METHANOL ROOT BARK EXTRACT OF ACACIA SEYAL AMELIORATES CHRONIC UNPREDICTABLE MILD STRESS INDUCED DEPRESSION-LIKE BEHAVIOUR: INVOLVEMENT OF BDNF AND CORTISOL

206 Nigerian Journal of Pharmaceutical and Biomedical Research Vol. 8 Issue.3 December, 2024. p-ISSN: 2579-1419 e-ISSN: 2814-1423 METHANOL ROOT BARK EXTRACT OF ACACIA SEYAL AMELIORATES CHRONIC UNPREDICTABLE MILD STRESS INDUCED DEPRESSION-LIKE BEHAVIOUR: INVOLVEMENT OF BDNF AND CORTISOL 1*Gambo, A.Y., 2Shehu, A., 2Magaji, M.G. and 2Danjuma, N.M. 1Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Maiduguri, Borno State, Nigeria 2Department of Pharmacology and Therapeutics, Ahmadu Bello University, Nigeria * Corresponding author: Tel: +234-803-8056502; Email: [email protected] http://doi.org/10.55639/607.phar.10301.003 Abstract Acacia seyal Del. (Fabaceae) is reported to be used in the management of depressive illnesses. The present study was aimed at determining the effect of the methanol root bark extract of Acacia seyal (AS) on Chronic Unpredictable mild stress (CUMS) induced brain derived neurotrophic factor and cortisol alterations in mice. Acute toxicity study (LD50) was carried out using OECD guideline 425. The Irwins test was carried out as described by Irwin (1968). The CUMS model was used to induce depression and behavioural tests such as sucrose preference test (SPT), Open Field test (OFT) and tail suspension test (TST) were carried out in mice. The plasma concentrations of cortisol and Brain derived neurotrophic factor (BDNF) were assessed using enzyme-linked immunosorbent assay kits.The LD50 of AS was found to be ≥5000 mg/kg. On CUMS-induced depression, AS significantly (p<0.05) reversed the weight loss, increased the line-crossing activity in OFT and decreased duration of immobility in TST. The AS extract significantly (p<0.05) increased the levels of BDNF and decreased the levels of plasma cortisol in mice. The plant AS ameliorates CUMS-induced depressive like behaviour possibly mediated via the neurotrophic and hypothalamic-pituitary-adrenal pathways. Keywords: Acacia seyal, BDNF, Cortisol, Acute toxicity, CUMS Introduction Depression is a common mental disorder that represents an important health problem worldwide as it is a known cause of global disease burden and disability (Melchior et al., 2007). According to the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), the diagnosis of a major depression disorder requires the expression of five or more symptoms for at least two weeks. One of the symptoms should be either a depressed mood or anhedonia (loss of interest). The other secondary symptoms are weight or appetite changes, sleep abnormalities (hypo or hypersomnia), psychomotor impairment, fatigue or a lack of energy, a lack of concentration, feelings of excessive guilt, and suicidal ideation or attempts (DSM-5). Chronic exposure to multiple intermittent stressors can initiate cumulative physiological stress responses, called the “allostatic load” (Doan, 2021). The allostatic load is associated with many systemic and psychological diseases (Guidi et al., 2020). 207 Chronic stress is a psychosomatic process and a crucial risk factor for many diseases such as depression (McEwen et al., 2015; Shehu et al., 2019) and cardiovascular diseases (Kivimaki & Steptoe, 2017). The Chronic Unpredictable Mild Stress (CUMS) model is commonly used to study depression in rodents, which was first described by Willner et al., 1987. Rodents in this model are chronically exposed to constant environmentally and psychologically unpredictable mild stressors, resulting in the initiation of behavioural changes and potentially inducing depression (Wilner, 1997; Wilner et al., 1992). This model provides a realistic depression model as it mimics daily stressors in human life and induces anhedonia, which is the core symptom of depressive disorder as mentioned in the Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV) (Guze, 1995; Antoniuk et al., 2018). Traditional medicinal plants have received recognition and patronage especially in the treatment of mental and psychiatric illnesses (Magaji et al., 2008), with plants like Acacia seyal reported to be used in the management of depression in traditional medicine (Shehu et al., 2017). Acacia seyal belongs to the family of Fabaceae (Mimosoideae). It is a small to medium‐ sized tree and up to 17 m tall and 60 cm in diameter, it is widespread in the semi‐arid zone of tropical Africa and the Red Sea and from the Nile valley south to Zambia (Kisoi, 2016; Singh &Thakur, 2016). Several studies conducted in recent decades revealed that extracts from the bark of Acacia seyal have antibacterial action (Eldeen & Van, 2007), antimalarial effect (Muthaura et al., 2015), antimycobacterial effect, cyclooxygenase inhibition effect (Eldeen & Van, 2008), and anticancer activities (Saeed et al., 2015). The aim of this study is to evaluate the effect of the methanol root bark extract of Acacia seyal (AS) on Chronic Unpredictable mild stress (CUMS). Materials and Methods: Animals Swiss Albino mice of either sexes were obtained from the Animal House Facility of the Department of the Pharmacology and Therapeutics, Ahmadu Bello University Zaria. They were housed in propylene cages and kept under natural day and light cycle. The animals were fed on standard laboratory animal diet and water ad libitum. All experiments were conducted according to the Ahmadu Bello University Animal Ethics Committee. Drugs and Chemicals The followings are some of the chemicals that were used for the experiment. Fluoxetine (Mebidos Laboratories Pvt. Ltd, India), Diazepam (Roche, France), Methanol (Fluka-Aldrich) Plant Preparation and Extraction The whole plant Acacia seyal was collected in March 2018 from Sabon Gari Local Government Area of Kaduna State. It was taken to Herbarium Section of the Department of Botany, Ahmadu Bello University, Zaria where it was authenticated by Dr Namadi Sanusi. A voucher specimen (no. 900347) was deposited in the Herbarium of Department of Botany, Ahmadu Bello University, 208 Zaria. The root bark extract of Acacia seyal collected was dried and sized reduced using mortar and pestle. Two hundred grams of the dried powdered material was extracted with 1500 milliliters of methanol by cold maceration technique for 2 days with occasional shaking. The resultant extract was evaporated to dryness over a water bath which gave a brown solid mass. All solutions for administrations were freshly prepared using distilled water. Acute Toxicity Studies Median lethal dose (LD50) determination was conducted using Organization for Economic Co-operation and Development (OECD 425) guidelines in rats and mice. In this method, two groups each of three mice were fasted 3 hours prior to dosing. The fasted body weight was determined for each mouse and dose calculated according to the body weight. The extract AS was administered in a single oral dose using a cannula. A start dose of 5000 mg/kg was used for one mouse and observed for 48 hours. The mouse survived, and an additional two mice were dosed and all three mice were observed individually during the first 30 minutes after dosing, periodically during the first 24 hours, and then daily for 14 days. Mice were observed for tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. Time of onset of toxic symptoms and disappearance were also noted. Treatments The mice were divided into 6 groups of 9 mice each, Group 1 mice were NO CUMS + distilled water (10 ml/kg), group 2 CUMS + distilled water (10 ml/kg), Group 3 CUMS + fluoxetine (20 mg/kg). Mice in groups 4, 5 and 6 received CUMS + methanol root bark extract of Acacia seyal at dose of 250, 500 and 1000 mg/kg per orally respectively. Irwin’s test The method described by Irwin (1968) was used. Four groups of six mice each were used. Group I was given distilled water (10 ml/kg). Groups II, III and IV received 250, 500 and 1000 mg/kg of the methanol root bark extract of Acacia seyal orally, respectively. Mice were observed for 48 hours, with more observation within first 4 hours. Observations include morphological, autonomic, neurological and behavioural changes and body weights were taken daily for two weeks. Chronic Unpredictable Mild Stress The chronic mild stress was induced by chronic unpredictable mild stress as described by Murua et al. (1991). Fifty-four mice were divided into six groups of 9 animals each, matched and assigned to stress and control groups. After three days acclimatization, mice in stressed groups were exposed to the following stressors daily for 4 consecutive weeks. The order of stressors used is described in Table 1. 209 Table 1: Order of Stressors used to induce Chronic Unpredictable Mild Stress DAY FD WD WB 2HI CS CT CR EC TP MON + + + TUES + + + WED + + + THUR + + + FRI + + + SAT + + + SUN + + + FD: 24-h Food deprivation, WD: 24-h Water deprivation, WB: Wet bedding, 2HI: 2-hour immobilization, CS: Cold Swim, TP: Tail pinch, CT: Cage tilt at 45⁰C CR: Cage reduction, EC: Empty cage. The stressors were applied alternatively so that the mice will not anticipate the occurring stress. The unstressed groups of animals were housed in one cage with access to food and water without disturbance except for necessary procedure such as weighing and cage cleaning. On day 27 (60 min after drug administration), animals were subjected to different behavioral (TST and OFT). For biochemical investigations, mice were sacrificed by decapitation and brains were harvested. Brains samples were then stored in phosphate buffer (0.1 M, pH 7.4) at −80 °C till further investigations. Sucrose consumption test The test was performed as described by Forbes et al. (1996). The test was carried out at 0, 2 and 4 weeks of stress. Animals were deprived of food and water for 21 hours after which they were exposed to drinking water and 2% sucrose. Sucrose preference consumption was calculated using the formula: (Sucrose intake / (water intake + sucrose intake) x 100. Tail suspension test Mice were suspended on the edge of the shelf 58 cm above a table top by adhesive tape placed approximately 1cm from the tip of the tail. The duration of immobility was recorded for a period of 6 minutes. Mice are considered immobile if hung passively and completely motionless (Steru et al., 1985). The test was carried out at 0, 2 and 4 weeks of stressors. Open field test Each mouse was placed in white wooden open field apparatus (70×70×35 cm, 210 length×breadth×height) of which one wall is plexiglass. And also, a plexiglass floor divided into 16 visible squares (15×15 cm) with a central square. Behaviour of each mouse such as peripheral and central square crossing was recorded for 5 minutes. Arena was cleaned with 10% ethanol between tests (Rex et al., 1996). The test was carried out at 0, 2 and 4 weeks after stressors. Sample Collection Blood samples were collected in plain bottles which were centrifuged at 1,000 x g at 8 °C for 30 minutes. Blood plasma and erythrocytes were separated for cortisol competitive ELISA detection method. Brain was homogenized in PBS buffer (0.01M, PH7.4) and centrifuged at 5000 x g to obtain the supernatant. Brain homogenates were used for sandwich Enzyme Linked Immunosorbent Assay. Measurement of brain derived neurotropic factor (BDNF) level Brain derived neurotrophic Factor (BDNF) level was measured using commercially available enzyme linked immunosorbent assay kit (ELISA) kit (Wuhan Fine Biotech Co., Ltd., Catalogue No. EM0020) according to the manufacturer’s instructions. The plates were washed 2 times before adding standard, sample and control (Zero) wells. Then 100 µL standard or sample was added to each well and incubated for 90 minutes at 37 oC. After which the plates were aspirated and washed 2 times. Then 100 µL Biotin labelled antibody working solution was added to each well and incubated for 30 minutes at 37 oC. Then plates were aspirated and washed 3 times. Then 100 µL SABC working solution was added into each well and incubated for 30 minutes at 37 oC. Plates were then removed aspirated and washed 5 times then 90 µL TMB substrate and incubated lastly for 15 minutes at 37oC. Plates were removed and 50 µL stop solution was added to each well. Absorbance was measured immediately at 450 nm using a micro plate reader (RaytoRT-2100C). The sensitivity of the assay was <2.0 pg/ml of BDNF. Measurement of serum cortisol level Serum cortisol level was measured using a commercially available Enzyme Linked Immunosorbent Assay (ELISA) kit (Wuhan Fine Biotech Co. Ltd, Catalogue No. EM1721) according to the manufacturer’s protocol. Plates were washed twice before standard, sample and control (Zero) wells were added, then 50 µL sample and standard solutions were added to already precoated antibody plate provided with the kit. After which 50 µL Biotinlabelled antibody was added into each well then incubated for 45 minutes at 37oC. Plates were removed, aspirated and washed 3 times followed by addition of 100 µL SABC working solution into each well then incubated for 30 minutes at 37 oC. Plates were further washed and aspirated and washed 5 times followed by the addition of 90 µL TMB substrate. Then incubated for 15-20 minutes at 37 oC. The reaction was stopped by adding 50 µL of stop solution and absorbance was read at 450 nm immediately using a microplate reader (Rayto-RT-2100C). The sensitivity of the assay was <0.234 ng/ml of cortisol. Data Analysis Results are presented as means ±SEM and median scores on tables or figures where necessary. Data for TST and OFT were analyzed using One Way ANOVA (Analysis of Variance) while weight variation were 211 analyzed using Repeated Measure ANOVA. Bonferoni post hoc test was used to assess significant differences. Data for Irwin’s test were analyzed using Kruskal Wallis test followed by Dunn’s post hoc test. Results were considered significant at p≤0.05 Results The Acute Toxicity Study of Methanol Root Bark Extract of Acacia seyal The median lethal dose (LD50) was estimated to be ≥5000 mg/kg orally in mice. Effect of Methanol Root Bark Extract of Acacia seyal on Irwin’s test The methanol root bark extract of Acacia seyal impaired changes on locomotor activity (Table 2), motor coordination (Table 3) and diarrhea (Table 3). No death was observed at all tested doses after 48 hours of administration in all the groups. Table 2: Effect of the Methanol Root Bark Extract of Acacia seyal on Behavioural activity Behaviour Time (h) Treatment (mg/kg) Locomotor activity Grooming Pain Vocalisation 0 D/W 10 ml/kg 4 4 4 0 AS 250 mg/kg 4 4 4 0 AS 500 mg/kg 4 4 4 0 AS 1000 mg/kg 4 4 4 0 1 D/W 10 ml/kg 4 4 4 0 AS 250 mg/kg 3.2 4 4 0 AS 500 mg/kg 2.8 4 4 0 AS 1000 mg/kg 2.0* 4 4 0 2 D/W 10 ml/kg 4 4 4 0 AS 250 mg/kg 3.2 4 4 0 AS 500 mg/kg 2.6 4 4 0 AS 1000 mg/kg 2* 4 4 0 3 D/W 10 ml/kg 4 4 4 0 AS 250 mg/kg 2.8 4 4 0 AS 500 mg/kg 2.6 4 4 0 AS 1000 mg/kg 2.2* 4 4 0 4 D/W 10 ml/kg 4 4 4 0 AS 250 mg/kg 2.6 4 4 0 AS 500 mg/kg 2.0* 4 4 0 AS 1000 mg/kg 1.8** 4 4 0 Each column represents the median of 5 animals. Data was analysed using Kruskall walli’s test followed by Dunn’s post hoc test, *p≤ 0.05, **p≤ 0.001, significantly different from distilled water treated group.AS=Acacia seyal, DW= 10 ml/kg of Distilled water 212 Table 3: Effect of the Methanol Root Bark Extract of Acacia seyal on Neurological Changes CoordinationNeurological Time (h) Treatment (mg/kg) Grip strength Body tone Motor coordination Staggering gait Tremor Convulsion Twitches 0 D/W 10 ml/kg 4 4 4 4 0 0 0 AS 250 4 4 4 4 0 0 0 AS 500 4 4 3 4 0 0 0 AS 1000 4 4 2.5* 4 0 0 0 1 D/W 10 ml/kg 4 4 4 4 0 0 0 AS 250 4 4 4 4 0 0 0 AS 500 4 4 3.5 4 0 0 0 AS 1000 4 4 3.0* 4 0 0 0 2 D/W 10 ml/kg 4 4 4 4 0 0 0 AS 250 4 4 4 4 0 0 0 AS 500 4 4 3.8 4 0 0 0 AS 1000 4 4 3.2* 4 0 0 0 3 D/W 10 ml/kg 4 4 4 4 0 0 0 AS 250 4 4 4 4 0 0 0 AS 500 4 4 4 4 0 0 0 AS 1000 4 4 4 4 0 0 0 4 D/W 10 ml/kg 4 4 4 4 0 0 0 AS 250 4 4 4 4 0 0 0 AS 500 4 4 4 4 0 0 0 AS 1000 4 4 4 4 0 0 0 Each column represents the median of 5 animals. Data was analysed using Kruskall walli’s test followed by Dunn’s post hoc test, *p≤ 0.05, significantly different from distilled water treated group.AS=Acacia seyal, DW= 10 ml/kg of Distilled water Table 3: Effect of Methanol Root Bark Extract of Acacia seyal on Autonomic Changes Autonomic Changes and Death Time (h) Treatment (mg/kg) Diarrhoea (Secretion excitation) Skin Colour (General) Death 0 D/W 10 ml/kg 4 0 AS 250 4 0 AS 500 4 0 AS 1000 4 0 1 D/W 10 ml/kg 4 0 AS 250 + 4 0 AS 500 + 4 0 AS 1000 + 4 0 2 D/W 10 ml/kg 4 0 AS 250 + 4 0 AS 500 + 4 0 AS 1000 + 4 0 213 3 D/W 10 ml/kg 4 0 AS 250 mg/kg 4 0 AS 500 mg/kg 4 0 AS 1000 mg/kg 4 0 4 D/W 10 ml/kg 4 0 AS 250 mg/kg 4 0 AS 500 mg/kg 4 0 AS 1000 mg/kg 4 0 Each column represents the median of 5 animals. Data was analysed using Kruskall walli’s test followed by Dunns post hoc test AS=Acacia seyal, DW= 10 ml/kg of Distilled water Chronic Unpredictable Mild Stress Effect of Chronic Unpredictable Mild Stress on Body weight Changes in body weight in the stressed group were statistically significant (p<0.05) as compared to the unstressed group for the duration of 4 weeks. There was slight improvement in body weight in the last two weeks of treatment but was not statistically significant (Figure 1). 0 5 10 15 20 25 WEEK 0 WEEK 1 WEEK 2 WEEK 3 WEEK 4 Body Weight (g) Treatment (mg/kg) NOCUMS CUMS CUMS+AS250 CUMS+AS500 CUMS+AS1000 CUMS+FTX20 * ** * 214 Figure 1: Weekly Body Weight of Mice Following Chronic Unpredictable Mild Stress-Induced Depression in Mice Results were presented as mean±S.E.M. n = 5-9; Data was analysed using Repeated Measure ANOVA followed by Bonferroni post hoc test *p<0.05 compared to the unstressed group. CUMS= Chronic Unpredictable Mild Stress + distilled water (10 ml/kg, po), NOCUMS= No chronic unpredictable mild stress, AS= Acacia seyal (250, 500 & 1000 mg/kg, po), FTX= Fluoxetine (20 mg/kg, po) 3.3.2 Effect of Methanol Root Bark Extract of Acacia seyal on Sucrose Consumption in Mild Stress There was no significant difference in sucrose consumption across all groups before stress. However, after two weeks the stressed mice showed significant decrease in sucrose consumption when compared to unstressed mice. After two weeks of treatment, AS (250, 500 and 1000 mg/kg) and FTX (20 mg/kg) did not significantly increase sucrose consumption when compared with distilled water/CMS group (Figure 2). 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