Abstract
Macaranga barteri is a plant used in traditional medicine to treat many illnesses. Previous studies showed the efficacy of the aqueous extract of leaf of this plant (AEMb) in the treatment of gastric ulcer within the dose range of 125–500 mg/kg body weight (b.w.). This study aims at evaluating the safety of AEMb on anthropometric and haematological parameters in wistar rats. Seventy rats were divided into seven groups of ten rats each, including five males and five females. The control group was repeatedly administered by gavage with distilled water at 1 ml/100 g for 28 days while test groups 2, 3, 4 and 5, were repeatedly gavaged with AEMb at the doses of 125, 250, 500 and 1000 mg/kg b.w. respectively. As for satellites (groups 6 and 7), they received daily and respectively distilled water at 1 ml/100 g b.w. and AEMb at the dose of 1000 mg/kg b.w. The results showed that AEMb caused no significant changes in the behaviour of rats and the weight of the organs removed (kidneys, liver, spleen, and heart) and their relative weights at the end of the 28 days of treatment. However, the body weight and the amount of food consumed by animals treated with AEMb at the doses of 250, 500 and 1000 mg/kg b.w. increased significantly (p < 0.05) from the third week compared to control group. Haematological analysis revealed a non-significant increase (p > 0.05) in leukocyte lineage and platelet level in female rats at the studied doses. However, a significant (p < 0.05) increase in platelet level was recorded in male rats at 1000 mg/kg b.w. A significant (p < 0.05) increase in erythrocyte and hemoglobin levels at the doses of 250, 500 and 1000 mg/kg b.w. in treated animals was also revealed. In conclusion, repeated administration of AEMb over 28 days to rats was safe on leucocyte lineage and most of erythrocyte indices at doses ranging from 125 to 1000 mg/kg b.w. Nevertheless, the use of this extract caused a transient increase of erythrocyte, hemoglobin and platelet levels 2 weeks after the end of AEMb administration, but these effects disappeared. So, the subacute oral administration of AEMb revealed few potential toxic effects overall.
Keywords: Macaranga barteri, Anthropometry, Haematology, Rat, Safety
Introduction
The world of plant which is rich in secondary metabolites, constitutes today a major source of traditional medicine [1]. According to World Health Organization (WHO) [2], more than 80% of the population in Africa still use traditional medicine for their healthcare needs. However, in spite of their natural origin, active substances of medicinal plants can at high or low dose and for prolonged administration or not, produce adverse or even harmful effects on vital organs [3]. Unfortunately, in developing countries, the effect of plants is always appreciated in an empirical way because of the ignorance of scientific information about plants, the lack of control of a definite posology and suitable instructions. The drawbacks are many adverse effects such as neurological, digestive, cardiovascular symptoms and many other clinical signs [4]. Many studies on medicinal plants in laboratory and field observation have demonstrated toxicity. For example, Momordica charantia, a known antidiabetic and antimalarial plant but also used in Ghana as an abortifactor [5, 6], reportedly caused fatal hypoglycaemia in children [7]. As for Lantana camara used against malaria and other diseases, it is endowed with hepatotoxic activity in several animal species [8]. WHO [2], thereby, recommends developing countries to initiate programs for identification, preparation, cultivation and conservation of medicinal plants but also to evaluate the quality, the efficacy and the safety of these plant extracts. That is why this study targeted Macaranga barteri Mull-Arg (Euphorbiaceae). The leaves of this plant are used in Côte d’Ivoire and Sierra Leone for the treatment of many pathologies [9, 10]. Studies undertaken by Ehilé et al. [11] on acute toxicity and gastric anti-ulcer activity of an aqueous extract of the leaves of M. barteri, showed that this extract possessed antiulcer effect at doses ranging from 62.5 to 500 mg/kg b.w. The dose of 500 mg/kg b.w. was found to be the most effective.The LD50 was higher than 5000 mg/kg b.w. according to the method of OECD 420 [12] and so the extract was considered as non toxic. On the other hand, no documented evidence concerning the safety of the prolonged use of this plant was mentioned. Therefore, this work was aimed to assess the potential toxic effects of a subacute administration of the aqueous extract of the leaves of M. barteri on anthropometric and haematological parameters in rats.
Material and methods
Material
Plant material
Fresh leaves of M. barteri were collected from the forest of Nangui Abrogoua University (Abidjan, Côte d’Ivoire) in August 2018. Taxonomic identification of these leaves was established by botanist of Nangui Abrogoua University. The species was then authenticated by the National Floristic Center of Felix Houphouët Boigny University of Cocody-Abidjan, Côte d’Ivoire, with voucher no. 14,735 in April 06, 1979 in Côte d’Ivoire National Herbarium.
Animals
Experiments were carried out on albino male and female Wistar rats weighing between 95 and 110 g, approximately 5–6 weeks old. They were bred in the Animal House of Laboratory of Physiology, Pharmacology and Pharmacopeia of Nangui Abrogoua University (Abidjan, Côte d’Ivoire) according to the principles for the care and use of laboratory animals of the Ethical Committee of the University. They were exposed to12 h dark/light cycle at room temperature (22–25 °C) and given standard food for rats and water ad libitum. Research was conducted in accordance with the internationally accepted principles for laboratory use and care as found in the European Community Guidelines [13].
Chemical substances
Ether (VWR International-Geldenaakfebaan 464-B-3001, Leuven, Belgium) and ethanol (Sigma Chemical Company, Saint Louis, MO, USA) were used for this study.
Methods
Preparation of the aqueous extract of leaves of Macaranga barteri
The fresh leaves of M. barteri were dried under shade (22–25 °C) and powdered with a grinding machine (RETSCH, type SM 100, Germany). One hundred grams (100 g) of the leaf powder of M. barteri were boiled in 1 l of distilled water for 15 min, filtered using a filter paper (Whatman no. 3) and then stored in desiccators (Mark Fruicell, France) at 45 °C for 2 days according to the method described by Zirihi [14]. 11.95 g of powder were obtained and corresponded to the aqueous extract of M. barteri (AEMb). It was dissolved extemporaneously in distilled water before oral administration to rats in all the experiments.
Subacute toxicity study
Grouping and treatment
The experiments were implemented according to the OECD test guideline 407 [13]. Thus, seven groups of rats were orally administered with different doses of AEMb once a day with one dose per group for 28 days.
Fifty (50) rats were randomly alloted into four test groups of ten rats each and a control group of ten rats. Each group included five males and five females. The control group (group 1) received orally distilled water at 1 ml/100 g b.w. Groups 2, 3, 4 and 5 received orally the aqueous extract of leaves of M. barteri at doses of 125, 250, 500 and 1000 mg/kg b.w., respectively. Two additional satellite groups (groups 6 and 7) of ten rats each were used. Group 6 (satelite control) was gavaged with distilled water and group 7 was administrered with 1000 mg/kg b.w. of AEMb for 28 days in order to check the persistence or late appearance of toxic effects 14 days after the end of the treatment.
Collection of blood samples
Animal were slightly anesthetized with cotton wool soaked in Ether. Blood samples from each fasted rat were collected by ocular puncture in tubes containing an anticoagulant (EDTA) at the end of the experiment (Day 28) according to the method described by Coles [15]. Thereafter they were taken for haematological analysis using automated cell counter (RAYTO-RT 7600S, China). Two weeks after the end of the treatment, the animals of the satellite groups were weighed, fasted and their blood samples were collected for haematological analysis.
Observation of clinical signs
Rats were observed individually once daily, at the same time during the 28 days of treatment to determine any signs of toxicity. Observations were focused on general condition, behavioural abnormalities (apathy, excitement, excessive grooming), skin and mucosal abnormalities, signs of partial or generalized nervous damage (coma, tremor, convulsion), signs resulting in gastrointestinal involvement (refusal of food, drink, change in the volume or appearance of feces), signs of corrosive and vascular effects (oral, nasal and anal bleeding), possible mortality. Constant observations were made as early as the first 4 h of administration of AEMb [13].
The amount of food and water consumed by the rats were recorded daily and weight was determined weekly to assess anthropometric parameters [13]. The relative organs weights of the liver, kidneys, heart and spleen were assessed by the following formular [16]
Determination of blood parameters
Blood samples were put in an automated cell counter (RAYTO-RT 7600S, China) which was programmed to evaluate red blood cell (RBC), hemoglobins (Hb), meam corpuscular hemoglobin concentraction (MCHC), meam corpuscular hemoglobin (MCH), mean corpscular volume (MCV), hematocrit (HCT), white blood cell (WBC), lymphocytes, monocytes, granulocytes, platelets.
Statistical analysis
Data were presented as mean ± standard error on mean (M ± SEM). Comparisons between treated groups and controls were made using one-way analysis of variance (ANOVA).
Values were considered statistically significant when p < 0.05. Graph Pad Prism 7.00 (San Diego, CA, USA) software was used for analysis and the different graphs. Tukey–Kramer was used as post-hoc test.
Result
Clinical signs
No particular clinical signs of toxicity were observed in rats within the 28 days treatment with AEMb at doses ranging from 125 to 1000 mg/kg b.w. compared to those of the control group.
Effect of the aqueous extract of leaves of Macaranga barteri on anthropometric parameters in rats
Effect of the leaves aqueous extract of Macaranga barteri on average food intake in rats
As shown in Fig. 1a and b, from the first to the fourth week of treatment, food intake increased in male as well as in female rats. This increase was non significant for male and female groups (p > 0.05) at the first and second week of treatment when compared to those of control group 1. On the other hand, from the third to the fourth week, male and female rats treated with AEMb (125, 250 and 500 mg/kg b.w.) experienced significant (p < 0.05) and high (p < 0.01) increases respectively when compared to control groups. The food intake reached 163.9 ± 8.23 g/day (male) and 148.5 ± 11.2 g/day (female) at 1000 mg/kg b.w compared to the respective controls (138.6 ± 9.50 g/day and 122.8 ± 11.2 g/day).
Fig. 1.
Effects of the aqueous extract of leaves of Macaranga barteri on rats food intake. a Effects of AEMb on male rats food intake. b Effects of AEMb on female rats food intake. ap < 0.05; bp < 0.01: significant difference between extract treated group and control (distilled water), n = 5. AEMb Aqueous Extract of Macaranga barteri; Control group received distilled water
Effect of the aqueous extract of leaves of Macaranga barteri on rats water intake
At the first week, the amount of water intake varied from 117 ± 9.46 to 122 ± 8.7 and from 117.2 ± 7.40 to 122 ± 10.60 ml/day in male and female rats respectively while control values were 119 ± 10.9 (male) and 118 ± 10.9 ml / day (female). From the second to the fourth week of treatment, a progressive and non significant (p > 0.05) rise in the amount of water intake was recorded in male and female rats treated with AEMb (125–1000 mg/kg b.w.) compared to control group 1 which received distilled water. This quantity of water intake remained maximum and constant in all the animals at the fourth week (Fig. 2).
Fig. 2.
Effect of the aqueous extract of leaves of Macaranga barteri on water intake in rats. a Effect of AEMb on water intake in male rats. b Effect of EAMb on water intake in female rats. There is no significant difference (p > 0.05) between the groups treated with AEMb and the control group; n = 5; AEMb Aqueous Extract of Macaranga barteri; Control group received distilled water
Effect of the aqueous extract of leaves of Macaranga barteri on rats body weight
Before repeated administration of AEMb to rats, the body weight of the animals was nearly similar and fluctuated between 102 ± 3.51 and 107 ± 3.94 g for male rats and between 103 ± 4.89 and 105 ± 3.47 g for female rats. The oral administration of AEMb (125–1000 mg/kg b.w) induced a non significant ponderal growth (p > 0.05) in both sexes when compared to those of the control groups at the first and second week of treatment as depicted in Fig. 3. From the third to the fourth week, significant augmentations of body weight were recorded in the AEMb treated animals.
Fig. 3.
Effect of the aqueous extract of leaves of Macaranga barteri on body weights in rats. a Effect of AEMb on male rats body weight. b Effect of AEMb on female rats body weight. ap < 0.05; bp < 0.01: significant difference between extract treated group and control (distilled water), n = 5. AEMb Aqueous extract from the leaves of Macaranga barteri; Control group received distilled water
Effect of the aqueous extract of leaves of Macaranga barteri on organ weights
The values of the organs weights (kidneys, liver, spleen, heart) and the relative weights were indicated in Tables 1, 2, 3 and 4. The observation of these tables revealed that no significant variation (p > 0.05) of the organ weights and the relative weights in male and female rats treated with AEMb (125–1000 mg/kg b.w) was recorded after 28 days administration of the extract compared to control.
Table 1.
Effect of the leaves aqueous extract of Macaranga barteri on organs weights in male rats
| Groups | Organ weights (g) | |||
|---|---|---|---|---|
| Heart | Liver | Kidneys | Spleen | |
| Control | 0.58 ± 0.40 | 5.80 ± 0.23 | 1.18 ± 0.03 | 0.50 ± 0.07 |
| AEMb 125 mg/kg | 0.60 ± 0.11 | 6.12 ± 0.43 | 1.21 ± 0.05 | 0.53 ± 0.09 |
| AEMb 250 mg/kg | 0.60 ± 0.25 | 5.56 ± 0.17 | 1.32 ± 0.07 | 0.51 ± 0.06 |
| AEMb 500 mg/kg | 0.55 ± 0.64 | 6.06 ± 0.24 | 1.30 ± 0.09 | 0.51 ± 0.23 |
| AEMb 1000 mg/kg | 0.62 ± 0.41 | 5.63 ± 0.11 | 1.11 ± 0.30 | 0.52 ± 0.62 |
| Satellite Control | 0.57 ± 0.13 | 5.81 ± 0.39 | 1.08 ± 0.38 | 0.50 ± 0.12 |
| Satellite (AEMb 1000 mg/kg) | 0.59 ± 0.23 | 5.63 ± 0.20 | 0.99 ± 0.21 | 0.58 ± 1.01 |
No statistical significant difference (p > 0.05) between values of the same column of the group treated with AEMb and control
AEMb Aqueous extract of the leaves of Macaranga barteri; Control and satellite control groups received distilled water
Table 2.
Effect of the aqueous extract of leaves of Macaranga barteri on organs weights in female rats
| Groups | Organ weights (g) | |||
|---|---|---|---|---|
| Heart | Liver | Kidneys | Spleen | |
| Control | 0.56 ± 0.19 | 5.21 ± 0.15 | 0.98 ± 0.12 | 0.40 ± 0.10 |
| AEMb 125 mg/kg | 0.52 ± 0.32 | 5.27 ± 0.12 | 0.95 ± 0.06 | 0.43 ± 0.13 |
| AEMb 250 mg/kg | 0.55 ± 0.20 | 5.21 ± 0.14 | 0.95 ± 0.05 | 0.48 ± 0.09 |
| AEMb 500 mg/kg | 0.55 ± 0.14 | 5.58 ± 0.27 | 0.96 ± 0.15 | 0.43 ± 0.19 |
| AEMb 1000 mg/kg | 0.53 ± 0.34 | 5.08 ± 0.13 | 0.98 ± 0.08 | 0.47 ± 0.14 |
| Satellite Control | 0.61 ± 0.15 | 6.08 ± 0.13 | 1.10 ± 0.34 | 0.46 ± 0.32 |
| Satellite (AEMb 1000 mg/kg) | 0.58 ± 0.16 | 5.53 ± 0.15 | 1.15 ± 0.08 | 0.52 ± 0.92 |
No statistical significant difference (p > 0.05) between values of the same column of the group treated with AEMb and control
AEMb Aqueous extract of the leaves of Macaranga barteri; Control and satellite control groups received distilled water
Table 3.
Effect of the aqueous extract of leaves of Macaranga barteri on relative organs weights in male rats
| Groups | Relative organ weights (%) | |||
|---|---|---|---|---|
| Heart | Liver | Kidneys | Spleen | |
| Control | 0.33 ± 0.22 | 3.32 ± 0.26 | 0.67 ± 0.16 | 0.34 ± 0.00 |
| AEMb 125 mg/kg | 0.36 ± 0.02 | 3.60 ± 0.21 | 0.70 ± 0.31 | 0.31 ± 0.01 |
| AEMb 250 mg/kg | 0.36 ± 0.05 | 3.34 ± 0.13 | 0.66 ± 0.02 | 0.31 ± 0.02 |
| AEMb 500 mg/kg | 0.34 ± 0.09 | 3.78 ± 0.22 | 0.70 ± 0.20 | 0.32 ± 0.02 |
| AEMb 1000 mg/kg | 0.36 ± 0.41 | 3.31 ± 0.13 | 0.69 ± 0.04 | 0.30 ± 0.05 |
| Satellite Control | 0.36 ± 0.19 | 3.24 ± 0.61 | 0.73 ± 0.15 | 0.29 ± 0.09 |
| Satellite (AEMb 1000 mg/kg) | 0.34 ± 1.09 | 3.55 ± 0.70 | 0.70 ± 0.52 | 0.35 ± 0.42 |
| 0.38 ± 0.17 | 3.72 ± 1.52 | 0.72 ± 0.30 | 0.31 ± 0.12 | |
No statistical significant difference (p > 0.05) between values of the same column of the group treated with AEMb and control
AEMb Aqueous extract of the leaves of Macaranga barteri; Control and satellite control groups received distilled water
Table 4.
Effect of the aqueous extract of leaves of Macaranga barteri on relative organs weights in female rats
| Groups | Relative organ weights (%) | |||
|---|---|---|---|---|
| Heart | Liver | Kidneys | Spleen | |
| Control | 0.36 ± 0.31 | 3.94 ± 0.14 | 0.66 ± 0.21 | 0.29 ± 0.08 |
| AEMb 125 mg/kg | 0.31 ± 0.12 | 3.65 ± 0.19 | 0.66 ± 0.12 | 0.30 ± 0.07 |
| AEMb 250 mg/kg | 0.33 ± 0.17 | 3.85 ± 0.20 | 0.66 ± 0.40 | 0.36 ± 0.03 |
| AEMb 500 mg/kg | 0.32 ± 0.11 | 3.87 ± 0.28 | 0.74 ± 0.34 | 0.32 ± 0.03 |
| AEMb 1000 mg/kg | 0.37 ± 0.20 | 3.74 ± 0.50 | 0.65 ± 0.11 | 0.30 ± 0.02 |
| Satellite Control | 0.32 ± 0.21 | 3.34 ± 0.56 | 0.69 ± 0.16 | 0.33 ± 0.11 |
| Satellite (AEMb 1000 mg/kg) | 0.38 ± 0.17 | 3.72 ± 1.52 | 0.72 ± 0.30 | 0.31 ± 0.12 |
No statistical significant difference (p > 0.05) between values of the same column of the group treated with AEMb and control
AEMb Aqueous extract of the leaves of Macaranga barteri; Control and satellite control groups received distilled water
Effect of the aqueous extract of leaves of Macaranga barteri on haematological profile in rats
Effect of the aqueous extract of leaves of Macaranga barteri on white blood cells and leukocyte indices in male rats
The average values of the white blood cells (WBC) and leukocyte differential count of male rats treated orally and daily for 28 days with AEMb at doses ranging from 125 to 1000 mg/kg b.w. are registered in Table 5. The analysis of this table revealed that the leukocyte rates and the leukocyte differential count of the animals that received AEMb (125, 250, 500 and 1000 mg/kg b.w.) varied in a non significant way (p > 0.05) compared to control group. Two weeks after the end of the treatment, there was no significant (p > 0.05) changes in WBC and leukocyte differential count in rats receiving 1000 mg/kg b.w. of AEMb compared to control of satellite group receiving distilled water.
Table 5.
Effect of the aqueous extract of leaves of Macaranga barteri on leukocyte indices of male rats
| Parameters | Dose (mg/kg b.w) | ||||||
|---|---|---|---|---|---|---|---|
| Treatment | Satellite | ||||||
| Control | AEMb 125 | AEMb 250 | AEMb 500 | AEMb 1000 | Control | AEMb 1000 | |
| WBC (103/μl) | 7.64 ± 0.68 | 8.46 ± 0.81 | 8.74 ± 0.63 | 8.88 ± 0.56 | 8.95 ± 0.49 | 8.58 ± 1.15 | 8.51 ± 1.08 |
| Lymphocytes (103/μl) | 6.70 ± 0.45 | 7.04 ± 0.80 | 7.19 ± 0.59 | 7.13 ± 0.81 | 7.35 ± 0.50 | 6.02 ± 0.91 | 6.98 ± 0.28 |
| Monocytes (103/μl) | 0.77 ± 0.12 | 0.75 ± 0.04 | 0.71 ± 0.20 | 0.78 ± 0.07 | 0.62 ± 0.05 | 0.75 ± 0.20 | 0.64 ± 0.06 |
| Granulocytes (103/μl) | 1.17 ± 0.11 | 0.93 ± 0.09 | 0.95 ± 0.13 | 0.97 ± 0.09 | 0.98 ± 0.09 | 0.81 ± 0.16 | 0.91 ± 0.57 |
There is no statistical significant difference (p > 0.05) between values of the same line of the groups treated with AEMb and the control group, n = 5.
WBC White Blood Cells, AEMb Aqueous extract from the leaves of Macaranga barteri. Control group received distilled water
Effect of the aqueous extract of leaves of Macaranga barteri on erythrocytes and erythrocyte indices in male rats
As exhibited in Table 6, analysis of the results unveiled a significant (p < 0.05) increase in red blood cell and hemoglobin levels at 250, 500 and 1000 mg/kg b.w. while no modification was observed at 125 mg/kg b.w compared to control group 1. This augmentation in red blood cell counts went up between 6.58 ± 0.31 and 6.78 ± 0.12 × 106/μl compared to 5.22 ± 0.16 × 106/μl in control group. As for the hemoglobin levels, it varied from 15.0 ± 0.38 to 15.5 ± 0.36 g/dl compared to 13.7 ± 0.24 g/dl in control group. The administration of EAMb elicited no changes in the other erythrocyte indices (hematocrit, MCV, MCH and MCHC) of rats compared to those of the control group.
Table 6.
Effect of the aqueous extract of leaves of Macaranga barteri on erythrocytes, hemoglobin and erythrocyte indices of male rats
| Paramètres | Dose (mg/kg b.w) | ||||||
|---|---|---|---|---|---|---|---|
| Treatments | Satellite | ||||||
| Control | AEMb 125 | AEMb 250 | AEMb 500 | AEMb 1000 | Control | AEMb 1000 | |
| RBC (106/μl) | 5.22 ± 0.16 | 5.75 ± 0.10 | 6.58 ± 0.31a | 6.52 ± 0.16a | 6.78 ± 0.12a | 5.95 ± 0,87 | 6.13 ± 0.32 |
| Hb (g/dl) | 13.7 ± 0.24 | 14.4 ± 0.11 | 15.0 ± 0.38a | 15.1 ± 0.23a | 15.5 ± 0.36a | 14.94 ± 0,40 | 15.6 ± 0.13 |
| HCT (%) | 39.9 ± 0.89 | 40.4 ± 1.87 | 41.9 ± 2.79 | 41.6 ± 1.73 | 42.7 ± 1.48 | 39.5 ± 4,6 | 42.6 ± 1.6 |
| MCV (fl) | 66.5 ± 1.18 | 67.6 ± 1.64 | 67.7 ± 2.61 | 66.8 ± 1.24 | 65.5 ± 0.91 | 67.8 ± 2.69 | 68.82 ± 0,31 |
| MCH (pg) | 24.6 ± 0.99 | 25.1 ± 0.58 | 25.6 ± 3.31 | 25.5 ± 0.33 | 24.4 ± 0.38 | 24.4 ± 0.94 | 25.5 ± 3.64 |
| MCHC (g/dl) | 37.10 ± 0.86 | 37.16 ± 0.15 | 37.13 ± 3.72 | 36.84 ± 0.90 | 36.38 ± 0.96 | 36.56 ± 0.36 | 37.63 ± 2.20 |
ap < 0.05; significant difference between extract treated group and control group (distilled water), n = 5
The values of the same line with the same letter are statistically different from those of the control group (p < 0.05)
Hb hemoglobin, RBC Red Blood Cells, HCT Hematocrit, MCV Mean Corpuscular Volume, MCHC Mean Corpuscular Hemoglobin Concentration, MCH Mean Corpuscular Hemoglobin, AEMb Aqueous extract from the leaves of Macaranga barteri; Control group received distilled water
Two weeks after the end of the treatment, no variation was recorded (p > 0.05) in erythrocyte and erythrocyte indices levels in animals treated with AEMb at 1000 mg/kg b.w. compared to those of the satellite control group.
Effect of the aqueous extract of leaves of Macaranga barteri on leukocyte profile of female rats
The white blood cells (WBC) and the leukocyte differential counts of the control and treated rats are compiled in Table 7. Oral repeated administration of AEMb (125, 250, 500 and 1000 mg/kg b.w.) to female rats caused no significant (p > 0.05) changes in leukocyte and leukocyte differential counts after 28 days of treatment compared to the control group.
Table 7.
Effect of aqueous extract of leaves of Macaranga barteri on leukocyte indices of female rats
| Parameters | Dose mg/kg (b.w) | ||||||
|---|---|---|---|---|---|---|---|
| Treatment | Satellite | ||||||
| Control | EAMb 125 | EAMb 250 | EAMb 500 | EAMb 1000 | Témoin | EAMb 1000 | |
| WBC (103/μl) | 6.61 ± 0.60 | 6.96 ± 0.41 | 7.80 ± 0.93 | 7.50 ± 0.89 | 7.66 ± 0.47 | 6.74 ± 0.51 | 7.72 ± 0.73 |
| Lymphocytes (103/μl) | 5.67 ± 0.47 | 5.40 ± 0.42 | 6.47 ± 0.88 | 5.45 ± 0.69 | 5.61 ± 0.74 | 6.06 ± 0.44 | 5.89 ± 1.36 |
| Monocytes (103/μl) | 0.42 ± 0.05 | 0.56 ± 0.02 | 0.52 ± 0.46 | 0.54 ± 0.27 | 0.56 ± 0.07 | 0.89 ± 0.17 | 0.69 ± 0.06 |
| Granulocytes ( 103/μl) | 0.82 ± 0.11 | 0.98 ± 0.07 | 0.80 ± 0.08 | 1.01 ± 0.15 | 0.98 ± 0.14 | 0.91 ± 0.1 | 0.82 ± 0.12 |
There is no significant difference (p > 0.05) between the values of the same line of the group treated with AEMb and those of the control group (distilled water), n = 5
WBC White Blood Cells, AEMb Aqueous extract from the leaves of Macaranga barteri, Control group received distilled water
Effect of the aqueous extract of leaves of Macaranga barteri on erythrocytes, hemoglobin and erythrocyte indices of female rats
Repeated administration of AEMb at doses ranging from 125 to 1000 mg/kg b.w. induced a significant (p < 0.05) increase in hemoglobin and erythrocyte rates at 250, 500 and 1000 mg/kg b.w when compared to the control group (Table 8). These increases attained 6.71 ± 0.13 × 106/μl (red blood cells) and 15.9 ± 0.43 μg/dl (hemoglobin) at 1000 mg/kg b.w. As for the other erythrocyte indices (Hematocrit, MCV, MCH and MCHC) of the animals treated with AEMb, no modification (p > 0.05) was found out compared to the rats of the control group. Two weeks after the end of the treatment, no significant (p > 0.05) changes in erythrocyte, hemoglobin and erythrocyte indices were recorded in animals receiving AEMb at 1000 mg/kg b.w. compared to satellite control group (Table 9).
Table 8.
Effect of aqueous extract of Macaranga barteri leaves on erythrocytes, hemoglobin and erythrocyte indices of female rats
| Parameters | Dose (mg/kg b.w) | ||||||
|---|---|---|---|---|---|---|---|
| Treatment | Satellite | ||||||
| Control | AEMb 125 | AEMb 250 | AEMb 500 | AEMb 1000 | Control | AEMb 1000 | |
| RBC (106/μl) | 5.19 ± 0.13 | 5.76 ± 0.42 | 6.66 ± 0.32a | 6.64 ± 0.07a | 6.71 ± 0.13a | 5.45 ± 0.12 | 6.93 ± 0.07 |
| Hb (g/dl) | 13.3 ± 0.23 | 14.6 ± 0.36 | 15.4 ± 0.69a | 15.8 ± 0.35a | 15.9 ± 0.43a | 14.9 ± 0.25 | 15.7 ± 0.22 |
| HCT (%) | 38.9 ± 1.11 | 39.7 ± 1.59 | 39.5 ± 2.01 | 41.8 ± 0.48 | 42.2 ± 0.68 | 38.1 ± 0.96 | 39.8 ± 0.60 |
| MCV (fl) | 64.4 ± 0.96 | 66.4 ± 0.68 | 65.3 ± 1.22 | 64.5 ± 1.47 | 63.9 ± 0.39 | 64.5 ± 0.60 | 64.1 ± 0.20 |
| MCH (pg) | 23.1 ± 0.21 | 22.9 ± 0.43 | 22.2 ± 0.22 | 25.5 ± 2.73 | 21.4 ± 0.38 | 20.06 ± 0.24 | 20.72 ± 0.31 |
| MCHC (g/dl) | 35.8 ± 0.81 | 34.5 ± 0.62 | 34.0 ± 0.82 | 37.5 ± 3.10 | 33.5 ± 0.54 | 33.9 ± 0.55 | 34.1 ± 0.35 |
ap < 0.05; significant difference between extract treated group and control group (distilled water), n = 5
The values of the same line with the same letter are statistically different from those of control (distilled water) at p < 0.05, n = 5
Hb hemoglobin, RBC Red Blood Cells, HCT Hematocrit, MCV Mean Corpuscular Volume, MCHC Mean Corpuscular Hemoglobin Concentration, MCH Mean Corpuscular Hemoglobin, AEMb Aqueous extract from the leaves of Macaranga barteri, Control group received distilled water
Table 9.
Effect of the aqueous extract of Macaranga barteri leaves on platelets of rats
| Parameters | Dose (mg/kg b.w) | ||||||
|---|---|---|---|---|---|---|---|
| Treatment | Satellites | ||||||
| Control | AEMb 125 | AEMb 250 | AEMb 500 | AEMb 1000 | Control | AEMb 1000 | |
| Platelets (103/μl) | Males | ||||||
| 675 ± 36.27 | 735 ± 63.4 | 732 ± 49.6 | 725 ± 50.8 | 757 ± 48.7a | 683 ± 68.4 | 714 ± 49.4 | |
| Platelets (103/μl) | Females | ||||||
| 682 ± 87.8 | 734 ± 26.2 | 744 ± 46.6 | 745 ± 70.7 | 750 ± 41.66 | 698 ± 22.2 | 706 ± 56.3 | |
ap < 0.05: significant difference between extract treated group and control (distilled water), n = 5
AEMb Aqueous extract from the leaves of Macaranga barteri, Control group received distilled water
Effect of the aqueous extract of Macaranga barteri leaves on rats platelets
Platelet counts of male and female rats are shown in Table 9. Oral administration of AEMb at doses between 125 and 1000 mg/kg b.w, caused a signficant (p < 0.05) rise in platelet levels in male rats at the dose of 1000 mg/kg b.w which amounted to 757 ± 48.7 × 103/μl. No significant effect was noticed in female rats which values increased from 734 ± 26.2 × 103/μl (AEMb 125 mg/kg) to 750 ± 41.16 103/μl (AEMb 1000 mg/kg) compared to the control groups at 28th day of the treatment. Two weeks after the end of the treatment, the platelet counts of the rats treated at 1000 mg/kg b.w. with AEMb showed no significant (p > 0.05) variation compared to the satellite control group.
Discussion
The aqueous extract of the leaves of M. barteri (AEMb) (Euphorbiaceae) is used in traditional medicine to treat gastric ulcer. The efficacy tests on this pathology showed that this extract is active against gastric ulcer at 500 mg/kg b.w. [11]. However, the potential toxic effects of administration of this extract over a long period have not been evaluated. Subacute toxicity study was undertaken to be assured of the safety of this extract. So, daily oral administration of AEMb for 28 days to rats within a dose range of 125 to 1000 mg/kg b.w., resulted in a modification of the anthropometric parameters by inducing a significant increase in food consumption from the third to the fourth week at 500 mg/kg b.w. AEMb caused a significant weight gain with this therapeutic dose in treated animals as compared to control rats. The observation of the ponderal gain in treated rats at 500 mg/kg b.w. suggested that AEMb did not have a negative impact in the treated animals since a weight lost could be a sign of toxicity of an administrered substance [17]. This increase in weight could be related to a stimulation of the appetite [18]. Indeed, the increase in food consumption by animals suggests that AEMb could shift their sense of taste. Morever, it could be hypothesized that presence of secondary metabolites in AEMb interfered with the nervous system and the gastric function and would involve consequently, the absorption of the nutriments in an efficient way [19]. It is well known that animal behaviour and survival are parameters that provide information on substance toxicity. Thus, the daily oral administration of AEMb to the rats during 28 days revealed that the treated animals survived and presented a behaviour similar to that of the control rats. This result suggests that AEMb would not be toxic at studied doses when administrered orally. This hypothesis is strenghened by the increase of food and water comsumption and significant body weight gain in all the treated animals. Similar results were obtained by Menegati et al. [20] and Porwal et al. [21]. with aqueous extract of Alibertia edulis (Rubiaceae) and ethanolic extract of the leaves of Marsdenia tenacissima (Asclepiadaceae) respectively which increased weight in rats treated during 28 days.
AEMb did not induced significant changes in organs (liver, spleen, heart and kidneys) weights and their respective relative weights. Organ weights and their relative weights are indicators of animal pathological or physiological state. According to Amresh et al. [22], organ weight and relative weight are an index that provides information on organs swelling, atrophy or hypertrophy. Raina et al. [23] stated that they indicate if the organ is the target of a drug or not. When these organs are altered, this indicates abnormal atrophy whereas if the value of this ratio in animals treated with the extract is much greater than that of the control, this suggests a hypertrophy of the organs according to Piao et al. [24]. The absence of significant variation observed for the weight of the organs and their relative weights suggests that AEMb is not toxic on vital organs (kidneys, liver, spleen and heart) and thereby does not induce any swelling and atrophy at studied doses. These results are similar to those of Gbogbo et al. [25] who showed that the administration of the aqueous extract of Spondias mombin (250–1000 mg/kg b.w) to rats did not have any effect on the relative weight of the vital organs.
This study also revelated that AEMb oral administration for 28 days did not fundamentally affect the hematopoietic system. Accordding to Muthuraman and Singh [26], the hematopoietic system is one of the most sensitive to toxic substances. This system is therefore important in the assessment of the toxicity of a substance. AEMb did not elevate significantly leucocyte lineage in male and female rats at the studied doses. Leukocytes play an essential role in immune system. They provide immunity to animal’s body against antigens invasion [27]. The increase in white blood cell counts and leukocyte differential counts suggests this extract stimulates immune response of treated animals. These results are similar to those obtained by Mezui et al. [28] with the aqueous extract of the leaves of Oxalis barrelieri (Oxalidaceae). Furthermore, 2 weeks after the end of the treatment, the modifications observed did not persist indicating that disturbances of leukocytes and leukocyte lineage caused by AEMb were transient. These results are in accordance with those obtained by Mezui et al. [29] with the aqueous extract of the leaves of Anthocleista schweinfurthii (Loganiaceae). AEMb administration significanly increased erythrocyte and hemoglobin levels in both male and female rats from 250 mg/kg b.w. to 1000 mg/kg b.w. and had no significant effect on hematocrit level. Therefore, the LOAEL (Lowest Observed Adverse Effect Level) of AEMb on erythrocyte and hemoglobin is 250 mg/kg b.w. and the NOAEL (No Observed Adverse Effect Level) is estimated to 125 mg/kg b.w. according to Bonvallot and Dor [30]. The augmentation of blood cells and hemoglobin levels suggests that AEMb could stimulate some factors involved in red blood cells production [31]. These results are similar to those obtained by Awotunde et al. [32], who carried out experiments on Terminalia schimperiana (Combretaceae) aqueous extract in rats. WHO [33] also reported that erythrocyte and hemoglobin rates respectively provide information about anemia level and iron intracellular quantity. The rise in these two rats’ parameters, in this study, showed that AEMb would not induce anemia or iron deficiciency and could even be beneficial in the treatment of anemia. Further studies with appropriate animal models could justify its use in traditional medicine by the populations of Côte d’Ivoire as anti anemic tonic [9]. These results corroborate those obtained by Ejere et al. [34] with the aqueous extract of the chalices of Hibiscus sabdariffa (Fabaceae). Erythrocyte indices such as MCV, MCH and MCHC of treated rats did not vary significantly. The realization of blood smears at studied doses in further works could confirm these observations. These results confirm those obtained by Dongmo et al. [35] who showed that administration of the aqueous extract of Tetrapleura tetrapteura (Fabaceae) to rats at 100 and 400 mg/kg b.w. over one-month period did not disturb erythrocyte indices. AEMb elicited a non significant increase in platelet count (125–1000 mg/kg b.w. in female rats and 125–500 mg/kg b.w in male rats) compared to distilled water control rats during 28 days of treatment. A significant rise was observed at the dose of 1000 mg/kg b.w in male rats. So, the AEMb dose of 500 mg/kg b.w constitutes the NOAEL according to Bonvallot and Dor [30]. The increase in the number of platelets observed can suggest that the extract acts on the coagulation system by improving and preventing haemorrhages. It could therefore have a stimulating effect on thrombopoietin, a physiological hormone that regulates platelet production [36]. Additional research should be undertaken to check if there is any possibility of procoagulant or prothrombic side effects of AEMb. These results are in accordance with those obtained by Ugwah-Oguejiofor et al. [37] with the aquous extract of the leaves of Caralluma dalzielii (Apocynaceae). After the end of AEMb treatment, a non significant variation was observed on the platelets level showing that these changes were transient. This study demonstrated that 28 days oral administration of the aqueous extract of M. barteri (AEMb) in rats did not present major toxic effects on anthropometric and haematological parameters. However, biochemical and histological studies need to be completed before comfirming the safety use of this extract.
Acknowledgements
The authors are thankful to Dr. OUSSOU N’Guessan Jean-Baptiste (Lecturer and Researcher at the Nangui Abrogoua University’s Laboratory of physiology, pharmacology and pharmacopoeia, Côte d’Ivoire) for his invaluable help in translating this manuscript and also, all the staff of the laboratory for their encouragement during these investigations.
Compliance with ethical standards
Conflict of interests
The authors have not declared any conflict of interests.
Ethical approval
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
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