芪灵胶囊清除超氧阴离子自由基的作用

目的:研究中药复方制剂芪灵胶囊及其组成中单味中药的体外清除超氧阴离子自由基的能力.方法:采用电子顺磁共振技术,观察芪灵胶囊及其组成中黄芪、淫羊藿、白豆蔻、鳖甲单味中药对模型体系中产生的O2·-量的影响.结果:在10%的相同浓度时,复方的作用优于单味中药;组分中黄芪、鳖甲对O2·-的清除作用与药物浓度成正比关系,黄芪的清除作用最强,鳖甲次之;淫羊藿在药物浓度为40%的时候,表现出最大的清除效果,豆蔻在80%浓度时清除效果为最佳,随着药物浓度的增加,它们的清除作用反而下降.结论:芪灵胶囊具有较高的体外清除O2·-的作用.     

Enzymatic oxidative activation of 5-iminodaunorubicin. Spectrophotometric and electron paramagnetic resonance studies

Horseradish peroxidase catalyzed oxidation of the antitumor agent 5-iminodaunorubicin by hydrogen peroxide was studied with both spectrophotometric and electron paramagnetic resonance methods. Kinetics of oxidation of the drug at pH 3, 6 and 8 were determined. Rapid formation of a nitrogen-centered free radical metabolite was demonstrated with electron paramagnetic resonance employing the 15N-labeled drug and by deuterium exchange techniques. This enzymatic oxidative activation of 5-iminodaunorubicin suggests an alternative mode of metabolism and mechanism of action of this less cardiotoxic anticancer agent. By contrast, the parent compound, daunorubicin, did not undergo oxidation by the horseradish peroxidase-hydrogen peroxide system.     

Effects of sodium valproate on mitochondrial membranes: electron paramagnetic resonance and transmembrane protein movement studies

Sodium valproate (VPA), the salt of a branched short-chain fatty acid, is a major antiepileptic whose mode of action, as yet unclear, may involve effects on the organization of membranes. VPA was either injected into rats whose liver and kidney mitochondria were then isolated, or was preincubated with isolated mitochondria. First, liver and kidney mitochondria were studied with paramagnetic probes. The electron paramagnetic resonance spectra of proteins of VPA-treated mitochondria spin-labeled with 4-maleimido-2,2,6,6-tetramethyl-1-pyrrolidinoxyl showed that the ratio of weakly immobilized to strongly immobilized SH groups was reduced with respect to control mitochondria, more so in liver than in kidney mitochondria of VPA-injected rats, and more so in kidney than in liver mitochondria for VPA-incubated mitochondria. Spectra of mitochondrial lipids spin-labeled with 5-doxyl stearic methyl ester showed that VPA had no significant effect on order parameters S. Second, the transmembrane movement of aspartate aminotransferase was studied by incubating liver mitochondria in a sucrose-succinate medium and then fractionating them. The translocation of aspartate aminotransferase from mitoplasts, vesicles formed of inner membrane and matrix, to the intermembrane fluid, was significantly higher in VPA-treated than in control mitochondria. Thus, VPA, at concentrations in the range of those used therapeutically, interacted with membranes by modifying the structural organization of the internal mitochondrial membrane, essentially the membrane protein conformation.     

Effect of surfactants on human stratum corneum: electron paramagnetic resonance study

Electron paramagnetic resonance (EPR) spectra of nitroxide spin probes are useful for studying biological membranes and chemical-membrane interactions. Recently, we established a stripping method to remove stratum corneum (SC) for this purpose. To assess this stripping method with EPR and correlate with standard methods, we quantified the irritant effects of three types of surfactants by measurements of visual score and transepidermal water loss (TEWL), SC hydration and chromametry and studied EPR spectra measurements of surfactant-treated cadaver SC (C-SC) and stripped off SC (S-SC) on patch tested sites. 5-Doxyl stearic acid was the spin label. The order parameter S obtained from the spectra of S-SC correlated with those of C-SC and TEWL values. The results suggest that this method is capable of evaluating the fluidity of SC and correlates with the above bioengineering parameters.     

Polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls in cetaceans from Korean coastal waters

Data on levels and accumulation profiles of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/ Fs) and dioxin-like polychlorinated biphenyls (PCBs) in cetaceans are scarce. This paper presents data on the concentrations and accumulation features of PCDD/Fs and dioxin-like PCBs in muscle of minke whales (Balaenoptera acutorostrata) and long-beaked common dolphins (Delphinus capensis) collected from Korean coastal waters. The residue levels in cetaceans were in the order of mono-ortho PCBs> non-ortho PCBs>PCDFs>PCDDs. Total toxic equivalent (TEQ) concentrations in muscle of minke whales and common dolphins from Korea were lower than those reported for cetaceans and seals from other countries. The accumulation profiles of PCDD/Fs and dioxin-like PCBs were different between the cetacean species. Concentrations of PCDD/Fs and dioxin-like PCBs in common dolphins were significantly higher than those measured in minke whales, due to differences in the habitat and diet. The relative contribution of individual chemical groups to total TEQs was different between the cetacean species, suggesting different exposures and metabolic activity.     

Intracellular hypoxia of tumor tissue estimated by noninvasive electron paramagnetic resonance oximetry technique using paramagnetic probes

Electron paramagnetic resonance (EPR) oximetry at 700 MHz operating frequency employing a surface coil resonator is used to assess tissue partial pressure of oxygen (pO(2)) using paramagnetic media whose linewidth and decay constant are related to oxygen concentration. Differences in extracellular and intracellular pO(2) in squamous cell carcinoma (SCC) tumor tissue were tested using several types of water-soluble paramagnetic media, which localize extracellularly or permeate through the cell membrane. The nitroxide carboxy-PROXYL (CxP) can only be distributed in blood plasma and extracellular fluids whereas the nitroxides carbamoyl-PROXYL (CmP) and TEMPOL (TPL) can permeate cell membranes and localize intracellularly. EPR signal decay constant and the linewidth of the intravenously administered nitroxides in SCC tumor tissues implanted in mouse thigh and the contralateral normal muscle of healthy mice breathing gases with different pO(2) were compared. The pO(2) in the blood can depend on the oxygen content in the breathing gas while tissue pO(2) was not directly influenced by pO(2) in the breathing gas. The decay constants of CmP and TPL in tumor tissue were significantly larger than in the normal muscles, and lower linewidths of CmP and TPL in tumor tissue was observed. The SCC tumor showed intracellular hypoxia even though the extracellular pO(2) is similar to normal tissue in the peripheral region.     

Management of electron paramagnetic resonance spectrometer in a university core laboratory

Electron Paramagnetic Resonance (EPR) is the only spectral method for direct detection of paramagnetic substances. EPR technology can be used for the qualitative and quantitative analysis of free radicals and transition metals in solid, liquid, gas, cell and living samples. It is widely used in chemistry, material science, life science, quantum physics and other researches. This report discusses the basic principle, instrument structure, common problem solving, daily maintenance, and management model of the electron paramagnetic resonance spectrometer (German Bruker A-200) in the State Key Laboratory of Natural and Biomimetic Drugs of Peking University Health Science Center, so that the instrument can be fully used for various scientific researches.     

Neurotoxicity of polychlorinated biphenyls: structure-activity relationship of individual congeners

Neurotoxicity of Polychlorinated Biphenyls: Structure-Activity Relationship of Individual Congeners, Shain, W., Bush, B., Seegal, R. (1991). Toxicol. Appl. Pharmacol. 111, 33-42. Experimental and epidemiological data indicate that polychlorinated biphenyls (PCBs) may function as neurotoxicants. The mechanism(s) of action of PCBs in the brain is not well understood. One reason for our lack of understanding of PCB action in the central nervous system is that, in general, commercial mixtures of PCBs have been used for these experiments. We used a homogeneous cell line, PC12 cells, to investigate the relative potency of 43 individual PCB congeners. The neurotoxicant action of PCB congeners was measured as a decrease in cell dopamine content. We first described the potency of individual congeners; 2,2'-dichlorobiphenyl was the most potent congener (EC50 = 65 microM). The structure-activity relationships described in these experiments indicated that (i) congeners with ortho- or ortho-, para-chlorine substitutions were most potent; (ii) chlorination in a meta position decreased cell dopamine content in ortho-substituted congeners, but had little effect in ortho-, para-substituted congeners; and (iii) increasing congener chlorination did not correlate with a decrease in potency, though total chlorination of a ring appeared to reduce potency. Second, we determined that potency did not correlate with either cellular PCB content or gas chromatographic retention time. Finally, experiments with 2,2'-dichlorobiphenyl indicated that PCBs and not their metabolites were the toxicants. Thus, PCB congeners decrease cell dopamine content by interaction at specific sites that have preference for ortho- or ortho-, para-substituted congeners. The neurotoxic action of PCBs may occur by a different mechanism than PCB hepato- and immunotoxicity since these effects are most sensitive to non-ortho-substituted, dioxin-like, congeners.     

电子顺磁共振技术研究一枝蒿总黄酮抗氧化作用

目的　探讨一枝蒿总黄酮清除自由基的作用。方法　采用电子顺磁共振技术 (EPR)分别检测一枝蒿总黄酮对超氧阴离子自由基 (O·2 ) ,羟自由基 (·OH)的清除作用。结果　一枝蒿总黄酮对超氧阴离子自由基、羟自由基具有不同程度的清除作用 ,且存在明显的药物浓度—清除效用关系。结论　一枝蒿总黄酮有明显的抗氧化作用     

Nanostructured lipid carriers as nitroxide depot system measured by electron paramagnetic resonance spectroscopy

Various nanometer scaled transport systems are used in pharmaceutics and cosmetics to increase penetration or storage of actives. Nanostructured lipid carriers (NLCs) are efficient drug delivery systems for dermatological applications. Electron paramagnetic resonance (EPR) spectroscopy was used for the determination of TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) distribution within the carrier and to investigate the dynamics of skin penetration. Results of ex vivo penetration of porcine skin and in vivo data - forearm of human volunteers - are compared and discussed to previously obtained results with invasomes under comparable conditions. W-band measurements show 35% of TEMPO associated with the lipid compartments of the NLC. Application of TEMPO loaded NLC to skin ex vivo increases the observation time by 12min showing a stabilisation of the nitroxide radical. Moreover, stabilisation is also seen with data generated in vivo. Thus, same as invasomes NLCs are a suitable slow release depot system.     

Stabilization of reactive nitroxides using invasomes to allow prolonged electron paramagnetic resonance measurements

The detection of the antioxidative capacity of the skin is of great practical relevance since free radicals are involved in many skin damaging processes, including aging and inflammation. The nitroxide TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxyl) in combination with electron paramagnetic resonance spectroscopy was found suitable for measuring the antioxidative capacity since its reaction with reducing agents is considerably fast. Yet, in order to achieve longer measurement times, e.g. in inflammatory skin diseases, the stabilizing effect of an invasome (ultraflexible vesicle/liposome) suspension with TEMPO was investigated ex vivo on porcine skin and in vivo on human skin. Invasomes increased the measurement time ex vivo 2-fold and the reduction was significantly slowed down in vivo, which is due to membrane-associated and therefore protected TEMPO. Furthermore, TEMPO accumulation in the membrane phase as well as the decreasing polarity of the ultimate surroundings of TEMPO during skin penetration explains the stabilizing effect. Thus, an invasome suspension with TEMPO exhibits stabilizing effects ex vivo and in vivo.     

Evaluation of the antioxidant activity of new carotenoid-like compounds by electron paramagnetic resonance

The antioxidant activity of a novel series of derivatives with a carotenoid-like structure was studied. These derivatives have recently been isolated chemically as a result of studies on the pigments present in a particular species of birds, namely parrots. These novel derivatives, which are also called parrodienes, have been proved to possess interesting biological properties that differ from those that carotenoids are known to have. The objective of this study was to demonstrate the ability of these novel compounds to inhibit the formation of reactive oxygen species, especially their ability to block the formation of hydroxyl radicals, which are among the most reactive products of oxygen reactions and which produce the greatest damage to cells and tissues. The technique used to assess this antioxidant capacity of parrodienes was electron paramagnetic resonance, which allows direct assessment of inhibition of hydroxyl radical formation (.OH). The results show that these derivatives, especially octatriene, are able to exert evident antioxidant activity, thus confirming that their antioxidant properties are important for their biological activity.     

Characterization of thermosensitive chitosan-based hydrogels by rheology and electron paramagnetic resonance spectroscopy.

Chitosan, an amino-polysaccharide, has been proposed as a promising biopolymer for tissue repair and drug delivery. Chitosan solutions containing glycerol-2-phosphate (beta-GP) have been described as injectable in situ gelling thermosensitive formulations, which undergo sol-gel transition at physiological pH and temperatures. This feature makes them suitable for the parenteral administration of drugs, especially for peptides and proteins. The aim of the present study was to get a deeper insight into the macro- and microstructure of chitosan/beta-GP systems. In addition to oscillating rheology, electron paramagnetic resonance (EPR) spectroscopy was applied to examine the microviscosity and pH inside the gels depending on the beta-GP concentration and to follow the loading and release of spin-labelled Insulin. All chitosan/beta-GP solutions showed a physiological pH ranging from 6.6 to 6.8 that did not change during gelation, irrespective of the proportion of beta-GP. The dynamics of the spin-labelled Insulin and its microviscosity inside the gels and during release were monitored by EPR spectroscopy. The results indicate that the Insulin was incorporated into the aqueous environment of the gel and was released in its native form. The in vitro drug release from the gels was governed by diffusion of drug from the gel matrix. A sustained release of Insulin was observed over a period of 2 weeks. Increasing the proportion of beta-GP increased the amount of released Insulin and the velocity thereof.     

The copper-induced deformability loss and echinocyte formation in human erythrocytes: an electron paramagnetic resonance study

The effects of incubating human erythrocytes with CuSO4 under prelytic conditions were investigated to clarify the cause of the decreased whole-cell deformability in flow, using electron paramagnetic resonance and the spin label methods. The effect of Cu(II) on erythrocytes was manifested by the dose-dependent echinocyte formation as well as by the decreased whole-cell deformability in flow. The deformability loss was attributed in part to the altered rheological characteristics due to echinocytosis. By using various reagents capable of reversing the effects of Cu(II) on morphology and deformability loss, it was shown that at least two types of damage sites can be distinguished. They include (1) the Cu(II) coordination sites which can be dissociated by chelating reagents and (2) the sites involving sulfhydryl groups in which disulfide bonds and probably Cu(II)-bridged strong coordination bonds are formed. The latter can be reversed by sulfhydryl reagents such as dithioerythritol, but not by EDTA or penicillamine. Pretreatment of erythrocytes with iodoacetate eliminates the variance in susceptibility to Cu(II) among donors and generally enhances the effects of Cu(II) by lowering the cellular glutathione level. A possible relation between the nature of the damage sites and the cellular glutathione level is discussed.     

Electron paramagnetic resonance spectrometry evidence for bioreduction of tirapazamine to oxidising free radicals under anaerobic conditions

Tirapazamine (SR 4233) is a bioreductive antitumour drug in Phase III clinical trial which is activated in hypoxic tumour regions to generate a cytotoxic species. Electron paramagnetic resonance (EPR) spectrometry was used to investigate directly the formation of free radicals as the result of tirapazamine reduction by NADPH-supplemented liver microsomes. Under anaerobic conditions, the tirapazamine nitroxide free radical EPR signal was not evident over a range of rat or human liver microsomal protein (1–5 mg) concentrations. However, in combination with 1,1′,5,5′-dimethylpyrolline-1-N-oxide (DMPO), a spin trap for short-lived free radicals, tirapazamine resulted in formation of a 1:1:1:1:1:1 spectrum with hyperfine splitting A_N = 15.8 G A_H = 22.3 G consistent with generation of DMPO-R, a carbon-centered radical adduct. Addition of DMSO increased the signal intensity of the carbon-centred radical by at least twofold. The hyperfine splitting constants associated with DMPO-R could be indicative of a tirapazamine carbon-centred radical per se or, more likely, carbon radicals from endogenous materials (or DMSO) in the biological matrix as a result of oxidative attack by the tirapazamine primary radical. Formation of DMPO-OH, the hydroxyl radical spin adduct, by tirapazamine in the absence of air indicates that liberation of a hydroxyl radical may be a consequence of tirapazamine bioreduction under anaerobic conditions. The reactivity of tirapazamine free radicals with endogenous microsomal substances to generate reactive carbon-centred radicals indicates that tirapazamine may disrupt a wide range of cellular activities.     

Polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls in breast milk from two cities in Ukraine.

Substantial environmental pollution has been alleged in Ukraine, but little information is available to allow an assessment of the possible impact on humans. To help remedy this lack of information, it was of interest to investigate whether certain polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), or coplanar polychlorinated biphenyls (PCBs) were elevated in people from Ukraine. Samples of breast milk were obtained from 200 women from the cities of Kyiv and Dniprodzerzhinsk; Kyiv is the capital and Dniprodzerzhinsk is a highly industrialized city. The samples were combined into four pools by city and age, and analyzed for 7 PCDDs, 10 PCDFs, and 2 coplanar PCBs (126 and 169). The total of the measured PCDDs, expressed as toxic equivalent, ranged from 5.1 to 7.6 pg/g lipid; for PCDFs from 3.6 to 5.2, and for PCBs from 11 to 18 pg/g lipid. Results from the two cities were similar; older women had slightly higher concentrations than did younger women. Levels of these compounds seen in Ukraine were similar to or lower than those seen in other recent studies from European and Asian countries.     

Influence of different anesthetics on skin oxygenation studied by electron paramagnetic resonance in vivo

The effects of two general anesthetics on skin oxygenation in mice are evaluated by electron paramagnetic resonance oximetry. Up to now no data on the effects of different anesthetics on skin oxygenation could be found. In this study animals were anesthetized with ketamine/xylazine or isoflurane, and partial pressure of oxygen (pO(2)) in the skin, heart rate and hemoglobin oxygen saturation were followed as a function of time and inhaled oxygen concentration. The skin pO(2) significantly increased continuously for about 60 min in mice anesthetized with isoflurane and remained constant after that. During ketamine/xylazine anesthesia, the pO(2) in the skin only slightly decreased. The skin pO(2) increased with higher inspired oxygen concentrations for both anesthetics groups. When breathing 21% oxygen, mice anesthetized with isoflurane had two-fold higher pO(2) in the skin compared to mice anesthetized with ketamine/xylazine. The heart rate was significantly lower in animals anesthetized with ketamine/xylazine, while hemoglobin saturation was almost the same in both groups at all inhaled oxygen concentrations. These results show that the type of anesthesia is an important parameter that needs to be considered in experiments where skin pO(2) is followed.