Evaluation of the photoprotective and antioxidant potential of an avobenzone derivative

Abstract

Solar radiation can cause damage to the skin, and the use of sunscreens is one of the main protective measures. However, photounstable ultraviolet (UV) filters can generate photoproducts and reactive oxygen species (ROS). Adding antioxidants, such as resveratrol, to enhance the action of UV filters in sunscreens is an interesting strategy for reducing the damage caused by UV radiation exposure. However, new compounds must have their stability, safety and efficacy guaranteed. Avobenzone, a commonly used UV filter, stands out as a promising candidate for structural modification to enhance its stability. Its molecular hybridization with other UV filters and antioxidants can lead to safer and more effective compounds. In this study, the photoprotective and antioxidant potential of a derivative of avobenzone, hybridized with resveratrol's molecule, was evaluated using in vitro models of cells in monolayer and reconstructed human skin (RHS). Phototoxic potential was assessed using fibroblasts, while the antioxidant activity was measured using the DCFH₂-DA probe in HaCaT keratinocytes and in-house RHS. The derivative exhibited UV absorption and demonstrated photostability. It did not exhibit any phototoxic nor photoreactivity potential. Additionally, it was able to photo stabilize a combination of photounstable UV filters, avobenzone and octyl methoxycinnamate, and to reduce their phototoxic potential. In terms of antioxidant activity, the derivative successfully protected against UVA-induced ROS production in the HaCaT keratinocytes model, showing statistical equivalence to the antioxidant control, quercetin (10 μg/mL). Furthermore, experiments conducted in the RHS model demonstrated a significant reduction of 30.7% in ROS generation compared to the irradiated control. This study demonstrated that structural modifications of avobenzone can lead to the development of a broad spectrum (absorbing UVB and UVA II radiation, as well as a portion of the UVA I radiation), non-phototoxic, non-photoreactive and photostable derivative for sunscreen and anti-aging formulations. This derivative enhances protection against oxidative stress induced by UV radiation and improves the effectiveness of sun protection. In addition to the monolayer model, the use of a standardized in-house RHS model was highly relevant for evaluating the effects of UV radiation and skin aging. This model closely mimics human physiological conditions and enables the testing of new compounds and the investigation of protective mechanisms against skin damage.

Keywords: antioxidant; avobenzone; photoprotective; reconstructed human skin; skin cells.

FIGURE 1

Molecular hybridization among resveratrol and avobenzone subunits to design a new photoprotective compound (AVO’s derivative).

FIGURE 2

Absorption spectra in the UV region (280–400 nm) of (A) The isolated UV filters avobenzone (AVO) and octyl methoxycinnamate (OMC), the antioxidant compound t-resveratrol (RESV) and avobenzone derivative (AVO’ derivative) (100 μg/mL); (B) The combinations Ca (avobenzone and octyl methoxycinnamate); Cb (avobenzone, octyl methoxycinnamate and avobenzone derivative); Cc (avobenzone, octyl methoxycinnamate and ethylhexyl methoxycrylene). Results are expressed as mean absorbance (n = 3).

FIGURE 3

Absorption spectra of the samples in solution before (nir) and after (ir) UVA irradiation. Ca: avobenzone and octyl methoxycinnamate; Cb: avobenzone, octyl methoxycinnamate and avobenzone derivative; Cc: avobenzone, octyl methoxycinnamate and ethylhexyl methoxycrylene. Results are expressed as mean absorbance (n = 3).

FIGURE 4

Dose-response curves of avobenzone derivative and the combinations Ca (avobenzone and octyl methoxycinnamate) and Cb (avobenzone, octyl methoxycinnamate and avobenzone derivative), and norfloxacin, used as positive control, obtained by the 3T3 NRU phototoxicity test and plotted using the Phototox Version 2.0 software. The blue and yellow dots refer to non-irradiated (−UV) and irradiated (+UV) substances, respectively.

FIGURE 5

Percentage of cell viability in HaCaT cells after treatment with different concentrations (μg/mL) of the avobenzone derivative. Sodium lauryl sulfate (SDS) was used as positive control. Avobenzone derivative was tested in the range of 31.25–1,000 μg/mL (constant factor: 2). Test substances below the 70% threshold (dotted line) are considered cytotoxic. The results are expressed as mean ± standard errors of the mean of three independent experiments (n = 3). *: Significantly different from the untreated non-irradiated control (NT -UV) (p < 0.05).

FIGURE 6

Protection against UVA-induced intracellular ROS production in HaCaT keratinocytes. The results are expressed as a percentage of fluorescence. NT -UV: untreated non-irradiated control; NT + UV: untreated irradiated control; Nor: norfloxacin (100 μg/mL); Quer: quercetin (10 μg/mL); Avobenzone derivative tested in the range of 31.25–1,000 μg/mL (constant factor: 2); Nir: non-irradiated cells; Ir: irradiated cells. Results are expressed as mean ± standard errors of the mean of three independent experiments (n = 3). °: Significantly different from the irradiated untreated control (NT + UV) control (p < 0.05); *: Statistically equal to quercetin (p > 0.05).

FIGURE 7

Histologic slide obtained by Hematoxylin-Eosin staining showing all layers of the in-house RHS. Scale bar: 200 μm.

FIGURE 8

UVA-induced intracellular ROS production in the RHS model. The results are expressed as a percentage of fluorescence in comparison to the NT + UV. Untreated non-irradiated control (NT -UV); Untreated irradiated control (NT + UV); Vehicle, PBS with ethanol 2%; Avobenzone derivative (200 μg/mL); Combination Cb (avobenzone, octyl methoxycinnamate and avobenzone derivative). Nir: non-irradiated tissue; Ir: irradiated tissue. Results are expressed as mean ± standard errors of the mean of three independent experiments (n = 3). *: Significantly different from the untreated irradiated control (NT + UV) (p < 0.05).

FIGURE 9

Fluorescence images obtained for confocal microscopy for each treatment: (A) Untreated non-irradiated tissue (NT -UV); (B) Untreated irradiated tissue (NT + UV); (C) Vehicle PBS with ethanol 2%; (D) Avobenzone derivative (200 μg/mL); (E) Combination Cb (avobenzone, octyl methoxycinnamate and avobenzone derivative) (4:8:5 w/w). Green fluorescence corresponds to the presence of ROS in the RHS.