In Vitro and In Vivo Toxicological Evaluation of Avicennia africana P: Beauv. (Avicenniaceae) Leaf Extract in a Rat Model

Avicennia africana is an important ethnomedicinal plant that has long been used to treat malaria and several other diseases. Despite the plant's antimalarial and other therapeutic properties, there is limited evidence-based data on its potential toxicity. Hence, the purpose of the current study was to assess the safety of A. africana leaf ethanolic extract (AAE). The study was designed to ascertain the cytotoxic effects of the crude extract on red blood cells (RBCs) as well as the acute and subacute toxicity in Wistar albino rats in accordance with Organization for Economic Co-operation and Development (OECD) guidelines “Test No. 423” and CPMW/SWP/1042/99. The pulverized, shade-dried plant leaves were sequentially macerated with 70% ethanol to obtain the crude extract (AAE). The extract's cytotoxic activity (CC50) against the uninfected human red blood cells (RBCs) was determined using the 3-(4,5-Dimethylythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. For the acute toxicity studies, the rats (male and female) were divided randomly into six groups of five rats (n = 5) and dosed orally once with the following dose levels: 100, 300, 1000, 3000, and 5000 mgkg−1, p.o. of the extracted AAE, with the control group receiving only the vehicle. In the repeated dose toxicity studies, the rats (both sexes) were orally administered daily with AAE at 100, 300, and 1000 mgkg−1 for 14 days. Rat body weights were measured, and blood samples were tested for haematological and biochemical markers. Internal organs like the heart, kidney, liver, and spleen were collected, inspected, and weighed, and histological examinations were performed. The median lethal dose (LD50) value is greater than 5000 mgkg−1 body weight, with no significant change in bodyweight or relative organ weight (ROWs) of the extract-treated groups or control group. The extract showed greater cytotoxicity activity (CC50), which was >100 μg/mL, compared to the reference drug (artesunate).The dosage groups of 100 and 300 mgkg−1﻿bwt had neutrophilia and lymphocytopenia (p < 0.05). However, changes in these haematological parameters may not be dose dependent and could be stress related. All the serum biochemical markers studied in rats given AAE did not show any significant change (p > 0.05). Histopathological examination of internal organs of AAE-treated rats did not show any significant abnormalities resulting from the extract treatment compared to the control group. Based on the findings in the present study, the LD50 value of AAE was found to exceed 5000 mgkg−1 in the acute toxicity test, while the no observed adverse effect level (NOAEL) in rats was 1000 mgkg−1 p.o. In the sub-acute toxicity tests. Histopathological analysis revealed no morphological abnormalities in the vital organs.


Introduction
Despite the availability of orthodox medications in Ghana, about 70% to 75% of Ghanaians are inclined to the use of plant-based medicine for general healthcare maintenance [1]. Te reliance on these natural products may be due to their availability, perceived efcacy, and safety as local medicinal plant preparations [2]. Medicinal plants have been suggested to have tremendous therapeutic potential over the years, and when exploited successfully, they could be incorporated into the traditional health care system [3]. Ghana's tropical forest cover is said to contain around 21,000 plant species. Most of these plants possess therapeutic properties and are utilized across the country to treat various diseases [4]. Te mangrove cover also provides useful resources and ofers a multipurpose livelihood support system to mangrove dwellers.
Avicennia africana is a member of the Avicenniaceae family and one of the eight noteworthy species of the genus Avicennia. Te plant grows along the shore in areas of seabed, streams, estuaries, and rivers, and has a world-wide distribution [5]. Many ethnomedicinal uses of various parts of the mangrove plant have been proposed. In folklore, the plant is used to remedy a variety of ailments, including cancer, asthma, and diabetes, as well as rheumatism, ringworm, lice, mange, and ulcers. Additionally, the plant is reported to treat some kinds of skin parasites [5], including malaria [6]. Te plant's usage by mangrove dwellers was allegedly attributed to a presumed potency and safety arising from a long folkloric history of use [7]. Plant-based medicines have long been thought to be safer than synthetic drugs because they are natural products [8]. Tis idea has been around for a long time, amid scant evidence-based research on the safety of plant-based medicines. Research has demonstrated that many medicinal plants are innately toxic because of their components and can result in inauspicious reactions if erroneously used [9]. To allow for rational discussion and clear up any doubt concerning the safety of the medicinal plants, it's anticipated that toxicity investigations may refect their traditional use. Even though the plant is widely used for the treatment of several diseases, its toxicity has not been scientifcally tested. Hence, the aim of this research was to evaluate the toxic efects of 70% ethanolic leaf extract of A. africana using in vitro and in vivo assays.

Plants Collection, Identifcation, Preparation, and
Extraction. Te plants (leaves) were collected from a mangrove forest near Elmina, Cape Coast in Ghana's Central Region. Te feld is located between latitude N 5°5 55.122 north of the equator and longitude W 1°19 22.277 west of the Greenwich Meridian, and it stretches along the University of Cape Coast's coastal boundary. A botanist identifed and authenticated the plant, and it was given the voucher specimen number CC3096 for future reference. Te specimen was stored in the herbarium of the University of Cape Coast's Department of Environmental Science, School of Biological Sciences. Te leaves were meticulously washed and dried under shade at room temperature. Afterwards, the dried leaves were pulverized into a powder form. Te pulverized plant material (900 g) was macerated with ethanol (70%, v/v) for 72 h [10]. Te fltrate was concentratedusing a rotary evaporator (R-114 SABITA) at 40 rpm and 40°C. Te resulting extract was stored in a desiccator at room temperature. Te residue of the plant material was remacerated to increase the extract yield. Te weight of 178.06 g (19.78%) of the crude extract was obtained and stored at −20°C in a freezer until used.

Animal Care and Treatment.
To evaluate the plant's toxicological efects in vivo, Wistar albino rats of either sex with a weight range of 151-216 g and aged 7 to 8 weeks were utilized in the experiment. Te rats were obtained from the University of Ghana Medical School's Animal Unit in Korle-Bu, Accra, Ghana. Tey were housed in metal cages and given commercially prepared rat food, acquired from Big Stars Ghana Ltd., as well as water daily. Before the experiment, the animals had to get used to the lab environment (acclimatization) for at least 7 days.

Cytotoxic Impact of A. africana Leaf Extract (AAE) on
RBCs. Te cytotoxic efects of AAE on erythrocytes were assessed using a slightly modifed version of the 3-(4,5-dimethylthiazol-2-yl)-5-diphenyltetrazolium bromide-MTT assay (Sigma Chemical Company, St. Louis, USA) as described by Ayisi et al. [9]. One hundred microliters (100 μL) of crude AAE in a two-fold serial dilution (100-6.25 μg/mL) was deposited into distinct wells of a 96-well microtiter plate in triplicate. Following that, each well received 100 μL of uninfected erythrocytes. Artesunate served as the positive control drug. Subsequently, the cultures were incubated at 37°C for 72 h in an atmosphere of 5% O 2 and CO 2 . After that, twenty microliters (20 μL) of MTT assay (7.5 mg/mL PBS) were dispensed into the wells, and the mixture was incubated for another 4 h. About 150 μL aliquot of the culture media was carefully removed and discarded from each of the wells. Te plates were then treated with acidifed isopropanol (200 μL) containing 1% Triton X-100, mixed very well, and stored under dark conditions for 24 h at ambient temperature to facilitate dissolution of formazan crystals. A plate reader was used to measure the optical density of the plates at 570 nm (Tecan Infnite M200, Austria). To determine the concentration of AAE that will cause the death/destruction of half of the red blood cell population (CC 50 values), the calculated percentage mean cell viability/RBC survival in the AAE-treated wells was plotted against the concentrations of the extract with the aid of Microsoft Excel 2013 software. Using the dose-response curves, we were able to extrapolate the concentrations of crude extract and artesunate that were most likely to cause cell death at 50% of the dose [11]. (30) healthy Wistar albino rats (weighing 151-216 g and aged 7-8 weeks, either sex) were used and grouped into six (6), with fve animals in each. Te animals were then marked and kept in separate cages for an additional seven days to acclimate to laboratory conditions before extract administration. Each rat in each of the fve groups received a single dose of the extract (AAE) orally. Te doses ranged from 100, 300, 1000, 3000, and 5000 mgkg −1 , with one group serving as a control (1-2 mL/ 100 g distilled water). As AAE was sticky, a few drops of 2% tween 20 were used to emulsify and dissolve in distilled water. Te average weight of the rats in each cage was used to calculate the dosage plan for oral administration, and individual rats were dosed based on their weight. Cage-side monitoring and observation were performed to check for mortality and signs of toxic efects in the frst 30 min and then hourly for the next 8 hours and the frst 24 hours post-AAE administration. Te rats were observed for an additional 13 days for symptoms of toxicity and death in the animals. Te tests for acute toxicity (Test No. 423) were done according to the guidelines of the Organization for Economic Co-operation and Development (OECD) [12].

Sub-acute Oral Toxicity Studies.
In the sub-acute toxicity study, twenty (20) healthy rats were employed, and the selected animals were grouped as described earlier. Te animals in this test were allowed seven days of acclimatization. Food and water were withheld overnight. To aid in dose calculation, the initial weights of the animals in all groups were determined, and each rat in all treatment groups received an extract ranging from 0.84 to 1.1 mL of AAE emulsifed with a few drops of 2% tween 20 and dissolved in distilled water. Based on the results of the acute toxicity studies, three dose levels (100, 300, and 1000 mgkg −1 ) were used in the sub-acute studies. Tis was similar to Wanjiru et al.'s study with slight modifcations [13].
Freshly prepared AAE preparations were administered orally and daily to rats in the treatment group for 14 days [14,15]. Te control group also received (1-2 mL distilled water containing 2% tween 20 p.o ) for the 14-day period. Te extract was administered at the same time (8 a.m.) every day. Cage-side observation and monitoring were done as described earlier and at least twice (10 a.m. and 4 p.m.) daily for toxicity signs such as salivation, piloerection, urinary incontinence, gait, tremors, convulsions, morbidity, and mortality.

Haematological Indices.
On the 15th day (one day after terminating the treatments), blood was collected (2 mL) via cardiac puncture from each of the rats in all study groups after the animals were given light anaesthesia of pentobarbitone (50 mgkg −1 , i.p). To examine the efects of AAE on haematological parameters, one millilitre (1 mL) of blood was quickly put into one-millilitre vacutainers containing K 2 EDTA anticoagulant and mixed well with a roll mixer. Te remaining 1 mL of the blood sample was put into a gelseparated vacutainer to obtain serum. Te whole blood samples were analysed using the URIT-5250Vet 5Part-Dif Auto Haematology Analyzer (Urit Medical Electronic Co., Ltd., PR, China) for haematological parameters. Te parameters tested include red blood cells (RBC), mean cell haemoglobin (MCH), packed cell volume (PCV), mean cell volume (MCV), haemoglobin (HGB), red cell distribution width (RDW), and mean cell haemoglobin concentration (MCHC). Te rest of the parameters that were analysed are: white blood cells (WBC), lymphocytes (LYM), neutrophils (NEU), monocytes (MON), eosinophils (EOS), basophils (BASO), platelets (PLT), mean platelet volume (MPV), and platelet distribution width (PDW).

Relative Organ Weight.
Te rats were killed in a humane way by dislocating their cervical spines, and the body weights were recorded. Following a necropsy, the heart, kidneys, liver, and spleen were inspected, isolated, and weighed. Te relative organ weight of each rat was determined as described earlier by both Rajeh et al. and Sharma et al. Te formula for calculating relative organ weight in this study is given as follows [14,16]: (1)

Histopathology of Vital
Organs. Each rat's vital organs were harvested, grossly examined for lesions, and preserved in formalin (10% v/v). Te organs were trimmed and placed in tissue cassettes for routine histopathology using a series of graded ethanol and xylene. Te processed tissues were then embedded in parafn wax blocks. A semiautomated rotary microtome was used to cut sections of 5.0 μm in thickness from the blocks. Sections were mounted on slides and stained with haematoxylin and eosin (H&E) [14]. A light microscope (Olympus, Japan) with a digital camera (AmScop 3.7 digital camera, MD500, USA) was used to examine the histological slides for lesions and other signs of toxicity. For a more detailed presentation, photomicrographs at x400 magnifcation were taken.

Te Cytotoxic Efect of AAE on Human RBCs.
Preclinical toxicological studies on all drugs, particularly plant-based medications, permit the collection of data regarding the safety and quality of drugs using in vitro as well as in vivo inquiries [17]. Te current study evaluated the cytotoxicity and acute as well as sub-acute toxicity profles of Avicennia africana extract (AAE) to establish preliminary safety data that can be used to corroborate or refute claims of safety from repeated folkloric applications. Te cytotoxicity of the extract was evaluated on uninfected erythrocytes alongside the artesunate drug in the MTT assay. In this study, the cytotoxicity activity of AAE wasdetermined as the cytotoxicconcentration (CC 50 ) >100 μg/mL. Te positive control (artesunate) also registered a CC 50 value of >100 μg/ mL. In this preliminary study, the cytotoxic efects of the crude extract were like those of the positive control, which showed that more RBCs survived. Tis is shown in Figure 1. Te use of cold maceration in this work was inspired by prior research on the plant. Te plant was reported to have a high concentration of highly polar compounds such as alkaloids, tannins, saponins, reducing sugars, and favonoids [18], which was confrmed by Ahmed [19].
An in vitro bioassay was deployed in this study to predict the presence of a potentially toxic compounds in the extract [20]. In the current study, the cytotoxicity of AAE was frst assessed using human erythrocytes in an MTT assay to ascertainthe toxicity at the cellular level. Te in vitro cytotoxicity test showed that both the extract and the control had negligible cytotoxicity on the erythrocytes. Te cytotoxic concentration of AAE required to kill 50% of uninfected red blood cells was not determined in the present study, indicating high erythrocyte survival outcomes in both the extract and artesunate. Te RBC survival values suggest that AAE and artesunate have negligible toxic efects on erythrocytes in the MTT assay. Tis assay was chosen for the determination of the cytotoxicity because it can transform the yellowish tetrazolium to formazan by some types of viable enzymes in erythrocytes [9]. Te conversion is made possible subject to the extracts' ability to maintain the RBCs at their normal morphologies, which is also linked to the extracts' intrinsic bioactive elements that protect the cells from cellmediated degradation. Te outcome of the cytotoxicity activity of AAE and artesunate from this assay demonstrated that AAE has inherent natural ingredients that preserve human erythrocytes against exogenous-mediated damage [21]. Tese results set the stage for acute and subacute investigations, with the predictable possibility that this plant may be of low toxicity.

Acute and Subacute Oral Toxicity Study.
In the acute toxicity tests, it was found that AAE did not cause any morbidity or mortality in the animals at doses up to 5000 mgkg −1 [22]. Te rats did not exhibit any toxic-related symptoms at a dose level of 5000 mgkg −1 p.o. In the acute toxicity test after being given AAE. Within 24 h and even up to 72 h after extract administration, no respiratory distress, aggression, diarrhoea, salivation, vomiting, or mortality were observed. Tere were no mortalities registered or observed to have occurred at the dose levels used up to 5000 mgkg −1 of AAE. Te LD 50 for A. africana crude extract was not determined in this study, suggesting that the LD 50 of the AAE is greater than 5000 mgkg −1 . Tus, the oral LD 50 of AAE appears to be more than 5000 mgkg −1 , indicating that dosage levels of up to 5000 mgkg −1 are tolerable and relatively safe. Our fndings agree with those of Tauheed et al., who found that drug candidates with an LD 50 of 5000 mgkg −1 or higher were safe for therapeutic use in acute toxicity studies [23]. In the sub-acute toxicity test, there was no fatality observed after the 14-day daily dose of extract (100-1000 mgkg −1 p.o.). Additionally, no physiological or behavioural abnormalities and several other toxicity indications, such as reduced feeding, tremors, or piloerection, were seen in the AAEtreated animals as opposed to the untreated animals.

Te Consequences of AAE on Haematological Indices.
Measurement of haematological biomarker thresholds forecasts the level of toxicity of the substance [22,24]. Moreover, haematological indicators in animal models are crucial for assessing toxicity concerns. Tus, any deviation from the normal blood variables in the cardiovascular system has a higher prognostic value for detecting haematotoxicity. Te outcome of the haematological test revealed that the red blood cells, white blood cells, monocytes, eosinophils, basophils, platelet count, platelet distribution width, haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, and red cell distribution width parameters in the extract-treated groups showed no substantial diference (p > 0.05) from those of the untreated group. In comparison of the haematological biomarkers, there were signifcant diferences between the control group and the rats given 100, 300, and 1000 mgkg −1 of the extract. Te neutrophils exhibited a signifcant increase in values (p < 0.05) for the AAE-treated animals in the 100 and 300 mgkg −1 groups as opposed to the 1000 mg/kg −1 and the control group. As demonstrated in Table 1, there was neutrophilia in rats given 100 and 300 mgkg −1 AAE (p � 0.002), compared with the 1000 mgkg −1 AAEtreated animals and the control group. Te variation in these parameters may not be dose dependent.
Previous research has shown that the efects of plant extracts on animals and humans in terms of cardiovascular toxicity, haemototoxicity, and gastrointestinal toxicity may be interconnected [25]. It is noteworthy that most of the haematological parameters (Table 1) evaluated in the present investigation had not changed signifcantly (p > 0.05). Although some parameters, such as neutrophils and lymphocytes, showed signifcant diferences in values (p < 0.05) among the AAE-treated animals (100 and 300 mgkg −1 as opposed to the control group), this outcome may not have been dose dependent since these parameters were normal at the dose level of 1000 mgkg −1 , which was the highest dose in this study. Plant extracts may cause neutropenia by suppressing growth factors, for example, granulocyte macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), which regulate neutrophil synthesis and deployment [26]. Neutropenia may be caused by infections due to microbial pathogens such as viruses, bacteria, protozoans, and fungi. Reduced neutrophils may also be triggered by an intrinsic disorder of proliferation and maturation of myeloid and stem cells [27]. Neutrophilia is a characteristic feature of infections or infammatory reactions [28].
Te pharmacological efect of AAE at 100 and 300 mgkg −1 may have increased GM-CSF production in rats, resulting in an increase in neutrophils. It is possible that the extract has a stimulating efect on the chemokines and cytokines that control their receptors. Tis would explain both the AAE efect and the increase in neutrophils [28]. Lymphocytopenia, on the other hand, can be caused by an active viral infection, or it could be caused by damage to the thymus or lymphoid architecture, which stops the body from making enough lymphocytes [29].
Te neutrophilia and lymphocytopenia (p < 0.05) recorded in the 100 and 300 mgkg −1 groups may be stress-induced in the animals during animal handling [30,31]. In the current study, there was no clear dose-response relationship, which might have resulted in the changes in these parameters by the extract that was tested. Overall, the haematological results of this investigation demonstrate that AAE has no signifcant detrimental impact on blood parameters.

Efect of AAE on Serum Biochemical Parameters.
All blood chemistry markers in rats, including aspartate aminotransferase, total proteins, albumin, alkaline phosphatase, alanine transaminase, total bilirubin, high-density lipoprotein, total cholesterol, very low-density lipoprotein, low-density lipoprotein, triglyceride, urea, creatinine, indirect direct bilirubin, and calcium, did not alter substantially (p > 0.05) following 14 days of daily AAE administration. Tere may be slight variations in all the blood chemistry parameters. However, up to a dosage level of 1000 mgkg −1 , these diferences are not statistically signifcant (Table 2). Tis study also found that none of the biochemical parameters that were measured changed at the highest dose level (1000 mgkg −1 ) because all the biomarkers in this study had normal values in all the profles that were analysed. Since AST, ALT, TB, DB, IDB, TP, ALP, ALB, and GGT levels did not change substantially (p > 0.05) in all extract-treated animals when compared to the untreated group, there is sufcient evidence to rule out liver damage (Table 2). Notably, ALT is a particular liver enzyme that, when elevated, indicates hepatocellular injury. However, in predicting liver disease, AST is considered a less specifc biomarker. It may be present in other organs but, when elevated, may suggest hepatic injury. Both ALT and AST are expected to rise dramatically in drug-induced toxicity [32]. High levels of ALP have been linked with biliary cirrhosis and obstructive jaundice [33]. Similarly, ALP levels are also pervasively elevated in bile duct obstruction. However, these enzymes did not change much in AAE-treated groups, which means that toxic-induced hepatobiliary disease is unlikely. Furthermore, histopathology excludes liver damage from the fndings of this study.
None of the kidney function test results changed substantially (p > 0.05) in any of the animals that took AAE. A decrease in glomerular fltration is the clinical manifestation of increased levels of CRE and urea, which are common in injured kidneys. In this study, the extract had no efect on kidney-related parameters like CRE and urea (p > 0.05) in animals that were treated with the extract compared with those in the control group (Table 2).
Several studies have linked increased levels of total cholesterol (T-Chol), triglycerides (Trig), and low-density lipoprotein (LDL) to a rising risk of coronary artery disease, cardiovascular disease, and ischemic stroke [34,35]. In this investigation, there was not enough evidence to suggest     Journal of Toxicology toxic-related cardiac damage in the AAE-treated rats. Tis is because the extract had no signifcant (p > 0.05) efect on any of the biochemical parameters tested.

Te Impact of AAE on Body
Weight. Alterations in body weight and relative organ weight in rats have been recommended as sensitive markers in toxicological evaluation to identify changes in organs that have been chemically exposed to toxicants [36]. Te impact of AAE on rats' body weight after 14 days of extract administration is presented in Table 3. Te variations in body weight of rats from pretreatment to postextract treatment were not statistically signifcant compared with the control animals during the 14day period. Body weight changes are also a factor in determining the overall health of laboratory animals and may be infuenced by exposure to potentially toxic substances [37]. In the current test, body weights of the extract-treated rats after the 14-day extract administration did not show any signifcant diferences (p > 0.05) when compared with the reference or control group (Table 3).

Efect of AAE on Relative Organ Weight (ROW).
Internal organs may undergo a variety of pathological changes because of the direct efects of toxic materials, causing weight changes. Internal organs, including the liver, kidney, heart, lungs, and spleen, are often the frst organs to be afected by toxic substance-induced metabolic reactions [38]. Table 4 illustrates the impact of AAE extract on the relative organ weights of rats following fourteen days of daily extract treatment. Te vital organs, including the kidneys, heart, spleen, and liver, did not show any signifcant changes in ROWs as opposed to the untreated group.
Te relative organ weights of AAE-treated animals had not changed signifcantly (p > 0.05) from the untreated group in this study (Table 4). Tus, repeated extract doses up to 1000 mgkg −1 during the 14-day period did not show any signifcant variation in organ weight, which means the extract did not harm the internal organs.

Histological Sections of Vital
Organs. Histopathology of selected internal organs was performed to corroborate the haematological and biochemical indices in the current preclinical toxicity studies as outlined in the 2002 OECD guidelines 423 [12,15]. Macroscopically, no morphological abnormalities were identifed in any of the vital organs of the rats dosed with AAE as opposed to the untreated animals. Tere were no histological abnormalities in the heart, kidney, and spleen, as these vital organs had the same normal architecture as those in the control group (Figures 2-5). Hepatocytes of the liver parenchyma were mostly normal looking, but some had large vesicular nuclei with an abundance of pink cytoplasm. Several sparsely scattered liver cells had dark, compact nuclei with more eosinophilic cytoplasm.    Journal of Toxicology At necropsy, the AAE-treated rats and untreated rats did not have hepatomegaly, hepatic vascular damage, interruption of bile production or fow, or ascites. However, there was a mild hepatocellular degeneration or death (necrosis) in the histopathology evaluation. Tis is consistent with a single-cell hepatocyte necrosis (death) amid many normal ones in the liver parenchyma. Since most hepatocytes are normal, single-cell necrosis of hepatocytes would have little efect on liver function. However, it has been suggested that until 75% of hepatocytes are dead, liver function does not decline. Te liver may be the only solid organ that utilizes regenerative processes to achieve complete recovery [39]. Depending on the degree of damage, liver injury is followed by vigorous hepatocyte multiplication to substitute dead cells and eventually induce spontaneous healing [40]. It is possible that the extract could not have caused the mild single-cell hepatocellular necrosis that was found in all groups of animals, as the same lesions were observed amongst the control animals. Because the changes in liver cells were minor, with a large proportion of the liver parenchyma being normal (Figure 4), they could not have had a signifcant impact on the overall outcome. Te results were consistent with the normal clinical chemistry parameters associated with a normal liver. On the other hand, the heart, kidneys, lung, and spleen had a normal architectural structure in the AAE-treated animals compared with the control group (Figures 2-5).

Conclusion
Te fnding shows that A. africana crude extract has no harmful efects on red blood cells. Te extract demonstrated no evident toxicity in both acute and sub-acute tests, up to a dose level of 5000 mgkg −1 . Te neutrophilia and lymphocytopenia observed in the 100 and 300 mgkg −1 animals were not dose dependent and could have been caused by stress. Tese fndings show that A. africana is hypothetically nontoxic for oral ingestion at doses up to 1000 mgkg −1 for repeated dosing in subacute toxicity studies. Further study is required to evaluate the chronic and subchronic toxicity of the plant extract.

Data Availability
Te underlying data supporting the results are included in this paper.

Additional Points
Highlights. (i) Traditionally, Avicennia species have been used as medicine for a wide range of diseases, including malaria. (ii) No potential toxicity signs were observed in all rats, demonstrating how safe the extract is. (iii) Te 70% ethanolic leaf extract of A. africana is potentially safe for oral consumption at doses of up to 1000 mg/kg bwt for repeated dosing.

Ethical Approval
Te research protocol was reviewed and approved (ID : UCCIRB/CHAS/2016/13) by the University of Cape Coast's Institutional Review Board. Te analysis was carried out in accordance with the guiding principles for the use of experimental animals as well as the Organization for Economic Cooperation and Development's good laboratory practises (GLP) [12,15,41].

Conflicts of Interest
Te authors declare that they have no conficts of interest.