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Plasmodium falciparum: Assessment of Selectivity of Action of Chloroquine,
Alchornea cordifolia, Ficus polita, and Other Drugs by a Tetrazolium-Based
Colorimetric Assay
Article · December 2011
DOI: 10.4061/2011/816250 · Source: PubMed
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Malaria Research and Treatment
Volume 2011, Article ID 816250, 7 pages
doi:10.4061/2011/816250
Research Article
Plasmodium falciparum: Assessment of Selectivity of Action of
Chloroquine, Alchornea cordifolia, Ficus polita, and Other Drugs
by a Tetrazolium-Based Colorimetric Assay
Nana Kofi Ayisi,1, 2 Regina Appiah-Opong,3 Ben Gyan,4 Kwasi Bugyei,5 and Fred Ekuban3
1Virology Department, Noguchi Memorial Institute for Medical Research (NMIMR), University of Ghana (UG), Legon, Ghana
2Department of Pharmacology, School of Medical Sciences, University of Cape Coast, Cape Coast, Ghana
3Department of Clinical Pathology, Noguchi Memorial Institute for Medical Research (NMIMR), University of Ghana (UG),
Legon, Ghana
4Department of Immunology, Noguchi Memorial Institute for Medical Research (NMIMR), University of Ghana (UG), Legon, Ghana
5Department of Pharmacology, University of Ghana Medical School, Korle Bu, Accra, Ghana
Correspondence should be addressed to Nana Kofi Ayisi, nana.ayisi@yahoo.co.uk
Received 28 July 2011; Accepted 12 September 2011
Academic Editor: Polrat Wilairatana
Copyright © 2011 Nana Kofi Ayisi et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
A tetrazolium-based colorimetric selective assay (MTT-based CSA) was developed to assess the selectivity of antimalarial drugs.
This in vitro assay, unlike all others, measures the ability of drugs to indirectly protect red blood cells (RBCs) from Plasmodium-
falciparum-induced destruction. Optimum incubation time and number of cells needed were 5 days and 23 × 106 RBCs per well,
respectively. A parasitemia range of 0.375% to 3% was found to be suitable for this assay. The MTT-based CSA determined anti-P.
falciparum strain DD2 activity of chloroquine at a higher 50% effective concentration (EC50) value (21.0 µg/mL) than the isotopic
microtest (10.0 µg/mL). Artesunate and oxytetracycline achieved 90% effect against DD2 with minimal or no toxicity to RBCs.
Against chloroquine sensitive strain 3D7, chloroquine and Alchornea cordifolia had EC50 values of 0.025 µg/mL and 4.9 µg/mL
respectively, and selective index (SI) values of >2,000 and >69.4 µg/mL, respectively.
1. Introduction parasite and the endpoints are directly parasite related.
Destruction of red blood cells (RBCs) is the end result of
The threat of malaria has continued to plague millions of malarial infection. None of the available in vitro assays uses
people living in most tropical countries despite numerous RBCs-related activity to determine endpoints. Furthermore,
strategies to bring this disease under control. Chloroquine the available in vitro assays do not test for drug selectivity
used to be the first drug of choice for the treatment of P. unless a separate assay on RBC toxicity is done. In this paper,
falciparum infections in Ghana and many African countries, we report on a tetrazolium-based colorimetric selective assay
but the emergence of drug-resistant strains has necessitated (MTT-based CSA) that uses indirect protection of RBCs
intensive search for new drugs [1–3]. Presently, Artesunate- from parasite destruction as an endpoint for measuring the
Amodiaquine and Artesunate-Lumefantrine combinations effect of drugs on parasites and also determines the possible
are first-choice drugs in Ghana. The standard World Health drug toxicity to the RBCs.
Organization (WHO) method for testing new antimalar-
ial drugs involves tedious microscopic parasite counting 2. Materials andMethods
method (MPCM). Other in vitro methods are haem poly-
merization inhibition assay (HPIA) [4], lactate dehydroge- 2.1. Drugs and Plant Extracts. Chloroquine diphosphate
nase assay (LDHA) [5, 6] and the isotopic microtest (IMT) (Sigma CQ) was obtained from SIGMA, St Louis, Mo, USA.
[7]. All these in vitro tests measure some activity of the Also tested in comparison were formulated chloroquine
2 Malaria Research and Treatment
(Troge Medical GmBH, Hamburg, Germany), formulated taken from each well and discarded. Two hundredmicroliters
chloroguanide (proguanil) (Zeneca Pharmaceuticals, UK), (200 µL) of 10% Triton X-100 in acidified isopropanol was
formulated artesunate (Guilin Pharmaceutical Works, Chi- added to each well in order to dissolve any formed formazan.
na), and formulated oxytetracycline (Bimeda, Ireland) pur- With the aid of a multichannel micropipettor, the contents
chased from a local pharmacy. Plant extracts GHX-26F and of each well were mixed several times, and the plates kept at
GHX-6L are aqueous extracts from the fruits of Alchornea room temperature in the dark for 24 h. The optical densities
cordifolia and the leaves of Ficus polita respectively. The of the wells were read by Emax Precision Microplate reader
aqueous extracts were freeze-dried. Both plants are effective (Molecular Devices Corporation, Menlo Park, Calif, USA) at
against human immunodeficiency virus in vitro [8] and 565 nm and 690 nm. Readings at 690 nm were automatically
are suspected to have antiprotozoal activities. All drugs and subtracted from readings at 565 nm by the microplate reader.
plant extracts were dissolved in culture medium and filtered The experiment was repeated once. The averages of all
through sterile 0.45 µm filters. readings were recorded for each dilution. A graph of cell
number versus differences in OD readings of uninfected and
2.2. Parasites. Strains 3D7 (chloroquine sensitive) and DD2 infected wells was then plotted to determine the optimum
(chloroquine resistant) of P. falciparum were obtained from cell number that will give the highest OD reading difference
Dr. Jorgehn Kurtzhals, Center for Medical Parasitology, between uninfected and infected wells.
University of Copenhagan, Denmark.
2.5. Determination of Optimum Parasitemia. Approximately,
2.3. Cell Cultures. P. falciparum infected and uninfected red 23× 106 cells per well were determined to give optimum dif-
blood cells (RBCs) were cultured in RPMI 1640 (Gibco, ference in OD readings between uninfected and P. falciparum
Parsley, UK) supplemented with 20mmol/mL L-glutamine infected cells in the above experiment. Twenty-three million
6
(Sigma-Aldrich Co. Ltd., UK), 20 µg/mL gentamicin and (23× 10 ) cells infected at various parasitemia ranging from
2.5 µg/mL fungizone (Gibco, Parsley, UK), 10% albumax II 0% to 6% were put into wells of 96-well microtitre plates and
(Gibco BRL, Grand Island, NY, USA), or 10% normal human incubated at 37◦C for 5 days. Each parasitemia was tested
AB serum (Sigma-Aldrich Co. Ltd., UK). The cultures were at 6 replicates. The cells were processed for MTT-based
flushed with a gas mixture consisting of 3%O2, 6%CO2, and colorimetric assay as described above. The average OD
91% N2, sealed and incubated at 37◦C. For experiments in readings at various levels of parasitemia were subtracted
96-well microtitre plates, the cultures were put in a desiccator from the average OD reading of control uninfected wells and
with a lit candle and nearly completely covered. As soon as the values plotted against % parasitemia.
the candle went off, the desiccator was completely closed.
2.6. Tetrazolium-Based Colorimetric Selective Assay (MTT-
2.4. Determination of Optimum RBC Number and Incubation Based CSA). From the results of the foregoing experiments,
Time. P. falciparum-strain-3D7-infected (1% parasitemia) optimum time of incubation, optimum number of RBCs,
and uninfected cultures were incubated as described above and optimum parasitemia were determined to be 5 days, 23×
6
and observed daily for color differences between the two cul- 10 cells per well, and 3% parasitemia, respectively. The drug
tures. The parasite-infected culture started becoming visually inhibition experiments therefore utilized these three values
pale from day 3. The difference in color between the infected in the testing of chloroquine, chloroguanide (proguanil),
and uninfected cultures increased up to day 5. There was no artesunate, oxytetracycline, and two plant extracts against P.
change in the observed paleness of the infected culture from falciparum infection in RBC cultures. In the case of Sigma
day 5 to day 6. Day 5 was therefore considered as the probable CQ, 1.5% parasitemia was used. Drug or plant extract was
optimum incubation time for visually determined optimum serially diluted, and a 100 µL of each dilution was put into six
parasite-induced damage to RBCs. The number of RBCs in wells of 96-well microtitre plates. One hundred microliters
culture that would give maximum difference between optical (100 µL) of uninfected RBCs was added to three replicate
densities (ODs) of uninfected and P. falciparum-infected drug dilutions. One hundred microliters (100 µL) of infected
RBCs was then determined as follows. The numbers of RBCs was added to three other replicate drug dilutions.
healthy RBCs in uninfected and parasite-infected stock cul- Drug-medium controls in the top and bottom outer wells
tures were determined by the trypan blue exclusion method were included in the experiments. The cultures were incu-
and adjusted to be the same per milliliter. Twofold serial dilu- bated in a desiccator as described in the previous experi-
tions of stock cell cultures (uninfected or 5% infected) were ments. TheMTT-based colorimetric test was then performed
made and 100 µL was put in wells of 96-well microtitre plates on the cultures. The percent protection of RBCs frommalaria
that already contained 100 µL of growth medium bringing parasite destruction by different drugs or plant extracts
the total volume per well to 200 µL. Each cell dilution was concentrations were determined by the following formula:
put in six replicate wells. The cultures were incubated in a Percent RBC Protection
desiccator as described above and after 5 days of incubation, { }
cell viability was determined by the MTT-based colorimetric (ODT) − (ODT) (1)Pf mock
method as previously described [9]. Twenty microliters = 1 + × 100,(ODC)mock − (ODC)Pf
(20 µL) of MTT (7.5mg/mL) was added to each well and
the plates incubated at 37◦C for another 2 h. One hundred where (ODT)Pf is the average OD measured for a given
and fifty microliters (150) µL of medium was then carefully concentration of the test drug in P. falciparum-infected
Malaria Research and Treatment 3
1
0.9 1
0.8 0.9
0.7 0.8
0.6 0.7
0.5 0.6
0.4 0.5
0.3 0.4
0.2 0.3
0.1 0.2
0 0.1
0 2.84 5.68 11.35 22.7 45.4 0
0 0.188 0.375 0.75 1.5 3 6
Number of RBCs ×106
Parasitemia (%)
(a) (b)
Figure 1: Effects of number of red blood cells (a) and parasitemia (b) on P. falciparum cytopathicity, that is, optical density of mock-infected
RBCs minus optical density of parasite-infected RBCs [(ODC)mock − (ODC)infected]. Infection was done with strain 3D7.
RBCs; (ODT)mock is the average OD measured for a given maximum lysis in infected RBCs with very little cell death
concentration of test drug in uninfected RBCs; (ODC)mock in uninfected control cultures. Figure 1(a) shows the effect
is the average OD measured for the control untreated of number of RBCs on the difference in OD readings
uninfected RBCs; (ODC)Pf is the average OD measured between infected and uninfected RBCs. The cell number
for the control untreated P. falciparum-infected RBCs. that produced the highest difference in OD readings between
The average percent RBC protection values for each three infected and uninfected RBCs was about 23× 106 per well.
replicates were plotted against the concentrations of drugs
and the concentrations needed to achieve 50% and 90% 3.2. Optimum Parasitemia. Figure 1(b) shows the effect of
RBC protection, that is, effective concentrations (EC50 and parasitemia on the ability of RBCs to metabolize tetrazolium
EC90) determined by regression analysis. The percent RBC to formazan. A parasitemia of 3% caused the optimum
survival determined the extent of drug toxicity and was damage to RBCs leading to high OD reading difference
calculated by the following formula: between uninfected and infected RBCs of 0.92. The lowest
− parasitemia at which enough damage to RBCs could be(ODT) (ODT)
Percent RBC Survival = mock cf × 100, detected was found to be 0.375% where the OD reading
(ODC)mock − (ODC)cf difference between uninfected and infected RBCs was 0.2.
(2)
where (ODT)cf is the average OD measured for cell-free
3.3. Comparative Activities of Drugs and Plant Extracts against
treated wells; and (ODC)cf is the average OD measured for
DD2 and 3D7 Strains of P. falciparum. The antimalarial
cell-free untreated wells. These values were plotted against activity of chloroquine diphosphate obtained from Sigma
the concentrations of the drugs and the concentrations was tested against strain DD2 in both the isotopic IMT and
needed to reduce RBC survival by 50%, that is, 50% the MTT-based CSA and the results are given in Figure 2
cytotoxic concentrations (CC50) determined by regression
and Table 1. Sigma CQ was slightly more active in the IMT
analysis. Anti-P. falciparum indices or selective indices (SI) than in the MTT-based CSA. The IMT unlike the MTT-
were then calculated as CC /EC . based CSA, does not include a toxicity test and is therefore50 50
not applicable to the determination of selective index of a
2.7. Isotopic Microtest (IMT) Assay. The effect of Sigma drug. In the MTT-based CSA, formulated CQ (Figure 3(a)
CQ on P. falciparum strain DD2 was tested in the IMT and Table 1) like Sigma CQ had moderate activity against
assay concurrently as in the MTT-based CSA. In the IMT this strain of P. falciparum. Proguanil (Figure 4(a)) and plant
assay, the incubation was interrupted after 24 hours for the extract GHX-26F (Figure 3(b) and Table 1) were minimally
addition of 0.5 µCi of 3H-hypoxanthine, and incubation con- effective against DD2. Formulated artesunate (Figure 4(b))
tinued for additional 24 hours before measurement of 3H- and oxytetracycline (Figure 4(c)) were very effective against
hypoxanthine uptake as published before [7]. The percent DD2. Formulated CQ and plant extract GHX-26F were
inhibition was plotted against concentration of drug and the very effective in the MTT-based CSA against chloroquine
EC and EC values determined by regression analysis. sensitive 3D7 with SI (CC50/EC50) values of >2,000 and50 90
>69.4, respectively (Table 1). Figure 5 is from a separate
3. Results experiment done for pictures to be taken to demonstrate that
the effects of drugs can be observed visually in the MTT-
3.1. Optimum Incubation Time and Cell Number. By visual based CSA. Clearly, formulated CQ and GHX-26F were very
examination, 5 days of incubation was found to give effective against strain 3D7, whereas GHX-6L had no activity.
(ODC)mock-(ODC)infected
(ODC)mock-(ODC)infected
4 Malaria Research and Treatment
120
100
80
120
100 60
80
60 40
40
20
20
0 0
0 0.39 0.78 1.56 3.13 6.25 12.5 25 50 100 0 0.39 0.78 1.56 3.13 6.25 12.5 25 50 100
Concentration of Σ CQ (µg/mL) −20 Concentration of Σ CQ (µg/mL)
RBC protection (%)
RBC survival (%)
(a) (b)
Figure 2: Anti-P. falciparum activity and toxicity of chloroquine (purchased from Sigma) tested in the IMT (a) and MTT-based CSA (b)
using parasite strain DD2.
140 120
120 100
100 80
80
60
60
40
40
20
20
0 0
0 0.01 0.02 0.07 0.21 0.62 1.85 5.56 16.7 50 0 1.33 2.66 5.31 10.63 21.25 42.5 85 170 340
Concentration of GHX-26F (µg/mL)
Concentration of formulated CQ (µg/mL)
RBC protection(3D7) (%) RBC protection(37D) (%)
RBC protection(DD2) (%) RBC protection(DD2) (%)
RBC survival (%) RBC survival (%)
(a) (b)
Figure 3: Anti-P. falciparum activity and toxicity of formulated chloroquine (a) and plant extract GHX-26F (b) tested in the MTT-based
CSA using parasite strains 3D7 and DD2.
4. Discussion to false-positive results. All available assays measure parasite-
related activity. Our MTT-based CSA measures RBC-related
Determination of in vitro selectivity is crucial in identifying activity ofmetabolism of tetrazolium into formazan. The end
new compounds with potentials as true antimalarial drugs. result is a measure of the ability of a compound to indirectly
There is no available in vitro antimalarial assay system that protect RBCs from P. falciparum-mediated lysis. The formula
determines drug selectivity. The term selectivity of action for calculating the percent RBC protection is the same as
in this paper refers to the extent to which the effect of drug published for antihuman immunodeficiency drugs [9]. The
is due to parasite events alone and not an indirect effect formula for calculating the percent RBC survival (toxicity)
resulting from effects on uninfected RBCs. This is very has however been modified to account for plant extracts and
important because a compound may destroy RBCs and other drugs whose color may increase the background OD
therefore prevent malaria parasite replication which will lead reading. The maximum difference in OD values between
Control (%) Inhibition (% )
Control (%) Control (%)
Malaria Research and Treatment 5
120 120
100 100
80 80
60 60
40 40
20 20
0 0
0 1 10 100 1000 0 0.5 5 50 500
Concentration of proguanil (µg/mL) Concentration of artesunate (µg/mL)
RBC protection(DD2) (%) RBC protection(DD2) (%)
RBC survival (%) RBC survival (%)
(a) (b)
120
100
80
60
40
20
0
0 0.08 0.31 1.22 4.88 19.53
Concentration of oxytetracycline (µg/mL)
RBC protection(DD2) (%)
RBC survival (%)
(c)
Figure 4: Anti-P. falciparum activity and toxicity of formulated proguanil (a), artesunate (b), and oxytetracycline (c) tested in theMTT-based
CSA using parasite strain DD2.
Table 1: Effects of drugs and plant extracts against P. falciparum strains DD2 (A) and 3D7 (B) infection in RBCs.
Drug EC50 µg/mL EC90 µg/mL CC50 µg/mL SI (CC50/EC50)
Sigma CQa∗ 10.0 20.4 NA NA
Sigma CQb∗ 21.0 77.9 82.7 3.9
(A) Formulated CQb 32.6 >50c >50c >1.5
GHX-6Lb >445c >445c >445c ND
GHX-26Fb 272.8 >340c >340c >1.2
Formulated CQb 0.025 0.05 >50c >2,000
(B) GHX-6Lb >445c >445c >445c ND
GHX-26Fb 4.9 16.2 >340c >69.4
a
Tested by IMT, bTested by MTT-based CSA, ∗Parasitemia used was 1.5%. For the other tests, parasitemia used was 3.0%, cHighest concentration tested, SI:
selective index, NA: not applicable, ND: not determined.
Control (%)
Control (%)
Control (%)
6 Malaria Research and Treatment
(a) (b)
(c)
Figure 5: Photographs of parasite strain 3D7-infected (bottom three replicates) and -uninfected (top three replicates) RBCs treated with
formulated chloroquine (a), plant extract GHX-6L (b), and plant extract GHX-26F (c) after the tetrazolium reaction. Concentrations are
given as µg/mL.
uninfected and infected RBCs may vary depending on the activity of the RBCs would be determined by the MTT-based
source of RBCs used as a result of differences in certain assay.
genetic factors involved in drug metabolism. As expected, chloroquine was not very effective against
We used a specific number of RBCs instead of a specific P. falciparum strain DD2 when tested in either the IMT or
hematocrit because the formation of formazan is dependent MTT-based CSA. There was very little difference between
on healthy and metabolically active RBCs. Even though unformulated chloroquine bought from SIGMA and formu-
approximately 23×106 RBCs/well was determined to give the lated chloroquine bought from a local pharmacy even though
maximum difference between OD readings of mock-infected the latter was tested at a higher parasitemia. Proguanil,
and parasite-infected RBCs, lower numbers of RBCs that give artesunate, and oxytetracycline were tested just to determine
OD reading difference of at least 0.2 can be considered as their effectiveness in inhibiting chloroquine-resistant DD2
a minimum requirement (Figure 1(a)). A similar case can induced RBC lysis. No attempt was made to find EC50 con-
be made for the parasitemia where reliable determinations centrations of these dugs since only four concentrations of
can be made using at least 0.375% parasitemia (Figure 1(b)). each drug were tested. Proguanil needs to be activated to
Chloroquine diphosphate (SIGMA CQ) was tested in the a triazine derivative in vivo to have any significant effect
IMT and MTT-based CSA using 1.5% parasitemia similar to on P. falciparum infection [10]. The minimal activity of
other tests. For the other drugs, 3% parasitemia was used in proguanil observed against chloroquine resistant strain DD2
order to be able to clearly visualize the effect of compounds may be due to the little intrinsic activity of the drug. Arte-
before and after the addition of tetrazolium without OD sunate and oxytetracycline were very effective against DD2
reading by microplate reader. The choice of not changing at concentrations with very little or no toxicity to RBCs.
the medium during the 5 days of incubation was to allow Artesunate is a peroxide antimalarial drug which is activated
for the death of the parasites so that only the metabolic by haem and through a series of processes, it is suspected
0.27
0.068
0.017
0.004
36.5
0.001
18.2
0
9.12 Infected Uninfected
4.56
2.28
850
1.14
170
0.57
34
0
6.8
Infected Uninfected
1.36
0.27
0.05
0
Infected Uninfected
Malaria Research and Treatment 7
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Acknowledgments
The authors thank Drs. Michael Aidoo (Centers for Disease
Control, Atlanta, USA), Vincent Tsiagbe (Department of
Pathology, New York University Medical School, USA),
Isabella Quakye (School of Public Health, University of
Ghana, Ghana), and Bill Rogers (Epidemiology Department,
NoguchiMemorial Institute forMedical Research, University
of Ghana, Ghana) for reading through this paper andmaking
valuable suggestions. This project was supported by the
Government of Ghana.
References
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585, 1992.
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