Acute and Repeated Dose 28-Day Oral Toxicity Study of the Aqueous Extracts from the Leafy Stem and Fruit of Pedalium murex D.Royen EX.L in Wistar Rats

Background Pedalium murex (P. murex) is used in folk medicine for treatment of male infertility. However, scientific data on its safety are limited. Objective This study was carried out to assess the acute and repeated dose 28-day oral toxicity of the aqueous extracts from P. murex leafy stem and fruit in Wistar rats. Methods The acute toxicity test was performed according to the line 423 of the Organization for Economic Cooperation and Development (OECD) guidelines. The rats were randomly divided into three groups (n = 3). The control group received distilled water, while the experimental groups were given at a single dose, 5000 mg/kg of each extract. The repeated dose 28-day oral toxicity was performed according to the line 407 of the OECD guidelines. 35 rats divided into 7 groups of 5 male rats each were daily treated for 28 days with each extract at 200 mg/kg, 400 mg/kg, and 800 mg/kg, respectively. The in-life parameters were recorded during the follow-up. At the end of this study, organ weights, hematology, biochemistry, and histology parameters were analyzed. Results In the acute oral toxicity test, there was no morbidity or mortality related to the treatments. Both extracts belong therefore to category 5 of the globally harmonized system (GHS) of classification. In the repeated dose 28-day oral toxicity test, both extracts did not alter animal's behavior. However, both extract administration led to proteinuria and renal damages. Conclusion P. murex leafy stem and fruit aqueous extracts exhibited potential nephrotoxicity. Therefore, care should be taken when they are used over an extended period.


Introduction
Infertility is defned by the World Health Organization (WHO) as the inability to conceive after at least 12 months of regular unprotected sexual intercourse [1]. Male factor is responsible for about 50% of infertility cases [2]. In 30% to 40% of cases known as idiopathic, the etiology of male infertility is not known [3].
Idiopathic male infertility treatment relies on assisted reproductive techniques and empirical medical therapy [4]. Te assisted reproductive technique cost makes it unafordable to most of the couples [5]. Te empirical medical therapy is based on low quality evidence and contradictory fndings [6,7]. Tis situation led infertile men to search for afordable, efective, and safe alternatives like phytotherapy [8].
Phytotherapy is the primary source of healthcare for about 80% of people in less developed countries [9]. Several medicinal plants are used to treat male infertility, including Pedalium murex (P. murex) [10]. Te common misconception on medicinal plant safeness favors their consumption despite the risks of toxicity inherent in their overdose or long-term use [11].
P. murex belongs to the Pedaliaceae family and is commonly called "azizafen" in Benin. P. murex is used as aphrodisiac, to improve appetite and useful to treat asthma, gonorrhea, heart troubles, urinary discharges, skin diseases, and impotency [12]. Te mucilaginous liquid resulting from shaking the leafy stem in water is used to treat seminal abnormalities and other male reproductive tract disorders. Te dried powder of the fruit mixed with porridge is also used to alleviate male reproductive diseases [13]. In our previous study, preliminary phytochemical analysis of the aqueous extracts of P. murex leafy stem and fruit has revealed presence of favonoids, tannins, alkaloids, coumarins, saponins, steroids, and lignans [14].
Diferent experimental models have confrmed the effectiveness of P. murex leafy stem or fruit folkloric use as aphrodisiac or male fertility booster [14][15][16]. Our prior study has also revealed the protective activity of P. murex leafy stem aqueous extract at 400 mg/kg against lead-induced testicular toxicity in Wistar rats [14]. Although phytochemicals could exhibit pharmacological efects, they could however be toxic [17].
A lot of data related to P. murex ethnomedicinal values and pharmacological activities are available [10]. However, there is a paucity of data on its safety and toxicity. It is the need to fll the gap on the potential side efects associated with the repeated oral treatment with the leafy stem and fruit of P. murex that prompted this work. Its aim was therefore to study the acute and repeated dose 28-day oral toxicity of the aqueous extracts of the leafy stem and fruit of P. murex in Wistar rats.

Preparation of Extracts
. 50 g of the leafy stem and fruit powder was subjected to maceration in 500 ml of distilled water for 24 hours. Te mixture was stirred continuously using an orbital magnetic stirrer. Te fltrate was concentrated in the rotavapor (IKA-RV8) and then dried in an oven at 40°C for 2 hours. Te extraction yields were equal to 15.56% ± 1.56 and 08.44% ± 0.92, respectively, for the leafy stem and fruit extract. Te dried extracts were stored at 4°C for further use.

Animals.
Nine-week-old male rats (168 ± 1.26 g) were purchased from the Husbandry of Histology, Biology of Reproduction, Cytogenetics, and Medical Genetics. Te rats were housed in plastic cages and maintained at a room temperature of 22°-24°C with a 12 h dark/light cycle. Te rats were fed with rodent chow (proteins: 18%, fat: 7.12%, carbohydrate: 14.14%, calcium: 1.16%, phosphorus: 0.80, and lysine: 0.82) and had ad libitum access to water.

Experimental Design.
Male rats were used due to the fact that this study aims to evaluate the toxicity associated with treatment of male infertility with P. murex. Tis study follows a frst one that has assessed the potential of the aqueous extracts from P. murex leafy stem and fruit in male infertility treatment [14].

Acute Oral Toxicity.
Te acute oral toxicity of the extracts was assessed according to the OECD (Organization for Economic Cooperation and Development) line 423 guidelines [18]. Nine male rats were randomly divided into 3 groups of 3 rats each. Te control group (Ctrl) received distilled water; groups LS5000 and FR5000 received, respectively, the leafy stem and fruit extracts at 5000 mg/kg. Te test was performed at 5000 mg/kg to increase knowledge on toxicity related to Pedalium murex overdosing in human. Treatments were given via gavage. Te rats were observed each day during 14 days for mortality, signs of acute toxicity, and behavioral changes. At the end of the experimental period, all animals were fasted overnight, weighed, and sacrifced under sodium thiopental anesthesia (50 mg/kg, IP). Te liver, kidneys, stomach, spleen, heart, and lungs of the animals were removed and weighed. Te liver and kidneys were fxed in 10% (v/v) formalin saline for histopathological analysis.

Repeated Dose 28-Day Oral Toxicity.
It was performed according to the 407 guidelines of OECD [19]. 35 male Wistar rats were randomly divided into 7 groups of 5 rats each. Te rats were daily treated via oral gavage with an intragastric canula. Te control group received distilled water; LS200, LS400, and LS800 groups were treated with P. murex leafy stem at 200 mg/kg, 400 mg/kg, and 800 mg/ kg, respectively; FR200, FR400, and FR800 groups were treated with the fruit of P. murex at 200 mg/kg, 400 mg/kg, and 800 mg/kg, respectively. Te highest dose (800 mg/kg) was determined by multiplying the pharmacologically active dose by 2-fold intervals [14]. Te rat behavior was observed during the follow-up. Te observations included clinical signs such as tremors, convulsion, salivation, diarrhea, lethargy, and coma. In the last week of the study, animals were housed in metabolic cages and their urine was collected for 24 hours (12 hours in light cycle/12 hours in the dark cycle). Urinalysis was performed using test strips [20].
At the 28 th day, all animals were fasted overnight prior to necropsy. Rats were anesthetized with 50 mg/kg of sodium thiopental. Blood was collected by carotid exsanguination in EDTA and plain tubes for hematological and biochemical analysis. Te liver, stomach, spleen, kidneys, lungs, heart, testes, and epididymis of the animals were weighed using an analytical scale (ESSE3). Te liver, kidneys, and testes were fxed in 10% (v/v) formal saline for histopathological analysis.
2.6. Statistical Analyses. Data were expressed as the mean-± Standard Error on the mean (SEM). Data of acute toxicity were analyzed with the nonparametric Kruskal-Wallis and Dunn's multiple comparison tests. Te data of the repeated dose 28-day oral toxicity test were analyzed through ANOVA followed by Dunnett's multiple comparison test. Values were considered statistically signifcant at p ≤ 0.05.

In-Life Observation.
Both extracts did not cause morbidity or mortality. Te treatment with fruit extract increased rat body weight at the end of the study when compared to the control group (Table 1). Both extracts increased the food intake when compared to the control group (Table 1). However, the body weight gain of both treated groups showed no change when compared to the control group (Table 1).

Relative Organ Weight.
Te relative organ weight showed no change between the treated and control groups ( Table 2).

Histopathology.
Te gross examination of the liver and kidneys of the treated groups revealed no diference when compared to the control group. Te treated groups showed normal liver and kidney histology when compared to the control group ( Figure 1).

In-Life Observation.
Te treatment with both extracts did not lead to any toxic symptoms in rats. Tere were no behavioral diferences between the treated and control groups. On the 3 rd week of the study, a higher mean body weight was noticed at 800 mg/kg in the leafy stem-treated group (Table 3). Te group treated with the fruit extract depicted a lower mean body weight than the control group. Tis diference reached statistical signifcance at 400 mg/kg during the last week (Table 3). A signifcant decrease in the body weight gain was noticed after treatment with the fruit extract at 400 mg/kg (Table 3). Te average food intake of the rats treated with the fruit extract at 400 mg/kg decreased signifcantly when compared to the control group during the 1 st , 2 nd , and 4 th weeks of the study (Table 4).

Relative Organ Weights.
At 400 mg/kg, the relative kidney weight increased signifcantly after treatment with both extracts. Te relative weight of the heart and testes showed a signifcant increase at 400 mg/kg after treatment with the fruit extract (Table 5).

Urinalysis.
Te urine from rats treated with the leafy stem showed proteinuria and leukocyturia at 400 mg/kg and 800 mg/kg (Table 6). Te rats treated with the fruit extract exhibited proteinuria and hematuria at 400 mg/kg and 800 mg/kg (Table 6).

Biochemistry.
AST activity decreased signifcantly at 200 mg/kg, while it increased at 400 mg/kg in the leafy stem treated group. ALT activity increased at 400 mg/kg and 800 mg/kg in the leafy stem-treated group (Table 7). Te fruit extract-treated rats showed a decrease in AST activity and an increase in ALT activity at 200 mg/kg. Te leafy stem decreased glucose at 200 mg/kg, 400 mg/kg, and 800 mg/kg (Table 7). Te leafy stem and fruit extracts increased the triglyceride amount at 400 mg/kg and at 800 mg/kg, respectively (Table 7).

Hematology.
Both extracts decreased the leukocyte count when compared to control, but statistical signifcance was reached only at 800 mg/kg. Te leafy stem extract increased the platelet count at 200 mg/kg while decreasing it at 400 mg/kg and 800 mg/kg. Te fruit extract led to a marked increase in the platelet count at 400 mg/kg and 800 mg/kg (Table 8).

Discussion
Te current treatment of idiopathic male infertility in orthodox medicine is unafordable and inefective [21,22]. Terefore, many couples do not hesitate to use medicinal plants including P. murex despite their potential toxicity. Tis study aimed to investigate the acute and repeated dose 28-day oral toxicity of P. murex leafy stem and fruit aqueous extracts. Te repeated treatment with both extracts revealed their potential nephrotoxicity.
Ideally, the toxicity of the xenobiotics to which people are exposed should be performed on humans. However, this experimental approach is unethical. Terefore, toxicity studies are preferentially performed on animals' models including the rat. Te rat is often used due to its metabolic rate similarity to humans, its small size, short lifespan, and gestation time [23].
Te acute toxicity of a chemical is estimated through its median lethal dose (LD 50 ). In this study, the single administration of the aqueous extracts from the leafy stem and fruit of P. murex at 5000 mg/kg did not induce morbidity and mortality. Hence, both extracts belong to category 5 of the globally harmonized system (GHS) of classifcation. Tis fnding agrees with prior data of Sharma et al. [15] and Gomathi et al. [24]. Our data revealed that the treatment with both extracts increased the food intake but did not modify the body weight gain. Tis fnding could be linked to the type of food predominant nutrient. Te chow given to the rat in this study was rich in dietary protein. But, according to Wu et al. [25], only the increment in food dietary fat was associated with the body weight gain in mice.
However, factors such as poor absorption and the frstpass efect may decrease extract bioavailability and mislead the appreciation of their toxicity [26]. Moreover, medicinal plants are often used for prolonged periods. Hence, it is important to assess their toxicity over long-term treatment.
Te repeated dose 28-day oral toxicity study provides data on the health risks associated with repeated exposure to a test substance over a limited period of time [19]. To our knowledge, this study is the frst that has evaluated the repeated dose 28-day oral toxicity of P. murex in rodents.
No behavioral changes, toxic signs, and mortality resulted from the treatment with the aqueous extracts from P. murex leafy stem and fruit. Body weight is used to monitor general health status [27]. Te fruit extract induced a loss of body weight and decreased the body weight gain at 400 mg/ kg (bw). Although the body weight loss has not reached statistical signifcance for other doses (200 mg/kg and 800 mg/kg), this decrease was higher than 5% when compared to the control group. According to Silva et al. [27], a body weight loss of 5% is predictive of organ injury. Tese data suggest that repeated treatment with the fruit of P. murex could be toxic.
Hematological parameters are the most sensitive to toxicants and have a high predictive value when data are extrapolated from animals to humans [28]. In this study, repeated treatment with both extracts showed a dose-dependent decrease in the leukocyte count. Tis fnding could indicate that animals are exposed to stress factors or are highly vulnerable to a toxicant [29]. Te leafy stem aqueous extract has led to a nondose-dependent variation in the platelet count. However, the fruit extract signifcantly increased the platelet count at 400 mg/kg and 800 mg/kg. Since a high platelet count increases the risk of thrombosis in humans, a more in-depth assessment through further studies of the efects of P. murex on coagulation is required to elucidate these fndings.
Organ weight changes are often correlated with treatment efects [30]. Te absolute organ weight changes with the animal body weight. Terefore, the relative organ weight is used to eliminate the bias associated with normal variation in animal growth [31]. In the present study, treatment with both extracts led to a nondose-dependent increase in the relative organ weight of the kidneys. Tis fnding could therefore be incidental. However, the organ weight data should be always analyzed in an integrated way with data of biochemistry, hematology, and histology [29].
Nephrotoxicity is usually evaluated through urinalysis, serum biochemistry, and kidney histopathology. Blood urea and creatinine are the classic indices of the glomerular fltration rate (GFR). Te increase in blood urea and creatinine above the reference range are inversely correlated to the GFR [32]. Both extract administration has not modifed urea and creatinine levels. However, urinalysis has revealed proteinuria after treatment with both extracts. Furthermore, several glomerular damages in the kidneys were noticed after treatment with both extracts. Terefore, both extracts depicted potential nephrotoxicity. Pedalium murex nephrotoxicity could be due to its steroidal saponins. Indeed, Pedalium murex contains several steroidal saponins notably diosgenin [10,33]. Pedalium murex is used as substitute of Tribulus terrestris by many practitioners of Ayurvedic medicine to cure sexual diseases [34]. Te features of renal damages induced by the ethanolic extract of Tribulus terrestris fruit (protodioscin) in rats were similar to our fndings. Tese data led us to hypothesize that P. murex leaf and fruit aqueous extract nephrotoxicity could be linked to their steroidal saponins. Contrary to our fndings, the ethanolic extract of P. murex fruit has showed protective efects against cisplatin-induced nephrotoxicity in Wistar rats [35,36]. In addition, fruit aqueous and ethanolic extracts have exhibited protective efects against cadmiuminduced nephrotoxicity in Wistar rats [36]. Te divergent results could stem from the diference in the study length. Our study takes 28 days while prior study length at most  Values are expressed as the mean ± SEM. p ≤ 5% value was considered signifcant using Kruskal-Wallis test followed by Dunn's multiple comparison test. Asterisk denotes diference compared with the control group. P. murex-treated rats without any change in creatinine and serum urea levels. Serum creatinine and urea are nonspecifc and insensitive markers for chronic kidney diseases. Tey are the later indicator of renal impairment since their levels increase signifcantly only when more than 50% of the glomerular fltration rate is reduced [37]. Also, in glomerular nephrotic syndrome, proteinuria may occur without any increase in the creatinine level [38]. Te activity of transaminases increased in response to hepatocellular damage. Te markers of hepatocellular injury are ALT and AST, with ALT being the most specifc [39]. In this study, the leafy stem extract decreased the ASTactivity at 200 mg/kg while the extract increased it at 400 mg/kg.  Values are expressed as the mean ± SEM. * p ≤ 0.05 value was considered signifcant using ANOVA followed by Dunett's multiple comparison test. Asterisk denotes diference compared with the control group. Values are expressed as the mean ± SEM. * p ≤ 0.05 value was considered signifcant using ANOVA followed by Dunett's multiple comparison test. Asterisk denotes diference compared with the control group.
Te aqueous extract of P. murex leafy stem showed a signifcant elevation of the ALT level at 400 mg/kg and 800 mg/ kg when compared to the control group. Te increase in the ALT activity suggests hepatocyte necrosis or transient changes in their membrane permeability [40]. However, the protein level, an indicator of liver function, was not altered by the leafy stem extract even at the highest dose (800 mg/ kg). Furthermore, liver histopathology did not show any sign of abnormalities. Te aqueous extract of the fruit showed a nonconsistent pattern of variation of AST and ALT. Treatment with the highest dose of the fruit extract did not alter the AST, ALT, total protein levels, and liver    Journal of Toxicology   histoarchitecture. Tese fndings evidenced the lack of toxicological relevance of the variations in the ALT activity and the need to assess P. murex toxicity over an extended period. Te testis is a target organ of P. murex according to its uses in folk medicine [10]. Te potential testicular toxicity of P. murex was evaluated via histopathology. Both extracts did not induce any deleterious efects on seminiferous tubule histoarchitecture. Te efects of extracts on seminal parameters were not assessed in this study due to the fact that spermatogenesis lasts about 52 days in rats [41]. Tese preliminary data indicate that P. murex does not exhibit testicular toxicity after repeated dose 28-day of oral treatment in Wistar rats.
Considering our data, the aqueous extract of P. murex leafy stem and fruit seems to be safe at 200 mg/kg. Terefore, the no-observed-adverse-efect level (NOAEL) for P. murex leafy stem and fruit aqueous extracts is found to be 200 mg/kg.

Conclusion
Te aqueous extracts from P. murex leafy stem and fruit do not present acute toxicity. However, both extracts should be used with caution when they are given over an extended period due to their potential nephrotoxicity. Studies over 90 days of exposure are needed to ensure the profle of safety and toxicity of P. murex.

Data Availability
Te majority of the data are included in this manuscript. Further data can be found from the corresponding author on reasonable request.

Ethical Approval
Te handling and use of animals for this study was approved by the institution's ethical committee for the use of animals in laboratory experiments (Reference No. 087/7/ 05/2021) and was in accordance with the National Institutes of Health guide for the use and handling of experimental animals.

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