NMR and HPLC-Q-Orbitrap-MS/MS-based profiling of pea (Pisum sativum L.) seedlings reveals secondary metabolites with in vitro anti-muscle atrophy activity

Pea ( Pisum sativum L.) seedlings are primarily consumed in Japan and China, and some studies have reported their metabolites and physiological activities. However, research into the structural and quantitative analysis of compounds in pea seedlings remains limited. Here, we performed a comprehensive metabolite profiling of pea seedlings and identified 14 major metabolites; due to the lack of comprehensive research linking the overall compound profile to physiological activity, pea seedling metabolites were isolated and purified using column chromatography techniques. Further, we conducted their structural characterization through nuclear magnetic resonance spectroscopy and high-resolution Orbitrap mass spectrometry and evaluated their effects on sarcopenia. Among the identified compounds, glycosylated flavonols, such as quercetin 3- O -sophorotrioside and kaempferol 3- O -sophorotrioside, and flavonoids conjugated with phenylpropanoid compounds, such as p -coumaric and sinapic acid, were predominant. Notably, a novel compound was discovered: kaempferol 3- O -sophorotrioside bearing a cis - p -coumaric acid moiety. Additionally, three soyasaponins and two phenolic compounds were identified. In in vitro experiments using C2C12 cells, pea seedling extract and six isolated flavonol compounds were found to inhibit dexamethasone-induced muscle atrophy. Furthermore, pea seedling extract ameliorated sarcopenia by inhibiting the muscle-specific ubiquitin ligases, atrogin-1 and MuRF-1, while activating the mTOR/AKT signaling pathway. This study presents comprehensive evidence from the metabolite analysis of pea seedlings and sarcopenia-ameliorating effects of both the extract and its compounds, thereby demonstrating the health-functional added value of pea seedlings. These results highlight the potential of pea seedlings as a functional food ingredient. • LC-Q-Orbitrap-MS/MS and NMR profiling revealed 14 metabolites in pea seedlings • A novel kaempferol glycoside with cis-p-coumaric acid was structurally identified • Pea seedling extract reduced dexamethasone-induced C2C12 muscle atrophy in vitro • Muscle protection was linked to AKT/mTOR activation and Atrogin-1/MuRF-1 inhibition • Six identified flavonol glycosides contributed to myotube recovery