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Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one), a naturally occurring flavonoid, was found to prevent human dermal fibroblast cell damage induced by reactive oxygen species such as hydrogen peroxide (H2O2), tert-butyl hydroperoxide (BuOOH) and superoxide anions (.O2-) in a concentration-dependent manner, and was more effective than the iron chelator, deferoxamine, hydroxyl radical (.OH) scavengers such as dimethyl sulfoxide (DMSO) and ethanol (EtOH), the lipid peroxidation chain blocker, alpha-tocopherol (Vit. E) and the xanthine oxidase inhibitor, allopurinol. To probe the mechanism of cell defense, the reaction of baicalein with oxygen free radicals was investigated using electron spin resonance (ESR) spectrometry. Baicalein decreased the signal intensities due to the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin adducts of .OH, .O2- and tert-butyl peroxyl (BuOO.) radicals in a concentration-dependent manner. The IC50 values, which are the 50% inhibition concentrations of baicalein for the free radicals, were 10, 45 and 310 microM, respectively. These results suggested that baicalein possesses free radical scavenging ability which prevents the fibroblast damage induced by these free radical species.

長期間にわたるUVA暴露は, 皮膚の「たるみ」や「深いしわ」に特徴付けられる光老化を引き起こす。このような臨床的症状は細胞外マトリックスの構造変化に由来すると考えられる。正常な真皮構造を維持するために, コラーゲン, エラスチンおよびグリコサミノグリカンなどの細胞外マトリックス成分は産生・分解を繰り返している。この産生・分解のアンバランスが真皮構造の変化の原因となると考えられる。光老化による真皮マトリックスの構造変化を阻止あるいは改善する方法として, われわれは紫外線暴露により活性が上昇する細胞外マトリックス成分分解酵素 (コラゲナーゼおよびエラスターゼ) の活性をコントロールすることに注目した。そこで, それぞれの酵素活性に対し阻害作用を有する有効成分を探索した。その結果, ユーカリ抽出物がI型コラゲナーゼおよびヒト線維芽細胞由来コラゲナーゼに対して, またセイヨウニワトコ抽出物が炎症に伴い浸潤してくる好中球由来エラスターゼ (セリンプロテアーゼ) の活性に対して高い阻害作用を示すことがわかった。これらのことはユーカリ抽出物がUVA暴露後に線維芽細胞が分泌するI型コラゲナーゼ (MMP-1) を阻害することによりI型コラーゲンの分解を阻害することを示唆する。さらに、セイヨウニワトコ抽出物は好中球由来エラスターゼ活性を阻害する成分として有効である。

毛髪の成長は多くの要因によって複雑に制御されている。男性型脱毛症の原因のひとつとして男性ホルモンの作用が挙げられる。男性ホルモンであるテストステロンは5α-ReductaseによってDHTに変換され, さらにDHTがレセプターに結合することによってホルモン作用を発揮する。そこで我々は80種類の植物抽出液を調整し, この中から5α-Reductase阻害作用を示す成分のスクリーニングを行った。その結果ホップ抽出液が高い阻害作用を示すことがわかった。さらに, ホップ抽出液には培養毛根由来細胞の増殖を促進する作用が認められた。この抽出液を用いてマウスを用いた育毛養毛試験を行ったところ, ミノキシジルと同等の発毛促進作用を示した。また, ヒト頭髪に用いた場合には毛髪成長速度を促進する作用があることも認められた。長期使用試験においても良好な結果が得られたことから, ホップ抽出液は有効な育毛養毛用化粧品原料であることが示された。

It has been reported by Von Scott that α-hydroxy acids have potent effects on wrinkle-repairing. MTT (3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium Bromide) method has been used for estimation of cytotoxicity, mitogenic activity and cell viability. We attempted the development of the cell activity estimating method by using MTT method and the investigation of α-hydroxy acids-wrinkle repairing mechanism on human dermis fibroblast culture system. It has been found that glycolic acid increased the activity which was estimated by the MTT method. Subsequently the proliferation effect of glycolic acid was examined. Glycolic acid showed high proliferating activity.Furthermore, the effect on collagen synthesis was examined.However, the enhancement on collagen synthesis by glycolic acid was not found.From these results, it was speculated that the wrinkle-repairing of glycolic acid was realized by the enhancement of proliferation in dermal fibroblasts.

It is recognized that reactive oxygen species (ROS) are responsible for skin damage due to UVB-radiation (UVB-R). However, the triggering substance(s) for ROS generation after UVB-R is uncertain with respect to the activation of NADPH oxidase (Nox), xanthine oxidase (XOD), and respiratory chain-chain reactions in mitochondria. As a first step in identifying the trigger(s) for UVB-induced ROS generation, we examined the relationship between Ca(2+) levels and ROS generation in HaCaT keratinocytes. UVB-R exposure of HaCaT keratinocytes resulted in an immediate elevation of ROS that recurred 7 hours later. This was accompanied by immediately elevated intracellular Ca(2+) . A Ca(2+) chelating agent, BAPTA, abolished the elevation of ROS after UVB-R completely. In addition, exogenous H(2)O(2) did not increase intracellular Ca(2+) levels. This suggests that intracellular Ca(2+) is the first trigger for UVB-induced ROS generation.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 50-52; doi:10.1038/jidsymp.2009.12.

The most important hair colorants on the market are the oxidation dyes because of their effective coloring. They consist of primary intermediates, oxidants, and couplers. In most cases, hydrogen peroxide is employed as the oxidants, but it has resulted in damage to hair. Therefore, there has been much hope of finding a better and safer hair colorant. In our studies, we have found a hair colorant in which the primary intermediates are oxidized in the presence of silver ions used as a catalyst without hydrogen peroxide.In order to dye hair, oxidation of the intermediates must take place within the hair. If the oxidative reaction is completed outside of the hair strand, the enlarged dye molecules will not diffuse within the hair strand. If the reaction proceeds slowly, it is too time-consuming for hair coloring. We have succeeded in controlling the elution rate of silver ion catalyzing the oxidation reaction by holding it within the zeolite. By controlling the contents of silver in zeolite, the contents of the silver-zeolite in dyeing system, and the composition of the solvent, the elution rate can be best controlled for dyeing the hair.The hair coloring products using silver-zeolite as an oxidation catalyst were shown to be safer in the mutagenicity tests which we have conducted. We have also found less damage of the dyed hair surface than with conventional hair deys using hydrogen peroxide.

Destruction of collagen is a hallmark of photoaging. The major enzyme responsible for collagen 1 digestion, matrix metalloproteinase-1 (MMP-1), is induced by exposure to sunlight. To study the molecular trigger for this induction, human skin was ultraviolet-B (UVB)-irradiated and treated with liposome-encapsulated DNA repair enzymes. The photolyase-mediated DNA repair of epidermal UV damage was associated with a reduction of MMP-1 mRNA and protein expression in both the epidermal and dermal compartments of the skin. The role of the epidermal cells in MMP-1 induction in the fibroblasts was examined when human epidermal keratinocytes were irradiated with UVB and their media were transferred to unirradiated human dermal fibroblasts. Transfer of media from irradiated keratinocytes to unirradiated fibroblasts enhanced MMP-1 mRNA and protein. Thus, UV damage to keratinocytes of the epidermis may participate in the destruction of collagen in the dermis by release of soluble mediators that signal fibroblasts to release MMP-1. The MMP-1 induction was reduced when the keratinocytes were treated with DNA repair enzymes T4 endonuclease V or UV endonuclease prior to transfer of the media to fibroblasts. This implies that UVB, which deposits most of its energy on the chromatin of the epidermal keratinocytes and to a lesser extent in the upper dermis, has a significant role in photoaging. DNA damage in the keratinocytes initiates one of the signals for MMP-1 release, and enhancing DNA repair can reduce MMP-1 expression in human skin cells and tissue.

Oxidative stress caused by ultraviolet (UV) radiation generates reactive oxygen species (ROS) in the skin, induces the secretion of melanocyte growth and activating factors from keratinocytes, which results in the formation of cutaneous hyper-pigmentation. Thus, increasing the anti-oxidative ability of skin cells is expected to be a good strategy for skin-lightening cosmetics. Metallothionein (MT) is one of the stress-induced proteins and is known to exhibit a strong anti-oxidative property. We previously reported that a zinc(II) complex with glycine (Zn(II)(Gly)(2)) effectively induces MT expression in cultured human keratinocytes. To determine its potential as a new skin lightening active, we examined whether Zn(II)(Gly)(2) regulates the release of melanocyte-activating factors from UVB-irradiated keratinocytes and affects melanin production in a reconstructed human epidermal equivalent. Conditioned medium from UVB-irradiated keratinocytes accelerated melanocyte proliferation to 110%, and that increase could be prevented by pre-treatment with Zn(II)(Gly)(2). In addition, Zn(II)(Gly)(2) significantly reduced both the production of prostaglandin E(2) and proopiomelanocortin expression in UVB-irradiated keratinocytes. Zn(II)(Gly)(2) also decreased melanin production in a reconstructed human epidermal equivalent. These results indicate that MT-induction in the epidermis effectively up-regulates tolerance against oxidative stress and inhibits the secretion of melanocyte growth and activating factors from keratinocytes. Thus, Zn(II)(Gly)(2) is a good candidate as a new skin-lightening active.

Summary Background Vitamin C, which is a strong anti-oxidant, plays an important role in maintaining physiological states. In dermatology, Vitamin C is used for treatment of various skin problems such as de-pigmentation of hyperpigmented spots. However, Vitamin C has limited stability and permeability, and development of a Vitamin C derivative with improved properties is needed. Objective We evaluated the effect of a lipophilic Vitamin C derivative, tetra-isopalmitoyl ascorbic acid (VC-IP), on ultraviolet (UV)-induced skin pigmentation, to determine its potential as a more effective form of Vitamin C. Methods The release of Vitamin C from VC-IP was examined using a reconstructed skin model following topical application of VC-IP. Anti-oxidative and anti-inflammatory activities of VC-IP were tested in cultured human keratinocytes. Subsequently, clinical test was done to clarify the effect of VC-IP on UVB-induced skin pigmentation. Results VC-IP released Vitamin C in physiological conditions and worked as pro-Vitamin C. In subsequent experiments, we found that VC-IP suppressed the elevation of intracellular peroxide after UVB irradiation, and enhanced cellular tolerance against UVB and reactive oxygen species such as hydrogen peroxide and tert-butyl hydroperoxide. Furthermore, VC-IP reduced the production of interleukin-1α and prostaglandin E2 in UVB-irradiated keratinocytes and suppressed melanocyte proliferation in conditioned culture medium prepared from UVB-irradiated keratinocytes. Finally, in a clinical study, topical application of a 3% VC-IP cream for 3 weeks suppressed pigmentation after UVB irradiation. Conclusions These results demonstrate that VC-IP is a precursor of Vitamin C, and effectively suppresses UVB-induced skin pigmentation, possibly through its anti-oxidative activity.

Summary Background It is well established that the alterations of dermal matrix contributes to skin aging characterized by wrinkles. On the other hand, physiological NO is useful to maintain skin homeostasis such as a vasodilatation. However, a role of NO on production of dermal matrix has been clarified. Objective In this study, we have attempted to analyze the role of NO on type I collagen synthesis of normal human dermal fibroblasts including expression of procollagen αI S(1) mRNA/protein and heat shock protein 47 (HSP47). Methods The effects of NO which was generated by two types of NO donors, sodium nitroprusside (SNP) and S -nitroso- N -acetylpenicillamine (SNAP), on type I collagen and HSP47 and their related mRNA expression were examined with ELISA and RT-PCR. Results NO was significantly accelerated the production of type I collagen by fibroblasts corresponding with up-regulation of procollagen αI (1) mRNA. Furthermore, NO increased both levels of HSP47 protein and mRNA in fibroblasts in a dose-dependent manner. Conclusions These results suggest that NO has dual effects on collagen synthesis by fibroblasts as follows; one is the direct stimulation of collagen synthesis due to the up-regulation of procollagen αI(1) mRNA, and the other is an indirect effect through the increase of HSP47 mRNA expression. This is the first report that exogenous NO stimulates HSP47 production by dermal fibroblasts.

Glycolic acid (GA), one of the alpha-hydroxy acids, is widely used as an agent for chemical peeling. Although there are several reports about the clinical effects of GA in the literature, its biological mechanism remains mostly unclear, and there are only a few reports about its effects on skin rejuvenation mediated by keratinocytes. The aim of this study was to investigate the effect of GA on the dermal matrix metabolism of keratinocytes and fibroblasts using in vitro and ex vivo systems. Our study shows that GA not only directly accelerates collagen synthesis by fibroblasts, but it also modulates matrix degradation and collagen synthesis through keratinocyte-released cytokines. We confirm that IL-1alpha is one of the primary mediators for matrix degradation released from keratinocytes after GA treatment. These results suggest that GA contributes to the recovery of photodamaged skin through various actions, depending on the skin cell type.

Advanced glycation end-products (AGEs) have been reported to accumulate in the dermal skin. However, it remains unknown whether the AGEs interact with the dermal fibroblasts and influence their function. Previously, we demonstrated that AGEs hastened photoaging of the skin by means of active oxygen species such as *O(2)(-), H(2)O(2), and *OH, generated during UVA irradiation. The purpose of the present study was to clarify the influence of AGEs on the functions of dermal fibroblasts under physiological conditions. It was found that AGEs decreased both hyaluronic acid (HA) synthesis and activity of elastase-type matrix metalloproteinase (ET-MMP). Because the reactions of both HA synthesis and ET-MMP were found to take place at the cell membrane region, it appeared that AGEs modulated cellular dysfunction through an interaction with the cell membrane. To clarify the mechanisms of these dysfunction in relation to AGEs, we examined the interaction between AGEs and cell membranes, and obtained the following results: (1) AGEs associated with the cell membranes and liposomal membrane prepared with phosphatidyl choline; (2) AGEs hydrophobically modified the circumstances of the cell membrane and liposome membrane as evaluated by experiments using a fluorescence probe; (3) AGEs increased the fluidity of the cell membrane and liposomal membrane as estimated by ESR spin-labeling using 5-doxylstearic acid; and (4) AGEs accelerated lactate dehydrogenase (LDH) leakage from the cells. On the basis of these experimental results, we proposed that AGEs modulated cell function through a nonspecific interaction with the membranes of dermal fibroblasts.

Our previous study reported that advanced glycation end-products (AGE)-modified BSA produced active oxygen species, *O2-, H2O2, and *OH under UVA irradiation and enhanced the cytotoxicity of UVA light. We examined whether pentosidine in AGE-modified BSA was involved in one of the mechanisms generating the active oxygen species. In biological investigations, fibroblasts exposed to UVA (20 J/cm2) in the presence of pentosidine-rich compounds (PRCs), which were prepared with L-arginine, L-lysine and glucose, showed a time-dependent leakage of the cytosolic enzyme LDH. In addition, release of LDH was suppressed by addition of DMSO and deferoxamine under UVA irradiation. From these results, it was determined that PRCs exposed to UVA damaged the plasma membrane of human dermal fibroblasts due to the conversion of *OH from H2O2 via a Fenton-like reaction. These features of PRCs exposed to UVA were consistent with those of AGE-modified BSA. In an ESR study, PRCs under UVA irradiation yielded DMPO-OH (DMPO-OH adduct) using DMPO as a spin-trapping reagent. *O2- generation from UVA-irradiated PRCs was also indicated by the combination of NBT reduction and SOD. When PRCs were exposed to UVA light controlled with a long-pass filter, WG-360, it was found that their production of *O2- was prohibited less than 50% in the NBT reduction assay. The *O2- production profile of PRCs depending on the wavelength of UVA light was similar to that of AGE-modified BSA. Furthermore, it was found that the H2O2 level was increased by PRCs exposed to UVA. These results indicated that pentosidine is an important factor of AGE-modified BSA in active oxygen generation under UVA irradiation.

Advanced glycation end-products (AGEs) have been reported to be accumulated in dermal skin. However, the role of AGEs in the photoaging of human skin remains unknown, and for this reason, we have examined the interaction between AGEs and ultraviolet A light (UVA) from both the chemical and biological aspects. Previously, we reported that exposing human dermal fibroblasts to UVA in the presence of AGEs that were prepared with bovine serum albumin (BSA) decreased the cell viability due to superoxide anion radical s (.O2(-)) and hydroxyl radicals (.OH) generated by AGEs under UVA irradiation, and active oxygen species are detected with ESR spin-trapping. To identify the active oxygen species in detail and to clarify the cell damaging mechanism, we performed several experiments and the following results were obtained. (1) In ESR spin-trapping, by addition of dimethyl sulfoxide and superoxide dismutase, ESR signals due to .O2(-) -derived DMPO-OOH and .OH-derived DMPO-OH adducts, respectively, were detectable. (2) UVA-irradiated AGEs elevated the lipid peroxide levels in both fibroblasts and liposomes. But the peroxidation in liposomes was inhibited by addition of deferoxamine. (3) Survival of fibroblasts exposed to UVA in the presence of AGEs was elevated by addition of deferoxamine. And finally, (4) survival of fibroblasts was found to be regulated by the level of H2O2. On the basis of these results, we propose a possible mechanism in which AGEs under UVA irradiation generate active oxygen species involving .O2(-), H2O2, and .OH, and the .OH species plays a harmful role in promoting cell damage.

The purpose of this study was to elucidate the differential contribution of catalase and glutathione peroxidase (GSH-Px) to H2O2 scavenging in cultured human dermal fibroblasts. Responses of the cells in terms of both enzyme activities were examined by using two sorts of inhibitors, 3-amino-1H-1,2,4-triazole (AT) for catalase and DL-buthionine-[S,R]-sulfoximine (BSO) for GSH-Px, under exposure to H2O2 or ultraviolet (UV) B radiation. AT treatment resulted in a decrease in H2O2 scavenging activity, while BSO treatment did not affect H2O2 scavenging. When fibroblasts were exposed to a low concentration of H2O2 (100 microM). AT treatment resulted in a significant decrease in cell survival, but BSO treatment did not affect survival. At higher concentrations of H2O2 ranging from 500 microM to 1 mM, BSO-treated fibroblasts showed reduced survival. In addition, AT treatment was much more cytotoxic in the presence of UVB than BSO treatment. The intracellular levels of H2O2 in fibroblasts treated with AT or BSO were also determined. BSO-treated cells showed similar H2O2 levels to control cells, but the intracellular H2O2 levels of AT-treated fibroblasts were 1.4-fold higher than found in control cells. These results with human dermal fibroblasts indicate that catalase acts as a primary defence against oxidative stress from exogenous or endogenous H2O2 at low concentrations. In contrast, GSH-Px helps protect the cell from damage during exposure to high concentrations of H2O2.

To clarify a possible role of advanced glycation end-products (AGE) on photoaging of human skin, the interaction between AGE and ultraviolet A light (UVA) was examined from both a biological and chemical perspective. Human dermal fibroblasts that were exposed to UVA in the presence of AGE bound with bovine serum albumin (AGE-BSA) exhibited a significant decrease of cell viability as compared to control cells, which were exposed to UVA in the absence of AGE-BSA. Further, UVA-irradiated AGE-BSA reduced nitroblue tetrazolium to its formazan, but the reaction was inhibited by addition of superoxide dismutase in the system. UVA dose-dependent formation of H2O2 in AGE-BSA was also observed. An ESR spin-trapping study revealed the generation of unstable free radicals in AGE-BSA under UVA irradiation. After addition of Fe2+ in the system, an ESR spectrum due to the formation of hydroxyl radicals was observed. On the basis of these results, the authors propose that AGE is an important factor for promoting photoaging in the skin via generation of active oxygen species involving .O2-, H2O2, and .OH.

Ergothioneine (EGT) is a sulfur-containing amino acid, and is presumed to function as a natural antioxidant. The purpose of this study was to identify the nature of the antioxidant activity and investigate the effects of EGT on UV-induced cellular response. In chemical studies, EGT scavenged the superoxide anion radical (*O(2)(-)) and singlet oxygen ((1)O(2)). In cultured fibroblasts, EGT suppressed TNF-alpha up-regulation by UVB irradiation. In addition, in fibroblasts exposed to UV-A, EGT suppressed the expression of matrix metalloproteinase 1 (MMP-1) protein by nearly 50% and reduced MMP-1 mRNA expression. From these results, we conclude that EGT scavenges reactive oxygen species generated by both Type I and Type II photosensitization and suppresses both TNF-alpha expression and MMP-1 at their transcriptional level. EGT may reduce skin anti-aging effects after UV irradiation by the scavenging of *O(2)(-) and (1)O(2), and reducing signals for protease and inflammatory activity.

In considering the effect of cosmetics, the moisturizing effect on the skin is most important.This consists of two components, one being the occlusivity which suppress water evaporation, and the other is water holding capacity. A new method for measuring the water evaporation rate (WER) has been developed by us. The present study was carried out in order to clarify the following: 1) The relationship between WER through the skin and skin condition. 2) The factors which determine occlusivity. In conclusion, it was recognized that the morphology of the skin surface become fine with decreasing WER and it was clarified that occlusivity could be explained in terms of lnorganic organic balance (10B), visvosity and water holding capacity.

In considering the effectiveness of cosmetics, the moisturizing effect on the skin is most important.This consists of two components, one being the occlusivity which supress water evaportion, and the other is water holding capacity. The new method for measuring the water evaporation rate has been developed by us. This method is made up by anhydrous cobalt chloride paper and high-speed spectrophotometer, Anhydrous cobalt chloride changes color from blue to pink when it absorbs water. The change in the reflectance was measured with a high-speed spectrophotometer. The detail of this method and examples measured were reported.

The sensory irritation such as biting appears after application of cosmetics. This irritation is the passing stimulation without inflammation. We investigated many cosmetic materials, and made researches on the substance and mechanism of this stimulation.In the results, the sensitivity among the population showed the individual differences. The intensity of irritation is directly related to the number of molecules, is effected by temperature and viscosity of vehicles.We considered that the causative substances must have the high affinity to the skin. This high affinity is dependent on suitable molecular size, polarity, structure and electronic property of the molecule.The detail of this stimulation was reported.