Furanocoumarins from Ruta chalepensis with Amebicide Activity.

Entamoeba histolytica (protozoan; family Endomoebidae) is the cause of amoebiasis, a disease related to high morbidity and mortality. Nowadays, this illness is considered a significant public health issue in developing countries. In addition, parasite resistance to conventional medicinal treatment has increased in recent years. Traditional medicine around the world represents a valuable source of alternative treatment for many parasite diseases. In a previous paper, we communicated about the antiprotozoal activity in vitro of the methanolic (MeOH) extract of Ruta chalepensis (Rutaceae) against E. histolytica. The plant is extensively employed in Mexican traditional medicine. The following workup of the MeOH extract of R. chalepensis afforded the furocoumarins rutamarin (1) and chalepin (2), which showed high antiprotozoal activity on Entamoeba histolytica trophozoites employing in vitro tests (IC50 values of 6.52 and 28.95 µg/mL, respectively). Therefore, we offer a full scientific report about the bioguided isolation and the amebicide activity of chalepin and rutamarin.

Brazilin Inhibits α-Synuclein Fibrillogenesis, Disrupts Mature Fibrils, and Protects against Amyloid-Induced Cytotoxicity.

The inhibitory effect of brazilin against α-synuclein (α-syn) fibrillogenesis, disruption effect against mature fibrils, and the following cytotoxicity were examined by systematical biochemical, biophysical, cellular biological, and molecular simulation experiments. It is found that brazilin inhibited α-syn fibrillogenesis and disrupted the performed fibrils with a concentration-dependent manner. Moreover, cellular experimental data showed that brazilin effectively reduced the cytotoxicity induced by α-syn aggregates. Finally, molecular dynamics simulations were performed to explore the interactions between brazilin and α-syn pentamer. It is found that brazilin directly interacts with α-syn pentamer, and the hydrophobic interactions are favorable for brazilin binding with the α-syn pentamer, while the electrostatic part provides adverse effects. Three binding regions were identified to inhibit α-syn fibrillogenesis or disrupt the preformed aggregates. Furthermore, six important residues (i.e., G51, V52, A53, E61, V66, and K80) of α-syn were also identified. We expected that brazilin is an effective agent against α-syn fibrillogenesis and associated cytotoxicity.

Kinetic aspects of humic substances derived from macrophyte detritus decomposition under different nutrient conditions.

Autochthonous particulate organic carbon (POC) is an important precursor of humic substances (HS), and macrophytes represent the major source of POC in tropical aquatic ecosystems. Autochthonous HS influence the carbon supply, light regime, and primary production within freshwater systems. This study addresses the conversion of POC from two macrophyte species into HS and their mineralization under different nutrient conditions (oligotrophic to hypereutrophic). A first-order kinetic model was adopted to describe the conversion routes. The POC conversion rate to HS for detritus derived from Paspalum repens was similar under different nutrient conditions, but eutrophication decreased the kR (global coefficient reaction) for detritus from Pistia stratiotes due to its high detritus quality (C:N:P ratio). Fulvic acids were the main fraction of HS in both plants. The mineralization of humic acids from P. stratiotes was inhibited at higher nutrient availability, while eutrophication increased the mineralization of fulvic acids from P. repens. The main route of POC cycling is humification through fulvic acid formation (up to 40% of POC). The intrinsic characteristics of the source detritus were the main forcing functions that stimulated the cycling of HS. In tropical aquatic ecosystems, the degradation of autochthonous carbon decreased due to eutrophication, thus contributing to the diagenetic process in the long term.

Brazilin Isolated from Caesalpinia sappan suppresses nuclear envelope reassembly by inhibiting barrier-to-autointegration factor phosphorylation.

To date, many anticancer drugs have been developed by directly or indirectly targeting microtubules, which are involved in cell division. Although this approach has yielded many anticancer drugs, these drugs produce undesirable side effects. An alternative strategy is needed, and targeting mitotic exit may be one alternative approach. Localization of phosphorylated barrier-to-autointegration factor (BAF) to the chromosomal core region is essential for nuclear envelope compartment relocalization. In this study, we isolated brazilin from Caesalpinia sappan Leguminosae and demonstrated that it inhibited BAF phosphorylation in vitro and in vivo. Moreover, we demonstrated direct binding between brazilin and BAF. The inhibition of BAF phosphorylation induced abnormal nuclear envelope reassembly and cell death, indicating that perturbation of nuclear envelope reassembly could be a novel approach to anticancer therapy. We propose that brazilin isolated from C. sappan may be a new anticancer drug candidate that induces cell death by inhibiting vaccinia-related kinase 1-mediated BAF phosphorylation.

Transport and metabolism behavior of brazilein during its entrance into neural cells.

Brazilein, a natural small molecule, shows a variety of pharmacological activities, especially on nervous system and immune system. As a potential multifunctional drug, we studied the distribution and the transport behavior and metabolic behavior of brazilein in vivo and in vitro. Brazilein was found to be able to distribute in the mouse brain and transport into neural cells. A metabolite was found in the brain and in the cells. Positive and negative mode-MS/MS and Q-TOF were used to identify the metabolite. MS/MS fragmentation mechanisms showed the methylation occurred at the 10-hydroxyl of brazilein (10-O-methylbrazilein). Further, catechol-O- methyltransferase (COMT) was confirmed as a crucial enzyme correlated with the methylated metabolite generation by molecular docking and pharmacological experiment.

The effect of extracellular pH changes on intracellular pH and nitric oxide concentration in endothelial and smooth muscle cells from rat aorta.

AIMS: It has been known for more than a century that pH changes can alter vascular tone. However, there is no consensus about the effects of pH changes on vascular response. In this study, we investigated the effects of extracellular pH (pHo) changes on intracellular pH (pHi) and intracellular nitric oxide concentration ([NO]i) in freshly isolated endothelial cells and cross sections from rat aorta. MAIN METHODS: The HCl was used to reduce the pHo from 7.4 to 7.0 and from 7.4 to 6.5; the NaOH was used to increase the pHo from 7.4 to 8.0 and from 7.4 to 8.5. The fluorescent dyes 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate (SNARF-1) and diaminofluorescein-FM diacetate (DAF-FM DA) were employed to measure the pHi and [NO]i, respectively. The fluorescence intensity was measured in freshly isolated endothelial cells by flow cytometry and in freshly obtained aorta cross sections by confocal microscopy. KEY FINDINGS: The endothelial and vascular smooth muscle pHi was increased at pHo 8.5. The extracellular acidification did not change the endothelial pHi, but the smooth muscle pHi was reduced at pHo 7.0. At pHo 8.5 and pHo 6.5, the endothelial [NO]i was increased. Both extracellular alkalinization and acidification increased the vascular smooth muscle [NO]i. SIGNIFICANCE: Not all changes in pHo did result in pHi changes, but disruption of acid-base balance in both directions induced NO synthesis in the endothelium and/or vascular smooth muscle.

Interaction of plant pigment brazilin with synthetic and natural DNA: spectroscopic and in silico perspective.

The interactions between the molecules and DNA shape up an avenue for DNA targeted therapeutics. For the first time, brazilin, a major component of Caesalpinia sappan L., has been investigated for its interaction with natural and synthetic DNA. Detailed analyses of the binding property of brazilin dye with DNA by UV-vis, FTIR and Circular Dichroism were carried out. In addition, in silico studies have been conducted via tools of energy minimization and ligand optimization using Yasara and Argus Lab softwares along with the molecular docking server integrating Auto Dock, Mavin and Mopac. Results show that brazilin dye has commendable proficiency in being moulded as a binder with DNA. The specificity of the dye to stain nuclei in tissue sections positively indicates its interaction with nucleic acid. As the intracellular target for the majority of anticancer and antibiotic drugs is DNA, the study on the interaction between molecules like brazilin and DNA has great significance and implications in several biological applications.

Changes in the corpora allata and epidermal proliferation along the fourth instar of the Chagas disease vector Triatoma infestans

Triatoma infestans, a blood-feeding insect, synchronises physiological mechanisms leading to moult with food intake. Since the corpora allata are important in moult and metamorphosis regulation, we have studied morphological changes in 4th instar nymphs (gland size, cell density, percent of animals showing mitoses and cell size). Changes were correlated with the effect of precocene II, epidermal proliferation, and with the extent of the [quot ]head critical period[quot ]. Based on morphological grounds, three stages can be defined in the gland along the 4th instar: Stage 1 (days 0-2 after feeding) showed small corpora allata, composed by a small number of cells, and in which mitoses were absent; Stage 2 (days 3-9) showed growing corpora allata, in which cell number was increasing and proliferation was apparent; and Stage 3 (days 10-13) showed no mitotic activity, and a sharply diminishing size of the gland, as a consequence of the diminishing size of their cells. The ability of precocene II to induce abnormal moulting disappeared during stage 2 correlating with the termination of the head critical period and suggesting that corpora allata are essential during days 3 to 5 to determine normal growth. Epidermal cell number was increasing as a consequence of more frequent mitotic activity, beginning after the finalization of the head critical period and after a first increment in the size of the gland.

Changes in the corpora allata and epidermal proliferation along the fourth instar of the Chagas disease vector Triatoma infestans

Triatoma infestans, a blood-feeding insect, synchronises physiological mechanisms leading to moult with food intake. Since the corpora allata are important in moult and metamorphosis regulation, we have studied morphological changes in 4th instar nymphs (gland size, cell density, percent of animals showing mitoses and cell size). Changes were correlated with the effect of precocene II, epidermal proliferation, and with the extent of the [quot ]head critical period[quot ]. Based on morphological grounds, three stages can be defined in the gland along the 4th instar: Stage 1 (days 0-2 after feeding) showed small corpora allata, composed by a small number of cells, and in which mitoses were absent; Stage 2 (days 3-9) showed growing corpora allata, in which cell number was increasing and proliferation was apparent; and Stage 3 (days 10-13) showed no mitotic activity, and a sharply diminishing size of the gland, as a consequence of the diminishing size of their cells. The ability of precocene II to induce abnormal moulting disappeared during stage 2 correlating with the termination of the head critical period and suggesting that corpora allata are essential during days 3 to 5 to determine normal growth. Epidermal cell number was increasing as a consequence of more frequent mitotic activity, beginning after the finalization of the head critical period and after a first increment in the size of the gland.(AU)

Changes in the corpora allata and epidermal proliferation along the fourth instar of the Chagas disease vector Triatoma infestans.

Triatoma infestans, a blood-feeding insect, synchronises physiological mechanisms leading to moult with food intake. Since the corpora allata are important in moult and metamorphosis regulation, we have studied morphological changes in 4th instar nymphs (gland size, cell density, percent of animals showing mitoses and cell size). Changes were correlated with the effect of precocene II, epidermal proliferation, and with the extent of the "head critical period". Based on morphological grounds, three stages can be defined in the gland along the 4th instar: Stage 1 (days 0-2 after feeding) showed small corpora allata, composed by a small number of cells, and in which mitoses were absent; Stage 2 (days 3-9) showed growing corpora allata, in which cell number was increasing and proliferation was apparent; and Stage 3 (days 10-13) showed no mitotic activity, and a sharply diminishing size of the gland, as a consequence of the diminishing size of their cells. The ability of precocene II to induce abnormal moulting disappeared during stage 2 correlating with the termination of the head critical period and suggesting that corpora allata are essential during days 3 to 5 to determine normal growth. Epidermal cell number was increasing as a consequence of more frequent mitotic activity, beginning after the finalization of the head critical period and after a first increment in the size of the gland.

Phenolic compounds profiles during ex vitro acclimatization of micropropagated Hypericum polyanthemum.

Accumulation of benzopyrans and total phenolic compounds were assessed in acclimatized field grown plants of Hypericum polyanthemum, an endemic species of southern Brazil, harvested at different developmental stages. The HPLC analysis of bioactive compounds 6-isobutyryl-5,7-dimethoxy-2,2-dimethylbenzopyran (HP1), 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran (HP2) and 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran (HP3) revealed that the three benzopyrans are accumulated both in the vegetative and reproductive parts with maximum contents observed after 18 weeks (in the former) and 20 weeks (in the later) of plant growth (1.92+/-0.085 g % DW and 2.62+/-0.13 g % DW in the vegetative and reproductive parts, respectively). Highest contents of HP1 (1.56+/-0.12 g % DW) and HP2 (0.19+/-0.01 g % DW) were quantified in the green floral buds of the plants, whereas HP3 reached the highest level (1.02+/-0.08 g % DW) in the overblown flowers. The evaluation of total phenolic compounds showed that the vegetative parts accumulated the highest levels of the metabolites (51.93+/-0.67 mg QE (g DW)(-1)) after 16 weeks of plant growth. Considering the reproductive parts, the open flowers accumulated the greatest levels of the bioactive compounds (75.99+/-0.95 mg QE (g DW)(-1)). The results show that H. polyanthemum can be efficiently propagated and acclimatized to produce benzopyrans and other phenolic compounds.

Chromenes from Peperomia serpens (Sw.) Loudon (Piperaceae).

Chromatographic separation of the CH2Cl2 extract from leaves of Peperomia serpens yielded two chromenes [5-hydroxy-8-(3',7'-dimethylocta-2',6'-dienyl)-2,2,7-trimethyl-2H-1-chromene (1) and 5-hydroxy-8-(3'-methyl-2'-butenyl)-2,2,7-trimethyl-2H-1-chromene-6-carboxylic acid (2)], besides the known chromene [methyl 5-hydroxy-2,2,7-trimethyl-2H-1-chromene-6-carboxylate (3)] and the flavonoid, dihydrooroxylin (4). Their structural elucidation were achieved by spectroscopic analyses. The antifungal activities of the CH2Cl2 extract and the isolated chromenes were measured bioautographically against Cladosporium cladosporioides and C. sphaerospermum, when it was found that the crude extract showed higher activity as compared to the pure compounds.

Formation of Cu(II)-brazilin complex in the presence of DNA and its activities as chemical nuclease.

Brazilin, a traditional medicine for the treatment of pain and inflammation, forms a complex with Cu(II) in the presence as well as the absence of DNA. The Cu(II)-brazilin complex exhibited the strand cleavage activity for the pBR322 supercoiled DNA, converting supercoiled form to nicked form. The presence of various scavengers for the oxygen species suppresses or reduces the cleavage activity of the complex, indicating that the DNA cleavage is oxidative. The binding mode of the Cu(II)-brazilin complex was studied by absorption and CD spectroscopy. While a large metal-to-ligand charge transfer (MLCT) band was apparent when Cu(II) and brazilin was mixed in the presence and absence of DNA, the CD did not show any signal in the same region in the presence of DNA, suggesting a weak interaction between the Cu(II)-brazilin complex and DNA bases.

Spectral imaging microscopy demonstrates cytoplasmic pH oscillations in glial cells.

Glial cells exhibit distinct cellular domains, somata, and filopodia. Thus the cytoplasmic pH (pH(cyt)) and/or the behavior of the fluorescent ion indicator might be different in these cellular domains because of distinct microenvironments. To address these issues, we loaded C6 glial cells with carboxyseminaphthorhodafluor (SNARF)-1 and evaluated pH(cyt) using spectral imaging microscopy. This approach allowed us to study pH(cyt) in discrete cellular domains with high temporal, spatial, and spectral resolution. Because there are differences in the cell microenvironment that may affect the behavior of SNARF-1, we performed in situ titrations in discrete cellular regions of single cells encompassing the somata and filopodia. The in situ titration parameters apparent acid-base dissociation constant (pK'(a)), maximum ratio (R(max)), and minimum ratio (R(min)) had a mean coefficient of variation approximately six times greater than those measured in vitro. Therefore, the individual in situ titration parameters obtained from specific cellular domains were used to estimate the pH(cyt) of each region. These studies indicated that glial cells exhibit pH(cyt) heterogeneities and pH(cyt) oscillations in both the absence and presence of physiological HCO(3)(-). The amplitude and frequency of the pH(cyt) oscillations were affected by alkalosis, by acidosis, and by inhibitors of the ubiquitous Na(+)/H(+) exchanger- and HCO(3)(-)-based H(+)-transporting mechanisms. Optical imaging approaches used in conjunction with BCECF as a pH probe corroborated the existence of pH(cyt) oscillations in glial cells.

Circadian rhythm of anti-fungal prenylated chromene in leaves of Piper aduncum.

Leaves of Piper aduncum accumulate the anti-fungal chromenes methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate (1) and methyl 2,2-dimethyl-8-(3'-methyl-2'-butenyl)-2H-1-chromene-6-carboxylate (2). The enzymatic formation of 2 from dimethylallyl diphosphate and 1 was investigated using cell-free extracts of the title plant. An HPLC assay for the prenylation reaction was developed and the enzyme activity measured in the protein extracts. The prenyltransferase that catalyses the transfer of the dimethylallyl group to C-2' of 1 was soluble and required dimethylallyl diphosphate as the prenyl donor. In the leaves, the biosynthesis of the prenylated chromene 2 was time-regulated and prenyltransferase activity depended upon circadian variation. Preliminary characterisation and purification experiments on the prenyltransferase from P. aduncum have been performed.

Temperature influence on Penicillium citrinum thom growth and citrinin accumulation kinetics.

To study the temperature influence on both Penicillium citrinum growth and citrinin accumulation, a 20, 25 and 30 degrees C. Radial and 30 degrees C. Radial growth rate and lag phase were determined from the increase in colony diameter with time. The optimal temperature for P. citrinum growth was 30 degrees C. Citrinin extracted from the agar medium was determined by thin layer chromatography. Citrinin accumulation kinetics were analyzed by fitting the data to curves generated by using a logistic function. The parameters obtained from this equation demonstrated, for all temperatures studied, that the maximum citrinin accumulation by P. citrinum on Czapek agar with maize extract was at about 30 degrees C. At 37 degrees C a rapid decrease in the citrinin concentration was observed after a maximal value was reached.

Application of current chemical concepts to metal-hematein and -brazilein stains.

Current chemical concepts were applied to Weigert's, M. Heidenhain's and Verhoeff's iron hemateins, Mayer's acid hemalum stain and the corresponding brazilein compounds. Fe bonds tightly to oxygen in preference to nitrogen and is unlikely to react with lysyl and arginyl groups of proteins. Binding of unoxidized hematoxylin by various substrates has long been known to professional dyers and was ascribed to hydrogen bonding. Chemical data on the uptake of phenols support this theory. Molecular models indicate a nonplanar configuration of hematoxylin and brazilin. The traditional quinonoid formula of hematein and brazilein was revised. During chelate formation each of the two oxy- groups of the dye shares an electron pair with the metal and contributes a negative charge to the chelate. Consequently, the blue or black 2:1 (dye:metal) complexes are anionic. Olation of such chelates affects the staining properties of iron hematein solutions. The color changes upon oxidation of hematoxylin, reaction of hematein with metals, and during exposure of chelates to acids can be explained by molecular orbital theory. Without differentiation or acid in dye chelate solutions, staining patterns are a function of the metal. Reactions of acidified solutions are determined by the affinities of the dye ligands. Brazilein is much more acid-sensitive than hematein. This difference can be ascribed to the lack of a second free phenolic -OH group in brazilein, i.e. one hydrogen bond is insufficient to anchor the dye to tissues. Since hematein and brazilein are identical in all other respects, their differences in affinity cannot be explained by van der Waals, electrostatic, hydrophobic or other forces.