Toxicological Characterization of Ten Medicinal Plants of the Beninese Flora Used in the Traditional Treatment of Diarrheal Diseases

The use of medicinal plants in traditional medicine is a common practice in developing countries. However, this unregulated or poorly rational use may present a dose-dependent risk of toxicity to humans. This study aimed to explore the phytochemical and toxicological characteristics of ten (10) plant species used in the traditional treatment of infectious diarrhea in Benin. The acute toxicity of aqueous and hydroethanolic extracts of Khaya senegalensis, Daniellia oliveri, Rauvolfia vomitoria, Vernonia amygdalina, Manihot esculenta, Ocimum gratissimum, Senna italica, Diospyros mespiliformis, Pterocarpus erinaceus, and Anacardium occidentale was evaluated following the OECD 423 protocol at a single dose of 2000 mg/kg. This safety test was complemented by a larval cytotoxicity test. Hematological and biochemical examinations, as well as a histological study of the liver and kidneys, were performed. Larval cytotoxicity was assessed by the sensitivity of Artemia salina larvae to different concentrations of the plant extracts studied. Testing for chemical compounds was performed on the basis of differential staining and precipitation reactions. The mean lethal concentration (LC50) was determined by the probit method. The qualitative phytochemical screening of the plants studied revealed the presence of catechic tannins, gallic tannins, flavonoids, anthocyanins and sterol-terpenes, alkaloids, saponosides, and reducing compounds. This composition varied according to the plants studied. Acute toxicity data indicated that there was no mortality and no structural and functional alterations of the liver and kidneys of treated animals. Larval cytotoxicity data suggest that the plants studied are not cytotoxic (LC50 ≥ 0.1 mg/mL). These observations reflect the safety of these plants and justify their use in traditional medicine in the treatment of many diseases including diarrheal diseases.


Introduction
Traditional medicine based on the use of medicinal plants is the first reflex of more than 80% of the world's population for primary health care [1][2][3]. In developing countries such as Benin, this widely accepted trend is an ancestral medical practice that is transmitted from generation to generation [4,5]. e real effectiveness, accessibility, and low cost of medicinal recipes are the main reasons for the perpetuation of this endogenous practice [6,7]. e plants used in the African pharmacopeia are directed against several diseases, particularly infectious ones. e prevalence of these infectious diseases is essentially linked to factors such as precarious water quality, unsanitary conditions, and poor food hygiene that are common to most developing countries [8,9]. Diarrheal diseases are among the most deadly infectious diseases, particularly among children. In fact, every year, it is estimated that there are 2.5 billion cases of diarrhea in children under the age of five [10]. Ugboko et al. [11] reported that childhood diarrhea affecting children five years old and below accounts for approximately 63% of the global diarrhea burden. 15% of these children die [12].
Benin is one of the developing countries in which diarrhea diseases are one of the main causes of morbidity [13,14]. Indeed, they have a direct impact on the costs associated with seeking health care, including several factors such as consultation, medication, and, in some cases, hospitalization which represents a burden on household expenditures [15]. e pathogens of diarrheal diseases are mainly bacteria [16]. Over the years, medical therapy based on the use of conventional antibiotics has shown not only its effectiveness but also its limitations. Indeed, most bacteria responsible for diarrheal episodes develop resistance to the antibiotics used in therapy. e phenomenon of multiresistance is today a real public health problem for the effective management of infectious diseases. is problem of bacterial resistance combined with the cost and sometimes difficult access to antibiotics by populations reinforces the use of herbal remedies by these populations. World Health Organization has supported this idea by launching a diarrheal disease control program based on traditional medicine practices and prevention approaches [17].
is can have valuable benefits in reducing child mortality rates in developing countries. In several African pharmacopeias, several ethnobotanical studies have provided information on the richness of African flora in the traditional management of diarrheal diseases [18][19][20]. Benin, a West African country, has a great floristic diversity to which is added a centuries-old traditional use of plants with unsuspected therapeutic virtues. Akoegninou et al. [21] estimated the ethnobotanical potential of Benin's flora at 2807 plant species. Khaya senegalensis, Daniellia oliveri, Rauvolfia vomitoria, Vernonia amygdalina, Manihot esculenta, Ocimum gratissimum, Senna italica, Diospyros mespiliformis, Pterocarpus erinaceus, and Anacardium occidentale are some of the medicinal plants of the Beninese flora used in the traditional treatment of diarrheal diseases. ese plants are also used in the traditional treatment of other diseases. For example, Khaya senegalensis, Ocimum gratissimum, Daniellia oliveri, and Manihot esculenta are used in the traditional treatment of metabolic diseases such as diabetes and blood disorders such as anemia [22][23][24][25]. e therapeutic virtues of these medicinal plants are attributed to secondary metabolites such as saponosides, flavonoids, coumarins, tannins, alkaloids, mucilages, volatile compounds, sterols, and terpenes identified in different parts of these plants [26]. However, despite the widespread use of traditional herbal remedies in the treatment of diarrheal diseases, very few scientific studies exist at this stage on the safety of antidiarrheal plants. It is, therefore, important, if not essential, to explore the toxicological characteristics of the main plants used by local communities in several African pharmacopeias in order to secure their use in traditional medicine. is study was initiated to produce recent data on the toxicological characteristics of selected plants as used in traditional medicine. It aimed to evaluate the larval cytotoxicity and acute toxicity of aqueous and hydroethanolic extracts of the selected plants on Wistar albino rats to predict their safety in the human species. Rats are recommended lower-level animals for toxicity studies to extrapolate to human biology according to Organization for Economic Cooperation and Development (OECD) safety study guidelines [27,28]. e finding of the study could also help to guide the optimization and validation of the traditional use of these antidiarrheal plants.

Plant Material.
e plant material used in this study consists of plant organs from 10 medicinal plants used in the traditional treatment of diarrheal diseases in Benin (Table 1). ese organs were harvested in various regions of Benin according to the indications of practitioners of traditional medicine. e plant samples once collected were authenticated at the National Herbarium of Benin using the analytical flora of Akoegninou et al. [21]. e botanical nomenclature of e PlantList available on the website http://www. ePlantList.com has been used to confirm this identification.

Animal Material.
e animal material consisted of eggs of Artemia salina and albino Wistar rats. Eggs of Artemia salina (ARTEMIO JBL D-67141 Gmbh Neuhofem) were used for the larval cytotoxicity test of selected medicinal plants.
ree-month-old Wistar albino rats weighing between 130 and 180 g, all nonpregnant and female, from the pet shop of the Institute of Applied Biomedical Sciences (ISBA) of the University of Abomey-Calavi were used for acute toxicity testing. Upon receipt, the rats were randomly assigned to groups of 3 in standard cages for a 2-week acclimatization period in the Research Unit in Applied Microbiology and Pharmacology of natural substances of the University of Abomey-Calavi prior to use. During this period, the animals had free access to food and water. Animal Research Review Panel and Animal Welfare Unit regulation of temperature and lighting systems was maintained with a room temperature of 20-26°C as well as regular light cycles of 12 hours light/dark. All methods and protocols used in this study were observed following established public health guidelines "Guide for Care and Use of Laboratory Animals" [29].

Phytochemical Analysis.
A qualitative phytochemical screening carried out according to the method based on precipitation and staining reactions described by Houghton and Raman [30] made it possible to detect the presence or absence of large chemical groups in the organs of the selected plants. ese searched chemical groups were tannins, gallic tannins, flavonoids, anthocyanins, leucoanthocyanins, alkaloids, mucilages, reducing compounds, sterols, terpenes, and saponosides.  [31]. ese were the aqueous extraction and the hydroethanolic extraction (50% water-ethanol; v/v). us, fifty (50) grams of powder were macerated in 500 mL of solvent (water and water-ethanol). e mixture was stirred continuously for 72 hours at room temperature. e homogenate obtained was filtered three times on absorbent cotton and once on Whatman No. 1 filter paper. e filtrate obtained was then evaporated at a temperature of 40°C in an oven (drying oven) until a dry mass was obtained which represents the extract. e extracts thus produced were placed in the refrigerator at 4°C and put back into solution during the various tests.

Larval Cytotoxicity Test.
e cytotoxic effect of the powders of the studied plants was evaluated on the Artemia salina larvae. e method described by Dougnon et al. [32] was adopted in this study. Artemia salina larvae were obtained by hatching 10 mg of Artemia salina eggs placed under continuous agitation in 1 L of seawater for 72 hours. Dilution series of order 2 of a stock solution of plant powder with a concentration of 20 mg/mL were carried out in order to have an increasing scale of concentration. To 1 mL of each of these diluted solutions was added 1 mL of seawater containing 16 live larvae. A control solution without the extract was prepared under the same conditions. All solutions were incubated under agitation for 24 hours. Counting the number of dead larvae in each solution under an optical microscope produced a representative curve of the number of surviving larvae versus the concentration of the extract. e data (concentration-response) were log-transformed, and the LC 50 (mean lethal concentrations) was determined. To assess the larval toxicity of the extract, the correlation grid associating the degree of toxicity to the LC 50 proposed by Moshi et al. [33] was used. According to this grid, if the LC 50 value is higher than 0.1 mg/mL, the extract is declared nontoxic. If this value is between 0.1 and 0.5 mg/mL, the extract is slightly toxic, and if the LC 50 is less than 0.1 mg/ mL, the extract is toxic.

Acute Toxicity Test.
e rats were divided into lots according to their weight. Since the extracts were administered orally, the method described in OECD guideline 423, the acute toxicity class method, was adopted. Since the selected plant species are commonly used by the population and no major toxic effects were reported, a toxicity limit test with a single dose of 2000 mg/kg body weight was used. Twelve hours before the toxicity tests were conducted, the animals were deprived of food and water. After weighing the rats, 21 groups of three rats were assembled and treated ( Table 2).
During the experiment, the animals were monitored and observed individually twice a day (morning and evening) over a period of 14 days. A data collection sheet was drawn up for each rat in order to collect possible signs of toxicity (skin and hair changes, the appearance of edema, walking backward, breathing difficulties, morbidity, and mortality). At the end of the treatment, the rats were deprived of food the last night before sampling. Blood samples (Day 0 and Day 14) were taken by puncture of the retroorbital sinus for all animals under ether anesthesia. e blood sample was collected in two types of tubes, one containing EDTA and the other without anticoagulant (dry tube). e samples from EDTA tubes were intended for hematological analysis. e dry tubes are centrifuged at 4000 rpm for 10 minutes, and the resulting serum is stored at − 20°C for analysis of biochemical parameters. After sampling, two animals per lot were sacrificed under anesthesia with ether for the collection of organs such as the liver and kidney. ese organs were rinsed with 0.9% saline and fixed in 10% buffered formalin.

Hematological and Biochemical Examinations.
ese examinations were performed at the Research Unit in Applied Microbiology and Pharmacology of natural substances (URMAPha). e hematological examinations included red and white blood cell counts, hemoglobin level, . e biochemical tests were concerned with the determination of urea, creatinine, aspartate aminotransferase (ASAT), and alanine aminotransferase (ALAT).

Histological Examinations.
Histological sections of the liver and kidneys were performed at the histopathology Laboratory of the Institute of Applied Biomedical Sciences (ISBA) of the University of Abomey-Calavi. e pathomorphological study consisted of hematoxylin-eosin staining of thin sections of 5 μm thicknesses. It is routine staining after which the nuclei, stained by hematoxylin, appear dark blue, and the cytoplasm, stained by eosin, appears pink. e microscopic observation of these sections was carried out with the ZEISS camera microscope at different magnifications so that only the most representative photographs were selected.

Data Analysis.
e obtained data were subjected to statistical analysis using SPSS 26.0. Quantitative variables were presented as mean and standard deviation. Qualitative variables were presented in percentages. e probit analysis was used for the LC 50 determination. e student test was used to compare the values of the different biochemical and hematological parameters of the treated animals with those of the control lot. e weight gain of the rats in each of the test lots was also compared to that of the control lot via Student's t-test. e significance threshold was set at 5%.

Results
e data obtained in this study are related to the phytochemical screening of the studied plants, larval cytotoxicity, and the different parameters of acute toxicity.

Weight Change of Animals in Different Lots.
Data collected on the body weight of the rats during the experiment are summarized in Table 5. From this table, a weight growth of the Wistar rats should be noted in all the groups. is weight growth was significant for the treated rats with Anacardium occidentale, Senna italica, and Manihot esculenta aqueous extract (p < 0.05). e influence of the different treatments on the biochemical parameters of treated and control rats is summarized in Table 6. From this table, it appears that the plant extracts

Effect of the Studied Plant Extracts on Hematological
Parameters . Tables 7 and 8 present, respectively, the influence of the studied plants on the parameters of the erythrocytic and leukocytic lineages. Statistical analysis of the data presented in these tables shows that, compared to the Wistar rats in the control group, all the extracts of the ten plants studied have no significant effect on these different hematological parameters of the animals (p > 0.05).

Histopathology Study.
From kidney and liver tissues, histological sections were performed to confirm the hematological and biochemical data. For all extracts, histological sections of the organs of treated rats show no structural abnormalities compared to controls. Figures 2 and  3 show, respectively, the hepatic and renal histology of extract-treated and control rats. e hepatic parenchyma of treated rats (B) has a typical appearance as observed in control rats (A). e hepatocytes (arrows) have a normal appearance and are arranged in Hydrochloric acid and ammonia at 50%   Evidence-Based Complementary and Alternative Medicine cords separated by sinusoids (S). e sinusoids drain into the centrilobular vein (V). e renal parenchyma of treated rats (B) has the typical architecture observed in control rats (A). e glomeruli (G), proximal tubes (TP), distal tubes (TD), and collecting channels (CC) are well identifiable. e extracts, therefore, did not affect the renal structures in any way.

Discussion
e present study aimed to explore the composition in secondary metabolites and reveal the toxicological characteristics of ten plants of the Beninese flora used in the traditional treatment of diarrheal diseases. e qualitative phytochemical screening of the plants studied revealed a varied richness in bioactive molecules. e presence of catechic tannins, gallic tannins, flavonoids, anthocyanins, and sterol-terpenes, alkaloids, saponosides, and reducing compounds were detected. Among these bioactive compounds, tannins are identified in all the plants studied. Similar observations are reported in the literature [34][35][36][37][38][39]. is presence of tannins as well as those of other identified bioactive molecules evokes the medicinal properties of the plants studied and their therapeutic uses in several pharmacopoeias. Indeed, plants containing tannins have astringent, hemostatic, antiseptic, and toning properties [40]. In addition, the tannins contained in medicinal plants are endowed with antimicrobial [41,42], antiviral, anti-inflammatory, antihypertensive, antimutagenic, immunostimulant, and antitumor properties [43][44][45]. Several reports have documented the efficacy of tannins in the treatment of diarrheal diseases [46][47][48]. As for flavonoids, it has been reported that plant extracts, which are rich in flavonoids, have antimicrobial activity [49,50]. A study has shown that flavonoids associated with saponins isolated from Anacardium occidentale, one of the medicinal plants in this study, are useful in the treatment of diarrhea [51]. Alkaloids, one of the metabolites identified in this study, are nitrogenous organic substances with antibacterial and antifungal properties [52]. e above evidence supports the use of the plants studied in the treatment of many diseases including diarrheal diseases. e safety of the plants was evaluated in vitro in the Artemia Salina model and by acute oral toxicity according to the OECD 423 protocol. e cytotoxic effect of the extracts evaluated according to the Artemia Salina model was used as a preliminary safety screening. e data obtained for this test indicate that, with the exception of Senna italica and Daniellia oliveri, all other plants studied are noncytotoxic to Artemia Salina larvae at the concentrations tested. Similar observations were reported by Dehou et al. [53] and Déguénon et al. [54] for Ocimum gratissimum and Soha et al. [55] for Khaya senegalensis. Lagarto Parra et al. [56] demonstrated a good correlation (r � 0.85; p < 0.05) between this larval toxicity test and toxicological effects on a whole animal. Some authors have confirmed this hypothesis in their work [57][58][59]. However, such an extrapolation is hotly debated [60]. In order to confirm the data of this preliminary screening, on the one hand, and to explore the safety of the studied plants on a whole organism, on the other hand, the acute oral toxicity test in Wistar albino rats was carried out.
e results obtained indicate that no mortality was recorded during the 14 days of the experiment. Similarly, no signs of apparent toxicity were observed. e plants studied thus  Evidence-Based Complementary and Alternative Medicine  [61]. In addition, during the experiment, the evolution of the body weight of the animals was monitored. e data obtained for this parameter indicate weight growth in all animals' lots, reflecting their good    [37] and Jintanaporn et al. [62] reported that, at 2000 mg/kg, hydroethanolic extract from the leaves of Anacardium occidentale induced no mortality or signs of apparent toxicity. e same observation is reported by Ahmadu et al. [63] for the ethanolic extract of Daniellia oliveri. Ebbo et al. [64] demonstrated the safety of the methanolic extract of the leaves of Diospyros mespiliformis at a dose of 5000 mg/kg in Wistar rats. Nadro and Modibo [65] have also proven the safety of aqueous and ethanolic extracts of Senna italica in their work. As for Pterocarpus erinaceus, Ajayi et al. [34] showed that the ethanolic extract of the leaves of this plant induced no apparent toxicity in Wistar rats at a dose of 5000 mg/kg. e study of Nkoua Badzi et al. [38] reached the same conclusion with regard to the aqueous extract of Rauvolfia vomitoria. Legba et al. [66] showed the safety of aqueous and ethanolic extracts of the leaves of Vernonia amygdalina used in Southern Benin. Ojo et al. [40] and Satish and Ranjana [67] reported the safety of ethanolic aqueous extracts of the leaves of Manihot esculenta and Ocimum gratissimum, respectively. For Khaya senegalensis, the data obtained from this study are consistent with those reported by Soha et al. [55].
Beyond the data on the apparent toxicity of medicinal plants, adverse effects can be observed at the tissue scale through functional and structural alterations of certain vital organs such as the liver and kidneys. ese aspects were taken into account in the present study by exploring renal, hepatic, and hematological parameters as well as a histopathological study of these organs. e exploration of the hepatic function concerned the measurement of the enzymatic activity of transaminases (ASAT, ALAT). ALAT and ASAT are the main markers of liver function generally explored in toxicological studies of medicinal plants. Under normal physiological conditions, these markers are present at low concentrations in serum. Elevated serum levels of these enzymes, particularly ALAT, are considered sensitive markers of liver damage [68,69]. In this study, no significant influence was noticed on the activity of these enzymes. However, it is reported that the nonsignificant increase in the levels of these enzymes observed in both the test and control lots is not related to any functional impairment of the liver but rather to stress due to manipulation. is hypothesis is reinforced by the histopathological study which revealed that no structural alterations of the liver were observed.
e renal parameters evaluated in the exploration of kidney function were uremia and creatininemia. Any increase in the level of these markers indicates probable kidney tissue damage [70]. In this study, it should be noted that no significant effect was induced by the treatments with the various plant extracts studied. ese data, which reflect an absence of functional alteration of the kidneys, are reinforced by the histological study which revealed an absence of structural alteration of this organ.
Moreover, the hematopoietic system is one of the most sensitive targets for toxic substances [71]. It represents an important marker of the physiological and pathological state of humans and animals. Any alteration in hematological parameters is perceived as a potential risk of anemia [72]. Data from the present study indicate that the plants studied had no significant effect on either erythrocyte or leukocyte lineage parameters. is suggests that the extracts of the ten plants studied were not responsible for any anemic disorders. However, the increase in the number of leukocyte lineage cells (white blood cells and monocytes) observed could be explained by the fact that there was a possible thrombocytosis coupled with hyperleukocytosis. is could be attributed to the use of the metallic gastric tube. Dougnon et al. [73] support this idea by reporting that since the test lots were force-fed and the control batch had free access to water, it is possible that the probe may have attacked the esophageal wall of these animals causing an acute syndrome.
From the abovementioned data, it appears that the different extracts of the studied plants were not responsible for any mortality and any alteration of biochemical and hematological parameters. ey do not present any risk of toxicity when used at a dose of 2000 mg/kg.

Conclusion
e purpose of this study was to generate recent data on the phytochemical and toxicological characteristics of ten (10) plants used in the traditional treatment of diarrheal diseases. Phytochemical screening of the plants studied revealed a varied richness of secondary metabolites. Two approaches were adopted for safety testing: acute oral toxicity and larval cytotoxicity.
e results obtained following the larval cytotoxicity model indicated that the plants studied are noncytotoxic. Acute toxicity data indicate that the plants studied did not induce mortality or structural and functional alterations of the liver and kidneys at the dose of 2000 mg/kg. ese observations justify the use of these plants in several African pharmacopeias. However, more in-depth toxicological studies (subacute, chronic, and subchronic toxicity) are necessary for a better knowledge of the toxicological profile of these plants.

Data Availability
e data used to support the findings of this study are fully available upon request from the corresponding author.