Molecular Docking, Metabolite Profile, In Vitro Antioxidant, Anticholinergic, Antidiabetic, and Antiglaucoma Properties of Royal Jelly

Background/Objectives: Royal jelly is a biologically active bee product with a complex chemical composition and diverse biological properties. This study aimed to comprehensively characterize the phytochemical composition of royal jelly and evaluate its antioxidant capacity, enzyme-inhibitory abilities, and potential molecular interactions with target enzymes. Methods: The phytochemical profile of royal jelly was characterized using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Total phenolic and flavonoid contents were determined spectrophotometrically. Antioxidant activity was evaluated using DPPH•, ABTS•+, and DMPD•+ scavenging assays, together with Fe3+, CUPRAC, and FRAP reducing methods. Enzyme-inhibitory abilities were assessed against human carbonic anhydrase I and II (hCA I and hCA II), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α-glucosidase, and α-amylase. Molecular docking analyses were subsequently performed to investigate the interactions of major identified compounds with enzyme active sites. Results: LC–MS/MS analysis revealed a diverse profile of phenolic and flavonoids, including quinic acid, caffeic acid, chlorogenic acid, p-coumaric acid, apigenin, luteolin, kaempferol, and amentoflavone. Royal jelly exhibited considerable antioxidant activity, with IC50 values of 29.33, 29.20, and 21.65 μg/mL in the DPPH•, ABTS•+, and DMPD•+ scavenging assays, respectively. It also demonstrated notable reducing capacity in Fe3+ reduction (0.702 μg/mL), CUPRAC (1.444 μg/mL), and FRAP (0.964 μg/mL) assays. Significant inhibitory ability was observed against hCA I (1.12 μg/mL), hCA II (1.33 μg/mL), AChE (1.44 μg/mL), BChE (1.52 μg/mL), α-glucosidase (2.04 μg/mL), and α-amylase (2.11 μg/mL). Docking analyses indicated that major constituents, particularly quinic acid, could interact with key catalytic residues through hydrogen bonding and electrostatic interactions. Conclusions: Royal jelly possesses a rich polyphenolic profile accompanied by substantial antioxidant and multi-target enzyme-inhibitory abilities. These findings provide biochemical and molecular evidence supporting the potential of royal jelly as a functional food and nutraceutical ingredient and highlight its value as a source of bioactive compounds for further pharmacological investigation.