Eleganolone, a Diterpene from the French Marine Alga Bifurcaria bifurcata Inhibits Growth of the Human Pathogens Trypanosoma brucei and Plasmodium falciparum

Organic extracts of 20 species of French seaweed have been screened against Trypanosoma brucei rhodesiense trypomastigotes, the parasite responsible for sleeping sickness. These extracts have previously shown potent antiprotozoal activities in vitro against Plasmodium falciparum and Leishmania donovani. The selectivity of the extracts was also evaluated by testing cytotoxicity on a mammalian L6 cell line. The ethyl acetate extract of the brown seaweed, Bifurcaria bifurcata, showed strong trypanocidal activity with a mild selectivity index (IC50 = 0.53 µg/mL; selectivity index (SI) = 11.6). Bio-guided fractionation led to the isolation of eleganolone, the main diterpenoid isolated from this species. Eleganolone contributes only mildly to the trypanocidal activity of the ethyl acetate extract (IC50 = 45.0 µM, SI = 4.0). However, a selective activity against P. falciparum erythrocytic stages in vitro has been highlighted (IC50 = 7.9 µM, SI = 21.6).


Introduction
Human African trypanosomiasis (sleeping sickness) is a fatal disease, if untreated. With fewer than 12,000 cases reported per year, trypanosomiasis belongs to the most neglected tropical diseases [1]. The causative agents are Trypanosoma brucei rhodesiense in East Africa and T. b. gambiense in West and Central Africa. Therapy of sleeping sickness remains a problem. The available drugs are outdated, complicated to administer and can cause severe adverse reactions [2]. Safe and effective drugs are urgently needed.
As a part of our continuous search for new natural antiprotozoal secondary metabolites, 35 polar (hydroalcoholic) and apolar (ethyl acetate) extracts from 20 species of seaweed from the Normandy coast (France), which we previously reported to have other antiprotozoal activities [6], were screened against cultured trypomastigotes of Trypanosoma brucei rhodesiense, as well as for cytotoxicity on a mammalian cell line (L6). Herein, we report the bio-guided fractionation of the most active extract and structure elucidation and antiprotozoal activity of its main constituent.

Selected Species
The sampling resulted in the selection of 20 species of seaweed ( Table 1). The samples were collected, as described previously [6]. Table 1. Marine algal species selected for the study [6].

In Vitro Trypanocidal Activity of the Selected Species
The trypanocidal activity of the resultant ethyl acetate and hydroalcoholic extracts were evaluated in vitro against Trypanosoma brucei rhodesiense trypomastigotes (STIB 900 strain). 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 IC 50 . Cytotoxicity to primary mammalian L6 cells was also evaluated to determine the selectivity of its activity. Table 2 presents the IC 50 values and selectivity indexes (SIs) of the active extracts (ratio of cytotoxic to trypanocidal activity). An SI value >10 is generally considered to indicate antiprotozoal activity not due to general cytotoxicity.  As shown previously for antiplasmodial and leishmanicidal activities [6], the active extracts were almost entirely ethyl acetate extracts (37%), while hydroalcoholic extracts were mainly inactive (2.8%).
Four ethyl acetate extracts showed activity under 10 µg/mL: B. bifurcata, D. dichotoma, P. canaliculata and S. muticum. B. Bifurcaria and P. canaliculata extracts showed selectivity indexes >10, suggesting these extracts could be selectively active against T. brucei rhodesiense. The remaining extracts were less active, with IC 50 values ranging from 10.8 to 29.7 µg/mL. Interestingly, the four most active species belong to the Heterokontophyta. Very little research has been carried out on the antiprotozoal potential of brown alga, and there are only few screening papers available on this subject [7][8][9][10]. Only one of them deals with the species studied here [7]. There were good similarities in the trypanocidal effect of Turkish [9], British [7] and French D. dichotoma. Also, French B. bifurcata, P. canaliculata and S. muticum show a similar activity profile to British ones, but with activity about twice as great [7].
B. bifurcata was the most active species, with an IC 50 value of 0.5 µg/mL for the ethyl acetate extract. To our knowledge, in addition to our study and that of Spavieri, there is no data available regarding the antiprotozoal properties of extracts derived from this species. In contrast, cytotoxic activities have been described for methanolic and chloroform/methanol extracts and compounds isolated from ether extracts of B. bifurcata [12][13][14].
As the ethyl acetate extract has shown significant activity against T. brucei rhodesiense, we subjected it to further fractionation in order to isolate and characterize the active ingredients.

Bio-Guided Fractionation of the Most Active Extract Obtained from B. bifurcata
The ethyl acetate extract of B. bifurcata was fractionated through column chromatography on silica gel using a polarity gradient to generate five main fractions. Bio-guided fractionation was followed by the isolation of the main constituent of the most active fraction. Each fraction or compound was evaluated for trypanocidal activity against T. brucei rhodesiense trypomastigotes. Fractions and purified compound were also evaluated for antiprotozoal activities against T. cruzi amastigotes and P. falciparum intraerythrocytic stages and for cytotoxic activity towards L6 mammalian cells to assess their selectivity of activity (Tables 3 and 4). Table 3. In vitro antiprotozoal activity of fractions obtained from the ethyl acetate crude extract of B. bifurcata. Data shown are means of two independent assays, which varied <±50%. SI: selectivity index; ratio of cytotoxic activity on L6 cells to antiprotozoal activity measured against a T. brucei rhodesiense, b T. cruzi and c P. falciparum. * Data from [6].

Fractions
The strong trypanocidal activity detected in the crude ethyl acetate extract (IC 50 = 0.5 µg/mL) was found in fraction 2 (IC 50 = 0.5 µg/mL), with a little increase in selectivity (SI = 12.4 and 15.4, respectively) ( Table 3). Other fractions were less active and less selective towards T. brucei rhodesiense.

Pure Compound
The main constituent of the fraction 2 was isolated by successive column chromatographies on silica gel and identified as eleganolone (Figure 1). Eleganolone was unambiguously identified by comparison of its spectral data with those described in the literature [21], on the basis of chemical and spectral evidence, including 1 H, 13   Antiprotozoal activity of eleganolone was then evaluated against the protozoa parasites to check whether or not eleganolone contribute to activity (Table 4).
Eleganolone was less active against T. brucei rhodesiense than the fraction from which it has been isolated (IC 50 = 13.7 µg/mL and 0.5 µg/mL, respectively). Indeed, eleganolone showed only mild trypanocidal activity against T. brucei rhodesiense, with a poor selectivity index (SI = 4.0). On the other hand, eleganolone exhibited antiplasmodial activity with a good selectivity (SI = 21.6). The trypanocidal activity, which was lost during the fractionation, could not be attributed to eleganolone, although it is the main component of the active fraction. However, B. bifurcata contains a large variety of structurally related oxygenated diterpenoids that could contribute synergistically to the activity of the crude extract. Nevertheless, the fractionation revealed that eleganolone was selectively active against P. falciparum, compared to other protozoa or mammalian cells. To the best of our knowledge, antiplasmodial activity has not been reported yet for eleganolone.

Algae Collection and Identification
The 20 algae species were collected between November 2005 and September 2007 at several locations on the coast of Basse-Normandie. Table 1 reports the collection dates and sites.
Taxonomic determination was performed by Dr. A.-M. Rusig, and voucher specimens of the algae are deposited in the Herbarium of the University of Caen.

Extraction and Compound Isolation
Crude extracts were prepared as described before [6]. Briefly, freeze-dried and ground thallus of B. bifurcata (250 g) was extracted at room temperature with ethyl acetate and concentrated to dryness under vacuum. The residue (12 g) was subjected to successive flash and column chromatographies over silica gel (230-400 mesh, Merck) eluting with a cyclohexane-ethyl acetate mixture of increasing polarity to yield five main fractions, labeled 1-5. Separations of fraction 2 (2.5 g) by repeated column chromatography on silica gel eluted with a cyclohexane-ethyl acetate mixture of increasing polarity yielded eleganolone (10.3 mg).

In Vitro Antiprotozoal Assays
The extracts were dissolved in dimethylsulfoxide (DMSO) to obtain a concentration of 10 mg/mL and screened for antiprotozoal activity against P. falciparum, T. cruzi and T. brucei rhodesiense and cytotoxicity against rat skeletal muscle myoblasts (L6 cells). The in vitro assays were conducted as described by Scala et al. [31]. A brief description is given below.

Activity against P. falciparum
In vitro activity against erythrocytic stages of P. falciparum was determined by a modified [ 3 H]-hypoxanthine incorporation assay with the chloroquine-and pyrimethamine-resistant K1 strain [32]. Briefly, parasite cultures incubated in Roswell Park Memorial Institute (RPMI) 1640 medium with 5% Albumax (without hypoxanthine) were exposed to serial drug dilutions in microtiter plates. After 48 h of incubation at 37 °C in a reduced oxygen atmosphere, 0.5 µCi [ 3 H]-hypoxanthine was added to each well. Cultures were incubated for a further 24 h before they were harvested onto glass-fiber filters and washed with distilled water. The radioactivity was counted with a Betaplate™ liquid scintillation counter (Wallac, Zurich, Switzerland). The results were recorded as counts per minute (CPM) per well at each drug concentration and expressed as the percentage of untreated controls. IC 50 values were calculated from graphically plotted dose-response curves by linear interpolation. Chloroquine (Sigma C6628) and artemisinin (Sigma 36,159-3) were used as positive references.

Activity against Trypanosoma cruzi
Rat skeletal myoblasts (L6 cells) were seeded in 96-well microtiter plates at 2000 cells/well in 100 μL RPMI 1640 medium with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. After 24 h, the medium was removed and replaced by 100 μL per well containing 5000 trypomastigote forms of T. cruzi Tulahuen strain C2C4 with the β-galactosidase (Lac Z) gene [33]. After 48 h, the medium was removed from the wells and replaced by 100 μL fresh medium with or without a serial drug dilution of seven 3-fold dilution steps covering a range from 90 to 0.123 μg/mL. After 96 h of incubation, the plates were inspected under an inverted microscope to assure growth of the controls and sterility. Then, the substrate CPRG/Nonidet (50 μL) was added to all wells. A color reaction developed within 2-6 h and could be read photometrically at 540 nm. The IC 50 values were calculated from the sigmoidal inhibition curves with SoftMax Pro software. Benznidazole (Roche) was used as a positive reference.

Activity against Trypanosoma brucei rhodesiense
The assays were performed according to the procedures described by [34]. For the extracts, working stock solutions of 180 µg/mL in serum containing culture medium according to Baltz et al. [35] were prepared. One-hundred microliters of the diluted extracts were pipetted in duplicate into the first row of a 96-well microtiter plate (Costar, USA). With the complete culture medium, three-fold serial dilutions were prepared. After the addition of Trypanosoma brucei rhodesiense bloodstream form trypanosomes from axenic culture, the concentrations of the extracts ranged from 90 µg/mL to 0.13 µg/mL and from 500 to 0.07 µg/mL for pure compounds. The total number of trypanosomes in each well was 2 × 10 3 /100 µL. The plate was then incubated for 72 h in a humidified atmosphere at 37 °C in 5% CO 2 . Ten microliters of resazurin solution (12.5 mg resazurin dissolved in 100 mL distilled water) were then added to each well, and incubation continued for a further 2-4 h. The plate was then read in a Spectramax Gemini XS microplate fluorometer (MolecularDevices Cooperation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and an emission wavelength of 588 nm [34]. Fluorescence development was measured and expressed as the percentage of the control. Melarsoprol (Arsobal) was used as a positive reference.

Cytotoxicity against L6 Cells
Assays were performed in 96-well microtiter plates, each well containing 100 L of RPMI 1640 medium supplemented with 1% L-glutamine (200 mM) and 10% fetal bovine serum and 4 × 10 4 L6 cells (rat skeletal myoblasts). Serial drug dilutions of seven 3-fold dilution steps covering a range from 90 to 0.123 μg/mL were prepared. After 72 h of incubation, the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. Then, 10 μL of a resazurin solution (12.5 mg resazurin dissolved in 100 mL distilled water) was added to each well and the plates incubated for another 2 h. They were then read with a Spectramax Gemini XS microplate fluorometer at an excitation wavelength of 536 nm and an emission wavelength of 588 nm. The IC 50 values were calculated from the sigmoidal inhibition curves with SoftMax Pro software. Podophyllotoxin (P4405, Sigma, Saint Louis, MO, USA) was used as a positive reference.

Calculation of IC 50
To measure antiplasmodial activity, the concentration of extract at which the parasite growth (=[ 3 H]hypoxanthine uptake) was inhibited by 50% (IC 50 ) was calculated by linear interpolation between the two concentrations above and below 50% [36]. To assess leishmanicidal, antitrypanosomal and cytotoxic activity, we transferred data into the graphic SoftMax Pro program (Molecular Devices), which calculated IC 50 values from the sigmoidal inhibition curve. The values given in Tables 3 and 4 are the means of two independent assays.

Conclusions
Among the marine macrophytes species tested in the current study against T. brucei rhodesiense, only B. bifurcata showed strong activity. Bio-guided fractionation led to the identification of eleganolone, the main linear diterpene described in this species. Eleganolone showed only mild trypanocidal activity, but better antiplasmodial activity, suggesting trypanocidal activity could be due to minor compounds or to the synergy of several compounds separated during fractionation. Minor compounds of the active fractions will be further characterized by LC-HRMS-Cap NMR.