Food Sci. Biotechnol. 23(2): 623-631 (2014)DOI 10.1007/s10068-014-0085-5

Preventive Effect of Fermented Gelidium amansii and Cirsium japonicum Extract Mixture against UVB-induced Skin Photoaging in Hairless MiceHyun Mee Kim, Dong Eun Lee, Soo Dong Park, Yong Tae Kim, Yu Jin Kim, Ji Woong Jeong, Jung-Hee Lee,Sung Sik Jang, Dae Kyun Chung, Jae-Hun Sim, and Chul-Sung Huh

Received July 31, 2013; revised September 24, 2013; accepted September 26, 2013; published online April 30, 2014© KoSFoST and Springer 2014

Abstract Chronic ultraviolet (UV) light causes skinphotoaging, characterized by fine and coarse wrinkleformation and dryness. In this study, the effect of fermentedGelidium amansii and Cirsium japonicum extract mixture(FGCM) with lactic acid bacteria on UVB-inducedphotoaging was evaluated in human dermal fibroblasts andSKH-1 hairless mice. In vitro, FGCM increased type Iprocollagen levels and suppressed UVB-induced matrixmetalloproteinase-1 (MMP-1) expression more effectivelythan G. amansii and C. japonicum extract mixture (GCM).In vivo, oral administration of FGCM significantlyinhibited UVB-induced the number and total depth ofwrinkles in the dorsal skin of mice. FGCM suppressedUVB-induced epidermal thickening, and attenuated UVB-

induced MMP-13 expression and MMP-2 and MMP-9activities in dermal tissue. Furthermore, FGCM increasedskin hydration and blocked transepidermal water loss in thedorsal skin of mice compared with the UVB-irradiatedgroup. These data indicate that FGCM exerts potent anti-photoaging activities by improving wrinkle formation anddryness.

Keywords: anti-wrinkle, Cirsium japonicum, Gelidiumamansii, photoaging, skin hydration

Introduction

Ultraviolet (UV) radiation is one of the most detrimentalenvironment factors resulting in a type of skin damageknown as photoaging (1). UV is classified in three regionsaccording to wavelength: UVA (320-400 nm), UVB (290-320 nm), and UVC (100-290 nm). Only UVA and UVBcan reach the Earth surface (1-3). UVB rays strongly affectthe epidermal layer, and cause several skin disordersincluding sunburn, cutaneous immunosuppression, cancer,and photoaging (4). Photoaging caused by continuousexposure to UV results in premature aging of the skin,which is characterized by dryness, coarse and fine wrinkleformation, laxity, and pigmentation (5). Numerous histologicalstudies have shown that photoaging is accompanied bystructural alterations, such as damage of collagen fibers,abnormal elastic fibers, and increased skin thickness (6-8).

Dermal fibroblasts in the skin mainly produce type Icollagen, which is the major structural protein in thedermis. Collagen destruction is considered as one of themajor factors contributing to the aged appearance of skin,and is caused by long-term sun exposure (9). The matrix

Hyun Mee Kim, Dong Eun Lee, Soo Dong Park, Yong Tae Kim, Yu JinKim, Ji Woong Jeong, Jung-Hee Lee, Sung Sik Jang, Jae-Hun SimKorea Yakult Co., Ltd., Yongin, Gyeonggi 446-901, Korea

Hyun Mee Kim, Dae Kyun ChungSchool of Biotechnology and Institute of Life Science and Resources,Kyunghee University, Yongin, Gyeonggi 449-701, Korea

Dae Kyun ChungSkin Bio Technology Center, Kyunghee University Yongin, Gyeonggi449-701, Korea

Dae Kyun ChungRNA Inc., College of Life Science, Kyunghee University, Yongin,Gyeonggi 449-701, Korea

Chul-Sung HuhDepartment of Food and Animal Biotechnology, College of Agricultureand Life Sciences, Seoul National University, Seoul 151-921, Korea

Chul-Sung Huh ( )Research Institute of Eco-friendly Animal Sciences, Institute of Green BioScience & Technology, Seoul National University, Pyeongchang, Gangwon232-916, Korea Tel: +82-33-339-5723; Fax: +82-33-339-5717E-mail: cshuh@snu.ac.kr

RESEARCH ARTICLE

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metalloproteinases (MMPs) are a family of zinc-dependentproteolytic enzymes that degrade components of theextracellular matrix (ECM) (10,11). Among these enzymes,MMP-1 (interstitial collagenase) primarily decomposesnative type I collagen in human skin (12). The collagencleaved by MMP-1 is further broken down into smallpeptides by MMP-9, resulting in ECM degradation, whichis the major factor contributing to wrinkle formation (13).Therefore, many researchers have shown great interest indeveloping natural anti-photoaging agents for regulatingthe expression of MMPs.

Seaweeds have shown various biological activities onthe skin, such as anti-photoaging (14-16), anti-inflammatory(17), and anti-cancer effects (18,19). In particular, Gelidiumamansii is one of the most abundant red alga species inmarine environments, and its extracted agar is popularlyconsumed as food material in South-East Asian countries,including Korea and Japan. A recent study showed that G.amansii extracts (GAE) exerted neurotrophic activity indeveloping rat hippocampal neurons in culture (20).However, the photoaging effects of G. amansii remainpoorly understood.

Cirsium japonicum is traditionally considered as medicinebecause of its various pharmacological functions, such asinhibition of hypertension and control of hemorrhage (21).Although C. japonicum extracts (CJE) are known tocontain several flavonoid compounds with various beneficialbiological activities (22-24), their anti-photoaging activitieshave not been widely studied.

Fermentation may cause various beneficial effects suchas improvement of flavors, enrichment of biologicalcomponents (e.g., proteins, essential amino acids, andvitamins), and improvement of bioavailability of nutrients(25). Therefore, in the present study, we investigated thepreventive effects of fermented G. amansii and C. japonicumextract mixture (FGCM) with lactic acid bacteria on UVB-induced photoaging by using human dermal fibroblasts andSKH-1 hairless mice.

Materials and Methods

Plant material G. amansii cultivated in Jeju Island waspurchased from the Miryang Agar-Agar Co., Ltd. (Miryang,Korea). C. japonicum grown in Jeju Island was purchasedfrom the Dispensary of Oriental Medicine, Insudang (Jeju,Korea).

Extraction and fermentation Dried G. amansii wasextracted with 50% fermentation ethanol (Daehan EthanolLife Co., Ltd., Seoul, Korea) for 5 h at 70oC. The obtainedextract was concentrated by evaporating ethanol. The driedC. japonicum was extracted with distilled water for 6 h at

100oC. The extracts of G. amansii and C. japonicum weremixed at a 4:1 ratio (w/w), which was then fermented withPediococcus pentosaseus BB (Korea Yakult Co., Ltd.,Yongin, Korea) for 24 h at 37oC without agitation. Thesupernatant of the fermentation product was collected bycentrifugation at 2,500×g for 15 min. This final product,containing 1.95 µg/mL syringin, was used for in vitro andin vivo tests.

Reagents Antibodies against MMP-1, MMP-13, and β-actin were purchased from Santa Cruz Biotechnology(Santa Cruz, CA, USA). All other chemicals werepurchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture Hs68 human dermal fibroblasts were purchasedfrom American Type Culture Collection (Manassas, VA,USA) and were cultured in monolayers at 37oC in a 5%CO2 incubator in Dulbecco’s modified Eagle’s medium(DMEM) containing 10% fetal bovine serum.

UVB irradiation UVB irradiation was performed usingUltraviolet Crosslinkers (UVP; Upland, CA, USA) withpeak emission at 302 nm.

Enzyme-linked immunosorbent assay (ELISA) Hs68cells were cultured in a 6-well plate (2×105 cell/well) andwere starved in serum-free DMEM for an additional 24 h.They were then treated with FGCM or G. amansii and C.japonicum extract mixture (GCM) (10 µg/mL) for 1 hbefore UVB (50 mJ/cm2) irradiation, and were incubatedfor 48 h. The cell culture medium was collected, and typeI procollagen and MMP-1 productions were quantifiedusing a procollagen type I C-peptide enzyme immunoassaykit (Takara, Shiga, Japan) and the Amersham MMP-1Biotrak activity assay system (GE Healthcare Biosciences,Piscataway, NJ, USA), respectively.

Western blot analysis For in vitro western blotting, afterthe cells (2×105) were cultured in a 6-well plate for 24 h,they were starved in serum-free DMEM for an additional24 h. They were treated with FGCM or GCM for 1 hbefore UVB (50 mJ/cm2) exposure, and were then incubatedfor 48 h. The harvested cells were lysed in radioimmuno-precipitation assay (RIPA) lysis buffer. The proteinconcentration was determined using a DC assay kit (Bio-Rad Corp., Hercules, CA, USA). Lysate protein (10 µg)was subjected to 10% SDS-PAGE, and then electrophoreticallytransferred to a polyvinylidene difluoride membrane. Afterblotting, the membrane was incubated with primary antibodiesagainst procollagen, MMP-1, and β-actin at 4oC overnight.Protein bands were visualized using a chemiluminescencedetection kit after hybridization with a horseradish peroxidase-conjugated secondary antibody. For the in vivo test, to

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isolate proteins from the mouse skin, the dorsal skin ofeach mouse was excised and blended in RIPA buffer usinga homogenizer. Proteins were analyzed as described abovefor the in vitro western blot assay. The relative amounts ofproteins associated with specific antibodies were quantifiedusing Image J (NIH, Bethesda, MD, USA).

Experimental animals and oral administration SKH-1hairless mice (5 weeks of age; mean body weight, 25 g)were purchased from Central Lab Animal Inc. (Seoul,Korea). Animals were maintained in climate-controlledquarters (24oC at 50% humidity) with a 12-h light/12-hdark cycle. The animal experimental protocol was approvedand experimental animals were maintained under specificpathogen-free conditions based on the guidelines establishedby the Ethics Committee at the R&BD Center of the KoreaYakult Company Limited. The mice were divided into acontrol group (n=8), a UVB-only treatment group (n=8),and a UVB and FGCM treatment group (n=8). The controland UVB-only treatment groups were orally administeredwith 200 µL of phosphate-buffered saline (PBS). UVB andFGCM treatment groups were daily orally administered200 µL of PBS containing 500 mg FGCM/kg body weight1 h before UVB irradiation.

UVB irradiation on mice The UVB radiation source(Ultraviolet Crosslinkers, UVP) emitted wavelengths withpeak emission at 302 nm. UVB radiation was applied tothe backs of the mice three times a week for eight weeks.The UVB dose was progressively increased in the followingmanner: 25 mJ/cm2 [1 minimal erythematous dose (MED)]the first week, 50 mJ/cm2 (2 MED) the second week, 75mJ/cm2 (3 MED) the third week, and 100 mJ/cm2 (4 MED)from the fourth to the eighth week. Body weights wererecorded weekly. Replica preparation and measurements ofskin hydration and transepidermal water loss (TEWL) wereperformed at the eighth week of radiation exposure.

Production of replicas and image analysis Replicas ofmice dorsal skin were constructed using SILFLO (CuDermCorporation, Dallas, TX, USA). The impression replicaswere set on a horizontal sample stand, and wrinkle imageswere obtained using a CCD camera and analyzed with SkinVisioLine 650 software (Courage & Khazaka ElectronicGmbH, Cologne, Germany).

Skin moisture measurement Skin hydration and TEWLof the dorsal skin were measured before the animals weresacrificed during the last week of the experiment. Skinhydration levels were quantified using Corneometer CM825(Courage & Khazaka Electronic GmbH), and TEWL wasevaluated using Tewameter TM300 (Courage & KhazakaElectronic GmbH).

Gelatin zymography The dorsal skin of each mousewas blended in RIPA buffer by using a homogenizer, andthen, equal amounts of the protein extract were mixed withnon-reducing sample buffer, incubated for 15 min at roomtemperature, and then resolved by 12% SDS-PAGEcontaining 1 mg/mL gelatin. The gels were washed with2.5% Triton X-100 twice for 30 min, rinsed three times for30 min with a 50 mM Tris-HCl buffer (pH 7.6) containing5 mM CaCl2, 0.02% Brij-35, and 0.2% sodium azide, andincubated overnight at 37oC. The gels were then stainedwith a 0.5% Coomassie brilliant blue R-250 solutioncontaining 10% acetic acid and 20% methanol for 30 min,and were destained with 7.5% acetic acid solution containing10% methanol. Areas of gelatinase activity were detectedas clear bands against the blue-stained gelatin background.Gelatinase activity was quantified by densitometricanalysis of the clear bands (as scanned JPEG images) usingImage J (NIH).

Statistical analysis Where appropriate, data are expressedas means±SD values, and the Student’s t-test was used formultiple statistical comparisons. A probability value ofp<0.05 was used as the criterion for statistical significance.

Results and Discussion

Synergistic effect of the mixture of GAE and CJE onUVB-reduced type I procollagen production in Hs68cells As the aberrant type I procollagen level is importantlyinvolved in skin wrinkle formation (26), the possiblerecovery effects of GAE, CJE, or GCM on the reduction oftype I procollagen production in response to UVB irradiationin Hs68 human dermal fibroblasts were first examined.GAE or CJE treatment slightly restored the level of type Iprocollagen secretion in culture media by up to 10 and11%, respectively, compared to UVB-irradiated cells (Fig.1A). On the other hand, the mixture of GAE and CJE at a4:1 ratio (w/w) synergistically increased the level of type Iprocollagen production compared to treatment with singleextracts. However, the combination at a ratio of 1:1 and 1:4(GAE:CJE, w/w) did not result in more effective activityrelative to that of a single extract (Fig. 1A). Therefore, wefurther compared the activities of GCM and FGCM usingthe mixture at 4:1 (GAE:CJE, w/w).

Comparison of the effect of GCM or FGCM on UVB-reduced type I procollagen secretion and expression inHs68 cells Previously, we screened the lactic acidbacteria promoting procollagen secretion in Hs68 cells, andselected P. pentosaseus BB for the best effect (unpublisheddata). Lactic acid bacteria can convert various componentsto more beneficial constituents using carbohydrates from in

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dietary materials. Therefore, it was next examined whetherthe fermentation of GCM with P. pentosaseus BB couldimprove its anti-photoaging activities in Hs68 cells. Thecells were pre-treated with FGCM or GCM for 1 h beforeUVB exposure and were analyzed by ELISA after 48 h.The ELISA results revealed that FGCM or GCM increasedthe amount of type I procollagen secreted in the culturemedium compared to UVB-irradiated cells (Fig. 1B).Compared with GCM, the same concentrations of FGCMtreatment more effectively inhibited type I procollagenreduction by UVB irradiation. We next investigated theeffects of FGCM or GCM on the reduction of type Iprocollagen expression induced by UVB exposure incultured Hs68 cells. The results of western blotting showedthat UVB irradiation caused a clear decrease in type Iprocollagen expression compared to non-irradiated controllevels in Hs68 cells. Consistent with the ELISA results,FGCM more effectively restored the type I procollagenexpression levels than did GCM (Fig. 1C).

FGCM suppresses UVB-induced MMP-1 expression inHs68 cells Since MMP-1 is well known as a collagenase,it was examined whether FGCM or GCM might block

UVB-induced MMP-1 secretion or expression in Hs68cells. The ELISA results showed that the activity of MMP-1 in Hs68 cells significantly increased after UVB exposurecompared with that of the control. In contrast, FGCMmarkedly suppressed UVB-induced MMP-1 activity, andthis effect was stronger than that of GCM (Fig. 2A). Wefurther investigated the effects of FGCM or GCM onUVB-induced MMP-1 expression in Hs68 cells. Consistentwith the ELISA results, the western blotting data showedthat FGCM down-regulated UVB-induced MMP-1expression more strongly than did GCM at the sameconcentration (Fig. 2B).

FGCM inhibits UVB-induced skin wrinkle formationin hairless mice skin To further confirm the anti-photoaging effect of FGCM in vivo, we studied the effectsof FGCM on UVB-induced photoaging using the hairlessmouse, a well-developed photoaging model (27). AfterUVB was repeatedly exposed to the dorsal skin of micethree times a week for eight weeks, the coarse and deepwrinkles were formed on the dorsal skin of mice. Bycontrast, oral administration of FGCM effectively reducedwrinkle formation. To further evaluate the preventive effect

Fig. 1. Synergistic effect of the GAE and CJE mixture (A) and Comparison of the effects of GCM and FGCM on UVB-reducedtype I procollagen secretion (B) and expression (C) in Hs68 cells. For A-C, the cells were treated with GAE, CJE, GCM, or FGCM at10 µg/mL for 1 h before exposure to UVB (50 mJ/cm2) for 48 h. Data are representative of three independent experiments. *,**Indicatesignificant differences (p<0.05 or p<0.01, respectively) between the group treated with UVB alone and the GAE-, CJE-, GCM-, orFGCM-treated groups. ##Indicates a significant difference (p<0.01) between the UVB+GAE-treated cells and the UVB+GCM 4:1mixture-treated cells. ###Indicates a significant difference (p<0.01) between the UVB+CJE treated cells and the UVB+GCM 4:1 mixture-treated cells.

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of FGCM on skin photoaging, replicas of mouse dorsalskin were prepared. The skin replicas were photographed,and the number and depth of wrinkles were quantitativelyanalyzed using the skin VisioLine 650 system. These datashowed that wrinkle formation was improved by oraladministration of FGCM prior to UVB irradiation (Fig.

3A). The number of wrinkles in the UVB-only treatedgroup was increased by 2.1-fold compared with the controlgroup. In contrast, FGCM administration reduced wrinklenumbers by 49% (Fig. 3B). In addition, the total depth ofwrinkles was attenuated by 74% compared with the UVB-only treated group (Fig. 3C).

Fig. 2. Comparison of the effects of GCM or FGCM on UVB-induced MMP-1 activity (A) and expression (B) in Hs68 cells. Effectof GCM or FGCM on UVB-induced MMP-1 activity (A) and expression (B) in Hs68 cells. Data are representative of three independentexperiments. *Indicate significant differences at p<0.05 between the group treated with UVB alone and the GCM- or FGCM-treatedgroups.

Fig. 3. Effect of oral administration of FGCM on UVB-induced wrinkle formation in hairless mice skin. (A) Photographs ofwrinkle replica of the dorsal mice skin at the end of the study. 100× magnification. (B) FGCM clearly reduced the number of wrinklescompared with the UVB only-treated group. (C) FGCM strongly inhibited the total wrinkle depth in SKH-1 hairless mice. The asterisksindicate a significant decrease in wrinkle formation between the group treated with UVB alone and the group treated with FGCM(**p<0.01).

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FGCM inhibits UVB-induced epidermal thickening inhairless mice skin Because epidermal hypertrophy isone of the critical factors causing wrinkle formation, theeffect of FGCM on UVB-induced epidermal thickeningwas evaluated. Hematoxylin and eosin staining resultsshowed that UVB-induced epidermal thickening of thedorsal skin was markedly increased by 3.5-fold due tochronic UVB exposure. This epidermal hypertrophy wasclearly attenuated by 52% (p<0.01 vs. UVB-only treatedgroup) by oral administration of FGCM prior to UVB

irradiation (Fig. 4).

FGCM inhibits UVB-induced activity or expression ofMMPs in hairless mice skin The effects of FGCM onthe expression or activity of key factors related to collagendegradation resulting in wrinkle formation were furtherexamined. Since MMP-13, but not MMP-1, acts as acollagenase in mice (28), we investigated the expression ofMMP-13 in mice skin. Chronic UVB irradiation on micedorsal skin induced MMP-13 expression, and FGCM

Fig. 5. Effect of oral administration of FGCM on UVB-induced MMPs activities (A) or expression (B) in hairless mouse skin. (A)FGCM inhibited the increase in MMP-9 or MMP-2 activities induced by UVB irradiation of mouse skin. (B) FGCM inhibited UVB-induced MMP-13 expression in mice skin. Data are representative of three independent experiments. Results are shown as means±SD(n=3). *,**Indicate a significant difference (p<0.05 and p<0.01, respectively) between the group exposed to UVB and that treated withFGCM.

Fig. 4. Effect of oral administration of FGCM on UVB-induced epidermal thickening in hairless mice skin. (A) Hematoxylin andeosin-stained images of UVB-irradiated mouse skin. Images are representative of results from eight tissue samples. 100× magnification.(B) FGCM prevented UVB-induced mice epidermal thickening. Bars represent the mean thickness (mm) of the epidermis from eightanimals (three measurements/section). Results are shown as means±SD (n=8). **Indicate significant differences at p<0.01 between thegroup treated with UVB alone and the group treated with FGCM.

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administration significantly attenuated it (Fig. 5A). Theskin collagen is primarily broken down by MMP-13, and isthen further degraded by MMP-9. Moreover, becauseMMP-2 digests type IV collagen, which is component ofthe epidermal basement membrane, we next examinedwhether FGCM might exert effects on UVB-inducedMMP-2 and MMP-9 activities. Zymography results showedthat oral administration of FGCM clearly suppressed theactivated MMP-2, proMMP-2, and MMP-9 activities inhairless mice skin (Fig. 5B).

FGCM regulates UVB-induced skin dryness in hairlessmice skin Photoaging is characterized as clinically fineand coarse wrinkling, and is accompanied by skin dryness(29). Thus, the moisturizing effect of FGCM on dorsalmice skin was investigated. The results showed thatchronic UVB exposure to the dorsal skin of mice overeight weeks decreased the skin hydration level by 28%(p<0.01), and increased the TEWL value by 223%(p<0.01) compared to control mice (Fig. 6). In contrast,oral administration of FGCM up-regulated the skinhydration level by 65% (p<0.01 vs. UVB-only treatedgroup) (Fig. 6A), and down-regulated the TEWL value by50% (p<0.01 vs. UVB-only treated group) (Fig. 6B).

Several recent studies have revealed that dietary seaweedsor their extracts exert a variety of biological activities.Extracts of the seaweed species Laminaria japonicashowed a moisturizing effect in vivo (30), and fucoxanthinisolated from edible brown seaweeds inhibited UV-inducedphotoaging in hairless mice (31). Fucoidan, a dietary fiberpurified from seaweed, improved atopic dermatitis-likelesions through control of the immune system (32).However, no reports on the bioactive components of G.amansii, a red alga species, or their biological functions areyet available. Nonetheless, several studies have reportedthat various oxygenated sterols, including chlorophyll, β-

carotene, and lutein, from various species of red algainhibited the growth of several cancer cell types (33,34).Even if these compounds can be considered as potentialbioactive compounds of G. amansii due to speciessimilarities, the roles of other yet unknown components,such as vitamins and minerals, cannot be excluded (35).Therefore, more studies on the identification of usefulbioactive substances of G. amansii are needed to betterunderstand its physiological functions.

It is well known that C. japonicum a variety ofphytochemicals, including luteolin, apigenin, and syringin(36-38). Recent studies reported that luteolin suppressedUVB-induced wrinkle formation and MMP-13 expressionin vivo (28), and that apigenin inhibited UVB-induced skininflammation in hairless mice (39). In addition, syringinshowed immunomodulatory activity by inhibiting tumor-necrosis factor and nitric oxide production (40). However,among these chemicals, we could only detect syringin byusing high-performance liquid chromatography analysis inC. japonicum water extract (data not shown). Therefore,further study is needed to confirm whether syringin hasanti-photoaging activity, and to investigate the bioactivecompounds of C. japonicum water extract.

Collagen is the major structural component of the skin,and marked degradation and degenerative changes ofcollagen in the dermis result in wrinkle formation (41). UVirradiation increased the expression of MMPs in humanskin, including MMP-1, MMP-2, and MMP-9 (42). MMP-1 initiates the cleavage of fibrillar collagen, and suchcleaved collagen can be further degraded by MMP-9 (10).The gelatinases MMP-2 and MMP-9 digest type IV andVII collagens specifically, which are components of theepidermal basement membrane (42). On the other hand,Inomata et al. (42) showed that acute UV exposure to theskin increased only MMP-9 levels, whereas chronic UVexposure caused increases of both MMP-2 and MMP-9

Fig. 6. Effect of oral administration of FGCM on UVB-induced dryness in hairless mouse skin. Water content (A) and TEWL (B)were evaluated at the end of the study. **Indicate a significant difference in the water content or TEWL value between the group treatedwith UVB alone and the group treated with FGCM (**p<0.01).

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levels in hairless mice. Therefore, MMP-2 might be acritical marker of chronically induced photoaging.

In the present study, we found that FGCM had a strongerinhibitory effect on UVB-induced MMP-1 expression, anda stronger recovery effect on UVB-reduced procollagenexpression in Hs68 cells compared to those of GCM. Oraladministration of FGCM also suppressed UVB-inducedMMP-13 expression and MMP-2 and MMP-9 activities,thereby inhibiting wrinkle formation and epidermal thickeningin the dorsal skin of hairless mice. FGCM significantlyblocked UVB-induced dryness through improving skinhydration and inhibiting TEWL in mice skin. These resultsdemonstrate that FGCM might be a good candidate as ananti-photoaging agent for skin. However, the detailedmolecular mechanisms and bioactive compounds responsiblefor the increased anti-photoaging effects of FGCM comparedwith GCM remain to be elucidated.

Acknowledgments This work was supported by theInterrelated Development Program (R-0000452) of Inter-Economic Regions, Ministry for Knowledge Economy,Republic of Korea.

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