- Email: [email protected]
Identification of HSP70-inducing activity in Arnica montana extract and purification and characterization of HSP70-inducers
Abstracts / Journal of Dermatological Science 84 (2016) e89–e180
P12-12[O3-17] A novel effects of a novel ascorbic acid derivative, 3-O-laurylglyceryl ascorbate, on improvement of skinbarrier Yushi Katsuyama 1,∗ , Tatsuya Tsuboi 1 , Sayaka Nakamura 1 , Norihisa Taira 1 , Hitoshi Masaki 2 , Masato Yoshioka 1 1 2
SEIWA KASEI CO., LTD, Osaka, Japan Tokyo University of Technology, Japan
Ascorbic acid (AsA) has an antioxidant ability originated from its strong reducing property. However, its water solubility attributes to lower penetration into the skin. Then, we developed a new amphipathic AsA derivative, 3-O-laurylglyceryl ascorbate (3LGA), which introduced a laurylglyceryl group into AsA. Firstly, we examined potentials of antioxidation on 3LGA. Although 3LGA was inferior as compared with AsA in chemical examinations, 3LGA showed significantly higher effects in biological examinations using cultured keratinocytes exposed extracellular stimuli. Then, we speculated a possibility that 3LGA stimulates cellular detoxification system, and examine mRNA expressions of ␥-glutamyl cysteine synthetase (␥-GCS) and catalase. Since 3LGA gave significant up-regulation of both mRNAs, we examined mRNA expression existing at the upstream like peroxisome proliferator activated receptor-␥ (PPAR-␥) and nuclear factor E2-related factor 2 (Nrf 2). 3LGA also upregulated both mRNAs. Gathering the results, it was suggested that 3LGA increases synthesis of GSH and catalase through stimulation of PPAR-␥ and Nrf-2 pathway. On the other hand, skin barrier function is very important to maintain skin water contents. It is known that elder people have the dry skin due to less property on skin moisture function. Then, we examined a contribution of ROS to the skin moisture function focusing on ceramide synthesis. Keratinocytes exposed to H2 O2 showed down-regulation of serine palmitoyltransferase (SPT) mRNA expression. In addition, H2 O2 exposed keratinocytes treated with 3LGA abolished the down-regulation of SPT mRNA. Taken together, it is expected that 3LGA improves barrier function in the skin which has less potentials on ceramide synthesis by exposing to excess ROS. http://dx.doi.org/10.1016/j.jdermsci.2016.08.504 P12-13[O3-18] Identification of HSP70-inducing activity in Arnica montana extract and purification and characterization of HSP70-inducers Keiko Usui 1,∗ , Tsuyoshi Ikeda 2 , Keiko Inoue 3 , Kenichi Hamada 3 , Tohru Mizushima 1 1
Department of Drug Discovery and Development, Faculty of Pharmacy, Keio University, Tokyo, Japan 2 Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan 3 Saishunkan Phamaceutical Co., Ltd., Japan The expression of heat shock proteins (HSPs), particularly HSP70, is receiving considerable attention in the field of cosmetics, particularly given our recent report that ultraviolet-induced melanin production, skin damage and wrinkle formation were all suppressed in transgenic mice expressing HSP70. In the present study, we searched for HSP70-inducers from a library of herbal extracts that have already been approved as quasi-pharmaceutical products in Japan. We selected an ethanol extract of Arnica montana (A. montana), based on its high HSP70-inducing activity and low cytotoxicity. From the extract, we purified and identified eight
e171
sesquiterpene lactones (AM1-8) as HSP70-inducers, among which AM-2 (helenalin 2-methylbutyrate) was selected due to its good HSP70-inducing properties and low cytotoxicity. Treatment of cultured mouse melanoma cells with AM-2 or A. montana extract up-regulated the expression of HSP70 in a dose-dependent manner. This treatment also activated heat shock factor-1, a transcription factor for hsp genes. Furthermore, pre-treatment of cells with AM-2 or A. montana extract decreased melanin production and expression and activity of tyrosinase. These results suggest that AM-2 and A. montana extract could be beneficial for use in hypopigmenting cosmetics as a consequence of their stimulatory effects on HSP70 expression. http://dx.doi.org/10.1016/j.jdermsci.2016.08.505 P12-14[O3-19] Carbonylated proteins in the stratum corneum generate reactive oxygen species under short wavelength visible light Taeko Mizutani ∗ , Yumiko Yamawaki, Yuri Okano, Hitoshi Masaki School of Bioscience and Biotechnology, Tokyo University of Technology, Japan Carbonylated proteins (CP) in the stratum corneum (SC) are detected more frequently in the outermost layer than in the inner layer. This fact implies that CP are induced by exogenous oxidative stimuli, for instance sunlight exposure. In addition, some epidemiological surveys have shown a negative correlation between the level of CP in the SC and skin moisture. We have demonstrated that protein carbonylation in porcine skin treated with acrolein lowers the water content and modulates the skin barrier function in an ex vivo study. Those facts suggest that CP in corneocytes may affect epidermal homeostasis. At the last meeting, we reported that CP can act as a photosensitizer involved in the generation of reactive oxygen species (ROS) in UV-exposed SC. Superoxide anion radicals are generated through a photosensitizing reaction (Type I reaction) in which one electron is transferred to molecular oxygen from an excited photosensitizer in the SC. To summarize our results, UV exposure promotes protein carbonylation through the generation of ROS by the photosensitizing reaction of CP itself. Recently, we found that the maximum excitation wavelength of carbonylated BSA was near 430 nm by in vitro spectroscopy analysis. The purpose of this study was to clarify the effect of CP under short wavelength visible light. Acrolein-treated fragments of the epidermal layer, including the SC, of porcine skin showed autofluorescence. As the peak of excitation spectrum gradually shifted from 350 nm to 430 nm, the emission spectrum more clearly exhibited a peak near 510 nm in an acrolein concentration-dependent manner. This result suggests that ROS generation through the photosensitizing reaction of CP in the SC is enhanced by short wavelength visible light. http://dx.doi.org/10.1016/j.jdermsci.2016.08.506
P12-12[O3-17] A novel effects of a novel ascorbic acid derivative, 3-O-laurylglyceryl ascorbate, on improvement of skinbarrier Yushi Katsuyama 1,∗ , Tatsuya Tsuboi 1 , Sayaka Nakamura 1 , Norihisa Taira 1 , Hitoshi Masaki 2 , Masato Yoshioka 1 1 2
SEIWA KASEI CO., LTD, Osaka, Japan Tokyo University of Technology, Japan
Ascorbic acid (AsA) has an antioxidant ability originated from its strong reducing property. However, its water solubility attributes to lower penetration into the skin. Then, we developed a new amphipathic AsA derivative, 3-O-laurylglyceryl ascorbate (3LGA), which introduced a laurylglyceryl group into AsA. Firstly, we examined potentials of antioxidation on 3LGA. Although 3LGA was inferior as compared with AsA in chemical examinations, 3LGA showed significantly higher effects in biological examinations using cultured keratinocytes exposed extracellular stimuli. Then, we speculated a possibility that 3LGA stimulates cellular detoxification system, and examine mRNA expressions of ␥-glutamyl cysteine synthetase (␥-GCS) and catalase. Since 3LGA gave significant up-regulation of both mRNAs, we examined mRNA expression existing at the upstream like peroxisome proliferator activated receptor-␥ (PPAR-␥) and nuclear factor E2-related factor 2 (Nrf 2). 3LGA also upregulated both mRNAs. Gathering the results, it was suggested that 3LGA increases synthesis of GSH and catalase through stimulation of PPAR-␥ and Nrf-2 pathway. On the other hand, skin barrier function is very important to maintain skin water contents. It is known that elder people have the dry skin due to less property on skin moisture function. Then, we examined a contribution of ROS to the skin moisture function focusing on ceramide synthesis. Keratinocytes exposed to H2 O2 showed down-regulation of serine palmitoyltransferase (SPT) mRNA expression. In addition, H2 O2 exposed keratinocytes treated with 3LGA abolished the down-regulation of SPT mRNA. Taken together, it is expected that 3LGA improves barrier function in the skin which has less potentials on ceramide synthesis by exposing to excess ROS. http://dx.doi.org/10.1016/j.jdermsci.2016.08.504 P12-13[O3-18] Identification of HSP70-inducing activity in Arnica montana extract and purification and characterization of HSP70-inducers Keiko Usui 1,∗ , Tsuyoshi Ikeda 2 , Keiko Inoue 3 , Kenichi Hamada 3 , Tohru Mizushima 1 1
Department of Drug Discovery and Development, Faculty of Pharmacy, Keio University, Tokyo, Japan 2 Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan 3 Saishunkan Phamaceutical Co., Ltd., Japan The expression of heat shock proteins (HSPs), particularly HSP70, is receiving considerable attention in the field of cosmetics, particularly given our recent report that ultraviolet-induced melanin production, skin damage and wrinkle formation were all suppressed in transgenic mice expressing HSP70. In the present study, we searched for HSP70-inducers from a library of herbal extracts that have already been approved as quasi-pharmaceutical products in Japan. We selected an ethanol extract of Arnica montana (A. montana), based on its high HSP70-inducing activity and low cytotoxicity. From the extract, we purified and identified eight
e171
sesquiterpene lactones (AM1-8) as HSP70-inducers, among which AM-2 (helenalin 2-methylbutyrate) was selected due to its good HSP70-inducing properties and low cytotoxicity. Treatment of cultured mouse melanoma cells with AM-2 or A. montana extract up-regulated the expression of HSP70 in a dose-dependent manner. This treatment also activated heat shock factor-1, a transcription factor for hsp genes. Furthermore, pre-treatment of cells with AM-2 or A. montana extract decreased melanin production and expression and activity of tyrosinase. These results suggest that AM-2 and A. montana extract could be beneficial for use in hypopigmenting cosmetics as a consequence of their stimulatory effects on HSP70 expression. http://dx.doi.org/10.1016/j.jdermsci.2016.08.505 P12-14[O3-19] Carbonylated proteins in the stratum corneum generate reactive oxygen species under short wavelength visible light Taeko Mizutani ∗ , Yumiko Yamawaki, Yuri Okano, Hitoshi Masaki School of Bioscience and Biotechnology, Tokyo University of Technology, Japan Carbonylated proteins (CP) in the stratum corneum (SC) are detected more frequently in the outermost layer than in the inner layer. This fact implies that CP are induced by exogenous oxidative stimuli, for instance sunlight exposure. In addition, some epidemiological surveys have shown a negative correlation between the level of CP in the SC and skin moisture. We have demonstrated that protein carbonylation in porcine skin treated with acrolein lowers the water content and modulates the skin barrier function in an ex vivo study. Those facts suggest that CP in corneocytes may affect epidermal homeostasis. At the last meeting, we reported that CP can act as a photosensitizer involved in the generation of reactive oxygen species (ROS) in UV-exposed SC. Superoxide anion radicals are generated through a photosensitizing reaction (Type I reaction) in which one electron is transferred to molecular oxygen from an excited photosensitizer in the SC. To summarize our results, UV exposure promotes protein carbonylation through the generation of ROS by the photosensitizing reaction of CP itself. Recently, we found that the maximum excitation wavelength of carbonylated BSA was near 430 nm by in vitro spectroscopy analysis. The purpose of this study was to clarify the effect of CP under short wavelength visible light. Acrolein-treated fragments of the epidermal layer, including the SC, of porcine skin showed autofluorescence. As the peak of excitation spectrum gradually shifted from 350 nm to 430 nm, the emission spectrum more clearly exhibited a peak near 510 nm in an acrolein concentration-dependent manner. This result suggests that ROS generation through the photosensitizing reaction of CP in the SC is enhanced by short wavelength visible light. http://dx.doi.org/10.1016/j.jdermsci.2016.08.506