Behavioural studies on the ethanol leaf extract of Grewia carpinifolia in Wistar rats

Background: Grewia carpinifolia is a plant commonly used in the tropics to manage various central nervous system (CNS) disorders. However, despite its widespread use no scientific work has been reported to validate these claims. Objectives: To evaluate the activity of G. carpinifolia as it affects behaviour using animal model. Methods: Twenty five adult Wistar rats were randomly divided into five groups (A-E). Group A served as control (given only distilled water), Groups B,C, D and E were administered with single oral dose of ethanol extract of G. carpinifolia leaf at 100, 200, 400 and 800 mg/kg body weight respectively for twenty eight days consecutively. Subsequently, open field test, negative geotaxis and hanging wire test were performed. Body and brain weights were measured and histological examination of the brain was also performed. Results: At the tested doses, the extract significantly increased the time spent on the hanging wire and decreased locomotor activity at 800 mg/kg. No significant difference was observed in body and brain weights of extract treated groups when compared with the control. No visible histological lesion was also observed. Conclusion: The plant extract may improve muscular strength at tested doses and possess CNS depressant activity at 800 mg/ kg.


Introduction
Recently herbs and extract of plants have been extensively studied in search of new medicine beneficial in improving medical disorders related to the central nervous system (CNS) such as Parkinson's disease, neuromuscular weakness etc. These plants which are used in both herbal and conventional medicine offer benefits that pharmaceutical drugs lack and thus provide a safe alternative 1 . In spite of the huge benefits derived over the years from the use of these medicinal plants, substantial research has shown the risk involved in the application of some of these plants due to lack of proper dosing, method of preparation and duration of usage 2 . Therefore, scientific evaluation of these medicinal plants is not only important to the discovery of new drugs but also to assess toxicity associated with the use of these herbal preparations.
Grewia carpinifolia belongs to the family Tiliaceae. This genus comprising shrubs and trees is distributed in the warmer parts of the world. Nearly 40 species of this genus are found throughout the globe. Different parts of species of the genus Grewia are used as folk medicine in different part of the globe. Grewia carpinifolia has been reported to have antiparasitic and antioxidant activities 3 . Members of this genus are known to elicit various CNS activities. For example, Grewia bicolor used in the treatment of skin lesions is also used as tranquilizer 4 . Ethanolic extract of stem bark of Grewia elastic, Grewia tenax and G.tiliaefolia have been reported to possess CNS depres-sant activity. The aerial parts of G. umbellifera exhibited CNS depressant, also hypotensive and diuretic receptors in the brain have been isolated from an extract of Grewia villosa used in treatment of tuberculosis 5 . However, despite the widespread use of Grewia carpinifolia in the management of various ailments, an extensive literature search revealed that no scientific work has been reported to evaluate and provide information on the CNS activity of the plant as well as its effect on behaviour using animal models.

Materials and methods Plant material and authentication
Fresh leaves of Grewia carpinifolia were collected from the botanical garden of the University of Ibadan. It was identified and authenticated at the Forestry Research Institute of Nigeria (FRIN) where herbarium specimen (voucher number FHI 109693) was deposited.

Experimental animals
Adult Wistar rats of both sexes were purchased and housed at the Laboratory Animal House, Department of Veterinary Physiology, Biochemistry and Pharmacology, University of Ibadan. The animals were housed under standard conditions of temperature (25 ± 2°C), and light, (approximately 12/12 h light-dark cycle), fed on standard diet and given water ad libitum. All the animals were acclimatized to laboratory conditions for two weeks before the commencement of the experiment. All experiments performed on the laboratory animals in this study followed the Organization for Economic Cooperation and Development (OECD) approved Standard Operation Procedures 6 .

Extract preparation
The leaves were cleaned to remove adhering dirt, air-dried for eight weeks and crushed into coarse powder using a pestle and mortar. Extraction was carried out by cold maceration of 500 g of the coarse powder with 2.5L of 100% v/v ethanol for 72 h, with constant shaking using the GFL shaker (no. 3017GBh, Germany). The resultant mixture was filtered using Whatman filter paper (No.1) and the filtrate was concentrated to dryness in vacuo at 40°C using rotary evaporator to give a yield of 20% w/w of the extract. Aliquot portions of the extract were weighed and dissolved in distilled water for use in this study.

Preliminary phytochemical screening
Chemical tests were carried out for preliminary phytochemical screening of the leaf extract of Grewia carpinifolia using standard procedures to identify the constituents as described 7,8,9,10 .

Experimental design
Twentyfive rats of both sexes were divided into five groups of five rats each. Group A, received distilled water and served as the control while rats in groups B, C, D and E were given 100, 200, 400 and 800 mg/kg bw of ethanol leaf extract of Grewia carpinifolia respectively in a single oral dose daily for 28 days. All the rats had free access to food and water throughout the duration of the experiment and were observed daily for general symptoms of toxicity and mortality.

Body weight change
Rats in all the groups were weighed on the first day of the experiment and thereafter weekly during the period of extract administration and on the last day of the study. Doses of the extract administered were adjusted accordingly.

Behavioural tests
Twenty four hours after the administration of the last dose of the extract, the following behavioural tests were carried out; open field test, the forelimb support (hanging wire) test, and for negative geotaxis as described 11,12,13,14 .
Open-field test (OFT) • Each rat was placed in the centre of a square cage (120x120cm). The floor was divided into 20cm squares drawn in black ink. At the beginning of each test, every animal was introduced to the same spot (the centre square) of the arena and was allowed to explore the arena freely for 5 minutes. The following observations were recorded: • Locomotion, that is, number of times a rat crossed from one square to another entering with at least its two front paws • Number of rearing that is, number of times rat stood on its hind legs • Number of grooms that is, sets of heterogeneous constituents comprising face washing, body licking, paw licking, head and body shaking, scratching and genital licking. The open field box was washed with 30% alcohol solution before placing the subsequent animals in it in order to avoid possible biasing effect due to odour clues left by previous rats 11 .

Forelimb support (hanging wire)
The testing procedure was as previously described 13 and was used to assess the muscle strength and balance. Each rat was suspended with both forepaws on a horizontal steel wire 80 cm long, diameter 2 mm. The animal was held in a vertical position when its front paws were placed in contact with the wire. When the rat grasped the wire, it was released, and the latency to fall was recorded with a stopwatch. Rats were randomly tested and each animal was given three trials with a 30 min inter-trial rest interval 14 .

Negative geotaxis
Each rat was placed in the middle of a slab, 30 o inclined to the surface plane, in a head down position and latency to turn 180 o to a head up position was observed and recorded 15 .

Animal sacrifice
The rats were sacrificed 24 hours following the last day of extract administration and their brains were excised, the weight were taken and recorded. Brains were quickly transferred to sample bottles containing 10% formol calcium and fixed for two days and processed for Haematoxylin and Eosin (H&E) staining technique. The sections were mounted and examined with a light mi-croscope (Olympus, Japan). Image acquisition was done using cameroscope 5.5 connected to a computer interface and mounted on the microscope. Relative brain-body weight ratio: The relative brain-body weight ratio (RBW) of each rat was calculated as follows: RBW = Absolute brain weight (g) Body weight of rats on sacrifice day (g)

Statistical analysis
All data are presented as mean ± Standard Error of mean (SEM). Data was subjected to one-way ANOVA and subsequently to the Bonferroni post-test using the Graph pad Prism version 5 (Windows® Graphpad software) to perform multiple comparisons in order to assess statistical significance of differences between all possible pairs of groups. The level of significance was P ≤ 0.05.

Phytochemical screening
Chemical tests were carried for preliminary qualitative phytochemical screening of the leaves extract. Tannins, saponins, flavonoids and alkaloids were strongly present in the extract, phlobatinins, terpenoids, cardiac glycosides and anthraquinones were only present in trace amount, however coumarin was found to be absent (Table 1).

Effect of Grewia carpinifolia leaves ethanol extract on body weight of Wistar rats
There were increases in the mean body weight in all the groups throughout the experimental period. The final mean body weight at the beginning of the study and point of autopsy did not differ significantly (P>0.05) in Grewia carpinifolia treated rats when compared with control group (Figure 1).

Effect of sub-chronic administration of Grewia carpinifolia leaves ethanol extract on behaviour
In the Open field test (OFT) there was a significant (p≤0.05) decrease in the number of new squares crossings in the 800 mg/kg group when compared to control group (Table 2), no significant difference was however observed at other tested doses. There was also a statistically significant (p≤ 0.05) increase in the time spent on the hanging wire in all the treated groups when compared with the control. Numbers of rearing, grooming, and faecal boli while in the open field box were not statistically different in the control and test groups. Effect of Grewia carpinifolia leaves on relative brain weights of Wistar rats There was no significant difference (P>0.05) between the relative brain weights at all tested doses; they were similar in the control and Grewia carpinifolia treated rats (Table 3).

Histopathology
The result of the histopathology of rats treated with the ethanol extracts of Grewia carpinifolia leaves on the brain of rats is shown in Figure 2. No visible lesion was observed in the brain section of the control and treated groups.
African Health Sciences Vol 16 Issue 1, March 2016

Discussion
Body weight changes serve as a sensitive indication of the general health status of animals 16 . The results of this study showed that the administration of leaf extract of G. carpinifolia did not interfere with growth irrespective of dose. Although the effect of extract of G. carpinifolia on body weight has not been earlier reported, the present observation suggests that G. carpinifolia did not interfere with body weight.
According to Moore et al 17  Locomotor activity has been used to represent a broad class of sensory, motor and integrative processes 19 , Rang et al., 20 reported that locomotor activity is mediated through dopamine and other neurochemical pathways.
A wide variety of agents have the capacity to excite the function of the CNS, such that calming or drowsiness (sedation) is inhibited 21 . In addition, central nervous system (CNS) depressants are known to inhibit locomotor activity of animals 22 .
Furthermore, inhibition of acetylcholinesterase (AchE), CNS activation of cholinergic motor inhibitory system and damage to the peripheral muscle due to necrosis of skeletal muscle fibres has been reported to reduce locomotor activity in animals 23  This indicates that the plant extract did not induce anxiety in treated rats. The decreased motor activity obtained at 800 mg/kg in the present study may suggest that the ethanol extract of Grewia carpinifolia had some CNS depressant activities at this dose. This type of activity profile can be compared with opium alkaloid which possesses stimulant effect at low dose initially and marked sedative effect after cumulative effect or after high dose. The phytochemical constituents may be responsible for this activity 25 . Gamma-aminobutyric acid (GABA A ) is the major inhibitory neurotransmitter in the central nervous system. Different anxiolytic, muscle relaxant, sedative-hypnotic drugs acts through GABA A receptors, therefore it is possible that extracts of G.carpinifolia may act by potentiating GAB A ergic inhibition in the CNS via direct activation of GAB A receptor or indirectly by membrane hyperpolarization which led to a decrease in the firing rate of critical neurons in the brain 26 . Polyphenolic compounds, like flavonoids, tannins, saponins and phenolic acids, commonly found in plants have been reported to possess multiple biological effects 27 and are useful in many CNS disorders 28 .
Many flavonoids and neuroactive steroids were found to be ligands for the GABA A receptors in the central nervous system; which led to the assumption that they can act as benzodiazepine-like molecules 29 . The presence of alkaloids, flavonoids, saponins and tannins in the extract in this study might thus be responsible for its CNS depressant activity. This is in consonance with investigations on other species of the genus; Jaspers et al. 30 , Hyo et al. 31 Tijani et al 32 found that extract from Grewia bicolor, G umbellifera and G. lasiodiscus respectively possesses central depressing activities.
The length of time a rat was able to hold the hanging wire is considered as an indirect measure of grip, muscle strength and co-ordination 33 which was significantly increased (p<0.05) at all the tested doses. This may suggest an increase grip strength which could be due to increased motor coordination and muscle tone 34 that occurred following the sub chronic administration of Grewia carpinifolia.
Hypertrophy of an organ is an indication of toxicity of chemical or biological substance 35 . However, no hypertrophy of the brain was observed in this study amongst all the groups studied. In addition, the microscopic examination revealed that the cerebellum from the extract treated rats showed no alteration in cell structure when viewed under the light microscope using multiple magnification powers. The cerebellum plays a significant role in behaviour, movement control and as well as sensory perception 36 . Anxiety, stress, neurotoxic agents amongst other factors have been reported to cause loss or damage of Purkinje cells leading to cerebellar damage which consequently affects movement coordination and other cerebellum-dependent cognitive function 37 . The intact (lack of lesion) structural integrity of the cerebellum in this study further corroborate the result from the behavioural tests. This demonstrates the safety of the plant extract on cerebellar functions at tested dosages.

Conclusion
From the findings of this study we conclude that subchronic oral administration of ethanol leaf extract of Grewia carpinifolia appear to be safe at tested doses and may increase muscle strength, it may also possess CNS depressing activity at a dose of 800 mg/kg. Therefore, this plant merits further attention. It is suggested that further study be carried out to isolate the bioactive principle(s) responsible for the observed activities and an in-depth study to determine the mechanism by which G. carpinifolia may act on the CNS.