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

<b>Background/Objectives</b><i>Galeopsis</i> spp. (<i>Lamiaceae</i>) 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 <i>Galeopsis</i> spp. (<i>G. bifida</i> Boenn., <i>G. speciosa</i> Mill., and <i>G. tetrahit</i> L.), along with their antioxidant and acetylcholinesterase (AChE) inhibitory potentials. <b>Methods</b>: 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-<i>bis</i>(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. <b>Results</b>: <i>Galeopsis</i> spp. exhibit significant polyphenol accumulation. Chlorogenic acid was the most abundant compound, with the highest levels detected in <i>G. tetrahit</i> leaves (22,347.907 ± 1117.395 μg/g), followed by <i>G. tetrahit</i> aerial parts (11,678.509 ± 583.925 μg/g) and <i>G. speciosa</i> leaves (8712.628 ± 435.631 μg/g). <i>G. tetrahit</i> 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 <i>G. speciosa</i> leaves also displaying a low IC₅₀ value of 0.789 ± 0.03 mg/mL, comparable to <i>G. tetrahit</i>. Aerial parts exhibited an intermediate effect, with <i>G. bifida</i> aerial parts showing an IC₅₀ of 8.102 ± 0.49 mg/mL, while <i>G. tetrahit</i> 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 <i>Galeopsis</i> spp. <i>G. tetrahit</i> leaves had the strongest inhibition, with an IC₅₀ of 4.002 ± 0.32 mg/mL, followed by <i>G. speciosa</i> leaves (6.92 ± 0.14 mg/mL) and <i>G. bifida</i> leaves (6.97 ± 0.68 mg/mL). <b>Conclusions</b>: Our study provides a comprehensive analysis of the phenolic acid content, in vitro antioxidant activity, and neuroprotective potential of three <i>Galeopsis</i> spp. (<i>G. bifida</i>, <i>G. speciosa</i>, and <i>G. tetrahit</i>) from the southwestern Romanian flora.