ARTEMISIA ANNUA

A whole against malaria

A program of local production for treatment of malaria in
poorest countries
M ONIMUS md1, J-M VOUILLOT2, G CLERC3

Artmisia Contre Paludisme

www.acp-paludisme.org

Amis Comtois des Missions Centrafricaines

www.acmc-ong.net

January 2013
1

Honorary professor of paediatric ortopaedic surgery, Besanon University, France. monimus@wanadoo.fr

Agricultural engineer ISARA Lyon, France.
3
Artemisia Contre Paludisme. acp-paludisme.org
2

FOREWORD

Tea preparation from leaves of Artemisia Annua is a hotly debated issue, unleashing
passionate controversies, sometimes not completely objective... Innumerable studies have
been dedicated to the subject.
It is more and more emerging from the currently published papers that not only artemisinin
but the entire plant used as tea preparation or powdered, is effective against malaria. Because
of that we intentionally changed the title of this booklet
The aim of this paper is to present this method of treatment of malaria in the more
comprehensive way as possible, considering all aspects, from the cultivation of the plant to its
effectiveness. Artemisia annua not only provides artemisinin, but also numerous other
molecules with synergistic action increasing the artemisinin effectiveness; however we are
quite aware of the disadvantages or limitations of the method. As presented, this study
probably includes deficiencies or inaccuracies; it will be our pleasure to correct them. We
would like this booklet to permit the reader to attempt Artemisia annua cultivation.

INTRODUCTION
Malaria is still a world health problem, responsible for millions of deaths every year,
including a very high proportion of children. It is unanimously admitted that the treatment
with artemisinin associated to slow-acting drugs (ACT = Artemisinin Combined Therapy) is
the more effective way for fighting falciparum malaria (80,82) and this is the recommended
guideline of the World Health Organization for the treatment of malaria (64). Artemisinin
clear peripheral parasitemia more quickly than any other antimalarial drug, with a rapid in
vitro effect suggesting a high parasitocidal action (3). Artemisinin is extracted from Artemisia
annua and semi-synthetic derivatives are commonly used. However the preparations made
from semi-synthetic derivatives (arthemeter, artesunate) are not routinely available in remote
rural areas, and they are still expensive and very frequently not easily affordable for poor
families.
Manirakariza (52) studied the availability and use of antimalarial drugs in Bangui (Central African Republic) ;
he observed that treatments with artemisinin or derivatives were available in 35 out of 82 assessed pharmacies
and drug shops ; moreover the medication the more commonly prescribed and used was chloroquin (66,7%),
arthemeter being used in 2,4% only. According to the WHO world malaria report in 2008 (88), in black Africa
38% of children with fever received antimalarial treatment, and 3% only had access to ACT.

Besides, the life of artemisinin and derivatives is short (32), not exceeding a few weeks,
particularly when stored in ambient temperature over 35. Moreother the preparations offered
on the local markets are occasionally imitations of uncertain origin or with insufficient
artemisinin content, or including other totally ineffective or even toxic molecules.
Newton (60) gathered 391 samples of uncertain drugs sold as artesunate in the south-east Asian. Half of them
(49, 9 %) did not contain artesunate or in insufficient quantity. Other molecules were present, some of them
potentially cancer-inducing. In another review about drugs sold as artesunate, Sengaloundeth (71) observed that
in 22 cases out of 25, pills were counterfeited without any trace of artesunate.

The increase in number of counterfeited drugs is becoming a public health scourge.

Meanwhile many studies have confirmed the immediate efficacy of artemisinin, which may
be a solution for resolving the tragedy of malaria in young children in endemic areas.
Tea preparation from Artemisia annua was used in traditional Chinese medicine for centuries;
it has been re-discovered by the Chinese in the seventies during the Vietnam War. It has been
proved to be effective and safe in treatment of all human malarial parasites, and notably
multidrug-resistant plasmodium falciparum. However, considering the recent literature, it
appears that the antimalarial potential of Artemisia annua is not exclusively related to
artemisinin. Several studies have shown that numerous other molecules present in the plant,
essentially flavonoids, have also an antimalarial action specific or synergetic with artemisinin,
3

and the importance given to the artemisinin may have been overestimated. These flavonoids
existing in the leaves and stem of Artemisia annua considerably enhance the interest of the
Artemisia annua tea preparation as a treatment of malaria (17,25,29,75). An alternative to the
tea preparation is the use of powdered leaves, mixed with milk or given in capsules, easier to
use than the tea, providing all molecules present in the leaves, usable for rectal administration,
but more costly.
Artemisia annua is easy to grow and may be available on the spot without any cost. However
the disadvantage is the rapidity of elimination of artemisinin, resulting in a short efficacy and
a risk of recurrence of malaria. This makes essential to continue the treatment during several
days.
The authors consider that, although not entirely satisfying, treatment of malaria by tea
preparations from leaves of Artemisia annua is an alternative to treatment by modern
antimalarial drugs, costly and occasionally non available (78). From a review of the literature
about the Artemisia annua cultivation and about its efficiency and from the analysis of some
personal observations treated with capsules or tea preparation, the authors assess this way of
treatment of malaria. They have developed a program of cultivation of Artemisia annua, the
aim being to provide seeds to local populations in endemic areas of undeveloped countries, to
favour local cultivation, to promote knowledge about Artemisia Annua, and to supply advices
and documents. They have created an association entitled Artemisine Contre Paludisme (acppaludisme.org), located in Longeville, Franche-Comt, East of France. There the plant is
cultivated at 700 m above sea level, without fertilization.

THE HISTORY OF ARTEMISIA ANNUA

Malaria was causing terrible losses among the north Vietnamese army during the Vietnam
war. Because of this, Mao Tse Toung initiated in 1967, during the Cultural Revolution, the
project 523 (so-called because it was launched on May 23rd, 1967). This was a secret
program on treatment of malaria developed from the Chinese traditional medicine (45). The
Chinese Traditional Medicine Academy entrusted this research to one of his members, Mrs
Youyou TU, young pharmacologist who at that time was 36 years old, and whose name
remained completely unknown to anybody until the last years (49). It should be remembered
that during the Cultural Revolution, the individual was far away behind the group and it could
be dangerous to draw too much attention to oneself...

Mrs Youyou TU, member

of the Chinese Traditional
Medicine Academy. She
was entrusted in 1967 a
research project on an
effective treatment for
malaria. She isolated the
Artemisinin molecule.

In the same way, one may notice that the first publications about artemisinin (90) did not
include any authors name... It is in 2005 that the name of Youyou TU was associated to the
artemisinin discovery, and that she became famous: I was taking part in a meeting gathering
the Chinese specialists of malaria in Shangai in 2005, and I asked who had discovered the
artemisinin, tells Louis Miller, a malaria researcher in the US National Institute of Health,
Rockville, Maryland. I was surprised to discover that nobody knew. In 2011 Youyou TU
was given the prestigious Lasker DeBakey Clinical Research award (59).
Youyou TU collected thousands of herbal receipts; she evaluated 380 extracts of different
plants, among which Artemisia annua (or Qinghao), which was known for centuries for its
effectiveness for treating recurrent fever. During the 4th century GE HONG described ancient
methods: Qing Hao, one bunch, take two sheng (0,2 x 2 l) of water for soaking it, wring it
out, take the juice, ingest it in its entirety. Another method involved soaking the plant in
urine rather than water and pounding the fresh herb to produce a juice... (89). Interestingly,
5

in these receipts fresh herb was used rather than dried herb, as well as whole plant rather than
the leaves only.
Later, during the 11th century, SHEN GUA recommended to use Artemisia apiacea (blue
green coloured during flowering) rather than Artemisia annua (light green coloured), because
of a greater efficacy (33). Now Artemisia apiacea contains a lot of flavonods, but few
artemisinin (43)

During the 11th century, SHEN GUA

recommended Artemisia apiacea
rather than Artemisia annua because
of a greater effectiveness.
However Artemisia apiacea contains a
lot of flavonods, but few artemisinin
(Qinghaosu)

The first results obtained by Youyou TU were disappointing, because the extracts were
obtained with boiling water which destroyed the active elements. In 1971 she made an
extraction with ether at a lower temperature, and she obtained extracts which were
experimentally evaluated on infected mice and monkeys; she observed a virtually 100 %
efficacy (92). Then, after checking on herself the harmlessness of the extract, she tested it on
21 patients with malaria, whose 90 % were cured. In 1972 she isolated a molecule which was
considered as the active element in Artemisia annua, and which was named artemisinin (or
Qinghaosu, the suffix su meaning the active substance). It should be noted that the first
positive trials had been done with Artemisia annua herb from the Beijing area, containing few
artemisinin. YouYou TU wanted to have a great quantity of artemisinin and afterwards she
used Artemisia annua herb from the Sichuan area, with a greater concentration in artemisinin,
and she observed similar clinical results, suggesting that the optimal concentration in
artemisinin is not necessarily the maximum concentration...
In 1975 Mrs Youyou TU established the molecular structure of artemisinin (lactone
sesquiterpene). In 1979 artemisinin started to be known after a paper was published in the
Chinese Medical Journal (90). In 1981, the 4th congress of the Scientific Workshop on
chemotherapy of malaria, sponsored by the UNDP, the Mondial Bank and the WHO was held
in Beijing, resulting in a worldwide knowledge of artemisinin; some big pharmaceutical
companies focused on it and produced semi-synthetic derivatives which are now largely used
(hydro soluble artesunate, lipo soluble arthemeter...). In 1986 the first artemisinin-based
medicines were available on the market. In 2004, considering the efficacy of artemisinin, the
WHO initiates a large scale promotion, recommending its association with other standard
molecules (Artemisinin Combined Therapy). Lastly in 2011, the WHO advised intra-venous
artesunate as a monotherapy instead of quinine salts for the treatment of severe malaria in
children.
6

CULTIVATION

Seedling: seeds are very thin (up to 10 000 seeds/gr) and should be mixed with fine sand (one
spoon of seeds for 5 to 6 spoons of sand) before sowing. The mixture should be sown in a
box filled with soften soil, and well-watered. Bedding out is necessary and should be
performed when the seedling is too dense, usually after 3 4 weeks. It should be done in
small pots or jars. Each young plant should be transferred in a pot.

Fig 1.The plant was grown in a pot and has got 5 leaves. It may be now transferred in open soil.

When the plant has grown 15 cm or has got 5 to 6 leaves (Fig 1 and 2), it should be again
transferred in open soil. This occurs after about 3 weeks. After softening the soil the whole
content of the pot should be planted out, preserving a few centimetres interval between each
plant. Coming up of the plant requires several weeks. Watering and weeding is necessary
In the authors experience at Longeville, with sowing during spring, at the beginning of April,
bedding out is performed during last week of April and the leaves are harvested at the end of
August. Under temperate climates, as in France, the plant may reach 1.5 to 2 m of height and
it is necessary to plant out with 80 cm interval. Five grams of seeds are sufficient for covering
one hectare in open soil. When days are invariably of the same duration, as in black Africa,
maturation is rapid and the growth usually does not exceed 20 to 80 cm, and harvesting
should be made as soon as the leaves are at maturation.
In Africa, cultivation of Artemisia annua is more difficult and some attempts have been
disappointing. In opposition to previous opinion broadcast sowing should be avoided; sowing
should be done in a box filled with soil carefully broken down, and above all copiously
watered. Seeds are photo sensitive and a better germination is obtained when they are kept
close to the surface. Otherwise, although the adult plant is repulsive for insects, young shoots
may be devastated by caterpillars (cutworm) and P Lutgen (pers com) advises to protect them
by treating with a 1% solution of oil or extract of neem, or even treating the soil with the same
solution before sowing. Ideally before sowing the soil should be prepared with termitary soil
and compost, and then slightly tamped down with a roll or a plank. Seeds should be kept in
7

humid atmosphere, and sprinkling with water should be abundant especially when sowing is
made during dry season. For retaining humidity, which is essential for germination, the
sowed area may be covered with a fine coat of soft soil or sand. Another way is to cover the
seedling with a thin transparent plastic leave. When bedding out is performed, a 30 60 cm
interval is sufficient between plants, as the growth potential is limited.

Fig 2.This cultivation was done in Madagascar. Plants were grown in small bags. They are ready for
transferring in open soil.

In an attempt of cultivation made in Senegal, budding occurred 3 months after seeding; the
plant got 60 cm of height. In another cultivation in Benin, bedding out was performed 3
weeks after sowing (in January); the maximum height of the plant was 1 m. In Burkina Faso
the height obtained was 20 30 cm. In African areas, sowing may be performed at any period
in the year, taking care to generously bring water during the dry season until germination
occurs or until transferring the plants.

HARVESTING
1) The time for harvesting the leaves is of great importance, as the concentration of
artemisinin in the leaves is maximum during budding and rapidly decreases after flowering.
In a study performed in Longeville, with budding occurring during last days of August, the
artemisinin content of the leaves was 0, 13% on August 16th ; it was 0, 31% on August 26th ; it
was again 0, 31% on September 2nd and it was 0, 25% on September 9th. Ideally the leaves
should be harvested just before flowering (Fig 3).

Fig 3. The plant is mature and leaves may be harvested

Determining the exact moment of transition between leaves and flowering may be difficult as
the leaves slightly vanish during budding; moreover the flowers are very small, yellowish
coloured, and may be difficult to differentiate from small leaves.
The simplest way for harvesting is to cut and collect the plants, then spread out on mesh
material for drying. The artemisinin content is markedly more important in dry leaves than in
green leaves, particularly after early harvesting (46) and drying is quite necessary.
The way of drying is debated in literature, exposure to the sun being harmful for some people
(Laughlin,41) and beneficial for others (Simonnet,73).
In a study performed by ACP-Paludisme, the artemisinin content is 0. 30% when plants are sun-dryed and 0.
36% when they are shadow-dryed, suggesting that avoiding sun exposure is preferable. Ferreira (24) compared
different ways of drying: shadow-drying during 1 to 3 weeks, sun-drying during 1to 3 weeks, owen-drying at 45
with ventilation during 12 to 24 hours, and drying by lyophilisation. He observed that shadow-drying or sundrying did not substantially change the artemisinin content.

On the other hand the owen-drying is harmful for the artemisinin content but is more effective
for preservation of the anti-oxidant leaves function. Whatever the way of drying, the
artemisinin concentration increases during the first three days as its biosynthesis is activated
by drying. When drying is completed (after 3-4 days), the leaves should be detached from the
stems as they contain artemisinin. However the small stems should be kept as well, as they
have a high concentration in flavonoids. This can be done by shaking or threshing the plants,
then sieving for eliminating the stems. Another way is to grasp the branch at the foot and pull
the leaves with fingers from bottom to top. These techniques can fragment or smash the
leaves. However this is not a disadvantage because the leaves can be crushed for tea
preparation. Then they should be stored away from light in opaque containers or plastic bags.
A different way for harvesting is to avoid cutting the plant and to directly pull out the leaves
one by one in the field (Fig 4), then to dry. This technique is more time-consuming, but the
leaves keep a more normal appearance. Lastly, after drying leaves should be stocked in mesh
bags in a shadowed and fresh area.
Leaves should imperatively be kept in a dark, cool and dry place.
9

According to an unpublished study (Vrije Universiteit Brussel) reported by P Lutgen, in leaves with an initial
artemisinin concentration of 1.08%, the concentration was unchanged after preserving during 7 weeks at 25C
with hygrometry 65%, and at 35C with hygrometry 75%. The artemisinin concentration was 0.78 % after
preserving during 39 months at 25C with hygrometry 65%, but collapsed after preserving at 35C with
hygrometry 75%. The artemisinin concentration was 0.90% after preserving in fridge at 4C.

A contrary to previous assertions (30), dry leaves are stable with a satisfying persisting
artemisinin concentration after three years, provided that they are kept in a dark and dry place.
On the other hand, once prepared the tea has a short life, and its efficiency is rapidly
decreasing after infusing, especially when kept at ambient temperature. Therefore one should
only use recently prepared tea, with a maximum 24 hours limit.
2) Harvesting the seeds should not be done too early, as they may not be grown up, and
should not be done too late, as the seeds may fall down on the ground and be lost for
gathering. In Longeville the best time for harvesting the seeds occurs in autumn just before
the first frost. The simplest way is to cut the stems and after drying thresh or shake them
against a stiff surface. Lastly, one should remember that harvesting the leaves at the right
time, before flowering, in order to have an optimal concentration of artemisinin, results in a
reduction of the number of seeds, and when gathering the leaves one should preserve a part of
the crop for later harvesting the seeds. One may identify some plants with any mark for their
preservation until flowering. When seeds are forming the flowers should be enclosed (without
tightening) in a small perforated plastic bag, in order to avoid dissemination of the seeds.

Fig 4. Harvesting green leaves is made one by one directly on the plant.

10

THE CYCLE OF THE PLASMODIUM FALCIPARUM

Abstract: When the mosquito bites a human, the parasite mixed with its saliva is injected and
pass into the blood of the person. First the parasite migrates to the liver where it silently
multiplies. Then the parasites spread into the blood; they invade and destroy the red blood
cells, causing the malaria attack. When an unaffected mosquito bites an infected human, it
gets infected and will transmit the disease to other humans when biting.
The development of the parasite (the more frequent is the Plasmodium Falciparum,
responsible for the more serious malaria) occurs in two cycles: an asexual one in human, and
a sexual one in the mosquito.
1) In Human: asexual cycle (schizogonic cycle).
When the mosquito bites a human, hundreds of parasites (or sporozotes) mixed with its saliva
pass into the blood of the person being bitten. The parasites migrate into the liver and enter
the hepatic cells where they multiply, swell, and at last cause rupture of the cell. Then they
pass again in the blood (and are called merozotes) and enter the red blood cells. Then they
are called trophozotes, the youngest stages with a ring form ; they get bigger, deteriorate the
haemoglobin of the red blood cells, and multiplicate in the red blood cells. At that stage they
are called schizonts. When mature, the schizonts break out, releasing new merozoites which
will invade intact red blood cells and continue the cycle. Duration of each cycle is about 48
hours, and rupture of red blood cells and schizonts is contemporary with febrile attack. Lastly,
after several cycles, sexual forms of the parasite appear in the red blood cells, i.e. male and
female gametocytes.
2) In Mosquitoe (anopheles female): sexual cycle (sporogonic cycle).
When biting an infected human, the mosquito absorbs schizonts, merozotes and gametocytes.
Schizonts and merozotes are digested in the mosquitos stomach. The female gametocyte is
inseminated by the male one, resulting in an egg (oocyst) which go through the stomach wall
and fix to it. Sporozotes develop in the oocyst and reach the salivary glands. Duration of
sexual cycle in mosquito is 10-20 days.
When the population is chronically exposed to a high risk of infection (as in sub-Saharan
Africa), the more serious malaria attacks are observed in children. A partial clinical immunity
is obtained during childhood, and adolescents and adults present only few malaria attacks,
even if the parasite is chronically present in their blood. This is an important fact clearly
specified in the 2006 WHO report on the treatment of malaria (64) which should be kept in
mind when evaluating the effectiveness of the treatments; that makes questionable the WHO
official position of considering both the clinical and the biologic results for evaluation of
treatments.

11

MECHANISM OF ACTION OF ARTEMISININ

The molecule of artemisinin was identified in 1972; its structure was determined in 1975: it is
a sesquiterpene lactone, containing an endoperoxide bridge (bridge joining two oxygen
atoms). It is a highly energetic molecule, unstable and rapid to react and release its energy
(35). Semi-synthetic derivatives were obtained from the molecule of artemisinin:
dihydroartemisinin, artemether, artesunate and arteether. These derivatives are not noticeably
more efficient than artemisinin, and they are quite rapidly ineffective compared to
artemisinin, after a few months for artesunate (32).
Two atoms of oxygen
are linked by the
endoperoxyde bridge

The artemisinin molecule.

The endoperoxide bridge (O-O)is
essential for the artemisinin activity.
In the presence of iron, the bridge is
open with formation of carbon free
radicals which interrupt the synthesis
of the parasite proteins(by inhibition
of the CA++ATPase)and kill the
parasite.

Artemisinin has an anti-inflammatory and antipyretic effect; it is effective not only on

Plasmodium Falciparum, but also on numerous bacteria and viruses and namely VIH (47), as
well as some parasitic diseases (leishmaniosis or schistosomiasis (70); it has been proved
effective for water disinfection (48) and it may be effective for slowing down the cancer cell
proliferation4 (25, 40, 58).
4

The anticancer activity of artemisinin is a burning question. This activity has been demonstrated many
times in vitro on multiple cancer cell lines: breast, lung, kidney, ovary, prostate, colon, leukemia (1, 2).
However no clinical studies have been reported and one may only rest on isolated observations. Various
pathways for anticancer activity include (3): apoptosis (death of cells), tumoral growth inhibition,
inhibition of angiogenesis, and disruption of cell migration. The mechanism of action is similar to that
observed in malaria (4,5): the artemisinin molecule is activated in the presence of iron, which open the
endoperoxid bridge, with formation of free radicals killing the cancer cells. Cancer cells require a large
amount of iron to proliferate and they are more sensitive to the cytotoxic effect of artemisinin than normal
cells. Experimentally the artemisinin has been tagged to transferrin which is a plasma iron-carrying
protein (10). The transferrin is endocytosed by the cancer cell, the iron is released and reacts with

12

Regarding malaria, artemisinin is effective on early trophozot stages (1) and prevent
evolution to later stages, with adherences of the parasite to the vascular endothelium; this
sequestration phenomenon occurs in the microcirculation of the vital organs and especially
the brain.
The sequestration stage is considered as inducing the complications of malaria attack, including cerebral malaria,
with adherences of the parasited red blood cells to the postcapillar veinulas in the brain, with inflammation and
haemorrhage around the vessels; the coma is attributed to accumulation in the cerebral capillary vessels of many
infected red blood cells, inducing metabolic troubles by toxins production.
artemisinin in the cell, with formation of free radicals killing the cell. The role of Calcium has also been
underscored (6). Actually in tea preparation artemisinin is not isolated. Many other molecules are present,
among which the flavonods. Flavonods are present in the leaves and also in big amount in the stems. The
flavonods have a specific anticancer action (7), some of them inhibiting angiogenesis, othersas limonen
inhibiting the cancer cell proliferation (8), and they have also a potential synergism with artemisinin: they
have antioxidant properties and they facilitate the activation of artemisinin and the release of free
radicals.
The cytotoxic effect of artemisinin has been observed in vitro on several cultures of cancer cell lines, as
breast (9), lung (10), lymphocytes (11,12). A specific anti-oestrogenic effect has been documented, the
artemisinin decreasing the level of oestrogen receptors in breast cancer cells culture (13).
In the absence of documented studies, no exact dosage is available for tea preparation; one liter per day of
tea preparation is usually recommended, prepared with a tablespoonful of leaves, but possibly one bowl
may be sufficient.
1. Efferth T, Dunstan H, Sauerbrey A, Miyachi H, Chitambar CR. The anti-malarial artesunate is also active
against cancer. Int J Oncol. 2001 ;18 : 767-73.
2. Efferth T. Antiplasmodial and antitumor activity of artemisinin--from bench to bedside. Planta Med.
2007 ; 73 : 299-309.
3. Firestone GL, Sundar SN. Anticancer activities of artemisinin and its bioactive derivatives. Expert Rev
Mol Med. 2009, 30;11: e32.
4. Lai H, Sasaki T, Singh NP. Targeted treatment of cancer with artemisinin and artemisinin-tagged ironcarrying compounds. Expert Opin Ther Targets. 2005 ; 9 : 995-1007.
5. Lai H, Singh NP. Oral artemisinin prevents and delays the development of 7,12dimethylbenz[a]anthracene (DMBA)-induced breast cancer in the rat. Cancer Lett. 2006 Jan 8;231(1):438.
6. Mu D, Zhang W, Chu D, Liu T, Xie Y, Fu E, Jin F. The role of calcium, P38 MAPK in dihydroartemisinininduced apoptosis of lung cancer PC-14 cells. Cancer Chemother Pharmacol. 2008 ; 61 : 639-45.
7. Ferreira JFS, Luthria DL, Sasaki T, Heyerick A. Flavonoids from Artemisia annua as antioxidants and
their potential synergism with artemisinin against malaria and cancer. Molecules 2010, 15, 3135-3170.
8. Manuele MG, Ferraro G, Anesini C. Effect of Tilia x viridis flower extract on the proliferation of a
lymphoma cell line and on normal murine lymphocytes: contribution of monoterpenes, especially
limonene. Phytother Res. 2008, 22:1520-6.
9. Singh NP, Lai H. Selective toxicity of dihydroartemisinin and holotransferrin toward human breast
cancer cells. Life Sci 2001 ; 70 : 49-56.
10. Lu YY, Chen TS, Qu JL, Pan WL, Sun L, Wei XB. Dihydroartemisinin (DHA) induces caspase-3-dependent
apoptosis in human lung adenocarcinoma ASTC-a-1 cells. Biomed Sci. 2009 ;16:16.
11. Zhou HJ, Wang Z, Li A. Dihydroartemisinin induces apoptosis in human leukemia cells HL60 via
downregulation of transferrin receptor expression. Anticancer Drugs. 2008 ; 19 : 247-55.
12. Lai H, Sasaki T, Singh NP, Messay A. Effects of artemisinin tagged holotransferrin on cancer cells. Life
Sci 2005 ; 76 : 1267-79.
13. Thangaiyan R, Anupam B. d-Limonene sensitizes docetaxel-induced cytotoxicity in human prostate cancer
cells: Generation of reactive oxygen species and induction of apoptosis. J Carcinog. 2009; 8: 9. Published online
2009 May 21. doi: 10.4103/1477-3163.51368
14. Sundar SN, Marconett CN, Doan VB, Willoughby WA, Firestone GL. Artemisinin selectively decreases
functional level of oestrogen receptor-alpha and ablates oestrogen-induced proliferation in human breast
cancer cells. Carconigenesis, 2008 ; 29 : 2252-58.

13

Artemisinin penetrates into infected red blood cells and precociously stops the schizonts
maturation. It has also a gametocytocid effect higher than standard antimalarials (Okell, 63),
decreasing the risk of transmission of the disease from human to mosquitoes. This
gametocytocid effect is of most importance as gametocytes may be latently persisting in the
blood, especially in children who may be contagious a long time after recovery from malaria
(Barnes, 11).
The mechanism of action is still not completely resolved. Several mechanisms are proposed
(27). Some of them are specific: interference with the protein metabolism of the parasite and
interference with the mitochondrial activity of the parasite. Others are not specific, as an
effect on immune system functions and an inhibition effect on angiogenesis.
Interference with the proteins metabolism of the parasite
The first proposed mechanism is inhibition of the Ca++ ATPase, which is an important
enzyme for the synthesis of cellular membranes proteins (20).
Ca++ATPase is present in the sarco-endoplasmic reticulum of the parasite and ensure the maintenance of
calcium ions concentration, which is essential for proteins processing. Artemisinin binds to the enzyme while
leaving exposed the peroxide bridge. This allows the bridge to be open by iron ions (as iron attracts electron of
oxygen), activating oxygen atoms which attract hydrogen ions, generating carbon-centered radicals, and leading
to inactivation of the Ca++-ATPase and parasite death. The origin of the iron is still debated: it may come from
the haem, i.e. the degraded haemoglobin secondary to the parasite infestation, with activation of artemisinin and
release of free radicals into the parasite, explaining the selective toxicity for parasite (Meshnick,54).

However the origin of iron may be different, because artemisinin is precociously active on the
parasite cycle, before degradation of the haemoglobin; furthermore artemisinin is found
mainly in cellular membranes of the parasite (endoplasmic reticulum, mitochondrial system)
and not in the cytoplasmic vacuoles, where the haem is present, suggesting other mechanisms.
Interference with the mitochondrial system of the parasite
Mitochondrial activity is necessary for the respiratory chain of both sexual and asexual stages
of Plasmodium Falciparum. Artemisinin is activated by iron contained in the mitochondria;
the reactive oxygen atoms interfere with the electron transport chain of the parasite, causing
depolarisation of the mitochondrial membrane, inhibiting the biosynthesis of the nucleic acids
and particularly the pyrimidin, and causing parasite death (Maeno,50).
Inhibition of angiogenesis
The artemisinin inhibits the inflammatory cellular proliferation secondary to the aggression of
the parasite with adhesion to the vascular endothelium in the capillaries. Artemisinin should
be efficient on oedema and haemorrhages due to inflammation and sequestration. This effect
is particularly beneficial in case of cerebral malaria.

14

EFFICACY OF ARTEMISIA ANNUA

There are multiple aspects of antimalarial effect of Artemisia annua, including a repulsive
effect on mosquitoes, related more to terpenoids than to artemisinin, and available by
fumigation or more simply by growing the plant close to the house, as well as a direct
parasitocid effect, due to the numerous components present in the plant. The literature
contains many papers about artemisinin and its action. The treatment of malaria with
extracted artemisinin or its semi-synthetic derivatives has been widely investigated and the
efficacy of tea preparation with leaves of Artemisia annua also has been documented in some
clinical series. Otherwise numerous studies are nowadays focusing on other molecules present
in Artemisia annua, of which the antimalarial power may have been underestimated. The
association of all these molecules results in a greater therapeutic effectiveness than that of
each isolated component.
1. ARTEMISININ
1.1. The leaves concentration in artemisinin depends upon many factors, including the time
of harvesting, the way of drying, the way and the duration of preserving the leaves, as well as
the variety of Artemisia Annua. Concentrations reported in literature spread from 0.10% to
1%.
In Madagascar we measured 0,50%. In Sngal different varieties were studied, including Brasilean, Anamed
and Mediplant. In all varieties the artemisinin content was over 0.8%, up to 1.3% with the Mediplant variety.
Concentration was 0,63% in Mueller study (56).

The upper leaves on the plant contain more artemisinin than the middle or lower leaves (61).
The artemisinin concentration is increased by hybridation of Artemisia annua. According to
the Jiang study (36) concentration was 0,90% in natural Artemisia annua and it was 1,45% in
transgenic Artemisia annua. However hybridation may jeopardize the seeds production.
Phosphorus fertilizer may have a potential role in improving the concentration of Artemisinin
(37).
The artemisinin concentration is of interest when considering commerce and business;
however it is known that a high concentration in artemisinin is not necessary for antimalarial
power of Artemisia annua: Omar Gueye (65) observed in vitro a similar efficacy with a
brasilian leaves-based tea preparation (with artemisinin concentration about 1%) and with a
luxembourg leaves-based tea preparation (with artemisinin concentration about 0,2%), this
being attributed to the presence of other molecules in the tea. Similarly, in the Malagon study
(51) the plasmodium reproduction was more than 98% inhibated with Artemisia ludoviciana
mexicana, which does not contain artemisinin.
1.2. The standard recommended amount of leaves for tea preparation is 9-10 grams of
dried leaves to be infused for 10-15 minutes in one liter of boiling water. Nevertheless similar
clinical results were observed by Blanke (13) and Mueller (57) with tea preparations
containing 5g/l only. Pouring boiling water onto the leaves seems more effective than adding
dries leaves to water. Continuing boiling after adding leaves should be strongly avoided, as
boiling considerably reduces the yield (two to three times less according to Rath (67) and
Mueller (56) after boiling from 5 to 30 minutes). If possible a plastic container or preferably a
15

glass container should be used for infusing; iron container should be avoided, as iron reacts
with artemisinin. After pouring boiling water onto the leaves, Rth advises a brief stirring
(with a woodcut spoon), then leaving the mixture to cool for 10-15 minutes, the container
being covered; then the leaves should be removed by filtration and gently squeezed to get
residual water.
Using this protocol, Rth got 94.5 mg/l of artemisinin with 9 grams of leaves. With 5 grams of leaves he got 60
mg/l. Using a similar protocol, Mueller measured 12 mg/l of artemisinin with 5g/l of leaves, 24 mg/l with 10
grams, 32 mg/l with 20 grams, and 64 mg/l with 40 grams of leaves. Rocha e Silva (69) obtained 40-46 mg with
5g/l of leaves. Jansen (34) reported 24.2 mg/l. According to Mueller 40% of artemisinin contained in the leaves
may be extracted by tea preparation, meaning 40 mg if one consider 1% content in 10 g of leaves.

When matching these figures with the usually recommended daily dose of extracted
artemisinin, from 200 mg to 500 mg, one may consider that the amount of artemisinin
delivered by the tea preparation is quite insufficient. However there is no definite study to
support this official 200-500 mg dosage. Moreover the usually recommended amount of
leaves may be excessive. Ashton (8) observed a similar efficacy with oral and rectal
artemisinin, although bioavailability after rectal relative to oral artemisinin was 30%,
suggesting that a 500 mg initial dose may be unnecessarily too high. So, one litre of tea
preparation provides a substantial amount of artemisinin. It is known that artemisinin has a
low aqueous solubility, and the high yield in tea preparation may be related to the presence of
other constituents in the leaves (flavonoids, saponins, polysaccharids), which might
improve the solubility of artemisinin in water by acting as detergent (81), or constituting
hydro-soluble complexes with artemisinin.
1.3. The maximum plasma concentration is rapidly obtained after intake of 500 mg of
extracted artemisin, between 2 and 3 hours, most often exceeding 200 g/l (51). It was 615
g/l in Alin study (4), 391g/l in Duc study (19), and 364 g/l in de Vries study (78).
The maximum plasma concentration after intake of a tea preparation with 9 grams of
Artemisia Annua leaves was 240 g/l in Rth study (67), suggesting a better absorption of the
artemisinin in tea preparation than the extracted artemisinin. Tea preparation contains other
compounds than artemisinin, possibly improving the absorption rate. The minimum
artemisinin concentration required for in vitro inhibition of 50% of growth (IC 50) of
Plasmodium falciparum is 3 to 30 g/l according to de Vries (78), and 10 g/l according to
Alin (3) and Heide (31), therefore quite lower than the measured blood concentration after
absorption of tea preparation.
The time necessary for inhibition of the Plasmodium Falciparum growth has been studied in
vitro as well as in vivo: in vitro, exposition to artemisinin during 3 hours induced a 63%
growth inhibition, the rate increasing up to 95% after 48 hours according to Alin. In vivo the
mean parasite clearance time is 36 hours: it was 26 hours in Alin study (5), 48 hours in
Mueller study (56), and 36 hours in de Vries study (78).
So the treatment of malaria with artemisinin is just about immediately effective, with
significant reduction of the parasitemia below detectable values, but without complete
elimination. The growth of the remaining parasites may result in early recrudescence of the
parasitaemia with or without recurrence of the malaria attack. This risk, combined to the
short mean half-life of artemisinin, averaging 2 and half hours3 hours (4,19,78), justifies
extending the treatment with tea preparation during at least 5 days (42). The usually
recommended duration is 7 days. Moreover this short half-life of artemisinin is sometimes
considered as beneficial, giving no chance to the parasite for developping resistance.
However the study on mice made by Atemnkeng (9) gives less optimistic results: the
parasitemia at the 4th day was 50% in the group treated with tea and it was 3% in the group
16

treated with artesunate; on the 11th day all mice treated with tea were dead whereas 83% of
mice treated with artesunate were alive.
1.4. The clinical efficacy of tea preparation in malaria treatment has been observed in
several isolated observations, all of them with rapid and complete improvement. The efficacy
has also been studied in some randomized series:
Chougouo Kengne (16) reported a study comparing tea preparation and artesunate alone or associated with
amodiaquine: the failure rate was 0% with tea taken during 7 days and 28% with tea taken during 5 days; failure
rate was 12% with artesunate alone and 14% with artesunate + amodiaquine. In a recent study on 3000 cases, of
whom 250 children were less than5 years, Willcox (85) reports a constant efficacy of the tea without noteworthy
adverse effects nor in children and in pregnant women (24 cases were treated during the first three months of
pregnancy). On the other hand difficulties in cultivation were present in nearly half of the plants (in Kenya and
in Ouganda). Fouda (26) reported a brasilean study on 72 patients treated with tea preparation (1 liter/day with
5g of leaves) during 7 days : the parasite eradication in blood was 4,3% on D+1, 47,7% on D+2, 85,9% on D+3,
and 93,7% on D+14. Blanke (13) reported 10 cases; Mueller (56,57) reported two series including 53 and 72
patients. According to these papers an important clinical improvement was observed right from the second or
third day of treatment. In all studies parasite clearance was observed in 70% of cases at the 7th day. Tiruneh (76)
reported a study on 473 patients from whom 92% were cured.

However the authors observed a recurrence of the parasitemia at the 28th day in a number of
cases, ranging from 70% to 90%. Recurrence of the parasitemia is usually considered as a
treatment failure, even without recurrence of clinical symptoms. However a low parasitemia
without clinical symptoms is quite common in endemic areas and may increase immunity and
provide a minimal protection against subsequent infestations.
According to the study of Bottius (14) on a population in endemic area, 50 % of the evaluated subjects had a
microscopic parasitemia, and the number was 90% with more sensitive techniques (polymerase chain reaction).
Similarly Mueller (56), Silachamroon (72), Swarthout (75), Whitty (81) have observed between the 4th and 6th
week a parasitemia varying from 20% to 50%. Moreover after 6 weeks it may be difficult to attribute the
parasitemia to a recurrence (and a failure of the treatment) or to a reinfestation (the distinction requiring a
genotypic study of the parasite). In the complete study of Ashley (6) including 2926 children treated with ACT,
the parasitemia increased in 942 cases between the 14 th and the 28th day, attributed to a recurrence in 12% of
cases and to a reinfestation in 78% of cases. Farnert (23) observed a great variation during day and night of
parasite density, as well as genotypic profiles and stages of maturation of the parasite in the same patient.

Eventually a low parasitemia may be considered as insignificant if not associated to

recurrence of clinical symptoms. Thus, treatment of malaria with artemisinin tea preparation
may be advised when no other option is available (68), the aim of the treatment being not to
definitively eradicate the disease, but to cure the malaria attack and to avoid its complications.
Besides, some studies suggest that Artemisia annua has not only a curative property, but also
a preventive effect on malaria. In a longitudinal study during 8 months on 300 patients taking
a tea bowl once a week, Ogwang (61) observed a 80% decrease of symptoms attributable to
malaria. For this author, in another randomized study, the risk for malaria attack is decreased
by 50% (62).
2. THE FLAVONOIDS
The numerous investigations done after the discovery of artemisinin were centered on this
molecule, which probably has wrongly been considered as the only available molecule for
fighting malaria. Actually artemisinin is not alone in Artemisia annua tea preparation, but is
associated in a cocktail containing several other molecules of flavonoids (scopoletin,
17

casticin, chrysoplenetin, chrysosphenol-D, cirsilineol) (15). Flavonoids are present in

leaves, but also in the stem (87) of the plant, perhaps at a higher concentration. Contrary to
artemisinin, flavonoids are resistant to boiling water.
Flavonoids are powerful antioxidants (being iron chelators, converting Fe+++ in Fe++). Antioxidants may
interfere in malaria prevention. Akpotuzor (2) and Metzger (55) observed a decrease in plasma concentration of
antioxidants (vitamin A and C, carotenoids) in children with acute malaria, as well as an increase when they
recovered from the attack.

With moderate concentration flavonoids synergize the effect of artemisinin, maybe by

inhibition of enzymes catalyzing artemisinin (25).
Ashton (7) observed that, with a treatment by pure artemisinin at the same daily dosage, the plasma
concentration decreased with time, from 34% on the 4 th day of treatment to 24% on the 7th day of treatment (this
decrease in plasma concentration in artemisinin may explain the recurrence of the parasitemia observed in 2% to
50% of cases on the 28th day after treatment with pure artemisinin); he suggested a possible induction by the
artemisinin of production of auto-destructive enzymes. According to Ferreira (25) these enzymes (and especially
CYP450) are destroyed by the flavonoids present in Artemisia annua. Thus the flavonoids favour the efficacy of
the artemisinin molecules which remain active a longer time. Moreover the flavonoids should be synergistic of
artemisinin by favouring the penetration of artemisinin through the cellular membrane of the parasite.

With high concentration flavonoids have a direct antimalarial action on Plasmodium

Falciparum (85,91).
Some flavonoids (quercetin, luteolin, apigenin, 1.8 cineol) precociously inhibit the growth of Plasmodium
Falciparum at the intraerythrocyt trophozot phase (44,74); other as terpens (limonene or famesol) inhibit the
Plasmodium Falciparum development by their antioxydative power (10,29).

Similarly an important place is presently attributed to the polysaccharids which are present in
Artemisia annua (91). They should favour the solubility of artemisinin and flavonoids. They
have an anti-inflammatory effect. The sulfated polysaccharids should prevent invasion of
erythrocytes by the parasite at an early merozoit stage. On the contrary, the mono or
disaccharids (glucose, lactose) should be avoided because they increase the development of
the parasite, by favouring its metabolism. Thus addition of sugar or honey in the tea
preparation should be avoided, although this has been considered for reducing the bitterness
of the tea, making it more acceptable for children.
Therefore the treatment of malaria with tea preparation is not a monotherapy, but rather is a
genuine polytherapy, and possibly the research has neglected this aspect of cocktail to the
benefit of artemisinin. As a polytherapy, the tea preparation may explain some paradoxes
existing between the use of pure artemisinin and the use of Artemisia annua:
- The doses of artemisinin in tea preparation are quite smaller than the recommended doses
with extracted artemisinin and nevertheless results are at least similar.
- It is known that the molecule of artemisinin has a very poor solubility in water and
nevertheless it is present in great concentration in the tea preparation.
- The plasmodium falciparum became resistant to artemisinin or its derivatives, even
associated to conventional slow-acting drugs (6,79). The first observations of acquired
resistance were observed in Cambodia with artesunate (18). On the contrary, no observation
of acquired resistance has been reported after treatment with tea preparation. This may
explain that the Plasmodium Falciparum did not develop any resistance to the tea after two
millenniums of use of tea preparation in China.

18

- The artemisinin concentration is very variable according to the different varieties of

Artemisia annua, from 0,1% up to 1% (for the bresilian variety), and nevertheless with similar
results in treatment of malaria.
So, there are several advantages to polytherapy. The different components act synergically, as
shown by de Donno (17), for whom the tea is three times more effective than should be the
same quantity of artemisinin alone. Moreover it seems that powdered Artemisia annua,
providing the whole plant, is more effective.
Elfawal and coll (22) observed that in mice fed with dried plant the artemisinin absorption is 40 times higher
than in mice fed with pure artemisinin. With a quantity of 24 mg/kg (dried plant), they observed an almost
complete decrease of the parasitemia 30 hours after absorption. During a recent surgical session in Central
Africa, we used powdered leaves in capsules as preventive treatment given before surgery during two and half
days in 27 patients, most of them children. The amount of powder was 400 to 500 mg. After 36 hours the
average parasitemia was reduced from 429 parasites/ml to 153 parasites/ml, i.e. 64% improvement. In this
experience, the amount of artemisinin was very low (0, 4 to 0, 5 mg per day, or about 16 mg/kg), and possibly a
higher dosage may have given a complete disappearance of the parasites in the blood. These results are in
agreement with those of the International Center of Insect Physiology and Ecology (84): 47 patients were treated
during 6 days with capsules containing 500 mg of Artemisia annua. The parasitemia which was positive in all
cases when the treatment started was equal to zero in 90% of cases between the 3 rd and 5th day.

These results confirm the advantages of administration of the whole plant, with a faster and
more complete absorption of artemisinin, with inhibition of hepatic and digestive enzymes
catalysing artemisinin, with antimalarial synergy between artemisinin and flavonoids, with
specific antimalarial action of the flavonoids.

19

ADMINISTRATION OF ARTEMISININ

1. The tea
1.1. The tea preparation.
The recommended quantity of leaves is 5 to 7 grams in one litre of boiling water (2 to 3
pinches or 2 teaspoons). A plastic or glass container should be used for infusing, avoiding an
iron container (because iron reacts with artemisinin). Boiling water should be poured on the
leaves (boiling the tea should be avoided because destroying the artemisinin), then stirring the
mixture with a wood spoon, then keep infusing during 10 minutes the container being
covered. Then the tea should be filtered, avoiding again an iron strainer, the leaves should be
squeezed, and lastly the tea should be left for cooling during 10 minutes.
However the duration of preservation of artemisinin in Artemisia annua tea preparation is
short once the tea has been prepared, especially if kept at ambient temperature (35), and tea
should be daily prepared for treatment.
1.2. The tea dosage
For adults, the recommended dosage is 1 litre per day divided in 4 doses of 250 ml..
For children, the dosage should be reduced to 15-20 ml/kg: i.e. for 30 kg weight, 500 ml
divided in 4 doses of 125 cc (or 4 times one full yogurt pot), or for 7 kg weight 120 ml
divided in 4 doses of 30 cc (i.e. 4 times yogurt pot).
The artemisinin is well tolerated and no adverse effect has been observed. There is no paper in
literature reporting complications after treatment by artemisinin or derivatives. Tea per os is
the simplest way for administration, according to the above described protocol. However,
when oral anti-malarial therapy cannot be given, as in uncompliant patients or patients with
cerebral malaria, rectal administration may be used.
This has been investigated by the World Health Organization (83). Artemisinin by rectal administration has
advantage to avoid any of the complications commonly observed after parenteral quinine treatment (66). Many
studies have confirmed the effectiveness of rectal artemisinin (8,12,28,38,39,53), rectal artemisinin initiating
parasite clearance more rapidly than oral treatment (parasite clearance time is about 24h), and being more active
and safer than parenteral quinine, even in severe illness. According to Koopmans (39) the artemisinin blood
concentration after rectal administration is higher than after oral administration. In the study of Gomes (28) made
on 1167 patients, a single massive dose was 5 times more effective than repeated moderate dosages for reducing
the parasitemia more than 90% at 24 hours. According to the meta-analysis of Karunajeewa (38) reviewing 39
papers, artemisinin by rectal administration appear to have an acceptable therapeutic efficacy, even if the clinical
outcome is not superior to that of conventional treatment. Thus, intrarectal artemisinin may be considered as a
potential standard treatment which may replace conventional parenteral quinine.

Suppositories of extracted artemisinin as well as its derivatives are currently available, but the
cost may be prohibitive for poor families, and extracted artemisinin may be replaced by tea
preparation, with a protocol similar to oral tea preparation, i.e for an adult 5-10 grams of
leaves infusing 10 minutes in one liter of boiling water, to repeat the following days. Tea
preparation is available at affordable price. It should be administered as a drop-by-drop, using
a rectal cannula, giving chance to rehydrate of the child, and resulting in a no-cost treatment.
20

2. The Artemisia annua powder

Administration of powdered leaves has advantage to deliver the whole plant, allowing
absorption of the totum, i.e all of the molecules present in stem and leaves.
2.1. The powder preparation : the plant should be very meticulously pulverised; this can be
done with a standard mixer, but is more difficult to achieve in an endemic area with the
traditional pestle.
2.2. The powder administration:
2.2.1. The powder may be administered in capsules, which are easy to ingest. In case of
cerebral malaria or in uncompliant patients, rectal administration of capsules may be used as
well. However capsules may be not routinely available in remoted areas and are more costly.
So the use of capsules is possible when they are prepared in advance and brought into the area
of distribution, but it is imperfectly fitted for a program of local production or use on a large
scale.
2.2.2. An alternative to the use of capsules is to directly administrate the powdered Artemisia
annua mixed to the aliments (yogurt, peanut butter...). The powdered Artemisia annua
probably is more effective than the tea preparation and this way of administration has to be
promoted.

21

CONCLUSION

It is universally admitted that extracted artemisinin is almost 100% effective in the treatment
of malaria. It is also admitted that the artemisinin production is insufficient for matching the
world needs, and lastly that the cost is unacceptable for most of endemic areas.
Some free-of-charge programs of distribution of ACT have been developed for children and
pregnant women, but these programs contribute in maintaining the targeted populations in a
situation of assistance... On the other hand treatments with Artemisia Annua are effective
more or less at 100%, either with tea or powdered leaves. Artemisinin tea preparation has
advantage to be inexpensive and economically acceptable for most of populations, and to be
easy to prepare and to take. Another advantage is the possibility of having locally grown herb,
avoiding the uncertainties of routing or dispatching and shortage in supply. Either oral or
rectal, either with tea or powdered leaves, Artemisia annua is acceptably effective for
treatment of falciparum malaria, provided that the duration of treatment is convenient. This is
an objection to be kept in mind as this necessary duration of the treatment may be poorly
compatible with the frequent low compliance of the patients or families. Despite its
imperfections, treatment of malaria with Artemisia Annua tea preparation is an acceptable
option, with advantages to the tea preparation for large scale diffusion.

22

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