Phytochemical composition and biological activities of native and in vitro-propagated Micromeria croatica (Pers.) Schott (Lamiaceae).

MAIN CONCLUSION: In vitro culture conditions and kinetin induced quantitative modifications in the production of the major volatile constituents in Micromeria croatica plantlets. Antimicrobial activity of methanolic extracts obtained from micropropagated and wild-growing plants was evaluated. Micromeria spp. are aromatic plants, many of which were shown to exhibit various biological effects. The present study aimed to determine the content and the composition of the essential oil of in vitro-cultured Micromeria croatica (Pers.) Schott and to evaluate the in vitro antimicrobial activity of its methanolic extract, in order to compare its phytochemical profile and biological activity with wild-growing plants. Shoots regenerated on MS medium without plant growth regulators (PGRs) or supplemented with kinetin were used for phytochemical analysis. Essential oils from both native plant material and in vitro-cultivated M. croatica plants, with a total of 44 identified constituents, were dominated by oxygenated monoterpenes. Borneol was the main component detected in wild-growing plants (25.28%) and micropropagated plants grown on PGR-free medium (20.30%). Kinetin treatment led to increased oil yield and favored the production of oxygenated monoterpenes, dominated by geranial (33.53%) and cis-p-mentha-1(7),8-dien-ol (23.69%). The percentage of total sesquiterpenoids in micropropagated plant material was considerably lower than in wild-growing plants. In vitro culture conditions and PGRs affected the production of essential oils, inducing quantitative modifications in the production of the major volatile constituents in M. croatica plantlets. The antimicrobial activity of M. croatica methanolic extracts was investigated using the broth microdilution method. Extracts obtained from in vitro cultures generally exhibited greater antibacterial potential, compared to wild-growing plants. Among six bacterial strains tested, Bacillus cereus and Staphylococcus aureus were the most sensitive microorganisms. The present study provided evidence that in vitro culture conditions might favorably affect the antimicrobial activity of M. croatica methanolic extracts.

Glandular trichomes and essential oil characteristics of in vitro propagated Micromeria pulegium (Rochel) Benth. (Lamiaceae).

MAIN CONCLUSION: In vitro conditions and benzyladenine influenced both content and composition of micropropagated Micromeria pulegium essential oils, with pulegone and menthone being the main essential oil components. The content and chemical composition of Micromeria pulegium (Rochel) Benth. essential oils were studied in native plant material at vegetative stage and in micropropagated plants, obtained from nodal segments cultured on solid MS medium supplemented with N(6)-benzyladenine (BA) or kinetin at different concentrations, alone or in combination with indole-3-acetic acid. Shoot proliferation was achieved in all treatments, but the highest biomass production was obtained after treatment with 10 µM BA. Phytochemical analysis identified up to 21 compounds in the essential oils of wild-growing and in vitro cultivated plants, both showing very high percentages of total monoterpenoids dominated by oxygenated monoterpenes of the menthane type. Pulegone and menthone were the main essential oil components detected in both wild-growing plants (60.07 and 26.85 %, respectively) and micropropagated plants grown on either plant growth regulator-free medium (44.57 and 29.14 %, respectively) or BA-supplemented medium (50.77 and 14.45 %, respectively). The percentage of total sesquiterpenoids increased in vitro, particularly owing to sesquiterpene hydrocarbons that were not found in wild-growing plants. Differences in both content and the composition of the essential oils obtained from different samples indicated that in vitro culture conditions and plant growth regulators significantly influence the essential oils properties. In addition, the morphology and structure of M. pulegium glandular trichomes in relation to the secretory process were characterized for the first time using SEM and light microscopy, and their secretion was histochemically analyzed.

Seasonal variations in the composition of the essential oils of Lavandula angustifolia (Lamiacae).

Seasonal variations in the composition of the essential oils obtained from the same individual (of the same genotype) of Lavandula angustifolia cultivated in Belgrade were determined by GC and GC/MS. The main constituents were 1,8-cineole (7.1-48.4%), linalool (0.1-38.7%), bomeol (10.9-27.7%), beta-phellandrene (0.5-21.2%) and camphor (1.5-15.8%). Cluster analysis showed that the 21 samples collected each month during the vegetation cycle were separable into three main clades with different compositions of essential oils. In the shoots with flowers, inflorescences and fruits of clade I, linalool is dominant, in the young leaves before flowering and old leaves of clade II, 1,8-cineole is dominant. In the young and incompletely developed leaves of clade III, beta-phellandrene is dominant. The composition of the essential oils of lavender depended on the plant part and the stage of development.

Cytotoxic effect of Potentilla reptans L. rhizome and aerial part extracts.

Potentilla reptans L. belongs to the least studied of the plants from Rosaceae family, Potentilla genus. There are no data on cytotoxicity of P. reptans extracts, though traditionaly it was used as antiinflammatory and antiinfective. The aim of these studies was to investigate potential antitumor activity of aqueous extracts (rhizome and aerial parts) of P. reptans on 4T1 mouse breast cancer cell line. _Aqueous extracts of rhizome and aerial parts of P. reptans were tested for cytotoxicity by the MTT colorimetric assay on 4T1 cancer cell line in concentration range 100-800 microg/mL. Aqueous extracts of P. reptans rhizome and aerial parts show concentration dependent cytotoxic effect in the range of tested concentrations. ICE50 value of P. reptans rhizome extract was 280.51 +/- 1.16 microg/mL. IC50 value of P. reptans aerial parts extract was 310.79 +/- 1.22 microg/mL. The significant difference in cytotoxicity among tested concentrations was observed. Aqueous extracts of P. reptans rhizome and aerial parts demonstrated weak cytotoxic activity on 4T1 mouse breast cancer cell line, which is in correlation with current cytotoxicity data for aqueous herbal extracts. Rhizome extract of P. reptans has slightly higher antitumor activity than aerial parts extract. The results represent the first report on cytotoxicity for this plant and further research on human cell lines is indicated.

Seasonal variations in the composition of the essential oils of rosemary (Rosmarinus officinalis, Lamiaceae).

Seasonal variations in the composition of the essential oils obtained from rosemary plants of the same genotype cultivated in Belgrade were determined by GC and GC/MS. The main constituents were camphor (18.2 - 28.1%), 1,8-cineole (6.4-18.0%), alpha-pinene (9.7-13.5%), borneol (4.4-9.5%), camphene (5.1-8.7%), beta-pinene (2.1-8.1%), beta-phellandrene (4.6-6.5%), myrcene (3.4-5.9%) and bornyl acetate (0.2-7.9%). Cluster analysis showed that 16 samples that had been collected each month during the vegetative cycle can be separated into three main clades with different compositions of essential oils. In the shoots with fruits ('fruits' - Clade I) and shoots with developed leaves ('old shoots' - Clade III) camphor is dominant. In shoots with young and incompletely developed leaves ('young shoot' - Clade II) camphor and 1,8-cineole had almost the same concentration. The fact that the same genotype during the growing seasons can synthesize oils that are so different that they can be classified as different chemotypes confirms the opinion that the chemical composition of essential oils sometimes critically depends on the time of collection. Also, for the definition of chemotypes it is not enough to base this on a chemical analysis of an oil from one phenophase only.

Environment-related variations of the composition of the essential oils of rosemary (Rosmarinus officinalis L.) in the Balkan Penninsula.

Composition of the essential oils of Rosmarinus officinalis of ten populations from the Balkan Peninsula were determined by GC/FID and GC/MS. The main constituents were 1,8-cineole, camphor, α-pinene, and borneol. Multivariate statistical analysis (UPGMA cluster analysis and principal-component analysis (PCA)) revealed two major types of rosemary oil, i.e., 1,8-cineole and camphor-type, and two intermediate types, i.e., camphor/1,8-cineole/borneol type and 1,8-cineole/camphor type. The regression analyses (simple linear regression and stepwise multiple regression) have shown that, with respect to basic geographic, orographic, and 19 bioclimatic characteristics of each population, bioclimatic factor temperature of habitat represented the dominant abiogenetic factor, which, in chemical sense, led to differentiation of populations in the studied region. Also, the regression analysis have shown that some constituents of essential oils are independent of any single bioclimatic factors. However, some constituents display statistically significant correlations with some abiotic factors.

Environmental and seasonal impacts on the chemical composition of Satureja horvatii Silic (Lamiaceae) essential oils.

The chemical composition of the essential oils of Satureja horvatii Silic from two natural habitats (Mt. Orjen and Mt. Lovcen in Montenegro) and from cultivated plants (Belgrade, Serbia) were characterized. For the latter, plants from the locus classicus, i.e., Orjenske Lokve (Mt. Orjen), were transferred to Belgrade and, after three years of cultivation, the chemical composition of their essential oils at different phenological stages was analyzed. The essential oils were obtained from the aerial parts of the plants by hydrodistillation and analyzed by GC and GC/MS. The yields and chemical compositions of the S. horvatii oils showed significant differences between the plants collected in the natural habitats and those from cultivation, as well as between the plants at different phenological stages. In the populations from the natural habitats, growing in Mediterranean conditions, the most abundant oil constituents were the phenols thymol (63.7% in the samples from Mt. Orjen) or carvacrol (68.1% in the samples from Mt. Lovcen), while the oils from the cultivated plants (Belgrade), growing in continental conditions, were dominated by linalool (up to 65.8 and 55.9% in average). The basic characteristics of the essential oil from plants at the early phenological stage (before flowering) were high percentages of linalool (37.4%), thymol (27.3%), and carvacrol (12.2%). At the stage of flowering, the percentage of linalool (56.6-57.5%) increased, while those of thymol (15.5-15.8%) and carvacrol (1.4-1.5%) significantly decreased. The essential oil of plants in the full stage of fruiting was characterized by the domination of linalool (58.4 and 65.8%) and lower percentages of thymol (7.6 and 1.3%) and carvacrol (0.7 and 0.1%). In conclusion, the oil composition of S. horvatii was found to depend on the pedoclimatic conditions of the habitat and the phenological stage of the plants.

Chemical composition and antimicrobial activity of the essential oil from Chaerophyllum aureum L. (Apiaceae).

The essential oils of the aerial parts and fruits of Chaerophyllum aureum L., collected from two mountains in Serbia, were analyzed by GC and GC/MS. Sabinene (18.5-31.6%), p-cymene (7.9-25.4%) and limonene (1.9-10.9%) were characterized as the main constituents. The oils were tested against six bacterial strains and one strain of yeast, Candida albicans. The highest antimicrobial activity was observed against the Gram-positive bacteria Staphylococcus aureus, S. epidermidis and Micrococcus luteus, while of the Gram-negative strains, Escherichia coli was the most sensitive.