Structure-activity studies on the protection of Trimetazidine derivatives modified with nitroxides and their precursors from myocardial ischemia-reperfusion injury

Trimetazidine, the known anti-anginal and anti-ischemic drug, was modified by pyrroline and tetrahydropyridine nitroxides and their hydroxylamine and sterically hindered secondary amine precursors. The synthesized new compounds proved to be better superoxide scavenger molecules compared to the parent Trimetazidine in an in vitro experiment. This reactive oxygen species (ROS) scavenging activity was further supported by ischemia/reperfusion (I/R) studies on Langendorff-perfused rat hearts pretreated with Trimetazidine and with the modified Trimetazidine derivatives before ischemia. Two of the investigated compounds, containing 2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole and 4-phenyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole substituents on the piperazine ring, provided significant protection from the cardiac dysfunction caused by I/R. The protective effect could be attributed to the combined anti-ischemic and antioxidant effects.     

Novel 2-substituted nitronyl nitroxides as free radical scavengers: synthesis, biological evaluation and structure-activity relationship

To develop more potent small molecules with enhanced free radical scavenger properties, we designed and synthesized a series of nitronyl nitroxide derivatives 4a-h. A lead compound 4f was discovered based on Ach-induced vascorelaxation assay. Further chemical modification based on this scaffold provided a new series of 2-substituted phenylnitronyl nitroxide derivatives 6a-s. The newly synthesized compounds 6a-s possess improved radical scavenger's activity based on PC12 cell survival assay. Compounds 6g,n,o, and s are some of the most potent compounds in terms of NO, H(2)O(2), and OH scavenging ability. 2-Substitued phenylnitronyl nitroxides had a higher radical scavenging activity with the electron-donating group (EDG). In contrast, the introduction of electron-withdrawing group (EWG) to the aromatic ring led to a dramatic decrease in its radical scavenging activity. These results suggest that the electron-donating group (EDG) of the aromatic ring may be an important factor influencing the radical scavenging behavior of these compounds, and the potency of free radical scavenging activity largely depended on the position and electronic properties of the phenyl ring substituents. The enhanced radical scavenging capacities of the novel 2-substituted nitronyl nitroxides may be potential drug leads against the deleterious action of ROS (reactive oxygen species)/RNS (reactive nitrogen species).     

Structure-activity relationship studies of protective function of nitroxides in Fenton system

The aim of this study was to evaluate the effect of piperidine nitroxides and their amine precursors on deoxyribose oxidation in the Fenton system. Protecting activity of nitroxides was found to be concentration-dependent and strongly influenced by ring substituents, while secondary amines did not provide any protection. The reported results suggest a mechanism of nitroxide action through iron oxidation rather than through direct scavenging of hydroxyl radicals. Moreover, presented data point to the danger of interference of nitroxides during the TBARS assay procedure.     

Reduction and destruction rates of nitroxide spin probes

A series of nitroxides was tested for rates of one-electron reduction in a chemical, a photochemical, and two biological systems by ESR assays. In all cases, piperidine and hydropyridine nitroxides were reduced consistently more rapidly than pyrroline and pyrrolidine nitroxides. Substituents on the nitroxides also affected reduction rates, although not as greatly as ring structure. One of the reduction systems, consisting of the photosensitizer FMN and the photoreductant EDTA, was used to study both anaerobic reduction and O2-dependent reoxidation of some of the nitroxides. Reduced piperidine and hydropyridine nitroxides were also oxidized more rapidly than the reduced pyrroline and pyrrolidine nitroxides. Reoxidation subsequent to reduction was partially inhibited by superoxide dismutase, indicating that superoxide radicals are involved in the process. Even after prolonged reoxidation, not all of the probe molecules were returned to their oxidized form, implying an irreversible "destruction" of the spin probe concomitant with its chemical reduction. Probe destruction was studied more specifically with a photochemical system for generating methyl radicals, which showed that these carbon-centered radicals destroyed different nitroxides at rates which were much less influenced by the nitroxide structures than one-electron reduction was.     

Superoxide Scavenging Activity of Spin-Labeled Nitrosourea and Triazene Derivatives

Superoxide scavenging activities (SSA) of newly synthesized spin-labeled nitrosourea and triazene derivatives, and their precursor nitroxides were investigated by the ESR/spin-trapping method using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and hypoxanthine/xanthine oxidase as the superoxide-generating system. The spin-labeled nitrosoureas, triazenes and their precursor nitroxides exhibited excellent SSA, whereas clinically used nitrosourea and triazene, which do not contain the nitroxide moiety, did not show any SSA. Furthermore, it was deduced that these nitroxides scavenge superoxide by redox cycling between nitroxide and corresponding hydroxylamine.     

Nitroxides inhibit peroxyl radical-mediated DNA scission and enzyme inactivation

Nitroxides are cell-permeable stable radicals that protect biomolecules from oxidative damage in several ways. The mechanisms of protection studied to date include removal of superoxide radicals as SOD-mimics, oxidation of transition metal ions to preempt the Fenton reaction, and scavenging carbon-centered radicals. However, there is no agreement regarding the reaction of piperidine nitroxides with peroxyl radicals. The question of whether they can protect by scavenging peroxyl radicals is important because these radicals are formed in the presence of oxygen abundant in biological tissues. To further our understanding of the antioxidative behavior of piperidine nitroxides, we studied their effect on biochemical systems exposed to the water soluble radical initiator 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH). AAPH thermally decomposes to yield tert-amidinopropane radicals (t-AP(*)) that readily react with oxygen to form peroxyl radicals (t-APOO(*)). It has recently been reported that piperidine nitroxides protect plasmid DNA from t-AP(*) though not from t-APOO(*). The present study was directed at the question of whether these nitroxides can protect biological systems from damage inflicted by peroxyl radicals. The reaction of nitroxides with AAPH-derived radicals was followed by cyclic voltammetry and electron paramagnetic resonance spectroscopy, whereas the accumulation of peroxide was iodometrically assayed. Assaying DNA damage in vitro, we demonstrate that piperidine nitroxides protect from both t-AP(*) and t-APOO(*). Similarly, nitroxides inhibit AAPH-induced enzyme inactivation. The results indicate that piperidine nitroxides protect the target molecule by reacting with and detoxifying peroxyl radicals.     

Computational Investigation on the ∙OOH Scavenging Sites of Gnetin C

Melinjo seed extract contains melinjo resveratrol compounds that has antioxidant activity. The radical-scavenging sites required for the antioxidant activity, however, is yet to be located. We report a computational study that aims to locate scavenging sites of the simplest resveratrol dimer, gnetin C. We consider the reaction of gnetin C and hydroperoxyl radical energetically with the basis of density-functional calculations, to be compared with the reaction of the resveratrol monomer, trans-resveratrol, and hydroperoxyl radical. The results show that OH group at the para position is the most reactive scavenging site for both molecules. Besides the OH group, gnetin C also provides two CH groups in the furan ring that are favorable as scavenging sites. Therefore, furan ring plays an important role in the scavenging activity, which is contrary to the experimental speculation that propose resorcinol ring. Our study shows the prospect of density-functional calculation for studying the radical-scavenging reaction.

     

The antioxidant effect of imine resveratrol analogues

Twenty five Imine resveratrol analogues (IRAs) were synthesized, replacing the CC bond in resveratrol with CN bond, as well as substitution modifications on aromatic rings. Radical scavenging activities against DPPH, along with singlet oxygen quenching capacities were evaluated, and further confirmed using density functional theory calculations (DFT). It was found that IRAs bearing ortho-OH on B ring have better radical scavenging activities against DPPH than resveratrol, these compounds were also found to be effective (1)O(2) quenchers. Theoretical studies on the reaction mechanism of these compounds with (1)O(2) suggest that the 1,3-addition to a double bond with a -OH group with the formation of allylic hydroperoxide is the most probable reaction route.     

Comparison of Antioxidant Activity Between Aromatic Indolinonic Nitroxides and Natural and Synthetic Antioxidants

In view of the possible employment of nitroxide compounds in various fields, it is important to know how they compare with other synthetic antioxidant compounds currently used in several industries and with naturally occurring antioxidants. To address this issue, the antioxidant activity of two aromatic indolinonic nitroxides synthesized by us was compared with both commercial phenolic antioxidants (BHT and BHA) and with natural phenolic antioxidants (α-hydroxytyrosol, tyrosol, caffeic acid, α-tocopherol). DPPH radical scavenging ability and the inhibition of both lipid and protein oxidation induced by the peroxyl-radical generator, AAPH, were evaluated. The results obtained show that overall: (i) the reduced forms of the nitroxide compounds are better scavengers of DPPH radical than butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) but less efficient than the natural compounds; (ii) the nitroxides inhibit both linolenic acid micelles and bovine serum albumin (BSA) oxidation to similar extents as most of the other compounds in a concentration-dependent fashion. Since the aromatic nitroxides tested in this study are less toxic than BHT, these compounds may be regarded as potential, alternative sources for several applications. The mechanisms underlying the antioxidant activity of nitroxides were further confirmed by UV–Vis absorption spectroscopy experiments and macroscale reactions in the presence of radicals generated by thermolabile azo-compounds. Distribution coefficients in octanol/buffer of the nitroxides and the other compounds were also determined as a measure of lipophilicity.     

Development of nitroxides for selective localization inside cells

The use of nitroxides to measure intracellular phenomena, especially oxygen concentrations, is a new and potentially important approach to a number of physiological and pathophysiological studies. This study provides data indicating the feasibility of developing nitroxides that localize selectively in the intracellular compartment; it is based on the use of readily hydrolysed ester linkages, such that the nitroxides become converted intracellularly to ionic derivatives that do not cross cell membranes readily. Up to 120-fold increased concentrations of intracellular nitroxides (and their one electron reduction product, the hydroxylamines) were obtained. The ESR spectra of the intracellular nitroxides were consistent with their conversion to the ionic species. Preliminary studies indicate that these nitroxides have the properties needed for their use as probes of intracellular concentrations of oxygen and that it should be feasible to synthesize nitroxides that will be even more effective for this purpose.     

Comparison of antioxidant activity between aromatic indolinonic nitroxides and natural and synthetic antioxidants

In view of the possible employment of nitroxide compounds in various fields, it is important to know how they compare with other synthetic antioxidant compounds currently used in several industries and with naturally occurring antioxidants. To address this issue, the antioxidant activity of two aromatic indolinonic nitroxides synthesized by us was compared with both commercial phenolic antioxidants (BHT and BHA) and with natural phenolic antioxidants (alpha-hydroxytyrosol, tyrosol, caffeic acid, alpha-tocopherol). DPPH radical scavenging ability and the inhibition of both lipid and protein oxidation induced by the peroxyl-radical generator, AAPH, were evaluated. The results obtained show that overall: (i) the reduced forms of the nitroxide compounds are better scavengers of DPPH radical than butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BLT) but less efficient than the natural compounds; (ii) the nitroxides inhibit both linolenic acid micelles and bovine serum albumin (BSA) oxidation to similar extents as most of the other compounds in a concentration-dependent fashion. Since the aromatic nitroxides tested in this study are less toxic than BHT, these compounds may be regarded as potential, alternative sources for several applications. The mechanisms underlying the antioxidant activity of nitroxides were further confirmed by UV-Vis absorption spectroscopy experiments and macroscale reactions in the presence of radicals generated by thermolabile azo-compounds. Distribution coefficients in octanol/buffer of the nitroxides and the other compounds were also determined as a measure of lipophilicity.     

Do stable nitroxide radicals catalyze or inhibit the degradation of hyaluronic acid?

Reactive oxygen-derived species and particularly OH radicals can degrade hyaluronic acid (HA), resulting in a loss of viscosity and a subsequent decrease in its effectiveness as a joint-lubricating agent. The production of OH in the vicinity of HA can be catalyzed by bound redox-active metals, which participate in the Haber-Weiss reaction. Damage to HA can also occur as a result of hypochlorite formed by myeloperoxidase (MPO). The protective reagents commonly used to inhibit oxidative stress-induced degradation of HA include antioxidative enzymes, such as SOD and catalase, chelators that coordinate metal ions rendering them redox-inactive, and scavengers of radicals, such as OH, as well as nonradical reactive species. In recent years, stable cyclic nitroxides have also been widely used as effective antioxidants. In many cases, nitroxide antioxidants operate catalytically and mediate their protective effect through an exchange between their oxidized and reduced forms. It was anticipated, therefore, that nitroxides would protect HA from oxidative degradation as well. On the other hand, nitroxides serve as catalysts in many oxidation reactions of alcohols, sugars and polysaccharides, including hyalouronan. Such opposite effects of nitroxides on oxidative degradation are particularly intriguing and the aim of the present study was to examine their effect on HA when subjected to diverse forms of oxidative stress. The results indicate that nitroxides protect HA from OH radicals generated enzymatically or radiolytically. The protective effect is attributable neither to the scavenging of OH nor to the oxidation of reduced metal, but to the reaction of nitroxides with secondary carbohydrate radicals-most likely peroxyl radicals.     

Radical scavenging activity of dicaffeoyloxycyclohexanes: contribution of an intramolecular interaction of two caffeoyl residues

Six regio- and stereoisomers of dicaffeoyloxycyclohexanes and 2,4-di-O-caffeoyl-1,6-anhydro-beta-D-glucose were synthesized as model compounds of dicaffeoylquinic acids, and their radical scavenging activity was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt) radical scavenging tests. Both DPPH and ABTS radical scavenging reactions of these compounds consisted of two different steps. In the first step, catechol moieties of the caffeoyl residues were rapidly converted to o-quinone structures and no significant difference in the reactivity was observed among the tested compounds. In the second step, however, the rate of the reaction increased as the intramolecular distance of the two caffeoyl residues decreased. A novel intramolecular coupling product, which could scavenge additional radicals, was isolated from the reaction mixture of trans-1,2-dicaffeoyloxycyclohexane and DPPH radical. The result suggests that the second step of the radical scavenging reaction is arising from an intramolecular interaction between the two caffeoquinone residues to regenerate catechol structures, and that the closer their distance is, the more rapidly they react. The radical scavenging activity of natural dicaffeoylquinic acids in a biological aqueous system might also depend on the positions of caffeoyl ester groups.     

Measurement of relative antioxidant activity of compounds: a methodological note

The relative activities of some hydrogen-donating antioxidants were assessed by comparing their activities with that of Trolox (Trolox equivalent antioxidant capacity, TEAC) for scavenging the ABTS radical cation (ABTS.+) generated in the aqueous phase. We have verified, however, that TEAC values may change with the concentration of compounds and with the measuring times used. Not withstanding, TEAC values do not differ significantly if the compounds have kinetic curves of ABTS.+ formation similar to that of Trolox. This is the case with ascorbic acid, whose TEAC values, determined by using five concentrations at three different measuring times, are very close. For the flavonoids studied (catechin, rutin, naringenin and silibinin) which have kinetic curves of ABTS.+ formation different from that of Trolox, the TEAC values decrease with increasing concentrations of the compounds for each measuring time, and increase with increasing measuring times for each concentration. In the present study, we conclude that, in order to evaluate relative antioxidant activities of compounds by the ABTS assay, it is essential to perform kinetic studies to assess scavenging of ABTS.+ by these compounds. Therefore, when the TEAC values of compounds are determined for more than one measuring time, we may be sure that all the antioxidant potential of compounds is being considered and whether or not it is possible to establish a hierarchy for their antioxidant activities.     

Effect of different C3-aryl substituents on the antioxidant activity of 4-hydroxycoumarin derivatives

The antioxidant activity of 4-hydroxycoumarin synthetic derivatives and 4-methylumbelliferone were determined taking 4-hydroxycoumarin as the reference compound. Six 3-aryl-4-hydroxycoumarin derivatives were synthesized from 4-hydroxycoumarin as precursor in order to evaluate changes in their antioxidant properties due to C3-aryl substituent nature. Free radical scavenging capacities of these compounds against two different species DPPH(·) and ABTS(·+) and the protecting ability towards the β-carotene-linoleic acid co-oxidation enzymatically induced by lipoxygenase were measured. In addition, the relationship between the activities of these molecules against DPPH radical and the bond dissociation energy of O-H (BDE) calculated using methods of computational chemistry was evaluated.     

Hispidin analogs from the mushroom Inonotus xeranticus and their free radical scavenging activity

Three new free radical scavengers were isolated from the methanolic extract of the fruiting bodies of Inonotus xeranticus (Hymenochaetaceae), along with the known compound davallialactone. Their structures were established as hispidin analogs by extensive NMR spectral data. Compounds 3 and 4 displayed significant scavenging activity against the superoxide radical anion, ABTS radical cation, and DPPH radical, while 1 and 2 exhibited potent antioxidative activity only against ABTS radical cation.     

Synthesis,antielastase, antioxidant and radical scavenging activities of 4-(aza substituted) methylene substituted dihydroxy coumarines

A series of some 4-(aza substituted) methylene substituted dihydroxy coumarines were evaluated for their antioxidant and antielastase activities. Different in vitro methodologies such as total reducing power, 1,1-diphenyl-2-picryl-hydrazil (DPPH·) free radical scavenging, ABTS radical scavenging activity were used as antioxidant activity. All the tested compounds exhibited potent free radical scavenging ability and antielastase activites.     

白藜芦醇脂质载体制备及体外抗氧化活性测定

白藜芦醇是一种天然功能性成分，具有美白、抗氧化等功效，可用于治疗改善多种皮肤病变，延缓细胞衰老。白黎芦醇在水中的溶解度低，稳定性较差，影响了其在医药及化妆品领域的应用。本研究制备了负载白黎芦醇的纳米结构脂质载体，提高了白藜芦醇的水中分散度、稳定性和功能活性。通过热高压均质法制备了白黎芦醇纳米结构脂质载体（NLC），确定了以单硬脂酸甘油酯为固态脂质，油酸为液态脂质，其比例为1：1，4%吐温80为乳化剂，均质压力为600 bar，循环3次时得到的白藜芦醇脂质载体粒径为179 nm，此时药物包封率为85.33%。MTT实验和ROS清除实验结果也表明白藜芦醇脂质载体浓度低于100 μmol·L^-1时都未对细胞产生明显毒性，并且有优于白藜芦醇原料药的抗氧化活性。     

Evaluation of in-vitro antioxidant and antibacterial properties of Commelina nudiflora L. extracts prepared by different polar solvents

The study explored on the commonly available weed plant Commelina nudiflora which has potential in-vitro antioxidant and antimicrobial activity. The different polar solvents such as ethanol, chloroform, dichloromethane, hexane and aqueous were used for the soxhlet extraction. The extracts were identified pharmacologically as important bioactive compounds and their potential free radical scavenging activities, and antimicrobial properties were studied. C. nudiflora extracts were monitored on their in-vitro antioxidant ability by DPPH and ABTS radical scavenging assay. Aqueous extract shows significant free radical scavenging activity of 63.4 mg/GAE and 49.10 mg/g in DPPH and ABTS respectively. Furthermore, the aqueous crude extract was used in antibacterial studies, which shows the highest inhibitory activity against Pseudomonas aeruginosa, Escherichia coli and Salmonella typhi. Among all the extracts, aqueous extract of C. nudiflora has significant control over free radical scavenging activity and inhibition of the growth of food pathogenic bacteria. Also, the aqueous extract contains abundance of phenolics and flavonoids higher than other extracts. This study explored weed plant C. nudiflora as a potential source of antioxidant and antibacterial efficacy and identified various therapeutic value bioactive compounds from GC–MS analysis.Abbreviations: ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid), DPPH, 2,2-diphenyl-1-picrylhydrazyl, GAE, gallic acid equivalent, GC–MS, gas chromatography and mass spectrometry     

Effects of alcoholic solvents on antiradical abilities of protocatechuic acid and its alkyl esters

DPPH radical scavenging reactions of protocatechuic acid and its methyl ester were investigated in various solvents. In alcoholic solvents, methyl protocatechuate rapidly scavenged more than four equivalents of the radical, whereas approximately two equivalents were consumed in aprotic solvents. Methyl, ethyl, butyl, isopropyl, and tert-butyl protocatechuates were examined for their DPPH radical scavenging abilities in methanol or ethanol. As a result, the radical scavenging equivalence of sterically bulky esters tended to decrease compared to that of methyl or ethyl ester. The ABTS radical scavenging ability of those esters in water also showed the same tendency. Since 2-methoxy derivatives were detected in the reaction mixture of methyl protocatechuate and DPPH radical in methanol, a nucleophilic attack of an alcoholic molecule on the o-quinone intermediate, which is sensitive to steric hindrance from alkyl groups of both esters and alcoholic solvents, must be crucial for total radical scavenging abilities.