# Biflavonoid Profiling of _Juniperus_ Species: The Influence of Plant Part and Growing Location
## 1 Introduction  
The _Juniperus_ genus, belonging to the Cupressaceae family, comprises evergreen, aromatic shrubs or trees predominantly found across the cold and temperate zones of the Northern Hemisphere, with certain species reaching as far south as tropical regions of Africa [1]. This genus includes approximately 75 species, although the precise number remains a topic of debate among taxonomists due to differing classification criteria [2]. Among these, _Juniperus communis_ L. (Figure 1), commonly referred to as juniper, is the most recognized and widely utilized species. Although commonly referred to as \"juniper berries,\" [3, 4]_Juniperus_ species do not produce true berries or fruits. Instead, they bear seed cones, which have a fleshy, berry-like appearance due to their unique structure [5]. Unlike the woody cones of many other conifers, juniper cones consist of fused, fleshy scales that enclose the seeds, giving them a soft, round shape that resembles a berry [5]. _Juniperus_ sp. are rich in bioactive compounds, including aromatic oils, invert sugars, resins, catechins, organic and terpenic acids, flavonoids, tannins, and alkaloids [6]. Traditionally used as a remedy for diuretic, anti-arthritis, anti-diabetes, and gastrointestinal issues, its essential oils and extracts exhibit antioxidant, antibacterial, antiviral, antifungal, anti-inflammatory, cytotoxic, hypoglycemic, and hypolipidemic properties [6; 7]. Essential oils have also found applications in the food industry in preserving meat quality and extending shelf life by reducing lipid peroxidation [1]. Plants from the _Juniperus_ genus, especially _J. communis_, are renowned for their aromatic essential oils, which have been extensively studied for their antimicrobial properties. In addition to essential oils, _Juniperus_ spp. contain various bioactive compounds, such as diterpenes, lignans, and flavonoids, that may further enhance the phytotherapeutic potential of their berries [8]. Specific for _Juniperus_ spp. is that they accumulate biflavonoids, dimeric form of flavonoids [8]. Biflavonoids are a unique subclass of polyphenolic compounds composed of two flavonoid units connected by a covalent bond [9]. Their precise distribution across the plant kingdom remains insufficiently documented, and they are generally less studied than their monomeric counterparts [10]. Despite this, biflavonoids exhibit a wide range of biological activities, including anti-inflammatory, antiviral, and cytotoxic effects, contributing to the therapeutic properties of various medicinal plants [11]. Notable sources of biflavonoids include medicinal plants such as _Ginkgo biloba_ L., _Hypericum_ species (commonly known as St. John's word), _Juniperus_ spp., etc. [10]. In _Juniperus_ spp., biflavonoids such as cupressoflavone, amentoflavone, hypoxiflavone, and other biflavones and methylbiflavones have been detected [3; 4], but their identification and quantification remain challenging. In a large quantity of the phytochemical studies related to _Juniperus_ spp., biflavonoids were not reported at all [12; 13; 14; 15; 16; 17; 18]. This is largely due to the limited commercial availability and high cost of biflavonoid standards, which restrict comprehensive phytochemical analyses. Consequently, there is relatively limited literature detailing the precise profile and concentrations of biflavonoids in _Juniperus_ spp. compared to other polyphenols. Furthermore, factors such as species-specific variations, growing  
Figure 1: Juniperus communis L. shrub at location Konjic in Bosnia and Herzegovina. location, and differences between plant parts and their influence on the biflavonoid profile have not been thoroughly investigated, to the best of our knowledge. To address this knowledge gap, we investigated the biflavonoid profiles of ripe and unripe cones and needles from _J. communis_, _Juniperus communis_ subsp. _nana_ [PERSON], and _J. oxycedrus_. To evaluate the influence of growing location on the biflavonoid composition, we also collected _J.