Essential Oils in Combination and Their Antimicrobial Properties

Essential oils (EOs) have been long recognized for their antibacterial, antifungal, antiviral, insecticidal and antioxidant properties. They are widely used in medicine and the food industry for these purposes. The increased interest in alternative natural substances is driving the research community to find new uses and applications of these substances. EOs and their components show promising activities against many food-borne pathogens and spoilage microorganisms when tested in vitro. In food systems, higher concentrations of EOs are needed to exert similar antibacterial effects as those obtained in in vitro assays. The use of combinations of EOs and their isolated components are thus new approaches to increase the efficacy of EOs in foods, taking advantage of their synergistic and additive effects. The purpose of this review is to provide an overview on the antimicrobial efficacy of these combinations. A survey of the methods used for the determination of the interactions and mechanisms involved in the antimicrobial activities of these combinations are also reported.

determine the type of interactions and the mechanisms involved in the antimicrobial activities of these combinations.

Interaction between Components of Essential Oils
The antimicrobial properties of EOs have been reported in several studies [6,11,21]. In many cases the activity results from the complex interaction between the different classes of compounds such as phenols, aldehydes, ketones, alcohols, esters, ethers or hydrocarbons found in EOs [11,22,23]. Though in some cases, the bioactivities of EOs are closely related with the activity of the main components of the oils [24]. Several studies have found that a number of these compounds exhibited significant antimicrobial properties when tested separately [21,[25][26][27][28][29][30].
Different terpenoid components of EOs can interact to either reduce or increase antimicrobial efficacy [13]. The interaction between EO compounds can produce four possible types of effects: indifferent, additive, antagonistic, or synergistic effects [11,[52][53][54]. An additive effect is observed when the combined effect is equal to the sum of the individual effects. Antagonism is observed when the effect of one or both compounds is less when they are applied together than when individually applied. Synergism is observed when the effect of the combined substances is greater than the sum of the individual effects [11] while the absence of interaction is defined as indifference.
Interestingly, phenolic monoterpenes and phenylpropanoids (typically showing strong antimicrobial activities) in combination with other components were found to increase the bioactivities of these mixtures. Most of the studies have focused on the interaction of phenolic monoterpenes (thymol, carvacrol) and phenylpropanoids (eugenol) with other groups of components, particularly with other phenols, phenylpropanoids and monoterpenes alcohols, while monoterpenes and sesquiterpenes hydrocarbons were used to a lesser extent ( Table 1). The combination of phenolics with monoterpenes alcohols produced synergistic effects on several microorganisms, in particular, the combination of phenolics (thymol with carvacrol, and both components with eugenol) were synergistically active against E. coli strains. Though other reports have observed additive [23] and antagonism effects [55] ( Table 1).   Bassole et al. [66] Mixtures of cinnamaldehyde with carvacrol or thymol yielded in most cases synergistic effects against E. coli and S. typhinurium, though in one case an additive effect was observed (Table 1). Other monoterpenes have also been tested, particularly the oxide 1,8-cineole that in combination with sesquiterpene and monoterpene hydrocarbons (e.g., aromadendrene and limonene) were found to have additive and synergistic effects, respectively. Other combinations including a monoterpene hydrocarbon (α-pinene) with limonene or linalool also showed additive and synergistic effects ( Table 1).
Mixture of EOs have also been shown to interact with each other acting as additive, synergistic and in a few cases antagonistic agents ( Table 1). The essential oil of oregano (Origanum vulgare) was the most used EO (rich in thymol and carvacrol) and combined with rosemary (Rosmarinus officinalis), thyme (Thymus vulgaris), basil (Ocimum basilicum), marjoram (O. majorana) and lemon balm (Melissa officinalis) ( Table 1). In most cases only additive effects were observed, only the combination with rosemary oil yielded synergistic effects (Table 1).
Most studies attributed additive and synergism effects to phenolic and alcohol compounds ( Table 1). Generally compounds with similar structures exhibit additive rather than synergistic effect. The occurrence of additive interaction of some essential oils has been related to their main phenolic compounds (carvacrol and thymol) [21,23,63]. Antagonistic effect has been attributed to the interaction between non-oxygenated and oxygenated monoterpene hydrocarbons [25,64].

Interaction Test Methods
The assessment of the interaction between essential oil components is based on using macro-or micro-dilution techniques. Checkerboard, Graphical and Time-kill methods are the most widely used procedures. The principles and practice of these methods are described in the literature. These methods were preliminary developed for the detection of drug synergism thus there is no standardized method developed for evaluating the interaction between essential oils or their components [67][68][69].
The checkerboard test requires determination of the fractional inhibitory concentration (FIC) or the effect of the combination index (EC index) of each agent [54,57,[70][71][72]. The FIC of a factor is the concentration that kills when used in combination with another agent divided by the concentration that has the same effect when used alone [73,74]. Generally, minimum inhibitory concentrations (MICs) or concentrations of maximal inhibition (C max ) are used as the reference concentrations [13,30,55,60,62,64]. The FIC index for the combination of A and B is the sum of their individual FIC values. Each checkerboard test generates many different combinations, and by convention, the FIC values of the most effective combination are used in calculating the FIC index. The FIC index defines the nature of the interaction. The values of the FIC index used for the definition of the nature of the interaction differs between publications and makes comparison between studies difficult ( Table 2). The definition of the reference concentration differs between publications and this is another obstacle at the time of comparing different research studies [11,54,64]. The effect of the combination index (EC index) is the absolute value of the difference between logarithms of the difference in population (DP) in the combination system and the single agent, respectively [54,57]. This index is used to determine synergistic effect of a combination on the basis on three principles: (1) The decrease in populations (DP > 90%): linked with the definition of DP, it was concluded that only when DP < 0.1 (log DP < −1) that the combinations of various reagents had significant antibacterial activity. (2) When there was significant difference (ANOVA) between the antibacterial activity of the combination and the individual components, respectively, it meant that the combination was effective. Some authors have mentioned that the results of the checkerboard assay can be represented graphically by plotting the FIC values on a graph known as an isobologram [73,74]. On the x-and y-intercepts the half maximal effective concentration (EC 50 ) are plotted or MIC values of the two agents when used alone [80]. Additive effects will produce straight lines while synergy will produce a concave curve and antagonistic effects a convex one [80].
The isobolograms of Salvia chamelaeagnea L. and Leonotis leonurus L. at various ratios against four pathogens showed that synergistic interactions were obtained against Gram-positive bacteria for nearly all ratios, while mostly antagonistic or additive interactions were observed with Gram-negative bacteria [81]. Delaquis et al. [13] defined synergistic effects of mixed fractions of dill, cilantro, coriander, and eucalyptus EOs when the isobologram showed concave shape.
The time-kill method evaluates combined antimicrobial action by measuring the effect of a subinhibitory concentration of one agent on the killing ability of another over time [74]. A synergistic interaction is observed when the killing ability of the first agent is increased by a sub-inhibitory concentration of the second agent. An antagonistic interaction is present if the antimicrobial effect of the first component is inhibited by the second. Because one agent is used at sub-inhibitory concentrations, this assay cannot distinguish additive interactions (combined activity equals the sum of individual activities) from indifferent interactions [80]. Mulyaningsih et al. [60] reported additive and synergistic effects of the combinations of 1,8-cineole and aromadendrene against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) and Enterococcus faecalis by using checkerboard and time-kill assays respectively. In addition to these methods, others have been reported. Fyfe et al. [82] considered the combined effects of plant volatile oils and benzoic acid derivatives against L. monocytogenes and S. enteritidis as synergistic when the combined components demonstrated ≥log10 higher inhibition than the sum of the inhibitory effects of the components used alone. Fu et al. [42] observed increased antifungal effects caused by combinations (1:5, 1:7 and 1:9) of essential oils of S. aromaticum (clove) and R. officinalis against C. albicans. Lambert et al. [23] reported that carvacrol and thymol in combination showed additive effects against S. aureus and P. aeruginosa by using half-fold dilutions within the Bioscreen plate.  Bassole et al. [66] However, due to the diversity of the methods, there is no an effective and standardized way to evaluate and quantify the synergistic effects of EOs combination, making it impossible to compare the results of the reports. Besides, the concentrations or ratios of the mixtures are not always provided by the authors so comparisons are not always possible (Tables 1 and 3).

Mechanism of Action
There are fewer reports on the mechanisms of action of combination of essential oils or their purified components on microorganisms [11,[83][84][85][86]. Some publications deal with the mode of action of the essential oil components in combination with other natural preservatives or antibiotics [58,[87][88][89][90][91][92][93][94][95][96][97][98][99][100]. There are limited numbers of papers dealing with the mechanism of action of combinations of the essential oils or their components. However, there are some generally accepted mechanisms of antimicrobial interaction that produce synergism. They include the sequential inhibition of a common biochemical pathway, inhibition of protective enzymes and use of cell wall active agents to enhance the uptake of other antimicrobials [19]. Synergism between carvacrol and some hydrocarbons monoterpenes (such as α-pinene, camphene, myrcene, α-terpinene and p-cymene) that typically showed low antimicrobial properties has been observed [58,63]. The capacity of hydrocarbons to interact with cell membrane facilitates the penetration of carvacrol into the cell [58,63,101]. Pei et al. [54] hypothesized that the synergistic effects of eugenol/carvacrol and eugenol/thymol might be due to the fact that carvacrol and thymol disintegrated the outer membrane of E. coli, making it easier for eugenol to enter the cytoplasm and combine with proteins. It was also observed the synergistic effect of eugenol/cinnamaldehyde is probable due to the interaction of these components with different proteins or enzymes.
The combination of pair of components showing synergistic effects will then reduce the concentration needed to yield the same microbial effect when compared with the sum of the purified components. Thus, the synergistic effects of cinnamaldehyde and thymol against E. coli had an effective reduction of concentration of 25% (Table 3), similar reduction at the same ratio was observed for T. typhinurium. The combination of cinnamaldehyde and reduced levels of eugenol generated a 50% reduction of the concentration. In the case of cinnamaldehyde and thymol the working ratio was of 1:1, while in the case of cinnamaldehyde and eugenol, lower levels of eugenol (1:4-1:8) were needed to reduce the concentration. Thymol and carvacrol at ratio of 1 to 1 also showed similar results (reduction of 25%), the use of thymo and eugenol at 1 to 4 further reduced the concentration to 50%. In the pair carvacrol/eugenol, the same ratios of 1 to 4 showed a reduction of 25%.
Zhou et al. [76] proposed two hypotheses to explain synergistic effects of cinnamaldehyde/thymol or cinnamaldehyde/carvacrol against S. typhimurium: -Thymol or carvacrol could increase the permeability of the cytoplasmic membrane, and probably enable cinnamaldehyde to be more easily transported into the cell. -Thymol or carvacrol could increase the number, size or duration of existence of the pores created by the binding of cinnamaldehyde to proteins in the cell membrane, so that a synergistic effect is achieved when these two components are used in combination.
These authors proposed three hypothesis that could explain the synergistic effect between thymol/carvacrol against S. typhimurium: (a) the antibacterial mechanism of thymol and carvacrol might be different; they act on the different targets of S. typhimurium; (b) the synergistic effect could be due to the similarity of their mechanism; and (c) the synergistic effect occurs only when they inhibit together S. typhimurium. More recently, Fei et al. [102] showed that the synergistic combinations of EOs of oregano/basil against E. coli, basil/bergamot against S. aureus, oregano/bergamot against B. subtilis and oregano/perilla against S. cerevisiae significantly disrupted the integrity of cell membranes when compared with control untreated membranes.
The practical implications of these observations are important at the time of using EOs components in food systems since the use of the lower concentration needed to yield a similar antibacterial activity will mean reduced flavor notes in foods products (Table 3). For certain foods, some EO components in high concentrations can impart undesirable notes to foods (e.g., eugenol).
Mechanisms of interaction that produced antagonistic effects were less studied. Some of the studies included combinations of bactericidal and bacteriostatic agents, use of compounds that act on the same target of the microorganism and chemical (direct or indirect) interactions among compounds such as the reduction of the active aqueous terpene solubility by non-aqueous monoterpene hydrocarbons [26,64].

Other Factors Affecting the Interaction of Components
There are limited number of studies on the effects of the test medium physical and chemical parameters on the interaction between essential oil components and their antimicrobial activities. Physical (temperature) and chemical (sodium chloride) parameters were also found to modulate the antimicrobial responses of the mixtures. Sodium chloride was found to have antagonistic effects when combined with carvacrol and p-cymene against B. cereus. It was also observed that carvacrol and p-cymene worked synergistically, but this effect was reduced when sodium chloride was added (1.25 g/L) [58]. It has been reported that the combination of cinnamon and clove EOs showed better antimicrobial activity in vapor phase than in liquid phase [64]. In the study of the combined effects of thymol, carvacrol and temperature on the quality of non conventional poultry patties by using a simplex centroid mixture design, the best effects were obtained when the patties were mixed with both compounds and stored at low temperature 0 to 3 °C [103].

Conclusions
Due to the limited number of studies and in order to optimize the synergy potential of mixtures, research should focus on: (a) the effects of intrinsic and extrinsic parameters of test medium (pH, fat, protein, water content, incubation time/temperature, packaging procedure, and physical structure) on the combinations of essential oils or their components and their antimicrobial properties; (b) the mechanism of action of the synergisms, additions or antagonisms to optimize the activity in food preservation, medicine and cosmetic; (c) possible toxicity of combined essential oils or components; (d) development of standardized methods for the evaluation of the interaction between essential oils or their components.
Essential oils are natural plant products containing complex mixture of components and thus having multiple antimicrobial properties. Most of the antimicrobial activity in EOs appears to derive from oxygenated terpenoids, particularly phenolic terpenes, phenylpropanoids and alcohols. Other constituents (e.g., hydrocarbons) that tipically showed low activities can be used in combinations to increase their bioactivities. Interactions between these components may lead to antagonistic, additive or synergistic effects. Checkerboard, graphical and Time-kill methods are the most widely used procedures to assess of the interaction of essential oil components. Investigations should be carried out on their mode of action and their probable toxicological effects in order to optimize their use.