Bioassay-Guided Fractionation, ESI-MS Scan, Phytochemical Screening, and Antiplasmodial Activity of Afzelia africana

Background Afzelia africana is a plant species with reported numerous medicinal potentials and secondary metabolites. Various parts of the plant have been applied for the treatment of hernia, rheumatism, pain, lumbago, malaria, etc. The study seeks to evaluate the phytochemical constituents, antiplasmodial, and ESI-MS scan of bioassay-guided fractions from the methanol extract of the bark of the plant. Aims The main aim of the study was to carry out bioassay-guided fractionation of the crude methanol extract of Afzelia africana in order to isolate fractions and to evaluate their antiplasmodial activities and ESI-MS fingerprints. Methods The methods employed include column chromatographic fractionation, phytochemical screening, antiplasmodial activity (malaria SYBER green assay (MSF)), and ESI-MS profile (full ESI-MS scan). Results The column chromatographic fractionation and phytochemical screening of the plant led to the separation of the following four fractions: 1 (flavonoids, phenolics, glycosides, terpenoids, and steroids), 2 (alkaloids, anthraquinones, flavonoids, phenolics, glycosides, terpenoids, and steroids), 3 (anthraquinones, flavonoids, phenolics, glycosides, terpenoids, and steroids), and 4 (alkaloids, flavonoids, phenolics, glycosides, terpenoids, and steroids). The antiplasmodial activities of the fractions were tested against the 3D7 strain of Plasmodium falciparum with reported stronger activities for 1 (IC50: 0.097 ± 0.034 μg/mL) and 3 (IC50: 1.43 ± 0.072 μg/mL), and weaker activities for 2 (IC50: >100 μg/mL) and 4 (IC50: 37.09 ± 6.14 μg/mL). The full ESI-MS fingerprint of fractions 1, 2, 3, and 4 revealed the presence of 14, 24, 34, and 37 major molecular ions or compounds in each fraction, respectively.


Introduction
Malaria is a parasitic disease transmitted by the female anopheles mosquito through the injection of the Plasmodium falciparum into the bloodstream of the host organism [1][2][3]. Statistical data from the WHO indicated a total of 229 and 228 million cases for 2018 and 2019, respectively. e number of deaths recorded within the duration stood at 411,000 and 409,000, respectively [4,5]. e infant-reported death cases accounted for about 67% (274,000) of the 2019 deaths. In view of the higher infant mortality, the WHO further recommended the use of the RTS, SAS01 malaria vaccine for children [4,5]. Previous intervention in the treatment of malaria involved single and combination therapy with known antiplasmodial compounds such as the derivatives of aminoquinoline and artesunate. However, antimalarial drug resistance has emerged as one of the challenges facing malaria treatment [4][5][6]. e biggest intervention in the chemical treatment regime for malaria has derived its source from herbal medicines [7]. e discovery of quinine and artesunate from natural sources rekindled giant steps into a rigorous search for secondary metabolites from plant sources. e utilization and derivation of treatment regimes from herbal medicines have gone through a two-way approach, that is, a rigorous approach through bioassay-guided fractionation to identify pure compounds and the evaluation of its antiplasmodial activity [8,9]. e second approach has gone through the formulation of herbal medicines directly from extracts from the plants after the evaluation of its biological activity. Furthermore, standard parameters are applied to standardize it for human patronage [10][11][12].
In the area of antimalarial drug discovery, where the contribution of ethnopharmacology has been well documented includes the discovery of quinine from the quincona plant, artemisinin from Artemisinin annua, and the isolation of cryptolepis from the West African plant, Cryptolepis sanguinolenta [13]. Over the last 20 years, 175 plant-derived antiplasmodial compounds have been published with several of them showing a nanomolar (nM) range of activity [14]. It is therefore evident that natural products from plants are endowed with thousands of secondary metabolites; however, the exact composition and the interactive effects of these secondary metabolites could be optimized to obtain them in different compositions [15]. Bioassay-guided isolation of bioactive compounds is a modern and efficient technique in metabolites screening.
rough bioassay-guided fractionation studies, secondary metabolites have been obtained in fractions containing compounds existing in different compositions in several mixtures. Such processes have yielded fractions that have generated biological activities not originally present in the crude extracts [16]. Currently, advanced analytical tools and techniques have helped to further fractionate, isolate, and purify compounds from plants, and their biological activities are evaluated. Analytical techniques that have been employed include chromatographic techniques (LC, HPLC, and TLC) and spectra techniques (mass spectrometry, infrared, ultraviolet/visible, nuclear magnetic resonance, X-ray) [17,18].
Afzelia africana is a plant species belonging to the Fabaceae family. e plant is commonly referred to as African mahogany and thrives well in the tropical regions of Africa. Various parts of the plant have been applied in traditional medicines such as analgesic, emetic, laxative, febrifuge, hemorrhagic, malaria, rheumatism, diuretic, paralysis, and lumbago. Furthermore, the pulverized mixture of the roots with millet beer is used to treat hernias and, whilst a decoction with pimento, is used as a remedy against gonorrhea and stomach ache. A leaf decoction, combined with Syzygium guineensis leaves and Xylopia fruit, forms a drink that is used to treat oedema [19][20][21][22]. e various reported ethnobotanical applications of the plant that have been authenticated by laboratory investigations include anthelmintic [23], antioxidant [24], anti-inflammatory/analgesic [25], and antitrypanocidal [26,27]. Furthermore, the ethnobotanical utilization of the plant as antiplasmodial agent has been confirmed against the 3D7 strain of Plasmodium falciparum (IC 50 : 2.97 μg/mL) [28]. e antimicrobial [29,30], antidiabetic, liver, and kidney protective ability (against alanine transaminase (ALT) and aspartate aminotransferase (AST), bilirubin, urea, and creatinine) are further reported with significant outcomes [31,32]. In view of the vast medicinal properties reported for the plant, further studies seek to carry out an extensive bioassayguided fraction of the methanol extract and the evaluation of its phytochemical and antiplasmodial activities. In this study, the ESI scan of the various fractions will be investigated to give a fair insight into the phytochemical constituents, number of compounds, or molecular ions present in them.

Extraction of Plant Material.
A 500 g of the bark was dried under shade for three (3) weeks to reduce the moisture content. e dried plant material was further pulverized and then cold macerated in 90% methanol. e mixture was then stored for 72 hours with occasional swirling and shaking. e extract was then filtered by a vacuum pump to obtain the filtrate. e filtration procedure was repeated two more times with fresh 90% methanol solution until enough filtrate was obtained. e filtrate was further concentrated using the rotavap and then dried to obtain a solid residue. e percentage yield of the successive extractions was 16% (80) g. e solid crude extract was stored at 2°C until further use.

Column Chromatography and Bioassay-Guided
Fractionation. Sixty grams (60 g) of the methanol extract was mixed with 100 g of silica gel 60 (0.05-0.2 mm (230-400 mesh)) in 100 mL methanol and then dried under reduced pressure. e adsorbed extract was then mounted on a column (open, 45 × 4.5 cm) containing 250 g silica gel (230-400 mesh size). e mounted column was eluted with the mobile phase solvent system comprising of petroleum ether, petroleum ether-ethyl acetate, and ethyl acetate through gradient elution. e procedure yielded 40 fractions based on TLC profiles using a solvent system of pet ether/ ethyl acetate (1 : 1). Spots were visualized by using saturated iodine vapour and anisaldehyde solution. e fractions were further pooled into four fractions based on thin-layer chromatography (TLC) profile to give 1 (200 mg, pale yellow powder), 2 (brown precipitate, 342 mg), 3 (300 mg, yellow powder), and 4 (500 mg, brown precipitate).

Phytochemical Screening of Fractions.
e phytochemical investigations of the fractions were achieved by adopting the procedure by Amir et al. [33] as described in the experimental procedures. e phytochemical classes that were tested for the fractions include alkaloids, flavonoids, saponins, coumarins, anthraquinones, steroids, and glycosides.

Antiplasmodial Screening of Fractions.
Antimalarial activities of the fractions were investigated by adopting the malaria SYBER green fluorescence (MSF) assay method by Kwansa-Bentum et al. [34] with slight modification. A highly synchronous ring-stage parasite was used in the assay.

In-Vitro Cultivation of Malaria
Parasite. Blood stages of the laboratory clones of chloroquine-sensitive 3D7 strain of P. falciparum were cultured in-vitro according to the method of Trager and Jensens. All culture steps were performed using the aseptic technique in the NuAire laminar flow class II safety cabinet. All glassware was autoclaved at 121°C (15 atmospheres) for at least 15 min. Malaria parasite was maintained in continuous culture with human packed red blood cells (blood group O + ) in RPMI 1640 medium supplemented with 10% human AB + serum, 25 mM N-2hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 25 mM sodium bicarbonate, and gentamicin sulfate (60 μg/ ml, pH 7.2). e culture was incubated at 37°C in an atmosphere consisting of 90% N 2 , 5% O 2 , and 5%. Parasite culture was synchronized to the ring stage by treatment with 5% (w/v) D-sorbitol.

Assessment of In Vitro Antimalarial Activity of
Fractions. An aliquot of parasite inoculum (50 μL) with 0.5% parasitaemia and 2% haematocrit was added into each well of the microtiter plate. e fraction dissolved in DMSO and diluted with RPMI 1640 to a final concentration of 1% was added to the malaria culture at six final concentrations of 100, 25, 6.25, 1.56, 0.39, and 0.098 μg/mL with artesunate (ART) as a standard antimalarial drug. Wells without the fractions but the culture at the same parasitaemia and haematocrit in distilled water were included on the 96-well plate as control. Using the candle jar method, the plate was put into a humidified chamber in an incubator at 37°C for 72 hours. e antiplasmodial activities of the fractions were deduced by the malaria SYBR green fluorescence (MSF) assay. After 72 hours of incubation, 100 μL of the SYBR green 1 fluorescence lysis buffer containing 20 mM Tris-Cl (pH 7.5), 5 mM EDTA, 0.008% saponin, and 0.08% triton-X 100 and SBR green were added to each prepared well. e plate was further covered with aluminum foil and then incubated in the dark at room temperature for 3 hours. e fluorescence generated by each well was read on the multiwell plate reader (Tecan Infinite M200). e experiment was repeated two more times (triplicate each). e evaluation of the percentage inhibition was done by inputting the parasitaemia of control and the parasitaemia of serially diluted concentrations of test samples into the relation.
e % inhibition versus the logarithm of the concentrations [LogC] of each fraction was plotted. e IC 50 (μg/ mL) of the fractions were further determined by employing the GraphPad Prism software package 6 (version 6.01) (San Diego, California USA) with non-regression (curve fit) using the log(inhibitor) vs response (three parameters).

Full Scan ESI-Ms Fingerprint of Fractions.
e ESI-MS fingerprint of the fractions was carried out using the Quantum Access MAX mass spectrometer equipped with an ESI source. Both the negative and the positive ion mode were applied with a source voltage of 100 V, the capillary temperature of 250°C, the nebulizing gas flow rate of 1.5 mL/ min, and collision energy of 100 V. e spectra data were obtained with full-scan mode for m/z in the range of 100 to 700. e solvent system for elution comprises of an isocratic volume of methanol/water, 50 : 50.

Statistical Analysis of Data.
e dose-response curves from the experimental data were analyzed by using the Graphpad Prism software package 6 for Windows (San Diego, California USA). e data were further analyzed by the one-way ANOVA using GraphPad Prism 6 software with a significant difference at P < 0.01. e data from the experiment were reported as mean and SD values from triplicate measurements.

Results and Discussion
e principle of bioassay-guided fractionation has played a significant role in understanding the phytochemistry, pharmacognosy, and pharmacology of herbal medicines [15]. e principle has further led to the linkage of various fractions from plants to their biological activity and specific compounds directly linked to their biological activities [35]. In the current study, the principle of column chromatographic fractionation was employed to obtain fractions that were bulked together based on a similar retention factor (R f ). e crude methanol extract of the A. africana was obtained by employing the cold maceration technique with an overall yield of 16%. e crude extract was subsequently mounted on the column and eluted with a solvent system comprising petroleum ether and ethyl acetate. e procedure led to the isolation of four fractions. Fraction 1 (comprising three spots on TLC (R f : 0.18, 0.36, 0.55)) was obtained as a yellow isolate with a solvent system of pet ether/ethyl acetate (85 : 15). Fractions 2, 3, and 4 were obtained with solvent systems of pet ether/ethyl acetate (75 : 25), pet ether/ethyl acetate (70 : 30), and pet ether/ethyl acetate (70 : 30), respectively. e fractions were bulked together based on the TLC profile (number of spots and their retentions times) and represented in Table 1. e biological activity and, specifically, the antiplasmodial activity of plants have been linked to the presence of secondary metabolites and their synergistic effects [36,37]. e existence of two or more secondary metabolites in their right compositions and permutations could be harnessed and optimized to yield resultant beneficial outcomes, notwithstanding, their associated toxic and adverse effects [36]. Phytochemical screening of secondary metabolites from plants is the first step in understanding the phytochemistry, pharmacognosy, and pharmacology of extracts from plants [15].
e phytochemical screening provides a rich source of information about the composition and structural varieties of compounds in extracts from plants [39]. Nevertheless, the test could further provide clues into biological activities, toxicities, and structures of active compounds for authenticating the ethnobotanical utilization of plant medicines [15]. As a result, well-standardized methods for screening alkaloids, terpenoids, steroids, flavonoids, phenolics, tannins, coumarins, anthraquinones are well documented [36]. e application of a standardized procedure for the screening of the phytochemical constituent of the plant is indicated in Table 2.
e presence of these secondary metabolites could account for the numerous medicinal properties reported for Afzelia africana. e phytochemical screening experiment indicating the presence of the phytochemicals in Afzelia africana further confirms similar results determined by other studies [31,40,41]. Further experimental procedures to justify the presence of the wide varieties of secondary metabolites in the four fractions were achieved by performing a full ESI-MS scan of the fractions. e single-stage full ESI-MS scanning of bioactive compounds in crude extracts from medicinal plants has attracted widespread interest and promoted a much better understanding of traditional herb medicines in recent years. Currently, liquid chromatography coupled with electrospray ionization (ESI) tandem mass spectrometry (LC-ESI-MS) has been widely used for the fast separation and structural identification of secondary metabolites without tedious isolation and purification of pure compounds from the complex sample matrix. Furthermore, several useful information is obtained from the MS experiment by MS full scan [39,42]. e full ESI-MS scans of fractions 1, 2, 3, and 4 are displayed in Figures 1-4 1, 181.1, 119.1, 125.1, 135.1,  161.0, 178.1, 179.1, 185.1, 193.1, 201.1, 209.1, 216.1, 219.2 (Table 3). In view of the presence of the wide varieties of secondary metabolites in the isolated fractions, they were submitted to in-vitro antiplasmodial testing against P. falciparum, 3D7 strain. A parasite culture was carried out as already described by Kwansa Bentum et al. [34]. e fractions were tested in triplicate, and the results are expressed as mean ± SD. e antiplasmodial activities of the isolated fractions were tested against 3D7 strain of Plasmodium falciparum through the malaria SYBER green fluorescence (MSF) assay method. e concentration range for the percentage inhibition assay was within the range of 100-0.098 μg/mL. e results for the plot of the percentage inhibition against the corresponding logarithm of the concentrations for the fractions and their IC 50 (s) are shown in Figure 5 and Table 3. e classification of the activity of the fractions was achieved by adopting the criteria, where IC 50 < 5 μg/mL was classified as very active; promising activity between 5 and 15 μg/mL; moderate activity between 15 and 50 μg/mL; and inactive at IC 50 > 50 μg/mL [43][44][45]. From the graph, the four fractions exhibited inhibitory activities for the various concentrations. For instance, fractions 1 and 3 exhibited stronger activity with percentage inhibitions within the range of (60-8)% and (65-8)% for the various concentrations. e fifty percentage inhibitory concentrations (IC 50 ) for the fractions reported values of 0.097 ± 0.034 and 1.43 ± 0.072 μg/mL for 1 and 3, respectively. Preliminary phytochemical screening for 1 and 3 indicated the presence of phenolics, steroids, coumarins, alkaloids, and flavonoids, and these could account for the stronger activities. e observation further corroborates the antiplasmodial activity reported for the crude methanol extracts with an IC 50 value of 2.97 μg/mL [28] and further supports the use of this plant in traditional medicine [46]. e fractions 2 and 4, however, respectively, recorded inactive and moderate inhibitory activities for the concentrations with inhibitory concentrations within the ranges of (up to 33) and (up to 47)%. e IC 50 values for 2 and 4 were found to be greater than 100 μg/ mL for fraction 2 and 37.09 ± 6.14 μg/mL for fraction 4. e negative inhibitory values of the percentage inhibitions for the fraction F4 could be due to the proliferation of the plasmodium species enhanced by the fractions. Several such observations are reported, and at specific concentrations, they achieve proliferation, and in other instances, they act as apoptosis [47]. In such instances, they significantly inhibit cell proliferation only at high concentrations but increase apoptosis at low concentrations. ese data demonstrate that the effects of the fraction are concentration-dependent [47,48]. Several classes of phytochemicals with such reported properties include resveratrol, epigallocatechin gallate, gingerol, phytosterol, and myricetin. ey are known to directly influence various molecular signal transduction pathways such as inflammation cascade, cell proliferation/ migration, oxidative stress, and metabolic disorders, which are involved in the development of several   Counts vs. Mass-to-Charge (m/z)       noncommunicable diseases [49]. Hence, the presence of a similar class of compounds in the fractions could contribute to the observation. e fractions could therefore be found to support accelerated cell divisions resulting in exponential cell proliferation faster than the positive control. For instance, several phytochemical classes such as triterpenoids, alkaloids, coumarins, flavonoids, steroids have been reported to contribute to the antiplasmodial activity of extracts [37,50,49]. Furthermore, Afzelia africana belongs to the Fabaceae family which has been known to exhibit promising antiplasmodial activities, with several active compounds isolated from them [52,53]. Furthermore, the activities of the fractions (2 and 4) were relatively weaker, in spite of the rich phytochemical constituents detected in them. e observation could be due to the activity of constituents being masked by other compounds when in combination. Such phenomenon is reported, where the coexistence of two or more secondary metabolites could either produce competitive or noncompetitive antagonism [54]. Furthermore, secondary metabolites present in plants are classified into various phytochemical classes such as phenolic, flavonoid, and terpenoids. However, a particular phytochemical class has vast structural diversities with different physicochemical properties (hydrophilicity, lipophilicity, concentration, pH value, solubility, and bioavailability in critical biological targets) that could enhance or reduce their respective biological activities [55]. erefore, the fact that the extracts have the same phytochemical composition or class does not mean they will register the same level of activity [55]. As a result, the current weaker activity cannot justify the rejection of the activities of the individual compounds in the fractions. It is therefore a good practice to carry out bioassay-guided fractionation in order to identify active fractions that could individually serve as drugs or in their combined state. Nevertheless, the active fractions could further be subfractionated in order to identify active compounds that could further serve as drugs or lead agents for further studies. e myriad of chemical (phytochemical, ESI-MS) and biological (antiplasmodial) data reported for the plant could therefore account for the ethnobotanical utilization of A. africana for the treatment of malaria. However, to account for the ethnobotanical utilization of A. africana as an antimalarial remedy as well as the myriad of promising antiplasmodial activity reported, a review of the mechanism of action of the phytochemical classes identified in the plant could provide further insight. Several literature suggests that the mechanism of action of most phytochemicals is dependent largely on the site of action. Moreover, phytochemicals have been known to exhibit their mechanism of biological activity by interfering with a biosynthetic pathway, cell wall destruction, DNA intercalation, interference with DNA transcription, and protein synthesis [56,57]. Furthermore, in order to account for the exact mechanism of action and the contribution of phytochemicals to the antiplasmodial activity of plants, the mechanism of action and drug-resistant activity of P. falciparum must be taken into account. Over the years, P. falciparum has developed several mechanisms for survival in the host organism as well as resisting drug activity, that is, drug resistance [58]. e parasite after infecting the host organism and degrading the RBC for its protein synthesis and other life processes (life stages: trophozoites, merozoites, and the schizonts) has been known to utilize the conversion of heme (Fe(II) protoporphyrin (IX): a hemoglobin metabolite) to hemozoin (a protective polymer) as a means of sequestering the toxic effect of the former (heme) [59]. Hence, targeting this mode of action involves the screening of compounds that could inhibit the synthesis of hemozoin in the parasite which subsequently leads to the accumulation of the toxic heme, leading to cytotoxicity and cell death. Phytochemicals with such inhibitory activity against heme-hemozoin conversion include alkaloids classes such as quinine, quinidine, cinchonine, and cinchonidine [60,61]. Furthermore, the antiplasmodial activities of several anthraquinones, phenolics, and flavonoids could be linked to their ability to form Biochemistry Research International π-π stacking interaction with heme leading to binding and subsequent inhibition of hemozoin formation in the parasite [62][63][64]. e major pharmacophore responsible for the activity of these phytocompounds is due to the presence of multiple phenolic systems in their structures. Specific phytocompounds identified to follow this mechanism of action include rufigallol, exifone, apigenin, ellagic acid, and derivatives [65][66][67]. e existence of alkaloids, flavonoids, phenolics, anthraquinones as well as other phytochemicals in the fractions could therefore account for their antiplasmodial activities.

Biochemistry Research International
In other studies, P. falciparum have been discovered to utilize glutathione for its inhibitory effect against drug activity. e glutathione-s-transferase enzyme (GST) has been a key enzyme in the biosynthesis of glutathione. In fact, the enzyme represents 1-10% of the cellular proteins of Plasmodium falciparum [68]. e enzyme has been implicated in the antimalarial drug-resistant capacity of P. falciparum. Hence, phytocompounds with inhibitory activity against the enzyme could provide a route for addressing drug resistance. Several classes of flavonoids and polyphenolic compounds isolated from plants have been found to inhibit the action of glutathione-s-transferase such as derivatives of the compounds of ellagic acid, quercetin, rutin, kaempferol, purpurogallin due to the presence of phenolic groups [69][70][71]. It is experimentally documented that flavonoids or compounds with α-β unsaturated carbonyl groups and polyphenolic systems form monoadduct with L-cysteine of the enzyme (glutathione-stransferase) through their α-β-unsaturated carbonyl moiety via the Michael addition mechanism [69][70][71]. e resultant effect could therefore lead to the inhibition of the protective capacity of the enzyme, leading to cytotoxicity and cell death [72]. As a result, the presence of flavonoids and phenolic compounds with similar structural motifs in fractions 1, 2, 3, and 4 ( Table 2) could therefore account for their reported antiplasmodial activity.
Other mechanisms of antimalarial activity of phytocompounds have been linked to their ability to antagonize folic acid's primary function. It is established that folic acid is involved in the biosynthesis of key biomolecules such as amino acids (e.g., serine, methionine) and DNA/ RNA components (e.g., thymidine and purine bases) [73]. Antiplasmodial compounds with the antifolate mechanism inhibit folic acid synthesis by antagonizing the main enzyme, dihydrofolate reductase (DHFR), which is involved in the folic acid synthesis. e inhibition of the enzyme further produces a series of cascading effects such as the impairment of DNA, RNA, and protein synthesis in P. falciparum leading to cell death [74]. Several phytochemicals such as alkaloids (piperine, berberine, dictamine reserpine), flavonoids (baicalein, biochanin, kaempferol, chalcone, catechin), coumarins, and terpenes have been found as potential folate synthesis inhibitors [56,75,76]. e phytochemical screening (Table 2) indicated the presence of a similar class of compounds in fractions 1, 2, 3, and 4 and could further account for the reported antiplasmodial and ethnobotanical application of A. africana as a malarial remedy.

Conclusion
e bioassay-guided fractionation of the bark of Afzelia africana led to the isolation of four distinct fractions with varying degrees of antiplasmodial activities against the 3D7 strain of P. falciparum. e study further reveals the presence of secondary metabolites such as alkaloids, anthraquinones, flavonoids, phenolic, glycosides, terpenoids, steroids, terpenoids, and steroids. e results of the study confirm the ethnobotanical utilization of A. africana as an herbal treatment for malaria and other diseases.
Data Availability e TLC profile (Table 1) data, the phytochemical screening ( Table 2) data, the ESI/MS (Figures 1-4) data, the antiplasmodial activity ( Figure 5) data, and the antiplasmodial activity IC50 (Table 3) data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.