Hindawi
e Scientific World Journal
Volume 2018, Article ID 4256782, 12 pages
https://doi.org/10.1155/2018/4256782
Research Article
Toxicological Assessment of Pseudospondias microcarpa
(A. Rich.) Engl. Hydroethanolic Leaf Extract in Rats:
Haematological, Biochemical, and Histopathological Studies
Donatus Wewura Adongo ,1 Priscilla Kolibea Mante,2
Kennedy Kwami Edem Kukuia ,3 Charles Kwaku Benneh ,4 Robert Peter Biney ,5
Eric Boakye-Gyasi ,2 Nicholas Akinwale Titiloye,6 and Eric Woode 2
1
Department of Pharmacology, School of Medicine, University of Health and Allied Sciences, Ho, Ghana
Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences,
Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
3
Department of Pharmacology and Toxicology, University of Ghana School of Pharmacy, College of Health Sciences,
University of Ghana, Accra, Ghana
4
Department of Pharmacology and Toxicology, School of Pharmacy, University of Health and Allied Sciences, Ho, Ghana
5
Department of Pharmacology, School of Medical Sciences, University of Cape Coast, Cape Coast, Ghana
6
Department of Pathology, School of Medical Sciences, College of Health Sciences,
Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
2
Correspondence should be addressed to Donatus Wewura Adongo; donatusadongo@yahoo.com
Received 11 October 2017; Accepted 10 April 2018; Published 20 May 2018
Academic Editor: Valdir Cechinel Filho
Copyright © 2018 Donatus Wewura Adongo et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Pseudospondias microcarpa is used traditionally for treating various diseases. However, although parts of the plant are extensively
used in African traditional medicine, no scientific study has been reported on its toxicity. Therefore, this study evaluated the acute
and subacute toxicity studies of the ethanolic extract of P. microcarpa in rats. Male Sprague-Dawley rats (120–150 g) were treated
orally with the extract (30, 100, 300, 1000, and 3000 mg kg−1 ) or distilled water (10 ml kg−1 ) for 2 weeks and observed daily for
general appearance and signs of toxicity. In addition, blood was collected for both biochemical and haematological assays. Sections
of tissues from liver, kidney, spleen, brain, and stomach were also used for histopathological examination. Administration of the
extract for 14 consecutive days caused no deaths, with an LD50 above 3000 mg kg−1 . Except for lymphocytes (%) that showed a
significant decrease (𝐹5,23 = 3.93, 𝑃 = 0.013), all other haematological parameters remained unaffected by the extract. The extract
at 100 mg kg−1 showed a significant decrease in the levels of triglyceride and very-low-density lipoproteins (both at 𝑃 < 0.05). Weight
change as well as histological evaluation of the organs indicated no toxicity. The study demonstrates that an ethanolic extract of P.
microcarpa given orally to rats is safe.
1. Introduction
Over the last few years, there has been a tremendous rise in
the acceptance and public interest in medicinal plants in both
developing and developed countries. It is estimated that about
75% of the world population, primarily those of developing
countries, rely on traditional remedies (mainly herbs) for the
healthcare of their people [1]. In these developing countries,
this has been attributed to easy access to herbal therapies,
as well as cultural and economic factors. Also, in developed
countries, herbal medicines are viewed as natural, timetested, and therefore safe compared with what are perceived
as synthetic drugs [2].
Although some herbal medicines have promising potential and are widely used, many of them remain untested and
their use is also not monitored. In this regard, knowledge
2
of their potential adverse effects is limited and identification
of the safest and most effective therapies is difficult [3].
Medicinal plants have been shown to be capable of producing
a wide range of undesirable or adverse reactions, with some
being capable of causing life-threatening conditions and even
death. It is therefore important to assess the toxicity of herbal
medicines.
Pseudospondias microcarpa is one of such plants used for
treating various diseases including central nervous system
(CNS) disorders, arthritis, rheumatism, eye problems, kidney
disorders, nasopharyngeal infections, stomach complaints,
malaria, and jaundice [4]. Various studies have also shown
the plant to possess antioxidant [5], antimicrobial [6], anticonvulsant [7], antidepressant [8, 9], anxiolytic [10], sedative,
analgesic [11] and cytotoxic and antiplasmodial effects [12]. As
demonstrated by Yondo et al. [5], we have shown in a previous
study that the leaves of the plant contain some phytochemical
constituents which may be responsible for its biologic activity
[11].
However, although various parts of P. microcarpa are
extensively used in African traditional medicine and despite
the fact that the plant has several pharmacological actions as
indicated previously, no scientific study has been reported on
its toxicity. The present study therefore assessed both acute
and subacute toxicity of P. microcarpa in rats.
2. Materials and Methods
2.1. Collection of Plant Material and Extraction. Fresh
leaves of P. microcarpa were collected from the campus
of Kwame Nkrumah University of Science and Technology
(KNUST), Kumasi (6∘ 40.626 N, 1∘ 34.041 W), and authenticated at the Department of Herbal Medicine, Faculty of
Pharmacy and Pharmaceutical Sciences, College of Health
Sciences, KNUST, Kumasi, Ghana. A voucher specimen
(KNUST/HM1/2013/L005) was kept at the herbarium of the
faculty.
Leaves of the plant were room-dried for seven days and
pulverised into fine powder. The powder was extracted by
cold percolation with 70% (v/v) ethanol in water over a period
of 72 h and the resulting extract concentrated into a syrupy
mass under reduced pressure at 60∘ C in a rotary evaporator. It
was further dried in a hot air oven at 50∘ C for a week and kept
in a refrigerator for use. The yield was 20.5% (w/w). In this
study, the crude extract is subsequently referred to as PME or
extract.
2.2. Animals. Male Sprague-Dawley rats (120–150 g) were
purchased from the Noguchi Memorial Institute for Medical
Research, Accra, Ghana, and kept in the animal house of the
Department of Pharmacology, Kwame Nkrumah University
of Science and Technology, Kumasi, Ghana. The animals
were housed in groups of 5 in stainless steel cages (34
× 47 × 18 cm3 ) with soft wood shavings as bedding and
housing conditions controlled: temperature maintained at
24-25∘ C, relative humidity of 60–70%, and 12 h light-dark
cycle. They had free access to tap water and food (commercial
pellet diet, GAFCO, Tema, Ghana). A period of at least
one week for adaptation to the laboratory facilities was
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allowed. The research was conducted in accordance with
accepted principles for laboratory animal use and care [13].
Approval for this study was obtained from the Faculty Ethics
Committee.
2.3. Acute Toxicity Study. Sprague-Dawley rats were orally
treated with the extract (30, 100, 300, 1000, and 3000 mg kg−1 )
or distilled water (10 ml kg−1 ) and placed in observation
cages. The rats were evaluated for general pharmacological
and physiological behaviours as well as mortality at 0, 15, 30,
60, 120, and 180 min, up to 24 h after treatment.
2.4. Subacute Toxicity. Sprague-Dawley rats were put into
six groups of 5 animals each. Five experimental groups
were given PME orally at doses of 30, 100, 300, 1000,
and 3000 mg kg−1 for 2 weeks. The control group received
distilled water orally at the volume of 10 mL kg−1 . During the
experimental period, animals were observed daily for general
appearance and signs of toxicity.
2.4.1. Preparation of Serum and Isolation of Organs. At the
end of the study, animals were fasted overnight and sacrificed
by cervical dislocation. About 1.5 mL of blood was collected
into vacuum tubes containing 2.5 𝜇g of ethylenediaminetetraacetic acid (EDTA) as an anticoagulant for haematological
assay and 3.5 ml into sample tubes containing a separating gel
for biochemical parameters. The blood for the biochemical
parameters was centrifuged (4000 rpm at 4∘ C for 10 min) to
obtain serum and stored at −20∘ C.
Organs harvested included liver, kidney, brain, stomach,
and spleen.
2.4.2. Haematological Assay. Haematological analysis was
performed with the haematological analyser, ABX micros ES
60 (HORIBA Medical Diagnostics, France). The parameters
examined included white blood cells (WBC), red blood cells
(RBC), haematocrit (HCT), haemoglobin (HGB), mean cell
volume (MCV), mean cell haemoglobin (MCH), mean cell
haemoglobin concentration (MCHC), lymphocytes (LMP),
platelet distribution width (PDW), red cell (erythrocyte
volume) distribution width, relative volume of thrombocytes
(PCT), platelets (PLT), and mean platelet (thrombocyte)
volume (MPV).
2.4.3. Biochemical Assay. Biochemical values were measured with a COBAS INTEGRA 400 (Hoffmann-La Roche
Ltd., Basel, Switzerland), which assessed levels of alkaline
phosphatase (ALP), alanine transaminase (ALT), aspartate transferase (AST), gamma-glutamyl transferase (GGT),
total protein, albumin, globulin, total bilirubin (T-Bil),
direct bilirubin (D-Bil), indirect bilirubin (I-Bil), creatinine,
urea, triglyceride (TG), cholesterol, high-density lipoproteins (HDL), low-density lipoproteins (LDL), and very-lowdensity lipoproteins (VLDL).
2.4.4. Body and Organ Weight Assessment. Brain, liver, kidney, stomach, heart, and spleen were isolated and weighed.
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Table 1: Effects of Pseudospondias microcarpa hydroethanolic leaf
extract in the primary observation test in rats.
Mortality D/T
0/5
0/5
0/5
0/5
0/5
0/5
Effects
No change
Sedation, analgesia
Sedation, analgesia
Sedation, analgesia
Sedation, analgesia
Sedation, analgesia
20
% weight change
Dose (mg kg−1 )
0
30
100
300
1000
3000
10
0
ctrl
D/T: dead/treated.
−10
Body weights of the rats were taken on days 0 and 15. Relative
organ weight (ROW) was then calculated:
ROW =
absolute organ weight (g)
body weight of animal on sacrifice day (g) (1)
× 100.
2.4.5. Histopathological Examinations. Sections of the tissue
from liver, kidney, spleen, brain, and stomach were used
for histopathological examination. Tissues were fixed in 10%
neutral buffered formalin (pH 7.2) and dehydrated through
a series of ethanol solutions, embedded in paraffin, and
routinely processed for histological analysis. Sections of
2 𝜇m thickness were cut and stained with haematoxylineosin for examination. The stained tissues were observed
through an Olympus microscope (BX-51) and photographed
by INFINITY 4 USB Scientific Camera (Lumenera Corporation, Ottawa, Canada).
2.5. Statistical Analysis. In all experiments, a sample size of 5
animals was utilized. Data are presented as mean ± SEM. The
presence of significant differences among means of groups
was determined by one-way ANOVA using GraphPad Prism
for Windows version 5 (GraphPad Software, San Diego, CA,
USA). Significant difference between pairs of groups was
calculated using the Newman-Keuls’ multiple comparison
test. 𝑃 < 0.05 was considered statistically significant.
3. Results
3.1. Acute Toxicity. Treatment of rats with the extract produced sedation and analgesia at all doses used (Table 1).
No deaths were recorded over the 24 h observation period,
indicating an LD50 above 3000 mg kg−1 .
3.2. Subacute Toxicity. No deaths were recorded after 14 days
of treatment with the extract. Other signs of toxicity were
absent except sedation, which was observed throughout the
treatment period.
3.2.1. Effect of Extract on Body Weight and Organ Weight. As
shown in Figure 1, the extract had no significant effect on
body weight change, although this parameter was decreased
at the highest dose (3000 mg kg−1 ). Treatment with the
extract increased weight of the spleen at 100, 300, and
30
100
300
1000
3000
−1
PME (mg EA )
Figure 1: Effect of 14-day treatment with PME on the % change in
body weights of rats in the subacute toxicity test. Data are expressed
as mean ± SEM (𝑛 = 5). Treated groups were compared to controls
with a one-way ANOVA followed by Newman-Keuls’ test. The
lower and upper margins of the boxes represent the 25th and 75th
percentiles, while the extended arms represent the 10th and 90th
percentiles, respectively.
1000 mg kg−1 (all at 𝑃 < 0.05) when compared to the control
group (Table 2).
3.2.2. Effect of Extract on Haematological Parameters. Except
for lymphocytes (%) that showed a significant decrease
(𝐹5,23 = 3.93, 𝑃 = 0.013), all other parameters remained
unaffected by the extract (Table 3).
3.2.3. Effect of Extract on Biochemical Parameters. Alanine
transaminase (ALT) and aspartate transferase (AST) levels
were decreased but the decrease was not statistically significant when compared to the control group (Table 4). Bilirubin
levels at the doses of 30 and 100 mg kg−1 were also decreased,
although ANOVA showed no significant difference. ANOVA
showed a significant decrease in the levels of triglyceride
(𝐹5,23 = 3.086, 𝑃 = 0.034) and VLDL (𝐹5,23 = 3.834,
𝑃 = 0.015) with Newman-Keuls’ post hoc analysis, revealing
significance at 100 mg kg−1 (both at 𝑃 < 0.05).
3.2.4. Histopathological Changes. Figures 2–6 show the photomicrographs of sections of the isolated organs of control
and PME-treated rats for the 14-day subacute toxicity study.
Histopathological evaluation of the organs isolated from rats
sacrificed at the end of the subacute toxicity study revealed no
significant extract-related morphological changes compared
to the control animals.
Sections from the splenic tissue (Figure 4) were essentially normal, showing preservation of the lymphoid follicles.
Few follicular enlargements as well as minimal dilation of
the sinusoids were observed. All sections of the kidneys
(Figure 2) showed essentially normal glomerulus, tubules,
and blood vessels. Inflammation and necrosis were also
absent. Furthermore, no casts in the tubules as well as
other deposits in the kidneys were observed. Brain sections
showed well-preserved brain tissue (Figure 5). No significant
morphological changes like necrosis and red neurons were
observed in all the doses of the extract as compared to
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Table 2: Effects of PME on relative organ weights (ROW) of rats in the subacute toxicity test.
Organs
Control
Liver
Kidney
Spleen
Stomach
Brain
3.03 ± 0.26
0.65 ± 0.01
0.48 ± 0.04
1.08 ± 0.05
0.93 ± 0.03
30
2.66 ± 0.11
0.62 ± 0.02
0.44 ± 0.02
1.04 ± 0.04
0.87 ± 0.02
100
2.90 ± 0.09
0.68 ± 0.01
0.65 ± 0.02∗
1.13 ± 0.08
0.94 ± 0.03
PME (mg kg−1 )
300
3.05 ± 0.18
0.72 ± 0.02
0.69 ± 0.09∗
1.17 ± 0.07
1.01 ± 0.06
Data are presented as mean ± SEM (𝑛 = 5). ∗ 𝑃 < 0.05 is considered statistically significant from control.
1000
3.05 ± 0.10
0.70 ± 0.02
0.69 ± 0.02∗
1.13 ± 0.04
1.00 ± 0.06
3000
2.75 ± 0.05
0.66 ± 0.02
0.45 ± 0.03
1.07 ± 0.05
0.88 ± 0.05
Table 3: Effect of 14-day treatment with PME on haematological parameters in rats.
Parameters
WBC (103 /mm3 )
LYM (%)
RBC (106 /mm3 )
HGB (g/dL)
HCT (%)
MCV (𝜇m3 )
MCH (pg)
MCHC (g/dL)
RDW (%)
PLT (103 /mm3 )
MPV (𝜇m3 )
PCT (%)
PDW (%)
Control
4.90 ± 1.66
90.33 ± 4.02
7.49 ± 1.07
13.08 ± 1.57
43.73 ± 5.27
59.00 ± 2.04
17.58 ± 0.51
30.33 ± 0.60
16.03 ± 0.87
883.3 ± 52.71
6.80 ± 0.30
0.60 ± 0.04
10.85 ± 0.66
30
4.63 ± 1.18
86.85 ± 1.12
7.21 ± 0.21
12.68 ± 0.33
41.83 ± 2.19
57.75 ± 1.65
17.58 ± 0.41
30.45 ± 0.91
16.25 ± 0.87
963.0 ± 165.7
7.43 ± 0.27
0.71 ± 0.12
10.85 ± 1.77
100
5.10 ± 1.32
77.0 ± 2.66∗
6.41 ± 0.19
11.43 ± 0.19
37.40 ± 1.43
58.25 ± 0.94
17.85 ± 0.29
30.70 ± 0.86
16.70 ± 0.68
800.0 ± 127.3
6.78 ± 0.39
0.54 ± 0.07
10.93 ± 0.96
PME (mg kg−1 )
300
4.68 ± 0.36
73.3 ± 2.07∗
6.33 ± 0.46
11.20 ± 0.88
37.78 ± 2.48
60.25 ± 2.06
17.68 ± 0.18
29.60 ± 0.99
16.68 ± 0.57
848.5 ± 103.8
6.73 ± 0.32
0.58 ± 0.09
11.63 ± 0.52
Data are presented as mean ± SEM (𝑛 = 5). ∗ 𝑃 < 0.05 is considered statistically significant from control.
1000
5.40 ± 1.19
75.7 ± 2.07∗
6.54 ± 0.39
12.00 ± 0.90
38.43 ± 0.66
59.25 ± 3.04
18.25 ± 0.48
31.10 ± 1.98
15.93 ± 0.45
875.5 ± 139.5
6.95 ± 0.33
0.62 ± 0.11
10.30 ± 2.52
3000
6.40 ± 1.28
74.8 ± 3.14∗
7.28 ± 0.23
12.58 ± 0.55
40.25 ± 1.67
55.25 ± 1.03
17.28 ± 0.44
31.20 ± 0.89
13.80 ± 1.15
850.8 ± 89.91
7.58 ± 0.25
0.64 ± 0.06
9.15 ± 2.30
Table 4: Effect of 14-day treatment with PME on biochemical parameters in rats.
Parameters
AST (U/L)
ALT (U/L)
ALP (U/L)
GGT (U/L)
Total pr. (g/L)
Albumin (g/L)
Globulin (g/L)
Bil. (𝜇mol/L)
D-Bil (𝜇mol/L)
I-Bil (𝜇mol/L)
Urea (mmol/L)
Creat. (mmol/L)
Chol. (mmol/L)
TG (mmol/L)
HDL (mmol/L)
LDL (mmol/L)
VLDL (mmol/L)
Coronary risk
Control
244.9 ± 43.44
122.4 ± 30.54
171.6 ± 28.38
0.97 ± 0.33
66.98 ± 0.47
43.83 ± 1.11
23.10 ± 1.14
1.00 ± 0.39
0.33 ± 0.13
0.67 ± 0.26
6.85 ± 0.94
47.25 ± 6.20
2.20 ± 0.14
0.60 ± 0.03
1.70 ± 0.02
0.20 ± 0.10
0.20 ± 0.01
1.30 ± 0.07
30
273.30 ± 9.17
103.1 ± 17.78
207.3 ± 38.76
0.78 ± 0.24
67.23 ± 2.63
45.33 ± 2.20
21.73 ± 1.51
0.62 ± 0.12
0.22 ± 0.05
0.40 ± 0.08
7.33 ± 0.47
48.75 ± 6.32
2.36 ± 0.16
0.57 ± 0.05
1.86 ± 0.10
0.23 ± 0.06
0.26 ± 0.02
1.25 ± 0.05
100
191.70 ± 11.1
77.70 ± 7.96
176.4 ± 14.01
1.45 ± 0.47
61.63 ± 2.60
41.83 ± 1.73
19.80 ± 0.91
0.48 ± 0.16
0.18 ± 0.05
0.30 ± 0.11
7.61 ± 0.67
46.25 ± 5.43
1.97 ± 0.13
0.4 ± 0.01∗
1.62 ± 0.10
0.18 ± 0.04
0.1 ± 0.01∗
1.22 ± 0.02
PME (mg kg−1 )
300
250.0 ± 23.06
106.6 ± 10.85
207.3 ± 31.67
1.20 ± 0.33
63.08 ± 1.78
41.95 ± 1.73
21.15 ± 0.79
1.13 ± 0.44
0.45 ± 0.24
0.68 ± 0.34
7.74 ± 0.40
53.75 ± 7.56
2.20 ± 0.28
0.56 ± 0.04
1.56 ± 0.10
0.39 ± 0.02
0.24 ± 0.02
1.42 ± 0.12
Data are presented as mean ± SEM (𝑛 = 5). ∗ 𝑃 < 0.05 is considered statistically significant from control.
1000
196.40 ± 8.28
86.15 ± 6.86
197.3 ± 35.09
0.95 ± 0.22
62.80 ± 2.25
39.88 ± 2.13
22.93 ± 3.81
0.88 ± 0.34
0.28 ± 0.11
0.60 ± 0.23
8.40 ± 0.87
41.75 ± 5.89
2.20 ± 0.17
0.61 ± 0.06
1.64 ± 0.13
0.29 ± 0.08
0.27 ± 0.02
1.35 ± 0.06
3000
200.90 ± 3.82
95.0 ± 23.82
168.7 ± 22.60
1.43 ± 0.43
64.18 ± 1.46
38.30 ± 1.13
25.88 ± 2.11
0.78 ± 0.26
0.20 ± 0.07
0.58 ± 0.19
8.54 ± 0.43
51.25 ± 5.39
1.89 ± 0.07
0.53 ± 0.02
1.57 ± 0.07
0.25 ± 0.15
0.23 ± 0.01
1.17 ± 0.02
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Bowman’s capsule
Glomerulus
Distal tubules
Bowman’s space
Proximal tubules
(a)
(b)
(c)
(d)
(e)
(f)
Figure 2: Photomicrograph of the sections of the kidney in control rats (a) and rats treated orally with 30 mg kg−1 (b), 100 mg kg−1 (c),
300 mg kg−1 (d), 1000 mg kg−1 (e), and 3000 mg kg−1 (f) of the extract for 14 days in the subacute toxicity study (H&E, ×400).
the control. With regard to the stomach (Figure 6), there
was essentially no ulceration and inflammation. In addition, the mucosa glands and muscularis propria showed no
abnormalities. At all the doses used, the extract showed no
tendency to induce gastritis. As shown in Table 5, the specific
morphological changes assessed in the liver included fatty
change, hydropic swelling, inflammation, and fibrosis. The
extract-induced inflammation was mainly restricted to the
portal triad with occasional spill over into the hepatocytes in
all the doses. Mild fibrosis (at 3000 mg kg−1 ) and mild fatty
change (at 1000 and 3000 mg kg−1 ) were observed in some
liver sections. In addition, hydropic swelling was observed
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Central vein
Sinusoids
Hepatocytes
(a)
(b)
(c)
(d)
(e)
(f)
Figure 3: Photomicrograph of the sections of the liver in control rats (a) and rats treated orally with 30 mg kg−1 (b), 100 mg kg−1 (c),
300 mg kg−1 (d), 1000 mg kg−1 (e), and 3000 mg kg−1 (f) of the extract for 14 days in the subacute toxicity study (H&E, ×400).
in all rats treated with PME at 1000 and 3000 mg kg−1 . The
toxic effect of the extract on the liver was relatively mild
and only trace evidence was seen at high doses (1000 and
3000 mg kg−1 ).
4. Discussion
Medicinal plants are often considered safe for human use due
to their natural origin. However, various reports suggest the
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White pulp
Red pulp
Central arteriole
(a)
(b)
(c)
(d)
(e)
(f)
Figure 4: Photomicrograph of the sections of the spleen in control rats (a) and rats treated orally with 30 mg kg−1 (b), 100 mg kg−1 (c),
300 mg kg−1 (d), 1000 mg kg−1 (e), and 3000 mg kg−1 (f) of the extract for 14 days in the subacute toxicity study (H&E, ×400).
potential risks involved with such plants [14]. Thus, there is
a need to assess the safety of these medicinal plants before
use. This study therefore assessed both acute and subacute
toxicity studies of Pseudospondias microcarpa hydroethanolic
leaf extract (PME) in rats.
In the acute toxicity study, rats treated with PME showed
signs of sedation and analgesia, suggesting possible central
nervous system (CNS) depressant and analgesic effects. This
confirms a previous study in our laboratory [11], as well as traditional use of the plant. The LD50 of the plant extract, given
orally, was found to be above 3000 mg kg−1 . At the relatively
high doses used, the plant extract caused no mortality and
appeared to cause no apparent toxicity, suggesting that PME
is relatively nontoxic.
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CA1
CA2
CA3
dentate gyrus
(a)
(b)
(c)
(d)
(e)
(f)
Figure 5: Photomicrograph of the sections of the brain in control rats (a) and rats treated orally with 30 mg kg−1 (b), 100 mg kg−1 (c),
300 mg kg−1 (d), 1000 mg kg−1 (e), and 3000 mg kg−1 (f) of the extract for 14 days in the subacute toxicity study (H&E, ×400).
In toxicological evaluation, it is always important to assess
subacute toxicity profile of test compounds, since repeated
dosing helps to evaluate morphological and physiological
changes in organs [15]. Therefore, a subacute toxicity study
was performed in rats and, similar to observations in the
acute toxicity test, treatment with the extract for 14 days
caused no mortality. In addition, water and food consumption was normal.
Body weight change of animals was also assessed, since
changes in the body weights can be used as an indicator
of adverse or toxic effects of drugs [16, 17]. In this study,
it was observed that the change in body weight of animals
treated with the extract was not significant when compared
to the control group, indicating absence of any severe adverse
effects. Aside the body weight change, the liver, spleen,
kidney, brain, and stomach are the primary organs affected
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muscularis mucosae
submucosa
pyloric gland
lamina propria
gastric pits
(a)
(b)
(c)
(d)
(e)
(f)
Figure 6: Photomicrograph of the sections of the stomach in control rats (a) and rats treated orally with 30 mg kg−1 (b), 100 mg kg−1 (c),
300 mg kg−1 (d), 1000 mg kg−1 (e), and 3000 mg kg−1 (f) of the extract for 14 days in the subacute toxicity study (H&E, ×400).
by metabolic reactions induced by toxicants. Therefore, the
relative organ weight is an important index of physiological
and pathological status in man and animals [18–20]. In this
study, PME treatment caused no significant difference in
organ-to-body ratio of the various organs when compared
to the control group, except for the spleen, which showed
an increase. However, gross pathological examination of the
spleen of rats treated with the extract did not reveal any
abnormalities, presence of lesions, or changes in colour.
Analysis of blood parameters in animal studies is important to evaluate the risk of alterations of the haematological
system in human toxicity and also to explain blood related
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Table 5: Histopathological results of the liver in rats orally treated
with PME for 2 weeks.
Dose
(mg kg−1 )
Saline
30
100
300
1000
3000
Fatty
change
Hydropic
swelling
Mild
inflammation
Mild
fibrosis
0/4
0/4
0/4
0/4
2/4
2/4
0/4
0/4
0/4
1/4
4/4
4/4
0/4
0/4
4/4
4/4
4/4
4/4
0/4
0/4
0/4
0/4
0/4
2/4
Results are expressed as the number of rats with pathological findings per
total number of rats sectioned.
functions of a plant extract or its isolates [21–23]. Thus,
various haematological indices were measured. No significant
difference was found in the majority of haematological
parameters between treated and control groups except for the
observed decrease in lymphocytes (%). Lymphocytes are the
main effector cells of the immune system and thus protect
the body from infections [24]. Decreased lymphocytes in the
present study may therefore suggest a low infection resistance, since the effector cells of the immune system might be
affected. Haematological parameters such as MCHC, MCH,
and MCV relate to individual red blood cells, whereas HGB,
RBC, PCV, and RCDW are linked to the total population of
red blood cells. Thus, the unchanged effect of the extract on
these parameters may imply that neither the incorporation
of haemoglobin into red blood cells nor the morphology
and osmotic fragility of the red blood cells were altered
[25, 26]. This therefore excludes the possibility of anaemia
or disturbance linked to erythrocytes and indicates nontoxic
effects of PME on the haematopoietic system.
Liver and kidney functions tests as well as serum lipid
profile are important parameters in determining the safety
of plant extracts or their isolates [27, 28]. To evaluate kidney
function, measurements of urea and creatinine levels in the
blood are usually performed [29]. These two parameters are
usually increased to four or five times the normal values in
control animals in cases of acute or chronic renal toxicity
[17]. This study shows that treatment with Pseudospondias
microcarpa extract in rats for 14 days does not produce
possible kidney malfunction, since the biomarkers of kidney
function (urea and creatinine) were not affected.
The activities of AST and ALT are the most sensitive
tests employed in the diagnosis of hepatic diseases [30].
With damaged liver cell plasma, various enzymes normally
located in the cytosol are released into the blood, resulting in
increased enzyme levels in the serum [31, 32]. Thus, increased
levels of ALT, AST, ALP, and GGT may be interpreted as a
result of liver cell destruction or changes in the membrane
permeability [30, 33]. Measurement of these enzymes in
the serum is therefore a useful quantitative marker of the
extent and type of hepatocellular damage. Treatment with the
extract had no effect on the levels of these serum enzymes,
indicating nontoxic effects on liver function.
An important physiologic role of the liver is the removal
of toxic endogenous and exogenous substances from the
blood. Thus, tests based on excretory functions of the liver
are related to bilirubin metabolism [34]. Bilirubin is the
product of haem following the breakdown of red blood cells
by phagocytic cells. It is carried by serum albumin to the
liver, where most of it is conjugated with glucuronide prior
to excretion into the bile. Increased levels in the blood result
in jaundice and could be due to increased haemolysis of red
blood cells, primary hepatocellular damage, or mechanical
biliary duct obstruction [35]. Therefore, this metabolite
serves as a good indicator to assess the functional capacity
of the liver. In this study, serum levels of direct, indirect,
and total bilirubin after treatment with the extract for 14
days were not elevated, indicating that PME did not have any
deleterious effects on hepatic metabolism or biliary excretion.
However, although not significantly, the extract decreased
levels of direct, indirect, and total bilirubin, especially at the
low doses used (30 and 100 mg kg−1 ). The bilirubin-lowering
effect as well as decreased ALT levels could suggest possible
hepatoprotective effects of the extract, confirming traditional
use of the plant for treating jaundice [4].
Cholesterol, apart from acting as a precursor for the
synthesis of bile acids, hormones, and vitamins, is also an
essential component of most biological membranes [36, 37].
However, an increase in its levels could lead to atherosclerosis,
hyperproteinaemia, cirrhosis, haemolytic jaundice, malnutrition, and increased risk of cardiovascular diseases [38, 39].
As the primary carrier of plasma cholesterol, low-density
lipoprotein (LDL) is often referred to as bad cholesterol,
since increased levels cause atherosclerosis [40]. In addition,
elevated LDL levels have been reported to be associated with
hepatic lesion and damage [31, 41]. Furthermore, increased
levels of serum triglyceride lead to hyperlipidaemia and
low levels imply that there is no risk factor related to
atherosclerosis [38]. In the present study, decreased levels
of triglycerides, LDL, and VLDL particularly at the low
dose of 100 mg kg−1 coupled with normal cholesterol levels
further confirm normal liver function and decreased risk
of atherosclerosis. The decreased levels of VLDL and LDL
cholesterols could also indicate hypolipidemic effects of the
extract.
Evaluation of pathological alterations induced in laboratory animals by novel drugs represents the basis of their
safety assessment before they can be used in the clinical
setting and this is largely based on conventional histopathological techniques [42, 43]. Therefore, histological analysis
was done to further examine the pathological state of the
various organs. Except for the liver that showed mild fatty
change, hydropic swelling, and mild inflammation at the
highest doses (1000 and 3000 mg kg−1 ), detailed gross and
histopathological examination of the various organs showed
no significant pathological changes. These changes in liver are
mild and may not be considered clinically significant, since
serum levels of hepatic enzymes (ALT and AST), which are
considered markers of liver function, were not significantly
elevated. However, caution should be taken in using this
extract beyond 3000 mg kg−1 . In addition, chronic toxicity
studies should be done in rats and other species to ascertain
the safety of the plant extract.
The Scientific World Journal
11
5. Conclusion
Results of this study show that oral administration of the
hydroethanolic leaf extract of Pseudospondias microcarpa is
relatively safe in rats. However, caution should be exercised
when extrapolating this result to man.
[11]
Conflicts of Interest
[12]
The authors declare that there are no conflicts of interest.
Acknowledgments
[13]
The authors are grateful to Messrs Thomas Ansah, Gordon
Darku, Prosper Akortia, Edmond Dery, and Prince Okyere
for their technical assistance.
[14]
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