Natural Substances for Prevention of Skin Photoaging: Screening Systems in the Development of Sunscreen and Rejuvenation Cosmetics

Abstract

Solar broadband UV irradiation is commonly regarded as a major causative reason for cutaneous photoaging. The pro-aging molecular pathways and cellular targets affected by UVA+UVB light in human skin have been extensively investigated. Notwithstanding growing knowledge in mechanisms of photoaging, research and development of clinically efficient, nontoxic, and sustainable topical preparations providing full physical, chemical, and biological photoprotection still remain a great challenge for pharmaceutical and cosmetic industries. In this study, we are proposing a panel of the in vitro methods for preselection of natural photoprotective substances with high photostability and low phototoxicity able of absorbing a broadband UVA+UVB irradiation (physical sunscreen), reducing UV-related overproduction of free radicals and loss of endogenous antioxidants (chemical protection), and attenuating UV-induced cytotoxicity and immune and metabolic responses (biological protection) in primary human epidermal keratinocytes and immortalized human keratinocyte cultures. Our data showed that secondary metabolites biosynthesized in plant cells in response to UV irradiation, such as phenylpropanoids and their glycosylated metabolites, aglycons and glycosylated flavonoids, and leontopodic acids, hold the best promise for complete natural topical prevention of photoaging and rejuvenation of photoaged skin. Meristem plant cell cultures elicited by solar simulating UV could be the most environmentally sustainable biotechnological source of polyphenols with combined photoprotective and antiaging properties.

Keywords: broadband sunscreens; human keratinocytes; photo-chemotoxicity; photostability; plant polyphenols; skin photoaging; solar UVA+UVB.

FIG. 1.

Chemical structures of plant-derived secondary metabolites interacting with solar irradiation. 1–4, photosensitizers which absorb solar light energy and transform it into energy of chemical/biochemical reactions (anoxic type I or oxygen-dependent type II). 5–10, substances with mainly photoprotective (sunscreen) properties which absorb solar light energy and dissipate it in the forms of thermal, fluorescence/phosphorescence, or oscillation energy.

FIG. 2.

Photostability of polyphenols. UV-visible absorption spectra of 50 μmol/L resveratrol (A) and verbascoside (B) in 0.1 M potassium phosphate buffer (pH 7.4) recorded (1) before and (2–5) 1, 3, 20, and 40 minutes after solar-simulated irradiation (UVA/UVB = 2/1, irradiance 25 W/m²).

FIG. 3.

Cytotoxicity versus photo-cytotoxicity of polyphenols. (A) Fluorescence photographs of HaCaT cells stained with acridine orange/ethidium bromide: I, control cells; II–III, cells 3 and 6 hours postirradiation (low-dose noncytotoxic UVA+UVB = 2 + 4 J/cm² for 40 minutes), respectively; IV–VI, cells similarly irradiated in the presence of 50 μM chalcone 3, 6, and 24 hours postirradiation, respectively; VII–IX, cells similarly irradiated in the presence of 50 μM resveratrol 3, 6, and 24 hours postirradiation, respectively. (B) For cytotoxicity determination, HaCaT cells were incubated with 50 μM polyphenols for 40 minutes. Cell viability was determined by a PrestoBlue viability assay 24 hours postincubation in a polyphenol- and serum-free medium in accordance with Materials and Methods section description. For photo-cytotoxicity determination, HaCaT cells were irradiated by low-dose UVA+UVB (2 + 4 J/cm²) in the absence or presence of 50 μM polyphenols for 40 minutes. Cell viability was determined by a PrestoBlue viability assay 24 hours postirradiation and incubation in a polyphenol- and serum-free medium. Results are expressed as mean ± SD of eight measurements in two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 versus UV or Control. ND, not done.

FIG. 4.

Effects of polyphenols on (UVA+UVB)-induced inflammatory and metabolic responses of primary human keratinocytes. (A) NFκB activation (measured by Western blot of phosphorylated form of p65, P-p65) in normal human epidermal keratinocytes by UVA+UVB (UVA 1.0 J/cm²+UVB 0.1 J/cm²) alone or in combination with 10 μM polyphenols (resveratrol, polydatin, rutin, quercetin, verbascoside, and epicatechin). Exposure time with UVA+UVB was 5, 10, and 20 minutes. Exposure to a combination of UV+polyphenol was 20 minutes. Actin was used in Western blots as a control for the protein loading. Quantification of P-p65 was done by densitometry, and data are shown at the bottom of blots. (B) mRNA induction of inflammatory cytokines (TNFα, IL1β, IL6, and IL8) and cytochrome P450 subfamilies (CYP1A1 and CYP1B1) by UVA+UVB (UVA 1.0 J/cm²+UVB 0.1 J/cm²) alone or in combination with 10 μM polyphenols in normal human epidermal keratinocytes. Real-time PCR analysis was carried out 6 hours postirradiation. Results are expressed as mean values and standard deviation from the mean (m ± SD) from nine measurements in three independent experiments. Control values are assumed as 1. *p < 0.05; **p < 0.01; versus UV irradiation in the absence of polyphenols.