Chemical and Bioactive Characterization of the Essential Oils Obtained from Three Mediterranean Plants

Cupressus sempervirens L., Juniperus communis L. and Cistus ladanifer L. are Mediterranean arboreal and shrub species that possess essential oils (EO) in their leaves and branches. This study aimed at characterizing the EOs obtained by steam distillation from the three species collected in different locations from Spain (Almazán, Andévalo, Barriomartín, Cerezal, Ermitas and Huéscar). For this purpose, volatiles composition was determined by GC-MS, and different bioactivities were evaluated. The highest content in terpenes was observed in C. sempervirens (Huéscar origin) followed by J. communis (Almazán origin), corresponding to 92% and 91.9% of total compounds, respectively. With exception of C. ladanifer from Cerezal that presented viridiflorol as the most abundant compound, all the three species presented in common the α-pinene as the major compound. The EOs from C. ladanifer showed high antibacterial potential, presenting MIC values from 0.3 to 1.25 mg/mL. Concerning other bioactivities, C. ladanifer EO revealed an oxidation inhibition of 83%, while J. communis showed cytotoxicity in the MCF-7 cell line, and C. sempervirens and C. ladanifer EOs exhibited the highest potential on NCI-H460 cell lines. Nevertheless, some EOs revealed toxicity against non-tumoral cells but generally presented a GI50 value higher than that of the tumor cell lines.


Introduction
For decades, the need to change from a fossil-based economy to bio-based systems has been discussed. It has been pushing the industry to transition towards a green, sustainable and circular economy, improving energy efficiency by using renewable raw materials [1,2]. In this sense, the use of lignocellulosic biomass, which represents 89.3% of the total biomass, is being explored as a potential substrate to obtain compounds of interest, such as bioactive molecules, through an integrated biorefinery approach.
Some crop and forest biomass resources can be an effective source of high-value compounds since this raw material is underused and naturally recycled into ecosystems. Thus, it is a low-cost feedstock that can assume a role of great importance for food, pharmaceutical and cosmetic industries due to beneficial effects, including antioxidant, antimicrobial, anti-inflammatory and anti-tumoral properties, that have been attributed to different compounds present in such agroforestry biomass [3,4].
The majority of the shrubs belonging to the Cupressaceae and Cistaceae families can be found all over the Mediterranean region and have the capacity of growing in open areas with deficient soils and harsh environments [5]. In addition, they are intensely aromatic plants due to the high content of essential oils (EOs) in their twigs and leaves [6]. In particular, the genus Cupressus and Juniperus (Cupressaceae) and Cistus (Cistaceae) are widespread include some species, such as Cupressus sempervirens L., Cistus ladanifer L. and Juniperus communis L., being their extracts or EOs used for many years in traditional medicine [7].
The species C. sempervirens L. and J. communis L. are widely planted as ornamental shrubs in parks and gardens, while C. ladanifer L. is frequently found in wild areas. However, these species present a high potential to be grown in marginal lands, which are not used either for other agricultural or forestry purposes, allowing for improving soil fertility and organic carbon stocks while simultaneously generating biomass that can be used for bio-based value chains.
In the BBI-JU BeonNAT project, some of these species have been selected to create new dedicated plantations that can be used to produce valuable EOs. EOs are complex mixtures of hydrocarbons and oxygenated hydrocarbons from the isoprenoid pathways, mainly mono-, di-, and sesqui-terpenes [13]. So far, different studies have investigated the chemical composition of C. ladanifer, C. sempervirens and J. communis EOs by gas chromatography coupled to mass spectrometry (GC-MS) [14][15][16][17], with α-pinene being frequently reported as the major component in these three species [10].
However, they mainly refer to the essential oil obtained by laboratory hydrodistillation from the leaves of C. ladanifer and C. sempervirens, as also from J. communis berries with few or non-existing data regarding other plant parts. Hydro and steam distillations are some of the most traditional ways to isolate volatile compounds from medicinal and aromatic plants [18]. The extraction method is a recognized factor that may greatly impact the quality of EOs. Moreover, the chemical composition of essential oils can be affected by other factors, such as environmental conditions [19].
Therefore, as part of the BBI-JU BeonNAT project, the present study aimed at evaluating the chemical composition of the EOs extracted by steam distillation from the crown biomass (with branches diameter < 50 mm) of these three species and studying their bioactive properties, namely antioxidant, antibacterial, cytotoxic and anti-inflammatory, to access its potential as ingredients for bio-based products development in different industries. Moreover, each species was collected from two different locations in Spain to look for different chemotypes associated with different geographical locations and/or elevations.

Essential Oil Yields
The extraction yield of the EOs obtained by steam distillation was higher for J. communis, followed by C. sempervirens and C. ladanifer, as can be observed in Figure 1. The obtained values are within the range reported in the literature for C. ladanifer (from 0.01 to 0.63%) [20][21][22][23] while being lower for C. sempervirens for which reported yields varied from 0.20 to 0.87% [24][25][26] and higher for J. communis (reported yields from 0.05 to 0.70%) [23,[27][28][29][30]. These differences are most probably related to (i) the used samples (crown biomass that includes twigs, leaves and fruits instead of leaves or berries); (ii) the location where the plant samples were collected; (iii) the date when these samples were obtained, and (iv) the EO extraction methodology. These factors are important since the extraction yield of essential oils depends on some variables like the part of the plant material, seasonal variations, environmental and cultivation conditions, plant age, harvesting time, and type of distillation [17]. and (iv) the EO extraction methodology. These factors are important since the extraction yield of essential oils depends on some variables like the part of the plant material, seasonal variations, environmental and cultivation conditions, plant age, harvesting time, and type of distillation [17].
2.3. Bioactive Evaluation 2.3.1. Antibacterial Activity Table 2 presents the results of the antibacterial capacity against a panel of bacteria selected according to their importance in public health. None of the essential oils showed the potential to inhibit the growth of gram-negative bacteria K. pneumoniae and P. mirabilis. On the other hand, the E. coli strain was sensitive to all the tested EOs. At the same time, M. morganii was only sensitive to C. ladanifer EOs, being notably inhibited by the EO from Cerezal location (MIC = MBC = 0.6 mg/mL). Among the samples, C. ladanifer EO was the only one that presented inhibitory potential against the gram-negative bacteria P. aeruginosa, which is frequently associated with nosocomial infections.
In general, gram-positive bacteria presented lower MIC values, being most susceptible compared with the gram-negative strains. E. faecalis was inhibited and killed by all the tested EOs, with MIC and MBC values ranging from 0.6 mg/mL to 2.5 mg/mL. The only sample that could not inhibit the growth of all the tested gram-positive bacteria was J. communis EO from Barriomartín location as it was not effective against L. monocytogenes.
In general, C. ladanifer exhibited the most potent antimicrobial potential, with samples from both locations showing the lowest MIC and MBC values for both gram-negative and gram-positive bacteria compared with other data described in the literature. Outstanding results were obtained against MRSA, a bacteria associated with nosocomial infections. According to Mohammed, Said, Fouzia, Kawtar, Zoubida, Abdelilah, Elhourri and Ghizlane [21], S. aureus shows high sensitivity (MIC = MBC = 6.25 mg/mL), and E. coli and P. aeruginosa respond very positively (MIC = MBC = 25 mg/mL for both strains) to the essential oil obtained from C. ladanifer stems and leaves. Curiously they found different compounds in major contents in the chemical characterization, suggesting that synergism between these compounds can occur. Identical results were reported by Benali, et al. [40] against S. aureus (MIC = MBC = 6.25 mg/mL), with much lower MIC and MBC values being observed for P. mirabilis (MIC = MBC = 0.19 mg/mL), which was the most sensitive strain when assessing the antimicrobial activity of C. ladanifer EO (aerial parts).
On the contrary, using a different method, the disk agar diffusion method, Tavares, Martins, Faleiro, Miguel, Duarte, Gameiro, Roseiro and Figueiredo [22] reported a weak antimicrobial activity for C. ladanifer essential oil against E. coli and S. aureus. Benayad, et al. [41] also studied the antimicrobial effect of C. ladanifer EO (full plant) and reported MIC values between 50-500 µg/mL, with the lower MIC being obtained against multiresistant S. aureus (MIC = 50 µg/mL). Although Benayad, Mennane, Charof, Hakiki and Mosaddak [41] obtained stronger activity with lower MIC values, they also verified that the EO was effective against both gram-positive and gram-negative bacteria, with better activity against the gram-positive, and no inhibition was observed for K. pneumoniae at the higher tested concentration. Although the studies reported by Benali, Bouyahya, Habbadi, Zengin, Khabbach, Achbani and Hammani [40] and Benayad, Mennane, Charof, Hakiki and Mosaddak [41] are in good agreement with the present ones, these authors did not analyze the chemical composition of the EO; thus it was not possible to corroborate the results with the chemical composition. Regarding C. sempervirens EO, different studies reported its inability to inhibit the growth of P. aeruginosa, either using the agar dilution [42] or the broth microdilution [43] methodologies. Hammer, Carson and Riley [43] achieved identical results for P. aeruginosa; however, the oil obtained from the leaves and twigs was able to inhibit the growth of E. coli, S. aureus and E. faecalis at the maximal concentration tested (MICs > 2.0% v/v) which is in line with the herein obtained results. Similar results were obtained by [44], who reported that the EO from the aerial parts of C. sempervirens showed an antimicrobial activity more pronounced against gram-positive than gram-negative bacteria, with MIC and MBC values of 0.07 µg/mL and 0.31 µg/mL for S. aureus and E. coli respectively. One of the main compounds identified was δ-3-carene in both studies. Nevertheless, in this study the authors also reported that P. aeruginosa was sensitive to C. sempervirens EO (MIC = MBC= 0.31 µg/mL). Contrarily to previous studies and the results herein obtained, Mazari, Bendimerad, Bekhechi and Fernandez [39] reported that the EO from C. sempervirens (leaves) was ineffective against S. aureus, E. faecalis, E. coli and P. aeruginosa at the highest concentration tested (10 µL/mL). In this study, the common major compound (carene) was not identified, which probably justifies these results.
Regarding the results herein obtained for J. communis EO, they are in good agreement with previous studies that also reported the capacity of the EO obtained from different parts of the plant against S. aureus, E. faecalis and E. coli while not presenting antimicrobial activity at the highest tested concentration (2% v/v in the broth dilution and 5 mg/mL in the agar disc diffusion methods) for K. pneumoniae, P. mirabilis and P. aeruginosa [6,43]. Angioni, Barra, Russo, Coroneo, Dessí and Cabras [27] reported contradictory results, which showed that the antimicrobial activity of the J. communis EO from berries and leaves was generally nonsignificant against S. aureus and E. coli at the highest concentration tested (900 µg/mL). Falcão, et al. [45] evaluated the antimicrobial activity of two commercial samples of J. communis EO and one obtained by hydrodistillation of the berries and found that all were able to inhibit the growth of S. aureus, Bacillus cereus, Bacillus subtilis, E. coli, E. faecalis and K. pneumonia. In contrast, only one commercial EO presented activity against P. mirabilis, P. aeruginosa and Salmonella Typhimurium. The wider range of activity of this EO was related to its different chemical composition compared to the other samples, namely its higher content in oxygenated monoterpenes, such as terpinene-4-ol and 1,8-cineole, which have been associated with antimicrobial properties. The herein studied J. communis EO present very low content of terpinene-4-ol and 1,8-cineol was not detected, what can possibly explain the lower activity of these oils.
Comparing the antibacterial potential of the analyzed EOs with common antibiotics, none of them could compete with these commercial drugs. Nevertheless, commercial drugs are isolated compounds, while EOs are a mixture of different compounds. Nonetheless, given the growing resistance to antibiotics, new antibacterial agents are needed, and the exploitation of novel sources of antibacterials is a major research topic worldwide.

Antioxidant Activity
Analyzing the antioxidant values obtained from the cellular-based assays (Table 3), all the samples revealed the capacity to inhibit the oxidation process, highlighting the sample J. communis from Almazán that inhibited about 78% of oxidation, presenting a GI 50 value of 324 ± 8 µg/mL, and C. ladanifer from Andévalo that inhibited about 83% of oxidation and exhibited a GI 50 of 336 ± 8 µg/mL.
To the best of our knowledge, data are scarce in the literature regarding the antioxidant properties of the studied EO, with most studies available relying on the use of the DPPH method. Boukhris, Regane, Yangui, Sayadi and Bouaziz [44] measured the antioxidant activity of C. sempervirens EO by the radicals-scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and reported an EC 50 value of 7.70 ± 0.70 µg/mL, however, higher IC 50 values (151 µg/mL and 290.09 µg/mL) have been reported for this species EO using the same methodology [25,26]. Additionally, using the DPPH assay, Höferl, Stoilova, Schmidt, Wanner, Jirovetz, Trifonova, Krastev and Krastanov [16] reported an IC 50 of 944 µg/mL for Juniper berry oil. On the contrary, using this assay, no activity was found for C. ladanifer EO at the highest tested concentration (100 µg/mL) while a value of 0.1 ± 0.06 AAE/g was reported using the Ferric Reducing Antioxidant Power (FRAP) methodology.

Cytotoxic and Anti-Inflammatory Activity
The effects of the oils obtained by steam distillation on the growth of four human tumor cell lines (MCF-7, NCI-H460, CaCo2 and AGS) and two non-tumoral cell lines (Vero and PLP2), represented as the concentration that caused 50% of cell growth inhibition (GI 50 ) are summarized in Table 3. The samples of C. ladanifer (both from Cerezal and Andévalo origins) exhibited higher potential in all the tested cell lines, presenting GI 50 values that ranged from 14 to 78 µg/mL in the NCI-H460 and AGS cells, respectively. The sample J. communis, presented GI 50 values of 30.88 ± 1.85 and 41.99 ± 3.60 µg/mL in MCF-7 cell line (from Almazán and Barriomartín, respectively), while C. sempervirens showed 20 ± 2 µg/mL in NCI-H460 cell-line (Ermitas origin). In general, all samples showed cytotoxicity in the non-tumor cell lines. However, in the majority of the cell lines, the value of GI 50 was higher than that of the tumor cell lines, meaning that for particular cases, these EOs can be used without toxicity. Additionally, it can be stated that in vivo studies are needed to verify the toxicity of these oils for specific applications.
All the tested oils showed anti-inflammatory capacity, which is in agreement with Murbach Teles Andrade, Nunes Barbosa, da Silva Probst and Fernandes Júnior [42], who reported that various essential oils exert an anti-inflammatory action by increasing interleukin-10 production. For J. communis the plant from Barriomartín showed the best results, but lower than the other species. C. ladanifer from Andévalo presented the strongest activity (19 µg/mL), while C. sempervirens collected in Ermitas exhibited the highest activity from all the tested samples. Najar, et al. [46] found that C. ladanifer EO exhibited cytotoxic activity at 90 ppm for the MCF-7 cell line. However, no further activity was found for the tested cell lines with the essential oils from J. communis and C. sempervirens.

Plant Material Collection and Conditioning
The plant material (crown biomass, with branches with a maximum stem diameter of 50 mm that included twigs, leaves and fruits) of each one of the species considered was collected in two different locations in Spain: C. ladanifer in Cerezal de Aliste and El Andévalo; C. sempervirens in Huéscar and Las Ermitas; and J. communis in Almazán and Barriomartín ( Figure 2 and Table 4). Samples were randomly taken from a minimum of 10 plants of a similar age, and the biomass of the different plants was mixed to obtain samples of 40 kg of green material from each species.
Previously to the steam distillation, fresh samples were air-dried in the shade at room temperature (10-15 °C) until moisture content was around 10-15%. Afterward, they were ground to a size of 20 mm using a shredder (90 kW, slow rotating single-shaft type, 70 rpm., SILMISA, Onil, Spain). Then, subsamples were taken to determine the moisture content following the standard ISO 18134-2:2017.

Essential Oils Extraction
The ground samples were distilled in a 50 L stainless steel still using steam produced in an electric boiler (ETE, Madrid, Spain). The steam conditions used for the extractions were 13 kg/h with a boiler pressure of 50 kPa. Batch extractions were performed, with two repetitions of 10 kg each per sample and an extraction duration of 2 h for each batch. Time was measured from the moment the first drop of distillate fell. The temperature inside the still was kept constant at 98 °C. The hydrolate and the essential oil were separated by density using a glass Florentine flask. The essential oil samples were then dried using anhydrous sodium sulfate and, after filtration, they were weighed and stored at 4 °C until further analysis. The oil yield for each sample was calculated as a percentage (w/w) on a biomass dry weight basis.

Gas Chromatography/Mass Spectrometry (GC-MS) Analyses
The EOs analysis was performed on a GC-MS Perkin Elmer system with a Clarus ® 580 GC and a Clarus ® SQ 8 S MS module, equipped with DB-5MS fused-silica column (30 m × 0.25 mm i.d., film thickness 0.25 µm; J&W Scientific, Inc., Folsom, CA, USA), according to Falcão, Bacém, Igrejas, Rodrigues, Vilas-Boas and Amaral [45]. The carrier gas was helium gas adjusted to a linear velocity of 30 cm/s. The oven temperature program was as follows: 40 °C for 4 min, raised at 3 °C/min to 175 °C, then at 15 °C/min to 300 °C  Samples were randomly taken from a minimum of 10 plants of a similar age, and the biomass of the different plants was mixed to obtain samples of 40 kg of green material from each species.
Previously to the steam distillation, fresh samples were air-dried in the shade at room temperature (10-15 • C) until moisture content was around 10-15%. Afterward, they were ground to a size of 20 mm using a shredder (90 kW, slow rotating single-shaft type, 70 rpm., SILMISA, Onil, Spain). Then, subsamples were taken to determine the moisture content following the standard ISO 18134-2:2017.

Essential Oils Extraction
The ground samples were distilled in a 50 L stainless steel still using steam produced in an electric boiler (ETE, Madrid, Spain). The steam conditions used for the extractions were 13 kg/h with a boiler pressure of 50 kPa. Batch extractions were performed, with two repetitions of 10 kg each per sample and an extraction duration of 2 h for each batch. Time was measured from the moment the first drop of distillate fell. The temperature inside the still was kept constant at 98 • C. The hydrolate and the essential oil were separated by density using a glass Florentine flask. The essential oil samples were then dried using anhydrous sodium sulfate and, after filtration, they were weighed and stored at 4 • C until further analysis. The oil yield for each sample was calculated as a percentage (w/w) on a biomass dry weight basis.

Gas Chromatography/Mass Spectrometry (GC-MS) Analyses
The EOs analysis was performed on a GC-MS Perkin Elmer system with a Clarus ® 580 GC and a Clarus ® SQ 8 S MS module, equipped with DB-5MS fused-silica column (30 m × 0.25 mm i.d., film thickness 0.25 µm; J&W Scientific, Inc., Folsom, CA, USA), according to Falcão, Bacém, Igrejas, Rodrigues, Vilas-Boas and Amaral [45]. The carrier gas was helium gas adjusted to a linear velocity of 30 cm/s. The oven temperature program was as follows: 40 • C for 4 min, raised at 3 • C/min to 175 • C, then at 15 • C/min to 300 • C and held for 10 min. The injector temperature was set at 260 • C, with a transfer line at 280 • C and an ion source at 220 • C. The ionization energy was 70 eV, and a scan range of 35-500 u with a scan time of 0.3 s was used. For each essential oil, 3 µL of sample diluted in HPLC grade n-hexane (1:100) was injected with a split ratio of 1:3. Identification of components was assigned by matching their mass spectra with NIST17 data and by determining the linear retention index (LRI) based on the retention times obtained for a mixture of n-alkanes (C8-C40, ref. 40147-U, Supelco) analyzed under identical conditions. When possible, comparisons were also performed with commercial standard compounds and published data. Quantification was performed using the relative peak area values obtained directly from the total ion current (TIC) values, and the results were expressed as the relative percentage of total volatiles.

Antibacterial Activity
To evaluate the antibacterial activity of C. ladanifer, C. sempervirens and J. communis EO, five gram-negative (Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Morganella morganii) and three gram-positive bacteria (Enterococcus faecalis, Listeria monocytogenes and methicillin-resistant Staphylococcus aureus (MRSA)) were used. The bacterial strains were clinical isolates obtained from the Northeastern local health unit (Bragança, Portugal) and Hospital Center of Trás-os-Montes and Alto Douro (Vila Real, Portugal). These microorganisms were incubated at 37 • C in an appropriate fresh medium for 24 h before analysis to maintain the exponential growth phase. The antibacterial activity was evaluated through the broth microdilution method, based on the methodology described by Pires, et al. [47], determining the MIC (minimum inhibitory concentration) and the MBC (minimum bactericidal concentration), expressed in mg/mL. Briefly, the samples were serially diluted to obtain the concentration ranges of 2.5 mg/mL to 0.078 mg/mL. Different controls were prepared, namely two negative controls: MHB with Tween 80 and another one with the extract. Two positive controls with MHB with Tween 80 and each inoculum and one culture medium, antibiotics and bacteria. Ampicillin and Imipenem were used for all the tested Gram-negative bacteria and Listeria monocytogenes, while ampicillin and vancomycin were selected for Enterococcus faecalis and MRSA. After serial dilution in a 96 well microplate, each bacterial inoculum was pipetted to each well (corresponding to 1.5 × 108 Colony Forming Unit (CFU)/mL). The microplates were covered and incubated in a stirring board at 37 • C for 24 h. The MIC values were detected following the addition (50 µL) of 0.2 mg/mL p-iodonitrotetrazolium chloride (INT) and incubation at 37 • C for 30 min. MIC was defined as the lowest concentration that inhibits the visible bacterial growth determined by changing the coloration from yellow to pink if the microorganisms are viable. For the determination of MBC, 10 µL of liquid from each well showed no change in color was plated on solid medium, Blood agar (7% sheep blood) and incubated at 37 • C for 24 h. The lowest concentration that yielded no growth determines the MBC. MBC was defined as the lowest concentration required to kill bacteria.

Antioxidant Activity
The reducing power (RP) Kostić, et al. [48] and cellular antioxidant activity (CAA) [14,49,50] assays were performed to determine the antioxidant potential of the EOs. The reducing power was evaluated by determining the capacity of the extract to reduce Fe 3+ to Fe 2+ by measuring the absorbance at 690 nm. The results were expressed as EC 50 values (µg/mL), corresponding to sample concentration with 0.5 of absorbance. For the CAA, the cells (RAW 264.7) were incubated with different EOs and AAPH (Azobis (2-methylpropionamidine) dihydrochloride), using DCFH-DA (2,7-Dichlorofluorescein diacetate) as a fluorescent marker [14,50]. DCF-DA is a compound that, once in the cell medium, is easily oxidized by peroxide radicals to a fluorescent compound, resulting in DCFH-DA. For the quantification of CAA, the efficacy of the antioxidant treatments was quantified by examining the percentage reduction in fluorescence, according to the formula: where AUC sample and AUC control corresponds to the Area Under the Curve of the sample and control, respectively, cells were immediately placed on a microplate reader (FLX800 Biotek, Winooski, VT, USA), where real-time fluorescence was read initially and then every 5 min for 40 min. Fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm.

Cytotoxicity
To determine the cytotoxic potential of the different EOs, the Sulforhodamine B (SRB) assay [51] was performed on four different human tumor cells obtained from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen: NCI-H460 (lung carcinoma), MCF-7 (breast carcinoma), AGS (gastric carcinoma) and CaCo (colon carcinoma) and two normal cell lines: PLP2 (porcine liver cells) and VERO (monkey kidney cells). For hepatotoxicity evaluation, the porcine liver primary culture was prepared from a freshly harvested porcine liver. Ellipticine was used as the positive control, and the results were expressed in GI 50 values (µg/mL), corresponding to the extract concentration that provides 50% of cell growth inhibition [52].

Anti-Inflammatory Activity
The anti-inflammatory activity was determined according to the method formerly reported by Mandim, et al. [53], in which the samples were tested for their capacity to inhibit the lipopolysaccharide (LPS)-induced NO (nitric oxide) production on a murine macrophage cell line (RAW 264.7). Dexamethasone was used as the positive control, and samples without LPS were used as a negative control. The results were expressed as IC 50 values (µg/mL), corresponding to the extract concentration responsible for 50% of NO production inhibition.

Statistical Analysis
Experimental results were expressed as mean value ± standard deviation (SD). The obtained data were subjected to ANOVA post-hoc Tukey's Honest Significant Difference (HSD) test, applied at p < 0.05, using the SPSS v.22.0 program. When less than three repetitions were available, the results were analyzed by the t-Student test as a form to determine the significant differences between two samples, with p = 0.05.

Conclusions
The obtained results suggest that the collection of these species in different geographical locations interfere with the essential oil's yield and respective chemical composition, which can vary in terms of individual compounds' contents, resulting in different chemotypes. Regardless, no relation was noticeable between the chemical composition and the location's elevation. Compared with other parts of the plant, in literature, the crown biomass is qualitatively similar in terpenes profile, particularly when taking into consideration the composition specified by the International Organization for Standardization (ISO) and/or the European Pharmacopoeia for the branches and leaves of C. sempervirens and J. communis berries.
The evaluated species showed to be a viable and low-cost source of EOs that can be used for bio-based products development in different industries, such as the food, cosmetic and medicinal industries. All tested EOs showed the potential to inhibit the growth of gram-negative bacteria, especially E. coli, while C. ladanifer EO from Cerezal was the only one with the potential to inhibit the growth of M. morganii.
Nevertheless, according to the target application, the toxicity exhibited by some of the EOs in the tested tumor cell lines must be deeper analyzed by verifying this condition in specific toxicity models for each industry/product. Therefore, further studies are recommended to deepen the knowledge on these EOs and respective compounds towards different applications.