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Melanin provides photoprotection against UV irradiation, but excessive melanin accumulation can cause several problems such as darkened skin, melasma, and freckles. Although many anti-melanogenic agents have been developed for skin-whitening cosmetics, safety issues have been raised regarding these products. As natural substances are often non-toxic and are considered by some to be safer than synthetic compounds, various attempts have been made to synthesize active derivatives from natural substances. In this study, we evaluated the anti-melanogenic effects of four synthesized derivatives (compound 2a, 2b, 2c, and 2d) of kojic and hydroxycinnamic acids which are natural anti-melanogenic substances. Three of these derivatives (2a, 2b, and 2c) inhibited tyrosinase, which catalyzes a rate-limiting step of melanogenesis. Additionally, cell viability and melanin formation were investigated using B16F10 melanoma cells. Compounds 2b and 2c exhibited cytotoxicity towards B16F10 melanoma cells, while compound 2a caused a significant decrease in melanin formation without cytotoxicity. Thus, compound 2a may be viable as a potent skin-whitening agent derived from natural compounds.

Adiponectin is an adipocytokine with insulin-sensitizing, anti-inflammatory, anti-atherosclerotic, and anti-aging properties. Compounds with the ability to promote adiponectin secretion are of interest for the development of anti-aging drugs to improve skin-aging phenotypes. In the phenotypic assay to measure adiponectin secretion during adipogenesis in human adipose tissue-derived mesenchymal stem cells (hAT-MSCs), kojyl cinnamate ester derivatives increased adiponectin secretion. A target identification study showed that the kojyl cinnamate ester derivatives competitively bound to peroxisome proliferator-activated receptor α/γ (PPARα/γ). The upregulation of adiponectin production induced by kojyl cinnamate ester derivatives was significantly correlated with PPARα and PPARγ binding activities. Kojyl cinnamate ester derivatives significantly increased the transcription of genes encoding cholesterol and fatty acid synthesizing enzymes in hAT-MSCs. Notably, the kojyl cinnamate esters upregulated the gene transcription of lipid metabolic enzymes in human epidermal keratinocytes, which are important in the integrity of skin permeability barrier. In addition, the kojyl cinnamate esters that function as PPARα/γ dual modulators inhibited ultraviolet B irradiation-induced inflammation in human epidermal keratinocytes. Therefore, kojyl cinnamate ester derivatives are a novel class of PPARα/γ dual agonists with the potential to improve skin-aging phenotypes.

Oxidoreductases are effective biocatalysts, but their practical use is limited by the need for large quantities of NAD(P)H. In this study, a whole-cell biocatalyst for NAD(P)H cofactor regeneration was developed using the economical substrate glycerol. This cofactor regeneration system employs permeabilized Escherichia coli cells in which the glpD and gldA genes were deleted and the gpsA gene, which encodes NAD(P)+-dependent glycerol-3-phosphate dehydrogenase, was overexpressed. These manipulations were applied to block a side reaction (i.e., the conversion of glycerol to dihydroxyacetone) and to switch the glpD-encoding enzyme reaction to a gpsA-encoding enzyme reaction that generates both NADH and NADPH. We demonstrated the performance of the cofactor regeneration system using a lactate dehydrogenase reaction as a coupling reaction model. The developed biocatalyst involves an economical substrate, bifunctional regeneration of NAD(P)H, and simple reaction conditions as well as a stable environment for enzymes, and is thus applicable to a variety of oxidoreductase reactions requiring NAD(P)H regeneration.

Lilium bulbs have long been used as Chinese traditional medicines to alleviate the symptoms of various human inflammatory diseases. However, mechanisms of Lilium bulb-mediated anti-inflammatory activity and the bioactive components in Lilium bulbs remain unknown. In the present study, the anti-inflammatory activity of Lilium bulbs and the underlying mechanism of action were investigated in macrophages using Lilium bulb ethanol extracts (Lb-EE). In a dose-dependent manner, Lb-EE inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and bone marrow-derived macrophages (BMDMs) without causing significant cytotoxicity. Lb-EE also down-regulated mRNA expression of inflammatory genes in LPS-stimulated RAW264.7 cells, which included inducuble nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), and tumor necrosis factor-[Formula: see text] (TNF-[Formula: see text]). Furthermore, Lb-EE markedly restored LPS-induced morphological changes in RAW264.7 cells to a normal morphology. HPLC analysis identified quercetin, luteolin, and kaempferol as bioactive components contained in Lb-EE. Mechanistic studies in LPS-stimulated RAW264.7 cells revealed that Lb-EE suppressed MyD88- and TRIF-induced NF-[Formula: see text]B transcriptional activation and the nuclear translocation of NF-[Formula: see text]B transcription factors. Moreover, Lb-EE inhibited IKK[Formula: see text]/[Formula: see text]-induced activation of the NF-[Formula: see text]B signaling pathway and IKK inhibition significantly reduced NO production in LPS-stimulated RAW264.7 cells. Taken together, these results suggest that Lb-EE plays an anti-inflammatory role by targeting IKK[Formula: see text]/[Formula: see text]-mediated activation of the NF-[Formula: see text]B signaling pathway during macrophage-mediated inflammatory responses.

Background/Aims: Excessive melanogenesis often causes unaesthetic hyperpigmentation. In a previous report, our group introduced a newly synthesized depigmentary agent, Melasolv™ (3,4,5-trimethoxycinnamate thymol ester). In this study, we demonstrated the significant whitening efficacy of Melasolv using various melanocytes and human skin equivalents as in vitro experimental systems. Methods: The depigmentary effect of Melasolv was tested in melan-a cells (immortalized normal murine melanocytes), α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 murine melanoma cells, primary normal human melanocytes (NHMs), and human skin equivalent (MelanoDerm). The whitening efficacy of Melasolv was further demonstrated by photography, time-lapse microscopy, Fontana-Masson (F&M) staining, and 2-photon microscopy. Results: Melasolv significantly inhibited melanogenesis in the melan-a and α-MSH-stimulated B16 cells. In human systems, Melasolv also clearly showed a whitening effect in NHMs and human skin equivalent, reflecting a decrease in melanin content. F&M staining and 2-photon microscopy revealed that Melasolv suppressed melanin transfer into multiple epidermal layers from melanocytes as well as melanin synthesis in human skin equivalent. Conclusion: Our study showed that Melasolv clearly exerts a whitening effect on various melanocytes and human skin equivalent. These results suggest the possibility that Melasolv can be used as a depigmentary agent to treat pigmentary disorders as well as an active ingredient in cosmetics to increase whitening efficacy.

Ethnopharmacological relevance Seed of Torreya nucifera (L.) Siebold & Zucc is used to treat several diseases in Asia. Reports document that T. nucifera has anti-cancer, anti-inflammatory, anti-oxidative activities. In spite of numerous findings on its pharmacological effects, the understanding of the molecular inhibitory mechanisms of the plant remains to be studied. Therefore, we aimed to explore in vitro anti-inflammatory mechanisms of ethyl acetate fraction (Tn-EE-BF) prepared from the seed of T. nucifera in LPS-stimulated macrophage inflammatory responses. Materials and methods For this purpose, we measured nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated macrophages. Additionally, using RT-PCR, luciferase reporter gene assay, immunoblotting analysis, and kinase assay, the levels of inflammatory genes, transcription factors, and inflammatory signal-regulatory proteins were investigated. Finally, the constituent of Tn-EE-BF was identified using HPLC. Results Tn-EE-BF inhibits NO and PGE2 production and also blocks mRNA levels of inducible NO synthase (iNOS), tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 in a dose dependent manner. Tn-EE-BF reduces nuclear levels of the transcriptional factors NF-κB (p65) and AP-1 (c-Jun and FRA-1). Surprisingly, we found that Tn-EE-BF inhibits phosphorylation levels of Src and Syk in the NF-κB pathway, as well as, IRAK1 at the protein level, part of the AP-1 pathway. By kinase assay, we confirmed that Src, Syk, and IRAK1 are suppressed directly. HPLC analysis indicates that arctigenin, amentoflavone, and quercetin may be active components with anti-inflammatory activities. Conclusion Tn-EE-BF exhibits anti-inflammatory activities by direct inhibition of Src/Syk/NF-κB and IRAK1/AP-1.

Lilium bulbs have long been used as Chinese traditional medicines to alleviate the symptoms of various human inflammatory diseases. However, mechanisms of Lilium bulb-mediated anti-inflammatory activity and the bioactive components in Lilium bulbs remain unknown. In the present study, the anti-inflammatory activity of Lilium bulbs and the underlying mechanism of action were investigated in macrophages using Lilium bulb ethanol extracts (Lb-EE). In a dose-dependent manner, Lb-EE inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and bone marrow-derived macrophages (BMDMs) without causing significant cytotoxicity. Lb-EE also down-regulated mRNA expression of inflammatory genes in LPS-stimulated RAW264.7 cells, which included inducuble nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), and tumor necrosis factor-[Formula: see text] (TNF-[Formula: see text]). Furthermore, Lb-EE markedly restored LPS-induced morphological changes in RAW264.7 cells to a normal morphology. HPLC analysis identified quercetin, luteolin, and kaempferol as bioactive components contained in Lb-EE. Mechanistic studies in LPS-stimulated RAW264.7 cells revealed that Lb-EE suppressed MyD88- and TRIF-induced NF-[Formula: see text]B transcriptional activation and the nuclear translocation of NF-[Formula: see text]B transcription factors. Moreover, Lb-EE inhibited IKK[Formula: see text]/[Formula: see text]-induced activation of the NF-[Formula: see text]B signaling pathway and IKK inhibition significantly reduced NO production in LPS-stimulated RAW264.7 cells. Taken together, these results suggest that Lb-EE plays an anti-inflammatory role by targeting IKK[Formula: see text]/[Formula: see text]-mediated activation of the NF-[Formula: see text]B signaling pathway during macrophage-mediated inflammatory responses.

Abutilon crispum L. Medik, better known as bladdermallow, is used as a traditional remedy in India, for its anti-inflammatory effect due to its high content of flavonoids. However, research about its anti-inflammatory effect at the molecular level has not been performed. In this study, we aimed to investigate the mechanism of Abutilon crispum methanol extract (Ac-ME) in inhibiting the inflammatory response by conducting several experiments including cellular and molecular assays. Ac-ME inhibited the production of nitric oxide (NO) in RAW264.7 cells during treatment of LPS and Pam3CSK4 without exhibiting cytotoxicity. Ac-ME also suppressed the mRNA expression of inducible nitric oxide (iNOS) and proinflammatory cytokines such as interleukin (IL)-1β and IL-6. Moreover, Ac-ME was shown to inhibit the NF-κB pathway, according to the luciferase reporter gene assay performed with a NF-κB-Luc construct containing NF-κB-binding promoter regions under MyD88 and TRIF overexpression conditions, and immunoblotting analysis by determining the phospho-form levels of IκBα, IKKα/β, and p85, a regulatory domain of phosphatidylinositide 3-kinase (PI3K). Finally, we observed that the level of phospho-p85 induced by the overexpression of spleen tyrosine kinase (Syk) and Src was decreased by Ac-ME at 200 μg/ml. Therefore, these results suggest that Ac-ME has an anti-inflammatory effect by targeting PI3K in the NF-κB signaling pathway.

Oxidoreductases are effective biocatalysts, but their practical use is limited by the need for large quantities of NAD(P)H. In this study, a whole-cell biocatalyst for NAD(P)H cofactor regeneration was developed using the economical substrate glycerol. This cofactor regeneration system employs permeabilized

Abutilon crispum L. Medik, better known as bladdermallow, is used as a traditional remedy in India, for its anti-inflammatory effect due to its high content of flavonoids. However, research about its anti-inflammatory effect at the molecular level has not been performed. In this study, we aimed to investigate the mechanism of Abutilon crispum methanol extract (Ac-ME) in inhibiting the inflammatory response by conducting several experiments including cellular and molecular assays. Ac-ME inhibited the production of nitric oxide (NO) in RAW264.7 cells during treatment of LPS and Pam3CSK4 without exhibiting cytotoxicity. Ac-ME also suppressed the mRNA expression of inducible nitric oxide (iNOS) and proinflammatory cytokines such as interleukin (IL)-1β and IL-6. Moreover, Ac-ME was shown to inhibit the NF-κB pathway, according to the luciferase reporter gene assay performed with a NF-κB-Luc construct containing NF-κB-binding promoter regions under MyD88 and TRIF overexpression conditions, and immunoblotting analysis by determining the phospho-form levels of IκBα, IKKα/β, and p85, a regulatory domain of phosphatidylinositide 3-kinase (PI3K). Finally, we observed that the level of phospho-p85 induced by the overexpression of spleen tyrosine kinase (Syk) and Src was decreased by Ac-ME at 200 μg/ml. Therefore, these results suggest that Ac-ME has an anti-inflammatory effect by targeting PI3K in the NF-κB signaling pathway.

Tyrosinase 1 is a copper-containing enzyme which is widely distributed in nature. This enzyme is associated with the production of melanin for the protection of the skin from solar irradiation. However, overproduction of melanin in the skin is of particular concern to woman, for example, in the conditions melasma and lentigo. 2 Tyrosinase catalyzes the first two steps of melanin biosynthesis, namely the hydroxylation of L-tyrosine to L-dopa and the subsequent oxidation of L-dopa to dopaquinone. Tyrosinase is contained in vegetables and fruits and is responsible for the undesirable enzymatic browning that occurs upon long term storage of these. 3 Therefore, tyrosinase inhibitors have attracted attention as important depigmenting agents for the treatment of hyperpigmentation and as anti-browning agents in vegetables and fruits. Kojic acid 4 (1) is a well known tyrosinase inhibitor. However, its inhibitory activity is not potent enough for the above purposes. To overcome this drawback, many semisynthetic kojic acid derivatives have been synthesized, usually by modification of the C-2 hydroxyl group. 5 Recently, we synthesized kojic acid derivatives containing thioether 6 and ester 7 linkers and evaluated their tyrosinase inhibitory activities. Among them, 2-((cyclohexylthio)methyl)-5-hydroxy-4H-pyran-4-one (2b), showed the most potent inhibitory activity. Compound 2b is composed of three parts, including the kojic acid moiety, a thioether linker, and a hydrophobic cyclohexane group. We synthesized and evaluated additional compounds to get more structural insight into the tyrosinase inhibitory activity of the kojic acid derivatives. The crystallographic structure of tyrosinase has recently been established, enabling closer examination of its three-dimensional structure, which revealed the presence of a hydrophobic protein pocket adjoining the binuclear copper active site. 8 We also conducted a molecular docking study to elucidate the structural importance of the kojic acid derivatives in binding with the active site of tyrosinase. The synthetic pathways of the kojic acid derivatives are shown in Scheme 1. Thioether derivatives (2a-2e) were synthesized by the condensation of kojyl chloride 4 with the potassium salts of thiols. Ester derivatives (3a-3e) were synthesized by the condensation of kojyl chloride 4 with the potassium salts of acids. The inhibitory activities of the kojic acid derivatives on mushroom tyrosinase were investigated using kojic acid as a positive control. The results are summarized in Table 1.

E-mail: snpark@seoultech.ac.krReceived April 22, 2014, Accepted June 5, 2014Key Words : Dimeric cinnamoylamide, Melanin, Depigmentation, TyrosinaseHuman skin color is primarily determined by melaninproduction. Melanin is a biopigment synthesized by melano-cytes in a specialized cellular organelle called the melano-some.