An ex vivo methodology to assess the lipid peroxidation in stratum corneum

Environmental risks, particularly UV radiation, provide a challenge to the function of the skin barrier. Protective measures such as the use of antioxidant products represent a possible method of providing protection to the skin.This paper reports the development of a non-invasive ex vivo method using tape strips of the outermost layers of stratum corneum (SC) from human volunteers in order to determine the effectiveness of an antioxidant emulsion topically applied to prevent lipid peroxidation (LPO) in the horny layer after an UV irradiation exposure. Two different formulations were used: formulation (A), containing Vitamin A, E and C, and formulation (B) containing fish extract. Both formulations were topically applied in vivo on volunteer forearms; then, a tape stripping of the SC of each volunteer was carried out. The lipid peroxidation was measured ex vivo after an UV irradiation of the SC samples. The amount of SC stripped to evaluate differences in lipid peroxidation, the UV irradiation intensity to form lipid peroxides and the accuracy of lipid peroxide analysis were optimized in this methodology using formulation (A). After an exposure application of seven days, a group of three strips of the outermost layers of SC of volunteers was irradiated with an intensity of 182.7 J/cm² to quantify the LPO inhibition.The percentage of LPO inhibition obtained after topical application of both formulations was in the range of 40–58% demonstrating the effectiveness of the formulations topically applied against lipid peroxidation on human SC. This methodology may be used as a quality control tool to determine ex vivo the percentage of the LPO inhibition on human SC for a variety of antioxidants topically applied.     

Photoprotective Effect of Black Tea Extracts Against UVB-induced Phototoxicity in Skin

In previous studies, we showed that green tea and black tea extracts and their major polyphenolic constituents protect against UVB light-induced carcinogenesis in murine skin. All of these studies required chronic administration of tea extracts or specific constituents either topically or orally. However, it is not known whether acute or subchronic administration of black tea extracts or constituents can ameliorate UVB-induced early effects in skin. In the present study, cultured keratinocytes and mouse and human skin were employed to assess the effect of both oral and topical administration of standardized black tea extract (SBTE) and its two major polyphenolic subfractions namely BTF1 and BTF2 against UVB-induced photodamage. In SKH-1 hairless mice, topical application of SBTE (0.2 mg/cm2) prior to UVB exposure (180 mJ/cm2) resulted in 40% reduced incidence and 64% reduced severity of erythema and 50% reduction in skinfold thickness by day 6 when compared to nontreated UVB-exposed animals. The SBTE was also effective in protecting against UVB-induced erythema in human volunteers. Administration of SBTE 5 min after UVB irradiation was similarly effective in reducing UVB-induced inflammation in both murine and human skin. The major polyphenolic subfractions, BTF1 and BTF2, were also effective in protecting in mouse skin. The SBTE subfractions inhibited UVB-induced tyrosine phosphorylation of epidermal growth factor receptor (EGFR). The UVB irradiation of human epidermoid carcinoma cells resulted in 3.3-fold induction of tyrosine phosphorylation of EGFR. Pretreatment with BTF1 and BTF2 reduced tyrosine phosphorylation of EGFR by 53% and 31%, respectively. The UVB-mediated enhanced expression of the early response genes, c-fos and c-jun in human epidermal keratinocytes was reduced in a dose-dependent manner by SBTE. Topical application of SBTE was also effective in reducing accumulation of c-fos and p53 proteins by 82% and 78%, respectively, in UVB-exposed mouse skin. These data provide evidence that constituents of black tea can abrogate UVB-induced erythema and associated early events in murine and human skin.     

Bucillamine Inhibits UVB-Induced MAPK Activation and Apoptosis in Human HaCaT Keratinocytes and SKH-1 Hairless Mouse Skin

Ultraviolet B (UVB) radiation is known as a culprit in skin carcinogenesis. We have previously reported that bucillamine (N-[2-mercapto-2-methylpropionyl]-L-cysteine), a cysteine derivative with antioxidant and anti-inflammatory capacity, protects against UVB-induced p53 activation and inflammatory responses in mouse skin. Since MAPK signaling pathways regulate p53 expression and activation, here we determined bucillamine effect on UVB-mediated MAPK activation in vitro using human skin keratinocyte cell line HaCaT and in vivo using SKH-1 hairless mouse skin. A single low dose of UVB (30 mJ cm⁻²) resulted in increased JNK/MAPK phosphorylation and caspase-3 cleavage in HaCaT cells. However, JNK activation and casaspe-3 cleavage were inhibited by pretreatment of HaCaT cells with physiological doses of bucillamine (25 and 100 µm ). Consistent with these results, bucillamine pretreatment in mice (20 mg kg⁻¹) inhibited JNK/MAPK and ERK/MAPK activation in skin epidermal cells at 6–12 and 24 h, respectively, after UVB exposure. Moreover, bucillamine attenuated UVB-induced Ki-67-positive cells and cleaved caspase-3-positive cells in mouse skin. These findings demonstrate that bucillamine inhibits UVB-induced MAPK signaling, cell proliferation and apoptosis. Together with our previous report, we provide evidence that bucillamine has a photoprotective effect against UV exposure.     

Inactivation of Enterobacter sakazakii of dehydrated infant formula by gamma-irradiation

Enterobacter sakazakii has been implicated as a causal organism in a severe form of neonatal meningitis, with reported mortality rates of 20%. The population at greatest risk is immunocompromised infants of any age. Dried infant formula has been identified as a potential source of the organism in both outbreaks and sporadic cases. The objective of this study was to investigate theirradiation effect of the inactivation on E. sakazakii (ATCC 29544) of a dehydrated infant formula. The D₁₀-values were 0.22–0.27 and 0.76 kGy for broth and dehydrated infant formula, respectively. The irradiation at 5.0 kGy was able to completely eliminate the E. sakazakii inoculated at 8.0 to 9.0 log CFU g⁻¹ onto a dehydrated infant formula. There was no regrowth for all samples during the time they were stored at 10 °C for 6 h after rehydration. The present results indicated that a gamma-irradiation could potentially be used to inactivate E. sakazakii in a dehydrated powdered infant formula.     

Acute Ultraviolet Radiation Perturbs Epithelialization but not the Biomechanical Strength of Full‐thickness Cutaneous Wounds

We hypothesized that priming of the skin with ultraviolet radiation (UVR) before being injured would enhance wound healing. Four groups, each comprising 20 immunocompetent hairless mice, were exposed to simulated solar irradiation in escalating UVR doses; 0 standard erythema dose (SED) = control, 1 SED, 3 SED and 5 SED. Twenty‐four hours after UV irradiation, inflammation was quantified by skin reflectance (erythema) and myeloperoxidase (MPO) tissue levels, and two 6 mm full‐thickness excisional wounds and one 3 cm incisional wound were inflicted. Epidermal hyperplasia was assessed by quantitative histology. Five days after wounding, wound coverage by neoepithelium and wound width of the excisional wounds was quantified in hematoxylin–eosin sections, and breaking strength was measured in strips from incisional wounds. Erythema (P < 0.001), MPO levels (P < 0.0005) and epidermal cell layers (P < 0.001) increased dose‐dependently by UV exposure of dorsal skin. In the excisional wounds, epithelial coverage decreased (P = 0.024) by increasing the UVR dose, whereas there was no significant difference (P = 0.765) in wound MPO levels. Neither wound width (P = 0.850) nor breaking strength (P = 0.320) differed among the groups. Solar‐simulated UVR 24 h before wounding impaired epithelialization but was not detrimental for surgical incisional wound healing.     

Skin Responses to Micro Scale Field Size of Solar‐Simulated Radiation – Preliminary Evaluation by Reflectance Confocal Microscopy in vivo

Erythema and pigment responses of human skin following an acute exposure to ultraviolet radiation (UVR) are frequently used to determine the photosensitivity of the skin. In this study we investigated the responses of the skin to a micro‐scale area of UVR exposure (MiR) and compared the responses to a macro‐scale area of exposure (MaR). Ten human volunteers were tested with solar‐simulated radiation on their upper arm or back using a beam size of 8 mm and 0.2 mm in diameter. The fluence required to produce a minimally perceptible erythema (MED) using the MiR was found to be higher than that for the MaR. The erythema response extended beyond the exposed area and this became pronounced when the beam size was microscopic. Reflectance confocal microscopy in vivo revealed that MiR induced cellular alterations within a confined area of smaller dimensions than the area of exposure. Pigment responses were confined within the areas of cellular damage. The erythema expression of exposed skin recovered faster for the sites receiving MiR even when the applied fluence was higher than the MED for the MaR. Through the use of MiR we were able to visualize spatially dissimilar skin responses of erythema and pigmentation suggesting different cellular mechanisms.     

Photoprotective Effects of a Formulation Containing Tannase-Converted Green Tea Extract Against UVB-Induced Oxidative Stress in Hairless Mice

Ultraviolet B (UVB) irradiation may induce the acceleration of skin aging. The purpose of this study was to develop an effective formulation containing tannase-converted green tea extract (FTGE) to inhibit UVB-induced oxidative damage. Significant (p < 0.05) prevention of the reduced form of glutathione (GSH) depletion was observed in mice treated with FTGE. The hydrogen peroxide levels of mice treated with FTGE were similar to those of UVB non-irradiated mice. No significant difference was observed between No UVB control and FTGE mice. Also, mice treated with FTGE had significant (p < 0.05) decreases in thiobarbituric acid-reactive substance levels by lipid peroxidation compared with No UVB control mice. Our data suggest that this formulation may be effective in protecting skin from UVB photodamage.     

A new approach to describe the skin surface physical properties in vivo

In the present paper, we describe a new mechanical method characterising the physico-chemical properties of human skin and their variations along with liquid exposure scenario to the skin surface. A specific bio-tribometer has been developed to study the physical properties of the skin in vivo by measuring the maximum adhesion force between the skin and the bio-tribometer. We showed that the lipidic film present on skin surface was responsible for skin adhesion due to capillary phenomena. The measure of pull-off force between skin and bio-tribometer has permitted to estimate the liquid/vapour surface tension of the lipidic film (γ_LV ≈ 6.3 mJ/m² in 30-year-old volunteer). The kinetic of sorption/desorption (sorption means indifferently adsorption and absorption process) of distilled water from the skin has been observed through the variation of the indenter/skin pull-off force versus time after distilled water application to the skin surface. This permits to follow in real time the variation of the skin physico-chemical properties after liquid application onto the skin surface. Finally, the increasing of skin friction coefficient after distilled water application onto skin surface was explained by the capillary adhesion force between the probe and the skin.     

Salt water bathing prior to UVB irradiation leads to a decrease of the minimal erythema dose and an increased erythema index without affecting skin pigmentation

The combination of salt water baths and solar radiation is known as an effective treatment for patients with psoriasis and atopic dermatitis. To determine whether increased susceptibility to UVB radiation may contribute to this therapeutic effect we have studied the effect of bathing the skin in salt water prior to UVB irradiation. Twelve subjects were phototested on the volar aspects of their forearms with increasing doses of UVB radiation. One forearm was exposed to 5% salt water prior to irradiation. The minimal erythema dose (MED) was determined and the erythema index and skin pigmentation were assessed by photometric measurement. The combination of salt water bath and irradiation yielded a significant decrease of the MED when compared to UVB alone (median 90 mJ/cm2 vs 130 mJ/cm2, P < 0.01). Analysis of variance showed a significant influence of salt water bath on erythema (P < 0.05) but not on skin pigmentation. Within the MED test area the erythema index of the salt water exposed forearms was elevated significantly (P < 0.05) while skin pigmentation was not affected. Thus, bathing the skin in salt water leads to a decreased threshold level for the elicitation of UVB-induced erythema and a selective increase of the erythemal response. This sensitization to the effects of shortwave UVB radiation may increase immunosuppressive effects of UVB radiation and may lead to an increased efficacy of UVB phototherapy. However, there is also an increased sunburn risk when salt water baths are followed by exposure to UV radiation.     

Cutaneous Photoprotective Activity of a Short‐term Ingestion of High‐Flavanol Cocoa: A Nutritional Intervention Study

In vivo and in vitro studies have shown that prolonged oral administration of flavanol‐rich cocoas extracts have photoprotective effects. The aim of the present study was to assess the photoprotective activity of short‐time administration of a new variety of naturally selected cocoa extract rich in bioactive compounds. We selected a cocoa powder particularly rich in polyphenols, flavanols, caffeine, theobromine and theophylline. We then investigated, in 10 healthy subjects, the photoprotective effects of one week of daily oral administration of two doses of such powder. Phototesting with solar simulated radiation was performed at baseline and after cocoa supplementation. Visual assessment of the minimal erythema dose and spectrophotometric measurement of the “a” parameter were done after 24 h from the irradiation. Oral daily supplementation of 1 g of high‐flavanol cocoa was not effective but a one‐week administration of 4–6 g of cocoa induced a statistically significant increase in the visual erythema threshold and a significant reduction in the “a” parameter. We demonstrated that a new variety of naturally selected cocoa extract, even when administered for a short time, retains a dose‐dependent photoprotective effect. These results are also indicative of the fact that topical sunscreens could be supplemented by a specific diet.     

Cinnamon zeylanicum bark extract and powder mediated green synthesis of nano-crystalline silver particles and its bactericidal activity

The exploitation of various plant materials for the biosynthesis of nanoparticles is considered a green technology as it does not involve any harmful chemicals. The present study reports the synthesis of silver (Ag) nanoparticles from silver precursor using the bark extract and powder of novel Cinnamon zeylanicum. Water-soluble organics present in the plant materials were mainly responsible for the reduction of silver ions to nano-sized Ag particles. TEM and XRD results confirmed the presence of nano-crystalline Ag particles. The pH played a major role in size control of the particles. Bark extract produced more Ag nanoparticles than the powder did, which was attributed to the large availability of the reducing agents in the extract. Zeta potential studies showed that the surface charge of the formed nanoparticles was highly negative. The EC₅₀ value of the synthesized nanoparticles against Escherichia coli BL-21 strain was 11 ± 1.72 mg/L. ThusC. zeylanicum bark extract and powder are a good bio-resource/biomaterial for the synthesis of Ag nanoparticles with antimicrobial activity.     

Dietary,but not topical,alpha-linolenic acid suppresses UVB-induced skin injury in hairless mice when compared with linoleic acids

Peroxidizability of fatty acids in the air is roughly proportional to the number of double bonds, but in vivo peroxidation proceeds in a more complex manner. Here, we compared the effects of dietary and topically applied oils enriched with linoleic acid (LA, 18:2n-6) or alpha-linolenic acid (ALA, 18:3n-3) on UV-induced skin injury in a strain of hairless mice. The UVB-induced erythema score was significantly lower in mice with topically applied creams containing LA and ALA than in mice with the basal cream; no significant increase in the score was detected in the ALA group compared with the LA group. However, dietary ALA inhibited the increase in erythema score after UVB irradiation compared with LA. The peroxidizability index of the skin total lipids was significantly higher, but UVB-induced prostaglandin E2 (PGE2) production was significantly lower in the group fed an ALA-rich diet compared with the group fed an LA-rich diet. The levels of thiobarbituric acid-reactive substances, estimated in the presence of butylated hydroxytoluene in the assay mixture, were not affected by UVB treatment or by the dietary fatty acids, but the severity of the skin lesion was associated with PGE2 levels. These results indicate that the type of fatty acids, n-6 or n-3, is critical for the suppression of UVB-induced skin lesion when the skin fatty acids are modified by dietary manipulation. Anti-inflammatory activity of dietary flaxseed oil with relatively high ALA and low LA contents was demonstrated in UVB-irradiated hairless mice.     

Effects of gamma irradiation on long-storage seeds of Oryza sativa (cv. 2233) and their surface infecting fungal diversity

We have reported an effective ⁶⁰Co gamma irradiation method for the removal and long-time prevention of contaminating fungi of Oryza sativa cv-2233, without the losses of seed viabilities. The fungal growth and their population on gamma treated seeds was found to decrease significantly (p<0.05) with the increasing irradiation dose and storage time. Immediately after irradiation, the depletion of fungal population on rice seed was noticed (>50%) at 2 kGy, whereas total inhibition was noticed at 3 kGy after 1.5 month.     

In vivo protective effect of beta-carotene against psoralen phototoxicity

The protective effect of β-carotene against PUVA- and UVB-induced erythema was determined in groups of albino female Osborne Mendel rats fed standard, placebo or β-carotene-fortified diets. After 13 weeks, the backs of selected animals were clipped and the animals received 8MOP orally, 20 mg/kg in corn oil, followed by 5 J/cm² UVA irradiation 2 h after dose. A significant β-carotene protective effect was observed against phototoxicity induced by 8MOP, as measured by erythema production. A correlation was noted between the observed protective effect and β-carotene serum and skin levels, determined by HPLC. No protective effect against erythema induced by UVB (0.7 J/cm²) was observed in animals fed the β-carotene-fortified diet. This report represents the first in vivo demonstration of a protective effect of dietary p-carotene against psoraten-induced phototoxicity. Possible mechanisms for the observed β-carotene-protective effect are discussed.     

Effects of gamma irradiation on the yields of volatile extracts of Angelica gigas Nakai

The study was carried out to determine the effects of gamma irradiation on the volatile flavor components including essential oils, of Angelica gigas Nakai. The volatile organic compounds from non- and irradiated A. gigas Nakai at doses of 1, 3, 5, 10 and 20 kGy were extracted by a simultaneous steam distillation and extraction (SDE) method and identified by GC/MS analysis. A total of 116 compounds were identified and quantified from non- and irradiated A. gigas Nakai. The major volatile compounds were identified 2,4,6-trimethyl heptane, α-pinene, camphene, α-limonene, β-eudesmol, α-murrolene and sphatulenol. Among these compounds, the amount of essential oils in non-irradiated sample were 77.13%, and the irradiated samples at doses of 1, 3, 5, 10 and 20 kGy were 84.98%, 83.70%, 83.94%, 82.84% and 82.58%, respectively. Oxygenated terpenes such as β-eudesmol, α-eudesmol, and verbenone were increased after irradiation but did not correlate with the irradiation dose. The yields of active substances such as essential oil were increased after irradiation; however, the yields of essential oils and the irradiation dose were not correlated. Thus, the profile of composition volatiles of A. gigas Nakai did not change with irradiation.     

Reduction of the IgE-binding ability and maintenance of immunogenicity of gamma-irradiated Dermatophagoides farinae

House dust mites, Dermatophagoides farinae (DF) and Dermatophagoides pteronyssinus, are major allergens in the most common indoor allergen and are important risk factor for asthma. The modified antigen has been studied to treat allergic disorder. This study was carried out to measure possibility of modified allergen using gamma irradiation to treat allergy such as asthma. DF solutions (2 mg/ml) as target allergen were irradiated with Co-60 at 50 and 100 kGy. Conformational alternation of irradiated DF was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Levels of anti-irradiated DF mouse IgGs (sub-isotypes) against intact DF were measured similar to that of anti-intact DF IgGs. The binding abilities of house dust mite-allergic patients’ IgE were reduced depending on radiation dose, and irradiation could inhibit the binding ability of patients’ IgE more than 40%. This study has shown that the binding ability of IgE was reduced by conformational alteration by irradiation and the irradiated DF had epitopes capable to induce immunogeniciy.     

The second order hyperpolarizability of cis azobenzene isomer

The second order hyperpolarizability of cis azobenzene isomer (γ_c) was obtained by measuring the third harmonic generation (THG) variation of an azobenzene doped polymer film when the film was optically pumped to create a large amount of cis isomers via photoisomerization. A steady state theory was developed to treat the THG intensity variation by considering the optical pump induced redistribution and reorientation of azobenzene in the polymer film and the contribution of cis isomer to the THG signal. The ratio of γ of cis and trans molecule, (γ_c/γ_t), was found to be 0.51. After the γ_t was obtained from the time-resolved optical Kerr effect (OKE) measurement, γ_c was deduced to be 5.6 × 10⁻³³ esu. The result shows that the optical nonlinearity of cis isomer is clearly not negligible.