Animals and treatment
Danio rerio stocks of the AB strain were maintained at 28.5 °C, on a 14-/10-h light/dark cycle under standard aquaculture conditions, and fertilized eggs were collected via natural spawning. Embryos were reared under standard light/dark cycle in embryo medium: pH 7.1–7.3, 17.4-µM NaCl, 0.21-µM KCl, 0.12-µM MgSO4 and 0.18-µM Ca(NO3)2 in an incubator at 28.5 °C. To the procedure, 5 days post fertilization (dpf) larvae (total number 48, 24 per group) were used, and all experiments were completed before 120-h post fertilization, before larvae start feeding independently. According to EU Directive, 2010/63/EU, there is no need of local ethics committee approval for larvae on this stage of development. Immediately after the experiment, larvae were killed by immersion in 15-μM tricaine solution.
Naive male Swiss mice (total number 48, 8 per group), 6 weeks old (25–30 g), delivered from the Centre of Experimental Medicine, Medical University of Lublin, Poland and kept under standard laboratory conditions (12-h light/dark cycle, room temperature 21 ± 1 °C, at least 1-week adaptation to the laboratory conditions) were used in experiments. Animals had free access to tap water and laboratory chow (Agropol, Poland). Animals were housed 4 per cage, in Individually Ventilated Cages (IVC)—Techniplast UK. Each experimental group consisted of 8 animals. All experiments were conducted in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and to the European Community Council Directive for the Care and Use of Laboratory Animals of 22 September 2010 (2010/63/EU) and were approved by the local ethics committee (63/2015). The experimenter was unaware of the animal’s group during experimentation. To minimize animal suffering, qualified and experienced experimenters handled the animals with utmost care. All animal experiments were performed between 9 a.m. and 2 p.m. Mice were subsequently euthanized with CO2.
XT was purified from dichloromethane extract of Pastinaca sativa L. (Apiaceae) fruits, collected in the Medical Plant Garden of the Department of Pharmacognosy, Medical University of Lublin (Poland). The location of the garden is as follows: Situation (position) 700 m N-W near Center of town Lublin, Latitude 51º 15′ 22″; Longitude 22º 33′ 51″; Altitude ca 185 m above sea level. Fruits were collected in the summer of 2014. The species was identified by specialists in botany—Mrs Krystyna Dąbrowska from Botanical Garden of Marie Curie University, Lublin and a voucher specimen (17/20) is kept in the Department of Pharmacognosy with the Medicinal Plant Unit.
Isolation was carried out by high-performance counter-current chromatography (HPCCC) with a two-phase solvent system composed of n-heptane, ethyl acetate, methanol and water with the ratio of 1:1:1:1 (v/v/v/v) according to a previously published method . The purity of XT was 98.6% and was checked by HPLC. Structure determination was confirmed with NMR.
MPTP hydrochloride (M103) and 6-OHDA hydrobromide (162,957) were delivered by Sigma-Aldrich (St. Louis, MO, USA). MPTP was dissolved in a saline solution (0.9% NaCl) and 6-OHDA was dissolved in embryo medium. For mice experiments, XT was suspended in a 50 µL of 1% solution of Tween 80 and dissolved in a saline solution to achieve a concentration of 5 mg/kg. Tween 80 can be employed safely as a vehicle for neuropsychopharmacological experiments in doses not exceeding 1 ml/kg . For zebrafish assay, XT was dissolved in DMSO (D8418, Sigma-Aldrich St. Louis, MO, USA) and diluted to proper concentration. The final concentration of DMSO in every sample was equal to 1%.
The doses of 6-OHDA (250 µM) was chosen based on the literature data . The doses of XT in zebrafish study was chosen after determination of the maximum-tolerated concentration (7.5 µM) according to the previously described protocol  and were 1.5, 3, 5, 7.5 µM. The doses of MPTP (4 × 20 mg/kg, 1 day) was chosen based on literature data [20, 21]. The doses of XT in rodent study were chosen on the basis of our previous study [8, 22] and were 15 and 25 mg/kg.
Locomotor activity in zebrafish larvae was measured using the dark chamber of an automated tracking device (ZebraBox system; Viewpoint, Lyon, France). Locomotor activity was calculated using ZebraLab software (Viewpoint, Lyon, France). The total distance moved was defined as the distance (in cm) that a larva moved during one 10-min session.
To evaluate the influence of XT on MPTP-induced hypokinesia, the Opto-Varimex-4 Auto-Track apparatus (Columbus Instruments, USA) was used. The locomotor cages are built from a transparent material (43 × 43 × 32 cm) with a lid. The cages are equipped with two rows of infrared emitters (each emitter has 16 laser beams), with detectors monitoring animal movements. Each mouse was placed individually into the cage for 30 min.
Experimental design PD
The 6-OHDA-induced PD symptoms in Danio rerio
5 dpf larvae were allocated into groups (24 larvae per group) as follows:
(1) Group I: control (1% DMSO solution in embryo medium); (2) Group II: 6-OHDA control (250 µM); (3) Group III: XT (1.5 µM); (4) Group IV: XT (3.0 µM); (5) Group V: XT (5.0 µM); (6) Group VI: XT (7.5 µM); (7) Group VII: 6-OHDA (250 µM) + XT (1.5 µM); (8) Group VIII: 6-OHDA (250 µM) + XT (3.0 µM); (9) Group IX: 6-OHDA (250 µM) + XT (5.0 µM); (10) Group X: 6-OHDA (250 µM) + XT (7.5 µM).
The neurodegeneration in larvae was induced by maintaining zebrafish from 2 to 5 dpf in medium containing 6-OHDA in the concentration of 250 µM. The larvae were maintained in groups of 30 individuals. Three times a day, medium with neurotoxin was exchanged and a new concentration of 6-OHDA was prepared directly before administration. 5-dpf larvae were transferred to 96-well plates, 1 larva per each well, and different concentrations (1.5, 3, 5, 7.5, µM) of XT were administrated by immersion method. The incubation with drug lasted 30 min (Fig. 1).
The MPTP-induced PD symptoms in mice
Mice were allocated into five groups, 8 mice per group: (1) Group I: control (saline, ip); (2) Group II: MPTP control (4 × 20 mg/kg × 1 day, ip); (3) Group III: XT (15 mg/kg; ip); (4) Group IV: XT (25 mg/kg; ip); (5) Group V: MPTP (4 × 20 mg/kg × 1 day, ip) + XT (15 mg/kg; ip); (6) Group VI: MPTP (4 × 20 mg/kg × 1 day, ip) + XT (25 mg/kg; ip).
On the day of the experiment, mice received four ip injections of MPTP (20 mg/kg) in saline at 2-h intervals, 4 doses a day; control mice received saline only. 14 days after MPTP treatment, XT was administered acutely, each animal received a single XT injection, and then the locomotor activity test was performed (Fig. 2).
Two-way ANOVA was used to perform the statistical analyses. Bonferroni’s post hoc test was calculated when appropriate. When the confidence limit was calculated as p < 0.05, the results were considered as statistically significant. For statistical analysis, means (distance in cm traveled by mice) ± SEM were used. GraphPad Prism version 7.0 (GraphPad Software Inc, San Diego, CA, USA) was used for the data analysis and for graphics.