Identification of novel target molecules of l-menthol

Abstract

The present study used a binding assay to identify novel target biomolecules of l-menthol ([-]-menthol) that promote mouse ambulation. Among 88 different ligands to specific biomolecules examined, 0.1 mM l-menthol inhibited the binding of 13 ligands with relatively high inhibition rates. The assays showed that l-menthol acts on calcium channels, sodium channels, γ-aminobutyric acid type A (GABA_A) receptor, GABA transporter, dopamine transporter, dopamine D4 receptor, adenosine A2a receptor, α2A-adrenergic receptor, histamine H2 receptor, bombesin receptor, angiotensin AT1 receptor, vasopressin V2 receptor, and leukotriene B4 receptor over a similar concentration range. The inhibition constant (K_i) for l-menthol inhibition of binding of [³H]-WIN35,428 to the human recombinant dopamine transporter was 6.15 × 10^-4 mol/L. The K_i for l-menthol inhibition of binding of [³H]-ethynylbicycloorthobenzoate (EBOB), a ligand of GABA_A receptor picrotoxin site, was 2.88 × 10^-4 mol/L. These results should aid future research by providing clues for investigating the mechanisms underlying l-menthol activities, including the ambulation-promoting effect. The present results suggest that the dopamine transporter, adenosine A2a receptor, dopamine D4 receptor, α2A-adrenergic receptor, and GABA_A receptor are promising candidate molecules that are involved in the mechanisms underlying the psychostimulant-like effect of l-menthol.

Keywords: Binding assay; Central nervous system stimulant; Locomotion; Target molecule; l-menthol.

Figure 1

Effect of l-menthol on ambulatory activity in mice. (a) Change in normalized ambulatory activity before and after subcutaneous administration of vehicle or 100–800 mg/kg l-menthol. Symbols show median values of normalized ambulatory activity for each 10-min period plotted against the midpoint of the measurement period, and vertical lines denote the first and third quartiles. Arrow indicates the time of vehicle or 100–800 mg/kg l-menthol administration. (b) Total normalized ambulatory activity for 60 min after administration of vehicle or 100–800 mg/kg l-menthol. Data are shown using a box plot. ∗P < 0.05 compared with vehicle control (n = 19–20 mice).

Figure 2

(a) Concentration-effect relationships for l-menthol–mediated inhibition of the binding of [³H]-WIN35,428 to the human recombinant dopamine transporter and for GBR12909. Tests were duplicated at each concentration, and data are expressed as the mean values of duplicate samples. (b) Linearized concentration-effect relationships for l-menthol–mediated inhibition of the [³H]-WIN35,428 binding and for GBR12909, prepared using logit transformation. Y = logit y = ln (y/1 – y); y = (B–N)/(B₀–N); B = the amount of radioactivity bound in the presence of the test compound, B₀ = the amount of radioactivity bound in the absence of the test compound, N = the amount of radioactivity nonspecifically bound: X = log x; x = the concentration of l-menthol or positive control substance.

Figure 3

(a) Concentration-effect relationships for l-menthol–mediated inhibition of the binding of [³H]-EBOB, a GABA_A receptor picrotoxin ligand, to rat cerebral cortex preparation and for picrotoxin. Tests were duplicated at each concentration, and data are expressed as the mean values of duplicate samples. (b) Linearized concentration-effect relationships for l-menthol–mediated inhibition of the [³H]-EBOB binding and for picrotoxin, prepared using logit transformation.