UPLC-MS/MS-based metabolomics and chemometrics unveil metabolic patterns and their regulatory pathways over the course of pumpkin seeds (Cucurbita maxima) germination

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While pumpkin seeds (Cucurbita maxima) are regarded as agro-industrial waste, they serve as powerhouses packed with a plethora of valuable compounds with potential nutraceutical significance. The present work offers an integrated view of metabolites dynamics and their regulatory pathways over the course of pumpkin seeds germination and their potential entanglements on Alzheimerʼs disease (AD) mitigation through Ultra Performance Liquid Chromatography-mass spectrometry (UPLC-MS/MS) analysis and chemometrics. A total of 76 compounds spanning amino acids, phenolic acid, flavonoids, lignans, vitamins, fatty acids, phospholipids and phytosterols were annotated revealing changing trends during germination process. Coincidentally, orthogonal projection to latent structures-discriminant analysis (OPLS-DA) demonstrated a clear separation trend among the five pumpkin samples assuring their chemical heterogeneity. Complementarily, OPLS-DA models were constructed in different timepoint comparisons to pick out the significant metabolic features which were subsequently imported into MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/) software for metabolic pathway enrichment analysis. Enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed biosynthesis of unsaturated fatty acids, phenylpropanoid biosynthesis, glycerophospholipid metabolism and phenylalanine, tyrosine and tryptophan biosynthesis as the top predictive metabolic pathways associated with significant phytochemical shifts during germination process. Importantly, our efficacy experimental results dissected that 5 d and 7 d sprouted samples exerted noteworthy inhibitory potentials against acetylcholine esterase (AChE) and monoamine oxidase (MAO-B) enzymes. OPLS-derived coefficients plots portrayed that docosahexaenoic acid, oleic acid, lirioresinol A and tocotrienols dramatically augmented during germination were probably ascribed to promising AChE and MAO-B inhibitory activities. These findings offer a turning point for using pumpkin sprouts with an appreciable biochemical content to pursue the concept of nutritional therapy and accelerate perpetual influx of novel bioproducts to rectify AD.