Journal of Ethnopharmacology 277 (2021) 114105

Contents lists available at ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Suppressive, curative, and prophylactic potentials of an antimalarial

polyherbal mixture and its individual components in Plasmodium
berghei-Infected mice
Stephenie C. Alaribe a, *, Akolade R. Oladipupo a, Goodness C. Uche a, Maryan U. Onumba a,
Duncan Ota d, Olufunsho Awodele b, Wellington A. Oyibo c
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Lagos, College of Medicine Campus, PMB 12003, Idi-araba, Lagos, Nigeria
b
Department of Pharmacology, Therapeutics & Toxicology, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Lagos, Nigeria
c
Department of Medical Microbiology & Parasitology, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Lagos, Nigeria
d
Department of Physiology, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Lagos, Nigeria

A R T I C L E I N F O A B S T R A C T

Keywords: Ethnopharmacological relevance: Malaria remains one of the most prevalent infectious diseases in tropical regions
Antiplasmodial activity of the world, particularly in sub-Saharan Africa, where it remains epidemiologically holoendemic. The absence of
Malaria chemosuppression effective vaccines and Plasmodium resistance to antimalarial drugs have been the major challenges to malaria
Malaria parasite clearance
control measures. An alternative strategy could be the application of validated and standardized herbal
Polyherbal decoction
Plasmodium berghei
formulations.
Aim of the study: To evaluate the antimalarial activity of a polyherbal mixture (APM) and compare it to those of
its individual constituent plants.
Methods: APM consisted of stem barks of Mangifera indica (MI), Azadirachta indica (AI), Nauclea latifolia (and
roots, NL) and roots of Morinda lucida (ML). Dihydroartemisinin-piperaquine (DHP) and pyronaridine-
artesunate (PA) served as positive controls. Antimalarial activity was evaluated using suppressive, curative
and prophylactic assays in mice infected with Plasmodium berghei.
Results: All the herbal mixtures, individually and in combination, showed significant (p < 0.05) antiplasmodial
activities in the various assays. They produced considerable parasite suppression (>50%), substantial clearance
(>70%), and notable prophylaxis (>60%, except for NL: 35%). APM (95.4–98.7%) and AI (92%), respectively,
elicited greater and comparable suppression relative to DHP (88%) and PA (87.3%). However, all the herbal
decoctions, individually (72–93.6%) and in combination (82.5–91%), showed lower parasite clearance than DHP
(100%) and PA (99.5%). Meanwhile, APM showed relatively greater suppression and prophylaxis than its
constituent plants, suggesting that the combination produced synergistic or additive effects.
Conclusion: These findings could substantiate the use of these plants, singly or in combination, as traditional
remedies for malaria. Further studies are recommended to evaluate their clinical usefulness.

1. Introduction concentrated in 13 countries, and over half in Nigeria, Congo, Ethiopia,

Malaria is one of the most virulent of the recognized infectious dis­
eases affecting man. This disease continues to wreak havoc to human
beings especially in tropical and subtropical regions—particularly in
sub-Saharan Africa, where it remains epidemiologically holoendemic
(Alaribe et al., 2020). World Health Organization (WHO) (2018) esti­
mated that 1.2 billion people are at high risk of malaria transmission,
half of which live in the African regions; 80% of such cases are
Tanzania and Kenya. Furthermore, recent World Malaria Report indi­
cated that malaria episodes and mortalities have decreased by approx­
imately 8% and 28%, respectively from 2010 to 2017 (WHO, 2018). This
suggests that some progress has been made in malarial control in this
past decade, which could be attributed to the different chemothera­
peutic and non-chemotherapeutic interventions. However, the latest
data also shows that there are still significant works to be done,
particularly if we are going to get anywhere close to meeting the WHO’s

* Corresponding author.
E-mail address: salaribe@unilag.edu.ng (S.C. Alaribe).

https://doi.org/10.1016/j.jep.2021.114105
Received 15 December 2020; Received in revised form 20 March 2021; Accepted 3 April 2021
Available online 5 May 2021
0378-8741/© 2021 Elsevier B.V. All rights reserved.
S.C. Alaribe et al.
(2015) ambitious target of “reducing the global malaria burden by 90%
by 2030”. The major challenges jeopardizing efforts in this regard are
the rapid development and spread of Plasmodium resistance to existing
and emerging antimalarial drugs on the one hand and mosquito resis­
tance to insecticides on the other hand. It has been widely articulated
that the best long-term control option is the development of vaccines,
which could effectively prevent the spread of the disease. Nevertheless,
all the promising vaccines developed so far are still undergoing clinical
trials (Bejon et al., 2008; Guinovart et al., 2009; Abdulla et al., 2013;
Moreno and Joyner, 2015; Butler and Stricker, 2019). Another approach
in malaria control is the application of traditional medicines, particu­
larly plant-based remedies for the treatment of malaria. In fact, this
practice is as old as malaria itself. Moreover, some of the current anti­
malarial drugs, including amodiaquine and artemisinin, were either
isolated from plants or developed using plant’s isolates as lead molecules
(Alaribe et al., 2020). Thus, it seems logical to continue to investigate
plants and other natural products, used traditionally for malaria treat­
ment, as potential source of novel antimalarial candidates. However,
since these natural products are traditionally often applied as
multi-component formulations, it is also important to investigate their
therapeutic properties in their traditional forms. In view of the forego­
ing, the attention that is accorded the conventional bioactivity-guided
isolation of individual molecules should be extended to validation and
standardization of multi-component herbal formulations for acceptance
for clinical use, particularly in malaria management. It could be spec­
ulated that owing to the complexity of these multi-component herbal
formulations, it may be unlikely that Plasmodium will develop resistance
to these multi-component herbal formulations, which has been the bane
of orthodox antimalarial drugs. Moreover, studies have shown that some
crude plant extracts exhibited greater antiplasmodial activity than in­
dividual antimalarial drugs, such as artemisinin, at an equivalent dose
(Wan et al., 1992; Wright et al., 2010; Rasoanaivo et al., 2011).
A large number of medicinal plants are used for treating malaria in
Nigeria, particularly in the Southern regions of the country, where rain
forests and humid tropical climate exist, which are ideal conditions for
persistent malaria transmission all year round (Adebayo and Krettli,
2011). One out of the many groups of herbal remedies used in Nigeria is
the antimalarial decoctions popularly called Agbo iba, which receive
great patronage, majorly in the Southern regions of the country (Alaribe
et al., 2018). The composition, method of preparation and dosage of
these antimalarial decoctions vary from vendor to vendor as earlier re­
ported (Alaribe et al., 2018), but usually, these decoctions are often
polyherbal, prepared in water and sometimes alcohol, which serves as
preservative and also as desired by vendors and consumers. In this study,
we comparatively evaluated the antimalarial activities of a polyherbal
mixture and individual plant of the polyherbal mixture with the stan­
dard antimalarial drugs on mice infected with chloroquine-sensitive
Plasmodium berghei (NK65 strain). The antimalarial polyherbal mixture
(APM) consists of the stem barks of Mangifera indica (MI), Azadirachta
indica (AI), and Nauclea latifolia (and roots, NL) as well as roots of
Morinda lucida (ML). These plants have been reported to be used for the
treatment of different diseases, including malaria (Awe et al., 1998;
Usha Devi et al., 2001; Umar et al., 2013; Alaribe et al., 2020).
2. Materials and methods
2.1. Collection and preparation of plant materials
The different plant materials: MI, AI, NL and ML were purchased
freshly from Mushin herbal market, Lagos, Nigeria. The plant materials
were identified and authenticated by a taxonomist, Dr. G.I. Nodza at the
Herbarium of the Department of Botany, Faculty of Science, University
of Lagos, Lagos, Lagos state. The herbarium specimens were deposited
and the following voucher numbers assigned: MI (LUH 8619), AI (LUH
8618), NL (LUH 8622) and ML (LUH 8621). The plant materials were air
dried at room temperature and ground to coarse powder using an impact
2
Journal of Ethnopharmacology 277 (2021) 114105
mill. The decoction of each component was prepared by boiling 100 g of
pulverized material in 1 L of distilled water for 24 h. For NL, 50 g each of
both stem barks and roots were used. For APM, the aforementioned
quantities of the different plant components were mixed together in the
ratio 1: 1: 1:1 and boiled in 4 L of distilled water for 24 h. The collected
liquid extracts were lyophilized and stored in a refrigerator at -20 ◦ C
until used.
2.2. Experimental animals and malaria parasite
Swiss albino mice (weighing 20–25 g; 4 weeks old) of either sex were
obtained from Animal Facility Centre of College of Medicine, University
of Lagos, Nigeria and maintained under standard environmental con­
ditions of temperature (≈25 ◦ C), relative humidity (≈50%) and illumi­
nation (≈12/12 h light/dark cycle). The animals were provided with
standard pellet feed and drinking water and allowed to acclimatize for 7
days prior to the commencement of the study. Animals were used in
accordance with the internationally accepted ‘Guide for the care and use
of laboratory animals’ (National Research Council, 1996). The study was
conducted with approval from the Health Research Ethics Committee of
College of Medicine, University of Lagos with document Number;
CMUL/HREC/03/18/341.
Chloroquine-sensitive Plasmodium berghei (NK65 strain) was ob­
tained from the National Institute of Medical Research (NIMR), Lagos
and was maintained by serial passage in different donor mice.
2.3. Parasite inoculation
Parasite inoculation was done as previously described (Fidock et al.,
2004). Briefly, mice previously infected with P. berghei (NK65 strain)
having a parasite load of 20–30% were used as donor. Blood samples
were collected from the donor mice through ocular puncture into hep­
arinized vacutainer tube. Subsequently, the blood was diluted with
physiological saline (0.9%) in such a way that 1 ml of the final sus­
pension contained 5 × 107 P. berghei-infected red blood cells (RBCs). All
the mice used for the antiplasmodial investigation were inoculated
intraperitoneally with 0.2 ml of infected blood (equivalent to 1 × 107
P. berghei-parasitized erythrocytes).
2.4. Grouping and dosing of animals
The animals were grouped and dosed for the antiplasmodial inves­
tigation as follows. For both suppressive and curative tests, separately,
50 mice were randomly grouped into 10 groups (I–X) of 5 mice each.
Groups I, II and III were orally administered APM at 100, 160 and 320
mg/kg/day (i.e., 0.162, 0.259 and 0.518 mg/kg human equivalent doses
(HED)), respectively. Groups IV, V, VI and VII were administered 100,
1000, 1200 and 1240 mg/kg/day (i.e., 0.162, 1.62, 1.94 and 2.0 mg/kg
HED) of NL, MI, ML and AI, respectively, based on their active doses and
LD50 values as previously reported (Okpanyi and Ezeukwu, 1981; Awe
et al., 1998; Usha Devi et al., 2001; Taïwe et al., 2011; Umar et al., 2013;
Adejo and Atawodi, 2014; Chidozie et al., 2014; Kouadio et al., 2014;
Ettebong et al., 2015; Alaribe et al., 2020). Group VIII, negative control,
received distilled water, while groups IX and X, positive controls, were
administered 2:18 and 4.5:1.5 mg/kg/day (i.e., 0.0032:0.0292 and
0.0073:0.0024 mg/kg HED) of dihydroartemisinin-piperaquine (DHP)
and pyronaridine-artesunate (PA), respectively. For the prophylactic
test, 40 mice were used and grouped into 8 groups (I–VIII) as described
above. The HEDs were calculated according to USFDA (2005)
guidelines.
2.5. Test for suppressive activity (Peter’s 4-day test)
Suppressive activities of APM and its components: MI, AI, NL and ML
were evaluated on early P. berghei infection in mice as described by
Knight and Peters (1980). The mice were inoculated, grouped and dosed
S.C. Alaribe et al.
as described in section 2.4. The different treatments were started 3 h
after infection and were administered as a single dose per day for four
consecutive days (days 0–3). On the fifth day (day 4 i.e., 96 h
post-infection), blood samples were collected from tail snips of the mice.
Thick and thin films were made on the same slide for each blood sample.
The thin films were fixed in methanol and air dried. The blood films
were stained with 3% Giemsa at a pH of 7.2 and examined under a light
microscope using × 100 objective lens (immersion oil). The number of
malaria parasites and leucocytes per high power field (HPF) were
counted and parasite density was determined, assuming an average
leucocyte count of 8000 per μl, using the following equation (Green­
wood and Armstrong, 1991).
Parasite density (No. of parasites/μl) = (No. of Parasites/No. of leucocytes
counted) x 8000
A slide was considered negative after 100 HPF have been examined.
Average percentage chemosuppression (or parasite clearance) of each
treatment was calculated from the average parasite density of the
negative control group (A) and the average parasite density of the test
group (B) using the following expression.
Average percentage (%) chemosuppression = [(A – B)/A] x 100 (2)
2.6. Test for curative activity (Rane’s test)
Curative activities of APM and its components: MI, AI, NL and ML
were evaluated on established P. berghei infection in mice as described
by Ryley and Peters (1970). The mice were inoculated, grouped and
dosed as stated in section 2.4. The different treatments were started 72 h
after the infection and were administered as a single dose per day for five
consecutive days. For the entire period of treatment (days 3–7), blood
samples were collected from tail snips of the mice daily and were made
into films. The blood films were developed for microscopic examination
to monitor their parasite densities as described above.
2.7. Test for prophylactic activity (Repository test)
Prophylactic activities of APM and its components: MI, AI, NL and
ML were assessed in mice using the method described by Peters (1965).
The mice were inoculated, grouped and dosed as described in section
2.4. The different treatments were administered as a single dose per day
for three consecutive days (days 0–2) before infection. On the fourth day
(day 3), the mice were inoculated and 72 h later (day 6), blood samples
were collected and the procedure completed as described above to
monitor their parasite densities.
2.8. Data analysis
All values are expressed as mean ± standard error of mean (SEM,
with n = 5) and were analysed on GraphPad Prism Version 6.0
(GraphPad Software, San Diego, USA). Statistical differences between
the controls and treated groups were evaluated using one-way analysis
of variances (ANOVA) with post hoc Tukey’s test; p values < 0.05 were
considered as being statistically significant.
3. Results
3.1. Suppressive activity
As shown in Fig. 1 (and supplementary file 1), APM; its components:
NL, MI, ML, and AI; and positive controls: DHP and PA all showed
chemosuppressive activities on P. berghei parasitaemia in mice. These
effects were statistically significant (p < 0.05) compared to the negative
control group. All doses of the polyherbal mixture (APM) significantly
Journal of Ethnopharmacology 277 (2021) 114105
(1)
Fig. 1. Suppressive activity of antimalarial decoctions and controls against
P. berghei infection in mice. Each bar represents the mean ± SEM; n = 5. Values
in each group are significantly different (p < 0.05) from the other groups.
suppressed parasitaemia than each of its components and the positive
controls. NL (100 mg/kg), MI (1000 mg/kg) and ML (1200 mg/kg),
having percentage chemosuppression of 54.5 ± 0.02, 76.9 ± 0.02, and
60.9 ± 0.01, respectively showed significantly lower suppressive ac­
tivities than DHP (2.18:17.45 mg/kg) and PA (4.58:1.53 mg/kg), with
percentage chemosuppression of 88.1 ± 0.02 and 87.3 ± 0.02, respec­
tively. However, the suppressive activity of AI (1240 mg/kg; percentage
chemosuppression of 92 ± 0.03) was comparable to those of DHP and
PA.
3.2. Curative activity
In established plasmodial infection in mice (72 h p. i.), all the herbal
decoctions: APM, NL, MI, ML, and AI; and positive controls: DHP and PA
showed chemosuppressive activities (Fig. 2 … (Fig. 2a and b). and
supplementary file 2). The different treatments significantly (p < 0.05)
reduced parasitaemia level from 24 h post administration to the end of
the treatment period (days 4–7) when compared to the negative control
group. Throughout this period, the activities of all the herbal decoctions
were significantly lower than those of DHP and PA. At the termination of
the treatment, DHP gave a total clearance (100%) of the parasites, PA,
AI, MI, ML, APM (320, 160, 100 mg/kg) and NL gave 99.5 ± 0.1%, 93.6
± 0.04%, 93.3 ± 0.05%, 91.9 ± 0.02%, 91 ± 0.05%, 85.1 ± 0.04%, 82.5
± 0.04%, and 72 ± 0.05% parasite clearance, respectively.
3.3. Prophylactic activity
All the herbal decoctions: APM, NL, MI, ML, and AI exerted signifi­
cant (p < 0.05) reduction in parasitaemia level relative to the negative
control in the prophylactic test (Fig. 3 and supplementary file 3). APM
suppressed parasitaemia level by 86.8 ± 0.03%, 79.1 ± 0.04%, and 60.8
± 0.04% at doses of 320, 160, and 100 mg/kg, respectively; while ML,
MI, AI, and NL produced suppressive effect of 74.6 ± 0.03%, 71.1 ±
0.02%, 67.1 ± 0%, and 35.8 ± 0.01%, respectively (see Fig. 3).
4. Discussion
Malaria remains one of the most prevalent infectious diseases in the
tropical regions of the world. More than a century after identification of
the causative parasites and more than half a century after finding
effective drugs and insecticides, this dreadful disease is still yet to be
eradicated (Mehta and Desai, 2013). While there have been continuous
efforts for development of novel antimalarial drugs and effective vac­
cines; at present there is no single drug effective for the treatment of
3
S.C. Alaribe et al. Journal of Ethnopharmacology 277 (2021) 114105

Fig. 2. (a). Curative effect of antimalarial decoctions and controls against established P. berghei infection in mice, showing parasite/μl. Each data point represents the
mean ± SEM; n = 5. Values in each group are significantly different (p < 0.05) from the other groups. (b). Curative effect of antimalarial decoctions and controls
against established P. berghei infection in mice, showing parasite clearance (%). Each data point represents the mean ± SEM; n = 5. Values in each group are
significantly different (p < 0.05) from the other groups.

multidrug resistant malaria; combination therapy has been the chemo­
therapeutic strategy. Unfortunately, there has been report of develop­
ment of Plasmodium resistance to the WHO recommended
artemisinin-based combination therapies (ACTs) in some parts of the
world (Dondorp et al., 2009). Some studies (Adepiti et al., 2014; Tar­
kang et al., 2014; Orabueze et al., 2018) have shown that polyherbal
preparations produced considerable antimalarial activities in animal
models; suggesting that such preparations could be considered for
further studies as an alternative strategy in our fight against malaria. In
this study we assessed and evaluated the suppressive, curative, and
prophylactic antiplasmodial activities of an antimalarial polyherbal
mixture (APM) in comparison to the activities of its individual constit­
uent plants and those of standard antimalarial drugs. The polyherbal and
individual plant decoctions were prepared in water and administered

4
S.C. Alaribe et al.
Fig. 3. Prophylactic activity of antimalarial decoctions and control against
P. berghei infection in mice. Each bar represents the mean ± SEM; n = 5. Values
in each group are significantly different (p < 0.05) from the other groups.
orally to maintain the traditional methods of preparation and adminis­
tration. Four weeks old mice were chosen for the study in order to
exclude the effects of anaemia, which is likely in older mice (Pierrot
et al., 2003). The in vivo models were adopted because they take into
consideration the possible prodrug effect and probable involvement of
the immune system in controlling the infection (Waako et al., 2005).
The four day suppressive test usually assesses the antimalarial ac­
tivity of candidates on early periods of infection (Verma et al., 2011). In
this test, all the herbal decoctions significantly suppressed parasite level.
The polyherbal mixture (APM) gave the highest effect and showed
percentage chemosuppression in the range of 95.4–98.7% at the doses of
100–320 mg/kg when administered 3 h after infection. Each of the in­
dividual constituent plants (NL, MI, ML, and AI) also showed consider­
able suppressive effect (>50%), corroborating previous findings on
these plants. Aqueous extract of N. latifolia roots was reported to exert
85.22% suppressive effect at 100 mg/kg in early infection of P. berghei
(Alaribe et al., 2020); while aqueous fraction of the stem bark exhibited
67.71% at 200 mg/kg (Ettebong et al., 2015). Similarly, MI, ML, and AI
produced 78.2%, 67.72%, and 66.47% at 400 mg/kg, 400 mg/kg, and
1000 mg/kg, respectively (Awe et al., 1998; Usha Devi et al., 2001;
Umar et al., 2013). These indicate that the decoction of either the in­
dividual plants or the combination could be useful in suppressing ma­
laria in its early stages. . In the curative test, both DHP and PA showed
remarkable activities. At the end of the treatment, DHP had totally
cleared the parasites, with no measurable parasite recorded, while PA
had elicited 99.5% parasite clearance. Typically, malaria parasite den­
sities are expected to be negative 48 h after treatment with an ACT
(White, 2008). In a previous study, out of 63 patients diagnosed with
P. falciparum, 37 (58.7%) showed cleared parasitaemia at day 2 or 3
after treatment with DHP (Lo et al., 2016). All the herbal decoctions
were also active in the curative test. Significant reduction in parasite
load was observed from day 2 of treatment and daily for the five days of
treatment for each of the decoctions. At the end of treatment, none of the
decoctions had completely cleared the parasites, however they all pro­
duced substantial clearance (>70%), indicating their potential useful­
ness against established plasmodial infection. Similarly, in the
prophylactic test, the herbal decoctions demonstrated significant che­
mosuppression. Although none of them completely suppressed para­
sitaemia, they all (except for NL; 35%) produced notable
chemosuppressive effect (>60%), implying that these decoctions could
be applied as prophylactics for malaria.
The suppressive, curative, and prophylactic antiplasmodial models
are the standard methods commonly used for screening antimalarial
candidates and in these methods, the determination of percentage
5
Journal of Ethnopharmacology 277 (2021) 114105
suppression of parasitaemia is the most reported parameter. A per­
centage suppression of parasitaemia ≥10% relative to the vehicle con­
trol usually indicates that the test candidate is active (Peter and Anatoli,
1998). This suggests that all the decoctions investigated in this study
could be considered as active antimalarial candidates. Furthermore, in
vivo antiplasmodial activity is classified as moderate, good, or very good
if an extract showed percentage parasitaemia suppression ≥50% at a
dose of 500, 250 and 100 mg/kg/day, respectively (Munoz et al., 2000).
Based on this classification, the polyherbal mixture (APM) showed very
good antiplasmodial activities, NL showed good activities while MI, ML
and AI could be considered to have shown moderate activities. The
suppressive and curative activities demonstrated might be through in­
direct boosting of the immune system (Waako et al., 2005), as evidenced
by the daily decrease in parasitaemia level observed in the negative
control group in the curative model. The prophylactic activity might be
due to inhibition of proliferation of plasmodial parasites as a result of
direct cytotoxicity (Golenser et al., 2006) and modulation of erythrocyte
membranes to prevent parasite invasion (Hansen, 2012).
These activities could be attributed to the bioactive compounds
present in the different decoctions. Studies have shown the presence of
alkaloids, terpenes, phenols, flavonoids, tannins, and saponins in NL,
MI, ML and AI (Okwu and Ezenagu, 2008; Bassey and Jude, 2014; Abu
et al., 2016; Nwali et al., 2018). These compounds could have elicited
the observed antiplasmodial activities either individually or in syner­
gistic interactions or multi-factorial effects between themselves (Duke
and Bogenschutz-Godwin, 1999; Gilbert and Alves, 2003); thus, isola­
tion and investigation of the individual compounds is often necessary.
Many compounds have been isolated from stem bark of M. indica, the
major component is the C-glucoside xanthone mangiferin (Fig. 4a). The
reported activities of mangiferin include immune-modulatory, antioxi­
dant and anti-inflammatory effects (Sanchez et al., 2000; Garcia et al.,
2002; Garrido et al., 2004). The immunostimulatory and
anti-inflammatory actions of mangiferin could have contributed to the
observed antimalarial effects of M. indica stem bark (Adepiti et al.,
2014). Compounds previously isolated from stem bark of A. indica
include the diterpenoids margolone (Fig. 4d), margolonone (Fig. 4e),
and isomargolonone (Fig. 4b), which were reported to possess antibac­
terial property (Ara et al., 1989). The limonoids nimbolide (Fig. 4f) and
gedunin (Fig. 4h) are the most promising compounds from A. indica with
antimalarial property, and were isolated from the seed oil (Rojanapo
et al., 1985; Khalid et al., 1989). Compounds belonging to different
phytochemical classes have been isolated from the stem bark and root of
N. latifolia. However, the alkaloids angustoline (Fig. 4c), naucleidinal
(Fig. 4g) and strictosamide (Fig. 4k) are the components reported to
exhibit antiplasmodial activity (Abreu and Pereira, 2001; Sun et al.,
2008). Anthraquinones rubiadin 1-methyl ether (Fig. 4i) and dam­
nacanthal (Fig. 4j) previously isolated from the root of M. lucida have
been reported to exhibit potent antiplasmodial activity, producing
98.7% and 100% inhibition of P. falciparum, respectively, at 30 μg/ml
(Koumaglo et al., 1992).
Wagner and Ulrich-Merzenich (2009) noted that individual com­
pounds rarely have the same degree of activity as the unrefined extract
at comparable concentrations or doses. This phenomenon could be
attributed to the heterogeneous nature of extracts in comparison to in­
dividual compounds. Rasoanaivo et al. (2011) identified that crude ex­
tracts could benefit from positive pharmacodynamic and/or
pharmacokinetic synergies between different constituents leading to
activity at different receptor targets and/or improved pharmacokinetic
profile of the main active principle(s). The authors further noted that
other beneficial interactions could include complementary mechanisms
of action (such as immunomodulation), reversal of resistance, and
modulation of adverse effects. These phenomena could explain the
greater activities of APM relative to its individual constituent plants in
both suppressive and prophylactic antiplasmodial models. Similar
studies have also shown that combining different medicinal plants or
orthodox drugs with herbal preparations could elicit beneficial effects
S.C. Alaribe et al.
Fig. 4. Structures of some compounds isolated from M. indica (a), A. indica (b, d, e, f, h), N. latifolia (c, g, k) and M. lucida (i, j)
(Mohd Ridzuan et al., 2007; Anagu et al., 2014; Adepiti et al., 2014,
2016). Nevertheless, such polyherbal or herb-drug combinations could
also elicit antagonistic or subtractive effect or produce no significant
difference, as previously reported (Gathirwa et al., 2008; Idowu et al.,
2014). Although our findings indicate that the polyherbal mixture
(APM) possessed antimalarial property and could be useful as treatment
or prophylactic for malaria. Further studies are needed to evaluate the
safety of this decoction and its activity in human. In the meantime, this
study provides evidence supporting the notion that the use of stan­
dardized herbal medicines could be a valid complementary approach for
malaria control, provided that the safety and efficacy of such formula­
tions are clinically validated.
5. Conclusion
The results of this study revealed that the Antimalarial polyherbal
mixture (APM) and its individual constituent plants (NL, MI, ML, and AI)
showed significant suppressive, curative and prophylactic antimalarial
activities. APM produced greater activities than its individual constitu­
ent plants in both suppressive and prophylactic models, suggesting that
the combination produced synergistic or additive effects. This could
serve as scientific basis for the traditional usage of these plants as
combined therapy for malaria. Further studies are recommended to
evaluate their safety, disposition and interactions in human.
Acknowledgement
We appreciate the technical assistance of Mrs Chinonye Nwosu
Anabike of the ANDI Centre of Excellence for Malaria Diagnosis,
Department of Medical Microbiology and Parasitology, College of
Medicine, University of Lagos.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
6
Journal of Ethnopharmacology 277 (2021) 114105
org/10.1016/j.jep.2021.114105.
Author statement
A:devised the project, the main conceptual ideas and supervised the
work
A&B:processed the experimental data, performed the analysis,
drafted the manuscript and designed the figures.
A.C.D:worked out almost all of the technical details and carried out
the experiment.
A .B. & E: Discussed the results, aided in interpreting the results and
worked on the manuscript.
F:Assisted with the technical staff on anti-plasmodial studies and
data interpretation.
References
Abdulla, S., Salim, N., Machera, F., Kamata, R., Juma, O., Shomari, M., 2013.
Randomized, controlled trial of the long term safety, immunogenicity and efficacy of
RTS,S/AS02(D) malaria vaccine in infants living in a malaria-endemic region. Malar.
J. 12 (11).
Abreu, P., Pereira, A., 2001. New alkaloids from Sarcocephalus latifolius. Nat. Prod. Lett.
15, 43–48.
Abu, N.E., Ezeomeke, S.I., Azegba, P., Davidson, G.I., 2016. Phytochemical, nutritional
and anti-nutritional properties of leaves, stems bark and roots of trees used in
popular medicine for the treatment of malaria in South Eastern Nigeria. J. Med.
Plants Res. 10 (38), 662–668.
Adebayo, J.O., Krettli, A.U., 2011. Potential antimalarials from Nigerian plants: a review.
J. Ethnopharmacol. 133, 289–302.
Adejo, G.O., Atawodi, S.E., 2014. Acute toxicity and genotoxic effects of all parts of
Morinda lucida benth on pUC18 plasmid DNA. Nat. Prod. Chem. Res. S1, 006.
Adepiti, A.O., Elujoba, A.A., Bolaji, O.O., 2014. In vivo antimalarial evaluation of MAMA
decoction on Plasmodium berghei in mice. Parasitol. Res. 113, 505–511.
Adepiti, A.O., Elujoba, A.A., Bolaji, O.O., 2016. Evaluation of herbal antimalarial MAMA
decoction-amodiaquine combination in murine malaria model. Pharm. Biol. 54 (10),
2298–2303.
Alaribe, C.S., Oladipupo, A.R., Musah, A.A., Coker, H.A.B., 2018. Investigation of
selected metallic impurities in antimalarial herbal decoctions ‘Agbo Iba’ collected
from four locations in Lagos state, Nigeria. UNILAG J. Med., Sci. Technol. 6 (1),
45–56.
S.C. Alaribe et al.
Alaribe, C.S., Oladipupo, A.R., Nani, M., Ijeoma, I., Olanipekun, B., Coker, H.A.B., 2020.
Suppressive and curative antiplasmodial properties of Nauclea latifolia root extract
and fractions against erythrocytic stage of mice-infective chloroquine-sensitive
Plasmodium berghei NK-65. J. Med. Plants Econ. Develop. 4 (1), 1–6 a72.
Anagu, O.L., Attama, A.A., Okore, V.C., Gugu, H.T., Ngene, A.A., Esimone, C.O., 2014.
Azadirachta indica extract-artesunic acid combination produces an increased cure
rate of Plasmodium berghei-infected mice. Pharm. Biol. 52, 883–889.
Ara, I., Siddiqui, B.S., Faizi, S., Siddiqui, S., 1989. Structurally novel diterpenoid
constituents from the stem bark of Azadirachta indica (melieceae). J. Chem. Soc.
Perkin. Trans. 2, 343–345.
Awe, S.O., Olajide, O.A., Oladiran, O.O., Makinde, J.M., 1998. Antiplasmodial and
antipyretic screening of Mangifera indica extract. Phytother Res. 12, 437–438.
Bassey, S.A., Jude, E.O., 2014. Phytochemical composition and antidiabetic activity of
ethanol root extract of Nauclea latifolia. J. Phytopharm. 3 (1), 52–56.
Bejon, P., Lusingu, J., Olotu, A., Leach, A., Lievens, M., Vekemans, J., 2008. Efficacy of
RTS,S/AS01E vaccine against malaria in children 5 to 17 months of age. N. Engl. J.
Med. 359 (24), 2521–2532.
Butler, D., Stricker, L., 2019. Promising Malaria Vaccine to Be Tested in First Large Field
Trial. http://www.nature.com/articles/d41586-019-01232-4. (Accessed 13
September 2020).
Chidozie, V.N., Adoga, G.I., Chukwu, O.C., Chukwu, I.D., Adekeye, A.M., 2014.
Antibacterial and toxicological effects of the aqueous extract of Mangifera Indica
stem bark on albino rats. Glob. J. Biol., Agricult. Health Sci. 3 (3), 237–245.
Dondorp, A.M., Nosten, F., Yi, P., Das, D., Phyo, A.P., Tarning, J., Lwin, K.M., Arley, F.,
Hanpithakpong, W., Lee, S.J., 2009. Artemisinin resistance in Plasmodium falciparum
malaria. N. Engl. J. Med. 361, 455–467.
Duke, J.A., Bogenschutz-Godwin, M.J., 1999. The synergy principle at work in plants,
pathogens, insects, herbivores and humans. In: Kaufmann, P.B., Cseke, L.J.,
Warbler, S., Duke, J.A., Brielmann, H.L. (Eds.), Natural Products from Plants. CRC
Press, Boca Raton, pp. 183–205.
Ettebong, E.O., Ubulom, P.M., Ekpenyong, C.E., Ekong, U.S., Akpan, O.E., Tambari, V.,
2015. In vivo antiplasmodial activities of Nauclea latifolia. Asian J. Med. Sci. 6 (3),
6–11.
Fidock, D., Rosenthal, J., Croft, S., Brun, R., Nwaka, S., 2004. Antimalarial drug
discovery: efficacy models for drug screening. Nat. Rev. Drug Discov. 3, 509–520.
Garcia, D., Delgado, R., Ubeira, F.M., Leiro, J., 2002. Modulation of rat macrophage
function by the Mangifera indica L extracts Vimang and mangiferin. Int.
Immunopharm. 2, 797–806.
Garrido, G., Delgado, R., Lemus, Y., Rodriquez, J., Garcia, D., Nunez-Selles, A.J., 2004. In
vivo and in vitro anti-inflammatory activity of Mangifera indica L extract (VIMANG).
Pharmacol. Res. 50, 165–172.
Gathirwa, J.W., Rukunga, G.M., Njagi, E.N., Omar, S.A., Mwitari, P.G., Guantai, A.N.,
Tolo, F.M., Kimani, C.W., Muthaura, C.N., Kirira, P.G., 2008. The in vitro
antiplasmodial and in vivo anti-malarial efficacy of combinations of some medicinal
plants used traditionally for treatment of malaria by the Meru community in Kenya.
J. Ethnopharmacol. 115, 223–231.
Gilbert, B., Alves, L.F., 2003. Synergy in plant medicines. Curr. Med. Chem. 10, 13–20.
Golenser, J., Waknine, J.H., Krugliak, M., Hunt, N.H., Grau, G.E., 2006. Current
perspectives on the mechanism of action of artemisinins. Int. J. Parasitol. 36,
1427–1441.
Greenwood, B.M., Armstrong, J.R.M., 1991. Comparison of two simple methods for
determining malaria parasite density. Trans. R Soc. Trop. 85 (2), 186–188.
Guinovart, C., Aponte, J.J., Sacarlal, J., Aide, P., Leach, A., Bassat, Q., 2009. Insights into
long-lasting protection induced by RTS,S/AS02 A malaria vaccine: further results
from a phase IIb trial in Mozambican children. PloS One 4, e5165.
Hansen, D.S., 2012. Inflammatory responses associated with the induction of cerebral
malaria: lessons from experimental murine models. PLoS Pathog. 8, e1003045.
Idowu, O.A., Babalola, A.S., Adenubi, O.T., Olukunle, J.O., 2014. Antiplasmodial
activities of combined Extracts of Morinda morindiodes, Morinda lucida and Vernonia
amygdalina in Plasmodium berghei infected mice. Zool. 11, 40–45.
Khalid, S.A., Duddeck, H., Gonzalez-Sierra, M., 1989. Isolation and characterization of
antimalerial agent of the neem tree, A. indica. J. Nat. Prod. 52, 922–927.
Knight, D.J., Peters, W., 1980. The antimalarial action of N-benzyloxy dihydrotriazines.
The action of cycloguanil (BRL50216) against rodent malaria and studies on its
mode of action. Ann. Trop. Med. Parasitol. 74, 393–404.
Kouadio, J.H., Bleyere, M.N., Kone, M., Dano, S.D., 2014. Acute and sub-acute toxicity of
aqueous extract of Nauclea latifolia in Swiss mice and in OFA rats. Trop. J.
Pharmaceut. Res. 13 (1), 109–115.
Koumaglo, K., Gbeassor, M., Nkabou, C.L., de Souza, C.C., Wenee, W., 1992. Effects of
three compounds extracted from Morinda lucida on Plasmodium falciparum. Planta
Med. 58, 533–534.
Lo, E., Nguyen, J., Oo, W., Hemming-Schroeder, E., Zhou, G., Yang, Z., Cui, L., Yan, G.,
2016. Examining Plasmodium falciparum and P. vivax clearance subsequent to
antimalarial drug treatment in the Myanmar-China border area based on
quantitative real-time polymerase chain reaction. BMC Infect. Dis. 16, 154.
Mehta, D., Desai, N.J., 2013. Laboratory diagnosis of malaria-various method and its
comparison. Natl. J. Integrated Res. Med. 4 (3), 138–143.
Journal of Ethnopharmacology 277 (2021) 114105
Mohd Ridzuan, M.A., Sow, A., NooRain, A., Mohd Ilham, A., Zakiah, I., 2007. Eurycoma
longifolia extract-artemisinin combination parasitaemia suppression of Plasmodium
yoelii infected mice. Trop. Biomed. 24, 111–118.
Moreno, A., Joyner, C., 2015. Malaria vaccine clinical trials: what’s on the horizon. Curr.
Opin. Immunol. 35, 98–106.
Munoz, V., Sauvain, M., Bourdy, G., Callapa, J., Bergeron, S., Rojas, I., 2000. A search for
natural bioactive compounds in Bolivia through a multidisciplinary approach. Part I.
Evaluation of the antimalarial activity of plants used by the Chacobo Indians.
J. Ethnopharmacol. 69, 127–137.
National Research Council NRC, 1996. Guide for the Care and Use of Laboratory
Animals, eight ed. The National Academies Press, Washington DC, USA.
Nwali, O.N., Idoko, A., Okolie, J.E., Ezeh, E., Ugwudike, P.O., Rita, O.N., Ezenwali, M.O.,
Odo, I.A., Ani, P.N., Okolie, S.O., 2018. Comparative analysis of the phytochemical
compositions of leaf, stem-bark and root of Azadirachta Indica (neem). Univers. J.
Pharm. Res. 3 (5), 46–50.
Okpanyi, S.N., Ezeukwu, G.C., 1981. Anti-Inflammatory and antipyretic activities of
Azadirachta indica. J. Med. Plants Res. 41, 34–39.
Okwu, D.E., Ezenagu, V., 2008. Evaluation of the phytochemical composition of mango
(Mangifera Indica linn) stem bark and leaves. Int. J. Chem. Sci. 6 (2), 705–716.
Orabueze, C.I., Adesegun, S.A., Ota, D.A., Coker, H.A., 2018. In vivo antiplasmodial
activities of four Nigerian plants used singly and in polyherbal combination against
Plasmodium berghei infection. Indian J. Tradit. Knowl. 17 (4), 716–723.
Peter, I.T., Anatoli, V.K., 1998. The Current Global Malaria Situation. Malaria Parasite
Biology, Pathogenesis, and Protection. ASM Press, Washington DC, USA, pp. 11–22.
Peters, W., 1965. Drug resistance in Plasmodium berghei. Vincke and Lips, 1948; I.
Chloroquine resistance. Exp. Parasitol. 17, 80–89.
Pierrot, C., Adam, E., Lafitte, S., Godin, C., Dive, D., Capron, M., Khalife, J., 2003. Age-
related susceptibility and resistance to Plasmodium berghei in mice and rats. Exp.
Parasitol. 104, 81–85.
Rasoanaivo, P., Wright, C.W., Willcox, M.L., Gilbert, B., 2011. Whole plant extracts
versus single compounds for the treatment of malaria: synergy and positive
interactions. Malar. J. 10 (Suppl. 1), S4.
Rojanapo, W., Suwanno, S., Somaree, R., Glinsukon, T., Thebtaranonth, Y., 1985.
Screening of antioxidants from some Thia vegetables and herbs. J. Sci. Thailand 11,
177–188.
Ryley, J.F., Peters, W., 1970. The antimalarial activity of some quinone esters. Ann. Trop.
Med. Parasitol. 84, 209–222.
Sanchez, G.M., Re, L., Giuliani, A., Nunez-Selles, A.J., Davison, G.P., Leon-Fernandez, O.
S., 2000. Protective effects of Mangifera indica L extract, mangiferin and selected
antioxidants against TPA-induced biomolecules oxidation and peritoneal
macrophage activation in mice. Pharmacol. Res. 42, 565–573.
Sun, J., Lou, J., Dai, S., Xu, H., Zhao, F., Liu, K., 2008. Indole alkaloids from Nauclea
officinalis with weak antimalarial activity. Phytochemistry 69, 1405–1410.
Taïwe, G.S., Ngo Bum, E., Talla, E., Dimo, T., Weiss, N., Sidiki, N., Dawe, A., Moto, F.C.
O., Dzeufiet, P.D., De Waard, M., 2011. Antipyretic and antinociceptive effects of
Nauclea latifolia root decoction and possible mechanisms of action. Pharm. Biol. 49
(1), 15–25.
Tarkang, P.A., Franzoi, K.D., Lee, S., Lee, E., Vivarelli, D., Freitas-Junior, L., Liuzzi, M.,
Tsabang, N., Ayong, L.S., Agbor, G.A., Okalebo, F.A., Guantai, A.N., 2014. In vitro
antiplasmodial activities and synergistic combinations of differential solvent extracts
of the polyherbal product. Nefang BioMed. Res. Int. 835013.
Umar, M.B., Ogbadoyi, E.O., Ilumi, J.Y., Salawu, O.A., Tijani, A.Y., Hassan, I.M., 2013.
Antiplasmodial efficacy of methanolic root and leaf extracts of Morinda lucida. J. Nat.
Sci. Res. 3 (2), 112–122.
US Food and Drug Administration USFDA, 2005. Guidance for Industry: Estimating the
Maximum Safe Starting Dose in Adult Healthy Volunteers. US Food and Drug
Administration, Rockville, MD.
Usha Devi, C., Valecha, N., Atul, P.K., Pilla, i C.R., 2001. Antiplasmodial effect of three
medicinal plants: a preliminary study. Curr. Sci. 80 (8), 917–919.
Verma, G., Dua, V.K., Agarwal, D.D., Atul, P.K., 2011. Anti-malarial activity of
Holarrhena antidysenterica and Viola canescens, plants traditionally used against
malaria in the Garhwal region of north-west Himalaya. Malar. J. 10 (1), 20.
Waako, P.J., Gumede, B., Smith, P., Folb, P.I., 2005. The in vitro and in vivo antimalarial
activity of Cardiospermum halicacabum and Momordica foetida. J. Ethnopharmacol.
99 (1), 137–143.
Wagner, H., Ulrich-Merzenich, G., 2009. Synergy research: approaching a new
generation of phytopharmaceuticals. Phytomedicine 16, 97–110.
Wan, Y.D., Zang, Y.D., Wang, J.S., 1992. Studies on the antimalarial action of gelatin
capsule of Artemisia annua. Zongguo ji sheng chong xue yu ji sheng chong bing za shi
(Chin. J. Parasitol. Parasitic Dis.) 10, 290–294.
White, N.J., 2008. Qinghaosu (artemisinin): the price of success. Science 320, 330–334.
World Health Organization WHO, 2015. Global Technical Strategy for Malaria 2016-
2030. World Health Organization, Geneva.
World Health Organization WHO, 2018. World Malaria Report, 2018. World Health
Organization, Geneva.
Wright, C.W., Linley, P.A., Brun, R., Wittlin, S., Hsu, E., 2010. Ancient Chinese methods
are remarkably effective for the preparation of artemisinin-rich extracts of Qing Hao
with potent antimalarial activity. Molecules 15, 804–812.