Essential Oils and Extracts of Juniperus macrocarpa Sm. and Juniperus oxycedrus L.: Comparative Phytochemical Composition and Anti-Proliferative and Antioxidant Activities

In this work, we conducted a comparative phytochemical, chemotaxonomic, and biological study of essential oils (EOs) and extracts (ethyl acetate and methanol) obtained from the leaves of Juniperus macrocarpa and J. oxycedrus. The dominant compounds of J. macrocarpa EO, analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), are α-pinene, sabinene, manoyl oxide, and germacrene D, whereas α-pinene, limonene, (Z,E)-farnesol, β-pinene, and γ-cadinene are the most representative volatiles of J. oxycedrus EOs. A multivariate analysis of EOs, included a selection of literature data comparing our samples to samples of J. oxycedrus/macrocarpa/deltoides from the Mediterranean area, was performed. As evident by high-performance liquid chromatography (HPLC) analyses, apigenin, (−)-epicatechin, and luteolin were abundant in J. oxycedrus extracts, while gallic acid, kaempferol-3-O-glucoside, and protocatechuic acid were the dominant constituents of J. macrocarpa extracts. EOs and extracts have been investigated for their potential antioxidant properties and anti-proliferative activity against lung adenocarcinoma (A549), breast cancer (MCF-7 and MDA-MB-231), and lung large cell carcinoma (COR-L23) human cell lines. The methanol and ethyl acetate extracts of J. oxycedrus exerted the most valuable antioxidant activity and exhibited the most promising activity against the COR-L23 cell line with an IC50 of 26.0 and 39.1 μg/mL, respectively, lower than that obtained with the positive control (IC50 of 45.5 μg/mL). To the best of our knowledge, this is the first report highlighting the anti-proliferative activity of J. oxycedrus and J. macrocarpa extracts against this lung cancer cell line. Our results indicate that J. oxycedrus may be considered a source of natural compounds with antioxidant and anti-proliferative effects that could be suitable for future applications.


Introduction
The genus Juniperus (Cupressaceae) is traditionally divided into three sections: J. sect. Caryocedrus Endl., J. sect. Juniperus, and J. sect. Sabina Spach [1]. The Juniperus oxycedrus group is included within J. sect. Juniperus [1] and includes two chemotaxonomically and phylogenetically close Mediterranean species: Juniperus oxycedrus L. and J. macrocarpa Sm. (Figure 1), with the latter often treated as a subspecies of the former. A third species included in this group, J. deltoids Adams, is hardly morphologically discernible from J. oxycedrus, but some molecular evidence suggests a late Miocene divergence [2]. Considering these features, we referred to Juniperus oxycedrus s.l. when the literature data were not sufficient to define an exact species. A literature survey found many studies that analysed the composition of the essential oils (EOs) from berries and leaves of Juniperus species and their bioactivities, mainly antibacterial, antiviral, antifungal, and antioxidant properties [3,7,[10][11][12][13][14][15][16][17][18][19][20][21]. These EOs are used for their biological activity but also in the food and cosmetic industries.
Cancer is one of the most important health problems of our community, and natural compounds are considered valuable candidates for the development of new intervention approaches to improve the therapeutic index and address the frequent occurrence of chemo-resistance to current anti-cancer therapies.
Over the last decades, scientific progress has highlighted the prominent role of reactive oxygen species (ROS) in the pathogenesis of inflammatory diseases, neurodegenerative diseases, and cancer [26]. Particularly, in the early stages of cancer, excessive ROS Juniperus oxycedrus L. (prickly juniper, plum juniper, cade juniper, red-berry juniper, cada) is a shrub or small tree native to the western Mediterranean region, from Morocco and Portugal eastward to southern Italy, growing on a variety of rocky sites from sea level up to 1600 m altitude. J. macrocarpa (maritime juniper), which is morphologically [3], molecularly [2], and genetically [4] differentiated from J. oxycedrus, is a Mediterranean species growing on coastal sand dunes. Juniperus false fruits, female cones-improperly called "berries"-are used as a spice, mainly in European cuisine; they are used in Northern European and particularly Scandinavian cuisine to impart a sharp, clear flavour to meat dishes [5]. J. oxycedrus s.l. berries have widely been used in traditional medicine for the treatment of gastrointestinal disorders, diabetes, common colds, as expectorant in cough, to treat calcinosis in joints, as diuretic to pass kidney stones, against urinary inflammations, haemorrhoids, and as hypoglycaemic; leaves and berries are applied externally for parasitic disease [5][6][7][8][9].
Cancer is one of the most important health problems of our community, and natural compounds are considered valuable candidates for the development of new intervention approaches to improve the therapeutic index and address the frequent occurrence of chemo-resistance to current anti-cancer therapies.
Over the last decades, scientific progress has highlighted the prominent role of reactive oxygen species (ROS) in the pathogenesis of inflammatory diseases, neurodegenerative diseases, and cancer [26]. Particularly, in the early stages of cancer, excessive ROS production and related oxidative stress are considered as important molecular hallmarks. Hence, the initial stages of carcinogenesis could be suppressed by antioxidants.
In spite of the extensive studies on the bioactivities of Juniperus EOs, the anti-proliferative activity of polar extracts is still less investigated. For this reason, as part of ongoing work to investigate medicinal plants as sources of antioxidant and anti-proliferative agents [27,28], the present study aimed to examine the anti-proliferative effects against human lung adenocarcinoma (A549), human breast cancer ER+ (MCF-7), triple negative breast adenocarcinoma (MDA-MB-231), and human lung large cell carcinoma (COR-L23) cell lines and the antioxidant activity of EOs and polar extracts from two Juniperus species with globally recognized health benefits from Italy, J. macrocarpa and J. oxycedrus, in relation to their chemical profiles analysed by gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography-diode-array detector (HPLC-DAD).
The main goal of our research was to support the use of these Juniperus species as valuable sources of bioactive compounds with potential benefits as anti-proliferative agents.

Chemical Composition of Essential Oils
J. macrocarpa and J. oxycedrus fresh leaves were subjected to hydro-distillation and exhaustive maceration by using two solvents at different polarities such as ethyl acetate and methanol.
Essential oils were obtained with yields of 0.2 and 0.3% for J. macrocarpa and J. oxycedrus, respectively. Analysing polar extracts, the highest extraction yield was obtained by using methanol with yields of 10.4 and 10.8% for J. macrocarpa and J. oxycedrus, respectively. Yields of 6.1 and 4.2% for J. macrocarpa and J. oxycedrus, respectively, were obtained by using ethyl acetate as solvent for maceration.
The essential oils of J. macrocarpa and J. oxycedrus were analysed by GC and GC-MS. Table 1 reports the main identified compounds listed in order of elution on an HP5 MS column with their retention index, identification methods, and percentage contribution. Forty-seven constituents (accounting for 90.2% of the total composition of the essential oil) were tentatively identified in the essential oil of J. macrocarpa in which the dominant compounds were monoterpene hydrocarbons (57.8%), followed by diterpenes (11.9%) and sesquiterpene hydrocarbons (8.7%). α-Pinene (25.3%), sabinene (8.2%), and manoyl oxide (6.6%) were the most abundant compounds.
Forty-four volatiles (accounting for 97.0% of the total composition of the essential oil) were identified in the essential oil of J. oxycedrus. α-Pinene (36.9%), limonene (6.5%), (Z,E)-farnesol (6.3%), and β-pinene (5.5%) were the most representative constituents. As for J. macrocarpa, the dominant class of constituents was represented by monoterpene hydrocarbons (57.5%). Sabinene was identified in the essential oil of J. macrocarpa but not in J. oxycredus. The amounts of α-pinene, β-pinene, limonene, γ-cadinene, δ-cadinene, and caryophyllene oxide detected in J. macrocarpa were lower than those characterizing the essential oil of J. oxycredus. Conversely, higher amounts of p-cymene, (E)-β-ocimene, terpinolene, α-terpineol, manoyl oxide, and germacrene D were found in J. macrocarpa. A multivariate analysis of essential oils included a selection of literature data comparing our samples to samples of J. oxycedrus/macrocarpa/deltoides from the Mediterranean area was performed. On the first two axes of principal coordinate analysis (PCoA, Figure 2) (explaining 71.9 and 8.9% of variability), J. deltoides was separated from the other taxa on the first axis, which showed most of the variability explained (71.9%).
paring our samples to samples of J. oxycedrus/macrocarpa/deltoides from the Mediterranean area was performed. On the first two axes of principal coordinate analysis (PCoA, Figure  2) (explaining 71.9 and 8.9% of variability), J. deltoides was separated from the other taxa on the first axis, which showed most of the variability explained (71.9%).
The analysis of J. oxycedrus and J. macrocarpa essential oils resulted in largely overlapping sections on the left side of the scatterplot, with no way to distinguish them.
Our J. oxycedrus sample was inside the variability of the species, whereas our sample of J. macrocarpa was shown to be differentiated from the J. oxycedrus/J. macrocarpa group on the second axis. However, the second axis, which explained 8.9% of the variability, did not reflect any particular taxonomic pattern, and it was linked to the variability of oils such as manoyl-oxide, abietatriene, and abieta-7,13-diene, which do not discriminate among taxa (p > 0.1). Cluster analysis confirmed the best grouping number of two ( Figure 3), which was also significant (silhouette average width = 0.75). The first cluster was formed by J. deltoides samples, the second by mainly J. oxycedrus/J. macrocarpa samples. The second cluster showed the first subcluster with our J. macrocarpa sample well-separated from the others The analysis of J. oxycedrus and J. macrocarpa essential oils resulted in largely overlapping sections on the left side of the scatterplot, with no way to distinguish them.
Our J. oxycedrus sample was inside the variability of the species, whereas our sample of J. macrocarpa was shown to be differentiated from the J. oxycedrus/J. macrocarpa group on the second axis. However, the second axis, which explained 8.9% of the variability, did not reflect any particular taxonomic pattern, and it was linked to the variability of oils such as manoyl-oxide, abietatriene, and abieta-7,13-diene, which do not discriminate among taxa (p > 0.1).
Cluster analysis confirmed the best grouping number of two ( Figure 3), which was also significant (silhouette average width = 0.75). The first cluster was formed by J. deltoides samples, the second by mainly J. oxycedrus/J. macrocarpa samples. The second cluster showed the first subcluster with our J. macrocarpa sample well-separated from the others and a second subcluster, which included all J. deltoides samples that were not included in the first cluster together with our J. oxycedrus sample.
In the third subcluster, all other J. oxycedrus/J. macrocarpa samples were included with no differentiation between the species.
Among selected essential oil constituents, limonene was the one most characteristic for J. deltoides (22.22 ± 6.8), and β-pinene for the J. oxycedrus/J. macrocarpa group. Higher content in sabinene and manoyl oxide, together with lower content in β-caryophyllene, distinguished the Calabrian J. macrocarpa sample from the J. oxycedrus/J. macrocarpa group. and a second subcluster, which included all J. deltoides samples that were not included in the first cluster together with our J. oxycedrus sample.
In the third subcluster, all other J. oxycedrus/J. macrocarpa samples were included with no differentiation between the species. Among selected essential oil constituents, limonene was the one most characteristic for J. deltoides (22.22 ± 6.8), and β-pinene for the J. oxycedrus/J. macrocarpa group. Higher content in sabinene and manoyl oxide, together with lower content in β-caryophyllene, distinguished the Calabrian J. macrocarpa sample from the J. oxycedrus/J. macrocarpa group.

The Chemical Profiles of Juniperus Polar Extracts
J. macrocarpa and J. oxycedrus were extracted by maceration using two solvents with different polarities. With the hypothesis that the constituents of these species could be efficiently extracted using solvents with different polarities, we evaluated the efficiency of ethyl acetate and methanol in the recovery of phytochemicals from Juniperus extracts.
J. oxycedrus and J. macrocarpa showed similar flavonoid and phenolic compound fingerprints, whereas they differed in terms of quantitative content. In light of the obtained data, the major identified constituents belonging to the flavonoid class were (-)-epicatechin, rutin, catechin, quercetin, quercetin-3-O-glucoside, and luteolin.

The Chemical Profiles of Juniperus Polar Extracts
J. macrocarpa and J. oxycedrus were extracted by maceration using two solvents with different polarities. With the hypothesis that the constituents of these species could be efficiently extracted using solvents with different polarities, we evaluated the efficiency of ethyl acetate and methanol in the recovery of phytochemicals from Juniperus extracts.

Antioxidant and Anti-Proliferative Properties
Juniperus species were investigated herein for their antioxidant and anti-proliferative properties. Cancer is a global challenge with a high impact on human health, causing morbidity and mortality. Although important advances have been obtained in early cancer diagnosis and cancer treatment, there is still a need for new compounds that contribute to and improve the therapeutic approaches in actual use.
Natural compounds represent a countless source of new molecules that can be used as anti-cancer agents once their activity, bioavailability, and toxicity are demonstrated to be acceptable. The ability of many natural compounds to act also as antioxidant agents could enhance their anti-cancer activity. In fact, several studies have demonstrated that antioxidant activities such as free radical scavenging, lipid peroxidation, and metal chelating activities from natural extracts can enhance the anti-cancer properties of many anticancer drugs [29,30].
The study of antioxidant properties, mainly of antioxidant agents that are multifunctional or mixtures that act in complex systems, cannot be adequately evaluated by a simple antioxidant assay without due regard for the many variables that may influence the results. Several procedures may be required to assess such antioxidant effects. For these reasons, the potential antioxidant activities of Juniperus species were analysed by using four in vitro assays, namely the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and β-carotene bleaching tests. A concentration-response relationship was observed for all tested samples. The IC 50 values are given in Table 3. Generally, J. oxycedrus was more active than J. macrocarpa. In particular, the ethyl acetate and methanol extracts exhibited promising ABTS radical scavenging activity with IC 50 values of 9.3 and 6.2 µg/mL, respectively.
Lower radical scavenging activity was observed in the DPPH tests, where IC 50 values of 19.7 and 20.6 µg/mL, for ethyl acetate and methanol, respectively, were found. Moreover, the ethyl acetate extract was shown to possess the ability to protect lipids from peroxidation, as evidenced by the β-carotene bleaching assay (IC 50 Table 4.  Lung cancer is the leading cause of about 18.6% of total cancer deaths worldwide. Non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) are the two major groups of lung cancer based on a histological classification. NSCLC represents 80% of all lung cancer cases and is subdivided into adenocarcinoma, large cell carcinoma, and squamous cell carcinoma. The management of the treatment of lung cancer is often difficult due to the development of drug resistance, non-specific targeting of the anti-cancer drugs, and/or drug-drug interactions. Therefore, the search for new and active compounds is necessary.
J. oxycedrus methanol extract was the only extract able to inhibit proliferation of human lung adenocarcinoma cell line (A549), with an IC 50 value of 87.9 µg/mL. J. oxycedrus EO was inactive against all tested cell lines.

Discussion
In this work, a comparative phytochemical, chemotaxonomic, and biological study of essential oils (EOs) and extracts (ethyl acetate and methanol) obtained from the leaves of Juniperus macrocarpa and J. oxycedrus was conducted.
Numerous studies are present in the literature on the chemical composition of J. macrocarpa EOs ( Table 5). The content of α-pinene in the J. macrocarpa EO analysed in our work was in agreement with that found by Valentini et al. [18], who reported α-pinene content of 22.8% in a sample collected in Puglia (southern Italy). The same authors also analysed J. macrocarpa aerial parts from the Abruzzo hills (central Italy) in two different months, emphasizing how the plant harvesting period, as well as the altitude at which it grew, could affect the content of active ingredients. Italy [18] Both samples collected in Abruzzo were rich in α-pinene, showing high percentages in the range 73.5-81.3%. Although similar in α-pinene content to the sample analysed by Valentini et al. [18], the Apulian oil showed some peculiarities especially regarding the identification of 1,8-cineole (9.1%), a compound not found in our sample, and α-terpineol (18.7%), a compound identified in our oil with a lower percentage of 2.9%. Interestingly, the Abruzzo J. macrocarpa oils revealed the presence of the sesquiterpene γ-muurolene (2.6%), which was not identified in either the Apulian oil or in our sample, suggesting that this sesquiterpene is characteristic of J. macrocarpa grown in the hills rather than at sea level. On the other hand, our J. macrocarpa essential oil was richer in p-cymene (13.2%) and sabinene (8.2%) compared with the oil samples analysed by Valentini et al. [18].
The chemical variability of J. macrocarpa essential oils from Italy is evident also in other countries such as Greece, Tunisia and Turkey [7,16,17,19]. The Greek essential oils showed α-pinene and cedrol as the dominant compounds, both differing in content by about double depending on whether they came from southern or south-eastern Greece. Voulrioti-Araopi et al. [19], analysing the essential oil of J. macrocarpa collected in Athens (south-eastern Greece), showed an α-pinene content of 58.0%, a value about double that described by Stassi et al. [17].
Medini et al. [16] analysed the EOs extracted from two samples of J. macrocarpa collected in north and central Tunisia (Laazib and Hawaria, respectively), not highlighting a notable difference in their phytochemical profiles, with α-pinene (15.9-22.8%) and sabinene (9.1-12.1%) as major compounds. Sezik et al. [7], who analysed three samples of EOs extracted from the leaves of J. macrocarpa collected in Turkey in three months (May, August, and October), confirmed the existence of seasonal variability in the phytochemical profile of this plant species. In particular, the authors highlighted that manoyl oxide (7.7-21.9%) was the main compound, followed by α-pinene (7.2-11.1%) and cedrol (2.3-9.7%). Except for α-pinene with the highest percentage (11.1%) in October, the highest values were observed in samples harvested in August.
Characteristic were the phytochemical profiles of J. macrocarpa oils from Croatia and Algeria [12,15]. In detail, the essential oil reported by Lesjak et al. [15] showed α-pinene (49.4%) and germacrene D (18.1%) as the main components, both present at very high percentages compared to the values shown in Table 5 in reference to the sample analysed by us, followed by β-phellandrene (3.8%), present in lower percentages in the samples from other countries and absent from the Italian samples. Djebaili et al. [12] highlighted germacrene D as the main component of the oil from Algeria, with a percentage of 21.3%, much higher than that found in the oil analysed by us.
Among the main components in the EOs from Spain, Adams et al. [9] highlighted α-pinene (22.6%), with a content very similar to the oils from Italy, and terpinen-4-ol (7.3%), the latter showing content clearly higher than that in the oil analysed by us. Sabinene showed the highest percentage content of 26.5%, a value about three times higher than that reported by us. Sabinene is a characteristic component of J. macrocarpa that is not found in J. oxycedrus, a peculiarity also observed by Valentini et al. [18].
From analysis of Table 5, it is also evident that there are other clear variations in the phytochemical profiles of these two plants. A first fundamental difference concerns the presence of different typical compounds in one or the other species. Eight compounds-sabinene, tricyclene, p-mentha-1,5-dien-8-ol, verbenone, (−)-carvone, piperitone, (−)-bornyl acetate, and β-cubebene-were exclusively present in J. macrocarpa. On the other hand, four compounds not found in J. macrocarpa were identified as distinctive of J. oxycedrus: myrtenal, α-gurjunene, trans-α-bergamotene, and (Z)-phythol. Besides these compounds, other interesting differences in the content of other constituents can be highlighted. Indeed, p-cymene showed a content about 13 times higher in J. macrocarpa compared to that in J. oxycedrus. The contents of α-terpineol, (E)-β-ocimene, manoyl oxide, and α-phellandrene in J. macrocarpa were also very high, each showing values about three times higher than in J. oxycedrus.
Limonene and α-pinene were also the main components in the J. oxycedrus essential oils.
Adams et al. [9] analysed and compared the phytochemical profiles of the essential oils extracted from the leaves of four J. oxycedrus samples, two of which were collected in Greece (north and south) and two in Spain. The oils were dominated by α-pinene and limonene, with moderate amounts of β-pinene, myrcene, p-cymene, β-phellandrene, and manoyl oxide. Adams et al. [9] evidenced a variability in the composition, especially in relation to the amounts of α-pinene, limonene, and δ-3-carene. In particular, the oil from southern Greece was found to be richer in α-pinene (42.7%) than that from northern Greece (25.3%), while the oils from Spain were found to be very similar, with α-pinene content of just over 40%. Both Spanish oils and southern Greek oils had content of this monoterpene very similar to that found by us (36.9%).
Interesting differences were reported for limonene content. In fact, the northern Greek oil showed a content of 27.7%, unlike the oil from southern Greece with a content of 17.1%. Spanish oils showed a percentage of limonene of 4.5%, similar to the percentage we observed in our sample. The greatest differences were found for the bicyclic terpene δ-3-carene, since the oil from northern Greece and Spain had small traces compared to the southern Greek oil, which showed a high percentage content of 13.7%. Of particular interest was the absence of (E,Z)-farnesol from the Greek and Spanish EOs examined by Adams et al. [9] as well as from those analysed in other countries.
Adams et al. [31], in a comparative study of the phytochemical profiles of the oils extracted from the leaves of J. oxycedrus, stated that generally, the oils of this species originating from western Mediterranean countries (Morocco, Spain, France, and Portugal) had a higher content of α-pinene (40-50%) than the oils originating from eastern Mediterranean countries (Italy, Greece, Croatia, Turkey) (20-40%).
Important differences were also present among plants of J. oxycedrus collected in different regions of Turkey. In this regard, Hayta and Bagci [14] analysed the essential oils extracted by hydro-distillation from different parts of J. oxycedrus, including the leaves.
Data showed also in this case that α-pinene and limonene were the most abundant monoterpenes. J. oxycedrus essential oil from Algeria showed trans-pinocarveol (7.0%) and cis-verbenol (6.3%) as characteristic compounds, not found in such quantities in samples from other Mediterranean regions [13]. In conclusion, J. oxycedrus oil from different regions of the Mediterranean is very variable in terms of chemical composition. Surely, this variability is related to the peculiar climatic characteristics that differ from region to region, and to factors related to the type of soil as well as to the altitude where this plant grows.
J. oxycedrus and J. macrocarpa showed similar flavonoid and phenolic compound fingerprints, whereas they differed in terms of quantitative content. In light of the obtained data, the major identified constituents belonging to the flavonoid class were (−)-epicatechin, rutin, catechin, quercetin, quercetin-3-O-glucoside, and luteolin.
The high (−)-epicatechin and quercetin-3-O-glucoside contents were previously demonstrated [15,32]. Taviano et al. [33] found high quantities of these compounds also in the fruits of J. macrocarpa. Protocatechuic acid was found to be the most abundant, with a content of 3355 µg/g.
There are numerous previous reports on the composition of the EOs, while few studies have investigated the composition of polar extracts of Juniperus species. Among them, Yaglioglu et al. [34] confirmed that rutin is one of the main phenolic compounds that characterize the methanol extract of the J. oxycedrus, followed by catechin.
In our methanol extract of J. oxycedrus, the amount of catechin was significantly less than that of rutin. This result is in disagreement with that reported by Yaglioglu et al. [34]. In fact, in this work, the methanol extract of J. oxycedrus showed about twice the catechin content (274.85 mg/g) compared to that of rutin (146.57 mg/g). This divergence was certainly due to differences in the locations where the plants were grown and collected, and, therefore, to climatic and weather differences to which the plants were exposed, as well as to the types of soil in which they grew. Apigenin was more abundant in J. oxycedrus extracts than in those from J. macrocarpa, in contrast to data obtained by Lesjak et al. [15] that found a content of 1407.21 µg/g. In our work, J. oxycedrus and J. macrocarpa extracts showed promising antioxidant effects. Previously, Živić et al. [35] investigated the DPPH radical scavenging activity of different extracts obtained from J. oxycedrus berries. The greatest ability to neutralize DPPH radicals was found with ethanol and ethyl acetate extracts, with IC 50 values of 2.55 and 106.40 µg/mL, respectively, whereas a lower activity was observed with chloroform extract (IC 50 of 257.66 µg/mL). It is evident that the extracts obtained with more polar solvents possessed higher antioxidant activity due to the high TPC. More recently, the antioxidant potential of J. phoenicea subsp. phoenicea and J. oxycedrus were analysed. In particular, the highest DPPH radical scavenging potential was observed with J. oxycedrus fruit essential oil, with an IC 50 value of 20.2 mg/mL, followed by J. phoenicea leaves (36.1 mg/mL). At a concentration of 9.2 mg/mL, all tested essential oils were able to reduce Fe 3+ to Fe 2+ [36].
The FRAP ability was confirmed also by Gök et al. [37], who investigated the J. macrocarpa branch and leaf ethyl acetate extracts, finding absorbance values of 3.12 and 3.19, respectively at 3 mg/mL, as well as methanol extracts (absorbance values of 3.31 and 3.02 for branch and leaf extracts, respectively). All extracts were characterized by higher absorbance values than that found for positive control ascorbic acid. A FRAP value of 71.50 mg of ascorbic acid equivalents/g of dried weight was recorded for J. macrocarpa leaf 80% aqueous methanol extract by Lesjak et al. [15], whereas no FRAP activity was observed with essential oil.
Several Juniperus species were found to be able to exert anti-proliferative activity, and different extracts were investigated. The cytotoxic activity of J. macrocarpa against the human lung adenocarcinoma cell line was confirmed by Calderón-Montaño et al. [38], who found IC 50 values of 3.7 and 146.1 mg/mL for ethanol/ethyl acetate/water extracts of the aerial parts and monosperma cones, respectively. Recently, Lai et al. [39] demonstrated that J. communis fruit extract exerted promising cytotoxicity activity against colorectal cancer cells with IC 50 values of 54.32 and 27.3 µg/mL for HT-29 and CT-26 after 72 h of exposure, respectively. Moreover, the phytocomplex demonstrated a synergistic effect when combined with 5-fluorouracil. An in vivo study confirmed that J. communis extract induced cell cycle arrest at the G 0 /G 1 phase via regulation of p53/p21 and CDK4/cyclin D1 and induced cell apoptosis via the extrinsic and intrinsic pathways.
A similar mechanism was observed also with J. communis extract against human oral squamous cancer cells (OECM-1) with an IC 50 value of 45.83 µg/mL after 72 h of exposure [40].
Yaglioglu et al. [42] investigated the essential oils obtained from the needles and cones of four Juniperus species from Turkey-J. oxycedrus ssp. oxycedrus, J. foetidissima, J. excelsa, and J. communis-for their anti-proliferative activities against HeLa (human cervical carcinoma) and C6 (rat brain tumour) cell lines. The results revealed that, generally, the needles had better anti-proliferative activity than the cones against both cell lines. However, the needles had strong anti-proliferative activities selective against the C6 cells. The essential oil of the needles of J. excelsa showed strong anti-proliferative activity against both HeLa and C6 cell lines. Recently, we analysed the anti-proliferative activity of nhexane and dichloromethane extracts of J. macrocarpa and J. oxycedrus [43]. Our results highlighted the promising activity of J. oxycedrus. In fact, both J. oxycedrus n-hexane and dichloromethane extracts exhibited cytotoxic activity against COR-L23 cells with IC 50 values of 26.9 and 39.3 µg/mL, respectively, while J. macrocarpa extracts were not active at the highest tested concentration, except for the n-hexane extract with an IC 50 value of 75.1 µg/mL.

Plant Materials and Extraction
The Fresh leaves of J. macrocarpa (480 g) and J. oxycedrus (480 g) were steam distilled for 3 h using a clevenger-type apparatus. White-yellow essential oils were obtained. The essential oils were dried over anhydrous sodium sulphate and stored in hermetically sealed brown glass bottles at 4 • C before analyses.
Fresh leaves of J. macrocarpa (180 g) and J. oxycedrus (180 g) were extracted by exhaustive maceration (700 mL, 4 × 72 h) using ethyl acetate and methanol as solvents. Two extracts for each Juniperus species were then obtained after the removal of the extraction solvents at reduced pressure, whose weights were 10.9 and 18.7 g for the ethyl acetate and methanol extracts of J. macrocarpa, respectively, and 7.6 and 19.4 g for the ethyl acetate and methanol extracts of J. oxycedrus, respectively. Dried extracts were kept in brown bottles at 4 • C before analyses.

Phytochemical Screening
Essential oils (EOs) of J. macrocarpa and J. oxycedrus were analysed using a Hewlett-Packard 6890 gas chromatograph and a Hewlett-Packard 5973 mass selective detector equipped with an HP-5 MS column (30 m × 0.25 mm i.d., film thickness 0.25 µm) [44]. The oven temperature was isothermally programmed at 50 • C for 5 min, rising from 50 to 250 • C at 13 • C/min, and then held isothermally at 250 • C for 10 min. The carrier gas was helium (1.0 mL/min), ionization of the sample components, EI (70 eV). The tentative identification of the compounds was based on the comparison of their retention indices, in relation to the retention times of a series of n-alkanes (C 9 -C 31 ), with those of the literature or with those of authentic compounds [45,46]. Further identification was made by comparison of their mass spectra with those stored in the Wiley 138 and NIST98 libraries or with published mass spectra. The GC analyses were performed using a Shimadzu GC17A apparatus controlled by Borwin Software and equipped with a flame ionization detector (FID) and an HP-5 column (30 m, 0.25 mm i.d., film thickness 0.25 µm) (Shimadzu, Milan, Italy). The oven temperature was programmed as reported for GC-MS analyses. The carrier gas was nitrogen (1.0 mL/min). The relative concentrations of the components were calculated based on the GC peak areas without using correction factors.
Apigenin, caffeic acid, (+)-catechin, chlorogenic acid, (−)-epicatechin, gallic acid, kaempferol, kaempferol-3-O-glucoside, luteolin, neochlorogenic acid, protocatechuic acid, quercetin, quercetin-3-O-glucoside, rutin, syringic acid, and vanillic acid were chosen as reference compounds and quantified. A standard mixture was prepared by adding an accurately weighed amount of each compound (100 mg) to a 100 mL volumetric flask and was brought to the mark with methanol (9:1). A straight calibration for each standard was obtained by analysing the standard solution diluted at different concentrations. All solutions were filtered through a 0.45 µm membrane filter (GMF Whatman) and injected into the HPLC system to determine retention times. Identification and quantification were carried out based on recorded retention times in comparison with authentic standards. Analyses were performed in triplicate.
In the β-carotene bleaching assay, a mixture of linoleic acid, β-carotene, and Tween 20 was prepared and after evaporation of the solvent and dilution with water, the obtained emulsion and Juniperus samples were added into tubes that were placed in a water bath at 45 • C [48]. The absorbance was measured at 470 nm at the initial time (t = 0) and after 30 and 60 min of incubation. Propyl gallate was used as a positive control.
In the ABTS test, ABTS radical cation was produced by the reaction of an ABTS solution and potassium persulphate [48]. The obtained solution was diluted with ethanol to an absorbance of 0.70 at 734 nm, and the ABTS scavenging ability was calculated according to the equation: [(A 0 − A 1 )/A 0 ] × 100, where A 0 is the absorbance of the control reaction and A is the absorbance in the presence of sample. Ascorbic acid was used as positive control.
The DPPH radical scavenging activity was measured at 517 nm, with ascorbic acid used as a positive control [48]. The DPPH radical scavenging activity, expressed as a percentage, was calculated using the formula: [(A 0 − A 1 )/A 0 ] × 100, where A 0 is the absorbance of the blank and A1 is the absorbance in the presence of the extract.
In the FRAP assay, based on the reaction that involves 2,4,6-tripyridyl-s-triazine)-Fe 3+ (TPTZ) complex, a mixture of 2.5 mL of 10 mM TPTZ solution in 40 mM HCl, 2.5 mL of 20 mM FeCl 3 , and 25 mL of acetate buffer (0.3 M, pH 3.6) was prepared [49]. The absorbance was measured at 595 nm. Data are expressed as µM Fe(II)/g. Butylated hydroxytoluene (BHT) was used as a positive control. . All media, buffers, trypsin, and dyes were filter-sterilized prior to use and warmed to 37 • C. For maintenance purposes, the MDA-MB-231 and CORL-23 cells were cultured in RPMI 1640 medium, while MCF-7 and A549 cells were cultured in DMEM. PC3 and A549 cells were cultured in DMEM/F12 supplemented with 10% foetal bovine serum (FBS), 2 mM L-glutamine, and 1% penicillin/streptomycin. The cell lines were cultured at 37 • C in 5% CO 2 in a humidified atmosphere. The cultures were passaged once a week by trypsinization using a 1:30 dilution of standard Trypsin-EDTA solution. Cell counts and viability were assessed using a standard trypan blue cell counting technique. All tested samples were dissolved in DMSO and diluted in the appropriate medium to obtain the working concentration.

Cell Viability Assay
Cell viability was determined using the protein-staining sulforhodamine B (SRB) assay as previously described [50]. Briefly, cells were trypsinized, counted, and placed in 96-well plates at optimal plating density of each cell line as determined over a range of 5-15 × 10 4 to ensure exponential growth throughout the experimental period and to ensure a linear relationship between absorbance at 490 nm and cell number. Cultures were analysed by the SRB assay and incubated to allow for cell attachment.
After 24 h, the cells were treated with serial dilutions of the samples to obtain the final concentrations ranging from 5 to 200 µg/mL for each sample. The final mixture used for treating the cells contained not more than 0.5% of the solvent (DMSO), the same as in the solvent-control wells. After 48 h of exposure, 100 µL of ice-cold 40% trichloroacetic acid (TCA) was added to each of the wells, which were left at 4 • C for 1 h and then washed with distilled water. The TCA-fixed cells were stained for 30 min with 50 µL of 0.4% (w/v) SRB in 1% acetic acid. Plates were washed with 1% acetic acid and air-dried overnight. For plate reading, the bound dye was solubilised with 100 µL of 10 mM tris base (tris[hydroxymethyl]aminomethane). The absorbance of each well was read on a Molecular Devices SpectraMax Plus Plate Reader (Molecular Devices, Celbio, Milan, Italy) at 490 nm. Cell survival was measured as the percentage absorbance compared to that of the untreated control. Vinblastine sulfate salt and taxol were used as a positive control.

Statistical Analysis
The concentration giving 50% inhibition (IC 50 ) was calculated by nonlinear regression with the use of GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA, USA). The concentration-response curve was obtained by plotting the percentage inhibition versus concentration. Differences within and between groups were evaluated by one-way analysis of variance test (ANOVA) followed by a multiple comparison Dunnett's test compared with the positive control. Data on oil chemical profiles of J. oxycedrus were retrieved from the literature ( Table 2). A first data matrix was built with 68 cases and 248 oils. We excluded cases when oil extraction was performed on fruits (two cases) or on material collected in the autumn/winter season (10 cases).
Furthermore, we excluded outliers by visual inspections of nonmetric multidimensional scaling, limiting cases to the core variability of J. oxycedrus, J. macrocarpa, and J. deltoides, and excluding cases widespread in the plot, resulting in a total of 40 cases. In our analysis, only oils that were present in at least 80% of cases for each species were included, resulting in a total of 30 oils. The final data matrix (40 cases × 30 oils) was subjected to multivariate analysis. Ordination was performed through principal coordinate analysis using the chord distance because of the frequent occurrence of double zero. Classification was performed using the unweighted pair group method with arithmetic mean (UPGMA) agglomerative method on chord distance. Silhouette analysis was used to obtain the best number of clusters (Silhouette score) and the relative significance (average distance), and an average distance over 0.5 was considered significant.

Conclusions
In recent decades, thanks to the growing number of studies on medicinal plants, their phytochemicals, and their potential applications, interest in natural products has increased. In this context, Juniperus species have been extensively studied and demonstrated the presence of a wide array of compounds with a variety of biological effects. Herein, essential oils and polar extracts of J. oxycedrus and J. macrocarpa aerial parts collected in southern Italy were subjected to a comparative study of their chemical profiles and anti-proliferative and antioxidant properties.
Previous studies described the anti-proliferative effects of some Juniperus species on several human cancer cell lines including lung cancer cells (A549) and breast cancer cells (MCF-7).
To the best of our knowledge, this is the first report highlighting the anti-proliferative activity of J. oxycedrus and J. macrocarpa methanol and ethyl acetate extracts against triple negative breast adenocarcinoma (MDA-MB-231) and human lung large cell carcinoma (COR-L23).
The most promising activities were evidenced with J. oxycedrus methanol and ethyl acetate extracts, which exerted good radical scavenging activity and exhibited remarkable activity against the large lung carcinoma cell line (COR-L23) with IC 50 values of 26.0 and 39.1 µg/mL, respectively, lower than that obtained with positive control vinblastine with an IC 50 value of 45.5 µg/mL. The World Health Organization (WHO) indicates that lung cancer is the most common type of cancer, with 2.09 million cases in 2019 [51]. Many natural compounds could specifically target different cell signalling pathways associated with cancer progression to provide a cytotoxic effect in the target cell. The importance of these compounds is emerging in many therapies developed with dual action often including a natural compound. Currently, there are many natural compounds or their derivatives in combination with synthetic drugs for lung cancer at different stages of clinical trials. The results of this study indicate that J. oxycedrus may be considered a source of natural compounds with antioxidant and anti-proliferative effects that could be suitable for future applications.