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NPC Natural Product Communications
DR. PAWAN K AGRAWAL
Natural Product Inc.
7963, Anderson Park Lane,
Westerville, Ohio 43081, USA
PROFESSOR ALESSANDRA BRACA
Dipartimento di Chimica Bioorganicae Biofarmacia,
Universita di Pisa,
via Bonanno 33, 56126 Pisa, Italy
PROFESSOR DEAN GUO
State Key Laboratory of Natural and Biomimetic Drugs,
School of Pharmaceutical Sciences,
Beijing 100083, China
PROFESSOR YOSHIHIRO MIMAKI
School of Pharmacy,
Tokyo University of Pharmacy and Life Sciences,
Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan
PROFESSOR STEPHEN G. PYNE
Department of Chemistry
University of Wollongong
Wollongong, New South Wales, 2522, Australia
PROFESSOR MANFRED G. REINECKE
Department of Chemistry,
Texas Christian University,
Forts Worth, TX 76129, USA
PROFESSOR WILLIAM N. SETZER
Department of Chemistry
The University of Alabama in Huntsville
Huntsville, AL 35809, USA
PROFESSOR YASUHIRO TEZUKA
Institute of Natural Medicine
Institute of Natural Medicine, University of Toyama,
2630-Sugitani, Toyama 930-0194, Japan
PROFESSOR DAVID E. THURSTON
Department of Pharmaceutical and Biological Chemistry,
The School of Pharmacy,
University of London, 29-39 Brunswick Square,
London WC1N 1AX, UK
Prof. Berhanu M. Abegaz
Prof. Viqar Uddin Ahmad
Prof. Øyvind M. Andersen
Prof. Giovanni Appendino
Prof. Yoshinori Asakawa
Prof. Lee Banting
Prof. Julie Banerji
Prof. Alejandro F. Barrero
Prof. Anna R. Bilia
Prof. Maurizio Bruno
Prof. César A. N. Catalán
Prof. Josep Coll
Prof. Geoffrey Cordell
Chicago, IL, USA
Prof. Cristina Gracia-Viguera
Prof. Duvvuru Gunasekar
Prof. A.A. Leslie Gunatilaka
Tucson, AZ, USA
Prof. Kurt Hostettmann
Prof. Martin A. Iglesias Arteaga
Mexico, D. F, Mexico
Prof. Jerzy Jaroszewski
Prof. Leopold Jirovetz
Prof. Karsten Krohn
Prof. Hartmut Laatsch
Prof. Marie Lacaille-Dubois
Prof. Shoei-Sheng Lee
Prof. Francisco Macias
Prof. Imre Mathe
Prof. Joseph Michael
Johannesburg, South Africa
Prof. Ermino Murano
Prof. M. Soledade C. Pedras
Prof. Luc Pieters
Prof. Peter Proksch
Prof. Phila Raharivelomanana
Tahiti, French Plynesia
Prof. Monique Simmonds
Prof. Valentin Stonik
Prof. Winston F. Tinto
Barbados, West Indies
Prof. Karen Valant-Vetschera
Prof. Peter G. Waterman
PROFESSOR GERALD BLUNDEN
The School of Pharmacy & Biomedical Sciences,
University of Portsmouth,
Portsmouth, PO1 2DT U.K.
Antiprotozoal, Antitubercular and Cytotoxic Potential of
Cyanobacterial (Blue-Green Algal) Extracts from Ireland
Barbara Broniatowskaa, Andrea Allmendingera, Marcel Kaiserb, Damien Montamat-Sicottec,
Suzie Hingley-Wilsonc, Ajit Lalvanic, Michael Guiryd, Gerald Blundene and Deniz Tasdemira,*
aDepartment of Pharmaceutical and Biological Chemistry, Centre for Pharmacognosy and
Phytotherapy, School of Pharmacy, University of London, London WC1N 1AX, UK
bDepartment of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health
Institute, CH-4002 Basel, Switzerland
cTuberculosis Research Unit, Department of Respiratory Medicine, National Heart and Lung Institute,
Imperial College London, London W2 1PG, UK
dAlgaeBase, Ryan Institute, National University of Ireland Galway, University Road, Galway, Ireland
eSchool of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, PO1 2DT, UK
Received: February 10th, 2011; Accepted: March 1st, 2011
Cyanobacteria (= blue-green algae) are prolific producers of structurally distinct and biologically active metabolites. In the continuation of
our search for new sources of anti-infective natural products, we have assessed the in vitro antiprotozoal (Plasmodium falciparum,
Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani) and antitubercular (Mycobacterium tuberculosis) potential of samples of
two terrestrial cyanobacteria, Nostoc commune (collected when desiccated and wet) and Rivularia biasolettiana. The cytotoxic potential of
the extracts was also evaluated against primary L6 cells. Except for T. cruzi and M. tuberculosis, the crude extracts were active against all the
organisms tested and showed no toxicity. The crude extracts were then partitioned between n-hexane, chloroform and aqueous methanol and
retested against the same panel of pathogens. The chloroform sub-extracts of both N. commune samples showed significant activity against
T. b. rhodesiense (IC50 values 2.0 and 3.5 μg/mL) and P. falciparum (IC50s 7.4 and 5.8 μg/mL), with low toxicity. This trend was also true for
R. biasolettiana extracts, and its chloroform sub-extract showed notable activity against all parasitic protozoa. There were differences in the
biological activity profiles of extracts derived from desiccated and hydrated forms of N. commune. To our knowledge, this is the first study
assessing the anti-infective activity of desiccated and hydrated forms of N. commune, as well as R. biasolettiana. Furthermore, the present
work reports such biological activity in terrestrial cyanobacteria from Ireland for the first time. These results warrant the further study of Irish
terrestrial cyanobacteria as a valuable source of new natural product leads for the treatment of parasitic protozoal infections.
Keywords: Cyanobacteria, Nostoc commune, Rivularia biasolettiana, Plasmodium, Trypanosoma, Leishmania, Mycobacterium.
Approximately one-third of the world’s population is
infected with tuberculosis (TB), the most prevalent
infectious disease worldwide that kills approximately two
million people annually . Resistant and/or extremely
drug-resistant strains of Mycobacterium tuberculosis are
emerging in both developing and developed countries to
pose a serious global health problem. Parasitic, mostly
vector-borne diseases, for example, malaria, leishmaniasis
and trypanosomiasis, also affect large populations in
impoverished tropical regions. The most severe form of
malaria is caused by the protozoan parasite Plasmodium
falciparum and is transmitted to humans by female
mosquitoes. Approximately 40% of the earth’s population
lives in malaria-endangered areas, and each year about
1 million people, mainly very young children, die of it .
The WHO estimates that more than 200 million people
are at risk of either leishmaniasis or trypanosomiasis
worldwide. Leishmaniasis is widespread across many
tropical regions, such as India, Sudan, and Brazil. Among
the three forms of leishmaniasis (cutaneous, mucosal, and
visceral), the last (VL) represents the greatest threat to
human health. VL is a sandfly-borne infection caused by
Leishmania donovani, L. infantum, and L. chagasi. If left
untreated, it can be 100% fatal within two years .
African trypanosomiasis (sleeping sickness), which is
transmitted to humans by tsetse flies, is caused by
Trypanosoma brucei rhodesiense and T. b. gambiense. It
affects about half a million people on the African
continent. Because the parasite is able to pass through the
blood-brain barrier, the chronic phases of the disease can
produce severe brain damage and death . American
trypanosomiasis (Chagas’ disease) is endemic to 21
countries in S. America. The causative agent of the disease
is T. cruzi, which is transmitted to humans by triatomine
NPC Natural Product Communications
689 - 694
690 Natural Product Communications Vol. 6 (5) 2011 Broniatowska et al.
insects. Chronic stage infection, if left untreated, results in
neurological disorders, intestinal damage and fatal cardio-
myopathy . Current treatment of all these infections is
limited to a few drugs, with serious drawbacks in efficacy,
toxicity and cost. More importantly, drug resistance is
becoming widespread and threatens their utility. These
facts, combined with the absence of an efficacious vaccine
and the lack of systemic vector control strategies, provide
the rationale in several academic settings for the
development of novel drugs against these diseases.
Cyanobacteria, an ancient photosynthetic group of
prokaryotic organisms, are prolific producers of a wide
range of natural products [6a-6d]. This capability has been
linked with the fact that these organisms face large
pressure from either grazers or competing organisms
forcing them to develop chemical defense strategies for
survival . Nostoc (Nostocales, Nostocaceae) is a diverse
genus of colonial cyanobacteria, commonly found in both
aquatic and terrestrial habitats. The genus currently
includes about 55 species . Nostoc species, in particular
N. commune, are well known for their capacity to
withstand long periods of desiccation, cold and UV
radiation that enable them to survive in most extreme
environments. In dry seasons, Nostoc species may lie
dormant for years, but readily recover when rehydrated.
The dehydrated colonies, which appear as a black and
brittle powder, swell into green-brown, gelatinous spheres
in the presence of humidity . The ability of Nostoc
colonies to tolerate excessive environmental stress,
including the long-term water deficit, has been attributed
to the production of a biochemically complex, extracellular
polysaccharide (glycan) matrix. The principal components
of this include water-soluble (mycosporine aminoacids)
and lipid-soluble (scytonemin) UV absorbing pigments, all
distributed within a high molecular weight extracellular
glycan unit .
Rivularia (Nostocales, Rivulariaceae) is a genus of about
70 species of free-living, colony-forming cyanobacteria
that can be found in both marine and freshwater
environments . Species of the genus are cosmopolitan,
often living in fast-flowing streams, seepages, salt
marshes, and particularly in the intertidal of rocky shores.
So far, Rivularia species have mainly remained unexplored
chemically, and there are only few published chemical and
biological activity studies.
Nostoc species have been used as both food and medicine
for centuries . Modern studies continue to confirm the
biomedical potential of Nostoc species and their chemical
constituents against a number of human diseases. A few
previous studies compared the chemical composition
(mainly fatty acids, lipids and carotenoids) of desiccated
and wet N. commune [9,12]; however, to our knowledge,
no study has been carried out previously to compare the
biological activity profile of dehydrated and hydrated
N. commune samples. The aim of this study was, therefore,
to evaluate and compare the in vitro antiprotozoal,
antitubercular and cytotoxic potential of terrestrial N.
commune collected from Ireland when desiccated (NC-D)
and when wet (NC-W). In addition, we screened another
terrestrial cyanobacterium, Rivularia biasolettiana (RB,
collected wet from Ireland) for the same biological
activities. The crude extracts of the cyanobacteria were
active against several protozoan parasites, and were thus
partitioned between n-hexane, CHCl3 and water, and
retested against the same panel of microorganisms. Herein,
we report the biological activity of the crude, as well as
solvent sub-extracts obtained from three crude extracts.
The crude extracts of the cyanobacteria were tested against
P. falciparum (erythrocytic stages of drug-resistant K1
strain), T. brucei rhodesiense (bloodstream forms), T. cruzi
(intracellular amastigotes in L6 rat skeletal myoblasts), L.
donovani (axenic amastigotes) and the tubercle bacillus
(M. tuberculosis strain H37Rv). The toxicity of the
extracts was also evaluated towards L6 cells, a primary
cell line derived from mammalian (rat) skeletal myoblasts,
in order to determine their selectivity. The IC50 and MIC
values of the extracts and the reference compounds are
displayed in Table 1.
None of the crude cyanobacterial extracts were active
against either the tuberculosis bacillus M. tuberculosis or
T. cruzi at the highest test concentrations. However, all
three crude extracts had moderate to significant ability to
inhibit the growth of the remaining protozoan parasites. In
particular, crude extracts of both desiccated (NC-D-CR)
and hydrated N. commune (NC-W-CR) had very promising
activities against T. brucei rhodesiense (IC50 values of 3.5
and 6.6 μg/mL) and P. falciparum (IC50 values 4.3 and 4.9
μg/mL). The antileishmanial activity of both crude Nostoc
extracts was only moderate, with the NC-D-CR extract
being more active (IC50 25.6 and 69.6 μg/mL). The
potency of the R. biasolettiana crude extract (RB-CR) was
also modest with IC50 values of 17.7 μg/mL (P.
falciparum), 27.8 μg/mL (T. b. rhodesiense) and 40.4
μg/mL (L. donovani). Both N. commune extracts were
found to lack any cytotoxic potential against L6 cells (IC50
> 90 μg/mL). The cytotoxic potential of R. bullata crude
extract was also negligible (IC50 84.3 μg/mL).
These results encouraged us to carry out a coarse
separation process on the crude extracts. Hence all extracts
were subjected to a liquid-liquid partition scheme to yield
n-hexane, chloroform and aqueous methanol sub-extracts.
All these semi-crude extracts were then subjected to the
same in vitro screening panel and the results are shown in
Table 1. No antimycobacterial effect was observed with
any of the sub-extracts (MIC values > 256 μg/mL). None
of the aqueous methanol sub-extracts had leishmanicidal
activity either (IC50 > 100 μg/mL).
Interestingly, the most potent trypanocidal (T. b.
rhodesiense) and plasmocidal activities were displayed by
the chloroform sub-extracts of both N. commune samples.
The same extracts also showed increased potencies, in
Anti-infective potential of cyanobacterial extracts Natural Product Communications Vol. 6 (5) 2011 691
Table 1: Anti-protozoal, anti-mycobacterial and cytotoxic activities of Irish cyanobacteria.
Sample T. brucei
All IC50 and MIC values are in μg/mL. IC50 values are mean values from at least 2 replicates of duplicates; MIC values are based on 3 replicates of triplicates (the
variation is max. 20%). Control drugs: amelarsoprol, bbenznidazole, cmiltefosine, dchloroquine, estreptomycin, fpodophyllotoxin.
comparison with the crude extracts, toward the remaining
parasites T. cruzi and L. donovani (Table 1). Starting with
the desiccated N. commune samples, NC-D-CHCl3 sub-
extract showed strong and comparable activities against T.
b. rhodesiense and P. falciparum, with IC50 values of 4.0
μg/mL and 5.8 μg/mL, respectively. Unlike NC-D-CR,
NC-D-CHCl3 exhibited some anti-Trypanosoma cruzi
activity (IC50 32.9 μg/mL), and had almost two-fold higher
antileishmanial activity (IC50 16.2 μg/mL). The n-hexane-
soluble portion of N. commune collected when dry (NC-D-
Hexane) was active against all four protozoan species with
IC50 values ranging between 28.6-80.6 μg/mL. As shown
in Table 1, some modest trypanocidal and antimalarial
activities were obtained with the aq. MeOH sub-extract
(NC-D-Aq. MeOH). In general, similar trends and IC50
values were obtained with the wet (hydrated) N. commune
(NC-W) sub-extracts. However, against T. b. rhodesiense,
NC-W-CHCl3 was twice as active (IC50 2.0 μg/mL) than
the NC-D-CHCl3 sub-extract. In contrast, its
antiplasmodial activity was slightly weaker (IC50 7.4
μg/mL). Also noteworthy was that the NC-W-Hexane
extract had slightly lower IC50 values against all four
parasites (IC50 17.8-65.0 μg/mL), whereas the NC-W-Aq.
MeOH extract showed only some marginal activity against
both trypanosomes with IC50 values around 70 μg/mL. A
similar trend was also observed with R. biasolettiana sub-
extracts. Interestingly, the RB-CHCl3 sub-extract showed
the best and almost identical IC50 values (14.8-19.4
μg/mL) against all four protozoan parasites. The n-hexane
sub-extract (RB-Hexane) also retained generally similar
IC50 values to those of the crude extract towards the
protozoan parasites (IC50 values 25.4 -56.8 μg/mL), except
that it had some modest potential against T. cruzi (IC50
value 54.1 μg/mL). The aq. MeOH extract (RB-Aq.
MeOH) displayed some plasmocidal effect (IC50 30.7
μg/mL) and very weak anti-T. cruzi activity (IC50 85.4
μg/mL). When tested for toxicity against primary cells,
only the CHCl3 sub-extracts of the cyanobacterial
collections exhibited weak potential with IC50 values
around 40 μg/mL, whereas all other sub-extracts were
devoid of any cytotoxicity (IC50 > 90 μg/mL).
In recent years, a number of cyanobacteria have been
studied for their anti-infective potential to yield potent,
novel natural products [6,13]. The promising biological
activities and intricate structures of these metabolites have
also led to several synthetic efforts [14,15]. Encouraged
by these studies, we decided to screen for biological
activity two terrestrial cyanobacterial species of Irish
origin. One major aim of the project was to evaluate,
comparatively, the biological potencies of both desiccated
and hydrated forms of N. commune, collected from the
same site at different times of the year. The results outlined
here indicate that both forms seem to have similar, but still
variable biological activities. This may lie in differences in
their chemical compositions in different seasons of
collection. Our initial TLC studies (data not shown) point
out similarities in the crude and semi-crude extracts
derived from both forms, but they do not appear identical.
Currently, we are trying to establish more sophisticated
chemical profiling techniques to compare the chemical
compositions of these crude and semi-crude extracts. One
trend shared by these cyanobacteria though was that the
bioactivity of the crude extracts was concentrated in the
CHCl3 sub-extracts. This is a common experience in our
laboratory, which is probably due to enrichment of
secondary metabolites in this middle polarity phase after
the removal of primary metabolites into the n-hexane (fats-
lipids) and the aqueous methanol (sugars) sub-extracts.
The medicinal value of Nostoc species was recognized as
early as 1500 BC, when they were used to treat gout,
fistula and cancer . Recent studies indicate their
antifungal  and acetylcholinesterase inhibitory 
effects. Species of Nostoc have been a valuable source of
biologically-active compounds, such as the cryptophycins
with potent anticancer activity. These potent tubulin
inhibitors have served as template compounds for a semi-
synthetic clinical candidate . A number of other
bioactive peptides, diterpenes and alkaloids have been
reported from Nostoc
antimicrobial, antiviral and antiprotozoal activities [6,20].
Nostocarboline, a quaternary beta-carbolinium alkaloid
derived from a Nostoc sp. and its synthetic derivatives
species with cytotoxic,
692 Natural Product Communications Vol. 6 (5) 2011 Broniatowska et al.
show potent antiprotozoal and antimycobacterial activities
[14,15]. The hydrated forms of Nostoc species, particularly
N. commune are consumed as foodstuff, primarily in Asia
and the Andes [21a,b]. It is a rich source of protein, fat,
carbohydrate, crude fiber and vitamins [22a,b]. However a
recent study showed the presence of beta-methylamino-L-
alanine (BMAA), a neurotoxic aminoacid in Nostoc
species [21a]. It would be of interest to test whether our
Irish N. commune samples contain BMAA.
A literature survey indicates that Rivularias species have
been poorly investigated. Several bromoindole alkaloids
have been isolated from R. firma [23a,b], and some
Rivularia species have been studied for their fatty acid
[24a,b] and sugar constituents . Antibacterial,
antifungal [24b,26] and algicidal  effects of some
species have also been assessed. To our knowledge, this is
the first study evaluating antiprotozoal, antitubercular and
cytotoxic potential of a Rivularia species.
The present investigation indicated the in vitro
antiprotozoal potential of
collected in the wild from Ireland. In particular, the
chloroform-soluble portion of these extracts displayed
highly interesting antiprotozoal activity. Because of the
promising IC50 values and low toxicity, these extracts of
cyanobacteria, particularly the chloroform sub-extracts,
merit further investigation for the isolation and
characterization of their active metabolites. This is the
subject of our current research. Antimalarial and
antiprotozoal activities of some alkaloids obtained from
Nostoc species have been reported [14,15], but this is the
first study comparing the antiprotozoal activity of N.
commune collected in desiccated and hydrated forms. It
also appears that the current work represents the first
antiprotozoal, antitubercular and cytotoxic assessment of
any Rivularia species. To our knowledge, this is also the
first study assessing such biological activity of Irish
Cyanobacterial samples: All cyanobacterial samples were
collected in the Burren near Finavarra, Co. Clare, Ireland.
The dry form of Nostoc commune Vaucher ex Bornet &
Flahault was collected in May 2007, whereas Rivularia
biasolettiana Meneghini ex Bornet and the wet form of N.
commune were collected in November and December
2007, respectively. Identifications were made by one of us
(M.G.) based on Whitton . Voucher samples of N.
commune are lodged in the Herbarium of the Hampshire
County Council Museums Service in Winchester,
Hampshire (accession number Bi 2000. 16. 375), and of
both N. commune and R. biasolettiana at the University of
London, School of Pharmacy (voucher # GB07-C1
Extraction and partition: The collected cyanobacteria
were immediately placed in i-PrOH and stored at -20˚C in
a freezer until work-up. Algal material was homogenized
in a household blender and filtered. The residue was
extracted overnight with CHCl3: MeOH mixtures (3:1 and
1:1) under continuous stirring at room temperature and
combined with the initial i-PrOH extract obtained above.
The combined crude extract was dissolved in 10 mL
EtOAc: MeOH (1:1) and centrifuged to remove insoluble
materials. The supernatant was removed, evaporated to
dryness in vacuo at 30˚C, and used for the initial biological
assays. A portion of each crude extract (1 g) was dissolved
in 10% water in MeOH (100 mL) and partitioned against
n-hexane (3x100 mL). The water content of the MeOH
phase was then adjusted to 30% by adding water before
partitioning against CHCl3 (3x100 mL). The n-hexane and
CHCl3 extracts were evaporated to dryness in vacuo at
30˚C, whereas the aq. MeOH sub-extract was freeze-dried
before using in the biological assays.
Trypanocidal activity against T. brucei rhodesiense and
cytotoxicity: Minimum Essential Medium (50 μL)
supplemented with 25 mM HEPES, 1g/L additional
glucose, 1% MEM non-essential amino acids (100 x),
0.2 mM 2-mercaptoethanol, 1mM Na-pyruvate and 15%
heat inactivated horse serum was added to each well of a
96-well microtiter plate . Serial drug dilutions of seven
3-fold dilution steps (90 - 0.123 μg/mL) were prepared.
Then 104 bloodstream forms of T. b. rhodesiense STIB 900
in 50 μL was added to each well and the plate incubated at
37°C under a 5% CO2 atmosphere for 72 h. Ten μL
Alamar Blue was then added to each well and incubation
continued for a further 2-4 h. The plates were read with a
Spectramax Gemini XS microplate fluorometer using an
excitation wavelength of 536 nm and an emission
wavelength of 588 nm. Data were analyzed using the
microplate reader software Softmax Pro (Molecular
Devices Cooperation, Sunnyvale, CA, USA). Cytotoxicity
was assessed using the same assay on rat skeletal
myoblasts (L6 cells). Melarsoprol and podophyllotoxin
were the control drugs.
Trypanocidal activity against T. 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% 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 containing the β-galactosidase (Lac Z) gene
. 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. After 96
h of incubation the plates were inspected under an inverted
microscope to assure growth of the controls and sterility.
The substrate CPRG/Nonidet (50 μL) was added to all
wells. A color reaction developed within 2-6 h, which was
read photometrically at 540 nm. Data were transferred into
the graphic program Softmax Pro, which calculated IC50
values. Benznidazole was the standard drug used.
Leishmanicidal activity against L. donovani: Amastigotes
of L. donovani strain MHOM/ET/67/L82 were grown in
Anti-infective potential of cyanobacterial extracts Natural Product Communications Vol. 6 (5) 2011 693
axenic culture at 37°C in SM medium at pH 5.4
supplemented with 10% heat-inactivated fetal bovine serum
under an atmosphere of 5% CO2 in air. Culture medium
(100 mL) with 105 amastigotes from axenic culture with or
without a serial drug dilution were seeded in 96-well
microtiter plates. Serial drug dilutions were prepared, and
after 72 h of incubation the plates were inspected under an
inverted microscope to assure growth of the controls and
sterile conditions. Ten μL of Alamar Blue was then added to
each well and the plates incubated for another 2 h .
Then the plates were read with a Spectramax Gemini XS
microplate fluorometer using an excitation wavelength of
536 nm and an emission wavelength of 588 nm. Data were
analyzed using the software Softmax Pro. Decrease of
fluorescence (= inhibition) was expressed as percentage of
the fluorescence of control cultures and plotted against the
drug concentrations. From the sigmoidal inhibition curves
the IC50 values were calculated. Miltefosine was used as a
Antimalarial activity against P. falciparum: In vitro
activity against erythrocytic stages of P. falciparum was
determined by a modified [3H]-hypoxanthine incorporation
assay using drug-resistant K1 strain and the standard drug
chloroquine. 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 37oC in a reduced oxygen
atmosphere, 0.5 μCi 3H-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. Radioactivity was measured using a
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. Briefly, Middlebrook 7H9 medium (100 μL),
supplemented with 10% oleic acid-albumin-dextrose-
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was added to each well of a 96-well flat-bottom plate. Serial
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well. The plate lid was then sealed with parafilm and the
plate incubated with gentle rocking (30 oscillations per min)
for 7 days at 37oC. Ten μL MTT (filter-sterilized at 5
mg/mL in dH2O) was added to each well and the plates were
then incubated for a further 24 h. MICs were recorded as the
lowest concentration at which a purple precipitate of
formazin did not appear in the wells. Streptomycin was used
as a positive control.
Acknowledgments – This project was partly supported by
the University of London Central Research Funds (DT),
together with funding from the Beaufort Marine
Biodiscovery Programme co-ordinated by the Marine
Institute (Ireland). We thank Alan and Anne Greenhouse
for help with the collection of the May sample of N.
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