A Practical Approach for the In Vitro Safety and Efficacy Assessment of an Anti-Ageing Cosmetic Cream Enriched with Functional Compounds

Abstract

(1) Background: Cosmeceuticals are topical products applied to human skin to prevent skin ageing and maintain a healthy skin appearance. Their effectiveness is closely linked to the compounds present in a final formulation. In this article, we propose a panel of in vitro tests to support the efficacy assessment of an anti-ageing cream enriched with functional compounds. (2) Methods: biocompatibility and the irritant effect were evaluated on reconstructed human epidermis (RHE) and corneal epithelium (HCE) 3D models. After a preliminary MTT assay, normal human dermal fibroblasts (NHDF) and keratinocytes (HaCaT) were used to evaluate the extracellular matrix (ECM) protein synthesis, and interleukin-6 (IL-6) and metalloproteinase-1 (MMP-1) production. (3) Results: data collected showed good biocompatibility and demonstrated the absence of the irritant effect in both 3D models. Therefore, we demonstrated a statistical increase in collagen and elastin productions in NHDF cells. In HaCaT cells, we highlighted an anti-inflammatory effect through a reduction in IL-6 levels in inflammatory stimulated conditions. Moreover, the reduction of MMP-1 production after UV-B radiation was demonstrated, showing significant photo-protection. (4) Conclusion: a multiple in vitro assays approach is proposed for the valid and practical assessment of the anti-ageing protection, anti-inflammatory and biocompatible claims that can be assigned to a cosmetic product containing functional compounds.

Keywords: 3D model; MMP-1; anti-ageing; ceramides; collagen; cosmeceuticals; elastin; in vitro; safety; skin irritation.

Figure 1

Cell viability (%) of HaCaT cells (in blue) and NHDF cells (in orange) after treatment with different concentrations of the cream (range between 0.312 and 5 mg/mL) with respect to untreated cells (n = 2; replicates = 3).

Figure 2

(a) Collagen levels and (b) elastin levels expressed as concentration (µg/mL) after treatment with the tested cream (0.625 and 1.25 mg/mL) in NHDF cells. * p values ≤ 0.05 were considered to be statistically significant compared with untreated cells (n = 3; replicates = 2).

Figure 3

Quantitative analysis of IL-6 levels (pg/mL) after treatment with the cosmetic cream at the concentrations of 1.25 and 2.5 mg/mL and stimulation with TNF-α; Ctrl (−): untreated cells; Ctrl (+): cells treated only with TNF-α. * p values ≤0.05 and *** p values ≤ 0.001 were considered to be statistically significant compared with respective controls (n = 2; replicates = 2).

Figure 4

Quantitative analysis of MMP-1 levels (ng/mL) after treatment with the cosmetic cream at the concentrations of 1.25 and 2.5 mg/mL. Ctrl (−): untreated cells; UVB (+): irradiated cells. * p values ≤0.05 and ** p values ≤ 0.01 were considered to be statistically significant compared with irradiated cells (n = 2; replicates = 2).

Figure 5

(a) Cell viability expressed as a percentage (%) of RHE treated with Ceramide Shield Cream. Ctrl (−): RHE treated with DPBS; Ctrl (+): RHE treated with SDS as the irritating stimulus. (b) IL-1α amount (IU/mL) in the medium after treatment with Ceramide Shield Cream; Ctrl (−): cells treated with DPBS; Ctrl (+): RHE treated with SDS. Values of *** p ≤ 0.001 were considered statistically significant compared with Ctrl (−) by one-way ANOVA statistical analysis followed by Fisher’s LSD test.

Figure 6

Cell viability expressed as a percentage (%) of HCE treated with Ceramide Shield Cream. Ctrl (−): HCE treated with DPBS; Ctrl (+): HCE treated with SDS as the irritating stimulus.