] ]. Chemical composition can vary, depending on the origin, storage of the clove buds, isolation and processing and storage [34Turek C, Stintzing FC. Impact of different storage conditions on the quality of selected essential oils. Food Res Int 2012a; 46: 341-53.
[http://dx.doi.org/10.1016/j.foodres.2011.12.028] -36Leela NK, Sapna VP. Clove. In: Parthasarathy VA, Chempakam B, Zachariah TJ, Eds. Chemistry of Spices. 2008; pp. 146-64.
[http://dx.doi.org/10.1079/9781845934057.0146] ], which in turn may affect the results of the in vitro antimicrobial studies.

The inhibitory activity of clove is due to the presence of several constituents, mainly eugenol, eugenyl acetate, beta-caryophyllene, 2-heptanone [33Chaieb K, Zmantar T, Ksouri R, et al. Antioxidant properties of the essential oil of Eugenia caryophyllata and its antifungal activity against a large number of clinical Candida species. Mycoses 2007; 50(5): 403-6.
[http://dx.doi.org/10.1111/j.1439-0507.2007.01391.x] [PMID: 17714361] ], and to a lesser extent because of their lower abundance- α-humulene, methyl salicylate, isoeugenol, methyl eugenol [37Giorgio P, Mario C, Paola M, Stefania Z, Antonella D, Bruno T. Chemical composition and antimicrobial activities of essential oil of Stachys glutinosa L. from Sardinia. Nat Prod Commun 2006; 1: 1133-6.].

Several studies have demonstrated potent antifungal [38Arina B, Iqbal A. In vitro fungitoxicity of the essential oil of Syzygium aromaticum. World J Microbiol Biotechnol 2012; 18(4): 317-9.-41Park MJ, Gwak KS, Yang I, et al. Antifungal activities of the essential oils in Syzygium aromaticum (L.) Merr. Et Perry and Leptospermum petersonii Bailey and their constituents against various dermatophytes. J Microbiol 2007; 45(5): 460-5.
[PMID: 17978807] ], antiviral [19Chaieb K, Hajlaoui H, Zmantar T, et al. The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): a short review. Phytother Res 2007; 21(6): 501-6.
[http://dx.doi.org/10.1002/ptr.2124] [PMID: 17380552] ] and antibacterial effects of clove [17López P, Sánchez C, Batlle R, Nerín C. Solid- and vapor-phase antimicrobial activities of six essential oils: susceptibility of selected foodborne bacterial and fungal strains. J Agric Food Chem 2005; 53(17): 6939-46.
[http://dx.doi.org/10.1021/jf050709v] [PMID: 16104824] , 42Cai L, Wu CD. Compounds from Syzygium aromaticum possessing growth inhibitory activity against oral pathogens. J Nat Prod 1996; 59(10): 987-90.
[http://dx.doi.org/10.1021/np960451q] [PMID: 8904847] -46Fu Y, Zu Y, Chen L, et al. Antimicrobial activity of clove and rosemary essential oils alone and in combination. Phytother Res 2007; 21(10): 989-94.
[http://dx.doi.org/10.1002/ptr.2179] [PMID: 17562569] ]. It has been proposed that the most active compounds contain the phenol functional group. These phenolic compounds sensitize the phospholipid bilayer of the microbial cytoplasmic membrane causing increased permeability, unavailability of vital intracellular constituents [47Juven BJ, Kanner J, Schved F, Weisslowicz H. Factors that interact with the antibacterial action of thyme essential oil and its active constituents. J Appl Bacteriol 1994; 76(6): 626-31.
[http://dx.doi.org/10.1111/j.1365-2672.1994.tb01661.x] [PMID: 8027009] ] and/or impairment of bacterial enzymes systems [48Farag RS. Antioxidant activity of some spice essential oils on linobic acid oxidation in aqueous media. JAOGS 1989; 66: 792-9.].

The 96-well micro-titre assay with resazurin [25Sarker SD, Nahar L, Kumarasamy Y. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods 2007; 42(4): 321-4.
[http://dx.doi.org/10.1016/j.ymeth.2007.01.006] [PMID: 17560319] ] was employed to evaluate the antimicrobial activity of clove essential oil and eugenol. Blue-coloured suspensions are consistent with no growth of microorganisms, and red-coloured suspension are consistent with higher levels of microorganisms proliferation. This method is a nonradiometric, rapid, high-throughput assay that allows for the detection of bacterial activity with a high degree of confidence [49Yajko DM, Madej JJ, Lancaster MV, et al. Colorimetric method for determining MICs of antimicrobial agents for Mycobacterium tuberculosis. J Clin Microbiol 1995; 33(9): 2324-7.
[PMID: 7494021] -51Kumar M, Khan IA, Verma V, Kalyan N, Qazi GN. Rapid, inexpensive MIC determination of Mycobacterium tuberculosis isolates by using microplate nitrate reductase assay. Diagn Microbiol Infect Dis 2005; 53(2): 121-4.
[http://dx.doi.org/10.1016/j.diagmicrobio.2005.05.011] [PMID: 16168616] ]. The assay is based on the oxidation–reduction dye resazurin as a general indicator of cellular growth [52OBrien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 2000; 267(17): 5421-6.
[http://dx.doi.org/10.1046/j.1432-1327.2000.01606.x] [PMID: 10951200] ]. In the presence of metabolic activity, the dye is reduced and undergoes a colour change with fluorescence.

The results for antimicrobial properties of extracts are shown in Table 3. Eugenol, as a main component of extracts, demonstrated very strong activity against all tested microorganisms, with lowest MIC of 0.049 μLmL^-1 for Bacillus subtilis ATCC 6633. With regard to antifungal activity, the clove extract was active against all the test fungal species (Table 3).The lowest MIC was for eugenol on Penicillium sp. (0.049 μLmL^-1). The antimicrobial properties of clove essential oil were tested and showed inhibitory activity to Listeria monocytogenes, Campylobacter jejuni, Salmonella enteritidis, Bacillus cereus, Escherichia coli and Staphylococcus aureus [53Cressy HK, Jerrett AR, Osborne CM, Bremer PJ. A novel method for the reduction of numbers of Listeria monocytogenes cells by freezing in combination with an essential oil in bacteriological media. J Food Prot 2003; 66(3): 390-5.
[http://dx.doi.org/10.4315/0362-028X-66.3.390] [PMID: 12636290] , 54Velluti A, Sanchis V, Ramos AJ, Egido J, Marín S. Inhibitory effect of cinnamon, clove, lemongrass, oregano and palmarose essential oils on growth and fumonisin B1 production by Fusarium proliferatum in maize grain. Int J Food Microbiol 2003; 89(2-3): 145-54.
[http://dx.doi.org/10.1016/S0168-1605(03)00116-8] [PMID: 14623380] ].

Table 3
Minimal Inhibitory Concentration (MIC) of eugenol, E. caryophyllata essential oils (EO), antibiotics and antimycotic against test microorganisms.

Essential oils of clove possess high antimicrobial properties [16Kalemba D, Kunicka A. Antibacterial and antifungal properties of essential oils. Curr Med Chem 2003; 10(10): 813-29.
[http://dx.doi.org/10.2174/0929867033457719] [PMID: 12678685] ]. Clove oil was effective against E. coli, L monocytogenes, S. enteric [55Friedman M, Henika PR, Mandrell RE. Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, and Salmonella enterica. J Food Prot 2002; 65(10): 1545-60.
[http://dx.doi.org/10.4315/0362-028X-65.10.1545] [PMID: 12380738] ]. The antibacterial activity of clove against two gram-negative bacteria, such as Pseudomonas fluorescens and Serratia liquefaciens, and four gram-positive bacteria, such as Brochothrix thermosphacta, Carnobacterium piscicola, Lactobacillus curvatus, and Lactobacillus sp., involved in meat spoilage, was found effective.

Generally, this study showed that clove oil was effective against both Gram-positive and Gram-negative bacteria, and yeasts and fungi, which is similar to other reports describing the use of essential oils. The present findings support, at least to some extent, the traditional uses of this plant, particularly its use for the treatment of bacterial and fungal infections and inflammation.

It was also found that the eugenol had the strongest effect against bacterial cultures, with the exception of E. coli, where the freshly isolated essential oil had the strongest bactericidal effect. Also, the freshly isolated essential oil had the strongest effect against the C. albicans, Important to note is that a similar study on antifungal activity of eugenol and clove essential oil on Candida and Aspergillus species was done by Pinto and co-workers [21Pinto E, Vale-Silva L, Cavaleiro C, Salgueiro L. Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus and dermatophyte species. J Med Microbiol 2009; 58(Pt 11): 1454-62.
[http://dx.doi.org/10.1099/jmm.0.010538-0] [PMID: 19589904] ]. Another peculiarity is that in their study they have used a commercial essential oil from Portugal, and based on their GC analysis they have not detected eugenyl acetate either. Taking into consideration that in our study the only major difference between the two EOs (freshly prepared and commercial) is the presence of eugenyl acetate, one is tempted to attribute the higher overall antimicrobial activity of the freshly hydrodistilled/dried EO to its presence. This assumption finds support in recent study by Chiaradia and co-workers [56Chiaradia V, Paroul N, Cansian RL, et al. Synthesis of eugenol esters by lipase-catalyzed reaction in solvent-free system. Appl Biochem Biotechnol 2012; 168(4): 742-51.
[http://dx.doi.org/10.1007/s12010-012-9814-5] [PMID: 22864649] ] who synthesized pure eugenyl acetate by lipase-catalyzed reaction in solvent-free system and tested its antimicrobial activity in parallel with eugenol employing diffusion disk technique. The eugenyl acetate led to an increase in the antimicrobial activity (16 bacteria tested) when compared to pure eugenol (i.e the acetylation of eugenol improved its antimicrobial properties). Further studies centered on the preparation and antimicrobial studies with surrogate EOs containing varying ratios of the three main components (eugenol, beta-caryophyllene and eugenyl acetate) are needed to establish the “true” contribution of each component. Also the minimal inhibitory concentration (MIC) for pure substances should be expressed in appropriate units, as to allow direct and unambiguous comparison of the antimicrobial effectiveness. The easiest way for comparison is to convert concentrations from μLmL^-1 and μgmL^-1 to μmol mL^-1. The antimicrobial activity of eugenol can now be directly and unambiguously compared to the activity of the commercial antibiotics and antimycotics. From Table 3. it can be clearly seen, based on micromolar amounts, that eugenol is especially potent antifungal agent and is superior to man-made antimycotics. If one takes into consideration the high EO yield, availability, affordability and high eugenol content, the flower buds of Eugenia caryophyllata are more than obvious source of natural antimicrobials.