Plant-Derived Antimalarial Agents: New Leads and Efficient Phytomedicines. Part II. Non-Alkaloidal Natural Products

Malaria is still the most destructive and dangerous parasitic infection in many tropical and subtropical countries. The burden of this disease is getting worse, mainly due to the increasing resistance of Plasmodium falciparum against the widely available antimalarial drugs. There is an urgent need for new, more affordable and accessible antimalarial agents possessing original modes of action. Natural products have played a dominant role in the discovery of leads for the development of drugs to treat human diseases, and this fact anticipates that new antimalarial leads may certainly emerge from tropical plant sources. This present review covers most of the recently-published non-alkaloidal natural compounds from plants with antiplasmodial and antimalarial properties, belonging to the classes of terpenes, limonoids, flavonoids, chromones, xanthones, anthraquinones, miscellaneous and related compounds, besides the majority of papers describing antiplasmodial crude extracts published in the last five years not reviewed before. In addition, some perspectives and remarks on the development of new drugs and phytomedicines for malaria are succinctly discussed.


Introduction
Among all parasitic agents causing disease in humans, malaria is undoubtedly the single most destructive and dangerous infectious agent in the developing world [1,2]. This vector-borne infectious disease is a classic example of one that affects the productivity of individuals, families and the whole society, since it causes more energy loss, more debilitation, more loss of work capacity and more economic damage than any other human parasitic diseases [3]. Malaria is commonly associated with poverty, but is also a cause of poverty and a major hindrance to economic development. There were an estimated 247 million malaria cases among 3.3 billion people at risk in 2006, causing nearly a million deaths, mostly of children under 5 years of age. It is widespread in tropical and subtropical regions, including parts of the Americas, Asia and Africa. A total of 109 countries were endemic for malaria in 2008, 45 within the WHO African region [4].
Although human malaria transmitted by female Anopheles mosquitoes has four Plasmodium species as its aetiological agents -P. falciparum, P. vivax, P. ovale and P. malariae, the most widespread and severe disease is caused by P. falciparum, which transiently infects the liver before invading red blood cells of the mammalian host ( Figure 1). Clinical manifestations occur at the erythrocytic stage and can include fever, chills, prostration and anaemia, as well as delirium, metabolic acidosis, cerebral malaria and multi-organ system failure, which may be followed by coma and death [5][6][7].
Quinine (1), an aminoquinoline alkaloid isolated from the bark of Cinchona species (Rubiaceae) in 1820 by Pelletier and Caventou, is one of the oldest and most important antimalarial drugs and is still used today. For almost three centuries, this alkaloid was the sole active principle effective against Plamodium falciparum, and it has been considered the responsible, after the Second World War, for the development of synthetic antimalarial drugs belonging to the classes of 4-and 8-aminoquinolines, such as chloroquine (2,1940) and primaquine (3,1952), among others. Until recently, chloroquine (2) was the only drug used for the treatment of malaria [8,9].
The appearance of drug-resistance P. falciparum strains since 1960, in particular to chloroquine, has made the treatment of malaria increasingly problematic in virtually all malarious regions of the world [2]. Several researchers have dedicated efforts to the development of new active compounds, especially from artemisinin (4), as an alternative to chloroquine (2). Currently no single drug is effective for treating multi-drug resistant malaria, and effective combination therapy includes artemisinin derivatives such as artesunate (5), or mixtures with older drugs such as the atovaquone (6) -proguanil (7) combination Malarone ® [2,10]. Unfortunately first reports on drug resistance to artemisinin-derivatives [11] and to drug combination therapies [12] have already appeared. So, in the absence of a functional, safe and widely available malaria vaccine, efforts to develop new antimalarial drugs continue being urgently needed now.
There is a consensus among the scientific community that natural products have been playing a dominant role in the discovery of leads for the development of drugs for the treatment of human diseases [13]. Indeed, the vast majority of the existing antimalarial chemotherapeutic agents are based on natural products, and this fact anticipates that new leads may certainly emerge from the tropical plant sources, since biological chemodiversity continues to be an important source of molecular templates in the search for antimalarial drugs [14][15][16]. Sporozoites are injected into human dermis through the bite of infected Anopheles mosquito. After inoculation, sporozoites migrate to liver cells to establish the first intracellular replicative stage. Merozoites generated from this exoerythrocytic phase then invade erythrocytes (RBCs), and it is during this erythrocytic stage that severe conditions of malaria occur. The life cycle is completed when sexual stages (gametocytes) are ingested by a mosquito. Some sporozoites deposited in the skin eventually penetrate capillaries or lymph vessels. Those entering the lymph vessels will penetrate lymph vascular endothelial cells in lymph nodes to establish a lymph node form, which appears not to continue the life cycle -but may be significant in priming an immune response. Adapted and reproduced by permission from Macmillan Publishers Ltd. [7].
Few reviews focused on natural products with antiplasmodial activities have been reported in the recent literature. Saxena and co-workers published in 2003 a review article providing a critical account of crude extracts, essential oils and antiplasmodial secondary metabolites with diverse chemical structures from higher plants, collected from the period 1993-2003. A total of 127 alkaloids, 18 quassinoids, 23 sesquiterpenes, 27 triterpenoids, 21 flavonoids/xanthones, nine quinones and 25 miscellaneous compounds were highlighted in their work [8]. The review published by Frederich and collaborators in 2008 covers 31 indole alkaloids isolated from natural sources with high antiplasmodial activity (in vitro and in vivo), most of them displaying IC 50 values under the micromolar range and with a good selectivity index [17]. Kaur and co-authors published in 2009 a review focusing on antimalarial compounds discovered during 1998-2008 from all natural sources, including crude plant and marine extracts. A total of 266 antiplasmodial natural products pertaining to the classes of alkaloids, terpenes, quassinoids, flavonoids, limonoids, chalcones, peptides, xanthones, quinones, coumarins and miscellaneous compounds, as well as 37 promising semisynthetic antimalarials, were listed in this compilation [18]. Fortunately, an increasing number of articles reporting antiplasmodial natural products from plants have been published recently. Considering that a review submitted by Oliveira and co-workers in 2009 deals with plant-derived antimalarial agents pertaining only to alkaloids [19], this review is intended as a continuation of that work, covering most of all recently-published reports of non-alkaloidal natural compounds with antiplasmodial and antimalarial properties from the period 2008-2009 not reviewed yet, in addition to recently published active crude extracts.
Regarding the criteria for considering the in vitro antiplasmodial activity of a given extract or compound as "good", "moderate", "low" or "inactive", earlier studies by Basco and co-workers [20,21] have adopted the following criteria: IC 50 < 10 µg/mL, good activity; IC 50 of 10-50 µg/mL, moderate activity; IC 50 of 50-100 µg/mL, low activity; and IC 50 > 100 µg/mL, inactive. On the other hand, Muriithi and collaborators have expressed their IC 50 values in µM and considered as inactive compounds showing IC 50 > 100 µM; of limited (moderate?) activity, those with IC 50 of 1-20 µM; and of low activity those displayng IC 50 of 20-60 µM [22]. In this paper, both criteria have been combined in order to establish the following criteria adopted for all compounds described in this review: IC 50

Recently-Published Crude Extracts with Antiplasmodial Activity
Although many crude plant extracts with in vitro and in vivo antiplasmodial activities have been reported in the recent literature, only those which were not referred to in previously published reviews [8,18,23,24] are listed in Table 1. In addition, articles with extensive results [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42] were not included in this Table. As it can be noted, the results often show only modest activity against the parasites in vitro or against malaria in mice, suggesting that the species in question probably have only a limited effect in man and that cure of the disease is unlikely. However, this may not necessarily mean that medicines made from these species are of no value, since partially effective treatments might be beneficial in those cases that the course of the disease is shortened by reducing anaemia and lowering the risk of death or serious illness from other anaemia-related diseases. Moreover, benefits may also include the alleviation of symptoms such as pain and fever and immunomodulation leading to increased immunity [43]. Finally, it is important to stress that plant extracts could also be effective against the parasite on hepatic stage.
As commented before, the more recent development of artemisinin derivatives has re-affirmed the potential of plant species to provide effective drugs for the treatment of malaria. The antiplasmodial activity of all plant extracts depicted in Table 1 confirms this statement and suggests that it would be worthwhile to invest more time and resources into further investigation on active plant species identified in these studies.

Antiplasmodial Non-Alkaloidal Natural Products
Although several classes of natural products are responsible for the antiplasmodial activity of many plant species used in traditional medicines for the treatment of malaria, the most important and diverse biopotency has been observed in alkaloids, quassinoids and sesquiterpene lactones [8]. Since alkaloids have been recently discussed in the literature [17,19], this review will highlight the majority of antiplasmodial non-alkaloidal natural products published in the period Jan/2008-May/2009 belonging to the classes of terpenes, limonoids, flavonoids, chromones, xanthones, anthraquinones, miscellaneous and related compounds.

Terpenes and related compounds
Combined use of bioassay-guided fractionation based on in vitro antiplasmodial assay and dereplication based on HPLC-PDA-MS-SPE-NMR methods led to isolation of the sesquiterpene lactones 8-11 ( Figure 3) as the main antiplasmodial constituents of the acetone extract of Distephanus angulifolius (Asteraceae), a scrambling shrub or climber commonly known as "Trailing Vernonia" due to its growth habit, which in South Africa is found from the Eastern Cape to Mozambique. The isolated compounds showed IC 50 values in the range 1.6-3.8 μM and 2.1-4.9 μM against chloroquine sensitive D10 and chloroquine resistant W2 P. falciparum strains, respectively. Compounds 8 and 9 show slightly better effect than 10, with 9 having the lowest resistance index. Moreover, compounds 9 and 10 show a selectivity index 2.8 and 3.6 times higher, respectively, than 8. In this work, resistance index has been defined as the ratio between the IC 50 values of a given compound on W2/D10 strains, while selectivity index has been conceptualized as the ratio between IC 50 values of such substance on D10 strain / Chinese Hamster Ovarian cell line. Under these concepts, a desirable chemotherapeutic lead structure should present both low resistance index and high selectivity index [62].

Figure 5. Antiplasmodial terpenes and related compounds 25-35.
A new indanone derivative 29 and two new diterpenoids 30 and 31 ( Figure 5), together with three known flavonoids, have been isolated from an ethanol extract of the leaves of Croton steenkampianus Gerstner (Euphorbiaceae), commonly known as "Marsh Fever-berry" and "Tonga Croton" in central and southern Africa. The isolated compounds were tested for their antiplasmodial activity and cytotoxicity. Antiplasmodial assays against chloroquine-susceptible strains (D10 and D6) and the chloroquine-resistant strains (Dd2 and W2) of P. falciparum showed that compound 30 gave good activities at 9.1-15.8 μM [66].
From the crude ethyl acetate extract of the medicinal plant Siphonochilus aethiopicus (Zingiberaceae), which showed a very good in vitro activity against the chloroquine-sensitive (D10) and chloroquine-resistant (K1) strains of P. falciparum with IC 50 values of 2.9 and 1.4 µg/mL, respectively, a bioassay-guided fractionation led to the isolation of three novel furanoterpenoids 36-38 (  very good in vivo activity, with a parasitemia reduction comparable to chloroquine. The compounds and crude extract were more active against the K1 strain than the D10 strain of P. falciparum [61]. As Salvia radula (Lamiaceae) exhibited the best antiplasmodial activity (IC 50 = 3.91 µg/mL) among 17 Salvia species used in traditional medicine in South Africa, it was selected for the bioassay-guided isolation of the active compounds. Fractionation of a 1:1 methanol/chloroform extract of this species afforded betulafolientriol oxide (39) and salvigenin (40) (Figure 6), which displayed similar or lower antiplasmodial activity [IC 50 values = 4.95 and 24.60 μg/mL (10.4 and 74.9 μM), respectively] when compared to the crude solvent extract. The loss of activity may be due to synergy that might account for the better activity of mixture than isolated compounds [68].
Pure natural monoterpenes geraniol (43)   From these results, the authors could anticipate arguing that friedelane derivatives might be interesting sources for new potential antimalarial leads [71]. The chloroform-soluble fraction obtained from the methanol extract of Carpesium rosulatum (Asteraceae) was found to have high in vitro antiplasmodial activity (IC 50 = 8.2 μg/mL) against the chloroquine-resistant D10 strain of P. falciparum, this activity being attributed to Ineupatorolide A (61) (Figure 8), which displayed a very impressive antiplasmodial activity (IC 50 = 0.019 µM) [72].
More recently, Korean researchers demonstrated that 61 shows potent in vivo antimalarial activity when tested against P. berghei in mice at doses of 2, 5 and 10 mg.kg -1 ·day -1 , exhibiting a significant blood schizontocidal activity in 4-day early infection, repository evaluation and in established infection with a significant mean survival time comparable to that of the standard drug, chloroquine (5 mg·kg -1 ·day -1 ). According to the authors, compound 61 possesses a promising antiplasmodial activity which can be exploited in malaria therapy [73].
Betulinic acid (62) and its derivative compounds betulonic acid (63), betulinic acid acetate (64), betulinic acid methyl ester (65) and betulinic acid methyl ester acetate (66) (Figure 8) have been evaluated for their antimalarial properties. These substances showed antiplasmodial activity against W2 chloroquine-resistant P. falciparum parasites in vitro, with IC 50 values of 9.89, 10.01, 5.99, 51.58 and 45.79 μM, respectively. Moreover, since betulinic acid acetate (64, SI = 9.6) displayed the best selectivity index among all substances tested, this compound was administered by intraperitoneal route to mice infected with P. berghei, causing a dose-dependent reduction of parasitemia of 70%, while mice treated with chloroquine had undetectable parasitemia. These results indicate that betulinic acid and its derivative compounds might be considered as potential lead compounds for the development of new antimalarial drugs, since the authors suggest that structural changes in the molecule significantly alter the anti-Plasmodium activity [74].

82
Cl Cl Cl Cl Cl followed by compound 80 which contains an aldehyde functional group at this position. In addition, almost all the other compounds were essentially inactive, including plocoralide A (85) which contains a 1-dichloromethyl moiety. The importance of a functional group at position 7 is further emphasized when considering the lack of antiplasmodial activity of other related halogenated monoterpenes [76]. Limonoids are highly oxidized secondary metabolites from Meliaceae species which are obtained through a unique biosynthetic route involving a tetranortriterpenoid nucleus. Numerous biological effects such as insecticidal, insect antifeedant, antibacterial, antifungal, antimalarial, anticancer and antiviral activities have been reported for them [77,78]. From the crude ethanol extract from the bark of Chisocheton ceramicus, Mohamad and co-workers isolated three new limonoids, named ceramicines B-D (89-91), together with ceramicine A (92) (Figure 10). Ceramicine B (89) showed a potent in vitro antiplasmodial activity against P. falciparum 3D7 (IC 50 = 0.56 µM), whereas ceramicines C (90) and D (91) exhibited a relative good activity (IC 50 = 4.83 µM and 5.06 µM, respectively) and ceramicine A (92) displayed a weak activity (IC 50 = 100.37 µM). The higher antiplasmodial activity of compounds 89-91 than that of 92 seems to be attributed to the presence of a tetrahydrofuran ring at C-4/C-6 and C-28 [79].
Starting from the first pharmacological screening of Cogniauxia podolaena (Cucurbitaceae) which confirmed that the roots of this species possesses antiplasmodial activity with IC 50 values lower than 27 µg/mL for all extracts obtained with different solvents, a phytochemical study of this plant was undertaken and three triterpenes have been isolated: cucurbitacin B (93), cucurbitacin D (94) and 20epibryonolic acid (95) (Figure 10). All isolated compounds were shown to have good antiplasmodial activity against the chloroquine-resistant FcM29 strain of P. falciparum, displaying IC 50  constituents responsible for the antiplasmodial activity of C. podolaena roots. In addition, compound 95 showed a better selectivity index than those from 93 and 94, indicating that this compound may be of therapeutic interest for further pharmacomodulation studies [80].
A new bischromone, chrobisiamone A (111) (Figure 11), with a good in vitro antiplasmodial activity against parasite P. falciparum 3D7 (IC 50 = 5.6 µM), has been isolated from the leaves of Cassia siamea, a Fabaceae species widely used in traditional medicine, particularly for the treatment of periodic fever and malaria in Indonesia [83].
In their studies on the screening of medicinal plants produced in Thailand with antimalarial activity against the multi-drug-resistant P. falciparum (K1 strain), Khaomek and collaborators have reported that an ethyl acetate (EtOAc) extract of the stem bark of Erythrina fusca Lour. showed significant antiplasmodial activity (IC 50 = 7.5 µg/mL).  concentration less than 12.5 µg/mL. Diprenylated flavanone 115 (IC 50 = 3.9 µM) displayed the most potent activity among the tested compounds, whereas 112 and 114 did not show such activity, even though these compounds also possess prenylated structures. This result postulates that appropriate introduction of prenyl groups into flavonoids may lead to more useful derivatives for construction of an antimalarial agent [55]. From the stem bark of Pentadesma butyracea Sabine (Clusiaceae), a multipurpose rain forest species commonly called "butter tree" and widely distributed in tropical West Africa including Cameroon, four new xanthones named butyraxanthones A-D (118-121), together with six known xanthones (122-127) ( Figure 13) and a triterpenoid (lupeol), were isolated and assayed in vitro for antiplasmodial activity against the P. falciparum chloroquine-resistant FcB1 strain. Except for compound 121 (IC 50 > 23.3 µM), all of these xanthones exhibited good antiplasmodial activity with IC 50 values ranging from 4.4 to 8.0 µM [84].     antiplasmodial activities against P. falciparum Dd2, with IC 50 values ranging from 1.0 to 5.9 µM, while compounds 135 (IC 50 = 1.7 µM), 136 (IC 50 = 7.9 µM) and 138 (IC 50 = 5.0 µM) also exhibited significant antiplasmodial activities against P. falciparum FCM29 strain [86].
Interactions of two phytochemicals, artemisinin (4) and licochalcone A (140) (Figure 14), have been studied against synchronized erythrocytic stages of chloroquine-sensitive 3D7 and chloroquineresistant RKL 303 strains of P. falciparum. These two compounds in combination showed synergistic antiplasmodial activity in vitro on these strains, provided by their antimalarial action on different metabolic pathways. According to the authors, artemisinin (4) but not licochalcone A (140) was shown to interfere with hemozoin formation, which could be either due to reduction in heme-detoxification or decrease in hemoglobin catabolism. The preferential inhibition of the bc1 complex of P. falciparum compared to that of mammals and the inhibition of new permeation pathways induced by the parasite in the host erythrocyte membrane have been suggested as the mode of action of licochalcone A (140) [87]. Neither of the phytochemicals alone or in combination obstructed sorbitol-induced hemolysis [88].
The effects of a range of common dietary flavonoids on the growth of two strains of P. falciparum have been investigated by Lehane and Saliba [89]. A chloroquine-sensitive (3D7) and a chloroquineresistant (7G8) strain were tested for in vitro susceptibility to a range of individual dietary flavonoids and flavonoid combinations. Of the eleven flavonoids tested, eight showed antiplasmodial activity against the 3D7 strain (with IC 50 values between 11 and 66 µM), and all showed activity against the 7G8 strain (with IC 50 values between 12 and 76 µM). The most active compound against both strains was luteolin (141) (Figure 14), with IC 50 values of 11 ± 1 µM and 12 ± 1 µM for 3D7 and 7G8, respectively. Luteolin was found to prevent the progression of parasite growth beyond the young trophozoite stage, and did not affect parasite susceptibility to the antimalarial drugs chloroquine or artemisinin. Combining low concentrations of flavonoids was found to produce an apparent additive antiplasmodial effect. The authors conclude that flavonoid combinations warrant further investigation as antiplasmodial agents [89].
Cassia siamea L. (Fabaceae), used traditionally in the southwest Nigerian ethnobotany as a laxative, for insomnia, diabetes and hypertension, has been recently identified as an antimalarial remedy [91,92]. This led to the bioassay-guided fractionation of the crude methanol stem bark extract from this plant, using the parasite lactate dehydrogenase assay and multi-resistant strain of P. falciparum (K1) for assessing the in vitro antimalarial activity. Emodin (143) and lupeol (144) ( Figure  14) were isolated from the ethyl acetate fraction by a combination of chromatographic techniques. Both compounds were found to be the active principles responsible for the antiplasmodial property of C. siamea, with IC 50 values of 18.5 µM for 143 and 11.7 µM for 144 [93].

Miscellaneous compounds
The bioassay-guided purification of CH 2 Cl 2 bark extract of Tapirira guianensis (Anacardiaceae) led to the isolation of cyclic alkyl polyol derivatives 148 and 149 (Figure 16), which were active against the chloroquine-sensitive F32 strain (IC 50 = 4.7 µM) and the chloroquine-resistant FcB1 strain (IC 50 = 5.4 µM) of P. falciparum. These results show that it is the 148-149 mixture which is mainly responsible for the activities of this plant extract, since other compounds isolated from T. guianensis did not show any interesting activity against the pathogens tested, although they were chemically quite similar [95].
Fractionation of dichloromethane extracts from the leaves of Piper heterophyllum and P. aduncum afforded three new prenylated hydroxybenzoic acids (181-183), along with six known compounds (184-189) ( Figure 18). Evaluation of the antiparasitic activity for all isolates showed that all the compounds were considered to be moderately active (IC 50 >> 10 µg/mL), except for compound 181, which exhibited a good activity against P. falciparum with an IC 50 of 7.0 µM, in comparison with chloroquine used as a positive control (IC 50 = 0.3 µM). In conclusion, a geranylgeranyl side chain along with a carboxyl acid group as in 181 seem to be relevant for the antiplasmodial activity [99].   Extracts from Holostylis reniformis were tested in vivo against P. berghei and in vitro against a chloroquine-resistant strain of P. falciparum. The hexane extract of the roots was the most active, causing 67% reduction of parasitemia in vivo. From this extract, six lignans 190-195 (Figure 18), including a new (7'R,8S,8'S)-3',4'-methylenedioxy-4,5-dimethoxy-2,7'-cyclolignan-7-one (195), were isolated and assayed in vitro against P. falciparum. The three most active lignans (190)(191)(192) showed IC 50 values of <0.32 µM (<0.12 µg/mL), lignan 192 being the most active compound (IC 50 = 0.20 µM). Lignan 194 exhibited a lower activity (IC 50 = 8.00 µM), whereas lignan 195 did not exhibit any activity under the same experimental conditions. An evaluation of minimum lethal dose (30%) values showed low toxicity for these lignans in a hepatic cell line (Hep G2A16). Therefore, in vivo assays and clinical studies need to be undertaken with these compounds in order to confirm them as potential candidates for the development of antimalarial drugs [100].
Based on previous work which revealed that petroleum ether extracts of Viola websteri (Violaceae) showed inhibition value of 31.7 as percentage of parasite inhibition at 25 μg/mL, this extract was investigated for its activity against the chloroquine-sensitive D10 strain of P. falciparum. Bioassayguided fractionation led to the identification of two structurally related compounds (196 and 197) ( Figure 18) which were shown to be responsible for the observed activity of the extract. The main antiplasmodial principles, 6-(8'Z-pentadecenyl)-salicylic acid (196) and 6-(8'Z, 11'Z, 14'Zheptadecatrienyl)-salicylic acid (197), have been isolated from V. websteri for the first time. In addition, this is the first report on the antiplasmodial activity of these compounds. Although compounds 196 (IC 50 = 10.1 ± 3.2 µM) and 197 (IC 50 = 13.3 ± 6.7 µM) are over 100 times less active than the positive controls, the data suggest that their antiplasmodial activity might not be due to general toxicity [101].
falciparum strains. In contrast with the crude extracts, these isolated compounds have not shown significant antiplasmodial activity against both strains, the lowest IC 50 value (60.5 µM) being obtained for talaumidin (201) against the Dd2-chloroquine resistant P. falciparum strain. These results might be explained by synergistic effects between the different constituents of the complex extract and, especially if the antimalarial activity remains being observed in clinical studies and/or animal models, they could suggest that a standardization of the bark extract might be the best solution to a rational use of this traditional antimalarial plant [102].

Perspectives and Remarks on the Development of New Drugs and Phytomedicines for Malaria
The research on new antimalarial agents presently faces two distinct challenges: the search for new chemical entities (NCE) of natural or synthetic origin, and the development of phytomedicines [19]. The antiplasmodial/antimalarial natural products focused in this review can be divided into two groups: one of highly active compounds, of complex structures, for which no possibility of practical synthesis can be forseen, and another one, of moderate to low activity but of relative simple structures and, therefore, their synthesis and/or of their analogues could be undertaken. Plant species producing compounds of the first group are potential candidates for the development of phytomedicines, while the second group would represent templates to synthetic drugs.
Will the research on traditional plants contribute for the discovery of new antimalarial drugs? There is no doubt about this possibility. Atovaquone, quinine, artemisinin and its semi-synthetic derivatives are remarkable examples of the diverse contribution of natural products for the development of effective antimalarial drugs, particularly valuable for the treatment of chloroquine-resistant parasites.
Indeed, several potent antiplasmodial natural products have been described, as shown in this review, most of them have only been evaluated by in vitro assays and few of them were evaluated for cytotoxicity and still smaller is the number of those assayed in vivo. Besides, many of them are found in low concentrations in the plant species and usually as part of complex mixtures making their isolation and purification highly expensive. These are typical situations which point to the development of phytomedicines, the known active compounds being useful as chemical-biological markers to guarantee product quality.
Validation of traditional plant remedies presents limitations such as prioritization of plant species for research, lack of informations on ethnobotany of these plants (location and abundance, parts used, form of use, duration of treatment), definition of dosages due to variation on the concentrations of active ingredients in a plant species [103]. It should also be stressed that a basic requirement for the validation of a medicinal plant is the standardization of extracts to be evaluated, including the identification and quantification of chemical and/or biological markers to assure the development of efficient and safe phytomedicines in a short time and at low cost. It is well known that the qualitative and quantitative contents of secondary metabolites in a plant are susceptible to marked variation that is regulated by intrinsic factors (ontogeny and phenology) as well by abiotic (e.g. light, moisture, nutrient availability) and biotic factors such as different physiological and growth stages [104], which make the standardization obligatory.

Conclusions
This work reviewed most recently-published non-alkaloidal natural compounds from plants with antiplasmodial and antimalarial properties, besides the majority of antiplasmodial crude extracts published in the last five years. Considering the many antiplasmodial activities observed for all crude extracts and natural compounds described here, it can be stated that in fact there are optimistic perspectives on the continuing investigation of plants used in traditional medicines for the treatment of malaria, and they will certainly lead the scientific community to the discovery of more new efficient molecular templates and phytomedicines for this disease. Malaria and other neglected diseases, such as Chagas' disease, leishmaniasis and African trypanosomiasis, among others, have a devastating impact on the world's poor. Unfortunately they have been progressively marginalized by those in charge of making research programme decisions both in public and private sectors, since people suffering from these diseases have not offered a market lucrative enough to attract any notable investment in research and development for new drugs. Thus, this work also intends to stimulate and bring together new and intensive efforts from all research communities of the world to the quest of efficient phytomedicines and novel potential drug candidates both for malaria and other neglected diseases.