International Journal of Medicinal Plants and Alternative Medicine Vol. 1(7), pp.

110-117, August 2013 Available online at http://academeresearchjournals.org/journal/ijmpam ISSN 2327-560X 2013 Academe Research Journals

Full Length Research Paper

Anti-photoaging potentials of ethanolic extracts of Curcuma aeruginosa and Plantago major

Dodi Darmakusuma1,2*, Wasmen Manalu3, Frans Umbu Datta4, Agik Suprayogi3, Nastiti Kusumorini3 and Amor T. Karyawati5
Department of Physiology and Pharmacology, Post Graduate School, Bogor Agricultural University, Bogor, Republic of Indonesia. 2 Department of Chemistry, University of Nusa Cendana, Kupang, Republic of Indonesia. 3 Department of Anatomy, Physiology and Pharmacology, Bogor Agricultural University, Bogor, Republic of Indonesia. 4 Department of Nutrition and Feed Science, University of Nusa Cendana, Kupang, Republic of Indonesia. 5 Department of Biology, University of Nusa Cendana, Kupang, Republic of Indonesia.
Accepted 12 July, 2013
1

The main purpose of this research is to evaluate the anti-photoaging potential of ethanolic extract of rhizome of Curcuma aeruginosa and ethanolic extract of leaf of Plantago major. This research evaluated the effect of extracts to inhibit UV-induced degeneration of type I Procollagen and inhibit UV-induced intracellular Reactive Oxygen Species (ROS) production in the Human Dermal Fibroblast (HDFs) cell. The result of this research shows that ethanolic extract of rhizome of C. aeruginosa and leaf of P. major were found potent to prevent UV-induced degeneration of type I procollagen. The mean relative percentage of expression levels of type I procollagen between vehicle group and extract group were significantly different (P<0.05, n=3). The mean percentage of expression levels of type I procollagen in P. major group (116.4925.349) was higher than that of the other group. Treatment of P. major extract increased expression levels of type I procollagen compared to the control group. This fact shows that the treatment of P. major extract is able to increase expression levels of type I procollagen on UV 2 exposure conditions at a dosage of 100 mJ/cm . UV radiation exposure decreased the expression levels of type I procollagen in vehicle group (23.9618.084) and C. aeruginosa group (62.4210.895), but the expression levels of type I procollagen in C. aeruginosa group was significantly higher than that of the vehicle group. These facts show that treatment of extracts inhibit UV-induced degeneration of type I procollagen in HDFs cell. The fold of DCFs fluorescence intensity at vehicle group and extracts group increased versus the control. The means of fold of DCFs uorescence intensity of extract group (2.640.016 at C. aeruginosa group, 2.410.024 at P. Major group) were significantly different and lower than that of the vehicle group (3.440.081, P<0.05, n=4). This fact shows that extracts inhibit UV-induced intracellular ROS production in the HDFs cell or it shows intracellular antioxidant effect of extracts. Based on the research result, the facts show that ethanolic extract of rhizome of C. aeruginosa and ethanolic extract of leaf of P. major have effect to inhibit UV-induced degeneration of type I procollagen and inhibit UV-induced intracellular ROS production in HDFs cells. Therefore, it can be concluded that ethanolic extract of rhizome of C. aeruginosa and ethanolic extract of leaf of P. major have potential to develop as anti-photoaging. The research results indicate that the extracts inhibit UV-induced degeneration of type I procollagen by inhibition of the UV-induced intracellular ROS production or any antioxidant effect of extracts. Key words: Curcuma aeruginosa, Plantago major, anti-photoaging, type I procollagen, reactive oxygen species (ROS), human dermal fibroblasts (HDFs).

INTRODUCTION Photoaging is skin aging that is caused by excessively exposure of the sun ultraviolet (UV) on the skin. It is very popular as premature aging of the skin, so that premature aging of the skin is often associated with photoaging.

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Photoaging has different characteristics from natural aging. One of the photoaging characteristics is the formation of deep wrinkling on the skin. The formation of deep wrinkles on the skin is caused by destruction and degeneration of collagen, particularly type I collagen. Destruction and degeneration of type I collagen on photoaging are caused by exposure of UV radiation (Yaar et al., 2001; Agius et al., 2007; Nishigori et al., 2003; Chung et al., 2001; Chung, 2003; Helfrich et al., 2008; Philips et al., 2009). The formation of deep wrinkles on the skin is a manifestation of the loss of structural integrity of the extracellular matrix (ECM) in the dermis. The loss of structural integrity of the ECM is caused by damaged collagen, particularly type I collagen as major component in the ECM of dermis. In the process of photoaging, ECM damage due to increased activity of matrix metalloproteinases (MMPs) causes damage of the collagen structure. Matrix metalloproteinase-1 (MMP-1) is a major MMP that damage type I collagen. In the process of photoaging, beside the damage process of type I collagen, there is also a process of type I collagen degeneration. Degeneration of type I collagen is caused by UV-induced degeneration of type I pro-collagen, a precursor of type I collagen. UV-induced degeneration of type I pro-collagen is caused by decrease in Transforming Growth Factor- (TGF-). Accumulation of destruction and degeneration of type I collagen caused a loss of structural integrity of the ECM that looks like deep wrinkles on the skin (Helfrich et al., 2008; Philips et al., 2009; Fisher et al., 2009; Kim et al., 2005, Dong et al., 2008). Mechanisms of photoaging take place in intracellular and extracellular space. Type I collagen is synthesized from type I procollagen by intracellular processes in the dermal fibroblasts cells. Type I procollagen is converted into type I collagen by a process in the extracellular space. In the process of photoaging, TGF- plays an important role in the formation of type I procollagen. UV radiation causes decrease in TGF-. Formation of ROS by UV exposure causes a decrease of TGF- activity. The decrease of TGF- activity on photoaging process inhibits formation of type I procollagen. Finally, UVinduced degeneration of type I procollagen causes degeneration of type I collagen in the extracellular space. Degeneration of type I collagen is one of the causes of formation of deep wrinkles in the skin as a photoaging characteristic (Helfrich et al., 2008; Lee et al., 2009; Philips et al., 2009; Huang et al., 2007; Fisher et al., 1999). The use of anti-photoaging is one of the ways to prevent photoaging. Prevention of the degeneration of type I collagen is one of the goals of anti-photoaging treatment. In this case, preventing the UV-induced degeneration of type I procollagen is an important step. One of the most popular ways today to prevent UV-induced degeneration of type I procollagen is the use of natural products.

The development of natural products to prevent UVinduced degeneration of type I procollagen is an important activity in anti-photoging research area. The natural products potency should be evaluated to prevent UV-induced degeneration of type I procollagen. Therefore, determination of the potential of a natural product to prevent UV-induced degeneration of type I procollagen is one of the important steps. Testing in vitro using cell culture is one of the important protocols in this step. Several previous publications have explained that the degeneration of type I procollagen occurred in the intracellular space. This process involves UV-induced ROS. The use of antioxidants is an effective strategy in cellular protection mechanism (Helfrich et al., 2008; Philips et al., 2009; Fisher et al., 2009, Traikovich, 1999; Lin et al., 2005). Therefore evaluation of antioxidant capacity is an important part in determination of the potency of natural products to prevent UV-induced degeneration of type I procollagen. The in vitro assays are efficient methods to evaluate the antioxidant capacity. The use of introduction screening to study the activity of anti MMP-1 from some ethanolic extracts of the Indonesian plants has found two ethanolic extracts that inhibit increase UV-induced MMP-1 in HaCaT cell culture. The extracts are ethanolic extract of rhizome of Curcuma aeruginosa (100 ppm) and ethanolic extract of leaf of Plantago major (100 ppm). This fact is not sufficient to infer potential anti-photoaging of the both extracts. Several previous publications have shown a correlation between the ability to inhibit MMP-1 and prevent the degradation of type I procollagen, particularly associated with decreased TGF- activity due to UV-induced ROS (Helfrich et al., 2008; Philips et al., 2009; Fisher et al., 2009). Therefore, evaluating the potential anti-photoaging of the both extracts needs further research to study the effect of both extracts in inhibiting UV-induced degeneration of type I procollagen and UV-induced intracellular ROS production. The main purpose of this research is to evaluate antiphotoaging potential of ethanolic extract of rhizome of C. aeruginosa and ethanolic extract of leaf of P. major. In this research, the effect of extracts on the inhibition of UV-induced degeneration of type I procollagen and UVinduced intracellular ROS production in HDFs cells were evaluated. MATERIALS AND METHODS Plant material and preparation of extracts Rhizome samples of C. aeruginosa were collected at Lembang Bandung, Indonesia. Leaf samples of P. major

*Corresponding author. E-mail: dodikimia@yahoo.com. Tel: +62 82375185471.

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were collected at the Ciampea Bogor, Indonesia. The plant samples were identified by the Research Center for Biology, Indonesian Institute of Sciences. Fresh samples were ground using commercial blender and extracted by maceration method using ethanol as a solvent for 72 h. Extracts were filtered and evaporated under vacuum. Soft extracts were dried by freeze dryer. Cell culture and extracts treatment The Human Dermal Fibroblast (HDFs) cell culture was originally obtained from Professor Jin Ho Chung (Laboratory for Cutaneous Aging Research, Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea). The passage 12 of HDFs cells 5 culture (10 cells/mL) were grown on 35 mm cell culture dishes in the Dulbeccos Modified Eagles Medium (DMEM, Gibco-BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS), 2 mM glutamine, penicillin (100 U/mL), and streptomycin (100 g/mL). The cells were incubated for 24 h at 37C in a humidified atmosphere containing 5% CO2. Thereafter, the cells were cultured in serum-free medium for 24 h. After incubation, the medium was removed and the cells were washed by warm phosphate buffered saline (PBS) for 2 times. The cells were randomly divided into different groups including the control group (normal cultural cells), vehicle group (UV-irradiated cells), and two extract groups (100 ppm extract pretreated and UV-irradiated cells). After 4 h extract pretreatment, the cells were washed twice with warm PBS, and 1 mL of PBS was added into each dish to keep the cells wet. The cells of the vehicle and extract groups were 2 exposed with 100 mJ/cm of UV (for 2 min 19 s of UV 2 irradiation at 0.72 W/cm ). Philips TL (Einthoven, Netherlands) 20W/12RS fluorescent sun lamps with an emission spectrum between 275 and 380 nm (peak, 310315 nm) were used as the UV source. Ultraviolet C (<290 nm) was blocked by Kodacel filter (TA401/407; Kodak, Rochester, NY). UV intensity was measured using Waldmann UV meter (Vehicle 585100; Waldmann, Villingen-Schwenningen, Germany). After UV irradiation, PBS was removed by DMEM medium and then the cells were incubated for 72 h at 37C in a humidified atmosphere containing 5% CO2. After incubation, 200 L of culture media were separated from cells culture for determination of expression levels of type I procollagen. The expression levels of type I procollagen were detected by Western Blotting methods. The cell viability was determined by 3-(4,5Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay method. Western blotting Western blot analyses were performed as described previously by Chung et al. (2001). The protein in the

culture media was separated by 10% SDS-PAGE, and then transferred to polyvinylidene fluoride membranes (Amersham Biosciences, Buckinghamshire, England). The blotted membranes were washed twice with Tris Buffer Saline Tween 20 (TBS/T: 20 mMTris-HCl, pH 7.6, 137 mMNaCl, 0.05% Tween-20). Membranes were subsequently blocked with 5% skim milk in TBS/T for 1 h in room temperature and incubated with the mouse monoclonal anti-procollagen type I primary antibodies (Laboratory for Cutaneous Aging Research, Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea; 1:1000) for overnight and then probed with the anti-mouse IgG-HRP conjugates secondary antibodies (Santacruz company; 1:5000) for 2 h at room temperature and then washed twice with TBST. The protein-antibody complexes were detected by using a SuperSignal West Pico chemiluminescent Substrate Detection Reagent (Thermo Scientific) and then followed by exposure to X-ray films (AFGA). The band of proteinantibody complexes was visualized by using a Kodak XOMAT 2000 Processor (Kodak). The band area was measured using ImageJ 1.43u software (Wayne Rasband National Institutes of Health, USA). The band area is a representation of the expression levels of type I procollagen. The expression levels of type I procollagen are described as relative percentage of expression levels of type I procollagen versus the control. Statistical significance was determined using the Student t test. Results are presented as the mean standard deviation (SD) of triplicates. All p values quoted are two-tailed and the significance was accepted when p was <0.05. MTT assay For this assay, 200 mL of MTT solution (5 mg / mL in PBS) was added to HDFS cell, and then incubated for 4 h at 37C in a humidified atmosphere containing 5% CO2. After incubation, MTT solution was discarded. MTformazan formed was dissolved by adding 2 mL of DMSO, and then the cells were placed on the rotor (50 rpm) for 5 min at room temperature. A total of 200 mL solution of MTT-formazan reaction products were placed in 96-well multi-well culture plate. Absorbance of MTTformazan was measured at a wavelength of 570 nm using a microplate reader and is a representation of the cell viability. The cell viability level is described as relative percentage of the cell viability versus the control. Statistical significance was determined using Student t test. Results are presented as the mean standard deviation (SD) of triplicates. All p values quoted are twotailed and the significance was accepted when p was <0.05. Intracellular ROS production assay This method adopted protocols of some methods described by Shin et al. (2005a), Ho et al. (2005) and

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Figure 1. The expression levels of type I procollagen in culture media of HDFs cells. The cells
were exposed by 100 mJ/cm2 of UV irradiation after 4 h extracts pretreatment. Philips TL lamps were used as UV sources at 0.72 W/cm 2 UV intensity for 2 min 19 s of time of UV irradiation. The expression levels of type I procollagen from culture media were analyzed 72 h after UV irradiation. 0 is for the vehicle group. C. aeruginosa is the extract group of pretreatment of C. aeruginosa extract. P. major is the extract group of pretreatment of P. major extract. (A) The expression levels of type I procollagen were analyzed by western blotting. This figure is representative of three replications. (B) The mean relative percentage of expression levels of type I procollagen is 23.9618.084 at vehicle group (a), 62.4210.895 at C. aeruginosa group (b), and 116.4925.349 at P. major group (c). There were significant differences of expression levels of type I procollagen between vehicle group and extract group (aP < 0.05, n=3). The mean relative percentage of expression levels of type I procollagen at C. aeruginosa group is significantly different than that of the vehicle and P. major groups (bP < 0.05, n=3). The mean percentage of expression levels of type I procollagen in P. major group is significantly different and higher than that of the other group (cP < 0.05, n=3).

Jeong et al. (2007). Intracellular ROS levels were determined by measuring 2,7-dichlororouorescein diacetate (DCF-DA) uorescence. This experiment used passage 14 of HDFs cells culture. A total of 1 mL 5 suspension of cells (10 cells/mL in DMEM with serum) were grown in each well of 12-Well Cell Culture Multiwell Plate. The cells were incubated at 37C in a humidified atmosphere containing 5% CO2. After 24 h, the media was removed, and the cultured cells were washed with PBS. A total of 1 ml of DMEM medium without serum and without phenol red was added, and then incubated for 24 h. After 24 h of the removal of the culture media, the cells were divided into different groups including the control group (normal cultural cells), vehicle group (UV-irradiated cells), and two extract groups (100 ppm extract pretreated and UV-irradiated cells). After 24 h extract pretreatment, the cells were washed twice with warm PBS. The cells were stained with 40 m of DCF-DA (Molecular Probes, Eugene, Oregon) and incubated for 20 min in a dark room at temperature of 37C. After incubation, the cells were washed with PBS 3 times, and 0.1 mL of PBS was added into each dish to keep the cells wet. The cells of the vehicle and extract groups were

exposed with 100 mJ/cm of UV (for 2 min 12 s of time of 2 UV irradiation at 0.76 W/cm ) to stimulate the formation of ROS. After 30 min, the cells were washed with PBS 3 times. The DCFs fluorescence intensity was measured by using a Victor3 multilabel plate counter vehicle 1420 (Perkin Elmer). The levels of intracellular ROS production are described as fold of DCFs fluorescence intensity versus the control group. Statistical significance was determined using the Student t test. Results are presented as the mean standard deviation (SD) of quadruplicates. All p values quoted are two-tailed and the significance was accepted when p was <0.05. RESULTS Extracts inhibition of UV-induced degeneration of type I procollagen The mean relative percentage of expression levels of type I procollagen is 23.9618.084 at vehicle group, 62.4210.895 at C. aeruginosa group, and 116.4925.349 at P. Major group (Figure 1). The mean relative percentage of expression levels of type I

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Figure 2. The relative cell viabilities of the HDFs cell. 0 is for the vehicle group. C.
aeruginosa is the extract group of pretreatment of C. aeruginosa extract. P. major is the extract group of pretreatment of P. major extract. The cells were exposed by 100 mJ/cm2 of UV irradiation after 4 h extracts pretreatment. Philips TL lamps were used as UV sources at 0.72 W/cm 2 UV intensity for 2 min 19 s of time of UV irradiation. The relative cell viabilities of the HDFs cells were measured using MTT assay after 72 h UV irradiation. The mean of relative percentage of cell viability is 99.990.055 at vehicle group (a), 99.990.094 at C. aeruginosa group (b), and 99.990.055 at P. major group (c). There were no significant difference between groups (P>0.05, n=3).

procollagen between the vehicle group and extract group are significantly different (P<0.05, n=3). The mean percentage of expression levels of type I procollagen in P. major group is higher than that of the other group. UV radiation exposure decreased the expression levels of type I procollagen in the vehicle group and C. aeruginosa group, but the expression levels of type I procollagen in C. aeruginosa group is significantly higher than that of the vehicle group. These facts show that the treatment of extracts inhibit UV-induced degradation of type I procollagen in HDFs cell. Treatment of P. major extract increases expression levels of type I procollagen compared to the control group. This fact shows that the treatment of P. major extract is able to increase expression levels of type I procollagen on UV exposure conditions at dosage of 100 2 mJ/cm . This suggests that P. major extract have the potential to increase metabolism of type I procollagen in conditions of UV exposure. The mean of relative percentage of cell viability is 99.990.055 at vehicle group, 99.990.094 at C. aeruginosa group, 99.990.055 at P. Major group (Figure 2). This fact shows that dosage of energy of UV exposure does not affect cell viability. The results of MTT assay show that the mean of relative percentage of cell viability between vehicle group and extract group are not significantly different (P>0.05, n=3), which in turn shows that the extract treatment does not affect cell viability.

These facts show that the difference of expression levels of type I procollagen between groups was not caused by difference of cell numbers. Extracts inhibition of UV-induced intracellular ROS production As shown in Figure 3, the fold of DCFs fluorescence intensity at vehicle group and extracts group increased versus the control. The mean of fold of the DCFs uorescence intensity is 3.440.081 at vehicle group, 2.640.016 at C. aeruginosa group, and 2.410.024 at P. Major group. The mean of fold of DCFs uorescence intensity between the vehicle and extract groups are significantly different (P<0.05, n=4). The mean of fold of DCFs uorescence intensity at vehicle group increases about more than 3 fold versus the control. This fact shows that UV radiation significantly increased ROS generation by more than 3 fold in vehicle group versus the control. However at extracts group, mean of fold of DCFs uorescence intensity is lower than that of the vehicle group. This fact shows that extracts inhibit UVinduced intracellular ROS production in the HDFs cell, and have an effect on intracellular antioxidant. DISCUSSION In this research, the HDFs cell was irradiated by 100

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Figure 3. DCFs fluorescence intensity of HDFs cells after exposure to UV radiation. 0 is for the
vehicle group. C. aeruginosa is the extract group of pretreatment of C. aeruginosa extract. P. major is the extract group of pretreatment of P. major extract. The cells were exposed by 100 mJ/cm 2 of UV irradiation after 24 h extracts pretreatment. Philips TL lamps were used as UV sources at 0.76 W/cm 2 UV intensity for 2 min 12 s of time of UV irradiation. The mean of fold of DCFs uorescence intensity is 3.440.081 at vehicle group (a), 2.640.016 at C. aeruginosa group (b), and 2.410.024 at P. major group (c). There were significant differences of fold of DCFs uorescence intensit y at vehicle group which were higher than those of the other groups (aP < 0.05, n=3). The mean of fold of DCFs uorescence intensity at C. aeruginosa group was significantly different than that of the vehicle and P. major groups (bP < 0.05, n=3), whereas the mean of fold of DCFs uorescence intensity at P. major group was significantly different and lower than that of the other groups (aP < 0.05, n=3).

mJ/cm of UV dosage levels. Previous studies (Shin et al., 2005b) used the same UV dosage levels to irradiate HDFs cell. Based on our preliminary study, the UV 2 dosage level on 100 mJ/cm does not inhibit viability of HDFs cell, but significantly inhibits synthesis of type I procollagen. Result of MTT assay after experiment shows 2 that 100 mJ/cm of UV dosage has no effect on viability of HDFs cell (Figure 2). The result of research shows that ethanolic extract of rhizome of C. aeruginosa and leaf of P. major were found potent to prevent UV-induced degeneration of type I procollagen. The ability of these extracts to prevent the UV-induced degeneration of type I procollagen is closely linked to the ability of the extract to reduce UV-induced intracellular ROS production. The result of this research suggests that the extracts inhibit UV-induced degeneration of type I procollagen by inhibition of UVinduced intracellular ROS production or any intracellular antioxidant effect of extracts. The UV irradiation causes an increase ROS production in the HDFs cell. Increasing ROS production causes a decrease of TGF-

activity, which in turn causes a decrease in the formation of procollagen-1 (Fisher et al., 1999; Quan et al., 2004; Dong et al., 2008; Helfrich et al., 2008; Lee, 2009). The ROS levels were measured after extracts treatment to investigate whether or not the extracts inhibit UV-induced degeneration of type I procollagen via an antioxidant effect. Antioxidant intracellular activity is expressed as the inhibitory activity of intracellular ROS formation caused by UV exposure in the test cell. This assay is based on inhibition of ROS formation by antioxidant compounds that are contained in the test sample. To determine the inuence of both extracts on ROS generation, the cell-permeable fluorogenic probe DCF-DA was used. HDFs cells were cultured in a 12-well cell culture plate, and then treated with extracts. After extracts treatments, the cells were pre-incubated with DCF-DA and diffused into cells. After incubation, the cells were irradiated by UV and DCF-DA was rapidly oxidized to highly fluorescent DCF by ROS. In the presence of ROS, DCF-DA was subsequently transferred to DCF and it emitted a green uorescent signal which could be

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detected on a standard fluorescence plate reader. The fluorescence levels are a representation of intracellular ROS in the HDFs cytosol. The higher the fluorescence levels, the higher the levels of intracellular ROS (Possel et al., 1997; Gunasekar et al., 1995; LaBel et al., 1992). Increasing of ROS in the cells caused by UV irradiation is one of the reasons that lead to photoaging (Peus et al., 1999). The accumulated ROS plays a critical role in the photoaging of human skin in vivo (Kawaguchi et al., 1996). The ROS in skin cells can be formed as a result of exposure to solar UV. UV radiation is a very potent initiator of photochemical reactions through excitation of electrons and this can result in energy transfer or chemical modification of the exposed molecule. The energy of the absorbed photon creates an excited state molecule, which is highly unstable under ambient conditions. In returning to the ground state, excited species transfer energy to adjacent intracellular chemical moieties particularly molecular oxygen and thereby convert it into ROS (Bickers and Athar, 2006; Svobodov et al., 2003). All the results suggested that treatment of extracts in HDFs prevents UV-induced degeneration of type I procollagen by inhibiting UV-induced intracellular ROS production, such that the extracts have potential to develop as natural anti-photoging materials. The results of this research show that the extract of P. major is able to maintain increase expression levels of procollagen-1 in the HDFs cells on UV exposure conditions at dosage of 2 100 mJ/cm . Based on the results of this research, we found facts that suggest the potential extract of P. major as an anti-photoaging better than the extract of C. aeruginosa. Conclusion This research has demonstrated for the first time the evaluation result of anti-photoaging potentials of ethanlic extracts of C. aeruginosa and P. major to inhibit UVinduced degeneration of type I procollagen. Based on the research result, the facts show that ethanolic extract of rhizome of C. aeruginosa and ethanolic extract of leaf of P. major have the potential to inhibit UV-induced degeneration of type I procollagen and UV-induced intracellular ROS production in HDFs cells. Therefore, it can be concluded that the ethanolic extract of rhizome of C. aeruginosa and ethanolic extract of leaf of P. major have the potential to develop as anti-photoaging. The research result indicates that the extracts inhibit UVinduced degeneration of type I procollagen by inhibition of the UV-induced intracellular ROS production or any antioxidant effect of extracts. ACKNOWLEDGMENTS The authors are grateful to Professor Jin Ho Chung for facility support and the excellent research guidance given

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