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.

Oxidative stress coping capacity (OSC) value: Development and validation of an in vitro measurement method for blood plasma using electron paramagnetic resonance spectroscopy (EPR) and vitamin C.

Oxidative stress as a driver of disease is reinforcing the trend towards supplementation with antioxidants. While antioxidants positively influence the redox status when applied at physiological doses, higher concentrations may have pro-oxidative effects. Precise assessment methods for testing the supply of antioxidants are lacking. Using in-situ-irradiation as stressor and electron paramagnetic resonance (EPR) spectroscopy as readout system for formed radicals, a stress response assessment method was developed, using protein solutions and plasma samples from transfusion medicine. The method was validated in a double-blind placebo-controlled in vivo cross-over pilot study in blood plasma samples of individuals before and after vitamin C supplementation. Reference measurements were performed for the exogenous antioxidants ß-carotene and vitamin C, and glutathione as an endogenous representative. Malondialdehyde was studied for oxidative stress indication. Protein solutions without antioxidants showed a linear increase in radical concentration during irradiation. The in-vitro-addition of vitamin C or plasma samples from subjects displayed two slopes (m1, m2) for radical production, whereby m1 represented the amount of antioxidants and proteins, m2 only the protein content. These two slopes in combination with the intervening transition area (T) were used to calculate the oxidative stress coping capacity (OSC), which correlated positively with vitamin C concentration in blood plasma, while oxidative stress biomarkers showed only fluctuations within their reference ranges. Furthermore, a selective radical quenching mechanism for vitamin C was observed: the proportion of reactive oxygen species (ROS) in the plasma samples was degraded in dependence to the vitamin C concentration ingested. The proportion of lipid oxygen species (LOS) remained stable while the ascorbyl radical increased with higher vitamin C intake. OSC may represent a sensitive method to detect treatment effects on the redox status in vivo in future validation and treatment studies, and potentially in clinical routine.

Cosmetic Formulations with Melaleuca alternifolia Essential Oil for the Improvement of Photoaged Skin: A Double-Blind, Randomized, Placebo-Controlled Clinical Study.

This aim of this study was to evaluate the penetration depth, antioxidant capacity and the clinical efficacy of Melaleuca alternifolia pure essential oil and in a nanoemulsion to prevent skin photoaging. For this, 2% of pure essential oil or 2% of this essential oil in a nanoemulsion were vehiculated in a formulation. The skin penetration was evaluated using confocal Raman microspectroscopy. The radical protection factor was evaluated using electron paramagnetic resonance spectroscopy. For a clinical study, 40 male participants, aged 18-28 years, were enrolled, being divided into three groups: vehicle formulation, M. alternifolia pure essential oil and M. alternifolia Nanoemulsion. All the participants also received a sunscreen SPF 50 to use during the day. Before and after 90 days of study, skin hydrolipidics and morphological characteristics were performed by skin imaging and biophysical techniques. The nanoemulsion presented a lower antioxidant capacity and a higher penetration through the stratum corneum, reaching the viable epidermis, improving the stratum granulosum morphology. The groups presented an increase in the papillary depth, improving in the dermis echogenicity and the collagen fibers. Melaleuca alternifolia essential provides the potential to improve photoaged skin, being the application of nanoemulsion able to reach deeper skin layers.

Safety and efficacy of combined essential oils for the skin barrier properties: In vitro, ex vivo and clinical studies.

OBJECTIVE: To evaluate the safety and the synergistic effects of tea tree, lavender, eucalyptus and tangerine essential oils in combination on the skin using in vitro, ex vivo and clinical studies. METHODS: The phototoxicity was predicted using 3T3 neutral red uptake phototoxicity test (OECD TG 432). Skin penetration was evaluated by confocal Raman microspectroscopy using direct application of essential oils to pig ears. For the clinical studies, 40 participants were enrolled and randomized in three groups: (1) lavender, eucalyptus and tangerine, (2) the same essential oils plus melaleuca and (3) placebo group. The skin was evaluated by noninvasive techniques before and after a 90-day period of topical use. RESULTS: The essential oils were non-phototoxic, but the tangerine oil showed dose-dependent cytotoxicity (IC50: 33.1 µg/ml), presenting 35% of penetration in the viable epidermis. On the contrary, 17.7 µg/ml in combination was applied per day in the clinical study and the penetration rate for the combinations (10%, 1.77 µg/ml achieving the viable epidermis) guaranteed the safety, since in the clinical study, the application of the four essential oils improved skin barrier and morphologic skin characteristics, as well as increased skin hydration and decreased sebum levels, with no unwanted effects reported. CONCLUSIONS: All essential oils studied were considered non-cytotoxic or non-phototoxic separately except tangerine, which present a dose-dependent cytotoxicity. Finally, the essential oils in combination in an appropriate amount were safe and effective in the improvement of the hydrolipidic balance and morphological properties of the skin.

OBJECTIF: évaluer la sécurité d'emploi et les effets synergiques des associations d'huiles essentielles d'arbre à thé, de lavande, d'eucalyptus et de mandarine sur la peau à l'aide d'études in vitro, ex vivo et cliniques. MÉTHODES: la phototoxicité a été prédite avec le test de phototoxicité de fixation du rouge neutre 3T3 (OCDE TG 432). La pénétration cutanée a été évaluée par microspectroscopie confocale de Raman grâce à l'application directe d'huiles essentielles sur les oreilles de cochons. Pour les études cliniques, 40 participants ont été inclus et randomisés dans trois groupes : (1) lavande, eucalyptus et mandarine, (2) les mêmes huiles essentielles plus melaleuca et (3) un groupe placebo. La peau a été évaluée par des techniques non invasives avant et après une période d'utilisation topique de 90 jours. RÉSULTATS: les huiles essentielles se sont avérées non phototoxiques, mais l'huile de mandarine a montré une cytotoxicité dose-dépendante (CI 50 : 33,1 µg/ml), représentant 35 % de pénétration dans l'épiderme viable. À l'inverse, dans l'étude clinique, une quantité de 17,7 µg/ml par jour en association a été appliquée, et le taux de pénétration des associations (10 %, soit 1,77 µg/ml atteignant l'épiderme viable) a garanti la sécurité d'emploi, puisque dans l'étude clinique, l'application des quatre huiles essentielles a amélioré la barrière cutanée et les caractéristiques morphologiques de la peau, et a entraîné une augmentation de l'hydratation cutanée et une diminution des taux de sébum, sans signalement d'effets indésirables. CONCLUSIONS: chacune des huiles essentielles étudiées a été considérée comme non cytotoxique ou non phototoxique, à l'exception de la mandarine, qui présente une cytotoxicité dose-dépendante. Enfin, l'association d'huiles essentielles en quantité appropriée a démontré sa sécurité d'emploi et son efficacité dans l'amélioration de l'équilibre hydrolipidique et des propriétés morphologiques de la peau.

In vivo confocal Raman microscopic determination of depth profiles of the stratum corneum lipid organization influenced by application of various oils.

BACKGROUND: The intercellular lipids (ICL) of stratum corneum (SC) play an important role in maintaining the skin barrier function. The lateral and lamellar packing order of ICL in SC is not homogenous, but rather depth-dependent. OBJECTIVE: This study aimed to analyze the influence of the topically applied mineral-derived (paraffin and petrolatum) and plant-derived (almond oil and jojoba oil) oils on the depth-dependent ICL profile ordering of the SC in vivo. METHOD: Confocal Raman microscopy (CRM), a unique tool to analyze the depth profile of the ICL structure non-invasively, is employed to investigate the interaction between oils and human SC in vivo. RESULTS: The results show that the response of SC to oils' permeation varies in the depths. All oils remain in the upper layers of the SC (0-20% of SC thickness) and show predominated differences of ICL ordering from intact skin. In these depths, skin treated with plant-derived oils shows more disordered lateral and lamellar packing order of ICL than intact skin (p<0.05). In the intermediate layers of SC (30-50% of SC thickness), the oils do not influence the lateral packing order of SC ICL (p>0.1), except plant-derived oils at the depth 30% of SC thickness. In the deeper layers of the SC (60-100% of SC thickness), no difference between ICL lateral packing order of the oil-treated and intact skin can be observed, except that at the depths of 70-90% of the SC thickness, where slight changes with more disorder states are measured for plant-derived oil treated skin (p<0.1), which could be explained by the penetration of free fatty acid fractions in the deep-located SC areas. CONCLUSION: Both oil types remain in the superficial layers of the SC (0-20% of the SC thickness). Skin treated with mineral- and plant-derived oils shows significantly higher disordered lateral and lamellar packing order of ICL in these layers of the SC compared to intact skin. Plant-derived oils significantly changed the ICL ordering in the depths of 30% and 70-90% of the SC thickness, which is likely due to the penetration of free fatty acids in the deeper layers of the SC.