Pharmacological Study of the Behavioural Effects of Chaenomeles Maulei in Experimental Animals

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Pharmacological Study of the Behavioural Effects of Chaenomeles Maulei in Experimental Animals Medical University of Varna Faculty of Pharmacy Department of pharmacology, toxicology and pharmacotherapy PHARMACOLOGICAL STUDY OF THE BEHAVIOURAL EFFECTS OF CHAENOMELES MAULEI IN EXPERIMENTAL ANIMALS Vesela Angelinova Borisova-Nenova, Pharm. D. Research supervisor: Prof. Stefka Valcheva-Kuzmanova, MD, PhD, DSc Varna 2020 1 I. Introduction According to a World Health Organization report, psychiatric disorders affect more than 264 million people worldwide and are a leading cause of disability for people of all ages. Depression is one of the most common psychiatric disorders, characterised by moderate or severe intensity and high mortality rates. The number of people suffering from neurological diseases is also growing worldwide, with more than 6 million people dying of stroke every year. There are over 45 million patients with dementia, and their number increases by 7.7 million each year. Alzheimer's disease is the most common cause of dementia and may contribute to about 70% of the cases. There is a significant correlation in morphological, molecular and biochemical mechanisms, which determine the clinical manifestation of psychiatric disorders and neurodegenerative diseases. All of these conditions are often accompanied by dysfunction of the corticosteroid system, abnormal activity of the monoamine system and the hypothalamus- pituitary axis, disorders of the neuroplasticity mechanisms and the presence of oxidative stress. The common pathogenetic mechanisms between neurodegenerative and psychiatric disorders may explain the high incidence of depression and anxiety in patients diagnosed with Alzheimer's disease or Parkinson's disease. Nowadays, the pharmacotherapeutic attention is focused on medicinal plants that might possess psychopharmacological effects and can therefore be used as an alternative to conventional drugs. The phytoproducts may also be beneficial in supplementing the primary pharmacological treatment. One of the top goals is to introduce a combination therapy of phytoproducts with drugs that would be associated with fewer side effects but preserved or improved clinical benefit. The literature data so far provide scarce information about the species Chaenomeles maulei. The main experimental studies of the properties and composition of the species concern only Chaenomeles Sinensis, Chaenomeles japonica and Chaenomeles speciosа. Chaenomeles maulei is classified as a synonymous species with Chaenomeles japonica. Based on the conducted research on the species of the Chaenomeles genus, some of the species’ properties have been revealed, including antioxidant, anti-inflammatory, and modulated noradrenergic, serotonergic and dopaminergic neurotransmission. These biologic activities may be valuable in the treatment of psychiatric and neurodegenerative disorders. 2 Currently, there is no literature data to prove the presence of unwanted or toxic effects of the species, regardless of their use – in the form of fruit extracts, fruit juice, essential oil, or individual components. On the contrary, the number of studies that demonstrate the organ- protective effects of the species is growing. After the chemical composition of the Chaenomeles maulei fruit juice was published and as the literature data on the other Chaenomeles species suggested, the fruit juice is expected to have beneficial effects on the anxiety and depressive-like behaviour of experimental animals. Although phytoproducts are perceived as a well-tolerated alternative to conventional therapy, they usually require some time for their clinical effects to manifest. It is therefore essential to assess the safety of the Chaenomeles maulei fruit juice. Several studies have shown that the main biological activity of the genus can be explained by the presence of polyphenols such as phenolic acids and flavonoids, vitamin C, polysaccharides, pectins and some organic acids. In Chaenomeles maulei fruit juice particularly high are the concentrations of phenolic acids such as vanillic and chlorogenic acids, flavonoids, and procyanidins, as well as vitamin C, which are responsible for its antioxidant activity. The results of the current experimental studies could help create medicinal products and/or food supplements with health-friendly properties. This includes phytoproducts that may support the primary therapy of diseases such as psychiatric and neurodegenerative disorders that still pose a challenge to modern medicine. II. Objectives and Tasks 1. Objective 3 This doctoral thesis aims to investigate the subchronic toxicity of Chaenomeles maulei fruit juice, as well as its psychopharmacological effects in young/healthy rats and experimental models of anxiety and depressive-like behaviour and impaired motor activity. 2. Tasks 1. Assessment of the effects of Chaenomeles maulei fruit juice after subchronic administration in young/healthy rats to: • motor activity and behaviour in the open field test; • anxiety in the elevated plus-maze and social interaction tests; • depressive-like behaviour in the forced swim test; • memory in the object recognition test. 2. Investigation of the effects of Chaenomeles maulei fruit juice on liver and kidney safety indices after subchronic administration. 3. Assessment of the effects of Chaenomeles maulei fruit juice in a model of reserpine- induced hypokinesia in rats to: • impaired motor activity in the open field test; • depressive-like behaviour in the forced swim test; • levels of oxidative stress in the brain. 4. Investigation of the effects of Chaenomeles maulei fruit juice in a model of impaired circadian rhythmicity in rats to: • locomotor activity; • social interaction; • depressive-like behaviour. III. Materials and methods 1. Experimental animals 4 The experiments were performed on young/healthy male Wistar rats (220±50 g), provided by the Vivarium of Varna Medical University. 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 had free access to food and drinking water. All procedures concerning animal treatment and experimentation were conducted in compliance with national and international laws and policies (2010/63/EU). 2. Chaenomeles maulei fruit juice Chaenomeles maulei fruit juice was produced from plants grown in the Balkan Mountains, Bulgaria, in the region of Troyan. After handpicking, fresh fruits were ground, crushed, and squeezed. The juice was filtered, preserved with potassium sorbate (1.0 g/l), and stored at 0°C until the experiments. The spectrophotometric Folin-Ciocalteu assay determined the total content of phenolic compounds. Absorbance was read at 760 nm. Gallic acid was used as a standard. The spectrophotometric assay showed a very high content of phenolic compounds – 8900.00 mg gallic acid equivalents per litre of juice. The high content of polyphenols was confirmed by an HPLC analysis that revealed the presence of phenolic acids and flavonoids. The phenolic acids were presented in the highest concentration by vanillic acid, caffeic acid and chlorogenic acid. The most abundant of the flavonoids were epicatechin, catechin and quercetin. 3. Experimental methods 3.1. Open field test (OFT) Open field test (OFT) OFT is a common measure of exploratory behaviour and general activity in rodents. It was performed for 5 min in an arena (100 × 100 × 40 cm) painted white except for 6 mm blue lines that divided the floor into 25 equal-size (20 × 20 cm) squares. Behaviours recorded were: number of crossings or horizontal activities (the number of lines crossed with the four paws), number of rearings or vertical activity (the number of times the animal stood on its hind limbs) and the time spent exploring the central region of the open field. Increased duration of central region exploration is considered an anxiolytic-like effect. 3.2. Social interaction test (SIT) 5 Rats were tested according to the method developed by Sandra and Hyde under conditions of high light, unfamiliar arena, and an unknown test partner to create a high level of anxiety. The two partners were matched by weight (difference of no more than 10 g). The square arena (100 × 100 × 40 cm) of the open field apparatus was used as a test box. The rats were gently placed at the opposite corners of the arena. The following behaviours were observed and scored during a 5 min session: sniffing, nipping, grooming, following, mounting, kicking, jumping on, and crawling under or over the partner. Passive contact (sitting or lying next to each other) was not considered as social interaction. The longer time for social contact showed a lower degree of anxiety. 3.3. Elevated plus maze (EPM) The experiments for the state of anxiety were carried out according to the method described by Pellow et al. The elevated plus maze consisted of two open arms (50 x 10 cm) facing each other and two closed arms (50 x 50 x 10 cm) with an open roof. The apparatus was elevated 50 cm above the floor and was illuminated by a 40 W bulb, positioned 50 cm above the apparatus. Each rat was then placed in the centre of the plus-maze facing one of the open arms. The following measurements were taken during the 5-min test period: • the number of entries into the open arms; • the time spent in the open arms; • the number of entries into the closed arms; • the time spent in the closed arms; • the total number of arm entries (the entries into the open arms and entries into the closed arms); • the ratio of the number of entries into the open arms/ total number of entries. 3.4. Object recognition test The test is based on discrimination between a familiar and a new object presented at a 1 h interval and is designed to measure working memory (18). The day before testing, rats were 6 submitted for 5 min to a habituation session in the apparatus, which was a wooden box (65 × 45 × 40 cm). The experimental session comprised of two trials. In the first trial (T1), one object (A) was placed near the rear wall of the box equidistant from the back corners. During the second trial (T2), a new object (B) was added. The objects were placed near the back corners. The object (A') presented during T2 was a duplicate of the sample presented in T1 (A).
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