Barbara Pilawa

Application of EPR spectroscopy to examination of gentamicin and kanamycin binding to DOPA-melanin

Electron paramagnetic resonance (EPR) spectroscopy was applied to measure the influence of two aminoglycoside antibiotics: gentamicin and kanamycin on free radical propertis of DOPA-melanin. DOPA-melanin was formed by oxidative polymerization of 3,4-dihydroxyphenylalanine. Different concentrations of gentamicin and kanamycin (from 1·10−4 to 1·10−2 M) were used. o-Semiquinone free radicals with ag factor of 2.0043 were found in all studied melanin samples. Their concentrations in the DOPA-melanin-drug complexes were higher than in DOPA-melanin, and increased with the increase of gentamicin and kanamycin concentration. A single EPR line of the analyzed samples (ΔBpp, 0.48-0.52 mT) indicates that aminoglycoside antibiotics do not create a new type of free radicals in DOPA-melanin. Microwave saturation behavior of the experimental lines indicates the homogeneous broadening of resonance absorption curves for DOPA-melanin and its complexes with aminoglycosides. The EPR lines saturate at low microwave powers. Slow spin-lattice relaxation processes were characteristic for all studied melanin samples.

EPR studies of free radicals decay and survival in gamma irradiated aminoglycoside antibiotics: sisomicin, tobramycin and paromomycin.

Radiation sterilization technology is more actively used now that any time because of its many advantages. Gamma radiation has high penetrating power, relatively low chemical reactivity and causes small temperature rise. But on the other hand radiosterilization can lead to radiolytic products appearing, in example free radicals. Free radicals in radiative sterilized sisomicin, tobramycin and paromomycin were studied by electron paramagnetic resonance (EPR) spectroscopy. Dose of gamma irradiation of 25kGy was used. Concentrations and properties of free radicals in irradiated antibiotics were studied. EPR spectra were recorded for samples stored in air and argon. For gamma irradiated antibiotics strong EPR lines were recorded. One- and two-exponential functions were fitted to experimental points during testing and researching of time influence of the antibiotics storage to studied parameters of EPR lines. Our study of free radicals in radiosterilized antibiotics indicates the need for characterization of medicinal substances prior to sterilization process using EPR values. We propose the concentration of free radicals and other spectroscopic parameters as useful factors to select the optimal type of sterilization for the individual drug. The important parameters are i.a. the τ time constants and K constants of exponential functions. Time constants τ give us information about the speed of free radicals concentration decrease in radiated medicinal substances. The constant K(0) shows the free radicals concentration in irradiated medicament after long time of storage.

Effect of oxygen on spin-spin and spin-lattice relaxation in DOPA-melanin. Complexes with chloroquine and metal ions

Electron paramagnetic resonance (EPR) studies of complexes of chloroquine with melanin synthesized from 3,4-dihydroxyphenylalanine (DOPA) confirmed an important role ofo-semiquinone free radicals during the drug binding by melanin. Chloroquine increases free radical concentration in DOPA-melanin. It has been also demonstrated that chloroquine binding to melanin is modified by metal ions. Co2+ ions reduce and Zn2+ ions increase the concentration of free radicals in both DOPA-melanin and melanin-chloroquine complexes. Free radical properties of melanin strongly depend on the presence of oxygen (O2) in the environment of the sample. Interactions of melanin and oxygen molecules cause a decrease of the free radical concentration in the samples and a narrowing of the EPR lines. Higher values of both spin-spin and spin-lattice relaxation times were determined for melanin samples in air than for evacuated samples. Application of the observed effects in oximetry is suggested.

E.p.r. studies of thermal decomposition of exinite and inertinite

Exinite and inertinite from Polish medium-rank coal (85.6 wt%C) heated in inert atmosphere at 300–650°C were investigated by electron paramagnetic resonance in the X-band (9.3 GHz). Numerical analysis of the line-shape of the e.p.r. spectra was performed. The g-factor, the linewidth of the components and their fraction in the total spectrum were determined. Three groups of paramagnetic centres with one Gauss and two Lorentz lines were detected for exinite. The e.p.r. spectra of inertinite were two superimposed Lorentz lines. During thermal decomposition the paramagnetic centres of exinite with broad Gauss and broad Lorentz lines and all the paramagnetic centres of inertinite were very active. The total concentration of paramagnetic centres in exinite considerably increased from 550 to 600°C and decreased at 650°C. A visible decrease in total concentration of paramagnetic centres in inertinite began at 500°C. The microwave saturation of all the measured e.p.r. lines depended on the heat-treatment temperature.

Electron spin resonance investigations of human retinal pigment epithelium melanosomes from young and old donors

Electron spin resonance (ESR) examinations of human retinal pigment epithelium melanosomes isolated from eyes of young and old donors were carried out. The examined ESR signal was a single line, which is characteristic for free radicals of eumelanin o-semiquinones. The content of free radicals related to melanosomes dry weight for samples from older donors (ages over 45 years) were higher than for sample from younger donors (between 14 and 22 years). Simultaneously, the content of free radicals calculated for one melanosome is constant and does not depend on age. The homogeneous broadening of the recorded ESR lines shows that there are no isolated spin packets in all investigated melanin samples. Slow spin-lattice (T1 approximately 10(-5) s) and fast spin-spin (T2 approximately 10(-8) s) relaxation processes occur in these samples. Saturation of the ESR lines at low microwave power was measured. High concentration of free radicals in melanosome samples was responsible for the fast spin-spin relaxation process.

Positive Effect of Propolis on Free Radicals in Burn Wounds

Concentration and properties of free radicals in the burn wounds treated with propolis were examined by the use of electron paramagnetic resonance spectroscopy. Magnetic spin-spin interactions and complex free radicals structures in wound beds were studied. The results were compared to those obtained for silver sulphadiazine used as a standard pharmaceutical agent. The changes of free radicals in the matrix of injury with time of exposition on these substances were tested. The aim of this study was to check the hypothesis about the best influence of propolis on the burn wounds healing. It was confirmed that a relatively lower concentration of free radicals exists in the burn wounds treated with propolis. The homogeneously broadened spectra and a complex free radical system characterize the tested tissue samples. The fastening of spin-lattice relaxation processes in the matrix of injury after treatment with propolis and silver sulphadiazine was observed. Practical usefulness of electron paramagnetic resonance spectroscopy in alternative medicine was proved.

Antioxidative properties of bee pollen extracts examined by EPR spectroscopy

Antioxidative Properties of Bee Pollen Extracts Examined by EPR Spectroscopy Bee pollen is a valuable and highly recognized source of exogenous antioxidants. The aim of these studies was to determine the antioxidant capacity of three types of bee pollen extracts: ethanol extracts of bee pollen, pepsin extracts of bee pollen and ethanol extracts of pepsin-digested bee pollen. Their antioxidant properties were determined with the use of electron paramagnetic resonance (EPR) and their ability to quench DPPH free radicals was estimated. The EPR results showed that ethanol extracts of pepsin-digested bee pollen (EEPP) had the highest antioxidative effect and the highest free radical DPPH scavenging potential. The pepsin extracts of bee pollen (PEP) had the weakest antioxidant capacity. The ability to quench DPPH free radicals was also the weakest one for this extract. An average antioxidative effect was recorded for ethanol extracts of bee pollen (EEP). Właściwości Antyoksydacyjne Ekstraktów z Pyłku Pszczelego Badane Metodą Spektroskopii EPR W ostatnim czasie dużo uwagi poświęca się substancjom o działaniu antyoksydacyjnym pochodzenia naturalnego. Cennym i uznanym źródłem egzogennych antyoksydantów jest pszczeli pyłek kwiatowy. W związku z powyższym, przedmiotem badań było oznaczenie aktywności antyoksydacyjnej trzech rodzajów ekstraktów z pyłku pszczelego: ekstraktu etanolowego pyłku pszczelego, ekstraktu pepsynowego pyłku pszczelego oraz ekstraktu etanolowego po uprzednim trawieniu enzymatycznym pepsyną pyłku pszczelego. Właściwości antyoksydacyjne oznaczono metodą elektronowego rezonansu paramagnetycznego (EPR) na podstawie zdolności redukcji wolnego rodnika DPPH. Najwyższą aktywnością antyoksydacyjną oznaczoną metodą EPR charakteryzował się ekstrakt etanolowy po uprzednim trawieniu enzymatycznym pepsyną pyłku pszczelego (EEPP). Dla tego rodzaju ekstraktu zdolność redukcji wolnego rodnika DPPH była najsilniejsza. Z kolei ekstrakt pepsynowy pyłku pszczelego (PEP) cechował się najniższą aktywnością antyoksydacyjną. Zdolność redukcji wolnego rodnika DPPH dla tego ekstraktu była najsłabsza. Pośrednią aktywność antyoksydacyjną spośród badanych ekstraktów wykazano dla ekstraktu etanolowego pyłku pszczelego (EEP).

Comparative e.p.r. analysis of interactions between macerals and atmospheric oxygen

Abstract Interactions of paramagnetic centres in exinite, vitrinite and inertinite from durain, vitrain and clarain of Polish medium-rank coal (85.6 wt% C) with atmospheric oxygen were studied by electron paramagnetic resonance in the X-band (9.3 GHz). The concentrations of paramagnetic centres, the number of component lines in the e.p.r. spectra and their linewidths, g -values and proportions in the total spectrum were determined. Total concentration of paramagnetic centres increases from exinite to inertinite. It decreases after oxygen evacuation from the exinite and vitrinite environment, whereas it increases for inertinite. Four groups of paramagnetic centres exist in these macerals, with e.p.r. lines of different widths: 0.78–0.85 mT (Gauss), 0.48–0.60 mT (Lorentz), 0.34–0.36 mT (Lorentz) and 0.14–0.20 mT (Lorentz). The linewidths of the broad Gauss line increases after oxygen evacuation. Narrowing of the Lorentz lines after evacuation was observed, g -Values were the same in air and in vacuum. Both decrease and increase in concentration of paramagnetic centres of the individual groups after oxygen evacuation were detected. The most active interactions with atmospheric oxygen were observed for paramagnetic centres with the narrowest Lorentz lines existing in all the types of macerals and for paramagnetic centres in exinite and vitrinite with the broadest Lorentz lines.

E.p.r. studies of interactions between paramagnetic centres of exinite, vitrinite and inertinite during thermal decomposition

Electron paramagnetic resonance in the X-band (9.3 GHz) was used for investigations of petrographically complex coal samples (densities 1.24-1.28 and 1.30-1.36 gcm -3) from Polish medium-rank coal (85.6 wt% C) heated in an inert atmosphere at 300-650°C. A strong increase in total concentration of paramagnetic centres in the sample of lower density at 300-350 and 450-550°C was measured. Relatively smaller changes in the total amount of paramagnetic centres in the heavier sample at 300-650°C were observed. There are three different types of paramagnetic centres in both samples, and their e.p.r, spectra are a superposition of one broad Gauss and two Lorentz (broad and narrow) lines. The most active group in the thermal decomposition of the two coal samples is formed by paramagnetic centres responsible for the broad Lorentz lines. Mainly dipolar interactions of unpaired electrons influence the linewidths of the e.p.r, signals. The microwave saturation power of all the e.p.r, lines detected increases with temperature. According to the e.p.r, results for pure macerals and the complex coal samples, the hypothesis of independent progress of reactions in individual macerals during the thermal decomposition of coal can be rejected. Copyright

Effect of Cu2+ and Zn2+ ions on DOPA-melanin structure as analyzed by pyrolysis-gas chromatography-mass spectrometry and EPR spectroscopy

Abstract Synthetic polymers, obtained by oxidative polymerization of 3,4-dihydroxyphenylalanine (DOPA) in the presence and in the absence of copper or zinc ions, were analyzed by pyrolysis–gas chromatography–mass spectrometry and electron paramagnetic resonance (EPR) methods. Pyrolytic profiles of DOPA–Cu 2+ - and DOPA–Zn 2+ -melanins were characterized by the abundance of pyridine- and pyrazine-derivatives and by considerably reduced levels of pyrrole and phenols that were predominant pyrolysis products of metal-free DOPA-melanin. It was found that incorporation of paramagnetic Cu 2+ ions and diamagnetic Zn 2+ ions into DOPA-melanin effects its free radical properties. It was concluded that the presence of Cu 2+ and Zn 2+ ions during DOPA-melanin synthesis modifies the structure of the biopolymer formed.