Neuropsychopharmacological profiling of scoparone in mice

Abstract

Scoparone (6,7-dimethoxycoumarin) is a simple coumarin from botanical drugs of Artemisia species used in Traditional Chinese Medicine and Génépi liquor. However, its bioavailability to the brain and potential central effects remain unexplored. We profiled the neuropharmacological effects of scoparone upon acute and subchronic intraperitoneal administration (2.5-25 mg/kg) in Swiss mice and determined its brain concentrations and its effects on the endocannabinoid system (ECS) and related lipids using LC-ESI-MS/MS. Scoparone showed no effect in the forced swimming test (FST) but, administered acutely, led to a bell-shaped anxiogenic-like behavior in the elevated plus-maze test and bell-shaped procognitive effects in the passive avoidance test when given subchronically and acutely. Scoparone rapidly but moderately accumulated in the brain (Cmax < 15 min) with an apparent first-order elimination (95% eliminated at 1 h). Acute scoparone administration (5 mg/kg) significantly increased brain arachidonic acid, prostaglandins, and N-acylethanolamines (NAEs) in the FST. Conversely, subchronic scoparone treatment (2.5 mg/kg) decreased NAEs and increased 2-arachidonoylglycerol. Scoparone differentially impacted ECS lipid remodeling in the brain independent of serine hydrolase modulation. Overall, the unexpectedly potent central effects of scoparone observed in mice could have toxicopharmacological implications for humans.

Figure 1

Chemical structures of scoparone (1) and its peripheral metabolites isofraxidin (2) scopoletin (3) and isoscopoletin (4).

Figure 2

Scoparone induces anxiogenic effects. Dose-dependent (bell-shaped) effect of scoparone administration on anxiety-like behavior in Swiss albino male mice in the elevated plus maze (EPM) test. Figures show mean values ± SEM of the percentage time spent on the open arms (a) and the percentage of open arm entries (b) measured 30 min after an acute injection of scoparone (2.5, 5, 12.5, 25 mg/kg, i.p.) or vehicle; n = 8–9; *p < 0.05, **p < 0.01, vs. vehicle-treated control group, Tukey's test.

Figure 3

Scoparone shows procognitive effects in male Swiss albino mice the passive avoidance (PA) test. (a) Effects of an acute scoparone administration on the latency index (LI) during the acquisition trial using the PA test. Scoparone (2.5, 5, 12.5, 25 mg/kg; i.p.) or vehicle were injected 30 min before the first trial and mice were re-tested 24 h later; n = 8–9; the means ± SEM; *p < 0.05, ***p < 0.001 vs. vehicle-treated control group; Tukey’s test. (b) Effects of subchronic scoparone administration on the latency index (LI) during the acquisition trial using the PA test in mice. Scoparone (2.5, 5, 12.5 and 25 mg/kg; i.p.) or vehicle were administered for the six days. On the seventh day scoparone was administered 30 min before the first trial and mice were re-tested 24 h later; n = 8–9; the means ± SEM; *p < 0.05; **p < 0.01 vs. vehicle-treated control group; Tukey’s test. (c) Effects of subchronic administration of scoparone (15 mg/kg, i.p.) on LPS (0.8 mg/kg)-induced impairment of memory acquisition trial using the PA test in mice. Mice were injected with scoparone 60 min after LPS administration and then consecutively for 6 days. On the seventh day, scoparone was injected 30 min before the first trial, and animals were retested 24 h after the last injection; n = 8–9; the means ± SEM; *p < 0.05, **p < 0.01 vs. vehicle-treated control group; Bonferroni’s test. (d) Effects of acute administration of scoparone on scopolamine-induced impairment of memory acquisition trial using the PA test in mice. Scoparone (5 and 12.5 mg/kg, i.p.) was administered 30 min and scopolamine (1 mg/kg, i.p.) 20 min before the first trial and animals were re-tested 24 h after the last injection; n = 8–9; the means ± SEM; *p < 0.05; ***p < 0.001 vs. vehicle-treated control group, ^p < 0.05, vs. scopolamine-treated group; Bonferroni’s post hoc test.

Figure 4

(a) Effects of acute administration of scoparone (5 and 12.5 mg/kg) on AChE brain levels induced by scopolamine administration in mice. Scoparone was administered 30 min and scopolamine (1 mg/kg, i.p.) 20 min before the first trial and animals were re-tested 24 h after the last injection; n = 8–9; the means ± SEM; ***p < 0.001 vs. vehicle-treated control group, ^p < 0.05, ^^^p < 0.001 vs. scopolamine-treated control group; Bonferroni’s test. (b) Effects of acute administration of scoparone on BChE level induced by scopolamine in mouse brain. Scoparone was administered 30 min and scopolamine (1 mg/kg, i.p.) 20 min before the first trial, and animals were re-tested 24 h after the last injection; n = 8–9; the means ± SEM; **p < 0.01 vs. vehicle-treated control group; Bonferroni’s test. (c) Effects of acute administration of scoparone on FRAP levels in mouse brain. Scoparone was administered 30 min and scopolamine (1 mg/kg, i.p.) 20 min before the first trial and animals were re-tested 24 h after the last injection, n = 8–9; the means ± SEM; **p < 0.01 vs. vehicle-treated control group, ^^^p < 0.001 vs. scopolamine-treated control group; Bonferroni’s test.

Figure 5

Pharmacokinetic analyses of scoparone in brain homogenates in Swiss albino male mice. (a) Time-dependent brain exposure upon administration of scoparone (i.p.) at 5 and 12.5 mg/kg and in combination with borneol (50 mg/kg) which significantly increased brain concentrations of scoparone. (b) Model of first order elimination of scoparone from mouse brain tissue upon administration of 12.5 mg/kg i.p. Data show mean values ± SD, n = 5.

Figure 6

Acute scoparone treatment (5 and 12.5 mg/kg i.p.) on endocannabinoids and related lipids in the forced swim test (FST) in male Swiss albino mice. scoparone was injected 30 min before the FST and subsequently mice were sacrificed, and brains were obtained for measurement; n = 5; *p < 0.05; **p < 0.01; ***p < 0.001 vs. vehicle-treated control group; Tukey’s test. AA arachidonic acid, SAG 1-stearoyl-2-arachidonoyl-sn-glycerol, 2-AG 2-arachidonoyl glycerol, PGE2/PGD2 prostaglandins, AEA anandamide, LEA linoloyl ethanolamide, PEA palmitoyl ethanolamide, OEA oleoyl ethanolamide, SEA stearoyl ethanolamide, 20:4 PE 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine. Data show mean values ± SD, n = 5.

Figure 7

Effects of subchronic scoparone treatment (2.5 mg/kg, i.p., for 14 days) on endocannabinoids and related lipids in the forced swim test (FST) and elevated plus maze (EPM) paradigms in male Swiss albino mice. On day 15, scoparone was injected 30 min before the FST or EPM test and subsequently mice were sacrificed, and brains were obtained for measurements; n = 5; *p < 0.05; **p < 0.01; ***p < 0.001 vs. vehicle-treated control group. Bars with black dots show vehicle treated mice and bars with blue dots show scoparone treated mice. AA arachidonic acid, SAG 1-stearoyl-2-arachidonoyl-sn-glycerol, 2-AG 2-arachidonoyl glycerol, PGE2/PGD2 prostaglandins, AEA anandamide, LEA linoloyl ethanolamide, PEA palmitoyl ethanolamide, OEA oleoyl ethanolamide, SEA stearoyl ethanolamide, 20:4 PE 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine. Data show mean values ± SD, n = 5.

Figure 8

Activity-based protein profiling showing serine hydrolase activity in membranes from Swiss albino mouse brain tissues. Scoparone did not inhibit enzymes activity of serine hydrolases fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), and alpha–beta hydrolase 6 (ABHD6). DMSO was used as vehicle control. Specific inhibitors were used as positive controls for FAAH (URB597), MAGL (JZL184), ABHD6 (THL), and ABHD6/12 (WWL70). Image shows a representative blot of three independent experiments.