Essential Oil Volatile Fingerprint Differentiates Croatian cv. Oblica from Other Olea europaea L. Cultivars.

Olive leaves are a highly available by-product from table olive and olive oil production. They are nowadays strongly valuable for their major bioactive compounds and their beneficial effects. To determine the differences between two Croatian domestic (Lastovka, Oblica) and two introduced (Leccino, Frantoio) cultivars, physical and chemical analysis of olive leaves were performed: surface area, color variability, total phenolic amounts, and essential oil volatile profiles were analyzed at three harvest periods. All cultivars greatly differed in surface area, with cv. Lastovka being the smallest. Color variability resulted in an overall decrease in darkness and amounts of green and yellow that could be attributed to a decrease in photosynthetic demand and chlorophyll content. The highest amount of total phenolic content occurred in the summer months, followed by a reduction until October. Essential oils volatiles were determined by GC-MS and showed great diversity not only amongst cultivars but also between harvest periods, with overall 45 compounds identified. Principal component analysis distinguished domestic cultivar Oblica from the other observed cultivars, mainly due to its essential oil volatile fingerprint. Compounds that differentiated cv. Oblica were aldehydes ((E,Z)-2,4-heptadienal, (E,E)-2,4-heptadienal, decanal), ketones ((E)-ß-damascone, dihydrodehydro-ß-ionone), sesquiterpenes (cyclosativene, α-copaene, α-muurolene) and saturated hydrocarbons (tetradecane, hexadecane). Essential oil volatile fingerprint attributed the highest to the biodiversity of domestic cv. Oblica through all three harvest periods.

Identification of Phenolic Compounds Present in Three Speedwell (Veronica L.) Species and Their Antioxidant Potential.

Extracts from Veronica species (speedwells) are known for the various biological activities they show, such as cytotoxic, antimicrobial, anti-inflammatory, and antioxidant activities. Also, the plants from this genus are known as medicinal plants used in traditional medicine worldwide. Phenolic compounds are specialized metabolites that contribute to biological activity the most. Therefore, the aim of this research is identification and quantification of phenolic compounds present in three Veronica species (Veronica anagallis-aquatica L., Veronica persica Poir., and Veronica polita Fr.) using the liquid chromatography-mass spectrometry (LC-MS/MS) technique. All extracts were tested for antioxidant activity with two methods: DPPH (2,2-diphenyl-1-picrylhydrazyl) and ORAC (oxygen radical absorbance capacity). Also, standards for compounds that were detected in the highest amount in all species were also tested for antioxidant activity. Three different solvents (pure methanol, 80% ethanol, and water) were used for the extraction of phenolic components and their comparison in order to test their antioxidant activity as a final goal. The main compounds present in the tested Veronica extracts were: p-hydroxybenzoic acid, vanillic acid, caffeic acid, gentisic acid, and apigenin. V. anagallis-aquatica contained the highest amount of phenolic components in comparison with the two other tested species, V. persica and V. polita. Caffeic acid showed the highest antioxidant activity in both studied methods with an IC50 value for DPPH activity of 1.99 µg/mL. For the plant extracts, in general, methanolic/ethanolic extracts showed higher activity than water extracts in both methods which was expected, as organic solutions extract more phenolic compounds. This research points to the potential application of extracts of different Veronica species for antioxidant activity.

Analysis of Post-COVID-19 Guillain-Barré Syndrome over a Period of One Year in the University Hospital of Split (Croatia).

The virus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is capable of attacking the nervous system in several ways and leading to neurological diseases such as GBS (Guillain-Barré syndrome) through the resulting neurotropism and immune response. The aim of this study is to show the relationship between Coronavirus disease (COVID-19) and GBS and to better understand the clinical symptoms to prevent poor outcomes. Data from 15 patients were extracted from the Department of Neurology, University Hospital of Split, Croatia, for the year 2021. The age of the patients ranged from 26 to 89 years, of whom 27% were women. Sixty seven percent of all GBS patients recovered from COVID-19 infection, whereas post-vaccinal polyradiculoneuritis was detected in 6%. Forty four percent of the patients who developed GBS had a severe form of COVID-19 infection. Forty percent of patients were treated with intravenous immunoglobulins (IVIG), followed by therapeutic plasma exchange (PLEX) in 27%. After the therapy, improvement was observed in 13 patients, while two patients died. The results suggest that SARS-CoV-2 triggers GBS because it follows a similar pattern of infection as the other viral and bacterial agents that contribute to the onset of GBS. There is no evidence that prior infection with COVID-19 worsens the clinical presentation of GBS.

Isolation of Volatile Compounds by Microwave-Assisted Extraction from Six Veronica Species and Testing of Their Antiproliferative and Apoptotic Activities.

This study was conducted to determine the differences in the chemical composition of the essential oils and hydrosols of six different Veronica species (V. agrestis, V. anagalloides, V. austriaca ssp. jacquinii, V. beccabunga, Veronica cymbalaria, and V. officinalis) and to test their antiproliferative and apoptotic activities, according to the authors' knowledge, because of insufficient research and lack of information. Also, the goal was to determine which obtained samples were better in achieving antiproliferative and apoptotic activities and due to which volatile components. Therefore, essential oils (EOs) and hydrosols (HYs) were isolated from the above-mentioned Veronica species by microwave-assisted extraction (MAE). Phytochemical identification of the free volatile compounds was performed using a GC equipped with a flame ionization detector and a mass spectrometer. Their antiproliferative and apoptotic activities against two human cancer cell lines, breast cancer cell line MDA-MB-231 and bladder cancer cell line T24, were determined. The main compounds identified in the studied Veronica EOs and HYs were terpinen-4-ol (0.34-6.49%), linalool (0.34-6.61%), (E)-caryophyllene (0.97-7.55%), allo-aromadendrene (0.18-2.21%), caryophyllene oxide (1.42-23.83%), benzene acetaldehyde (0.26-13.34%), and ß-ionone (1.08-16.53%). In general, HYs of the tested Veronica species showed higher antiproliferative activity (IC50 13.41-42.05%) compared to EOs (IC50 158.1-970.4 µg/mL) on MDA-MB-231 and T24 cancer cell lines after 48 and 72 h. V. agrestis EO showed the best apoptotic effect among the EOs on the MDA-MB-231 cancer cell line (10.47 ± 0.53% and 9.06 ± 0.74% of early/late apoptosis, compared with control 3.61 ± 0.62% and 0.80 ± 0.17% of early/late apoptosis, respectively) and among the HYs V. cymbalaria showed 9.95 ± 1.05% and 3.06 ± 0.28% of early/late apoptosis and V. anagalloides 8.29 ± 1.09% and 1.95 ± 0.36% of early/late apoptosis compared with control (for EO was 7.45 ± 1.01% and 0.54 ± 0.25%, and for HY was 4.91 ± 1.97% and 0.70 ± 0.09% of early/late apoptosis, respectively) on the T24 cancer cell line. Future research will include other Croatian species of the genus Veronica to gain a more complete insight into the biological activity of the volatile products of this genus for potential discovery of drugs based on natural plant extracts.

In the Beginning Was the Bud: Phytochemicals from Olive (Olea europaea L.) Vegetative Buds and Their Biological Properties.

Even though Olea europaea L. is one of the most important and well-studied crops in the world, embryonic parts of the plants remain largely understudied. In this study, comprehensive phytochemical profiling of olive vegetative buds of two Croatian cultivars, Lastovka and Oblica, was performed with an analysis of essential oils and methanol extracts as well as biological activities (antioxidant, antimicrobial, and cytotoxic activities). A total of 113 different volatiles were identified in essential oils with hydrocarbons accounting for up to 60.30% and (Z)-3-heptadecene being the most abundant compound. Oleacein, oleuropein, and 3-hydroxytyrosol had the highest concentrations of all phenolics in the bud extracts. Other major compounds belong to the chemical classes of sugars, fatty acids, and triterpenoid acids. Antioxidant, antimicrobial, and cytotoxic activities were determined for both cultivars. Apart from antioxidant activity, essential oils had a weak overall biological effect. The extract from cultivar Lastovka showed much better antioxidant activity than both isolates with both methods (with an oxygen radical absorbance capacity value of 1835.42 µM TE/g and DPPH IC50 of 0.274 mg/mL), as well as antimicrobial activity with the best results against Listeria monocytogenes. The human breast adenocarcinoma MDA-MB-231 cell line showed the best response for cultivar Lastovka bud extract (IC50 = 150 µg/mL) among three human cancer cell lines tested. These results demonstrate great chemical and biological potential that is hidden in olive buds and the need to increase research in the area of embryonic parts of plants.

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.

Hydrosols of Veronica Species-Natural Source of Free Volatile Compounds with Potential Pharmacological Interest.

In this study, free volatile compounds (FVCs) were isolated from the water fractions (hydrosols) of 10 Croatian Veronica species obtained by hydrodistillation (HD) and microwave-assisted extraction (MAE). The use of different isolation techniques is important for the analysis of the influence of extraction conditions on the qualitative and quantitative composition of the isolated constituents. The composition of the hydrosols was analyzed using gas chromatography and mass spectrometry. The compounds ß-ionone and benzene acetaldehyde were detected in all 10 Veronica hydrosols studied. E-caryophyllene was also identified in all isolates except the MAE isolate of V. arvensis L. Caryophyllene oxide was isolated in all isolates apart from the HD isolate of V. catenata Pennell. (E)-ß-Damascenone is significantly present in all isolates except the MAE isolates of V. catanata and V. cymbalaria Bodard. In these two species, α-muurolol was identified in a high percentage. The same basic phytochemical constituents and compounds characteristic of a given Veronica species suggest the importance of further research. Antioxidant activity was tested for all extracts using two methods, ORAC and DPPH. Therefore, it is crucial to identify as many specialized metabolites from Veronica species as possible, especially hydrosols, which are natural products of potential pharmacological interest.

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.

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.

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.

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.

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.