Antimalarial potential of Moringa oleifera Lam. (Moringaceae): A review of the ethnomedicinal, pharmacological, toxicological, and phytochemical evidence

Abstract Several regions of the world frequently use the species Moringa oleifera Lam. (Moringaceae) in traditional medicine. This situation is even more common in African countries. Many literature reports point to the antimalarial potential of this species, indicating the efficacy of its chemical compounds against malaria-causing parasites of the genus Plasmodium. From this perspective, the present study reviews the ethnobotanical, pharmacological, toxicological, and phytochemical (flavonoids) evidence of M. oleifera, focusing on the treatment of malaria. Scientific articles were retrieved from Google Scholar, PubMed®, ScienceDirect®, and SciELO databases. Only articles published between 2002 and 2022 were selected. After applying the inclusion and exclusion criteria, this review used a total of 72 articles. These documents mention a large use of M. oleifera for the treatment of malaria in African and Asian countries. The leaves (63%) of this plant are the main parts used in the preparation of herbal medicines. The in vivo antimalarial activity of M. oleifera was confirmed through several studies using polar and nonpolar extracts, fractions obtained from the extracts, infusion, pellets, and oils obtained from this plant and tested in rodents infected by the following parasites of the genus Plasmodium: P. berghei, P. falciparum, P. yoelii, and P. chabaudi. Extracts obtained from M. oleifera showed no toxicity in preclinical tests. A total of 46 flavonoids were identified in the leaves and seeds of M. oleifera by different chromatography and mass spectrometry methods. Despite the scarcity of research on the antimalarial potential of compounds isolated from M. oleifera, the positive effects against malaria-causing parasites in previous studies are likely to correlate with the flavonoids that occur in this species.


Background
Human malaria is an infectious disease caused by five species of Plasmodium (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi, and Plasmodium malariae) [1]. Its transmission between humans occurs through the bite of female Anopheles mosquitoes infected with Plasmodium spp. [2]. Among the species of parasites that infect humans, Plasmodium falciparum is the main cause of the severe form of the disease, which can lead to death and is responsible for 99.7% of infections in sub-Saharan Africa [3]. Malaria symptoms usually include high fever, headache, muscle aches, vomiting, chills, and fatigue [4]. Despite major advances in the control of this disease, estimates for 2021 suggest a total of about 247 million clinical cases and 619,000 deaths from malaria worldwide [5].
Despite the modest reduction in the number of cases over the past 20 years, malaria remains a global health problem [6], even with the production of the Mosquirix TM vaccine, which is not yet widely available [7]. Increasing resistance to drugs currently used in the treatment of this disease is also a serious threat to global malaria control efforts [8]. In this way, the discovery of antimalarial drugs is driven by the need to obtain new therapeutic alternatives to treat infections and save lives in a context of a constantly evolving drug resistance [9]. Herbal medicine has been considered the backbone of malaria treatment for thousands of years. The first antimalarial drug (quinine) was isolated from the bark of a tree of the family Rubiaceae belonging to the genus Cinchona [10].
Medicinal plants are viable alternatives for the isolation and screening of active phytochemicals that may be responsible for antiplasmodial activity in in vitro and in vivo assays [11]. In in vivo tests, rodents are infected with four different species of Plasmodium (P. berghei, P. chabaudi, P. yoelii, and P. vinckei) and used in research aimed at discovering new antimalarial drugs [12]. Flavonoids apigenin, kaempferol, rutin, and quercetin occur in several plant species and showed promising antimalarial activity in in vitro and in vivo experiments [13][14][15][16][17][18]. In addition, the flavonoids are phytoconstituents with the ability to scavenge free radicals and act as antioxidants [19]. These properties make flavonoids very promising for antimalarial activity, as during malaria infections both the host and the parasites are under severe oxidative stress [20]. In summary, the infected host presents an exacerbated production of free radicals. These free radicals produced in large quantities cause damage to the vascular endothelium, increasing vascular permeability and platelet adhesion, known to be associated with severe cerebral malaria [21,22]. In this context, flavonoids are promising antioxidants to reverse this clinical condition, deserving a highlight compared to other phytochemicals in the treatment of malaria.
Moringa, the single genus in the family Moringaceae, is one of the most phenotypically varied groups of angiosperms [23,24]. With only 13 species, Moringa occurs in arid regions of Africa, Madagascar, the Arabian Peninsula, and India [23,25]. The Moringa genus has high antioxidant activity mainly due to its high content of flavonoids. Most of the flavonoids present in the genus are in the flavanol and glycoside form [26]. Moringa oleifera Lam., popularly known as drumstick and horseradish tree, is native to sub-Himalayan areas of the Indian subcontinent and has been introduced in many tropical countries [25,27,28]. Researchers attribute the medicinal, nutritional, and industrial properties of this plant to the constituents that occur in its roots, bark, leaves, flowers, fruits, and seeds [29,30].
Ethnobotanical and ethnopharmacological surveys carried out in African and Asian countries have reported the use of M. oleifera for the treatment of malaria in traditional communities [31][32][33][34][35][36][37][38]. These ethnomedicinal uses have been confirmed through in vivo and in vitro assays using different products obtained from the leaves and seeds of M. oleifera against several malaria-causing species of Plasmodium [39][40][41][42][43][44]. It is important to emphasize that the phytochemicals isolated from this species have not yet had their antimalarial activity evaluated in scientific research.
Considering that malaria still causes several deaths around the world [5] and that the discovery of new antimalarial drugs is of great importance in assisting in the treatment of infections caused by parasites of the genus Plasmodium [9], the present study reviews the ethnobotanical, pharmacological, toxicological, and phytochemical (flavonoids) evidence of M. oleifera, focusing on the treatment of malaria.

Inclusion and exclusion criteria
Only scientific articles published between 2002 and 2022 addressing the following information about M. oleifera were selected: 1) Ethnomedicinal uses of M. oleifera by traditional communities in different regions of the world; 2) In vitro and in vivo antimalarial activity of extracts, fractions, oils, and other products obtained from M. oleifera; 3) Toxicity and biological safety of products obtained from this plant; 4) Flavonoids isolated and identified in M. oleifera that have already been reported in the literature. As for the exclusion criteria, review articles, e-books, book chapters, undergraduate theses, Masters' theses, Ph.D. theses, and works published in technical or scientific events were excluded.

Data screening and categorization of information
A total of 130 scientific articles were selected from the databases ( Figure 1). Subsequently, 58 documents that did not meet the criteria of this review were excluded. Finally, the present study considered 72 articles containing data on ethnomedicinal uses, pharmacological activities, and toxicological and phytochemical (flavonoids) investigations of M. oleifera focusing on the treatment of malaria ( Figure 1, Additional file 1). The results were grouped in tables and represented in graphs when necessary. The general information described in the "Results" section has been categorized by: "Botanical aspects of Moringa oleifera", "Ethnomedicinal uses of Moringa oleifera for the treatment of malaria", "In vivo and in vitro antimalarial activity of Moringa oleifera", "Toxicity of Moringa oleifera", and "Flavonoids identified in Moringa oleifera".

Botanical aspects of Moringa oleifera
Moringa oleifera is native to northwest India and adapted to arid and semiarid environments. This plant has gained popularity in certain developing countries due to its medicinal, industrial, and nutritional properties [29]. African, South American, Central American, and Asian countries currently cultivate M. oleifera commercially (Figure 2) [45]. This is a medium-sized, fast-growing evergreen tree about 10 to 12 m tall. The bark of mature trees is gray-white while young shoots have a purplish or greenish-white bark [27]. It has a more conventional trunk and fibrous and resistant roots [46]. The fruits are long, woody pods, which when ripe open into three valves, containing trivalve seeds with longitudinal wings. Its pinnate leaves are divided into leaflets arranged on a rachis. The flowers are zygomorphic with five petals, five sepals, five functional stamens, and several staminodes. In addition, the flowers have pedicels and axillary inflorescences [47].

Ethnomedicinal uses of Moringa oleifera for the treatment of malaria
The following African countries use Moringa oleifera as a traditional medicine for the treatment of malaria: Nigeria, Uganda, Benin, Ghana, Togo, Tanzania, Cameroon, Kenya, Ethiopia, Mozambique, and Cote d'Ivoire. Regarding Asia, ethnobotanical and ethnopharmacological studies have reported the use of this plant for the treatment of malaria in Indonesia, India, and Pakistan ( Figure 3). The fact that medicinal indications of M. oleifera occur mainly in African countries may correlate directly with the high incidence of cases of this disease in sub-Saharan Africa. In recent years, researchers have carried out several studies on the impact, progression, and control of malaria in this region [48][49][50].
Traditional communities mainly use M. oleifera leaves (63%) for the treatment of malaria. The other most used parts are seeds (13%), roots (9%), flowers (5%), and stems (4%) ( Figure 4 and Figure 5). These plant parts are used for the preparation of decoction, maceration, infusion, paste, and cataplasm. Table 1 shows further information on the forms of administration of herbal medicines. The literature often reports a wide use of plant leaves for the treatment of malaria, confirming the findings of this study [51][52][53].     Ethnobotanical and ethnopharmacological surveys carried out in Nigeria have highlighted the constant use of M. oleifera leaves for the treatment of malaria by traditional communities in the country [54][55][56][57][58][59][60][61]. Other African countries that also stood out in the use of M. oleifera leaves were Uganda [36,62,63], Benin [31,64,65], and Ghana [66][67][68]. Scientific research shows that extracts from the leaves of this plant had in vivo antimalarial activity [69][70][71][72], confirming its use in traditional medicine.

In vivo and in vitro antimalarial activity of Moringa oleifera
Several studies have reported the in vivo and in vitro antimalarial activity of M. oleifera (Table 2). Researchers mostly used in vivo methods to evaluate the potential of polar and nonpolar extracts, fractions obtained from extracts, infusions, pellets, and oils obtained from this plant and tested in rodents infected by the following parasites of the genus Plasmodium: P. berghei, P. falciparum, P. yoelii, and P. chabaudi. Leaves were the most used parts to obtain the evaluated products. Regarding in vitro tests, only two studies reported the potential of M. oleifera against the parasite P. falciparum. This parasite infects humans and causes the most severe form of malaria [40,73].
According to Orman et al. [69], the parasitic suppression of the aqueous extract of M. oleifera leaves was not entirely dose-dependent in mice. This is because the two lowest doses, 250 mg/kg (69.31% of suppression) and 500 mg/kg (77.26% of suppression), exhibited better suppression of P. berghei (NK65) than the two highest doses, 750 mg/kg (25.28% of suppression) and 1000 mg/kg (7.12% of suppression). In turn, Ogundapo et al. [74] observed in their in vivo antimalarial studies that the methanolic extract of M. oleifera leaves (50 and 100 mg/ kg) was able to suppress 42.37 and 55.30 %, respectively, the parasitemia induced by P. berghei. Somsak et al. [71] reported that the aqueous extract of M. oleifera leaves at doses of 500, 1000, and 2000 mg/kg showed antimalarial activity of 35, 40, and 50%, respectively, against P. berghei.
Dondee et al. [70] observed that the aqueous extract of M. oleifera leaves significantly inhibited parasitemia in mice infected with P. berghei in a dose-dependent manner. Percent inhibitions of 42.86, 71.43, and 85.71% occurred at doses of 100, 500, and 1000 mg/kg of the extract, respectively. Dondee et al. [75] also reported results similar to these, but evaluated doses 100, 1000, and 2000 mg/kg. Despite the concentration-dependent behavior, it can be inferred that due to the low variation between the doses  [41]. According to Obediah and Obi [42], doses of 200 mg/kg (68.93% of suppression), 300 mg/kg (72.56% of suppression), and 500 mg/kg (67.01% of suppression) of the ethanol extract of M. oleifera seeds showed good chemosuppression of P. berghei multiplication in relation to the negative control. According to Shrivastava et al. [77], extracts of M. oleifera flowers and leaves showed dose-dependent suppression in mice infected with the parasite P. yoelii (N-67). At the lowest dose (125 mg/kg), the flower and leaf extracts suppressed infection by 40.74 and 31.85 %, respectively, after four days of experiment [77].
In an in vitro experiment, Daskum et al. [40] tested different extracts of M. oleifera leaves against the P. falciparum strain 3D7. According to these authors, although some extracts were more potent than others, all were biologically active with the following IC 50 values: hexane extract IC 50 = 3.36 µg/mL; methanolic extract IC 50 = 3.44 µg/mL; aqueous extract IC 50 = 4.09 µg/mL. It is important to highlight that the most severe form of malaria and the mortality rate in humans often correlate with infections caused by P. falciparum [78][79][80]. Thus, studies focusing on the evaluation of new drugs against this specific parasite are of great importance for public health.

Toxicity of Moringa oleifera
Researchers evaluated the toxicity of different products obtained from M. oleifera in experimental rodent models [39,42,70,71,75,77]. These studies regarded the extracts obtained from this species as biologically safe since the evaluated animals did not present relevant behavioral or physiological changes during the acute and subacute toxicity experiments. However, despite the extracts being considered safe, a recent study by Abdulahi et al. [44] reported that precautions should be taken when administering M. oleifera seed oil at a dose greater than 200 mg/kg, as this product may be mildly toxic.
According to Somsak et al. [71], the aqueous extract of M. oleifera leaves orally administered in a single dose of up to 4000 mg/kg showed no visible signs of toxicity (paw licking, salivation, stretching, urination, lacrimation, hair erection, and reduction in feeding activity) in mice. Additionally, no mortality occurred within the observation period of 30 days. Dondee et al. [70] observed similar results, reporting that the aqueous extract of M. oleifera leaves administered orally at a dose of up to 4000 mg/kg also did not cause mortality in mice over the seven days of observation. When orally administering the single dose of 2000 mg/kg of the aqueous extract of M. oleifera leaves to mice, Dondee et al. [75] reported the absence of lethal effect in the animals up to one week after the experiment. However, at the dose of 4000 mg/kg, these authors observed tremors and drowsy activities after 24 hours of extract administration.
In a study carried out by Mulisa et al. [39], the acetone extract of M. oleifera leaves did not result in animal death at the dose of 2000 mg/kg. This implies that the lethal dose (LD 50 ) of the extract was greater than 2000 mg/kg. Moreover, Obediah and Obi [42] reported that the ethanol extract of M. oleifera seeds was not considered toxic at the highest dose of 911 mg/kg administered to albino rats. According to Shrivastava et al. [77], the results of the subacute toxicity evaluation indicated that both methanolic extracts (flower and leaves) were not considered toxic to mice, even at the highest dose level (3000 mg/kg), in the first 24 h, as well as in the following 14 days of the experiment.

Discussion
Despite the many reports of the use of M. oleifera in traditional medicine for the treatment of malaria in several countries and the evaluation of its extracts for their antimalarial potential in in vitro and in vivo experiments, this study identified some research gaps. Initially, it is important to note the absence of studies on the antimalarial activity of phytochemicals isolated from this plant. This fact makes it difficult, for example, to elucidate the mechanisms of action of compounds isolated from M. oleifera against malaria-causing parasites of the genus Plasmodium.
Pan et al. [90] report the isolation of several antimalarial compounds from plants during the last decade, with many of these compounds showing significant in vitro activity against P. falciparum. These studies are essential for the discovery of new antimalarial drugs. When evaluating the performance of an extract instead of isolated molecules, you have a phytocomplex, which can have synergistic compounds, as well as interfering compounds (PAINs). For the use of these extracts, standardization is recommended based on biomarkers that can be correlated with pharmacological activity, such as the flavonoids present in this species.
In addition to the metabolic composition, recent studies have pointed to a potential role for the microRNA of this species in the production of key molecules capable of justifying the bioactivity of this plant [91,92]. In this perspective, studies of in vitro cell culture and callus of M. oleifera have already been carried out for the massive production of secondary metabolites and microRNA that present biological properties [93]. These studies bring perspectives for optimizing and obtaining pharmacologically active metabolites.
Furthermore, the present study did not find reports of randomized clinical trials of products obtained from M. oleifera that can be used in the treatment of malaria. Randomized clinical trials are needed before herbal remedies can be recommended on a large scale. As these studies are expensive and time-consuming, it is important to prioritize new drugs for clinical investigation according to existing data from sociological, ethnobotanical, pharmacological studies, and preliminary clinical observations [94]. Moreover, the observed in vitro studies were not carried out with resistant strains of Plasmodium, providing yet another gap in this study.
Therefore, considering the widespread use of M. oleifera by traditional communities for the treatment of malaria and the vast scientific evidence on its antimalarial potential in preclinical studies, it is important to carry out in vitro assays with resistant strains and clinical trials to ensure the effective and safe use of products obtained from this plant in humans.

Conclusion
Africans and Asians make large use of Moringa oleifera for the treatment of malaria. The leaves of this plant are the main parts used in the preparation of herbal medicines. The in vivo antimalarial activity of M. oleifera was confirmed through several studies using polar and nonpolar extracts, fractions obtained from the extracts, infusion, pellets, and oils obtained from this plant and tested in rodents infected by the following parasites of the Plasmodium genus: P. berghei, P. falciparum, P. yoelii, and P. chabaudi. Extracts obtained from M. oleifera showed no toxicity in preclinical tests. By using different chromatography and mass spectrometry methods, researchers identified a total of 46 flavonoids in M. oleifera leaves and seeds. Despite the scarcity of studies on the antimalarial potential of compounds isolated from M. oleifera, the positive effects against malaria-causing parasites observed in previous studies are likely to correlate with the flavonoids that occur in this species.

Availability of data and materials
Not applicable.

Funding
The present study was supported by National Council for Scientific and Technological Development (CNPq), Brazil.