New in vitro findings about halogenated boroxine cytotoxicity and deregulation of cell death-related genes in GR-M melanoma cells.

Anti-proliferative effects of halogenated boroxine - K2(B3O3F4OH) (HB) - have been confirmed in multiple cancer cell lines, including melanoma, but the exact mechanism of action is still unknown. This study aimed to determine its cytotoxic effects on human Caucasian melanoma (GR-M) cell growth in vitro as well as on the expression of cell death-related genes BCL-2, BECN1, DRAM1, and SQSTM1. GR-M and peripheral blood mononuclear (PBM) cells were treated with different HB concentrations and their growth inhibition and relative gene expression profiles were determined using the Alamar blue assay and real-time PCR. HB significantly inhibited cell growth of both GR-M and PBM cells but was even more effective in GR-M melanoma cells, as significant inhibition occurred at a lower HB concentration of 0.2 mg/mL. GR-M BCL-2 expression was significantly downregulated (P=0.001) at HB concentration of 0.4 mg/mL, which suggests that HB is a potent tumour growth inhibitor. At the same time, it upregulated BCL-2 expression in normal (PBM) cells, probably by activating protective mechanisms against induced cytotoxicity. In addition, all but the lowest HB concentrations significantly upregulated SQSTM1 (P=0.001) in GR-M cells. Upregulated BECN1 expression suggests early activation of autophagy at the lowest HB concentration in SQSTM1 cells and at all HB concentrations in PBM cells. Our findings clearly show HB-associated cell death and, along with previous cytotoxicity studies, reveal its promising anti-tumour potential.

Effects of Different Methods of Isolation on Volatile Composition of Artemisia annua L.

In order to determine influence of extraction method on volatile oil composition of Artemisia annua L., steam distillation, hydrodistillation, organic solvent extraction, and headspace sampling have been applied. The relative abundance of volatile compounds from the odorous aerial parts of A. annua, obtained by different extraction techniques, was analyzed by GC-MS. Exactly fifty constituents were identified. The leaf and flower essential oil yield ranged from 0.9 to 2.3% (v/w). Oxygenated monoterpenes were predominant in all samples ranged from 42.6% for steam-distilled fraction of petroleum ether extract to 70.6% for headspace of plant material. Essential oils isolated by steam distillation and hydrodistillation indicate that A. annua belongs to artemisia ketone chemotype with its relative content of 30.2% and 28.3%, respectively. The principal constituent in headspace sample of plant material was also artemisia ketone (46.4%), while headspace of petroleum ether extract had camphene (25.6%) as the major compound. The results prove the combined approaches to be powerful for the analysis of complex herbal samples.

Acetylcholinesterase Inhibition and Antioxidant Activity of N-trans-Caffeoyldopamine and N-trans-Feruloyldopamine.

Phenolic acids and their derivatives found in nature are well-known for their potential biological activity. In this study, two amides derived from trans-caffeic/ferulic acid and dopamine were synthesized and characterized by Fourier-transform infrared spectroscopy (FTIR), mass spectrometry, proton and carbon-13 nuclear magnetic resonance spectroscopy. The compounds were tested for the inhibition of acetylcholinesterase (AChE) from Electrophorus electricus and for antioxidant activity by scavenging 2,2-diphenyl-1-pycrylhydrazyl free radical (DPPH•) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS•+), reducing ferric ions, and ferrous ions chelation. N-trans-Feruloyldopamine displayed the highest inhibitory effect on AChE with half-maximal inhibitory concentration (IC50) values of 8.52 μM. In addition, an in silico study was done to determine the most favorable AChE cluster with the synthesized compounds. Further, these clusters were investigated for binding positions at the lowest free binding energy. Both synthesized hydroxycinnamates were found to be better antioxidants than the parent acids in in vitro tests applied. N-trans-Caffeoyldopamine showed the best antioxidant activity in the three tested methods—against non-biological stable free radicals IC50 5.95 μM for DPPH•, 0.24 μM for the ABTS•+ method, and for reducing power (ascorbic acid equivalent (AAE) 822.45 μmol/mmol)—while for chelation activity against Fe2+ ions N-trans-feruloyldopamine had slightly better antioxidant activity (IC50 3.17 mM).

HPLC-ED Analysis of Phenolic Compounds in Three Bosnian Crataegus Species.

The aim of this work was the qualitative and quantitative determination of selected phenolic compounds in three Crataegus species grown in Bosnia. Crataegus plants are consumed for medicinal purposes and as foodstuff in the form of canned fruit, jam, jelly, tea, and wine. Two samples of plant material, dry leaves with flowers, and berries of three Crataegus species—Crataegus rhipidophylla Gand., Crataegus x subsphaericea Gand., and Crataegus x macrocarpa Hegetschw.—were analyzed. Twelve ethanolic extracts were isolated from the selected plant material using Soxhlet and ultrasound extraction, respectively. Soxhlet extraction proved to be more effective than ultrasound extraction. A simple and sensitive method, high-performance liquid chromatography with electrochemical detection, HPLC-ED, was used for the simultaneous determination of phenolic acids and flavonoids in Crataegus species. The content of gallic acid in the extracts ranged from 0.001 to 0.082 mg/g dry weight (DW), chlorogenic acid from 0.19 to 8.70 mg/g DW, and rutin from 0.03 to 13.49 mg/g DW. Two flavonoids, vitexin and hyperoside, commonly found in chemotaxonomic investigations of Crataegus species, were not detected in the examined extracts. In general, leaves with flowers samples are richer in gallic acid and rutin, whereas the berries samples are richer in chlorogenic acid. Distinct similarities were found in the relative distribution of gallic acid among the three species. Extracts of C. x macrocarpa had the highest content of all detected compounds, while significant differences were found in rutin content, depending on the plant organ. To the best of our knowledge, this is the first study reporting content of phenolic compounds in Crataegus rhipidophylla Gand., Crataegus x subsphaericea, and Crataegusxmacrocarpa from Bosnia.

Essential Oil Profile, Phenolic Content and Antioxidant Activity of Geranium kikianum.

This work presents the first phytochemical investigation of Geranium kikianum Kit Tan & G. Vold (Geraniaceae), a species endemic to the southern Peloponnese, Greece. The essential oil from aerial parts of the plant was isolated by hydrodistillation and its chemical composition characterized by GC-MS. A total of 26 compounds were identified, constituting 98.3% of the oil composition. Oxygenated sesquiterpenes were the main group of constituents (75.4%). The major component of the oil was the sesquiterpene ketone germacrone (45.6%). Eleven alcohols represented the most diverse chemical class in the volatile oil (36.3%), and phytol was the most abundant acyclic diterpene alcohol (11.4%). The volatile compounds and residual water remaining after hydrodistillation were screened for their radical-scavenging activity by the DPPH method. IC50 values ranged from 0.2±0.03 mg/mL for the undiluted decoction to 69.7±0.5 mg/mL for the essential oil. The significant antioxidant activity of the residual water, comparable with that of the widespread synthetic antioxidant BHT. (0.2±0.01 mg/mL), and almost tenfold higher than thymol (1.90 t 0.04 mg/mL), correlates with a high content of total phenolic compounds (100.2±1.7 mg GAE/g), and flavonoids (11.9±1.2 mg GAE/g).

Impact of calcium ion on cytotoxic effect of the boroxine derivative, K2[B3O3F4OH].

The effect of Ca2+ ions on the cytotoxic ability of boron heterocyclic compound dipotassium-trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]), on in vitro tumor cells (mammary adenocarcinoma 4T1, melanoma B16F10 and squamous cell carcinoma SCCVII) and non-tumoral fibroblast cells (mouse dermal L929 and hamster lung V79) was examined. At small concentrations of Ca2+ ions (0.42 mM), K2[B3O3F4OH] (3.85 mM) has a very strong cytotoxic effect on all cancer cells tested (89.1, 85.6 and 84.6%) and significantly less effect on normal cells (19.5 and 24.2%), respectively. Applying larger concentrations of Ca2+ ions (9.42-72.42 mM), at the same concentration of K2[B3O3F4OH], no significant cytotoxic effect was detected on cancer cells and normal cells investigated. The selective ability of K2[B3O3F4OH], in the medium with a low concentration of Ca2+ ions has a strong cytotoxic effect on cancer cells and very weak effect in normal cells, opens up the possibility of its application in antitumor therapy.

Volatiles of Helichrysum italicum (Roth) G. Don from Croatia.

Helichrysum italicum (Roth) G. Don is a flowering plant of the family Asteraceae. It is rich in oil that is used for different medicinal purposes and in fragrance industry. Volatile profile of four populations of H. italicum, collected from natural habitat in Dalmatia (Croatia), was analysed by capillary GC-MS. Sample from BraC Island had α-trans-bergamotene (10.2%) and ß-acoradiene (10.1%) as the majors, whereas sample collected on Biokovo Mt. was rich in neryl acetate (8.1%). ß-Acoradiene was also the main constituent of sample collected near Tijarica, whereas rosifoliol (8.5%) was the most abundant constituent in sample collected near Makarska. Presented results show the influence of environmental conditions on chemical differentiation of the volatiles of H. italicum from Croatia.

Total phenolic and flavonoid contents, antioxidant and antimicrobial activities of Alnus glutinosa (L.) Gaertn., Alnus incana (L.) Moench and Alnus viridis (Chaix) DC. extracts.

The objective of this study was to determine total phenolic and flavonoid contents, antioxidant and antimicrobial activities of methanolic extracts from the leaves and barks of three Alnus species. The phenolic and flavonoid contents of extracts were determined spectrophotometrically using Folin-Ciocalteau and aluminium chloride methods, respectively. In addition, antioxidant activity of the extracts was determined using 1,1-diphenyl-2-picrylhydrazyl radical scavenging method. The antimicrobial activity was performed by disc diffusion assay against six reference bacterial strains including Gram-negative and Gram-positive bacteria and two fungal strains. Extract of Alnus viridis bark contained the highest amounts of total phenolics (780 mg CAT/g), while extract of A. viridis leaves had the highest amount of flavonoids (30.01 mg RUT/g). All extracts showed antioxidant activity higher than thymol, which was used as a positive probe. The largest diameters of inhibition zone (25 mm) were recorded with Bacillus subtilis 168 M and Staphylococcus aureus ATCC 6538.

Cytotoxic and genotoxic activity of some Helleborus species.

Despite their known toxic properties, various Helleborus species are used as medicaments in folk medicine to treat some diseases and health conditions. As the main mechanism of many cytostatic drugs is based on their cytotoxic activity, there is potential for the toxicity of hellebore to be used in anticancer therapy. This study tested the geno- and cytotoxic effects of extracts of three hellebore taxa (Helleborus odorus, Helleborus multifidus and Helleborus hercegovinus) on meristemic onion (Alliumcepa L.) cells and human lymphocytes. Treatments with Helleborus extracts induced cytotoxic and cytostatic effects in meristemic onion cells as well as in cultivated cytokinesis-blocked human lymphocytes. Cytokinesis-block micronucleus cytome assay indicated that treatments with hellebore extracts induce genotoxic effects in human lymphocytes, and that the significant mechanism of their antiproliferative activity is apoptosis induction.

Volatile constituents, phenolic compounds, and antioxidant activity of Calamintha glandulosa (Req.) Bentham.

BACKGROUND: Calamintha glandulosa (Req.) Bentham is an aromatic perennial plant belonging to the family Lamiaceae, mostly found on rocky pastures, dry meadows, and abandoned places of the Mediterranean area. Plants belonging to this genus are known as highly aromatic and to possess significant antimicrobial and antifungal properties. The aim of this study was to provide clear picture of the volatiles of this plant species, and, for the first time, to present C. glandulosa from Croatia in terms of its antioxidant activity. RESULTS: The essential oil and headspace obtained from odorous parts of C. glandulosa were subjected to capillary gas chromatography-mass spectrometry analysis. More than 50 volatile compounds were identified in six samples obtained using different extraction techniques. The most abundant components in all the samples examined were oxygenated monoterpenes, with piperitone (19.9-59.5%) and piperitenone (7.1-42.6%) as the main representatives. The total phenolic content of extracts obtained by successive Soxhlet extraction was measured, and the scavenging potency of the samples, indicated as IC50 values, were examined using four different spectrophotometric and spectrofluorimetric methods. In all cases the essential oil showed the lowest antioxidant activity, while the aqueous extract showed the highest. This can be explained by the levels of the phenolic compounds in the samples examined. CONCLUSIONS: A clear picture of aroma profile of C. glandulosa is presented, and the results obtained differ from those published previously. The high antioxidant potential of C. glandulosa from Croatia was established for the first time. Results from the present study suggest further analysis on this plant species in order to define its medicinal properties.

Antioxidative and antitumor properties of in vitro-cultivated broccoli (Brassica oleracea var. italica).

CONTEXT: Broccoli [Brassica oleracea L. var. italica Plenck. (Brassicaceae)] contains substantial quantities of bioactive compounds, which are good free radical scavengers and thus might have strong antitumor properties. Enhancing production of plant secondary metabolites could be obtained with phytohormones that have significant effects on the metabolism of secondary metabolites. In that manner, in vitro culture presents good model for manipulation with plant tissues in order to affect secondary metabolite production and thus enhance bioactive properties of plants. OBJECTIVE: Estimation of the antioxidative and antitumor properties of broccoli cultivated in different in vitro conditions. MATERIALS AND METHODS: In vitro germinated and cultivated broccoli seedlings, as well as spontaneously developed calli, were subjected to Soxhlet extraction. Antioxidative activity of the herbal extracts was determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH(•)) radical method. Antitumor properties of the extracts were determined using crown-gall tumor inhibition (potato disc) assay. RESULTS: Three, 10, 20, and 30 days old broccoli seedlings, cultivated in vitro on three different Murashige-Skoog media, two types of callus, and seedlings from sterile filter paper were used for extraction. In total, 15 aqueous extracts were tested for antioxidative and antitumor potential. Three day-old seedlings showed the highest antioxidative activity. Eleven out of 15 aqueous extracts demonstrated above 50% of crown-gall tumor inhibition in comparison with the control. Tumor inhibition was in association with types and concentrations of phytohormones presented in growing media. DISCUSSION AND CONCLUSIONS: It is demonstrated that phytohormones in plant-growing media could affect the bioactive properties of broccoli either through increasing or decreasing their antioxidative and antitumor potential.

Antioxidant and antiproliferative activities of Helleborus odorus Waldst. & Kit, H. multifidus Vis. and H. hercegovinus Martinis.

This study was undertaken in order to evaluate possible antioxidative and antiproliferative activities of three Helleborus taxa. The dry leaves and roots of three Helleborus taxa were extracted with ethanol and water. A phytochemical evaluation of the selected extracts was performed using spectrophotometric methods and a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity assay was used for measuring the antioxidative activity of extracts. The antiproliferative activity of the three Helleborus taxa was studied using Burkitt's lymphoma B cells (BJAB) cell lines. The phytochemical evaluation showed that the leaves contain high levels of total phenolic and flavonoid content. Results from the DPPH assay indicated that the activity of the ethanol and water extracts of the leaves was higher than that of positive control (thymol). Extracts from the roots of H. odorus also displayed higher antioxidant activity than the positive probe, while H. mulifidus and H. hercegovinus root extracts were less effective. A statistically significant correlation between total phenolic content and antioxidative properties indicates that these compounds contribute to the antioxidant activity. The highest percentage of cell growth inhibition was observed when testing the water root extracts of H. multifidus (50.14%) and H. hercegovinus (49.04%). In contrast, the water leaf extract of H. hercegovinus exhibited the lowest inhibition of cell growth (8.59%), although it showed strong antioxidant activity.

Effects of dipotassium trioxohydroxytetrafluorotriborate (K2[BO3F4OH]) on genetic material and inhibition of cell division in human cell cultures.

We have examined antiproliferative, cytotoxic, and genotoxic potential of a halogenated boroxine dipotassium trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]). The impact on cell growth was evaluated by alamarBlue assay in basal cell carcinoma culture. Cytostatic, cytotoxic, and genotoxic potential were evaluated in lymphocytes culture, applying cytokinesis-block micronucleus cytome assay and chromosome aberrations analysis. Tested concentrations (0.05, 0.1, 0.2, and 0.4 mg/mL) were correlated with inhibition of cell growth in basal cell carcinoma culture and with the lymphocytes proliferation. Clastogenic activity has been confirmed, without evidences of aneugenic activity, in human lymphocytes.

Volatile constituents of two rare subspecies of Thymus praecox.

Hydrodistilled essential oil and the corresponding headspace volatiles of Bosnian wild growing Thymus praecox ssp. polytrichus (A. Kern. Ex Borbàs) Jalas and Thymus praecox ssp. skorpilii (Velen.) Jalas were subjected to capillary GC-MS analysis. This work presents a detailed essential oil analysis of these two rare Thymus subspecies from Bosnia, as well as the very first report on their headspace composition. Eighty-seven volatile constituents were identified in four samples. Two alcohols were the major constituents in the essential oil of T. praecox ssp. polytrichus, a monoterpene, linalool (13.9%), and a sesquiterpene, (E)-nerolidol (10.4%), while linalyl acetate (36.7%) and linalool (22.7%) were the most abundant volatiles in the corresponding headspace sample. Oxygenated monoterpenes (57.5%) predominate in the essential oil of T. praecox ssp. skorpilii with linalyl acetate (28.7%) and linalool (14.4%) as the main representatives. Headspace sample of this subspecies also showed richness in linalyl acetate (52.4%), while the second most abundant compound was alpha-pinene (14.5%), a monoterpene hydrocarbon.

Chemical composition of the essential oil of Stachys menthifolia Vis.

Stachys menthifolia Vis. (Lamiaceae) is an endemic species from the Balkan Peninsula spread throughout Albania, Greece, Montenegro, and Croatia. This article presents the first investigation of the essential oil composition of this species from Croatia. Aerial parts of the plant were collected from three different natural habitats in the region of Biokovo Mountain. The studied populations showed similarity in qualitative, but not in quantitative, composition of their essential oils. Hydrodistilled volatile oil obtained from the plant material of S. menthifolia was subjected to gas chromatographic analysis coupled to mass spectrometry. More than 100 compounds were identified in the three samples, representing 86.8-90.8% of the total oil. The terpene profile of S. menthifolia is characterized by a high content of oxygenated sesquiterpenes (48.4-58.9%) and diterpene hydrocarbons (3.5-25.2%), with 8-alpha-acetoxyelemol (6.9-21.3%), abietatriene (3.5-21.1%), and 4'-methoxyacetophenone (4.5-17.0%) as the main constituents.

Influence of the continental climatic conditions on the essential-oil composition of Salvia brachyodon Vandas transferred from Adriatic Coast.

Essential-oil profile of Salvia brachyodon Vandas, an endemic Dinaric species transferred from Adriatic Coast to the continental climatic conditions, was determined. Hydrodistilled oils obtained from the plant material collected in three-year field trial were subjected to the detailed GC/MS analysis. Hundred and fifty volatile compounds were identified in four samples. Comparison of the chemical composition of the isolated essential oils showed that population collected one year after transfer preserved sesquiterpene character of its oil (74.3%), while all subsequent samples gave the oils of monoterpene type with 1,8-cineole as the principal constituent (22.2-42.3%). The high degree of variation in the qualitative and quantitative composition of the volatile constituents revealed the strong influence of environmental conditions on the nature of plant chemical composition that has an important role in a plant adaptation.

The essential oil of Thymus aureopunctatus (Beck) K. Malý.

Essential oil of Thymus aureopunctatus (Beck) K. Malý, obtained by hydrodistillation and headspace technique, was subjected to capillary GC-MS analysis, and its volatile composition was compared with essential oil profile of Thymus serpyllum L. and a botanically undetermined thyme species, Thymus spp., all growing wild in the same habitat in Bosnia and Herzegovina. This paper presents the first report on phytochemical analysis of the rare T. aureopunctatus species, and also the first report on headspace analysis of plants belonging to the genus Thymus. One hundred and two volatile constituents were identified. The investigated Thymus species of Bosnian origin clearly belong to the thymol-chemotype with relatively abundant level of this main constituent varying from 30.5% for T. serpyllum, and 34.5% for T. aureopunctatus, to 44.2% for Thymus spp., while their corresponding headspace samples contain very high percentage of p-cymene, thymol biosynthesis precursor, in the range from 29.1% to 68.1%.

In vitro acetylcholinesterase inhibitory properties of thymol, carvacrol and their derivatives thymoquinone and thymohydroquinone.

The aim of this study was to examine in vitro the inhibitory activity exerted by the main constituents of essential oil obtained from the aromatic plant Thymus vulgaris L. on acetylcholinesterase (AChE). The total essential oil and selected compounds, specifically linalool and thymol, carvacrol and their derivatives thymoquinone and thymohydroquinone, were tested for AChE inhibition. Thymohydroquinone exhibited the strongest AChE inhibitory effect over the range of concentrations. The AChE inhibitory potential decreased in the following order: thymohydroquinone > carvacrol > thymoquinone > essential oil > thymol > linalool. It is interesting that the AChE inhibitory effect exerted by carvacrol was 10 times stronger than that exerted by its isomer thymol, although thymol and carvacrol have a very similar structure.