Without being aware of their chemical composition, many cultures have used herbaceous peony roots for medicinal purposes. Modern phytopreparations intended for use in human therapy require specific knowledge about the chemistry of peony roots and their biological activities. In this study, ethanol-water extracts were prepared by maceration and microwave- and ultrasound-assisted extractions (MAE and UAE, respectively) in order to obtain bioactive molecules from the roots of Paeonia tenuifolia L., Paeonia peregrina Mill., and Paeonia officinalis L. wild growing in Serbia. Chemical characterization; polyphenol and flavonoid content; antioxidant, multianti-enzymatic, and antibacterial activities of extracts; and in vitro gastrointestinal digestion (GID) of hot water extracts were performed. The strongest anti-cholinesterase activity was observed in PT extracts. The highest anti-ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical potential was observed in PP extracts, whereas against DPPH (2,2-diphenyl-1-picrylhydrazyl radicals), the best results were achieved with PO extracts. Regarding antibacterial activity, extracts were strongly potent against Bacillus cereus. A molecular docking simulation was conducted to gather insights into the binding affinity and interactions of polyphenols and other Paeonia-specific molecules in the active sites of tested enzymes. In vitro GID of Paeonia teas showed a different recovery and behavior of the individual bioactives, with an increased recovery of methyl gallate and digallate and a decreased recovery of paeoniflorin and its derivatives. PT (Gulenovci) and PP (Pirot) extracts obtained by UAE and M were more efficient in the majority of the bioactivity assays. This study represents an initial step toward the possible application of Paeonia root extracts in pharmacy, medicine, and food technologies.

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