Thin layer chromatography of certain preformed metal complex dyes used in biological staining.

Thin-layer chromatographic systems are described for the analysis of various performed metal complex dyes (aluminon-chromium (III), carminic acid-aluminum, carminic acid-chromium (III), carminic acid-iron (III), celestine blue-chromium (III), gallamine blue-chromium (III), gallocyanin-chromium (III), hematein-aluminum, hematein-chromium (III), purpurin-aluminum) and their parent dyes. Certain of these dyes have also been analysed by agar-gel electrophoresis or gel-filtration chromatography. The merits of the three analytical methods are discussed.     

Notes on Technic: Amyl Acetate as a Clearing agent for Refractory Plant Materials

Thin-layer chromatographic systems are described for the analysis of various preformed metal complex dyes (aluminon-chromium(III), carminic acid-aluminum, carminic acid-chromium(III), carminic acid-iron(III), oelestine blue-chromium(III), gallamine blue-chromium(III), gallocyanin-chromium(III), hematein-aluminum, hematein-chromium(III), purpurin-aluminum) and their parent dyes. Certain of there dyes have also been analysed by agar-gel electrophoresis or gel-filtration chromatography. The merits of the three analytical methods are discussed.     

The red insect dyes: carminic,kermesic and laccaic acids and their derivatives

Three groups of insect dyes are described: three cochineal dyes, the kermes dye and the lac dye. The major color components are carminic acid, kermesic acid and laccaic acids, respectively. These dyes are red anthraquinone derivatives. The chemical structures are described. All of these dyes have extensive histories that are related briefly; however, only American cochineal is of commercial importance today. Two manufactured derivatives of cochineal, carmine and acid-stable carmine (4-aminocarminic acid) are described in some detail including the chemical identity, toxicity, stability, and staining and non-staining applications.     

Separation and recovery of food coloring dyes using aqueous biphasic extraction chromatographic resins

Aqueous biphasic systems (ABS) and aqueous biphasic extraction chromatographic (ABEC) resins are currently under investigation for their utility in the removal of color from textile plant wastes. The structures of several widely used food colorings, suggest that these dyes would also be retained on the resins. In work currently in progress, we have begun to investigate the retention and resolution of several common food colorings including indigo carmine, amaranth, carminic acid, erythrosin B, tartrazine and quinoline yellow. The relationship between the uptake of these dyes on ABEC resins in terms of the binding strengths and capacities of the resins and their partitioning behavior in ABS is illustrated. Some possible theoretical and practical approaches to the prediction of the partitioning and retention behavior is discussed.     

A method for determining identity and relative purity of carmine, carminic acid and aminocarminic acid.

Carmine is one of the few dyes currently certified by the Biological Stain Commission that is not assayed for dye content. Existing assay methods are complex and do not differentiate the three cochineal derivatives carmine, carminic acid and aminocarminic acid. The latter dye is relatively new to the food trade as an acid-stable red colorant and may eventually enter the biological stains market. The assay proposed here is a two-step procedure using quantitative spectrophotometric analysis at high pH (12.5-12.6) followed by a qualitative scan of a low pH (1.90-2.10) solution. Carmine is distinct at high pH, and the remaining dyes are easily distinguished at low pH. Four instances of mislabeling are documented from 18 commercial products, but the mislabeled dyes were not certified dyes. Samples from nearly all lots of carmine certified by the Biological Stain Commission from 1920 to 2004 proved to be carmine, but they varied widely in dye content. Batches from 1920 through the 1940s were significantly richer in dye content. Variability has been extreme since 2000, and most of the poorest lots have been submitted since 1990.     

A method for determining identity and relative purity of carmine,carminic acid and aminocarminic acid

Carmine is one of the few dyes currently certified by the Biological Stain Commission that is not assayed for dye content. Existing assay methods are complex and do not differentiate the three cochineal derivatives carmine, carminic acid and aminocarminic acid. The latter dye is relatively new to the food trade as an acid-stable red colorant and may eventually enter the biological stains market. The assay proposed here is a two-step procedure using quantitative spectrophotometric analysis at high pH (12.5–12.6) followed by a qualitative scan of a low pH (1.90–2.10) solution. Carmine is distinct at high pH, and the remaining dyes are easily distinguished at low pH. Four instances of mislabeling are documented from 18 commercial products, but the mislabeled dyes were not certified dyes. Samples from nearly all lots of carmine certified by the Biological Stain Commission from 1920 to 2004 proved to be carmine, but they varied widely in dye content. Batches from 1920 through the 1940s were significantly richer in dye content. Variability has been extreme since 2000, and most of the poorest lots have been submitted since 1990.     

A method for determining identity and relative purity of carmine, carminic acid and aminocarminic acid

Carmine is one of the few dyes currently certified by the Biological Stain Commission that is not assayed for dye content. Existing assay methods are complex and do not differentiate the three cochineal derivatives carmine, carminic acid and aminocarminic acid. The latter dye is relatively new to the food trade as an acid-stable red colorant and may eventually enter the biological stains market. The assay proposed here is a two-step procedure using quantitative spectrophotometric analysis at high pH (12.5-12.6) followed by a qualitative scan of a low pH (1.90-2.10) solution. Carmine is distinct at high pH, and the remaining dyes are easily distinguished at low pH. Four instances of mislabeling are documented from 18 commercial products, but the mislabeled dyes were not certified dyes. Samples from nearly all lots of carmine certified by the Biological Stain Commission from 1920 to 2004 proved to be carmine, but they varied widely in dye content. Batches from 1920 through the 1940s were significantly richer in dye content. Variability has been extreme since 2000, and most of the poorest lots have been submitted since 1990.     

Enhancement of the antiviral and interferon-inducing activities of poly r(A-U) by carminic acid

Experiments have been designed to systematically examine the effects of carminic acid (CAR) on the antiviral/interferon-inducing activity of poly r(A-U), using the human foreskin fibroblast-vesicular stomatitis virus bioassay system. Modulation of the antiviral/interferon-inducing activity of poly r(A-U) by carminic acid was examined at fixed poly r(A-U) concentrations of 0.05 mM or 0.2 mM while varying the carminic acid concentrations to produce variable CAR/ribonucleotide ratios ranging from 1:16 to 2:1. Carminic acid and poly r(A-U) were tested individually at the concentrations employed in the CAR/poly r(A-U) combinations. Neither the carminic acid alone nor poly r(A-U) alone were effective antiviral agents/interferon inducers. The antiviral/interferon-inducing activity of poly r(A-U) was potentiated twelve-fold at CAR/ribonucleotide ratios in the region of 1/6 to 1/4. These results suggest a synergism between the poly r(A-U) and the carminic acid at the concentrations employed in this study.     

Anthraquinone dyes decolorization capacity of anamorphic Bjerkandera adusta CCBAS 930 strain and its HRP-like negative mutants

Cultures of the anamorphic fungus Bjerkandera adusta CCBAS 930 decolorizing, in stationary cultures, 0.01 % solutions of carminic acid and Poly R-478, were characterised by a strong increase in the activity of the horseradish peroxidase (HRP-like) and manganese-dependent peroxidase (MnP) at a low activity of lignin peroxidase. Genotypically modified mutants of B. adusta CCBAS 930: 930-5 and 930-14, with total or partial loss of decolorization capabilities relative to anthraquinonic dyes, showed inhibition of the activity of HRP-like peroxidase and MnP. Whereas, compared to the parental strain, in the mutant cultures there was an increase in the activity of lignin peroxidase and laccase. The paper presents a discussion of the role of the studied enzymatic activities in the process of decolorization of anthraquinonic dyes by the strain B. adusta CCBAS 930.     

Revised procedures for the certification of carmine (C.I. 75470, Natural red 4) as a biological stain

Carmine is one of the original dyes certified by the Biological Stain Commission (BSC). Until now it has lacked both an assay procedure for dye content and a means to positively identify the dye. The methods for testing carmine in the laboratory of the BSC have been revised to include spectrophotometric examination at pH 12.5-12.6 to determine that the dye is carmine (λ_max=530-335 nm). The maximum absorbance of a solution containing 100 mg of dye per liter of water, adjusted to pH 12.5-12.6, which provides a relative measure of dye content, should lie in the range 1.2 to 1.8. If the dye is not carmine, spectrophotometry at pH 1.9-2.1 shows whether it is carminic acid (λ_max=490-500 nm) or 4-aminocarminic acid (λ_max=525-530 nm). The latter two dyes, which are also called carmine when sold as food colorants, have physical properties different from those of true carmine. The functional tests for carmine as a biological stain are Orth's lithium-carmine method for nuclei, Southgate's mucicarmine method for mucus, and Best's carmine method for glycogen.     

Decolorization of water and oil-soluble azo dyes by <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> and <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis>

The capability of Lactobacillus acidophilus and Lactobacillus fermentum to degrade azo dyes was investigated. The bacteria were incubated under anaerobic conditions in the presence of 6 μg/ml Methyl Red, Ponceau BS, Orange G, Amaranth, Orange II, and Direct Blue 15; 5 μg/ml Sudan I and II; or 1.5 μg/ml Sudan III and IV in deMann–Rogosa–Sharpe broth at 37°C for 36 h, and reduction of the dyes was monitored. Both bacteria were capable of degrading all of the water-soluble azo dyes to some extent. They were also able to completely reduce the oil-soluble diazo dyes Sudan III and IV but were unable to reduce the oil-soluble monoazo dyes Sudan I and II to any significant degree in the concentrations studied. Growth of the bacteria was not significantly affected by the presence of the Sudan azo dyes. Metabolites of the bacterial degradation of Sudan III and IV were isolated and identified by liquid chromatography electrospray ionization tandem mass spectrometry analyses and compared with authentic standards. Aniline and o-toluidine (2-methylaniline), both potentially carcinogenic aromatic amines, were metabolites of Sudan III and IV, respectively.     

Transforming Benzophenoxazine Laser Dyes into Chromophores for Dye‐Sensitized Solar Cells: A Molecular Engineering Approach

The refunctionalization of a series of four well‐known industrial laser dyes, based on benzophenoxazine, is explored with the prospect of molecularly engineering new chromophores for dye‐sensitized solar cell (DSC) applications. Such engineering is important since a lack of suitable dyes is stifling the progress of DSC technology. The conceptual idea involves making laser dyes DSC‐active by chemical modification, while maintaining their key property attributes that are attractive to DSC applications. This molecular engineering follows a stepwise approach. First, molecular structures and optical absorption properties are determined for the parent laser dyes: Cresyl Violet ( 1 ), Oxazine 170 ( 2 ), Nile Blue A ( 3 ), Oxazine 750 ( 4 ). These reveal structure‐property relationships which define the prerequisites for computational molecular design of DSC dyes; the nature of their molecular architecture (D‐π‐A) and intramolecular charge transfer. Second, new DSC dyes are computationally designed by the in silico addition of a carboxylic acid anchor at various chemical substitution points in the parent laser dyes. A comparison of the resulting frontier molecular orbital energy levels with the conduction band edge of a TiO₂ DSC photoanode and the redox potential of two electrolyte options I^?/I₃^? and Co^(II/III)tris(bipyridyl) suggests promise for these computationally designed dyes as co‐sensitizers for DSC applications.     

Specific Staining of Glycogen with Haematoxylin and Certain Anthraquinone Dyes

Specific staining of glycogen in rat liver fixed in chilled 80% alcohol, chilled formol alcohol or 10% neutral formalin has been accomplished with acid alizarin blue SWR, alizarin brilliant blue BS, alizarin red S, gallein, haematein, and haematoxylin solutions. TO prepare a staining solution, 1 gm dye, 1 gm K₂CO₃ and 5 gm KCl were dissolved by heating in 60 ml of water. Concentrated NH₄OH (0.880 sp.gr.), 15 ml, followed by 15 ml of dry methanol were added to 20 ml of the cooled solution. Paraffi sections were stained for 5 min, rinsed in dry methanol, cleared in xylene, and mounted in D.P.X. The high specificity obviated the need for counterstaining: nuclei and cytoplasm were unstained. Precipitation of stain onto the slide was rare. As all the dyes carried, like carminic acid, numerous groups capable of forming hydrogen bonds, it is suggested that the staining mechanism involved hydrogen bonding.     

Comparison of historic Grübler dyes with modern counterparts.

Seventeen Grübler dyes produced in Germany between 1880 and 1939 were examined in this study. These dyes were: fuchsin-bacillus, diamond fuchsin, fuchsin S acid, rubin S, safranin O water soluble, safranin yellowish water soluble, methyl eosin, Sudan III, scarlet R, auramine, orange G, aniline blue, pyronin, carmine, lithium carmine, hematein and aurantia. Spectrophotometry and staining characteristics were used to determine the maximum absorbance and efficacy of each dye in common staining techniques. The spectral curves and staining characteristics of these dyes compared well with modern dyes used as controls. Fuchsin bacillus and diamond fuchsin are synonyms for basic fuchsin. Fuchsin S acid and rubin S are synonyms for acid fuchsin. The scarlet R sample was the same as the Sudan III. The two safranins were the same. The basic fuchsin samples were unsuitable for preparation of Schiff's reagent. Both basic fuchsin and pyronin samples were less concentrated than modern counterparts. It is noteworthy that the dyes worked well after up to 100 years in storage, and this observation indicates that dyes can have a long shelf life when stored in cool, dry, air-tight conditions.     

Hair Trace Element Levels in Han and Indigenous Hualien Inhabitants in Taiwan

The boron content was determined in 42 different foods consumed in Istanbul, Turkey. Eleven species of fruit, ten species of vegetable, eight species of food of animal origin, four species of grain, two species of nuts, two species of legume, and five other kinds of foods were included to this study. They were analyzed by two methods: Inductively coupled plasma mass spectrometry (ICP-MS) technique and carminic acid assay, and the results of two methods were also compared. Boron concentration in foods ranged between 0.06–37.2 mg/kg. Nuts had the highest boron content while foods of animal origin had the lowest. A strong correlation was found between the results of the carminic acid assay and the ICP-MS technique (p = 0.0001, Pearson correlation coefficient: r = 0.956). Bland Altman analysis also supported this correlation. ICP-MS is one of the most common, reliable, and powerful method for boron determination. The results of our study show that spectrophotometric carminic acid assay can provide similar results to ICP-MS, and the boron content in food materials can be also determined by spectrophotometric method.

     

Interaction of cibacron blue 3G-A and related dyes with nucleotide-requiring enzymes

Cibacron Blue 3G-A (I), the chromophore in Blue Dextran, its structural isomer Cibacron Brilliant Blue BR-P (II), and two other structural analogs (III, IV) were used to probe the nucleotide binding sites of selected kinases and dehydrogenases. Inhibition studies indicate that the portion of the dye molecule necessary for effective inhibition of nucleotide binding is a structure similar to 1-amino-4(4′-aminophenylamino)-anthraquinone-2,3′-disulfonic acid (ASSO; III). The strong inhibition exhibited by these dyes is likely to be due to interaction with specific nucleotide binding sites, irrespective of the presence of a “dinucleotide fold” in the proteins' supersecondary structure.     

Evaluation of metabolism of azo dyes and their effects on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> metabolome

Dyes containing one or more azo linkages are widely applied in cosmetics, tattooing, food and drinks, pharmaceuticals, printing inks, plastics, leather, as well as paper industries. Previously we reported that bacteria living on human skin have the ability to reduce some azo dyes to aromatic amines, which raises potential safety concerns regarding human dermal exposure to azo dyes such as those in tattoo ink and cosmetic colorant formulations. To comprehensively investigate azo dye-induced toxicity by skin bacteria activation, it is very critical to understand the mechanism of metabolism of the azo dyes at the systems biology level. In this study, an LC/MS-based metabolomics approach was employed to globally investigate metabolism of azo dyes by Staphylococcus aureus as well as their effects on the metabolome of the bacterium. Growth of S. aureus in the presence of Sudan III or Orange II was not affected during the incubation period. Metabolomics results showed that Sudan III was metabolized to 4-(phenyldiazenyl) aniline (48%), 1-[(4-aminophenyl) diazenyl]-2-naphthol (4%) and eicosenoic acid Sudan III (0.9%). These findings indicated that the azo bond close to naphthalene group of Sudan III was preferentially cleaved compared with the other azo bond. The metabolite from Orange II was identified as 4-aminobenzene sulfonic acid (35%). A much higher amount of Orange II (~90×) was detected in the cell pellets from the active viable cells compared with those from boiled cells incubated with the same concentration of Orange II. This finding suggests that Orange II was primarily transported into the S. aureus cells for metabolism, instead of the theory that the azo dye metabolism occurs extracellularly. In addition, the metabolomics results showed that Sudan III affected energy pathways of the S. aureus cells, while Orange II had less noticeable effects on the cells. In summary, this study provided novel information regarding azo dye metabolism by the skin bacterium, the effects of azo dyes on the bacterial cells and the important role on the toxicity and/or inactivation of these compounds due to microbial metabolism.