- Interleukin 4 induced 1 (IL41I) transcription [2]
- Aryl hydrocarbon receptor (AHR) signaling pathway [2]
- Voltage-gated potassium channel (Kv) beta subunit KCNAB [4]
- Transient Receptor Potential Ankyrin Subtype 1 channel (TRPA1) with an IC50 of 104 μmol for skeletal muscle sodium channel and 149 μmol for neuronal sodium channel. [1][3]

- In a lipid system consisting of a purified fraction of triacylglycerols of lard (TGL) and sunflower oil (TGSO), thymol at concentrations of 0.02, 0.05, 0.10 and 0.20% inhibited both TGL and TGSO oxidation. [1]
- Thymol at 10 and 20 μg/ml interfered with the release of elastase by human neutrophils stimulated with FMLP in a concentration-dependent manner by inhibiting calcium entry. [1]
- Thymol showed a half-maximum blocking concentration (IC50) of 104 μmol for the skeletal muscle sodium channel and 149 μmol for the neuronal sodium channel. [1]
- Thymol at 10-1000 μM inhibited L-type Ca2+ currents and K+ currents in canine and human ventricular cardiomyocytes, causing negative inotropic actions. At 10 μM, it removed the notch of the action potential; at 100 μM or higher, it shortened action potential duration. [1]
- Thymol at 300 and 1000 μM significantly reduced the contraction evoked by phorbol dibutyrate (1 μM) in isolated rat aorta, inducing endothelium-independent relaxation. [1]
- Thymol at 100, 200, and 400 μM induced the inhibition of gastric cell growth in a dose-dependent manner, prolonging the sub-G1 phase and inducing apoptosis. [1]
- Thymol showed MIC values ranging from 0.03 to 0.06% v/v against 22 S. aureus and 21 S. epidermidis clinical isolates. [1]
- Thymol had MIC values of 1.0 mmol/L against S. typhimurium and 1.2 mmol/L against E. coli. [1]
- Thymol showed MIC of 0.31 mg/ml against S. aureus ATCC 68380 and 5.00 mg/ml against E. coli ATCC 15221. [1]
- Thymol at 2.5 mM inhibited S. aureus, E. coli and S. typhimurium, and at 0.31 mM inhibited S. pyogenes. [1]
- Thymol showed MIC of 62.5 μg/ml for S. aureus, 125 μg/ml for S. mutans, and 250 μg/ml for E. coli. [1]
- Thymol showed MIC of 0.010 mg/ml for E. coli and 0.156 mg/ml for S. aureus, with MBC of 0.156 mg/ml for E. coli and 0.313 mg/ml for S. aureus. [1]
- Thymol showed MIC of 0.5 g/L and MBC of 1 g/L against E. coli O157:H7 and L. monocytogenes. [1]
- MIC values for thymol against M. luteus was 1250 μg/ml. [1]
- Thymol at a concentration of 750 mg/L showed a time-dependent decrease in CFU count against S. typhimurium. [1]
- Thymol showed MIC values ranging from 0.125 - 1 μg/ml against E. coli, M. catarrhalis, S. aureus, E. faecalis, B. cereus, C. albicans and C. tropicalis. [1]
- Thymol showed antifungal MIC of 100-500 mg/ml against various fungi including Cladosporium spp., Aspergillus spp., A. flavus, and A. ochraceus (MIC: 100 mg/ml) and A. fumigatus (MIC: 150 mg/ml). [1]
- Thymol showed MIC of 39 μg/ml for C. albicans and C. krusei, and 78 μg/ml for C. tropicalis. [1]
- Thymol showed MIC of 187.5 μg/ml for E. coli, and was active against M. tuberculosis (MIC 0.78 μg/ml) and M. bovis (MIC 2.02 μg/ml). [1]
- Silencing IL4I1 in LLC cells significantly increased the proportion of CD8+ T cells, granzyme B+ CD8+ T cells, and M1-like macrophages, while decreasing Tregs and M2-like macrophages. It also blocked the nuclear translocation of AHR. [2]
- Thymol (5 μM for LLC cells, 20 μM for A549 cells) inhibited IL4I1 expression in a dose- and time-dependent manner, blocked nuclear translocation of AHR, and decreased mRNA levels of AHR target genes (IL6, IL10, IL22, IL1b, Tiparp, Mmp13, Cyp1b1). [2]
- Thymol (5 μM for LLC cells, 20 μM for A549 cells) inhibited the migration ability and epithelial-mesenchymal transition (increased epithelial markers, decreased mesenchymal markers) but did not affect proliferation. [2]
- Thymol (4.3 mM) for 2 minutes showed a long-lasting cytotoxic effect in Cal27 and HeLa cells, with no colony formation after 9 days. [3]
- Thymol induced a concentration-dependent reduction in cell viability in OSCC cell lines (Cal27 GC50: 300 μM; SCC4 GC50: 500 μM; SCC9 GC50: 550 μM) and other cancer cell lines (HeLa, H460, MDA-231, PC3 GC50s: 350-500 μM). [3]
- Thymol (200-800 μM) induced a concentration-dependent increase in cleaved PARP (c-PARP) and depolarization of mitochondrial membrane potential (Ψm) in Cal27 and HeLa cells. [3]
- Thymol (400 μM) induced calcium influx in Cal27 cells (reversed by TRPA1 inhibitor HC030031), but not in HeLa cells. [3]
- Thymol (200 μg/mL) induced conidial apoptosis in A. flavus, as evidenced by chromatin condensation, phosphatidylserine externalization, DNA damage (TUNEL positive), mitochondrial depolarization (JC-1), and caspase 9 activation. [4]
- Thymol (200 μg/mL) activated aldo-keto reductase activity of the purified recombinant KCNAB protein in vitro. [4]
- Thymol stimulated a transient K+ efflux in A. flavus conidia and protoplasts, as determined by automated patch-clamp, showing activation of voltage-gated K+ channels (Kv). [4]
- Blocking K+ efflux with 4-aminopyridine (4-AP) significantly alleviated thymol-mediated conidial apoptosis. [4]

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In A549 cells, thymol (20 μM, 24-48 hours) affects the expression of IL4I1 (24 hours) and the aromatic channel receptor (AHR) signaling pathway (48 hours) [2]. Thymol (5 μM, 48 hours) A549 Thymol (0-600 μM, 48 hours) modulation suppresses the epithelial-to-mesenchymal transition, motor activity, and cell proliferation in several binary types (Cal27, SCC4, SCC9, HeLa, H460, MDA-231, and PC3 cells) [2]. In A, thymol (200 μg/mL) causes conidia acetone that is reliant on caspase. flavus [4].

- Thymol, as the main constituent (32.68%) of L. gracilis essential oil (200 mg/kg), reduced carrageenan-induced edema formation and leukocyte migration in mice, showing anti-inflammatory activity. [1]
- Thymol (10, 30, and 100 mg/kg) decreased edema and leukocyte influx in rodent models. Collagen-based films containing thymol showed significantly bigger wound retraction rates after 7 and 14 days of treatment. [1]
- Silencing IL4I1 in LLC cells dramatically repressed tumors and enhanced survival in C57BL/6 mice treated with anti-PD-1 antibody (10 mg/kg, i.p., every 1 week). [2]
- Thymol (75 mg/kg, i.p., every 2 days) significantly suppressed the progression of LUAD in an orthotopic mouse model. Combination treatment of thymol (75 mg/kg, i.p., every 2 days) with anti-PD-1 antibody (10 mg/kg, i.p., every 1 week) dramatically suppressed tumor progression and greatly enhanced survival. [2]
- Thymol (75 mg/kg) treatment increased CD8+ T cells, GZMB+ T cells, and M1-like macrophages, and decreased Tregs and M2-like macrophages in tumors. [2]
- Thymol (4.3 mM) injected every other day for 26 days significantly inhibited Cal27-derived xenograft tumor growth in athymic nude mice, with mean tumor volumes of 96 mm³ (treated) vs 423 mm³ (control). Significant reduction was seen by day 16 (p<0.05). [3]
- Thymol (4.3 mM) injected every other day for 22 days significantly inhibited HeLa-derived xenograft tumor growth, with mean tumor volumes of 537 mm³ (treated) vs 903 mm³ (control). Significant reduction was seen by day 18 (p<0.05). [3]
- Thymol treatment (4.3 mM) significantly reduced the number of proliferating cells (Ki67 positive) and increased the number of apoptotic cells (TUNEL positive) in Cal27-derived tumors. [3]

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In an orthotopic mouse lung adenocarcinoma (LUAD) model, thymol (75 mg/kg, i.p.) alone and in combination with anti-PD-1 antibody (10 mg/kg, i.p.) both decrease LUAD progression and make animals more susceptible to anti-PD-1 antagonist therapy [2]. In Cal27-derived xenograft mice, thymol (4.3 mM in 50 μL of sterile saline, intratumoral injection, daily) demonstrated anticancer efficacy.

- The aldo-keto reductase activity of the purified recombinant KCNAB protein was determined. The reaction mixture contained 0.4 mM NADPH, 10 mM 4-formylbenzonitrile, and 300 μg/mL KCNAB protein in phosphate buffer (pH 7.4). The mixture was incubated at 37°C for 2 hours. The absorption at 340 nm was measured to indicate reductase activity, shown as a decrease in OD340nm (1 U = 0.01). Blank controls without KCNAB were used to subtract background NADPH consumption. [4]
- No detailed description of other enzyme or target binding assays (e.g., SPR, ITC, HTRF) was found in the provided literature.

- Cell Viability Assay (MTS): Cells were plated and treated for 48 hours with thymol at indicated concentrations (final DMSO concentration 0.1%). Cell Titer 96® Aqueous Non-Radioactive Cell Proliferation Assay was performed according to the manufacturer's protocol. Absorbance values of test groups were compared to vehicle-treated controls (n=4). [1][3]
- Clonogenic Assay: 300 cells per plate were allowed to adhere in 60 mm³ tissue culture dishes and subsequently treated with vehicle (final 0.5% DMSO) or thymol (4.3 mM) for 2 minutes. Cells were washed with sterile PBS, fresh growth medium was added, and cells were allowed to grow and form colonies for 9 days. Following incubation, cells were fixed and stained with 0.5% crystal violet in 10% methanol, washed with PBS, and quantified. [3]
- Flow Cytometry Analysis of Tumor-Infiltrating Leukocytes: Tumors were minced and incubated with digestion buffer for 30 min at 37°C. Red Blood Cells Lysis Buffer was used to lyse erythrocytes. Cells were rinsed with PBS containing 1% FBS and filtered using a 40-μm nylon filter. Cells were labeled with specified antibodies for 30 minutes for cell-surface marker staining. For macrophage and MDSC analysis, cells were Fc-blocked with anti-CD16/32 before antibody staining. For intracellular staining, surface-stained cells were fixed, permeabilized, and then stained with indicated antibodies. [2]
- Detection of Conidial Apoptosis in A. flavus: Conidia were stained with Hoechst 33342 (10 μg/mL) and propidium iodide (PI, 1 μg/mL) for 30 min at 4°C in the dark. Apoptotic cells were observed with an inverted fluorescence microscope and a flow cytometer. [4]
- Phosphatidylserine Externalization Assay (Annexin V-FITC): Conidia (2×10⁶) exposed to 200 μg/mL thymol for 6 hours were collected. Annexin V-FITC apoptosis detection kit was used according to the manufacturer's instructions. Flow cytometry and fluorescence microscopy were utilized to monitor spores with positive FITC fluorescence. [4]
- TUNEL Assay for DNA Damage: Conidia (2×10⁶) exposed to 200 μg/mL thymol for 6 hours were collected. A one-step TUNEL apoptosis assay kit was used according to the manufacturer's instructions. Flow cytometry and fluorescence microscopy were utilized to monitor spores with positive FITC fluorescence. [4]
- Mitochondrial Membrane Potential (Δψm) Assay (JC-1): The fluorescent dye JC-1 was used. JC-1 accumulates in mitochondria in a potential-dependent manner. At high Δψm, JC-1 forms aggregates (red fluorescence); at low Δψm, it exists as monomers (green fluorescence). Mitochondrial depolarization is indicated by an increase in the green (529 nm)/red (590 nm) fluorescence intensity ratio, measured by a microreader and fluorescence microscope. [4]
- Caspase 9 Activity Assay: Conidial caspase 9 activity was detected using a kit containing the substrate Ac-LEHD-pNA. Spore suspension (2×10⁶/mL) was exposed to 200 μg/mL thymol for 6 hours. Spores were washed with PBS, suspended in lysis buffer, and sonicated on ice for 20 min. After centrifugation at 16000g at 4°C for 15 min, the supernatant was used to determine the increase in absorbance at 400 nm, indicating caspase-9 activity (1 U = 1 μM pNA released). [4]
- Western Blot Analysis: Cells were treated for 48 hours with vehicle control or thymol (final DMSO concentration 0.1%), then harvested and lysed in 1% Triton-PBS. Cell lysate concentration was determined by A280 reading. Lysates containing equal protein concentration were separated using 10% SDS-PAGE. Proteins were transferred to PVDF membrane and blocked in 5% milk solution. Primary antibodies (anti-cPARP, anti-α/β-tubulin) were diluted in 1% milk in PBS-0.1% Tween-20 and incubated overnight at 4°C. The membrane was washed with PBS-Tween-20 and incubated with ECL Plus detection solution for 1 min. Signal was detected by exposure to radiograph film for 30 seconds. [3]
- Calcium Imaging: Cells were incubated with 2 μM Oregon Green 488 BAPTA-1, AM at 37°C for 25 min, then treated with thymol (200 μM, 400 μM, or 1 mM; n=4 per group). 3 μM ionomycin was used as a positive control. To evaluate specific TRP channel activity, cells were pretreated with TRPA1 inhibitor HC030031 (10 μM), TRPM8 inhibitor RQ00203078 (10 mM), or TRPV1 inhibitor capsazepine (10 μM) followed by thymol. Images were acquired on a confocal microscope with a 40× oil immersion objective for 5 min recordings in recording media at 37°C. Images were analyzed, and background-corrected fluorescence was normalized. [3]
- Dual-Luciferase Reporter Assay: The IL4I1 promoter region (-2760 to -200) was cloned into the pGL4.17[luc2/Neo] plasmid. A549 cells were stably transfected with this firefly luciferase reporter plasmid and a renilla luciferase control reporter plasmid. Cells were treated with 20 μM of compounds from a drug library for 24 hours, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System. Compounds inhibiting reporter gene expression to less than 30% were considered effective. [2]
- RNA Extraction and qRT-PCR: Total RNA was extracted using TRIzol reagent and converted to cDNA using M-MLV reverse transcriptase. qRT-PCR was performed using SYBR Green SuperMix on a LightCycler96 system. The program consisted of an initial step at 95°C for 300 s followed by 45 cycles of 30 s at 95°C and 45 s at 60°C. The 2⁻ΔΔCt method was used to calculate relative gene expression with β-actin for normalization. [2]
- Immunofluorescence (IF) Staining: Cells were fixed and stained with primary antibodies against IL4I1 (red) and AHR (green), and DAPI (blue) for nuclear staining. Images were acquired, and the fluorescent intensity of AHR in the nucleus versus cytoplasm was quantified. [2]
- Intracellular K+ Assay: The K+ fluorescent indicator PBFI-AM was used. The relative fluorescence intensity at 500 nm excited by 340 and 380 nm was time-course recorded by a microreader. The ratio between them was calculated. Flow cytometry was also used. For K+ distribution, double staining with PBFI and JC-1 was performed, and images were evaluated using a confocal laser scanning microscope. [4]

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Western blot analysis [2]
Cell Types: A549 cells
Tested Concentrations: 20 μM incubation.
Incubation Duration: 48 h
Experimental Results: Inhibition of IL4I1 protein levels and AHR nuclear translocation. diminished overall AHR level.

- For the orthotopic LUAD model, 1×10⁶ luciferase-labeled LLC cells suspended in 50 μL cold PBS mixed with 50 μL cold Matrigel were injected into the middle lobe of the right lungs of female C57BL/6 mice. From day 5 post-injection, mice were intraperitoneally injected with 10 mg/kg of mouse anti-PD-1 antibody dissolved in 100 μL PBS every 7 days. Thymol was dissolved in a solvent containing 40% PEG300, 5% Tween-80, 10% DMSO, and 45% saline. 75 mg/kg thymol was intraperitoneally injected every 2 days. Tumor growth was detected by bioluminescence imaging. [2]
- For the subcutaneous xenograft model, 6-week-old female athymic nude mice were used. Cal27 or HeLa cells were injected subcutaneously. When tumors reached approximately 90 mm³, mice were treated every other day with thymol (4.3 mM) or vehicle control. Tumor volumes were calculated using the formula: 1/2(length × width²). At experimental conclusion, tumors were fixed for histological analysis (H&E, TUNEL, Ki67). Body weight was monitored throughout the study. [3]

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Animal/Disease Models: Orthotopic mouse LUAD model [2]
Doses: Thymol 75 mg/kg, anti-PD-1 antibody 10 mg/kg
Route of Administration: anti-PD-1 antibody: intraperitoneal (ip) injection, once every 1 week. Day 5 after tumor injection. Thymol: intraperitoneally (ip) (ip), every 2 days starting on day 5 after tumor injection.
Experimental Results: Inhibited the progression of LUAD and improved the survival rate of mice. Reduce IL4I1 levels in tumors. Improve the efficacy of anti-PD-1 antibodies.

Absorption, Distribution and Excretion
Aromatic herbs are increasingly attracting attention as animal feed additives, but data on the metabolism of aromatic compounds in animals is very scarce. This study used thyme (Thymus vulgaris, leaves and flowers only, excluding stems) as a raw material to feed broiler diets. Five groups of broilers were fed thyme at addition levels of 0%, 0.1%, 0.2%, 0.3%, and 1% (w/w) for 35 days. Solid-phase microextraction-gas chromatography/mass spectrometry was used to determine the growth performance of the animals and the concentration of thymol, the main essential oil component of thyme, in intestinal contents, plasma, liver, and muscle tissue. There were no significant differences in feed intake, daily weight gain, feed conversion ratio, and slaughter weight among the groups. Thymol was detected in intestinal contents, plasma, liver, and muscle tissue. Compared with other groups, the intestinal thymol concentration was significantly higher in the group with 1% thyme (P<0.05). The thymol content in liver and muscle tissue is close to the limit of quantification. Data indicate that thyme has no positive effect on animal production performance. When high doses of thyme are added to the diet, the concentration of thymol in intestinal contents and plasma increases, but the concentration in edible tissues such as liver and muscle remains low. Thymol is readily absorbed from the gastrointestinal tract after oral administration. It is primarily excreted in the urine within 24 hours of absorption. Only a small amount of the absorbed substance is excreted in the urine as hydroxylated compounds. Thymol is primarily excreted in its original form and in the form of glucuronide and sulfate conjugates. Substituted monophenols, such as thymol…are found in plant essential oils, especially thyme essential oil, where they…are conjugated with glucuronic acid and sulfate. Known human metabolites of thymol include p-cymene-8-en-3-ol, p-cymene-2,3-diol, thymol sulfate, thymol O-glucuronide, and thyquinone.

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- Thymol has low water solubility and low palatability. [1]
- A single dose of thymol (0.5 g/kg) was rapidly absorbed after oral administration and slowly eliminated within 24 h. The time to reach maximum concentration (Tmax) was 30 min, while the half-life of the absorption phase (t1/2) was approximately 0.3 h. [1]
- Concentrations of thymol recovered in organs such as the liver, lungs, kidneys, and muscles were quite low. A higher amount was detected in the intestines, indicating it is not totally absorbed. [1]
- When complexed with β-cyclodextrin, the in vivo absorption of thymol increased, while the half-life remained long. Liposomal encapsulation did not increase solubility. A co-drug design with diacerein enhanced lipophilicity and bioavailability. [1]

Toxicity Summary
Identification and Uses: Thymol forms colorless crystals, usually large, or as a white crystalline powder. It is used as an insecticide (pesticide, fungicide, rodenticide, antimicrobial agent); also in fragrance manufacturing; as a fungicide; for microscopic examination; as a preservative; an antioxidant; a flavoring agent; and as a laboratory reagent. Thymol is included in a U.S. Food and Drug Administration (FDA)-approved over-the-counter drug as an antimicrobial and antifungal agent and is approved as an excipient. Human Exposure and Toxicity: Given the wide range of uses of thymol, primary skin irritation and sensitization are caused in humans, alone or as an ingredient in compound preparations, in very rare cases. Thymol is a mild local irritant. Its systemic effects are similar to phenol, but less toxic, partly due to its lower solubility. It can cause stomach pain, nausea, vomiting, central nervous system hyperactivity (e.g., polyphagia), and occasionally seizures, coma, and cardiopulmonary failure. Thymol has no genotoxicity against the human colon cancer cell line Caco-2. Animal studies: Acute oral administration of thymol is harmful, while acute dermal administration is virtually non-toxic. In rabbits, thymol is corrosive to the skin and eyes. Rats tolerated 10,000 ppm of thymol after 19 weeks of subchronic administration in their diet. Thymol did not increase the incidence of spontaneous lung tumors in mice. In chicken embryos, injection of thymol into the yolk sac or cyst resulted in various malformations. In vivo studies showed that oral administration of thymol did not induce micronuclei in mice, even at toxic doses. Thymol did not exhibit mutagenic effects in the Salmonella/microsome assay. However, thymol has been reported to be positive in the UDS (liquid scintillation counting) assay and the SCE assay in Syrian hamster embryonic cells. Although no strict dose-response relationship was found, these results were statistically significant. Other effects of thymol include cytotoxicity, antitumor activity, antibacterial activity, fungicide activity, anti-inflammatory activity, antispasmodic activity, and other pharmacodynamic effects. Ecotoxicity Study: Researchers investigated the effects of thymol on olfactory memory and brain gene expression in bees previously exposed to thymol. The results showed that the bees' specific responses to the conditioned stimulus disappeared 24 hours after learning. The study also indicated that genes encoding thymol cellular targets can be rapidly regulated after exposure to the molecule. Beekeepers use essential oils (including thymol) to control Varroa mites infesting bee colonies.

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Interactions
This study used the human hepatocellular carcinoma cell line HepG2 to investigate the protective effect of thymol (TOH) against HgCl2-induced cytotoxicity and genotoxicity. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide (MTT) assay confirmed that TOH pretreatment effectively reduced HgCl2-induced cytotoxicity. TOH pretreatment inhibited HgCl2-induced genotoxicity, mitochondrial membrane depolarization, oxidative stress, and mitochondrial superoxide levels. Interestingly, thymol alone (TOH, 100 μM) increased basal intracellular glutathione S-transferase (GST) levels, and TOH pretreatment eliminated the decreases in glutathione, GST, superoxide dismutase, and catalase levels even after HgCl₂ poisoning. Furthermore, TOH inhibited HgCl₂-induced apoptosis and necrosis, as confirmed by flow cytometry analysis of Annexin-FITC/propidium iodide double-stained cells. These results clearly demonstrate that TOH has a cytoprotective effect against HgCl₂-induced toxicity, which may be attributed to its ability to scavenge free radicals, thereby helping to reduce oxidative stress and mitochondrial damage, and ultimately inhibiting cell death. Thymol is a natural monoterpene phenol, mainly found in thyme, oregano, and orange peel. Studies have shown that it possesses anti-inflammatory properties both in vivo and in vitro. This study investigated the anti-inflammatory effects of thymol on lipopolysaccharide (LPS)-stimulated mouse mammary epithelial cells (mMECs). mMECs were stimulated with LPS in the presence or absence of thymol (10, 20, 40 μg/mL). The concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β in the culture supernatant were determined by enzyme-linked immunosorbent assay (ELISA). The expression levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), nuclear factor-κB (NF-κB), and NF-κB inhibitor (IκB) were detected by Western blot. The results showed that thymol significantly inhibited the production of TNF-α and IL-6 in LPS-stimulated mMECs. Furthermore, thymol also inhibited the expression of iNOS and COX-2 in a dose-dependent manner. More importantly, thymol blocked the phosphorylation of IκB, NF-κB p65, ERK, JNK, and p38 mitogen-activated protein kinase (MAPK) in LPS-stimulated mMECs. These results indicate that thymol exerts its anti-inflammatory effect in LPS-stimulated mMECs by interfering with the activation of the NF-κB and MAPK signaling pathways. Therefore, thymol may be a potential therapeutic agent for mastitis.

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Non-human toxicity values
Oral LD50 in rats: 980 mg/kg
Oral LD50 in mice: 640 mg/kg
Intravenous LD50 in mice: 100 mg/kg
Dermal LD50 in rats: >2000 mg/kg

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Toxicity/Toxicokinetics: - The use of thymol in drugs is limited by its moderate cytotoxicity that was shown both in vitro on human and animal cells, and in experimental animal studies. [1]
- Thymol is used in small concentrations (less than 1 mM) for cosmetic formulations, foods, external drugs, and biocidal products. [1]
- Thymol at 100 and 1000 μM induced cardiac arrhythmias in canine ventricular cardiomyocytes via inhibition of K+ and Ca2+ currents. [1]
- No obvious toxicity was observed in mice under the treatment of thymol (75 mg/kg, i.p., every 2 days) by monitoring body weight change, pathologic changes of major organs (heart, liver, kidney, brain by H&E staining), and serum biochemical indexes. [2]
- No adverse reactions were observed in athymic nude mice treated with thymol (4.3 mM every other day). Adjacent non-cancerous tissues appeared healthy with no erythema, swelling, or ulceration, and no nociceptive behaviors or weight loss was noted. Body weight slightly increased with no significant differences between treated and control animals. [3]

[1]. Antibacterial and antifungal activities of thymol: A brief review of the literature. Food Chem. 2016 Nov 1;210:402-14.

[2]. Thymol targeting interleukin 4 induced 1 expression reshapes the immune microenvironment to sensitize the immunotherapy in lung adenocarcinoma. MedComm (2020). 2023 Aug 30;4(5):e355.

[3]. Thymol inhibits oral squamous cell carcinoma growth via mitochondria-mediated apoptosis. J Oral Pathol Med. 2018 Aug;47(7):674-682.

[4]. Thymol Induces Conidial Apoptosis in Aspergillus flavus via Stimulating K+ Eruption. J Agric Food Chem. 2018 Aug 15;66(32):8530-8536.

- Thymol is biosynthesized by the aromatization of γ-terpinene to p-cymene followed by hydroxylation of p-cymene. [1]
- The essential oils containing thymol have been used in the food industry as flavouring and preservative agents. Thymol is registered by the European Commission as a flavouring in foodstuffs and is on the FDA's 'generally recognized as safe' (GRAS) list. [1]
- Thymol is used in commercial mosquito repellent formulations. It repels Culex pipiens pallens and has a toxic effect on its larvae. It also exhibits insecticidal and genotoxic activities on Drosophila melanogaster. [1]
- Thymol has been traditionally used as a medicinal plant extract for the treatment of headaches, coughs, diarrhea, warts, and worms. [1]
- The combination of thymol with specific antimicrobial agents (e.g., itraconazole, clarithromycin) produced synergism against Pythium insidiosum (up to 96%). Thymol also showed synergistic effects with nystatin against C. albicans, reducing MIC values by 87.4% (FIC I 0.25). [1]
- Thymol is a known TRP channel agonist (TRPA1, TRPV3, and weakly TRPM8). [3]
- Thymol is a redox inhibitor that interferes with electron transport. Its cytotoxicity is reversible with the addition of Vitamin C, Vitamin E, or the antioxidant N-acetyl-cysteine (NAC). [3]

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Thymol is a phenolic compound, a natural monoterpene derivative of cymene. It is a volatile oil component. It belongs to the phenolic and monoterpene classes. It is derived from the hydride of cymene. Thymol is a phenolic compound extracted from thyme oil or other volatile oils. It can be used as a stabilizer in pharmaceutical preparations. It has been used for its antiseptic, antibacterial, and antifungal properties, and was previously used as an insect repellent. (Dorland, 28th edition) Thymol has been reported in Australian erythrorhizon (Acanthospermum australe), hops (Humulus lupulus), and other organisms with relevant data. Thymol is a phenolic compound extracted from thyme oil or other volatile oils and can be used as a stabilizer and preservative (antibacterial or antifungal agent) in pharmaceutical preparations. See also: Paeonia suffruticosa root (partial); Lysimachia christinae root (partial); Eucalyptol; Thymol (component)... See more...

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Mechanism of Action
We assessed the important role of the natural compound thymol in regulating macrophage activity by measuring all the sequential steps involved in phagocytosis. We found that spleen cell proliferation was significantly increased in the presence of thymol, and thymol was shown to be a good mitogen. Thymol treatment enhanced macrophage uptake due to increased cell membrane fluidity, and also increased macrophage lysosomal activity. Superoxide anion generation data indicated that thymol is involved in the respiratory burst, as it enhanced this property of macrophages at a concentration of 150 μM. Compared with lipopolysaccharide-treated cells, the secretion levels of TNF-α, IL-1β, and PGE₂ in the thymol-treated group decreased to 154 pg/mL, 736.1 pg/mL, and 151 pg/mL, respectively, while the levels of these cytokines were significantly increased in the lipopolysaccharide-treated group. We also determined the anticomplement activity of thymol, which was found to be more effective than rosmarinic acid. Therefore, the results of this study suggest that thymol may serve as a natural immunostimulant for the treatment of various immune diseases.

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Therapeutic Uses
Anti-infective; Topical anti-infective; Antifungal
Exploratory Therapy: Thymol is a naturally occurring monocyclic phenolic compound derived from thyme (Lamiaceae family), and has been reported to possess anti-inflammatory properties both in vivo and in vitro. However, the mechanism of action of thymol remains unclear. This study aimed to investigate the effects of thymol on ovalbumin (OVA)-induced allergic inflammation in mice with asthma and its mechanism of action. A mouse asthma model was established using OVA induction. One hour before OVA challenge, mice were orally administered thymol at doses of 4, 8, and 16 mg/kg body weight, respectively. Twenty-four hours after the last challenge, mice were sacrificed, and data were collected using various experimental methods. Results showed that thymol pretreatment reduced ovalbumin (OVA)-specific IgE levels, inhibited the recruitment of inflammatory cells to the airways, and decreased the levels of IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid (BALF). Furthermore, thymol pretreatment significantly improved lung tissue pathology and effectively inhibited goblet cell proliferation. Additionally, thymol reduced the development of airway hyperresponsiveness and blocked NF-κB pathway activation. All data suggest that thymol may improve airway inflammation in OVA-induced asthma in mice by inhibiting NF-κB activation. These findings suggest that thymol may be a potential alternative treatment for allergic asthma.
Exploring treatments for obesity has become a global health problem. Most synthetic anti-obesity drugs have failed to effectively control obesity due to poor efficacy or significant side effects. Therefore, research on the main chemical components in herbal medicines used to treat obesity has greatly increased. The main objective of this study was to investigate the effects of thymol on a high-fat diet (HFD)-induced obesity model in mice. Male Wistar rats were fed HFD for 6 weeks to induce obesity. Subsequently, the HFD-fed rats were orally administered thymol (14 mg/kg) twice daily for 4 weeks. Changes in body weight gain, visceral fat pad weight, and serum biochemical parameters were assessed. At the end of the study, compared with rats fed a normal diet, the HFD-fed rats showed significantly higher levels of body weight gain, visceral fat pad weight, blood lipids, alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), glucose, insulin, and leptin (p<0.001). Thymol treatment significantly reduced body weight gain, visceral fat pad weight, blood lipids, ALT, AST, LDH, BUN, glucose, insulin, and leptin levels in HFD-induced obese rats (p<0.001). Furthermore, thymol treatment significantly reduced serum lipid peroxidation levels and increased antioxidant levels in HFD-induced obese rats. Thymol prevents HFD-induced obesity in mouse models through multiple mechanisms, including reducing visceral fat accumulation, lowering blood lipids, improving insulin and leptin sensitivity, and enhancing antioxidant capacity.
Exploratory Treatment: Mast cells play a crucial role in inflammatory skin diseases by releasing pro-inflammatory mediators; however, therapies directly targeting these cells are rare. In 1878, the topical application of thymol (a now recognized potent agonist of transient receptor potential channels) to treat eczema and psoriasis was first described. This study aimed to determine the mechanism by which thymol alters skin inflammation. Methods: This study used a mast cell-dependent passive cutaneous anaphylaxis (PCA) model and cultured mast cells in vitro to investigate the effects of topical thymol on IgE-dependent responses. Results showed that topical application of thymol 24 hours before antigen stimulation dose-dependently inhibited PCA, but direct application of thymol induced ear swelling. This direct effect is associated with local mast cell degranulation and disappears in histamine-deficient mice. However, unlike the PCA response, delayed swelling was not observed. In vitro experiments showed that thymol directly triggers intracellular calcium ion influx in mast cells via transient receptor potential channel activation, accompanied by degranulation and cytokine transcription. However, no cytokine protein production was observed. Conversely, thymol significantly increases apoptosis, a phenomenon observed both in vitro and in vivo. The authors suggest that thymol's efficacy in reducing IgE-dependent responses is achieved by promoting mast cell activation-induced apoptosis, which may explain the clinical benefits observed in earlier clinical reports. For more complete data on the therapeutic uses of thymol (8 in total), please visit the HSDB record page.

C10H14O

150.22

150.104

C, 79.96; H, 9.39; O, 10.65

89-83-8

Thymol-d13;1219798-93-2

6989

White to off-white solid powder

1.0±0.1 g/cm3

233.0±0.0 °C at 760 mmHg

48-51 °C(lit.)

102.2±0.0 °C

0.0±0.4 mmHg at 25°C

1.523

3.28

1

1

1

11

120

0

CC1=CC(=C(C=C1)C(C)C)O

MGSRCZKZVOBKFT-UHFFFAOYSA-N

InChI=1S/C10H14O/c1-7(2)9-5-4-8(3)6-10(9)11/h4-7,11H,1-3H3

5-methyl-2-propan-2-ylphenol

NSC-11215; THYMOL; 89-83-8; 2-Isopropyl-5-methylphenol; 5-Methyl-2-(1-methylethyl)phenol; Thyme camphor; NSC 11215; Thymol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~832.11 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (13.85 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (13.85 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (13.85 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)