Plant material

The leaves of C. febrifuga species were used. Several plant leaves of the same species growing in the same place were collected in July 2019 in Kimbedi locality, Department of Pool (Congo). Botanical identification of the plant material was done by Mousamboté, botanist systematist of Higher Normal School of Agronomy and Forestry (HNSAF) and confirmed at the herbarium of the National Institute for Research in  Exact  and  Natural  Sciences (NIRENS) in which a collected sample was compared to a reference samples (n° 8012). After identification, the plant material was dried at room temperature (27±50°C) for 14 days in the Laboratory of Pharmacodynamics and Experimental Physiopathology (L2PE), and then pulverized with a mortar. 5 g of C. febrifuga powder was mixed in 50 mL of distilled water. The whole was left under magnetic stirring for 24 h. The macerate (10%) obtained after filtration with the help of cotton wool was kept in a sterile bottle for study.

Animal material

Albino rats (100 to 150 g) and albino mices (20 to 30 g) of either sex aged 3 months were used. These animals were provided to us by the animal house of the Laboratory of Pharmacodynamics and Experimental Physiopathology (L2PE) of the Faculty of Science and Technical of the Marien Ngouabi University. They were fed with a standard food and with running water. They were acclimatized during one week before experimentation and were housed under standard conditions (12 h light and 12 h dark) and at the temperature of 27 ± 1°C. The rules of ethics published by the International Association for the Study of Pain have been considered (Zimmermann, 1980).

Acute toxicity of aqueous extract of C. febrifuga 

The acute toxicity of the aqueous extract of the leaves of C. febrifuga was evaluated according to OECD no. 423 guideline adopted on December 17, 2001. Mice were fasted for 24 h before the treatment and then divided into three groups of three mice each and treated with different doses of distilled water (control group, 10 mL/kg) and aqueous extract of the leaves of C. febrifuga at the doses of 2000 and 5000 mg/kg. After rectal administration, macroscopic observations including mobility, ptosis, alertness, piloerection, aggressiveness, stool status, vomiting was evaluated at ½, 1, 2, 3 and 8^th. Mortality was evaluated in 48 h. 

Laxative effect of aqueous extract of C. febrifuga in non-constipated rats

Method reported by Meité et al. (2010) was used. Five (5) groups of 5 rats each were formed and treated with the differents doses of aqueous extract of C. febrifuga (100, 200 and 400 mg/kg), Normacol* (standard drug, 2 mL/rat) and distilled water (control group, 10 mL/kg) were administered rectally to groups. After that, the animals of each groups were placed individually in metabolic cages and the following parameters were observed, onset, frequency, and the quantity of stool emission were evaluated for 8 h.

Laxative effect of aqueous extract of C. febrifuga in loperamide hydrochloride (Imoduim*) induced constipation

The method reported by Wintola et al. (2010) was used. Before the administration of the test products, the animals were selected. Constipation was induced in thirty (30) normal rats by oral administration of loperamide hydrochloride (3 mg/kg body weight in 0.9% sodium chloride) for three days. However, five rats were treated only with distilled water (control). During these three days, they were fed standard food and water. Thus, food consumption, water intake, number of faecal pellets, water content of pellets, weight  of  pellets  and  weight  gain  were  determined every day at eight hours for three days. The water content was calculated as the difference between the dry weight and the wet weight of the stools were determined. Three days later, animals showing signs of constipation (presence of hard or dry stools) were placed in metabolism cages and grouped into five (5) groups of five (5) rats each treated rectally for one week (7 days) with different doses of distilled water (control group, 0.5 mL/100 g), Normacol* (standard group, 2 mL/rat) and with aqueous extract of C. febrifuga leaves (100, 200 and 400 mg/kg). Food consumption, water intake, number of faecal pellets, water content of pellets, weight of pellets and weight gain were calculated every day at 8^th. for the duration of the experiment (7 days). The onset, the frequency and the quantity of stools excreted was determined each day for eight hours of observation of the animals after administration of the test products.

Evaluation of the effect of aqueous extract of C. febrifuga on intestinal fluid accumulation in mice

The method reported by Meité et al. (2010) was used. Mice were divided into groups of five mice each treated rectally with different doses of distilled water (control group, 0.5 mL/100 g), Sodium dihydrogen phosphate dihydrate (Normacol*, standard drug, 2 mL/rat), and aqueous extract of C. febrifuga leaves (100, 200 and 400 mg/kg). One hour after the administration of the products, the mice were treated with extract of C. febrifuga at the doses of 100, 200 and 400 mg/kg respectively received 0.5 mL/mouse of Normacol® 2 h after, the mice were sacrificed by cervical dislocation. The small intestine was removed and weighed (W1), then emptied of its contents and reweighed (W2) and its length (L) measured. The difference between the weights divided by the length of the total intestine gives the net quantity (Q) of accumulated stool:

Enteric contents of each group have been analyzed to define ions concentrations, sodium (Na⁺), (potassium) K⁺ by the aid of a spectrophotometer of mark Micro Touch Biochemistry Analyser.

Chemical profile

It was carried out using the classical tube reaction tests to identify the major chemical groups (Clarke, 1975).

Statistical analysis

All values were expressed as mean ± standard error of the mean (SEM). An analysis of variance using Excel version 2016 software followed by Student-Fischer t test “t” was performed. The significance level was set at p<0.05

Acute toxicity of the aqueous extract of C. febrifuga leaves

The results of macroscopic observation show that the aqueous extract of the leaves of C. febrifuga administered at a single dose of 2000 mg/kg rectally did not cause  any change in general behavior compared to the control group. However, the aqueous extract of the leaves of C. febrifuga administered rectally at a single dose of 5000 mg/kg modified the general behavior characterized by immobility of the mice following the rectal administration of the products. Furthermore, mortality was observed after the dose of 5000 mg/kg with a calculated LD50≥3500 mg/kg.

Effect of aqueous extract of C. febrifuga leaves on the onset, frequency and quantity of excreted sells in normal rats

Tables 1 to 3 show the effect of aqueous extract of C. febrifuga leaves on onset appearance of sells, frequency and quantity excreted sells in normal rats respectively. They show that animals mice treated with Normacol* (2 mL/rat) as well as those treated with the aqueous extract at increasing doses of 100, 200 and 400 mg/kg significantly (p<0.001) reduced the onset of sells emission compared to the control group (Table 1). On the other hand, Normacol* (2 mL/rat) as well as aqueous extract (100, 200 and 400 mg/kg) significantly (p<0.05, p<0.01, and p<0.001) increased the frequency and quantity of sells emission (Tables 2 and 3) during 6 h of experimentation compared to control group. However, no significant change in the frequency of excreted sells was observed at 8 h (Table 2).

Effect of aqueous extract of C. febrifuga in constipated rats

The loperamide induced constipation in all normal animals during the three days of treatment (Table 4). Thus, there is a significant decrease (p ?0.01 and p?0.001) in the faecal parameters (food consumption, water intake, number of faecal pellets, water content of the pellets, weight of stool excreted), except body weight of the animals which increased significantly (p ?0.01) compared to the control group (Non-constipated rats). In addition, Normacol* (standard drug) and the aqueous extract of the leaves of C. febrifuga at the doses used (100, 200 and 400 mg/kg) significantly increase (p?0.001) food consumption, water intake, number of faecal pellets, water content of the pellets, weight of stool excreted) and body weight of constipated rats during the seven days of treatment (Table 5) compared to the control group.

Phytochemical analysis of the aqueous extract of C. febrifuga leaves

Phytochemical analysis of the macerated leaves of C. febrifuga by tube reactions revealed the presence of anthraquinones, alkaloids, flavonoids, saponosides, tannins, reducing sugars and mucilages (Table 7). 

This work was initiated to justify the laxative properties of C. febrifuga used in traditional Congolese medicine. The results show that the aqueous extract of the leaves has sedative properties with high dose toxicity (5000 mg/kg). Moreover, by rectal administration at the doses used (100, 200 and 400 mg/kg), the aqueous extract exhibits laxative properties such as normacol* (reference laxative). Phytochemically, these properties are due to the presence of secondary metabolites highlighted in this plant. These results could justify the use of C. febrifuga leaves in the traditional treatment of constipation.

The authors declare that they have no conflict of interest.