Fast Blue RR Research Articles

Color determination method and evaluation of methods for the detection of cannabinoids by thin-layer chromatography (TLC).

Cannabis sativa is the drug of abuse most cultivated, trafficked, and consumed worldwide. One of several techniques used to detect cannabinoids is based on the thin-layer chromatography (TLC). However, the designation of the colors observed can be inaccurate and not reproducible. The designation of colors goes beyond physical and physiological aspects, because what is conventionally called color is a socio-cultural construction. Thus, the objective of this paper was to evaluate the different TLC methods to detection of cannabinoids, and apply standardization method in naming of colors. TLC analysis performed using silica gel 60 F254 as a stationary phase. Three mobile phase compositions [hexane:chloroform (8:2 v:v), hexane:ethyl ether (8:2 v:v), and chloroform:hexane (8:2 v:v)], as well as, two different solutions of Fast Blue B salt (FBBS, Azoic Diazo No. 48) and Fast Blue RR (FBRR, Azoic Diazo No. 24) were evaluated. Determination of colors names was realized through the Sci-Chromus® software. The best resolution was obtained using hexane:ethyl ether (8:2 v:v) as a mobile phase. It was observed that although the cannabidiol (CBD), delta-9-tetrahydrocannabinol (Δ9 -THC), cannabinol (CBN), and cannabigerol (CBG) were detect using both the FBBS- and FBRR-acidified solutions, the best visualization was achieved using the latter reagent. To the best of our knowledge, this is the first study that applied and demonstrated a method for standardization and denomination of colors in the TLC analysis of cannabinoids. This method was able to reduce the subjectivity in naming the colors observed and presented several application possibilities.

Identification of Carboxylesterase, Butyrylcholinesterase, Acetylcholinesterase, Paraoxonase, and Albumin Pseudoesterase in Guinea Pig Plasma through Nondenaturing Gel Electrophoresis.

Drugs to protect against nerve agent toxicity are tested in animals. The current preferred small animal model is guinea pigs because their plasma bioscavenging capacity resembles that of NHP. We stained nondenaturing polyacrylamide slab gels with a variety of substrates, inhibitors, and antibodies to identify the esterases in heparinized guinea pig plasma. An intense band of carboxylesterase activity migrated behind albumin. Minor carboxylesterase bands were revealed after background activity from paraoxonase was inhibited by using EDTA. The major butyrylcholinesterase band was a disulfide-linked dimer. Incubation with the antihuman butyrylcholinesterase antibody B2 18-5 shifted the butyrylcholinesterase dimer band to slower migrating complexes. Carboxylesterases were distinguished from butyrylcholinesterase by their sensitivity to inhibition by bis-p-nitrophenyl phosphate. Acetylcholinesterase tetramers formed a complex with the antihuman acetylcholinesterase antibody HR2. Organophosphorus toxicants including cresyl saligenin phosphate, dichlorvos, and chlorpyrifos oxon irreversibly inhibited the serine esterases but not paraoxonase. Albumin pseudoesterase activity was seen in gels stained with α- or β-naphthyl acetate and fast blue RR. We conclude that guinea pig plasma has 2 types of carboxylesterase, butyrylcholinesterase dimers and 5 minor butyrylcholinesterase forms, a small amount of acetylcholinesterase tetramers, paraoxonase, and albumin pseudoesterase activity. A knockout mouse with no carboxylesterase activity in plasma is available and may prove to be a better model for studies of nerve agent toxicology than guinea pigs.

Histochemical localization of urushiols in stems and leaflets of Schinopsis lorentzii and S. marginata using diazonium salts

The trees Schinopsis lorentzii and S. marginata produce alk(en)ylcatechols, also known as urushiols, which function in plant defense as antifungals, antifeedants and cause contact dermatitis in human beings. Here, we propose the use of the diazonium salts Fast Blue B ½ZnCl2 and Fast Blue RR ZnCl2 for the in situ histochemical localization of urushiols in stems and leaflets of S. lorentzii and S. marginata. The main surface and internal anatomical features of the aerial plant parts were visualized with cresyl violet and astra blue safranin. Extracts from leaflets and stems were separated by TLC and revealed with aqueous solutions of 0.5% Fast Blue B ½ZnCl2 in 5% acetic acid (FBB), 0.5% Fast Blue RR ZnCl2 in 5% KOH (FBRR) and 5% FeCl3. Fresh cross sections of leaflets and stems were also stained with these chromogenic reagents. Mature leaflets and stems showed few glandular trichomes in their surfaces. Well developed phloem schizogenous ducts were uniformly distributed in the leaflet mesophylls and radially organized in the cross sections of the stems. TLC of the extracts revealed with FBB, FBRR and 5% FeCl3 gave bluish black spots for urushiols. Very few constituents of the extracts other than the urushiols revealed the same colour after spraying with the diazonium salts. FBB and FBRR indicated the presence of urushiols in both the cavity of the phloem schizogenous ducts and their surrounding epithelial cells. Urushiols were not visualized neither in the cuticle of the aerial parts nor in the glandular trichomes. FBB was coupled to more individual polyphenol species than FBRR whereas FeCl3 was by far a less specific chromogenic reagent. Nevertheless, FBRR was more selective towards the urushiols than FBB. The exposure to urushiols should be possible only after the breakdown of leaflets and stems of Schinopsis trees.

Modulation of Osteogenesis in MC3T3-E1 Cells by Different Frequency Electrical Stimulation.

Electrical stimulation (ES) is therapeutic to many bone diseases, from promoting fracture regeneration to orthopedic intervention. The application of ES offers substantial therapeutic potential, while optimal ES parameters and the underlying mechanisms responsible for the positive clinical impact are poorly understood. In this study, we assembled an ES cell culture and monitoring device. Mc-3T3-E1 cells were subjected to different frequency to investigate the effect of osteogenesis. Cell proliferation, DNA synthesis, the mRNA levels of osteosis-related genes, the activity of alkaline phosphatase (ALP), and intracellular concentration of Ca2+ were thoroughly evaluated. We found that 100 Hz could up-regulate the mRNA levels of collagen I, collagen II and Runx2. On the contrary, ES could down-regulate the mRNA levels of osteopontin (OPN). ALP activity assay and Fast Blue RR salt stain showed that 100 Hz could accelerate cells differentiation. Compared to the control group, 100 Hz could promote cell proliferation. Furthermore, 1 Hz to 10 Hz could improve calcium deposition in the intracellular matrix. Overall, these results indicate that 100Hz ES exhibits superior potentialities in osteogenesis, which should be beneficial for the clinical applications of ES for the treatment of bone diseases.

Fast Blue RR-Siloxane Derivatized Materials Indicate Wound Infection Due to a Deep Blue Color Development.

There is a strong need for simple and fast methods for wound infection determination. Myeloperoxidase, an immune system-derived enzyme was found to be a suitable biomarker for wound infection. Hence, alkoxysilane-derivatized Fast Blue RR was immobilized via simple hydrolytic polymerization. The resulting enzyme-responsive siloxane layers were incubated with myeloperoxidase, wound fluid or hemoglobin. The reaction was monitored via HPLC measurements and the color development quantified spectrophotometrically. Myeloperoxidase was indeed able to oxidize immobilized Fast Blue RR leading to a blue colored product. No conversion was detected in non-infected wound fluids. The visible color changes of these novel materials towards blue enable an easy distinction between infected and non-infected wound fluids.

Chemical variability and antifungal activity of phenolic lipids from several Gramineae

An important class of phenolic lipids is alkylresorcinol (AR)-related compounds, which have been identified in many plants, fungi taxa, bacteria and some animals. Furthermore, there are several studies indicating that AR exhibit potent antimicrobial activity in plant defense possibly by their amphiphilic nature. This type of compounds can be found in large proportions in cereal grains such as wheat, rye, triticale and barley, whose occurrence is mainly located within the outer cuticle of the seed. As part of our research on antifungal compounds, the chemical profile and antifungal activity of extracts obtained from a set of 21 grasses were investigated. The chemical profile was determined by measuring total AR content (TAC) using Fast Blue RR protocol and by LC-DAD and LC-MS analysis of the resulting extracts for comparative drives by chemometrics through principal component analysis (PCA). TAC values indicated that triticale bran and wheat bran had the highest AR amounts with 45.0 and 33.1 mg AR/g dried material, respectively. In addition, the acetone-soluble extracts were evaluated in vitro against Fusarium oxysporum. The highest inhibition percentage against F. oxysporum was found for triticale bran-derived extract (81.9 and 76.9% at 10.0 and 1.0 µg/mL, respectively). The LC-DAD and LC-MS profiling for the 21 extracts led us to identify the some AR, involving AR with alkyl and alkenyl side chains possessing 13 – 19 carbons (tetradecenyl and hydroxytetradecenylresorcinol as main AR). Multivariate analysis (PCA) was performed on chromatographic data for all extracts. PCA-derived score plot showed that there is a relationship between the content of ARs and the antifungal activity. A bioguided fractionation leading to the isolation of the active AR is currently in the course.

Identification of oxidized protein hydrolase as a potential prodrug target in prostate cancer

BackgroundEsterases are often overexpressed in cancer cells and can have chiral specificities different from that of the corresponding normal tissues. For this reason, ester prodrugs could be a promising approach in chemotherapy. In this study, we focused on the identification and characterization of differentially expressed esterases between non-tumorigenic and tumorigenic prostate epithelial cells.MethodsCellular lysates from LNCaP, DU 145, and PC3 prostate cancer cell lines, tumorigenic RWPE-2 prostate epithelial cells, and non-tumorigenic RWPE-1 prostate epithelial cells were separated by native polyacrylamide gel electrophoresis (n-PAGE) and the esterase activity bands visualized using α-naphthyl acetate or α-naphthyl-N-acetylalaninate (ANAA) chiral esters and Fast Blue RR salt. The esterases were identified using nanospray LC/MS-MS tandem mass spectrometry and confirmed by Western blotting, native electroblotting, inhibition assays, and activity towards a known specific substrate. The serine protease/esterase oxidized protein hydrolase (OPH) was overexpressed in COS-7 cells to verify our results.ResultsThe major esterase observed with the ANAA substrates within the n-PAGE activity bands was identified as OPH. OPH (EC 3.4.19.1) is a serine protease/esterase and a member of the prolyl oligopeptidase family. We found that LNCaP lysates contained approximately 40% more OPH compared to RWPE-1 lysates. RWPE-2, DU145 and PC3 cell lysates had similar levels of OPH activity. OPH within all of the cell lysates tested had a chiral preference for the S-isomer of ANAA. LNCaP cells were stained more intensely with ANAA substrates than RWPE-1 cells and COS-7 cells overexpressing OPH were found to have a higher activity towards the ANAA and AcApNA than parent COS-7 cells.ConclusionsThese data suggest that prodrug derivatives of ANAA and AcApNA could have potential as chemotherapeutic agents for the treatment of prostate cancer tumors that overexpress OPH.

Development and validation of a micromethod for fast quantification of 5-n-alkylresorcinols in grains and whole grain products

A 96-well plate micromethod was developed to measure 5-n-alkylresorcinols (5nARs) in cereal grains and food derived products. The 5nARs reacted in alkaline alcoholic medium with Fast Blue RR ½ZnCl2 salt to yield coloured azo-derivatives. The highest sensitivity for 5nARs was obtained at 490nm with 0.025% ethanolic Fast Blue RR and 5% K2CO3. This reaction showed good linearity for olivetol (0.05–0.20μg). Contents of 5nARs determined in cereal grains and derived products by the new Fast Blue RR micromethod were highly correlated (R2=0.9944) with those obtained by a Fast Blue B method currently used. A Bland–Altman analysis indicated a small positive bias near to zero (R2=0.0401), suggesting that the methods can be interchangeably used. The new reaction is completed in 15min and the coloured products are read within the 15min after completion. The micromethod offers a fast analysis of 5nARs in cereal grains and derived products with low consumption of reagents and solvents.

Analysis of myeloperoxidase activity in wound fluids as a marker of infection

Neutrophilic polymorphonuclear leukocytes play a crucial role in the host defence against bacterial and fungal infections. They participate in the inflammatory response through the liberation of peptides and enzymes like myeloperoxidase (MPO). Therefore, MPO has a potential as a marker enzyme for the diagnosis of wound infection. Substrate specificities and reaction pathways of MPO were investigated for new MPO substrates: crystal violet, leuco crystal violet, fast blue RR (4-benzoylamino-2,5-dimethoxybenzenediazonium chloride hemi(zinc chloride) salt) and various systematically substituted model substrates based on 2,7-dihydroxy-1-(4-hydroxyphenylazo)naphtalene-3,6-disulphonic acid. In addition, fast blue RR was covalently bound to siloxanes allowing immobilization of the substrate, while cellobiosedehydrogenase was integrated for generation of hydrogen peroxide required by MPO. Elevated concentrations of MPO were found in infected wounds compared with non-infected wounds (92.2 ± 45.0 versus 1.9 ± 1.8 U/mL). Various soluble and immobilized substrates were oxidized by MPO in wound samples and the influence of substrate structure and reaction pathways were elucidated for selected compounds. Incubation of different MPO substrates with infected wound fluid samples resulted in a clear colour change in the case of elevated MPO concentrations, thus allowing early diagnosis of wound infection.

Twisted intramolecular charge transfer effects on fast violet B and fast blue RR: Effect of HP-α- and HP-β-cyclodextrins

Absorption, steady state and time resolved fluorescence spectra of fast blue RR (FBRR) and fast violet B (FVB) were studied in hydroxypropyl-α-cyclodextrin (HP-α-CD) and hydroxypropyl-β-cyclodextrin (HP-β-CD). Dual emission observed in both CDs suggested that twisted intramolecular charge transfer (TICT) is present in both molecules. The normal Stokes shifted bands originated from the locally excited state and the large Stokes shifted bands were due to the emission from a TICT state. The ratio of the TICT to the normal emission increased with HP-β-CD concentration, while it was constant upon addition of HP-α-CD. This behavior was in accordance with CD dependent decay times of the TICT and normal emissions, indicating the formation of different 1:1 FVB/CD and FBRR/CD inclusion complexes. The benzoyl moiety of FVB/FBRR was deeply encapsulated in the HP-β-CD cavity, whereas in HP-α-CD, it was exposed to the water phase. Semiempirical quantum optimization was also carried out to assign the encapsulation of the FBRR and FVB.

Dual Fluorescence of Fast Blue RR and Fast Violet B: Effects of Solvents and Cyclodextrin Complexation

Absorption and steady-state and time-resolved fluorescence spectra of fast blue RR (FBRR) and fast violet B (FVB) were studied in solvents with different polarities and in the presence of α- and β-cyclodextrins (CDs). Dual emission observed in nonpolar solvents suggested that the energy of the intramolecular charge transfer (ICT) state is lower than that of the locally excited state. The normal Stokes-shifted band originated from the locally excited state, and the large Stokes-shifted band was due to the emission from a planar intramolecular charge transfer (PICT) state. The ratio of the PICT emission to the normal emission increased with β-CD concentration, whereas it was constant upon addition of α-CD. This behavior is in accordance with CD-dependent decay times of PICT and normal emissions, indicating the formation of different 1:1 FBRR/CD inclusion complexes. The rise time for the PICT emission increased with β-CD concentration, whereas no rise time was observed in the case of the α-CD complex. The size of the dimethoxyaniline ring suggested that the orientation of FBRR in the β-CD complex was different from that in the α-CD complex. The benzamido moiety of FBRR is deeply encapsulated in the CD cavity, whereas the aniline ring is exposed to the hydrophilic part. Semiempirical quantum-mechanical (ΔE, ΔG, ΔH, ΔS, and HOMO-LUMO) calculations were also carried out to assign the encapsulation of FBRR and FVB.

Lipase activity in Thermus thermophilus HB8: Purification and characterization of the extracellular enzyme

In this study, the lipolytic activity of Thermus thermophilus HB8 was examined. The addition of various oils increased the production of extracellular lipolytic activity, while a combination of olive oil and glucose increased both extracellular and intracellular lipolytic activity. The oxygen transfer rate had a significant influence on both biomass and production of extra- or intra-cellular lipolytic activity. The formation of white halos due to the hydrolysis of oleic acid ester (Tween 80) in agar plates containing Nile Blue and the formation of Ca2+-oleate indicated the secretion of lipase. When the cell-free supernatant of cells grown in basal reach medium or the corresponding intracellular extract were electrophoresed under denatured and renatured conditions, using α-naphthyl acetate and Fast Blue RR, major bands at 56 kDa or 62 and 32 kDa were observed, respectively. The 56 kDa extracellular enzyme was partial purified and characterized. Its peak of activity occurred at 80°C and pH 7.0, while the T1/2 was 1 h at 100°C. The Km of the partial purified enzyme was 1 mM and the Vmax was 0.044 U/mL/min when using p-nitrophenyl laurate as substrate. The presence of Ca2+ and Hg2+ stimulated lipase activity, whereas Zn2+, Co2+, or EDTA inhibited lipase activity. The highest activity was observed in the presence of coconut oil and p-nitrophenyl laurate (pNPL). Purified lipase was the most stable in the presence of various organic solvents, such as pentanol, chloroform and n-dodecane. Because of the superior thermostability and stability in the presence of organic solvents of T. thermophilus extracellular lipase, this lipase holds great promise for use in industrial applications.

Potential of Trametes trogii culture fluids and its purified laccase for the decolorization of different types of recalcitrant dyes without the addition of redox mediators

Trametes trogii BAFC 463 culture fluids (containing 110 U ml −1 laccase; 0.94 U ml −1 manganese peroxidase), as well as its purified laccase were capable of decolorizing azoic, indigoid, triphenylmethane, anthraquinonic and heterocyclic dyes, in the absence of redox mediators. Six dyes: RBBR, Indigo Carmine, Xylidine, Malachite Green, Gentian Violet and Bromophenol Blue were almost completely degraded (more than 85% decolorization after 1 d) by either laccase or T. trogii itself in culture, proving the role of the enzyme in dye decolorization. The purified laccase also decolorized 65% of Fast Blue RR and 30% of Azure B and Methylene Blue after 24 h. The use of redox mediators significantly increased the decolorization rates (90% decolorization of Azure B after 1 h). 1-hydroxybenzotriazole resulted the best redox mediator, but the natural mediator p-hydroxybenzoic acid also demonstrated its efficiency for dye decolorization. Due to their ability to decolorize recalcitrant dyes without addition of redox mediators, high laccase activities, high thermostability and efficient decolorization at 70 °C and pH 7.0, even in the presence of high concentrations of heavy metals (100 mM Cu +2, Pb +2 or Cd +2) or in a synthetic dyebath, T. trogii culture fluids could be effectively used to decolorize synthetic dyes from effluents.

Development and characterization of a voltammetric carbon-fiber microelectrode pH sensor.

This work describes the development and characterization of a modified carbon-fiber microelectrode sensor capable of measuring real-time physiological pH changes in biological microenvironments. The reagentless sensor was fabricated under ambient conditions from voltammetric reduction of the diazonium salt Fast Blue RR onto a carbon-fiber surface in aprotic media. Fast-scan cyclic voltammetry was used to probe redox activity of the p-quinone moiety of the surface-bound molecule as a function of pH. In vitro calibration of the sensor in solutions ranging from pH 6.5 to 8.0 resulted in a pH-dependent anodic peak potential response. Flow-injection analysis was used to characterize the modified microelectrode, revealing sensitivity to acidic and basic changes discernible to 0.005 pH units. Furthermore, the modified electrode was used to measure dynamic in vivo pH changes evoked during neurotransmitter release in the central nervous system of the microanalytical model organism Drosophila melanogaster.

Replica plate screening method for detecting phosphatase activity in basidiomycetes using 1-Napthyl Phosphate as a chromogenic substrate

A chromogenic substrate depended replica plate assay that can be used to screen phosphatase activity in basidiomycetes is described. 1-napthyl phosphate can be incorporated directly into the agar plates used to culture the organisms of interest. The approach offers several advantages over current methodologies including the ability to monitor the development of phosphatase activity with colony development, and the ability to distinguish between activity arising from cell-bound and cell-free enzyme. Phosphatase activity was confrrmed by staining the replica plate with fast blue RR salt. This enzyme probe was successfully used to detect phosphatase producing basidiomycetes. KEY WORDS: Phosphatase activity, Basidiomycetes, 1-napthyl phosphate, Fast Blue RR

Meloidogyne chitwoodi and Meloidogyne fallax

<i>Meloidogyne chitwoodi</i> and <i>Meloidogyne fallax</i>

A new colorimetric method for determination of alkylresorcinols in ground and whole-cereal grains using the diazonium salt Fast Blue RR

Abstract A fast and inexpensive method was developed to determine the content of alkylresorcinols (ARs) in ground and whole-cereal grains. This method is based on the ability of ARs to couple with Fast Blue RR salt in alkaline medium, yielding coloured azo-derivatives that can be quantified colorimetrically. Good linearity was observed for olivetol in the range of 1–10 μg with methanol as solvent (λmax = 480 nm) and 1–7 μg with butanol as solvent (λmax = 530 nm). Sensitivity obtained in butanol was comparable to that obtained in the Fast Blue B based method (methanol as solvent, λmax = 520 nm). In the new colorimetric method described here, incubation time was reduced to 20 min and the stability of the reaction products was as long as 3 h. The method appears promising for the analysis of 1,3-dihydroxybenzene derivatives in samples from plant breeding and food analyses.

A study of the pathogenesis of Rosacea: how angiogenesis and mast cells may participate in a complex multifactorial process

In the present study we evaluated, in involved and clinically uninvolved skin of Rosacea, microvessels density (MVD) and total vascular area (TVA) in addition to multiple morphologic characteristics of microvessels and also mast cells (MCs) number. We examined also the relationship between angiogenesis, MCs number and disease clinicopathological data. The study included 69 patients with Rosacea. A skin biopsy with a 4-mm punch was performed from clinically involved skin in each case. In nine randomly selected patients, facial biopsy specimens were obtained from both involved and clinically uninvolved skin. Histological sections, immunostained for factor VIII, were evaluated by image analysis for the quantification of MVD, TVA and several morphometric parameters related to the vessel size or shape. MCs detection in the dermis was carried out using the chloracetate esterase method (Fast Blue RR) in parafin sections. Serum antibodies against H.pylori were detected. Statistically important differences concerning the factors of angiogenesis between lesional and clinically non-lesional skin were demonstrated. A statistical important correlation was found also between high vascular density, PPR clinical type and the presence of ocular manifestations. MVD or TVA showed no correlation with the degree of solar elastosis or inflammation and with the Demodex density as well. However, high MVD values were found to correlate with granuloma formation in the dermis. MCs number were significantly greater in lesional compared to clinically non-lesional skin. Statistical significance was shown between MCs density and disease duration. However, no correlation between MCs number and blood vessel density was found. Angiogenesis seems to play an important role in the pathogenesis especially of the more severe clinical form of Rosacea. MCs seem to participate in evolution to disease chronicity by contributing to inflammation, angiogenesis and tissue fibrosis.

Phoma tracheiphila

<i>Phoma tracheiphila</i>

Separation and Identification of Oxidized Terpenoids by Thin‐Layer Chromatography

A simple, fast and sensitive thin-layer chromatography (TLC) method for analysis of the oxidation of geranylacetone and several related compounds is reported. This method can be used to monitor chemical and biochemical reactions. Visualization of the spots on silica get under different conditions allows distinguishing either between different epoxides or between epoxides and allylic alcohols and therefore enables the quite rapid screening of oxygenases with different regio- and chemoselectivity. While spray reagents like potassium permanganate or Fast Blue RR Salt are able to detect both, epoxides and alcohols, ultra violet scanning at 254 nm is appropriate only for detection of allylic alcohols.