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In vitro anti-malarial interaction and gametocytocidal activity of cryptolepine

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DOI: 10.1186/s12936-017-2142-z

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 Malaria Journal
Forkuo et al. Malar J (2017) 16:496
https://doi.org/10.1186/s12936-017-2142-z

RESEARCH Open Access

In vitro anti‑malarial interaction

and gametocytocidal activity of cryptolepine
Arnold Donkor Forkuo1*, Charles Ansah1, Kwesi Boadu Mensah1, Kofi Annan1, Ben Gyan2, Anjo Theron3,
Dalu Mancama3 and Colin W. Wright4

Abstract 
Background:  Discovery of novel gametocytocidal molecules is a major pharmacological strategy in the elimination
and eradication of malaria. The high patronage of the aqueous root extract of the popular West African anti-malarial
plant Cryptolepis sanguinolenta (Periplocaceae) in traditional and hospital settings in Ghana has directed this study
investigating the gametocytocidal activity of the plant and its major alkaloid, cryptolepine. This study also investigates
the anti-malarial interaction of cryptolepine with standard anti-malarials, as the search for new anti-malarial combina-
tions continues.
Methods:  The resazurin-based assay was employed in evaluating the gametocytocidal properties of C. sanguinolenta
and cryptolepine against the late stage (IV/V) gametocytes of Plasmodium falciparum (NF54). A fixed ratio method
based on the SYBR Green I fluorescence-based assay was used to build isobolograms from a combination of cryptole-
pine with four standard anti-malarial drugs in vitro using the chloroquine sensitive strain 3D7.
Results:  Cryptolepis sanguinolenta ­(IC50 = 49.65 nM) and its major alkaloid, cryptolepine ­(IC50 = 1965 nM), showed
high inhibitory activity against the late stage gametocytes of P. falciparum (NF54). In the interaction assays in asex-
ual stage, cryptolepine showed an additive effect with both lumefantrine and chloroquine with mean ΣFIC50s of
1.017 ± 0.06 and 1.465 ± 0.17, respectively. Cryptolepine combination with amodiaquine at therapeutically relevant
concentration ratios showed a synergistic effect (mean ΣFIC50 = 0.287 ± 0.10) whereas an antagonistic activity (mean
ΣFIC50 = 4.182 ± 0.99) was seen with mefloquine.
Conclusions:  The findings of this study shed light on the high gametocytocidal properties of C. sanguinolenta and
cryptolepine attributing their potent anti-malarial activity mainly to their effect on both the sexual and asexual stages
of the parasite. Amodiaquine is a potential drug partner for cryptolepine in the development of novel fixed dose
combinations.
Keywords:  Gametocytocidal, Malaria, Anti-malarial drug combinations, Cryptolepis sanguinolenta, Cryptolepine

Background Despite the successes seen in the approach to reduce

Malaria still remains a disease of public health impor- the number of deaths associated with malaria, this dis-
tance with an estimated 3.3 billion people at risk globally ease still remains a grave public health problem. The
and 214 million new cases with 438,000 deaths reported majority of the currently used anti-malarial drugs were
in 2015. The sub-Saharan region of Africa accounts for an intended to target the symptom-causing pathogenic
estimated 90% of all deaths due to malaria, with children blood stages in man and to contend with the continu-
under 5 years of age accounting for 78% of these [1]. ous risk of drug resistance [2]. Nonetheless, to achieve
the global objective of malaria eradication, medicines
with activity against parasite transmission [3] and the
*Correspondence: forkuoarnold@yahoo.com
1
Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical
hepatic stages responsible for disease relapse also need to
Sciences, College of Health Sciences, Kwame Nkrumah University be developed. The reported development of Plasmodium
of Science and Technology, Kumasi, Ghana falciparum resistance to the most potent anti-malarial
Full list of author information is available at the end of the article

© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Forkuo et al. Malar J (2017) 16:496 Page 2 of 9

agents (the artemisinins) in the Thai–Cambodia border in distilled water prior to use in the gametocytocidal
puts the progress achieved under serious threat [4, 5]. assay.
This scenario requires the urgent need to accelerate the
discovery and development of novel anti-malarial leads Drugs/chemicals
and combinations that have activity against both the sex- Lumefantrine, chloroquine, mefloquine and amodi-
ual and asexual stages of the parasite. aquine were purchased from Sigma-Aldrich (St. Louis,
For this reason, the popular West African anti-malar- MO, USA). Gentamicin was obtained from Invitrogen
ial plant Cryptolepis sanguinolenta (Periplocaceae) and Life Technologies Inc. (Carlsbad, CA, USA). RPMI-
it major alkaloid, cryptolepine were assessed against the 1640 medium, streptomycin/penicillin, l-glutamine and
transmissible stages of the human malaria parasite. The HEPES were obtained from Gibco BRL Life Technologies
aqueous root extract of C. sanguinolenta has been used (Grand Island, NY, USA). Cryptolepine hydrochloride
for decades in West Africa for the treatment of malaria (purity 98.9%) was isolated from the root of C. sanguino-
[6] and currently, several herbal preparations registered lenta as described by Kuntworbe et al. [10].
for use in Ghanaian orthodox clinics contain the aque-
ous root extract of the plant. The wide usage of the plant In vitro cultivation of asexual stage Plasmodium falciparum
may be attributed to the safety and high cure rate of a tea The in  vitro cultivation method of P. falciparum drug-
bag formulation (Phyto-laria) of the plant against chloro- sensitive strain NF54 was adapted from Reader et al. [11].
quine resistant strains of P. falciparum in human clinical Parasite cultures were maintained in human erythrocytes
trials [7], supporting their usage as alternative tools to the (5% haematocrit, A rhesus positive) suspended in com-
standard anti-malarial interventions or as complemen- plete parasite medium (CPM) (RPMI 1640 medium sup-
tary treatments in the absence of standard anti-malarial plemented with 25  mM HEPES, 0.2% d-glucose, 24  μg/
drugs. Cryptolepine, the major indoloquinoline alkaloid mL gentamicin, 0.2% sodium bicarbonate (pH  =  7.3),
isolated from the plant has the most potent antiplasmo- 200 μM hypoxanthine with 10% human serum (A rhesus
dial activity [8] and has been shown to exhibit potent positive) and flushed with 90% ­N2, 5% O
­ 2, and 5% C
­ O2 in
in vitro activities against both chloroquine-sensitive and humidified modular chambers at 37 °C. Parasite medium
chloroquine-resistant P. falciparum [9]. was changed daily and fresh CPM introduced. Parasitae-
With lessons learnt from the widely used artemisinin mia of Giemsa-stained slides were monitored daily with
derivatives, thus their isolation from the medicinal plant light microscopy.
Artemisia annua, directed the exploration of the popu-
larly used anti-malarial plant, C. sanguinolenta and its Induction of gametocytogenesis and maintenance
major alkaloid, cryptolepine for possible gametocytocidal of gametocyte cultures
effects on P. falciparum. The in vitro anti-malarial inter- Combined conditions of low haematocrit and nutri-
action of cryptolepine with chloroquine, lumefantrine, ent starvation were employed in the induction of game-
amodiaquine and mefloquine (Fig.  1) were also ascer- tocytogenesis. To trigger gametocytogenesis, asexual
tained in this study. cultures of 6–10% parasitaemia were diluted to 0.5%
parasitaemia at 6% haematocrit and then introduced into
Methods a glucose-free medium. The medium was kept at 37  °C
Plant material gassed with 90% ­N2, 5% ­O2, and 5% ­CO2 without shaking.
The sun-dried cut roots of C. sanguinolenta used in this The medium was changed daily. After 72  h, the haema-
study were collected at the Centre for Scientific Research tocrit was dropped to 3% (day 0). Gametocytogenesis
into Plant Medicine (CSRPM), Mampong-Akwapim, was monitored daily before medium (glucose-free) was
Ghana in August 2012 and were identified by the Plant changed. To eliminate residual asexual parasites, cultures
Development Centre of the Institution. Its authenticity were treated on days 6–9 by continuous treatment with
was confirmed by Dr. Kofi Annan of the Department of 50  mM N-acetyl glucosamine (NAG). The medium was
Pharmacognosy, KNUST and subsequently compared then fortified with 0.2% glucose from day 10 onwards.
to a voucher specimen KNUST/HM1/2008/L056 at the The gametocyte levels were monitored daily by micros-
herbarium of the Department of Pharmacognosy/Herbal copy until they were predominantly (> 90%) stage V and
Medicine, College of Health Sciences. these gametocytes were employed in the resazurin-based
The powdered roots (650  g) were boiled for 30  min assay.
with 5  L of distilled water, decanted and filtered. The
filtrate was freeze dried to obtain a freeze-dried sam- Gametocyte viability assays
ple of the crude extract (yield  =  11.08% w/w) referred The in  vitro gametocytocidal activities of cryptolepine
to as cryptolepis (CPS). Cryptolepis was reconstituted and the aqueous root extract of C. sanguinolenta were
Forkuo et al. Malar J (2017) 16:496 Page 3 of 9

a
b OH CH3
HO
N
H N CH3
HN

F
N
F
F Cl N
F F
F

d Cl

c Cl
CH3
CH3

N CH3
Cl
HN
H

HO N

Cl N
CH3

CH3

e
CH3

N
H

HCl
Fig. 1  Structure of anti-malarial agents used in the interaction assay. a Mefloquine, b amodiaquine, c chloroquine, d lumefantrine, e crytolepine
hydrochloride

measured by assessing gametocyte survival after drug concentration of 5% haematocrit, 2% gametocytaemia in

exposure using resazurin-based assay. The resazurin- a total incubation volume of 100 µL.
based assay was based on method described by Tanaka Dihydroartemisinin (10  µM) was used as a reference
et  al. [12]. Eleven drug concentrations of cryptolepine, standard in the drug assay. The drug plate was placed on a
the aqueous root extract of C. sanguinolenta and dihy- mechanical shaker for 20 s before being encased in an air-
droartemisinin ranging from 10  µM to 1.69  ×  10−4 µM tight chamber and gassed for 5 min with a 5% ­O2, 5% ­CO2
(threefold serial dilutions) prepared in distilled water and a balanced ­N2 mixture. The plates were incubated at
were placed in triplicate in a transparent 96-well flat 37 °C for 48 h after which 10 µL of resazurin-based assay
bottom plates. Parasitized RBCs were added to a final reagent was added to each well and the plate was shaken
Forkuo et al. Malar J (2017) 16:496 Page 4 of 9

for 20 s. The plate was left to incubate for 2 h and then desired final concentrations for the assay. The final haem-
centrifuged at 120×g for 1 min. The supernatant (70 µL) atocrit and parasitaemia of the culture media were 2 and
was transferred to a clean 96-well plate before being read 1%, respectively. The assay plates were gassed with a mix-
in a multiwell spectrophotometer (Infinite F500, Tecan, ture of gases containing 92% ­N2, 5% ­CO2 and 3% ­O2 for
USA) by fluorescence detection at 535/612 nm. Gameto- 6 min and carefully arranged in a modular incubator (Bil-
cyte survival in each test culture after treatment was cal- lups-Rothenberg Inc, USA). The plates were incubated
culated relative to the control wells. The experiment was at 37 °C for 48 h. Post incubation, the plates were stored
performed in triplicate on two separate occasions. at −  30  °C wrapped in aluminium foil. The plates were
thawed and subsequently mixed with 100  μL of Malaria
Antiplasmodial interaction assay SYBR Green 1 fluorescent (MSF) lysis buffer containing
In vitro malaria parasite cultivation SYBR Green. The plates were incubated at room tem-
Plasmodium falciparum laboratory strain 3D7 was cul- perature in the dark for an hour and fluorescence data
tured according to method described by Trager and (relative fluorescence unit, RFU) were acquired using a
Jensen [13] with slight modifications as described. Human fluorescence multi-well plate reader (Tecan Infinite M200
erythrocytes (O rhesus positive) fortified in complete Pro) with emission and excitation wavelength at 535 and
culture medium (pH 7.3) served as host cells for parasite 485  nm, respectively. The experiment was performed in
maturation. The complete culture medium consisted of triplicate.
filter-sterilized RPMI 1640 solution supplemented with
0.5% AlbuMAX II, 0.72% HEPES (N-2-hydroxyethyl- Statistical analysis
piperazine-N-2-ethanesulfonic acid) and hypoxanthine The performance of the gametocyte assay was assessed
buffered with 0.4% sodium bicarbonate (­NaHCO3). Gen- by estimating the Z factor statistical parameter described
tamicin (0.005 mg/mL) was added to the final solution. by Zhang et al. [15]. Gametocyte viability was calculated
A controlled experimental environment with a gas using the relation below
supply of 92% ­N2, 5% ­CO2 and 3% ­O2 at 37 °C was used
% Parasite viability
to grow the parasites in culture flasks. Parasite growth,  
Fluorescencecompound − Mean fluorescencebackground
viability and stages were monitored daily by light micros- =   × 100
Mean fluorescencepositive − Mean fluorescencebackground
copy of Giemsa-stained cultures after changing the cul-
ture media. Parasitaemia levels were kept between 2 and The ­IC50 (50% inhibitory concentration) served as a
8%, with a 5% haematocrit. measure of anti-malarial activity and was determined
by plotting and analysing dose–response curves using
In vitro antiplasmodial interaction assay GraphPad Prism (GraphPad 6 Software, San Diego,
The SYBR Green I-based fluorescence assay was USA). The assessment of drug interaction was based on
employed to investigate the in  vitro anti-malarial inter- calculating the sum of the fractional inhibitory concen-
action between cryptolepine and the four standard tration (ΣFIC50s) at the given effective concentration by
anti-malarial agents against P. falciparum (chloroquine the formula below
sensitive, 3D7). Cryptolepine (Drug A, CPE), lumefan-
trine (LUM), mefloquine (MFQ), chloroquine (CQ) and  
IC50 of cryptolepine in combination

amodiaquine (AMQ) stock solutions were prepared in FIC50 =
IC50 of cryptolepine alone
ethanol at 1  mM. Concentrations ranging from 32.5 to  
2080  nM for CPE, 2 to 640  nM for LUM, 3.2 to 83  nM IC50 of Drug B in combination
+
for CQ, 5 to 800 nM for MFQ and 1 to 128 nM for AMQ IC50 of Drug B alone
were used for the interaction assays. A fixed ratio interac-
tion assay as described previously by Fivelman et al. [14] The nature of interaction was explained using ΣFIC50s.
was employed in this assay. Values <  0.8 denote synergism, 0.8–1.4 denotes addi-
For each assay, fixed drug ratios (4:1, 3:2, 2:3 and 1:4) tive interaction, and ≥ 1.4 denotes antagonism [16]. The
were prepared in a 10 µL volume for cryptolepine (Drug overall nature of the anti-malarial interaction was based
A) and any of the standard agents (Drug B). A twofold on the mean ΣFIC50s.
serial dilution followed each fixed ratio in a well ensur- A Microsoft Excel datasheet was used to calculate
ing the ­IC50 of each drug alone (5:0 and 0:5) fell approxi- percentage inhibition in relation to the control results.
mately at the mid-point of the serial dilution. Parasite Figures were made using GraphPad Prism for Windows
culture (90  µL) was added to each well to obtain seven version 6.0 (GraphPad Software, San Diego, CA, USA).
Forkuo et al. Malar J (2017) 16:496 Page 5 of 9

Results P. falciparum. The assay assesses gametocyte survival

Gametocyte viability assay after drug exposure using resazurin-based assay, and
Data from the late stage gametocyte assay indicates a indicated a potent loss of viability of the late stage game-
preferential inhibition of the viability of mature sexual- tocyte with both C. sanguinolenta (49.65 nM) and cryp-
stage parasites by the aqueous extract of C. sanguino- tolepine (1965 nM). This result is not surprising as earlier
lenta and its major alkaloid cryptolepine. The reduction work [18] demonstrated potent in  vitro loss of viability
in gametocytes was in a dose-dependent fashion in all of the late stage gametocytes by local polyherbal anti-
drug treatments and statistically significant. I­C50 values malarial products containing C. sanguinolenta. Despite
of 49.65 and 1965  nM were recorded for CPS and CPE, the demonstrated activity, the major alkaloid from the
respectively (Fig. 2). The Z-factor for the assay was calcu- plant known to possess the most efficacious in  vitro
lated to be 0.98, indicating that the assay performed very antiplasmodial activity showed less gametocytocidal
well. Cryptolepis sanguinolenta however, demonstrated activity compared to the root extract of the plant. This
more potent activity against the late stage gametocytes indicates the presence of other alkaloids in the aqueous
compared to its major alkaloid, cryptolepine. The refer- root extract of the plant contributing to the high activity
ence standard, dihydroartemisinin (10–1.69 × 10−4 µM) against gametocytes. Given the demonstrated high game-
exhibited an ­IC50 of 15  nM (Fig.  2) falling within the tocytocidal properties of CPS and CPE, their prominent
range normally expected for the resazurin-based assay antiprotozoal activity may be attributed mainly to their
and other gametocytocidal assay platforms, as described effect on both the asexual and sexual stages of the Plas-
by Reader et al. [11]. modium parasite.
Taken together, C. sanguinolenta (CPS) and it major Our current study also sheds light on the in vitro anti-
alkaloid, cryptolepine (CPE) have potent inhibitory malarial interaction of cryptolepine with four standard
effects on the late stage gametocyte of P. falciparum anti-malarial agents. The steady rise in the resistance of
strain NF54 (Fig. 2). P. falciparum to current anti-malarial drugs, has directed
current therapeutic strategies toward the development of
In vitro antiplasmodial interaction combination therapies. Cryptolepine is a promising anti-
The in vitro ­IC50s of cryptolepine and the standard anti- malarial compound effective against both chloroquine-
malarial drugs in the asexual stages of P. falciparum sensitive and chloroquine-resistant P. falciparum [9].
3D7 are presented in Table 1. In the in vitro interaction It has however been shown to possess DNA intercalat-
assays, cryptolepine’s combination with chloroquine or ing and topoisomerase II inhibitory effect [19, 20]. This
lumefantrine showed additivity with a mean ΣFIC50 of has directed research into structural modification of the
1.342  ±  0.34 and 1.017  ±  0.45, respectively (Table  2). compound to synthesize relatively safer derivatives. Pre-
Cryptolepine combination with amodiaquine at all vious pharmacokinetic reports of cryptolepine showed a
therapeutically relevant ratios showed a mean ΣFIC50 rapid disappearance from the plasma and localization in
(0.235  ±  0.15) of less than 0.8 suggesting synergism. various tissues except the CNS with possible hepatobil-
Antagonism (mean ΣFIC50 = 4.182 ± 0.68) was observed iary clearance pathway [21]. Aldehyde oxidase has been
when cryptolepine was combined with mefloquine. shown to be involved in the metabolism of cryptolepine
Isobolograms indicating various cryptolepine-based [22]. Nanoformulation of cryptolepine hydrochloride
combinations are presented in Fig. 3. demonstrated a better in  vivo antiplasmodial chemo-
suppression, superior bioavailability and an increased
Discussion half-life compared with the free compound [23]. A can-
The aqueous root extract of C. sanguinolenta has gained didate drug partner is sought for the parallel develop-
wide usage as an anti-malarial agent in both herbal and ment of cryptolepine, comparable to ACT encouraged
orthodox hospitals in Ghana and other West African by the WHO. This approach will provide cryptolepine
countries. This coupled with the plant’s established effi- with potential drug partners for further development to
cacy against the asexual stages of P. falciparum [7, 17] reduce the risk of drug resistance to P. falciparum. A new
motivated this study to evaluate possible gametocyto- anti-malarial combination should preferably target both
cidal properties of the plant and its major alkaloid, cryp- the rapidly replicating asexual stages and the less meta-
tolepine. In the current study, the stage-specific in  vitro bolically active, nonreplicating mature gametocytes.
gametocytocidal activities of C. sanguinolenta (CPS) The in  vitro anti-malarial interaction of cryptole-
and its major alkaloid cryptolepine (CPE) were tested pine using the SYBR Green fluorescent assay demon-
against the late-stage NF54 strain of the malaria parasite, strated varied interaction with these standard agents.
Forkuo et al. Malar J (2017) 16:496 Page 6 of 9

a
100

CPS
80
% Gametocyte viability

60

40

20

0
-4 -3 -2 -1 0 1 2 3
Log conc ( M)

b 100

CPE
80
% Gametocyte viability

60

40

20

0
-4 -3 -2 -1 0 1 2 3

Log conc ( M)

c 100

DHA
80
% Gametocyte viability

60

40

20

0
-4 -3 -2 -1 0 1 2 3
Log conc ( M)
Fig. 2  Gametocyte viability of CPS and CPE. Gametocytocidal activity of CPS (a), CPE (b) and DHA (c) against late stage gametocytes. Bars represent
mean gametocyte activity at each compound concentration (with standard deviation [SD])
Forkuo et al. Malar J (2017) 16:496 Page 7 of 9

Table 
1 In vitro anti-malarial activity against asexual membrane damage and parasite death [9, 26, 27]. On
blood stages this background it is not surprising that antagonism was
Drugs Mean ­IC50 ± S.E.M (nM) observed when cryptolepine was combined with meflo-
quine, possibly following a similar pathway of antago-
Amodiaquine 12.63 ± 1.66 nism demonstrated by mefloquine when combined with
Mefloquine 12.98 ± 0.47 chloroquine. In the case of cryptolepine-lumefantrine
Lumefantrine 10.80 ± 0.97 combination at therapeutically relevant concentration
Chloroquine 11.05 ± 1.79 ratios, an additivity effect (mean ΣFIC  =  1.017  ±  0.45)
Cryptolepine 603.82 ± 75.57 was observed.
IC50 values of cryptolepine, lumefantrine, mefloquine, and chloroquine against The combination of cryptolepine with amodiaquine
Plasmodium falciparum strain 3D7 are expressed as mean ± standard error of showed synergy in vitro in P. falciparum strain 3D7 and
mean of the results of at least three independent assays
this combination provides a dual action with both agents
inhibiting haemoglobin digestion in the asexual blood
Cryptolepine showed varied interaction with the 4-ami- stages and amodiaquine inhibiting gametocyte matu-
noquinolines, amodiaquine and chloroquine. The ration/gamete exflagellation by different mechanisms
combination of cryptolepine with amodiaquine had a [28]. Such combinations with dual action are relevant in
synergistic effect in  vitro (mean ΣFIC  =  0.235  ±  0.15) malaria endemic regions, where infections are usually
whereas an additive effect (mean ΣFIC  =  1.342  ±  0.34) asymptomatic with clinical symptoms developing late
was seen with chloroquine. The 4-aminoquinolines in the course of the disease, permitting the maturation
are considered to share, in principle, the same mode of of gametocytes and hence disease transmission [29, 30].
action. However, a different interactive profile was found The enhanced anti-malarial activity of cryptolepine with
with cryptolepine. It is therefore wrong to equate amo- amodiaquine may possibly result in low-dose treatment
diaquine to chloroquine in terms of activity. The synergy regimens and hence reduce toxicity. However, it should
observed with amodiaquine may be due to the Man- be noted that these assay results may vary in different
nich base structure in amodiaquine. A similar synergis- plasmodium strains and further investigations is recom-
tic effect attributed to the Mannich base side chain in mended in the early stages of gametocytes and in several
pyronaridine and amodiaquine has been reported with P. falciparum strains and/or rodent malaria models to
artemisinin [24]. An antagonistic effect was observed accentuate the interactions established.
when cryptolepine was combined with mefloquine.
Mefloquine has been shown to inhibit the uptake of chlo- Conclusions
roquine as well as chloroquine’s ability to cause the accu- Cryptolepis sanguinolenta and its major alkaloid, cryp-
mulation of undigested haemoglobin [25]. tolepine exhibited high activities against the late-stage
Mefloquine (and possibly quinine) has also been gametocyte of P. falciparum NF54, attributing their pri-
hypothesized to inhibit endocytosis of the erythrocyte mary targets as the mature gametocytes and intraeryth-
cytosol by the parasite, resulting in a lowered free haem rocytic parasite. Lumefantrine and chloroquine both
concentration, to which chloroquine binds, in the diges- showed additivity when each drug was combined with
tive vacuole [25]. Cryptolepine, an indoloquinoline has cryptolepine whereas antagonism was observed when
been shown to act similarly to chloroquine by inhibit- cryptolepine was combined with mefloquine at thera-
ing the biomineralization of the toxic waste material peutically relevant concentration ratios. Cryptolepine
haem into an insoluble complex, haemozoin, leading to in combination with the Mannich base, amodiaquine,

Table 2  In vitro interaction of cryptolepine combinations against 3D7 P. falciparum strains

Drug combination ΣFIC50 Mean ΣFIC50 Interaction
4:1 3:2 2:3 1:4

Cryptolepine + amodiaquine 0.640 ± 0.08 0.0234 ± 0.11 0.0631 ± 0.07 0.421 ± 0.12 0.287 ± 0.10 Synergism

Cryptolepine + mefloquine 3.015 ± 0.91 3.013 ± 1.01 5.106 ± 1.13 5.595 ± 0.91 4.182 ± 0.99 Antagonism
Cryptolepine + lumefantrine 2.1108 ± 0.17 1.6865 ± 0.01 0.1475 ± 0.02 0.1245 ± 0.04 1.017 ± 0.06 Additivity
Cryptolepine + chloroquine 1.95 ± 0.21 1.775 ± 0.36 1.66 ± 0.04 0.475 ± 0.05 1.465 ± 0.17 Additivity
The ratios 4:1, 3:2, 2:3 and 1:4 refer to fixed dosage ratios for drug A (cryptolepine) to drug B (amodiaquine, mefloquine, lumefantrine, chloroquine). Values are the
means from ≥ 3 experiments
Forkuo et al. Malar J (2017) 16:496 Page 8 of 9

Fig. 3  Effects of combinations of cryptolepine with standard anti-malarial drugs on Plasmodium falciparum growth in vitro (3D7 strain). Isobolo-
grams illustrating the effect of combinations of cryptolepine with amodiaquine (a), mefloquine (b), and chloroquine (c). The interaction between
cryptolepine and amodiaquine, mefloquine, chloroquine or lumefantrine against ring stage parasites was determined using the SYBR Green I
fluorescence-based drug sensitivity assay with the fixed ratio method. Each combination was set up in triplicate for 48 h. The F­ IC50 concentrations
were used for plotting the isobologram

showed synergism. On this basis, the cryptolepine– lumefantrine; AMQ: amodiaquine; CQ: chloroquine; nM: nano-molar; mM:
milli-molar; CPM: complete parasite medium; RBC: red blood cell; MSF: malaria
amodiaquine combination certainly deserves atten- SYBR Green I fluorescence.
tion for further studies in regards to the synergy
demonstrated. Authors’ contributions
ADF, DM, AT participated in the study design, carried out the experiments, per-
formed the statistical analysis, and drafted the manuscript; CA, KBM, and BG
participated in the study design and the execution of the experiments; ADF
Abbreviations
conducted the in vitro anti-malarial interaction assays. CW isolated cryptole-
FIC: fractional inhibitory concentrations; Mean ΣFICs: mean sums of fractional
pine hydrochloride from the plant, Cryptolepis sanguinolenta; KA, CA, CW, KBM
inhibitory concentrations; IC50: 50% inhibitory concentration; WHO: World
and BG contributed to the study design and revision of the manuscript. All
Health Organization; ACT: artemisinin-based combination therapy; CPE:
authors read and approved the final manuscript.
cryptolepine; CPS: aqueous root extract of Cryptolepis sanguinolenta; LUM:
 Forkuo et al. Malar J (2017) 16:496 Page 9 of 9

Author details 11. Reader J, Botha M, Theron A, Lauterbach SB, Rossouw C, Engelbrecht D,
1
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