In vitro studies to evaluate the wound healing properties of Calendula officinalis extracts.

Calendula officinalis (pot marigold) flower extracts have a long-lasting tradition in ethnopharmacology. Currently, the European Medicines Agency (EMA) has approved its lipophilic and aqueous alcoholic extracts as traditional medicinal products for the treatment of minor inflammation of the skin and as an aid in the healing of minor wounds. The purpose of this study was to analyse the molecular mechanism of the wound healing effects of Calendula extracts, which may reflect the phytomedicines currently used in the market. The effect of three different extracts from Calendula flowers (n-hexanic, ethanolic, aqueous) on the inflammatory phase of wound healing was studied in human immortalized keratinocytes and human dermal fibroblasts. An electrophoretic mobility shift assay on NF-κB-DNA binding, qRT-PCR and ELISA experiments were performed. The effect of Calendula extracts on the new tissue formation phase of wound healing was evaluated by studying the migratory properties of these extracts, triterpene mixtures and single compounds in human immortalized keratinocytes using the scratch assay. Finally, the effect of the extracts on the formation of granulation tissue in wound healing was studied using bacterial collagenase isolated from Clostridium histolyticum and the determination of soluble collagen in the supernatant of human dermal fibroblasts. The n-hexanic and the ethanolic extracts from Calendula flowers influence the inflammatory phase by activating the transcription factor NF-κB and by increasing the amount of the chemokine IL-8, both at the transcriptional and protein level, in human immortalized keratinocytes. The migration of the keratinocytes during the new tissue formation phase was only marginally influenced in the scratch assay. However, it can be assumed that the granulation tissue was affected, as the ethanolic extract inhibited the activity of collagenase in vitro and enhanced the amount of collagen in the supernatant of human dermal fibroblasts. Our results contribute to a better understanding of the wound healing properties of the traditional medicinal plant Calendula officinalis. However, further studies are necessary to evaluate which of its known constituents are responsible for these effects. Triterpenes seem to play only a marginal role, but carotene and xanthophyll derivatives should garner more attention in future studies.