Antiplasmodial Potential of selected Medicinal Plants Extracts on mice Infected with Plasmodium Berghei

Introduction

Malaria remains one of the most significant parasitic diseases affecting humans and animals in tropical and subtropical regions of the world, where transmission is sustained by suitable climatic and ecological conditions [18]. The disease is caused by protozoan parasites belonging to the genus Plasmodium, which are transmitted to humans and other vertebrate hosts through the bite of infected female Anopheles mosquitoes. Malaria continues to be a major public health concern, particularly in sub-Saharan Africa, where climatic conditions favor the breeding of mosquito vectors and the transmission of the parasite [19]. According to the World Health Organization, malaria accounted for over 240 million cases and more than 600,000 deaths globally in recent years, with the majority of these cases occurring in African countries such as Nigeria [17].  Children under five years of age and pregnant women are the most vulnerable populations affected by this disease.

The malaria parasite undergoes a complex life cycle involving both the mosquito vector and the vertebrate host. When an infected mosquito bites a host, sporozoites are introduced into the bloodstream and subsequently migrate to the liver, where they multiply and develop into merozoites [16]. These merozoites are then released into the bloodstream and invade red blood cells, leading to clinical manifestations such as fever, chills, anemia, headache, and general weakness [5]. If not treated promptly and effectively, malaria can lead to severe complications and death.

Over the years, several synthetic and plant-derived antimalarial drugs have been developed for the treatment and control of malaria [2]. Historically, natural products have played an important role in antimalarial drug discovery. For example, Quinine, one of the earliest effective treatments for malaria, was isolated from the bark of Cinchona species. Similarly, Artemisinin, a potent antimalarial compound, was isolated from Artemisia annua, a medicinal plant traditionally used in Chinese medicine [14]: [7]. Despite these advances, the emergence of resistance of malaria parasites to commonly used drugs has become a serious global health challenge. The increasing resistance to conventional antimalarial drugs has stimulated the search for new and effective therapeutic agents, particularly from medicinal plants.

In many African countries, including Nigeria, traditional medicine plays a vital role in primary healthcare delivery [15]. A large proportion of the population relies on herbal remedies derived from indigenous plants for the treatment of malaria and other infectious diseases [1]. Ethnobotanical knowledge passed down through generations has identified several plants that are believed to possess antimalarial properties [10]. Scientific evaluation of such plants is therefore essential in order to validate their therapeutic potential and identify bioactive compounds that could lead to the development of new antimalarial drugs.

Among the plants commonly used in Yoruba traditional medicine for the treatment of malaria fever and malaria-related symptoms are Rauvolfia vomitoria commonly as African snake root  (Asofeyeje), Gongronema latifolium commonly known as bush buck (Arokeke), Senna alata  commonly known as candle bush (Asuwon), Newbouldia laevis commonly known as tree of life (Akoko), Chromolaena odorata commonly known as christmas bush (Akintola) and Spondias mombin commonly known as hog plum (Iyeye). These plants are widely distributed across tropical Africa and are known to contain various phytochemicals such as alkaloids, flavonoids, tannins, saponins, and terpenoids, which have been associated with antimicrobial, anti-inflammatory, antioxidant, and antiplasmodial activities [12] : [9]. For instance, Rauvolfia vomitoria contains indole alkaloids that exhibit significant pharmacological activities, while Chromolaena odorata and Senna alata have been reported to possess antimicrobial and anti-inflammatory properties. Similarly, Gongronema latifolium and Spondias mombin are traditionally used for the treatment of fever, infections, and gastrointestinal disorders, which are common symptoms associated with malaria.

Therefore, this study was designed to investigate the antiplasmodial potential of selected medicinal plants traditionally used in Yoruba ethnomedicine. Extracts obtained from (Rauvolfia vomitoria), (Gongronema latifolium), (Senna alata), (Newbouldia laevis), (Chromolaena odorata) and (Spondias mombin) were administered to malaria-infected mice in order to evaluate their effectiveness in reducing parasitemia and improving the health status of the animals. The findings from this experiment may contribute to the scientific validation of these traditional medicinal plants and provide useful information for the development of novel plant-based antimalarial therapies.

Materials And Methods

Collection of Plant samples

The fresh barks of Rauvolfia vomitoria (Egbesi), Gongronema latifolium (Ahun), Senna alata (Asofeyeje), Newbouldia laevis (Ogano), Chromolaena odorata (Awopa), and Spondias mombin (Iyeye) were purchased at Oba market in Ado- Ekiti and kept in a sterile containers until used.

Preparation of Herbal Extract

The bark of the plants was air-dried separately at room temperature (28 – 30^oC) and reduced to coarse powder by grinding. 200g of the powdered bark was soaked in 4L of distilled water for 48hrs with intermittent agitation, after which it was filtered using muslin cloth. The filtrate was concentrated using rotary evaporator followed by drying in vacuum oven at 45⁰C. The dry thick slurry was transferred to dry sample bottles and stored at -5^oC until used.

Experimental animals

20g – 31g of Swiss albino mice were used in the study. The mice were kept in cages at room temperature, fed with standard mouse cubes from Ladokun feed Nigeria limited with clean drinking water. The animals were divided into nine groups consisting of five mice per group. 

Source of Parasite (Plasmodium berghei)

The malaria parasite (Plasmodium berghei NK65) utilized in this study was procured from the university of Ibadan teaching hospital, Oyo state, Nigeria. A sterile insulin syringe was used to collect blood from the tail vein of the mouse infected with Plasmodium berghei (NK65 strain). Sufficient volume of blood containing the parasite was collected poured into a sterile tube containing anti-coagulant and normal saline. The uninfected mice were gently restrained; the collected blood was injected intraperitonially into the uninfected mice using a sterile syringe and needle. It was done carefully to prevent injury of the mice. All mice were observed for signs of infection and health status.

Plant extract administration

The dosage calculation and preparation of the stock solution of the plant extract for experimental animals was conducted. Each herbal extract was delivered orally to the mice using an oral cannula, with extract at dosage of 40mg/kg for 10 days. The study utilized 9 groups consisting of 5 mice in each group. Group 1 received herbal combination of Rauvolfia vomitoria and Gongronema latifolium, 2 received herbal combination of Senna alata and Spondias mombin, 3 received herbal combination of Chromolaena odorata and Newbouldia laevis. Group 4 received only Spondias mombin, Group 5 received Chromolaena odorata, Group 6 received Newbouldia laevis, Group 7 received Senna alata, Group 8 received Gomgronema latifolium and Group 9 received Rauvolfia vomitoria.

Determination of packed cell volume (PCV)

The infected mice were gently restrained using a handling technique that keeps them calm. The PCV of the mice in each group was determined according the [3]. The tail area of the mice was cleared with an alcohol swab to disinfect the skin and minimize the risk of infection. A sterile needle was used to make a small puncture in the tail tip (approximately 2mm deep). A drop of blood was allowed to form at the punctured site. The base of the tail was massaged gently to encourage blood flow. The capillary tube was placed quickly into the drops of blood to fill it. It was filled to an appropriate level (usually two- third full) of the capillary tube. The end of the capillary tube was sealed using plasticine to prevent leakage. The filled capillary tube was centrifuged for 15 minutes to determine the packed cell volume of the infected mice. The centrifuged capillary tube was read using a haematocrit reader.

Determination of parasitemia

 Parasitemia level was determined according to the method of [6]. The tail area of the mice was cleaned with an alcohol swab to disinfect the skin and minimize the risk of infection. A sterile needle was used to puncture the tail and a drop of blood was placed on a clean microscope slide. Another slide was used to spread the blood drop into a thin film. The smear was allowed to air dry completely. The dried blood smear was fixed in methanol for five minutes. The slide was stained with 20% Giemsa stain. The slide was then rinsed gently with distilled water and was allowed to dry. The stained smear was examined under a light microscope using a low magnification (x 100) to locate the parasites. The number of parasitized red blood cells (RBCs) were counted.  The percentage parasitemia was calculated and the results were recorded.

Percentage Parasitemia =  × 100

Parasite suppression was also calculated by using the formula below:

% Parasite Suppression =  × 100

Statistical Analysis

The results obtained from the study were analyzed by using descriptive statistical methods and one way analysis of variance (ANOVA) SPSS version 22 at 95% confidence level.

Results and Discussion

The average PCV of mice in each group is shown in Table 1, the treatment groups differed in their ability to preserve red blood cells. Group 7 recorded the highest PCV (40.5%), indicating that Senna alata extract was the most effective in preventing malaria-induced destruction of erythrocytes or promoting recovery from anemia. Group 8 which received herbal extract of Gomgronema latifolium have PCV of (38.2%) while group 2 which received herbal combination of Senna alata and Spondias mombin  and group 4 which was treated with Spondias mombin extract had 37.5% and 37.3% respectively, these showed relatively high PCV values, suggesting good antimalarial and hemato-protective activity. In contrast, Group 9 which was treated with Rauvolfia vomitoria had the lowest PCV (34.0%), indicating the greatest degree of anemia and the least protective effect. The lower PCV in Groups 3 (35.2%) and 5 (35.6%) also suggests that these treatments were less effective in preventing the hematological damage caused by P. berghei infection. P. berghei infects and destroys red blood cells, leading to a reduction in PCV. Therefore, treatments that maintain or restore PCV are considered protective against malaria-induced anemia. The maintenance of higher PCV values in treated groups suggests that some of the plant extracts possess antimalarial properties capable of reducing the severity of malaria-induced anemia. The higher PCV observed in Groups 7 and 8 indicates stronger hematological protection, while the reduced PCV in Group 9 reflects more severe malaria-associated anemia. These results support the potential use of Senna alata, Gomgronema latifolium and Spondias mombin extracts as antimalarial agents with protective effects against red blood cell loss. This result is in line with Atanu et al. (2022) where the authors observed that Senna alata extract improved the packed cell volume (PCV) level of mice infected with P. berghei.

 Table 2 shows the average parasitaemia of the different experimental groups. Parasitaemia represents the percentage of red blood cells infected with P. berghei, a lower value generally indicates a lower parasite burden. Group 9 recorded the highest average parasitaemia (38.07%), indicating the highest parasite burden. Group 8 followed with 27.51%, while Group 6 had 24.01% and Group 4 had 23.65%. Groups 1, 2, 3 and 5 had relatively similar values of 21.01%, 21.16%, 20.60%, and 20.18%, respectively. Group 7 recorded the lowest parasitaemia (10.65%), suggesting the greatest reduction in parasite burden among the groups. The results show considerable variation in parasitaemia among the groups. Group 7 appears to have the most effective treatment, assuming the groups represent different treatments, because it had the lowest average parasitaemia. In contrast, Group 9 had the poorest outcome, with the highest parasitaemia. This suggests that the treatment (Senna alata) administered to Group 7 had a stronger antiplasmodial effect than those administered to the other groups. The result corroborates the findings of [3] where the test for curative effect showed that S. alata significantly suppressed parasitemia to 4.50 ± 1.05% compared to untreated group 29.83 ± 3.49%.

Figure 1 represents parasite suppression percentage which provides a clearer comparison of the antimalarial effectiveness of the treatments administered to each experimental group. Parasite suppression was calculated by comparing the parasitemia levels of the treated groups with that of the negative control group.

Among all the groups, Group 7 recorded the highest parasite suppression of approximately 72%, indicating strong antiplasmodial activity. According to commonly used criteria in in-vivo antimalarial screening studies, parasite suppression values above 50% are generally considered to indicate significant antimalarial activity [13]. This result is in line with the findings of [8] where the authors observed that Senna alata extract significantly suppressed parasitemia to 4.50 ± 1.05% in mice infected with Plasmodium berghei.

Groups 3 and 5 also demonstrated relatively high parasite suppression values of approximately 45–47%, indicating moderate antimalarial effects. Groups 1 and 2 showed similar levels of suppression, suggesting that the plant extracts administered to these groups possessed some degree of inhibitory effect on parasite growth.

On the other hand, Groups 4 and 6 exhibited lower suppression percentages of about 37–38%, indicating weaker antimalarial activity. Group 8 showed the lowest suppression among the treated groups, suggesting minimal parasite inhibition. As expected, Group 9 recorded 0% suppression, confirming that it functioned as the negative control group where parasite growth was not inhibited.

The line graph illustrates the progression of parasitemia in mice infected with Plasmodium berghei across the different experimental groups over the observation period. The results show a general increase in parasitemia levels in most groups as the infection progressed. Initially, on the first observation day, parasitemia levels were relatively low in most groups, indicating the early stage of infection. However, as the experiment progressed, several groups experienced a sharp increase in parasitemia, reflecting rapid multiplication of the malaria parasite within the red blood cells of the mice.

Group 9 showed the highest increase in parasitemia over time, reaching the greatest parasite density by the final observation day. In contrast, Group 7 maintained the lowest parasitemia levels during the early phase of the experiment, suggesting that the treatment administered to this group may have inhibited parasite replication more effectively than the treatments in other groups.

Groups 3 and 5 showed moderate parasitemia progression, indicating partial suppression of parasite growth. However, Groups 4 and 6 exhibited sharp increases in parasitemia by the third day, suggesting that the treatments administered to these groups were less effective in controlling parasite multiplication.

The parasite reduction trend graph illustrates how parasitemia levels changed over the course of the experiment in response to the treatments administered. Ideally, effective antimalarial treatments should result in a progressive decline in parasitemia levels over time.

In the present experiment, only a few groups demonstrated evidence of parasite suppression. Group 7 showed the most promising pattern, with relatively low parasitemia during the early stages of infection. This suggests that the treatment administered to this group may contain bioactive compounds capable of inhibiting parasite development.

However, most groups exhibited an overall increase in parasitemia with time, indicating that the treatments administered were either insufficient to completely eliminate the parasite or were only partially effective. The steady rise in parasitemia observed in Group 9 further confirms the natural progression of malaria infection in the mice. The variations observed among the groups may be attributed to differences in the phytochemical composition of the plant extracts used, dosage levels, or the physiological response of the mice to the treatments [4].

Conclusion and Recommendations

The study provides strong evidence that some of the plant extracts tested in this study possess antiplasmodial activity against Plasmodium berghei in infected mice. Among the treatments evaluated, the extract administered to Group 7 demonstrated the highest level of parasite suppression and the most promising therapeutic potential. Similarlly, extract administered to groups 1, 2, 3 and 5 also showed high level of parasite suppression. These findings support the traditional use of medicinal plants in the treatment of malaria and suggest that certain plant extracts such as Senna alata, combination of Rauvolfia vomitoria and Gongronema latifolium, combination of Senna alata and Spondias mombin, combination of Chromolaena odorata and Newbouldia laevis and Chromolaena odorata may serve as potential sources of new antimalarial compounds. Further studies involving phytochemical analysis and toxicity evaluation would be necessary to identify the active constituents responsible for the observed antimalarial effects.

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