Red algae extract improves barrier recovery

Skin has evolved several mechanisms to combat water evaporation, including lipid coating of the skin’s surface, inter-cellular space occlusion through the formation of tight junctions, production in the upper layers of the epidermis of highly hygroscopic substances (filaggrinderived fragments), and accumulation of water-retaining substances within epidermal cells (osmolytes such as taurine or glycyl-betaine). Water loss is the primary consequence of epidermal impermeability impairment, and skin dryness (xerosis) is associated with several physio-pathological conditions where barrier function is weakened, such as psoriasis, atopic dermatitis and ageing. An important recent finding was that superficial dryness tampers with some mechanisms in charge of barrier function restoration. To combat dehydration and improve barrier function recovery, we have designed a specific red algae extract (trade name Arct’Alg) rich in humectants (including osmolytes) and energetic metabolites. The algae was exposed to low temperature and limited illumination during a long period of time (‘over-wintering’ in cultivation tanks on the Eastern shores of Canada), conditions that were reported to induce the accumulation of antioxidants, cryoprotective osmolytes, and energy storage compounds in red algae.1,2 Among other metabolites, a characteristic nitrogen-rich dipeptide, L-citrullinyl-L-arginine, which is an energy storage metabolite for the algae, was found to accumulate up to 10% of algae dry weight (Fig. 1). This dipeptide was regarded as of particular interest for moisturisation since citrulline is a constituent of skin natural moisturisation factor (NMF). Considering the metabolites’ accumulation profiles, the algae was harvested at the end of Canadian winter, before it enters into its proliferative phase which causes rapid expenditure of energy stores. Also, an extraction procedure was designed for maximum recovery of highly polar metabolites, with the exclusion of carrageenan polyuronides whose strong gelling properties is troublesome for cosmetic formulation. In addition to the dipeptide L-citrullinyl-L-arginine and related amino acids, we extracted high amounts (2%-3% in the extract) of galactosylglycerol conjugates floridoside and iso-floridosides (Fig. 2), known as osmotic and anti-stress molecules seasonally accumulated in various red algae.3,4 This unique combination was tested for its skin moisturising and anti-dehydrating properties. The study was carried out on human skin explants, whose epidermal barrier was disrupted by acetone. This method was reported to induce corneocyte disorganisation and surface lipid removal5,6 leading to increased transepidermal water loss (TEWL). In our model system, stratum corneum lipids’ partial withdrawal mimics surface lipids’ decrease associated with skin ageing7,8 and seasonal stress (winter xerosis).8 Here, we present the results of an histological study showing a sequence of early and late events triggered by epidermal barrier disruption with acetone, and the benefits from topical application of the red algae extract. In this model, early markers filaggrin and aquaporin-3 (monitoring three hours after stress) were mainly indicative for the moisturising properties of the extract, while late markers transglutaminase and Ki67 nuclear protein (monitoring 24 hours after stress) gave information on the effect of the extract on adaptive response to barrier disruption.

Material and methods

Human skin explants (epidermis/dermis) (Biopredic) obtained from mammary plastic surgery of young Caucasian women less than 45 years old were processed less than 48 hours after the operation. Skin explants surface was exposed during two minutes to an acetone-soaked blotting paper. Test substance was applied topically immediately after removal of the acetone-containing blotting paper. Controls were exposed to PBS-soaked blotting papers. Skin fragments were snap-frozen and then processed for immunofluorescence (IF) studies three hours or 24 hours after barrier disruption, depending on the marker. Primary antibodies used for IF staining were anti-filaggrin Ab-1 (Microm France), anti-aquaporin 3 (Chemicon), antitransglutaminase 1 (Harbor), anti-Ki67 (MIB1, Dako). Sections were then incubated with the appropriate fluorescein isothiocyanate-labelled secondary antibody for revelation. Utracruz mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) was used to visualise nuclei. For each marker, several representative fields were selected from three punch biopsies, and three images were taken from each biopsy. Computer-assisted image analysis was achieved with the Cell F software (Olympus, Japan). Fluorescence intensity was expressed in arbitrary units (a.u.), taking into account the number of pixels within the measurement frame and the level of illumination (256 discrete gray levels). Cell counts for Ki67-positive cells in the basal layer of epidermis were reported to total number of nuclei in the basal layer.