Metabolism / Metabolites
This study aims to characterize the metabolism of alpha-thujone in human liver preparations in vitro and to identify the role of cytochrome P450 (CYP) and possibly other enzymes catalyzing alpha-thujone biotransformations. With a liquid chromatography-mass spectrometry (LC-MS) method developed for measuring alpha-thujone and four potential metabolites, it was demonstrated that human liver microsomes produced two major (7- and 4-hydroxy-thujone) and two minor (2-hydroxy-thujone and carvacrol) metabolites. Glutathione and cysteine conjugates were detected in human liver homogenates, but not quantified. No glucuronide or sulphate conjugates were detected. Major hydroxylations accounted for more than 90% of the primary microsomal metabolism of alpha-thujone. Screening of alpha-thujone metabolism with CYP recombinant enzymes indicated that CYP2A6 was principally responsible for the major 7- and 4-hydroxylation reactions, although CYP3A4 and CYP2B6 participated to a lesser extent and CYP3A4 and CYP2B6 catalyzed minor 2-hydroxylation. Based on the intrinsic efficiencies of different recombinant CYP enzymes and average abundances of these enzymes in human liver microsomes, CYP2A6 was calculated to be the most active enzyme in human liver microsomes, responsible for 70-80% of the metabolism on average. Inhibition screening indicated that alpha-thujone inhibited both CYP2A6 and CYP2B6, with 50% inhibitory concentration values of 15.4 and 17.5 uM, respectively.
alpha-Thujone is quickly metabolized in vitro by mouse liver microsomes with NADPH (cytochrome P450) forming 7-hydroxy-alpha-thujone as the major product plus five minor ones (4-hydroxy-alpha-thujone, 4-hydroxy-beta-thujone, two other hydroxythujones, and 7,8-dehydro-alpha-thujone), several of which also are detected in the brain of mice treated i.p. with alpha-thujone. The major 7-hydroxy metabolite attains much higher brain levels than alpha-thujone but is less toxic to mice and Drosophila and less potent in the binding assay. The other metabolites assayed are also detoxification products. Thus, alpha-thujone in absinthe and herbal medicines is a rapid-acting and readily detoxified modulator of the GABA-gated chloride channel.
Essential oils containing alpha- and beta-thujones are important herbal medicines and food additives. The thujone diastereomers are rapidly metabolized convulsants acting as noncompetitive blockers of the gamma-aminobutyric acid-gated chloride channel. Synthesis and analysis of the metabolites are essential steps in understanding their health effects. Oxidation of alpha- and beta-thujones as their 2,3-enolates with oxodiperoxymolybdenum(pyridine)(hexamethylphosphoric triamide) gave the corresponding (2R)-2-hydroxythujones assigned by (1)H and (13)C NMR and X-ray crystallography. Alpha-Thujone was converted to 4-hydroxy-alpha- and -beta-thujones via the 3,4-enol acetate on oxidation with peracid and osmium tetroxide, respectively. Ozonation provided 7-hydroxy-alpha- and -beta-thujones, and by dehydration provided the 7,8-dehydro compounds. 4,10-Dehydrothujone was prepared from sabinene via sabinol. The hydroxy and dehydro derivatives are readily identified and analyzed by GC/MS as the parent compounds and trimethylsilyl and methyloxime derivatives. A separate study established that all of these compounds are metabolites of alpha- and beta-thujones.
Metabolism of thujone has been elucidated both in vitro and in vivo in several species and in vitro in human liver preparations. CYP2A6 is the principal metabolic enzyme, followed by CYP3A4 and, to a lesser extent, CYP2B6. CYP-associated metabolism may give rise to some potential pharmacogenetic and metabolic interaction consequences.
For more Metabolism/Metabolites (Complete) data for alpha, beta-Thujone (7 total), please visit the HSDB record page.

Interactions
The action of beta-hydroxybutyric-acid (BHA) and diacetone-alcohol on thujone induced convulsions was investigated in rabbits. Rabbits were injected intravenously (iv) with 0.4 cubic centimeters (cc) of 1 percent thujone to produce convulsions, followed in 5 minutes by iv injection of 0.5cc BHA per kilogram (kg). No effect was observed after BHA treatment. Diacetone-alcohol (05 cc/kg) was fed by stomach tube to thujone treated rabbits at 1, 5, and 24 hours. A definite anticonvulsant effect was observed at 5 hours and diminished by 24 hours. A similar procedure using 50 milligrams (mg)/kg phenobarbital-sodium showed a definite anticonvulsant effect at 6 hours and at 24 hours. A 500 mg/kg dose of phenobarbital-sodium caused a comatose like state, which was followed in 1 hour by thujone treatment. The thujone convulsing dose had no effect. The author concludes that diacetone-alcohol administered by mouth is much less toxic than phenobarbital but required a higher dose to obtain similar anticonvulsant effect. It is the diacetone part of the molecule that has anticonvulsant properties.

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Non-Human Toxicity Values
LD50 Mice i.p. 1200 mg/kg /Essential oil of East Mediterranean sage/
LD50 Mice i.p. 839 mg/kg /Essential oil of East Mediterranean sage/
LD50 Mice oral 230 mg/kg
LD50 Rats oral 192 mg/kg
LD50 Guinea pigs oral 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
EXPL THER ... The aim of this investigation was to study the cytotoxicity of Salvia officinalis L. (sage) essential oil. Salvia officinalis essential oil was gained by aqueous extraction from plant material and subsequently analyzed by gas chromatography. The cytotoxicity of the essential oil on the squamous human cell carcinoma cell line of the oral cavity (UMSCC1) was assessed with the XTT assay. These experiments revealed the half maximal inhibitory concentration (IC(50)) of the essential oil. It was used in the microarray-based analysis of gene expression of UMSSC1 cells. The results were submitted to a signaling pathway analysis. The main constituents of Salvia officinalis essential oil include the monoterpenes thujone, beta-pinene, and 1,8-cineol. Low concentrations of the essential oil increased vitality of the UMSCC1 cells. Beyond the concentration of the IC(50) of 135 ug/mL, sage essential oil reduced UMSSC1 cells viability to a minimum. In the microarray gene expression analysis, genes involved in cancer, cellular growth and proliferation, cell death, cell morphology, cell cycle, gene expression, and DNA repair were the most prominent. The three most significantly regulated pathways by sage were aryl hydrocarbon receptor signaling, cell cycle (G1/S checkpoint) regulation, and p53 signaling. To the best of our knowledge, this study suggests for the first time the ability of Salvia officinalis essential oil to inhibit human HNSCC cell growth. The therapeutic potential of sage essential oil might exceed that of its common use in otorhinolaryngology. /Salvia officinalis essential oil/
EXPL THER In the present study, the chemical constituents of Artemisia fukudo essential oil (AFE) were investigated using GC-MS. The major constituents were alpha-thujone (48.28%), beta-thujone (12.69%), camphor (6.95%) and caryophyllene (6.01%). /Investigators/ also examined the effects of AFE on the production of nitric oxide (NO), prostaglandin E(2) (PGE(2)), tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6, in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Western blotting and RT-PCR tests indicated that AFE has potent dose-dependent inhibitory effects on pro-inflammatory cytokines and mediators. /the authors/ investigated the mechanism by which AFE inhibits NO and PGE(2) by examining the level of nuclear factor-kappaB (NF-kappaB) activation within the mitogen-activated protein kinase (MAPK) pathway, which is an inflammation-induced signal pathway in RAW 264.7 cells. AFE inhibited LPS-induced ERK, JNK, and p38 phosphorylation. Furthermore, AFE inhibited the LPS-induced phosphorylation and degradation of Ikappa-B-alpha, which is required for the nuclear translocations of the p50 and p65 NF-kappaB subunits in RAW 264.7 cells. /These/ results suggest that AFE might exert an anti-inflammatory effect by inhibiting the expression of pro-inflammatory cytokines. Such an effect is mediated by a blocking of NF-kappaB activation which consequently inhibits the generation of inflammatory mediators in RAW264.7 cells. AFE may be useful for treating inflammatory diseases.
EXPL THER The antimetastatic potential of thujone, a naturally occurring monoterpene, was evaluated. Metastasis was induced in C57BL/6 mice by injecting highly metastatic B16F-10 melanoma cells through the lateral tail vein. Administration of thujone (1 mg/kg body weight), prophylactically and simultaneously with tumor induction, inhibited tumor nodule formation in the lungs by 59.45% and 57.54%, respectively, with an increase in the survival rate (33.67% and 32.16%) of the metastatic tumor bearing animals. These results correlated with biochemical parameters such as lung collagen hydroxyproline, hexosamine and uronic acid contents, serum sialic acid and gamma-glutamyl transpeptidase levels, and histopathological analysis. Treatment with thujone downregulated the production of proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and granulocyte-monocyte colony-stimulating factor. Thujone administration downregulated the expression of matrix metalloproteinase (MMP)-2, MMP-9, extracellular signal-regulated kinase (ERK)-1, ERK-2, and vascular endothelial growth factor (VEGF) and also upregulated the expression of nm-23, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 in the lung tissue of metastasis-induced animals. Treatment with thujone inhibited the activity of MMP-2 and MMP-9 in gelatin zymographic analysis. Thujone treatment significantly inhibited the invasion of B16F-10 melanoma cells across the collagen matrix in a Boyden chamber. Thujone also inhibited the adhesion of tumor cells to collagen-coated microtire plate wells and the migration of B16F-10 melanoma cells across a polycarbonate filter in vitro. These results indicate that Thujone can inhibit the lung metastasis of B16F-10 cells through inhibition of tumor cell proliferation, adhesion, and invasion, as well as by regulating expression of MMPs, VEGF, ERK-1, ERK-2, TIMPs, nm23, and levels of proinflammatory cytokines and IL-2 in metastatic animals.
EXPL THER Thujone is thought to be the main constituent of medicinal herbs that have antidiabetic properties. Therefore, we examined whether thujone ameliorated palmitate-induced insulin resistance in skeletal muscle. Soleus muscles were incubated for < or = 12 hr without or with palmitate (2 mM). Thujone (0.01 mg/mL), in the presence of palmitate, was provided in the last 6 hr of incubation. Palmitate oxidation, AMPK/acetyl-CoA carboxylase (ACC) phosphorylation and insulin-stimulated glucose transport, plasmalemmal GLUT4, and AS160 phosphorylation were examined at 0, 6, and 12 hr. Palmitate treatment for 12 hr reduced fatty acid oxidation (-47%), and insulin-stimulated glucose transport (-71%), GLUT4 translocation (-40%), and AS160 phosphorylation (-26%), but it increased AMPK (+51%) and ACC phosphorylations (+44%). Thujone (6-12 hr) fully rescued palmitate oxidation and insulin-stimulated glucose transport, but only partially restored GLUT4 translocation and AS160 phosphorylation, raising the possibility that an increased GLUT4 intrinsic activity may also have contributed to the restoration of glucose transport. Thujone also further increased AMPK phosphorylation but had no further effect on ACC phosphorylation. Inhibition of AMPK phosphorylation with adenine 9-beta-d-arabinofuranoside (Ara) (2.5 mM) or compound C (50 muM) inhibited 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 (< or = 20%) by Ara or compound C. Thus, while thujone, a medicinal herb component, rescues palmitate-induced insulin resistance in muscle, the improvement in fatty acid oxidation cannot account for this thujone-mediated effect. Instead, the rescue of palmitate-induced insulin resistance appears to occur via an AMPK-dependent mechanism involving partial restoration of insulin-stimulated GLUT4 translocation.
For more Therapeutic Uses (Complete) data for alpha, beta-Thujone (11 total), please visit the HSDB record page.

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Drug Warnings
There are no preclinical or clinical studies which would permit reliable scientific assessment of potential consequences regarding exposure of sensitive groups (i.e. pregnant women, children etc). Thus the use of thujone-containing herbal medicinal products in these groups should be minimised.
Human intoxications by thujone-containing preparations have indicated that animal studies are of relevance to the human situation. However, dose-effect comparisons are uncertain. It seems that low doses (of the order of 1.5 to 3.85 mg) have no or very little effects whereas higher doses (15 mg) clearly affected CNS measures.

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.)