Effects of peppermint oil on rat liver and cultured human hepatoma cells   King, R.G., Chan, D., Vo, L.T. (Introduced by Summers, R.J.) Department of Pharmacology, Monash University, Vic 3800, Australia.

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Peppermint oil is used for the treatment of irritable bowel syndrome. It contains menthol and pulegone, which in high doses can affect liver function. The aim of this study was to investigate acute and chronic effects of peppermint oil on rat liver, and acute effects on cultured human hepatoma cells.

Experiments were approved by a Committee for Ethics in Animal Experimentation. Adult male Long Evans rats (250-330 g) received peppermint oil (Sunspirit Pty Ltd, Australia, in olive oil) as a single oral dose of 8.3, 83 or 830µl/kg^-1, or as daily oral doses of 83µl/kg^-1 for 28 days, or vehicle. 24 h after the last dose, rats were anaesthetised (Nembutal, 0.06 mg g^-1 i.p.), the bile duct was cannulated, and bile flow rate measured. A 1.5ml blood sample was taken for liver function testing. FITC-dextran (M.W.167kDa, 10mg/ml, 25mg/kg) was given i.v., and in vivo confocal fluorescent microscopy performed on exposed liver (excitation 488nm, detection >515nm). Image analysis software was used to measure sinusoidal area of confocal images. Rats were killed by overdose of anaesthetic, and livers removed for histology (4µm paraffin sections, haematoxylin eosin stained). Human liver hepatoma cells (HG2P128) were cultured and incubated for 1 hour at 37⁰C with 0.05 or 0.5µl ml^-1 peppermint oil, and stained with ethidium homodimer-1 and DioC5 for dead and live cells, respectively. Live and dead cells were identified by confocal microscopy and the percentage dead cells calculated. Statistical analysis was performed by ANOVA and Dunnett's test, and s.e. means (n=5-7 in all cases) are shown throughout.

Bile flow was unaffected by any treatment except for an increase in flow after acute high-dose peppermint oil (p<0.05). Bile flow rates were as follows: 17+1, 20+2, 34+3µl min^-1 (for 8.3, 83 and 830µl kg^-1 acute peppermint oil), 16+µl min^-1 (for 83µl kg^-1 chronic peppermint oil) versus 20+2 and 14+2µl min^-1 (acute and chronic vehicle controls, respectively).

No change in liver enzyme activity was found except for an increase in alkaline phosphatase after chronic peppermint oil. Levels for vehicle and chronic peppermint oil (83µl kg^-1) respectively were: bilirubin (µM) 23±9, 13±4; alanine transaminase (µ L^-1) 55±8, 73±7; gamma glutamyl transpeptidase (µ L^-1) 4.6±0.9, 3.8±0.6; alkaline phosphatase (µ L^-1) 181±18, 262±21 (p<0.05).

No change in sinusoidal area in vivo or in histology was found following any of the above treatments, although pre-treatment with carbon tetrachloride (rats maintained on drinking water with 0.5g L^-1 phenobarbitone for 7 days, followed by a single oral dose of 0.25 ml kg^-1 CCl₄) reduced sinusoidal bed area to 10±1% (vs control of 28±4%, p<0.05) and produced histological damage (inflammatory cell infiltration and fatty changes). Incubation of human hepatoma cells with 0.5ul/ml (but not 0.05µl ml^-1) peppermint oil resulted in increased cell death (0.5µl ml^-1, 56±7% versus vehicle 13±5% p<0.01).

This study demonstrated in vitro toxicity of high doses of peppermint oil in cultured human hepatoma cells, and, at doses 2-3 orders of magnitude greater than those recommended for human use, an increase in rat bile flow after acute peppermint oil and an increase in alkaline phosphatase after chronic peppermint oil.