Protozoal diseases, especially malaria, leishmaniasis, and Chagas disease, are major causes of mortality in various tropical and subtropical regions [1]. Moreover, treatment failure is increasingly common with current drugs. The resistance of Plasmodium falciparum, for example, to the former first-line antimalarials, chloroquine and sulfadoxine/pyrimethamine, has reached critical levels in many malaria-endemic regions and is producing deleterious effects on human health, wealth, and lifespans. Recent reports that malaria parasites have begun to develop tolerance to the artemisinin-based combination therapies threaten the last mainstay for treating uncomplicated malaria in endemic countries [2,3]. Furthermore, no new class of antimalarials has been introduced into clinical practice since 1996 [4]. Worse, chemotherapy of visceral leishmaniasis and Chagas disease still relies on first-line drugs that are old and far from satisfactory because of their major side effects, limited efficacy, and difficulty of administration. New more effective chemotherapeutic agents with novel modes of action are needed, and marine biodiversity might be a source of chemodiversity for that purpose.

Marine biodiversity contains resources composed of a variety of biologically active compounds [5–7]. Marine macrophytes produce an array of secondary compounds [8] still underexploited for their biomedical potential although they are a particularly available biomass. Studies of tropical marine macrophytes have shown their extensive biological activity, including antiprotozoal [9–12], but very little is known about Western seaweed species.

As a part of a screening program to search for new natural antiprotozoal products, we report here the in vitro screening of 35 polar (hydroalcoholic) and apolar (ethyl acetate) extracts from 20 species of seaweeds from the Normandy (France) coast against cultured protozoa responsible for human malaria, visceral leishmaniasis, and Chagas disease.

The sampling resulted in the selection of 20 species of seaweeds that were brown (8), red (9) and green algae (3). The species were collected from rocky habitats along the Normandy coast in northern France (Table 1).

The potential antiplasmodial activity of the resultant ethyl acetate and hydroalcoholic extracts was evaluated in vitro against erythrocytes infected by a resistant K1 strain of P. falciparum, as well as against T. cruzi trypomastigotes and Leishmania donovani amastigotes (Table 2). Extracts were first screened at two concentrations (1.6 and 9.7 μg/mL), and parasite growth inhibition was measured. Extracts for which parasite growth inhibition was greater than 50% at the concentration of 9.7 μg/mL were subsequently assayed to determine their IC₅₀. An extract was considered as active if the IC₅₀ value was less than 5 μg/mL. Cytotoxicity to primary mammalian L6 cells was also evaluated to determine the selectivity of its activity. Table 3 presents the IC₅₀ values and selectivity indexes (ratio of cytotoxic to antiprotozoal activity).

The active extracts were almost entirely (97%) ethyl acetate extracts, while the hydroalcoholic extracts were mainly inactive. This finding suggests that the active antiprotozoal compounds were relatively apolar, except for the hydroalcoholic extract of C. crispus, which was quite active against L. donovani (95% inhibition of parasite growth at 9.7 μg/mL).

P. falciparum was the pathogen most responsive to these extracts: 40% of the extracts showed activity against this protozoon. L. donovani was less sensitive (11% of the extracts were active against it) and T. cruzi quite insensitive (3%). Red and brown seaweeds were almost equally active against P. falciparum and L. donovani, but green seaweeds were inactive (Figure 1).

Few studies have reported antiprotozoal screening of marine algae, and those that did looked at tropical and Asian species [13,14], most often individually [5,6]. Trypanocidal and leishmanicidal activity was recently assessed in British and Irish species [15,16]. Those studies found that organic extracts from several British green algae species were active, although weakly, against the same strains of T. cruzi and L. donovani that we used here [16]. U. lactuca, the only green algae common to their study and ours, was inactive in ours at 9.7 μg/mL, despite similar experimental conditions. This discrepancy may stem either from the different extraction solvents or from their different phytochemical compositions, probably due to the different habitats of the two samples.

Brown tropical algae have previously been shown to be moderately active against L. mexicana promastigotes, while red and green algal organic extracts were almost inactive, as in our study [14]. Here, the most active extract against L. donovani axenic amastigotes was the ethyl acetate extract of B. bifurcata, which had an IC₅₀ value of 3.9 μg/mL. Nevertheless, the selectivity index of 1.6 found for this extract seems to indicate general toxicity. Moreover, crude organic extracts of Irish B. bifurcata were recently shown to be active against both L. donovani and T. brucei rhodesiense, the protozoan parasite responsible for sleeping sickness [15]. B. bifurcata contains arrays of terpenoids [17–19] with cytotoxic activity [19,20]. The ethyl acetate that we used here efficiently extracts this type of compound. Their presence could explain the absence of selectivity.

Our results provide further evidence that marine seaweeds may be active against protozoa, especially Plasmodium species. Investigations of red algae species are rare, still more so against P. falciparum. We showed here, for the first time, in vitro antiplasmodial activity by eight French species of red algae: C. jubata, C. crispus, D. carnosa, G. latifolium, G. gracilis, G. turuturu, H. flosculosus, and M. stellatus. Moreover, the IC₅₀ values measured for these extracts reflect activity similar to that reported for such reference extracts as ethanolic crude extracts of Artemisia annua (Quinguao) and of Azadirachta indica (Neem) in the same in vitro microdilution test under similar experimental conditions [21,22].

Interestingly, Plasmodium parasites contain a cellular peculiarity, called the apicoplast, a rudimentary plastid that was acquired long ago by secondary endosymbiosis between a free-living ancestor of Apicomplexans and a red algal species [23]. Accordingly, this organelle probably shares several metabolic pathways and housekeeping processes with red algae. We can hypothesize that some algal compounds contained in these extracts kill malaria parasites by interfering with these common pathways or processes.

The most active and interesting extract was the ethyl acetate extract of M. stellatus, which had the best IC₅₀ and SI values (2.8 μg/mL and >30, respectively). Its selectivity towards P. falciparum was also good, compared with the other protozoan parasites we tested. To our knowledge, no antiplasmodial activity has previously been shown for Mastocarpus species. Moreover, phytochemical data for these genera are sparse. Nevertheless, some tropical species of red algae have been shown to contain such antiplasmodial compounds as halogenated diterpenes [6] or bromophycolides [5,24]. Bioguided fractionation associated with dereplication techniques should allow us to identify new antiplasmodial compounds from this species and thus help us to determine its mode of action.

Almost all the marine macrophytes species tested in the current study showed activity against at least one protozoan. Some of them may be promising sources for further bio-guided isolation of new active components. Most of these components are probably apolar compounds. The most interesting species, in terms of antiplasmodial activity and selectivity, is the red algae M. stellatus. Bioguided fractionation is underway.