Phenolic Acid Investigation and In Vitro Antioxidant and Antiacetylcholinesterase Potentials of Galeopsis spp. (Lamiaceae) from Romanian Flora

Background/Objectives Galeopsis spp. (Lamiaceae) are widely distributed across extensive areas in Romania, being used mainly for their sedative, neuroprotective, antioxidant, anti-inflammatory, expectorant, astringent, and diuretic properties. The paper reports for the first time the investigation of the total phenolic content (TPC), total flavonoid content (TFC), and phenolic acid profile in the roots, aerial parts, and leaves from three wild-grown Galeopsis spp. (G. bifida Boenn., G. speciosa Mill., and G. tetrahit L.), along with their antioxidant and acetylcholinesterase (AChE) inhibitory potentials. Methods: The ultra-high-performance liquid chromatography/ultraviolet/mass spectrometry (HPLC/UV/MS) method was used for the identification and quantification of key phenolic acids. The spectrophotometric method was applied for the determination of TPC, TFC, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities and also the ferric-reducing antioxidant power (FRAP). High-performance thin-layer chromatography (HPTLC) was employed for the assessment of in situ antioxidant (DPPH assay) and AChE inhibitory potentials. Results: Galeopsis spp. exhibit significant polyphenol accumulation. Chlorogenic acid was the most abundant compound, with the highest levels detected in G. tetrahit leaves (22,347.907 ± 1117.395 μg/g), followed by G. tetrahit aerial parts (11,678.509 ± 583.925 μg/g) and G. speciosa leaves (8712.628 ± 435.631 μg/g). G. tetrahit leaves had the highest DDPH radical scavenging activity, with a half-maximal inhibitory concentration (IC50) of 0.458 ± 0.03 mg/mL, demonstrating a markedly stronger antioxidant effect. Leaves consistently showed the strongest DPPH activity across all species, with G. speciosa leaves also displaying a low IC50 value of 0.789 ± 0.03 mg/mL, comparable to G. tetrahit. Aerial parts exhibited an intermediate effect, with G. bifida aerial parts showing an IC50 of 8.102 ± 0.49 mg/mL, while G. tetrahit aerial parts demonstrated stronger activity at 1.511 ± 0.11 mg/mL. AChE inhibition activity increased progressively from the roots to aerial parts to leaves, with leaves consistently exhibiting the strongest inhibitory effects across all Galeopsis spp. G. tetrahit leaves had the strongest inhibition, with an IC50 of 4.002 ± 0.32 mg/mL, followed by G. speciosa leaves (6.92 ± 0.14 mg/mL) and G. bifida leaves (6.97 ± 0.68 mg/mL). Conclusions: Our study provides a comprehensive analysis of the phenolic acid content, in vitro antioxidant activity, and neuroprotective potential of three Galeopsis spp. (G. bifida, G. speciosa, and G. tetrahit) from the southwestern Romanian flora.