Antiplasmodial and Genotoxic Study of Selected Ghanaian Medicinal Plants
Ethnopharmacological Relevance. Development of resistance to antimalarial drugs by Plasmodium falciparum is still rampant, and there is an urgent need for novel drugs to either standalone or to partner artemisinin for treatment of malaria. Traditionally, plants have, over the years, been a good source of antimalarial drugs. Efficacy and safety of such plants need to be scientifically authenticated. Aims, Materials, and Method. This study investigated the in vitro antiplasmodial activity, cytotoxicity, and genotoxicity of aqueous extracts of Acanthospermum hispidum DC, Alstonia boone (De Wild), Cocos nucifera L, Cymbopogon citratus (DC.) Stapf, Morinda lucida Benth, Psidium guajava, Phyllanthus niruri L, and Senna siamea Lam. Results. Five out of the eight plants, A. boonei stem bark, S; siamea Lam root, M. lucida Benth leaves, P. niruri, and A. hispidum DC whole plants, showed varying degrees of antiplasmodial activity against the asexual stage of the parasite. The most active extract against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) P. falciparum strains is the A. hispidum extract which yielded a mean inhibitory concentration at 50% (IC50) of 3.66 µg/ml and 3.71 µg/ml for 3D7 and Dd2, respectively. This was followed by S. siamea Lam with 3.95 µg/ml for 3D7 and 4.47 µg/ml for Dd2. The IC50 values of the A. boonei extract against 3D7 and Dd2 P. falciparum parasites were 5.13 µg/ml and 3.62 µg/ml, respectively. For the M. lucida Benth extract, the least IC50 value was 6.46 µg/ml. All five extracts exhibited dose-dependent antiplasmodial activity. Assessment of the genotoxic effects the A. hispidum extract by the comet assay revealed substantial damage to P. falciparum DNA. Conclusion. This study demonstrates that the crude extract of A. hispidum DC, one of the plants used traditionally to treat malaria, inhibits the growth of P. falciparum in vitro and could be a potential source of antimalarial drug. The report has highlighted genotoxic and cytotoxic effects of the selected plant extracts on human leukocytes as well.
Malaria in humans is caused by five species of haemospozoan parasites, namely, Plasmodium falciparum, P. malariae, P. ovalae, and P. vivax and, lately, the Zoonotic P. knowlesi . Of these, P. falciparum is the most widespread and causes the most lethal infection with the female mosquito of the Anopheles gambiae complex being responsible for the transmission of the disease . The disease threatens billions of people globally. Infection with the parasite may result in asymptomatic or clinical malaria  with about 2% of the clinical cases resulting in severe disease  that accounts for thousands of deaths attributed annually to malaria . More than 90% of these deaths occur in sub-Saharan Africa of which children are in the majority. Mortality in children under five years of age is quite high, but the disease affects all age groups, including pregnant women and nonimmune adults either residing in or visiting endemic areas. Low birth weight, spontaneous abortion, and stillbirth that are associated with placental malaria are some of the other effects of malaria on humans .
To control the global malaria havoc, preventive and curative measures were initiated in the 1950s to eradicate the disease following the discoveries of the causative parasite, mode of transmission of the parasite, the vector, and antimalaria drugs. Although some successes were chalked by the campaign in wiping out the disease in the temperate regions, it failed in tropical countries and was abandoned in 1969. The disease still lingers on, and infection is on the increase probably due to the emergence of resistance of the parasite to most of the common and affordable drugs such as chloroquine .
Until recently, chloroquine was the first-line drug for treating and preventing malaria in Ghana and many other countries. However, there is widespread of P. falciparum in circulation that is resistant to chloroquine and other drugs such as sulfadoxine-pyrimethamine and proguanil . The worsening resistance [4, 5] has resulted in a change from chloroquine to artemisinin-based combination therapies such as Coaterm, but these are very expensive and, hence, not readily accessible to most of the rural folks. This, coupled with a lack of an efficacious malaria vaccine, has necessitated the search for novel, well-tolerated, newer, cheaper, accessible, more efficient, and safer antimalarial drugs .
Natural products are important sources of biologically active compounds that could be potential sources of novel antimalarial drugs. The WHO estimates that 80% of the world population use herbal medicine for some aspect of primary health care . Herbal medicine is, thus, a major component of all indigenous peoples’ traditional medicine and is a common element in homeopathic, naturopathic, traditional healing, and native medicine .
In Ghana and some other African countries, herbal medicinal preparations from plants such as Triclisia leaves, Jatropha spp, M. lucida Benth, Cryptolepis sanguinolenta (Lindl) Schlt, Khaya senegalensis, and Azadirachta indica are commonly used either alone or in combination with other plants for the treatment of fevers and other conditions such as malaria [9–15]. While some medicinal plants have shown a great deal of antimalarial activities, either as schizontocidal and/or prophylactic agents [16, 17], others have not been investigated scientifically. In this study, we report on the efficacy, cytotoxicity, and genotoxicity of extracts from eight medicinal plants that have been reported to possess antimalarial properties or used by traditional healers in the treatment of malaria [9, 11, 12, 15, 18].
2.1. Collection of Plants
The root of Senna siamea Lam and Cocos nucifera L, the stem bark of Alstonia boonei (De Wild), leaves Morinda lucida Benth and of Psidium guajava Linn, and whole plant of Phyllanthus niruri L, Acanthospermum hispidum DC, and Cymbopogon citratus (DC.) Stapf were collected between June and July 2008 from the University of Ghana’s botanical garden at Legon. Each plant was identified with the help of Mr Patrick P. Ekpe, a Taxonomist at the Department of Plant and Environmental Biology, University of Ghana, Legon. The plants, Acanthospermum hispidum DC (DEDE/2008/001), Alstonia boonei (DEDE/2008/002), Cocos nucifera L (DEDE/2008/003), Cymbopogon citratus (DC.) Stapf (DEDE/2008/004), Morinda lucida Benth (DEDE/2008/005), Phyllanthus niruri L (DEDE/2008/006), Psidium guajava (DEDE/2008/007), and Senna siamea Lam (DEDE/2008/008), were collected and assigned collection numbers as indicated in the parentheses mentioned above. The voucher specimen of each plant was prepared and deposited in the Ghana Herbarium at the Department of Plant and Environmental Biology, University of Ghana, Legon, Ghana.
2.1.1. Preparation of the Crude Extracts
The parts of A. boonei, S. siamea Lam, M. lucida Benth, P. guajava, P. niruri L, A. hispidum DC, and Cymbopogon citratus (DC.) Stapf were washed thoroughly with potable water, followed by distilled water, and air-dried at room temperature. For standardization purposes, 200 g of each sample was weighed and boiled in 800 ml distilled water for an hour to obtain the extracts. The aqueous extracts were collected and centrifuged twice at 2000 rpm for 10 minutes to remove particulate materials. Each of the supernatants was, then, freeze-dried in a Hepto Power Dry LL3000 machine (Jouan Nordic, Denmark) and stored in a dedicated refrigerator until later use. Twenty milligrams (20 mg) of each of the freeze-dried plant extract was reconstituted in one millilitre of sterile distilled water (1 ml) to obtain a concentration of 20 mg/ml. Each of the solutions was filtered successively through membranes of 0.45 µm pores and 0.22 µm pores (Millipore Corp., Bedford) in a laminar flow biosafety cabinet. Two-fold serial dilutions of each of the plant extracts were prepared to start from 833.3 µg/ml to 6.5 µg/ml in a parasite culture medium (RPMI 1640, L-glutamine, Gentamycin, and Albumax). All dilutions, including that of artesunate which was used as the positive control, were prepared fresh on the day of the assays.
2.1.2. Growth Inhibition, Cytotoxicity, and Genotoxicity Assays
Parasites were cultured in O+ and sickle-negative human blood and coincubated with different concentrations of the various extracts and growth inhibition determined as described earlier . The cytotoxic and genotoxic effects of the extracts on both parasites and human peripheral mononuclear cells (PBMCs) were evaluated by coculturing the parasites with the extracts at the predetermined concentrations. The cytotoxic effect of the extract on the PBMCs was assessed by the colorimetric determination of human lactate dehydrogenase released into the supernatant of the culture media . The genotoxic effect of the extracts on either the parasites or the PBMCs was evaluated by the comet assay  using a commercially available Comet assay kit (4250-050-K, Trevigen, USA). The parasite DNA was stained with silver stain, while the DNA of the PBMCs was dyed with Sybr green.
2.1.3. Statistical Analysis
GraphPad Prism version 8.4 (GraphPad Software, San Diego, CA, USA) and Microsoft Excel software were used to analyze the data and plot graphs. Mean values of repeated experiments presented as percentage growth inhibition of the various extracts were evaluated with the 50% growth inhibitory concentration (IC50) for the various extract calculated by a nonlinear regression analysis. Differences among three or more groups were assessed with ANOVA followed by Turkey post hoc correction analyses, while differences between any two groups were evaluated by Student’s T-test analysis at .
Two different laboratory strains of the P. falciparum parasite (3D7 which is chloroquine-sensitive and Dd2, chloroquine-resistant strain) were successfully cultured and used to evaluate the in vitro efficacy of seven selected medicinal plant extracts. All the extracts were screened with the parasites, but three did not show any appreciable antiplasmodial activities and were dropped from further in vitro analyses.
3.1. Effect of Extracts on the Growth of Plasmodium falciparum 3D7 and Dd2 Strains
The inhibitory characteristics of the various extracts and the standard drug, artesunate, on the growth of the two strains of P. falciparum are presented in Figure 1 with the IC50 presented in Table 1. The least concentration 6.51 µg/ml of each extract significantly inhibited the growth of either strain of P. falciparum used compared to the control (). The P. falciparum growth inhibition by A. boonei was concentration-dependent which ranges from 18.86% to 71.67% (Figure 1(a)). The highest concentration of 833.3 µg/ml inhibited the growth of the 3D7 parasite up to 71.67%, while in the case of Dd2, the percentage growth inhibition reached a maximum of 66.67%. In the case of the S. siamea Lam extract, the percentage growth inhibition of the 3D7 strain of P. falciparum ranges from 17.29% for the minimum 6.5 µg/ml to 79.11% (for a maximum of 833.3 µg/ml concentration used, respectively) (Figure 1(b)). The Morinda lucida Benth extract inhibited the growth of both strains of P. falciparum parasites used (Figures 1(a) and 1(b)). The least concentration of 6.5 µg/ml inhibited the growth of 3D7 up to 18.06% with the maximum concentration of 833.3 µg/ml inhibiting the parasite growth up to 61.94% (Figures 1(a)). Similarly, the same minimum and maximum concentration of the extract inhibited the growth of Dd2 from 20.63% to 57.59% (Figures 1(b)). The Phyllanthus niruri L extract also inhibited the growth of both strains of P. falciparum (Figures 1(a) and 1(b)). The growth inhibition ranges from 27.29% to 62.19%. The most concentrated preparation at 833.3 µg/ml inhibited the growth of chloroquine-sensitive parasite strain (3D7) up to 62.19%, and in the case of chloroquine-resistant strain (Dd2), the percentage growth inhibition was about the same (Figures 1(a) and 1(b)). Similarly, the A. hispidum DC extract showed growth inhibition of chloroquine-sensitive strain of P. falciparum ranging from 17.91% at 6.5 µg/ml to 90.95% at 833.3 µg/ml for the maximum concentration, whiles in the case of chloroquine-resistant strain, the minimum and maximum percentage growth inhibition were 16.61% and 84.22%, respectively (Figure 1). In general, the degree of P. falciparum strain 3D7 growth inhibition increased significantly with increasing extract concentrations up to 26.042 µg/ml () for A. boonei and () for S. siamea but to only 13.021 µg/ml in the case of A. hispidum () and M. lucida Benth (). The rest, including data on Dd2, increased gradually and never reached significant levels (Figure 1). There was no significant difference amongst the IC50 when the IC50 values obtained for all the extracts for 3D7 () or Dd2 () were compared. Similarly, no differences were detected between the IC50 for the two Plasmodium falciparum strains (Table 1).
The percentage growth inhibition of ring and schizont developmental stages of 3D7 strain of the Plasmodium parasite with time is represented in Figure 2. The percentage growth inhibition of ring developmental stage by the A. hispidum DC extract increased gradually from the 6th (21.41%) to the 48th hour (63.88%) when the assay was terminated (Figure 2(c)). Growth inhibition leveled off from 18 hours to 24 hours, followed by an increase to 67.47% at 36 hours. It, then, dropped sharply to 52.12% at 48 hours. There was no significant difference between the growth inhibitory effects of A. boonei (De Wild) and S. siamea Lam extracts on the rings and schizont developmental stages of the malaria parasites (Figure 2). With the chloroquine-resistant strains (Dd2), growth inhibition by A. boonei (De Wild), S. siamea Lam, and A. hispidum DC extracts increased with time (Figures 2(a)–2(c)). The increase in growth inhibition for the developmental stages was from the 6th to the 36th hour. Growth inhibition, then, dropped from 44.11% to 41.13% for S. siamea Lam extracts and from 58.71% to 45.57% for A. hispidum DC. There was, however, a gradual increase of growth inhibition for the A. boonei (De Wild) extract from the 36th to the 48th hour when the assay was terminated (Figure 2).
3.2. Effect of Extracts on Plasmodium DNA
The effect of the plant extracts on P. falciparum DNA is presented in Figures 3. Undamaged cells appeared as intact nuclei without tails (negative control Figure 3(a)), whereas damaged cells had different appearances (B–F), with some appearing as comet (Figure 3(f)), unwinding, or distortion of cells DNA. A visual scoring and quantification of the extent of DNA damage produced an average score of 1.5 for the lowest diluted extract corresponding to 42% DNA in the tail of the comet. The highest score of 4 which translated to 75% DNA in the comet tail was recorded for the highest concentration of the A. hispidum DC extract (Table 2). The Acanthospermum hispidum DC extract also had a comet score of 4 (75% of DNA in the tail of the comet). The Senna siamea Lam extract, on the hand, had a score of 3 (corresponding to 65% of parasite DNA in comet tail (Table 2).
Visual examination and scoring of the comet were carried out according to the guidelines provided by the manufacturer [22, 23]. The results are presented as average comet score and average percentage scores. The higher the score or percentage score is, the more destructive the extract is to the DNA.
3.3. Cytotoxicity of the Extracts to Human Peripheral Blood Mononuclear Cells
Cytotoxicity of the three most active extracts determined by LDH leakages from the human peripheral blood mononuclear cells (PBMCs) is presented in Table 3. The table shows that A. boonei (De Wild), S. siamea Lam, and A. hispidum DC extracts-mediated hLDH release was concentration-dependent (Table 3). Comparing the three extracts, A. hispidum DC seems to cause more LDH leakage. The Alstonia boonei (De Wild) extract caused the least release of LDH (Table 3). Based on the percentage LDH leakage, IC50 was determined using the regression curve. The IC50 values estimated were 7.58 μg/ml, 7.65 μg/ml, and 3.46 μg/ml for extracts A. boonei (De Wild), S. siamea Lam, and A. hispidum DC, respectively. All the three extracts were active and selectively toxic to the parasites than mammalian cells with selective toxicity indices ranging from 93 to 209 (Table 4).
The table shows the percentage of leakage of human lactate dehydrogenase from human peripheral mononuclear cells when the cells were subjected to various concentrations of the extracts. The greater the percentage leakage, the greater the cytotoxicity of the extract at the concentration.
3.3.1. Qualitative and Quantitative Interpretation of Results
The results of exposure of PBMCs to extracts are presented in (Figure 4). Undamaged cells have their nuclei intact without comet tails or diffused DNA as in the negative control panel (Figure 4(a)), whereas damaged cells have different appearances (B–F). This includes unwinding or distortion of DNA in the cells. Alstonia boonei (De Wild), S. siamea Lam, and A. hispidum DC extracts had the highest comet score of 3, and the percentage DNA in the comet tail seen was 75% for A. hispidum and 70% for S. siamea Lam of DNA in the comet tail (Table 5). Extract A. boonei (De Wild) recorded 65% DNA in the comet tail.
The widespread resistance of the Plasmodium falciparum parasite to drugs being used to treat malaria poses a lot of public health challenges. Should the P. falciparum develop resistance to artemisinin, the last surviving most potent antimalarial drug, the havoc will be unprecedented. There is, therefore, an urgent need for novel and affordable drugs to either standalone or partner artemisinin for management of malaria; hence, this investigation into selected medicinal herbs that are used in treating malaria in Ghana and some other countries in Africa [9, 11–15, 24, 25]. Aqueous extracts from all the plants tested showed various degrees of antiplasmodial activities against both chloroquine-sensitive 3D7 and chloroquine-resistant Dd2 laboratory strains of P. falciparum. The observed sensitivity of both parasite lines to the extract lends more credence to their use traditionally as antimalarial plants. From rankings of the IC50s of the extracts, the A. hispidum extract was the most efficacious, closely followed by A. boonei (De Wild) and S. siamea Lam. They inhibited parasite growth in a concentration-dependent manner, and this seems to result from tempering of the integrity of P. falciparum parasite DNA by the plant extracts. This observation is consistent with other studies that have demonstrated that herbal preparations [26, 27], as well as chloroquine  disrupt P. falciparum parasite DNA.
Alstonia boonei (De Wild) displayed high activity against chloroquine-resistant P. falciparum parasites. This confirms earlier in vitro and in vivo  reports which suggested the plant as a source of potentially useful lead substances for future antimalarial drugs. Currently, artemisinin, a plant-derived drug is the most potent drug against chloroquine-resistant parasites . To minimize the development of resistance by P. falciparum parasites to artemisinin, artemisinin combination therapy has been recommended by the World Health Organization. The exact-active antimalarial agent in A. boonei (De Wild) could, therefore, be identified and developed into an antimalarial drug that may be used as a standalone or be in combination with artemisinin to treat malaria.
The Senna siamea Lam extract displayed high antiplasmodial activity in this study. This corroborates reports of the use of the plant in countries in Central and West Africa, including Ghana for the management of malaria [31–33]. Interestingly, the observed IC50 value of 4.48 µg/ml is about five-folds lower than what was reported by Sanon et al. . This might have arisen from the plant part used and the type of solvent used, as well as the duration of extraction of the active agent relative to other products during the preparation of the extract. The aqueous extraction employed in the current report was made from the root of the plant by boiling at 100°C for 60 minutes. However, Sanon et al. made their aqueous extraction from the leaves of the plant and the duration of extraction was shorter . It is, thus, possible that the active antiplasmodial ingredient in the plant was more concentrated in the root bark. These notwithstanding, it is very clear that extracts from either part of Senna siamea Lam have antiplasmodial activity that can be isolated and developed into a novel, a readily available, and affordable antimalaria drug.
Morinda lucida Benth and P. niruri L extracts separately showed relatively low antiplasmodial activity in vitro. Even though this is in agreement with an earlier report , it contrasts sharply with others [12, 33]. The difference in the current and the earlier reports might be due to the solvent used in the extraction as it has been well documented that plants might show no activity against Plasmodium species when extracted with polar solvents but exhibit extensive antimalarial/antiplasmodial activity when they are extracted with organic solvents such as ether or chloroform [32, 33]. This situation may be due to the polarity of the bioactive agent and, hence, its solubility in solvents with varying polarities. The current data, together with that from Chithambo and colleagues , therefore, suggest that the main antiplasmodial agent in these plants might be more soluble in organic solvents than polar ones such as water. Comparatively, the A. hispidum extract showed a more effective growth inhibitory effect on the two strains of P. falciparum parasites than the rest of the extracts. The high level of antiplasmodial activity of this extract against both strains of P. falciparum agrees with earlier findings [32, 33] by Sanon and his colleagues .
Extracts of C. Nucifera L, P. guajava, and C. citratus, which were reported as useful for the management of malaria [10, 18, 35, 36], did not demonstrate any appreciable antiplasmodial activity in this study. These studies were performed in vitro and it is possible that the constituents in the plants might require bioactivation in vivo to exhibit antiplasmodial activity .
Assessment of the extracts by the comet assay to evaluate their genotoxic effects suggested that the plants act via damaging parasite DNA. This is akin to the mechanism by which chloroquine inhibits parasite growth , although the selective indices of these plants are not as high as values reported earlier for chloroquine. This is not surprising because whereas chloroquine is a pure substance, the plant materials used are total extracts, which included phytoconstituents that may not have antiplasmodial activity. The genotoxic effects observed are in line with an earlier report that A. hispidum is toxic in both in vitro and in vivo , which extends to its antimalarial or antimicrobial activity as per these and other findings .
It is not clear which phytoconstituents are responsible for the antiplasmodial activities observed in these studies. However, phytochemical studies have shown that alkaloids, saponins tannins, flavonoids, steroids and glycosides, terpenes, anthraquinones, and glycoside abound in the plants under study [39, 40] which have been suggested to possess varying degrees of antimalarial activities depending upon extraction media and parasite strains/isolates used [39–41]. It is, therefore, reasonable to postulate that these phytoconstituents, either singly or in combinations may be responsible for the observed antiplasmodial activities recorded.
In conclusion, the Alstonia boonei, Senna siamea Lam, and Acanthospermum hispidum extract showed good antiplasmodial activity which warrants further investigations to ascertain their usefulness as good antimalarial agents. It will, therefore, be useful to carry out activity-guided fractionation studies to identity the antiplasmodial active phytoconstituents in the extracts for further development.
The data used to support the findings of this study are included within the article.
Conflicts of Interest
None of the authors has any conflicts of interest to declare.
AKN, DE, and MOF conceived and designed the experiments. DE, PKE, RHA, and SA performed the experiments. DE, MFO, RHA, and SA analyzed the data. AKN and MFO contributed reagents/materials/analysis tools. DE and SA wrote the paper. AKN, DAE, DE, MFO, PKE, RHA, and SA participated in the discussion of the result. Selorme Adukpo and Doris Elewosi contributed equally.
The authors are grateful to the Technical staff of the Immunology Department and the Department of Animal Biology and Conservation Science, University of Ghana, for their enormous technical support. The study was jointly supported through a Post-Doctoral grant awarded to Dr Michael F. Ofori by Danida through the Danida-RUF Research grant and University of Ghana.
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