The present study was designed to assess the biological properties of Terminalia mantaly and Terminalia superba as sources of antiplasmodial agents with potential to be further investigated for anti-malarial drug development. The results achieved are summarized and discussed below.
Antiplasmodial activity and cytotoxicity of crude extracts from Terminalia mantaly and Terminalia superba
A total of 12 crude extracts were prepared from the leaves, stem bark and roots of Terminalia mantaly and Terminalia superba by maceration in distilled water and methanol and further assessed for biological activities. Table 1 summarizes the results of crude extracts preparation and biological screening against P. falciparum INDO and 3D7 strains and HEK239T mammalian cells. The dose–response curves schematizing the activity of crude extracts are presented in Fig. 2.
Table 1 Crude extracts preparation yields and activity parameters
Results from Table 1 show that plant extraction yields varied from 7.45 to 35.50% for Terminalia mantaly parts and from 2.27 to 17.18% for Terminalia superba parts. Yields were dependent upon plant species, parts and solvent of extraction. In general, leaves as source and water as extractant gave higher yields than was the case with methanol. Water is environment-friendly, making it an ideal and cheap solvent for the extraction of bioactive and safe principles from any source.
The antiplasmodial activity (expressed as IC50) of crude extracts ranged from 0.26 to > 25 µg/mL with nine extracts showing very promising potency (IC50 < 5 µg/mL). The aqueous extract from Terminalia mantaly stem bark (Tmsbw) showed the best antiplasmodial activity and selectivity, respectively, on PfINDO and Pf3D7 strains with IC50 values of 0.26 µg/mL (SIPfINDO > 769.23) and 1.03 µg/mL (SIPf3D7 > 194.17). For Terminalia superba, the aqueous extract from leaves (Tslw) also showed high antiplasmodial activity and selectivity with IC50 values of 0.57 µg/mL (SIPfINDO > 350.88) and 1.26 µg/mL (SIPf3D7 > 158.73) on PfINDO and Pf3D7 strains, respectively.
With most values hovering around one, resistance index data on crude samples (Table 1) suggests that the antiplasmodial metabolites present in the plant extracts may be equipotent against both chloroquine-sensitive and -resistant strains of P. falciparum. However, Tmsbw (RI: 0.25), and Tslw (RI: 0.45) were clearly three to fourfold more active against PfINDO than Pf3D7, respectively, suggesting interesting possibilities of unique and novel drug targets in the resistant strain. The selectivity indices of crude extracts ranging from > 28 to > 769 µg/mL suggest that the crude extracts not only exhibit potent antiplasmodial potency but also possess great selectivity in their action.
Antiplasmodial activity and cytotoxicity of fractions from the most promising extracts
Four crude extracts showing promising antiplasmodial activity profile were selected and fractionated via successive solid–liquid extractions using three different organic solvents (n-hexane, chloroform, ethyl acetate). These were the aqueous extracts from leaf and stem bark of Terminalia mantaly (Tmlw: IC50PfINDO: 2.09 µg/mL, SIPfINDO > 95; IC50Pf3D7: 2.66 µg/mL, SIPf3D7 > 75; and Tmsbw: IC50PfINDO: 0.26 µg/mL, SIPfINDO > 769; IC50Pf3D7: 1.03 µg/mL, SIPf3D7 > 194), and the aqueous and methanolic extracts from leaf and root of Terminalia superba (Tslw: IC50PfINDO: 0.57 µg/mL, SIPfINDO > 350; IC50Pf3D7: 1.26 µg/mL, SIPf3D7 > 158; and Tsrm: IC50PfINDO: 2.38 µg/mL, SIPfINDO > 84; IC50Pf3D7: 2.28 µg/mL, SIPf3D7 > 87).
Thus, this fractionation led to 3 fractions and 1 final residue for each crude extract, for a total of 12 fractions and 4 final residues that were tested for biological activities as reported in Table 2. The dose–response curves of the tested fractions against P. falciparum INDO and 3D7 are given in Fig. 3a, b.
Table 2 Yields of crude extracts fractionation and activity parameters
Overall, 11 fractions showed antiplasmodial potential against both resistant and sensitive strains of P. falciparum, with IC50 values ranging from 0.36 to 14.09 µg/mL. Four out of those fractions codified TmlwChl, TmsbwChl, TmsbwEA, and TsrmEA exhibited highly potent antiplasmodial activity with IC50 values ≤ 2 µg/mL on both strains, and high selectivity (SI > 109).
The chloroform fraction (TmlwChl) from the leaf aqueous extract of Terminalia mantaly (Tmlw) showed the highest antiplasmodial activity, with IC50PfINDO = 0.36 µg/mL and IC50Pf3D7 = 0.96 µg/mL. Two fractions from the stem bark aqueous extract of Terminalia mantaly (Tmsbw) exhibited high activity mainly against the resistant PfINDO strain, namely the chloroform fraction (TmsbwChl, IC50PfINDO: 0.56 µg/mL against IC50Pf3D7: 1.12 µg/mL), and the ethyl acetate fraction (TmsbwEA, IC50PfINDO: 0.68 µg/mL against IC50Pf3D7: 1.35 µg/mL). These two fractions were found to be about twofold more active against the resistant PfINDO strain than the sensitive Pf3D7, and also showed very good selectivity with SI > 148. The ethyl acetate fraction (TsrmEA) of the root methanolic extract of Terminalia superba was also quite promising (IC50PfINDO: 1.82 µg/mL, IC50Pf3D7: 1.65 µg/mL, SI > 109). Three other fractions from both plants (Terminalia mantaly and Terminalia superba) showed significant activity profiles with IC50 values ranging (2.26–4.93 µg/mL), viz. TmsbwH (IC50PfINDO: 4.50, IC50Pf3D7: 4.40 µg/mL), TmsbwR3 (IC50PfINDO: 2.80, IC50Pf3D7: 2.44 µg/mL), and TsrmChl (IC50PfINDO: 2.26, IC50Pf3D7: 4.93 µg/mL). However, these latter fractions showed moderate selectivity (8 < SI > 71) compared to the more potent ones.
Crude extract fractionation led to fractions and final residues with varied activities against P. falciparum parasites. The overall more potent fraction TmlwChl (IC50PfINDO: 0.36 µg/mL and IC50Pf3D7: 0.96 µg/mL; SI > 208) with an average 4.3-fold activity magnification derived from the crude extract Tmlw (IC50PfINDO: 2.09 µg/mL and IC50Pf3D7: 2.66 µg/mL; SI > 75). Fractionation of Tsrm led to no significant change in activity. Fractionation of Tmsbw (IC50PfINDO: 0.26 µg/mL and IC50Pf3D7: 1.03 µg/mL; SI > 194) slightly reduced the antiplasmodial potency (but led to promising and selective fractions—IC50PfINDO: 0.39–4.50 µg/mL and IC50Pf3D7: 1.12–4.40 µg/mL), whereas fractionation of extract Tslw (IC50PfINDO: 0.57 µg/mL and IC50Pf3D7: 1.26 µg/mL) negatively impacted its potency leading to only two moderately active fractions (TslwH and TslwEA-IC50: 6.89–14.09 µg/mL).
The thresholds for the in vitro antiplasmodial activity of the plant extracts/fractions were based on the classification according to Gessler et al. [15] where the promise of the extract is based on its potency: IC50 < 10 µg/mL (very good); 10–50 µg/mL (moderate) and > 50 µg/mL (low activity). Based on this classification, results from this study indicate that most of the tested extracts and fractions exerted very good activities against both sensitive and resistant strains of P. falciparum.
Very few reports are available in the literature on the activity of the studied plants against P. falciparum. Among those, Ngemenya et al. [16] previously reported the antiplasmodial activity (IC50 of 19.5 µg/mL) of methanolic extract from the leaf of Terminalia superba against the chloroquine-sensitive P. falciparum F32 strain. This value is about five to ninefold greater than that of similar extract tested in this study against PfINDO and Pf3D7. This activity discrepancy might be explained by the difference in parasite strains and approaches used and the specific features of parasites relating to drug susceptibility. Indeed, there might be a relationship between in vitro adaptation to culture of P. falciparum and drug-resistant characteristic of the strain. There is also the possibility of the emergence of a drug-resistant sub-population or of changes in the metabolic pathways during the course of in vitro routine culture maintenance [17]. Likewise, Adewunmi et al. [18] investigated the activity of root and stem of Terminalia superba against Trypanosoma congolense IL 1180 and reported IC50 values of 56.1 µg/mL (root ethanolic extract), 91.73 and 55.26 µg/mL for stem hexane and ethanolic extracts. These findings further highlight the potential of Terminalia superba as a source of anti-protozoan principles.
The antiplasmodial activities of extracts, fractions and isolated compounds from many Terminalia species have been previously reported. However, this study is reporting for the first time the antiplasmodial activity of extracts from Terminalia mantaly. Moreover, it is the first report on the antiplasmodial activity of Terminalia species against P. falciparum chloroquine-resistant INDO strain.
Muganga et al. [19] reported the antiplasmodial activity of Terminalia mollis crude methanolic extract (IC50: 3.84 µg/mL), aqueous extract (IC50: 4.66 µg/mL), ethyl acetate fraction (IC50: 2.10 µg/mL), aqueous fraction (IC50: 19.72 µg/mL) and isolated ellagic acid (IC50: 0.17 µg/mL) against P. falciparum 3D7 strain. Mohd Abd Razak et al. [20] reported the antiplasmodial activity of Terminalia catappa aqueous extract (IC50: 4.28 µg/mL), methanolic extract (IC50: 5.19 µg/mL) and dichloromethane extract (IC50: 5.29 µg/mL) on P. falciparum K1. Abiodun et al. [21] also reported the antiplasmodial activity of hexane, ethyl acetate and methanolic extracts from Terminalia catappa on P. falciparum K1 (IC50: 10.10, 3.05 and 7.42 µg/mL, respectively) and P. falciparum NF54 (IC50: 21.93, 6.68 and 9.40 µg/mL, respectively). Sanon et al. [22] reported the antiplasmodial activity of Terminalia avicennioides aqueous, methanolic and dichloromethane extracts from leaf and stem bark with IC50 values ranging from 1.60 to 7.40 µg/mL on P. falciparum K1. Ouattara et al. [23] reported the activity of Terminalia avicennioides ethyl acetate and butanol crude extracts against P. falciparum K1, while ellagic acid isolated from the leaf showed potent antiplasmodial activity with an IC50 of 0.52 µM. Bavagan et al. [24] previously reported the antiplasmodial activity of Terminalia chebula hexane, ethyl acetate, acetone, and methanolic extracts on P. falciparum 3D7 with IC50 values of 51.91, 67.45, 4.76, 42.98 µg/mL, respectively. They equally highlighted the antiplasmodial potential of aqueous extracts from stem bark and stem wood of Terminalia spinosa on P. falciparum chloroquine-resistant ENT36 (IC50: 29.50 and 49.20 µg/mL) and chloroquine-sensitive K67 (IC50: 9.90 and 35.90 µg/mL) strains.
The findings in this report, together with all previous data, emphasize the potential of Terminalia species to produce secondary metabolites with potent antiplasmodial activity. Furthermore, recent reports on phytochemical studies of Terminalia mantaly mainly showed the presence of phenols, flavonoids, tannins, saponins, and steroids [25, 26]. Many compounds belonging to these classes of phytochemicals have been found to be highly potent against several sensitive and resistant strains of P. falciparum [4, 19, 27,28,29,30]. Moreover, studies on their potential mechanisms of action revealed that phenolic compounds and derivatives are very active as enzymes inhibitors. Examples of such enzymes inhibited by phenolic compounds and derivatives are aspartic proteases, xanthine oxidase, 1,5-lipoxygenase, α-glucosidase, glucose-6-phosphate dehydrogenase, carbonic anhydrase and glutathione-S-transferase [26, 30, 31]. It is noteworthy that nowadays some of the abovementioned enzymes including aspartic proteases, glutathione-S-transferase are clearly identified as potential new targets for drug discovery against malaria and several others metabolic dysfunctions of public health significance including cancer, obesity, epilepsy and gout.