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Artemisia annua : A New Version of a Traditional Tea under Randomized, Controlled Clinical Trial for the Treatment of Malaria

Advances in Bioscience and Biotechnology, 2016, 7, 545-563
ISSN Online: 2156-8502
ISSN Print: 2156-8456
DOI: 10.4236/abb.2016.712049 December 12, 2016
Artemisia annua: A New Version of a Traditional
Tea under Randomized, Controlled Clinical Trial
for the Treatment of Malaria
Pedro Melillo de Magalhães1, Glyn Mara Figueira1, José Maria de Souza2,
Ana Maria Revorêdo Ventura2, Maria Deise de Oliveira Ohnishi2, Darci Rodrigues da Silva2,
Luiz Alberto Gonçalves Lobo2, Fabrício Bezerra Eleres2, Rosana Libonati3, Merlin Willcox4,
Eloisa Cavassani Pimentel5
1University of Campinas, Campinas, Brazil
2Evandro Chagas Institute, Belém, Brazil
3Federal University of Pará, Belém, Brazil
4University of Oxford (Research Initiative for Traditional Antimalarial Methods)
Oxford, UK
5Campinas Municipal Centre of Health, Campinas, Brazil
The traditional antimalarial tea
Artemisia annua
, indicated for cent
ries in China to treat fevers, is
again arousing interest for the treatment of malaria
due to improvements attained in the plant composition by a few Institutions thro
ughout the world, including the State University of Campinas (UNICAMP), Brazil,
increasing its principal component by more than 100 times as from standard vari
ties, giving 1% in artemisinin and an expressive biomass yield such as 2 tons of dried
Clinical trials carried out with this material in African countries have
proven its therapeutic potential for a new generation of Artemisia tea in the trea
ment of falciparum malaria. In addition to artemisinin, recent studies have identified
and quantified other compounds present in the crude extract and characterized their
contributions to the anti-malarial efficacy, including their action against chloroquine
resistant strains. The majority of the clinical trials carried out with Artemisia tea in
African countries have shown that the control of the parasitaemia is efficient in the
initial treatment period, but few trials have followed the patients up to the 28th
This first clinical trial carried out in Brazil with the
A. annua
infusion, after toxic
logical trials that defined the safety of this form of medication.
The ther
peutic efficacy of the tea was measured in patients with falciparum malaria
over 28
days, comparing it with the current first-line treatment namely artemether-lume
fantrine (Coartem®). The trial was carried out in controlled groups according to offi-
How to cite this paper:
de Magalhães
.M., Figueira, G.M., de Souza, J.M., Ven-
, A.M.R., de Oliveira Ohnishi, M.D.,
, D.R., Lobo, L.A.G., Eleres, F.B., Li-
bonati, R
., Willcox, M. and Pimentel, E.C.
Artemisia annua
: A New Version of
Traditional Tea under Randomized, Con-
trolled Clinical Trial for the Treatment of
Advances in Bioscience and Bi
, 545-563.
October 10, 2016
December 9, 2016
December 12, 2016
Copyright © 201
6 by authors and
Research Publishing Inc.
This work is licensed under the Creative
Commons Attribution International
License (CC BY
Open Access
P. M. de Magalhães et al.
cial protocol approved by the National Ethics in Research Committee (CONEP: 77/
2011) and a rigorous control of the 17 patients with non-
serious cases of falciparum
malaria, recruited in the following three municipalities of the State of Pará, Brazil:
Tucuruí, Goianésia do Pará and Anajás. The
tea group
received the infusion pr
pared in the proportion of 1.25 g of dry leaves of the variety CPQBA with 1% artem
sinin for 250 mL of just boiled water, taken every 6 hours for 7 days, giving a total of
approximately 175 mg of artemisinin, whilst the artemether-lumefantrine (Coar
group received a total of 525 mg of artemisinin equivalent to artemether.
The parasitaemia by the tea treatment became negative in the firs
t days, even though
it was administered with a dose that was one third of the recommended dose of a
temisinin. However, as in the case artemisinin or artesunate
monotherapies, 57.1% of
the patients treated with the
A. annua
tea presented type I resistance, with a return of
the parasitaemia around the 14th or 21st
day. The other patients in the tea group
showed type II/III resistance without manifestation of any serious signs or sym
toms. In these cases, according to the protocol, the patients were redirected for trea
ment with artemether-lumefantrin (Coartem®) with subsequent negativity of the p
The fact that the efficacy of the tea with 1/3 of the dose of a
temisinin was similar to that of the full
dose of this medication infers that other
compounds present in the crude extract, probably flavonoids,
had contributed to the
negativity of the parasitaemia at the start of the treatment. Considering that the pos
tive control group, where the compound derived from artemisinin (artemether) was
associated with another antimalarial agent (lumefantrine), presented excellent eff
cacy throughout the entire control of the cure, future trials with the
A. annua
should use the same strategy of association with another antimalarial agent, prefer
bly from
A. annua
itself, in order to extend its therapeutic
action during the whole
control period. The
Artemisia annua
tea in the form standardized and used in this
research, should not substitute the most efficient treatment, but could be considered
as an emergency therapeutic resource in the first hours of symp
toms as a function of
its availability, anti-
inflammatory action and lack of side effects. Other regimes and
standardizations deserve investigation, mainly those with a high content of artea
nuin B, as occurs in the initial cultivation phase.
Malaria, Herbal Medicine,
Artemisia annua
, Clinical Trial
1. Introduction
The species
Artemisia annua
L., Asteraceae, native of Asia, has been indicated since an-
cient times as an anti-fever agent, and was described by Ge Hong in 340 A.D. in his
Manual of Prescriptions for Emergences [1] (Zhou Hou Bei Ji Fang). The traditional
form of preparation shows variations, both infusions of the leaves in hot water and
their immersion in water at room temperature being cited. In the 1970s, based on
prescriptions from Traditional Chinese Medicine, the Chinese researcher Tu Youyou
P. M. de Magalhães et al.
(Nobel Prize for Medicine, 2015) isolated the molecule of artemisinin from
A. annua
leaves [2], and this became the safest and most efficient chemical structure discovered
to date to combat the parasite
, causer of malaria. Artemisinin is a sesqui-
terpene lactone containing an endoperoxide group (Figure 1), which is decisive in the
control of the protozoa
, a parasite which infects the red blood corpus-
As from this discovery, the pharmaceutical industry dedicated itself to the develop-
ment of antimalarial agents containing pure artemisinin extracted from
A. annua
and eventually developed more stable and efficient artemisinin derivatives by semi-
synthesis, such as sodium artesunate and artemether. The search for the base molecule,
artemisinin, led to genetic selection projects aimed at increasing the content of the ac-
tive principle in the leaves and per area, with a view to satisfying the demand and im-
proving industrial extraction, isolation and purification yields. Throughout the world,
the following two institutions were pioneers in this agronomical development, pro-
moting, in addition to hybrids and populations 100 times richer in artemisinin in rela-
tion to the original populations, the necessary technology for the cultivation and post-
harvest processing of the species: Mediplant in Switzerland and the State University of
Campinas (UNICAMP) in Brazil. In addition, some important research scientists, such
as John Laughlin (Australia), Jorge Ferreira (USA), Pierre Lutgen (Luxemburg) and
James Simon (USA) also acted in the agro-technology of
A. annua
resulting in impor-
tant contributions. More recently the University of York (UK) developed plants even
richer in artemisinin. Although
A. annua
is the only herbal raw material for the pro-
duction of artemisinin, significant technological advances have been made by the Uni-
versity of California, Amyris, USA for the total synthesis of the molecule as from arte-
misininic acid, which is produced by yeasts [3]. However, it has been estimated that
there will always be a market for both sources of the molecule, since the plant manu-
factures other compounds of interest in malarial therapy.
In the use of artemisinin or its derivatives in malaria therapy, which are also efficient
in the control of
P. falciparum
strains resistant to the antimalarial agents previously
used, the fact that the parasite could also acquire resistance to artemisinin is of concern.
Figure 1. Artemisinin: the most efficient molecule against
P. falciparum.
P. M. de Magalhães et al.
This could occur either by way of mutant genes that could acquire tolerance, or by a
population constituted of artemisinin-resistant genotypes resulting from selection by
the antimalarial agent itself. These two processes are present in malaria therapy, and
inexorably promote, with time, a resistant population to a determined antimalarial
agent. However, in the case of artemisinin, these processes occur with greater difficulty
due to the mode of action of the molecule, which acts in a physicochemical way on the
. In fact, artemisinin is one of those rare molecules found in nature that
have an endo-peroxide bridge in their structure with a double bond between oxygens,
which, when broken, produces a sort of “micro-explosion” capable of destroying the
membrane of a red blood corpuscle (RBC) parasitized by
. However, this
“micro-explosion” that destroys both the RBC and the parasite, only occurs around in-
fected RBC, since these produce a reducing ambient on their surface which causes the
double bond of the peroxide bridge of the artemisinin to break. It is an extremely selec-
tive mechanism, since the artemisinin in the blood stream will get together with all the
RBC due to its affinity for iron, but will only destroy infected ones. As a consequence,
will also be destroyed since its membrane will be broken. However
even with this mechanism in place there is the possibility that some infected RBC will
not be destroyed, either because they are resistant to the physicochemical effect of ar-
temisinin, or because they are surrounded by other healthy RBC that neutralize the re-
ducing ambient, forming agglomerates known as “rosettes”. Thus in the case of mono-
therapy with artemisinin or its derivatives the threat exists of the development of pop-
ulations resistant to these pure antimalarial agents due to the selective process and not
because they acquired resistance. The action of artemisinin or even its derivative Arte-
sunate when used in monotherapy, was shown not to be efficient throughout the whole
cure period, that is, the phenomenon of recrudescence occurred, signaling the danger
of resistance, since tolerant or resistant genes could be present in the parasitic popula-
tion. However this is not the only hypothesis for the occurrence of recrudescence, since
the phenomenon can occur by other means, such as: the artemisinin was already de-
graded before acting against all the parasites; that some parasites were protected by the
action of the antimalarial agent in microcirculation cells, or; by red blood cells adhered
one to another forming structures known as rosettes, protecting infected RBC and pre-
venting the action of artemisinin or its derivatives on its membranes, as mentioned ear-
lier. As a function of this behavior and aiming to prolong the longevity of the best an-
timalarial therapeutic agent to date, the World Health Organization (WHO) preconized
the use of
A. annua
antimalarial preparations always in association with other antima-
larial agents with distinct action modes and/or with a longer half-life in the blood stream
[4]. Thus the product Coartem (arthemeter + lumefantrine) and other associated anti-
malarial agents (ACTs-Artesunate Combined Treatment) have been on the market for
more than 10 years, with high efficacy and safety.
Although the registered ACTs show known efficiency and safety, they are not always
available in the endemic regions of poor countries where mortality is still to the order
of 1 million people per year. In fact, for the treatment of malaria, apart from efficiency
P. M. de Magalhães et al.
and safety, availability of the medication is fundamental, which, for its part, involves
adhesion, distribution, price, and governmental policies.
These fundamental characteristics for the efficacy of an antimalarial agent are present
in the
A. annua
tea, including the aspect of association, since the artemisinin is extracted
from the trichomes of the leaves together with other molecules that have antimalarial
action, or, at least, specific synergic action with the artemisinin by various mechanisms,
such as: anti-inflammatory action [5]; more efficient transport through the intestinal
barrier [6]; or an increase in susceptibility of
by way of arteannuin B.
However, one cannot affirm if the
A. annua
tea favors or avoids the process of resis-
tance to artemisinin. Nevertheless, is reinforced the hypothesis that the synergism be-
tween artemisinin and certain other compounds present in the
A. annua
tea make the
development of resistance difficult, even though the total amount of artemisinin admi-
nistered in the treatment with the infusion is below that that was recommended for the
treatment with pure artemisinin. During the process for the commercial development
of antimalarial agents from isolated and purified artemisinin, the other substances
produced by the plant are discarded. However, recent studies have reevaluated the
composition of the crude extract, this time using selected plants, and identified and
quantified the principal components and their antimalarial, antagonistic or synergistic
action with artemisinin. Some compounds, such as arteannuin B, show important syn-
ergism with the artemisinin, improving its efficiency, including that against chloro-
P. falciparum
strains [7] [8]. The molecule arteannuin B is the precur-
sor of artemisinin.
The present clinical study englobes earlier multidisciplinary research carried out over
25 years with the species at CPQBA-UNICAMP, since it evaluates the maximum finali-
ty, which is: the therapeutic efficacy of the
A. annua
infusion prepared with the leaves
of an artemisinin-rich genotype and in combination with other compounds of interest
in the plant. When a medication reaches the phase of clinical research, be it synthetic or
of natural origin, this opens the opportunity for a complete evaluation, including even
adjustments to the dosage, formulation and therapeutic regime. It is hence a decisive
step in the improvement of its use, and in this case, represents an important step for
other plants of the Brazilian flora to be evaluated. Without doubt, the tea obtained from
the variety UNICAMP-1, with 1% artemisinin, is highly distinct from that prepared with
the leaves from wild plants, where the artemisinin content was to the order of 0.005% -
The idea to test the efficiency of the tea from an improved
A. annua
plant was born
in 1995, when Magalhães
et al
. showed that the infusion extracted approximately 60%
of the total artemisinin from the leaves [9]. The first institution to vulgarize this thera-
peutic form was the German NGO ANAMED-Action for Natural Medicine, as from
promising results obtained in various African countries since 1996. Subsequent clinical
trials carried out in African countries demonstrated the therapeutic potential of Arte-
misia tea, despite the occurrence of some cases of recrudescence which can be partly
explained by the short half-life of artemisinin in the plasma [10] [11] [12]. Apart from
P. M. de Magalhães et al.
ANAMED, other European non-governmental organizations acting in African coun-
tries started recommending the use of
A. annua
infusions, gathering together successful
cases of the treatment of falciparum malaria with the tea made from
A. annua
including: the ICEI-Istituto Cooperazione Economica Internazionale, and IFBV-Tech-
niques Durables et Solidaires. In recent years, renewed scientific interest has been
shown in the pharmacological potential of the tea made from genotypes of
A. annua
containing high artemisinin contents, as related to malaria [13] [14] [15] and other in-
fectious-parasitic diseases, such as toxoplasmosis, schistosomiasis and also anticancer
action [16] [17] [18] besides anti-HIV [19]. The anti-inflammatory action detected by
Prof. Yves-Jaques Schneider’s group at the University of Louvain-la-Neuve (Belgium)
and the facilitated passage of artemisinin through the intestinal barrier when
A. annua
tea is taken, open new horizons, demonstrating that compounds of important pharma-
cological action for malaria, such as the anti-inflammatory action, are present in the
tea. However, many of these earlier results were not conclusive with respect to the real
efficacy of the tea from new
A. annua
genotypes in the treatment of human malaria,
since they did not follow the official WHO protocol for the control of the patients as
done in this clinical research. It seemed to us to be fundamental for scientists involved
in therapies for malaria, to carry out a clinical trial within the criteria accepted by the
scientific community, and rigorously following the guidelines of the World Health Or-
ganization for the evaluation of antimalarial medication. Thus we did not start from the
pretext that the tea could be more efficient than the therapeutic option currently in first
place, Coartem, but aimed to evaluate the efficiency of the infusion in relation to Coar-
tem. In agreement with this posture was the fact that there was absolutely no commer-
cial interest in the
A. annua
2. Material and Methods
2.1. Raw Material
A. annua
variety CPQBA-UNICAMP was cultivated in Paulínia-SP-Brazil, Lat.
22˚48'02.38''S, long. 47˚06'43.10''W, alt. 612 m, irrigated by spraying and the weeds
controlled by manual weeding until the disappearance of the lines between the plants
due to their development. The soil in the experimental area is classified as typical clayey
eutroferric red latosol. The seedlings were formed in sleeves under screening with 50%
interception in the period between August and September in the years 2009, 2010 and
2011, and cultivated in the field for approximately 4 months as from October of the
respective years, with a spacing of 0.5 × 1.0 m. Cultivation was carried out during the 3
years of the project aimed at always treating the recruited patients with new leaves,
within the stipulated shelf life of 12 months. With the objective of always obtaining raw
material with the highest possible artemisinin content, harvesting was done in the state
preceding flowering and only the top third of the plant was harvested, resulting in a ar-
temisinin content between 1.0% and 1.1% [20].The leaves were sun-dried with periodic
turning over, followed by manual separation of the stalks and thick twigs. All the
batches of
A. annua
used in the project were cultivated at CPQBA-UNICAMP follow-
P. M. de Magalhães et al.
ing the same cultivation and drying procedures and their artemisinin content remained
in the range from 0.9% to 1.1% in relation to the dry weight of the leaves harvested
(Figure 2).Thus the final raw material consisted only of leaves, and was sent to Fiocruz-
Far manguinhos, where it was ground and packed into individual sachets of aluminized
paper, each containing exactly 1.25 g of dried ground leaves (Figure 3).
2.2. Protocol and FCTC
The proposal to carry out the clinical phase II trial in Brazil was approved in the Pro-
nex-Rede Malaria note in 2009, the study being financed exclusively by the Brazilian
Government Organ CNPq-MS and FAPESP, as well as the support of the following in-
stitutions: State University of Campinas, Farmanguinhos, Campinas Health Secretariat,
the Tucuruí, Anajás and Goianésia hospitals in the State of Pará, Brazil, and, principal-
ly, the Evandro Chagas Institute in Belém do Pará, Brazil. The protocol for the clinical
trial, the Free and Clarified Term of Consent (FCTC) and the questionnaire used in the
Figure 2. Manual harvest and early drier of
A. annua
leaves at CPQB-
Figure 3. Aluminized sachets, exclusively prepared to this clinical trial,
each containing 1.25 g dried
A. annua
leaves with 1, 0% artemisinin.
P. M. de Magalhães et al.
patient interviews, were all approved by the Ethics in Research Committee of the Evan-
dro Chagas Institute and by the National Committee for Ethics in Research (CONEP),
n 77/2011.
The clinical research protocol and the Free and Clarified Term of Consent were ela-
borated rigorously following the recommendations found in the WHO guidelines,
number 05.1 of 2005, entitled “Assessment and monitoring of antimalarial drug efficacy
for the treatment of uncomplicated falciparum malaria”, with adaptations and CONSORT
statement [21]. Based on previous research carried out by Mueller
et al
. (2000) and
et al
. (2004) which showed the efficacy of
A. annua
tea to be about 74% in the
therapy of falciparum malaria, the number of patients necessary was calculated as being
two unilateral samples of 61 patients. This would give statistical certainty to demon-
strate differences with 80% of power at 5% of significance, calculated by the proportion
test. Considering an estimated a rate of loss of 10% during the procedure, the number
of patients in each sample (Coartem and tea) was defined as 70 (Total number of pa-
tients = 140). The research was directed at uncomplicated cases of malaria in patients
infected by
P. falciparum
. Recovery of the patients with Coartem was planned in the
case of any eventual therapeutic failure or resistance of any type, as also specific medi-
cal attention in these cases.
2.3. Inclusion and Exclusion Criteria
The patients were recruited according to the following inclusion criteria: age between
18 and 50, weight between 40 and 80 Kg, parasitaemia below 50,000 asexual forms/mm3
of blood with no dysfunction of vital organs, absence of other serious concomitant
diseases, and a declaration of consent. The exclusion criteria were defined according to
the presence of, at least, one of the following danger signals: incapacity to eat or drink,
more than 2 episodes of vomiting in the last 24 hours, unconsciousness, showing the
incapacity to sit down or stand up without help, parasitaemia above 50,000/mm3 blood,
or presenting severe malaria as defined by the WHO criteria: Severe malaria is charac-
terized by presenting: alterations in respiratory rate (respiratory rate above 20 and the
use of accessory muscles), jaundice (clinically diagnosed), clinically severe anemia, Hb
< 5 g/dl, or evidence of paleness, alterations in respiratory function, tachycardia, high
blood pressure during the exams, presenting spontaneous mouth or gum bleeding, sys-
tolic blood pressure below 80 mm Hg with signs of low peripheral perfusion, and being
incapable of knowing where he/she is or give an appropriate verbal response to a pain-
ful stimulus or suffer a generalized convulsion in the previous 30 minutes. The exclu-
sion criteria also include: being pregnant as determined by the B-HCG-urine test, being
allergic to artemisinin and/or lumefantrine, and having used an antimalarial agent in
the last 20 days.
2.4. Recruitment and Logistics (Epidemiological Survey—External
Monitoring; Adjustment of Criteria, Home Care, Changing
Municipalities and Changes in Tea Preparation)
The location chosen for installation of the research was defined considering: recent
P. M. de Magalhães et al.
epidemiological history, profile of the patients (avoiding gold digging regions since these
patients are difficult to control), the infrastructure of the local hospital, and access to
allow assessment by health professionals from the Evandro Chagas Institute in Belém-
PA-Brazil. The first region chosen according to these criteria was the municipality of
Tucuruí-PA. By way of complex logistics, the following were installed in the Tucur
Regional Hospital for use in the project: a microscope, a nurse, and a health agent/
driver, all from the Evandro Chagas Institute (IEC). Periodic visits were also made by
doctors from the IEC, responsible for all the medical-scientific activities of the project
and by the project coordinator and external assessors. The municipality of Tucuruí has
4 public health clinics, and, on agreeing to take part in the research, the patients re-
cruited in these clinics signed the FCTC and were sent to the Tucuruí Regional Hospital
where they were hospitalized throughout the whole treatment (7 days) independent of
the randomly chosen treatment. Control of the cure and parasitaemia was carried out
after 14, 21 and 28 days. In addition to parasitaemia, the control of the patients in the
two groups evaluated the biochemical parameters on days D-0, D-3, D-7, D-21 and
D-28. The laboratory analyses were: CBC (red blood corpuscles, platelets, leucocytes,
segmented cells, lymphocytes, eosinophils, monocytes and reticulocytes); coagulation
(bleeding time, coagulation time, coagulum retraction, prothrombin time, prothrombin
activity and thromboplastin time); aspartate aminotransferase (AST), alanine amino-
transferase (ALT), urea, creatine, uric acid, glycaemia, high sensitive C-reactive protein
(Hs-CRP), blood group and urine (volume).
2.5. Preparation of the Treatments and Follow-Up of the Patients
In the majority of cases the patients were diagnosed, treated and controlled in the Tu-
curuí Hospital, where they were hospitalized for 7 days. In the other recruitment loca-
tions, Goianésia do Pará and Anajás, the initial diagnosis was done in the hospital,
whereas treatment and control were done at home. A temperature-controlled room was
made available in each of the hospitals (Tucuruí, Goianésia do Pará and Anajás) for
storage of the
A. annua
tea sachets, for the documentation of the patients, and, for
preparation of the treatments according to the experimental protocol. Every 6 hours for
7 days, 300 mL of filtered water was heated to boiling point in a stainless steel recipient,
the recipient removed from the heat, and the dry
A. annua
leaves added, stirred briefly
and then left to rest for 15 minutes in a covered cup. The tea was then filtered and given
to the patient to drink. The members of the control group, who were treated with
Coartem, followed the dosage and therapeutic regime of this medication, and were also
hospitalized for 7 days for a comparative accompaniment involving the use of the same
laboratory analyses for the two groups, as well as using the same accommodation and
catering program. However, the patients recruited in Anajás received their
A. annua
prepared in a different way: after reaching boiling point, the leaves were added and
boiled for 2 minutes before removing from the heat and leaving to rest at room tem-
perature for 15 minutes. This alteration was based on information that the amount of
artemisinin extracted was significantly greater when the leaves were boiled for 2 mi-
P. M. de Magalhães et al.
nutes [22].
3. Results
3.1. Raw Material
The batches were submitted to an analysis for microbial contamination during storage,
but were all considered innocuous [23]. This infers that the high camphor content of
the essential oil and the low moisture content of the stored leaves inhibited microbial
3.2 Therapeutic Efficacy
The main parameter followed in the therapeutic treatment of malaria was the monitor-
ing of parasitaemia, together with a clinical follow-up of the patient.
3.2.1. Parasitaemia Results
As shown in Table 1, parasite clearance by Coartem was excellent and all the patients
showed negative results between the second and third days of treatment, with no oc-
currence of recrudescence up to the 28th day. This justifies the comparative proposal of
this research, using the most efficient medication as the positive control. The reduced
number of patients is explained by the early closure of recruitment due to the successive
occurrence of unsatisfactory efficiency of the treatment with Artemisia tea as shown in
Table 2.
Despite the fact that 50% of the patients showed negative results for parasitaemia in
the first days of treatment with Artemisia tea, recrudescence occurred after about two
or three weeks, when the patients were rescued using appropriate medicine, in this case
Coartem. In the case of therapeutic failure, R2 and R3, this behavior coincided with the
change in region and modification of the tea-making procedure. The change in region
to Anajás on the Isle of Marajó was necessary due to the low endemic level of cases of
falciparum malaria in Tucuruí and Goianésia do Pará, which made recruitment difficult
Table 1. Parasitaemia of patients infected with
P. falciparum
and treated with Coartem.
Parasitaemia against 200 leucocytes
Patient 1 85 0 0 0
Patient 2 15 0 0 0
Patient 3 8 2 0 0
Patient 4 76 22 0 0
Patient 5 122 25 0 0
Patient 6 20 3 0 0
Patient 7 253 99 14 0
Patient 8 294 177 2 0
P. M. de Magalhães et al.
Table 2. Control of patients treated with an infusion of
Artemisina annua
var. CPQBA.
Parasitaemia against 200 leucocytes
Patient 1 19 168 R3
Patient 2 177 307 304 R3
Patient 3 10 6 4 0 0 D10 R1
Patient 4 52 21 1 0 0 D14 R1
Patient 5 149 104 35 9 0 D21 R1
Patient 6 105 48 13 2 0 D14 R1
Patient 7 18 224 R3
Patient 8 92 38 105 R2
within the designated project time. In order to maintain the same quality of the Arte-
misia leaves with an artemisinin content of 1.1%, new cultures were developed at CPQBA-
UNICAMP, such that the material was never more than 6 months old. The whole har-
vesting and post-harvest processing procedure was rigorously standardized for the 3
cultures. The change in tea-making procedure was due to a publication by Kooy &
Verpoorte (2011), who showed that more artemisinin was extracted when the infusion
was prepared by boiling the leaves for exactly two minutes. The possibility remains that
this procedure could have degraded other substances, such as arteannuin B, and, in this
way, have reduced the parasite control efficiency. Although the phenomenon of recru-
descence could have been expected in the tea group, considering the relatively short
half-life of the compounds in the blood stream, one should also consider the possibility
that the
Plasmodium falciparum
from the Isle of Marajó could have been genetically
more sensitive to artemisinin in relation to the same parasite found in Goianésia do
Pará and in Tucuruí.
3.2.2. Bio-Clinical Analyses
The laboratory analyses were: CBC (red blood corpuscles, platelets, leucocytes, segmented
cells, lymphocytes, eosinophils, monocytes and reticulocytes); coagulation (bleeding time,
coagulation time, coagulum retraction, prothrombin time, prothrombin activity and
thromboplastin time); aspartate aminotransferase (AST), alanine aminotransferase (ALT),
urea, creatine, uric acid, glycaemia, high sensitive C-reactive protein (Hs-CRP), blood
group and urine (volume). Despite the limited number of patients, there were signifi-
cant differences for the parameters: coagulation time, bleeding time and prothrombin
time. Figures 4-6 show the means obtained for these parameters in the groups treated
Artemisia annua
tea and with Coartem.
These data show that in the tea group there was less alteration in the coagulation
factors with respect to the bleeding, coagulation and prothrombin times.
P. M. de Magalhães et al.
Figure 4. Coagulation time (minutes).
Figure 5. Bleeding time (minutes).
Figure 6. Prothrombin time (minutes).
Coagulation time (minutes)
Coagulation time with CoArt
Coagulation time with tea
Bleeding time (minutes)
Bleeding time with CoArt
Bleeding time with tea
Rep. 1
Rep. 2
Rep. 3
Rep. 4
Prothrombin time (minutes)
Prothrombin time with CoArt
Prothrombin time with tea
P. M. de Magalhães et al.
The normal times are: for bleeding, between 2 - 8 minutes (Ivy test), for prothrom-
bin, between 80% - 100% or from 11 to 14.6 seconds, and for coagulation, from 4 to 10
min. It can be seen that, in general, the bleeding and coagulation times were within the
normal ranges for both groups, although the tea-taking patients showed shorter times
than those in the Coartem group. With respect to the prothrombin time, the Coartem
patients showed a time that was more prolonged than normal. In fact, the longer the
prothrombin time, the smaller its concentration in the blood. This parameter evaluates
the extrinsic coagulation route, and one of the factors that lead to its alteration is con-
sumption coagulopathy (disseminated intravascular coagulation). The bleeding time
evaluates the efficiency of the platelets. The coagulation time evaluates the intrinsic
coagulation route, and, if altered, confirms defects in the coagulation factors. The pa-
tients in the Coartem group presented a mean value for initial parasitaemia much higher
and a mean platelets count much lower than the tea group, which, despite not being
significantly different, could have contributed to these results.
3.2.3. Interviews with Tea Group Patients after Treatment
After treatment, the patients who took the Artemisia tea reported that they felt good
throughout the entire treatment and that the bitter taste was tolerable. Some of them
referred to this taste as “the more bitter, the better”, showing an empiric sensitivity
concerning the drugs traditionally recommended for malaria therapy. No patient re-
ported feeling sick or vomiting. Even those who showed recrudescence did not present
symptoms of the disease, and questioned the need for a rescue treatment with Coartem.
Nevertheless, according to Brazilian policies for the control of falciparum malaria, with
a view to reducing transmission, cases of recrudescence are treated with Coartem for
the total control of the infection, even if, as in these cases, they were asymptomatic (In
some African countries a patient is not obliged to treat asymptomatic infections since
they consider that this immune response of the monocytes, even if temporary, can be
an advantage for the host. Nevertheless, he is collaborating with the maintenance of the
focus of the disease and its transmission. Currently the reduction in
P. vivax parasitae-
is considered in Brazil, treating the cases of resistance even when the infection is
asymptomatic. This procedure is essential in the context of eliminating malaria). In
many African countries the therapy of promoting a reduction in parasitaemia until the
symptoms are eliminated is accepted in the control of malaria. However, this does not
necessarily signify that the infection has been controlled, since asymptomatic recrudes-
cence could have occurred. It can be said that the asymptomatic patient has acquired a
degree of immunity, and has the right to continue the treatment required to completely
control the parasitaemia, or otherwise. As a consequence, such an asymptomatic pa-
tient collaborates with an increase in transmission if bitten by the vector. In Brazil, the
policy of treating malaria preconizes the total control of parasitaemia, aimed exactly at
reducing transmission. For this reason, and also to assure that no parasite remains that
could promote the selection of a population resistant to the antimalarial agents, the
control of the cure of malaria falciparum should demonstrate that the parasitaemia re-
mains negative up to the 28th day.
P. M. de Magalhães et al.
Despite the low number of patients recruited, a higher value was observed for the
monocytes in the group treated with
A. annua
tea (non-statistically significant differ-
4. Discussion
Important advances and changes in the paradigms were made in this research, which
included work led by UNICAMP with the support of various partner entities over 28
years, in addition to reaching the specific objective of knowing the efficiency of
sia annua
tea in malarial therapy, after innovating the raw material. Four large seg-
ments, united in this proposal, can be highlighted: agronomy, chemistry, biology and
medicine. The studies in the ambience of agronomy concentrated on the genetic im-
provement of the plant, adapting it to the inter-tropical and tropical conditions, where
malaria occurs. By monitoring the chemical composition and the use of biological tri-
als, the plant can be improved with respect to its yield of active substances (content and
biomass), not only of artemisinin, but also of molecules that take part in the biological
actions under investigation, such as the case of the flavonoids. Knowledge of the plant
physiology and also of the parameters related to post-harvest processing involving the
drying, storage and making of the tea, were decisive points in the standardization of the
raw material. However, it was the carrying out of this clinical research together with the
accompanying of the data in the literature that allowed for an integrated understanding
of the various segments. As a result, we now know the current efficiency of this form of
medication and the direction in which to carry out adjustments aimed at improve-
ments, as occurs in the development of all pharmaceutical products. The innovation of
developing medication to treat malaria as from a plant opens the possibility for new
studies using the same model, with other vegetable species which have the advantage of
counting with traditional use, and hence with strong adhesion by the populations who
live in endemic areas. Traditional medication can have new versions and amplify its
benefits if its development is based on the same integrated scientific arrangement used
in this research, making the most of what Nature produced. This research fits into the
context of a clinical study of medicinal plants from the Brazilian flora, with the poten-
tial to improve the treatment of malaria in the form of simple preparations, albeit safe
and efficient, which are well accepted by the local population [24].
Another innovative aspect, intrinsic to the use of the crude extract, was the evalua-
tion of the “active complex”, in contrast to conventional studies focused on a single
“active principal”. In fact, the plant produces an arsenal of substances with specific
functions, frequently synergetic with the main active molecule. What could have been
lost with the use of the pure substance? One must consider there are no reports of re-
sistance to the tea or of any side effects, and that the tea contains various substances
many of which are antimalarial, representing an association of antimalarial substances
thus complying with the strategy of not using the pure substance, however efficient it
may be.
Although this research was initially considered polemic, since it dealt with a disease
P. M. de Magalhães et al.
of risk and generated concern about the development of resistance, the proposed project
guaranteed that it would be carried out in a controlled group, following the official
protocol approved by CONEP, and hence received financial support from the main
Brazilian governmental organs as an expression of its scientific legitimacy and without
conflicts of interests.
Two facts should be considered with respect to the concern about the development of
resistance. However, first one should clarify the possible types of resistance in order to
give support to the discussion. The following types of resistance exist: 1) resistance
caused by the selection of a
population, that is, some individuals resistant
to the drug remain as the only survivors and multiply, forming a resistant population,
2) some individuals were not exposed to the drug, either because they were in micro-
circulation or because they were protected by other RBC (rosettes, cyto-adhesion) and
multiply after the drug has already degraded, and 3) by mutation of
starts interacting with the drug. With respect to the latter case, which deserves more at-
tention, the fact that the tea was used for more than a thousand years in Asia in the tra-
ditional way, without any reports of resistance, is encouraging. With the current know-
ledge concerning the action mechanism of artemisinin in destroying
which is predominantly physical, breaking the endo-peroxide bridges, there is a strong
argument justifying the absence of resistance to Artemisia tea and the anti-malarial
drugs based on artemisinin. However, the second fact that collaborates with the under-
standing that Artemisia tea does not favor the development of resistance, and, to the
contrary, reverts this process, are the findings of Suberu
et al
. (2013), who showed that
the compound Arteannuin B, present in the tea, interacts with the artemisinin for the
chloroquine resistant strains. This fact also justifies the evaluation of Artemisia tea for
the therapeutic treatment of malaria caused by
P. vivax
, which currently represents
from 5% to 8% of the resistance attributed to chloroquine-resistant parasites. In addi-
tion, with the information of Nair
et al
. [25] that the leaves harvested after only one
month of cultivation in the field have 2.9 times more Arteannuin B than artemisinin,
one can prepare a tea from a blend of two harvests, or even start treatment with a batch
with a high artemisinin content (harvested after 4 months) and then continue with
another batch richer in Arteannuin B (harvested after 2 months).
The fact that
A. annua
tea promotes a drastic reduction in the initial parasitaemia is a
great, but not excellent, result, making it necessary to search for adjustments in the me-
dication and regime. The sooner the parasetemiais quelled, the better, since one more
day of infection represents one more day of transmission by the vector. In its present
stage of development, the
A. annua
tea could be an important resource for first aid.
Due to the consecutive occurrence of recrudescence in the patients recruited into the
A. annua
tea group and rigorously following the protocol without any modifications
that could improve the efficiency of this treatment, the clinical research had to be ter-
minated before reaching the predicted number of patients. One should therefore con-
sider the results in order to provide recommendations and directions for future re-
P. M. de Magalhães et al.
Thus we understand that an adequate direction for the next actions with
A. annua
should involve: 1) not encouraging its use as a substitute for the treatment with arte-
mether + lumefantrin since, although the patients showed an excellent clinical response,
recrudescence (type I resistance), or no decrease or even an increase in parasitaemia
(type II/type III resistance) did occur; and 2) evaluate the efficacy of the
A. annua
tea in
association with other antimalaric species or compounds from a plant like Arteannuin
B, maintaining the principal of simple and available medication; 3) evaluate the efficacy
of the
A. annua
tea in a new therapeutic regime, including a dose on day14, aimed at
avoiding recrudescence; 4) during the follow up evaluate eventual inflammatory altera-
tions (PCR-US) in the patients and also the monocyte content when used concomi-
tantly with artemether + lumefantrin; and 5) evaluate the efficacy of the
A. annua
when associated with the conventional malaria vivax treatment. 6) Evaluate the nutritional
status of the patients in both groups, and also the blood test (CBC) parameters.
5. Conclusions and Perspectives
As in the case of artemisinin or artesunate monotherapies [26] [27] [28], 57.1% of the
patients treated with the
A. annua
tea presented type I resistance, with a return of the
parasitaemia around the 14th or 21st days of the cure control.
This fact demonstrates that the Artemisia tea is still not a final product, but on the
other hand should not be considered inefficient and hence banned, since it is a product
under development and its current efficiency is already important for the initial fight
, in the context of the first therapeutic actions. It is evident that its
current efficiency does not allow it to substitute the official medication, but it could
eventually be considered as part of the therapy, principally because the official medica-
tion is not always immediately available. In fact, the ease of access and acceptance of the
Artemisia tea by the population, added to the absence of collateral effects, characterizes
an important drug resource. Efforts to improve its efficiency by way of associations and
adjustments to the regime are part of the development of this important therapeutic
tool against malaria. Direction of future research: administer the dose on the 14th day;
take it associated with the conventional medication focusing on the anti-inflammatory
property (controlling the exacerbated inflammation); take a mixture or concomitantly
with other species, for example:
Pogostemon cablin
Chenopodium ambrosioides
dens pilosa
Curcuma longa
Baccharis dracunculifolia
(nerolidol); test the
A. annua
in vivax malaria using the same principals used here.
This was the first clinical Brazilian research with phyto-medication directed at the
treatment of malaria. In addition to the scientific results, the teams were capacitated
and specific points in the research with the raw material identified, mainly with respect
to standardization.
The authors thank State University of Campinas, Evandro Chagas Institute (Belém),
FIOCRUZ-farmanguinhos, Campinas Health Secretariat, Tucuruí Regional Hospital,
P. M. de Magalhães et al.
Goianésia do Pará Hospital, and Anajás Hospital.
Author’s Note
During this research, the present author was infected with malaria by
P. knowlesi
in the
region of Kota Kinabalu-Malasia, and was cured using
Artemisia annua
tea. This result
is not part of the data presented.
This work was supported by CNPq-MS, FAPESP, and FAEPEX.
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In the first part of this chapter antimalarial drugs derived from natural products are mentioned. The mentioned drugs are quinine and other quinolone derivatives, derivatives of artemisinin, tetracyclines, macrolides, lincosamide and malarone derived from naturally occurring naphthoquinones. The recently registered drugs bulaquine and tafenoquine are also included. The mechanisms of action, clinical effects and development of resistance are discussed. In the second part are results from clinical trials of traditional medicines marketed as antimalarial drugs discussed. Clinical trials on drug derived from Argemone mexicana, Artemisia annua, Vernonia amygdalina and Cochlospermum species are described.
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Artemisinin from the plant Artemisia annua (A. annua) L, and used as artemisinin combination therapy (ACT), is the current best therapeutic for treating malaria, a disease that hits children and adults especially in developing countries. Traditionally, A. annua was used by the Chinese as a tea to treat "fever". More recently, investigators have shown that tea infusions and oral consumption of the dried leaves of the plant have prophylactic and therapeutic efficacy. The presence of a complex matrix of chemicals within the leaves seems to enhance both the bioavailability and efficacy of artemisinin. Although about 1000-fold less potent than artemisinin in their antiplasmodial activity, these plant chemicals are mainly small molecules that include other artemisinic compounds, terpenes (mainly mono and sesqui), flavonoids, and polyphenolic acids. In addition, polysaccharide constituents of A. annua may enhance bioavailability of artemisinin. Rodent pharmacokinetics showed longer T1/2 and Tmax and greater Cmax and AUC in Plasmodium chabaudi-infected mice treated with A. annua dried leaves than in healthy mice. Pharmacokinetics of deoxyartemisinin, a liver metabolite of artemisinin, was more inhibited in infected than in healthy mice. In healthy mice, artemisinin serum levels were > 40-fold greater in dried leaf fed mice than those fed with pure artemisinin. Human trial data showed that when delivered as dried leaves, 40-fold less artemisinin was required to obtain a therapeutic response compared to pure artemisinin. ACTs are still unaffordable for many malaria patients, and cost estimates for A. annua dried leaf tablet production are orders of magnitude less than for ACT, despite improvements in the production capacity. Considering that for > 2000 years this plant was used in traditional Chinese medicine for treatment of fever with no apparent appearance of artemisinin drug resistance, the evidence argues for inclusion of affordable A. annua dried leaf tablets into the arsenal of drugs to combat malaria and other artemisinin-susceptible diseases.
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Significance Evolution of malaria parasite drug resistance has thwarted efforts to control this deadly disease. Use of drug combinations has been proposed to slow that evolution. Artemisinin is a favorite drug in the global war on malaria and is frequently used in combination therapies. Here we show that using the whole plant ( Artemisia annua ) from which artemisinin is derived can overcome parasite resistance and is actually more resilient to evolution of parasite resistance; i.e., parasites take longer to evolve resistance, thus increasing the effective life span of the therapy.
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Artemisia annua hot water infusion (tea) has been used in in vitro experiments against P. falciparum malaria parasites to test potency relative to equivalent pure artemisinin. High performance liquid chromatography (HPLC) and mass spectrometric analyses were employed to determine the metabolite profile of tea including the concentrations of artemisinin (47.5±0.8 mg L(-1)), dihydroartemisinic acid (70.0±0.3 mg L(-1)), arteannuin B (1.3±0.0 mg L(-1)), isovitexin (105.0±7.2 mg L(-1)) and a range of polyphenolic acids. The tea extract, purified compounds from the extract, and the combination of artemisinin with the purified compounds were tested against chloroquine sensitive and chloroquine resistant strains of P. falciparum using the DNA-intercalative SYBR Green I assay. The results of these in vitro tests and of isobologram analyses of combination effects showed mild to strong antagonistic interactions between artemisinin and the compounds (9-epi-artemisinin and artemisitene) extracted from A. annua with significant (IC50 <1 μM) anti-plasmodial activities for the combination range evaluated. Mono-caffeoylquinic acids, tri-caffeoylquinic acid, artemisinic acid and arteannuin B showed additive interaction while rosmarinic acid showed synergistic interaction with artemisinin in the chloroquine sensitive strain at a combination ratio of 1:3 (artemisinin to purified compound). In the chloroquine resistant parasite, using the same ratio, these compounds strongly antagonised artemisinin anti-plasmodial activity with the exception of arteannuin B, which was synergistic. This result would suggest a mechanism targeting parasite resistance defenses for arteannuin B's potentiation of artemisinin.
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In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths. The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers. A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker's yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid. Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.
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Ethnopharmacological relevance: Artemisia annua contains the well-known antimalarial compound artemisinin, which forms the backbone of the global malaria treatment regime. In African countries a tea infusion prepared from Artemisia annua has been used for the treatment of malaria only for the past 10-20 years. Several informal claims in Africa exist that the Artemisia annua tea infusions are also able to inhibit HIV. Since HIV is a relatively newly emerged disease, the claims, if substantiated, could provide a very good example of "ethnopharmacology in overdrive". The objective of this study was to provide quantitative scientific evidence that the Artemisia annua tea infusion exhibits anti-HIV activity through in vitro studies. A second objective was to determine if artemisinin plays a direct or indirect (synergistic) role in any observed activity. This was done by the inclusion of a chemically closely related species, Artemisia afra, known not to contain any artemisinin in our studies. Materials and methods: Validated cellular systems were used to test Artemisia annua tea samples for anti-HIV activity. Two independent tests with different formats (an infection format and a co-cultivation format) were used. Samples were also tested for cellular toxicity against the human cells used in the assays. Results: The Artemisia annua tea infusion was found to be highly active with IC(50) values as low as 2.0 μg/mL. Moreover we found that artemisinin was inactive at 25 μg/mL and that a chemically related species Artemisia afra (not containing artemisinin) showed a similar level of activity. This indicates that the role of artemisinin, directly or indirectly (synergism), in the observed activity is rather limited. Additionally, no cellular toxicity was seen for the tea infusion at the highest concentrations tested. Conclusion: This study provides the first in vitro evidence of anti-HIV activity of the Artemisia annua tea infusion. We also report for the first time on the anti-HIV activity of Artemisia afra although this was not an objective of this study. These results open the way to identify new active pharmaceutical ingredients in Artemisia annua and thereby potentially reduce the cost for the production of the important antimalarial compound artemisinin.
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The traditional use of the medicinal plant Artemisia annua for the treatment of malaria entails the preparation of a tea infusion. In the scientific literature there have been some discrepancies on the quantity of the active principle, artemisinin, in the tea infusion. Due to these discrepancies, we decided to quantify artemisinin in tea infusions prepared according to different methods. We also studied the water solubility of pure artemisinin at room temperature and at 100  °C and compared it to the solubility of artemisinin from the plant material. We found that the extraction efficiency is very sensitive to temperature and that efficiencies of above 90 % can be reached. We also showed that the solubility of artemisinin is not improved by other components in the extract but that a supersaturated solution of artemisinin might be formed, which is stable for at least 24 hours.
In 2003, a study in Mali showed that 87% of episodes of uncomplicated malaria were first treated at home. We investigated whether treatment-seeking patterns in Mali had changed 10 years later. In 2013, we repeated the retrospective treatment-outcome study on 400 children with presumed malaria in the same area. Most children with reported uncomplicated malaria were still first treated at home (76% [196/258] in 2013 vs 85% in 2003; p=0.006), rather than in modern health centres (20% [52/258] in 2013 vs 12% in 2003; p=0.01). Overall, 58% of children with uncomplicated malaria were treated with herbal medicine alone, a significant increase from 24% 10 years earlier (p<0.001). This was associated with an increase in use of Argemone mexicana decoction from 8% to 26% (p<0.001), with a reported cure or improvement in 100% of cases among those aged >5 years. For severe malaria, first treatment was sought less often from a traditional healer compared with 10 years earlier (4% vs 32%; p<0.001) and more often from a modern health centre (29% vs 17%; p=0.04). Two trends that emerged are that there is a greater use of modern health facilities for treatment of severe malaria, and a greater use of traditional medicine alone for treatment of uncomplicated malaria. © The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail:
The efficacy of artemisinin monotherapy was studied in 227 patients with uncomplicated falciparum malaria. They all received artemisinin at t = 0 hr, t = 8 hr, and thereafter once daily; treatment was extended at random until they had taken either 5 days of artemisinin followed by 2 days of placebo (A5), or 7 days (A7) of artemisinin. The adult artemisinin dose was 500 mg; children aged < 15 years received 10 mg/kg per dose. The median (range) parasite clearance time was 39 (8-112) hr for A5 and 43 (38-104) hr for A7 (P = 0.085). The recrudescence rates were similar between the groups. The lowest parasite count achieved during treatment (Pterm) was associated with the occurrence of recrudescence (P = 0.046, Cox regression model); it was lower for patients with a radical cure or late recrudescence than for early recrudescence (P = 0.034, t-test). Artemisinin monotherapy may offer rapid recovery and fast parasite clearance, but recrudescence is frequent. Extending the duration of monotherapy from 5 days to 7 days does not reduce recrudescence.
In an attempt to understand the beneficial health effects of Artemisia annua other than its anti-malaria properties, extracts from different cultivars prepared as tea infusions were investigated using Caco-2 cells on the intestinal inflammation and cytochrome P450 (CYP) activities. The characterisation of their phenolic compound (PC) profile revealed rosmarinic and chlorogenic acids as the main PCs. The extracts, assayed on Caco-2 cells at a plausible intestinal concentration, significantly decreased the secretion of pro-inflammatory cytokines, IL-8 and IL-6. This effect could be attributable at least to their content in rosmarinic acid, detected as a potent anti-inflammatory compound. The extracts also inhibited the activity of CYP3A4, whose expression was induced by 1,25-dihydroxyvitamin D(3), and of CYP1A1, induced by benzo(a)pyrene. Our results highlight the advantage of drinking A. annua infusions for their potent anti-inflammatory effect, linked to PC content, which could synergise their antimalarial activity.
Artemisia annua L. (sweet wormwood, qinhao) has traditionally been used in Chinese medicine. The isolation of artemisinin from Artemisia annua and its worldwide accepted application in malaria therapy is one of the showcase success stories of phytomedicine during the past decades. Artemisinin-type compounds are also active towards other protozoal or viral diseases as well as cancer cells in vitro and in vivo. Nowadays, Artemisia annua tea is used as a self-reliant treatment in developing countries. The unsupervised use of Artemisia annua tea has been criticized to foster the development of artemisinin resistance in malaria and cancer due to insufficient artemisinin amounts in the plant as compared to standardized tablets with isolated artemisinin or semisynthetic artemisinin derivatives. However, artemisinin is not the only bioactive compound in Artemisia annua. In the present investigation, we analyzed different Artemisia annua extracts. Dichloromethane extracts were more cytotoxic (range of IC₅₀: 1.8-14.4 μg/ml) than methanol extracts towards Trypanosoma b. brucei (TC221 cells). The range of IC₅₀ values for HeLa cancer cells was 54.1-275.5 μg/ml for dichloromethane extracts and 276.3-1540.8 μg/ml for methanol extracts. Cancer and trypanosomal cells did not reveal cross-resistance among other compounds of Artemisia annua, namely the artemisinin-related artemisitene and arteanuine B as well as the unrelated compounds, scopoletin and 1,8-cineole. This indicates that cells resistant to one compound retained sensitivity to another one. These results were also supported by microarray-based mRNA expression profiling showing that molecular determinants of sensitivity and resistance were different between artemisinin and the other phytochemicals investigated.