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Identification of natural antimutagens with modulating effects on DNA repair.

The results of a study of bioantimutagenesis, with emphasis on natural antimutagens from plant extracts with modulating effects on DNA repair in Escherichia coli bacteria are presented in this chapter. Comparative screening for spontaneous or induced mutagenesis, as well as expression of the SOS gene, sfiA was accomplished. Antimutagenic capacity was obtained with nontoxic concentrations of the plant extracts; the same plant extract may decrease or increase the mutation rate, or even be ineffective, depending on the bacterial strain used and the concentration of the extract applied. Since antimutagenic effects may be the consequence of either stimulation of error-free repair, inhibition of error-prone repair, or involvement of multiple mechanisms, the effects of several plant extracts on the level of UV-induced beta-galactosidase were screened (to monitor SOS induction in cells). Reduction of the enzyme activity induced by UV was observed following addition of St. John's wort extract, while there was not reduction after thyme, aloe, camomile, or lime-tree and the level of UV-induced enzyme was even higher with sage extract. Our results indicate that the antimutagenic effect of St. John's wort is probably due to suppression of error-prone repair. Moreover, we assume that an antimutagenic effect obtained with thyme, mint, and sage under certain conditions may be due to enhanced error-free repair.

Antimutagenic Agents↗

Effect of some spices as preservatives for storage of lentil (Lens esculenta L.) seeds.

The high antimycotic activity of four kinds of spices, viz, Chinese cassia, cinnamon, clove and thyme, were tested as preservatives for agricultural commodities. Clove, thyme and a mixture of the four kinds of spice completely inhibited aflatoxin production on lentil seeds during eight weeks of incubation. Chinese cassia and cinnamon partially inhibited the aflatoxin production.

Aflatoxin B1↗

Comparison of different extraction methods for the determination of essential oils and related compounds from aromatic plants and optimization of solid-phase microextraction/gas chromatography.

Different extraction methods for the subsequent gas chromatographic determination of the composition of essential oils and related compounds from marjoram (Origanum majorana L.), caraway (Carum carvi L.), sage (Salvia officinalis L.), and thyme (Thymus vulgaris L.) have been compared. The comparison was also discussed with regard to transformation processes of genuine compounds, particularly in terms of expenditure of time. Hydrodistillation is the method of choice for the determination of the essential oil content of plants. For investigating the composition of genuine essential oils and related, aroma-active compounds, hydrodistillation is not very useful, because of discrimination and transformation processes due to high temperatures and acidic conditions. With cold solvent extraction, accelerated solvent extraction, and supercritical fluid extraction, discrimination of high and non-volatile aroma-active components as well as transformation processes can be diminished, but non-aroma-active fats, waxes, or pigments are often extracted, too. As solid-phase microextraction is a solvent-free fully automizable sample preparation technique, this was the most sparing to sensitive components and the most time-saving method for the rapid determination of the aroma compounds composition in marjoram, caraway, sage, and thyme. Finally, solid-phase microextraction could be successfully optimized for the extraction of the aroma components from the plants for their subsequent gas chromatographic determination.

Acetates↗

Purification and characterization of the monoterpene cyclase gamma-terpinene synthase from Thymus vulgaris.

The monoterpene cyclase, gamma-terpinene synthase, from Thymus vulgaris (thyme) leaves was purified to apparent homogeneity by isoelectric focusing and dye-ligand, anion-exchange, hydrophobic interaction, and gel permeation chromatography. The enzyme has a native molecular weight of 96,000 as determined by gel permeation chromatography, and exhibited a specific activity of 538 nmol/h.mg protein (turnover number of approximately 0.01/s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the enzyme to be composed of two apparently identical subunits of Mr approximately 55,000. The protein was very hydrophobic, and possessed a pI value of 4.85 as determined by isoelectric focusing. Maximum activity was observed at pH 6.8 in the presence of 20 mM Mg2+; 5 mM Mn2+ could support catalysis, albeit at a much lower rate. The Km value for the substrate, geranyl pyrophosphate, was 2.6 microM. Cyclase activity was inhibited by cysteine- and histidine-directed reagents. Purified gamma-terpinene synthase also possessed the ability to cyclize geranyl pyrophosphate to small amounts of alpha-thujene and to lesser quantities of myrcene, alpha-terpinene, limonene, linalool, terpinen-4-ol, and alpha-terpineol, all of which appear to be coproducts of the reaction sequence leading to gamma-terpinene. In general properties, the gamma-terpinene synthase from thyme leaves resembles other monoterpene cyclases as well as sesquiterpene and diterpene cyclases.

Alkyl and Aryl Transferases↗

Preliminary study on immunological and behavioural effects of Thymus broussonetii Boiss., an endemic species in Morocco.

In the present work, we had tried to evaluate the immunotropic and behavioural effects of Thymus broussonetii Boiss. So, we tested the neurostimulant effects of four extracts. This preliminary study allowed to identify both the immunostimulant and the neurotropic antistress effects of the studied extracts. Among the four extracts, only the aqueous and ethyl acetate ones showed an apparent effect on the tested biological activities, whereas the butanolic extract and the essential oil did not show any interesting effect (data not shown). These results showed that the aqueous and ethyl extracts of this endemic species are of interest for two reasons: stimulation of the immunizing system and protection against the stress by a neurotropic activity. Thyme extracts increased in vivo the number of leucocyte categories studied including polynuclears, total lymphocytes, TCD4+, TCD8+ and NK cells. These data suggest that the intraperitoneal administration of Thymus broussonetii extract has a potent direct effect on leucocytes in vivo. The elevation of leucocyte and thrombocyte counts produced by thyme in the peripheral blood was already reported in the literature. These results could be of practical importance in the field of phytotherapy in the treatment of some cases of human immunodeficiency such as cancer, leukaemia and AIDS.

Animals↗

Testing various herbs for antithrombotic effect.

OBJECTIVE: Prevention of arterial thrombotic diseases has high priority in developed countries. Because people who eat inappropriate food are prone to thrombotic events, regular intake of an antithrombotic diet may offer a convenient and effective way of prevention. The aim of the present study was to find herbs that have antithrombotic effects. METHODS: Herbs were crushed and centrifuged and the juice obtained was filtered. The effect of filtrate on platelet-rich thrombus formation was assessed by a shear-induced in vitro platelet function test (hemostatometry). Filtrates showing significant antithrombotic activity were further assessed by using a laser-induced in vivo thrombosis test in mice. The effect of orally administered herb filtrates on flow-mediated vasodilation was also measured. RESULTS: Common thyme and rosemary showed significant antithrombotic activity in vitro and in vivo. Neither herb affected flow-mediated vasodilation. CONCLUSION: Some herbs possess antithrombotic effects in experimental models of thrombosis. The mechanism of the antithrombotic effect of common thyme and rosemary may involve a direct inhibitory effect on platelets.

Animals↗

Essential oils--their antimicrobial activity against Escherichia coli and effect on intestinal cell viability.

Essential oils are known to possess antimicrobial activity against a wide spectrum of bacteria. The main objective of this study was to evaluate possible harmful effects of four commonly used essential oils and their major components on intestinal cells. Antimicrobial activity of selected plant extracts against enteroinvasive Escherichia coli was dose dependent. However, doses of essential oils with the ability to completely inhibit bacterial growth (0.05%) showed also relatively high cytotoxicity to intestinal-like cells cultured in vitro. Lower doses of essential oils (0.01%) had only partial antimicrobial activity and their damaging effect on Caco-2 cells was only modest. Cell death assessment based on morphological and viability staining followed by fluorescence microscopy showed that essential oils of cinnamon and clove and their major component eugenol had almost no cytotoxic effect at lower doses. Although essential oil of oregano and its component carvacrol slightly increased the incidence of apoptotic cell death, they showed extensive antimicrobial activity even at lower concentrations. Relatively high cytotoxicity was demonstrated by thyme oil, which increased both apoptotic and necrotic cell death incidence. In contrast, its component thymol showed no cytotoxic effect as well as greatly-reduced ability to inhibit visible growth of the chosen pathogen in the doses used. On the other hand, the addition of all essential oils and their components at lower doses, with the exception of thyme oil, to bacterial suspension significantly reduced the cytotoxic effect of E. coli on Caco-2 cells after 1h culture. In conclusion, it is possible to find appropriate doses of essential oils showing both antimicrobial activity and very low detrimental effect on intestinal cells.

Caco-2 Cells↗

Acaricidal activity of plant essential oils against Dermanyssus gallinae (Acari: Dermanyssidae).

The acaricial activity of 56 plant essential oils against poultry house-collected adult Dermanyssus gallinae De Geer was examined using direct contact and fumigation methods. In a filter paper contact bioassay, 100% mortality at 0.07 mgcm-2 was observed in bay, cade, cinnamon, clove bud, coriander, horseradish, lime dis 5F, mustard, pennyroyal, pimento berry, spearmint, thyme red and thyme white oils, whereas the mortality of these oils was significantly decreased at 0.02 mgcm-2. In fumigation tests with adult D. gallinae at 0.28 mgcm-2, cade, clove bud, coriander, horseradish and mustard oils were more effective in closed containers than in open ones, indicating that the effect of these essential oils was largely due to action in the vapour phase. Plant essential oils described herein merit further study as potential D. gallinae control agents.

Animals↗

Carbohydrate and nitrogenated compounds in liquid smoke flavorings.

Commercial smoke flavorings were extracted with dichloromethane and the remaining aqueous phase was evaporated at room temperature; the residues obtained were dissolved in methanol and studied by gas chromatography/mass spectrometry. The composition of these residues was totally different from that of the dichloromethane extracts, constituting a small number of compounds that were also detected in the dichloromethane extract, as well as a large number of compounds not described before as components of either smoke for food smoking or smoke flavorings. Among those compounds not previously described there are some furan, pyran, and phenolic derivatives, as well as some pyridine and carbohydrate derivatives. The main component of these fractions is 1,6-anhydro-beta-D-glucopyranose, or levoglucosan. Likewise, aqueous residues of liquid smoke flavorings, prepared at a laboratory scale from beech, vine shoots, thyme, and sage, were studied in the same way. These contained compounds of the same groups cited above, but showed clear differences. The aqueous residue of beech smoke flavoring was the most similar to that of the commercial smoke flavorings. The aqueous residues of vine shoots, thyme, and sage smoke flavorings contained a lower number of furan, pyran, and carbohydrate derivatives and a higher number of nitrogenated derivatives. Instead of levoglucosan, the main component was an unidentified compound, present in all samples, included in the carbohydrate derivatives group. In the samples studied, the influence of the vegetal source on the composition of the aqueous fraction has been shown. In the future, attention must be paid to the functionality of these smoke components.

Carbohydrates↗

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.

Chromatography, Gas↗

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.

Acyclic Monoterpenes↗

GC-MS analysis of essential oils from some Greek aromatic plants and their fungitoxicity on Penicillium digitatum.

The isolated essential oils from seven air-dried plant species were analyzed by gas chromatography-mass spectrometry (GC-MS). Thymus vulgaris (thyme), Origanum vulgare (oregano), and Origanumdictamus (dictamus) essential oils were found to be rich in phenolic compounds representing 65.8, 71.1, and 78.0% of the total oil, respectively. Origanum majorana (marjoram) oil was constituted of hydrocarbons (42.1%), alcohols (24.3%), and phenols (14.2%). The essential oil from Lavandula angustifolia Mill. (lavender) was characterized by the presence of alcohols (58.8%) and esters (32.7%). Ethers predominated in Rosmarinus officinalis (rosemary) and Salvia fruticosa (sage) essential oils, constituting 88.9 and 78.0%, respectively. The radial growth, conidial germination, and production of Penicillium digitatum were inhibited completely by oregano, thyme, dictamus, and marjoram essential oils at relatively low concentrations (250-400 microg/mL). Lavender, rosemary, and sage essential oils presented less inhibitory effect on the radial growth and conidial germination of P. digitatum. Conidial production of P. digitatum was not affected by the above oils at concentrations up to 1000 microg/mL. Apart from oregano oil, all essential oils were more effective in the inhibition of conidial germination than of radial growth. The monoterpene components, which participate in essential oils in different compositions, seem to have more than an additive effect in fungal inhibition.

Antifungal Agents↗

Antifungal activity of volatile compounds generated by essential oils against fungi commonly causing deterioration of bakery products.

AIMS: To investigate the volatile fractions of 16 essential oils for activity against the more common fungi causing spoilage of bakery products, Eurotium amstelodami, E. herbariorum, E. repens, E. rubrum, Aspergillus flavus, A. niger and Penicillium corylophilum. METHODS AND RESULTS: The study applied 50 microl of pure essential oils in a sterilized filter paper, were carried out at pH 6 and at different water activity levels (0.80-0.90). First, a wheat flour based agar medium was used, where cinnamon leaf, clove, bay, lemongrass and thyme essential oils where found to totally inhibit all microorganisms tested. These five essential oils were then tested in sponge cake analogues, but the antifungal activity detected was much more limited. CONCLUSION: Five essential oils showed potential antifungal capacity against all species tested, over a wide range of water availability. Their activity, however, seems to be substrate-dependent. More research is needed to make them work in real bakery products, as in the preliminary study limited effectiveness was found. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the cinnamon leaf, clove, bay, lemongrass and thyme essential oils against species belonging to Eurotium, Aspergillus and Penicillium genus has been demonstrated.

Agar↗

Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens.

The antimicrobial properties of 21 plant essential oils and two essences were investigated against five important food-borne pathogens, Campylobacter jejuni, Salmonella enteritidis, Escherichia coli, Staphylococcus aureus and Listeria monocytogenes. The oils of bay, cinnamon, clove and thyme were the most inhibitory, each having a bacteriostatic concentration of 0.075% or less against all five pathogens. In general, Gram-positive bacteria were more sensitive to inhibition by plant essential oils than the Gram-negative bacteria. Campylobacter jejuni was the most resistant of the bacteria investigated to plant essential oils, with only the oils of bay and thyme having a bacteriocidal concentration of less than 1%. At 35 degrees C, L. monocytogenes was extremely sensitive to the oil of nutmeg. A concentration of less than 0.01% was bacteriostatic and 0.05% was bacteriocidal, but when the temperature was reduced to 4 degrees, the bacteriostatic concentration was increased to 0.5% and the bacteriocidal concentration to greater than 1%.

Bacteria↗

Inhibitory effects of spice essential oils on the growth of Bacillus species.

A series of essential oils of 11 Turkish plant spices [black thyme, cumin, fennel (sweet), laurel, marjoram, mint, oregano, pickling herb, sage, savory, and thyme], used in foods mainly for their flavor, aromas, and preservation, in herbal tea, in alternative medicines, and in natural therapies, were screened for antibacterial effects at 1:50, 1:100, 1:250, and 1:500 dilutions by the paper disc diffusion method against six Bacillus species (Bacillus amyloliquefaciens ATCC 3842, Bacillus brevis FMC 3, Bacillus cereus FMC 19, Bacillus megaterium DSM 32, Bacillus subtilis IMG 22, and B. subtilis var. niger ATCC 10). All of the tested essential oils (except for cumin) showed antibacterial activity against one or more of the Bacillus species used in this study. Generally, the essential oils at 1:50 and 1:100 levels were more effective. Only one essential oil (laurel) was not found effective against the tested bacteria. The bacterium most sensitive to all of the spice essential oils was B. amyloliquefaciens ATCC 3842. Based on the results of this study, it is likely that essential oils of some spices may be used as antimicrobial agents to prevent the spoilage of food products.

Anti-Bacterial Agents↗

Inhibition of listeriolysin O and phosphatidylcholine-specific production in Listeria monocytogenes by subinhibitory concentrations of plant essential oils.

Successful infection by Listeria monocytogenes is dependent upon a range of bacterial extracellular proteins including a cytolysin termed listeriolysin O and phosphatidylcholine-specific phospholipase C. Five plant essential oils--bay, clove, cinnamon, nutmeg and thyme--significantly reduced the production of listeriolysin O by L. monocytogenes. The greatest change was observed after culture with oil of thyme, which reduced haemolysis to 52.1 haemolytic units (HU)/ml compared with 99.8 HU/ml observed with the control. Oil of clove was the only oil that also significantly reduced phosphatidylcholine-specific phospholipase C activity. These changes were observed despite the oils causing no change to the final bacterial concentration or total extracellular protein concentration.

Bacterial Toxins↗

Occupational asthma caused by aromatic herbs.

We describe a subject with occupational asthma caused by several aromatic herbs: thyme, rosemary, bay leaf, and garlic. The diagnosis was confirmed by several inhalation challenges in the laboratory. Although immediate skin reactivity was demonstrated to the herbs the subject reacted to by inhalation, RAST showed that garlic was the most potent allergen by weight, the other herbs showing less reactivity. These aromatic herbs, bay leaf, thyme, and rosemary, should be included among agents causing occupational asthma in the food industry.

Adult↗

Sensitivity of heat-stressed yeasts to essential oils of plants.

Eight strains of yeasts (Candida lipolytica, Debaryomyces hansenii, Hansenula anomala, Kloeckera apiculata, Lodderomyces elongisporus, Rhodotorula rubra, Saccharomyces cerevisiae, and Torulopsis glabrata) were examined for changes in sensitivity to eight essential oils of plants (allspice, cinnamon, clove, garlic, onion, oregano, savory, and thyme) after being sublethally heat stressed. With the exception of garlic oil for all test yeasts, onion oil for S. cerevisiae, and oregano oil for R. rubra, the essential oils at concentrations of up to 200 ppm in recovery media did not interfere with colony formation by unheated cells. However, some oils, at concentrations as low as 25 ppm in recovery media, reduced populations of sublethally heat-stressed cells compared to populations recovered in media containing no test oils. This demonstrates that the yeasts were either metabolically or structurally damaged as a result of being exposed to elevated temperatures and that essential oils prohibited repair of injury. The size (diameter) of colonies produced on oil-supplemented recovery agar by heat-stressed cells was reduced compared to that observed on unsupplemented agar. Pigment production by heated R. rubra was inhibited by oils of oregano, savory, and thyme, but enhanced by garlic and onion oils. The influence of essential oils on survival of yeasts in thermally processed foods and in the enumeration of stressed cells in these foods should not be minimized.

Condiments↗