Bioactivity and chemical profiling of the Juniperus excelsa, which support its usage as a food preservative and nutraceutical

ABSTRACT

Leaves and cones of the genus Juniperus, regularly used as a spice, could be a candidate for new food preservative with significant nutraceutical qualities. The main goal of this study was to evaluate the bioactivity and chemical profiling of the Juniperus excelsa leaves and cones in order to support its usage as a health-promoting food additive. The content of 44 phenolics was determined using Liquid chromatography tandem-mass spectrometry (LS-MS/MS), with catechin and quercitrin being the most dominant, especially in leaves (234 and 142 mg/g of leaves dry weight, respectively). Gas chromatography–mass spectrometry (GC-MS) analysis showed a simple terpene composition both in leaves and in cones, with α-pinene (31 and 77%), cedrol (37 and 8%), and limonene (15 and 6%) as the most abundant compounds, respectively. J. excelsa showed a noteworthy antioxidant effect comparable with butylated hydroxyanisole (BHA), especially leaves extract towards HO^• and lipid peroxidation (LP) inhibition, which is particularly important in food-quality maintenance. Essential oil of leaves showed significant anti-inflammatory activity by means of inhibition of eicosanoids production and considerable antimicrobial activity against the clinically relevant and/or foodborne illness-causing bacteria. Due to its considerable bioactivity and high content of phenolics with proven health benefits, usage of J. excelsa as a food additive with valuable nutraceutical properties should be supported.

KEYWORDS:

• Bioactivity
• Essential oils
• Juniperus excelsa
• Nutraceutical
• Phenolics

Introduction

Foodstuffs are highly perishable and require additional preservation to be resistant to spoilage during preparation, storage, and distribution. On the one hand, foodstuffs need to have a longer shelf life, while on the other hand, there is a growing demand for minimally processed, easily prepared, and ready-to-eat fresh foodstuffs. In order to obtain satisfactory levels of food safety and quality and meet the consumer demand, the food industry is facing a lot of challenges, among which contamination by microorganisms and food auto-oxidation are the most significant.^[1,2,3,4]

Foodstuffs are often contaminated by bacteria and fungi, which can cause severe foodborne illnesses or diminish its sensory qualities.^[5] Antiseptics used in foodstuffs processing are decreasing in popularity mainly due to their insufficient effectiveness and potential health concerns.^[2] Consequently, there is an urgent need for the identification of alternative antimicrobial agents, preferably those of natural origin. Essential oils are the likely targets, since they are generally recognized as flavouring agents for animal and human consumption and have significant antimicrobial activity against numerous microorganisms, among which are the foodborne bacteria.^[2]

Inhibition of lipid peroxidation (LP) and rancidification are also great challenges for the food industry.^[4] LP is the most common process resulting in the oxidative damage of lipids, which results in off-flavour and off-odour of the foodstuffs. LP is caused by the formation of free radicals, primarily hydroxyl radical (HO^•), and antioxidants that can successfully prevent radical formation and are of great interest to the food industry.^[1] The popularity of food synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene, is dramatically decreasing due to the existing doubts over their toxic and carcinogenic effects.^[6] Therefore, phenolics and terpenes are particularly interesting as food additives with powerful antioxidative properties, especially because they are anyway consumed on a daily basis as a part of foodstuffs of plant origin.^[1]

Nutraceuticals are defined as compounds isolated or purified from food sources that possess health-promoting properties such as anti-inflammatory, anticancer, neuroprotective, cardioprotective, etc.^[7] Moreover, inflammation in the tumour tissue has been recognized as an integral part of cancer. Inflammation mediators, such as eicosanoids, which are the arachidonic acid (AA) metabolites, are believed to be a missing link between inflammation and cancer. Research on suppression of the eicosanoids synthesis targeted at inhibiting tumour-associated inflammation is mainly focused on the cyclooxygenase (COX) and lipoxygenase (LOX) pathways.^[8] Thus, the popularity of nutraceuticals with potency to alter the AA metabolism is growing.^[7].

Having in mind that all of the factors previously mentioned and that plants are regularly used in food preparation processes, such as spices, it is clear that they are potential candidates for new food preservatives with antimicrobial, antioxidant properties and a significant nutraceutical quality. Plants from the Juniperus L. genus could be the possible targets, especially because J. phoenicea has already been used as a preservative in food production.^[9] Additionally, plants from the genus Juniperus are widely used in cooking as a spice, preferably for pickling game birds and meat. In some traditional recipes, such as the cabbage dishes “bigos” and “sauerkraut”, the Polish sausage “kiełbasa jałowcowa”, and jam from Turkey, common juniper (J. communis) seed cones are an important ingredient. In addition, they are an indispensable additive in the manufacture of gin, the Italian liquor “Gineprino”, the Serbian brandy called “Klekovača”, and the Polish beer “piwo kozicowe”.^[10,11] Furthermore, species from the genus Juniperus, mainly the extracts and essential oils of needles or leaves (flake leaves), seed cones, and wood, are well-known for their numerous pharmacological properties in the traditional medicine of ancient civilizations of Europe and Asia.^[11,12]

In spite of the widespread use of the Juniperus species in food preparation and beverage manufacture, and their great healing power, there are very few reports concerning their chemical compositions and biological activities, which could further support their use as food preservative and nutraceutical. One of the Juniperus species whose bioactivity and chemical profiling have been poorly investigated is the Juniperus excelsa M. Bieb. (syn. Juniperus excelsa M. Bieb. subsp. excelsa), which is the main focus of this study. Specifically, J. excelsa was broadly used in traditional recipes as a distinctive flavouring agent and to increase appetite, while in traditional medicine it was used for treating hypertension, diarrhoea, fever, headache, rheumatism, arthritis, tuberculosis, asthma, bronchitis, etc.^{[10,13,14,15]}

Keeping in mind the widespread use of J. excelsa in traditional cooking and medicine and its proven high non-toxic dose determined for mice (10 g/kg), it is worthwhile to further investigate this species and propose it as a potentially new food preservative with nutraceutical properties.^[14] In this study, phenolics and terpenoid profiles, together with the antioxidant, anti-inflammatory, and antimicrobial potential of the extracts and essential oils of leaves and juicy seed cones of J. excelsa, were investigated in depth.

Methods

Plant material

Branches (≤5 mm in diameter) of the female J. excelsa with flaked leaves and juicy seed cones were collected on the carbonate rocks of island Golem Grad in the Great Prespa Lake, located between Greece, Albania, and FYR Macedonia. Plant material was collected from three different trees during November 2014. The voucher specimen (No. 2–1875) was prepared, identified, and deposited at the Herbarium of the Department of Biology and Ecology (BUNS Herbarium), Faculty of Sciences, University of Novi Sad, Republic of Serbia.

Extracts preparation

Air-dried and finely ground, particle size ≤0.2 mm in diameter, leaves (flake leaves with tiny branch) and seed cones of the J. excelsa weighing 30 g were extracted separately by maceration with 80% aqueous methanol (300 mL) during 72 h, with constant shaking at 120 rpm/min at room temperature. Extracts of each of the leaves and seed cones were prepared separately in triplicate. After filtration, the solvent was evaporated in vacuo at 45°C and the crude residue was resuspended in hot distilled water (1 g/mL). In order to remove the nonpolar compounds, the extracts were washed three times exhaustively with 25 mL of petroleum ether (fraction 40–60°C) and concentrated to dryness under vacuum, yielding 9.16% and 8.49% for leaves and seed cones extracts, respectively. Dried extracts were dissolved in 80% aqueous methanol to obtain 200 mg/mL or in DMSO to obtain 300 mg/mL stock solutions for the antioxidant and anti-inflammatory studies, respectively. Furthermore, dried extracts were dissolved in a mixture of 0.5% formic acid and methanol (in the ratio of 7:3) to obtain 2 mg/mL (0.2%) stock solutions for HPLC–MS/MS analysis.

Essential oils preparation

Air-dried and finely ground, particle size ≤0.2 mm in diameter, leaves and seed cones of J. excelsa plant sample were separately subjected to hydro-distillation using a Clevenger-type apparatus. Essential oils of each of the leaves and seed cones were prepared separately in triplicate. Three hundred grams of each of the plant’s organ were distilled for 4 h with 1200 mL of distilled water, followed by the recipient solvent removal (hexane) under reduced pressure. The yields of essential oils were 1.29% and 0.89% for leaves and seed cones, respectively. The essential oils were clear, slightly yellowish, and with a strong odour. The essential oils were dissolved (0.05%, v/v) in hexane before GC-MS analysis.

LC-MS/MS analysis of the selected phenolics in J. excelsa extracts

Quantification of the 44 selected phenolics in the leaves and seed cones extracts of J. excelsa was carried out according to the previously published procedure.^[16]. In brief, samples and standards (prepared in serial dilutions, ranging from 1.53 to 25.0 × 10³ ng/mL, dissolved in a mixture of 0.5% formic acid and methanol (in 1:1 ratio)) were analysed using Agilent Technologies 1200 Series HPLC coupled with Agilent Technologies 6410A QqQ mass spectrometer with electrospray ionization source, and controlled by Agilent Technologies MassHunter Workstation software (ver. B.03.01). Injection volume was 5 μL. Separation was performed using Zorbax Eclipse XDB-C18 (Agilent Technologies) column, 50 mm × 4.6 mm, 1.8 μm, held at 50°C. The mobile phase, consisting of 0.05% aqueous formic acid (phase A) and methanol (B) was delivered at a flow rate of 1 mL/min in gradient mode (0 min 30% B, 6 min 70% B, 9 min 100% B, 12 min 100% B, post time 3 min). Ion source parameters were: nebulization gas pressure 40 psi, drying gas flow 9 L/min, temperature 350°C, and capillary voltage 4000 V. All compounds were detected in the negative mode, using dynamic selected reaction monitoring with optimized compound-specific parameters (retention time, precursor ion, product ion, fragmentor voltage, collision voltage). Concentrations of standard compounds in extracts were determined from the peak areas by using the equation for linear regression obtained from the calibration curves.

GC-MS analysis of J. excelsa essential oils

Essential oils of leaves and seed cones of J. excelsa were analysed by gas chromatography with electron-ionization mass-selective detector, according to the previously published procedure.^[17] In brief, 1 μL of the sample dissolved in hexane was injected into a split/split-less inlet at 250°C, with a split ratio of 1:10. Helium (purity 99.999%) was used as a carrier, with a constant flow of 1 mL/min. The separation was achieved on the Agilent Technologies HP-5 ms column (30 m × 0.25 mm × 0.25 μm), using the following temperature program: start at 50°C, 8°C/min to 120°C, 15°C/min to 230°C, 20°C/min to 270°C (total run time 16.9 min). The eluate was delivered to the mass spectrometer via a transfer line held at 280°C. The ion source temperature was 230°C, electron energy was 70 eV, and the quadrupole temperature was 150°C. Data were acquired in scan mode (m/z range 35–400). The compounds were identified by comparison of mass spectral with data libraries (Wiley Registry of Mass Spectral Data, 7th ed. and NIST/EPA/NIH Mass Spectral Library 05) and confirmed by comparison of the Kovats retention indices with the literature data.^[18] Diesel oil, consisting of a mixture of C₈–C₂₈ n-alkanes corresponding to 800–2800 Kovats retention indices, was used as a standard for the determination of retention indices. Relative amounts of components, expressed in percentages, were calculated by normalization measurement according to the peak area in total ion chromatogram.

Estimation of J. excelsa antioxidant potential

The antioxidant potential of J. excelsa extracts was determined by using various assays adapted for 96-well microplates related to the free radical (2,2-diphenyl-1-picrylhydrazyl radical (DPPH^•) (11), reactive oxygen (HO^•, O₂^•–),^[9] and reactive nitrogen species (^•NO)^[11] scavenging ability. In addition, the inhibitory potentials of the examined extracts towards LP^[17] and their reducing power (ferric reducing antioxidant power (FRAP))^[11] were determined. Furthermore, the total phenolics and flavonoid contents of the extract examined were studied in this section using the previously described procedures.^[19] Additionally, the antioxidant potential of J. excelsa essential oils was determined towards DPPH^• scavenging ability and LP inhibitory potential using the above-mentioned procedures. Taking into account the DPPH^• and HO^• scavenging ability, the LP inhibitory potential, and the reducing power ability, the results were compared with the synthetic antioxidant BHA. Essential oils and BHA were examined by a limited number of antioxidant assays due to their low solubility in aqueous systems. All of the applied assays and methods for the determination of the antioxidant potential have not been described in detail in this section. Instead, only the reference is given, as they are carried out by the globally accepted protocols without modifications.

Estimation of J. excelsa anti-inflammatory activity

The anti-inflammatory activity of J. excelsa was determined using an in vitro assay based on the inhibitory potential of samples on the biosynthesis of 12(S)-hydroxy (5Z,8E,10E)-heptadecatrienoic acid (12-HHT), thromboxane B₂ (TXB₂), prostaglandin E₂ (PGE₂), and 12(S)-hydroxy-(5Z, 8Z, 10E, 14Z)-eicosatetraenoic acid (12-HETE), in human platelets^[17], inflammation mediators derived from AA metabolism by COX-1 and 12-LOX enzymes.

Estimation of J. excelsa essential oils antimicrobial activity

The antimicrobial activity of the essential oils was tested against the reference bacterial strains from The American Type Culture Collection (ATCC; Microbiologics Inc., St. Cloud, MN, USA): Clostridium perfringens (ATCC 13124), Staphylococcus aureus (ATCC 11632), Escherichia coli (ATCC 25922), Salmonella enterica serovar. Enteritidis (ATCC 13076), Pseudomonas aeruginosa (ATCC 9027), and Acinetobacter baumannii (ATCC BAA–747). Disc diffusion assay was employed to preliminarily determine the effect of the essential oils on bacterial growth, while the agar dilution method was applied for further confirmation of the antibacterial activity. Ceftriaxone and amikacin were used as positive controls. Neither of the two methods has been described in detail, and only the reference is given^[11] as they are carried out by the globally accepted protocols without modifications.

Statistical analysis

Detailed statistical analysis of the results was previously explained.^[11] In brief, the results were expressed as mean ± standard error (SD) of the three measurements. The significant statistical difference between the two results throughout the study was determined using the Student’s two-tailed unpaired t-test. Statistical significance was set at p ≤ 0.05.

Results and discussion

Phenolic profile of J. excelsa extracts

Comprehensive quantitative and qualitative studies of the 44 phenolic compounds in J. excelsa extracts were performed using the state-of-the-art LC-MS/MS technique. It should be noted that these data are particularly valuable as they present the first in-depth study of the phenolic profile of J. excelsa species. The contents of the phenolics determined in the leaves and seed cones extracts of J. excelsa are presented in Table 1.

Table 1. Phenolic profile of J. excelsa extracts.

	Content in J. excelsa extracts (μg/g of dw)
Compound	Leaves	seed cones
Phenolic acids
p-Hydroxybenzoic acid	415 ± 18^ab	(1.27 ± 0.06) × 10^3a
2,5-Dihydroxybenzoic acid	568 ± 23.3b	804 ± 43.6a
Protocatechuic acid	396 ± 6.98b	569 ± 29.0a
Vanillic acid	177 ± 2.13b	479 ± 24.2a
Gallic acid	295 ± 8.93b	351 ± 3.41a
Syringic acid	61.5 ± 1.34a	6.29 ± 0.31b
Cinnamic acid	30.6 ± 0.98b	68.4 ± 5.21a
o-Coumaric acid	<5	<5
p-Coumaric acid	302 ± 18.2b	456 ± 42.4a
Caffeic acid	21.3 ± 0.98b	46.2 ± 3.15a
Ferulic acid	241 ± 17.9b	694 ± 4.21a
3,4-Dimethoxycinnamic acid	29.8 ± 0.95	<25
Sinapic acid	<15	<15
5-O-caffeoylquinic acid	84.3 ± 6.45a	9.31 ± 0.45b
Flavonoids
Apigenin	(3.56 ± 0.09) × 10^3b	(6.99 ± 0.05) × 10^3a
Apigenin-7-O-glucoside	(1.39 ± 0.10) × 10^3b	(2.63 ± 0.09) × 10^3a
Apiin	<10	<10
Vitexin	117 ± 10.3b	162 ± 11.9a
Amentoflavone	(4.93 ± 0.20) × 10^3a	(1.09 ± 0.08) × 10^3b
Baicalein	<1000	<1000
Baicalin	<4	<4
Daidzein	<15	<15
Genistein	<50	<50
Isorhamnetin	6.00 ± 0.56a	3.11 ± 0.22b
Kaempferol	14.0 ± 0.98a	6.94 ± 0.07b
Kaempferol-3-O-glucoside	405 ± 32.1a	106 ± 2.00b
Chrysoeriol	33.7 ± 0.53b	50.2 ± 0.10a
Luteolin	65.7 ± 1.24a	64.7 ± 2.23a
Luteolin-7-O-glucoside	623 ± 42.9b	(1.46 ± 0.06) × 10^3a
Quercetin	151.1 ± 0.98a	34.7 ± 0.56b
Quercitrin	(142 ± 0.44) × 10^3a	(50.0 ± 0.36) × 10^3b
Quercetin-3-O-glucoside	(1.66 ± 0.04) × 10^3b	(2.30 ± 0.05) × 10^3a
Hyperoside	<5	<5
Rutin	(14.6 ± 1.3) × 10^3a	(3.87 ± 0.21) × 10^3b
Myricetin	67.8 ± 3.00a	2.28 ± 0.09b
Naringenin	22.6 ± 1.29b	46.6 ± 0.96a
Epicatechin	(11.1 ± 0.19) × 10^3a	(7.36 ± 0.08) × 10^3b
Catechin	(234 ± 0.44) × 10^3a	(56.5 ± 0.43) × 10^3b
Epigallocatechin gallate	<400	<400
Coumarins
Umbelliferone	404 ± 3.43a	3.45 ±0.08b
Aesculetin	1.74 ± 0.08b	5.68 ± 0.11a
Scopoletin	<5	<5
Lignans
Matairesinol	122 ± 9.11b	(4.94 ± 0.08) × 10^3a
Secoisolariciresinol	41.1 ± 0.80a	<35

^a Values are expressed as means ± SD of three measurements. Values within each row with different letters (a, b) differ significantly (p ≤ 0.05).

Namely, this study resulted in the chemical profile determination of significant 41.8% of leaves and 13.7% of seed cones extract dw, respectively, where the flavonoids were the most abundant (41.5% and 12.7% of dw, respectively). The following six flavonoids were present in considerable amounts in both extracts: catechin, quercitrin, epicatechin, rutin, apigenin, and amentoflavone. Phenolic acids were observed in small amounts (0.26% and 0.48% of dw of leaves and seed cones, respectively), with 2,5-dihydroxybenzoic and p-hydroxybenzoic acid being the most prevalent. Furthermore, lignans, mainly matairesinol, were present in moderate quantities, primarily in seed cones, while coumarins were found in minute amounts in both J. excelsa extracts. Generally, the leaves extract of J. excelsa was significantly richer in content of the most examined phenolics compared with the seed cones .

The results presented in this study are not in agreement with the only and limited study of the qualitative phenolics profile of the seed cones extract of J. excelsa.^[20] Specifically, Kilic et al.^[20] investigated 16 phenolics in J. excelsa seed cones collected in Turkey by using GC-MS. However, they did not detect p-hydroxybenzoic acid and matairesinol, while catechin was present in the 282-fold reduced amount compared with the result in this study. The disagreement between the results is probably due to the application of inappropriate methodology for phenolics detection. Apart from Kilic et al.,^[20] to date, there has been no other study of the phenolic profile of J. excelsa species, making this study the first one to reveal the phenolic profile of needles.

Taking into account that the determined dominant phenolics express great bioactivity, that it is confirmed that they prevent several diseases such as cancer, cardiovascular ailments, neurodegenerative diseases, obesity, and infectious disease, and that the juniper is already introduced into diet, the juniper-fortified food products could be considered as potent nutraceuticals in general health promotion.^[21,22,23] Furthermore, a considerable content of catechin, rutin, and quercetin indicates J. excelsa as a natural preservative for stopping food spoilage by bacteria and fungi or LP during preparation, storage, and distribution.^[1,24,25].

Chemical profile of J. excelsa essential oils

The chemical profile of the J. excelsa essential oils of leaves and juicy seed cones is shown in Table 2. A total of 22 compounds were identified in both essential oils, among which three compounds present in minute amounts in leaves were only tentatively identified as sesquiterpenes. Essential oils of J. excelsa needles and seed cones had a similar qualitative profile, dominated by monoterpene hydrocarbons. The most prevailing monoterpene hydrocarbons in both oils were α-pinene and limonene. Sesquiterpene hydrocarbons were present in oils in a lower percentage compared with monoterpene hydrocarbons, but noteworthy, with cedrol being the most abundant.

Table 2. Chemical profile of J. excelsa essential oils.

		Relative concentration (%)
LRI	Compound	Leaves	Seed cones
935	α-pinene	30.9	76.7
979	Β-pinene	0.7	2.4
989	Β-myrcene	0.9	2.5
1012	Δ-3-carene	3.9	0.8
1031	limonene	14.8	5.8
1062	Γ-terpinene	0.4	0.7
1093	α-terpinolene	0.5	0.7
1295	bornyl acetate	nd	0.5
1425	β-funebrene	2.2	0.6
1433	sesquiterpene	0.6	nd
1443	thujopsene	0.5	nd
1459	muurola-3,5-dien	0.2	nd
1480	trans-cadina-1(6),4-diene	0.4	nd
1491	germacrene D	1.1	nd
1491	tran-–muurola-4(14),5-diene	nd	0.7
1503	sesquiterpene	1.0	nd
1524	sesquiterpene	0.5	nd
1529	δ–cadinene	0.8	nd
1612	salvia-4(14)-en-1-one	2.4	0.4
1625	cedrol	37.4	7.7
1645	1-epi-cubenol	0.3	nd
2135	abietadien	0.2	0.5
	monoterpene hydrocarbons	52.1	89.6
	oxygenated monoterpenes	0.0	0.5
	sesquiterpene hydrocarbons	41.5	7.7
	oxygenated sesquiterpene	5.9	1.7
	diterpenes	0.2	0.5
	total	99.7	100.0

nd, not detected.

The composition of J. excelsa leaves and seed cones essential oils, originating from different parts of the Holarctic (i.e. Azerbaijan, Greece, Iran, Lebanon, Turkey), has been previously examined in numerous studies.^{[10,13,26,27,28,29,30,31]} The chemical profile of the J. excelsa essential oils presented in this work is generally in a good correlation with the references listed by others. However, slight differences in composition came as no surprise as it is well known that the chemical composition of volatile compounds is not simply genetically regulated and largely depends on non-genetic factors such as environmental conditions, growth, and collecting period.^[28,30,31] The chemical profiling of J. excelsa might help in its usage as a possible source of therapeutic agent, especially in traditional medicine. As an example, α-pinene and cedrol-rich oil could be effectively used as a local antiseptic.^[32] Moreover, J. excelsa oils could be used as naturally derived preservatives due to the limonene-rich content. Namely, limonene is capable of reducing microbial contamination and preventing food spoilage in a wide range of food, but without adversely altering the taste or function.^[.33]

Antioxidant potential of J. excelsa

All of the antioxidant activities determined in this study were concentration dependent, and the corresponding IC₅₀ values are shown in Table 3. Also worth mentioning is that while comparing the antioxidant, anti-inflammatory, and antimicrobial activities of extracts, essential oils, and standards, it was taken into account that the concentration of extracts and standards expressed in μg/mL corresponded to the concentration of essential oils expressed in nL/mL. Overall, our results demonstrated that both the leaves and the seed cones of J. excelsa showed noteworthy antioxidant effects, comparable to BHA. It is worth emphasising that the extracts and essential oils of both plant organs showed great potential to inhibit LP, while the leaves extract was significantly better in the neutralization of HO^• compared with BHA. These facts are particularly important with respect to food preservation, especially because HO^• is the most potent radical to initiate LP. In general, leaves appeared to be more potent then the seed cones in all of the assays applied. They also exhibited the higher overall contents of phenolics and flavonoid. It should be noted that, while the essential oils either showed weak activity in the DPPH test or could not be assayed at all using other tests due to the low solubility in aqueous media, they still exhibited a significant activity in the lipophilic environment. Due to the fact that not all of the assays applied were optimized for both polar and nonpolar substrates, the results are inconclusive as to whether the extracts or essential oils have shown a higher antioxidant capacity.

Table 3. Total phenolics and flavonoid content, antioxidant, and anti-inflammatory activities of extracts and essential oils of J. excelsa and standard compounds.

		J. excelsa extracts		J. excelsa essential oils
		leaves	seed cones	leaves	seed cones	Standards
Content	Total phenolics (mg gallic acid equivalents/g of dw)	187.73 ± 16.99^aa	94.71 ± 8.16 b	NA^b	NA	–
	Total flavonoids (mg quercetin equivalents/g of dw)	48.81 ± 4.18a	30.54 ± 2.92 b	NA	NA	–
Antioxidant activity	IC50 values (μg/mL for extracts and standard; nL/mL for essential oils)	Leaves	seed cones	leaves	seed cones	BHA
	DPPH^•	3.45 ± 0.21a	5.28 ± 0.16 b	(16.10 ± 1.01) × 10^3 d	(20.36 ± 2.62) × 10^3 d	12.39 ± 1.15 c
	HO^•	191.36 ± 7.71a	649.51 ± 44.79 c	na^c	na	301.05 ± 22.63 b
	O2^•–	41.45 ± 2.81a	54.50 ± 0.71 b	na	na	na
	^•NO	(0.51 ± 0.05) × 10^3a	(0.55 ± 0.03) × 10^3 a	na	na	na
	LP	40.41 ± 4.12b	249.16 ± 17.14 c	43.89 ± 5.10 b	59.82 ± 9.00 b	24.12 ± 2.52 a
	reducing power
	FRAP (mg of ascorbic acid equivalents /g of dw)	132.18 ± 11.25b	86.73 ± 2.95c	na	na	1057.24 ± 78.10 a
Anti–inflammatory activity	IC50 values (μg/mL for extracts and standard; nL/mL for essential oils)	leaves	seed cones	leaves	seed cones	aspirin	quercetin
	COX–1 pathway
	12–HHT	(2.74 ± 0.03) × 10^3 f	(1.57 ± 0.49) × 10^3 e	154.70 ± 7.83 c	490.94 ± 12.36 d	4.98 ± 0.43 a	22.45 ± 2.12 b
	TXB2	na	na	186.66 ± 8.24 d	408.57 ± 1.07 c	4.98 ± 0.06 a	53.69 ± 2.47 b
	PGE2	na	na	220.78 ± 4.20 d	517.30 ± 10.92 c	5.58 ± 0.53 a	12.75 ± 0.26 b
	12–LOX pathway
	12–HETE	(2.68 ± 0.26) × 10^3 e	(1.41 ± 0.16) × 10^3 d	132.14 ± 5.98 b	467.54 ± 0.64 c	na	7.44 ± 0.65 a

^a Values are means ± SD of three measurements. Means within each row with different letters (a–f) differ significantly (p ≤ 0.05).

^b NA, not applicable.

^c na, 50% inhibition not achieved.

This study presents a considerable contribution to knowledge about the antioxidant capacity of J. excelsa. To date, J. excelsa extracts and essential oils were examined using their reducing power, ability to scavenge DPPH^•, 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic radical and O₂^•–, or to inhibit LP and β-carotene bleaching, as well as the total phenolics and flavonoid contents.^{[12,13,29,34]} In most of the reports listed, the extracts and essential oils of J. excelsa expressed a lower antioxidant capacity compared with the results of this study. Moreover, our study is the first to evidence the ability of J. excelsa to stop LP or neutralize HO^•, which is crucially important for food-quality maintenance. Therefore, the antioxidant activity of the Juniperus species can enhance its benefits as nutraceuticals and food additive with considerable preservative potential.

Anti-inflammatory potential of J. excelsa

The extracts and essential oils of J. excelsa demonstrated a concentration-dependent inhibitory potential towards the synthesis of 12-HHT, TXB₂, PGE₂, and 12-HETE inflammatory mediators (Table 3). Surprisingly, terpene mixtures – essential oils, especially those of leaves, demonstrated a significantly superior anti-inflammatory potential with the phenolic mixtures – extracts. While the IC₅₀ values of J. excelsa essential oils were significantly higher than those of aspirin and quercetin, it should be noted that essential oils represent raw mixtures of active components and inactive bulk matter, and hence the expressed activity of essential oils is considered significant. This report is the first to evidence a noteworthy anti-inflammatory capacity of the J. excelsa essential oils and a general potency of J. excelsa to affect AA metabolism. Namely, the anti-inflammatory activity of the extracts of J. excelsa was previously reported in only one study,^[15] but not towards the inhibition of eicosanoid production. Due to the fact that eicosanoids induce inflammation and support tumorigenesis and that J. excelsa is shown to be particularly potent in inhibition of the eicosanoid biosynthesis, usage of this species as nutraceutical is firmly supported by this study.

Antimicrobial potential of J. excelsa

The antimicrobial activity of the J. excelsa essential oils was evaluated against the Gram-positive and Gram-negative bacterial strains, which are clinically relevant and/or cause foodborne illnesses.^[5] The antimicrobial activity of the essential oils against the Gram-negative bacteria was moderate, while in preliminary disc diffusion assay only the leaves showed considerable inhibition zones (Table 4). On the contrary, both essential oils were effective in the growth inhibition of the examined Gram-positive bacteria, i.e. S. aureus and C. perfringens (Table 4). S. aureus was more sensitive to J. excelsa compared with C. perfringens. Furthermore, the leaves showed better antimicrobial activity than the seed cones oil. Antibacterial activity of the J. excelsa essential oils against the foodborne pathogenic bacteria C. perfringens was previously reported only in one study, while it had earlier been studied more extensively against S. aureus and E. coli.^{[27,29,34,35]} Generally, the essential oils from our study revealed better antimicrobial activity towards the examined bacteria strains compared with the earlier mentioned studies. Furthermore, this study is the first to evidence the antimicrobial activity of J. excelsa towards the clinically relevant bacteria A. baumannii and S. Enteritidis. Considering the fact that the investigated essential oils showed a considerable effect against the Gram-positive bacteria, which cause diarrhoea or intoxication by enterotoxins, further investigations are necessary for determining the terpene constituents responsible for the activity observed, especially against the growth of S. aureus. Nevertheless, the proven antimicrobial activity of the Juniperus species against the examined strains can contribute to their general application as a preservative in the food industry, as well as a nutraceutical.

Table 4. Antimicrobial activity of J. excelsa essential oils.

				Test microorganisms
Applied assay		Tested sample		S. aureus	C. perfringens	A. baumannii	E. coli	S. Enteritidis
Disc diffusion assay	Inhibition zone diameter (mm)	Essential oil	leaves	22.0 ± 0.0^a c	24.0 ± 0.0 b	8.0 ± 1.6 c	8.7 ± 1.5 c	8.7 ± 0.6 c
			seed cones	14.7 ± 1.2 d	18.7 ± 0.6 d	abs^b	abs	abs
		ceftriaxone^c		32.0 ± 0.0 a	30.5 ± 0.4 a	14.3 ± 0.3 b	27.7 ± 1.9 a	24.7 ± 0.3 a
		amikacin^c		28.0 ± 1.0 b	21.0 ± 0.0 c	21.3 ± 0.3 a	20.0 ± 0.0 b	19.7 ± 0.3 b
Agar dilution assay	Minimal inhibitory concentration (μL/mL for essential oil; mg/mL for amikacin)	Essential oil	leaves	4 b	16 b	> 16	> 16	> 16
			seed cones	8 c	16 b	> 16	> 16	> 16
		amikacin		0.004 a	0.008 a	0.004	0.004	0.002

^a Values are means ± SD of three measurements. Means within each column with different letters (a–d) differ significantly (p ≤ 0.05).

^b abs, absence of inhibition zone.

^c disc activity 30 µg.

Conclusion

Nowadays, there is a growing interest in the usage of plant-derived antioxidants mostly due to the existing concerns over possible adverse health effects caused by the use of synthetic antioxidants. Thanks to the proven bioactivity and chemical profile of the leaves and seed cones, the J. excelsa species could be successfully used not only as a spice in enhancing the flavour of a particular food, but also to extend the shelf life of the various food products. Because of its high antioxidant and antimicrobial properties, it could be particularly suitable as a preservative for foodstuffs highly susceptible to LP or spoilage due to microbial contamination, such as meat, mayonnaise, dressings, frying oils, bakery and dairy products, beverages, etc. Finally, due to its considerable bioactivity, especially in bacterial growth inhibition and eicosanoid biosynthesis, as well as the high content of phenolics with proven health benefits, the use of J. excelsa as a food additive with valuable nutraceutical properties should be supported.

Acknowledgements

We wish to thank the Institute for Blood Transfusion of Vojvodina, Novi Sad, Serbia, for providing platelets. We are thankful to Gordana Vlahović, MSc, for editorial assistance.

Additional information

Funding

This research work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant No. 172058).