MATERIALS AND METHODS

Plant collection: Fresh leaves of Morinda lucida (Brimestone tree) were collected from Oba-ile, Olorunda Local Government Area of Osun State Nigeria between the months of May and June, 2008. The plant was identified by a Botanist at the National Institute for Pharmaceutical Research and Development, Abuja, Nigeria. The whole study was carried out in the Biochemistry laboratory of Federal University of Technology, Minna, Nigeria.

Experimental animals: Albino mice, used for screening, were purchased from the Biochemistry and Chemotherapy division of the National Institute for Trypanosomiasis and Onchocerciasis Research, Vom, Plateau State, Nigeria. The animals were acclimatized in the Department of Biochemistry laboratory, Federal University of Technology, Minna for minimum two weeks prior to study. All experiments involving the animals were conducted in compliance with the internationally accepted principles for laboratory animal use and care, as contained in the Canadian Council on Animal Care guidelines on animal use protocol review in 1997 and as described by Kabiru et al. (2010).

Parasites: Trypanosoma brucei brucei was obtained from the National Institute for Trypanosomiasis and Onchocerciasis Research (NITER), Kaduna, Kaduna State, Nigeria and subsequently maintained in the laboratory of the Biochemistry Department, Federal University of Technology, Minna, Niger State, Nigeria, by serial passage in mice.

Preparation of plant extracts: The hot extraction was carried out by subjecting 50 g of powdered sample to successive extraction with hexane, ethyl acetate, ethanol and water under reflux. For the cold extraction 50 g of the sample was directly soaked in 400 mL of distil water for 72 h. Both extracts were filtered using cheese cloth and solvent was removed using rotary evaporator followed by drying on steam bath. The dry extract was transferred into a sterile sample bottle and stored in a refrigerator until required for use.

Infection of animals: The animals were inoculated using the method described by Ogbadoyi et al. (2007). Blood from a highly infected mouse was obtained by cardiac puncture and collected with EDTA-coated syringe. The blood was appropriately diluted with physiological saline to serve as inoculums. Healthy mice of weight range 25-35 g were infected intraperitoneally with 0.1 mL of the inoculums containing about 1x10³ trypanosomes.

Preparation of stock solution of extract: The stock solution was prepared just before use by dissolving 1 g of the aqueous extract in 10 mL physiological saline.

Administration of crude extract and monitoring the course of parasitemia: Rapid matching method of Herbert and Lumsden (1976), as described by Atawodi et al. (2003), Atawodi (2005), was used to estimate parasite in the blood of the infected animals. The method involves a matching technique where the microscopic fields were compared with a range of standard logarithmic values. Counting of parasites per field was done in blood approximately diluted with physiological saline. A drop of blood was obtained on a slide by pinching the tip of the pre-sterilized tail with a sterile needle, immediately covered with a cover slip and then observed under the microscope at 40X magnification. The number of trypanosomes per microscopic field was compared with the table of logarithmic values. The logarithmic values which matched the microscopic observation were then converted to antilogarithm, from where the absolute number of trypanosomes per milliliter of blood was obtained.

The first phase of the experiment was the preliminary screening for trypanocidal activity of aqueous extract of M. lucida (leaf) using five groups; with each consisting of three infected mice. Groups A, B, C, D and E were administered intraperitoneally 100, 200, 300, 400 and 500 mg kg^-1 body weight per day of the extracts, respectively for 14 consecutive days commencing 72 h post infection while group D was infected but treated with physiological saline of the highest dose equivalent (500 mg kg^-1 b.wt.) to serve as negative control.

The second and third phases of the experiment included the screening of the most effective dose of the extract from the first phase of the experiment. Therefore, 400 mg kg^-1 b.wt. of both hot and cold aqueous leaf extracts were given intraperitoneally to infected mice along with a single dose of berenil (3.5 mg kg^-1 b.wt., to the positive control group) and 400 mg kg^-1 b.wt. of physiological saline to the negative control group. Another group was left uninfected but treated with 1000 mg kg^-1 b.wt. of both extracts to establish the toxicity of the extract at higher dose.

Estimation of percentage packed cell volume: In the second and third phases of the experiment, the blood samples of the animals were collected into heparinized capillary tubes with one end of the tube sealed with plasticine and were spun at 2000G for 5 min in a micro-haematocrit centrifuge. The PCVs were determined in the first and second week with the aid of a micro-haematocrit reader and the values expressed as percentages.

Determination of body weight: Body weights of all the mice in each group of the second and third phase of the experiments were taken before the commencement of the experiment, after first and second week of medications and compared with their initial weights.

Statistical analysis: Data was analyzed using analysis of variance (ANOVA). Significant differences between means were determined at 5% level using Duncan Multiple Range Test (SPSS16version).

RESULTS

Preliminary screening of trypanocidal activities of hot aqueous leaf extract of Morinda lucida: The trypanocidal activity of the extract at various doses used is represented in Fig. 1. It shows that the group treated with 400 mg kg^-1 b.wt., survived for 33 days with the least level of parasitemia (15.2x10⁶ mL^-1) while negative control group died five days into the study.

Trypanocidal activities of the most effective dose (400 mg kg^-1 b.wt.) hot aqueous leaf extract of Morinda lucida: The most effective dose (400 mg kg^-1 b.wt.) of hot aqueous leaf extract of Morinda lucida in Fig. 2 indicate the clearing of parasite from circulation after 2 weeks of treatment with live span of 34 days.

Trypanocidal activity of cold aqueous leaf extract of Morinda lucida: Trypanocidal activity of cold aqueous leaf extract of Morinda lucida was observed. The trypanocidal activity of 400 mg kg^-1 b.wt. of cold aqueous leaf extract of Morinda lucida is shown in Fig. 3. The parasites were cleared from circulation after 2 weeks of treatment with live span extending to 60 days post infection.

Effect of cold and hot aqueous leaf extract of Morinda lucida on body weight: The effect of both hot and cold aqueous leaf extracts of Morinda lucida on body weight of the treated animals are shown in Table 1. Significant (p<0.05) reduction in body weight was observed in the negative control group on day 7 while the treated groups experienced increase in weights after the 14th day of treatment.

Fig. 1:	Preliminary trypanocidal screening of aqueous leaf extract of Morinda lucida

Fig. 2:	Trypanocidal activity of hot aqueous leaf extracts of Morinda lucida in T. brucei brucei infected mice

Fig. 3:	Trypanocidal properties of aqueous leaf cold extract of Morinda lucida in T. brucei brucei infected mice

Table 1:	Effect of hot and cold aqueous leaf extract of Morinda lucida on body weight
Values are Mean±SD, n = 3. Values with different alphabetical superscripts along the same column are significantly different at p<0.05. A: 400 mg kg-1 b.wt., B: 1000 mg kg-1 b.wt. (not infected), C: 3.5 mg kg-1 b.wt. berenil, D: 400 mg kg-1 b.wt. Normal saline (Negative control)

Table 2:	Effect of hot and cold aqueous leaf extract of Morinda lucida treatment on percentage packed cell volume (%PCV)
Values are Mean±SD, n = 3. Values with different alphabetical superscripts along the same column are significantly different at p<0.05. A: 400 mg kg-1 b.wt., B: 1000 mg kg-1 b.wt. (not infected), C: 3.5 mg kg-1 b.wt. berenil, D: 400 mg kg-1 b.wt. Normal saline (Negative control)

Effect of hot and cold aqueous leaf extract of Morinda lucida treatment on percentage Packed Cell Volume (%PCV): The percentage packed cell volume of the animals treated with hot and cold aqueous leaf extracts of Morinda lucida are shown in Table 2. The %PCV was drastically reduced on day 7 compared with the initial level but there was significant improvement on the 14th day of the treatment. The %PCV stabilized afterwards.

DISCUSSION

The results obtained in this study, for both the first and second stages, demonstrated the efficacy of the hot and cold aqueous extracts of Morinda lucida in clearing the parasites from the blood of Trypanosoma brucei brucei-infected mice, especially when administered at a dose of 400 mg kg^-1 body weight/day. It also demonstrated the fact that the treatment period must be extended to two weeks to accomplish complete clearance of parasites and elongation of life span (Fig. 1-3). We could infer from these observations that the efficacy of the extracts is dose and time dependent. Antitrypanosomal activities of plants of different families have been reported by Freiburghaus et al. (1996).

The extracts were also found to reverse two principal symptoms of trypanposomiasis, viz., weight loss and anemia (characterized by reduction in %PCV). Mice treated with 400 mg kg^-1 body weight per day had their body weights and %PCV stabilized after treating them for two weeks. Although, the extracts did not perform to the same level with the standard drug, their activity against trypanosomes in circulation is appreciable. It provides a lead to further investigation of the plant for drug development.

The actual mechanism by which the extract exhibited their trypanocidal activity is not known but Sepulveda-Boza and Cassels (1996) suggested that many natural products exhibit their trypanocidal activity through interference of redox balance of the parasite acting on their respiratory chain or their cellular defense against oxidative stress. This may be so because some natural products possess constituents, capable of generating radicals that may cause peroxidative damage to trypathione reductase that is very sensitive to alteration in the redox balance. It is also known that some agent act by binding with the kinetoplast DNA of the parasite.

The extract was found to be safe and free of toxicity at a dose level of 1000 mg kg^-1 body weight, thus justifying its local use by traditional herbalists at doses that are unquantified.

CONCLUSION

The results of this study demonstrated that Morinda lucida possesses considerable trypanocidal activity with no observable toxicity. The plant can be further exploited for use in the treatment of trypanosomiasis.

ACKNOWLEDGMENT

This study was funded by a research grant to Emmanuel O. Ogbadoyi by the Organization for the Prohibition of Chemical Weapons, The Hague.