Putative Mycobacterial Efflux Inhibitors from the Seeds of <i>Aframomum melegueta</i>

In order to identify new putative efflux pump inhibitors that represent an appropriate target in antimycobacterial chemotherapy, nine paradol- and gingerol-related compounds (<b>1</b>–<b>9</b>) isolated from the seeds of A<i>framomum melegueta</i> were assessed for their potential to inhibit ethidium bromide (EtBr) efflux in a <i>Mycobacterium smegmatis</i> model. Five of the compounds from <i>A. melegueta</i> and NMR spectroscopic data of the diketone 6-gingerdione (<b>2</b>) and its enolic tautomers, methyl-6-gingerol (<b>5</b>) and <i>rac</i>-6-dihydroparadol (<b>7</b>), are presented herein for the first time. After determination of their antimycobacterial activities and modulatory effects on the MIC of antibiotics as well as their synergistic effects in combination with antibiotics against <i>M. smegmatis</i> mc² 155, their impact on EtBr accumulation and efflux was evaluated using a microtiter plate-based fluorometric assay. The compounds exhibited moderate to weak antimycobacterial activities, and the best modulators induced a 4- to 16-fold decrease of the MICs of EtBr and rifampicin as well as a reduction of the MIC of isoniazid with fractional inhibitory concentration index values indicating synergistic activities in some cases. 6-Paradol (<b>3</b>), 8-gingerol (<b>6</b>), and <i>rac-</i>6-dihydroparadol (<b>7</b>) were the most potent EtBr efflux inhibitors in <i>M. smegmatis</i> mc² 155, displaying EtBr efflux inhibiting activities comparable to reference inhibitors

Metabolic profiling of rhizomes of native populations of the strictly endemic Croatian species <i>Iris adriatica</i>

<p><i>Iris adriatica</i> Trinajstić ex Mitić (Iridaceae L.) is a strictly endemic taxon from Croatia. It is a rhizomatous dwarf plant from the <i>I. pumila</i> complex with a distribution area limited to the Croatian part of the Mediterranean area, mainly central Dalmatia. The genus <i>Iris</i> is known for its richness in isoflavonoids which also play a significant role in chemotaxonomy and biological activity. Hence, in the current study, different plant batches of <i>I. adriatica</i> collected in early spring of 2016 were analysed for their phytochemical profiles and qualitatively compared. UHPLC-PDA-ESI-MS analyses of methanolic rhizome extracts were performed. Altogether, 36 compounds, representing isoflavonoids (including 6,7-methylendioxy derivatives), benzophenones and xanthones were found as aglycones or in glycosidically bound form to be the main constituent groups of <i>I. adriatica</i> rhizomes. Qualitative results were identical between different batches of plant material from collection sites in central Dalmatia, they differed only in quantity. For some phenolic compounds of <i>I. adriatica</i>, chemotaxonomic relevance was detected.</p

Antiproliferative Carvotacetones from <i>Sphaeranthus africanus</i>

Five carvotacetone derivatives, including two known ones, 3,5-diangeloyloxy-7-hydroxycarvotacetone (<b>1</b>) and 3-angeloyl­oxy-5-[2″,3″-epoxy-2″-methylbutanoyloxy]-7-hydroxycarvotacetone (<b>2</b>), along with three new compounds, 3-angeloyloxy-5-[3″-chloro-2″-hydroxy-2″-methylbutanoyloxy]-7-hydroxycarvotacetone (<b>3</b>), 5-angeloyloxy-7-hydroxy-3-tigloyloxycarvotacetone (<b>4</b>), and 3-angeloyl­oxy-5,7-dihydroxycarvotacetone (<b>5</b>), were isolated from the aerial parts of <i>Sphaeranthus africanus</i> collected in Vietnam. Bioassay-guided fractionation was monitored by the antiproliferative activity on CCRF-CEM human cancer cells. The structures of compounds <b>1</b>–<b>5</b> were determined on the basis of NMR spectroscopic and mass spectrometric data. Activities of compounds <b>1</b>–<b>5</b> were evaluated in vitro against the human cancer cell lines CCRF-CEM, MDA-MB-231, U-251, and HCT-116. All compounds exhibited significant antiproliferative activity against all four cancer cell lines. CCRF-CEM was most sensitive to the compounds, with IC₅₀ values ranging from 0.6 to 1.5 μM. Compounds <b>3</b> and <b>4</b> possessed the highest activity, with IC₅₀ values in the four cell lines ranging from 0.6 to 2.9 μM and 1.3 to 2.5 μM, respectively. These compounds also showed inhibitory activity toward the HEK-293 human embryonic kidney cells with IC₅₀ values ranging from 2.5 to 5.5 μM. This is the first time that antiproliferative activity of <i>S. africanus</i> has been reported, and <b>1</b>–<b>5</b> are the most cytotoxic carvotacetone derivatives reported so far

Naphthoquinones from <i>Onosma paniculata</i> Induce Cell-Cycle Arrest and Apoptosis in Melanoma Cells

Activity-guided fractionation of a petroleum ether-soluble extract of the roots of <i>Onosma paniculata</i>, which has been shown to affect the cell cycle and to induce apoptosis in melanoma cells, led to the isolation of several shikonin derivatives, namely, β-hydroxyisovalerylshikonin (<b>1</b>), acetylshikonin (<b>2</b>), dimethylacrylshikonin (<b>3</b>), and a mixture of α-methylbutyrylshikonin and isovalerylshikonin (<b>4</b>+<b>5</b>). All compounds exhibited strong cytotoxicity against eight cancer cell lines and MRC-5 lung fibroblasts, with <b>3</b> found to possess the most potent cytotoxicity toward four melanoma cell lines (SBcl2, WM35, WM9, and WM164). Furthermore, <b>3</b> and the mixture of <b>4</b>+<b>5</b> were found to interfere with cell-cycle progression in these cell lines and led to an increasing number of cells in the subG1 region as well as to caspase-3/7 activation, indicating apoptotic cell death

Polyyne Hybrid Compounds from <i>Notopterygium incisum</i> with Peroxisome Proliferator-Activated Receptor Gamma Agonistic Effects

In the search for peroxisome proliferator-activated receptor gamma (PPARγ) active constituents from the roots and rhizomes of <i>Notopterygium incisum</i>, 11 new polyacetylene derivatives (<b>1</b>–<b>11</b>) were isolated. Their structures were elucidated by NMR and HRESIMS as new polyyne hybrid molecules of falcarindiol with sesquiterpenoid or phenylpropanoid moieties, named notoethers A–H (<b>1</b>–<b>8</b>) and notoincisols A–C (<b>9</b>–<b>11</b>), respectively. Notoincisol B (<b>10</b>) and notoincisol C (<b>11</b>) represent two new carbon skeletons. When tested for PPARγ activation in a luciferase reporter assay with HEK-293 cells, notoethers A–C (<b>1</b>–<b>3</b>), notoincisol A (<b>9</b>), and notoincisol B (<b>10</b>) showed promising agonistic activity (EC₅₀ values of 1.7 to 2.3 μM). In addition, notoincisol A (<b>9</b>) exhibited inhibitory activity on NO production of stimulated RAW 264.7 macrophages

Proposed molecular binding mode of falcarindiol in the PPARγ LBD.

<p>Predicted binding mode of falcarindiol shown as 3D depiction in which predicted hydrogen-bonds are shown as dashed lines (<b>A</b>), and 2D depiction including chemical features of the interaction pattern derived from the docking pose (<b>B</b>). Chemical features in the 2D depiction are color-coded: green arrow – hydrogen-bond donor; magenta – hydrophobic contacts.</p

PPARγ-mediated transactivation activity as well as receptor binding activity of falcarindiol.

<p>(<b>A</b>) HEK-293 cells, transiently transfected with a human PPARγ expression plasmid, a luciferase reporter plasmid (tk-PPREx3-luc) and EGFP as internal control, were treated with different concentrations of pioglitazone or falcarindiol (0.1–30 µM) for 18 h. Luciferase activity was normalized by the EGFP-derived fluorescence, and the result is expressed as fold induction compared to the solvent vehicle control (DMSO, 0.1%). The data points shown are means ± SEM of three independent experiments each performed in quadruplet. (<b>B</b>) Cells were transfected and treated as indicated above. Pioglitazone was applied at 5 µM, falcarindiol at 10 µM, and the PPARγ antagonist T0070907 at 1 µM. The data shown are means ± SD of six independent experiments each performed in quadruplet. ***<i>p</i><0.001 (ANOVA/Bonferroni). (<b>C</b>) The cells were prepared as indicated above and treated for 18 h with different concentrations of falcarindiol, always in the presence of 1 µM pioglitazone. The data shown are means ± SD of three independent experiments each performed in quadruplet. (<b>D</b>) Dilutions of the two investigated compounds were prepared in DMSO and mixed with a buffer containing the hPPARγ LBD tagged with GST, terbium-labelled anti-GST antibody, and fluorescently-labelled PPARγ agonist. After 6 h of incubation, the ability of the test compounds to bind to the PPARγ LBD and thus to displace the fluorescently labelled ligand was estimated from the decrease of the emission ratio 520 nm/495 nm upon excitation at 340 nm. Each data point represents the mean ± SEM from four independent experiments performed in duplicate.</p

Assessment of adipogenicity and glucose uptake-enhancing properties of falcarindiol.

<p>(<b>A</b>) 3T3-L1 preadipocytes were differentiated to adipocytes as described in the <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061755#s2" target="_blank">Materials and Methods</a></i> section for 10 days in the presence of pioglitazone, falcarindiol, solvent vehicle (0.1% DMSO), or a standard differentiation mixture (MDI; containing 10% FBS, 500 µM IBMX, 500 nM dexamethasone, and 1 µg/ml insulin) as a positive control. For an estimate of accumulated lipids, and thus for the adipogenic potential of the test compounds, Oil Red O staining and photometric quantification at 550 nm was performed. The data shown are means ± SD of three independent experiments. ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05 (ANOVA/Bonferroni). (<b>B</b>) 3T3-L1 mature adipocytes were incubated with falcarindiol (10 µM) or solvent vehicle (0.1% DMSO) for 48 h and 2-deoxy-D-(1H³)-glucose cellular uptake was determined for 10 min in the presence or absence (basal uptake) of insulin (500 pM). The data shown are means ± SEM of five independent experiments. *<i>p</i><0.05 (compared to the solvent vehicle group; two-tailed paired <i>t</i>-test).</p

Activity of the isolated polyacetylenes towards the three subtypes of human PPAR (α, β/δ, γ) in a luciferase reporter transactivation assay.

<p>n.d. not detected up to 30 µM.</p><p>HEK-293 cells transiently co-transfected with the respective PPAR subtype expression plasmids, a luciferase reporter plasmid (tk-PPREx3-luc), and EGFP as internal control were treated with six different concentrations of the polyacetylenes (0.1–30 µM) for 18 h. The luciferase activity was expressed as fold induction above the vehicle control (DMSO, 0.1%) after normalization to the EGFP-derived fluorescence. To verify the specificity of the performed assays, GW7647, GW0742, and pioglitazone were used as a selective agonist for PPARα, PPARβ/δ, and PPARγ, respectively. EC50 and maximal fold activation were determined with a non-linear regression in the GraphPad Prism software version 4.03 (GraphPad Software Inc, USA). The data shown are means of three independent experiments performed in triplicate.</p