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.

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.

Progress in genetic engineering of peanut (Arachis hypogaea L.)--a review.

Peanut (Arachis hypogaea L.) is a major species of the family, Leguminosae, and economically important not only for vegetable oil but as a source of proteins, minerals and vitamins. It is widely grown in the semi-arid tropics and plays a role in the world agricultural economy. Peanut production and productivity is constrained by several biotic (insect pests and diseases) and abiotic (drought, salinity, water logging and temperature aberrations) stresses, as a result of which crop experiences serious economic losses. Genetic engineering techniques such as Agrobacterium tumefaciens and DNA-bombardment-mediated transformation are used as powerful tools to complement conventional breeding and expedite peanut improvement by the introduction of agronomically useful traits in high-yield background. Resistance to several fungal, virus and insect pest have been achieved through variety of approaches ranging from gene coding for cell wall component, pathogenesis-related proteins, oxalate oxidase, bacterial chloroperoxidase, coat proteins, RNA interference, crystal proteins etc. To develop transgenic plants withstanding major abiotic stresses, genes coding transcription factors for drought and salinity, cytokinin biosynthesis, nucleic acid processing, ion antiporter and human antiapoptotic have been used. Moreover, peanut has also been used in vaccine production for the control of several animal diseases. In addition to above, this study also presents a comprehensive account on the influence of some important factors on peanut genetic engineering. Future research thrusts not only suggest the use of different approaches for higher expression of transgene(s) but also provide a way forward for the improvement of crops.

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.

Production and characterization Te-peptide by induced autolysis of Saccharomyces cerevisiae.

Recently, the interest in mimicking functions of chalcogen-based catalytic antioxidants like selenoenzymes, has been increased. Various attempts had been done with selenium, but very few attempts were carried out with tellurium. Bio-complex formation and characterization of tellurium was not tried earlier by using any organism. The present study was focused on tellurium peptide production, characterization, and bioactivity assessment especially Mimetic to glutathione peroxidase (GPx). The production was achieved by the autolysis of total proteins obtained from Saccharomyces cerevisiae ATCC 7752 grown with inorganic tellurium. The GPx-like activity of the hydrolyzed tellurium peptide was increased when prepared by autolysis, but decreased when prepared by acid hydrolysis. Tellurium peptide produced by autolysis of the yeast cell showed increased GPx-like activity as well as tellurium content. Tellurium peptide showed little toxicity, compared to highly toxic inorganic tellurium. The results showed the potential of tellurium peptide as an antioxidant that can be produced by simple autolysis of yeast cells.

Isolation and characterization of Pseudoalteromonas sp. from fermented Korean food, as an antagonist to Vibrio harveyi.

The microbial intervention for sustainable management of aquaculture, especially use of probiotics, is one of the most popular and practical approaches towards controlling pathogens. Vibrio harveyi is a well-known pathogenic bacterium, which is associated to a huge economic loss in the aquaculture system by causing vibriosis. The present study is crafted for screening and characterization of anti-Vibrio strains, which were isolated from various traditional fermented Korean foods. A total of 196 strains have been isolated from soybean paste (78 strains), red chili paste (49 strains), soy sauce (18 strains), jeotgal-a salted fish (34 strains), and the gazami crab-Portunus trituberculatus (17 strains). Fifteen strains showed an inhibitory effect on the growth of V. harveyi when subjected to coculture condition. Among the strains isolated, one has been identified as a significant anti-Vibrio strain. Further biochemical characterization and 16S rDNA sequencing revealed it as Pseudoalteromonas aliena, which had been deposited at the Korean Culture Center of Microorganisms (KCCM), Korea and designated as KCCM 11207P. The culture supernatants did not have any antimicrobial properties either in pure or in coculture condition. The culture supernatant was not toxic when supplemented to the swimming crab, Zoea, and Artemia larvae in aquaculture system. The results were very encouraging and showed a significant reduction in accumulated mortality. Here, we reported that pathogenic vibriosis can be controlled by Pseudoalteromonas sp. under in vitro and in vivo conditions. The results indicated that the biotic treatment offers a promising alternative to the use of antibiotics in crab aquaculture.

Production and characterization of low molecular weight sophorolipid under fed-batch culture.

The present study was designed for the production and optimization of the C12-C14 sophorolipid, using the yeast Candida bombicola ATCC-22214. The fermentation was carried under fed-batch culture conditions i.e., maintaining 15% coconut oil and 10% glucose as hydrophobic and hydrophilic carbon sources, respectively. A maximum yield 54.0 g/L (in 234 h) was achieved. A significant antimicrobial activity, surface activity, and emulsion ability were recorded. The native sophorolipid was found as enhancer of detergent efficacy of commercial detergent, tested on complex, smudge and oil contaminated clothes. Molecular weight of the C12 (605/623) and C14 (633/651) sophorolipids were determined by LC-MS which revealed it as diacetylated sophorolipid. This study is being important in terms of yield, which is better than the previously reported.

Medicinal and cosmetic potentials of sophorolipids.

Sophorolipids, a kind of glycolipids, regarded as the most promising bio-surfactant, are synthesized by a selected number of yeast species. Instead of offering, as an environmental friendly alternative to the petrochemical based surfactants, it also exhibits various bioactivities. Sophorolipids in particular are on the brink of being incorporated into several cosmetic application. The potential of this molecule is also screened in various industries like the cosmetics, pharmaceuticals, and other medical applications. In this review, our aim is to provide an overview and updates on basic and applied research on Sophorolipid especially in terms of cosmetics and medicinal uses.

In silico study and validation of phosphotransacetylase (PTA) as a putative drug target for Staphylococcus aureus by homology-based modelling and virtual screening.

Staphylococcus aureus, a Gram-positive bacterium, can cause a range of illnesses from minor skin infections to life-threatening diseases, such as bacteraemia, endocarditis, meningitis, osteomyelitis, pneumonia, toxic shock syndrome and sepsis. Due to the emergence of antibiotic resistance strains, there is a need to develop of new class of antibiotics or drug for this pathogen. The phosphotransacetylase enzyme plays an important role in the acetate metabolism and found to be essential for the survival of the S. aureus. This enzyme was evaluated as a putative drug target for S. aureus by in silico analysis. The 3D structure of the phosphotransacetylase from S. aureus was modelled, using the 1TD9 chain 'A' from Bacillus subtilis as a template at the resolution of 2.75 Å. The generated model has been validated by PROCHECK, WHAT IF and SuperPose. The docking was performed by the Molegro virtual docker using the ZINC database generated ligand library. The ligand library was generated within the limitation of the Lipinski rule of five. Based on the dock-score, five molecules have been subjected to ADME/TOX analysis and subjected for pharmacophore model generation. The zinc IDs of the potential inhibitors are ZINC08442078, ZINC8442200, ZINC 8442087 and ZINC 8442184 and found to be pharmacologically active antagonist of phosphotransacetylase. The molecules were evaluated as no-carcinogenic and persistent molecule by START programme.

Virtual screening and evaluation of Ketol-Acid Reducto-Isomerase (KARI) as a putative drug target for Aspergillosis.

Aspergillus is a leading causative agent for fungal morbidity and mortality in immuno-compromised patients. To identify a putative target to design or identify new antifungal drug, against Aspergillus is required. In our previous work, we have analyzed the various biochemical pathways, and we found Ketol Acid Reducto-Isomerase (KARI) an enzyme involves in the amino acid biosynthesis, could be a better target. This enzyme was found to be unique by comparing to host proteome through BLASTp analysis. A homology based model of KARI was generated by Swiss model server. The generated model had been validated by PROCHECK and WHAT IF programs. The Zinc library was generated within the limitation of the Lipinski rule of five, for docking study. Based on the dock-score six molecules have been studied for ADME/TOX analysis and subjected for pharmacophore model generation. The Zinc ID of the potential inhibitors is ZINC00720614, ZINC01068126, ZINC0923, ZINC02090678, ZINC00663057 and ZINC02284065 and found to be pharmacologically active agonist and antagonist of KARI. This study is an attempt to Insilco evaluation of the KARI as a drug target and the screened inhibitors could help in the development of the better drug against Aspergillus.

Algal fucoidan: structural and size-dependent bioactivities and their perspectives.

Fucoidan is a complex-sulfated polysaccharide distributed in various marine organisms, and the brown algae are reported as the major producer. The fucoidan is important for their high bioactive properties, like antibacterial, anticoagulant, antiviral, anti-tumor, etc., and many more to be explored. There is a strong archival support for the bioactivity and promising properties of this molecule, which creates a hope for this molecule as future drug against thrombosis and some kind of cancers. Reports other than the above bioactive properties have also been a matter of interest for the design of signal or enzyme-arrested new class of drugs. In the past three decades, the research on isolation, molecular characterization, and screening of biological applications has significantly increased. One major issue associated with this molecule is the higher size and seasonal variation in their chemical composition; to resolve the issue and maintain its bioactivity, a prioritized and literal hydrolysis process is required to be developed. Here, in this mini-review, we have tried to summarize the algal fucoidan research and the bioactivities influenced by their molecular size.

In silico characterization of alkaline proteases from different species of Aspergillus.

A total of 49 protein sequences of alkaline proteases retrieved from GenBank representing different species of Aspergillus have been characterized for various physiochemical properties, homology search, multiple sequence alignment, motif, and super family search and phylogenetic tree construction. The sequence level homology was obtained among different groups of alkaline protease enzymes, viz alkaline serine protease, oryzin, calpain-like protease, serine protease, subtilisin-like alkaline proteases. Multiple sequence alignment of alkaline protease protein sequence of different Aspergillus species revealed a stretch of conserved region for amino acid residues from 69 to 110 and 130-204. The phylogenetic tree constructed indicated several Aspergillus species-specific clusters for alkaline proteases namely Aspergillus fumigatus, Aspergillus niger, Aspergillus oryzae, Aspergillus clavatus. The distributions of ten commonly observed motifs were analyzed among these proteases. Motif 1 with a signature amino acid sequence of 50 amino acids, i.e., ASFSNYGKVVDIFAPGQDILSCWIGSTTATNTISGTSMATPHIVGLSCYL, was uniformly observed in proteases protein sequences indicating its involvement with the structure and enzymatic function. Motif analysis of acidic proteases of Aspergillus and bacterial alkaline proteases has revealed different signature amino acid sequences. The superfamily search for these proteases revealed the presence of subtilases, serine-carboxyl proteinase, calpain large subunit, and thermolysin-like superfamilies with 45 representing the subtilases superfamily.

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.

A stimulatory effect of Cassia occidentalis on melanoblast differentiation and migration.

In vitiligo, the active melanocytes in the epidermis are totally missing, whereas melanoblast cells in the outer root sheath of hair follicles are not affected. In an attempt to find potent repigmenting agents for vitiligo therapy, pod extracts of Cassia occidentalis was found to be effective in inducing differentiation and migration of mouse melanoblast cell line. Methanolic extract redissolved in DMSO at 12.5 µg/ml was found to cause 3.5- to 3.8-fold melanin induction in melb-a melanoblast cells after 4 days in treatment medium. In addition it induced the tyrosinase activity and altered melb-a cell morphology. Transwell migration assay showed the potential of this herbal candidate to induce direct migration of treated cells. To the best of our knowledge, this is the first report investigating the effect of Cassia occidentalis on the differentiation and migration of melanoblast cells. The findings of present study are significant in designing preclinical and clinical studies on the efficacy of C. occidentalis as a stimulant for skin repigmentation in vitiligo.

Development of a protein chip to measure PKCß activity.

Phosphorylation of proteins by kinases plays an important role in regulating cellular processes including melanin production in the skin cells. Protein kinase C ß (PKCß) is known to be involved in phosphorylating tyrosinase, the key enzyme of melanin production, regulating the skin pigmentation process. In melanogenesis, PKCß activates the tyrosinase by phosphorylation of its two serine residues. In this study, phosphorylation activity by PKCß was monitored on a protein chip for the screening of depigmenting agents. As a tyrosinase mimic, 11 or 30 amino acids of the C-terminal of tyrosinase was fused with maltose-binding protein (MBP). After immobilizing the MBP-fused PKCß substrate peptide on epoxy-treated slide surface, PKCß reaction mix was applied over the immobilized MBP-fused PKCß substrate peptide. Phosphorylation was detected with anti-phosphoSer/Thr antibodies, followed by fluorescence-labeled second antibodies. Phosphorylation of MBP-30aa was observed on a protein chip, and this phosphorylation was inhibited by the PKC inhibitor (GF109203X). These results indicate the potential of PKCß protein chip as a high-throughput screening tool in the screening of depigmenting agents.

Ipomoea batatas attenuates amyloid ß peptide-induced neurotoxicity in ICR mice.

In this study, the protective effects of 17 Korean native plants against amyloid ß peptide (Aß)-induced oxidative stress were screened using the 2',7'-dichlorofluorescin diacetate assay and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Ipomoea batatas exerted the highest protective effects against oxidative stress and was selected for further investigation. To confirm the protective activity of this extract, the I. batatas extract was fed to ICR mice that had been injected with Aß to induce neuronal deficits. In these experiments, the extract of I. batatas significantly reversed Aß-induced neurotoxicity as assessed by the passive avoidance test, a behavioral experiment. Moreover, I. batatas administration reduced the level of lipid peroxidation and increased catalase activities in biochemical studies using the brain tissue of mice. These results indicate that I. batatas might be beneficial against Alzheimer's disease, especially by limiting oxidative stress in the brain.

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.