In vivo method to evaluate antioxidative activity using UVA-induced carbonylated protein on human skin.

BACKGROUND: Skin is continuously exposed to oxidative stress caused by reactive oxygen species (ROS) produced by the ultraviolet (UV) light, and it is important to evaluate the antioxidant activity. Carbonylated proteins (CPs) are candidate markers of oxidative modification as a result from the ROS. We aimed to develop the CP-based method to assess the efficacy of antioxidants in human skin. METHODS: Ten healthy females were enrolled in the study to determine the UVA dosage for CP production, and another 10 females were included to evaluate the antioxidative activity. The stratum corneum was collected from test skin using D-Squame tape, and CPs from the SC were stained by fluorescence labeling and observed using a fluorescence microscope. RESULTS: CP level significantly increased with UVA irradiation from 15J/cm2 to 50J/cm2 compared to the control (non-UVA) area. CP production significantly increased by 34.38% and 35.22% in UVA irradiation and squalene (vehicle) areas. 5% α-tocopherol and ß-carotene significantly increased the CP production by 20.77% and 19.34% after 2 hours of 30J of UVA irradiation compared to control area. Inhibition rate of CPs in 5% α-tocopherol and 5% ß-carotene showed 41.45% and 45.37% after 2 hours of UVA irradiation. CONCLUSION: This study developed the simple, visual, and direct in vivo method to evaluate the antioxidative activity for products in human skin by measuring the CP level as an oxidative modification caused by UVA-induced ROS generation.

A Combination of Egg Yolk IgY and Phosvitin Inhibits the Growth of Enterotoxigenic Escherichia coli K88 and K99.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is the main cause of fatal diarrhea in piglets during the first week of life and over the time of weaning. Pathogenesis of ETEC-causing diarrhea involves intestinal colonization mediated by fimbriae. Although, both IgY and egg yolk phosvitin (PV) possess antimicrobial activity, their combined activity has not been explored. A combination of IgY specific for ETEC and metal-chelating PV may show synergistic effect in reducing the growth of ETEC by inhibiting bacterial proliferation and stipulating protection against ETEC infection. OBJECTIVE: The goal of this study was to determine the effects of anti-ETEC IgY and PV on in vitro growth inhibition of ETEC strains possessing K88 and K99 fimbriae prevalent in the porcine population. METHODS: Anti-K88 and -K99 IgY antibodies were obtained from egg yolks of 23-week-old Single- Comb White Leghorn hens immunized with K88 and K99 fimbriae of ETEC, respectively, with high titres sustained over 6 to 8 weeks of the immunization period. Specific IgY, PV, and PV-hydrolysate from alcalase-hydrolysis under high hydrostatic pressure (PVH-Alc-HHP) alone or in combination, were used to treat ETEC K88 and K99 cultures at optimal concentrations of 100 µg/mL, 1 mg/mL, and 1 mg/mL, respectively, for 24 h. RESULTS: PVH-Alc-HHP demonstrated the highest degree of hydrolysis, 38.9%. Combined use of IgY and PVH-Alc-HHP showed the highest bactericidal effect resulting in ETEC K88 and K99 growth inhibition of 2.8 and 2.67 log CFU/mL, respectively. CONCLUSION: Combined IgY-PVH effectively control ETEC, therefore holds a great potential for microbial control in veterinary pharmaceutical industry.

High Hydrostatic Pressure-Assisted Enzymatic Treatment Improves Antioxidant and Anti-inflammatory Properties of Phosvitin.

BACKGROUND: Phosvitin (PV) is a highly-phosphorylated metal-binding protein in egg yolk. Phosphoserine clusters make PV resistant to enzymatic digestion, which might be nutritionally undesirable. OBJECTIVE: This study was designed to determine the effects of high hydrostatic pressure and enzymatic hydrolysis (HHP-EH) on the antioxidant and anti-inflammatory properties of PV hydrolysates (PVHs). METHODS: PV was hydrolyzed by alcalase, elastase, savinase, thermolysin, and trypsin at 0.1, 50, and 100 MPa pressure levels. PVHs were evaluated for degree of hydrolysis, molecular weight distribution patterns, antioxidant and anti-inflammatory properties in chemical and cellular models. The effect of PVH on gene expression of pro-inflammatory cytokines (TNF-α and IL-1ß) was also evaluated using real time-PCR. The hydrolysate with most potent antioxidant and anti-inflammatory properties was subjected to LC-MS/MS analysis to identify the peptide sequence. RESULTS: Hydrolysates produced at 100 MPa exhibited higher degree of hydrolysis and greater reducing power and free radical scavenging activity compared to those obtained at atmospheric pressure. After adjusting the phosphate content, alcalase- and trypsin-digested PVHs showed superior iron chelation capacity (69-73%), regardless of pressure. Both alcalase- and trypsin-digested PVHs significantly inhibited nitric oxide production by RAW264.7 macrophage cells. LPS-stimulated up-regulation of proinflammatory cytokines was also suppressed by alcalase-digested PVH. CONCLUSION: The HHP-EH method could play a promising role in the production of bioactive peptides from hydrolysis-resistant proteins. HHP-assisted PVH may be useful in preparing a potential pharmaceutical with antioxidant and anti-inflammatory properties.