UVA-induced ROS generation inhibition by Oenothera paradoxa defatted seeds extract and subsequent cell death in human dermal fibroblasts

Highlights

• Oenothera paradoxa defatted seeds extract lowered UVA cytotoxicity in fibroblasts.

• The extract decreased ROS production and lipid peroxidation in irradiated cells.

• UVA induced cell apoptosis was decreased by the extract.

• The extract increased the number of viable fibroblasts.

Abstract

UVA radiation stimulates the production of reactive oxygen species (ROS), which react with lipids, proteins and other intracellular molecules leading to oxidative stress, cellular damage and ultimately cell death. There is, therefore, a growing need for substances exhibiting antioxidant activity, which may support repair mechanisms of the skin. This study evaluates the protective effect of the aqueous Oenothera paradoxa Hudziok defatted seeds extract, rich in polyphenolic compounds, against UVA (25 and 50 J/cm²)-induced changes in normal human dermal fibroblasts (NHDFs). The tested extract (0.1–10 μg/ml) has decreased, in a concentration-dependent fashion, the UVA-induced release of lactate dehydrogenase (LDH) into the culture medium, the ROS production (with the use of 2′,7′-dichlorodihydrofluorescein diacetate) and lipid peroxidation (utilizing redox reactions with ferrous ions) as compared to the control cells (incubated without the extract). Moreover, the extract increased the number of viable (calcein positive) cells decreasing the number of cells in late apoptosis (annexin V-FITC and propidium iodide positive). Thus our results show that O. paradoxa defatted seeds extract may be beneficial for the prevention of UVA skin damage.

Introduction

UVA (315–400 nm), further divided into UVA1 (340–400 nm) and UVA2 (315–340 nm) as well as UVB (280–315 nm) are ultraviolet radiation ranges that reach the surface of the skin, having a significant impact on its diseases and mutagenesis. Excessive skin exposure to UV radiation induces inflammation, immunosuppression, genetic mutations and, eventually, the malignant transformation of skin cells. Since the ozone layer blocks most UVB radiation, UVA makes up about 95% of the UV radiation that reaches the earth. UVA passes through the layer of the epidermis, dermis and blood vessels, injuring cells indirectly by increased production of reactive oxygen species (ROS). ROS such as superoxide anion (${\text{O}}_2^{-}$), hydrogen peroxide (H₂O₂) and hydroxyl radical (HO) interact with lipid-rich membranes, enzymes and cellular DNA, leading to oxidative stress and, consequently, cell death [1]. Furthermore, UVA may trigger the formation of ROS long after the exposure [2].

In the last decades there has been a significant increase in exposure of the human population to UVA radiation. This is due to the popularity of UVA tanning salons and efficient UVB-absorbing sunscreens which block erytherma, allowing prolonged periods of sunbathing [3]. By the fact that for a long time, UVB radiation was considered to be predominantly responsible for UV-induced skin changes, there exist only a few substances proven to be useful in cell protection against UVA-induced oxidative damage. These include epicatechin [4], epigallocatechin-3-gallate [5], [6], Polypodium leucotomos extract [7], flavonolignans from Silybum marianum [8], [9], Prunella vulgaris extract [10], rosmarinic and gallic acid [11], [12]. Therefore, there is a growing need for substances exhibiting antioxidant activity, which may support repair mechanisms of the skin damaged by UVA irradiation.

Oenothera paradoxa Hudziok (Oenotheraceae) is cultivated in Europe for the production of seeds rich in essential fatty acids, including γ-linolenic acid (GLA). The production of evening primrose oil (EPO) used in the pharmaceutical and cosmetic industries, generates significant amounts of defatted seeds. This waste material contains polyphenolic compounds [13], estimated in quantity as comparable to that of green tea or grape seeds [14]. O. paradoxa defatted seeds extracts, rich in polyphenols, exhibit strong antioxidant potential [15]. Ex vivo studies have revealed that the aqueous extract of O. paradoxa defatted seeds and its constituents: gallic acid, (+)-catechin, ellagic acid, penta-O-galloyl-β-d-glucose and polymeric procyanidins decrease levels of ROS in human neutrophils due to the scavenging of ${\text{O}}_2^{-}$ and H₂O₂ oxygen species [16]. We have also recently revealed that high concentrations of O. paradoxa defatted seeds extract exhibit anticancer activity on skin melanoma cells [17], [18]. However, the photoprotective potency of O. paradoxa defatted seeds extract has not been studied yet.

The purpose of the present study was to investigate the UVA protective activity of the aqueous O. paradoxa defatted seeds extract, on normal human dermal fibroblasts (NHDFs) by ROS generation, lipid peroxidation and apoptosis vs necrosis assessment.