Comparative Study on Phytochemical Alterations and Immunomodulatory Activity of the Bulgarian Endemic Plant Betonica bulgarica Degen & Nejceff During the Process of Ex Situ Conservation

Betonica bulgarica Degen & Nejceff is an endemic medicinal plant found only in Bulgaria. It is a valuable source of phenylethanoid glycosides and iridoids and is associated with antioxidant and anti-inflammatory activities. In the present study, we investigated whether phytochemical variations occurring during ex situ conservation of the species influence its immunomodulatory activity on pro-inflammatory response. Methanolic extracts obtained from in vitro-cultivated and ex vitro-adapted plants were evaluated for their effects on complement activity and on LPS-stimulated macrophages by assessing nitric oxide, TNF-α, IL-6, IL-12 production, and COX-2 expression. Comparative NMR metabolite profiling revealed the presence of verbascoside and the iridoids allobetonicoside and 8-O-acetylharpagide in in vitro- and ex vitro-adapted plants. Both variants exhibited anti-complement activity through binding to IgG-C1q complexes. The nitric oxide and TNF-α levels were reduced by extracts from in vitro-cultivated plants: 38.68 μM NO2 and 208.64 pg/mL, respectively, whereas extracts from ex vitro plants showed stronger suppression of IL-6 (168 pg/mL). None of the variants had an effect on IL-12 production. COX-2 expression was slightly reduced by the extract of in vitro-adapted plants. Overall, these findings demonstrate that phytochemical alterations associated with environmental adaptation during ex situ conservation influence the immunomodulatory activity of B. bulgarica, highlighting the dynamic relationship between plant metabolism and biological activity.