Metabolism / Metabolites
(R)-(+)-Menthone is a monoterpene ketone and the main component of peppermint oil. Ingestion of high doses of peppermint oil can lead to severe toxicity and even death. Studies have shown that the metabolites of menthone are the cause of this toxicity. Previous metabolic studies used near-lethal doses and separation and analytical techniques that may lead to the degradation of certain metabolites. To elucidate these issues and further explore its metabolic pathways, this study administered 14C-labeled menthone to F344 rats at doses ranging from 0.8 to 80 mg/kg. High-performance liquid chromatography (HPLC) analysis of collected urine revealed that the metabolism of menthone is extensive and complex. Fourteen metabolites were isolated by HPLC and characterized by nuclear magnetic resonance (NMR), ultraviolet spectroscopy (UV), and mass spectrometry (MS). The results showed that menthone is mainly metabolized through three pathways: 1) hydroxylation to form monohydroxymenthone, followed by glucuronidation or further metabolism; 2) reduction of the carbon-carbon double bond to form diastereomers of menthone/isomenthone, followed by hydroxylation and glucuronidation; 3) a Michael addition reaction between glutathione and menthone, followed by further metabolism to form diastereomers of 8-(N-acetylcysteine-S-yl)menthone/isomenthone. This 1,4-addition reaction occurs not only in vivo but also in vitro under the catalysis of glutathione S-transferase or a weak base. In addition, several hydroxylation products of two thiouric acids were observed. Contrary to previous studies, the major metabolites identified in this study, except for one, were all phase II metabolites, and most of the free metabolites were structurally different from the previously identified phase I metabolites. Menthone is a monoterpene that protects plants from predators and is also a hepatotoxic component in peppermint oil, a traditional abortifacient. In rats, menthone significantly reduced glutathione levels in liver tissue and plasma, and its toxicity was significantly enhanced in animals treated with sulfoxide-3-butyrate. Inhibition of cytochrome P-450 by piperonyl butyl ether weakened the glutathione-depleting effect of menthone. Furthermore, the authors found no evidence of glutathione binding to unchanged menthone in vitro. Administration of menthol furan (a known oxidizing and hepatotoxic metabolite of menthone) had only a slight effect on glutathione levels in plasma and liver, and sulfoxide-3-butyrate did not enhance its toxicity. These results indirectly demonstrate that cytochrome P-450-catalyzed bioactivation of menthone occurs through at least two independent pathways: 1) menthone is generated and subsequently activated to menthol furan; 2) menthone (but not menthol furan) generates active intermediates, which can be detoxified through mechanisms requiring reduced glutathione. (R)-(+)-Menthone is a monoterpenoid component of peppermint oil and is a hepatotoxic substance that, despite its potential lethality, is still used in folk medicine as an abortifacient. Menthone is metabolized by human hepatic cytochrome P-450 to menthofuran, a direct hepatotoxic metabolite of menthone. We tested the ability of expressed human hepatic cytochrome (CYP) P-450s (1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4) to catalyze the oxidation of menthone and menthofuran. The expressed CYP2E1, CYP1A2, and CYP2C19 can oxidize menthone to menthofiuran, with Km and Vmax values of 29 μM and 8.4 nmol/min/nmol P-450, respectively; CYP1A2 94 μM and 2.4 nmol/min/nmol P-450; and CYP2C19 31 μM and 1.5 nmol/min/nmol P-450. The human liver P-450 involved in menthone metabolism is the same as that of menthone metabolism, except for the addition of CYP2A6. These P-450s can oxidize menthofiuran to a newly discovered metabolite, 2-hydroxymenthofiuran, which is an intermediate in the formation of the known metabolites mentholactone and isomenthofiuran. Based on studies of (18)O2 and H2(18)O, 2-hydroxymenthofiuran mainly originates from the dihydrodiol formed from furan epoxides. The Km and Vmax values of CYP2E1, CYP1A2, and CYP2C19 for oxidizing mentholatum were as follows: CYP2E1: 33 μM and 0.43 nmol/min/nmol P-450; CYP1A2: 57 μM and 0.29 nmol/min/nmol P-450; and CYP2C19: 62 μM and 0.26 nmol/min/nmol P-450.
Using intake-related metabolism (MICA) experiments, the major metabolites of (S)-(-)-menthone in the human body were newly identified as 2-(2-hydroxy-1-methylethyl)-5-methylcyclohexanone (8-hydroxymenthone, M1) and 3-hydroxy-3-methyl-6-(1-methylethyl)cyclohexanone (1-hydroxymenthone, M2). Based on mass spectrometry combined with synthesis and nuclear magnetic resonance experiments, 3-methyl-6-(1-methylethyl)cyclohexanol (menthol) and E-2-(2-hydroxy-1-methylethylidene)-5-methylcyclohexanone (10-hydroxymenthol, M4) were identified. Minor metabolites included 3-methyl-6-(1-methylethyl)-2-cyclohexenone (piperone, M5) and α,α,4-trimethyl-1-cyclohexen-1-methanol (3-p-menthene-8-ol, M6). Menthol furan was not a major metabolite of menthol and is likely an artifact generated during post-processing from known (M4) and/or unknown precursors. The difference in toxicity between (S)-(-)- and (R)-(+)-menthol is attributed to the significantly reduced enzymatic reduction ability of the double bonds in (R)-(+)-menthol. This may lead to further oxidative metabolism of 10-hydroxymenthone (M4), generating more undetected metabolites that may explain the hepatotoxicity and pulmonary toxicity observed in humans. For more complete data on the metabolism/metabolites of menthone (25 in total), please visit the HSDB record page.

Interactions
Intraperitoneal injection of R-(+)-menthone (menthone), the main component of peppermint oil, caused extensive liver damage in ddY mice, characterized by elevated serum alanine aminotransferase (ALT) activity and central lobular necrosis of hepatocytes. Treatment of mice with the cytochrome P-450 enzyme inhibitor SKF-525A, metheprone, piperonyl butyl ether, and carbon disulfide (CS2) prevented or significantly reduced the hepatotoxicity of menthone. These results are consistent with the view that certain metabolites of menthone are responsible for liver damage in mice. Ingestion of peppermint oil is associated with severe hepatotoxicity and death. Its main component, R-(+)-menthone, is metabolized by hepatic cytochrome P450 into toxic intermediates. This study aimed to evaluate whether the specific cytochrome P450 inhibitors disulfiram and cimetidine could reduce hepatotoxicity in mice exposed to toxic doses of R-(+)-menthone. Female BALB/c mice (20 g) were pre-treated with intraperitoneal injection of 150 mg/kg cimetidine, 100 mg/kg disulfiram, or a combination of both. One hour later, the mice were intraperitoneally injected with 300 mg/kg menthone and sacrificed 24 hours later. Data were analyzed using ANOVA. Post-hoc t-tests were performed with Bonferroni correction. Compared with the R-(+)-menthone group, serum alanine aminotransferase (ALT) levels tended to decrease in the disulfiram and cimetidine groups. The differences between the cimetidine group and the disulfiram-cimetidine combination group and the R-(+)-menthone group were statistically significant. Pretreatment with disulfiram and cimetidine most effectively reduced R-(+)-menthone-induced hepatotoxicity. Given the limitations of the pretreatment animal model, the combination of cimetidine and disulfiram significantly reduced the toxic effects of menthone, and the effect was superior to either drug alone. These data suggest that R-(+)-menthone metabolism appears to be more important via CYP1A2 than via CYP2E1 in the formation of hepatotoxic metabolites. Glutathione plays a role in the detoxification of menthone. At hepatotoxic doses, menthone depletes glutathione in the liver, and in mice, intraperitoneal injection of menthone followed by a reduction in glutathione levels with diethyl maleate enhances its toxicity. No such increase in toxicity was observed in (R)-(+)-menthrenofuran. Studies have shown that, similar to acetaminophen, saturation of the glutathione pathway leads to a higher proportion of doses being metabolized to active metabolites (e.g., 8-menthrenofuranaldehyde). However, other studies have shown that glutathione can react with menthofuran epoxide (a precursor to 8-menthrenofuranaldehyde). The isolation of glutathione conjugates (including mixed glutathione glucuronide) from the bile of rats injected intraperitoneally with menthone suggests that glutathione conjugates may play an important role in the detoxification of active metabolites ((R)-(+)-menthrenfuran or γ-ketoenal) produced by cytochrome P450. This evidence indicates that the metabolic activation of menthone that occurs in animals also occurs in humans, ultimately leading to (R)-(+)-menthrenfuran. At high concentrations, (R)-(+)-menthrenfuran is a direct hepatotoxic product, but hepatotoxicity may not be observed if the concentration of menthone metabolites is insufficient to deplete glutathione within hepatocytes. The role of cytochrome P450 in the metabolic activation of (R)-(+)-menthone has been confirmed by the following observations: various P450 inhibitors reduce the toxicity of (R)-(+)-menthone and (R)-(+)-menthfuran, while phenobarbital pretreatment enhances the toxicity of both. Therefore, oxidation appears to enhance the toxicity of (R)-(+)-menthone and (R)-(+)-menthfuran, consistent with the fact that (R)-(+)-menthone is converted to (R)-(+)-menthfuran via 9-hydroxymenthone, with active 8-menthone aldehyde being the final toxicant. There is evidence that menthone can be oxidized to 9-hydroxymenthone via a free radical mechanism. This evidence comes from the observation that treatment with the free radical scavenger C-phycocyanin reduces the hepatotoxicity of menthone in rats.

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Non-human toxicity values
LD50 Rat (Wistar, male) Intraperitoneal injection 819 mg/kg (24 hours) / Peppermint oil USP /
LD50 Rat Oral administration 470 mg/kg / R-(+)-Menthone /
LD50 Rat Intraperitoneal injection 150 mg/kg
LD50 Dog Intravenous injection 330 mg/kg
LD50 Mouse Subcutaneous injection 1,709 mg/kg

[1]. Calamintha nepeta (L.) Savi and its Main Essential Oil Constituent Pulegone: Biological Activities and Chemistry. Molecules. 2017 Feb 14;22(2).

[2]. Involvement of nociceptive transient receptor potential channels in repellent action of pulegone. Biochem Pharmacol. 2018 May;151:89-95.

According to California labor law, menthone may be carcinogenic. (+)-Menthone is the (5R)-enantiomer of p-menth-4(8)-en-3-one. Menthone has been reported in soft peppermint (Minthostachys mollis), perilla (Perilla frutescens), and other organisms with relevant data. See also: Birch leaf (partial). Mechanism of Action: In rats, intraperitoneal injection of menthone (300 mg) caused central venous dilation and sinusoidal dilation within 6 hours, and centrilobular necrosis was observed after 12 hours. Electron microscopy after 24 hours showed endoplasmic reticulum degeneration, mitochondrial swelling, and nuclear alterations. Studies have shown that metabolites of (R)-(+)-menthone inactivate cytochrome P450 by modifying the prosthetic group hem or apolipoprotein. In vitro, in human liver microsomes, (R)-(+)-menthofuran specifically inhibits CYP2A6, and adducts with this enzyme have been isolated. CYP1A2, CYP2D6, CYP2E1, or CYP3A4 did not show similar inactivation. (R)-(+)-menthone and its metabolite (R)-(+)-menthofuran are both hepatotoxic, producing similar hepatotoxic effects after intraperitoneal injection in mice. These effects are similar to those reported in humans after peppermint oil poisoning.
Therapeutic Uses
The therapeutic indications for peppermint oil and peppermint oil are primarily related to the common cold and gastrointestinal discomfort, and it is presumed that the vast majority of these products are used for self-medication. Therefore, reports of side effects may be insufficient.

C10H16O

152.24

152.12

89-82-7

442495

Colorless to light yellow liquid

0.9±0.1 g/cm3

224.0±0.0 °C at 760 mmHg

< 25 °C

82.2±0.0 °C

0.1±0.4 mmHg at 25°C

1.470

2.56

0

1

0

11

197

1

C(=C1/CC[C@@H](C)CC/1=O)(/C)\C

NZGWDASTMWDZIW-MRVPVSSYSA-N

InChI=1S/C10H16O/c1-7(2)9-5-4-8(3)6-10(9)11/h8H,4-6H2,1-3H3/t8-/m1/s1

(5R)-5-methyl-2-propan-2-ylidenecyclohexan-1-one

NSC-15334; NSC 15334; Pulegone

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 : ≥ 270 mg/mL (~1773.63 mM)

Solubility in Formulation 1: ≥ 2.25 mg/mL (14.78 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 22.5 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: ≥ 2.25 mg/mL (14.78 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 22.5 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: ≥ 2.25 mg/mL (14.78 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 22.5 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.)