An Agilent high performance liquid chromatography system (1100 series, Santa Clara, CA, USA), equipped with a photodiode-array detector (DAD, G1315), was used. The qualitative and quantitative determination of polyphenols in HFE was carried out as previously reported. This kind of activity should be further potentiated by stimulation of keratinocyte mobilization, revealed by our scratch wound assay, suggesting a support to skin repair following injury or degenerative processes. robertianum, used at proper doses, could support skin redox balance processes, thus contributing to prevent or counteract oxidative-related dysfunctions, especially those leading to skin ageing. However, polyphenols are also widely known to exert antioxidant effects, as fully confirmed in this study by the panel of specific assays. The lack of a pan-antioxidant protection could reflect the known tendency of some polyphenols, e.g., flavonoids, to elicit pro-oxidant activities under specific redox conditions. Significant protection of keratinocytes against injurious effects has been found after double exposure to HFE and H 2O 2 as the oxidant agent, but only at high HFE doses. In parallel with phytochemical characterization and antioxidant evaluation, skin-preserving properties have been clearly suggested by the complex of our bioassays. However, despite its protected status, the species has been found to expand its presence across the Mediterranean distributional range. In Italy it is a protected entity at national level and is covered by total regional protection in different areas ( ). The species is included in the Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), and as Least Concern on the French red list (UICN France et al. It is called ‘giant orchid’, as it exceeds most European wild orchids by its large size, reaching a height of 50–80 cm. The plant grows up to an altitude of 1700 m, and blooms from January to April. Typical habitats include poor grassland, garrigue, scrub, and open woodland. robertianum is a Mediterranean orchid with scattered occurrence and large distribution area, spanning from Portugal and Morocco to Anatolia. Barlia robertiana) due to its large inflorescences, and because it can be easily propagated in vitro, making it exploitable as a source of bioactive substances without damaging natural populations. We chose to investigate Himantoglossum robertianum (Loisel.) P. robertianum flowers for skin-preserving, repair, and anti-aging applications. In summary, HFE counteracted different free radicals, while protective properties were shown by cell-free and cell-based bioassays, suggesting the possible use of H. Biological assays showed elastase and collagenase inhibition (up to 42% and 78%, respectively), improvement of HaCaT cell viability after treatment with 500 μM H 2O 2 (from 30% to 84% of control), and stimulation of cell migration rate up to 210% of control. Antioxidant assays showed strong activities, rating ORAC > FRAP > TEAC > β-carotene bleaching > DPPH > iron-chelation. Flavonoids (flavones and flavan-3-ols) represented the most abundant compounds (42.91%), followed by scopoletin (33.79%), and phenolic acids (23.3%). Microscopy showed epidermal cells containing anthocyanins in the flower labellum. Antioxidant assays, in vitro collagenase and elastase inhibition, and MTT and cell motility assays on HaCaT keratinocytes were done. HFE was characterized for total phenols, flavonoids and proanthocyanidins, and for polyphenol profile by RP-LC-DAD. Flowers were analyzed by light and SEM microscopy and used to obtain a polyphenol-rich, hydroalcoholic flower extract (HFE). Delforge is a Mediterranean orchid whose propagation in vitro has been achieved, making it eligible as a source of bioactive substances.
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