BioFactors 30 (2007) 149–157 149

IOS Press

Brazilin and the extract from

Caesalpinia sappan L. protect oxidative injury
through the expression of heme oxygenase-1

Byung-Min Choia , Ju-A. Leea , Shang Shang Gaoa , Sang Yong Euna, Young-Sup Kimb,
Shi-Yong Ryub , Yeon-Hee Choib , Raekil Parkc , Dae Young Kwond and Bok-Ryang Kima,∗
a Vestibulocochlear Research Center and Department of Biochemistry, School of Medicine, Wonkwang
University, Chonbuk, Republic of Korea
b Phytochemistry Research Laboratory, Korea Research Institute of Chemical Technology, Daejeon,

Republic of Korea
c Vestibulocochlear Research Center and Department of Microbiology, School of Medicine, Wonkwang

University, Chonbuk, Republic of Korea

d Functional Food Research Center, Korea Food Research Institute, Gyungki-do, Republic of Korea

Received 20 October 2007

Revised 11 December 2007
Accepted 26 December 2007

Abstract. In this study, we examined the protective effects of Caesalpinia sappan L. and its major component, brazilin, against
tert-butylhydroperoxide (t-BHP)-induced cell death in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. We found that
the extract of C. sappan L. and brazilin induced antioxidant response element (ARE)-luciferase activity and heme oxygenase-1
(HO-1) expression in a concentration-dependent manner. The inductive effect of brazilin was more potent than the extract of
C. sappan L. and the expression of HO-1 reached a peak at 12 h after brazilin treatment. The extract and brazilin protected the
cells against t-BHP-induced cell death. Their protective effects were abrogated by zinc protoporphyrin IX (ZnPP IX), a HO
inhibitor. These results demonstrate that the extract of C. sappan L. and brazilin induce the expression of HO-1 and the enzyme
diminishes t-BHP-induced cell death in HEI-OC1 cells.

Keywords: Caesalpinia sappan, brazilin, heme oxygenase-1, oxidative stress, zinc protoporphyrin IX

Abbreviations: ARE, antioxidant response element; HEI-OC1, House Ear Institute-Organ of Corti 1; FBS, fetal bovine serum;
GST, glutathione S-transferase; HO-1, heme oxygenase-1; ROS, reactive oxygen species; t-BHP, tert-butylhydroperoxide; ZnPP
IX, zinc protoporphyrin IX

∗
Address for correspondence: Bok-Ryang Kim, Ph.D., Department of Biochemistry, School of Medicine, Wonkwang
University, Iksan, Chonbuk 570-749, Republic of Korea. Tel.: +82 63 850 6756; Fax: +82 63 841 1616; E-mail: bokim@
wonkwang.ac.kr.

0951-6433/07/$17.00  2007 – IUBMB/IOS Press and the authors. All rights reserved
150 B.-M. Choi et al. / Brazilin and the extract from Caesalpinia sappan L. protect oxidative injury

1. Introduction

Caesalpinia sappan L. has been reported to have pharmacological activities such as modulation of
immune function [3], depressing effects on the central nervous system [24], anitcomplemention [26],
antinflammation [10], and vasorelaxation [34]. Brazilin, the major component of C. sappan L., is a
natural red pigment and usually used for histological staining. Brazilin exhibited various biological ac-
tivities including hypoglycemic activity [21], anti-platelet aggregation [11], induction of immunological
tolerance [20], and protection of hepatocytes against BrCCl 3 -induced toxicity [22].
A cellular defensive mechanism against electrophiles and oxidants relies on detoxification by phase
2-detoxifying enzymes, antioxidants enzymes, and stress response proteins [15,30,31]. Coordinated ex-
pression of these enzymes, such as glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase,
and heme oxygenase-1 (HO-1) are regulated at the transcriptional level through a cis-acting enhancer
sequence known as antioxidant response element (ARE) [25]. The ARE-mediated gene expression has
been implicated as a pivotal protection mechanism against various stimuli such as reactive oxygen species
(ROS) and carcinogenic intermediates [18].
HO-1 is a critical protein in the response to oxidative injury whose main function is associated with the
degradation of heme to biliverdin, iron, and carbon monoxide [14,33]. Recent studies have demonstrated
that HO-1 functions as part of a cytoprotective mechanism derived from the antioxidant activities [8],
anti-inflammatory- [13], antiproliferative- [9], and antiapoptotic-properties [5]. Thus, in light of the
cytoprotective role of HO-1, the upregulation of HO-1 gene expression by a pharmacological modulator
may represent a novel target for therapeutic treatment of various diseases.
In this study, we examined whether the extract of C. sappan L. and brazilin induce ARE-related genes
and those could protect House Ear Institute-Organ of Corti 1 (HEI-OC1) cells against oxidative damage.
Our results demonstrated that the extract of C. sappan L. and brazilin induced the expression of HO-1
and its induction played an important role in the protective effects of C. sappan L. against oxidative
injury.

2. Materials and methods

2.1. Reagents

tert-Butyl hydroperoxide (t-BHP) was purchased from Sigma-Aldrich (St. Louis, MO). Anti-HO-1 and
β -actin antibodies were purchased from Calbiochem (San Diego, CA, USA). Unless indicated otherwise,
all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Plant materials

The wood of C. sappan L. was purchased at Kyungdong market in Seoul, and identified by Dr. Sung-
Joo Park, Department of Herbology. A voucher specimen was deposited at 4 ◦ in the Department of
Herbology, College of Oriental Medicine, Wonkwang University, Iksan, South Korea.

2.3. Extraction and isolation

The dried heartwood of C. sappan L. was extracted with methanol, and then was purified according
to the methods described by Oh et al. [26]. The chemical structure of brazilin was identified by Korea
 B.-M. Choi et al. / Brazilin and the extract from Caesalpinia sappan L. protect oxidative injury 151

HO

OH

O
OH
OH

Fig. 1. Chemical structure of brazilin.

Research Institute of Chemical Technology (Young-Sup Kim, Ph.D.) (Fig. 1). Brazilin, [a] 25 D +93.7
−1
(MeOH; c 1.44), UV λmax nm: 274 , IRv : 3393, 2926, 2852, 1622, 1509 cm , EI-MS (m/z): 286
MeOH

[M]+ C16 H14 O5 , 1 H-NMR (500MHz, DMSO-d6 ): δ 7.18 (1H, d, J = 8.4 Hz, H-1), 6.70 (1H, s, H-8),
6.59 (1H, s, H-11), 6.46 (1H, dd, J = 8.3, 2.4 Hz, H-2), 6.29 (1H, d, J = 2.4 Hz, H-4), 3.96 (1H, br
s, H-12), 3.92 (1H, dd, J = 1.2, 11.3 Hz, H-6a), 3.69 (1H, d, J = 11.2 Hz, H-6b), 3.02 (1H, d, J =
15.6 Hz, H-7a), 2.77 (1H, d, J = 15.6 Hz, H-7b) 13 C-NMR (125MHz, DMSO-d6 ): δ 43(C-4a), 51(C-
3), 71(C-9), 78(C-10), 104(C-11a), 110(C-1), 112(C-7a), 113,(C-1a) 115(C-8), 131(C-11), 132(C-2),
137(C-4), 145(C-6a), 145(C-6), 155(C-12), 158(C-7).

2.4. Cell culture

HEI-OC1 cells have recently established and characterized from long-term cultures of Immorto-mouse
cochlea [12]. Expression of outer hair cell (OHC) specific markers, including Math1 and Myosin 7a,
suggests that HEI cells may represent OHC precursors [27]. Thus HEI-OC1 cells provide a powerful
tool for the in vitro study of the mechanisms of auditory cells. Cells were maintained in high-glucose
Dulbecco’s modified Eagle’s medium (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal
bovine serum (FBS; GIBCO BRL, Grand Island, NY) at 33 ◦ in a humidified incubator with 5% CO2 .

2.5. MTS assay for cell viability

Cells were subcultured in 96-well plates at a density of 5 × 10 4 cells/well. Cells were treated with
t-BHP in the presence or absence of the extract of C. sappan L. or brazilin for 12 h. The MTS assay was
performed with the CellTiter 96 Aqueous nonradioactive cell proliferation assay kit (Promega Corp.,
Madison, WI), according to the manufacturer’s instruction. The absorbance was read at 490 nm on an
ELISA reader, and the percentage of cell survival was obtained.

2.6. Transient transfection and luciferase assay

A day before transfection, cells were subcultured at a density of 1 × 10 5 cells in 12 well plates to
maintain approximately 70–75% confluency. The cells were transiently transfected using lipofectamine
with a plasmid containing ARE sequence,according to the instruction given by the manufacturer (GIBCO-
BRL). After overnight transfection, cells were incubated with the extract of C. sappan L. and brazilin
for 24 h. For luciferase assay, cell were washed twice with PBS and lysed with reporter lysis buffer
(Promega). After 20 µl of the cell extract was mixed with 100 µl of the luciferase assay reagent at room
temperature, the mixture was placed in a luminometer to measure the light produced.
152 B.-M. Choi et al. / Brazilin and the extract from Caesalpinia sappan L. protect oxidative injury

A 10
B 10

8 8

Luciferase Activity
Luciferase Activity

(fold induction)
(fold induction)
6 6 *

*
4 * 4
*
2 2

0 0
M)
(25 µM)

M)
(25 µM)
DMSO

DMSO
1 5 10 20 1 5 10 20

SFN
SFN

Extract ((µg/ml)
g/ml) Brazilin (µM)
( M)

Fig. 2. Effects of the extract and brazilin from C. sappan L. on ARE-luciferase reporter gene activity in HEI-OC1 cells. Cells
were transfected with an ARE-luciferase plasmid. After 16 h, cells were maintained in low serum medium and then stimulated
with various concentration of the extract (A) and brazilin (B) for 24 h. Cells were lysed and analyzed for luciferase activities.
The data represent the means ± SD of three independent experiments. *, P < 0.05 compared with treated DMSO.

2.7. Western blot analysis

Western blot analysis was performed as follows. Briefly, cells were harvested, washed twice with
ice-cold PBS, and resuspended in 20 mM Tris-HCl buffer (pH 7.4) containing a protease inhibitor
mixture (0.1 mM phenylmethylsulfonyl fluoride, 5 µg/mL aprotinin, 5 µg/mL pepstatin A, and 1 µg/mL
chymostatin). Protein concentration was determined with a Lowry protein assay kit. Samples were
subjected to electrophoresis in a 12% SDS-polyacrylamide gel and then transferred to a nitrocellulose
membrane. The membrane was probed with a specific antibody (diluted 1:1,000), followed by incubation
with secondary antibody (diluted 1:5,000) for specific band detection. Autoradiographic signals were
quantified by a densitometric scanning (Molecular Dynamics, Sunnyvale, CA). All densitometric values
obtained for the HO-1 protein were normalized to values of β -actin obtained on the same blot. The HO-1
protein level in treated cells was expressed in densitometric absorbance units, normalized to control
untreated samples, and expressed as fold induction compared to controls.

2.8. Statistical analysis

Differences in the data among the groups were analyzed by one-way ANOVA, and all values were
expressed as means ± S.D. The differences between groups were considered to be significant at P <
0.05.

3. Results

3.1. Inductions of ARE-luciferase reporter gene and HO-1 gene by the extract and brazilin from C.
sappan L. in HEI-OC1 cells

HEI-OC1 cells, which were transfected with ARE-luciferase reporter plasmid, were exposed to the
extract of C. sappan L. and brazilin for 24 h and the resulting luciferase activities were measured.
 B.-M. Choi et al. / Brazilin and the extract from Caesalpinia sappan L. protect oxidative injury 153

A Extract ((µg/ml)
g/ml) B Brazilin (µM)
( M)

0 1 5 10 20 0 1 5 10 20

HO-1 HO-1

b-actin b-actin

C 25
D 25

Relative Fold Induction

Relative Fold Induction

20 20

15 15

10 10

5 5

0 0
0 1 5 10 20 0 1 5 10 20
Extract (µg/ml) Brazilin (µM)

Extract (20 µg/ml) Brazilin (10 µM)

E g/ml)
F M)

Time (h) 0 6 12 18 24 Time (h) 0 6 12 18 24

HO-1 HO-1

b-actin b-actin

Fig. 3. Effects of the extract and brazilin from C. sappan L. on HO-1 protein expression in HEI-OC1 cells. Cells were incubated
with various concentrations of the extract (A and C) and brazilin (B and D) from C. sappan L. for 12 h. The HO-1 expression
was measured at various time points after treatments of the extract (E) and brazilin (F). Cell lysates (40 µg) were prepared and
subjected to Western blot analysis with antibodies against HO-1 or β-acitn. (E and D) Relative fold induction of HO-1 protein
level was quantified as described under “Materials and Methods.” The figure represents one of three similar experiments.

As shown in Fig. 2, each induction of ARE-dependent gene by the extract from C. sappan L. and
brazilin was 3.7 and 5.6 fold of control, respectively. These induction potentials were 44% and 67% of
25 µM sulforaphane which is one of the most powerful inducer for ARE-related genes [16]. HEI-OC1
cells were exposed to the extract and brazilin for 12 h then the endogenous HO-1 protein expression
was investigated. As shown in Fig. 3, the extract and brazilin induced the expression of HO-1 in a
concentration-dependent manner. The level of maximal induction of HO-1 protein was 24 fold of control
at 20 µM brazilin. Treatment of the cells with the extract and brazilin resulted in a time-dependent
increase in HO-1 expression (Fig. 3E and F). The induction of HO-1 by brazilin was evident as early as
6 h, and reached a maximum at 12 h, in the meanwhile the induction by the extract reached a plateau at
18 h.
154 B.-M. Choi et al. / Brazilin and the extract from Caesalpinia sappan L. protect oxidative injury

A 120
* * B 120
* *
100 100

Cell Viability (%)

Cell Viability (%)
80 80

60 60

40 40

20 20

0 0
_ _ _ _
t-BHP (100 mM) + + + + + t-BHP (100 mM) + + + +
Extract (mg/ml) 0 0 1 5 10 20 20 Brazilin (mM) 0 0 5 10 20 20

120
C
100 * *
Cell Viability (%)

80

60

40

20

0
_
t-BHP + + + + +
Extract (mg/ml) 0 0 10 10 0 0
Brazilin (mM) 0 0 0 0 10 10
ZnPP (mM) 0 0 0 10 0 10

Fig. 4. HO-1 induced by the extract and brazilin from C. sappan L. protects t-BHP-induced cell death. Cells were pretreated
with the indicated doses of the extract (A) and brazilin (B) from C. sappan L. for 12 h and then incubated with 100 µM t-BHP
for 12 h. Cells were pretreated with the extract and 10 µM brazilin for 12 h in the absence or presence of 10 µM ZnPP and then
incubated with 100 µM t-BHP for 12 h (C). Cell viability was measured by MTS assay. The data represent the means ± SD of
three independent experiments. *, P < 0.05.

3.2. Cytoprotection of the extract and brazilin from C. sappan L. against oxidative injury by their
inductive effect of HO-1

We examined whether HO-1 expression was responsible for the protection afforded by the extract
of C. sappan L. and brazilin against oxidative injury. As shown in Fig. 4, the incubation of the cells
with t-BHP (100 µM) for 12 h resulted in a marked reduction of cell viability by 40%. However, the
pre-treatment of the cells with the extract and brazilin for 12 h diminished t-BHP-induced cell death
in a concentration manner (Fig. 4A and B). The involvement of HO-1 in the protective effects of the
extract and brazilin were confirmed using an inhibitor of HO activity, ZnPP IX. ZnPP IX abolished
their protective effects against t-BHP-induced cell death (Fig. 4C). These results demonstrated that the
observed protective effects of the extract C. sappan L. and brazilin on t-BHP-induced cell death were
due to their inductive effects of HO-1.
 B.-M. Choi et al. / Brazilin and the extract from Caesalpinia sappan L. protect oxidative injury 155

4. Discussion

Oxidative stress is one of major underlying contributors to the development of many pathological
states, including cancer, cardiovascular disease, brain dysfunction, and cataract [1]. In this study, we
found that the extract of C. sappan L. and its major component, brazilin, induced HO-1 expression by
activating ARE in HEI-OC1 cells (Figs 2 and 3). The protein can play a vital role in the prevention of
cell dysfunction as a result of oxidative stress [6]. The extract of C. sappan L. and brazilin have been
antioxidant properties by the inhibitory effect on xanthine oxidase activity as well as scavenging effect
on superoxide anion and hydroxyl radicals [2,28]. However, the induction of HO-1 by the extract and
brazilin are not reported yet. The antioxidant properties of the extract and brazilin can be prolonged
by their inductive effects of HO-1 enzyme. The content of brazilin was 8.7–22.2% w/w of the extract
of C. sappan L. [32]. The report showed that 20 µg/ml of the extract of C. sappan L. contains about
1.74 µg/ml – 4.4 µg/ml (about 5.7 µM – 14.5 µM) of brazilin. Therefore, the most HO-1 induction
potential by the extract of C. sappan L. seems to be driven by its content of brazilin. Because each the
highest concentration of the extract and brazilin in this study was 20 µg /ml and 20 µM, respectively.
We found that the treatment of the cells with the extract and brazilin resulted in resistance to t-
BHP-induced cell death (Fig. 4A and B). ZnPP IX abrogated the protective effect of the extract and
brazilin on t-BHP-induced cell death (Fig. 4C). Several plant constituents have been reported to induce
HO-1 expression, including curcumin [23], quercetin [7], carnosol [19], and piperine [6], and HO-1
was shown to mediate the antioxidant/cytoprotective properties of the components. Our previous study
indicated that HO-1 induction participates in the protective effects of piperine against cisplatin-induced
cell death [6]. Chen et al. also demonstrated that resveratrol-induced HO-1 expression participated in
augmenting cellular antioxidant defense capacity in PC12 cells from oxidative stress [4]. Although
antioxidant activities of brazilin and C. sappan L. have been reported [2,28], these results provide the
first evidence to indicate that the induction of HO-1 expression by the extract and brazilin may in part
participate in protection against t-BHP-induced cell death. Oxidative stress has been implicated as a
major cause of hair cell loss through ROS generation [29]. Indeed, antioxidants have shown efficacies in
the attenuation of noise-induced hearing loss, significantly protecting auditory outer hair cells [17]. Our
results demonstrate that the induction of HO-1 expression by the extract and brazilin may serve as one
of the important mechanisms for the protective effect on hair cell loss through ROS generation.

5. Conclusion

The present results suggest that the extract of C. sappan L. and brazilin induce HO-1 expression by
activating ARE and the expression could contribute to cellular defense mechanism against t-BHP-induced
cell death in HEO-OC1 cells. It suggests that the extract and brazilin could be beneficial for the treatment
of several diseases associated with oxidative stress.

Acknowledgments

This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the
Vestibulocochlear Research Center (VCRC) and the Biofoods Research Program, Ministry of Science &
Technology.
156 B.-M. Choi et al. / Brazilin and the extract from Caesalpinia sappan L. protect oxidative injury

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