Sudan I Research Articles

Development and validation of rapid disequilibrium enzyme-linked immunosorbent assays for the detection of Methyl Yellow and Rhodamine B dyes in foods

Sensitive and specific enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of two illegal synthetic dyes: Methyl Yellow (MY) and Rhodamine B (RB) in food. Polyclonal antibodies were raised against synthesised immunogens and employed in unique direct disequilibrium ELISAs. The time of the assays was only twenty minutes (five minutes for each incubation step with sample and enzyme conjugate and ten minutes with enzyme substrate). The IC(50) for MY was in the range 1.4-4.2 ng mL(-1) and for RB 0.1-0.5 ng mL(-1). A simple sample preparation method was developed for the analysis of a range of sauces. In the case of spices a dispersive solid phase extraction was applied to purify the extracts. The testing of twenty samples took approximately one and a half hours (including sample preparation and analysis). Both assays were validated according to the Commission Decision 2002/657/EC criteria for use in sauces and spices. The detection capability for MY in sauces and spices was determined to be less than 15 ng g(-1) and 50 ng g(-1), respectively and for RB, 10 ng g(-1) for both types of food samples. The precision of the developed assays was determined in a repeatability study. The intra- and inter-assay coefficients of variation were less than 25% for both tests and matrix types. The simplicity and performance of both assays indicate that they will be very reliable screening methods for the detection of the illegal dyes MY and RB in a range of food products.

Efficacy of ethanolic spore extract of <i>Lycopodium clavatum</i> in reducing induced hepatotoxicity and genotoxicity in mice

Ethanolic extract of spores of Lycopodium clavatum L., reportedly has profound effect against liver disorders, but lacks adequate experimental validation. To test this claim, healthy inbred Swiss albino mice, Mus musculus, were divided into different groups: Gr.I mice were fed normal diet (negative control); Gr.II - fed normal diet plus ethanol; Gr.III - fed two carcinogens of liver, [0.06% p-dimethyl aminoazobenzene (initiator) and 0.05% phenobarbital (promoter)] known to induce hepatotoxicity and genotoxicity; Gr.IV- mice fed ethanol plus both the carcinogens, and Gr.V- fed carcinogens plus spore extract of Lycopodium clavatum. They were sacrificed at day 90 and 120 for histological studies of liver, assay of cytotoxicity markers and assessment of genotoxicity using endpoints such as chromosome aberrations, micronuclei, mitotic index in bone marrow cells and sperm head anomaly. Additionally, western blot for p53 protein expression and matrix metalloproteinase (MMP) activity in liver was compared among different groups of treated and control mice to evaluate its therapeutic potentials. Compared to Gr.III and IV, less number of mice developed tumors in Gr.V along with significant reduction in hepatotoxicity and genotoxicity, thereby validating its potential use against liver ailments as a herbal remedy.Keywords: p-dimethyl aminoazobenzene, phenobarbital, plant extract, amelioration.

Investigation of Tetramethylenesulfonated Calix[4]resorcinarene Interactions with Azo Dyes in Aqueous Solution

The interaction of a macrocycle's aggregates with a guest molecule has special interest due to the double role, both of macrocycle cavity and self-associates, in the binding of the guests. Here, we report on the interactions of nonaggregated methyl-substituted (SCA1) and aggregated penthyl-substituted (SCA2) tetramethylenesulfonated calix[4]resorcinarenes with pH indicators methyl yellow (MY) and methyl orange (MO) in aqueous solutions. It was found that the pH of aqueous solutions of SCA1 and SCA2 depends on their concentration; besides, variation of the concentration of SCA1 and SCA2 results in a shift of the absorption maxima and of pH-sensitive azo dyes. Association of the marocycles with azo dyes was demonstrated to follow a proton-transfer mechanism accompanied by protonation of the dyes; it was found that excess of the macrocycle in solution with the universal buffer background shifts the pK(a) values and stabilizes the protonated form of the dyes. Consideration of interactions of small dye molecules with large molecular associates, containing both individual and multimolecule binding sites, gives a closer approximation of synthetic biomimetics to their natural prototypes.

Intrinsic apoptosis and NF-κB signaling are potential molecular targets for chemoprevention by black tea polyphenols in HepG2 cells in vitro and in a rat hepatocarcinogenesis model in vivo

Antiproliferative and apoptosis inducing effects of black tea polyphenols (Polyphenon-B) on HepG2 cells in vitro and in a rat hepatocarcinogenesis model in vivo were investigated. Viability of HepG2 cells was evaluated by the MTT assay, and apoptosis by AO–EB and DAPI staining, cell cycle analysis, and annexin V-PI assay. For the in vivo study, male Sprague–Dawley rats treated with dimethylaminoazobenzene (DAB) (0.06%) were used. The expression of Bcl-2 and NF-κB family members were analyzed by immunoblotting. Administration of Polyphenon-B induced dose-dependent inhibition of growth of HepG2 cells and reduced tumor incidence in DAB administered animals. HepG2 cells also exhibited morphological features characteristic of apoptotic cell death. In addition, administration of Polyphenon-B increased the expression of Bax, tBid, Smac/Diablo, cytochrome C, Apaf-1, caspases, and IκB with PARP cleavage, and decreased the expression of Bcl-2, Bcl-xL, pBad, NF-κB, p-IκB-α, IKKβ and Ub in both HepG2 cells and in DAB-treated animals. These results provide evidence that Polyphenon-B effectively inhibits proliferation and induces apoptosis both in vitro and in vivo by inhibiting NF-κB, and inducing intrinsic apoptosis by modulating the expression of a network of interrelated molecules eventually culminating in caspase-mediated cell death.

Helianthin induces antiproliferative effect on human glioblastoma cells in vitro

A major focus of brain cancer research today is to translate understanding of glioma biology into advances in treatment, by exploring the potential of target therapy. Here we investigated the ability of three compounds belonging to the chemical class of azo dyes (methyl red, methyl yellow, and helianthin) to inhibit glioblastoma (GB) cell growth in vitro. Our results showed that helianthin induced cytotoxicity in two GB cell cultures, cell lines 18 and 38, whereas methyl red and methyl yellow were not cytotoxic. The effect of helianthin on EGFR, IGF-1R, and their common intracellular signaling via PI3-K and ERK1/2 was also analyzed. Treatment with helianthin down-regulated EGFR and IGF-1R activity in both cell lines. Helianthin treatment blocked ERK1/2 phosphorylation without affecting PI3K activity in cell line 18 and reduced both PI3K and ERK1/2 in GB 38cell line. The cell death was accompanied by degradation of PARP without affecting BCL2 expression in both GB cell cultures. Because of the genetic heterogeneity of malignant gliomas, we tested the effect of helianthin on other two primary GB lines (11 and 15) and two early-passage GB cultures (BT1GB and BT2GB), to assess the general nature of the anti-tumor effect of the drug in GB cells. We found that helianthin treatment induced cell death in all the GB cell cultures analyzed. To our knowledge, this is the first report indicating that helianthin can reduce GB cell growth.

Chelidonium majus 30C and 200C in induced hepato-toxicity in rats

Homeopathy is a popular form of complementary and alternative medicine and is used to treat for certain liver ailments. To analyze the efficacy of homeopathic Chelidonium majus (Chel) 30C and 200C in amelioration of experimentally induced hepato-toxicity in rats. Rats were randomized into six sub-groups: negative control; negative control+EtOH; positive control; positive control+EtOH group; Chel 30; Chel 200. Rats were sacrificed at day 30, 60, 90 and 120; various toxicity biomarkers and pathological parameters were evaluated. Gelatin zymography for determination of metalloproteinases activity and Western blot of p53 and Bcl-2 proteins were also employed. All analyses were observer blind. Chronic feeding of p-dimethyl amino azo benzene (p-DAB) and phenobarbital (PB) elevated the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transferase (GGT), lactate dehydrogenase (LDH), triglyceride, cholesterol, creatinine and bilirubin and lowered the levels of glutathione (GSH), glucose-6-phosphate dehydrogenase (G-6-PD), catalase and HDL-cholesterol. There were statistically significant modulations of these parameters in the treated animals, compared to positive controls. In both treated groups, there was downregulation of metalloproteinases, p53 and Bcl-2 proteins compared to over-expression in the positive control groups. Both the potencies of Chel exhibited anti-tumor and anti-oxidative stress potential against artificially induced hepatic tumors and hepato-toxicity in rats. More studies are warranted.

Azo-hydrazo Tautomerism and Inclusion Complexation of 1-phenylazo-2-naphthols with Various Solvents and β-cyclodextrin

Spectral characteristics of sudan I (SDI), sudan II (SDII) and mordant violet-5 (MV5) have been studied in various solvents and beta-cyclodextrin (beta-CD). The inclusion complex of the above molecules with beta-CD was analyzed by UV-visible, fluorometry, and DFT methods. The solvent study shows that azo-hydrazo tautomer is present in these molecules. The increase in the fluorescence intensity and a large bathochromic shift in the S(1) state indicate these molecules forms 1:1 inclusion complex with beta-CD.

Multivariate image analysis-thin layer chromatography (MIA-TLC) for simultaneous determination of co-eluting components

This paper addresses the solution of peak overlapping, as a fundamental problem in TLC, by multivariate analysis of the images recorded by a digital camera. We report the results of our study on the application of multivariate image analysis (MIA) for simultaneous determination of several species on thin layer chromatography (TLC) sheet for the first time. An imaging system, composed of a dark cabinet, a digital camera and a multivariate image analysis program, was prepared for recording the images of TLC plates after development of a multi-component solution. The written program was able to produce 2- and 3-dimensional chromatograms of the solutions, which were subsequently used as inputs of partial least squares, as an efficient multivariate calibration method. The ability of the proposed MIA-TLC method for simultaneous determination of the co-eluting components was validated by analysis of ternary synthetic mixtures of indicators of highly overlapped chromatograms (i.e., methyl yellow, bromocresol green and creseol red) and a real mixture of nifedipine and its photo-degradation product. By application of different strategies like principal component analysis and variable selection, models were obtained that could estimate the concentration of indicators in the external prediction set with relative errors of lower than 10% and in most cases lower than 5%.

アゾベンゼン系フォトメカニカル結晶

Molecular machines based on the conversion of changes in molecular geometry to mechanical motion in macroscale materials are interesting from the perspective of basic research and offer a wide range of potential applications. Such machines are amenable to remote operation with external stimuli such a photon impingement and do not require direct contact. Azobenzenes are typical chromophores that undergo trans-cis photoisomerization. We have recently found photomechanical bending of trans-4-(dimethylamino)azobenzene microcrystals. Upon UV irradiation, the platelike microcrystal bent quickly in the direction opposite to the light source, reaching the maximum deflection after 0.5 s. The microcrystal returned to its initial flat shape 30 s after the illumination was stopped. This mechanical motion was reversible over repeated 100 cycles of UV irradiation. The bending effect was attributed to a gradient in the extent of UV-induced trans-cis photoisomerization as a function of light penetration, causing the expansion of the irradiated crystal surface to result in a bent macrostructure.

Lycopodine from Lycopodium clavatum extract inhibits proliferation of HeLa cells through induction of apoptosis via caspase-3 activation

Crude ethanolic extract of the plant Lycopodium clavatum has long been used in complementary and alternative medicine for treating various liver ailments and Alzheimer's disease. It has also been claimed to have potential anti-cancer properties in vivo in mice chronically fed liver carcinogens, p-dimethylamino azobenzene (initiator) and phenobarbital (promoter). Incidentally, crude ethanolic extract of Lycopodium clavatum is a mixture of some 201 alkaloids. In order to ascertain if any major fraction can be attributed to have pronounced anti-cancer effect, we examined this major fraction by eluting the crude extract in petroleum ether:ethyl aetate (17:3 vol/vol;) solvent and tried to understand its underlying mechanism. Studies on morphological changes, cell viability and cytotoxicity by microscopy and FACS, Western blot and immunofluorescence of Bcl-2, Bax, cytochrome c, caspase-3 were conducted. Lycopodine was found to induce chromatin condensation, inter-nucleosomal DNA fragmentation and enhanced cell population in sub-G1 region along with increase in reactive oxygen species generation and mitochondrial membrane potential depolarization, release of cytochrome c and activation of caspase-3 which are the events closely involved in apoptosis. An overall analysis of results showed that Lycopodine considerably inhibited growth of HeLa cells which indicates its potential use in chemotherapy.

Molecular dynamics simulation study of probe diffusion in liquid n-alkanes

We performed molecular dynamics simulations for the probe diffusion and friction dynamics of Lennard-Jones (LJ) particles modelled for methyl yellow (MY) in liquid n-alkanes of C12–C200 at temperatures of 318, 418, 518 and 618 K. Two LJ particles are chosen: MY1 with a mass of 114 g/mol, LJ parameters of σ = 4.0 Å and ϵ = 0.4 kJ/mol, and MY2 with a mass of 225 g/mol, σ = 6.0 Å and ϵ = 0.6 kJ/mol. We observed a clear transition in the power law dependence of MY2 diffusion on the molecular weight of n-alkanes at lower temperatures of 318 and 418 K. The sharp transitions occur near n-dotriacontane (C32). However, no such transition is found for MY1 at all the temperatures and for MY2 at higher temperatures of 518 and 618 K. We also calculated the friction constants of both MY probe molecules in liquid n-alkanes. For the larger probe molecule (MY2), at lower temperatures, a large deviation of slope from the linear dependence of the friction of MY2 on the chain length of n-alkane is observed, which indicates a large reduction of friction in longer chains when compared with the shorter chains, enhancing the diffusion of the probe molecules (MY2).

Preliminary note on the specific gravity of liver during the course of liver cancer production by butter yellow feeding.

Preliminary note on the specific gravity of liver during the course of liver cancer production by butter yellow feeding.

Visible-Light Photocontrol of (E)/(Z) Isomerization of the 4-(Dimethylamino)azobenzene Pseudo-Nucleotide Unit Incorporated into an Oligonucleotide and DNA Hybridization in Aqueous Media

We demonstrate significant photoresponsivity in aqueous media to visible light of pseudo-oligonucleotides possessing 4-(dimethylamino)azobenzene (4-DMAzo) side chains. The spectrum of the 4-DMAzo moiety during 436 nm light irradiation at pH >9 was clearly different from that of the all (E)-form, indicating the presence of the (Z)-form. Thermal (Z)-to-(E) recovery isomerization was faster at pH 9 (k Z -E = ∼101 s−1) than at pH 11; however, addition of 50% ethanol significantly slowed the thermal recovery isomerization at pH 9 (k Z -E = ∼2 s−1) and increased the magnitude of the spectral changes. Significant photoregulation of DNA hybridization by visible light was demonstrated under this condition.

Chromic materials for responsive surface-enhanced resonance Raman scattering systems: a nanometric pH sensor

The use of chromic materials for responsive surface-enhanced resonance Raman scattering (SERRS) based nanosensors is reported. The potential of nano-chromic SERRS is demonstrated with the use of the halochrome methyl yellow to fabricate an ultrasensitive pH optical sensor. Some of the challenges of the incorporation of chromic materials with metal nanostructures are addressed through the use of computational calculations and a comparison to measured SERRS and surface-enhanced Raman scattering (SERS) spectra is presented. A strong correlation between the measured SERRS and the medium's proton concentration is demonstrated for the pH range 2-6. The high sensitivity achieved by the use of resonance Raman conditions is shown through responsive SERRS measurements from only femtolitres of volume and with the concentration of the reporting molecules approaching the single molecule regime.

Biochemical studies in the course of the development of liver cancer by butter yellow

Biochemical studies in the course of the development of liver cancer by butter yellow

Asparaginase activity in tissues of the rats fed with carcinogens, p-dimethylaminoazobenzene and 2-acetylaminofluorene.

Asparaginase activity in tissues of the rats fed with carcinogens, p-dimethylaminoazobenzene and 2-acetylaminofluorene.

Diffusion of Probe Molecule in Small Liquid n-Alkanes: A Molecular Dynamics Simulation Study

The probe diffusion and friction constants of methyl yellow (MY) in liquid n-alkanes of increasing chain length were calculated by equilibrium molecular dynamics (MD) simulations at temperatures of 318, 418, 518 and 618 K. Lennard-Jones particles with masses of 225 and 114 g/mol are modeled for MY. We observed that the diffusion constant of the probe molecule follows a power law dependence on the molecular weight of nalkanes, DMY ~M −γ well. As the molecular weight of n-alkanes increases, the exponent γ shows sharp transitions near n-dotriacontane (C32) for the large probe molecule (MY2) at low temperatures of 318 and 418 K. For the small probe molecule (MY1) DMY1 in C12 to C80 at all the temperatures are always larger than Dself of n-alkanes and longer chain n-alkanes offer a reduced friction relative to the shorter chain n-alkanes, but this reduction in the microscopic friction for MY1 is not large enough to cause a transition in the power law exponent in the log-log plot of DMY1 vs M of n-alkane. For the large probe molecule (MY2) at high temperatures, the situation is very similar to that for MY1. At low temperatures and at low molecular weights of n-alkanes, DMY2 are smaller than Dself of n-alkanes due to the relatively large molecular size of MY2, and MY2 experiences the full shear viscosity of the medium. As the molecular weight of n-alkane increases, Dself of n-alkanes decreases much faster than DMY2 and at the higher molecular weights of n-alkane, MY2 diffuses faster than the solvent fluctuations. Therefore there is a large reduction of friction in longer chains compared to the shorter chains, which enhances the diffusion of MY2. The calculated friction constants of MY1 and MY2 in liquid n-alkanes supported these observations. We deem that this is the origin of the so-called “solventoligomer” transition.

A highly sensitive and selective immunonanogold resonance scattering spectral assay for sudan I

A sensitive and selective resonance scattering spectral (RSS) assay was proposed for the determination of sudan I (SDI), using 10 nm nanogold to label the antibody against sudan I (anti-SDI Ab) to obtain a RSS probe for SDI. The immunonanogold reaction between nanogold-labelled anti-SDI Ab and SDI took place in pH 4.92 KH2PO4–Na2HPO4 buffer solution and in the presence of polyethylene glycol (PEG)-6000, and the intensity of resonance scattering peak at 580 nm decreased greatly. The decreased intensity ΔI580 nm was proportional to the concentration of SDI in the range of 0.23–45.0 ng mL−1. The linear regression equation was calculated as ΔI580nm = 1.20c + 2.01 (R = 0.9975, n = 6), with a detection limit (3σ) of 0.13 ng mL−1. The SDI in egg samples was assayed, with satisfactory results.

How I became a biochemist

How I became a biochemist

Methyl orange removal over Zn-incorporated TiO 2 photo-catalyst

This study focuses on the removal of the methyl orange (sodium p-dimethyl amino azo benzene sulfonate) of a representative dye compound in a liquid photo-system using nanometer-sized Zn-incorporated TiO 2 synthesized by a solvothermal method. The Zn-incorporated TiO 2 particles exhibited a mixed structure of anatase of TiO 2 and ZnTiO 3 with a particle size of below 20 nm. The absorbance was shifted to the higher wavelength on 2.0 mol% Zn-incorporated TiO 2 compared to pure TiO 2. In the XRD pattern, the structure of ZnTiO 3 was seen at 2 θ = 31.75. The XPS result of O 1s confirmed that 2.0 mol% Zn–TiO 2 had a higher hydrophilic property (Me–OH/Me–O = 0.46) than pure TiO 2 (Me–OH/Me–O = 0.25) did. Based on these results, the photodecomposition for methyl orange in liquid reaction was enhanced over Zn-incorporated TiO 2 compared with pure TiO 2: methyl orange of 10 ppm was completely decomposed after 120 min over 2.0 mol% Zn-incorporated TiO 2.