The Composition of Volatiles and the Role of Non-Traditional LOX on Target Metabolites in Virgin Olive Oil from Autochthonous Dalmatian Cultivars.

The lipoxygenase pathway has a significant influence on the composition of the volatile components of virgin olive oil (VOO). In this work, the influence of the maturity index (MI) on the activity of the lipoxygenase enzyme (LOX) in the fruits of the autochthonous Dalmatian olive cultivars Oblica, Levantinka and Lastovka was studied. The analysis of the primary oxidation products of linoleic acid in the studied cultivars showed that LOX synthesises a mixture of 9- and 13-hydroperoxides of octadecenoic acid in a ratio of about 1:2, which makes it a non-traditional plant LOX. By processing the fruits of MI~3, we obtained VOOs with the highest concentration of desirable C6 volatile compounds among the cultivars studied. We confirmed a positive correlation between MI, the enzyme activity LOX and the concentration of hexyl acetate and hexanol in cultivars Oblica and Lastovka, while no positive correlation with hexanol was observed in the cultivar Levantinka. A significant negative correlation was found between total phenolic compounds in VOO and LOX enzyme activity, followed by an increase in the MI of fruits. This article contributes to the selection of the optimal harvest time for the production of VOOs with the desired aromatic properties and to the knowledge of the varietal characteristics of VOOs.

Inhibition of Satellite RNA Associated Cucumber Mosaic Virus Infection by Essential Oil of Micromeria croatica (Pers.) Schott.

The present results dealing with the antiphytoviral activity of essential oil indicate that these plant metabolites can trigger a response to viral infection. The essential oil from Micromeria croatica and the main oil components ß-caryophyllene and caryophyllene oxide were tested for antiphytoviral activity on plants infected with satellite RNA associated cucumber mosaic virus. Simultaneous inoculation of virus with essential oil or with the dominant components of oil, and the treatment of plants prior to virus inoculation, resulted in a reduction of virus infection in the local and systemic host plants. Treatment with essential oil changed the level of alternative oxidase gene expression in infected Arabidopsis plants indicating a connection between the essential oil treatment, aox gene expression and the development of viral infection.

Composition and antiviral activity of the essential oils of Eryngium alpinum and E. amethystinum.

The chemical compositions of the essential oils obtained by hydrodistillation of the aerial parts of Croatian Eryngium alpinum L. and E. amethystinum L. were characterized by GC-FID and GC/MS analyses. The main components identified were the sesquiterpene ß-caryophyllene (19.7%) in the oil of E. amethystinum and the oxygenated sesquiterpene caryophyllene oxide (21.6%) in the oil of E. alpinum. Overall, 32 and 35 constituents were detected in the essential oils of the aerial parts of E. alpinum and E. amethystinum, respectively, representing 92.4 and 93.1% of the total oil compositions. The essential oils of both Eryngium species were proved to reduce the number of lesions in the local host Chenopodium quinoa infected with cucumber mosaic virus and an associated satellite. This is the first investigation of antiphytoviral activity of essential oils of Eryngium species.

Significant Benefits of Environmentally Friendly Hydrosols from Tropaeolum majus L. Seeds with Multiple Biological Activities.

Tropaeolum majus L. is a traditional medicinal plant with a wide range of biological activities due to the degradation products of the glucosinolate glucotropaeolin. Therefore, the goals of this study were to identify volatiles using gas chromatography-mass spectrometry analysis (GC-MS) of the hydrosols (HYs) isolated using microwave-assisted extraction (MAE) and microwave hydrodiffusion and gravity (MHG). Cytotoxic activity was tested against a cervical cancer cell line (HeLa), human colon cancer cell line (HCT116), human osteosarcoma cell line (U2OS), and healthy cell line (RPE1). The effect on wound healing was investigated using human keratinocyte cells (HaCaT), while the antibacterial activity of the HYs was tested against growth and adhesion to a polystyrene surface of Staphylococcus aureus and Escherichia coli. Antiphytoviral activity against tobacco mosaic virus (TMV) was determined. The GC-MS analysis showed that the two main compounds in the HYs of T. majus are benzyl isothiocyanate (BITC) and benzyl cyanide (BCN) using the MAE (62.29% BITC and 15.02% BCN) and MHG (17.89% BITC and 65.33% BCN) extraction techniques. The HYs obtained using MAE showed better cytotoxic activity against the tested cancer cell lines (IC50 value of 472.61-637.07 µg/mL) compared to the HYs obtained using MHG (IC50 value of 719.01-1307.03 µg/mL). Both concentrations (5 and 20 µg/mL) of T. majus HYs using MAE showed a mild but statistically non-significant effect in promoting gap closure compared with untreated cells, whereas the T. majus HY isolated using MHG at a concentration of 15 µg/mL showed a statistically significant negative effect on wound healing. The test showed that the MIC concentration was above 0.5 mg/mL for the HY isolated using MAE, and 2 mg/mL for the HY isolated using MHG. The HY isolated using MHG reduced the adhesion of E. coli at a concentration of 2 mg/mL, while it also reduced the adhesion of S. aureus at a concentration of 1 mg/mL. Both hydrosols showed excellent antiphytoviral activity against TMV, achieving100% inhibition of local lesions on the leaves of infected plants, which is the first time such a result was obtained with a hydrosol treatment. Due to the antiphytoviral activity results, hydrosols of T. majus have a promising future for use in agricultural production.

A Plant Worthy of Further Study-Volatile and Non-Volatile Compounds of Portenschlagiella ramosissima (Port.) Tutin and Its Biological Activity.

New and detailed data are presented on the phytochemical composition of the volatile and non-volatile organic compounds of the Mediterranean endemic species Portenschlagiella ramosissima (Port.) Tutin. Both the essential oil and hydrosol were obtained from the air-dried plant by hydrodistillation and analyzed by gas chromatography-mass spectrometry. The volatile compounds from the fresh and air-dried plants and from the hydrosol were isolated for the first time by headspace solid-phase microextraction using two fibres of different polarity. The benzene derivative group was the predominant group in all samples, with myristicin being the most abundant component of all. The non-volatile compounds of the methanol extract were analyzed by ultra-high-performance liquid chromatography-high-resolution mass spectrometry with electrospray ionisation, and three flavonoid glycosides, one anthocyanidin glycoside, and lipid derivatives were detected. Both the chemical composition and biological activities of this plant have been described in a very limited number of publications, making it an interesting source for further study. The antiphytoviral activity of the essential oil and hydrosol showed that both extracts significantly reduced the number of lesions on the leaves of local host plants infected with tobacco mosaic virus. Moderate antiproliferative activity of the methanol extract was detected in three cancer cell lines, cervical cancer cell line, human colon cancer cell line and human osteosarcoma cell line, using the MTS-based cell proliferation assay. Based on the results, we highlight this plant as a new source of bioactive compounds and natural phytotherapeutic agent that deserves further investigation.

New Membrane Active Antibacterial and Antiviral Amphiphiles Derived from Heterocyclic Backbone of Pyridinium-4-Aldoxime.

Quaternary ammonium salts (QAS) are irreplaceable membrane-active antimicrobial agents that have been widely used for nearly a century. Cetylpyridinium chloride (CPC) is one of the most potent QAS. However, recent data from the literature indicate that CPC activity against resistant bacterial strains is decreasing. The major QAS resistance pathway involves the QacR dimer, which regulates efflux pump expression. A plausible approach to address this issue is to structurally modify the CPC structure by adding other biologically active functional groups. Here, a series of QAS based on pyridine-4-aldoxime were synthesized, characterized, and tested for antimicrobial activity in vitro. Although we obtained several potent antiviral candidates, these candidates had lower antibacterial activity than CPC and were not toxic to human cell lines. We found that the addition of an oxime group to the pyridine backbone resulted in derivatives with large topological polar surfaces and with unfavorable cLog P values. Investigation of the antibacterial mode of action, involving the cell membrane, revealed altered cell morphologies in terms of corrugated and/or disrupted surface, while 87% of the cells studied exhibited a permeabilized membrane after 3 h of treatment at 4 × minimum inhibitory concentration (MIC). Molecular dynamic (MD) simulations of the interaction of QacR with a representative candidate showed rapid dimer disruption, whereas this was not observed for QacR and QacR bound to the structural analog CPC. This might explain the lower bioactivity of our compounds, as they are likely to cause premature expression of efflux pumps and thus activation of resistance.

Hydrodistillation and Microwave Extraction of Volatile Compounds: Comparing Data for Twenty-One Veronica Species from Different Habitats.

Free volatile compounds were isolated from 21 Croatian Veronica species studied by hydrodistillation (HD) and microwave extraction (ME) and analyzed by gas chromatography coupled with mass spectrometry. Principal Component Analysis (PCA) distinguished some clusters based on the relative proportion of major compounds, such as hexadecanoic acid, hexahydrofarnesyl acetone, phytol, E-caryophyllene, and caryophyllene oxide, which were identified in all species studied by both isolation methods. In addition to these compounds, germacrene D, δ-selinene, and eicosane were also identified in five samples from dry habitats isolated using ME. Allo-aromadendrene and ß-ionone are particularly abundant in five species from wet habitats isolated by both methods. The peculiarities of Veronica species from moderate habitats isolated with HD are benzene acetaldehyde, n-nonanal, and the identification of significant compounds from the hydrocarbon class, while the peculiarity of ME is (E)-ß-damascenone. In this article, we present new results on the phytochemical characterization of Veronica species from different habitats. The biological potential of these compounds should be further investigated for a better understanding and utilization of the specialized plant metabolites.

Antiphytoviral activity of sesquiterpene-rich essential oils from four croatian teucrium species.

The purpose of this study was to compare the essential oil profiles of four Croatian Teucrium species (Lamiaceae), as determined by GC and GC/MS, with their antiphytoviral efficiency. A phytochemical analysis showed that T. polium, T. flavum, T. montanum and T. chamaedrys are characterized by similar essential oil compositions. The investigated oils are characterized by a high proportion of the sesquiterpene hydrocarbons ß-caryophyllene (7.1-52.0%) and germacrene D (8.7-17.0%). Other important components were ß-pinene from T. montanum and α-pinene from T. flavum. The investigated essential oils were proved to reduce lesion number in the local host Chenopodium quinoa Willd. infected with Cucumber Mosaic Virus (CMV), with reductions of 41.4%, 22.9%, 44.3% and 25.7%, respectively.

Antioxidant Capacity of Free Volatile Compounds from Olea europaea L. cv. Oblica Leaves Depending on the Vegetation Stage.

Previous research on specialized metabolites of olive leaves has focused on the phenolic components and their biological role. The research in this article focuses on the metabolites that form free volatile compounds (FVCs). The composition of FVCs is divided into compounds isolated in the oil phase (essential oils; EO) and in the aqueous phase (hydrosols; Hy) from leaves of Olea europaea L. cultivar Oblica. Plant material was collected from the same olive tree over a six-month period, from December to May, and analyzed by gas chromatography-mass spectrometry (GC-MS). The compounds ß-caryophyllene, α-humulene, allo-aromadendrene, docosane, hexadecanoic acid and oleic acid were identified in all EO study periods. In the Hy in all studied periods, the major compounds are α-pinene, ß-ionone, myristicin, docosane, 1-hexanol, oleic acid and (E)-ß-damascenone. The differences in the qualitative composition of FVC are directly related to the phenological development of the leaves. Antioxidant capacity of the EOs and hydrosols was measured with two methods, ORAC and DPPH. Hydrosol extracts showed higher capacity than the EOs in all methods.

Not Only a Weed Plant-Biological Activities of Essential Oil and Hydrosol of Dittrichia viscosa (L.) Greuter.

With the increasing interest in obtaining biologically active compounds from natural sources, Dittrichia viscosa (L.) Greuter (Asteraceae) came into our focus as a readily available and aromatic wild shrub widely distributed in the Mediterranean region. This work provides a phytochemical profile of D. viscosa in terms of parallel chemical composition in the lipophilic fraction (essential oil) and the water fraction (hydrosol). GC-MS analysis identified 1,8-cineole, caryophyllene oxide, α-terpenyl acetate, and α-muurolol as the major components of the essential oil, while in the hydrosol p-menth-1-en-9-ol, 1,8-cineole, linalool, cis-sabinene hydrate, and α-muurolol were the major volatile components. 3,4-Dihydroxybenzoic acid was found to be the predominant compound in the hydrosol composition by HPLC analysis. The antimicrobial potential of both extracts was evaluated against thirteen opportunistic pathogens associated with common skin and wound infections and emerging food spoilage microorganisms. The antimicrobial activity of the essential oil suggests that the volatiles of D. viscosa could be used as novel antimicrobial agents. The antiproliferative results of D. viscosa volatiles are also new findings, which showed promising activity against three cancer cell lines: HeLa (cervical cancer cell line), HCT116 (human colon cancer cell line), and U2OS (human osteosarcoma cell line). The decrease in GSH level observed in hydrosol-treated HeLa cells suggests oxidative stress as a possible mechanism of the antiproliferative effect of hydrosol on tumor cells. The presented results are also the first report of significant antiphytoviral activity of hydrosol against tobacco mosaic virus (TMV) infection. Based on the results, D. viscosa might have the potential to be used in crop protection, as a natural disinfectant and natural anticancer agent.

Phytochemical Characterization and Screening of Antioxidant, Antimicrobial and Antiproliferative Properties of Allium × cornutum Clementi and Two Varieties of Allium cepa L. Peel Extracts.

Onions are one of the most widely grown vegetable crops. As production increases, so does the generation of waste from various parts of the onion, raising the need for efficient ecological disposal and use of such waste products. However, onion waste products are a rich source of antioxidants with a range of biological properties, therefore, they could potentially be used in food and pharmaceutical industries. In the present study, we identified the main flavonols and anthocyanins in peel extracts of Allium × cornutum Clement ex Visiani, 1842, and two varieties of Allium cepa L. and tested their antioxidant, antimicrobial and antiproliferative properties. Quercetin 3,4'-diglucolside, quercetin 4'-monoglucoside and quercetin are the most abundant flavonols in all onion extracts detected by high-performance liquid chromatography (HPLC) method. The composition of anthocyanins varied in all extracts. 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) assays showed that the triploid onion A. × cornutum had the highest antioxidant power. Evaluation of antimicrobial activity by broth microdilution assay also showed that A. × cornutum had higher antimicrobial activity compared to the red and yellow onion varieties. Comparable antiproliferative activity was confirmed for all onion extracts tested on three cancer cell lines: Hela (cervical cancer cell line), HCT116 (human colon cancer cell line) and U2OS (human osteosarcoma cell line). The most abundant onion flavonols (quercetin 3,4'-diglucoside and quercetin 4'-monoglucoside) showed weaker antimicrobial as well as antiproliferative properties compared to the extracts, leading to the conclusion that other phytochemicals besides flavonols contribute to the biological activity of onion peel extracts. The results demonstrate the antioxidant and antimicrobial properties of onion peels, which have promising potential as cancer cell proliferation inhibitors.

Chemical Composition and New Biological Activities of Essential Oil and Hydrosol of Hypericum perforatum L. ssp. veronense (Schrank) H. Lindb.

The chemical profile, antiproliferative, antioxidant and antiphytoviral activities of the species Hypericum perforatum ssp. veronense (Schrank) H. Lindb. (Clusiaceae) were investigated. Free volatiles were isolated and the chemical composition was determined in the lipophilic fraction (essential oil) and for the first time in the water fraction (hydrosol). The aim is to provide phytochemical data for H. perforatum ssp. veronense useful for distinguishing ssp. veronense from ssp. angustifolium, as there are taxonomic disagreements between them and the composition of the secretory products may be helpful in this respect. In the essential oil, the most abundant compounds identified were α-pinene and n-nonane, while in the hydrosol, myrtenol, carvacrol and α-pinene were the most abundant. Overall, the class of monoterpenes and oxygenated monoterpenes dominated in the EO and hydrosol samples. The essential oil showed high antioxidant activity, in contrast to the antiproliferative activity, where the hydrosol showed exceptional activity against three cancer cell lines: Hela (cervical cancer cell line), HCT116 (human colon cancer cell line) and U2OS (human osteosarcoma cell line). Both the essential oil and hydrosol showed antiphytoviral activity against tobacco mosaic virus infection on the local host plants. This is the first report dealing with biological activities of hydrosol of H. perforatum ssp. veronense, and the obtained results suggest that this traditional medicinal plant is a valuable source of volatiles with promising antiproliferative, antioxidant and antiphytoviral activities.

Free Volatile Compounds of Veronica austriaca ssp. jacquinii (Baumg.) Eb. Fisch. and Their Biological Activity.

The composition of free volatile compounds of essential oils (EO) and hydrosols (Hy) from four different localities of the species Veronica austriaca ssp. jacquinii (Baumg.) Eb. Fisch. were analyzed by gas chromatography coupled with mass spectrometry. In the EOs, the most abundant compounds identified were hexahydrofarnesyl acetone (23.34-52.56%), hexadecanoic acid (palmitic acid, 26.71-58.91%) and octadecanol acetate (0-6.24%). The hydrosols were characterized by high abundance of methyl eugenol (23.35-57.93%), trans-p-mentha-1(7),8-dien-2-ol (5.24-7.69%) and thymol (3.48-9.45%). Glandular trichomes were analyzed using SEM (Scanning Electron Microscopy), as they are the sites of synthesis of free volatile compounds. We have detected glandular trichomes, consisting of a one stalk cell and two elliptically shaped head cells, and non-glandular (unbranched, bi-cellular to multicellular) trichomes on stems, leaves and the sepals. Data for volatile compounds from EOs and hydrosols were analyzed using Principal Component Analyses (PCA) to demonstrate variations in the composition of the volatile compounds identified. Isolated samples of EO and hydrosols were analyzed for their antioxidant activity using two methods, DPPH (2,2-diphenyl-1-picrylhydrazyl) and ORAC (Oxygen Radical Absorbance Capacity). The essential oils showed higher antioxidant activity than the hydrosols in ORAC method, but lower activity by the DPPH method. The isolates were also tested for their antiproliferative activity on different types of cancer cells and also on two lines of healthy cells, and the results showed that the extracts were not toxic to the cell lines tested. Total polyphenols, total tannins, total flavonoids and total phenolic acids were also analyzed and determined spectrophotometrically. The free volatile compounds of Veronica austriaca ssp. jacquinii can be considered as a safe natural product.

Endemic Veronica saturejoides Vis. ssp. saturejoides-Chemical Composition and Antioxidant Activity of Free Volatile Compounds.

Chemical profile and antioxidant activity of the species Veronica saturejoides Vis. ssp. saturejoides (Plantaginaceae)-which is endemic to Croatia, Bosnia and Herzegovina and Montenegro -were investigated. Volatile compounds produced by glandular trichomes (composed of one stalk cell and two elliptically formed head cells according to scanning electron microscope investigation) were isolated from the plants collected in two locations. Additionally, as a part of specialized metabolites, total polyphenols, total tannins, total flavonoids and total phenolic acids were determined spectrophotometrically. In the lipophilic volatile fractions-essential oils, the most abundant compounds identified were hexahydrofarnesyl acetone, caryophyllene oxide and hexadecanoic acid. In total, the class of oxygenated sesquiterpenes and the group of fatty aldehydes, acids and alcoholic compounds dominated in the essential oils. In the hydrophilic volatile fractions-hydrosols, the most abundant compounds identified were trans-p-mentha-1(7),8-dien-2-ol, allo-aromadendrene and (E)-caryophyllene. A group of oxygenated monoterpenes and the sesquiterpene hydrocarbons dominated in the hydrosols. Antioxidant activity of essential oils and hydrosols was tested with two methods: 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC). Essential oils showed higher antioxidant activity than hydrosols and showed similar antioxidant activity to Rosmarinus officinalis essential oil. Obtained results demonstrate that this genus is a potential source of volatiles with antioxidant activity.

Chemical composition and antiphytoviral activity of essential oil of Micromeria graeca.

Phytochemical analysis of the essential oils of Micromeria graeca (L.) Rchb from Vis and Komiza on the island of Vis (Croatia) showed them to have a similar chemical composition. The oxygenated sesquiterpene alpha-bisabolol was a major component of both oils with percentages of 13.9% in the oil from Vis and 15.5% in the oil from Komiza. A comparison of the mean number of lesions on the essential oil-treated Chenopodium quinoa plants infected with satellite-associated cucumber mosaic virus (satCMV) with the corresponding control showed that the oil from Vis caused delay in the development and reduction of the number of lesions by 59.3%. The essential oil of M. graeca from Komiza was not effective in the reduction of satCMV infection.

Antiphytoviral activity of essential oil from endemic species Teucrium arduini.

The essential oil of Teucrium arduini L. was characterized by a high concentration of sesquiterpene hydrocarbons (43.8%) of which beta-caryophyllene (19.9%) being the major compound, followed by oxygenated sesquiterpenes (19.6%) of which caryophyllene-oxide (14.6%) was dominant. When applied to plants of Chenopodium amaranticolor and Ch. quinoa for two successive days prior inoculation, the oil was effective in reducing lesion numbers on plants infected with Tobacco mosaic virus (25.7%) and Cucumber mosaic virus (21.9%). The main components of oil, beta-caryophyllene and caryophyllene oxide showed potent antiviral activity against CMV, but weak activity against TMV infection.