Evidence of the protective effect of anti-pollution products against oxidative stress in skin ex vivo using EPR spectroscopy and autofluorescence measurements.

The concentration of air pollution is gradually increasing every year so that daily skin exposure is unavoidable. Dietary supplements and topical formulations currently represent the protective strategies to guard against the effects of air pollution on the body and the skin. Unfortunately, there are not yet enough methods available to measure the effectiveness of anti-pollution products on skin. Here, we present two ex vivo methods for measuring the protective effect against air pollution of different cream formulations on the skin: Electron paramagnetic resonance (EPR) spectroscopy and autofluorescence excited by 785 nm using a confocal Raman microspectrometer (CRM). Smoke from one cigarette was used as a model pollutant. EPR spectroscopy enables the direct measurement of free radicals in excised porcine skin after smoke exposure. The autofluorescence in the skin was measured ex vivo, which is an indicator of oxidative stress. Two antioxidants and a chelating agent in a base formulation and a commercial product containing an antioxidant mixture were investigated. The ex vivo studies show that the antioxidant epigallocatechin-3-gallate (EGCG) in the base cream formulation provided the best protection against oxidative stress from smoke exposure for both methods.

Establishment of a method to expose and measure pollution in excised porcine skin with electron paramagnetic resonance spectroscopy.

Health problems associated with the amount of air pollutants are increasing worldwide. Pollution damages not only the lungs; it also has an impact on skin health and is co-responsible for the development of skin diseases. Anti-pollution products are on the rise in the cosmetic market but so far, there is no established method to directly assess the impact of pollution on the skin and to test the efficacy of anti-pollution products. To address this problem, two different chambers were developed for the reproducible exposure to realistic air pollutant concentrations. One chamber for the exclusive use of excised skin and hair samples, the second chamber for ex vivo and in vivo measurements. Measurements of nicotine next to the investigated skin area allow conclusions to be drawn on the particle concentration to which the skin is exposed. Electron paramagnetic resonance spectroscopy, which enables the detection of free radicals in different systems, was applied to assess the hazard potential of pollution in the skin. A direct proof of the formation of free radicals in the skin by the model pollutant cigarette smoke could be demonstrated. An additional application of UV irradiation even increased the formation of free radicals in the skin seven-fold (sum parameter). Depending on the question of interest, the use of different spin probes allows various assessments of the radical formation in skin: the amount of radicals but also the antioxidant status of the microenvironment can be estimated. Using two exposure chambers, the direct formation of oxidative stress by cigarette smoke on ex vivo skin, with and without additional UV exposure, could be reproducibly examined. This measurement method is promising for the assessment of anti-pollution products and could allow a direct causal connection between pollutant, effect on the skin and the protective function of skin care products.

Anti-pollution effects of two antioxidants and a chelator-Ex vivo electron spin resonance and in vivo cigarette smoke model assessments in human skin.

BACKGROUND: Skin damage arising from pollutants in gaseous and particulate matter forms is mainly mediated by oxidative stress. The pollutants directly or indirectly generate free radicals on and in the skin, leading, for example, to MMP up-regulation and damage of collagen fibers. Antioxidants and chelators are used in anti-pollution cosmetics to reduce the harmful effects of free radical generation. MATERIALS AND METHODS: We investigated the efficacy of two antioxidants and one chelator in an anti-pollution cigarette smoke model. Free radical generation was measured directly after UV and cigarette smoke exposure ex vivo on pig skin (slaughterhouse waste), by use of Electron Spin Resonance (ESR). Effects of cigarette smoke were compared to those of Urban Dust (NIST-standard). ESR was also used to measure the copper chelation activity of the test products. Following cigarette smoke application in vivo, two markers of lipid peroxidation malondialdehyde (MDA), and squalene monohydroperoxide (SQOOH), were measured from swab solutions taken from the smoke-exposed skin sites. RESULTS: EDTA generated no effect and the non-chelator antioxidant Tocopherol only small antioxidant effects after exposed to cigarette smoke ex vivo as well as in vivo. Only the hydrophilic phenylethanoid H1 showed significant effects. A clear reduction of free radicals ex vivo and further a significant reduction of in vivo lipid peroxide formation was measured. CONCLUSION: The cigarette smoke model is an ideal method for in vivo assessment of anti-pollution efficacy of topical products with close relation to the real situation of subjects exposed to urban pollution. Further research is required to better understand the role of chelators in anti-pollution cosmetics.

One-time intrathecal triamcinolone acetonide application alters the redox potential in cerebrospinal fluid of progressive multiple sclerosis patients: a pilot study.

INTRODUCTION: Cerebrospinal fluid analysis may provide insight into the interplay between chronic inflammation and response to treatment. OBJECTIVES: To demonstrate the impact of one intrathecal triamcinolone injection on the redox potential and on ascorbyl radical appearance in the cerebrospinal fluid of chronic progressive multiple sclerosis patients. METHODS: A total of 16 patients received 40 mg triamcinolone. Electron-spin resonance spectroscopy measured the oxidation range after copper ion [Cu (II)] addition and ascorbyl-radical bioavailability. RESULTS: There was an increase of Cu (II) ion absorption, which reflects an augmented content of reduced proteins. Ascorbyl radicals were present in contrast to healthy controls according to the literature. CONCLUSION: Intrathecal steroid application alters the redox potential in cerebrospinal fluid. Our findings support the beneficial role of steroids on oxidative stress generally demonstrated by ascorbyl radical appearance. Reactive oxygen species decline is necessary for an upregulated production of reduced proteins.

Effects of water-filtered infrared-A and of heat on cell death, inflammation, antioxidative potential and of free radical formation in viable skin--first results.

The effects of water-filtered infrared-A (wIRA) and of convective heat on viability, inflammation, inducible free radicals and antioxidative power were investigated in natural and viable skin using the ex vivo Bovine Udder System (BUS) model. Therefore, skin samples from differently treated parts of the udder of a healthy cow were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, by prostaglandin E2 (PGE2) measurement and by electron spin resonance (ESR) spectroscopy. Neither cell viability, the inflammation status, the radical status or the antioxidative defence systems of the skin were significantly affected by wIRA applied within 30 min by using an irradiance of 1900 W m(-2) which is of relevance for clinical use, but which exceeded the maximum solar IR-A irradiance at the Earth's surface more than 5 times and which resulted in a skin surface temperature of about 45 °C without cooling and of about 37 °C with convective cooling by air ventilation. No significant effects on viability and on inflammation were detected when convective heat was applied alone under equivalent conditions in terms of the resulting skin surface temperatures and exposure time. As compared with untreated skin, free radical formation was almost doubled, whereas the antioxidative power was reduced to about 50% after convective heating to about 45 °C.

Reduction in the free radical status and clinical benefit of repeated intrathecal triamcinolone acetonide application in patients with progressive multiple sclerosis.

BACKGROUND: Previous open trials performed repeated intrathecal application of the sustained release steroid triamcinolone acetonide every third day in patients with progressive multiple sclerosis and described enhanced walking abilities. OBJECTIVES: The objectives of this study were to demonstrate the efficacy of 5 triamcinolone administrations every other day and to describe their effects on the amount of inducible free radicals in cerebrospinal fluid. SUBJECTS/METHODS: Clinical ratings, determinations of maximum walking distance, and execution of an instrumental peg insertion test were performed at baseline and on each day after a triamcinolone injection in 21 patients with progressive multiple sclerosis. Induction of free radicals was assessed in cerebrospinal fluid before each triamcinolone application by electron spin resonance spectroscopy. RESULTS: Scores for multiple sclerosis improved, walking distance increased, and necessary intervals for the peg insertion procedure were shortened. The amount of inducible free radicals decreased. CONCLUSIONS: Repeat triamcinolone application improves dysfunction of upper and lower extremities even when administered 5 times only and in series every other day. The declined potential for free radical synthesis may be caused by the anti-inflammatory effect of triamcinolone. It may contribute to suppress the smoldering, chronic inflammation, particularly in spinal lesions of patients with progressive multiple sclerosis. The enhanced arm function hypothetically reflects the effect on cervical and brain lesions due to the hypobaric features of triamcinolone.

Ex vivo evaluation of radical sun protection factor in popular sunscreens with antioxidants.

BACKGROUND: UVA induces tissue damage via the production of radical oxygen species. Adding antioxidants to UV filters in sunscreens is a novel photoprotective strategy. The topical application of antioxidants in sunscreen can potentially neutralize the UVA-induced free radicals. OBJECTIVES: We sought to assess the degree of free radical protection offered by sunscreens with antioxidants and attempted to differentiate the contribution of free radical protection from that of the UV filters. METHOD: Twelve sunscreen products were purchased. The degree of UVA protection (UVA-PF) was measured via an in vitro assay according to a European guideline (Colipa). In addition, an electron spin resonance (ESR) spectroscopy-based assay was used to measure the radical skin protection factor (RSF) and antioxidant power (AP) of each product. RESULTS: The sun protection factor (SPF) values of the sunscreens ranged from 15 to 55, and the UVA-PF values ranged from 2.4 to 28.2. The RSF values ranged from 2.4 to 27.1. There is a high correlation between RSF and UVA-PF. The AP values for nearly all the products were 0, and two products (#4 and #9) had very low AP values of 16 and 12, respectively. LIMITATIONS: The study only evaluated a small number of sunscreen products, and only ex vivo and in vitro methods were used to assess the products. CONCLUSIONS: The idea of combining UV filters with antioxidants is appealing. Current sunscreen products on the market offer free radical protection, but the majority of the radical protection is from UV filters rather than antioxidants.

The role of melanin as protector against free radicals in skin and its role as free radical indicator in hair.

Throughout the body, melanin is a homogenous biological polymer containing a population of intrinsic, semiquinone-like radicals. Additional extrinsic free radicals are reversibly photo-generated by UV and visible light. Melanin photochemistry, particularly the formation and decay of extrinsic radicals, has been the subject of numerous electron spin resonance (ESR) spectroscopy studies. Several melanin monomers exist, and the predominant monomer in a melanin polymer depends on its location within an organism. In skin and hair, melanin differs in content of eumelanin or pheomelanin. Its bioradical character and its susceptibility to UV irradiation makes melanin an excellent indicator for UV-related processes in both skin and hair. The existence of melanin in skin is strongly correlated with the prevention against free radicals/ROS generated by UV radiation. Especially in the skin melanin (mainly eumelanin) ensures the only natural UV protection by eliminating the generated free radicals/ROS. Melanin in hair can be used as a free radical detector for evaluating the efficacy of hair care products. The aim of this study was to investigate the suitability of melanin as protector of skin against UV generated free radicals and as free radical indicator in hair.

The antioxidative power AP--A new quantitative time dependent (2D) parameter for the determination of the antioxidant capacity and reactivity of different plants.

In the last decade, naturally occurring antioxidants continue to play an important role in the food-supplement industry. The content of antioxidants in a plant depends on the species, temperature, humidity, period of growth, harvest month, part of the plant used and many other variables. Herein, we present a new method able to determine the all over antioxidative power (AP) of plant extracts or lyophilised plant parts based on the reducing activity against a stable test radical. The method is performed by ESR spectroscopy and is based on the well-known 1,1-diphenyl-2-picryl-hydrazil (DPPH) method with the major difference that both the antioxidative capacity and the antioxidative activity are used to characterise an antioxidant. The resulting antioxidative power is expressed in antioxidative units (AU), where 1AU corresponds to the activity of a 1 ppm solution of Vitamin C as a benchmark. This method allows a rapid, unexpensive and general applicable technique for the measurement of the antioxidative power of very different kinds of substances. The inclusion of the kinetic behaviour of the reducing process of the antioxidant for the determination of the AP allows the identification of the main antioxidant present in a sample. Herein, we present the application example of seeds, sprouts and adult parts of dandelion, amaranth, quinoa, fenugreek, broccoli, red clover and mugwort, where the AP method permits to characterise the plants with the highest antioxidant capacity and reaction velocity. The method permits to select active plant extracts for the food and nutrition industry.