Milka Jadranin

Isolation and Biological Evaluation of Jatrophane Diterpenoids from Euphorbia dendroides

From the Montenegrin spurge Euphorbia dendroides, seven new diterpenoids [jatrophanes (1-6) and a tigliane (7)] were isolated and their structures elucidated by spectroscopic techniques. The biological activity of the new compounds was studied against four human cancer cell lines. The most effective jatrophane-type compound (2) and its structurally closely related derivative (1) were evaluated for their interactions with paclitaxel and doxorubicin using a multi-drug-resistant cancer cell line. Both compounds exerted a strong reversal potential resulting from inhibition of P-glycoprotein transport.

Study of forced degradation behavior of Eletriptan hydrobromide by LC and LC–MS and development of stability-indicating method

The objective of the present study was to report the stability profile of novel antimigrain drug Eletriptan hydrobromide based on the information obtained from forced degradation studies. The drug was subjected to acid (0.1-1 mol L(-1) HCl), neutral and base (0.1-1 mol L(-1) NaOH) hydrolysis and to oxidative decomposition (3-15% (v/v) H(2)O(2)). Photolysis and thermo degradation at 75 degrees C were carried out in methanol solution and in solid state with both Eletriptan hydrobromide bulk drug and the tablet formulation. The products formed under different stress conditions were investigated by LC and LC-MS. The experimental conditions for LC were chosen by employing experimental design and multicriteria decision making methodology. These powerful tools enabled the accomplishment of satisfactory resolution with the shortest possible analysis time. Analytes were separated on a C(18) column (XTerra, 150 mm x 3.9 mm, 5 microm) with the mobile phase composed of methanol-water solution of TEA (pH 6.52, 1%, v/v) (30:70, v/v) pumped at 1 mL min(-1) flow rate. The column temperature was set at 50 degrees C and the detection at 225 nm using DAD detector. The LC method was suitably modified for LC-MS analysis which was further used to characterize the arisen degradation products. The possible degradation pathway was outlined based on the results. The drug appeared to be instable towards every stress condition but oxidation. The stability was not jeopardized even under more exaggerated conditions such as increased temperature of the solutions to 75 degrees C, increased strength of acid/alkali solutions and prolonged testing period. Validation of the LC-DAD method was carried out in accordance with ICH guideline. The method met all required criteria and was applied when testing the commercially available tablets.

Effect of four lichen acids isolated from Hypogymnia physodes on viability of rat thymocytes.

Four lichen acids, physodalic acid (F1), physodic acid (F2), 3-hydroxyphysodic acid (F3), and isophysodic acid (F4), were isolated from Hypogymnia physodes methanol extract using preparative reversed-phase high performance liquid chromatography and their structures were determined by UV, MS, (1)H NMR and (13)C NMR. This is the first report on the isolation of F4 from H. physodes. Isolated rat thymocytes were cultivated with increasing F1-F4 concentrations (0.1, 1, 10μg/well) and proliferative activity, viability, ROS (reactive oxygen species) production and MMP (mitochondrial membrane potential) disturbances were evaluated. Obtained results show significantly decreased thymocytes proliferation was observed when cells were treated with F1 (1μg, p<0.05; 10μg; p<0.001), F2 (10μg, p<0.05) and F3 compound (10μg, p<0.05). Significantly increased cytotoxicity was detected when cells were incubated with F1 (1μg, p<0.05; 10μg, p<0.01), F2 (10μg, p<0.05) and F3 compound (10μg, p<0.001). Increased H2DCF-DA fluorescence intensity, when cells were treated with F1 (1μg, p<0.001; 1μg, p<0.01; 10μg, p<0.001) and F2 (1μg, p<0.05; 10μg, p<0.01) compound, indicating the increase of intracellular ROS production. Simultaneously, increased ROS levels were followed with significantly decreased MMP when thymocytes were cultivated with F1 (0.1μg, p<0.001; 1μg, p<0.001; 10μg, p<0.001) and F2 compound (10μg, p<0.001). Thymocytes exposure to increased (0.1, 1, 10μg) concentrations of F3 and F4 compounds did not result with significant alterations in MMP and intracellular ROS production. We have shown that higher F1 and F2 concentrations induce thymocytes toxicity mainly through induction of oxidative stress, while cytotoxicity effect of F3 is followed with altered antioxidant/oxidant balance. The rigid 11H-dibenzo[b,e][1,4]dioxepin-11-one ring in the depsidone structure may play a important role for the examined biological activities.

Chemical composition, cytotoxic and antioxidative activities of ethanolic extracts of propolis on HCT-116 cell line.

BACKGROUND Propolis is a complex resinous sticky substance that honeybees collect from buds and exudates of various plants. Owing to its versatile biological and pharmacological activities, propolis is widely used in medicines, cosmetics and foods. The aim of this study was to evaluate the cytotoxic and antioxidative effects of various ethanolic extracts of propolis (EEPs) on human colon cancer cell line HCT-116 and compare them with their composition determined by HPLC-DAD. RESULTS The most abundant flavonoids in all samples were chrysin, pinocembrin and galangin (12.697-40.811 µg mg⁻¹), while the main phenolic acids were caffeic acid, ferulic acid and isoferulic acid. Dose- and time-dependent inhibition of growth of HCT-116 cells was observed for all propolis samples, with IC₅₀ values ranging from 26.33 to 143.09 µg mL⁻¹. Differences in cytotoxic activity of propolis samples were associated with differences in their composition. All EEP samples reduced both superoxide anion radical and nitrite levels and also had strong DPPH-scavenging activity. CONCLUSION All tested propolis samples had pronounced cytotoxic and antioxidative activities.

New anti-cancer characteristics of jatrophane diterpenes from Euphorbia dendroides.

Jatrophane diterpenes were shown to be inhibitors of P-glycoprotein (P-gp). There are also evidences on their microtubule-interacting activity in cancer cells. We evaluated new anti-cancer characteristics of two jatrophane type compounds from Euphorbia dendroides. For that purpose, the model system of sensitive non-small cell lung cancer cell line (NCI-H460) and its resistant counterpart (NCI-H460/R) was used. Although both jatrophanes showed inhibitory effect on cancer cell growth, they were non-toxic for peripheral blood mononuclear cells (PBMC). We examined their effects in combination with paclitaxel (PTX), a well-known mitotic spindle interacting chemotherapeutic. Jatrophanes overcome PTX resistance in concentration-dependent manner in MDR cancer cell line (NCI-H460/R). We observed that this synergistic effect is not caused merely by P-gp inhibition. In combination with PTX, jatrophanes induce cell killing and change cell cycle distribution leading to G2/M arrest. Furthermore, they exert an anti-angiogenic effect by decreasing the vascular endothelial growth factor (VEGF) secretion. The reduction of the level of mdr1 mRNA expression in sensitive cells, suggests that these compounds could not contribute to the development of resistance. In conclusion, present study provides a rational basis for the new cancer treatment approach with jatrophanes that are non-toxic to normal cells and have new favorable anti-cancer characteristics.

Defensive Secretions in Three Species of Polydesmids (Diplopoda, Polydesmida, Polydesmidae)

Nine compounds were detected in three different millipede species: Polydesmus complanatus (L.), Brachydesmus (Stylobrachydesmus) avalae Ćurčić & Makarov, and Brachydesmus (Stylobrachydesmus) dadayi Verhoeff. Benzaldehyde, benzyl alcohol, benzoylnitrile, benzyl methyl ketone, benzoic acid, benzyl ethyl ketone, mandelonitrile, and mandelonitrile benzoate were identified by GC-FID and GC-MS analyses. Hydrogen cyanide was detected qualitatively by the picric acid test. Benzyl ethyl ketone, benzyl methyl ketone, and benzyl alcohol were detected for the first time in polydesmidan millipedes. Benzoylnitrile was the major component in all three hexane extracts. These compounds are suspected to be active in the defensive secretions of these millipede species.

In vitro antitumor actions of extracts from endemic plant Helichrysum zivojinii

BackgroundThe aim of this research was to determine the intensity and mechanisms of the cytotoxic actions of five extracts isolated from the endemic plant species Helichrysum zivojinii Černjavski & Soška (family Asteraceae) against specific cancer cell lines. In order to evaluate the sensitivity of normal immunocompetent cells implicated in the antitumor immune response, the cytotoxicity of extracts was also tested against healthy peripheral blood mononuclear cells (PBMC).MethodsThe aerial parts of the plants were air-dried, powdered, and successively extracted with solvents of increasing polarity to obtain hexane, dichloromethane, ethyl-acetate, n-butanol and methanol extracts. The cytotoxic activities of the extracts against human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562, human breast adenocarcinoma MDA-MB-361 cells and PBMC were evaluated by the MTT test. The mode of HeLa cell death was investigated by morphological analysis. Changes in the cell cycle of HeLa cells treated with the extracts were analyzed by flow cytometry. The apoptotic mechanisms induced by the tested extracts were determined using specific caspase inhibitors.ResultsThe investigated Helichrysum zivojinii extracts exerted selective dose-dependent cytotoxic actions against selected cancer cell lines and healthy immunocompetent PBMC stimulated to proliferate, while the cytotoxic actions exerted on unstimulated PBMC were less pronounced. The tested extracts exhibited considerably stronger cytotoxic activities towards HeLa, Fem-x and K562 cells in comparison to resting and stimulated PBMC. It is worth noting that the cytotoxicity of the extracts was weaker against unstimulated PBMC in comparison to stimulated PBMC. Furthermore, each of the five extracts induced apoptosis in HeLa cells, through the activation of both intrinsic and extrinsic signaling pathways.ConclusionExtracts obtained from the endemic plant Helichrysum zivojinii may represent an important source of novel potential antitumor agents due to their pronounced and selective cytotoxic actions towards malignant cells.

Jatrophane diterpenoids from the latex of Euphorbia dendroides and their anti-P-glycoprotein activity in human multi-drug resistant cancer cell lines

Thirteen jatrophane diterpenoids (1-10, 13-15), three previously isolated (11, 12, 16) and a known tigliane (17) were isolated from the latex of Euphorbia dendroides. The structures and relative configurations of compounds were elucidated by spectroscopic techniques. The P-glycoprotein (P-gp) inhibiting activities of the representative set of jatrophanes (1-6 and 11-16) have been assessed. Jatrophanes 2 and 5 demonstrated the most powerful inhibition of P-gp, higher than R(+)-verapamil and tariquidar in colorectal multi-drug resistant (MDR) cells (DLD1-TxR).

Lipophilicity of some guaianolides isolated from two endemic subspecies of Amphoricarpos neumayeri (Asteraceae) from Montenegro.

In this study 10 guaianolide-type sesquiterpene gamma-lactones named amphoricarpolides, isolated from the aerial parts of two endemic subspecies of Amphoricarpos neumayeri (ssp. neumayeri and ssp. murbeckii Bosnjak), were investigated by means of reversed-phase thin-layer chromatography. Methanol-water and tetrahydrofuran-water binary mixtures were used as mobile phase in order to determine lipophilicity parameters R (0) (M) and C(0). Some of the investigated compounds were screened for their cytotoxic activity against HeLa and B16 cells. Chromatographically obtained lipophilicity parameters were correlated with calculated logP values and IC(50) values. Principal component analysis identified the dominant pattern in the chromatographically obtained data.

Chemodiversity of Nonacosan-10-ol and n-Alkanes in the Needle Wax of Pinus heldreichii

This is the first report of individual variability and population diversity of the contents of nonacosan‐10‐ol and n‐alkanes in the needle cuticular waxes of Bosnian pines originated from Montenegro, regarded as Pinus heldreichii var. leucodermis, and from Serbia, regarded as P. heldreichii var. pančići. The amount of nonacosan‐10‐ol varied individually from 27.4 to 73.2% (55.5% in average), but differences between the four investigated populations were not statistically confirmed. The size of the n‐alkanes ranged from C18 to C33. The most abundant n‐alkanes were C23, C27, and C25 (12.2, 11.2, and 10.8% in average, resp.). The carbon preference index (CPI) of the n‐alkanes ranged from 0.8 to 3.1 (1.6 in average), while the average chain length (ACL) ranged from 20.9 to 26.5 (24.4 in average). Long‐chain and mid‐chain n‐alkanes prevailed (49.6 and 37.9% in average, resp.). It was also found that the populations of P. heldreichii var. leucodermis had predominantly a narrower range of n‐alkanes (C18C31) than the trees of the variety pančići (C18C33). Differences between the varieties were also significant for most of the other characteristics of the n‐alkane pattern (e.g., most abundant n‐alkanes, CPI, ACL, and relative proportion of short‐, mid‐, and long‐chain n‐alkanes). The principle component and cluster analyses of eleven n‐alkanes confirmed the significant diversity of these two varieties.