Introduction

Trypanosomosis, an important blood protozoan zoonotic disease, is caused by flagellate parasites of the genus Trypanosoma. Many causative agents are responsible for trypanosomosis in both animals and humans with devastating consequences in all its ramifications (Stich et al., 2002; Seed,2001; WHO, 2004)..

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., 2009). 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 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).

This study is a follow-up to the previous reported trypanocidal potential of T. belirica dried fruits in this journal (Shaba et al. 2009). In this present paper, MPE of T. belirica dried fruits was evaluated for its in vitro (on different medium) and in vivo antitrypanosomal effects.

Materials and methods

Chemicals

Plant Material

Preparation of extract

Thin layer chromatography (TLC) plates

Solvent System

Animals

Test Organism

Parasite counts

Estimation of parasite counts was carried out according to Lumsden et al. (1973).. 10 -15 fields of each drop of a blood or incubated media and parasites in triplicate were counted using glass slide under inverted microscope (400X) and an average means parasites count was taken as number of parasites per field.

In vitro tryponocidal activity

Infectivity assessment

In vivo antitrypanosomal activity

Statistical Analysis

Results

Methanol was suitable (as previously reported by the same group on different medium during preliminary screening of the T. belirica dried fruits) in detecting the presence of bioactive constituents as observed on TLC plates. This is in accordance to the mobility of applied aliquots of extract on the plates (plates not shown). Solvent system methanol/chloroform (20: 80) was suitable in development of TLC plates. In vitro antitrypanosomal activity of MPE of T. belirica dried fruits were as given in Table 1. There was immobilization, reduction and complete killing of trypanosomes at different concentrations. At 250 µg/ml, trypanosomes were apparently killed at 4 h of incubation. Bioassay status of MPE extract 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 extract and negative control. (P ≤ 0.05). In infectivity assessment, group of mice inoculated with contents of wells with apparently killed trypanosomes survived while, the other group of mice with reduced trypanosomes died of parasitemia. In vivo, mice treated with test extract of T. belirica dried fruits demonstrated trypanostatic effect (Table 2). At 200 mg/kg body weight test extract, mice in this group survived for 7days in comparison to 3 days in untreated negative control group 48 h post onset of paraitemia and commencement of treatment. .

Figure 1

Table 1. trypanocidal activity of methanolic extract of dried fruits on Vero cell line.

Antitrypanosomal activity were expressed as mean ± SEM with significant difference (P<0.05)

Figure 2

Table 2 trypanocidal activity of methanolic extract of dried fruits in mice

At dose rate of 200 mg/kg body weight, the mice in this group survived for 7 days post on set of parasitaemia. There was degree of significant difference (P ≤ 0.05 ) between treated groups with test material in comparison to negative control that survived for only 3 days

Discussion

African trypanosomosis (either in animals or humans) has been described as neglected disease in our generation. Antitrypanosomal drugs currently in used were developed before 1950 when stringent laws on drugs development and uses were not in placed as today. These drugs are very toxic to the body system (Denis and Barret, 2001).

In this report, Presence of bioactive constituents on TLC plates and subsequent suitable solvent system used in development of TLC plates is comparable to extraction and development of Terminalia chebula dried fruits (Shaba et al., 2007). At 250 µg/ml, trypanosomes in MPE of test extract were apparently killed at 4 h ,which was equivalent to diminazine aceturate (50 µg/ml) at 4 h after incubation. Sluggish movements and clumps formation of trypanosomes at the bottom of the wells indicated impact of the test extract. This result is comparable to in vitro trypanocidal activity of some screened American plants extracts against T. cruzi at 250 µg/ml (Muellas-Serrano et al., 2000) and. MPE of Picrorrhiza korroa rhizomes where trypanosomes were completely killed at 500 µg/ml. (Shaba et al., 2007). Mice inoculated with contents of wells with apparently killed trypanosomes during infectivity assessment 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) and MPE of Picrorrhiza korroa rhizomes (Shaba et al., 2007). 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). Gallic acid, which has been isolated from extract of T. belrica dried fruits may be partly responsible for the antitrypanosomal activity observed. This is because gallic acid has been reported to possessed anttrypanosomal activity (Koide et al., 1998). Trypanostatic effect was observed during in vivo testing of MPE of T. belirica dried fruits in mice. The in vitro antitrypanosomal activity result was not repeated during in vivo testing of extract in mice. There could be release of trypanosomes lodged in the body tissues that were not affected by the extract. Those trypanosomes would have been relapsed after blood plasma level of the test extract might have waned. This observation was previously reported by Benson, 1988 and Madubunyi, 1995, respectively. The differences in physiological state of mice body system compare to artificial in vitro medium may account for disparity in results.. Also, there may be possibility of ease of degradation of the test extract thereby unable to attain sustained maximum therapeutic blood plasma level for effective antitrypanosomal activity. There could be possibility of test extract binding to proteins in the mice leading to no availability of it for effective antitrypanosomal activity. This result is comparable to trypanocidal activities of the ethanolic extract of Nauclea latifolia root bark at 40-100 mg/kg body weight with decreased in parasitemia in infected- extract treated mice at dose-dependent manner (Madubunyi, 1995). Significant maximum trypanocidal activity of N. latifolia extract was recorded at 100 mg/kg body weight but later rise in parasitemia of mice was observed (Madubunyi, 1995).

It can be concluded from the present study that T. belirica dried fruits possess antitrypanosomal compound(s). Further studies (e.g. bioassay-guided purification to isolate pure compound(s) responsible for its antitrypanosomal activity is needed. The pure compound(s) to be obtained may prolong and enhance antitrypanosomal activity

Acknowledgements