Biflavonoids are an important group of flavonoids found in Juniperus species, yet their distribution and accumulation patterns remain insufficiently explored. In this study, we applied a method for the simultaneous quantification of seven biflavonoids to analyze different plant parts of J. communis, J. communis subsp. nana, and J. oxycedrus. In order to determinate the influence of growing location, we also analyzed J. communis samples collected from different locations. Four biflavonoids—cupressuflavone, amentoflavone, bilobetin, and hinokiflavone—were detected. In both analyzed J. communis varieties, amentoflavone was the predominant biflavonoid in cones and needles, while in J. oxycedrus, cupressuflavone was the most abundant in cones, with amentoflavone dominating in needles. Overall, biflavonoid content was significantly higher in needles than in cones, with total biflavonoid levels in needles exceeding 5 mg/g dw, highlighting the tissue-specific nature of biflavonoid biosynthesis within Juniperus species. Additionally, our results suggest that in J. communis, biflavonoid accumulation is significantly influenced by growing location.

Biflavonoids are dimeric flavonoids recognized for their diverse biological activities and significant pharmacological potential, with ginkgo (Ginkgo biloba L.) serving as a primary natural source. This study presents a comprehensive spatiotemporal characterization of the biflavonoid profile across a diverse population of 90 trees. High-resolution chromatographic analysis quantified five major biflavonoids, revealing a consistent hierarchical abundance: sciadopitysin > isoginkgetin > ginkgetin > bilobetin > amentoflavone. Notably, sciadopitysin emerged as the predominant constituent (1532.89 ± 544.13 µg/g dw). To decode the complex drivers of metabolite accumulation, we integrated Principal Component Analysis (PCA) with Piecewise Linear Regression (PLR). PCA confirmed a robust chemical structure, explaining 71.5% of the total variance, where Factor 1 represents a general biflavonoid gradient and Factor 2 captures localized environmental influences. The PLR models (R² = 0.75–0.83) identified tree age as a primary negative regulator, showing a significant decline in total biflavonoids as trees mature beyond the 30-year reproductive threshold. While sexual dimorphism and location exhibited compound-specific nonlinear effects, younger trees (10–30 years) demonstrated the highest biosynthetic plasticity and potency. These findings establish a predictive framework for optimizing the pharmaceutical harvest of ginkgo leaves, highlighting that age-related physiological shifts, rather than gender or broad geography, are the critical determinants of biflavonoids yield.

Biflavonoids, or flavonoid dimers, are characteristic phytochemicals of ginkgo associated with various biological activities, yet they remain far less studied than monomeric flavonoids. For their effective industrial application, optimization of extraction conditions is essential. This study investigated the effect of ethanol–water ratio (0, 10, 30, 50, 70, and 96% ethanol) on the extraction efficiency of major ginkgo biflavonoids (amentoflavone, bilobetin, ginkgetin, isoginkgetin, and sciadopitysin). Three ginkgo tissue types, green leaves, yellow leaves, and sarcotesta, previously reported to accumulate biflavonoids, were analyzed. Biflavonoids were quantified by HPLC-DAD, and total polyphenol content and antioxidant activity were also determined. Biflavonoids were most abundant in yellow leaves, with sciadopitysin identified as the dominant compound. No biflavonoids were detected in water or 10% ethanol extracts, while 30% ethanol extracts contained detectable biflavonoids only in yellow leaves at low concentrations. For most biflavonoids, the highest concentrations were obtained using 70% and 96% ethanol. Considering comparable extraction efficiency and lower toxicity, 70% ethanol was identified as the most suitable solvent. These findings highlight the importance of tissue type and solvent composition for efficient biflavonoid extraction from ginkgo.