Transcriptomic variation in proanthocyanidin biosynthesis pathway genes in soybean (Glycine spp.).

BACKGROUND: Proanthocyanidins are oligomeric or polymeric end products of flavonoid metabolic pathways starting with the central phenylpropanoid pathway. Although soybean (Glycine spp.) seeds represent a major source of nutrients for the human diet, as well as components for the cosmetics industry as a result of their high levels of flavonoid metabolites, including isoflavonoids, anthocyanins and proanthocyanidins, the genetic regulatory mechanisms underlying proanthocyanidin biosynthesis in soybean remain unclear. RESULTS: We evaluated interspecific and intraspecific variability in flavonoid components in soybean using 43 cultivars, landraces and wild soybean accessions. We performed transcriptomic profiling of genes encoding enzymes involved in flavonoid biosynthesis using three soybean genotypes, Hwangkeum (elite cultivar), IT109098 (landrace) and IT182932 (wild accession), in seeds. We identified a Glycine max landrace, IT109098, with a proanthocyanidin content as high as that of wild soybean. Different homologous genes for anthocyanidin reductase, which is involved in proanthocyanidin biosynthesis, were detected as differentially expressed genes between IT109098 and IT182932 compared to Hwangkeum. CONCLUSION: We detected major differences in the transcriptional levels of genes involved in the biosynthesis of proanthocyanidin and anthocyanin among genotypes beginning at the early stage of seed development. The results of the present study provide insights into the underlying genetic variation in proanthocyanidin biosynthesis among soybean genotypes. © 2017 Society of Chemical Industry.

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.

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.

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.

Analysis of the Maillard reaction in human hair using Fourier transform infrared spectroscopic imaging and a focal-plane array detector.

The Maillard reaction has been well researched and used in the food industry and the fields of environmental science and organic chemistry. Here, we induced the Maillard reaction inside human hair and analyzed its effects by using Fourier transform infrared spectroscopy with a focal-plane array (FTIR-FPA) detector. We used arginine (A), glycine (G), and D-xylose (X) to generate the Maillard reaction by dissolving them in purified water and heating it to 150 °C. This label-free process generated a complex compound (named AGX after its ingredients) with a monomer structure, which was determined by using nuclear magnetic resonance (NMR) and FTIR-FPA. This compound was stable in hair and substantially increased its tensile strength. To our knowledge, we are the first to report the formation of this monomer in human hair, and our study provides insights into a new method that could be used to improve the condition of damaged or aging hair.

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.

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.

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

BACKGROUND/PURPOSE: 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. METHOD: One hundred Korean female subjects aged 20 to 60 were recruited in this study. Facial image was taken using light source adjusted VISIA-CR(®) and 3-dimensional wrinkle depth on the NL area was evaluated by Phase shift rapid in vivo measuring of human skin (PRIMOS(®)). The pixel number of NL area and the angle were obtained using processed images. The severity of NL was assessed by visual score. Skin elasticity was measured by Cutometer(®) MPA580. Statistical significance was determined at P < 0.05. RESULT: The optical images obtained by light source adjusted VISIA-CR(®) were easy to distinguish NL and significantly increased age-dependently. And three parameters of elasticity (R2, R5, and R7) on NL area were gradually decreased with age. The Pearson correlation coefficient was -0.756 (P < 0.01) between R7 parameter and ages. Also the pixel number of NL area, angle, wrinkle depth on the NL area (Ra), and visual score were decreased elasticity-dependently. The pixel number of NL area was highly related to Ra (r = 0.567, P < 0.01) and visual score (r = 0.647, P < 0.01). CONCLUSION: This study has shown that NL severity is related to decrease of dermal elasticity and age using quantitative new method by processing optical images.

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,ß-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.

Epigallocatechin-3-O-(3-O-methyl)-gallate-induced differentiation of human keratinocytes involves klotho-mediated regulation of protein kinase-cAMP responsive element-binding protein signaling.

(-)-Epigallocatechin-3-O-gallate (EGCG) has long been known as a potent inducer of keratinocyte differentiation. Although its molecular mechanisms have been extensively studied, its actions on human skin remain to be elucidated. In this study, we demonstrated that methylated EGCG and EGCG increase the expression of klotho, and that klotho functions as a downstream target of EGCG and methylated EGCG in keratinocyte differentiation. We demonstrated that methylated EGCG3 and EGCG induce morphological changes in normal human epidermal keratinocytes (NHEKs) that are related to up-regulation of klotho expression. We also demonstrated that a klotho-induced keratinocyte differentiation marker in NHEKs is inhibited by H-89, a protein kinase (PKA) inhibitor. These results suggest that methylated EGCG and EGCG may function as inducers of keratinocyte differentiation via transcriptional regulation of the klotho protein.

Metabolomics approach for understanding geographical dependence of soybean leaf metabolome.

The soybean plant (Glycine max) is widely used as an ingredient in various foods, nutraceuticals and cosmetics, due to their diverse bioactive compounds. Their metabolic compositions are likely affected by environmental conditions during growth. To investigate the influence of different environmental conditions on the metabolite composition of soybean leaves, we cultivated soybean (G. max Sinhwa) in the southernmost island and volcanic region of Korea, and in the central section and limestone region of the Korean peninsula. Comprehensive metabolite variations of their leaves were analyzed through 1H NMR-based metabolomics approach. With marked differences in soil compositions and climatic conditions between the two growing areas, differences in accumulations of pinitol and diverse flavonoids were noted between the soybean leaves, reflecting the distinct metabolism of soybean plants for physiological adaptation toward different environmental conditions. Therefore, the current study highlights the geographical dependences of diverse soybean leaf metabolites for developing biofunction-enhanced soybean products.

Development of efficient adeno-associated virus (AAV)-mediated gene delivery system with a phytoactive material for targeting human melanoma cells.

We exploited the emerging potential of gene therapy strategies to design a powerful therapeutic system that combines two key components-AAV vector and [6]-gingerol. In this study, we created an AAV2 construct expressing the proapoptotic protein BIM, which uses HSPG as its primary receptor, to target HSPG-overexpressing melanoma cells. This combination treatment showed promising results in vitro, inducing apoptosis in human melanoma cells. This new platform technology will make a significant contribution to numerous therapeutic applications, most notably for melanoma, including overcoming resistance to conventional anticancer therapies.

Effect of BMP-2 Delivery Mode on Osteogenic Differentiation of Stem Cells.

Differentiation of stem cells is an important strategy for regeneration of defective tissue in stem cell therapy. Bone morphogenetic protein-2 (BMP-2) is a well-known osteogenic differentiation factor that stimulates stem cell signaling pathways by activating transmembrane type I and type II receptors. However, BMPs have a very short half-life and may rapidly lose their bioactivity. Thus, a BMP delivery system is required to take advantage of an osteoinductive effect for osteogenic differentiation. Previously, BMP delivery has been designed and evaluated for osteogenic differentiation, focusing on carriers and sustained release system for delivery of BMPs. The effect of the delivery mode in cell culture plate on osteogenic differentiation potential was not evaluated. Herein, to investigate the effect of delivery mode on osteogenic differentiation of BM-MSCs in this study, we fabricated bottom-up release and top-down release systems for culture plate delivery of BMP-2. And also, we selected Arg-Gly-Asp- (RGD-) conjugated alginate hydrogel for BMP-2 delivery because alginate is able to release BMP-2 in a sustained manner and it is a biocompatible material. After 7 days of culture, the bottom-up release system in culture plate significantly stimulated alkaline phosphate activity of human bone marrow-mesenchymal stem cells. The present study highlights the potential value of the tool in stem cell therapy.

New approach to the immobilization of glucose oxidase on non-porous silica microspheres functionalized by (3-aminopropyl)trimethoxysilane (APTMS).

The immobilization and encapsulation of glucose oxidase (GOD) onto the mesoporous and the non-porous silica spheres prepared by co-condensation of tetraethylorthosilicate (TEOS) and (3-aminopropyl)trimethoxysilane (APTMS) in the water-in-oil (W/O) emulsion system were studied. The terminal amine group was used as the important functionality for GOD immobilization on the silica substrate. When only TEOS is used as a silica source, the disordered mesoporous silica microspheres are obtained. As the molar ratio of APTMS to TEOS (R(AT)) increases, the surface area and pore volume of the silica particles measured by nitrogen adsorption and desorption method and SEM decrease rapidly. Particularly, the largest change of the surface morphology is observed between R(AT)=0.20 and R(AT)=0.25. The amount and the adsorption time of immobilized enzyme were measured by UV spectroscopy. About 20wt% of GOD was immobilized into the silica substrates above R(AT)=0.60 and was completely adsorbed into the substrate of R(AT)=0.80 with lapse of 4h after addition. In the measurement of the thermal stability, GOD dissolved in buffer solution loses nearly all of its activity after 30 min at 65 degrees C. In contrast, GOD immobilized on the surface-modified silica particles still retains about 90% of its activity after the same treatment. At this temperature, the immobilized glucose oxidase retained half of its initial activity after 4h. It is shown that the suitable usage of functionalizing agent like APTMS as well as the control of surface morphology is very important on the immobilization of enzyme.

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, ß-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.

A novel adamantyl benzylbenzamide derivative, AP736, inhibits melanogenesis in B16F10 mouse melanoma cells via glycogen synthase kinase 3ß 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 element­binding protein (CREB) signaling pathway. Thus, we examined another pathway of melanogenesis involving the effects of AP736 on the glycogen synthesis kinase 3ß (GSK3ß) 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 GSK3ß was assessed following treatment with AP736. AP736 induced GSK3ß phosphorylation (inactivation), but it did not alter the level of ß-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 GSK3ß phosphorylation.

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.