Journal of Medicinal Plants

An Overview on Two Valuable Natural and Bioactive Compounds, Thymol and Carvacrol, in Medicinal Plants

Naghdi Badi H (Ph.D.)1, Abdollahi M (Ph.D. student)2, Mehrafarin A (Ph.D.)1*, Ghorbanpour M (Ph.D.)3, Tolyat M (Ph.D.)1, Qaderi A (Ph.D.)1,Ghiaci Yekta M (M.Sc.)1

1- Medicinal Plants Research Centre, Institute of Medicinal Plants, ACECR, Karaj, Iran 2- Department of Horticulture, Science and Research Branch, Islamic Azad University (IAU), Tehran, Iran 3- Department of Medicinal Plants, Faculty of Agriculture and Natural Resources, Arak University, Arak, Iran *Corresponding author: Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, 55th Kilometer of Tehran-Qazvin Freeway, Karaj, Iran Tel: +98-26-34764010-19, Fax: +98-26-34764021 E-mail: [email protected]

Received: 29June 2016 Accepted: 7 Aug. 2017

Abstract

Thymol (2-isopropyl-5-methylphenol), and carvacrol (5-isopropyl-2-methylphenol) are the main components of the essential oils of some Laminaceae,Verbenaceae and Ranunculaceae members such as oregano, thyme, savory, etc. The formation of thymol and carvacrol is thought to involve hydroxylation of γ-terpinene and p-cymene precursors. They are produced by the plant species as a chemical defense mechanism upon exposure to pathogens, pests, herbivores, or environmental stresses. Accordingly, the potent antimicrobial and fungi toxic properties of these compounds against various plant pathogens have been demonstrated. They have antibacterial, antifungal, insecticidal, and anti-oxidative properties which are the basis for the wide use of these compounds in the cosmetic, food, and pharmaceutical industries.

Keywords: Thymol, Carvacrol, Biological activity, Biosynthesis pathway, Terpenes

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Introduction Two monoterpenes of the Lamiaceae that have Plants are sophisticated light-driven attracted much attention are thymol and “green” factories able to synthesize an carvacrol which are often found in thyme and immense number of bioactive natural products oregano. These two phenolic monoterpenes are [1]. These natural products are also referredto especially known for their anti-herbivore, as secondary metabolites since they are not antimicrobial, and antioxidant activities [9-13]. directly essential for thebasic processes of growth and development [2]. They can be Biosynthesis of terpenes divided into two major classes, the first class Terpenes (also known as terpenoids or formed by nitrogen-containing substances, isoprenoids) form the largest group of natural such as alkaloids, amines, cyanogenic products with more than 30,000 different glycosides, non-protein amino acids and structures [14]. Terpenes play important roles glucosinolates, and the second class consisting in almost all the basic processes, including of nitrogen-free substances which are plant growth, development, reproduction and represented by polyketides, poly-acetylenes, defense [8]. The oxidized monoterpenes, saponins, phenolics and terpenes [3]. thymol and carvacrol, are most likely derived The family Lamiaceae contains many from one of the initial cyclic products, aromatic plants of great scientific and γ-terpinene, by oxidation [15]. γ-terpinene economic interest such as rosemary, sage, formed from geranyl pyrophosphate (GPP) by mint, marjoram, oregano and thyme. The a monoterpene synthase, OvTPS2 [16]. aroma associated with these plants arises from Several evidences are supporting the the essential oil found in palate glandular involvement of cytochrome P450s in thymol trichomes on the aerial parts of the plant. and carvacrol biosynthesis. Eleven new These glandular trichomes consist of highly cytochrome P450 gene sequences are specialized secretory cells which the described from oregano, thyme and marjoram components of the essential oil are synthesized which were assigned to five new cytochrome and accumulate subsequently in subcuticular P450 genes. The expression levels of these storage cavity [4, 5]. The mono and sesqui- genes, quantified by relative and absolute terpenes are main compounds of essential oils quantitative real-time PCR, then correlated of oregano, thyme and marjoram [6]. These with thymol and carvacrol biosynthesis in substances are responsible for the aroma and Origanum vulgare L., Thymus vulgaris L., and flavor of these herbs, and the extracted Origanum majorana L. essential oils are used for the manufacturing of The mevalonic acid (MVA) pathway is perfumes and cosmetics as well as for located in the cytosol in peroxisomes and medicinal purposes as antimicrobial or endo-plasmatic reticulum (ER). It starts and antiseptic agents [6, 7]. Mono- and sesqui- overs with three units of acetyl-CoA and terpenes are also thought to help defend the farnesyl pyrophosphate (FPP) respectively, plant against herbivores and pathogens [8]. which is the precursor molecule for all sesqui-

2 Journal of Medicinal Plants, Volume 16, No. 63, Summer 2017 Naghdi Badi & et al. terpenes. The methyl-erythritol-phosphate three of these units are fused to form farnesyl (MEP) pathway is located in the plastids and pyrophosphate (FPP) the precursor of most the initial substrates are glyceraldehyde-3- sesqui-terpenes. Next, the linear carbon phosphate (GA3P) and pyruvate. Geranyl skeletons of GPP and FPP are converted to the pyrophosphate (GPP) is the precursor for all basic terpene skeletons by terpene synthases, a monoterpenes and geranyl geranyl- widespread class of enzymes responsible for the pyrophosphate (GGPP) is the precursor for huge structural diversity of monoterpenes and diterpenes. Carotenoids are derived from two sesqui-terpenes [20]. Terpene synthase genes units of GGPP. Dimethylallyl pyrophosphate have been isolated and characterized from (DMAPP) is the backbone to which different several Lamiaceae species, including the genus numbers of the isomer isopentenyl Mentha where a limonene synthase, an (E)-β- pyrophosphate (IPP) are added to form GPP, farnesene synthase, and a cis-muuroladiene FPP or GGPP. Ubiquinone is formed in synthase have been identified [21, 22]. From mitochondria [17, 18]. Terpenes are formed other Lamiaceae, terpene synthase genes are from the universal five-carbon building blocks, known from Salvia officinalis, Salvia pomifera isopentenyl pyrophosphate (IPP) and its and Salvia fruticosa [23], Rosmarinus isomer dimethylallyl pyrophosphate officinalis [24], Lavandula angustifolia [25] (DMAPP), which are both synthesized by the and Ocimum basilicum [26]. The initial plastidic methylerythritol pathway and the terpene synthase products (olefins and cytosolic mevalonate pathway (Figure 1) [8]. monoalcohols) are often further oxidized or The reaction starts by ionization of GPP conjugated. In Mentha, the biosynthesis of and the resulting geranyl cation is isomerized menthol and carvone involves hydroxylation to a linalyl intermediate capable of steps catalyzed by cytochrome P450 cyclizations. The initial cyclic α-terpinyl monooxygenases [21, 22, 26]. cation is the central intermediate and can be Plant terpene biosynthesis is often restricted subject to further cyclizations, hydride shifts to special morphological structures like and rearrangements before the reaction is idioblasts (e.g. oil cells in Laurus sp.) or ducts terminated by deprotonation or water capture. (e.g. resin ducts in Pinus sp.) or trichomes The number of carbon atoms in intermediates (e.g. glandular trichomes in Lamiaceae and and products corresponds to GPP [19]. Asteraceae) that can store these lipophilic Within the secretory cells, the five-carbon compounds in high concentrations (Figure 2) structure blocks for terpenes, isopentenyl [27, 28]. Studies on Lamiaceae species such as pyrophosphate and its isomer dimethylallyl mint (Mentha sp.) and sweet basil (Ocimum pyrophosphate, are synthesized by the basilicum) have provided many insights about plastidial methylerythritol pathway and the essential oil biosynthesis. The essential oil in cytosolic mevalonate pathway [8]. Two of mint and sweet basil is produced in glandular these five-carbon units are fused to form GPP, trichomes situated on the aerial parts of the the usual precursor of the monoterpenes, and plants. These glandular trichomes consist of a

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An Overview on … cluster of secretory cells covered by a oil accumulates [4, 5]. subcuticular storage cavity where the essential

Figure 1- Biosynthesis pathway of terpenes in the plant cell. Abbreviations: CDP-ME, 4-(cytidine 5’-diphospho)-2-C- methyl-D-erythritol; CDP-MEP, CDP-ME 2-phosphate; DMAPP, dimethylallyl diphosphate; DXP, 1-deoxy-D-xylulose 5- phosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; GPP, geranyl diphosphate; HBMPP, 1-hydroxy- 2-methyl-2-butenyl 4-diphosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl CoA; IPP, isopentenyl diphosphate; MEcPP, 2- C-methyl-D-erythritol 2,4-cyclodiphosphate; MEP, 2-C- methyl-D-erythritol 4-phosphate; MVA, mevalonic acid; MVP, 5- phosphomevalonate; MVPP, 5-diphosphomevalonate [8]

Figure 2-Schematic of peltate glandular trichomes of Lamiaceae. Schematic drawing of a peltate glandular trichome with a basal cell (b) and the head cells (h) based on one stalk cell (s). Secreted terpenes are stored in the subcuticular cavity (sc) as an oil drop (o). Eight larger cells are arranged around four smaller inner cells [28].

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Oxidation of monoterpenes peroxide. The first is Fenton reaction Chemical transformation of abundant and mechanism which is free radical mechanism cheap products into novel and more valuable (homolytic pathway) and second is the compounds can be achieved by liquid-phase peroxide complex mechanism (heterolytic oxidation reactions using hydrogen peroxide pathway) [17]. Oxidations with H2O2 can as clean oxidant and zeolite encapsulated involve homolytic pathways via free radical metal complexes as heterogeneous catalyst intermediates and/or heterolytic oxygen [29]. Hydrogen peroxide is a clean oxidant transfer processes [30]. because it is easy to handle and its reaction Carvacrol is used as reactant which is the produces only water as by-product [30]. major monoterpene component of essential oil. Catalytic oxidation of aromatic monoterpenes In the presence of transition metal complexes with hydrogen peroxide is a reaction of oxidation reactions with hydrogen peroxide industrial importance [31]. are direct activation (unproductive oxidation) Activation of hydrogen peroxide can be and productive oxidation reactions as seen in divided into two parts: Catalytic activation by Figure 3, and water is only by product. transition metal complexes and direct Catalysts (transition metal complexes) are activation (unproductive reaction), Fenton transferred oxygen from hydrogen peroxide to chemistry [32]. For these activations, two the reactant or substrate by a homolytic general types of mechanisms have been cleavage of the metal oxygen bond [17]. postulated for decomposition of hydrogen

Figure 3- Oxidation of carvacrol by hydrogen peroxide by using metal complex [31].

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Cytochrome P450s - a widespread enzyme explained [37]. The biosynthesis of thymol family and carvacrol is thought to involve Cytochrome P450s monooxygenases hydroxylation of γ-terpinene and p-cymene (P450s) form an ubiquitous class of enzymes precursors. Researcher found that an known from all kinds of organisms including Origanum vulgare L. (oregano) cDNA library animals, bacteria, fungi, archaea, protists and for sequences similar to those of known even viruses. Cytochrome P450 cytochrome P450 monoterpene hydroxylases monooxygenases are hemi-dependent mixed [38] (Figure 4). function oxidases that utilize NADH (Nicotinamide adenine dinucleotide Production of thymoquinone from thymol phosphate) and/or NADPH to reductively and carvacrol cleave atmospheric dioxygen producing a Thymoquinone has a commercial value functionalized organic product and a molecule considerably higher than its precursors of water [33]. All plant cytochrome P450s (thymol and carvacrol) found in thyme described to date are bound to membranes of essential oil. It can be obtained by catalytic the endoplasmic reticulum through a short oxidation of thymol and carvacrol. Essential hydrophobic segment of their N-terminus [34, oil rich in carvacrol and thymol were easily 35]. P450s need to be coupled to an electron oxidized to oil containing thymoquinone as the donating protein, a cytochrome P450 reductase main component in the presence of Fe (III) or a cytochrome b5, which is also anchored to porphyrin and phtylocyanine complexes [39]. the endoplasmic reticulum by its N- or C- The carvacrol oxidation with hydrogen terminus [33, 35]. γ-terpinene is the most peroxide was also studied using Mn (III) likely converted to thymol by the action of one porphyrin complexes and keggin-type or more cytochrome P450 oxidases, catalyzing tungstoborates [40, 41]. Oxidation of carvacrol a hydroxylation similar to (-)-S-limonene in yielded a mixture of benzoquinones containing menthol biosynthesis in Mentha sp. [26, 36]. a small amount of thymoquinone for keggin- Hydroxylation reactions catalyzed by type tungstoborates whereas for Mn (III) cytochrome P450s [33] which is important porphyrin complexes oxidation of carvacrol since the position of hydroxylation can selectively yielded thymoquinone. determine the downstream fate of the products Thymoquinone can be obtained in carvacrol in biosynthetic pathways. In mint, the oxidation reactions catalyzed by zeolite oxygenation pattern of monoterpenes is encapsulated metal complexes. The oxidation determined by the region specificity of P450 of carvacrol (<25% conversion) and thymol mediated (-)-S-limonene-hydroxylation either (<18% conversion) gave thymoquinone with at carbon C3 or C6. The structural similarity of 100% selectivity. However, leaching of the these highly region specific limonene porphyrin complex from the zeolitic matrix hydroxylases to those enzymes forming occurred in the presence of H2O2 [42]. thymol and carvacrol was previously

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Figure 4-Chemical structures of all substrates and major products formed by CYP71D178, CYP71D180v1 and CYP71D181 in vitro. (A) All three enzymes form mainly p-cymene from γ-terpinene, α-terpinene and (-)-R-α-phellandrene as indicated by the arrows. CYP71D178 forms thymol and carvacrol from γ-terpinene while CYP71D180v1 and CYP71D181 from only carvacrol. CYP71D181 forms carvacrol also from α-terpinene. (B) Major products formed from (-)-S-limonene and (+)-R-limonene. Mainly carveol is formed from (-)-S-limonene by all three enzymes. CYP71D180v1 and CYP71D181hydroxylate (+)-R-limonene only at carbon C6 while CYP71D178 catalyzes a hydroxylation at C3. Hydroxyl groups at carbon C2 in carvacrol corresponds to that designated as C6 in carveol. The numbering differs because of different substituent priorities in the p-cymene vs. limonene carbon skeleton [16].

Plants containing carvacrol and Thyme volatile phenolic oil has been reported thymol to be among the top 10 essential oils, with Carvacrol, thymol and p-cymene are antibacterial, antimycotic, antioxidative, natural p-menthane type of aromatic monoterpenes, food preservative, and mammalian age delaying which can be found in the essential oils of properties [46, 47]. Thymol and its chemical aromatic plants [40]. These two phenolic relative carvacrol are found not only in thyme monoterpenes are especially known for their and oregano, but also in many other plant anti-herbivore, antimicrobial, pharmaceutical species in different families [7, 48, 31, 49]. and antioxidant activities [43, 10, 44, 45, 13].

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Thymol γ-terpinene to thymol and carvacrol [16]. Thymol (also known as 2-isopropyl-5- Heterologous expression of two of them in methylphenol, IPMP) is a natural yeast resulted in active proteins catalyzing the monoterpenephenol derivative of cymene, formation of p-cymene, thymol and carvacrol C10H14O, isomeric with carvacrol. Thymol is from γ-terpinene. Since p-cymene itself was only slightly soluble in water at neutral pH, not accepted as a substrate, it is likely that but it is extremely soluble in alcohols and γ-terpinene is directly converted to thymol and other organic solvents. It is also soluble in carvacrol with p-cymene as a side product. strongly alkaline aqueous solutions due to The properties and sequence motifs of these deprotonation of the phenol. Thymol not P450s are similar to those of well- directly formed by the characterized terpene characterized monoterpene hydroxylases synthases. It is the aromatic monoterpene isolated from mint. The general outline of which is predicted to be synthesized from monoterpene biosynthesis is well known [8]. γ-terpinene (a product of OvTPS2) via First, the ubiquitous C10 intermediate, geranyl p-cymene [63, 64, 65]. pyrophosphate (GPP), is converted by Analysis of the terpene content of the enzymes known as monoterpene synthases to inbreed lines ff2 and ff7 (Figure 4) supports cyclic or acyclic products. Then, oxidized this hypothesis that no thymol is found in lines monoterpenes are formed from these initial that lack the γ-terpinene and p-cymene. cyclic or acyclic products by reactions γ-terpinene is most likely converted to thymol catalyzed frequently by cytochrome P450s by action of one or more cytochrome P450 monooxygenases [69]. The oxidized oxidases similar to (-)-S-limonene in menthol monoterpenes, thymol and carvacrol, are most biosynthesis in Mentha sp. [66, 22]. likely derived from one of the initial cyclic Conversion of γ-terpinene to thymol might products, γ-terpinene, by oxidation [64]. proceed via a p-cymene intermediate which The possible positions of 13C atoms are was detected in minor amounts in in vitro highlighted as colored bars and small boxes, assays of the γ- terpinene synthase OvTPS2. respectively (Figure 5); the color depends on However, these low levels of p-cymene may the metabolic pathway which leads to a certain be instead due to spontaneous conversion of metabolite. 3-phosphoglycerate (PGA) and γ-terpinene into p-cymene [67]. glyceraldehydes 3-phosphate (GAP) are built up in the Calvin cycle which generate either Biosynthesis pathway of thymol molecules with two 13C atoms (pink bar) or The biosynthesis pathway of thymol has molecules carrying three 13C atoms (red bars). been studied and coworkers identified thymol A label at position 1 exclusively is also as a terpenoid biosynthetic product despite the possible. The synthesis of monoterpenes is fact that it is aromatic [68]. It was possible via the MEP pathway (left side) based demonstrated the involvement of cytochrome on pyruvate and GAP which leads to a labelled P450 mono oxygenases in the conversion of

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13 13 Figure 5- Incorporation of C atoms derived from labelling experiments with CO2 into various metabolites during the plant metabolism [70]

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C2 block originating from pyruvate (dark red Carvacrol is an isomer and derivative of bar and dark green bar, respectively) and/or a phenol, the chemical formula of carvacrol C2 or C3 block originating from GAP carrying (cymophenol) is C6H3CH3(OH)(C3H7), a either two 13C atoms (pink and light green bar, monoterpenoid phenol[73,74]. Carvacrol is a respectively) or three 13C atoms (red/green bar liquid and has the same taste of thymol.The and red/green box, respectively), respectively. density of carvacrol ranges from 0.976 g/cm3 Red color marks carbon atoms coming from at 20 °C to 0.975 g/cm3 at 25°C. The boiling IPP and green color marks carbon atoms point of carvacrol is 237~238 °C. It can coming from DMAPP. On the right side the bevolatile with steam. Carvacrol is highly predicted labelling pattern of thymol arising lipophilic and insoluble in water [75]. Yadav from the mevalonate pathway based on acetyl- and Kamble (2009) reported that formation of CoA is shown. This path does not generate carvacrol could be resulted from alkylation of monoterpenes carrying three 13C atoms. Boxed o-cresol with propylene or isopropyl alcohol thymol is displayed with the labelling pattern (IPA) over solid acid catalysts [75]. Alkylation observed by NMR spectroscopy [70]. of o-cresol with propylene or IPA over solid acid catalysts results in the formation of Carvacrol carvacrol. Carvacrol does not have many long- Carvacrol (2-methyl-5-isopropylphenol) is term genotoxic risks. The cytotoxic effect of a phytochemical derived from aromatic plants carvacrol can make it an effective antiseptic of the genera oregano, thyme and satureja. and antimicrobial agent. Carvacrol has been Brownish liquid practically is insoluble in found to show antioxidant activity [76]. water and very soluble in ethanol (96%). Carvacrol is a monoterpene phenolic Biological activity of thymol and constituent of essential oils of various aromatic carvacrol plants. The essential oils obtained from the Effects of thymol on melanoma cells genera Origanum, Thymbra, Cordiothymus, Aromatic monoterpene, thymol, shows Satureja, and Lippia are rich sources of several beneficial activities, such as an anti- carvacrol (CRL). It is synthesized by the oxidative effect. However, the mechanism of specialized secretory cells in the aerial parts of their toxicity remains to be fully defined. the plants and is stored in a subcuticular Thymol was characterized as a melanin storage cavity. The essential oils containing formation inhibitor in an enzymatic system. the active substances are then extracted from The antioxidant actions of thymol generate a the plant materials by mechanical pressing or stable phenoxy radical intermediate, which steam distillation or by using organic solvents generates reactive oxygen species and quinone [71, 72].Carvacrol (C10H14O), is represented oxide derivatives. Thus, it is proposed that the by the synonyms: isopropyl-o-cresol, p- primary mechanism of thymol toxicity at high cymen-2-ol, 2-hydroxy-p-cymene, 5- doses is due to the formation of antioxidant- isopropyl-2-methylphenol and iso-thymol. related radicals [77] (Figure 6).

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Figure 6-Effects of thymol on melanoma cells.

Biological activity of carvacrol activities besides inhibiting lipid peroxidation Several in vitro and in vivo studies in broiler chicks [36]. described different bioactivity of carvacrol, including antibacterial, antioxidant, antiseptic, Antimicrobial activity antispasmodic, growth promoter, antifungal, The antimicrobial effects of essential oils antiviral, anti-inflammatory, expectorant, have been due to the presence of phenolic antitussive, immunomodulatory and chemo compounds, such as carvacrol, thymol, preventive as well as modifier of rumen eugenol, curcumin and cinnamaldehyde which microbial fermentation and reduction of are presented in essential oils of oregano, methane emission [9, 36, 78, 79]. Carvacrol thyme, clove, turmeric and cinnamon, plays a critical role as natural antioxidant in respectively [81-84]. Sikkema et al. (1995), the reduction of lipid peroxidation which Adam et al. (1998), Ben-Arfa et al. (2006) and leading to oxidative destruction of cellular Nostro and Papalia (2012) mentioned that the membranes [80]. Moreover, the deleterious beneficial inhibitory effects of phenolic effect of these compounds may lead to compounds could be attributed to the increase in the production of toxic metabolites interactions between the effective compounds (free radicals) and also to apoptosis (Figure 7) and cell membrane of microorganisms and is [79-81]. usually associated with the hydrophobicity of these compounds [84-87]. Improving nutrients bioavailability and Arsi et al. (2014) showed that numbers of growth/productive performance Campylobacter were reduced with 1% Researcher reported that feed carvacrol supplementation, or a combination supplementation with thymol + carvacrol of both thymol and carvacrol at 0.5% [88]. mixture by 60, 100, and 200 mg/kg of diet Friedman et al. (2002), Nastro et al. (2004) improved growth performance, digestive and Baser (2008) found antimicrobial enzyme activities, and antioxidant enzyme

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Figure 7-Modes of action and biological activities of carvacrol [79-81]

influences of carvacrol against many species and antimicrobial activity [93, 94, 95]. Thymus of microbes such as Pseudomonas, daenensis essential oils have significant Aspergillus, Salmonella, Streptococci, antifungal properties against three pathogens: Listeria, Bacillus and Fusarium [89, 90, 71]. Rhizoctonia solani, Fusarium solani and Also as carvacrol supplement as antimicrobial Alternaria solani. In all samples, the component has a significant impact on harmful maximum antifungal activity of the essential bacteria including Escherichia coli and oil was observed in flowering stage but this Salmonella numbers in chickens, this effect different was not significant. The results may be attributed to inhibit the growth of showed that, the highest thymol content was pathogenic bacteria by carvacrol vapor [89- obtained in flowering stage and the greatest 91]. antifungal activity was observed in this period. The amount of thymol contents was lower in Antifungal activity of thymol and carvacrol pre flowering period than flowering and an Thymol is normally the major phenolic antifungal activity was reduced in this period component in common thyme [92]. Zataria [93, 94, 95]. Ahmadi et al. (2015) reported that multiflora effectively inhibited growth of phonological and ontogenetically variation Aspergillus niger [49]. Antifungal activity caused changed in the main components in T. against A. niger were reported by Siddiqui et daenensis essential oil and these components al. (1996) [92]. The high phenolic contents of can be effect in antifungal properties in this Z. multiflora such as thymol, p-cymene and plant [55]. According to Saez (1998) reported carvacrol may account for its strong antifungal that essential oil of T. hymalis possessed a

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An Overview on … high level of p-cymene and γ-terpinene in and plant extracts containing antioxidants flowering stage [63]. Also, Gergis et al. (1990) including phenolic compounds may prevent and Panizzi et al. (1993) were reported that from free radical damage [101, 102, 103]. The essential oils rich in phenolic compounds antioxidant activity of plant extracts were (carvacrol and thymol) possessing high levels assessed by the DPPH (2, 2-diphenyl-1- of antifungal activity [95, 96]. Karaman et al., picrylhydrazyl) free radical scavenging (2001) reported that T. revolutus Celak method and ferric reducing antioxidant power essential oil contains high level of carvacrol, (FRAP) assay [104]. The highest antioxidant p-cymene and γ-terpinene and has antibacterial activity of T. daenensis was observed in and antifungal activity [97]. flowering stage, while the lowest value was The marked difference between the detected in preflowering stage. Also the metabolism of carvacrol and thymol by highest FRAP value was observed in flowering Colletotrichum acutatum may reflect the stage than preflowering stage. Also it was higher toxicity of thymol against this founded the coefficient correlation between pathogenic fungus. In the same way, small total phenolic content and radical scavenging differences between the metabolisms of both capacity. These results suggest that the major compounds by Botryodiplodia theobromae are part of the antioxidant activity in T. daenensis consistent with the observed similarities in the results from the phenolic compounds. antifungal activity against this fungus. Based According to, their results showed that on the structures of the metabolites, metabolic increasing of phenolic content, caused increase pathways for the biotransformation of thymol in antioxidant activity in different stages of by C. acutatum and B. theobromae were harvesting times in T. daenensis. However, the proposed [31]. antioxidant activity in plants is not caused only by phenols and may also come from the Antioxidant activity and scavenging of free presence of other antioxidant secondary radicals metabolites such as volatile oils, carotenoids Phenols are a class of compounds, which and vitamins. Iranian T. daenensisare strong act as free radical scavengers and are radical scavengers and can be considered as a responsible for the antioxidant activity in good source of natural antioxidant for many medicinal plants [61, 98, 99]. Free traditional and medicinal uses [104]. The radicals may cause many disease conditions antioxidant activity by radical scavenging such as heart diseases and cancer [46, capacity of Iranian and British T. vulgaris was 100].These compounds that can delay or shown in Table 4. The highest antioxidant prevent the oxidative damage of lipids or other activity was observed in Iranian T. vulgaris molecules caused by free radicals, can (7.76 μg/mL), while the lowest value was 8.05 probably be protective against the μg/mL in British T. vulgaris [105]. This result development of major diseases such as suggests that the major part of the antioxidant coronary heart disease and cancer in human activity in T. vulgaris results from the phenolic

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compounds. This is in line with the observation feed conversion ratio, immune response, of other authors who found similar correlations antioxidant status, carcass traits and quality, between total phenolic content and antioxidant and lowered morbidity and mortality rates activity of various plants [106, 58]. [111-114, 82]. In fact, carvacrol component is Free radicals or reactive oxygen added to various ingredients, such as intermediates are generated by cells during the nonalcoholic beverages (28.54 mg/kg), baked normal metabolism. When free radicals are goods (15.75 mg/kg), chewing gum (8.42 accumulated excessively, this leads to damage mg/kg), etc. [115]. However, the mode of in tissue and privation of many cellular action of this compound was unknown by functions. Carvacrol as an antioxidant protects many researchers. A good knowledge of the cells against free radicals. Moreover, carvacrol mode of action is very required antioxidants inhibit prostaglandin synthesis regarding application in nutritional systems. and induct drug-metabolizing enzymes in Formerly, Ultee et al. (1998) reported the addition to many biological activities as antimicrobial activity of carvacrol on pathogen reported by Azirak and Rencuzogullari (2008) Bacillus cereus [115]. Carvacrol is a [107]. Some studies assured the efficiency of hydrophobic compound and has an effective carvacrol in scavenging free radicals i.e. nitric impact on biological membranes. Carvacrol oxide, superoxide radicals, peroxyl radicals plays a key role as antiviral component against and hydrogen peroxide [79, 108-111]. The human rotavirus (RV). On the same context, existence of hydroxyl group (OH) which Mexican oregano (Lippia graveolens) extract linked to aromatic ring is suggested to be the and oil as well as carvacrol component are reason for the highly antioxidant activity of able to reduce/inhibit the viral diseases in carvacrol either in vitro or in vivo as explained animal and human. Specifically, the antiviral by Aeschbach et al. (1994) and Guimaraes et activity of oregano and its phenolic al. (2010) [109, 110]. The reaction of components on acyclovir resistant herpes carvacrol with a free radical is facilitated due simplex virus type 1 (ACVRHHV-1) and to its weak acid character, so donating human respiratory syncytial virus (HRSV) and hydrogen atoms to an unpaired electron, of carvacrol on RV have been documented producing another radical that is stabilized by [116-118]. Acamovic and Brooker (2005) and electron scattering generated at a molecule Silveira et al. (2013) reported that herbs rich in resonance structure [111]. flavonoids such as thyme and carvacrol could improve the immune functions through acting Anti-carcinogenic and antiplatelet effects as antioxidants and extending the activity of Several studies have been reported the vitamin C [118]. addition of some phytogenic additives or their products such as cold pressed oil, essential oil Anti-obesity effect of carvacrol or extracts to animal and poultry diets that Obesity is a medical condition in which improved live body weight, body weight gain, excess body fat accumulates to the extent that

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An Overview on … it may have a negative effect on health, inflammatory agents by preventing free leading to increased health problems. It is like radicals and hazardous compounds from other chronic diseases such as hyperlipidemia, interacting with cellular DNA and its ability to cancers and diabetes. Umaya and Manpal change the gut microflora, improving digestion (2013) stated that the main factor in enhancing coefficient and absorption of nutrient obesity and attributed to the metabolic diseases compounds [112, 122]. in humans and animal models is the consumption of high levels of fat in the diet Application of thymol and carvacrolin and they found that carvacrol caused an postharvest technology inhabiting of fat accumulation between cells Food safety is one of the major issues and adipocyte differentiation in mouse embryo related to fresh fruits and vegetables. More 3T3- L1 cells [119]. Also, results showed that degradable natural compounds that can be diet high in fat and supplemented with regarded as safe to human health and carvacrol decreased total visceral fat, plasma environment are alternatives for synthetic and liver total cholesterol, HDL-cholesterol, chemicals used to control postharvest fruit triglyceride and free fatty acids of mice. rots. Results of recent studies on control of Moreover, carvacrol decreased the expression fungal rots using natural substances indicate of adipogenesis related genes- fibroblast that demands for research and development of growth factor receptor in visceral adipose natural fungicides are just gaining momentum tissues. Carvacrol decreased the expression of globally [53]. Essential oil not only improved receptors which stimulates the intake of fat the storage life of lime fruits, but also caused rich diet such as galanin receptor 1 and 2 no undesirable change in the appearance and [119]. Wieten et al. (2010) and Cho et al. quality of the fruits. Lime fruits treated with (2012) found that free fatty acid levels and the essential oil of Z. multiflora did not show any mRNA and protein levels of toll-like receptors change in taste and odor even after 50 days of were reduced by carvacrol [120, 121]. Free storage, a property that makes this essential oil fatty acids in high levels are reported in obese an acceptable candidate for controlling animals, because of their release either from postharvest lime fruit rot [50, 53]. high fat diet or from adipose tissues. Carvacrol as anti-obese drug needs for more detailed Various factors affecting thymol and studies to be recommended for this purpose. carvacrol contents in plants Such properties could be due to its ability as In nature several factors affect production of antimicrobial, antioxidant, antifungal, secondary metabolites in commercially immunomodulatory, anticancer and anti- important plants [123] (Figure 8).

16 Journal of Medicinal Plants, Volume 16, No. 63, Summer 2017 Naghdi Badi & et al.

Figure 8- Factors affecting production of secondary metabolites in plant cells [81-123]

Origin of plant showed a high variability, with carvacrol, According to Alizadeh et al. (2013), fresh thymol or α-terpineol as the major components and dry weight of British Thymus vulgaris was [124]. higher than those of Iranian T. vulgaris. Moreover, the essential oil yield and efficiency Environmental conditions of the aerial parts in British T. vulgaris The biosynthesis of secondary metabolites, significantly increased compared with Iranian although controlled genetically, is affected T. vulgaris. These variations may be attributed strongly by environmental factors. to the different origins (Esfahan and London) Agricultural practices have a critical effect on of cultivated seed [105]. Alizadeh et al. (2013) quantitative and qualitative characteristics of and Saez (1998) reported that, diversity of plant-derived metabolites, which finally result plant origins, seasonal variations or different in plant growth and yield increment [125]. One ontogenesis and environmental conditions way of increasing the consumption of health- affected on essential oil yield and composition promoting secondary plant metabolites in the in T. daenensis, T. vulgaris and T. hymalis diet would be by increasing metabolite levels [105, 63].In a study by Santos et al., (2005), in the fruit and vegetables themselves [57]. the essential oils of Thymus caespititius from Upgrading vegetables and fruits in this respect populations collected in mainland Portugal and could be done by breeding, genetic on Madeira were characterized to be α- engineering, or modification of secondary terpineol, whereas those obtained from metabolite biosynthesis by elicitor populations collected on nine Azorean islands applications, which provides immediate

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An Overview on … response. In the latter case identification of the acting as feeding deterrents against herbivores, key enzymes or key genes targeted by these pollination attractants, protective compounds elicitor treatments is necessary. Several against pathogens or various abiotic stresses, elicitors including chemical, physical, and antioxidants and signaling molecules. Low biological elicitors for influencing levels of UV-B radiation can cause distinct glucosinolate biosynthesis has been applied. changes in the plant’s secondary metabolism Piccaglia and Marotti (1993) reported that resulting in the accumulation of a broad range during their two-year study differences on of secondary plant metabolites [127]. relative amounts of thymol, carvacrol, γ-terpinene, and p-cymene in essential oils of Light Satureja montana which is grown in Italy, In order to find the light effects on amount could be attributed to the effects of of essential oil, Thymus was put under several environmental conditions [125]. intensity of light (sunshade, cloudy, 15, 27, 45 and 100% light) and recognized that the UV radiation maximum essential oil concentration and According to the international commission thymol found in 100% sun light, and length of on illumination, ultraviolet (UV) wavelength leaf decreased with reduction of light levels. (200-400 nm) is a small part of the solar Amount of essential oil not only depended on radiation reaching the Earth’s surface, which is grow stage of plant but also temperature, divided into UV-A (315-400 nm), UV-B (280- moisture, quality and quantity of light, rainfall 315 nm) and UV-C (200-280 nm), and it are influenced on it [128]. affects negatively all living organisms. The harmful effect of UV radiation increases Temperature towards the shorter wavelengths. Solar Omer (1998) on Origanum syriacum radiation in the UV-B range corresponds to a obligated that the phenolic compounds minor percentage of the total solar energy but increase in hot seasons at the expense of their it is potentially harmful because these short preceding precursors. In other words, the wave lengths are capable of causing relative percentage of carvacrol was higher in deleterious effect in cells. Plants are the second cut than in the first cut. This may vulnerable to increased UV-B radiation be attributed to the effect of the environmental because many cellular components such as factors especially non-edaphic factors, since nucleic acids, proteins, lipids and quinones can these plants grew in summer months under absorb UV-B radiation directly [126]. The high temperatures and received more solar treatment with UV light, particularly from the energy than those grown in the spring UV-B range, is an example for effective summers. These conditions accelerate the elicitor application. Secondary plant transformation of terpinene and p-cymene to metabolites mediate many aspects of the phenolic compounds [129]. interaction of plants with their environment by

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Drought and salinity stress Plant growth By increasing drought stress, essential oil Both moisture and the plant growth phase percentage and essential oil components had had a significant effect on the plant material no significant difference, but there was a production. The crop yield increased significant difference between essential oil significantly with increasing moisture and age yields (P≤0.01). Because essential oil yield is of the Mexican oregano plants [133]. obtained from dry yield multiply to essential Although, older plants contained less oil than oil percentage [75]. Salinity stress had no the younger plants, the differences were not effect on essential oil percentage and essential statistically significant. Total thymol and oil components but decreased essential oil carvacrol contents of oregano oils obtained yield [130, 131]. When irrigation volume from younger plants were higher than that of decreased from 50 to 16 L/m2 Satureja the mature plants. The amount of water khuzistanica and S. bachtiarica dry matter received by the plant did not have a significant were decreased up to 56 and 54%, effect on the thymol and carvacrol content of respectively. Thus, essential oil yield the oil isolated from Mexican oregano [133]. decreased (> 50%). S. bachtiarica and S. khuzistanica essential oil had 14 and 11 Harvesting time (phenological stages) components, respectively. Water stress caused Study about the effects of phenological an increase in oil production of thyme [57, stages such as harvesting times on herbage 132]. It has been suggested that increased oil yield and quantity and quality of essential oil gland density, accompanied with higher is very important goal to obtain maximum absolute number of gland production during productivity of valuable medicinal plants [134, stress, could be the reasons behind 135]. Harvest of Thyme is the critical point in accumulation of essential oil in some plant agricultural management of this plant. Stahl- species. Other factors could be the net Biskup and Saez (2005) reported that the best assimilation or the partitioning of assimilates time of harvest is early and middle of among growth and differentiation processes. flowering period to get maximum effective Sometimes, the decline in the primary material [65]. The yield of plant materials, metabolism of plants during stress could lead essential oil composition and secondary to the accumulation of certain intermediate metabolites in medicinal and aromatic plants products, which get channelized to form are strongly influenced by harvest time, secondary metabolites like essential oil. In ecological and climatic conditions, plants with decreased levels of essential oil ontogenetically variation and the others under stress, the lowering could be due to the environmental factors such as soil type and overall anabolism which gets inhibited on nutrition [54, 136-138, 64, 45]. Knowledge of exposure to saline conditions [123, 130-132]. the factors that influence on yield and essential

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An Overview on … oil composition in medicinal plants is stage [140]. Ozguven and Tansi (1998) insufficient, and these factors seems to play an reported that increasing day-length and important role in drug yield and essential oil sunlight significantly affected fresh and dry composition of medicinal and aromatic plants weight of Thymus vulgaris [141]. Essential oil [138-140]. Sefidkon et al (2009) studied the yield and efficiency data are reported in Table effects of harvest stages and various methods 2. Essential oil yield and efficiency of of hydro-distillation on essential oil efficiency T. daenensis increased at flowering stage, but of garden thyme, phenological stages had not significant. This is due to high yields of significant effect on essential oil efficiency fresh and dry biomass and content of oil and [138]. Mean comparison results showed that thymol in this stage. the highest efficiency (1.18%) belonged to beginning of blooming and vegetative phase Fertilizers had the lowest (0.86 %) value. Also, some Physical, biological and chemical properties studies showed that the highest herbage yield of soil have effect on grow and effective and essential oil of garden thyme were materials of medicinal plants [55]. With obtained in lower elevations and in full fertilizing of medicinal plants, initial and blooming stage [138]. Khorshidi et al (2010b) necessary elements of grow such as nitrogen, investigated four various stages in two regions phosphorus and potassium are supplied. Foliar to assess the effect of climate and phenological application of nitrogen significantly can be stages on essential oil percentage of Denaian increased the plant height of thyme thyme (T. daenensis Celak.) [54]. Results (T. vulgaris), percentage of thymol and showed higher essential oil percentage in full essential oil [69-70, 128, 132]. blooming and fruit set stage in both regions. Golparvar (2013) recommended harvesting Nitrogen Kouhi thyme (T. kotschyanus) in fruit set stage Nitrogen is essential and vital element for to obtain the highest amount of essential oil plant growth and development. Review of and thymol yield as well as fresh and dry literature showed that the increasing amount of herbage [134]. nitrogen fertilizer caused an increase in Effects of different harvesting times on Thymus yield. Fertilizer treatments did not fresh and dry weights (g/plant) are presented effect on amount of essential oil and thymol, in Table 2. Thymus daenensis fresh weight was but with attention to effect of it on plant yield, increased at flowering stage but this is not it can increase thymol and oil yield [38, 132]. significant. Dry weight of Thymus daenensis Research showed that the application of was significantly increased at flowering stage. nitrogen produced more thymol yield in It seems that increasing day-length, Thyme oil [8]. Also, it has been reported that temperature and sunlight in flowering stage nitrogen increased Thymus vulgaris oil yield compared to pre-flowering stage causing an and thymol content [132, 142, 143]. increase in fresh and dry weight of flowering

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Table 2- Means of T. daenensis oil content at different stages of plant growth by hydro-distillation [140] Plant growth stage Essential oil content (%) Thymol (%) Carvacrol (%) Full flowering 2.28a 73.4 2.2 Beginning of flowering 2.04b 72.6 2.3 Before flowering 1.77b 70.1 3.1

Biological fertilizers an experiment conducted on the medicinal Now a day, use of beneficial soil plant Rosado Paraguayan garlic, Arguello et al microorganisms called biological fertilizers as (2006), have shown that application of vermi- the most natural and desirable solution for compost may cause a considerable increase in keeping the soil alive and active is widely the height of the shrub of the plant [145]. considered. Biological fertilizers like Similar results have also been obtained on Azotobacter absorb and increasing the fennel and Chamomile [146, 147]. concentration of essential elements such as nitrogen, phosphorus, potassium, zinc, magnesium, iron and protein in crops [57]. Conclusion Natural plant chemicals are the potential Effects of animal fertilizers source of agrochemicals, flavors, and Akbarnia et al. (2004) have stated that pharmaceuticals, known as secondary chemical fertilizers have no effect on the oil metabolites, which are synthesized by plants content of the seeds of Bishop's weed (also when exposed to different elicitors and/or known as Carom, Ajowan, Ajwain), however, inducer molecules. Thymol (2-isopropyl-5- application of animal fertilizers results in a methylphenol), a natural monoterpenephenol dramatic increase in the seed yield and oil derivative of cymene, isomeric with carvacrol content of the plant [144]. Furthermore, (5-isopropyl-2-methylphenol), are found as application of animal fertilizers up to 20 tons major constituents of essential oil in members per hectare results in a substantial increase in of the Laminaceae, Verbenaceae and the percentage of thymol in the oil while no Ranunculaceae. Biosynthesis of thymol and effect can be observed on the percentages of carvacrol is thought to involve hydroxylation gamma-terpene and p-cymene. Mallanagoula of γ-terpinene and p-cymene precursors. These (1995) has reported the positive effects of components are known as biocides, with a animal fertilizers on the improvement of wide spectrum of antimicrobial, anti- quality in medicinal herbs [36]. Animal inflammatory, anti-leishmanial, antioxidant, fertilizers due to having great advantages such hepato-protective and anti-tumoral activities, as maintenance of water in the soil and which have been the subject of several inclusion of nutritive substances can help in investigations in vitro and in vivo. The the increase of the essential oil content of the presence, content and constituents of these plants by augmentation of vegetative growth, compounds in medicinal and aromatic plants which comprises the most oil content. Through may be affected in a number of ways, from

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An Overview on … their formation in the plant cell to their final benefits and biological properties may play isolation. These include: (a) plant origin (b) critical role in their beneficial applications in environmental conditions (c plant growth and different industries by providing further physiological variations (d) phenological understanding of the health usages and stages (e) and agronomical practices, etc. improving performance parameters in Exploration of thymol and carvacrol modes of agriculture species. Further experiments may action like pharmacological, nutritional, health be worthy of evaluation for future.

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