Different wavelengths of LED irradiation promote secondary metabolite production in Pycnoporus sanguineus for antioxidant and immunomodulatory applications.

Pycnoporus sanguineus is a fungus of the phylum Basidiomycota that has many applications in traditional medicine, modern pharmaceuticals, and agricultural industries. Light plays an essential role in the metabolism, growth, and development of fungi. This study evaluated the mycelial growth and antioxidant and anti-inflammatory activities in P. sanguineus fermentation broth (PFB) cultured under different wavelengths of LED irradiation or in the dark. Compared to the dark cultures, the dry weight of mycelia in red- and yellow-light cultures decreased by 37 and 35% and the yields of pigments increased by 30.92 ± 2.18 mg and 31.75 ± 3.06 mg, respectively. Compared with the dark culture, the DPPH free radical scavenging ability, ABTS+ free radical scavenging capacity, and reducing power of yellow-light cultures increased significantly, and their total phenolic content peaked at 180.0 ± 8.34 µg/mL. However, the reducing power in blue-light cultures was significantly reduced, though the total phenol content did not vary with that of dark cultures. In LPS- and IFN-γ-stimulated RAW 264.7 cells, nitrite release was significantly reduced in the red and yellow light-irradiated PFB compared with the dark culture. In the dark, yellow-, and green-light cultures, TNF-α production in the inflamed RAW 264.7 cells was inhibited by 62, 46, and 14%, respectively. With red-, blue-, and white-light irradiation, TNF-α production was significantly enhanced. Based on these results, we propose that by adjusting the wavelength of the light source during culture, one can effectively modulate the growth, development, and metabolism of P. sanguineus.

Suppression of Propionibacterium acnes-stimulated proinflammatory cytokines by Chinese bayberry extracts and its active constituent myricetin in human sebocytes in vitro.

Myrica rubra Sieb. et Zucc. (Myricaceae), known as Chinese bayberry, is traditionally used as folk medicine in Asian countries. The interaction of Propionibacterium acnes signalling with sebocytes is considered important in the pathogenesis of acne. In the present study, extracts and active compounds of Chinese bayberry were used to determine chemical antioxidant activity and anti-inflammatory effects in P. acnes-stimulated human SZ95 sebocytes. A high-performance liquid chromatography with electrochemical detection system was used to analyse the phenolic composition of bayberry extracts. Accordingly, the flavonols, myricitrin and myricetin, were found to be abundant in the unhydrolysed and hydrolysed extracts of Chinese bayberry fruits, respectively. The anthocyanin cyanidin-3-glucoside was also predominantly found in the unhydrolysed extracts. Quantification of human inflammatory cytokines indicated that cell-free extracts of P. acnes stimulated IL-8 and IL-6 production, which was inhibited by myricetin, rather than its glycoside or anthocyanin. Myricetin also exhibited inhibitory effects in P. acnes-stimulated gene expression of Toll-like receptor (TLR) 2 and protein phosphorylation of p70 S6 kinase. In conclusion, myricetin shows a suppressive effect on P. acnes-induced cytokine production through regulation of the TLR and mammalian target of rapamycin pathways. Myricetin goes beyond previous research findings to potentially modulate inflammatory signalling in human sebocytes. These results will be valuable in developing anti-inflammatory agents against skin acne.

A 24.7-kDa copper-containing oxidase, secreted by Thermobifida fusca, significantly increasing the xylanase/cellulase-catalyzed hydrolysis of sugarcane bagasse.

Thermobifida fusca is a moderately thermophilic soil bacterium belonging to Actinobacteria. It has been known for its capability to degrade plant cell wall polymers except lignin and pectin. To know whether it can produce enzymes to facilitate lignin degradation, the extracellular proteins bound to sugarcane bagasse were harvested and identified by liquid chromatography tandem mass spectrometry. Among the identified proteins, a putative copper-containing polyphenol oxidase of 241 amino acids, encoded by the locus Tfu_1114, was thought to presumably play a role in lignin degradation. This protein (Tfu1114) was thus expressed in E. coli and characterized. Similarly to common laccases, Tfu1114 is able to catalyze the oxidation reaction of phenolic and nonphenolic lignin related compounds such as 2,6-dimethoxyphenol and veratryl alcohol. More interestingly, it can significantly enhance the enzymatic hydrolysis of bagasse by xylanase and cellulase. Tfu1114 is stable against heat, with a half-life of 4.7 h at 90 °C, and organic solvents. It is sensitive to ethylenediaminetetraacetic acid and reducing agents but resistant to sodium azide, a potent inhibitor of laccases. Atomic absorption spectroscopy indicated that the ratio of copper to the protein monomer is 1, instead of 4, a feature of classical laccases. All these data suggest that Tfu1114 is a novel oxidase with laccase-like activity, potentially useful in biotechnology application.

Comparative study on the photostability of arbutin and deoxy arbutin: sensitivity to ultraviolet radiation and enhanced photostability by the water-soluble sunscreen, benzophenone-4.

Arbutin and deoxy arbutin may release hydroquinone under some conditions. We therefore investigated the photostability of arbutin and deoxy arbutin in an aqueous solution. The results revealed arbutin and deoxy arbutin to be photolabile in an aqueous solution. Deoxy arbutin was less stable than arbutin when exposed to UV radiation. The hydroquinone concentration was also increased during the radiation period in both solutions. Benzophenone-4 could clearly improve the photostability of arbutin during the period of UV radiation, but only slightly enhance the photostability of deoxy arbutin.

Organic ultraviolet filters regulate hyaluronan metabolism in human epidermal keratinocytes through the phosphatidylinositol 3-kinase pathway.

Chronic exposure of skin to ultraviolet (UV) radiation is responsible for skin ageing, which includes degradation of the epidermal and dermal layers. Filtering UV light is key in the sunscreen industry. We studied the effects of organic UV filters on hyaluronan (HA) metabolism and skin hydration in human HaCaT keratinocytes. The gene expression of HA receptors, HA synthase (HAS), hyaluronidase (HYAL), and water channel aquaporin 3 (AQP3) was evaluated by quantitative RT-PCR. The state of oxidative stress was determined by measuring the intracellular levels of reactive oxygen species (ROS). The results showed that five organic UV filters reduced the extracellular contents of HA, and a phosphatidylinositol 3-kinase (PI3K) inhibitor partially restored the decreased HA levels after octinoxate, octocrylene, and oxybenzone treatment. The expression levels of HA receptors, including cluster of differentiation 44 (CD44), receptor for hyaluronic acid-mediated motility (RHAMM), and toll-like receptors (TLRs), were determined. Avobenzone, octinoxate, oxybenzone, and padimate O exerted inhibitory effects on RHAMM expression. Oxybenzone led to a significant increase in CD44 and AQP3 expression. Both octinoxate and octocrylene increased TLR4 expression but decreased ROS accumulation by activating the PI3K pathway. However, the organic UV filters differentially regulated the mRNA expression of HAS and HYAL. Taken together, these results suggest that certain organic UV filters regulate HA metabolism in human keratinocytes in a PI3K pathway-dependent manner.

Attenuation of the extract from Moringa oleifera on monocrotaline-induced pulmonary hypertension in rats.

The purpose of this study was to determine the effects of an extract from Moringa oleifera (MO) on the development of monocrotaline (MCT)-induced pulmonary hypertension (PH) in Wistar rats. An ethanol extraction was performed on dried MO leaves, and HPLC analysis identified niaziridin and niazirin in the extract. PH was induced with a single subcutaneous injection of MCT (60 mg/kg) which resulted in increases in pulmonary arterial blood pressure (Ppa) and in thickening of the pulmonary arterial medial layer in the rats. Three weeks after induction, acute administration of the MO extract to the rats decreased Ppa in a dose-dependent manner that reached statistical significance at a dose of 4.5 mg of freeze-dried extract per kg body weight. The reduction in Ppa suggested that the extract directly relaxed the pulmonary arteries. To assay the effects of chronic administration of the MO extract on PH, control, MCT and MCT+MO groups were designated. Rats in the control group received a saline injection; the MCT and MCT+MO groups received MCT to induce PH. During the third week after MCT treatment, the MCT+MO group received daily i.p. injections of the MO extract (4.5 mg of freeze-dried extract/kg of body weight). Compared to the control group, the MCT group had higher Ppa and thicker medial layers in the pulmonary arteries. Chronic treatments with the MO extract reversed the MCT-induced changes. Additionally, the MCT group had a significant elevation in superoxide dismutase activity when normalized by the MO extract treatments. In conclusion, the MO extract successfully attenuated the development of PH via direct vasodilatation and a potential increase in antioxidant activity.

Gum Arabic in combination with IFN-γ promotes the M1 polarization in macrophage.

Gum Arabic, a mixture of polysaccharide and glycoprotein, is used as an emulsifying stabilizer in the food industry. It might have immunomodulatory effects. We hypothesized that the combination of IFN-γ and Gum Arabic promotes the production of pro-inflammatory factors in RAW 264.7 cells. Treatment of RAW 264.7 cells with the combination of 3% Gum Arabic and 40 ng/mL IFN-γ resulted in a drastic increase (320%) in nitric oxide production compared with that induced by IFN-γ alone. PGE-2 was produced after the cells were treated with 3% Gum Arabic and 40 ng/mL IFN-γ for 6 h. Gum Arabic and IFN-γ increased the production of iNOS and COX-2 proteins, and triggered TNF-α release. Apart from TNF-α, the release of both G-CSF and IL-6 increased by more than 100 times. The release of IL-3, RANTES, and IL-10 increased by more than ten times. Gum Arabic and IFN-γ also increased the secretion of IL-10, IL-1α, IL-1ß, IL-13, KC, IL-5, IL-4, IL-12, Eotaxin, IL-9, MCP-1, and ROS. Cytokines associated with M1 polarization of macrophages such as TNF-α, IL-1ß, IL-12, NO, and ROS were induced by Gum Arabic and IFN-γ. Our findings help to explore the inflammatory reaction caused by Gum Arabic in cosmetics.

Antimicrobial, antioxidant, and anti-inflammatory activities of essential oils from five selected herbs.

Eucalyptus bridgesiana, Cymbopogon martinii, Thymus vulgaris, Lindernia anagallis, and Pelargonium fragrans are five species of herbs used in Asia. Their essential oils were analyzed by GC-MS, and a total of 36 components were detected. The results of our study indicated that, except for the essential oil of P. fragrans, all of the essential oils demonstrated obvious antimicrobial activity against a broad range of microorganisms. The C. martinii essential oil, which is rich in geraniol, was the most effective antimicrobial additive. All of the essential oils demonstrated antioxidant activities on 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, ß-carotene/linoleic acid assay, and nitric oxide radical scavenging assay. Furthermore, the T. vulgaris essential oil, which possesses plentiful thymol, exhibited the highest antioxidant activity. For P. acnes-induced secretion of pro-inflammatory cytokines, the essential oils of P. aeruginosa, C. martinii, and T. vulgaris reduced the TNF-α, IL-1ß, and IL-8 secretion levels of THP-1 cells.

Selective determination of arbutin in cosmetic products through online derivatization followed by disposable electrochemical sensor.

An online derivatization followed by a disposable electrochemical sensor was used for the determination of arbutin (AR) in cosmetic products. The AR was chemically oxidized by MnO2 and subsequently reduced at inexpensive screen-printed carbon electrodes using a low detection potential which improved the selectivity of the method. The effects of various parameters, such as solution pH, detection potential, and flow rate of the mobile phase, were studied in detail. Under optimal conditions [pH 1.6 (0.1 M H3PO4), detection potential 0.0 V (versus Ag/AgCl), flow rate 0.6 mL/min], the linear range for AR was 0.1-1500 ppm (r2 = 0.999) with LOD of 30.06 ppb (S/N = 3). The practical application of the proposed method was demonstrated by the determination of arbutin concentration in commercial cosmetic products.

Study on the stability of deoxyArbutin in an anhydrous emulsion system.

The skin-whitening agent, deoxyArbutin, is a potent tyrosinase inhibitor that is safer than hydroquinone and arbutin. However, it is thermolabile in aqueous solutions, where it decomposes to hydroquinone. Pharmaceutical and cosmetic emulsions are normally oil-in-water (o/w) or water-in-oil (w/o) systems; however, emulsions can be formulated with no aqueous phase to produce an anhydrous emulsion system. An anhydrous emulsion system could offer a stable vehicle for compounds that are sensitive to hydrolysis or oxidation. Therefore, to enhance the stability of deoxyArbutin in formulations, we chose the polyol-in-silicone, anhydrous emulsion system as the basic formulation for investigation. The quantity of deoxyArbutin and the accumulation of hydroquinone in both hydrous and anhydrous emulsions at various temperatures were analyzed through an established high performance liquid chromatographic (HPLC) method. The results indicated that water increased the decomposition of deoxyArbutin in the formulations and that the polyol-in-silicone, oil-based, anhydrous emulsion system provided a relatively stable surrounding for the deoxyArbutin that delayed its degradation at 25 °C and 45 °C. Moreover, the composition of the inner hydrophilic phase, containing different amounts of glycerin and propylene glycol, affected the stability of deoxyArbutin. Thus, these results will be beneficial when using deoxyArbutin in cosmetics and medicines in the future.

Heterologous expression of thermostable acetylxylan esterase gene from Thermobifida fusca and its synergistic action with xylanase for the production of xylooligosaccharides.

The axe gene which encodes an acetylxylan esterase from Thermobifida fusca NTU22, was cloned, sequenced and expressed in Escherichia coli. The gene consists of 786 base pairs and encodes a protein of 262 amino acids. The deduced amino acid sequence of the acetylxylan esterase axe exhibited a high degree of similarity with BTA-hydrolase from T. fusca DSM43793, esterase from Thermobifida alba and lipase from Streptomyces albus. The optimal pH and temperature of the purified esterase were 7.5 and 60°C, respectively. Cooperative enzymatic treatment of oat-spelt xylan by transformant xylanase and acetylxylan esterase significantly increased the xylooligosaccharides production compared with the xylanase or acetylxylan esterase action alone. The synergy of transformant acetylxylan esterase and xylanase cannot increase the production of reducing sugars from lignocellulolytic substrate, bagasse.

Heterologous expression of Thermobifida fusca thermostable alpha-amylase in Yarrowia lipolytica and its application in boiling stable resistant sago starch preparation.

A gene encoding the thermostable alpha-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced, and cloned into Yarrowia lipolytica P01g host strain using the vector pYLSC1 allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 730 U/l in the Hinton flask culture broth. It is higher than that observed in P. pastoris expression system and E. coli expression system. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis and this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60 degrees C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60 degrees C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 72-h treatment, the DP(w) of raw sago starch obviously decreased from 830,945 to 237,092. The boiling stable resistant starch content of the sago starch increased from 8.3 to 18.1%. The starch recovery rate was 71%.

Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis.

A gene encoding the thermostable raw starch digesting alpha-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into Pichia pastoris X-33 host strain using the vector pGAPZalphaA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 510 U/l in the Hinton flask culture broth. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-beta-N-acetylglycosaminidase H, and this agrees with the predicted size based on the nucleotide sequence. About 75% of the original activity remained after heat treatment at 60 degrees C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60 degrees C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 48-h treatment, the DPw of raw sago starch obviously decreased from 830,945 to 378,732. The surface of starch granules was rough, and some granules displayed deep cavities.

Determination of the thermodegradation of deoxyArbutin in aqueous solution by high performance liquid chromatography.

Tyrosinase is the key and rate-limiting enzyme responsible for the conversion of tyrosine into melanin. Competitive inhibition of tyrosinase enzymatic activity results in decreased or absent melanin synthesis by melanocytes in human skin. DeoxyArbutin (4-[(tetrahydro-2H-pyran-2-yl)oxy]phenol), a novel skin whitening agent, was synthesized through the removal of hydroxyl groups from the glucose side-chain of arbutin. DeoxyArbutin not only shows greater inhibition of tyrosinase activity but is also safer than hydroquinone and arbutin. Hence, deoxyArbutin is a potential skin whitening agent for cosmetics and depigmenting drugs; however, stability of this compound under some conditions remains a problem. The lack of stability poses developmental and practical difficulties for the use of deoxyArbutin in cosmetics and medicines. Improving the thermostability of deoxyArbutin is an important issue for its development. In this research, we established an analytical procedure to verify the amount of deoxyArbutin in solutions using a high performance liquid chromatographic (HPLC) method. The results indicate that this novel skin whitening agent is a thermolabile compound in aqueous solutions. Additionally, the rate constant for thermodegradation (k) and the half-life (t(1/2)) of deoxyArbutin were determined and can be used to understand the thermodegradation kinetics of deoxyArbutin. This information can aid in the application of deoxyArbutin for many future uses.

Foam properties and detergent abilities of the saponins from Camellia oleifera.

The defatted seed meal of Camellia oleifera has been used as a natural detergent and its extract is commercially utilized as a foam-stabilizing and emulsifying agent. The goal of this study was to investigate the foam properties and detergent ability of the saponins from the defatted seed meal of C. oleifera. The crude saponin content in the defatted seed meal of C. oleifera was 8.34 and the total saponins content in the crude saponins extract was 39.5% (w/w). The foaming power of the 0.5 crude saponins extract solution from defatted seed meal of C. oleifera was 37.1 of 0.5 SLS solution and 51.3% to that of 0.5% Tween 80 solution. The R5 value of 86.0% represents good foam stability of the crude saponins extracted from the defatted seed meal of the plant. With the reduction of water surface tension from 72 mN/m to 50.0 mN/m, the 0.5% crude saponins extract solution has wetting ability. The sebum-removal experiment indicated that the crude saponins extract has moderate detergency. The detergent abilities of the saponins from C. oleifera and Sapindus mukorossi were also compared.

Constitutive expression of Thermobifida fusca thermostable Acetylxylan Esterase gene in Pichia pastoris.

A gene encoding the thermostable acetylxylan esterase (AXE) in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into the Pichia pastoris X-33 host strain using the vector pGAPZαA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular AXE production, as high as 526 U/mL in the Hinton flask culture broth. The purified enzyme showed a single band at about 28 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-ß-N-acetylglycosaminidase H; this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60 °C for three hours. The optimal pH and temperature of the purified enzyme were 8.0 and 60 °C, respectively. The properties of the purified AXE from the P. pastoris transformant are similar to those of the AXE from an E. coli transformant.

Synthesis and structure-activity relationships of fenbufen amide analogs.

The previous discoveries of butyl fenbufen amide analogs with antitumor effects were further examined. The amide analogs with 1, 3, 4 and 8 carbons chains were prepared in 70-80% yield. Fenbufen had no cytotoxic effects at concentrations ranging from 10 to 100 µM. Methyl fenbufen amide had significant cytotoxic effects at a concentration of 100 µM. As the length of the alkyl amide side chain increased, the cytotoxic effects increased, and the octyl fenbufen amide had the greatest cytotoxic effect. After treatment with 30 µM octyl fenbufen amide, nearly seventy percent of the cells lost their viability. At the concentration of 10 µM, fenbufen amide analogs did not show cytotoxicity according to the MTT assay results. The NO scavenging activities of the fenbufen amide analogs were not significantly different from those of fenbufen.

Purification and properties of an acetylxylan esterase from Thermobifida fusca.

An acetylxylan esterase from Thermobifida fusca NTU22 was purified 51-fold as measured by specific activity from crude culture filtrate by ultrafiltration concentration, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. The overall yield of the purified enzyme was 14.4%. The purified enzyme gave an apparent single protein band on an SDS-PAGE. The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sepharose CL-6B was found to be 30 and 28kDa, respectively, indicating that the acetylxylan esterase from T. fusca NTU22 is a monomer. The pI value of the purified enzyme was estimated to be 6.55 by isoelectric focusing gel electrophoresis. The N-terminal amino acid sequence of the purified esterase was ANPYERGP. The optimum pH and temperature for the purified enzyme were 8.0 and 80°C, respectively. The Zn(2+), Hg(2+), PMSF and DIPF inhibited the enzyme activity. The K(m) value for p-nitrophenyl acetate and acetylxylan were 1.86µM and 0.15%, respectively. Co-operative enzymatic degradation of oat-spelt xylan by purified acetylxylan esterase and xylanase significantly increased the acetic acid liberation compared to the acetylxylan esterase action alone.

Locust bean gum galactomannan hydrolyzed by thermostable ß-d-mannanase may reduce the secretion of pro-inflammatory factors and the release of granule constituents.

Locust bean gum (LBG) galactomannan has been claimed to have applications in the biopharmaceutical field. However, the effects of LBG galactomannan on immunomodulatory aspects are not yet clear. The purpose of this study was to over-express thermostable ß-d-mannanase from the thermophilic actinomycete Thermobifida fusca BCRC 19214 using a Pichia pastoris expression system. The maximum intracellular ß-d-mannanase activity obtained from the cell-free extract was approximately 40.0U/mL after 72h of cultivating a P. pastoris transformant (pPICZ-man) induced with methanol. Hydrolysis of native LBG galactomannan with 8U/mL ß-d-mannanase for 24h significantly decreased the weight-average molecular weight of LBG galactomannan from 5,580,010 to 3188. Native and hydrolyzed LBG galactomannan in a range of 0-0.2% did not trigger significant cytotoxicity after 24h of treatment compared with the control. The native LBG galactomannan stimulated RAW 264.7 cells to produce cytokine TNF-α dose-dependently, but there was no significant IL-1ß or nitric oxide production. The native LBG galactomannan also stimulated ß-hexosaminidase secretion in RBL-2H3 cells. After the native LBG galactomannan was hydrolyzed with ß-d-mannanase, all of the immunological properties disappeared. These results suggest the possible immunomodulatory effects of native LBG galactomannan.

Production of xylooligosaccharides from xylans by extracellular xylanases from Thermobifida fusca.

Xylooligosaccharides are produced for use as a valuable food sweetener or additive. They have many beneficial biomedical and health effects. In this study, a process for producing xylooligosaccharides from lignocellulolytic agricultural waste was developed. Bagasse, corncob, wheat bran, and peanut shell were used as carbon sources for production of xylanolytic enzymes from Thermobifida fusca NTU22. When using bagasse as the carbon source, the xylanolytic enzymes that simultaneously accumulated in the broth in a 500 mL Hinton flask after 72 h of cultivation at 50 degrees C were measured as xylanase (14.0 U/mL), beta-xylosidase (74.1 mU/mL), and acetyl esterase (29.1 mU/mL). The optimum pH and temperature for xylanases were 6.0-8.0 and 70 degrees C, respectively. Six proteins with xylanase activity were identified by zymogram analysis of isoelectric focusing gel. This was followed by heat treatment at 70 degrees C for 30 min that eliminated 90% of the beta-xylosidase activity. The xylanase and acetyl esterase activities were still 100%. Two percent of xylan extracted from the bagasse was then hydrolyzed by heat-treated crude xylanase preparation at 60 degrees C, pH 7.0, for 10 h. The xylooligosaccharides that accumulated in the broth were about 23.7%. After the purification process by activated charcoal chromatography, the purity of xylooligosaccharides was 71.4%.