Viola hondoensis (Violaceae) distributed in southern partof Korea. In traditional medicine, the herb has been used asan expectorant, a diuretic, and an antiinflammatory for bron-chitis, rheumatism, skin eruptions, and eczema.1,2) Previousphytochemical studies on Viola species have revealed themto be a rich source of cyclotides3—5) and several flavone gly-cosides.6,7) Matrix metalloproteinases (MMPs) are a familyof zinc-dependent endoproteinases that play pivotal roles inthe dynamic remodeling of extracellular matrix. Based onsubstrate preference and structural homology, MMPs aresub-classified into functional groups: collagenases, gelati-nases, stromelysins, matrilysins, membrane type-MMPs(MT-MMPs) and other non-classified MMPs.8) MMPs arefrequently overexpressed by the various extracellular stimuliincluding growth factors, cytokines, tumor promoters, andultraviolet (UV), and this increase in MMP-related activitiesmay be involved in the pathogenesis of diseases such as can-cer and inflammation as well as in physiologic processes.9,10)

It has been also reported that up-regulation of some MMPs isresponsible for the enhanced degradation of dermal collagenduring chronological and UV-induced skin aging.11—14)

Therefore, the regulation of MMP activity might be a poten-tial strategy for prevention and/or treatment of UV-inducedskin damage. Recently, Kim et al. reported the isolation ofthe styraxjaponoside B from the stem bark of Styrax japonicato regulate MMP-1 expression.15) In this study, we investi-gated the effect of 2�,4�,7-trihydroxyisoflavone, Isoflavonoidfrom Viola hondoensis, on the expression of UV-inducedMMP-1 and MMP-2 mRNA and protein in human skin fi-broblasts.

MATERAL AND METHODS

General Procedure IR spectra were obtained with aPerkin Elmer 1710 spectrophotometer. The NMR spectrawere 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 spec-trometer. TLC was carried out on silica gel 60 F254 and RP-

18 F254 plates (Merck, Germany). Column chromatographywas 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. JongHwan 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 ofthe authors, Dr. Joongku Lee. A voucher specimen of thisraw material has been deposited at the herbarium of theSeoul National University (SNU-04-04-13).

Isolation of 2�,4�,7-Trihydroxyisoflavone The driedwhole plants (413 g) of Viola hondoensis were extracted fivetimes 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 distilledwater and partitioned with n-hexane, CHCl3, EtOAc, and n-BuOH. The EtOAc fraction (1.3 g) was subjected to silicagel column chromatography using CHCl3–EtOAc gradientsystem (9 : 1→1 : 1) to provide 11 fractions (fractions 1—11).From fraction 7, compound 1 (2.1 mg) was isolated using asilica gel column chromatography (CHCl3–EtOAc, 21 : 2),and then purified by semipreparative RP-HPLC (YMC J’-sphere-H80, 4 mm, 250�10 mm, MeOH–H2O�3 : 2.5).Compound 1 (1.3 mg) was also separated from fraction 8 bythe same procedure as those of the fraction 7.

Cell Culture Primary human foreskin dermal fibrob-lasts were established by outgrowth from biopsies of healthydonors of 3—12 years and cultured in Dulbecco’s modifiedEagle’s media (DMEM; Life Technologies, Rockville, MD,U.S.A.) supplemented with glutamine (2 mM), penicillin(400 U/ml), streptomycin (50 mg/ml), and 10% fetal bovineserum (FBS; HyClone, Logan, UT, U.S.A.) in a humidified5% CO2 atmosphere at 37 °C. The cells from passages 6—10were used for the experiments.

UV Irradiation The UV light source was anF75/85W/UV21 fluorescent sun lamps, having an emissionspectrum between 285—350 nm (peak at 310—315 nm) aspreviously described.16) Subconfluent cells were serum-

May 2005 Notes Biol. Pharm. Bull. 28(5) 925—928 (2005) 925

∗ To whom correspondence should be addressed. e-mail: himun@snu.ac.kr, Jhchung@snu.ac.kr © 2005 Pharmaceutical Society of Japan

Isoflavonoid from Viola hondoensis, Regulates the Expression of MatrixMetalloproteinase-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 AgingResearch, Clinical Research Institute, Seoul National University Hospital; Seoul, 110–744, Korea: b Korea ResearchInstitute of Bioscience and Biotechnology; Daejeon, 305–333, Korea: and c College of Pharmacy, Sung Kyun KwanUniversity; Suwon, 440–746, 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 ofteninduces chronic skin diseases such as skin cancer as well as photoaging (premature skin aging), and the mecha-nisms 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 Violahondoensis (Violaceae) on the expression of MMPs in UV-irradiated human skin fibroblasts in vitro. 2�,4�,7-Tri-hydroxyisoflavone markedly reduced UV-induced MMP-1 expression, but not MMP-2, at the both mRNA andprotein levels in a dose-dependent manner. Our report is the first description for the ability of 2�,4�,7-trihydroxy-isoflavone to regulate MMP-1 expression specifically.

Key words Viola hondoensis; 2�,4�,7-trihydroxyisoflavone; Matrix Metalloproteinase (MMP)-1; MMP-2; human skin fibroblasts

starved for 24 h to synchronize cells in G1/G0 phase of thecell cycle. Fresh phosphate-buffered saline (PBS) was addedto the quiescent cells prior to UV light exposure (0—100mJ/cm2). After UV irradiation, the cells were washed withPBS, and incubated for the indicated time in DMEM with orwithout compound.

RT-PCR Total RNA was extracted using the TRIZOL

reagent (Life Technologies). The integrity and size of RNAwere examined by agarose gel electrophoresis and ethidiumbromide staining. The extract of total RNA was reverse tran-scribed using a first strand cDNA synthesis kit for reversetranscription-polymerase chain reaction (RT-PCR) (MBI Fer-mentas, Lithuania). Semiquantitative PCR was performedusing primers for human MMP-1 (forward, 5�-ATTCTACT-GATATCGGGGCTTTGA-3�; reverse, 5�-ATGTCCTTGGG-GTATCCGTGTAG-3�). MMP-2 (forward, 5�-GGC CAAGTG GTC CGT GTG-3�; reverse, 5�-GAG GCC CCA TAGAGC TCC-3�). The PCR condition used was as follows: ini-tial denaturation (for 5 min at 94 °C), 28 cycles of amplifica-tion (for 1 min at 94 °C, for 1 min at 60 °C, and for 1 min at72 °C), and final extension (for 10 min at 72 °C). Primer sequences and PCR conditions for GAPDH were describedpreviously.16) Reaction products were electrophoresed in2.0% agarose gels and visualized with ethidium bromide.Following normalization by GAPDH intensity the results arepresented as the percentage of basal MMP-1, 2 mRNA expression. The signal strengths were quantified using a den-sitometric program (TINA; Raytest Isotopenme gerate, Ger-many).

Western Blot Analysis To determine the amounts ofMMP-1 protein secreted into culture media, equal aliquots ofconditioned culture media from an equal number cells wereresolved by 10% SDS-PAGE, transferred to Hybond ECLmembrane (Amersham Biosciences, England), and analyzedby Western blotting with specific antibody against MMP-1(Oncogene Research Products, San Diego, CA, U.S.A.).

Zymography Zymography was used for the semi-quan-titative analysis of gelatinase (MMP-2) levels. Equal aliquotsof conditioned culture media from equal number cells wereapplied to 10% zymogram gels containing gelatin, accordingto the manufacturer’s instructions (NOVEX, San Diego, CA,U.S.A.). After electrophoresis, the gels were incubated in renaturing buffer (50 mM Tris–HCl [pH 7.4], 2% (v/v) TritonX-100) for 30 min at room temperature and then incubatedovernight in developing buffer (50 mM Tris–HCl [pH 8.0],2.5 mM CaCl2, 0.02% (w/v) sodium azide) at 37 °C. Prote-olytic bands were visualized by staining in 30% methanol–10% glacial acetic acid solution containing 0.5% (w/v)Coomassie blue and destaining in 30% methanol–10%glacial acetic acid solution. Signal strengths were quantifiedusing a densitometric program (TINA; Raytest Isotopenmegerate, Germany).

RESULTS AND DISCUSSION

2�,4�,7-Trihydroxyisoflavone (1), isoflavonoid from thewhole plants of V. hondoensis, was verified by comparingNMR spectral data with those of previous reports17) (Fig. 1).Recently, we demonstrated the effect of triterpenoids on theregulation of MMP-1 and type I procollagen expression inhuman skin fibroblasts and suggested the possibility of drug

development for the treatment and recovery of UV-inducedskin damages through MMP-1 regulation.18,19) However,specificity of those compounds toward other MMPs remainsto be determined. It has been also reported that compound 1 exhibits marginal cytotoxic activity against tumor celllines.20) These observations have led us to investigate theability of compound 1, to regulate a specific MMP activity inhuman skin fibroblasts. To examine the effect of compound 1on the expression of MMP-1 and MMP-2 in primary humanskin fibroblasts, we exposed cultured fibroblasts to100 mJ/cm2 using UV light, and measured the MMP-1 andMMP-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 mM) completely in-hibited 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 notobserved and these findings are consistent with previous report.11) Compound 1 also failed to change the expression ofMMP-2 mRNA (Fig. 2B). Genistein treatment (0.01, 1 mM:refer compound) completely inhibited UV-induced MMP-1mRNA levels to the control levels at 1 mM, and these findingsare consistent with previous report21) (Fig. 2A), but, changesin MMP-2 mRNA levels by UV irradiation (Fig. 2B). In vitrocytotoxicity assay was performed to determine whether theinhibitory effect of compounds on MMP-1 mRNA expres-sion were attributable to nonspecific cytotoxicity, as previ-ously reported.22) In the presence of up to 1 mM, compoundsdid 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 lev-els, we next examined MMP-1 and MMP-2 levels by West-ern blot and zymogram analysis, respectively. Cells were exposed to UV 100 mJ/cm2 for 30 min and further incubatedfor 72 h in the absence or presence of compounds (0.01,1 mM), and the conditioned culture media were collected andused for analysis. As expected, compound 1 markedly inhib-ited UV-induced MMP-1 expression to that of control, anddid not alter MMP-2 protein levels (Fig. 2C). Genistein(1 mM) markedly inhibited UV-induced MMP-1, 2 proteinlevel expression to that of control (Fig. 2C). These resultsdemonstrate that compound 1 specifically reduces the expres-sion of MMP-1, but not of MMP-2, at both the mRNA andprotein levels. It is well established that the UV irradiation ofcultured human skin fibroblasts in vitro or human skin in vivoinduces the expression of MMPs which play important rolesin the degradation of extracelluar matrix components duringpremature skin aging (photoaging).12—14) Therefore, the de-velopment of MMP inhibitors has been considered to be

926 Vol. 28, No. 5

Fig. 1. Chemical Structure of Isofalvonoid Compounds

May 2005 927

Fig. 2. 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 means�S.E. ofdata from three independent experiments (C) (∗ p�0.05, n�5). Conditioned media of fibroblasts at 72 h time point following exposure to UV light were collected and Western blot-ted with anti-MMP-1 antibodies, and also subjected to gelatin zymography for MMP-2 expression. Data shown are the representative of four independent experiments.

promising strategy for therapy of skin cancer and photoag-ing. However, most synthetic MMP inhibitors have broad-spectrum activity against MMPs. This lack of specificity to-ward MMPs family restricts their use in clinical trials.23) Re-cently, it is widely appreciated that natural products frommedicinal plants are a potential source for the developmentof selective MMP inhibitors. We have recently reported thatcompounds isolated from natural plants may prevent andtreat UV-induced skin aging process through inhibition ofMMP-1 expression.15,18,19) Here, we investigated the effectsof compound 1 on the expressions of MMP-1 and MMP-2protein in cultured human skin fibroblasts. Our results showthat specifically inhibits the expression of UV-induced MMP-1 mRNA and protein, but not those of MMP-2. However, themolecular mechanisms of 2�,4�,7-trihydroxyisoflavone inhi-bition on UV-induced MMP-1 expression have remained un-explored. In conclusion, this report is the first descriptionthat 2�,4�,7-trihydroxyisoflavone specifically inhibits UV-in-duced MMP-1 expression in human skin fibroblasts and sug-gest the possibility of 2�,4�,7-trihydroxyisoflavone as a thera-peutic agent for the chronological and UV-induced skinaging.

Acknowledgements This study was supported by grantof the Korea Health 21 R&D Project, Ministry of Health &Welfare, Republic of Korea (03-PJ1-PG1-CH14-0001).

REFERENCES

1) Schuele S., Lederman R. J., Mov. Disord., 19, 43—48 (2004).2) Tennstedt D., Comphaut P., Dooms-Goossens A., Lachapelle J. M.,

Derm. Beruf. Umwelt., 27, 165—169 (1979).3) Lindholm P., Goransson U., Johansson S., Claeson P., Gullbo J., Lars-

son R., Bohlin L., Backlund A., Mol. Cancer Ther., 1, 365—369

(2002).4) Svangard E., Goransson U., Smith D., Verma C., Backlund A., Bohlin

L., Claeson P., Phytochemistry, 64, 135—142 (2003).5) Goransson U., Svangard E., Claeson P., Bohlin L., Curr. Protein Pept.

Sci., 5, 317—329 (2004).6) Carnat A. P., Carnet A., Fraisse D., Lamaison J. L., Heitz A., Wylde

R., Teulade J. C., J. Nat. Prod., 61, 272—274 (1998).7) Xie C., Veitch N. C., Houghtn P. J., Simmonds M. S., Chem. Pharm.

Bull., 51. 1204—1207 (2003).8) Visse R., Nagase H., Crc. Res., 92, 827—839 (2004).9) Sternlicht M. D., Werb Z., Annu. Rev. Cell Dev. Biol., 17, 463—516

(2001).10) Stetler-Stevenson W. G., Yu A. E., Semin Cancer Biol., 11, 143—152

(2001).11) Fisher G. J., Datta S. C., Talwar H. S., Wang Z. Q., Varani J., Kang S.,

Voorhees J. J., Nature (London), 379, 335—339 (1996).12) Rittie L., Fisher G. J., Ageing Res. Rev., 1, 705—720 (2002).13) Brenneisen P., Sies H., Scharffetter-Kochanek K., Ann. N.Y. Acad. Sci.,

973, 31—43 (2002).14) Chung J. H., Hanft V. N., Kang S., J. Am. Acad. Dermatol., 49, 690—

697 (2001).15) Kim M. R., Moon H. I., Chung J. H., Moon Y. H., Hahm K. S., Woo E.

R., Chem. Pharm. Bull., 52, 1466—1469 (2004).16) Seo J. Y., Lee S. H., Youn C. S., Choi H. R., Rhie G. E., Cho K. H.,

Kim K. H., Park K. C., Eun H. C., Chung J. H., J. Invest. Dermatol.,116, 915—919 (2001).

17) Markham K. R., Geiger H., “The Flavonoids: Advances in ResearchSince 1986,” Chapman & Hall, London, 1994, pp. 441—497.

18) Moon H. I., Chung J. H., Lee J. K., Zee O. P., Arch. Pharm. Res., 27,730—733 (2004).

19) Moon H. I., Lee J., Zee O. P., Chung J. H., Arch. Pharm. Res., 27,1060—1064 (2004).

20) Alhasan S. A., Aranha O. M., Sarkar F. H., Clin. Cancer Res., 7,4174—4181 (2001).

21) Watanabe H., Shimizu T., Nishihira J., Abe R., Nakayama T.,Taniguchi M., Sabe H., Ishibashi T., Shimizu H., J. Biol. Chem., 16,1676—1683 (2004).

22) Mosmann T., J. Immunol. Methods, 65, 55—63 (1983).23) Coussens L. M., Fingleton B., Matrisian L. M., Science, 295, 2387—

2392 (2002).

928 Vol. 28, No. 5