Toxicological assessment of the hydroethanolic leaf extract of clerodendrum capitatum in Wistar rats

Introduction Clerodendrum capitatum (Willd) Schumach. & Thonn (Lamiaceae) is used in African traditional medicine for the treatment of malaria, hypertension, obesity, jaundice and diabetes however there is lack of experimental data on its possible toxicity. This study investigated the acute and 28 days sub-chronic toxicity of C. capitatum in Wistar rats. Methods In acute toxicity tests, a single administration of the hydroethanolic C. capitatum leaf extract (5 g/kg) was given orally to 5 female rats. The general behavior, adverse effects and mortality were recorded for up to 14 days post treatment. On the 15th day, the rats were weighed and euthanized for necropsy. In sub-chronic toxicity tests, the extract (4, 8 and 16 g/kg/day) was given orally to both male and female rats for 28 days. The animal body weight was recorded throughout the experiment, while hematological and biochemical parameters of blood and relative organs weights were evaluated on the 29th day. Results Clerodendrum Capitatum did not cause any death or any hazardous symptoms of acute toxicity, showing an LD50 higher than 5 g/kg. Sub-chronic administration of C. capitatum resulted in no noticeable changes in weight gain and water or food consumption. White blood cells and hemoglobin increased while urea concentration, liver enzymes, total cholesterol and glucose concentrations significantly decreased in treated animals. No changes in macroscopical aspect of organs were observed in the animals. Conclusion These results showed that acute or sub-chronic oral administration of the hydroethanolic leaf extract of Clerodendrum capitatum may be considered as relatively free of toxicity.


Introduction
The use of medicinal plants to treat various ailments is as old as the world. It is well known throughout history and has always been part of human culture. The World Health Organization (WHO) estimates that up to 80% of the world's population relies on traditional medicinal system for some aspects of primary health care [1]. In African ethnomedicines, it is well known that traditional healers make use of a large variety of herbs in the treatment of parasitic diseases including malaria and a wide proportion of herbal remedies dispensed by traditional healers are widely believed by their clients to be effective [2,3]. Such plants can play an important role in drug discovery and their studies are logical research strategies in the search for new drugs [4]. In some countries, government encourages the use of indigenous forms of medicine rather than expensive imported drugs. In the USA, nearly one in three people uses some kind of alternative medical treatment [5]. Previous studies have reported the effectiveness of medicinal plants in treating of various disease conditions such as liver problems [6], circulatory and respiratory problems [7], febrile illnesses [8] and diarrhea [9]. However, despite the profound therapeutic advantages possessed by some of the plants, some constituents of medicinal plants have been shown to be potentially toxic, carcinogenic and teratogenic [10,11]. Moreover, it is known that the consumption of medicinal plants without evaluating their efficacy and safety can result in unexpected or toxic effects that may affect the physiology of different organs in the human body. Liver and kidney are the first targets in toxicological evaluation because they are involved in the metabolism and excretion of chemical compounds. Renal damage has also been associated with the use of medicinal plants in the treatment of various diseases [12].
Clerodendrum capitatum (Willd) Schumach. & Thonn (Lamiaceae) is an indigenous tropical Africa perennial undershrub, fast growing, erect, well branched, and grows up to 0.5-2 m high [13]. In Togo the leaves of the plant are traditionally used in the treatment of malaria [14]. Based on ethno botanical report in the maritime region of Togo, C. capitatum is frequently used to treat high blood pressure related to hypertension and also to alleviate obesity, jaundice and constipation. In Nigeria, the plant is used to treat diabetes mellitus, obesity and high blood pressure [15].
Pharmacological studies of the Clerodendrum genus demonstrated biological activities such as antitumorgenic [16,17], hypoglycemic, and hypolipidemic [18]. Despite the widespread use of C. capitatum, there is lack of experimental data on its possible toxicity. Thus, the present investigation was undertaken to provide data on the safety of C. capitatum by focusing on the acute toxicity and 28 days subchronic toxicity of the hydroethanolic leaf extract of C. capitatum given orally to male and female Wistar rats.

Plant material and extraction
Fresh leaves of C. capitatum were collected in August 2013 from Adidigomé, a locality at North West of Lomé (Togo). Botanical authentication was confirmed at the Department of Botany, University of Lomé, where a voucher specimen of C. capitatum was deposited at the herbarium (N° TG12806). The leaves were washed, air-dried under air conditioner for three days and were coarsely powdered. The powder (300 g) was macerated at room temperature with 3 L of ethanol-water (8:2, v/v) for 72 h. The solvent was removed using a rotary vacuum evaporator (Buchi, Japan) at 40°C and the crude extract, representing a yield of approximately 12.7% (w/w), was stored at -4°C until administrated doses preparation, when it was dissolved in sterilized distilled water.

Animals and treatment
Wistar rats of either sex weighting 145-150 g (males) and 120-125 g (females) used in the present study were provided by animals

Acute toxicity
Healthy female Wistar rats were used in this study according to the Organization for Economic Cooperation and Development (OECD) revised up-and-down procedure for acute oral toxicity testing [19].
Page number not for citation purposes 3 Female rats were selected for the test because they are frequently more sensitive to the toxicity of test compounds than males. The extract was dissolved in sterilized distilled water in a volume of 10 mL/kg body weight of experimental animal by oral administration.
All the animals were fasted overnight, but with free access to water and they were weighed before the extract administration. Animals were randomly divided in two groups (n = 5 per group). The first group (control group) received distilled water orally. The second group (acute toxicity group) was treated as followed: a dose limit of 5 g/kg was given to the first animal which was observed for mortality, signs of acute toxicity and behavioral changes (unusual aggressiveness, unusual vocalization, restlessness, sedation and somnolence, twitch, tremor, ataxia, catatonia, paralysis, convulsion, fasciculation, prostration and unusual locomotion and asphyxia) for the first thirty minutes and the first hour, then hourly for 5 h and, finally periodically until 48 h. If this first animal survived, then four additional animals were to be given the same 5 g/kg dose sequentially at 48 h intervals. All the experimental animals were individually observed daily for general behavioral and body weight changes, hazardous symptoms and mortality for a period of 14 days post treatment. The LD50 was predicted to be above 5 g/kg if three or more rats survived. At the end of the experimental period, all animals were weighed and sacrificed by cervical dislocation, and the organs were excised for necropsy.

Experimental design
The administered doses in this study were chosen based on the pharmacologically effective doses of the hydroethanolic extract from earlier hepatoprotective effects studies carried out in our laboratory in males Wistar rats. A total of 40 rats of either sex were used in this study. Rats were divided into 4 groups of 10 each (n = 10; 5 males and 5 females per group) and their weights were recorded.
Before treatment, rats were individually handled and carefully examined for abnormal behavior and appearance. The extract of C. capitatum, dissolved in sterilized distilled water, was administered orally once a day for 28 consecutive days in single doses of 0.4 g/kg (group I), 0.8 g/kg (group II) or 1.6 g/kg (group III), while the control rats (group IV) received sterilized distilled water. Animals were observed daily during the experimental period for mortality or morbidity, changes in posture, changes in skin fur, eyes, mucous membranes and behaviors. At the end of each week, the body weights of all the rats were recorded and doses of the extract were adjusted accordingly. At the end of the 28 days of administration, animals were fasted overnight, but allowed free access to water. On the 29th day they were anesthetized with ether and blood samples were collected via retro-orbital puncture using capillary tubes for hematological and biochemical studies respectively [20]. Blood samples for hematological analysis were collected in tubes containing Ethylene Diamine Tetra Acetic acid (EDTA) as anticoagulant whereas samples for biochemical analysis were collected into tubes without anticoagulant and centrifuged within one hour. After blood collection, the rats were sacrificed by clavicle dislocation. Organs such as liver, kidneys, sex organs (testes, seminal vesicles and epididymis for male rats; ovaries and uterus for female rats), brain, spleen, lungs, and heart were excised, washed immediately in NaCl (0.9%), weighed individually and examined macroscopically.

Relative organ weights ratio
The excised organs were weighed and the weight of each organ was multiplied by 100 and divided by the weight of the animal before sacrifice to obtain the relative organs weight (%) [ GmbH. D-65205, Wiesbaden, Germany.

Statistical analysis
The results are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test to evaluate significant differences between groups. Results were considered to be significant at p < 0.05. All statistical analyses were performed using GraphPad Prism 5 Software Inc., USA.

Results
In acute toxicity study, oral administration of hydroethanolic leaf extract of C. capitatum at the dose of 5 g/kg did not cause mortality or any clinical signs of acute toxicity in rats observed for a short period of 48 h and a long period of 14 days. All five female rats survived until the end of the observation period. No abnormality was found in organs at necropsy.

Sub-chronic toxicity
In sub-chronic toxicity study, daily oral administration of the hydroethanolic leaf extract of C. capitatum at the doses of 0.4 g/kg, 0.8 g/kg and 1.6 g/kg for 28 consecutive days resulted in no noticeable changes in the general behavior of treated rats compared to controls. No lethality was recorded in either sex in control and treated groups at any of the doses administered during the 28 days of extract administration. No differences in food and water consumption were observed between the groups of rats. Likewise, no significant differences in body weight gain (Figure 1 and Figure   2) or relative organ weights ( Table 1, Table 2) were observed between control and treated groups. Macroscopic observations of organs of treated animals did not show any significant change in color and texture between control and treated groups. Both the control and treated rats appeared uniformly healthy at the end of and throughout the 28-day treatment period. Similarly, sub-chronic oral administration of the extract did not cause any significant change in hematological profile of male and female rats ( Table   3, Table 4). However, white blood cells and hemoglobin rate increased in treated animals. The extract did not cause any significant change in serum creatinine, total and conjugated bilirubins, total proteins, triglycerides, phosphorus, potassium, chlorine, sodium and uric acid in both male and female rats ( Table   5, Table 6). However, at 1.6 g/kg the extract decreased significantly (p<0.05) blood concentration of urea in both male and female rats as compared to control. In the same way, total cholesterol and glucose concentrations decreased significantly in both male and female rats. Liver enzymes (AST, ALT and ALP) also decreased in treated animals ( Table 5, Table 6). This suggests that the median acute toxicity value (LD50) of the extract is above 5 g/kg body weight. According to Kennedy et al. [24], substances that present LD50 higher than 5 g/kg by oral route may be considered practically non-toxic. Therefore, it can be suggest that acute toxicity of the hydroethanolic leaf extract of C.

Discussion
capitatum is devoid of acute oral toxicity. Similar results were observed by Mirtes et al. [25] and Caroline et al. [26] with other plants using the same toxicological method.
A good extrapolation has been reported between toxicological insults in rats and humans; this is in contrast weaker when comparing humans and mice [27]. Therefore, oral intake of the leaf Page number not for citation purposes 5 of C. capitatum may be considered as safe. However, in an acute oral toxicity study by Adeneye et al. [15], C. capitatum aqueous fresh leaves extract was documented to be non-lethal in rats at 5 g/kg but caused rapid-onset increased somatomotor activity, tachypnea, bilateral narrowing of the eyelids, tremor, piloerection, and increased feeding pattern in rats. Sub-chronic treatment did not produce any death or clinical signs of toxicity. There were no statistically significant differences (p > 0.05) in the body weight gain by the animals throughout the course of extract administration in all the doses compared with the control animals. This observation may indicate that extract did not alter the metabolic processes of the treated animals which may subsequently affect the hormones and body weight [28]. An important index to diagnose whether an organ has been exposed to injury is to calculate the organ-to-body weight ratio [22]. Hence, if rats are exposed to toxic substances the weight of the damaged organ will either increase or decrease as will the organ-to-body weight ratio. In the present study there were no significant changes in the relative weights of organs between the control and treated rats. This suggests no glossy toxic effect from the extract. Analysis of blood parameters is relevant to risk evaluation as any changes in the hematological and biochemical systems have a higher predictive value for human toxicity, when data are translated from animal studies [27]. In this study, the hematological profile of treated rats showed no significant difference with control group, except white blood cells and hemoglobin which increased in the groups treated with the hydroethanolic leaf extract of C. capitatum. Increase in white blood cells directly indicates the strengthening of the organism defense [29,30]. This elevation in total leucocytes count suggests that the extract contains biologically active compounds that have the ability to boost the immune system through increasing the population of defensive white blood cells.
However, a global increase was observed in red blood cell count, hematocrit and hemoglobin concentration, implying that there may be a possible increase in erythropoiesis. Kidney functions were evaluated by means of serum urea, creatinine, sodium, potassium, chloride, uric acid and inorganic phosphorus. Increase blood creatinine is a good indicator of negative impact in kidney functions [31,32]. Moreover, serum creatinine and urea concentrations are used for the assessment of renal sufficiency [33]. Thus, higher than normal levels of serum creatinine and urea are indications of deficiency in renal function. In the present study, there were no significant changes in blood creatinine, uric acid, sodium, potassium, chloride and inorganic phosphorus. These results suggest that the kidney functions are not altered in animals treated with the extract.
However, serum urea decreased in rats treated at the dose of 1.6 g/kg. The decrease in serum urea concentration in the treated rats confirms that functioning of the kidney is normal. The increase levels of AST and ALT in the blood are associated with damage of hepatic cells [34,35]. Emerson et al. [36] also have reported that enhancement in the level of serum proteins is an indication of tissue injury and reflection of hepatic toxicity. Significant changes in such classical enzymes as ALP, ALT and AST suggest liver impairment, since these are reliable indices of liver toxicity [37], or altered integrity of cellular membrane as well as cell lyses or death [38]. As one of the biochemical parameters analyzed, AST is normally found in the cytoplasm and mitochondria of many cells, primarily in cardiac muscle, liver and skeletal muscle. Its concentration is much lower, however, in the kidney, pancreas and erythrocytes. Therefore, an increase in the serum levels of ALT, AST, ALP or total bilirubin indicates hepatic toxicity [39].

Conclusion
In conclusion, we summarize that acute (5 g/kg) and sub-chronic

What is known about this topic
• Clerodendrum capitatum exerts hypoglycemic and hypolipidemic effects.
• Some constituents of medicinal plants have been shown to be potentially toxic.
• Consumption of medicinal plants without evaluating their safety can result in unexpected or toxic effects.

What this study adds
• Clerodendrum capitatum may be considered as relatively free of toxicity when administrated orally during 28 days.
• Clerodendrum capitatum leaf extract may contain biologically active compounds that have the ability to boost the immune.

Competing interests
The authors declare no competing interest.

Authors' contributions
All authors participated in reading the paper and its drafting. All the author's read and agreed to the final manuscript. Table 1: effect of CECC on mean relative organ weight (%) in male rats Table 2: effect of CECC on mean relative organ weight (%) in female rats Table 3: effect of CECC on hematological profile of male rats Table 4: effect of CECC on hematological profile of female rats      Values are expressed as Mean ± SEM (n = 5)