ASSESSING ANXIETY AND EXPLORATORY BEHAVIOUR IN ALBINO RATS EXPOSED TO MIXED EFFECTS OF ENERGY DRINK WITH CAFFEINE

234 Nigerian Journal of Pharmaceutical and Biomedical Research Vol. 8 Issue.3 December, 2024. p-ISSN: 2579-1419 e-ISSN: 2814-1423 ASSESSING ANXIETY AND EXPLORATORY BEHAVIOUR IN ALBINO RATS EXPOSED TO MIXED EFFECTS OF ENERGY DRINK WITH CAFFEINE Yahaya Hassan A. Gandawa* Fatima S. Dibal, Uthman G. Sadiq, Zulaihat Surajudeen Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Maiduguri, Borno State, Nigeria. Corresponding Author Email: yahay[email protected] (07031514757). http://doi.org/10.55639/607.phar.10501.005 Abstract Energy drinks, which are highly caffeinated and sugary beverages have grown in popularity, especially among teenagers and young adults with the potential for high caffeine intake, which can lead to health issues. This study was carried out to evaluate the mixed effects of Energy drink (Predator® ) and Caffeine (King Café®) on Anxiety and Exploratory level in Albino Rats. Twenty-four Albino rats of both sexes, were divided into four groups (n=6). Group I served with distilled water, while groups II, III, and IV were served with a graded doses of mixed EDWC through oral gavage at the doses of 5 ml/kg (Low-dose), 7.5 ml/kg (Medium-dose) and 10 ml/kg (High-dose) respectively. Their anxiety and exploratory like behaviour were observed in Hole Board Test and Open Field Test. The findings of this study revealed that the mixture of EDWC lead to decreased anxiety and exploratory like behaviour in Albino Rats. Research on the behavioural effects of energy drinks mixed with Caffeine, particularly concerning anxiety and exploratory behaviour using Hole Board Test and Open Field Test, is limited. Key: Energy drink, Caffeine, Albino Rats, Anxiety, neuro-behaviour. Introduction Energy drinks (EDs) are soft drinks that are highly caffeinated, containing stimulant compound, first marketed as physical and mental performance in Europe and Asia in the 1960's (Finnegan, 2003). However, EDs compositions vary between different brands, but caffeine is the most common recipe (Gandawa et al., 2024), followed by guarana (a natural ingredients that contain caffeine), significant quantities of sugar and contain other sweeteners, ginseng, amino acids, B-Vitamins, and herbal extracts (Tarragon, 2023). More than 500 new EDs were launched worldwide in 2006 and over 31 different brands of EDs are sold in the open market in Nigeria (Saeed et al., 2017). Energy drinks are sold in numerous places (supermarket, kiosk, vendors and shops) and are easily accessed by children, adolescents, and young adults (Petit et al., 2012). The consumption of energy drinks and caffeine has become increasingly popular worldwide particularly among young individuals and college students. Energy drinks and coffee are commonly consumed for their stimulant effects, primarily to enhance alertness and combat fatigue (Gandawa et al., 2024: Arria et al., 2010). A great public health concern is related to the trend among young people 235 to consume alcohol-mixed energy drinks particularly in occasion of social events or dance partying. Energy drinks are also consumed by young people often being combined with illicit substances such as amphetamines and marijuana (Arria et al., 2010). EDs consumption has also been associated with serious health complains such as arrhythmias, tachycardia, strokes, psychotic symptoms/mania, seizures, and even death (Seifert et al., 2011). The caffeine content in EDs varies between 50 and 505 mg per can or bottle (Keaver et al., 2017). Caffeine is soluble in water and lipids, easily crosses the blood-brain barrier, and can be found in all body fluids including saliva and cerebrospinal fluid (Temple et al., 2017). Extremely large doses of caffeine can cause acute psychosis (Iyer et al., 2016). Fatalities from caffeine intoxication although rare continue to occur (Kerrigan et al., 2005, Mrvos et al., 2005). Manufacturers of EDs do not usually mention the caffeine content in the list of the ingredients. Thus, the actual caffeine dose in a single serving may exceed even when listed (Seifert et al., 2011). Aside from being added to beverages, caffeine is now being added to chocolates, food products such as potato chips, and bottled water, which confirms its growing popularity (Temple, 2009). Since the introduction of Red Bull in 1987, the energy drink market has grown extensively, with hundreds of different brands of varying caffeine content now available (Reissig et al., 2009: Gandawa et al., 2024). However, the combined effects of energy drinks and caffeine on behaviour, specifically anxiety levels and exploratory behaviour (Andrew and Gareth, 2018) have not been extensively investigated. Understanding the behavioural consequences of these commonly consumed beverages is crucial, as it may have implications for mental health and overall well-being. Materials and Methods Energy drinks (Predator®) and Caffeine (King Café®) The energy drinks were purchased from retailer shop at Maiduguri Monday market of Maiduguri Borno State. The Energy drink used for the experiment is Predator® (Gold Strike) a product of Monster Energy with batch number P2011 23AK3 and expiry date of 20/05/2025. A 400 ml of Predator energy drink contains: carbonated water, sucrose, acids (citric acid, tartaric acid), acidity regulator (sodium citrate), nature identical and artificial pineapple, and carbohydrate (56 g), of which sugars were 54 g, sodium (0.2 g), niacin (12.8 mg, 85 %), vitamin B6 (1.2mg, 92 %), and caffeine (120mg), flavour, inositol (1 mg/100 ml), taurine (100 mg/100 ml), and preservatives (potassium sorbate and sodium benzoate) and negligible fat, saturates, and proteins. The coffee (King Café®) was also purchased from the same shop with expiration date of 01/08/2025. King Café is a product of an India 100% instant coffee made from unique blend of Arabic and Robust. Animal Care and Treatments doses The animals (Swiss albino rats) weighing between 90-120 grams of both sexes, aged 12-16 weeks were procured from Faculty of Pharmaceutical Sciences animal house, Bayero University Kano (BUK), Kano State. The rats were kept in standard condition (12 hours` day cycle), in a transparent cage, in the laboratory of Pharmacology and Toxicology Faculty of 236 Pharmacy, University of Maiduguri. They were given free access to rat feed (TOPFEEDS®) and water daily for a period of fifteen (15) days under standard laboratory conditions of temperature (34±2°C) and optimum ventilation. After acclimatization period, a total of twentyfour (24) adult albino rats of both sexes were randomly selected and divided into four groups (6 per group). Control group: received distilled water (10 ml/kg), low dose, medium dose and high dose groups, were treated with mixed Energy drink with Caffeine (EDWC) orally by the aid of nasogastric tube for fifteen (15) days. Treatment and administration of EDWC A bottle of Predator which contains 400mL that is been taken by an average man weighing 70kg were considered as the low dose, two bottles as the medium dose and three bottles as the high dose (Matsko and Osborne, 2024). A caffeine dose of 20mg, 40mg and 60mg was used based on body weight (Tataru et al., 2016) which were dissolved in the energy drink. The dose of the ED administered was low dose (5 ml/kg), medium dose (7.5 ml/kg), and high dose of (10 ml/kg) respectively (Ferreira et al., 2013). Hole Board Test The hole-board test (HBT) is a widely used behavioural assay in preclinical research to evaluate anxiety and exploratory behaviour in rodents, particularly albino rats. This test is simple, reliable, and provides a balance between anxiety-related and exploratory parameters. The apparatus typically consists of a flat board with evenly spaced holes. Rats are placed on the board, and their interactions with the holes, such as head dips, are observed and recorded (Casarrubea et al., 2023). Each rat was placed at the centre of the apparatus and allowed to roam about. The number of holes each rat dipped its head within 5 minutes was counted and recorded for each day following a continuous daily administration of EDWC starting from the last day of training (Day 10) up to fifteen days. Open Field Test The open field test (OFT) is a widely used behavioural assay to evaluate anxiety and exploratory behaviour in rodents, including albino rats. The test involves placing a rat in a square or circular open arena with defined boundaries and observing its movement and interactions with the environment. The test relies on the natural conflict between a rat’s innate tendency to explore a novel environment and its fear of open, unprotected spaces. The OFT is frequently used to test the effects of anxiolytic and anxiogenic substances, providing insights into behavioural pharmacology. It is suitable for assessing both anxiety-related behaviours and general locomotor activity in a single test (Horka et al., 2024). Each rat was placed at the middle of a plain board with squares of various sizes drawn on it. The rat was allowed to roam about and the number of each square entered by the rat within 2 minutes was counted and recorded for each day following a continuous daily administration of EDWC starting from the last day of training (Day 10) up to fifteen days (Patil et al., 2009). Data Analysis 237 Data were collected and statistically analysed using Statistical Package for the Social Sciences (SPSS) version 20. Categorical data were expressed as Mean and Standard Deviation (M and SD), Repeated Measure analysis of variance (RM-ANOVA) was applied for mean comparison of quantitative variables followed by Post hoc Tukey test for multiple comparisons of these groups. The statistical significance was set at a pvalue of equal or less than 0.05 was considered. Ethical approval Approval was sought and received from National Health Research Ethics Committee (NHREC) to conduct this research(NHREC/01/01/200722/01/2021) Results Table 1: The OFT analysis revealed significant differences in locomotors activity between the control group and the treated groups across various days. On Day 3, the low-dose (L/Dose) group displayed significantly reduced activity compared to the control group (t = -2.93, p = 0.015). Similarly, on Day 4, the highdose (H/Dose) group exhibited a significantly lower number of squares entered (t = -4.56, p = 0.001), and on Day 6, the medium-dose (M/Dose) group showed a comparable reduction in activity (t = -3.36, p = 0.008). Conversely, the H/Dose group demonstrated increased activity on Day 7 (t = 2.49, p = 0.032) and Day 10 (t = 2.62, p = 0.034). The L/Dose group also displayed elevated activity on Day 11 (t = 3.36, p = 0.007), while the M/Dose group exhibited a similar increase on Day 14 (t = 2.52, p = 0.032). However, on Day 15, the H/Dose group once again showed significantly reduced activity compared to the control group (t = 4.21, p = 0.003). These findings highlight doseand time-dependent effects of the treatment on locomotors behaviour. Table 2: At baseline, the low-dose (L/Dose) group exhibited significantly higher defecation rates compared to the control group (p < 0.05), while the medium-dose (M/Dose) and high-dose (H/Dose) groups showed no significant differences. On Day 5 and Day 7, the L/Dose group maintained a significantly higher defecation rate than the control (p < 0.05). However, by Day 13, both the M/Dose and H/Dose groups showed significantly lower defecation rates compared to the control (p < 0.05), a trend that persisted on Day 14. By Day 15, all treated groups (L/Dose, M/Dose, and H/Dose) demonstrated significantly reduced defecation rates compared to the control group (p < 0.05), indicating a doseand time-dependent inhibitory effect on bowel activity. Table 3: The high-dose (H/Dose) group exhibited significantly elevated urination rates from the outset, starting at 25.2 ± 1.5 compared to the control (4.2 ± 0.9), reflecting a strong diuretic effect of the energy drink. On Day 1, the H/Dose group showed a dramatic increase (32 ± 0.8), far exceeding the control, while the medium-dose (M/Dose) and low-dose (L/Dose) groups also exhibited higher urination rates (7 ± 1 and 5.2 ± 1.2, respectively). From Days 2 to 5, the H/Dose group experienced a gradual decline in urination, though it remained substantially elevated, suggesting an adaptive physiological response. Between Days 6 and 10, urination in the H/Dose 238 group remained consistently high, peaking on Day 9 (23.4 ± 0.7), while the L/Dose and M/Dose groups showed rates closer to the control, indicating reduced diuretic effects at lower doses. By Day 15, the H/Dose group’s urination decreased significantly to 9.6 ± 0.2 but remained higher than the control, suggesting renal adaptation. Meanwhile, the L/Dose and M/Dose groups exhibited minimal urination, comparable to the control, indicating no prolonged diuretic impact at these doses. Table 4: At baseline, the high-dose (H/Dose) group exhibited significantly higher hole-poking behaviour (25.2 ± 1.5) compared to the control group (4.2 ± 0.9), indicating heightened exploratory activity, while the medium-dose (M/Dose) and low-dose (L/Dose) groups showed moderate increases (6.8 ± 1.02 and 5.2 ± 0.9, respectively). On Day 1, the H/Dose group demonstrated a dramatic increase in hole poking (32 ± 0.8), far exceeding the control and other treated groups, reflecting significant hyperactivity or arousal. The M/Dose and L/Dose groups also showed elevated activity but to a lesser extent. From Days 2 to 5, the H/Dose group’s activity gradually declined but remained significantly higher than the control, while the M/Dose and L/Dose groups approached control levels. During Days 6 to 10, the H/Dose group maintained elevated exploratory behaviour, peaking on Day 9 (23.4 ± 0.7), whereas the M/Dose and L/Dose groups exhibited behaviour closer to the control, indicating diminished effects at lower doses. By Day 15, the H/Dose group showed a marked reduction in activity (9.6 ± 0.2) but remained above the control (1.4 ± 0.5), while the M/Dose and L/Dose groups exhibited minimal exploratory behaviour, comparable to or below control levels, suggesting negligible sustained effects at these doses. Table 1 showing the number of squares entered by the rats following daily administration of EDWC on the Open field test apparatus. Control L/Dose M/Dose H/Dose Baseline 26±1.7 20.4±2.3 24.6±1.7 25.2±1.4 Day 1 38.4±2.9 37.8±1.7 41.6±1.3 38±2.3 Day 2 18.4±3.2 24.4±4.2 17±4.3 32±4.1 Day 3 10.8±3 14.4±4.1 25.2±9.2 26.6±2.4 Day 4 14.6±2.2 16.6±4 26.4±4.3 20.8±4.5 Day 5 23.5±3 28.8±4.5 28.8±4 20.6±2.4 Day 6 24.8±6 22.2±2.1 20.8±2 18.4±4 Day 7 21.6±2.2 20.4±4 17.6±2.5 18.8±3.4 Day 8 22.8±2.3 18.4±3 19.2±4 16±2 Day 9 23.4±3 30.8±5.2 19.6±3 17.2±2 Day 10 23.4±2.04 20±2.3 21.4±3.4 23.4±2 .1 Day 11 20.20±3 21.1±3.4 16.2±3 20.8±2.2 Day 12 21.2±3 18±3.3 17.8±2.3 20.4±2 Day 13 18.4±1.03 18±2 16±1.03 19.4±2 239 Day 14 19.8±3 19.6±1.03 16±2 18.4±2.4 Day 15 20.2±1.2 20±3 16.6±3 14.2±2.3 Fig 1: Assessment of the number of squares entered by the rats in the Open Field test Table 2 showing the number of defecations by the rats following daily administration of predator for 15days Control L/Dose M/Dose H/Dose Baseline -2.8±0.4 3.6±0.4 2.4±1 2.2±1 Day 1 2.6±0.8 1.4±0.5 2.6±0.4 5.6±1.5 Day 2 3.2±0.9 2.8±0.9 2±0.7 2.4±0.7 Day 3 1.8±0.8 1.8±0.7 2.4±0.2 2±0.5 Day 4 2.2±0.6 1.2±0.8 2.4±0.9 3±1 Day 5 2.6±0.8 4.8±1.9 3.6±1.03 2±0.7 Day 6 3±0.6 2.4±0.7 1.8±0.8 2±0.7 Day 7 1.8±0.6 4.4±1.3 1±0.7 2.2±0.6 Day 8 3±1 2.6±0.7 1±0.7 2.2±0.4 Day 9 2.4±0.5 3.2±0.4 2.2±0.7 3±07 Day 10 2±0.6 2.4±0.7 2±0.6 2.2±0.5 Day 11 2.2±0.8 2.4±0.7 2.2±0.7 1.4±0.6 Day 12 2.4±1.3 2±0.7 1.6±0.5 1.2±0.6 Day 13 3.6±0.7 2.4±1 1±0.4 0.6±0.4 Day 14 2.6±0.8 1.8±0.8 1±0.5 0.6±0.4 Day 15 1.4±0.8 1±0.4 0.8±0.4 0.6±0.4 0 20 40 60 80 100 120 140 160 Day 1Day 2Day 3Day 4Day 5Day 6Day 7Day 8Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Control Low dose Medium dose High dose 240 Table 3 showing the number of urinations per day following daily administration of predator energy drink for 15 days Control L/Dose M/Dose H/Dose Baseline 4.2±0.9 5.2±0.9 6.8±1.02 25.2±1.5 Day 1 2.6±0.5 5.2±1.2 7±1 32±0.8 Day 2 5.2±0.7 4.2±1.5 15.2±9.2 26.6±5 Day 3 5±1.7 1.4±0.7 7.4±0.4 20.8±1 Day 4 3.2±2 3.2±2 4.61.3 20.6±2.3 Day 5 1.8±0.7 3.2±0.9 3.4±1.03 18.4±0.3 Day 6 3.8±0.7 3.4±1.2 3.2±0.9 18.8±1.2 Day 7 3.6±1.2 1.8±0.4 3.6±0.8 16±0.6 Day 8 2±0.7 2.6±1.07 1±0.7 17.2±1 Day 9 3±1 3.2±0.5 3.2±0.9 23.4±0.7 Day 10 2±0.7 1.8±0.4 1.4±0.5 20.8±0.7 Day 11 2.4±0.5 1.8±0.7 1.4±0.6 20.4±0.5 Day 12 2.2±0.6 1.4±0.5 1.2±0.5 19.4±0.2 Day 13 1±0.4 1.2±0.4 1±0.4 18.4±0.6 Day 14 1.4±0.5 0.6±0.4 1±0.42 14.2±0.3 Day 15 1.4±0.5 1±0.3 1.8±0.4 9.6±0.2 Table 4 showing the number of the hole poking by the rats following daily administration of predator on the Hole board test apparatus for 15 days Control L/Dose M/Dose H/Dose Baseline 4.2±0.9 5.2±0.9 6.8±1.02 25.2±1.5 Day 1 2.6±0.5 5.2±1.2 7±1 32±0.8 Day 2 5.2±0.7 4.2±1.5 15.2±9.2 26.6±5 Day 3 5±1.7 1.4±0.7 7.4±0.4 20.8±1 Day 4 3.2±2 3.2±2 4.61.3 20.6±2.3 Day 5 1.8±0.7 3.2±0.9 3.4±1.03 18.4±0.3 Day 6 3.8±0.7 3.4±1.2 3.2±0.9 18.8±1.2 Day 7 3.6±1.2 1.8±0.4 3.6±0.8 16±0.6 Day 8 2±0.7 2.6±1.07 1±0.7 17.2±1 Day 9 3±1 3.2±0.5 3.2±0.9 23.4±0.7 Day 10 2±0.7 1.8±0.4 1.4±0.5 20.8±0.7 Day 11 2.4±0.5 1.8±0.7 1.4±0.6 20.4±0.5 Day 12 2.2±0.6 1.4±0. 1.2±0.5 19.4±0.2 Day 13 1±0.4 1.2±0.4 1±0.4 18.4±0.6 Day 14 1.4±0.5 0.6±0.4 1±0.42 14.2±0.3 Day 15 1.4±0.5 1±0.3 1.8±0.4 9.6±0.2 241 Fig 2: Assessment of hole poking by the rats on the Hole Board apparatus Discussion Anxiety is an unpleasant state of inner turmoil, mostly associated with nervous behaviour, such as forth and pacing back, somatic complaints and rumination (Saeed et al., 2017). From the above result of Open Field Test (OFT), the initial reduction in locomotor activity across different doses at varying times by the EDWC suggests an anxiogenic effect, consistent with caffeine's known properties of inducing stress-like responses at certain doses (Bhattacharya et al., 1997: Hughes and Hancock, 2017). Conversely, the subsequent increase in activity observed later in all groups reflects a stimulatory phase, potentially associated with reduced anxiety (Hilbert et al., 2014: Hughes 2013). The biphasic nature of these effects emphasises caffeine's dual role (Mendaros, 2018), inducing stress at the onset and promoting stimulation or habituation with prolonged exposure. In this study the high-dose group displayed signs of fatigue or a rebound anxiogenic effect by last day, indicating potential overstimulation at high doses (Lu et al., 2024). This emphasizes the importance of dosage and time in modulating caffeine's behavioural effects, highlighting its complex interaction with anxiety and exploration (Alasmari 2020). The defecation patterns from the current study revealed increased defecation in the low-dose group early in the study points to increased anxiety affecting exploratory behaviour. Increased defecation is a welldocumented physiological marker of anxiety in rodents (Hughes and Hancock, 2019). Reduced defecation in the mediumand high-dose groups later in the study may signify habituation to the stimulant or anxiolytic effects (Kang and Yan, 2024). The transition from anxiogenic responses at low doses to possible habituation or reduced anxiety at higher doses reflects a doseand timedependent behavioural modulation, supporting caffeine's complex and context-dependent effects on anxiety (Pechlivanova et al., 2010). 0 10 20 30 40 50 60 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Number of hole poking Number of days Control Low Dose Medium Dose High Dose 242 Elevated urination in the high-dose group, persisting early in the study, reflects acute stress and increased anxiety, likely driven by increased sympathetic activity. This correlates with reduced exploratory behaviour, as increased stress can inhibit exploration (Spritzler, 2023). The normalization of urination in the mediumand low-dose groups over time indicates minimal sustained anxiety at these doses (Staack et al., 2022). The decline in urination by last day in the high dose group suggests decrease in acute stress responses over time, further supporting the biphasic effects of caffeine (Pechlivanova et al., 2010). From the result of Hole Board Test (HBT), there is progressive reduction in hole poking, especially under high dose, might reflect increased anxiety, reduced exploratory drive, or habituation to the effects of EDWC. Low and Medium Dose groups display intermediate activity levels, with less fluctuation than the High Dose group but a similar overall reduction trend (Yacoubi et al., 2000). The data suggest a dose-dependent suppression of exploratory behaviour, with the High Dose group showing initial hyperactivity followed by a significant decline. This pattern aligns with heightened anxiety or stress-induced behavioral changes, which reduce exploration as the exposure continues (Hilakivi et al., 1989). From this study it is observed that administration of caffeine in high doses has shown to heightened anxiogenic behaviour in animals. There is also evidence that caffeine in lower doses has anxiolytic effect in humans (Hughes and Hancock, 2017). Caffeine is known to act as a nonselective receptor antagonist on adenosine receptors, and thereby block the actions of endogenous adenosine (Snyder et al., 1981). This is probably the reason why caffeine promote anxiety – like behaviour (Shen and Chen, 2009). The combined results across all tests highlight the complex interaction between EDWC, anxiety, and exploratory behaviour: Caffeine's effects shift from anxiogenic at low doses or early exposure to stimulatory and potentially anxiolytic at higher doses or with habituation. The findings suggest that while EDWC can modulate anxiety and exploration, the outcomes are highly dependent on dose and duration of exposure. Excessive doses may lead to overstimulation and fatigue, while lower doses may have transient anxiogenic effects without sustained behavioural disruption. Further Studies on EDWC Effects should focus on examining the structural and cellular changes in brain regions associated with anxiety and exploration, such as the hippocampus, amygdala, and prefrontal cortex, following EDWC exposure. Also, the potential neurodegenerative or neuroprotective effects of prolonged EDWC use, focusing on dose-related impacts. Then, analyse the neurochemical pathways influenced by EDWC, particularly changes in neurotransmitters like dopamine, serotonin, and GABA, to elucidate the mechanisms underlying its anxiogenic and stimulatory effects. Studies combining behavioural, histological, and biochemical analyses to provide a comprehensive understanding of EDWC's effects on anxiety and exploratory behaviour should be given attention.