Neuropsychopharmacological profiling of scoparone in mice

Coumarins are an independent class of shikimate-derived natural products with a 2H-chromen-2-one core scaffold. In profiling studies, various coumarins show a surprisingly broad range of biological activities in vitro and in vivo 1 . In addition, different coumarins, both synthetic and natural, have demonstrated effects on the central nervous system (CNS) in preclinical in vivo experiments 1 , 2 , suggesting their penetration through the blood–brain barrier (BBB). However, only a few studies have addressed the bioavailability and pharmacokinetics of natural coumarins in the brain, and there is a gap between the reported descriptive behavioral effects and the insights generated from bioanalytical measurements. Scoparone (6,7-dimethoxycoumarin) is widely present in mugwort (also wormwood) plant species [ Artemisia species (spp.)]. Regarding Traditional Chinese Medicine (TCM), the pharmacology of scoparone has been extensively studied in preclinical liver inflammation mouse models 3 . As non-terpenoid volatile, scoparone is present in essential oils 4 and is a major natural product in Génépi liquor produced from Artemisa spp . plants, including A. genepi . Using high-performance countercurrent chromatography (HPCCC) we isolated scoparone as a major constituent from the extracts of the alpine wormwood species Artemisia umbelliformis Lam. (white genepì) used in the distillation of Génépi, a well-known traditional herbal liqueur or aperitif originating from the Alpine regions of Europe 5 , 6 . Although the bitter sesquiterpene lactones and the ketone monoterpene, thujone are the best-known psychoactive constituents of Artemisia spp. 6 – 8 , we hypothesized that scoparone might contribute to the alleged psychoactive or neurotoxic effects of mugwort-based alcoholic drinks 5 , 9 . To date, the potential pharmacological effects of scoparone in the CNS have not been studied. To our knowledge, the only report related to the central effects refers to pilocarpine and methylscopolamine-induced seizures in mice, where relatively high doses of scoparone (25–100 mg/kg) injected intraperitoneally ( i.p. ) attenuated the seizures' duration and decreased inflammatory processes in both the hippocampus and prefrontal cortex 10 . Although a recent comprehensive study on the rapid elimination of scoparone in humans and its metabolism in mouse, rat, pig, dog, and rabbit liver microsomes highlighted species-related differences in phase I metabolism 11 , the brain penetration and central effects of scoparone are unclear 12 . In the present study, we specifically assessed the neuropharmacological effects of scoparone (Fig. 1 ) in a test battery of complex behavioral paradigms, including (a) depression-like behavior in the forced swim test (FST), (b) anxiety-like behavior in the elevated plus-maze (EPM), and (c) learning and memory in the passive avoidance test (PA) after acute and subchronic, (i.p.) injections of scoparone (2.5–25 mg/kg) in Swiss albino mice. In addition, the PA paradigm was applied to assess scoparone's procognitive/neuroprotective effects in both lipopolysaccharide (LPS) and scopolamine-induced mouse impaired learning and memory 13 , 14 . Figure 1 Chemical structures of scoparone (1) and its peripheral metabolites isofraxidin (2) scopoletin (3) and isoscopoletin (4). We aimed to correlate the central effects with the concentration of scoparone and its major metabolites (Fig. 1 ) in mouse brain tissue when the compound was administered alone or in combination with borneol, a natural monoterpene shown to improve drug delivery to the brain 15 possibly via p-glycoprotein inhibition 14 . It has already been shown that borneol promotes the accumulation of other drugs in brain tissue 15 , 16 . However, there is little evidence that it can enhance the brain permeability of coumarins. Thus, one of the aims was to check whether borneol increases the level of scoparone in the CNS. This is relevant because there is already a well-established TCM pure natural preparation derived from plants and marketed in China promoting neurogenesis, where borneol is co-administered with herbal drugs from the Apiaceae family 17 . Using LC–MS/MS-based targeted lipidomics, we measured the effects of scoparone on the brain lipidome associated with the endocannabinoid system (ECS), a major lipid signaling network that regulates complex behaviors and stress responses in neuropsychiatric disorders 13 .

Scoparone was also tested in the PA paradigm to assess its effect on learning and memory (Fig. 3 ). A single injection of scoparone (i.p.) had a statistically significant effect on latency index (LI) values for memory acquisition in the PA test (F (4, 33) = 7.486; p = 0.0002) (Fig. 3 a). The post-hoc Tukey’s test confirmed that the treatment with scoparone at the doses 2.5, 5, and 12.5 mg/kg significantly increased LI values in male mice compared to those in the saline-treated control group ( p < 0.05, p < 0.001, p < 0.05, respectively) (Fig. 3 a), indicating that scoparone at these used doses improved learning or memory acquisition. No effect was observed when scoparone was delivered at dose of 25 mg/kg. Thus, scoparone exhibited nonlinear dose–response effects showing no significant effects at the higher doses. Since this effect was noticed after a single injection of scoparone , we decided to evaluate the effects of subchronic scoparone treatment (Fig. 3 b). One-way ANOVA analysis showed that subchronic (14-days) administration of scoparone (2.5, 5, 12.5, and 25 mg/kg, i.p.) had a statistically significant effect on LI values for memory acquisition in the PA test (F (4, 37) = 0.0074; p = 0.0021). The post-hoc Tukey’s test confirmed that scoparone treatment significantly increased LI values in mice compared to those in the saline-treated control group ( p < 0.01 for the dose of 5 mg/kg, and p < 0.05 for the dose of 12.5 mg/kg). Upon either acute or subchronic administration of scoparone, the dose of 5 mg/kg exerted the most pronounced effect (Fig. 3 a,b). Noteworthy, a bell-shaped dose–response was recorded in the PA test after both acute and subchronic scoparone treatment, similar to those observed in the EPM (see above).

We next assessed whether scoparone could attenuate the memory deficits induced by scopolamine injection in the PA test. As expected, we observed a statistically significant memory impairment caused by scopolamine (1 mg/kg) treatment (F (1, 44) = 65.27; p < 0.0001). A significant effect of memory improvement (F (2, 44) = 25.91; p < 0.0001) and an interaction between scoparone and scopolamine (F (2, 44) = 33.61; p < 0.0001) was observed. The post-hoc Bonferroni’s test confirmed that scopolamine at the dose of 1 mg/kg significantly decreased LI values in mice in the PA test in comparison to the vehicle-treated mice, which confirms the amnesic effect of this drug ( p < 0.05). It was also confirmed that scoparone, at the doses of 5 and 12.5 mg/kg, improved acquisition of memory and learning in this assay ( p < 0.001, p < 0.05, respectively). Additionally, scoparone (12.5 mg/kg) prevented the amnesic-like effect of scopolamine ( p < 0.05) as compared to scopolamine-treated mice (Fig. 3 d). Because low and high scoparone doses (2.5 and 25 mg/kg) were inactive in the PA test, we did not consider these doses in the scopolamine memory impairment model.

We next quantified the concentrations of scoparone and its known metabolites (Fig. 5 ) in total mouse brain tissue using a quantitative LC–MS/MS method. Concentrations of scoparone [ng/g] were measured at different time points after acute i.p. injection of 5 mg/kg and 12.5 mg/kg (Fig. 5 A). We did not detect scopoletin, isoscopoletin, and isofraxidin (limit of detection ≤ 0.2 ng/g brain tissue). Because the monoterpene borneol has been previously shown to increase brain concentrations of certain lipophilic natural products 15 , 16 , 18 , we performed an experiment, where both scoparone and borneol (50 mg/kg) were injected. Borneol was administered 60 min before scoparone (5 and 12.5 mg/kg, i.p.). The brain tissue was isolated 30 min after scoparone injection. As shown in Fig. 5 A, borneol (50 mg/kg) increased the brain concentrations of scoparone by a factor 3. Interestingly, in this experiment we could also detect traces of isofraxidin in the brain, but only upon i.p. injection of 12.5 mg/kg scoparone in combination with 50 mg/kg borneol (data available on request). As summarized in Fig. 5 B, scoparone was effectively eliminated from the mouse brain tissue showing an apparent first-order elimination kinetics. After 1 h, only traces of scoparone (2–6 ng/g brain tissue) could be detected under all conditions, independently on the C max achieved. 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.

The apolar coumarin scoparone (6,7-dimethoxycoumarin) is mainly associated with the consumption of Génépi liquors that are very popular in the Alpine regions of Europe 6 . Distillation recipes of the French apéritif quina and some liquors like the Swedish-made Jeppson’s Malört use Artemisia species containing scoparone in Amaro. We hypothesized that scoparone might contribute to the psychoactive activity of liquors mentioned above and influence the brain and behavioral changes. We found that scoparone is a major coumarin in Artemisia umbelliformis Lam., a plant frequently used to distill mentioned above liquors, with an estimated amount of around 0.1–0.5% of the dried aerial parts. For the peripheral effects, various mechanisms of action of scoparone have been proposed in vitro, mainly at concentrations in the micromolar concentration range 19 – 21 . However, these concentrations are not achieved in the brain. Thus, despite the wealth of data related to the peripheral anti-inflammatory and liver-protective effects of scoparone, it was hitherto unclear whether this coumarin also affects the brain. Here we show that scoparone exerts potent differential neuropharmacological effects in mice. We used quantitative LC–MS/MS to measure scoparone in total brain homogenates at different time points after the FST and EPM assays. Because the administration of 5 or 12.5 mg/kg gave similarly low scoparone concentrations in the brain after 1 or 2 h (< 2 ng/g), respectively, we conclude that this coumarin is rapidly metabolized in the periphery, in agreement with previous literature. A fast metabolism of scoparone was described in the periphery after oral administration of herbal drugs 22 . After 30 min, around 0.1 µg/ml of scoparone was measured that further decreased to 0.01 µg/ml after 2 h. Overall, the bioavailability of coumarins was limited by liver metabolism by CYP1A2 enzymes that demethylate scoparone 23 . Scoparone was easily O-demethylated to scopoletin in the liver 24 , and different metabolites were generated 22 . Our study did not detect the accumulation of the major metabolites (Fig. 1 ) in the brain. However, after treatment with borneol and the highest dose of scoparone (12.5 mg/kg), traces of isofraxidin were detected. In the experiment in which borneol was co-administered with scoparone, no modulation of the scoparone-induced memory improvement was observed (Supplementary Fig. S2 ). Co-administration of 50 mg/kg of borneol, a monoterpene reported previously to specifically increase brain permeability of different drugs, hypothetically via p -glycoprotein inhibition 18 , increased scoparone concentrations in the brain by approximately 300%. The effect of borneol enantiomers on the pharmacokinetics of the simple coumarin osthole was previously investigated. Co-administration of these two compounds increases the bioavailability of osthole even by up 270% 25 . We found a similar acute administration of 12.5 mg/kg of scoparone yielded approximately 3–4 times the brain exposure of scoparone at 5 mg/kg. Our data thus suggest that scoparone could be eliminated from the brain via p -glycoprotein associated mechanisms. The concentration of scoparone (5 and 12.5 mg/kg) in the brain was highest at the earliest time point measured, after 15 min from the injection, and decreased in a time-dependent manner showing first-order kinetics. Despite the low central bioavailability of scoparone the neuropharmacological profiling in different behavioral paradigms revealed an apparent effect specificity. Scoparone administered acutely (Supplementary Table S2 ) and subchronically (Supplementary Table S3 ) did not affect stress and depressive-like behaviors in the FST in both sexes, even though it exerted significant effects on endocannabinoids and related lipids (see below). We employed imipramine as a positive control in this model. Contrary to our expectations, we did not observe sex differences in behavior and acute drug action in the FST. Upon acute administration of scoparone, in the EPM paradigm we observed anxiogenic-like effects in a bell-shaped curve of changes in the percentage of open arms time and entries (Fig. 2 ). Interestingly, the anxiogenic-like effect was not observed upon subchronic administration of scoparone (Supplementary Table S4 ). Unlike the acutely injected mice, these mice were more stressed (receiving repetitive daily injections) as judged by the corticosterone levels between vehicle control groups. Lipidomic analyses showed that subchronic administration of scoparone significantly reduced NAEs, including the endocannabinoid anandamide (AEA) reported to mediate anxiolytic effects in different preclinical models like the EPM 26 . The reversal of stress-induced anandamide deficiency is a postulated mechanism subserving the therapeutic effects of FAAH inhibition 27 . Conversely, acute scoparone administration led to a very strong and unexpected increase of AA, prostaglandins, but also NAEs like ananda

Swiss albino mice (total number 372 mice) were housed 4 per cage group in the 12 h light/dark cycles with free access to food and water (room temperature 21 ± 1 °C). Each experimental group was comprised of 9–10 animals randomly allocated to prevent bias. 10-weeks old male Swiss albino mice (35–40 g) were used for the LPS model of cognitive impairment in the PA test. 6-weeks old male Swiss mice (25–30 g) were used for all other experiments, except the FST in which both males and females were tested for sex differences in response to the scoparone treatment.

0.9% sterile sodium chloride solution for injections (0.9% NaCl) was used to prepare solutions of scopolamine, borneol, LPS, and imipramine (Sigma-Aldrich, St. Louis, MO, USA). Solutions of scoparone were prepared by suspension in one drop of 1% Tween 80 solution and gradually dissolving in the saline. Scoparone was obtained as described above. Injections (i.p.) were made at the volume of 10 ml/kg of mice body weight, and fresh solutions were prepared every day before administration. The scopolamine LPS and imipramine doses were chosen based on literature data 39 – 41 and our previous studies 14 . The doses of scoparone (2.5, 5, 12.5, 15, 25 mg/kg) were chosen based on our pilot study and literature data 42 . 15 mg/kg of scoparone was used in the LPS-model of memory impairment (PA test) based on our previous study 43 . The doses of borneol (50 mg/kg) were chosen based and our preliminary study. Control groups were injected with 0.9% sterile sodium chloride solution for injections (0.9% NaCl) each time. In the acute model of administration, mice received a single scoparone injection 30 min before the tests (PA, FST, EPM). In the extended subchronic experiments, scoparone was administered once a day for 14 days at 9 a.m. (FST, EPM, and PA tests). On the 15th day of the treatment, 30 min after scoparone injection, a behavioral test was performed. LPS- and scopolamine-induced memory deficits, as well as borneol treatment (48 mice), were described below and in the Supplementary Information.

The acquisition of memory processes was evaluated in the PA test (88 mice) using the same procedure described previously 14 , 45 . For the scopolamine-induced model of memory impairment, scoparone (5 and 12.5 mg/kg) was administered 30 min before the pre-test, and 10 min after coumarin injection, mice (48 mice) were treated with scopolamine (1 mg/kg, i.p.), or saline in the control group. The model of LPS memory impairment was established experimentally by our group 43 (40 mice). Scoparone (15 mg/kg, i.p.), or saline in the control group were administered 60 min after a single injection of LPS (0.8 mg/kg), and then only coumarin was injected once daily for 6 consecutive days. On the seventh day, scoparone or saline was administered once (9.00 a.m.), 30 min before the first trial (memory acquisition) and re-tested after 24 h.

A hybrid triple quadrupole API 4000 QTRA P (Sciex) was used with Shimadzu UHPLC. Chromatography was performed on a Synergi™ 4 µm Fusion-R P 80 Å C18, 50 × 2 mm (Phenomenex). The column temperature was maintained at 45 °C, and subsequently, a gradient of 0.1% (v/v) formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B) was used as follows: gradient of 95% A to 5% A during 0.2–4.2 min, then linear 95% A at 4.40–5.00. The flow rate was 0.8 ml/min, and the autosampler was cooled at + 4 °C. The MS detector was operated in the positive electrospray ionization (ESI) mode using multiple reaction monitoring (MRM) transitions (Supplementary Table S1 ). There were used the ion spray voltage (4,000 V), ion spray temperature (450 °C), curtain gas (30 psi, GS1 65 psi, and GS2 35 psi). Data were acquired and processed using Analyst software. Supplementary Fig. S1 shows m/z in QI ESI scans. Calibration curves in the biological matrix were generated by plotting the peak area ratio (y) of all coumarins to IS versus its nominal concentration (x) using a weighted (1/ × 2) linear regression model. A coefficient of correlation (r ≥ 0.9900) was obtained for all compounds. In the applied method, scopoletin and isoscopoletin were not distinguished. To estimate the elimination kinetics of scoparone from the brain the software PCModfit Version 6 was used, assuming negligible background from brain vasculature and that a constant fraction of the substance in the brain is eliminated per unit time.

Determination of AChE and BChE levels in brain homogenate The intact brain without the cerebellum, dissected immediately after behavioral tests, was homogenized in ice-cold PBS (10% w/v) and centrifuged at 10,000 rpm at 4 °C for 15 min. The supernatant was collected and stored at − 80 °C for further analysis of AChE and BChE. The PierceTM BCA Protein Assay Kit determined protein concentration. For AChE and BChE determination, the ELISA kit (Cloud-Clone Corp., USA) was used, and all measurements were done according to the manufacturer’s protocol. Absorbance was read at 450 nm, and obtained data were presented as pg/mg protein.

The ABPP was chosen to investigate possible effects on the activity of serine hydrolases in the mouse brain. ABPP experiments were performed by using membrane preparations obtained from mouse brains as described by Ref. 47 , with minor modifications. In brief, brain samples (25 µg of total membrane proteins) were incubated with the serine hydrolase targeting probe FP-TAMRA (ActivX™) at a final concentration of 500 nM and incubated for 15 min at RT. To visualize the possible modulation of the activity of the crucial lipid-metabolizing serine hydrolases that regulate endocannabinoid degradation (FAAH; MAGL, ABHD6, and ABHD12), brain samples were preincubated with scoparone and the positive controls (all from Cayman Chemical, UK), namely URB597 (1 µM); JZL184 (1 µM); WWL70 (10 µM); THL (10 µM) or DMSO (as vehicle control) for 30 min at 37 °C. After applying the FP-TAMRA probe for 15 min, the incubation the reaction was stopped by 4 × Lämmli Buffer, and the samples were boiled at 70 °C for 10 min. Samples were cooled down to RT and loaded into an SDS-PAGE gel as was previously described 47 .

For statistical analysis GraphPad Prism 8.3.1 was used. When appropriate, one or two- way ANOVA analyses were performed for statistical analysis with post hoc Bonferroni’s or Tukey’s test. All data are shown as means (± SEM). The results of p < 0.05 were considered statistically significant. Outliers were calculated using Grubbs' test, alpha = 0.05. The index of latency (IL) was calculated to evaluate changes in memory acquisition and consolidation. IL = (TL2 − TL1)/TL1 where TL1 was the time to enter the dark compartment during the training and TL2 was the time to re-enter the dark compartment during the retention.