Phytonutrient profiling of spinach leaves using LC-ESI-QTOF/MS: Linking polar and non-polar extracts to enzyme inhibitory properties

The study investigated the chemical composition and bioactivity of spinach leaves, focusing on two-phase extraction using 15 different solvents to obtain lipophilic and hydrophilic fractions. The extracts were analyzed for tocopherols, pigments (xanthophylls, carotenoids, chlorophylls, and pheophytins), color parameters (CIELab), and ORAC. In vitro enzymatic activity was assessed for anticholinergic (AChE, BuChE), anti-inflammatory (15-LOX), and antihypertensive (ACE) effects. Notably, this study is the first to report ACE inhibition in polar spinach extracts. Additionally, significant potential for cholinesterase inhibition was revealed. In the residual material after pre-extraction, phenolic compounds were analyzed by LC-MS, along with procyanidin polymers. Free amino acid content was also determined. The residual extract exhibited significant ACE inhibitory activity, reinforcing its potential for antihypertensive applications. These findings highlight the untapped bioactive potential of spinach, especially in cholinesterase inhibition and antihypertensive activity. By selecting the appropriate solvent, the composition of the extract can be tailored through fractional extraction, thereby influencing its potential health benefits and creating opportunities for further targeted applications.