# Biflavonoid Profiling of _Juniperus_ Species: The Influence of Plant Part and Growing Location
## 4 Discussion  
In the analyzed _Juniperus_ species, we detected and quantified cupressuflavone, amentoflavone, bilobetin, and hinokiflavone, which aligns with previously reported juniperus biflavonoids in the literature. [PERSON] et al. [3] identified in _J. communis_ six biflavonoids--amentoflavone, hinokiflavone, cupressuflavone, and three unidentified methylated biflavonoids. Similarly, [PERSON] et al. [4] detected cupressuflavone, amentoflavone, bilobetin, and hinokiflavone in _J. communis_ var. _communis_ (Jcc) and _J. communis_ var. _saxatitis_ Pall. cones, while also annotating one compound as a biflavonoid and two as methylated biflavonoids. [PERSON] et al. [22] in _J. oxycedrus_ reported cupressuflavone, amentoflavone, two additional biflavones, and methyl-biflavone. In all these studies, the authors did not use commercially available standards for biflavonoid quantification. They relied solely on amentoflavone as a reference compound, making accurate quantification challenging. We utilized commercially available standards and successfully identified bilobetin--particularly in green cones--which was likely annotated as a methylated biflavonoid in previous studies. Bilobetin was present in the green cones of both _J. communis_ varieties but was absent in _J. oxycedrus_ samples. In ripe cones, bilobetin was detected only at a few locations, suggesting its levels may decrease during cone ripening. While [PERSON] et al. [3] reported the presence of methylated biflavonoids in unripe but not in ripe _Juniperus_ berries, they did not identify bilobetin. Given their lack of a bilobetin standard, it remains unclear whether the detected methylated biflavonoid corresponds to bilobetin. In our study the ratio and content of cupressuflavone and amentoflavone in unripe and ripe cones _Juniperus_ species remain comparable, while we detected hinokiflavone in unripe green cones but not in ripe cones of _J. oxycedrus_. A recent review article on phytochemicals in _J. oxycedrus_[23] lists hinokiflavone, cupressuflavone, and amentoflavone as characteristic biflavonoids in berries and needles, which aligns with our findings. We analyzed needle samples from _J. communis_ and _J. oxycedrus_ and generally detected significantly higher concentrations of biflavonoids compared to cones. In _J. communis_, amentoflavone was the most abundant biflavonoid, followed by cupressuflavone and hinokiflavone, while bilobetin was detected in only a few samples. Similarly, in _J. oxycedrus_, amentoflavone was the predominant biflavonoid, followed by cupressuflavone and hinokiflavone, whereas bilobetin was not detected. Our findings also indicate that _J. oxycedrus_ contains higher levels of cupressuflavone compared to _J. communis_, which is consistent with the study by [PERSON] et al. [24], who compared polyphenolic compounds in needle extracts of five _Juniperrus_ species and also identified amentoflavone as the most abundant biflavonoid. However, literature data are not entirely consistent, as [PERSON] et al. [22] reported cupressuflavone as the predominant biflavonoid in _J. oxycedrus_ subsp. _macrocarus_ (Sm.) [PERSON]. As evident from our results, and consistent with the literature, the biflavonoid profile in _Juniperrus_ species differs from that in other plants. To date, ginkgo has been the most extensively studied, with five predominant biflavonoids identified: amentoflavone, ginkgetin, isoginkgetin, bilobetin, and sciadopitysin [25; 26]. In ginkgo leaves, for example, amentoflavone is the least abundant biflavonoid, typically present at around 30-70 \(\upmu\)g/g dry weight (dw) [25]. In contrast, our research shows that in _Juniperrus_ species, the concentration of amentoflavone is much higher--reaching up to approximately 3000 \(\upmu\)g/g dw in the needles of _J. oxycedrus_. This suggests that Juniperrus needles could serve as a valuable natural source of amentoflavone for potential medicinal applications.