In vitro anti-canine distemper virus activity of fucoidan extracted from the brown alga Cladosiphon okamuranus

Canine distemper virus (CDV) is a morbillivirus related to measles virus that infects dogs and other carnivores. CDV has a significant global impact on animal health; however, there is no current antiviral treatment for CDV infection. In recent years, it has been demonstrated that sulfated polysaccharides exhibit antiviral properties both in vivo and in vitro, despite their low cytotoxicity to host cells. Fucoidan is a sulfated polysaccharide found in the cell wall matrix of brown algae. In this study, we evaluated in vitro anti-CDV activity of fucoidan, which was derived from Cladosiphon okamuranus. Fucoidan actively inhibited CDV replication in Vero cells at a 50% inhibitory concentration (IC50) of 0.1 lg/ml. The derived selectivity index (SI50) was[20,000. This polysaccharide likely inhibits viral infection by interference in the early steps and by inhibiting CDV-mediated cell fusion. Fucoidan may be useful in development of pharmacological strategies to treat and control CDV infection.

Characterization of silver nanoparticles prepared by wet chemical method and their antibacterial and cytotoxicity activities

Purpose: To investigate the efficiency of silver nanoparticles synthesized by wet chemical method, and evaluate their antibacterial and anti-cancer activities. Methods: Wet chemical method was used to synthesize silver nanoparticles (AgNPs) from silver nitrate, trisodium citrate dehydrate (C6H5O7Na3.2H2O) and sodium borohydride (NaBH4) as reducing agent. The AgNPs and the reaction process were characterized by UV–visible spectrometry, zetasizer, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) equipped with energy dispersive spectroscopy (EDS). The antibacterial and cytotoxic effects of the synthesized nanoparticles were investigated by agar diffusion method and MTT assay respectively. Results: The silver nanoparticles formed were spherical in shape with mean size of 10.3 nm. The results showed good antibacterial properties, killing both Gram-positive and Gram-negative bacteria, and its aqueous suspension displayed cytotoxic activity against colon adenocarcinoma (HCT-116) cell line. Conclusion: The findings indicate that silver nanoparticles synthesized by wet chemical method demonstrate good cytotoxic activity in colon adenocarcinoma (HCT-116) cell lines and strong antibacterial activity against various strains of bacteria.

Phytochemical screening and evaluation of cytotoxicity of stem bark extracts of Genus dolichocarpa and Duguetia chrysocarpa (Annonaceae)

Purpose: To investigate the phytochemistry and cytotoxic activity of stem bark extracts from Genus dolichocarpa and Duguetia chrysocarpa - two species of the Annonaceae family. Methods: The crude ethanol bark extracts (EtOH) of the plants were obtained by maceration. The crude extracts were suspended in a mixture of methanol (MeOH) and water (H2O) (proportion 3:7 v/v) and partitioned with hexane, chloroform (CHCl3) and ethyl acetate (AcOEt) in ascending order of polarity to obtain the respective fractions. The extracts were evaluated on thin layer chromatography (TLC) plates of silica gel to highlight the main groups of secondary metabolites. Cytotoxicity was tested against human tumor cell lines - OVCAR-8 (ovarian), SF-295 (brain) and HCT-116 (colon) - using 3- (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Results: The screening results demonstrated that all the extracts were positive for the presence of flavonoids and tannins. The presence of alkaloids also was detected in some extracts. The hexane extract of A. dolichocarpa showed the strongest cytotoxicity against HCT-116 with cell growth inhibition of 89.02 %. Conclusion: The findings demonstrate for the first time the cytotoxic activity of the extracts of A. dolichocarpa and D. chrysocarpa, thus providing some evidence that plants of the Annonaceae family are a source of active secondary metabolites with cytotoxic activity.

Solid Lipid Nanoparticles For Hydrophilic Biotech Drugs: Optimization And Cell Viability Studies (caco-2 & Hepg-2 Cell Lines).

Insulin was used as model protein to developed innovative Solid Lipid Nanoparticles (SLNs) for the delivery of hydrophilic biotech drugs, with potential use in medicinal chemistry. SLNs were prepared by double emulsion with the purpose of promoting stability and enhancing the protein bioavailability. Softisan(®)100 was selected as solid lipid matrix. The surfactants (Tween(®)80, Span(®)80 and Lipoid(®)S75) and insulin were chosen applying a 2(2) factorial design with triplicate of central point, evaluating the influence of dependents variables as polydispersity index (PI), mean particle size (z-AVE), zeta potential (ZP) and encapsulation efficiency (EE) by factorial design using the ANOVA test. Therefore, thermodynamic stability, polymorphism and matrix crystallinity were checked by Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD), whereas the effect of toxicity of SLNs was check in HepG2 and Caco-2 cells. Results showed a mean particle size (z-AVE) width between 294.6 nm and 627.0 nm, a PI in the range of 0.425-0.750, ZP about -3 mV, and the EE between 38.39% and 81.20%. After tempering the bulk lipid (mimicking the end process of production), the lipid showed amorphous characteristics, with a melting point of ca. 30 °C. The toxicity of SLNs was evaluated in two distinct cell lines (HEPG-2 and Caco-2), showing to be dependent on the concentration of particles in HEPG-2 cells, while no toxicity in was reported in Caco-2 cells. SLNs were stable for 24 h in in vitro human serum albumin (HSA) solution. The resulting SLNs fabricated by double emulsion may provide a promising approach for administration of protein therapeutics and antigens.

Silk Fibroin And Sodium Alginate Blend: Miscibility And Physical Characteristics.

Films of silk fibroin (SF) and sodium alginate (SA) blends were prepared by solution casting technique. The miscibility of SF and SA in those blends was evaluated and scanning electron microscopy (SEM) revealed that SF/SA 25/75 wt.% blends underwent microscopic phase separation, resulting in globular structures composed mainly of SF. X-ray diffraction indicated the amorphous nature of these blends, even after a treatment with ethanol that turned them insoluble in water. Thermal analyses of blends showed the peaks of degradation of pristine SF and SA shifted to intermediate temperatures. Water vapor permeability, swelling capacity and tensile strength of SF films could be enhanced by blending with SA. Cell viability remained between 90 and 100%, as indicated by in vitro cytotoxicity test. The SF/SA blend with self-assembled SF globules can be used to modulate structural and mechanical properties of the final material and may be used in designing high performance wound dressing.

Graphene And Carbon Nanotube Nanocomposite For Gene Transfection.

Graphene and carbon nanotube nanocomposite (GCN) was synthesised and applied in gene transfection of pIRES plasmid conjugated with green fluorescent protein (GFP) in NIH-3T3 and NG97 cell lines. The tips of the multi-walled carbon nanotubes (MWCNTs) were exfoliated by oxygen plasma etching, which is also known to attach oxygen content groups on the MWCNT surfaces, changing their hydrophobicity. The nanocomposite was characterised by high resolution scanning electron microscopy; energy-dispersive X-ray, Fourier transform infrared and Raman spectroscopies, as well as zeta potential and particle size analyses using dynamic light scattering. BET adsorption isotherms showed the GCN to have an effective surface area of 38.5m(2)/g. The GCN and pIRES plasmid conjugated with the GFP gene, forming π-stacking when dispersed in water by magnetic stirring, resulting in a helical wrap. The measured zeta potential confirmed that the plasmid was connected to the nanocomposite. The NIH-3T3 and NG97 cell lines could phagocytize this wrap. The gene transfection was characterised by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Before application, we studied GCN cell viability in NIH-3T3 and NG97 line cells using both MTT and Neutral Red uptake assays. Our results suggest that GCN has moderate stability behaviour as colloid solution and has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity and good transfection efficiency.

Carbon Nanoparticles For Gene Transfection In Eukaryotic Cell Lines.

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

Design, Synthesis And Biological Evaluation Of Hybrid Bioisoster Derivatives Of N-acylhydrazone And Furoxan Groups With Potential And Selective Anti-trypanosoma Cruzi Activity.

Hybrid bioisoster derivatives from N-acylhydrazones and furoxan groups were designed with the objective of obtaining at least a dual mechanism of action: cruzain inhibition and nitric oxide (NO) releasing activity. Fifteen designed compounds were synthesized varying the substitution in N-acylhydrazone and in furoxan group as well. They had its anti-Trypanosoma cruzi activity in amastigotes forms, NO releasing potential and inhibitory cruzain activity evaluated. The two most active compounds (6, 14) both in the parasite amastigotes and in the enzyme contain the nitro group in para position of the aromatic ring. The permeability screening in Caco-2 cell and cytotoxicity assay in human cells were performed for those most active compounds and both showed to be less cytotoxic than the reference drug, benznidazole. Compound 6 was the most promising, since besides activity it showed good permeability and selectivity index, higher than the reference drug. Thereby the compound 6 was considered as a possible candidate for additional studies.

Chitosan/tripolyphosphate Nanoparticles Loaded With Paraquat Herbicide: An Environmentally Safer Alternative For Weed Control.

Paraquat is a fast acting nonselective contact herbicide that is extensively used worldwide. However, the aqueous solubility and soil sorption of this compound can cause problems of toxicity in nontarget organisms. This work investigates the preparation and characterization of nanoparticles composed of chitosan and sodium tripolyphosphate (TPP) to produce an efficient herbicidal formulation that was less toxic and could be used for safer control of weeds in agriculture. The toxicities of the formulations were evaluated using cell culture viability assays and the Allium cepa chromosome aberration test. The herbicidal activity was investigated in cultivations of maize (Zea mays) and mustard (Brassica sp.), and soil sorption of the nanoencapsulated herbicide was measured. The efficiency association of paraquat with the nanoparticles was 62.6 ± 0.7%. Encapsulation of the herbicide resulted in changes in its diffusion and release as well as its sorption by soil. Cytotoxicity and genotoxicity assays showed that the nanoencapsulated herbicide was less toxic than the pure compound, indicating its potential to control weeds while at the same time reducing environmental impacts. Measurements of herbicidal activity showed that the effectiveness of paraquat was preserved after encapsulation. It was concluded that the encapsulation of paraquat in nanoparticles can provide a useful means of reducing adverse impacts on human health and the environment, and that the formulation therefore has potential for use in agriculture.

Chemical Composition And Free Radical-scavenging, Anticancer And Anti-inflammatory Activities Of The Essential Oil From Ocimum Kilimandscharicum.

The essential oil from the leaves of Ocimum kilimandscharicum (EOOK), collected in Dourados-MS, was investigated for anticancer, anti-inflammatory and antioxidant activity and chemical composition. The essential oil was extracted by hydrodistillation, and the chemical composition was performed by gas chromatography-mass spectrometry. The essential oil was evaluated for free radical-scavenging activity using the DPPH assay and was tested in an anticancer assay against ten human cancer cell lines. The response parameter (GI50) was calculated for the cell lines tested. The anti-inflammatory activity was evaluated using carrageenan-induced pleurisy in mice. The chemical composition showed 45 components with a predominance of monoterpenes, such as camphor (51.81%), 1,8 cineole (20.13%) and limonene (11.23%). The EOOK exhibited potent free radical-scavenging activity by the DPPH assay with a GI50 of 8.31 μg/ml. The major constituents, pure camphor (IC50=12.56 μg/ml) and mixture of the limonene: 1, 8 cineole (IC50=23.25 μg/ml) displayed a potent activity. The oral administration of EOOK (at 30 and 100 mg kg(-1)), as well as the pure camphor or a mixture of 1,8 cineole with limonene, significantly inhibited the carrageenan (Cg) induced pleurisy, reducing the migration of total leukocytes in mice by 82 ± 4% (30 mg kg(-1) of EOOK), 95 ± 4% (100 mg kg(-1) of EOOK), 83 ± 9% (camphor) and 80 ± 5% (mixture of 1,8 cineole:limonene 1:1). In vitro cytotoxicity screening against a human ovarian cancer cell line displayed high selectivity and potent anticancer activity with GI50=31.90 mg ml(-1). This work describes the anti-inflammatory, anticancer and antioxidant effects of EOOK for the first time. The essential oil exhibited marked anti-inflammatory, antioxidant and anticancer effects, an effect that can be attributed the presence of majorital compounds, and the response profiles from chemical composition differed from other oils collected in different locales.

Effects Of Sterilization Methods On The Physical, Chemical, And Biological Properties Of Silk Fibroin Membranes.

Silk fibroin has been widely explored for many biomedical applications, due to its biocompatibility and biodegradability. Sterilization is a fundamental step in biomaterials processing and it must not jeopardize the functionality of medical devices. The aim of this study was to analyze the influence of different sterilization methods in the physical, chemical, and biological characteristics of dense and porous silk fibroin membranes. Silk fibroin membranes were treated by several procedures: immersion in 70% ethanol solution, ultraviolet radiation, autoclave, ethylene oxide, and gamma radiation, and were analyzed by scanning electron microscopy, Fourier-transformed infrared spectroscopy (FTIR), X-ray diffraction, tensile strength and in vitro cytotoxicity to Chinese hamster ovary cells. The results indicated that the sterilization methods did not cause perceivable morphological changes in the membranes and the membranes were not toxic to cells. The sterilization methods that used organic solvent or an increased humidity and/or temperature (70% ethanol, autoclave, and ethylene oxide) increased the silk II content in the membranes: the dense membranes became more brittle, while the porous membranes showed increased strength at break. Membranes that underwent sterilization by UV and gamma radiation presented properties similar to the nonsterilized membranes, mainly for tensile strength and FTIR results.

Design And Synthesis Of N-acylated Aza-goniothalamin Derivatives And Evaluation Of Their In Vitro And In Vivo Antitumor Activity.

Herein we describe the synthesis of a focused library of compounds based on the structure of goniothalamin (1) and the evaluation of the potential antitumor activity of the compounds. N-Acylation of aza-goniothalamin (2) restored the in vitro antiproliferative activity of this family of compounds. 1-(E)-But-2-enoyl-6-styryl-5,6-dihydropyridin-2(1H)-one (18) displayed enhanced antiproliferative activity. Both goniothalamin (1) and derivative 18 led to reactive oxygen species generation in PC-3 cells, which was probably a signal for caspase-dependent apoptosis. Treatment with derivative 18 promoted Annexin V/7-aminoactinomycin D double staining, which indicated apoptosis, and also led to G2 /M cell-cycle arrest. In vivo studies in Ehrlich ascitic and solid tumor models confirmed the antitumor activity of goniothalamin (1), without signs of toxicity. However, derivative 18 exhibited an unexpectedly lower in vivo antitumor activity, despite the treatments being administered at the same site of inoculation. Contrary to its in vitro profile, aza-goniothalamin (2) inhibited Ehrlich tumor growth, both on the ascitic and solid forms. Our findings highlight the importance of in vivo studies in the search for new candidates for cancer treatment.

The Neuromuscular Activity Of Bothriopsis Bilineata Smaragdina (forest Viper) Venom And Its Toxin Bbil-tx (asp49 Phospholipase A2) On Isolated Mouse Nerve-muscle Preparations.

The presynaptic action of Bothriopsis bilineata smaragdina (forest viper) venom and Bbil-TX, an Asp49 PLA2 from this venom, was examined in detail in mouse phrenic nerve-muscle (PND) preparations in vitro and in a neuroblastoma cell line (SK-N-SH) in order to gain a better insight into the mechanism of action of the venom and associated Asp49 PLA2. In low Ca(2+) solution, venom (3μg/ml) caused a quadriphasic response in PND twitch height whilst at 10μg/ml the venom additionally induced an abrupt and marked initial contracture followed by neuromuscular facilitation, rhythmic oscillations of nerve-evoked twitches, alterations in baseline and progressive blockade. The venom slowed the relaxation phase of muscle twitches. In low Ca(2+), Bbil-TX [210nM (3μg/ml)] caused a progressive increase in PND twitch amplitude but no change in the decay time constant. Venom (10μg/ml) and Bbil-TX (210nM) caused minor changes in the compound action potential (CAP) amplitude recorded from sciatic nerve preparations, with no significant effect on rise time and latency; tetrodotoxin (3.1nM) blocked the CAP at the end of the experiments. In mouse triangularis sterni nerve-muscle (TSn-m) preparations, venom (10μg/ml) and Bbil-TX (210nM) significantly reduced the perineural waveform associated with the outward K(+) current while the amplitude of the inward Na(+) current was not significantly affected. Bbil-TX (210nM) caused a progressive increase in the quantal content of TSn-m preparations maintained in low Ca(2+) solution. Venom (3μg/ml) and toxin (210nM) increased the calcium fluorescence in SK-N-SH neuroblastoma cells loaded with Fluo3 AM and maintained in low or normal Ca(2+) solution. In normal Ca(2+), the increase in fluorescence amplitude was accompanied by irregular and frequent calcium transients. In TSn-m preparations loaded with Fluo4 AM, venom (10μg/ml) caused an immediate increase in intracellular Ca(2+) followed by oscillations in fluorescence and muscle contracture; Bbil-TX did not change the calcium fluorescence in TSn-m preparations. Immunohistochemical analysis of toxin-treated PND preparations revealed labeling of junctional ACh receptors but a loss of the presynaptic proteins synaptophysin and SNAP25. Together, these data confirm the presynaptic action of Bbil-TX and show that it involves modulation of K(+) channel activity and presynaptic protein expression.