Acute and Subchronic Toxicity Studies on the Aqueous Extract of the Plant Mixture (Bidens pilosa and Cymbopogon citratus Aerial Parts) in Rat Model

Bidens pilosa (B. pilosa) and Cymbopogon citratus (C. citratus) are plants used individually or in combination in the traditional treatment of several ailments such as cardiovascular disorders. In order to valorise their traditional use, a toxicological study was conducted on the aqueous extract of the mixture of aerial parts of B. pilosa and C. citratus. The acute and subchronic toxicity studies were conducted according to the OECD 425 and 407 guidelines. Regarding the acute study, the aqueous extract of the mixture of B. pilosa and C. citratus 50 : 50 (2000 and 5000 mg/kg) was administered once to rats of both sexes. In the subchronic study, the aqueous extract of the mixture of B. pilosa and C. citratus (200, 400 and 800 mg/kg) was administered once daily to rats for 28 days. The aqueous extract of the mixture of B. pilosa and C. citratus (2000 and 5000 mg/kg) did not cause death and did not induce any apparent sign of toxicity during the 14 days of observation. The DL50 of the extract is therefore greater than 5000 mg/kg. Taken daily for 28 days, the extract had no significant effect on selected parameters (creatinine, AST, ALT, urea, and uric acid) of renal and hepatic function, as well as on the number of some blood cells. However, the aqueous extract of the mixture of B. pilosa and C. citratus (200 and 400 mg/kg) caused a significant (p  <  0.05; p  <  0.001, respectively) decrease in creatinine levels in male rats as compared to normal control animals. In females, the aqueous extract of the mixture of B. pilosa and C. citratus (200 and 400 mg/kg) resulted in a significant (p  <  0.05) increase in total cholesterol levels as compared to normal control animals. The study showed that the aqueous extract of the mixture of B. pilosa and C. citratus has a low toxicity and does not cause any injury to the liver, kidney, lungs, or spleen.


Introduction
Te variety of plant species in the Cameroonian fora is very interesting. Indeed, these plants are important for the balance of ecosystems and are used for the survival of the population and their daily activities, by providing some natural resources. Teir contribution for food security and primary health care is of high importance for the developing countries' population [1]. Today, the practice of herbal medicine combined with conventional medicine has attracted more attention and is increasingly accepted worldwide [2]. Tough generic medicines are available, many of them remain fnancially inaccessible to the economically disadvantaged population. Tis leads people to turn to the more available and efective bioactive molecules [3]. However, there is no direct link between the traditional use of a plant for therapeutic purposes and its safety [4]. While many studies have demonstrated the efectiveness of many plants, investigations about their safety are seriously lacking. Te main goal of the toxicity study is to regulate the use of the substance studied, and this passed through several pharmacological tests. Tese tests are arranged in some conventional experimental protocols to check the administration route as well as potential injuries and mortality.
Many plants have already proven their efects on various diseases. For example, Bidens pilosa (B. pilosa) and Cymbopogon citratus (C. citratus) are medicinal plants belonging to the Asteraceae and Poaceae families, respectively. Tese plants commonly called black Jack and fever grass have been traditionally used to manage a number of diseases. B. pilosa is used for infuenza, malaria, ear infections, hepatitis, haemorrhoids, and heart problems [5]. C. citratus is used to treat gastrointestinal pain, herpes, fever, headaches, and heart problems [6]. Previous studies have confrmed the traditional uses of these plants [7][8][9][10][11] and assessed the toxicity of each plant [6,[12][13][14]. Several scientifc studies have already been carried out on plant mixtures [15][16][17][18]. Tese two plants in mixture are also used to treat cardiovascular disorders (hypertension) in the central region of Cameroon but no data on the toxicity of the mixture are available. Tis gap prompted the implementation of the present work, which intends to study the toxicity of the aqueous extract of the mixture of the aerial parts of B. pilosa and C. citratus in rats. Te main purpose has been to highlight the potential dangers to human health associated with its consumption. More specifcally, the work aims to study the acute and subchronic toxicity of the plant's mixture extract in rats.

Plant Material.
Te aerial parts of C. citratus (leaves and stems) and B. pilosa (leaves, stems, and fowers) were collected in Yaoundé (central region of Cameroon), more precisely in Messa-ssi, in February 2018. C. citratus was identifed by comparison with the botanical collection of D. Dang N°202, registered at the National Herbarium of Yaoundé under N°18626/SFRcam. B. pilosa was identifed by comparison with the botanical collection of B. Pollar N°2 register at the National Herbarium of Yaoundé under N°60447/HNC.

Preparation of the Aqueous
Extract of the Mixture of B. pilosa and C. citratus. Te aerial parts of B. pilosa and C. citratus were washed, dried in the shade, and then crushed to obtain the powder of each plant. Following the protocol described by Tcheutchoua et al. [17], briefy, 125 g of B. pilosa and 200 g of C. citratus powder were mixed in 3.25 L of hot distilled water. Te mixture was left until cool and then fltered with Whatman paper N°3 and evaporated in an oven (45°C). Tis made 33 g of aqueous extract form the mixture of B. pilosa and C. citratus (50 : 50), giving a yield of 10.15%. From the previous study conducted by Tcheutchoua et al. [17], the highest dose of this plant mixture, which was 200 mg/kg was multiplied by 2 and 4 in order to obtain the doses of 400 and 800 mg/kg. Tese three doses were therefore used in the subchronic study.

Animal Material.
Toxicological studies were performed with male and female Wistar albino rats, aged 6-8 weeks and weighing 140-160 g at the start of the experimentation. Females (nulliparous and nonpregnant) and males were separated in cages throughout the studies. Te animals were reared in the animal house of the Faculty of Science of the University of Yaoundé I (Cameroun) in Plexiglas cages. Te animals were acclimatized for two weeks before the start of the experiment. Te animals had free access to tap water as drinking water and to a standard diet (for a 50 kg bag, containing 40% maize, 20% wheat bran, 24% fsh meal, 7% palm kernel meal, 4% peanut meal, 2% cottonseed meal, 2% bone meal, 1% table salt, and 20 g vitamin complex). Every efort was made to minimise animal sufering and reduce the number of animals used. Te rats were housed in groups of 3 and 5 per cage, respectively, for the acute and subchronic toxicity studies under room temperature (25 ± 3°C), sufcient ventilation, and natural light cycle (12 h/12 h). All procedures and protocols involving animals and their care were conducted in accordance with institutional guidelines and approved by the National Ethics Committee of Cameroon (Reg. No. FWA-IRB00001954).

Evaluation of the Acute Toxicity of the Aqueous Extract of
the Mixture of B. pilosa and C. citratus. Te acute toxicity of aqueous extract of the mixture of B. pilosa and C. citratus was carried out according to the modifed OECD guideline 425 [19]. Eighteen rats (9 males and 9 females) were used and divided into 3 batches of 3 females and 3 males each (two test batches and one control batch). Te test batches were treated by gavage with the aqueous extract of the mixture of B. pilosa and C. citratus at doses of 2000 and 5000 mg/kg, respectively, in a single administration. Te control lot was given distilled water (10 mL/kg). Te animals were observed individually at least once during the frst 4 hours. Ten, regularly during the frst 24 hours and for 14 days after the administration, behaviour (locomotion, aggression, sensitivity to touch, and sound), appearance of faeces and coat condition, and mortality of the animals were observed continuously. Te animals were weighed every three days throughout the experimentation period. Te weight of the animals obtained was used to calculate the percentage body weight gain change from their initial weights. Weight gain (%) � ((mim)/m × 100; mi � mass on day i (g); and m � initial mass (g).
At the end of the experiment, the rats were fasted for 12 hours but had free access to tap water. Tey were anaesthetised by ether inhalation and sacrifced by cervical dislocation. Te rats were then dissected, and organs such as heart, liver, lungs, kidneys, brain, and spleen were well examined, collected, and weighed for the assessment of relative organ weight using the following formula [13]: where Wr is the relative organ weight; wo is the organ weight (g); and wb is the rat body weight (g).

Evaluation of the Subchronic Toxicity of the Aqueous
Extract of the Mixture of B. pilosa and C. citratus. Tis test was performed according to the modifed OECD guideline 407 [20]. Six batches of 10 animals each (5 males and 5 females) were used for each dose. Batch 1 received distilled water (10 mL/kg) and batches 2, 3, and 4 received the aqueous extract of the mixture of B. pilosa and C. citratus (200, 400, and 800 mg/kg), respectively. Animals in batches 5 and 6 (satellite control and satellite batches) were treated by gavage with distilled water (10 mL/kg) and an aqueous extract of the mixture of B. pilosa and C. citratus at a dose of 800 mg/kg, respectively. Tose two batches were observed two more weeks after the other groups. During treatment (28 days), the body weights of the animals were assessed weekly to determine the percentage change in body weight gain, and the signs of toxicity were carefully examined.

Blood and Organ Collection.
At the end of the experiment, the rats were made to fast for 12 hours but had free access to tap water. Tey were then anaesthetised by ether inhalation and sacrifced by decapitation and a part of the arteriovenous blood (around 4 mL) was collected in dry tubes without anticoagulant and allowed to stand for 30 min at room temperature and then centrifuged at 3000 rpm for 15 minutes. Te serum obtained was stored in the freezer at −20°C for biochemical analysis. Te other part of the arteriovenous blood (around 1 mL) was collected in EDTA (ethylene diamine tetra acetate) tubes for haematological studies. Te rats were then dissected, and organs such as the heart, liver, lungs, kidneys, brain, and spleen were collected, rinsed in saline (0.9%), blotted on absorbent paper, and weighed for the assessment of relative organ weights according to the formula previously described in acute toxicity. A portion of these organs was fxed in bufered formalin (4%) for histological analysis.

Biochemical Analysis.
Total cholesterol (TC), highdensity lipoprotein cholesterol (HDL-Chol), triglyceride (TG) concentrations, uric acid, urea, albumin, and bilirubin levels in serum were determined using commercial diagnostic kits (LABKIT and CHEMELEX S. A. Barcelona) by the colorimetric method of Jafé [21]. Te concentrations of creatinine and activities of alanine and aspartate aminotransferases (ALT and AST) were also determined spectrophotometrically using commercial diagnostic kits (LABKIT and CHEMELEX S. A. Barcelona) by the kinetic method UV-IFCC optimized [21]. 2.9. Histological Analysis. For microscopic evaluation, the organs studied were dehydrated and embedded in parafn for microscopic examination according to routine laboratory procedures [22]. Parafn sections of 4 μm were prepared and stained with haematoxylin and eosin (H and E) for histological examination. Morphometric measurements of artery thickness were performed using Image J 1.3 software.

Statistical Analysis.
Results were expressed as mean-± SEM. Te diference between the groups was compared using a one-way analysis of variance (ANOVA) followed by Turkey's post-test using Graphpad Prism version 8.01. Te diferences were considered signifcant from p < 0.05.

Efects of the Plant Mixture Aqueous Extract on the Behaviour and Mortality of Rats.
Te efects of the aqueous extract of the mixture of B. pilosa and C. citratus on the behaviour and mortality of female and male rats are recorded in Table 1. Administration of the aqueous extract of the mixture of B. pilosa and C. citratus at doses of 2000 and 5000 mg/kg, compared to the control animals, did not induce any apparent signs of toxicity either during the frst 4 hours of administration or during the 14 days of experimentation. Te extract did not cause any visible changes in coat appearance, aggressiveness, mobility, sensitivity to noise and touch, and the appearance of the faeces and eyes of the test animals as compared to the normal control group. All the animals that started the test were found alive and sound, two weeks after the beginning of the acute toxicity evaluation.

Efects of the Plant Mixture Aqueous Extract on the Relative Organ Weights and Weight Evolution of Rats.
Te efects of the aqueous extract of the mixture of B. pilosa and C. citratus on the relative weight of some organs (A and B) and on weight evolution (C and D) in male and female rats, respectively, are summarised in Figure 1. A single oral dose of the extract at 2000 and 5000 mg/kg in healthy animals of both sexes did not afect signifcantly the body weight and the relative organ weight of the organs as compared to the normal control.      a signifcant decrease in the concentration of creatinine 13.16% (p < 0.05) and 21.93% (p < 0.001), respectively, compared to normal control animals.

Efects of the Plant Mixture Aqueous Extract on the
Elements of the Blood. Te efects of the aqueous extract of the mixture of B. pilosa and C. citratus on some of blood elements are recorded in Tables 6 (female) and 7 (male). Table 6 shows that administration of the aqueous extract of the mixture of B. pilosa and C. citratus for 28 days did not afect some blood components. However, the aqueous extract of the mixture of B. pilosa and C. citratus at a dose of 200 mg/kg induced a signifcant decrease in white blood cell count (p < 0.05) in females, compared to normal controls. In males, administration of the aqueous extract for 28 days at doses of 200 and 400 mg/kg resulted in a signifcant (p < 0.05) increase in monocyte count compared to normal controls (Table 7).

Efects of the Plant Mixture Aqueous Extract on the
Architecture of the Liver, Kidneys, Spleen, and Lungs. Figure 3 shows the efects of the aqueous extract of the mixture of B. pilosa and C. citratus on the architecture of the liver, kidneys, spleen, and lungs in female (A) and male (B) rats. Histological sections of the liver of animals in the normal control group and in the groups treated with the aqueous extract of the mixture of B. pilosa and C. citratus at doses of 200, 400, and 800 mg/kg, as well as those of the satellite groups, showed a normal architecture of the liver parenchyma with a distinct centrilobular vein and hepatocytes. Histology of the kidney showed normal parenchyma with a distinct glomerulus and urinary space. In all treated groups, lung histology showed distinct lung epithelium, alveolar sacs, and muscle wall. Histology of the spleen showed a distinct white and red pulp. TN: normal rats received distilled water (10 mL/kg); TSAT: normal satellite observed 14 more days after stopping every treatment; BC 200, BC 400, and BC 800: animals receiving the aqueous extract of the mixture of B. pilosa and C. citratus at the respective doses of 200, 400, and 800 mg/kg; SAT: satellite extract at the dose of 800 mg/kg observed 14 days after stopping every treatment. Liver: Pv � portal vein; He � hepatocyte; Bc � bile canaliculus; kidney: G � glomerulus; Us � urinary space; lungs: Mw � muscle wall; As � alveolar sac; Pe � pulmonary epithelium; spleen: Wp � white pulp; Rp � red pulp. n � 5; X : 100X (spleen and lungs), 200X (kidneys and liver).

Discussion
Studies of toxicity on animals are used to evaluate the potential health risk humans face to unwanted intrinsic efects caused by plant extracts [23]. Tese secondary efects can appear in the form of biochemical, haematological, histological, or anthropometric alterations [24]. Te present study was designed to evaluate the toxicology profle of the aqueous extract of the mixture of B. pilosa and C. citratus in male and female rats.
One of the problems of phytotherapy is the dose used. Potential toxic efects of natural substances have been reported in several published research works [25,26]. It is thereby necessary to characterize possible biological efects of every plant used in traditional medicine and their side efects. Terefore, one of our objectives was to verify the toxicity of the aqueous extract of the mixture of B. Pilosa and C. citratus in acute administration.
Te acute toxicity study is the basis for classifcation and labelling of substances, and the acute toxicity study is the basis for classifcation and labelling of substances and helps to decide on the dose of compounds to be given in animal studies [26]. In the present work, the acute toxicity focused on the observation of behavioural and physiological changes in rats. Te administration of a single dose of the aqueous extract of the mixture of B. pilosa and C. citratus (2000 and 5000 mg/kg) to rats did not induce any visible signs of toxicity either during the frst 4 hours of administration or during the 14 days of experimentation. Te fact that all the animals were found alive and sound at the end of the experimental period suggests that the lethal dose 50 (LD 50 ) of the plant mixture aqueous extract is above 5000 mg/kg. Tese fndings are in agreement with those of Yinyang Jacques et al. [3] who obtained similar results in the acute toxicity study of the combination of the aqueous extracts of the trunk bark of Musanga cecropioides and the fruits of Picralima nitida. Te abovementioned assessment is not completely true as far the plants are taken alone in the extract. In fact, the acute toxicity of C. citratus (2000 mg/kg) was accompanied with symptoms such as torpor, nose, and eyelid bleeding in Wistar rats [6]. Histological examination revealed atrophy of the stomach mucosa and necrosis of the hepatocytes [6], whereas the LD 50 of C. citratus was 3250 and 3500 mg/kg in rats and male Swiss mice, respectively [27]. On the other hand, with B. Pilosa extract (3500 mg/kg), there were no changes in behaviour, sensory nervous system   responses, and gastrointestinal efects in the experimental animals [14]. All the mice survived beyond 24 h of observation; the LD 50 of the extract was then above 3.5 g/kg [14]. When compared to the extract of isolated plants, the mixture appeared to be more saved. Tis could be explained with the inhibition of the side efects of C. citratus compounds by some compounds found in B. Pilosa. According to Hodge and Sterner, the observations in the present study implies that the extract of the combination can be classifed as less toxic [28]. Since no toxic efects were observed in the acute study, an additional study was conducted to assess the subchronic toxicity [19] of the aqueous extract of the B. pilosa and C. citratus mixture during a 28-day experiment on rats. In  that second study, the weight loss observed in female rats treated with the aqueous extract of the plant mixture can be explained with the fact that C. citratus present in this plant mixture is well known to induce weight loss [10]. Generally, its decoction is advised to pregnant women who are overloaded. Te interference with some female hormones could also be incriminated, since no efect on body weight gain was observed in male rats. A sensitive indicator in toxicity studies is the relative organs weight [29]. In the present study, no signifcant change was observed in the relative organ (heart, liver, spleen, kidney, and lungs) weight of all the rats. Tese results may then confrm the low toxicity of the aqueous extract of the mixture of B. pilosa and C. citratus.
Te liver is involved in the body detoxifcation, while the kidneys participate in the purifcation of the blood and the elimination of waste [30]. Te assessment of liver and kidney integrity and function is therefore very important in the study of the toxicity of drugs and plant extracts, since they are used for the welfare of the organism [29]. Tus, biochemical analysis carried out in the present study could reveal possible injuries to the liver and kidney induced by the ingestion of the extract. Te increase in TC on female rats treated with the aqueous extract of the plant mixture (200 and 400 mg/kg) correlated to the increase in HDL-C can therefore be taken as an improvement for lipid profle. It is true that Hanisa et al. [31] found no signifcant changes in serum markers of lipid profle in the subchronic toxicity of C. citratus. Nonetheless, the observations in the present study are in agreement with our previous study showing that the extract of the mixture improves the lipid profle of hypertensive rats [17]. Transaminases (ALT and AST) are good markers of liver integrity and function [32]. Normally, AST and ALT are enzymes of mitochondrial and cytoplasmic origin. But any cell necrosis, destruction of the hepatic parenchyma or an increase in membrane permeability of the hepatocytes leads to the fow of these enzymes in the blood. Tat in turn will increase their serum levels [34]. According to Adewale et al. [34], a low level in the liver enzymes (AST and/or ALT) indicates a hepato-protective efect of the plant, which could explain the results observed. In fact, Hanisa et al. [31] observed no signifcant changes in serum biochemical markers of liver function after 28 days of C. citratus infusion administration. Te same observations were made in the subchronic toxicity of B. pilosa [12]. Te results observed in the biochemical markers were confrmed with the histology of the liver. Moreover, renal function can be assessed by testing some biochemical markers such as urea, creatinine, uric acid, and electrolytes [32]. Indeed, these markers have high values in case of renal alteration [25]. Te decrease in creatinine levels in male rats having received the aqueous extract of the mixture plants (200 and 400 mg/kg) confrmed therefore the nephroprotective efects already demonstrated in this extract [17]. Tese results were confrmed by histological analysis which showed no alteration of the renal structure.
Toxicity may manifest itself in the haematopoietic system, in the form of a decrease in the number of circulating cells, functional and structural alterations, and, more rarely, changes in morphology [35]. Terefore, the evaluation of haematological parameters is essential to establish the efects of plant extracts on animal's blood system. Blood parameter analysis is relevant because it provides much information. It can reveal haematopoietic function (myeloid lineage cells), allergies occurrence (white blood cells), or intravascular efects such as haemolysis [36]. Te decrease in white blood cells in females and the increase in monocyte count in males having received the aqueous extract of the mixture plant at the dose of 200 mg/kg suggest that the extract did not cause a signifcant change in haematopoietic function. Tis could therefore explain why no sign of infammation was observed in the structure of all investigated organs, suggesting a minor cause of the above diference in blood cells.

Conclusion
Te work in the present study focused on the evaluation of the acute and subchronic toxicity of the aqueous extract of the mixture of B. pilosa and C. citratus. No sign of toxicity was observed in acute toxicity study with the oral administration of the dose 5000 mg/kg or less. No morphological and behavioural signs of toxicity were observed for both acute and subchronic toxicity. In subchronic toxicity, the extract stimulated weight growth in male. Te extract did not induce any change in relative organ weights at all doses tested. Furthermore, it did not induce any signifcant changes in liver and kidney function; nonetheless, it improved the lipid profle. Tis study supports the production of an improved traditional medicine after preclinical testing.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare no conficts of interest in the present research.