Cytoprotective effect of tectorigenin, a metabolite formed by transformation of tectoridin by intestinal microflora, on oxidative stress induced by hydrogen peroxide

Article abstract of DOI:10.1016/j.ejphar.2005.06.043

In the present study, the antioxidative properties of tectorigenin, a metabolite formed by transformation of tectoridin by intestinal microflora, were investigated. Tectorigenin was found to scavenge intracellular reactive oxygen species, and 1,1-diphenyl

Full text of DOI:10.1016/j.ejphar.2005.06.043

European Journal of Pharmacology 519 (2005) 16 – 23
www.elsevier.com/locate/ejphar
Cytoprotective effect of tectorigenin, a metabolite formed by
transformation of tectoridin by intestinal microflora,
on oxidative stress induced by hydrogen peroxide
a
Kyoung Ah Kang , Kyoung Hwa Lee , Sungwook Chae , Rui Zhang , Myung Sun Jung ,
a
a
a
a
b
b
c
So Young Kim , Hee Sun Kim , Dong Hyun Kim , Jin Won Hyun
a,
*
a
Department of Biochemistry, College of Medicine and Applied Radiological Science Research Institute, Cheju National University,
Jeju-si 690-756, Republic of Korea
b
Department of Neuroscience, College of Medicine, Ewha Womans University, Seoul 110-783, Republic of Korea
c
Department of Microbial Chemistry, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
Received 13 January 2005; received in revised form 27 June 2005; accepted 28 June 2005
Available online 15 August 2005
Abstract
In the present study, the antioxidative properties of tectorigenin, a metabolite formed by transformation of tectoridin by intestinal
microflora, were investigated. Tectorigenin was found to scavenge intracellular reactive oxygen species, and 1,1-diphenyl-2-picrylhydrazyl
(
DPPH) radical, and thus prevented lipid peroxidation. The radical scavenging activity of tectorigenin protected the viability of Chinese
hamster lung fibroblast (V79-4) cells exposed to hydrogen peroxide (H O ) via activation of extracellular signal regulated kinase (ERK)
2
2
2 2 2
pathway. Furthermore, tectorigenin reduced the apoptotic cells formation and cell cycle arrest at G /M phase induced by H O . Tectorigenin
increased the activities of cellular antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase, and also increased their
protein level. Taken together, these findings suggest that tectorigenin protected V79-4 cells against H O damage, by enhancing the
2
2
antioxidative activity and by activating ERK pathway.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Tectorigenin; Reactive oxygen species; Antioxidant enzyme
1
. Introduction
transformed by intestinal bacteria before being absorbed
from gastrointestinal tract (Akao et al., 1994; Kim et al.,
1998a). Tectoridin, a biologically active component, iso-
lated from the flowers of P. thunbergiana was transformed
into tectorigenin by human intestinal bacteria (Bae et al.,
1999). The transformed tectorigenin showed more potent
hypoglycemic activity, cytotoxicity against tumor cells, and
anaphylaxis inhibitory activity than tectoridin (Bae et al.,
1999; Park et al., 2004). These results suggested that
tectoridin is a prodrug, which can be transformed into the
active compounds by human intestinal bacteria.
Isoflavones are biologically active compounds, which
occur naturally in a variety of plants, with relatively high
levels in soybean. Recently, Park et al (1999) have isolated a
series of isoflavonoids; glycitin, tectoridin, 6-O-xylosyltec-
toridin, and 6-O-xylosylglycitin from the flowers of
Pueraria thunbergiana (Leguminosae), which have been
used in Chinese medicine. As most of the traditional
medicines are administered orally, their components inevi-
tably come into contact with intestinal microflora in the
alimentary tract. Finally, most of the components are
Reactive oxygen species are known to cause oxidative
modification of DNA, proteins, lipids and small cellular
molecules and are associated with tissue damage and are the
contributing factors for inflammation, aging, cancer, arterio-
sclerosis, hypertension and diabetes (Cooke et al., 1997;
*
Corresponding author. Tel.: +82 64 754 3838; fax: +82 64 726 4152.
E-mail address: jinwonh@cheju.ac.kr (J.W. Hyun).
0014-2999/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2005.06.043

K.A. Kang et al. / European Journal of Pharmacology 519 (2005) 16–23
17
Darley-Usmar and Halliwell, 1996; Farinati et al., 1998;
Laurindo et al., 1991; Nakazono et al., 1991; Palinski et al.,
rate, 1.0 ml/min; detection wavelength, 280 nm). The retention
time of tectorigenin was 11.45 min.
The characteristics of tectorigenin are: yellow amorphous
1
995; Parthasarathy et al., 1992). Cells have developed a
powder; mp 230–233 -C; UV (MeOH)max 264 nm (loge 4.11),
variety of antioxidant defense mechanisms, for protecting
themselves against the detrimental effects of reactive oxy-
gen species. Enzymatic defense mechanisms involve super-
oxide dismutase, which catalyzes dismutation of superoxide
anion to hydrogen peroxide; catalase, which converts
hydrogen peroxide into molecular oxygen and water, and
glutathione peroxidase, which destroys toxic peroxides.
In the present study, we have investigated the protective
effect of tectorigenin on cell damage induced by hydrogen
peroxide and its protective mechanism.
À1
2
37 nm (loge 3.18); IR (KBr)max 3447, 2921, 1648, 1023 cm
2
.2. Reagents
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2V,7V-
dichlorodihydrofluorescein diacetate (DCF-DA), and Hoechst
33342 were purchased from Sigma Chemical Company, St. Louis,
USA, and thiobarbituric acid from BDH Laboratories, England.
The other chemicals and reagents were of analytical grade. Primary
rabbit polyclonal anti-ERK1/2 (44 kDa/42 kDa ERK) and
-phospho-ERK1/2 (phosphorylated 44 kDa/42 kDa ERK) (Thr
2
02/Tyr 204) antibodies were purchased from Cell Signaling
Technology (Beverly, USA). Primary sheep monoclonal super-
oxide dismutase, and catalase antibodies were purchased from
Biodesign International Company (Maine, USA).
2
. Materials and methods
2
.1. Preparation of tectoridin and tectorigenin
2
.3. Cell culture
Tectoridin was isolated from dried flowers of P. thunbergiana
according to previous method (Lee et al., 2003). To obtain the
metabolites of tectoridin by human intestinal bacteria, a reaction
mixture was prepared containing 2 g of tectoridin and Bacteriodes
spercoris HJ-15, a human intestinal bacterium, in a final volume of
The Chinese hamster lung fibroblasts (V79-4) were obtained
from the American Type Culture Collection. The V79-4 cells were
maintained at 37 -C in an incubator, with a humidified atmosphere
of 5% CO , and cultured in Dulbecco’s modified Eagle’s medium
2
5
00 ml of anaerobic dilution medium in an anaerobic glove box
containing 10% heat-inactivated fetal calf serum, streptomycin
(
Coy Laboratory Products Inc., MI, USA). The reaction mixture
(
100 Ag/ml) and penicillin (100 units/ml).
was incubated at 37 -C for 24 h and was extracted three times with
ethyl acetate. The ethyl acetate-soluble portion of the reaction
mixture was dried on a rotary evaporator under reduced pressure
and subjected to silica gel column chromatography (2.5Â15 cm)
2
.4. Intracellular reactive oxygen species measurement
The DCF-DA method was used to detect the levels of
3
with CHCl /MeOH (10:1–10:2) as a mobile phase. Both tectoridin
intracellular reactive oxygen species (Rosenkranz et al., 1992).
DCF-DA diffuses into cells, where it is hydrolyzed by intra-
cellular esterase to polar 2V,7V-dichlorodihydrofluorescein. This
non-fluorescent fluorescein analog gets trapped inside the cells
and is oxidized by intracellular oxidants to a highly fluorescent,
and tectorigenin (0.19 g) were obtained from these fractions (Fig.
) and were identified according to the previously reported method
Park et al., 1999). The purity of tectorigenin (>98%) was analyzed
1
(
by HPLC system (A-Bondapak C18 (3.9Â300 mm); elution
solvent, methanol/water/glacial acetic acid (55:42.5:2.5); elution
2
V,7V-dichlorofluorescein. The V79-4 cells were seeded in a 96-
5
well plate at 1Â10 cells/ml. Sixteen hours after plating, the cells
were treated with various concentrations of tectoridin and
G1cO
O
O
2 2
tectorigenin and 30 min later, 1 mM H O was added to the
plate. The cells were incubated for an additional 30 min at 37 -C.
The fluorescence of 2V,7V-dichlorofluorescein was detected at 485
nm excitation and at 535 nm emission, using a PerkinElmer LS-
OCH
3
OH
OH
5
B spectrofluorometer.
Tectoridin
2
.5. DPPH radical scavenging activity
Bacteriodes spercoris HJ-15
in human intestine
Various concentrations of tectoridin and tectorigenin were
À4
added to a 1Â10 M solution of DPPH in methanol, and the
reaction mixture was shaken vigorously. After 5 h, the amount of
remaining DPPH was determined at 520 nm using a spectropho-
tometer (Lo et al., 2004).
HO
O
OCH
2.6. Lipid peroxidation inhibitory activity
3
OH
O
OH
Lipid peroxidation was assayed by thiobarbituric acid reaction
Tectorigenin
(
Ohkawa et al., 1979). V79-4 cells were seeded in a culture dish
5
at 1Â10 cells/ml. Sixteen hours after plating, the cells were
Fig. 1. Metabolic formation of tectorigenin. Tectorigenin is formed from the
transformation of tectoridin by Bacteriodes spercoris HJ-15, a human
intestinal bacterium.
treated with various concentrations of tectorigenin. One hour
2 2
later, 1 mM H O was added to the plate, and was incubated for

18
K.A. Kang et al. / European Journal of Pharmacology 519 (2005) 16–23
further 1 h. The cells were then washed with cold phosphate-
buffered saline (PBS), scraped and homogenized in ice-cold
2 2
of tectorigenin and after further incubation for 1 h, 1 mM H O
was added to the culture. After 24 h, 1.5 Al of Hoechst 33342
(stock 10 mg/ml), a DNA specific fluorescent dye, was added to
each well (1.5 ml) and incubated for 10 min at 37 -C. The stained
cells were then observed under a fluorescent microscope, which
was equipped with a CoolSNAP-Pro color digital camera, in order
to examine the degree of nuclear condensation.
1
.15% KCl. One hundred microliters of the cell lysates was
mixed with 0.2 ml of 8.1% sodium dodecylsulfate, 1.5 ml of 20%
acetic acid (adjusted to pH 3.5) and 1.5 ml of 0.8% thiobarbituric
acid. The mixture was made up to a final volume of 4 ml with
distilled water and heated to 95 -C for 2 h. After cooling to room
temperature, 5 ml of n-butanol and pyridine mixture (15:1, v/v)
was added to each sample, and the mixture was shaken well.
After centrifugation at 1000Âg for 10 min, the supernatant
fraction was isolated, and the absorbance was measured
spectrophotometrically at 532 nm.
2.10. Superoxide dismutase activity
5
The V79-4 cells were seeded at 1Â10 cells/ml, and 16 h
after plating, the cells were treated with various concentrations of
tectorigenin for 1 h. The harvested cells were suspended in 10
mM phosphate buffer (pH 7.5) and then lysed on ice by
sonicating twice for 15 s. 1% of Triton X-100 was then added
to the lysates and incubated for 10 min on ice. The lysates were
separated, by centrifugation at 5000Âg for 30 min at 4 -C in
order to remove the cellular debris. The protein content of the
supernatant was determined by the Bradford method (Bradford,
1976), with bovine serum albumin as the standard. The super-
oxide dismutase activity was used to detect the level of
epinephrine auto-oxidation inhibition (Misra and Fridovich,
1972). Fifty micrograms of the protein was added to 500 mM
of the phosphate buffer (pH 10.2) containing 0.1 mM EDTA and
2
.7. Cell viability
The effect of tectorigenin on the viability of the V79-4 cells
was determined using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium] bromide (MTT) assay, which is based on the
reduction of a tetrazolium salt by mitochondrial dehydrogenase in
the viable cells (Carmichael et al., 1987). To determine the
2 2
cytoprotective effect of tectorigenin in H O treated V79-4 cells,
5
cells were seeded in a 96-well plate at 1Â10 cells/ml. Sixteen
hours after plating, the cells were treated with various concen-
2 2
trations of tectorigenin. One hour later, 1 mM H O was added to
the plate and incubated at 37 -C for an additional 24 h. Fifty
microliters of the MTT stock solution (2 mg/ml) was then added
into each well to attain a total reaction volume of 200 Al. After
incubating for 4 h, the plate was centrifuged at 800Âg for 5 min
and the supernatants were aspirated. The formazan crystals in each
well were dissolved in 150 Al of dimethylsulfoxide and A540 was
read on a scanning multi-well spectrophotometer. To determine
the effect of tectorigenin on the viability of V79-4 cells during
A
100
Tectoridin
Tectorigenin
80
60
40
20
0
serum starvation, cells in 10% fetal calf serum were seeded in a
5
9
6-well plate at 1Â10 cells/ml. Sixteen hours after plating, cells
were serum starved (0.1% fetal calf serum), and then treated with
various concentrations of tectorigenin. The plate was incubated at
3
7 -C for 24 h and the cell viability was measured using MTT
test.
0
0.1
1
10
2
.8. Flow cytometry analysis
[µg/ml]
B
Flow cytometry was performed to determine the content of
apoptotic sub-G hypo-diploid cells and cell distributions at each
100
1
phase of a cell cycle (Nicoletti et al., 1991). The V79-4 cells were
5
Tectoridin
Tectorigenin
80
60
40
20
0
placed in a 6-well plate at 1Â10 cells/ml. Sixteen hours after
plating, the cells were treated with 10 Ag/ml of tectorigenin. After a
further incubation for 1 h, 1 mM H O was added to the culture.
2 2
After 24 h, the cells were harvested, and fixed in 1 ml of 70%
ethanol for 30 min at 4 -C. The cells were washed twice with PBS,
and then incubated for 30 min in dark at 37 -C in 1 ml of PBS
containing 100 Ag propidium iodide and 100 Ag RNase A. Flow
cytometric analysis was performed using a FACSCalibur flow
cytometer (Becton Dickinson, Mountain View, USA). The
0.1
1
10
proportion of sub-G
1
hypo-diploid cells and cell distributions at
[µg/ml]
each phase of cell cycle was assessed by the histograms generated
using the computer program, Cell Quest and Mod-Fit.
Fig. 2. Effect of tectoridin and tectorigenin on scavenging intracellular
reactive oxygen species and DPPH radicals. (A) The intracellular reactive
oxygen species generated was detected by DCF-DA method. (B) The
amount of DPPH radicals was determined spectrophotometrically at 520
nm. The measurements were made in triplicate (n =3/group) and values are
expressed as meansTS.E.M. *Significantly different from control
( p <0.05).
2
.9. Nuclear staining with Hoechst 33342
5
The V79-4 cells were placed in a 24-well plate at 1Â10 cells/
ml. Sixteen hours after plating, the cells were treated with 10 Ag/ml

K.A. Kang et al. / European Journal of Pharmacology 519 (2005) 16–23
19
50
40
30
20
10
electrophoresed in 10% sodium dodecysulfate-polyacrylamide
gel. Blots in the gels were transferred onto nitrocellulose
membranes (Bio-Rad, Hercules, USA), which were then
incubated with primary rabbit monoclonal-ERK1/2, -phospho
ERK1/2, primary sheep monoclonal superoxide dismutase, and
-catalase antibodies. The membranes were further incubated with
goat anti-rabbit or rabbit anti-sheep immunoglobulin G–horse-
radish peroxidase conjugates (Pierce, Rockland, USA), and then
exposed to X-ray film. Protein bands were detected using an
enhanced chemiluminescence Western blotting detection kit
0
(Amersham, Little Chalfont, USA).
0
0.1
[
1
10
µg/ml]
Fig. 3. Effect of tectorigenin on inhibition of lipid peroxidation. Lipid
peroxidation was assayed by measuring the amount of malondialdehyde
formation. The measurements were made in triplicate (n =3/group) and
values are expressed as meansTS.E.M. *Significantly different from
control ( p <0.05).
A
1
00
8
6
4
2
0
0
0
0
0
0
.4 mM epinephrine. Epinephrine rapidly undergoes auto-
oxidation at pH 10 to produce adrenochrome, which is a pink-
colored product, measured at 480 nm using a UV/VIS spec-
trophotometer in kinetic mode. Superoxide dismutase inhibits the
auto-oxidation of epinephrine. The rate of inhibition was
monitored at 480 nm and one unit of enzyme activity was
defined as the amount of enzyme required to produce 50%
inhibition of enzyme activity. The superoxide dismutase activity
is expressed as units/mg protein.
0
0.1
1
10
[
µg/ml]
B
4
3
2
0
0
0
2
.11. Catalase activity
Fifty micrograms of protein was added to 50 mM of phosphate
2 2
buffer (pH 7) and 100 mM of H O (v/v). This reaction mixture
was incubated for 2 min at 37 -C and the absorbance was
monitored at 240 nm for 5 min. The change in absorbance is
proportional to the breakdown of H
2
O
2
(Carrillo et al., 1991). The
10
0
catalase activity is expressed as units/mg protein.
0.1
1
10
2
.12. Glutathione peroxidase activity
[
µg/ml]
Fifty micrograms of the protein was added to 25 mM of the
C
phosphate buffer (pH 7.5), 1 mM EDTA, 1 mM NaN
3
, 1 mM
60
glutathione, 0.25 unit of glutathione reductase, and 0.1 mM
NADPH. After incubation for 10 min at 37 -C, H O was added
2
2
to the reaction mixture at a final concentration of 1 mM. The
absorbance was monitored at 340 nm for 5 min. The glutathione
peroixdase activity was measured as the rate of NADPH oxidation
at 340 nm (Paglia and Valentine, 1967). The glutathione peroixdase
activity is expressed as units/mg protein.
4
0
0
0
2
2
.13. Western blot
0
.1
1
10
5
The V79-4 cells were placed in a plate at 1Â10 cells/ml.
[µg/ml]
Sixteen hours after plating, the cells were treated with 10 Ag/ml
of tectorigenin. The cells were harvested at the indicated times,
and washed twice with PBS. The harvested cells were then
lysed on ice for 30 min in 100 Al of a lysis buffer [120 mM
NaCl, 40 mM Tris (pH 8), 0.1% NP 40] and centrifuged at
Fig. 4. Protective effect of tectorigenin on H
induced oxidative damage of V79-4 cells. (A) The viability of V79-4 cells
on H treatment was determined by MTT assay. (B) The intracellular
2 2
O or serum starvation
2 2
O
reactive oxygen species generated by serum starvation was detected by
DCF-DA method. (C) The viability of V79-4 cells on serum starvation was
determined by MTT assay. The measurements were made in triplicate
(n =3/group) and values are expressed as meansTS.E.M. *Significantly
different from control ( p <0.05).
1
3,000Âg for 15 min. Supernatants were collected from the
lysates and protein concentrations were determined. Aliquots of
the lysates (40 Ag of protein) were boiled for 5 min and

20
K.A. Kang et al. / European Journal of Pharmacology 519 (2005) 16–23
2
.14. Nuclear extract preparation and electrophoretic mobility
were resolved on 5% polyacrylamide gel and the bands were
shift assay
visualized by autoradiography (Kim et al., 1998b).
5
The V79-4 cells were placed in plate at 1Â10 cells/ml. Sixteen
hours after plating, the cells were treated with 10 Ag/ml of
tectorigenin. The cells were harvested at the indicated times, and
were then lysed on ice with 1 ml of lysis buffer (10 mM Tris–HCl,
pH 7.9, 10 mM NaCl, 3 mM MgCl and 1% NP-40) for 4 min. After
2.15. Statistical analysis
All the measurements were made in triplicate and all values
were represented as meansTS.E.M. The results were subjected to
an analysis of variance (ANOVA) using the Tukey test to analyze
the difference. p <0.05 were considered as significant.
2
,
1
5
1
0 min of centrifugation at 3000Âg, the pellets were resuspended in
0 Al of extraction buffer (20 mM HEPES, pH 7.9, 20% glycerol,
2
.5 mM MgCl , 0.2 mM EDTA, 1 mM DTT, and 1 mM PMSF),
incubated on ice for 30 min, and centrifuged at 13,000Âg for 5 min.
The supernatant was then harvested as nuclear protein extracts and
stored at À70 -C after determination of protein concentration.
Oligonucleotides containing transcription factor activator protein-1
3. Results
3.1. Radical scavenging activity of tectoridin and tectorigenin
(
AP-1) consensus sequence (5V-CGC TTG ATG ACT CAG CCG
The radical scavenging effect of tectoridin and tectorigenin on
the intracellular reactive oxygen species and DPPH free radical
scavenging activities was compared. The intracellular reactive
oxygen species scavenging activity of tectoridin was 12.1T2.3% at
0.1 Ag/ml, 25.1T1.7% at 1 Ag/ml, and 36.2T1.3% at 10 Ag/ml
(n =3/group). In case of tectorigenin, it was 22.9T3.3% at 0.1 Ag/
ml, 42.3T2.5% at 1 Ag/ml, and 63.2T2.3% at 10 Ag/ml (n =3/
3
2
GAA-3V) were annealed, labeled with [g- P] ATP using T4
polynucleotide kinase, and used as probes. The probes (50,000
cpm) were incubated with 6 Ag of the nuclear extracts at 4 -C for 30
min in a final volume of 20 Al containing 12.5% glycerol, 12.5 mM
HEPES (pH 7.9), 4 mM Tris–HCl (pH 7.9), 60 mM KCl, 1 mM
EDTA, and 1 mM DTTwith 1 Ag of poly(dI–dC). Binding products
A
G1
1
20
00
S
G2/M
1
80
60
40
20
0
Control
H O
Tectorigenin
2
2
B
Control
H O
Tectorigenin
2
2
C
Control
H O
Tectorigenin
N-acetylcysteine
2
2
Sub-G : 2%
Sub-G : 22%
Sub-G : 14%
Sub-G : 4%
1
1
1
1
M1
M1
M1
M1
0
200 400 600 800 1000
DNA content
0
200 400 600 800 1000
DNA content
0
200 400 600 800 1000
DNA content
0
200 400 600 800 1000
DNA content
2 2
Fig. 5. Effect of tectorigenin on cell cycle arrest and apoptosis induced by H O (A) The cell distribution at each phase of cell cycle was determined by flow
cytometry. (B) Apoptotic body formation was observed under a fluorescent microscope after Hoechst 33342 staining and (C) apoptotic sub-G
was detected by flow cytometry after propidium iodide staining. Apoptotic bodies are indicated by arrows.
1
DNA content

K.A. Kang et al. / European Journal of Pharmacology 519 (2005) 16–23
21
group) (Fig. 2A). N-acetylcysteine, which was used as a positive
of S phase to 71%, when compared with 65% of the cells exposed
only to H O . In order to study the cytoprotective effect of
control, showed 83% inhibition of reactive oxygen species at 2 mM
2
2
(
data not shown). The reactive oxygen species scavenging activity
tectorigenin on apoptosis induced by H O , nuclei of V79-4 cells
2 2
of both the compounds was consistent with its DPPH radical
scavenging activity (Fig. 2B). The DPPH radical scavenging
activity of tectoridin showed 8.1T0.8% at 0.1 Ag/ml, 17.3T1.1%
at 1 Ag/ml, and 39.3T1.3% at 10 Ag/ml (n =3/group), and for
tectorigenin, it was 9.0T1.2% at 0.1 Ag/ml, 31.5T3.4% at 1 Ag/ml,
were stained with Hoechst 33342 for microscopy and with
propidium iodide for flow cytometric analysis. The microscopic
pictures in Fig. 5B show the control cells with intact nuclei, and the
2 2
H O treated cells showed significant nuclear fragmentation, a
characteristic of apoptosis. However, when the cells were treated
with tectorigenin for 1 h prior to H O treatment, a dramatic
5
4.3T2.3% at 10 Ag/ml (n =3/group), when compared with 90% of
2
2
N-acetylcysteine. However, the radical scavenging effect of
tectorigenin in both the experiments was more effective when
compared to tectoridin. From these results, we selected tectorigenin
as active compound for further studies on radical scavenging effect.
decrease in nuclear fragmentation was observed. In addition to the
morphological evaluation, the protective effect of tectorigenin
against apoptosis was also confirmed by flow cytometry. As shown
in Fig. 5C, an analysis of the DNA content in the H
revealed an increase of 22% of apoptotic sub-G
2
O
2
treated cells
1
DNA content.
3
.2. Effect of tectorigenin on lipid peroxidation
Treatment with 10 Ag/ml of tectorgenin decreased the apoptotic
sub-G DNA content to 14%, showing 4% of the apoptotic sub-G
1
1
The ability of tectorigenin to inhibit lipid peroxidation in H
2
O
2
DNA content in N-acetylcysteine treated cells. These results
suggest that tectorigenin protects cell viability by inhibiting
H O induced apoptosis.
treated V79-4 cells was also tested. The generation of malondial-
dehyde, and related substances that react with thiobarbituric acid,
was inhibited by tectorigenin. The activities of tectorigenin were
2
2
2
8.9T1.2% at 0.1 Ag/ml, 32.9T2.1% at 1 Ag/ml, and 34.9T3.4% at
0 Ag/ml as compared to 21.7T0.3% in untreated group (n =3/
3.5. Activation of ERK and AP-1 by tectorigenin
1
group) (Fig. 3). However, inhibitory effect of N-acetylcysteine
31%) on lipid peroxidation was not so effective, when compared
to its scavenging activity against H O and DPPH radical.
To better understand the protective mechanism of tectorigenin
on V79-4 cells, the activation of ERK by Western blot analysis
with phospho-ERK specific antibody was examined. As shown in
Fig. 6A, tectorigenin activated phosphorylated ERK dramatically,
within 3 h. However, there was no change in the total ERK
protein level. Subsequently, we examined the effect of tector-
igenin on DNA binding activity of AP-1, which is the down-
stream target of phospho-ERK pathway (Karin et al., 1997;
Mukhopadhyay et al., 2001). As shown in Fig. 6B, AP-1 activity
in tectorigenin treated cells was significantly increased. The
(
2
2
3
.3. Effect of tectorigenin on cell damage induced by H
2 2
O and
serum starvation
2 2
The protective effect of tectorigenin on cell survival in H O
treated V79-4 cells was also measured. Cells were treated with
tectorigenin at various concentrations for 1 h, prior to the addition
2 2
to H O . The cell survival was determined 24 h later by the MTT
assay. As shown in Fig. 4A, treatment with tectorigenin induced a
dose-dependent increase in the cell survival; 34.8T1.1% at 0.1
Ag/ml, 49.3T2.3% at 1 Ag/ml, and 73.7T2.1% at 10 Ag/ml (n =3/
group). It is reported that serum starvation produces a marked
accumulation of reactive oxygen species and results in cell death
A
0
3
6
12h
PhosphoERK
ERK
(
Kang et al., 2003). We also examined whether tectorigenin
showed the ROS scavenging effect and the protective effect on
serum starved cells. The ROS scavenging effect by tectorigenin
was determined after 6 h of serum starvation. As shown in Fig.
B
0
3
6
12 24h
4
9
B, tectorigenin showed the ROS scavenging activity of
.1T0.4% at 0.1 Ag/ml, 13.4T0.6% at 1 Ag/ml, and 20.5T1.6%
at 10 Ag/ml (n =3/group). The cell survival was determined after
4 h of serum starvation. As shown in Fig. 4C, tectorigenin
2
increased the cell survival: 15.1T1.5% at 0.1 Ag/ml, 31.7T1.9%
at 1 Ag/ml, and 44.6T1.6% at 10 Ag/ml (n =3/group). These
results suggest that tectorigenin protects the cell damage induced
by oxidative stress.
3
.4. Effect of tectorigenin on cell cycle arrest and apoptosis
induced by H
2 2
O
Ratio
1
2.1 1.4 1.3 1.3
The protective effect of tectorigenin was confirmed as cell
distribution at cell cycle using flow cytometry. As shown in Fig.
Fig. 6. Effect of tectorigenin on ERK and AP-1 activity (A) Cell lysates
were electrophoresed and proteins of ERK1/2 and phospho-ERK1/2 were
detected by their respective specific antibodies. (B) AP-1 specific
oligonucleotide–protein complexes were detected by electrophoresis
mobility shift assays. The values represent the normalized intensities of
the AP-1 band against intensities of control band measured by bio-image
analyzer system.
5A, the cells exposed to H O for 24 h induced cell cycle arrest at
2 2
the G
2
/M phase in 29%, when compared with 3% of control. The
treatment reduced
the number of cells at the G /M phase to 20%. Cells pretreated with
2 2
cells pretreated with tectorigenin prior to H O
2
2 2
tectorigenin prior to H O treatment recovered the cell population

22
K.A. Kang et al. / European Journal of Pharmacology 519 (2005) 16–23
pattern of AP-1 activity was consistent with the pattern of
activated ERK.
4. Discussion
In general, hydrophilic compounds with sugar moieties
are not so easily permeable to the cellular membrane (Li et
al., 1993), and hence glycosides are not easily absorbed
form the gastrointestinal tract. In this study, tectorigenin
3
.6. Effect of tectorigenin on the intracellular antioxidant systems
In order to investigate whether the radical scavenging activity
of tectorigenin was mediated by the activities of antioxidant
enzymes, the activities of superoxide dismutase, catalase and
glutathione peroxidase in tectorigenin treated V79-4 cells were
measured. Tectorigenin increased the activities of these three
enzymes (Fig. 7A); in the superoxide dismutase activity,
(aglycon), a metabolite formed from the transformation of
tectoridin (glycoside) by intestinal microflora, showed more
potent antioxidant effect than tectoridin, suggesting that
aglycon form may show active biological activity. Although
some reports suggest that tectorigenin exhibit the antiox-
idant effect on free radical (Lee et al., 1999, 2000; Park et
al., 2002), there is lack of evidence to show whether
tectorigenin inhibits formation of reactive oxygen species on
H O induced cells. In our study, tectorigenin was found to
23.5T1.2 U/mg protein at 0.1 Ag/ml, 24.5T2.1 U/mg protein at
1 Ag/ml, and 26.9T3.4 U/mg protein at 10 Ag/ml, as compared to
13.5T0.3 U/mg protein of the control (n =3/group); in the
catalase activity, 14.6T1.2 U/mg protein at 0.1 Ag/ml, 17.3T1.1
U/mg protein at 1 Ag/ml, and 24.7T3.1 U/mg protein at 10 Ag/
ml, as compared to 7.5T0.4 U/mg protein of the control (n =3/
group); in the glutathione peroxidase activity, 12.5T1.1 U/mg
protein at 0.1 Ag/ml, 15.4T2.6 U/mg protein at 1 Ag/ml, and
2
2
decrease intracellular generation of reactive oxygen species
and DPPH radical level, suggesting the enhanced viability
of V79-4 cells in the presence of tectorigenin upon exposure
1
9.6T0.9 U/mg protein at 10 Ag/ml, as compared to 11.7T0.5 U/
to H O and on serum starvation. This shows that
2
mg protein of the control (n =3/group). To confirm the activation
of superoxide dismutase and catalase by tectorigenin in terms of
protein expression, the Western blot analysis was performed. As
shown in Fig. 7B, the protein expressions of superoxide
dismutase and catalase by tectorigenin were found to increase
in a dose-dependent manner. The protein levels of the enzymes,
superoxide dismutase (16 kDa) and catalase (42 kDa), induced by
tectorigenin were consistent with the enzyme activity. The results
show that the enhancement in antioxidant enzyme activities by
tectorigenin may be associated with the inhibition of the
production of reactive oxygen species.
2
tectorigenin scavenges intracellularly generated reactive
oxygen species. The cells exposed to H exhibited
distinct morphological features of apoptosis, such as nuclear
O
2 2
fragmentation and an increase in 22% of sub-G hypo-
1
diploid cells. However, cells that were pretreated with
tectorigenin had significantly reduced percentage of apop-
totic cells, as shown by morphology and 14% reduction in
sub-G DNA content. Our results are also consistent with
1
the antioxidant activity of N-acetylcysteine, which also
prevents H O induced apoptosis, indicating that the
2
2
inhibition of formation of reactive oxygen species may be
important for cells to protect themselves against oxidative
damage. The activation of ERK pathway induces cell
proliferation, which leads to the induction of AP-1 activity,
one of the transcription factors (Pages et al., 1991; Karin et
al., 1997). Tectorigenin was found to significantly protect
V79-4 cells against H O induced damage, and the level of
A 6
0
0
0
1
1
ug/ml
.1 ug/ml
ug/ml
0 ug/ml
4
0
0
2
2
*
phosphorylated ERK and AP-1 activity was elevated in
time-dependent manner, suggesting that the protective effect
of tectorigenin on cells may be involved in activation of
ERK pathway. Antioxidant enzymes like superoxide dis-
mutase, catalase, and glutathione peroxidase play significant
roles in effective augmentation of antioxidant defenses in
cells. Superoxide dismutase converts superoxide radicals to
hydrogen peroxide and subsequently to water by catalase
and glutathione peroxidase. Tectorigenin was found to
increase superoxide dismutase, catalase, and glutathione
peroxidase activities and their protein levels, suggesting that
the scavenging of reactive oxygen species may be related to
the increased antioxidant activity. Therefore, the effects of
tectorigenin on cell viability might involve dual actions:
direct action on oxygen radical scavenging, as shown by
DPPH radical scavenging, and indirect action through the
induction of antioxidative enzymes.
*
*
*
*
2
*
*
0
Superoxide dismutase Catalase Glutathione peroxidase
0.1 10 g/ml
B
0
1
Superoxide
dismutase
Catalase
Actin
Fig. 7. Effect of tectorigenin on activity of antioxidant enzymes and its
protein expression. (A) The detection methods for activities of antioxidant
enzymes are described in Materials and methods. (B) Cell lysates were
electrophoresed and proteins of superoxide dismutase and catalase were
detected by their respective specific antibodies. The measurements were
made in triplicate (n =3/group) and values are expressed as meansTS.E.M.
In conclusion, tectorigenin, a metabolite of tectoridin,
exerted intracellular reactive oxygen species and DPPH
radical scavenging activity, promoted cell viability via
*
Significantly different from control ( p <0.05).

K.A. Kang et al. / European Journal of Pharmacology 519 (2005) 16–23
23
Lee, K.T., Sohn, I.C., Kim, D.H., Choi, J.W., Kwon, S.H., Park, H.J., 2000.
Hypoglycemic and hypolipidemic effects of tectorigenin and kaikasa-
ponin III in the streptozotocin-induced diabetic rat and their antioxidant
activity in vitro. Arch. Pharm. Res. 23, 461–466.
activation of ERK pathway, inhibited H O induced
2
2
apoptosis, and enhanced the effects of antioxidant enzymes.
Lee, H.W., Choo, M.K., Bae, E.A., Kim, D.H., 2003. Beta-glucuronidase
inhibitor tectorigenin isolated from the flower of Pueraria thunbergi-
ana protects carbon tetrachloride-induced liver injury. Liver Int. 23,
Acknowledgements
2
21–226.
This study was supported by a grant from Korea Science
and Engineering Foundation (KOSEF), and Ministry of
Science and Technology (MOST), Korean government.
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