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

Olamide E. Adebiyi1, Funmilayo E. Olopade2, James O. Olopade3, Funsho O. Olayemi1

1. Department of Veterinary Physiology, Biochemistry & Pharmacology, Faculty of Veterinary Medicine,

University of Ibadan, Ibadan, Nigeria.
2. Department of Anatomy, College of Medicine, University of Ibadan, Ibadan, Nigeria.
3. Department of Veterinary Anatomy, Faculty of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria.

Abstract
Background: Grewia carpinifolia is a plant commonly used in the tropics to manage various central nervous system (CNS) disor-
ders. 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 ac-
tivity 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.
Keywords: Grewia carpinifolia, negative geotaxis test, locomotor activity, Wistar rats.
DOI: http://dx.doi.org/10.4314/ahs.v16i1.45
Cite as: Adebiyi OE, Olopade FE, Olopade JO, Olayemi FO. Behavioural studies on the ethanol leaf extract of Grewia carpinifolia in Wistar
rats. Afri Health Sci. 2016;16(1): 339-346. http://dx.doi.org/10.4314/ahs.v16i1.45

Introduction the use of these medicinal plants, substantial research

Recently herbs and extract of plants have been exten- has shown the risk involved in the application of some
sively studied in search of new medicine beneficial in im- of these plants due to lack of proper dosing, method of
proving medical disorders related to the central nervous preparation and duration of usage2. Therefore, scientific
system (CNS) such as Parkinson's disease, neuromuscular evaluation of these medicinal plants is not only important
weakness etc. These plants which are used in both herbal to the discovery of new drugs but also to assess toxicity
and conventional medicine offer benefits that pharma- associated with the use of these herbal preparations.
ceutical drugs lack and thus provide a safe alternative1.
In spite of the huge benefits derived over the years from 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 ge-
nus are found throughout the globe. Different parts of
Corresponding author: species of the genus Grewia are used as folk medicine in
Olamide E. Adebiyi, different part of the globe. Grewia carpinifolia has been
Department of Veterinary Physiology, reported to have antiparasitic and antioxidant activities3.
Biochemistry & Pharmacology, Members of this genus are known to elicit various CNS
Faculty of Veterinary Medicine, activities. For example, Grewia bicolor used in the treat-
University of Ibadan, Ibadan, Nigeria ment of skin lesions is also used as tranquilizer4. Ethano-
Phone: +2348035624918 lic extract of stem bark of Grewia elastic, Grewia tenax and
E-mail: [email protected] G.tiliaefolia have been reported to possess CNS depres-

African Health Sciences Vol 16 Issue 1, March 2016 339

sant activity. The aerial parts of G. umbellifera exhibited Preliminary phytochemical screening
CNS depressant, also hypotensive and diuretic receptors Chemical tests were carried out for preliminary phyto-
in the brain have been isolated from an extract of Grewia chemical screening of the leaf extract of Grewia carpinifo-
villosa used in treatment of tuberculosis5. lia using standard procedures to identify the constituents
However, despite the widespread use of Grewia carpinifo- as described7,8,9,10.
lia in the management of various ailments, an extensive
literature search revealed that no scientific work has been Experimental design
reported to evaluate and provide information on the CNS Twentyfive rats of both sexes were divided into five
activity of the plant as well as its effect on behaviour us-groups of five rats each. Group A, received distilled water
ing animal models. 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
Materials and methods leaf extract of Grewia carpinifolia respectively in a single
Plant material and authentication oral dose daily for 28 days. All the rats had free access to
Fresh leaves of Grewia carpinifolia were collected from the food and water throughout the duration of the experi-
botanical garden of the University of Ibadan. It was iden- ment and were observed daily for general symptoms of
tified and authenticated at the Forestry Research Institute toxicity and mortality.
of Nigeria (FRIN) where herbarium specimen (voucher
number FHI 109693) was deposited. Body weight change
Rats in all the groups were weighed on the first day of
Experimental animals the experiment and thereafter weekly during the period
Adult Wistar rats of both sexes were purchased and of extract administration and on the last day of the study.
housed at the Laboratory Animal House, Department of Doses of the extract administered were adjusted accord-
Veterinary Physiology, Biochemistry and Pharmacology, ingly.
University of Ibadan. The animals were housed under
standard conditions of temperature (25 ± 2°C), and light, Behavioural tests
(approximately 12/12 h light-dark cycle), fed on standard Twenty four hours after the administration of the last
diet and given water ad libitum. All the animals were ac- dose of the extract, the following behavioural tests were
climatized to laboratory conditions for two weeks before carried out; open field test, the forelimb support (hanging
the commencement of the experiment. All experiments wire) test, and for negative geotaxis as described11, 12,13, 14.
performed on the laboratory animals in this study fol- Open-field test (OFT)
lowed the Organization for Economic Cooperation and • Each rat was placed in the centre of a square cage
Development (OECD) approved Standard Operation (120x120cm). The floor was divided into 20cm squares
Procedures6. drawn in black ink. At the beginning of each test, ev-
ery animal was introduced to the same spot (the centre
Extract preparation square) of the arena and was allowed to explore the arena
The leaves were cleaned to remove adhering dirt, air-dried freely for 5 minutes. The following observations were re-
for eight weeks and crushed into coarse powder using corded:
a pestle and mortar. Extraction was carried out by cold • Locomotion, that is, number of times a rat crossed
maceration of 500 g of the coarse powder with 2.5L of from one square to another entering with at least its two
100% v/v ethanol for 72 h, with constant shaking using front paws
the GFL shaker (no. 3017GBh, Germany). • Number of rearing that is, number of times rat stood
The resultant mixture was filtered using Whatman filter on its hind legs
paper (No.1) and the filtrate was concentrated to dryness • Number of grooms that is, sets of heterogeneous con-
in vacuo at 40°C using rotary evaporator to give a yield of stituents comprising face washing, body licking, paw lick-
20% w/w of the extract. Aliquot portions of the extract ing, head and body shaking, scratching and genital licking.
were weighed and dissolved in distilled water for use in The open field box was washed with 30% alcohol solu-
this study. tion before placing the subsequent animals in it in order

340 African Health Sciences Vol 16 Issue 1, March 2016

to avoid possible biasing effect due to odour clues left by croscope (Olympus, Japan). Image acquisition was done
previous rats11. using cameroscope 5.5 connected to a computer interface
and mounted on the microscope.
Forelimb support (hanging wire)
The testing procedure was as previously described13 and Relative brain-body weight ratio: The relative brain-body
was used to assess the muscle strength and balance. Each weight ratio (RBW) of each rat was calculated as follows:
rat was suspended with both forepaws on a horizontal
steel wire 80 cm long, diameter 2 mm. The animal was RBW = Absolute brain weight (g)
held in a vertical position when its front paws were placed
in contact with the wire. When the rat grasped the wire, Body weight of rats on sacrifice day (g)
it was released, and the latency to fall was recorded with
a stopwatch. Rats were randomly tested and each animal Statistical analysis
was given three trials with a 30 min inter-trial rest inter- All data are presented as mean ± Standard Error of mean
val14. (SEM). Data was subjected to one-way ANOVA and sub-
sequently to the Bonferroni post-test using the Graph
Negative geotaxis pad Prism version 5 (Windows® Graphpad software) to
Each rat was placed in the middle of a slab, 30 inclined perform multiple comparisons in order to assess statisti-
o

to the surface plane, in a head down position and latency cal significance of differences between all possible pairs
to turn 180o to a head up position was observed and re- of groups. The level of significance was P ≤ 0.05.
corded15.
Results
Animal sacrifice Phytochemical screening
The rats were sacrificed 24 hours following the last day Chemical tests were carried for preliminary qualitative
of extract administration and their brains were excised, phytochemical screening of the leaves extract. Tannins,
the weight were taken and recorded. Brains were quickly saponins, flavonoids and alkaloids were strongly present
transferred to sample bottles containing 10% formol cal- in the extract, phlobatinins, terpenoids, cardiac glycosides
cium and fixed for two days and processed for Haema- and anthraquinones were only present in trace amount,
toxylin and Eosin (H&E) staining technique. however coumarin was found to be absent (Table 1).
The sections were mounted and examined with a light mi-

Table 1 Qualitative phytochemical analysis of ethanol leaves

extract of Grewia carpinifolia.

Phytochemical Qualitative
Tannins ++
Phlobatinins +
Saponins ++
Flavonoids ++
Terpenoids +
Cardiac glycosides +
Coumarin -
Alkaloids ++
Anthraquinones +
++ = strongly positive + = trace - = not detected

African Health Sciences Vol 16 Issue 1, March 2016 341

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

Effect of sub-chronic administration of Grewia car- observed at other tested doses. There was also a statisti-
pinifolia leaves ethanol extract on behaviour cally significant (p≤ 0.05) increase in the time spent on
In the Open field test (OFT) there was a significant the hanging wire in all the treated groups when compared
(p≤0.05) decrease in the number of new squares cross- with the control. Numbers of rearing, grooming, and fae-
ings in the 800 mg/kg group when compared to control cal boli while in the open field box were not statistically
group (Table 2), no significant difference was however different in the control and test groups.

Table 2: Mean values for the behavioural tests on day 28 following administration
of Grewia carpinifolia leaves ethanol extract

Control 100 mg/kg 200 mg/kg 400 mg/kg 800 mg/kg

Rearings 20.60±2.30 30.80±1.04 26.7±1.90 31.78±1.90 19.30±2.30
Groomings 10.10±1.40 9.20±1.08 6.40±2.08 10.03±1.72 9.97±1.48
Number of new 25.40 ± 4.99a 18.80 ± 5.56 18.00 ± 6.43 17.60 ± 5.38 13.75 ±
b
squares 2.66
Faecal bolus 3.00 ± 1.14 5.00 ± 1.45 4.67 ± 2.91 4.00 ± 1.27 3.75 ± 1.38
n=5; Mean ± S.E.M (Standard Error of Mean); Means with different superscripts within rows are significantly
different at p<0.05
342 African Health Sciences Vol 16 Issue 1, March 2016
Effect of Grewia carpinifolia leaves on relative brain relative brain weights at all tested doses; they were similar
weights of Wistar rats in the control and Grewia carpinifolia treated rats (Table 3).
There was no significant difference (P>0.05) between the

Table 3: Effect of Grewia carpinifolia leaves on relative brain weights of

Wistar rats treated for 28days

Relative brain weights (x10-3)

Control 7.60 ± 0.08
100 mg/kg 7.72 ± 0.08
200 mg/kg 7.96 ± 0.02
400 mg/kg 7.85 ± 0.05
800 mg/kg 7.88 ± 0.02
n=5; Mean ± S.E.M (Standard Error of Mean)

Histopathology of rats is shown in Figure 2. No visible lesion was ob-

The result of the histopathology of rats treated with the served in the brain section of the control and treated
ethanol extracts of Grewia carpinifolia leaves on the brain groups.

A B

C D

Figure 2 The brain section (Purkinje cells of the cerebellum) of rat (H&E stain x100 magnification).
A: the control group showing no visible lesion
B: The brain section (Purkinje cells of the cerebellum) of rat in the group B administered with 100 mg/kg of etha-
nol leaves extract of Grewia carpinifolia showing no visible lesion (H&E stain x100 magnification).
C: The brain section (Purkinje cells of the cerebellum) of rat in the group C administered with 200 mg/kg of etha-
nol leaves extract of Grewia carpinifolia showing no visible lesion (H&E stain x100 magnification).
D: The brain section (Purkinje cells of the cerebellum) of rat in the group E administered with 800 mg/kg of etha-
nol leaves extract of Grewia carpinifolia showing no visible lesion (H&E stainx100 magnification).

African Health Sciences Vol 16 Issue 1, March 2016 343

Discussion doses showed no significant increase in number of rear-
Body weight changes serve as a sensitive indication of ings, groomings and time spent in the centre.
the general health status of animals16. The results of this
study showed that the administration of leaf extract of This indicates that the plant extract did not induce anxi-
G. carpinifolia did not interfere with growth irrespective ety in treated rats. The decreased motor activity obtained
of dose. Although the effect of extract of G. carpinifolia at 800 mg/kg in the present study may suggest that the
on body weight has not been earlier reported, the present ethanol extract of Grewia carpinifolia had some CNS de-
observation suggests that G. carpinifolia did not interfere pressant activities at this dose. This type of activity profile
with body weight. can be compared with opium alkaloid which possesses
stimulant effect at low dose initially and marked sedative
According to Moore et al17, an increase in absolute/ rela- effect after cumulative effect or after high dose. The phy-
tive organ weight is an indication of inflammation while a tochemical constituents may be responsible for this ac-
reduction in the same parameter can be adduced to cellu- tivity25. Gamma-aminobutyric acid (GABAA) is the major
lar constriction. Oral administration of 100, 200, 400 and inhibitory neurotransmitter in the central nervous system.
800 mg/kg bw of G. carpinifolia leaves extracts also re- Different anxiolytic, muscle relaxant, sedative-hypnotic
vealed no significant (P<0.05) difference in brain weight drugs acts through GABAA receptors, therefore it is pos-
of extract treated animals when compared with controls. sible that extracts of G.carpinifolia may act by potentiating
GABAergic inhibition in the CNS via direct activation of
The open field test is used to evaluate the emotional GABA receptor or indirectly by membrane hyperpolariza-
state and locomotor activity of an animal. The open field tion which led to a decrease in the firing rate of critical
model examines anxiety related behaviour characterized neurons in the brain26. Polyphenolic compounds, like fla-
by the normal aversion of the animal to an open area. vonoids, tannins, saponins and phenolic acids, commonly
Thus, animals removed from their acclimatized cage and found in plants have been reported to possess multiple bi-
placed in environment express anxiety and fear, by show- ological effects27 and are useful in many CNS disorders28.
ing alteration in all or some parameters18. Many flavonoids and neuroactive steroids were found to
be ligands for the GABAA receptors in the central ner-
Locomotor activity has been used to represent a broad vous system; which led to the assumption that they can
class of sensory, motor and integrative processes19, Rang act as benzodiazepine- like molecules29. The presence of
et al.,20 reported that locomotor activity is mediated alkaloids, flavonoids, saponins and tannins in the extract
through dopamine and other neurochemical pathways. in this study might thus be responsible for its CNS de-
A wide variety of agents have the capacity to excite the pressant activity. This is in consonance with investiga-
function of the CNS, such that calming or drowsiness tions on other species of the genus; Jaspers et al.30, Hyo et
(sedation) is inhibited21. In addition, central nervous sys- al.31 Tijani et al32 found that extract from Grewia bicolor, G
tem (CNS) depressants are known to inhibit locomotor umbellifera and G. lasiodiscus respectively possesses central
activity of animals22. depressing activities.

Furthermore, inhibition of acetylcholinesterase (AchE), The length of time a rat was able to hold the hanging
CNS activation of cholinergic motor inhibitory system wire is considered as an indirect measure of grip, muscle
and damage to the peripheral muscle due to necrosis of strength and co-ordination33 which was significantly in-
skeletal muscle fibres has been reported to reduce loco- creased (p<0.05) at all the tested doses. This may suggest
motor activity in animals23. The effects of Grewia carpinifo- an increase grip strength which could be due to increased
lia on spontaneous activities were studied by rearing and motor coordination and muscle tone34 that occurred fol-
the number of lines crossed. The rearing (vertical move- lowing the sub chronic administration of Grewia carpini-
ment) is an index of the locomotor activity24 while the in- folia.
creased number of line crossed (horizontal movement) is Hypertrophy of an organ is an indication of toxicity of
an indication of the central nervous system stimulant or chemical or biological substance35. However, no hyper-
depressant properties. Rats treated with extract at varying trophy of the brain was observed in this study amongst

344 African Health Sciences Vol 16 Issue 1, March 2016

all the groups studied. In addition, the microscopic ex- 6. Organization of Economic Co-operation and Devel-
amination revealed that the cerebellum from the extract opment (OECD) The OECD Guideline for Testing of
treated rats showed no alteration in cell structure when Chemicals: 420 Acute Oral Toxicity-Fixed Dose Proce-
viewed under the light microscope using multiple mag- dure. Paris, France: OECD; 2001.
nification powers. The cerebellum plays a significant role 7. Wallis TE. Textbook of Pharmacology, 5th ed, CBS
in behaviour, movement control and as well as sensory Publishers & Distributors, Delhi, 1985; 561-565
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of Purkinje cells leading to cerebellar damage which con- 9. Sofowora A. Medicinal plants and Traditional Medicine
sequently affects movement coordination and other cer- in Africa. Spectrum Books, Ibadan. 1993; 150.
ebellum-dependent cognitive function37. The intact (lack 10. Harborne, JB. Pytochemical methods: A guide to
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Conflict of interest statement differences in motor and memory performance. Can. J.
We have no conflict of interest to declare. Physiol. Pharmacol. 2012; 90: 379-386.
15. Carter RJ, Morton AJ, Dunnet SB. Motor coordina-
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