Antibacterial Activity

Based on a broad spectra of antibacterial activity, oregano seems to be one of the most inhibitory spices tested. However, when considering the reported data, questions on the accuracy of the interpreted information often arise. When assessing the antimicrobial potency of herbs, spices and medicinal plants, that are used traditionally in gastronomy for preservative reasons or in prevention of human—plant diseases, one has to consider many factors which influence the efficacy of an extract or essential oil. These include the plant species used and its origin, concentration and composition of an extract/essential oil, the mode of dispersal of extract/oil to the medium, the concentration of tested organism in the growing medium, susceptibility of bacterial strains, the method used — in vitro or in vivo — pH, temperature . . .). Moreover, Skandamis et al. (2000) have stressed the importance of the fluidity of the culture medium for accurate estimation of the potency of essential oils against microorganisms. Different culture media (liquid culture or gel matrix) were found to influence the rate of consumption of glucose, thus influencing the growth of bacteria as well as their susceptibility toward extracts/essential oils.

Altogether, 52 essential oils (including O. vulgare and O. majorana essential oils) and extracts of different plant genera have been investigated (Hammer et al., 1999) for their activity against Acinetobacter baumanii, Aeromonas veronii biogroup sobria, C. albicans, Streptococcus faecalis, E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype typhimurium, Serratia marcescens and S. aureus. It was found that O. vulgare (Australian origin) yielded one of the most potent antibacterial agents, which considerably inhibited the growth of all tested microorganisms. The lowest minimum inhibitory concentration of O. vulgare essential oil was 0.12 per cent (v/v) and 0.25 per cent (v/v) of O. majorana. Among the tested bacteria, the most resistant was Pseudomonas aeruginosa, that was inhibited by O. vulgare essential oil at 2 per cent (v/v), but not by O. majorana oil.

Screening of Italian Medicinal Plants for their antibacterial activity using the in vitro paper disk diffusion method (paper disks Whatman No. 1) revealed the strong activity of O. vulgare L. dimethylsulphoxide (DMSO) extracts against Gram-positive (Bacillus subtilis, S. aureus, Streptococcus haemolyticus) and Gram-negative bacteria (E. coli 7075, Klebsiella pneumoniae, Proteus mirabilis, Salmonella typhi H) (Izzo et al, 1995). The minimal inhibitory concentration (MIC) of applied extracts towards all tested bacteria was less than 4 pg/disk, with the exception of Pseudomonas aeruginosa, which was not susceptible. The essential oils of Origanum onites L. of Sicilian origin and of commercial oregano (which was found to be a mixture of two species, i.e. O. vulgare L. and O. majorana L.) exhibited bactericidal or bacteriostatic activity against a variety of Gram-positive

(S. aureus, Streptococcus faecalis, Micrococcus luteus and Bacillus subtilis) and Gram-negative bacteria (Proteus vulgaris, E. coli, Hafnia alvei) (Biondi et al, 1993). GC analysis revealed the carvacrol (61.68 per cent) as the main component of O. onites essential oil, while the commercial oregano sample consisted of terpinene-4-ol (24.87 per cent), y-terpinene (15.91 per cent) and thymol (11.61 per cent) as leading compounds. When comparing the two species, O. onites essential oil was found to be more effective, inducing bactericidal effects against all G(_) tested organisms and against S. aureus and bacteriostatic effects against Micrococcus luteus and Bacillus subtilis at dilutions (in absolute ethanol) of 1:10 (final concentration of essential oil 1 pl/disk). The bactericidal effect of essential oil, distilled from commercial oregano sample showed bactericidal effects only against Streptococcus faecalis (dilution 1:2, final conc. of EO 5 pl/disk), E. coli and Proteus vulgaris (dilution 1:5, final conc. of EO 2 pl/disk), whilst bacteriostatic effects (dilution 1:10) were observed against other tested bacteria. Pseudomonas aeruginosa was not susceptible to any of the tested oils or concentrations. Similar results were obtained by Paster et al. (1990), who found that Pseudomonas aeruginosa was not affected by oregano (O. vulgare L.) essential oil at concentrations of up to 500 pg/ml. Under aerobic conditions the O. vulgare oil was very effective against Campylobacter jejuni (microaerophile) and Clostridium sporogenes (anaerobe) at 250 pg/ml (Paster et al, 1990). Also, good bacterio-static (at concentration of 225 mg/l) and bactericidal (at concentration of 900 mg/l) effects in vitro against Erwinia amylovora were observed with O. vulgare essential oil (Scortichini and Rossi, 1989; Scortichini and Rossi, 1993).

Origanum vulgare essential oil (agar dilution method: 10 pl oil/Petri dish), characterised by high thymol (32.4 per cent) and carvacrol (16.7 per cent) content, showed a strong inhibitory (inhibition zone >20 mm) effect against a broad spectrum of tested bacteria, that were both G( + ) or G(_) (Alcaligenes faecalis, Bacillus subtilis, Beneckea natriegens, Brevibacterium linens, Brocothrix thermosphacta, Citrobacter freundii, Clostridium perfringens, Enterobacter aerogenes, Erwinia carotovora, Klebsiella pneumoniae, L. plantarum, Leuconostoc cremoris, Moraxella spp., Proteus vulgaris, Salmonella pullorum, Serratia marcescens, S. aureus, Streptococcus faecalis, Yersinia enterolitica) (Baratta etal, 1998a). Good inhibitory activity (inhibition zone >10 mm <20 mm) was observed also against E. coli, Flavobacterium suaveolens, Micrococcus luteus, Pseudomonas aeruginosa, whilst two bacteria (Acinetobacter calcoaceticus, Aeromonas hydrophila) were not susceptible to oregano essential oil. In affected organisms, the origin of the bacterial strain or the fact of being G( + ) or G(—) did not influence their susceptibility towards the oil. This is in agreement with the observations of Deans (Deans and Ritchie, 1987; Deans et al, 1992), who found that volatile oils of O. vulgare ssp. hirtum were equally effective against both G( + ) and G(_) microorganisms.

Using the same bacterial strains and test conditions, the essential oils of O. majorana — consisting prevalently of terpinen-4-ol (20.8 per cent), y-terpinene (14.1 per cent) and a-terpinene (14.1 per cent) — showed similar, but less potent antibacterial activity than O. vulgare. However, O. majorana exhibited a strong inhibitory effect against Acinetobacter calcoaceticus and Aeromonas hydrophila (Baratta etal, 1998b), against Beneckea natriegens, Erwinia carotovora and Moraxella spp. (Deans and Svoboda, 1990) but was not active against Brevibacterium linens and Leuconostoc cremoris.

Essential oils of O. vulgare of Turkish origin exhibited still more potent antibacterial activity as observed by Kivang and Akgül (1986), who have studied the bactericidal effects of essential oils by both the agar diffusion and the serial dilution methods. Essential oils of O. vulgare showed pronounced bacteriostatic (dilution levels of

1:20—1:160) and bactericidal effects (dilution level of 1:20) against seven bacteria (Aerobacter aerogenes, Bacillus subtilis, E. coli, Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus albus, Staphylococcus aureus).

The growth of a wide range of bacteria (Clostridium sporogenes, Enterobacter, E. coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella pullorum, S. aureus, Streptococcus faecalis, Yersinia enterolitica) was strongly inhibited (zone inhibition >31.5 mm <71.2 mm), when grown in an agar medium supplemented with essential oil of Origanum officinalis (dilution 1:10), a special breed from Israel, that was selected for elevated oil yields (Deans et al, 1992).

Origanum majorana L. essential oil (Vojvodina origin), at concentration of 0.15 per cent showed moderate inhibitory activity against four bacteria (E. coli, Proteus vulgaris, Salmonella enteritidis, Pseudomonas fluorescens), that are frequently present as undesirable flora in the meat-processing industry (Sirnik and Gorisek, 1983). Deans and Ritchie (1987) reported, that Origanum majorana L. essential oil had a broad spectrum of antibacterial activities (at dilution level of 1:10) against bacteria of animal or human origin (E. coli, Salmonella pullorum, Streptococcus faecalis, S. aureus, Clostridium sporogenes) against soil bacteria (Bacillus subtilis, Serratia marcescens), plant pathogen (Erwinia carotovora) and aquatic bacteria (Beneckea natriegens). At dilution level of 1:5 of O. majorana essential oil, a remarkable effect was detected against Yersinia enterocolica and Pseudomonas aeruginosa (agar dilution method).

In a way comparable to antifungal activity, the antibacterial effects of oregano essential oils, as experienced in O. vulgare ssp. hirtum, O. dictamnus and commercial Greek Origanum oil, were mainly due to the presence of phenolic constituents of essential oils (carvacrol and/or thymol), whilst their biosynthetic precursors y-terpinene and p-cymene were inactive (Pellecuer et al, 1980; Gergis et al, 1990; Panizzi et al, 1993; Sivropoulou et al, 1996; Adam et al, 1998). Hence, synergistic antibacterial activities of carvacrol and thymol were reported (Didry et al., 1993; Sivropoulou et al., 1996). According to Sivropoulou and co-workers (1996) P. aeruginosa exhibited resistance to all three tested essential oils as well as towards the compounds tested (carvacrol, thymol, y-terpinene, p-cymene), although later findings of Dorman and Deans (2000) confirmed good inhibitory effects of O. vulgare essential oils and of carvacrol against this G(—) bacteria.

The essential oils of O. vulgare ssp. hirtum and O. dictamnus were extremely bactericidal (in S. aureus) at 1:4000 dilution, and even at dilutions as high as 1:50000 caused considerable decrease in bacterial growth rates. Essential oils of O. vulgare, rich in carvacrol (49.1 per cent) and of Thymus vulgaris L., rich in thymol (67.3 per cent), showed approximately the same range of antibacterial efficiency (MIC: 1:2000—1:3000) against E. coli, S. aureus, Bacillus megaterium, Salmonella hadar (Remmal et al., 1993). Dorman and Deans (2000) have studied the effects of essential oils of O. vulgare ssp. hirtum and of Thymus vulgaris toward a range of G( + ) and G( —) bacteria. When comparing the relative efficacy of the two species, they found that thyme was generally more effective against the majority of G( + ) bacteria (with the exception of Acinetobacter calcoacetica and Yersinia enterolitica) and against all G(_) bacteria (especially toward Alcaligenes faecalis, Flavobacterium suaveolens, Klebsiella pneumoniae, Proteus vulgaris, Salmonella pullorum and Serratia marcescens), but not toward Pseudomonas aeruginosa. This was more sensitive to essential oil of O. vulgare. The same results have been obtained by isolated phenolic compounds, showing a more pronounced effect of thymol against G( + ) bacteria (with the exception of Clostridium sporogenes, that was well inhibited by O. vulgare and carvacrol) or G(_) bacteria (with the exception of Pseudomonas aeruginosa). This might indicate that the relative position of the hydroxyl group in the phenolic structure might contribute to the antibacterial potency of essential oil components (Dorman and Deans, 2000).

While most of the reported studies on the antimicrobial activity of oregano involved pathogenic bacteria, only a limited number of authors studied the inhibitory effects of oregano on lactic bacteria. Authors generally agree that, like all susceptible bacteria, lactic bacteria are also inhibited by Origanum essential oil in a concentration-dependent manner. High sensitivity of Vibrio parahaemolyticus was observed in media containing oregano (Beuchat, 1976). Zaika and Kissinger (1981) have found that oregano extracts were bactericidal toward lactic acid bacteria (at 8 g/l against L. plantarum, 4 g/l against Pediococcus cerevisiae), but these organisms became resistant toward the toxic effects of oregano, when sublethal concentrations (3 g/l in L. plantarum, 2 g/l in P. cerevisiae) were applied to the starter culture of bacteria. Instead of inhibition, low concentrations of oregano in culture medium stimulated the growth and production of acid production in resistant bacteria. Moreover, a phenomenon of cross-resistance of bacteria against different herb species was observed (Zaika et al, 1983). This means that bacteria which had acquired a resistance to one herb species (oregano) were also resistant to other herbs (sage, rosemary, thyme). So far, the mechanism by which the starter cultures acquire their resistance is not known. Kivanij and co-workers (1991) studied the effects of Origanum onites leaves and essential oil on growth and acid production of L. plantarum and L. mesenteroides. They found that oregano leaves (0.5 per cent, 1.0 per cent or 2.0 per cent) had no significant influence on the growth of L. plantarum, but after 2 days of fermentation in vitro they stimulated its acid production. By contrast, the growth and acid production of L. plantarum were strongly inhibited by O. onites essential oil (150, 300 or 600 ppm). When considering Leuconostoc mesenteroides, both the leaves and the essential oil of O. onites at all tested concentrations inhibited the growth and acid production.

The stimulative effects of extracts of O. majorana on the growth of non-lactic acid bacteria have been reported by Adlova et al. (1998). They observed that the phenolic fraction of water extracts of O. majorana, present in media at low concentration (0.0001 per cent), exhibited a stimulating effect on E. coli and Streptococcus pyogenes, but had no influence on the growth of Corynebacterium xerosis. Vokou and Liotiri (1999) established that essential oil of O. vulgare L. ssp. hirtum, when added to soil samples (0.1 ml per 150 g of soil) of Mediterranean ecosystems, could be used by soil bacteria as a carbon and energy source, and that it stimulated soil microbial activity. Dose-dependent bactericidal (at mmol/l) or bacteriostatic (MIC = 0.75 mmol/l) effects of isolated carvacrol on the food-borne pathogen Bacillus cereus were detected in vitro (Ultee et al, 1998). The pH of the medium and the growth temperatures (8 °C or 30 °C) considerably influenced the bactericidal activity. Sensitivities recorded at pH 5.5 and 8.0 were two- and six-fold higher than those at pH 7.0, where the lowest sensitivity of B. cereus was detected. The study of the mechanism of action showed that the inhibitory effect of carvacrol was due to the interaction with membranes of B. cereus, changing their permeability for cations (K+, H+). The dissipation of the ion gradient influenced the membrane transport and led to impairment of cell essential processes and finally to cell death (Ultee et al., 1999). However, a considerable decrease in sensitivity of B. cereus against carvacrol was observed after growth in the presence of sublethal concentrations of carvacrol (0.4 mmol/l). Concomitantly, a lower membrane fluidity of adapted cells was detected, indicating the changes in the fatty acid composition and rearrangement of the phospholipid bilayer in bacterial cell membranes during adaptation process (Ultee et al., 2000). Pol and Smid (1999) report on the synergistic effects of carvacrol and nisin (bactericidal peptide, used as a biopreservative in certain foods) against Bacillus cereus and Listeria monocytogenes in vitro, which resulted in a much higher sensitivity of both pathogens (at 20 °C) to combined exposure (B. cereus: MICcombination = 0.63 mmol/l carvacrol and 1.25 pg/ml nisin, L. monocytogenes: MICcombination = 1.25 mmol/l carvacrol and 0.63 pg/ml nisin) than to individual compounds (B. cereus: MICcarvacrol = 1.25 mmol/l or MICnisin = 10 pg/ml, L. monocytogenes: MICcarvacrol = 2.50 mmol/l or MICnisin = 10 pg/ml). This means that lower concentrations of both carvacrol and nisin are needed for effective decrease in the number of colony-forming units of food-borne pathogens.

Oregano essential oils have been considered as an alternative natural additive in gastronomy and in the food processing industries. It was found that O. vulgare essential oil was effective in inactivation of E. coli in concentrations as low as 0.7 per cent, acting synergistically with the pH and storage temperatures, thus contributing to the intrinsic safety of home-made eggplant salad (Skandamis and Nychas, 2000). Also, Dorman and Deans (2000) believe that volatile oils (but not spices as integral ingredients) may have the greatest potential use as food preservatives. Due to their high antimicrobial potency they could be added to foodstuffs in small quantities and would cause no loss of organoleptic properties of the food. However, when assessing the potential use of essential oils in food and the food industries, one has to consider the whole system (food, essential oil, processing, storage temperatures, marketable/sensory characteristics of the processed food) to be able accurately to judge the usable value of plant essential oils/extracts. A considerable reduction in antimicrobial activity, when plant essential oils or extracts were evaluated in food systems/actual foods, was found by several authors (Shelef etal, 1984; Ismaiel and Pierson, 1990b; Hao et al, 1998). Similar observations were reported by Aureli et al. (1992), who found that the high antilisteric activities (4 strains of L. monocytogenes, one strain of Listeria innocua) of Origanum or Thymus essential oil (dilution 1:5 and 1:50 in absolute ethanol) in vitro were significantly reduced when essential oils were tested in a meat matrix (minced pork meat). Accordingly, the leaf extract of O. majorana exhibited no remarkable antilisteric effect, when assayed in cooked chicken breasts (Hao et al, 1998). By contrast, a significant inhibitory activity of 0.8 per cent (v/w) oregano essential oil against L. monocytogenes on naturally contaminated beef meat fillets was observed under various packaging conditions at 5 °C (Tsigarida etal., 2000). Based on the findings on the sporostatic and growth inhibiting activities of Origanum essential oil (at 150 and 200 ppm) against food contaminant Clostridium botulinum in vitro (TYG: thiotone yeast extract glucose medium) (Ismaiel and Pierson, 1990a), the same authors examined the potential antibotulinal effect of Origanum oil in minced pork. They observed considerable diminishing of antibotulinal activity of Origanum oil in the meat system. Origanum essential oil was effective only when used at 400 ppm and in combination with 50—100 ppm of sodium nitrite, depending on the spore inocula. The absence of inhibition by oregano oil in the meat system, in contrast to TYG medium, could be due to the high solubility of the oil components in the lipid fraction of the meat. Such concentrations (>400 ppm) of Origanum essential oil are questionable with regard to the possible effects on marketable characteristics of cured meat (flavour, colour, taste, and structure), although these were not assessed in the study of Ismaiel and Pierson (1990b). In view of practical implications in the preservation of food products, where the antimicrobial efficacy and sensory attributes of food have to be considered, the approach of combining different food-preservative compounds, proposed by Pol and Smid (1999), seems the most appropriate.

190 Dea Baricevic and Tomaz Bartol ANTIOXIDANT ACTIVITY

A large number of reports on the antioxidant effects of Origanum species have been published. A survey of the potential use of Origanum or oregano based preparations, that would replace synthetic substances such as BHT, as protectors of highly unsaturated lipids in foodstuffs has been made by numerous research groups. However, limited industrial applications are often ascribed to the characteristic oregano aroma and flavour, that influence the sensorial characteristics of processed food, so deodorization steps would be required (Nguyen et al., 1991; Moure et al., 2001). Dietary supplies of antioxidants from Origanum species were also considered as effective scavengers of the free radicals that are generated by metabolic pathways in the body, and in sufficient amounts prevent cellular damages and human diseases.

In these studies different methods, test/model systems (lard, bulk oils, emulsions, meat products, human cells as oxidation substrates), different plant preparations (whole spices, essential oils, hydrophilic or hydrophobic extracts, isolated phenolic compounds, etc.) have been used in the quantification of antioxidative potential (Madsen and Bertelsen, 1995; Madsen et al, 1997; Pearson et al, 1997; Moure et al, 2001). Comparison between the different reports, which is often difficult due to a high variability in experimental design, mostly refer to the different potency of antioxidant activity of tested Origanum species or their compounds. This is why several authors (Laughton et al., 1989; Frankel et al., 1994; Pearson et al, 1997) claim that a variety of testing systems is required when assessing the antioxidant potential of a substance, since a substance exhibiting high antioxidant activity in one system may have a prooxi-dant effect in another system. In the current literature, relatively little information is available on mechanisms of the antioxidative action, although phenolic compounds are most frequently cited as active ingredients, responsible for the antioxidant effect (Madsen etal, 1997; Moure et al, 2001). Comparison of the antioxidant activity of model phenolic compounds has shown that polymeric phenolic compounds are generally more potent antioxidants than simple monomeric phenolics (Moure et al, 2001). Yamaguchi et al. (1999) observed that the degree of polymerisation of flavanols correlates with the superoxide-scavenging capacity. The antioxidant activity relies also on the polarity of tested compounds, depending on the type and polarity of the extracting solvent. It was found that hydrophilic antioxidants are generally more effective in bulk oil, whereas lipophilic antioxidants exhibit more potent effects in emulsions ('polar paradox' phenomenon) (Moure et al, 2001).

Investigations on the antioxidative activity of herbs and spices date about 40 years ago, when Chipault with co-workers (Chipault etal., 1952; Chipault etal., 1955; Chipault etal., 1956) screened the effects of 32 different spices in various model systems/substrates, measuring their persistence with antioxidant index (AI). This was defined as a ratio between substrate, containing herb ingredient, and substrate without herb addition. In order to investigate the stabilising capacity of O. vulgare in different substrates, these were exposed to autooxidation at substrate-specific temperature regimes: lard (at 99 °C), egg yolk (at 63 °C), oil in water emulsion (at 40 °C), minced pork (at —5 °C) and mayonnaise (at 20 °C). In all tested substrates dry oregano (at concentration of 0.1 per cent in o/w emulsion, at 0.25 per cent in minced pork and at 0.2 per cent in all other substrates) displayed high antioxidant activity, the AI being between 2.7 (egg yolk) and 8.5 (mayonnaise). The length of the induction period in autooxidation of lard, which was used as an indicator of antioxidative potency, showed a higher antioxidant potency of dry oregano

(O. vulgare) when compared with that of marjoram (O. majorana), although Saito etal. (1976) had found marjoram to be a more potent antioxidant herb than oregano at the same concentration tested (Gerhardt and Schröter, 1983).

Methanol extracts of O. vulgare and of O. majorana exhibited strong hydroxyl radical-scavenging activity, inhibiting the oxidation of 2-deoxyribose by more than 50 per cent at the 1 pg/ml concentration, however, the scavenging effect was much less evident when the antioxidant potential was measured by benzoic acid hydroxylation method (Chung et al, 1997). By contrast with the high antioxidant activity of O. vulgare methanol extracts, as observed in the lard test system (Herrmann et al, 1981; Banias et al, 1992), oregano and marjoram extracts showed only scarce antioxidant effect in the ^-carotene model system (Dapkevicius etal., 1998).

Essential oil of O. vulgare showed the ability to form stable free radicals upon reaction with potassium superoxide (Deighton etal., 1993). The essential oil monophenols, car-vacrol and thymol, were identified as molecules which react with the superoxide anion (O2), probably through hydrogen atom donation, and form stable paramagnetic species (free radicals) as found by EPR spectroscopy.

Origanum vulgare essential oil (Italian origin) demonstrated protective antioxidant properties in an egg yolk assay. At high concentrations (1000 ppm and 750 ppm), the antioxi-dative potency of oregano oil was higher than those of butylated hydroxytoluene (BHT) or of «-tocopherol. However, in a rat liver assay the antioxidative effect of oregano essential oil was much lower, at 750 ppm exhibiting the same range of antioxidant activity as a-tocopherol at 250 ppm (Baratta et al, 1998a). Similar results were observed by Lagouri et al. (1993), who studied the antioxidative potency of O. vulgare L. ssp. hirtum Ietswaart and Origanum onites L. essential oils (at 1000 ppm) by measuring the autooxidation rate (peroxide value) of lard stored at 35 °C. The antioxidant activities of essential oils were attributed to their high phenol moiety (carvacrol and thymol) and were comparable to BHT (at 200 ppm). Still more potent activity was observed with O. majorana essential oil, the antioxidant activity being much higher than that of a-tocopherol and comparable to that of BHT at all concentration levels (100 ppm, 250 ppm, 500 ppm, 750 ppm, 1000 ppm) (Baratta etal., 1998b).

A survey of the antioxidant activity of methanol extracts of Origanum species of Greek origin showed that O. vulgare, O. dictamnus and O. majorana at concentration of 0.02 per cent significantly prolonged the induction period of lard autooxidation at 75 °C and slightly decreased the rate of peroxide formation. However, their relative antioxidant efficiencies (oregano > dittany > marjoram) in comparison to those of BHT or rosemary extracts were much lower (Economou et al, 1991).

Turkish oregano (O. vulgare L.) and Chilean oregano (Origanum onites L.) showed high antioxidative effects, measured by the oxygen depletion method as well as by EPR spectroscopy. The study of oregano water extracts, using both methods, allowed Madsen and co-workers (1996) to find that the antioxidant activity of oregano was due to at least two different antioxidative mechanisms. The activity might be due both to the non-phenolic group of compounds and to the phenolic group. The group of non-phenolic compounds act as scavengers of free radicals and are effective in early stages of oxidation. The group of phenolic compounds were effective in interrupting the chain processes responsible for oxygen consumption by a mechanism similar to that for toco-pherols. Among the phenolic compounds that have been isolated from oregano, there were isolated at least five different groups of substances which were highly antioxida-tive active (rosmarinic acid, water soluble phenolic glycosides, flavonoids, carvacrol and thymol) (Madsen and Bertelsen, 1995). The ESR spin-trapping assay showed that the free radical scavenging capacity of the O. dictamnus water and methanol extracts correlated with the content of phenolics. When compared to acetone or ethanol extracts (poor in phenolics), the aqueous and methanol extracts (rich in phenolic compounds) were also the most effective in reducing oxygen consumption and thus had high chain-breaking properties, as evidenced by the oxygen depletion assay (M0ller et al., 1999). The ability of aqueous extracts of O. dictamnus to inhibit development of secondary lipid oxidation products was also confirmed in model food systems (turkey thigh meat homogenate), where dittany dose-dependently (from 0.0018 mg dittany/g meat upwards) inhibited development of thiobarbituric reactive substances.

Vekiari et al. (1993a) have studied the extracts of O. vulgare of different polarity, to find the active compounds responsible for the antioxidative effect of oregano. The main antioxidant factor of the non-polar hexane extract was isolated by repeated fraction-ations, and consisted mainly of terpene derivatives. Among polar compounds that were extracted from O. vulgare leaves, the most effective in stabilising lard against oxidation, with potency equal to BHT, were flavonoids (flavanone eriodictyol, the dihydroflavonols dihydrokaempferol and dihydroquercetin and flavone apigenine) (Vekiari et al, 1993b). These compounds also showed marked antioxidant activity when tested on vegetable oils (corn, soybean and olive) under storage or frying conditions.

Nakatani (1997) reports that both the polar and non-polar fractions of oregano leaves significantly retarded oxidation of linoleic acid, measured by the ferric thiocianate (FTC) and thiobarbituric acid (TBA) methods. From a water-soluble fraction of methanol extracts, phenolic compounds with high antioxidant activities have been purified, the most potent being derivative of rosmarinic acid (2-caf-feoyloxy-3-[2-(4-hydroxybenzyl)-4,5-dihydroxy] phenylpropionic acid and a new glycoside of protocatechuic acid ester, identified as 4-(3,4-dihydroxybenzoyl-oxymethyl)phenyl-^-D-glucopyranoside (Nakatani and Kikuzaki, 1987; Kikuzaki and Nakatani, 1989; Nakatani, 1992). These were more effective against linoleic acid oxidation than the natural a-tocopherol, and are comparable to the synthetic antioxidants, BHA (butylated hydroxyanisole) or BHT (butylated hydroxytoluene). Similar polyphenolic compounds were found in O. majorana leaves, but these also contain compounds such as 6-O-4-hydoxybenzoyl arbutin and 2-hydroxy-3-(3,4-dihydr-oxyphenyl)propionic acid, which possess moderate antioxidant activity (Nakatani, 1997). These findings are in agreement with Herrmann (1994), who observed that antioxidant active plant phenols often possess a 3,4-dihydroxybenzoyl- or 3-methoxy-4-hydroxybenzoyl group in their structure. Lamaison and co-workers (Lamaison et al, 1990; Lamaison etal, 1991; Lamaison etal, 1993), who extensively studied the antioxidant activity of members of Lamiaceae, reported that the content of rosmarinic acid and of total hydroxycinnamic derivatives in hydroalcoholic extracts of Origanum taxa (O. onites, O. tytthantum, O. vulgare ssp. hirtum) was only partly correlated with their antioxidant effect, estimated by measuring the free radical scavenger effect on DPPH (1,1-diphenyl-2-picrylhydrazyl). They stressed the importance of flavonoid content for oregano antioxidant activity.

The antioxidant activity of isolated carvacrol and thymol in liposomal systems was confirmed by Aeschbach et al. (1994), and in biological systems (human aortic endothelial cells, HAEC) by Pearson et al. (1997). The potency of antioxidant activity of thymol (ID50 = 4.02 pM), measured as per cent of inhibition of HAEC-mediated human LDL oxidation, was significantly higher than that of carvacrol (ID50 = 5.53 pM), but it was found that both monophenols (thymol or carvacrol) had much lower antioxidant activities than rosmarinic acid (ID50 = 0.74 pM) (Pearson et al, 1997).

It was also found that combinations of spices or compounds with high antioxidant activities exhibited synergistic antioxidant effects, which would practically result in a better protection of foods from oxidation (Madsen et al., 1996). However, no practically important synergistic effects were observed, when O. vulgare methanol extracts — which were highly effective in stabilising lard (at concentration 0.02 per cent) stored at 75 °C — were mixed in lard with less potent antioxidants (methanol extracts of thyme, marjoram, spearmint, basil) (Economou et al, 1991).

In the study of Banias etal. (1992), combinations of methanol extracts of oregano, dittany or marjoram with primary antioxidants were used in the lard autooxidation process. The results showed that significant positive synergism in antioxidant activity existed in combinations of oregano or marjoram (0.1 per cent) with BHT (0.005 per cent) and in a combination of dittany (0.1 per cent) with ascorbyl palmitate (0.01 per cent). By contrast, high negative synergism was observed in combinations of oregano (0.1 per cent) or marjoram (0.1 per cent) with propyl gallate (PG) (0.01 per cent) and in a combination of oregano (0.1 per cent) or marjoram (0.1 per cent) with a-toco-pherol. Milos et al. (2000) have studied the antioxidant activity of volatile aglycons, that are bound glycosidically in dry O. vulgare plants, in comparison to that of essential oil. Although, the total content of volatile aglycons in plant material (0.002 per cent) was significantly lower than the content of essential oil (2.9 per cent), a mixture of volatile aglycons (thymoquinone, benzyl alcohol, eugenol, thymol, carvacrol) showed similar antioxidant activity to that of essential oil. They inhibited hydroperoxide formation in lard stored at 60 °C even after 80 days and were significantly more effective than a-tocopherol. Thymoquinone, which was found to be a potent inhibitor of membrane lipid peroxidation (Houghton et al, 1995; Jerkovic et al, 2001) and the major component (40.2 per cent) among aglycons (Milos et al., 2000), as well as pure thymol as the major component (40.4 per cent) of essential oil, were much less active than a mixture of aglycons or essential oil of O. vulgare. These results indicated the importance of mixtures and their synergistic power in the antioxidant activity of O. vulgare.

In addition to numerous studies, where potent or moderate antioxidant effects of oregano were established in theoretic model systems, practical considerations on the use of oregano as stabilisers of edible oils (vegetable or fish oils) or of finished meat products have been made by several research groups. Generally, authors confirm the protective role of different Origanum taxa (O. vulgare, O. compactum, O. majorana) against the autooxidation process over time, although the potencies of oregano antioxidative effects are lower than those reported for rosemary or sage (Ozcan and Akgül, 1995; Antoun and Tsimidou, 1997). Ozcan and Akgül (1995) studied the antioxidant effects of methanol extracts and essential oils of numerous Turkish spices on sunflower oil, stored at 70 °C, and found that methanol extracts (including O. vulgare and O. majorana) exhibited higher antioxidant activity compared with essential oils. The increased delay in the onset of autooxidation might be due to the improved preservation of a-tocopherol, an internal antioxidative microcomponent principle of sunflower oil (Yanishlieva and Marinova, 1998; Beddows etal, 2000). When compared to hexane and ethyl acetate extracts, the ethanol extracts of several species of the Lamiaceae family were the most active in retarding the autooxidation process of sunflower oil exposed to 100 °C. However, O. vulgare ethanol extracts (at 0.08 per cent) showed only low antioxidant effect comparable to that of 0.02 per cent BHT (Yanishlieva and Marinova,

1995), or else did not improve the oxidation stability of sunflower oil, as is evident from the later study by Marinova and Yanishlieva (1997). Only a moderate stabilising effect of O. vulgare leaves in sunflower oil, exposed to autooxidation at room temperature, was observed also by de Felice et al. (1993), who measured the quality characteristics of the oil in the time period of 16 weeks.

By contrast, ground oregano (O. vulgare) inhibited lipid oxidation of fish/mackerel (Scomber scombrus) oil stored at 40° in dark at concentrations of 0.5 per cent and at 1 per cent as effectively as 200 ppm BHA and 200 ppm TBHQ (tertiary butylhydroquinone), respectively (Tsimidou et al, 1995). Dry leaves of O. vulgare ssp. hirtum (at concentration of 2 per cent) or essential oil of O. compactum (at concentrations 0.05 per cent and 0.1 per cent) also showed a high antioxidant activity in olive oil and, besides their stabilising effect, the organoleptic quality of the olive oil was significantly improved by addition of oregano, as assessed by Mediterranean consumer acceptability studies (Antoun and Tsimidou, 1997; Charai etal, 1999). A significant increase in the oxidative stability of fried chips, measured as the rate of peroxide formation during storage at 63 °C, was achieved both by addition of ground O. vulgare (1 per cent, after frying) or its petroleum ether extracts (1.1 per cent, before frying) (Lolos et al, 1999). The oregano antioxidant activity was almost as effective as that of TBHQ up to 6 days of observation, although the peroxide value of cottonseed oil, extracted from oregano-treated potato chips increased after one week. However, the results of this study indicated that ground oregano or its extract might be used to extend the storage life of potato chips as they decrease the oxidative deterioration of the oil absorbed into the chips.

The importance of the testing substrate (bulk oil or o/w emulsion) in the evaluation of oregano antioxidant potency has been shown in the study of Abdalla and Roozen (1999), who found that acetone extract of O. vulgare effectively inhibited the autooxidation process of sunflower oil at both 600 ppm and 1200 ppm, but exhibited only moderate antioxidant activity when tested in a 20 per cent sunflower o/w emulsion. It has been also shown that oregano extracts acted as pro-oxidants in both oil and emulsions, when exposed to light (Abdalla et al., 1999).

When assessing the antioxidative potential of O. majorana or O. vulgare in preventing rancidity of meat products, only limited practical significance has been documented. El-Alim et al. (1999) report that O. majorana and O. vulgare, and especially their ethanol extracts, had a strong antioxidant activity, inhibiting lipid peroxidation both in fresh chicken meat as well as in heat-treated pork. Because of their shelf time prolonging properties, oregano and oregano- based preparations have been recommended for use in semi-prepared meat products. However, other studies show less optimistic results. Ground O. majorana was added at a concentration of 0.2 per cent to the laboratory and industrial prepared sausage model systems, that were exposed to ripening. Only a low antioxidative effect was observed on the basis of the redox potential reduction of the marjoram-supplemented model compared to the control (Palic et al, 1993). Korczak and co-workers (1988) have found relative low antioxidant efficacy of O. majorana (at 0.5 per cent) in minced meat model systems when compared to those of rosemary or sage. Hence, the pro-oxidising activity of marjoram, which is probably influenced by elevated temperature, diminishes its practical value as a natural additive in meat processing (Korczak et al, 1988).

The antioxidative effects of O. vulgare drug plant (Origani herba) have been studied in the light of both direct use as stabilisers of fat and, indirectly, as feed additives in order to improve the shelf-life of meat and fat-containing food (Vichi et al, 2001).

In contrast with the significant antioxidative and stabilising effects of oregano extracts in lard (measured by photochemiluminescence), no effect on the quality or shelf life of the fat obtained from animals fed with oregano additives was observed.

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