Chaenomeles maulei fruit juice was produced from plants grown in the Balkan Mountains, Bulgaria, in the region of Troyan. The freshly handpicked fruits were grinded, crushed, and squeezed. The juice was filtered, preserved with potassium sorbate (1.0 g/l), and stored at 0°C until experiments. The total content of phenolic compounds in CMFJ proved to be very high - 8900.00 mg gallic acid equivalents per liter of juice. It was determined by spectrophotometric Folin-Ciocalteu assay.^[18] Absorbance was read at 760 nm. Gallic acid was used as a standard. The HPLC analysis confirmed the high content of polyphenols, especially phenolic acids and flavonoids. The phenolic acids were presented in the highest concentration by vanillic acid (149.1 mg/l), caffeic acid (144.8 mg/l), and chlorogenic acid (110.0 mg/l).^[19] The most abundant flavonoids in the fruit juice were epicatechin (5.59 mg/l), catechin (52.5 mg/l), and (quercetin 35.8 mg/l).^[19]

We used healthy male Wistar rats with mean weight of 220±30 g, bred in the Animal Centre of Medical University of Varna. The animals were housed in plastic cages in a well-ventilated room maintained at 22±1°C and on a 12/12 light/dark cycle. They received standard rodent pelleted diet and water ad libitum. All procedures concerning animal treatment and experimentation were conducted in conformity with the national and international laws and policies (EU Directive 2010/63/ EU for animal experiments) and were approved by Bulgarian Food Safety Agency (No. 141/23.06.2016).

In this study, 50 male Wistar rats were divided into five groups, each consisting of 10 animals. The groups were respectively the control, ICR, ICR+CMFJ_2.5, ICR+CMFJ₅, and ICR+CMFJ₁₀.

Treatment of the animals was carried out as follows: the animals from the control and ICR groups were treated once daily with distilled water (10 ml/kg) through an orogastric cannula. The animals from the ICR+CMFJ_2.5 group received once daily 2.5 ml/kg of CMFJ diluted with distilled water to 10 ml/kg. The animals from the ICR+CMFJ₅ group were treated once daily with CMFJ at a dose of 5 ml/kg, diluted to 10 ml/kg with distilled water. The animals from the ICR+CMFJ₁₀ group received CMFJ at a dose of 10 ml/kg once a day.

The administration period was 14 days during which the animals from groups ICR, ICR+CMFJ_2.5, ICR+CMFJ₅ and ICR+CMFJ₁₀ were subjected to impaired circadian rhythmicity (ICR) through exposure to continuous light. The control animals had a normal lighting regime throughout the duration of the experiment. Fifteen days after the experiment started, one hour after the oral treatment, the open field test was performed. At 16 days, 1 hour after the oral treatment, the social interaction test was conducted. On the next two days, 1 hour after the oral treatment, a training and experimental session of the forced swim test were carried out.

The OFT is one of the experimental paradigms initially introduced to estimate the locomotor activity and willingness of animals to explore.^[20] The test allows measuring the behavior of an animal after it is released into an open, novel arena.^[21] The open field was a wooden arena (100×100×40 cm) painted white. The floor was divided with blue paint into 25 equal-size squares. The duration of the test session for each animal was 5 min. The measure for the horizontal activity was the number of squares crossed with four paws (crossings). The vertical activity was measured by the times the animal stood on its hind limbs (rearings).

The test consists in placing unfamiliar pairs of animals under conditions of bright light and unfamiliar arena, according to the method described by File and Hyde.^[22] The square arena of the open field apparatus (100×100×40 cm) was used. The partner animals were matched by weight (difference of no more than 10 g was allowed). The two rats were gently placed at two opposite corners of the arena. The duration of the test period was 5 minutes during which the following behaviors were recorded: sniffing, nipping, grooming, following, mounting, kicking, jumping on, and crawling over or under the partner (active interaction). Passive interactions like lying or sitting next to each other was not considered a sign of social contact. The prolonged involvement in active interactions indicated reduced anxiety.

The forced swim test, also known as the Porsolt test, was carried out in two sessions in two consecutive days.^[23] The training session took place 24 hours after the SIT and the test session took place exactly 24 hours after the training session. Because of its relative simplicity, the FST has become a widely used procedure in the screening of antidepressant drugs. FST measures coping behavior to an inescapable stress and allows the assessment of ‘behavioral responses’.^[24] According to the protocol, the animals were tested one by one in a transparent glass cylinder (17 cm in diameter and 60 cm in height). The cylinder was filled partially with water (22±1°C), so that there was some space left, but not allowing the animals to escape. Each animal was dropped down in water and its activity was documented for 5 min. The immobility time, which was the time during which the animal assumed an immobility posture with only minimal movements necessary to keep his head above the water, during the test session was recorded. The longer duration of the immobility time is related to increased behavioral despair while anti-depressant drugs decrease the immobility time.^[25]

The results obtained were expressed as mean ± SEM. The data were analyzed by one-way ANOVA, followed by Dunnett’s multiple comparison post hoc test. A level of p<0.05 was considered significant. All analyses were performed using GraphPad Prism statistical software.

The results from the OFT are presented in Fig. 1. The animals in the ICR group showed a statistically significant increase (p<0.05) in the locomotion (crossings 63.4±6.8, rearings 22.4±2.3) in comparison with the control group (crossings 39.7±6.25, rearings 12.9±1.9). The number of crossings and rearings of the ICR+CMFJ_2.5 and ICR+CM‌FJ₅ animals did not show a significant difference when compared to the controls or the ICR group. The crossings (29.8±5.2) and rearings (14.3± 2.2) of ICR+CMFJ₁₀ animals were significantly decreased (p<0.05) in comparison with the ICR results.

The social interaction time of ICR rats (31.8±4.0 sec) was slightly decreased compared to that of the control group (36.8±10.3 sec). Administration of CMFJ_2.5 produced an increase in the time spent in social interaction (52.98±2.14) when compared to the ICR group (p<0.01). The animals from the ICR+CMFJ₁₀ group also showed a significant increase (p<0.05) in the social interaction time (51.6±6.84) in comparison with the ICR animals. The social interaction time of the animals belonging to ICR+CMFJ5 group was not significantly different from that of the control and ICR rats (Fig. 2).

The immobility time of the ICR animals (115.6±11.4 sec) was slightly but not significantly increased when compared with the control (105.8±15.1 sec). The administration of CMFJ at doses of 2.5 and 10 ml/kg produced a significant reduction of the immobility time in comparison with the ICR group. The results obtained were 84.9±7.7 sec for the ICR+CMFJ_2.5 group (p<0.01 vs. ICR) and 73.8±11.7 SEC for the ICR+CMFJ₁₀ group, respectively (p<0.05). The administration of CMFJ at the dose of 5 ml/kg resulted in an immobility time of 100.3±18.9 sec which was not significantly different from that of the control and ICR rats (Fig. 3).