Protective Effects of Standardized Siegesbeckia glabrescens … · 2017-02-24 · Evaluation of...

J. Microbiol. Biotechnol.

J. Microbiol. Biotechnol. (2017), 27(2), 242–250https://doi.org/10.4014/jmb.1610.10050 Research Article jmbReview

Protective Effects of Standardized Siegesbeckia glabrescens Extractand Its Active Compound Kirenol against UVB-Induced Photoagingthrough Inhibition of MAPK/NF-κB Pathways Jongwook Kim, Mi-Bo Kim, Jun Gon Yun, and Jae-Kwan Hwang*

Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea

Introduction

Skin aging occurs because of exposure to extrinsic factors,

such as ultraviolet (UV) irradiation, physical stimulation,

and various chemicals; skin aging due to prolonged

exposure to UV irradiation is termed photoaging. The

photoaging symptoms include oxidative stress, dryness,

wrinkles, and hyperpigmentation [1, 2]. Once UVB-

induced reactive oxygen species (ROS) are formed in the

skin, the extracellular matrix (ECM) components, including

collagens, gelatins, elastic fibers, and glycosaminoglycans,

are broken down and disorganized [3, 4].

Increased ROS in UVB-irradiated skin induce matrix

metalloproteinase (MMP) expression and collagen

degradation in the ECM, resulting in photodamaged skin.

UVB-induced oxidative stress stimulates protein kinase

cascades, such as mitogen-activated protein kinases

(MAPKs), activated protein 1 (AP-1), and nuclear factor

kappa B (NF-κB), ultimately enhancing MMP secretion and

collagen degradation. Thus, ROS-scavenging antioxidants

in natural materials have been proposed as photoprotective

agents [5, 6].

The ECM in the skin is broken down by MMPs containing

zinc-binding catalytic domains [7]. Depending on the

substrates, MMPs can be categorized into various subgroups,

including collagenases, gelatinases, and stromelysins.

Collagenases (MMP-1 and -13) degrade native collagens,

whereas gelatinases (MMP-2 and -9) cleave the basement

membrane. On the other hand, stromelysins (MMP-3 and -

10) confer broader substrate specificity than that of the first

two subgroups mentioned [8].

UV-induced photoaging stimulates MMPs by several

pathways, including the MAPK and NF-κB pathways [9].

UV irradiation activates three MAPKs: extracellular

signal-regulated kinase (ERK), p38, and c-Jun N-terminal

kinase (JNK) [10]. The MAPK activation results in the

heterodimeratization of c-Jun/c-Fos and the formation of

the AP-1 complex, which promotes collagen degradation

by regulating MMP transcription [11]. Furthermore, UV

irradiation activates the NF-κB pathway, stimulating the

Received: October 21, 2016

Revised: November 7, 2016

Accepted: November 15, 2016

First published online

November 23, 2016

*Corresponding author

Phone: +82-2-2123-5881;

Fax: +82-2-362-7265;

E-mail: [email protected]

pISSN 1017-7825, eISSN 1738-8872

Copyright© 2017 by

The Korean Society for Microbiology

and Biotechnology

Anti-photoaging effects of standardized Siegesbeckia glabrescens extract (SGE) and its major

active compound kirenol were investigated using Hs68 human dermal fibroblasts and hairless

mice, respectively. UVB-irradiated hairless mice that received oral SGE (600 mg/kg/day)

showed reduced wrinkle formation and skinfold thickness compared with the UVB-irradiated

control. Furthermore, SGE treatment increased the mRNA levels of collagen synthesis genes

(COL1A1, COL3A1, COL4A1, and COL7A1) and activated antioxidant enzyme (catalase),

while suppressing matrix metalloproteinase (MMP-2, -3, -9, and -13) expression. In Hs68

fibroblasts, kirenol also significantly suppressed MMP expression while increasing the

expression of COL1A1, COL3A1, and COL7A1. Collectively, our data demonstrate that both

SGE and kirenol attenuated UVB-induced photoaging in hairless mice and fibroblasts through

inhibition of the mitogen-activated protein kinases and nuclear factor kappa B pathways,

suggesting that SGE has potential to serve as a natural anti-photoaging nutraceutical.

Keywords: Kirenol, MAPK/NF-κB pathways, photoaging, Siegesbeckia glabrescens

Anti-Photoaging Effects of SGE and Kirenol 243

February 2017⎪Vol. 27⎪No. 2

production of the proinflammatory cytokines, such as

interleukin (IL)-6 and -8. Then, IL-6 and -8 can upregulate

MMP expression, epidermal proliferation, inflammation,

and various UV-induced cellular responses in human skin

[12, 13].

Siegesbeckia glabrescens, a subspecies in Herba Siegesbeckiae,

has been traditionally used to treat inflammation, arthritis,

and hypertension [14]. It is known that S. glabrescens has

antiasthma [15], anticancer [16], and antibacterial effects

[17]; however, its anti-photoaging effect remains to be

elucidated. Kirenol (Fig. 1) is a diterpenoid component found

in Herba Siegesbeckiae [18], exhibiting anti-adipogenesis

[19], antiarthritic [20], and anti-inflammatory effects [21];

however, its anti-photoaging activity remains unknown.

Here, the anti-photoaging effects of S. glabrescens extract

and kirenol were investigated in UVB-induced photoaging

in vitro and in vivo model, respectively.

Materials and Methods

Preparation of Standardized S. glabrescens Extract (SGE)

The edible aerial parts of S. glabrescens were purchased from

Kyungdong market (Korea). Dried edible aerial parts were

ground and extracted with 95°C hot water for 3 h. Then, a vacuum

rotary evaporator (Laborata 4000-efficient; Heidolph Instruments

GmbH & Co. KG., Germany) was used to concentrate the extract

with a yield of 12% (w/w), including 2% (w/w) of kirenol as a

bioactive compound.

Chemical Reagents

Kirenol was obtain from the Natural Product Bank of the

Institute for Korea Traditional Medical Industry (Korea).

Dulbecco’s modified Eagle’s medium (DMEM) was supplied from

Hyclone Laboratories, Inc. (USA). Fetal bovine serum (FBS) was

supplied from Gibco (USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) and protease inhibitor

cocktails were purchased from Sigma-Aldrich (USA). Primary

antibodies directed against MMP-1, -2, -9, phospho- and total-

ERK, phospho- and total-JNK, phospho- and total-p38, phospho-

and total-c-Jun, c-Fos, catalase, NF-κB, and α-tubulin were

purchased from Cell Signaling Technology (USA). Horseradish

peroxidase-linked secondary anti-rabbit and anti-mouse antibodies

were purchased from Bethyl Laboratories (USA).

Animal Experiments

Fifteen 5-week-old female albino hairless mice (SKH-1; Daehan

Biolink Ltd., Korea) were maintained in a humidity-, temperature-,

and light-controlled specific pathogen-free room (55 ± 10%

humidity, 23 ± 2°C, and 12 h light/dark cycle) at Yonsei

Laboratory Animal Research Center (Korea). All protocols for

animal experiments were in compliance with the guideline of the

Institutional Animal Care and Use Committee at Yonsei

University (Permit No. 201404-147-02). The hairless mice were

randomly grouped into three groups (5 mice/group): (i) normal

group (Normal), (ii) UVB-irradiated control group (UVB), and (iii)

UVB-irradiated and 600 mg/kg/day SGE treatment group (UVB +

SGE 600). Mice received SGE daily for 13 weeks by oral gavage

and were concurrently exposed to UVB irradiation on their dorsal

surface. UVB irradiation was carried out as described in our

previous publication [9]. UVB radiation was performed using a

CL-1000M UV Crosslinker (UVP, USA) which delivered UVB

energy at a wavelength of 302 nm. Mice were exposed to UVB

radiation at a dose of 75 mJ/cm2 during the first week. The dose of

UVB radiation was increased weekly from 1 minimal erythema

dose (MED) to 3 MEDs, which was maintained until the end of the

experiment. The mice were intraperitoneally euthanized with a

1:1 mixed solution of rompun and zoletil (One Bio, Korea). The

dorsal skin sections of the hairless mice were collected and kept at

-80°C.

Evaluation of Skin Wrinkle Formation

Replicas of mouse dorsal skin were prepared using the Silflo kit

(CuDerm Corporation, USA) and analyzed with Visioline VL650

(Courage + Khazaka Electronic GmbH, Germany). The skin

wrinkle parameters were measured in terms of total wrinkle area,

length, depth, and number.

Evaluation of Skinfold Thickness and Skin Elasticity

Skinfold thickness of the dorsal skin was measured at the mid-

back level with a caliper (Ozaki MFG Co., Ltd., Japan) after 13

weeks. The dorsal skin of hairless mice was picked up by finger

by pinching at the neck and base of the tail, and skinfold thickness

was measured at the mid-back level. More than three

measurements were taken at close proximity, and the mean value

was calculated. Skin elasticity was measured with the Cutometer

MPA580 (CK Electronics GmbH).

Histological Analysis

Skin samples of the mice were fixed with 10% formalin for 24 h.

Hematoxylin and eosin (H&E) staining for measuring skin thickness,

Fig. 1. Chemical structure of kirenol.

244 Kim et al.


Masson’s trichrome (M&T) staining for measuring collagen fiber,

and Verhoeff-van Gieson’s staining for measuring elasticity were

performed on skin tissue sections. The stained skin area was

examined using the Eclipse TE2000U Inverted Microscope with

twin CCD cameras (Nikon, Japan).

Hydroxyproline Assay

The hydroxyproline content was determined using a

hydroxyproline content assay kit of Quickzyme Biosciences

(Netherlands), according to the manufacturer’s instructions.

Cell Culture and UVB Irradiation

Hs68 human skin fibroblasts (American Type Culture Collection,

USA) were maintained in DMEM supplemented with 10% FBS

and 1% antibiotics (penicillin and streptomycin) under a

humidified atmosphere of 5% CO2 at 37°C. The fibroblasts were

pretreated in a serum-free medium with or without kirenol for

24 h. After pretreatment, the fibroblasts were exposed to 302 nm

UVB light (25 mJ/cm2) by using the UV crosslinker CL-1000M. To

determine the anti-photoaging activity of kirenol in vitro, the cells

were additionally treated with or without kirenol in serum-free

medium for 24 h.

Cell Viability Assay

Cell viability was assessed by MTT assay. Hs68 human skin

fibroblasts were treated to various doses of kirenol (0 to 60 μM).

The solution of MTT (0.5 mg/ml) was added to with or without

kirenol-treated cells and then incubated at 37°C for 4 h. Thereafter,

the MTT solution was suctioned using a suction pump, and 200 μl

of dimethyl sulfoxide was applied to dissolve the violet-colored

formazan crystals. The UV-Vis absorbance of the formazan

crystals at 540 nm was quantified by spectrophotometry, using a

VERSAmax turnable microplate reader (Molecular Devices Co.,

USA). No significant cytotoxicity was shown at various concentrations

of kirenol ranging from 10 to 40 μM (data not shown). The

following experiments were conducted with kirenol concentrations

upto 40 μM.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Analysis

Total RNA of homogenized skin sections and Hs68 cells was

isolated with Trizol reagent (Invitrogen, USA). cDNA was

synthesized with 1 μg of total RNA, oligo(dT), and Reverse

Transcription Premix (ELPIS-Biotech, Korea) in a 20 μl reaction

mixture. The sequences of primer were as follows: mouse COL1A1

(forward, 5’-GTC CCC AAT GGT GAG ACG TG-3’; reverse, 5’-

GCA CGG AAA CTC CAG CTG AT-3’), mouse COL3A1

(forward, 5’-AGC GGC TGA GTT TTA TGA CG-3’; reverse, 5’-

AGC ACA GGA GCA GGT GTA GA-3’), mouse COL4A1

(forward, 5’-GCC AAA GCC AAA CCC ATT CC-3’; reverse, 5’-

TGG TAC GTG TGG TAA CTT CTC-3’), mouse COL7A1

(forward, 5’-AAG CCG AGA TTA AGG GCT GG-3’; reverse, 5’-

CAC CAA ATG GAG CAC AGC AG-3’), mouse MMP-3 (forward,

5’-TAG CAG GTT ATC CTA AAA GCA-3’; reverse, 5’-CCA GCT

ATT GCT CTT CAA T-3’), mouse MMP-13 (forward, 5’-CAT CCA

TCC CGT GAC CTT AT-3’; reverse, 5’-GCA TGA CTC TCA CAA

TGC GA-3’), mouse β-actin (forward, 5’-GCT CCG GCA TGT

GCA A-3’; reverse, 5’-AGG ATC TTC ATG AGG TAG T-3’),

human COL1A1 (forward, 5’-GGG AGT TTC TCC TCG GGG TC-

3’; reverse, 5’-GTC ATC GCA CAA CAC CTT GC-3’), human

COL3A1 (forward, 5’-TGG TGC CCC TGG TCC TTG CT-3’;

reverse, 5’-TAC GGG GCA AAA CCG CCA GC-3’), human

COL7A1 (forward, 5’-ACC GTG AGC ACC CTA TTT GG-3’;

reverse, 5’-CAA CTG GTA GCG GGT CAC AT-3’), human IL-6

(forward, 5’-ATG AGG AGA CTT GCC TGG TG-3’; reverse, 5’-

ACA ACA ATC TGA GGT GCC CA-3’), human IL-8 (forward, 5’-

CCA GGA AGA AAC CAC CGG AA-3’; reverse, 5’-CCT CTG

CAC CCA GTT TTC CT-3’), and human GAPDH (forward, 5’-

CTC CTG TTC GAC AGT CAG CC-3’; reverse, 5’-TCG CCC CAC

TTG ATT TTG GA-3’). PCR was carried out in a Gene Amp PCR

System 2700 (Applied Biosystems, USA). The products of PCR

were separated by 1.5% agarose gel electrophoresis and detected

by UV illumination with the G:BOX Image Analysis System

(Syngene, UK).

Western Blot Analysis

Homogenized skin tissues and Hs68 cells were lysed with RIPA

buffer (ELPIS-Biotech) containing protease inhibitor cocktails.

Total protein concentrations were quantified by the Bradford

assay. Equal 30 μg amounts of protein were size-fractionated by

10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) and transferred to membranes. The membranes were

incubated for 1 h in blocking solution with Tris-buffered saline

with Tween-20 containing 5% skim milk. They were then incubated

overnight with primary antibodies at 4°C. The membranes-bound

primary antibodies were again incubated with horseradish

peroxidase-linked secondary anti-rabbit or anti-mouse antibodies

for 2 h. Antibody signals were visualized with ECL detection

solution (GE Healthcare, Sweden) and were detected using the

G:BOX Image Analysis System (Syngene).

Statistical Analysis

Descriptive results are reported as the mean ± standard

deviation (SD). Statistical analysis was performed with SPSS21.0

(SPSS, Inc., USA). The differences of groups were evaluated by

unpaired Student’s t-test for in vivo data. One-way analysis of

variance followed by Scheffe’s test was performed for in vitro

data. ##p < 0.01, *p < 0.05, and **p < 0.01 were considered to be

statistically significant.

Results and Discussion

Effect of SGE on Wrinkle Formation and Skin Thickness

In Vivo

UV-irradiated skin exhibits clinical and histological



changes, including wrinkling, elastosis, and skin thickening.

Clinical symptoms of UV-irradiated skin photoaging include

dryness, roughness, hyperpigmentation, coarse elastosis,

and laxity [9]. This study investigated whether SGE

protects UVB-induced photoaging by assaying histological

and clinical changes in hairless mice. UVB irradiation

increased the formation of wrinkles in the dorsal skin of

control mice; however, SGE treatment significantly reduced

it, as seen in the UVB + SGE 600-treated mice (Fig. 2A). To

quantitate wrinkle formation, we measured the total wrinkle

area, length, depth, and number (Fig. 2B). UV irradiation

stimulates MMP production and inhibits collagen synthesis

in the ECM, which results in increasing skin thickness by

excessive epidermal proliferation and wrinkle formation

by degradation of dermal ECM components [22]. In contrast,

SGE treatment led to decreased abnormal skinfold

thickness (Fig. 2C) and inhibition of abnormal elastic fiber

accumulation compared with those observed in the UVB-

irradiated control group, thereby preventing a decrease in

skin elasticity (Fig. 2D). Thus, SGE could be beneficial in

the prevention of photoaging by reducing wrinkle formation

and skin thickness, and preventing abnormal elastic fiber

accumulation.

Effect of SGE on Collagen Synthesis and MMP Expression

In Vivo

Collagen, a predominant component of the skin’s

connective tissue, regulates functional properties of skin in

the dermis [9]. In the present study, UVB markedly decreased

collagen in the UVB-irradiated mice, as indicated by the

decreased hydroxyproline contents (total collagen levels)

(Fig. 3A) and the reduced blue-stain section (Fig. 3B),

compared with that in the normal mice, whereas SGE

treatment significantly increased collagen synthesis. The

Fig. 2. Effect of Siegesbeckia glabrescens extract (SGE) treatment on wrinkle formation, skin thickness, and skin elasticity in vivo.

Mice received SGE (600 mg/kg/day) for 13 weeks by oral gavage and were concurrently exposed to UVB irradiation on their dorsal surface. (A)

Wrinkle formation. (B) Wrinkle value. (C) Skin thickness. (D) Skin elasticity. Data represent the mean ± SD from five mice per group. ##p < 0.01

(Normal vs. UVB control) and **p < 0.01 (UVB control vs. SGE-treated mice).

246 Kim et al.


mRNA expression of COL1A1, COL3A1, COL4A1, and

COL7A1 was remarkably elevated in the SGE-treated mice

compared with that in the control group (Fig. 3C). UVB

irradiation increased the protein levels of MMP-2 and -9

and the mRNA levels of MMP-3 and -13, in the control

group, whereas SGE treatment inhibited MMP protein and

mRNA expression (Figs. 3D and 3E). Most collagen fibers

in the dermis primarily consist of type I and III collagens,

which are degraded by MMP-3 and -13. Type VII collagen,

which is crucial in forming the dermal epidermal junction,

is cleaved by MMP-3 [4, 9]. In contrast, types IV and VII

collagens are degraded by MMP-2 and -9 [9]. SGE greatly

suppressed MMP-3 expression in UVB-damaged skin of

hairless mice. MMP-3 is a known activator of other MMPs,

such as MMP-1, -2, and -9 [23], suggesting that SGE has the

potential to inhibit the activation of downstream MMPs.

Thus, the protective effect of SGE against the collagen

degradation may be regulated by the suppression of MMP

expression in UVB-irradiated skin tissue.

Effect of SGE on Catalase and the MAPK/NF-κB Pathways

In Vivo

Accumulation of intracellular ROS by UVB irradiation

causes inactivation of major antioxidant enzymes, such as

catalase, glutathione peroxidase, and superoxide dismutase

[24, 25]. UVB irradiation reduced catalase expression in the

UVB-irradiated control mice, whereas SGE treatment

significantly increased the expression of catalase (Fig. 4A).

The antioxidant enzyme catalase is a key differentiation

marker in the epidermal layer of UVB-induced oxidative

skin, playing a preventive role against photooxidative stress

[9, 26]. Thus, SGE may act as an effective ROS scavenger by

increasing the catalase expression.

This study showed that the SGE treatment group

Fig. 3. Effect of Siegesbeckia glabrescens extract (SGE) treatment on collagen synthesis and MMP expression in vivo.

Mice received SGE (600 mg/kg/day) for 13 weeks by oral gavage and were concurrently exposed to UVB irradiation on their dorsal surface. (A)

Hydroxyproline content. (B) Collagen fibers. (C) mRNA expression of collagen synthesis gene assessed by RT-PCR. (D) Protein expression of

MMP-2 and -9 assessed by western blotting. (E) mRNA expression of MMP-3 and -13 assessed by RT-PCR. β-Actin was utilized as a loading

control for mRNA levels and α-tubulin for protein levels. Data represent the mean ± SD from five mice per group. ##p < 0.01 (Normal vs. UVB

control) and **p < 0.01 (UVB control vs. SGE-treated mice).



Fig. 4. Effect of Siegesbeckia glabrescens extract (SGE) treatment on catalase and the MAPK/NF-κB pathways in vivo.

Mice received SGE (600 mg/kg/day) for 13 weeks by oral gavage and were concurrently exposed to UVB irradiation on their dorsal surface. The

protein expression of (A) catalase, (B) p-ERK, t-ERK, p-JNK, t-JNK, p-p38, and t-p38, (C) p-c-Jun, t-c-Jun, c-Fos, and (D) NF-κB was assessed by

western blotting. α-Tubulin was utilized as a loading control for protein levels. Data represent the mean ± SD from five mice per group. ##p < 0.01

(Normal vs. UVB control) and **p < 0.01 (UVB control vs. SGE-treated mice).

Fig. 5. Effect of kirenol treatment on collagen synthesis and MMP expression in vitro.

Human dermal fibroblasts were pretreated with or without kirenol for 24 h. The cells were exposed to UVB light (25 mJ/cm2) and then additionally

treated with or without kirenol for 24 h. (A) mRNA expression of COL1A1, COL3A1, and COL7A1 assessed by RT-PCR. (B) Protein expression of

MMP-1, -2, and -9 assessed by western blotting. GAPDH was utilized as a loading control for mRNA levels and α-tubulin for protein levels. Data

represent the mean ± SD from triplicate experiments. ##p < 0.01 (Normal vs. UVB control); *p < 0.05 and **p < 0.01 (UVB control vs. kirenol-treated

cells).

248 Kim et al.


demonstrated lower UVB-induced phosphorylation of

MAPK components, including ERK, JNK, and p38, than the

control group (Fig. 4B). Furthermore, the SGE treatment

decreased the protein levels of AP-1 complex components,

such as c-Jun and c-Fos, compared with the UVB-irradiated

control (Fig. 4C). Wrinkle formation is in close relation

with MMP production regulated by c-Jun and c-Fos, which

are components of the AP-1 complex. Activated JNK

simulates the transcriptional activity of c-Jun through

phosphorylation [22].

SGE dramatically suppressed the UVB-induced inflammatory

pathway by downregulating NF-κB expression (Fig. 4D).

UVB-induced inflammation is another mechanism by which

the skin photoages. It is well evidenced that oxidative

stress induces NF-κB associated with MMP expression in

the skin [9]. It was reported that S. glabrescens, which has

been long used as an herbal medicine to treat inflammatory

diseases, inhibited inducible nitric oxide synthase and

cyclooxygenase-2 expression in lipopolysaccharide-activated

RAW 264.7 macrophages [27]. Accordingly, it is conceivable

that SGE prevents UVB through activation of the MAPK

and NF-κB pathways.

Effect of Kirenol on Collagen Synthesis and MMP

Expression In Vitro

Collagen degradation in UV-damaged skin is a result of

the upregulation of MMP expression, leading to an aged

appearance of skin [28]. In the present study, UVB reduced

the mRNA levels of COL1A1, COL3A1, and COL7A1,

whereas kirenol significantly increased the expression of

these collagen genes in Hs68 human dermal fibroblasts

(Fig. 5A). UVB increased the protein levels of MMP-1, -2,

and -9; however, kirenol reduced these levels dose-

dependently (Fig. 5B). Because MMPs are crucial in the

pathophysiological photoaging mechanisms of skin, natural

biomaterials inhibiting MMP expression in UV-damaged

Fig. 6. Effect of kirenol treatment on catalase and the MAPK/NF-κB pathways in vitro.

Human dermal fibroblasts were pretreated with or without kirenol for 24 h. The cells were exposed to UVB light (25 mJ/cm2) and then

additionally treated with or without kirenol for 24 h. The protein expression of (A) catalase, (B) p-ERK, t-ERK, p-JNK, t-JNK, p-p38, and t-p38, (C)

p-c-Jun, t-c-Jun, c-Fos, and (D) NF-κB was assessed by western blotting. (E) mRNA expression of IL-6 and IL-8 assessed by RT-PCR. GAPDH was

utilized as a loading control for mRNA levels and α-tubulin for protein levels. Data represent the mean ± SD from triplicate experiments. ##p < 0.01

(Normal vs. UVB control); *p < 0.05 and **p < 0.01 (UVB control vs. kirenol-treated cells).



skin can successfully prevent photoaging [9, 22]. Thus,

kirenol can mediate its anti-photoaging activity by promoting

collagen synthesis and inhibiting MMP expression.

Effects of Kirenol on Catalase and MAPK/NF-κB Pathways

In Vitro

Protein expression of catalase was reduced after UVB

irradiation; however, catalase was highly elevated after

kirenol treatment, indicating that kirenol has strong

antioxidant activity (Fig. 6A). These data suggest that

kirenol may mediate protection against photoaging, in

part, by upregulation of catalase. As shown in Fig. 6B,

kirenol reduced the phosphorylation of ERK, JNK, and p38

induced by UVB. The protein levels of c-Jun and c-Fos were

markedly upregulated in the control cells, whereas kirenol

significantly decreased the expression of c-Jun and c-Fos

(Fig. 6C). The UVB-irradiated control cells expressed high

levels of inflammatory mediators (NF-κB, IL-6, and IL-8);

however, kirenol effectively inhibited their expression

dose-dependently (Figs. 6D and 6E). These data demonstrate

that kirenol protects fibroblasts from UVB-induced

inflammation. It was reported that kirenol was also found

to confer anti-inflammatory activity, as it reduced

proinflammatory cytokine secretion and increased anti-

inflammatory cytokine production of type II collagen-

specific lymphocytes from collagen-induced arthritic rats

[20]. Thus, kirenol inhibits the activation of the MAPK/NF-

κB pathways and prevents UVB-induced inflammation in

Hs68 human dermal fibroblasts.

This study was the first to demonstrate that SGE, and its

active compound kirenol, prevents UVB-induced photoaging

by regulating MMP expression and collagen degradation, in

addition to suppressing oxidative stress and the associated

intracellular MAPK/NF-κB pathways. It is anticipated that

S. glabrescens and the active compound kirenol can be

employed as effective natural anti-photoaging agents. Further

human studies are required to clarify whether SGE is

clinically effective as a natural anti-photoaging supplement.

Acknowledgment

This work was supported in partly by the World Class

300 Project R&D Program (S2435140) funded by the Small

and Medium Business Administration (SMBA, Republic of

Korea).

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