Extract of Ettlia sp. YC001 Exerts Photoprotective Effects against UVB Irradiation in Normal Human Dermal Fibroblasts

Abstract

The identification of novel reagents that exert a biological ultraviolet (UV)-protective effect in skin cells represents an important strategy for preventing UV-induced skin aging. To this end, we investigated the potential protective effects of Ettlia sp. YC001 extracts against UV-induced cellular damage in normal human dermal fibroblasts (NHDFs). We generated four different extracts from Ettlia sp. YC001, and found that they exhibit low cytotoxicity in NHDFs. The ethyl acetate extract of Ettlia sp. YC001 markedly decreased UVB-induced cytotoxicity. Additionally, the ethyl acetate extract significantly inhibited the production of hydrogen peroxide-induced reactive oxygen species. Moreover, it inhibited UVB-induced thymine dimers, as confirmed by luciferase assay and thymine dimer dot-blot assay. Thus, the study findings suggest Ettlia sp. YC001 extract as a novel photoprotective reagent on UVB-induced cell dysfunctions in NHDFs.

Introduction

The skin is the largest organ of the human body, and dermal fibroblasts generate collagen and elastin responsible for maintaining its firmness and elasticity [8,23]. The main function of skin is protection from harmful environmental factors, such as microbes, pollutants, and ultraviolet (UV) radiation [23]. As the interface between the human body and the environment, skin tissue is continuously vulnerable to damage from UV radiation, which is a major extrinsic factor in skin aging [7]. UV irradiation, including UVA, UVB, and UVC, induces generation of reactive oxygen species (ROS), which are associated with DNA damage, matrix metalloproteinase synthesis, loss of cell growth, and apoptosis and senescence in dermal fibroblasts [23]. Furthermore, continuous exposure to UV radiation is considered a major causative factor for photodermatoses, actinic keratosis, and skin cancer [26]. Unlike the inevitable aging of skin associated with intrinsic factors (a genetically programed process that occurs with age), UV radiation-mediated skin aging and disease can be prevented and repaired by regulating exposure to UV radiation and, therefore, UV-induced damage [6]. Thus, the identification of novel reagents exerting a biological UV-protective effect in skin cells is an important future strategy for preventing UV-induced skin aging.

Microalga Ettlia sp. YC001 has been shown to have high growth rates and lipid productivity under high CO2 [27]. The diverse strains of microalgae suggest the potential to produce a variety of molecules exhibiting diverse targets and levels of efficacy. However, studies of such microalgae and their products have generally been limited to the field of biodiesel research [5,12]. Although a relationship between microalgae and anti-aging effects has not been demonstrated, recent studies have begun to show the possibility of UV-photoprotective effects of molecules derived from microalgae. Astaxanthin, which is a carotenoid derived from Haematococcus pluvialis, is a promising antioxidant molecule, and has therapeutic potential for protecting human cells against oxidative stress [21], which is a main cytotoxic result of UV radiation [20]. Additionally, we previously reported that an extract of the microalga Chlorella vulgaris can protect against UV-induced reduction in cell growth in normal human dermal fibroblasts (NHDFs) [15]. These reports indicate that a function of microalgae-derived molecules is related to UV protection in cells; therefore, the present study examines whether extracts of Ettlia sp. YC001 exert inhibitory effects on UV-induced cellular and DNA damage in human skin dermal fibroblasts.

 

Materials and Methods

Extracts of Ettlia sp. YC001 and Reagents

Solvent extraction of microalgal biomass is widely used for extraction of pigment, total lipid, and essential fatty acids. Hexane, ethanol, ethyl acetate, and distilled water can be used as solvents for various cell extracts [19]. An aliquot (50 mg) of lyophilized Ettlia sp. YC001 biomass was homogenized using a pre-chilled mortar and pestle. The total cell extract was repeatedly extracted with ethanol, ethyl acetate, hexane, and distilled water using an ultrasonicator (Vibra Cell, Sonics & Materials, Inc., CT, USA) until the biomass became colorless, after which it was centrifuged at 5,000 rpm at 4℃ for 5 min. The extract was filtered through a 0.45 μm membrane filter (Whatman/GE Healthcare Life Sciences, PA, USA). The solvent was removed by a speed vacuum concentrator (Hanil Science, Inc., Korea) for 6 h. The extract powders were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) and stored at −20℃ until use.

LC-MS Analysis of Extract of Ettlia sp. YC001

The samples were analyzed using an analytic UtiMate 300 HPLC system (Dionex, CA, USA) equipped with an UtiMate 300 autosampler column compartment, an HPG3200 binary pump, an UtiMate 3000 UV detector, and using Chromeleon software. Carotenoid analysis was performed using an YMC C30 carotenoid column (150 mm × 4.6 mm, particle size of 3 μm; Waters Corp., MA, USA).

HPLC-coupled mass spectrometry was conducted using a Thermo Scientific LCQ Fleet ion trap spectrometer (Thermo Fisher Scientific, CA, USA) equipped with an APCI interface operating in the positive-ion mode. The extract was dissolved in ethanol and filtered through a 0.22 μm membrane of a nylon filter. The solution was directly introduced into the APCI source by flow injection at 5 μl/min, using a syringe pump. The APCI capillary temperature was 150℃, APCI vaporizer temperature 450℃, sheath gas flow 58 l/min, auxiliary gas flow 10 l/min, and source current 5 μA.

Cell Culture

NHDFs were purchased from Lonza (Basel, Switzerland) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 at 37℃ [4].

WST-1-Based Viability Assay

Cell viability was evaluated via water-soluble tetrazolium salt-1 (WST-1) assay (EZ-Cytox cell viability assay kit; Itsbio, Seoul, Korea) [4]. NHDFs (4 × 103) were seeded into 96-well culture plates and incubated for 24 h under normal growth conditions. Cells were then treated with several doses of Ettlia extracts for 24 h. After treatment, the NHDFs were mixed with 100 μl of WST-1 solution followed by incubation at 37℃ for 0.5 h. Cell viability was determined after measuring the absorbance at 450 nm with a reference filter at 620 nm using an iMark plate reader (Bio-Rad, Hercules, CA, USA). All results are presented as mean percentage ± standard deviation (SD) of three independent experiments.

UVB Irradiation and Treatment with Ettlia sp. YC001 Extracts

UVB irradiation of NHDFs was performed as described previously [3]. Briefly, NHDFs (4 × 103 or 8 × 105) were seeded and incubated under normal growth conditions. The NHDFs were then pretreated with DMSO or Ettlia sp. YC001 extracts for 6 and 12 h. After treatment, the medium was replaced with phosphatebuffered saline followed by UVB (30 mJ/cm2) irradiation without the culture plate cover. Following UVB irradiation, the NHDFs were further incubated with growth medium containing DMSO or extracts of Ettlia sp. YC001 for 12, 24, and 48 h, respectively.

Luciferase Assay

Luciferase reporter plasmid pGL3-Luc (Promega, Madison, WI, USA) was cotransfected with pSV-β-galactosidase plasmid (as a transfection control) into NHDFs using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). The transfected cells were treated with the extracts of Ettlia sp. YC001 prior to UVB irradiation. After the treatments, DNA damage was determined by means of the luciferase assay (Promega). The results are normalized to β-galactosidase activity and presented as percentages of control values (mean ± SD). The results shown are the means of three independent experiments.

Immunoblotting of Thymine Dimers

NHDFs (8 × 105) were seeded in 60 mm culture dishes and cultured overnight under normal growth conditions. The NHDFs were pretreated with the indicated concentrations of ethyl acetate extract of Ettlia sp. YC001 for 12 h and then irradiated with 30 mJ/cm2 UVB. Following irradiation, the cells were harvested, and genomic DNA was extracted using an Exgene Cell SV Genomic DNA Extraction Kit (GeneAll Biotechnology, Seoul, Korea) according to the manufacturer’s instructions. Each purified genomic DNA sample (100 ng) was denatured by incubation in denaturation buffer (0.4 M NaOH and 10 mM EDTA) and boiled at 100℃ for 10 min. Samples were neutralized by addition of neutralization buffer (2 M ammonium acetate, pH 7.0) and blotted onto nitrocellulose membrane using a Bio-Dot Microfiltration Apparatus (Bio-Rad) according to the manufacturer’s instructions. The blotted membrane was briefly rinsed with 2× saline sodium citrate buffer (0.3 M NaCl and 30 mM sodium citrate) and baked at 80℃ for 2 h. The blots were then placed in blocking solution (10% skimmed milk) for 1 h, and incubated with anti-thymine dimer clone H3 antibody (Sigma-Aldrich) for an additional 1 h. For thymine dimer detection, horseradish peroxidase-conjugated anti-mouse secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) were used, followed by enhanced chemiluminescence (ECL; Pierce/Thermo Fisher Scientific, Waltham, MA, USA) and autoradiography.

ROS Scavenging Assay

Levels of intracellular ROS scavenging effect were determined by measuring the fluorescence intensity of the 2’7’-dichlorofluorescein diacetate (DCF-DA) probe, as previously described [16]. NHDFs (8 × 105) were pretreated with the extract of Ettlia sp. YC001 for 12 h, and then with 800 M H2O2 for 3 h. The cells were then mixed with 20 μM DCF-DA solution and incubated at 37℃ for 1 h. Fluorescence was measured using a flow cytometer (DB FACSCalibur; BD Biosciences, San Jose, CA, USA). The mean of DCF fluorescence intensity was calculated based on measurements of 10,000 cells using the FL1-H channel. The M1 range was calculated as the percentage of each subpopulation of cells showing increased DCF-DA fluorescence.

Statistical Analysis

The data were analyzed using the two-tailed Student’s t-test, and p-values <0.05 were considered statistically significant.

 

Results

Analysis of Carotenoid Compositions of Extract of Ettlia sp. YC001

Carotenoids are the major products of microalgae, and have been shown to exert UVB-protective and antioxidant effects [11,22,24]. First, we evaluated the carotenoid composition of Ettlia sp. YC001 using LC-MS analysis, as described in the Materials and Methods. The ethanol extract of Ettlia sp. YC001 was found to have six different carotenoids: four known (neoxanthin, cis-neoxanthin, lutein, and cis-lutein) and two unknown (Fig. 1 and Table 1). Of note, the peaks of lutein, cis-neoxanthin, and unknown carotenoid (peak c) were highest, indicating that those three carotenoids were relatively abundant in the ethanol extract (Fig. 1).

Fig. 1.HPLC chromatogram of Ettlia sp. YC001 extract. Total scan from the photo diode array detector. Full spectrum from LC-MS APCI measurement in the positive-ion mode. Peaks a, unidentified carotenoid; b, cis-neoxanthin; c, unidentified carotenoid; d, neoxanthin; e, lutein; and f, cis-lutein.

Table 1.aRT, retention time; bUV/VIS, ultraviolet-visible; cMS, mass spectrometry.

It has been reported that the carotenoid concentration and composition vary according to the type of solvent [2]. Therefore, before investigating the UVB-protective effects of the ethanol extracts in NHDFs, we further prepared three additional extracts of Ettlia sp. YC001 using ethyl acetate, hexane, and water as solvents; these are commonly used for extraction of carotenoids from algae, and have been reported as “recommended” solvents [17,19]. We then tested whether the four different extracts of Ettlia sp. YC001 exhibited UVB-protective activity in NHDFs.

Extracts of Ettlia sp. YC001 Have Minimal Cytotoxicity in NHDFs

Prior to investigating the protective effects of the four extracts of Ettlia sp. YC001 on UVB-induced cytotoxicity, we first determined the dose range of the extracts that caused cytotoxicity in NHDFs. With the exception of the highest dose (50 μg/ml), no significant differences in cell viability were detected between cells treated with 1–20 μg/ml of the extracts, and the control cells treated with DMSO (Fig. 2A). Of note, at 20 μg/ml extract concentration, the viability of cells treated with the ethanol, hexane, ethyl acetate, and distilled water extracts was 98.14 ± 3.4%, 106.12 ± 4.34%, 90.13 ± 2.31%, and 92.76 ± 4.50%, respectively. At the highest dose, the extracts showed less than 20% decline in cell viability (10.87 ± 3.1%, 14.69 ± 5.6%, 18.69 ± 4.1%, and 15.77 ± 3.9%, respectively). These results indicate that the extracts of Ettlia sp. YC001 were not cytotoxic at concentrations ≤20 μg/ml in NHDFs; therefore, the extracts were used within that concentration range in the subsequent experiments.

Fig. 2.Ettlia sp. YC001 extracts have minimal cytotoxicity and photoprotective effect on UVB-induced cytotoxicity in NHDFs. (A) NHDFs were seeded into 96-well culture plates and treated with the indicated doses of ethanol (EtOH), hexane, ethyl acetate (EtOAC), and distilled water (D.W.) extracts of Ettlia sp. YC001. After 24 h incubation, WST-1 solution was added to evaluate cell viability. (B) Before and/or after exposure to 30 mJ/cm2 UVB, cells were treated for 6 and 24 h, respectively, with the indicated concentrations of the four extracts of Ettlia sp. YC001. Cell viability was analyzed using the WST-1 assay. (C) Cells were pretreated with the indicated concentrations of ethyl acetate extract of Ettlia sp. YC001 for 12, 24, and 48 h, followed by 30 mJ/cm2 UVB irradiation. After UVB irradiation, the cells were post-treated with DMSO or the extract for 12, 24, and 48 h. Cell viability was assessed by the WST-1 assay. Data represent the mean ± SD from three independent experiments. *p < 0.05 compared with DMSO-treated control group. apre_12 indicates that the extract was pretreated for 12 h before UVB irradiation; bpost_12 indicates that the extract was post-treated for 24 h after UVB irradiation. cpre_12 + post_12 indicates that the extract was pre- and post-treated for 12 h before and after UVB irradiation.

Extract of Ettlia sp. YC001 Suppresses UVB-Induced Cell Damage in NHDFs

We next examined the protective effects of the Ettlia sp. YC001 extracts against UVB-induced cytotoxicity in NHDFs. To investigate the potential protective effect of the extracts more precisely, we established three experimental groups based on treatment regimen. The first group of cells was pretreated with the extracts prior to UVB irradiation; the second group was post-treated following irradiation; and the last group was irradiated pre- and post-treatment. The cells were seeded and treated with DMSO or 0–20 μg/ml of the four extracts for 12 h. Following treatment, the cells were irradiated with 30 mJ/cm2 UVB radiation, and then post-treated with the same concentrations of extracts for an additional 24 h. WST-1 assay was then performed to assess the cell viability in each group. As shown in Fig. 2A, in the pretreatment subgroup of cells administered the ethyl acetate extract, the UVB-induced loss of cell viability was reversed in a dose-dependent manner. Specifically, pretreatment with 10 and 20 μg/ml of the ethyl acetate extract restored cell viability compared with the UVB irradiated-only control by 5.14 ± 2.56% and 12.53 ± 2.60%, respectively (Fig. 2B). We also found that cell viability in the post-treatment subgroups administered 10 and 20 μg/ml ethyl acetate extract was restored by 4.83 ± 1.42% and 6.92 ± 2.03%, respectively, compared with controls (Fig. 2B). Finally, viability was restored in the subgroups pre- and post-treated with these two doses of ethyl acetate by 8.37 ± 2.1% and 19.44 ± 2.56%, respectively, compared with controls. Therefore, the ethyl acetate extract of Ettlia sp. YC001 blocked the UVB-induced decrease in cell viability of NHDFs.

Pre- and Post-Treatment with Ethyl Acetate Extract of Ettlia sp. YC001 Has Maximum Photoprotective Effect against UVB Radiation in NHDFs

Next, we attempted to maximize the protective effect of the ethyl acetate extract against UVB radiation by varying the duration of treatment in NHDFs. First, the duration of pretreatment with the extract was increased from the original 6 h to 12, 24, and 48 h. Second, the duration of post-treatment with the extract was changed from the original 24 h to 12 and 48 h. Therefore, the set times of treatments were 12, 24, and 48 h for pretreatment, and 12, 24, and 48 h for post-treatment before and after UVB irradiation of NHDFs. As shown in Fig. 2C, the protective effect of the extract increased in a time-dependent manner. Of note, combined pre- and post-treatments with 20 μg/ml extract for 48 h showed maximum protective effect (29.85 ± 3.80%) against UVB-induced cell damage compared with the UVB-irradiated control, and the protective effect of the extract was more strongly induced in pre-treated than in post-treated cells (Fig. 2C). These results indicate that treatment with the ethyl acetate extract of Ettlia sp. YC001 significantly reverses UVB-induced damage in NHDFs.

Ethyl Acetate Extract of Ettlia sp. YC001 Inhibits UVB-Mediated DNA Damage

To further examine the protective effect of the ethyl acetate extract against UV-induced DNA damage in NHDFs, two biochemical assays were conducted: luciferase assay to assess UVB-induced DNA damage, and immunoblot analysis of thymine dimers. A previous study employed a luciferase reporter assay to demonstrate that UV radiation induces DNA damage in promoter regions and inhibits transcription [9]. Therefore, we simply tested whether the extract inhibits UVB-induced loss of luciferase expression in cells after transfection with a pGL3-promoter plasmid and pSV-β-galactosidase control plasmid. As shown in Fig. 3A, 30 mJ/cm2 UVB irradiation greatly reduced the luciferase activity, to 13.30 ± 2.39 fold, compared with nonirradiated control cells, which showed 27.49 ± 3.1 fold activity relative to negative control cells expressing pSV-β-gal only. Interestingly, the UVB-induced decrease in luciferase activity significantly restored the activity compared with the UVB-irradiated group, to 20.61 ± 3.34 fold (Fig. 3A).

Fig. 3.Ethyl acetate extract of Ettlia sp. YC001 has protective effect against UVB-induced DNA damage in NHDFs. (A) Cells were seeded in 60 mm culture plates and transfected with control plasmid pSV-β-galactosidase and/or pGL3 luciferase reporter plasmid. The cells were then pretreated with DMSO (vehicle control) or 20 μg/ml ethyl acetate extract of Ettlia sp. YC001 for 12 h and exposed to 30 mJ/cm2 UVB radiation, followed by post-treatment with DMSO or the extract for 48 h. The level of DNA damage was determined by luciferase assay. A β-galactosidase assay was performed for normalization. (B) Cells were treated with DMSO or 20 μg/ml ethyl acetate extract of Ettlia sp. YC001 before and after UVB irradiation. Cells were lysed and genomic DNA was extracted. The induced level of thymine dimers was analyzed by immunoblotting with anti–thymine-dimer antibody. Data represent the mean ± SD from three independent experiments. *p < 0.05 compared with DMSO-treated control group and UVB-irradiated group.

We further tested whether the extract regulated the level of cyclobutane pyrimidine dimers (CPDs), which are directly induced by UV radiation in skin cells [14]. As shown by immunoblot analysis, 30 mJ/cm2 UVB irradiation clearly increased the level of CPDs in NHDFs, whereas treatment with 20 μg/ml extract significantly inhibited this increase (Fig. 3B). Taken together, these results suggest that the ethyl acetate extract of Ettlia sp. YC001 reduced UVB-induced DNA damage in NHDFs.

Carotenoid Composition and Antioxidant Activity of Ethyl Acetate Extract of Ettlia sp. YC001

The UVB-protective effect of the ethyl acetate extract of Ettlia sp. YC001 that we observed in NHDFs prompted us to investigate whether the carotenoid composition of this extract differed from that of the ethanol extract shown in Fig. 1. HPLC analysis showed nine different carotenoid peaks in the ethyl acetate extract (Fig. 4A). Comparison with the ethanol extract shown in Fig. 1 showed lutein as a common carotenoid, whereas the others had different peak patterns, indicating that the carotenoid composition of the ethyl acetate extract was markedly different from that of the ethanol extract.

Fig. 4.Carotenoid composition and antioxidant property of ethyl acetate extract of Ettila sp. YC001. (A) HPLC chromatogram of ethyl acetate of Ettlia sp. YC001. (B) Cells were treated with the ethyl acetate extract for 6 h, and then with H2O2. After further incubation for 3h, cells were collected and stained with DCF-DA solution for 1 h. Levels of intracellular ROS were measured by flow cytometry. Quantification of the percentage of cells in the M1 range. Data represent the mean ± SD from three independent experiments. *p < 0.05 compared with control group or H2O2-treated sample for indicated pairs. aEtOAC extract, ethyl acetate extract of Ettlia sp. YC001.

To investigate the antioxidant effect of the ethyl acetate extract, in vivo ROS scavenging assay was carried out using DCF-DA staining and flow cytometry. Intracellular levels of ROS were not increased in the ethyl-acetate-treated NHDFs, but were increased to 33.56 ± 4.41 in the H2O2-treated NHDFs (Fig. 4B). Of note, the high levels of ROS induced by H2O2 were markedly reduced to 7.2 ± 0.67 in the NHDFs pre-treated with H2O2 (Fig. 4B). Taken together, these results suggest that the ethyl acetate extract has intracellular ROS-scavenging effect in NHDFs.

 

Discussion

UV protection of skin is important to prevent skin aging and disease such as melanoma [7]. Therefore, there is a growing need to understand how UV radiation induces skin damage, and how to efficiently block radiation of skin tissue, which is the most exposed tissue of the human body. The common significant effects of UV radiation are ROS production, DNA damage (such as pyrimidine dimer formation), and DNA mutations that induce cell senescence and apoptosis if not repaired [18,23]. Previous reports, which dealt with an extract or single chemical (including astaxanthin) purified from microalgae, have shown that microalgae-derived materials are effective against skin wrinkles and as ingredients in sunscreen [10,21]. Therefore, we chose to investigate whether the extract of the microalga Ettlia sp. YC001 modulated UV-induced cellular damage and showed potential as an anti-UV reagent. We found that the ethyl acetate extracts of Ettlia sp. YC001 displayed minimal cytotoxicity and significantly restored UVB-induced losses of cell growth in NHDFs.

The negative effects of UV radiation on cell growth and chromosome stability should be prevented or repaired in order to prevent aging-related skin damage and disease [7,20]. Our results reveal that application of the extract prior to UVB irradiation significantly inhibited radiation-induced loss of cell growth in NHDFs. We also found that, although pretreatment application had greater inhibitory effect on UV-induced damage, post-treatment application also significantly inhibited radiation-induced loss of cell growth, indicating that the extract potentially has both protective and reparative effects on UV-induced cell damage in NHDFs. Furthermore, the extract-mediated inhibitory effect against UVB was maximized by combined pre- and post-irradiation application in NHDFs.

We suspect that the photoprotective action of the ethyl acetate extract is related to protection against both UVB-mediated DNA damage and ROS production, because UV-induced DNA damage and ROS production lead to reductions in either cell growth or division [16,20]. Indeed, our results clearly indicate that UVB increased the level of thymine dimers, a specific sign of UV-induced DNA damage, and decreased the level of luciferase expression, whereas the extract clearly reversed these effects. Additionally, we found that the extract markedly decreased H2O2-induced intracellular ROS production.

The protective effect of the extract appears to predominantly derive from its antioxidant effect, because UV is also known to induce oxidative stress through the production of ROS that attack DNA, resulting in DNA damage [13]. However, our results showed that ROS-independent and UV-dependent DNA damage such as cyclobutane pyrimidine dimers [13] were markedly inhibited by the ethyl acetate extract. Therefore, the UVB-protective effect mediated by the ethyl acetate extract might be induced by two different mechanisms of antioxidant-dependent and - independent actions in NHDFs. Moreover, comparative analysis of the carotenoid compositions between ethanol and ethyl acetate extracts clearly demonstrated their differing compositions (with the exception of lutein, their one common carotenoid). These data suggest that the ethyl acetate extract-mediated photoprotective action might be induced not by lutein, but by other carotenoids that were shown in the extract. Further studies are needed to elucidate which of the carotenoids in the extract specifically regulate UVB-induced damage in NHDFs.

A recent report showed that the onset of UV-induced melanoma was delayed by the application of UVB sun protection factor 50 on mouse skin, but that only partial protection was achieved, indicating that UV light can penetrate sunscreen to some extent and cause DNA damage in skin [25]. An earlier report also showed that irregular application of sunscreen, with even one instance of unprotected exposure to UV irradiation, significantly increases the level of thymine dimer formation in human skin [1]. These reports indicate that there are limits to the physical protection provided by sunscreens against UV-induced cellular and DNA damage. Our study shows that the extract of Ettlia sp. YC001 has biological protective and reparative effects on UVB-induced cellular and DNA damage, and is therefore a potentially important reagent for preventing UV-induced skin damage.

In summary, the results presented here suggest that Ettlia sp. YC001 extract protected NHDFs against UVB-induced reduction in cell growth rate, and also inhibited UVB-induced thymine dimer generation. These findings suggest Ettlia sp. YC001 extract as a potential reagent for use in products intended to prevent UV-induced skin aging.