The Constituents of Essential Oil: Antimicrobial and Antioxidant Activity of Micromeria congesta Boiss. & Hausskn. ex Boiss. from East Anatolia

Plant material and isolation procedures

The aerial parts of M. congesta were collected during the flowering stage in June to July 2009, in the Germüş village (elevation 550 m) of Şanlıurfa. Dr. Ali Celik further identified all the collected plants. The voucher specimens are deposited at the herbarium of the Pamukkale University, Faculty of Science & Art, Biology Department (herbarium no. AÇE 2547). The samples were air-dried and stored in polyethylene bags until use. The essential oil of dried parts of M. congesta was obtained via hydrodistillation by using a Clevenger type apparatus for 8 h. The oil samples were dried over a hydrous sodium sulfate and stored at +4°C until a further analysis (yield 3.2%).

Gas chromatography–mass spectrometry analysis

The essential oil was analyzed by gas chromatography–mass spectroscopy (Agilent 5973) in the Ankara Test and Analysis Laboratory (TUBITAK). A gas chromatography–mass spectrometry (GC-MS) apparatus equipped with a fused silica HP-5 capillary column (30 m×0.25 mm×0.25 μm film thickness) was used. The oven temperature was maintained at 40°C for 4 min and then programmed at 240°C with the rate of 6°C·min⁻¹. The carrier gas was helium, at a flow rate of 1 mL·min⁻¹, and the injection volume was 1 μL. In mass spectrometry electron-impact, ionization was performed at electron energy of 70 eV. The results were also confirmed by comparison of the compound's elution order with their relative retention indices on nonpolar phases as reported by Adams. ¹⁴ Relative amount of individual components was performed on the basis of their GC peak areas.

Microbial strains

M. congesta essential oil was investigated for its in vitro antimicrobial activity on a panel that included Gram-positive and Gram-negative bacteria. The tested microorganisms were Bacillus subtilis NRRL B-3711, Staphylococcus aureus ATCC 25923, S. aureus ATCC 33862, Escherichia coli 0157H7, Enterococcus faecalis ATCC 19433, Bacillus cereus NRRL B-3711, Micrococcus luteus NRLL B-4375, Proteus vulgaris RSKK 96026, Yersinia enterecolitica RSKK 1501, and Candida albicans ATCC 1023.

Evaluation of antibacterial activity

The disc diffusion method ¹⁵ was employed to determine the antimicrobial activity of the essential oil. Bacterial strains were cultivated on the Mueller Hinton Broth (Oxoid, Unipath Limited). Briefly, a suspension of the tested microorganism (0.1 mL of 10⁸ cells/mL) was spread on the solid media plates. Sterilized discs of 6 mm (Schleicher and Schuell, No. 2668) were soaked with 0.1 and 0.25 μL of the pure liquid oil placed on the inoculated plates and, after staying at 4°C for 2 h, were incubated at 37°C for 24 h. The diameters of the inhibition zones were measured in millimeters. Tetracycline and streptomycin were used as positive controls: 1 mg of tetracycline or streptomycin was dissolved in 1 mL of sterilized and distilled water, and then the sterilized disc was soaked with 25 μL of this solution. All tests were carried out in duplicate.

Determination of total phenol content

Total phenol content (TPC) was determined by a modified method of Skerget et al. ¹⁶ For this purpose, a 200-mL sample was taken and mixed with 1000 mL of 10-fold diluted Folin-Ciocalteu reagent, 800 mL of sodium bicarbonate solution (7.5% w/w) was added, incubated at room temperature for 2 h, and then the absorbance was read at 750 nm in the spectrophotometer. About 1 mM gallic acid was used in the standard preparation, and the results were expressed in mmol gallic acid equivalent (GAE)/L.

Determination of total antioxidant activity

The total antioxidant activity (TAA) of the samples was measured by the method described by Erel. ¹⁷ The method is based on the decolorization of 2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), a radical cation that stays stable for a long time in the acetate buffer solution. While it is diluted with a more concentrated acetate buffer solution at high pH values, the color is spontaneously and slowly bleached. Antioxidants present in the sample accelerate the bleaching rate to a degree proportional to their concentrations, which can be monitored in the spectrophotometer, and the bleaching rate is inversely related to TAA of the sample. The reaction rate is calibrated with Trolox, a widely used standard for TAA measurement assays, and the assay results are expressed in mmol Trolox equivalent (TE)/L.

Determination of total oxidant status

The total oxidant status (TOS) of the samples was determined by the assay ¹⁸ based on the oxidation of ferrous ion to ferric ion in the presence of various oxidant species in an acidic medium, and the measurement of the ferric ion by xylenol orange. In this method, ferrous–odianisidine complex is oxidized to ferric ion making a colored xylenol orange–ferric ion complex. The color intensity is related with the oxidant strength of the sample. The values are given as mmol H₂O₂ equivalent (E)/L.

Chemical composition of the essential oil

The yield of the essential oil of M. congesta on a dry weight basis was 3.2% (v/w). The oil was colorless, with a weak perfume odor. The percentage of the main components is given in Table 1. Sixty-six components of the essential oil identified by the GC-MS analysis represent 95.18% of the oil. As it can be seen from Table 1, the major components of the essential oil were piperitone oxide, linalool oxide, and veratrole (39.18%); pulegone, dihydro carvone, and naphthalene (24.25%); and trans-piperitone epoxide, cis-piperitone epoxide, and octadecene (4.93%). 18 of these 66 compounds were also found previously ¹² with some variations in the percentages. These differences could be resulted from the fact that the chemical composition of essential oils vary with different climatic, seasonal, and geographic conditions, harvest period, and distillation technique. ¹³

According to the reports ^{11,12,19 –24} on the chemical composition of the oil isolated from the plants belonging to the genus Micromeria, pulegone, p-menthone, α-pinene, isomenthone, limonene, linalol, β-pinene, p-cymene, α-terpinene, γ-terpinene, α-terpineol, camphene, β-bourbonene and borneol are the most common essential components. In our study, linalool oxide, veratrole, and piperitone oxide being in the form of monocyclic terpene compose the considerable part (39.18%) of the volatile oil. Monocyclic terpenes of pulegon and dihydrocarvone and a sesquiterpene of naphthalene exist in the oil in a rate of 24.25%. Monocyclic terpenes of limonene and silvestrene, and careen, which partially resemble to limonene in structure and found in the form of bicyclic terpene, are of importance with respect to flavor and odor of the volatile oil.

Antibacterial activity of the essential oil

The antibacterial activity of the M. congesta oil has been evaluated in vitro against 12 bacterial species (Table 2) that are known to cause infections in humans. As summarized in the Table, the oil exhibited the antibacterial activity against all of the bacteria. While low concentrations (30 μL) of the essential oil inhibited growth in many of the tested bacteria, a strong antimicrobial activity was observed at higher concentrations (50 μL) for all the bacteria tested. In the present study, the essential oil of M. congesta exhibited a high antimicrobial activity on all the bacteria tested, especially on B. subtilis NRRL B-3711, S. aureus ATCC 12598, and S. aureus ATCC 25923.

When comparing the antibacterial activity of the essential oil to that of reference antibiotics, the inhibitory potency of the essential oil could mostly be considered as significant. It is evident that the antibacterial activity depends on many factors, including the type, composition and concentration of essential oil, the type and concentration of the target microorganism, the composition of the substrate, the processing and the storage conditions ²⁵ so, it is important to consider all these factors to make a comparison properly. The antimicrobial activity of plants well recognized with their antiallergenic and anti-inflammatory properties, along with their antimutagenic action. ²⁶ The results of this study are important as they provide information about potential natural antimicrobial sources because there is an increasing demand to naturally occurring antimicrobials for food preservation due to consumer awareness of natural food products, and a growing concern of microbial resistance toward conventional preservatives.

TPC, TAA, and TOS of the essential oil

TPC, TAC, and TOS of the M. congesta essential oil were studied in this study. Since the phenolic compounds are very important constituents of plants and known as powerful chain-breaking antioxidants, TPC of the oil was investigated and expressed as mmol GAE/L. The amount of total phenolics was found as 3.35 mmol GAE/L (equal to 570 mg GAE/L). This value of TPC is higher than that of many plant-derived beverages ²⁷ (juices of apple, white grape, and orange, and white wines) and essential oils of some widely used spice plants such as rosemary and sage. ²⁸ On the other hand, TPC of M. congesta is lower than black tea and red grape juice, ²⁷ the essential oil of some widely used spice plants as thyme and clove. ²⁸ Phenolic substances have proven to possess an antioxidant activity, so, they are supposed to contribute to the antioxidant properties of the extract. For it is well known that the total phenolic content measured by the Folin–Ciocalteu procedure does not give a full picture of the quality or quantity of the phenolic constituents in the extracts, there is a need for activity-guided fractionation to isolate the compounds that are responsible for the antioxidant activity of the extract,.

The mean TAA value of the essential oil of M. congesta is 2.52 mmol TE/L representing a good antioxidant effectiveness and is comparable with those of artificial antioxidants of butylated hydroxyanisole (BHA) and butylhydroxytoluene, and Trolox, which were previously found ²⁹ by the ABTS scavenging method to have radical scavenging activity (IC₅₀) values of 10.94, 10.23, and 8.00 μM, respectively. In another study, ³⁰ the antioxidant activity of BHA was found as 5.84 mmol TE/L that means, the essential oil of M. congesta is comparable well to some well-known antioxidant compounds with respect to TAA. In the present study, the total antioxidant and oxidant parameters were studied instead of individual antioxidant compounds, which act in combination with each other affecting the total antioxidant capacity, producing synergistic or antagonistic effects because the antioxidant activity may be due to different mechanisms, such as prevention of chain initiation, decomposition of peroxides, prevention of continued hydrogen abstraction, free-radical scavenging, reducing capacity, and binding of transition metal ion catalysts. ³¹

TOS found in this study is 0.202 mmol H₂O₂ E/L which is also an important data for understanding the antioxidant–oxidant balance of the plant essential oil. Because the existence of both of the antioxidants and oxidants was reported to make it evident that there are mechanisms controlling the balance of oxidant and antioxidant species. ^32,33