Ex vivo and in vitro inhibitory potential of Kava extract on monoamine oxidase B activity in mice

Abstract

Background and aim: This study investigated the effect of Kava extract (Piper methysticum), a medicinal plant that has been worldly used by its anxiolytic effects, on monoamine oxidase (MAO) activity of mice brain after 21 days of treatment as well as anxiolytic and locomotor behavior. Furthermore, the in vitro inhibitory profile of Kava extract on MAO-B activity of mouse brain was evaluated.

Experimental procedure: Mice were treated with Kava extract (10, 40, 100 and 400 mg/kg) for 21 days by gavage. After behavioral analysis (plus maze test and open field), MAO activity in different mouse brain structures (cortex, hippocampus, region containing the substantia nigra and striatum) were performed. MAO-B inhibitory profile was characterized in vitro.

Results: The treatment with Kava extract (40 mg/kg) increased the percentage of entries of mice into the open arms. Ex vivo analysis showed an inhibition on MAO-B activity caused by Kava extract in cortex (10 mg/kg) and in the region containing the substantia nigra (10 and 100 mg/kg). In vitro, Kava extract also reversibly inhibited MAO-B activity with IC₅₀ = 14.62 μg/mL and, increased Km values at the concentrations of 10 and 30 μg/mL and decreased Vmax value at 100 μg/mL.

Conclusion: Kava extract showed different effects on MAO-B isoform depending on the brain structure evaluated. Therefore, the use of Kava extract could be promissory in pathologies where MAO-B is the pharmacological target.

Keywords: 4-HQ, 4-hydroxyquinoline; 5-HT, serotonin; AMPH, amphetamine; Anxiety; CEUA, Ethic Committee on Animal Use; CNS, central nervous system; CONCEA, National Council of Control of Animal Experimentation; DA, dopamine; GABA, gamma-aminobutyric acid; HPLC, High performance liquid chromatography; Kynuramine; MAO, Monoamine oxidase; NE, norepinephrine; Open field; Piper methysticum; Plus maze.

Graphical abstract

Fig. 1

Behavioral assays. (A) the percentage of the time spent on the open arms, (B) the percentage of the number of entries into the open arms and (C) the number of head dips in plus maze test, along with the number of (D) crossing and (E) rearing in the open field test were evaluated in mice, both for 5 min. The animals were treated with vehicle or Kava extract (10, 40, 100, 400 mg/kg) for 21 days. Data are expressed as mean + standard error of mean (n = 8). ∗p < 0.05, compared to control group. ^#p < 0.05, compared to kava extract (40 mg/kg) group.

Fig. 2

MAO-A activity in (A) cortex, (B) hippocampus, (C) region containing the substantia nigra and (D) striatum in treated mice with vehicle or Kava extract (10, 40, 100, 400 mg/kg) for 21 days. Data were analyzed by one-way ANOVA followed by Tukey's post hoc test. Data are expressed as mean + standard error of mean (n = 5).

Fig. 3

MAO-B activity in (A) cortex, (B) hippocampus, (C) region containing the substantia nigra and (D) striatum in treated mice with vehicle or Kava extract (10, 40, 100, 400 mg/kg) for 21 days. Data were analyzed by one-way ANOVA followed by Tukey's post hoc test. Data are expressed as mean + standard error of mean (n = 5). ∗p < 0.05, compared to control group.

Fig. 4

Inhibitory potential in vitro of Kava extract (10, 30, 100 μg/mL) on (A) MAO-A and (B) MAO-B activities in mouse brain homogenates. Values are expressed as mean + standard error of mean (n = 3–4). MAO activity was analyzed by one-way followed by Tukey's post hoc test. ^&p < 0.05, compared to Kava extract 3 and 10 μg/mL ∗p < 0.05, compared to control and ethanol group. ^#p < 0.05, compared to Kava extract 3 μg/mL (C) reversibility of MAO-B inhibition caused by Kava extract (100 μg/mL) after 24 h of dialysis in mouse brain homogenates. Values are expressed as mean + standard error of mean (n = 3–4). Reversibility was analyzed by two-way ANOVA followed by Tukey's post hoc test. ^$p < 0.05, compared to nondialyzed control. (D) Substrate concentrations curve for in vitro MAO-B activity in the absence or presence of Kava extract (10, 30, 100 μg/mL). Km (μM) and Vmax values were calculated by nonlinear regression using the Michaelis–Menten equation (n = 6).