BACKGROUND:The interaction between antioxidants and free radicals has become increasingly important in cosmetic research. This review is not a conventional literature overview but is primarily based on our own experimental data and methodological developments. SUMMARY:The first part describes noninvasive spectroscopic techniques, such as resonance Raman spectroscopy are highlighted as robust tools for the in vivo determination of carotenoid concentrations, serving as indirect markers of the skin's antioxidant status. The next section addresses the detection of free radicals. Electron paramagnetic resonance (EPR) spectroscopy is presented as the only direct and quantitative method for detecting free radicals in biological systems. Its application in vitro, ex vivo, and in vivo enables controlled assessment of radical formation and antioxidant efficacy, including under defined irradiation conditions. The introduction of the radical protection factor (RPF) provides a standardized parameter to compare the scavenging capacity of individual compounds and complex formulations, thereby supporting product development and claim substantiation. In the last section, in vivo and ex vivo measurements are compared. It is shown that the amounts of free radicals generated in the UV and infrared spectral ranges of solar radiation differ significantly between different skin types. For sun protection, this means that the protective effect of a sunscreen must extend across the entire spectral range of solar radiation, since 50% of the free radicals generated by the sun originate in the visible and infrared spectral ranges. KEY MESSAGES:These findings led to the development of a sunscreen important for Asian skin (skin type IV).

Cutaneous exposure to food allergens prior to oral exposure has been associated with sensitization and the development of food allergies (Lack 2008). Similarly, the prevalence of food allergies has been linked to the frequency of massage in babies (Perkin et al. 2021). However, the mechanism by which environmental food allergens come into contact with the immune cells responsible for sensitization in the skin or how skin massage may facilitate this process is currently unknown. This study aimed to investigate whether massage-induced skin stretching facilitated the percutaneous permeation of food allergens into the skin. Hydrophilic and hydrophobic proteins were extracted from raw and roasted peanuts and fluorescently labelled with Sulfo Cyn5 NHS ester. Franz diffusion cells mounted with full-thickness mice skin (Balb/C, 8–12 weeks old) were employed to assess the skin penetration of the peanut proteins with [stretch (+)] and without [stretch (−)] skin stretching. Peanut roasting led to significant changes in the peanut protein composition compared with raw peanuts with hydrophilic protein Ara h −1 aggregate formation and reduced hydrophobic olesion and caleosin protein levels. When applied to the tissue without stretching, a small amount of the peanut protein passed into and through the tissue regardless of peanut type or processing. However, stretching increased the peanut penetration of all the extracts applied to the skin. The raw hydrophilic and roasted hydrophobic peanut extracts showed the most extensive tissue penetration at 9.5 ± 8.4 µg cm−2 and 7.2 ± 5.9 µg cm−2, respectively. Increasing skin tension by 4–5 fold, akin to the skin stretching experienced during baby massage, enhances percutaneous permeation of peanut proteins into the skin. Therefore, skin stretching may contribute to the process of cutaneous allergen sensitization. Further work is needed to understand the clinical relevance of hydrophilic, hydrophobic and aggregated peanut proteins entering the skin during stretching.

Topical antioxidant-containing creams have been investigated as adjunctive treatments for atopic dermatitis, but clinical efficacy remains inconsistent. In this study, a plant extract mixture containing epigallocatechin gallate, kaempferol, quercetin, and phlorizin was incorporated into two different base formulations and compared with their respective vehicles using ex vivo, in vitro and in vivo approaches. Skin penetration was assessed by confocal Raman microscopy, formulation structure by multiphoton microscopy and clinical efficacy in patients with moderate atopic dermatitis using clinical scores, non-invasive biophysical measurements, and laser scanning microscopy. Despite significant differences in cutaneous penetration between formulations, no clinically relevant differences were observed between antioxidant-containing creams and their vehicles. The water-in-oil emulsion showed a non-significant trend toward greater improvement in pruritus and SCORAD, likely related to occlusive properties, but was not superior to the lighter formulation. Laser scanning microscopy did not demonstrate antioxidant-specific effects. Instead, formulation-dependent effects were observed, with the lighter cream tending to support deeper epidermal recovery, while more occlusive formulations mainly improved stratum corneum hydration. Overall, antioxidant enrichment did not confer additional clinical benefit beyond vehicle effects under the conditions studied. The findings highlight the dominant role of the base formulation and suggest that future studies should optimize formulation design and carefully consider disease severity to detect effects beyond vehicle-related improvements.

Amazonian fruit residues like piquia shells are often discarded despite their antioxidant potential for sustainable cosmetic use. This study evaluated the photostability, phototoxicity, and photoprotection of hydroalcoholic piquia shell extract (PqSE) combined with UV filters in solutions and cosmetic formulations. PqSE formulations were photostable, even stabilizing photounstable UV filters. Phototoxicity tests (OECD TG 432) showed no phototoxic potential (MPE < 0.15) and reduction in the phototoxic potential of UV filters, while ocular irritation potential via HET-CAM assay indicated no irritant effects. The extract combined with UV filters enhanced protection against UVA-induced reactive oxygen species (ROS) production, achieving 60.9% effectiveness, outperforming commercial photostabilizers. Against UVB radiation, it showed cellular viability above 80%, comparable to benzophenone-3. PqSE formulations exhibited a radical protection factor (RPF) nine times higher than controls and reduced radical production by 64% after visible/near-infrared (VIS/NIR) irradiation on porcine skin, compared to 38% for controls. Confocal Raman microspectroscopy showed penetration depths below 12 µm for all time points. This study highlights the potential of reusing fruit residues like PqSE as sustainable, effective ingredients in sunscreen formulations, offering enhanced photoprotection and reduced environmental waste.

Ultraviolet A (UVA) irradiation significantly impacts skin health by generating free radicals, including reactive oxygen species (ROS), lipid oxygen species (LOS), and carbon-centered radicals (CCR), contributing to oxidative stress. Electron paramagnetic resonance (EPR) spectroscopy enables the direct detection of these radicals, using spin traps like 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO). While DMPO is suitable for detecting short-lived species such as hydroxy (•OH) and alkoxy radicals, BMPO offers greater stability, particularly for superoxide (O2-) and hydroperoxyl radicals (•OOH). This study refines EPR-based protocols for radical detection in UVA-irradiated skin by comparing DMPO and BMPO, revealing a shift from short-lived ROS to more stable LOS with increasing UVA-exposure. For the first time, the glutathione (GSH)-mediated conversion of O2- to •OH was directly quantified via spin trapping in skin tissue. Although GSH functions as a central antioxidant in skin, it indirectly promotes •OH formation via the Fenton reaction under UV-induced oxidative stress, potentially contributing to tissue damage. BMPO's enhanced stability as a spin trap for O2- in skin tissue enables precise detection of this GSH-dependent radical transformation, offering new insights into protective and damaging mechanisms under oxidative conditions. A standardized protocol for ex situ UVA irradiation of skin and subsequent radical measurement was developed, establishing a foundation for future studies with other stress factors. This research refines spin trapping methodologies and advances the understanding of UVA-induced oxidative processes, offering a framework for future dermatological and photobiological investigations.

BACKGROUND:In Europe, 1-2% of children are diagnosed with a peanut allergy. Atopic dermatitis (AD) is a significant risk factor for food allergy development, with cutaneous allergen exposure playing a causative role in allergic sensitization, particularly in early childhood. OBJECTIVES:To investigate the primary and secondary skin-to-skin transfer of peanut proteins and the impact of hand washing in reducing allergen transfer. METHODS:Primary transfer and secondary skin-to-skin transfer of fluorescently labelled peanut proteins was determined with and without hand washing, using two commonly used soaps containing ionic or nonionic surfactants, in 11 healthy volunteers. Tape stripping and fluorescence quantification of the peanut proteins on the tapes were used to analyse skin transfer. The depth of peanut protein deposition in the skin was determined in situ by confocal laser scanning microscopy. RESULTS:Over 50% of the peanut proteins penetrated the upper horn layer during primary transfer, with aqueous solutions being significantly more effective [mean (SD) 70.5% (7.6)] compared with glycerol [mean (SD) 53.8% (11.2); P ≤ 0.001]. Approximately 10% of the proteins underwent secondary transfer, with higher values observed for glycerol than for water (9.8% vs. 7.8%). This was related to the amount of peanut applied after primary transfer (P ≤ 0.05 for water; P ≤ 0.01 for glycerol). Hand washing removed up to 90% of the applied peanut proteins. For proteins dissolved in water, nonionic soap was more effective (87.6%) than anionic soap (63.7%) in removing the applied peanut proteins from the palm (P ≤ 0.01). When proteins were dissolved in glycerol, both soaps were similarly effective in clearing protein contamination from the palm (approximately 97-99%; P ≤ 0.01). CONCLUSIONS:We demonstrate the rapid contamination of hands with allergenic peanut protein and the potential for skin-to-skin transfer. Hand hygiene prevented allergen transmission by up to 90%. Soaps based on nonionic surfactants were more robust to external factors than classical anionic soaps, and this may be particularly important for infants with AD.

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.

Melanin, the most abundant skin chromophore, is produced by melanocytes and is one of the key components responsible for mediating the skin’s response to ultraviolet radiation (UVR). Because of its antioxidant, radical scavenging, and broadband UV absorbing properties, melanin reduces the penetration of UVR into the nuclei of keratinocytes. Despite its long-established photoprotective role, there is evidence that melanin may also induce oxidative DNA damage in keratinocytes after UV exposure and therefore be involved in the development of melanoma. The present work aimed at evaluating the dependence of UV-induced DNA damage on melanin content and distribution, using reconstructed human epidermis (RHE) models. Tanned and light RHE were irradiated with a 233 nm UV-C LED source at 60 mJ/cm2 and a UV lamp at 3 mJ/cm2. Higher UV-mediated free radicals and DNA damage were detected in tanned RHE with significantly higher melanin content than in light RHE. The melanin distribution in the individual models can explain the lack of photoprotection. Fluorescence lifetime-based analysis and Fontana–Masson staining revealed a non-homogeneous distribution and absence of perinuclear melanin in the tanned RHE compared to the in vivo situation in humans. Extracellularly dispersed epidermal melanin interferes with photoprotection of the keratinocytes.

Cold atmospheric plasma (CAP) enables painless tissue treatment by producing reactive species including excited molecules and charged particles and is of great interest for medical applications. Medical CAP sources work in contact with air at ambient pressure, resulting in the generation of substantial amounts of reactive oxygen and nitrogen radicals. These radicals have a significant influence on cellular biochemistry, are crucial components of the immune system, and play a central role in wound therapy. CAP has a variety of applications, with a particular emphasis on tissue treatment in dermatology. It eradicates microorganisms by preventing biofilm formation so that wounds can be effectively disinfected and treated antiseptically.Using both in vitro and ex vivo methods, a comprehensive preclinical assessment of a novel battery-operated cold plasma handheld device with a reusable, and autoclavable glass cylinder was performed. The objectives were to evaluate the potential impact of single CAP application on radical formation with and without wound dressing, by directly measuring radicals in skin, to investigate the influence of CAP application on antimicrobial activity and cytotoxicity in vitro, and to assess skin tolerance ex vivo.The direct effect of CAP on the formation of radicals in the skin after plasma application at different levels with and without wound dressing was demonstrated quantitatively for the first time using electron paramagnetic resonance spectroscopy. Free radicals were measured in the skin as a function of the duration of CAP treatment. Furthermore, it was found that an alginate or wound plaster dressing does not significantly inhibit radical formation in skin compared to application without a dressing. In vitro and ex vivo data showed no cytotoxic potential with simultaneous efficacy against bacteria strains and no risk of temperature rise, pH change, skin barrier or DNA damage. These results show a high potential for wound healing applications in vivo.

BACKGROUND:Human life is based on oxygen respiration and an enzymatic, free radical-dependent water chemistry, whose billions of parallel reactions take place at pH ∼7.4 and a temperature of 37°C, in accordance with the laws of chemistry. The cellular metabolic processes occur over time periods covered by the half-lives of reactive oxygen species (ROS) for °OH to over 10 s for lipid oxygen species (LOS), indicating that mixtures of free radicals form the basic components for these processes. SUMMARY:The main source of radicals is the mitochondrial conversion of 1-5% oxygen into "primary" ROS and "secondary" LOS. Every endogenous and exogenous radical generation, triggered by "natural background radiation," "natural environment," or "solar radiation" leads to qualitatively similar mixtures of "primary" ROS and "secondary" LOS or RNS (reactive nitrogen species). A Multilevel Antioxidant Regulation, Repair and Protection System (MARRPS) keeps these radical mixtures in a steady state. Depending on the total number of free radicals, different areas of radical action are defined. The Free Radical Ground State (FRGS) with "homeostasis" and "adaptive homeostasis," the Free Radical Threshold Value (FRTV), and Free Radical Pathological Conditions (FRPC). The quantitative ratio ROS > LOS comprehensively characterizes the "'homeostasis" and "adaptive homeostasis" area of the FRGS. The total number of free radicals cannot be measured directly in the "homeostasis" area. "Adaptive homeostasis" is achieved when excess radicals are stable produced beyond "homeostasis" of the FRGS. The quantity that remains controllable in this range is a maximum of ∼3.58 × 1012 radicals/mg, the value of the body constant FRTV. The sensitized MARRPS provides "semi-stable homeostatic" states characterized by dual stability with ROS > LOS and a stable total ROS/LOS and RNS count beyond the basal FRGS "homeostasis." If the total number of all radicals exceeds the FRTV, where LOS > ROS, this initiates uncontrolled radical chain reactions. The partial failure of the MARRPS in the FRPC area leads to pathological processes which are the starting point for a hundred different diseases. KEY MESSAGES:The universal body constant FRTV is the basis for all regular life processes. The design principle described by this simple model applies universally to all aerobic life.

Excessive exposure to ultraviolet (UV) light leads to acute and chronic UV damage and is the main risk factor for the development of skin cancer. In most countries with western lifestyle, the topical application of sunscreens on UV-exposed skin areas is by far the most frequently used preventive measure against sunburn. Further than preventing sunburns, increasing numbers of consumers are appreciating sunscreens with a medium- to high-level sun protective factor (SPF) as basis for sustainable-skin ageing or skin cancer prevention programs. However, recent investigations indicate that clinically significant DNA damages as well as a lasting impairment of cutaneous immunosurveillance already occur far below the standard of one minimal erythema dose (MED) sunburn level, which contributes to the current discussion of the clinical value of high-protective SPF values. Ex vivo investigations on human skin showed that the application of SPF30 reduces DNA damage for a day long sun exposure (24 MED) drastically by about 53% but is significantly surpassed by SPF100 reducing DNA damage by approx. 73%. Further analysis on different SPF protection levels in UV-exposed cell culture assays focusing on IL-18, cell vitality and cis/trans-urocanic acid support these findings. Whereas SPF30 and SPF50+ sunscreens already offer a solid UVB cover for most indications, our results indicate that SPF100 provides significant additional protection against mutagenic (non-apoptotic-) DNA damage and functional impairment of the cutaneous immunosurveillance and therefore qualifies as an optimized sunscreen for specifically vulnerable patient groups such as immunosuppressed patients, or skin cancer patients.

The inactivation of multi resistant pathogens is an important clinical need. One approach is UV-C irradiation, which was previously not possible in vivo due to cytotoxicity. Recently, far UV-C irradiation at λ < 240 nm was successfully used on skin with negligible damage. A potential application site is the nasal vestibule, where MRSA accumulates and cannot be treated using antiseptics. We irradiated 3D mucosa models and excised human mucosa with 222 and 233 nm far UV-C in comparison to 254 nm and broadband UV-B. Eradication efficiency was evaluated by counting colony forming units; irritation potential was evaluated by hen’s egg-chorioallantoic membrane assay and trans epithelial electrical resistance; cell viability was assessed by MTT. DNA damage and cell protective mechanisms were evaluated immunohistopathologically. On mucosa models, MRSA reduced by ≈ 5 log10 for 60 mJ/cm2 irradiation at 233 nm. A slightly increased cell viability was observed after 24 h. Lower doses showed lower irritation potential than the positive controls or commercial mouthwash, while 80 mJ/cm2 had strong irritation potential. DNA damage occurred only superficially and decreased after 24 h. On excised human mucosa, < 10% of keratinocytes were affected after 150 mJ/cm2 222 nm or 60 mJ/cm2 233 nm.

The application of a far-ultraviolet C (UVC) light emitting diode (LED) of 233 nm showed significant bactericidal efficacy at an applied dose between 20 and 80 mJ cm-2 as reported recently. In addition, only minor epidermal DNA lesions were observed in ex vivo human skin and in vitro epidermal models <10% of the minimal erythema dose of UVB radiation. To broaden the potential range of applications of such systems, e.g. to include postoperative application on wounds for the purpose of decontamination, we assessed how a disruption of normal anatomic skin structure and function influences the skin damage induced by light from 233 nm far-UVC LEDs. Thus, we induced superficial skin wounds by mechanical detachment of the stratum corneum in ex vivo human skin. Barrier-disruption of the skin could be successfully determined by measuring an increase in the transepidermal water loss (TEWL) and the stratum corneum loss could be determined morphologically by 2-photon microscopy (2-PM). After far-UVC irradiation of the skin, we screened the tissue for the development of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). The abundance of DNA lesions was elevated in wound skin in comparison to intact skin after irradiation with far-UVC. However, no increase in DNA lesions was detected when artificial wound exudate consisting of cell culture medium and serum was applied to the disrupted skin surface prior to irradiation. This effect agrees with the results of ray tracing simulations of the absorption of far-UVC light incident on a superficial skin wound. Interestingly, no significant deviations in radical formation between intact skin and superficially wounded skin were detected after far-UVC irradiation as analyzed by electron paramagnetic resonance (EPR) spectroscopy. In conclusion, 233 nm LED light at a dose of 60 mJ/cm2 could be applied safely on superficial wounds for the purpose of skin antisepsis as long as the wounds are covered with wound fluid.

Far‐UVC radiation sources of wavelengths 222 nm and 233 nm represent an interesting potential alternative for the antiseptic treatment of the skin due to their high skin compatibility. Nevertheless, no studies on far‐UVC‐induced DNA damage in different skin types have been published to date, which this study aims for. After irradiating the skin with far‐UVC of the wavelengths 222 and 233 nm as well as broadband UVB, the tissue was screened for cyclobutane pyrimidine dimer‐positive (CPD+) cells using immunohistochemistry. The epidermal DNA damage was lower in dark skin types than in fair skin types after irradiation at 233 nm. Contrary to this, irradiation at 222 nm caused no skin type‐dependent differences, which can be attributed to the decreased penetration depth of radiation. UVB showed the relatively strongest differences between light and dark skin types when using a suberythemal dose of 3 mJ/cm2. As melanin is known for its photoprotective effect, we evaluated the ratio of melanin content in the stratum basale and stratum granulosum in samples of different skin types using two‐photon excited fluorescence lifetime imaging (TPE‐FLIM) finding a higher ratio up to skin type IV–V. As far‐UVC is known to penetrate only into the upper layers of the viable skin, the aforementioned melanin ratio could explain the less pronounced differences between skin types after irradiation with far‐UVC compared to UVB.

Antioxidants can reduce free radical formation in deeper skin layers where typical sunscreen filters may no longer be effective. Here, a general method is presented to pre-select optimum combinations of antioxidants and physical filters. The radical production of selected common physical filters after UV irradiation, the capacity of different antioxidants and the interaction between these compounds was investigated in solution by optical measurement of DPPH scavenging, allowing a theoretical calculation of the antioxidant amount necessary to scavenge UV-induced radicals. Furthermore, the antioxidant capacity and the scattering properties were determined. All physical filters induced different amounts of radicals in suspensions depending on the coating. ZnO coated with polydimethylsiloxane and myristic acid (ZnOpolymyr) showed the lowest radical formation. Epigallocatechin-gallate (EGCG) provided the highest antioxidant capacity. Different formulations with different ratios of selected physical filters and antioxidants were prepared. It turned out that the high radical protection factor (RPF) of cream formulations, which originally did not contain any physical filters, was reduced when such filters were added. The data demonstrates that the addition of physical filters to antioxidant-containing formulations lowers their reduction capacity, but to varying degrees. An optimal combination of physical filters and antioxidants must be pre-selected in order to incorporate them into a formulation and verify their effect on skin.

Air pollution is increasing worldwide and skin is exposed to high levels of pollution daily, causing oxidative stress and other negative consequences. The methods used to determine oxidative stress in the skin are invasive and non-invasive label-free in vivo methods, which are severely limited. Here, a non-invasive and label-free method to determine the effect of cigarette smoke (CS) exposure on skin ex vivo (porcine) and in vivo (human) was established. The method is based on the measurement of significant CS-exposure-induced enhancement in red- and near-infrared (NIR)-excited autofluorescence (AF) intensities in the skin. To understand the origin of red- and NIR-excited skin AF, the skin was exposed to several doses of CS in a smoking chamber. UVA irradiation was used as a positive control of oxidative stress in the skin. The skin was measured with confocal Raman microspectroscopy before CS exposure, immediately after CS exposure, and after skin cleaning. CS exposure significantly increased the intensity of red- and NIR-excited skin AF in a dose-dependent manner in the epidermis, as confirmed by laser scanning microscopy AF imaging and fluorescence spectroscopy measurements. UVA irradiation enhanced the intensity of AF, but to a lower extent than CS exposure. We concluded that the increase in red- and NIR-excited AF intensities of the skin after CS exposure could clearly be related to the induction of oxidative stress in skin, where skin surface lipids are mainly oxidized.