The potential of anti-malarial compounds derived from African medicinal plants, part I: a pharmacological evaluation of alkaloids and terpenoids

Traditional medicine caters for about 80% of the health care needs of many rural populations around the world, especially in developing countries. In addition, plant-derived compounds have played key roles in drug discovery. Malaria is currently a public health concern in many countries in the world due to factors such as chemotherapy faced by resistance, poor hygienic conditions, poorly managed vector control programmes and no approved vaccines. In this review, an attempt has been made to assess the value of African medicinal plants for drug discovery by discussing the anti-malarial virtue of the derived phytochemicals that have been tested by in vitro and in vivo assays. This survey was focused on pure compounds derived from African flora which have exhibited anti-malarial properties with activities ranging from “very active” to “weakly active”. However, only the compounds which showed anti-malarial activities from “very active” to “moderately active” are discussed in this review. The activity of 278 compounds, mainly alkaloids, terpenoids, flavonoids, coumarines, phenolics, polyacetylenes, xanthones, quinones, steroids, and lignans have been discussed. The first part of this review series covers the activity of 171 compounds belonging to the alkaloid and terpenoid classes. Data available in the literature indicated that African flora hold an enormous potential for the development of phytomedicines for malaria.


Background
Malaria is an infectious disease with ravaging effects in the world. The World Health Organization (WHO) has published statistics which reveal that half the world's population is at risk of malaria and that one to two million annual deaths can be attributed to malaria alone [1,2]. Four protozoan species of the genus Plasmodium (Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax) are responsible for this infection, although the majority of fatal cases are caused by P. falciparum [3]. Malaria has been treated with quinine, chloroquine, mefloquine, and artemisinin ( Figure 1), among other drugs. However, the protozoans have developed resistance against many of the current treatment regimens [4]. In the quest to identify new anti-malarial chemotherapeutic agents, many research groups have resorted to plant sources [3,5,6]. This is because of the use of many of these plant materials in the treatment of malaria and fevers in African traditional medicine (ATM) [7]. There has been a general call for the use of natural products as drugs for malaria or as sources of inspiration for the development of novel antimalarials [8][9][10][11] in order to possibly avoid problems related to drug resistance [12].
The African continent is very rich in floral biodiversity and its plant materials are endowed with natural products (NPs) with intriguing chemical structures and promising biological activities. Therefore, the next generation antimalarials or the scaffolds necessary for their synthesis may be found in plants currently used in ATM [13,14]. It should also be mentioned that malaria mostly affects the populations of Africa, Asia and Latin Africa. Asia has offered artemisinin to humanity while Latin America has offered quinine. Many researchers are therefore of the opinion that it is Africa's turn to offer a new antimalarial drug to humanity. Why do we not yet find a (real) anti-malarial drug from Africa? This brings us to the need to have an overview of the anti-malarial/antiplasmodial activity of compounds from bitter African plants (alkaloids and terpenoids). Several research groups in Africa have been involved in the bioassayguided fractionation of plant extracts, leading to the isolation, purification and characterization of a significant number of NPs, some with remarkable anti-malarial activities. The literature survey reported in this work has led to the identification of several vast screening efforts of crude extracts derived from plants used in ATM, harvested from the following countries, just to mention a few: the Democratic Republic of Congo [15,16], Nigeria [17][18][19], Mozambique, Cape Verde, Guinea-Bissau, São Tomé and Príncipe and Angola [20], Mali and São Tomé and Príncipe [21], Madagascar [22][23][24], Congo [25], Benin [26], Burkina Faso [27], South Africa [28], Ivory Coast [29], West African countries [30], Tanzania [31], Kenya [32], and East African countries [33,34].
The potential of plant-derived NPs for anti-malarial drug discovery has been examined in a number of review papers [3,[35][36][37][38][39][40][41]. Other review articles have concentrated on anti-malarials from specific countries/ regions in Africa [19,20,[42][43][44][45][46]. However, there has been no review offering coverage of promising anti-malarials from the entire African continent in the last ten years [13]. In this review series, the potential of plant-derived NPs that could be developed into drugs have been discussed, by giving an overview of the most pertinent in vitro and in vivo screening results reported in the literature.
The compound 17-O-acetyl,10-hydroxycorynantheol (10) was isolated from Strychnos usambarensis (harvested in Rwanda), along with isostrychnopentamine (18), the main alkaloid responsible for the anti-plasmodial activity of the plant, by Cao et al. [48]. The study showed that compound 10 is one of the most promising, monomeric indole alkaloids known to date, showing an in vitro activity against P. falciparum close to 5 μM and a high selectivity.

Naphthoisoquinolines
These compounds are characterized by the C5/C8' linkage between the naphthalene and the isoquinoline portions of these alkaloids (Figure 3). They have been isolated from Ancistrocladus (Acistrocladaceae), Triphyophyllum, Dioncophyllum, and Habropetalum (Dioncophyllaceae) species. The chemical significance of naphthylisoquinoline alkaloids rests on their unique structure and their biological activities [45,46].
The anti-malarial properties of some of these species have been investigated by Bringmann et al. [57][58][59][60][61][62][63][64][65][66]. Regarding the Acistrocladaceae-derived naphthoisoquinolines, compounds 20 to 24, derived from the stems and leaves of Ancistrocladus robertsoniorum growing in Kenya, exhibited moderate anti-malarial activities (IC 50 values from 2.0 to 15.9 μM) against the K-1 and NF54 strains of P. falciparum [57], meanwhile the Tanzanian species, Ancistrocladus tanzaniensis, gave compounds 25 to 29 with IC 50 values ranging from 0.1 to 3.6 μg mL -1 against the K1 strain and between 1.9 and 34.1 μg mL -1 against the 3D7 strain [58]. Habropetaline A (30) and 5′-Odemethyl-dioncohylline A (31) were derived from the roots of Triphyophyllum peltatum, harvested in the Parc de Taï, in west Ivory Coast [59,60]. Both naphthoisoquinolines exhibited interesting anti-plasmodial activities against drug-sensitive and drug-resistant strains of the parasite. Habropetaline A (30) showed very good effect against P. falciparum, without cytotoxicity, with respective IC 50 values of 5.0 and 2.3 ng mL -1 for the strains K1 (chloroquine and pyrimethamine resistant) and NF54 (sensitive to all known drugs). Compound 30 was almost as active as artemisinin (K1: 1.2 ng mL -1 , NF54: 1.2 ng mL -1 ) and is known to be one of the most potent NPs used against P. falciparum [59]. On the other hand, 5′-O-demethyl-dioncophylline A (31) showed improved in vitro anti-malarial activity (IC 50 = 0.340 μg mL -1 ) against the erythrocytic forms of P. falciparum [60]. Jozipeltine A (32), the dimer of the highly hydroxylated  naphthylisoquinoline alkaloid dioncopeltine A (36), was derived from a mixture of root and bark of Triphyophyllum peltatum and Dioncophyllum thollonii, along with twigs of Habropetalum dawei (Dioncophyllaceae), harvested from different regions on the continent [61]. Although this compound showed some in vitro anti-plasmodial activity against P. falciparum (K1 = 875 ng mL -1 , NF54 = 2530 ng mL -1 ), it is significantly less active than its monomeric precursor, dioncopeltine A (36) (K1 = 4.8 ng mL -1 , NF54 = 3.3 ng mL -1 ). This observation could lead to the conclusion that only naphthoisoquinolines containing one phenolic OH group each (such as dioncophylline A (36) and ancistrocladine (28)), could easily undergo the required dimerization reaction, implying that doubling of the number of free OH groups would increase the anti-plasmodial activity [61]. Dioncophyllines A (33), B (34) and C (35) and dioncopeltine A (36) were also active in the in vivo rodent model [66], with dioncophylline C (35) exhibiting a 50% effective dosage (ED 50 ) of 10.71 mg kg -1 day -1 . Four daily treatments with 50 mg kg -1 day -1 were needed to achieve radical cure, one oral dose being sufficient to kill 99.6% of the parasites. Intravenous application of dioncophylline C was shown to be even more effective, with an ED 50 of 1.90 mg kg -1 day -1 and no noticeable toxic effects. Compound 35 also suppressed more established Plasmodium berghei infections when orally applied at day 3 after infection. It should be mentioned that rodent malaria is a well-known animal model for testing new compounds and plant extracts. However, trial in human being is decisive to identify a "hit" as "a real hit"; and this is a good way to assess toxicity and safety. Both dioncopeltine A (36) and dioncophylline C (35) were active against the chloroquine-resistant P. berghei Anka CRS parasites. The naphthoisoquinolines are also known to exhibit other biological activities, e.g. dioncophylline A (33), is the main cytotoxin in Ancistrocladus letestui [67]. The above observations all point to the fact that naphthylisoquinoline alkaloids are promising lead compounds for the development of anti-malarial drugs.

Furoquinolines
This subclass of alkaloids is easily identified with the Vepris, Toddalia and Teclea genera of the Rutaceae family. From the roots of Vepris uguenensis, Cheplogoi et al. isolated flindersiamine (37) and maculosidine (38) [68]. Although compound 37 lacked anti-malarial efficacy against all tested strains, maculosidine (38) exhibited moderate anti-malarial activity against two strains of P. falciparum, with IC 50 values of 29.2 and 40.4 μg mL -1 against the chloroquine-susceptible 3D7 and the chloroquineresistant FCM29 strains respectively. Nitidine (39) has been derived from the roots of Toddalia asiatica harvested in Kenya and modified to yield the reduced derivative 5,6-dihydronitidine (40) [69]. Even though nitidine is mostly known for its potential anticancer properties, the investigations of Gakunju et al. showed the alkaloidal extract of the roots of this plant to have high activity against the chloroquine-resistant K39 strain of P. falciparum, with an IC 50 value of 0.04 μg mL -1 . Further phytochemical analysis on the extract by these authors yielded nitidine as a major compound. In vitro screening against the K39 strain of P. falciparum revealed that nitidine exhibited high antiplasmodial activity, with an IC 50 of 0.045 μg mL -1 , in addition to its known cytotoxic property. In order to remove toxicity, synthetic modification led to 5,6-dihydronitidine (40), with a much weaker anti-malarial activity (IC 50 of 1.03 μg mL -1 , 23 times weaker than nitidine). Evoxine (41), derived from Teclea gerrardii (Rutaceae) harvested from Durban, South Africa, displayed moderate anti-plasmodial activity against the CQS D10 strain of P. falciparum, with IC 50 value 24.5 μM [70].
Another study by Paulo et al. on the roots of Cryptolepis sanguinolenta harvested from Guinea-Bissau led to the isolation of cryptolepinoic acid (62) and methyl cryptolepinoate (63) in addition to 53, 54 and 56 from the ethanol and chlorophorm extracts of the leaves [81]. The isolated compounds and extracts were tested in vitro against P. falciparum K1 (multidrug-resistant strain) and T996 (chloroquine-sensitive clone). All extracts had 90% inhibition of P. falciparum K1 growth at concentrations <23 μg mL -1 . Cryptolepine (53) was the most active alkaloid tested with IC 50 values (0.23 μM to K1; 0.059 μM to T996), compared to chloroquine (0.26 μM to K1; 0.019 μM to T996). The indolobenzazepine alkaloid cryptoheptine (57) was the second most active with IC 50 values of 0.8 μM (K1) and 1.2 μM (T996). Cryptolepinoic acid (62) showed no significant activity while its ethyl ester derivative (63′) was active against P. falciparum K1 (IC 50 = 3.7 μM). All the indoloquinoline alkaloids showed cross-resistance with chloroquine but not the indolobenzazepine cryptoheptine (57). It was noticed that alkaloids with weakly basic characteristics were active whereas other structurally related alkaloids with different acid-base profiles were inactive. These observations are in agreement with the anti-malarial mechanism of action for quinolines. According to Hadden et al., the unusual incorporation of the isopropyl group at the 11-position of the indolo [3,2-b] quinoline nucleus in 11-isopropylcryptolepine (56) is suggestive of a mixed biosynthetic origin for the alkaloid [82].

Bisnorterpenes
Bisnorterpenes with interesting anti-plasmodial properties were purified from the roots of Salacia madagascariensis (Celastraceae), a shrub found in East Africa whose roots are used in the treatment of malaria, fever and menorrhagia specifically in Tanzania [92]. This plant is a rich source of bisnortriterpenes with potent antiprotozoal activity [45]. Four bisnortriterpenes; isoiguesterin (100), 20-epi-isoiguesterinol (101), isoiguesterinol (102) and 6-oxoisoiguesterin (103), were reported from the roots of this plant [92], Figure 11. However, only the first two showed high activity, with respective IC 50 values of 200 and 68 ng mL -1 against the D6 strain of P. falciparum, and 170 and 68 ng mL -1 (against the W2 strain of P. falciparum), respectively.

Acyclic triterpenes
The most active acyclic triterpenes have been found in the stem back of Ekebergia capensis (Zingiberaceae) by Murata et al. [91]. Four triterpenes from the stem bark of this species, comprising two new acyclic triterpenoids, namely ekeberin D4 (104) and D5 (105) (108) isolated from the seeds of Aframomum escapum [87], is an important constituent of essential oils used in the treatment of malaria. This compound is also found in Artemisia herba alba and in lemon grass, and is able to arrest development of the intraerythrocytic stages of the parasite. Compound 108 was identified as the active constituent leading to 100% growth inhibition at the schizont stage [93].

Sesquiterpenes and sequiterpene lactones
Sequiterpenes derived from Vernonia sp. are known to have interesting anti-plasmodial activities. The compounds include vernodalin (121), vernodalol (122), vernolide (123), hydroxyvernolide (124), derived from the leaves of Vernonia amygdalina by Ohigashi et al. [99], in addition to 16,17-dihydrobrachycalyxolide (125) isolated from the leaves of the sister species, Vernonia brachycalyx, as a major anti-plasmodial compound, by Oketch-Rabah et al., Figures 15 and 16 [100]. These compounds exhibited moderate anti-plasmodial activity against the multidrug-resistant K-1 strain of P. falciparum, vernodalin (121)  Quantitative analysis showed that young leaves of this species have a higher concentration of compound 121 than the other derived compounds, suggesting that the anti-malarial efficacy of the leaf extracts of this species may be partly due to the high content of this NP. It has also been reported that dry leaves of Vernonia brachycalyx contain 0.2-0.4% of the sesquiterpene dilactone 125. This compound exhibited moderate to high anti-plasmodial activity against the K39, 3D7, V1/S and Dd2 P. falciparum strains, with IC 50 values of 4.2, 13.7, 3.0, and 16 μg mL -1 , respectively [100]. In spite of the anti-plasmodial activity of this compound, it also had higher toxicity against human lymphocytes, indicating that the anti-plasmodial activity may have been due to the general toxicity the compound had on cells. Despite these observations, the leaves of this species are still used in the treatment of malaria and parasitic infections in East Africa [45]. Ajugarin-1 (126) is another sesquiterpene, which has been reported from aerial parts of Ajuga remota, harvested in Kenya [101]. The compound has exhibited moderate antimalarial properties against the chloroquine-sensitive FCA20/GHA strain of P. falciparum, with an IC 50  causing a delayed effect. In vivo acute (2000 mg kg -1 ) and sub-acute (1000 mg kg -1 ) toxicity tests of the crude acidic water extract did not show toxicity. Moreover, the crude acidic water extract, fractions and pure isolated compounds from Acanthospermum hispidum showed promising in vitro anti-plasmodial activity. Despite the fact that this study did not show in vivo acute and subacute toxicities of the crude acidic water extract, its weak in vivo anti-malarial activity and the in vitro cytotoxicity of pure compounds and enriched extracts containing 130 and 131 indicate that the aerial parts of this plant should be used with caution for malaria treatments [103].
The combined use of bioassay-guided fractionation based on in vitro anti-plasmodial assay and dereplication based on HPLC-PDA-MS-SPE-NMR by Pederson et al. [104], led to isolation of (6S,7R,8S)-14-acetoxy-8-  , revealed the prospect of cultivating Artemisia and eventually using the active principle to offer the population of Burundi a fundamental resource in a country where malaria is endemic [106]. Standard phytochemical analysis techniques, including solvent-solvent extraction, thin-layer-and column chromatography, were used by Becker et al. to isolate a eudesmanolide-type sesquiterpene lactone, dehydrobrachylaenolide (138), as the main active constituent of Dicoma anomala subsp. gerrardii from the Brits region of North West Province of South Africa [107]. The compound demonstrated an in vitro IC 50 of 1.865 μM against a chloroquine-sensitive strain (D10) of P. falciparum. The biological activities of synthetic analogues of compound 138 showed that a methylene lactone group must be present in the eudesmanolide before any significant antimalarial activity could be observed. This feature is absent in the artemisinins and suggests that eudesmanolide-type sesquiterpene lactones have a different mode of action from artemisinins. This hypothesis was further confirmed by microarray gene ontology analysis [107]. The ether extract from aerial parts of Tithonia diversifolia collected in São Tomé and Príncipe demonstrated good antiplasmodial activity (IC 50 of 0.75 μg mL -1 against the FCA strain) and fractionation of this extract yielded the sesquiterpene lactone tagitinin C (139) as an active compound against P. falciparum (IC 50 of 0.33 μg mL -1 against the FCA strain) [108].

Beilshmiedic acid derivatives
Beilschmiedic acid derivatives exhibiting antibacterial and anti-plasmodial activities were obtained from Beilschmiedia cryptocaryoides (Lauraceae) collected from Madagascar ( Table 5). The work of Talontsi et al. [111] led to the isolation of four new beilschmiedic acid derivatives, cryptobeilic acids A − D (149 to 152), and tsangibeilin B (153), Figure 19. Compounds 149 to 153 exhibited anti-plasmodial activity against erythrocytic stages of chloroquine-resistant P. falciparum strain NF54 (with IC 50 values ranging from 5.35 to 17.70 μM) and weak cytotoxicity against L6 cell lines (with IC 50 values ranging from 20.4 to 61.0 μM), the most promising antiplasmodial activity being shown by compound 150.
Another lupane-type triterpene, lupeyl docosanoate (162), was isolated from the bark extract of Hymenocardia acida (Phyllanthaceae) collected in Chad, along with lupeol (161) and β-sitosterol by Mahmout et al. [114]. The anti-malarial property of compound 162 justifies the ethnobotanic use of the plant in the treatment of malaria. Cassia siamea (Fabaceae) was identified from an ethnobotanical survey of southwest Nigeria as a remedy for febrile illness. Bioassay-guided fractionation of stem bark of the plant extract, using the parasite lactate dehydrogenase assay and multi-resistant strain of P. falciparum (K1) for assessing the in vitro anti-malarial activity led to the isolation of emodin and lupeol (161) from the ethyl acetate extract [115]. Both compounds were found to be the active principles responsible for the anti-plasmodial property with IC 50 values of 5 μg mL 1 , for each compound. The compounds 22-hydroxyhopan-3-one (163) and 24-methylene cycloartenol (164) from the stem bark of Entandrophragma angolense (Meliaceae) had moderate activities against P. falciparum W2 [90]. Zofou et al. evaluated the anti-plasmodial activity of betulinic acid (165) from the stem bark of the African St John's wort, Hypericum lanceolatum (Hypericaceae). The compound had an IC 50 of 2.05 μg mL -1 [116]. The n-hexane extract of Psorospermum glaberrimum from Cameroon showed good anti-plasmodial activity against the P. falciparum W2 strain, with IC 50 of 0.87 μg mL -1 [117]. Lenta et al. isolated betulinic acid (165) and friedelan-3-ol (166) from this extract. The measured in vitro activity of compound 165 against the P. falciparum W2 strain gave an IC 50  oic acid (168) was isolated by Zofou et al. [119] from the stem bark of Kigelia africana (Bignoniaceae). This compound exhibited an IC 50 of 0.90 μg mL -1 against the W2 strain of P. falciparum. Cogniauxia podolaena (Cucurbitaceae) is traditionally used in Congo Brazzaville for the treatment of malaria. The anti-plasmodial activity of the plant and some of the isolated compounds responsible for its activity were assessed by Banzouzi et al. [120]. Cucurbitacin B (169), cucurbitacin D (170) and 20-epibryonolic acid (171) were assayed for anti-plasmodial activity (on FcM29, a chloroquine-resistant strain of P. falciparum) and cytotoxicity (on KB and Vero cell lines). The compounds showed respective IC 50 values of 1.6, 4.0 and 2.0 μg mL -1 on FcM29. Compounds 169 and 170 both showed high cytotoxicity whereas 171 showed a better selectivity index.

Conclusions
In this review an attempt has been made to document anti-malarial activities of NPs derived from African medicinal plants. It covers results published until the time of submission of the article. The first part of the review involves naturally occurring, anti-plasmodial/anti-malarial alkaloids and terpenoids while the second part of the review focuses on the remaining classes of compounds. Some of the compounds have been isolated from plants reputed to have a long history of usage in ATM, inferring that knowledge from ATM could be very useful in drug discovery efforts from African medicinal plants. From every indication, recent research efforts on new anti-malarial agents should focus on two main areas: the search for new chemical entities (NCEs) of natural/semisynthetic origin, and the development of phytomedicines [37]. It should be mentioned that African researchers have, knowingly or unknowingly, blown the former avenue out of proportion. This is basically as a result of the fact that most of the research activities on medicinal plants going on in Africa are carried out by academic research groups and the focus is on publications, not application. This calls for the need to develop the necessary applications required to turn acquired knowledge on NPs derived from African medicinal plants into concrete applications in phytomedicine, within an industrial setting. It has been noticed that among the anti-malarials mentioned in this review, most have never been tested for cytotoxicity and very few have been tested for in vivo antoplasmodial activity. Another limitation is the, often small, quantities of compounds isolated from the plants which frustrate ambitions of large-scale screening efforts. Since some complex anti-malarial mixtures derived from plant extracts sometimes loose their anti-malarial properties when pure compounds are isolated, due to synergism of molecules in mixture, the trend towards the development of total extracts into phytomedicines or improved traditional preparations is to be encouraged. Moreover, the isolation and characterization of NPs is an expensive endeavour, not within the reach of the average African research group. However, the attempt to validate ATM remedies as drugs will also face a number of limitations, among which are dosage determinations, variations of the concentration of the active ingredients in the plants with seasonal variations, the rapid loss of tropical forests and the extinction of key species, intellectual property rights management, the intervariability of plant species, quality control, and the conservation of biodiversity. The reconciliation between academic-oriented research and the development of phytomedicines could be feasible with the establishment of African centres of excellence in drug discovery [121], an initiative of the African Network for Drugs and Diagnostics Innovation (ANDI) [122], ATM being a major hub in this endeavour. In order to enhance modern drug discovery efforts from phytochemicals derived from the African flora, a recent effort by the authors of this paper has been to develop virtual libraries including NPs derived from African medicinal plants that have been reported in the literature, for computer-aided drug discovery (CADD). These include the CamMedNP database, containing three-dimensional structures of NPs derived from Cameroonian medicinal plants [123], the ConMedNP database, which covers ten countries in the Central African geographical region, converging the Congo Basin [124] and the AfroDb database, which is a select highly potent dataset, covering compounds with remarkable activities derived from plants across the entire continent [125]. Such databases could serve as starting points for virtual screening (VS) and CADD, leading to the identification of in silico hits, followed by validation by biological assays. These efforts have been in line with the prediction of DMPK profiles of the NPs, with a view to prioritizing hit selection during VS campaigns [125][126][127].