Amination of Epigallocatechin Gallate and Dihydromyricetin in a Caenorhabditis elegans Culture Medium Causes Experimental Artifacts by Reducing the Free Amino Acid Content.

Abstract

Pyrogallol-type polyphenols undergo Strecker degradation with amino acids in cell culture media, yet whether such chemistry measurably alters the nutritional composition of Caenorhabditis elegans growth medium and affects worm phenotypes remains unclear. Using a tert-butyldimethylsilyl derivatization-based GC-MS workflow, we analyzed five flavonoids with distinct B-ring hydroxylation patterns in the nematode growth medium (NGM): epigallocatechin gallate (EGCG), dihydromyricetin (DHM), quercetin, luteolin, and apigenin. Within 2 h, 400 μM EGCG and DHM reduced multiple amino acids in NGM, with concurrent formation of the aminated derivatives N-EGCG and N-DHM identified by LC-MS/MS. This effect persisted over 72 h in OP50-seeded NGM and propagated to worms, lowering endogenous amino acids together with lactate and pyruvate, and ultimately delaying growth, development, and reproductive capacity. These findings highlight that polyphenol bioactivity studies should not overlook chemical reactions between polyphenols and medium constituents to avoid erroneous conclusions.