Antimicrobial effects of thyme essential oils and thyme preparations
The first researcher who attributed antibacterial properties to thyme (without specifying the species) was Chamberlain in 1887, after observing the antibacterial effect of its “vapours” on Bacillus anthracis. Since then, numerous studies with essential oils of different species of Thymus have been carried out. They were shown to inhibit a broad spectrum of bacteria, generally Gram-positive bacteria being more sensitive than Gram-negative bacteria. This became obvious in some screening studies administering Thymus oils to a variety of bacteria.
Recently the antibacterial activity of thyme (Thymus vulgaris) oil against some important food-borne pathogens, namely Salmonella enteritidis, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Campylobacter jejuni, was tested. The latter was found to be the most resistant of the bacteria investigated. In another study it was shown that the essential oil of thyme and especially its phenols, thymol and carvacrol, have antibacterial acivity against periodontopathic bacteria including Actinobacillus, Capnocytophaga, Fusobacterium, Eikenella, and Bacteroides species, and may therefore be suitable for plaque control, although few essential oils have been found to possess clinical efficacy. Furthermore, the essential oil of thyme showed a wide antibacterial activity against microorganisms that had developed resistance to antibiotics such as methicillin-resisting Staphylococcus aureus and vancomycin-resisting Enterococcus faecium ().
Several studies have focused on the antimicrobial activity of the essential oils of thyme in order to identify the responsible compounds. Thymol and carvacrol seem to play an outstanding role. These terpene phenols join to the amine and hydroxylamine groups of the proteins of the bacterial membrane altering their permeability and resulting in the death of the bacteria. In addition, thymol and carvacrol were shown to induce a decrease of the intracellular adenosine triphosphate (ATP) pool of Escherichia coli and an increase of the extracellular ATP. Antibacterial activity was also observed for the aliphatic alcohols, especially geraniol, and ester components. A variety of activities was presented by the esters, in some cases they were more active than their corresponding free alcohols, but sometimes less active.
Crespo et al. () have evaluated the antimicrobial activity exhibited by the main chemical groups found in the essential oil of Thymus serpylloides ssp. gadorensis including hydrocarbons, alcohols, acetates, and phenols (Table 10.1). Again the phenols turned out to be the most effective against all microorganisms tested, the activity of the alcohols was on lower levels. Hydrocarbons proved to be effective only against Bacillus megaterium and Mycobacterium phlei, against the latter also the acetates showed weak activity. The higher sensitivity of Bacillus megaterium and Mycobacterium phlei to the essential oil of T. serpylloides ssp. gadorensis may be interpreted as the joint effectiveness of three and four active fractions respectively.
Table Antibacterial activity of individual components of thyme oil
|Components||Pseudomonas aeruginosa||Escherichia colt||Staphylococats attreus||Bacillus cereus|
Results are expressed as growth inhibition in an agar overlay technique assay.
(-) area of inhibition minor than 7 mm.
(+) area of inhibition between 7-10 mm.
(++) area of inhibition between 11-16 mm.
(+++) area of inhibition greater than 16 mm.
Studies on the structure—activity relationships of 32 terpenoids resulted in the following observations (): (a) α-isomers were inactive as opposed to the β-isomers which showed a pronounced activity, e.g. α-pinene; (b) cis-isomers proved to be inactive contrary to the active tr-isomers, e.g. geraniol versus its cis-isomer nerol; (c) compounds with a methyl-isopropyl cyclohexane ring like some alcohols and ketones were the most active, e.g. pulegone; (d) unsaturation of the cyclohexane ring further increased the antimicrobial activity, e.g. terpinolene and ct-terpineol which proved to be the most active of the compounds examined against all the bacteria of the test. Negative results were found in case of α- or cis-isomers or when the compounds lack the common terpene C10-structure, e.g. citronellol or nerolidol.
With respect to the botanical species one can classify the essential oils of thyme, in general terms, into two main groups (): (i) The first group contains those species in which phenols (thymol and carvacrol) are the predominant components. These oils show remarkable antimicrobial activities, (ii) In the oils of the second group phenols are scarce or lacking, whereas other components, such as monoterpene hydrocarbons, non-phenolic oxygenated monoterpenes or even sesquiterpene hydrocarbons, predominate. Such oils usually demonstrate lower antimicrobial activities than those in the first group.
The results obtained by the evaluation of the antimicrobial activity of a non-phenolic essential oil of thyme from Thymus granatensis may serve as an example of the above statement. Although this essential oil presented activity against all the germs tested, with the exception of Escherichia colt, it proved to be only weakly active, in some cases practically inactive as was the case of Candida albkans and Pseudomonas fluorescens (Table Antimicrobial activity of essential oils from Thymus granatensis and Thymus serpylloides ssp. gadorensis). Similar results were obtained when other non-phenolic essential oils of thyme were tested, e.g. T. hyemalis (), Thymus longiflorus () and Thymus baeticus (). A remarkably stronger antimicrobial activity was observed when typically phenolic oils, such as e.g. from T. serpylloides ssp. gadorensis were administered, a fact which was confirmed by studies with further phenolic essential oils such as that from Thymus zygis () and from Thymus orospedanus ().
Table Antimicrobial activity of essential oils from Thymus granatensis and Thymus serpylloides ssp. gadorensis
|Test micro-organisms||Thymus granatensis||Thymus serpylloides ssp. gadorensis|
Results are expressed as growth inhibition in an agar overlay technique assay.
(-) area of inhibition minor than 7 mm.
(+) area of inhibition between 7—10 mm.
(++) atea of inhibition between 11—16 mm.
(+++) area of inhibition greater than 16 mm.
Two research groups evaluated the different antimicrobial (antibacterial and anti-fungal) effects of the essential oils of the seven chemotypes of Thymus vulgaris containing 1,8-cineole, geraniol/geranyl acetate, linalool, α-terpineol/α-terpinyl acetate, thymol, carvacrol, and tr-sabinene hydrate/cis-myrcenol-8 respectively as main compounds. Logically the oils of these chemotypes vary in their minimal inhibitory concentration (MIC) values obtained. The most active oil was proven to be that of the thymol chemotype followed by the carvacrol and geraniol type; the linalool type showed similar activity to that of the geraniol type. The oils of the other chemotypes were much less active.
When accepting that the chemistry of the essential oils is responsible for their antimicrobial activities, as was explained above, it becomes obvious that all factors influencing the chemical composition of essential oils within the plant indirectly influence their antimicrobial activity. Climatic conditions are known to modify the chemical composition of the essential oils and therefore, climatic conditions indirectly influence the antibacterial activities. For example, Kowal and Kuprinska found large differences in the MIC values for the essential oils of T. pulegioides from different regions in Croatia. Also Cabo have found considerable differences in the antimicrobial activity of the essential oils obtained from different populations of Thymus granatensis collected in southeastern Spain. Another factor determining the variability of the oil composition of thyme is the stage of the plant growth during harvest. Arras and Grella studied the influence of the harvest time of T. capitatus (Thymbra capitata) on the fungistatic effects of its essential oil.
Although the antimicrobial action is to a large extent attributed to the essential oils, non-volatile constituents also have been described to possess antimicrobial activity, such as saponins and resins. These two constituents from T. capitatus (Thymbra capitata) inhibited the growth of several bacteria and fungi. It has recently been demonstrated that the watery extract of thyme (Thymus vulgaris) showed a strong inhibition of Helicobacter pylory reducing both, its growth and potent urease activity. Clinical trials confirming this property would be of significant interest, since this microorganism plays a role in the etiology of gastroduodenal ulcers.
Finally we will refer to the techniques commonly used to evaluate antibacterial activity. They can be classified depending on whether they require a homogeneous dispersion in water or not. (a) The agar overlay technique does not require a homogeneous dispersion in water. Discs, holes or cylinders are used as reservoirs containing the essential oils to be tested and are brought into contact with an inoculated medium and, after incubation, the diameter of the transparent zone around the reservoir (inhibition diameter), where the microorganisms have been destroyed by the action of the essential oil, is measured. The method can be modified when the reservoir is placed in the lid of the Petri dish, thus excluding transport by diffusion. (b) Dilution techniques require a homogeneous dispersion of the oil. Since essential oils are insoluble in the watery liquid culture medium, non-ionic emulsifying agents such as Tween or Spans are needed. Although the addition of an emulsifying agent introduces an extra component with respect to activity and possible interactions, this method has proved easily reproducible. However, other dilution methods that avoid the use of tensioactives have been proposed, such as the solution of the essential oil in DMSO, or in the formation of a stable suspension for 24 h in a sterile watery solution of agar.
The techniques to determine the qualitative bacteriostatic activity are based on the agar overlay technique. Quantification of the antimicrobial activity is generally established by determining the MIC values. The MIC values can be determined either by using the agar overlay technique or the dilution techniques.
Several in vitro and in vivo screenings have shown that volatile oils, especially those of the genus Thymus, may be used against fungal diseases. Different screenings focused on the essential oil of Thymus vulgaris and its effect on food spoiling yeasts, especially Aspergillus (), on various dermatophytes, and on some phytopathogenic fungi, e.g. Rhizoctonia solani, Pythium ultimum, Fusarium solani, and Calletotrichum lindemthianum (). Not only the oil of Thymus vulgaris but also the oils of other Thymus species showed antifungal activity, e.g. that of Thymus zygis against Botrytis cinerea. The oil of Thymus serpyllum was found to be highly active against various species of Penicillium, Fusarium and Aspergillus (). Various oils, namely the oils of Thymus zygis (), T. hyemalis (), Thymus vulgaris (Menghini etal., 1987), and Thymus baeticus (), inhibited the growth of Candida alhicans.
The essential oil of Thymus vulgaris inhibits both mycelial growth and aflatoxin synthesis by Aspergillus parasiticus () at only 0.1 per cent in the medium. Therefore it is used as a preservative in agriculture, completely inhibiting aflatoxin production on lentil seeds up to eight weeks of incubation. In addition, the essential oil of Thymus vulgaris exerts a protective effect in corn against Aspergillus flavus, without producing phytotoxic effects on germination or corn growth.
According to Agarwal and Mathela the antifungal activity of the essential oil of Thymus serpyllum is attributable to thymol and carvacrol. They cause the degeneration of the fungal hyphae which seems to empty their cytoplasmic content. The terpenic alcohols as well as the aldehydes, ketones and some esters, also presented considerable activities, whereas the hydrocarbons showed only low activities. Terpenic alcohols which display monoterpenic structure and hydroxyl group at terminal carbon (i.e. geraniol, nerol and citronellol) have shown the highest activity. No difference was observed in the antifungal activity between cis or trans isomer forms of these molecules. The components which display carbonyl groups were also active in inhibiting fungal growth, showing the aldehydes (i.e. citral and citronellal) a higher activity than ketones. Similarly in terpenic alcohols, this effect could be attributed to the presence of the functional group at a terminal carbon.
Only few studies demonstrate the antiviral effects of thyme extracts. In 1967, Herrmann and Kucera reported on the antiviral effects of Thymus serpyllum and Spanish and French thymes (Thymus sp.) against Newcastle disease virus (NDV). The antiviral activity was concentrated in the tannin fraction although non-tannin extracts also showed effects attributed to the polyphenol precursor compounds of tannins. However the activity was smaller compared to that observed with Melissa officinalis extracts. More recently other studies failed to demonstrate antiviral effects of Thyme extracts against Rubella virus. The antiviral activity recently observed in other members of the Labiatae family has been attributed to new di- and tri-terpenoid compounds that appear to be specific inhibitors of HIV-1 protease. Those compounds have not yet been detected in the genus Thymus.
It was only in the 1970s that scientists realised that the human body constantly creates free radicals and eliminates them by a series of antioxidant defense mechanisms. When free-radical generation exceeds the capacity of antioxidant defenses, the result is “oxidative stress”. It occurs in many human diseases and sometimes makes a significant contribution to their pathogenesis. In literature several publications are dedicated to the antioxida-tive effects of plant extracts with phenolic compounds. Recently Chung studied the effects of methanol extracts from 51 plant species on OH-radical scavenging. Mustard varieties, thyme, oregano, and clove all exhibited strong scavenging activity.
There are several papers showing that both the essential oil and the flavonoids of Thymus vulgaris are potent antioxidant agents. Dorman studied the antioxidant properties of the essential oil of Thymus vulgaris (0.75—100 ppm) among others. The antioxidant properties were evaluated in three avian thiobarbituric acid reactive substances (TBARS) assays using egg yolk, one-day-old chicken liver or muscle from mature chicken. Thymus vulgaris was one of the most effective antioxidants in the egg yolk assay besides Monarda citriodora var. citriodora and Myristica fragrans.
Deans investigated the protection of polyunsaturated fatty acids within the liver of old mice by ingestion of culinary and medicinal plant volatile oils obtained by hydrodistillation. This protection effectively reverses the normal trend in polyunsaturated fatty acid metabolism during aging where a decrease in level is concomitant with a reduction in tissue function and integrity. The essential oil of thyme was overall the most effective agent in this protective effect.
Ternes showed that carvacrol, thymol and p-cymene-2,3-diol, all the three components of thyme oil, exhibit antioxidant activities. The authors determined the concentration of each substance in different foodstuffs containing thyme extracts as well as their stabilities at different temperatures. Schwarz assessed the antioxidant activity of thyme phenols (Rancimat method 110°C, Schal test 60 °C) and showed that p-cymene-2,3-diol was the most active one being more active than α-tocopherol and butylated hydroxyanisole. Five thyme species (Thymus vulgaris, T. pseudolanuginosus, T. citriodorus, Thymus serpyllum and T. doerfleri) were analysed by means of High-Performance Liquid Chromatography (HPLC) for all 3 compounds. The highest amounts were found in Thymus vulgaris.
Pearson investigated the potential antioxidant activity of various plant phenolics, namely carnosic acid, carnosol, and rosmarinic acid (in rosemary extracts), thymol (in thyme extracts), carvacrol (in origanum extracts), and zingerone (in ginger extracts), using aortic endothelial cells to mediate the oxidation of low-density lipoprotein (LDL). The extent of oxidation was determined spectrometrically by measuring the absorbance at 234 nm of the conjugated dienes. Their relative antioxidant activities decreased in the order carnosol > carnosic acid approximate to rosmarinic acid > thymol > carvacrol > zingerone.
Flavonoids are abundant in diverse species of thyme. They are potent antioxidants which are responsible for many of the beneficial effects of these plants. The electron-donating properties of flavonoids have been repeatedly emphasised as the basis of their antioxidant action. Common antioxidative flavonoids, like luteolin and quercetin, isolated from various species of thyme, have shown potent antioxidant activity in vivo as much as in vitro ().
From leaves of Thymus vulgaris Haraguchi isolated two antioxidative components by a bioassay-directed fractionation: a biphenyl compound, 3,4,3′,4′-tetra-hydroxy-5,5 -diisopropyl-2,2 -dimethylbiphenyl, and a flavonoid, eriodictyol. Their antioxidant effects on biological systems were studied in three different biological systems: inhibition of superoxide anion production in the xanthine/xanthine oxydase system, inhibition of microsomal peroxydation, inhibition of hemolysis of human erythrocytes. Both the new biphenyls as well as eriodictyol showed outstanding antioxidant effects.
An extract of Thymus vulgaris shows antiparasitic properties against Leishmania mexkana (), because it inhibits the mitochondrial DNA polymerase (IC50 value was 0.82mg/ml). Thymol was mainly responsible for this effect). The essential oil of this species was also active against diverse phytonematodes, the oxygenated compounds being partially responsible for the nematicidal effects.
Perrucci evaluated the in vitro ascaricidal properties of some natural monoterpenoid constituents of several essential oils against rabbit mange mite (Psoroptes cuniculi) by direct, external contact and by inhalation. The natural terpenoids assayed were: hydrocarbons (limonene, myrcene, 7-terpinene), alcohols (linalool, geraniol, nerol, terpinen-4-ol, α-terpineol) and phenols (thymol, eugenol), an ester (linalyl acetate) and an ether (estragole). Because the test components represent different chemical classes, it was also possible to discern in a preliminary fashion a correlation between chemical structure and ascaricidal activity. All the monoterpene hydrocarbons, either acyclic (i.e. myrcene) or cyclic (i.e. limonene and 7-terpinene) did not show any miticidal activity at the doses tested (1.0 per cent, 0.25 per cent and 0.125 per cent). The double-bond position and/or number seems to be unimportant for this kind of biological activity.
In contrast, the terpene alcohols, such as linalool, geraniol, nerol, menthol, terpinen-4-ol, and α-terpineol, were able to kill nearly 100 per cent of the mites at the dose tested. Therefore, the oxygenated functional groups potentiate the ascaricidal properties among these compounds. Neither the acyclic (i.e. linalool, geraniol, nerol) nor cyclic (i.e. menthol, terpinen-4-ol, α-terpineol) nature of the compound appeared to influence the miticidal activity. Similarly, the site of linkage to the ring or to a side chain, as well as the nature of the hydroxyl group (primary, secondary, or tertiary), does not influence the activity. The cis!trans isomerism represented by nerol and geraniol seems to be important.
Thymol and eugenol killed nearly 100 per cent of the parasites at all dosages assayed in the direct contact test, indicating that a phenolic function can enhance the miticidal characteristics of terpenes. The low susceptibility of mites to linalyl acetate, particularly at the lowest doses, could be related to the esterification of the oxygenated function. Estragole, structurally close to eugenol, but with a methylated phenolic group, exhibited, at a concentration of one per cent, an activity comparable to that of the same dose of eugenol. However, this action decreased (63 per cent) at 0.25 per cent and disappeared completely at 0.125 per cent. These results indicate that the best miticidal activity of the monoterpenes examined in the direct contact test can be related to compounds with free alcoholic or phenolic functional groups.
Recently it was demonstrated that aromatic plants present a double insecticidal effect: by direct toxicity on adult insects and by inhibiting reproduction. The most efficient plant in this regard belongs to the Labiatae family. Therefore one can profit using the essential oils of Thymus vulgaris and Thymus serpyllum in addition to a fumigant against Anathoscelides ohtectus Say (Coleoptera, Bruchidae), a frequent pest that damages its host plant, the kidney bean (Phaseolus vulgaris L.) in the field and during storage. The oils have a toxic effect on adult insects and also inhibit the reproduction through ovicidal and larvicidal effects. This insecticidal action is also produced by other components of the species such as non-volatile phenols, non-proteinic amino acids, and flavonoids.
The essential oil of Thymus vulgaris and thymol shows activity against Tetranychus urticae. Thymol was shown to be more potent than thyme oil as a deterrent factor for reducing egg laying by the mite. Mortality percentage reached 100 per cent with both materials used; however, at low concentrations the effect again was more pronounced applying thymol than applying thyme oil.
Karpouhtsis have demonstrated the genotoxic effect of thymol on the somatic mutation and recombination test on Drosophila and that this effect could contribute to the insecticidal action of essential oils such as Thymus vulgaris. Other components of the oils such as carvacrol, 7-terpinene and p-cymene have been found to be ineffective. Another pest species sensitive to the essential oil of Thymus vulgaris is Spodoptera littoralis. Feeding larvae with leaves treated with the essential oil reduced the successful development and egg production.
Lee evaluated the acute toxicity of 34 naturally occurring monoterpenoids against three important arthropod pest species: the larvae of the Western corn rootworm, Diabrotka virgifera LeConte, the adult two-spotted spider mite, Tetranychus urticae Koch, and the adult house fly, Musca domestica L. Thymol was the most topically toxic against the house fly, and citronellol and thujone were the most effective on the Western corn rootworm. Most of the monoterpenoids were lethal to the two-spotted spider mite at high concentrations; terpinen-4-ol was especially effective.
Selections from the book: “Thyme. The genus Thymus”. Edited by Elisabeth Stahl-Biskup. Series: “Medicinal and Aromatic Plants — Industrial Profiles”. 2002.