Abraham and Ahmad
Bulletin of the National Research Centre
(2021) 45:226
https://doi.org/10.1186/s42269-021-00686-9
Bulletin of the National
Research Centre
Open Access
RESEARCH
Preliminary sub-acute toxicological
assessment of methanol leaves extract
of Culcasia angolensis (Araceae) in Wistar rats
Idagu Godwin Abraham1
and Mubarak Hussaini Ahmad2*
Abstract
Background: The plant Culcasia angolensis (Araceae) has diverse ethnomedicinal uses, including the management
of rheumatic pain, healing of cuts, dislocations, and bruises. Despite its potential therapeutic uses, the toxicity profile
of Culcasia angolensis has not been evaluated. This study assessed the sub-acute toxicity effects of Culcasia angolensis leaves extract (CAE). The phytochemical determination of the CAE was conducted as per the standard protocols.
The median lethal dose (LD50) was determined using the Organization for Economic Cooperation and Development
(OECD) 423 guideline. Besides, the sub-acute toxic effects of the CAE (125, 250, and 500 mg/kg) were investigated
following administration of the CAE daily for 28 consecutive days as per the OECD 407 guideline. The weekly body
weights were recorded. The animals were euthanized on the 29th day, and blood samples were obtained for haematological and biochemical investigations. The heart, kidney, liver, and lungs were collected for histological examinations. Besides, the relative organ weights (ROW) were determined.
Results: The CAE contains cardiac glycosides, alkaloids, tannins, flavonoids, steroids, saponins, and terpenoids. The
oral LD50 was above 5 g/kg. There was a remarkable decline in the weekly body weight at all the CAE doses. The CAE
increased the lymphocytes, aspartate transaminase, and urea. However, the levels of alanine transaminase and alkaline phosphatase were elevated remarkably. The histological studies did not reveal any serious organs abnormalities.
Conclusion: The CAE is relatively safe on acute administration. However, it may be slightly toxic on sub-acute administration, especially to the liver and kidney.
Keywords: Biochemical parameters, Weekly body weight, Culcasia angolensis, Haematological parameters, Relative
organ weight, Sub-acute toxicity
Background
The plant-derived products have been used for nutritional and therapeutic purposes to manage various
pathological conditions and form the major source of
medicinal products (Jiménez-estrada et al. 2013; Miekus
et al. 2020). About 80% of the global population use
plants products as a basis for their primary health needs,
particularly in developing countries (Ahmad et al. 2021a,
*Correspondence: mubarakhussainiahmad@gmail.com
2
Department of Pharmacology and Therapeutics, Ahmadu Bello
University, Zaria, Kaduna, Nigeria
Full list of author information is available at the end of the article
b). Several scientific studies have reported promising
therapeutic properties of medicinal plants that could
guide drug development and discovery (Anand et al.
2019; Bernardini et al. 2018). The urgent requirement to
discover new drugs, global attention in herbal products,
and the high price of orthodox medications result in an
upsurge in the utilization of herbal preparations from traditional practice (Ouedraogo et al. 2012). Besides, there
has been a general perception that medicinal plants are
without adverse effects (Seremet et al. 2018). However,
many of them have been reported to be toxic (Kharchoufa et al. 2018; Nasri and Shirzad 2013; Ndhlala et al.
2013). Besides, limited data exist on the clinical and
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Abraham and Ahmad Bulletin of the National Research Centre
(2021) 45:226
safety profile of many medicinal plants (Zhou et al. 2013).
Therefore, it is essential to document scientific information on the toxic concerns of herbal preparations, including the plant Culcasia angolensis to increase confidence
in therapeutic use and to discover effective medicinal
products (Ahmad et al. 2021a, b; Ukwuani et al. 2012).
The plant Culcasia angolensis is a part of the family
Araceae. It is a robust forest climber tree with thick and
tough stems (about 6 cm in diameter) that grow more
than 30 m in length and attaches to its host by clasping
roots (Burkill 1985). The plant is widely available in tropical African nations, including Sierra Leone, Cameroon,
and Angola. The whole plant is harvested from the wild
environment for use in traditional medicine. The Culcasia angolensis leaves have been in use against menstrual
problems, pain, and inflammation in Africa, including
Nigeria (Bown 2000). However, safety information on
the plant is not available in the literature. Therefore, this
research was intended to check the safety profile of Culcasia angolensis leaves extract (CAE) after the acute and
sub-acute administration via oral route to stimulate more
research and discover novel, effective, and safe medicinal
compounds.
Page 2 of 10
with chloroform and euthanized by cervical dislocation,
after which they were appropriately buried in accordance with the University guide of disposing the remains
of experimental animals.
Plant extraction
The Culcasia angolensis leaves were shade-dried with frequent weighing until a constant weight was achieved and
powdered into fine particles with mortar and pestle. The
dried and finely powdered leaves (1500 g) were extracted
with methanol (70%v/v) using Soxhlet apparatus for 72 h.
The CAE was concentrated at reduced pressure at a temperature of 45 °C on a water bath and kept in a closed
container tightly. The percentage yield of the extract was
calculated as follows:
Percentage yield (% )
=
Weight of the Culcasia angolensis leaves extract g
× 100.
Weight of the dried and powdered Culcasia angolensis leaves g
Phytochemical investigation
The phytochemical analysis to determine the secondary
metabolites present in CAE was carried out as per the
method previously reported by Sofowora (1993).
Methods
Plant collection and authentication
Acute toxicity
The Culcasia angolensis was obtained from Ngaring Nok,
Jaba Local Government of Kaduna State, Nigeria, in April
2020 and authenticated by Mallam Namadi Sanusi at the
Herbarium section of the Botany Department, Faculty of
Life Sciences, Ahmadu Bello University (ABU), Nigeria.
The comparison of the plant was with a specimen previously kept at the herbarium. The voucher sample number
was 01676.
The acute toxic actions of the CAE were evaluated in
rats as per the Organization of Economic Co-operation
and Development (OECD) 423 guideline (OECD 2001).
The median lethal dose (LD50) following oral administration was evaluated in nulliparous and non-pregnant
female rats. Two groups with three rats were fasted
before extract administration. (Food was withheld overnight for rats and 3 h for mice with the provision of water
sufficiently.) In the first phase, 2000 mg/kg of the CAE
was administered to each rat and observed for 48 h for
any sign of toxicity and mortality. In the second phase,
5000 mg/kg of the CAE was administered to the rats and
checked for signs of adverse effects once every 30 min
within the first 4 h and subsequently for 14 consecutive
days.
Laboratory animals
Both genders of adult Wistar rats (160–200 g) employed
for the experiment were obtained from the laboratory animal section of Pharmacology and Therapeutics
Department, ABU, Zaria, Nigeria. The animals were kept
in clean, dried, adequately ventilated cages with sufficient
and standard laboratory feed (Vital feed, Jos, Nigeria) and
water provision adequately. They were kept at optimum
laboratory conditions (temperature 22 ± 3 0C, relative
humidity 30–70% with 12 h light and 12 h dark). The animals were acclimatized for two weeks to the laboratory
environment before the experimental work commenced.
The permission to conduct the experiment was given
by ABU Ethical Committee on Animal Use and Care
Research Policy (ABUCAUC) with a permission number (ABUCAUC/2019/006) as per the ARRIVE (Animal
Research: Reporting of In Vivo Experiments) guidelines.
At the end of the experiment, the rats were anaesthetized
Sub‑acute toxicity investigation
The Organization for Economic Co-operation and Development (OECD) test guideline 407 was used (OECD
2008). Twenty-four rats (both genders) were categorized into four groups, with 3 males and 3 female rats
per group. (The males were separated from the females.)
The rats were orally treated daily with CAE (125, 250, and
500 mg/kg) and distilled water (1 ml/kg) for 28 days. The
weekly body weight was determined, and signs of harmful effects and deaths were monitored. On day 29, they
Abraham and Ahmad Bulletin of the National Research Centre
(2021) 45:226
were deprived of food with free access to water for 24 h
and euthanized (inhaled chloroform). The blood samples
were obtained from each rat through the cardiac puncture into ethylenediaminetetraacetic acid (EDTA) and
plain containers for haematological and biochemical
investigations, respectively. Some organs (liver, kidney,
heart, and lung) were dissected out free from adjoining
supportive tissues, gently rinsed in normal saline, blotted
with filter paper, and weighed. The relative organ weight
(ROW) of the organs was determined using the following
relation:
Absolute organ weight g
× 100.
ROW =
Final body weight of the animal g
Haematological analysis
Page 3 of 10
Results
Percentage yield
A sticky-black solid residue weighing 107.6 g with a mild
smell was obtained from a 1500 g crude plant of Culcasia
angolensis powdered sample representing 7.2% w/w as the
percentage yield.
Phytochemical constituents
Preliminary phytochemical determination of CAE
showed cardiac glycosides, triterpenes, tannins, flavonoids, alkaloids, saponins, and steroids.
Acute toxicity study
Acute oral toxicity results showed that the CAE has no
adverse effect in rats. Besides, no mortality was observed
at the dose levels tested. Therefore, the oral LD50 of the
CAE could be above 5000 mg/kg.
The haematological investigation was conducted to
determine any possible changes in the levels of haematocrit (HCT), platelet count (PLT), red blood cell (RBC)
count, haemoglobin (HB), white blood cell (WBC) count,
monocytes (MON), lymphocytes (LYMPH), neutrophils
(NTP), and eosinophils (ENP) (OECD 2008).
The CAE significantly declined the rats’ body weight at
all the doses tested. Figure 1 displays the results of the
28-day oral administration of CAE on the weekly body
weight.
Biochemical analysis
Relative organ weight
The non-heparinized blood samples were stored at room
temperature for 1 h to clot and centrifuged at 3000 revolutions per minute for 10 min. The plasma obtained was
used to determine any changes in the biochemical biomarkers, including alanine transaminase (ALT), alkaline
phosphatase (ALP), aspartate transaminase (AST), total
protein, albumin urea, and creatinine (OECD 2008).
The CAE produced no remarkable change in the ROW of
the selected organs at all the doses as shown in Table 1.
The liver, kidneys, lung, and heart of each animal were
fixed in 10% formalin. The sections of the organs were
cut 4–5 µm with rotary microtone, stained with haematoxylin and eosin, and analysed at a magnification of
250× for any histopathological changes by a consultant
histopathologist.
Statistics
All the values were displayed as mean values ± SEM
in figures and tables. One-way analysis of variance
(ANOVA) was used to analyse the ROW, biochemical,
and haematological parameters, whereas repeated measure ANOVA was employed to analyse the weekly body
weight. Dunnett’s post hoc test was employed to compare
the means. The p ≤ 0.05 values were taken as significant.
Haematological parameters
There were no significant alterations in the levels of HCT,
HGB RBC, PLT, WBC, MON, ENP, and NTP in relation
to the control class. On the contrary, a remarkable elevation in lymphocytes was detected at 500 mg/kg as shown
in Table 2.
180
control
Body weight of rats (g)
Histopathology
Weekly body weight
CAE 125mg/kg
160
CAE 250mg/kg
140
*
*
*
*
3
4
*
*
*
CAE 500 mg/kg
*
120
100
0
1
2
Time (weeks)
Fig. 1 Weekly body weight of rats following 28-day repeated
administration Culcasia angolensis leaves extract (CAE). The values
were displayed as mean ± SEM; *p ≤ 0.05 in relation to the control
group (repeated measure ANOVA followed by Dunnett’s post hoc
test, n = 6, CAE Culcasia angolensis leaves extract)
Abraham and Ahmad Bulletin of the National Research Centre
(2021) 45:226
Page 4 of 10
Table 1 Relative organ weight of rats following 28-day repeated
administration of Culcasia angolensis leaves extract (CAE)
Table 3 Hepatic parameters of rats following 28-day repeated
administration of Culcasia angolensis leaves extract (CAE)
Organs
Parameters Treatment (mg/kg)
Treatment groups (mg/kg)
DW (1 ml/kg)
CAE (125)
CAE (250)
CAE (500)
DW (1 ml/
kg)
CAE (125)
CAE (250)
CAE (500)
15.1 ± 1.57*
16.3 ± 2.07*
Liver
7.56 ± 0.64
7.71 ± 0.22
7.68 ± 0.09
7.54 ± 0.14
Kidney
0.74 ± 0.45
0.77 ± 0.78
0.81 ± 0.92
0.76 ± 0.07
ALT (IU/L)
26.2 ± 4.11
27.2 ± 7.20
37.67 ± 0.92
41.68 ± 0.58* 44.80 ± 1.67* 59.66 ± 2.28*
Lungs
2.29 ± 0.04
2.33 ± 0.03
2.38 ± 0.04
2.27 ± 0.06
AST (IU/L)
Heart
0.68 ± 4.42
0.70 ± 9.81
0.72 ± 4.62
0.75 ± 9.72
ALP (IU/L)
22.76 ± 3.38
19.68 ± 4.32
TP (mg/dL)
6.54 ± 0.24
6.69 ± 0.32
6.44 ± 0.30
6.93 ± 0.34
ALB (mg/dL)
3.14 ± 0.07
3.17 ± 0.08
3.08 ± 0.06
3.03 ± 0.09
The values were documented as mean ± SEM (one-way ANOVA followed by
Dunnett’s post hoc test)
DW distilled water, p.o per oral, CAE Culcasia angolensis leaves extract, n = 6
15.80 ± 3.24* 19.21 ± 4.03
The values were tabulated as mean ± SEM, *p ≤ 0.05 in relation to control group
(one-way ANOVA followed by Dunnett’s post hoc test), n = 6
DW distilled water, ALT alanine transaminase, AST aspartate transaminase, ALP
alkaline phosphatase, TP total protein, ALB albumin, CAE Culcasia angolensis
leaves extract
Table 2 Haematological parameters of rats following 28-day
repeated administration of Culcasia angolensis leaves extract
(CAE)
Parameters
Treatment (mg/kg)
DW (1 ml/
kg)
WBC
(× 109/L)
3.85 ± 0.27
CAE (125)
4.07 ± 0.22
CAE (250)
4.27 ± 0.10
CAE (500)
3.85 ± 0.15
HGB (g/dL)
12.72 ± 0.33
RBC (× 106/
µL)
5.85 ± 0.17
5.98 ± 0.15
5.70 ± 0.14
5.93 ± 0.19
PLT (× 103/
µL)
7.20 ± 0.10
7.18 ± 0.10
7.18 ± 0.11
7.08 ± 0.06
HCT (%)
37.16 ± 1.86
MON (%)
2.33 ± 0.56
LYMPH (%)
78.17 ± 1.17
ENP (%)
2.17 ± 0.30
NTP (%)
17.33 ± 2.08
12.62 ± 0.97 11.15 ± 0.69 11.12 ± 0.51
36.67 ± 3.18 35.00 ± 1.97 35.17 ± 1.30
1.67 ± 0.21
1.50 ± 0.34
2.00 ± 0.37
77.67 ± 0.72 78.50 ± 0.99 81.33 ± 1.38*
1.66 ± 0.33
2.00 ± 0.36
1.83 ± 0.31
19.00 ± 0.97 18.00 ± 1.15 15.00 ± 0.97
The values were tabulated as mean ± SEM, *p ≤ 0.05 in relation to the control
group (one-way ANOVA followed by Dunnett’s post hoc test), n = 6
DW distilled water, CAE Culcasia angolensis leaves extract, WBC white blood cell,
RBC red blood cells, HGB haemoglobin, HCT haematocrit, PLT platelets, LYMP
lymphocytes, MON monocytes, ENP EOSINOPHILS, NTP neutrophils
Hepatic parameters
The ALT was remarkably reduced at 250 and 500 mg/
kg. Besides, ALP was reduced in the category that
received the 250 mg/kg of the CAE. However, there was
an elevation of AST in relation to the control group. No
alteration was observed in the protein and albumin levels. The effects of the CAE on hepatic parameters are
displayed in Table 3.
Kidney parameters
The result showed a remarkable elevation in the plasma
urea level in the group that received 500 mg/kg of the
CAE related to the normal group. On the contrary, no
remarkable change in the creatinine levels and serum
electrolytes (sodium, potassium, chlorine, and bicarbonate) was observed as shown in Table 4.
Histopathology
There were no histopathological alterations in the hepatic
tissue of the group treated with 125 mg/kg of the CAE.
In contrast, the groups that received the extract at 250
and 500 mg/kg showed moderate hepatic necrosis (HN)
(Fig. 2). Besides, the groups that received the lowest
(125 mg/kg) and highest (500 mg/kg) doses revealed
slight tubular necrosis (TN), whereas lymphocyte hyperplasia (LH) was observed at 250 mg/kg (Fig. 3). However,
no histopathological abnormalities were observed in the
heart muscles of rats in all the treated categories (Fig. 4).
Slight alveoli congestion (AC) was observed in the categories that received the lower doses (125 and 250 mg/kg),
whereas the CAE (500 mg/kg) revealed nuclei hardening
and pyknosis (HP) (Fig. 5).
Discussion
The herbal products have been used in a traditional practice because they have chemical agents with potential
therapeutic actions for treating various human diseases
(Hosseinzadeh et al. 2015). However, experimental investigations have shown that some of the herbal products
are toxic, which necessitates the need to examine the toxicological effect of plants with medicinal values (Ndhlala
et al. 2013). In fact, international regulatory agencies
such as Food and Drug Administration (FDA) have
encouraged taking effective strategies against the use of
herbal products with no scientific and toxicological information (De Smet 2004; Kale et al. 2019).
Abraham and Ahmad Bulletin of the National Research Centre
(2021) 45:226
Page 5 of 10
Table 4 Kidney parameters of rats following 28-day repeated administration of Culcasia angolensis leaves extract (CAE)
Parameters
Treatment group (mg/kg)
DW (1 ml/kg)
Urea (mmol/L)
Creatinine (mmol/L)
Sodium (mmol/L)
Potassium (mmol/L)
CAE (125)
32.2 ± 1.22
CAE (250)
CAE (500)
30.80 ± 0.89
31.66 ± 2.70
0.80 ± 0.05
0.85 ± 0.11
0.88 ± 0.12
38.40 ± 1.82*
0.90 ± 0.60
220.32 ± 7.44
221.56 ± 2.99
225.08 ± 6.91
222.98 ± 3.24
7.90 ± 0.10
6.76 ± 1.20
7.30 ± 0.36
6.60 ± 2.27
Chloride (mmol/L)
96.67 ± 5.39
98.67 ± 5.26
87.83 ± 3.02
89.40 ± 1.50
Bicarbonate (mmol/L)
88.8 ± 3.30
88.60 ± 2.11
94.50 ± 3.71
89.84 ± 1.82
The values were tabulated as mean ± SEM; *p ≤ 0.05 compared to control group (one-way ANOVA followed by Dunnett’s post hoc test), n = 6
DW distilled water, CAE Culcasia angolensis leaves extract
Fig. 2 Photomicrographs of liver sections of rats following 28-day oral administrations of Culcasia angolensis leaves extract (CAE) (haematoxylin
and eosin-stained at ×250 magnification). A Control (distilled water); B CAE (125 mg/kg); C CAE (250 mg/kg); D CAE (500 mg/kg), H (normal
hepatocytes); HN (moderate hepatocyte necrosis)
Plants possess bioactive chemicals that serve as lead
in drug discovery (Susanto et al. 2017). Besides, documenting the safety data of medicinal plants is important
for the subsequent pharmacological screening (Momin
et al. 2014). The secondary metabolites’ investigation in
the current work shows that the CAE possesses cardiac
glycosides, triterpenes, tannins, flavonoids, alkaloids,
saponins, and steroids. Some of these phytochemical compounds have various pharmacological actions
(Kpemissi et al. 2020). However, despite their promising therapeutic actions, they could have toxicity effects
(Kpemissi et al. 2019). For instance, cardiac glycosides
are associated with cardiovascular toxicity such as heart
muscles lesions and arrhythmias (Botelho et al. 2018),
tannins could cause mild hepatic and renal disturbances
(Ekambaram et al. 2018), and flavonoids cause hepatic
failure, haemolytic anaemia, hypoglycaemia (Galati and
Brien 2004). Besides, alkaloids and saponins are associated with hepatic failure (Qin et al. 2009; Wiedenfeld
2011), and steroids have cardiovascular and hepatic toxicity as well as immune suppression effects (Amsterdam
et al. 2010; Heming et al. 2018).
The study of acute harmful effects of biological
agents is utilized to check the toxic potential of bioactive agents following single-dose administration for the
short term and is the starting point in determining the
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(2021) 45:226
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Fig. 3 Photomicrographs of kidney sections of rats following 28-day oral administrations of Culcasia angolensis leaves extract (CAE) (haematoxylin
and eosin-stained at ×250 magnification). A Control (distilled water); B CAE (125 mg/kg); C CAE (250 mg/kg); D CAE (500 mg/kg), T (normal kidney
tubules and glomerulus); TN (slight tubular necrosis); LH (lymphocyte hyperplasia)
Fig. 4 Photomicrographs of heart sections of rats following 28-day oral administrations of Culcasia angolensis leaves extract (CAE) (haematoxylin
and eosin-stained at ×250 magnification). A Control (distilled water); B CAE (125 mg/kg); C CAE (250 mg/kg); D CAE (500 mg/kg), M (normal cardiac
muscles)
pharmacological actions of unknown agents (Kpemissi
et al. 2020; Musila et al. 2017). Additionally, toxicity
assessment plays an essential part in determining the
LD50 of compounds (Ugwah-oguejiofor et al. 2019).
Hence, the lack of obvious toxic signs and mortality by
the CAE after the single administration showed that the
LD50 could be above 5000 mg/kg. The result concurs
with the report on hesperidin sourced from orange peel
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Fig. 5 Photomicrographs of lung sections of rats following 28-day oral administrations of Culcasia angolensis leaves extract (CAE) (haematoxylin
and eosin-stained at ×250 magnification). A Control (distilled water); B CAE (125 mg/kg); C CAE (250 mg/kg); D CAE (500 mg/kg), A (normal lung
alveoli); AC (slight alveoli congestion); HP (nuclei hardening and pyknosis)
(Li et al. 2019) and the leaves of Combretum hypopilinum (Ahmad et al. 2020a, b).
The determination of sub-acute harmful actions is
used to document an information on the safety profile of
chemical agents following 28-day repeated oral administration in rodents (Christapher et al. 2017). In addition, it
shows the cumulative effects of the agents to certain tissues and organs (Loha et al. 2019). The parameters that
are used in checking the long-term toxicological data
of herbal products include animals’ general behaviour,
body weight, biochemical and haematological indices,
and histopathological outcome (Jothy et al. 2011). Similar to the acute toxicity results in the current work, the
CAE did not produce any clear signs of toxicity and death
throughout the experimental period (28 days).
An alteration in the animals’ body weight indicates
harmful actions after being exposed to harmful agents
due to fat accumulation, loss of appetite, and low caloric
consumption (Prasanth et al. 2015). Likewise, reduction in organ weight expresses toxicity from toxic agents.
The toxic effects of herbal preparations usually target key
organs such as the kidney, liver, heart, and spleen (Unuofin
et al. 2018). The body weight reduction caused by the
CAE in this study could be attributed to loss of appetite,
which may reduce food intake and interfered with nutrient absorption. Besides, the presence of phytocomponents
such as saponins and tannins in the CAE could have produced antinutritional effects by interfering with nutrients
absorption (Nguenang et al. 2020). The declined body
weight in the current research is in line with the reduced
ALP levels observed in the hepatic biomarkers which
could have resulted in malnutrition, vitamins, and mineral deficiency due poor intestinal absorption (El Kabbaoui
et al. 2017; Ray et al. 2017). Previous studies have shown
that various plant extracts such as Bridelia ferruginea
(Bakoma et al. 2013) and Epigynum auritum (Yang et al.
2019) reduced the animals’ body weight. However, other
plant extracts such as Campomanesia velutina (Araújo
et al. 2017) and Lycopersicon esculentum (Nguenang et al.
2020) have no effect on body weight.
Several toxic agents target the haematopoietic system,
which is a key indicator for health status (El Kabbaoui
et al. 2017). An alteration in the blood parameters including RBC, HGB, HCT, MCH, MCV, MCHC is related to
blood disorders, especially anaemia and heart-related
diseases (Olorunnisola et al. 2012), whereas WBC including lymphocytes acts against infectious agents, inflammatory processes, and tissue injury (Hervé et al. 2020). The
present result has shown that the CAE may not interfere
with erythropoiesis and could be devoid of heart-related
complications. The outcome concurs with the cardiac
histology in the current experiments that revealed a lack
of cardiotoxicity of the extract. Besides, the remarkable
increase in the lymphocyte level shows that the CAE
may contain biologically active agents that activate the
immune system. The result corroborates with the work of
Nguenang et al. (2020), which shows that the Lycopersicon esculentum leaves extract possesses immune-stimulatory actions.
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The principal organ where the biotransformation of
drugs and other bioactive agents takes place is in the liver
(Nguenang et al. 2020). The ALT, AST, and ALP are liver
biomarkers that determine the liver metabolic activities
(El Kabbaoui et al. 2017). They are used to evaluate and
manage hepatic disorders (Kim et al. 2012; Villela-nogueira et al. 2005). After a hepatic cellular injury, the levels
of these enzymes increase in the serum due to changes in
cell membrane permeability (Li et al. 2019), whereas their
reduction is associated with chronic kidney disease that
could complicate hepatic injury (Cavalcanti et al. 2012).
The ALP is also important in evaluating biliary duct diseases, and its reduction is a result of vitamin C and B12
deficiency, malnutrition, hypothyroidism, hypophosphatasia, as well as magnesium and zinc deficiency (El Kabbaoui
et al. 2017; Ray et al. 2017). Therefore, the dose-dependent
elevation of the AST in the present study shows that the
CAE could be hepatotoxic which is evident from the histopathological results of the liver in this research that indicates hepatic necrosis. Besides, the extract may adversely
affect renal function as shown by the elevated urea levels
and tubular necrosis from the outcomes of renal parameters and kidney histopathological examinations respectively. The findings have also shown that the CAE could
cause malnutrition, mineral, and vitamin (C and B12)
deficiency as a result of the reduced ALT and ALP, respectively. The malnutrition and vitamins and mineral deficiency produced by the CAE in the present study could be
related to the reduction in the animals’ body weight due
to improper absorption. Previous research has shown the
hepatic effects of Psidium guajava (Manekeng et al. 2019).
The kidney is responsible for maintaining vital physiological processes such as regulation of acid–base, electrolytes, and blood pressure (Bencheikh et al. 2021).
Renal toxicity arises as a result of the inability of the kidney to sufficiently detoxify and remove toxicants (Kim
and Moon 2012). Creatinine and urea are metabolic byproducts removed from the body by glomerular filtration
and used as an index for nephrotoxicity (Aprioku et al.
2014). In kidney disorder, the plasma urea levels increase
due to the increased production, which is often used as a
reliable indicator for assessing renal function (Oyagbemi
et al. 2013). Therefore, the elevated plasma urea level at
500 mg/kg in this study shows that the CAE could lead
to kidney damage due to enhanced urea production that
exceeded its clearance. The outcome corroborates with
the renal histopathology that shows tubular and glomerular necrosis. Other plant extracts such as Terminalia
schimperiana (Awotunde et al. 2019), Simarouba glauca
(Osagie-Eweka et al. 2021), and Caralluma dalzielii
(Ugwah-oguejiofor et al. 2019) revealed a possible renal
effect by remarkably increasing the plasma urea levels.
Page 8 of 10
The toxicological assessment of bioactive agents comprises histopathological examination of vital organs
(Traesel et al. 2016). Hepatic necrosis is an essential
marker for hepatotoxic studies. Hepatocytes necrosis
usually arises due to inflammatory response, neutrophils,
and mononuclear cells recruitment into the hepatic tissues. Besides, hepatic necrosis is accompanied by hepatic
nuclear pyknosis and eosinophilic infiltration (Sharifudin
et al. 2013). The slight hepatic necrosis observed in this
work further showed the possible hepatotoxic actions of
the CAE, which concurs with the elevated AST observed.
The slight tubular necrosis of the kidney observed at 250
and 500 mg/kg could be related to the delivery of harmful agents from the systemic circulation to the kidney and
cause renal tubular system malfunction. It was reported
that severe glomerular and renal tubular damage could
impair renal tubular reabsorption, glomerular filtration,
and electrolyte absorption which could be associated with
the accumulated urea levels beyond the excretory ability of the kidney (Zhao et al. 2007). The alveoli congestion
observed in the group administered with 125 and 500 mg/
kg indicates that the CAE could impair oxygen diffusion
and other gases across the alveoli epithelium and into the
pulmonary circulation. The absence of histological alteration in the heart architecture shows that the CAE may not
possess cardiotoxicity. Besides, the delivery of blood to the
cardiac muscles may not be affected as shown by the noninterference of the extract with RBC production.
Conclusions
The outcome of the study has shown that the CAE could
be relatively safe on acute administration. However, it
may be slightly toxic on sub-acute administration, especially to the liver and kidney. Therefore, more research to
determine the chronic toxicity effects of the plant should
be conducted. Besides, traditional herbal practitioners should be educated on the possible harmful effects
related to the long-term intake of Culcasia angolensis.
Abbreviations
ABU: Ahmadu Bello University; ABUCAUC: ABU Ethical Committee on Animal
Use and Care Research Policy; ALP: Alkaline phosphatase; ALT: Alanine
transaminase; ALT: Aspartate transaminase; ANOVA: One-way analysis of
variance; ARRIVE: Animal Research: Reporting of In Vivo Experiments; CAE:
Culcasia angolensis leaves extract; EDTA: Ethylenediaminetetraacetic acid; ENP:
Eosinophils; HB: Haemoglobin; HCT: Haematocrit; LD50: Median lethal dose;
LYMPH: Lymphocytes; MON: Monocytes; NTP: Neutrophils; OECD: Organization
of Economic Co-operation and Development; PLT: Platelet; RBC: Red blood
cell; ROW: Relative organ weight; WBC: White blood cell.
Acknowledgements
The authors thank all the Pharmacology and Therapeutics Department staff,
ABU, Nigeria, for the support throughout the research period.
Abraham and Ahmad Bulletin of the National Research Centre
(2021) 45:226
Authors’ contributions
IGA contributed to conceptualization, investigation, writing—original draft,
and data analysis. MHA was involved in writing, review, and editing. All the
authors read and approved the final manuscript.
Funding
Not applicable.
Availability of data and materials
The datasets generated during and/or analysed during the current study are
available with the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
The permission for the experiment was given by the Ahmadu Bello University
Ethical Committee on Animal Use and Care Research Policy (permission
number: ABUCAUC/2019/006) and carried out as per the Animal Research
Reporting of In Vivo Experiments (ARRIVE) protocols.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Pharmacology and Toxicology, Faculty of Pharmacy, University
of Calabar, Calabar, Nigeria. 2 Department of Pharmacology and Therapeutics,
Ahmadu Bello University, Zaria, Kaduna, Nigeria.
Received: 10 September 2021 Accepted: 11 December 2021
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