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.

Antioxidant and Antimicrobial Properties of Veronica spicata L. (Plantaginaceae).

Tea made from Veronica spicata L. (syn. Pseudolysimachion spicatum (L.) Opiz, family Plantaginaceae) herb is used in traditional medicine as expectorant for cough and throat rinsing. To get insight into chemical compounds of V. spicata, the essential oil content (analysed by GC-FID and GC-MS), the content of phenolic compounds (analysed by HPLC), the content of macroelements and trace elements (analysed by ICP-AES), quantity of total phenols and total flavonoids (analysed by UV/Vis spectrophotometer), and antioxidant and antimicrobial properties were investigated. The main compounds of the essential oil were phytol (21.13%), heptacosane (10.22%) and pentacosane (8.91%). The most abundant investigated macroelement was K (8261 mg/kg) while Fe was the most represented element (32.49 mg/kg) among investigated micronutrients. Ten phenolic compounds (chrysin, rutin, quercitrin, quercitrin, and cichoric, ferulic, protocatehuic, rosmarinic, syringic and tannic acid) were identified and quantified. Additionally, V. spicata extract demonstrated notable radical-scavenging and chelating properties. The bacterial and fungal strains used in study were found to be susceptible toward methanolic and ethyl-acetate extracts with MIC values between 1.25 and 5.00 mg/mL using microdilution method. Aquose extracts were found to be antimicrobial inactive.

Morphological, genetic and phytochemical variation of the endemic Teucrium arduini L. (Lamiaceae).

Analysis of the morphological traits of leaves, genetic variability (analyzed by AFLP) and chemical composition of essential oils (analyzed by GC-MS) was conducted on eleven populations of the endemic Illyric-Balcanic species Teucrium arduini L. in Croatia, Bosnia and Herzegovina, and Montenegro. Average blade length and width ranged from 20.00 to 31.47mm and from 11.58 to 15.66mm, respectively. Multivariate analysis (PCA, UPGMA) of morphological traits distinguished two continental Bosnian populations from the remaining populations. AFLP analysis separated the investigated populations into two groups based primarily on geographical distance. Essential oil analysis showed a total of 52 compounds, with two chemotypes distinguished based on the essential oil profile. The first was a sesquiterpene chemotype, with ß-caryophyllene, germacrene D or caryophyllene oxide as the major compounds, while the second was an oxygenated monoterpene chemotype, with pulegone and piperitone oxide as the main components. The Mantel test showed a stronger correlation between the morphological traits and AFLP than between the essential oil profile and AFLP. The test also showed a stronger association between the essential oil profile and geographical position than between the morphological traits and geographical position.

Anti-Legionella activity of essential oil of Satureja cuneifolia.

The essential oil of Satureja cuneifolia Ten. was characterized by a high concentration of the phenolic compounds carvacrol (21.3%) and thymol (9.2%). The in vitro activity of the essential oil against Legionela pneumophila serogroups (SG) I and 2-15 and Legionella spp. from different sources, using microdilution, showed that L. pneumofila is sensitive to the oil, with MICs ranging from 0.12 to 0.5%, v/v, and a MBC at 0.5 to 1%, v/v. The essential oil of S. cuneifolia was effective in the reduction of Legionellosis infections.

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.

Investigation of chemical compounds, antioxidant and antimicrobial properties of teucrium arduini L. (lamiaceae).

In this paper chemical composition of the essential oil (analysed by GC and GC-MS), the content of phenolic compounds (analysed by HPLC), quantity of total phenols and total flavonoids (analysed by UV/Vis spectrophotometer), antioxidant and antimicrobial activities of ethanolic extracts were investigated in endemic Teucrium arduini L. in population of Mt Biokovo (Croatia). The oil was characterized by a high concentration of sesquiterpene hydrocarbons (70.4%) of which ß-caryophyllene (35.2%) and germacrene D (18.7%) being the major compounds. Three phenolic compounds (quercetin, ferulic acid and rosmarinic acid) were identified and quantified in ethanolic extract of T. arduini using HPLC. The results also showed that T. arduini is a source of polyphenolic and other antioxidants with radical-scavenging and chelating properties. The ethanol extracts prepared from the leaf of T. arduini showed broad spectrum of antimicrobial activity on Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Aspergillus brasiliensis, which are susceptible on concentration below or equal to 4.00 mg/mL, whilst Microsporum gypseum was resistant at investigated concentrations.

Chemotaxonomic and micromorphological traits of Satureja montana L. and S. subspicata Vis. (Lamiaceae).

Satureja montana and S. subspicata are used as spice, pepper substitute, for preparing tea, juice, and as a medicine. Fourteen populations (seven per species) of Satureja montana L. and S. subspicata Vis. growing in Croatia were examined to determine the chemical composition of the essential oil (analyzed by GC-FID and GC/MS), the content of macroelements (Na, K, Ca, Mg) and trace elements (B, Fe, Cu, Mn, Zn, Al, Pb, Cr, Cd, Ni, Hg, As) analyzed by ICP-AES, antioxidant compounds (analyzed by UV/VIS spectrophotometer), and the types and distribution of trichomes (analyzed by scanning electron microscopy). The main constituents of the essential oil were carvacrol and thymol in S. montana (all populations belong to one phenol chemotype), while α-eudesmol, ß-eudesmol, and spathulenol dominated in S. subspicata (three chemotypes could be distinguished). Both species possess considerably higher quantities of Ca and Mg, and moderate concentrations of K and Na, while Hg and As levels were below the limit of quantification. Non-glandular trichomes, peltate trichomes, and three types of capitate trichomes were observed on leaves, stem, calyx, and corolla.

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.

Micromorphological and chemotaxonomical traits of Micromeria croatica (Pers.) Schott.

A study on the types and distribution of trichomes, pollen morphology, chemical composition of essential oil (analyzed by GC-FID and GC/MS), and the content of macroelements (Na, K, Ca, and Mg) and trace elements (B, Fe, Cu, Mn, Zn, Al, Pb, Cr, Cd, Ni, Hg, and As) analyzed by ICP-AES (=inductively coupled plasma atomic emission spectroscopy) was conducted on Micromeria croatica (Pers.) Schott. Non-glandular trichomes, peltate trichomes (typical hairs of Lamiaceae), small capitate trichomes (composed of one basal epidermal cell and one head cell), and larger capitate trichomes (composed of one basal epidermal cell, two to three stalk cells, and one head cell with subcuticular space) were observed on leaves, stems, the calyx, and corolla. Pollen of M. croatica had six apertures (hexacolpate) which were set in the equatorial pollen belt (zonocolpate) and showed medium reticulate ornamentation (hetrobrochate type). The essential oil contained from 27 to 39 constituents, the most representative of which were caryophyllene oxide and E-caryophyllene. Among the investigated macroelements, the content of K was highest (8730-10080 mg/kg). The content of trace elements ranged from 0.12 mg/kg (Cr) to 78.00 mg/kg (Fe), while the content of Cd, Ni, Hg, and As were lower than the limit of quantification.

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 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.

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.

Essential oil composition and internal transcribed spacer (ITS) sequence variability of four South-Croatian Satureja species (Lamiaceae).

The purpose of this study was to compare the essential oil profiles of four South-Croatian Satureja species, as determined by GC/FID and GC/MS, with their DNA sequences for an internal transcribed spacer (ITS1-5.8S-ITS2) of the nuclear ribosomal DNA. A phylogenetic analysis showed that S. montana and S. cuneifolia, characterized by a similar essential oil composition, rich in the monoterpene hydrocarbon carvacrol, clustered together with high and moderate bootstrap support. On the contrary, S. subspicata and S. visianii, characterized by quite unique essential oil compositions, clustered together with the moderate bootstrap support. All four Croatian Satureja species clustered in one clade, separately from Macaronesian S. hortensis,although it had essential oil composition similar to that of S. montana and S. cuneifolia. This is the first report on the comparison between the phytochemical and DNA sequence data in Satureja species and useful contribution to the better understanding of interspecies relationships in this genus.

Antibacterial activity of Achillea clavennae essential oil against respiratory tract pathogens.

The essential oil of Achillea clavennae was investigated for its antibacterial activity against some respiratory tract pathogens. Maximum activity was observed against Klebsiella pneumoniae and penicillin-susceptible and penicillin-resistant Streptococcus pneumoniae. The oil also exhibited strong activity against Gram (-) Haemophilus influenzae and Pseudomonas aeruginosa. Gram (+) Streptococcus pyogenes was the most resistant to the oil.

Phytochemical analysis and in vitro antimicrobial activity of two Satureja species essential oils.

The phytochemical GC[sol ]MS analysis and in vitro antimicrobial activity of the essential oils of the aerial parts of Satureja montana L. and Satureja cuneifolia Ten., collected in Croatia were performed. The major compound of S. montana oil was the phenolic monoterpene carvacrol (45.7%). Other important compounds were the monoterpenic hydrocarbons p-cymene (12.6%), gamma-terpinene (8.1%) and the oxygen-containing compounds carvacrol methyl ether, borneol, thymol and thymol methyl ether. The volatile oil of S. cuneifolia was characterized as beta-cubebene (8.7%), limonene (8.3%), alpha-pinene (6.9%), spathulenol and beta-caryophyllene. The antimicrobial effects of S. montana and S. cuneifolia oils were found to have a broad spectrum activity against multidrug-resistant pathogens by the broth microdilution method. These oils were active against all the test strains, with the exception of Pseudomonas aeruginosa. Compared with S. cuneifolia, savory oil exhibited greater antimicrobial activity. The maximum activity of savory oil was observed against Escherichia coli, methicillin-resistant Staphylococcus aureus and against the yeast (Candida albicans). The essential oil of S. cuneifolia was also found to inhibit the growth of medically important pathogens such as S. aureus and E. coli. Fungicidal activity for both oils against C. albicans and S. cerevisiae was also observed.

Composition and antimicrobial activity of Achillea clavennae L. essential oil.

The volatile constituents of Achillea clavennae L. (Asteraceae), rare plant of Europe, have been analysed using GC/MS. Twenty- five components making up 81.6% of the oil were characterized with camphor (29.5%), myrcene (5.5%), 1,8-cineole (5.3%), beta-caryophyllene (5.1%) and linalool (4.9%) being the major constituents. The essential oil was evaluated for antibacterial and antifungal activities. The screening of the antimicrobial activity of essential oil was conducted by a disc diffusion test against Gram-positive (Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Streptococcus faecalis), Gram-negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis) and fungal organisms (Aspergillus niger, Aspergillus fumigatus, Candida albicans). The activity was more pronounced against Gram-negative and fungal organisms than against Gram-positive bacteria. A. clavennae oil was found to possess antimicrobial activity against Klebsiella pneumoniae, Pseudomonas aeruginosa and all fungal organisms.