Headspace/GC-MS Analysis and Investigation of Antibacterial, Antioxidant and Cytotoxic Activity of Essential Oils and Hydrolates from Rosmarinus officinalis L. and Lavandula angustifolia Miller.

In this work, essential oils (EOs) and hydrolates (Hys) of Rosmarinus officinalis L. and Lavandula angustifolia Mill., grown in Tuscany (Italy), were studied to describe their chemical composition and biological activities. The aromatic profile of the EOs liquid phase was carried out by gas chromatography-mass spectrometry (GC-MS), while the volatile composition of vapor phase EOs and Hys was performed by headspace (HS)/GC-MS. The obtained results show that monoterpene hydrocarbons (71.5% and 89.5%) were the main compounds, followed by oxygenated monoterpenes (26.0% and 10.5%) in the liquid and vapor phase of R. officinalis EO, respectively. The oxygenated monoterpenes were the main components of L. angustifolia EO, reaching 86.9% in the liquid phase and 53.7% in the vapor phase. Regarding Hys, they consisted only of oxygenated monoterpenes, and 1,8-cineole (56.2%) and linalool (42.9%), were the main components of R. officinalis and L. officinalis Hys, respectively. Their cytotoxicity was investigated on an SHSY5Y neuroblastoma cell line by thiazolyl blue tetrazolium bromide (MTT) test, showing a notable effect of the EOs with a time-independent manner of activity and half maximal effective concentration (EC50) values quite similar for the two plant species (from 0.05% to 0.06% v/v for the three time points evaluated). A measurable activity of Hys was also obtained although with higher EC50 values. The antibacterial activity against Escherichia coli ATCC® 25922, Pseudomonas fluorescens ATCC® 13525, Acinetobacter bohemicus DSM 102855 as Gram-negative bacteria and Kocuria marina DSM 16420, Bacillus cereus ATCC® 10876 as Gram-positive bacteria, was evaluated by the agar disk-diffusion method and the VPT (vapor phase test) to determinate the MIC (minimal inhibitory concentration) and the MBC (minimal bactericidal concentration) values. Both EOs possessed a high activity against all the bacterial strains with MIC values ranging from 0.19% to 3.13% v/v. Unlike EOs, Hys did not show an inhibition of the bacterial growth at the tested concentrations. Furthermore, antioxidant power was measured by 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt-based (ABTS•+) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays, showing a remarkable ability to reduce radicals by both EOs; Hys were slightly less active. The findings highlighted that R. officinalis and L. angustifolia EOs and Hys have a chemical composition rich in bioactive molecules, which can exert different biological activities.

Liquid and Vapor Phase of Four Conifer-Derived Essential Oils: Comparison of Chemical Compositions and Antimicrobial and Antioxidant Properties.

In this study, the chemical composition of the vapor and liquid phase of Pinus cembra L., Pinus mugo Turra, Picea abies L., and Abies Alba M. needles essential oils (EOs) was investigated by Headspace-Gas Chromatography/Mass Spectrometry (HS-GC/MS). In the examined EOs, a total of twenty-eight components were identified, most of which belong to the monoterpenes family. α-Pinene (16.6-44.0%), ß-pinene (7.5-44.7%), limonene (9.5-32.5%), and γ-terpinene (0.3-19.7%) were the most abundant components of the liquid phase. Such major compounds were also detected in the vapor phase of all EOs, and α-pinene reached higher relative percentages than in the liquid phase. Then, both the liquid and vapor phases were evaluated in terms of antibacterial activity against three Gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, and Acinetobacter bohemicus) and two Gram-positive bacteria (Kocuria marina and Bacillus cereus) using a microwell dilution assay, disc diffusion assay, and vapor phase test. The lowest Minimum Inhibitory Concentration (MIC) (13.28 mg/mL) and Minimal Bactericidal Concentration (MBC) (26.56 mg/mL) values, which correspond to the highest antibacterial activities, were reported for P. abies EO against A. bohemicus and for A. alba EO against A. bohemicus and B. cereus. The vapor phase of all the tested EOs was more active than liquid phase, showing the inhibition halos from 41.00 ± 10.15 mm to 80.00 ± 0.00 mm for three bacterial strains (A. bohemicus, K. marina, and B. cereus). Furthermore, antioxidant activities were also investigated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis (3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays, and a concentration-dependent antioxidant capacity for all EOs was found. P. mugo EO showed the best antioxidant activity than the other Pinaceae EOs. The four Pinaceae EOs could be further investigated for their promising antibacterial and antioxidant properties, and, in particular, α-pinene seems to have interesting possibilities for use as a novel natural antibacterial agent.

Chemical Investigation and Screening of Anti-Proliferative Activity on Human Cell Lines of Pure and Nano-Formulated Lavandin Essential Oil.

Lavandin essential oil (LEO), a natural sterile hybrid obtained by crossbreeding L. angustifolia × L. latifolia, is mainly composed by active components belonging to the family of terpenes endowed with relevant anti-proliferative activity, which can be enhanced by proper application of nanotechnology. In particular, this study reports the chemical characterization and the screening of the anti-proliferative activity on different human cell lines of pure and nano-formulated lavandin essential oil (EO). LEO and its formulation (NanoLEO) were analyzed by HS/GC-MS (Headspace/Gas Chromatography-Mass Spectrometry) to describe and compare their chemical volatile composition. The most abundant compounds were linalool and 1,8-cineole (LEO: 28.6%; 27.4%) (NanoLEO: 60.4%; 12.6%) followed by α-pinene (LEO: 9.6%; NanoLEO: 4.5%), camphor (LEO: 6.5%; NanoLEO: 7.0%) and linalyl acetate (LEO: 6.5%; NanoLEO: 3.6%). The cytotoxic effects of LEO and NanoLEO were investigated on human neuroblastoma cells (SHSY5Y), human breast adenocarcinoma cells (MCF-7), human lymphoblastic leukemia cells (CCRF CEM), human colorectal adenocarcinoma cells (Caco-2) and one normal breast epithelial cell (MCF10A) by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)-assay. Caco-2, MCF7 and MCF10A normal cells resulted more resistant to the treatment with LEO, while CCRF-CEM and SHSY5Y cells were more sensitive. The antiproliferative effect of LEO resulted amplified when the essential oil was supplied as nanoformulation, mainly in Caco-2 cells. Scanning and transmission electron microscopy investigations were carried out on Caco-2 cells to outline at ultrastructural level possible affections induced by LEO and NanoLEO treatments.

Antimicrobial Testing of Schinus molle (L.) Leaf Extracts and Fractions Followed by GC-MS Investigation of Biological Active Fractions.

Schinus molle (L.) is a dioecious plant of the Anacardiaceae family, originating in South America and currently widespread in many regions throughout the world. In this work leaf extracts and derived low-pressure column chromatography (LPCC) fractions of S. molle L. male and female plants were investigated for the antimicrobial activity. Leaf extracts were tested on microbes Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Candida albicans and Bacillus subtilis. Furthermore, the extracts showing antimicrobial activity were fractionated by LPCC and the obtained fractions tested on the same microorganism strains. Positive fractions were investigated by gas-chromatography/mass spectrometry (GC-MS) and were seen to be rich in sesquiterpenes, sesquiterpenoids and other terpens. The obtained effects highlighted the antimicrobial properties of S. molle (L.) leaf compounds and revealed their importance as a source of bioactive molecules of potential pharmaceutical interest. To our knowledge, this is the first paper reporting investigations on the chemical composition of the extracts and derived positive fractions from Schinus molle (L.) plants grown in central Italy.

Apoptotic Effects on HL60 Human Leukaemia Cells Induced by Lavandin Essential Oil Treatment.

Recent scientific investigations have reported a number of essential oils to interfere with intracellular signalling pathways and to induce apoptosis in different cancer cell types. In this paper, Lavandin Essential Oil (LEO), a natural sterile hybrid obtained by cross-breeding L. angustifolia × L. latifolia, was tested on human leukaemia cells (HL60). Based on the MTT results, the reduced cell viability of HL60 cells was further investigated to determine whether cell death was related to the apoptotic process. HL60 cells treated for 24 h with LEO were processed by flow cytometry, and the presence of Annexin V was measured. The activation of caspases-3 was evaluated by western blot and immunofluorescence techniques. Treated cells were also examined by scanning and transmission electron microscopy to establish the possible occurrence of morphological alterations during the apoptotic process. LEO main compounds, such as linalool, linalyl acetate, 1,8-cineole, and terpinen-4-ol, were also investigated by MTT and flow cytometry analysis. The set of obtained results showed that LEO treatments induced apoptosis in a dose-dependent, but not time-dependent, manner on HL60 cells, while among LEO main compounds, both terpinen-4-ol and linalyl acetate were able to induce apoptosis.

Liquid and Vapour Phase of Lavandin (Lavandula × intermedia) Essential Oil: Chemical Composition and Antimicrobial Activity.

Essential oils from Lavandula genus and the obtained hybrids are widely used for different purposes such as perfume production in the cosmetic field and for its biological properties. This is the first study on the liquid and vapour phase of Lavandula × intermedia "Grosso" essential oil grown in the Lazio Region, Italy, investigated using headspace coupled to gas chromatography and mass spectrometry (HS-GC/MS). The results showed the most abundant components were linalool and linalyl acetate, followed by 1,8-cineole and terpinen-4-ol, while lavandulyl acetate and borneol were identified as minor compounds, maintaining the same proportion in both the liquid and vapour phase. Furthermore, we tested lavandin liquid and vapour phase essential oil on gram-negative bacteria (Escherichia coli, Acinetobacter bohemicus, and Pseudomonas fluorescens) and gram-positive bacteria (Bacillus cereus and Kocuria marina).

A mass spectrometric study of the acid-catalysed d-fructose dehydration in the gas phase.

5-hydroxymethylfuraldehyde (5-HMF) and simpler compounds, such as levulinic acid (LA) and glyceraldehyde, are platform molecules produced by the thermal acid-catalyzed dehydration of carbohydrates coming from biomass. Understanding sugar degradation pathways on a molecular level is necessary to increase selectivity, reduce degradation by-products yields and optimize catalytic strategies, fundamental knowledge for the development of a sustainable renewable industry. In this work gaseous protonated d-fructose ions, generated in the ESI source of a triple quadrupole mass spectrometer, were allowed to undergo Collisionally Activated Decomposition (CAD) into the quadrupole collision cell. The ionic intermediates and products derived from protonated d-fructose dehydration were structurally characterized by their fragmentation patterns and the relative water-loss dehydration energies measured by energy-resolved CAD mass spectra. The data were compared with those obtained from protonated d-glucose decomposition in the same experimental conditions. In the gas phase, d-fructose dehydration leads to the formation of a mixed population of isomeric [C6H6O3]H(+) ions, whose structures do not correspond exclusively to 5-hydroxymethyl-2-furaldehyde protonated at the more basic aldehydic group.

Acid-catalysed glucose dehydration in the gas phase: a mass spectrometric approach.

Understanding on a molecular level the acid-catalysed decomposition of the sugar monomers from hemicellulose and cellulose (e.g. glucose, xylose), the main constituent of lignocellulosic biomass is very important to increase selectivity and reaction yields in solution, key steps for the development of a sustainable renewable industry. In this work we reported a gas-phase study performed by electrospray triple quadrupole mass spectrometry on the dehydration mechanism of D-glucose. In the gas phase, reactant ions corresponding to protonated D-glucose were obtained in the ESI source and were allowed to undergo collisionally activated decomposition (CAD) into the quadrupole collision cell. The CAD mass spectrum of protonated D-glucose is characterized by the presence of ionic dehydrated daughter ion (ionic intermediates and products), which were structurally characterized by their fragmentation patterns. In the gas phase D-glucose dehydration does not lead to the formation of protonated 5-hydroxymethyl-2-furaldehyde, but to a mixed population of m/z 127 isomeric ions. To elucidate the D-glucose dehydration mechanism, 3-O-methyl-D-glucose was also submitted to the mass spectrometric study; the results suggest that the C3 hydroxyl group plays a key role in the reaction mechanism. Furthermore, protonated levulinic acid was found to be formed from the monodehydrated D-glucose ionic intermediate, an alternative pathway other than the known route consisting of 5-hydroxymethyl-2-furaldehyde double hydration.

The mechanism of 2-furaldehyde formation from D-xylose dehydration in the gas phase. A tandem mass spectrometric study.

The mechanism of reactions occurring in solution can be investigated also in the gas phase by suited mass spectrometric techniques, which allow to highlight fundamental mechanistic features independent of the influence of the medium and to clarifying controversial hypotheses proposed in solution studies. In this work, we report a gas-phase study performed by electrospray triple stage quadrupole mass spectrometry (ESI-TSQ/MS) on the dehydration of D-xylose, leading mainly to the formation of 2-furaldehyde (2-FA). It is generally known in carbohydrate chemistry that the thermal acid catalyzed dehydration of pentoses leads to the formation of 2-FA, but several aspects on the solution-phase mechanism are controversial. Here, gaseous reactant ions corresponding to protonated xylose molecules obtained from ESI of a solution containing D-xylose and ammonium acetate as protonating reagent were allowed to undergo collisionally activated decomposition (CAD) into the triple stage quadrupole analyzer. The product ion mass spectra of protonated xylose are characterized by the presence of ionic intermediates arising from xylose dehydration, which were structurally characterized by their fragmentation patterns. As expected, the xylose triple dehydration leads to the formation of the ion at m/z 97, corresponding to protonated 2-FA. On the basis of mass spectrometric evidences, we demonstrated that in the gas phase, the formation of 2-FA involves protonation at the OH group bound to the C1 atom of the sugar, the first ionic intermediate being characterized by a cyclic structure. Finally, energy resolved product ion mass spectra allowed to obtain information on the energetic features of the D-xylose→2-FA conversion. ᅟ

Gas-phase basicity of 2-furaldehyde.

2-Furaldehyde (2-FA), also known as furfural or 2-furancarboxaldehyde, is an heterocyclic aldehyde that can be obtained from the thermal dehydration of pentose monosaccharides. This molecule can be considered as an important sustainable intermediate for the preparation of a great variety of chemicals, pharmaceuticals and furan-based polymers. Despite the great importance of this molecule, its gas-phase basicity (GB) has never been measured. In this work, the GB of 2-FA was determined by the extended Cooks's kinetic method from electrospray ionization triple quadrupole tandem mass spectrometric experiments along with theoretical calculations. As expected, computational results identify the aldehydic oxygen atom of 2-FA as the preferred protonation site. The geometries of O-O-cis and O-O-trans 2-FA and of their six different protomers were calculated at the B3LYP/aug-TZV(d,p) level of theory; proton affinity (PA) values were also calculated at the G3(MP2, CCSD(T)) level of theory. The experimental PA was estimated to be 847.9 ± 3.8 kJ mol(-1), the protonation entropy 115.1 ± 5.03 J mol(-1) K(-1) and the GB 813.6 ± 4.08 kJ mol(-1) at 298 K. From the PA value, a ΔH°(f) of 533.0 ± 12.4 kJ mol(-1) for protonated 2-FA was derived.