Characterization of Phenolic Profiles Using UPLC-Q-TOF-MS/MS and NMR in the Biofunctional Fraction of Korean Winter Spinach (Spinacia oleracea L.) Leaves: Evaluation of Major Phenolics and Their Bioactivities Under Optimized Extraction Conditions

초록

This study is the first to demonstrate fluctuations in major phenolics and biofunctional properties under various extraction conditions of Korean winter spinach (Allseason cultivar) leaves. In contrast to earlier reports on summer- or greenhouse-cultivated spinach, which mainly relied on HPLC-DAD or LC-MS profiling and one or two bioactivity assays, the present work combines UPLC-Q-TOF-MS/MS with NMR-based structural confirmation of three major flavone glucuronides (11-13) and integrates five complementary bioactivity assays (DPPH, ABTS, FRAP, DNA protection, and tyrosinase inhibition) within a single optimization framework. A 50% methanol extract yielded twelve phenolics (patuletin, spinacetin, spinatoside, jaceidin, and methylenedioxyflavone-glucuronide derivatives) elucidated by UPLC-Q-TOF-MS/MS, with the isolated major phenolics 11-13 further verified by NMR. Total phenols and total flavonoids of biofunctional characteristics varied significantly depending on the solvent system. The optimal extraction conditions (50% methanol, 72 h, 35 degrees C) resulted in the highest phenolic levels of phenolics 11-13 (total: similar to 6.5 mg/g) and bioactivities (DNA protection > ABTS > tyrosinase inhibition > FRAP > DPPH, at 500 mu g/mL). PCA and hierarchical clustering distinguished extraction profiles, with 50-70% methanol extracts forming clear clusters. Among the isolated phenolics, phenolic 12 showed the strongest antioxidant activity (DPPH IC50 = 57.6 mu M; ABTS IC50 = 21.9 mu M). These findings suggest that spinach leaves are a valuable source of bioactive phenolics for nutraceutical applications under optimized extraction conditions.

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