Antimalarial activity of Mangifera indica aqueous extract in Plasmodium berghei’s apicoplast

Purpose: To investigate the in vivo antiplasmodial effect of Mangifera indica aqueous extract in Plasmodium berghei-infected mice, and to characterize its phytochemical constituents and their mechanism of action. Methods: The plant leaves (250.71 g) and stem bark (509.34 g) were weighed, macerated in 5 L of boiled, distilled water for 72 h, and filtered. In vivo antiplasmodial evaluation of the extract was carried out using Plasmodium berghei-infected mice. The leaf and stem bark extracts were subjected to gas chromatography-mass spectroscopy (GC-MS) analysis for characterization of their chemical constituents. Results: The leaf/stem bark (1:2) extract (88.27 %) exhibited chemosuppressive activities that was better than artesunate (88.04 %) and artemisinin-based combination therapy (ACT, 84.60 %). Its schizonticidal activities (98.63 %) revealed a better therapeutic response when compared with artesunate (92.34 %). Spectroscopic analysis showed linoleic acid esters and other compounds. Conclusion: Therapeutic response of leaf/stem bark (1:2) extract exhibit superior schizonticidal activity to ACT and artesunate. Furthermore, the linoleic acid esters in Mangifera indica aqueous extract may contribute to strong antimalarial activity by inhibiting fatty acid biosynthesis.


INTRODUCTION
Malaria is a terrible disease that affects almost 0.5 percent of the world's population [1]. A study by a group of scientists [2] revealed that about 40 percent of human race is likely to contract malaria; half of this population is resident in Africa, with almost 80 percent of such cases found in Nigeria, Tanzania, Congo, Kenya, and Ethiopia [2]. Malaria is caused by plasmodium parasite, and there are reports of resistance by the parasite against chemotherapies like  [3], and this is a limitation to malaria prevention and elimination in Nigeria. Over the years, phytomedicines have been reported as an alternative in the management of malaria in some countries. Mangifera indica (mango) is a pharmacologically rich and diverse plant [4]. Some of its reported pharmacological potentials are anti-inflammatory, antiplasmodial, and antioxidant in different parts [4]. Many metabolites have been isolated from the plants using different techniques, although some of their mechanisms of action are unknown.
Fatty acids are one of the metabolites present in plants. They inhibit biosynthesis in the Plasmodium parasite through the alteration of metabolic activities in the parasites' apicoplasts [5]. The apicoplast is where the generation of isoprenoids through the mevalonate-independent 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway takes place [5]. This pathway is very essential in the growth of malaria parasites. Moreover, polyunsaturated fatty acid (PUFA) is implicated in the inhibition of fatty acid biosynthetic machinery of Plasmodium parasite [6]. While linoleic and linolenic acids have medicinal properties, n-3 and n-6 PUFA have antimalarial properties.
However, antiplasmodial activity of the herbal formulation of Mangifera indica administered to Plasmodium berghei-infected mice was compared with that of artesunate and ACT. Its antiplasmodial constituent was characterized and their possible mechanisms of action was determined.

EXPERIMENTAL Sourcing and authentication
The stem bark and leaves of Mangifera indica were obtained in April 2021 from Arthur Jarvis University, Nigeria. The plant parts' identity was determined by their ascertained number, UCH432G, which was in the department's herbarium after authentication by Taxonomist Dr. Godwin Eteng.

Animal handling
One hundred and sixty-five Wistar (13 -27 g) mice of both sexes were sourced from the Animal House, Department of Pharmacology and Toxicology, University of Uyo, Nigeria, and used for the antiplasmodial study. The NIH guideline for handling and usage of experimental animals (the ARRIVE guidelines in compliance with NIH publications no. 8023, revised 1978) was judiciously followed. The mice were given the freedom to access a standard pellet diet, water, and were housed at ambient temperature. The Animal Ethics Committee of the Faculty of Pharmacy, University of Uyo, Nigeria approved the study (approval no. UUFPHARM/01933) and ensured strict compliance with the experimental protocols.

Determination of acute toxicity
Lethal dose (LD50) and effective doses (ED50) were calculated by using Lorke's method [7] which involved phases I and II. Rats were observed for 24 hours while the number of deaths were recorded in order to establish the medial dosage of the herbal formulation of the plant as well as the one that might induce toxicity in a short term in mice.

Parasites
The National Institute of Medical Research (NIMR), Nigeria, provided the Plasmodium berghei (NK-65) infected donor mice used in this study, and mice were acclimatized according to the method described by some researchers [8].

Inoculum preparation
The inoculum of the parasite-infected erythrocytes with about 0.2 mL peripheral parasitemia was obtained from the donor mice via cardiac puncture and emptied into an anticoagulant-coated tube. Percentage of parasites in blood and concentration of the original inoculum were calculated according to the method described by some researchers [9].

Drug administration
Antiplasmodial screening followed the dissolution of tablets of ACT (560 mg) in distilled water (100 mL) and was given orally at doses of 8 mg/kg body weight of experimental animals, whereas that of artesunate (100 mg) was orally given at doses of 5 mg/kg body weight of mice after dissolution in 20 mL of distilled water.

In vivo antiplasmodial determination
A method by Asanga et al [8] was slightly modified and used for the evaluation of in vivo antimalarial properties of herbal formulation of the plant.

Microscopic examination
Each slide was prepared by measuring blood (2 μL) for the thin film as well as 6 μL for the thick film according to an earlier report [8], whereas their parasite densities and % growth inhibition of Plasmodium berghei were calculated and expressed as earlier reported by [8,10].

Gas chromatography-mass spectroscopy (GC-MS)
The extracts of Mangifera indica's herbal formulation were each weighed (10 mg), dissolved in DMSO, and subjected to GC-MS analysis according to the method described by [8,11]. Two (2) µL (split ratio 10:1; split flow 12 mL/min) was injected into an Agilent system consisting of a model 7890 N gas chromatograph and a model mass detector Triple Quad 7000 A in EI model at 70 eV (m/z range 40 -600 amu; Agilent Technologies, Santa Clara, California. USA. GC column was an HP-5ms fused silica capillary with a 5 % phenyl-methyl polysiloxane stationary phase (30 m x 250 μm x 0.25 μm). The carrier gas was helium with a column head pressure of 9.7853 psi and flow rate of 1.2 mL/min. Inlet temperature was 250 o C and mass selective detector temperature was 250 o C.
The GC oven temperature programming used was as follows: 50 o C initial temperature was held for 10 minutes; increased at 6 o C/min to 190 o C for 20 minutes; increased 7 o C/min to 290 o C for 30 minutes. Compounds were identified based on their retention indices and by comparing their mass spectral fragmentation patterns with the National Institute of Standards and Technology (NIST) database/ChemStation data system.

Statistical analysis
GraphPad Prism version 9.0 (GraphPad Software, Incorporated, CA, USA) was used to analyze the statistics and presented as mean ± standard error of the mean (SEM). The differences that were significant within and between the different treatment groups were analyzed using one-way ANOVA, and then their means were compared using Turkey's post hoc test. Differences at a confidence level of 95 % were considered to be statistically significant.

RESULTS
The results (  Results from GC-MS spectra showed the presence of many compounds with different retention times and percentage abundance (area, %), as illustrated in Table 4 and Table 5. Mass spectrometer (MS) analyzed the eluted compounds at varying times to characterize their chemical structures, nature,

DISCUSSION
Repository assay of drugs is very critical in antiplasmodial research. The results showed that the parasite densities were significantly reduced in the entire group as compared with the negative control group; therefore, all the treatment groups had remarkable repository activity against malaria parasites, nevertheless, with varying capacities. Increased inhibition (%) of parasitemia in this study was consistent with an earlier report [12]. Plasmodium berghei inhibition (%) by leaves and stem bark (1:2), stem bark, and leaves had better repository activities than artesunate. However, this could be due to the linoleic acid esters in the extract, as some researchers posited that the linolenic and linoleic acids were more potent against P. berghei in mice [5].
Furthermore, the prophylactic potentials of Mangifera indica's herbal formulation are indicative of their potential in the destruction of the various stages of the parasite, whereas some drugs' mechanisms of action involve the destruction of the first stage of parasite development in the hepatic tissue or the suppression of the emerging asexual stages, as well as the destruction of hypnozoites to avert relapses [13]. Thus, the consumption of      Mean survival time (MST) is a pharmacological parameter used in the evaluation of drug efficacy; it gives clues on the parasitemia clearance time in the host system. The longer the MST of a drug, the higher its therapeutic response; hence, the better its efficacy. The significant increase in MST of the entire groups treated with Mangifera indica's herbal formulation when compared to the negative control suggests that the plant has good repository activity against malaria infection. Nevertheless, with respect to MST, it was only the stem bark (300 mg/kg) of this plant that compared favourably with that of ACT. This suggests a similarity in the metabolites of this extract to the compounds that enhance parasitemia clearance and recovery from malaria infection. The chemosuppressive potential of the herbal formulation (Table 2) showed that all the treatment models produced significant reductions in their parasite densities as compared with the group of negative controls; hence, the extracts greatly suppressed Plasmodium berghei-induced infection in the animal model used. Therefore, the chemosuppressive activity of the herbal formulations against the parasite could be attributed to the esters of linoleic acid present in the plant. Some researchers [13,14] reported that some plant extracts exert antimalarial action via the mitigation of fatty acid and protein biosynthesis in the parasites.
The results on the significant increases in % growth inhibition of plasmodium parasites in all the treatment models as compared with the group of negative controls are an indication that the herbal formulation had excellent chemosuppressive potentials and were in tandem with an earlier report by some researchers [14]. Consequently, the chemosuppressive abilities against malaria infection of the leaves and stem bark (1:2) and stem bark/artesunate compared favourably with artesunate and ACT, respectively. Furthermore, all the groups except leaf, leaves/stem bark (1:1), and leaf/stem bark (2:1) showed significant increases in their MST as compared with the group of the negative control. Therefore, the results of the above-listed treatment groups corroborated their growth inhibition of parasites as well as parasite densities, suggesting they were not effective in parasitemia clearance. However, other treatment groups were remarkable in malaria treatment.
In addition, most antimalarial drugs' mechanisms of action target the inhibition of the growth and replication of Plasmodium parasites. The increase in growth inhibition of the malaria parasite in all the groups as compared with the group of negative controls were consistent with an earlier report by some researchers [8] on Nauclea latifolia root extract and fractions (150 mg/kg). Kumaratilake et al [15] reported that fatty acids induce direct destruction of malaria parasites, whereas others act through neutrophil priming. Therefore, the presence of esters of linoleic acid and other compounds in this herbal formulation may have aided the destruction of parasites. More so, the role of long-chain fatty acids in the inhibition of P. berghei's enoyl-ACP reductase (FabI), an enzyme that catalyzes the fatty acid elongation cycle in P. berghei was reported [16]. Therefore, the mechanism of action of Mangifera indica's herbal formulation may be as result of the inhibition of the biosynthesis of fatty acids in the parasites' apicoplast due to the esters of linoleic acid. A similar finding was reported by some scientists [8] on the significant increases in MST in mice where it was proposed that the prolonged MST for the parasitized mice resulted from higher parasitemia clearance by the herbal formulation, leading to higher schizonticidal activities.

CONCLUSION
There is a significant therapeutic response to leaves and stem bark (1:2) extracts against Plasmodium berghei-induced malaria in mice, as corroborated by its stronger repository, chemosuppressive, and schizonticidal activities than ACT and artesunate. Moreover, linoleic acid esters, shown to be present by GC-MS data, have previously been reported to act through the inhibition of fatty acid synthase type II and enzymes of mevalonate-independent 1-deoxy-Dxylulose-5-phosphate (DOXP) pathway in Plasmodium berghei's apicoplast.