Studies on the Antioxidant and Free Radical Scavenging Properties of Idb 1016 A New Flavanolignan Complex

Silybin has been complexed in 1:1 ratio with phosphatidyl choline to give IdB 1016 in order to increase its bioavailability. The antioxidant and free radical scavenger action of this new form of silybin has beenn evaluated.

One hour after the intragastric administration to rats of IdB 1016 (1.5g/kg b.wt.) the concentration of silybin in the liver microsomes was estimated to be around 2.5°g/mg protein corresponding to a final concentration in the microsomal suspension used of about 10°M. At these levels IdB decreased by about 40% the lipid peroxidation induced in microsomes by NADPH, CC1₄ and cumene hydroperoxide, probably by acting on lipid derived radicals. Spin trapping experiments showed, in fact, that the complexed form of silybin was able to scavenge lipid dienyl radicals generated in the microsomal membranes. In addition, IdB 1016 was also found to interact with free radical intermediates produced during the metabolic activation of carbon tetrachloride and methylhydrazine.

These effects indicate IdB 1016 as a potentially protective agent against free radical-mediated toxic damage.     

Studies on the Antioxidant and Free Radical Scavenging Properties of Idb 1016 A New Flavanolignan Complex

Silybin has been complexed in 1:1 ratio with phosphatidyl choline to give IdB 1016 in order to increase its bioavailability. The antioxidant and free radical scavenger action of this new form of silybin has beenn evaluated.

One hour after the intragastric administration to rats of IdB 1016 (1.5g/kg b.wt.) the concentration of silybin in the liver microsomes was estimated to be around 2.5°g/mg protein corresponding to a final concentration in the microsomal suspension used of about 10°M. At these levels IdB decreased by about 40% the lipid peroxidation induced in microsomes by NADPH, CC1₄ and cumene hydroperoxide, probably by acting on lipid derived radicals. Spin trapping experiments showed, in fact, that the complexed form of silybin was able to scavenge lipid dienyl radicals generated in the microsomal membranes. In addition, IdB 1016 was also found to interact with free radical intermediates produced during the metabolic activation of carbon tetrachloride and methylhydrazine.

These effects indicate IdB 1016 as a potentially protective agent against free radical-mediated toxic damage.     

Antimalarial Drugs Exacerbate Rat Liver Microsomal Lipid Peroxidation in the Presence of Oxidants

The study was undertaken to evaluate the effect of prior treatment of rats with the antimalarial drugs amodiaquine (AQ) mefloquine (MQ) and halofantrine (HF) on rat liver microsomal lipid peroxidation in the presence of 1 mM FeSO4, 1 mM ascorbate and 0.2 mM H₂O₂ (oxidants). Ingestion of -tocopheral, a radical chain-breaking antioxidant was also included to assess the role of antioxidants in the drug treatment. In the presence of oxidants AQ, MQ and HF elicited 288%, 175% and 225% increases in malondialdehyde (MDA) formation while the drugs induced 125%, 63% and 31% increases in the absence of oxidants respectively. Similarly, AQ, MQ and HF induced lipid hydroperoxide formation by 380%, 256%, 360% respectively in the presence of oxidants and 172%, 136% and 92% in the absence of exogenously added oxidants respectively. -tocopherol reduced AQ, MQ and HF-induced MDA formation by 40%, 55% and 52% respectively and lipid hydroperoxide formation by 53%, 59% and 54% respectively. Similarly, -tocopherol attenuated the AQ, MQ and HF-induced MDA formation by 49%, 51% and 51% in the presence of oxidants and lipid hydroperoxide formation by 61%, 62% and 47% respectively. The results indicate that rat liver microsomal lipid peroxidation could be enhanced by antimalarial drugs in the presence of reactive oxygen species and this effect could be ameliorated by treatment with antioxidants.     

On the Characteristics of the Visible Chemiluminescence Following Free Radical Lipid Peroxidation

The characteristics of the visible luminescence that follows the lipid peroxidative process were investigated either in the autoxidation of rat brain homogenates or in the azo-bis-arnidinopropane initiated lipid peroxidation of erythrocyte plasma membranes and liver microsomes. In these systems the luminescence decay observed after total inhibition of the lipid peroxidation is not an iron-catalyzed process, and follows a complex kinetics comprising fast and slow components. The slow component of the decay lasts for several hours at 27°C and amounts to nearly half of the total intensity measured prior to the inhibition of the oxidative process by propyl gallate. The addition of thiols (diethyldithiocarbamate, penicillamine or dithiothreitol) to a lipid peroxidizing system inhibits the chain oxidation and catalyzes the dark decomposition of one (or several) of the luminescence precursors, following first order kinetics. The effect of temperature on the slow luminescence decay corresponds to an activation energy of 18.5kcal/mol.     

On the Characteristics of the Visible Chemiluminescence Following Free Radical Lipid Peroxidation

The characteristics of the visible luminescence that follows the lipid peroxidative process were investigated either in the autoxidation of rat brain homogenates or in the azo-bis-arnidinopropane initiated lipid peroxidation of erythrocyte plasma membranes and liver microsomes. In these systems the luminescence decay observed after total inhibition of the lipid peroxidation is not an iron-catalyzed process, and follows a complex kinetics comprising fast and slow components. The slow component of the decay lasts for several hours at 27°C and amounts to nearly half of the total intensity measured prior to the inhibition of the oxidative process by propyl gallate. The addition of thiols (diethyldithiocarbamate, penicillamine or dithiothreitol) to a lipid peroxidizing system inhibits the chain oxidation and catalyzes the dark decomposition of one (or several) of the luminescence precursors, following first order kinetics. The effect of temperature on the slow luminescence decay corresponds to an activation energy of 18.5kcal/mol.     

Effect of Free Radical Scavengers and Metal Ion Chelators on Hydrogen Peroxide and Phenylhydrazine Induced Red Blood Cell Lipid Peroxidation

Desferoxamine a well-known iron chelator was found to decrease hydrogen peroxide and phenylhy-dra/ine induced lipid peroxidation of red blood cell membranes assessed by hydrocarbon gas release and loss of polyunsaturated fatty acids. Hydroxyl radical scavengers like mannitol and thiourea and proteins like albumin were unable to reduce peroxidative reactions to our system. Addition of uric acid (in an unphysiological concentration of 5mM) to the incubation medium resulted in a slight reduction in H,0₂/phenyIhydrazine mediated break-down of arachidonic (20:4) and docosahexaenoic acid (22:6) in the erythrocyte membrane and consequently in a decreased alkane release and haemolysis.     

菟丝子黄酮体外清除自由基活性的研究

采用DPPH体系、羟基自由基体系、烷基自由基引发的亚油酸氧化体系、超氧阴离子自由基体系对菟丝子黄酮的抗氧化活性进行研究,并同Vc、BHT进行了比较。结果表明:在试验质量浓度范围内(0.008 mg/mL~1 mg/mL),菟丝子黄酮对DPPH.、.OH自由基、O2-.自由基的清除率可达91.56%、89.39%和87.65%;其对烷基自由基的抑制率为84.22%,且其抗氧化活性强于Vc和BHT,表明菟丝子黄酮是一种天然有效的自由基清除剂。     

Lipid Peroxides in the Free Radical Pathophysiology of Brain Diseases

1. Polyunsaturated fatty acids are essential for normal neural cell membrane functioning because many membrane properties, such as fluidity and permeability, are closely related to the presence of unsaturated and polyunsaturated side chains. Lipid peroxidation results in loss of membrane polyunsaturated fatty acids and oxidized phospholipids as polar species contributing to increased membrane rigidity.2. Polyunsaturated fatty acids are released from membrane phospholipids by a number of enzymic mechanisms involving the receptor-mediated stimulation of phospholipase A₂ and phospholipase C/diacylglycerol lipase pathways.3. The overstimulation of excitatory amino acid (EAA) receptors stimulates the activities of lipases and phospholipases, and this stimulation produces changes in membrane phospholipid composition, permeability, and fluidity, thus decreasing the integrity of plasma membranes.4. Alterations in properties of plasma membranes may be responsible for the degeneration of neurons seen in neurodegenerative diseases. Two major processes may be involved in neuronal injury caused by the overstimulation of EAA receptors. One is a large Ca²⁺ influx and the other is an accumulation of free radicals and lipid peroxides as a result of neural membrane phospholipid degradation. It is suggested that calcium and free radicals act in concert to induce neuronal injury in acute trauma (ischemia and spinal cord injury) and in neurodegenerative diseases.     

用清除有机自由基DPPH法评价植物抗氧化能力

几种抗氧化剂的浓度与其清除1,1-二苯基苦基苯肼(DPPH)能力呈显著的线性相关.不同抗氧化剂清除DPPH能力差异明显.抗坏血酸与DPPH反应的灵敏性高于其抑制肾上腺素氧化的能力.用DPPH法和亚油酸氧化法同时测定了生长在不同光强下植物叶片抗氧化能力的变化,两种方法所得结论相一致.结果表明清除有机自由基法是一种快速、简便、灵敏的评估植物抗氧化能力的可行方法.     

库克诺你果汁提取物体外清除自由基及抗氧化活性研究

本文对诺你果汁多糖、乙醇溶出物和乙酸乙酯萃取物体外对超氧阴离子(O2·)、羟自由基(·OH)、DPPH和脂质过氧化(LPO)的抑制作用进行了研究。超氧阴离子(O2·)由邻苯三酚自氧化产生;羟自由基(·OH)由Fenton反应产生;利用Fe2 诱发卵黄脂蛋白产生丙二醛(MDA),TBA法测定。所有测定均为分光光度法。结果表明,与已知抗氧化剂L抗坏血酸相比,乙醇溶出物和乙酸乙酯萃取物均有明显的捕捉自由基和抗氧化能力,而多糖捕捉自由基和抗氧化能力很低,且对O2·没有抑制作用,反而会增加其生成速度。     

Inhibition of Lipid Peroxidation by Dihydroquinoline-Type Antioxidant (CH 402)

The in vitro effect of a non-toxic, water soluble, low molecular weight, stable dihydroquinoline-type antioxidant, CH 402 (Na (2,2-dimethyl-1,2-dihydroquinoline-4-yl) –- methane sulphonic acid) was studied on free radical reactions in brain subcellular fractions. Experiments were performed using rat and mouse brain homogenate and microsomal fractions. Non … enzymatically induced lipid peroxidation by ascorbic acid was studied in correlation with ascorbic acid and CH 402 concentrations and incubation time. Malondialdehyde production during lipid peroxidation was measured by the thiobarbituric acid test. In a concentration range of 10^?2–10^?5 M CH 402 dose - dependently inhibited the ascorbic acid induced in vitro lipid peroxidation in mouse and rat brain subcellular fractions.     

Review: Alzheimer's Amyloid β-Peptide-Associated Free Radical Oxidative Stress and Neurotoxicity

Alzheimer's disease, the major dementing disorder of the elderly that affects over 4 million Americans, is related to amyloid β-peptide, the principal component of senile plaques in Alzheimer's disease brain. Oxidative stress, manifested by protein oxidation and lipid peroxidation, among other alterations, is a characteristic of Alzheimer's disease brain. Our laboratory united these two observations in a model to account for neurodegeneration in Alzheimer's disease brain, the amyloid β-peptide-associated oxidative stress model for neurotoxicity in Alzheimer's disease. Under this model, the aggregated peptide, perhaps in concert with bound redox metal ions, initiates free radical processes resulting in protein oxidation, lipid peroxidation, reactive oxygen species formation, cellular dysfunction leading to calcium ion accumulation, and subsequent neuronal death. Free radical antioxidants abrogate these findings. This review outlines the substantial evidence from multiidisciplinary approaches for amyloid β-peptide-associated free radical oxidative stress and neurotoxicity and protection against these oxidative processes and cell death by free radical scavengers. In addition, we review the strong evidence supporting the notion that the single methionine residue of amyloid β-peptide is vital to the oxidative stress and neurotoxicological properties of this peptide. Further, we discuss studies that support the hypothesis that aggregated soluble amyloid β-peptide and not fibrils per se are necessary for oxidative stress and neurotoxicity associated with amyloid β-peptide.     

Oxidative Glycation and Free Radical Production: A Causal Mechanism of Diabetic Complications

Glucose may oxidise under physiological conditions and lead to the production of protein reactive ketoaldehydes, hydrogen peroxide and highly reactive oxidants. Glucose is thus able to modify proteins by the attachment of its oxidation derived aldehydes, leading to the development of novel protein fluoro-phores, as well as fragment protein via free radical mechanisms.

The fragmentation of protein by glucose is inhibitable by metal chelators such as diethylenetriamine pentaacetic acid (DETAPAC) and free radical scavengers such as benzoic acid, and sorbitol. The enzymic antioxidant, catalase, also inhibits protein fragmentation.

Protein glycation and protein oxidation are inextricably linked. Indeed, using boronate affinity chromatography to separate glycated from non-glycated material, we demonstrate that proteins which arc glycated exhibit an enhanced tryptophan oxidation. Our observation that both glycation and oxidation occur simultaneously further supports the hypothesis that tissue damage associated with diabetes and ageing has an oxidative origin.     

Oxidative Glycation and Free Radical Production: A Causal Mechanism of Diabetic Complications

Glucose may oxidise under physiological conditions and lead to the production of protein reactive ketoaldehydes, hydrogen peroxide and highly reactive oxidants. Glucose is thus able to modify proteins by the attachment of its oxidation derived aldehydes, leading to the development of novel protein fluoro-phores, as well as fragment protein via free radical mechanisms.

The fragmentation of protein by glucose is inhibitable by metal chelators such as diethylenetriamine pentaacetic acid (DETAPAC) and free radical scavengers such as benzoic acid, and sorbitol. The enzymic antioxidant, catalase, also inhibits protein fragmentation.

Protein glycation and protein oxidation are inextricably linked. Indeed, using boronate affinity chromatography to separate glycated from non-glycated material, we demonstrate that proteins which arc glycated exhibit an enhanced tryptophan oxidation. Our observation that both glycation and oxidation occur simultaneously further supports the hypothesis that tissue damage associated with diabetes and ageing has an oxidative origin.     

Prophylactic Effects of Purple Shoot Green Tea on Cytokine Immunomodulation through Scavenging Free Radicals and NO in LPS-Stimulated Macrophages

Polyphenols and flavonoids from non-fermented green tea and fully-fermented black tea exhibit antioxidant abilities that function as natural health foods for daily consumption. Nonetheless, evidence regarding prophylactic effects of purple shoot tea on immunomodulation remains scarce. We compared the immunomodulatory effects of different tea processes on oxidative stress and cytokine expressions in lipopolysaccharide (LPS)-stimulated macrophages. Major constituents of four tea products, Taiwan Tea Experiment Station No.12 (TTES No. 12) black and green tea and purple shoot black and purple shoot green tea (TB, TG, PB and PG, respectively), were analyzed to explore the prophylactic effects on expressions of free radicals, nitric oxide (NO), monocyte chemoattractant protein-1 (MCP-1), interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in LPS-activated RAW264.7 cell models. PG contained abundant levels of total polyphenols, flavonoids, condensed tannins and proanthocyanidins (371.28 ± 3.83; 86.37 ± 1.46; 234.67 ± 10.1; and 24.81 ± 0.75 mg/g, respectively) contributing to excellent free radical scavenging potency. In both the LPS-activated inflammation model and the prophylactic model, all tea extracts suppressed NO secretion in a dose-dependent manner, especially for PG. Intriguingly, most tea extracts enhanced expressions of IL-6 in LPS-stimulated macrophages, except PG. However, all teas disrupted downstream transduction of chemoattractant MCP-1 for immune cell trafficking. In the prophylactic model, all teas inhibited inflammatory responses by attenuating expressions of IL-6 and TNF-α in a dose-dependent manner, especially for TG and PG. Our prophylactic model demonstrated PG exerts robust effects on modulating LPS-induced cytokine expressions of MCP-1, IL-6 and TNF-α through scavenging free radicals and NO. In light of the prophylactic effects on LPS-related inflammation, PG effectively scavenges free radicals to modulate cytokine cascades that could serve as a functional beverage for immunomodulation.     

Studies on the Effects of Antioxidants and Inhibitors of Radical Generation on Free Radical Production in the Reperfused Rat Heart Using Electron Spin Resonance Spectroscopy

Reperfusion of the heart after a period of ischaemia can precipitate ventricular arrhythmias and lead to an exacerbation of tissue injury. Direct evidence to suggest the involvement of free radicals has been obtained using electron spin resonance (esr) spectroscopy and the spin trap N-tert. butyl-α-phenyl nitrone (PBN). In the present study, we have used esr spectroscopy and PBN to examine the individual effects of superoxide dismutase (SOD), catalase. allopurinol or desferal on radical production in the isolated. reperfused rat heart. A burst of radical production was observed in the control group during the first 5 minutes of reperfusion; the peak occurred during the first minute, when signal intensity had increased by almost 300%. but returned to the baseline by 15 minutes of reperfusion. The esr signals were consistent with the trapping of either alkoxyl or carbon-centered radicals (a_N = 13.6 and a_H = 1.56G). In the desferal-treated group, a burst of radical production was observed during the first five minutes of reperfusion; this was maximal during the second minute, when signal intensity had increased by almost 200%, but had returned to the baseline value by 30 minutes of reperfusion. In the SOD-treated group, a burst of radical production was observed during the first 10 minutes of reperfusion; signal intensity was maximal during the tenth minute of reperfusion, when signal intensity had increased by almost 200%. but had returned to the baseline value by 30 minutes of reperfusion. In the allopurinol- and catalase-treated groups, no significant burst of radical production could be detected. These data further support the concept that cytotoxic, oxygen-derived species are formed upon reperfusion and that hydrogen peroxide and/or hy-droxyl radicals, are likely to be involved.     

黄鳝清除氧自由基作用的研究

采用化学发光法建立四个活性氧体外模型分析黄鳝粘液、血液、粗多糖清除氧自由基和抑制脂质过氧化作用。结果表明:黄鳝粘液、血液和粗多糖具有清除超氧阴离子自由基(O2·)、羟自由基(·OH)、过氧化氢(H2O2)和抑制脂质过氧化(LPO)作用。清除O2·的IC50分别为5.10±2.68μg/mL、3.62±1.56μg/mL、7.19±1.19μg/mL;清除·OH的IC50分别为5.86±1.54μg/mL、3.36±1.36μg/mL、7.93±0.50μg/mL;清除H2O2的IC50分别为6.91±1.29μg/mL、5.92±0.39μg/mL、8.21±0.61μg/mL;抑制LPO的IC50分别为8.11±0.83μg/mL、6.90±0.51μg/mL、7.62±1.01μg/mL。提示黄鳝血液清除氧自由基作用最明显,粘液次之,最弱为粗多糖。     

平菇多糖清除O_2~-及对红细胞膜自由基氧化的影响

平菇经热水抽提 ,Sevag法去蛋白后 ,用乙醇沉淀 ,得平菇粗糖。上SephadexG - 10 0层析 ,可得精制多糖。在体外反应产生O2 -,此体系中 ,加入不同浓度的平菇多糖。平菇多糖在较低浓度时 (<2 0 0mg/L)具有清除O2 -作用 ,而在较高浓度 (>2 0 0mg/L)时 ,作用不明显。同时 ,用荧光法研究平菇多糖对小鼠红细胞脂质过氧化的影响 ,结果表明平菇多糖能够抑制小鼠红细胞的脂质过氧化     

The Free Radical Scavenger, α-Lipoic Acid, Protects Against Cerebral Ischemia-Reperfusion Injury in Gerbils

-Lipoic acid (thioctic acid) was tested for its neuroprotective activity in a Mongolian gerbil model of forebrain ischemia/reperfusion. Adult gerbils were treated for 7 days with two intraperitoneal injections per day of -lipoic acid (20 mg/kg), vehicle or saline and on the 7th day the animals were subjected to 5 min of forebrain ischemia. Ischemic injury was assessed by monitoring the increases in locomotor activity and from the extent of damage to the CA1 hippocampal pyramidal cell layer after 5 days of recovery. By both criteria, -lipoic acid was neuroprotective against ischemia/reperfusion evoked cerebral injury.     

Inhibition of Membrane Lipid Peroxidation by a Radical Scavenging Mechanism: a Novel Function for Hydroxyl-Containing Ionophores

In the present study we show that K⁺/H⁺ hydroxyl-containing ionophores lasalocid-A (LAS) and nigericin (NIG) in the nanomolar concentration range, inhibit Fe²⁺-citrate and 2,2'-azobis(2-amidinopropane) di-hydrochloride (ABAP)-induced lipid peroxidation in intact rat liver mitochondria and in egg phosphatidyl-choline (PC) liposomes containing negatively charged lipids—dicetyl phosphate (DCP) or cardiolipin (CL)—and KCl as the osmotic support. In addition, monensin (MON), a hydroxyl-containing ionophore with higher affinity for Na⁺ than for K⁺, promotes a similar effect when NaCl is the osmotic support. The protective effect of the ionophores is not observed when the osmolyte is sucrose. Lipid peroxidation was evidenced by mitochondrial swelling, antimycin A-insensitive O₂ consumption, formation of thiobarbituric acid-reactive substances (TBARS), conjugated dienes, and electron paramagnetic resonance (EPR) spectra of an incorporated lipid spin probe. A time-dependent decay of spin label EPR signal is observed as a consequence of lipid peroxidation induced by both inductor systems in liposomes. Nitroxide destruction is inhibited by buty-lated hydroxytoluene, a known antioxidant, and by the hydroxyl-containing ionophores. In contrast, vali-nomycin (VAL), which does not possess alcoholic groups, does not display this protective effect. Effective order parameters (S_eff), determined from the spectra of an incorporated spin label are larger in the presence of salt and display a small increase upon addition of the ionophores, as a result of the increase of counter ion concentration at the negatively charged bilayer surface. This condition leads to increased formation of the ion-ionophore complex, the membrane binding (uncharged) species. The membrane-incorporated complex is the active species in the lipid peroxidation inhibiting process. Studies in aqueous solution (in the absence of membranes) showed that NIG and LAS, but not VAL, decrease the Fe²⁺-citrate-induced production of radicals derived from piperazine-based buffers, demonstrating their property as radical scavengers. Both Fe²⁺-citrate and ABAP promote a much more pronounced decrease of LAS fluorescence in PC/CL liposomes than in dimyristoyl phosphatidyl-choline (DMPC, saturated phospholipid)-DCP liposomes, indicating that the ionophore also scavenges lipid peroxyl radicals. A slow decrease of fluorescence is observed in the latter system, for all lipid compositions in sucrose medium, and in the absence of membranes, indicating that the primary radicals stemming from both inductors also attack the ionophore. Altogether, the data lead to the conclusion that the membrane-incorporated cation complexes of NIG, LAS and MON inhibit lipid peroxidation by blocking initiation and propagation reactions in the lipid phase via a free radical scavenging mechanism, very likely due to the presence of alcoholic hydroxyl groups in all three molecules and to the attack of the aromatic moiety of LAS.