Composition of the essential oils of Thymus and Origanum species from Algeria and their antioxidant and antimicrobial activities

The composition of the essential oils of Origanum and Thymus species restricted to Algeria and the North Africa region was determined. Antioxidant and antibacterial activities of the isolated essential oils were also determined. The oils of oregano plants were strongly characterized by p-cymene (16.8-24.9%), gamma-terpinene (16.8-24.9%), thymol (8.4-36.0%), and carvacrol (1.1-29.7%), a thymol chemotype for Origanum floribundum and a alpha-terpineol chemotype for Thymus numidicus being described for the first time. The strains of Listeria monocytogenes tested were relatively resistant to the action of essential oils of either Origanum or Thymus species. All essential oils possessed antioxidant activity, but this was dependent on the specific chemical composition and the method employed to determine such activity.     

The main citral-geraniol and carvacrol chemotypes of the essential oil of thymus pulegioides L. growing wild in vilnius district (Lithuania).

Thymus pulegioides L. with lemon and carvacrol odor form the major part of plants growing wild in all 10 investigated localities during 1995-1997. The main components of the citral-geraniol chemotype of lemon-scented essential oil are the following (%): geraniol (14.9-30. 8), geranial (trans-citral, 9.7-19.7), beta-caryophylene (6.0-11.4), nerol (4.1-11.8), and neral (cis-citral, 0.1-9.5). The essential oil of carvacrol chemotypes contain more compounds that are characteristic of the thyme genus (%): carvacrol (16.0-22.2), beta-bisabolene (11.1-20.2), beta-caryophyllene (11.1-19.1), gamma-terpinene (5.8-16.2), p-cimene (5.5-10.4), thymol (3.3-9.8), and carvacrol methyl ether (5.6-8.6). The correlation between the odor and composition of the essential oil will help the users of wild thyme to choose the necessary chemotype for their purposes.     

Antimicrobial and antioxidant activity of the essential oil and methanol extracts of Thymus pectinatus Fisch. et Mey. Var. pectinatus (Lamiaceae)

The essential oil, obtained by using a Clevenger distillation apparatus, and water-soluble (polar) and water-insoluble (nonpolar) subfractions of the methanol extract of Thymus pectinatus Fisch. et Mey. var. pectinatus were assayed for their antimicrobial and antioxidant properties. No (or slight) antimicrobial activity was observed when the subfractions were tested, whereas the essential oil showed strong antimicrobial activity against all microorganisms tested. Antioxidant activities of the polar subfraction and the essential oil were evaluated using 2,2-diphenyl-1-picrylhydrazyl, hydroxyl radical, superoxide radical scavenging, and lipid peroxidation assays. The essential oil, in particular, and the polar subfraction of the methanol extract showed antioxidant activity. The essential oil was analyzed by GC/MS, and 24 compounds, representing 99.6% of the essential oil, were identified: thymol, gamma-terpinene, p-cymene, carvacrol, and borneol were the main components. An antimicrobial activity test carried out with fractions of the essential oil showed that the activity was mainly observed in those fractions containing thymol, in particular, and carvacrol. The activity was, therefore, attributed to the presence of these compounds. Other constituents of the essential oil, such as borneol, gamma-terpinene, and p-cymene, could be also taken into account for their possible synergistic or antagonistic effects. On the other hand, thymol and carvacrol were individually found to possess weaker antioxidant activity than the crude oil itself, indicating that other constituents of the essential oil may contribute to the antioxidant activity observed. In conclusion, the results presented here show that T. pectinatus essential oil could be considered as a natural antimicrobial and antioxidant source.     

Antibacterial and antioxidant activities of essential oils isolated from Thymbra capitata L. (Cav.) andOriganum vulgare L

Antilisterial activities of Thymbra capitata and Origanum vulgare essential oils were tested against 41 strains of Listeria monocytogenes. The oil of T. capitata was mainly constituted by one component, carvacrol (79%), whereas for O. vulgare three components constituted 70% of the oil, namely, thymol (33%), gamma-terpinene (26%), and p-cymene (11%). T. capitata essential oil had a significantly higher antilisterial activity in comparison to O. vulgare oil and chloramphenicol. No significant differences in L. monocytogenes susceptibilities to the essential oils tested were registered. The minimum inhibitory concentration values of T. capitata essential oil and of carvacrol were quite similar, ranging between 0.05 and 0.2 microL/mL. Antioxidant activity was also tested, the essential oil of T. capitata showing significantly higher antioxidant activity than that of O. vulgare. Use of T. capitata and O. vulgare essential oils can constitute a powerful tool in the control of L. monocytogenes in food and other industries.     

Modulating effects of thyme and its major ingredients on oxidative DNA damage in human lymphocytes

The present study was carried out to investigate the modulating effects of thyme and its major components against the oxidative DNA damage induced by H(2)O(2). The human lymphocytes with thymol, carvacrol, and gamma-terpinene incubated with or without 0.1 mM H(2)O(2) for 30 min at 37 degrees C and the DNA damage were evaluated by singe cell gel electropheresis (comet assay). Concentrations above 0.1 mM thymol and gamma-terpinene and 0.05 mM carvacrol significantly induced DNA damage in human lymphocytes, but at the smaller concentrations no additional DNA strand breakage has been observed. At the all concentrations studied, gamma-terpinene did not show any protective effect against H(2)O(2) induced oxidative DNA damage, but the phenolic compounds thymol and carvacrol at concentrations below 0.2 and 0.1 mM, respectively, significantly reduced the oxidative DNA damage (p < 0.001). The n-hexane and ethyl acetate fractions prepared from the methanolic extracts of Thymus spicata also were found to inhibit DNA damage.     

Effect of thyme essential oils (Thymus hyemalis and Thymus zygis) and monensin on in vitro ruminal degradation and volatile fatty acid production

The effect of the essential oils of thyme on the in vitro ruminal degradability of a barley seed/alfalfa hay substrate was studied. Two essential oils were used, one from Thymus hyemalis (TH), rich in carvacrol, and the other from Thymus zygis (TZ), rich in thymol. Four experimental treatments of in vitro degradability, using the Daisy II(200/220) incubator, were conducted including a negative control (CO), a positive control at 7.5 microg/mL of monensin (MO), and two treatments with essential oils (TH or TZ) at 1.35 microL/mL. The material was incubated at 39.5 degrees C for various lengths of time. At each time, the disappearance of dry matter, crude protein, and neutral detergent fiber was measured. Volatile fatty acids (VFAs) were determined after 48 h of incubation. CO and MO provided (p < 0.01) higher values of potential degradability (a + b) of DM than the TH and TZ treatments (72.6 and 70.8 vs 53.2 and 48.2%, respectively). Also, crude protein degradability was lowest in the essential oil treatments. The CO treatment showed the highest potential degradability of NDF. The values of VFA production obtained (p < 0.001) with CO and MO treatments were higher than those obtained with TH and TZ treatments (21.0 and 19.1 vs 11.2 and 10.1 mM). The essential oils decreased the molar proportion of propionate, increasing the acetate/propionate ratio. In conclusion, the effects of essential oils at assayed doses would not be nutritionally beneficial to the ruminal energetic metabolism.     

Effect of maturation on the composition and biological activity of the essential oil of a commercially important Satureja species from Turkey: Satureja cuneifolia Ten. (Lamiaceae)

The essential oil obtained by hydrodistillation from aerial parts of Satureja cuneifolia Ten., collected in three different maturation stages such as preflowering, flowering, and postflowering, were analyzed simultaneously by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Thymol (42.5-45.2%), p-cymene (19.4-24.3%), and carvacrol (8.5-13.2%) were identified as the main constituent in all stages. At the same time, the essential oils and main components were evaluated for their antimicrobial activity using a microdilution assay resulting in the inhibition of a number of common human pathogenic bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and the yeasts Candida albicans and Candida tropicalis. The minimum inhibitory concentrations (MIC) varied between 62.5 and 250 microg/mL within a moderate antimicrobial activity range. Furthermore, the antioxidant capacity of the essential oils and major components thymol and carvacrol were examined in vitro. The essential oils obtained from S. cuneifolia in three different stages and its main components were interacted with 1,1-diphenyl-2-picrylhydrazyl (DPPH (*)) as a nitrogen-centered stable radical, resulting in IC 50 = 1.6-2.1 mg/mL. In addition, the effects on inhibition of lipid peroxidation of the essential oils were assayed using the beta-carotene bleaching method. All of the tested oils inhibited the linoleic acid peroxidation at almost the same level as butylated hydroxytoluene (BHT) (93.54-94.65%). BHT and ascorbic acid were used as positive controls in the antioxidant assays.     

Watering level effect on Thymus hyemalis Lange essential oil yield and composition

Thymus hyemalis Lange (chemotype thymol) was cultivated as an experimental crop under different watering level conditions in order to achieve 80, 60, 40, and 20% of the local potential evapotranspiration (Eto). Two harvesting periods were considered, winter and spring. As a consequence of the great variability among plants, essential oil yield percentages did not show statistically significant differences among seasons and different levels of water supply. Capillary GC-MS analysis of the essential oils permitted the detection of 84 volatile components. Among them, 54 are described for the first time as volatile constituents of the chromatographic profile of this Thymus species. Winter harvesting showed high concentrations in thymol percentage (25.92 +/- 4.39), the 40% Eto watering level being the best with respect to obtaining the optimum quality of this essential oil. However, in spring this thyme species needs a greater water supply (80% Eto) to achieve the same amount of thymol in the essential oil (29.20 +/- 2.83). From this, it was concluded that winter harvesting could be used for the extraction of the essential oil, with a low level of water supply, whereas spring harvesting could be employed for collection of leaves as a food condiment.     

Antioxidative properties of Thymus vulgaris leaves: comparison of different extracts and essential oil chemotypes

Thyme (Thymus vulgaris L., Lamiaceae) is a subshrub from the Lamiaceae family with plants that are rich in essential oils and antioxidative phenolic substances. Twelve accessions originating from southern France and the variety 'Deutscher Winter' were grown in an experimental field in eastern Austria. Leaf samples from these plants as well as from a commercial thyme rich in thymol were analyzed for their essential oil and the antioxidative potential in various extracts. The assays for antioxidative activity were the total phenolics according to the Folin-Ciocalteu method, DPPH decoloration, and Fe(3+) reduction (FRAP). Both extraction techniques used, in the water bath at 40 degrees C and in the ultrasonic bath at room temperature, proved to be efficient. The best results were obtained with 60% ethanol as extractant. In the comparison of the different accessions the less active and the most active of these extracts differed by factors of 2.1 and 2.6 in the total phenolics and FRAP assay, respectively, and by factors 1.5-2.0 in the DPPH assay. Rosmarinic acid accounted for 22-55% of the antioxidant activity in the ethanolic extracts. Essential oils with high proportions of the phenolic components thymol and/or carvacrol showed the highest antioxidant activity. Ethanolic extracts from the residues after distillation were considerably lower in antioxidant activity than the respective extracts from the dried leaves. Extracts with CH2Cl2 in the ultrasonic bath contained volatiles in proportions close to the essential oil but displayed very low antioxidant activity.     

Correlation between the chemical and genetic relationships among commercial thyme cultivars

The essential oil composition and genetic variability of six commercial cultivars of thyme (Thymus vulgaris L.), a Mediterranean medicinal and aromatic plant, were analyzed by GC-MS and randomly amplified polymorphic DNA (RAPD), respectively. All evaluated cultivars belong to the thymol chemotype, with differences in the concentrations of thymol, gamma-terpinene, p-cymene, and other minor components. The comparison of the oil components concentration by multivariate analysis allowed separation of the cultivars into two groups. All of the cultivars exhibited characteristic RAPD patterns that allowed their identification. On the basis of the RAPD patterns, the cultivars could be divided into two clusters, which coincides with results obtained by oil GS-MS analysis, with a correlation coefficient of -0.779.     

In vitro antimicrobial and antioxidant activities of the essential oils and various extracts of Thymus eigii M. Zohary et P.H. Davis

This study was designed to examine the in vitro antimicrobial and antioxidant activities of the essential oil and various extracts obtained from aerial parts of Thymus eigii. The essential oil was particularly found to possess stronger antimicrobial activity, whereas other nonpolar extracts and subfractions showed moderate activity and polar extracts remained almost inactive. GC-MS analysis of the oil resulted in the identification of 39 compounds, representing 93.7% of the oil; thymol (30.6%), carvacrol (26.1%), and p-cymene (13.0%) were the main components. The samples were also subjected to a screening for their possible antioxidant activity by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and beta-carotene-linoleic acid assays. In the former case, the polar subfraction of the methanol extract was found to be superior to all extracts tested, only 16.8 microg/mL of which provided 50% inhibition, whereas all extracts, particularly the polar ones, seem to inhibit the oxidation of linoleic acid in the latter case. These data were further supported by total phenolics analysis, indicating that the antioxidative potential of the extracts was closely related to their phenolic constituents.     

Lipolytic effects of citrus peel oils and their components

This study was conducted to determine the lipolytic effects of eight kinds of citrus peel oils and their components. All of the citrus peel oils revealed lipolytic effects on olive oil model solution ranging from 10.9 to 73.8%. Hakyul (Citrus natsudaidai Hayata) showed the highest lipolytic effect (73.8%), followed by yuza (Citrus junos Sieb. ex Tanaka, 68.1%) and lemon (Citrus limonium, 63.4%), and their effects were comparable with or stronger than that of 5 mM raspberry ketone (p < 0.05). Among 17 authentic compounds relating to citrus peel oils, octanal (78.6%) showed the highest lipolytic effect, followed by gamma-terpinene (76.3%), limonene (75%), terpinen-4-ol (70.7%), nerol (69.9%), p-cymene (67.7%), and geranyl acetate (67.2%), and their effects were stronger than that of 5 mM raspberry ketone (p < 0.05). Ethyl acetate, alpha-pinene, myrcene, citronellal, linallyl acetate, and citronellol exhibited poor lipolytic effect in the model solution. Lipolytic effect was found to be high when the oils included a higher content of gamma-terpinene and p-cymene. Limonene showed potential lipolytic effect, and its effect is likely to be enhanced by the presence of gamma-terpinene and p-cymene. It is considered that monoterpene hydrocarbons consisting of one or two double bonds would have stronger lipolytic effect than those having three double bonds.     

Essential oils of Satureja, Origanum, and Thymus species: chemical composition and antibacterial activities against foodborne pathogens

The chemical composition of the essential oils obtained from the species restricted to Greece and the eastern Mediterranean region, Satureja spinosa L. and Thymus longicaulis L.; species endemic to central and south Greece, Satureja parnassica ssp. parnassica Heldr. and Sart ex Boiss.; species endemic to the island of Crete, Origanum dictamnus L.; and species widely distributed in the Mediterranean region, Satureja thymbra L. and Origanum vulgare L. subsp. hirtum, were determined by gas chromatography (GC) and GC/mass spectrometry (MS) analysis. The in vitro antibacterial activities of the essential oils were evaluated against a panel of five foodborne bacteria (Escherichia coli 0157:H7 NCTC 12900, Salmonella enteritidis PT4, Staphylococcus aureus ATCC 6538, Listeria monocytogenes ScottA, and Bacillus cereus FSS 134). The analytical data indicated that various monoterpene hydrocarbons and phenolic monoterpenes constitute the major components of the oils, but their concentrations varied greatly among the oils examined. The antibacterial assay results showed that 5 muL doses of the essential oils extracted from the endemic Satureja species in Greece possess remarkable bactericidal properties, which are clearly superior as compared to those of Origanum and Thymus species essential oils. Therefore, they represent an inexpensive source of natural mixtures of antibacterial compounds that exhibit potentials for use in food systems to prevent the growth of foodborne bacteria and extend the shelf life of the processed food.     

Antibacterial activities of plant essential oils and their components against Escherichia coli O157:H7 and Salmonella enterica in apple juice

We evaluated 17 plant essential oils and nine oil compounds for antibacterial activity against the foodborne pathogens Escherichia coli O157:H7 and Salmonella enterica in apple juices in a bactericidal assay in terms of % of the sample that resulted in a 50% decrease in the number of bacteria (BA(50)). The 10 compounds most active against E. coli (60 min BA(50) range in clear juice, 0.018-0.093%) were carvacrol, oregano oil, geraniol, eugenol, cinnamon leaf oil, citral, clove bud oil, lemongrass oil, cinnamon bark oil, and lemon oil. The corresponding compounds against S. enterica (BA(50) range, 0.0044-0.011%) were Melissa oil, carvacrol, oregano oil, terpeineol, geraniol, lemon oil, citral, lemongrass oil, cinnamon leaf oil, and linalool. The activity (i) was greater for S. enterica than for E. coli, (ii) increased with incubation temperature and storage time, and (iii) was not affected by the acidity of the juices. The antibacterial agents could be divided into two classes: fast-acting and slow-acting. High-performance liquid chromatography analysis showed that the bactericidal results are related to the composition of the oils. These studies provide information about new ways to protect apple juice and other foods against human pathogens.     

Chemical composition and antibacterial activity of essential oils from Thymus spinulosus Ten. (Lamiaceae)

The chemical composition of essential oils from aerial parts of Thymus spinulosus Ten. (Lamiaceae) is reported. Four oils from plants growing in different environmental conditions were characterized by GC and GC-MS methods; the oils seem to indicate a new chemotype in the genus Thymus. Influences of soil and altitude characteristics on the essential oil composition are discussed. The oils showed antibacterial activity against Gram-positive (Staphylococcus aureus, Streptococcus faecalis, Bacillus subtilis, and Bacillus cereus) and Gram-negative (Proteus mirabilis, Escherichia coli, Salmonella typhimuium Ty2, and Pseudomonas aeruginosa) bacteria.     

Interaction of four monoterpenes contained in essential oils with model membranes: implications for their antibacterial activity

The present article reports the antimicrobial efficacy of four monoterpenes (thymol, carvacrol, p-cymene, and gamma-terpinene) against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli. For a better understanding of their mechanism of action, the damage caused by these four monoterpenes on biomembranes was evaluated by monitoring the release, following exposure to the compounds under study, of the water-soluble fluorescent marker carboxyfluorescein (CF) from large unilamellar vesicles (LUVs) with different lipidic composition (phosphatidylcholine, PC, phosphatidylcholine/phosphatidylserine, PC/PS, 9:1; phosphatidylcholine/stearylamine, PC/SA, 9:1). Furthermore, the interaction of these terpenes with dimyristoylphosphatidylcholine multilamellar vesicles as model membranes was monitored by means of differential scanning calorimetry (DSC) technique. Finally, the results were related also with the relative lipophilicity and water solubility of the compounds examined. We observed that thymol is considerably more toxic against S. aureus than the other three terpenes, while carvacrol and p-cymene are the most inhibitory against E. coli. Thymol and carvacrol, but not gamma-terpinene and p-cymene, caused a concentration-dependent CF leakage from all kinds of LUVs employed; in particular, thymol was more effective on PC and PC/SA LUVS than on PC/PS vesicles, while carvacrol challenge evoked a CF leakage from PC/PS LUVs similar to that induced from PC/SA LUVs, and lower than that measured with PC vesicles. Concerning DSC experiments, these four terpenes caused a decrease in Tm and (especially carvacrol and p-cymene) DeltaH values, very likely acting as substitutional impurities. Taken together, our findings lead us to speculate that the antimicrobial effect of thymol, carvacrol, p-cymene, and gamma-terpinene may result, partially at least, from a gross perturbation of the lipidic fraction of the plasmic membrane of the microorganism. In addition to being related to the physicochemical characteristics of the compounds (such as lipophilicity and water solubility), this effect seems to be dependent on the lipidic composition and net surface charge of the microbic membranes. Furthermore, the compounds might cross the cell membranes, thus penetrating into the interior of the cell and interacting with intracellular sites critical for antibacterial activity.     

Thymus zygis subsp. Gracilis: watering level effect on phytomass production and essential oil quality

Thymus zygis subsp. gracilis (chemotype thymol) was evaluated on the basis of its phytomass production and essential oil quality. Three different watering levels were assayed to achieve 63, 44, and 30% of the local potential evapotranspiration (Eto). According to the statistical analysis, a water supplement equivalent to 44% Eto in this cultivation area was optimal for maximum plant dry matter production and essential oil yield. Capillary GC-MS analysis of the essential oil allowed the identification of 86 volatile components. Among them, 30 are described for the first time as volatile constituents of the essential oil in this thyme subspecies and chemotype. The watering level effect on essential oil composition was noticeable, because the application of a water supplement equivalent to the 63% Eto favored the production of an essential oil richer in low molecular weight components. However, the greatest thymol concentrations were obtained under the 30 and 44% Eto watering levels.     

Essential oils from Mediterranean lamiaceae as weed germination inhibitors

The essential oils obtained from rosemary (Rosmarinus officinalis L.), thyme (Thymus vulgaris L.), and savory (Satureja montana L.) and the four monoterpenes that are their major constituents have been analyzed by GC and GC-MS and tested for their allelopathic properties on the seeds of three different annual weeds (Chenopodium album, Portulaca oleracea, and Echinochloa crus-galli) and three crops (Raphanus sativus, Capsicum annuum, and Lactuca sativa), with the aim to evaluate in vitro their potential as germination inhibitors. The essential oil composition varied with the species, thymol being the main constituent (44%) of thyme and carvacrol (57%) that of savory oil. Differences in essential oil composition were observed within two different rosemary ecotypes, type A, with alpha-pinene (37%) and 1,8-cineole (23%), and type B, characterized by a 2-fold content of 1,8-cineole (47%). This latest essential oil inhibited completely the germination of weeds while concurrently displaying little effect on pepper. The other two oils showed less selective action. S. montana essential oil, with 57% carvacrol, is the most active compound, completely inhibiting germination both of crops and weeds. Borneol, one of the main constituents of the oil of rosemary type B, showed an activity comparable to that of the whole oil. Crop and weed seeds treated with 1,8-cineole showed germination values that were not significantly different from controls, even if a slowing of the germination process expressed in terms of a significant increase in mean germination time was observed. Monoterpene compounds also present in the essential oils mainly represented the volatile fraction released from the crops and their residues into the soil.     

Volatile components and key odorants of fennel (Foeniculum vulgare Mill.) and thyme (Thymus vulgaris L.) oil extracts obtained by simultaneous distillation-extraction and supercritical fluid extraction

Volatile oil extracts of fennel seeds (Foeniculum vulgare Mill.) and thyme leaves (Thymus vulgaris L.) were obtained by simultaneous distillation-extraction (SDE) and supercritical fluid extraction (SFE) and analyzed by gas chromatography-mass spectrometry (GC-MS). In general, fennel oil extracted by SDE and SFE showed similar compositions, with trans-anethole, estragole, and fenchone as the main components. In contrast, thymol and p-cymene, the most abundant compounds in thyme leaves, showed big differences, with generally higher amounts of monoterpenes obtained by SDE. However, in this case, the differences between the extracts were higher. Key odorants of fennel seeds determined by gas chromatography-olfactometry (GC-O) showed similar patterns when applying SDE and SFE. trans-Anethole (anise, licorice), estragole (anise, licorice, sweet), fenchone (mint, camphor, warm), and 1-octen-3-ol (mushroom) were the most intense odor compounds detected in fennel extracts. Thymol and carvacrol, with oregano, thyme, and spicy notes, were identified as key compounds contributing to the aroma of thyme leaves.     

Chemical constituents, antifungal and antioxidative effects of ajwain essential oil and its acetone extract

GC and GC-MS analysis of ajwain essential oil showed the presence of 26 identified components which account for 96.3% of the total amount. Thymol (39.1%) was found as a major component along with p-cymene (30.8%), gamma-terpinene (23.2%), beta-pinene (1.7%), terpinene-4-ol (0.8%) whereas acetone extract of ajwain showed the presence of 18 identified components which account for 68.8% of the total amount. The major component was thymol (39.1%) followed by oleic acid (10.4%), linoleic acid (9.6%), gamma-terpinene (2.6%), p-cymene (1.6%), palmitic acid (1.6%), and xylene (0.1%). Moreover, the oil exhibited a broad spectrum of fungitoxic behavior against all tested fungi such as Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Aspergillus ochraceus, Fusarium monoliforme, Fusarium graminearum, Pencillium citrium, Penicillium viridicatum, Pencillium madriti, and Curvularia lunata as absolute mycelial zone inhibition was obtained at a 6-microL dose of the oil. However, the acetone extract showed better antioxidative activity for linseed oil as compared with synthetic antioxidants such as butylated hydroxyl toluene and butylated hydroxyl anisole.