Ivica Blažević

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 β-caryophyllene (7.1–52.0%) and germacrene D (8.7–17.0%). Other important components were β-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.

Phytochemical Analysis and Antimicrobial Activity of Cardaria draba (L.) Desv. Volatiles

Two different volatile isolates from the aerial parts of Cardaria draba (L.) Desv., obtained either by hydrodistillation (Extract I) or by CH2Cl2 extraction subsequent to hydrolysis by exogenous myrosinase (Extract II), were characterized by GC‐FID and GC/MS analyses. The main volatiles obtained by hydrodistillation, i.e., 4‐(methylsulfanyl)butyl isothiocyanate (1; 28.0%) and 5‐(methylsulfanyl)pentanenitrile (2; 13.8%), originated from the degradation of glucoerucin. In Extract I, also volatiles without sulfur and/or nitrogen were identified. These were mostly hexadecanoic acid (10.8%), phytol (10.2%), dibutyl phthalate (4.5%), and some other compounds in smaller percentages. Extract II contained mostly glucosinolate degradation products. They originated from glucoraphanin, viz., 4‐(methylsulfinyl)butyl isothiocyanate (3; 69.2%) and 5‐(methylsulfinyl)pentanenitrile (4; 4.5%), glucosinalbin, viz., 2‐(4‐hydroxyphenyl)acetonitrile (5; 7.2%), and glucoerysolin, viz., 4‐(methylsulfonyl)butyl isothiocyanate (6; 5.0%). Moreover, the volatile samples were evaluated for their antimicrobial activity using the disc‐diffusion method and determining minimum inhibitory concentrations (MIC). All volatile isolates expressed a wide range of growth inhibition activity against both Gram‐positive and Gram‐negative bacteria and fungi. The MIC values varied between 4 and 128 μg/ml.

Hydrodistillation-adsorption method for the isolation of water-soluble, non-soluble and high volatile compounds from plant materials.

Proposed method of hydrodistillation-adsorption (HDA) on activated carbon and hydrodistillation (HD) with solvent trap were compared for the isolation of water-soluble, non-soluble and high volatile compounds, such as acids, monoterpenes, isothiocyanates and others from carob (Certonia siliqua L.), rosemary (Rosmarinus officinalis L.) and rocket (Eruca sativa L.). Isolated volatiles were analyzed by GC and GC/MS. The main advantages of HDA method over ubiquitous HD method were higher yields of volatile compounds and their simultaneous separation in three fractions that enabled more detail analyses. This method is particularly suitable for the isolation and analysis of the plant volatiles with high amounts of water-soluble compounds. In distinction from previously published adsorption of remaining volatile compounds from distillation water on activated carbon, this method offers simultaneous hydrodistillation and adsorption in the same apparatus.

Hedge Mustard (Sisymbrium officinale): Chemical Diversity of Volatiles and Their Antimicrobial Activity

Volatile compounds of hedge mustard (Sysimbrium officinale) have been investigated for the first time. Forthy‐two compounds were identified after hydrodistillation (without or upon autolysis) after gas chromatography and gas chromatography/mass spectrometry analyses. In addition, after decoction and hydrolysis of O‐glycosides, 18 volatile O‐aglycones were identified. In general, the main volatiles found in hydrodistillates were: isopropyl isothiocyanate (27.6–48.9%), 2‐methylpropanenitrile (0.5–18.8%), (Z)‐hex‐3‐en‐1‐ol (0.5–18.0%), sec‐butyl isothiocyanate (4.9–9.4%), (E)‐hex‐2‐enal (3.5–8.6%), (Z)‐hex‐2‐en‐1‐ol (0.3–8.4%), octanoic (0.5–8.6%) and dodecanoic acid (0–5.0%), 2‐methylbutanenitrile (0–4.6%), dibutyl phthalate (0–4.5%), and ethyl linolenate (0–3.6%). The main volatile O‐aglycones were: 2‐phenylethyl alcohol (21.5%), 6,7‐dehydro‐7,8‐dihydro‐3‐oxo‐α‐ionol (9.3%), eugenol (8.3%), benzyl alcohol (7.0%), ethyl vanillate (5.2%), 6‐(tert‐butyl)‐5‐methylphenol (5.1%), vanillin acetone (4.7%), ethyl 4‐hydroxybenzoate (4.3%), and 2‐hydroxy‐β‐ionone (3.8%). All hydrodistillates exhibited great potential of antibacterial activity against five Gram‐positive bacteria, nine ampicillin‐resistant Gram‐negative bacteria, and four fungi at a concentration of 500 μg/ml using the disc diffusion method.

Chemical Composition and Antimicrobial Activity of Volatiles from Degenia velebitica, a European Stenoendemic Plant of the Brassicaceae Family

Free and glucosidic bound leaf volatiles of Degenia velebitica were isolated and fractionated simultaneously into H2O‐soluble, H2O‐insoluble, and highly volatile compounds by hydrodistillation–adsorption (HDA) and analyzed by GC/MS. Among the 24 constituents identified, the main compounds obtained by the HDA method were S‐ and/or N‐atom containing compounds, i.e., 6‐(methylsulfanyl)hexanenitrile (10; 26.78%), dimethyl trisulfide (6; 26.35%), 3,4,5‐trimethylpyrazole (17; 13.33%), hex‐5‐enenitrile (2; 10.11%), dimethyl tetrasulfide (8; 4.93%), and pent‐4‐enyl isothiocyanate (7; 4.45%). In addition, O‐glycosidically bound volatiles and free volatiles were isolated by solvent extraction. Sixteen volatile O‐aglycones and twelve free volatile components were identified. The main O‐aglycones were eugenol (19; 24.15%), 2‐methoxy‐4‐vinylphenol (11; 11.50%), and benzyl alcohol (20; 9.49%), and the main free volatiles were (9Z,12Z)‐octa‐9,12‐dienic acid (38.35%), hexadecanoic acid (22.64%), and phytol (5.80%). The H2O‐soluble volatile fraction obtained by HDA, containing mostly glucosinolate degradation products and 3,4,5‐trimethylpyrazole (17), was evaluated for antimicrobial activity by determining inhibition zones with the diffusion method as well as minimal inhibitory concentrations (MIC) and minimal microbicidal concentrations (MMC) with the micro‐dilution method. The fraction expressed activity against the tested Gram‐positive and Gram‐negative bacteria as well as against yeast, with MIC values equal to or lower than 16.7 μg/ml.

Glucosinolate Profiling and Antimicrobial Screening of Aurinia leucadea (Brassicaceae)

Glucosinolates (GLs) were characterized in various aerial parts (stems, leaves, and flowers) of Aurinia leucadea (Guss.) C. Koch and quantified according to the ISO 9167‐1 official method based on the HPLC analysis of desulfoglucosinolates. Eight GLs, i.e., glucoraphanin (GRA), glucoalyssin (GAL; 1), gluconapin (GNA; 2), glucocochlearin (GCC), glucobrassicanapin (GBN; 3), glucotropaeolin (GTL), glucoerucin (GER), and glucoberteroin (GBE) were identified. The total GL contents were 57.1, 37.8, and 81.3 μmol/g dry weight in the stems, leaves, and flowers, respectively. The major GL detected in all parts of the plant was 2, followed by 1 and 3. GC/MS Analysis of the volatile fractions extracted from the aerial parts of fresh plant material either by hydrodistillation or CH2Cl2 extraction showed that these fractions mostly contained isothiocyanates (ITCs). The main ITCs were but‐3‐enyl‐ (55.6–71.8%), pent‐4‐enyl‐ (7.6–15.3%), and 5‐(methylsulfinyl)pentyl ITC (0–9.5%), originating from the corresponding GLs 2, 3, and 1, respectively. The antimicrobial activity of the volatile samples was investigated by determining inhibition zones with the disk‐diffusion method and minimal inhibitory concentrations (MIC) with the microdilution method. They were found to inhibit a wide range of bacteria and fungi, with MIC values of 2.0–32.0 μg/ml, indicating their promising antimicrobial potential, especially against the fungi Candida albicans and Rhizopus stolonifer as well as against the clinically important pathogen Pseudomonas aeruginosa.

Glucosinolate Distribution in Aerial Parts of Degenia velebitica

The glucosinolates present in the leaf, stem, and seed extracts of Degenia velebitica (Degen) Hayek were characterized and quantified according to the ISO 9167–1 method, which is based on the HPLC analysis of desulfoglucosinolates. The stems contained glucoalyssin (3a) as the major compound as well as glucoberteroin (1a) and glucoaubrietin (4a). The leaves contained three glucosinolates, the major one being 3a, followed by glucobrassicanapin (2a) and 1a. Glucoberteroin (1a) was the major glucosinolate in the seeds, along with the two minor glucosinolates 3a and glucoerucin (5a). The content of 1a in the whole, non‐defatted seeds amounted to 4% (w/w). The compound was characterized as its desulfo counterpart by spectroscopic techniques.

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.

Abstract 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 (Sušac 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 (Palagruža 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.

Isothiocyanates: cholinesterase inhibiting, antioxidant, and anti-inflammatory activity

Abstract Finding a new type of cholinesterase inhibitor that would overcome the brain availability and pharmacokinetic parameters or hepatotoxic liability has been a focus of investigations dealing with the treatment of Alzheimer’s disease. Isothiocyanates have not been previously investigated as potential cholinesterase inhibitors. These compounds can be naturally produced from their glucosinolate precursors, secondary metabolites widely distributed in our daily Brassica vegetables. Among 11 tested compounds, phenyl isothiocyanate and its derivatives showed the most promising inhibitory activity. 2-Methoxyphenyl ITC showed best inhibition on acetylcholinesterase with IC50 of 0.57 mM, while 3-methoxyphenyl ITC showed the best inhibition on butyrylcholinesterase having 49.2% at 1.14 mM. Assessment of the antioxidant efficacy using different methods led to a similar conclusion. The anti-inflammatory activity was also tested using human COX-2 enzyme, ranking phenyl isothiocyanate, and 3-methoxyphenyl isothiocyanate as most active, with ∼99% inhibition at 50 μM.