Background
In the Amazon region, the occurrence of malaria is related to demographic, ecological, socio-economic, and cultural changes, especially in the first half of the twentieth century. In this period, relatively few plants from this region were used to treat this disease [
1,
2]. Over time, the co-existence of traditional populations with this disease caused a search for new therapeutic resources in the Amazonian environment, especially among plants, to treat the symptoms of malaria. Nowadays, this traditional knowledge, available through ethnopharmacological studies, is the most often used means to target plants for the discovery of new bioactive substances. The ethnopharmacological approach has led to the saving of time and financial resources as compared to other approaches, such as chemosystematic or random plant selection [
3,
4]. The chemosystematic approach for the selection of anti-malarial plants for study is also valid especially where ethnopharmacological studies have shown a plant family, or a particular genus, to contain anti-malarial extracts and chemical constituents.
Natural products are the origin of approximately two-thirds of all drugs introduced in the past 30 years [
5]. Plants are recognized as important sources of antiprotozoal compounds for the development of drugs against many tropical diseases, including malaria. Examples of anti-malarial natural products are (1) quinine, present in western Amazonian
Cinchona spp., (2) quassinoids and limonoids in plants of the Simaroubaceae and Meliaceae families, respectively, and, (3) artemisinin from
Artemisia annua, among others [
6,
7].
The Amazon region is megadiverse. Screening for in vitro and in vivo anti-malarial activity of extracts of traditionally used plants from this region is a strategy for the discovery of new anti-malarial substances [
8‐
10]. Studies on the anti-malarial activity of plant species from countries of the Amazon region such as Bolivia [
11‐
15], Brazil [
16‐
29], Colombia [
30], French Guiana [
31‐
34], and Peru [
35‐
38] have demonstrated the potential of local traditional medicinal practices as sources of potent extracts and anti-malarial substances.
Krettli and collaborators performed ethnobotanic surveys of anti-malarial plants across the Brazilian Amazon region and applied the ethnopharmacological approach to the study of these plants for the first time [
1,
39]. Ethnopharmacology provided a relatively large number (4 in 22 plants or 18 %) of plants exhibiting extracts with in vivo efficacy against
Plasmodium berghei compared to an approach based on random selection of plants (two active plants in 273 tested or 0.7 %) [
40,
41].
In the present work, after a systematic literature search, 11 Amazonian plants were selected based on their use as anti-malarials or based on the proven anti-malarial activity of the plant genus. No previous report on the activity against
Plasmodium parasites was found for the selected plant species. Their extracts were assayed for in vitro and in vivo anti-malarial activity and cytotoxicity. The aim of this study was to discover Amazonian plant extracts exhibiting important in vitro and in vivo anti-malarial activity as a first step towards bioguided isolation of active principles. The plant species studied are shown in Table
1.
Table 1
Information on plant species, voucher specimens, traditional remedies and ethnobotanic sources indicating anti-malarial use
Anacardium occidentale
| Anacardiaceae | INPA 57941 | Cajueiro | Bark, leaves, fruit infusions, decoction (10 drops 2×/day of trunk bark alcohol extract) | |
Andropogon leucostachyus
| Poaceae | INPA 250467 | Capim-colchão | Whole plant decoction | |
Clidemia bullosa
| Melastomataceae | INPA 250466 | Caiuia | Not founda
| Not founda
|
Croton cajucara
| Euphorbiaceae | EAFM 315 | Sacaca | Bark and leaves infusions | |
Derris floribunda
| Fabaceae | INPA 15562 | Timbó | Branches | |
Miconia nervosa
| Melastomataceae | INPA 250467 | Miraúba | Decoction (part not specified) | |
Parkia nítida
| Fabaceae | INPA 152124 | Faveira | Not specified | |
Paullinia cupana
| Sapindaceae | INPA 122001 | Guaraná | Leaves, branches, roots, seeds | |
Stigmaphyllon sinuatum
| Malpighiaceae | INPA 205629 | Cipó asa de gafanhoto | Leaves decoction | |
Xylopia amazonica
| Annonaceae | INPA 183108 | Envira sarassará | Not foundb
| Not foundb
|
Zanthoxylum djalma-batistae
| Rutaceae | INPA 210077 | Tamanqueira | Not foundc
| Not foundc
|
Discussion
Several strategies are available for the discovery of new anti-malarial drugs. In vitro screening for inhibitory activity against
P. falciparum and identification of traditionally used plant extracts exhibiting IC
50 values less than 10 µg/mL are important first steps in the search for new anti-malarial plant extracts. Similar approaches have led to the identification of extracts for chemical composition studies and the discovery of potent plant natural products, such as artemisinin and nimbolide [
56,
57].
In traditional medicine,
Andropogon
leucostachyus whole plant decoctions are ingested as a treatment for malaria [
58]. In this work,
Andropogon leucostachyus aerial part decoctions exhibited low in vitro activity. Methanol extraction was the most efficient process for concentrating the in vitro (IC
50 = 7.1 ± 3.3 µg/mL against
P. falciparum) anti-malarial activity and selectivity of
Andropogon leucostachyus. In fact,
Andropogon leucostachyus aerial part methanol extracts exhibited the highest selectivity index (SI = 28.2) of all extracts evaluated herein. The concentration of anti-malarial components in these extracts is further attested to by the in vivo result (71 % suppression of
P. berghei). Interestingly, the leaf decoctions of a related species,
Andropogon schoenanthus, are ingested (with large amounts of sugar) to treat malaria fevers. Inhalation of the vapours from the boiling decoction is also used to treat malaria [
59]. Very little is known about the chemical composition of
Andropogon leucostachyus.
C-glycosylflavones, the
O-methyl flavone tricin and the flavanol luteoforol have been described in the leaves of
Andropogon leucostachyus [
60]. No anti-malarial activity has been reported for these flavonoids in the literature. In silico docking studies have explored the potential of tricin as a parasite dihydrofolate reductase inhibitor however it was found to interact less favorably with this enzyme than other compounds [
61].
Croton cajucara is a cultivated plant that has red and white varieties (a reference to the coloration of young leaves). It occurs in Bolivia, Brazil, Guyana and Venezuela.
Croton cajucara trunk bark or leaf infusions are used in traditional medicine to treat malaria according to many sources [
62‐
68]. Herein,
Croton cajucara extracts of both varieties were active or moderately active in vitro. Red variety leaf chloroform extract exhibited the highest in vitro inhibitory activity against
P. falciparum W2 clone (IC
50 = 6.4 ± 1.2 µg/mL). This extract was further evaluated for in vivo oral activity against
P. berghei in infected mice, however, it exhibited low in vivo anti-malarial activity (Table
3). Synergism among the bioactive components that comprise an extract could explain in vitro antiplasmodial activity however in vivo these chemical constituents may have a lessened effect due to their metabolism (biotransformation), low bioavailability and physiological factors in the host [
69].
A number of
Croton species have been found in previous studies to exhibit significant in vitro and in vivo anti-malarial activity.
Croton leptostachyus aerial part ethanol extracts exhibited high in vitro activity against
P. falciparum (IC
50 = 2.1 ± 0.2 µg/mL), however, this extract was toxic to mice [
30]. Several
Croton zambesicus root extracts and fractions exhibited in vivo anti-malarial activity (79–86 % parasitaemia suppression at doses of 27–81 mg/kg/day) against
P. berghei in rodents [
70]. Also,
Croton mubango stem bark water extracts inhibited
P. falciparum in vitro (IC
50 = 3.2 µg/mL) and suppressed
P. berghei ANKA by 77 % at oral doses of 200 mg/kg/day [
71]. Significant dose dependency in the suppression of
P. berghei in mice has been observed for
Croton macrostachyus water and methanol extracts (200, 400 and 600 mg/kg) [
72]. Similar results were obtained for crude extracts and chloroform, methanol and water fractions of this same species wherein the chloroform fraction exhibited the best result [
73].
A number of antiplasmodial diterpenes have been isolated from
Croton species. 8,9-secokaurane was isolated from
Croton kongensis and inhibited
P. falciparum K1 strain (IC
50 = 1–2.8 µg/mL) [
74] and geranyl geraniol was isolated from
Croton lobatus extracts and inhibited
P. falciparum (IC
50 = 3.7 µM) [
64]. Steenkrotin A, was isolated from
Croton steenkampianus leaf ethanol extracts and exhibited IC
50 = 15.8, >30, 9.4 and 9.1 µM against
P. falciparum D10, D6, Dd2 and W2 clones, respectively [
66].
Miconia nervosa is used traditionally in the treatment of malaria as a decoction as are
Miconia laevigata and
Miconia willdenowii [
58,
62,
75,
76]. Herein,
Miconia nervosa leaf extracts exhibited in vitro activity against
P. falciparum W2 clone. No previous report on the antiplasmodial activity of extracts of a species of
Miconia is available in the literature. Interestingly, other species from this genus,
Miconia fallax and
Miconia stenostachya, are known to produce triterpene compounds that inhibit the protozoa
Trypanosoma cruzi [
77].
Xylopia amazonica was revealed herein as a plant whose crude extracts have anti-malarial potential. Two of its extracts were active and a third was moderately active. Among these, the leaf chloroform extracts exhibited good in vitro antiplasmodial activity (IC
50 = 7.3 µg/mL) and no significant toxicity to human fibroblasts or melanoma cells was observed. Notwithstanding, these extracts exhibited low selectivity (SI = 4.6), which is an indication that chloroform extraction concentrates specific toxicity to
P. falciparum and murine macrophages. Interestingly, cytotoxicity has been observed for the extracts of
Xylopia aromatica trunk chloroform–methanol extracts against NCI-H460, KM-12 and SF-268 cell lines and cancer cell line RPMI-8226 [
78]. Also,
Xylopia aromatica wood hexane extracts exhibited IC
50 = 5–20 µg/mL against several tumour cell lines (SF-295, HCT-8, MDA-MB-435 and HL-60) [
79].
In several countries, the macerated or infused fruit and/or trunk bark of at least a dozen
Xylopia species are used to treat malaria [
30,
58,
80‐
85]. The extracts of several of these plants exhibit in vitro antiplasmodial activity according to previous studies.
Xylopia phloiodora and
Xylopia aethiopica extracts inhibit
P. falciparum in vitro (IC
50 = 18 µg/mL) [
80].
Xylopia emarginata leaf [
81], root bark, trunk bark and wood [
86] extracts exhibit IC
50 = 3–11 µg/mL against
P. falciparum Palo Alto or FcB1 strains. The ethanol extract of the aerial parts of
Xylopia aromatica strongly inhibit
P. falciparum in vitro (IC
50 <1 µg/mL) [
30] while root, root bark and trunk bark hexane extracts do so to a lesser extent (IC
50 = 4.7, 6.8 and 15.3 µg/mL, respectively) against
P. falciparum FcB1 strain [
86].
Besides the in vitro antiplasmodial activity observed,
Xylopia amazonica leaf chloroform extracts administered orally were able to suppress (52 %)
P. berghei in mice at daily doses of 250 mg/kg herein. For a related species,
Xylopia aromatica, it was found that aerial part ethanol extracts strongly inhibited
P. falciparum in vitro (IC
50 <1 µg/mL) but were inactive in vivo [
30].
Xylopia amazonica is known to produce kaurene diterpenes and aporphine alkaloids. From the wood and or bark, the diterpenes beyerene,
ent-kauran-16β-ol,
ent-kaur-16-en-19-oic acid (kaurenoic acid) and 4-
epi-kaurenoic acid have been isolated [
87,
88] as have the aporphine alkaloids liriodenine, dicentrinone [
87], oxoglaucine, (+)-glaucine, lirioferine and (+)-laurotetanine [
87,
88].
Importantly, several of the natural products isolated from
Xylopia amazonica have been isolated from other plant species and found to exhibit in vitro antiplasmodial activity. Thus, (+)-laurotetanine (isolated from
Nectandra salicifolia) exhibited IC
50 values of 3.9 and 2.5 μg/mL against
P. falciparum D6 and W2 parasite clones [
89]. Dicentrinone from another species inhibited
P. falciparum K1 strain (IC
50 = 1.2 μg/mL) [
90]. Liriodenine inhibited
P. falciparum D6, D10 and W2 clones (IC
50 = 1.3, 4.1 and 2.4 μg/mL, respectively) [
91,
92] whereas oxoglaucine exhibited low activity [
92]. The diterpene
ent-kaur-16-en-19-oic acid was not active against chloroquine-sensitive
P. falciparum D10 clone (IC
50 = 31.8 μg/mL) [
93].
Clidemia hirta known as soap bush or Koster’s curse, is used as an anti-malarial among the traditional peoples of the Peruvian Amazon [
38]. Herein, three
Clidemia bullosa extracts exhibited moderate in vitro antiplasmodial activity. There is no information referring specifically to the traditional anti-malarial use of this species. However,
Clidemia bullosa,
Clidemia hirta and another species are closely related and occur together [
94]. No information is available on the chemical composition of
Clidemia bullosa, however, recent work on the chemical composition of the related species,
Clidemia hirta, revealed the presence of hydrolysable tannins, derivatives of ellagic acid and the triterpene arjunolic acid [
95].
Most
Paullinia cupana (guaraná) extracts did not exhibit antiplasmodial activity. Bark and fruit chloroform extracts of this plant exhibited only moderate antiplasmodial activity. Guaraná extracts are widely consumed in the form of soft drinks and other beverages in Brazil. This plant is used in anti-malarial remedies in different locations in Latin America [
58,
63,
96,
97]. In the branch bark, catechin and epicatechin have been detected [
98]. These compounds may contribute, together with other compounds, to the moderate antiplasmodial properties observed [
99].
Zanthoxylum djalma-
batistae leaf infusion and branch chloroform extracts exhibited moderate antiplasmodial activity (IC
50 = 15.6 ± 2.9 and 17.4 ± 1.3 µg/mL, respectively) herein. More than a dozen
Zanthoxylum species are reported to be used as anti-malarials in several countries [
33,
59,
84,
85,
100‐
110] and antiplasmodial activity has been observed for extracts of these plants. Thus,
Zanthoxylum
chalybeum extracts inhibit
P. falciparum in vitro (IC
50 <10 μg/mL) [
100,
102,
104,
107]. Also,
Zanthoxylum
usambarense trunk bark and trunk wood methanol extracts inhibit
P. falciparum NF54 strain (IC
50 <5 µg/mL) [
103].
There is no information on the chemical composition of
Zanthoxylum
djalma-
batistae. Antiplasmodial benzophenanthridine alkaloids such as nitidine are found in
Zanthoxylum and other Rutaceae species. Nitidine has been isolated from
Zanthoxylum
usambarense and
Zanthoxylum rhoifolium [
33,
111] and exhibits submicromolar IC
50 values against
P. falciparum [
33,
112].
Use of cashew tree (
Anacardium occidentale) trunk bark, leaves or fruit in the treatment of malaria symptoms is practiced by traditional peoples in Brazil, Colombia, Nigeria and Peru [
58,
68,
82,
96,
113].
Anacardium occidentale leaves are reported to contain anacardic acid and cardol [
58,
114]. Anacardic acid is believed to alter parasite gene expression and inhibit parasite development in vitro through enzyme inhibition. In vitro this compound was inactive (IC
50 = 30.4–34.8 µM) against a number of
P. falciparum strains (3D7, D10, 7G8 and Dd2) [
115].
Anacardium occidentale extracts were in general inactive in the present study.
Surprisingly,
Derris floribunda extracts were inactive herein despite the traditional use of timbó as an anti-malarial and previous reports on the antiplasmodial activity of
Derris species.
Derris amazonica extracts have been previously shown to inhibit
P. falciparum F32 strain in vitro (IC
50 = 3.2 µg/mL) [
15] and lupifolin has been isolated from
Derris trifoliata seed pod extracts and inhibits
P. falciparum D6 and W2 strains (IC
50 = 2.6–3.7 µg/mL) [
116].
In animal models of malaria, large experimental variability of the results is associated with drug, parasite and host interactions. For ethical reasons, the numbers of animals used may not be increased to more accurately characterize the antiparasitic effects. Despite this low experimental reproducibility, the murine malaria model used herein is an important tool in anti-malarial drug discovery and development programmes.
Methods and criteria vary among research groups that investigate the anti-malarial potential of plants using rodent models. Extract doses of 300–650 mg/kg/day providing 47.0–84.5 % parasitaemia suppressions have been considered evidence of important anti-malarial activity [
22,
117‐
119]. Also, extracts providing >60 % suppression of parasitaemia at oral doses of 100–250 mg/kg/day in the rodent model have been deemed active or highly active and suppression >30 % at these doses has been deemed moderate activity [
120‐
123]. In the present work, oral doses of 250 mg of plant extract per kg of body weight per day were used for evaluation of plant extracts and detection of relevant parasitaemia suppression (>30 %) on the fifth and or seventh days in the rodent model.
Herein, significant in vivo oral suppression of
P. berghei by
Andropogon leucostachyus and
Xylopia amazonica extracts was demonstrated. Significant differences were not observed in the mean survival of animals treated with these extracts and untreated controls. This is true even for
Andropogon leucostachyus extracts that were responsible for the largest suppression of parasitaemia observed. In the antiplasmodial extracts tested in vivo, the substances responsible for the suppressive activity may be present in low amounts and may exhibit short half-lives, thus not attaining the concentrations necessary for parasite suppression after the end of treatment. This together with rapid parasite metabolism may provide extract-treated and control groups exhibiting equal parasitaemia after only a few parasitic cycles, which for
P. berghei is 24 h [
124].
The in vitro inhibition of P. falciparum and selectivity demonstrated by several plant extracts and oral suppression of P. berghei by Andropogon leucostachyus and Xylopia amazonica extracts are significant findings. Bioguided fractionation of several of the extracts revealed in this study is now underway and should reveal the anti-malarial chemical constituents of these plant species in the future.