Toxicology and Environmental Health Sciences

https://doi.org/10.1007/s13530-023-00176-7

ORIGINAL ARTICLE

Sub‑acute toxicity study of the aqueous extract from leaves

and flowers of Acmella caulirhiza on female albino Wistar rats
Huiny Miriane Fotso Tienoue1 · Françoise Raïssa Ntentie1,2 · Mary‑Ann Angie Mbong1 ·
Ferdinand Larvin Ebouel Edoun1,3 · Inelle Makamwe1 · Janvier Aimé Fotso Youovop1 · Enyong Julius Oben1

Accepted: 23 May 2023

© The Author(s), under exclusive licence to Korean Society of Environmental Risk Assessment and Health Science 2023

Abstract
Object Acmella caulirhiza is a medicinal plant traditionally widely used in Cameroon for the management of several patholo-
gies, hence the need for confirming its pharmacological properties. The objective of this study was to evaluate the subacute
toxicity of the aqueous extract of leaves and flowers of A. caulirhiza (AE-AC) on Wistar rats.
Methods Three groups of female rats received the aqueous extract of A. caulirhiza (AE-AC) at 100, 250, or 500 mg k­ g−1 Bw
doses respectively while a normal group (NG) received distilled water by oral intubation at 10 mL ­kg−1 Bw daily for 28 days.
Animals were weighed every 4 days, death and general toxicity signs were recorded. At the end, rats were fasted for 12 h and
after diazepam and ketamine anaesthesia, they were sacrificed; blood was collected for blood count and biochemical analysis.
The liver integrity was assessed through transaminase activities, total protein, total cholesterol, and glucose levels, and the
kidney integrity through the evaluation of uric acid, and electrolytes level. Histology of some vital organs was also carried out.
Results Administration of the extract did not result in death or any observable deleterious effects in rats. No difference in
body weight variation of the animals was noted. At 100 and 250 mg/kg Bw doses, AE-AC induced hepatic (through the
decrease in transaminase activities and total cholesterol level) and nephroprotective effects (through the decrease in creati-
nine, uric acid and electrolyte levels) and no change of microarchitecture among treated rats compared to the control group.
AE-AC led to an increase in the relative weight of the brain, uterus, and ovaries as well as a change in some haematological
parameters compared to normal rats.
Conclusion Results indicate that AE-AC had immune-stimulatory effects on rats but could have deleterious effects at 500 mg/
kg Bw.

Keywords Sub-acute toxicity · A. caulirhiza · Aqueous extract · Organ’s integrity

Abbreviations Hcte Hematocrit

AE-AC The aqueous extract of Acmella caulirhiza Lymp Lymphocytes
ALAT Alanine Amino Transferase MCHC Mean Corpuscular Hemoglobin Concentration
ASAT Aspartate Amino Transferase MCH Mean Corpuscular Hemoglobin
Hb Hemoglobin MCV Mean corpuscular volume
Mon Monocytes
NIH National Institutes of Health
* Françoise Raïssa Ntentie OECD Organization for Economic Cooperation and
franc_ntentie@yahoo.fr Development
1
Laboratory of Nutrition and Nutritional Biochemistry, PCV Packed cell volume
Department of Biochemistry, Faculty of Sciences, University PLT Platelet
of Yaounde 1, P.O Box: 812, Yaounde, Cameroon RBC Red blood cell
2
Department of Biological Science, Higher Teachers’ Training WBC White blood cells
College, University of Yaounde 1, P.O Box: 47, Yaounde,
Cameroon
3
Centre for Food and Nutrition Research, Institute of Medical
Research and Medicinal Plant Studies, MINRESI, P.O
Box 13033, Yaounde, Cameroon

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 Toxicology and Environmental Health Sciences

Introduction exhibits various pharmacological activities such as anti-

oxidant, anti-inflammatory, hypolipidemic, and antibacte-
Phytotherapy is receiving more attention nowadays and rial [17–20]. Phytochemical studies have shown that A.
is increasingly being used for the management of several caulirhiza contains polyphenols, flavonoids, terpenoids,
diseases worldwide [1]. According to WHO, about 80% of saponins, coumarins, spilanthol and even sterols [21].
the population are oriented to traditional medicine (espe- Plants that possess such phytochemical content have been
cially those in developing countries) where plant extracts demonstrated to prevent or treat many pathologies like
and their derivatives are used to overcome many diseases diabetes and cancer [22] and/or their related complica-
[2, 3]. Medicinal plants have a wide variety of bioactive tions [23, 24]. But some of those metabolites due to their
molecules involved in the discovery of new drugs [4, 5], structure and quantity can induce deleterious effects [25]
as the activities of most of the natural products used in tra- such as hematotoxicity and hepatotoxicity, depressive
ditional medicine have already been proven scientifically activity on the central nervous system, mutagenesis, and
[6]. Whatever the usefulness of these natural products, it carcinogenesis [22, 26]. Thus, to validate the biological
is important to determine whether they have deleterious activities of A. caulirhiza and to promote its pharmaco-
effects [7]. Several studies indicate that many medicinal logical use against several diseases, toxicity studies of its
plants have adverse effects [8–11]. In order to avoid pos- extracts are necessary. To this end, Azame et al. observed
sible poisoning and to protect the health, it is impera- that the aqueous extract of A. caulirhiza in acute assess-
tive to evaluate the safety of these plants before their use ment at 5000 mg ­kg−1 Bw caused no apparent deleterious
[12]. Thus, the systematic evaluation of the toxicity of effects and no death in rats 2 weeks after exposure [22].
plants is a preliminary step towards the validation of their This implies that the lethal dose 50 (LD50) is greater than
regular therapeutic use. A. caulirhiza is a flowering plant 5000 mg/kg Bw. The acute toxicity study provides infor-
belonging to the Compositae or Asteraceae family [13]. mation only on the lethal dose of this extract [27] and no
It is called hen’s eye in the West Region of Cameroon information about the effect on organs. Considering the
where it is used by population against several diseases. wide traditional use of A. caulirhiza in the management
The whole plant, once macerated, is used to fight tonsil- of several diseases, it is important to evaluate the adverse
litis and respiratory diseases (colds, asthma) [14] while effects of its extracts on some vital organs after prolonged
the leaves are used against babies' diaper rash and dental exposure to better identify its therapeutic doses. Thus, the
caries [15]. In addition, fresh leaves of A. caulirhiza are present study aimed at evaluating the subacute toxicity
crushed and infused in cold water and administered orally of the aqueous extract of A. caulirhiza (AE-AC) among
to the patient as a breast cancer treatment [16]. Previous female albino Wistar rats.
studies revealed that aqueous extract of the whole plant

Table 1  General appearance and behavioral observations after

28 days
Parameters Groups
Normal AE-AC 100 AE-AC 250 AE-AC 500

Motricity Normal Normal Normal Normal

Aggressivity Not present Not present Not present Not present
Audition Normal Normal Normal Normal
Weakness Not present Not present Not present Not present
Pain sensibility Normal Normal Normal Normal
Rate of respi- Normal Normal Normal Normal
ration
Change in skin No effect No effect No effect No effect Fig. 1  Effect of A. caulirhiza on rats’ body weight variation. AE-AC
100: Aqueous Extract of A. caulirhiza at the dose of 100 mg/kg.Bw;
Diarrhea Not present Not present Not present Not present AE-AC 250: Aqueous Extract of A. caulirhiza at the dose of 250 mg/
Coma Not present Not present Not present Not present kg.Bw; AE-AC 500: Aqueous Extract of A. caulirhiza at the dose of
Death Alive Alive Alive Alive 500 mg/kg.Bw. * indicates a significant difference at p < 0.05 com-
pared to the control group. AE-AC 100: Aqueous Extract of A. cau-
AE-AC 100: Aqueous Extract of A. caulirhiza at the dose of 100 mg/ lirhiza at the dose of 100 mg/kg.Bw; AE-AC 250: Aqueous Extract
kg Bw; AE-AC 250: Aqueous Extract of A. caulirhiza at the dose of of A. caulirhiza at the dose of 250 mg/kg.Bw; AE-AC 500: Aqueous
250 mg/kg Bw; AE-AC 500: Aqueous Extract of A. caulirhiza at the Extract of A. caulirhiza at the dose of 500 mg/kg.Bw. * indicates a
dose of 500 mg/kg Bw significant difference at p < 0.05 compared to the control group

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Table 2  Effect of A. caulirhiza on relative organ weight after 28 days toxicity was noted compared to the animals of the normal
Organs Relative organ weight (g/100 g of Bw)
group (Table 1).

Normal AE-AC 100 AE-AC 250 AE-AC 500

Effect of aqueous extract of A. caulirhiza on rats’ body
Liver 3.61 ± 0.19 3.40 ± 0.29 3.49 ± 0.25 3.80 ± 0.18 weight change
Kidneys 0.65 ± 0.02 0.65 ± 0.05 0.62 ± 0.04 0.69 ± 0.01
Lungs 0.46 ± 0.02 0.58 ± 0.04* 0.62 ± 0.06* 0.63 ± 0.02* The oral daily administration of AE-AC at different doses to
Heart 0.31 ± 0.01 0.28 ± 0.02 0.29 ± 0.02 0.30 ± 0.02 the rats did not induce a change in animal weight compared
Spleen 0.45 ± 0.10 0.46 ± 0.04 0.46 ± 0.06 0.53 ± 0.06 to the normal group throughout the experiment as recorded
Brain 0.72 ± 0.04 0.82 ± 0.09 0.83 ± 0.05 0.86 ± 0.01* in Fig. 1.
Stomach 0.64 ± 0.02 0.69 ± 0.06 0.66 ± 0.06 0.66 ± 0.01
Uterus 0.23 ± 0.01 0.21 ± 0.01 0.21 ± 0.01 0.26 ± 0.02* Effect of AE‑AC on relative organ weight after 28 days
Ovaries 0.05 ± 0.01 0.05 ± 0.01 0.07 ± 0.01* 0.07 ± 0.01* of daily exposure

AE-AC 100 Aqueous Extract of A. caulirhiza at the dose of 100 mg/

No difference was noted in relative organ weight between
kg Bw; AE-AC 250 Aqueous Extract of A. caulirhiza at the dose of
250 mg/kg Bw; AE-AC 500 Aqueous Extract of A. caulirhiza at the the AE-AC treated groups and the normal group at the level
dose of 500 mg/kg Bw. * indicates a significant difference at p < 0.05 of kidney, liver, heart, spleen, and stomach, while for lungs,
compared to the control group an increase was noted at all doses. In addition, administra-
tion of AE-AC at 500 mg ­kg−1 Bw was associated with an
increased weight of the brain, uterus, and ovaries (Table 2).
Results and discussion
Effect of A. caulirhiza on some haematological makers
Results of experimental animals

Effect of AE‑AC on animal behaviour and general signs Except for MCHC, compared to the normal group, the
of toxicity over time extract caused a decrease of monocytes, red blood cells,
and haemoglobin (p < 0.05) at different doses. In addition,
After 4 weeks of experimentation, animals treated with a decrease in lymphocytes at 100 and 250 mg k­ g−1 Bw was
AE-AC at different doses showed no death and no unu- recorded. The doses of 250 and 500 mg k­ g−1 Bw resulted
sual changes in behaviour or locomotor activity. No sign of in a decrease in platelets compared to normal rats. Also,
a significant increase in white blood cells at doses of 250
and 500 mg ­kg−1 Bw and an increase of mean corpuscular

Table 3  Effect of A. caulirhiza Parameters Groups

on some haematological
markers Normal AE-AC 100 AE-AC 250 AE-AC 500
3 −1
WBC(10 µL ) 11.66 ± 0.11 6.45 ± 0.76* 14.32 ± 0.64 16.56 ± 2.23*
RBC ­(103 µL−1) 8.54 ± 0.18 7.13 ± 0.27 7.45 ± 0.65 7.48 ± 0.36
Hb (g/dL) 14.25 ± 1.22 12.17 ± 1.31 11.33 ± 1.53 12.81 ± 0.75
Hcte 49.05 ± 0.65 41.53 ± 0.42* 42.25 ± 5.14 42.85 ± 2.26
MCV 56.66 ± 1.36 56.03 ± 1.41 56.90 ± 6.14 57.40 ± 0.26
MCH 16.80 ± 0.26 17.10 ± 0.49 16.66 ± 2.08 17.07 ± 0.31
MCHC 29.66 ± 1.07 30.53 ± 0.14* 32.10 ± 0.95 29.26 ± 1.12
PCV 0.48 ± 0.04 0.40 ± 0.02* 0.35 ± 0.02* 0.44 ± 0.02
PLT ­(103 µL−1) 800.00 ± 20.00 732.00 ± 9.90 681.33 ± 15.50* 646.00 ± 53.11*
Lymp (%) 8.41 ± 0.18 5.15 ± 0.17* 6.41 ± 0.11* 12.40 ± 0.42*
Mon (%) 1.23 ± 0.13 0.52 ± 0.12* 0.41 ± 0.06* 0.51 ± 0.01*

WBC: White blood cell; RBC: Red blood cell; Hb: Hemoglobin; Hcte: Hematocrit; PLT: Platelet; Lymp:
Lymphocytes; Mon: Monocytes; IG: Antibody; MCV: Mean corpuscular volume; MCH: Mean corpuscular
hemoglobin; MCHC: Mean corpuscular hemoglobin; PCV: Packed cell volume; AC 250: Aqueous Extract
of A. caulirhiza at the dose of 250 mg/kg Bw; AEAC 500: Aqueous Extract of A. caulirhiza at the dose of
500 mg/kg. Bw. * indicates a significant difference at p < 0.05 compared to the control group

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Fig. 3  Effect of A. caulirhiza on creatinine and uric acid level.

Fig. 2  Effect of A. caulirhiza on transaminases activities. ALAT: AE-AC 100: Aqueous Extract of A. caulirhiza at the dose of 100 mg/
Alanine Amino Transferase; ASAT: Aspartate Amino Transferase; kg.Bw; AE-AC 250: Aqueous Extract of A. caulirhiza at the dose of
AE-AC 100: Aqueous Extract of A. caulirhiza at the dose of 100 mg/ 250 mg/kg.Bw; AE-AC 500: Aqueous Extract of A. caulirhiza at the
kg.Bw; AE-AC 250: Aqueous Extract of A. caulirhiza at the dose of dose of 500 mg/kg.Bw. * indicates a significant difference at p < 0.05
250 mg/kg.Bw; AE-AC 500: Aqueous Extract of A. caulirhiza at the compared to the control group
dose of 500 mg/kg.Bw. * indicates a significant difference at p < 0.05
compared to the control group
(b) Effect of A. caulirhiza on some metabolic markers
of the liver function
haemoglobin and lymphocytes at 500 mg ­kg−1 Bw was
observed as compared to the normal group (p < 0.05), The metabolic function of the liver was evaluated through
(Table 3). the levels of glucose, total proteins, total cholesterol, and
triglycerides. The administration of AE-AC revealed a
Effect of A. caulirhiza on some markers of liver toxicity high glucose level (p < 0.05) and a significant decrease of
triglycerides level (dose–response) at different doses. No
significant difference was observed in total cholesterol and
(a) Effect of A. caulirhiza on some markers of liver total protein levels between the extract-treated animals and
integrity normal group rats (Table 4).

Liver integrity was assessed through transaminase activities. Effect of A. caulirhiza on some markers of kidney toxicity
The administration of AE-AC at doses of 250 and 500 mg
­kg−1 Bw induced a decrease (p < 0.05) in ALAT and ASAT The kidney function was assessed through the plasma levels
activities compared to normal rats (Fig. 2). of uric acid, creatinine, and some electrolytes. The AE-AC
did not induce any change in creatinine (at all tested doses)
and uric acid level at 100 and 250 mg k­ g−1Bw doses, but
at 500 mg ­kg−1Bw, significant decreased of uric acid was

Table 4  Effect of A. caulirhiza Parameters Groups

on some metabolic markers of
the liver function Normal AE-AC 100 AE-AC 250 AE-AC 500

Glucose (mg/dL) 0.79 ± 0.01 0.90 ± 0.10* 1.01 ± 0.02* 0.90 ± 0.05*
Total cholesterol (mg/dL) 36.01 ± 2.31 33.25 ± 1.71 33.67 ± 3.25 29.73 ± 2.87
Triglycerides (mg/dL) 83.12 ± 2.57 47.54 ± 1.86* 61.02 ± 3.09* 66.81 ± 1.21*
Total Proteins (g/L) 212.28 ± 1.53 208.23 ± 3.36 212.57 ± 2.16 218.83 ± 1.33

AE-AC 100: Aqueous Extract of A. caulirhiza at the dose of 100 mg/kg Bw; AE-AC 250: Aqueous Extract
of A. caulirhiza at the dose of 250 mg/kg Bw; AE-AC 500: Aqueous Extract of A. caulirhiza at the dose of
500 mg/kg Bw; * indicates a significant difference at p < 0.05 compared to the control group

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Table 5  Effect of A. caulirhiza Parameters Groups

on plasmatic electrolytes level
Normal AE-AC 100 AE-AC 250 AE-AC 500
2+
Ca (mmol/L) 2.11 ± 0.43 2.70 ± 0.24 2.57 ± 0.09 2.45 ± 0.15
Na+ (mmol/L) 120.39 ± 3.61 122.51 ± 2.36 122.30 ± 4.14 123.78 ± 3.15
K+ (mmol/L) 8.47 ± 0.46 9.15 ± 0.57 8.42 ± 0.91 9.79 ± 0.77
Cl− (mEq/L) 39.08 ± 1.34 36.36 ± 1.21 39.57 ± 2.44 40.17 ± 3.53

AE-AC 100: Aqueous Extract of A. caulirhiza at the dose of 100 mg/kg Bw; AE-AC 250: Aqueous Extract
of A. caulirhiza at the dose of 250 mg/kg Bw; AE-AC 500: Aqueous Extract of A. caulirhiza at the dose of
500 mg/kg Bw.* indicates a significant difference at p < 0.05 compared to the control group

Fig. 4  Effect of A. caulirhiza on

organs microarchitecture. Pv:
portal vein (rounds); Pa: Pulmo-
nary alveolus; IGs: Intra Glo-
merular space; H: Hepatocytes
(these are blackheads, short
arrow); Hu: uterine height;
G: Glomerulus (long arrow);
M: Myocardia; S: Sinusoids
(long arrow); T: Tubules (short
arrow); NG: Normal Group;
AE-AC 100: Aqueous Extract of
A. caulirhiza at the dose of 100
mg/kg.Bw; AE-AC 250: Aque-
ous Extract of A. caulirhiza
at the dose of 250 mg/kg.Bw;
AE-AC 500: Aqueous Extract
of A. caulirhiza at the dose of
500 mg/kg.Bw

observed compared to the normal group (Fig. 3). Also, no space was noted in animals receiving 500 mg/kg Bw dose
change was noted with the electrolyte’s levels ­(Ca2+, ­K+, of AE-AC with inflammation characterized by larger tubules
­Na+, and ­Cl−) between groups (Table 5). compared to those in the control, AE-AC 100 and AE-AC
250 groups. The lung sections of the animals revealed a
Effect of A. caulirhiza on organ microarchitecture normal architecture of the pulmonary alveoli (Pa) in the
normal and AE-AC treated groups, as well as the architec-
The results in Fig. 4 represent the architecture of ture of the myocardia (M) of the animals. Concerning the
some organs collected at the end of the experiment. Histo- uterus, the extract at the doses of 250 and 500 mg/kg body
pathological studies of liver sections from the control group weight caused a decrease in the uterine heights (Uh) com-
showed a normal appearance of the portal vein (Pv), normal pared to the normal rats.
sinusoids (S), and hepatocytes (H). Rats treated with the
extract at doses of 250 and 500 mg/kg body weight showed Discussion
mild hepatic necrosis characterized by vascular congestion
of the portal vein and moderate degeneration of hepatocytes. Medicinal plants around the world are increasingly being
Regarding to kidney integrity, a reduction in glomerular used as primary care for various human diseases. [28].

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Despite their high use in developing countries, there is no important analgesic but equally leads to severe dehydration
more data on their toxicity and side effects [2]. For this rea- in animals causing an imbalance in immune system cells
son, systematic analysis is imperative to predict the toxic [25].
risks of plants and to identify safe doses for human beings The liver, although the most involved organ in metabo-
[3]. The objective of this work was to evaluate the subacute lism, is also the site of biotransformations and detoxification
toxicity of AE-AC in female Wistar rats. The data showed of drugs in the body. Its integrity can be assessed through
that no mortality and no notable behavioural changes were transaminase activities and some metabolic markers such
physically observed in the animals receiving AE-AC at dif- as glucose, total protein, total cholesterol and triglycerides
ferent doses (Table 1). Equally, no important variation in levels as well as the assessment of liver histology [40]. Tate-
body weight was observed among different groups after fuji et al. have shown that abnormal levels of total proteins
4 weeks of experimentation (Fig. 1). The organ weights of would be related to liver infection or chronic inflammation
experimental animals were also assessed given that drug [41]. Administration of AE-AC not affected the total plas-
metabolism could lead to physiological and possible changes matic protein level (Table 4). A significant increase in glu-
in the weights of certain organs [29]. The oral administration cose levels was noted among animals treated with the extract
of AE-AC leads to an increase in the relative weight of the (Table 4). The ability of the AE-AC to activate the metabolic
lungs, and brain at different doses, and the uterus at 500 mg/ function of the liver can be pointed out. Indeed, the liver has
kg Bw (Table 2). There were no changes in the key organs a key role in the homeostasis of glucose. In fasting condi-
of metabolism and detoxification as confirmed by the results tions, the decrease of insulin level stimulates the activation
represented in Fig. 2. of pancreatic α-cells which in turn secrete glucagon which
Haematological analysis can provide highly predictive in order to cover the energy needs, will start the glycog-
information on the risk of toxicity [30]. The results showed enolysis pathway or the de novo pathway and thus release
that the oral administration of the AE-AC caused a signifi- hepatic glucose which will be redistributed [42]. Also, it is
cant reduction of platelets (PLT) at doses of 250 and 500 mg well known that the liver is the key organ in the synthesis
­kg−1 (Table 3). This can be explained by the presence at and excretion of cholesterol. Therefore, any type of obstruc-
these doses of a higher content of metabolites such as sapo- tion in the liver, either intra- or extra-hepatic, will cause an
nins and glycosides in the extract [20], which may lead to increase in the total cholesterol level in the plasma. A sig-
a high risk of haemorrhage due to thrombocytopenia [31]. nificant decrease in total cholesterol and triglycerides was
Platelet balance is regulated by thrombopoietin, a glycopro- noted in animals receiving the AE-AC at all the doses which
tein produced by the liver and kidneys, which is involved could thus testify its hepatoprotective effect.
in the regulation of platelet production [31–33]. RBC and Traditionally, liver injury is assessed by evaluating the
Hb were lowered in rats treated with the AE-AC at 500 mg serum activity of enzymes such as alanine aminotransferase
­kg−1 but not significantly. This implies that at a high dose, (ALAT), aspartate aminotransferase (ASAT), and glutamate
the AE-AC could induce anaemia. However, this anaemia dehydrogenase (GLDH). These enzymes are released from
would be normocytic because normal levels of MCV, MCH, hepatocytes when cell membrane integrity is lost as a result
and MCHC maintain erythropoiesis [33]. The decrease in of cell necrosis or inflammation [43]. AE-AC administered
monocytes and lymphocytes here implicates the main role showed a decrease in transaminase activities (ALAT and
of phagocytes which is to protect the body against foreign ASAT) among groups treated with 250 and 500 mg/kg Bw
agents and destroy them, thus contributing to the inflamma- (Fig. 3). The literature has shown that some polyphenols,
tory response [34, 35]. like flavonoids, possess hepatoprotective activities acting
This reaction would be expressed by the recruitment of through several mechanisms. They enhance the enzymatic
white blood cells (WBC) as could be confirmed by treat- antioxidant defence system via the mediation of the expres-
ment with 500 mg/kg Bw (Table 3). The increase in WBC sion of the second nuclear erythroid-related factor (Nrf2)/
number could be associated with the activation of leucocy- cytochrome P450 2E1 (CYP2E1), slowing of the inflamma-
tosis leading to the induction of a better defence antiinfec- tion through inhibition of mitogens (MAPK)/nuclear fac-
tion mechanism. Consequently, they could have phagocytic tor kappa B (NF-κB), and reduction of apoptosis through
immuno-sensitizing effects on the experimental animals regulation of B-cell lymphoma 2 (Bcl-2)/protein kinase B
[36], but this reaction is most often immunosuppressive [37]. (AKT)/caspase expression [44, 45]. At the histological level,
This reaction can also be justified by the presence of alka- the administration of the extract did not lead to any percep-
loids contained in AE-AC. Indeed, some studies reported tible hepatocellular damage at lower doses (100 and 250 mg
that although alkaloids are endowed with several pharma- ­kg−1 Bw) but at 500 mg k­ g−1 Bw dose, the liver sections
cological activities, they remain toxic [38, 39]. Thus, Lang- showed histological disturbances characterized by vascular
kilde et al. have shown that the glycoalkaloids content of the congestion when compared to normal rats. This suggests that
crude alkaloid extract of leaves of Solanum tuberosum is an

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Toxicology and Environmental Health Sciences

AE-AC has hepatoprotective properties at 100 and 250 mg Preparation of aqueous extract of A. caulirhiza
­kg−1 Bw.
The kidneys are responsible for the excretion of meta- The plant material was sorted, washed, and dried in the pro-
bolic products and toxins through the urine [46]. Clinical cessing room until a constant weight was obtained before
assessment of renal function is based on the measurement of being powdered and kept at room temperature in an amber
serum urea and creatinine as markers of reduced glomerular bottle before extraction. The extraction protocol was closer
capacity. Creatinine undergoes tubular secretion, whereas to those of Azame et al. [22] but the volume of water was
urea is not secreted but reabsorbed by the renal tubules with slightly high and this increased the contact area for a bet-
the help of electrolytes [47]. In this study, renal function was ter extraction. Thus, 100 g of A. caulirhiza powder were
evaluated using some predictive biomarkers (uric acid, cre- macerated in 1200 mL of distilled water with the extraction
atinine, and electrolyte levels) and histology. The AE-AC at ratio of 1:12 (mass: volume) for 24 h at room temperature
different doses resulted in a decrease (p < 0.05) in creatinine (25 °C). The liquid extract obtained was separated from the
and uric acid levels compared to the control group (Table 5), solid residue by filtration on Whatman ­No 3 filter paper. The
and this decrease was significant with uric acid at 500 mg filtrate was lyophilized using SMH 45 USIFROID (Lagep).
­kg−1 Bw, this is in line with the findings of Christian et al.
[48]. Creatinine and uric acid are nitrogenous final products Sub‑acute toxicity study
of protein degradation which are continuously eliminated.
Their increased level in the serum generally translates a Animals
decrease in glomerular filtration rate (the main parameter
for assessing renal function). These results may indicate that Twenty adult nonpregnant and nulliparous female Wistar
the extract at these doses is nephroprotective and may be rats (due to the ethical conditions associated with their
due to the terpenoids contain of the plant extract that has greater resistance (due to the hormonal factors to which they
proven to be renal antiapoptotic and anti-inflammatory via are subjected), unlike males) between 8 and 10 weeks old
mitochondrial caspase signalling pathways [49]. Increased with an average weight of 160 ± 10 g were provided by the
­Na+, ­K+, and C
­ l− levels are usually related to renal failure or LNNB of the Faculty of Sciences, University of Yaounde I,
a decrease in glomerular filtration. As a result, the sodium Cameroon. They were maintained in polypropylene cages
retained in the blood will lead to water retention, increasing (containing white wood chips that were changed daily) with
blood volume, and therefore an increase in blood pressure freedom of movement and acclimatized for one week before
[50]. In this study, there was no significant difference in the beginning of the study. They were kept under a tempera-
these electrolytes between groups. ture of 20–25 °C and a regular 12 h light/12 h dark cycle
Lung sections from animals treated with AE-AC at throughout the experimental period and under appropriate
500 mg/kg Bw revealed more developed pulmonary alveoli; ventilation conditions. They were fed with standard feed for
this could be an indicator of diffuse alveolar damage which rodents and received water ad libitum.
may lead to various complications in the body including
hypoxia, hypercapnia, pulmonary hypertension, and cardi-
orespiratory arrest [51, 52]. Experimental design

All experimental protocols were conducted under the gen-

Materials and methods eral OECD guidelines of October 2008 Nº407 for the safety
and use of experimental animals with some modifications
Plant material and preparation of extract [53]. Animals were assigned randomly to four groups of five
each (n = 5 per group). Once the animals were acclimatized,
Collection of plant material they were distributed as follows:

The flowers and leaves of A. caulirhiza were harvested in • A normal group (NG): receiving distilled water;
October 2018 in Bandjoun (West Region, Cameroon). The • A test group 1 (AE-AC 100): receiving AE-AC at
plant identification was done at the National Herbarium of 100 mg/kg of Bw;
Cameroon (NHC) under voucher number 602 in compari- • A test group 2 (AE-AC 250): receiving AE-AC at
son with the specimen from the collection of the Herbarium 250 mg/kg of Bw;
N° 57,420/NHC. The plant material was then transferred to
the Laboratory of Nutrition and Nutritional Biochemistry
(LNNB) of the Faculty of Sciences, University of Yaounde I.

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• And test group 3 (AE-AC 100): receiving AE-AC at Histomorphological changes were evaluated under an Axi-
500 mg/kg of Bw. oskop 40 microscope connected to a computer where the
image was transferred using MRGrab1.0 and Axio Vision 3.1
The extract at different doses and distilled water were software (Zeiss, Hallbergmoos, Germany). The examination
administered to the corresponding animal groups by oral has been realized microscopically at × 400 in a blind manner
intubation at 10 mL/kg.Bw daily with a curved ball-tipped by a pathologist [55].
stainless steel feeding needle for 4 weeks. The rat weight
was measured every 4 days using a precision scale (SF-400, Haematology analysis
China). Thereafter, water and a standard normal diet were
free to access by each animal. Toxicity signs, notably death, Haematological analysis was performed using an Auto
changes in physical appearance and behaviour (aggres- Hematology Analyzer (Mindray BC-2800, Shenzhen Mind-
siveness, pain sensitivity, motor activity, tremor, convul- ray, Bio-Medical Electronics Co., Ltd). Red blood cell count
sions, salivation, and diarrhoea), and signs of distress were (RBC), hematocrit (Hcte), haemoglobin (Hb), mean corpus-
recorded daily. At the end of the experiment, the overnight cular volume (MCV), mean corpuscular haemoglobin con-
fasted (12 h) rats were weighed. They were anaesthetized by centration (MCHC), mean corpuscular haemoglobin (MCH),
intramuscular injection of Diazepam (10 mg k­ g−1 Bw) and white blood cell count (WBC), lymphocytes (Lymp), mono-
Ketamine (50 mg.kg−1 Bw) to minimize pain and damage cytes (Mon) and platelet count (PLT) of treated rats were
before they were sacrificed. Blood was collected in EDTA estimated and compared to the normal rats. The packed cell
anticoagulant tubes via cardiac puncture. For each animal, volume (PCV) was then calculated using the formula of
about 1 mL of blood was collected in the first tube for hae- Brian et al. as follows [56]:
matological analyses and the rest of the blood was collected
for plasma preparation in the second tube. Plasma was Packed Cell Volume (as a fraction)
obtained after centrifugation of the blood sample at 1500 g Haemoglobin (g∕dL)
=
for 10 min at 4 °C. The supernatant was then aliquoted into Mean Corpuscular Haemoglobin Concentration (g∕dL)
Eppendorf tubes and conserved at − 20 °C for biochemical
parameters analysis. Biochemical analysis

Effect of the AE‑AC on some toxicity makers Liver integrity was assessed by transaminases activity
and some vital organs in rats after 28 days [57], while plasma creatinine [58], uric acid levels [59],
of exposure and electrolytes (potassium, sodium, calcium, and chlo-
rine ions estimated using Biorex kits following the manu-
Organ relative weight and Histopathology facturer's protocols) were used to explore renal function.
Blood glucose [60], total protein [61], total cholesterol
Organs including the heart, liver, kidneys, stomach, lungs, [62], and triglycerides [63] were used to assess hepatic
spleen, uterus, and ovaries were surgically isolated, cleaned metabolic function.
with ice-cold saline solution, placed on absorbent paper,
then weighed and registered as absolute organ weight in
grams. The relative organ weight (ROW) of each animal Statistical analysis
was then calculated using the formula below [54]:
[ ] Results were expressed as mean ± Standard Deviation (SD)
ROW = Absolute organ weight (g) ∕
[ ] and as a percentage (%) of variation. Statistical analyses
Body weight of rat on sacrifice day (g) × 100 were performed using the Statistical Package for Social
Science (SPSS) software version 20.0 for Windows. Stu-
The lungs, heart, uterus, liver, and kidneys were then
dent’s t-test (two-tailed, type 2) was used to compare
fixed in 10% formalin solution for histological analy-
means between the test and normal groups. Significatively
sis. Tissue samples were dehydrated in a graded series of
was considered at p values less than 0.05. Microsoft Excel
ethanol (70–100%), washed in toluene, and finally embed-
Spreadsheet 2016 was used to plot graphs.
ded in paraffin. Thereafter, Thin 5-µm sections were pre-
pared with a microtome (Leica RM2235) and stained with
hematoxylin and eosin before microscopic examination.

13
Toxicology and Environmental Health Sciences

Conclusion Pharm Sci 8(06):147–155. https://​doi.​org/​10.​7324/​JAPS.​2018.​

8619
5. Masoko P (2017) Phytochemical analysis, antioxidant and anti-
Oral daily exposure to the AE-AC at doses of 100, 250 and bacterial properties of Spilanthes mauritiana used traditionally
500 mg.kg−1.Bw during 28 days does not cause apparent in Limpopo Province, South Africa. J Evid Based Compl Altern
deleterious effects on female Wistar rats. It has a hepato- Med 22(4):936–943. https://d​ oi.o​ rg/1​ 0.1​ 177/2​ 51569​ 0X177​ 46774
6. Musila MN, Ngai DN, Mbiri JW, Njagi SM, Mbinda WM, Ngugi
protective, nephroprotective and immuno-sensitizing effect MP (2017) Acute and subchronic oral toxicity study of methanolic
at 100 and 250 mg.kg.−1.Bw but at 500 mg.kg.−1.Bw dose, extract of Caesalpinia volkensii (Harms). J Drug Metab Toxicol
the aqueous extract of A. caulirhiza could have deleterious 8(1):1–8. https://​doi.​org/​10.​4172/​2157-​7609.​10002​22
effects. A sub-chronic toxicity study of this extract will 7. Tang R, Tian R, Cai J, Wua J, Shen X, Hu Y (2017) Acute and
sub-chronic toxicity of Cajanus cajan leaf extracts. Pharm Biol
help to have more information on its adverse effects. 55(1):1740–1746. https://​doi.​org/​10.​1080/​13880​209.​2017.​13095​
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Acknowledgements The authors are thankful to the Laboratory of
8. Ochoa M, Reyes V, Sánchez A, Guzmán M, Noguera P, Angeles
Animal Physiology of the University of Yaounde 1 and the University
E, Alba-Hurtado F (2014) Subchronic toxicity study in rats of two
Hospital of Yaounde 1 for their technical support in histopathology
new ethyl-carbamates with ixodicidal activity. Biomed Res Int.
analysis.
https://​doi.​org/​10.​1155/​2014/​467105
9. Etame RME, Mouokeu RS, Ngane RAG, Assam Assam JP,
Author contributions HMFT, MAAM and FRN designed of the study.
Masoohe AM, Tientcheu R, Hopogap ML, Etoa FX (2017) Acute
HMFT, IM and FLEE realized the experimentation. HMFT and JAFY
and sub-acute toxicity of Harungana madagascariensis LAM
have analyzed and interpreted the results. HMFT and FLEE have writ-
(Hypericaceae) stem bark methanol extract. J Appl Pharm Sci
ten the manuscript. MAAM, FRN, and EJO revised it. All authors have
7(03):160–167. https://​doi.​org/​10.​7324/j.​JAPS.​2017.​70326
read and agreed to the final draft.
10. Bedoor AS, Muhsin SGA, Ausama AJ (2022) Sub-chronic effects
of mefenamic acid alone or in combination with diclofenac on the
Funding No funding was provided for this study.
female reproductive system in albino rats. Toxicol Environ Heal
Sci 14:319–326. https://​doi.​org/​10.​1007/​s13530-​022-​00145-6
Data availability The data employed to support the conclusions of the
11. Youovop FJA, Takuissu GRN, Ngondi JL, Oben JE (2022) Acute
study are available upon request to the corresponding author.
and sub-chronic toxicity studies of aqueous bark extract of Detar-
ium microcarpum guill. and perr in albinos wistar rat. Toxicol
Declarations Environ Health Sci. https://​doi.​org/​10.​1007/​s13530-​022-​00139-4
12. Yuet K, Darah I, Chen Y, Sreeramanan S, Sasidharan S (2013)
Conflict of interest Conflict of interest Huiny Miriane Fotso Tienoue, Acute and subchronic toxicity study of Euphorbia hirta L, metha-
Françoise Raïssa Ntentie, Mary-Ann Angie Mbong, Ferdinand Larvin nol extract in rats. Biomed Res Int. https://​doi.​org/​10.​1155/​2013/​
Ebouel Edoun, Inelle Makamwe, Janvier Aimé Fotso Youovop, and 182064
Enyong Julius Oben declare that we have no conflict of interest. 13. Kidane B, Tinde A, Laurentis JGM, Zemde A (2014) Use and
management of traditional medicinal plants by Maale and Ari
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tee for Animal and Human Bioethics, CRFD-SVSE of the University of https://​doi.​org/​10.​1186/​1746-​4269-​10-​46
Yaoundé I. The animals were treated following the Institute's guidelines 14. Yimta F, Mahiou-Leddet V, Nguimatsia F, Mbenkum T,
of the National Institutional Ethics Committee of Cameroon (Council Wouessidjewe D, Evelyne O, et al. (2017) Ethnobotanical survey
EEC 86/609), which has adopted all the protocols recommended by the of medecinal plants used in the western region of cameroon for
European Union on the safety of animals in scientific research. the treatment of various infectious diseases
15. Ndjouondo GP, Ngene JP, Ngoule CC, Kidik MC, Ndjib RC, Dib-
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