Exploring Antimalarial Herbal Plants across Communities in Uganda Based on Electronic Data

Malaria is one of the most rampant diseases today not only in Uganda but also throughout Africa. Hence, it needs very close attention as it can be severe, causing many deaths, especially due to the rising prevalence of pathogenic resistance to current antimalarial drugs. The majority of the Ugandan population relies on traditional herbal medicines for various health issues. Thus, herein, we review various plant resources used to treat malaria across communities in Uganda so as to provide comprehensive and valuable ethnobotanical data about these plants. Approximately 182 plant species from 63 different plant families are used for malaria treatment across several communities in Uganda, of which 112 plant species have been investigated for antimalarial activities and 96% of the plant species showing positive results. Some plants showed very strong antimalarial activities and could be investigated further for the identification and validation of potentially therapeutic antimalarial compounds. There is no record of an investigation of antimalarial activity for approximately 39% of the plant species used for malaria treatment, yet these plants could be potential sources for potent antimalarial remedies. Thus, the review provides guidance for areas of further research on potential plant resources that could be sources of compounds with therapeutic properties for the treatment of malaria. Some of the plants were investigated for antimalarial activities, and their efficacy, toxicity, and safety aspects still need to be studied.


Introduction
Malaria, a dangerous and life-threatening disease caused by Plasmodium parasites is spread to humans through bites of infected female Anopheles mosquitoes [1]. It is one of the most widespread diseases today not only in Uganda but also throughout Africa. Hence, careful monitoring of malaria is required as the disease can be severe and can cause many deaths, especially due to the increasing prevalence of resistance to current antimalarial drugs. Among the five parasitic species that cause malaria to humans, Plasmodium falciparum and Plasmodium vivax are the deadliest [2,3]. P. falciparum and P. vivax being the most prevalent malaria parasites in sub-Saharan Africa and regions of the Americas, respectively, were responsible for about 99.7% and 74.1% of malaria cases in 2017 [4]. In Southeast Asia, Plasmodium knowlesi is the most common cause of malaria, accounting for up to 70% of malaria cases, although it has been known to infect Old-World monkeys more [5]. Two other species of Plasmodium, Plasmodium malariae and Plasmodium ovale, generally cause mild fevers. Approximately 216 million malaria cases were registered in 2016, with a death toll of up to 445,000 [1]. According to the World Health Organization [6], the incidence of malaria in Uganda, at 47.8%, was the highest worldwide in 2005. According to Njoroge and Bussman [7], malaria is responsible for one to two million deaths annually in Africa. Typical symptoms of malaria include high fever, fatigue, headache, muscle ache, nausea, abdominal discomfort, and profuse sweating. However, in extreme cases and cases of prolonged illness without treatment, brain tissue injury, pulmonary edema, kidney failure, severe anemia, yellow discoloration of the skin, and low blood sugar may be noted ( Figure 1) [1,2]. In Uganda, malaria is one of the major causes of illness and death [7]. Statistically, it accounts for 46% of children's sicknesses, almost 40% of outpatient visits to hospitals and clinics, 25% of hospital admissions, 14% of inpatient deaths, and approximately 23% of infant mortalities [7].
In different parts of the world, the use of herbs and herbal extracts in the management and treatment of malaria is very common since herbs are cheap and readily available besides being effective. In fact, the use of herbal medicine for treatment worldwide is on the rise. Over 80% of the Ugandan population relies directly on herbal plants for their health care primarily [8]. A great majority of the population uses traditional herbal medicines because of their confirmed therapeutic value [8]. e increase in preference for herbal remedies coupled with resistance exhibited by pathogenic strains, including Plasmodium species, to the modern drugs available is the driving force behind researchers' interest in herbal plants for possible alternatives for more effective antimalarial drugs [9,10].
is review was aimed at providing comprehensive ethnobotanical information about various plant resources with antimalarial properties that are primarily used to manage and treat malaria across communities in Uganda, based on which further evaluation of these plants such as those of their efficacy and safety for the treatment of malaria may be based.

Methods and Materials
In the review, the data search processes employed by Komakech et al. [11] were modified to gather information on herbal plants for malaria treatment in Uganda from peerreviewed articles in English published in scientific journals and other verifiable databases, with a focus on plant species and families, plant parts used, antimalarial activities of the extracts from herbal plants, and mechanisms of action of novel antimalarial phytochemicals and derivatives. Electronic literature databases such as PubMed, Medline, Scopus, SciFinder, Google Scholar, and Science Direct were carefully searched for suitable information. e following words were used as key search terms: ("Herbal medicine in Uganda" OR "Herbs in Uganda" OR "Traditional remedies in Uganda" OR "Natural remedies in Uganda" OR "Antimalarial herbs in Uganda" OR "Anti-malarial plants in Uganda" OR "Ugandan herbs" OR "Ugandan ethnomedicine" OR "Ugandan phyto-medicine"), AND ("antiplasmodial activities" OR "anti-malarial activities" OR "anti-plasmodial effects" OR "anti-malarial effects" OR "malaria treatment" OR "malaria management") OR ("Malaria in Uganda" AND "prevalence" OR "occurrence" OR "distribution" OR "herbal treatment" OR "herbal remedies" OR "phyto-medicine" OR "phyto remedy" OR "plant parts used for treatment") OR (Phytochemicals for malaria treatment OR Artemisinins OR Quinine OR Noble anti-malarial compounds OR Plant derived anti-malarial compounds AND mechanisms of action OR modes of action) OR ("Malaria herbal medicine in Uganda" OR "Herbal medicine in Uganda" OR "Herbal malaria remedy in Uganda" OR "Natural malaria medicine in Uganda" OR "Traditional malaria herbal medicine" OR "Malaria herbal recipe" AND "dosage" OR "dose" OR "dose given" OR "mode of administration" OR "means of traditional extraction" OR "traditional extraction" OR "Toxicity" OR "Safety and toxicity" OR "Policy framework" OR "other ethno-pharmacological uses" OR "other ethno-pharmacological utilizations" OR "other ethno-medicinal uses"). e information gathered was verified separately for its reliability; any discrepancies discovered were resolved by discussions between the authors. ereafter, these data were summarized and analyzed, and comparisons were made to draw conclusions.

Prevalence of Malaria
Malaria in Uganda is highly endemic because the climate is favorable for its consistently stable and year-round transmission in about 99% of the country, with the country's entire population being at risk for contraction [12]. e most vulnerable groups of people at great risk for malaria are expectant mothers and young children under the age of 5 years [12]. e malarial parasite, P. falciparum, is most commonly the cause of malaria throughout Uganda, accounting for over 90% of malaria cases. However, Betson et al. [13] have warned of the potential for the emergence of infections due to P. malariae and P. ovale spp. as well, since there is much focus on countering P. falciparum infections. In 2016, Larocca et al. [14] indicated that Uganda was one of the leading countries in the world with malaria incidence rate as high as 478 cases per 1,000 population per year. Specifically, overall registered death cases caused by malaria in children were between 70,000 and 100,000 annually in Uganda [14]. Tremendous effort has been made to control malaria in Uganda by the government-headed Uganda Malaria Reduction Strategic Plan and Mass Action Against Malaria.
ese efforts have greatly reduced the malaria burden and incidence from 272 cases per 1000 population in 2016/17 to 191 cases per 1000 population in 2017/18 [12]. Although there has been a general reduction in the incidence of malaria, studies indicate that malaria prevalence along lakes, for example, Lake Victoria, and in remote areas of the country (villages) as well as areas closer to forests are much higher, with over 450 malaria cases per 1000 population ( Figure 2) [12,13,15]. Communities around lakeshores in Uganda have always had high prevalence of malaria among children and especially the young ones despite routine treatments [12,16]. rough the government initiative to control malaria, the prevalence in some districts remained as low as 4.3% in 2018 [12]. Malaria control strategies including indoor residual spraying along with house to house distribution of mosquito nets treated with insecticides resulted in a remarkable reduction in malaria burdens in many parts of the country [17]. Raouf et al. [18] observed that significant reductions in the levels of malaria in Uganda cannot be sustained if the current control measures are terminated.

Mechanisms of Actions of Novel Phytochemicals in Malaria Treatment
Herbal plants are extremely rich in phytochemicals that are highly efficacious in the treatment of malaria, such as sesquiterpenes and sesquiterpene lactones, fluoroquinolones, chalcones, flavanones, phenolics, quinones, coumarins, and alkaloids (Table 1) [35,36]. e herbal plants that are used as prophylactic measures to prevent malaria as well contain some of these compounds (Table 2). From these groups of compounds, active metabolites including quinine and artemisinin have been derived and the most successful antimalarial drugs to date have been obtained. Artemisinins from Artemisia annua a plant belonging to the family Asteraceae have actually been an integral part of the fight against malaria, with artemisinin-based combination therapy contributing enormously to modern day treatments [36]. ey have been effective against all strains of P. falciparum including multi-drug-resistant ones [36,37]. e mechanism of action of artemisinin is widely debated but the most accepted theory is that of activation of the molecule by heme, which enables it to produce free radicals that then destroy the proteins needed for parasite survival [36]. e presence of an uncommon chemical peroxide linkage bridge in artemisinin, a sesquiterpene lactone, is the most probable reason for its antimalarial effects. Cleavage of the peroxide linkage bridge in the presence of iron (II) ions (from heme) forms very reactive free radicals that undergo rapid rearrangement to form more stable carbon-centered radicals, which chemically modify the parasite and inhibit various processes within the parasite molecules, resulting in its death [36]. Artemisinin acts on primarily the trophozoite parasitic phase and prevents disease progression. It kills circulating ring-stage parasites, thus increasing the therapeutic response [37]. Mok et al. [38] suggested that artemisinin is linked to the upregulation of unfolded protein response pathways, which leads to decreased parasitic growth and development. Shandilya et al. [39] suggested that artemisinin is activated by iron, which then functionally inhibits PfATP6, a calcium pump, by terminating phosphorylation, nucleotide binding, and actuator domains, eventually leading to a functional loss of PfATP6 of the Plasmodium parasite and its death. A study by Mbengue et al. [40] indicated that artemisinin strongly inhibits phosphoinositide-3-kinase (PfPI3K), an enzyme important in cellular activities including growth, multiplication, differentiation, and survival in P. falciparum.
Cinchona tree bark, from which quinine was isolated, has been used to treat malaria since 1632 [41]. e World Health Organization listed quinine as one of the important medicines needed in a health system [42]. It is however only used to treat malaria caused by chloroquine-resistant strain of P. falciparum in the absence of artemisinins [43]. A popular hypothesis about the mechanism of action of quinine is based on chloroquine, another quinoline drug which is closely linked to quinine and has been comprehensively Evidence-Based Complementary and Alternative Medicine studied. Quinine inhibits the pathway of biocrystallization of hemozoin, resulting in the accumulation of the free cytotoxic heme which eventually kills the parasite [44].
Most of the plants used in the treatment of malaria in Uganda contain alkaloids greatly implicated in antiplasmodial activity (Table 3). A number of alkaloids target apicoplast, an organelle in the Plasmodium parasite, while others such as benzylisoquinoline alkaloids in Cissampelos mucronata, a plant belonging to the family Menispermaceae inhibits protein synthesis in the parasite [99].
Flavonoids in a vast number of plants used for malaria treatment in Uganda are common to plants in the family Asteraceae such as B. longipes, A. conyzoides, and A. africana although other herbal plants from different families including C. roseus in Apocynaceae and A. zygia and A. nilotica in Mimosaceae also have them as active antiplasmodial constituents (Table 3). Flavonoids exhibit great antiplasmodial activity against different strains of the malaria parasite although the mechanism of antimalarial action is not clear [99]. Some studies suggest that flavonoids impede the influx of myoinositol and L-glutamine in erythrocytes that are infected [99]. Some flavonoids increase the level of oxidation of erythrocytes and inhibit protein synthesis in malaria parasites [99]. Furthermore, flavonoids are believed to inhibit fatty acid biosynthesis (FAS II) in Plasmodium [102].
Artemisinin resistance in P. falciparum has been reported in Vietnam, Cambodia, Muang Lao, and ailand. A report published in 2018 showed over 30 separate cases in Southeast Asia of artemisinin resistance [36]. In case of resistance, parasitic clearance is slowed down and gametocytemia increases, resulting in greater selective pressure on other partner drugs to which resistance increases, thereby posing a great health threat. us, it is very important that the discovery of other drugs with novel mechanisms of action be prioritized by extensive exploration of the huge medicinal plant resources in Africa, which have been used by locals for effective malaria treatment yet have never been scientifically investigated for their antimalarial potential. Amoa Onguéné et al. [35] emphasized that it was indeed Africa's turn to offer a new antimalarial drug to humanity since artemisinin was discovered in Asia and quinine in Latin America.   Evidence-Based Complementary and Alternative Medicine  [8,25] Evidence-Based Complementary and Alternative Medicine 5  [8,19] Evidence-Based Complementary and Alternative Medicine 7   [8,10] Evidence-Based Complementary and Alternative Medicine 9 10 Evidence-Based Complementary and Alternative Medicine Evidence-Based Complementary and Alternative Medicine 11

Herbs and Plant Parts Used to Manage and Treat Malaria across Communities in Uganda
About 182 plant species from about 63 different plant families are used to treat malaria across several communities in Uganda (Table 1). Of the 63 plant families, species within the family Asteraceae are most widely used in the country to treat malaria, constituting up to 15% of all plant species used (Figure 3(a)). is is followed by species from Fabaceae (9%), Lamiaceae (8%), Euphorbiaceae (6%), and Mimosaceae (4%)    Aloin, anthraquinones, aloe-emodin [56] Evidence-Based Complementary and Alternative Medicine 13  14 Evidence-Based Complementary and Alternative Medicine Tamarindus indica L.

Stem bark
Water Hot water 25.1% chemo suppressive activity at 10 mg/kg (P.

berghei)
Saponins (leaves), tannins (fruits) [78] Flacourtiaceae Trimeria bakeri Gilg. Quinones including bazouanthrone, feruginin a, harunganin, harunganol a [70] Evidence-Based Complementary and Alternative Medicine 15  Polyphenolic curcumin [101] Evidence-Based Complementary and Alternative Medicine  [19,26,32] 18 Evidence-Based Complementary and Alternative Medicine  (3) Low cytotoxicity [97] Evidence-Based Complementary and Alternative Medicine  (Figure 3(a)). e remaining families contribute only 49% of the total plant species used for malaria treatment (Figure 3(a)). e plant parts greatly used to treat malaria are leaves (54.4%) followed by roots (17.4%) and bark (16%); whole plants and other plant parts are used less commonly (Figure 3(b)). A particular herbal plant is commonly used singly though some times in combination with other herbs. e most common way of use is by boiling the medicinal plant part in water and then drinking the decoction; ingestion of fresh extracts and powdered forms of the herbs is also practiced ( Table 1).
Different herbal remedies are used in different communities in different parts of the country depending on the geographical distribution of the medicinal plant species, for example, Warburgia ugandensis is particularly used in the eastern part of Uganda. However, herbal plant species such as Bidens pilosa L. are spread throughout the country and thus well known for malaria treatment across the country. In a study conducted by Ssegawa and Kasenene [20], no tree species in the forests of southern Uganda were more useful than Hallea rubrostipulata and Warburgia ugandensis in the treatment of malaria. ese medicinal plants are known by different local names in different parts of the country as Uganda has diverse ethnic groups, including the Luo, Baganda, Itesots, and Banyankole/Bakiga. Among all communities in Uganda, some measures are taken to control malaria, including draining of stagnant water, clearing and burning bushes, sleeping under insecticide-treated mosquito nets, and house spraying with insecticides.

Mode of Preparation and Use of Herbs in
Treatment of Malaria in Uganda e mode of preparation and use of herbs among different communities vary depending on the nature of the herb and plant parts used for malaria treatment [10]. Most commonly, the herbal medicines are prepared as water extracts in the form of decoction and infusion or as steam baths (Table 1) [19,23]. e herbal plant water extract is made mostly by boiling a handful of the medicinal plant parts such as leaves in a litre of water and then given to the patient to take orally (Table 1) [23]. e dose of the extract given is dependent on the age of the patient and the "strength" of the herbal medicine although occasionally the weight of the patient [19,23]. e quantity of extract given ranges from 100 to 500 ml, 100 to 250 ml, and 1 to 3 tea or tablespoons for adults, older children, and young children below 5 years of age, respectively, between 1 and 3 times a day for about a week or until when patient has recovered [19,25]. e extracts are mostly prepared from single herbal plants or from combination of two herbal plants, for example, a decoction of Tamarindus indica and Mangifera indica is common [25].
In some cases, the medicinal plant parts are dried then pulverized to powder and 2-5 tablespoons of the power added to water and boiled to make a decoction. Some medicinal plant parts such as bark of M. indica stem and roots of V. lasiopus and their powders are boiled for long until the water is half the initial amount [25]. e herbal plant powder can also be added to cold or hot water and stirred and then drunk as recommended [10].
Medicine for malaria treatment from a herb such as B. pilosa can be made by squeezing a handful of its freshly picked leaves and drinking 1-3 teaspoons of the extract a day (Table 1) [23]. Occasionally, malaria herbal medicines can be obtained by preparing different plant parts in combination, for example, an infusion can be made from fresh leaves and pounded fresh roots of V. amygdalina [25].
is is then taken orally in a recommended dose. A handful of medicinal plant parts such as leaves can be squeezed and mixed with cold or warm water for bath, for example, leaves of B. adoensis [25]. Some common herbs are also eaten as vegetables as a prophylactic measure against malaria while others are planted in pots around houses or burnt to drive away mosquitoes (Table 2).

Antimalarial Activities and Toxicity of Herbs Used in Uganda for Malaria Treatment
Some studies have been performed on antiplasmodial/antimalarial activities of some of the herbal plants used in Uganda to treat malaria by using various strains of malarial parasites to confirm effectiveness as malaria treatment [26,28]. Furthermore, a broad range of phytochemicals responsible for biological activities in some of the antimalarial herbs have been isolated and identified [23]. Of the 182 plant species used in Uganda for the treatment of malaria, 112 plant species (64%) have been investigated for antimalarial activities, of which 108 plants showed positive results and only four plant species did not give positive results when tested for antimalarial activities (Table 1). For about 70 plant species (39%) that are used among different communities in Uganda for the treatment of malaria, there was no record of investigation for antimalarial activities (Table 1). e antimalarial activity of herbal plants is due to the presence of a number of metabolically active compounds [23]. ese compounds may occur in the form of alkaloids, sesquiterpenes, quinones, triterpenoids, flavonoids, quassinoids, limonoids, terpenes, chalcones, coumarins, or other miscellaneous forms [85]. e solvent of extraction largely determines the concentrations of the active metabolites in the extract. For example, methanolic extracts of the herbal plants are in general more active in vitro than water extracts probably due to the presence of higher amounts of more active lipophilic compounds (Table 3) [54].
e levels of activity of the antimalarial plant extracts depend on the concentration of the active antimalarial secondary metabolites [54]. For example, gedunin, a very active compound against Plasmodium present in leaves of A. indica had an IC 50 of 0.02 μg/ml against P. falciparum, but its concentration in the plant is in very low and thus moderate activity of its extract (Table 3) [23,54].
Evidence-Based Complementary and Alternative Medicine e synergistic effect of the interaction of the different active secondary metabolites is a main contributing factor to the high levels of antiplasmodial activity of some of the herbal plant extracts, for example, in A. afra, none of the isolated flavonoids and sesquiterpenes had a high activity, yet the plant extract had an IC 50 of 3.9 μg/ml against P. falciparum suggesting a synergistic effect of the compounds in the extract [54]. e presence of particular active compounds in the herbal plant extracts is key in enhancing its antimalarial property. e compound 6Egeranylgeraniol-19-oic-acid a diterpene isolated from M. pyrifolia aqueous extract was considered responsible for its antiplasmodial activity; nitidine isolated from Z. chalybeum had an IC 50 as low as 0.17 μg/ml against P. falciparum 3D7 [10]; and pristimerin with an IC 50 0.5 mg/ml against P. falciparum was the main active ingredient in M. senegalensis extract, making it have a very high antiplasmodial activity [54]. e presence of a moderate amount of a minimum of two secondary metabolites in the extract could explain the efficacy of the herbal extracts for malaria treatment [10]. e pathogenic strains used may be different for different in vitro studies; thus, resistance of the parasite to the active metabolites could cause a variation in the level of antimalarial activity of the extracts [10]. Herbal plants with no antiplasmodial activity suggest the absence of the metabolically active compounds against the Plasmodium parasites in their extracts [23]. Table 4 indicates a list of herbal plants used for malaria treatment in Uganda with high antiplasmodial activities (IC 50 < 5 μg/ ml in one of its solvent extracts or high percentage inhibition of plasmodia) that could be potentially investigated further.
Although herbs are generally considered safer when used for treatment compared to conventional drugs, some of the herbs used traditionally to treat malaria in Uganda may be efficacious, but there is a need to have them used with caution as some may be toxic (Table 4). ere is a variation in degree of toxicity depending on the sensitivity of animals, tissue or cells used, type of extract, nature of the test substance, dose, and mode of administration [114]. According to Lacroix et al. [32] one third of the herbs for malaria treatment in Uganda they investigated had significant antiplasmodial activity with low toxicity. Some of the plant parts with good antiplasmodial/antimalarial activities with no or low toxicity include leaves of A. annua, leaves of A. africana, S. pinnata whole plant, leaves of C. papaya, and leaves of F. virosa amongst others (Table 4).
ere are however extracts of some plants used for malaria treatment with very good activity against Plasmodium but with high toxicity; such plant extracts include petroleum ether leaf extract of V. amygdalina and dichloromethane leaf extract of M. pyrifolia (Table 4) [32,55]. Clerodendrum rotundifolium is on those plants that have very good antimalarial/antiplasmodial activities but have not been investigated for their toxicity (

Traditional Health Care Practice and Policy
Framework in Uganda e health care system of Uganda consists of the public, private-profit oriented, and private-nonprofit oriented sectors. ere is quite a large sector of informal health care including traditional medicine practitioners, drug shops, medicine vendors, and complementary and alternative practitioners. e contribution of traditional health practitioners to Uganda's health care system was not valued until lately [115]. e negative perspective could be traced back to the colonial times when culture including use of traditional medicine such as herbs for treatment was considered primitive and so discouraged [115]. Efforts are now being made to promote the use of traditional medicine since the government has realized that traditional health practitioners are key contributors to its primary health care system [115].
e Ministry of Health created a public-private partnership with the traditional health practitioners following a recommendation that they be brought into the mainstream health system [115,116].
A policy on Traditional and Complementary Medicine was created to regulate traditional medicine practice focusing on research and development while emphasizing the propagation, protection, and sustainable use of medicinal plant resources [115,116]. For collaboration between the mainstream health care sector and traditional health practitioners, the Ministry of Health submitted a bill for the creation of the National Council of Indigenous and Complementary Medicine Practitioners, a semiautonomous body that shall as well protect their intellectual property rights [115,116].
e National Drug Authority (NDA) is a body that ensures quality control of all medical products including herbal medicines in Uganda under the government statute and policy of 1993 [117]. In Uganda, there is no special regulatory measure for herbal medicines in that the same laws and policies for conventional pharmaceuticals also apply to the herbal medicinal products. A policy was introduced in 2002 to have herbal medicines registered, but so far, no registration of any herbal medicine has been made [117].
Herbal medicines though vastly used in Uganda are not sufficiently regulated. A system to license and track traditional health practitioners or their products is still lacking in the country, and the efforts to have the TCM integrated in the mainstream health care system is still a long way from being realized.

Conclusion
Uganda is rich in indigenous plant resources that are used by its people to treat malaria. Communities in different regions of the country use different herbs within their geographical range, though a few common herbs are used by different communities across the country. Many herbs used for 22 Evidence-Based Complementary and Alternative Medicine malaria treatment among several communities have not been investigated for their efficacy, and yet they could be potential sources for antimalarial remedies including drugs. Few studies have been conducted to document herbs for malaria treatment in the country, especially in the northern region. Some of the plants investigated for antimalarial/ antiplasmodial activities have been found to lack efficacy, toxicity, and safety study aspects. Some plants used in the local communities had very strong antimalarial activities and could be investigated further for the identification and validation of the potential therapeutic antimalarial compounds. is review is critical in that it clearly highlights herbal plants documented in Uganda for malaria treatment but have never been investigated for their antimalarial potential, thus providing guidance for further research on potential natural plant resources that could be sources of novel compounds with therapeutic properties for the treatment of malaria.

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