SALI NI TY STRESS

Effect of Chemical and Physical Stress Conditions on the

Concentration and Composition of Essential Oil Components
in Leaves of Full-Grown Artemisia annua L.
E. Ivarsen
1
, A. Kjr
2
, M. Jensen
2
, K. Grevsen
2
, L. P. Christensen
1
& X. Frett e
1
1 Department of Chemical Engineering, Biotechnology and Environmental Technology, Faculty of Engineering, University of Southern Denmark,
Odense, Denmark
2 Department of Food Science, Faculty of Science and Technology, Aarhus University, Aarslev, Denmark
Keywords
Artemisia annua; chemical stress; essential oil
components; gas chromatography; mass
spectrometry; physical stress
Correspondence
E. Ivarsen
Department of Chemical Engineering,
Biotechnology and Environmental
Technology, Faculty of Engineering,
University of Southern Denmark,
Niels Bohrs Alle 1, DK-5230 Odense,
Denmark
Tel.: +4565507361
Fax: +4565507354
Email:eli@kbm.sdu.dk
Accepted April 16, 2013
doi:10.1111/jac.12030
Abstract
Full-grown Artemisia annua plants were subjected to chemical and physical stress
conditions, and the effect of these on the concentration and chemical composi-
tion of essential oil components (EOC) in the leaves was studied. The chemical
stress treatments were performed by foliar application of NaCl, H
2
O
2
, salicylic
acid and chitosan oligosaccharide (COS). The EOC of the leaves were extracted
with n-hexane and identied and quantied by GCMS and GCFID, respec-
tively. Approximately 96 % of EOC in the extracts were identied and quantied
of which b-pinene, camphene, germacrene D, camphor, coumarin and dihydro-
epi-deoxyarteannuin B were the major EOC accounting for about 75 % of the
total content of EOC in the extracts. The physical stress treatment, sandblasting
of the plants resulted in a signicant enhancement in the content of a-pinene,
camphene, coumarin and dihydro-epi-deoxyarteannuin B. The total yield of
identied EOC in non-treated plants (control) was 86.2 13.8 lg g
1
fresh
weight (FW) compared with 104.0 9.1 lg g
1
FW in sandblasted plants. The
chemical stress treatments did not affect the composition of EOC signicantly.
The results indicate that chemical stress treatments do not affect the concentra-
tion and composition of EOC in full-grown A. annua plants to the same extent
as physical stress treatment by sandblasting.
Introduction
Artemisia annua L. (A. annua) also known as Sweet Worm-
wood, is an annual herb belonging to the Asteraceae family,
which has been used for centuries in Chinese traditional
medicine for the treatment for cerebral fever and malaria,
but was falling-out of common use, until its use was redis-
covered in 1970s, which led to the discovery of the anti-
malarial sesquiterpene artemisinin and its semisynthetic
derivative dihydroartemisinin (Ferreira et al. 2005, Brown
2010). Artemisinin and dihydroartemisinin are effective
antimalarial drugs against both chloroquine-resistant and
chloroquine-sensitive strains of Plasmodium falciparum as
well as against cerebral malaria (Ferreira et al. 2005, Brown
2010). Artemisia annua is well known for its high content of
essential oil components (EOC) as well as for its high con-
tent of artemisinin (Ferreira et al. 2005, Lommen et al.
2006, Brown 2010, Kjr et al. 2012). These secondary
metabolites are accumulated in glandular trichomes located
on the outside of leaves and owers of the plants. Although
A. annua is native to Asia, it is now cultivated in many
parts of the world and even successfully in temperate
regions such as Denmark and the Netherlands (Ferreira
et al. 2005, Liu et al. 2010, Damtew et al. 2011). The con-
tent of essential oil (EO) in A. annua is variable and has
been reported in values of 0.020.5 % of dry weight (Ferre-
ira et al. 2005). The content depends on the plant material
(cultivar, variety), age, growing conditions, as well as sea-
sonal and geographical variations (Olsson et al. 2009). Fac-
tors such as temperature changes and stress treatments can
be applied to affect the content of secondary metabolites of
plants (Malik et al. 2009). It has been proposed that the
majority of EOC are produced in the glandular trichomes
together with artemisinin (Goel et al. 2007, Khodakov and
2013 Blackwell Verlag GmbH, 199 (2013) 395404 395
J Agro Crop Sci (2013) ISSN 0931-2250
Kotikov 2009). The EO of A. annua has been thoroughly
studied, and hundreds of components have been identied
where the major components are sesquiterpenes (e.g. ger-
macrene D, b-caryophyllene), monoterpenes (e.g. artemisia
ketone, camphor, 1,8-cineole) and phenylpropanoid deriva-
tives (e.g. coumarin, scopoletin, eugenol) (Lommen et al.
2006, Brown 2010). The EO of A. annua is used as pharma-
ceuticals due to its fungicidal and antimicrobial properties
(

Cavar et al. 2012, Engberg et al. 2012). The EO of

A. annua has shown signicant inhibition on several bacte-
ria strains among these are Staphylococcus aureus (Mini-
mum inhibitory concentrations (MIC) = 15.5 ppm),
Escherichia coli (MIC = 31.3 ppm), Bacillus substillis
(MIC = 7.8 ppm) and B. thuringiensis (MIC = 15.6 ppm)
(Li et al. 2011). The EO tested against these four bacteria
strains contained 50 % terpenoids (Li et al. 2011); however,
the components responsible for the antibacterial effects have
not been determined, but the activity is probably caused by
a combination of different EOC that exert their antibacterial
effect due to synergistic interactions.
Plants respond to stress by various biochemical defence
mechanisms, for example by producing secondary metabo-
lites, which are toxic to insects, micro-organisms and/or
herbivore repellent. It has been demonstrated that applied
stress can change the composition of EOs of medicinal
herbs such as Mentha piperita, Origanum vulgare and Oci-
mum basilicum (Banchio et al. 2005, Karray-Bouraoui
et al. 2009, Taarit et al. 2009, Khadhri et al. 2011). Stress
treatments in these experiments were selected to represent
a broad range of stress-inducing agents. Physical stress was
applied by sandblasting resulting in mechanical damage of
the plants mimicking a possible predation by sucking or
leaf eating insects. Chemical stress was applied with four
different chemicals, mimicking four different stress condi-
tions of the plants. Sodium chloride (NaCl) was applied to
affect the plants by osmotic stress. The elicitor chitosan oli-
gosaccharide (COS) was applied to mimic stress conditions
by triggering the recognition of molecules associated with
insect and fungus attacks (Zheng et al. 2010, Lei et al.
2011). Hydrogen peroxide (H
2
O
2
) was applied to mimic
the bursts of reactive oxygen species (ROS) triggered by the
plants under stress (Neill et al. 2002, Mittler et al. 2011),
and the hormone salicylic acid was applied because it is
naturally directly involved in the internal stress manage-
ment of the plants (Pu et al. 2009). Salicylic acid is a com-
mon chemical elicitor used on a large variety of plants to
modify their production of secondary metabolites. Salicylic
acid has been reported to affect the EO composition of Oci-
mum basilicum and Majorana hortensis. In these two spe-
cies, the yield of terpenes and sesquiterpenes was increased
(Gharib 2007), whereas Mentha piperita treated with sali-
cylic acid decreased the terpenoid and the total EO content
(Zheljazkov and Astatkie 2012). Foliar application of NaCl
increased the total amount of EO of Pelargonium sp. (Pra-
sad et al. 2008); Salvia ofcinalis and Mentha pulegium
grown in medium with low salt concentration were also
shown to increase their production of EO (Karray-Boura-
oui et al. 2009, Taarit et al. 2009) in contrast to Origanum
majorana grown in medium with high salt concentration,
which had a decreased EO production (Ba^atour et al.
2011). COS, at a maximum concentration of 0.4 %, has
been shown to increase the total amount of linalool, 1,8-
cineole and six other terpenoids in Ocimum basilicum with
45.0 % (Kim et al. 2005). In Origanum vulgare ssp. hirtum,
it has been demonstrated that treatment with H
2
O
2
or COS
has a tendency to increase the production of secondary
metabolites, but the effect was not signicant (Yin et al.
2012). Mechanical wounding has been shown to change the
composition of volatile components in Ocimum minimum
leaves. After 24 h treatment, the content of linalool was
increased by 2.4 % while camphor and eugenol decreased.
A suggestion for the decrease is that they undergo catabo-
lism after wounding (Zabaras and Wyllie 2001). Stress
experiments on A. annua have been conducted on young
plants, and the main focus of applying stress to A. annua
has been to enhance the production of artemisinin in
young and/or in the corresponding full-grown plants,
which have been carried out successfully with increases in
artemisinin content up to 50 % (Aftab et al. 2010a,b, Guo
et al. 2010, Lei et al. 2011). The effect of the selected stress
treatments on the content of EOC in A. annua has not pre-
viously been reported in young and/or in full-grown plants.
The yield of secondary metabolites per hectare is much
higher in full-grown compared with young plants primarily
due to large differences in size of the plants, and thus, it
would be interesting to investigate the effect of stress treat-
ment on the production of secondary metabolites in full-
grown A. annua plants. Based on the results from various
experiments with stress treatments on the production of
artemisinin in A. annua plants, it is hypothesized that
stress treatments may also lead to an increased concentra-
tion of, for example EOC in full-grown A. annua plants.
The purpose of the present study was to investigate the
above-mentioned hypothesis, which was carried out by
examining the effect of different chemical stress treatments
(NaCl, salicylic acid, H
2
O
2
, COS) and one physical stress
treatment (sandblasting) on the content of EOC in full-
grown A. annua plants. After stress treatments for 5 weeks
of full-grown A. annua plants, the main stem leaf material
of the plants was extracted with n-hexane and the extracts
investigated for their EO constituents and their content of
EOC compared with non-treated plants grown under exact
the same conditions (controls). The EOC in extracts were
identied by gas chromatographymass spectrometry
(GCMS) and quantied by GCame ionization detection
(FID).
2013 Blackwell Verlag GmbH, 199 (2013) 395404 396
Ivarsen et al.
Materials and Methods
Chemicals and reagents
Chitosan oligosaccharides powder (degree of polymeriza-
tion = 210, degree of deacetylation >95 %) was obtained
from Dalian GlycoBio Co., Ltd. (Dalian, China). Salicylic
acid was purchased from SigmaAldrich (S7401, Stein-
heim, Germany). Ten percent Hydrogen peroxide (H
2
O
2
)
was purchased from Matas (Alleroed, Denmark). n-Hexane
was analytical grade purchased from Rathburn Chemicals
Ltd. (Walkerburn, Scotland). n-Octadecane (GC-standard
grade, >99.8 %) and authentic standards were purchased
from SigmaAldrich.
Plant material
From a eld population of seed propagated Artemisia
annua (cv. Artemis, F2 seeds, Mediplant, Switzerland), two
plants were randomly selected as mother plants for clonal
propagation by cuttings, and sufcient plants were
obtained during two rounds of multiplication carried out
in a greenhouse. Tip cuttings were ca. 10 cm long and con-
sisted of 45 internodes longer than 1 cm. Cuttings had
rooted after 23 weeks and were potted in 3.5 L containers
with Pindstrup No. 2 peat moss (Pindstrup Mosebrug,
Denmark). Plants were transferred from the nursery to the
experimental greenhouse and allowed to acclimatize for
7 days before the onset of treatments. Plants were 9 weeks
from propagation and 80 to 110 cm in height at the onset
of the treatments and 14 weeks and 150 to 190 cm in
height at sampling. During the experiment, plants were
drip-irrigated twice diurnally with a liquid fertilizer. The
liquid fertilizer was developed specically for cultivation of
Asteraceae plants, for example Asters, and had the follow-
ing main characteristics: pH 5.7; EC: 2.16 l Siemens; N:
206 ppm (NH

4
: 40 ppm, NO

3
: 165 ppm), P: 44 ppm, K:
257 ppm, Mg: 11 ppm, Ca: 138 ppm, SO
2
4
: 37 ppm, Fe:
0.91 ppm, Mn: 0.84 ppm, B: 0.33 ppm, Sn: 0.3 ppm and
Mo: 0.08 ppm. Night temperatures ranged from 8 to 12 C
and day temperatures from 10 to 32 C. Humidity was
medium to high, following patterns of air exchange with
fresh air controlled by temperature set points. Only natural
sunlight was supplied.
Greenhouse experiment
The experiment was carried out during April and May,
2010 at Department of Food Science, Aarslev, Denmark.
The experimental setup of plants and stress treatments was
carried out as described in literature (Kjr et al. 2012,
2013) and is briey repeated here. A randomized complete
block experimental design with subsampling was set up in
a greenhouse divided into two self-contained compart-
ments. In each compartment, three beds of plants were
established, each bed consisting of 3 9 23 plants of one of
the two clones. Plants were interspaced by 50 cm, and indi-
vidual beds were separated by 100 cm of gravel. Every sec-
ond row of three plants were left as guard plants, allowing
for 11 treatable subplots of three plants in each bed (2
blocks 9 2 clones 9 3 plants = 12 subplots per treatment).
Treatments were performed weekly for 5 weeks and
included NaCl at 10 g L
1
aqueous solutions, salicylic acid
at 1.0 g L
1
aqueous solutions, H
2
O
2
at 1.0 %, COS at
1.0 g L
1
, sandblasting (Mini Sandblaster, Model 260-3,
Badger Airbrush Co., IL, USA) and control plants with no
treatment. A manual garden vaporizer (Gardena) delivered
1 mL liquid per spray, and 24 sprays covered the majority
of a plant in a water lm. To compensate for the growth
during the 5 week treatment period, two additional sprays
were added each week (32 sprays at last treatment). Sand-
blasting was carried out for 2 9 15 s at the onset of the
experiment and 2 9 20 s at the last treatment. Treatments
were carried out during the late afternoon to minimize any
sun scalding effect from water droplets on the leaf surface.
Seven days after the last treatment, an upper leaf was
dened as the rst leaf below the apex on the main stem
with internodes longer than 2 cm. This upper leaf and the
two leaves below were collected for other studies (Kjr
et al. 2012, 2013), and for the present study, the 45 leaves
below were collected, vacuum-packed and frozen at -20C
until extraction. The entire plants, including the collected
leaves from each treatment, were treated with stress chemi-
cals or directly treated physically by sandblasting.
Extraction of essential oil components
EOC were extracted from fresh-frozen leaf samples of
A. annua with n-hexane. Approximately 2 g leaves from
each sample (accurate weight determined for each sample)
were weighed in a 10-mL vial and extracted with n-hexane
(4.0 mL) and then subjected to sonication for 99 min and
stored at 5 C for 12 h until analysis. The extracts were l-
tered (0.45 lm, nylon), and the internal standard n-octa-
decane (140.8 lg per sample) was added before GC and
GCMS analysis. The extraction method was validated after
analysing the content of the main EOC after 3 successive
extractions with n-hexane or dichloromethane in triplicate.
According to these preliminary extraction experiments,
more than 95.0 % of the EOC were extracted after the rst
extraction with n-hexane. Similar extraction experiments
with dichloromethane demonstrated that n-hexane
extracted more of the EOC; hence, n-hexane was chosen as
the extraction solvent for EOC from A. annua leaves. Fur-
thermore, it was demonstrated that the standard deviation
of the used extraction method was <5.0 %.
2013 Blackwell Verlag GmbH, 199 (2013) 395404 397
Effect of Stress Treatments on Essential oil Components in A. annua
Identication of essential oil components in leaf extracts
by gas chromatographymass spectrometry (GCMS)
The GCMS analysis of the EOC was performed with a
Thermo Scientic DSQ II single-quadrupole mass spec-
trometer coupled to a trace GC ultra gas chromatograph
(Thermo Fisher Scientic, Waltham, MA, USA) operating
in EI mode at 70 eV, tted with a Zebron ZB WAXplus
TM
column (60.0 m 9 0.25 mm 9 0.25 lm, Phenomenex,
Torrance, USA). Temperature programme was as follows:
4560 C at 4 C min
1
, 6064 C at 2 C min
1
, 64
90 C at 2.6 C min
1
, 90110 C at 4 C min
1
, 110
150 C at 1.50 C min
1
with an isothermal step at 150 C
for 2 min and nally from 150 to 230 C at 3 C min
1
with an isothermal step at 230 C for 15 min. The carrier
gas was He, with a ow rate of 50 mL min
1
. The injection
volume was 1 lL with a split ratio of 50:1. Identication
was based on comparison with the relative retention indices
(RI) of the EOC, with literature values (Brown 1992, Mar-
ques et al. 2006, Khodakov and Kotikov 2009, Lei et al.
2011), mass spectra from the National Institute of Stan-
dards and Technology Database (NIST 2008) and by com-
parison with authentic standards except for germacrene D
and dihydro-epi-deoxyarteannuin B.
Quantication of essential oil components in leaf extracts
by gas chromatographyame ionization detection
(GCFID)
For quantication of the EOC, capillary GC was carried
out using a Hewlett-Packard (model 6890) chromato-
graphic system with a ame ionization detector (FID),
equipped with a Zebron ZB WAXplus
TM
column
(60.0 m 9 0.25 mm 9 0.25 lm, Phenomenex, Torrance,
CA, USA). The method used was the same as described for
GCMS. All samples were run with n-octadecane as inter-
nal standard (IS). The relative concentrations of the EOC
were quantied from the area of the IS and expressed as
lg g
1
plant fresh weight. The response factor was set to 1
for all compounds. The intraday variation was 4.5 %, and
interday variation was 2.5 %.
Statistical analysis
Each plant treatments were performed on 6 plants from
each clone, giving a total of 12 replicates. The mean was
compared by analysis of variance (ANOVA) followed by mean
separation by Students t-test. The difference between indi-
vidual means was dened to be signicant at P < 0.05. All
analyses were performed using the statistical analysis sys-
tem (SAS) JMP 9 software (SAS institute, Inc., Cary, NC,
USA). Standard error of means (S.E.M.) was calculated
using (SAS) JMP 9 software.
Results and Discussion
Essential oil components in leaf extracts
A representative GCFID chromatogram from the analysis
of an n-hexane leaf extract of Artemisia annua is shown in
Fig. 1. A total of 20 EOC were detected and quantied in
control plants and in different stress-treated samples of
A. annua; however, linalool and cis-sabinene hydrate could
not be fully separated and were quantied as one peak, and
p-cymene and 1,8-cineole showed large variation between
individual plants, which is also reected in the relatively
large variation of these constituents both in control plants
and stress-treated plants (Table 1, Fig. 1). The EOC in the
extracts were identied by comparison of their mass spec-
tral data with those from reference compounds or mass
spectra suggested by NIST database and GC retention indi-
ces (Table 1). The EOC have been reported previously as
constituents of A. annua (Khodakov and Kotikov 2009,
Brown 2010). The EOC consisted mainly of monoterpenes
and sesquiterpenes. The identied and quantied monot-
erpenes were tricyclene, a-pinene, camphene, b-pinene,
1,8-cineole, p-cymene, chrysanthenone, camphor, linalool,
cis-sabinene hydrate, linalyl acetate, borneol and myrtenol,
and the sesquiterpenes were b-caryophyllene, b-farnesene,
germacrene D, bicyclogermacrene, a-bisabolol and the ses-
quiterpene lactone dihydro-epi-deoxyarteannuin B
(Table 1, Fig. 2). The identied monoterpenoids and ses-
quiterpenoids accounted for approximately 51.5 % and
35.0 % of the total content of EOC, respectively. In addi-
tion, coumarin was identied as a major constituent
accounting for approximately 8.8 % of the total content of
EOC. The quantity of the identied EOC in leaves of
A. annua and their retention index are summarized in
Table 1.
Effect of stress treatments on essential oil components
In the present study, the chosen doses of stress-inducing
agents and frequencies of treatments reected what had
previously been shown to cause changes in glandular tri-
chome morphology or content of artemisinin and related
secondary metabolites in young A. annua plants (Pu
et al. 2009, Aftab et al. 2010a, Lei et al. 2011). However,
doses were increased in proportion to the physically lar-
ger plants, that is, full-grown A. annua plants. No toxic
effects on the plants were observed at the applied concen-
trations of the stress-inducing agents as shown previously
(Kjr et al. 2012, 2013). The effect of the various stress
treatments on individual EOC and total content of EOC
are shown in Table 1. The stress treatments in the pres-
ent study only showed limited effect on the content of
EOC. Plants treated with NaCl showed no signicant
2013 Blackwell Verlag GmbH, 199 (2013) 395404 398
Ivarsen et al.
effect in the content of EOC (Table 1). However, a
decrease in EOC has been observed in Origanum major-
ana treated with NaCl (Ba^atour et al. 2011). A decrease
in the content of EOC in the NaCl treatment may be
explained by an increased osmotic pressure, which may
affect the uptake of water and nutrients for the plant,
thus changing the physiological and biochemical potential
of the plant and hence the production of secondary
metabolites (Marschner 2012). Plants treated with COS
did not produce signicantly larger quantities of EOC as
compared with untreated controls (Table 1). Higher EOC
quantities have previously been demonstrated for COS-
treated Ocimum basilicum and Origanum vulgare (Kim
et al. 2005, Yin et al. 2012). Plants treated by sandblast-
ing did not produce higher quantities of total EOC,
monoterpenes or sesquiterpenes than the controls, but
the contents were signicantly different on single com-
pound level, for the compounds: a-pinene (P = 0.024),
camphene (P = 0.0128), coumarin (P < 0.0001) and
dihydro-epi-deoxyarteannuin B (P = 0.0220). In Ocimum
minimum, mechanical wounding signicantly decreased
the content of camphor and eugenol, but increased the
content of linalool (Zabaras and Wyllie 2001).
Glandular trichomes are found on the surface of many
plant species, including A. annua, and are known to store
and secrete both volatile and non-volatile compounds,
which primarily serve as protection against herbivores
and/or attraction of pollinators (Werker 1993, Tissier
2012). Glandular trichomes exhibit a great diversity, both
in their shape and the compounds they produce. This
diversity is expressed between species but also within spe-
cies or even individual plants (Tissier 2012). The latter
may explain the variation observed in EOC between indi-
vidual plants used in the present study and thus may
cover over small signicant increases in EOC in, for
example, the chemical treatments of individual plants.
The biosynthesis of EOC stored in glandular trichomes
takes place in secretory cells in the trichomes (Gershen-
zon et al. 1992). Therefore, an increase in the concentra-
tion of EOC due to external stress could arise from either
an increased activation of the biosynthesis of EOC in the
secretory cells and/or in the size and shape of the glandu-
lar trichomes. Recently, it has been shown that the stress
treatments used in the present study of full-grown
A. annua plants were not able to induce larger glandular
trichomes or increase the number of glandular trichomes
(Kjr et al. 2012). This may explain why the concentra-
tion of artemisinin and related compounds was not
increased in full-grown A. annua plants subjected to
external stress (Kjr et al. 2013). This indicates that an
increased production of EOC in full-grown A. annua
plants subjected to the physical stress treatment sandblast-
ing was most likely due to an increased activation of the
biosynthesis of EOC in secretory cells of the glandular
trichomes. The enhanced production of artemisinin
observed in young A. annua plants subjected to both
chemical and physical stress treatments could indicate
that young plants which have not fully established mature
glandular trichomes are more susceptible towards external
stress compared with full-grown A. annua plants. A
higher metabolic activity in the glandular trichomes of
young plants compared with full-grown plants may there-
fore be a plausible explanation of the differences observed
in the production of artemisinin in elicitor-treated young
A. annua plants compared with elicitation of full-grown
plants (Aftab et al. 2010a,b, Guo et al. 2010, Lei et al.
2011, Kjr et al. 2013). An increased production of arte-
misinin results from an increased biosynthesis of some
terpenoid precursors (Brown 2010, Lei et al. 2011, Kjr
et al. 2013). As the majority of EOC in A. annua are
Fig. 1 GCFID chromatogram of essential oil
components detected in A. annua. Peak num-
bers refer to those in Table 1. IS represents the
internal standard n-octadecane.
2013 Blackwell Verlag GmbH, 199 (2013) 395404 399
Effect of Stress Treatments on Essential oil Components in A. annua
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2013 Blackwell Verlag GmbH, 199 (2013) 395404 400
Ivarsen et al.
terpenoids (Table 1), it is here proposed that treatment
of young A. annua plants with external stress may also
result in relative larger increases in EOC compared with
full-grown A. annua plants.
The precise physiological roles of most plant monoterp-
enes have not been determined, but there is a general agree-
ment on that they play important roles in the interaction
between the plant and the environment. It has been dem-
onstrated that plants belonging to the Coniferophyta divi-
sion such as pines and cypresses secrete a complex mixture
of terpenes when they are attacked by insect predators. This
mixture includes the pinenes and myrcene that are toxic to
insects (Mahmoud and Croteau 2002). Furthermore, it has
also been demonstrated that linalool, camphene and 1,8-
cineole are involved in pollinator attraction and are
allelochemicals, the latter being a part of the plants defence
system against other plants (Mahmoud and Croteau 2002).
The enhancement seen in the concentration of monoterp-
enes in plants treated by sandblasting is probably compara-
ble with the response of plants to herbivore attack. It has,
for example, been shown that jasmonic acid and ethylene
are inducing the biosynthesis of volatile terpenoids in
barrel clover (Medicago truncalata) by herbivore attack and
thus these signalling pathways may be triggered by sand-
blasting leading to the increased production of terpenoid
EOC in A. annua (Arimura et al. 2008, Garms et al. 2008).
Fig. 2 Essential oil components in n-hexane
extracts of aerial parts of A. annua identied
and quantied in the present study.
2013 Blackwell Verlag GmbH, 199 (2013) 395404 401
Effect of Stress Treatments on Essential oil Components in A. annua
The increase in EOC observed with sandblasting treatment
is interesting as the EO of A. annua has clearly shown anti-
bacterial activity in vitro, against a wide spectrum of micro-
organisms (Li et al. 2011, Engberg et al. 2012). Thus, it
appears that some stress treatments may be able to increase
the concentration of secondary metabolites in A. annua
plants such as volatile terpenoids (Malik et al. 2009) as well
as antimalarial compounds, for example artemisinin (Liu
et al. 2010). This suggests that stress treatment can improve
the concentration and yield of bioactive compounds being
used as chemotherapeutic agents against infectious diseases
in humans and animals caused by micro-organisms and/or
parasites.
Dihydro-epi-deoxyarteannuin B was also signicantly
increased in sandblasted plants. Dihydro-epi-deoxyartean-
nuin B is one of the products from the metabolism of di-
hydroartemisinic acid in A. annua and has been suggested
to be a precursor to artemisinin. However, recent experi-
ments using dihydro-epi-deoxyarteannuin B labelled with a
stable isotope and fed to an intact plant system have not
shown any detectable incorporation into artemisinin
(Brown 2010). The precursor of dihydro-epi-deoxyartean-
nuin B and artemisinin appears to be the same, namely
dihydroartemisinic acid (Brown 2010), and therefore, an
increased production of dihydro-epi-deoxyarteannuin B
could indicate an increased production of artemisinin. This
is supported by the observation that mechanical wounding
of A. annua by damaging leaves and stems with surgical
blades has shown an enhanced production of artemisinin
(Liu et al. 2010). However, experiments designed to inves-
tigate the effect of stress treatments on artemisinin and its
biosynthetic precursors, in the same full-grown A. annua
plants as in the present study, did not show any signicant
increases in the level of these components in any of the
applied stress treatments including sandblasting. Within
the pool of artemisinin and related compounds, the treat-
ments caused a small shift in the conversion of precursors
towards artemisinin (Kjr et al. 2013). The mechanism by
which the plant responds to sandblasting wounding is not
yet known, and a compositional change in the EO of
A. annua, as a result of stressing the plants by sandblasting
has not previously been reported. Dihydro-epi-deoxyarte-
annuin B has been shown to exhibit intense antiulcerogenic
activity in rat ulcer models induced by indomethacin and
methanol (Marques et al. 2006), but its bioactivity in rela-
tion to plant defence, that is, its toxicity to insects and
micro-organisms is to the best of our knowledge unknown.
Salicylic acid and H
2
O
2
had no signicant effects on the
content of EOC (Table 1). The differences in the response
to stress treatments observed in the present study and com-
pared with former experiments on other plant species,
demonstrate that it is very difcult to predict how different
plants respond to various stress treatments. Thus, it is not
possible to compare or predict the results of stress treat-
ments of plants with regard to their production of second-
ary metabolites. Therefore, eld experiments have to be
performed for each individual plant species to elucidate
how the plant responds to stress treatments. In addition,
other parameters such as climate, temperature, soil, season,
development stage etc. probably also inuences the plants
response to stress treatments.
In conclusion, full-grown A. annua plants treated with
sandblasting showed signicantly higher concentrations
of a-pinene, camphene, coumarin and dihydro-epi-deox-
yarteannuin B, but not signicantly higher total content
of EOC compared with controls. Furthermore, no signif-
icant changes in the composition of EOC were observed
after applying chemical stress to full-grown A. annua
plants. The limited effect of the stress treatments could
be due to the developmental stage, because the leaves
that were analysed in this experiment had most likely
already developed glandular trichomes, at the time of the
treatments (Kjr et al. 2012, 2013). Hence, the experi-
ments in this study have demonstrated that the synthesis
of secondary metabolites in full-grown A. annua plants
may be difcult to alter signicantly by elicitation, indi-
cating that the trichomes might have reached their
potential for production and biosynthesis of EOC at the
development stage where the plants are full-grown. Thus,
the results from the applied stress treatments does not
support the hypothesis that elicitation lead to an
increased production of EOC in full-grown A. annua
plants. Instead, the present results indicate that EOC
and/or other secondary metabolites in the glandular tric-
homes are more or less stored after they have been bio-
synthesized, being ready to be released when the plants
are exposed to external stress. This conclusion is sup-
ported by stress experiments on young A. annua plants
demonstrating the ability to enhance the production of
artemisinin that can also be observed in the correspond-
ing full-grown plants, but is apparently not the case
when the elicitor treatment is only performed when the
plants are at the full-grown development stage (Aftab
et al. 2010a,b, Guo et al. 2010, Lei et al. 2011, Kjr
et al. 2013). Thus, it would be interesting to perform a
similar study as the one described here on younger
plants in order to investigate whether the content of
EOC is also enhanced signicantly in young plants upon
stress treatment, and if so, whether the plants maintain
an increased production of some EOC during growth
and whether this increased production of EOC can be
observed in full-grown plants. However, based on the
present results, it is not recommended to elicitor-treat
full-grown A. annua plants as this will not increase the
production of EOC and other secondary metabolites.
Results from other studies on artemisinin production in
2013 Blackwell Verlag GmbH, 199 (2013) 395404 402
Ivarsen et al.
A. annua indicates that it may be benecial to elicitor-
treat young A. annua plants until they reach maturity
and then harvest the increased secondary metabolite
content at the full-grown development stage. To elicitor-
treat young plants and then harvest the increased metab-
olite content of the young plants is not recommended
due to the lower yield of biomass and thus lower yield
of metabolites. Information on whether the yield of EOC
can be improved in A. annua by elicitation and/or selec-
tion could be of outmost importance for growers of
A. annua plants due to an increased interest of using
EOs and EOC in the treatment of infectious diseases in
both humans and animals.
Acknowledgements
We greatly acknowledge nancial support from The Danish
Council for Strategic Research (Project no. 2101-08-0048).
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