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.

Phytochemical and Cytogenetic Study of Two Centaurea Species from Croatia: the Particular Case of Diploid and Tetraploid C. salonitana.

Natural wild populations of C. rupestris and C. salonitana were studied to determine possible relationships between the volatile oil (VO) composition and ploidy level. The chemical composition of the volatile oil was investigated using the GC/MS technique. The predominant components of the VO of diploid and tetraploid C. salonitana were hexadecanoic acid and α-linoleic acids, while in C. rupestris they were germacrene D and ß-caryophyllene in one population and heptacosane and germacrene D, in another. The nuclear DNA amounts (2 C DNA), determined by flow cytometry, were 3.54 pg for C. rupestris, 3.39 pg for the diploid and 6.79 pg for the tetraploid population of C. salonitana. Evidence that the degree of ploidy solely influences the chemical composition of the essential oil of C. salonitana was not found. The results presented are the first data to be reported on the DNA content of the studied Centaurea populations from Croatia, as well as on the chemical composition of C. salonitana volatile oil.

Glucosinolates of Sisymbrium officinale and S. orientale.

Glucosinolates (GSLs) from Sysimbrium officinale and S. orientale were analyzed qualitatively and quantitatively by their desulfo-counterparts using UHPLC-DAD-MS/MS. Eight GSLs were identified in S. officinale, including Val-derived (glucoputranjivin) and Trp-derived (4-hydroxyglucobrassicin, glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin) as the major ones followed by Leu-derived (Isobutyl GSL), Ile-derived (glucocochlearin) and Phe/Tyr-derived (glucosinalbin). Different S. orientale plant parts contained six GSLs, with Met-derived (progoitrin, epiprogoitrin, and gluconapin) and homoPhe-derived (gluconasturtiin) as the major ones, followed by glucosinalbin and neoglucobrassicin. GSL breakdown products obtained by hydrodistillation (HD) and microwave-assisted distillation from S. officinale, as well as isopropyl isothiocyanate, as the major volatile in both isolates, were tested for their cytotoxic activity using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Generally, all volatile isolates showed similar activity toward the three cancer cell lines. The best activity was shown by isopropyl isothiocyanate at a concentration of 100 µg/mL after 72 h of incubation, with 53.18% for MDA-MB-231, 56.61% for A549, and 60.02% for the T24 cell line.

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.

Lepidium graminifolium L.: Glucosinolate Profile and Antiproliferative Potential of Volatile Isolates.

Glucosinolates (GSLs) from Lepidium graminifolium L. were analyzed qualitatively and quantitatively by their desulfo-counterparts using UHPLC-DAD-MS/MS technique and by their volatile breakdown products-isothiocyanates (ITCs) using GC-MS analysis. Thirteen GSLs were identified with arylaliphatic as the major ones in the following order: 3-hydroxybenzyl GSL (glucolepigramin, 7), benzyl GSL (glucotropaeolin, 9), 3,4,5-trimethoxybenzyl GSL (11), 3-methoxybenzyl GSL (glucolimnanthin, 12), 4-hydroxy-3,5-dimethoxybenzyl GSL (3,5-dimethoxysinalbin, 8), 4-hydroxybenzyl GSL (glucosinalbin, 6), 3,4-dimethoxybenzyl GSL (10) and 2-phenylethyl GSL (gluconasturtiin, 13). GSL breakdown products obtained by hydrodistillation (HD) and CH2Cl2 extraction after hydrolysis by myrosinase for 24 h (EXT) as well as benzyl ITC were tested for their cytotoxic activity using MTT assay. Generally, EXT showed noticeable antiproliferative activity against human bladder cancer cell line UM-UC-3 and human glioblastoma cell line LN229, and can be considered as moderately active, while IC50 of benzyl ITC was 12.3 µg/mL, which can be considered as highly active.

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.

Micromorphological Traits of Balcanic Micromeria and Closely Related Clinopodium Species (Lamiaceae).

A study of the trichomes types and distribution and pollen morphology was carried out in nine Micromeria taxa (M. cristata ssp. cristata, M. cristata ssp. kosaninii, M. croatica, M. graeca ssp. graeca, M. graeca ssp. fruticulosa, M. juliana, M. kerneri, M. longipedunculata and M. microphylla) and five closely related Clinopodium species (C. dalmaticum, C. frivaldszkyanum, C. pulegium, C. serpyllifolium and C. thymifolium) from the Lamiaceae family of the Balkan Peninsula. By scanning electron microscope, non-glandular trichomes, peltate and capitate trichomes were observed on the calyx, leaves and stem of the studied species. Two subtypes of capitate trichomes were observed in Micromeria species: subtype 1 (consisting of a basal epidermal cell and an elliptically shaped head cell) and subtype 2 (consisting of a basal epidermal cell, two to three stalk cells and a round head cell). In Clinopodium species, three types of capitate trichomes were observed: subtype 1, subtype 3 (consisting of a basal epidermal cell, a short peduncle cell, and a single round head cell), and subtype 4 (consisting of a basal epidermal cell, a stalk cell, and an elongated head cell). These results support the recent transfer of Micromeria species from the section Pseudomelissa to the genus Clinopodium.

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.

Bunias erucago L.: Glucosinolate Profile and In Vitro Biological Potential.

Bunias erucago belongs to the Brassicaceae family, which represents a forgotten crop of the Euro-Mediterranean area. The aim of the present study was to determine the glucosinolate profile in different plant parts and biological properties (antioxidant, anticholinesterase, and cytotoxic activities) of the isolates containing glucosinolate breakdown products. The chemical profiles were determined by using HPLC-PDA-MS/MS of desulfoglucosinolates and GC-MS of glucosinolate degradation products. The analysis of B. erucago showed the presence of seven glucosinolates: gluconapin (1), glucoraphasatin (2), glucoraphenin (3), glucoerucin (4), glucoraphanin (5), glucotropaeolin (6), and glucosinalbin (7). The total glucosinolate content ranged from 7.0 to 14.6 µmol/g of dry weight, with the major glucosinolate glucosinalbin in all parts. The antioxidant activity of all volatile isolates was not notable. At a tested concentration of 227 µg/mL, flower hydro-distillate (FH) showed good AChE inhibition, i.e., 40.9%, while root hydro-distillate (RH) had good activity against BChE, i.e., 54.3%. FH showed the best activity against both tested human bladder cancer cell lines, i.e., against T24 after 72 h, which have IC50 of 16.0 µg/mL, and against TCCSUP after 48 h with IC50 of 7.8 µg/mL, and can be considered as highly active. On the other hand, RH showed weak activity against tested cancer cells.

Phytochemical and Cytogenetic Characterization of Centaurea solstitialis L. (Asteraceae) from Croatia.

The cytogenetic characterization of Centaurea solstitialis L. (Asteraceae) showed a chromosome number of 2n = 16. Karyotype is composed by four pairs of metacentric, two pairs of submetacentric and two pairs of subtelocentric chromosomes. Physical mapping of two rDNA probes revealed two loci of 35S and one locus of 5S rRNA genes. Chromomycin fluorochrome banding revealed that all rDNA loci were GC rich. The genome size (2C-value) of 1.95 pg classes this species in the group of very small genomes. Chemical composition of C. solstitialis volatile oil (VO) from Croatia, studied with gas chromatography-mass spectrometry showed dominant components as it follows: hexadecanoic acid, α-linolenic acid, germacrene D and heptacosane. Antioxidant capacity, measured by ferric reducing power assay and 2,2-diphenyl-1-picrylhydrazyl methods, as well as inhibition of acetyl- and butyrylcholinesterase of VO was lower comparing to a standard solutions. Volatile oil tested with disc diffusion method showed good inhibitory potential against Pseudomonas aeruginosa, Escherichia coli and all tested fungi: Candida albicans, Penicillium funiculosum and Aspergillus fumigatus. The microdilution method showed best activity against Chronobacter sakazakii and A. fumigatus.

LC-MS profiling of glucosinolates in the seeds of Brassica elongata Ehrh., and of the two stenoendemic B. botteri Vis and B. cazzae Ginzb. & Teyber.

The glucosinolates (GLs) present in seed extracts of Brassica elongata Ehrh., B. botteri Vis and B. cazzae Ginzb. & Teyber from Croatia were identified by LC-MS. 4-Hydroxyindol-3-ylmethyl GL (3) was the major GL in the seeds of B. elongata, along with the four minor GLs 2-(R)-hydroxy-3-butenyl- (1), 3-butenyl- (2), 4-pentenyl- (4) and indol-3-ylmethyl (5). The seeds of B. botteri (Vis island) and B. cazzae (Susac island) contained 2 as the major GL as well as 1, 3, 5 and 4-methoxyindol-3-ylmethyl GL (6). However, the GLs in B. botteri (Palagruza island) differed from other varieties having 2-propenyl GL (7) as the major GL in the seeds, and the four minor GLs 2, 3, 5 and 6. This first report of the GL content in the seeds of B. elongata, B. botteri and B. cazzae indicates that the unique GL profiles could be specific to the geographical origin of the plant.

Micromorphological traits and essential oil contents of Micromeria kerneri Murb. and M. juliana (L.) Benth. (Lamiaceae).

The chemical composition of the essential oil (analysed by GC and GC-MS), the types and distribution of trichomes and pollen morphology (analysed by scanning electron microscopy) were investigated in two closely related species, Micromeria kerneri Murb. and Micromeria juliana (L.) Benth. (Lamiaceae) from Southeast Europe as a contribution to their taxonomy. The essential oil of M. kerneri was characterized by a high concentration of oxygenated sesquiterpenes, with caryophyllene-oxide as the major compound. Caryophyllene-oxide was also the major component of the essential oil of M. juliana from all localities, except from Mt Krivosije (Montenegro), where piperitone oxide was the major constituent. Non-glandular trichomes, peltate trichomes, and two types of capitate trichomes (type 1 composed of one basal epidermal cell, and one head cell with subcuticular space; type 2 composed of one basal epidermal cell, two stalk cells, and one head cell with subcuticular space) were observed on leaves, the calyx and on the stem. Pollen of both species had six apertures (hexacolpate) set in the equatorial pollen belt (zonocolpate) and showed medium reticulate ornamentation. Multivariate analysis (PCA and UPGMA) of essential oil components clearly separated the investigated M. kerneri and M. juliana populations, and confirmed the opinion that they are different taxa. On the other hand, micromorphological traits between these species were the same. Nevertheless, definitive conclusions about the taxonomic relationships among these species will require genetic analysis.

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.

Campanula portenschlagiana ROEM. et SCHULT.: chemical and antimicrobial activities.

The phytochemical profile and the antimicrobial effects of the volatile oil and the aqueous extract of Campanula portenschlagiana, a wild growing plant endemic to Croatia, were described. In the volatile oil, 53 compounds were identified by GC-FID and GC/MS analyses. Diterpene alcohols constituted the major compound class with labda-13(16),14-dien-8-ol as the main compound. The aqueous extract was characterized by the total phenolic content. The antimicrobial potential of the volatile oil and the aqueous extract was evaluated against a diverse range of microorganisms comprising food-spoilage and food-borne pathogens. The volatile oil exhibited interesting and promising antimicrobial effects against the tested species, which were generally more pronounced against Gram-negative bacteria. In addition, the inhibitory effect of this volatile oil was also evaluated against eleven extended-spectrum ß-lactamase (ESBL)-producing isolates. The results suggest that the C. portenschlagiana volatile oil might be used as antimicrobial agent against ESBL-producing isolates and Gram-negative bacteria.

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.

Phytochemical and micromorphological traits of endemic Micromeria pseudocroatica (Lamiaceae).

Micromeria pseudocroatica Silic is an endemic species distributed in southern Croatia. Chemical composition of the essential oil (analysed by GC and GC-MS), and the content of macroelements and trace elements analysed by ICP-AES was conducted. Additionally, a study on the types and distribution of trichomes was done by scanning electron microscopy. The essential oil was characterized by a high concentration of oxygenated monoterpenes, whose major compounds are borneol (22.7% and 24.8%) and camphor (16.1% and 13.9%). The content of Ca was highest (13202.69 mg/kg and 13223.83 mg/kg) among the investigated macroelements, while Fe was the most represented element (100.68 and 326.48 mg/kg) among the micronutrients. The content of potentially toxic elements that is Se, Cu, and Hg was below the limit of quantification. Non-glandular trichomes, peltate trichomes, and two types of capitate trichomes (type 1: one basal epidermal cell, one head cell with subcuticular space; type 2: one basal epidermal cell, two stalk cells, and one head cell with subcuticular space) were observed on leaves, bracteoles, calyx, corolla and stem.

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.

Chemical composition and antimicrobial activity of the essential oil of endemic Dalmatian black pine (Pinus nigra ssp. dalmatica).

The chemical composition and the antimicrobial activity of the essential oil isolated from the needles of endemic Dalmatian black pine (Pinus nigra ssp. dalmatica) from Croatia were investigated. The chemical composition of the essential oil was determined by GC and GC/MS analyses, and the main compounds identified were α-pinene, ß-pinene, germacrene D, and ß-caryophyllene. Disc-diffusion and broth-microdilution assays were used for the in vitro antimicrobial screening. The Dalmatian black pine essential oil exhibited a great potential of antibacterial activity against Gram-positive bacteria (MIC=0.03-0.50% (v/v)) and a less pronounced activity against Gram-negative bacteria (MIC=0.12-3.2% (v/v)). The volatile compounds also inhibited the growth of all fungi tested, including yeast.