John Hwan Lee

Distinctive Metabolism of Flavonoid between Cultivated and Semiwild Soybean Unveiled through Metabolomics Approach

Soybeans are an important crop for agriculture and food, resulting in an increase in the range of its application. Recently, soybean leaves have been used not only for food products but also in the beauty industry. To provide useful and global metabolite information on the development of soy-based products, we investigated the metabolic evolution and cultivar-dependent metabolite variation in the leaves of cultivated (Glycine max) and semiwild (G. gracilis) soybean, through a (1)H NMR-based metabolomics approach, as they grew from V (vegetative) 1 to R (reproductive) 7 growth stages. The levels of primary metabolites, such as sucrose, amino acids, organic acids, and fatty acids, were decreased both in the G. gracilis and G. max leaves. However, the secondary metabolites, such as pinitol, rutin, and polyphenols, were increased while synthesis of glucose was elevated as the leaves grew. When metabolite variations between G. gracilis and G. max are compared, it was noteworthy that rutin and its precursor, quercetin-3-O-glucoside, were found only in G. gracilis but not in G. max. Furthermore, levels of pinitol, proline, β-alanine, and acetic acid, a metabolite related to adaptation toward environmental stress, were different between the two soybean cultivars. These results highlight their distinct metabolism for adaptation to environmental conditions and their intrinsic metabolic phenotype. This study therefore provides important information on the cultivar-dependent metabolites of soybean leaves for better understanding of plant physiology toward the development of soy-based products.

Different effects of five depigmentary compounds, rhododendrol, raspberry ketone, monobenzone, rucinol and AP736 on melanogenesis and viability of human epidermal melanocytes

Numerous medications are used to treat hyperpigmentation. However, several reports have indicated that repeated application of some agents, such as rhododendrol (RD), raspberry ketone (RK) and monobenzone (MB), can be toxic to melanocytes. Although these agents had severe side effects in human trials, no current in vitro methods can predict the safety of such drugs. This study assessed the in vitro effects of five depigmentary compounds including leukoderma‐inducing agents. In particular, we determined the effects of different concentrations and exposure times of different depigmentary agents on cell viability and melanogenesis in the presence and absence of ultraviolet B (UVB) radiation. Concentrations of RD, RK and MB that inhibit melanogenesis are similar to concentrations that are cytotoxic; however, concentrations of rucinol (RC) and AP736 that inhibit melanogenesis are much lower than concentrations that are cytotoxic. Furthermore, the concentrations that cause toxic effects depend on exposure duration, and prolonged exposure to RD, RK and MB had more cytotoxic effects than prolonged exposure to RC and AP736. The cytotoxic effects of RD and RK appear to be mediated by apoptosis due to increased expression of caspase‐3 and caspase‐8; UVB radiation increased the cytotoxicity of these agents and also increased caspase activity. Our results indicate that different leukoderma‐inducing compounds have different effects on the viability of normal epidermal melanocytes and suggest that the in vitro assay used here can be used to predict whether an investigational compound that induces leukoderma may lead to adverse effects in human trials.

The natural phytochemical dehydroabietic acid is an anti-aging reagent that mediates the direct activation of SIRT1

Dehydroabietic acid (DAA) is a naturally occurring diterpene resin acid of confers, such as pinus species (P. densiflora, P. sylvestris) and grand fir (Abies grandis), and it induces various biological actions including antimicrobial, antiulcer, and cardiovascular activities. The cellular targets that mediate these actions are largely unknown yet. In this report, we suggest that DAA is an anti-aging reagent. DAA has lifespan extension effects in Caenorhabditis elegans, prevents lipofuscin accumulation, and prevents collagen secretion in human dermal fibroblasts. We found that these anti-aging effects are primarily mediated by SIRT1 activation. Lifespan extension effects by DAA were ameliorated in sir-2.1 mutants and SIRT1 protein expression was increased, resulting in the deacetylation of SIRT1 target protein PGC-1α. Moreover, DAA binds directly to the SIRT1 protein independent of the SIRT1 substrate NAD(+) levels. Through a molecular docking study, we also propose a binding model for DAA-SIRT1. Taken together, our results demonstrate that the anti-aging effects are the first identified biological property of DAA and that the direct activation of SIRT1 enzymatic activity suggests the potential use of this natural diterpene, or related compounds, in age-related diseases or as a preventive reagent against the aging process.

Kojyl cinnamate ester derivatives promote adiponectin production during adipogenesis in human adipose tissue-derived mesenchymal stem cells

The subcutaneous fat tissue mass gradually decreases with age, and its regulation is a strategy to develop anti-aging compounds to ameliorate the photo-aging of human skin. The adipogenesis of human adipose tissue-mesenchymal stem cells (hAT-MSCs) can be used as a model to discover novel anti-aging compounds. Cinnamomum cassia methanol extracts were identified as adipogenesis-promoting agents by natural product library screening. Cinnamates, the major chemical components of Cinnamomum cassia extracts, promoted adipogenesis in hAT-MSCs. We synthesized kojyl cinnamate ester derivatives to improve the pharmacological activity of cinnamates. Structure-activity studies of kojyl cinnamate derivatives showed that both the α,β-unsaturated carbonyl ester group and the kojic acid moiety play core roles in promoting adiponectin production during adipogenesis in hAT-MSCs. We conclude that kojyl cinnamate ester derivatives provide novel pharmacophores that can regulate adipogenesis in hAT-MSCs.

A novel adamantyl benzylbenzamide derivative, AP736, inhibits melanogenesis in B16F10 mouse melanoma cells via glycogen synthase kinase 3β phosphorylation

Recently, much effort has been made to develop effective dermatological depigmenting compounds. In this study, we investigated the novel candidate compound, AP736 (an adamantyl benzylbenzamide derivative), and its effects on melanogenesis in B16F10 melanoma cells, as well as the mechanisms involved. AP736 has been reported to exert anti-melanogenic effects in melanocytes in vitro and in artificial skin equivalents through the inhibition of key melanogenic enzymes and the suppression of the cAMP-protein kinase A (PKA)-cAMP response element‑binding protein (CREB) signaling pathway. Thus, we examined another pathway of melanogenesis involving the effects of AP736 on the glycogen synthesis kinase 3β (GSK3β) pathway. Melanin content and tyrosinase activity were measured using a spectrophotometer after the cells were treated with AP736. The AP736-induced activation of signaling pathways was examined by western blot analysis. We confirmed that AP736 decreased melanin production in a dose-dependent manner; however, it did not directly inhibit tyrosinase, the rate-limiting melanogenic enzyme. The expression of microphthalmia-associated transcription factor, tyrosinase, and related signal transduction pathways was also investigated. The Wnt signaling pathway is deeply involved in melanogenesis; therefore, phosphorylation by GSK3β was assessed following treatment with AP736. AP736 induced GSK3β phosphorylation (inactivation), but it did not alter the level of β-catenin. Furthermore, the expression of α-melanocyte-stimulating hormone-induced tyrosinase was downregulated by AP736. Our data suggest that AP736 exerts hypopigmentary effects through the downregulation of tyrosinase via GSK3β phosphorylation.

Isolation and identification of Malassezia species from Chinese and Korean patients with seborrheic dermatitis and in vitro studies on their bioactivity on sebaceous lipids and IL-8 production.

We investigated the distribution of Malassezia yeast in 120 Chinese (20 patients from each of six cities) and 20 Korean patients with scalp seborrheic dermatitis (SD) and dandruff (SD/D) using ITS1 and ITS2 polymerase chain reaction‐restriction fragment length polymorphism. Bioactivity was studied by quantifying sebum lipid production by human primary sebocytes and inflammatory cytokine, interleukin‐8 (IL‐8) production was studied by exposing HaCaT keratinocytes with extracts of five standard Malassezia strains; M. globosa, M. restricta, M. sympodialis, M. dermatis and M. slooffiae. M. restricta and M. globosa were the most frequently encountered species from both Chinese and Korean patients. These two Malassezia species also promoted neutral lipid synthesis although the result was not statistically significant and induced significant increase in IL‐8 production among the five Malassezia species studied. The study suggests a possible role of these organisms in the pathogenesis of SD/D.

A quantitative evaluation method using processed optical images and analysis of age-dependent changes on nasolabial lines

Nasolabial lines (NL) and wrinkles of the face are major features of aging. Wrinkles have been studied widely by morphological methods using 3‐dimensional (3D) photographic analysis instrument, but NL were evaluated by visual scoring usually. To evaluate NL quantitatively, another method is needed. This study is purposed to find out quantitative method for evaluation of NL.

Coumestrol Down-Regulates Melanin Production in Melan-a Murine Melanocytes through Degradation of Tyrosinase

Pigmentation reflects skin darkening caused by melanin production, but excessive melanin synthesis may cause problems, such as melasma, solar lentigo, dark spots, and freckles. Considerable effort has been devoted to alleviating these undesired symptoms through the development of safe and effective depigmenting agents. Coumestrol, a plant-derived natural isoflavone with an estrogen-like structure and actions, is known to have anti-aging ability, but its potential depigmenting efficacy has not been evaluated. In the present study, we investigated the effects of coumestrol on melanin synthesis in normal melan-a murine melanocytes. Coumestrol significantly reduced melanin synthesis in a concentration-dependent manner up to a concentration of 25 µM without causing cytotoxicity. It also brightened tissue in an artificial skin model (MelanoDerm) that incorporates both human keratinocytes and melanocytes. Interestingly, although coumestrol did not inhibit tyrosinase activity or transcript level in melan-a cells, it clearly decreased the expression level of tyrosinase protein at a concentration of 25 µM. This coumestrol-induced reduction in tyrosinase protein levels was prevented by pretreatment with the proteasome inhibitor MG-132 or the lysosomal proteolysis inhibitor chloroquine. Collectively, our findings indicate that coumestrol exerts an inhibitory effect on melanin synthesis in melan-a cells, at least in part, through degradation of tyrosinase. These findings suggest that coumestrol is a good candidate for use in depigmentary reagents from a cosmetic and clinical perspective.

Flavokawains B and C, melanogenesis inhibitors, isolated from the root of Piper methysticum and synthesis of analogs.

The ethanolic extract of the root of Piper methysticum was found to inhibit melanogenesis in MSH-activated B16 melanoma cells. Flavokawains B and C were isolated from this extract based on their anti-melanogenesis activity and found to inhibit melanogenesis with IC50 values of 7.7μM and 6.9μM, respectively. Flavokawain analogs were synthesized through a Claisen-Schmidt condensation of their corresponding acetophenones and benzaldehydes and were evaluated in terms of their tyrosinase inhibitory and anti-melanogenesis activities. Compound 1b was the most potent of these with an IC50 value of 2.3μM in melanogenesis inhibition assays using MSH-activated B16 melanoma cells.

Antimelanogenic Efficacy of Melasolv (3,4,5-Trimethoxycinnamate Thymol Ester) in Melanocytes and Three-Dimensional Human Skin Equivalent

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.