Abstract: Fifteen plants were screened for in vivo antimalarial activity in albino mice. The plants are Mormodica balsamina, Artemisia maciverae, Xylopia aethiopica, Cyperus articulatus, Guiera senegalensis. Syzygium aromaticum, Zingiber officinale, Thonningea sanguinea, Sorghum sp., Securinega virosa B, Chrozophora senegalensis, Feretia apodanthera, Diospyrous mespiliformis, Centaturea perrottetti and Acacia nilotica Del. The petroleum ether, chloroform and methanol extracts from the various parts of the plants were screened for in vivo antimalarial activity in mice experimentally infected with Plasmodium berghei. Three days after inducing the malaria, the plant extracts were administered intraperitoneally to the mice daily for four days, while chloroquine was used as a standard drug control. Parasitaemia was monitored microscopically in all the groups for four days using thick and thin blood films obtained from tail vein of each mouse. At the end of this study, it was observed that the chloroform extracts of Artemisia maciverae (whole plant), Xylopia aethiopica (fruits) and Acacia nilotica Del (Leaves) have antimalarial activity. The methanol extracts of Syzygium aromaticum (cloves) and Zingiber officinale (tuber stem) showed slight antimalarial activity, while the rest of the plant extracts earlier listed showed no noticeable activity. These results suggest that many plants used as recipes in ethnomedical preparation for malaria, have no direct antimalarial activity.
INTRODUCTION
Malaria is a mosquito-borne disease caused by the parasite plasmodium. Four identified species of this parasite, which cause different types of human malaria include Plasmodium vivax, Plasmodium falciparum, Plasmodium ovale and Plasmodium malariae, all of which are transmitted by the female anopheles mosquito (Hardman and Limbird, 2001). Although all the four species of malaria parasites can infect humans and cause illness, only the malaria caused by Plasmodium falciparum is known to be potentially life threatening in humans. Infection with P. falciparum is therefore a medical emergency. About 2% of persons infected with Falciparum malaria die usually because of delayed treatment (Peter and Anatoli, 1998).
The present global situation indicates a recent resurgence in the severity of the disease and that malaria could still be described as one of the most important diseases, with an annual incidence of 300-500 million clinically manifest cases and a death toll of 1-2 million people (Martin et al., 2004; Miller et al., 1994; More, 2002; David et al., 2004). Mortality and morbidity due to malaria are a matter of great concern throughout the whole world, especially in tropical and subtropical regions. Even though casualty in children below the age of 5 years is very high, the disease affect all age groups (Bickii et al., 2000).
In earlier times, the common oral treatment for uncomplicated falciparum malaria was limited to chloroquine and quinine sulfate, mefloquine, doxcycline, sulfadoxine/pyrimethamine (SP) in chloroquine resistant cases (Williams et al., 2004; Laxminarayan, 2004).
One of the most important factors limiting success in the treatment of malaria, whether for preventive or for curative purposes, is the varying response of individuals parasites to the drug used. Drug resistance has emerged as one of the greatest challenges facing malaria control today (Trape, 2002). It has been implicated in enhanced mortality from malaria in hyper and holoendemic areas and in the development of new and expanding foci of falciparum malaria, but above all, it has been identified as a factor in the economic constraint of malaria control. Emerging and spreading resistance to an increasing number of antimalarial drugs has been a major concern especially in Asia, Africa and South America (Sibley, 2001). Chloroquine, though effective as a blood schizontocidal drug, is ineffective or partially effective in resistant cases. The emergence of strains of Plasmodium falciparum resistant to chloroquine and many other drugs in succession has stimulated efforts to identify new antimalarial agents (Bickii et al., 2000).
The need for an alternative drug initiated intensive efforts for developing new antimalarials from indigenous plants (Wright and Phillipson, 1990). This led to the diversification of drug research into medicinal plants. For example, in vitro antimalarial activity of leaf extracts of Guiera senegalensis on Plasmodium yoelii nigeriensis (Iwalewa et al., 1990), as well as in vivo and in vitro antimalarial activity of two crude extracts of Cassia occidentalis leaf (Iwalewa et al., 1997) had been reported. In the present study, fifteen plants grown in Nigeria were screened in vivo for antimalarial activity.
MATERIALS AND METHODS
Plant Collection and Sample Preparation
Fifteen plants commonly found in Maiduguri, Nigeria were used for this study.
The plants are: Mormodica balsamina, Artemisia maciverae, Xylopia
aethiopica, Cyperus articulatus, Guiera senegalensis, Syzygium
aromaticum, Zingiber officinale, Thonningea sanguinea, Sorghum
sp., Securinega virosa B, Chrozophora senegalensis, Feretia
apodanthera, Diospyrous mespiliformis, Centaturea perrotteti and
Acacia nilotica Del. The various parts of these plants ie leaves, stem
bark, roots, fruits and whole plants were collected from Maiduguri, North-Eastern
Nigeria. All the plants were identified by a Botanist from the Department of
Biological Sciences, University of Maiduguri, Nigeria. This study was carried
out at the Biochemistry Department of Ahmadu Bello University Zaria, Nigeria,
between April and October, 2006.
All the plant parts were air dried and ground into powdered form before extraction in the following order: ie petroleum ether-chloroform-methanol. The extracts of the various plant parts were kept in a tightly closed glass bottle in a refrigerator at 4°C until used for antimalarial testing.
Plasmodium berghei Parasite
The Plasmodium berghei used for this study was obtained from the
Biochemistry Department of Nigerian Institute of Medical Research, Yaba, Lagos,
Nigeria. The parasite was maintained by sub-passaging into healthy mice on a
weekly basis throughout the duration of the study.
The infection of the recipient mice was initiated by needle passage of the above mentioned parasite preparation, from the donor to healthy test animals via an intraperitoneal route (David et al., 2004; Peter and Anatoli, 1998). That is P. berghei infected red blood cells were intraperitoneally injected into the mice f rom the blood diluted with Phosphate Buffered Saline (PBS) so that each 0.2 mL had approximately 106-107 infected red cells (parasite per kg of body weight). Each mouse was infected with single inoculums of 0.2 mL blood.
In vivo Antimalarial Tests
Tests were performed using a 4-day curative standard test (David et al.,
2004; Peter and Anatoli, 1998) and employing the rodent malaria parasite- Plasmodium
berghei.
The mice were divided into groups. Three mice were used for each of the 69 test/treatment groups, chloroquine (CQ) control and negative control groups. All the mice were infected with the malaria parasites as described above.
Seventy two hours after infecting the mice with the malaria parasites, the plant extracts were administered to the test groups at a dose level of 100 mg kg-1 body weight daily for four days. Chloroquine was administered to the CQ standard (control) group at the standard dose of 25 mg kg-1 b.wt. for four days. The negative control group were not treated. All drug administration was done through the intraperitoneal route. The dose level of 100 mg kg-1 b.wt. of the extract was selected from a pilot study carried out in mice and earlier studies (Teferi and Heinz, 2002).
The extracts were dissolved to the indicated suitable dose level in solution and suspension, the later requiring total dissolution in 3% v/v Tween 80. Treatments were performed daily for 4 consecutive days starting 72 h after infection, receiving a total of 4 intraperitoneal doses (David et al., 2004).
The parasitemia was monitored in all the groups for four days using thick and thin smears of blood films made from tail vein of mice (David et al., 2004; WHO, 1980). The smears were stained with 10% Giemsa at pH 7.2 for 15 min and examined under the microscope to access the level of parasitemia. The percentage parasitemia was calculated according to the method outlined by Iwalewa et al. (1997) as:
All the results were compared with the untreated control using student t-test.
RESULTS
Results obtained showed that normal mice infected with P. berghei and not treated died within 7-10 days. While infected mice treated with chloroquine survived. On the other hand, all infected mice treated with the chloroform medicinal plant extracts of Artemisia maciverae (whole plant), Xylopia aethiopica (fruits) and Acacia nilotica Del (Leaves) showed clearance of the parasite to a higher level when compared with the untreated control (Table 2), though the parasitemia was not completely cleared (Table 1). This did not lead to their survival, they died after about four weeks of drug administration.
The methanol extracts of Syzyguim aromaticum (cloves) and Zingiber officinale (tuberstem) showed slight antimalarial activity when compared with untreated control (Table 3). Their extracts cleared the parasites to a minimal level. The remaining plant extracts showed no activity at all (Table 1, 2, 3).
| Table 1: | In vivo screening of petroleum ether extract for anti-malarial activity |
| All values were compared with the untreated control at p = 0.05, n = 3 | |
| Table 2: | In vivo screening of chloroform extract for anti-malarial activity |
| All values were compared with the untreated control at p = 0.05, n = 3. All values with the superscript a are statistically different from the untreated control bc | |
| Table 3: | In vivo screening of methanol extract for anti-malarial activity |
| All values were compared with the untreated control at p= 0.05, n = 3. All values with the superscript d are statistically different from the untreated control ef | |
DISCUSSION
The antimalarial activity observed with the chloroform extracts of the whole plant of Artemisia maciverae, fruits of Xylopia aethiopica and Leaves of Acacia nilotica Del might be attributed to the presence of some active ingredients in these plants. Also the slight antimalarial activity observed with the methanol extracts of Syzyguim aromaticum (cloves) and Zingiber officinale (tuber stem) might be due to the presence of minute quantity of some active ingredients. Some phytochemicals have been reported to be present in the above mentioned plant extracts. In the extracts of the Artemisia maciverae, the presence of artemisinin and 1,2,4- trioxane analogs have been reported (Rajendra et al., 2002). Diterprene kaurenoids have been reported to be present in Xylopia aethiopica fruits (Samova et al., 2001). Saponins and triterpenes have equally been reported to be present in the extracts of Acacia nilotica Del (Kamalijit et al., 2002). In Syzygium aromaticum clove, the presence of eugenol, thymol and benzyl alcohols were reported (Lee and Shibamoto, 2001), while Zingiber officinale was found to contain antioxidant cyclic diaryheptanoid (He, 2001). The presence of these phytochemicals in these plants might be responsible for the antimalarial activity exhibited by them. This can be supported by the studies carried out by Badman et al. (1988) and Klayman (1985) which show conclusively that artemisinine the antipyretic principle/ingredient of plant Artemisia annua, possess antimalarial activity.
The slight antimalarial activity shown by the methanol extract of Syzyguim aromaticum (cloves) after about 10 days of drug administration has stopped might be attributed to the fact that the extract boosted the immune response of the animals thereby giving the capacity to handle the parasites (Abebe et al., 2003; Kiseko et al., 2000).
In a similar study carried out by Ajaiyeoba et al. (1999) on two Nigerian simaroubaceae plants, Quassia amara L. and Quassia undulata were screened in vivo in mice for antimalarial properties using hexane and methanol extracts. The extracts at a concentration of 100 mg kg-1 body weight (b.wt.) of mouse showed significant antimalarial activities in the 4 day suppressive in vivo antimalarial assay in mice inoculated with red blood cells parasitized with Plasmodium berghei. This is in support of the antimalarial activity observed with the chloroform extracts of the whole plant of Artemisia maciverae, fruits of Xylopia aethiopica, leaves of Acacia nilotica Del and methanol extracts of Syzygium aromaticum (cloves) and Zingiber officinale at a dose concentration of 100 mg kg-1 b.wt.
Mice infected with the parasite and not treated, showed fulminant parasitemia which resulted in death 7-10 days later. This is in agreement with the work of Agomo et al. (1992).
The results suggest that many plants used as recipes in ethnomedical preparation for malaria, have no direct antimalarial activity and have also indicated that some plants grown in Nigeria were not previously in the list of antimalarial plants are now known to have antimalarial effect. That plant may be sources of potent antimalarial agents have previously been established by other workers (Iwalewa et al., 1997).
There may be geographical, regional and specie differences in medicinal efficacy of plants. This justifies our inclusion of the plant Artemisia maciverae in our list of plants, although Artemisia annua, another specie within the genus has been extensively studied in Asia, particularly in China. Currently we are conducting a detailed study to establish the antimalarial potency, toxicity and possible mechanism of action of Artemisia maciverae and other plants that have showed significant antimalarial activity in this study.
CONCLUSION AND RECOMMENDATIONS
The results of the preliminary studies carried out with the above mentioned plants are encouraging. The scope for developing antimalarials from indigenous plants depends on screening of a large number of medicinal plants from different geographical regions, especially from the tribal/rural areas, where usage of these medicinal plants is more common. Once the anti-plasmodial effect of the plant is confirmed, the active ingredients could be isolated by different extraction methods.
Since the chloroform extracts of Artemisia macivae (whole plant), Xylopia aethopica (fruits) and Acacia nilotica Del (Leaves) showed a high level of antimalarial activity, more studies are currently being undertaken by our team to establish the antimalarial component ie active ingredient distribution and quantity and toxicological potential in malaria therapy.