Abstract
Seventy-eight crude organic fractions from nineteen species of marine algae collected from the coast of South Africa were screened for activity against a chloroquine sensitive strain of Plasmodium falciparum (D10), Staphylococcus aureus, Klebsiella pneumoniae, Mycobacterium aurum and Candida albicans. Fifteen crude fractions showed good antiplasmodical activity (IC50 <10 μg/mL). The dichloromethane fraction from Sargassum heterophyllum (Turner) C. Agardh (Sargassaceae) was the most active with an IC50 value of 2.8 μg/mL. Eleven extracts showed MIC values ≤ 1 mg/mL against at least one of S. aureus, K. pneumoniae, M. aurum and C. albicans. The broadest spectrum of antimicrobial activity was exhibited by the aqueous-HP20 fraction of Polysiphonia incompta Harvey (Rhodomelaceae). This study shows that marine algae not only produce antimicrobial compounds but also may be a source of antimalarial lead compounds.
Introduction
Infectious diseases such as malaria and tuberculosis are widespread in developing countries, causing millions of deaths annually. In addition, widespread resistance to commonly used chemotherapeutic agents presents a continuous cause for concern. The discovery and development of new antibacterials and therapies for the treatment of infectious diseases is therefore vitally important. Although terrestrial plants and microorganisms have traditionally been an important source of new drugs, several new natural products with potent and selective activity against a number of infectious disease organisms have been isolated from marine invertebrates and algae (CitationMayer et al., 2007). A number of studies have explored the antibacterial activity of marine algal extracts (CitationReichelt & Borowitzka, 1984; CitationVlachos et al., 1997), however, only limited information is available on these plants as a source of antimalarial or antituberculosis leads (CitationOrhan et al., 2006).
With more than 800 species of marine algae having been identified from the South African coast (CitationStegenga et al., 1997), we have initiated a research program to evaluate the biomedical potential of this rich resource. In this preliminary study, 78 extracts of 19 species of marine macroalgae were screened for activity toward a chloroquine sensitive (D10) strain of Plasmodium falciparum, two clinically important strains of bacteria (Staphylococcus aureas and Klebsiella pneumoniae), a fungus (Candida albicans) and Mycobacterium aurum.
Materials and methods
Collection of algae
In January 2006, 19 species of algae (14 red, 2 green, 3 brown) were collected () during low tide at Kalk Bay on the west coast, and Kenton-on-Sea and Noordhoek/Port Elizabeth on the east coast of South Africa. The algae were transported to the laboratory on ice where they were carefully sorted and stored frozen until extraction. All algae were identified by J.J.B. and voucher specimens are kept at the Faculty of Pharmacy, Rhodes University, Grahamstown, South Africa.
Table 1. Marine algae collected and screened for antiplasmodial, antibacterial, antifungal and antimycobacterium activities.
Extraction of algae
The frozen algae were extracted sequentially with methanol and methanol-dichloromethane (1:2). The combined extracts were concentrated and prefractionated by solvent partitioning as follows. The concentrated extract was reconstituted in methanol-water (9:1) and extracted with hexane (fraction A). The water component of the aqueous methanol partition layer was increased to 30% and extracted with dichloromethane (fraction B). The methanol was removed under reduced pressure and the aqueous mixture extracted with ethyl acetate (fraction C). The remaining aqueous mixture was passed through a column containing HP20 resin and the adsorbed organic material eluted with methanol (fraction D) and dichloromethane (fraction E).
Sample preparation for the antiplasmodial and cytotoxicity assays
Stock solutions of 2 mg/mL prefractionated extract (A–D) were prepared in 10% MeOH or 10% DMSO depending on solubility. A 2 μg/mL stock solution of chloroquine diphosphate (CQ) (Stigma-Aldrich, Schnelldorf, Germany) was used as the reference drug for the antiplasmodial assay, while a 2 mg/mL stock solution of emetine dihydrochloride (Stigma-Aldrich, Schnelldorf, Germany) was used as the reference drug in the cytotoxicity assay. All stock solutions were stored at -20ºC until use. Samples were further diluted to the various concentrations in complete medium on the day of the experiment.
Antiplasmodial assay
All samples were tested in duplicate against a chloroquine sensitive (CQS) strain of Plasmodium falciparum (D10). Continuous in vitro cultures of asexual erythrocyte stages of P. falciparum were maintained using a modified method of CitationTrager and Jensen (1976). The parasites were maintained at a 5% hematocrit in complete medium which contains RPMI 1640 medium (BioWhittaker, Walkers-ville, MD, USA) supplemented with Albumax II (GibcoBRL, Gaithers- burg, MD) (5 g/L), Hypoxanthine (0.088 g/L), HEPES (N-[2-hydroxyethyl]-piperazine-N1-[2-ethansulphonic acid]) (Stigma-Aldrich, Schnelldorf, Germany) (6 g/L), glucose (4 g/L), gentamycin (Stigma-Aldrich, Schnelldorf, Germany) (0.05 g/L) and 5% sodium bicarbonate solution (Sigma-Aldrich). The parasite cultures were maintained below 10% parasitemia by the addition of O+ human erythrocytes which were washed with medium.
Quantitative assessment of in vitro antiplasmodial activity was determined via the parasite lactate dehydrogenase assay using a modified method described by CitationMakler et al. (1993). Percentage parasite viability for test samples was initially tested at three concentrations (50, 25 and 12.5 μg/mL), while CQ was tested at 30, 15 and 7.5 ng/mL. Full dose-response curves were performed on the most active extracts starting at 100 μg/mL followed by two-fold dilutions in complete medium to a final concentration of 0.195 μg/mL. CQ was tested at a starting concentration of 100 ng/mL in these experiments. The highest concentration of solvent (0.5%) to which the parasites were exposed had no measurable effect on the parasite. The 50% inhibitory concentration (IC50) values were obtained using a non-linear dose-response curve fitting analysis via Microsoft Excel and GraphPad Prism v.4.0 software.
Cytotoxicity assay
All extracts were tested in triplicate using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT)-assay (Mossman, 1983; CitationSieuwerts et al., 1995). The cells were cultured in complete medium containing Dulbeco’s Modified Eagle’s medium; Hams F-12 supplemented with 10% heat inactivated fetal calf serum and gentamycin (0.05 g/L). All reagents for the medium were obtained from Highveld Biological, South Africa. Monolayers of the cells were maintained in culture flasks and incubated at 37ºC in a 5% CO2 humidified atmosphere.
The highest concentration of solvent (0.5%) to which the cells were exposed to had no measurable effect on the cell viability. Full dose-response curves were done for the selected extracts with a starting concentration of 100 μg/mL, which was serially diluted in complete medium with 10-fold dilutions to give five concentrations, with the lowest concentration at 0.01 μg/mL. The starting concentration of emetine was 100 μg/mL, which was serially diluted in complete medium with 10-fold dilutions to give 6 concentrations, with the lowest at 0.001 μg/mL.
The 50% inhibitory concentration (IC50) values were obtained using a non-linear dose-response curve fitting analysis via Microsoft Excel and GraphPad Prism v.4.0 software.
Antimicrobial assays
Only selected extracts were screened for antimicrobial activity due to limited samples. The extracts were prepared to a concentration of 16 mg/mL in 10% DMSO. Each sample was tested against a Gram-negative bacterium (Klebsiella pneumoniae ATCC 13883), a Gram-positive bacterium (Staphylococcus aureus ATCC 12600), a fungus (Candida albicans ATCC 90028) and a mycobacterium (Mycobacterium aurum A+) in duplicate in each of two separate broth micro-dilution assays. The assays are based on visualization of red formation of p-iodonitrotetrazolium salt (INT) in the presence of organism growth and results are expressed as minimum inhibitory concentrations (MIC). Sample concentrations ranged from 31.25 μg/mL to 4 mg/mL. S. aureus and K. pneumoniae were tested as described by CitationEloff (1998) in a 96-well plate using tryptone soya broth (TSB, Oxoid) and were incubated at 37ºC overnight, while the M. aurum was similarly tested, using Middlebrook 7H9 (Becton Dickinson) supplemented with OADC (oleic acid, albumin, dextrose, catalase) (Becton Dickinson) as described by Chung et al. (1995), with minor variations. Briefly, 100 μL sterile distilled water was added to all the wells after which 100 μL of the test samples were added and serially diluted. The organism was prepared in double strength Middlebrook 7H9 supplemented with 20% OADC and 100 μL added to each well. The plates were incubated at 37ºC for 72 hours. C. albicans was tested similarly, but with double strength RPMI (pH 7) and the plates were incubated at 35ºC for 48 hours. After incubation, 40 μL of 0.4 mg/mL INT was added to each plate and the results were obtained after 6 hours.
Results and discussion
In vitro antiplasmodial activity against P. falciparum
For clarity, only antiplasmodial activities of crude fractions measured as “parasite viability” at an extract concentration of 50 μg/mL are shown in . Twenty eight of the 78 extracts tested (36%) killed 90% of parasites at 50 μg/mL. Significantly, this activity is distributed between 15 (59%) of the 22 algae tested with 50% of this activity present primarily in the dichloromethane partition fractions, while the rest is distributed evenly between the hexane, ethyl acetate and HP20 fractions. Full dose-response experiments were performed to determine the IC50 values of these 28 extracts. Fifteen extracts (19%); representing nine different algae (41%), exhibited IC50 values less than 10 μg/mL and are being considered for further study (). Of these extracts, the dichloromethane fraction of Sargassum heterophyllum was the most active (IC50 2.8 μg/mL) followed by the hexane fraction of Plocamium corallorhiza (Turner) J.D. Hooker & Harvey (Plocamiaceae) which showed and IC50 value of 3.5 μg/mL.
Figure 1. In vitro antiplasmodial activity of hexane, dichloromethane, ethyl acetate and aqueous partitions against P. falciparum CQS D10 strain
Table 2. Antiplasmodial and cytotoxic activities of the most active extracts.
Table 3. Antimicrobial activity of algal extracts against S. aureus, K. pneumoniae, M. aurum and C. albicans.
In order to assess the selectivity of the 15 most active extracts for P. falciparum, these extracts were also evaluated for cytotoxicity toward Chinese hamster ovary (CHO) cells and the selectivity index (SI = IC50 CHO/IC50 P. falciparum) for each extract was calculated. A selectivity index of 10 or more is suggestive of a selective mode of action. The dichloromethane fraction of S. heterophyllum showed potent cytotoxicity toward CHO cells (IC50 3.7 μg/mL) and a corresponding low selectivity index (SI = 1.3). It is therefore likely that the antiplasmodial activity of this extract may be due to general cytotoxicity. However, this can only be confirmed by isolating the active metabolites. More promising selectivities (SI > 10) were obtained for the HP20 fraction of Pterosiphonia cloiophylla (C. Agardh) Falkenberg (Rhodomelaceae) (SI = 16), the hexane fraction of Plocamium corallorhiza (2) (SI = 10), and the dichloromethane fractions of Plocamium cornutum (Turner) Harvey (Plocamiaceae) (SI = 12.6) and Amphiroa ephedraea (Lamarck) Decaisne (Corallinaceae) (SI >12.1).
Antimicrobial activity
Several studies have reported on the antibacterial activity of marine algal extracts, including a study by CitationVlachos et al. (1997) on South African marine algae. However, it is difficult to compare the results from these studies due to the different extraction, storage and testing methods employed (CitationCronin et al., 1995). In this study we investigated the antimicrobial effects of marine algal extracts against a panel of four clinically important pathogens. For the purpose of this study, minimum inhibitory concentrations (MIC) of 1 mg/mL or less were considered active. Eleven of the 24 extracts tested (46%) were active against one or more of the pathogens. This activity was distributed between eight (67%) out of the 12 algae. As with the antiplasmodial assay, the dichloromethane extracts were the most active (72% of active extracts). These results compare favorably with those previously reported from marine algae (CitationReichelt & Borowitzka 1984; CitationVlachos et al., 1997).
Interestingly, the Gram-negative K. pneumoniae was the most resistant to the antimicrobial effect of the crude extracts with only one alga (Pterosiphonia cloiophylla) showing activity. This is also in accordance with the results obtained from previous studies, indicating that Gram-positive organisms were more susceptible to the antimicrobial affects of algal extracts (CitationVlachos et al., 1997). Interestingly, four algae (33%) produced at least one extract that was active against M. aurum, while four algae also produced antifungal extracts. The broadest spectrum of activity was found in the HP20 fraction of Polysiphonia incompta (1) (active against S. aureus, M. aurum and C. albicans).
In conclusion, the lack of detailed ethnobotanical information on the medicinal uses of marine algae makes the screening of marine algal extracts an important first step in the selection and prioritization of algae for further chemical and pharmacological investigation. Our results have shown that South African marine algae may be a useful source of natural products, showing activity against a number of clinically relevant microorganisms. Further studies on the isolation and characterization of these metabolites are in progress.
Acknowledgements
This study was funded by the National Research Foundation, the Medical Research Council and Rhodes University.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References
- Cronin G, Lindquist N, Hay ME, Fenical W (1995): Effects of storage and extraction procedures on yields of lipophilic metabolites from the brown seaweeds Dictyota ciliolata and D. menstrualis. Mar Ecol Prog Ser 119: 265–273.
- Eloff JN (1998): A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Med 64: 711–713.
- Makler MT, Ries JM, Williams JA, Bancroft JE, Piper RC, Gibbins BL, Hinrichs DJ (1993): Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. Am J Trop Med Hyg 48: 739–741.
- Mayer AMS, Rodríguez AD, Berlinck RGS, Hamann MT (2007): Marine pharmacology in 2003–4: Marine compounds with anthelmintic antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiplatelet, antiprotozoal, antituberculosis, and antiviral activities; affecting the cardiovascular, immune and nervous systems, and other miscellaneous mechanisms of action. Comp Biochem Physiol Part C Pharmacol Toxicol 145: 553–581.
- Mosmann T (1983): Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55–63.
- Orhan I, Sener B, Atici T, Brun R, Perozzo R, Tasdemir D (2006): Turkish freshwater and marine macrophyte extracts show in vitro antiprotozoal activity and inhibit FabI, a key enzyme of Plasmodium falciparum fatty acid biosynthesis. Phytomedicine 13: 388–393.
- Reichelt JL, Borowitzka MA (1984): Antimicrobial activity from marine algae: Results of a large-scale screening programme. Hydrobiol 116/117: 158–168.
- Sieuwerts AM, Klijn JGM, Peters HA, Foekens JA (1995): The MTT Tetrazolium salt assay scrutinized: How to use this assay reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50-values and cell survival. Eur J Clin Chem Clin Biochem 33: 813–823.
- Stegenga H, Bolton JJ, Anderson RJ (1997): Seaweeds of the South African west coast: Contributions from the Bolus Herbarium. Cape Town, Creda Press, 655 pp.
- Trager W, Jensen JB (1976): Human malaria parasite in continuous culture. Science 193: 673–675.
- Vlachos V, Critchley AT, von Holy A (1997): Antimicrobial activity of extracts from selected southern African marine macroalgae. S Afr J Sci 93: 328–332.