Int. J. Mol. Sci. 2011, 12, 3871-3887; doi:10.3390/ijms12063871
International Journal of
Butin (7,3′,4′-Trihydroxydihydroflavone) Reduces Oxidative
Stress-Induced Cell Death via Inhibition of the
Mitochondria-Dependent Apoptotic Pathway
Rui Zhang 1,†, In Kyung Lee 2,†, Mei Jing Piao 1, Ki Cheon Kim 1, Areum Daseul Kim 1,
Hye Sun Kim 2, Sungwook Chae 3, Hee Sun Kim 4 and Jin Won Hyun 1,*
1 School of Medicine and Applied Radiological Science Research Institute, Jeju National University,
Jeju-si 690-756, Korea; E-Mails: firstname.lastname@example.org (R.Z.);
email@example.com (M.J.P.); firstname.lastname@example.org (K.C.K.);
2 Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-799, Korea;
E-Mails: email@example.com (I.K.L.); firstname.lastname@example.org (H.S.K.)
3 Aging Research Center, Korea Institute of Oriental Medicine, Daejeon 305–811, Korea;
4 Department of Neuroscience, College of Medicine, Ewha Womans University, Seoul 110-783,
Korea; E-Mail: email@example.com
† These authors contributed equally to this study.
* Author to whom correspondence should be addressed; E-Mail: firstname.lastname@example.org;
Tel.: +82-64-754-3838; Fax: +82-64-702-2687.
Received: 7 April 2011; in revised form: 16 May 2011 / Accepted: 31 May 2011 /
Published: 10 June 2011
Abstract: Recently, we demonstrated that butin (7,3′,4′-trihydroxydihydroflavone) protected
cells against hydrogen peroxide (H2O2)-induced apoptosis by: (1) scavenging reactive
oxygen species (ROS), activating antioxidant enzymes such superoxide dismutase and
catalase; (2) decreasing oxidative stress-induced 8-hydroxy-2'-deoxyguanosine levels via
activation of oxoguanine glycosylase 1, and (3), reducing oxidative stress-induced
mitochondrial dysfunction. The objective of this study was to determine the cytoprotective
effects of butin on oxidative stress-induced mitochondria-dependent apoptosis, and
possible mechanisms involved. Butin significantly reduced H2O2-induced loss of
Int. J. Mol. Sci. 2011, 12
mitochondrial membrane potential as determined by confocal image analysis and flow
cytometry, alterations in Bcl-2 family proteins such as decrease in Bcl-2 expression and
increase in Bax and phospho Bcl-2 expression, release of cytochrome c from mitochondria
into the cytosol and activation of caspases 9 and 3. Furthermore, the anti-apoptotic effect of
butin was exerted via inhibition of mitogen-activated protein kinase kinase-4, c-Jun
NH2-terminal kinase (JNK) and activator protein-1 cascades induced by H2O2 treatment.
Finally, butin exhibited protective effects against H2O2-induced apoptosis, as demonstrated
by decreased apoptotic bodies, sub-G1 hypodiploid cells and DNA fragmentation. Taken
together, the protective effects of butin against H2O2-induced apoptosis were exerted via
blockade of membrane potential depolarization, inhibition of the JNK pathway and
mitochondria-involved caspase-dependent apoptotic pathway.
Keywords: butin; oxidative stress; mitochondria-dependent apoptotic pathway
Oxidative stress mediated by reactive oxygen species (ROS) has been implicated as a major cause
of cellular damage and contributes to inflammation, aging, cancer, arteriosclerosis, hypertension and
diabetes [1–3]. Persistent ROS elevation is a result of an imbalance between ROS production and
scavenging by endogenous antioxidants that directly or indirectly disturb physiological functions of
many cellular macromolecules, such as DNA, proteins and lipids. Excessive ROS ultimately induce
cell death, either by apoptosis or necrosis . Mitochondrial dysfunction results in increased ROS
production that enhances oxidative stress if the cellular defense systems are overwhelmed . Previous
studies have indicated that ROS might alter intracellular redox states, change the inner mitochondrial
membrane potential (m) and release soluble inter-membrane proteins, including cytochrome c, from
mitochondria into the cytosol [6,7]. It is also well known that ROS plays a crucial role in triggering the
mitochondria-mediated apoptotic pathway, which is associated with activation of the caspase cascade
and the family of Bcl-2 proteins [8–10].
Flavonoids are a group of naturally occurring polyphenolic compounds found ubiquitously in fruits
and vegetables, and represent substantial constituents of the non-energetic part of the human diet.
Butin (7,3′,4′-trihydroxydihydroflavone, Figure 1), one of the most widely distributed flavonoids, is
reported to be a potent antioxidant against oxidative stress-related diseases, such as cancer, aging, liver
diseases and diabetes [11–14]. In previous reports, Zhang et al. demonstrated that butin protected cells
against hydrogen peroxide (H2O2)-induced apoptosis by scavenging ROS and activating antioxidant
enzymes , decreased oxidative stress-induced 8-hydroxy-2'-deoxyguanosine levels via activation
of oxoguanine glycosylase 1 (OGG1) , and reduced oxidative stress-induced mitochondrial
dysfunction via scavenging of ROS . Considering mitochondria, the intracellular organelles
producing the largest amount of ROS in cells, play a major role in the development of oxidative stress
under both physiological and pathological conditions [18,19], mitochondrial dysfunction is most likely
Int. J. Mol. Sci. 2011, 12
to be responsible for oxidative stress-induced apoptosis . To extend our previous investigations, we
focused on the effect of butin on mitochondria-mediated caspases dependent apoptotic pathway which
is induced by oxidative stress in this study.
Figure 1. Chemical structure of butin (7,3′,4′-trihydroxydihydroflavone).
2. Results and Discussion
2.1. Effect of Butin on H2O2-Induced m Depolarization
In a previous report, we have indicated that butin protected against H2O2-induced apoptosis .
Change in m was examined to improve understanding of butin’s protection mechanism for
H2O2-induced apoptotic process in terms of mitochondrial involvement. JC-1 is a cationic dye that
indicates mitochondrial polarization by shifting its fluorescence emission from green (~525 nm) to
red (~590 nm). As shown in Figure 2A, control cells and butin-treated cells exhibited strong red
fluorescence (JC-1 aggregated form, indicative of mitochondrial polarization) in the mitochondria.
However, H2O2 resulted in reducing red fluorescence and increasing green fluorescence (JC-1
monomer form, indicative of mitochondrial depolarization) in the mitochondria. Butin treatment
blocked reducing red fluorescence and increasing green fluorescence in H2O2-treated cells. Image
analysis data was consistent with flow cytometric data; the level of m loss was increased in
H2O2-treated cells, as substantiated by an increase in fluorescence with JC-1 dye. However, butin
recovered the level of m loss (Figure 2B), suggesting that butin partially inhibited loss of m in
response to H2O2 treatment.
Int. J. Mol. Sci. 2011, 12
Figure 2. Effects of butin on H2O2-induced m depolarization. m was analyzed by
(A) confocal microscope and (B) flow cytometer after staining cells with JC-1. FI indicated
the fluorescence intensity of JC-1.
2.2. Effect of Butin against H2O2-Induced Apoptosis
In order to confirm the cytoprotective impact of butin on H2O2-induced apoptosis, cell nuclei were
stained with Hoechst 33342 for visualization by microscopy. The microscopic images in Figure 3A
demonstrate that the control cells had intact nuclei, whereas H2O2-treated cells showed significant
nuclear fragmentation, a characteristic of apoptosis. However, butin-pretreated cells exhibited a
dramatic decrease in nuclear fragmentation induced by H2O2 treatment. In addition to morphological
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evaluation, the protective effect of butin against apoptosis was also confirmed by apoptotic sub-G1
DNA analysis. As shown in Figure 3B, an analysis of DNA content in H2O2-treated cells revealed
a 36% increase in the apoptotic sub-G1 DNA content. However, butin decreased the apoptotic
sub-G1 DNA content to 16%. Furthermore, H2O2-treated cells increased the levels of cytoplasmic
histone-associated DNA fragmentations as compared to control, and butin significantly decreased the
level of DNA fragmentation (Figure 3C).
Figure 3. Effects of butin on H2O2-induced apoptosis. (A) Apoptotic body formation was
observed under a fluorescence microscope and quantitated after Hoechst 33342 staining.
Arrows indicate apoptotic bodies; (B) The apoptotic sub-G1 DNA content was detected by
a flow cytometry after propidium iodide staining; (C) DNA fragmentation was quantified
by ELISA kit. * Significantly different from control cells (p < 0.05). ** Significantly
different from H2O2-treated cells (p < 0.05). N = 3 and “n” indicates the number of repetitions.
Int. J. Mol. Sci. 2011, 12
Figure 3. Cont.
To further understand the protection mechanism of butin on H2O2-induced apoptotic process, we
detected the protein expressions involved in mitochondria related apoptosis. Beforehand, changes in
Bcl-2 expression, an anti-apoptotic protein, and Bax expression, a pro-apoptotic protein, were
examined. As shown in Figure 4A, butin showed an increase in Bcl-2 expression and a decrease in Bax
expression in H2O2-treated cells. It has been reported that Bcl-2 fails to inhibit cell apoptosis when
inactivated via phosphorylation . We noticed that butin also decreased phosphorylation of Bcl-2 (Ser 87)
induced by H2O2 treatment. During the apoptotic process, Bcl-2 prevented the opening of the
mitochondrial membrane pore, whereas Bax induced the opening of membrane pore . Pore opening
induces loss of m, which in turn induces the release of cytochrome c from the mitochondria . As
shown in Figure 4B, butin inhibited the release of mitochondrial cytochrome c. Next, caspase 9
activity was examined by Western blot since it is known that this enzyme is activated due to
mitochondrial membrane disruption . As shown in Figure 3C, treatment of cells with butin
inhibited H2O2-induced active form of caspase 9 (39 and 37 kDa) and caspase 3 (19 and 17 kDa), a
target of caspase 9. These results suggest that butin protects cells from apoptosis by inhibiting the
caspase dependent pathway via mitochondria.
Int. J. Mol. Sci. 2011, 12
Figure 4. Effects of butin on mitochondrial apoptosis related proteins. Western blot
analysis was performed. Cell lysates were electrophoresed and (A) Bax, Bcl-2, phospho
Bcl-2; (B) cytochrome c; (C) active caspase 9, and active caspase 3 proteins were detected
by their specific antibodies.
2.3. Effect of Butin on the SEK1-JNK-AP-1 Signaling Pathway
The JNK signal pathway plays an important role in oxidative stress-induced apoptosis  and JNK
translocates to the mitochondrial, then phosphorylates Bcl-2, and presumably inactivates them . In
addition, JNK induces the mitochondrial pathway of apoptosis by activating Bax , thus we tested
whether butin regulates this signaling pathway. As shown in Figure 5A, butin inhibited JNK activation
in H2O2-treated cells at 12 h. Moreover, SEK1 is known to be an upstream component in the JNK
signaling pathway .
To investigate whether this upstream kinase plays a role in H2O2-induced JNK activation, SEK1
phosphorylation was determined by Western blot analysis. As shown in Figure 5B, SEK1
phosphorylation levels were increased in H2O2-treated cells at 6 h. However, treatment of cells with
butin inhibited H2O2-induced SEK1 phosphorylation. AP-1 is a downstream target of the phospho JNK
pathway, and activated AP-1 is involved in cell death including apoptosis . Subsequently, we
examined the effect of butin pretreatment on the DNA binding activity of AP-1 after H2O2 treatment at
Int. J. Mol. Sci. 2011, 12
24 h. As shown in Figure 5C, AP-1 DNA binding activity was increased in H2O2 treated cells, whereas
treatment of cells with butin inhibited AP-1 activity.
The transcriptional activity of AP-1 was also assessed using a promoter construct containing AP-1
binding DNA consensus sequences, which were linked to a luciferase reporter gene. As shown in
Figure 5D, butin inhibited the transcriptional activity of AP-1 induced by H2O2. These results suggest
that butin inhibits H2O2-induced apoptosis via suppression of the SEK1-JNK-AP-1 pathway.
Figure 5. Effects of butin on H2O2-induced SEK1-JNK-AP-1 activation. Cell lysates were
electrophoresed and the cell lysates were immunoblotted using (A) anti-JNK, phospho JNK
and (B) -phospho SEK1 and -SEK1 antibodies; (C) AP-1 specific oligonucleotide-protein
complexes were detected by the electrophoresis mobility shift assay; (D) The transcriptional
activity of AP-1 was assessed using plasmid containing an AP-1 binding site-luciferase
construct. * Significantly different from control (p < 0.05) and ** significantly different from
H2O2-treated cells (p < 0.05). N = 3 and “n” indicates the number of repetitions.
Int. J. Mol. Sci. 2011, 12
Figure 5. Cont.
3. Experimental Section
Butin was purchased from Wako Pure Chemical Ind., Ltd. (Tokyo, Japan). 5,5′,6,6′-Tetrachloro-
1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) was purchased from Invitrogen (Carlsbad,
CA, USA). The primary anti-B-cell lymphoma 2 (Bcl-2), -Bcl-2-associated x protein (Bax), -phospho
Bcl-2, and -cytochrome c antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz,
CA, USA). Primary anti-caspase 9, -caspase 3, -c-Jun N-terminal kinases (JNK), -phospho JNK,
-mitogen-activated protein kinase kinase-4 (SEK1), and -phospho SEK1 antibodies were purchased
from Cell Signaling Technology (Beverly, MA, USA). A plasmid containing activator protein-1 (AP-1)
binding site-luciferase construct was a generous gift from Professor Young Joon Surh of Seoul
National University (Seoul, Korea). Propidium iodide and Hoechst 33342 were purchased from the
Sigma Chemical Company (St. Louis, MO, USA).
3.2. Cell Culture
Chinese hamster lung fibroblasts (V79-4 cells) from the American type culture collection were
maintained at 37 C in an incubator, with a humidified atmosphere of 5% CO2 and cultured in
Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal calf serum,
streptomycin (100 g/mL) and penicillin (100 units/mL).
3.3. Mitochondrial Membrane Potential (m) Analysis
m analysis was determined by confocal image analysis and flow cytometer. The V79-4 cells were
seeded at a concentration of 1 105 cells/mL, and 16 h after plating, were treated with butin at
10 g/mL, and after 1 h, 1 mM of H2O2 was added to the plate, and the mixture was incubated for 12 h.
Cells were then harvested, and after changing the media, JC-1 was added to each well and was
incubated for an additional 30 min at 37 °C. After washing with PBS, the stained cells were mounted
onto microscope slide in mounting medium (DAKO, Carpinteria, CA, USA). Microscopic images
Int. J. Mol. Sci. 2011, 12
were collected using the Laser Scanning Microscope 5 PASCAL program (Carl Zeiss, Jena, Germany)
on confocal microscope . In addition, m analysis was also determined by flow cytometer. The cells
were harvested, washed and suspended in phosphate buffered saline (PBS) containing JC-1 (10 g/mL).
After incubation for 15 min at 37 °C, the cells were washed and were suspended in PBS and were
analyzed by flow cytometer .
3.4. Western Blot Analysis
Cells were seeded at a concentration of 1.5 105 cells/mL and 16 h after plating, cells were treated
with butin at 10 g/mL, and after 1 h, 1 mM of H2O2 was added. After 6, 12 or 24 h, cells were
harvested, washed twice with PBS, lysed on ice for 30 min in 100 L of a lysis buffer (120 mM NaCl,
40 mM Tris (pH 8), 0.1% NP 40) and then centrifuged at 13,000 g for 15 min. The supernatants were
collected from the lysates and the protein concentrations determined. Aliquots of the lysates (40 g of
protein) were boiled for 5 min and electrophoresed in 10% sodium dodecysulfate-polyacrylamide gel.
The blots in the gels were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA),
which were then incubated with the primary antibodies. The membranes were further incubated with
the secondary immunoglobulin-G-horseradish peroxidase conjugates (Pierce, Rockford, IL, USA). Protein
bands were detected using an enhanced chemiluminescence Western blotting detection kit (Amersham,
Little Chalfont, Buckinghamshire, UK), and then exposed onto X-ray film.
3.5. Nuclear Staining with Hoechst 33342
Cells were seeded at a concentration of 1 105 cells/mL, and 16 h after plating, were treated with
butin at 10 g/mL. After 1 h, 1 mM of H2O2 was added to the plate and the mixture was incubated for
24 h. 1.5 L of Hoechst 33342 (stock 10 mg/mL), a DNA specific fluorescent dye, was added to each
well 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. The percentage of apoptotic cells (apoptotic index) was assessed by
counting 3 random fields in triplicate wells.
3.6. Detection of Apoptotic Sub-G1 Hypodiploid Cells
The amount of apoptotic sub-G1 hypodiploid cells was determined using flow cytometer . Cells
were seeded at a six-well plate at a concentration of 1 105 cells/mL, and 16 h after plating, were
treated with butin at 10 g/mL. After 1 h, 1 mM of H2O2 was added to the plate and the mixture was
incubated for 24 h. Cells were harvested and fixed in 1 mL of 70% ethanol for 30 min at 4 C. The
cells were then washed twice with PBS, and incubated for 30 min in the dark at 37 C in 1 mL of PBS
containing 100 g of propidium iodide and 100 g of RNase A. A flow cytometric analysis was
performed using a FACS Calibur flow cytometer. Sub-G1 hypodiploid cells were assessed based on
histograms generated by the Cell Quest and Mod-Fit computer programs.
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3.7. DNA Fragmentation
Cells were seeded at a concentration of 5 104 cells/mL, and 16 h after plating, cells were treated
with butin at 10 g/mL. After 1 h, 1 mM of H2O2 was added to the plate and the mixture was incubated
for 24 h. Cellular DNA-fragmentation was assessed by analyzing cytoplasmic histone-associated DNA
fragmentation, using a kit from Roche Diagnostics according to the manufacturer’s protocol.
3.8. Preparation of the Nuclear Extract and Electrophoretic Mobility Shift Assay
Cells were seeded at a concentration of 1.5 105 cells/mL, and 16 h after plating, cells were treated
with butin at 10 g/mL. After 1 h, 1 mM of H2O2 was added to the plate and the mixture was incubated for
24 h. After 24 h, cells were harvested, and subsequently lysed on ice with 1 mL of lysis buffer (10 mM
Tris-HCl, pH 7.9, 10 mM NaCl, 3 mM MgCl2, and 1% NP-40) for 4 min. After 10 min of
centrifugation at 3000 g, the pellets were re-suspended in 50 L of extraction buffer (20 mM HEPES,
pH 7.9, 20% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, and 1 mM PMSF), incubated on
ice for 30 min and centrifuged at 13,000 g for 5 min. The supernatant (nuclear protein) was stored at
−70 °C after determining the protein concentration. Oligonucleotides containing transcription
factor AP-1 consensus sequence (5'-CGC TTG ATG ACT CAG CCG GAA-3') were annealed, labeled
with [-32P] ATP using T4 polynucleotide kinase and used as probes. The probes (50,000 cpm) were
incubated with 6 g of the nuclear extracts at 4 °C for 30 min, to reach a final volume of 20 L,
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 DTT with 1 g of poly (dI-dC). The binding products were resolved on 5%
polyacrylamide gel and the bands were visualized by autoradiography.
3.9. Transient Transfection and AP-1 Luciferase Assay
Cells were seeded at a concentration of 1.0 105 cells/mL, and 16 h after plating, cells were
transiently transfected with plasmid harboring the AP-1 promoter using DOTAP as the transfection
reagent, according to the manufacturer’s protocol (Roche Diagnostics, Portland, OR, USA). Following
overnight transfection, cells were treated with 10 g/mL of butin, and after 1 h, 1 mM of H2O2 was
added to the plate for 24 h. Cells were washed twice with PBS and lysed with reporter lysis
buffer (Promega, Madison, WI, USA). Following vortex mixing and centrifugation at 12,000 g for
1 min at 4 C, the supernatant was stored at −70 C for the luciferase assay. After mixing 20 L of cell
extract with 100 L of luciferase assay reagent at room temperature, the mixture was placed in an
illuminometer to measure the light produced.
3.10. Statistical Analysis
All measurements were performed in triplicate and all values were represented as the mean ± standard
error of the mean (SEM). The results were subjected to an analysis of variance (ANOVA) using the
Tukey’s test to analyze difference. P < 0.05 were considered statistically significant.
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In this study, treatment of cells with H2O2 resulted in significant collapse of m, however,
treatment with butin recovered H2O2-induced depolarization of m. In addition, H2O2 treatment
induced a dramatical increase in Bax expression and decrease in Bcl-2 expression, suggesting that
changes in the pro-apoptotic and anti-apoptotic Bcl-2 family proteins may contribute to apoptosis.
Moreover, elevation of phospho Bcl-2 by H2O2 treatment further helps to reduce its ability to bind with
Bax and enhance translocation of Bax from the cytosol to mitochondria, leading to an enhanced
susceptibility of the cells to apoptosis . Butin significantly restored these changes induced by H2O2.
These results confirmed that butin inhibited H2O2-induced apoptosis associated with regulation of
Bcl-2 family proteins. Changes in caspases 9 and 3 protein expressions were evaluated for the
underlying mechanisms, as cleaved caspases 9 and 3 represent downstream signals of apoptosis and
the Bcl-2 protein can prevent activation of caspases during apoptosis. Butin inhibited H2O2-induced
activation of caspases 9 and 3. Treatment of cells with butin showed anti-apoptotic effects in cells
exposed to H2O2, as shown by apoptotic body formation, sub G1-hypodiploid cells levels and
Various studies have suggested possible mechanisms for the JNK pathway also relate to
mitochondrial depolarization and apoptosis induction. It has been reported that JNK translocates to the
mitochondria, then phosphorylates Bcl-2 and Bcl-XL, anti-apoptotic members of Bcl-2 family, and
presumably inactivates them . In addition, SEK1-JNK-AP-1 activation has been suggested as a
critical component in the oxidative stress-induced apoptosis process . Butin inhibited H2O2-induced
JNK phosphorylation, resulting in a decrease of AP-1 activity. H2O2-induced phosphorylation of SEK1,
an upstream regulator of JNK, was also attenuated by butin treatment. These results demonstrated that
butin attenuated H2O2-induced apoptosis through the SEK1-JNK-AP-1 pathway. Our previous study
has demonstrated that PI3K-Akt pathway also involved in cytoprotective effect of butin against
oxidative stress-induced cell damage. Butin induced OGG1, DNA base repair enzyme, via regulation
of PI3K-Akt pathway .
The structural requirement for effective radical scavenging criteria in flavonoids is the presence of a
3′,4′-orthodihydroxy group (catechol structure) in the B ring or in the A ring, and the C2-C3 double
bond conjugated with a carbonyl group in the C ring. The existence of C2-C3 double bond in the
C ring is important for electron delocalization from the B ring, enhancing radical-scavenging
capacity . The unique feature of butin as compared to flavonoid is partially consistent with these
criteria as mentioned above. The absence of C2-C3 double bond in the C ring of butin might be
expected to have weak antioxidant activity. Nevertheless, butin increased antioxidant activity via
radical scavenging activities and enhancing the effects of antioxidant enzymes . Comparing the
structural criteria with radical scavenging activity among flavonoids, including butin, remains a subject
for further study. To the best of our knowledge, the exact cellular mechanism of butin on cells has not
been well understood. Thus in the present study, we focused on butin effects on mitochondria-
dependent apoptosis induced by oxidative stress and it is the first report on cytoprotective mechanisms
of butin. Many of different clinical mechanisms of flavonoids have been related with their antioxidant
Int. J. Mol. Sci. 2011, 12
properties, either through their reducing capacities or influences on intracellular components. The
precise mechanisms by which flavonoids exert their beneficial or toxic actions remain unclear [34,35].
Although Maruta et al. reported that quercetin and kaempferol were mutagenic to hamster
fibroblasts , however there is increasing interest in research on flavonoids, due to growing
evidence of their health benefits through epidemiological studies. We have reported that morin
(2′,3,4′,5,7-pentahydroxy-flavone) protected against oxidative stress-induced cellular damage in lung
fibroblast cells [37,38]. In addition, myricetin (3,3′,4′5,5′,7-hexahydroxylflavone) prevented cells from
oxidative stress-induced apoptosis via regulation of PI3K/Akt and MAPK signaling pathways in lung
fibroblast cells . In the present study, the evaluation of butin on lung protection induced by
oxidative stress was not performed in vivo. However, baicalin (5,6,7-trihydroxyflavone), similar
compound to butin, showed protective effect on lipopolysaccharide-induced lung damage in rats with
administration of 20 mg/kg , and an in vivo study of butin and its underlying metabolism (absorption,
distribution, metabolism, excretion) remains for further study.
Many flavonoids are shown to have antioxidant activity, coronary heart disease prevention,
anti-inflammation, oestrogenic activity, anticancer activity, and other biological activities [41–43]. As
such research progresses, potential application of flavonoids in either foods or pharmaceutical
supplements will expand. Considering butin’s reduction of mitochondria-dependent apoptosis, it might
give a hopeful picture for oxidative stress related diseases such as aging, diabetes, and neurological
diseases for the initial step of clinical trial and development of raw materials of medicine. Accordingly,
an appropriate system for assessment of intake of butin needs to be developed for further study.
Taken together, the protective effect of butin against H2O2-induced apoptosis was exerted via
blockage of membrane potential depolarization, inhibition of the JNK and mitochondria involved
caspase-dependent apoptosis pathways. Therefore, we suggest that inhibition of these pathways by
butin may provide oxidative stress protection (Figure 6).
Figure 6. A proposed cyto-protective pathway of butin, which explains its properties
against oxidative stress-induced mitochondrial involved apoptosis.
Int. J. Mol. Sci. 2011, 12
This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded
by the Korea government [2010-0017184].
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