(2019) 18:431
Camara et al. Malar J
https://doi.org/10.1186/s12936-019-3071-9
Malaria Journal
Open Access
RESEARCH
Terminalia albida treatment improves
survival in experimental cerebral malaria
through reactive oxygen species scavenging
and anti-inflammatory properties
Aissata Camara1,2* , Mohamed Haddad1, Karine Reybier1, Mohamed Sahar Traoré2,3, Mamadou Aliou Baldé2,3,
Jade Royo1, Alpha Omar Baldé2,3, Philippe Batigne1, Mahamane Haidara4, Elhadj Saidou Baldé2,3, Agnès Coste1,
Aliou Mamadou Baldé3 and Agnès Aubouy1
Abstract
Background: The development of Plasmodium resistance to the last effective anti-malarial drugs necessitates the
urgent development of new anti-malarial therapeutic strategies. To this end, plants are an important source of new
molecules. The objective of this study was to evaluate the anti-malarial effects of Terminalia albida, a plant used in
Guinean traditional medicine, as well as its anti-inflammatory and antioxidant properties, which may be useful in
treating cases of severe malaria.
Methods: In vitro antiplasmodial activity was evaluated on a chloroquine-resistant strain of Plasmodium falciparum
(K-1). In vivo efficacy of the plant extract was measured in the experimental cerebral malaria model based on Plasmodium berghei (strain ANKA) infection. Mice brains were harvested on Day 7–8 post-infection, and T cells recruitment
to the brain, expression levels of pro- and anti-inflammatory markers were measured by flow cytometry, RT-qPCR and
ELISA. Non-malarial in vitro models of inflammation and oxidative response were used to confirm Terminalia albida
effects. Constituents of Terminalia albida extract were characterized by ultra‐high performance liquid chromatography coupled with high resolution mass spectrometry. Top ranked compounds were putatively identified using plant
databases and in silico fragmentation patterns.
Results: In vitro antiplasmodial activity of Terminalia albida was confirmed with an IC50 of 1.5 μg/mL. In vivo, Terminalia albida treatment greatly increased survival rates in P. berghei-infected mice. Treated mice were all alive until Day
12, and the survival rate was 50% on Day 20. Terminalia albida treatment also significantly decreased parasitaemia by
100% on Day 4 and 89% on Day 7 post-infection. In vivo anti-malarial activity was related to anti-inflammatory properties, as Terminalia albida treatment decreased T lymphocyte recruitment and expression of pro-inflammatory markers
in brains of treated mice. These properties were confirmed in vitro in the non-malarial model. In vitro, Terminalia albida
also demonstrated a remarkable dose-dependent neutralization activity of reactive oxygen species. Twelve compounds were putatively identified in Terminalia albida stem bark. Among them, several molecules already identified
may be responsible for the different biological activities observed, especially tannins and triterpenoids.
*Correspondence: aichali2004@yahoo.fr
1
UMR152 PHARMADEV, IRD, UPS, Université de Toulouse, Toulouse,
France
Full list of author information is available at the end of the article
© The Author(s) 2019. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
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permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco
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zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Camara et al. Malar J
(2019) 18:431
Page 2 of 15
Conclusion: The traditional use of Terminalia albida in the treatment of malaria was validated through the combination of in vitro and in vivo studies.
Keywords: Terminalia albida, Malaria, Experimental cerebral malaria, Inflammatory response, Oxidative stress, UHPLCHRMS
Background
Malaria continues to be one of the primary medical concerns in many countries worldwide in terms of number
of cases and deaths. In 2017, there were approximately
219 million malaria cases and 435,000 deaths worldwide,
91% of these in sub-Saharan Africa [1]. Today, there are
few efficient anti-malarial treatments, and all are derived
from artemisinin. However, Plasmodium falciparum has
developed mechanisms of resistance to artemisinin and
its derivatives, particularly in Southeast Asia [2]. Recent
studies report increasing time for parasite clearance
after treatment in a few parasite isolates originating from
West Africa [3, 4]. The development of new treatments
based on effective molecules using mechanisms of action,
which are different to artemisinin and its derivatives, is
thus urgently needed.
Malaria occurs in different forms; it can be uncomplicated, or it may lead to more severe pathologies, particularly cerebral malaria (CM). This is the deadliest form of
malaria, with a mortality rate of approximately 15–25%
[5]. The cerebral complications are related to a preferential localization of infected erythrocytes (iEs) in the
brain through interactions between parasite proteins
expressed on the surface of infected red blood cells and
brain endothelium [6]. The mechanical obstruction of
brain blood flow due to accumulation of iEs and rosetting leads to ischemia, hypoxia and activation of cerebral
endothelium [7]. Activated endothelium produces proinflammatory cytokines and chemokines involved in the
recruitment of immune cells. While the role of monocytes, macrophages and dendritic cells is to remove iEs
by phagocytosis, they also produce pro-inflammatory
cytokines that activate cytotoxic T cells involved in
blood–brain barrier damage [8]. Degradation of haemoglobin by the parasite generates large quantities of toxic
free haem and reactive oxygen species (ROS), causing
cell damage to the host [9]. In addition, ROS production
by monocytes/macrophages is an efficient host defence
mechanism, although these oxidative processes lead to
an imbalance between pro- and anti-oxidant responses,
resulting in oxidative stress [10].
Plants are a significant source of molecules for the
development of new drugs. Many plants from traditional
medicine have provided opportunities for the identification of anti-malarial molecules. Artemisia annua (Asteraceae), a plant used in traditional Chinese medicine, is
currently the basis of the last effective treatment strategy
[11]. However, specific research of active natural products against severe malaria is scarce. Among the biologically active plant species, Terminalia albida, a member
of the Combretaceae family traditionally used in Guinea
to treat malaria, has shown promising anti-malarial
effects against P. falciparum (clone Pf-K1, IC50 = 0.6 µg/
mL) [12]. It was previously demonstrated that treatment
with Terminalia macroptera, another species of the same
genus, facilitated increased survival in experimental
cerebral malaria (ECM) [13]. Therefore, this study was
designed to assess the potential anti-malarial and antiplasmodial effects of Terminalia albida in the murine
model of ECM. Anti-inflammatory and anti-oxidant
mechanisms of Terminalia albida treatment, which may
resolve neuro-inflammation, were also investigated both
in vitro and in the murine model of ECM. The phytochemical content of the crude extract was then explored
by a dereplication approach in order to understand the
origin of the bioactivity.
Methods
Plant collection
The stem bark of Terminalia albida was collected in
Danaya, Préfecture of Dubréka, Guinea. The plant was
collected at maturity in a forest area. Authorization for
collection of plant materials was obtained prior to collection at the Institut de Recherche et de Développement des Plantes Médicinales et Alimentaires de Guinée
(IRDPMAG), Dubréka, Guinea. The identification and
authentication was previously carried out by the Department of Botany of IRDPMAG where a voucher specimen
(38HK457) was deposited [14].
Preparation of Terminalia albida extract
The stem barks of the plant were shade dried for 2 weeks
and ground into powder. The powdered material (600 g)
was macerated with 2 L of pure methanol (> 99%) for
72 h. The macerate was then filtered and evaporated
under reduced pressure (Buchi® rotary evaporator, model
R-200). The dried extracts were stored at − 20 °C until
use. Before in vitro and in vivo experiments, stock solutions were dissolved in distilled water to provide working
concentrations.
Camara et al. Malar J
(2019) 18:431
In vitro antiplasmodial activity
The in vitro antiplasmodial activity of Terminalia albida
was investigated using the SYBR Green I-based fluorescence assay with P. falciparum chloroquine-resistant sensitive strain K1, as described before [15]. Chloroquine
was used as the positive control. Briefly, K1 cultures were
maintained at 2% haematocrit in RPMI 1640 containing
10% human serum, 3 g/L of glucose, 45 µg/L of hypoxanthine, and 50 µg/L of gentamicin, and incubated at
37 °C under a gas mixture of 5% O2, 5% CO2, and 90%
N2. A suspension of sorbitol-synchronized, infected red
blood cells was adjusted to 0.5% parasitaemia and 4%
haematocrit in complete medium and added to the wells
in a 96-well plate. Several wells containing non-parasitized erythrocytes at 4% haematocrit served as reference controls. Stock solutions (at 10 mM in ethanol for
plant extracts and in water for chloroquine) were diluted
serially in complete medium to test final concentrations
ranging from 0 to 10−5 M in triplicates in the 96-well
plate. Test plates were incubated at 37 °C for 48 h and 100
µL of SYBR Green I in lysis buffer was added to the wells.
After 1 h of incubation in the dark at room temperature,
fluorescence data were acquired on a Cytofluor II fluorescence multiwell plate reader (PerSeptive Biosystems,
Framingham, USA) with excitation and emission wavelength bands at 485 and 530 nm, respectively, and a gain
setting equal to 50. After subtraction of background values, the counts were plotted against the logarithm of the
drug concentration and curve fitting by nonlinear regression (sigmoidal dose–response/variable slope equation)
to yield the 50% inhibitory concentration (IC50) that
served as a measure of the anti-malarial activity.
Animal studies
Female healthy C57BL/6 mice aged 12 weeks and
weighing 20–22 g were obtained from Janvier Laboratories (Toulouse, France). For all in vivo experiments described below, the mice were kept in standard
and constant laboratory conditions (23–25 °C, relative humidity around 60%, and light/dark cycles, i.e.,
12/12 h) with unlimited access to food and tap water.
Animal welfare requirements were strictly followed during the experiments as required by the Midi-Pyrénées
ethics committee for animal experiments in Toulouse,
France. The study was authorized with permit number
APAFIS#5921-2016070118008477v3.
Acute oral toxicity assessment
Acute oral tests were performed as described before
with slight modifications [13]. Mice were randomly
divided in 2 groups of 3 mice and treated by oral route
with a single dose of Terminalia albida (2000 mg/kg) or
Page 3 of 15
water (20 mL/kg) to follow the limit test proposed by
the Organization for Economic Cooperation and Development (OECD) [16]. Animals were observed for the
first 4 h after treatment to record immediate deaths,
and once daily for 20 days to record deaths and behavioural changes. To evaluate the effect of Terminalia
albida treatment on body weight, the weight was taken
at Days 7, 14 and 20 and compared to Day 0 for both
groups (Terminalia albida and water).
In vivo antiplasmodial and anti‑malarial effects
of Terminalia albida in ECM
The rodent malaria parasite, P. berghei (strain ANKA)
(kindly given by A. Berry, CPTP research unit, Toulouse) was used throughout the study. Mice were
infected by intraperitoneal injection with 200 µL of
infected blood containing 1 × 106 parasitized erythrocytes on Day 0 (D0). The infected mice were randomly divided into 3 groups of 6 mice and treated by
oral gavage for 4 consecutive days with 100 mg/kg of
Terminalia albida crude extract. The positive control
group received chloroquine (5 mg/kg) and the negative control group received distilled water (25 mL/
kg). The extracts were administered orally once a day.
Weight, parasitaemia, survival and neurological symptoms were followed daily. To evaluate the ability of
treatments to prevent weight loss due to infection, the
percentages of weight loss as compared to D0 were
calculated. Parasitaemia was followed daily through
thin blood smears from tail blood from the D3. Blood
smears were fixed and stained with a fast-acting variation of May-Grünwald Giemsa staining (RAL 555 kit,
RAL Diagnostics). Parasitaemia was determined by
light microscopy using a 100 × objective lens as follows: % parasitaemia = 100 × (number of parasitized
RBC/total number of RBC counted). Average percentage of antiparasite activity was calculated at D4, D6,
D7 and D8 as [(A − B)/A] × 100 where A is the average
percentage parasitaemia in the negative control group
(H2O) and B is the average percentage parasitaemia in
the test group. To monitor the onset of neurological
symptoms, 10 parameters were assessed daily between
D3 and D7 to determine a rapid murine cerebral behaviour scale (RMCBS) as described by Caroll et al. [17].
The parameters measured were: gait, balance, exploratory behaviour, grooming, body position, limb strength,
tactile escape reflex, ear pavilion reflex, toe reflex and
aggressiveness. Each parameter was scored 0 to 2, with
a 2 score correlating with the highest function. Survival
was monitored twice daily. The percentage of survival
was determined over a period of 20 days post-infection
and compared between groups.
Camara et al. Malar J
(2019) 18:431
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After 7 days of infection, mice brains were removed and
crushed in PBS−/− (3 mice per group), centrifuged, filtered (100 μM) and mononuclear cells were separated
over a Lymphoprep gradient (Alere Technologies AS,
Oslo, Norway), and total leucocytes per brain counted.
Isolated brain leucocytes were labelled with antibodies coupled to fluorochromes (all from Miltenyi Biotec,
France) to distinguish the different T cell populations:
CD3-PE (lymphocytes), CD4-VioBright FITC (CD4 T
Lymphocytes), CD8-VioGreen (CD8 T Lymphocytes)
and Live dead-Violet (dead cells). Cells were collected on
a LSR Fortessa cytometer (BD Biosciences) using FACSDiva™ software.
RNA Minipreps Super Kit, Bio Basic). The brains were
crushed in a FastPrep®-24 (MP) and centrifuged for
10 min. mRNA was prepared with the EZ-10 Spin Column Total RNA Minipreps Super Kit (Bio Basic) using
the manufacturer’s protocol. Synthesis of cDNA was
performed according to the manufacturer’s recommendations (Verso kit, Thermo Scientific). RT–qPCR was
performed on a LightCycler 480 system using LightCycler SYBR Green I Master Mix (Roche Diagnostics).
The primers (Eurogentec) were designed with the software Primer 3. GAPDH mRNA was used as the invariant control. Serially diluted samples of pooled cDNA
were used as external standards in each run for the
quantification. The primers used are listed in Table 1.
Gene expression of pro‑ and anti‑inflammatory markers
in mice brains
Levels of cytokines in mice brains
Brain cell analysis by flow cytometry
To measure the effect of Terminalia albida treatment
on the inflammatory response in P. berghei-infected
mice, the mice were divided into 3 groups of 6 mice,
infected and treated with Terminalia albida crude
extract, chloroquine or distilled water, as before. Mice
were sacrificed 7 or 8 days after infection (D7 or D8).
Mice brains were dissected and divided longitudinally into two equal parts to be used for RT-qPCR and
ELISA experiments. Half of the brain was placed in lysing matrix tubes (MP Biomedicals) containing 650 μL
of RLT lysis buffer (from the EZ-10 Spin Column Total
The remaining half of the brain was crushed in PBS
between two glass slides and filtered through a 100 µM
sterile filter. After centrifugation (1 min at 10,000g and
room temperature), the remaining pellet was homogenized in 5 mL IGEPAL, a protease inhibitor solution,
and stored at − 80 °C until use. The levels of IL-6, TNF,
IL-1β, and IL-10 were determined by enzyme-linked
immunosorbent assay (ELISA) using commercially
available OptiEIA murine kits (BD Biosciences) according to the manufacturers’ instructions.
Table 1 Sequences of murine primers used in quantitative RT-PCR experiments
Gene
Sense
Antisense
Product
size (bp)
GAPDH
5′-ACA-CAT-TGG-GGG-TAG-GAA-CA
5′-AAC-TTT-GGC-ATT-GTG-GAA-GG
222
151
IL-1β
5′-GAT-CCA-CAC-TCT-CCA-GCT-GCA
5′-CAA-CCA-ACA-AGT-GAT-ATT-CTC-GAT-G
TNF
5′-CTC-CCT-TTG-CAG-AAC-TCA-GG
5′-AGC-CCC-CAG-TCT-GTA-TCC-TT
211
HO-1
5′-CCA-GAG-TGT-TCA-TTC-GAG-CA
5′-CAC-GCA-TAT-ACC-CGC-TAC-CT
174
IL-12
5′-TGG-TTT-GAT–GAT-GTC-CCT-GA
5′-AGG-TCA-CAC-TGG-ACC-AAA-GG
172
IFNg
5′-TGA-GCT-CAT-TGA-ATG-CTT-GG
5′-ACT-GGC-AAA-AGG-ATG-GTG-AC
236
169
CD11b
5′-AGA-TCG-TCT-TGG-GAG-ATG-CT
5′-GAC-TCA-GTC-AGC-CCC-ATC-AT
TLR2
5′-TGT-AAC-GCA-ACA-GCT-TCA-GG
5′-TGC-TTT-CCT-GCT-GGA-GAT-TT
196
ICAM-1
5′-AGC-TTG-CAC-GAC-CCT-TCT-AA
5′-AGC-ACC-TCC-CCA-CCT-ACT-TT
159
GranzB
5′-GCT-TCA-CAT-TGA-CAT-TGC-GC
5′-AGA-ACA-GGA-GAA-GAC-CCA-GC
172
NFkB
5′-ACC-GAA-GCA-GGA-GCT-ATC-AA
5′-GCG-TAC-ACA-TTC-TGG-GGA-GT
178
PGEs
5′-CAG-CCT-ATT-GTT-CAG-CGA-CA
5′-CCT-AGG-CTT-CAG-CCT-CAC-AC
157
TGFb
5′-GAC-TCT-CCA-CCT-GCA-AGA-CC
5′-ACG-CGG-GTG-ACC-TCT-TTA G
246
206
CD36
5′-GAG-CAA-CTG-GTG-GAT-GGT-TT
5′-GCA-GAA-TCA-AGG-GAG-AGC-AC
iNOS
5′-ACA-AGG-CCT-CCA-ATC-TCT-GC
5′-TCC-TGG-ACA-TTA-CGA-CCC-CT
95
VEGF
5′-GCT-GTA-ACG-ATG-AAG-CCC-TG
5′-CGC-TCC-AGG-ATT-TAA-ACC-GG
236
P. berghei
5′-TCA-TTG-GGC-TCT-CAA-AGG-GT
5′-CAA-TTG-GAG-GGC-AAG-TCT-GG
209
Camara et al. Malar J
(2019) 18:431
Gene expression of pro‑ and anti‑inflammatory markers
by murine macrophages after LPS/IFNγ activation
and Terminalia albida treatment
Peritoneal macrophages were harvested from 6 healthy
mice. The peritoneal cavity of each mouse was washed
with 5 mL of sterile NaCl 0.9% before harvesting resident peritoneal cells. Collected cells were centrifuged at
1500 rpm for 10 min and the cell pellet was suspended
in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with glutamine (Invitrogen), penicillin, streptomycin (Invitrogen) and 5% heat-inactivated fetal calf
serum. Cells (5.105/well) were left to adhere for 2 h at
37 °C and 5% CO2, and non-adherent cells were removed
by washing with PBS. After washing, adherent macrophages were immediately stimulated with 10 ng/mL
lipopolysaccharide (LPS) and 40 UI/mL IFNγ (Clinisciences) during 2 h. After stimulation, cells were washed
and treated with 10 µg/mL Terminalia albida crude
extract diluted in medium or with medium alone for 4 h
at 37 °C and 5% CO2. The dose of 10 µg/mL was previously determined by an LDH test showing that this concentration is not toxic to mouse peritoneal macrophages
(Additional file 1: Fig. S1). Each condition was tested in
triplicate. mRNA extraction, cDNA synthesis and RT–
qPCR were performed as described above. The primers
used are listed in Table 1.
Levels of cytokines produced by murine macrophages
after LPS/IFNγ activation and Terminalia albida treatment
After LPS/IFNγ stimulation (10 ng/mL LPS and 40 UI/
mL IFNγ) during 2 h, murine peritoneal macrophages
were treated with Terminalia albida (10 µg/mL) or
medium alone for 24 h at 37 °C and 5% CO2. Each condition was tested in triplicate. The supernatants were then
assayed to quantify cytokines (TNF, IL-1β, IL-6, IL-10)
by ELISA using commercially available OptiEIA murine
kits (BD Biosciences) according to the manufacturers’
instructions.
In vitro ROS production model and Terminalia albida
scavenging activity
The Light-Up Cell System (LUCS) was used to evaluate
the ability of Terminalia albida to inhibit the production of ROS by HepG2 cells, as previously described [18].
Briefly, Terminalia albida methanolic extract was solubilized in DMSO and 9 serial dilutions were prepared from
1.92 mg/mL to 0.0075 mg/mL. Since DMSO 4% was used
to dilute the highest concentration of extract, DMSO
4% was used as negative control. No toxic effect was
observed on HEpG2 cells at this concentration. HepG2
cells (75,000/well) were cultured for 24 h in DMEM complemented with 10% FBS and 1% penicillin–streptomycin solution at 37 °C and 5% CO2. The experiments were
Page 5 of 15
carried out in DMEM without SVF. At least two independent experiments were performed on 96-well plates,
each in triplicate. Cells were then incubated for 4 h in
the presence or not of increased concentrations of Terminalia albida extract. Extract dilutions were added by
replacing the media. Cells were then treated with the fluorescent marker thiazole orange at 4 µM for 1 h and fluorescence was measured at 535 nm (emission) during 20
recurrent irradiations at 480 nm (excitation). Antioxidant
index (AI) was calculated from the kinetics using the
formula AI = 100 – 100 (0∫12 RFUn sample/0∫12 RFUn
control). Dose/response curves were obtained by combining AI as a function of the logarithm (10) of the sample concentration and processed by fit sigmoid according
to
the
equation
AI = AImin + (AImax − AImin)/
(1 + 10(Log(EC50−CE)*HS)) to determine EC50value.
UHPLC‑HRMS profiling
Acquisition of metabolite profiles of Terminalia albida
methanol extract (1 mg/mL), data processing and statistical analysis were performed as previously described [19].
Statistical analysis
Results were expressed as mean ± SEM (standard error
of mean) and analysed using Graph Pad Prism software
version 6. Comparisons were performed by a one-way
analysis of variance (ANOVA) followed by the means
multiple comparison method of Bonferroni–Dunnett.
Mann–Whitney U-test was used for two-by-two comparisons. Differences were considered significant if P < 0.05.
Results
Acute oral toxicity assessment and determination
of the test dose
Terminalia albida administration to C57BL/6 mice at
a dose of 2000 mg/kg did not cause mortality or major
behavioural changes among experimental animals during
the 20 days of follow-up. Mice body weight was not modified by the administration of Terminalia albida (Additional file 2: Fig. S2). Thus, LD50 of Terminalia albida is
greater than 2000 mg/kg in C57BL/6 mice. Terminalia
albida crude extract, as prepared in this work, can therefore be classified as category 5 and considered non-toxic
orally, according to the OECD’s Globally Harmonized
System of Classification [16]. Based on these results, antimalarial activity in ECM was assessed at 100 mg/kg.
In vitro and in vivo antiplasmodial activity
Terminalia albida crude extract presented an IC50 of
1.5 μg/ml and IC50 for chloroquine was 0.08 µM on
the chloroquine-resistant K1 strain of P. falciparum.
In vivo, mice that received water were parasitized
from D4, while mice treated with Terminalia albida
Camara et al. Malar J
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were positive from D7. Mice that received water were
all dead at D8. At D4, parasite chemosuppression by
Terminalia albida was 100% and this complete inhibition lasted until D6. At D7, Terminalia albida treatment resulted in parasite suppression of 89% compared
to water (Fig. 1a, b, P < 0.0001). However, Terminalia
albida treatment did not limit parasite multiplication
since parasitaemia increased until 7% at D20. Conversely, chloroquine treatment abolished parasite multiplication (Fig. 1a, b).
Effect of Terminalia albida treatment on survival, weight
and neurologic symptoms in ECM
Terminalia albida treatment greatly improved survival
as chloroquine and Terminalia albida treatments were
statistically similar until D20 although Fig. 2a suggests
that Terminalia albida treatment was less effective than
chloroquine from D12 onwards. At D20, chloroquine
was statistically more effective than Terminalia albida
treatment (Fig. 2a, P = 0.045). Terminalia albida treatment prevented death up to D12 and maintained a 50%
survival rate at D20 (Fig. 2a. P = 0.003 at D8, P = 0.0005
from D9 to D13, and P < 0.05 until D20 for the comparison between Terminalia albida and water). Terminalia
albida treatment improved the overall condition of the
mice since the treated group lost less weight than the
water group (the groups were compared up to D7 only
because the group that received water had only one living mouse left on D8. P = 0.037 at D7. Figure 2b). The
comparison of RMCBS between groups showed a rapid
deterioration of cerebral functions in untreated mice
from D5 (Fig. 2c). Although mice treated with Terminalia albida had slightly impaired brain functions at
D6 and D7, there was no statistical difference between
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the scores of the CQ and Terminalia-treated mice at D6
and D7 (Fig. 2c).
Effect of Terminalia albida on T cell infiltration in the brain
of Plasmodium berghei‑infected mice
Leucocyte recruitment and activation in the brain is one
of the key mechanisms of ECM, particularly CD8 effector T cells [20]. To assess such mechanism of P. bergheiinfected mice according to the treatment received
(chloroquine or Terminalia albida or H2O), the percentages of the different T cell populations were compared
in brains after 7 days of infection by flow cytometry.
Interestingly, the percentages of CD3 T and CD3 CD8 T
cells in brain was lower in mice treated with chloroquine
or Terminalia albida extract, compared to mice that
received water (Fig. 3a, b). Conversely, the percentage
of CD4 T cells was higher in chloroquine and Terminalia albida-treated mice, compared to water-treated mice
(Fig. 3c, P = 0.04 for both comparisons).
Absence of pro‑inflammatory response in brains
of Terminalia albida‑treated mice during ECM
To assess the immunological state of brains from Terminalia albida-treated mice during ECM, mRNA levels
of pro- and anti-inflammatory markers were measured
in the 3 groups of mice (Terminalia albida, chloroquine
and H2O). Plasmodium berghei mRNA was undetectable at D7 in brains of chloroquine and Terminalia
albida-treated mice (Fig. 4a). The expression of VEGF, a
marker of vascular permeability and endothelial activation, was lowered in the chloroquine group compared to
the water group (Fig. 4a). Conversely, the expression of
ICAM-1, another marker of endothelial activation was
diminished in brains of mice treated with Terminalia
albida (Fig. 4a, P = 0.002). Among the pro-inflammatory
Fig. 1 Antiplasmodial activity of Terminalia albida treatment in ECM model. C57BL/6 mice were infected with P. berghei ANKA and treated 2 h
later with chloroquine (5 mg/kg), Terminalia albida crude extract (100 mg/kg) or water (25 mL/kg) from Day 0 to Day 3. a Mean parasite densities
during infection. b Percentages of parasite suppression according to the treatment, calculated by comparison to H2O treated mice. **P < 0.005,
***P < 0.0005 compared Terminalia albida to H2O group
Camara et al. Malar J
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Fig. 2 Effect of Terminalia albida treatment on survival, weight and cerebral symptoms in ECM model. C57BL/6 mice were infected with P. berghei
ANKA and treated 2 h later with chloroquine (5 mg/kg), Terminalia albida crude extract (100 mg/kg) or water (25 mL/kg) from Day 0 to Day 4. a
Percentage of survival. b Percentage of weight loss at each day compared to Day 0. c Rapid murine cerebral behavior scale (RMCBS) between D3
and D7. *P < 0.05 compared Terminalia albida to H2O groups at D7. *P < 0.05 and **P < 0.005 compared Terminalia albida to H2O groups. §P < 0.05
compared Terminalia albida to chloroquine groups
Fig. 3 Effect of Terminalia albida on cell infiltration in the brain of Plasmodium berghei-infected mice. C57BL/6 mice were infected with P. berghei
and treated 2 h later with chloroquine (5 mg/kg), Terminalia albida crude extract (100 mg/kg) or water (25 mL/kg) from Day 0 to Day 4. Brains were
analysed by flow cytometry at D7 post-infection. Percentages of cell populations were compared between groups for CD3+ (a), CD8+ (b) and CD4+
T lymphocytes (c). *P < 0.05, **P < 0.005, ***P < 0.0005
cytokines tested, IFNγ expression was completely abolished in mice treated with Terminalia albida similarly
to the chloroquine group, in comparison to untreated
mice (Fig. 4b. P = 0.006 and P = 0.005 for Terminalia
albida versus H2O and chloroquine versus H2O, respectively). Interestingly, Terminalia albida treatment also
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(See figure on next page.)
Fig. 4 Effect of Terminalia albida treatment on the expression of pro- and anti-inflammatory markers in brains of Plasmodium berghei-infected mice.
C57BL/6 mice were infected with P. berghei and treated with chloroquine (5 mg/kg), Terminalia albida (100 mg/kg) or water. Brains were harvested
at D7 (for P. berghei and VEGF) or D8 post-infection (for the other markers). Gene expression was measured by RT-qPCR and cytokine levels were
measured by ELISA. Gene expression levels of (a) P. berghei, VEGF, ICAM and Granzyme B; b pro-inflammatory cytokines, c other pro-inflammatory
markers, and d anti-inflammatory markers. e Cytokine levels in pg/mL. *P < 0.05, **P < 0.005, ***P < 0.0005. Data presented are mean ± standard
deviation
significantly limited the expression of granzyme B similarly to chloroquine (Fig. 4a. P = 0.003 and 0.002 for H2O
versus Terminalia albida and versus chloroquine). For
TNF, IL-1β and IL-12 expression, the effect of Terminalia
albida treatment compared to chloroquine or H2O was
not significant (Fig. 4b). Among the other pro-inflammatory markers tested, Terminalia albida treatment also
diminished CD11b expression similarly to chloroquine
(Fig. 4b, P = 0.006). Finally, for TLR2, NFκB, PGES and
anti-inflammatory markers (TGFβ, CD36 and haem oxygenase 1 named HO-1), Terminalia albida treatment had
no significant effect (Fig. 4c, d). Cytokine levels were also
measured in brains on D8. However, cytokine levels were
similar regardless of the treatment received by the mice
(Fig. 4e).
Anti‑inflammatory properties of Terminalia albida
in a non‑malarial context
To further confirm the anti-inflammatory properties of
Terminalia albida assessed in the ECM model, additional
experiments were performed by stimulating the peritoneal macrophages of C57BL/6 healthy mice with LPS
and IFN and treating them with the methanolic extract of
Terminalia albida. The expression of several pro-inflammatory markers was decreased following treatment of
the cells with Terminalia albida compared to untreated
cells: TNF (P = 0.04), IL12 (P = 0.003), NFkB (P = 0.005)
and inducible nitric oxide synthase (iNOS) (P = 0.001)
(Fig. 5a, c). Notably, CD36 and HO-1, two anti-inflammatory markers, were more expressed by Terminalia
albida-treated cells (Fig. 5d, P = 0.001 and P = 0.01,
respectively). In addition, a significant decrease was also
obtained in the enzymatic quantification of TNF, IL-6
and IL-1β for Terminalia albida treated-cells (Fig. 5e,
P = 0.001, P = 0.02 and P = 0.03, respectively).
Anti‑oxidative properties of Terminalia albida
The evaluation of Terminalia albida’s ability to neutralize intracellular radical species of the human HepG2
cells showed a remarkable dose-dependent antioxidant
activity (Fig. 6a). The antioxidant index (AI) calculated
from the area under curves was 997 at concentrations
greater than or equal to 60 μg/mL (Fig. 6b). The EC50
of the extract was 10.2 μg/mL. Finally, the absence of a
fluorescent signal higher than the control at time 0 indicates an absence of cytotoxicity at 4 h of treatment.
Phytochemical characterization of the Terminalia albida
crude extract
After maceration with methanol for 72 h, 253.5 g of crude
extracts were obtained. The extracts were qualitatively
analysed by UHPLC-HRMS. Metabolite profiling of Terminalia albida was acquired in positive and negative
ionization mode. Qualitative analysis by UHPLC-HRMS
of Terminalia albida stem bark enabled the putative
identification of 16 known compounds through HRMS
and MS/MS fragmentation patterns using MzMine, MSDIAL and MS-FINDER software (Table 2). Among the
top 12 annotated compounds, 6 are tannins (flavogallonic acid, flavogallonic acid-Me ester, ellagic acid, ellagic
acid 3,8-di-Me ether, corilagin, ellagic acid; 2,3,7-tri-me
ether), 4 are triterpenoids (23-O-galloylarjunolic acid
28-O-β-d-glucopyranosyl ester, 23-galloylarjunolic acid,
23-galloylterminolic acid, 7-beta-hydroxy-23-deoxojessic
acid), 1 is phenol glucoside gallate (vanillic acid 4-(6-galloylglucoside)) and 1 is cycloartanols and derivatives
(ouadrangularic acid F).
Discussion
In this study, the anti-malarial and antiparasite activity of Terminalia albida found respectively in ECM and
in vitro was high. In vivo, mice survived until D12, the
limit for neurological symptoms in this model, and there
was a 50% rate after 20 days of infection, while untreated
mice were all dead by D8. Such a result obtained with a
crude plant extract is quite remarkable when compared
to many other similar studies (performed with crude
plant extracts at doses ≤ 300 mg/Kg) in which mean mice
survival often did not exceed 9 days [21–23]. Antiparasite
activity was also very high in vivo, 100% at D4 and until
D6, 89% at D7, and 61% at D8. Interestingly, mice treated
with Terminalia albida have developed a parasitaemia
that remained below 8% until D20, while the average
parasitaemia of untreated mice reached 10%. This result
may be explained by the pharmacokinetics of the active
principle(s) responsible for the antiparasite activity. Further bio-guided fractionation is needed to decipher the
reasons of such activity.
Camara et al. Malar J
(2019) 18:431
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Camara et al. Malar J
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Fig. 5 Assessment of in vitro anti-inflammatory properties of Terminalia albida. Murine macrophages were activated by LPS and IFNγ in the
presence or not of 10 µg/mL of Terminalia albida. Gene expression levels of a pro-inflammatory cytokines, b other pro-inflammatory markers,
c pro-inflammatory signalling pathways, and d anti-inflammatory markers. e Cytokine levels measured in supernatants, in pg/mL. *P < 0.05,
**P < 0.005, ***P < 0.0005. Data are presented as mean ± SD
Camara et al. Malar J
(2019) 18:431
Fig. 6 LUCS anti-oxidant assay. a Kinetics of fluorescence emission
recorded under illumination for different concentrations of
extract (7.5 10−3 mg/mL–1.92 mg/mL) using the LUCS assay. b
Corresponding antioxidant index calculated from RFU values as
follows: AI = 1000–1000 (0∫12 RFUn sample/0∫12 RFUn control)
In addition, antiparasite activity found in vitro was
also interesting with an IC50 of 1.5 µg/ml. This value
is slightly higher than that of 0.6 µg/mL, previously
found by Traore et al. in 2014 [12]. This difference may
be due to the disparity of harvesting locations, harvest
time, and extraction methods that influence plant biological active compounds [24]. In addition, Traore et al.
used the lactate dehydrogenase assay to measure antiplasmodial activity whereas a Sybr green assay was used
in the present study. These different methods may also
explain the small disparities between the two studies.
In this research, the aim was to elucidate whether,
in addition to its antiparasitic properties, Terminalia
albida presents anti-inflammatory and anti-oxidant
activities useful for the resolution of cerebral malaria.
Page 11 of 15
During malaria, the first immune responses (oxidative
and inflammatory) induced by monocytes are essential
to control parasite multiplication. However, excessive
and inappropriate activation of the immune system is
detrimental to the host and contributes to the severe
form that can lead to death [25]. Mechanisms leading to
ECM involve the recruitment of T lymphocytes to the
brain, particularly CD8 T cells known to be responsible
for lethal neuropathology [8]. In the ECM model, treatment with Terminalia albida, similarly to chloroquine,
greatly restrained the recruitment of T cells and more
specifically of CD8 T cells in mice brains after 7 days
of infection. It seems logical since at D7, the parasitaemia of the treated mice was very low and the parasite
mRNA undetectable.
Here, brains from infected mice treated with the methanolic extract of Terminalia albida showed reduced
expression of IFNγ, CD11b, ICAM-1 and granzyme B.
The secretion of IFNγ by NK cells contributes to the CM
pathogenesis by increasing the expression of endothelial
receptors including ICAM-1 and VCAM involved in the
sequestration of parasitized red blood cells in the murine
model [26]. This phenomenon induces the activation of
endothelial cells by excessive production of pro-inflammatory cytokines such as TNF, IL-6, IL-1β, IL-12 and the
recruitment of inflammatory leukocytes in the cerebral
micro-vessels that damage the blood–brain barrier [25].
CD11b constitutes a marker of brain endothelium disruption, as described before [27]. In addition, CD8 + T
cells have been identified as a major mediator in death
during ECM by their capacity to produce granzyme B
able to kill P. berghei antigen-presenting endothelial cells
and to damage neuronal cells [8]. Thus, during ECM, Terminalia albida treatment not only limits parasitaemia
but also reduces the expression of mediators involved in
pathogenesis. Such anti-inflammatory properties were
confirmed in vitro in a non-malarial context. After LPS/
IFNγ stimulation in murine peritoneal macrophages,
Terminalia albida treatment led to a significant inhibition of the expression of TNF, IL-1β, IL-6 and IL12, four
pro-inflammatory cytokines. NFkB, the transcription
factor regulating the inflammatory response has been
suggested as therapeutic target to treat severe malaria
[28]. In the present study, NFkB was also less expressed
by cells treated with the plant compared to untreated
cells. Similarly, CD36, an important marker for malaria,
was higher in treated cells compared to untreated ones
(Fig. 4). Through PPARγ activation, the scavenger receptor CD36 is known for its implication in P. berghei elimination through non-opsonic phagocytosis [29].
Terminalia albida also presented highly interesting antioxidant properties in vitro. LPS/IFNγ-activated
cells treated with Terminalia albida showed a lower
Camara et al. Malar J
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Page 12 of 15
Table 2 Putative identified features (m/z × RT pairs) using HRMS and MS/MS fragmentation patterns using MzMine,
MS-finder and DNP database
ID
RT (min)
m/z
Formula finder
Δ Da
Putative ID
Ontology
Scoring
1
3.59
481.0977 [M–H]−
C21H22O13
0.0010643
Vanillic acid 4-(6-galloylglucoside)
Phenolic glucoside gallate
7.7481
2
4.11
447.0561 [M–H]−
C20H16O12
0.0007995
Eschweilenol C
Hydrolyzable tannins
7.5965
3
5.57
329.0296 [M–H]−
C16H10O8
0.0006908
Ellagic acid-3,8-Di-Me ether
Hydrolyzable tannins
7.5676
4
3.08
633.0719 [M–H]−
C27H22O18
0.0014374
Corilagin
Hydrolyzable tannins
7.5558
5
4.27
300.9984 [M–H]−
C14H6O8
0.0078670
Ellagic acid
Hydrolyzable tannins
7.4150
6
5.90
801.405 [M–H]−
C43H62O14
0.0016802
23-O-Galloylarjunolic acid 28-O-β-Dglucopyranosyl ester
Triterpenoids
7.3505
7
3.97
483.0196 [M–H]−
C22H12O13
0.000914
Flavogallonic acid-Me ester
Hydrolyzable tannins
7.3463
8
2.69
469.0038 [M–H]−
C21H10O13
0.0010639
Flavogallonic acid
Hydrolyzable tannins
7.1290
9
7.26
639.3527 [M–H]−
C37H52O9
0.0011568
23-Galloylarjunolic acid
Triterpenoids
7.0819
10
6.92
655.3481 [M–H]−
C37H52O10
0.0006714
23-Galloylterminolic acid
Triterpenoids
6.9868
501.3589 [M–H]−
C31H50O5
− 0.0003518
7-beta-Hydroxy-23-deoxojessic acid
Triterpenoids
6.7122
547.3273 [M–H]−
C31H48O8
0.000342
Quadrangularic acid F
Cycloartanols and derivatives
6.7025
11
12
10.7
7.71
expression of iNOS and higher HO-1. In addition, Terminalia albida neutralized intracellular radical species
of the human HepG2 cells in a dose-dependent antioxidant activity. It is largely accepted that oxidative stress
is involved in the pathogenesis of severe malaria [30].
Nitric oxide is produced via the enzyme NOS and the
substrate l-arginine. However, its beneficial or unfavourable effect is controversial in the experimental model [31,
32]. In the present study, Terminalia albida limits iNOS
expression in vitro and increases survival in ECM. HO-1
is a key protective gene in the host infected by Plasmodium, able to catabolize toxic free haem into iron, biliverdin and carbon monoxide. HO-1 induction has been
shown to prevent blood–brain barrier disruption, brain
microvasculature congestion and neuro-inflammation
in the murine model [33]. Thus, Terminalia albida presents antiplasmodial, anti-inflammatory and anti-oxidant
properties. However, these activities are not sufficient to
limit parasitic multiplication as chloroquine does. These
results suggest that the anti-inflammatory and anti-oxidant activities of the extract limit neuro-inflammation
secondary to infection, but that the antiplasmodial activity is not sufficient to cancel the multiplication of the parasite in this model.
Terminalia species are rich sources of secondary
metabolites including cyclic triterpenes and their derivatives, and polyphenols (flavonoids, phenolic acids and
tannins). Several Terminalia extracts or fractions have
been tested for their anti-Plasmodium activities [34–42]
but most of these studies lack chemical characterization of the studied extracts, except Muganga et al. [36]
who isolated the active compounds, through a bioguided fractionation [36]. Among all the compounds
isolated, ellagic acid, already know to have a strong
anti-malarial activity (IC50 between 90 and 175 ng/mL)
[43], was found to be the main antiplasmodial compound
(IC50 = 0.175 μg/mL) in Terminalia mollins, while ellagic
acid derivatives were inactive, suggesting the crucial role
of the free hydroxyl groups in antiplasmodial activity. In
addition, gallic acid and some condensed tannins, such
as catechin, gallocatechin and epigallocatechin, have
been isolated and found to have a lower antiplasmodial
activity (IC50 > 25 μg/mL) than the one found for ellagic
acid, and a mixture of punicalagin A and B (anomeric
isomers) did not present any interesting antiplasmodial
activity. Akanbi et al. [44] evaluated the anti-malarial
activity of total saponins extracted from Terminalia avicennioides and concluded that the Terminalia’s saponins
may be able to increase the level of immunity against
malaria infection, responsible for the antiplasmodial
activity displayed by the extract [44]. However, saponin
isolation was based on a phytochemical extraction followed by TLC analysis and a non-specific colorimetric
test. No complementary analyses such as IR, UV, LC–
MS/MS dereplication or NMR, were carried out to confirm the composition of this extract, which makes these
results questionable. In addition, saponins are characterized by a strong haemolytic activity and may be toxic
[45]. Finally, 23-galloylarjunolic acid, a triterpene of the
galloyl group type, has shown interesting antiplasmodial activity against the chloroquine-sensitive (D6) and
chloroquine-resistant (W2) strains with respective IC50s
of 4.5 µg/mL and 2.8 µg/mL and selectivity index greater
than 1 [46]. Thus, in Terminalia albida extract, ellagic
acid and 23-galloylarjunolic acid may be responsible, at
least in part, for the antiplasmodial activity.
Phenolic compounds are widely described in the literature for their potential biological activity such as
Camara et al. Malar J
(2019) 18:431
anti-inflammatory and immunomodulatory effects [47,
48]. They are excellent anti-oxidants due to the presence
of a hydroxyl group capable of capturing oxygen free radicals [49]. In addition to anti-Plasmodium activity, ellagic
acid has known anti-oxidant and anti-inflammatory activity that could explain the higher survival rate obtained
after Terminalia albida treatment in ECM [47, 50]. In a
model of carrageenan-induced inflammation, ellagic acid
prevented the production of pro-inflammatory mediators
and promoted the production of anti-inflammatory IL-10
and antioxidant glutathione [51]. Eschweilenol C has also
been reported for anti-inflammatory activity in the aqueous extract of Terminalia fagifolia by inhibition of NFkB
pathway in LPS-activated microglial cells [48]. Corilagin
has also been studied for its anti-inflammatory activity based on the inhibition of the NFkB signaling pathway and even tested as a treatment in a model of sepsis
[52–54]. Anti-inflammatory and anti-oxidant effects of a
derivative of vanillic acid, vanillic acid 4-(6-galloylglucoside), were also reported both in vitro and in vivo in the
carrageenan-based inflammation murine model [55, 56].
Finally, cyclic triterpenes are also a phytochemical group
with various biological activities, particularly in inflammatory and oxidative diseases [57]. Nevertheless, further
bioassay-guided fractionation will be necessary to confirm the origin of the antiplasmodial, anti-inflammatory
and anti-oxidant activities of Terminalia albida, including synergistic potential between tannins, lignans and
terpenoids found in this plant.
Conclusion
Terminalia albida displays highly interesting antimalarial, anti-inflammatory and anti-oxidant properties.
UHPLC-HRMS analysis showed that 12 compounds are
found in Terminalia albida and may be implicated in its
biological activities. However, further investigations into
the long-term toxicity, prophylactic effects and antiparasite mechanisms are necessary before recommending the
use of Terminalia albida or its constituents for malaria
treatment or prevention.
Supplementary information
Supplementary information accompanies this paper at https://doi.
org/10.1186/s12936-019-3071-9.
Additional file 1: Fig. S1. Terminalia albida cytotoxicity against healthy
murine peritoneal macrophages by the lactate deshydrogenase (LDH)
test. Cells (2.105/well) were left to adhere for 2 h at 37 °C and 5% CO2, and
non-adherent cells were removed by washing with PBS. Adherent cells
were treated with serial dilutions of Terminalia albida extract (100 μg/mL,
50 μg/mL, 25 μg/mL, 10 μg/mL, 5 μg/mL) and incubated at 37 °C and 5%
CO2 for 24 h. LDH leakage from the cells was determined using a commercial LDH cytotoxicity detection kit according to the manufacturer’s
protocols (Cytotoxicity detection Kit, Roche, France). Absorbance was
measured at 490 nm with a Wallac Victor 2 1420 Multilabel Counter. Cell
Page 13 of 15
mortality was calculated as a percentage: (absorbance of wells treated
with extract *100/absorbance of wells treated with triton).
Additional file 2: Fig. S2. Acute oral toxicity test in vivo: effect of Terminalia albida treatment on body weight. Mice were treated by oral route
with a single dose of Terminalia albida (2000 mg/kg) or water (20 mL/
kg). To evaluate the effect of Terminalia albida treatment on body weight,
the weight was taken at D7, D14 and D20 and compared to D0 for both
groups (Terminalia albida and water).
Abbreviations
CD: cluster of differentiation; CPTP: Centre de Physiopathologie de Toulouse
Purpan; ECM: experimental cerebral malaria; HepG2: liver hepatocellular cells
G2; HO-1: haem oxygenase-1; IC50: 50% inhibitory concentration; ICAM-1:
intercellular adhesion protein 1; iEs: infected erythrocytes; IFNγ: interferon
gamma; IL: interleukin; iNOS : nitric oxide synthase; LC/MS: liquid chromatography–mass spectrometry; LD50: 50% lethal dose; LPS : lipopolysaccharide; NFkB: nuclear factor-kappa B; NK: natural killer; OECD: Organization for
Economic Cooperation and Development; PPARγ: peroxisome proliferatoractivated receptor; ROS: reactive oxygen species; TNF: tumor necrosis factor;
VCAM-1: vascular cell adhesion protein 1.
Acknowledgements
we thank Melissa Parny who kindly provided us with ELISAs reagents.
Authors’ contributions
AMB, AA and AiC designed the study and wrote the publication with the help
of AC. MST, AOB and ESB performed the field work. AiC carried out most of
the in vitro and in vivo lab experiments with the help of JR. MAB evaluated the
in vitro anti-malarial activity of the extracts and analysed the results. PB and
MaH helped for murine models. MoH managed the dereplication approach
and KR the Light-Up Cell System to measure Terminalia albida scavenging
activity. All authors read and approved the final manuscript.
Funding
A. Camara received funding for her PhD research from the French Embassy in
Guinea (Grant No. 001) and from the Guinean Ministry of Higher Education
and Scientific Research (Grant No. 001).
Availability of data and materials
The datasets used and/or analysed during the current study are available from
the corresponding author on reasonable request.
Ethics approval and consent to participate
Mice studies (oral toxicity tests and anti-malarial in vivo evaluation) were
approved by the Midi-Pyrénées ethic committee for animal experimentation in Toulouse, France. The study was authorized with the permit number
APAFIS#5921-2016070118008477 v3.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
UMR152 PHARMADEV, IRD, UPS, Université de Toulouse, Toulouse, France.
2
Institute for Research and Development of Medicinal and Food Plants
of Guinea (IRDPMAG), Dubréka, Guinea. 3 Department of Pharmacy, University
Gamal Abdel Nasser of Conakry, Conakry, Guinea. 4 Department of Pharmacy,
University of Sciences, Technics and Technologies (USTTB) of Bamako, Bamako,
Mali.
Received: 11 September 2019 Accepted: 11 December 2019
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