The biflavonoid extraction from ginkgo (Ginkgo biloba L.) leaves using solvent-based extraction with 70% ethanol, alone and in combination with enzyme-assisted, ultrasound-assisted, mechanical-assisted, and chemically assisted methods was investigated and the influence of extraction duration was explored. The total content of polyphenols, flavonoids, and phenolic acids in the extracts was determined spectrophotometrically, while individual biflavonoids were identified and quantified using HPLC-DAD. Amentoflavone, bilobetin, ginkgetin, isoginkgetin, and sciadopitysin were identified in all our extracts. Among these, sciadopitysin emerged as the most prevalent biflavonoid with an amount above 1 mg g−1 dw, followed by isoginkgetin. Comparative analysis of the extraction methods revealed that, except for chemically assisted extraction, similar levels of compounds were obtained after 45 min of extraction. However, enzymatic (EAE) and mechanical-assisted extraction (MAE) exhibited significantly higher individual (EAE: 19–41% higher; MAE: 22–67% higher) and total biflavonoid (EAE: 29% higher; MAE 50% higher) levels after just 5 min, suggesting their potential to abbreviate extraction duration and facilitate the efficient retrieval of target compounds. However, as extraction efficiency varies between individual biflavonoids, our findings also underscore the importance of considering specific compounds and extraction kinetics in the optimization of ginkgo leaf extraction processes.
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