Chemistry and Multibeneficial Bioactivities of Carvacrol (4-Isopropyl-2

Review pubs.acs.org/JAFC

Chemistry and Multibeneficial Bioactivities of Carvacrol (4-Isopropyl2-methylphenol), a Component of Essential Oils Produced by Aromatic Plants and Spices Mendel Friedman Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710, United States ABSTRACT: Aromatic plants produce organic compounds that may be involved in the defense of plants against phytopathogenic insects, bacteria, fungi, and viruses. One of these compounds, called carvacrol, which is found in high concentrations in essential oils such as oregano, has been reported to exhibit numerous bioactivities in cells and animals. This integrated overview surveys and interprets our present knowledge of the chemistry and analysis of carvacrol and its beneficial bioactivities. These activities include its antioxidative properties in food (e.g., lard, sunflower oil) and in vivo and the inhibition of foodborne and human antibiotic-susceptible and antibiotic-resistant pathogenic bacteria, viruses, pathogenic fungi and parasites, and insects in vitro and in human foods (e.g., apple juice, eggs, leafy greens, meat and poultry products, milk, oysters) and food animal feeds and wastes. Also covered are inhibitions of microbial and fungal toxin production and the anti-inflammatory, analgesic, antiarthritic, antiallergic, anticarcinogenic, antidiabetic, cardioprotective, gastroprotective, hepatoprotective, and neuroprotective properties of carvacrol as well as metabolic, synergistic, and mechanistic aspects. Areas for future research are also suggested. The collated information and suggested research might contribute to a better understanding of agronomical, biosynthetic, chemical, physiological, and cellular mechanisms of the described health-promoting effects of carvacrol, and facilitate and guide further studies needed to optimize the use of carvacrol as a multifunctional food in pure and encapsulated forms, in edible antimicrobial films, and in combination with plant-derived and medical antibiotics to help prevent or treat animal and human diseases. KEYWORDS: carvacrol, biosynthesis, chemistry, analysis, antioxidative effects, anti-inflammatory effects, antimicrobial activities, antiviral effects, antifungal properties, antibiotic resistance, health-promoting potential, mechanisms, food and feed safety, food animals, animal waste odors, human health, research needs

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INTRODUCTION In 2002, we published a quantitative study on the relative bactericidal activities (inhibitory potencies) of 96 plant essential oils and 23 pure compounds against four foodborne pathogenic bacteria.1 We found that, compared to other evaluated formulations, oregano oil and its major constituent carvacrol seemed to exhibit exceptionally high antimicrobial properties under the test conditions. This study seems to have stimulated worldwide interest in evaluating the most active compounds in laboratory media and in different food categories, as indicated by more than 425 citations in the Scopus database. Because carvacrol exhibits strong antioxidative properties as well as both hydrophobic properties associated with the substituted aromatic ring and hydrophilic properties associated with the phenolic OH group, numerous studies report on its antioxidative, anti-inflammatory, antibacterial, antiviral, antifungal, antiprotozoal, anticarcinogenic, antidiabetic, antinociceptive, cardioprotective, and neuroprotective properties, as indicated by more than 2,500 citations in the Scopus database that included reviews.2−4 The general objective of this review is to integrate and interpret the widely scattered literature on the beneficial properties of carvacrol in cells, animals, and humans. Possible mechanisms of the beneficial effects are discussed, as is the potential use of carvacrol to protect human foods and animal feeds against bacteria, viruses, fungi, and parasitic organisms. © 2014 American Chemical Society

The potential value of carvacrol to prevent and treat major diseases, and areas for the future, is also covered. The aim is to facilitate progress in fundamental and applied research by bringing together viewpoints and expertise from different research areas. An objective is to increase the awareness of scientists from related disciplines of one another’s results; results from practical applications need to be shared along with the problems and experiences of basic studies. For example, food microbiologists need to be aware that carvacrol-containing foods not only have the potential to inhibit the growth of bacteria but also might concurrently exhibit numerous other beneficial bioactive effects after consumption. We hope and expect everyone to profit from a broader overview. Table 1 lists the carvacrol-induced bioactivities discussed in the text.

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CHEMISTRY Biosynthesis. The biosynthesis of carvacrol has been studied. Figure 1 illustrates a general biosynthetic scheme for enzyme-catalyzed formation of carvacrol and thymol in green aromatic plants via the mevalonate pathway.5−8 Briefly, the

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the individual transformations are given in the cited references. The discovery of genes that govern the biosynthesis of carvacrol and other monoterpenes via the γ-terpinene intermediate is currently an active area of research.9 Ramak et al.10 discovered that inhibition of the mevalonate pathway enhances the biosynthesis of carvacrol in shoot cultures of the Iranian plant Satureja khuzistanica. They found that expression of the methylerythritol-4-phosphate (MEP) pathway provides the precursor isopentenyl diphosphate (IPP) for the multistep biosynthetic transformation to carvacrol with a yield of ∼90%. The authors suggest that a better understanding of the regulation of carvacrol biosynthesis could result in an increased yield of carvacrol in this and other plants. Carvacrol is present in large amounts in numerous aromatic essential oil producing plants.11−17 Although the growing season seems to influence yields, chemical composition, and antioxidant properties of Turkish oregano (Origanum onites L.) plants, the carvacrol content of the oils from plants harvested in four different seasons was >80%.18 An investigation of the biotransformation of carvacrol in cultured plant cells of Eucalyptus perriniana showed that the monoterpene is transformed to glycoside derivatives that are accumulated in the cells.19 It would be of interest to determine antimicrobial and other bioactivities of the carvacrol glycosides, which are probably more soluble than carvacrol in water and body fluids. Carvacrol is not only produced by plants, but its presence in the soil seems to act as a selective agent on different plant species, with the response ranging from negative to neutral to positive, suggesting that carvacrol and other thyme monoterpenes can influence plant−plant interactions that might affect the dynamics and stability of plants.20 Physical Properties. Carvacrol (liquid; mol wt, 150.22; boiling point, 237−238 °C; density, 0.967 mL/g; melting point, ∼0 °C; UV max in 95% ethanol, 277.5 nm; log extinction coefficient ε, 3.262; pKa phenolic OH group, ∼10.9; practically insoluble in water; soluble in ethanol; oral rat LD50, 810 mg/ kg; oral rabbit LD50, 100 mg/kg) is a major constituent of oregano oil, widely used as a salad dressing, and numerous other essential oils with concentrations up to ∼86%.21−25 Carvacrol has a pleasant tangy taste and smells like marjoram.10 Carvacrol is considered to be a generally accepted-as-safe (GRAS) compound used commercially as a food flavor.26 Standard pure carvacrol for analysis characterized by quantitative NMR (qNMR) is available from a commercial source.27 Carvacrol is included in the list of chemical flavorings by the European Commission28 and approved by the Food and Drug Administration for use as a flavoring in food.29 Synthesis. Yadav and Kamble,30 describe a kinetically controlled synthesis of carvacrol with an 82% yield without the use of solvent (green chemistry) that involves a one-step alkylation of ortho-cresol with propylene or isopropyl alcohol over solid acid catalysts. Synthetic carvacrol may be less expensive than the plant-derived natural form. Several studies report on the synthesis and bioactivities of carvacrol derivatives. These include the following: (a) antioxidative and antimicrobial hydroxymethyl carvacrols;31 (b) the anti-inflammatory and antinociceptive carvacryl acetate formed by esterification of the carvacrol OH group;32 (c) other antimicrobial carvacryl esters formed by esterification of the phenolic OH group with different organic acids;33 (d)

Table 1. Carvacrol Bioactivities Described in the Text Listed Alphabetically Bioactivity acaricidal analgesic antiallergic antiarthritic antibiotic, animal feed antibiotic, animal waste antibiotic, food bacteria antibiotic, human bacteria antibiotic, resistant bacteria anticarcinogenic anticholesterol antidiabetic antiendotoxemic antifungal anti-inflammatory antileishmanial antimicrobial films antioxidant antitoxins antiviral cardioprotective gastroprotective hepatoprotective insecticidal nematocidal neuroprotective trypanocidal

References 183, 184 2, 32, 202, 206, 209 59 213 59, 148, 150−152 156−158 55, 63, 64, 70, 71, 78, 79, 96, 102, 103, 150, 152, 257, 258 37, 60, 78, 94, 130, 141 90, 91, 93−95, 99, 100, 103 4, 7, 31, 37, 215, 216, 223−225, 227−229 147, 148, 159, 231, 237 237, 238, 250 240, 241 4, 11, 14, 34, 112, 164, 165, 167−172, 176, 215 32, 198−202, 208, 213, 239, 245 185, 186, 189 45, 55, 70−74, 100, 139 13, 16, 18, 31, 35, 46, 50, 51, 53, 54, 56−58, 69, 85, 86, 138, 139, 159, 181, 195, 248 79, 167, 174−176, 257, 258 11, 55, 103, 141, 177−182, 257, 258 229, 232, 234−236 2, 243 2, 4, 159, 194, 200, 213, 217−219, 221, 224, 228, 231, 245−248, 258 34, 160−163 187 204, 227, 250, 251, 255, 256 188, 189

Figure 1. Biosynthesis of carvacrol in aromatic plants. Adapted from refs 5−7.

major steps involve cleavage of glucose to phosphoenolpyruvate followed by decarboxylation and acetylation to acetyl coenzyme A (acetyl CoA) and transformation to the key intermediate, mevalonic acid. The latter is then transformed to γ-terpinene, which then undergoes aromatization to p-cymene and hydroxylation to carvacrol and thymol. Not all intermediates are shown in Figure 1. The enzymes and cofactors that catalyze 7653

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antimicrobial apple films using an HPLC system that consisted of a Beckman 110B pump, a Thermo Separation Products AS3500 Autosampler, a UV 3000HR scanning detector with both deuterium and tungsten lamps, and an eluent consisting of 50% acetonitrile, 50 mM ammonium phosphate, and 0.05% phosphoric acid at pH 3.1 (Figure 2). Hadad et al.46 measured

antifungal and insecticidal hybrid molecules of carvacrol and benzoyl urea/thiourea prepared by structurally modifying carvacrol with benzoyl urea;34 (e) Schiff’s bases with high antioxidant activity formed by introduction of an NH2 group into the benzene ring of carvacrol followed by reaction with structurally different aldehydes;35 and (f) antimicrobial chlorocarvacrol formed by chloroperoxidase-catalyzed halogenation of carvacrol.36 The carvacrol derivatives need to be evaluated for safety before use in agriculture, food, and medicine. It is also worth noting that the phosphate form of carvacrol solubilized in liposomes has been reported to inhibit tumor growth in a murine tumor model.37 Analysis. Several HPLC and GC/MS analytical methods are described to measure the carvacrol content in plants, essential oils, food, pharmaceuticals, and plasma. Here, we briefly mention highlights of selected analyses. Viñas et al.38 found that a monolithic HPLC column with fluorometric detections and gradient elution with an acetonitirile−water mixture permitted good separation of chromatographic peaks of carvacrol and thymol in different types of honey with a limit of detection (LOD) of 1−4 ng/g. Hajimehdipoor et al.12 validated an HPLC method for the analysis of carvacrol and thymol in the medicinal plant Thymus vulgaris using an ACE C18 column and isocratic acetonitrile water (50:50, v/v) at a flow rate of 1 mL/min as the elution solvent. The authors suggest that the method can be used for quality control of pharmaceutical samples for which GC methods may not be effective. Aleksseva39 developed a chromatographic system consisting of a reverse-phase (RP) Diasorb 130-C18T column eluted with MeOH:H2O:THF (50:50:22, v/v) for the analysis of carvacrol and thymol and in plant and medicinal samples. Horváth et al.40 describe a method for analysis of carvacrol and other essential oil compounds based on direct bioautography with TLC. The method was used to screen for microorganisms that grow on the TLC plate, suggesting that bioautography can quickly determine which component of an essential oil exhibits antimicrobial activity. Cantalapiedra et al.17 developed a reversed-phase highperformance liquid chromatographic (RP-HPLC) method for the simultaneous determination of phenolic compounds (carvacrol, eugenol, thymol, and vanillin) of aromatic plants using spectrophotometric detection with diode-array and electrochemical detection with amperometric and coulometric detectors, suggesting that the low detection limits (LODs) obtained make the method more useful for quality control than more expensive GC-MS methods. In related studies, Chaieb et al.11 and Vale-Silva et al.14 used GC/MS to characterize multiple components of essential oils, Kirkin et al.41 used the same technique to study the gammairradiation-induced degradation of carvacrol and other monoterpenes, and Nostro et al.42 used gas chromatography to determine residual carvacrol in carvacrol-treated microbial biofilms. Hallier et al.43 describe a GC/MS method without the use of solvents to determine carvacrol, thymol, cinnamaldehyde, and dially disulfide residues in cow milk. Lejonklev et al.44 describe a GC/MS method for analysis of the transfer of carvacrol and other terpenes from oregano and caraway essential oils into cow milk by gastrointestinal or respiratory exposure. Other studies used HPLC for carvacrol analysis: Du et al.45 determined the storage stability of carvacrol in edible

Figure 2. HPLC chromatogram of carvacrol extracted from a carvacrol-containing antimicrobial apple-based film. Adapted with permission from ref 45. Copyright 2008 American Chemical Society.

carvacrol and other compounds in black seed phytopharmaceuticals. Keawchaoon and Yoksan47 measured carvacrol released from chitosan nanoparticles using UV−vis spectroscopy over the wavelength range 250−400 nm. Shakeri et al.48 determined carvacrol in polyhydroxylate nanoparticles at 275 nm. Finally, Fiori et al.49 used GC/MS and headspace solidphase microextraction to measure carvacrol and thymol in plasma and milk of dairy cows with an LOD of 0.5 and 2.0 ng/ mL, respectively. Sensitive analytical methods seem to be available for the analysis of carvacrol in different milieus. Antioxidative Properties. A kinetic investigation of the antioxidant effects showed that carvacrol and thymol inhibit autoxidation in purified triacylglycerols, lard, and sunflower oil at room temperature, suggesting their possible use to extend the shelf life of lipid-containing food.50 Related studies found that carvacrol was highly effective in inhibiting the oxidation of sunflower seeds,51 that it protected blueberries against storageinduced spoilage,52,53 and that carvacrol grafted to chitosan nanoparticles via the Schiff’s base reaction exhibited greater antioxidant and antibacterial activities and was less cytotoxic to mammalian cells than free carvacrol, suggesting that the method can be applied to chitosan films, membranes, and fibers for biomedical and food packaging applications.54,55 In another study, carvacrol and thymol were shown to exhibit moderate antioxidant activity in vitro and no clastogenic effects in vivo at biologically relevant concentrations.56 In addition, DPPH antioxidant values of 73 active essential oils ranged from 14 to 340 μg/mL, suggesting that supplementing of food with spices containing high levels of essential oils may counteract and retard oxidative damage and lipid oxidation and enhance 7654



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the antioxidant effects of the food products. These findings suggest the need to determine which components provide maximal antioxidative effects with minimal toxicity and whether combinations of oil components exhibit synergistic effects.57 Antioxidant properties of essential oil components often differ from those of the oils from which they are derived.58 Carvacrol in chicken feed exhibited in vivo antioxidative and immunostimulating (antiallergic) properties.59

surface of raw chicken, suggesting the vapors diffused into the agar and into the moisture layer and/or cell membranes on the surface of the chicken substrate, possibly affecting the flavor and aroma of the chicken breast.68 Studies have also been conducted on the use of carvacrol in antimicrobial films. An HPLC method that was used to measure the stability of carvacrol in apple-based antimicrobial films during storage showed that optimal antimicrobial effects against E. coli occurred with a carvacrol concentration of ∼1.0% added to the apple purees prior to film preparation and that carvacrol in the films and the film weights remained unchanged for up to 7 weeks of storage, suggesting that the apple films merit use as antimicrobial food coatings.45 El Abed et al.69 showed that Thymus capitata essential oil (0.25−1.0%) with a carvacrol content of 89.9% inhibited Listeria monocytogenes in contaminated minced beef. Apple films containing carvacrol or cinnamaldehyde also inactivated E. coli and Salmonella inoculated on chicken breast and L. monocytogenes inoculated on ham, suggesting that the food industry could use the edible antimicrobial apple films as wrappings to control surface contamination by pathogenic microorganisms.70 Related studies found that apple, carrot, and hibiscus edible carvacrol-containing films were effective against L. monocytogenes on ham and bologna,71 and against Salmonella on iceberg lettuce in sealed plastic bags, which could perhaps lead to their potential use in commercial salad bags.72 As far as palatability is concerned, chicken wrapped with antimicrobial apple and tomato films was acceptable to a human sensory panel.73 Carvacrol released from microencapsulated gum arabic polymer films inactivated a broad spectrum of pathogenic bacteria, suggesting that gum arabic has good edible-filmforming and flavor-enhancing potential.74 Carvacrol facilitated the simultaneous heat inactivation of E. coli and inhibition of formation carcinogenic heterocyclic amines in grilled ground beef patties (hamburgers), suggesting that the compound can impart dual health benefits to meat products cooked at lower temperatures.75 Combinations of carvacrol cinnamaldehyde, eugenol, and thymol acted synergistically against E. coli at physiological pH, indicating that using them in combination might minimize the unpleasant flavors of some of the individual compounds.76 Carvacrol exhibited dose-dependent inhibition against Vibrio cholerae in inoculated carrot juice more efficiently at 25 °C than at 4 or 15 °C, suggesting its potential value to mitigate the virulent cholera disease.77 Subinhibitory levels of carvacrol inhibited C. jejuni motility and infection of epithelial cells by interfering with flagellar function, suggesting the potential use of the compound in infection prevention strategies.78 As mentioned, carvacrol can act against many pathogens. It has been shown that the efficacy of carvacrol against cultures of multiple pathogens in corn flour dough is influenced by the complexity of the microbial populations and the relationship between individual populations.79 Carvacrol and lauric alginate acted synergistically in reducing Salmonella counts in ground turkey by 4 log cfu/g, a much greater value than was found with the individual compounds.80 Predictive models can be useful when considering the action of antimicrobials on food and in vitro. For example, a threefactor polynomial predictive model can be used to estimate the processing times and temperatures needed to achieve recommended 7.0-log reduction of Salmonella by carvacrol and cinnamaldehyde in ground chicken.81 A related in vitro

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INHIBITION OF FOODBORNE PATHOGENS Generalities. Foodborne diseases result from ingesting food that is contaminated with either infectious microorganisms or toxins produced by the organisms. Foodborne pathogenic bacteria include Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium perf ringens, Escherichia coli, Listeria monocytogenes, Salmonella enterica, and Vibrio cholerae. Bacteria can exert adverse effects in animals and humans by adhering to cells as biofilms and releasing cellular toxins. Understanding the molecular basis of bacteria and toxins will facilitate the development of food-compatible approaches to mitigate the burden of pathogenic bacteria. A key objective of worldwide research efforts in this area is to develop unexplored ways to reduce the human pathogen burden of foods with the aid of naturally occurring safe compounds. The ultimate goal is to develop a better understanding of the structural features that govern antimicrobial activities as well as to devise food formulations that use the active compounds and plant extracts to reduce pathogens in foods, feeds, animals, and humans after consumption. Studies with food need to assess safety and palatability and the effect of the food matrix and storage conditions on antibacterial activities. Here, we present brief overviews of reported efforts to define the potential of carvacrol to meet these needs. Inhibition of Foodborne Pathogenic Bacteria in Vitro and in Food. Carvacrol has been shown to have broadspectrum antibiotic effects against bacteria involved in upper respiratory tract infections,60 and it also inhibits Shigella sonnei and S. f lexneri in an agar well diffusion assay with MIC values of 0.1−1.0%, suggesting it has the potential to be used as a disinfectant in the washing water of minimally processed vegetables.61 It was also highly active against multiple pathogens in an in vitro bactericidal assay,1 and it inhibited Mycobacterium avium subsp. paratuberculosis that causes the virulent Johne’s disease in livestock, with a MIC value of 72.2 μg/mL.62 With these activities in mind, carvacrol-containing recipes for antimicrobial wine marinades against four foodborne pathogens have the potential to improve the microbial safety of food.63 Indeed, carvacrol could also improve the safety and shelf life of fruit juices because it has been shown to inactivate E. coli and Salmonella in freshly prepared apple juices.64,65 Many studies have been carried out on the antimicrobial properties of carvacrol on meat and meat products. The bacterium Clostridium perf ringens is a common cause of foodborne illness. Carvacrol and cinnamaldehyde were shown to inhibit spore germination and outgrowth of C. perf ringens in ground turkey during chilling, suggesting their value for inhibiting C. perf ringens in commercial products.66 The two antimicrobials also facilitated the thermal destruction of E. coli in ground beef, suggesting that their use could allow shorter times and lower temperatures for heating beef products safely.67 In another study, carvacrol vapor inhibited the growth of S. enterica and inhibited and eliminated the pathogen on the 7655



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study describes carvacrol-induced death kinetics of L. monocytogenes.82 Carvacrol and citral potentiated the antibiotic activity of bacitracin, colistin, and erythromycin by reducing MIC values of Listeria strains, suggesting that such combinations may help reduce the required dosages of antibiotics and may be of value against both Gram-negative and Gram-positive bacteria.83,84 Carvacrol microencapsulated in hydroxypropyl-β-cyclodextrin inhibited E. coli and Salmonella at lower concentrations than free carvacrol, suggesting that encapsulation can enhance antimicrobial action and decrease the concentration of antimicrobial for inhibition, and indicating that carvacrol inclusion complexes could be useful antimicrobial delivery systems.85 It is also worth noting that combinations of compounds may have detrimental effects. For example, combinations of carvacrol with the food antioxidant BHA (2-isobutyl-4methxyphenol) exhibited antagonistic antimicrobial effects against S. aureus, suggesting that these interactions could affect food safety.86 A possible explanation is that carvacrol and structurally similar BHA compete for the same microbial membrane receptor sites. The cited studies demonstrate worldwide interest in defining the antimicrobial potential of carvacrol in vitro, in antimicrobial films, in encapsulated form, and in food. It seems that combinations of carvacrol with other plant-derived antimicrobials and with medical antibiotics enhance activity and require lower amounts of each component for the same effect, with the additional possible benefit of better sensory properties. This promising aspect merits further study with foods, feeds, and medicine. Carvacrol Inhibits Antibiotic-Resistant Bacteria. Antibiotics are widely used as animal feed supplements to fight infection, to promote growth of livestock and poultry, and to reduce production costs as well as to treat human infections. Resistant microorganisms that often arise from use of antibacterial soaps and sanitizers, administration of subtherapeutic levels and overprescription of antibiotics, and widespread use in livestock are a worldwide concern.87−90 In the United Sates, infections of about 2 million people annually cannot be treated with antibiotics, resulting in about 23,000 deaths.88 Because of the need to develop new alternatives to standard antibiotics that can be effective against antibioticresistant bacteria, we evaluated several natural products, including carvacrol, for their ability to inhibit resistant Bacillus cereus, E. coli, and S. aureus.91 All substances were active against the resistant organisms, suggesting that natural compounds can provide candidates to reduce both susceptible and resistant pathogens. Here, we briefly mention published studies on the efficacy of carvacrol against resistant bacteria in vitro and in food. An evaluation of the susceptibility of methicillin-susceptible and methicillin-resistant Staphylococci by Nostro et al.92 to oregano oil, carvacrol, and thymol showed that all three substances inhibited both types of pathogens to the same extent, with MIC values for carvacrol of 0.015−0.03%. In another study, it was shown that essential oils from the Korean Thymus and their components carvacrol and thymol inhibited both susceptible and resistant strains of Streptococcus pneumoniae, S. aureus, S. Enteritidis, and S. Typhimurium with MIC values ranging from 0.125 to 8 mg/mL.93 Carvacrol inhibited seven nalidixic acid-resistant bacteria species, with MIC values of 125−500 μg/mL.94 Combinations

of carvacrol or methyl gallate isolated from an essential oil were more effective than the individual compounds, suggesting that the two natural compounds have the potential to restore the efficacy of nalidixic acid against resistant bacteria. Carvacrol and thymol were highly effective in reducing the resistance of microbes, including Salmonella and E. coli, to several medicinal antibiotics; some combinations of the plant and medical antibiotics acted synergistically, suggesting that natural antibiotics can decrease the MIC of a diverse group of drug-resistant bacteria.95 A subinhibitory concentration of five natural antimicrobials including carvacrol individually and synergistically increased the susceptibility of multidrug resistant S. Typhimurium to five medical antibiotics, suggesting the potential value of the natural compounds as feed supplements to reduce the antibiotic resistance of Salmonella in food animals.96 In other studies, it was found that tea tree oil did not induce bacterial resistance to antimicrobial agents including carvacrol,97 and that Thymus maroccanus essential oil and its major components carvacrol and thymol modified the susceptibility of resistant bacteria to antibiotics by a membrane-associated mechanism, thereby conferring protection to bacteria.98 Carvacrol and cinnamaldehyde rapidly inactivated susceptible and resistant Campylobacter jejuni strains, suggesting that the same mechanism probably governs the inactivation of both types of pathogens.99 These two compounds also inhibited resistant Campylobacter on chicken breast,100 and resistant S. enterica on celery and oysters,101 suggesting that these plant antimicrobials have the potential to be used to decontaminate resistant pathogens on seafood, salads, and oysters. Carvacrol, cinnamaldehyde, and eugenol decreased a five-strain mixture of nalidixic-acid-resistant Salmonella on shell eggs from log 8.0 cfu/mL to undetectable levels, suggesting that the natural antimicrobials could be used as a wash treatment to reduce Salmonella Enteritidis on shell eggs.102 Our studies also indicate that the antimicrobial efficacy of carvacrol is similar to that of oregano oil with a ∼80% carvacrol content, suggesting that the minor components of the oil do not seem to contribute to the bioactivity.1,103 The availability of plant-derived antibiotics provides more options for the treatment of livestock and poultry and reduces the exposure of humans to resistant bacteria. Mechanism of Antimicrobial Effects. Here, we briefly mention results of experimental approaches designed to define possible mechanisms that might govern the antimicrobial effects of carvacrol. It has been shown that exposure of B. cereus to carvacrol results in the depletion of the intracellular ATP pool, a change in membrane potential, and an increase in the permeability of the cytoplasmic to membrane for protons and potassium ions.104 The loss of the ion gradient impairs essential metabolic processes in the cell and consequently leads to cell death. We used autofluorescence spectra to determine the effect of carvacrol on E. coli.103 The autofluorescence spectra and data showed significant changes at much lower concentrations of carvacrol (0.01 mM) than changes in membrane potential or release of ATP (ATP flux) after disruption of the bacterial cell membrane (1−2 mM), suggesting that autofluorescence detects physiological responses to carvacrol more efficiently than do changes in membrane potential or release of ATP. Difference spectra also make it possible to differentiate between autofluorescence associated with native bacterial cells and those exposed to carvacrol. 7656



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while planktonic bacterial pathogens are easily controlled by low levels of washing water disinfectant, the degree to which leafy greens are sanitized is highly limited and is dependent on diverse intrinsic factors and on the effectiveness of the commercial unit. Washing with hypochlorite or other sanitizing agents can achieve only limited pathogen reduction (1−2 logs) and therefore cannot ensure microbial safety; greater reduction of the pathogen is not possible, owing to strong microbial attachment and internalization of the organisms and biofilm formation.119 Bodur et al.120 and Shen et al.121 describe biofilms on stainless steel surfaces, and Gião et al.122 describe biofilms in tap water. GC-MS methods show that the biofilm matrix of Streptococcus pneumoniae is composed of extracellular DNA, proteins, and polysaccharide, suggesting that the web-like biofilm matrix links bacterial cells to one another and to their underlying substrate. We do not know to what extent the composition of biofilms varies among different pathogens.117 Continuous exposure to carvacrol was as effective as a commercial sanitizer in inhibiting biofilm formation by Staphylococcus aureus and S. enterica pathogens.123 In a series of studies, Nostro et al.42,124,125 found that (a) the concentrations of carvacrol and thyme required to eradicate biofilms produced by S. aureus and Staphylococcus epidermidis were 2- to 4-fold greater than those required to inhibit planktonic groups and that subinhibitory concentrations of the antimicrobials attenuated biofilm formation on polystyrene microtiter plates; (b) acidic formulations of carvacrol were more effective than neutral ones in inhibiting the formation of the biofilms; and (c) carvacrol- and cinnamaldehyde-containing polymeric films progressively released the antimicrobial that then inhibited both the bacteria and biofilm formation by Staphylococci and E. coli, possibly by reducing attachment ability and interfering with flagellar motility. In a related study, Zodrow et al.126 found that a biodegradable FDA-approved polymer containing carvacrol or cinnamaldehyde delayed biofilm formation by Gram-positive S. aureus, Gram-negative E. coli, and spoilage bacteria Pseudomonas aeruginosa, suggesting that these films might be used in polymer coatings for medical and other devices to delay colonization by bacteria. Subinhibitory concentration ratios of carvacrol, transcinnamaldehyde, eugenol, and thymol inhibited biofilm formation and inactivated mature biofilms of Listeria monocytogenes on microtiter plates and stainless steel coupons, suggesting that these compounds could be used to control the biofilms in food processing environments.127 A GFP-promoter fusion library with 79 Salmonella biofilm genes was used to study gene expression in the biofilms under experimental conditions, and that might facilitate study of biofilm inhibition in different environments.128 Carvacrol, thymol, and carvacrol−thymol combinations limited biofilm formation in paper mill machines, suggesting that the natural (green) compounds are promising alternatives or supplements to the currently applied treatments.129 A test of the effect of carvacrol against the oral pathogens Fusobacterium nucleatum, Porphyromonas gingivalis, and Streptococcus mutans and their preformed biofilms on a titanium disc surface showed that carvacrol has the potential to serve as a useful hygiene formulation to prevent periodontal disease in implanted patients.130 Inhibition of biofilm formation by Chromobacterium violaceum, S. enterica, and S. aureus but not by P. aeruginosa on polystyrene microplates by subinhibitory concentrations of carvacrol seems to be related to the

A Langmuir trough equipped with a computer-controlled microbalance was used to measure the effect of carvacrol and four other natural antimicrobials on monolayers of model membranes composed of bacterial phospholipids.105 Surface pressure−area (π−A) and surface potential−area (Δψ−A) isotherms were measured to monitor changes in the thermodynamic and physical properties of the lipid monolayers. Results indicated that carvacrol modified the three lipid monolayer structures by integrating into the monolayer, forming aggregates of antimicrobial−lipid complexes, reducing the packing effectiveness of the lipids, increasing the membrane fluidity, and altering the total dipole moment in the monolayer membrane model. These results provide insight into the mechanism of the molecular interactions between naturally occurring antimicrobial compounds and phospholipids of the bacterial cell membrane that govern activities. Related studies reported that (a) exposure of B. cereus to subinhibitory, nonlethal concentrations of carvacrol resulted in increased resistance of the bacteria to inhibition that was accompanied by a lowering of membrane fluidity associated with changes in their fatty acid and headgroup composition;106 (b) similar changes in membrane fatty acid profiles observed by Di Pasqua et al.107 and by Luz Ida et al.108 suggest that the mechanism of the antimicrobial effect is associated with an alteration in membrane lipid profiles and damage to the cell envelope; (c) a whole-cell biosensor assay suggests that the antimicrobial mechanism of action of carvacrol and related compounds involves damage to cell membranes and disruption to cellular metabolism and energy production in the evaluated five pathogens;109 (d) carvacrol and low doses of γ-radiation synergistically inhibited B. cereus via disruption of cell membranes, suggesting that the combined treatment could be effective in contaminated food;110,111 (e) carvacrol and thymol seem to induce antimicrobial activity via a mechanism that involves production of formaldehyde and its reaction products;112 (f) bacterial membrane proteins are promising therapeutic targets of potential antibacterial therapy;98,113,114 and (g) carvacrol and thymol bind to DNA via H-bonding of the OH group to the guanine N7, cytosine N3, and backbone phosphate group, with binding constants K = 1.55 × 103/M and 2.43 × 103/M, respectively, suggesting that the mechanism of formation of the DNA complexes can serve as a model for drug−DNA interactions.115 A better understanding of the mechanisms that govern the antimicrobial activities of carvacrol will help in the design of better approaches to inhibit pathogenic bacteria in different environments. Inhibition of Microbial Biofilms. Biofilms occur both at infection sites and during environmental growth of bacteria. They inhibit the diffusion and penetration of antibiotics into the microbial cells, and in vivo, they inhibit the penetration of immune cells to the microbes.116 The composition of biofilms is a mixture of extracellular polymeric matrices mostly synthesized by the microorganisms that are responsible for the cohesion and three-dimensional architecture of the biofilms.117 The presence of biofilms on food and on food contact surfaces negatively impacts food quality and safety, and compared to nonattached cells, biofilm-associated bacteria are more resistant to inactivation by antimicrobial compounds and biocides.118 Annous et al.119 describe biofilm formation by E. coli and other pathogens on fruits and vegetables, including on leafy greens, apples, and cantaloupes. The authors note that 7657



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colonization during oral iron therapy. The cited observations suggest that carvacrol inhibits dental caries, indicating the need for further human studies. Impact of Carvacrol on Animal Feed, Milk Production, and Waste Odors. The following observations suggest carvacrol can improve the quality and safety of animal feed. (a) The addition of up to 200 mg of carvacrol + thymol to a viscose poultry feed resulted in a decrease in digesta viscosity, in improvement of feed conversion and growth performance, and in reduced serum cholesterol level, suggesting that monoterpenes might alleviate the negative effects of viscous compounds in poultry diets.147 (b) Carvacrol-containing oregano leaves improved the growth performance of turkeys.148 (c) A mixture of carvacrol, capsicum, and cinnamaldehyde added to a poultry diet increased feed efficiency and carcass protein but not fat retention, suggesting that the additives improved the metabolic utilization of absorbed nutrients, possibly by inhibiting pathogenic bacteria.149,150 (d) Carvacrol inhibited the growth of Brachyspira intermedia, which chronically infects laying hens, causing spirochetosis.151 (e) Four natural antimicrobials including carvacrol acted synergistically with two antibiotics in inhibiting the growth of antibioticresistant microorganisms from animal feed, suggesting that the natural antimicrobials have the potential to control the development of antibiotic-resistant bacteria as substitutes for antibiotics.152 In the area of milk production, a study has shown that carvacrol/thymol and carvacrol/thyme/p-cymene mixtures inhibit the growth of multiple bacterial strains responsible for livestock mastitis. The incidence of mastitis, an inflammatory reaction of the mammary gland induced by pathogens in the udder that produce toxins, can be a problem for livestock and milk production, so the essential oil compounds might have use as a preventive herbal treatment.153 Hallier et al.43 developed a method to determine carvacrol and other essential residues in cow milk samples, enabling a measure of the transfer of the compounds from feed to milk and to evaluate their effect on sensory perception on raw milk and milk processing. No residues of these compounds were detected in the milk samples. By contrast, Lejonklev et al.44 found that carvacrol and other terpenes are transferred unaltered into milk within hours of exposure, regardless of whether the animals receive the compounds by gastrointestinal or respiratory exposure. Giannenas et al.154 found that supplementation of the feed with essential oils resulted in improved feed utilization and in increased production of milk by dairy ewes (sheep), suggesting the need for long-term trials to validate these results on dairy sheep. A study on goats found that supplementation of the diet with carvacrol resulted in reduced methane emissions.155 Carvacrol and thymol reduced livestock and swine waste odors and pathogens.156−158 A study on mice revealed that dietary Thymbra spicata essential oil containing 44.1% carvacrol reduced total and LDL and increased HDL serum cholesterol as well as GSH and oxidative enzyme levels, explaining the ethnomedical use of this oil.159 Will this oil also beneficially impact livestock and human cholesterol? Insecticidal Activities. A fumigation bioassay found that carvacrol and related compounds exhibited good larvicidal activity against Lycoriella ingénue, similar to that of the synthetic pesticide dichlorovos.160 Carvacrol was an effective fumigant against Culex pipiens pallens adult insects161 and showed a

disruption of quorum sensing, an essential part of biofilm formation, not by bactericidal activity.131

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INHIBITION OF HUMAN PATHOGENIC BACTERIA Although foodborne pathogens cause human infections, carvacrol also inhibits nonfoodborne pathogens. This section presents brief overviews of reported studies on the inhibition of growth and/or destruction of a variety of pathogenic foodborne and human pathogenic bacteria that can cause human illness. These pathogens are listed in alphabetical order. Campylobacter jejuni. C. jejuni is a widely distributed human pathogen that is implicated in abortion of sheep and induces emetic and diarrheal syndromes in humans. There also seems to be an apparent association between outbreaks of Campylobacter-induced diarrhea and the development of Guillain−Barre syndrome in humans.132 As mentioned previously, carvacrol inhibited the motility of C. jejuni and thus infection of epithelial cells.78 Helicobacter pylori. This bacterium in gastric mucosa might contribute to gastritis and peptic ulcer. Carvacrol and 15 essential oils inhibited the bacterium in vitro with MIC values ranging from 0.02 to 0.1 g/L but were not effective when administered orally to infected mice, suggesting that essential oils are unlikely to be efficient anti-Helicobacter compounds.133 Another in vitro study using a disc diffusion assay found that essential oils from the Iranian Thymus caramanicus plant inhibited ten clinical isolates of H. pylori with MIC values that ranged from 14.5 to 58.0 μg/mL.134 Klebsiella pneumoniae. Several studies reported that carvacrol-containing plant essential oils inhibited pneumoniacausing bacterium K. pneumoniae.135−139 Iten et al.140 found that an increase in carvacrol concentration in the oils enhanced the kill-rate and that the combination of carvacrol and linalool has additive antimicrobial effects. Mycobacterium tuberculosis. Carvacrol-containing aromatic and medicinal plants from Columbia exhibited antitubercular activity,141 and carvacrol derivatives inhibited the Mycobacterium tuberculosis chorismate mutase enzyme,142 suggesting that these substances might help alleviate tuberculosis symptoms. Salmonella enterica. Oral iron therapy can increase the level of bacterial pathogens in the large intestine of African children. To help overcome this problem, Kortman et al.143 discovered that carvacrol inhibited iron-induced epithelial intestinal cell adhesion of S. enterica at high iron concentrations, suggesting its potential value to prevent pathogenic overgrowth and colonization in the large intestine during oral iron therapy. Oral Pathogenic Bacteria. Several studies describe the anticariogenic effects of carvacrol. For example, the essential oil from Lippia sidoides and its major components carvacrol and thymol exhibited potent antimicrobial activity against the cariogenic species Streptococcus mutans and Candida albicans, with MIC values from 0.625 to 10.0 mg/mL, suggesting their value in combating oral microbial growth.144 In addition, mucoadhesive patches consisting of bilayers containing carvacrol, and the antibiotic tetracycline exhibited antimicrobial activity against oral bacteria and the yeast C. albicans, indicating the potential use of carvacrol in combination to treat local mouth bacterial infections and candidiasis.145 Related studies reported that carvacrol inhibited oral biofilms and planktonic cultures,130 that a carvacrol gel protected rats against induced periodontitis,146 and that carvacrol has the potential to serve as a dietary supplement to prevent pathogenic overgrowth and 7658



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and trans-cinnamaldehyde substantially reduced toxin production by this pathogen in Vero cells, down-regulated toxin genes, and did not inhibit the growth of beneficial gut bacteria, suggesting the potential of the plant compounds to reduce C. dif f icile virulence.174 A structure−activity study of ten natural phenolic compounds including carvacrol found that a mathematical expression obtained was able to predict the release of the highly toxic mycotoxin fumonisin from the Fusarium verticillioides fungal strain, suggesting that lipophilicity is the key property that allowed the target molecule to reach inside the fungal cells.175 The authors suggest that the mathematical analysis can be used to predict the antifumonisin activity of structurally related molecules, so it will be interesting to find out whether carvacrol can inactivate the biological (toxicological) activities of these and other pure toxins.176 Antiviral Activities. Viral contamination of food and infection of animals and humans is a major cause of numerous diseases. The mechanism of antiviral action of plant compounds is based on their abilities to act as antioxidants, to inhibit enzymes, to disrupt cell membranes, to prevent viral binding and penetration into cells, and to induce the host cell defense mechanism. Here, we present brief summaries of reported studies on the antiviral activities of carvacrol against several pathogenic viruses. Carvacrol and thymol had significant antiviral activity with an IC50 value of 7 μM against herpes simplex virus type 1 (HSV1) that causes orofacial and genital infections, suggesting that the topical application of these monoterpenes for the treatment of the viral infections merits study.177 An evaluation of thymolrelated monoterpenes showed that the aliphatic side chains had a minor effect, while the hydrophilic OH group on the benzene ring was sufficient for activity. Carvacrol and thymol were reported to be responsible for the antiviral activity against HSV1 in supercritical carbon dioxide oregano and sage oil extracts.178 Mexican oregano oils exhibited high antiviral activity against acyclovir-resistant HSV-1, bovine herpesvirus type 2 (BoHV-2), bovine viral diarrhea virus (BVDV), and human rotavirus.179 Carvacrol inactivates murine norovirus, a human norovirus surrogate after 1 h of exposure via direct action on the viral capsid followed by the RNA.180 Carvacrol at concentrations of ∼0.5% induced an up to 3.87 log reduction within 1 h, acting directly on the virus capsid and then on the RNA. Surprisingly, oregano oil was less effective than carvacrol. Sökmen et al.181 and Schnitzler et al.182 describe viricidal effects of monoterpenes derived from essential oils and essential oil extracts. An unanswered question is whether carvacrol in pure form and in carvacrol-containing essential oils will inactivate pathogenic viruses in contaminated food and water and in a clinical setting. Acaricidal, Antileishmanial, Nematicidal, and Trypanocidal Effects. Treatment with carvacrol (2.5 μL/mL) induced 100% death of larvae of the cattle parasite Rhipicephalus microplus and the tropical horse tick parasite Dermacentor nitens, suggesting the potential of the monoterpene to control these two tick species in the field.183 A related study on the acaricidal efficacy of carvacrol and related compounds on larvae of nymphs of Amblyomma cajennense (Cayenne tick) and Rhipicephalus sanguineous parasites that afflict horses and other animals found that similar low concentrations of carvacrol and thymol (2.5 μL/mL) also cause 100% lethality in these parasites, indicating the value of further studies to evaluate these acaricides under natural conditions.184

concentration-dependent inhibition of nicotine binding in a membrane preparation of housefly heads containing the nicotinic acetylcholine receptor, suggesting that insect Nacetylcholine receptors could serve as a novel target for monoterpenoid insecticides.162,163 Antifungal Activities. The following capsule summaries of published studies show that low concentrations of carvacrol inhibit the growth or inactivate phytopathogenic and pathogenic fungi. Carvacrol was effective against both susceptible (MIC = 75− 90 mg/L) and fluconazole-resistant (MIC = 75−100) Candida albicans and other fungal isolates.164 Thymol was shown to have similar antifungal effects, suggesting that both compounds could serve as an alternative fluconazole therapy for the treatment and prevention of human candidiasis. A study on fruit decay found that an application of carvacrolor thymol-containing waxes to lemons inoculated with Penicillium digitatum and P. italicum fungi reduced the decay of the citrus fruit, indicating that perhaps such waxes could be used in citrus packing lines to replace synthetic fungicides.165 Candida lusitaniae is a pH-tolerant yeast that contaminates fruit juices. Carvacrol at concentrations >1 mM inhibited the growth of C. lusitaniae, suggesting carvacrol could be used for preserving minimally processed food.166 Carvacrol inhibited the following pathogenic and toxinogenic fungi with MIC50 values of 37−76 μg/mL and MIC100 values of 131−262 μg/mL: Aspergillus f lavus, A. f umigatus, Fusarium oxysporum, F. verticillioides, Penicillium brevicompactum, and P. expansum.167 Carvacrol and thymol were the most promising of the 22 tested compounds, indicating their potential as effective fungicides. A study on the inhibition of 11 food-decaying fungi found that carvacrol exhibited high efficacy with an average MIC value of 154.5 μg/mL, suggesting that the in vitro study may serve as a guide for future studies of the inhibition of the foodborne fungal pathogens on/in food.168 Essential oils from Origanum vulgare from Southern Italy showed both significant compositional variability and antifungal activity, and their antifungal potencies increased with carvacrol content. All the oils were the carvacrol/thymol chemotypes. At 1000 ppm, the three oils completely inhibited fungal growth of Monilinia laxa, M. fructicola, and M. fructigena species without any hemolytic effect on the cell membranes of bovine erythrocytes.169 A quantitative structure−activity relationship (QSAR) analysis of natural phenolic compounds including carvacrol against fluconazole-resistant Candida species based on steric descriptors can be used to predict the anti-Candida activity of new phenolic compounds.170 Related studies on the effects of carvacrol against Candida albicans are described by Ahmad et al.171 and Lima et al.172 In a study of fungal infection of the tree Ulmus minor, it was shown that carvacrol imparts resistance to U. minor xylem tissues against the fungal pathogen Ophiostoma novo-ulmi, the cause of Dutch elm disease in trees.173 The cited studies indicate that carvacrol and thymol, as well as essential oils that contain high levels of carvacrol, strongly inhibit both human pathogenic and phytopathogenic fungi. Further studies are needed to define their usefulness in fungalcontaminated food. Inhibition of Microbial and Fungal Toxin Production. Hospital-acquired Clostridium dif f icile is the most common cause of antibiotic-associated colitis and diarrhea that affects about 300,000 individuals in the United States.132 Carvacrol 7659



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Leishmaniasis caused by the tropical parasite Leishmania amazonensis affects more than 12 million people worldwide. A comparison of the antileishmanial activity of the essential oil from Chenopodium ambrosioides and its main components carvacrol and caryophyllene oxide in mice showed that oil prevented lesion development, the individual compounds were not effective, and the mixture of the pure compounds caused death of the animals after 3 days of treatment, suggesting the superiority of the oil against experimental leishmaniasis.185 An in vitro study found that carvacrol and thymol from the essential oil Lippia sidoides inhibited Leishmania chagasi with IC50 values of 58.4 and 74.1 μg/mL, respectively, suggesting that this oil is a promising source of leishmanicidal compounds.186 Carvacrol also showed antileishmanial activity against experimental cutaneous leishmaniasis caused by L. amazonensis.185 At a concentration of 1000 μL/L, 12 of 27 essential oils and active components evaluated immobilized more than 80% of juveniles of the root-knot nematode Meloidogyne javanica and inhibited nematode hatching and root galling of cucumber seedlings in pot experiments, indicating that the oils and components could serve as nematicides. Carvacrol immobilized the juveniles and inhibited hatching at 125 μL/L.187 Human African trypanosomiasis (sleeping sickness) is caused by Trypanosoma brucei and poses a risk of infection for about 60 million people living in sub-Saharan countries.188 The cited investigators discovered that Hagenia abyssinica essential oils and their bioactive components including carvacrol strongly inhibited the parasite with an IC50 value of 11.25 μg/mL, with minimal toxicity on human leukemia cells. The order of decreasing trypanocidal effects of phenolic monoterpenes, carvacrol > thymol > eugenol, paralleled acute oral toxicities in rats (LD50 in mg/kg): carvacrol, 810; thymol, 980; eugenol, 2680. The relative potencies are similar to those we reported for antimicrobial activities of these compounds.1 The most active compounds have the potential to protect against or treat human and animal trypanosomiasis. Another study189 reported that 13 essential oils from different species of Columbian Lippia plants containing an average of 46.2% carvacrol inhibited Leishmania chagasi and Trypanosoma cruzi parasites with IC50 values of 5.5−12.2 μg/ mL. The corresponding values for pure carvacrol were 3.0−38.0 μg/mL, suggesting that some of oils and carvacrol could be useful in the treatment and prevention of Chagas and leishmaniasis protozoan diseases, which are a major public health problem in Latin America. Carvacrol and nootkatone suppressed Ixodes scapularis and Amblyomma americanum parasites in a Lyme disease endemic in New Jersey.190

Figure 3. Metabolism and urinary excretion of carvacrol in rats. Adapted from refs 191 and 192.

methyl groups of carvacrol. These compounds seem to be excreted as the glucoronide and sulfate derivatives. Using human microsomes, Dong et al.193 detected two new hydroxylated metabolites that resulted from the cytochrome isoform CYP2A6-induced oxidation of carvacrol to new hydroxylated metabolites, suggesting that carvacrol could impact pharmacokinetic and clinical outcomes when coadministered with other compounds undergoing CYP2A6 metabolism. Related studies found that (a) carvacrol acted in a dose-dependent manner as a liver mitochondrial antioxidant and protected against reactive-oxygen-species (ROS)-generating mitochondrial enzymes and DNA damage in D-galactosamine-induced hepatotoxicity in rats, suggesting that the antioxidant effect of carvacrol could be responsible for the observed hepatoprotective effects;194 (b) in vitro and parallel in vivo studies in the hippocampus of mice revealed that synthetic carvacrol acetate reduced lipid peroxidation, GSH levels, catalase activity, and nitrite content and that the antioxidative potential correlated between these two parameters;195 and (c) plant enzymes in germinating wheat seeds partly converted carvacrol and other monoterpenes to corresponding less toxic oxidation and reduction products and partly to degradation products, suggesting that such changes might also take place in other foods.196 Noma and Asakawa197 comprehensively reviewed biotransformation pathways of monoterpenes by microorganisms, insects, and mammals. Anti-inflammatory Mechanisms. The strong anti-inflammatory properties of carvacrol provide a scientific basis for many of its beneficial bioactivities. Here, we will mention some of the cellular events that seem to be associated with these properties. An investigation on the cytokine modulation induced in paw inflammation in mice showed that carvacrol at 100 μg/kg attenuated the paw edema and reduced the pro-inflammatory COX-2 and IL-1ß mRNA expression and enhanced the levels of IL-10 and IL-10 mRNA anti-inflammatory cytokines, suggesting that the treatment reduced the production of inflammatory mediators, possibly through induction of IL-10 release.198 Carvacrol did not produce an antidermatogenic effect in IL-10 deficient mice. In related studies, Silva et al.199 found that carvacrol induced reduction of mouse ear edema and significantly up-regulated mRNA and protein expressions of TNF-α, IL-6, iNOS, COX-2, and NF-κB in D-galactosamine-induced hepatotoxic rats and

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ADDITIONAL BIOACTIVITIES AND HEALTH-PROMOTING PROPERTIES OF CARVACROL Metabolism. After consumption, carvacrol is metabolized by cytochrome enzymes, mostly hydroylated derivatives that has unknown safety. Here, we briefly mention selected studies on carvacrol metabolism. A metabolic study of carvacrol in rats showed that only a small amount is excreted in urine 24 h after oral administration.191,192 Figure 3 shows the structures of the characterized metabolic products that result from the in vivo enzyme-catalyzed hydroxylation of the benzene ring and hydroxylation and oxidation of the aliphatic isopropyl and 7660



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down-regulated the expression of these genes,200 confirming the mechanistic basis for the anti-inflammatory activity of carvacrol. A review on anti-inflammatory effects of monoterpenes further expands on the proposed mechanisms that seem to govern anti-inflammatory effects of monoterpenes.201 Analgesia. Pain that occurs in response to tissue injury results from activation of peripheral pain receptors (nociceptors) and their sensory fibers. Many studies have been designed to demonstrate the antinociceptive (analgesic) properties of carvacrol. For example, carvacrol attenuates carrageenaninduced hypernociception and inflammation in mice, suggesting that carvacrol is a candidate in the treatment of painful conditions associated with inflammation.202 The beneficial effects seem to be associated with inhibition of proinflammatory cytokine TNF-α production and NO release. Related studies found that (a) carvacrol enhanced warmth and heat pain on the human tongue;203,204 (b) synthetic carvacrol propionate attenuates nociception, mechanical hyperalgesia, and inflammation in male Swiss mice through inhibition of cytokines;205 (c) oral administration of carvacrol in mice models of pain resulted in significant inhibition of nociception without impairment of motor performance, suggesting that the antinociceptive activity might not act through the opioid system;206 (d) results of a study of orofacial analgesic-like activity of carvacrol in rodents suggest that the compound might represent an important tool for the treatment of orofacial pain;207 (e) carvacrol in drinking water was more effective than the steroidal drug dexamethasone in mitigating adverse effects in sensitized guinea pigs, suggesting the monoterpene could be used to treat asthma in humans;208 and (f) Guimarães et al.209 reviewed mechanisms that might govern analgesic aspects of structurally different monoterpenes. These observations indicate that carvacrol could benefit individuals suffering from inflammatory-related allergies. Arthritis. The folate antagonist methotrexate is an antiinflammatory drug with many side effect used to treat human rheumatoid arthritis and cancer. We previously reported that both folic acid and methotrexate protected frog embryos against organ malformations (teratogenicity) induced by the potato glycoalkaloid α-chaconine.210,211 The following observations suggest that carvacrol has the potential to ameliorate adverse side effects of methotrexate: (a) carvacrol overcame methotrexate-induced oxidative stress and the pro-inflammatory response in the sciatic nerve tissue of the rat, indicating that carvacrol has the potential to reduce the toxic side effects;212 (b) carvacrol enhanced the antiarthritic action and reduced liver toxicity of methotrexate in a rat model, suggesting that carvacrol might promote the safe use of methotrexate in the management of arthritis;213 and (c) carvacrol partially abrogated methotrexate-induced kidney damage in male rats.214 The cited observations suggest the need to evaluate the potential of carvacrol to ameliorate methotrexate-associated toxicity in human patients. Cancer. Cancer that can develop in any tissue organ at any age is an unregulated proliferation of cells due to loss of normal control, resulting in unregulated growth and, often, metastasis.132 Because multiple genes are involved in carcinogenesis, there is a need for therapies that target multiple signaling pathways. The following observations suggest that carvacrol might act additively or synergistically with other cancer cell inhibitors. Incubation of human nonsmall cell lung cancer cells (A549) with carvacrol in DMSO for 24 h resulted in a decrease in cell

number and protein content and in degeneration of cell morphology, suggesting that the monoterpene is a potent inhibitor cell associated with about 75% of lung cancers.215 Carvacrol also induced a 30% decrease of 3,4-benzopyreneinduced in vivo carcinogenicity in Wistar rats and exhibited anticarcinogenic effects against the cells in vitro, with IC50 values of 90 μM and 67 μM after 24 and 48 h incubations.216 In a series of studies, Slameňová et al.217−220 found that carvacrol protected human hepatoma (HepG2) and human colonic (Caco-2) and other cell lines against DNA strand breaks induced by hydrogen peroxide and methylene blue, and reduced DNA lesions in hepatocytes and testicular cells in rats given carvacrol in drinking water for 7 days, suggesting that the beneficial effects might be associated with an increase in antioxidant activity in liver and testicular cells in the animals. Carvacrol was also shown to inhibit human hepatoma HepG2 cell growth by inducing apoptosis by the direct activation of the mitochondrial and mitogen-activated protein kinases (MAPK) pathways; thus, it might help prevent liver cancer.221 Carvacrol inhibited the proliferation of porcine lymphocytes in the MTT assays that measure cell viability, with an IC50 value of 182 μM, suggesting that the reduced lymphocyte proliferation was due to apoptotic cell death, as determined by Annexin-V binding and caspase-3 activation.222 These authors also mention that because the antimicrobial concentrations of carvacrol (5−100 mM) are much higher than concentrations that are toxic to leucocytes and epithelial cells (100−1000 μM), caution should be exercised when using carvacrol as a feed additive in pigs. In another cancer cell death study, carvacrol exhibited antiproliferative effects in metastatic breast cancer, MDAMB231, cells with an IC50 value of 100 μM, suggesting that carvacrol could be a potent antitumor molecule against metastatic breast cancer cells.223 The induction of apoptosis seems to have been mediated by cell cycle arrest at S phase, increases in Annexin V positive cells, caspase activity, cytochrome c release from mitochondria, decreases in mitochondrial membrane potential and in the Bcl2Bax protein ratio, cleavage of PARP, and fragmentation of DNA. Carvacrol (15 mg/kg BW) protected rats against diethylnitrosamine-induced hepatocellular carcinoma. Reduction in liver cancer progression was associated with prevention of liver peroxidation, hepatic cell damage, and protection of the liver antioxidant system.224 Carvacrol-induced ROS-associated apoptosis in OC2 human oral cancer cells was triggered by caspase-3 activity and the release of calcium ions from the endoplasmic reticulum.225 A related study on periodontal inflammation used a focused microarray analysis of apoptosis-related genes, and the results suggest that caspase-3 is a target protein for carvacrol in the inhibition of periodontitis apoptosis of HaCaT epithelial cells.226 On the basis of the results from a study of the effect of carvacrol on healthy neurons and N2a neuroblastoma cells, Aydin et al.227 conclude that the efficacy of anticancer chemotherapy seems to be limited by the cytotoxic effect on healthy cells, owing to the lack of selectivity of carvacrol and poor uptake by the neuroblastoma cells. Overall, the in vitro and rodent studies suggest that the food ingredient carvacrol alone or in combination with cancer drugs such as methotrexate and doxorubicin merits further study for the potential to mitigate human breast, liver, and other cancers. 7661



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Gastroprotection. The protection of drug-induced gastric lesions in mice and rats seems to be mediated by endogenous prostaglandins, K(ATP) channel openings, NO synthase activation, and antioxidant properties, suggesting that carvacrol has the potential to be an effective gastroprotective agent.243 A related study found that carvacrol helps heal induced gastric ulcers in mice by interfering with the release and/or synthesis of pro-inflammatory mediators.199 By contrast, an in vitro study found that exposure of human intestinal Caco-2 cells to carvacrol and thymol resulted in vacuolated cytoplasm, altered organelles, and other evidence of morphological cellular damage.244 Hepatoprotection. Pretreatment with carvacrol ameliorates thioacetamide-induced liver injury in rats. This is mediated by the inhibitory effect of carvacrol on nuclear factor NFκB activation of Bax and Bcl-2 protein expression, and the restoration of histopathological changes, suggesting that the beneficial effect is associated with the inhibition of apoptosis.245 Related studies show that carvacrol: (a) increases the regeneration rate of rat livers undergoing partial hepatectomy;246 (b) protects rat livers against hepatic ischemia, a risk factor in liver surgery and transplantation;247and (c) protects rats against D-galactosamine-induced hepatotoxicity.231,248 It seems that carvacrol has the potential to help overcome liver injuries in clinical settings. Neuroprotection. Carvacrol was 10 times stronger than the isomeric thymol in inhibiting acetylcholinesterase, a key compound involved in neurotransmission.249 An investigation of the effect of carvacrol on diabetes-associated cognitive deficit (DACD) in a rat model showed that the compound dosedependently prevented behavioral, biochemical, and molecular changes associated with diabetes, indicating that it might be useful for treating diabetes and DACD.250 The cognitive impairment was associated with oxidative stress, inflammation, and apoptotic cascades. An investigation of the effect of carvacrol on induced cerebral edema in a mouse model showed that the molecule reduced the edema and neurological deficits, suggesting that it might protect against intracerebral hemorrhage injury by ameliorating aquaporin-4 (AQP4)-mediated edema.251 Carvacrol also enhanced the recovery of traumatic brain injury in mice by altering permeable calcium ion channels that are involved in neuronal cell death, suggesting the monoterpene should be further evaluated in animals and humans.252 Related studies reported that carvacrol protected against methotrexate-induced neurotoxicity in rats,212 and alleviated cognitive impairment in scopolamine-treated rats caused by increased amyloid-ßpathology (Aß) levels associated with Alzheimer’s disease or cholinergic hypofunction.253 Orally administered carvacrol, up to 50 mg/kg BW, induced antidepressant effects that seem to be dependent on an interaction with the dopaminergic brain pathways.254 The cited and related studies255,256 imply that carvacrol merits further study in humans to establish its potential to treat neurological disorders, including Alzheimer’s disease and depression.

Moreover, because other widely consumed food ingredients and foods are also reported to inhibit cancer cells in vitro and in vivo, combinations of carvacrol with anticarcinogenic tea catechins and theaflavins,228 the tomato compounds lycopene and α-tomatine,229 and pigmented rice brans230 also merit study. Cardioprotection. The protective effect of carvacrol against risk factors associated with heart disease has also been reported. For example, the administration of carvacrol for 21 days to hepatotoxic rats increased levels of good very low density (VLDL) and low density lipoprotein (LDL) and decreased bad high density lipoprotein (HDL) serum levels, suggesting that carvacrol affords a significant hepatoprotective and hypolipidemic effect against induced hepatotoxicity.231 Carvacrol induces relaxation of the arteries by activating TRPV cation channels and suppressing calcium ion currents in the endothelium, which may account for some of the cardioprotective effects of the Mediterranean diet.232−234 Carvacrol has also been shown to decrease the heart rate and blood pressure of anesthetized rats235 and protected rats against myocardial infarction,236 suggesting that the compound seems to be a promising cardioprotective agent for the treatment of myocardial infarction. The beneficial effects of carvacrol seem to be modulated by its antioxidative and antiapoptotic properties. Diabetes and Obesity. Here, we briefly examine the beneficial properties of carvacrol against obesity and diabetes in rodents. Orally administered carvacrol at doses of 25 and 50 mg/BW to diabetic rats for 7 days after the onset of streptozotocininduced diabetes did not significantly reduce serum glucose or insulin levels. The treatments did, however, have a protective effect against the negative effects of streptozotocin on liver enzymes, suggesting that the compound would be useful in the diet of diabetics.237 Co-administration of carvacrol (20 mg/kg BW) and the antidiabetic drug rosiglitazone (4 mg/kg BW) for 35 days to rats on a high-fat diet prevented changes in metabolic enzymes associated with obesity and diabetes, thus indicating that the combination had a better antihyperglycemic effect than the individual compounds.238 The results of the histopathology of the islet cells of the pancreas support the biochemical findings. It has also been reported that mice fed a 0.1% carvacrolsupplemented diet had reduced body weight gain, visceral fadpad weights, and plasma lipid levels compared with mice fed a high-fat diet.239 Inhibition of adipogenesis seems to occur by suppression of bone marrow protein-, fibroblast growth factor 1-, and galanin-mediated signaling and reduction of the of proinflammatory cytokines in visceral adipose tissues by inhibiting toll-like receptor 2 (TLR2)- and TLR4-mediated signaling. The cited findings suggest that carvacrol might help prevent obesity in humans. Endotoxemia. Endotoxemia is an infection-related virulent disease with a high mortality rate.240 Intraperitoneal administration of carvacrol improved the survival of mice during induced lethal endotoxemia and attenuated lung injury.241 The protective effect seems to be related to inhibition of activation of NF-κB and MAPK signaling pathways, thereby inhibiting inflammatory cytokines TNF-α, IL-6, and IL-1ß; thus, it seems that carvacrol might be useful in preventing endotoxemiaassociated adverse effects. Possible synergistic antiendotoxemia effects of carvacrol with other natural compounds240,242 merit study.

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OUTLOOK Carvacrol-containing aromatic plants and spices are a major source of essential oils that are widely used in the human diet. Carvacrol can be isolated from a large number of essential oil producing plants. The results of the cited studies indicate that the antioxidative properties associated with the phenolic OH group, the hydrophobic properties associated with the benzene 7662



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(5) Sell, C. Chemistry of essential oils. In Handbook of Essential Oils: Science, Technology, and Applications; Baser, K. H. C., Buchbauer, G., Eds.; CRC Press, Taylor & Frances Group: Boca Raton, FL, 2010; pp 121−150. (6) Nhu-Trang, T. T.; Casabianca, H.; Grenier-Loustalot, M. F. Deuterium/hydrogen ratio analysis of thymol, carvacrol, gammaterpinene and p-cymene in thyme, savory and oregano essential oils by gas chromatography-pyrolysis-isotope ratio mass spectrometry. J. Chromatogr., A 2006, 1132, 219−227. (7) Loza-Tavera, H. Monoterpenes in essential oils: biosynthesis and properties. Adv. Exp. Med. Biol. 1999, 464, 49−62. (8) Smith, C. A.; Wood, E. J. Biosynthesis; Chapman & Hall: London, 1992; p 226. (9) Mendes, M. D.; Barroso, J. G.; Oliveira, M. M.; Trindade, H. Identification and characterization of a second isogene encoding γterpinene synthase in Thymus caespititius. J. Plant Physiol. 2014, 171, 1017−1027. (10) Ramak, P.; Kazempour Osaloo, S.; Ebrahimzadeh, H.; Sharifi, M.; Behmanesh, M. Inhibition of the mevalonate pathway enhances carvacrol biosynthesis and DXR gene expression in shoot cultures of Satureja khuzistanica Jamzad. J. Plant Physiol. 2013, 170, 1187−1193. (11) Chaieb, K.; Hajlaoui, H.; Zmantar, T.; Kahla-Nakbi, A. B.; Rouabhia, M.; Mahdouani, K.; Bakhrouf, A. The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): a short review. Phytother. Res. 2007, 21, 501−506. (12) Hajimehdipoor, H.; Shekarchi, M.; Khanavi, M.; Adib, N.; Amri, M. A validated high performance liquid chromatography method for the analysis of thymol and carvacrol in Thymus vulgaris L. volatile oil. Pharmacogn. Mag. 2010, 6, 154−158. (13) Hadad, G. M.; Salam, R. A.; Soliman, R. M.; Mesbah, M. K. High-performance liquid chromatography quantification of principal antioxidants in black seed (Nigella sativa L.) phytopharmaceuticals. J. AOAC Int. 2012, 95, 1043−1047. (14) Vale-Silva, L.; Silva, M. J.; Oliveira, D.; Gonçalves, M. J.; Cavaleiro, C.; Salgueiro, L.; Pinto, E. Correlation of the chemical composition of essential oils from Origanum vulgare subsp. virens with their in vitro activity against pathogenic yeasts and filamentous fungi. J. Med. Microbiol. 2012, 61, 252−260. (15) Ghassemi, N.; Ghanadian, M.; Ghaemmaghami, L.; Kiani, H. Development of a validated HPLC/photodiode array method for the determination of isomenthone in the aerial parts of Ziziphora tenuior L. Jundishapur J. Nat. Pharm. Prod. 2013, 8, 180−186. (16) Jaberian, H.; Piri, K.; Nazari, J. Phytochemical composition and in vitro antimicrobial and antioxidant activities of some medicinal plants. Food Chem. 2013, 136, 237−244. (17) Cantalapiedra, A.; Gismera, M. J.; Sevilla, M. T.; Procopio, J. R. Sensitive and selective determination of phenolic compounds from aromatic plants using an electrochemical detection coupled with HPLC method. Phytochem. Anal. 2014, 25, 247−254. (18) Ozkan, G.; Baydar, H.; Erbas, S. The influence of harvest time on essential oil composition, phenolic constituents and antioxidant properties of Turkish oregano (Origanum onites L.). J. Sci. Food Agric. 2010, 90, 205−209. (19) Shimoda, K.; Kondo, Y.; Nishida, T.; Hamada, H.; Nakajima, N.; Hamada, H. Biotransformation of thymol, carvacrol, and eugenol by cultured cells of Eucalyptus perriniana. Phytochemistry 2006, 67, 2256− 2261. (20) Jensen, C. G.; Ehlers, B. K. Genetic variation for sensitivity to a thyme monoterpene in associated plant species. Oecologia 2010, 162, 1017−1025. (21) Yannai, S. Dictionary of Food CompoundsAdditives, Flavors, and Ingredients; Chapman & Hall/CRC Press: Boca Raton, FL, 2004; p 835. (22) O’Neil, M. J. The Merck Index, 15th ed; Royal Society of Chemistry: Oxford, U.K., 2013. (23) Teuscher, E. Medicinal SpicesA Handbook of Culinary Herbs, Spices, Spice Mixtures and Their Essential Oils; Medpharm Scientific Publishers: Stuttgart, Germany, 2006; p 459.

ring and the methyl and isopropyl substituents, the binding affinity to the guanine moiety of DNA, and the antiinflammatory and apoptotic and cell membrane disruptive properties of carvacrol all seem to contribute to the observed diverse bioactivities. These bioactivities have the potential to contribute to the prevention and therapy of several chronic human diseases, including allergic response, cancer, diabetes, and infectious, cardiovascular, and neurological syndromes. Future studies should further explore the beneficial synergistic properties of combinations of carvacrol with other natural antimicrobials, including tea compounds,257 seashell-derived chitosans,55 and wine flavonoids.258 Such dietary combinations in which the individual components might exert their effects by different mechanisms could make it possible to use lower amounts of each component, thus minimizing possible adverse sensory properties, antibiotic resistance, and toxicity.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS I am most grateful to my colleagues whose names appear on the cited references for excellent scientific collaboration, to Carol E. Levin for facilitating the preparation of the manuscript, and to journal reviewers for helpful comments.

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ABBREVATIONS USED ATP, adenosine triphosphate; Bcl2/Bax, cancer biomarker signaling proteins; BoHV-2, bovine herpesvirus type 2; BVDV, bovine viral diarrhea virus; BW, body weight; COX-2, cyclooxygenase-2; CYP2A6, cytochrome P450 2A6; DPPH, 2,2-diphenyl-1-picrylhydrazyl; GFP, green fluorescent protein; DACD, diabetes-associated cognitive deficit; HDL, high-density lipoprotein; HSV-1, herpes simplex virus type 1; IL, interleukin; LD50, concentration of test substance that kills 50% of the animals; LDL, low-density lipoprotein; LOD, limit of detection; GC/MS, gas chromatography/mass spectrometry; iNOS, inducible nitric oxide synthase; MIC, minimum inhibitory concentration; MAPK, mitogen-activated protein kinases; MTT, tetrazol 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide; NF-κB, nuclear factor kappa B; NO, nitric oxide; PARP, poly(ADP-ribose) polymerase; RT-HPLC, reversedphase high-performance liquid chromatography; QSAR, quantitative structure−activity relationship; RNA, ribonucleic acid; ROS, reactive oxygen species; TNF-α, tumor necrosis factor alpha; TRPV, transient receptor potential vanilloid; VLDL, very low density lipoprotein

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