Photoprotective Effect of Calluna vulgaris Extract Against UVB-Induced Phototoxicity in Human Immortalized Keratinocytes

Abstract

There is an increasing interest in the use of natural antioxidants as photoprotective agents against skin damages produced by ultraviolet radiation. The aim of our study was to investigate the protective effect of a Calluna vulgaris extract in human keratinocytes (HaCaT) exposed to ultraviolet B (UVB) radiation. HaCaT cells were treated with C. vulgaris extract 30 minutes prior to irradiation with UVB. The protective effect was evaluated by assessing cell viability using tetrasolium salt (MTT) assay; the generation of lipid peroxides was evaluated using malondialdehide assay (MDA); and DNA damage was evaluated using the comet assay and the quantification by ELISA of specific DNA photolesions [i.e., cyclobutane-pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs)]. After irradiation with cytotoxic doses of UVB (300 and 500 mJ/cm(2)), HaCaT cells pretreated with C. vulgaris extract (50 μg GAE/ml) showed significantly increased viability compared to control cells exposed to UVB only. Irradiation alone increased MDA levels in a dose-dependent fashion. Pretreatment with 12 μg GAE/ml extract lowered MDA levels both at 100 mJ/cm(2) (ρ<0.01) and 300 mJ/cm(2) (ρ<0.001). Treatment with C. vulgaris extract before exposure to UVB also reduced DNA damage: Lesion scores in a comet assay were significantly reduced at UVB doses of 50 mJ/cm2 (ρ<0.01) and 100 mJ/cm(2) (ρ<0.05), while CPDs and 6-4PPs (via ELISA) were significantly lower after irradiation with 100 mJ/cm(2) in the protected cells (ρ<0.05 for CPDs and ρ<0.001 for 6-4PPs). These results recommend the use of the C. vulgaris extract as photoprotective agent, in combination with sunscreens and/or other natural products with similar or complementary properties.

Modulation of the UV-B-induced Oxidative Stress and Apoptosis in HaCaT Cell Line with <i>Calluna vulgaris</i> Extract

“…These effects are concentration-dependent and time-dependent and, at least to a certain extent, are due to the modulation of Bax-α and Bcl-XL proteins. These results are in concordance to others in literature (Olteanu et al, 2012;Olteanu et al, 2014;Perde-Schrepler et al, 2011) and support further studies to identify other molecules and pathways whereby the CV extract may exert its beneficial protective effects. These findings also suggest that the CV extract could be used as a potential photoprotective agent.…”

“…The UV-B's cytotoxicity on the HaCaT cells was previously determined (Perde-Schrepler et al, 2011) and the half maximal inhibitory concentration (IC50) (217 mJ/cm 2 ) was considered for the selection of the dose used in this study. Therefore, a dose within the physiological range of UVR exposure usually experienced by human skin and approximately equivalent to the minimal erythema dose (Pathak et al, 1987), was chosen (100 mJ/cm 2 ).…”

“…Proanthocyanidins represented 3.15 mg/ml and monomer anthocyanins 5.81 mg/ml. The most important biologically active compounds were evidenced by highperformance liquid chromatography which indicated the presence of hyperozid, quercetin, isoquercetin, kampferol and smaller amounts of eulic and p-cumaric acids, rutozid and luteolin (Perde-Schrepler et al, 2011). The antioxidant activity of CV extract was established at 0.211 ± 0.08 mM/mM 2,2-diphenyl-1-picrylhydrazyl (DPPH), respectively 38.92 ± 0.01 mM Trolox eq .…”

“…The CV extract's protective effect against UV-B-induced oxidative stress and apoptosis, evaluated through sunburn cells formation (Olteanu et al, 2012) and caspase 3 activity (Olteanu et al, 2014), were demonstrated earlier on SKH-1 nude mice and on endothelial cells, respectively. Also, the extract's preventive effect on the UV-B-induced DNA lesions in HaCaT cells was previously established (Perde-Schrepler et al, 2011).…”

“…Assessment of the plant extract's cytotoxicity on the HaCaT cells The cytotoxicity of the CV extract on HaCaT cells was determined earlier (Perde-Schrepler et al, 2011), and the calculated IC50 value (74.2 µg EqGA/ml) was considered as guide-mark for the tested concentrations. Aiming to identify protective effects concentrations in the non-toxic range were used, below the measured IC50 values.…”