Advances in Biological Chemistry, 2011, 1, 49-57
doi:10.4236/abc.2011.13007 Published Online November 2011 (http://www.SciRP.org/journal/abc/ ABC ).
Published Online November 2011 in SciRes. http://www.scirp.org/journal/ABC
Gentian extract induces caspase-independent and
mitochondria-modulated cell death
Mika Ogata1, Kazushige Matsukawa2, Kiyomi Kogusuri1, Tetsur o Yamashita2, Takashi Hikage2,3,
Kikukatsu Ito1,2, Yasushi Saitoh1,2, Ken-ichi Tsutsu mi 1,2*
1Cryobiofrontier Research Center, Iwate University, Morioka, Japan;
2United Graduate School of Agricultural Sciences, Iwate University, Morioka, Japan;
3Hachimantai City Floricultural Research and Development Center, Hachimantai, Japan.
Received 23 September 2011; revised 25 October 2011; accepted 3 November 2011.
ABSTRACT
Extracts from the dried roots of gentian plant, Gen-
tiana triflora, exhibit an antiproliferative activity a-
gainst cultured and implanted tumor cells. However,
the underlying mechanism has been unclear. In the
present study, we show that the cell death induced by
the extract occurs caspase-independently and de-
pends on metabolic status of mitochondrial respira-
tion. We observed that sensitivity to the extract was
considerably lower in HeLa cells, which have a low
rate of mitochondrial respiration, in comparison to
Y3-Ag1.2.3 cells, which have a higher rate of respira-
tion. Furthermore, sensitivity of HeLa cells to the
extract increased significantly when they were forced
to switch their energy dependency from glycolysis to
mitochondrial respiration. These results indicate that
the gentian extract targets on mitochondrial respira-
tion. Consequently, different respiratory activities in
mitochondria confer cells to have different suscepti-
bilities to the extract-induced cell death.
Keywords: Antiproliferative Activity; Cell Death; Mi-
tochondria; Respiration; Gentiana triflora
1. INTRODUCTION
As a typical form of cell death, apoptosis has been ex-
tensively characterized. However, reports concerning to
additional types of cell death are accumulating, and the
pathways for cell death are now thought to be far more
diverse than predicted [1-3]. Despite such diverse path-
ways, the mitochondria act in many cases as a relay
point that transmits different signaling for cell death,
with the final outcome of the mitoch o ndria depend ing on
the cell types. Accordingly, the metabolic condition of
the mitochondria modulates both apoptotic [4-7] and
non-apoptotic cell death [8-12]. Highly proliferative can-
cer cells derive most their energy from glycolysis ra-
ther than from mitochondrial respiration [13-19]. Those
cancer cells are adapted for survival by using various
pathways to avoid cell death [20,21]. Upregulated gly-
colysis, for example, activates the serine/threonine ki-
nase Akt, conferring resistance to antiproliferative drugs
[6,22]. Susceptibility of cells to antipro liferative drugs is
also modulated by mitochondrial respiration. A high con-
centration of glucose in culture strongly inhibits mito-
chondrial respiration in most cancer cells, a phenomenon
called the Crabtree effect [23]. Those cells exhibit resis-
tance to mitochondrial toxicants such as antimycin and
oligomycin. Circumventing the Crabtree effect by grow-
ing in glucose-deprivated galactose media [24,25], cells
become susceptible to the toxicants [26]. Collectively,
glycolysis and mitochondrial respiration are directly or
indirectly involved in cell death and survival signaling.
Previously, we reported that extracts from dried roots
of Gentiana triflora, an ingred ient in Chinese herbal me-
dicine [27], exhibit cell death-inducing activity against
cultured tumor cells and antiproliferative activity agains t
implanted tumor cells [28]. Th e underlining mechanism,
however, has been unknown. Here, we show that the ex-
tract-induced cell death occurs caspase-independently
and depends on mitochondrial state of energy metabo-
lism.
2. MATERIALS AND METHODS
2.1. Plant Materials and Extract Preparation
The gentian plant used in this study was Gentiana tri-
flora var. japonica, which was provided by the Hachi-
mantai City Floricultural Research and Development
Center, Hachimantai, Iwate 028-7592, Japan. Prepara-
tion of gentian root extract and its cell antiproliferative
activity has been described previously [28]. Dried roots
were powdered and extracted sequentially with 10 times
volume (w/v) of chloroform at 50˚C, methanol at 50˚C,

M. Ogata et al. / Advances in Biological Chemistry 1 (2011) 49-57 50
and boiling water for 30 min. Each extraction was re-
peated twice. After cooling, the extract was vacuum-
dried, dissolved in methanol or water (for boiling-water
extract) at a concentration of 20 mg/ml, and centrifuged
at 14.000 ×g for 30 min to discard insoluble fibrou s ma-
terials. The boiling-water extract was autoclaved for 20
min at 121˚C, and passed through a 0.22 mm filter. The
extracts were stored at 4˚C in the dark.
2.2. Cells and Culture Conditions
Y3-Ag1.2.3 cells derived from rat myeloma were pur-
chased from Health Science Research Resources Bank
(JCRB9091, Osaka, Japan) and were cultured in Dul-
becco’s modified Eagle’s medium (DMEM) containing
25 mM glucose and supplemented with 5% fetal bovine
serum (FBS). Human HeLa and rat dRLh84 cells were
cultured in DMEM supplemented with 10% FBS and
newborn bovine serum (NBS), respectively. Daudi and
3Y1-B1-6 cells were cultured in DMEM supplemented
with 5% FBS. For culturing HeLa and Y3-Ag1.2.3 cells
in galactose, the glucose in each medium was replaced
by 10 mM galactose and supplemented with 1 mM so-
dium pyruvate [18]. Cells were maintained in the galac-
tose medium for more than 10 passages prior to the ex-
periments.
2.3. Cell Proliferation Assay
Cell proliferation was measured either by counting vi-
able cells with a trypan blue exclusion assay, or by a
WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetra-
zolio]-1, 3-benzene disulfonate) assay according to the
supplier’s protocol (Cell Counting Kit, DOJINDO, Ja-
pan). Briefly, cells (1 to 2 × 104) were seeded in 100 ml
of medium in a 96-well plate in the presence or absence
of gentian root extracts. After cultur ing for 24 to 48 hr at
37˚C in a CO2 incubator, 10 ml of WST-1 reagent was
added, followed by additional 2 to 4 hr of incubation at
37˚C. After that, colorimetric measurements were per-
formed at 450 nm and 650 nm (for reference) using a
plate reader apparatus (Multiscan JX, Thermob io Analy-
sis Co., Tokyo). The relative number of viable cells was
expressed as a percentage of the control, based on the
absorbance at 450 nm.
2.4. Inhibition of Caspase Activity and Detection
of Cytochrome C
The pan-caspase inhibitor Z-VAD-fmk [benzyloxycar-
bonylvalyl-alanyl-aspartic acid (O-methyl)-fluoro-me-
thylketone, Peptide Institute Inc., Osaka, Japan] was
used to determine the caspase dependency of cell death.
Cells were plated at a density of 1 to 2 × 104 per 100 ml
in a 96-well plate and cultured in th e presence of gentian
extract and 0 to 50 mM Z-VAD-fmk for the indicated
times in a CO2 incubator. The number of viable cells was
measured with either the WST-1 assay or trypan blue
exclusion, as described above. Cytoplasmic and mito-
chondrial cytochrome c were detected by a Cytochrome
C Release Apoptosis Assay Kit (Oncogene, San Diego,
USA), according to the manufacturer’s protocol. Equal
amounts (10 mg) of cytoplasmic and mitochondrial pro-
teins were subjected to SDS-polyacrylamide gel elec-
trophoresis, followed by Western blotting using an
anti-cytochrome c antibody, and the ECL Western Blot-
ting Analysis System (Amersham Pharmacia Biotech).
2.5. Flow Cytometric Analysis of Mitochondrial
Transmembrane Potential () and
Mitochondrial Mass m
Mitochondrial transmembrane poten tial () and mi-
tochondrial mass were analyzed by staining cells with
200 nM MitoTracker Red CMXRos [Molecular Probes
Inc. (Invitrogen)] and 200 nM MitoTracker Green FM
(Molecular Probes Inc.), respectively, according to the
manufacturer’s protocols. MitoTracker Red accumulates
in the mitochondrial matrix under the influence of the
changes in mitochondrial membrane potential. Mito-
Tracker Green selectively accumulates in the mitochon-
drial matrix where it binds covalently to mitochondrial
proteins. Mitochondrial
m
m

 and mass-related fluo-
rescence was measured using an FL1 photomultiplier
and a FACS Calibur flow cytometer. The measurements
were performed at least in triplicate for each dye, and the
values were expressed as arbitrary scanning units of
fluorescence at the peak of cell populations.
2.6. Measurements of Oxygen Consumption
Oxygen consumption in intact cells was measured as an
activity of mitochondrial respiration as previously de-
scribed [29]. After the growth under different culture
conditions, 1 to 5 × 106 cells were resuspended in 1 ml
of fresh culture medium and placed in the sealed respira-
tion chamber equipped with a thermostat and micro-
stirrer. Oxygen consumption was measured polaro-
graphically at 37˚C using the Clark-type oxygen elec-
trode disk, according to the procedures recommended by
the manufacturer (Oxy1, Hansatech Instrument Inc.,
UK). The respiration rate was expressed as nmoles of O2
consumed per 106 cells as a function of time (min).
2.7. Statistical Analysis
In most cases, data are expressed as mean ± S.D. from at
least three independent experiments. Statistical com-
parisons were performed by one-way ANOVA. The re-
sults were considered significant at a value of p < 0.05.
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M. Ogata et al. / Advances in Biological Chemistry 1 (2011) 49-57 51
3. RESULTS AND DISCUSSION to different activities of mitochondrial toxicants against
HepG2 cell with different energy metabolism [26].
3.1. Antiproliferative Activity of Gentian Extract
Differs among Tumor Cell Types In the following experiments, Y3-Ag1.2.3 and HeLa
cells were used as typical cancer cell lines exhibiting
high and low sensitiv ity against the extract, respectively. Gentian extracts were prepared by sequential extraction
with chloroform, methanol, and boiling-water. The anti-
proliferative activity was found in boiling-water extract
and, to a lesser extent, methanol extract. In the following,
all experiments were carried out using the boiling-water
extract.
3.2. Cell Death Induced by Gentian Extract is
Caspase-Independent
To elucidate the mechanism of the antiproliferative ac-
tion of the extract, characteristic features of apoptosis
were examined using Y3-Ag1.2.3 cells. Firstly, to see
whether the activity depends on caspase or not, the effect
of a pan-caspase inhibitor, Z-VAD-fmk, on extract- in-
duced cell death was examined (Figure 2(a)). As a con-
trol experiment, rescue of apoptosis by the inhibitor was
monitored in parallel, using the cells treated with a
known apoptosis inducer, campthotecin (CPT) [30]. The
results showed that, while 50 mM Z-VAD-fmk almost
completely inhibited CPT-induced apoptosis, it did not
affect the extract-induced cell death. Thus, the extract-
induced cell death could be caspase-independent. Change
in the cellular localization of cytochrome c was also ex-
amined (Figure 2(b)). Equal amounts of proteins from
mitochondrial and cytoplasmic fractions of the extract-
or CPT-treated cells were subjected to Western blotting
using anti-cytochrome c antibodies. Cytoplasmic cyto-
chrome c increased after the cells were treated with 10
mM CPT for 3 to 7 hr. In contrast, even at higher con-
centrations (5 mg/ml), the g entian extract did not induce
the release of cytochrome c from mitochondria within
the time examined. It is not clear at present whether the
extract contains a factor(s) that inhibits release of cyto-
chrome c from mitochondria, similar to that reported for
aqueous extract of the Chinese medicine, Danggui-Shao-
yao-San [31].
The antiproliferative activity of the boiling-water ex-
tract from gentian roots was assayed for different cell
types. As shown in Figure 1, the activity differed among
target cell types. For example, Y3-Ag1.2 .3 (rat myelo ma)
and Daudi cells (human Burkitt’s lymphoma) exhibited
high sensitivities to th e extract, while HeLa (human cer-
vical cancer), dRLh84 (rat hepatoma), and 3Y1 B1-6 (rat
embryonic fibroblast) cells showed considerably lower
sensitivities by comparison to Y3-Ag1.2.3 cells. Such a
cell type-dependent susceptibly to antiproliferation has
not been observed so far for gentian extract. Why the
activity is distinctive for different cell types is unknown
at present. One possibility may arise that metabolic
status of the cells correlates with their sensitivities to
antiproliferative or death-inducing compound(s), similar
We next examined the change in mitochondrial trans-
membrane potential (m


m
) during the process of the
extract-induced cell death. Cells treated with CPT or oli-
gomycin were also examined. Oligo mycin is an inh ibitor
of the F1Fo-ATPase, which is integrated in the inner mi-
tochondrial membrane; it inhibits proton transport from
intermembrane space into matrix and ATP synthesis, re-
sulting in increased

 (Figure 2(c), right). As
shown in Figure 2(c) (left), increased in
Y3-Ag1.2.3 cells after treatment with the extract, while
that of cells treated with the apoptosis-inducer CPT sig-
nificantly decreased (Figure 2(c), right). These results
implied that gentian extract acts on mitochondrial energy
production in a manner similar to oligomycin. Oligomy-
cin has been known to induce caspase-independent cell
death via mitochondria-emanating effectors and Bax BH3
domain [32], as well as to inhibit F1Fo-ATPase. However,
such an oligomycin-like activity has not, to to our knowl
m
Figure 1. Antiproliferative activity of gentian extract against
different cancer cell lines. HeLa (human), 3Y1B1-6 (rat, show-
n as 3Y1), dRLh84 (rat), Daudi (human) and Y3-Ag1.2.3 (rat,
shown as Y3Ag) cells were plated at a density of 1 × 104 cells
per 100 l medium and cultured for 48 hr in the presence of in-
creasing concentrations of the gentian extract. The relative nu-
mber of viable cells was determined by staining with trypan
blue, and expressed as a percentage of the control (without
extract). Data are averages of three independent experiments.
Error bars represent value ranges.
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M. Ogata et al. / Advances in Biological Chemistry 1 (2011) 49-57 52
Figure 2. Cell death induced by gentian extract does not proceed via typical apoptotic pathways. (a) Effect of a pan-caspase inhibitor,
Z-VAD-fmk, on the extract-induced cell death. Y3-Ag1.2.3 cells (Y3Ag) were cultured for 48 hr in the presence of gentian extract
(extract) and Z-VAD-fmk at the concentrations indicated. As a control experiment, Y3-Ag1.2.3 cells were treated with a known
apoptosis inducer, campthotecin (CPT), at 10 M for 24 hr in the presence or absence of Z-VAD-fmk. The relative number of viable
cells was determined by a WST-1 assay and expressed as a percentage of the control. (b) Release of cytochrome c from mitochondria
after incubation with gentian extract (left) or CPT (right). Y3-Ag1.2.3 cells were cultured in the presence or absence of gentian ex-
tract or CPT for the indicated times. Equal amounts (10 g) of proteins from mitochondrial (M) and cytoplasmic (c) fractions (see
Materials and methods) were subjected to Western blotting with an anti-cytochrome c antibody. (c) Changes in mitochondrial trans-
membrane potential () in Y3-Ag1.2.3 cells after growth in the presence of gentian extract, camptothecin (CPT) and oligomy-
cin. Cells were cultured in the presence of 0.6 mg/ml and 2 mg/ml gentian extract for 21 hr, 10 M CPT for 7 hr, and 25 M oligomycin
for 3 hr. m was measured using MitoTracker Red CMXRos as described in Materials and methods. Data are averages of four to six
independent experiments, and are expressed as mean ± S.D. *Statistically significant, P < 0.05, ***P < 0.001.50.
m
edge, been reported for extracts/compounds from plants.
The results obtained from the HeLa cells were co nsisten t
with a previous report [25]. We examined whether mito-
chondrial energy metabolism, such as oxidative phospho-
rylation, affects the antiproliferative activity of the extract.
3.3. Acti vation of Mitocho ndrial Respiration Af ter
Adaptation t o Gr ow in Galactose Medium
Cancer cells are generally characterized by a high gly-
colytic activity and a low oxidative phosphorylation
(respiration) in mitochondria [13,14,24]. From the re-
sults described above, we hypothesized that low levels
of mitochondrial energy metabolism affect the suscepti-
bility of cells to extract-induced death. To test this, the
sensitivities of the cells to the extract were examined
after mitochondrial respiratory activity was forced to
increase by culture conditions. This experiment was
conducted based on the observations that the lowered
activity of mitochondrial respiration in cancer cells can
be reactivated by growth in a medium in which glucose
is replaced by gal actose [25,26].
To confirm that growth in galactose medium alters
mitochondrial functions in HeLa and Y3-Ag1.2.3, the
cells grown in galactose were assayed for their growth
rates, oxygen consumption, mitochondrial mass and tran-
smembrane potential (m

). As shown in Figure 3,
HeLa cells showed different growth rates when cultured
in glucose and galactose media (about 2.6-fold prolong-
ed doubling time in galactose). However, Y3-Ag1.2.3
cells exhibited a similar doubling time in glucose and
galactose during the exponentially growing phase.
The rate of oxygen consumption differed between HeLa
and Y3-Ag1.2.3 cells grown in standard glucose medium
(Figure 4(a)). Y3-Ag1.2.3 cells exhibited a higher rate
of oxygen consumption than HeLa cells by about
1.7-fold. When grown in glucose-deprivated galactose
medium, oxygen consumption rate increased by about
2.1- and 1.3-fold in HeLa and Y3-Ag1.2.3 cells, respec-
tively. Galactose-grown HeLa cells reached to a similar
range in the oxygen consumption rate to that of glu-
cose-grown Y3-Ag1.2.3 cells. Thus, the cells grown in
galactose medium enhanced their aerobic metabolism in
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M. Ogata et al. / Advances in Biological Chemistry 1 (2011) 49-57 53
Figure 3. Proliferation of cells in different sugar media. HeLa (left) and Y3-Ag-
1.2.3 cells (right) were grown in DMEM containing glucose (Glc, circle) or galac-
tose (Gal, triangle). Glucose concentrations in media for HeLa and Y3-Ag1.2.3
cells were 5.5 mM and 25 mM, respectively. Galactose concentration was 10 mM
for both cell lines. The number of viable cells was counted by trypan blue staining,
and the presented values are the averages of three independent experiments. Error
bars denote value ranges.
Figure 4. Mitochondrial activities of HeLa and Y3-Ag1.2.3 cells grown in glucose (Glc) or ga-
lactose media (Gal). Concentrations of glucose and galactose were as described in the legend to
Figure 3. Oxygen consumption (a), mitochondrial transmembrane potential (m

, b), and
mitochondrial mass (c) were measured as described in Materials and Methods. Mitochondrial
and mass-related fluorescences were measured by FCM using an FL1 photomultiplier.
Presented values were the averages of three to six independent experiments, and are expressed as
mean ± S.D. *Statistically significant, P < 0.05, ***P < 0.001.
m
mitochondria. The results obtained from the HeLa cells
were consistent with a previous report [25].
m in galactose-grown HeLa cells increased slig-
htly, while Y3-Ag1.2.3 cells showed no significan t chan-
ge, as compared to their glucose-grown counterparts (Fi-
gure 4(b)). Cells grown in glucose and galactose had no
statistically significant difference in their mitochondrial
masses (Figure 4(c)).
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M. Ogata et al. / Advances in Biological Chemistry 1 (2011) 49-57 54
3.4. Cells with Increased Respiration Rates
Become More Sensitive to Gentian Extract
To determine whether an increased rate of mitochondrial
respiration affects the susceptibility of the cells to ex-
tract-induced death, HeLa and Y3-Ag1.2.3 cells were
cultured in glucose and in galactose media in the pres-
ence or absence of the extract. As shown in Figure 5
(left), HeLa cells cultured in galactose medium exhibited
a significantly higher susceptibility to extract-induced
cell death, as compared with those cultured in glucose.
The 50% effective concentration (EC50) of the extract for
HeLa cells in glucose media was calculated to be ap-
proximately 2.0 mg/ml. This value was significantly re-
duced (0.6 mg/ml - 0.7 mg/ml) after the cells were ada-
pted to grow in galactose. Based on the EC50 values, the
sensitivity of HeLa cells to the extract-triggered death in
galactose medium had increased nearly 3-fold higher than
that in glucose medium. The increased sensitivity of
HeLa cells is due to altered energy metabolism in galac-
tose medium, since the extract by itself did not affect
oxygen consumption in galactose medium (data not
shown). Proliferation of Y3-Ag1.2.3 cells was also in-
hibited more when the cells were grown in galactose
medium. However, this increased sensitivity was seen
only at higher concentrations of the extract, and the lev-
els of increase were not as high as that of HeLa cells
(Figure 5, right).
The results presented here indicated that the gentian
extract does neither induce release of cytochrome c from
the mitochondria, decrease in mitochondrial transmem-
brane potential, n or activation of caspases. Previous data
showed that the extract triggers large-scale chromosomal
fragmentation but not oligonucleosomal DNA fragmen-
tation (DNA ladder) [28]. These observations do not
entirely exclude a possibility that the extract ind uces cell
death via apoptosis, since several caspase-independent
apoptotic pathw ays have also b een kno wn [33,34 ]. How-
ever, increase in m

 (Figure 2(c)) implies the ex-
tract-induced cell death to be other than apoptosis, be-
cause, apoptotic pathway involves dissipation (not in-
crease) of m

, which means mitochondrial transmem-
brane permeabilization, i.e., one of the biochemical as-
pects of distinct modalities of apoptosis [35].
Using HeLa and Y3-Ag1.2.3 cells as the representa-
tive cell lines with low and high sensitivity to th e extract,
respectively, correlation of the antiproliferative activity
of the extract to the metabolic circumstances in the mi-
tochondria was examined. The sensitivity of HeLa cells
was approximately 5-fold lower than Y3-Ag1.2.3 cells.
However, HeLa cells become more sensitive after adap-
tation for growth in galactose medium, a conditio n under
which mitochondrial respiration (oxidative phosphoryla-
tion) increased by about two-fold. Thus, the resistance of
HeLa cells to the extract seems to correlate with low
levels of oxidative phosphorylation. The extract most
Figure 5. Effect of gentian extract on the growth of cells cultured in different sugar
media. HeLa (left) or Y3-Ag1.2.3 cells (right) were grown in glucose or galactose
media for 48 hr in the presence of increasing concentrations of the extract. The rela-
tive number of viable cells was counted by trypan blue exclusion. Values were the
averages of three to four independent experiments, and were expressed as a per-
centage of the control. Error bars represent value ranges.
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M. Ogata et al. / Advances in Biological Chemistry 1 (2011) 49-57 55
likely abrogated a step(s) in the mitochondrial energy me-
tabolism, and the extract- induced cell death proceeded via
increased in a way similar to the action of oligo-
mycin [36]. Therefore, the extrac t did not efficiently act on
cells with low levels of oxidative phosphorylation.
m
In contrast, the cell death-inducing effects of the ex-
tract on Y3-Ag1.2.3 cells increased only slightly after
the cells were adapted for growth in galactose medium.
The following explanation may be plausible for this. As
described earlier, oxygen consumption in Y3-Ag1.2.3
cells is considerably higher than in HeLa cells in stan-
dard growth conditions (glucose medium), and the in-
crease of that in galactose medium is less significant as
compared to that in HeLa cells. In addition, the rate of
oxygen consumption in Y3-Ag1.2.3 cells in glucose is in
a similar range to that in HeLa cells in galactose medium.
Thus, Y3-Ag1.2.3 cells per se appear to have an innate
physiological state in mitochondrial energy metabolism,
which is responsible for a nearly maximal level of sus-
ceptibility to the gentian extract, hence the limited in-
crease in susceptibility to death in galactose medium.
Critical roles for the mitochondrial respiratory chain
in cell death have been demonstrated by several groups.
A recent report showed that defects in the ability to carry
out oxidative phosphorylation render HepG2 cells be-
come more resistant to mitochondrial toxicants such as
rotenone, antimycin and oligomycin [26]. In cell death
triggered by ER stress and DNA damage, oxidative pho-
sphorylation is required for the activation of Bax and
Bak, which are essential for alteration of mitochondrial
membrane permeability [37]. Consequently, cancer cells
with very low levels of oxidative phosphorylation are
unable to activate Bax and Bak, and thus, are able to
evade cell death signals. Kwong et al. [7] also reported
that cells lacking respiratory chain were protected
against both mitochondrial- and ER stress-induced apo-
ptosis. The action of the gentian extract on HeLa and
Y3-Ag1.2.3 cells does not contradict the observations
described above.
The responsive gentian compound is yet to be identi-
fied, although it has small molecular mass and is wa-
ter-soluble. Gentian plants contain several characteristic
small compounds, including gentiopicrosides, gentisic
acid, oligosaccharides (gentianose and gentiobiose), and
glucosides. Those are known for various activities such
as inhibitor of aldose reductase, inhibitor of platelet ac-
tivating factor (PAF), and scavenger of free radicals [27].
However, cell death-inducing or antiproliferative activity
has not been reported for those compounds. Chroma-
tographic behavior and mass spectroscopic analysis su-
ggested that the cell death -inducing compound relates to
gentiopicroside or its derivatives. However, commercia-
lly available gentiopicroside did not show such an a-
ctivity. Structure of the responsive compound (s) remains
to be elucidated in the future.
4. CONCLUSIONS
The gentian extract-induced cell death occurs in a cas-
pase-independent pathway and in a manner depending
on energy metabolism, i.e., cells with low rate of mito-
chondrial respiration had lower susceptibility to the ex-
tract-induced cell death as compared to those with higher
level of mitochondrial respiration. This was confirmed
by the fact that sensitivity to the extract increased sig-
nificantly when cells were forced to switch their energy
dependency from glycolysis to mitochondrial resp iration.
Thus, the extract targets on mitochondrial respiration , so
that different mitochondrial activities confer cells to
have different susceptibilities to the extract-induced cell
death.
5. ACKNOWLEDGEMENTS
We thank the Hachimantai City Floricultural Research and Develop-
ment Center for providing gentian plants and dried roots. We also
thank Dr. R. Tsutsumi at Iwate Medical University School of Medicine
for her help and discussion.
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