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Article

Biflavonoid Profiling of Juniperus Species: The Influence of Plant Part and Growing Location

by
Barbara Medvedec
1,†,
Iva Jurčević Šangut
1,†,
Armin Macanović
2,
Erna Karalija
3 and
Dunja Šamec
1,*
1
Department of Food Technology, University North, Trg. Dr. Žarka Dolinara 1, 48 000 Koprivnica, Croatia
2
Center for Ecology and Natural Resources “Academician Sulejman Redžić”, Department of Biology, Faculty of Science, University of Sarajevo, 71000 Sarajevo, Bosnia and Herzegovina
3
Department for Biology, Faculty of Science, University of Sarajevo, 71000 Sarajevo, Bosnia and Herzegovina
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Appl. Sci. 2025, 15(13), 7082; https://doi.org/10.3390/app15137082 (registering DOI)
Submission received: 29 April 2025 / Revised: 10 June 2025 / Accepted: 17 June 2025 / Published: 24 June 2025
(This article belongs to the Special Issue Advances in Phytochemicals: Extraction, Isolation, and Identification)
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Versions Notes

Abstract

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.

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 wort), Juniperus spp., etc. [10].
In Juniperus spp., biflavonoids such as cupressoflavone, amentoflavone, hynokiflavone, 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 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 Syme, and J. oxycedrus. To evaluate the influence of growing location on the biflavonoid composition, we also collected J. communis samples from 10 different locations. For the identification and quantification of biflavonoids, we employed the HPLC-DAD method, which simultaneously separates and detects seven biflavonoids: cupressuflavone, amentoflavone, bilobetin, ginkgetin, isoginkgetin, hinokiflavone, and sciadopitysin.

2. Materials and Methods

2.1. Plant Material

Plant material was collected during July of 2023 at locations in Bosnia and Herzegovina, as shown in Figure 2. A detailed list of the locations and geolocations is presented in Supplementary Material Table S1. The altitude at the investigated sites ranges from 352 to 1607 m.
Juniper cones do not ripen simultaneously, due to the plant’s extended and irregular reproductive cycle. Juniper species typically produce cones that take one to three years to mature, depending on the species and environmental conditions [19], resulting in the presence of ripe and unripe cones on the same plant (Figure 3). We collected separately ripe cones, unripe cones, and needles from each plant. Species were determined based on morphological species markers according to the relevant keys for determination of plant species by Armin Macanovic. After harvesting, the plant material was transferred to the laboratory and air-dried, and a voucher specimen was deposited at the University of Sarajevo.

2.2. Extraction

The plant material was air-dried and subsequently homogenized into fine powder using Omni Bead Ruptor (OMNI International, Kennesaw, GA, USA). According to our previous study, drying method does not influence biflavonoid content [20]. For plant extract preparation, 30 mg of dried plant material in three replicates was weighed (Adam Equipment, Maidstone, UK), 1 mL of 70% ethanol was added and immediately vortexed (V-1 plus, Biosan, Riga, Latvia). After incubation in an ultrasonic bath for 10 min (DU-100, Argo Lab, Carpi, Italy), samples were transferred to a centrifuge for 45 min (Bio RS-24, Biosan, Riga, Latvia). After 5 min centrifugation of the extract at 3800 rpm (LMC-4200R, Biosan, Riga, Latvia), supernatants were filtered through hydrophobic PTFE syringe filters (13 mm/0.4 µm) for HPLC preparation.

2.3. Biflavonoid Profiling

A total of seven biflavonoids were included in the analysis. Amentoflavone, bilobetin, ginkgetin, isoginkgetin, and sciadopitysin were obtained from PhytoLab (Vestenbergsgreuth, Germany), cupressuflavone from Extrasynthese (Genay, France), and hinokiflavone from Sigma-Aldrich (Darmstadt, Germany). All reference standards were of HPLC grade purity. Standard stock solutions of seven biflavonoids were prepared individually in DMSO at a concentration of 1000 µg/mL. Four working solutions were prepared for each standard (concentrations 1.0, 10, 50, and 100 µg/mL) and were injected into an Agilent 1260 Infinity II high-performance liquid chromatography system (Agilent, Santa Clara, CA, USA) with a diode array detector (DAD). Separation of all seven biflavonoids was performed on a Zorbax 300Extend-C18 column (Agilent, Santa Clara, CA, USA) maintained at 40 °C, and was successfully achieved under the following conditions: mobile phase A–0.1% formic acid solution and acetonitrile as mobile phase B, with a flow rate of 1 mL/min. The gradient elution profile was set as follows: 0 min–98% A, 10 min–79% A, 15 min–77% A, 20 min–75% A, 25 min–64% A, 30 min–62% A, 35 min–51% A, 40 min–25% A, 43 min–8% A, and 45 min–98% A. The injection volume for both samples and standards was 10 µL. Data acquisition and subsequent processing were performed using Agilent OpenLAB CDS software (version 2.6, Agilent, Santa Clara, CA, USA). Biflavone identification was performed by comparing the sample spectra with those of the reference standards. Chromatograms were recorded at 330 nm. The concentrations of 3′–8″ biflavones in the samples were determined using standard calibration curves (Table S2) and expressed in µg/mg of dry weight (dw). The chromatogram of all seven biflavonoids at 330 nm is presented in Figure 4. Representative chromathograms for analyzed samples are presented in Supplementary Material Figures S1 and S2.

2.4. Statistical Analysis

For analysis, various parts of Juniperus species were collected from different locations across Bosnia and Herzegovina. After extraction, the samples were analyzed using HPLC-DAD in three replicates. The results are presented as the mean value ± standard deviation (SD). To present the data by species, results for the same species from different locations are averaged. Statistical analysis was performed using the free software PAST (version 4.15) [21]. A one-way ANOVA was applied, followed by a post hoc Tukey’s HSD test, with statistical significance set at p < 0.05.

3. Results

We applied a method for the simultaneous separation, quantification, and identification of seven biflavonoids (Figure 4) in Juniperus species samples. In Juniperus species, ginkgetin, isoginkgetin, and sciadopitysin were not detected, while cupressuflavone, amentoflavone, bilobetin, and hinokiflavone (Figure 5) were present in varying amounts depending on the geographical origin and plant part analyzed. Representative chromatograms of ripe and unripe cones from all species are shown in Supplementary Figure S2.

3.1. Species-Specific and Organ-Specific Biflavonoid Accumulation

In unripe cones of J. communis and J. communis subsp. nana, we detected cupressuflavone, amentoflavone, bilobetin, and hinokiflavone (Figure 6). In contrast, unripe cones of J. oxycedrus contained cupressuflavone, amentoflavone, and hinokiflavone. The most abundant biflavonoid in J. communis subsp. nana was amentoflavone (709.90 ± 93.23 µg/g dw), while in J. communis, amentoflavone was present at much lower concentrations, comparable to bilobetin. In J. oxycedrus unripe cones, cupressuflavone was the most abundant biflavonoid.
The biflavonoid profiles of ripe cones were similar to those of unripe cones, with some notable differences (Figure 7). Bilobetin was not detected in J. communis subsp. nana and was present only in small amounts in J. communis, detected in some locations but not consistently across all samples. Additionally, in J. oxycedrus ripe cones, hinokiflavone was absent, despite being present in unripe cones.
We also analyzed needle samples of J. communis and J. oxycedrus, with their biflavonoid profiles presented in Figure 8. Cupressuflavone, amentoflavone, and hinokiflavone were detected in the needles of both species, while bilobetin was found only in small quantities and only in certain J. communis locations. The biflavonoid content in needles was significantly higher than in both ripe and unripe cones. In both species, the most abundant biflavonoid was amentoflavone, with concentrations of 2965.49 ± 437.62 µg/g dw in J. communis and 3014.88 ± 68.06 µg/g dw in J. oxycedrus. In J. communis needles, as in the cones, amentoflavone was the most abundant biflavonoid, followed by cupressuflavone, hinokiflavone, and bilobetin. In contrast, while cupressuflavone was the most abundant biflavonoid in both ripe and unripe cones of J. oxycedrus, the needles of this species contained the highest levels of amentoflavone, followed by cupressuflavone and hinokiflavone.
Figure 9 presents the total biflavonoid content in different plant parts across the three analyzed species. As evident from the results, the needles contain the highest biflavonoid levels, with 5125.22 ± 132.43 µg/g in J. communis and 5578.60 ± 115.45 µg/g in J. oxycedrus. In J. communis, this represents a 6.6-fold and 12-fold increase compared to unripe and ripe cones, respectively. Similarly, in J. oxycedrus, the needles contain 5.3 times more biflavonoids than unripe cones and 41.1 times more than ripe cones. In both J. communis and J. oxycedrus, unripe cones generally had higher biflavonoid levels than ripe cones, whereas in J. communis subsp. nana, the biflavonoid content was comparable between unripe and ripe cones.
To explore the relationships among Juniperus species based on their biflavonoid content, the data were analyzed using principal component analysis (PCA). The distribution of Juniperus species and plant parts, as well as the associations among the measured biflavonoids, are illustrated in the PCA biplots shown in Figure 10. The first two principal components (F1 and F2) accounted for over 99% of the total variance observed in the biflavonoid profiles (see also Supplementary Material). The clear separation of J. communis and J. oxycedrus needle samples on the right side of the PCA plot indicates their higher biflavonoid content, which is also evident in Figure 8 and Figure 9.

3.2. Geography-Dependent Biflavonoid Accumulation in J. communis

Samples of J. communis we collected at different locations and the results for biflavonoid profile are presented in Table 1.
Cupressuflavone and amentoflavone were detected in samples from all locations. Bilobetin was present in all unripe (green) cone samples but was only found in ripe cones from three locations (Proskok, Igman, and Trebović) and in four out of six locations where needle samples were collected. The cupressuflavone content ranged from 4.69 µg/g (Nahorevo ripe cones) to 1987.54 µg/g (Trnovo needles). Amentoflavone concentrations varied between 107.29 µg/g (Visočica ripe cones) and 3486.68 µg/g (Nahorevo needles). Bilobetin levels ranged from undetectable to 388.01 µg/g (Trebović unripe cones), while hinokiflavone was detected in amounts from 0 µg/g (Trebović unripe cones) to 602.58 µg/g (Nahorevo needles). Interestingly, in unripe cones from the Trebović location, we did not detect hinokiflavone. However, these cones contained the highest amounts of cupressuflavone and bilobetin among all unripe cone samples. In needles, the concentrations of cupressuflavone, amentoflavone, and hinokiflavone were significantly higher than in both unripe and ripe cones. This reflects the total biflavonoid content, which ranged from 201.03 µg/g (ripe cones from Konjic) to 6033.24 µg/g (needles from Trnovo). Notably, samples from Konjic had significantly lower levels of all detected biflavonoids compared to other locations, with particularly reduced amounts of amentoflavone and hinokiflavone.
The biflavonoid content of samples collected from different locations was also subjected to principal component analysis (PCA), with the results presented in Figure 10. In this analysis, the first two principal components accounted for 99.7% of the total variance, indicating a strong representation of the data structure. The distinct separation of needle samples on the right side of the PCA plot highlights their significantly different biflavonoid profiles compared to other analyzed plant parts, a trend that is also evident in Table 1. These findings suggest that plant part has a greater influence on biflavonoid accumulation than geographic 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. Innocenti et al. [3] identified in J. communis six biflavonoids—amentoflavone, hinokiflavone, cupressuflavone, and three unidentified methylated biflavonoids. Similarly, Miceli et al. [4] detected cupressuflavone, amentoflavone, bilobetin, and hinokiflavone in J. communis var. communis (Jcc) and J. communis var. saxatilis Pall. cones, while also annotating one compound as a biflavonoid and two as methylated biflavonoids. Taviano 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 Innocenti 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 Miceli et al. [24], who compared polyphenolic compounds in needle extracts of five Juniperus species and also identified amentoflavone as the most abundant biflavonoid. However, literature data are not entirely consistent, as Taviano et al. [22] reported cupressuflavone as the predominant biflavonoid in J. oxycedrus subsp. macrocarpa (Sm.) Ball. As evident from our results, and consistent with the literature, the biflavonoid profile in Juniperus 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 µg/g dry weight (dw) [25]. In contrast, our research shows that in Juniperus species, the concentration of amentoflavone is much higher—reaching up to approximately 3000 µg/g dw in the needles of J. oxycedrus. This suggests that Juniperus needles could serve as a valuable natural source of amentoflavone for potential medicinal applications. Amentoflavone is known for its significant biological activities, including strong anticancer, neuroprotective, antiviral, and anti-inflammatory effects [27].
The total biflavonoid content in both analyzed species, J. communis and J. oxycedrus, exceeded 5 mg/g, which is comparable to the amount we previously reported for yellow ginkgo leaves [25]. In cones, the total biflavonoid content was lower for both J. communis and J. oxycedrus, ranging from 0.13 to 1.3 mg/g dw. A general trend was observed where green cones accumulated more biflavonoids than ripe cones, similar to findings by Innocenti et al. [3] for some J. communis berries. However, this trend was not consistent across all locations. Innocenti et al. [3] reported a total biflavonoid content in J. communis berries ranging from 0.14 to 1.38 mg/g of fresh weight. Our results suggest that biflavonoids are important phytochemicals in Juniperus species, particularly in needles, which should be included in phytochemical studies. However, they have often been neglected or omitted in previous studies.
To investigate how growing location influences biflavonoid accumulation, we collected J. communis samples from various locations. As shown in Table 1, the growing location significantly affected both individual and total biflavonoid accumulation. In some locations, bilobetin was not detected at all, which may be directly related to environmental factors. However, since our samples were collected from wild plants, where environmental conditions were not controlled, it is challenging to determine which specific factors influence biflavonoid accumulation. According to the literature, biflavonoid accumulation is influenced by tissue type, with plant parts directly exposed to the environment containing higher levels, while inner tissues lack detectable biflavonoids, as observed in Ginkgo biloba L. [26]. Popescu et al. [16] investigated how harvesting time and geographic area affect polyphenolic compounds in J. communis cones from Romania. While they did not detect biflavonoids, they found that total polyphenol content was significantly higher in areas with greater altitude and in samples collected in November compared to September and October. Similarly, Rawat et al. [28] compared the total flavonoid content in J. communis berries from four locations in India and reported a significant influence of growing location. To the best of our knowledge, our study is the first to report biflavonoid content in J. communis across different locations, and our findings align with expectations. Although the exact role of biflavonoids in plants remains unclear, their localization within plant tissues and biological activity suggest a potential role in plant–environment interactions [11]. However, to identify the precise environmental factors affecting biflavonoid accumulation, further controlled laboratory experiments are needed.
As evident from the PCA plots in Figure 10, the plant part—particularly needle tissue—emerges as the dominant factor influencing biflavonoid accumulation, surpassing both species identity and geographic origin in its impact. This clear separation of needle samples underscores the tissue-specific nature of biflavonoid biosynthesis within Juniperus species.

5. Conclusions

This study focused on biflavonoids, a group of flavonoids present in Juniperus species. We used a method for the simultaneous quantification of seven biflavonoids and successfully detected four—cupressuflavone, amentoflavone, bilobetin, and hinokiflavone—in samples of J. communis, J. communis subsp nana, and J. oxycedrus. In both analyzed J. communis varieties, amentoflavone was the predominant biflavonoid in ripe and unripe cones, as well as in needles. In J. oxycedrus, cupressuflavone was the most abundant biflavonoid in cones, while amentoflavone dominated in needles. Overall, biflavonoid content was significantly higher in needles compared to cones, with total biflavonoid levels in needles exceeding 5 mg/g of dry weight in both species. Our findings highlight that biflavonoids are especially concentrated in needles, suggesting a potential ecological or physiological role unique to this organ. These results emphasize the importance of tissue-specific metabolic profiles over environmental variation in determining biflavonoid content. Consequently, future phytochemical studies on Juniperus should incorporate biflavonoid profiling, particularly in needle tissues, to ensure a more comprehensive understanding of the species’ chemical composition. This supports the view that biflavonoids are important phytochemicals in Juniperus species. However, the exact factors in addition to organ specific accumulation driving this variation remain unknown. Further research should focus on identifying these factors through controlled studies to better understand the environmental influences on biflavonoid accumulation.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/app15137082/s1: Figure S1: Representative chromatograms recorded at 330 nm for green cones of Juniperus species.; Figure S2: Representative chromatograms recorded at 330 nm for ripe cones of Juniperus species; Table S1: Species, collected plant part and location; Table S2: Retention times (RT) and curve equation for a standards.

Author Contributions

Conceptualization, D.Š.; investigation, B.M. and I.J.Š.; resources, A.M. and E.K.; writing—original draft preparation, D.Š.; writing—review and editing, B.M., I.J.Š., A.M., E.K. and D.Š.; visualization, B.M., I.J.Š., A.M. and D.Š.; supervision, D.Š.; project administration, D.Š.; funding acquisition, D.Š. All authors have read and agreed to the published version of the manuscript.

Funding

This work has been supported by Croatian Science Foundation project “Biflavonoids role in plants: Ginkgo biloba L. as a model system” under project no. UIP-2019-04-1018.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Juniperus communis L. shrub at location Konjic in Bosnia and Herzegovina.
Figure 1. Juniperus communis L. shrub at location Konjic in Bosnia and Herzegovina.
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Figure 2. Map with the locations of sampling.
Figure 2. Map with the locations of sampling.
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Figure 3. Juniperus communis L. ripe and unripe cones and needles.
Figure 3. Juniperus communis L. ripe and unripe cones and needles.
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Figure 4. Biflavonoid standard chromatogram at 330 nm.
Figure 4. Biflavonoid standard chromatogram at 330 nm.
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Figure 5. Structure of the biflavonoids detected in Juniperus sp.
Figure 5. Structure of the biflavonoids detected in Juniperus sp.
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Figure 6. Concentration (µg/g) of biflavonoids in unripe (green) cone of J. communis and J. communis subsp. nana and J. oxycedrus. Values marked with different letters are significantly different (p < 0.05) according to one-way ANOVA followed by Tukey’s HSD post hoc test.
Figure 6. Concentration (µg/g) of biflavonoids in unripe (green) cone of J. communis and J. communis subsp. nana and J. oxycedrus. Values marked with different letters are significantly different (p < 0.05) according to one-way ANOVA followed by Tukey’s HSD post hoc test.
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Figure 7. Concentration (µg/g) of biflavonoids in ripe cone of J. communis and J. communis subsp. nana and J. oxycedrus. Values marked with different letters are significantly different (p < 0.05) according to one-way ANOVA followed by Tukey’s HSD post hoc test.
Figure 7. Concentration (µg/g) of biflavonoids in ripe cone of J. communis and J. communis subsp. nana and J. oxycedrus. Values marked with different letters are significantly different (p < 0.05) according to one-way ANOVA followed by Tukey’s HSD post hoc test.
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Figure 8. Concentration (µg/g) of biflavonoids in needles of J. communis and J. oxycedrus. Values marked with different letters are significantly different (p < 0.05) according to one-way ANOVA followed by Tukey’s HSD post hoc test.
Figure 8. Concentration (µg/g) of biflavonoids in needles of J. communis and J. oxycedrus. Values marked with different letters are significantly different (p < 0.05) according to one-way ANOVA followed by Tukey’s HSD post hoc test.
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Figure 9. Total biflavonoid content in different plant parts of analyzed Juniperus spp. Values marked with different letters are significantly different (p < 0.05) according to one-way ANOVA followed by Tukey’s HSD post hoc test.
Figure 9. Total biflavonoid content in different plant parts of analyzed Juniperus spp. Values marked with different letters are significantly different (p < 0.05) according to one-way ANOVA followed by Tukey’s HSD post hoc test.
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Figure 10. Principal component analysis (PCA) of detected biflavonoids in different juniperus species and plant part: needles (red), green cones (black), and ripe cones (violet). For J. communis subsp. nana, we use the abbreviation J. nana.
Figure 10. Principal component analysis (PCA) of detected biflavonoids in different juniperus species and plant part: needles (red), green cones (black), and ripe cones (violet). For J. communis subsp. nana, we use the abbreviation J. nana.
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Table 1. Concentration (µg/g) of biflavonoids in unripe cones, ripe cones, and needles of J. communis collected at different locations. Values marked with different letters are significantly different at p < 0.05.
Table 1. Concentration (µg/g) of biflavonoids in unripe cones, ripe cones, and needles of J. communis collected at different locations. Values marked with different letters are significantly different at p < 0.05.
Plant PartLocationCupressuflavone
[µg/g dw]		Amentoflavone
[µg/g dw]		Bilobetin
[µg/g dw]		Hinokiflavone
[µg/g dw]		Total Biflavonoids
[µg/g dw]
unripe conesNahorevo136.29 ± 32.56 ghi342.63 ± 61.14 d358.63 ± 43.00 a166.83 ± 16.24 e1004.38 ± 152.93 d
Trebević264.02 ± 5.93 e291.89 ± 13.09 d 388.01 ± 116.81 and943.91 ± 135.83 de
Visočica97.62 ± 2.14 ghi111.18 ± 1.86 d139.41 ± 14.81 c72.07 ± 3.59 fgh420.28 ± 22.39 fg
Bradina108.15 ± 0.33 ghi309.47 ± 17.61 d219.25 ± 17.09 b167.43 ± 11.22 e804.30 ± 46.25 def
Konjic76.65 ± 6.44 hi189.06 ± 22.22 d263.43 ± 14.97 b49.15 ± 7.27 gh594.00 ± 50.91 defg
ripe conesProskok118.63 ± 19.89 ghi239.01 ± 41.03 d43.15 ± 4.17 d94.90 ± 10.13 fg495.69 ± 66.88 efg
Igman162.29 ± 70.95 fg233.70 ± 95.64 d36.87 ± 12.90 d85.95 ± 39.07 fg518.80 ± 218.56 efg
Trnovo219.52 ± 4.69 ef245.64 ± 0.91 dnd108.82 ± 0.86 f573.97 ± 4.63 defg
Nahorevo4.69 ± 0.55 fghi205.84 ± 5.69 dnd99.90 ± 1.08 fg442.93 ± 6.22 fg
Ajdinovici101.51 ± 43.47 ghi141.53 ± 38.52 dnd50.48 ± 15.76 gh293.52 ± 97.75 g
Međeđa134.32 ± 24.57 ghi206.27 ± 14.70 dnd15.76 ± 7.02 fgh408.93 ± 46.30 fg
Trebević120.24 ± 0.89 ghi159.17 ± 4.55 d27.17 ± 1.79 d68.33 ± 1.29 fgh374.91 ± 4.16 fg
Visočica139.43 ± 28.49 fgh107.29 ± 14.22 dnd69.07 ± 8.80 fgh315.79 ± 51.50 g
Bradina86.45 ± 0.10 ghi238.32 ± 1.29 dnd97.82 ± 0.09 fg422.59 ± 1.29 fg
Konjic51.40 ± 16.14 i116.52 ± 30.69 dnd33.11 ± 11.46 h201.03 ± 58.29 g
needles Trnovo1987.54 ± 88.24 a3267.78 ± 319.03 a23.94 ± 0.98d528.39 ± 42.23 b6033.24 ± 448.52 a
Nahorevo1825.59 ± 9.27 b3486.68 ± 91.71 and602.58 ± 9.11 a5914.86 ± 91.55 a
Visočica2059.95 ± 90.38 a2790.53 ± 139.81 b52.62 ± 28.56 d433.29 ± 6.35 c5336.39 ± 14.52 b
Bradina1360.87 ± 9.14 c2998.96 ± 382.28 b21.79 ± 4.13 d548.66 ± 45.76 b4930.28 ± 433.04 b
Trebević1385.09 ± 80.91 c3030.85 ± 496.13 b21.62 ± 2.47 d445.61± 62.07 c4883.18 ± 636.63 b
Konjic1124.42 ± 7.91 d2218.14 ± 290.28 cnd250.80 ± 41.57 d3593.36 ± 339.76 c
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MDPI and ACS Style

Medvedec, B.; Jurčević Šangut, I.; Macanović, A.; Karalija, E.; Šamec, D. Biflavonoid Profiling of Juniperus Species: The Influence of Plant Part and Growing Location. Appl. Sci. 2025, 15, 7082. https://doi.org/10.3390/app15137082

AMA Style

Medvedec B, Jurčević Šangut I, Macanović A, Karalija E, Šamec D. Biflavonoid Profiling of Juniperus Species: The Influence of Plant Part and Growing Location. Applied Sciences. 2025; 15(13):7082. https://doi.org/10.3390/app15137082

Chicago/Turabian Style

Medvedec, Barbara, Iva Jurčević Šangut, Armin Macanović, Erna Karalija, and Dunja Šamec. 2025. "Biflavonoid Profiling of Juniperus Species: The Influence of Plant Part and Growing Location" Applied Sciences 15, no. 13: 7082. https://doi.org/10.3390/app15137082

APA Style

Medvedec, B., Jurčević Šangut, I., Macanović, A., Karalija, E., & Šamec, D. (2025). Biflavonoid Profiling of Juniperus Species: The Influence of Plant Part and Growing Location. Applied Sciences, 15(13), 7082. https://doi.org/10.3390/app15137082

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