Evaluation of in vivo Antitrypanosomal activity of aqueous and methanol leaf extracts of Clutia abyssinica (Euphorbiaceae) against Trypanosoma congolense

Research Article

Austin J Pharmacol Ther. 2014; 2 (5). 1029

Evaluation of in vivo Antitrypanosomal activity of aqueous and methanol leaf extracts of Clutia abyssinica (Euphorbiaceae) against Trypanosoma congolense

Mergia E1, Terefe G2, Teklehaymanot T3 and Shibeshi W1*

1Department of Pharmacology and Clinical Pharmacy, Addis Ababa University, Ethiopia

2Department of Parasitology and Pathology, Addis Ababa University, Ethiopia

3Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Ethiopia

*Corresponding author: : Shibeshi W, Department of Pharmacology and Clinical Pharmacy, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia

Received :June 19, 2014; Accepted: July 31, 2014; Published: Aug 04, 2014


Introduction: African trypanosomiasis is a major disease of economic and public health importance affecting agricultural and human development. The search for alternative compounds against African trypanosomiasis is essential as existing chemotherapeutic agents have several drawbacks. The objective of the study reported was to evaluate aqueous and methanol leaf extracts of Clutia abyssinica for in vivo activity in Trypanosoma congolence infected mice.

Materials and Methods: The in vivo antitrypanosomal efficacy of the aqueous and methanol extracts was evaluated in Swiss albino mice infected with T. congolense isolated from naturally infected cattle. The leaf extracts were administered 12 days post-infection at doses of 100, 200 and 400 mg/kg by intraperitoneal injection once daily for 7 days. Parasitaemia, packed cell volume (PCV), mean survival time and change in body weight were used as indices for monitoring the efficacy of the extracts by comparing with the positive control (28 mg/kg dose of diminazene aceturate) and negative control (2% tween 80) treated groups.

Results: Highly significant (p<0.001) reduction in pre-treatment parasitaemia by 3.91% (7.38 ± 0.18), increase in PCV by 1.12% (48.66 ± 0.20), body weight improvement by 1.36% (22.34 ± 0.27) and mean survival time of 39.20 ± 0.37 days was observed in the group treated with 400 mg/kg methanol leaf extract of C. abyssinica on day 14 of treatment while the mice treated with aqueous extracts of C.abyssinica at 400 mg/kg dose had low parasitaemia on day 6 (p<0.01), day 8, 10 and 14 (p<0.001) of treatment as compared to the negative control.

Conclusion: The results obtained confirm ethno-pharmacological usefulness of the plant in treatment of trypanosomiasis and possible indications in development of alternative drugs.

Keywords: Clutia abyssinica; In vivo antitrypanosomal activity; Mice; Trypanosoma congolense


T. congolense: Trypanosoma Congolense; C. abyssinica: Clutia Abyssinica; CAAE: C. Abyssinica Aqueous Extract; CAME= C. Abyssinica Methanol Extract; DA: Diminazine Aceturate


African trypanosomosis is a protozoan disease of humans and livestock caused by trypanosomes and transmitted by tsetse fly vector. While Trypanosoma rhodiense and Trypanosoma gambiense cause African Human Trypanosomosis, Trypanosoma brucei brucei, Trypanosoma vivax, Trypanosoma congolense, Trypanosoma evansi and Trypanosoma equiperdum are the agents of African Animal Trypanosomosis (AAT) [1].

The economic impacts of trypanosomosis in Africa are diverse and complex, with direct effects on animal production and human health, as well as indirect effects on settlement patterns, land use, animal husbandry and farming [2]. Chemotherapy, the main means of controlling the disease is under threat due to parasite resistance [3] and toxicity of the trypanocidal drugs [4]. The poor prospect for a vaccine due to antigenic variation of the parasite is further compounded by unwillingness of the pharmaceutical industry to develop new compounds because of uncertain and unprofitable market or perhaps the localized nature of the disease.

The few commercial trypanocides (diminazene aceturate, isometamidium and homidium) have been in use for well over 40 years. Thus, the search for medicinal plants with trypanocidal activities continues to generate a lot of research interest [5,6]. Although recent reports indicate antitrypanosomal activity exists in some medicinal plants [7-11], the potentials of many other plants used in folkloric medicine in Ethiopia are yet to be investigated. Euphorbiaceae is a large and fascinating family of about 300 genera and 8,000-10,000 species, mostly found in the tropics of both hemispheres.

Clutia is a genus within a family Euphorbiaceae, having about 60 species. Clutia abyssinica called by the Amharic name ‘fyele fej’ is herb 1-2 m high [12]. Traditionally it is used in treatment of venereal and skin diseases, chest problems, cancer [13]; Skin fungal infections [14,15]; yellow fever and malaria; management of ear, nose and throat diseases [16]; diarrhoea [17]; gonorrhea, cough and fever, headache, toothache, menstrual pain, burns, pneumonia, enlarged spleen and kidney, shock, abdominal problems- as a laxative and to expel intestinal worms, elephantiasis, diarrhoea and tachycardia [15]. The maceration of the crushed leaves of C. abyssinica given orally has been traditionally used for the treatment of animal trypanosomosis [18]. Therefore, this study was aimed to evaluate the in vivo antitrypanosomal activity of Clutia abyssinica in mice infected with T. congolense isolated from natural infection of cattle.

Materials and Methods

Chemicals and drugs

The chemical reagents used were diminazine aceturate (Ceva Santé Animale, France; batch number- 719A1), Giemsa stain, tween-80 (BDH Ltd, England), 40% glucose (Pharmacure, Ethiopia), methanol (Carlo ebra reagents, Italy).

Test organism

The test organism T. congolense was isolated from infected cattle in Sebategna kebele, Bedele town, Dabo Hana woreda, 480 km south west of Addis Ababa. The presence of T. congolense in the screened cattle was detected from blood samples collected from the ear vein of the animals. The slide was examined for T. congolense based on their type of motility in the microscopic field 40x objective and confirmations of T. congolense species by morphological characteristics was done by staining with Giemsa stain, and examination under a microscope using oil immersion 100x objectives [19,20]. Then the infected blood was collected from the jugular vein of the animal using EDTA coated tubes and heavily inoculated to laboratory mice and transported to laboratory at Akililu Lemma Institute of Pathobiology, Addis Ababa University for subsequent serial passage to other mice.

Experimental animals

Healthy Swiss albino mice (weighing 20–30 gm and age of 8–12 weeks) were obtained from the animal house of the Ethiopian Health and Nutrition Research Institute and School of Pharmacy, Addis Ababa University. Animals were housed in polypropylene cages (6–10 animals per cage), maintained less than 12 hr light and 12 hr dark cycle and allowed free access to pellet diet and clean water ad libitum. All procedures complied with the guide for the care and use of laboratory animals [21]. The experimental protocols and use and handling of animals were approved by research and ethics committee of Department of Pharmacology and clinical pharmacy.

Preparation of plant materials

The leaves of C. abyssinica were collected from Debre Libanos Monastery in Amhara regional state, Ethiopia. The fresh leaves were wrapped by plastic sheets during transportation. Taxonomic identification was done and a voucher specimen was deposited (Collection EM/001) at the National Herbarium, College of Natural sciences, and Addis Ababa University. The leaves were washed with distilled water and were dried under shade. The dried leaves were pulverized using mortar and pestle. For preparation of extracts, 200g of dried leaf powder of C. abyssinica was separately macerated with 1000 ml of distilled water and absolute methanol for 48 hours with frequent agitation in orbital shaker and the resulting liquid was filtered using Whatman No. 3 filter paper (Whatman Ltd., England). Extraction was repeated three times and the filtrates of all portions were pooled in one vessel. The aqueous extract was placed in a petridish and lyophilized for one week to yield a solid residue, while the methanol extract was concentrated using Rota vapor (BÜCHI Rota-vapor, Switzerland) at no more than 40°C in order to obtain dry extract. Then the resulting dried mass was then powdered and weighed resulting in yield of 12.92% and 17.21% for aqueous and methanol extracts respectively.

Acute toxicity study

The acute toxicity study was conducted in accordance with the Lorke’s [22] method. The study was conducted for each extract in two phases using female Swiss albino mice after 7 days of adaptation. In the first phase, nine mice were divided into 3 groups (n=3). Each group was given 10, 100, and 1000 mg/kg body weight of the test substance respectively. In the second phase, further specific doses (1600, 2900, and 5000 mg/kg) of each extract were administered to nine mice to estimate the lethal dose (LD50) value. The control group received the reconstituting solvent 2% tween 80 in sterile water. The extract was dissolved in 2% tween 80 in sterile water and given through intraperitoneal route. All animals were kept under strict observation for behavioral, neurological, autonomic or physical changes such as alertness, motor activity, restlessness, convulsions, coma, diarrhea and lacrimation for 24 h. These observations continued for further 14 days for any signs of overt toxicity. Then the lowest dose which killed one mouse (minimum toxic dose) and the highest dose which had not killed any mouse (maximum tolerated dose) were noted, and the geometric mean of these two doses gave LD50. The detailed procedures for determining the doses for testing acute toxicity are shown in annex 1.

Parasite inoculation and extract administration

Healthy Swiss albino mice that are infected intraperitoneally with 0.2 ml of T. congolense infected blood (~104 trypanosomes/ ml) collected by cardiac puncture from donor mice was divided into eight groups (n =5): C. abyssinica aqueous extract (CAAE 100, CAAE 200, CAAE 400), C. abyssinica methanol extract (CAME 100, CAME 200, CAME 400), diminazene aceturate (DA28), and 2% tween 80 (TW80). Treatment with the extracts began on the 12th day post-infection (day 0 of treatment), when the infected mice showed peak parasitaemia of (~108 trypanosomes/ml). On each day of drug administration, the aqueous and methanol extracts of C. abyssinica were freshly prepared by dissolving in 2% Tween-80 in sterile water for injection and administered intraperitoneally daily at 9 am for seven days at the doses of (100, 200 and 400 mg/kg). The doses were selected based on the acute toxicity study. For the positive control, diminazine aceturate (DA28), dissolved in sterile water as recommended by the manufacturer was administered at the dose of 28 mg/kg intraperitoneally based on previous reports of Moti et al. [23], Feyera et al. [11], while for the negative control, 2% tween 80 in sterile water (TW80), was administered intraperitoneally. Volume administered was 2 ml/100 gm of body weight of the animal (OECD, 2001).

Determination of parasitaemia

Parasitaemia was monitored every other day by microscopic examination of blood obtained from the tail of each mouse that was pre-sterilized with methylated spirit. The tail tip was cut to extrude blood and drop of blood was placed on microscope slide and covered with a cover-slide. The blood was examined microscopically at 400x total magnification. The degree of parasitaemia was determined using the “Rapid Matching” method of Herbert and Lumsden [24]. Wet smear were prepared in triplicates from each animal and the mean value of slide counts were taken per sample examined microscopically. Logarithm values of these counts were obtained by matching with the table given by Herbert and Lumsden [24].

Determination of packed cell volume (PCV)

PCV was measured [25] to predict the effectiveness of the test extracts in preventing hemolysis resulting from increasing parasitaemia associated with trypanosomosis. It was monitored before infection and three times till the 14th day (on day 0, 7 14). Briefly, blood was collected from tail of each mouse in heparinized microhaematocrit capillary tubes filled up to 3/4th of their length. The tubes were then sealed immediately by cristal seal and centrifuged in a microhaemtocrit centrifuge (Hettich Haematokrit, Germany) for 5 min at 12,000 rpm. After centrifugation, the height of the red blood cell column were measured by use of haematocrit reader and compared to the total height of the column of the whole blood [26]. The effect of extracts in improving PCV of treated animals was compared with the controls.

Determination of body weight

The body weight of each mouse in all groups was measured before infection, on the day treatment commenced (day 0) and every other day up to day 14.

Determination of mean survival time

Mortality was monitored daily and the number of days from the time of inoculation of the parasite up to death was recorded for each mouse in the treatment and control groups throughout the follow up period for six weeks.

Statistical analysis

Values of the data obtained from the study were summarized and expressed as mean ± standard error of mean (SEM). Data analysis was performed using Statistical Package for Social Science (SPSS), version 17.0. To compare the results obtained from different groups, one way ANOVA followed by Tukey’s multiple comparison tests were performed to determine statistical significance. P values less than 0.05 were considered significant.


Acute toxicity test

The acute toxicity bioassay had shown that the lethal dosage (LD50) of the aqueous and methanol leaf extracts of C. abyssinica was above 2000 mg/kg and there were no evidence of acute toxicity at the doses tested indicating good safety margin. As shown on annex 1, the lethal dosage (LD50) for aqueous and methanol extract of C.abyssinica were 2154.065 mg/kg and 3807.886 mg/kg body weight respectively.

Effect of extracts on parasitaemia

Mice treated with aqueous extract of C. abyssinica at 400 mg/ kg dose had low parasitaemia on day 6 (p<0.01), day 8, 10 and 14 (p<0.001) (Table 1), while mice treated with the methanol extract at 200 and 400 mg/kg dose had statistically significant low parasitaemi on day 6, 8, 10 and 14 (p<0.001) of treatment as compared to the negative control group (Table 2). Comparison of the percentage change in parasitaemia on day 14-0 showed that only 400mg/kg dose of the methanol extract significantly (p<0.05) reduced parasitaemia by 3.91%. The lowest mean parasitaemia value (5.94+0.24) was observed in the same group on day 8 of treatment which was highly significant (p<0.001) when compared to the 100 and 200mg/kg dose. Animals treated with diminazene have cleared the parasites from circulation though parasitemia relapsed as of day 12 of treatment.

Effect of extracts on packed cell volume

The animals treated with 400 mg/kg of the aqueous extract of C. abyssinica, and with 200 and 400 mg/kg dose of the methanol extract had significantly (p<0.001) higher PCV value as compared to the negative control groups on day 14 of treatment (Table 3). Analysis of change in percentage of PCV from day 7 to day 14 of treatment also showed that the methanol extract at 200 and 400 mg/kg doses had significantly increased PCV value of treated animals by 1.29 and 1.12%, respectively as compared to the negative control groups which had a drop in PCV by 9.38 % from day 7 to 14 of treatment (Table 4). This finding was consistent with the effect shown by the extracts on parasitaemia of T. congolense infected mice (Figure 2). Animals treated with diminazene showed significantly improved PCV compared to all groups at days 7 and 14 of treatment (Figure 1).