EGCG NEUROPROTECTION AGAINST MPP⁺-INDUCED DOPAMINERGIC INJURY IS REAL BUT PARTLY ARTIFACTUAL: A FOUR-CONDITION MEDIA DESIGN FOR CONCENTRATION-RESOLVED CORRECTION

Abstract

(−)-Epigallocatechin-3-gallate (EGCG) auto-oxidises in standard bicarbonate-buffered culture media, generating hydrogen peroxide as a by-product — a confound documented in the methodological literature for over two decades but rarely quantified in disease-relevant cellular neuroprotection assays. We addressed this directly in a dopaminergic model of Parkinson's disease. Differentiated SH-SY5Y cells were challenged with MPP⁺ (500 µM, calibrated against a half-maximal effective concentration of 320 µM) and treated with EGCG across a concentration ladder (0.1–100 µM) under four media conditions crossing the presence or absence of sodium pyruvate with the presence or absence of exogenous catalase. Catalase, which destroys extracellular hydrogen peroxide but cannot cross the cell membrane, removed 51.3% of EGCG's apparent protection at 10 µM, 38.0% at 25 µM, 21.3% at 50 µM, and 9.6% at 100 µM — a gradient that declined steadily as EGCG concentration rose. In the artifact-controlled condition (pyruvate-free medium with catalase), genuine protection still reached 79.2% viability at 50 µM, confirming that a real cellular effect persists once the peroxide confound is removed. These results indicate that roughly half of the apparent neuroprotection recorded for EGCG at low micromolar concentrations in standard culture medium reflects medium chemistry rather than cellular pharmacology, while protection at higher concentrations is far less affected by this artifact. We propose the four-condition design used here as a practical, low-cost addition to cellular polyphenol-protection assays, and report a per-concentration discount factor that can be applied in re-reading protection magnitudes from the existing EGCG-Parkinson's literature, much of which was generated without any control for this confound.