Isolation and characterization of bioactive constituents from <i>Withania coagulans</i> Dunal with antioxidant and multifunctional enzyme inhibition potential, supported by docking, MD, and DFT studies

<i>Withania coagulans</i> Dunal is a medicinal plant with potential therapeutic applications in neurodegenerative and metabolic disorders. This study aimed to isolate, structurally characterize, and evaluate the biological potential of withanolide-type compounds from <i>Withania coagulans</i> Dunal, with a focus on their antioxidant and enzyme inhibitory activities relevant to neurodegenerative and metabolic disorders. Two new withanolides (WTH1 and WTH2) and one known compound (WTH3) were isolated from <i>W. coagulans</i>. The methanol extract and its fractions were assessed for total phenolic and flavonoid contents, antioxidant capacity (DPPH, FRAP, phosphomolybdenum assays), and enzyme inhibitory activities against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), lipoxygenase, α-glucosidase, and tyrosinase. The methanol extract exhibited the highest total phenolic (71.53 mg GAE per g) and flavonoid (64.32 mg QE per g) contents, correlating with strong antioxidant activity (DPPH: 91.2%, FRAP: 678.3 µmol Fe²⁺ per g, phosphomolybdenum: 4.2 mmol TE per g). It showed significant inhibitory effects against AChE (4.10 mg GALAE per g), BChE (3.71 mg GALAE per g), lipoxygenase, α-glucosidase, and tyrosinase, with the ethyl acetate fraction displaying the strongest α-glucosidase inhibition (3.51 mmol ACAE per g). <i>In silico</i> docking revealed strong binding affinities of WTH1 and WTH3 toward AChE (-11.616 and -11.438 kcal mol^-1, respectively), while WTH3 also interacted effectively with BChE (-9.30 kcal mol^-1), surpassing the standard drug physostigmine (-5.85 kcal mol^-1). Pharmacokinetic evaluation of WTH1 predicted high gastrointestinal absorption (97.65%), moderate oral bioavailability (0.55), and absence of hepatotoxicity or AMES toxicity. DFT analysis indicated a stable HOMO-LUMO energy gap (9.923 eV), and binding free energy calculations confirmed strong interaction of WTH1 with AChE using PB (-29.731 kcal mol^-1) and GB (-43.54 kcal mol^-1) methods, outperforming the reference drug (-15.08 kcal mol^-1). The findings demonstrate that <i>W. coagulans</i> methanol extract, particularly the isolated new withanolide WTH1, exhibits potent antioxidant and enzyme inhibitory activities with promising pharmacokinetic properties. These results support further pharmacological and clinical evaluation of <i>W. coagulans</i> as a natural source of therapeutic agents against neurodegenerative and metabolic disorders.