The sun provides fundamental energy for the survival of humans and other living beings. However, excessive exposure to solar radiation causes various damages, such as sunburn, premature aging, and even skin cancer. Regular use of sunscreens is one of the main protective measures (Krutmann et al., 2021). Organic UV filters absorb photons from solar radiation and, as a result, the electron is promoted from the ground state to the singlet excited state of higher energy. This molecule in the singlet excited state may return to its ground state or may reach a triplet excited state of lower energy, which can trigger photochemical reactions that lead to the formation of photoproducts (Shaath, 2010; Bonda et al., 2016).

Among the most used UV filters, avobenzone stands out for its ability to absorb in the UVB (280–320 nm) region and mainly in the UVA II (320–340 nm), being one of the few UV filters approved by the Food and Drug Administration (FDA) for UVA-I protection (340–400 nm) (Shaath, 2010; Balogh et al., 2011; Chaudhuri et al., 2016). Nevertheless, it is important to acknowledge that avobenzone undergoes significant degradation in the presence of radiation. Therefore, the photostabilization of UV filters becomes imperative, achieved through their combination with other UV filters like octocrylene and octyl methoxycinnamate, alongside the incorporation of antioxidants such as ubiquinone and trans-resveratrol (Gaspar and Maia Campos, 2006; Silva Scarpin et al., 2021; Afonso et al., 2014; Freitas et al., 2015).

Considering its great importance as an UV filter, avobenzone has become an excellent candidate for structural modification in an attempt to obtain more stable and compatible derivatives with the other components of the formulation (Santana Reis et al., 2014). In light of this, some studies have focused on the discovery of new UV filters and raw materials capable of increasing avobenzone’s photostability and neutralizing the ROS formed during its photofragmentation (Chaudhuri et al., 2006; Jesus et al., 2022).

Reactive oxygen species (ROS) directly affect cellular components as mutagens, as well as indirectly as messengers and regulators in various aspects of cell biology. The term “ROS” encompasses a wide variety of oxidant molecules with several properties and biological functions, including cell signaling and cell damage (Checa and Josep, 2020; Sies et al., 2022). Exposure to UV radiation increases ROS generation, which in turn depletes the levels of the endogenous antioxidant system (Farris and Valacchi, 2022) and enhances cell damage. These damages involve increased permeability of the lysosomal membrane, reduction in the pH gradient, and other changes that gradually become irreversible. As a result, there is a decreased uptake and binding of the neutral red vital dye, allowing for the distinction between viable and damaged or dead cells (OECD TG 432).

Additionally, it is known that UV filters do not effectively protect against ROS. Therefore, the inclusion of antioxidants in photoprotective formulations can offer new ways to prevent oxidative damage caused by ROS (Afaq, 2011; Matsui, 2016; Silva Scarpin et al., 2021). Several studies demonstrate that polyphenols are highly promising antioxidant agents for the prevention of skin damage, particularly skin cancer (Afaq, 2011; Souto et al., 2019; Sajadimajd et al., 2020). Resveratrol, a natural polyphenol, is capable of absorbing radiation across the entire UVB spectrum and part of the UVA spectrum. In addition to its action on oxidative stress, it also exhibits immunomodulatory properties and reduces the levels of inflammation (Nichols and Katiyar, 2010; Afaq, 2011; Hecker et al., 2021; Cui et al., 2022).

Resveratrol acts as an antioxidant compound primarily by neutralizing ROS through the transfer of hydrogen atoms or electrons, followed by the transfer of protons (Constantinescu and Mihis, 2023). Additionally, it can inhibit the oxidation process through enzymatic inhibition (Fauconneau et al., 1997; Acquaviva et al., 2002). Studies demonstrate that resveratrol regulates the levels of cyclooxygenase, lipoxygenase and xanthine oxidase, inhibiting their activities and preventing several diseases (Baur and Sinclair, 2006; Hecker et al., 2021). Furthermore, some analogues of resveratrol have also undergone analysis to prove its photoprotective activity due to their ability to absorb high-energy UV rays and convert them into less energetic molecules (Hudson, et al., 2013). Therefore, the development of new derivatives of UV filters that also possess antioxidant potential is an interesting strategy in the search for new photoprotective compounds. This approach minimizes the cutaneous effects resulting from exposure to UV radiation and simultaneously reduces the damage caused by the formation of ROS (Santana Reis et al., 2014).

With that in mind, new molecular modifications such as hybridization of avobenzone and resveratrol are proposed in order to obtain more effective, stable and safe compounds. One example is the derivative studied in this study {[(E)-4-hidroxi-N’-(4-hydroxybenzylidene) benzohydrazide) (LogP: 1.32 ± 0.52)]}, presented in Figure 1. The structure of this derivative comprises a carbonyl connected to a phenol in the para position (A1) and a phenol group in the R1 hydroxyl linked to the aromatic group in the para position. The N-acylhydrazone subunit serves as the connecting element between both subunits (A1 and R1), facilitating conjugation extension. Each subunit was selected for the synthesis process due to the presence of phenol groups that contribute to the antioxidant activity of this derivative (Santana Reis et al., 2014).

Cell culture systems are crucial tools in various biomedical and clinical studies worldwide, particularly in the development of drugs. The use of 3D reconstructed skin tissues in cell culture systems approximates in vivo functionality, providing a cost-effective and efficient alternative to animal testing for preclinical trials. This approach not only reduces the failure rate in drug discovery but also standardizes testing conditions, minimizing experimental variability and project costs. Advances in science and technology are continuously improving these models, increasing their complexity to better reflect the intricate interactions between cells, tissues and organs (de Vries et al., 2015; Amelian et al., 2017; De Vecchi et al., 2018).

Therefore, this study is highly relevant to the cosmetic field because it addresses the evaluation of the photoprotective and antioxidant potential of a derivative of avobenzone, hybridized with a resveratrol molecule, using in chemico and in vitro models. These models utilize skin cell lines, i.e., fibroblasts and keratinocytes, as well as an in-house standardized reconstructed human skin (RHS) model.

Photoprotective formulations must effectively protect the skin against the harmful effects of UV radiation without posing any health risks to consumers. Therefore, achieving broad-spectrum absorption in both the UVA and UVB regions, as well as ensuring the photostability of UV filters, is crucial. Photodegradation reactions can generate ROS, compromising the safety and efficacy of the formulation. Thus, the addition of antioxidants, such as resveratrol, in sunscreens to enhance the action of UV filters is an interesting strategy for reducing the damage caused by UV radiation exposure (Freitas et al.,. 2015). Various molecular modification strategies have been explored to obtain derivatives with broad-spectrum absorption in the UV region and higher antioxidant potential (Santana Reis et al., 2014), leading to safer and more effective compounds. In this study, an avobenzone derivative was evaluated for its photoprotective and antioxidant potential using cells in monolayer and RHS models. The synergistic effects of the derivative in combination with a photounstable combination of UV filters, avobenzone and octyl methoxycinnamate, was also examined.

The UV absorption profile of the avobenzone derivative was investigated as an initial step in characterizing its photoprotective properties. The derivative exhibited an absorption primarily in the UVB (280–320 nm) and UVA II (320–340 nm) regions as well as a portion of the UVA I (340–360 nm) region. This absorption profile is similar to that of resveratrol and octyl methoxycinnamate, while avobenzone itself absorbs in the UVA region (320–400 nm) (Rangel et al., 2020; Silva Scarpin et al., 2021). The absorption spectra for avobenzone derivative can be explained by its chemical structure, since it is formed by aromatic rings, conjugated double bonds, and electron donor and acceptor groups resulting from the hybridization process with avobenzone and resveratrol molecules, as described by Santana Reis et al. (2014). Notably, the addition of the avobenzone derivative to the photounstable combination of UV filters resulted in a significant increase in absorption in the UVB region.

The combination of avobenzone and octyl methoxycinnamate exhibited broad-spectrum UV absorption and was found to be photounstable, both in solution (Ca) and formulation (Fa), as described by Rangel et al. (2020) and Silva Scarpin et al. (2021). The addition of the avobenzone derivative and ethylhexyl methoxycrylene to the formulations (Fb and Fc, respectively) improved the photostability of the UV filters.

It is important to emphasize the correlation between the photodegradation/photoinstability of a compound and its potential harmful effects on biological systems. The use of photounstable molecules can compromise the safety and efficacy of the formulations. These toxicity effects can manifest through the formation of reactive intermediates and degradation products, or even through the degradation of active molecules and modifications to their chemical structure (Gaspar and Maia Campos, 2006). Therefore, it is recommended to perform photoreactivity and phototoxicity tests (ICH, 1996; Damiani et al., 2007; Silva Scarpin et al., 2021; Teixeira, et al., 2021).

Photosensitizing compounds can become toxic when exposed to UV radiation. The first step in this process is the absorption of photons that excite the chromophore. This excitation promotes an electron from the ground state to the higher energy singlet excited state. This molecule in the singlet excited state can either return to its ground state or reach a lower energy triplet excited state, which can trigger photochemical reactions leading to the formation of photoproducts (Shaath, 2010) and the generation of ROS. These ROS reactions can involve the superoxide anion (SA) through type I reaction, or singlet oxygen (SO) through type II reaction, which are the main intermediate species in the phototoxic response. Therefore, the photoreactivity test was based on the generation of ROS, SO and SA, by avobenzone derivative after UVA irradiation (OECD, 2019b).

The avobenzone derivative, formed by the groups A1 (avobenzone), carbonyl connected to a phenol in the para position, and a phenol group in the R1 (resveratrol) hydroxyl linked to the aromatic group in the para position, was more photostable to UVA radiation than avobenzone itself. It was also determined to be non-photoreactive, precisely due to absence of keto-enol group present in avobenzone, which is responsible to the formation of photoproducts (in this case SO), as discussed by Schwack and Rudolph, (1995), Lhiaubet-Vallet et al. (2010), Shaath (2010) and Freitas et al. (2015).

Due to the photoinstability of avobenzone and octyl methoxycinnamate, it is likely that their photodegradation products or increased ROS levels are responsible for their phototoxicity in the prediction model, damaging the fibroblasts and decreasing cell viability after UVA irradiation (OECD TG 432) (Benevenuto et al., 2015; Kawakami and Gaspar, 2015; Silva Scarpin et al., 2021). The avobenzone derivative, on the other hand, was considered non-phototoxic, which can be correlated to its photostability. When the avobenzone derivative was added to the combination of the photounstable UV filters, it was able to photo stabilize them and consequently decrease their phototoxicity potential. The phototoxicity assay is considered a stand-alone test for negative results due to its high sensitivity, being, generally, the only test required in cases of acute toxicity, when the compound under study is not predicted as phototoxic (Liebsch et al., 2005). It is also the first step of the biological assessment for photosafety prediction, considering that photoreactive compounds, when exposed to UV light, may generate photo irritation, photoallergy and photogenotoxicity reactions (OECD, 2019a).

In the evaluation of the antioxidant activity of the avobenzone derivative, it is important to consider that oxidative stress is a complex process. As a result, complementary methodologies are needed to assess the antioxidant potential of the compounds of interest, taking into account different species of ROS (Alam et al., 2013). For the initial screening of antioxidant activity, in chemico methods such as DPPH free radical scavenging are commonly used. This assay is simple, sensitive and low-cost (Chen et al., 2013; Lópes-Alarcón and Denicola, 2013). In this specific methodology, the avobenzone derivative exhibited promising antioxidant potential, with statistically similar IC₅₀ values to the antioxidant control, quercetin, a molecule well-known for its antioxidant activity (Song, 2020).

To complement the evaluation of avobenzone derivative’s antioxidant potential, in addition to analytical assays, in vitro cell based methodologies were used. This is because a single chemical method does not take into account important biological parameters, such as lipophilicity and bioavailability. Therefore, the ability of compounds to exhibit antioxidant activity in cell culture should also be assessed, as the involvement of different cellular components is essential during oxidative stress (Lópes-Alarcón and Denicola, 2013). The assays involved UVA exposure, since UVA radiation is relevant for ERO production (Zastrow et al., 2009; Vandersee et al., 2015).

In this study, the UVA protective effect of avobenzone derivative was assessed by detecting intracellular ROS immediately after UVA radiation using the probe DCFH₂-DA, firstly in keratinocytes HaCaT and after in a RHS model. This probe is commonly used to evaluate intracellular ROS, such as hydroxyl radical, hydrogen peroxide, nitrite and carbonate anion. It undergoes hydrolysis by cellular esterases and, in the presence of these ROS, DCFH is converted into its fluorescent derivative DCF, which can be measured using various fluorescence-based techniques (Kalyanaraman et al., 2012). The results showed that the avobenzone derivative, tested at 125, 250, and 500 μg/mL, was able to protect HaCaT cells against ROS generation. However, at the highest tested concentration (1,000 μg/mL) it exhibited a potential pro-oxidant effect. Similar pro-oxidant effects have been reported in the literature for other antioxidant compounds, such as tocopherols, polyphenols and nitric oxide, which may exhibit pro-oxidant properties at concentrations above a certain threshold (Veskoukis, et al., 2019; Bacchetti, et al., 2020).

As mentioned earlier, the avobenzone derivative consists of carbonyl connected to a phenol in the para position (A1) and a phenol group in the R1 (resveratrol) hydroxyl linked to the aromatic group in the para position, and in between them, an N-acylhydrazone subunit, facilitating conjugation extension. According to observations made by Spiegel et al., (2020), the antioxidant potential of a compound can be enhanced, depending on the position of phenoxyl groups (hydroxyl groups attached to the aromatic ring). These groups play a critical role in the delocalization process, which stabilizes the molecule in the presence of ROS. The authors observed that when two or more phenoxyl groups are in ortho or para position (as seen in the avobenzone derivative), the molecule has greater stabilization and higher antioxidant activity. There are also reports in the literature suggesting the amine and amide groups (which are present in the avobenzone derivative) are associated with the action of glutathione peroxidase, an enzyme that protects bio membranes and cellular components from oxidative stress. As a result, these groups contribute to the antioxidant potential of this molecule (Bhabak and Mugesh, 2009; Mamgain et al., 2022).

Since the RHS model mimics the biological functions of the skin, with a differentiated epidermis, with stratification and a living dermis (Roguet et al., 1994; Afaq et al., 2009; Lee et al., 2017), it serves as a valuable tool in assessing the antioxidant potential of the avobenzone derivative through the detection of UVA-induced intracellular ROS production. The results showed that the avobenzone derivative was able to protect the RHS model, resulting in a 30.7% reduction of ROS production, confirming its antioxidant potential in monolayer cells. When combined with the photounstable combination of UV filters, avobenzone and octyl methoxycinnamate, the avobenzone derivative reduced ROS production by 39.5%, further confirming its photo stabilizing effect and ability to minimize ROS generation in tissues.

Various methods, including the use of natural and synthetic matrices, have been employed to develop artificial skin models to replace animal testing. These skin models serve as valuable tools for understanding the functional mechanisms of the skin and for conducting transdermal drug and chemical testing (Kumamoto, et al., 2018; Yun, et al., 2018; Hausmann, et al., 2019; Jordão et al., 2024). The RHS model used in this study is particularly important for assessing UV damage, being positioned between monolayer cell cultures, animal models and clinical studies. In this case, we have investigated the effects of a single dosage of UVA radiation accompanied by a single 1 h of treatment with antioxidant compounds. However, it is important to note that repeated exposures and longer treatment durations would provide a more realistic representation of in vivo conditions and real-life situations (Lelièvre et al., 2024).

Overall, the avobenzone derivative was evidenced to possess a potential as a promising ingredient for sunscreens, as it exhibits UV booster properties. It effectively photostabilizes the combination of photounstable UV filters, avobenzone and octyl methoxycinnamate, both in solution and in formulation, reducing their phototoxic potential. Additionally, it primarily also absorbs UVB (280–320 nm) and UVA II (320–340 nm) radiation, as well as a portion of the UVA I (340–360 nm) radiation. Furthermore, it exhibits significant antioxidant potential, both in chemico and in vitro, protecting against DPPH free radical formation and UVA-induced intracellular ROS formation in HaCaT keratinocytes and a RHS model at concentrations ranging from 125 to 500 μg/mL. While the methodologies employed in this study have certain limitations, the use of skin cells and the RHS model adds relevance to the evaluation of photoprotective potential of novel UV filter derivatives. These findings offer significant insights for the development of new topical materials aiming to protect the skin against UV damage. Although acknowledging the current limitations, further validation and testing could enhance the applicability of these methodologies in real-life situations.