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Cretovi su danas među najugroženijim staništima u Hrvatskoj. Unatoč tomu što su velikim dijelom stavljeni pod različite kategorije zaštite, oni i dalje ubrzano propadaju. S ciljem pronalaska načina na koji zaštititi neke od posljednjih cretova na Velebitu provedena je inventarizacija vaskularnih biljnih svojti na bazofilnim cretovima Ljubica i Raketa smještenim na području naselja Baške Oštarije na srednjem Velebitu. Utvrđeno je prisustvo 140 biljnih svojti na području creta Ljubica te 85 svojti na području creta Raketa, od čega se 52 svojte pojavljuju na oba creta. Očuvanje ovih cretova moguće je uz sprječavanje daljnjeg smanjenja dotoka vode na cret Ljubica te uz uklanjanje agresivnijih biljnih svojti i invazivnih vrsta koje na oba creta onemogućavaju opstanak svijetloljubivih cretnih svojti. S obzirom na snažan utjecaj čovjeka koji je doveo do značajnog smanjenja dotoka vode na područje creta jedini način da se cret Ljubica pokuša zaštiti je da ga se proglasi botaničkim rezervatom. Za praćenje učinkovitosti predloženih mjera zaštite potrebno je kontinuirano pratiti prisutnost i pokrovnost biljnih svojti karakterističnih za bazofilne cretove., Fens are among the most endangered habitats in Croatia today. Despite the fact that they are largely placed under different categories of protection, they continue to deteriorate rapidly. With the aim of finding a way to protect some of the last fens on Velebit Mountain, an inventory of vascular plants was carried out on the basophilic fens Ljubica and Raketa located in the area of the settlement Baške Oštarije on the middle part of Velebit Mountain. During the inventory, the presence of 140 plant taxa was determined in the Ljubica fen and 85 taxa in the Raketa fen. Of these, 52 taxa appear on both fens. Preservation of both fens is possible by preventing a further decrease in water flow to the Ljubica fen and by removing more aggressive plant taxa and invasive species that disable the survival of heliophilic fen taxa. Considering the strong influence of human, which led to a considerable reduction of the water flow in the fen area, the only way to try to protect the Ljubica fen is to declare it a botanical reserve. To see if the proposed measures produce results, it is necessary to continuously monitor the presence and coverage of plant species characteristic for basophilic fens.

The aim of this study was to identify the volatile compounds in hydrosols of different Veronica species (Veronica cymbalaria, V. anagalloides- aquatica, V. agrestis, V. jacquinii, V. officinalis, and V. beccabunga) and to examine their cytotoxic and apoptotic activities on the bladder cancer cell line T24. Hydrosols were obtained by microwave-assisted extraction (MAE) using the Milestone ETHOS X apparatus for 30 min at 800 W (98 °C). The volatile compounds contained in them were analyzed by gas chromatography and mass spectrometry (GC-MS). Cell viability and proliferation were assessed by measuring cell metabolism using the MTT assay. Hydrosols were tested at different dilutions (50%, 40%, 30%, 20%, and 10%) for cytotoxic activity at 4, 24, 48, and 72 hours. For apoptosis, cells were treated with hydrosols for 48 hours. The FITC Annexin V Apoptosis Detection Kit with PI was used to detect cell apoptosis by flow cytometry. The major compounds in all investigated Veronica species were phenylacetaldehyde, caryophyllene oxide and hexadecanoic acid. The hydrosols of the analyzed Veronica species showed inhibitory activity on the bladder cancer cell line T24. The results of the study show that Veronica species are an excellent source of volatile compounds with various biological activities and future research should focus on their hydrosols.

Composition of volatile compounds and antioxidant activity of hydrosols of five Veronica species is presented in this research: V. acinifolia, V. anagallis-aquatica, V. anagalloides, V. catenata and V. arvensis. Hydrodistillation (HD) and microwave-assisted extraction (MAE) were methods used for the extraction of free volatile compounds (FVCs) in hydrosols of the chosen species. These extraction methods yielded two layers, the lipophilic layer or essential oil and the aqueous layer - hydrosols. In general, research on the aroma profiles of hydrosols and their biological efficacy is limited but in the last decade, hydrosols have been increasingly explored due to higher acceptance towards using natural products in daily life. The compounds β - ionone and benzene acetaldehyde were detected in all five species regardless of the extraction method. Except these two compounds, (E)-β-damascenone was detected in all HD hydrosols and caryophyllene oxide in all MAE hydrosols. Hydrosols from both extraction techniques, were tested by two methods: ORAC and DPPH. Comparing the antioxidant activity of all hydrosols, it can be concluded that V. anagallis-aquatica HD hydrosol and V. anagalloides HD hydrosol showed highest but similar antioxidant activity in ORAC method. For DPPH method, V. arvensis HD hydrosol showed highest antioxidant activity. Both V. anagalloides HD and V. anagallis-aquatica HD hydrosols had benzene acetaldehyde in high percentages. Veronica arvensis HD extract also had benzene acetaldehyde together with (E)-β-damascenone as a major compound so these compounds could be responsible for the shown antioxidant activity of these extracts.

European speedwell is a widespread and well known species among the population. It is commonly known to be edible in the period before flowering. Therefore, the aim of this study was to compare the composition of free volatile compounds extracted in the lipophilic part and in the aqueous part (hydrosol) of the aerial parts of Veronica beccabunga L. (Plantaginaceae). This comparison is important for the selection of the extract for further biological studies. Two extraction methods were used: the classical one (hydrodistillation, HD) and a new technique (microwave-assisted water extraction, MAE). Gas chromatography-mass spectrometry was used to identify the volatile compounds in two phases of the two extractions, i.e., in four samples. The main components in the hydrosol HD sample are: piperitone (28.15%), benzene-acetaldehyde (13.23%) and trans-p-mentha- 1(7), 8-dien-2-ol (10.3%), while in the MAE hydrosol sample piperitone is the main compound with 79.86%. The oxygenated diterpene phytol (27.31%) and the two hydrocarbons, hexacosane (16.21%) and heptacosane (14.89%), are the predominant compounds in the lipophilic HD extract. Piperitone (29.28%) and phytol (34.54%) are the predominant compounds in the lipophilic MAE extract. Comparing these two extraction methods, the method MAE is more environmentally friendly due to the working conditions. From the obtained results, it is clear that hydrosols should not be classified as extraction waste, as they are a valuable source of bioactive compounds. Due to the richness of volatiles, Veronica beccabunga is important for further research. Considering the wide distribution of this plant, the material is easy accessible for research and application.

New methods for the extraction of free volatile compounds (FVCs) are being introduced because future use of these compounds will require greener solutions that consume less energy in the extraction processes. Evaluating the differences between the compositions of FVCs derived from different extraction techniques are valuable for estimating the quality of the product at the end of the extraction process and for the future use of this components. FVCs as part of specialized metabolites are a source of valuable biologically active compounds. To gain insight into the differences between hydrodistillation (HD) and microwave extraction (ME), FVCs extracted from the aerial parts of Veronica officinalis L. were investigated. It is precisely because of the phytochemical constituents of the studied species, that V. officinalis L. is used in the traditional medicine. HD and ME extracts consist of an oil part and the water part (hydrosols). The FVCs of both parts were analyzed by gas chromatography combined with mass spectrometry. In the obtained HD oil extract, β­ionone (18.11%) and hexadecanoic acid (13.21%) are the most abundant components. The most abundant compounds identified in the oil portion isolated by ME were also hexadecanoic acid (12.40%) and 3­hexen­1­ol (22.04%). This species is commonly used in herbal tea, so the presence of 3­hexen­1­ol was expected since this compound is widely present in fresh tea leaves. In the hydrosols obtained by HD, 2­methoxy­4­vinylphenol (11.12%) and β­ionone (12.78%) dominated. In the composition of hydrosol obtained by ME, caryophyllene oxide (15.46%) and squalene (15.15%) dominated. From our results, it is clear that the extraction methods used isolate different compounds in the same extract (from the same plant material). Therefore, despite the fact that ME is the more environmentally friendly choice, it is important to gain insight into the composition of the extract obtained, especially when the extract is further used.

Rod Veronica L. (čestoslavice) iz obitelji Plantaginaceae obuhvaća oko 500 vrsta koje su nešto više rasprostranjene po sjevernoj hemisferi [1]. Najviše predstavnika roda Veronica raste u područjima s mediteranskom klimom, od razine mora do visokog alpskog područja [2]. Ovo istraživanje opisuje fitokemijsku karakterizaciju hlapivih komponenti izoliranih klasičnom hidrodestilacijom (HD) i zelenom mikrovalnom ekstrakcijom (ME) iz pet vrsta Veronica rasprostranjenih u Hrvatskoj, sa staništa s umjerenom količinom raspoložive vode. Proučavane su vrste: V. acinifolia, V. chamaedrys, V. serpyllifolia, V. urtifolia i V. polita. Cilj ovog rada je dobiti nove podatke za nedovoljno proučene hlapive tvari te steći uvid u razlike i sličnosti u sadržaju hlapivih spojeva među vrstama dobivenih različitim metodama izolacije. E-kariofilen, kariofilen oksid, heksahidrofarnezil aceton, fitol, βionon, heksadekanska kiselina i dokosan identificirani su u svih pet izolata dobivenih HD. V. acinifolia je bogata β-iononom (17, 01%), zatim spojevima heksahidrofarnezil acetonom (15, 37%) i kariofilen oksidom (7, 71%). Vrste V. chamaedrys, V. polita, V. serpyllifolia i V. urticifolia bogate su fitolom, posebno V. urtifolia koja sadrži 47, 55% navedene tvari. Iste glavne komponente identificirane u izolatima pomoću HD također dominiraju u sastavu hlapivih komponenti izolata dobivenih ME. Rezultati ovog istraživanja pokazuju da su heksahidrofarnezil aceton, heksadekanska kiselina, fitol, E-kariofilen i kariofilen oksid komponente identificirane u obje metode ekstrakcije. Kako su ovi spojevi izolirani u svim vrstama, mogli bi se smatrati kemofenetskim markerima za rod Veronica. Buduća istraživanja koja uspoređuju skupine temeljene na hlapljivim spojevima i skupine proizašle iz genetičkih istraživanja mogla bi razriješiti ovu hipotezu. Gledajući rezultate za izolirane hlapljive spojeve za rod Veronica i činjenicu da se svi glavni spojevi ekstrahiraju s obje metode, pri ekstrakciji hlapljivih spojeva treba uzeti u obzir mikrovalnu ekstrakciju jer je to prihvatljivi izbor manje štetan za okoliš koji koristi manje vode i energije.

Introduction. Veronica species have been used in traditional medicine for the treatment of various diseases, including influenza, respiratory diseases, and cancer. In this study, a qualitative analysis of various polyphenolic components and a quantitative analysis of phenolic acids were carried out on selected Veronica L. species growing in Croatia (V. longifolia, V. cymbalaria, V. chamaedrys, V. hederifolia, V. polita, V. persica, V. serpyllifolia, and V. arvensis) to evaluate their phytotherapeutic potential. Materials and methods. The presence of polyphenols was investigated using appropriate chemical reactions and thin-layer chromatography (TLC). Quantitative analysis of total phenolic acids (TPA) was performed spectrophotometrically, according to official pharmacopoeial method for determination of hydroxycinnamic derivatives. Results. The polyphenols were detected in all investigated plant samples by general chemical reactions of developing colored products and precipitates. The strongest intensity of the reaction was observed for the sample of V. persica, which indicates a higher content of polyphenols in that sample. TLC analysis proved the presence of phenolic acids in methanolic extracts of all investigated Veronica species. Spectrophotometrically determined TPA content varied from 0.44% (V. cymbalaria) to 3.59% (V. chamaedrys), when expressed as rosmarinic acid (at 505 nm), and from 0.81% (V. cymbalaria) to 7.18% (V. chamaedrys), when expressed as chlorogenic acid (at 525 nm). Conclusions. This study is a contribution to the research of Veronica species and increases the previous knowledge about the phytotherapeutic potential of the analyzed plant species, especially in relation to the significant content of bioactive phenolic substances.

Plants of the genus Veronica (family Plantaginaceae) are used in traditional medicines in countries around the world. This sparked interest in studying these plants in terms of their chemical composition and biological activity. Veronica officinalis L., and Veronica urticifolia Jacq. which are the subject of this study, are traditionally used in the medicine of Balkan peoples. The objective of this study was to investigate free volatile compounds (FVCs) of Veronica urticifolia and. V. officinalis species especially in the terms of comparing FVCs from essential oil (EO) and from water residues (hydrolates, HY) and also discussing differences and similarities in the composition considering different plant material collection locations. Hydrolates are condensed water vapours containing dissolved molecules of EOs and more water-soluble (polar) FVCs. For the purposes of this research, EOs and HY were isolated by water distillation of dry plant material of the species V. urticifolia and V. officinalis in a Clevenger type apparatus. These plant species were collected in different areas in the territory of the Republic of Croatia. The phytochemical composition of FVCs was determined by GC and GC-MS analysis of isolated EO and HY, and the relative percentage of the identified compounds were calculated. The individual peaks for all samples were identified by comparison of their retention indices of n-alkanes to those of authentic samples and literature. The most abundant compound In EO of V. officinalis collected in Lokve - Mali raj (Site 1) was hexadecanoic acid (49.5%) whereas in the material collected in Zagreb (Site 2) heptacosane (25.1%), hexahydropharnesyl acetone (phyton) (21.8%) and phytol (17.1%) were the main components. The most common compounds in HY of V. officinalis from the Sites 1 and 2 were hexadecanoic acid (34.1% ; 30.3%), phytol (17.3% ; 23.3%) and phyton (14.5%: 18.5%), respectively. In the EO of V. urticifolia collected in Mala Kapela (Site 1) and Zelin Crnoluški (Site 2) the most common compounds were hexadecanoic acid (28.3% ; 30.3%), phyton (20.2% ; 18.5%), heptacosan (17.9%, 17.2%) and phytol (15.4% ; 20.3%), respectively. The most common compounds in the HY of this species from the mentioned localities were phyton (35.9% ; 40.3%), hexadecanoic acid (22.9%) and phytol (20.3% ; 21.1%), respectively.

The genus Veronica L. is the largest genus of the Plantaginaceae family and in Croatia about 40 species can be found in various habitats. Some species of this genus, especially V. officinalis have been used in folk medicine for the treatment of colds, respiratory diseases, laryngopharyngitis, cough, hernia, cancer, as a diuretic and in the treatment of wounds [1]. According to previous studies, plants of the genus Veronica are a potential source of nutrients and functional substances with a variety of biological effects [2]. The aim of this study was to investigate differences in the composition of free volatile compounds of essential oils and hydrosols of the species Veronica austriaca ssp. jacquinii (Baumg.) Eb. Fish. and Veronica saturejoides Vis. ssp. saturejoides and to determine the antioxidant activity of the isolated extracts. From the essential oils of selected species, we isolated and identified free volatiles and statistically analyzed the results by using PCA to see which of the isolated extracts were more similar to each other and to relate the results with antioxidant activity. The components of the essential oils and hydrosols were analyzed by gas chromatography (GC-MS and GC-FID instrument) [3]. In both species the major component of the oil is hexahydropharnesyl acetone, while in the hydrosols of Veronica saturejoides Vis. ssp. saturejoidesspecies, the major component wastrans-p-mentha-1 (7), 8-dien-2-ol and in Veronica austriaca ssp. jacquinii, it was methyl eugenol. For each isolate, antioxidant activity was examined using two methods, ORAC (Oxygen Radical Absorbance Capacity Assay) and DPPH (2, 2-diphenyl-1-picrylhydrazyl) solution) [3]. Samples of essential oils isolated from Veronica saturejoides Vis. ssp. saturejoides showed stronger antioxidant activity by both tested methods at concentration of 10 mg of essential oil per mL of acetone. The antioxidant activity of the hydrosols proved to be similar in all tested samples for both tested plants according to both ORAC and DPPH methods. It is expected that the antioxidant activity of the hydrosol is lower than that of the oil, since hydrosols contain less active substances than the essential oils.

Within the research project entitled as “Taxonomy, Ecology and Utilization of Carob tree (Ceratonia siliqua L.) and Bay laurel (Laurus nobilis L.) in Croatia” acronym: TEUCLIC, grant number: IP-11-2013-3304-TEUCLIC, financed by Croatian Science Foundation, a phytochemical characterization of Bay laurel (Laurus nobilis L.) essential oils was studied in seven distinct populations of Croatian Adriatic region, such as: Lovran, Cres, Split, Hvar, Mljet, Dugi Otok and Žirje. The results of gas chromatography–mass spectrometry (GC-MS) analyses shows the highest content of 1, 8-Cineole within the analysed samples of all populations, and its content vary between maximally 58.13 to minimally 38.94 %. Than follows linalool whose content vary between maximally 18.33 to minimally 1.99 %, terpinyl acetate, methyl eugenol, α-terpineol and terpinen-4-ol, β-pinen, etc. On the other hand, the content of myrcene is lower than 1 % and varies from only 0.26 % to maximally 0.69 %. Generally, the southern populations of Bay laurel have a higher content of the same components of essential oils in the comparison with the northern populations. However, the content of essential oils depends not just of geographic position of the population, but also of the season and date of leaves sampling.

Genus Veronica L. is divided into 13 subgenuses according to morphological traits and it belongs to the Plantaginaceae family. There are 40 species of Veronica in Croatia. They are cosmopolitan and ecologically diverse species spread on a variety of habitats from aquatic, marshy and forest to rock, rock cracks, fields and ruderal habitats. The investigated Veronica jacquinii belongs to Southeast- European- Mediterranean hemicryptophytes. Plant material was collected from Ličko polje (Croatia) and was air dried and water distilled. Composition of volatile compounds was analyzed by GC and GC/MS (Adams, 2007) using VF-5ms capillary column. The peaks appeared from 30 – 59 minutes. Thirteen compounds were determined representing 89% of the total oil. Composition of this essential oil is characterized by a high concentration of hydrocarbon derivatives: hexadecanoic acid (63.5%), followed by octadecanoic acetate (8.9%), oleic acid (6.2%) and heptatriacotanol (3.5%). In our previous research in the oil of V. spicata one of the most abundant hydrocarbon compound was heptacosane (Dunkić et al., 2015). According to a literature review GC-MS studies have been performed only on Veronica thymoides subsp. pseudocinerea and the most abundant constituent was hexatriacontene (21%) (Ertas et al., 2015). The present study gives additional knowledge about volatile compounds of the genus Veronica.

Within research project “Taxonomy, Ecology and Utilization of Carob tree (Ceratonia siliqua L.) and Bay laurel (Laurus nobilis L.) in Croatia” (HRZZ-IP-11-2013-3304), a total of 1200 plant samples were collected from 12 distinct populations of bay laurel (Laurus nobilis L.) on Croatian Adriatic area. After morphometric analyses of leafs, AFLP analyses of genetic variability, and variability of total essential oils content in leafs of bay laurel populations, significant correlation between leaf surface and essential oils content (Spearman’s Rank Order Corr. Coeff. rs = 0.15 ns) was not found, while the correlation between latitude and essential oils content was strongly negative (Spearman’s Rank Order Corr. Coeff. rs = - 0.78 ; p0.05). However, the correlation between longitude and essential oils content was strongly positive (Spearman’s Rank Order Corr. Coeff. rs = 0.73 ; p0.05). On the other hand, the results of Mantel test showed low, but positive and highly significant correlation between AFLP variability of populations and essential oils content (r = 0.39 ; p0.01), while the significant correlation between AFLP variability of populations and leaf lamina surface (i.e. expectedly the most influential factor on accumulation of essential oils) was not obtained. Obtained results of these matrix correlations (i.e. Spearman’s Rank Order Correlations and Mantel test) corresponds with the results Friedman’s ANOVA and Kendall’s Coeff. of Concordance for variability of total essential oils content between the populations (ANOVA Chi Square = 21.88 ; p = 0.025 and Kendall’s Coeff. of Concordance = 0.99 ; Aver. rank r = 0.98). According to these results, it is possible to conclude that the populations of bay laurel from locations of South East Croatian Adriatic islands and coastal area accumulate higher quantity of essential oils in the comparison with the populations of North West islands and coastal area.

Genus Veronica L. is divided into 13 subgenuses according to morphological traits and it belongs to the Plantaginaceae family. There are 40 species of Veronica in Croatia. They are cosmopolitan and ecologically diverse species spread on a variety of habitats from aquatic, marshy and forest to rock, rock cracks, fields and ruderal habitats. Investigated Veronica arvensis L. belongs to terophytes. Literature review showed that the most investigated secondary metabolites for Veronica arvensis and generally for Veronica genus include iridoid glucosides, phenylethanoids and flavonoid glycosides, so this is the first study of the chemical composition of the essential oil from this species. Plant material was collected from Velebit (Croatia), in the spring (June) 2013. and was air dried. Water distilled essential oil from aerial parts of investigated plant have been analyzed by GC (gas chromatography) and GC/MS (gas chromatography – mass spectrometry) using VF-5ms capillary column and the first peaks appear after 30 minutes. The total yield of oil was 0.1%, based on dry weight of samples. Nine compounds were determined representing 78, 4% of the total oil. Results of the GC/MS showed that the most abundant compound is phytone represented with 38, 1% followed by hexadecanoic acid (26, 3%) and hexahydrofarnesyl acetone (4, 4%). In our previous research in the oil of V. spicata phytol was the most abundant compound represented with 21.3%. In our review, we found one more species, Veronica thymoides subsp. pseudocinerea, that was investigated with the same methodology (GC-MS). Compound peaks for Veronica thymoides also appeared after 30 minutes of the GC-MS analysis and the most abundant constituent was hexatriacontene (21%). The present study gives additional knowledge about volatile compounds of the genus Veronica.