Isolation of a Melanoblast Stimulator from Dimocarpus longan, Its Structural Modification, and Structure-Activity Relationships for Vitiligo.

A novel melanoblast stimulator (1) was isolated from Dimocarpus longan. Its analogs were also synthesized to support a new furan-based melanoblast stimulator scaffold for treating vitiligo. Isolated 5-(hydroxymethyl)furfural (HMF, 1) is a well-known compound in the food industry. Surprisingly, the melanogenic activity of HMF (1) was discovered here for the first time. Both HMF and its synthetic analog (16) promote the differentiation and migration of melanoblasts in vitro. Typically, stimulator (1) upregulated MMP2 expression, which promoted the migration of melanoblasts in vitro.

Electrification of Sonoluminescing Single Bubble.

The charge of microbubbles in water is considered to be negative at the liquid-bubble interface. We investigated, for the first time, the charge of a single bubble sonoluminescence (SBSL bubble) that exhibits spatiotemporally stable light emission. When negative DC voltage was applied, the SBSL bubble was attracted to a hot electrode. Conversely, the SBSL bubble was repelled by the hot electrode when positive DC voltage was applied. The translation of the SBSL bubble under an electric field suggests that it is positively charged, and the bubble moved to an equilibrium position to balance the primary Bjerknes force and the electrostatic force. The amount of bubble translation under an electric field depended on the elapsed time of sonoluminescence, suggesting that the products generated inside the SBSL bubble affect the mechanism of bubble charging. Furthermore, we measured the electric field effects on bubble expansion and contraction by a light scattering technique. Applying a positive voltage decreased the maximum bubble diameter and also the intensity of the SBSL. Conversely, applying a negative voltage increased the maximum bubble diameter and also the intensity of the SBSL. The present study revealed that a SBSL bubble is positively charged.

Water-molecular emission from cavitation bubbles affected by electric fields.

Orange emission was observed during multibubble sonoluminescence at 1 MHz in water saturated with noble gas. The emission arose in the vicinity of the peeled ground electrode of a piezoceramic transducer exposed to water, suggesting that cavitation bubbles were affected by the electric fields that leaked from the transducer. The spectrum of the emission exhibited a broad component whose intensity increased towards the near-infrared region with peaks at 713 and 813 nm. The spectral shape was independent of the saturation gas of He, Ne, or Kr. The broad component was attributed to the superposition of lines due to vibration-rotation transitions of water molecules, each of which was broadened by the high pressure and electric fields at bubble collapse. An emission mechanism based on charge induction by electric fields and the charged droplet model is proposed.

Na emission and bubble instability in single-bubble sonoluminescence.

Na emission in single-bubble sonoluminescence (SBSL) was observed from 0.1mM sodium dodecyl sulfate (SDS) solution containing a dissolved noble gas at a low acoustic pressure, at which a continuous spectral component was negligible. High-speed shadowgraph movies were captured at a frame rate of 30,000fps, which indicated that bubble dancing is responsible for the Na emission. The measured bubble path length was well correlated with the Na intensity. The disintegration of a daughter bubble followed by immediate coalescence was frequently observed, which may have been the cause of the bubble dancing. A comparison of the Na spectra obtained in SBSL and multibubble SL showed that the conditions under which Na emission is generated are twofold. A narrow component was observed in the Na spectrum in SBSL, while narrow and broad components were observed in MBSL.

Kojic Acid Peptide: A New Compound with Anti-Tyrosinase Potential.

BACKGROUND: Kojic acid was used for decades in the cosmetic industry as an antimelanogenic agent. However, there are two major drawbacks of Kojic acid, one is cytotoxicity and second are instability on storage. These limitations led the scientist to synthesize the active Kojic acid peptides. OBJECTIVE: In the present study, we synthesize and investigate the effect of five Kojic acid peptides to overcome the limitation of Kojic acid. METHODS: The peptide was analyzed and purified by high-performance liquid chromatography and matrix-assisted laser desorption ionization time of flight mass spectroscopy. Further, the tyrosinase activities of the Kojic acid and Kojic acid peptides were compared. The toxicity was measured and the melanin content is recorded in B16F10 mouse melanoma cells. RESULTS: Maximum tyrosinase activity was measured by Kojic acid peptides. Therefore, Kojic acid peptides were subjected to melanin assay and cytotoxicity assay and finally the stability of the Kojic acid peptide was measured. CONCLUSION: It was observed that this newly synthesized Kojic acid peptide is stable and potent to inhibit the tyrosinase activity and melanin content of B16F10 mouse melanoma cells without exhibiting cell toxicity. Together, these preliminary results suggest that a further exploration is being needed to establish Kojic acid peptide as antimelanogenic agent.

Unrevealing the role of P-protein on melanosome biology and structure, using siRNA-mediated down regulation of OCA2.

The pink-eyed dilution protein (P-protein) plays a critical role in melanin synthesis in melanocytes and retinal pigment epithelium cells. Mutation in this protein may cause complete or partial albinism. Role of the P-protein ranges in melanin synthesis to maturation and trafficking of the melanosomes. The aim of the present study was to evaluate the effect of P-protein inhibition on melanosome biology by comparing the shape, size, count, and types of melanosomes in melan-a melanocytes. The cells were extensively examined by the transmission electron microscopy. The P-protein inhibition was carried by P-protein-siRNA transfection to melan-a melanocytes, B16F10 mouse melanoma, and melan-p1 cells. Measurement of melanin contents, cellular tyrosinase, and different tyrosinase related proteins were also determined to investigate the effect of P-protein siRNA transfection on melanocytes. Results suggested that the inhibition of P-protein can significantly change the melanosomal morphology, types and their respective numbers, and provided a novel strategy for the control of melanin synthesis.

Homology modelling and virtual screening of P-protein in a quest for novel antimelanogenic agent and in vitro assessments.

An adequate knowledge on molecular mechanism of melanogenesis provides an opportunity to find the novel molecular targets for the discovery and development of new cosmetics. Among various genes, the OCA2 is being essential for proper melanin synthesis, and mutation or deletion of this gene leads to oculocutaneous albinism type 2. Thus, for this study, the product of this gene, that is P-protein, was targeted in quest for novel inhibitors as antimelanogenic agents. Based on pattern search of amino acid sequence and homology analysis, the protein structure was modelled. The role of this protein has been predicted as a tyrosine transporter of melanosomes. Thus, the molecular library was generated on the basis of tyrosine transporter inhibitor. Based on the dock score, 20 molecules have been considered as putative inhibitors for P-protein. Among these compounds, five molecules (compound #1, #4, #8, #13 and #17) were found to be quite effective as antimelanogenic without having any toxicity. Further investigations to establish the mechanism of action, the indirect methods such as tyrosinase assay, analysis for eumelanin and pheomelanins and investigation of mRNA levels were being carried out. The results from the studies offered a new lead in antimelanogenic therapy and may be very useful for further optimization work in developing them as novel depigmenting agents.

Acoustic power dependences of sonoluminescence and bubble dynamics.

The decreasing effect of sonoluminescence (SL) in water at high acoustic powers was investigated in relation to bubble dynamics and acoustic emission spectra. The intensity of SL was measured in the power range of 1-18W at 83.8kHz for open-end (free liquid surface and film-covered surface) and fixed-end boundaries of sound fields. The power dependence of the SL intensity showed a maximum and then decrease to zero for all the boundaries. Similar results were obtained for sonochemiluminescence in luminol solution. The power dependence of the SL intensity was strongly correlated with the bubble dynamics captured by high-speed photography at 64kfps. In the low-power range where the SL intensity increases, bubble streamers were observed and the population of streaming bubbles increased with the power. At powers after SL maximum occurred, bubble clusters came into existence. Upon complete SL reduction, only bubble clusters were observed. The subharmonic in the acoustic emission spectra increased markedly in the region where bubble clusters were observed. Nonspherical oscillations of clustering bubbles may make a major contribution to the subharmonic.

Design and optimization of SPR-based binding assay for evaluation and screening of MITF-E-box binding inhibitor.

Melanin synthesis is a complex phenomenon which involves about 192 known gene products. Among them, MITF is a key transcription factor for tyrosinase, Trp1 and Trp2 proteins, which are essential for melanin biosynthesis. Thus, intervening inhibitor for the MITF-E-box complex formation can downregulate melanin synthesis. The focus of the present study is to develop a surface plasmon resonance-based system to screen the MITF-E-box complex inhibitor. The standardization of the MITF and E-box binding assay was calibrated for kinetics and specificity, in the presence of a pre-incubated 22 mer sequence containing mutated E-box (CTTGAG) along MITF. The binding assay with C17 was optimized and the steady-state kinetics was evaluated. C17 was identified as inhibitor to MITF-E-box, by virtual screening followed by in vitro assessment and EMSA assay. The k(a) and k(d) were found to be 5.5 9 103 M⁻¹ s⁻¹ and 0.0014 s⁻¹, respectively, while the steady-state association constant (K(A)) was 3.928 9 106 M⁻¹. The resonance variations after inhibition were quantified and analyzed to develop the standard method for screening of microphthalmia transcription factor-E-box binding inhibitor.

Isolation and anti-inflammatory effect of astragalin synthesized by enzymatic hydrolysis of tea seed extract.

BACKGROUND: The application of tea seed extract (TSE) has been widely investigated because of its biological activities. In this paper, two flavonol triglycosides in TSE-camelliaside A (CamA) and camelliaside B (CamB)-were subjected to hydrolysis in the presence of two commercial enzyme complexes (Pectinex™ series): Smash and Mash. RESULTS: Smash hydrolyzed only the xylosyl moiety of CamB, and the main product was kaempferol diglycoside (nicotiflorin, NF). On the other hand, Mash induced the hydrolysis of both CamA and CamB, and kaempferol monoglycoside (astragalin, AS) was found to be a main product. Pure AS with > 96% purity was prepared by enzymatic hydrolysis of TSE using Mash, and the chemical structure of AS was confirmed by (1)H- and (13)C-nuclear magnetic resonance analyses. The prepared pure AS showed anti-inflammatory activities by significantly inhibiting cellular nitrite oxide (IC(50) = 363 µg mL(-1)), prostaglandin E(2) (IC(50) = 134 µg mL(-1)) and interleukin-6 production (IC(50) = 289 µg mL(-1)) by lipopolysaccharide -stimulated RAW 264.7 cells. CONCLUSION: It was concluded that pure AS can be prepared by enzymatic partial hydrolysis of TSE and employed as an anti-inflammatory material. This is the first study to address the preparation of pure AS from natural sources.

Water-retentive and anti-inflammatory properties of organic and inorganic substances from Korean sea mud.

Sea mud has been popularly used as an effective base in cosmetic preparations although its biologically-active materials and mechanisms on skin have not yet been fully determined. We isolated humic substances as the major organic substance of the sea mud from a tidal flat in Korea, and investigated their water-retentive properties. Among the three isolated humic substances, humic acid (HA) showed the highest water retentive property (approximately 50 % mass increase from water uptake). Based on the observations that mud pack therapy has been traditionally used to soothe UV-irradiated skin, we examined the antiinflammatory property of the sea mud on UVB-irradiated human keratinocytes (HaCaT cells) by measuring PGE2 levels produced by keratinocytes in the presence of either the total water or methanol extracts of the mud. The water extract showed higher inhibition of PGE2 production from HaCaT cells (30% inhibition) than the methanol extract at 200 ppm (microg/g). We further fractionated the water extract to determine the major components responsible for its anti-inflammatory effect. It was found that the minerals in the mud inhibited PGE2 production by 83 % at 200 ppm, which is comparable with the inhibitory effect of 1 microM indomethacin. No mud extract showed cytotoxicity at the tested concentrations. The mineral compositions of the mineral extract were determined by ICP-MS, revealing that the sea mud consisted of more than 19 different mineral components, rich in Na+, Mg2+, and Zn2+. These results imply that the anti-inflammatory effect of the sea mud is largely due to the minerals in the mud. Our research suggests the potential use of the organic and inorganic substances from the sea mud in various skin products as safe biological substances for skin protective purposes.

Isolation and characterization of nicotiflorin obtained by enzymatic hydrolysis of two precursors in tea seed extract.

Two flavonol triglycosides, camelliaside A (CamA) and camelliaside B (CamB), of tea seed extract (TSE) were subjected to enzymatic hydrolysis. Among five kinds of glycosidases investigated, beta-galactosidase (Gal) induced selective hydrolysis of CamA. On the other hand, pectinase (Pec) and cellulase (Cel) induced hydrolysis of CamB. For Gal and Pec, only kaempferol diglycoside (nicotiflorin, NF) was produced; on the other hand, significant amounts of kaempferol monoglycoside (astragalin, AS) and kaempferol (KR) were also detected for Cel. The combination of the use of Gal and Pec in the enzymatic hydrolysis of TSE afforded NF with high specificity. Crude NF with 22% purity was recovered from the enzymatic reaction mixture by extraction with organic solvent, and pure NF with >95% purity was obtained by crystallized in water. The chemical structure of NF was confirmed by (1)H and (13)C NMR analyses.

Visualization of the melanosome transfer-inhibition in a mouse epidermal cell co-culture model.

Transfer of melanin-containing melanosomes from melanocytes to neighboring keratinocytes results in skin pigmentation. To provide a more practical method of visualizing melanosomes in melanocytes as well as in keratinocytes, we attempted to use murine cell lines instead of human primary cells. We generated various fluorescent fusion proteins of tyrosinase, a melanin synthesis enzyme located in the melanosome, by using green fluorescent protein and red fluorescent protein. The intracellular localization of tyrosinase was then examined by fluorescence and confocal microscopy. Co-culture of murine melanocytes and keratinocytes was optimized and melanosome transfer was either stimulated with alphaMSH or partially inhibited by niacinamide. To the best of our knowledge, this is the first study showing that a murine co-culture model, in addition to human primary cell co-culture, can be a good tool for depigmenting agent screening by monitoring melanosome transfer.

Depigmentation of melanocytes by (2Z,8Z)-matricaria acid methyl ester isolated from Erigeron breviscapus.

To discover an active skin depigmenting agent, we isolated a novel inhibitor of melanin biosynthesis from the methanol extract of Erigeron breviscapus using a bioactivity-guided fractionation and identified it as (2Z,8Z)-matricaria acid methyl ester by means of spectroscopic analysis. The compound showed strong whitening activity in melan-a cell. Compared with arbutin (IC(50)=4.0 mM) as a positive control, the depigmentation IC(50) value for (2Z,8Z)-matricaria acid methyl ester was 25.4 muM in B16F10 melanoma cell. Moreover, its inhibitory effect on tyrosinase, the key enzyme of melanogenesis, was examined by in vivo and in vitro tyrosinase assay and Western blot. The results indicate that (2Z,8Z)-matricaria acid methyl ester isolated from Erigeron breviscapus is a promising compound that could be useful for treating hyper-pigmentation as skin-whitening agents.

Production of calcium-stearate by lipase using hydrogenated beef tallow.

Calcium-stearate has been traditionally produced by chemical methods, producing wastes and requiring high energy because of high temperature operation. To achieve enzymatic production of calcium-stearate at unfavorable conditions, i.e., pH 10 and 60 degrees C, suitable lipase was selected and reaction conditions were optimized using calcium hydroxide and hydrogenated beef tallow as substrates. Under optimum conditions, 95% of beef tallow, in 2.5 h, was converted into calcium-stearate by using commercial lipase SDL 451. Investigation of the time-course reaction revealed that fatty acid was initially produced by lipase, followed by conversion into calcium-stearate. The fatty acid production rate was faster than that of the conversion into calcium-stearate at the beginning of the reaction. Alkaline pH, originating from the addition of calcium hydroxide, increased the converting reaction. This is the first report demonstrating that chemical production of calcium-stearate can be replaced by enzymatic reaction, thereby creating a cleaner process.

Inhibition of melanogenesis by dioctyl phthalate isolated from Nigella glandulifera Freyn.

Although a number of melanogenesis inhibitors have recently been reported and used as cosmetic additives, none is completely satisfactory, leaving a need for novel skin-depigmenting agents. Thus, to develop a novel skin-depigmenting agent from natural sources, the inhibition of melanogenesis by Chinese plants was evaluated. A methanolic extract of Nigella glandulifera Freyn was found to inhibit the melanin synthesis of murine B16F10 melanoma cells by 43.7% and exhibited a low cytotoxicity (8.1 %) at a concentration of 100 microg/ml. Thus, to identify the melanogenesis-inhibiting mechanism, the inhibitory activity towards tyrosinase, the key enzyme of melanogenesis, was further evaluated, and the results showed inhibitory effects on the activity of intracellular tyrosinase yet not on mushroom tyrosinase. Finally, to isolate the compounds with a hypopigmenting capability, activity-guided isolation was performed, and Dioctyl phthalate identified as inhibiting melanogenesis.