Food and Chemical Toxicology 50 (2012) 4260–4269

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Food and Chemical Toxicology

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Michelia alba extract attenuates UVB-induced expression of matrix

metalloproteinases via MAP kinase pathway in human dermal fibroblasts
Hsiu-Mei Chiang, Hsin-Chun Chen, Tsen-Jung Lin, I-Chen Shih, Kuo-Ching Wen ⇑
Department of Cosmeceutics, China Medical University, Taichung, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: Solar ultraviolet (UV) radiation can cause skin photoaging by inducing secretion of matrix metallopro-
Received 1 March 2012 teinases (MMPs). It has been reported that MMPs, especially MMP-1, -3 and -9, reduce elasticity of the
Accepted 9 August 2012 dermis by degrading collagen. Polyphenols are a group of compounds that exist mainly in glycosides
Available online 17 August 2012
in the plants and they may transform to aglycone after hydrolysis. Polyphenols can inhibit MMP expres-
sion and elastase activity. In this study, we investigated the effects of Michelia alba extract (MAE) on
Keywords: expression and activity of MMPs in human skin fibroblast cultures after UVB exposure. The results
Michelia alba
showed that MAE and its hydrolysates (MAH) inhibited collagenase and elastase activities. In addition,
Matrix metalloproteinase
ERK
MAE exhibited antioxidant activity, elevated hyaluronic acid content and inhibited UVB-induced MMP-
JNK 1, MMP-3 and MMP-9 expression. In addition, the zymography assay revealed that MAE also inhibited
Photoaging MMP-9 activity. We also found that MAE inhibited UVB-induced ERK and JNK kinase but not p38 kinase
Type I procollagen expression, suggesting that MAE may regulate the UVB-induced expression of MMP-1, MMP-3 and MMP-
9 via the ERK and JNK kinase pathway. MAE could restore total collagen synthesis reduced by UVB. The
results also suggest that MAE treatment may prevent UVB-induced extracellular matrix damage by inhib-
iting the expression of MMP-1, MMP-3 and MMP-9 through the MAP kinase pathway. Our findings imply
that MAE is an effective agent against UVB-induced photodamage.
Ó 2012 Published by Elsevier Ltd.

1. Introduction D-glucosamine with retaining water, stimulating collagen synthe-

Wrinkles, laxity, and hyperpigmentation are characteristics of
aging (Jenkins, 2002). Skin aging can be divided into two basic pro-
cesses, intrinsic and extrinsic aging. Extrinsic aging is generally re-
ferred to as photoaging and is characterized by severe wrinkling
and pigmentary changes, such as solar lentigo and mottled pig-
mentation on exposed areas such as the face, neck, and forearm.
The most abundant structural protein in skin connective tissue is
type I collagen, which is synthesized primarily by fibroblasts and
is responsible for conferring strength and resiliency (Gelse et al.,
2003). Besides collagen, hyaluronic acid is associated with skin
aging. Hyaluronic acid is an extracellular matrix glycosaminogly-
can polysaccharide composed of D-glucuronic acid and N-acetyl-
Abbreviations: AAPH, 2,20 -azobis dihydrochloride; DPPH, 2,2-diphenyl-1-pic-
rylhydrazyl; ECM, extracellular matrix; MAE, Michelia alba extracts; MAH, hydroly-
sates of MAE; MAP kinase, mitogen-activated protein kinase; MMP,
metalloproteinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide; UV, ultraviolet.
⇑ Corresponding author. Address: Department of Cosmeceutics, China Medical
University, 91 Hsueh-Shih Road, Taichung 404, Taiwan. Tel.: +886 4
22053366x5302; fax: +886 4 22078083.
E-mail address: kcwen0520@mail.cmu.edu.tw (K.-C. Wen).
sis, repairing tissue and maintaining intercellular space activities
(Andre, 2004; Miyazaki et al., 2002; Nawrat et al., 2005). Hyalu-
ronic acid is synthesized by keratinocytes and fibroblasts, and it
will decrease as age increasing and then causes wrinkles and a de-
crease in skin elasticity and thickness (Longas et al., 1987).
UV-induced skin damage principally manifests as degradation
of extracellular matrix (ECM) proteins, including type I collagen,
elastin, proteoglycans, and fibronectin (Fisher et al., 1997; Rittie
and Fisher, 2002). The disarrangement and fragmentation of those
proteins by UV irradiation accelerate the skin aging process. UV
irradiation leads to the formation of reactive oxygen species
(ROS), up-regulates the mitogen-activated protein (MAP) kinase
signaling pathway, and induces the expression of matrix metallo-
proteinases (MMPs), namely MMP-1, -3, and -9 in human skin
in vivo (Fisher et al., 1996; Rittie and Fisher, 2002; Shin et al.,
2005). It is generally believed that MMP-mediated collagen
destruction is the major cause of the connective tissue damage that
occurs in photoaging. Thus, preparations from natural products or
compounds with the ability to stimulate collagen synthesis or inhi-
bit the major collagen-degrading enzymes, namely MMPs, would
be beneficial in the development of a useful anti-aging agent.
Magnolia officinalis, Magnolia obovata, Michelia alba (MA), and
Michelia champaca, members of the Magnoliaceae family of

0278-6915/$ - see front matter Ó 2012 Published by Elsevier Ltd.

http://dx.doi.org/10.1016/j.fct.2012.08.018
 H.-M. Chiang et al. / Food and Chemical Toxicology 50 (2012) 4260–4269 4261

flowering plants, are well known for their aroma constituents and
bioactivities. For example, M. obovata and M. officinalis are used in
Chinese medicine as anti-inflammatory agents to treat cramps and
abdominal pain (Lee et al., 2005). In addition, M. obovata and its ac-
tive components magnolol and obovatol have been shown to pre-
vent photoaging by inhibiting the expression of MMPs (Choi et al.,
2007; Tanaka et al., 2007). Flowers of M. alba and M. champaca are
aromatics and a source of essential oils. Dichloromethane extracts
of M. champaca have been shown to possess anti-microbial activity
against Salmonella enteritidis, Enterobacter cloacae, and Bacillus sub-
tilis (Rangasamy et al., 2007). In addition, fractions of the leaves of
that species of Magnoliaceae exhibit a broad spectrum of antibac-
terial and antifungal activities (Khan et al., 2002). The flower of
MA, with its pleasant aroma and flavor, has been shown to be
effective in the treatment of bronchitis and prostatitis (Shang
et al., 2002). The bark of MA was used for fever, syphilis and malar-
ia (Asaruddin et al., 2003). Constituents of MA leaves include
aporphines, oxoaporphines, sesquiterpenes, terpenes, benzenoids,
steroids, and lignans as well as some aliphatic compounds such
as palmitic acid, stearic acid, and linoleic acid (Chen et al., 2008).
One of the constituent, michephyll A, exhibited antioxidant activity
(Wang et al., 2010). Polyphenols such as epigallocatechin-3-gallate
(EGCG) (Bae et al., 2008) and lignans such as macelignan (Ang-
gakusuma et al., 2010) have been shown to inhibit photoaging by
preventing the expression of MMPs. In our previous study, we
found that polyphenol-rich extracts of Coffea arabica (Chiang
et al., 2011b) and Terminalia catappa L. (Wen et al., 2012) protected
against anti-photoaging by inhibiting the expression of MMPs and
MAP kinases. In addition, in our preliminary study, the DPPH scav-
enging assay revealed that M. alba leaves extract (MAE) was an
effective antioxidant. Therefore, we hypothesized that MAE pre-
vent against photoaging and photocarcinogenesis by inhibiting
UV-induced ROS generation. Polyphenols exist in plants mainly
in glycosides form and they may be hydrolyzed by acid or bacteria
to more hydrophobic form, aglycone. Aglycones may be more skin
penetration than glycoside.
The aim of this study was to investigate whether MAE and
their hydrolysates (MAH) can attenuate UVB-induced expression
of MMPs, elastase, hyaluronic acid and type I procollagen in
human dermal fibroblasts. We found that MAE and their

A 140
MAE

120

100
Inhibition (%)

80

60

40

20

0
control Ascorbic 12.5 100 500 1000 2000 (µg/mL)

B 120
1.5 h
2h
100 3h
4h

80
Inhibition (%)

60

40

20

-20
0 10 50 100 500 (µg/mL)

Concentration
Fig. 1. The antioxidant activities of MAE. (A) DPPH radical scavenging activity of Michelia alba extracts. (B) Time–course study demonstrating the inhibitory effect of MAE on
AAPH-induced lysis of rat erythrocytes. MAE inhibited erythrocyte hemolysis in a dose-dependent manner (50–500 lg/mL). (n = 4; ⁄p < 0.05; ⁄⁄p < 0.01;⁄⁄⁄p < 0.001).
4262 H.-M. Chiang et al. / Food and Chemical Toxicology 50 (2012) 4260–4269

hydrolysates were potent antioxidants and that they protected by Bio-Rad Laboratories (CA, USA). Elastase substrate IV and porcine elastase were
purchased from Calbiochem (San Diego, CA, USA). Human foreskin fibroblasts were
dermal fibroblasts against photo-damage by suppressing the
obtained from the Bio-resource Collection and Research Center (Hsinchu, Taiwan).
expression of MMPs and by inhibiting the activation of MAP
kinase pathways.
2.2. Preparation of MAE and MAH

2. Materials and methods
2.1. Chemicals
The leaves of MA were harvested in Changhua County, Taiwan. 2,20 -Azobis dihy-
drocholoride (AAPH), 2,2-diphenyl-1-picrylhydrazyl (DPPH), gelatin, agarose, hydro-
chloric acid, methanol, dimethyl sulfoxide (DMSO), doxycycline hyclate, calcium
chloride (CaCl2), and DL-dithiothreitol were purchased from Sigma–Aldrich Chemicals
(St. Louis, MO, USA). Fetal bovine serum (FBS), penicillin–streptomycin, trypsin–EDTA,
and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Gibco, Invit-
rogen (Carlsbad, CA, USA). Coomassie blue R-250, dibasic sodium phosphate, lgepalTM
CA-630, tris and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) were purchased from USB (Cleveland, OH, USA). Collagenase was purchased
from Calbiochem, Merck (Darmstadt, Germany). Fluorogenic Peptide Substrate I was
purchased from R&D systems (Wiesbaden, Germany). Bradford Reagent was supplied
The dried leaves were ground and then extracted twice with a 30-fold volume
of methanol for 1 h by ultrasonication. The supernatant was filtered and then the
filtrate was evaporated to dryness in vaccuo to obtain MAE. Acid hydrolysis of
MAE was carried out at 80 °C in the presence of 1.2 N HCl (2 mL) for 30 min and
2.4 N HCl (2 mL) for 60 min. After hydrolysis, the solution was partitioned with
ethyl acetate (EA). The EA layer was evaporated to dryness in vacuo. The abbrevia-
tions and hydrolytic conditions of MAH are as follows: MAH1, 1.2 N HCl, 0.5 h;
MAH2, 1.2 N HCl, 1 h; MAH3, 2.4 N HCl, 0.5 h; MAH4, 2.4 N HCl, 1 h. MAE and MAHs
were stored at 20 °C before use.
2.3. Total phenolic content of MAE
Total phenolic content was determined by the Folin–Ciocalteu reaction as pre-
viously reported with slight modifications (Chiang et al., 2011a,b). In brief, a 100-lL
aliquot of MAE was mixed with 200 lL of 10% Folin–Ciocalteu phenol reagent. The
mixture was allowed to stand at room temperature for 5 min and then 800 lL so-

*** *** *** ***

100 *** ***

80
Inhibition (%)

60

40

20

0
PG DC100 MAE MAH1 MAH2 MAH3 MAH4 (1000 µ g/mL)

MAE
***
100 *** ***

80
Inhibition (%)

60
***
***
40

20

0
PG DC100 50 100 500 1000 µ g/mL

Fig. 2. The inhibition of collagenase activity in cells exposed to MAE and MAH. (A) MAE and MAH 1000 lg/mL (B) MAE 50–1000 lg/mL (MAH 1: hydrolyzed by 1.2 N HCl for
30 min; MAH 2: 1.2 N HCl for 60 min; MAH 3: 2.4 N HCl for 30 min; MAH 4: 2.4 N HCl for 60 min) (⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001).
 H.-M. Chiang et al. / Food and Chemical Toxicology 50 (2012) 4260–4269 4263

dium carbonate (700 mM) was added to the mixture. The resulting blue complex
was then measured at 760 nm. Gallic acid was used as a standard for the calibration
curve. The phenolic compound contents were calibrated using a linear equation
based on the calibration curve. The contents of phenolic compounds are expressed
as lg gallic acid equivalent/mg MAE dry weight.
2.4. The antioxidant effects of MAE
2.4.1. DPPH radical scavenging activity
Reaction mixtures containing a methanolic solution of 200 lM DPPH (100 lL)
and serial dilutions of sample (concentration of sample ranging from 25 to
1000 lg/mL) were placed in a 96-well microplate at room temperature in the dark
for 30 min. After incubation, the absorbance was read at 492 nm by an ELISA reader.
Ascorbic acid was used as a positive control. Scavenging activity was determined by
the following equation:
2.6. Cell culture
Human foreskin fibroblasts (Hs68) were maintained in DMEM supplemented
with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin in
a humidified atmosphere of 5% CO2 at 37 °C. Before being passaged, Hs68 cells were
cultured until they reached 90% confluence.
2.7. Cell viability test
The fibroblasts were plated at a density of 104 cells/well in 96-well plates and
then treated with various concentrations of extracts dissolved in DMSO (<0.1%)
for 24 h. Mitochondrial dehydrogenase activity, which can be used as an index of
cell viability, was assessed using the MTT assay as previously described (Chiang
et al., 2011a,b). Viability was quantified by measuring the absorbance at 570 nm
using a microplate reader (Tecan, Grodig, Austria).

% scavenging activity ¼ ½1  ðAsample =Acontrol Þ  100

2.8. Quantitative determination of hyaluronic acid

2.4.2. Preparation of erythrocyte suspensions and hemolysis assay MAE was incubated with cells in a 96-well plate for 48 h, after which 100 lL
Whole blood was obtained from male SD rats via cardiopuncture and collected medium from each well was taken for hyaluronic acid measurement. The quantity
in EDTA-containing tubes as previously described (Chiang et al., 2011a,b). This ani- of hyaluronic acid in culture supernatant was determined by Hyaluronan Enzyme-
mal study adhered to The Guidebook for the Care and Use of Laboratory Animals Linked Immunosorbent Assay Kit (HA-ELISA) according to the manufacturer’s
(Published by The Chinese Society for Laboratory Animal Science, Taiwan). In order instruction (Echelon Biosciences Inc., UT, USA). The amount of hyaluronic acid
to induce free radical chain oxidation in the erythrocytes, aqueous peroxyl radicals (HA) was calculated using the formula [(HAsample/HAcontrol)]  100.
were generated by thermal decomposition of AAPH in oxygen. An erythrocyte sus-
pension at 5% hematocrit was incubated with PBS (control) or preincubated with 2.9. UV irradiation
MAE (10–500 lg/mL) at 37 °C for 30 min, followed by incubation with or without
25 mM AAPH in PBS at pH 7.4. The reaction mixture was shaken gently while being Cells were cultured until 80% confluent, washed twice with phosphate-buffered
incubated for a fixed interval at 37 °C. A 200-lL aliquot of the reaction mixture was saline (PBS), and then exposed to UVB irradiation (302 nm, CL-1000M, UVP, USA). In
removed and centrifuged at 3000g for 2 min, with absorbance of the supernatant our previous study, an 80 mJ/cm2 dose of UVB irradiation was determined to induce
determined at 540 nm. Reference values were determined using the same volume
of erythrocytes in a hypotonic buffer (5 mM phosphate buffer at pH 7.4; 100%
MAE
hemolysis). The percentage of hemolytic cells was calculated using the formula
MAH1
[(Asample/Acontrol)]  100. MAH2
MAH3
MAH4
2.5. Screening of MMPs and elastase inhibition
A 100
2.5.1. Gelatin digestion assay
Agarose solution (1%) was prepared in collagenase buffer with 0.15% porcine
gelatin (Sigma Aldrich, Cat. G-2500) and allowed to solidify. Various concentrations 80
of MAE and MAH (10 lL) dissolved in 50% propylene glycol (PG) were incubated
Inhibition (%)

with 10 lL of bacterial collagenase-1 (0.1 mg/mL) in 80 lL of collagenase buffer

for 1 h at room temperature. The samples (40 lL) were loaded onto paper disks, 60
placed on gelatin-agarose gel, and incubated for 18 h at 37 °C. The degree of gelatin
digestion in agarose gel was determined by the intensity of Coomassie Blue staining
after removal of the paper disks. Following destaining, the area of the light translu- 40
cent zone over the blue background was determined by a densitometric program to
estimate gelatinase activity. Doxycycline hyclate was used as a positive control.
20
2.5.2. Fluorescent MMP activity assays
Enzyme activity assays were performed in 50 mM tris buffer (pH 7.8), 0.15 M
NaCl, and 10 mM CaCl2. Various concentrations of MAE and MAH were tested for
0
their ability to digest a synthetic fluorogenic substrate (a general MMP substrate). 10 50 100 500 µg/mL)
(µ
Each concentration of MAE and MAH was incubated with 1 lM substrate at 37 °C
for 20 h. Fluorescence intensity was measured at 328 nm (excitation) and 393 nm
(emission) with a fluorescence reader. The rate of collagenase inhibition was calcu-
B MAE
MAH1
lated by the following equation: 30 *** MAH2
  MAH3
Acontrol  Asample MAH4
Inhibitionð%Þ ¼  100 ***
Acontrol 25

2.5.3. Measurement of elastase activity 20

Inhibition (%)

Elastase inhibition was investigated using elastase from porcine pancreas. Elas-
tase (500 U) was dissolved in 5 mL of 10 mM tris buffer solution (pH 6.0) and 5 mg
elastase substrate IV was dissolved in 5 mL of 100 mM tris buffer solution (pH 8.0). 15
**
To measure elastase activity, 100 lL of 100 mM tris buffer solution (pH 8.0), 25 lL **
of elastase substrate IV solution, 50 lL of sample solution, and 25 lL of elastase 10
solution were dispensed into each well of a 96-well plate and then incubated for
20 min at room temperature. Elastase activity was quantified by measuring light
absorbance at 405 nm by a microplate reader (Tecan, Grodig, Austria). Each assay 5
was carried out in triplicate.
The inhibition rate of elastase was calculated by the following equation:
0
ðC  DÞ 10 50 100 500 (µg/mL)
Inhibitionð%Þ ¼ 1   100
ðA  BÞ
Fig. 3. (A) The inhibition (%) of bacterial collagenase activity in cells exposed to
where A indicates the absorbance with enzyme but without sample, B indicates the MAE and MAH. MAE and MAH significantly inhibited the activity of bacterial
absorbance without enzyme and sample, C indicates the absorbance with enzyme collagenase-1. (B) The inhibition (%) of porcine elastase activity in cells exposed to
and sample, and D indicates the absorbance without enzyme but with sample. MAE and MAH. (⁄⁄P < 0.01; ⁄⁄⁄P < 0.001).
4264 H.-M. Chiang et al. / Food and Chemical Toxicology 50 (2012) 4260–4269
the expression of MMPs without being cytotoxic (Chiang et al., 2011a,b). Subse-
quently, cells were incubated for 24 h at 37 °C in a humidified atmosphere of 5%
CO2 in serum-free DMEM containing various concentrations of MAE and MAH.
2.10. Western blotting for MMPs, MAP kinase, and type I procollagen
Cells were harvested and homogenized with lysis buffer. All reactions were per-
formed in triplicate. Protein concentration in the culture medium was measured
using Bradford reagent (Bio-Rad, Hercules, CA, USA) with bovine serum albumin
as the standard. Cell lysates containing equal amounts of total protein were sepa-
rated by electrophoresis on SDS–polyacrylamide gel and then transferred to a PVDF
membrane (Hybond ECL, Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA).
Non-specific binding was blocked using non-fat milk in TBST. The membrane was
incubated with goat polyclonal antibodies against MMP-1 (1:500) and type I procol-
lagen (1:500), and mouse polyclonal antibodies against MMP-3 (1:500), MMP-9
(1:500), ERK (1:500), JNK (1:500), p38 (1:500), p-ERK (1:500), p-JNK (1:500) and
p-p38 (1:500) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Anti-immuno-
globulin G-horseradish peroxidase (Santa Cruz Biotechnology Inc.) was used as the
secondary antibody. Immunoreactive proteins were detected with the ECL Western
blotting detection system (Fujifilm, LAS-4000, Japan). Signal strengths were quanti-
fied using a densitometric program (multi Gauge V2.2).
2.11. Zymography for MMP-9
Cell-free medium (30 lL) after UVB exposure was mixed with tris–glycine SDS
sample buffer without reducing agent and electrophoresed. After electrophoresis,
the gels were incubated with zymogram renaturing buffer (Invitrogen, USA) for
30 min at room temperature. Zymogram developing buffer (Invitrogen, USA) was
then added to the gel. After incubation of the gels at 37 °C for 24 h, the gels were
stained with 0.05% Commassie Blue G-250 and destained in 10% acetic acid and
40% methanol until proteinase bands were clearly visible in a blue background.
The area of light translucent zones over the blue background was determined by
a densitometric program to estimate gelatinase activity.
2.12. Measurement of total collagen synthesis
Total collagen synthesis in fibroblasts after UVB exposure was measured by the
Sircol™ soluble collagen assay kit (Biocolor Ltd., UK) according to the manufac-
turer’s protocol. Briefly, cell culture medium was mixed with Sircol dye reagent
and incubated at room temperature for 30 min. After centrifugation, ice-cold
acid-salt washing reagent was added to the precipitate and then centrifuged. The
precipitate was dissolved with Alka reagent and the absorption was determined
at 555 nm by ELISA reader (Tecan, Austria).
2.13. Statistical analysis
Each experiment was performed in triplicate and all data are presented as
mean ± SD. Significant differences between groups were analyzed by ANOVA fol-
lowed by the Scheffe’s test. A p-value <0.05 was considered significant.
140
*
120 **
* *
* *
Cell viability (% of control)
* * **
* collagenase-1 for 20 h in the presence of different concentrations
100
80
60
40
20
0
µg/mL)
0 1 10 100 1000 (µ
Fig. 4. Cell viability (%) of human fibroblasts exposed to MAE and MAH. (⁄P < 0.05;
⁄⁄
P < 0.01).
3. Results
3.1. Extraction yield and total phenolic content of MA
The extraction yield of MA was about 16.9%. The total phenolic
content, expressed as lg gallic acid equivalents per mg of dry
weight (MA leaves), was 2.7 ± 0.2 lg/mg.
3.2. Antioxidant effects of MAE
3.2.1. Scavenging of DPPH radicals
The free radical scavenging activities of MAE (100–5000 lg/mL)
and ascorbic acid (12.5 lg/mL) are shown in Fig. 1A; MAE exhib-
ited DPPH radical scavenging activity in a dose-dependent manner.
3.2.2. Erythrocyte hemolysis assay
The influence of MAE (10–500 lg/mL) on in vitro erythrocyte
hemolysis was examined by incubating rat erythrocytes in the
presence of 25 mM AAPH. As shown in Fig. 1 B, MAE (50–500 lg/
mL) exhibited a strong dose-dependent inhibitory effect against
erythrocyte hemolysis.
3.3. Screening of MMPs and elastase inhibition
MMPs degrade extracellular matrix proteins such as collagen
and elastin. This study screened for the activity of MMPs using a
gelatin digestion assay and fluorometric analysis of bacterial colla-
genase-1.
3.3.1. MAE and MAH inhibit bacterial collagenase-1 (gelatin digestion
assay)
An indirect assay was developed using bacterial collagenase-1 to
visualize the inhibitory effect of MAE and MAH on expression of
MMPs. Following incubation of bacterial collagenase-1 with various
concentrations of MAE and MAH, the inhibition of enzyme activity
was compared with enzyme activity of the control. As shown in
Fig. 2, MAE and MAH inhibited gelatinolytic activity in a dose-depen-
dent manner. The rates of collagenase-1 inhibition were 97.3 ± 0.02%
for doxycycline (100 lg/mL), 0.4 ± 0.92% for 50% PG (blank),
100.0 ± 0.03% for MAE (1000 lg/mL), 99.3 ± 0.13% for MAH1,
99.1 ± 0.14% for MAH2, 99.2 ± 0.12% for MAH3, and 93.7 ± 0.40% for
MAH4.
MAE 3.3.2. MAE and MAH inhibit bacterial collagenase-1 (fluorometric
MAH1 assay)
MAH2
MAH3 Fluorescence-conjugated gelatin was used to measure the
MAH4 inhibitory effect of MAE on bacterial collagenase-1 activity.
Fluorescence-conjugated substrate was incubated with bacterial
of MAE, MAH, or doxycycline hyclate (positive control) at 37 °C.
MAE and MAH significantly inhibited the activity of bacterial colla-
genase-1. As shown in Fig. 3A, treatment with 10 lg/mL of MAE
decreased the activity of bacterial collagenase-1 by 93%. MAH
preparations also showed a concentration-dependent inhibitory
effect (range, 10–500 lg/mL).
3.3.3. The effect of MAE on elastase activity
This assay measures the synthesis and activity of elastase in
cells exposed to MAE. As shown in Fig. 3B, the effect of MAE and
MAH2 (1.2 N HCl, 1 h) on elastase activity was significant at
500 lg/mL; the effect of MAH4 (2.4 N HCl, 1 h) on elastase activity
was significant at P500 lg/mL.
 H.-M. Chiang et al. / Food and Chemical Toxicology 50 (2012) 4260–4269 4265

3.4. Cytotoxicity of MAE and MAH on Hs68 cells of cells at concentrations P10 lg/mL. The effect of MAH1 was sim-
ilar to that of MAE. In addition, MAH3 and MAH4 significantly stim-
A MTT assay was performed to determine whether MA prepara- ulated cell growth (120%) at concentrations >100 lg/mL.
tions are cytotoxic. Hs68 cells were treated with various concentra-
tions of MAE and MAH and cell viability was measured. As shown in 3.5. MAE and MAH inhibit MMP expression
the survival curve in Fig. 4, MAE (1–1000 lg/mL) and MAH (1–
1000 lg/mL) did not inhibit the proliferation of cells (cell viability UVB irradiation of untreated Hs68 cells up-regulated the
>90% of controls). Furthermore, MAE promoted the proliferation expression of MMPs; however, MAE treatment suppressed the

C MAE
2.5 MMP-3

Relative density (of control)

2.0
A
* **
1.5

1.0

0.5

0.0
control UV 5 10 25 (µg/mL)

B MAE D MAE
MMP-1 MMP-9
2.5 5

*
Relative density (of control)
Relative density (of control)

2.0 4
**
*
1.5 3 ***

***
1.0 2

0.5 *** 1

0.0 0
control UV 5 10 25 µg/mL)
(µ control UV 5 10 25 (µg/mL)

E MAE
1.2 Type I procollagen

1.0
Relative density (of control)

0.8

0.6

0.4

0.2

0.0
control UV 5 10 25 (µg/mL)

Fig. 5. Effects of MAE on UVB-induced expression of MMP-1, -3, and -9 and type I procollagen in human fibroblasts. (A) Western blot, (B) MMP-1, (C) MMP-3, (D) MMP-9, (E)
type I procollagen, (F) Zymography assay showing the effect of MAE on MMP-9 activity in human fibroblasts, (G) Effect of MAE on total collagen synthesis, and (H) effect of
MAE on relative amount of hyaluronic acid in Hs68 cells. (⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001).
4266 H.-M. Chiang et al. / Food and Chemical Toxicology 50 (2012) 4260–4269

G
60
*
*

Total collagen (mg/mL)

40
F

20

0
MAE
350 MAE 0 0 10 25 50 100 200 (µg/mL)
UVB 0 80 80 80 80 80 80 (mJ/cm2)
300
***
H MAE
250 140
*
Activity (%)

Relative amount of hyaluronic acid (%)

200 120 *
***
150 100

100 80

50 *** 60
***

0 40
no UV UV 5 10 50 µg/mL)
100 (µ
20

0
0 25 50 100 200 (µg/mL)

Fig. 5 (continued)

UVB-induced up-regulation of MMPs (Fig. 5A–E). MAE at concen-
trations of 10 lg/mL and higher significantly decreased the expres-
sion of MMP-1 and -3 to basal levels (Fig. 5A–C). Treatment with
5 lg/mL MAE resulted in a 1.5-fold decrease in MMP-9 expression
relative to untreated UVB-exposed cells (Fig. 5A and D).
3.9. MAE elevated hyaluronic acid content
MAE showed a stimulation effect on hyaluronic acid production
in dose dependent manner, and the content of hyaluronic acid was
significant increase after high concentration of MAE (>100 lg/mL)
treatment (Fig. 5H).

3.6. MAE and MAH inhibit MMP-9 activity

3.10. Effect of MAE on MAP kinase expression

UVB irradiation of untreated Hs68 cells led to the activation of

The inhibitory effect of MAE (5–25 lg/mL) on UVB-induced
MMP-9; however, treatment with MAE at concentrations higher
MAP kinase expression is shown in Fig. 6. The inhibitory effect of
than 5 lg/mL suppressed the UVB-induced activation of that ma-
MAE (5–25 lg/mL) on JNK phosphorylation was dose-dependent
trix metalloproteinase (Fig. 5F). In addition, the level of MMP-9
(Fig. 6A and B) and the expression level of p-JNK returned to the
in Hs68 cells treated with MAE at concentrations over 50 lg/mL
basal level at 25 lg/mL. In addition, treatment with MAE at a dose
was lower than that in control cells.
of 25 lg/mL suppressed the activation of p-ERK but did not signif-
icantly inhibit the phosphorylation of p38 (Fig. 6A, C and D).

3.7. MAE and MAH did not inhibit type I procollagen expression
Fibroblasts were treated with MAE (5–25 lg/mL) for 24 h after
exposure to UVB (80 mJ/cm2). MAE did not significantly affect the
expression of type I procollagen (Fig. 5A and E).
3.8. MAE restored UVB-reduced total collagen synthesis
Fibroblasts were pretreated with MAE (10–200 lg/mL) for 1 h,
exposed to UVB, and then treated with MAE for 24 h. As shown
in Fig. 5G, MAE treatment resulted in a dose-dependent restoration
of collagen.
4. Discussion
Collagen in skin is predominantly type I collagen (85–90%), which
is synthesized by dermal fibroblasts. UV irradiation stimulates the
synthesis and activation of MMPs, which lead to connective tissue
damage and skin aging (Pillai et al., 2005; Uitto, 2008). The amount
of UVB from sunshine is less than that of UVA, but its intensity is
strong enough to cause photoaging and skin cancer (Rogers and Gil-
chrest, 1990). Aging is a complex phenomenon that is modulated by
multiple factors. Any agent that is capable of inhibiting collagenase
would be a potential therapeutic candidate for preventing photoag-
ing and wrinkle formation (Inomata et al., 2003). The primary MMPs
 H.-M. Chiang et al. / Food and Chemical Toxicology 50 (2012) 4260–4269 4267

MAE
p-ERK
A C 3.5

3.0

Relative density (of control)

2.5

2.0

**
1.5

1.0

0.5

0.0
control UV 5 10 25 (µg/mL)
B MAE D
p-JNK MAE
p-p38
2.0
1.8 5
Relative density (of control)

1.6
Relative density (of control)
4
1.4
*
1.2 **
3
1.0 **

0.8
2
0.6
0.4 1
0.2
0.0 0
control UV 5 10 µg/mL)
25 (µ control UV 5 10 25 (µg/mL)

Fig. 6. Effect of MAE on UVB-induced expression of MAP kinases in human fibroblasts. (⁄P < 0.05; ⁄⁄P < 0.01). Activation of p-ERK and p-JNK, but not p38 phosphorylation, was
suppressed by MAE.

that are expressed in response to UVB irradiation include MMP-1, not exhibited in dose-dependent manner. To further investigate
which initially degrades types I and III fibrillar collagens, MMP-9, the effect of MAE on MMP-9, we measured the MMP-9 activities
which further degrades collagen fragments generated by collagenas- on degrading gelatin by zymography assay. The results clearly
es, and MMP-3, which activates proMMP-1 (Rittie and Fisher, 2002). showed that MAE reduced MMP-9 activities up to 90% and were,
Therefore, topical MMP inhibitors may be effective at preventing in a dose-dependent manner. The activities of MMP family are well
UVB-induced wrinkle formation. known for their inter-connectivities. That is the presence of one
Botanical extracts appear to be effective anti-photoaging and MMP will affects the activities of other MMPs. Previous studies
anti-photocarcinogenic agents. In our previous studies, polyphe- (Toth et al., 2003) indicated that the activity of MMP-9 is regulated
nol-rich extracts of C. arabica (Chiang et al., 2011b) and T. catappa by MMP-2 and MMP-3. Also some post-translational modification
L. (Wen et al., 2012) prevented skin cells from UVB-induced photo- of MMP-9 must occur to obtain its full gelatinase activity
aging by inhibiting the activation of MMPs and MAP kinases. In (Symowicz et al., 2007). Therefore, though there is no dose-depen-
addition, obovatol from Magnolia extract has been shown to inhi- dent manner observed in the amount MMP-9 produced but impor-
bit MMP-3 in UVB-irradiated human fibroblasts (Choi et al., 2007; tantly, there are significant reduced MMP-9 activities detected in a
Tanaka et al., 2007); water extracts of Rosmarinus officinals have dose-dependent manner by zymography assay. Only fully pro-
been shown to inhibit UV-induced MMP-1 expression (Martin cessed MMP-9 with proper gelatinase activity could degrade colla-
et al., 2008); and berberine from Coptis chinensis has been shown gen, therefore, the zymography results were more comparable
to decrease the expression of MMP-1 in UVB-exposed cells (Kim with MMP-9 activity under UV treatment. In addition, our results
and Chung, 2007). It has also been shown that the polyphenol epi- indicate that MAE inhibits the expression of MMPs by suppressing
gallocatechin-3-gallate (EGCG) in green tea inhibits UVB-induced MAP kinase signaling pathways. MAE appears to be a potent MMP
expression of MMP-1, MMP-8, and MMP-13 (Bae et al., 2008), inhibitor, and therefore, an effective anti-photoaging agent. By
and that equol from soy prevents against UV-induced expression inhibiting MMP-1, MAE protects against the degradation of types
of MMP-1 and type I procollagen (Kim et al., 2004a,b; Reeve I and III collagens. In addition, by inhibiting MMP-9, MAE prevents
et al., 2005). In this study, we found that MAE significantly the degradation of collagen fragments generated by MMP-1, and
inhibited the expression of MMP-1 at 10 lg/mL, the expression inhibits the activation of proMMP-1. MAE did not influence the
of MMP-3 at 25 lg/mL, and the expression of MMP-9 at 5 lg/mL expression of type I procollagen, but elevated total collagen syn-
(Fig. 5). The results of western blotting suggested that MAE re- thesis. These results indicated that MAE significantly evaluated
duced UVB-induced MMP-9 expression (by 9.8–37.8%), although collagen, the observed effect may on the stimulation of total
4268 H.-M. Chiang et al. / Food and Chemical Toxicology 50 (2012) 4260–4269
UV
MAE ROS
MAP kinases phosphorylation
(ERK/JNK)
C-Jun C-Fos
COLTA2 promotor MMP promotor
MMP-1, -3, -9
Type I procollagen
degradation
ECM degradation
(elastase)
Photoaging
Fig. 7. The mechanisms modulating the protective effect of MAE against UVB-
induced photodamage.
collagen synthesis, but not on the protein expression of type I
procollagen.
Some studies have shown that the antioxidant activity of poly-
phenols can reduce the risk of skin diseases, such as skin cancer (de
la Roche et al., 2010; Domingo et al., 2010). Our results indicate
that MAE possesses antioxidant activity as demonstrated by its
ability to scavenge DPPH free radicals and prevent AAPH-induced
hemolysis. Our findings also suggest that MAE may protect against
the degradation of dermal collagen by preventing the generation of
ROS and by inhibiting the activity of collagenases. Three distinct
MAP kinase signal transduction pathways are activated in response
to UV irradiation: the extracellular signal regulated kinases 1 and 2
(ERK1/2), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (Xia
et al., 1995; Su and Karin, 1996). In this study, MAE decreased the
production of collagenolytic MMP and protected against MMP-
mediated photoaging by modulating the UVB-activated MAP ki-
nase signaling pathways. MAE and MAH hampered the activation
of JNK and ERK1/2 and may have down-regulated the down stream
proteins c-Fos, c-Jun, and AP-1, transcription factors and proto-
oncogenes known to be involved in photoaging. We propose that
the inhibition of MAP kinase and MMPs by MAE is related to its
antioxidant activity.
Polyphenols are predominantly present as glycosides in plants
and the amount of alycones increase noticeably after hydrolysis
by acid or enzymes (Chiang et al., 2009). It has been reported that
the activity of the aglycone component is superior to that of its gly-
cone component (Kim et al., 2004a,b). Aglycone is hydrophobic and
more easily absorbed through the skin. Although many plant ex-
tracts have been shown to suppress collagenases (Leu et al.,
2006; Lim and Kim, 2007), few studies have investigated the effect
of aglycones on collagenase activity. This study attempted to inves-
tigate the inhibitory effect of MAE on collagenase before and after
acid hydrolysis. The leaves of MAE contain aporphines such as
()-N-formylanonaine, (+)-nornuciferine and lysicamine, chloro-
phylls such as pheophorbide a, aristophyll-C and michephyll A,
and lignin including yangambin, (+)-syringaresinol (Chen et al.,
2008; Wang et al., 2010). The latter two lignans, yangambin and
(+)-syringaresinol, posses hydroxyl groups which are prone to form
glycosides. O-glycosides can be hydrolyzed by enzymes or acid;
therefore, MAH may contain their aglycones after hydrolysis. The
results of this study indicated that the effects of MAH on collage-
nase were similar to MAE.
Elastin also plays an important role in dermis (Labat-Robert and
Robert, 1988). UV exposure has been shown to cause elastin degra-
dation by activating elastase (Getie et al., 2005; Seite et al., 2006).
In this study, we evaluated the inhibitory effect of MAE on elastase
activity by measuring elastase activity and peptide synthesis. As
shown in Fig. 3 B, the effect of MAE and MAH2 (1.2 N HCl, 1 h)
on elastase activity was significant at a concentration of 500 lg/
mL and that of MAH4 (2.4 N HCl, 1 h) was significant at concentra-
tions of 500 lg/mL and higher. Hyaluronic acid is a moisturizer in
the skin. The result indicated that MAE elevated the hyaluronic
acid content in skin cells; therefore, MAE may play an important
role in skin hydration for preventing skin aging.
Our results show that MAE protects UVB-exposed Hs68 cells
against collagen degradation and collagenolytic MMP production
(Fig. 7). The relative contributions of the MAP kinase family appear
to be responsible for the anti-apoptotic effects of MAE in fibro-
blasts after exposure to UVB irradiation. Consequently, MAE could
be promising in the design and development of new treatment
strategies aimed at limiting sunlight-induced cellular oxidative
damage.
5. Conclusions
MAE attenuated MMP-1, MMP-3 and MMP-9 expression in
fibroblasts exposed to UVB irradiation by inhibiting UVB-induced
activation of p-JNK and p-ERK resulting in collagen synthesis.
Our results indicate that MAE is a promising agent for the preven-
tion of photodamage.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This study was supported in part by the National Science
Council (NSC99-2622-B-039-001-CC3; NSC100-2622-B-039-002-
CC3), Taipei, and the China Medical University (CMU99-S-39), Tai-
chung, Taiwan.
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