Toxoplasma gondii is zoonotic protozoa that is very prevalent worldwide and a major cause of abortions and neonatal deaths in sheep and other livestock, resulting in significant economic losses [1]. Infected livestock meat and products act as a source of T. gondii infection in humans [2]. Despite these challenges, there is currently no medicine that can eliminate T. gondii infection, particularly the encysted stage. Thus, there is urgent need to develop a generation of safe and efficacious drugs for use in controlling T. gondii infections. Marine algae contain significant amounts of bioactive compounds [3] and as a result has been shown to possess potent antifungal and antibacterial properties [4]. However, there has been no reports on its anti-protozoal properties. Therefore, in this study we endeavored to evaluate the in vitro anti-Toxoplasma activity of green algae extracts.

Dried, pure cultured green algae, Chlorophyceae was generously provided by Dr. Wei Liao of the Anaerobic Digestion Research and Education Center, Michigan State University. Dried algae samples were finely milled and soaked in methanol or n-hexane for 24 hours with agitation. The extraction solution was filtered, evaporated to dryness and the extracts reconstituted in dimethyl sulfoxide (DMSO) to desired concentration. T. gondii RH strain, engineered to constitutively express cytosolic yellow fluorescent protein (RH-YFP) [5] was cultured in human foreskin fibroblasts (HFF) maintained in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% (v/v) heat inactivated fetal bovine serum (Life Technologies), 1% (v/v) Glutamax and 1% (v/v) penicillin-streptomycin-fungizone (Life Technologies) at 37˚C with 5% CO₂. T. gondii tachyzoites were extracted from HFF cells by passing the cell suspension twice through a 25-gauge needle, and extruded parasites isolated from cell debris by passing through a 3 μM filter, followed by washing the filtered parasites in PBS.

To determine the effect of the algae extracts on the growth of T. gondii , HFF cells were grown to confluence in 96-well plates. Prior, to infection, old medium was replaced with fresh medium, and freshly extracted T. gondii added to the HFF cultures at 1000 tachyzoites per well. Test algae extracts at increasing concentrations were added immediately after parasite inoculation. Cultures without compounds (but with similar volume of DMSO) were maintained as controls. At, 24, 48 and 72 hour post-infection, parasite proliferation was determined using an image Xpress micro automated fluorescence microscope by measuring the parasite YFP fluorescence followed by quantification of YFP intensity using ImageJ version 1.37v software (NIH). We found that at 72 hours of culture, the methanol and nhexane extracts of Chlorophyceae algae exhibited dose-dependent inhibitory effect on the intracellular growth of T. gondii tachyzoites (Figure 1A and 1B). However, at earlier times of 24 and 48 hours postinfection, there was no significant difference in parasite growth between the algae extract-treated and the DMSO-treated parasites. This suggested that the bioactive compounds from algae could have acted by inhibiting the invasion of host cells by egressed parasites, assuming that during the first 48 hours post-infection, the parasites would mostly be growing only in cells infected initially. Between 48 and 72 hours post-infection, it is expected that the first egress would have occurred and the egressed parasites would have infected new host cells and started growing intracellularly [6], thereby increasing the number of parasites in the culture substantially. However, in the presence of the algae bioactive compound, the egressed parasites would be directly exposed and internalize considerable amounts of the compounds, which would then compromise the parasites’ infectivity and ability to proliferate in host cells.

Figure 1: Growth curves for T. gondii tachyzoites in cultures treated with varying concentrations of Chlorophyceae extracts. Confluent human foreskin fibroblasts (HFF) were inoculated with T. gondii tachyzoites and immediately treated with varying concentrations (μg/ml) of (A) n-hexane extract, and (B) methanol extract for 72 hours. The fluorescence generated by the parasites is proportional to the amount of parasites in culture and is depicted as the mean fluorescence on the Y-axis. The data shown represent means of three independent experiments with standard error bars.

By nonlinear regression analysis, using Graph Pad Prism software version 6.0 program (CA, USA), we found that the T. gondii growth inhibitory concentration (IC₅₀) values for the methanol and n-hexane Chlorophyceae extracts were, 4.43 ± 1.26 μg/mL and 23.32 ± 3.97 μg/mL, respectively. This indicated that, the methanol extract had more potent activity than the n-hexane extract, suggesting that the methanol extract was more enriched in bioactive compounds content than the n-hexane extract. We, therefore, performed massspectrometry analysis to determine the major constituents in the nhexane and methanol. The extracts were analyzed by High Performance Liquid Chromatography (HPLC) and Electrospray Ionization-Mass spectrometry (ESI-MS) at the Mass Spectrometry facility, University of Illinois at Urbana-Champaign. Briefly, equal amounts of the extracts were run on HPLC using a 2.1 mm ID reverse phase C-18 column with the mobile phase composed of 5% acetonitrile/95% water/0.1% formic acid, followed by ESI-MS analysis in negative mode, operated by Masslynx software v.4.1. Based on the elemental composition of the extracts, as determined by ESI-MS analysis, the methanol extract of Chlorophyceae contained higher content of flavonoids/polyphenols, alkaloids (elaeocarpidine and auramine), and artemisic acid with mass numbers of 256.95, 2667.17 and 296.24, respectively, than the n-hexane extract (Figure 2A and 2B).

Figure 2: ESI-LC/MS chromatogram of Chlorophyceae extracts. (A) Methanol extract of Chlorophyceae contained high intensity of flavonoids, alkaloids (Eaeocarpidine/auramine) and artemisic acid that have been reported to have antiprotozoal activity. (B) The n- Hexane extract contained detectable levels of alkaloids (Eaeocarpidine/auramine) but not flavanoids nor artemisic acid.

Consistent with our findings in the present study, in our previous study, we have found that Sorghum bicolor red leaf extract fraction that was rich in flavonoids content, had potent in vitro activity against T. gondii at dose levels that were non-toxic to mammalian cells [7]. Corroborating our observations in the present study, alkaloids have also been previously shown to have anti-Toxoplasma activity in vitro [8]. Additionally, artemisic acid has been documented to have potent activity against a broad range of protozoan parasites including Plasmodium, Leishmania, Trypanosoma, Toxoplasma, Neospora, Eimeria, Acanthamoeba, Naegleria, Cryptosporidium, Giardia and Babesia [9]. Therefore, the anti-Toxoplasma activities of the methanol Chlorophyceae extracts can be attributed to the high content of flavonoids, alkaloids and artemisic acid.

To determine the cytotoxic IC₅₀ values of the Chlorophyceae extracts in mammalian cells, HFF cells were cultured in supplemented IMDM medium (without red phenol) in 96-well plates and varying concentrations of the extracts added. Wells to which equivalent volumes of DMSO only were added were included as negative control. At 72 hours of culture, a colorimetric assay using the cell proliferation reagent WST-1 (Roche) for the quantification of cell viability was performed on the cultures by adding 20 μL of the WST-1 reagent to each well. After mixing, the plates were wrapped in aluminum foil and incubated for 1 hour at 37 ˚C with 5% CO₂. After 1 hour of incubation, 150 μL of the medium from each well was transferred to a new 96-well plate and quantification of the formazan dye produced by metabolically active cells was read as absorbance at a wavelength of 420 nm using a scanning multi-well spectrophotometer (Spectra Max 250; Molecular Devices). We generated dose–response curves using GraphPad PRISM software and found that the HFF cytotoxicity values for the methanol and n-hexane Chlorophyceae extracts were 150.83 ± 12.94 and 159.52 ± 15.71, respectively. Thus, the n-hexane and methanol Chlorophyceae extracts had T. gondii IC₅₀ values that were 34-fold and 7-fold lower than their respective cytotoxic IC₅₀values in HFF host cells. This indicated that both extracts have potent in vitro inhibitory activity against T. gondii parasites at concentrations that are non-toxic to mammalian cells suggesting that they may not have adverse side-effects in vivo . Together, our findings demonstrate that green algae, Chlorophyceae , contains significant amounts of bioactive compounds that possess potent anti-Toxoplasma activity.

This study was funded in part by the University of Illinois at Urbana-Champaign. We are grateful to Dr. Furong Sun of the Mass Spectrometry facility, School of Chemical Sciences at the University of Illinois at Urbana-Champaign for the help in ESI-MS analysis of the of the extracts used in this study. We thank Dr. Wei Liao of the Anaerobic Digestion Research and Education Center, Michigan State University, for generously providing the Chlorophyceae algae.