Octahedral ruthenium and magnesium naringenin 5-alkoxide complexes: NMR analysis of diastereoisomers and in-vivo antibacterial activity against Xylella fastidiosa.

Although many copper-based antimicrobial compounds have been developed to control pathogenic bacteria and fungi in plants and applied for crop protection, there is evidence that several plant pathogens have developed resistance to copper-based antimicrobial compounds, including some Xanthomonas species. Xylella is a bacterial genus belonging to the Xanthomonas family; and X. fastidiosa, which is responsible for citrus variegated chlorosis (CVC) in sweet orange, may develop resistance to one or more copper-based antimicrobials. Because of the time required for the development and approval of new antimicrobials for commercial use, the discovery of novel bactericidal compounds is essential before the development of resistance to the antimicrobials currently in use becomes widespread. Here, we explored the antimicrobial potential of two newly synthesized antimicrobials complexes and one natural compound against X. fastidiosa. Several nuclear magnetic resonance (NMR) assays with high resolution and sensitivity were developed to identify new diastereoisomers in the context of octahedral ruthenium - [Ru(narin)(phen)2]PF6-and magnesium naringenin 5-alkoxide - [Mg(narin)(phen)2]OAc - complexes, obtained in the present work. The NMR assays proved to be powerful tools for the identification of isomers in metal complexes. Moreover, a protocol for the in-vivo determination of the effects of these complexes against X. fastidiosa was developed. The main trunks of X. fastidiosa infected plants were injected with the two complexes as well as with the limonoid azadirachtin using a syringe; the number of bacterial cells in the plants following treatment was estimated via real-time quantitative PCR (qPCR). Importantly, the administration of both complexes and of azadirachtin drastically reduced the number of X. fastidiosa cells in vivo.

Biochemical and morphological biomarker responses in the gills of a Neotropical fish exposed to a new flavonoid metal-insecticide.

The flavonoid metal-insecticide [Mg(hesp)2(phen)], denominated MgHP, has high potential for controlling agricultural pests. If applied in large scale, it may reach aquatic ecosystems and be harmful to the biota. This study evaluated the effects of MgHP in the gills of the Neotropical fish, Prochilodus lineatus by determining the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione s-transferase (GST), and the levels of glutathione (GSH) and lipid peroxidation (LPO) after 24 and 96 h exposure to 0, 1, 10, 100 and 1000 µg L-1. The histopathological changes with emphases to mitochondria-rich cells (MRC) were evaluated as well. After 24 h exposure the enzyme activities and the GSH and LPO levels were unchanged however, after 96 h exposure to high MgHP concentration (1000 µg L-1), the GST activity and GSH levels increased. Oxidative stress measured as LPO levels did not occur after MgHP exposure in both periods. Gill tissue alterations increased after MgHP exposure to 10, 100 and 1000 µg L-1. Cellular atrophy, pillar cells changes, filament epithelium hyperplasia and hypertrophy, lamellar epithelium hyperplasia were the most frequent histopathology. MRC in the filament epithelium decreased after exposure to 24 h and increased after 96 h indicating possible transitory osmo-ionic disruption. P. lineatus exhibited high tolerance to MgHP. The increased GST activity and GSH levels after 96 h exposure suggested possible MgHP accumulation and concentration- and time-dependent response. Histopathology in the gills of exposed fish occurred at high MgHP concentrations. These results suggested that the MgHP into water, at high concentrations, affect the gills by changing GST activity, GSH levels and histology being useful biomarkers for MgHP water contamination.

Multi-biomarkers approach to access the impact of novel metal-insecticide based on flavonoid hesperidin on fish.

Aquatic ecosystem health is the main concern to increasing pesticides application to control agricultural pests as it is the ultimate receptor of such materials. This study evaluated the impact of new metal-insecticide, the [Mg(hesp)2(phen)], referred as MgHP, on fish using physiological, genetic, biochemical, and morphological biomarkers. The fish, Prochilodus lineatus, was exposed to 0 (control), 1, 10, 100, 1000 µg L-1 MgHP, for 24 and 96 h. MgHP was not lethal but caused genotoxicity, altered hematological variables and, the activity of antioxidant and biotransformation enzymes and histology of liver, depending on concentration and time exposure. Hematocrit and erythrocyte number (RBC) increased without change hemoglobin content resulting in changes in hematimetric indexes after 24 h; after 96 h, only RBC was changed. Erythrocyte nuclear abnormalities and crenate cells increased after 24 h but, not after 96 h. Erythrocytes and hepatocytes indicated instability in DNA integrity however, the absence of micronuclei suggested DNA damage repairment. After 24 h, the antioxidant defense system and the phase II biotransformation enzyme was responsiveness and catalase activity decreased at high MgHP concentrations; the antioxidant response was triggered after 96 h. Hepatocyte hypertrophy, intracellular cytoplasmic substances, cytoplasm degeneration, melanomacrophage and hyperemia increased in fish exposed from 10 µg L-1 to higher MgHP concentrations; the organ alteration index increased as MgHP concentration increased showing dose-dependence. Most of hematological and genotoxic effects occurred after 24 h exposure evidencing potential recover capability of organism by activation of the antioxidant defense system and DNA repairment mechanisms. Nevertheless, the histopathological changes in the liver was maintained over time at high MgHP concentrations, a concentration usually no environmental relevant. In conclusion, this data reinforced the importance of continuing research on MgHP effects in other organisms considering the promising use of such compound to control the leaf-cutter ants and other insects.

Antineoplastic activity of a novel ruthenium complex against human hepatocellular carcinoma (HepG2) and human cervical adenocarcinoma (HeLa) cells.

Novel metal complexes have received much attention recently because of their potential anticancer activity. Notably, ruthenium-based complexes have emerged as good alternatives to the currently used platinum-based drugs for cancer therapy, with less toxicity and fewer side effects. The beneficial properties of Ru, which make it a highly promising therapeutic agent, include its variable oxidative states, low toxicity, and high selectivity for cancer cells. The present study evaluated the cytotoxic effects of a ruthenium complex, namely cis-[Ru(1,10-phenanthroline)2(imidazole)2]2+ (RuC), on human hepatocellular carcinoma (HepG2) and human cervical adenocarcinoma (HeLa) cells and analyzed metabolic parameters. RuC reduced HepG2 and HeLa cell viability at all tested concentrations (10, 50, and 100 nmol/L) at 48 h of incubation, based on the MTT, Crystal violet, and neutral red assays. The proliferation capacity of HepG2 cells did not recover, whereas HeLa cell proliferation partially recovered after RuC treatment. RuC also inhibited all states of cell respiration and increased the levels of the metabolites pyruvate and lactate in both cell lines. The cytotoxicity of RuC was higher than cisplatin (positive control) in both lineages. These results indicate that RuC affects metabolic functions that are related to the energy provision and viability of HepG2 and HeLa cells and is a promising candidate for further investigations that utilize models of human cervical adenocarcinoma and mainly hepatocellular carcinoma.

Biochemical and genotoxic biomarkers and cell cycle assessment in the zebrafish liver (ZF-L) cell line exposed to the novel metal-insecticide magnesium-hespiridin complex.

The flavonoid metal-insecticide magnesium-hesperidin complex (MgHP) has recently been considered as a novel insecticide to replace some persistent pesticides. However, it is important to evaluate its action on non-target species, mainly those living in an aquatic environment, as these ecosystems are the final receptors of most chemicals. Reactive oxygen species, antioxidant and oxidative stress biomarkers, genotoxicity as well as cell cycle was evaluated in the liver cell line from zebrafish (Danio rerio; ZF-L) exposed to 0, 0.1, 1, 10, 100 and 1000 ng mL-1 MgHP. MgHP affected cell stability by increasing reactive oxygen species (ROS) in both exposure times (24 and 96 h) at high concentrations. Catalase (CAT) activity decreased after 24 h exposure, and glutathione and metallothionein values increased, avoiding the lipid peroxidation. Genotoxicity increased as MgHP concentration increased, after 24 h exposure, exhibiting nuclear abnormalities; it was recovered after 96 h exposure, evidencing possible stimulation of DNA repair mechanisms. The alteration in the cell cycle (increasing in the Sub-G1 phase and decreasing in the S-phase) was associated with chromosomal instability. In conclusion, the responses of ROS and the antioxidant defense system depended on MgHP concentration and time exposure, while DNA exhibited some instability after 24 h exposure, which was recovered after 96 h.

Mitochondrial and lysosomal dysfunction induced by the novel metal-insecticide [Mg(hesp)2(phen)] in the zebrafish (Danio rerio) hepatocyte cell line (ZF-L).

The development of novel agrochemical compounds to reduce the use of pesticides with high ecological impact is urgently needed. A complex of Mg with two flavonoids hesperidin and phenanthroline [Mg(hesp)2(phen)], referred to as MgHP, results in high insecticidal activity against urban, agricultural and forest insect pests. In vitro cytotoxicity biomarkers were used to assess the mechanism of action MgHP on fish cells, as this insecticide can reach the aquatic environment and affect its biota. The cytotoxic effects of MgHP were evaluated at different concentrations (0, 0.1, 1, 10, 100 and 1000 ng mL-1) in a zebrafish hepatocyte cell line (ZF-L). Twenty-four hours of exposure to high concentrations (10 and 1000 ng mL-1) of MgHP affected cell confluence and morphology. Mitochondrial activity and lysosomal retention ability decreased as the MgHP concentration was increased. Cell membrane injury, apoptosis, and necrosis were not induced. These results suggested that toxicity to ZF-L cells was due loss of organellar activity caused by MgHP, which may also include activation of an alternative cell death mechanism. However, after 96 h of exposure, the toxic effects of MgHP may be mitigated, even at high concentrations, enabling cellular population recovery. These data provide important information on the mechanism of action of MgHP on hepatocyte fish cells and stimulate analyses to elucidate the cellular responses to MgHP.

The antioxidant gallic acid induces anxiolytic-, but not antidepressant-like effect, in streptozotocin-induced diabetes.

The physiopathology of anxiety or depression related to diabetes is still poorly understood. The treatment with antidepressant drugs is a huge challenge due to theirs adherence low rate and many adverse effects. Thus, the seeking for a better treatment for these associated diseases is of utmost importance. Given that the oxidative stress in different tissues occurs in diabetes and anxiety or depression as well, the antioxidant gallic acid becomes an interesting compound to be investigated. Thus, the effects of long-term treatment with gallic acid (0, 10, 20 and 40 mg/kg; gavage) were evaluated in diabetic (DBT) animals submitted to the elevated plus-maze (EPM), the light-dark transition (LDT) tests and modified forced swim test (mFST). Also, indirect parameters of oxidative stress, lipid peroxidation (LPO) and reduced glutathione (GSH) levels were evaluated in the hippocampus (HIP) and prefrontal cortex (PFC). The results showed that DBT animals presented a decrease in the spent time in the open arms, in the end arm exploration and head dips when evaluated in the EPM test; moreover, a decrease in the spent time in the lit compartment of LDT test was observed, suggesting an anxiogenic-like behavior. During the mFST, an increase in the mean counts of immobility and a decrease in the mean counts of swimming and climbing were observed, indicating a depressive-like behavior. These aversive behaviors were more pronounced when compared to normoglycemic (NGL) animals and streptozotocin-treated animals that not become DBT. In addition, DBT rats showed an increase in the oxidative stress parameters in the HIP and PFC that was reversed by the gallic acid treatment (lowest dose - 10 mg/kg), i.e., the treatment decreased the elevated LPO levels and increased the reduced GSH in the HIP and PFC. Also, gallic acid treatment was able to produce an anxiolytic-like effect in the EPM and LDT tests, but not antidepressant-like effect in the FST. Taken together, the results suggest that the antioxidant/neuroprotective effect of gallic acid treatment in HIP and PFC of DBT animals may be essential to the anxiolytic-like effect.

Ruthenium complex exerts antineoplastic effects that are mediated by oxidative stress without inducing toxicity in Walker-256 tumor-bearing rats.

The present study evaluated the in vivo antitumor effects and toxicity of a new Ru(II) compound, cis-(Ru[phen]2[ImH]2)2+ (also called RuphenImH [RuC]), against Walker-256 carcinosarcoma in rats. After subcutaneous inoculation of Walker-256 cells in the right pelvic limb, male Wistar rats received 5 or 10mgkg-1 RuC orally or intraperitoneally (i.p.) every 3 days for 13 days. A positive control group (2mgkg-1 cisplatin) and negative control group (vehicle) were also used. Tumor progression was checked daily. After treatment, tumor weight, plasma biochemistry, hematology, oxidative stress, histology, and tumor cell respiration were evaluated. RuC was effective against tumors when administered i.p. but not orally. The highest i.p. dose of RuC (10mgkg-1) significantly reduced tumor volume and weight, induced oxidative stress in tumor tissue, reduced the respiration of tumor cells, and induced necrosis but did not induce apoptosis in the tumor. No clinical signs of toxicity or death were observed in tumor-bearing or healthy rats that were treated with RuC. These results suggest that RuC has antitumor activity through the modulation of oxidative stress and impairment of oxidative phosphorylation, thus promoting Walker-256 cell death without causing systemic toxicity. These effects make RuC a promising anticancer drug for clinical evaluation.

Copper (II) and zinc (II) complexes with flavanone derivatives: Identification of potential cholinesterase inhibitors by on-flow assays.

Metal chelates strongly influence the nature and magnitude of pharmacological activities in flavonoids. In recent years, studies have shown that a promising class of flavanone-metal ion complexes can act as selective cholinesterase inhibitors (ChEIs), which has led our group to synthesize a new series of flavanone derivatives (hesperidin, hesperetin, naringin, and naringenin) complexed to either copper (II) or zinc (II) and to evaluate their potential use as selective ChEIs. Most of the synthesized complexes exhibited greater inhibitory activity against acetylcholinesterase (AChE) than against butyrylcholinesterase (BChE). Nine of these complexes constituted potent, reversible, and selective ChEIs with inhibitory potency (IC50) and inhibitory constant (Ki) ranging from 0.02 to 4.5µM. Copper complexes with flavanone-bipyridine derivatives afforded the best inhibitory activity against AChE and BChE. The complex Cu(naringin)(2,2'-bipyridine) (11) gave IC50 and Ki values of 0.012±0.002 and 0.07±0.01µM for huAChE, respectively, which were lower than the inhibitory values obtained for standard galanthamine (IC50=206±30.0 and Ki=126±18.0µM). Evaluation of the inhibitory activity of this complex against butyrylcholinesterase from human serum (huBChE) gave IC50 and Ki values of 8.0±1.4 and 2.0±0.1µM, respectively. A Liquid Chromatography-Immobilized Capillary Enzyme Reactor by UV detection (LC-ICER-UV) assay allowed us to determine the IC50 and Ki values and the type of mechanism for the best inhibitors.

Copper (II) and 2,2'-bipyridine complexation improves chemopreventive effects of naringenin against breast tumor cells.

Cancer is the second leading cause of death worldwide and there is epidemiological evidence that demonstrates this tendency is emerging. Naringenin (NGEN) is a trihydroxyflavanone that shows various biological effects such as antioxidant, anticancer, anti-inflammatory, and antiviral activities. It belongs to flavanone class, which represents flavonoids with a C6-C3-C6 skeleton. Flavonoids do not exhibit sufficient activity to be used for chemotherapy, however they can be chemically modified by complexation with metals such as copper (Cu) (II) for instance, in order to be applied for adjuvant therapy. This study investigated the effects of Cu(II) and 2,2'-bipyridine complexation with naringenin on MDA-MB-231 cells. We demonstrated that naringenin complexed with Cu(II) and 2,2'-bipyridine (NGENCuB) was more efficient inhibiting colony formation, proliferation and migration of MDA-MB-231 tumor cells, than naringenin (NGEN) itself. Furthermore, we verified that NGENCuB was more effective than NGEN inhibiting pro-MMP9 activity by zymography assays. Finally, through flow cytometry, we showed that NGENCuB is more efficient than NGEN inducing apoptosis in MDA-MB-231 cells. These results were confirmed by gene expression analysis in real time PCR. We observed that NGENCuB upregulated the expression of pro-apoptotic gene caspase-9, but did not change the expression of caspase-8 or anti-apoptotic gene Bcl-2. There are only few works investigating the effects of Cu(II) complexation with naringenin on tumor cells. To the best of our knowledge, this is the first work describing the effects of Cu(II) complexation of a flavonoid on MDA-MB-231 breast tumor cells.

The effects of the substitution on the imidazole ligand on the photochemical properties of fac-[Mn(CO)3(phen)(Imidazole)](SO3CF3) complexes.

Photochemical and photophysical data are reported for a series of fac-[Mn(CO)(3)(phen)(Im-R)](SO(3)CF(3)) complexes, where phen is 1,10-phenanthroline and Im is imidazole. Intraligand and metal-to-ligand charge transfer (MLCT) transitions are observed in the electronic absorption spectra of these complexes and are sensitive to the nature of the ligand substituent. At room temperature the emission spectra show a clear progression from broad structureless MLCT to highly structured pi-pi* emission on going from R = -H, -CH(3), -C(6)H(5), to -Metro, where Metro is 2-methyl-5-nitroimidazole. Even at low temperatures the latter complexes show only the pi-pi* emission. The trend in the photophysical properties found in the emission spectra parallels the changes in the photochemical properties with the electron-donating or electron-withdrawing power of the substituent on the imidazole ligand. Although MLCT irradiation of the complexes with R = -H, -CH(3) leads to the mer-[Mn(CO)(3)(phen)(Im-R)](+) isomers, the complexes with the imidazole ligand substituted by -C(6)H(5) or -Metro release the Im-R ligand and produce the stereoretentive fac-[Mn(CO)(3)(phen)(S)](+) complexes. The stereochemical fate and mechanistic implications of the photolysis reactions are discussed in terms of the nature of ligand substitution.

Reactivity of radicals generated on irradiation of trans-[Ru(NH3)4(NO2)P(OEt)3](PF6).

The electronic absorption spectrum of trans-[Ru(NH(3))(4)(NO(2))(P(OEt)(3)](+) in aqueous solution is characterized by a strong absorption band at 334 nm (lambda(max) = 1800 mol(-1) L cm(-1)). On the basis of quantum mechanics calculations, this band has been assigned to a MLCT transition from the metal to the nitro ligand. Molecular orbital calculations also predict an LF transition at 406 nm, which is obscured by the intense MLCT transition. When trans-[Ru(NH(3))(4)(NO(2))(P(OEt)(3)](+) in acetonitrile is irradiated with a 355 nm pulsed laser light, the absorption features are gradually shifted to represent those of the solventocomplex trans-[Ru(NH(3))(4)(solv)(P(OEt)(3)](2+) (lambda(max) = 316 nm, epsilon = 650 mol(-1) L cm(-1)), which was also detected by (31)P NMR spectroscopy. The net photoreaction under these conditions is a photoaquation of trans-[Ru(NH(3))(4)(NO(2))(P(OEt)(3)](+), although, after photolysis, the presence of the nitric oxide was detected by differential pulse polarography. In phosphate buffer pH 9.0, after 15 min of photolysis, a thermal reaction resulted in the formation of a hydroxyl radical and a small amount of a paramagnetic species as detected by EPR spectroscopy. In the presence of trans-[Ru(NH(3))(4)(solv)P(OEt)(3)](2+), the hydroxyl radical initiated a chain reaction. On the basis of spectroscopic and electrochemical data, the role of the radicals produced is analyzed and a reaction sequence consistent with the experimental results is proposed. The 355 nm laser photolysis of trans-[Ru(NH(3))(4)(NO(2))(P(OEt)(3)](+) in phosphate buffer pH 7.4 also gives nitric oxide, which is readily trapped by ferrihemeproteins (myoglobin, hemoglobin, and cytochrome C), giving rise to the formation of their nitrosylhemeproteins(II), (NO)Fe(II)hem.