Metabolism / Metabolites
(R)-(+)-Pulegone, a monoterpene ketone, is a major component of pennyroyal oil. Ingestion of high doses of pennyroyal oil has caused severe toxicity and occasionally death. Studies have shown that metabolites of pulegone were responsible for the toxicity. Previous metabolism studies have used high, near lethal doses and isolation and analysis techniques that may cause degradation of some metabolites. To clarify these issues and further explore the metabolic pathways, a study of (14)C-labeled pulegone in F344 rats at doses from 0.8 to 80 mg/kg has been conducted. High-pressure liquid chromatography (HPLC) analysis of the collected urine showed the metabolism of pulegone to be extensive and complex. Fourteen metabolites were isolated by HPLC and characterized by NMR, UV, and mass spectroscopy. The results demonstrated that pulegone was metabolized by three major pathways: 1) hydroxylation to give monohydroxylated pulegones, followed by glucuronidation or further metabolism; 2) reduction of the carbon-carbon double bond to give diastereomeric menthone/isomenthone, followed by hydroxylation and glucuronidation; and 3) Michael addition of glutathione to pulegone, followed by further metabolism to give diastereomeric 8-(N-acetylcystein-S-yl)menthone/isomenthone. This 1,4-addition not only took place in vivo but also in vitro under catalysis of glutathione S-transferase or mild base. Several hydroxylated products of the two mercapturic acids were also observed. Contrary to the previous study, all but one of the major metabolites characterized in the present study are phase II metabolites, and most of the metabolites in free forms are structurally different from those previously identified phase I metabolites.
Pulegone, a monoterpene that protects source plants against predators, is a hepatotoxic constituent of the folklore abortifacient pennyroyal oil. In the rat, pulegone extensively depleted glutathione measured in both liver tissue and plasma, and its toxicity was markedly enhanced in animals treated with buthionine sulfoximine. The glutathione-depleting effect of pulegone was compromised following inhibition of cytochrome P-450 by piperonyl butoxide. In addition, /the authors/ found no evidence for conjugation of glutathione to unchanged pulegone in vitro. Administration of menthofuran, a known oxidative and hepatotoxic metabolite of pulegone, only marginally affected glutathione levels in plasma and liver, and toxicity was not augmented by buthionine sulfoximine. These results provide indirect evidence for cytochrome P-450-catalyzed bioactivation of pulegone via at least two independent pathways: 1) the formation and subsequent activation of menthofuran from pulegone; and 2) the formation of reactive intermediate(s) from pulegone, but not menthofuran, which can be detoxified through a mechanism requiring reduced glutathione.
(R)-(+)-Pulegone, a monoterpene constituent of pennyroyal oil, is a hepatotoxin that has been used in folklore medicine as an abortifacient despite its potential lethal effects. Pulegone is metabolized by human liver cytochrome P-450s to menthofuran, a proximate hepatotoxic metabolite of pulegone. Expressed human liver cytochrome (CYP) P-450s (1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4) were tested for their ability to catalyze the oxidations of pulegone and menthofuran. Expressed CYP2E1, CYP1A2, and CYP2C19 oxidized pulegone to menthofuran, with respective Km and Vmax values of 29 uM and 8.4 nmol/min/nmol P-450 for CYP2E1, 94 uM and 2.4 nmol/min/nmol P-450 for CYP1A2, and 31 uM and 1.5 nmol/min/nmol P-450 for CYP2C19. The human liver P-450s involved in the metabolism of menthofuran are the same as pulegone except for the addition of CYP2A6. These P-450s were found to oxidize menthofuran to a newly identified metabolite, 2-hydroxymenthofuran, which is an intermediate in the formation of the known metabolites mintlactone and isomintlactone. Based on studies with (18)O2 and H2(18)O, 2-hydroxymenthofuran arises predominantly from a dihydrodiol formed from a furan epoxide. CYP2E1, CYP1A2, and CYP2C19 oxidized menthofuran with respective Km and Vmax values of 33 uM and 0.43 nmol/min/nmol P-450 for CYP2E1, 57 uM and 0.29 nmol/min/nmol P-450 for CYP1A2, and 62 uM and 0.26 nmol/min/nmol P-450 for CYP2C19.
The major in vivo metabolites of (S)-(-)-pulegone in humans using a metabolism of ingestion-correlated amounts (MICA) experiment were newly identified as 2-(2-hydroxy-1-methylethyl)-5-methylcyclohexanone (8-hydroxymenthone, M1), 3-hydroxy-3-methyl-6-(1-methylethyl)cyclohexanone (1-hydroxymenthone, M2), 3-methyl-6-(1-methylethyl)cyclohexanol (menthol), and E-2-(2-hydroxy-1-methylethylidene)-5-methylcyclohexanone (10-hydroxypulegone, M4) on the basis of mass spectrometric analysis in combination with syntheses and NMR experiments. Minor metabolites were be identified as 3-methyl-6-(1-methylethyl)-2-cyclohexenone (piperitone, M5) and alpha,alpha,4-trimethyl-1-cyclohexene-1-methanol (3-p-menthen-8-ol, M6). Menthofuran was not a major metabolite of pulegone and is most probably an artifact formed during workup from known (M4) and/or unknown precursors. The differences in toxicity between (S)-(-)- and (R)-(+)-pulegone can be explained by the strongly diminished ability for enzymatic reduction of the double bond in (R)-(+)-pulegone. This might lead to further oxidative metabolism of 10-hydroxypulegone (M4) and the formation of further currently undetected metabolites that might account for the observed hepatotoxic and pneumotoxic activity in humans.
For more Metabolism/Metabolites (Complete) data for Pulegone (25 total), please visit the HSDB record page.

Interactions
Intraperitoneal injection of R-(+)-pulegone (pulegone), the main constituent of pennyroyal oil, to ddY mice caused extensive liver injury as characterized by an increase in serum glutamic pyruvic transaminase (GPT) activity and centrilobular necrosis of hepatocytes. Treatments of mice with the cytochrome P-450 enzyme inhibitors, SKF-525A, metyrapone, piperonyl butoxide, and carbon disulfide (CS2), prevented or markedly alleviated the hepatotoxicity of pulegone. These results are compatible with the view that some metabolite of pulegone is responsible for the liver injury in mice.
Pennyroyal oil ingestion has been associated with severe hepatotoxicity and death. The primary constituent, R-(+)-pulegone, is metabolized via hepatic cytochrome P450 to toxic intermediates. The purpose of this study was to assess the ability of the specific cytochrome P450 inhibitors disulfiram and cimetidine to mitigate hepatotoxicity in mice exposed to toxic levels of R-(+)-pulegone. 20-g female BALB/c mice were pretreated with either 150 mg/kg of cimetidine intraperitoneal (IP), 100 mg/kg of disulfiram IP, or both. After one hour, mice were administered 300 mg/kg of pulegone IP and were killed 24 hours later. Data were analyzed using ANOVA. Post-hoc t-tests used Bonferroni correction. There was a tendency for lower serum glutamate pyruvate transaminase in the disulfiram and cimetidine groups compared with the R-(+)-pulegone group. The differences were significant for both the cimetidine and the combined disulfram and cimetidine groups compared with the R-(+)-pulegone group. Pretreatment with the combination of disulfiram and cimetidine most effectively mitigated R-(+)-pulegone-induced hepatotoxicity. Within the limitations of a pretreatment animal model, the combination of cimetidine and disulfiram significantly mitigates the effects of pennyroyal toxicity and does so more effectively than either agent alone. These data suggest that R-(+)-pulegone metabolism through CYP1A2 appears to be more important in the development of a hepatotoxic metabolite than does metabolism via CYP2E1.
Glutathione plays a role in the detoxication of pulegone. At hepatotoxic doses, pulegone depletes glutathione in the liver and the toxicity of pulegone on i.p. injection in mice is enhanced by treatment with diethyl maleate to decrease glutathione levels. No such increase in toxicity was seen with (R)-(+)-menthofuran . It was suggested that pulegone is analogous to acetaminophen in that saturation of the glutathione pathway leads to a higher proportion of the dose being metabolised to reactive metabolites (such as 8-pulegone aldehyde). However, in other studies, glutathione has been shown to react with menthofuran epoxide, the precursor to 8-pulegone aldehyde. Glutathione conjugation may play a major role in the detoxication of the reactive metabolite produced by cytochrome P450 ((R)-(+)-menthofuran or the gamma-ketoenal) as indicated by the isolation of glutathione conjugates, including a mixed glutathionyl glucuronide, from the bile of rats treated i.p. with pulegone. The evidencesuggests that the metabolic activation of pulegone occurring in animals also occurs in humans, resulting in the formation of (R)-(+)-menthofuran. At high concentrations, (R)-(+)-menthofuran is a proximate hepatoxic product but if the concentration of metabolites of pulegone is not sufficient to deplete hepatocellular concentrations of glutathione, hepatotoxicity may not be observed.
The role of cytochrome P450s in metabolic activation of (R)-(+)-pulegone has been demonstrated by the observations that a variety of P450-inhibitors decreased the toxicity while pretreatment with phenobarbital enhanced the toxicity of (R)-(+)-pulegone and (R)-(+)- menthofuran. Thus, oxidation appears to enhance the toxicity of (R)-(+)-pulegone and (R)-(+)-menthofuran, which is consistent with the fact that (R)-(+)-pulegone is converted to (R)-(+)-menthofuran via 9-hydroxypulegenone, and the reactive 8-pulegone aldehyde is an ultimate toxicant. Evidence that pulegone is oxidized to 9-hydroxypulegone via a free radical mechanism is provided from the observation that treatment with the free radical scavenger C-phyocyanin decreased the hepatotoxicity of pulegone in rats.

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Non-Human Toxicity Values
LD50 Rats (Wistar, male) i.p. 819 mg/kg (24 hr)/Peppermint Oil USP/
LD50 Rats oral 470 mg/kg /R-(+)-pulegone/
LD50 Rats ip 150 mg/kg
LD50 Dogs iv 330 mg/kg
LD50 Mice sc 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.

Pulegone can cause cancer according to California Labor Code.
(+)-pulegone is the (5R)-enantiomer of p-menth-4(8)-en-3-one.
Pulegone has been reported in Minthostachys mollis, Perilla frutescens, and other organisms with data available.
See also: Agathosma betulina leaf (part of).

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Mechanism of Action
In rats pulegone (300 mg i.p.) caused dilation of the central veins and distension of sinusoidal spaces within 6 hours and centrilobular necrosis was observable starting at 12 hours. Electron microscopy after 24 hours showed degeneration of endoplasmic reticulum, swelling of mitochondria and nuclear changes. It has been suggested that metabolites of (R)-(+)-pulegone deactivate cytochrome P450s by modifying the prosthetic hem group or the apoprotein . In human liver microsomes in vitro, (R)-(+)-menthofuran specifically inhibits CYP2A6 and adducts with this enzyme have been isolated. CYP1A2, CYP2D6, CYP2E1 or CYP3A4 were not similarly inactivated.
(R)-(+)-Pulegone and its metabolite, (R)-(+)-menthofuran, are hepatotoxic and produce similar effects following i.p. injection in mice. These effects are similar to those reported following human intoxication with pennyroyal oil.

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Therapeutic Uses
The therapeutic indications for peppermint oil and mint oil are mainly related to common cold and gastrointestinal disturbances and presumably the vast majority of these products are used in selfmedication. An underreporting of side effects may be suspected.

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.)