Reduction of Ginkgotoxin and Ginkgolic Acids in Ginkgo biloba Seed Extracts Using a Multistep Liquid–Liquid Extraction Approach

Ginkgo biloba seeds are a rich source of flavonoids and the unique terpene lactones—ginkgolides and bilobalide, known for their neuroprotective and cognitive-improving effects. However, unlike the widely used leaves, the seeds contain substantial levels of ginkgolic acid and ginkgotoxin (4′-O-methylpyridoxine), an antivitamin B6 compound. At high concentrations, ginkgotoxin exhibits neurotoxicity, potentially inducing seizures, respiratory distress, and loss of consciousness, thus limiting the safe application of Ginkgo seed-derived products. This study aimed to develop a simple yet effective extraction protocol that reduces ginkgotoxin levels in Ginkgo biloba seed extracts while preserving their beneficial phytochemicals. A multistep liquid–liquid extraction approach employing sequential polar and non-polar solvents was implemented. Following each extraction stage, fractions were analyzed using ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC–MS). The concentrations of flavonoids, ginkgolides, bilobalide, ginkgolic acid, and ginkgotoxin were quantified to evaluate detoxification efficiency and phytochemical retention. Compared with conventional single-step extraction using 70% methanol, this multistep protocol markedly reduced ginkgotoxin and ginkgolic acid to near-undetectable levels, while preserving detectable concentrations of major flavonoids and terpene trilactones. The findings demonstrate that multistep extraction represents a promising and practical strategy for minimizing ginkgotoxin in Ginkgo biloba seed extracts without compromising their beneficial phytochemical composition. This approach provides a sound basis for developing safer, functionally active Ginkgo-based products.