The Antimalarial Potential of Three Ghanaian Medicinal Plants

Objective: Malaria is a major public health problem in Ghana and many indigenes, especially those in rural areas, resort to the use of medicinal plants to treat the disease. The plants: Persea americana Mill. (Lauraceae), Theobroma cacao L. (Malvaceae) and Tridax procumbens (L.) L. (Compositae) are used solely or in combination with other medicinal plants to manage malaria and its associated conditions. The leaves of the plants which are normally the main parts employed, were studied for their phytochemistry and antiplasmodial activity to establish their chemical profile and verify the antimalarial claim. Methods: Plant materials were subjected to basic phytochemical screening to identify the major secondary metabolites. The aqueous extracts were evaluated against chloroquine-sensitive 3D7 P. falciparum and chloroquine-resistant W2 P. falciparum strains, using the fluorescence-based SYBR® green I method to determine their antiplasmodial activity. Results: Basic phytochemical screening of the leaves revealed the presence of tannins, flavonoids and alkaloids in all three plant materials. T. cacao and P. americana, in addition, contained purine base alkaloids, triterpenoids including saponins. The aqueous extracts of the leaves showed antiplasmodial activity against the chloroquine-sensitive 3D7 P. falciparum (9.50 ± 1.38 ≤ IC50 ≤ 10.15 ± 0.45 μg/mL) and against chloroquine-resistant W2 P. falciparum strains (6.40 ± 1.94 ≤ IC50 ≤ 44.94 ± 1.12 μg/mL). The aqueous extract of T. cacao was the most active and was more active against W2 than 3D7 P. falciparum. Only T. procumbens displayed cytotoxicity (CC50<25 μg/mL). Conclusion: T. cacao, T. procumbens and P. americana possess antiplamodial activity. The activity illustrates their antimalarial potential, and provides rationale for their use in traditional malaria therapy in Ghana. It thus paves the way for further study of these plants for antiplasmodial lead compound(s).


Introduction
The use of medicinal plants in the treatment of diseases has a long history worldwide. In developing countries, about 80% of the population relies on traditional medicine for the treatment of various ailments including life-threatening ones such as malaria. In Sub-Saharan Africa, medicinal plants, which constitute the mainstay of traditional medicines, have significant medicinal usages, though with little or no supportive scientific data. Plants used in malaria and fever account for 6% of the medicinal plants of the Ghanaian domestic market [1] and some of these plants have been formulated into commercial phytomedicines [2]. According to Komlaga et al. [2], about 6%, 8% and 7% of herbalists in the Bosomtwe and Sekyere East Districts of Ghana, respectively, employ Persea americana Mill. (Lauraceae), Theobroma cacao L. (Malvaceae) and Tridax procumbens (L.) L. (Compositae) in the traditional treatment of malaria. We therefore evaluated the antiplasmodial activity of the aqueous extracts of the three plants in order to provide scientific validation for their antiplasmodial use in traditional medicine. Persea americana is cultivated for its nutritious fruit which contains vitamins and minerals [3]. The leaf is used for the treatment of malaria [3][4][5], dysentery, coughs, liver obstructions and high blood pressure [6]. It is used to enhance menstrual flow and reduce high levels of uric acid in the body [6]. The skin of the fruit is used to treat intestinal parasitic worms infestation, cancer of the labia and other tumours [3]. The fruit pulp has emollient and carminative properties, and lowers blood cholesterol levels. The mashed fruit pulp has aphrodisiac properties, and the unripe fruit used to induce abortion. The pulp is used externally against suppurating wounds and for hair growth. The seed ointment is used to treat various skin diseases including scabies, purulent wounds, lesions of the scalp and dandruff. The seed oil has astringent properties [6]. The leaf extract induced weight reduction, displayed antihepatotoxic activity, antiulcer and anticonvulsant effects [7]. Aqueous extract of the leaf showed hypoglycaemic activity [8] and inhibitory effect on acyclovir and PAA-resistant herpes simplex virus [9]. Methanol extracts of the leaf exhibited antimycobacterial activity [10] while the leaf and stem bark extracts showed antibacterial activity and the fruit extract wound healing activity [11]. Theobroma cacao has been used for an array of medicinal purposes. The unfermented seeds and the seed coat are used to treat diabetes, digestive and chest complaints. Cocoa powder from fermented cocoa seeds is used to prevent heart disease. Cocoa butter helps lower cholesterol levels [12]. The leaf extracts showed antiproliferative activity [13] and blocked glycolytic pathway of glucose oxidation resulting in accumulation of glucose and pyruvate [14]. Tridax procumbens is used as food and medicine [15]. It is used to treat high blood pressure, bronchial catarrh, malaria, dysentery, diarrhoea, stomach ache, headache and wound healing [2,16,17]. It also prevents hair loss and stops hemorrhage from cuts and bruises [16,17]. The aqueous extract of the plant exhibited antiplasmodial activity [18], hypotensive activity and bradycardia in normal rats [19]. Various extracts showed anti-inflammatory [20], anti-arthritic activities [21] and antimicrobial activity [22]. The aqueous extract did not show cytotoxicity against HepG2 cells [23].  (Table 1) deposited in the same Department. The harvested plant materials were air dried under shade at ambient temperature (28-35°C) for 7 days. They were then coarsely powdered using an electric mill and stored in airtight containers under room temperature until required for use.

Extraction of plant materials
For each plant material, 100 g of powder was boiled in 2 L of water in accordance with traditional preparations for 30 minutes. The decoction was then strained through double-layered white cotton material and subsequently through filter paper. The filtrate was lyophilized and the dry material kept at 4°C until needed for analysis.

Preliminary phytochemical analysis of materials
Plant materials were subjected to qualitative phytochemical screening to determine the presence of the major phytochemical constituents. The screening was performed according to standard procedures [21,22].

Plasmodium culture maintenance
Parasite cultures consisting of chloroquine-sensitive 3D7 P. falciparum and chloroquine-resistant W2 P. falciparum were maintained according to the method described by Trager & Jensen [24,25]. The parasites were separately cultivated at 37°C in a candle jar on complete culture medium of RPMI 1640, containing 2.5% hematocrit, hypoxanthine, HEPES, glucose, albumax II, and buffered with NaHCO 3 . Fresh culture was maintained for at least 96 hours (2 complete life cycles) before being used for assays. Cultures were synchronized with 5% D-sorbitol (Sigma) and at least 90% ring forms were obtained before assays were run. Prior to the launch of assays, the level of parasitaemia of an aliquot of a stock culture was determined by light microscopy following Giemsa staining [26].

Antiplasmodial activity
The antimalarial activity of aqueous extracts was evaluated against 3D7 P. falciparum and W2 P. falciparum strains using the fluorescence-based SYBR® green I approach in 96-well microplates as described by Smilkstein et al. [27] with some modifications. Positive control wells for each assay contained no inhibitor while negative control contained chloroquine (CQ). Experiments were run in duplicate with both test and control drugs employed at varying concentrations. Test solutions were prepared in dimethyl sulfoxide (DMSO) and diluted with culture medium to give a maximum DMSO concentration of 0.5% in a final well volume of 200 μL, containing 1% parasitemia and 2.5% haematocrit. Extracts and negative control [chloroquine (CQ)] were prepared by twofold dilution, in a dose-titration range of 0.098 -100 µg/mL, to obtain 11 concentrations each in triplicate. After 42 h incubation in a candle jar at 37°C, the plates were frozen at -80°C overnight. The frozen plates were then taken through a 3 freeze-thaw cycles of 1 h thawing at room temperature, 5 minutes homogenization and 1 h freezing at -80°C. Five microlitres (5 µL) of homogenate was added to 45 µL of SYBR® green I in lysis buffer in a PCR plate. The PCR plate was incubated at room temperature for an hour and the fluorescence measured using a multi-well plate reader (Eppendorf realplex mastercycler epgradient S) with excitation and emission wavelength bands centered at 485 and 530 nm, respectively. The IC 50, defines as drug concentration that inhiits the growth of P. falciparum by 50%, was estimated using the web based antimalarial estimator, ICEstimtor, at http://www. antimalarial-icestimator.net/MethodIntro.htm [26].

Cytotoxicity on HUVEC using MTT assay
Human umbilical vein endothelial cells (HUVECs) were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12) medium in the presence of fetal bovine serum (FBS; 10%) plus streptomycin (1%) and incubated in 5% CO 2 at 37°C [28,29]. The cytotoxicity of aqueous plant extracts was evaluated using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) colorimetric method [30]. HUVEC were seeded in a 96 well plate at 15000 cells/ well and incubated for 24 h when cells reached >80% confluence. After discarding the old medium, the cells were incubated in a medium containing aqueous extracts of the plant materials in a dose-titration range of 0.78-100 μg/mL. After 24 h incubation, 20 μL MTT (5 mg/mL) was added to each well and cells were incubated for another 3 h. Finally, the culture medium containing MTT solution was removed and the formazan crystals were dissolved in 100 μL of dimethylsulfoxide (DMSO). Absorbance was read with an Eppendorf plate reader at 546 nm. Drug concentration that reduced the number of viable cells by 50%, CC 50 , was calculated using GraphPad Prism Software (Version 5.0, San Diego, CA, USA).

Phytochemical screening
All three plant materials tested positive for flavonoids, tannins, and alkaloids. However, Theobroma cacao and Persea americana also showed the presence of purine base alkaloids and terpenoids (

In vitro antiplasmodial activity of aqueous extracts
The extracts were assessed at concentrations up to 100 μg/ mL against 3D7 and W2 strains of Plasmodium falciparum. All three extracts showed antiplasmodial activity. Activity against 3D7 P. falciparum strain was generally around 10 µg/mL. The extract of T. cacao was particularly more active against the chloroquine-resistant W2 P. falciparum strain (IC 50 : 6.40 ± 1.94 µg/ mL). However, P. americana and T. procumbens extracts were respectively 3 and 4 folds less active when compared to their activity against 3D7 P. falciparum. Resistant index (RI) was generally low and exceptionally low (RI<1) for T. cacao. Only T. procumbens demonstrated cytotoxicity (CC 50 : 24.89 ± 3.68). The extracts of P. americana and T. cacao generally showed high selectivity index (SI>2.9) whereas T. procumbens displayed largely low SI (Table 3).

Discussion
The use of medicinal plants in malaria therapy and also, in the therapy of many other diseases is commonplace at the primary healthcare level in many developing countries. In Ghana, medicinal plants have been used traditionally to manage malaria; some without scientific evidence of efficacy or safety. Theobroma cacao, Persea americana and Tridax procumbens, examples of such plants, were evaluated for their phytochemical profile, antiplasmodial activity and cytotoxicity.
The leaves contained various secondary metabolites including, tannins, alkaloids, flavonoids, and terpenoids (Table 2). Many compounds belonging to these phytochemical groups had shown antiplasmodial activity [31] and may indicate the same for constituents of the plants studied.
The aqueous extracts of the three plants demonstrated varying degrees of activity against chloroquine-sensitive 3D7 and chloroquine-resistant W2 strains of P. falciparum ( Table 3). According to Philippe et al. [32], the antiplasmodial activity of plant extracts can be classified as highly active (IC 50 ≤ 5 µg/mL); moderately active (5<IC 50 ≤ 15 µg/mL); weakly activity (15<IC 50 ≤ 50 µg/ mL) and inactive (IC 50 >50 µg/mL). Against this backdrop, all the extracts were considered to be moderately active against 3D7 P. falciparum. On the other hand, only the leaf extract of T. cacao showed moderate activity against W2 P. falciparum; that of P. americana and T. procumbens were only weakly active against the same strain. P. americana and T. cacao leaf extracts exhibited no cytotoxicity against HUVECs up to the highest concentration (100 µg/mL) tested, and displayed good selectivity for the parasite (SI>2.9). Conversely, T. procumbens showed significant cytotoxicity and very low selectivity especially for the chloroquine-resistant parasite ( Table 2). T. procumbens can therefore be described as generally cytotoxic since it demonstrated cytotoxicity to both parasite and human cells. This extract needs further investigation regarding potential toxicity and evaluation of benefit/risk ratio in man. Until this is done, T. procumbens should be used in traditional treatment of malaria with caution since it could be harmful to the host especially in high doses. Extracts with high selectivity index offer the potential of safer therapy [27,28]. Therefore with appropriate dosage regimen, T. cacao in particular could serve as an important malaria therapy. However, preclinical studies and clinical studies would be required to ascertain potential usefulness.
The resistance index (RI) was determined to assess the levels of resistance demonstrated by the resistant strain of the parasite against the respective extracts. The chloroquine-resistant W2 P. falciparum displayed no resistance to T. cacao while it showed low level of resistance to P. americana and T. procumbens extracts. This may suggest T. cacao a more appropriate remedy in the traditional treatment of malaria particularly at a time that P. falciparum resistance is a challenge in the treatment of the disease [33,34].
Appiah-Opong et al. [18] reported an inferior antiplasmodial activity (IC 50 >500 µg/mL) for the aqueous extract of T. procumbens against chloroquine-resistant Dd2 P. falciparum strain while in this study, a more stronger activity (IC 50 : IC 50 44.94 ± 1.12 µg/mL) was established against the chloroquine-resistant W2 P. falciparum strains. Many factors could account for this difference among which could be the strain of P. falciparum used and plantassociated factors such as the geographical source of plant, the season of collection, the age when collected, preparation and storage condition (where applicable) of extract.
The aqueous extract of P. americana leaf is reported for the first time for antiplasmodial activity; likewise, the leaf aqueous extract of T. cacao. However, the bean (cocoa) powder has been shown to have antiplasmodial activity [35].
Aside the direct effects of drugs on the malaria parasite in   [36] reported the analgesic and anti-inflammatory activities of the leaf aqueous extract of P. americana. Also T. procumbens has shown anti-inflammatory [20] and anti-arthritic activities [21]. These symptomatic non-parasite specific effects of the extracts together with their antiplasmodial activity could account for the relief from the disease during therapy. This study thus justifies the traditional use of these plants in malaria treatment in Ghana.

Conclusion
The aqueous extracts of Theobroma cacao (leaf), Tridax procumbens (whole plant) and Persea americana (leaf) possess antimalarial effect. They were active against both chloroquinesensitive 3D7 and chloroquine-resistant W2 P. falciparum. Only T. procumbens displayed cytotoxicity. The study supports the tra-ditional use of the three plants as a therapy for malaria in Ghana, and provides scientific justification for further study of the plants [36].
P. falciparum has displayed resistance to existing antimalarial drugs including artermisinin and its derivatives [37], emphasizing the need for new effective but affordable antimalarials. The three plants could be considered for formulations into standardized antimalarial phytomedicines using the 'reversed pharmacology' approach [38] to afford cheaper alternatives to malaria therapy. Also, different organic solvent extracts of the plant materials could be evaluated to identify solvent specific extracts with promising antiplasmodial activity. Such extracts can become a subject of activity-directed fractionation to isolate active compounds. Compound showing significant activity could be modified chemically to afford molecule with high efficacy and safety. Isolated compounds can also be assessed in combination with themselves and classical antimalarial drugs for synergistic effect against P. falciparum.