Antiplasmodial, Antitrypanosomal, and Cytotoxic Effects of Anthonotha macrophylla, Annickia polycarpa, Tieghemella heckelii, and Antrocaryon micraster Extracts

Malaria and trypanosomiasis are protozoan diseases which pose a devastating challenge to human health and productivity especially, in Africa where their respective vectors (female Anopheles mosquito and tsetse fly) abound. Various medicinal plants are used to treat these parasitic diseases. However, the scientific basis of their use and toxicological profiles have not been assessed. We have, therefore, evaluated the antiplasmodial, antitrypanosomal, and cytotoxic activities of four African medicinal plant extracts namely, Anthonotha macrophylla leaf (AML), Annickia polycarpa leaf (APLE), Tieghemella heckelii stem bark (THBE), and Antrocaryon micraster stem bark (AMSBE) extracts in vitro against P. falciparum (W2mef laboratory strain), T. brucei (GUTat 3.1 strain), and mammalian RAW 264.7 macrophage cell line, respectively. The most active antiplasmodial extract was AML (IC50 = 5.0 ± 0.08 μg/mL with SI of 21.9). THBE also, produced the most effective antitrypanosomal activity (IC50 = 11.0 ± 0.09 μg/mL and SI of 10.2) among the extracts. In addition, none of the extracts produced toxic effect in the RAW 264.7 macrophage cell line except APLE which was moderately cytotoxic and also produced the least SI in both antitrypanosomal and antiplasmodial assays. These results suggest that AML and THBE could offer safe and alternative therapy for malaria and trypanosomiasis. This is the first study to report the antitrypanosomal and in vitro antiplasmodial activities of these four plants/plant parts. The cytotoxicity of the plant parts used is also being reported for the first time except for the T. heckelii stem bark.


Introduction
Malaria is caused by several species of protozoan parasites from the genus Plasmodium. In sub-Saharan Africa, the most pathogenic species is the Plasmodium falciparum that causes more than 90% of all malaria cases [1]. e World Malaria Report reported estimated 241 million malaria infections and 627 000 malaria deaths globally in 2020, which denotes about a 14 million increase in cases and 69 000 additional deaths, in 2020 as compared to 2019 [1]. Even though the development of resistance, by the Plasmodium parasite, to artemisinin and its combination therapies (ACTs) in sub-Saharan Africa is slower than that in places like Asia and South America, the burden of malaria in the former region remains high [1][2][3].
African trypanosomiasis, a tsetse-transmitted disease of humans and livestock caused by protozoan parasites of the genus Trypanosoma, is also of serious health and economic concern in various sub-Saharan African countries [4,5]. In the absence of vaccines, chemotherapy remains the only practical means to control African trypanosomes [6]. However, development of resistance to present drugs, side effects, and difficulty in regimen application pose serious challenges to chemotherapy [7][8][9][10].
Macrophages are mononuclear phagocytes that form critical components of the mammalian immune system. RAW cells are macrophages originally established from an ascitic tumor induced in a male BALB/c mouse by intraperitoneal injection of the Abelson leukemia virus [11]. RAW 264.7 cell lines are useful in the evaluation of the bioactivity of natural products as well as envisage their probable in vivo outcomes [12] and toxicity. us, they provide useful advantages in drug discovery research.
Folkloric medicinal plants have proven to be the most versatile source of crude and purified drugs for mankind [13]. Furthermore, it has been documented that approximately 60% of pharmaceutical drugs being used currently were directly or indirectly derived from plants and other natural origins [14]. Due to the challenges associated with current antimalarial and antitrypanosomal drugs, the need to find alternate and new medications to combat these diseases, especially, from folkloric medicinal plants has become a focus of the global scientific research.
Four antimalaria folkloric African medicinal plants have been selected for this study. Malaria and trypanosomiasis are both protozoan diseases. Hence, the extracts were tested against both parasites. e selected medicinal plants are discussed below.
Although the in vivo antimalaria activity of A. micraster stem bark, A. polycarpa stem bark, and leaf in addition to the in vitro antimalaria activity of the A. macrophylla stem bark have been reported, the antitrypanosomal activity of the four plants and/or plant parts is not known. In addition, the in vitro antiplasmodial activity and the selectivity index (SI) of the selected plant parts have also not been reported. is study, therefore, sought to evaluate the in vitro antiplasmodial, antitrypanosomal, and cytotoxic activities of the A. macrophylla leaf (AML), A. micraster stem bark (AMSBE), A. polycarpa leaf (APLE), and T. heckelii stem bark (THBE).

Identification, Collection, Processing, and Extraction of Plant Materials.
e plants were identified and collected by Mr. Jonathan Dabo, a botanist at the Forestry Research Institute of Ghana (FORIG), Kumasi. e A. macrophylla leaf, A. polycarpa leaf, and T. heckelii stem bark were collected from the Bobiri Forest Reserve in the Ashanti Region of Ghana. e A. micraster stem bark was collected from the Asubima Forest Reserve in the Ahafo Region of Ghana. A. polycarpa leaf, T. heckelii, and A. micraster stem barks were assigned voucher specimen numbers (FORIG 0012, FORIG 0013, and FORIG 0014, respectively), and deposited at the herbarium of FORIG. A. macrophylla leaf was given a voucher specimen number of CPMR 4937 at the herbarium of the Centre for Plant Medicine Research, Mampong-Akwapim, Ghana. e plant materials were sun dried for 8 days and pulverized into a coarse powder. Extraction was performed using the protocol of Kumatia et al. [20]. Briefly, the powdered stem bark of A. micraster (200 g) was extracted with 70% ethanol (2 L × 2) at room temperature for four days each filtered and combined. Ethanol in the extract was removed by concentrating the filtrate at low pressure in a rotary evaporator (Eyeler N1110, Tokyo-Japan). e aqueous part was lyophilized to obtain a powder-coded AMSBE. A similar procedure was applied to the other plant materials to obtain solid extracts coded APLE, THBE, and AML for A. polycarpa leaf, T. heckelii stem barks, and A. macrophylla leaf, respectively. e extracts were stored in air-proof containers at 4°C until required.

Phytochemical Screening of the
Extracts. An aliquot of each extract (50 mL) was analyzed for the presence or absence of phytochemical compounds such as flavonoids, alkaloids, triterpenes, phenolic compounds, saponins, polyuronoids, antracenosides, reducing sugars, phytosterols, and cyanogenic glycosides as per the methods described by Fong et al. [38].

Culturing of Plasmodium falciparum.
W2mef laboratory strain of P. falciparum was cultured with human group O + erythrocytes using standard methods [39] with slight modifications. P. falciparum parasites were cultured to >5% parasitemia of ring-stage parasites. Using 5% sorbitol treatment, a synchronized culture of ring-stage parasites [40] was obtained and diluted to 1% parasitemia and 2% hematocrit for the growth inhibition assays.

Evaluation of Antiplasmodial
Activity. Extracts were tested against P. falciparum activity in the SYBR Green I fluorescence assay [41]. Extracts at concentrations of 10 mg/ mL were diluted with culture media to a starting working concentration of 100 µg/mL. Serial dilutions (1 : 2) were made to yield 7 final concentrations of each of the extracts (100-0.78 µg/mL). An aliquot of 10 µL of each concentration was dispensed into test wells of the 96-well plate in triplicates with each tested well already containing 90 µL of 2% hematocrit and 1% parasitemia. Wells containing RBCs at 2% hematocrit and 1% parasitemia were used. e final volume per well was 100 µL. Plates were incubated for 48 h, and an aliquot of 100 µL of 4x buffered SYBR Green I (0.25 µL of 10000x SYBR Green I/mL of 1x phosphate buffer saline) was added to each well after the incubation period and incubated again in the dark for 30 min at 37°C. e presence and the amount of infected red blood cells (RBCs) were detected using the BD FACS LSRFortessa ™ X-20 flow cytometer and analyzed with BD FACSDiva Software (v8.0.1). A total of 5,000 RBCs were counted to determine the number of infected RBCs (SYBR Green I positive cells) present. Chloroquine (CHL) was used as the reference antiplasmodial drug.

Evaluation of Antitrypanosomal Activity.
e assay was performed as described previously [42]. Cells were seeded at a density of 3.0 × 10 5 cells/ml in 96-well plates in a two-fold dilution of extracts. Extracts were incubated in a two-fold dilution with the cells for another 24 h. Alamar blue dye (resazurin, 10% v/v) was then added to all wells and incubated for another 24 h. Spectrophotometric absorbance was recorded at a wavelength of 570 nm. Diminazene aceturate (DA) was used as a positive antitrypanosomal control drug.

Culturing of Mouse Macrophages (RAW 264.7) Cell Line.
Mouse macrophages (RAW 264.7 cell lines) were cultivated in vitro to the logarithm phase using Dulbecco's modified Eagle media (DMEM, ermo Fisher Scientific) with 10% foetal bovine serum at 5% CO 2 and 37°C.

Evaluation of Cytotoxity of the Extracts against Mouse
Macrophages (RAW 264.7) Cell Line. Cell lines were plated at a density of 3.0 × 10 5 cells/mL for 48 h to allow for sufficient adherence to plates. Extracts were incubated in a two-fold dilution with the cells for another 24 h. Alamar blue dye (resazurin, 10% v/v) was then added to all wells and incubated for another 24 h to allow for a complete color development. Spectrophotometric absorbance was recorded at a wavelength of 570 nm.

Selectivity Index (SI).
e SI was calculated for each extract in each test to determine their effectiveness and toxicity in their use as an antiplasmodial or antitrypanosomal agent using the following formula: SI � IC50 obtained for the extract in the RAW macrophage cell IC50 obtained for the extract against the protozoan parasite . (1)

Statistical Analysis.
Algorithms obtained from flow cytometry (FACS) were analyzed using regression equations of best fit of plotted growth inhibition versus concentration curves. GraphPad Prism for Windows version 7.0 (Graph Pad Software, San Diego, CA, USA) was used for all statistical analyzes. Cell viability of parasites was estimated from the fluorescence of SYBR Green I or absorbance of resazurin and represented as a percentage of treated to untreated cells. IC 50 values were calculated from a nonlinear regression model. Data were presented as the mean ± S.E.M.

Yield and Nature of Crude
Extracts. e weight, yield, and nature of the extracts obtained are described in Table 1.
e result shows that the stem barks produced higher yields of extracts than the leaves. e stem bark extracts yielded more extracts than the leaves under the same conditions. is was due to the fact that most trees have very thick and malleable stem barks. THBE produced the highest yield of the extract (21.88% w/ w). is was followed closely by AMSBE. AML produced the least yield of the extract (4.72% w/w). is indicates that less plant material is needed to produce more extracts for the plant parts that gave high yield and vice versa.

Classes of Phytochemical Constituents in the Extracts.
e results of the phytochemical analysis of the extracts are tabulated in Table 2. e results show that all the extracts contained saponins, phenolics, and reducing sugars. Phytosterols were found in all the extracts except AMSBE. AML and THBE also contained flavonoids in addition to the other four constituents. Alkaloids were also present in APLE, which contained a greater number of phytochemical constituents among the extracts. e yield, nature, and phytoconstituents obtained for the extracts were similar to those reported elsewhere [20,26,31] because it was the same bath of the plant material used in both studies.
e results of the in vitro antiplasmodial, antitrypanosomal, and cytotoxic activities of the extracts are shown in Table 3. e extracts produced antiplasmodial activity with IC 50s of 5.0 ± 0.08-110.1 ± 0.12 μg/mL and that of CHL was 0.146 ± 0.05 μg/mL. AML was the most active extract. e extracts can be arranged in increasing order of antiplasmodial activity as follows: APLE < AMSBE < THBE < AML. According to Zirihi et al. [15], the in vitro antiplasmodial activity of the plant extracts is classified as good, weak, or inactive using their IC 50 values. e IC 50 value of greater than 50 μg/mL, 15-50 μg/mL, or less than 15 μg/mL indicates inactive, weak, or good antiplasmodial activity, respectively. us, AML (IC 50 � 5.0 ± 0.08 μg/mL) produced good antiplasmodial activity. THBE and AMSBE (IC 50 � 28.8 ± 0.16 and 24.5 ± 0.21 μg/mL, respectively) ( Table 3), produced weak in vitro antiplasmodial activity. However, APLE (IC 50 � 110.1 ± 0.12 μg/mL) was inactive. AMSBE and APLE were shown to demonstrate significant antimalaria activity in mice in vivo [20,26]. However, these extracts were inactive against P. falciparum in vitro. is suggests that the extracts act as prodrugs in vivo where their metabolites might be responsible for their observed antimalaria action.
e most important parameter in the cytotoxic test is the IC 50 value that is defined as the concentration of a test substance, which can inhibit the viability of 50% of a cell under specific test conditions. IC 50 values are used to measure the toxicity or safety of chemical agents in cytotoxic studies using cell lines. e smaller the IC 50 value of a substance, the more toxic is the substance. Conversely, the higher the IC 50 value of a test substance, the safer/less toxic is that substance [1]. In this study, AMSBE, AML, and THBE gave IC 50 values of 98.5 ± 0.24-112.0 ± 0.32 μg/mL which are relatively high. Hence, they are nontoxic (Table 3). APLE and the reference drug DA gave somewhat low IC 50 values of 76.5 ± 0.22 and 74.1 ± 0.31 μg/mL, respectively (Table 3).
is makes them relatively toxic.

Selectivity Index (SI) of the Extracts.
e activity and toxicity profiles in the parasites and macrophages resulted in the list of selectivity profiles are shown in Table 4. e SI is the ratio of the IC 50 value in mice macrophage RAW 264.7 to the IC 50 value in the respective parasite (P. falciparum or T. brucei).
e higher SI of a substance   suggests the likelihood that the substance could produce innocuous treatment. And the smaller the SI of a substance, the more likely it is for that substance to produce ineffective and harmful treatment. e results obtained from this study, therefore, indicates that AML (antiplasmodial SI = 21.9) and THBE (antitrypanosomal SI = 21.9) recorded the highest SI against P. falciparum and T. brucei, respectively, among the extracts as the most promising in vitro antiplasmodial and antitrypanosomal agents, respectively. APLE emerged as the least promising antitrypanosomal and antiplasmodial agent since it has the least SI of 1.4 and 0.7, respectively, in both tests. It was suggested that a substance with an SI value <2 might produce wide-ranging toxicity [43]. erefore, the extracts are safe in vitro except for APLE that could be classified as generally toxic in vitro. e phytochemical analysis results showed that only AML and THBE have flavonoids present in them, whereas three to four other constituents were common to all the extracts. AML and THBE are the most active extracts in this study. is indicates that flavonoids may be responsible for the antiplasmodial and antitrypanosomal activity demonstrated by the two extracts in this study. Flavonoids such as apigenin, genkwanin, scutellarein, and diosmetin were reported to produce antitrypanosomal activity against T. brucei in vitro with IC 50 of 5.1, 8.0, 4.6, and 6.1 μg/mL, respectively [44].

Conclusion
e results from this study indicate that AML possessed good antiplasmodial activity, weak antitrypanosomal activity, andhighest SI among the extracts in the antiplasmodial test. THBE, also, produced good antitrypanosomal activity with the highest SI of 10.2 among the extracts. None of the extracts showedcytotoxic effect in the RAW 264.7 macrophage cell line except for APLE which was moderately cytotoxic and also produced the least SI in both antitrypanosomal and antiplasmodial tests. ese results suggest that AML and THBE could offer safe and alternative therapy for malaria and trypanosomiasis. is is the first study to report the antitrypanosomal and in vitro antiplasmodial activities of these four plants.
e activity of AML and THBE may be due to their chemical constituents such as flavonoids, which were absent in the other extracts. e AML and THBE could also be fractionated to isolate their constituents for preclinical, antiplasmodial, and antitrypanosomal studies.
Data Availability e data can be obtained from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that they have no conflicts of interest. Advances in Pharmacological and Pharmaceutical Sciences 5