Introduction

In Africa, the estimated losses as a result of trypanosomosis in agricultural production amounted to 3 billion pounds annually (Hursey, 2000). In addition, it is estimated that 50 million cattle are at risk of being infected with trypanosomes leading to more than 3 million livestock deaths yearly, looses in calving, reduction in livestock number, drop in meat and milk off take and reduced work efficiency of draft animals and profitability of mixed farming (Mahmoud and Gray, 1980; Hursey,2000; Seed,2001)..

Human African trypanosomosis (HAT) is caused by Trypanosoma brucei rhodesiense and other species and transmitted by tsetse flies, which are equally spreading to other part of Africa (.Doua and Yapo 2001). At present, over 60 million people living in 36 sub-Saharan countries are at risk of contacting the disease (WHO, 2000). It is estimated that currently about 300-500,000 people are infected with 50,000 deaths annually (WHO, 2000). The above mentioned facts stress the zoonotic importance of trypanosomosis.

Chemotherapy and chemoprophylaxis are the only means of combating the menace of the disease. Chemotherapy of trypamosomosis is faced with problems such as limited choice of trypanocides in the market, high cost, toxicity, and emergence of drug-resistant trypanosome strains that have been reported (Freiburghaus et al., 1996a; Denise and Barret, 2001). Recent ethnopharmacology and ethnomedicine revealed that several medicinal plants possess trypanocidal compounds, which may hold the key for a future potential trypanocides (Lopez et al. 1998;; Wurochekke and Nok, 2004: Shaba et al., 2006) More so, several semi-synthetic and synthetic drug derivatives were originally isolated from natural compounds (Cragg et al., 1997; Soerjatta, 1996)

Terminalia belirica (Combretaceae) dried fruits have been used immemorial in traditional medicinal herb of India. It is an ingredient of Indian Ayurvedic drug ‘triphala’ used for the treatment of digestion and liver disorders (Nadkami, 1954). Active principle such as gallic acid (3,4,5-trihydroxybenzoic acid) has been isolated. T. belirica has been evaluated against carbon tetrachloride I (Jando et al., 2006)

Thus, new approach and trypanocides are highly needed to combat trypanosomes. Based on this, Terminalia belirica dried fruits were screened for possible antitrypanosomal activity.

Materials and methods

Chemicals

Plant Material

Preparation of extract

Solvent System

Thin layer chromatography (TLC) plates

Animals

Test Organism

In vitro tryponocidal activity

Infectivity assessment

Cytoxicity test

Statistical Analysis

Results

The results of trypanocidal activity of T. belirica dried fruits using different solvents of polarities were as shown in Tables 1-4. The order of trypanocidal activity was methanol, aqueous, chloroform and hexane. Bioassay status of all solvents used indicated that an average mean parasites count of 37.67± 0.58 is statistically critical value. Average mean trypanosomes counts from 37.67± 0.58 and below is significant between the tested extracts and negative control. (P ≤ 0.05). In vitro cytotoxicity test on Vero cell line, Table 5, indicated that the concentrations of MPE of T. belirica and diminazine aceturate (Berenil) were cytotoxic to Vero cells except at 1.56 and 6.25 µg/ml .

Figure 1

Table 1: trypanocidal activity of comparative extraction of dried fruits with solvents of different polarities agains Alsevier medium.

Hexane extract

Bioassay status: significant reduction of trypanosomes average counts from concentrations (250-1000 µg/ml and but no complete killing of trypanosomes in any well throughout hours of observation. An average mean trypanosomes count of 37.67± 0.58 is statistically critical value.

Figure 2

Table 2: Chloroform extract

Bioassay status: significant reduction of trypanosomes counts from concentrations (250-1000 µg/ml and but no complete killing of trypanosomes in any well through out hours of observation. An average mean trypanosomes parasites count of 37.67± 0.58 is statistically critical value.

Figure 3

Table 3: Methanolic extract

Bioassay status: significant reduction of trypanosomes counts from concentration of 250 µg/ml and complete killing of trypanosomes at same concentration at 4^th hour of observation. An average mean trypanosomes count of 37.67± 0.58 is statistically critical value.

Figure 4

Table 4: Aqueous extract

Bioassay status: significant reduction of trypanosomes counts from concentration of 250 µg/ml and complete killing of trypanosomes at same concentration at 5^th hour of observation. An average mean trypanosomes count of 37.67± 0.58 is statistically critical value.

Figure 5

Table 5: Cytotoxic effect of methanolic extract. of dried fruit on Vero cell line compared to diminazine aceturate (Berenil)

Terminalia belirica and diminazine aceturate were toxic to Vero cell line except at concentrations of 1.56 and 6.25-1.56 µg/ml

Same concentrations were used for both extracts and diminazine aceturate (Berenil)

Methanolic extract was chosen for cytotoxicity test due to its maximum trypanocidal activity

Discussion

Methanol extracted more of bioactive constituents than other solvents as observed on TLC plates in accordance to the mobility of applied aliquots of extracts on the plates (plates not shown) Solvent system methanol/chloroform (20: 80) was suitable in development of TLC plates. Also, methanolic extract exhibited antitrypanosomal activity than other solvents. Antitrypanosomal activity ranged from immobilization and killing of trypanosomes. At 250 µg/ml, trypanosomes in methanolic extract were apparently killed at 4 h which was equivalent to diminazine atceturate (50 µg/ml). This results are comparable to comparative antitrypanosomal activity of Terminalia chebula against T. evensi with minimum activity at 250 µg/ml in methanolic extract (Shaba et al., 2007), in vitro trypanocidal activity of some screened American plants extracts against T. cruzi at 250 µg/ml (Muellas-Serrano et al. 2000) and comparative in vitro extraction of some Nigeria medicinal plants with most higher activities in methanolic extracts (Atawodi, 2005). Mice inoculated with contents of wells with complete killing of trypanosomes, methanolic extract, survived for more than 60 days, while other group died of parasitemia. Infectivity assessment of antitrypanosomal activity is comparable to antitrypanosomal effect of the aqueous extract of Brassica oleracea (Igweh et al, 2002). Both MPE and diminazine aceturate were cytotoxic to Vero cells in all concentrations with effects such as distortion, swelling and sloughing of cells in the affected wells. These effects are comparable to cytotoxic effects of in vitro comparative antitrypanosomal activity of methanolic extract of Terminalia chebula dried fruits against T. evansi on Vero cells with almost same pattern of effects (Shaba et al., 2007) and extract of Terminalia arjuna bark with distortion and apoptosis of cells on human hepatoma cell line (HEPG2) (Sarveswaran et al., 2006)..Antrypanosomal activity could be due to intercalation of extracts with DNA leading to death of trypanosomes, blockage of glycolysis pathway and interference with flagella which temporally immobilizes trypanosomes (.Madubunyi, 1995; Sepulveda- Boza et al. 1996, Nok and Nock, 2002)

In conclusion, results of current investigation shows a positive promise of trypanocidal compounds depicted by different degrees of extraction of bioactive constituents by solvents with corresponding antirypanosomal activity. Further studies (e.g. bioassay-guided purification and in vivo testing) are required.

Acknowledgements