May 2005

Notes

Biol. Pharm. Bull. 28(5) 925928 (2005)

925

Isoflavonoid from Viola hondoensis, Regulates the Expression of Matrix

Metalloproteinase-1 in Human Skin Fibroblasts
Hyung-In MOON,*,a Joongku LEE,b Jong Hwan KWAK,c Ok-Pyo ZEE,c and Jin Ho CHUNG*,a
a

Department of Dermatology, Seoul National University College of Medicine and Laboratory of Cutaneous Aging
Research, Clinical Research Institute, Seoul National University Hospital; Seoul, 110744, Korea: b Korea Research
Institute of Bioscience and Biotechnology; Daejeon, 305333, Korea: and c College of Pharmacy, Sung Kyun Kwan
University; Suwon, 440746, Korea. Received December 13, 2004; accepted February 7, 2005
Long term and repeated exposure of ultraviolet (UV) light, a harmful environmental stress, on the skin often
induces chronic skin diseases such as skin cancer as well as photoaging (premature skin aging), and the mechanisms of these skin damages are closely associated with up-regulation of matrix metalloproteinases (MMPs)
activities. Here we investigated the effect of 2
,4
,7-trihydroxyisoflavone isolated from the whole plants of Viola
hondoensis (Violaceae) on the expression of MMPs in UV-irradiated human skin fibroblasts in vitro. 2
,4
,7-Trihydroxyisoflavone markedly reduced UV-induced MMP-1 expression, but not MMP-2, at the both mRNA and
protein levels in a dose-dependent manner. Our report is the first description for the ability of 2
,4
,7-trihydroxyisoflavone to regulate MMP-1 expression specifically.
Key words Viola hondoensis; 2
,4
,7-trihydroxyisoflavone; Matrix Metalloproteinase (MMP)-1; MMP-2; human skin fibroblasts

Viola hondoensis (Violaceae) distributed in southern part

of Korea. In traditional medicine, the herb has been used as
an expectorant, a diuretic, and an antiinflammatory for bronchitis, rheumatism, skin eruptions, and eczema.1,2) Previous
phytochemical studies on Viola species have revealed them
to be a rich source of cyclotides35) and several flavone glycosides.6,7) Matrix metalloproteinases (MMPs) are a family
of zinc-dependent endoproteinases that play pivotal roles in
the dynamic remodeling of extracellular matrix. Based on
substrate preference and structural homology, MMPs are
sub-classified into functional groups: collagenases, gelatinases, stromelysins, matrilysins, membrane type-MMPs
(MT-MMPs) and other non-classified MMPs.8) MMPs are
frequently overexpressed by the various extracellular stimuli
including growth factors, cytokines, tumor promoters, and
ultraviolet (UV), and this increase in MMP-related activities
may be involved in the pathogenesis of diseases such as cancer and inflammation as well as in physiologic processes.9,10)
It has been also reported that up-regulation of some MMPs is
responsible for the enhanced degradation of dermal collagen
during chronological and UV-induced skin aging.1114)
Therefore, the regulation of MMP activity might be a potential strategy for prevention and/or treatment of UV-induced
skin damage. Recently, Kim et al. reported the isolation of
the styraxjaponoside B from the stem bark of Styrax japonica
to regulate MMP-1 expression.15) In this study, we investigated the effect of 2
,4
,7-trihydroxyisoflavone, Isoflavonoid
from Viola hondoensis, on the expression of UV-induced
MMP-1 and MMP-2 mRNA and protein in human skin fibroblasts.
MATERAL AND METHODS
General Procedure IR spectra were obtained with a
Perkin Elmer 1710 spectrophotometer. The NMR spectra
were taken on a JEOL LA 300 (1H, 300 MHz; 13C, 75 MHz).
EIMS spectra were obtained on a VG Trio-2 spectrometer.
FAB-MS spectra were obtained on a JMS AX505WA spectrometer. TLC was carried out on silica gel 60 F254 and RP To whom correspondence should be addressed.

18 F254 plates (Merck, Germany). Column chromatography

was performed over silica gel 60 (Merck, particle size 230
400 mesh) and Sephadex LH-20 (Pharmacia, Sweden).
Genistein was obtained from one of the authors (Dr. Jong
Hwan Kwak).
Plant Material The whole plants of Viola hondoensis
(Violaceae) were collected in April 2004 at Ullung Island,
Korea and the botanical identification was made by one of
the authors, Dr. Joongku Lee. A voucher specimen of this
raw material has been deposited at the herbarium of the
Seoul National University (SNU-04-04-13).
Isolation of 2
,4
,7-Trihydroxyisoflavone The dried
whole plants (413 g) of Viola hondoensis were extracted five
times with 80% MeOH in an ultrasonic apparatus for 3 h.
This residue was evaporated in vacuo to yield the total
extract (30 g). This extract was then suspended in distilled
water and partitioned with n-hexane, CHCl3, EtOAc, and
n-BuOH. The EtOAc fraction (1.3 g) was subjected to silica
gel column chromatography using CHCl3EtOAc gradient
system (9 : 11 : 1) to provide 11 fractions (fractions 111).
From fraction 7, compound 1 (2.1 mg) was isolated using a
silica gel column chromatography (CHCl3EtOAc, 21 : 2),
and then purified by semipreparative RP-HPLC (YMC Jsphere-H80, 4 m m, 25010 mm, MeOHH2O3 : 2.5).
Compound 1 (1.3 mg) was also separated from fraction 8 by
the same procedure as those of the fraction 7.
Cell Culture Primary human foreskin dermal fibroblasts were established by outgrowth from biopsies of healthy
donors of 312 years and cultured in Dulbeccos modified
Eagles media (DMEM; Life Technologies, Rockville, MD,
U.S.A.) supplemented with glutamine (2 mM), penicillin
(400 U/ml), streptomycin (50 m g/ml), and 10% fetal bovine
serum (FBS; HyClone, Logan, UT, U.S.A.) in a humidified
5% CO2 atmosphere at 37 C. The cells from passages 610
were used for the experiments.
UV Irradiation The UV light source was an
F75/85W/UV21 fluorescent sun lamps, having an emission
spectrum between 285350 nm (peak at 310315 nm) as
previously described.16) Subconfluent cells were serum-

e-mail: himun@snu.ac.kr, Jhchung@snu.ac.kr

2005 Pharmaceutical Society of Japan

926

starved for 24 h to synchronize cells in G1/G0 phase of the

cell cycle. Fresh phosphate-buffered saline (PBS) was added
to the quiescent cells prior to UV light exposure (0100
mJ/cm2). After UV irradiation, the cells were washed with
PBS, and incubated for the indicated time in DMEM with or
without compound.
RT-PCR Total RNA was extracted using the TRIZOL
reagent (Life Technologies). The integrity and size of RNA
were examined by agarose gel electrophoresis and ethidium
bromide staining. The extract of total RNA was reverse transcribed using a first strand cDNA synthesis kit for reverse
transcription-polymerase chain reaction (RT-PCR) (MBI Fermentas, Lithuania). Semiquantitative PCR was performed
using primers for human MMP-1 (forward, 5
-ATTCTACTGATATCGGGGCTTTGA-3
; reverse, 5
-ATGTCCTTGGGGTATCCGTGTAG-3
). MMP-2 (forward, 5
-GGC CAA
GTG GTC CGT GTG-3
; reverse, 5
-GAG GCC CCA TAG
AGC TCC-3
). The PCR condition used was as follows: initial denaturation (for 5 min at 94 C), 28 cycles of amplification (for 1 min at 94 C, for 1 min at 60 C, and for 1 min at
72 C), and final extension (for 10 min at 72 C). Primer
sequences and PCR conditions for GAPDH were described
previously.16) Reaction products were electrophoresed in
2.0% agarose gels and visualized with ethidium bromide.
Following normalization by GAPDH intensity the results are
presented as the percentage of basal MMP-1, 2 mRNA
expression. The signal strengths were quantified using a densitometric program (TINA; Raytest Isotopenme gerate, Germany).
Western Blot Analysis To determine the amounts of
MMP-1 protein secreted into culture media, equal aliquots of
conditioned culture media from an equal number cells were
resolved by 10% SDS-PAGE, transferred to Hybond ECL
membrane (Amersham Biosciences, England), and analyzed
by Western blotting with specific antibody against MMP-1
(Oncogene Research Products, San Diego, CA, U.S.A.).
Zymography Zymography was used for the semi-quantitative analysis of gelatinase (MMP-2) levels. Equal aliquots
of conditioned culture media from equal number cells were
applied to 10% zymogram gels containing gelatin, according
to the manufacturers instructions (NOVEX, San Diego, CA,
U.S.A.). After electrophoresis, the gels were incubated in
renaturing buffer (50 mM TrisHCl [pH 7.4], 2% (v/v) Triton
X-100) for 30 min at room temperature and then incubated
overnight in developing buffer (50 mM TrisHCl [pH 8.0],
2.5 mM CaCl2, 0.02% (w/v) sodium azide) at 37 C. Proteolytic bands were visualized by staining in 30% methanol
10% glacial acetic acid solution containing 0.5% (w/v)
Coomassie blue and destaining in 30% methanol10%
glacial acetic acid solution. Signal strengths were quantified
using a densitometric program (TINA; Raytest Isotopenme
gerate, Germany).
RESULTS AND DISCUSSION
2
,4
,7-Trihydroxyisoflavone (1), isoflavonoid from the
whole plants of V. hondoensis, was verified by comparing
NMR spectral data with those of previous reports17) (Fig. 1).
Recently, we demonstrated the effect of triterpenoids on the
regulation of MMP-1 and type I procollagen expression in
human skin fibroblasts and suggested the possibility of drug

Vol. 28, No. 5

Fig. 1.

Chemical Structure of Isofalvonoid Compounds

development for the treatment and recovery of UV-induced

skin damages through MMP-1 regulation.18,19) However,
specificity of those compounds toward other MMPs remains
to be determined. It has been also reported that compound
1 exhibits marginal cytotoxic activity against tumor cell
lines.20) These observations have led us to investigate the
ability of compound 1, to regulate a specific MMP activity in
human skin fibroblasts. To examine the effect of compound 1
on the expression of MMP-1 and MMP-2 in primary human
skin fibroblasts, we exposed cultured fibroblasts to
100 mJ/cm2 using UV light, and measured the MMP-1 and
MMP-2 mRNA levels at 24 h by semi-quantitative RT-PCR.
UV irradiation markedly induced MMP-1 mRNA levels,
however, compound 1 treatment (0.01, 1 m M) completely inhibited UV-induced MMP-1 mRNA levels to the control
levels in a dose-dependent manner (Fig. 2A). In contrast,
changes in MMP-2 mRNA levels by UV irradiation were not
observed and these findings are consistent with previous
report.11) Compound 1 also failed to change the expression of
MMP-2 mRNA (Fig. 2B). Genistein treatment (0.01, 1 m M:
refer compound) completely inhibited UV-induced MMP-1
mRNA levels to the control levels at 1 m M, and these findings
are consistent with previous report21) (Fig. 2A), but, changes
in MMP-2 mRNA levels by UV irradiation (Fig. 2B). In vitro
cytotoxicity assay was performed to determine whether the
inhibitory effect of compounds on MMP-1 mRNA expression were attributable to nonspecific cytotoxicity, as previously reported.22) In the presence of up to 1 m M, compounds
did not show cytotoxicity in these human skin fibroblasts
(data not shown).
To further ascertain whether compounds regulates the
expression of MMP-1 and MMP-2 protein at the protein levels, we next examined MMP-1 and MMP-2 levels by Western blot and zymogram analysis, respectively. Cells were
exposed to UV 100 mJ/cm2 for 30 min and further incubated
for 72 h in the absence or presence of compounds (0.01,
1 m M), and the conditioned culture media were collected and
used for analysis. As expected, compound 1 markedly inhibited UV-induced MMP-1 expression to that of control, and
did not alter MMP-2 protein levels (Fig. 2C). Genistein
(1 m M) markedly inhibited UV-induced MMP-1, 2 protein
level expression to that of control (Fig. 2C). These results
demonstrate that compound 1 specifically reduces the expression of MMP-1, but not of MMP-2, at both the mRNA and
protein levels. It is well established that the UV irradiation of
cultured human skin fibroblasts in vitro or human skin in vivo
induces the expression of MMPs which play important roles
in the degradation of extracelluar matrix components during
premature skin aging (photoaging).1214) Therefore, the development of MMP inhibitors has been considered to be

May 2005

Fig. 2.

927

Effects of 2
,4
,7-Trihydroxyisoflavone and Genistein on the Expression of MMP-1 and MMP-2 in Human Skin Fibroblasts

Quiescent primary human foreskin fibroblasts were exposed to UV 100 mJ/cm2 and collected at indicated time points. (A) and (B) RT-PCR analysis of MMP-1 and MMP-2.
Compounds suppressed UV-induced MMP-1 mRNA expression at 24 h. Expression levels of MMP-1 were normalized to GAPDH control. Values represent the meansS.E. of
data from three independent experiments (C) ( p0.05, n5). Conditioned media of fibroblasts at 72 h time point following exposure to UV light were collected and Western blotted with anti-MMP-1 antibodies, and also subjected to gelatin zymography for MMP-2 expression. Data shown are the representative of four independent experiments.

928

promising strategy for therapy of skin cancer and photoaging. However, most synthetic MMP inhibitors have broadspectrum activity against MMPs. This lack of specificity toward MMPs family restricts their use in clinical trials.23) Recently, it is widely appreciated that natural products from
medicinal plants are a potential source for the development
of selective MMP inhibitors. We have recently reported that
compounds isolated from natural plants may prevent and
treat UV-induced skin aging process through inhibition of
MMP-1 expression.15,18,19) Here, we investigated the effects
of compound 1 on the expressions of MMP-1 and MMP-2
protein in cultured human skin fibroblasts. Our results show
that specifically inhibits the expression of UV-induced MMP1 mRNA and protein, but not those of MMP-2. However, the
molecular mechanisms of 2
,4
,7-trihydroxyisoflavone inhibition on UV-induced MMP-1 expression have remained unexplored. In conclusion, this report is the first description
that 2
,4
,7-trihydroxyisoflavone specifically inhibits UV-induced MMP-1 expression in human skin fibroblasts and suggest the possibility of 2
,4
,7-trihydroxyisoflavone as a therapeutic agent for the chronological and UV-induced skin
aging.
Acknowledgements This study was supported by grant
of the Korea Health 21 R&D Project, Ministry of Health &
Welfare, Republic of Korea (03-PJ1-PG1-CH14-0001).
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