# Syringic acid protective role: Combatting oxidative stress induced by UVB radiation in L-929 fibroblasts

Karine Campos Nunes, Danielle Lazarin-Bidoia, Tânia Ueda-Nakamura, Sueli de Oliveira Silva Lautenschlager, Raphaël Michel, Rachel Auzély-Velty, Celso Vataru Nakamura

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Karine Campos Nunes, Danielle Lazarin-Bidoia, Tânia Ueda-Nakamura, Sueli de Oliveira Silva Lautenschlager, Raphaël Michel, et al.. Syringic acid protective role: Combatting oxidative stress induced by UVB radiation in L-929 fibroblasts. Journal of Photochemistry and Photobiology B: Biology, 2025, 264, pp.113104. 10.1016/j.jphotobiol.2025.113104. hal-05343617

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Syringic Acid Protective Role: Combating Oxidative Stress Induced by UVB Radiation in L-929 Fibroblasts

Karine Campos Nunes¹, Danielle Lazarin-Bidoia¹, Tânia Ueda-Nakamura¹, Sueli de Oliveira Silva Lautenschlager¹, Raphaël Michel², Rachel Auzély-Velty², Celso Vataru Nakamura¹*

Afilliation

¹Post-Graduate Program in Pharmaceutical Sciences, State University of Maringá, Maringá, Paraná, Brazil.

²Université Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France

*Corresponding author: cvnakamura@uem.br
ABSTRACT

Neglecting proper skin care and repeated exposure to ultraviolet (UV) radiation can have serious consequences, including skin burns, photoaging and even the development of skin cancer. UV radiation-induced damage is mediated by highly unstable and reactive molecules, named reactive oxygen species (ROS). To counteract ROS, the skin has an endogenous antioxidant system. Considering that, many sunscreens incorporate antioxidant substances to ensure additional photochemioprotective action in the formulation. Syringic acid (SA) is classified as a phenolic acid derived from hydroxybenzoic acid. It has antioxidant properties, which can reduce oxidative stress, and has shown potential to prevent skin cancer. The aim of this study was to assess the ability of SA to protect L-929 fibroblasts from UVB radiation by evaluating oxidative stress biomarkers. As a result, we demonstrated the antioxidant activity of SA through four methodologies, and confirmed the photochemioprotective activity of SA by attenuating the cytotoxicity of UVB radiation in L-929 fibroblasts. The mechanisms involved in the photoprotection of SA include a significant reduction in total ROS, maintenance of mitochondrial membrane potential, decrease in lipid peroxidation, preservation of endogenous antioxidant system enzymes and reduced glutathione (GSH) levels, thereby mitigating the ultrastructural damage caused by UVB. Additionally, SA showed promising results in wound healing. Considering such properties, SA emerges as a strong candidate for incorporation into photoprotective and multifunctional formulations.

**KEY WORDS:** Photoprotection; UV Radiation; Oxidative Stress; Syringic Acid
<table>
  <tr>
    <td>87</td>
    <td>Abbreviations:</td>
  </tr>
  <tr>
    <td>88</td>
    <td>ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)</td>
  </tr>
  <tr>
    <td>89</td>
    <td>BHT: Butylated Hydroxytoluene</td>
  </tr>
  <tr>
    <td>90</td>
    <td>CAT: Catalase</td>
  </tr>
  <tr>
    <td>91</td>
    <td>CCCP: Carbonyl 3-chlorophenylhydrazone of cyanide</td>
  </tr>
  <tr>
    <td>92</td>
    <td>CO2: Carbon dioxide</td>
  </tr>
  <tr>
    <td>93</td>
    <td>DMEM: Dulbecco's modified medium</td>
  </tr>
  <tr>
    <td>94</td>
    <td>DMSO: Dimethyl sulfoxide</td>
  </tr>
  <tr>
    <td>95</td>
    <td>DNA: Deoxyribonucleic acid</td>
  </tr>
  <tr>
    <td>96</td>
    <td>DPI: Diphenyleneiodonium</td>
  </tr>
  <tr>
    <td>97</td>
    <td>DPPH*: 2,2-diphenyl-1-picrylhydrazyl</td>
  </tr>
  <tr>
    <td>98</td>
    <td>DPPP: Diphenyl-1-pyrenylphosphine</td>
  </tr>
  <tr>
    <td>99</td>
    <td>EDTA: Ethylene diamine tetra acetic acid</td>
  </tr>
  <tr>
    <td>100</td>
    <td>FBS: Fetal Bovine Serum</td>
  </tr>
  <tr>
    <td>101</td>
    <td>FeCl3: Ferric chloride</td>
  </tr>
  <tr>
    <td>102</td>
    <td>FRAP: The Ferric Reducing Antioxidant Power</td>
  </tr>
  <tr>
    <td>103</td>
    <td>GSH: Reduced Glutathione</td>
  </tr>
  <tr>
    <td>104</td>
    <td>H2DCF-DA: 2,7-dichlorodihydrofluorescein diacetate</td>
  </tr>
  <tr>
    <td>105</td>
    <td>H2O2: Hydrogen peroxide</td>
  </tr>
  <tr>
    <td>106</td>
    <td>HBSS: Hank's Balanced Salt Solution</td>
  </tr>
  <tr>
    <td>107</td>
    <td>HCl: Hydrochloric acid</td>
  </tr>
  <tr>
    <td>108</td>
    <td>MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide</td>
  </tr>
  <tr>
    <td>109</td>
    <td>Nac: N-acetylcysteine</td>
  </tr>
  <tr>
    <td>110</td>
    <td>NC: Negative control</td>
  </tr>
  <tr>
    <td>111</td>
    <td>O2⁻: Superoxide anion</td>
  </tr>
  <tr>
    <td>112</td>
    <td>°C: Degrees Celsius</td>
  </tr>
  <tr>
    <td>113</td>
    <td>OPT: O-phthalaldehyde</td>
  </tr>
  <tr>
    <td>114</td>
    <td>PBS: Phosphate Buffered Saline</td>
  </tr>
  <tr>
    <td>115</td>
    <td>QT: Quercetin</td>
  </tr>
  <tr>
    <td>116</td>
    <td>ROS: Reactive Oxygen Species</td>
  </tr>
  <tr>
    <td>117</td>
    <td>RPM: Rotations per minute</td>
  </tr>
  <tr>
    <td>118</td>
    <td>SA: Syringic Acid</td>
  </tr>
  <tr>
    <td>119</td>
    <td>SOD: Superoxide Dismutase</td>
  </tr>
  <tr>
    <td>120</td>
    <td>TE/g: Equivalent of mmol of Trolox per gram of sample</td>
  </tr>
  <tr>
    <td>121</td>
    <td>TEM: Transmission Electron Microscopy</td>
  </tr>
  <tr>
    <td>122</td>
    <td>TMRE: Tetramethylrhodamine ethyl ester</td>
  </tr>
  <tr>
    <td>123</td>
    <td>TPTZ: 2,4,6-tri(2-pyridyl)-1,3,5-triazine 1:3 with p-Toluenesulfonic acid</td>
  </tr>
  <tr>
    <td>124</td>
    <td>TROLOX: 2-carboxylic acid-6-hydroxy-2,5,7,8-tetramethylchroman</td>
  </tr>
  <tr>
    <td>125</td>
    <td>UV: Ultraviolet Radiation</td>
  </tr>
  <tr>
    <td>126</td>
    <td>XOD: Xanthine Oxidase</td>
  </tr>
  <tr>
    <td>127</td>
    <td>Δψm: Mitochondrial Membrane Potential</td>
  </tr>
  <tr>
    <td>128</td>
    <td></td>
  </tr>
  <tr>
    <td>129</td>
    <td></td>
  </tr>
</table>
# 1) Introduction

The skin is an organ that acts as a barrier, defending our organism from external pathogens. Because it is continuously exposed to the environment, some extrinsic factors can make it vulnerable [1-4]. One of these factors is the exposure to sunlight through ultraviolet (UV) radiation [2,5,6], that can be classified into subgroups according to its wavelength: UVA (320-400 nm), UVB (280-320 nm) and UVC (100-280 nm) [7,8]. Because it has a longer wavelength, UVA penetrates deeper into the dermis, while UVB mainly affects the epidermis [9]. The intense UV exposure can cause oxidative stress, which may lead to damage in DNA, proteins and lipids, resulting in negative consequences such as photoaging and carcinogenesis [10-13].

Oxidative stress can be defined as an exacerbated production of Reactive Oxygen Species (ROS), in which it cannot be contained by the action of antioxidants, generating a disturbance of the cellular redox balance [9,14]. Examples of ROS include singlet oxygen, peroxyl radicals, superoxide radicals, hydroxyl radicals, and some non-radical oxidizing agents that can be converted into oxygen radicals, such as hydrogen peroxide, ozone and hypochlorous acid [9,15,16]. Oxidative stress can lead to oxidation of structurally important proteins and lipids, increasing plasma membrane permeability, and altering transmembrane ion gradients, which leads to loss of secretory functions and inhibition of cellular metabolic processes. The oxidative damage can be reduced through antioxidants that can neutralize ROS [12,15].

To counteract the deleterious effects of oxidizing molecules, the human body is equipped with a variety of antioxidant components. The antioxidants present in the body can be classified as enzymatic and non-enzymatic [17,18]. The main enzymatic antioxidants are the superoxide dismutase (SOD) enzymes, catalase and glutathione peroxidase. In addition to these main enzymes, there is also the enzyme heme oxygenase-1 and some redox proteins, such as thioredoxins, peroxiredoxins and glutaredoxins, which act as an antioxidant defense [17,19,20].

Despite the wide arrays of naturally occurring antioxidants, ROS can still cause significant damage to the skin in case of exposure to UV [14,17]. Thus, the use of components that have antioxidant functions in photoprotective formulations can help reduce oxidative stress, prevent skin aging and provide additional protection to the skin [21]. Some findings contribute to the advancement of clinical skin studies, leading to
162 effective cosmetic products, including sunscreens, after-sun care, and anti-aging
163 formulations [22-25].

164 Syringic acid (SA) is classified as a phenolic acid derived from hydroxybenzoic
165 acid and it presents antioxidant activity [26]. Its chemical structure is shown in Figure 1:

<figure><img src="image_2.png" /><figcaption>**Fig. 1:** Chemical structure of syringic acid [27].</figcaption></figure>

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188
189 **1. Material and Methods**

190 **1.1 Chemicals**

191 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-
192 picrylhydrazyl (DPPH), 2,4,6-tri(2-pyridyl)-1,3,5-triazine 1:3 with p-toluenesulfonic acid
193 (TPTZ), 2,7-dichlorodihydrofluorescein diacetate (H₂DCF-DA), 2-carboxylic acid-6-
194 hydroxy-2,5,7,8-tetramethylchroman (Trolox), carbonyl 3-chlorophenylhydrazone of
195 cyanide (CCCP), catalase, diphenyleneiodonium (DPI), glutaraldehyde, Hank's balanced
196 salt solution (HBSS), n-acetylcysteine (NAC), O-Phthalaldehyde (OPT), reduced
197 glutathione (GSH), syringic acid (SA), tetramethylrhodamine ethyl ester (TMRE),
198 xanthine oxidase and xanthine were obtained from Sigma-Aldrich (St. Louis, USA).
199 Potassium persulfate and sodium acetate were purchased from Labsynth (Diadema,
200 Brazil). Ethylenediaminetetraacetic acid (EDTA) was purchased from F. Maia (Belo
201 Horizonte, Brazil), while ferric chloride was from Dinâmica (São Paulo, Brazil).
202 Hydrochloric acid and penicillin were acquired from Biotec (São José dos Pinhais,
203 Brazil). Fetal bovine serum (FBS) from Nova Biotecnologia (Cotia, Brazil), hydrogen
204 peroxide (H₂O₂) from Biorad (California, USA) and luminol from Invitrogen (Oregon,
205 USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was
206 purchased from Chemgold (Sydney, Australia), while Dulbecco's modified eagle medium
207 (DMEM) and streptomycin were obtained from Gibco (Grand Island, USA). Cacodylate
208 was bought from Koch (São Paulo, Brazil), diphenyl-1-pyrenylphosphine (DPPP) came
209 from Molecular Probes (Oregon, USA), and epoxy resin from EMS (Domat/EMS,
210 Switzerland).

211
212 2.2 Antioxidant Activity

213 2.2.1 DPPH* assay

214 SA is synthetically derived, with purity greater than 95% as indicated by the
215 supplier. In order to confirm the antioxidant activity of SA, the DPPH method was
216 employed. This method provides antioxidant capacity by reducing DPPH* to hydrazine,
217 and generates a light-yellow solution [33]. To achieve that, 100 µL of DPPH* (65 µM)
218 was added to 100 µL of SA (630-10 µM) in a 96-well plate. Then, the resulting mixture
219 was incubated in a place without light for 30 min. The reading was taken in a
220 spectrophotometer at 517 nm (Bio Tek - Power Wave XS). The absorbance was quantified
221 and the EC50 (concentration of sample required to be effective in neutralizing DPPH* by
50%) was determined through linear regression. For all experiments of antioxidant activity, quercetin was used as a positive control. Quercetin (QT) is a substance with promising bioactive effects, including its well-known antioxidant activity [34–36].

### 2.2.2 ABTS<sup>+</sup> assay

The ABTS<sup>+</sup> radical naturally exhibits a bluish-green hue, but when Trolox or an antioxidant sample is introduced, the radical undergoes reduction, causing the solution to lose color and absorbance at 734 nm to decrease [42]. For this method, the ABTS<sup>+</sup> was obtained by mixing 5 mL of ABTS solution (7.0 mmol/L) and 88 µL of potassium persulfate (2.45 mmol/L) for 12 h, in the absence of light. In a 96-well transparent microplate, 7 µL of previously diluted SA (630-10 µM) and 200 µL of ABTS<sup>+</sup> solution was added. Then, the mixture was homogenized, and after 6 min of reaction, the reading was performed at 734 nm in a microplate reader (Bio Tek – Power Wave XS). Trolox was used as a standard antioxidant and the results expressed in mmol TE/g (equivalent of mmol of Trolox per gram of sample).

### 2.2.3 FRAP assay

FRAP values are determined by quantifying the rise in absorbance at 595 nm and establishing a correlation with a standard solution of ferrous ions or an antioxidant standard solution. The alteration in absorbance is directly proportional to the cumulative FRAP value of antioxidants present in the sample [41]. Firstly, the FRAP reagent was prepared with 300 mM acetate buffer (pH 3.6), TPTZ reagent (10 mmol/L) in HCl solution (40 mmol/L) and FeCl₃ solution (20 mmol/L), in a 10:1:1 ratio. In a 96-well transparent microplate, 30 µL of SA (630-10 µM) or Trolox standard (3200–160 µM) and 150 µL of the prepared FRAP reagent were added. This solution was incubated for 30 min at 37°C, and the reading was performed at 595 nm in a microplate reader (Bio Tek – Power Wave XS). Trolox was used as a standard antioxidant and the results were expressed in mmol TE/g.

### 2.2.4 Xanthine/luminol/xanthine oxidase assay

To perform the xanthine/luminol/xanthine oxidase (XOD) assay, a reagent solution was prepared with 400 µL of glycine buffer (0.1 mol/L and 1 mol/L EDTA, pH 9.4), 10 µL of luminol (0.6 mmol/L) and 150 µL of xanthine (6 mmol/L). SA was diluted in hydroethanolic solution (50:50, v/v) and added to the reagent solution (10 µL). To start
the reaction, 100 µL of XOD (20 mU/mL) was added to the mixture and, after 30 seconds,
the reading was performed in 96-well plates in a luminescence reader (SpectraMax L
Microplate Reader). The sample concentration required to inhibit 50% of O₂⁻ production
(IC₅₀) was calculated by linear regression.

2.3 *In vitro* Photochemioprotective Potential

2.3.1 Cell Culture and Ultraviolet Irradiation

L-929 cell line (ATCC® CCL-1™, Manassas, USA) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), streptomycin (100 µg/mL) and penicillin (100 U/mL) at 37 °C in 5% CO₂ atmosphere. To select the best dose of UVB radiation to be used, a dose curve was created, varying doses from 200 to 1200 mJ/cm².

The dose that decreased cell viability by approximately 50% was chosen. A UVB lamp was used in all experiments involving irradiation (TL40W/12RS; Philips®; peak intensity at a wavelength of 312 nm) and a UV spectrum radiometer (VLX-3W, Vilber Lourmat®) to monitor UV radiation levels.

2.3.2 Cell Viability and Photochemioprotective Effect assays

From cell culture was possible to evaluate the cell viability of L-929 fibroblasts in the presence or absence of UVB radiation. To verify the cytotoxicity of SA alone on the cells, they were plated in 96-well plates (2.5 × 10⁵ cells/mL) and treated with SA at concentrations of 1-200 µM for 24 h. To verify the cytoprotective effect, cells were plated in 24-well plates (2.5 × 10⁵ cells/mL), pretreated (100, 50, 25 and 10 µM of SA) and irradiated (500 mJ/cm²) or treated after the irradiation for 1 h. Both assays were performed using MTT, a yellow compound that turns dark blue when its substrate is broken down by mitochondrial enzymes [37]. MTT (2 mg/mL) was added and incubated for 4 h at 37°C under an atmosphere of 5% CO₂. Subsequently, the blue formazan crystals were dissolved with DMSO and the absorbance was measured at 570 nm (BIO-TEK Power Wave XS).

2.4 Reactive Species Oxygen (ROS) and Microscopy

To quantify ROS and to visualize them in microscopy, the probe H₂DCF-DA was
used. Upon detecting ROS, the non-fluorescent H₂DCF-DA converts to a fluorescent DCF
compound [22]. Firstly, the cells were plated (2.5 × 10⁵ cells/mL) in 96-well plates for
quantitative measurement and in 24-well plates for microscopy. As a positive control, we
used the H₂O₂ (10 mM, 1 h) in both experiments. For pretreatment, SA was used at concentrations of 100, 50, 25 and 10 µM for 1h. Then, the probe H₂DCF-DA (5 µM) was added for 30 min, in the dark at 37°C. Then, that probe was removed, HBSS was added and the cells were irradiated with UVB radiation (500 mJ/cm²).

To compare the SA activity, we used two known active substances in 10 µM: Quercetin (QT) and Diphenyleneiodonium (DPI). Total ROS quantification was determined using a VICTOR equipment (Excitation: 490 nm/Emission 535 nm) right away and 1, 2 and 3 h later. The cell normalization was performed by protein dosage (Bradford method) [38]. For microscopy, a fluorescence microscope (Olympus BX51) with a coupled camera (Olympus UC30) was used, equipped with a green filter.

## 2.5 Mitochondrial Membrane Potential (ΔΨm) and Lipid Peroxidation

Mitochondrial Membrane Potential (Δψm) and intracellular Lipid Peroxidation were determined in L-929 cells. Both methodologies followed the same initial protocol: Firstly, the cells were plated in black 96-well plates (2.5 × 10⁵ cells/mL) for 24 h. Then, they were treated with SA in HBSS (100, 50, 25 and 10 µM), or known active substances for 1 h: QT (10 µM) for both methods, DPI (10 µM) for Δψm, N-acetylcysteine (Nac, 2 mM) for Lipid Peroxidation, and irradiated with UVB in pure buffer (500 mJ/cm²).

### 2.5.1 (ΔΨm) measurement

The mitochondrial membrane potential (Δψm) was evaluated using the TMRE probe (Tetramethyl Rhodamine ethyl ester), which has negative charges and accumulates in active mitochondria [39]. After pretreatment and irradiation, the plate was incubated for 24 h. As a positive control, the CCCP (100 µM) treatment for 1 h was used, that is, a compound that alters the proton gradient of membranes, destabilizing ΔΨm [22]. Then, the TMRE probe (0.025 µM) was added, and the cells were incubated at 37 °C for 30 min. The reading was performed in VICTOR equipment (Excitation: 540 nm/ Emission: 595 nm). Finally, the cells were normalized with protein dosage (Bradford Assay) [38].

### 2.5.2 Lipid Peroxidation measurement

Lipid peroxidation was determined using the DPPP probe at 20 mM. DPPP undergoes a reaction with lipid hydroperoxides, resulting in the formation of the highly fluorescent
314

DPPP oxide [40]. As a positive control, a non-radical ROS capable of generating lipid peroxidation was used: H₂O₂ (1 mM). The DPPP probe was added, incubated for 30 min, and the reading was performed on a VICTOR equipment (Excitation: 351 nm / Emission: 460 nm). To normalize the assay, protein dosage was performed (Bradford method) [38].

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## 2.6 Endogenous Enzymatic System

319

To determine the levels of reduced glutathione (GSH) and catalase, L-929 cells were first plated (4 x 10⁵ cells/mL) in a 6-well plate and incubated for 24 h. Cells were pre-treated with SA (25 and 10 µM) for 1 h and irradiated with UVB radiation (500 mJ/cm²). A cell lysate was prepared by moving the L-929 cells with the aid of a scraper into a lysis buffer [10 mM tris-HCl (pH 7.4), 1% triton X-100], followed by sonication for 60 s (4C15, Branson Ultrasonics®) and centrifugation at 14000 RPM, in 4 °C for 10 min. Subsequently, the supernatant was collected, and protein quantification was performed (Bradford method) [38].

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### 2.6.1 Reduced Glutathione

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For the GSH experiment, 10 µL of o-phthalaldehyde (OPT, 1 mg/mL in methanol), 10 µL of cell lysate were added along 180 µL of sodium phosphate buffer [100 mM, 5 mM EDTA (pH 8)]. Then, the solution was incubated for 15 min at room temperature. Fluorescence was measured in the Victor ® X3 equipment (Excitation: 350 nm/Emission: 420 nm). Results were expressed as U GSH/mg protein and were based on the standard GSH calibration curve.

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336

### 2.6.2 Catalase activity

337

To calculate catalase activity, cell lysate was mixed with H₂O₂ (30 mM) and potassium phosphate (50 mM, pH 7.0). Absorbance was read at 240 nm for 5 min (Shimadzu, UV-1700). To determine catalase activity, the drop in absorbance and the molar extinction coefficient of H₂O₂ (39.4 M⁻¹cm⁻¹) were used. Results were expressed as U Catalase/mg protein and were based on a catalase standard curve.

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342

## 2.7 Wound Healing assay

343

To perform this assay, L-929 cells were plated (2.5 × 10⁵ cells/mL) in 24-well plates and kept in the oven at 37°C for 24 h. The next day, the cell monolayer was wounded in the

344
center of the well with a sterile 20 µL pipette tip, washed with PBS and treated with SA in
supplemented DMEM (0.5% FBS) at concentrations of 25 and 10 µM. To evaluate the
wound-healing activity of SA under UVB irradiation, cells were pretreated with SA (25 µM
and 10 µM) for 1 hour, irradiated in HBSS buffer, and the treatment was re-applied for more
23h. Wound healing was observed at 0 and 24h using an inverted phase contrast microscope
(Olympus CKX41; 4× magnification). To quantify the open area of the wound, the ImageJ
software was used, and the percentage of open wound area was calculated in comparison
with the initial wound (at t = 0 h).

## 2.8 Microscopy analysis

In order to better assess the morphology of cells subjected to radiation, as well as the
photochemioprotection of SA, cells were observed under an inverted microscope and by
transmission electron microscope (TEM). For both analyses the initial protocol was: the cells
were plated (2.5 × 10⁵ cells/mL) in 24-well plates for phase contrast inverted microscope
analysis, and in 6-well plates for TEM and incubation for 24 h. Afterwards, the cells were
treated with the chosen concentrations (25 and 10 µM) of SA for 1h and irradiated in Hank's
Balanced Buffer (500 mJ/cm²). Subsequently, the buffer was replaced with DMEM.

### 2.8.1 Phase Contrast Inverted Microscope

After the initial protocol described in 2.8 section, cells were photographed on a phase
contrast inverted microscope (40×, CKX41, Olympus).

### 2.8.2 Transmission Electron Microscopy

After completing the initial procedure, a fixing solution (2.5% glutaraldehyde in 0.1 M cacodylate buffer) was applied to the cells, and the plate was incubated for 24 h. After this step, the cells were gently scraped and then centrifuged at 1000 RPM for 5 min. Subsequent post-fixation involved the use of potassium ferrocyanide and osmium tetroxide. Then, cells underwent dehydration through progressively increasing concentrations of acetone (30, 50, 70, 90, 95, and 100%). The cells were then embedded in epoxy resin (Polybed® 812) and polymerized in an oven at 60 °C for 72 h. Thin sections (measuring 60 nm in thickness) were stained using a 5% (w/v) uranyl acetate and 2% (w/v) lead citrate solution before being analyzed using a transmission electron microscope (JEOL, JEM 1400).

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## 2.9 Statistical analysis

To carry out the statistical analyzes in the various experiments, the Prism 5 software (GraphPad, San Diego, CA, United States) was used. Data were expressed as mean ± standard deviation of 3 independent experiments. The results were analyzed by Unpaired t-test; one- or two-way analysis of variance by ANOVA method with Tukey's test, and a p value < 0.05 (statistically significant) was considered.

# 3. Results

## 3.1 Antioxidant Activity

In order to validate the free radical scavenging capacity of SA, as previously described in the literature [41], various methodologies to assess antioxidant activity were employed. The ability of SA to neutralize some radicals was evaluated, comparing it with a standard and well-known compound, quercetin (QT) [35]. The results showed low values of EC₅₀ in DPPH⁺ assay, and IC₅₀ in xanthine/luminol/XOD assay for both substances, demonstrating their high antioxidant activity (Table 1).

<table><caption>**Table 1:** Antioxidant activity of Syringic Acid (SA) and Quercetin (QT) using 4 antioxidant methods.</caption><thead><tr><th rowspan="2">Subst.</th><th colspan="4">ANTIOXIDANT ACTIVITY</th></tr><tr><th>DPPH<sup>•</sup><br>EC₅₀ (µM) ± SD</th><th>XOD<br>IC₅₀ (µM) ± SD</th><th>ABTS<sup>•+</sup><br>(mmol TE/g) ± SD</th><th>FRAP<br>(mmol TE/g) ± SD</th></tr></thead><tbody><tr><td>SA</td><td>14.87<sup>a</sup> ± 2.86</td><td>1.46<sup>c</sup> ± 0.00</td><td>8.34<sup>e</sup> ± 0.66</td><td>6.16<sup>g</sup> ± 0.23</td></tr><tr><td>QT</td><td>9.46<sup>b</sup> ± 0.52</td><td>0.67<sup>d</sup> ± 0.01</td><td>12.15<sup>f</sup> ± 1.45</td><td>12.88<sup>h</sup> ± 1.36</td></tr></tbody></table>

Results are expressed as means ± SD (n=3). Different letters in the column indicate significant differences between means according to Unpaired t-test (p < 0.05).

The results of ABTS and FRAP experiments are expressed in millimoles of Trolox equivalent per gram of sample. Trolox is a water-soluble analog of vitamin E and serves as a widely used standard for assessing the antioxidant capacity of compounds in various laboratory assays [42]. Those results are related to the Trolox antioxidant potential, that means, the higher this value, the more antioxidant the analyzed sample is [43]. The results showed that SA achieved approximately 68.6% of the antioxidant activity of quercetin by the ABTS method and 47.8% by FRAP method. The antioxidant activity results demonstrated the high antioxidative capacity of SA through four methods, comparing it with QT.
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## 3.2 Cell viability assays

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After confirming the high antioxidant activity of SA, as previously reported in the literature [26,41,44], we employed the MTT method to assess the cellular viability of L-929 fibroblasts in the presence of this substance. The results indicated that SA was nontoxic to the cells at concentrations ranging from 1 to 200 µM (Fig. 2A).

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Subsequently, it was crucial to identify the optimal dose of UVB to proceed with the experiments on fibroblastic cells. Consequently, an experiment was conducted to assess cellular viability in the presence of UVB radiation, ranging from 200 to 1200 mJ/cm². The results demonstrated a dose-dependent curve, in which the lowest dose resulted in nearly 40% cell death, while the highest dose caused almost 70% of fibroblast death (Fig. 2B).

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**Fig. 2:** (A) Effect of SA treatment on L-929 cell viability. Untreated cells (NC), SA at concentrations of 1-200 µM. (B) Cell viability in dose-response curve of UVB radiation on L-929 fibroblasts; doses in mJ/cm² (200-1200). (C) Effect of SA treatment on the viability of L-929 cells irradiated with 500 mJ/cm² of UVB radiation. NC: untreated cells, PRE: pretreatment, POST: posttreatment, SA at 10-100 µM concentrations. ##p < 0.01, ###p < 0.001 compared to UVB, *p < 0.05, **p < 0.01, ***p < 0.001 compared to NC (one-way ANOVA, post-Tukey test).
The dose chosen for subsequent experiments was 500 mJ/cm², which was able to reduce the cell viability in untreated fibroblasts by 50%. Other studies described in the literature consider 50% cell viability as a suitable choice for photochemioprotection tests, in which it is possible to observe that the analyzed compounds shielded the cells from the radiation's effects [22,24].

To assess the photochemioprotective effect of SA, both pretreatment and posttreatment were tested, meaning cells were either treated and subsequently irradiated or irradiated with UVB and then treated with SA. A broad spectrum of SA concentrations ranging from 10 to 100 µM was tested.

In pretreatment, all four concentrations demonstrated the ability to protect cells and prevent cell death, in particular the higher concentrations. While untreated and irradiated cells achieved only 50% cell viability, pretreatment with 100 µM prevented cell death, equaling to the negative control (untreated and non-irradiated cells). The two highest concentrations used in pretreatment exhibited no significant differences compared to the negative control (NC). Still in pretreatment, all SA concentrations demonstrated significant differences when compared to untreated and irradiated cells (p<0.001) (Fig. 2C).

Posttreatment yielded a favorable result only at the highest concentration (100 µM), when cell viability with 100 µM treatment was statistically distinct from the absence of SA treatment and irradiated UVB (Fig. 2C). Hence, pretreatment was chosen to proceed with the other photoprotection experiments.

### 3.5 Reactive Species Oxygen (ROS) total and Microscopy

ROS is a broad term that encompasses a diverse array of oxidative molecules characterized by distinct properties and biological roles, ranging from signaling to inducing cellular damage [45]. As antioxidants play a crucial role in neutralizing ROS and thereby protecting cell [21], we assessed whether SA could reduce intracellular ROS levels. The method for measuring total ROS may lack specificity, as it detects multiple types of reactive oxygen species without distinguishing between them. Nevertheless, it provides a general overview of the oxidative status in the cells, offering valuable insight into the overall level of oxidative stress [22].
As observed in the fluorescence microscopy images (Fig. 3), SA led to a reduction in ROS, which is evident through reduced fluorescence intensity in cells across the four tested concentrations (10, 25, 50, and 100 µM).

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** ROS production in L-929 cells irradiated with UVB and pretreated with syringic acid (SA). L-929 cells were treated with SA for 1 h (10, 25, 50 and 100 µM) and irradiated with UVB 500 mJ/cm². NC: untreated, non-irradiated cells; UVB: cells untreated and irradiated with UVB radiation, H₂O₂: cells treated with 10 mM H₂O₂. H₂DCFDA fluorescence (5 µM) was measured and analyzed by fluorescence microscopy. Black images show fluorescence (green filter). Gray images show differential interference contrast (DIC).</figcaption></figure>

Quantitative assessment of total ROS generation was conducted at 0, 1, 2, and 3 h, with untreated and non-irradiated cells (NC), untreated and irradiated cells (UVB), cells treated with SA, standard antioxidants (QT and DPI), and H₂O₂ (a ROS generator). Quantitative analysis corroborated the microscopy findings, demonstrating that SA treatment led to a dose-dependent reduction in ROS levels, which significantly differs from untreated and irradiated cells. Over the 3-h period, H₂O₂ induced a progressive increase in ROS levels, while NC maintained a low baseline ROS value (Fig. 4).
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** ROS production in L-929 cells irradiated with UVB and pretreated with syringic acid (SA). L-929 cells were treated with SA (10, 25, 50 and 100 µM). QT: cells treated with quercetin (10 µM). DPI: cells treated with diphenyleneiodonium (10 µM); those three substances were treated for 1h and irradiated with UVB 500 mJ/cm². NC: untreated, non-irradiated cells. UVB: cells not treated and irradiated with UVB radiation. H₂O₂: cells treated with 10 mM H₂O₂. H₂DCFDA fluorescence (5 µM) was performed on a spectrofluorometer. The time points of 0, 1, 2, and 3 h refer to the measurement times. ##p < 0.01, ###p < 0.001 compared to UVB, ***p < 0.001 compared to NC (one-way ANOVA, post-Tukey test).</figcaption></figure>

Furthermore, ROS levels from SA treatment exhibited no statistical distinction when compared to the known antioxidants QT and DPI. It can be inferred that ROS reduction is a potential mechanism for SA's photochemioprotection, which remained consistent throughout the 3-h period (Fig. 4).

## 3.6 Mitochondrial Membrane Potential (ΔΨm) and Lipid Peroxidation measurements

With UV radiation exposure, L-929 cells might have their mitochondrial membrane potential (ΔΨm) reduced [22], as observed also with the CCCP treatment, that is, a substance capable of destabilizing the ΔΨm. Some compounds can inhibit the depolarization of the mitochondrial membrane caused by UVB [23], like QT and DPI, and those findings can be also observed at the highest concentration of SA, 100 µM (Fig. 5A). All those three substances showed approximately a 36% difference from UVB ΔΨm. The lower concentrations of SA did not show significant differences in relation to ΔΨm with UVB irradiated and non-treated cells (Fig. 5A).

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<figure><img src="image_9.png" /><figcaption>**Fig. 5.** (A) Evaluation of mitochondrial membrane potential (ΔΨm) and (B) Lipid Peroxidation in L-929 fibroblasts irradiated with UVB. L-929 cells were pretreated with SA (10, 25, 50 and 100 µM). QT: cells treated with quercetin (10 µM). DPI: cells treated with diphenyleneiodonium; the three substances were treated for 1 h and irradiated with UVB 500 mJ/cm². As a control method, CCCP (100 µM) was used for ΔΨm, and H₂O₂ (1 mM) for lipid peroxidation. NC: untreated, non-irradiated cells. UVB: not treated cells and irradiated with UVB radiation. #p < 0.05, ##p < 0.01, ###p < 0.001 compared to UVB; *p < 0.05, **p < 0.01 compared to NC (one-way ANOVA, post-Tukey test).</figcaption></figure>

One of the markers of oxidative damage caused by UV radiation is the lipid peroxidation [46]. Treatment with SA significantly reduced the lipid peroxidation induced by UVB. (Fig. 5B) SA was effective at all tested concentrations, with no statistically significant differences between them and between the two known antioxidants: QT and Nac. When compared to irradiated and untreated cells, the treatment with antioxidants, including SA, decreased lipid peroxidation by around 42%. On the other hand, as expected, H₂O₂ led to a high level of peroxidation.

## 3.7 Endogenous Enzymatic System: Intracellular Catalase and Reduced Glutathione Levels

SA had its photoprotective activity confirmed by preventing the decrease in cell viability in the presence of UVB (see Fig. 2). Additionally, it significantly reduced ROS and lipid peroxidation at the four concentrations involved (see Fig. 4 and 5). Therefore, the two lower concentrations were chosen to proceed with subsequent experiments. These concentrations (25 and 10 µM) were not cytotoxic to L-929 cells, and they were effective in antioxidant activity experiments.

Antioxidants found within the body can be categorized as enzymatic and non-enzymatic, and the UV radiation can affect these species [14,46]. By analyzing our measurements of catalase and GSH, we can observe that there was a depletion of approximately 50% in both enzyme and antioxidant in the UVB-irradiated and untreated
cells (Fig. 6). Meanwhile, in irradiated cells treated with 25 µM of SA, these levels remained at 86.5% and 97.7%, respectively (Fig. 6).

<figure><img src="image_10.png" /></figure>

**Fig. 6.** Effect of UVB radiation (500 mJ/cm²) and SA treatment on the endogenous antioxidant system of L-929 fibroblasts. (A) Catalase activity (CAT) evaluated by H₂O₂ consumption (U CAT/mg of protein). (B) Reduced glutathione (GSH) level quantified using O-phthal dialdehyde fluorescence. NC: untreated, non-irradiated cells. UVB: cells not treated and irradiated with UVB radiation. Cells treated with SA (10 and 25 µM). ***p < 0.001, significant difference in relation to NC. ###p < 0.001, significant difference in relation to UVB (one-way ANOVA, post Tukey test).

After the treatment with SA, the levels of the enzyme and the antioxidant did not show significant differences compared to the negative control group. This suggests that the SA treatment prevented the reduction of these species, matching to the non-irradiated cells. Furthermore, there were no significant differences between the two concentrations of SA utilized.

## 3.8 Wound Healing Assay

In wound healing, there is cell migration, which depends on low levels of ROS. Thus, an exacerbated increase in ROS, characteristic of oxidative stress, can interfere with effective healing [49]. Therefore, antioxidants can help maintain healing, as UV radiation can slow this process [22]. The wound healing activity of SA was evident at both tested concentrations. After 24 hours, untreated cells (NC) exhibited approximately 86% of the open wound area, while cells treated with SA (25 and 10 µM) showed a significantly reduced open wound area, around 76%, indicating greater wound closure (Fig. 7).

Under UVB irradiation, untreated cells had a larger wound area after 24 h (~96%), indicating that UVB radiation impairs the wound healing process. In contrast, cells treated with 25 µM SA under UVB irradiation displayed significantly improved healing compared to the untreated group (Fig. 7).
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<figure><img src="image_12.png" /><figcaption>**Fig. 7.** Evaluation of wound healing in L-929 fibroblasts treated with SA for 24 h, with and without UVB irradiation (500 mJ/cm²). (A) Images were examined using an inverted microscope with phase contrast (4x). (B) The acquired images were quantified through ImageJ. Untreated cells or treated with SA (10 and 25 µM). ###p < 0.001, significant difference in relation to the 0h untreated cells. **p < 0.01 and ***p < 0.001, significant difference compared to 24h untreated cells. αp < 0.05, significant difference compared to 24h untreated and UVB-irradiated cells (one-way ANOVA, post Tukey test).</figcaption></figure>

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## 3.9 Microscopy analysis

### 3.9.1

#### Phase Contrast Inverted Microscope

We also evaluated the effect of SA on the morphology of L-929 cells using an inverted phase contrast microscope. This experiment revealed that UVB radiation reduced the number of cells and affected their morphology by making them more rounded and spaced out (Fig. 8). The negative control (untreated and non-irradiated cells) and the irradiated SA-treated cells exhibited an elongated and flattened shape, characteristic of cells from this line. Furthermore, the decrease in the quantity of cells in the irradiated and untreated case aligns with the cytotoxicity results which displayed a decrease in cell viability of approximately 50% (Fig. 2B and 2C).

<figure><img src="image_13.png" /><figcaption>**Fig. 8.** Effect of syringic acid (SA) treatment on the L-929 cells irradiated with 500 mJ/cm² of UVB radiation. Cells untreated and non-irradiated show a normal morphology and elongated and flattened appearance as well as the SA treated and irradiated cells. Cells untreated and irradiated with UVB exhibit a rounded appearance and smaller quantities. Cells were observed and photographed on a phase contrast inverted microscope (40× magnification).</figcaption></figure>
### 3.9.2 Transmission Electron Microscopy (TEM)

When analyzed by TEM, the untreated and non-irradiated cells displayed a typical ultrastructure, characterized by a prominent nucleus and cellular membranes with well-preserved structures (Fig. 9A). With UV radiation and without SA treatment, there was an increase in the size of the nuclei and intense disruption of the cytoplasmic membrane, which resulted in the leakage of intracellular content (Fig. 9B). Furthermore, the irradiation caused a significant cellular disorganization. It can be observed that the treatment with SA mitigated these structural modifications, with cells exhibiting preserved membranes, more organized intracellular environment (Fig. 9C and 9D), and elongated and flattened shape (9D).

<figure><img src="image_14.png" /><figcaption>**Fig. 9.** Effect of syringic acid (SA) treatment on L-929 cells subjected to 500 mJ/cm² of UVB radiation observed through Transmission Electron Microscopy. (A) Untreated and non-irradiated cells displaying a standard ultrastructure, characterized by a prominent nucleus (black asterisk) and well-preserved cellular membranes. (B) Untreated cells irradiated with UVB, exhibiting an enlarged nucleus (white asterisk) and severe disruption of the cytoplasmic membrane (arrow), resulting in the release of intracellular contents and significant cellular disarray (arrowhead). (C) Cells treated with SA at a concentration of 10 µM (nucleus: black asterisk) (D) Cells treated with SA at a concentration of 25 µM, showing a more elongated morphology. Treated cells demonstrated an attenuation of ultrastructural modifications, with cells featuring preserved membranes and a more organized intracellular environment.</figcaption></figure>
608 **4. Discussion**

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The exposure to the UV radiation can lead to various skin damages, primarily mediated by the formation of ROS. The excessive elevation of intracellular ROS results in oxidative stress, causing the activation of specific signaling pathways, which directly influences processes and leads to cell death. The main ROS include free radicals and unstable molecules that can harm cellular structures, thus causing premature aging and increased risk of skin cancer [12,14,50]. Antioxidants can interact with free radicals, interrupting the chain reaction and converting them into harmless molecules by donating an electron [18,47]. SA exhibits antioxidant activity, by reducing oxidative stress [31–32]. It has also some effects in the treatment of depression [30], skin cancer [29], and myocardial infarction [28]. In this work, we evaluated for the first time the photochemioprotective potential of SA on L-929 fibroblasts.

Our initial experiments demonstrated the high antioxidant potential of SA, which neutralized DPPH radicals. The results were very similar to those of a derivative phenolic acid and even superior to ascorbic acid, a well-known antioxidant [46]. According to Reynertson et al. (2005), substances with EC₅₀ values lower than 50 µg/mL are considered potent antioxidants [52]. This is a case of SA as can be seen in Table 1.

The antioxidative capacity of SA was also demonstrated by the ABTS•⁺ and FRAP methods, and compared with the well-known substance quercetin. Quercetin is a flavonol antioxidant that belongs to the category of flavonoids, being even more potent than vitamins C and E [36]. Despite SA not exhibiting higher antioxidant activity than quercetin by the analyzed methodologies, it showed better results than the synthetic antioxidant Butylated Hydroxytoluene (BHT), as previously described in the literature [25]. The xanthine/luminol/XOD assay, a cell-free system assay, indicated that SA can reduce oxidative processes by scavenging superoxide anion [21,67].

After investigating the antioxidant activity of SA, we initiated tests involving fibroblasts, cells previously documented in the literature in studies related to photochemioprotection [53]. Cytotoxicity assays demonstrated that SA was non-toxic to L-929 cells and protected them from cellular death caused by UVB radiation. The higher concentrations of SA were comparable to the negative control, that is, untreated and non-irradiated cells, which demonstrates reversal of the deleterious effect of radiation on the cells. The amount of UV radiation influences the quantity of ROS produced by keratinocytes and fibroblasts in a dose-dependent manner, that is, the higher the dose of

Our initial experiments demonstrated the high antioxidant potential of SA, which neutralized DPPH radicals. The results were very similar to those of a derivative phenolic acid and even superior to ascorbic acid, a well-known antioxidant [46]. According to Reynertson et al. (2005), substances with EC₅₀ values lower than 50 µg/mL are considered potent antioxidants [52]. This is a case of SA as can be seen in Table 1.

The antioxidative capacity of SA was also demonstrated by the ABTS•⁺ and FRAP methods, and compared with the well-known substance quercetin. Quercetin is a flavonol antioxidant that belongs to the category of flavonoids, being even more potent than vitamins C and E [36]. Despite SA not exhibiting higher antioxidant activity than quercetin by the analyzed methodologies, it showed better results than the synthetic antioxidant Butylated Hydroxytoluene (BHT), as previously described in the literature [25]. The xanthine/luminol/XOD assay, a cell-free system assay, indicated that SA can reduce oxidative processes by scavenging superoxide anion [21,67].

After investigating the antioxidant activity of SA, we initiated tests involving fibroblasts, cells previously documented in the literature in studies related to photochemioprotection [53]. Cytotoxicity assays demonstrated that SA was non-toxic to L-929 cells and protected them from cellular death caused by UVB radiation. The higher concentrations of SA were comparable to the negative control, that is, untreated and non-irradiated cells, which demonstrates reversal of the deleterious effect of radiation on the cells. The amount of UV radiation influences the quantity of ROS produced by keratinocytes and fibroblasts in a dose-dependent manner, that is, the higher the dose of
radiation applied, the more ROS is formed, which results in more damage to the cell [24,54]. Our findings have demonstrated that ROS levels in irradiated cells progressively increase over time (up to 3 h), meaning that even after irradiation ceases, intracellular ROS levels continue to rise, as previously reported in the literature [48].

The excessive generation of ROS results in an imbalance between the production of ROS and the capacity of a biological system to neutralize these reactive compounds or repair the consequent oxidative harm [55,56]. Thereat, one of the primary protective mechanisms against UV radiation is the reduction of total ROS levels [48,57,58]. SA significantly decreased total ROS levels in irradiated cells in a dose-dependent manner, and consistently maintained low ROS levels in cells, indicating its continued protective effect. Additionally, SA exhibited very similar results to the antioxidant substances QT and DPI, as well as to Nac [46] and 2-acetylphenothiazine [22]. Studies report that the effectiveness of phenolic acids in free radical scavenging depends on the presence of multiple hydroxyl groups linked to the aromatic ring of benzoic or cinnamic acid molecules [41]. The rise in ROS triggered by UV radiation initiates a redox imbalance in skin cells, a range of oxidative damage, which can lead to a lipid peroxidation [24,59], alterations in the Δψm [46,58] and a mitochondrial dysfunction [63].

While various oxidant compounds, such as H₂O₂, are widely recognized as mediators and triggers of oxidative stress, lipid hydroperoxides are crucial mediators of processes linked to cell death and diseases. Since lipids are the primary component of cell membranes, they play an essential role in preserving the structural integrity of cells [59,60]. Excessive lipid oxidation alters the physical characteristics of cell membranes and can lead to the covalent modification of nucleic acids and proteins [13,61,62].

Regarding lipid peroxidation, it is known that lipid peroxides cause damage through two main mechanisms. First of all, excessive lipid oxidation affects the properties of cell membranes, leading to changes in their organization and composition. Furthermore, due to their high reactivity, lipid peroxides can generate more ROS and transform them into compounds that interact with DNA and proteins [5,59]. Some natural compounds exhibit photoprotective effects by reducing ROS and lipoperoxidation in skin cells [24], which was observed with quercetin (QT) [64] and was also confirmed with SA thanks to the present study (see Fig. 6 and 7).

To regulate the levels of ROS, the endogenous antioxidant system relies on numerous enzymes [60,65]. The primary enzymatic antioxidants include the enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase [17,60,66]. The
enzyme SOD catalyzes the dismutation of O₂⁻ in O₂ and H₂O₂, and catalase acts by decomposing H₂O₂ into O₂ and H₂O [20,50]. In addition to catalase, the enzyme glutathione peroxidase (GPx) catalyzes H₂O₂ in the presence of the reduced form of glutathione (GSH), an antioxidant molecule [21,61,67].

There are also antioxidant molecules that deactivate ROS, thereby preventing damage to DNA and other macromolecules [68]. GSH, for example, functions as a non-enzymatic defensive element, serving as a co-factor for various antioxidant enzymes and directly scavenges ROS. As a result, strategies aiming at averting the generation of ROS and maintaining GSH levels have been firmly established to mitigate skin injuries [45,60]. The ability of SA to reduce the depletion of GSH and catalase in UVB-irradiated cells, as evidenced in our study (Fig. 8), is another key attribute of SA for photochemioprotection. To protect the cell, catalase, SOD, and peroxidase participate in the catalytic removal of free radicals, while GSH, vitamin E (tocopherol), vitamin C (ascorbic acid), for example, reduce free radicals as they serve as electron donors [19,21,60,65].

Overexposure to UVB radiation can also lead to a reduction in wound healing rate [49]. After an injury, for example, the skin must be promptly restored to ensure the fulfillment of its functions. In this process, cytokines, chemokines, growth factors, resident skin cells, peripheral blood mononuclear cells, and the extracellular matrix play a crucial role in the wound healing process [69]. Certain antioxidant substances have the ability to assist in this process, that is, they have wound-healing properties [22,70–72].

After 24 h, our results demonstrated that SA was able to enhance wound healing compared to the negative control (untreated cells). Furthermore, the SA treatment effectively counteracted the negative impact of UVB on wound healing. Studies have reported other phenolic acids with wound-healing properties, such as the ferulic acid. The use of ferulic acid ointment during the wound healing process also increased antioxidant enzymes such as catalase and superoxide dismutase, and inhibited lipid peroxidation [73]. Vanillic acid, a phenolic acid of the hydroxybenzoic acid (similar to SA), is described in the literature as effective in wound healing and possesses antioxidative effects [74].

Substances capable of promoting antioxidative effects in cells can significantly reverse the biological and physiological damages that occur in the cell after extensive ROS generation [45,75]. Microscopy experiments revealed that treatment with SA prompted cells to closely resemble the negative control. Untreated irradiated cells showed more damage with ruptured cell membranes, an increase in nucleus size, and disorganization of the cellular interior. To prevent these damages, effective
photochemioprotective formulations are of the utmost importance [76], since the deleterious effects of oxidative stress are strongly related to photoaging [21,53,56], immunosuppression [10,53,66] and skin cancer [11,29,55]. Photochemioprotective substances can be incorporated into various formulations, acting as an adjuvant in sun protection [77–79]. In this way, SA is a great candidate for inclusion in sunscreens and multifunctional formulations.

## 5. Conclusion

We have demonstrated the antioxidant activity of SA and its photochemioprotection in fibroblasts exposed to UVB radiation. SA neutralizes ROS, prevents lipid peroxidation, inhibits the depolarization of mitochondrial membrane caused by UVB and reduces GSH and catalase depletion, which contribute to the maintenance of the cellular endogenous antioxidant system. SA also proved to accelerate wound healing and preserve the ultrastructural characteristics of the cell, by protecting them from damage caused by oxidative stress (Fig. 10). Therefore, SA's effectiveness in preventing the deleterious effects of UVB make it a valuable component in products dedicated to skin health and protection, such as sunscreens and multifunctional formulations.

<figure><img src="image_15.png" /><figcaption>**Fig. 10.** Syringic acid reduces oxidative stress in fibroblastic cells irradiated with UVB radiation through different biochemical mechanisms.</figcaption></figure>
735

736 **Author statement**

737 The conceptualization of the project was done by K.C.N. and C.V.N.

738 K.C.N. carried out the experiments. The data curation and the writing of the

739 original draft were performed by K.C.N. The funding acquisition was done by S.O.S.,

740 T.U-N. and C.V.N. The review and the project administration were organized by D.L-B.,

741 R.M., R.A.V. and C.V.N. The authors collectively analyzed the findings and designed the

742 final manuscript.

743

744 **Declaration of Competing Interest**

745 The authors have no conflicts of interest to declare.

746

747 **Acknowledgment**

748 The authors acknowledge the financial support provided by Coordenação de

749 Aperfeiçoamento de Pessoal de Nível Superior (CAPES-COFECUB), CNPq, FINEP,

750 Fundação Araucária.

751
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