Cyclohexanediol bis-ethylhexanoate inhibits melanogenesis of murine B16 melanoma and UV-induced pigmentation in human skin.

The role of cyclohexane diester analogues in the formation of melanin has been recently reported. In the present study, we investigated the inhibitory effect of cyclohexanediol bis-ethylhexanoate (CHEH) on melanogenesis in B16 melanoma cells and on UV-B-induced pigmentation in human skin. CHEH significantly reduced the melanin content in a dose-dependent manner, without cytotoxic effects at the effective concentrations. Moreover, CHEH dose-dependently inhibited tyrosinase activity in B16 melanoma cells, as confirmed by Western blot analysis of the tyrosinase protein levels. However, tyrosinase transcript levels remained unchanged under the same experimental conditions. These results indicate that CHEH inhibited melanogenesis in B16 melanoma cells by regulating tyrosinase activity at the post-transcriptional level. On the other hand, in a cell-free system, CHEH did not inhibit tyrosinase activity. This indicated that CHEH suppressed the pigmentation of melanocytes by indirectly regulating tyrosinase activity. Finally, in a clinical trial, a cream containing 1.0% CHEH showed good whitening effect on UV-induced pigmented human skin without adverse effects. In conclusion, we suggest that CHEH may be an effective inhibitor of melanogenesis and useful effects in the treatment of hyperpigmented disorders.

The Chloroform Fraction of Carpinus tschonoskii Leaves Inhibits the Production of Inflammatory Mediators in HaCaT Keratinocytes and RAW264.7 Macrophages.

Inflammation is the immune system's response to infection and injury-related disorders, and is related to pro-inflammatory factors (NO, PGE2, cytokines, etc.) produced by inflammatory cells. Atopic dermatitis (AD) is a representative inflammatory skin disease that is characterized by increasing serum levels of inflammatory chemokines, including macrophage-derived chemokine (MDC). Carpinus tschonoskii is a member of the genus Carpinus. We investigated the anti-inflammatory activity of C. tschonoskii by studying the effects of various solvent fractions prepared from its leaves on inflammatory mediators in HaCaT and RAW264.7 cells. We found that the chloroform fraction of C. tschonoskii inhibited MDC at both the protein and mRNA levels in HaCaT cells, acting via the inhibition of STAT1 in the IFN-γ signaling pathway. In addition, the chloroform fraction significantly suppressed the expression of inflammatory factors induced by lipopolysaccharide stimulation, except COX-2 and TNF-α. These results suggest that the chloroform fraction of C. tschonoskii leaves may include a component with potential anti-inflammatory activity.

Empetrum nigrum var. japonicum extract suppresses γ-ray radiation-induced cell damage via inhibition of oxidative stress.

The ethylacetate fraction of Empetrum nigrum var. japonicum (ENE) was shown to reduce intracellular reactive oxygen species (ROS) generated by γ-radiation and activate antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and gluthathion peroxidase (GPx). ENE protected cells against radiation-induced cellular DNA damage, membrane lipid peroxidation, and protein modification, which are the main points of radiation-induced damage. In addition, ENE recovered cell viability by inhibiting apoptosis after cells were treated with radiation. ENE treatment also reduced γ-radiation induced Bax, and caspase 9 and 3 expression in irradiated cells. However, irradiated cells with ENE recovered Bcl-2 expression, which was reduced by radiation. This anti-apoptotic effect of ENE was due to the inhibition of mitogen-activated protein kinase kinase-4 (MKK4/SEK1)-c-Jun NH(2)-terminal kinase (JNK) cascades induced by γ-radiation. In summary, these results suggest that ENE protects cells against γ-radiation-induced oxidative stress via the reduction of ROS and attenuation of apoptosis.

Oenothera laciniata inhibits lipopolysaccharide induced production of nitric oxide, prostaglandin E2, and proinflammatory cytokines in RAW264.7 macrophages.

We elucidated the pharmacological and biological effects of Oenothera laciniata extracts on the production of inflammatory mediators in macrophages. The CH(2)Cl(2) fraction of O. laciniata extract effectively inhibited LPS-induced NO, PGE(2), and proinflammatory cytokine production in RAW264.7 cells. These inhibitory effects of the CH(2)Cl(2) fraction of O. laciniata were accompanied by decreases in the expression of iNOS and COX-2 proteins and iNOS, COX-2, TNF-alpha, IL-1beta, and IL-6 mRNA. Asiatic acid and quercetin were present in the HPLC fingerprint of the O. laciniata extract. We tested the potential application of O. laciniata extract as a cosmetic material by performing primary skin irritation tests. In New Zealand white rabbits, primary irritation tests revealed that application of O. laciniata extracts (1%) did not induce erythema or edema formation. Human skin primary irritation tests were performed on the normal skin (upper back) of 30 volunteers to determine if any material in O. laciniata extracts had irritation or sensitization potential. In these assays, O. laciniata extracts did not induce any adverse reactions. Based on these results, we suggest that O. laciniata extracts be considered possible anti-inflammatory candidates for topical application.

Risk reduction of ethyl acetate fraction of Empetrum nigrum var. japonicum via antioxidant properties against hydrogen peroxide-induced cell damage.

Reactive oxygen species (ROS) produce damage to all major cellular constituents. The antioxidant properties of the ethyl acetate fraction of Empetrum nigrum was assessed against hydrogen peroxide (H(2)O(2))-induced cell damage. Empetrum extract was found to scavenge (1) intracellular ROS in cell system, (2) hydroxyl radicals generated by the Fenton reaction (FeSO(4) + H(2)O(2)), and (3) superoxide radicals generated by xanthine/xanthine oxidase in a cell-free system as detected by electron spin resonance (ESR) spectrometry. Cell damage was produced by H(2)O(2) treatment as evidenced by DNA damage, lipid peroxidation, and increased protein carbonyl formation; however, Empetrum extract prevented H(2)O(2)-induced damage to these parameters. Empetrum extract increased viability of Chinese hamster lung fibroblast (V79-4) cells exposed to H(2)O(2), as evidenced by decreased apoptotic nuclear fragmentation, and lower sub G(1) cell population. Further, Empetrum extract restored the cellular antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and heme oxygenase-1 (HO-1), which were reduced by H(2)O(2) treatment. In conclusion, Empetrum extract protected cells against H(2)O(2)-induced cell damage via antioxidant properties by scavenging ROS and enhancing antioxidant enzyme activities.

Inhibitory effects of triphlorethol-A on MMP-1 induced by oxidative stress in human keratinocytes via ERK and AP-1 inhibition.

Oxidative stress is known to generate reactive oxygen species (ROS) in cells, which subsequently induce the synthesis of matrix metalloproteinases (MMP) and an aging phenomenon. The protective effects of triphlorethol-A, derived from Ecklonia cava, were investigated against hydrogen peroxide (H(2)O(2))-induced damage using human skin keratinocytes. Data showed that triphlorethol-A inhibited ROS formation, induced catalase expression, inhibited DNA damage, and increased cell viability in keratinocytes. Triphlorethol-A treatment significantly reduced MMP-1 expression and production, compared to H(2)O(2)-treated cells. In addition, triphlorethol-A abrogated the activation of extracellular signal regulated protein kinase (ERK), which originates upstream of MMP-1 expression, and was induced by H(2)O(2) treatment. Moreover, triphlorethol-A inhibited DNA binding activity of activator protein-1 (AP-1), a downstream transcription factor of ERK. Data indicate that the antioxidative properties of triphlorethol-A involve the inhibition of MMP-1 via ERK and AP-1 inhibition.

Protective effects of Castanopsis cuspidate through activation of ERK and NF-kappaB on oxidative cell death induced by hydrogen peroxide.

The protective effect of Castanopsis cuspidate var. sieboldii was examined on H2O2-induced cell damage. The ethanol extract of Castanopsis cuspidate was found to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and reduce intracellular reactive oxygen species (ROS) generation, and thus prevent lipid peroxidation and cellular DNA damage induced by H2O2. As a result, Castanopsis extract reduced H2O2-induced cell death of V79-4 cells via inhibition of apoptosis. Castanopsis extract was also found to increase catalase activity and its protein expression. Further molecular mechanistic studies revealed that Castanopsis extract enhanced phosphorylation of extracellular signal regulated kinase (ERK) and activity of nuclear factor kappa B (NF-kappaB). Taken together, the results suggest that Castanopsis extract protects V79-4 cells against oxidative damage by enhancing catalase activity and modulating the ERK and NF-kappaB signal pathway.

Triphlorethol-A from Ecklonia cava protects V79-4 lung fibroblast against hydrogen peroxide induced cell damage.

In the present study, triphlorethol-A, a phlorotannin, was isolated from Ecklonia cava and its antioxidant properties were investigated. Triphlorethol-A was found to scavenge intracellular reactive oxygen species (ROS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and thus prevented lipid peroxidation. The radical scavenging activity of triphlorethol-A protected the Chinese hamster lung fibroblast (V79-4) cells exposed to hydrogen peroxide (H2O2) against cell death, via the activation of ERK protein. Furthermore, triphlorethol-A reduced the apoptotic cells formation induced by H2O2. Triphlorethol-A increased the activities of cellular antioxidant enzymes like, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Hence, from the present study, it is suggestive that triphlorethol-A protects V79-4 cells against H2O2 damage by enhancing the cellular antioxidative activity.

Proteomic analysis of carbonylated proteins in two-dimensional gel electrophoresis using avidin-fluorescein affinity staining.

A method for detecting carbonylated proteins in two-dimensional electrophoresis (2-DE) was developed using biotinylation and avidin-fluorescein isothiocyanate (FITC) affinity staining. The method was used to examine oxidatively modified proteins associated with oxidative stress. Carbonyl formation in proteins was first examined in a model system by subjecting bovine serum albumin (BSA) and ribonuclease A (RNase A) to metal-catalyzed oxidation (MCO). Carbonyl group formation was found to occur at multiple sites along with a small amount of polypeptide chain cleavage. In vivo studies were conducted in yeast cell cultures using 5 mM hydrogen peroxide to induce oxidative stress. Biotinylation of yeast protein was accomplished during extraction at 4 degrees C in a lysis buffer containing 5 mM biotin-hydrazide. Biotin-hydrazide forms a Schiff base with a carbonyl group on an oxidized protein that is subsequently reduced before electrophoresis. Proteins were separated by either 2-DE or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biotinylated species were detected using avidin-FITC affinity staining. Detection sensitivity with biotinylated proteins was five times higher than achieved by silver staining. The limit of detection with avidin-FITC staining approached 0.64 pmol of protein-associated carbonyls. Twenty carbonylated proteins were identified in the proteome of yeast following oxidative stress with hydrogen peroxide. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis of tryptic peptides was used to identify peptides extracted from gels. Aconitase, heat shock protein SSA1 and SSC1, pyruvate decarboxylase isozyme 1, pyruvate kinase 1, enolase 1 and 2, phosphoglycerate kinase, fructose-bisphosphate aldorase, and glyceraldehyde-3-phosphate dehydrogenase were among the major targets of oxidative stress.