Metabolism / Metabolites
This study aimed to characterize the metabolism of α-thujone in human liver preparations in vitro and to determine the role of cytochrome P450 (CYP) and other enzymes that may catalyze the biotransformation of α-thujone. A developed liquid chromatography-mass spectrometry (LC-MS) method was used to determine α-thujone and four potential metabolites. Results showed that human liver microsomes produce two major metabolites (7-hydroxythujone and 4-hydroxythujone) and two minor metabolites (2-hydroxythujone and carvacrol). Glutathione and cysteine conjugates were detected in human liver homogenates, but were not quantified. No glucuronide or sulfate conjugates were detected. Major hydroxylation reactions accounted for over 90% of the primary microsomal metabolism of α-thujone. Screening of α-thujone metabolism using recombinant CYP enzymes revealed that CYP2A6 is primarily responsible for the major 7- and 4-hydroxylation reactions, while CYP3A4 and CYP2B6 have lower involvement. CYP3A4 and CYP2B6 also catalyze minor 2-hydroxylation reactions. Based on the intrinsic efficiency of different recombinant CYP enzymes and their average abundance in human liver microsomes, CYP2A6 was calculated to be the most active enzyme in human liver microsomes, responsible for an average of 70-80% of metabolism. Inhibition screening showed that α-thujone can inhibit CYP2A6 and CYP2B6, with half-maximal inhibitory concentrations (IC50) of 15.4 μM and 17.5 μM, respectively.
α-Thujone is rapidly metabolized in vitro by mouse liver microsomes using NADPH (cytochrome P450), with the major product being 7-hydroxy-α-thujone, and five minor products (4-hydroxy-α-thujone, 4-hydroxy-β-thujone, two other hydroxythujones, and 7,8-dehydro-α-thujone). Several of these minor products were also detected in the brain tissue of mice injected intraperitoneically with α-thujone. The concentration of the major 7-hydroxy metabolite in brain tissue was much higher than that of α-thujone, but it showed lower toxicity in mice and fruit flies, and lower potency in binding assays. The other detected metabolites were also detoxification products. Therefore, α-thujone in absinthe and herbs is a rapidly acting and easily detoxified GABA-gated chloride channel modulator.
Essential oils containing α- and β-thujone are important herbal and food additives. Thujone diastereomers are rapidly metabolized spasmodics that act as non-competitive blockers of γ-aminobutyric acid-gated chloride channels. The synthesis and analysis of the metabolites are crucial steps in understanding their health effects. The 2,3-enolates of α- and β-thujone were oxidized with oxodiperoxymolybdate (pyridine) (hexamethylphosphoramide) to yield the corresponding (2R)-2-hydroxythujone, whose structures were identified by ¹H and ¹³C NMR and X-ray crystallography. α-thujone was oxidized by acid and osmium tetroxide, followed by conversion to 4-hydroxy-α-thujone and 4-hydroxy-β-thujone via 3,4-enol acetate, respectively. Ozonolysis yielded 7-hydroxy-α-thujone and 7-hydroxy-β-thujone, which were then dehydrated to give 7,8-dehydro compounds. 4,10-Dehydrothujone was prepared from safflowerene via safflower alcohol. Hydroxyl and dehydrogenated derivatives can be easily identified and analyzed by GC/MS, and their parent compounds, as well as trimethylsilyl and methoxyoxime derivatives, can be identified. Another study showed that all these compounds are metabolites of α-thujone and β-thujone. The metabolism of thujone has been elucidated in vitro and in vivo experiments in various animals and in vitro experiments on human liver specimens. CYP2A6 is the major metabolic enzyme, followed by CYP3A4, with CYP2B6 playing a minor role. CYP-related metabolism may lead to several potential pharmacogenetic and metabolic interaction consequences. For more complete metabolite/metabolite data on α,β-thujone (7 metabolites in total), please visit the HSDB record page.

Interactions
This study investigated the effects of β-hydroxybutyric acid (BHA) and diacetone alcohol on thujone-induced seizures in rabbits. Seizures were induced by intravenous injection of 0.4 mL of 1% thujone solution into rabbits, followed by intravenous injection of 0.5 mL/kg body weight of BHA 5 minutes later. No effect was observed after BHA treatment. Thujone-treated rabbits were administered 0.5 mL/kg body weight of diacetone alcohol via gastric tube at 1, 5, and 24 hours. A significant anticonvulsant effect was observed at 5 hours, which diminished after 24 hours. Using a similar method, 50 mg/kg body weight of phenobarbital sodium showed significant anticonvulsant effects at 6 and 24 hours. After administration of 500 mg/kg phenobarbital sodium, patients developed a coma-like state, followed by thujone treatment 1 hour later. However, the convulsant dose of thujone was ineffective. The authors concluded that oral diacetone-alcohol is far less toxic than phenobarbital, but higher doses are required to achieve a similar anticonvulsant effect. The diacetone moiety in the molecule possesses anticonvulsant properties.

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Non-human toxicity values
Mouse intraperitoneal injection LD50: 1200 mg/kg /Eastern Mediterranean sage oil/
Mouse intraperitoneal injection LD50: 839 mg/kg /Eastern Mediterranean sage oil/
Mouse oral LD50: 230 mg/kg
Rat oral LD50: 192 mg/kg
Guinea pig oral LD50: 396 mg/kg

[1]. Toxicology and carcinogenesis studies of alpha,beta-thujone (CAS No. 76231-76-0) in F344/N rats and B6C3F1 mice (gavage studies). Natl Toxicol Program Tech Rep Ser. 2011, 570.

Therapeutic Uses
This study aimed to investigate the cytotoxicity of sage (Salvia officinalis L.) essential oil. Sage essential oil was extracted from plant material via water extraction and subsequently analyzed by gas chromatography. The XTT assay was used to assess the cytotoxicity of this essential oil against the oral squamous cell carcinoma (UMSCC1) cell line. The results determined the half-maximal inhibitory concentration (IC50) of the essential oil. Subsequently, gene expression in UMSCC1 cells was analyzed using microarray technology, and signaling pathway analysis was performed. The main components of sage essential oil include monoterpenoids thujone, β-pinene, and 1,8-cineole. Low concentrations of this essential oil enhanced the viability of UMSCC1 cells. At concentrations exceeding the IC50 value of 135 μg/mL, sage essential oil reduced the viability of UMSCC1 cells to a minimum. In gene chip expression analysis, genes associated with cancer, cell growth and proliferation, cell death, cell morphology, cell cycle, gene expression, and DNA repair were most prominent. Sage essential oil significantly regulates three pathways: the aryl hydrocarbon receptor signaling pathway, cell cycle (G1/S checkpoint) regulation, and the p53 signaling pathway. To our knowledge, this study is the first to reveal the ability of sage essential oil to inhibit the growth of human head and neck squamous cell carcinoma (HNSCC) cells. The therapeutic potential of sage essential oil may exceed its common uses in otolaryngology. /Sage Essential Oil/
Exploring Therapeutic Effects This study used gas chromatography-mass spectrometry (GC-MS) to analyze the chemical composition of Artemisia fukuto (AFE) essential oil. The main components were α-thujone (48.28%), β-thujone (12.69%), camphor (6.95%), and caryophyllene (6.01%). Researchers also examined the effects of AFE on the production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Western blotting and RT-PCR showed that AFE had a significant dose-dependent inhibitory effect on pro-inflammatory cytokines and mediators. The authors investigated the mechanism by which AFE inhibited NO and PGE₂ by examining the activation level of nuclear factor-κB (NF-κB) in the mitogen-activated protein kinase (MAPK) pathway. The MAPK pathway is an inflammation-induced signaling pathway in RAW 264.7 cells. AFE inhibited LPS-induced phosphorylation of ERK, JNK, and p38. Furthermore, AFE also inhibited LPS-induced phosphorylation and degradation of IκB-α, which is essential for nuclear translocation of the NF-κB p50 and p65 subunits. These results suggest that AFE may exert its anti-inflammatory effect by inhibiting the expression of pro-inflammatory cytokines. This effect is mediated by blocking NF-κB activation, thereby inhibiting the production of inflammatory mediators in RAW 264.7 cells. AFE may be effective in treating inflammatory diseases.
Exploratory Treatment: This study evaluated the anti-metastatic potential of the natural monoterpene compound thujone. Metastasis was induced in C57BL/6 mice by tail vein injection of highly metastatic B16F-10 melanoma cells. Prophylactic administration of thujone (1 mg/kg body weight) and concurrent administration of thujone inhibited lung tumor nodule formation by 59.45% and 57.54%, respectively, and improved the survival rate of mice with metastatic tumors (33.67% and 32.16%, respectively). These results were correlated with biochemical indicators such as lung collagen hydroxyproline, hexosamine, and uronic acid content, serum sialic acid and γ-glutamyl transferase levels, and histopathological analysis results. Thujone treatment downregulated the production of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, interleukin-6, and granulocyte-monocyte colony-stimulating factor. Thujone administration downregulated the expression of matrix metalloproteinase-2, MMP-9, extracellular signal-regulated kinase-1, ERK-2, and vascular endothelial growth factor (VEGF) in lung tissue of metastasis-induced animals, while upregulating the expression of nm-23, tissue inhibitor of metalloproteinases-1, and TIMP-2. Gelatin zymography analysis showed that thujone treatment inhibited the activity of MMP-2 and MMP-9. Boyden chamber assays demonstrated that thujone treatment significantly inhibited the invasion of B16F-10 melanoma cells through the collagen matrix. Thujone also inhibited tumor cell adhesion to collagen-coated microplate pores and suppressed the migration of B16F-10 melanoma cells across polycarbonate membranes in vitro. These results indicate that thujone can inhibit lung metastasis of B16F-10 cells by suppressing tumor cell proliferation, adhesion, and invasion, as well as regulating the expression of MMPs, VEGF, ERK-1, ERK-2, TIMPs, nm23, and the levels of pro-inflammatory cytokines and IL-2 in metastatic animals.
Experimental Treatment: Thujone is considered a major component of medicinal plants with anti-diabetic properties. Therefore, we investigated whether thujone could improve palmitate-induced insulin resistance in skeletal muscle. Soleus muscle was incubated with or without palmitate (2 mM) for ≤12 hours. In the presence of palmitate, thujone (0.01 mg/mL) was added during the last 6 hours of incubation. Palmitate oxidation, AMPK/acetyl-CoA carboxylase (ACC) phosphorylation, insulin-stimulated glucose transport, and plasma membrane GLUT4 and AS160 phosphorylation were measured at 0, 6, and 12 hours. After 12 hours of palmitate treatment, fatty acid oxidation decreased by 47%, insulin-stimulated glucose transport decreased by 71%, GLUT4 translocation decreased by 40%, and AS160 phosphorylation decreased by 26%, but AMPK phosphorylation increased by 51% and ACC phosphorylation increased by 44%. Thujone (6–12 hours) completely restored palmitate oxidation and insulin-stimulated glucose transport, but only partially restored GLUT4 translocation and AS160 phosphorylation, suggesting that increased intrinsic GLUT4 activity may also have promoted the recovery of glucose transport. Thujone further increased AMPK phosphorylation, but had no further effect on ACC phosphorylation. Inhibition of AMPK phosphorylation with adenine 9-β-D-arabinofuranoside (Ara) (2.5 mM) or compound C (50 μM) suppressed the thujone-induced improvement in insulin-stimulated glucose transport, GLUT4 translocation, and AS160 phosphorylation. In contrast, the thujone-induced improvement in palmitate oxidation was only slightly inhibited (≤20%) by arabinogalactosidase (Ara) or compound C. Therefore, while thujone, a medicinal plant ingredient, can improve palmitic acid-induced insulin resistance in muscle, the improvement in fatty acid oxidation does not explain this thujone-mediated effect. Instead, the improvement in palmitic acid-induced insulin resistance appears to be achieved through an AMPK-dependent mechanism involving the partial restoration of GLUT4 translocation stimulated by insulin. For more complete data on the therapeutic uses of α,β-thujone (11 in total), please visit the HSDB record page. Drug Warnings: Currently, there are no reliable preclinical or clinical studies to assess the potential consequences of exposure to this drug in sensitive populations (e.g., pregnant women, children). Therefore, the use of herbal products containing thujone in these populations should be minimized. Cases of human toxicity from thujone preparations suggest that animal studies are relevant to human cases. However, dose-response comparisons are uncertain. Low doses (approximately 1.5 to 3.85 mg) appear to have no or minimal effects, while higher doses (15 mg) significantly affect central nervous system parameters.

C10H16O

152.23

152.12

76231-76-0

10931629

Colorless or almost colorless liquid

0.925 g/mL at 25 °C (lit.)

84-86 °C/17 mmHg (lit.)

64°C

2.257

0

1

1

11

207

2

C[C@@H]1[C@H]2C[C@]2(CC1=O)C(C)C.C[C@H]1[C@H]2C[C@]2(CC1=O)C(C)C

USMNOWBWPHYOEA-VWHDNNRLSA-N

InChI=1S/C10H16O/c1-6(2)10-4-8(10)7(3)9(11)5-10/h6-8H,4-5H2,1-3H3/t7?,8-,10+/m1/s1

(1S,5R)-4-methyl-1-propan-2-ylbicyclo[3.1.0]hexan-3-one

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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