LC-ESI-MS/MS-MRM Profiling of Polyphenols and Antioxidant Activity Evaluation of Junipers of Different Origin

This study was aimed at identifying new efficient antioxidant juniper species and their metabolites, which are responsible for this activity. About 30 juniper representatives were assayed for antioxidant activity (DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) radical scavenging) and total polyphenol content (TPC). The most active species were identified, and their most abundant polyphenols were quantified by the LC-electrospray ionization (ESI)-MS/MS-multiple reaction monitoring (MRM) method. In the group of studied species, J. ashei (mountain cedar) leaf extract was outlined as the best antioxidant with the highest TPC. Catechin was revealed as the most abundant polyphenol in the J. ashei extract, contributing to its superior antioxidant properties. An in-depth analysis of antioxidant capacity was also performed. The higher metal-chelating activity was observed in the case of J. sibirica (0.83 mg DE/mL), whereas the lowest was observed for J. communis (3.2 mg dry extract (DE)/mL) extracts. All efficient antioxidant extracts were also able to inhibit lipoxygenase. EC50 values ranged from 1.77 to 2.44 mg DE/mL. The most effective inhibitors were J. ashei and J. formozana extracts, which acted as uncompetitive lipoxygenase (LOX) inhibitors. The presented results have potential application in the pharmacy and cosmetics for the generation of antioxidant compositions based on naturally derived lead compounds for the prevention of oxidative-stress associated organ-degenerative diseases, cancer, or other free radical-induced disorders.


Introduction
The genus Juniperus L. (Cupressaceae) includes a great diversity of evergreen magnificent trees or small shrubs belonging to the Pinophyta division (Coniferophyta, Coniferae) of gymnosperm cone-bearing seed plants. This genus consists of about 50-67 species, depending on the taxonomic classification, and it includes over 220 cultivars. Junipers produce nearly 580 secondary metabolites, including anticancer lignans (podophyllotoxin and other derivatives), sesquiterpenes, diterpenes the multiple reaction monitoring (MRM) scan mode, which ensures high sensitivity and selectivity of the analysis. Therefore, this method was employed as a very reliable and suitable tool for analyses of complex mixtures, e.g., plant extracts. Moreover, this technique enabled a fast distinguishing of compounds having the same parent ions but giving different fragment ions [18,19]. The present detailed study of the antioxidant properties of plenty of junipers around the world revealed the genus Juniperus as an invaluable natural source of efficient antioxidant agents. As a result, after the screening of a diversity of Juniperus species of various origin, extracts with excellent antioxidant activity and polyphenol content were distinguished and the metabolites responsible for these properties were identified. To our knowledge, Juniperus ashei J. Buchholz (mountain cedar) was determined for the first time as the representative with superior antioxidant activity and highest total polyphenol content in the group of assayed representatives of the genus Juniperus. The results of the present study have potential application in the generation of natural antioxidant compositions for the prevention of oxidative-stress associated organ-degenerative or other radical-induced diseases.

Extraction Procedure
The collected plant material was kept in vacuum bags in the freezer at −20 • C until extraction. Then, leaves or galbuli were separated, homogenized, weighed (5 g), and mixed with methanol (50 mL, 80% v/v) in an Erlenmeyer flask with a stopper. The suspension was stirred for 1.5 h in a shaker water bath at 20 • C. The mixture was filtered, and the extract was collected. The remaining solid material was subjected to a second extraction for 1.5 h with a new portion of 80% methanol (50 mL). After filtration, the solid mass was stirred again for 1.5 h in 80% methanol (25 mL). The extracts were combined and concentrated by a vacuum evaporator. The remaining residue was freeze-dried (24 h, at −50 • C, 0.2 mbar) and kept in the freezer at −20 • C until analyses.

Total Polyphenol Content Determination
The total content of phenolic compounds (TPC) of the corresponding juniper extracts was determined according to the Folin-Ciocalteu method [21] with minor modifications. In brief, 20 µL of the extract [5 mg/mL in 80% (v/v) methanol] were mixed with distilled water (1.58 mL) and FC-reagent (100 µL) was added. The control sample contained the same reagents but without plant extract. After 3-5 min, 300 µL of sodium carbonate (20% w/v) were added, and the samples were kept at room temperature for 2 h. The absorbance at 765 nm was registered on a spectrophotometer.
The calibration curve was prepared using gallic acid standard. The TPC of the extracts was expressed in GAE (Gallic Acid Equivalents) according to the formula C = c.V/m, where C is the concentration of phenolic compounds in mg GAE per gram dry extract (DE); c is the gallic acid concentration [mg/mL] from the calibration curve; m is the weight of plant extract [g]; V is the volume of plant extract [mL]. The TPC of each extract was determined by 2 independent analyses and was given as an average value ±SD.

DPPH Radical Scavenging Method for Antioxidant Activity Determination
The antiradical activity was determined by the DPPH method [22]. One ml of the extract (at various concentrations) was mixed with 4 mL of DPPH solution (0.004% w/v) in a test tube. The control sample was prepared with the same reagents but without plant extract. The blank sample contained 80% (v/v) ethanol. The solutions were kept at room temperature for 1 h in the dark, and then, the absorption at 517 nm was measured. The percentage of the DPPH-scavenging was assessed according to the formula: where Ac is the absorbance of the control and As is the absorbance of the sample. The half maximal inhibitory concentration of the extracts (DPPH-EC 50 ) was determined by interpolation of the dose-response curves. These EC 50 values were calculated at fitted models as the concentration of the tested extract that gave 50% of the maximum inhibition of the initial DPPH concentration, based on a dose-dependent mode of action.

Ability to Quench ABTS Radicals
The ABTS free radical scavenging potential was determined according to Re et al. [23] with some modifications. The ability of samples to quench the ABTS free radical was assessed according to Formula (1).
The half maximal inhibitory concentration of the extracts (ABTS-EC 50 ) was determined by interpolation of the dose-response curves. These EC 50 values were calculated at fitted models as the concentration of the tested extract that gave 50% of the maximum inhibition, based on a dose-dependent mode of action.

Metal Chelating Activity (CHP)
Metal chelating ability was determined according to Guo et al. [24]. The extract samples (100 µL) have been mixed with 20 µL of 2 mM FeCl 2 solution and 40 µL 5 mM ferrozine. The mixture was shaken vigorously and left standing at room temperature for 10 min. Then, absorbance of the solution was measured spectrophotometrically at 562 nm The chelating power was assessed using the formula: where Ac is the absorbance of the control and As is the absorbance of the sample. Antioxidant activity was determined as EC 50 -the extract concentration providing 50% of activity was based on a dose-dependent mode of action.

Inhibition of Lipoxygenase Activity (LOXI)
Lipoxygenase activity was determined according to method described by Axelrod et al. [25] adapted for a microplate reader (Epoch 2 Microplate Spectrophotometer, BioTek Instruments, Winooski, VA, USA) [26]. An increase in absorbance of 0.001 per minute at 234 nm was assumed as one unit of LOX activity. Antioxidant activity was expressed as EC 50 -extract concentration provided 50% of activity was based on dose-dependent mode of action.

LC-ESI-MS/MS-MRM Analyses
The identification and quantification of phenolic acids, flavonoid aglycones, and flavonoid glycosides was carried out using liquid chromatography coupled to electrospray ionization triple quadrupole mass spectrometry. The method for simultaneous analysis of all mentioned polyphenol groups was partly founded on the basis on previous experiments [19,27]. The LC-MS system consisted of a 1200 Series LC system (Agilent Technologies, Santa Clara, CA, USA), which was coupled with a 3200 QTRAP mass spectrometer with Turbo V™ source and an electrospray ionization (ESI) probe (AB Sciex, Redwood City, CA, USA). The sample or standards' solutions at a volume of 3 µL were injected on an Eclipse XDB-C18 column (4.6 mm × 150 mm; 5 µm; Agilent Technologies, Santa Clara, CA, USA), whose temperature was maintained at 25 • C. The mobile phases were 0.1% HCOOH in milli-Q water (solution A) and 0.1% HCOOH in acetonitrile (solution B). The gradient elution (flow rate 400 µL min −1 ) was used: 0-1.5 min 13% B; 1.5-2 min 13-20% B; 2-4.5 min 20% B; 4.5-5 min 20-25% B; 5-8 min 25% B; 8-9 min 25-33% B; 9-11 min 33% B; 11-13 min 33-60% B; 13-16 min 60% B; 16-18 min 60-80% B; 18-21 min 80% B; 23-28 min conditioning with 13% B. QTRAP worked in multiple reaction monitoring (MRM) mode. Optimal mass analyzer conditions and the selection of product ions were determined experimentally (results are given in Table S1). MS parameters were set as a capillary temperature of 500 • C, negative ionization mode source voltage −4500 V, curtain gas at 30 psi, nebulizer gas at 55 psi. MS data acquisition and processing was performed using Analyst 1.5 was software (AB Sciex, Redwood City, CA, USA) analytes. The calibration curves were generated using peak areas of the most intense MRM transitions. The linearity ranges, LOD (limit of detection; at a signal-to-noise ratio of 5:1) and LOQ (limit of quantification; at a signal-to-noise ratio of 10:1) values for all analytes were established (Table S2). LC-MS assay was performed in triplicate for each standard solution and sample.

Statistical Analysis
Obtained data were presented as means ± standard deviations. Moreover, one-way ANOVA and Tukey's test were performed (α = 0.05) for the data obtained from 3 independent extracts in 3 parallel experiments (n = 3). Statistica 6.0 software (StatSoft, Inc., Tulsa, OK, USA) was used for the analysis.

Results
The present study was designed to analyze the total polyphenol content and antioxidant activity of juniper extracts in order to reveal species of different origin around the world, exhibiting the highest activity, and to identify their secondary metabolites responsible for these properties. The first step of the study aimed at identifying juniper extracts with the highest total polyphenol content, which was correlated with their half-maximum DPPH-radical scavenging concentrations (DPPH-EC 50 ). The comparison of these experimental variables revealed that the leaf extracts demonstrated higher antioxidant properties than the corresponding galbuli extracts, as it was shown by analysis of the leaf and galbuli extracts of J. chinensis L., J. virginiana L., as well as cultivars J. virginiana 'Glauca' and J. virginiana 'Grey Owl'. Using the Folin-Ciocalteu method for quantitative polyphenol determination, several juniper leaf extracts were found to exhibit highest TPC values: J. ashei J. Buchholz > J. pinchotii Sudw. ≈ J. communis Laxa ≥ J. formosana Hayata. The original sources summaries of the 31 studied juniper samples are shown in Table 1.
The DPPH-EC 50 values of all studied extracts were correlated with their TPC values, deriving a polynomial function, where greater TPC and lower DPPH-EC 50 denote higher antioxidant activities ( Figure 1). The juniper species from which the leaf extracts were characterized as the best sources of antioxidants and a rich polyphenol source (based on their TPC and DPPH-EC 50 values) were subjected to LC-ESI-MS/MS (liquid chromatography/electrospray ionization mass spectrometry) metabolite analysis. The method designed for the simultaneous analysis of flavonoid aglycones, flavonoid glycosides, and phenolic acids was partly founded on the basis of the previous experiments [10,18]. The LC-MS conditions are presented in Tables S1 and S2 (in the Supplementary Material). The optimization of the experimental method allowed the separation of 23 different compounds belonging to several polyphenol structural subclasses (hydroxybenzoic and hydroxycinnamic acids, dihydroflavonols, flavanones, flavan-3-ols, flavonols, flavones) ( Table 2). Polyphenolic profiles of the studied species were qualitatively and quantitatively differentiated. It was observed that phenolic acids constitute a relatively small part of all polyphenols, with protocatechuic acid as a dominant representative (up to 43.3 ng/mg DE). It can be seen that the majority of them belong to hydroxybenzoic acid derivatives (salicylic, gallic, protocatechuic, 4-OH-benzoic). Only one hydroxycinnamic acid derivative was found, i.e., p-coumaric acid. The highest amount of phenolic acids was found in J. sibirica extract. BQL-below the quantification limit (peak detected, concentration > limit of detection, but < limit of quantification). Means (n = 9) followed by the different lowercase letters (a-y) in each group of compounds are significantly different at p < 0.05.
As a result of the comparative LC-ESI-MS/MS analysis of the best antioxidant extracts, catechin was revealed as the abundant flavonoid aglycone (1013.00-2227.50 ng/mg DE), with the highest amount found in the J. ashei leaves extract. The contents of other individual aglycones (apigenin, quercetin, luteolin, taxifolin, myricetin and eriodictyol) were much lower (to 114.50 ng/mg). It can be noticed that in the case of the studied juniper samples, the total aglycone contents and profiles are species-specific. The greatest differentiation was observed in phenolic glycosides. The highest amount of these metabolites was determined in the leaves of J. communis, J. sibirica, J. formozana, and J. sabina. In the case of J. ashei and J. pinchotii, glycosides represented a relatively small portion of all polyphenols. Quercetin derivatives (e.g., rutin, isoquercetin, quercitrin) were found to be predominant reaching concentrations ≥2375.00 ng/mg. Rutin was the major flavonoid glycoside in J. communis 'Laxa' and J. sibirica, while quercitrin was dominant in J. formozana, J. excelsa, and J. sabina var. balkanensis. Several other glycosides including luteolin, kaempferol, isorhamnetin, apigenin, and naringenin derivatives were detected in the samples. However, J. ashei and J. pinchotii extracts did not have isorhamnetin glycosides and there was only a trace amount of one kaempferol glucoside (astragalin). In addition to the above-mentioned and confirmed analytes, some isobars of naringenin 7-glucoside, apigenin-7-glucoside, isorhamnetin-3-glucoside, astragalin, luteolin-7-glucoside, kaempferol-3-rutinoside, kaempferol, quercetin, naringenin, myricetin, catechin, and isorhamnetin were detected during LC-MS analyses. However, their reliable identification could not be performed.

In-Depth Analysis of Antioxidant Activity
All tested samples showed strong antiradical activity. Taking into account the ability to scavenge ABTS free radicals, samples can be ordered as follows: J. ahei > J. sibirica > J. excelsa > J. formozana ≈ J. pinchotii > J. communis ≈ J. sabina. The activity (expressed as EC 50 ) ranged from 331.64 ± 5 µg DE/mL (J. ahei) to 798.71 ± 10 µg DE/mL (J. sabina) (Figure 2A). All tested extracts were also able to chelate metal ions. The higher activity was observed in the case of J.sibirica (0.83 mg DE/mL) whereas the lowest was observed for J. communis (3.2 mg DE/mL). In terms of this activity, samples can be ordered as follows: J. sibirica > J. formozana > J. sabina > J. pinchotii > J. excelsa > J. ashei > J. communis ( Figure 2B). All extracts were also able to inhibit LOX. In the case of this activity, the smallest differences between extracts were observed. EC 50 values ranged from 1.77 mg DE/mL to 2.44 mg DE/mL. The most effective inhibitors were extracts from J.ashei and J. formozana, whereas the lowest activity was observed in the case of J. communis extract ( Figure 2C). Interestingly, despite the similar inhibitory activity, the extracts have different modes of action. The most effective samples acted as uncopettitive LOX inhibitors (Figure 3). Extracts from J. pinchotii and J. excelsa demonstrated a non-competitive mode of action, whereas the weakest inhibitor-extract from J. communis-acted as a competitive LOX inhibitor ( Figure 3D,F). Interestingly, despite the similar inhibitory activity, the extracts have different modes of action. The most effective samples acted as uncopettitive LOX inhibitors (Figure 3). Extracts from J. pinchotii and J. excelsa demonstrated a non-competitive mode of action, whereas the weakest inhibitor-extract from J. communis-acted as a competitive LOX inhibitor ( Figure 3D,F).

Discussion
Polyphenols contribute to a wide range of biological properties of herbal samples. They can be major active ingredients or a valuable addition, demonstrating a synergistic effect with other natural molecules or synthetic drugs [28]. Therefore, sources of bioactive polyphenols are still searched and evaluated. Moreover, the content and profile of polyphenols is an important phytochemical and chemotaxonomic information as well. The present study was designed to analyze the total polyphenol content and antioxidant activity of juniper extracts in order to reveal species of different origin, exhibiting highest activity, and to identify the phytochemicals (secondary metabolites) responsible for these properties. Antioxidant activity, particularly antiradical activity, is very important due to the deleterious role of free radicals in biological systems and food. Plants rich in secondary metabolites, including phenolic compounds, have antioxidant activity related to their redox properties. The DPPH screening is one of the most widely used tests for the evaluation of the antioxidant activity of the extracts [29]. Thus, the first step of the study was aimed at identifying juniper extracts with highest TPC content correlated with high antiradical potential measured as DPPH-radical scavenging ability.
This screening enabled the designation of the most promising juniper extracts for further detailed studies of polyphenolic profile and biological assays. It was found that all samples contain large amount of polyphenols (45-263 mg GAE/g of dry extract (DE)). For several juniper leaf extracts, TPC values were found to be outstanding (>180 mg/g DE): J. ashei J. Buchholz > J. pinchotii Sudw. > J. communis Laxa ≥ J. formosana Hayata. > J. sibirica Burgsd. It could be noticed that the leaf extracts demonstrated higher antioxidant properties than the corresponding galbuli extracts, as it was shown by analysis of the leaves and galbuli extracts of J. chinensis L., J. virginiana L., as well as cultivars J. virginiana 'Glauca' and J. virginiana 'Grey Owl'. TPC values were significantly higher than those reported for Juniperus phoenicea L. leaves (6.3 and 9.6 mg/g) by Ghouti et al. [17]. On the other hand, the TPC values found for galbuli ("berries") were lower than those reported for J. communis and J. oxycedrus [29]. It can be noticed that the TPC values of juniper samples are strictly species and organ-dependent. Moreover, differences in phenolic content may be related to the different growing or/and climate conditions. All samples had radical scavenging action in the DPPH radical scavenging assay test (Table 1). Determined EC 50 values ranged from 49 to 352 µg DE/mL. The lowest values (indicating the highest activity) were found for J. ashei, J. pinchotii, and J. formosana (49-86 µg DE/mL). Juniperus samples (e.g., J. communis, J. formosana, and J. sibirica ethanolic extracts) were previously reported to be effective radical scavengers with EC 50 21.39-28.55 µg/mL [29,30]. Interestingly, it was observed that juniper ethanol extracts are more effective than essential oils obtained from the corresponding plant materials [30].
After identification of the best antioxidant plants in the group of all assayed junipers, the LC-ESI-MS/ MS-MRM technique was employed for the qualitative and quantitative determination of the secondary metabolites (phenolic acids, flavonoid aglycones, flavonoid glycosides), which is responsible for the highest activity of best antioxidant extracts. It was observed that phenolic acids constitute a relatively small part of all polyphenols, with protocatechuic acid as a dominant representative (up to 43.3 ng/mg DE). The majority of them belong to hydroxybenzoic acid derivatives (4-OH-benzoic, salicylic, protocatechuic, gallic). Only one hydroxycinnamic acid derivative was found, i.e., p-coumaric acid. The highest amount of phenolic acids was found in J. sibirica extract, whereas J. excelsa contained only trace amount of these compounds. Mrid et al. [15] have also observed that hydroxybenzoic derivatives constitute the biggest part of Juniperus oxycedrus leaf phenolic acids. However, they found that salicylic and 4-OH benzoic acid were the most abundant ones.
We did not find free kaempferol, naringenin, or myricetin glucosides previously identified in some species [11,13,31]. However, we have detected free myricetin. Moreover, kaempferol 3-O-rutinoside was found in J. communis 'Laxa', J. formozana, J. sibirica, J. excelsa, and J. sabina var. balkanensis, and kaempferol 3-glucoside (astragalin) with naringenin glucoside was found in all the studied species. All the studied juniperus species are very diverse in terms of polyphenolic profile. Moreover, the polyphenolic profile established for J. communis 'Laxa' differed greatly from the one recently reported by Dziedzinski et al. [31] for J. communis shoot collected in Poland. Similarly, polyphenolic profiles of other studied juniper extracts are very diverse in terms of composition [6,11,17]. Therefore, it can be assumed that the species, variety, and origin strongly influence the phytochemical composition of juniper plant material. The major chemical components in the J. excels leaves were monoterpene hydrocarbons, sesquiterpene hydrocarbons, and oxygen-containing sesquiterpenes [32].
Other major phenolic constituents found in extracts of J. excels species are coumarins, lignans, sesquiterpenes, abietane, labdane and pimarane diterpenes, flavonoids, flavone glycosides, and tannins [33]. In leaves and seed cones extracts of J. excels, catechin, quercitrin, epicatechin, rutin, apigenin, and amentoflavone were present in substantial amounts. Phenolic acids were found in small amounts (0.26% and 0.48% of dry weight (DW) of leaves and seed cones, respectively); the dominant compounds were 2,5-dihydroxybenzoic and p-hydroxybenzoic acid. Furthermore, lignans, mainly matairesinol, were present in moderate amounts, primarily in seedcones, while coumarins were found in small amounts in both J. excelsa extracts [34].
The antioxidant activity of a given plant extract depends on the plant's pchytochemical profile. Some of the phenolics (anthocyanidin, catechins, flavonoids), tannins (ellagic and gallic acids), phenyl isopropanoids (caffeic, coumaric, ferulic acids), lignans, catechol, and many others act as antioxidants [35]. There are many reports in the available literature on the antioxidant activity of juniper fruit [5]. The aqueous and ethanolic extracts from J. communis L. fruits demonstrated a strong total antioxidant capacity at the concentrations of 20, 40, and 60 µg/mL. At these concentrations, both extracts of juniper fruit demonstrated effective reducing power, metal-chelating activity, as well as antiradical potential in various scavenging assays. These fruit extracts effectively inhibited the peroxidation of the linoleic acid [36]. However, most studies on the antioxidant potential of juniper concern essential oils [37,38]. The in vitro antioxidant capacity of juniper oil has been observed using various free radical scavenging tests. It is mainly attributed to electron transport, which makes juniper essential oil a powerful antioxidant. Due to the increased activity of various antioxidant enzymes, the possibility of blocking the oxidation process was also confirmed in living models [39].
The mechanisms of inflammation mediated by metabolites of lipoxygenase and nitric oxide play key roles in physiological immune response [40].
Lipoxygenases (LOXs) are enzymes involved in the production of inflammatory mediators such leukotrienes and prostaglandins [41]. Human LOX isoenzymes (5-, 12-, and 15-lipoxygenases) are extremely unstable at physiological temperature in the cell-free lipoxygenase assay; therefore, a good solution is to use commercially available and the physiological temperature-stable LOX derived from Glycine max L. for the LOX inhibitory assay [42].
LOX inhibition is a prospective method for the treatment of inflammation [43]. Furthermore, the additional advantage of this type of experiment is avoidance of the undesirable in vivo tests on experimental animals (carrageenan-induced paw edema in rats) [44]. The LOX-inhibitory potential of pure chemicals and plant extracts is well known and extensively studied [45,46]. Samples tested in our study are able to inhibit LOX. EC 50 values ranged from 1.77 to 2.44 mg DE/mL. The most effective inhibitors were extracts from J. ashei and J. formozana, whereas the lowest activity was observed in the case of J. communis extract ( Figure 2C). Extracts from J. pinchotii and J. excelsa demonstrated a non-competitive mode of action, whereas the weakest inhibitor-extract from J. communis-acted as a competitive LOX inhibitor (Figure 3). The extracts from J. sibirica Burgsdorf. extracts demonstrated 12-LOX inhibitory activity (IC50 = 4.85 mg/mL for needles and 1.34 mg/mL for cones). The cones extract showed a better anti-inflammatory activity compared to the needles [44].
Our results indicate that all tested species exhibit a strong antioxidant activity and sufficient ability to inhibit the LOX enzyme included in inflammation processes. No correlation was found between the total phenolics content and the anti-inflammatory capacity, which suggests a synergistic effect of compounds contained in the extracts. The literature data also suggest a synergistic action of various compounds and do not attribute the biological effects (antioxidant, antifungal, or anti-inflammatory) to a single compound [6].
In the group of all assayed juniper species, the leaf extract of Juniperus ashei J. Buchholz (mountain cedar) was distinguished as the representative with highest total polyphenol content and best antioxidant properties. To our knowledge, this is the first determination of J. ashei as a juniper representative with superior antioxidant activity in the genus Juniperus. The mountain cedar is a dioecious plant that is found naturally in north-eastern Mexico and south-central USA, including Missouri and central Texas. The original source of J. ashei in this study is collected from a wild representative in the Murray County, Oklahoma, United States (Table 1). Using LC-ESI-MS/MS-MRM analysis, catechin was revealed as its most abundant antioxidant metabolite than in the other studied juniper extracts, and therefore, it was considered as the metabolite contributing to the superior antioxidant properties of the J. ashei J. Buchholz leaves extract.

Conclusions
To our knowledge, this is the first detailed antioxidant activity evaluation of Juniperus L. representatives of various origins around the world, which was accomplished with a quantitative and qualitative analysis of the polyphenol compounds of selected best antioxidant samples. As a result of the study, the most promising species for potential application were identified. For the first time, J. ashei J. Buchholz was distinguished as the species with superior antioxidant activity in the group of studied junipers. The identified juniper extracts and polyphenols with superior antioxidant activity have potential application in the pharmacy and cosmetics as sources of lead compounds for the generation of antioxidant compositions for the prevention of oxidative-stress-associated organ-degenerative diseases. We hope our findings will accelerate interest from the wider research community in the involvement of the Juniperus organs in different plant-related products. It should be mentioned that the observed differences in the biological activity and polyphenol content may be partially related to the collection site, climate, altitude, sampling season, soil parameters, etc. Therefore, once the most promising species have been identified, the impact of pedoclimatic conditions and sampling season on their chemical constituents and bioactivity should be investigated. Moreover, the safety of the application of juniper-based natural products should be established.