Turkish freshwater and marine macrophyte extracts show in vitro antiprotozoal activity and inhibit FabI, a key enzyme of Plasmodium falciparum fatty acid biosynthesis.

Abstract

The ethanolic extracts of a number of Turkish freshwater macrophytes (Potamogeton perfoliatus, Ranunculus tricophyllus and Cladophora glomerata) and marine macroalgae (Dictyota dichotoma, Halopteris scoparia, Posidonia oceanica, Scinaia furcellata, Sargassum natans and Ulva lactuca) were assayed for their in vitro antiprotozoal activity. Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum were used as test organisms. The cytotoxicity of the extracts was also assessed against primary rat skeletal myoblasts (L6 cells). Whereas none of the extracts were active against T. cruzi, all crude extracts displayed appreciable trypanocidal activity against T. brucei rhodesiense, with S. natans being the most active one (I[C.sub.50] 7.4 [micro]g/ml). Except for the marine alga H. scoparia, all extracts also possessed leishmanicidal potential. The best antileishmanial activity was exerted by U. lactuca and P. oceanica (I[C.sub.50]'s 5.9 and 8.0 [micro]g/ml, respectively). Five extracts that demonstrated inhibitory activity towards P. falciparum (I[C.sub.50]'s 18.1-48.8 [micro]g/ml) were simultaneously assayed against FabI, a crucial enzyme of the fatty acid system of P. falciparum, to find out whether FabI was their target. The extracts of C. glomerata and U. lactuca efficiently inhibited the FabI enzyme with I[C.sub.50] values of 1.0 and 4.0 [micro]g/ml, respectively. None of the extracts were cytotoxic towards mammalian L6 cells. This work reports for the first time antiprotozoal activity of some Turkish marine and freshwater algae, as well as a target-based antiplasmodial screening for the identification of P. falciparum FabI inhibitors from aquatic and marine macrophytes.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Algae; Trypanosoma; Leishmania; Plasmodium; Fatty acid synthase; Enoyl-ACP reductase (FabI)

Introduction

Parasitic protozoans are single-celled organisms causing serious tropical diseases worldwide in both humans and animals. Malaria, trypanosomiasis and leishmaniasis are among the major parasitic diseases distributed throughout the world. Trypanosoma brucei rhodesiense is responsible for African trypanosomiasis (sleeping sickness), that affects half a million people on the African continent, while Trypanosoma cruzi is the causative parasite for Chagas' disease, which is widespread in South America (World Health Organization WHO, 1998). Leishmaniasis is a vector-borne infection afflicting 14 million people worldwide. Leishmania donovani is the causative agent of visceral leishmaniasis (VL), the most severe form of leishmaniasis, which is nearly always fatal if left untreated (Campos-Ponce et al., 2005). The World Health Organization (WHO, 2000) estimates that there are at least 300 million new cases of malaria annually and that 43% of the world population lives in malaria-endemic areas. Therapeutic treatment of parasitic diseases has made considerable progress in the last few decades; however, the presently used drugs possess adverse side effects, and do not provide complete eradication (Keiser et al., 2001). Moreover, resistance development by the parasites to the present drugs has become a major problem. Therefore, new chemotherapeutic agents and novel targets are urgently needed. One such new biological target is the fatty acid biosynthesis.

The recent discovery of the apicoplast, a cryptic plastid organelle in Plasmodium and a number of other Apicomplexan parasites (McFadden et al., 1996) has opened a new door for the discovery of novel antiparasitic agents. Type II fatty acid biosynthesis (FAS-II) is the first identified anabolic process that takes place in the apicoplast of P. falciparum (Waller et al., 1998). This biochemical process has been identified also in T. brucei (Morita et al., 2000) and probably exists in Leishmania sp. (Roberts et al., 2003). FAS-II is characterized by the presence of individual enzymes that are responsible for catalyzing one single step of fatty acid biosynthesis. This differs from the type I system (FAS-I) found in mammals (including human) and fungi, where the entire fatty acid biosynthesis is accomplished by a single, multifunctional fatty acid synthase enzyme. The significant organizational and structural differences between the malaria parasite and human make the type II FAS system an attractive target for malaria drug discovery. The unique enzyme enoyl-ACP reductase (FabI) of Plasmodium commits the final reduction step during the fatty acid elongation process, therefore representing an excellent target for the development of novel antiplasmodial agents (Surolia and Surolia, 2001).

Aquatic macrophytes comprise a phylogenetically diverse class of the plant kingdom. These organisms, particularly of marine origin, have gained increasing attention from researchers due to chemically interesting secondary metabolites with miscellaneous biological activities (Mayer and Hamann, 2002; Donia and Hamann, 2003). The goal of this study was to determine in vitro antitrypanosomal, antileishmanial and antiplasmodial activities of the ethanolic extracts of nine species of freshwater and marine algae found in Turkish waters, namely Potamogeton perfoliatus, Ranunculus tricophyllus, Cladophora glomerata, Dictyota dichotoma, Halopteris scoparia, Posidonia oceanica, Scinaia furcellata, Sargassum natans and Ulva lactuca. The extracts were also tested for their cytotoxicity on primary mammalian (L6) cells. Those extracts with antimalarial activity were further assayed for their inhibition potential against the FabI enzyme of Plasmodium falciparum. Two extracts were identified to target plasmodial FabI. This is the first study reporting the broad spectrum antiprotozoal and FabI inhibiting activities of the freshwater and marine algae extracts.

Material and methods

Plant material and extraction

The marine brown algae of the Mediterranean Sea, namely Halopteris scoparia (L.) Sauvagau (syn. Stypocaulon scoparium (L.) Kutz) (Stypocaulaceae) and Dictyota dichotoma (Huds.) Lam. (Fucophyceae) were collected in August, 1999 from Alanya, Turkey. The other marine brown alga Scinaia furcellata (Turn.) J. Agardh (Galaxauraceae) was gathered from Tekirdag province located at the coast of the Marmara Sea, in September 2000. Samples of the marine brown alga Sargassum natans (L.) Gaill. (syn. Fucus natans L., Sargassum bacciferum (Turn.) Agardh) (Sargassaceae), the marine green alga Ulva lactuca L. (Ulvaceae), and the sea grass Posidonia oceanica L. Del. (Potamogetonaceae) were collected from the shore of Davutlar village, Kusadasi (the Aegean Sea), in August 2000. The freshwater plants, Potamogeton perfoliatus L. (Potamogetonaceae) and Ranunculus trichophyllus Chaix (Ranunculaceae) were collected from Mogan Lake, Ankara, in April 1999. The freshwater green alga Cladophora glomerata (Dilw.) Kutz (Chlorophyceae) was scooped from Ceyhan River, Elbistan in April 1999 and Beysehir Lake, Konya, in May 1999, respectively. The taxonomical identification of the organisms was done by one of us (T.A.) and the voucher specimens are kept at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Ankara, Turkey. Following collection and air-drying at room temperature, each species was accurately weighed (5.0 g) and macerated with ethanol (95[degrees]) for 2 days. After filtration, the ethanolic extracts were dried under vacuum and used in the bioassays.

Bioassays

Trypanocidal activity against Trypanosoma brucei rhodesiense and cytotoxicity tests

T. brucei rhodesiense STIB 900 strain and the standard drug melarsoprol were used for the assay. Minimum Essential Medium (50 [micro]l) supplemented according to Baltz et al. (1985) with 2-mercaptoethanol and 15% heat-activated horse serum was added to each well of a 96-well microtiter plate. Serial drug dilutions were added to the wells. Then [10.sub.4] bloodstream forms of T. brucei rhodesiense in 50 [micro]l were added to each well and the plate incubated at 37[degrees]C (under a 5% C[O.sub.2] atmosphere) for 72h. Rezasurin (12.5 mg dissolved in 11 distilled water) was then added to each well and incubation continued for a further 2-4h (Raz et al., 1997). The plates were read in a microplate fluorescence scanner (Spectramax Gemini XS by Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and emission wavelength of 588 nm. From the sigmoidal inhibition curve, I[C.sub.50] values were calculated. Cytotoxicity was assessed by the same assay (Sperandeo and Brun, 2003) using rat skeletal muscle myoblasts (L6 cells) and the standard compound, phodophyllotoxin.

Trypanocidal activity against Trypanosoma cruzi

Rat skeletal muscle myoblasts (L6 cells) were seeded in 96-well microtiter plates at 2000 (cells/well) /100 [micro]l RPMI 1640 medium with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. After 24 h, 5000 trypomastigotes of T. cruzi (Tulahuen strain C2C4 containing the [beta]-galactosidase (Lac Z) gene) was added. After 48 h, the medium was removed from the wells and replaced by 100 [micro]l of fresh medium with or without a serial drug dilution. Seven 3-fold dilutions were used covering a range from 90 to 0.123 [micro]g/ml. The plates were incubated at 37[degrees]C in 5% C[O.sub.2] for 4 days. Then the substrate CPRG/Nonidet (50 ml) was added to all wells. The color reaction that developed during the following 2-6 h was read photometrically at 540 nm. From the sigmoidal inhibition curve, I[C.sub.50] values were calculated. Benznidazole was the standard drug used.

Antileishmanial activity

The assay for L. donovani (strain MHOM/ET/67/L82) was done using the Alamar Blue assay as described for T. brucei rhodesiense. Briefly, axenic amastigotes were grown in SM medium (Cunningham, 1977) at pH 5.4 supplemented with 10% FBS. One hundred micro liters of the culture medium with [10.sup.5] amastigotes from axenic culture with or without a serial drug dilution were seeded in 96-well microtiter plates. After 72 h of incubation, 10 [micro]l of Alamar Blue (12.5 mg resazurin dissolved in 100 ml distilled water) was added to each well and the plates incubated for another 2 h. Then the plates were read with a microplate fluorometer as previously described (Sperandeo and Brun, 2003). Miltefosine was used as a standard drug.

Antiplasmodial activity

In vitro activity against erythrocytic stages of P. falciparum was determined by a modified [[.sup.3]H]-hypoxanthine incorporation assay (Matile and Pink, 1990), using the chloroquine- and pyrimethamine-resistant K1 strain and the standard drug artemisinin. Briefly, parasite cultures incubated in RPMI 1640 medium with 5% Albumax (without hypoxanthine) were exposed to serial drug dilutions in microtiter plates. After 48 h of incubation at 37[degrees]C in a reduced oxygen atmosphere, 0.5 [mu]Ci [.sup.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 using a Betaplate[TM] liquid scintillation counter (Wallac, Zurich, Switzerland). The results were recorded as counts per minute (CPM) per well at each drug concentration and expressed as percentage of the untreated controls. I[C.sub.50] values were calculated from the sigmoidal inhibition curves using Microsoft Excel.

P. falciparum enoyl-ACP reductase (FabI) inhibition assay

The FabI enzyme assay was performed as previously described (Kirmizibekmez et al., 2004; Tasdemir et al., 2005a, b). Briefly, the extracts were dissolved in DMSO and tested at 100 [micro]g/ml concentration in the presence of 1 [micro]g (20 nM) enzyme and 200 [micro]M NADH. All measurements were performed on a Uvikon 941 Plus spectrophotometer (Kontron Instruments) in 1 ml of 20 mM Tris/HCI pH 7.4 and 150 mM NaCl. The reaction was started by addition of 50 [micro]M crotonoyl-CoA. The reaction mixture was read spectrophotometrically for 1 min by following the oxidation of NADH to NA[D.sup.+] at 340 nm ([epsilon] = 6.3 m[M.sup.-1][cm.sup.-1]). The I[C.sub.50] values were estimated from graphically plotted dose-response curves. Triclosan was used as a positive control (I[C.sub.50] 50 nM = 14 ng/ml).

Results

The in vitro growth inhibitory activity of the ethanolic extracts of a number of macrophytes of both marine and aquatic origin, namely H. scoparia, D. dichotoma, S. furcellata, S. natans, U. lactuca, C. glomerata, P. oceanica, P. perfoliatus and R. trichophyllus were evaluated against four parasitic protozoa (T. brucei rhodesiense, T. cruzi, L. donovani and P. falciparum). Table 1 displays the I[C.sub.50] values determined for each extract as well as the standard compounds (artemisinin, benznidazole, melarsoprol and miltefosine). In order to determine the selectivity, the extracts were also tested on rat skeletal myoblasts (L6 cells) by using podophyllotoxin as positive control (Table 1). None of the extracts displayed activity versus T. cruzi even at the highest test concentrations (I[C.sub.50]'s > 90 [micro]g/ml). However, all nine extracts were found to possess inhibitory activity against the trypomastigote forms of T. brucei rhodesiense. The best trypanocidal activity was demonstrated by the marine brown alga S. natans (I[C.sub.50] 7.4 [micro]g/ml), followed by freshwater green alga C. glomerata (I[C.sub.50] 11.5 [micro]g/ml), marine brown algae D. dichotoma (I[C.sub.50] 17.1 [micro]g/ml) and S. furcellata (I[C.sub.50] 17.9 [micro]g/ml). The remaining extracts possessed milder trypanocidal activity with I[C.sub.50] values ranging between 22.3 and 43.9 [micro]g/ml. Except for the Mediterranean brown alga H. scoparia, all extracts also had leishmanicidal potential against axenic amastigotes of L. donovani. The most potent activities were shown by the marine green alga, U. lactuca (I[C.sub.50] 5.9 [micro]g/ml) and the sea grass P. oceanica (I[C.sub.50] 8.0 [micro]g/ml). The freshwater plant P. perfoliatus also displayed moderate antileishmanial effect (I[C.sub.50] 22.9 [micro]g/ml), whereas the remaining extracts had only weak activity. Five extracts (C. glomerata, D. dichotoma, S. furcellata, S. natans and U. lactuca) exhibited some antiplasmodial activity, with the marine brown alga S. natans being the most potent (I[C.sub.50] 18.1 [micro]g/ml). These five extracts were also evaluated for their ability to inhibit the recombinant FabI enzyme of P. falciparum in vitro. Two of them, C. glomerata and U. lactuca, efficiently inhibited the FabI enzyme with I[C.sub.50] values of 1.0 and 4.0 [micro]g/ml, respectively (data not shown). The other three extracts had no impact on FabI, indicating that their target was not the FabI enzyme of Plasmodium FAS-II system. All extracts were found to be safe for mammalian L6 cells as they did not possess any cytotoxicity (I[C.sub.50]'s>90 [micro]g/ml) even at the highest concentrations tested.

Discussion

In the continuation of our search for antiprotozoal plants from Turkey, we selected nine macrophytes found in Turkish waters and studied their antiparasitic activities against four protozoa. Our results revealed P. oceanica and U. lactuca extracts to display remarkable inhibitory activity in antileishmanial assay. Besides, S. natans was highly active against both T. brucei rhodesiense and P. falciparum without any toxicity. FabI is a major point of regulation for type II fatty acid synthesis in P. falciparum and as such, a potential drug target for antimalarial drug discovery. Very recently, we reported the results of very first FabI enzyme-based antimalarial screening study performed on Turkish medicinal plants (Tasdemir et al., 2005a). This strategy also yielded the very first natural products, luteolin 7-O-glucoside and (-)-ningpogenin, targeting the plasmodial FabI enzyme (Kirmizibekmez et al., 2004; Tasdemir et al., 2005b). In the current study, we employed the same approach for the first time on freshwater and marine macrophyte extracts. Two green algae, C. glomerata and U. lactuca, have emerged as potent inhibitors of the P. falciparum FabI enzyme in vitro. The moderate antiplasmodial activity of these extracts are most likely due to their poor penetration into the target organelle (the apicoplast) in parasite cells.

A review of the literature indicates that the most bioactive algal extracts show some overlap in their chemistry and biological activity. The brown alga Sargassum has been reported to be rich in polysaccharides, fatty acids, particularly dihomo-[gamma]-linolenic acid, which comprise approximately 10% of this alga (Zhukova and Svetashev, 1999; Orhan et al., 2003; Zhu et al., 2004). Sulfated polysaccharides obtained from several Sargassum sp. demonstrated strong antiviral and antitumor activities (Fujihara et al., 1984; Zhu et al., 2004). The genus Ulva is best known for bromophenolic compounds, but also proteins, lipids and polysaccharides (Abdel-Fattah and Sary, 1987; Lahaye and Ray, 1996; Wahbeh, 1997; Flodin et al., 1999; Pengzhan et al., 2003; Sanina et al., 2004) have been reported. The n-BuOH extract of U. lactuca (Immanuel et al., 2004) as well as a sphingosine-type compound isolated from U. fasciata (Garg et al., 1992) have pronounced antiviral effects. The Cladophora sp. are environmentally very important, as they accumulate and metabolize (detoxification) heavy metals and carcinogenic polycyclic aromatics (Vymazal, 1990; Kirso and Irha, 1998). A number of biological activities, e.g. antibacterial, antimycobacterial, antimycotic and cytotoxic, have been described from the genus (Demina and Mal'dov, 1981; Orhan et al., 2002; Kamenarska et al., 2004). The Cladophora is the source of native cellulose of high quality (Ek et al., 1998) and also contain sulphated polysaccharides (Ramana and Rao, 1991; Shanmugam et al., 2001), steroids, volatiles (Kamenarska et al., 2004; Abrahamsson et al., 2003) and fatty acids (Carballeira et al., 1997; Orhan et al., 2003). The sea grass P. oceanica is an endemic species to the Mediterranean Sea. The P. oceanica meadows are incredibly important for the local ecosystem since many other species find their nutrients and housing in them. Chemically, P. oceanica is characterized by the phenolic compounds (Cuny et al., 1995; Agostini et al., 1998) and long-chain ([C.sub.22]-[C.sub.24]) fatty acids (Nichols et al., 1982; Viso et al., 1993). We reported previously P. oceanica to contain shorter-chain fatty acids, e.g. palmitic ([C.sub.16]), oleic ([C.sub.18]) and stearic ([C.sub.18]) acids (Orhan et al., 2003). All these chemical constituents might underlie the antiprotozoal and FabI inhibitory activity of the algal extracts.

Our literature survey also elucidated that although marine organisms are prominent sources of biologically active compounds, the list of marine extracts and/or natural products that have been evaluated for antiprotozoal properties is quite short. To our knowledge, this is the first report on antiprotozoal activity of marine and freshwater algae investigated here. The present study is also the first application of the FabI-target-based malaria screening on macroalgal extracts. We believe that the ethanolic extracts of S. natans, U. lactuca, C. glomerata and P. oceanica might represent alternative sources in the search of new antiprotozoal agents with novel mechanism of actions, i.e. FabI enzyme inhibition. The isolation and characterization of the active principle(s) of these macrophytes are in progress in our laboratory.

Acknowledgment

Deniz Tasdemir acknowledges the financial support provided by the Dr. Helmut Legerlotz Foundation of the University of Zurich.

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I. Orhan (a), B. Sener (a), T. Atici (b), R. Brun (c), R. Perozzo (d), D. Tasdemir (e,*)

(a) Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, TR-06330 Ankara, Turkey

(b) Department of Biology, Faculty of Education, Gazi University, TR-06500 Ankara, Turkey

(c) Department of Medical Parasitology and Infection Biology, Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland

(d) Department of Pharmaceutical Biochemistry, School of Pharmaceutical Sciences, University of Geneva, Quai Ernest-Ansermet 30, CH-1211 Geneve 4, Switzerland

(e) Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190. CH-8057 Zurich, Switzerland

*Corresponding author. Tel.: [phone]; fax: [phone].

E-mail addresses: [e-mail], [e-mail] (D. Tasdemir).

Table 1. In vitro antiprotozoal activity of the ethanolic extracts of
Turkish macrophytes (I[C.sub.50] values are in [micro]g/ml)

Plants                   T. brucei rhodesiense  T. cruzi    L. donovani

Cladophora glomerata     11.5                   >90           39.2
Dictyota dichotoma       17.1                   >90           52.0
Halopteris scoparia      43.9                   >90         >100
Posidonia oceanica       34.0                   >90            8.0
Potamogeton perfoliatus  30.8                   >90           22.9
Ranunculus tricophyllus  27.8                   >90           52.3
Scinia furcellata        17.9                   >90           64.4
Sargassum natans          7.4                   >90           90.9
Ulva lactuca             22.3                   >90            5.9
Standards                 0.0098 (a)              1.06 (b)     0.102 (e)

Plants                   P. falciparum  Cytotoxicity (L6 cells)

Cladophora glomerata      33.7          >90
Dictyota dichotoma        33.9          >90
Halopteris scoparia      >50            >90
Posidonia oceanica       >50            >90
Potamogeton perfoliatus  >50            >90
Ranunculus tricophyllus  >50            >90
Scinia furcellata         41.3          >90
Sargassum natans          18.1          >90
Ulva lactuca              48.8          >90
Standards                  0.0022 (d)     0.08 (e)

Standard compounds: (a) melarsoprol, (b) benznidazole, (c) miltefosine,
(d) artemisinin, (e) phodophyllotoxin.