In tropical Africa, protozoan parasites cause several diseases of social and economic importance. One of the most devastating is trypanosomiasis which is caused  by infection with trypanosomes. Trypanosomes are transmitted by tsetse flies to people, domestic livestock and wildlife. The disease constrains agricultural development on over a third of the African continent by causing livestock production losses due to poor weight gains, stunted growth, poor milk production, reproductive failure and finally death (Olukunle et al., 2010)

Morinda lucida Benth (Rubiaceae) is a medium-sized tree about 15 m tall (Yinusa et  al., 2005). It is known as Sangogo or Bondoukou alongua in Cote d’ Ivoire; Twi, kon kroma or Ewe amake in Ghana; Ewe amake or atak ake in Togo and Oruwo in South Western Nigeria (Adeneye and Agbaje, 2008). In many countries   different parts of the plants are used in different ways. Cold decoction of the plant leaves is used for the treatment of fever in Cameroon; the bitter water decoction of the plant bark, root and leaf are used as bitter tonic and as astringent for dysentery, abdominal colic and intestinal worm infestation (Adeneye and Agbaje, 2008). Oliver-Bever (1986) reported the use of weak decoction of the stem bark in the treatment of jaundice. There are documented in vitro antimalarial activity of M. lucida leaf extract Koumaglo et al. (1992) and stem bark extract (Bello et al., 2009) against Plasmodium falciparum and antimalarial activity of M. lucida against Plasmodium berghei berghei in mice (Obih et al., 1985). Methanolic extract of M. lucida leaf extract have been reported to possess trypanocidal activity (Asuzu and Chineme, 1990) and aortic vasorelaxant effect (Ettarh and Emeka, 2004). Oliver-Bever (1986) documented the use of a weak decoction of the stem bark to treat severe jaundice.

Tridax procumbens (Asteraceae) is known for several potential therapeutic activities like antiviral, antibiotic efficacies, wound healing activity, insecticidal and anti-inflammatory activity (Suseela et al., 2002). Some reports from tribal areas in India state that the leaf juice can be used to cure fresh wounds, to stop bleeding. The plants has been extensively used in Ayurvadic system of medicine for various ailments and is shown to possess significant antiinflamatory, hepatoprotective, wound healing and antimicrobial properties (Diwan et al., 1989; Pathak et al., 1991; Saraf et al., 1991; Udupa et al., 1991; Taddei and Rosas, 2000). 

The entire plant is used by indigenous people in Gautemala for the treatment of protozoal infections (malaria, leishmaniasis, vaginitis, dysentry) and gastrointestinal disorders (colic/stomach pains, gastritis/ enterocolitis) (Caceres et al., 1998; Berger et al., 1998). Ethnobotanically, in Gautemala the whole plant of Tridax procumbens is used by the population for topical applications to treat chronic ulcers caused by leishmaniasis (Caceres et al., 1998).  

The present study is designed to evaluate anti-trypanosomal activities of M. lucida (leaf  and  stem  bark)  extract and T. Procumbens singly and in combination using various solvents.

Phytochemical analysis

The crude methanol and ethyl acetate extracts of T. procumbens was subjected to phytochemical analysis using standard analytical methods described by Sofowora (1979) (Table 1).

Infection of animals

Blood from heavily infected donor mouse was obtained by cardiac puncture and collected with EDTA coated syringe to avoid clotting. The blood was immediately diluted with physiological saline to give 1.0 × 10⁷ parasites per ml to obtain inoculums. Healthy mice were then infected intraperitoneally with 0.1 ml of the inoculum containing about 10⁶ trypanosomes (Herbert and Lumsden, 1976).

Administration of extracts

Crude extracts dissolved in (physiological buffered saline PBS) for aqueous extract or in little Dimethyl sulphoxide (DMSO) and made up with PBS for organic solvent extract in varied concentrations were administered on infected animals via the intraperitoneal route. Parasitaemia in the blood of infected animals was monitored daily by obtaining blood from the tail end of mice and observing the wet smear under light microscope set at 40X magnification for parasite per field. The number of parasites per ml of blood is then estimated using “Rapid Matching Method” described by Herbert and Lumsden (1976).

Initial screening of the extracts for antitrypanosomal activities

In T. procumbens, the ethyl acetate, methanol and aqueous extracts of the whole plants were subjected to the screening against T. b. brucei infected mice. For each extract, there are 7 groups (A - G) of 3 mice each. Groups A - F were inoculated with T. b. brucei  and with the appearance of parasitaemia, animals in Groups A - D were each treated intraperitoneally with the extract at 100, 200, 300 and 400 mg/kg body weight respectively for 14 consecutive days respectively. Mice in Group E were treated once intraperitoneally with berenil at 3.5 mg/kg body weight while Groups F was untreated and Group G was neither infected nor treated and served as control. Similarly in M. lucida, the hexane, ethyl acetate, methanol and aqueous extracts of the stem bark and leaves were screened for antitrypanosomal activity against T. b. brucei in mice. For each extract of leaves and stem  bark,  there  are  7  groups  (A - G)  of  3  mice each. Groups A - F were inoculated with the parasite and with the appearance of parasitaemia, animals in Groups A - D were each treated intraperitoneally with the extract at 100, 200, 300 and 400 mg/kg body weight respectively for 14 consecutive days respectively. Mice in Group E were treated once intraperitoneally with berenil at 3.5 mg/kg body weight while Groups F was untreated and Group G was neither infected nor treated and served as control.

Confirmatory screening with effective doses of T. procumbens and M. lucida

In order to ascertain the efficacy and reproducibility of the doses that demonstrated appreciable antitrypanosomal activities in the initial screening, eight groups of mice (A - H) each containing three mice, were set up. Group A, B and C were administered with methanolic leaves, methanolic stem bark and ethyl acetate extracts of M. lucida at 400, 200 and 100 mg/kg body weight respectively. Groups D, E and F were administered with ethyl acetate, methanol and aqueous extracts of Tridax procumbens at 200, 300 and 300 mg/kg body weight respectively. All the administration was through intraperitoneal route for 14 consecutive days. Groups G was infected untreated while Group H was neither infected nor treated and served as controls. Parasitemia was monitored three times weekly.

Combination therapy

The possibility of synergistic action of different extracts in varying combinations was investigated using a modified method of Gerardo et al. (2007). To establish this, different extracts that gave highest antitrypanosomal activity in the initial screening were combined in varying ratios and was screened against T. b. brucei infected mice. Thus, T. procumbens ethyl acetate extract at 200 mg/kg body weight and M. lucida methanolic leaves extract at 400 mg/kg body weight were combined in ratios 1:1, 2:1 and 1:2. Each ratio was used treat 3 groups of T. b. brucei infected mice comprising of 3 animals each at 200, 300 and 400 mg/kg body weight respectively. In all cases, parasitaemia was monitored daily and means (±SD), maximum / minimum survival was calculated.

Screening of M. lucida leaf extract

The result for the screening of leaf extract of Morinda lucida is presented in  Figures  1  to  4. The parasitaemia was lowered with methanol extract (Figure 3) having a means prolongation of life by 9.7

days at 400 mg/kg body weight (Table 2). Other solvent extracts also extend the life  of   treated   groups  and  the  least  was  the  hexane extract. The result for screening stem extract of M. lucida was presented on Figures 5 to 8. The methanol extract at a dose of 200 mg/kg recorded the longest mean life prolongation by 7.0 days beyond the

infected untreated control. The active principle may be less shielded and hence the desire effect was observed at low dosage. The least was the animals treated  with  ethyl  acetate  extract (Table 3). In all, the parasitaemia kept fluctuating until the animals died of acute infection.

Screening of T. procumbens extracts

Treatment with the T. procumbens at all the dose levels resulted to lowering of the parasitaemia leading to prolongation of life (Figures 9 to 11). Ethyl acetate and methanol extract have the best trypanostatic effect resulting to significant means prolongation life by 11.7 and  14.3  days  respectively  (P<0.05).  Aqueous  extract has the least effect (Table 4).

Confirmatory test

Confirmatory test for the initial screening with the extracts

at different doses and a subsequent repeated screening showed that 400 mg/kg body weight of methanol extract of M. lucida leaves and 200 mg/kg body weight of T. procumbens ethyl acetate extracts gave consistent antitrypanosomal activities. The means prolongation of life  was  almost  the  same  with  the  result  of  the  initial screening. However, the methanol stem bark extract of M. Lucida and methanol extract of T. procumbens at 200 mg/kg body weight could not reproduce the result of the initial screening (Table 5). The control animals that were untreated died one week post infection.

Combination of extracts

The combination of T. procumbens and M. lucida at 1:2 gave significant means survival  of  10  days  (P<0.05)  at

200 mg/kg body weight, while the least was when combined in ratio 1:1 (Figures 12 to 14). It is possible that there is less interference of other compounds with active principle in the extract, hence the observed effect at low dosage. 

This was possible because there was suppression of parasitaemia which was more in group treated at 400 mg/kg body weight. The ethyl acetate treated group was the second best in terms of mean survival and parasitaemia. The ethyl acetate extract is more polar than hexane extract and less than methanol. The effect of hexane and aqueous extract on the parasitaemia and prolongation of   life   beyond   the   control   are  minimal (P>0.05).

The stem bark extract showed a similar activity with leaves extract although to a lesser extent. Therefore, the antitrypanosomal property of leaves methanol extract  supersede that of  the stem bark methanol extract both in term of mean survival and prolongation of life beyond the untreated control.

The transient trypanocidal activity of methanol extracts of Morinda lucida leaves and stem bark is in agreement with the earlier studies by Asuzu and Chineme (1990). They reported the suppression of the T. b. brucei infected animal when treated with 50% methanol dry leaves extract and 1000 mg/kg produced the highest suppression. In this study, the extracted leaves of M. lucida were in absolute methanol and the dose  level  that gave highest suppression of parasite was 400 mg/kg body weight. Furthermore, Asuzu and Chineme (1990) obtained the best trypanocidal activity when treatment with M. lucida extract commenced simultaneously with trypanosome inoculation. In this study the best anti-trypanosomal activity was observed when treatment commenced at 24 h  post  parasite inoculation. Therefore the efficacy of treament is dependent on the number of parasite inoculated and time the treatment commenced.  This studies and many other investigators (Asuzu and Chineme, 1990; Aguiyi et al., 1999; Onyekwelu and Okwuasaba, 2006;

Ogbadoyi et al., 2011) all observed that the trypanocidal activity of the extracts from the various plants was not sustained and parasites were not cleared from the peripheral circulation. The phyto-chemicals present in M. lucida was earlier reported to be alkaloids, saponins, tannins. Doughari (2012) reported that plant-derived alkaloids in clinical use include the analgesics,  the   muscle   relaxant,  the   antibiotics,   the anticancer agent, the antiarrythmic, the pupil dilator, and the sedative.

M. lucida and T. procumbens have trypanostatic effect and prolonged the life of T. b. brucei infected, treated animals. There is synergy in combination of the 2 plants extracts which result in significant prolongation of life than when used singly. 

Authors have not declared any conflict of interest.

The Authors are grateful to the Director General and Management of Nigerian Institute for Trypanosomiasis Research, Kaduna for sponsoring this project.