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In vitro and in vivo anti-photoaging effects of an isoflavone extract from soybean cake
Journal of Ethnopharmacology 126 (2009) 108–113
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
In vitro and in vivo anti-photoaging effects of an isoflavone extract from soybean cake Tsu-Man Chiu a,1 , Chieh-Chen Huang b,c,1 , Tzu-Ju Lin d , Jia-You Fang e , Nan-Lin Wu f , Chi-Feng Hung b,d,∗ a
Department of Dermatology, Changhua Christian Hospital, Changhua Hsien, Taiwan, ROC School of Medicine, Fu Jen Catholic University, Taipei Hsien, Taiwan, ROC Department of Dermatology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan, ROC d Graduate Institute of Basic Medicine, Fu-Jen Catholic University, Taipei Hsien, Taiwan, ROC e Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan, ROC f Department of Dermatology, Mackay Memorial Hospital, Hsinchu, Taiwan, ROC b c
a r t i c l e
i n f o
Article history: Received 28 October 2008 Received in revised form 22 June 2009 Accepted 31 July 2009 Available online 11 August 2009 Keywords: Isoflavone Genistein UVB COX-2 PCNA Catalase
a b s t r a c t Ethnopharmacological relevance: Soy has been used in traditional Chinese medicine for thousands of years for its health and nutritional benefits, as well as to treat and care for the skin. Advanced skin care research has shown that soy isoflavone and genistein are effective in reducing damage to the skin from the sun. Aim of the study: To study the protective effects of isoflavone extract from soybean cake against the UVB-induced skin damage. Materials and methods: The in vitro effects and possible mechanisms of soybean extract on UVB protection were determined in HaCaT cells. In the in vivo study, ICR-Foxn/nu mice were irradiated with UVB. The epidermal thickness, catalase and the expressions of cyclooxygenase-2 (COX-2) and proliferating cell nuclear antigen (PCNA) were detected to evaluate the antioxidant and anti-inflammatory activities of the isoflavone extract. Results: Our in vitro studies showed that UVB-induced HaCaT cell death and the phosphorylation of p38, JNK, and ERK1/2 decreased in the presence of isoflavone extract. In the in vivo studies, we found that the topical application of isoflavone extract before UVB irritation decreased the epidermal thickness and the expressions of COX-2 and PCNA and increased catalase concentration. These results showed anti-photoaging effect of isoflavone extract from soybean cake involved the inhibition of UVB-induced apoptosis and inflammation. Conclusions: It is shown that isoflavone extract from soybean cake may be functional cosmeceutical candidate for skin photoaging. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Exposure of the skin to ultraviolet (UV) radiation, particularly its UVB component (280–320 nm) from sunlight, might results in erythema, edema, hyperplasia, hyperpigmentation, sunburn cells, immunosuppression, photoaging, and tumorigenesis in the skin (Afaq and Mukhtar, 2006). Botanical antioxidants have been reported to have good potential as photoprotective agents (Berson, 2008). For example, dietary grape seed proanthocyanidins resulted in inhibition of the expressions of proliferating cell nuclear antigen
∗ Corresponding author at: School of Medicine, Fu Jen Catholic University, No. 510, Chung-Cheng Road, Hsinchuang, Taipei Hsien, Taiwan, ROC. Tel.: +886 2 29052292; fax: +886 2 29052096. E-mail address: [email protected] (C.-F. Hung). 1 These authors contributed equally. 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.07.039
(PCNA) and cyclooxygenase-2 (COX-2) in the skin (Sharma et al., 2007). Flavonoids are the largest group among plants with active properties; more than 5000 flavonoids have been extracted and indentified (Arct and Pytkowska, 2008). Soybeans are a rich source of those kinds of flavonoids called isoflavones, the most potent ones being genistein and daidzein. Genistein (4 ,5,7-trihydroxyisoflavone) is primarily present in soy, ginkgo biloba extract, Greek oregano, and Greek sage (Afaq and Mukhtar, 2006). Genistein has been shown to possess antioxidant and anti-carcinogenic effects in skin (Wei et al., 1995; Wei et al., 2003). In vitro and in vivo studies have revealed that isoflavones inhibited UV-induced damage (Wei et al., 2003; Kim et al., 2004; Afaq and Mukhtar, 2006; Chiang et al., 2007). Some reports also demonstrated that topical application of genistein before UV-B radiation reduced c-fos and c-jun expression in the SENCAR mouse skin in a dose-dependent manner (Wang et al., 1998). Genistein also had a powerful potential to reduce the inflammatory edematous reaction and suppress contact
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hypersensitivity induced by moderate doses of solar UV irradiation (Widyarini et al., 2001). Soybean cake, a byproduct obtained during the processing of soybean oil, has been shown to be a rich source of isoflavones and other functional components such as saponins and phenolic compounds. Kao and coworkers isolated four isoflavone extracts from soybean cake by chromatography (Kao et al., 2005). They found that the isoflavone extracts showed better antioxidant activities than the isoflavone standards (Kao et al., 2005). Moreover, isoflavone powder produced from soybean cake was effective in decreasing the leukocyte number in mouse blood and lowering the secretions of interleukin (IL)-1, IL-6, nitric oxide (NO), and PGE2 in peritoneal exudate cell supernatant and peritoneal exudate fluid (Kao et al., 2007). Therefore, the aim of this study was to evaluate the in vitro and in vivo anti-photoaging effects of an isoflavone extract including 12 isoflavones from soybean cake. In the in vitro study, we examined the protective effects of isoflavone extract on UVB-induced keratinocyte damage, including its effects on UVB-induced apoptosis, mitogen-activated protein kinase (MAPK) signaling (e.g., extracellular-regulated kinase (ERK1/2), p38, and JNK) in keratinocytes. In the in vivo study, catalase activity, the expressions of COX-2 and PCNA, the epidermal thickness, transepidermal water loss (TEWL), and erythema were assayed to evaluate the anti-photoaging activity of the isoflavone extract.
2.3. Cell viability assay (MTT assay)
2. Materials and methods
2.5. Western blotting
2.1. Chemicals
The analysis of JNK, ERK and p38 was determined by Western blot as previously described (Huang et al., 2005; Wu et al., 2006).
Genistein, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), aprotinin, leupeptin, phenylmethylsulfonyl fluoride (PMSF), sodium fluoride (NaF), and sodium orthovanadate were purchased from Sigma Chemical (St. Louis, MO). The antibody (Ab) raised against p-ERK1/2 was from Santa Cruz Biotechnology (Santa Cruz, CA). Abs raised against p38 and p-JNK were from Cell Signaling Technology (Beverly, MA). Abs raised against JNK, ERK1/2 and p-p38 were from R&D System (Minneapolis, MN). The catalase assay kit was purchased from Molecular Probes (Eugene, OR). COX-2 and PCNA Abs were respectively purchased from Lab Vision (Fremont, CA) and Santa Cruz Biotechnology.
2.2. Extraction of soybean isoflavones Isoflavone extract including 12 isoflavones (ISO-1) were prepared by a method previously described by Kao et al. (2007). Briefly, a 50 g soybean cake sample was mixed with 150 mL of ethanol/water (1:1, v/v) and shaken at room temperature for 2 h. Then the extracted sample was centrifuged at 6000 rpm for 20 min at 25 ◦ C. The supernatant was collected, filtered through a glass filter paper, and then dried in a freeze-drying system. A powdered product of 9.5 g was produced with a level of total isoflavone of 43,839 g/g by high-performance liquid chromatographic (HPLC) analysis. A portion (0.52 g) of powder was dissolved in 6.9 mL of deionized water to obtain the total isoflavone content at 3.33 mg/mL. The isoflavone extract from soybean cake is water-soluble, so the tested isoflavone was dissolved in water and genistein was dissolved in 1:1 ethanol:propylene (Wei et al., 2002) for the in vitro and in vivo experiments. A control group was maintained without exposure to UVB irradiation for each of the isoflavone extract and genistein experiments. On the other hand, we considered the transepidermal absorption that the higher concentration (1 and 3 mg/mL) was used in vivo experiments than in vitro experiments (30 g/mL).
Human immortalized keratinocytes (HaCaT cells) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (GibcoBRL, Invitrogen Life Technologies, Carlsbad, CA), 100 units/mL penicillin, and 100 g/mL streptomycin (Sigma). Briefly, vehicle-, isoflavone-, or genistein-pretreated cells were exposed to UVB and incubated for an additional 24 h. Cells were irradiated in a Bio-Sun system illuminator from VL (Vilber Lourmat, France) with a UV peak at 312 nm. The UVB irradiation dose was 50 mJ/cm2 which took approximately 44–48 s to administer (at an irradiance of 1.26–1.29 mW/cm2 ). After a brief wash with medium, MTT (0.5 mg/mL in DMEM) was used to quantify living metabolically active cells. Mitochondrial dehydrogenases metabolize MTT to a purple formazan dye, which was measured photometrically at 550 nm. Cell viability was proportional to the absorbance measured. 2.4. Flow cytometric analysis of the cell cycle Cell-cycle determination was performed using a Partec CyFlow ML flow cytometer (Partech, Munster, Germany). Flow cytometric histograms were analyzed by defining borders between the preG1 , G1 , S, and G2 + M phases from an asynchronous population and using these boundaries to analyze the experimental samples.
2.6. Animals and measurement of physiological skin functions Six-week-old male ICR-Foxn/nu mice were obtained from the National Laboratory Animal Center, Taipei, Taiwan. Mice were randomized into five groups for the experiments and then animals were housed eight per cage with controlled temperature (21–25 ◦ C), humidity (60 ± 5%), and light (12/12 h light/dark cycle) for 1 week. During the week, animals were allowed free access to water and food. ICR-Foxn/nu mouse dorsal skin was treated with different concentrations of the isoflavone extract or genistein before 150 mJ/cm2 UVB irradiation (Gali-Muhtasib et al., 1998; Moison et al., 2001) for 7 days. The TEWL, erythema, melanin and blood flow were measured before the first UVB irradiation and 24 h after the UVB irradiation. The surface changes in the dorsal skin were recorded by photography. The level of TEWL, erythema, melanin, and blood flow were regularly measured with MPA-580 (Courage & Khazaka, Cologne, Germany) and FLO-N1 (Omegawave, Tokyo, Japan). 2.7. Histology and immunostaining Skin specimens were excised for histological examination. They were immediately fixed in 10% neutral buffered formaldehyde and frozen in optimal cutting temperature (OCT) compound (Thermo, Waltham, MA) for hematoxylin and eosin staining as well as immunohistochemistry. Vertical sections of 7-m thickness were cut with a cryotome (Leica CM3050, Wetzlar, Germany). The following primary Abs were used: affinity-purified rabbit polyclonal COX-2 Ab and rabbit polyclonal PCNA Ab. All incubations with primary Abs were performed in blocking buffer overnight at 4 ◦ C. After incubation with the primary Ab, samples were incubated with biotinylated secondary Abs (anti-rabbit IgG antibody, PerkinElmer) for 2 h at RT. Images from immunostaining were obtained using an Olympus IX70 Inverted System microscope (Tokyo, Japan) and SPOT Cam software (Sterling Heights, MI). Quantification was made
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highly fluorescent oxidation product, resorufin. Therefore, as catalase activity increases, the resorufin signal decreases. The results are typically plotted by subtracting the observed fluorescence from that of a no-catalase control. 2.9. Statistical analysis Values are presented as a percentage of the control group and shown as the mean ± S.E. Student’s t-test was used to assess the statistical significance between the groups. We considered p values of <0.05 as statistically significant. 3. Results 3.1. In vitro study 3.1.1. Isoflavone extract inhibited UVB-induced cell death As shown in Fig. 1(A), treatment with the isoflavone extract (30 g/mL) resulted in 95.2 ± 5.5% of cell viability compared with the non-UVB irradiation group. However, cell viability dosedependently decreased after treatment with the genistein standard (85.3 ± 8.5%, 69.1 ± 4.7%, and 44.9 ± 4.3% at 3, 10, and 30 g/mL of genistein, respectively). The data indicates that isoflavone extract from soybean cake is relatively safe for keratinocytes. To clarify the photoprotective effects of the isoflavone extracts on keratinocytes, cells were treated with isoflavone extract (3–30 g/mL) and 50 mJ/cm2 UVB irradiation. Our data showed that UVB irradiationinduced cell death decreased after treatment with the isoflavone extract (Fig. 1(A)). Treatment with 10 g/mL of the isoflavone extract had a maximum effect. Since the induction of apoptosis by UVB irradiation may be mediated through regulation of the cell cycle, we next examined the effect of the isoflavone extract on cell cycle perturbations by flow cytometry. As shown in Fig. 1(B), UVB irradiation resulted in an appreciable arrest in the pre-G1 phase and a peculiar pre-G1 peak in DNA content. Treatment with the soybean extract at 3, 10, and 30 g/mL before UVB irradiation resulted in a decrease in apoptotic DNA oligonucleosomic strand-breaks (Fig. 1(B)).
Fig. 1. (A) The ISO-1 was not cytotoxic to keratinocytes: () ISO-1; () genistein. UVB irradiation-induced cell death decreased after treatment with the ISO-1 by MTT assay. (B) ISO-1 inhibited UVB irradiation-induced cell death by flow cytometry. Results in (A) and (B) were expressed as a percentage of control and the mean ± S.E. (n = 3). * p < 0.05 vs. the control. (C) Effects of ISO-1 on UVB-induced ERK1/2, p38, and JNK phosphorylation. HaCaT cells were preincubated with ISO-1 for 24 h, followed by UVB irradiation (50 mJ/cm2 ). After further incubation, cells were collected and lysates were analyzed by Western blot analysis.
by counting the various positive cells in an arbitrarily selected field at 400× magnification and the thickness of the epidermis was quantified by Image J 1.3.4s. 2.8. Catalase assay Skin homogenates were centrifuged at 5000 rpm for 10 min, and the supernatant fractions were used. Catalase was analyzed according to the manufacturer’s protocol. Briefly, catalase first reacts with H2 O2 to produce water and oxygen (O2 ). Next the Amplex Red reagent reacts with a 1:1 stoichiometry with any unreacted H2 O2 in the presence of horseradish peroxidase (HRP) to produce the
3.1.2. Isoflavone extract inhibited the UVB-induced MAPK signaling pathway UVB irradiation is known to activate ERK1/2, JNK, and p38 kinase (Katiyar et al., 2001; Wu et al., 2006), which may contribute to skin cell damage. Thus, we examined if these isoflavone extract affected UVB-induced MAPK activation. We observed that ERK1/2, JNK, and p38 phosphorylations were obviously increased in UVBirradiated keratinocytes and could be inhibited by treatment with the isoflavone extract (3–30 g/mL) (Fig. 1(C)). 3.2. In vivo study 3.2.1. Isoflavone extract attenuated the level of erythema and TEWL after UVB exposure We found that the wrinkles in UVB-exposed skin increased after 7 days compared with non-exposed skin. UVB-exposed skin was desquamated and this manifestation was inhibited by the treatments of isoflavone extract and genistein (data not shown). Furthermore, TEWL and erythema in UVB-exposed skin increased after 7-day UVB exposure compared with non-exposed skin (Fig. 1). However, UVB-induced erythema was inhibited significantly by the treatment of isoflavone extract or genistein (Fig. 2(A) and (B)). The TEWL level was also inhibited by the treatment of isoflavone extract and genistein (Fig. 2(C) and (D)). Moreover, our data showed that the treatment of isoflavone extract inhibited the TEWL more potently than the treatment of genistein. The inhibitory effect on erythema was similar between the isoflavone extract and genistein
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Fig. 2. Change in skin surface physiology parameter after treatments with genistein and isoflavone extract. (A and B) Change in erythema after treatments with isoflavone extract (ISO-1) and genistein. (C and D) Change in transepidermal water loss (TEWL) after treatments with ISO-1 and genistein.
groups (Fig. 2). There were no significant differences in melanin or blood flow between the UVB-irradiated group and those treated with the isoflavone extracts or genistein before UVB irradiation (data not shown). 3.2.2. Isoflavone extract decreased the epidermal thickness In the control group, the epidermal thicknesses of the dorsal skin of mice were 10.4 ± 1.3 m (genistein group) and 12.6 ± 2.7 m (isoflavone extract group). We found that the thickness of the epidermis increased after UVB exposure. The average of epidermal thicknesses was 33.3 ± 3.4 and 33.6 ± 3.5 m in the genistein and isoflavone extract groups, respectively. After treatment with the isoflavone extract or genistein before UVB irradiation, the epidermal thickness was significantly lower than that of the UVB irradiation group (p < 0.05). The average of epidermal thicknesses in the 1 and 3 mg/mL isoflavone extract-treated groups were 21.3 ± 3.0 and 23.6 ± 3.2 m, respectively, and were 23.5 ± 3.5 and 25.0 ± 5.2 m in the genistein-treated groups (Table 1). 3.2.3. Isoflavon extract attenuated the expression of COX-2 and PCNA after UVB exposure In our study, COX-2 expression was observed in epidermal cells of the dorsal skin of mice after UVB exposure. Topical application with the isoflavone extract or genistein significantly reduced COX2 expression (Fig. 3(A)). Furthermore, the results showed that the treatment with isoflavone extract inhibited COX-2 expression more potently than the treatment with genistein. As shown in Fig. 3(B), PCNA expression, a marker of DNA repair and indirectly as an indicator of UVB-induced damage, was higher in the UVB group than
in the control group. PCNA expression was inhibited by treatment with the isoflavone extract and genistein (Fig. 3(B)). 3.2.4. Isoflavone extract decreased the depletion of catalase after UVB exposure Catalase removes UVB-induced H2 O2 and converts it to oxygen and water. Thus it contributes to reduction of oxidative stress. UVB irradiation for 7 days resulted in significant depletion of catalase in the dorsal skin of ICR-Foxn/nu mice. As shown in Fig. 4, we found that the activity of catalase almost decreased 50% after UVB irradiation (day 7). Interestingly, isoflavone extract-treated, irradiated mice exhibited higher catalase activity compared with vehicletreated, irradiated animals. However, genistein-treated, irradiated mice did not differ compared with vehicle-treated, irradiated animals (Fig. 4). 4. Discussion In our study, we found that isoflavone extract from soybean cake attenuated UVB-induced keratinocyte death. Several studies indicated that the activation of MAPK correlated with skin cell death in response to UVB (Assefa et al., 2005; Wu et al., 2006; Huang et al., 2007). Moreover, previous studies demonstrated that reactive oxygen species (ROS), in particular H2 O2 , acted as mediators to activate MAP kinase pathway and induced cellular responses following UVB exposure (Peus et al., 1999). In our study, we found that UVB-induced the activation of ERK1/2, p38 and JNK in keratinocytes could be inhibited by isoflavone extract. The attenuation of catalase level after UVB exposure could recover with the treatment of
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Table 1 The thickness of the epidermis increased after UVB exposure, and the thickening could be attenuated by pretreatments with genistein and isoflavone extract (ISO-1).
Genistein Isoflavone extract *
Normal control (m)
Drug control (m)
UVB control (m)
1 mg/mL (m)
3 mg/mL (m)
10.4 ± 1.3 12.6 ± 2.7
11.7 ± 0.4 9.2 ± 0.8
33.3 ± 3.4 33.6 ± 3.5
21.3 ± 3.0 23.5 ± 3.5*
23.6 ± 3.2* 25.0 ± 5.2*
*
p < 0.05 compared to the UVB control group.
Fig. 3. (A) UVB irradiation increased cyclooxygenase-2 (COX-2) expression, which was inhibited by the treatments of genistein and isoflavone extract (ISO-1). (B) UVB irradiation increased proliferating cell nuclear antigen (PCNA) expression, which was inhibited by the treatments of genistein and ISO-1.
Fig. 4. Effects of genistein and the isoflavone extract (ISO-1) on catalase activity. Data was shown as the mean ± S.E. (n = 4). * p < 0.05 compared to the control group.
isoflavone extract. These results indicated that isoflavone extract could reduce the UVB-induced damage by its antioxidant activity and thus protect skin from the UVB-induced injury. In addition, early activation of signaling pathways in response to UV radiation may be involved in the erythema and inflammatory reactions, photoaging various photodermatoses, and carcinogenesis (Katiyar et al., 2001). Several studies have shown that acute UVB exposure caused TEWL (Haratake et al., 1997), erythema (Afaq and Mukhtar, 2006; Herr et al., 2007), decreased catalase activity (Erden Inal et al., 2001), epidermal proliferation (Moore et al., 2004), and COX-2 (Mahns et al., 2004; Chun and Langenbach, 2007; Rundhaug et al., 2007) and PCNA (Ahsan et al., 2007) expression in skin. In our study, epidermal proliferation, and the expressions of COX-2 and PCNA after UVB exposure were inhibited after treatment with the isoflavone extract. Thus, we also suggest that the decrease of phosphorylation of ERK1/2, JNK and p38 contributes to these protective effects against the UVB-induced skin damages. On the other hand, we found that the inhibitory effect of TEWL after treatment with isoflavone extract was better than that with the genistein. There was no difference in the inhibitory effect on erythema after
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treatments with isoflavone extract and genistein. In present data, we cannot explain the reasons and need more studies to explore the exact mechanisms. In summary, our results reveal that isoflavone extract from soybean cake could decrease the early activation of signaling pathway in response to UVB. Furthermore, isoflavone extract prevents skin cell apoptosis, erythema and inflammation reactions. Thus, the isoflavone extract from soybean cake is a good candidate for an anti-photoaging agent in skin care. Developing skin care products containing an isoflavone extract from soybean cake has many advantages, such as convenience, economy, and environmental protection. Acknowledgements This work was supported by research grants from Changhua Christian Hospital, Shin Kong Wu Ho-Su Memorial Hospital and the National Science Council of Taiwan. References Afaq, F., Mukhtar, H., 2006. Botanical antioxidants in the prevention of photocarcinogenesis and photoaging. Experimental Dermatology 15, 678–684. Ahsan, H., Reagan-Shaw, S., Eggert, D.M., Tan, T.C., Afaq, F., Mukhtar, H., Ahmad, N., 2007. Protective effect of sanguinarine on ultraviolet B-mediated damages in SKH-1 hairless mouse skin: implications for prevention of skin cancer. Photochemistry and Photobiology 83, 986–993. Arct, J., Pytkowska, K., 2008. Flavonoids as components of biologically active cosmeceuticals. Clinical Dermatology 26, 347–357. Assefa, Z., Van Laethem, A., Garmyn, M., Agostinis, P., 2005. Ultraviolet radiationinduced apoptosis in keratinocytes: on the role of cytosolic factors. Biochimica et Biophysica Acta 1755, 90–106. Berson, D.S., 2008. Natural antioxidants. Journal of Drugs in Dermatology 7, s7–12. Chiang, H.S., Wu, W.B., Fang, J.Y., Chen, B.H., Kao, T.H., Chen, Y.T., Huang, C.C., Hung, C.F., 2007. The UVB-protective effects of isoflavone extracts from soybean cake in human keratinocytes. International Journal of Molecular Sciences 8, 651–661. Chun, K.S., Langenbach, R., 2007. A proposed COX-2 and PGE(2) receptor interaction in UV-exposed mouse skin. Molecular Carcinogenesis 46, 699–704. Erden Inal, M., Kahraman, A., Koken, T., 2001. Beneficial effects of quercetin on oxidative stress induced by ultraviolet A. Clinical and Experimental Dermatology 26, 536–539. Gali-Muhtasib, H.U., Perchellet, J.P., Khatib, S.H., 1998. Inhibitory effects of plant tannins on ultraviolet light-induced epidermal DNA synthesis in hairless mice. Photochemistry and Photobiology 67, 663–668. Haratake, A., Uchida, Y., Schmuth, M., Tanno, O., Yasuda, R., Epstein, J.H., Elias, P.M., Holleran, W.M., 1997. UVB-induced alterations in permeability barrier function: roles for epidermal hyperproliferation and thymocyte-mediated response. The Journal of Investigative Dermatology 108, 769–775. Herr, H., Cho, H.J., Yu, S., 2007. Burns caused by accidental overdose of photochemotherapy (PUVA). Burns 33, 372–375. Huang, C.C., Fang, J.Y., Wu, W.B., Chiang, H.S., Wei, Y.J., Hung, C.F., 2005. Protective effects of (−)-epicatechin-3-gallate on UVA-induced damage in HaCaT keratinocytes. Archives of Dermatological Research 296, 473–481.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
In vitro and in vivo anti-photoaging effects of an isoflavone extract from soybean cake Tsu-Man Chiu a,1 , Chieh-Chen Huang b,c,1 , Tzu-Ju Lin d , Jia-You Fang e , Nan-Lin Wu f , Chi-Feng Hung b,d,∗ a
Department of Dermatology, Changhua Christian Hospital, Changhua Hsien, Taiwan, ROC School of Medicine, Fu Jen Catholic University, Taipei Hsien, Taiwan, ROC Department of Dermatology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan, ROC d Graduate Institute of Basic Medicine, Fu-Jen Catholic University, Taipei Hsien, Taiwan, ROC e Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan, ROC f Department of Dermatology, Mackay Memorial Hospital, Hsinchu, Taiwan, ROC b c
a r t i c l e
i n f o
Article history: Received 28 October 2008 Received in revised form 22 June 2009 Accepted 31 July 2009 Available online 11 August 2009 Keywords: Isoflavone Genistein UVB COX-2 PCNA Catalase
a b s t r a c t Ethnopharmacological relevance: Soy has been used in traditional Chinese medicine for thousands of years for its health and nutritional benefits, as well as to treat and care for the skin. Advanced skin care research has shown that soy isoflavone and genistein are effective in reducing damage to the skin from the sun. Aim of the study: To study the protective effects of isoflavone extract from soybean cake against the UVB-induced skin damage. Materials and methods: The in vitro effects and possible mechanisms of soybean extract on UVB protection were determined in HaCaT cells. In the in vivo study, ICR-Foxn/nu mice were irradiated with UVB. The epidermal thickness, catalase and the expressions of cyclooxygenase-2 (COX-2) and proliferating cell nuclear antigen (PCNA) were detected to evaluate the antioxidant and anti-inflammatory activities of the isoflavone extract. Results: Our in vitro studies showed that UVB-induced HaCaT cell death and the phosphorylation of p38, JNK, and ERK1/2 decreased in the presence of isoflavone extract. In the in vivo studies, we found that the topical application of isoflavone extract before UVB irritation decreased the epidermal thickness and the expressions of COX-2 and PCNA and increased catalase concentration. These results showed anti-photoaging effect of isoflavone extract from soybean cake involved the inhibition of UVB-induced apoptosis and inflammation. Conclusions: It is shown that isoflavone extract from soybean cake may be functional cosmeceutical candidate for skin photoaging. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Exposure of the skin to ultraviolet (UV) radiation, particularly its UVB component (280–320 nm) from sunlight, might results in erythema, edema, hyperplasia, hyperpigmentation, sunburn cells, immunosuppression, photoaging, and tumorigenesis in the skin (Afaq and Mukhtar, 2006). Botanical antioxidants have been reported to have good potential as photoprotective agents (Berson, 2008). For example, dietary grape seed proanthocyanidins resulted in inhibition of the expressions of proliferating cell nuclear antigen
∗ Corresponding author at: School of Medicine, Fu Jen Catholic University, No. 510, Chung-Cheng Road, Hsinchuang, Taipei Hsien, Taiwan, ROC. Tel.: +886 2 29052292; fax: +886 2 29052096. E-mail address: [email protected] (C.-F. Hung). 1 These authors contributed equally. 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.07.039
(PCNA) and cyclooxygenase-2 (COX-2) in the skin (Sharma et al., 2007). Flavonoids are the largest group among plants with active properties; more than 5000 flavonoids have been extracted and indentified (Arct and Pytkowska, 2008). Soybeans are a rich source of those kinds of flavonoids called isoflavones, the most potent ones being genistein and daidzein. Genistein (4 ,5,7-trihydroxyisoflavone) is primarily present in soy, ginkgo biloba extract, Greek oregano, and Greek sage (Afaq and Mukhtar, 2006). Genistein has been shown to possess antioxidant and anti-carcinogenic effects in skin (Wei et al., 1995; Wei et al., 2003). In vitro and in vivo studies have revealed that isoflavones inhibited UV-induced damage (Wei et al., 2003; Kim et al., 2004; Afaq and Mukhtar, 2006; Chiang et al., 2007). Some reports also demonstrated that topical application of genistein before UV-B radiation reduced c-fos and c-jun expression in the SENCAR mouse skin in a dose-dependent manner (Wang et al., 1998). Genistein also had a powerful potential to reduce the inflammatory edematous reaction and suppress contact
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hypersensitivity induced by moderate doses of solar UV irradiation (Widyarini et al., 2001). Soybean cake, a byproduct obtained during the processing of soybean oil, has been shown to be a rich source of isoflavones and other functional components such as saponins and phenolic compounds. Kao and coworkers isolated four isoflavone extracts from soybean cake by chromatography (Kao et al., 2005). They found that the isoflavone extracts showed better antioxidant activities than the isoflavone standards (Kao et al., 2005). Moreover, isoflavone powder produced from soybean cake was effective in decreasing the leukocyte number in mouse blood and lowering the secretions of interleukin (IL)-1, IL-6, nitric oxide (NO), and PGE2 in peritoneal exudate cell supernatant and peritoneal exudate fluid (Kao et al., 2007). Therefore, the aim of this study was to evaluate the in vitro and in vivo anti-photoaging effects of an isoflavone extract including 12 isoflavones from soybean cake. In the in vitro study, we examined the protective effects of isoflavone extract on UVB-induced keratinocyte damage, including its effects on UVB-induced apoptosis, mitogen-activated protein kinase (MAPK) signaling (e.g., extracellular-regulated kinase (ERK1/2), p38, and JNK) in keratinocytes. In the in vivo study, catalase activity, the expressions of COX-2 and PCNA, the epidermal thickness, transepidermal water loss (TEWL), and erythema were assayed to evaluate the anti-photoaging activity of the isoflavone extract.
2.3. Cell viability assay (MTT assay)
2. Materials and methods
2.5. Western blotting
2.1. Chemicals
The analysis of JNK, ERK and p38 was determined by Western blot as previously described (Huang et al., 2005; Wu et al., 2006).
Genistein, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), aprotinin, leupeptin, phenylmethylsulfonyl fluoride (PMSF), sodium fluoride (NaF), and sodium orthovanadate were purchased from Sigma Chemical (St. Louis, MO). The antibody (Ab) raised against p-ERK1/2 was from Santa Cruz Biotechnology (Santa Cruz, CA). Abs raised against p38 and p-JNK were from Cell Signaling Technology (Beverly, MA). Abs raised against JNK, ERK1/2 and p-p38 were from R&D System (Minneapolis, MN). The catalase assay kit was purchased from Molecular Probes (Eugene, OR). COX-2 and PCNA Abs were respectively purchased from Lab Vision (Fremont, CA) and Santa Cruz Biotechnology.
2.2. Extraction of soybean isoflavones Isoflavone extract including 12 isoflavones (ISO-1) were prepared by a method previously described by Kao et al. (2007). Briefly, a 50 g soybean cake sample was mixed with 150 mL of ethanol/water (1:1, v/v) and shaken at room temperature for 2 h. Then the extracted sample was centrifuged at 6000 rpm for 20 min at 25 ◦ C. The supernatant was collected, filtered through a glass filter paper, and then dried in a freeze-drying system. A powdered product of 9.5 g was produced with a level of total isoflavone of 43,839 g/g by high-performance liquid chromatographic (HPLC) analysis. A portion (0.52 g) of powder was dissolved in 6.9 mL of deionized water to obtain the total isoflavone content at 3.33 mg/mL. The isoflavone extract from soybean cake is water-soluble, so the tested isoflavone was dissolved in water and genistein was dissolved in 1:1 ethanol:propylene (Wei et al., 2002) for the in vitro and in vivo experiments. A control group was maintained without exposure to UVB irradiation for each of the isoflavone extract and genistein experiments. On the other hand, we considered the transepidermal absorption that the higher concentration (1 and 3 mg/mL) was used in vivo experiments than in vitro experiments (30 g/mL).
Human immortalized keratinocytes (HaCaT cells) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (GibcoBRL, Invitrogen Life Technologies, Carlsbad, CA), 100 units/mL penicillin, and 100 g/mL streptomycin (Sigma). Briefly, vehicle-, isoflavone-, or genistein-pretreated cells were exposed to UVB and incubated for an additional 24 h. Cells were irradiated in a Bio-Sun system illuminator from VL (Vilber Lourmat, France) with a UV peak at 312 nm. The UVB irradiation dose was 50 mJ/cm2 which took approximately 44–48 s to administer (at an irradiance of 1.26–1.29 mW/cm2 ). After a brief wash with medium, MTT (0.5 mg/mL in DMEM) was used to quantify living metabolically active cells. Mitochondrial dehydrogenases metabolize MTT to a purple formazan dye, which was measured photometrically at 550 nm. Cell viability was proportional to the absorbance measured. 2.4. Flow cytometric analysis of the cell cycle Cell-cycle determination was performed using a Partec CyFlow ML flow cytometer (Partech, Munster, Germany). Flow cytometric histograms were analyzed by defining borders between the preG1 , G1 , S, and G2 + M phases from an asynchronous population and using these boundaries to analyze the experimental samples.
2.6. Animals and measurement of physiological skin functions Six-week-old male ICR-Foxn/nu mice were obtained from the National Laboratory Animal Center, Taipei, Taiwan. Mice were randomized into five groups for the experiments and then animals were housed eight per cage with controlled temperature (21–25 ◦ C), humidity (60 ± 5%), and light (12/12 h light/dark cycle) for 1 week. During the week, animals were allowed free access to water and food. ICR-Foxn/nu mouse dorsal skin was treated with different concentrations of the isoflavone extract or genistein before 150 mJ/cm2 UVB irradiation (Gali-Muhtasib et al., 1998; Moison et al., 2001) for 7 days. The TEWL, erythema, melanin and blood flow were measured before the first UVB irradiation and 24 h after the UVB irradiation. The surface changes in the dorsal skin were recorded by photography. The level of TEWL, erythema, melanin, and blood flow were regularly measured with MPA-580 (Courage & Khazaka, Cologne, Germany) and FLO-N1 (Omegawave, Tokyo, Japan). 2.7. Histology and immunostaining Skin specimens were excised for histological examination. They were immediately fixed in 10% neutral buffered formaldehyde and frozen in optimal cutting temperature (OCT) compound (Thermo, Waltham, MA) for hematoxylin and eosin staining as well as immunohistochemistry. Vertical sections of 7-m thickness were cut with a cryotome (Leica CM3050, Wetzlar, Germany). The following primary Abs were used: affinity-purified rabbit polyclonal COX-2 Ab and rabbit polyclonal PCNA Ab. All incubations with primary Abs were performed in blocking buffer overnight at 4 ◦ C. After incubation with the primary Ab, samples were incubated with biotinylated secondary Abs (anti-rabbit IgG antibody, PerkinElmer) for 2 h at RT. Images from immunostaining were obtained using an Olympus IX70 Inverted System microscope (Tokyo, Japan) and SPOT Cam software (Sterling Heights, MI). Quantification was made
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highly fluorescent oxidation product, resorufin. Therefore, as catalase activity increases, the resorufin signal decreases. The results are typically plotted by subtracting the observed fluorescence from that of a no-catalase control. 2.9. Statistical analysis Values are presented as a percentage of the control group and shown as the mean ± S.E. Student’s t-test was used to assess the statistical significance between the groups. We considered p values of <0.05 as statistically significant. 3. Results 3.1. In vitro study 3.1.1. Isoflavone extract inhibited UVB-induced cell death As shown in Fig. 1(A), treatment with the isoflavone extract (30 g/mL) resulted in 95.2 ± 5.5% of cell viability compared with the non-UVB irradiation group. However, cell viability dosedependently decreased after treatment with the genistein standard (85.3 ± 8.5%, 69.1 ± 4.7%, and 44.9 ± 4.3% at 3, 10, and 30 g/mL of genistein, respectively). The data indicates that isoflavone extract from soybean cake is relatively safe for keratinocytes. To clarify the photoprotective effects of the isoflavone extracts on keratinocytes, cells were treated with isoflavone extract (3–30 g/mL) and 50 mJ/cm2 UVB irradiation. Our data showed that UVB irradiationinduced cell death decreased after treatment with the isoflavone extract (Fig. 1(A)). Treatment with 10 g/mL of the isoflavone extract had a maximum effect. Since the induction of apoptosis by UVB irradiation may be mediated through regulation of the cell cycle, we next examined the effect of the isoflavone extract on cell cycle perturbations by flow cytometry. As shown in Fig. 1(B), UVB irradiation resulted in an appreciable arrest in the pre-G1 phase and a peculiar pre-G1 peak in DNA content. Treatment with the soybean extract at 3, 10, and 30 g/mL before UVB irradiation resulted in a decrease in apoptotic DNA oligonucleosomic strand-breaks (Fig. 1(B)).
Fig. 1. (A) The ISO-1 was not cytotoxic to keratinocytes: () ISO-1; () genistein. UVB irradiation-induced cell death decreased after treatment with the ISO-1 by MTT assay. (B) ISO-1 inhibited UVB irradiation-induced cell death by flow cytometry. Results in (A) and (B) were expressed as a percentage of control and the mean ± S.E. (n = 3). * p < 0.05 vs. the control. (C) Effects of ISO-1 on UVB-induced ERK1/2, p38, and JNK phosphorylation. HaCaT cells were preincubated with ISO-1 for 24 h, followed by UVB irradiation (50 mJ/cm2 ). After further incubation, cells were collected and lysates were analyzed by Western blot analysis.
by counting the various positive cells in an arbitrarily selected field at 400× magnification and the thickness of the epidermis was quantified by Image J 1.3.4s. 2.8. Catalase assay Skin homogenates were centrifuged at 5000 rpm for 10 min, and the supernatant fractions were used. Catalase was analyzed according to the manufacturer’s protocol. Briefly, catalase first reacts with H2 O2 to produce water and oxygen (O2 ). Next the Amplex Red reagent reacts with a 1:1 stoichiometry with any unreacted H2 O2 in the presence of horseradish peroxidase (HRP) to produce the
3.1.2. Isoflavone extract inhibited the UVB-induced MAPK signaling pathway UVB irradiation is known to activate ERK1/2, JNK, and p38 kinase (Katiyar et al., 2001; Wu et al., 2006), which may contribute to skin cell damage. Thus, we examined if these isoflavone extract affected UVB-induced MAPK activation. We observed that ERK1/2, JNK, and p38 phosphorylations were obviously increased in UVBirradiated keratinocytes and could be inhibited by treatment with the isoflavone extract (3–30 g/mL) (Fig. 1(C)). 3.2. In vivo study 3.2.1. Isoflavone extract attenuated the level of erythema and TEWL after UVB exposure We found that the wrinkles in UVB-exposed skin increased after 7 days compared with non-exposed skin. UVB-exposed skin was desquamated and this manifestation was inhibited by the treatments of isoflavone extract and genistein (data not shown). Furthermore, TEWL and erythema in UVB-exposed skin increased after 7-day UVB exposure compared with non-exposed skin (Fig. 1). However, UVB-induced erythema was inhibited significantly by the treatment of isoflavone extract or genistein (Fig. 2(A) and (B)). The TEWL level was also inhibited by the treatment of isoflavone extract and genistein (Fig. 2(C) and (D)). Moreover, our data showed that the treatment of isoflavone extract inhibited the TEWL more potently than the treatment of genistein. The inhibitory effect on erythema was similar between the isoflavone extract and genistein
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Fig. 2. Change in skin surface physiology parameter after treatments with genistein and isoflavone extract. (A and B) Change in erythema after treatments with isoflavone extract (ISO-1) and genistein. (C and D) Change in transepidermal water loss (TEWL) after treatments with ISO-1 and genistein.
groups (Fig. 2). There were no significant differences in melanin or blood flow between the UVB-irradiated group and those treated with the isoflavone extracts or genistein before UVB irradiation (data not shown). 3.2.2. Isoflavone extract decreased the epidermal thickness In the control group, the epidermal thicknesses of the dorsal skin of mice were 10.4 ± 1.3 m (genistein group) and 12.6 ± 2.7 m (isoflavone extract group). We found that the thickness of the epidermis increased after UVB exposure. The average of epidermal thicknesses was 33.3 ± 3.4 and 33.6 ± 3.5 m in the genistein and isoflavone extract groups, respectively. After treatment with the isoflavone extract or genistein before UVB irradiation, the epidermal thickness was significantly lower than that of the UVB irradiation group (p < 0.05). The average of epidermal thicknesses in the 1 and 3 mg/mL isoflavone extract-treated groups were 21.3 ± 3.0 and 23.6 ± 3.2 m, respectively, and were 23.5 ± 3.5 and 25.0 ± 5.2 m in the genistein-treated groups (Table 1). 3.2.3. Isoflavon extract attenuated the expression of COX-2 and PCNA after UVB exposure In our study, COX-2 expression was observed in epidermal cells of the dorsal skin of mice after UVB exposure. Topical application with the isoflavone extract or genistein significantly reduced COX2 expression (Fig. 3(A)). Furthermore, the results showed that the treatment with isoflavone extract inhibited COX-2 expression more potently than the treatment with genistein. As shown in Fig. 3(B), PCNA expression, a marker of DNA repair and indirectly as an indicator of UVB-induced damage, was higher in the UVB group than
in the control group. PCNA expression was inhibited by treatment with the isoflavone extract and genistein (Fig. 3(B)). 3.2.4. Isoflavone extract decreased the depletion of catalase after UVB exposure Catalase removes UVB-induced H2 O2 and converts it to oxygen and water. Thus it contributes to reduction of oxidative stress. UVB irradiation for 7 days resulted in significant depletion of catalase in the dorsal skin of ICR-Foxn/nu mice. As shown in Fig. 4, we found that the activity of catalase almost decreased 50% after UVB irradiation (day 7). Interestingly, isoflavone extract-treated, irradiated mice exhibited higher catalase activity compared with vehicletreated, irradiated animals. However, genistein-treated, irradiated mice did not differ compared with vehicle-treated, irradiated animals (Fig. 4). 4. Discussion In our study, we found that isoflavone extract from soybean cake attenuated UVB-induced keratinocyte death. Several studies indicated that the activation of MAPK correlated with skin cell death in response to UVB (Assefa et al., 2005; Wu et al., 2006; Huang et al., 2007). Moreover, previous studies demonstrated that reactive oxygen species (ROS), in particular H2 O2 , acted as mediators to activate MAP kinase pathway and induced cellular responses following UVB exposure (Peus et al., 1999). In our study, we found that UVB-induced the activation of ERK1/2, p38 and JNK in keratinocytes could be inhibited by isoflavone extract. The attenuation of catalase level after UVB exposure could recover with the treatment of
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Table 1 The thickness of the epidermis increased after UVB exposure, and the thickening could be attenuated by pretreatments with genistein and isoflavone extract (ISO-1).
Genistein Isoflavone extract *
Normal control (m)
Drug control (m)
UVB control (m)
1 mg/mL (m)
3 mg/mL (m)
10.4 ± 1.3 12.6 ± 2.7
11.7 ± 0.4 9.2 ± 0.8
33.3 ± 3.4 33.6 ± 3.5
21.3 ± 3.0 23.5 ± 3.5*
23.6 ± 3.2* 25.0 ± 5.2*
*
p < 0.05 compared to the UVB control group.
Fig. 3. (A) UVB irradiation increased cyclooxygenase-2 (COX-2) expression, which was inhibited by the treatments of genistein and isoflavone extract (ISO-1). (B) UVB irradiation increased proliferating cell nuclear antigen (PCNA) expression, which was inhibited by the treatments of genistein and ISO-1.
Fig. 4. Effects of genistein and the isoflavone extract (ISO-1) on catalase activity. Data was shown as the mean ± S.E. (n = 4). * p < 0.05 compared to the control group.
isoflavone extract. These results indicated that isoflavone extract could reduce the UVB-induced damage by its antioxidant activity and thus protect skin from the UVB-induced injury. In addition, early activation of signaling pathways in response to UV radiation may be involved in the erythema and inflammatory reactions, photoaging various photodermatoses, and carcinogenesis (Katiyar et al., 2001). Several studies have shown that acute UVB exposure caused TEWL (Haratake et al., 1997), erythema (Afaq and Mukhtar, 2006; Herr et al., 2007), decreased catalase activity (Erden Inal et al., 2001), epidermal proliferation (Moore et al., 2004), and COX-2 (Mahns et al., 2004; Chun and Langenbach, 2007; Rundhaug et al., 2007) and PCNA (Ahsan et al., 2007) expression in skin. In our study, epidermal proliferation, and the expressions of COX-2 and PCNA after UVB exposure were inhibited after treatment with the isoflavone extract. Thus, we also suggest that the decrease of phosphorylation of ERK1/2, JNK and p38 contributes to these protective effects against the UVB-induced skin damages. On the other hand, we found that the inhibitory effect of TEWL after treatment with isoflavone extract was better than that with the genistein. There was no difference in the inhibitory effect on erythema after
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treatments with isoflavone extract and genistein. In present data, we cannot explain the reasons and need more studies to explore the exact mechanisms. In summary, our results reveal that isoflavone extract from soybean cake could decrease the early activation of signaling pathway in response to UVB. Furthermore, isoflavone extract prevents skin cell apoptosis, erythema and inflammation reactions. Thus, the isoflavone extract from soybean cake is a good candidate for an anti-photoaging agent in skin care. Developing skin care products containing an isoflavone extract from soybean cake has many advantages, such as convenience, economy, and environmental protection. Acknowledgements This work was supported by research grants from Changhua Christian Hospital, Shin Kong Wu Ho-Su Memorial Hospital and the National Science Council of Taiwan. References Afaq, F., Mukhtar, H., 2006. Botanical antioxidants in the prevention of photocarcinogenesis and photoaging. Experimental Dermatology 15, 678–684. Ahsan, H., Reagan-Shaw, S., Eggert, D.M., Tan, T.C., Afaq, F., Mukhtar, H., Ahmad, N., 2007. Protective effect of sanguinarine on ultraviolet B-mediated damages in SKH-1 hairless mouse skin: implications for prevention of skin cancer. Photochemistry and Photobiology 83, 986–993. Arct, J., Pytkowska, K., 2008. Flavonoids as components of biologically active cosmeceuticals. Clinical Dermatology 26, 347–357. Assefa, Z., Van Laethem, A., Garmyn, M., Agostinis, P., 2005. Ultraviolet radiationinduced apoptosis in keratinocytes: on the role of cytosolic factors. Biochimica et Biophysica Acta 1755, 90–106. Berson, D.S., 2008. Natural antioxidants. Journal of Drugs in Dermatology 7, s7–12. Chiang, H.S., Wu, W.B., Fang, J.Y., Chen, B.H., Kao, T.H., Chen, Y.T., Huang, C.C., Hung, C.F., 2007. The UVB-protective effects of isoflavone extracts from soybean cake in human keratinocytes. International Journal of Molecular Sciences 8, 651–661. Chun, K.S., Langenbach, R., 2007. A proposed COX-2 and PGE(2) receptor interaction in UV-exposed mouse skin. Molecular Carcinogenesis 46, 699–704. Erden Inal, M., Kahraman, A., Koken, T., 2001. Beneficial effects of quercetin on oxidative stress induced by ultraviolet A. Clinical and Experimental Dermatology 26, 536–539. Gali-Muhtasib, H.U., Perchellet, J.P., Khatib, S.H., 1998. Inhibitory effects of plant tannins on ultraviolet light-induced epidermal DNA synthesis in hairless mice. Photochemistry and Photobiology 67, 663–668. Haratake, A., Uchida, Y., Schmuth, M., Tanno, O., Yasuda, R., Epstein, J.H., Elias, P.M., Holleran, W.M., 1997. UVB-induced alterations in permeability barrier function: roles for epidermal hyperproliferation and thymocyte-mediated response. The Journal of Investigative Dermatology 108, 769–775. Herr, H., Cho, H.J., Yu, S., 2007. Burns caused by accidental overdose of photochemotherapy (PUVA). Burns 33, 372–375. Huang, C.C., Fang, J.Y., Wu, W.B., Chiang, H.S., Wei, Y.J., Hung, C.F., 2005. Protective effects of (−)-epicatechin-3-gallate on UVA-induced damage in HaCaT keratinocytes. Archives of Dermatological Research 296, 473–481.
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