Effects of Citrus sunki Peel Extract on Matrix Metalloproteinase-1 Expression
Effects of Citrus sunki Peel Extract on Matrix Metalloproteinase-1 Expression
Journal of Life Science. 2013. Dec, 23(12): 1553-1556
Copyright © 2013, Korean Society of Life Science
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : October 24, 2013
  • Accepted : November 21, 2013
  • Published : December 30, 2013
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구슬, 한
선령, 이

Flavonoids are one of the major components found in the peels of citrus fruits. Present evidence has suggested that polymethoxyflavonoids, including nobiletin and tangeretin isolated from Citrus sunki , have many biological properties, such as anti-inflammatory, anti-oxidant, and anti-obesity capabilities. Here, we investigated the effect of Citrus sunki peel extract and its possible mechanisms on oxidative stress-induced MMP-1 expression, a major marker of skin photoaging. H 2 O 2 induced MMP-1 expression in a dose- and time-dependent manner. Extract of Citrus sunki peel (1-25 μg/ml) dose-dependently decreased MMP-1 mRNA levels. When H 2 O 2 was combined with Citrus sunki peel extract, the phosphorylation of ERK was further decreased compared to a single treatment with H 2 O 2 alone. Moreover, U0216, an MEK inhibitor, markedly prevented the production of MMP-1. These data suggest that Citrus sunki peel extract has demonstrated protective activity against oxidative damage on MMP-1 expression, and ERK MAP kinase may be involved.
Human skin is exposed to potentially harmful compounds and/or irradiation and becomes aged gradually. It is well known that ultraviolet irradiation, one of potent factors for skin aging, induce oxidative stress through the generation of reactive oxygen species (ROS) such as superoxide anion radical (O2-) and hydrogen peroxide (H 2 O 2 ) in human dermal cells. Oxidative damage induced by ROS is responsible for inflammatory disorders and skin aging [ 2 , 8 , 12] . Aged skin shows a decrease in collagen synthesis, which is related to matrix metalloproteinases (MMPs) regulation [6] . MMPs are a family of matrix degrading enzymes consisting of different members with substrate specificity that play important roles in various destructive processes by remodeling extracellular matrix (ECM) [10] . Among these, MMP-1, known as collagenase, is a key enzyme involved in collagen degradation due to oxidative stress, which is mediated by mitogen activating protein kinase (MAPK) - extracellular signal-regulated kinase (ERK)/p38/JUN-N-terminal kinase (JNK) activation [ 3 , 8] . Especially, ERK and JNK are known to be involved in MMP-1 expression via activation of the MMP-1 promoter by the transcription factor AP-1 and NF-kB [ 2 , 4 , 8] .
The peel of Citrus fruit contains a wide range of flavonoids constituents has been used as a traditional medicine in Korea. Flavonoids isolated from Citrus peel are categorized into two types. One is flavones glycosides including naringin, the other is polymethoxylated flavones (PMF) including nobiletin and tangeretin [11] . Following a report analyzing the composition of Citrus peel, the peel of Citrus sunki Hort. ex Tanaka is a rich source of polymethoxylated flavones [9] . A number of biological activities of polymethoxylated flavonoids isolated from Citrus sunki Hort. ex Tanaka such as anti-inflammation and anti-obesity activities have been suggested [ 5 , 9] . In this study, we investigated the effect of Citrus sunki Hort. ex Tanaka peel extract including polymethoxylated flavones on the expression of MMP-1 against oxidative stress in human skin fibroblast. Citrus sunki peel extract was found to prevent oxidative stress-induced MMP-1 expression by inhibiting ERK MAPK signaling. These results indicate that Citrus sunki peel extract may be a useful product for skin anti-aging.
Materials and Methods
- Preparation ofCitrus sunkipeel extract
Citrus sunki Hort. ex Tanaka peel extract was isolated as described previously [9] .
- Cell culture
HS-68 cells obtained from Korea Cell Bank (KCLB) were cultured in DMEM containing 10% FBS (fetal bovine serum) and 1% antibiotics at 37℃ in humidified atmosphere of 5% CO 2 .
- Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was isolated using easy blue kit and RT-PCR reaction was carried out by Maxime RT-PCR PreMix kit. The conditions for MMP-1 and GAPDH were as follows: 30 cycles of 94℃ for 1 min, 68℃ for 1 min, 72℃ for 1 min. Forward and reverse primers were 5’-ATTCTACTGATATCGGGGCTTTGA-3’ and 5’-ATGTCCTTGGGGTATCCGTGTAG-3’, respectively, for MMP-1 and 5’-ACCACAGTCCATGCCATCAC-3’ and 5’-TCCACCAC CCTGTTGCTGTA-3’, for GAPDH. Amplified product was separated through 1.5% agarose gel and visualized on an UV transillumunator.
- Western blot
Cells were lysed in buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1mM β-Glycerolphospate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM PMSF) and centrifuged at 14,000 rpm for 10 min at 4℃. Protein (30 µg) was separated in 10% SDS-PAGE and transferred to nitrocellulose membrane. The blots were incubated with anti- ERK, anti-pERK and anti-actin antibody, followed by incubation with peroxidase-conjugated secondary antibodies and then detected by enhanced chemiluminescence kit.
Results and Discussion
- Protective effect of Citrus sunki peel extract on MMP-1 expression
Oxidative stress due to the ROS production is related to the skin aging. Oxidative stress-induced skin damage results from MMPs up-regulation, which causes collagen degradation [ 1 , 2 , 6] . Since MMPs-induced collagenous matrix damage is a main phenomenon of aged skin, MMPs inhibition is thought be a useful method for skin anti-aging. It has been reported that natural product plays a role on MMPs down-regulation in UV-irradiated cells [ 2 , 3 , 13 , 15] . Polymethoxyflavonoids have been shown to exhibit a broad spectrum of pharmacological actions including anti-cancer, anti-inflammation, anti-oxidant and anti-obesity [ 5 , 7 , 14] . Recently, Kang et al. reported that extract isolated from Citrus sunki Hort. ex Tanaka peel was a rich resource of polymethoxyflavonoid such as nobiletin and tangeretin [9] . In this study, we examined that effect of Citrus sunki Hort. ex Tanaka peel extract on MMP-1 inhibition in oxidative stress-induced skin aging model. To check up-regulation of MMP-1 by oxidative stress, H 2 O 2 was treated to the HS-68 cells and RT-PCR analysis was performed. As shown in Fig. 1A and 1B , H 2 O 2 induced MMP-1 expression in a dose and time dependent manner. For further studies, 500 µM of H 2 O 2 were chosen as an optimal dose. Next, we investigated whether Citrus sunki peel extract might affect oxidative stress-induced MMP-1 expression. H 2 O 2 induced increase in MMP-1 level was significantly reduced by treatment with 25 µg/ml Citrus sunki peel extract ( Fig. 1C ). These results indicate that Citrus sunki peel extract inhibits the H 2 O 2 -induced production of MMP-1 in human skin fibroblasts.
PPT Slide
Lager Image
Protective effect of Citrus sunki peel extract (CPE) on MMP-1 expression. Cells were exposed to H2O2 with indicated dose (A) or time (B), respectively. (C) Cells were treated with each indicated dose of CPE for 30 min prior to H2O2 treatment and harvested 24 hr later. MMP-1 mRNA expression was analyzed by RT-PCR. GAPDH was used as an internal control.
- Involvement of ERK on MMP-1 expression
The activation of mitogen activating protein kinase (MAPK) is well known to be associated with MMP-1 regulation. Up-regulation of MMP-1 by UV irradiation as a potent factor of skin aging, which causes collagen degradation, is mediated by MAPK signaling via activation of transcription factor AP-1 and/or NF-kB [ 2 , 3 , 4 , 8] . To investigate involvement of MAPK signaling cascades on Citrus sunki peel extract -induced MMP-1 down-regulation, total and the phosphorylation levels of ERK was measured by Western blot analysis. Incubation of cells with H 2 O 2 induced ERK phosphorylation. Citrus sunki peel extract significantly inhibited the phosphorylation of ERK compared with single treatment of H 2 O 2 ( Fig. 2A ). However, Citrus sunki peel extract did not affect the phosphorylation and expression of p38 and JNK (data not shown). Moreover, pretreatment of U0216, a MEK inhibitor, blocked the H 2 O 2 induced increase MMP-1 expression markedly ( Fig. 2B ). These results suggest that protective effect of Citrus sunki peel extract on H 2 O 2 -induced MMP-1 expression is regulated by ERK inhibition.
PPT Slide
Lager Image
Involvement of ERK on MMP-1 regulation.
In conclusion, MMP-1 inhibition is an important strategy for skin anti-aging. Our results demonstrate that Citrus sunki peel extract inhibits oxidative stress -induced MMP-1 expression via suppression of ERK signaling, suggesting that Citrus sunki peel extract could potentially be used in the prevention of skin aging through MMP-1 inhibition.
This research was supported by the 2013 scientific promotion funded by Jeju National University.
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