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ABSTRACT: Fluorescence assays employing semisynthetic or commercial dansyl-polymyxin B have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary γ-amines on diaminobutyric acid residues of polymyxin B are potentially derivatized with dansyl-chloride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra-dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]¹polymyxin B₃. The affinity of polymyxin for purified gram-negative LPS and whole bacterial cells was investigated. The affinity of dansyl[Lys]¹polymyxin B₃ for LPS was comparable to polymyxin B and colistin, and considerably greater (K(d)<1 μM) than for whole cells (K(d)∼6-12μM). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]¹polymyxin B₃ to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]¹polymyxin B₃-LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]¹polymyxin B₃-LPS complex due to steric hindrance from the dansyl[Lys]¹ fluorophore; this corresponded with diminished antibacterial activity (MIC≥16μg/mL). Dansyl[Lys]¹polymyxin B₃ may prove useful as a screening tool for drug development.
Analytical Biochemistry 11/2010; 409(2):273-83. · 3.00 Impact Factor
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Journal of Antimicrobial Chemotherapy 02/2009; · 5.07 Impact Factor
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ABSTRACT: Objectives The purpose of this study was to develop a specific, sensitive, accurate and reproducible high-performance liquid chromatographic (HPLC) method to measure polymyxin B in human plasma. Methods Derivatization of polymyxin B with fluorescent 9-fluorenylmethyl chloroformate (FMOC-Cl) was performed in the same solid-phase extraction C18 cartridge used for the sample pre-treatment. Reversed-phase HPLC was employed with fluorometric detection. The summed peak areas of polymyxin B1 and B2 derivatives were used for quantification. Stability of polymyxin B FMOC derivatives was examined at room temperature for 6 days. Specificity was investigated against seven potentially co-administered antibiotics. Accuracy and reproducibility of the HPLC assay were determined by inter- and intra-day validation. Results The derivatives of polymyxin B2 and B1 were well resolved and had retention times of 4.75 and 5.55 min, respectively. Good linearity (r(2) > 0.99) was obtained between 0.125 and 4.00 mg/L polymyxin B in human plasma with good accuracy and reproducibility at the limit of quantification (0.125 mg/L). Intra- and inter-day validation demonstrated good accuracy and reproducibility for quality control samples with nominal concentrations of 0.30 and 3.00 mg/L. FMOC derivatives of polymyxin B were stable for at least 3 days at room temperature. None of the possibly co-administered antibiotics tested interfered with the chromatographic analysis of the polymyxin B FMOC derivatives. Conclusions A rapid, specific, sensitive, accurate and reproducible HPLC method has been developed and validated to measure polymyxin B in human plasma. The method is suitable for clinical pharmacokinetic studies.
Journal of Antimicrobial Chemotherapy 09/2008; · 5.07 Impact Factor
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ABSTRACT: Two isoflavones in vivo metabolites, genistein-7-O-beta-D-glucuronic acid (G7G) and daidzein-7-O-beta-D-glucuronic acid (D7G) were synthesised chemically. The ability of these metabolites to scavenge an organic radical was measured by the trolox equivalent antioxidant capacity (TEAC) assay, while their reducing ability was measured by the ferric reducing antioxidant power (FRAP) assay. The TEAC and FRAP values of G7G were 45 and 51% of that of genistein, while those of D7G were 52 and 77% of that of daidzein, respectively. A direct assessment of G7G and D7G antioxidant activity by their ability to delay copper(II)-mediated lipid oxidation of human LDL showed that these metabolites retained the ability to prevent oxidation in the lipid phase, but activity was diminished compared to their corresponding aglycones. However, G7G and D7G also decreased the rate of lipid oxidation to 53 and 86% of control without isoflavones, respectively, indicating a continuous exchange of antioxidants between the aqueous environment and the LDL lipid phase during the whole oxidation period.
Molecular Nutrition & Food Research 09/2008; 52(12):1457-66. · 4.30 Impact Factor
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ABSTRACT: Fructus lycii was extracted with 95% ethanol and the flavonoid content was determined to be 1.56 mg quercetin-equivalents/g extract. The following antioxidative activities were determined in the extract: (a) radical-scavenging activity (80 umol trolox equivalent antioxidant capacity/g), measured by its ability to scavenge 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical, (b) reducing capacity (301 umol trolox equivalent reducing capacity/g), measured by its ability to directly donate an electron in the reduction of Fe(III) to
Fe(II), and (c) chelating activity (2.5 umol ethylenediamine-tetraaceticacid equivalents/g), measured by its ability to remove Fe(II) ion from complexation with ferrozine. Three flavonol species in the extract were identified and quantitated by reversed-phase HPLC and their structures confirmed by electrospray ionization-mass spectroscopy: kaempferol (135 ug/g), quercetin (296 ug/g) and myricetin (247 ug/g). These results showed that F. lycii contains substantial amounts of the three antioxidative activities and is rich in flavonoids.
The three flavonols accounted for 43% of total flavonoid content.
Food Chemistry 01/2007; 105:353. · 3.65 Impact Factor
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ABSTRACT: Fructus lycii was extracted with 95% ethanol and the flavonoid content was determined to be 1.56 mg quercetin-equivalents/g extract. The following antioxidative activities were determined in the extract: (a) radical-scavenging activity (80 μmol trolox equivalent antioxidant capacity/g), measured by its ability to scavenge 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical, (b) reducing capacity (301 μmol trolox equivalent reducing capacity/g), measured by its ability to directly donate an electron in the reduction of Fe(III) to Fe(II), and (c) chelating activity (2.5 μmol ethylenediamine-tetraaceticacid equivalents/g), measured by its ability to remove Fe(II) ion from complexation with ferrozine. Three flavonol species in the extract were identified and quantitated by reversed-phase HPLC and their structures confirmed by electrospray ionization-mass spectroscopy: kaempferol (135 μg/g), quercetin (296 μg/g) and myricetin (247 μg/g). These results showed that F. lycii contains substantial amounts of the three antioxidative activities and is rich in flavonoids. The three flavonols accounted for 43% of total flavonoid content.
Food Chemistry.
