Pharmacological Properties and Molecular Mechanisms of Thymol: Prospects for Its Therapeutic Potential and Pharmaceutical Development.

Thymol, chemically known as 2-isopropyl-5-methylphenol is a colorless crystalline monoterpene phenol. It is one of the most important dietary constituents in thyme species. For centuries, it has been used in traditional medicine and has been shown to possess various pharmacological properties including antioxidant, free radical scavenging, anti-inflammatory, analgesic, antispasmodic, antibacterial, antifungal, antiseptic and antitumor activities. The present article presents a detailed review of the scientific literature which reveals the pharmacological properties of thymol and its multiple therapeutic actions against various cardiovascular, neurological, rheumatological, gastrointestinal, metabolic and malignant diseases at both biochemical and molecular levels. The noteworthy effects of thymol are largely attributed to its anti-inflammatory (via inhibiting recruitment of cytokines and chemokines), antioxidant (via scavenging of free radicals, enhancing the endogenous enzymatic and non-enzymatic antioxidants and chelation of metal ions), antihyperlipidemic (via increasing the levels of high density lipoprotein cholesterol and decreasing the levels of low density lipoprotein cholesterol and low density lipoprotein cholesterol in the circulation and membrane stabilization) (via maintaining ionic homeostasis) effects. This review presents an overview of the current in vitro and in vivo data supporting thymol’s therapeutic activity and the challenges concerning its use for prevention and its therapeutic value as a dietary supplement or as a pharmacological agent or as an adjuvant along with current therapeutic agents for the treatment of various diseases. It is one of the potential candidates of natural origin that has shown promising therapeutic potential, pharmacological properties and molecular mechanisms as well as pharmacokinetic properties for the pharmaceutical development of thymol.

Thymol, chemically known as 2-isopropyl-5-methylphenol is a colorless crystalline monoterpene phenol. It is one of the most important dietary constituents in thyme species. For centuries, it has been used in traditional medicine and has been shown to possess various pharmacological properties including antioxidant, free radical scavenging, anti-inflammatory, analgesic, antispasmodic, antibacterial, antifungal, antiseptic and antitumor activities. The present article presents a detailed review of the scientific literature which reveals the pharmacological properties of thymol and its multiple therapeutic actions against various cardiovascular, neurological, rheumatological, gastrointestinal, metabolic and malignant diseases at both biochemical and molecular levels. The noteworthy effects of thymol are largely attributed to its anti-inflammatory (via inhibiting recruitment of cytokines and chemokines), antioxidant (via scavenging of free radicals, enhancing the endogenous enzymatic and nonenzymatic antioxidants and chelation of metal ions), antihyperlipidemic (via increasing the levels of high density lipoprotein cholesterol and decreasing the levels of low density lipoprotein cholesterol and low density lipoprotein cholesterol in the circulation and membrane stabilization) (via maintaining ionic homeostasis) effects. This review presents an overview of the current in vitro and in vivo data supporting thymol's therapeutic activity and the challenges concerning its use for prevention and its therapeutic value as a dietary supplement or as a pharmacological agent or as an adjuvant along with current therapeutic agents for the treatment of various diseases. It is one of the potential candidates of natural origin that has shown promising therapeutic potential, pharmacological properties and molecular mechanisms as well as pharmacokinetic properties for the pharmaceutical development of thymol.
Keywords: thymol, antioxidant, free radical scavenger, cancer, animals, drug discovery, phytochemicals, natural compounds INTRODUCTION Extensive epidemiological and experimental studies have suggested the health benefits of medicinal herbs as well as their constituents against various human ailments (Sofowora et al., 2013). The therapeutic importance of plants have been quoted in the ancient cultures and traditions of many countries and societies and they are believed to be cost effective and safe. Since ancient times, plants and their products are used as a culinary preparation or as a remedy in different traditional medicine for many diseases (Rahmani et al., 2014). Turmeric, oregano, thyme, olives and dates, to name a few, have been used extensively for culinary purposes in diets and are also believed to possess beneficial effects against numerous diseases (Rahmani et al., 2014). Among these spices, the Greeks, Romans, and Egyptians have used thyme as a preservative, odorant and flavoring agent in foods. It is a small subshrub abundantly used as a traditional medicine in the western Mediterranean region and its leaves are often used as herbal medicinal products and food additives (Zarzuelo and Crespo, 2002). Thyme possesses potent antibacterial, antifungal, sedative, antiseptic, antioxidative, expectorant, antispasmodic, antifungal, antivirotic, antihelminthic, carminative and diaphoretic effects (Rustaiyan et al., 2000;Soliman and Badeaa, 2002). Thyme contains abundant amount of terpenoids, flavonoids, glycosides and phenolic acids (Vila, 2002).
Thymol is biosynthesized by the hydroxylation of p-cymene after the aromatization of γ-terpinene to p-cymene (Poulose and Croteau, 1978). The lethal dose (LD 50 ) value of thymol for rats and guinea pigs is 980 mg/kg and 88 mg/kg, body weight, respectively (Jenner et al., 1964). For male and female ddY mice the LD 50 is 1200 and 1050 mg/kg, respectively (Hasegawa et al., 1989), whereas the LD 50 for cats, rabbits and mice are 250 mg/kg, 750 mg/kg and 640 mg/kg, respectively (Instituto Superiore di Sanita, 1999). The time-tested safety and activity of thymol can also be attested to by the use of thymol for centuries in different cultures and civilizations. One example is the use of thymol containing herbs by the ancient Egyptians for the preservation of mummies. According to the Environmental Protection Agency, there are no known adverse effects with respect to thymol when used in animals and humans. Thymol is cataloged by the United States Food and Drug Administration as 'Generally Recognized As Safe' for use as a food additive, therefore it is considered to be safe with negligible toxicity.
An extensive literature survey has revealed some excellent reviews on essential oils regarding their phytochemical and pharmacological activities based on their various pharmacological properties. There is a short review describing the therapeutic and pharmacological effects of thymol (Parasei et al., 2016) and a detailed one presenting the antimicrobial properties of thymol (Marchese et al., 2016). But there are no reviews that have been published focusing on the multipharmacological properties of thymol against various human ailments until now. Thus, thist review aims to reveal the various pharmacological activities and therapeutic potential of thymol as well as prospects for its pharmaceutical development followed by its mechanism of action demonstrated in both in vivo and in vitro studies.

Chemistry and Physical Characteristics of Thymol
Thymol (2-isopropyl-5-methylphenol) is a white crystalline substance that gives thyme its strong flavor, pleasant aromatic odor and strong antiseptic property. Its density at 25 • C is 0.96 g/cm 3 with a melting point ranging from 49 • C to 51 • C (322-324 K; 120-124 • F) (Jordan et al., 1991;Lide and Frederikse, 1996). It is highly soluble in alcohols, alkaline solutions and other organic solvents due to the deprotonation of phenol but it is slightly soluble in water at neutral pH and it absorbs maximum UV radiation at 274 nm (Norwitz et al., 1986;Wade and Reynolds, 1997). It has low solubility in water and its unpleasant taste and smell makes it less palatable . It also has low solubility in the hydrophobic domain of the bacterial cytoplasmic membrane due to its hydrophobicity (Trombetta et al., 2005). For the past few decades, the synthesis of thymol has been achieved using the reaction between p-cymene and m-cresol with isopropyl alcohol or propene and by the use of supercritical CO 2 (Amandi et al., 2005;Nagle et al., 2013). Thymol is an important agent of natural origin and has generated interest in the scientific community in pharmacological studies for its therapeutic potential in different diseases. The present review presents an overview of its preclinical data, pharmacokinetics, pharmacological properties and therapeutic potential in different human diseases.

Pharmacokinetics of Thymol
Absorption Previous reports have revealed the rapid absorption of thymol following oral administration and its degradation in the stomach or intestine (Michiels et al., 2008;Anderson et al., 2012). A report from Schroder and Vollmer (1932) has evidenced the presence of thymol in the stomach, intestine, and urine after its oral administration with sesame oil at a dose around 500 mg in rats and 1-3 g in rabbits. A single dose of thymol (1 or 3 g) encapsulated in gelatin capsule administered to dogs showed the presence of thymol conjugates in urine (22 or 34%) after 3-4 h following urine and fecal analysis (Robbins, 1934). Oral administration of a single dose of thymol (50 mg/kg) was rapidly absorbed and slowly eliminated approximately within 24 h . The maximum concentration (T max ) was reached after 30 min, while approximately 0.3 h was needed for the half-life of the absorption phase (t 1/2 ). The lower concentrations of thymol were recovered in the liver, lungs, kidneys, and muscles while its higher concentrations were detected in the mucosa and other inner contents of the intestines indicating its partial absorption . According to the report of Kohlert et al. (2002), after the intake of one Bronchipret R TP tablet that is equivalent to 1.08 mg of thymol, the plasma concentrations of thymol metabolites were detectable after 20 min. The rapid absorption of thymol indicates that it's mainly absorbed in the upper component of the gut. In healthy volunteers, the oral administration of one Bronchipret R TP tablet resulted in a peak plasma concentration (C max ) of 93.11 ng/ml, T max of 1.97 h, t 1/2 of 10.2 h, area under time curve from time 0 to clast (AUC 0→clast of 837.3 ngh/ml, mean residence time after extravascular administration (MRT abs ) of 12.6 h and a mean absorption time (MAT) of 0.53 h (Kohlert et al., 2002). It also had a total body clearance of 1.2 l/h, volume of distribution at steady state of 14.7 l and a volume of distribution during the elimination phase of 17.7 h was reported in healthy humans (Kohlert et al., 2002).

Distribution
Free thymol is usually not detectable in human plasma. It is circulated as thymol sulfate, not glucuronide, in the blood stream as detected by liquid chromatography-mass spectrophotometry/mass spectrophotometry (LC-MS/MS). Thymol sulfate has been detected in plasma 20 min after administration. The maximum plasma levels (93.1 ± 24.5 ng/ml) of thymol were reported after 1.97 ± 0.77 h of administration. After administration the bioavailability of thymol measured in plasma as thymol sulfate was found to be about 16%. It is eliminated by kidneys and is measured in the urine as thymol conjugates. The volume of distribution (Vdss/f) of 14.7 L has revealed that thymol sulfate mainly resides in the extracellular space (Kohlert et al., 2002).

Metabolism
Thymol undergoes glucuronidation by uridine 5 -diphosphoglucuronosyltransferase (UGT) following secretion into the proximal tubule (Raoof et al., 1996;Shipkova et al., 2001). The absence of thymol glucuronide in plasma could be due to the lower activity of hepatic UGT compared to sulfotransferase and the formation of glucuronide was shown only at much higher doses (Ogata et al., 1995). In healthy human volunteers, thymol (0.6 g/kg) was metabolized to thymol sulfate, thymol glucronide and thymol thymohydroquinone sulfate and it was excreted in urine (Takada et al., 1979).

Elimination
The elimination of thymol conjugates in urine was detectable for the first 24 h, with the majority being eliminated after 6 h. The combined amount of both thymol sulfate and glucuronide excreted in urine during the first 24 h was 16.2 ± 4.5% of thymol intake. The renal clearance was calculated to be 0.271 ± 0.7 L/h (Kohlert et al., 2002). Takada et al. (1979) studied the metabolism of thymol in rabbits and humans, wherein thymol (0.5 g/kg) fed to rabbits metabolized to thymol glucuronide as the main metabolite of thymol and eliminated as glucuronic acid and sulfuric acid metabolites (Takada et al., 1979). Austgulen et al. (1987) reported the rapid excretion of thymol and its metabolites in the urine of male albino Wistar rats after thymol was dosed by gavage (1 mM/kg) as analyzed by capillary gas chromatographymass spectrometry (GC-MS). Williams (1959) summarized the previous reports on the metabolism of thymol and reported its excretion as sulfate and glucuronide conjugates in the urine of dogs, rats and humans. Around 1g (one third) of the dose was excreted in the urine of dogs while feces were found to be devoid of thymol (Robbins, 1934).

Antioxidant Properties
The antioxidant properties of thymol have been well documented in various preclinical studies including cell lines and animal models. At high rate constants, it effectively scavenged the hydroxyl free radicals thereby producing major transient species named phenoxyl radicals. The generated adducts from the phenoxyl radicals undergo dehydration which can be accelerated by an alkaline medium. The addition of hydroxyl radicals at the ortho position (C6 atom) of the phenolic group yields the phenoxyl radical after dehydration. The attack at the ortho position is more favorable energetically while the attack at the para position is also expected to occur. Furthermore, additions at the ortho positions occur without any precomplex formation. The non-toxicity and redox potential of the thymol•/thymol couple makes it a promising antioxidant (Venu et al., 2013).
One of the most studied effects of thymol includes the scavenging of free radicals by increasing the activities of several endogenous antioxidant enzymes levels viz. superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione-S-transferase (GST) along with non-enzymatic antioxidants such as vitamin C, vitamin E and reduced glutathione (GSH) (Nagoor Meeran and Prince, 2012). A comparative study revealed that thymol has superior reducing power, DPPH, superoxide and hydroxyl radical scavenging activity and bestows protection against oxidative damage to lipids (Nagoor Meeran et al., 2015b). The various supportive evidences for the antioxidant activity of thymol are detailed below.

In Vitro Studies
Thymol was shown to exhibit potent superoxide anion, hydroxyl and DPPH radical scavenging and reducing capacity in a concentration dependent manner (Nagoor Meeran and Prince, 2012;Nagoor Meeran et al., 2015b, 2016b. Thymol possesses SOD like activity in removing superoxide radicals in vitro (Kruk et al., 2000). It has also shown moderate antioxidant activity in V79 Chinese hamster lung fibroblast cells (Undeger et al., 2009). Thymol (25 µg/ml) showed potent antioxidant activity by modulating the activities of enzymatic antioxidants and decreased lipid peroxidation in gamma ray induced V79 Chinese hamster cells (Archana et al., 2011b). Thymol (0.02-0.20%) showed better antioxidant capacity than its isomer carvacrol in lipid systems due to its greater steric hindrance and it was shown that at room temperature it inhibited the autoxidation of two lipid systems namely triacylglycerols of lard (TGL) and triacylglycerols of sunflower oil (TGSO). Furthermore, it also inhibited the oxidation of TGL and TGSO, with potent antioxidant activity against TGSO. Thymol increased chain initiation during the oxidation of TGSO more so than during the oxidation of TGL (Yanishlieva et al., 1999). In the intestinal Caco-2 cell line, thymol (250 µM) attenuated oxidative stress induced by hydrogen peroxide (H 2 O 2 ) (Cabello et al., 2015). Thymol (250 and 500 µM) strongly inhibited nicotinamide adenine dinucleotide phosphate reduced (NADPH)-cytochromec reductase mediated lipid peroxidation isolated from detergent solubilized liver microsomes of rats (Kamataki et al., 1978). Thymol attenuated the production of reactive oxygen species (ROS) and showed myeloperoxidase inhibitory activity in human neutrophils (Perez-Roses et al., 2016).

Anti-inflammatory Properties
Thymol (150 µM) has been shown to ameliorate LPS-induced inflammation in murine macrophage cell lines (Chauhan et al., 2014). Thymol (84 µg/ml) treatment attenuated lipopolysaccharide (LPS) and interferon gamma (IFN-γ) induced macrophage inflammation in vitro by inhibiting messenger RNA expression of inducible nitric oxide (NO) in J774A.1 cell lines (Vigo et al., 2004). In human polymorphonuclear neutrophils (PMNs), thymol (10 and 20 µg/ml) inhibited the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced release of elastase, a marker of inflammatory diseases and a serine proteinase released by activated human neutrophils in a concentration-dependent manner (Braga et al., 2006). Thymol (100 µM) has been reported to alter prostaglandin catalyzed biosynthesis by inhibiting both isoforms of cyclooxygenase (COX), with the most active being against COX-1 with an IC 50 value of 0.2 µM. These studies suggest the potential of thymol as an anti-inflammatory drug and indicate that it could be used in a similar fashion to non-steroidal antiinflammatory drugs (Marsik et al., 2005). Thymol (1.1 µg/ml) exhibited inhibitory effects against arachidonic-acid-induced blood coagulation and platelet aggregation in vitro (Enomoto et al., 2001). Thymol (50-150 µM) attenuated bleomycin induced genotoxicity in human ovarian cells (SKOV-3) by virtue of its antioxidant and anti-inflammatory properties (Arab et al., 2015). Ku and Lin (2013) reported the anti-inflammatory nature of thymol by its inhibiting of the T cell immune response and improved T-helper cells-1 (Th1) (interleukin-2 (IL-2) and IFN-γ/T-helper cells-2 (Th2) (interleukin-4 (IL-4), interleukin-5 (IL-5) and interleukin-10 (IL-10) ratio in mouse primary splenocytes. Thymol (40 µg/ml) inhibited the LPS stimulated inflammatory response in mouse mammary epithelial cells mediating the down regulation of mitogen-activated protein kinases (MAPK) and nuclear factor-kB (NF-κB) signaling pathways . Thymol (7.5 mg/kg) abrogated the induction of inflammation in isoproterenol (ISO) challenged rats, an animal model of myocardial infraction (MI), which had developed myocardial necrosis. (Nagoor Meeran et al., 2015b). Thymol isolated from essential oils of Lippia gracilis leaves has been shown to inhibit carrageenan-induced edema formation in the paws (administered at the dose of 200 mg/kg) similar to the activity of positive control acetylsalicylic acid (300 mg/kg) (Mendes et al., 2010). Furthermore, treatment with this essential oil at the dose of 50, 100, and 200 mg/kg abrogated leukocyte migration into the peritoneal cavity in carrageenan-challenged experimental animals. Treatment with the essential oil also inhibited the occurrence of abdominal writhes in experimental animals induced by acetic acid.
Thymol isolated from leaf essential oils of Lippia gracilis (32.68%) is believed to be primarily responsible for its antinociceptive and anti-inflammatory actions (Mendes et al., 2010). It has been shown to inhibit the release of arachidonic acid, COX and the biosynthesis of prostaglandins such as prostaglandin E2 (PGE2) in the visceral pain model (Mendes et al., 2010). Monteiro et al. (2007) has demonstrated the antiinflammatory effect of thymol present in the Lippia sidoides essential oil administered at the doses of 1 and 10 mg/ear in the acute ear edema model induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA) in mice as evidenced by reduced edema (a 45.93 and 35.26% reduction, respectively). Thymol (100 mg/kg) attenuated inflammation and promoted wound healing in several rodent models via inhibiting the influx of leucocytes to the injured areas and thus preventing edema (Riella et al., 2012). Thymol exhibited potent anti-inflammatory activity by diminishing the release of inflammatory mediators viz. prostanoids, interleukins and leukotrienes in the buccal sites of young adults (Skold et al., 1998;Yucel-Lindberg et al., 1999). Thymol (10-250 µg/pellet) also elicited potent anti-inflammatory and antiangiogenic action in chorioallantoic membrane (CAM) assay using the experiment model of CAM of the fertilized hen's egg (Demirci et al., 2004).
Thymol (50 µg/ml) increased the mean fluorescence intensity (MFI) of cluster of differentiation 40 (CD40), cluster of differentiation 86 (CD86) and major histocompatibility complex-II (MHCII) expressions determined by flow cytometric analysis in the dendritic cells isolated from spleen of BALB/c mice . Thymol inhibited ROS (IC 50 = 3 µg/ml), reactive nitrogen species (RNS) (IC 50 = 4.7) and significantly reduced generation of NO and H 2 O 2 as well as activities of nitric oxide synthase (NOS) and nicotinamide adenine dinucleotide reduced oxidase (NADH oxidase) in LPSinduced murine macrophages . A report from Hejazian (2006) has revealed that thymol present as an important constituent in the essential oil of Carum copticum (20 mg/kg) significantly diminished pain sensation in the inflammatory phase of the formalin test in mice. Lorente et al. (1989) revealed an important pharmacological advantage of the potentiation of anti-inflammatory activity of α and β-pinene mixtures (80 mg/kg) upon use in conjunction with thymol (1 mg/kg) in female Wistar rats. Thymol (10 and 20 µg/ml) reduced inflammatory responses through modulation of the expression of c-Jun N-terminal kinase (JNK), stress-activated protein kinases (STAT-3), activator protein-1 (AP-1) and nuclear factors of activated T-cells (NFATs) in LPS treated macrophages . Thymol (600 µM) reduced immunoglobulin-E (IgE)-dependent responses in mast cells through the activation of apoptotic cell death in bone marrowderived mast cells (BMMCs) and BALB/c mice (Wechsler et al., 2014).

Immunological Properties
Thymol (25-200 mg/kg) was shown to modulate the immune system in cyclosporine-A treated Swiss albino mice by enhancing the expressions of cluster of differentiation 4 (CD4), cluster of differentiation 8 (CD8) and Th1 cytokines via upregulation of IFN-γ expression and enhanced secretion of interleukin-12 (IL-12) (Chauhan et al., 2010). Khajeali et al. (2012) reported that thymol produced a significant increase in antibody titers against the Newcastle disease virus in broiler chickens. Thymol feed supplementation (0-200 mg/kg) improved the activities of digestive enzymes, growth indices and antioxidant status with declined MDA levels. It also improved the immune response via increasing tolerance to hypersensitivity and immunoglobulin G (IgG) in broiler chickens (Hashemipour et al., 2013). It improved innate immunity (Giannenas et al., 2012). Thymol diet supplementation (1%) enhanced the levels of immunoglobulin A (IgA) and immunoglobulin M (IgM) in the sera of weaning pigs challenged with Salmonella typhimurium (Trevisi et al., 2010). Thymol treatment in low-weight growing-finishing pigs raised the percentage of CD4+, CD8+, and MHC-II in their peripheral blood and it also down regulated tumor necrosis factor-α (TNFα) expression in the stomach of post-weaned pigs (Taranu et al., 2012).
Thymol (IC 50 = 7.69 µg/ml) suppressed the adhesion and superoxide production on isolated ovine neutrophils and also anti-inflammatory property on sheep neutrophils (Farinacci et al., 2008). Thymol has been shown to improve the immune system by increasing the levels of IgA and IgM in the pig's gut (Li et al., 2012). Thymol (10 µg/ml) attenuated the maturation of dendritic cells and inhibited the mitogenic and allogenic T cell responses along with the secretion of IFN-γ and IL-4 cytokines . Thymol (10 µg/ml) increased phagocytosis by enhancing the membrane fluidity of macrophages and suppressed the inflammatory responses by downregulating the secretion of pro-inflammatory cytokines by its potent immunostimulating effect (Chauhan et al., 2014).

Antimicrobial Properties
There are a convincing number of studies that reveal that thymol alone or thymol in plants along with other metabolites possess potent antimicrobial, antifungal, antibacterial, and antiparasitic properties. Marchese et al. (2016) has comprehensively reviewed the antimicrobial properties of thymol. Thymol (32.55%) present in the essential oil of Thymus vulgaris L. showed bacteriostatic activity against most of the gram positive and negative bacteria (Marino et al., 1999). A report from Olasupo et al. (2003) revealed the antibacterial effect of thymol with the lowest minimum inhibitory concentration (MIC) values of 1.0 mmol/L (S. typhimurium) and 1.2 mmol/L (Escherichia coli). Thymol possesses antimicrobial activity against S. aureus (MIC: 0.31 mg/ml) and E. coli (MIC: 5.00 mg/ ml) by the perturbation of the lipid fraction of the bacterial plasma membrane resulting in the leakage of intracellular materials (Trombetta et al., 2005). The thymol chemotypte of the essential oils of T. zygis and T. vulgaris has shown antibacterial effect against various gram negative and positive bacterial strains with MIC ≤ 0.2 µl/ml (Rota et al., 2008). Thymol (200 mg/ml) could inhibit the growth of E. coli. by inducing the permeabilization and depolarization of the cytoplasmic membrane (Xu et al., 2008). Thymol at 2.5 mM inhibits the growth of S. aureus, E. coli and S. typhimurium. Furthermore, a synergistic interaction was found for thymol with all antibiotics tested against E. coli, S. typhimurium, S. aureus, and S. pyogenes (Palaniappan and Holley, 2010). Thymol ester derivatives were found to be more effective against streptococcus species (Mathela et al., 2010). Thymol was found to possess antibacterial activity against selected verocytotoxigenic E. coli (Rivas et al., 2010). Thymol (0.12%) possess antifungal activity against C. albicans MTCC 227 biofilm inhibition (Pemmaraju et al., 2013). Gelatin films containing different thymol concentrations (1-8%) produced inhibitory zones ranging from 30 to 46 mm against several bacteria. Thymol was more effective against Gram positive strains (Kavoosi et al., 2013). Thymol (15 and 30 mg/kg) was shown to possess cytotoxic and antileishmanial activities in hamsters experimentally infected with Leishmania (Viannia) panamensis (Robledo et al., 2005). Thymol derivative named benzoyl-thymol was the best inhibitor (8.67 ± 0.28 µg/mL) against Leishmania infantum chagasi (de Morais et al., 2014).

Other Pharmacological Properties
The blocking effect of thymol on voltage-activated sodium currents has been investigated in the in vitro setup using experimental cell models of animal and human origin. For skeletal muscle and the neuronal sodium channel, it showed a half-maximum blocking concentration (IC 50 ) of 104 and 149 µM, respectively. Blockade of voltage-operated sodium channels were attributed to confer the antinociceptive and anesthetic effects (Haeseler et al., 2002). In rat skeletal muscle fibers isolated enzymatically, thymol (30-600 µg) treatment suppressed both calcium (Ca 2+ ) and potassium (K + ) currents in a concentration-dependent manner with half-maximal effect (EC 50 ) values of 193 ± 26 and 93 ± 11 µM and Hill coefficients of 2.52 ± 0.29 and 1.51 ± 0.18 respectively (Szentandrassy et al., 2003). Thymol has been shown to accelerate K + -induced contracture in skeletal muscle and inhibit Ca 2+ -binding by the fragmented sarcoplasmic reticulum thus causing the suppression of relaxation (Ebashi, 1965). Thymol (224 µM) was able to invoke the release of Ca 2+ with an EC 50 value of 158 ± 16 µM and activate ryanodine receptors in heavy sarcoplasmic reticulum vesicles isolated from skeletal muscle which were loaded with Ca 2+ (Sarkozi et al., 2007). Thymol (30 µM) was found to increase the depolarization-induced release of Ca 2+ from the sarcoplasmic reticulum in rodents (Szentesi et al., 2004).
Thymol, at micro concentrations, reduced calcium dependent adenosine triphosphatase (Ca 2+ -ATPase) activity and increased the permeability of Ca 2+ in the sarcoplasmic membrane and it was found to increase the Ca 2+ concentrations of neurons or of smooth muscle preparations (Hisayama and Takayanagi, 1986;Kostyuk et al., 1991). Thymol has agonistic effects for the adrenergic receptors (α1, α2, and β) on the circular smoothmuscle strips (SMAs) isolated from stomach and vena portae of guinea pigs. Thymol (10 −4 M) inhibits spontaneous contractile activity of the SMAs (100%) and diminishes the excitatory effect of acetylcholine chloride to 35%. Thymol via its influence on nerve cell α2-adrenergic receptors showed an analgesic effect (Beer et al., 2007). Thymol (500 µM) was shown to activate the transient receptor potential channel (TRPV3) of the tongue and nasal epithelium (Boudry and Perrier, 2008). In transient receptor potential ankyrin1 (hTRPA1) expressing human embryonic kidney cells (HEK293 cells), thymol (6.25 and 25 µM) activated the response of the membrane potential and increased intracellular Ca 2+ (Lee et al., 2008). Piglets fed diets supplemented with a combination of thymol (100 and 200 mg/kg) and benzoic acid promoted nutrient digestion and absorption, reduced diarrhea and maintained a favorable micro-environment in the gut (Diao et al., 2015). Thymol (10 −3 M) lysed dissociated mouse pancreatic acinar cells as evidenced by increased amylase secretion and the secretion of lactate dehydrogenase (LDH) by 315% (Singh, 1980). A report from Manabe et al. (1987) revealed the biphasic effects of thymol on hypnotic hemolysis. Thymol had a protective effect on erythrocytes at 0.06-1 mM whereas at 2-4 mM it showed a lytic effect on erythrocytes. At a concentration of 1 mM, thymol showed maximum protection for erythrocytes. Thymol (0.2-4 mM) increased the leakage of glutamic oxaloacetate transaminase (GOT) in hepatocytes isolated from male Sprague-Dawley rats. At 33 • C, thymol (1 mM) depressed the phase transition temperature and thereby possessed a significant effect on membrane fluidity and it reduced the surface tension from 72 to 53 dye/cm (Manabe et al., 1987). Thymol (0.75-2 mM) triggered the production of superoxide radicals in blood leukocytes in a concentration dependent manner (Suzuki et al., 1987). In guinea pig neutrophils, thymol (1 mM) stimulated superoxide radical production and this was dependent on the initial density of the binding sites and the initial intracellular adenosine triphosphate (ATP) concentrations (Suzuki and Furuta, 1988). Thymol (300 µg/ml) stimulated cytotoxicity whereas treatment with thymol (30-300 µg/ml) dose dependently inhibited the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and protein in cultured mammalian cells (Arai, 1988). Thymol (5%) treatment increased the passive transport of leutinizing hormone-releasing hormone (LHRH) in the porcine epidermis (Bhatia and Singh, 1998). The partition coefficient of thymol was assessed to be 1.65 ± 0.01 × 10 −2 and at the same concentration it enhanced the permeability of tamoxifen through the porcine epidermis (Gao and Singh, 1998). Thymol (<0.05 mM) suppressed the action potential and reduced membrane resistance and potential in the stomach of guinea pigs at higher concentrations. Thymol (1 mM) inhibited the generation of spikes, hyperpolarized the membrane and resistance in the rectum and ileum (Ito and Kuriyama, 1974).
Thymol (0.002 and 0.00015 mol/L) has been shown to induce nerve blocking action in the phrenic nerve of rats (Seeman et al., 1974). In rabbit white muscle, thymol (0.6 mM) induced accumulation of Ca +2 in the sarcoplasmic reticulum (Takishima et al., 1979). Thymol in 0.4% aqueous ethanol has been shown to reduce contractions (ED 50 = 0.86 × 10 −4 M) in the guinea pig ileum induced by acetylcholine (Van den Broucke and Lemli, 1980). Thymol, a Ca 2+ antagonist at the concentration of 1 × 10 −4 M, also reduced the contractions of rat vas deferens induced by 1-noradrenaline via blocking nerve fiber conductions and inhibited the contractions of guinea pig ileum induced by carbachol, histamine and dimethyl phenyl piperazinium (Van den Broucke and Lemli, 1982). Viana et al. (1981) reported that thymol (2 mg/kg) enhanced the contractions of isolated phrenic-diaphragm and muscles in rats. The same authors revealed that thymol (100 µg) showed potent spasmolytic effects by decreasing the amplitude of peristaltic movements and muscle tone in rabbit duodenum. Thymol (10-300 mg/L) attenuated the force as well as rate of atrial contractions in guinea pigs and at a concentration of 10-300 µg/ml it decreased the aortic contractions isolated from New Zealand white rabbits. Thymol (0.001-0.01%) possessed the ability to relax tension in rabbit intestinal muscles (RIFM 2001, unpublished). Thymol (20 or 40 mg/day) dissolved in olive oil showed clear thyroid activation as detected by oxygen consumption and histological examinations in guinea pigs (Moller, 1939). Thymol (30-300 µg/L) showed a concentration dependent inhibition of DNA, RNA and proteins in V79 cells (Instituto Superiore di Sanita, 1999). Thymol (0.005 M) was shown to possess the ability to convert toxic metals into their non-toxic forms by forming metal complexes or by converting them into their reduced forms by virtue of its antioxidant property (Kishwar et al., 2013).
Thymol (200 mg/kg) attenuated Chang's disease in male Balb/c mice by reducing parasitemia, trypomastigotes, heart amastigotes and inflammatory infiltrates by its anti-Trypanosomaruzi effect (Juan et al., 2015). Supplementation of thymol rich sources like sage, rosemary extracts and pepper improved the digestibility of feed and the final performance in production (Hernandez et al., 2004). Thymol (2 and 3 g/kg) administration improved various growth parameters such as food conversion ratio, final weight, body growth and composition of tissues (whole body lipids, fibers and proteins) (Ahmadifar et al., 2011). Zheng et al. (2009) reported that thymol has a positive effect on the growth performance of channel catfish (Ictalurus panctatus). The presence of thymol might be responsible for the antispasmodic effect of the thyme extract (Engelbertz et al., 2012). Thymol (0-250 µM) showed a weak genotoxic effect in L5178Y/Tk ± cells as analyzed by the micronucleus (MN) test and mouse lymphoma (MLA) assays (Maisanaba et al., 2015). Thymol (100 µM) blocked voltage-gated sodium channels in stably transfected HEK 293 cells expressing α-subunit of rat brain IIA or hSkM1 sodium channels and this is attributed to its antinoceptive and anesthetic properties (Haeseler et al., 2002).

THYMOL IN CANCER CELLS
Thymol showed anticancer properties in different types of cell lines mimicking human cancer and it demonstrated its potential as a chemopreventive or anticancer agent in various types of cancers. The protective effect of thymol against various types of cancers is represented in Table 1 and the schema of the protective effects of thymol shown in the studies is represented in Figure 1. The major mechanisms of anticancer actions of thymol include induction of apoptosis, anti-proliferation, inhibition of angiogenesis and migration as well as the diminution of umourigenesis by modulating the activity of carcinogen metabolizing enzymes.

Glioblastoma Cells
In human glioblastoma cells, thymol (200-600 µM) produced a rise in (Ca 2+ ) i levels by prompting release of phospholipase C and protein kinase C-dependent Ca 2+ from the endoplasmic reticulum (ER) and entry of Ca 2+ via non-store-operated Ca 2+ channels. Furthermore, the cell death induced by thymol was found to involve apoptosis and necrosis as observed in Annexin V/PI staining . Further, thymol (6.0 ± 0.11 mg/g) present in the Zataria multiflora extract possessed radio sensitizing effect in human glioblastoma cells (Aghamohammadi et al., 2015).

Breast Cancer Cells
In breast cancer cells (MCF-7 cells), thymol (0.05-1.25 µM) stimulated cytotoxicity by arresting the cell cycle in the G0/G1 phase (Jaafari et al., 2012). Thymol triggered cytotoxicity in MCF-7 breast cancer cell lines with an LC 50 of 2.5 µg/mL (Melo et al., 2014). In another study, Thymol present in the essential oil of T. lanceolatus (IC 50 = 304.81 µg/ml) was shown to induce cytotoxicity and proliferation in MCF-7 cells (Khadir et al., 2016). . The ability of thymol to stop the cell cycle in G0/G1 phase of the K-562 cells seems to be due to its anti-tumor activity (Jaafari et al., 2012). Thymol (5-100 µM) triggers cell death and cell cycle arrest at the sub G0/G1 phase by genomic DNA fragmentation pattern on acute promyelotic leukemia cells (HL-60 cells). Thymol increased the production of ROS and mitochondrial H 2 O 2 thereby depolarizing mitochondrial membrane potential.

Leukemia Cells
Thymol treatment induced caspase dependent apoptosis by up-regulating Bcl-2 associated X protein (Bax) expression and down-regulating B-cell lymphoma (Bcl-2) expression in a dosedependent manner. It further augmented the activation of caspase-3, 8, and 9 concomitant to Poly ADP ribose polymerase (PARP) cleavage that is the hallmark of caspase-dependent apoptosis. Furthermore, it also promoted the translocation of apoptosis inducing factor (AIF) from the mitochondria to cytosol and to nucleus, which shows its ability to induce caspaseindependent apoptosis. Altogether, the observations indicate that thymol-induced cell death includes both caspase-dependent and caspase-independent pathways (Dutta et al., 2011).

Osteosarcoma Cells
In human osteosarcoma cells (MG63 cells), thymol (400 µM/L) treatment induced a rise in the levels of (Ca 2+ ) i by triggering phospholipase C-dependent Ca 2+ release from the ER and promoting protein kinase-C sensitive store-operated Ca 2+ channels mediated entry of Ca 2+ . Thymol also triggered ROS mediated apoptotic cell death via mitochondrial pathways in MG63 cells .
Thymol (IC 50 = 497 and 266 mM) was shown to induce DNA damage by increasing the levels of lipid peroxidation products; MDA and 8-hydroxy deoxyguanozine (8-OHdG) in parental and drug resistant human non-small cell lung carcinoma cells (H1299 cell lines). Thymol (<IC 50 = 497 and 266 mM) treatment elicit protection against H 2 O 2induced cytotoxicity and showed stabilizing effects on membrane and DNA damage in H1299 cells (Ozkan and Erdogan, 2012). Thymol (25-100 µM) also inhibited acetaminopheninduced cytotoxicity in HepG2 cells as evidenced by improved antioxidant activity and reduction in levels of the proinflammatory cytokines such as IL-1β and TNF-α (Palabiyik et al., 2016).

Cervical Cancer Cells
Cervical cancer is a cancer arising from the cervix due to abnormal cell growth that possesses the ability to invade or spread to other parts of the body. Thymol (30.5-244 ng/ml) induced cytotoxicity by inhibiting the growth of HeLa cells in a concentration-dependent manner. The observed inhibition at the concentration 30.5 ng/ml was 74.06-87.25%. This study has revealed that thymol possesses strong antitumor activities by inducing cytotoxicity and decreasing the mitotic index at higher concentrations in HeLa cell lines (Reema, 2011). Thymol present in the essential oil of T. lanceolatus (IC 50 = 134.29 µg/ml) was shown to induce cytotoxicity in HeLa cells (Khadir et al., 2016).

Gastric Carcinoma Cells
In human gastric AGS cells, Thymol (100-400 µM) showed a change in cell morphology due to chromatin condensation, cleavage of DNA, cytoplasm shrinkage, and membrane blebbing. The beneficial effects in these cells were attributed to the generation of intracellular ROS, depolarization of mitochondrial membrane potential, apoptosis and impeding cell growth via intrinsic mitochondrial pathway and the activation of proapoptotic mitochondrial proteins; caspases, Bax and PARP (Kang et al., 2016).

Neuroblastoma Cells
Thymol (400 mg/L) decreased cell proliferation in cultured neuroblastoma cells (N2a cells) whereas thymol (19, 25, and 50 mg/L) increased the total antioxidant capacity in rat neurons but not in N2a cells. This report clearly revealed that thymol is a potent anticancer and antiproliferative agent (Aydin et al., 2016).

Other Studies
Thymol (50 and 100 µM) has been reported to inhibit bleomycin induced genotoxicity in human lymphocytes by its chemoprotective effect. It was also shown that thymol pretreatment in bleomycin treated human ovarian carcinoma cells (SKOV-3 cells) neither enhanced cell neither death nor cell protective effects but it prevented bleomycin induced DNA damage in normal cells. This study recommended the combination of thymol with various chemotherapeutic agents to minimize its toxicity on normal cells and to improve the effectiveness of cancer treatment (Arab et al., 2015).
Thymol triggered cytotoxicity with an IC 50 value of 400 µM (60.09 µg/mL) along with oxidative stress in B16 melanoma cells. Thymol generates a phenoxy radical intermediate by its potent antioxidant effect followed by the production of ROS and quinine oxide derivatives. The toxicity of thymol at higher doses is due to the formation of antioxidant-related free radicals (Satooka and Kubo, 2012). Thymol (IC 50 = 20-40 µM) showed protective effect against H 2 O 2 induced DNA double strand breaks in HepG2, human colonic cells (Caco-2 cells) and hamster lung cells (V79 cells) . Thymol (0.24%) present in the essential oil of Origanum compactum showed a strong inhibitory effect on indirectacting mutagen in urethane (URE) induced mutagenicity in Drosophila melanogaster as investigated by the somatic mutation and recombination test (SMART test). Thymol suppressed the mutations by 43% (Mezzoug et al., 2007). Thymol (0.1 mM) significantly decreased DNA double strand breaks in 2-amino-3methylimidazo(4,5-f)-quinoline (IQ) and mitomycin C (MMC) induced DNA damage in human lymphocytes and at higher concentrations of about 0.2 mM, thymol itself induced DNA damage in lymphocytes (Aydin et al., 2005). In the SOSchromotest and the DNA-repair test the genotoxic potential of thymol was found to be very weak (Stammati et al., 1999).

THYMOL IN CARDIOMETABOLIC DISEASES
The protective effects of thymol in various cardiovascular related disorders such as MI, hyperlipidemia and several others are represented in Tables 2, 3 and the schema of the protective effects of thymol shown in the studies is represented in Figures 2, 3.

Myocardial Infarction
Thymol (7.5 mg/kg) was shown to inhibit the occurrence of oxidative stress in rats challenged with ISO, an agent which commonly induces myocardial necrosis. The benficial effects were attributed to decreased levels of lipid peroxidation products such as thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LOOH) and conjugated dienes (CDs) in plasma. Further, it also normalized non-enzymatic antioxidants such as vitamin-C, vitamin-E and GSH in the plasma due to its potent antioxidant action (Nagoor Meeran and Prince, 2012). Furthermore, thymol attenuates altered lipid metabolism [decreased the levels/concentrations of serum and heart lipids such as total cholesterol, triglycerides (TGs) and free fatty acids (FFAs)], reinstating the normal levels of lipoproteins (increased HDL-C with decreased LDL-C and VLDL-C levels in the circulation) in ISO-induced myocardial infarcted rats. Thymol was shown to attenuate the alterations in the activities of lipid marker enzymes such as 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA reductase) and lecithin-cholesterol acyltransferase (LCAT) in the liver, inhibiting tachycardia (increased heart rate), decreasing atherogenic index, and the levels of serum cardiac troponins, altered electrocardiographic patterns (ST segment elevation), cardiac hypertrophy (decreased heart weight and left ventricular weight/body weight) and apoptosis (increased expression of myocardial Bcl-2 gene and decreased expression of Bax-gene in ISO-induced myocardial infracted rats) (Nagoor Meeran et al., 2015b). Also, thymol has been shown to attenuate inflammation of the myocardium by inhibiting the release of lysosomal enzymes (β-glucuronidase, β-galactosidase, cathepsin-B and cathepsin-D) from the heart to the circulation by decreasing the levels of lysosomal TBARS, release of inflammatory marker such as high sensitive C-reactive protein (hsCRP) and down regulating the myocardial expressions of pro-inflammatory cytokines such as TNF-α, interleukin-6 (IL-6) and IL-1β genes in ISO-induced myocardial infracted rats. The transmission electron microscopic findings revealed preservation of lysosomal architecture and histopathological salvage in concurrence with the biochemical observations (Nagoor Meeran et al., 2015b).
Oral administration of thymol abrogates myocardial membrane destabilization by inhibiting myocardial oxidative stress (decreased concentrations of lipid peroxidations products in heart and improved activities of antioxidant enzymes), reduced leakage of the cardiac marker enzyme LDH into the circulation, decreasing the activity of Ca 2+ ATPase and increasing the activity of sodium/potassium dependent adenosine triphosphatase (Na + /K + ATPase) in ISO-induced infarcted rats. Furthermore, thymol also increased K + concentrations and enhanced sodium (Na + ) and Ca 2+ concentrations in the heart. Also, thymol significantly diminished the myocardial infarct size as analyzed by 2,3,5-triphenyl tetrazolium chloride (TTC) assay due its potent membrane stabilizing property (Nagoor Meeran et al., 2015c). Thymol was shown to inhibit mitochondrial dysfunction in ISO-induced myocardial necrosis in rats. Pre and co-treatment with thymol showed decreased heart mitochondrial lipid peroxidation products (TBARS and LOOH), lipids (cholesterol, TGs, FFAs and phospholipids (PLs), Ca 2+ and significant increase in the activities of heart mitochondrial antioxidants (SOD, catalase, GPx, GSH) and mitochondrial marker enzymes such as isocitrate dehydrogenase (ICDH), malate dehydrogenase (MDH), α-ketoglutarate dehydrogenase (α-KGDH), reduced nicotinamide adenine dinucleotide dehydrogenase (NADH dehydrogenase) and cytochrome-Coxidase) in ISO-induced MI in rats. It also enhanced the ATP levels and diminshed the mitochondrial swelling. Transmission electron microscopic study on heart mitochondria confirmed the biochemical findings of the study. This study revealed the ability of thymol in protecting the heart mitochondria against ISO induced oxidative stress in rats (Nagoor Meeran et al., 2016b).
Thymol has been shown to decrease the levels of plasma uric acid and glycoprotein components viz. hexose, hexosamine, fucose and sialic acid in ISO-induced rats due to its potent antioxidant property (Nagoor Meeran et al., 2014). Thymol was shown to inhibit apoptosis by decreasing oxidative stress in ISO-induced myocardial infracted rats. Thymol treatment decreased the concentrations of lipid peroxidation products and increased the status of antioxidants in the myocardium such as GPx, GSH, vitamin-C and vitamin-E. It also decreased the myocardial gene expressions of caspase-8, 9 and Fas genes and increased the expressions of B-cell lymphoma extra-large (BcL-xL) gene. Histopathological and the in vitro ferric reducing antioxidant power (FRAP) assay confirmed the biochemical observations. This study revealed the protective effect of thymol against apoptotic cell death in the heart by attenuating oxidative stress (Nagoor Meeran et al., 2016a). In all these studies, thymol pre-and co-treatment in rats appear devoid of any deleterious effects which is suggestive of its safety. These preclinical studies recommended the clinical trials to reveal the exact dosage of thymol against MI in humans.

Doxorubicin Induced Cardiotoxicity
Thymol has been shown to abrogate oxidative stress, inflammation and apoptosis in doxorubicin induced cardiotoxicity in rats. Thymol (20 mg/kg), in pre-and cotreated rats, was shown to decrease the levels of serum LDH, aspartate transaminase (AST), creatine kinase (CPK), creatine kinase-MB (CK-MB), cardiac troponin-I and TNF-α with decreased concentrations of caspase-3 and MDA in the heart. The activities of antioxidants such SOD, catalase and GSH were  shown to increase in thymol pre-and co-treated doxorubicininduced cardiotoxic rats. This study has shown that the combined treatment of thymol and carvacrol revealed a much better effect than the treatment with thymol and carvacrol alone in doxorubicin-induced cardiotoxic rats. But, thymol possesses a more superior effect than its isomer carvacrol in the same model and the actions are attributed to the antioxidant, antiinflammatory, and antiapoptotic activity of thymol (El-Sayed et al., 2016). A report from Cardoso et al. (2016) has revealed that thymol (10-100 mg/kg) attenuates inflammation and recovers skeletal muscle from cardiotoxicity in mice.

Atheroscelerosis
A report from Yu et al. (2016) showed that thymol attenuates oxidative stress, aortic intimal thickening, and inflammation by regulating gene expression in hyperlipidemic rabbits. Thymol (3 and 6 mg/kg) supplementation has been shown to decrease the levels of TGs, total cholesterol, LDL-C, MDA, high sensitive C-reactive protein, intimal thickening of aorta with increased levels of HDL-C and total antioxidant capacity in hyperlipidemic rabbits induced by giving a high fat diet. Furthermore, thymol (3 and 6 mg/kg) was shown to decrease the mRNA expressions of IL-1β, IL-6, TNF-α, TNF-β, Vascular cell adhesion protein 1 (VCAM-1), monocyte chemo attractant protein-1 (MCP-1) and MMP-9 in hyperlipidemic rabbits. Thymol (121.4 µM) effectively scavenged DPPH and 2,2 -azinobis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radicals which revealed its potent antioxidant and free radical scavenging properties. Finally, thymol administration lowered serum lipids and attenuated oxidative stress followed by an inflammatory response in hyperlipidemic rabbits. This study recommended further studies to reveal the mechanism of action of thymol on endothelial dysfunction and smooth muscle cell migration . Thymol (5-25 µg/mL) administration showed decreased expressions of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) with increased expression of IL-10 that inhibited translocation of NF-κB into the nucleus in the oxidative-LDL induced THP-1 macrophages, a cellular model of inflammation/atherogenesis (Ocana-Fuentes et al., 2010;Ocana and Reglero, 2012). In human aortic endothelial

Hypertension
Thymol has been shown to exhibit vasorelaxant activities in the isolated rat aorta. Thymol showed relaxation on aortic ring preparations in a concentration dependent manner using potassium chloride (KCl) or using phenylephrine (PHE) (IC 50 value of 64.40 ± 4.41 and 78.80 ± 11.91 µM) and (PHE, 0.1 µM) (IC 50 value of 106.40 ± 11.37 and 145.40 ± 6.07 µM). In isolated rat aorta, endothelium-independent relaxation induced by thymol occurs via release of Ca 2+ from the sarcoplasmic reticulum diminishing the sensitivity of contractile elements to Ca 2+ and preventing the influx of Ca 2+ across the membrane (Peixoto-Neves et al., 2010). Thymol (1-10 mg/kg) showed a dose dependent decline in blood pressure and heart rate in rats. Also, it decreased the force and rate of atrial contractions in spontaneously beating atria (Aftab et al., 1995). Thymol (10-300 µg/ml) (IC 50 = 100 µg/ml, 0.1 mM) dose dependently triggered the relaxation of potassium and norepinephrine induced contractions in the rabbit aorta. Thymol by virtue of its Ca 2+ channel blocking effect expressed its hypotensive and bradycardiac effects in various animal studies (Aftab et al., 1995). Thymol (1, 3, and 10 mg/kg) administration decreased the blood pressure and heart rate of Wistar rats whereas thymol (5 mg/kg) attenuated blood pressure in rabbits (RIFM, 2001, unpublished).

Cardiac Arrythmias
Thymol (10 and 100 µM) induced cardiac arrhythmias via concentration-dependent inhibition of K + and Ca 2+ currents in canine ventricular cardiomyocytes using microelectrode and patch clamp techniques. Thymol (10 µM) ablated the action potential notch whereas thymol (100 µM) decreased the duration of the action potential, reduced maximum velocity (V max ) and the depression of the plateau. These results are found in line with the activity of thymol in ventricular myocytes isolated from healthy human hearts (Magyar et al., 2002). Thymol (10-1000 µM) inhibits the effect of L-type Ca 2+ currents in human and canine ventricular myocytes using the 'patch clamp technique' in the 'whole-cell' configuration on the inactivation of the channel machinery . Thymol triggers negative inotropic actions in canine and guinea pig preparations in a concentration-dependent manner. At lower concentrations, thymol reduced intracellular Ca 2+ transients without altering the contractile function whereas Ca 2+ transients and at higher concentrations suppressed contractions in guinea pig hearts. Thymol reduced the activity of Ca 2+ pump by inducing rapid release of Ca 2+ in canine sarcoplasmic reticular vesicles .

THYMOL IN METABOLIC DISORDERS
The protective effects of thymol in metabolic disorders such as diabetes mellitus and obesity are represented in Table 3 and the schema of the protective effects of thymol shown in the studies are depicted in Figure 4.

Diabetes Mellitus
Thymol was shown to protect against various metabolic disorders. A report from Saravanan and Pari (2015) has revealed the anti-hyperglycemic and hyperlipidemic activity of thymol in high fat diet induced type-2 diabetes in C57BL/6J mice. Thymol (40 mg/kg) administration was shown to reduce final body weight, HOMA of insulin resistance (HOMA-IR), glycosylated hemoglobin (HbA1c), plasma insulin and blood glucose in high fat diet induced type-2 diabetes in C57BL/6J mice. Thymol suppressed plasma and hepatic levels of total cholesterol, TGs, FFAs, PLs, LDL-C and significantly increased the levels of HDL-C in high fat diet induced mice. Furthermore, thymol treatment increased the levels of adiponectin and decreased the levels of leptin in high fat diet (HFD) mice. Also, thymol inhibited alterations in the activities of lipid metabolizing enzymes (significant increase in the activities of LCAT, lipoprotein lipase (LPL) and decrease in the activities of HMG-CoA reductase in HFD mice). Thymol treatment reduced the levels of fatty acid β-oxidation and the activities of glucose 6-phosphate dehydrogenase (G6PD), fatty acid synthase (FAS) along with increased activities of carnitine palmitoyl transferase (CPT), malic enzyme (ME) and phosphatidate phosphohydrolase (PAP) in HFD mice (Saravanan and Pari, 2015). Another study reported by the same group, has revealed that thymol abrogated diabetic nephropathy in HFD-induced diabetes in C57BL/6J mice (Saravanan and Pari, 2016). Thymol (40 mg/kg) treatment for a period of 5 weeks reduced blood glucose level and improves the parameters of renal function. Thymol treatment also suppressed the activation of vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1) and down regulated expression of sterol regulatory element binding protein-1c (SREBP-1c) and reduced lipid accumulation in the kidneys. Extracellular mesangial matrix expansion and glomerulosclerosis were suppressed also by thymol in HFD induced mice as evidenced in histological studies and it also enhanced antioxidant status and inhibited lipid peroxidation in erythrocytes and kidneys. Thymol (0.5-2.0 mg/ml) has been shown to protect red blood cells (RBCs) from 2,2-azo-bis(2-amidinopropane) dihydrochloride (AAPH) induced hemolysis in diabetic patients due to its potent antioxidant and free radical scavenging effect (Aman  (2012), thymol reduced NO, H 2 O 2 production along with NOS, NADH-oxidase (NOX) activities in human monocytes cultured in the presence of 20 mM glucose. Thymol present in the methanolic extract of Thymus quinquecostatus showed inhibitory effect on the enzymes α-amylase and α-glucosidase responsible for breakdown of carbohydrates and further intestinal absorption (IC 50 = 4.39 ± 0.22 µg/ml) (Hyun et al., 2014). The findings demonstrate that thymol has promising potential in the treatment of hyperglycemia and associated complication.

Obesity
Obesity is defined as excessive adiposity and is one of the major health and socioeconomic burdens which leads to a number of chronic diseases such as coronary heart disease (Sedova et al., 2004), diabetes (Lazar, 2005;Sanchez-Castillo et al., 2005), hyperlipidemia (Jeusette et al., 2005) and various cancers (Stunkard and Allison, 2003a,b;Stunkard et al., 2003). Thymol (30 mg/kg) was shown to inhibit the accumulation of visceral fats, enhance insulin and leptin sensitivity and improve lipid lowering action as well as augment antioxidant status in HFD-induced obesity in murine models (Haque et al., 2014). Thymol (20 µM) has been shown to promote the biogenesis of mitochondria and increase the expression of brown fat-specific markers along with improved expressions of peroxisome proliferator activated receptor-γ (PPARγ), peroxisome proliferator activated receptor-δ (PPARδ), phospho AMP-activated protein kinase (pAMPK), pampk; Phospho acyl-CoA carboxylase (pACC), hormone-sensitive lipase (HSL), perilipin (PLIN), carnitine palmitoyltransferase-1 (CPT1), acyl-coenzyme A oxidase-1 (ACO), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and uncoupling protein 1 (UCP1) in the browning of white adipocytes (3T3-L1 white adipocytes) which play an important role in glucose homeostasis and lipid metabolism. Altogether, the findings reveal that thymol has the potential to regulate oxidation of fatty acids, lipolysis augmentation, lipolysis reduction and thermogenesis. Thymol possesses the ability to activate the β3-adrenergic receptor along with AMPK-activated protein kinase (AMPK), protein kinase-A (PKA), and p38 mitogen-activated protein kinase (p38 MAPK) pathways and it could be the reason for its ability to trigger UCP1 expression in other brown fat-specific markers (Choi et al., 2016).

THYMOL IN RENAL DISEASES
The protective effects of thymol in renal diseases are represented in Table 3 and the schema of the protective effects of thymol shown in the studies are represented in Figure 4. Thymol (20 mg/kg) was shown to inhibit cisplatin-induced renal injury by attenuating oxidative stress, inflammation and apoptosis in male adult Swiss Albino rats (El-Sayed et al., 2014). Thymol (200-500 µM) induced Ca 2+ release from the ER which facilitated the entry of Ca 2+ via store-operated Ca 2+ entry in Madin-Darby canine kidney (MDCK) renal tubular cells. Thymol triggers cell death by promoting apoptosis mediated by ROS in MDCK renal tubular cells (Chang et al., 2014). Thymol's (50 and 150 mg/kg) beneficial effect on cisplatin-induced renal injury in mice was also demonstrated by quantitative renal dimer captosuccinic acid ( 99m Tc-DMSA) uptake concomitant to potent antioxidant and anti-inflammatory properties. 99m Tc-DMSA uptake per gram tissue of kidneys in %ID/g was 65.02 ± 32.21 and 88.46 ± 20.46 in the thymol (50 and 150 mg/kg) treated mice induced with cisplatin. Furthermore, Thymol administration increased the level of %ID/g .

THYMOL IN GASTROINTESTINAL DISORDERS
The protective effects of thymol in gastrointestinal disorders are represented in Table 4. Nowadays, the prevalence of inflammatory diseases in the intestines are rising as a serious problem in humans. The increased expressions of proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-8, TNF-α, IL-12, and IFNγ were reported in the inflamed intestinal mucosa of both animal and humans (Rogler and Andus, 1998;Wirtz and Neurath, 2000;Bertevello et al., 2005;Raddatz et al., 2005;Bukovska et al., 2007). Thymol present in thyme and oregano oils (0.05-0.4%) was shown to inhibit 2,4,6trinitrobenzenesulfonic acid-induced colitis by decreasing the mRNA expressions of pro inflammatory cytokines (IL-1β, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and TNF-α) and protein expressions of IL-1β and IL-6 in mice (Bukovska et al., 2007). A report from Platel and Srinivasan (2004) demonstrated the ability of thymol to prompt secretion of salivary amylase in humans and of bile acids, gastric and pancreatic enzymes such as lipase, amylase and proteases and intestinal mucosa in rats. Thymol administration increased the activities of pancreatic amylase, maltase and trypsin in broiler chickens (Jang et al., 2007).
Thymol (100 mg/kg) has been shown to attenuate acute and chronic ulcers induced by various agents such as ethanol, indomethacin and acetic acid by attenuating the inflammatory process, i.e., infiltration of inflammatory cells and edema. This gastroprotective effect of thymol is believed to be due to increased mucus secretion, prostaglandins and ATP-sensitive K + channels (Ribeiro et al., 2016). Thymol (10-100 µM) induced the secretion of chloride (Cl − ) and bicarbonate (HCO3 − ) ions in piglets' intestinal epithelial cells via the nervous pathway likely through the activation of nervous nicotinic receptors (Boudry and Perrier, 2008). Thymol (0-100 µM) showed concentration dependent antispasmodic effects by inhibiting K + induced tracheal contractions in rats (43% at 100 µM thymol). It also inhibits Barium chloride (BaCl 2 ) induced tracheal contractions in a concentration dependent manner where the EC 50 of thymol is in the range of 70 µg/mL. Also, Thymol (1.3 mg/kg) increased the mucociliary clearance in mouse trachea in situ (Begrow et al., 2010). Thymol (10 mg/kg) administered orally inhibited ethanol induced gastric mucosal injury by up-regulating the status of antioxidants and down regulating MMP-9 protein expressions (Chauhan and Kang, 2015). Thymol (0.072%) in 434 µg/ml extract of Thymus vulgaris concentration dependently antagonized the contractions in guinea pig trachea brought by four different spasmogens (Meister et al., 1999).
Thymol (IC 50 = 2.85 × 10 −2 ± 1.2 × 10 −2 µg/mL) isolated from A. phleoides essential oil has been shown to induce antispasmodic activity in charcoal meal mice (Astudillo et al., 2014). Thymol present in the extract of Trachyspermum ammi (0.01%) showed anti-spasmolytic and anti-spasmodic action against contractions in the isolated rat's ileum induced by acetylcholine (Hejazian et al., 2014). Thymol (0-2 mM) exerted its relaxant effect on smooth muscle cells by opposing Ca 2+ activation and ATP dependent process by its potent anti-spasmodic effect (Tamura and Iwamoto, 2004). Thymol (50 mg/kg) was shown to influence gastric maturation and function via stimulation of gastric proliferative activity and the control of digestive activity in weaned pigs .

THYMOL IN MODELS OF LIVER DISEASES
The protective effects of thymol in liver diseases are represented in Table 4.

Hepatotoxicity
Thymol (30 mg/100 g) has been shown to inhibit oxidative stress in hydrocortisone-induced hepatotoxicity in rats by attenuating lipid peroxidation and enhancing antioxidant defense in the liver. Thymol treatment reinstated the activities of liver marker enzymes attributed to its potent free radical scavenging and antioxidant activity (Aboelwafa and Yousef, 2015). Thymol (300 mg/kg) has been shown to attenuate carbon tetrachloride induced liver injury in mice. Thymol treatment reduced lipid peroxidation and increased the status of antioxidants thereby preventing oxidative stress mediated hepatic injury in mice. Liver function tests and histological studies confirmed the other biochemical findings of the study (Al-Malki, 2010). In carbon tetrachloride (CCl 4 ) (20 µl/kg) induced liver injury, thymol (300 mg/kg) abrogated lipid peroxidation and reinstated the normal activities of hepatic marker enzymes in the liver due to its potent free radical scavenging property (Alam et al., 1999).
Thymol (150 mg/kg) showed to inhibit paracetamol induced hepatotoxicity in mice by preventing the alterations in the activities of hepatic marker enzymes (Janbaz et al., 2003). Thymol (50 µg/ml) inhibited oxidative damage to liver cells by inhibiting ROS overproduction, ameliorating lipid peroxidation, preventing apoptosis and increasing antioxidant levels in tertbutyl hydroperoxide (t-BHP) induced Chang liver cells (Kim  (Jimenez et al., 1993). Thymol (1 ml/kg and 5.6 ml/kg) from thyme tincture and syrup inhibited CCl 4 induced liver injury by reducing lipid peroxidation mediated oxidative stress and it maintained the levels of hepatic markers in Wistar rats (Raskovic et al., 2015). Thymol (50-200 mg/kg) increased the activities of phase I enzymes such as 7-ethoxycoumarin O-deethylase (ECOD) and phase II enzymes such as GST and quinone reductase (QR) along with raised activities of GST alpha and GST micro in mouse liver (Sasaki et al., 2005). In t-BHP induced Chang liver cells, thymol (50 µg/ml) inhibited lipid peroxidation and apoptosis by increasing the status of the antioxidants . These results revealed that thymol imparts a hepatoprotective effect on t-BHP-induced oxidative injury by mediating antioxidant activity . Thymol (25-100 µM) increased both enzymatic and nonenzymatic antioxidants and inhibited lipid peroxidation against paracetamol-induced toxicity in human HepG2 cells (Palabiyik et al., 2016).

THYMOL IN MODELS OF CNS DISEASES
The protective effects of thymol in CNS diseases are represented in Table 5.

Alzheimer's Disease (AD)
Alzheimer's disease is the most common cause of age associated dementia that leads decline in cognitive function following memory deterioration. Nowadays, treatment strategies have been developed for the management of AD with the use of acetylcholinesterase (AChE) inhibitors (an enzyme principally involved in the hydrolysis of acetylcholine) (Jukic et al., 2007). Thymol (EC 50 = 0.74 mg/mL) was shown to possess acetylcholine esterase inhibitory activity but much less than its isomer carvacrol (Jukic et al., 2007). In elderly patients, AD is associated with oxidative stress, inflammation and it is also characterized by the deposition of amyloid beta (Aβ) proteins in the central nervous system (CNS) which results in the formation of amyloid plaques, neurofibrillary tangles and area specific neuronal loss and synaptic changes in the brain (Duyckaerts et al., 2009). Thymol (0.5-2 mg/kg) has been shown to inhibit cognitive impairments caused by increased Aβ levels or cholinergic hypofunction in Aβ (25-35) or scopolamine treated rats attributed to its antioxidant, anti-inflammatory and anticholine esterase properties (Azizi et al., 2012). Thymol (0.39-25 µg/mL) has been shown to inhibit H 2 O 2 induced oxidative stress in PC-12 cells whereas thymol (100 and 1000 µg/ml) also inhibited both AChE and butyrylcholinesterase (BChE) in a dose dependent manner (Lee et al., 2015).

Anxiety
It is one of the most common mental disorders that is characterized by a disturbance in mood or emotional tone due to excessive fear. Thymol (5-20 mg/kg) has been shown to promote anti-anxiety activity in mice on both elevated plus-maze (EPM) and light/dark exploration test (LDT) behavioral models. This effect of thymol could be due to the possible modulation of the 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA) and nitric oxide-cyclic guanosine 3 ,5 -monophosphate (NO-cGMP) pathways (Bhandari and Kabra, 2014).

Depression
Depression is a life threatening illness and the changes induced by inflammatory cytokines in monoamine neurotransmitters is a primary pathway of depression (Miller and Timmie, 2009). Thymol (15 and 30 mg/kg) has been shown to up regulate the levels of central neurotransmitters and inhibit the expressions of proinflammatory cytokines in unpredictable mild stress (CUMS) mice model (Deng et al., 2015).

Epilepsy
Epilepsy is a devastating neurological disease characterized by spontaneous recurrent seizures affecting millions of people all over the world (Bhutada et al., 2010). Thymol (100 mg/kg) decreased the duration of the hind limb extension (HLE) in maximal electroshock (MES)-induced seizures.
In pentylenetetrazole (PTZ)-induced seizure model, thymol (100 mg/kg) prolonged the onset of myoclonic jerk, onset of clonic seizures, onset of HLE and onset of death. Thymol (50 and 100 mg/kg) showed improved activity compared to diazepam in prolonging clonic seizure and the onset of myoclonic jerks. Furthermore, thymol (100 mg/kg) significantly prolonged the onset of death and reduced convulsions in the strychnine (STR) induced mouse model. Thymol (25 mg/kg, i.p.) significantly reduced seizure score, MDA levels and enhanced the levels of glutathione in the animal model of PTZ induced kindling (Sancheti et al., 2014). The authors revealed the antiepileptogenic potential of thymol by its Na + channel blocking effect, positive modulation of GABA A receptor and antioxidant property and they also concluded that it could be a potential candidate to treat epileptic patients (Sancheti et al., 2014). Thymol (10-50 mg/kg) attenuated PTZ (i.p. administration) induced epileptic stages in kindled rats via inhibiting oxidative stress markers in the serum MDA and with increased SOD activity. It also decreased the hippocampal pro-inflammatory cytokines viz. TNF-α and IL-1β released from astrocytes and microglia during and after the seizure induction in rats (Turrin and Rivest, 2004;Aliabadi et al., 2016). Thymol (ED 50 = 35.8 mg/kg) elicited inhibitory activity in the MES, sc Metrazol (scMET) and corneal-kindled models (Mishra and Baker, 2014).

Cholinergic Dysfunction and Other Neurodegenerative Disorders
Cholinergic dysfunction is manifested in a plethora of neurodegenerative and psychiatric disorders such as Alzheimers, Parkinsons, and Huntington's diseases. Thymol (10-100 ppm) in combination with gamma terpinene or para-cymene attenuated cholinergic dysfunction by enhancing synaptic levels of acetyl choline (Ach) and the responsiveness of nicotinic acetylcholine receptor (nAchR) in the Caenorhabditis elegans model (Sammi et al., 2016). Thymol (100 µM) was shown to possess GABAergic activity and it potentiates GABAA-mediated inhibition of synaptic transmission in vitro (Marin et al., 2011). Thymol (0-1 mM) enhanced GABA-induced (5 mM) chloride influx at concentrations lesser than those revealing direct activity in the absence of GABA (EC 50 = 12 µM and 135 µM, respectively) in  Aydin et al., 2016 primary cultures of mouse cortical neurons (Garcia et al., 2006). A diet rich in thymol has been reported to enhance antioxidant defense and to maintain polyunsaturated fatty acid levels in aging rat brains Deans, 1999, 2000). Thymol has been reported to interact explicitly with synaptic neural functions and block the action of neuronal Na + channels (Haeseler et al., 2002).
Thymol raised the action of chloride channels in oocytes and the cell lines expressing GABA A receptor subunits (Mohammadi et al., 2001;Priestley et al., 2003). Recently, Sanchez et al. (2004) described the ability of thymol to integrate itself into the artificial membranes and enhance the binding affinity of (3H)flunitrazepam to GABA A receptors in synaptosomal membranes that is indicative of thymol's GABA A receptor agonist/modulator property. Thymol (1 mM) has been shown to activate TRPA1 channels and increase the frequent release of L-glutamate on substantia gelatinosa (SG) neurons while generating an outward current without transient receptor potential (TRP) activation in adult rat spinal cord slices by its potent antinoceptive effect (Xu et al., 2015). Thymol (2.7 mM) (IC 50 = 0.34 mM) inhibited the peak amplitude in compound action potentials (CAP) in frog sciatic nerves (Kawasaki et al., 2013). Thymol (200 µM) potentiated the release of (3H)-GABA (EC 50 = 170 µM/L) probably by its antagonistic effect on GABA b autoreceptors in rat neocortical slices (Parker et al., 2014). Thymol (0-400 mg/L) was shown to trigger cytotoxicity in N2a neuroblastoma cells (Aydin et al., 2016).

THYMOL IN LUNG DISEASES
The protective effects of thymol in pulmonary diseases are represented in Table 6.

Asthma
Asthma is an inflammatory disorder characterized by the infiltration of inflammatory cells into lung tissues, hypersecretion of the mucus by goblet cells, airway hypereactivity (AHR), Th2 mediated cytokines and their over-expressions including IL-4, IL-5 and interleukin-13 (IL-13) (Rogerio et al., 2010). Thymol (4, 8, and 16 mg/kg) has been shown to abrogate hyperresponsiveness (AHR) and allergic airway inflammation by attenuating infiltration of inflammatory cells, Th2 cytokines and ovalbumin (OVA)-specific IgE and suppressing the pathological changes due to its NF-κB activation blocking property in OVA-induced allergic mice (Zhou et al., 2014). Thymol (0.7 µg/ml/kg) attenuated ovalbumin induced bronchial allergic asthma by inhibiting oxidative stress in male Wistar albino mice (Al-Khalaf, 2013). In OVA induced mice, thymol (80 mg/kg) suppressed the antigen-specific immune response by inducing reductions T H cells [T H 1, T H 2 and T-helper cell 17 (T H 17)]related cytokines and key transcription factors, revealed their potential to modulate over-activation of T-cells and the associated destructive immune responses . Thymol (50 mg/kg) attenuated oxidative stress mediated bronchial asthma in OVA-Alum induced rat erythrocytes by increasing the status of antioxidants (Mottawie et al., 2011). These findings suggest that thymol possesses the potential to be used as an agent for therapeutic benefits in asthma. However, for the clinical usage, comprehensive safety and efficacy studies are further required (Zhou et al., 2014).
Thymol present in the leaf extract of Ocimum gratissimum Linn (100, 200, and 400 mg/kg) suppressed coughing in OVA induced bronchial asthma by reducing tracheal fluid secretion in rodents through its anti-asthmatic and antitussive effects (Ozolua et al., 2016). A previous report from Gavliakova et al. (2013) has revealed that nasal administration of thymol has been associated with the reduction of cough in asthma patients by an olfactory mechanism. Intake of one bronchipret (around 1.08 mg of thymol) for about a month improved the compliance, pulmonary pressure and airway resistance in the lungs of horses (Van den Hoven et al., 2003). Thymol at higher concentrations (10 −4 -10 −2 M) showed bronchodilatory effects in guinea-pig tracheal preparations (Astudillo et al., 2014). Combined treatment with carvacrol/thymol (200 µM, equal to 30 µg/mL) inhibited the effects of chitin induced asthma by suppressing type 2-promoting release of cytokines and Src Homology 2 (SH2) domain-containing inositol polyphosphate 5 phosphatase 1 (SHIP1), toll like receptors (TLRs), cytokine signaling 1 (SOCS1) and micro RNAs expressions. It also reduced the toll like receptor 4 (TLR4), toll like receptor 2 (TLR2) protein levels and increased the SHIP1 and SOCS1 protein levels (negative regulators of total knee replacement (TKR) mediated immune response) in immortalized human bronchial epithelial cells (BEAS-2B cells). This study revealed the inhibitory effects of carvacrol/thymol treatment against chitin induced epithelial cell pro-inflammatory responses (Khosravi and Erle, 2016).

Pleurism
Thymol (750 mg/kg) has been shown to abrogate carrageenan induced pleurisy by inhibiting the accumulation of inflammatory exudates in the pleural cavity of the lungs (Fachini-Queiroz et al., 2012).

THYMOL IN RADIATION INDUCED TOXICITY
The protective effects of thymol in radiation-induced toxicity are represented in Table 7. Radiotherapy for the treatment of various cancers has been shown to induce serious damage to both tumors and normal cells. A report from Archana et al. (2011b) has revealed that thymol (0-100 µg/mL) diminished radiation-induced genotoxicity, apoptosis and necrosis in V79 cells primarily by the free radical scavenging and modulation of oxidative stress. Thymol treatment prevents the collapse of mitochondrial membrane potential (MMP) and protects the cells from apoptotic and necrotic cell death (Archana et al., 2011b). The radioprotective and anticlastogenic potential of a phenol derivative monoterpene thymol has been reported in whole-body gamma radiation induced Swiss albino mice (Archana et al., 2011a). The antioxidant, anticlastogenic and radioprotective potential of thymol is attributed to the stabilization of intracellular antioxidant levels and free radical scavenging activities by thymol (Archana et al., 2011a). The radioprotective potential of thymol is also demonstrated by increased LD50/30 by 2.17 gray (Gy) which resulted in a dose reduction factor (DRF) of 1.25 (Archana et al., 2011a). Thymol (5 µg/ml) has been shown to abrogate radiation induced cytotoxicity by inhibiting the levels of lipid peroxidation and increasing the status of antioxidants in V79 cells grown in vitro (Archana et al., 2011a). Thymol (1 µg/ml) inhibited UV radiation A (UVA) and UV radiation B (UVB) induced genotoxicity via inhibiting oxidative stress in the NCTC 2544 cell line (Calo et al., 2015). Thymol (1 µg/ml) attenuated UV radiation induced genotoxic damage in ex vivo human skin models by its potent anti-cancer properties (Cornaghi et al., 2016).

THYMOL IN AUTOIMMUNE DISEASES
The protective effect of thymol against autoimmune diseases is represented in Table 8. Rheumatoid arthritis, an autoimmune disease is characterized by chronic and progressive inflammation of the synovial joints and erosive destruction of the articular tissue (Feldmann et al., 1996;Feldmann, 2002;Choi et al., 2009). Thymol (100 mg/kg) was shown to inhibit collagen induced arthritis by decreasing lipid peroxidation mediated oxidative stress by increasing the status of antioxidants in male Wistar rats. Thymol also stopped the activity of elastase, a marker for collagen degradation and prevented the invasion of inflammatory cells to the injured site by blocking the Ca 2+ channels (Braga et al., 2006;Umar et al., 2012). The physical mixture of diacerein and thymol (DTH) (50 + 20.4 mg/kg) abrogated Freund's complete adjuvant (FCA) induced arthritis in male albino Wistar rats. This combined treatment decreased oxidative stress, ulcer index and synovitis in arthritic rats by its potent antioxidant property. DTH administration also improved the histoarchitecture as evidenced by decreased necrosis in bones, cellular infiltration, connective tissue proliferation and the involvement of adjacent tissues (Dhaneshwar et al., 2013). In Jurkat leukemia cells as an in vitro T cell model, thymol (25 µg/ml) modulated T-cell activity by reducing IL-2 and IFN-γ production via down regulation of AP-1 and nuclear factors of activated T-2 (NFAT-2) transcription factors showing its capacity in reducing the overactivity of T-cells in immune mediated diseases (Gholijani et al., 2015).

THYMOL IN REPRODUCTIVE DISORDERS
The protective effects of thymol in reproductive disorders are represented in Table 8.

Contraceptive
Male infertility refers to the inability of males to cause pregnancy in females usually due to reduced sperm quantity and quality (Cooper et al., 2009). Thymol (400 mg/kg) decreased fertility in male albino Wistar rats. Thymol decreased the weight of testis, sperm count and motility and increased the amount of abnormal sperms in rat testis (Surendra Kumar et al., 2011). Chikhoune et al. (2015) revealed the anti-fertility effect of thymol in human spermatozoa. Thymol (100-500 µg/ml) dose dependently decreased sperm count, sperm motility, sperm vitality in human sperm. These two studies have revealed that thymol could be used as a standard contraceptive agent in humans.

THYMOL IN METAL INDUCED TOXICITY
The protective effects of thymol in metal induced toxicity are represented in Table 8.

Chromium
Chromium is a naturally occurring, highly toxic transition metal due to its strong ability to oxidize cellular components through its passive entry via cellular membranes into cells (O'Brien et al., 2003). Thymol (2.5 µg/ml) has been shown to inhibit hexavalent chromium induced oxidative damage in rat erythrocytes. Thymol treatment significantly decreased MDA levels, hemolysis, erythrocyte destabilization and increased the activities of antioxidants enzymes and improved the levels of glutathione in rat erythrocytes (Abd-Elhakim and Mohamed, 2016).

Arsenic and Mercury
Arsenic and mercury are toxic metals found in nature in soil, in industrial and agrochemicals as well as pharmaceuticals. Upon exposure, they are known to cause acute and chronic disease and mainly affect smooth muscles of the cardiovascular and respiratory systems. Thymol (0-200 µM/L) abrogated arsenic and mercury induced hyper contraction of both aortic and tracheal smooth muscles by inhibiting Ca 2+ influx at low concentrations. It also neutralizes ROS and inhibits Ca 2+ influx at higher concentrations (Kundu et al., 2016). Thymol (100 µM) was shown to protect against cytotoxicity and genotoxicity induced by mercuric chloride in the human HepG2 cell line due to its potent free radical scavenging ability that in reflected in the attenuation of mitochondrial and oxidative damage (Shettigar et al., 2015). Thymol present in the essential oil of T. lanceolatus (IC 50 = 256.17 µg/ml) was shown to induce cytotoxicity and cell proliferation in HepG2 cells (Khadir et al., 2016).

PHARMACEUTICAL DEVELOPMENT OF THYMOL
Nowadays, the focus on natural products is to develop their formulation with improved bioavailability, favorable pharmacokinetics and minimal adverse effects. Various attempts have also been made to develop thymol formulation with improved drug delivery options for the treatment of various human diseases. The pharmacokinetic and physiochemical properties of thymol including absorption, bioavailability, elimination rate, solubility are the major barricades in the drug design and delivery of thymol. There are lot of techniques such as structural modification (Mastelic et al., 2008), microparticles using cellular derivatives (Zamani et al., 2015), encapsulation , solid dispersion (Roost et al., 2015), complexations  and nanoparticle formulation (Pan et al., 2014;Zhang et al., 2014) which could pave the way to advance drug delivery options for thymol and these are mentioned below.

STRUCTURAL MODIFICATION FOR FUTURE DRUG DEVELOPMENT
The structural alterations into the phenol structures, like introducing a polar hydroxymethyl moiety, could enhance its antioxidant activity compared to parent compounds (Torres de Pinedo et al., 2007). The derivative of thymol, named 4-(hydroxymethyl)-2-isopropyl-5-methylphenol, was synthesized by the hydroxymethylation of thymol (Mastelic et al., 2008). The phenolates as nucleophiles reacted with methanol which yielded hydroxymethylphenols at alkaline pH. This might be due to the delocalization of the phenolate ion charge between the phenolate oxygen and its respective ortho and para-carbons. The steric effect of the isopropyl group was believed to confer improved antioxidant activity and reduced mitochondrial activity of thymol derivative in HeLa cells in a concentration dependent manner (Mastelic et al., 2008). A set of new thymol derivatives invented and patented recently showed potent antitumor activity against A549, SKOV-3, human melanoma cells (SK-MEL-2), cellosaurus cells (XF498) and colorectal adenocarcinoma cells (HCT15 cells) (Zee et al., 2001). Thymol analogues such as 4-morpholinomethyl-2-isopropyl-5methylphenol (THMO) and 4-Pyrrolidinomethyl-2-isopropyl-5methylphenol (THPY) were synthesized by the reaction between thymol and formaldehyde with morpholine or pyrrolidine (Shen et al., 2005). These two analogs of thymol showed a potent superoxide anion scavenging effect in vitro and in human blood neutrophils and also possess a superior lipid peroxidation inhibitory effect via the attenuation of enzymes involved in antioxidant defense. In the two thymol analogs, THMO revealed potent antioxidant activity with IC 50 values of 21.72 and 61.29 µM for the inhibition of xanthine oxidase and lipid peroxidation (Shen et al., 2005). THMO (10 mM) also decreased the peak amplitude of L-type inward current of Ca 2+ (I Ca,L ) in NG108-15 cells as analyzed by the patch-clamp technique. These reports have revealed that the antioxidative action of the thymol analogs is linked with its capacity of inhibiting Ca 2+ current (Shen et al., 2005). This study suggests that THMO could be a suitable candidate for the treatment of free radical related disorders by virtue of its antioxidant and Ca 2+ ion current inhibition activity.

MICROENCAPSULATION FOR DRUG DELIVERY
Microencapsulation is a tool frequently used in pharmaceutical, food, cosmetic, and agrochemical industries. The encapsulation of thymol in microspheres made up of natural polymers such as methylcellulose and hydroxylpropyl methylcellulose phthalate can serve to obtain efficient delivery of this phytochemical as adjuvants or current medications for the treatment of infectious diseases and compensate the limited bioavailability due to its lower solubility . The core-shell or matrix particle encapsulation of essential oils has been investigated to determine their controlled release (Martins et al., 2014). The encapsulation of thymol into methylcellulose microspheres by spray drying remarkably increases the bioavailability compared to free thymol and it can be suggested to be used for the treatment of intestinal infections .
The synthetic, natural and semisynthetic polymers play a crucial role in drug release formulations and nowadays these are used as efficient drug carriers (Nayak et al., 2009). Cellulose derivatives have been used for sustained release matrices, delayed release dosage forms, binders in granules and tablets and they also have many other applications (Chambin et al., 2004). Zamani et al. (2015) has structured the matrix polymer encapsulation of thymol by the emulsion solvent evaporation method with hydroxy propyl methyl cellulose (HPMC) and ethyl cellulose (EC) to increase the duration of action of thymol. Both polymers have shown a considerable effect on drug release behavior, efficiency of drug entrapment, drug loading and particle size, whereas the formulation F6 revealed a controlled effect compared to the other formulations in in vitro release (Zamani et al., 2015).

CO-ADMINISTRATION FOR DRUG DELIVERY
A co-drug DTH has been developed recently by Dhaneshwar et al. (2013). Diacerein, an IL-1β inhibitor possesses anti-arthritic and moderate anti-inflammatory properties (Dhaneshwar et al., 2013). This mutual prodrug was developed by the covalent linkage of thymol with the carboxylic acid (−COOH) group of diacerein and this prodrug showed lessened irritant effect, improved absorption, prolonged drug release with improved anti-inflammatory effect. The hydrophobic nature of thymol enhanced the lipophilicity of diacerein which could be responsible for its enhanced bioavailability and its better absorption. The synthesis of DTH was done by the dicyclohexylcarbodiimide (DCC) coupling method (Holmberg and Hansen, 1979) and its physicochemical characterization was evaluated using spectral analysis. DTH was very stable in acidic pH conditions of the stomach and complete diacerein release was observed in phosphate buffer (91-94%) (pH 7.4) and in the small intestine.
Thymol has been used as an antioxidant agent in this design and its selection was justified after the promising anti-inflammatory and anti-arthritic effect of DTH against osteoarthritis compared to standard drugs which is attributed to the pharmacological effects of thymol. DTH attenuated FCA induced chronic synovitis in rats by its marked antiarthritic effect compared to the moderate effect of diacerein and mild effect of thymol. The authors suggested that the combination of diacerein with thymol could be promising therapeutic approach in inflammation related diseases (Dhaneshwar et al., 2013).

CONCLUDING REMARKS
The need for alternative therapies with less toxic effects for various human ailments is evident. The findings from various studies reviewed herein showed the role of thymol in the prevention of various types of diseases through its multipharmacological properties from antioxidant to anti-tumor ones.
Thymol containing plants have been used in traditional medicine for management of various diseases such as many cancer types, cardiovascular diseases, diabetes, and neurodegenerative diseases. Multiple pharmacological and molecular mechanisms of action for its preventive and therapeutic effects have been demonstrated based on its molecular targets identified in numerous studies. While a great number of in vitro studies for numerous diseases including cancer and cardiovascular diseases have been reported, more in vivo studies should be undertaken to confirm the in vitro findings. In addition, there is a contradiction between in vitro concentrations and in vivo doses in certain types of cancer. Thus, pharmacokinetics and pharmaceutical studies are needed to interpret the inconsistency between in vitro and in vivo results. These reported features along with the minimal side effects, cost effectiveness and easy access made thyme and its constituent thymol an effective therapeutic agent for the management of numerous chronic diseases. Furthermore, thymol, being abundantly and ubiquitously present in numerous plants, could be available for dietary use. Its administration and benefits could be achieved in a simpler way through normal daily diet. However, the vast majority of the data is preclinical, and further clinical studies are warranted. Furthermore, comprehensive toxicological studies should be conducted to support the safety of thymol in animal models to progress for clinical studies. Though, taking together all the studies, it is significant to say that research on thymol as a drug candidate is progressive and encouraging. This has been well demonstrated by the publication patterns year after year. Hence, thymol is one of the most powerful contenders in the race of phytochemicals of natural origin with polypharmacological properties displaying potent preventive and therapeutic properties against various human diseases.

AUTHOR CONTRIBUTIONS
SO and MN conceptualized and outlined the study. MN drafted the manuscript. HAT, SA, HJ, and SO edited and reviewed the manuscript. SO and MN throughly re-reviewed it and all authors approved it.