The Scientific World Journal
Volume 2012, Article ID 343652, 16 pages
NewInsights inStaging andChemotherapyof
Paul F.Seke Etet1,2and M.FawziMahomoodally3
1Department of Neurological Sciences (DNNMMS), University of Verona, Via Delle Grazie 8, 37134 Verona, Italy
2Department of Neurology, Yaound´ e Central Hospital, Rue Henri Dunant, P.O. Box 87, Yaound´ e, Cameroon
3Department of Health Sciences, Faculty of Science, University of Mauritius, Reduit 230, Mauritius
Correspondence should be addressed to M. Fawzi Mahomoodally, firstname.lastname@example.org
Received 10 October 2011; Accepted 16 November 2011
Academic Editors: A. Casulli and G. Hide
Copyright © 2012 P. F. Seke Etet and M. Fawzi Mahomoodally. This is an open access article distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
Human African trypanosomiasis (HAT) is a fatal if untreated fly-borne neuroinflammatory disease caused by protozoa of the
species Trypanosoma brucei (T.b.). The increasing trend of HAT cases has been reversed, but according to WHO experts, new
epidemics of this disease could appear. In addition, HAT is still a considerable burden for life quality and economy in 36 sub-
Saharan Africa countries with 15–20 million persons at risk. Following joined initiatives of WHO and private partners, the fight
and pathogenesis and the new insights in the development of accurate tools and tests for disease staging and severity monitoring in
the field. Also, we elaborate herein the promising progresses made in the development of less toxic and more efficient trypanocidal
drugs including the potential of medicinal plants and related alternative drug therapies.
Human African trypanosomiasis (HAT) or sleeping sick-
ness is a severe fly-borne disease caused by protozoan of
the species Trypanosoma brucei (T.b.). This disease was first
described by European explorers by the late 1800s and
early 1900s even if this disease has probably existed in
Africa for many centuries . The disease occurs in foci in
ranging from the Sahara to the Kalahari Desert equivalent to
“the combined size of the United States, India and Western
Europe” where these flies have their habitat [2–5]. Three
major epidemics of HAT occurred in Africa during the
last century, of which the most devastating (which killed
millions of persons) occurred from the 1930s to the 1960s
. The colonial administrations established mobile teams
which systematically screened people in the endemic areas,
curing those found with the disease. This initiative resulted
in a significant roll back of the disease. In the early 1960s,
HAT ceased to be a public health problem, and was no more
considered . From the 1970s to the 1990s, favored by dra-
matic events such as wars and population movements, HAT
re-emergedand becameanongoing epidemic.WHO,private
partners, and local governments took action, resulting in
a significant decrease of the number of new cases reported
in 50 years .
Despite these encouraging results, HAT is still a consid-
erable burden for life quality and economy in many sub-
Saharan Africa countries, where there may be 200 foci and
15–20 million persons at risk , as a large number of new
infections may remain unreported or undiagnosed because
of remote accessibility of many areas of the endemic region
and ongoing wars [9–11]. Besides, it is generally assumed
that new epidemics of HAT could occur, originating from
these uncontrolled areas where there still are very active
foci , as illustrated in Figure 1. HAT affects poor and
remote rural populations dependent on agriculture, fishing,
2The Scientific World Journal
(a) Countries reporting the higher number of foci
(b) Countries at war from about 10 years
Figure 1: African trypanosomiasis repartition and sociopolitical instability. (a) Illustration of the geographical repartition of the countries
reporting the higher number of foci of both T.b. subspecies causing HAT. (b) Illustration of the geographical repartition of the countries at
war from more than 10 years. Note the correlation between countries at war and the localization of foci of T.b. gambiense. HAT cases occur
more often in countries with conflict, high political terror, or civil war, with a lag of about 10 years between the conflict beginning and peak
in incidence . Epidemiological data are from .
or hunting. Until very recently, this disease was receiving
development were inadequate to the need . In the last 50
years, only one drug, eflornithine, has been developed even
the current drugs used to cure HAT are expensive, highly
toxic, need parenteral administration, and parasites increas-
ing resistance has been observed [14, 15]. Therefore, less
toxic, more efficient, easy-to-administer and nonexpensive
drugs are urgently needed in the field. WHO and some
private partners have been recently multiplying initiatives,
offering funding for research activities for this purpose.
Some encouraging results have already been reported. The
research activities have been also aiming at developing new
field suitable, easy to-use, and cheap tools to solve the HAT
diagnosis, staging, and follow-up issues observed in the
HAT occurs in two forms: the Gambian or West African
form (caused by T.b. gambiense) and the Rhodesian or
East African form (caused by T.b. rhodesiense). The two
forms differ in their clinical course, which is chronic (with
a course from months to years) in the Gambian form,
which represents about 95% of cases, and acute or subacute
(with a course from weeks to months) in the Rhodesian
form, which represents a minority of cases [7, 8]. After
infection and a relatively long (Gambian form) or short
(Rhodesian form) latency time, HAT evolves in two stages:
into a meningoencephalitic stage (second stage or stage
2) irreversibly followed by death if untreated [17, 18].
The hemolymphatic stage entails bouts of fever, headaches,
adenopathy, joint pains, and itching. The trypanosomes
proliferate at the site of infection and then spread to
where they continuously multiply and from which they
invade the peripheral organs. Evidence from animal models
shows that at this stage of the disease, the parasites already
reside in the brain, but only in the structures located outside
the blood-brain barrier (BBB) such as circumventricular
organs . The meningoencephalitic stage starts when the
trypanosomes cross the BBB and invade the central nervous
system parenchyma, excluding then the possibility to cure
patients with drugs used in the first stage, as they do not
cross the BBB in amounts sufficient to kill the parasites.
The second stage is marked by a complex neuropsychiatric
syndrome characterized by changes of behavior, confusion,
sensory disturbances, poor coordination, a disruption of
the sleep-wake cycle, and an alteration of the sleep structure
[20, 21]. These qualitative alterations of sleep gave to HAT
its alternative name of sleeping sickness. While the drugs
The Scientific World Journal3
in use to cure the first stage are relatively safe, the drugs
in use to cure the second stage are highly toxic, and
resulting undesired effect include death in about 5% of cases
In a general way, the clinical features of HAT do not
suffice for a precise diagnosis, and HAT is usually confused
with another endemic and more frequent sub-Saharan
Africa disease: malaria caused by the apicomplexan parasite
Plasmodium falciparum . Therefore, For HAT diagnosis
in the field, physical examination for posterior cervical
lymphadenopathy (Winterbottom’s sign) is performed, and
a venous blood sample is taken from the subjects .
The blood is screened for the presence of specific antibodies
against the parasite with the card agglutination test for
trypanosomiasis (CATT). CATT is used in the field for mass
screening of HAT because of its sensitivity and ease of use
have been reported in areas of low endemicity , and in
several foci in West Africa were found some T.b. gambiense
strains lacking the gene that encodes the surface glycoprotein
LiTat 1.3 , main gene targeted by the agglutination
process [28, 29]. To solve this problem, in addition to
CATT are performed microscopic examinations of lymph
aspirated from enlarged cervical lymph nodes or of blood
films to assess the presence of the parasite in the lymph or
blood . If the CATT remains positive at a dilution of
1 in 8 or greater and trypanosomes are seen in the blood
film (or lymph), the subject is diagnosed with HAT [31,
32]. However, the occurrence of cases with positive CATT
without parasites is common, and considering the high
toxicity of the trypanocidal drugs (even those used to cure
the first stage), the management of such cases is debated and
constitutes an important issue . Therefore, as possible
replacement of low sensitivity current parasite detection
methods has been suggested, molecular methods are far
more sensitive [33, 34].
Generally, T.b. gambiense loads in the blood are low,
and molecular methods request concentration techniques
to increase the detection of these parasites . Such
(mAECT) [35, 36]. The latter was recently improved and is
the most sensitive technique for trypanosome detection of
the blood . The mAECT technique consists in the sepa-
ration of trypanosomes by anion exchange chromatography
on diethylaminoethyl cellulose and low-speed centrifugation
to concentrate the eluted trypanosomes. The parasites can
then be detected by direct microscopic examination of
the sediment in a transparent collector tube [25, 33]. This
test presents the advantage of applicability in the field
conditions and is robust and less cumbersome than previous
versions which required mounting a collector tube in water
neck and issues presented by this test as presently formulated
are the need of qualified personnel to perform it [39, 40], the
short-time stability (1 year maximum at 37◦C), as glucose is
incorporated in the column buffer , and the need of very
specific apparatus rarely found in the hospital of rural areas
where the disease is endemic .
positive subjects is represented by the “immune trypanolysis
test,” a technique assessing the absence of nonspecific
trypanolytic activity in the plasma . Studies evaluating
this technique were performed on plasma collected from
CATT-positive subjects with diverse epidemiological status
Burkina Faso HAT foci. This test appeared to be a marker
for contact with T.b. gambiense, suggesting its possible use as
a tool in the field to identify nonparasitologically confirmed
T.b. gambiense and should be followed up .
Also of interest are the polymerase chain reaction (PCR)
and nucleic acid sequence-based amplification techniques
modified by coupling to oligochromatography for easy
and fast visualization of products . These techniques
appeared to be very sensitive and specific for diagnosis of
T.b. gambiense in studies performed on blood samples from
DRC HAT patients. However, they failed to be as sensitive
and specific for T.b. rhodesiense detection on blood samples
from Uganda HAT patients .
Of high interest for the development of less invasive
HAT diagnosis tests are the encouraging results from studies
performed on saliva samples from T.b. gambiense HAT
linked immunosorbent assay (ELISA) antibody detection
technique. As ELISAs performed on serum and CATT
performed on whole blood or serum, ELISAs performed
on saliva appeared to be more than 90% sensitive and
specific for the detection of trypanosome-specific antibodies
in the saliva [43–45]. Contrarily to CATT which cannot
be successfully performed with saliva due to its insufficient
analytical sensitivity and the occurrence of unspecific agglu-
tination reactions, ELISA presents the advantage of a high
specificity . Unfortunately, ELISA is not applicable for
mass screening of the population at risk in sub-Saharan
Africa rural areas, as this technique requires large volumes
of pure water, pipettes, and many secondary antibodies and
conjugates that are not stable at ambient temperatures .
Besides, the test takes a few hours .
for T.b. rhodesiense infection in the field, where the same
tests used for T.b. gambiense are used with less accurate
results, and unfortunately, there is no promising laboratory
breakthrough to solve this problem. On the other hand,
powerful molecular techniques have been successfully tested
for the diagnosis of T.b. gambiense HAT. However, up to
now, many of these techniques still need to be modified and
adapted to field conditions in order to reach the patients.
The development of simple and standardized tests applicable
to field conditions from these findings is considered .
As previously stated and discussed in Section 2, the drugs
used to cure the HAT first stage poorly cross the BBB, and
other drugs, which are far more toxic, are used to cure stage
2 patients. Therefore, after HAT diagnosis, the disease stage
determination is a crucial step to decide the treatment to
4The Scientific World Journal
administer. After treatment, patients must be followed up to
early detect and cure the relapses.
3.1. Who Criteria. The commonly accepted criteria in
used in the field for HAT staging are from 1998 WHO
recommendations  modified in 2006 . According
to these recommendations, HAT stage 2 is marked in
the CSF by the presence of trypanosomes, by alterations of
the total protein level (different cutoffs have been proposed
and vary from 250 to 450mg/L), and by elevated white
blood cell (WBC) counts (cut-offs are Stage I < 5 cells/μL,
“intermediate” stage with 5–20 cells/μL, early Stage II at 20
cells/μL) [22, 38]. The study of cells in the CSF of HAT
patients for disease staging was justified by the postmortem
findings . However, WHO cut-off criteria seem to have
been decided arbitrarily, resulting in malfunction of these
criteria in the field . Overall, these criteria are debated
[7, 11, 17], and African trypanosomes are rarely found in
the CSF of patients, even in the late HAT stage 2 [32, 48].
For posttreatment followup, WHO recommends that
treated HAT patients be followed for up to 2 years before
a decision on treatment outcome can be taken . Treated
patients’ blood and CSF are to be examined every 6 months
[22, 43]. However, due to the generally low sensitivity of
the available parasite detection tests, a substantial number
of relapsing patients is not detected early and thus not
given treatment. This results in prolonged suffering and even
death of patients and also has major consequences at the
T.b. gambiense . Additionally, many patients are afraid of
lumbar puncture and as soon as they feel better, they cease
coming to follow-up appointments [17, 23].
Therefore, less invasiveness and better criteria for disease
staging are needed, considering the importance of such cri-
teria for the treatment and followup of patients. Less invasive
and sensitive diagnostic tools are also needed for disease
severity monitoring for relapses detection and management.
3.2.1. Infiltrating Inflammatory Cells. Knowledge about the
inflammatory cells infiltration and brain damage in HAT
originates mostly from animal studies, as only a few clinical
studies reporting observations on deceased HAT patients
have been published . In T.b. gambiense-infected vervet
monkeys, perivascular cuffing, meningitis, and encephalitis
have been described, with inflammatory infiltrate comprised
mononuclear cells, lymphocytes, plasma cells, and Mott cells
. In this model, as well as in T.b. rhodesiense-infected
mice and in T.b. gambiense-infected rats, trypanosomes
appeared to spread together with inflammatory cells, being
first located in the choroid plexus, then spreading to
the perivascular space and finally to the brain parenchyma,
resulting in a triphasic meningoencephalitic inflammatory
disease [51, 52]. The 3 phases which have been envisaged are
(i) a chronic meningitis with plasma cells, lymphocytes, and
monocytes in the subarachnoid and pial connective tissue,
(ii) a progressive neuroinflammation from the meninges to
the cerebral vessels entering the brain, and (iii) the develop-
ment of encephalitis.
Overall, the human post-mortem material examination
has revealed a pattern of neuroinflammation similar to that
observed in animal models though with some differences
in the severity of inflammatory reaction features .
The hallmark of CNS pathology in autopsies of HAT fatal
cases is a generalized meningoencephalitis with marked
cellular proliferation seen in the leptomeninges together
with diffuse perivascular infiltration of white matter with
lymphocytes, plasma cells and macrophages, and activated
adjacent parenchyma .
To enter the brain parenchyma, T.b. as well as WBC cross
the BBB. This physical barrier situated between the lumen
of the cerebral blood vessels and the brain parenchyma
is formed by tight junctions of the endothelial cells of
blood vessel walls surrounded by basement membrane and
astrocyte endfeet [54, 55]. To cross the BBB and enter
the brain parenchyma, leukocytes establish loose connec-
tions with endothelial cells via selectin-integrin interactions,
which allow them to roll along the endothelial cell barrier
with the flowing blood. Leukocyte transmigration occurs
in response to the presence of surface-bound luminal
chemokines following a chemotactic gradient. If these
chemokines are fixed by leukocyte chemokine receptors, sig-
naling pathways within the leukocyte are activated resulting
to high-affinity binding to the endothelial cell via adhesion
junction, and through that junction, they extend protru-
sions, sampling for abluminal chemokines. After crossing
the endothelial cell layer, leukocytes are sequestered in
the perivascular space between the endothelial cell base-
ment membrane and the parenchymal basement membrane.
For the completion of the transmigration into the brain
metalloproteinases is needed [56, 57].
In the presence of elevated amounts of the proin-
flammatory cytokine tumor necrosis factor-(TNF-)alpha,
the binding of leukocytes to cellular adhesion molecules
and their transmigration across the blood-CSF barrier are
increased . This cytokine level is high all over the course
of African trypanosomiasis, and this finding could explain at
least part of the observations made on the brains of deceased
HAT patients. On this basis, studies were recently performed
in the blood and CSF of T.b. gambiense HAT patients in
Angola and Gabon to determine the number and types
of leukocyte immunophenotypes present along the disease
course. From this studies emerged that the number of B cells
severity . Other studies, on basis of the investigation of
the CSF from T.b. gambiense patients at different stages of
as indicator of HAT stage and severity . Even if the
application of this approach would still require the invasive
and “frightening” lumbar puncture, B cells rosettes are
easily detected in field conditions  and would therefore
constitute a good replacement for WBC count.
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3.2.2. Inflammatory Mediators. The precise mechanisms by
which T.b. enter the brain and how this parasite and the
infiltrating inflammatory cells interact between them and
with resident cells to produce the alterations resulting in the
specific meningoencephalitis observed in African trypanoso-
miasis are still to be unraveled. However, the proinflam-
matory cytokine interferon- (IFN-) gamma is likely to play
a critical role for the traversal of the BBB by T.b. [61, 62].
Numerous studies in animal models indicate that the
expression of inflammatory mediators (cytokines, chemok-
ines, and adhesion molecules) change with the course of the
infection, with a central role played by the balance between
pro- and anti-inflammatory mediators in the outcome of
the disease (see Kristensson et al., 2010 for review). Findings
have pointed to an association between cytokine expression,
particularly IFN-gamma and TNF-alpha, and the onset
and development of the neuroinflammatory reaction. The
CSF levels of the chemokines CXCL-2, CCL-5, CCL-3, and
CCL-2 have been reported to increase in the brain early after
infection; the early source of these inflammatory mediators
appeared to be the brain resident cells astrocytes and
microglia, with T cells and macrophages taking the pro-duc-
suggests that the initial steps in the development of the neu-
roinflammatory disease are controlled from within the CNS.
Such factors may be responsible for initiating inflammatory
cell and T.b. infiltration to the brain parenchyma, that is, the
beginning of African trypanosomiasis stage 2 .
In contrast to the cytokine profiles derived from rodent
models, no significant changes in TNF-alpha or IFN-gamma
CSF concentrations were reported in humans [43, 64].
Such a discrepancy could reflect either divergences between
the cytokines present in the brain and the CSF, or variations
in the sensitivity of the assay systems used .
In terms of clinical data, a correlation between IFN-
gamma concentration in the plasma and disease progression
in the CNS has been shown in HAT patients in Uganda
(T.b. rhodesiense), but no significant changes were found in
CSF levels of TNF-alpha or IFN-gamma [64, 65]. In the CSF
of these patients were also found significant increases of
interleukin- (IL) 10 and IL-6 levels. In DR Congo T.b. gambi-
serum/CSF concentration quotients indicated an intrathecal
synthesis of IL-10 in 29% of patients [65, 66].
On the basis of the hypothesis that brain damage
and inflammation-related proteins could individually or
in combination indicate the CNS invasion by T.b., many
studies aiming at the determination of markers for efficient
in the CSF by proteomic analyses. CSF samples from T.b.
gambiense patients, diagnosed on the basis of CSF WBC
counts and presence of parasites, have been used to study the
levels of 3 brain damage-related proteins (H-FABP, GSTP-
1, overexpressed in post-mortem CSF, and S100b, marker
of BBB and neuronal damage) and 13 inflammation-related
proteins (IL-1-alpha, IL-1-beta, IL-6, IL-9, IL-10, G-CSF,
VEGF, IFN-gamma, TNF-alpha, CCL2, CCL4, CXCL8, and
CXCL10). The findings indicated that CXCL10 could distin-
guish stage 1 from stage 2 patients, with a sensitivity of 84%
and 100% specificity, and a panel characterized by CXCL10,
HAT stage 2 patients . These analyses were performed on
a relatively limited sample of patients from the same cohort,
and still are to be validated in a larger multicentric cohort,
but other experimental evidence from animal models and
HAT patients confirmed these findings [67, 68].
4.Treatment andVaccine Development
4.1. Presently Available Drugs. Four trypanocidal drugs are
mainly in use in the field: pentamidine and suramin,
which are efficient in the early stage of the disease, and
melarsoprol and eflornithine, which are efficient in the late
stage. The field drugs, particularly those used in the second
stage of the disease, have severe side effects and may even be
fatal [14, 32, 69, 70].
4.1.1. Suramin. Pioneering work of the German researcher
iology or Medicine in 1908, demonstrated that naphthalene
dyes, trypan red, and trypan blue have trypanocidal activity
due to selective accumulation by trypanosomes. Following
Ehrlich’s observations, suramin, a colorless polysulphonated
symmetrical naphthalene derivative drug, was developed in
the 1920s . This drug has also been used against the
immunodeficiency virus, and other human viruses, and
against different types of cancer have been performed [72,
3–7 days, over a 4-week period, is used to cure HAT [14, 69].
The trypanocidal action of suramin is still unclear, and
many hypotheses have been proposed. (i) Suramin could
impede uptake of serum proteins or inhibit endocytosis
and key enzymes in metabolic pathways such as glycolysis
thanks to its negative charge and the chemical properties
derived . Thus, suramin could act by the formation
of complexes with LDL impeding the receptor-mediated
uptake of LDL, carrier of cholesterol required for parasite
growth. (ii) Suramin could accumulate inside the lysosomes
and inhibit some key enzymes such as 3?-nucleotidase or
protein kinase (which both bind to the plasma membrane
of the trypanosome), acid phosphatase or acid pyrophos-
phatase (in the flagellar pocket), or phospholipase A1.
(iii) Suramin could also inhibit the high positive-charged
glycolytic enzymes located inside the glycosome on the
African trypanosomes [69, 75].
fatty acid and cholesterol, the development of resistance to
suramin in the field is unlikely considering the important
role of LDL in the growth and proliferation of these
parasites [69, 75]. However, reports of treatment failures
from foci of the Gambian form of HAT in the 1950s led
the use of this drug mainly for the Rhodesian form of HAT
. In veterinary use, resistance has been noted in some
of resistance are still to be unraveled.
6The Scientific World Journal
A considerable amount of suramin binds to serum
proteins, and consequently, the suramin half-life in serum
is very long (44–54 days in the study of Collins et al.,
1986). Although HAT regimens are considered short enough
to offer safety and tolerability, the US Food and Drug
Administration blocked the approval of suramin for use in
prostate cancer because of the adverse effects reported .
4.1.2. Pentamidine. Pentamidine (1,5-bis (4-amidi-phenox-
ypentane]) is a diamidine, that is, an aromatic diamine,
which has been used for several decades in the chemotherapy
of African trypanosomiasis, leishmaniasis, and against Pneu-
mocystis carinii pneumonia in acquired immunodeficiency
syndrome patients .
As for suramin, the pentamidine mode of trypanocidal
action remains uncertain. Overall, diamidines act directly
against the parasites independently of their physiological
action against the host, and the transport of these drugs
across the cell membrane is a necessary first step to antipar-
asitic action [69, 70]. The trypanosomes accumulate large
amounts of pentamidine via P2 aminopurine permease .
In trypanosomatids of the Leishmania species, close relatives
of trypanosomes, fluorescent analogues of pentamidine have
been shown to accumulate mostly in the mitochondria
resulting in the permanent damage of these organelles
and cell death [78, 79]. In addition, in Leishmania, the
pentamidine resistance correlates with a reduction in the
mitochondrial membrane potential [80, 81]. This is due
to the fact that pentamidine interacts electrostatically with
cellular polyanions, binding DNA including the kinetoplast.
This latter organelle is a characteristic of kinetoplastid flag-
ellates and is constituted by a unique intercatenated network
genome [82, 83]. However, whether the localization of
fluorescent analogues of pentamidine correlates with activity
is not certain, and, in addition, the mammal bloodstream
form of T.b. can survive kinetoplast DNA disintegration
A high-affinity and a low-affinity pentamidine trans-
uptake. These transporters explain, at least in part, the
efficacy of this drug also against melaminophenyl arsenical-
resistant parasites that lack the P2 transporter [70, 79]. A
retained activity of the P2 transporter has been shown in an
African trypanosome laboratory line selected for pentami-
dine resistance [69, 77]. Furthermore, lack of HAPT1 trans-
porter has been observed in another pentamidine resistant
line also lacking the P2 transporter . On this basis, it has
been suggested that the resistance to pentamidine may be
due to the lack of pentamidine transporters . However,
as these pentamidine-resistant lines displayed much reduced
development of resistance to pentamidine is associated with
substantial fitness costs, therefore rendering the propagation
of resistant lines in the field unlikely [14, 70].
4.1.3. Melarsoprol. The mechanism of action of the arsenical
compound melarsoprol has been recently reviewed [13, 86].
This drug is still the most widely used to cure the late stage
of HAT despite its extremely toxic side effects , as it is the
only drug effective in the second stage of the Rhodesian form
of HAT, and as it is far less expensive than the other drugs
used in the second stage of Gambian form of the disease [22,
The uptake of melarsoprol in the trypanosomes is
accomplished by purine transporters, as this drug acts as a
competing ligand for the purine site on the transport protein
. Purine transport is highly developed in trypanosomes
as they directly acquire nucleic acids from their hosts. The
trypanosomes lyse rapidly when exposed to melarsoprol
. T.b. thiol-containing enzymes (such as glycerol-3-
phosphate dehydrogenase) could be the targets of melarso-
prol, as it has been reported that trypanocidal analogues of
this drug (such as cymelarsan used to treat nagana) bind
strongly to these enzymes . Functional alterations of
these enzymes could underlie the lysis of trypanosomes, as
they lead to inhibition of glycolysis, and therefore to the loss
of ATP, although these cells seems to lyse before ATP supplies
are seriously depleted [14, 87].
The active metabolites of melarsoprol contain a trivalent
arsenic element with a markedly reactive arsenoxide group,
which confers the physicochemical ability of lipid solubility
that allows the passage of the drug across the BBB [89,
90]. In addition to this transport function, the arsenoxide
group probably mediates the killing of trypanosomes in
the cerebrospinal fluid (CSF). This is suggested by the fact
that modifications of the melarsoprol parent ring have a
significant impact on its trypanocidal action. The trivalent
derivatives of melarsoprol, such as melarsen oxide and
phenylarsine, are highly active even in relatively low con-
less active, and its nonarsenical chemical constituents are
completely inactive against T.b. [69, 88].
Melarsoprol was introduced to replace tryparsamide,
anotherarsenical,and thedrug regimens werenot supported
by pharmacokinetic studies . After recent assessment of
pharmacological properties and profile of melarsoprol, the
treatment schedule has been improved. Much of the drug
has been found to bind to plasma protein with a mean
serum half-life of active metabolite of 3.5–3.8h and a very
slow elimination time from the CSF with a half-life of 120h
. The drug regimen used nowadays is a standardized
10-day course with 2.2mg/kg once a day instead of 3 series
of 4 intravenous injections (at a dose of 3.6mg/kg), with
[32, 89]. This drug regimen reduces drastically the time of
exposure to melarsoprol but fails to show improvements of
the severe side effects of this drug, particularly the lethal
reactive encephalopathy .
In the field, failures of HAT treatment with melarsoprol
have reached 30% of the treated cases in several foci [21,
22]. Most parasites selected for resistance to melamine-
based arsenicals in the laboratory and several parasites
isolated from relapse cases in the field have been shown
to have lost the P2 aminopurine transporter [14, 87].
However, trypanosomes from which this transporter has
based arsenicals compared to wild-type cells . This
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suggests the existence of secondary routes of uptake and
indicates that the loss of P2 transporter must be coupled
with the loss of these secondary routes for high-level
resistance . Melarsoprol resistance has also been shown
in trypanosomes with ectopic overexpression of the tbmrpa
gene that encodes a P-glycoprotein type pump .
4.1.4. Eflornithine. Eflornithine (D,L-a-difluoromethylor-
nithine) is an analogue of the amino acid ornithine first
developed as a potential antineoplastic agent . This drug
is efficient against T.b. gambiense but not T.b. rhodesiense
Eflornithine has similar affinity for both the mammalian
and trypanosomal polyamine biosynthetic enzyme ornithine
decarboxylase (ODC) and acts as an inhibitor of this enzyme
. T.b. gambiense ODCs are degraded and replenished
much more slowly than in the mammalian counterpart, and
therefore, eflornithine deprives trypanosomes of polyamine
synthesis for a prolonged period compared with mammalian
cells. This polyamine biosynthesis inhibition is accompanied
by an increase in cellular levels of S-adenosyl methionine,
which causes inappropriate methylation of proteins, nucleic
acids, lipids, and other cell components [14, 95, 96]. At
variance with T.b. gambiense, T.b. rhodesiense present a rapid
turnover of that enzyme, rendering eflornithine noneffective
against this parasite . A diminution of trypanothione
levels was also observed after eflornithine treatment [14, 98],
and this may increase the susceptibility of T.b. gambiense to
oxidative stress and other immunological insults.
Eflornithine passive diffusion across the plasma mem-
brane was proposed to account for the uptake of eflornithine
in both bloodstream forms of T.b.  even if evidence from
genomic studies suggests the presence in T.b. of genes encod-
ing amino acid transporters which could carry eflornithine
[100, 101]. Doses beyond 100mg/kg of eflornithine given per
os fail to increase the drug level in the plasma, suggesting that
the drug is accumulated by a saturable transporter . It
is also probable that a transporter carries the drug across
the BBB, from where the “y system” (the more important
cationic amino acid transport system in mammals) takes
over [102, 103].
Little serum protein binding of eflornithine occurs,
and, accordingly, the mean half-life in plasma following
intravenous injection of eflornithine is about 3-h, with up to
Thus, the drug regimen is very fastidious, as large doses are
given by prolonged intravenous infusion [22, 32].
Eflornithine resistance of T.b. procyclic forms has been
shown to be related to a reduction of drug uptake [99, 105],
suggesting that resistance could be related to loss or changes
of eflornithine transport into cells.
4.2. New Combination Therapy and Drugs in Clinical Trial
Nifurtimox is a drug used to treat another trypanosomal
illness, Chagas disease or American trypanosomiasis caused
by T. cruzi. The organization “Medecins Sans Fronti` eres” has
conducted 2 sequential clinical drug-combination studies at
HAT treatment sites in northern Uganda from 2001 to 2004,
which have reported that NECT is highly effective and well
tolerated [76, 79]. NECT was added to the “WHO Essential
Medicines List for the treatment of second-stage Gambian
HAT” in April, 2009, on the basis of key advantages over the
previous therapeutic options such as high efficacy and good
safety profile consistently observed .
NECT is easier to administer, requires fewer human and
and currently stands as the most promising first-line treat-
ment for second-stage Gambian HAT . NECT requires
14 intravenous infusions of eflornithine over 7 days and oral
administration of nifurtimox 3 times per day for 10 days,
while eflornithine monotherapy requires 56 intravenous
infusions over 14 days [76, 106]. However, the training needs
not yet used eflornithine .
NECT has been suggested to be less susceptible to gen-
erate parasitic resistance, as this treatment strategy combines
two drugs with different modes of action .
4.2.2. Diamidines. Several thousands of diamidine deriva-
tives with a broad range of trypanocidal activity and sur-
prisingly diverse pharmacokinetic profiles have been devel-
oped . One of these derivatives approved by the US
FDA for the treatment of Pneumocystis jiroveci pneumonia,
pafuramidine (DB289), demonstrated equal efficacy and less
overt toxicity with/than pentamidine in a multicenter phase
3 trial involving 273 HAT patients.
Interestingly, some aza analogs of DB289 have shown
similar in vitro profiles against different T.b. strains, melar-
knockout strain (AT1KO) . Some of these compounds,
as DB75, show a higher trypanocidal activity , and
others as DB868 have been reported not only to kill the
trypanosomes in the peripheral organs and in the blood
compartment, but also, interestingly, to cross the BBB in
levels sufficient to kill trypanosomes in the HAT stage 2
mouse model, suggesting efficiency in both stages of the
infection [107, 109]. CPD-0802, a compound of this group,
is currently under consideration for clinical development for
stage 2 HAT .
4.2.3. Nitroheterocycles. The discovery in T.b. metabolism of
an unusual bacterial type 1 nitroreductase enzyme capable
of the reductive activation of nitro compounds, that is not
found in mammals , has “added impetus to the quest as
did the introduction of novel nitroheterocycles into clinical
trials for tuberculosis, anaerobic protozoan and helminth
infections” . Among the numerous compounds tested,
fexinidazole showed to be efficient with oral dosing in
the mouse model of stage 2 HAT, and the drug proved
be metabolized to trypanocidal sulphoxide and sulphone
metabolites. In 2009, fexinidazole entered the phase I clinical
trials which are presently ongoing .
4.3. Emerging Challenges for Vaccine Development. The
emerging challenges for the development of a vaccine
8The Scientific World Journal
against African trypanosomiasis were recently reviewed [29,
110], and in this section, they will be briefly discussed.
The cell surface of the procyclic and epimastigote forms
of T.b. (found in the fly) is covered with an invariant
glycoprotein coat composed of about 10 million copies of
two isoforms of a protein named procyclin. These isoforms,
named accordingly to their amino acid repeats in their C-
and GPEET-procyclin (which has 5-6 Gly-Pro-Glu-Glu-Thr
repeats followed by 3 EP repeats), [4, 111]. Both protein
113]. When epimastigotes differentiate into the metacyclic
form (the form inoculated by the fly), the EP-procyclin and
GPEET-procyclin coat is replaced by about 10 million copies
of a single VSG. Once in the host bloodstream, the parasite
keeps the metacyclic VSGs for up to 7 days and then switches
to the expression of nonmetacyclic VSGs [112, 114]. In
the bloodstream parasites, about 1,000 genes are coding for
VSGs , thanks to which African trypanosome species
their surface coat, sequentially expressing different forms
of VSG at a rate of 10−2to 10−7switches/doubling time
of 5−10h [4, 113, 116]. Such escape mechanism confirms
the adaptation of the parasite to its hosts and constitutes
the main difficulty for the development of a vaccine against
African trypanosomiasis and also because of the incomplete
understanding of the control and execution of this immune
evasion strategy in trypanosomes [114, 117, 118].
The VSG coat challenge has led to the question of
the development of a non-VSG-based vaccine. African try-
panosomes express numerous nonvariable surface antigens.
In the recent years, many non-VSG candidates have been
used for experimental vaccination schemes for trypanosomi-
asis; most reports prove promising, but not a single strategy
was effective enough for the development of an effective
vaccine . Of particular interest has been the flagellar
pocket, an organelle specialized in endocytosis and exocyto-
sis containing relatively well-conserved receptors , and
cytoskeleton proteins, as an interesting group of nonvariable
of all these strategies was suggested to be the fact that
immunization against these proteins might never result in
significant B cell memory-based protection in experimental
model systems that are characterized by an excessively high
parasite burden early on in infection, as most of the models
used up to know for vaccine development . In order
for a vaccine targeting trypanosomes to act, it should have
the ability to eliminate all circulating trypanosomes before
they trigger mechanisms of B cell memory suppression or
destruction . Thus, these parasites seem, up to now,
to have always been able to modulate the B cell memory
response in their advantage, impeding the B cell response
that aims to eliminate them, rendering further more difficult
the realization of a vaccine.
Interestingly, a positive note derives from research
attempting another approach: the development of vaccines
aimed to reduce T.b. transmission through immunization
against insect parasite stages which express an invariant
glycoprotein coat, that is, blocking the parasitemia onset in
the host, as the successful antitick vaccine . Several
antigens have been already proposed as candidates for
such experimental vaccination schemes and are being tested
5.The Potentialof MedicinalPlants in
5.1. Medicinal Plants as Alternative Drugs. Interest in higher
plant extracts exhibiting antimicrobial activity has increased
in recent years, and several reports on this subject have been
published. Indeed, the use of and search for drugs derived
from plants have accelerated in recent years [121–128],
whereby ethnopharmacologists, botanists, microbiologists,
and natural-product chemists are combing the earth for
phytochemicals and “leads” which could be developed
for the treatment of various ailments. WHO has esti-
mated that 80% of the population of developing countries
relies on traditional medicines, mostly plant drugs, for
their primary health care needs [129–132]. For instance,
the use of herbs and medicinal plant products has become
a mainstream phenomenon over the past two decades in
many countries, where herbs and phytomedicines (herbal
remedies) have become one of the fastest growing segments
in retail pharmacies and supermarkets [121, 128]. It is of
no denying that medicinal herbs now constitute the most
rapidly growing segment of the total US pharmaceuti-
cal market and are now used by approximately 20% of
the population [133, 134]. Available reports tend to show
that about 25% of all prescriptions sold in the US are from
natural products, while another 25% are from structural
modifications of a natural product [134, 135]. In other
reports [136, 137], it is proposed that 3 in 10 Americans
use botanical remedies in a given year giving rise to
a whole new industry referred to as “nutraceuticals” and
Indeed, it is clear from available literature that modern
pharmacopoeia still contains at least 25% drug derived from
plants, and many others, which are synthetic analogues,
built on prototype compounds isolated from plants [125,
135]. Despite the availability of different approaches for
the discovery of therapeuticals, natural plant products still
remain as one of the best reservoirs of new structural types.
Concurrently, many people in developing countries have
begun to turn to alternative therapies as cheap sources
of complex bioactive compounds and evidence of the
beneficial therapeutic effects of these medicinal herbs is
seen in their continued use [124, 127, 134, 139]. The
importance of medicine of natural product molecules lies
not only in their pharmacological or chemotherapeutic
effects, but also in their role as template molecules for the
production of new drug molecules. It is of no denying
that knowledge gained from the use of medicinal herbs
and their active ingredients has served as the foundation
for much of modern pharmacology, and many modern
drugs have their origin in ethnopharmacology. Additionally,
the development of modern chemistry has permitted the
isolation of chemicals from medicinal herbs which have
The Scientific World Journal9
served as drugs or starting materials for the synthesis of
many important commercially important drugs used today
[139, 140]. Drugs such as aspirin, digitalis, morphine, met-
formin, and quinine amongst others were all originally iso-
lated or synthesized from materials derived from plants
Medicinal plants, unlike pharmacological drugs, com-
monly have several chemicals working together catalyti-
cally and synergistically to produce a combined effect that
surpasses the total activity of the individual constituents.
The combined action of these substances increases the
activity of the main, medicinal constituent by speeding up
or slowing down its assimilation in the body. Secondary
substances from plant origins might increase the stability
of the active compound(s) or phytochemicals, minimize
the rate of undesired side effects, and have an additive,
potentiating, or antagonistic effect [125, 126].
With the exception of antimalarials and as mentioned
above, there are currently only four drugs approved to treat
HAT. However, eflornithine and pentamidine are ineffective
against sleeping sickness caused by T.b. rhodesiense. Treat-
ment with melarsoprol, the only generally effective first-line
drug, required lengthy parenteral administration and can
result in up to 10% mortality. Additionally, the toxicity and
the upsurge in the number of patients failing to respond
to melarsoprol because of drug resistance reflects the need
for discovery of new chemotherapeutic agents against HAT
. To this effect, the insufficiency of current therapies
for the treatment and management of trypanosomiasis,
combined with both a lack of trust in conventional medical
treatment and an inability of the economy to absorb the cost
of pharmaceuticals, have created a growing public interest in
alternative natural drugs from botanicals.
5.2. Phytotherapy for HAT. Drug-screening activities from
plants have started decades back, and an emerging number
of studies have now been developed and reported so far to
trypanosomiasis. The main aim has been geared towards
alternatives to conventional drugs with fewer side effects but
A plethora of studies has been conducted to investigate
the effect of some traditionally used medicinal plants in
alleviating the cellular changes in vivo produced during
the T.b. brucei infections of rats. Traditional knowledge
was the basis for the selection of plants, and one study
included Momordica balsamina pulp, Aloe vera pulp, Annona
On the basis of folk medicines, they were claimed to possess
antiprotozoal activity and alleviate one or many of the
clinical symptoms such as intermittent fever, immunosup-
pression, anemia, jaundice, and hepatomegaly commonly
associated with trypanosomiasis. Interestingly, it was found
that these plants had the potential in the management of
HAT due to the fact that T.b. brucei. Momordica balsamina,
and S. longipenduculata were found to possess the highest
potential, since they are able to control anemia by resisting
sudden drop in packed cell volume values .
In another study, it was showed that the extracts of
Hymenocardia acida stem bark exhibited significant try-
panocidal activity, whereas Gardenia erubescens and Lophira
lanceolata were effective at minimum inhibitory concentra-
tion (MIC) of 20mg/mL . Nigerian plants were also
evaluated in vitro for trypanocidal activity against T.b. brucei
and T. congolense at concentrations of 4mg/mL, 0.4mg/mL,
and 0.04mg/mL. It was found that extracts of Khaya sene-
galensis, Piliostigma reticulatum, Securidaca longepeduncu-
lata, and Terminalia avicennoides were strongly trypanocidal
to both organisms while extracts of Anchomanes difformis,
Cassytha spp, Lanneakerstingii,Parkia clappertioniana,Striga
spp, Adansonia digitata, and Prosopis africana were try-
panocidal to either T.b. brucei or T. congolense. Kigelia
africana, from Kenya, was also evaluated in vivo, and it was
found that the dichloromethane fruits extract of K. africana
tested at a dose of 2000mg/kg was effective, curing 60% of
the Swiss white mice that had previously been inoculated
with T.b. rhodesiense KETRI 3798 [145, 146].
the population of immune cells during a 28-day experimen-
tal T. brucei infection in rabbits. The result obtained showed
the absolute number of circulating lymphocytes followed
by a progressive decrease in total WBC and all WBC sub-
types (lymphocytes, monocytes, and granulocytes) although
the percentage of lymphocytes remained consistently higher
than normal throughout the study period. These changes
were consistent with the development of trypanosome-
induced immunosuppression in their mammalian host, and
interestingly, treatment with S. dulcis at a daily oral dose
of 25mg/Kg body weight was found to significantly reduce
the severity of the observed lesions when compared with
untreated infected animals. Thus, S. dulcis was classified as
a potential herb that had demonstrated significant potency
in protecting against the parasite-induced decrease in the
population of immunologically active cells .
From the above key investigations, it is clear that these
findings provide strong evidence of the potential beneficial
panosomiasis which could be subsequently developed into
a cost-effective alternative drug to complement treatment of
5.3. Possible Mechanism of Phytochemicals against Trypanoso-
that the plant parts differ significantly in their activity.
gest the susceptibility of the test microorganism to various
secondary metabolites present in these medicinal plants. In
general, discussions pertaining to anti-Trypanosoma agents
from plants center on plant secondary metabolites, that is,
nonubiquitous constituents with no known essential role
in the plant’s metabolism. However, it has been postulated
that bioactive plant secondary metabolites may play a role
in chemical defense mechanisms and are likely molecules for
the antiparasitic agents in these plants [125, 126].
Recently, it has been postulated that the enormous diver-
10The Scientific World Journal
products but specialized secondary metabolites involved in
the relationship of the organism with the environment,
for example, as attractants of pollinators, signal products,
defensive substances against predators and parasites, or in
resistance against pests and diseases . Indeed, the com-
position of these secondary metabolites in turn varies from
species to species, climatic conditions, and the physiological
state of developments of the endemic plants . Available
reports tend to show that alkaloids and flavonoids are the
responsible compounds for the antimicrobial activities, and
anti-Trypanosoma in higher plants . Moreover, it is
also claimed that secondary metabolites such as tannins and
other compounds of phenolic nature are classified as active
antimicrobial compounds [150, 151].
Nonetheless, several investigations tend to suggest that
it is often difficult to speculate and decipher the exact
mode of action by which these plants extracts exhibit their
trypanocidal action. Indeed, the possible mechanisms by
which these plants extracts and phytochemicals therein
carry out this role remain a subject of great speculations
and debate in the scientific community. Several possible
mechanisms working separately or in concert may account
for the observed effect .
In one study, it was suggested that different phyto-
compounds could be responsible and operate in a syn-
ergistic effect for the observed antitrypanocidal activities.
Interestingly, preliminary phytochemical screening of potent
plants against trypanosome showed the presence of biolog-
ical known active compounds such as saponins, tannins,
flavonoids, and alkaloids in the crude plant extracts tested.
Several authors have also identified or isolated tannins and
phenolic compounds, flavonoids, and alkaloids in plants that
showed significant trypanocidal activities .
Accumulated evidence also suggest that many natural
products exhibit their trypanocidal activity by virtue of
their interference with the redox balance of the parasites
acting either on the respiratory chain or on the cellular
defenses against oxidative stress. For instance, the observed
trypanocidal activity of K. africana extract was justified due
to the increase of oxygen consumption and stimulation
of hydrogen peroxide production in the protozoan cell.
Trypanosoma do not have the same biochemical mecha-
nism as mammalian cells for dealing with excess peroxide
and consequent oxygen free radicals . Furthermore,
it is proposed that natural products possess structures
capable of generating radicals that may cause peroxidative
damage to trypanothione reductase that is very sensitive
to alterations in redox balance. It is also known that
some agents act by binding with the kinetoplast DNA of
On the other hand, the result of  has clearly
indicated that different solvent extracts of the same plant
may exhibit different trypanocidal activity just as extracts of
different parts of the same plants. Therefore, the statement
that a plant is trypanocidal or not should be taken within
the context of the solvent used and the parts investigated.
On the other hand, out of the 40 plant extracts tested
by [154, 155], the dichloromethane extract from stem
bark of Warburgia salutaris (claimed to be used against
many pathologies in many parts of Africa) was found
to exhibit the most potent trypanocidal activity. The try-
panocidal activity was suggested to be due to the drimane
sesquiterpenoids (warburganal and polygodial). Concerning
the mechanism, it was proposed that the two sesquiterpene
aldehydes, warburganal and polygodial, formed covalent
bonds with amino groups of proteins and affect a vast
number of cellular activities. In another groundbreaking
in vitro study, the authors were able to isolate the pregnane
glycosides from genus Caralluma (C. Penicillata, C. tubercu-
lata, and C. russelliana) and evaluated for the trypanocidal
activity. It was found that the penicilloside E to possess
the highest anti-Trypanosoma activity followed by caratu-
berside C, which exhibited the highest selectivity index
Studies have shown that it is probable that the etiology of
trypanosome-induced leucopenia in rabbit may be similar to
the case with trypanosome-induced anemia. There has been
striking indications that the onset of anemia in HAT may
be strongly related to disruption of erythrocyte membrane
caused directly by parasite attack on red cells. It has also
been suggested that products secreted by the parasite may
play a significant role in the disruption of red cell mem-
brane. Reduction in red cell membrane sialoglycoprotein
secondary to elevated activity of plasma sialidases promotes
the rapid destruction of erythrocytes. A role for parasite
and macrophage-derived free radicals and proteases in the
pathogenesis of trypanosome-induced anemia has also been
postulated . The possibility that S. dulcis or certain
components of the herb may help stabilize the membrane
of blood cells cannot be outrightly dismissed. Specifically,
it is not out of place to suggest that the antioxidant or
free radical scavenging properties of S. dulcis may play
vital roles in this regard especially against the backdrop of
the role of free radicals in the pathogenesis of T. brucei
infection and also probable that increased production of
blood cells helps in replenishing of these cells. In the
absence of any evidence of possible trypanocidal activity
for the herb, it does not seem an attractive option to
speculate that the higher level of immunological cells in
treated animals could be due to the destruction of the
parasite by agents native to the plant . On the other
hand,  have showed that Psidium guajava leaf extract
has trypanocidal properties and has attributed these effects
in parts to the broad antimicrobial and iron chelating
activity of flavonoids and tannins, respectively. They have
also proposed that iron chelation is an effective way of
killing trypanosomes and the prime target is the enzyme
ribonucleotide reductase whose activity is central to DNA
Moreover, a plant with high in vitro trypanocidal activity
may have no in vivo activity and vice versa because of
peculiarities in the metabolic disposition of the plant’s
chemical constituents. Therefore, plants found to be active
in the above-mentioned investigations must be tested in vivo
and tested clinically before a definite statement can be made
on their trypanocidal potentials .
The Scientific World Journal 11
Once neglected, HAT has returned in the center of
the attentions of the scientific community, resulting in
the recent reversal of the trend of cases which was increasing.
However, as illustrated in Figure 1, studies have shown that
in the last 30 years, HAT cases occurred more often in
countries with conflict, high political terror, or civil war,
with an interval as long as 10 years between the start of
conflict events and a peak in incidence , and considering
that unfortunately, these events still are current in HAT
endemic regions, the risk of future epidemics is consider-
able. Fortunately, a new approach for vaccine development
targeting the insect parasite stages is being tested and bear
higher chances of success than the precedent approaches. In
addition, powerful molecular tools analyzing inflammatory
mediators are also proving very efficient for HAT diagnos-
tic staging and followup. Therefore, even if in the field,
the problem of proper diagnostic and staging, together with
the posttreatment followup remain the adaptation of such
molecular techniques to field conditions that will solve these
The research re-engagement also has produced promis-
ing antitrypanocidal molecules from the diamidine and
nitroheterocycle pharmacological classes which are under
further tests and considered for clinical trials. Prodrugs able
to cure both phases may even have been found. Overall,
developing new drugs to replace the very toxic ones still in
use is crucial, together with more reliable tools for disease
staging and treatment followup. In the field, even if only one
drug, eflornithine, has been developed in the last 50 years,
a new combination therapy involving that drug and another
one developed for Chagas disease, nifurtimox, is proving a
good replacementforthehighlytoxic melarsoprol whichwas
previously the exclusive drug able to cure stage 2 patients.
But this combination of antitrypanocidal drugs appears to
be efficient only against T. gambiense, leaving the treatment
of T. rhodesiense stage 2 patients to the latter arsenic-derived
drug. To this effect, the active principles of traditional
pharmacopoeia and medicinal plants of African countries
have been scrutinized over the past decade as prospective
alternative trypanocides due to less toxicity and side effects.
Indeed, the recent quest for novel anti-HAT pharmacophore
has been geared mainly towards traditional medicines,
local knowledge, and ethnopharmacology. Phytotherapies,
besides their traditional and holistic values, also hold great
public and medical interest worldwide as cheap sources of
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