Impressive properties make graphene-based materials (GBMs) promising tools for nanoelectronics and biomedicine. However, safety concerns need to be cleared before mass production of GBMs starts. As skin, together with lungs, displays the highest exposure to GBMs, it is of fundamental importance to understand what happens when GBMs get in contact with skin cells. The present study was carried out on HaCaT keratinocytes, an in vitro model of skin toxicity, on which the effects of four GBMs were evaluated: a few layer graphene, prepared by ball-milling treatment (FLG), and three samples of graphene oxide (GOs, a research-grade GO1, and two commercial GOs, GO2 and GO3). Even though no significant effects were observed after 24 h, after 72 h the less oxidized compound (FLG) was the less cytotoxic, inducing mitochondrial and plasma-membrane damages with EC₅₀s of 62.8 μg/mL (WST-8 assay) and 45.5 μg/mL (propidium iodide uptake), respectively. By contrast, the largest and most oxidized compound, GO3, was the most cytotoxic, inducing mitochondrial and plasma-membrane damages with EC₅₀s of 5.4 and 2.9 μg/mL, respectively. These results suggest that only high concentrations and long exposure times to FLG and GOs could impair mitochondrial activity associated with plasma membrane damage, suggesting low cytotoxic effects at the skin level.

By means of a bioassay-oriented fractionation of the CO₂ extract of Calendula flowers, the triterpenoids are shown as the most important anti-inflammatory principles of the drug. Among them, the faradiol monoester appears to be the most relevant principle for the activity of the drug, due to its quantitative prevalence. The unesterified faradiol, not present in the extract, is the most active of the tested compounds and equals indomethacin in activity, whereas the monools ψ-taraxasterol, lupeol, taraxasterol, and β-amyrin are less active than the free diol. The anti-inflammatory activity of different CO₂ extracts is proportional to their content of faradiol monoester, which can be taken as a suitable parameter for the quality control of Calendula preparations.