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South African Journal of Botany 75 (2009) 97 – 103

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Elicitation of 7-methyljuglone in Drosera capensis

S.M. Ziaratnia a,⁎, K.J. Kunert a,b , N. Lall a
a
Department of Plant Science, University of Pretoria, Pretoria 0002, South Africa
b
Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria 0002, South Africa

Received 29 March 2008; received in revised form 8 July 2008; accepted 4 August 2008

Abstract

Drosera capensis L. (Droseraceae) is an important source of pharmacologically active 1,4-naphthoquinones including 7-methyljuglone
which has been shown to have significant antimicrobial and antifungal as well as antituberculosis activity. In this study, we report on the
production of 7-methyljuglone in D. capensis under in vivo conditions by salicylic acid and jasmonic acid elicitation and by applying different
strengths of media as well as different levels of total inorganic nitrogen (N) and ratios of nitrate (NO−3 ) to ammonium (NH+4 ) to a plant tissue
culture medium. The amount of 7-methyljuglone produced was highest at 60 mM total nitrogen and at a 50:50 nitrate to ammonium ratio, which is
ten-times higher when compared to the normal basal Murashige and Skoog growth medium. Elicitation of 7-methyljuglone in greenhouse grown
plants using salicylic acid and jasmonic acid showed that the amount of 7-methyljuglone was the highest in the shoots of plants elicited with
50 µM of salicylic acid and jasmonic acid after 48 h and 3 h respectively. In roots, the highest amount of 7-methyljuglone was found in plants
treated with 50 µM of salicylic acid and 100 µM of jasmonic acid after 1.5 h. The 7-methyljuglone concentration is generally higher in the roots
than in shoots.
© 2008 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: 7-Methyljuglone; Drosera capensis; Elicitation; Jasmonic acid; Salicylic acid; Tissue culture

1. Introduction The major naphthoquinones found in the Drosera genus

The most widespread genus of carnivorous plants is Drosera
(Sundew) with about 130 species found worldwide (Didry et al.,
1998; Bekesiova et al., 1999; Hook, 2001; Kawiak et al., 2003).
Drosera plants grow mostly on acidic soils that are often
deficient in nitrogen and phosphorous (Chandler and Anderson,
1976; Stewart and Nielsen, 1992; Adamec, 1997). They are rich
in naphthoquinones (Hirsikorpi et al., 2002) and also contain
glucosides of quinones (Budzianowski, 2000; Hirsikorpi et al.,
2002; Panichayupakaranant and Tewtrakul, 2002; Kämäräinen
et al., 2003) of which some are used as antispasmodic agents in
the treatment of respiratory tract ailments (Didry et al., 1998;
Hook, 2001; Kawiak et al., 2003). The naphthoquinone
concentration in plants, however, is dependent on the growth
season and differs between plant parts (Bonnet et al., 1984;
Hook et al., 1997; Kämäräinen et al., 2003).
⁎ Corresponding author. Tel.: +27 12 4203770; fax: +27 12 3625099.
E-mail address: mahdi.ziaratnia@fabi.up.ac.za (S.M. Ziaratnia).
0254-6299/$ - see front matter © 2008 SAAB. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.sajb.2008.08.001
are plumbagin (2-methyl-5-hydroxy-1, 4-naphthoquinone) and 7-
methyljuglone (7-MJ, 7-methyl-5-hydroxy-1, 4-naphthoquinone)
(Hirsikorpi et al., 2002). 7-Methyljuglone has shown to be
inhibitory to several insects and highly toxic to fungal pathogens
(Seigler, 1998). The antifungal activity of 7-MJ isolated from Eu-
clea has already been reported (Watt and Breyer-Brandwijk, 1962;
Steffen and Peschel, 1975). 7-MJ also has significant antitubercu-
losis activity (Lall et al., 2005; Bapela et al., 2006; Van der Kooy
et al., 2006) and the compound is a potent inhibitor of 12-
hydroxyeicosatetraenoic acid, which is a critical signalling molecule
in tumour metastasis (Tang and Honn, 1994) and atherosclerotic
processes (Nakao et al., 1982).
7-MJ production has been investigated in cell suspensions of
Drosera capensis (Fig. 1) (Hook et al., 1997; Hook, 2001). Cell
suspensions grown in bioreactors, can be an effective system to
produce bioactive compounds (Yanpasian et al., 1999; Walker
et al., 2002). More over in vitro condition is also a good
opportunity for modification of environmental conditions,
including medium ingredients, to obtain the optimal condition.
98 S.M. Ziaratnia et al. / South African Journal of Botany 75 (2009) 97–103
Fig. 1. Drosera capensis.
However, there is no information available on elicitation studies
or optimization of tissue or cell suspension culture system for
targeting of the 7-MJ enhancement in D. capensis.
We have therefore investigated in this study the elicitation of
7-MJ production in D. capensis under in vivo and modified in
vitro conditions to increase 7-MJ production in D. capensis. For
that purpose, the effect of different factors was studied on 7-MJ
production including, elicitation of 7-MJ production by salicylic
acid (SA) and jasmonic acid (JA) in in vivo experiments. The
application of different nitrogen concentrations in combination
with ratios of nitrate (NO3−) to ammonium (NH4+) in in vitro
conditions was also studied in vitro.
2. Materials and methods
2.1. Plant material
Greenhouse grown plants and seeds of D. capensis were
collected from the Botanical Garden of the Plant Science
Department, University of Pretoria. Seeds were stored at 4 °C
prior to use.
2.2. D. capensis seed germination
For the determination of the 7-MJ concentration in in vitro
grown plants, seeds were germinated after surface-sterilization
(70% ethanol for 30 s, 0.5% sodium hypochlorite for 30 min)
and rinsing (3 times with sterile distilled water). Seeds were
germinated on water agar medium containing 0.5% sucrose as
the only ingredient and agar (0.7% w/v). The medium was
adjusted to pH 5.7 before autoclaving for 20 min at 1.05 kg/cm2
and 121 °C. Seeds were then incubated for germination at 25 °C
under a 16/8 h photoperiod with light intensity of 30 µE m− 2 s− 1.
Twenty five seeds were germinated in each Petri dish.
2.3. In vitro nitrogen treatments and media modification
10-day old seedlings from the germination study were used
in this experiment. All media were supplemented with MS
vitamins. The pH of the media was adjusted to 5.7 prior to
autoclaving for 20 min at 1.05 kg/cm2 and 121 °C. The cultures
were incubated at 25 °C under a 16/8 h photoperiod with light
intensity of 30 µE m− 2 s− 1. Six plants were used for each
treatment.
2.3.1. Medium modification
The seedlings were cultured on hormone-free 1/3, 1/2 and full-
strength MS (Murashige and Skoog, 1962) and B5 (Gamborg
et al., 1968) media in culture vessels (Magenta No 7). The cultures
were incubated as described before. After an eight week
incubation period the plant samples were harvested, dried at
50 °C and extracted with dichloromethane and analyzed with
HPLC to determine the concentration of the 7-MJ content.
2.3.2. Nitrogen content modification in MS medium
Since Drosera plants grow in poor nutrient-conditions and
they gain their nutrients, especially nitrogen and phosphorous
from captured insects (Kämäräinen et al., 2003), another experi-
ment was performed to determine the response of Drosera
plants to different quantities and sources of inorganic nitrogen.
In this experiment the inorganic nitrogen content in combina-
tion with the ratio of ammonium to nitrate in MS basal medium
was modified. Two inorganic nitrogen levels, 30 and 60 mM
and four NO3− :NH4+ ratios (50:50, 67:33, 75:25 and 100:0) were
used according to Niedz (1994). MS medium contains 60 mM
of inorganic nitrogen at a ratio of NO3−:NH4+ (67:33) was used as
control medium. The result medium modification experiment
showed that the concentration of 7-MJ was very low after
8 weeks and we therefore decided to harvest the plants in this
experiment after 3 months.
2.4. In vivo elicitor treatment
Greenhouse grown plants were used for the elicitation of 7-MJ
experiment in vivo. 3-month old Drosera plants were grown in
pots filled with peat and washed sand (2:1) and were irrigated
with distilled water only. SA and JA were applied as elicitors
separately at different concentrations (0, 20, 50, 100 µM) on
greenhouse grown plants. A stock solution of SA was prepared in
deionised water. JAwas first dissolved in ethanol and then diluted
with deionised water (Biondi et al., 2000). For elicitor treatment,
shoot tips of plants were excised with a scalpel and 2 µl of elicitor
solution was placed on the excised surface of the shoot tips.
Three individual plants were used for each treatment. Control
plants were treated with distilled water in a similar procedure to
the treated plants. The shoots and roots of treated and control
plants were harvested separately at 1.5, 3.0, 6.0, 12.0, 24.0 and
48.0 h after treatment, dried in oven at 50 °C and weighed.
The concentration of 7-MJ was measured three times by HPLC
analysis.
2.5. Measurement of 7-MJ concentration
At the time of harvest the plant material of all replicates was
pooled together according to Kirakosyan et al. (2004).
Dichloromethane extracts of pooled samples were air-dried
and dissolved in acetonitrile to a concentration of 10 mg/ml.
Samples (10 µl) were subjected to an isocratic elution on a
reverse phase HPLC system (5 µ, C18 column, 150 × 4.6 mm)
 S.M. Ziaratnia et al. / South African Journal of Botany 75 (2009) 97–103 99

Fig. 2. Comparison of 7-MJ contents in intact plants of Drosera capensis grown in different media in vitro with those in green house plants (control). Values of the bars
with different letters are significantly different (p b 0.01) according to ANOVA test (n = 6).

(Phenomenex Co, USA). The chromatographic system con-
sisted of a 9012 pump (Varian, USA) connected to a manual
sample injector 7161 (Rheodyne, USA) (Walker et al., 2002).
The absorbance at 430 nm was measured by a UV6000LP
photodiode array variable UV/VIS detector (tPS, USA). The
mobile phase consisted of 62.5% acetonitrile, 32.5% water, and
5.0% aqueous citric acid and the flow rate was 1 ml/min. 7-MJ
was quantified at 430 nm by comparison with a defined
standard (Van der Kooy and Meyer, 2006) based on the peak
area. PC 1000 software was used for peak integration analysis.
2.6. Statistical analysis
Analysis of variance (ANOVA) was used to determine the
significance of variance. Tukey's significant difference test was
applied to determine the groups according to Compton (1994).
Mean differences with a p value greater than 5% were regarded
as non-significant.
3. Results and discussion
The production of 7-MJ in D. capensis plants under in vitro
conditions was influenced by MS medium strengths after eight
weeks of incubation. The lowest 7-MJ production in the in vitro
grown plants was found in the plants that were grown on 1/3-
strength MS medium (p b 0.01). No significant difference in the
7-MJ concentration was found when plants were grown on full-
strength MS, 1/2 MS or B5 medium (Fig. 2). Although the basal
MS medium has previously been reported as a medium for the
tissue culture of the Drosera genus (Crouch et al., 1990), the
results of this study showed that full and 1/2 strength MS and
full strength of B5 medium are also suitable for the 7-MJ
production in D. capensis under in vitro conditions. Drosera
species usually grow in nutrient-poor habitats, low in nitrogen
and phosphorous. Insects are a rich source of nutrients,
particularly in nitrogen (Juniper et al., 1989). It has also been
reported that Drosera plants are not an obligate insectivore, but

Fig. 3. Effect of different levels of total nitrogen, 30 and 60 mM and ratios of nitrate to ammonium on 7-MJ concentration in intact grown Drosera capensis plants. The
ratio of nitrate to ammonium in original MS basal medium is 67:33, which is considered as the control in this experiment. Values of the bars not followed by the same
letter are significantly different (p b 0.01) according to ANOVA test (n = 3).
100 S.M. Ziaratnia et al. / South African Journal of Botany 75 (2009) 97–103

that insectivory may provide the plant with an alternative source
of nutrients (Givnish et al., 1984). In another study D. binata
and D. capensis were fertilised with nitrogen and phosphorous
and it was observed that both species benefited from soil
nutrient addition (Stewart and Nilsen, 1993).
The concentration of 7-MJ after three months was 0.07 g/kg
dry weight when 60 mM total inorganic nitrogen and NO3−:NH4+ at
a ratio of 50:50 was used (Fig. 3). This is more than ten-fold
higher when compared to basal full-strength MS medium (control
plants) containing 60 mM total inorganic nitrogen with a NO3−:
NH4+, 67:33 ratio, where the concentration of 7-MJ was found to
be 0.006 g/kg dry weight. In contrast, among the treatments with
30 mM total inorganic nitrogen, 7-MJ content was found highest
in plants treated with a ratio of 100:0, NO3−:NH4+. There is
evidence of a link between the forms of N uptake and N
assimilation (NO3− and NH4+) and the formation of intercellular H+
and OH− (Kirkby and Knight, 1977; Van Beusichem et al., 1988).
Inorganic N uptake can strongly influence the pH of the medium
since the uptake of NO3− and NH4+ changes the pH in the medium
in opposite directions (Niedz, 1994). The absorption of the NO3−
anion results in an increase in pH to maintain charge neutrality,
generally by the extraction of HCO3−. Conversely, the absorption
of NH4+ cations results in the production of H+ which is excreted
into the medium. This lowers the pH and reduces any further
cation uptake by competitive effects (Kirkby and Mengel, 1967).
Ammonium in the medium can be consumed faster than nitrate,
and during this time the pH in the medium decreases (De Block,
1990; Fracago and Echeverrigaray, 2001). In sweet orange callus,
93% of the variation in pH of the medium was dependent on NO3−:
NH4+ ratios (Niedz, 1994). The role of pH as a necessary factor in
the synthesis and composition of naphthoquinones has been
demonstrated (Kurobane et al., 1980; Medentsev and Akimenko,

Fig. 4. Effect of different concentrations of salicylic acid on 7-MJ synthesis in Drosera capensis shoots and roots at different intervals. Values of the bars not followed
by the same letter are significantly different (p b 0.05) according to the ANOVA test (n = 3).
 S.M. Ziaratnia et al. / South African Journal of Botany 75 (2009) 97–103 101

1998). Obtaining the highest amount of 7-MJ in this study under
the 60 mM total inorganic nitrogen and NO3−:NH4+ at a ratio of
50:50 condition can be attributed to the decreased pH. In another
study the plumbagin content in Plumbago rosea was increased by
using double the amount of (NH4)2SO4 in B5 medium
(Panichayupakaranant and Tewtrakul, 2002). In fungi of the
genus Fusarium the biosynthesis of naphthoquinone pigments
occurred at pH 2.8–4.0, by using (NH4)2SO4 in the medium as
nitrogen source (Medentsev et al., 2005).
The concentration of 7-MJ in the shoots of D. capensis
plants, which were elicited with SA showed that after 48 h of
treatment of 50 µM SA, shoots produced the highest level of
7-MJ (0.013 g/kg dry weight). This was 2.1-times higher than
the 7-MJ contents in the shoots of untreated control plants at
the same time (0.006 g/kg dry weight) (Fig. 4-shoot). The
highest amount of 7-MJ in the roots was observed in plants
elicited with 50 µM SA after 1.5 h treatment (0.028 g/kg dry
weight). The 7-MJ concentration in this sample was 2.3-fold
higher when compared to the 7-MJ contents in the roots of
control plants with 0.012 g/kg per dry weight after 1.5 h
treatment (Fig. 4-root). Overall, we found that SA was effective
in the enhancement of 7-MJ production in both roots and
shoots, but it is more effective in eliciting 7-MJ production in
the roots. In contrast to our results, it has been reported earlier

Fig. 5. Effect of different concentrations of jasmonic acid on 7-MJ synthesis in Drosera capensis shoots and roots. Values of the bars not followed by the same letter are
significantly different (p b 0.05) according to the ANOVA test (n = 3).
102 S.M. Ziaratnia et al. / South African Journal of Botany 75 (2009) 97–103
that in cell cultures of Hypercum perforatum, the application of
SA had no effect on the concentration of another naphthoqui-
none, hypercin (Walker et al., 2002).
The amount of 7-MJ was the highest (Fig. 5-shoot) in shoots of
plants elicited with 50 µM of JA after 3 h of treatment (0.017 g/kg
dry weight). This was 2.8-fold higher than the 7-MJ content after
3 h of treatment in the shoots of untreated control plants. Under
these conditions, the highest amount of 7-MJ was synthesized in
the roots (0.028 g/kg dry weight) after 1.5 h of treatment, which
was 2.3-times higher than the 7-MJ in the roots of control plants at
the same time (Fig. 5-root). In plants elicited with different
concentrations of JA, the content of 7-MJ in the roots reduced
significantly (p b 0.05) between 1.5 and 6 h of treatment and after
that, the content of 7-MJ stayed relatively constant until 48 h,
except for a significant increase at 24 h (p b 0.05). The elicitor, JA
and its derivate MeJA are considered to be involved in a part of the
signal transduction pathway that activates particular enzymes
catalyzing biochemical reactions to form defence compounds of
low molecular weight in plants, such as polyphenoles, alkaloids,
quinones, terpenoids, and polypeptides (Mizukami et al., 1993;
William et al., 1996; Ding et al., 2004). JA and MeJA do not
appear to be specific to a particular class of secondary metabolites,
since a wide spectrum of compounds is elicited after exposure to
exogenous JA or MeJA (Gundlach et al., 1992). The results of this
study therefore confirm previous results (Walker et al., 2002;
Ding et al., 2004) where an enhancing effect of JA and its
derivative, MeJA on hypercin, a naphthoquinone, has been found
in cell cultures of H. perforatum and also the red naphthoquinone
compounds of cultured Onosma paniculatum cells.
Overall, the present study has demonstrated for the first time
that the elicitation of 7-MJ by SA and JA at the concentrations
used in this study is an effective way to increase the amount of
7-MJ in both roots and shoots of D. capensis. The results of this
study also showed that the yield of 7-MJ in plants treated with
SA and JA was higher than intact plants (no wounding, no
elicitation) whereas the 7-MJ contents in the shoots and roots
were 0.008 and 0.009 g/kg dry weight respectively (This sample
was prepared with the same procedure to the other samples in
this study). The increased 7-MJ can be attributed to the stressed
condition in plants, which was derived by applying SA and JA
on D. capensis plants. Further, accumulation of 7-MJ in the
roots of D. capensis is generally higher than in shoots.
However, there is a significant interaction between concentra-
tion of elicitor (SA or JA) and time of harvesting (p b 0.01) for
the 7-MJ concentration in the shoots and roots of D. capensis.
The results of this study also showed that the production of
7-MJ can be influenced by the nitrogen concentration and the
ratio of nitrogen sources. Although we cannot directly compare
our in vitro and in vivo results, treating D. capensis with
inorganic nitrogen in combination with ratio of nitrogen
sources (nitrate and ammonium) is an effective way to increase
7-MJ production. Using nitrogen would be less expensive and
therefore a more cost-effective strategy for elicitation than
using chemical elicitors like SA and JA in a commercial
approach for the 7-MJ production. This experiment still has to
be carried out in vivo to confirm the positive effect of nitrogen
and its sources on 7-MJ synthesis in D. capensis.
Acknowledgements
Authors are grateful to Dr Frank Van der Kooy for providing
the 7-MJ standard and special thanks to Susan Myburgh and
Daniel Bilankulu for supplying the plants. We also thank the
National Research Foundation (NRF) of South Africa for the
financial support.
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