Cytoprotective, antioxidant and anti-migratory activity of Pistacia lentiscus L. supercritical carbon dioxide extract on primary human endothelial cells

Cytoprotective, antioxidant and anti-migratory activity of Pistacia lentiscus L. supercritical carbon dioxide extract on primary human endothelial cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cytoprotective, antioxidant and anti-migratory activity of Pistacia lentiscus L. supercritical carbon dioxide extract on primary human endothelial cells Roberta Giordo, Annalisa Cossu, Maria Cristina Porcu, Roberto Cappuccinelli, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1265639/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Green chemistry is emerging as a useful tool for producing valuable chemicals from biomass. However, extracted compounds need to be tested for safety, quality, and efficacy before their use in humans. Here we investigate the chemical composition and biological effects of leaves Pistacia lentiscus L . essential oil (EO) extract obtained with supercritical carbon dioxide (SCCO 2 ). Extract's phytoconstituents profiling was performed by GC-MS/MS, while antioxidant activity was evaluated on human primary endothelial cells (ECs) using the reactive oxygen species (ROS) probe H 2 DCFDA. Potentil extract toxicity and protective effect against H 2 O 2 -induced oxidative stress were investigated using LDH-leakage and BrdU-proliferation tests. Extract’s effect on ECs migration was determined by trans-well assay. Terpenes represented the main extraction process fraction yielding 0.14% of EO. Germacrene D (11.18%), delta-cadinene (10.54%), and alpha-pinene (8.7%) were the most abundant OE molecules. Challenged with ECs, increasing extract concentrations failed to affect cell proliferation or promote cell death. ROS assessment in unstressed and H 2 O 2 -treated ECs demonstrated an extract dose-dependent antioxidant activity. The exposure of H 2 O 2 -treated ECs to increasing extract concentrations dose-dependently rescued cells from the H 2 O 2 -induced impairments of cell proliferation and death. Extract was able to significantly counteract fetal calf serum-induced ECs migration. For the first time, we report that a SCCO 2 extract obtained from PL leaves is safe on ECs and may be a useful source of valuable compounds with vasculoprotective properties. Pistacia lentiscus supercritical CO2 human endothelial cells reactive oxygen species antioxidants Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Statement Of Novelty Here we report that a Pistacia lentiscus supercritical CO 2 extract is safe on human primary ECs and may be a useful source of valuable compounds capable of modulating endothelial cell functions of paramount importance in preventing or counteracting cardiovascular diseases. 1. Introduction Biomass exploitation is emerging as an important component in the production of chemicals from renewable sources [ 1 – 6 ]. In the regard, wild aromatic plants are invaluable sources of new potential drugs. Indeed, plant essential oils (EO) and their constituents, as well as products from secondary plants metabolism, have been widely used in the pharmaceutical, cosmetic, food and beverage industries [ 7 ]. Different beneficial properties, such as antioxidant, anti-inଂammatory, antiviral, antibacterial, antidiabetic and anticancer have been reported for EO [ 8 ], which are increasingly employed in the practice of complementary therapies, such as aromatherapy [ 8 ]. Among aromatic plants, Pistacia lentiscus L (PL) has found extensive use in folk medicine for several therapeutic uses including anti-hypertensive, anti-inflammatory, and antiseptic [ 9 ]. PL is an aromatic bush indigenous to Italy and other Mediterranean and Middle East countries [ 10 ]. PL leaves extracts have shown antimicrobial, antifungal, and antioxidant activity [ 11 , 12 ]. Mastic oil from Pistacia lentiscus var. Chia has also been reported to possess antioxidant and cytoprotective potential [ 13 ] and the ability to inhibit the growth and survival of human K562 leukemia cells [ 14 ]. Antiproliferative effects of PL fixed oil and its phenolic extract have also been reported on BHK21 cancer cells [ 15 ]. PL EO extraction can be performed with different techniques including steam distillation, solvent extraction, CO 2 extraction, maceration, enfleurage, cold press extraction, and water distillation. In this regard, supercritical CO 2 (SCCO 2 ) is emerging as an excellent extraction technology since it can be performed at low temperatures, thus protecting matrices components from thermal degradation [ 16 ]. Many studies have specifically indicated that SCCO 2 extraction provides the most desirable solvent for separating natural products used in foods and medicines because of its inertness, non-toxicity, low cost, critical temperature, and low pressure [ 4 , 16 – 18 ]. Cardiovascular diseases (CVD) and vascular complications linked to diabetes, rheumatic, pulmonary and inflammatory diseases are still the leading cause of morbidity and mortality worldwide [ 19 – 22 ]. In this regard, oxidative-induced endothelial damage has emerged as one of the most common triggers underlying the onset and progression of several disease-associated vascular complications [ 23 – 27 ]. Reactive oxygen species (ROS) are generally recognized as normal byproducts of the aerobic metabolism and essential second messengers that play a crucial role in regulating vital cellular functions, including proliferation, differentiation, and migration. ROS's physiological levels are finely tuned by the orchestrated action of ROS-generating enzymes and cellular antioxidants mechanisms [ 28 ]. However, dysregulation of the above-mentioned redox homeostasis can generate ROS increase, ultimately promoting oxidative stress, a phenomenon that has been linked to several pathological conditions [ 23 , 25 – 27 , 29 , 30 ]. The linkage between oxidative stress and disease-associated vascular complications suggests that counteracting oxidative stress with antioxidants might prevent diseases occurrence or ameliorate its associated vascular complications [ 31 ]. In this light, a great deal of attention is now directed on the employment of naturally occurring antioxidants as potential candidates for disease prevention and/or treatment. However, although plant-derived compounds are recognized as useful adjuvants or therapeutic tools in preventing and treating various pathological conditions [ 32 – 34 ], their efficacy and safety in humans are still major concerns [ 35 – 40 ]. Moreover, the quality of the extracted compounds may be affected by several factors, including the technological process employed; it is therefore, essential to test their safety, efficacy, and quality before they potential therapeutic utilization [ 41 ]. To our knowledge, no previous reports have investigated the effects of PL leaves SCCO 2 extract on primary human ECs. In this light, the present work aims to examine the safety and potential cytoprotective, antioxidant and anti-migration properties of a SCCO 2 extract obtained from PL on primary human ECs as an in vitro vascular model 2. Materials And Methods 2.1 Raw material preparation and characterization. Preparation and characterization of PL extract was performed as previously described [ 4 ]. Briefly, Pl leaves samples were purchased from by the Consorzio Officinerbe Sardigna (Monti, Sassari, Sardinia, Italy). Leaves were collected during full blossom and then dried to increase the ratio of essential oil to vegetable matter. This procedure also allows improving the extraction yield and preventing the risk of clogging due to the ice formation in the SCCO 2 extraction plant. The final moisture content was 11% (w/w). The dried leaves were milled and sieved to a particle size of ≤4 mm diameter and then vacuumed stored until use, at 4°C. All the highest purity solvents and reagents used in this work were purchased from Sigma-Aldrich, Steinheim, Germany. Chemical constituents of the oil were identified by comparison with reference compounds [Fluka, Acros Organics (Geel, Belgium) and Sigma-Aldrich (Steinheim, Germany)] 2.2 Pilot apparatus for the supercritical CO 2 extraction. As previously described [ 4 ], the scale-up of the SCCO 2 extraction process was performed in a pilot plant (Superfluids-5/3SEP/COL; Proras S.r.l., Rome, Italy) with a maximum allowable pressure of 700 bar. This was equipped with a 5.0 L extractor vessel, three separator vessels in series and a CO 2 recycling system. The dried vegetable matter was loaded into the extractor in a stainless-steel basket. In all the SCCO 2 extractions, the CO 2 flow was ca. 16 L/h. The dried and triturated PL leaves were submitted to a single-step extraction process, made at 90 bars and 50°C for 4 h. 2.3 Extract Analysis. Analysis of essential oil was carried out by gas chromatography (GC) and by gas chromatography-mass spectrometry (GC-MS) as previously reported [ 3 ]. Analytical GC was carried out in an Agilent 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with HP GC ChemStation data handling system, equipped with a single injector and two flame ionization detectors (FID). A Graphpak divider (Agilent Technologies) was used for simultaneous sampling to two Supelco fused silica capillary columns (Supelco Inc., Bellefonte, PA, USA) with different stationary phases: SPB-1 (polydimethylsiloxane 30 m × 0.20 mm I.D., film thickness 0.20 µm) and SUPELCOWAX 10 (polyethylene glycol 30 m × 0.20 mm I.D., film thickness 0.20 µm). Oven temperature was settled at 70° C, raising at 3°C min -1 to 220°C and then held 15 min at 220°C; injector temperature: 250°C; carrier gas: helium, adjusted to a linear velocity of 30 cm/s; splitting ratio 1:40; detector temperature: 250°C. GC-MS analyses were carried out in an Agilent 6890 gas chromatograph fitted with a HP1 fused silica column (polydimethylsiloxane 30 m × 0.25 mm I.D., film thickness 0.25 µm), interfaced with a Hewlett Packard mass selective detector 5973 (Agilent Technologies) operated by Agilent Enhanced ChemStation software. GC parameters as above; interface temperature: 250°C; MS source temperature: 230°C; MS quadrupole temperature: 150°C; ionization energy: 70 eV; ionization current: 60 µA; scan range: 35-350 u; scans/sec: 4.51. The components' identity was assigned by comparing mass spectra and retention indices for two different chromatographic stationary phases, calculated by linear interpolation to the retention of a series of n-alkanes. Experimental data were compared with corresponding data of reference oils and commercially available standards banked at a home-made library or from literature data [ 42 , 43 ]. 2.4 Cell culture and treatments. Human umbilical vein endothelial cells (HUVECs), were obtained from Innoprot, (Bizkaia, Spain) and cultured as previously described [ 44 ]. Cells were cultured in 25 cm2 flasks at 37°C, 90% humidity and 5% CO 2 in a complete Medium 199, supplemented with 100 U/mL penicillin, 25 µg/mL streptomycin, 0.85% amphotericin, 2 mM glutamine, 10% fetal calf serum and 10% new-born calf serum. For the experiments, ECs were used within 3 passages at an apparent confluence of 80%. The PL extract was kept in the dark at room temperature; immediately before use, a stock containing 1% extract (solubilized in the culture medium containing 1% dimethyl sulfoxide, DMSO) was prepared and sterilized by 0.45µm filter. Unless specified in the text, cells were plated in 96-well plates (Corning, Lowell, MA, USA) at a concentration of 105 cells/ml and processed for experiments in a complete medium as indicated in figure legends. Concentrations of 50, 150, and 600 \(\mu\) g/ml of PL extract were tested according to previous studies [ 4 , 45 ]. PL extract working solution contained 0.1% DMSO, which was used as control. Potential extract toxicity was tested over a cells exposition time of 24 hrs. Potential extract protective effect against H 2 O 2 -induced ROS increase and oxidative cell damage was investigated by pretreating the cells for 3 hrs with different extract concentrations before exposure to H2O 2 . Potential extract anti-migratory effect against FCS-induced cell migration was investigated by pretreating the cells for 3 hrs with different extract concentrations before exposure to FCS. 2.5 Measurements of intracellular reactive oxygen species. Intracellular ROS levels were determined using the ROS molecular probe H 2 DCFDA. Within the cell, esterases cleave the acetate groups on H 2 DCFDA, thus trapping the reduced form of the probe (H2DCF). Intracellular ROS oxidize H 2 DCF, yielding the fluorescent product, DCF. Before the treatments, the cells were incubated for 30 min with PBS plus (PBS with 0.5 mM CaCl2, 1 mM MgCl2, 30 mM glucose) containing 1 µM H2DCFDA, and then washed with PBS. Fluorescence was measured after the treatments using a GENios plus microplate reader (Tecan Mannedorf, Switzerland). The excitation and emission wavelengths used for fluorescence quantification were 485 nm and 535 nm, respectively. Treatment-induced variations in the fluorescence were measured kinetically, every minute for 15 consecutive minutes. All the fluorescence measurements were corrected for background fluorescence and protein concentrations. Using untreated cells as a reference, the antioxidant and prooxidant outcomes were evaluated by comparison of five measurements, and then expressed as a percentage of untreated controls [ 46 ] 2.6 Measurement of cell viability. As previously described, HUVECs cell viability was assessed after the treatments by checking the leakage of the cytoplasmatic lactate dehydrogenase (LDH) from cells with a damaged membrane [ 47 ]. The amount of LDH released in the medium by death cells was assessed using the kit CytoTox-ONE™ (Promega, Madison, WI). A standard curve with definite amounts of cells (200µ/well) was made, and the release of LDH in the medium was measured after applying lysis solution (4 µl/well). Plates containing samples were removed from the incubator and equilibrated to 22°C. Then the release of LDH from dead cells was measured by supplying lactate, NAD+, and resazurin as substrates in the presence of the enzyme diaphorase. Generation of the fluorescent resorufin product, which is proportional to the amount of LDH, was measured using a GENios plus microplate reader (Tecan Mannedorf, Switzerland) with excitation and emission of 560 nm and 590 nm, respectively. Using a standard curve, the amount of LDH release in treated and untreated cells was conversed in the number of cells per well. Data are representative of four independent experiments and are shown as the percent of untreated control cells. 2.7 Measurement of cell proliferation. Cell proliferation was assessed after the treatments using the BrdU assay (Roche CH), a chemiluminescent immunoassay based on the determination of BrdU incorporation during DNA synthesis [ 48 ]. When cells are exposed to BrdU, the compound is incorporated into the newly synthesized DNA strands of actively proliferating cells. DNA-incorporated BrdU can be measured using anti-BrdU antibodies, allowing the assessment of the DNA synthesizing cells. BrdU was added 12 hrs before the end of the experiments, then the supernatant was removed, and cells were fixed for 30 min with a Fixing-Denaturating solution (Fix-Denat). At the end of incubation time, the Fix-Denat solution was discarded, and the cells were incubated for 90 minutes with a horseradish peroxidase-conjugated anti-BrdU antibody (anti-BrdU-POD). Following three rinsing with washing buffer, the substrate solution was added and allowed to react for 3-10 min at room temperature. Within this time window, the horseradish peroxidase catalyzes the oxidation of diacylhydrazide, and the reaction product, decaying from its excited state, yields light. Finally, a GENios Plus microplate reader (Tecan Mannedorf, Switzerland) was used to read the light emission of treated cells. Results were expressed as a means ± SD of the relative light units/sec (RLU/s) values. Data are representative of four independent experiments and are shown as the percent of untreated control cells. 2.8 Migration Assay. HUVEC migration assays were performed in 24 wells trans-well as previously described [ 49 ]. Near confluent cells were serum-starved overnight, and then 40,000 cells were plated onto the top chamber of the cell culture inserts. Cells in the upper part of the chamber were treated with PL extract in serum free medium (Medium 199 with antibiotics), whereas the complete medium was added to the lower chamber. The assembled cell culture chamber was then incubated at 37°C, 5% CO 2 for 12 hrs. At the end of incubation time, the upper surfaces of the membranes were gently wiped with cotton swabs to remove the non-migratory cells. The membranes were then fixed with paraformaldehyde, PAF (4%), stained with the fluorophore Hoechst 33342 (10 µg/ml), and the fluorescence was measured using a GENios plus microplate reader (Tecan Mannedorf, Switzerland). The excitation and emission wavelengths used for fluorescence quantification were 340 nm and 485 nm, respectively. 2.9 Statistical analysis. One-way analysis of variance (ANOVA), followed by a post-hoc Newman-Keuls for multiple comparisons, was used to detect differences among studied groups. All statistical analyses were performed using GraphPad Prism version 9.00 for Windows (GraphPad Software, San Diego, CA, USA), and p-values < 0.05 were considered statistically significant. 3. Results 3.1 Essential oils composition Percentages of individual components of PL extract were calculated based on gas chromatography (GC) peak areas without flame ionization detection (FID) response factor correction. Chromatogram and main chemical components of PL essential oil are reported respectively in figure 1 and Table 1 . The analytical results were consistent with those reported in the literature concerning terpene compounds, representing the more important fraction with antioxidant activity. The total essential oil yield, after 4 hours of extraction was 0.14% and the main abundant constituents included germacrene D (11.18%), delta-Cadinene (10.54%), alpha-pinene (8.7%), beta-caryophyllene (5.74%), myrcene (4.5%), beta-phellandrene (4.33%), terpin-4-ol (4.306), epi-alpha-muurolol (3.262) and beta-pinene (3.00%). The complete profiling of the essential oil components is reported in Table 1 . Table 1 Chemical components of the Pistacia Lentiscus L . SCCO2 extract obtained by GC-GM n RIK Tr (min) Compound % 1 926 5.01 Tricyclene 0.096 2 931 5.11 Alpha-Thujene 0.219 3 938 5.29 Alpha-Pinene 8.692 4 953 5.66 Camphene 0.453 5 977 6.29 Sabinene 2.857 6 980 6.39 Beta-Pinene 3.025 7 993 6.76 Myrcene 4.562 8 1007 7.17 Alpha-Phellandrene 2.170 9 1019 7.55 Alpha-Terpinene 1.661 10 1028 7.79 Para-Cymene 0.649 11 1032 7.95 Beta-Phellandrene 4.332 12 1051 8.56 Beta(E)-Ocimene 0.135 13 1059 8.83 Isopentil N-Butanoate 0.245 14 1062 8.94 Gamma-Terpinene 2.533 15 1090 10 Terpinolene 0.830 16 1093 10.12 2-Nonanone 0.248 17 1101 10.43 Linalool 0.388 18 1107 10.63 Isopentyl Isovalerate 0.207 19 1180 13.47 Terpin-4-Ol 4.306 20 1191 13.98 Alpha-Terpineol 0.874 21 1253 16.49 N.I. 0.440 22 1256 16.61 N.I. 0.218 23 1286 17.95 Bornyl Acetate 0.118 24 1294 18.32 2-Undecanone 1.362 25 1302 18.65 N.I. 0.110 26 1351 20.61 Alpha-Cubebene 0.262 27 1377 21.69 Alpha-Copaene 1.330 28 1390 22.29 Beta-Cubebene 0.353 29 1392 22.38 Beta-Elemene 1.288 30 1408 23.04 N.I. 0.138 31 1420 23.48 Beta-Caryophyllene 5.738 32 1429 23.85 Beta-Gurjunene 0.213 33 1438 24.18 N.I. 0.746 34 1451 24.71 N.I. 0.759 35 1454 24.82 Alpha-Humulene 2,097 36 1461 25.1 Allo-Aromadendrene 0.907 37 1463 25.2 Muurola-4(14),5-Diene 0.198 38 1474 25.66 Cadina-1(6),4-Diene 1.297 39 1478 25.82 Gamma-Muurolene 2.610 40 1482 26 Germacrene-D 11.182 41 1486 26.15 Beta-Selinene 0.279 42 1491 26.36 Muurola-4(15),5-Diene 1.025 43 1495 26.55 Alpha-Selinene 1.507 44 1499 26.73 Alpha-Muurolene 2,313 45 1503 26.88 N.I. 0.294 46 1506 26.99 N.I. 0.183 47 1509 27.09 Beta-Bisabolene 0.924 48 1513 27.25 Gamma-Cadinene 1.186 49 1525 27.68 Delta-Cadinene 10.542 50 1533 27.97 Trans-Cadina-1(2)-4-Diene 0.791 51 1538 28.16 Alpha-Cadinene 0.234 52 1544 28.4 N.I. 0.340 53 1550 28.61 Elemol 0.178 54 1556 28.86 N.I. 0.116 55 1559 28.98 Elemicin 0.100 56 1565 29.21 E-Nerolidol 0.117 57 1576 29.65 Spatutenol 0.248 58 1582 29.87 Caryophyllene Oxide 0.251 59 1584 29.97 N.I. 0.155 60 1590 30.19 N.I. 0.052 61 1613 31.07 N.I. 0.104 62 1616 31.17 N.I. 0.158 63 1627 31.58 N.I. 1.247 64 1631 31.7 Gamma-Eudesmol 0.236 65 1642 32.11 Epi-Alpha-Muurolol 3.262 66 1646 32.25 Alpha-Muurolol 0.828 67 1649 32.36 Beta-Eudesmol 0.121 68 1654 32.56 Alpha-Cadinol 2.626 69 1683 33.64 Epi-Alpha-Bisabolol 0.506 70 1685 33.71 Alpha-Bisabolol 0.148 71 1694 34.05 N.I. 0.086 Retention indices (RIK), retention times (Tr) and chromatographic area percentages (%) of the most abundant compounds found in the essential oil extract obtained by supercritical CO 2 (SCCO 2 ) extraction at 90 bar, 50°C from Pistacia lentiscus L . 3.2 Biological activity Using primary human endothelial cells as an in vitro vascular model, we investigated the PL extract ability to modulate different biological cell functions, including proliferation, migration, and intracellular redox status. 3.2.1 PL extract does not affect endothelial cell viability and proliferation Compounds safety is of paramount importance for their potential therapeutic employment; therefore, we first investigated the potential toxicity of the obtained extract by assessing its effects on cell viability, cell proliferation, and intracellular ROS production. Indeed, intracellular ROS generation is closely related to cell survival, proliferation and apoptosis [ 50 , 51 ]. Based on previously reported data concerning the effect of PL essential oil on different cultured cells [ 45 ], we evaluated the possible harmful effects of three concentrations [50, 150, and 600 µg/ml] of the obtained PL extract on HUVEC viability and proliferation. To this end, cells were treated for 24 hrs with the PL extract, then cell viability and proliferation were assessed as reported in the material and methods sections. As reported in Figure 2 A, the PL extract had no toxic effect at any of the tested concentrations indicating safety of both the extract and the extraction process applied. Likewise, the data in figure 2 B indicate that the tested extract concentrations failed to induce any detrimental effects on HUVEC proliferation, further confirming the extract's safety. 3.2.2 PL extract showed antioxidant effect against H 2 O 2 -induced oxidative stress The antioxidant properties of PL leaves and fruits compounds have been reported in vitro by measuring their scavenging ability or assessing their anti-lipid peroxidation potential [ 52 , 53 ]. However, whether PL SCCO 2 extract is able to exert antioxidant effects in biological models by protecting cells from oxidative stress remains to be elucidated. Therefore, we sought to investigate whether the obtained PL extract could counteract oxidative stress in human primary endothelial cells oxidatively stressed with H 2 O 2 . For this purpose, H 2 DCFDA-loaded cells were pretreated for 3 hrs with the indicated extract concentrations and then incubated for 6 hrs in the presence or absence of 75 µM H 2 O 2 . The data derived from five pooled measurements were expressed as percentages of the untreated cells and compared with the vehicle control (0.1% DMSO). As depicted in figure 3 A, exposure of H 2 O 2 -treated cells to increasing concentrations of PL extract showed a significant dose-dependent antioxidant effect compared to cells treated with only H 2 O 2 . We next wondered whether the PL extract per se could exert any antioxidant or prooxidant effect in the absence of oxidative insults. As reported in figure 3 B, the exposure of unstressed cells to increasing extract concentrations induced a significant antioxidant effect at 150 and 600 µg/ml, while failing to affect the intracellular redox state at 50 µg /ml. These findings are in agreement with the cell viability and proliferation results showing no extract toxicity up to 24 hrs of cell treatment (Fig. 2 A-B) 3.2.3 PL extracts showed protective effect against H 2 O 2 -induced oxidative stress Oxidative-induced endothelial damage appears to trigger and sustain cardiovascular diseases [ 51 ]. Therefore, a great deal of research is now focused on finding natural antioxidants capable to prevent or counteract CVD-associated ROS increases. To determine whether the observed PL extract antioxidant effect could be protective against the H 2 O 2 -induced oxidative damage, we measured HUVEC viability by assessing the cellular membrane integrity. To this end, cells were exposed for 3 h to increasing concentrations of PL extract and then incubated for 24 h in the absence or presence of 75 µM H 2 O 2 . Cell were then processed for cell viability as reported in material and methods. As indicated in figure 4 , cells exposition to increasing doses of PL extract provided a significant dose-dependent cytoprotective effect with respect to the H2O2-induced cell damage. Indeed, all the tested PL concentrations were able to significantly counteract H2O2-induced cell damage. Consonant with these findings are the data in figure 3 B, reporting the extract's ability to dose-dependently prevent the detrimental effect on HUVEC proliferation elicited by H 2 O 2 . 3.2.4 PL extracts showed antimigratory effect against serum-induced migration Migration, the cell movement in response to chemical and/or mechanical signals, beside playing an essential role in physiologic processes, such as embryonic development, wound healing, tissue regeneration, it is also a critical process in pathological conditions such as tumor growth, blinding eye diseases, diabetic retinopathy and arthritis [ 54 ]. Endothelial cell migration is an essential step of the angiogenic process; indeed, endothelial cells, which normally are maintained in a quiescent state, are stimulated to degrade the basement membrane and migrate into the perivascular stroma in response to either proangiogenic factors or by the downregulation of antiangiogenic factors [ 55 ]. For this reason, we investigated the ability of PL extract to modulate endothelial cells migration using the matrigel transfilter cell invasion assays, a modification of the Boyden chamber migration assay [ 56 ]. These three-dimensional assays are based on the migration of endothelial cells, placed on top of a filter containing 8 µm diameter pores, which allow only active passage of the cells towards an attractant placed in the lower chamber [ 57 ]. Migrated endothelial cells through modified Boyden chambers were measured by reading the fluorescent product of Hoechst 33342 resulted from the metabolism of live cells migrated through the micropores of the upper chamber. Figure 5 demonstrated that the PL extract dose-dependently counteracted the serum-induced cell migration, eliciting a significant reduction at the doses of 150 and 600. 4. Discussion The genus Pistacia belongs to the Anacardiaceae family and comprises about 70 genera and over 600 species [ 9 , 58 ]. The species PL is a dioecious evergreen shrub or small tree, from 1 to 5 m high, with a strong smell of resin, growing in dry and rocky areas in Mediterranean Europe. The aromatic resin, ivory colored, is harvested as a spice from the cultivated mastic trees. The genus Pistacia has shown many interesting biological activities in vivo and in vitro, such as antimicrobial activity [ 59 ], antifungal and anticancer activity [ 60 ]. It has also been shown to inhibit pro-inflammatory substances production and protect the cardiovascular system by lowering total serum cholesterol, low-density lipoprotein, and triglycerides in rats. In addition, it resulted able to preserve low-density lipoproteins from oxidation in humans.[ 61 , 62 ]. Although the antioxidant, antimicrobial and anti-inflammatory activities of the PL. gum and the fixed oil have been previously tested [ 12 , 63 – 66 ]; this is the first report showing the effects of the SCCO 2 technique-obtained PL essential oil on primary human endothelial cells. However, we need to clarify that we cannot discriminate whether the biological effects observed in this work are attributable to a single molecule's type present in the analyzed PL extract or to an entire chemical class of secondary metabolites, the terpenes (beta-caryophyllene, germacrene D, alpha-pinene, myrcene, beta-phellandrene, and alpha-humulene). A great number of plants produce terpenes and many studies have demonstrated how this fraction represents the more important component with antioxidant activity [ 67 ]. In this regard, an interesting review shows how the terpene, lycopene, may protect against atherosclerosis, inhibiting ROS production in vitro and protecting LDL from oxidation [ 68 ]. Furthermore, several studies showed that sesquiterpene lactones, another class of terpenes, possess strong anti-inflammatory, anti-tumor and antimicrobial activities [ 69 – 71 ]. Since then, this class of phytochemicals has attracted the attention of researchers towards their potential medicinal properties. Several sesquiterpene lactones such as artemisinins, thapsigargins, parthenolide etc., have demonstrated the ability to inhibit angiogenesis in vitro and in vivo by suppressing HUVECs proliferation and migration, microvessel formation, and vascular endothelial growth factor (VEGF) expression. In addition, they resulted also able to prevent osteolytic bone metastasis by suppressing the growth and migration of mammary carcinosarcoma cells [ 72 – 74 ]. Therefore, based on the literature data, we are of the opinion that our current data might be the result of the synergistic effect produced by the components present in the extract, for which antioxidant and antinflammatory activities have been already reported [ 63 , 75 , 76 ]. This synergic mechanism is not fully understood, and it may involve the compounds’ action toward different pharmacological targets. In the present study, we investigated the SCCO 2 -obtained PL extract cytoprotective, antioxidant, and anti-migratory potential, three aspects essential for maintaining an optimal vascular structure and functionality. According to previously reported data [ 45 ], HUVECs were treated with increasing PL extract concentrations [50, 150 and 600 µg/ml], and no effect on the cell viability and proliferation was observed, indicating that the applied process was able to provide a safe extract that lack of cell cytotoxicity. Then we investigated potential biological properties harbored by the obtained extract. To this end, the same PL extract concentrations were employed to assess their antioxidant potential against H 2 O 2 -induced oxidative stress in primary human endothelial cells, along with their possible protective effect toward oxidative stress-induced cell death. All the tested extract doses were able to counteract H 2 O 2 -induced ROS increase and oxidative-induced cell death, confirming that the possible synergistic effect of its components confers antioxidant properties to the PL extract. In recent years, it was established that natural antioxidants might modulate HUVECs migration [ 77 ]. As already mentioned, PL extract GC–MS data demonstrated a predominance of terpenes (monoterpenes and sesquiterpenes). In this regard, previous studies showed that sesquiterpenes owned anti-inଂammatory properties [78,79], and the contained-monoterpene in plant-derived EO effectively inhibited carrageenan-induced edema and neutrophil migration [ 78 ]. Cell migration is a highly integrated, multi-step process that plays an important role in the progression of various diseases, including cancer, atherosclerosis, and arthritis. In this regard, the two higher PL concentrations tested were able to significantly reduce serum-induced HUVECs migration, suggesting a direct effect of the extract on the migration process. These results were in line with those related to PL extract antioxidant activity as a confirmation of the link between oxidative stress and endothelial cells migration. In fact, ROS produced via NADPH oxidase activation stimulate various redox signaling pathways leading to angiogenic responses, including endothelial cell migration. In particular, activated VEGF increases ROS production via Rac1-dependent NADPH oxidase, and ROS in turn are involved in VEGF-induced autophosphorylation of VEGFR-2 [ 55 ]. We know that further investigations are needed to better understand the molecular events elicited by PL extract on this cellular model. Nevertheless, our data provide novel insight concerning the use of SCCO 2 as useful green chemistry process to obtain valuable products harboring essential biological process form biomass 5. Conclusions Whether PL extract obtained by SCCO 2 is safe and possesses biological activities against CVD remains to be investigated. Here we report that SCCO 2 -derived PL extract is safe, shows cytoprotective effects against oxidative damage, and modulates ROS production and HUVECs migration. Our results indicate SCCO 2 as a secure green chemistry process to exploit biomasses to obtain precious material containing molecules capable of modulating cell functions of paramount importance in preventing or counteracting CVD. Credit Authorship Contribution Statement RG, GP, AMP: Conceptualization. RG, AC, MCP, RC, GB, JSR, AMP: Methodology. RG, AC, MCP, RC, GB, AMP: Investigation. GKN, LP, GP: resources. RG, GP, AMP: writing—original draft preparation. RG, AC, MCP, RC, GB, JSR, GKN, LP, GP, AMP: writing—review and editing. GP: supervision. LP, GP: project administration. GKN, LP, GP: funding acquisition Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data Availability Statement The data presented in this study are available in this article. Acknowledgements This work has been made possible thanks to grants from the University of Sharjah (Seed 2001050151 and collaborative 2101050160) to GP; Qatar University (IRCC-2019-007) to GKN and GP; (Fondo UNISS di Ateneo per la Ricerca 2020) to GP. References Cho, E.J., Trinh, L.T.P., Song, Y., Lee, Y.G., Bae, H.-J.: Bioconversion of biomass waste into high value chemicals. Bioresour. Technol. 298 , 122386 (2020) Posadino, A.M., Cossu, A., Giordo, R., Piscopo, A., Abdel-Rahman, W.M., Piga, A., Pintus, G.: Antioxidant Properties of Olive Mill Wastewater Polyphenolic Extracts on Human Endothelial and Vascular Smooth Muscle Cells. Foods 10 (4), 800 (2021) Posadino, A.M., Biosa, G., Zayed, H., Abou-Saleh, H., Cossu, A., Nasrallah, G.K., Giordo, R., Pagnozzi, D., Porcu, M.C., Pretti, L.: Protective effect of cyclically pressurized solid–liquid extraction polyphenols from Cagnulari grape pomace on oxidative endothelial cell death. 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Pintus","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYFACxgYQKccgASQlGA4QqQWozpgULUAAVJfYIAFjEgLy0c1tjz+21aXPn9387INFzR0G/vYDjB8+4NFieOdgu8HBtsO5G+4cM54hcewZg8SZBGbJGfi0zEhskzjYdiB3g0QC0D9shxkYbjAwSPMQ1lKXLj8j/TODxL/DDPI3GJh//8HnFwmwFuYEhhs5xgySbYcZDG4wsEnj876BzME2iTPnDhtuuJFTzCDZd5jH8Exim2UPPltmtz+TqCirkwc6bDOzxLfDcnLHDx++8QOfLTeQOMzAuOGBpQfctiAHJyO+6BgFo2AUjIKRCwBbTlJlk8+UAAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-3031-7733","institution":"University of Sharjah","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Gianfranco","middleName":"","lastName":"Pintus","suffix":""},{"id":78581092,"identity":"1c05bbe6-1363-4021-92fd-6bb4c2351d53","order_by":9,"name":"Anna Maria Posadino","email":"","orcid":"","institution":"University of Sassari: Universita degli Studi di Sassari","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anna","middleName":"Maria","lastName":"Posadino","suffix":""}],"badges":[],"createdAt":"2022-01-16 12:12:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1265639/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1265639/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":17811214,"identity":"934c633f-2cef-4655-89f0-73e209b9eb50","added_by":"auto","created_at":"2022-01-31 16:42:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12924,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of the SCCO\u003csub\u003e2\u003c/sub\u003e extract obtained at 90 bar, 50°C from \u003cem\u003ePistacia lentiscus L\u003c/em\u003e. leaves.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1265639/v1/dafd8df2e9636395c03aac5e.png"},{"id":17811212,"identity":"cd8d6758-7fb4-4ee8-a37a-4313553fe526","added_by":"auto","created_at":"2022-01-31 16:42:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33299,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003ePistacia lentiscus L\u003c/em\u003e extract on HUVEC viability and proliferation. Cells were exposed for 24 h in the absence (CTRL) or presence to the indicated concentrations of \u003cem\u003ePistacia lentiscus L\u003c/em\u003e extract. Cell viability and proliferation were assessed as reported in the “Materials and Methods’’ section. CTRL, untreated cells; PL, \u003cem\u003ePistacia lentiscus L\u003c/em\u003e. extract. Values are shown as mean ± SD and expressed as a percentage of the vehicle CTRL equal to 100% (0.1% DMSO), (n = 4).\u003c/p\u003e","description":"","filename":"f2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1265639/v1/f4a26e72f20848ecae6551ee.jpg"},{"id":17811014,"identity":"f5cb2b41-83f1-40a7-a5c1-f8a561a52c19","added_by":"auto","created_at":"2022-01-31 16:39:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39858,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant dose-response effect of \u003cem\u003ePistacia lentiscus L\u003c/em\u003e extract against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress. A) Dose-response effect of \u003cem\u003ePistacia lentiscus \u003c/em\u003eextract on intracellular ROS levels in HUVEC treated with 75µM of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Cells were exposed for 3 hrs to the indicated concentrations of PL extract and then incubated for 6 hrs in the absence or presence of 75 µM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. B) Dose-response effect of \u003cem\u003ePistacia lentiscus L\u003c/em\u003e extract on intracellular ROS levels in unstreased HUVECs. Cells were exposed for 3 hrs to the indicated concentrations of \u003cem\u003ePistacia lentiscus L\u003c/em\u003e extracts and then incubated for 6 hrs before ROS determination. Intracellular ROS levels were assessed, as reported in the “Materials and Methods’’ section. CTRL, untreated cells; H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, hydrogen peroxide; PL, \u003cem\u003ePistacia lentiscus L\u003c/em\u003e extract. Values are shown as mean ± SD and expressed as a percentage of the vehicle CTRL equal to 100% (0.1% DMSO). *Significantly different from the CTRL (p \u0026lt; 0.05). #, Significantly different from H2O2 (p \u0026lt; 0.05), (n = 5).\u003c/p\u003e\u003cp\u003e3.2.3 PL extracts showed protective effect against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress\u003c/p\u003e\u003cp\u003eOxidative-induced endothelial damage appears to trigger and sustain cardiovascular diseases [51]. Therefore, a great deal of research is now focused on finding natural antioxidants capable to prevent or counteract CVD-associated ROS increases. To determine whether the observed PL extract antioxidant effect could be protective against the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage, we measured HUVEC viability by assessing the cellular membrane integrity. To this end, cells were exposed for 3 h to increasing concentrations of PL extract and then incubated for 24 h in the absence or presence of 75 µM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Cell were then processed for cell viability as reported in material and methods. As indicated in figure 4, cells exposition to increasing doses of PL extract provided a significant dose-dependent cytoprotective effect with respect to the H2O2-induced cell damage. Indeed, all the tested PL concentrations were able to significantly counteract H2O2-induced cell damage. Consonant with these findings are the data in figure 3B, reporting the extract's ability to dose-dependently prevent the detrimental effect on HUVEC proliferation elicited by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"f3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1265639/v1/bb287093a674d636b7a8add3.jpg"},{"id":17811211,"identity":"dc7dbd56-c6e4-45e3-9809-b77d9b756429","added_by":"auto","created_at":"2022-01-31 16:42:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":43977,"visible":true,"origin":"","legend":"\u003cp\u003eCytoprotective dose-response effect of \u003cem\u003ePistacia lentiscus L\u003c/em\u003e extract against H2O2-induced cell death. Dose-response effect of \u003cem\u003ePistacia lentiscus L\u003c/em\u003e extract on cell viability in HUVEC treated with 75µM of H2O2. Cells were exposed for 3 hrs to the indicated concentrations of \u003cem\u003ePistacia lentiscus L\u003c/em\u003e extracts and then incubated for 24 hrs in the absence or presence of 75 µM H2O2. Cell viability was assessed as reported in the “Materials and Methods’’ section. CTRL, untreated cells; H2O2, hydrogen peroxide; PL, \u003cem\u003ePistacia lentiscus L\u003c/em\u003e extract. Values are shown as mean ± SD and expressed as a percentage of the vehicle CTRL equal to 100% (0.1% DMSO). *Significantly different from CTRL (p \u0026lt; 0.05). #, Significantly different from H2O2 (p \u0026lt; 0.05), (n = 4).\u003c/p\u003e\u003cp\u003e\u0026nbsp;\u003c/p\u003e\u003cp\u003e3.2.4 PL extracts showed antimigratory effect against serum-induced migration\u003c/p\u003e\u003cp\u003eMigration, the cell movement in response to chemical and/or mechanical signals, beside playing an essential role in physiologic processes, such as embryonic development, wound healing, tissue regeneration, it is also a critical process in pathological conditions such as tumor growth, blinding eye diseases, diabetic retinopathy and arthritis [54]. Endothelial cell migration is an essential step of the angiogenic process; indeed, endothelial cells, which normally are maintained in a quiescent state, are stimulated to degrade the basement membrane and migrate into the perivascular stroma in response to either proangiogenic factors or by the downregulation of antiangiogenic factors [55]. For this reason, we investigated the ability of PL extract to modulate endothelial cells migration using the matrigel transfilter cell invasion assays, a modification of the Boyden chamber migration assay [56]. These three-dimensional assays are based on the migration of endothelial cells, placed on top of a filter containing 8 μm diameter pores, which allow only active passage of the cells towards an attractant placed in the lower chamber [57]. Migrated endothelial cells through modified Boyden chambers were measured by reading the fluorescent product of Hoechst 33342 resulted from the metabolism of live cells migrated through the micropores of the upper chamber. Figure 5 demonstrated that the PL extract dose-dependently counteracted the serum-induced cell migration, eliciting a significant reduction at the doses of 150 and 600.\u003c/p\u003e","description":"","filename":"f4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1265639/v1/cf790eddd94ea40de3a56468.jpg"},{"id":17811017,"identity":"e9ac3a75-d280-4403-833e-91de21fc1248","added_by":"auto","created_at":"2022-01-31 16:39:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":18928,"visible":true,"origin":"","legend":"\u003cp\u003eAntimigratory dose-response effect of \u003cem\u003ePistacia lentiscus L\u003c/em\u003e extract against serum-induced cell migration. Dose-response effect of \u003cem\u003ePistacia lentiscus L\u003c/em\u003e extract on cell migration in HUVEC treated with 10% fetal calf serum. Cells were exposed for 3 hrs to the indicated concentrations of \u003cem\u003ePistacia lentiscus L\u003c/em\u003e extracts in serum free medium, then cell migration was determined for 12 hrs as reported in the ‘‘Materials and Methods’’ section. CTRL, untreated cells; PL, \u003cem\u003ePistacia lentiscus L\u003c/em\u003e extract. Values are shown as mean ± SD and expressed as a percentage of the vehicle CTRL equal to 100% (0.1% DMSO). *Significantly different from CTRL (p \u0026lt; 0.05), (n = 4).\u003c/p\u003e","description":"","filename":"f5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1265639/v1/d84b3366a9466244c6ed2f16.jpg"},{"id":20672907,"identity":"2e8e02b2-531d-4110-af7c-86233cb94d9f","added_by":"auto","created_at":"2022-04-22 21:19:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":481998,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1265639/v1/21330e14-2544-42b7-9197-dd395c27f5c7.pdf"}],"financialInterests":"","formattedTitle":"Cytoprotective, antioxidant and anti-migratory activity of Pistacia lentiscus L. supercritical carbon dioxide extract on primary human endothelial cells","fulltext":[{"header":"Statement Of Novelty","content":"\u003cp\u003eHere we report that a \u003cem\u003ePistacia lentiscus\u003c/em\u003e supercritical CO\u003csub\u003e2\u003c/sub\u003e extract is safe on human primary ECs and may be a useful source of valuable compounds capable of modulating endothelial cell functions of paramount importance in preventing or counteracting cardiovascular diseases.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eBiomass exploitation is emerging as an important component in the production of chemicals from renewable sources [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In the regard, wild aromatic plants are invaluable sources of new potential drugs. Indeed, plant essential oils (EO) and their constituents, as well as products from secondary plants metabolism, have been widely used in the pharmaceutical, cosmetic, food and beverage industries [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Different beneficial properties, such as antioxidant, anti-inଂammatory, antiviral, antibacterial, antidiabetic and anticancer have been reported for EO [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], which are increasingly employed in the practice of complementary therapies, such as aromatherapy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Among aromatic plants, \u003cem\u003ePistacia lentiscus L\u003c/em\u003e (PL) has found extensive use in folk medicine for several therapeutic uses including anti-hypertensive, anti-inflammatory, and antiseptic [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. PL is an aromatic bush indigenous to Italy and other Mediterranean and Middle East countries [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. PL leaves extracts have shown antimicrobial, antifungal, and antioxidant activity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Mastic oil from \u003cem\u003ePistacia lentiscus var. Chia\u003c/em\u003e has also been reported to possess antioxidant and cytoprotective potential [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and the ability to inhibit the growth and survival of human K562 leukemia cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Antiproliferative effects of PL fixed oil and its phenolic extract have also been reported on BHK21 cancer cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. PL EO extraction can be performed with different techniques including steam distillation, solvent extraction, CO\u003csub\u003e2\u003c/sub\u003e extraction, maceration, enfleurage, cold press extraction, and water distillation. In this regard, supercritical CO\u003csub\u003e2\u003c/sub\u003e (SCCO\u003csub\u003e2\u003c/sub\u003e) is emerging as an excellent extraction technology since it can be performed at low temperatures, thus protecting matrices components from thermal degradation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Many studies have specifically indicated that SCCO\u003csub\u003e2\u003c/sub\u003e extraction provides the most desirable solvent for separating natural products used in foods and medicines because of its inertness, non-toxicity, low cost, critical temperature, and low pressure [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCardiovascular diseases (CVD) and vascular complications linked to diabetes, rheumatic, pulmonary and inflammatory diseases are still the leading cause of morbidity and mortality worldwide [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this regard, oxidative-induced endothelial damage has emerged as one of the most common triggers underlying the onset and progression of several disease-associated vascular complications [\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Reactive oxygen species (ROS) are generally recognized as normal byproducts of the aerobic metabolism and essential second messengers that play a crucial role in regulating vital cellular functions, including proliferation, differentiation, and migration. ROS's physiological levels are finely tuned by the orchestrated action of ROS-generating enzymes and cellular antioxidants mechanisms [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, dysregulation of the above-mentioned redox homeostasis can generate ROS increase, ultimately promoting oxidative stress, a phenomenon that has been linked to several pathological conditions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe linkage between oxidative stress and disease-associated vascular complications suggests that counteracting oxidative stress with antioxidants might prevent diseases occurrence or ameliorate its associated vascular complications [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In this light, a great deal of attention is now directed on the employment of naturally occurring antioxidants as potential candidates for disease prevention and/or treatment. However, although plant-derived compounds are recognized as useful adjuvants or therapeutic tools in preventing and treating various pathological conditions [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], their efficacy and safety in humans are still major concerns [\u003cspan additionalcitationids=\"CR36 CR37 CR38 CR39\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Moreover, the quality of the extracted compounds may be affected by several factors, including the technological process employed; it is therefore, essential to test their safety, efficacy, and quality before they potential therapeutic utilization [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo our knowledge, no previous reports have investigated the effects of PL leaves SCCO\u003csub\u003e2\u003c/sub\u003e extract on primary human ECs. In this light, the present work aims to examine the safety and potential cytoprotective, antioxidant and anti-migration properties of a SCCO\u003csub\u003e2\u003c/sub\u003e extract obtained from PL on primary human ECs as an in vitro vascular model\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cp\u003e2.1 Raw material preparation and characterization.\u003c/p\u003e \u003cp\u003ePreparation and characterization of PL extract was performed as previously described [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Briefly, Pl leaves samples were purchased from by the Consorzio Officinerbe Sardigna (Monti, Sassari, Sardinia, Italy). Leaves were collected during full blossom and then dried to increase the ratio of essential oil to vegetable matter. This procedure also allows improving the extraction yield and preventing the risk of clogging due to the ice formation in the SCCO\u003csub\u003e2\u003c/sub\u003e extraction plant. The final moisture content was 11% (w/w). The dried leaves were milled and sieved to a particle size of \u0026le;4 mm diameter and then vacuumed stored until use, at 4\u0026deg;C. All the highest purity solvents and reagents used in this work were purchased from Sigma-Aldrich, Steinheim, Germany. Chemical constituents of the oil were identified by comparison with reference compounds [Fluka, Acros Organics (Geel, Belgium) and Sigma-Aldrich (Steinheim, Germany)]\u003c/p\u003e \u003cp\u003e2.2 Pilot apparatus for the supercritical CO\u003csub\u003e2\u003c/sub\u003e extraction.\u003c/p\u003e \u003cp\u003eAs previously described [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], the scale-up of the SCCO\u003csub\u003e2\u003c/sub\u003e extraction process was performed in a pilot plant (Superfluids-5/3SEP/COL; Proras S.r.l., Rome, Italy) with a maximum allowable pressure of 700 bar. This was equipped with a 5.0 L extractor vessel, three separator vessels in series and a CO\u003csub\u003e2\u003c/sub\u003e recycling system. The dried vegetable matter was loaded into the extractor in a stainless-steel basket. In all the SCCO\u003csub\u003e2\u003c/sub\u003e extractions, the CO\u003csub\u003e2\u003c/sub\u003e flow was ca. 16 L/h. The dried and triturated PL leaves were submitted to a single-step extraction process, made at 90 bars and 50\u0026deg;C for 4 h.\u003c/p\u003e \u003cp\u003e2.3 Extract Analysis.\u003c/p\u003e \u003cp\u003eAnalysis of essential oil was carried out by gas chromatography (GC) and by gas chromatography-mass spectrometry (GC-MS) as previously reported [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Analytical GC was carried out in an Agilent 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with HP GC ChemStation data handling system, equipped with a single injector and two flame ionization detectors (FID). A Graphpak divider (Agilent Technologies) was used for simultaneous sampling to two Supelco fused silica capillary columns (Supelco Inc., Bellefonte, PA, USA) with different stationary phases: SPB-1 (polydimethylsiloxane 30 m \u0026times; 0.20 mm I.D., film thickness 0.20 \u0026micro;m) and SUPELCOWAX 10 (polyethylene glycol 30 m \u0026times; 0.20 mm I.D., film thickness 0.20 \u0026micro;m). Oven temperature was settled at 70\u0026deg; C, raising at 3\u0026deg;C min -1 to 220\u0026deg;C and then held 15 min at 220\u0026deg;C; injector temperature: 250\u0026deg;C; carrier gas: helium, adjusted to a linear velocity of 30 cm/s; splitting ratio 1:40; detector temperature: 250\u0026deg;C. GC-MS analyses were carried out in an Agilent 6890 gas chromatograph fitted with a HP1 fused silica column (polydimethylsiloxane 30 m \u0026times; 0.25 mm I.D., film thickness 0.25 \u0026micro;m), interfaced with a Hewlett Packard mass selective detector 5973 (Agilent Technologies) operated by Agilent Enhanced ChemStation software. GC parameters as above; interface temperature: 250\u0026deg;C; MS source temperature: 230\u0026deg;C; MS quadrupole temperature: 150\u0026deg;C; ionization energy: 70 eV; ionization current: 60 \u0026micro;A; scan range: 35-350 u; scans/sec: 4.51. The components' identity was assigned by comparing mass spectra and retention indices for two different chromatographic stationary phases, calculated by linear interpolation to the retention of a series of n-alkanes. Experimental data were compared with corresponding data of reference oils and commercially available standards banked at a home-made library or from literature data [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e2.4 Cell culture and treatments.\u003c/p\u003e \u003cp\u003eHuman umbilical vein endothelial cells (HUVECs), were obtained from Innoprot, (Bizkaia, Spain) and cultured as previously described [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Cells were cultured in 25 cm2 flasks at 37\u0026deg;C, 90% humidity and 5% CO\u003csub\u003e2\u003c/sub\u003e in a complete Medium 199, supplemented with 100 U/mL penicillin, 25 \u0026micro;g/mL streptomycin, 0.85% amphotericin, 2 mM glutamine, 10% fetal calf serum and 10% new-born calf serum. For the experiments, ECs were used within 3 passages at an apparent confluence of 80%. The PL extract was kept in the dark at room temperature; immediately before use, a stock containing 1% extract (solubilized in the culture medium containing 1% dimethyl sulfoxide, DMSO) was prepared and sterilized by 0.45\u0026micro;m filter. Unless specified in the text, cells were plated in 96-well plates (Corning, Lowell, MA, USA) at a concentration of 105 cells/ml and processed for experiments in a complete medium as indicated in figure legends. Concentrations of 50, 150, and 600 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu\\)\u003c/span\u003e\u003c/span\u003eg/ml of PL extract were tested according to previous studies [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. PL extract working solution contained 0.1% DMSO, which was used as control. Potential extract toxicity was tested over a cells exposition time of 24 hrs. Potential extract protective effect against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced ROS increase and oxidative cell damage was investigated by pretreating the cells for 3 hrs with different extract concentrations before exposure to H2O\u003csub\u003e2\u003c/sub\u003e. Potential extract anti-migratory effect against FCS-induced cell migration was investigated by pretreating the cells for 3 hrs with different extract concentrations before exposure to FCS.\u003c/p\u003e \u003cp\u003e2.5 Measurements of intracellular reactive oxygen species.\u003c/p\u003e \u003cp\u003eIntracellular ROS levels were determined using the ROS molecular probe H\u003csub\u003e2\u003c/sub\u003eDCFDA. Within the cell, esterases cleave the acetate groups on H\u003csub\u003e2\u003c/sub\u003eDCFDA, thus trapping the reduced form of the probe (H2DCF). Intracellular ROS oxidize H\u003csub\u003e2\u003c/sub\u003eDCF, yielding the fluorescent product, DCF. Before the treatments, the cells were incubated for 30 min with PBS plus (PBS with 0.5 mM CaCl2, 1 mM MgCl2, 30 mM glucose) containing 1 \u0026micro;M H2DCFDA, and then washed with PBS. Fluorescence was measured after the treatments using a GENios plus microplate reader (Tecan Mannedorf, Switzerland). The excitation and emission wavelengths used for fluorescence quantification were 485 nm and 535 nm, respectively. Treatment-induced variations in the fluorescence were measured kinetically, every minute for 15 consecutive minutes. All the fluorescence measurements were corrected for background fluorescence and protein concentrations. Using untreated cells as a reference, the antioxidant and prooxidant outcomes were evaluated by comparison of five measurements, and then expressed as a percentage of untreated controls [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e2.6 Measurement of cell viability.\u003c/p\u003e \u003cp\u003eAs previously described, HUVECs cell viability was assessed after the treatments by checking the leakage of the cytoplasmatic lactate dehydrogenase (LDH) from cells with a damaged membrane [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The amount of LDH released in the medium by death cells was assessed using the kit CytoTox-ONE\u0026trade; (Promega, Madison, WI). A standard curve with definite amounts of cells (200\u0026micro;/well) was made, and the release of LDH in the medium was measured after applying lysis solution (4 \u0026micro;l/well). Plates containing samples were removed from the incubator and equilibrated to 22\u0026deg;C. Then the release of LDH from dead cells was measured by supplying lactate, NAD+, and resazurin as substrates in the presence of the enzyme diaphorase. Generation of the fluorescent resorufin product, which is proportional to the amount of LDH, was measured using a GENios plus microplate reader (Tecan Mannedorf, Switzerland) with excitation and emission of 560 nm and 590 nm, respectively. Using a standard curve, the amount of LDH release in treated and untreated cells was conversed in the number of cells per well. Data are representative of four independent experiments and are shown as the percent of untreated control cells.\u003c/p\u003e \u003cp\u003e2.7 Measurement of cell proliferation.\u003c/p\u003e \u003cp\u003eCell proliferation was assessed after the treatments using the BrdU assay (Roche CH), a chemiluminescent immunoassay based on the determination of BrdU incorporation during DNA synthesis [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. When cells are exposed to BrdU, the compound is incorporated into the newly synthesized DNA strands of actively proliferating cells. DNA-incorporated BrdU can be measured using anti-BrdU antibodies, allowing the assessment of the DNA synthesizing cells. BrdU was added 12 hrs before the end of the experiments, then the supernatant was removed, and cells were fixed for 30 min with a Fixing-Denaturating solution (Fix-Denat). At the end of incubation time, the Fix-Denat solution was discarded, and the cells were incubated for 90 minutes with a horseradish peroxidase-conjugated anti-BrdU antibody (anti-BrdU-POD). Following three rinsing with washing buffer, the substrate solution was added and allowed to react for 3-10 min at room temperature. Within this time window, the horseradish peroxidase catalyzes the oxidation of diacylhydrazide, and the reaction product, decaying from its excited state, yields light. Finally, a GENios Plus microplate reader (Tecan Mannedorf, Switzerland) was used to read the light emission of treated cells. Results were expressed as a means \u0026plusmn; SD of the relative light units/sec (RLU/s) values. Data are representative of four independent experiments and are shown as the percent of untreated control cells.\u003c/p\u003e \u003cp\u003e2.8 Migration Assay.\u003c/p\u003e \u003cp\u003eHUVEC migration assays were performed in 24 wells trans-well as previously described [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Near confluent cells were serum-starved overnight, and then 40,000 cells were plated onto the top chamber of the cell culture inserts. Cells in the upper part of the chamber were treated with PL extract in serum free medium (Medium 199 with antibiotics), whereas the complete medium was added to the lower chamber. The assembled cell culture chamber was then incubated at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e for 12 hrs. At the end of incubation time, the upper surfaces of the membranes were gently wiped with cotton swabs to remove the non-migratory cells. The membranes were then fixed with paraformaldehyde, PAF (4%), stained with the fluorophore Hoechst 33342 (10 \u0026micro;g/ml), and the fluorescence was measured using a GENios plus microplate reader (Tecan Mannedorf, Switzerland). The excitation and emission wavelengths used for fluorescence quantification were 340 nm and 485 nm, respectively.\u003c/p\u003e \u003cp\u003e2.9 Statistical analysis.\u003c/p\u003e \u003cp\u003eOne-way analysis of variance (ANOVA), followed by a post-hoc Newman-Keuls for multiple comparisons, was used to detect differences among studied groups. All statistical analyses were performed using GraphPad Prism version 9.00 for Windows (GraphPad Software, San Diego, CA, USA), and p-values \u0026lt; 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1 Essential oils composition\u003c/p\u003e \u003cp\u003ePercentages of individual components of PL extract were calculated based on gas chromatography (GC) peak areas without flame ionization detection (FID) response factor correction. Chromatogram and main chemical components of PL essential oil are reported respectively in figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The analytical results were consistent with those reported in the literature concerning terpene compounds, representing the more important fraction with antioxidant activity. The total essential oil yield, after 4 hours of extraction was 0.14% and the main abundant constituents included germacrene D (11.18%), delta-Cadinene (10.54%), alpha-pinene (8.7%), beta-caryophyllene (5.74%), myrcene (4.5%), beta-phellandrene (4.33%), terpin-4-ol (4.306), epi-alpha-muurolol (3.262) and beta-pinene (3.00%). The complete profiling of the essential oil components is reported in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChemical components of the \u003cem\u003ePistacia Lentiscus L\u003c/em\u003e. SCCO2 extract obtained by GC-GM\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRIK\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTr (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e926\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTricyclene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.096\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e931\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlpha-Thujene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.219\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e938\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlpha-Pinene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.692\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e953\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCamphene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.453\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e977\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSabinene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.857\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e980\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta-Pinene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e993\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMyrcene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.562\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlpha-Phellandrene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.170\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlpha-Terpinene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.661\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePara-Cymene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.649\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta-Phellandrene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.332\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1051\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta(E)-Ocimene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.135\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1059\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIsopentil N-Butanoate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.245\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1062\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGamma-Terpinene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.533\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1090\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTerpinolene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.830\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2-Nonanone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.248\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLinalool\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.388\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIsopentyl Isovalerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.207\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTerpin-4-Ol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.306\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1191\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlpha-Terpineol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.874\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1253\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.440\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.218\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1286\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBornyl Acetate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.118\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2-Undecanone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.362\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.110\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1351\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlpha-Cubebene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.262\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlpha-Copaene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.330\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta-Cubebene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.353\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1392\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta-Elemene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.288\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1408\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.138\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1420\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta-Caryophyllene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.738\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1429\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta-Gurjunene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.213\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1438\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.746\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1451\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.759\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1454\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlpha-Humulene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2,097\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1461\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAllo-Aromadendrene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.907\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1463\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMuurola-4(14),5-Diene\u0026lt;Cis\u0026gt;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.198\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1474\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCadina-1(6),4-Diene\u0026lt;Trans\u0026gt;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.297\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGamma-Muurolene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.610\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1482\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGermacrene-D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.182\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1486\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta-Selinene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.279\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1491\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMuurola-4(15),5-Diene\u0026lt;Trans\u0026gt;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1495\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlpha-Selinene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.507\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1499\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlpha-Muurolene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2,313\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1503\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.294\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1506\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.183\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta-Bisabolene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.924\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1513\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGamma-Cadinene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.186\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1525\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDelta-Cadinene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.542\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1533\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans-Cadina-1(2)-4-Diene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.791\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1538\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlpha-Cadinene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.234\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1544\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.340\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1550\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eElemol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.178\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1556\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.116\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1559\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eElemicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1565\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eE-Nerolidol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.117\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1576\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSpatutenol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.248\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1582\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCaryophyllene Oxide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.251\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1584\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.155\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1590\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.052\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1613\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.104\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1616\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.158\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1627\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.247\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1631\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGamma-Eudesmol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.236\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1642\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEpi-Alpha-Muurolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.262\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1646\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlpha-Muurolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.828\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1649\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta-Eudesmol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.121\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1654\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlpha-Cadinol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.626\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1683\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEpi-Alpha-Bisabolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.506\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1685\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlpha-Bisabolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.148\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1694\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.I.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.086\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eRetention indices (RIK), retention times (Tr) and chromatographic area percentages (%) of the most abundant compounds found in the essential oil extract obtained by supercritical CO\u003csub\u003e2\u003c/sub\u003e (SCCO\u003csub\u003e2\u003c/sub\u003e) extraction at 90 bar, 50\u0026deg;C from \u003cem\u003ePistacia lentiscus L\u003c/em\u003e.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e3.2 Biological activity\u003c/p\u003e \u003cp\u003eUsing primary human endothelial cells as an in vitro vascular model, we investigated the PL extract ability to modulate different biological cell functions, including proliferation, migration, and intracellular redox status.\u003c/p\u003e \u003cp\u003e3.2.1 PL extract does not affect endothelial cell viability and proliferation\u003c/p\u003e \u003cp\u003eCompounds safety is of paramount importance for their potential therapeutic employment; therefore, we first investigated the potential toxicity of the obtained extract by assessing its effects on cell viability, cell proliferation, and intracellular ROS production. Indeed, intracellular ROS generation is closely related to cell survival, proliferation and apoptosis [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Based on previously reported data concerning the effect of PL essential oil on different cultured cells [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], we evaluated the possible harmful effects of three concentrations [50, 150, and 600 \u0026micro;g/ml] of the obtained PL extract on HUVEC viability and proliferation. To this end, cells were treated for 24 hrs with the PL extract, then cell viability and proliferation were assessed as reported in the material and methods sections. As reported in Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the PL extract had no toxic effect at any of the tested concentrations indicating safety of both the extract and the extraction process applied. Likewise, the data in figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB indicate that the tested extract concentrations failed to induce any detrimental effects on HUVEC proliferation, further confirming the extract's safety.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.2.2 PL extract showed antioxidant effect against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress\u003c/p\u003e \u003cp\u003eThe antioxidant properties of PL leaves and fruits compounds have been reported in vitro by measuring their scavenging ability or assessing their anti-lipid peroxidation potential [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. However, whether PL SCCO\u003csub\u003e2\u003c/sub\u003e extract is able to exert antioxidant effects in biological models by protecting cells from oxidative stress remains to be elucidated. Therefore, we sought to investigate whether the obtained PL extract could counteract oxidative stress in human primary endothelial cells oxidatively stressed with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. For this purpose, H\u003csub\u003e2\u003c/sub\u003eDCFDA-loaded cells were pretreated for 3 hrs with the indicated extract concentrations and then incubated for 6 hrs in the presence or absence of 75 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The data derived from five pooled measurements were expressed as percentages of the untreated cells and compared with the vehicle control (0.1% DMSO). As depicted in figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, exposure of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated cells to increasing concentrations of PL extract showed a significant dose-dependent antioxidant effect compared to cells treated with only H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. We next wondered whether the PL extract per se could exert any antioxidant or prooxidant effect in the absence of oxidative insults. As reported in figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, the exposure of unstressed cells to increasing extract concentrations induced a significant antioxidant effect at 150 and 600 \u0026micro;g/ml, while failing to affect the intracellular redox state at 50 \u0026micro;g /ml. These findings are in agreement with the cell viability and proliferation results showing no extract toxicity up to 24 hrs of cell treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.2.3 PL extracts showed protective effect against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress\u003c/p\u003e \u003cp\u003eOxidative-induced endothelial damage appears to trigger and sustain cardiovascular diseases [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Therefore, a great deal of research is now focused on finding natural antioxidants capable to prevent or counteract CVD-associated ROS increases. To determine whether the observed PL extract antioxidant effect could be protective against the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage, we measured HUVEC viability by assessing the cellular membrane integrity. To this end, cells were exposed for 3 h to increasing concentrations of PL extract and then incubated for 24 h in the absence or presence of 75 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Cell were then processed for cell viability as reported in material and methods. As indicated in figure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, cells exposition to increasing doses of PL extract provided a significant dose-dependent cytoprotective effect with respect to the H2O2-induced cell damage. Indeed, all the tested PL concentrations were able to significantly counteract H2O2-induced cell damage. Consonant with these findings are the data in figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, reporting the extract's ability to dose-dependently prevent the detrimental effect on HUVEC proliferation elicited by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.2.4 PL extracts showed antimigratory effect against serum-induced migration\u003c/p\u003e \u003cp\u003eMigration, the cell movement in response to chemical and/or mechanical signals, beside playing an essential role in physiologic processes, such as embryonic development, wound healing, tissue regeneration, it is also a critical process in pathological conditions such as tumor growth, blinding eye diseases, diabetic retinopathy and arthritis [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Endothelial cell migration is an essential step of the angiogenic process; indeed, endothelial cells, which normally are maintained in a quiescent state, are stimulated to degrade the basement membrane and migrate into the perivascular stroma in response to either proangiogenic factors or by the downregulation of antiangiogenic factors [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. For this reason, we investigated the ability of PL extract to modulate endothelial cells migration using the matrigel transfilter cell invasion assays, a modification of the Boyden chamber migration assay [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. These three-dimensional assays are based on the migration of endothelial cells, placed on top of a filter containing 8 \u0026micro;m diameter pores, which allow only active passage of the cells towards an attractant placed in the lower chamber [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Migrated endothelial cells through modified Boyden chambers were measured by reading the fluorescent product of Hoechst 33342 resulted from the metabolism of live cells migrated through the micropores of the upper chamber. Figure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e demonstrated that the PL extract dose-dependently counteracted the serum-induced cell migration, eliciting a significant reduction at the doses of 150 and 600.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe genus \u003cem\u003ePistacia\u003c/em\u003e belongs to the Anacardiaceae family and comprises about 70 genera and over 600 species [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The species PL is a dioecious evergreen shrub or small tree, from 1 to 5 m high, with a strong smell of resin, growing in dry and rocky areas in Mediterranean Europe. The aromatic resin, ivory colored, is harvested as a spice from the cultivated mastic trees. The genus \u003cem\u003ePistacia\u003c/em\u003e has shown many interesting biological activities in vivo and in vitro, such as antimicrobial activity [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], antifungal and anticancer activity [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. It has also been shown to inhibit pro-inflammatory substances production and protect the cardiovascular system by lowering total serum cholesterol, low-density lipoprotein, and triglycerides in rats. In addition, it resulted able to preserve low-density lipoproteins from oxidation in humans.[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Although the antioxidant, antimicrobial and anti-inflammatory activities of the PL. gum and the fixed oil have been previously tested [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR64 CR65\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]; this is the first report showing the effects of the SCCO\u003csub\u003e2\u003c/sub\u003e technique-obtained PL essential oil on primary human endothelial cells. However, we need to clarify that we cannot discriminate whether the biological effects observed in this work are attributable to a single molecule's type present in the analyzed PL extract or to an entire chemical class of secondary metabolites, the terpenes (beta-caryophyllene, germacrene D, alpha-pinene, myrcene, beta-phellandrene, and alpha-humulene). A great number of plants produce terpenes and many studies have demonstrated how this fraction represents the more important component with antioxidant activity [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. In this regard, an interesting review shows how the terpene, lycopene, may protect against atherosclerosis, inhibiting ROS production in vitro and protecting LDL from oxidation [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Furthermore, several studies showed that sesquiterpene lactones, another class of terpenes, possess strong anti-inflammatory, anti-tumor and antimicrobial activities [\u003cspan additionalcitationids=\"CR70\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Since then, this class of phytochemicals has attracted the attention of researchers towards their potential medicinal properties. Several sesquiterpene lactones such as artemisinins, thapsigargins, parthenolide etc., have demonstrated the ability to inhibit angiogenesis in vitro and in vivo by suppressing HUVECs proliferation and migration, microvessel formation, and vascular endothelial growth factor (VEGF) expression. In addition, they resulted also able to prevent osteolytic bone metastasis by suppressing the growth and migration of mammary carcinosarcoma cells [\u003cspan additionalcitationids=\"CR73\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Therefore, based on the literature data, we are of the opinion that our current data might be the result of the synergistic effect produced by the components present in the extract, for which antioxidant and antinflammatory activities have been already reported [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. This synergic mechanism is not fully understood, and it may involve the compounds\u0026rsquo; action toward different pharmacological targets. In the present study, we investigated the SCCO\u003csub\u003e2\u003c/sub\u003e-obtained PL extract cytoprotective, antioxidant, and anti-migratory potential, three aspects essential for maintaining an optimal vascular structure and functionality. According to previously reported data [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], HUVECs were treated with increasing PL extract concentrations [50, 150 and 600 \u0026micro;g/ml], and no effect on the cell viability and proliferation was observed, indicating that the applied process was able to provide a safe extract that lack of cell cytotoxicity. Then we investigated potential biological properties harbored by the obtained extract. To this end, the same PL extract concentrations were employed to assess their antioxidant potential against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress in primary human endothelial cells, along with their possible protective effect toward oxidative stress-induced cell death. All the tested extract doses were able to counteract H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced ROS increase and oxidative-induced cell death, confirming that the possible synergistic effect of its components confers antioxidant properties to the PL extract. In recent years, it was established that natural antioxidants might modulate HUVECs migration [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. As already mentioned, PL extract GC\u0026ndash;MS data demonstrated a predominance of terpenes (monoterpenes and sesquiterpenes). In this regard, previous studies showed that sesquiterpenes owned anti-inଂammatory properties [78,79], and the contained-monoterpene in plant-derived EO effectively inhibited carrageenan-induced edema and neutrophil migration [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Cell migration is a highly integrated, multi-step process that plays an important role in the progression of various diseases, including cancer, atherosclerosis, and arthritis. In this regard, the two higher PL concentrations tested were able to significantly reduce serum-induced HUVECs migration, suggesting a direct effect of the extract on the migration process. These results were in line with those related to PL extract antioxidant activity as a confirmation of the link between oxidative stress and endothelial cells migration. In fact, ROS produced via NADPH oxidase activation stimulate various redox signaling pathways leading to angiogenic responses, including endothelial cell migration. In particular, activated VEGF increases ROS production via Rac1-dependent NADPH oxidase, and ROS in turn are involved in VEGF-induced autophosphorylation of VEGFR-2 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. We know that further investigations are needed to better understand the molecular events elicited by PL extract on this cellular model. Nevertheless, our data provide novel insight concerning the use of SCCO\u003csub\u003e2\u003c/sub\u003e as useful green chemistry process to obtain valuable products harboring essential biological process form biomass\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eWhether PL extract obtained by SCCO\u003csub\u003e2\u003c/sub\u003e is safe and possesses biological activities against CVD remains to be investigated. Here we report that SCCO\u003csub\u003e2\u003c/sub\u003e-derived PL extract is safe, shows cytoprotective effects against oxidative damage, and modulates ROS production and HUVECs migration. Our results indicate SCCO\u003csub\u003e2\u003c/sub\u003e as a secure green chemistry process to exploit biomasses to obtain precious material containing molecules capable of modulating cell functions of paramount importance in preventing or counteracting CVD.\u003c/p\u003e"},{"header":"Credit Authorship Contribution Statement","content":"\u003cp\u003eRG, GP, AMP: Conceptualization. RG, AC, MCP, RC, GB, JSR, AMP: Methodology. RG, AC, MCP, RC, GB, AMP: Investigation. GKN, LP, GP: resources. RG, GP, AMP: writing\u0026mdash;original draft preparation. RG, AC, MCP, RC, GB, JSR, GKN, LP, GP, AMP: writing\u0026mdash;review and editing. GP: supervision. LP, GP: project administration. GKN, LP, GP: funding acquisition\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in this study are available in this article.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work has been made possible thanks to grants from the University of Sharjah (Seed 2001050151 and collaborative 2101050160) to GP; Qatar University (IRCC-2019-007) to GKN and GP; (Fondo UNISS di Ateneo per la Ricerca 2020) to GP.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCho, E.J., Trinh, L.T.P., Song, Y., Lee, Y.G., Bae, H.-J.: Bioconversion of biomass waste into high value chemicals. 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Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives \u003cb\u003e14\u003c/b\u003e(4), 240\u0026ndash;244 (2000)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pistacia lentiscus, supercritical CO2, human endothelial cells, reactive oxygen species, antioxidants","lastPublishedDoi":"10.21203/rs.3.rs-1265639/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1265639/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGreen chemistry is emerging as a useful tool for producing valuable chemicals from biomass. However, extracted compounds need to be tested for safety, quality, and efficacy before their use in humans. Here we investigate the chemical composition and biological effects of leaves \u003cem\u003ePistacia lentiscus L\u003c/em\u003e. essential oil (EO) extract obtained with supercritical carbon dioxide (SCCO\u003csub\u003e2\u003c/sub\u003e). Extract's phytoconstituents profiling was performed by GC-MS/MS, while antioxidant activity was evaluated on human primary endothelial cells (ECs) using the reactive oxygen species (ROS) probe H\u003csub\u003e2\u003c/sub\u003eDCFDA. Potentil extract toxicity and protective effect against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress were investigated using LDH-leakage and BrdU-proliferation tests. Extract\u0026rsquo;s effect on ECs migration was determined by trans-well assay. Terpenes represented the main extraction process fraction yielding 0.14% of EO. Germacrene D (11.18%), delta-cadinene (10.54%), and alpha-pinene (8.7%) were the most abundant OE molecules. Challenged with ECs, increasing extract concentrations failed to affect cell proliferation or promote cell death. ROS assessment in unstressed and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated ECs demonstrated an extract dose-dependent antioxidant activity. The exposure of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated ECs to increasing extract concentrations dose-dependently rescued cells from the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced impairments of cell proliferation and death. Extract was able to significantly counteract fetal calf serum-induced ECs migration. For the first time, we report that a SCCO\u003csub\u003e2\u003c/sub\u003e extract obtained from PL leaves is safe on ECs and may be a useful source of valuable compounds with vasculoprotective properties.\u003c/p\u003e","manuscriptTitle":"Cytoprotective, antioxidant and anti-migratory activity of Pistacia lentiscus L. supercritical carbon dioxide extract on primary human endothelial cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-01-31 16:39:21","doi":"10.21203/rs.3.rs-1265639/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e664bbaf-7407-4391-afc6-8c9be1982e24","owner":[],"postedDate":"January 31st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-04-22T21:19:34+00:00","versionOfRecord":[],"versionCreatedAt":"2022-01-31 16:39:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1265639","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1265639","identity":"rs-1265639","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}