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Skin transparency is a cosmetic asset highly considered by Asian women. Resulting from complex light interactions within the skin, but still not fully understood, there is no simple method to measure it objectively. In this study, skin parameters from digital images were analysed to build a model predicting transparency.Initially, 71 Japanese women (between ages 50 and 60 years) were recruited. This group was then extended to 262 women (between ages 21 and 60 years). Pictures of their faces were taken with the ColorfaceIn the initial group of 71 subjects, 109 parameters correlated with transparency. Half of them are from the cheek and relate to colour or colour homogeneity. If the cheek presented the largest proportion of correlated parameters, best correlations were usually found in other facial regions. Multiple regressions from some cheek parameters can predict up to 80% of transparency. Stepwise regression on parameters from 262 subjects led to a six-parameter model, which is highly correlated (R = 84.1%) with transparency. It combines skin texture, colour, colour homogeneity and gloss parameters. If half of them are from the cheek, the others are from the tear trough, the full face and the cheekbone.Using parameters from digital pictures exclusively, we propose a model that accurately reflects transparency. Including parameters previously shown to relate to transparency, this model should be useful for future dermatology and cosmetic research.

Residues of olive fruit (ROF) after the extraction of oils are an increasing source of industrial waste, because olive oil is becoming more popular as a healthy food. It has been reported that olives have some polyphenols that have an antioxidation capability. On the other hand, excess oxidative stress disrupts epidermal barrier function. This study was conducted to determine whether ROF could be utilized as an antioxidant source to reduce industrial wastes and to identify possible active materials to maintain healthy skin. Olive fruits are categorized into two groups depending on the time of harvest, young fruit (YF) and mature fruit (MF). Thus, we examined the antioxidant potentials of extracts from YF and from MF to remove reactive oxygen species (ROS) from biological and chemical aspects. HaCaT keratinocytes cultured with extracts of YF or MF had reduced levels of intracellular ROS in spite of the relatively low chemical capability against ROS scavenging. The biological effects of the YF extract were superior to those of the MF extract. The YF extract showed effective reductions of intracellular ROS and carbonylated proteins that were elevated by the stress-related hormone cortisol. In addition, the YF extract reinforced the intracellular antioxidation capability through the activation of Nrf2 signaling. Taken together, the YF extract was an effective source to reinforce the intracellular antioxidation capability. We conclude from these results that utilizing ROF would lead to the reduction of industrial wastes and would supply active materials to maintain healthy skin.

Although extracellular carbonylated proteins (CPs) are found at higher levels in sun-exposed skin, their impact on the cellular functions of fibroblasts and their involvement in the progression of photoaging skin are not fully clarified. In our previous study, we reported that extracellular CPs increase levels of intracellular oxidative stress and result in the accumulation of newly synthesized CPs in normal human dermal fibroblasts (NHDF). Furthermore, fibroblasts exposed to CP-BSA, which is a model of extracellular CPs, had upregulated expression levels of mRNAs encoding matrix metalloproteinase-1 (MMP-1) and interleukin-8/CXCL8 (IL-8/CXCL8). These facts suggested the possibility that extracellular CPs induce a fragile structure in the dermis through the degradation of collagen and elastin. The purpose of this study was to characterize the efficacy of natural carotenoids, such as astaxanthin analogs, produced by Hematococus pluvialis (CHPs) to improve the impaired functions of fibroblasts exposed to CPs. CHPs suppressed the intracellular CP levels elevated by CP-BSA, restored mRNA expression levels of factors involved in the formation and assembly of collagen and elastin fibers and improved the formation of those fibers impaired by CP-BSA. We conclude that CHPs function as antiaging substances due to their restoration of the impaired formation of collagen and elastin fibers caused by extracellular soluble CPs.

Photoaged skin is characterized by the appearance of pigmented spots such as solar lentigos, deep wrinkles and sags, and progresses due to chronic sun exposure. Among the wavelengths of sunlight, UVA is responsible for the appearance of wrinkles and sags that originate from structural alterations in the dermis of photoaged skin such as the depletion of collagen fibers. Thus, improving and restoring collagen fibers is an effective approach to reduce skin photoaging and maintain a youthful appearance. This study was conducted to evaluate the potential of an extract of Ocimum basilicum (OC), which contains rosmarinic acid (RA), as an anti-photoaging material focusing on the capacity to restore collagen fibers that are disrupted due to intracellular oxidative stress. In spite of their relatively low capacities for chemical scavenging of reactive oxygen species (ROS), both OC and RA showed efficient removal of biological oxidative stress by reducing levels of intracellular ROS and carbonylated proteins (CPs) in fibroblasts following exposure to single or repetitive UVA irradiations. Fibroblasts irradiated with repetitive UVA as a model for chronic sun-exposed cells showed significant increases in matrix metalloproteinase-1 and decreases in type I collagen synthesis and formed reduced numbers of collagen fibers. Since both OC and RA restored the adverse phenomena caused by repetitive UVA irradiation, we conclude that OC containing RA is an effective anti-photoaging material.

Sacran, a polysaccharide isolated from the alga Aphanothece sacrum (Suizenji-nori), has unique physical and physiological characteristics. In a previous study, we reported that sacran improves skin conditions in individuals who suffer from atopic dermatitis (AD), focusing on its trapping function against extrinsic stimuli compared with hyaluronic acid (HA). First, we examined the penetration of sacran through stratum corneum (SC) with an impaired barrier function using immature reconstructed human epidermal equivalents. Sacran penetrates the SC to living cell layers of the epidermis, which suggested that sacran would attenuate adverse influences in keratinocytes caused by extracellular factors such as irritants or proinflammatory cytokines such as interleukin 1α (IL-1α). Sacran markedly reduced the cell damage induced by a nonionic detergent, sodium lauryl sulfate (SLS). Moreover, sacran restored the elevation of intracellular reactive oxygen species (ROS) levels stimulated by SLS and by IL-1α. These effects of sacran were superior to those of HA. In order to investigate the restoration effects of sacran, the influence of sacran on the physical properties of lipid bilayers was evaluated by measuring the order parameter using the ESR spin-labeling method. Because sacran failed to cause changes in the order parameters of liposomes and HaCaT keratinocytes, these results indicate that sacran does not interact with lipid bilayers although it restored changes in the order parameter caused by SLS. The sum of these results demonstrates that sacran reduces the influence of extracellular stimuli by its trapping effects. We conclude that the improving action of sacran is based on its trapping effect.

The formation of skin pigmentation requires multiple steps, namely the activation of melanocytes, the synthesis of melanin, the transport of melanosomes to the tips of melanocyte dendrites and the transfer of melanosomes from melanocytes to surrounding keratinocytes. Recently, we reported that melanosomes accumulate in melanocytes when melanosome transport is disrupted and that they are then degraded by the autophagy system. In this study, we examined whether 3-O-glyceryl-2-O-hexyl ascorbate (VC-HG) suppresses melanogenesis through the activation of autophagy since VC-HG interferes with melanosome transport through the down-regulated expression of MyosinVa and Kinesin. The results demonstrate that VC-HG-treated B16 cells show an activation of autophagy through an increased expression level of Microtubule-associated protein 1 light chain 3 (LC3)-II and a decreased expression level of p62. Furthermore, the decrease of melanin content elicited by VC-HG was partially abolished by hydroxychloroquine or pepstatin A which are inhibitors of autophagy. Taken together, we conclude that VC-HG suppresses melanogenesis by activating the autophagy system.

Sodium lauryl sulfate (SLS), a representative anionic surfactant, is well-known to induce rough skin following single or multiple topical applications. The mechanism by which SLS induces rough skin is thought to result from the disruption of skin moisture function consisting of NMF and epidermal lipids. However, a recent study demonstrated that topically applied SLS easily penetrates into the living cell layers of the epidermis, which suggests that physiological alterations of keratinocytes might cause the SLS-induced rough skin. This study was conducted to clarify the effects of SLS on keratinocytes to demonstrate the contribution of SLS to the induction of rough skin. In addition, the potentials of other widely used anionic surfactants to induce rough skin were evaluated. HaCaT keratinocytes treated with SLS had increased levels of intracellular ROS and IL-1α secretion. Application of SLS on the surface of a reconstructed epidermal equivalent also showed the increased generation of ROS. Further, SLS-treated cells showed an increase of intracellular calpain activity associated with the increase of intracellular Ca2+ concentration. The increase of intracellular ROS was abolished by the addition of BAPTA-AM, a specific chelator of Ca2+. In addition, IL-1α also stimulated ROS generation by HaCaT keratinocytes. An ESR spin-labeling study demonstrated that SLS increased the fluidity of membranes of liposomes and cells. Together, those results indicate that SLS initially interacts with cell membranes, which results in the elevation of intracellular Ca2+ influx. Ca2+ stimulates the secretion of IL-1α due to the activation of calpain, and also increases ROS generation. IL-1α also stimulates ROS generation by HaCaT keratinocytes. We conclude from these results that the elevation of intracellular ROS levels is one of the causes of SLS-induced rough skin. Finally, among the other anionic surfactants tested, sodium lauryl phosphate has less potential to induce rough skin because of its lower generation of ROS.

The formation of skin pigmentation requires multiple steps, namely the activation of melanocytes, the synthesis of melanin, the transport of melanosomes to the tips of melanocyte dendrites and the transfer of melanosomes from melanocytes to surrounding keratinocytes. Recently, we reported that melanosomes accumulate in melanocytes when melanosome transport is disrupted and that they are then degraded by the autophagy system. In this study, we examined whether 3-O-glyceryl-2-O-hexyl ascorbate (VC-HG) suppresses melanogenesis through the activation of autophagy since VC-HG interferes with melanosome transport through the down-regulated expression of MyosinVa and Kinesin. The results demonstrate that VC-HG-treated B16 cells show an activation of autophagy through an increased expression level of Microtubule-associated protein 1 light chain 3 (LC3)-II and a decreased expression level of p62. Furthermore, the decrease of melanin content elicited by VC-HG was partially abolished by hydroxychloroquine or pepstatin A which are inhibitors of autophagy. Taken together, we conclude that VC-HG suppresses melanogenesis by activating the autophagy system.

一酸化窒素 (NO) は皮膚血流量の調節や血管の弛緩などに重要な役割を果たしていることが知られている。NOは生体内でNO合成酵素 (NOS) の作用によりL-arginine (L-Arg) から合成される。われわれはヒト血管内皮細胞において, (1) L-ピロリドンカルボン酸 (L-PCA) は濃度依存的に, NO産生量を促進する, (2) L-PCAは血管内皮細胞へのL-Argの取り込みを促進する, そして (3) この促進効果はカチオン性アミノ酸トランスポーター (CAT) 阻害剤であるL-NMMAによって抑制されることを見出した。これらの結果にもとついて, L-PCAはCATを介したL-Argの細胞への取り込みを促進することにより, NO生成量を適度に増加させ血流を調節していることがわかった。しかし, これらのNO合成促進およびL-Arg取り込み促進効果はL-PCAにのみ見られ, D-体には見られなかった。さらに, ヒト前腕内側部での閉塞パッチテストにおいても, L-PCAは皮膚の赤み・血流量を一時的に増加させることが認められた。これまでL-PCAは天然保湿因子の主要な成分の一つであることから保湿を目的としたスキンケアに広く用いられてきたが, 血流促進を目的とした用途においても有用であることが明らかとなった。

We previously reported that epidermal glycation causes an increase in saturated fatty acid (FA) content in a differentiated reconstructed skin model and HaCaT cells. However, the relationship between ceramides (CERs) and glycation and their effects on stratum corneum (SC) barrier function was not elucidated. In this study, we investigated the effect of glycation on lipid content in 6-day-old cultured reconstructed skin. We used the EPISKIN RHE 6D model and induced glycation using glyoxal. In addition to transepidermal water loss, content of CERs, cholesterol and FA in the reconstructed epidermal model were analyzed by high performance thin layer chromatography. Expression of genes related to ceramide metabolism was determined by real time RT-PCR. Membrane fluidity of stratum corneum lipid liposomes (SCLL) that mimic glycated epidermis was analyzed using an electron spin resonance technique. It was found that FA was significantly increased by glycation. CER[NS], [AP], and cholesterol were decreased in glycated epidermis. Expression of ceramide synthase 3 (CERS3) was significantly decreased while fatty acid elongase 3 was increased by glyoxal in a dose dependent manner. Membrane fluidity of SCLL mimicking the lipid composition of glycated epidermis was increased compared with controls. Therefore, disruption of CER and FA content in glycated epidermis may be regulated via CERS3 expression and contribute to abnormal membrane fluidity.

皮膚の老化をより印象づける, 「しわ」や「たるみ」といった現象は, 種々の真皮マトリックスの構造変化に由来すると考えられている。真皮マトリックスの中でもグリコサミノグリカンは, 膠原線維 (collagen) や弾性線維 (elastin) などの構造基盤を成す成分の間隙を埋めるものであり, 皮膚の水分保持や柔軟性に関わりが深い。今回われわれはこれらグリコサミノグリカンの一種であるヒアルロン酸 (HA) に注目した。線維芽細胞を用いたin vitroの系でHA合成を促進させる植物抽出物の探索を行ったところ, 数種の植物抽出物に有効なHA合成促進効果を認め, その中でもキダチアロエに高い活性を認めた。成分分画を行い活性を追ったところ, 比較的高極性画分に活性が移行することを確認した。また, 分画に伴いその活性は増大した。さらに線維芽細胞にその存在が認められているHA合成酵素 (HAS) のmRNA発現について検討を行い, 分画後のフラクションにHAS 2 mRNA発現量の増加を確認した。

シワは加齢に伴って現れる皮膚の老化の特徴であり, シワを改善することは化粧品の大きな課題である。シワの予防効果や改善効果を評価するためには, シワ深さを定量化することが必要となる。しかし, これまではシワ深さ定量法は試験者各様の手法で行われており, 個々の手法の測定条件などの違いにより安定した測定値を得ることが非常に困難であった。そこで今回, われわれは標準スケールを用いたシワ深さの定量法について検討した。その結果, 光量や照射角度などの画像撮影条件や, 撮影した画像からシワを抽出する画像処理条件にかかわらず, 再現性の高いシワ深さ定量値を得ることができたので報告する。

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.

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.