| Summary: | <i>Stachys thracica</i> Davidov is a Balkan endemic species distributed in Bulgaria, Greece, and Turkey. In Bulgaria, it is classified as “rare” and is under the protection of the Bulgarian biodiversity law. The aim of our study was to develop an efficient protocol for ex situ conservation of <i>S. thracica</i> and to perform comparative NMR-based metabolite profiling and bioactivity assays of extracts from in situ grown, in vitro cultivated, and ex vitro acclimated plants. Micropropagation of <i>S. thracica</i> was achieved by in vitro cultivation of mono-nodal segments on basal MS medium. Ex vitro adaptation was accomplished in the experimental field with 83% survival while conserved genetic identity between in vitro and ex vitro plants as shown by the overall sequence-related amplified polymorphism marker patterns was established. Verbascoside, chlorogenic acid, and trigonelline appeared the main secondary metabolites in in situ, in vitro cultivated, and ex vitro acclimated <i>S. thracica</i>. High total phenolic and flavonoid content as well as antioxidant and radical scavenging activity were observed in in situ and ex vitro plants. Further, the anti-inflammatory activity of <i>S. thracica</i> was tested by hemolytic assay and a high inhibition of the complement system was observed. Initiated in vitro and ex vitro cultures offer an effective tool for the management and better exploitation of the <i>Stachys</i> secondary metabolism and the selection of lines with high content of bioactive molecules and nutraceuticals.
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