Biotechnol Lett (2007) 29:11431146

DOI 10.1007/s10529-007-9368-8

ORIGINAL RESEARCH PAPER

Improvement of artemisinin production by chitosan in hairy

root cultures of Artemisia annua L.
Waraporn Putalun Wanwimon Luealon
Wanchai De-Eknamkul Hiroyuki Tanaka
Yukihiro Shoyama

Received: 4 December 2006 / Revised: 26 February 2007 / Accepted: 1 March 2007 /

Published online: 11 April 2007
Springer Science+Business Media B.V. 2007

Abstract Artemisinin production by hairy roots of

Artemisia annua L. was increased 6-fold to
1.8 mg mg 1 dry wt over 6 days by adding
150 mg chitosan l 1. The increase was dose-dependent. Similar treatment of hairy roots with methyl
jasmonate (0.2 mM) or yeast extract (2 mg ml 1)
increased artemisinin production to 1.5 and
0.9 mg mg 1 dry wt, respectively.
Keywords Artemisia annua L.
Artemisinin

Elicitors
Hairy roots

Introduction
Artemisia annua L. (wormwood or sweet wormwood), an important medicinal plant of the family

W. Putalun (&)
W. Luealon

Faculty of Pharmaceutical Sciences, Khon Kaen
University, Khon Kaen 40002, Thailand
e-mail: waraporn@kku.ac.th
W. De-Eknamkul
Faculty of Pharmaceutical Sciences, Chulalongkorn
University, Bangkok 10330, Thailand
H. Tanaka
Y. Shoyama
Graduate School of Pharmaceutical Sciences, Kyushu
University, 3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan

Asteraceae, contains an antimalarial sesquiterpene

endoperoxide, artemisinin, which is effective
against both chloroquinine-resistant and -sensitive
strains of Plasmodium falciparum, and is thus
useful for the treatment of cerebral malaria (Klayman
1985). However, the low concentration (0.01
0.6% dry wt) of artemisinin in A. annua limits its
commercialization. Therefore, enhancement of artemisinin production, either in tissue culture or in the
breeding of A. annua, is the aim of many research
groups. As the concentration of artemisinin in hairy
roots transformed with Agrobacterium rhizogenes
remained low (Weathers et al. 1994; Paniego and
Giuliette 1996), the productivity of artemisinin may
depend on the choice of transformed root clones,
basal media and culture conditions. One approach
is the promotion of plant tissue metabolism by
elicitors. Elicitors induced accumulation of secondary metabolites have received wide acceptance
because of its ability to improve productivity of
the plant tissue cultures significantly (Wang et al.
2002; Zhao et al. 2005; Ali et al. 2006). Liu et al.
(1999) reported that elicitors, derived from mycelial
extracts of Penicillium chysogenum enhanced artemisinin production in hairy roots of A. annua
1.2-fold higher than that of the control experiment.
In this study, the effects of chitosan, methyl
jasmonate and yeast extract on artemisinin accumulation in A. annua hairy root cultures have been
investigated.

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Materials and methods

Chemicals and immunochemicals
Human serum albumin (HSA) was provided by
Pierce (USA). Peroxidase-labeled anti-mouse IgG
was provided by ICN Pharmaceuticals (USA). Artemisinin was purchased from Sigma. All other chemicals were standard commercial products of analytical
grade.
Plant materials and hairy roots induction
Artemisia annua seeds were washed with sterile,
distilled water and were surface-sterilized in 10% (w/v)
sodium hypochlorite for 20 min. After being washed
three times with sterilized water, the seeds were
immersed in 70% (v/v) ethanol for 1 min and then
germinated on hormone-free Murashige and Skoog
(MS) medium containing 3% (w/v) sucrose, pH 5.5 at
25 18C for 16 h under a fluorescent light. Plantlets
were used as material for induction of hairy roots.
The derived stems were infected with Agrobacterium
rhizogenes ATCC 15834 and cultured on MS at 258C
for 2 days and then transferred to half-strength MS
with Cefotaxime, 500 mg l 1. After three passages on
to new antibiotic-containing medium at 2 weeks
intervals, the bacterial-free hairy roots were transferred into half-strength MS liquid medium. The
medium was shaked (100 rpm) at 258C with light for
16 h day 1. The hairy roots were subcultured every
3 weeks into fresh medium.
Growth of hairy roots and artemisinin content
Fully growth hairy roots were subcultured in 125 ml
flasks containing 30 ml half-strength MS liquid
medium. Hairy roots were harvested every 5 days
to determine the dry weight and the artemisinin level
by ELISA using anti-artemisinin monoclonal
antibody (MAb). Each experiment was done with
triplicate.
Effect of elicitors on artemisinin production
in hairy roots
Elicitation was carried out with yeast extract, methyl
jasmonate and chitosan. Each elicitor in the following
final concentration was added: (1) methyl jasmonate

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Biotechnol Lett (2007) 29:11431146

(MJ) 50, 100 and 200 mM; (2) yeast extract (YE) 0.5,
1 and 2 mg ml 1; (3) chitosan 50, 100 and
150 mg l 1. Various concentrations of elicitors were
added to 21 day-old hairy root cultures and were
harvested every 2 days from 2 to 6 days. Each
experiment was done in triplicate.
Sample preparation and analysis
Hairy root cultures were dried and ground. Hairy
roots powder, 30 mg, was extracted with 400 ml
petroleum ether five times using an ultrasonic bath
for 15 min. After filtration and evaporation to
dryness, the residue was resuspended in 1 ml methanol. Artemisinin equivalents in sample solutions
were determined by a competitive ELISA using a
monoclonal antibody against artemisinin (Putalun
et al. 2006). Artemisinin-HSA (100 ml, 1 mg ml 1)
was adsorbed onto 96-well immunoplate and then
treated with 300 ml phosphate buffered saline (PBS)
containing 5% (w/v) skim milk (S-PBS) for 1 h. Fifty
ml of various concentrations of artemisinin, or
samples that had been diluted in 20% (v/v) methanol,
were incubated with 50 ml monoclonal antibody
solution for 1 h. The plate was washed three times
with PBS containing 0.05% (v/v) Tween 20 (T-PBS),
and then incubated with 100 ml of a 1,000-fold
dilution of peroxidase-labeled goat anti-mouse IgG
for 1 h. After washing the plate with T-PBS, 100 ml of
substrate solution [0.1 M citrate buffer, pH 4.0,
containing 0.003% (v/v) H2O2 and 0.3 mg ml 1
ABTS] was added to each well and incubated for
15 min. The absorbance was measured by microplate
reader at 405 nm. All reactions were carried out at
378C.

Results and discussion

Hairy roots were emerged from infection sites and the
percentage of transformed root was about 80% after
2 weeks. The growth rate of A. annua hairy roots and the
artemisinin production are shown in Fig. 1. The hairy
roots grew faster over 15 days and gave the highest dry
weight (105 mg flask 1) at day 30. The artemisinin
contents increased from day 10, and reached its
maximum level (0.28 0.02 mg mg 1 dry wt) at day
25. These results resembled the observation of Liu et al.
(1999) who showed that the maximum peak of

140
0.30
120
0.25
100
0.20
80
0.15

60

0.10

40

0.05

Dry weight
Artemisinin

20
0
3

15

20

25

Control
YE 0.5 mg ml-1
YE 1.0 mg ml-1
YE 2.0 mg ml-1

0.8
0.6
0.4
0.2

Day 2

Day 4

Day 6

Fig. 3 Effect of yeast extract (YE) on artemisinin production

in hairy roots. Yeast extract (0.5, 1 and 2 mg ml 1) was added
to 21-day-old hairy root cultures and cultivated at 258C with
light 16 h day 1 for a further 2, 4 or 6 days. Values are means
of triplicate results and error bars show standard deviations

2.0

.2.5
Control
MJ 50 M
MJ 100 M
MJ 200 M

Artemisinin ( g mg -1 dry wt)

1.6

1.0

30

Fig. 1 Time-course for growth and artemisinin production in

A. annua hairy roots cultured at 258C with light 16 h day 1.
Values are means of triplicate results and error bars show
standard deviations

1.8

1.2

0
10

Time (day)

Artemisinin ( g mg -1 dry wt)

Artemisinin (g mg -1 dry wt)

1145

Artemisinin ( g mg -1 dry wt)

Hairy roots (mg dry wt flask-1)

Biotechnol Lett (2007) 29:11431146

1.4
1.2
1.0
0.8
0.6
0.4
0.2

Control

2.0

Chitosan 50 mg l-1
Chitosan 100 mg l-1
Chitosan 150 mg l-1

1.5

1.0

0.5

0
Day 2

Day 4

Day 6

Fig. 2 Effect of methyl jasmonate (MJ) on artemisinin

production in hairy roots. Methyl jasmonate (50, 100 and
200 mM) was added to 21-day-old hairy root cultures and
cultivated at 258C with light 16 h day 1 for a further 2, 4 or
6 days. Values are means of triplicate results and error bars
show standard deviations

artemisinin (450 mg l 1) accumulation was induced in

hairy roots at day 24. After 25 days, the dry weight
decreased due to the depletion of nutrient. The stronger
stimulation of secondary metabolite formulation by an
elicitor probably occurs in the late growth stage (Chen
et al. 2006). In this study, we choose the 21-day hairy
root cultures for elicitor experiments.
Figure 2 shows the effect of methyl jasmonate on
the artemisinin production in hairy roots. Methyl
jasmonate at 200 mM gave the highest concentration
of artemisinin (1.52 0.32 mg mg 1 dry wt) after
4 days which was 5-fold higher than the control hairy
roots. Ali et al. (2006) has also reported the induction
of ginsenosides (triterpene saponin) in Panax ginseng

Day 2

Day 4

Day 6

Fig. 4 Effect of chitosan on artemisinin production in hairy

roots. Chitosan (50, 100 and 150 mg l 1) was added to 21-dayold hairy root cultures and cultivated at 258C with light
16 h day 1 for a further 2, 4 or 6 days. Values are means of
triplicate results and error bars show standard deviations

hairy roots by methyl jasmonate. Yeast extract at

2 mg ml 1 also increased artemisinin production
(0.95 0.01 mg mg 1 dry wt) after 6 days in a dosedependent manner (Fig. 3).
The effect of chitosan (50150 mg l 1) on the
artemisinin production in hairy roots is shown in
Fig. 4. Chitosan at 150 mg l 1 gave the highest
content of artemisinin (1.84 0.02 mg mg 1 dry wt)
after 6 days in a dose-dependent which was 6-fold
higher than the control hairy roots. A direct correlation between incubation times (26 days) and the
artemisinin yield was observed.
Several studies have reported that artemisinin
production is stimulated by elicitors such as a fungal
eilicitor and (22S, 23S)-homobrassinolide (Lui et al.

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1146

1999; Wang et al. 2002). However, we have now

established that chitosan, methyl jasmonate and
yeast extract can act as stimulants. Artemisinin
production in hairy roots was increased 6-fold to
1.84 0.02 mg mg 1 dry wt over 6 days by adding
150 mg chitosan l 1. Hairy roots treatment with
methyl jasmonate at 200 mM or yeast extract at
2 mg ml 1 also increased artemisinin production to
1.52 0.32 and 0.95 0.01 mg mg 1 dry wt, respectively. From a biotechnological point of view, the
productivity increment after chitosan treatment is of
practical value. Our present work could be recommended approach for artemisinin production in a large
scale cultures of A. annua hairy roots in bioreactor.
Acknowledgements This work was supported by a grant
from the National Center for Genetic and Biotechnology
(BIOTEC), Thailand and Asian Core Program (Medicinal Plant
Breeding Division) under the Japan Society for the Promotion
of Science (JSPS).

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Biotechnol Lett (2007) 29:11431146

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