Phytochemistry and Micromorphology of Epicuticular Waxes in Four Juniper Species With Fragmented Distribution in the Balkans.

The cuticle is important in the interaction between the plant and its environment, especially in the dry areas. Four species of junipers from the section Sabina wild growing in the Balkans were selected to study leaf wax composition using GC/MS and GC-FID and its surface morphology under SEM to understand the correlation between the distribution and/or habitat of these species and their cuticles. SEM micrographs showed continuous, smooth epicuticular layers with crusts in all species but with a species-specific distribution of different 3D crystalloid types and different cuticle thickness. n-C33 alkane was the most abundant compound, followed by n-C29, n-C31, and n-C35, depending on the species and the site. The average chain length (N) was the lowest in J. phoenicea, but with the greatest dispersion around it. At the same time, the two most continental species (J. foetidissima and J. excelsa) show the N with the lowest dispersion around it. The statistical analyses confirmed the significance of climate on the evolution of the specific epicuticular wax composition in studied junipers.

Insights on Juniperus phoenicea Essential Oil as Potential Anti-Proliferative, Anti-Tyrosinase, and Antioxidant Candidate.

In this study, the anti-cancer, anti-tyrosinase, and antioxidant activities of essential oils (EOs) of berries and leaves of Juniperus phoenicea grown wild in North of Tunisia were investigated. The EO yields from leaves and berries were 1.69% and 0.45%, respectively. GC-MS analysis revealed that α-pinene is the predominant component in both EOs (44.17 and 83.56%, respectively). Leaves essential oil presented high levels of ß-phellandrene (18%) and camphene (15%). The EOs displayed cytotoxic effects against MCF-7 breast cancer cell, HT-29 colon cancer, and the normal cells H9C2 cardiomyoblasts. Leaves oil strongly inhibited colon cell line proliferation (IC50 of 38 µg/mL), while berries essential oil was more potent against breast cancerous cells MCF-7 (IC50 of 60 µg/mL). Interestingly, berries essential oil exhibited high ability to inhibit melanin synthesis by inhibiting enzyme mono and diphenolase activities. Overall, the results suggested that the two oils are significant sources of healthy natural chemicals.

Isolation, characterization, and antimicrobial activity of communic acid from Juniperus phoenicea.

OBJECTIVES: A mixture of Z and E communic acid is isolated for the first time from the cones of Juniperus phoenicea. Its biological activity was studied. METHODS: The plant material was extracted in a Soxhlet apparatus with n-hexane, the resulting extract was subjected to column chromatography (CC) on silica gel. The structure elucidation of the constituents of the isolated fraction was identified by comparison of its spectroscopic properties 1H and 13C NMR data with those reported in the literature. The antimicrobial assay of hexanic extract and isolated compounds was carried out by the disc diffusion and micro-dilution methods. RESULTS: A mixture of two diterpene acids isomers was isolated, with a high yield (68%). Their chemical structures were confirmed after comparing their spectral data with published reports. These natural products exhibited a significant antibacterial and antifungal activity against the tested strains. Indeed, for Bacillus cereus, Staphylococcus aureus, and Pseudomonas aeruginosa, the inhibition zone diameters (36-37 mm) was better than penicillin, novobiocin, and amoxicillin. For Candida albicans activity, it show that the mixture possess an activity similar to that of Metrazol. Against Escherichia coli, the inhibitory activity was found less than Amoxicillin. This is the first report of isolation of communic acid from J. phoenicea. CONCLUSIONS: These results showed that the cones of J. phoenicea were an important source of communic acid, and its hexanic extract had the greatest potential antibacterial activity against both Gram-negative and Gram-positive bacteria and C. albicans.

Acaricidal activity against Ixodes ricinus nymphs of essential oils from the Libyan plants Artemisia herba alba, Origanum majorana and Juniperus phoenicea.

Ixodes ricinus (L.) (Acari: Ixodidae) is a major vector for the transmission of several important human pathogens. The aim of the present study was to evaluate the in vitro efficacy of different concentrations of essential oils (Eos) on I. ricinus tick nymphs. Oils were obtained from the leaves of three plants native to Libya: white wormwood (Artemisia herba alba Asso), marjoram (Origanum majorana L.) and Arâr (Juniperus phoenicea L., English common name Phoenician juniper). Assays were done using the "open filter paper method". Two concentrations from each oil, 0.5 and 1 µl/cm, were tested. The acaricidal effect was measured in terms of the lethal concentrations (LC50, LC95) and lethal time (LT50, LT95). Mortality rates were obtained by counting the surviving nymphs every 30 min for the first five hours and then at 24, 48 and 72 h. A mortality of 100% was recorded at the higher concentration of oils (1 µl/cm2) from A. herba alba and J. phoenicea at the first 2 h of exposure. Exposure to O. majorana led to 100% mortality on the third day (72 h), and this effect decreased noticeably with 0.5 µl/cm2 oil at the same exposure time. However, 50% of ticks showed a paralysis effect and less movement after 2 h. The LC50 of mortality was reached within the first 24 h of exposure time at 0.5 µl/cm2 of O. majorana, which produced 60% tick's mortality. Chemical composition of the essential oils was elucidated by gas chromatography-mass spectrometry analyses. These results suggest that essential oils deserve further investigation as components of alternative approaches for I. ricinus tick control.

Past, present, and future geographic range of the relict Mediterranean and Macaronesian Juniperus phoenicea complex.

AIM: The aim of this study is to model the past, current, and future distribution of J. phoenicea s.s., J. turbinata, and J. canariensis, based on bioclimatic variables using a maximum entropy model (Maxent) in the Mediterranean and Macaronesian regions. LOCATION: Mediterranean and Macaronesian. TAXON: Cupressaceae, Juniperus. METHODS: Data on the occurrence of the J. phoenicea complex were obtained from the Global Biodiversity Information Facility (GBIF.org), the literature, herbaria, and the authors' field notes. Bioclimatic variables were obtained from the WorldClim database and Paleoclim. The climate data related to species localities were used for predictions of niches by implementation of Maxent, and the model was evaluated with ENMeval. RESULTS: The potential niches of Juniperus phoenicea during the Last Interglacial period (LIG), Last Glacial Maximum climate (LGM), and Mid-Holocene (MH) covered 30%, 10%, and almost 100%, respectively, of the current potential niche. Climate warming may reduce potential niches by 30% in RCP2.6 and by 90% in RCP8.5. The potential niches of Juniperus turbinata had a broad circum-Mediterranean and Canarian distribution during the LIG and the MH; its distribution extended during the LGM when it was found in more areas than at present. The predicted warming in scenarios RCP2.6 and RCP8.5 could reduce the current potential niche by 30% and 50%, respectively. The model did not find suitable niches for J. canariensis during the LIG and the LGM, but during the MH its potential niche was 30% larger than at present. The climate warming scenario RCP2.6 indicates a reduction in the potential niche by 30%, while RCP8.5 so indicates a reduction of almost 60%. MAIN CONCLUSIONS: This research can provide information for increasing the protection of the juniper forest and for counteracting the phenomenon of local extinctions caused by anthropic pressure and climate changes.

In situ approaches show the limitation of the spoilage potential of Juniperus phoenicea L. essential oil against cold-tolerant Pseudomonas fluorescens KM24.

The present study aimed to elucidate the effect of subinhibitory concentrations (sub-MICs) of juniper essential oil (EO), α-pinene, and sabinene on the quorum-sensing (QS)-mediated proteolytic and lipolytic properties of Pseudomonas fluorescens KM24. These activities were verified under in situ conditions, in which sub-MICs of the agents altered the morphology of KM24 cells. RNA-Seq studies revealed key coding sequences (CDSs)/genes related to QS and the proteolytic/lipolytic activities of pseudomonads. In this work, all the examined agents decreased autoinducer synthesis and influenced the mRNA expression of the encoding acyltransferase genes lptA, lptD, and plsB. The highest reduction on the 3rd and 5th days of cultivation was observed for the genes lptD (-5.5 and -5.61, respectively) and lptA (-3.5 and -4.0, respectively) following treatment with EO. Inhibition of the lptA, lptD, and plsB genes by singular constituents of EO was on average, from -0.4 to -0.7. At 5 days of cultivation the profile of AHLs of the reference P. fluorescens KM24 strain consisted of 3-oxo-C14-HSL, 3-oxo-C6-HSL, C4-HSL, and N-[(RS)-3-hydroxybutyryl]-HSL, the concentrations of which were 0.570, 0.018, 3.744, and 0.554 µg ml-1, respectively. Independent of the incubation time, EO, α-pinene, and sabinene also suppressed the protease genes prlC (-1.5, -0.5, and -0.5, respectively) and ctpB (-1.5, -0.7, and -0.4, respectively). Lipolysis and transcription of the lipA/lipB genes were downregulated by the agents on average from -0.3 to -0.6. α-Pinene- and sabinene-rich juniper EO acts as an anti-quorum-sensing agent and can repress the spoilage phenotype of pseudomonads. KEY POINTS: Juniper EO, α-pinene, sabinene exhibited anti-QS potential toward KM24. RNA-Seq revealed key CDSs/genes related to QS/proteolytic/lipolytic activities of KM24. Agents at sub-MIC levels influenced the mRNA expression of QS/lipase/protease genes.

Protective Role of Juniperus phoenicea L. Leaves Extract against Gamma-irradiation-induced Oxidative Stress.

BACKGROUND AND OBJECTIVE: Radiation exposure can cause several harmful effects in biological systems due to free radical production. Several antioxidants have been tested as potential hepatoprotective agents against ionizing radiation as they lower oxidative stress in normal cells induced by Reactive Oxygen Species (ROS). The present study was conducted to evaluate the possible ameliorative effects of Juniperus phoenicea L. MATERIALS AND METHODS: Aqueous leaves extract on different biochemical and histopathological parameters against whole body gamma-irradiation-induced oxidative stress, organ dysfunction and metabolic disturbances in experimental Swiss Albino rats. After a single dose of gamma-radiation (6 Gy), there was a significant reduction in albumin, total protein and globulin levels and a significant increase in the liver enzymes (ALT, AST, ALP and GGT) and lipid profile parameters (cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol) in gamma-irradiated rats unlike in normal controls. RESULTS: The gamma-irradiated rats pre-treated with J. phoenicea leaf extracts, however, showed a significant increase in albumin, total protein and globulin levels and a significant reduction in liver enzymes and lipid profile parameters as opposed to the untreated ones. The gamma-irradiated rats showed toxic changes in the liver, whereas, the rats pre-treated with J. phoenicea leaves extract demonstrated a protective effect. Additionally, gamma- irradiation caused myocardial degenerative changes, interstitial edema between muscle fibers, necrosis and inflammatory cells infiltration and fibrotic and cellular damages to the heart, but J. phoenicea leaves extract were found to ameliorate the gamma-irradiation-induced changes in the heart. CONCLUSION: The results suggested that treatment with J. phoenicea leaves extract is possibly safe and can ameliorate gamma-irradiation-induced oxidative damage and tissue injury in rats. The leaves of J. phoenicea could serve as a potential source of therapeutic antioxidants.

Chemical Composition, Antioxidant, Anti-Tyrosinase, Anti-Cholinesterase and Cytotoxic Activities of Essential Oils of Six Algerian Plants.

In this study, the essential oils (EOs) of six Algerian plants (Artemisia campestris L., Artemisia herba-alba Asso, Juniperus phoenicea L., Juniperus oxycedrus L., Mentha pulegium L. and Lavandula officinalis Chaix) were obtained by hydrodistillation, and their compositions determined by GC-MS and GC-FID. The antioxidant activity of the EOS was evaluated via 2,2'-diphenyl-1-picrylhydrazyl (DPPH), ferric-reducing/antioxidant power (FRAP) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assays. Moreover, their cytotoxic effect was evaluated-as well as their tyrosinase, acetyl- and butyryl-cholinesterase (AChE and BuChE) inhibitory activities. The chemical analyses detected 44, 45, 51, 53, 26 and 40 compounds in EOs of A. campestris, A. herba-alba, J. phoenicea, J. oxycedrus, M. pulegium and L. officinalis, respectively. A. campestris EO was mainly composed of ß-pinene (20.7%), while A. herba-alba EO contained davanone D (49.5%) as the main component. α-Pinene (41.8%) was detected as the major constituent in both J. phoenicea (41.8%) and J. oxycedrus (37.8%) EOs. M. pulegium EO was characterized by pulegone as the most abundant (76.9%) compound, while linalool (35.8%) was detected as a major constituent in L. officinalis EO. The antioxidant power evaluation revealed IC50 values ranging from 2.61 to 91.25 mg/mL for DPPH scavenging activity, while the FRAP values ranged from 0.97-8.17 µmol Trolox equivalents (TX)/g sample. In the ABTS assay, the values ranged from 7.01 to 2.40 µmol TX/g sample. In the presence of 1 mg/mL of the samples, tyrosinase inhibition rates ranged from 11.35% to 39.65%, AChE inhibition rates ranged from 40.57% to 73.60% and BuChE inhibition rates ranged from 6.47% to 72.03%. A significant cytotoxic effect was found for A. herba-alba EO. The obtained results support some of the traditional uses of these species in food preservation and for protection against several diseases.

Greater sensitivity to hotter droughts underlies juniper dieback and mortality in Mediterranean shrublands.

Drought-induced dieback episodes have been globally reported. However, few studies have jointly examined the role played by drought on growth of co-occurring shrub and tree species showing different dieback and mortality. Here, we focused on dieback events affecting Mediterranean shrublands dominated by the Phoenician juniper (Juniperus phoenicea) since the middle 2000s in three sites across a wide geographical and climatic gradient in Spain. We compared their growth responses to climate and drought with coexisting tree species (Pinus pinea, Pinus pinaster and Quercus faginea), which did not show dieback in response to drought. We characterized the major climatic constraints of radial growth for trees, surviving and dead junipers by quantifying climate-growth relationships. Then, we simulated growth responses to temperature and soil moisture using the process-based VS-Lite growth model. Growth of shrubs and trees was strongly reduced during extreme droughts but the highest negative growth responsiveness to climate and drought was observed in trees followed by dead junipers from the most xeric and cold sites. Growth of dead junipers responded more negatively to droughts prior to the dieback than co-occurring, living junipers. Growth was particularly depressed in the dead junipers from the warmest site after the warm and dry 1990s. The growth model showed how a steep precipitation reduction in the 1980s triggered soil moisture limitation at the driest sites, affecting growth, particularly in the case of dead junipers and mainly in warm and dry sites. The asynchrony in the simulated seasonal timing of drought events caused contrasting effects on growth of co-occurring shrubs and tree species, compromising their future coexistence. Junipers were particularly vulnerable to hotter droughts during the early growing season. The presented projections indicate that de-shrubification events in response to hotter droughts will be common but conditioned by site conditions. Our modelling approach provides tools to evaluate vulnerability thresholds of growth under similar drought-induced dieback and mortality processes.

Growth inhibitory activity of biflavonoids and diterpenoids from the leaves of the Libyan Juniperus phoenicea against human cancer cells.

Three biflavonoids [cupressuflavone (1), amentoflavone (2), and sumaflavone (3)], four diterpenoids [13-epi-cupressic acid (4), imbricatholic acid (5), 3-hydroxy-sandaracopimaric acid (6), and dehydroabietic acid (7)], and one lignan [ß-peltatin methyl ether (8)] were isolated from the cytotoxic fractions of the extracts of the leaves of the Libyan Juniperus phoenicea L. The structures of these compounds were elucidated by spectroscopic means. Cytotoxicity of compounds 1-6 were assessed against the human lung cancer cell line A549 using the MTT assay. Compounds 1 and 3 showed cytotoxicity against the A549 cells (IC50  = 65 and 77 µM, respectively), whereas compound 2 did not show any activity. Diterpenes 4-6 exhibited weak cytotoxicity against the A549 cells with the IC50 values of 159, 263, and 223 µM, respectively. The cytotoxicity of each compound was compared with the anticancer drug, etoposide (IC50  = 61 µM). Cupressuflavone (1) was evaluated also for cytotoxicity against both the human PC3 cancer cell line and the normal prostate cell line (PNT2), and this compound revealed a high degree of cytotoxic selectivity towards the prostate cancer cells (PC3), with IC50 value of 19.9 µM, without any evidence of cytotoxicity towards the normal prostate cell line (PNT2).

Potential antioxidant properties and hepatoprotective effects of Juniperus phoenicea berries against CCl4 induced hepatic damage in rats.

OBJECTIVE: To investigate the antioxidant and hepatoprotective properties of Juniperus phoenicea (J. phoenicea) berries against CCl4-induced oxidative damage in rats. METHODS: Hepatotoxicity was induced in albino Wistar rats by single dose of CCl4 dissolved in olive oil (1 mL/kg BW, 1/1 in olive oil, i.p.). Aqueous extract of J. phoenicea berries (AEJP) was administered at the dose of 250 mg/kg/day by gavage for 12 days. RESULTS: Obtained results revealed that administration of CCl4 caused a significant increase in plasma ASAT, ALAT, ALP and LDH activities and total bilirubin concentration, compared to the control group. While, albumin and total protein concentration were significantly lower. Additionally, a significant decrease in the level of hepatic GSH, GPx and GST activities associated with a significant increase of MDA content in CCl4 group than those of the control. However, the treatment of experimental rats with AEJP prevented these alterations and maintained the antioxidant status. The histopathological observations supported the biochemical evidences of hepatoprotection. CONCLUSIONS: The results of the present investigation indicate that J. Phoenicea possesses hepatoprotective activity and this effect was may be due to its antioxidant properties.

Activity of Six Essential Oils Extracted from Tunisian Plants against Legionella pneumophila.

The aim of this study was to investigate the composition of six essential oils extracted from Tunisian plants, i.e., Artemisia herba-alba Asso, Citrus sinensis (L.) Osbeck, Juniperus phoenicea L., Rosmarinus officinalis L., Ruta graveolens L., and Thymus vulgaris L., and to evaluate their activity against Legionella pneumophila (microdilution assays). Eight Legionella pneumophila strains were studied, including the two well-known serogroup 1 Lens and Paris strains as controls and six environmental strains isolated from Tunisian spas belonging to serogroups 1, 4, 5, 6, and 8. The essential oils were generally active against L. pneumophila. The activities of the A. herba-alba, C. sinensis, and R. officinalis essential oils were strain-dependent, whereas those of the J. phoenicea and T. vulgaris oils, showing the highest anti-Legionella activities, with minimum inhibitory concentrations (MICs) lower than 0.03 and lower than or equal to 0.07 mg/ml, respectively, were independent of the strains' serogroup. Moreover, the microorganisms treated with T. vulgaris essential oil were shorter, swollen, and less electron-dense compared to the untreated controls. Isoborneol (20.91%), (1S)-α-pinene (18.30%) ß-phellandrene (8.08%), α-campholenal (7.91%), and α-phellandrene (7.58%) were the major components isolated from the J. phoenicea oil, while carvacrol (88.50%) was the main compound of the T. vulgaris oil, followed by p-cymene (7.86%). This study highlighted the potential interest of some essential oils extracted from Tunisian plants as biocides to prevent the Legionella risk.

Protective role of Juniperus phoenicea and Cupressus sempervirens against CCl(4).

AIM: To investigate the role of Cupressus sempervirens (C. sempervirens) and Juniperus phoenicea (J. phoenicea) extracts as therapeutic effect against CCl(4) with biochemical, histopathological evaluations. METHODS: A single intraperitoneal dose of 10% CCl(4) in olive oil (1 mL/kg body weight) was administered to a group of female Wister rats, sacrificed after 24 h (as the injury group). The other groups were given CCl(4) as described above and divided as follows: two groups of ten rats each were orally administered either J. phoenicea extract or C. sempervirens extract three times per week for six weeks and a further group administered CCl(4) was left for six weeks to allow self-recovery. At the end of experiment, the rats from all groups were sacrificed for sampling and for biochemical and histological analysis. RESULTS: Remarkable disturbances were observed in the levels of all tested parameters. On the other hand, rats injected with the toxic agent and left for one and a half month to self recover showed moderate improvements in the studied parameters while, treatment with both medicinal herbal extracts ameliorated the levels of the disturbed biochemical parameters. The group treated with J. phoenicea extract showed a remarkable improvement in comparison to the CCl(4) treated group. The C. sempervirens group revealing an even more remarkable effect showing histopathological liver& kidney profiles close to those of the control group. CONCLUSION: C. sempervirens and J. phoenicea leaf extracts show a remarkable effect in enhancing liver and kidney functions and may thus be of therapeutic potential in treatment hepatotoxicity and nephrotoxicity.

Chemical composition, cytotoxic activity and antimicrobial activity of essential oils of leaves and berries of Juniperus phoenicea L. grown in Egypt.

Hydrodistillation of berries and leaves of Juniperus phoenicea grown in Sinai yielded volatile oils in the yield of 0.36 and 1.96%, respectively. Using gas chromatography/mass spectrometry technique, fifty eight compounds were identified in berry oil representing 99.2% of the oil composition. alpha-Pinene was the major compound in berry oil (39.30%) followed by sabinene (24.29%). Berry oil composed mainly of monoterpenoids which amounted to 90.53%, of which 72.85% was monoterpene hydrocarbons. The sesquiterpenoids accounted for about 8% of the total oil composition. Leaf oil was composed of about 66 compounds representing 99.16% of the total composition of the oil. alpha-Pinene was the major constituent of leaf oil at concentration of 38.22%, followed by alpha-cedrol (31.23%). The monoterpene hydrocarbon was the predominant chemical group (41.29%) followed by the oxygenated sesquiterpenes (32.21%). Both oils showed very high cytotoxic activities against all cell line tested. They showed equal activities against brain (0.6 microg/ml) and cervix (5.0 microg/ml) human cell lines, while berry oil was slightly more active than leaf oil against lung (0.6 and 0.7 micro/ml, respectively), liver (0.7 and 0.9 microg/ml, respectively) and breast human cell lines (0.8 and 1. microg/ml, respectively).The antimicrobial activity and minimum inhibitory concentration (MIC) of leaf and berry oils were also determined. The oils showed high activity against most of the tested strains.