Plant Cell Tiss Organ Cult (2013) 112:87–93

DOI 10.1007/s11240-012-0218-6

ORIGINAL PAPER

Stimulation of Rg3 ginsenoside biosynthesis in ginseng hairy

roots elicited by methyl jasmonate
Ok Tae Kim • Nam Hee Yoo • Gum Soog Kim •
Young Chang Kim • Kyong Hwan Bang •
Dong Yun Hyun • Seung Hye Kim • Min Young Kim

Received: 11 March 2012 / Accepted: 6 August 2012 / Published online: 26 August 2012
Ó Springer Science+Business Media B.V. 2012

Abstract It has been recognized that ginsenoside Rg3 is
not naturally produced in ginseng although this ginsenoside
can accumulate in red ginseng as the result of a thermal
process. In order to determine whether or not Rg3 is syn-
thesized in ginseng, hairy roots were treated with methyl
jasmonate (MJ). From HPLC analysis, no peak for Rg3 was
observed in the controls. However, Rg3 did accumulate in
hairy roots that were MJ-treated for 7 days. Rg3 content
was 0.42 mg/g (dry weight). To gain more insight into the
effects of MJ on UDP-glucosyltransferase (UGT) activity,
we attempted to evaluate ginsenoside Rg3 biosynthesis by
UGT. A new peak for putative Rg3 was observed, which
was confirmed by LC-MS/MS analysis. Our findings
Ok Tae Kim and Nam Hee Yoo equally contributed to this paper.
Electronic supplementary material The online version of this
article (doi:10.1007/s11240-012-0218-6) contains supplementary
material, which is available to authorized users.
O. T. Kim (&)
Planning and Coordination Division, National Institute
of Horticultural & Herbal Science, RDA, Suwon
440-706, South Korea
e-mail: kimot99@hanmail.net
N. H. Yoo
Industrial Cooperation Foundation, Chonbuk National
University, Jeonju 561-756, South Korea
G. S. Kim  Y. C. Kim  K. H. Bang  D. Y. Hyun  S. H. Kim
Department of Herbal Crop Research, National Institute
of Horticultural and Herbal Science, RDA,
Eumseong 369-871, South Korea
M. Y. Kim
Medicinal Crop Seed Supply Center, Jeollanamdo Development
Institute for Traditional Korean Medicine, Jangheung 529-851,
South Korea
indicate that the proteins extracted from our hairy root lines
can catalyze Rg3 from Rh2. This suggests that our ginseng
hairy root lines possess Rg3 biosynthesis capacity.
Keywords Ginsenosides  Methyl jasmonate  Rg3 
Rh2  Panax ginseng
Introduction
Panax ginseng C.A. Meyer, commonly referred to as Korean
ginseng, is a perennial herb native to Northeast Asia
including Korea and China. Ginseng has multiple pharma-
cological effects including antifatigue, anticancer, cardio-
protective, immunomodulatory, and anti-oxidant activities
(Briskin 2000). Its major components are ginsenosides, and
35 types of ginsenoside have previously been isolated from
this plant (Ma et al. 2005). In particular, the ginsenoside Rg3
is a strong candidate for an anti-cancer compound because of
its anti-angiogenic, anti-proliferative, apoptosis and che-
motherapy-protective effects, both in vitro and in vivo (Liu
et al. 2000; Min et al. 2006; Zhang et al. 2008).
After the cyclization of oxidosqualene, subsequent region
and stereospeific hydroxylation of the triterpene skeleton
generates a number of triterpene sponins (Fig. 1). Two tri-
terpene aglycons, protopanaxadiol (PPD) and protopanaxa-
trial (PPT), were synthesized by the consecutive reaction of
two cytochrome P450-dependent hydroxylases. Recently,
Han et al. (2011) has isolated a gene (CYP716A47) encoded
a protopanaxadiol synthase, but until now the gene encoded a
protopanaxatriol synthase has not been reported. Although
there are 35 kinds of ginsenosides in P. ginseng, the mech-
anism by which a variety of structures can be produce has yet
to be clearly elucidated. This may be the result of the innu-
merable glucosyltransferases encoded in a plant. Taking into

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Fig. 1 Putative biosynthetic pathway for ginsenosides of the protopanaxadiol (PPD) group. Glc b-D-glucopyranosyl, Arap a-L-arabiopyranosyl,
Araf a-L-arabinofuranosyl
account the chemical structure, it is likely that Rg3 biosyn-
thesis occurs in ginseng, because this ginsenoside is the
precursor of Rd which is involved in biosynthesis of other
ginsenosides of the protopanaxadiol (PPD) group. Never-
theless, it has been reported that ginsenosides Rg3 and Rh2
are not naturally present in P. quinquefolius and P. ginseng
(Popovich and Kitts 2004). When ginseng roots are steamed
at 98–100 °C, it is referred to as ‘‘Red ginseng’’. Rg3 and
Rh2 can only accumulate in red ginseng by way of a thermal
process. When ginseng roots are heated to a high tempera-
ture, Rg3 is generated by the loss of the glucose group linked
with the C-20 of ginsenoside Rc, Rd, Rb1, or Rb2 (Fig. 2).
Depending on how often the heating process is repeated, Rc,
Rd, Re, Rb1, Rb2 and Rb3 contents decrease and Rg3 and
Rh2 increases (Sun et al. 2010b). However, the presence of
an Rg3 biosynthesis mechanism has not been described.
Methyl jasmonate (MJ) is a ubiquitous signaling molecule
which mediates not only plant responses to environmental
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Plant Cell Tiss Organ Cult (2013) 112:87–93
stress such as wounding, insect and pathogen attack
(Wasternack 2007) but also the enhancement of a variety of
secondary metabolites (Bonfill et al. 2011; Hu et al. 2011; Qu
et al. 2011). In ginseng, several papers have reported the
accumulation of several ginsenosides in cell suspension,
adventitious root, and hairy root cultures elicited by MJ
(Lu et al. 2001; Kim et al. 2004; Kim et al. 2009). These
results all demonstrated that ginsenosides of the PPD group,
such as Rb1, Rb2, Rb3, Rc, and Rd accumulated at far higher
levels than members of the PPT group, such as Rg1, Re, Rf,
and Rg2, in response to MJ. Although ginsenoside Rg3 is a
member of the PPD group, no study has yet reported the
effects of MJ on the biosynthesis of ginsenoside Rg3 in
ginseng. As mentioned by above, ginsenoside Rg3 is not
detected in wild P. ginseng. According to the chemical
structure of ginsenoside Rg3, we hypothesized that Rh2 is
the precursor of Rg3. Hu and Zhong (2007) have reported the
manipulation of ginsenoside heterogeneity, resulting that
Plant Cell Tiss Organ Cult (2013) 112:87–93
Fig. 2 Production of ginsenoside Rg3 in red ginseng. Arrowheads indicate the loss position of the glucose linked with the C-20 of ginsenosides
by a thermal process
ginsenoside Rb1 was synthesized from the reaction mixture
with UDP-glucose, ginsenoside Rd and proteins extracted
from cell cultures of P. notoginseng. To elucidate ginseno-
side Rg3 biosynthesis, the measurement of ginsenoside Rg3
acitivity (UDP-glucose:ginsenoside Rh2 glucosyltransfter-
ase) is necessary.
The principal objective of this study was to determine
whether or not the production of the ginsenoside Rg3 was
stimulated in ginseng hairy roots elicited by MJ without the
thermal process. In HPLC analysis, we evaluated the
changes in ginsenoside content in hairy roots elicited by
treatment with 0.1 mM MJ. UDP-glucose:ginsenoside Rh2
glucosyltransferase (UGRh2GT) activity was also mea-
sured in hairy roots elicited by MJ. The final product of the
reaction mixture with Rh2, UDP-glucose, and proteins
extracted from hairy roots was confirmed by LC/MS/MS
analysis.
Materials and methods
Plant materials
A P. ginseng hairy root culture was generated from the
explant of the 4-year old root by infection with Agrobac-
terium rhizogenes strain A4, as reported previously
(Hwang and Ko 1989). Induced hairy roots were cultured
on hormone-free half-strength MS liquid medium (Mu-
rashige and Skoog 1962) at 23 °C in darkness. The hairy
89
roots were sub-cultured at 4-week intervals. After 5 weeks
of pre-cultivation, 0.1 mM methyl jasmonate (Tokyo
Chemical Industry, Tokyo, Japan) was introduced to the
medium. The MJ-treated hairy roots were harvested after
7 days and immediately frozen with liquid nitrogen and
freeze-dried at -80 °C.
HPLC analysis of ginsenosides
For each sample, 100 mg of powdered dry hairy roots was
extracted using 2 ml of solvent (90 % methanol) for 3 h at
room temperature. After centrifugation (15,0009g) for
10 min, the aqueous layer was collected and filtered
through 0.45-lm PVDF membrane (Watman, NJ, USA).
Crude ginsenosides from the aqueous layer were used for
HPLC analysis. Quantitative determinations of the contents
of 11 ginsenosides were achieved by HPLC using a Cap-
cell-pak C18 MG (4.6 9 250 mm) column (Shiseido,
Tokyo, Japan). The HPLC conditions were as follows:
gradient elution, with the eluents being 0–10 min, 18–18 %
acetonitrile, 10–24 min, 18–19 %, 24–35 min, 19–25%,
35–54 min, 25–25 %, 54–71 min, 25–38 %, 71–100 min,
38–100 %, 100–105 min, 100–100 %, at a constant flow
rate of 1 ml/min, at 40 °C; and detection was performed at
203 nm. Authentic samples of ginsenosides Rg1, Re, Rf,
Rh1, Rg2, Rb1, Rb2, Rc, Rd, Rg3, and Rh2 were purchased
from Chromadex (CA, USA). Each experiment was carried
out in triplicate, and three different sets of experiments
reproduced the same result.
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UGRh2GT assay
For the UGRh2GT activity assay, proteins were prepared as
described by Yue and Zhong (2005). The hairy roots (1 g)
were ground in liquid nitrogen and resuspended in 2 ml of
50 mM Tris-HCl (pH 8.5), 2 mM EDTA, 10 mM
2-mercaptoethanol and 1 mM phenylmethylsulfonyl fluoride.
The tissue homogenates were centrifuged at 20,000 9 g for
20 min at 4 °C. Protein concentrations were determined by
the Bradford method (Bradford 1976). Aliquots of the crude
protein extract were assayed in 50 mM Tris-HCl (pH 8.5)
containing 5 mM uridine 50 -diphosphoglucose (Sigma-
Aldrich, CA, USA), 0.5 mM ginsenoside Rh2 (dissolved in
methanol), and 100 lg of protein. The reaction mixture was
incubated for 30 min at 35 °C. An equal volume of butanol
(200 ll) was added to stop the reaction. The butanol layer was
collected and evaporated under vacuum, after which the res-
idue was re-dissolved in 200 ll of methanol. The HPLC
conditions for the isolation of ginsenoside Rg3 were per-
formed as described above. A kat indicates a unit of catalytic
activity, defined as the conversion of one mol substrate per
second under the assay conditions (1 pkat/mg = 60 pmol/
min/mg).
LC-ESI/MS analysis of ginsenoside Rg3
To determine the product of the enzymatic reaction, ginse-
noside Rg3 was purified by the method established previ-
ously by Ko et al. (2007). The extract was concentrated and
applied to an Agilent 3300 Ion Trap LC/MS/MS equipped
with an electrospray ionization (ESI) apparatus. Negative-
ion ESI was conducted using an ion source voltage of 4.0 kV.
The optimized parameters were as follows: sheath gas, 50
arbitrary units; auxiliary gas, 10 arbitrary units; capillary
voltage, 12 V; tube lens offset voltage, 40 V; capillary
23 nA; nebulizing gas pressure, 45 psi; dry gas flow,
12 l/min; drying gas temperature 350 °C. All mass data were
acquired using Analysis Scripstarter software with an Agi-
lent ESI-MS.
Results and discussion
Effect of MJ on ginsenoside Rg3 biosynthesis
In ginseng, it has been reported previously that ginseno-
sides accumulated in hairy roots elicited by MJ, and its
elicitation resulted in the upregulation of transcriptional
expression of genes related to ginsenoside biosynthesis
(Kim et al. 2009). However, no studies showed Rg3
accumulation in ginseng hairy root cultures elicited by MJ.
It has been previously thought that Rg3 and Rh2 accu-
mulate in red ginseng only as a result of the thermal
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Plant Cell Tiss Organ Cult (2013) 112:87–93
process. If Rg3 accumulates in ginseng elicited by MJ, this
may be evidence that the mechanism of Rg3 biosynthesis
exists in ginseng. Therefore, in an attempt to evaluate the
effects of MJ elicitation on Rg3 biosynthesis, ginsenoside
contents in hairy roots after elicitation were evaluated by
HPLC analysis. The results demonstrated that the levels of
most ginsenosides were enhanced in MJ-treated hairy
roots, and the increases in the PPD group members were
much greater than those of the PPT group (Fig. 3). These
results were consistent with those of previous studies
(Wang and Zhong 2002; Kim et al. 2004, 2009). After
7 days of MJ elicitation, a peak of Rg3 was detected
(Fig. 4) and quantified in the hairy roots (0.42 ± 0.02 mg/
g, dry wt.). The identity of the signal was confirmed by
spiking with an internal Rg3 standard in our supplementary
experiments (Figure 1S, Online Resource 1). Our data
demonstrated that Rg3 in the hairy roots accumulated as
the result of MJ elicitation. To our best knowledge, this
study is the first to demonstrate that MJ elicitation induces
ginsenoside Rg3 formation in hairy root cultures. Inter-
estingly, a peak for Rh2, which showed a retention time of
82.4 min, was detected in the controls (Fig. 4), albeit at
very low levels (0.11 ± 0.01 mg/g, dry wt.). This result
clearly demonstrated that this hairy root line had an inde-
pendent Rh2 biosynthesis capacity even in the absence of
the thermal process. We attempted to confirm the presence
of Rh2 in other hairy root lines generated by A. rhizogenes-
mediated transformation. All four of the hairy root lines
possessed this Rh2 biosynthesis capacity, but Rg3 was not
observed at 77.4 min (data not shown). After 7 days of MJ
elicitation, the content (0.14 ± 0.02 mg/g, dry wt.) of Rh2
in MJ-treated hairy roots increased to 1.27 times that of the
controls. Compared to the levels of Rg3 accumulation, the
increase in the level of Rh2 in the MJ-treated hairy roots
was rather low. In another study, the levels of compounds
such as a-amyrin, b-amyrin and lupeol, positioned up
stream in triterpene biosynthesis, were low in spite of
elicitation by MJ, because triterpene aglycone is considered
Fig. 3 Ginsenoside contents in ginseng hairy roots elicited by
0.1 mM MJ for 7 days. The values are expressed as mean of three
independent samples with standard deviations
Plant Cell Tiss Organ Cult (2013) 112:87–93
Fig. 4 HPLC analysis of ginsenosides. Three chromatograms of
HPLC analysis, authentic ginsenoside Rb1, Rc, Rb2, Rd, Rg3 and
Rh2 (a), the extract isolated from hairy roots not treated with MJ (b),
the extract isolated from hairy roots treated with 0.1 mM MJ for
7 days (c), indicating the increase of ginsenoside content with MJ
elicitation. Two arrows indicate the peak of ginsenoside Rg3 and Rh2,
respectively
to be a rate limiting-step. However, both gene transcription
levels and protein activities associated with triterpene
biosynthesis were much higher than those of the control
(Hayashi et al. 2003). Therefore, Rh2 as a precursor may
rapidly supply carbon flow into downstream biosynthesis
of ginsenoside instead of accumulating. It appears likely
that Rh2 biosynthesis may be a more rate limiting-step than
Rg3 biosynthesis. However, until now there has been no
direct evidence about a rate limiting-step of Rh2 and Rg3
biosynthesis.
Detection of UGRh2GT activity
In an effort to gain greater insight into the effects of MJ on
UGT activity, we attempted to evaluate ginsenoside het-
erogeneity for Rg3 caused by UGT activity. The intro-
duction of glucose into ginsenoside Rh2, leading to Rg3, is
a binding reaction catalyzed by a glucosyltransferase.
As shown in Fig. 5b, incubation of Rh2 and UDP-glu-
cose with the boiled proteins did not result in a peak.
However, a novel peak for putative Rg3 was noted
(Fig. 5c, d). In order to characterize this product with
greater precision, we conducted LC-MS/MS analysis. As
shown in Fig. 6, one of the major MS/MS fragments was
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Fig. 5 HPLC analysis of reaction mixture of proteins from ginseng
hairy root elicited by 0.1 mM MJ for 7 days. a Chomatogram of
ginsenoside Rg3 standard; b no formation of Rg3 from reaction
mixture by heat-inactived proteins (at 100 °C for 5 min); c the
reaction mixture from the control not treated with MJ; d the reaction
mixture from hairy roots treated with MJ. Arrows indicate the peak of
ginsenoside Rg3
m/z 783 [M–H]-, similar to that of ginsenoside Rg3. Other
fragment ions were seen at m/z 621 [M–Glc–H]- and m/z
459 [M–Glc–Glc–H]-, similar to those of Rh2 and proto-
panaxadiol of the Rh2 aglycone, respectively. The mass
difference between m/z 783, m/z 621 and m/z 459 was 162,
which suggests the loss of a total of two glucoses. Addi-
tionally, these fragment profiles were exactly consistent
with the fragmentation pattern of Rg3 extracted from North
American ginseng (Popovich and Kitts 2004). The mass
fragmentation pattern of the Rg3 standard was consistent
with that of the purified product extracted from the enzyme
reaction mixture. This result showed that a new peak cor-
responding to Rg3 was formed by the enzyme reaction,
thus suggesting that the ginseng hairy roots independently
possess an Rg3 biosynthesis capacity in the absence of a
thermal process.
On the other hand, the peak for UGRh2GT activity was
also detected in the controls which were not treated with
MJ elicitation (Fig. 5c), although Rg3 was not detected in
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Fig. 6 LC-ESI-MS/MS spectra of ginsenoside Rg3 in negative ion
mode. a The authentic sample of Rg3, b The Rg3 obtained from the
reaction mixture with UDP-glucose, ginsenoside Rh2 and proteins
extracted from hairy roots elicited by 0.1 mM MJ
0.3
Con
UGRh2GT activity (pkat)
0.25 MJ
0.2
0.15
0.1
0.05
0
12 h 24 h 3d 7d
Time course after MJ treatment
Fig. 7 UGRh2GT activity in ginseng hairy roots treated with
0.1 mM MJ. Each value represents mean ± standard deviation from
three independent samples
the controls, as shown by the results of HPLC chromatog-
raphy (Fig. 4b). Therefore, we measured the UGRh2GT
activity to compare the controls with the MJ-treated samples.
As illustrated in Fig. 7, UGRh2GT activity in the hairy roots
treated with MJ elicitation for 7 days was 1.63-times greater
than that of the controls. A low level of UGRh2GT activity
was maintained in the controls. It appears likely that Rg3
contributes as a precursor to the biosynthesis of other PPD-
type ginsenosides, including ginsenosides Rb1, Rb2, Rb3,
Rc, and Rd. Although Rg3 was not detected in the control,
UGRh2GT activity was maintained. This in conjunction
with other evidence might suggest that Rg3 biosynthesis is
present in ginseng. In addition, our results showed that the
proteins extracted from our hairy root lines can catalyze Rg3
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Plant Cell Tiss Organ Cult (2013) 112:87–93
from Rh2, thereby suggesting that our ginseng hairy root
lines possessed Rg3 biosynthesis capacity. Recently,
expressed sequence tags (EST) from several cDNA libraries
of ginseng have been sequenced and analyzed to discover
novel genes related to ginsenoside biosynthesis, due to the
substantial commercial and medicinal importance of this
plant (Jung et al. 2003; Kim et al. 2006). In particular, glu-
cosyltransferase (GT), among the genes associated with
ginsenoside biosynthesis, plays a regulatory role in ginse-
noside formation. It has been previously reported that 235
GT genes exist in American ginseng (Sun et al. 2010a).
Among these genes, four GTs that evidenced tissue-specific
expression patterns similar to those of the dammarenediol-II
synthase gene, were selected as strong candidates. Although
a number of candidates for GT genes associated with gin-
senoside biosynthesis have been reported thus far (Choi et al.
2005; Kim et al. 2009; Sun et al. 2010a), no information
regarding these functions is currently available.
Conclusion
From the results obtained, it can be inferred that Rg3 was
produced in ginseng hairy roots without a thermal process,
when the cultures were elicited by MJ for 7 days. More-
over, the fact that the protein extract from hairy roots
catalyzed the reaction synthesizing Rg3 from Rh2, result-
ing in ginsenoside heterogeneity. Our results, showing that
Rg3 biosynthesis exists in ginseng, will provide us with
basic information needed to discover GT genes associated
with Rg3 biosynthesis in ginseng. The discovery of the GT
gene for Rg3 biosynthesis and its function will make
possible the manipulation of ginsenoside biosynthesis.
Finally, Rg3 and Rh2 will be biosynthesized in transgenic
ginseng without the thermal process.
Acknowledgments This work was supported by the Cooperative
Research Program for Agricultural Science & Technology Develop-
ment (Project No. 200901AFT143782304), RDA, Republic of Korea.
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