High level extracellular production of a recombinant alkaline catalase in E. coli BL21 under ethanol stress and its application in hydrogen peroxide removal after cotton fabrics bleaching.

The effects of induction parameters, osmolytes and ethanol stress on the productivity of the recombinant alkaline catalase (KatA) in Escherichia coli BL21 (pET26b-KatA) were investigated. The yield of soluble KatA was significantly enhanced by 2% ethanol stress. And a certain amount of Triton X-100 supplementation could markedly improved extracellular ratio of KatA. A total soluble catalase activity of 78,762U/mL with the extracellular ratio of 92.5% was achieved by fed-batch fermentation in a 10L fermentor, which was the highest yield so far. The purified KatA showed high stability at 50°C and pH 6-10. Application of KatA for elimination of H2O2 after cotton fabrics bleaching led to less consumption of water, steam and electric power by 25%, 12% and 16.7% respectively without productivity and quality losing of cotton fabrics. Thus, the recombinant KatA is a promising candidate for industrial production and applications.

New Whitening Constituents from Taiwan-Native Pyracantha koidzumii: Structures and Tyrosinase Inhibitory Analysis in Human Epidermal Melanocytes.

Nontoxic natural products useful in skin care cosmetics are of considerable interest. Tyrosinase is a rate-limiting enzyme for which its inhibitor is useful in developing whitening cosmetics. Pyracantha koidzumii (Hayata) Rehder is an endemic species in Taiwan that exhibits tyrosinase-inhibitory activity. To find new active natural compounds from P. koidzumii, we performed bioguided isolation and studied the related activity in human epidermal melanocytes. In total, 13 compounds were identified from P. koidzumii in the present study, including two new compounds, 3,6-dihydroxy-2,4-dimethoxy-dibenzofuran (9) and 3,4-dihydroxy-5-methoxybiphenyl-2'-O-ß-d-glucopyranoside (13), as well as 11 known compounds. The new compound 13 exhibited maximum potency in inhibiting cellular tyrosinase activity, the protein expression of cellular tyrosinase and tyrosinase-related protein-2, as well as the mRNA expression of Paired box 3 and microphthalmia-associated transcription factor in a concentration-dependent manner. In the enzyme kinetic assay, the new compound 13 acted as an uncompetitive mixed-type inhibitor against the substrate l-3,4-dihydroxyphenylalanine and had a Km value against this substrate of 0.262 mM, as calculated using the Lineweaver-Burk plots. Taken together, our findings show compound 13 exhibits tyrosinase inhibition in human melanocytes and compound 13 may be a potential candidate for use in cosmetics.

Membrane treatment of alkaline bleaching effluents from elementary chlorine free kraft softwood cellulose production.

This paper reports experimental results on the sequential use of ultrafiltration (UF), nanofiltration (NF) and reverse osmosis (RO) to fractionate alkaline extraction bleaching effluents from kraft cellulose production. The aim was to unveil the way key pollutants are distributed when subjected to sequential UF/NF/RO membrane separation processes. Alkaline bleaching effluents were obtained from a local pinewood-based mill, featuring elementary chlorine free bleaching to produce high-brightness cellulose. The experimental system was based on a laboratory-scale membrane system, DSS LabStak® M20 Alfa Laval, using Alfa Laval UF and NF/RO membranes, operated at a constant transmembrane pressure (6 bar for UF membranes and 32 bar for NF/RO membranes), at 25°C. Results show that 78% chemical oxygen demand (COD) and total phenols, 82% adsorbable organic halogens (AOX) and 98% colour were retained by UF membranes which have molecular weight cut-off (MWCO) above 10 kDa. In all, 16% of original COD, total phenols and AOX, and the remaining 2% colour were retained by UF membranes within the 1 to 10 kDa MWCO range. Chloride ions were significantly present in all UF permeates, and RO was required to obtain a high-quality permeate with a view to water reuse. It is concluded that UF/NF/RO membranes offer a feasible option for water and chemicals recovery from alkaline bleaching effluents in kraft pulp production.

Antioxidant capacity of leaf extracts from two Stevia rebaudiana Bertoni varieties adapted to cultivation in Mexico.

The recent introduction of the cultivation of Stevia rebaudiana Bertoni in Mexico has gained interest for its potential use as a non-caloric sweetener, but some other properties of this plant require studies. Extracts from two varieties of S. rebaudiana Bertoni adapted to cultivation in Mexico were screened for their content of some phytochemicals and antioxidant properties. Total pigments, total phenolic and flavonoids contents of the extracts ranged between 17.7-24.3 mg/g, 28.7-28.4 mg/g, and 39.3-36.7 mg/g, respectively. The variety "Criolla" exhibited higher contents of pigments and flavonoids. Trolox equivalent antioxidant capacity ranged between 618.5-623.7 mM/mg and DPPH decolorization assay ranged between 86.4-84.3%, no significant differences were observed between varieties. Inhibition of ß-carotene bleaching ranged between 62.3-77.9%, with higher activity in the variety "Criolla". Reducing power ranged between 85.2-86% and the chelating activity ranged between 57.3-59.4% for Cu²âº and between 52.2-54.4% for Fe²âº, no significant differences were observed between varieties. In conclusion, the results of this study showed that polar compounds obtained during the extraction like chlorophylls, carotenoids, phenolic compounds and flavonoids contribute to the antioxidative activity measured. The leaves of S. rebaudiana Bertoni could be used not only as a source of non-caloric sweeteners but also naturally occurring antioxidants.

La reciente introducción del cultivo de Stevia rebaudiana Bertoni en México ha ganado interés debido a su potencial uso como fuente de edulcorantes no calóricos, pero otras propiedades de esta planta aun requieren de estudios. Extractos de hojas de dos variedades de S. rebaudiana Bertoni adaptadas al cultivo en México fueron evaluados en cuanto a su contenido de algunos fitoquímicos y sus propiedades antioxidantes. El contenido de pigmentos, fenoles totales y flavonoides en los extractos, osciló entre 17.7-24.3 mg/g, 28.7-28.4 mg/g, y 39.3-36.7 mg/g, respectivamente. La variedad "Criolla" exhibió los mayores contenidos de pigmentos y flavonoides. La capacidad antioxidante equivalente de Trolox osciló entre 618.5-623.7 mM/mg y el ensayo de decoloración del radical DPPH osciló entre 86.4-84.3%, no observándose diferencias significativas entre ambas variedades. La inhibición de la decoloración del -caroteno osciló entre 62.3-77.9%, siendo mayor en la variedad "Criolla". El poder reductor osciló entre 85.2-86%, las capacidades quelantes de cobre y hierro oscilaron entre 57.3-59.4% y 52.2-54.4%, respectivamente, no observándose diferencias significativas entre ambas variedades. En conclusión, los resultados de este estudio demuestran que los compuestos de naturaleza polar obtenidos durante la extracción, tales como pigmentos clorofílicos, carotenoides, compuestos fenólicos y flavonoides contribuyen a la actividad antioxidante. Las hojas de S. rebaudiana Bertoni podrían ser empleadas no solo como fuente de edulcorantes no calóricos, sino también como fuente de antioxidantes de origen natural.

The decontamination of bleaching effluent by pilot-scale solar Fenton process.

A solar Fenton process was applied as post-treatment to selectively eliminate organic pollutants and toxicants in bleaching effluents of kraft pulp mills. Experiments were conducted to study the effect of system parameters (pH, initial concentration of H2O2, molar ratio of Fe2+/H2O2 and solar-UV irradiance) on the removals of chemical oxygen demand and colour. The results showed 92.8% of COD and 99.6% of colour were removed at pH 3.5, H2O2 30 mM/ L, Fe2+/H2O2 1:100, solar-UV irradiance 11070 mW/m2, reaction time 120 min. The first-order kinetic model was used to study the dependence of the reaction rate on solar-UV irradiance: a linear relationship was shown to exist between reaction rate constants and solar-UV irradiance. The results of gas chromatography mass spectrometry analysis showed that the toxicity of the bleaching effluents was mainly derived from the presence of mononuclear aromatics, polycyclic aromatic hydrocarbons and organochlorides, which were all degraded into harmless organic acids under the attack of hydroxyl radicals generated from the solar Fenton reaction.

Inhibitory effects of Sargassum polycystum on tyrosinase activity and melanin formation in B16F10 murine melanoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Sargassum polycystum, a type of brown seaweed, has been used for the treatment of skin-related disorders in traditional medicine. AIM OF THE STUDY: The aim of the present study is to investigate the antimelanogenesis effect of Sargassum polycystum extracts by cell-free mushroom tyrosinase assay followed by cell viability assay, cellular tyrosinase assay and melanin content assay using B16F10 murine melanoma cells. MATERIALS AND METHODS: Sargassum polycystum was extracted with 95% ethanol and further fractionated with hexane, ethyl acetate and water. The ethanolic crude extract and its fractionated extracts were tested for their potential to act as antimelanogenesis or skin-whitening agents by their abilities to inhibit tyrosinase activity in the cell-free mushroom tyrosinase assay and cellular tyrosinase derived from melanin-forming B16F10 murine melanoma cells. The tyrosinase inhibitory activity was correlated to the inhibition of melanin production in α-MSH-stimulated and unstimulated B16F10 cells. RESULTS: Sargassum polycystum ethanolic extract and its fractions had little or no inhibitory effect on mushroom tyrosinase activity. However, when tested on cellular tyrosinase, the ethanolic extract and its non-polar fraction, hexane fraction (SPHF), showed significant inhibition of cellular tyrosinase activity. In parallel to its cellular tyrosinase inhibitory activity, SPHF was also able to inhibit basal and α-MSH-stimulated melanin production in B16F10 cells. CONCLUSIONS: Our findings showed that (i) cellular tyrosinase assay is more reliable than mushroom tyrosinase assay in the initial testing of potential antimelanogenesis agents and, (ii) SPHF inhibited melanogenesis by inhibiting cellular tyrosinase activity. SPHF may be useful for treating hyperpigmentation and as a skin-whitening agent in cosmetics industry.

Partially purified components of Nardostachys chinensis suppress melanin synthesis through ERK and Akt signaling pathway with cAMP down-regulation in B16F10 cells.

UNLABELLED: Ethnopharmacological relevance Nardostachys chinensis has been used in folk medicine to treat melasma and lentigines in Korea. We investigated the inhibitory activities of Nardostachys chinensis in melanogenesis and its related signaling pathway. MATERIALS AND METHODS: Bioassay-guided fractionation of Nardostachys chinensis using solvent partitioning and purification with octadecylsilane open-column chromatography resulted in partial purification. The active 20% methanol chromatographic fraction from the ethyl acetate layer (PPNC) was used to investigate melanogenesis by melanin synthesis, tyrosinase activity assay, cAMP assay, Western blot and flow cytometric analyses in B16F10 mouse melanoma cells. RESULTS: PPNC markedly inhibits melanin synthesis and tyrosinase activity in a concentration-dependent manner. We also found that PPNC decreases microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (TRP)-1, and dopachrome tautomerase (Dct) protein expressions and MITF and tyrosinase mRNA levels. Moreover, PPNC reduces intracellular cAMP levels and activates mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt expression in B16F10 cells. The specific MEK/ERK inhibitor PD98059 and PI3K/Akt inhibitor LY294002, block the PPNC-induced hypopigmentation effect, and abrogate the PPNC-suppressed expression of melanogenic proteins such as MITF, tyrosinase, TRP-1, and Dct. Using flow cytometry, we elucidated whether PPNC directly induces ERK phosphorylation at the level of an intact single cell. PPNC shows marked expression of phosphorylated ERK in live B16F10 cells and abrogates PPNC-induced phosphorylated ERK by PD98059 treatment. CONCLUSIONS: PPNC stimulates MEK/ERK phosphorylation and PI3K/Akt signaling with suppressing cAMP levels and subsequently stimulating MITF and TRPs down-regulation, resulting in melanin synthesis suppression.

Screening of marine algae for potential tyrosinase inhibitor: those inhibitors reduced tyrosinase activity and melanin synthesis in zebrafish.

In order to find new anti-browning and whitening agents in this study, we investigated 43 indigenous marine algae for tyrosinase inhibitory activity. The extracts from Endarachne binghamiae, Schizymenia dubyi, Ecklonia cava (EC) and Sargassum silquastrum (SS) evidenced potent tyrosinase inhibitory activity similar to that of positive control, kojic acid. Among those marine algae, EC and SS are distributed abundantly on Jeju Island. Therefore, we selected those two species for further studies. Our results evidenced that both species reduced cellular melanin synthesis and tyrosinase activity. On the other hand, we utilized zebrafish as an alternative in vivo model. All the tested samples evidenced excellent inhibitory effects on the pigmentation of zebrafish, most likely due to their potential tyrosinase inhibitory activity. In simultaneous in vivo toxicity tests, no toxicity was observed in either algal species, on the other hand, toxicity was observed in positive controls. These results provided that EC and SS extract could be used as an ingredient for whiting cosmetics and that zebrafish is an alternative in vivo model.

Sulfur-containing constituents and one 1H-pyrrole-2-carboxylic acid derivative from pineapple [Ananas comosus (L.) Merr.] fruit.

Two sulfur-containing compounds, (S)-2-amino-5-((R)-1-carboxy-2-((E)-3-(4-hydroxy-3-methoxyphenyl)allylthio)ethyl-amino)-5-oxopentanoic acid (1) and (S)-2-amino-5-((R)-1-(carboxymethylamino)-3-((E)-3-(4-hydroxyphenyl)allylthio)-1-oxopropan-2-ylamino)-5-oxopentanoic acid (2), and one 1H-pyrrole-2-carboxylic acid derivative, 6-(3-(1H-pyrrole-2-carbonyloxy)-2-hydroxypropoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-carboxylic acid (3), together with eighteen known phenolic compounds, were isolated from the fruits of pineapple. Their structures were elucidated by a combination of spectroscopic analyses. Some of these compounds showed inhibitory activities against tyrosinase. The half maximal inhibitory concentration values of compounds 1, 4, 5, 6, 7 are lower than 1 mM. These compounds may contribute to the well-known anti-browning effect of pineapple juice and be potential skin whitening agents in cosmetic applications.