www.idosr.

org Tabakwot et al
©IDOSR PUBLICATIONS
International Digital Organization for Scientific Research ISSN: 2579-0781
IDOSR JOURNAL OF EXPERIMENTAL SCIENCES 8(1) 48-59, 2022.
Evaluation of the Effects of Galinsoga parviflora Aqueous Leaf Extract on
Motor Coordination (muscular strength and balance) on Adult Male Mice
after Mercury Chloride Exposure.

John Ayuba Tabakwot, Okesina Akeem Ayodeji and Usman Ibe M.

Department of Human Anatomy, Kampala International University, Uganda.

ABSTRACT
The cerebellum forms a major part of the brain and its function is to control all muscular
movements and coordination. Injury or damage to the cells of the cerebellum may lead to
conditions like; ataxia, Joubert syndrome, and many others. These injuries may be a result
of traumatic brain injuries or toxic agents such as mercury, tin and aluminum. For
example,mercury exposure has been reported to be part of our daily lives, therefore it is
important to understand the pathological implication that might follow these exposures
and also with the help of a local herb; Galinsoga parviflora (GP) to find a cheap and readily
available management solution to mercury toxicity in the cerebellum. This study was
designed to evaluate the effects of Galinsoga parviflora aqueous leaf extract on motor
coordination (muscular strength and balance) on adult male mice after mercury chloride
exposure.Twenty-five adult male mice of an average weight of 25g were randomly divided
into 5 groups of 5 rats each (n=5). The animals in Groups 1-5 received oral administration
of distilled water, 2.3mg/kg body weight of HgCl2, 2.3 mg/kg HgCl2 followed by GP extract
(800mg/kg), GP extract (800mg/kg) followed by 2.3 mg/kg HgCl2 and GP extract
(800mg/kg) followed by 2.3 mg/kg HgCl2 concurrently respectively. These treatments were
followed by behavioral tests. The animals exhibited abnormal neuromuscular function and
uncoordinated balance and posture. In conclusion, the study revealed that mercury
exposure in the cerebellum of mice caused loss of neuromuscular function, loss of balance
and coordination. However, the introduction of Galinsoga parviflora in the cerebellum
acted as a therapeutic agent against mercury toxicity in the cerebellum of adult male mice.
Keywords: Galinsoga parviflora, motor coordination, muscular strength and balance.

INTRODUCTION
There are three functional areas of the involvement in neuronal damage and
cerebellum the cerebrocerebellum, neurodegenerative diseases, suggested
spinocerebellum and vestibulocerebellum that mercury is dangerous to human
[1]. The spinocerebellar lobes play a role health and in most cases associated with
in the regulation of muscle tone, behavior deficit. Teixeira et al., [5]
vestibulocerebellum lobes play a role in studied the effects of chronic intoxication
the maintenance of posture and balance with inorganic mercury on memory and
and cerebrocerebellum plays a role in the motor control in rats; then found that
coordination of voluntary motor activity long-term exposure to mercury chloride
[1]. Any possible damage to any parts of in the rat can cause motor function
the cerebellum can affects the above damage, anxiety-related disorder, and
mention parts which can lead to memory impairment. A study by [5] on
conditions such as ataxia, nystagmus, the effect of inorganic mercury exposure
dysdiadochokinesia, and dysmetria [2]. on the motor cortex, indicated that
Behavioral changes constitute a chronic exposure to inorganic mercury
significant hallmark associated with decreases balance and motor
nervous system disturbance in mercury coordination, an effect likely linked to
exposure [3]. A review by [4] on mercury cellular and molecular damages in some

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areas of the cerebellum. The enormity of derivatives. Several flavonoids and
the behavioral consequence of mercury polyphenolic compounds have been
exposure raises the need for possible reported to possess antioxidant
therapeutic and ameliorative intervention properties both in in-vivo and in-vitro
[5]. According to [6] who reported that studies [9]. A study by [11] on the
mice with cortical impact lesions often differential effects of alkaloids on
exhibit contralateral slipping on the beam memory in rodents, suggested that
and might be due to cortical impact alkaloids have the potential to improved
lesion. A study by [7] on grip strength is learning and memory function. Morales-
potentially an early indicator of age- García et al., [12] on the alkaloids of
related decline in mice, indicated that Banisteriopsiscaapi, the plant source of
there was decreased in the strength of the Amazonian hallucinogen Ayahuasca,
male mice 12 months of age compared to stimulate adult neurogenesis in vitro. An
8-month-old mice, and continued a robust alkaloid which is one of the
decline to 20 months and then 28 months phytochemicals of GP has been
of age when the study was terminated. established that its modulation of
The decline was not related to lean antidepressant effects. Possible
muscle mass, but extensive age-related behavioral improvement following
carpal and digital exostosis could help administration of GP during mercury
explain the decreased grip strength times exposure may be linked with the presence
with increasing age. A report by [8] on the of heavy metal chelators and antioxidants
use of a grip strength meter to assess the (flavonoid) as one of its very important
limb strength of MDX mice, indicated that components [13,14,15,16,17,18]. A review
the measure the disease progression of by [2] emphasized the importance of
neuromuscular disorders can measure by various phytochemicals in the
the time the animal used to finish the management of diverse behavioral
tasks. alterations. Their review also laid discreet
Ethnomedicinal use of plants has emphasis on the neuroprotective
gradually moved to the fore in the potentials of various phytochemicals such
struggle to curtail the impact of most as fatty acids, phenols, alkaloids,
environmental neurotoxicants including flavonoids, saponins, and terpenes,
those associated with mercury exposure therefore buttressing our choice of GP in
[9]. However, there is a paucity of the management of mercury exposure to
literature on the effect of GP on the our Wistar rat model [2,19,20,21].
cerebellum in mercury, therefore the need Aim of the Study
for the present studies. Most The aim of this study was to evaluate the
ethnopharmacological interventions effects of Galinsoga parviflora aqueous
targeting possible leaf extract on motor coordination
protection/amelioration against mercury- (muscular strength and balance) on adult
induced behavioral changes are hinged on male mice after mercury chloride
the abundance of very important exposure.
phytochemical composition of different Research Question
plant materials [9]. A recent review by What are the effects of Galinsoga
[10] on phytochemical and parviflora aqueous leaf extract on motor
pharmacological properties of GP, coordination (muscular strength and
suggested that GP contains aromatic balance) on adult male mice after mercury
esters, flavonoids, and phenolic acid chloride exposure?
METHODOLOGY
Study Location Western Campus (KIU-WC); which is
The study was conducted in the animal situated in Western Uganda in Ishaka
house of the Kampala International Municipality, Bushenyi District 350KM
University, Institute of Biomedical from Kampala, Uganda.
Research (IBR) and Histology Laboratory
of Kampala International University,

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Ethical Consideration
Ethical approval (KIU-2021-14) was
obtained from the Kampala International
University Research Ethics Committee
before conducting the research and
register in Uganda National Council for
Science and Technology. The
experimental study was carried out at the
Institute of Biomedical Research
Laboratory (IBRL) Kampala International
University-Western Campus (KIU-WC) and
the laboratory animal experiments were
conducted from the Kampala International
University Animal House. The 3Rs
(Reduction, Replacement, and Refinement)
were considered in determining the
number of mice to be used in this study
as well as the handling of animals
(European Medicine Agency, 2016). A total
of 25 mice were used in this study. The
study will use an animal model system
and not an in vitro system to determine
the effect of Galinsoga parviflora on the
cerebellum after mercury chloride is
introduced then the expression of
antioxidant biomarkers in cells is
determined. The report by ‘resource
equation’ was considered when chosen
the number of animals. The animals were
grouped into 5 groups with five animals
per group and with groups, three and four
receiving the same doses but in reverse
method and also a suitable dose was used
according to literature to avoid using too
many animals so that to be able to
achieve the reduction principle. The
researcher obtains training on how to
handle laboratory animals before the
study and work under the supervision of
his supervisors. All procedures and works
on animals were conducted following a
guide for the care and use of laboratory
animals (IACUC, 2010). The animals were
treated in a humane way to prevent them
from any discomfort like excessive pain
and stress. The animals were housed five
per group and there was fed on a daily
basic morning and evening and also their
beddings were changed weekly so that the
animals were comfortable to avoid the
fight and so allow to respond to treatment
in a ventilated environment. At the end of
42 days, all animals were sacrificed
humanely by subjecting them to light
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Tabakwot et al
ether anesthesia followed by cervical
dislocation. At the end of the study, all
sacrificed animals were incinerated.
Collection and Preparation of Plant
Extract
Galinsoga parviflora was obtained from
the local garden in Ishaka- Bushenyi
District, Uganda. A small sample of the
leaves was taken to Mbarara University of
Science and Technology, Faculty of
Science Herbarium Unit, Mbarara, for
botanical identification and deposition of
voucher number. Extraction of the plant
was carried out in the Biochemistry
Departmental laboratory, Kampala
International University, Western Campus,
Ishaka, Uganda. The plant’s leaves were
dried at room temperature; grounded with
a grinding machineand followed by
extraction. Sixteen grams (16g) of the
powdered leaves were dissolved in 300ml
of distilled water and left to stand for
three days, shaken frequently until
complete extraction of plant materials.
Then the extracts were filtered through a
filter into a funnel and the extract was
kept in an oven at 400 C for three days to
solidified [13].
Purchase of Chemical
Mercuric chloride (May and Bakers,
England) was purchased from a chemical
store in Ishaka town, Western Uganda.
Animals
The animals were procured from the
animal facility of Kampala International
University Western Campus (KIU-WC)
twenty-five (25) adult male mice were
used for this study weighing 20-35g. All
mice were housed in a well-ventilated
plastic cage; the animals were left to
acclimatize for two weeks with free
access to food and water.
Study Sample Size
‘The resource equation’ approach was
used to arrive at adequate sample size.
According to the ‘resource equation’
method a value “E or DF” is measured. DF
= degree of freedom of analysis of
variance (ANOVA).
DF can be measured by the following
formula:
DF= N – k
n = DF/k + 1
www.idosr.org Tabakwot et al
Where: N = total subjects number; k = of numbers was randomly used to direct
number of groups; n = number of subjects how the animals will place in groups
in a group. Minimum DF range = 10 and (table 1). After the randomization, the
maximum DF range = 20 [14]. micewere placed in five groups
The sample size for the study will be: N = comprising of 5 mice each labeled
25 male mice; K = 5 groups mercury and gallant soldier dose (MG),
DF = 25 – 5 = 25, n = DF/k+1 = 20/5+1 = gallant soldier and mercury (GM), mercury
5 (five mice will be placed per group) and gallant soldier dose
Sampling Technique concurrently(MGC), positive control
The mice were sampled via simple (mercury dose) (PC) and negative control
random sampling procedures. A sequence (NC)(table 2).

Table 1: Randomization assignment sequence

Original 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 4 4 4 4 4 5 5 5 5 5
Randomiz 2 4 3 5 1 3 1 4 3 2 2 4 4 5 3 2 1 3 5 4 4 1 3 2 5
ed

Table 2: Group placement after randomization

Group MG GM MGC PC NC

Randomized mice 2, 1, 3, 5,1 3, 1, 4, 3,2 2, 4,4,5,2 2, 1,3,5,4 4, 1, 3, 2,

5

Experimental
Dosage and Administration
Mercury chloride:2.3mg/kg of Mercury
Chloride was administered. The stock
solution was prepared daily to prevent
the risk of degradation [15]. The dilution
factor was 50mg in 100ml.
Galinsoga parviflora
800mg/kg of GP was administered. The
stock solution was prepared daily to
prevent the risk of degradation [16]. The
dilution factor was 100mg in 1ml.
Mercury chloride and GP was
administered orally for three weeks
respectively
Reconstitution of Galinsoga parviflora
Extract mercury Chloride for Animal
Administration
A suitable dose of GP was administered to
the animals. The right amount of the
extract which was guided by the OECD’s
(organization of economic corporation
Design
and developments) guidelineswas
reconstituted and the calculated doses
shall be administered according to each
animal's body weight daily (between 9am
and 10am). The mercury chloride on the
other hand was constituted for
administration on the mice according to
the appropriate dose which was
800mg/kg as describe by [15]. The dosage
of GP was prepared by dissolving in an
appropriate volume of distilled water not
exceeding 20 ml/kg [17]. The mercury
chloride was guided by the OECD’S
guideline and the powder be dissolved in
distilled water daily. The mixture was
kept in the refrigerator at 60C pending
daily administration to the animals
according to their body weight.
Dosage and preparation of stock solution
will be calculated using the formula:

Dosage (mg) = Bodyweight animals (g) x dose (mg)

1000g
Dose volume = weight of rat (g) x appropriate volume (10ml) or maximum volume (20ml)
1000g

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Groupings
Animals were randomly assigned to five (5) groups of five (5) animals in each group i.e. N=5
per group.
Table 3: Animals Grouping
GROUP DOSAGE
Group 1 Receive distilled water only
Group 2 Receive mercury chloride only (2.3mg/kg)
Group 3 Receive mercury chloride (2.3mg/kg) followed byGalinsoga
parviflora (800mg/kg)
Group 4 Receive Galinsoga parviflora (800mg/kg) followed by mercury
chloride (2.3mg/kg)
Group 5 Receive mercury chloride (2.3mg/kg) and Galinsoga parviflora
(800mg/kg) concurrently

Motor Coordination apparatus a 120cm by 50cm wire forearm

Beam walking test: This test was grip apparatus. The wire was placed
conducted to assess motor coordination horizontally and the mice were placed in
and balance. 1m long narrow wooden the center to balance the time it takes to
beam (1-2cm wide) suspended from its reach the other end or falls will be
ends at an elevation of 30cm to the recorded. The training was done for a
darkroom. The animal was gently be week once a day before administration
placed in the center of the Beam, facing and the test was conducted three times a
one of the ends. The rat was allowed to week [18].
walk to the end of the darkroom; the Statistical Analysis
procedure was repeated three times, the All the data of behavior and the
animals were trained for a week once a biochemical study was statistically
day before administration and the test evaluated with SPSS 25 using ANOVA to
was conducted three times a week and the compare the mean. Where necessary,
time to reach the end was recorded [18]. Tukey post hoc test was used to find
Forearm grip test where the significant difference lies
The neuromuscular/skeletal muscle across the groups. All the results were
functions were determined by the expressed as mean ± SD or SEM value was
maximal peak force developed by the significant at p≤0.05.
mice. The mice were placed on the
RESULTS
Behavioral Studies of Adult Male Mice During Administration of Galinsoga Parviflora
(Cav) Aqueous Leaf Extract on the Cerebellum After Mercury Chloride Exposure
Beam Walking Test of adult male mice was a reduction time in test time (Table
Group 3 revealed there was a significant 4). Group 5,revealed a reduction in time
increase in the time taken for the mice to when compared with groups 1 and 2
finished the apparatus especially at the during test time (Table 4).
beginning of thetest time, while during Forearm Grip Test of adult male mice
the end of the test time there was a Group 3 revealed there was a significant
significant reduction in time when decrease in timein both reaching and
comparedto group 1(P < falling timewhen compared to group 1(P <
0.05).Although,when compared to group 2 0.05). While when compared to group 2
there was an increase in time during test there was a decrease in time in both
time (Table 4). Group 4 revealed an reaching and falling time(Table 5).In
increase in time taken for the mice to group 4, both the falling and reaching
finished the apparatus during the time revealed that there was a significant
beginning of test time and a significant decrease in time when compared to group
reduction in time at the end of test 1(P < 0.05). When compared with group 2
timewhen compared to group 1 (P < 0.05), revealed a reduction in both falling and
while when compared with group 2 there reaching time (Table 5).The falling time in

52
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group 5 revealed a significant decrease in
time (P < 0.05). Although, reaching time
revealed an increase in time in week 1
then reduced from week 2 to 3 when
compared with group 1. When compared
with group 2 it revealed a reduction in
falling time. While reaching time revealed
an increase in groups 1 and 2 then
reduced in week 3 (Table 5).

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Table 4:Beam walking test ofadult male mice during administration of Galinsoga parviflora (cav) aqueous leaf extract on the cerebellum
after mercury chloride exposure
Groups TN 1(sec) TN 2 (sec) TN3 (sec) TN 4 (sec) TN 5(sec) TS 1 (sec) TS 2 (sec) TS 3 (sec) TS 4 (sec)
Group 15.67±1.87 10.92±0.47 12.67±1.35 20.10±2.34 11.10±1.93 15.80±1.71 10.81±0.98 15.80±1.71 10.81±0.98
1
Group 13.81±1.88 11.79±1.30 11.43±1.62 20.75±2.60 16.02±1.29 19.80±3.25 15.68±3.40
2
Group3 17.50±1.96 12.70±1.84 11.50±1.19 21.77±3.69 20.42±2.48a 26.72±5.73 25.23±5.06a 10.30±2.39 20.99±4.43a
Group 17.18±3.59 12.10±2.26 10.41±1.12 17.46±3.53 12.28±1.86 14.27±4.29 11.87±1.75 23.41±1.22a 9.87±1.22
4
Group 12.29±1.36 12.67±1.34 15.63±2.36 19.50±3.26 13.90±1.67 14.04±3.12 8.74±1.73
5
F value 0.969 0.225 1.596 0.265 3.809 1.907 4.507 11.175 8.681
P value 0.446 O.921 0.214 0.897 0.018 0.149 0.009 0.002 0.005

N=5, TN=Training time, TS=Test time. The value was considered significantly different at p<0.05. b: indicates significance when test
group is compared against Group 2, a: indicates significance when test group is compared against Group 1.

54
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Table 5: Forearm Grip test of adult male mice during the administration of Galinsoga parviflora(cav) aqueous leaf extract on the
cerebellum after mercury chloride exposure
Group WBA W1 W2 W3 W4 W5
s

RT1(sec) FT1(sec RT2(se FT2(sec RT3(se FT3(sec RT4(se FT4(sec RT5(se FT5(sec RT6(se FT6(sec
) c) ) c) ) c) ) c) ) c) )

GP 1 24.2±8.0 109.8±3 8.0±3.5 131.2±3 61.8±10 180.0±0 29.4±10 174.0±6 42.0±6. 180.0±0 42.0±9. 180.0±0
3 7.33 1 2.96 .28 .00 .53 .00 63 .00 57 .00

GP 2 45.0±10. 180.0±0 33.0±17 164.0±1 20.4±5. 124.2±2 12.8±3. 84.0±26

82 .00 .07 6.0 96 6.91 07 .88

GP 3 24.2±16. 102.6±3 21.6±6. 96.6±31 36.8±29 79.0±36 5.0±2.5 43.4±2 19.6±9. 39.2±1 5.2±2.3 102.0±3
59 4.10 97 .98 .15 .68 3a 1.12a 79 4.04a 3a 6.43

GP 4 23.4±10. 116.2±3 20.6±5. 91.4±36 16.6±7. 85.2±29 12.8±4. 75.6±31 9.4±1.0 36.2±1 15.4±8. 38.4±1
70 9.07 33 .86 27 .93 61 .17 3a 5.51a 69 4.73a

GP 5 20.2±5.5 89.2±37 44.0±32 73.0±31 21.8±8. 80.4±19 8.4±3.6 59.6±2

2 .78 .97 .29 19 .99 1 5.76a

F 0.822 1.127 0.633 1.396 1.559 2.821 2.719 4.560 5.920 46.270 6.281 9.772
value
0.526 0.372 0.644 0.271 0.224 0.053 0.059 0.009 0.016 0.000 0.014 0.003
P
value
N=5, WBA=Before Administration, W: Weak, RT=Reaching Time, FT=Falling Time. The value was considered significantly different at
p<0.05. b: indicates significance when test group is compared against Group 2, a: indicates significance when test group is compared
against Group 1

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DISCUSSION
This study was centered on assessment
effects of Galinsoga parviflora (cav)
aqueous leaf extract on the cerebellum
during mercury exposure in adult male
mice, using neurobehavioral to observe
the specific effect of this chemical on the
brain using the mice as a model. The
specific objective of the study was
neurobehavioral studies using forearm
grip and beam walk apparatus to assess
locomotor function and motor
coordination
The morphometric study indicated that
groups treated with mercury chloride had
a decrease in weight when compared with
the control (group1) might be due to the
presence of damage of the cells, will
those who have been treated with GP
show an increase in weight might be due
to the presence of phytochemicals
present in the plant such as alkaloid,
carbohydrate, flavonoid, polyphenol and
glycoside. While organ body ratio did not
show any significant difference
The forearm gripapparatus is used to
measureneuromuscular/skeletal muscle
functions [18,21,22,23,24]. The forearm
grip test showed a significant decrease
(p˂0.05) in treatment groups (3,4 and 5)
compared to the control(group 1) in both
falling and reaching a time of theforearm
grip test and also a decrease in time when
compared to group 2.Thisresult suggests
that mercury chloride may have the
potential to decrease
neuromuscular/skeletal muscle
functions.Teixeira et al., [19] studied the
effects of chronic intoxication with
inorganic mercury on memory and motor
control in rats; then found that long-term
exposure to mercury chloride in the rat
can cause motor function damage,
anxiety-related disorder, and memory
impairment. A report by [20,25,26,27] on
the use of a grip strength meter to assess
the limb strength of mice, indicated that
the measure the disease progression of
neuromuscular disorders can measure by
CONCLUSION
In conclusion, the study revealed that
mercury exposure in the cerebellum of
mice caused loss of neuromuscular
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Tabakwot et al
the time the animal used to finish the
tasks. This means that a decrease in the
neuromuscular function of the cerebellum
may be a result of damage of the Purkinje
cells as reported by [5; 20,28,29,30,31].
Fine motor coordination and balance can
be assessed by the beam walking test.
This is useful for detecting subtle deficits
in motor skills and balance that may not
be detected by other motor tests, such as
the Rotarod [18]. In this study, the beam
walk test was used to assess fine motor
coordination and parameters such as the
time it will take the mice to reach the
darkroom.Results showed a significant
increase in time in groups treatedwith
mercury chloride when compare to a
group treated with GP, which suggests
poor fine motor coordination in treatment
groups given mercury chloride
[32,33,34,35,36]. The treatment groups
had increase time of the group given
mercury chloride on the beam walk when
compared to the control, this suggests
poor balance which may be due to cortical
impact lesion. This is in line with an
earlier study reported by [21; 6] who
reported that mice with cortical impact
lesions often exhibit contralateral
slipping on the beam. The poor fine
motor coordination and poor balance
observed in the beam walk test suggests
motor deficits that may have been caused
by CNS lesions. The motor deficits
observed in the neurobehavioural studies
may be due to the pyknosis, degenerating
myelinobserved in the histomorphological
studies of the cerebellum. The poor fine
motor coordination and poor balance
observed in the beam walk test suggests
motor deficits that may have been caused
by CNS lesions as aresult of mercury
exposure. The motor deficits observed in
the neurobehavioural studies may be due
to the pyknosis, degenerating myelin
earlier observed in the
histomorphological studies of the
cerebellum.
function, loss of balance and
coordination. However, the introduction
of Galinsoga parviflora in the cerebellum
www.idosr.org Tabakwot et al
acted as a therapeutic agent against adult male mice.
mercury toxicity in the cerebellum of
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