Plant Cell Tiss Organ Cult (2011) 104:61–67
DOI 10.1007/s11240-010-9804-7
ORIGINAL PAPER
Production of centellosides and phytosterols in cell suspension
cultures of Centella asiatica
Mercedes Bonfill • Susana Mangas •
Elisabeth Moyano • Rosa M. Cusido •
Javier Palazón
Received: 19 May 2010 / Accepted: 17 July 2010 / Published online: 9 August 2010
Ó Springer Science+Business Media B.V. 2010
Abstract Centella asiatica is a herbaceous plant used in
medicine for its wound-healing and anti-inflammatory
properties. Its bioactive compounds are ursane-type triter-
pene saponins known as centellosides. With the aim of
increasing the biotechnological production of these com-
pounds, C. asiatica cell suspensions were established and
treated with two concentrations (100 and 200 lM) of methyl
jasmonate (MeJA). The maximum centelloside production
was observed in the stationary growth phase, reaching
0.16 mg g-1 dry weight (DW) at day 25 of the culture in the
control and 1.11 mg-1 g DW at day 15 in the MeJA-elicited
cultures. The elicitor did not change the centelloside pattern,
with madecassoside being the main compound, followed by
asiaticoside. Reverse transcription polymerase chain reac-
tion (RT-PCR) analysis of the b-amyrin synthase gene
(CabAS, the specific oxidosqualene cyclase that leads to
centelloside formation) showed higher levels of expression
in the elicited cultures than in the control. The maximum
content of centellosides was obtained at day 15, with a time
lag between gene activation and centelloside biosynthesis.
In the cultures elicited with 200 lM MeJA, the centelloside
production did not increase compared to the control. Both
elicitor concentrations decreased the content of phytoster-
ols. Thus, MeJa elicitation in this type of culture was
M. Bonfill (&)  S. Mangas  R. M. Cusido  J. Palazón
Laboratorio de Fisiologı́a Vegetal, Facultad de Farmacia,
Universidad de Barcelona, Avda. Diagonal 643, 08028
Barcelona, Spain
e-mail: mbonfill@ub.edu
E. Moyano
Departament de Ciències Experimentals i de la Salut, Universitat
Pompeu Fabra, Avda. Dr. Aiguader 80, 08003 Barcelona, Spain
dose-dependent and its inducing role was apparent at low
concentrations.
Keywords Asiaticoside  Cell suspension cultures 
Centella asiatica  Centellosides  Madecassoside  Methyl
jasmonate
Introduction
Centella asiatica (Gotu kola, Pegaga) is a herbaceous plant
widely used in the health, food and cosmetic industries.
Its active metabolites, centellosides such as asiaticoside,
madecassoside, asiatic acid and madecassic acid, are
reported to have many medicinal and therapeutic properties,
used for diseases associated with the skin, nervous system
and blood (Skopińska-Rózewska et al. 2002). Centellosides
are ursane-type triterpene saponins resulting from the cyc-
lisation of 2,3-oxidosqualene by amyrin synthase. Although
Kim et al. (2005a) have cloned the CabAS gene, the last
steps of this biosynthetic pathway are still unknown.
Due to the growing commercial importance of plant
secondary metabolites, there is great interest in enhancing
their production through biotechnology. In vitro plant cell
cultures offer the possibility of obtaining desirable medici-
nal compounds as well as ensuring a sustainable conserva-
tion and rational utilisation of biodiversity (Phillipson
1990). Specifically, cell suspension cultures can be used for
the large-scale culturing of plant cells from which secondary
metabolites are extracted. The advantage of this method is
that it can ultimately provide a continuous and reliable
source of natural products.
The biosynthetic activity of cultured cells can be
enhanced by methyl jasmonate (MeJA) (Suzuki et al. 2005;
Yoon et al. 2000). Jasmonates are molecules known to be
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62
efficient elicitors for a wide range of secondary metabolites
from different plant origins (Gundlach et al. 1992;
Memelink et al. 2001). An increase in centelloside content
has been observed in C. asiatica in vitro plants and hairy
root cultures elicited with 100 lM MeJA (Mangas et al.
2006; Kim et al. 2004, 2007).
We have previously experimented with different culture
media to obtain high rates of induction, good growth and
friable calli of C. asiatica (Mangas et al. 2008, 2009). In the
work presented here, our aim was to study the effect of
elicitor concentration on the production of centellosides and
phytosterols. Thus, cell suspension cultures of C. asiatica
established from friable calli were treated with 100 and
200 lM MeJA. Moreover, to achieve a better understanding
of the biosynthetic pathway of these secondary metabolites,
we compared the production of centellosides and phytos-
terols in the C. asiatica cell suspension cultures with the
expression level of genes codifying for the following
enzymes: b-amyrin synthase (CabAS), the specific oxido-
squalene cyclase for centelloside production; cycloartenol
synthase (CaCYS), which directs the pathway toward phy-
tosterols; and squalene synthase (CaSQS), which directs the
pathway toward both phytosterols and centellosides.
Materials and methods
Establishment of callus and cell suspension cultures
Calli were obtained as described by Mangas et al. (2008),
using Murashige and Skoog (MS, Murashige and Skoog
1962) medium supplemented with 30 g l-1 of sucrose,
2 mg l-1 of 6-benzyladenine (BA) and 2 mg l-1 of
1-naphthaleneacetic acid (NAA), which provided good
callus induction. As a growth medium, we used MS med-
ium supplemented with 30 g l-1 of sucrose, 2 mg l-1 of
2,4-dichlorophenoxyacetic acid (2,4-D) and 3 mg l-1 of
N1-(2-chloro-4-pyridyl)-N2 phenylurea (4PU-30) to obtain
large and friable calli (Mangas et al. 2009).
To establish cell suspension cultures, callus pieces
(20 g) were inoculated into 500-ml flasks containing
200 ml of MS liquid medium supplemented with 30 g
sucrose, 2,4-D (2 mg l-1) and BA (0.1 mg l-1) and placed
in a rotary shaker at 100 rpm in the dark at 25°C. Cell
cultures were passed through a 60-lm nylon filter every
2 weeks for 6 months, as described by Wickremesinhe and
Arteca (1994), to obtain a fine cell suspension culture. For
the cell suspension assays, 20 g of cells (fresh weight, FW)
were inoculated into 200 ml of the aforementioned liquid
medium in 500-ml flasks, with or without the elicitor, and
were maintained in a rotary shaker at 100 rpm in the dark
at 25°C.
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Plant Cell Tiss Organ Cult (2011) 104:61–67
Treatment with methyl jasmonate (MeJA)
At day 12, MeJA was added to the liquid medium at two
concentrations (100 and 200 lM), prior to autoclaving. The
culture was maintained for 30 days.
Determination of growth and cell viability
Control and MeJA-elicited cell samples were taken every
5 days over a period of 30 days to determine growth and
cell viability. Each sample was washed, weighed for FW
and lyophilised to obtain dry weight (DW). Cell viability
was studied by the fluorescein diacetate staining technique
(Widholm 1972). The cells (0.2 g FW) were incubated in
fresh growth medium (5 ml) containing fluorescein diace-
tate (0.1 mg ml-1) for 30 min at room temperature. Cell
fluorescence was observed with a fluorescence microscope
at 520 nm and the percentage of fluorescent cells in rela-
tion to the total was assessed.
Extraction and analysis of centellosides
and phytosterols
The quantification of the four centellosides (asiaticoside,
madecassoside, asiatic acid and madecassic acid) was car-
ried out every 5 days over a period of 30 days in the cell
suspensions with and without MeJA. Lyophilised cells (1 g)
were extracted as reported by Bonfill et al. (2006). The dried
extract was dissolved in 1 ml of methanol and filtered
through a 0.22-lm filter (Waters Millipore, Billerica, MA,
USA) for high-performance liquid chromatography (HPLC)
analysis. HPLC-UV analysis was performed following the
method of Inamdar et al. (1996), modified as described in
our previous paper (Mangas et al. 2006). The chromato-
graphic analysis was performed at room temperature with a
Spherisorb 51 ODS2 (250 9 4-mm) column (Waters,
Milford, MA, USA) using gradient elution, the eluents
being acetonitrile (A) and water with ammonium dihydro-
gen phosphate 10 mM (pH 2.5 with orthophosphoric acid)
(B) according to the following profile: 0–15 min, 80% A;
15–30 min, 62% A; 30–37 min, 30% A; 37–40 min, 80%
A. The flow rate was 1 ml min-1 and the detector was set at
214 nm. Asiaticoside, madecassoside, asiatic acid and
madecassic acid were obtained from ChromaDex Inc.
(Irvine, CA, USA). The calibration curves were obtained
from a stock solution with the four centellosides at
10 mg ml-1 in HPLC-grade methanol. From this stock
solution, five solutions were prepared ranging from 0.025
to 1 mg ml-1 and 20 ll was injected three times for each
concentration.
Free sterols (b-sitosterol, stigmasterol, campesterol
and cholesterol) were extracted using the same method as
for centellosides (Bonfill et al. 2006) and analysed by
Plant Cell Tiss Organ Cult (2011) 104:61–67 63

HPLC-UV as previously reported (Mangas et al. 2006). Table 1 Sequences of the primers used to amplify the housekeeping
Campesterol was obtained from ChromaDex Inc. (Irvine, gene (5.8S rRNA) and the genes coding for squalene synthase
(CaSQS), cycloartenol synthase (CaCYS) and b-amyrin synthase
CA, USA). b-Sitosterol, stigmasterol and cholesterol were
(CabAS)
obtained from Sigma-Aldrich (St. Louis, MO, USA). The
calibration curves for the four phytosterols were obtained Genes Sequences Amplicon
size (bp)
from a stock solution in methanol. From this stock solution,
five solutions of 5–75 mg ml-1 were prepared and 20 ll CaCYS Forward primer 50 -GAATCCACGC 421
was injected three times for each concentration. The HPLC CATGAAGTCT-30
system consisted of a Pharmacia LKB-HPLC 2150 pump Reverse primer 50 -ACCACCATGA
(Pharmacia LKB Biotechnology AB, Uppsala, Sweden), an TCCAGAATCC-30
LC 2152 Controller (Pharmacia LKB Biotechnology AB, CabAS Forward primer 50 -TGGTTGGGGA 302
GAAAGTCTTG-30
Uppsala, Sweden), an HPLC autosampler 465 (Kontron
Reverse primer 50 -ACAAGCGTTTG
Instruments, Eching, Germany), a 2141 Variable Wave- CGGTACTCT-30
length Monitor (Pharmacia LKB Biotechnology AB, CaSQS Forward primer 50 -TGGGTTAGGGT 324
Uppsala, Sweden) and a Biodacs integrator (Pharmacia TGTCAAAGC-30
LKB Biotechnology AB, Uppsala, Sweden). Reverse primer 50 -CGGAAGATAGC
AGGATCTCG-30
RT-PCR analysis 5.8S rRNA Forward primer 50 -CGGCAACGGAT 201
ATCTCGGCTCT-30
Total RNA was isolated from frozen cells to check the Reverse primer 50 -TCCGCCCCGACC
CCTTTC-30
expression level of genes codifying for b-amyrin synthase
(CabAS), cycloartenol synthase (CaCYS), squalene syn-
thase (CaSQS) and the housekeeping gene (5.8S rRNA), as phase of the culture began on day 10 and the elicitor was
described by Mangas et al. (2008). One microgram of total added on day 12 because secondary rather than primary
RNA from each sample was reverse-transcribed by First- metabolites are generally synthesised in this phase (James
Strand cDNA synthesis using M-MLV RT (Invitrogen, et al. 2008). As shown in Fig. 1, the growth curve of the
Carlsbad, CA, USA), according to the manufacturer’s elicited cells was similar to that of the control cells,
instructions. although with a shorter stationary phase. The DW of con-
Polymerase chain reaction (PCR) amplification was trol cells increased three-fold on day 10, remaining largely
performed as described by Mangas et al. (2008). Three unchanged until day 25, when it began to decrease, probably
replicates of each biological sample were used for reverse due to the depletion of nutrients in the culture medium.
transcriptase PCR (RT-PCR) analysis. The primer sequen- Similar growth courses have been previously described for
ces corresponding to the three genes under study (CabAS, C. asiatica cell cultures (Nath and Buragohain 2005; James
CaCYS and CaSQS) were cloned and sequenced by Kim et al. 2008; Hernández-Vázquez et al. 2010). The elicited
et al. (Kim et al. 2005a, b, c, respectively) and to the cells (100 and 200 lM MeJA) showed a similar growth
housekeeping gene (5.8S rRNA) were chosen using the BTI
Software GeneTool Lite (version 1.0.0.1) and are given in
Table 1. 120 20

Statistical analyses 100

16
Dry weight (g l -1)
Cell viability (%)

80
Analysis of variance (ANOVA; 0.05 significance level) 12
and Tukey tests were used for the statistical analyses. 60
8
40 Viability Control
Viability MeJA
Results and discussion 4
20 Dry weight Control
Dry weight MeJA
Viability and growth in control and elicited cell 0 0
suspensions 0 5 10 15 20 25 30
days
After establishing the cell suspension cultures and deter-
Fig. 1 Growth curves and viability of Centella asiatica cell suspen-
mining the growth as described in the previous section, we sion cultures with and without the elicitor methyl jasmonate (MeJA)
obtained the growth curve shown in Fig. 1. The stationary 100 lM

123
64 Plant Cell Tiss Organ Cult (2011) 104:61–67

pattern to the control but with lower values and a decrease with and without MeJA (Mangas et al. 2006, 2009). Cen-
from day 15. The higher elicitor concentration resulted in telloside production in the unelicited cell suspensions was
the least growth (data not shown). 2–2.5 times lower than in the calli, but after treatment with
The viability of control cells began to decrease slightly at 100 lM MeJA, it was more than two-fold higher, reaching
day 10 (when it was 90%), and in the last 10 days of culture, the same level as the lowest found in the aerial parts of
was reduced by half (from 86% at day 20 to 48% at day 30). C. asiatica in vitro plants. A lower level of centelloside
In cells treated with 100 lM MeJA, viability was lower production in cell suspensions than in calli has also been
than in the control, with 83% living cells at day 15 and 40% observed by James et al. (2008) in two phenotypes of
at day 30. The lowest viability was observed in cells treated C. asiatica from Southern Africa, at a ratio of 1.5–2.
with 200 lM MeJA, being 20% at day 30 (data not shown). The centelloside profile in the elicited cell suspension
These results show that MeJA negatively affects C. asiatica cultures (both 100 and 200 lM) was the same as in calli
cell cultures, as well as Centella whole plants (Kim et al. (Mangas et al. 2009). Thus, the main compound was
2004; Mangas et al. 2006), the effect increasing with the madecassoside followed by asiaticoside, with very low
elicitor concentration. quantities of asiatic and madecassic acids (data not shown).
In a previous study of 100 lM MeJA-treated whole
Centelloside production in control and elicited cells C. asiatica in vitro plants (Mangas et al. 2006), the cen-
telloside profile also remained the same as in the unelicited
The highest production of centellosides was achieved dur- plants, although in that case, the main compound in
ing the stationary growth phase in the 100 lM MeJA-elic- the aerial parts was asiaticoside and madecassoside in the
ited cultures (1.11 mg g-1 DW at day 15), being seven-fold roots. In short, 100 lM MeJA treatment did not change the
higher than the maximum production in the control centelloside pattern in any of our C. asiatica cultures
(0.16 mg g-1 DW at day 25) (Fig. 2). Thus, the elicitor (aerial parts and roots of C. asiatica in vitro plants and cell
effect was greatest during the first 4 days of treatment. We suspensions).
can say that elicitation with MeJA 200 lM had a negative
effect on the centelloside pathway, since it resulted in a Free sterol production in control and elicited cells
lower centelloside production than that of the control (data
not shown). As shown in Fig. 3, the content of free sterols (b-sitosterol,
We compared the centelloside production of the cell stigmasterol, campesterol and cholesterol) was higher in the
suspensions with that of our other C. asiatica in vitro cul- control than in the 100 lM MeJA-treated cells. A decrease
tures, including calli, aerial parts and roots of in vitro plants in phytosterol content was also observed in plants elicited
with MeJA 100 lM in relation to control plants (Kim et al.
1.2
2005b; Mangas et al. 2006). Since 100 lM MeJA directly
or indirectly increases the biosynthesis of centellosides, the
asiaticoside
1 decrease in phytosterols suggests a competition between the
madecassoside
Centelloside content (mg g -1 DW)

0.8 0.3
Total contents of free sterols mg g-1 DW

Control
0.6 0.25 MeJA 100
MeJA 200
0.2
0.4

0.15
0.2

0.1
0
Control

MeJA

Control

MeJA

Control

MeJA

Control

MeJA

0.05

Day Day 15 Day 20 Day 25 Day 30 0

12* 0 5 10 12 15 20 25 30
days
Fig. 2 Centelloside production in control and elicited cells of
C. asiatica. MeJA: methyl jasmonate 100 lM. Asterisk indicates Fig. 3 Free sterol production in control and elicited cells of C.
the day of addition of MeJA asiatica. MeJA: methyl jasmonate

123
Plant Cell Tiss Organ Cult (2011) 104:61–67
two pathways conducting the carbon flow through the
centelloside pathway. As occurred with centellosides, the
lowest phytosterol content was found in cells elicited with
200 lM MeJA.
Expression levels of CabAS, CaCYS and CaSQS genes
and centelloside production
Expression levels of the CabAS gene (encoding the specific
oxidosqualene cyclase for centelloside production) were
monitored for a 31-day period in the control and 100 and
200 lM MeJA-treated cell suspensions. As shown in
Fig. 4, CabAS gene expression was higher in the elicited
cells (100 lM) than in the control, achieving the highest
level at 20 h of treatment.
Kim et al. (2007) reported that CaAS gene expression in
C. asiatica hairy roots was detectable at significant levels
within 12 h of 100 lM MeJA treatment and was maintained
for 14 days. Thus, our results show that, despite the sig-
nificant differences between undifferentiated cell cultures
and hairy roots, CabAS gene transcripts in both systems
begin to increase at a similar time after MeJA elicitation,
although the up-regulation of high levels of CabAS tran-
scripts in C. asiatica hairy roots was sustained for longer
than in C. asiatica cell suspensions. Interestingly, in the
100 lM MeJA-elicited cells, the highest centelloside con-
tent appeared at day 15 (Fig. 4), which shows a delay
between the activation of this gene and centelloside pro-
duction, suggesting that, although CabAS is a key enzyme,
140000
Cab AS mRNA expression level
120000
100000
80000
60000
40000
20000
0 0
12 d ∗ 1 h 2h 4h
MeJA 100
centelloside
production
Control
Fig. 4 Centelloside production and expression level of b-amyrin
synthase (CabAS) mRNA in control and elicited C. asiatica cells
using two concentrations of methyl jasmonate (MeJA). Asterisk
indicates the day of addition of MeJA
65
CaCYS mRNA expression level
120000
Control
MeJA 100
80000
MeJA 200
40000
0
12 1h 2h 4h 8h 20 h 48 h 15 d 20 d 25 d 30 d
Fig. 5 Expression level of cycloartenol synthase (CaCYS) mRNA in
control and elicited cells of C. asiatica using two concentrations of
methyl jasmonate (MeJA)
it does not represent a limiting step in the centelloside
biosynthetic pathway. It is also noteworthy that the carbon
skeleton of centellosides like asiaticoside and madecasso-
side is more related with the a-amyrin skeleton than the
b-amyrin skeleton (Hernández-Vázquez et al. 2010; Kim
et al. 2010). Therefore, the up-regulation of CabAS gene
expression after MeJA treatment could play a major role in
the biosynthesis of other centellosides, such as centellasa-
ponin A, asiaticoside B etc. However, as MeJA strongly
induces the expression of several genes associated with the
triterpene pathway (Suzuki et al. 2005; Kim et al. 2009,
2010), it could also induce the over-expression of a more
specific oxidosqualene cyclase (multi-functional triterpene
synthase) and the consequent enhancement in the asiatico-
side and madecassoside content.
In the cell suspensions treated with 200 lM MeJA, an
increase in CabAS expression was observed 8 h after the
addition of the elicitor (Fig. 4) but to a lesser extent than
with 100 lM MeJA, and the centelloside content was
1.2
lower than in control cells (data not shown). These results
show that, in C. asiatica cell suspension cultures, the
1
Total centellosides (mg g -1 DW)
elicitation depends on the MeJA dose applied, the lower
ones producing a better response from the cells.
0.8
Expression levels of the CaCYS gene (encoding the
enzyme that directs the pathway towards phytosterol bio-
0.6
synthesis) decreased after elicitation with both MeJA
concentrations (Fig. 5) and, consequently, the free sterol
0.4
content also decreased (Fig. 3). Transcript levels of the
CaSQS gene (encoding the enzyme that directs the path-
0.2
way towards both phytosterol and centelloside biosynthe-
sis) increased with 100 lM MeJA and decreased with
8 h 20 h 48 h 15 d 20 d 30 d 200 lM MeJA (Fig. 6).
MeJA 100
Conclusion
MeJA 200
Our data shows that the centelloside content in Centella
asiatica cells was increased by treatment with 100 lM of
methyl jasmonate (MeJA), while the phytosterol content
decreased, which indicated that the elicitor boosts the
123
66
180000
CaSQS mRNA expression level
MeJA 100
160000 centelloside
140000 production
Control
120000
MeJA 100
100000
MeJA 200 μM
80000
60000
40000
20000
0 0
12 d ∗ 1 h 2h 4h
Fig. 6 Centelloside production and expression level of squalene
synthase (CaSQS) mRNA in control and elicited cells of C. asiatica
using two concentrations of methyl jasmonate (MeJA). Asterisk
indicates the day of addition of MeJA
centelloside biosynthetic pathway and represses that of
phytosterols, which are more related to cell growth. There
was a simultaneous increase in the transcript levels of the
two genes (CabAS and CaSQS) involved in the centello-
side biosynthetic pathway and a decrease in the CaCYS
gene, which leads to phytosterol biosynthesis. However,
considering that the carbon skeletons of asiaticoside
and madecassoside are more related to a-amyrin than to
b-amyrin, the expression levels of a more specific oxido-
squalene cyclase need to be studied.
The centelloside pattern was not changed by 100 lM
MeJA treatment, madecassoside being the most abundant
centelloside in roots, calli and cell suspension cultures, and
asiaticoside being the most abundant in the aerial parts of
the in vitro plants.
Elicitation with 200 lM MeJA decreased the content of
both phytosterols and centellosides, since this concentra-
tion was more toxic for the cells. The expression levels of
CaSQS and CaCYS also decreased. Although the transcript
levels of CabAS increased, the centelloside content did not,
suggesting that this concentration of the elicitor can inhibit
the translation to the enzyme.
We can conclude that MeJA elicitation in this type of
cell suspension culture was dose-dependent and its induc-
ing role was apparent at low concentrations. Comparing the
centelloside content in the elicited cells with in-vitro-elic-
ited plants (*1.5 mg g-1 DW), we can observe that
100 lM MeJA elicitation resulted in similar values of
production in both systems, with cell suspension cultures
having the advantages of permitting the optimisation of the
culture conditions and a shorter time of culture.
Acknowledgements This work was partially supported by grants
from the Spanish Ministerio de Ciencia e Innovación (BIO2008-
01210) and the Generalitat de Catalunya (2009SGR1217).
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Plant Cell Tiss Organ Cult (2011) 104:61–67
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