Structure-activity relationship analysis of antioxidant ability and neuroprotective effect of gallic acid derivatives

Gallic acid and its derivatives are a group of naturally occurring polyphenol antioxidants which have recently been shown to have potential healthy effects. In order to understand the relationship between the structures of gallic acid derivatives, their antioxidant activities, and neuroprotective effects, we examined their free radical scavenging effects in liposome and anti-apoptotic activities in human SH-SY5Y cell induced by 6-hydrodopamine autooxidation. It was found that these polyphenol antioxidants exhibited different hydrophobicity and could cross through the liposome membrane to react with 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical in a time and dose-dependent manner. At the same time, the structure-antioxidant activity relationship of gallic acid derivatives on scavenging DPPH free radical in the liposome was also analyzed based on theoretical investigations. Analysis of cell apoptosis, intracellular GSH levels, production of ROS and the influx of Ca(2+) indicated that the protective effects of gallic acid derivatives in cell systems under oxidative stress depend on both their antioxidant capacities and hydrophobicity. However, the neuroprotective effects of gallic acid derivatives seem to depend more on their molecular polarities rather than antioxidant activities in the human SH-SY5Y cell line. In conclusion, these results reveal that compounds with high antioxidant activity and appropriate hydrophobicity are generally more effective in preventing the injury of oxidative stress in neurodegenerative diseases.     

Effect of natural exogenous antioxidants on aging and on neurodegenerative diseases

Abstract

Aging and neurodegenerative diseases share oxidative stress cell damage and depletion of endogenous antioxidants as mechanisms of injury, phenomena that are occurring at different rates in each process. Nevertheless, as the central nervous system (CNS) consists largely of lipids and has a poor catalase activity, a low amount of superoxide dismutase and is rich in iron, its cellular components are damaged easily by overproduction of free radicals in any of these physiological or pathological conditions. Thus, antioxidants are needed to prevent the formation and to oppose the free radicals damage to DNA, lipids, proteins, and other biomolecules. Due to endogenous antioxidant defenses are inadequate to prevent damage completely, different efforts have been undertaken in order to increase the use of natural antioxidants and to develop antioxidants that might ameliorate neural injury by oxidative stress. In this context, natural antioxidants like flavonoids (quercetin, curcumin, luteolin and catechins), magnolol and honokiol are showing to be the efficient inhibitors of the oxidative process and seem to be a better therapeutic option than the traditional ones (vitamins C and E, and β-carotene) in various models of aging and injury in vitro and in vivo conditions. Thus, the goal of the present review is to discuss the molecular basis, mechanisms of action, functions, and targets of flavonoids, magnolol, honokiol and traditional antioxidants with the aim of obtaining better results when they are prescribed on aging and neurodegenerative diseases.     

Synthesis, stereochemical assignments, and biological activities of homoisoflavonoids

A series of four naturally occurring homoisoflavonoids and eight analogs have been synthesized starting from an appropriately substituted phenol through chroman-4-one, in four steps. The products were assigned as E-isomers based on NMR spectroscopic data. The E-isomers were converted into Z-isomers by photoisomerization. The E- and Z-isomers showed distinct chemical shifts and the differences between (E) and (Z)-homoisoflavonoids in the proton NMR spectra afford a useful method for ascertaining the stereochemistry. The antioxidant activity of homoisoflavonoids was determined by superoxide (NBT) and DPPH free radical scavenging methods. The analog 7-hydroxy-3-[(3,4,5-trihydroxyphenyl)methylene]chroman-4-one displayed excellent activity followed by sappanone A in both the methods and were several times potent than the commercial antioxidants like BHA, BHT, etc. These compounds were evaluated in vitro for their inhibitory activities against 5-lipoxygenase (5-LOX) enzyme. The analog 7-hydroxy-3-[(N,N-dimethylaminophenyl)methylene]chroman-4-one was found to possess potent inhibitory activity and was comparable to that of the standard, nordihydroguiaretic acid. These results suggest that these homoisoflavonoids, with their potent antioxidant and 5-LOX inhibitory activities, may have useful applications as antioxidants and lead compounds for asthma and inflammatory diseases.     

Antioxidant effects of ubiquinones in microsomes and mitochondria are mediated by tocopherol recycling

Ubiquinones and tocopherols (vitamin E) are intrinsic lipid components which have a stabilizing function in many membranes attributed to their antioxidant activity. The antioxidant effects of tocopherols are due to direct radical scavenging. Although ubiquinones also exert antioxidant properties the specific molecular mechanisms of their antioxidant activity may be due to: (i) direct reaction with lipid radicals or (ii) interaction with chromanoxyl radicals resulting in regeneration of vitamin E. Lipid peroxidation results have now shown that tocopherols are much stronger membrane antioxidants than naturally occurring ubiquinols (ubiquinones). Thus direct radical scavenging effects of ubiquinols (ubiquinones) might be negligible in the presence of comparable or higher concentrations of tocopherols. In support of this our ESR findings show that ubiquinones synergistically enhance enzymic NADH- and NADPH-dependent recycling of tocopherols by electron transport in mitochondria and microsomes. If ubiquinols were direct radical scavengers their consumption would be expected. Further proving our conclusion HPLC measurements demonstrated that ubiquinone-dependent sparing of tocopherols was not accompanied by ubiquinone consumption.     

Free radical involvement in the generation of trans‐root potential

The identity of the naturally occurring compounds that accept electrons from plasma membrane-bound redox systems in vivo is obscure. We analysed the effect of ascorbate, oxygen, iron, as well as their free radical forms, and also the free radical-generating and -quenching systems on the trans-root electrical potential, which had previously been shown to be coupled to plasma membrane-bound redox systems. The material was the primary root of 8-day-old maize (Zea mays L.) seedlings. Trans-root electrical potential difference was measured across excised roots. Different ascorbate (ascorbate, dehydroascorbate and ascorbate free radical) and oxygen redox forms (superoxide and hydroxide radicals and hydrogen peroxide), as well as scavenging agents of oxygen species (superoxide dismutase, catalase, mannitol), and ferric and ferrous ions were added to the solution flowing around the root. Ascorbate free radical induced the greatest depolarization of the trans-root potential when compared to other ascorbate redox forms, which is consistent with its suggested role as a natural electron acceptor. Addition of xanthine oxidase, with or without xanthine, also produced depolarizing effects. The presence of SOD magnified this effect both with ascorbate free radical and xanthine oxidase. When ferric or ferrous chloride and ferric EDTA were applied to the bathing medium, only free ferric ion produced a very pronounced depolarization. The magnitude and kinetics of trans-root potential depolarization, induced by the ascorbate redox forms and systems for the generation and scavenging of oxygen species, argue in favour of the mutually competing electron transfer role of ascorbate free radicals and superoxide radicals in the extracellular space of the root. These results provide evidence that at least a part of the electrical potential difference occurring across plant roots arises from current flow from the symplast, via the plasma membrane-bound redox systems, to naturally occurring compounds in the apoplast, and that this transfer is achieved through the mediation of their free radical forms.     

A novel method for evaluating free radical scavenging abilities of antioxidants using ultraviolet induction of bacteriophage lambda

A novel biological method used to evaluate free radical scavenging abilities of antioxidants using ultraviolet (UV) induction of bacteriophage lambda in lysogenic Escherichia coli kappa12 (lambda+) has been developed. This method is based on the induction of bacteriophage lambda from lysogenic cycle to lytic cycle by ultraviolet irradiation, and formation of free radicals during the course of induction. In the experiments, 10(8)cells/ml and 30s (39J/m2) were determined as the cell density of the lysogenic bacterium and UV irradiation time, respectively. The reliability of this method was demonstrated by electron paramagnetic resonance (EPR) spectroscopy and spin trapping with DMPO. This method had good reproducibility with intra-day variations (RSD, %) of less than 4% and inter-day variations (RSD, %) of less than 5%, respectively. By this method, the free radical scavenging abilities (ID50) of well-known antioxidants such as glutathione, superoxide dismutase (SOD), catalase (CAT) and carotenoids were determined quantitatively. The results were consistent with the ones obtained by conventional methods for evaluating free radical scavenging abilities. This developed method is reliable and uses common instruments and inexpensive, stable reagents, thus it could be utilized as a routine laboratory quantitative assay to screen a large number of substances that have potential to scavenge the free radical.     

Ferulic acid antioxidant protection against hydroxyl and peroxyl radical oxidation in synaptosomal and neuronal cell culture systems in vitro: structure-activity studies

In this study, free radical scavenging abilities of ferulic acid in relation to its structural characteristics were evaluated in solution, cultured neurons, and synaptosomal systems exposed to hydroxyl and peroxyl radicals. Cultured neuronal cells exposed to the peroxyl radical initiator AAPH die in a dose-response manner and show elevated levels of protein carbonyls. The presence of ferulic acid or similar phenolic compounds, however, greatly reduces free radical damage in neuronal cell systems without causing cell death by themselves. In addition, synaptosomal membrane systems exposed to oxidative stress by hydroxyl and peroxyl radical generators show elevated levels of oxidation as indexed by protein oxidation, lipid peroxidation, and ROS measurement. Ferulic acid greatly attenuates these changes, and its effects are far more potent than those obtained for vanillic, coumaric, and cinnamic acid treatments. Moreover, ferulic acid protects against free radical mediated changes in conformation of synaptosomal membrane proteins as monitored by EPR spin labeling techniques. The results presented in this study suggest the importance of naturally occurring antioxidants such as ferulic acid in therapeutic intervention methodology against neurodegenerative disorders such as Alzheimer's disease in which oxidative stress is implicated.     

Antioxidant strategies in the treatment of stroke

Excessive production of free radicals is known to lead to cell injury in a variety of diseases, such as cerebral ischemia. In this review, we describe some of the numerous studies that have examined this oxidative stress and the efficiency of antioxidant strategies in focal cerebral ischemia. Besides using genetically modified mice, these strategies can be divided into three groups: (1) inhibition of free radical production, (2) scavenging of free radicals, and (3) increase of free radical degradation by using agents mimicking the enzymatic activity of endogenous antioxidants. Finally, the clinical trials that have tested or are currently testing the efficiency of antioxidants in patients suffering from stroke are reviewed. The results presented here lead us to consider that antioxidants are very promising drugs for the treatment of ischemic stroke.     

Phenolics as potential antioxidant therapeutic agents: mechanism and actions

Accumulating chemical, biochemical, clinical and epidemiological evidence supports the chemoprotective effects of phenolic antioxidants against oxidative stress-mediated disorders. The pharmacological actions of phenolic antioxidants stem mainly from their free radical scavenging and metal chelating properties as well as their effects on cell signaling pathways and on gene expression. The antioxidant capacities of phenolic compounds that are widely distributed in plant-based diets were assessed by the Trolox equivalent antioxidant capacity (TEAC), the ferric reducing antioxidant power (FRAP), the hypochlorite scavenging capacity, the deoxyribose method and the copper-phenanthroline-dependent DNA oxidation assays. Based on the TEAC, FRAP and hypochlorite scavenging data, the observed activity order was: procyanidin dimer > flavanol > flavonol > hydroxycinnamic acids > simple phenolic acids. Among the flavonol aglycones, the antioxidant propensities decrease in the order quercetin, myricetin and kaempferol. Gallic acid and rosmarinic acid were the most potent antioxidants among the simple phenolic and hydroxycinnamic acids, respectively. Ferulic acid displayed the highest inhibitory activity against deoxyribose degradation but no structure–activity relationship could be established for the activities of the phenolic compounds in the deoxyribose assay. The efficacies of the phenolic compounds differ depending on the mechanism of antioxidant action in the respective assay used, with procyanidin dimers and flavan-3-ols showing very potent activities in most of the systems tested. Compared to the physiologically active (glutathione, -tocopherol, ergothioneine) and synthetic (Trolox, BHA, BHT) antioxidants, these compounds exhibited much higher efficacy. Plant-derived phenolics represents good sources of natural antioxidants, however, further investigation on the molecular mechanism of action of these phytochemicals is crucial to the evaluation of their potential as prophylactic agents.     

咖啡和绿茶的抗氧化活性研究

茶和咖啡是全世界消费最多的饮料。该工作利用电子自旋共振(ESR)技术和ABTS〔2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid)〕等方法,研究和比较了3种绿茶(龙井、碧螺春和毛峰)和3种咖啡的抗氧化活性,以及对ABTS 、DPPH、超氧阴离子、羟基自由基、单线态氧的清除作用和对脂质过氧化的抑制作用。利用高压液相色谱(HPLC)分析了绿茶中的抗氧化成分之一EGCG((-)-epigallocatechin gallate)和咖啡中的抗氧化成分之一绿原酸(chlorogenic acid5,CQA)的含量。结果发现,如果按每克剂量比较,对于清除ABTS 的能力而言,茶和咖啡水提取液对ABTS 清除能力各有千秋,咖啡水提取液的比龙井、毛峰水提取液的高,但与碧螺春无显著差异,咖啡B比咖啡A和C强;按每杯茶(3g/200mL)和咖啡(2.4g/200mL)对ABTS 的清除能力进行比较,咖啡的比龙井和毛峰的高但比碧螺春低,咖啡B比咖啡A和C强。对超氧阴离子、羟基和DPPH自由基及单线态氧的清除作用和对脂质过氧化的抑制作用的研究表明,如果按每克剂量比较,茶和咖啡水提取液没有明显区别;按每杯量进行比较时,茶水提取液比咖啡水提取液对自由基的清除能力稍强。HPLC结果表明,EGCG含量在毛峰中最高,5CQA含量在咖啡B中最高。该研究结果表明茶和咖啡中均含有丰富的抗氧化成分,并都具有较强的抗氧化活性和清除自由基能力。     

Pyrogallol-mediated toxicity and natural antioxidants: Triumphs and pitfalls of preclinical findings and their translational limitations

Pyrogallol, a potent anti-psoriatic drug, produces toxicity due to its ability to generate free radicals, besides its beneficial effects. Oxidative stress is implicated in pyrogallol-mediated toxicity in general and hepatotoxicity in particular. Naturally occurring antioxidants including, resveratrol and silymarin have been proposed as potential supplements to counteract pyrogallol-mediated toxicity, without reducing its efficacy. Due to increase in the popularity of natural antioxidants in combating pyrogallol-mediated toxicity, a literature-based survey was performed to assess their role in experimental studies and possible implications in real life situations. Although preclinical studies revealed the boons of naturally occurring antioxidants in attenuating/abolishing the undesirable effects of pyrogallol exposure, limited studies have been conducted to evaluate their role in clinics. In this review, an update on the recent development in assessing the potential of natural antioxidants in pyrogallol-mediated toxicity in preclinical interventions, triumphs and pitfalls of such investigations, their translational challenges and future possibilities are discussed.     

Kinetic evaluation of lipophilic inhibitors of lipid peroxidation in DLPC liposomes

The authors have developed a kinetic method that allows one to obtain relative reactivity constants for lipophilic antioxidants in free radical systems. Two experimental model systems were developed: (a) a methanolic solution using AMVN as the free radical initiator and linoleic acid as the substrate, and (b) a multilamellar vesicle system composed of dilinoleoylphosphatidylcholine and AAPH as the substrate and the initiator, respectively. The use of these two systems allows researchers not only to determine the intrinsic reactivity of a potential antioxidant, but also to evaluate its potency in a membranous system where the contribution of the physical properties of the antioxidant to the inhibition of lipid peroxidation is important. These results show that all antioxidants tested acted in these systems as free radical scavengers, and they validate the synergism between intrinsic scavenging ability and membrane affinity and/or membrane-modifying physical properties in the inhibition of lipid peroxidation.     

Effects of antioxidants on X-ray- or hyperthermia-induced apoptosis in human lymphoma U937 cells

Hydroxyl radicals (.OH) and superoxide anion radicals (O2.-) are known to play cardinal roles in cell killing and various types of cell damage. In order to elucidate the mechanism of the involvement of both free radicals on apoptosis, the correlation between anti-apoptotic effects and free radical scavenging abilities of anti-oxidants was studied. As an indicator of anti-apoptotic effects, C1/2 (antioxidant concentration to inhibit DNA fragmentation by 50%) was evaluated in human lymphoma cell line U937 cells 6 hr after X-ray (10 Gy) or hyperthermia (44 degrees C, 30 min) treatment. Rate constants of the reactions between antioxidants and .OH or O2.- were calculated as the scavenging ability of the antioxidants with graded concentration estimated by EPR spectroscopy. No apparent correlation between C1/2 obtained in apoptosis induced by X-rays or hyperthermia and the rate constants of antioxidants for .OH or O2.- was observed. On the other hand, the partition coefficients in 1-octanol/water of the antioxidants, an indicator of hydrophobicity, revealed a correlation with the C1/2 of the agents with hyperthermia, but not with X-ray irradiation. These results indicate that the prevention of apoptosis by an antioxidant is not simply associated with its scavenging ability for .OH or O2.-. The hydrophobicity of the antioxidant, among other possible factors, is involved in the inhibition of hyperthermia- induced apoptosis.     

4-Mercaptoimidazoles derived from the naturally occurring antioxidant ovothiols 1. Antioxidant properties

4-Mercaptoimidazoles derived from the naturally occurring family of antioxidants, the ovothiols, were assayed for their antioxidant properties. These compounds are powerful HOCI scavengers, more potent than the aliphatic thiol N-acetylcysteine. They react slowly with hydrogen peroxide with second order rate constants of 0.13-0.89 M^-1 s^-1. Scavenging of hydroxyl radical occurs at a diffusion-controlled rate (k = 2.0-5.0 × 10¹⁰ M^-1 s^-1) for the most active compounds, which are also able to inhibit copper-induced LDL peroxidation. The combination of radical scavenging and copper chelating properties may explain the inhibitory effects on LDL peroxidation. Two molecules of mercaptoimidazole can chelate a copper ion and form a square planar complex detected by EPR. Compounds bearing an electron-withdrawing group on position 2 of the imidazole ring are the most potent antioxidant molecules in this series.     

Geometrical isomerism of monounsaturated fatty acids: thiyl radical catalysis and influence of antioxidant vitamins

Thiyl radicals generated either from thiols or disulfides act as the catalyst for the cis-trans isomerization of a variety of monounsaturated fatty acid methyl esters in homogeneous solution. Similar results have also been obtained using alpha-lipoic acid and its reduced form. The effectiveness of the isomerization processes in the presence of the most common antioxidants has been addressed. The ability of thiyl radical scavenging was found to increase along the series alpha-tocopherol < ascorbic acid < all-trans retinol. The cis-trans isomerization of fatty acid residues in multilamellar vesicles of dioleoyl phosphatidyl choline by thiyl radical, in the absence and presence of the various antioxidants, has also been studied in detail. The influence of the isomerization process on the phospholipid bilayer has been tested by permeability measurements of vesicles and it is clearly shown that trans fatty acid-containing membranes have intermediate properties between those formed by all-cis and saturated components. This study contributes to the understanding of radical processes that can alter or protect the naturally occurring cis geometry of unsaturated lipids in cell membranes and demonstrates a new role of essential antioxidants.     

Antioxidant activities of enzymatic extracts from brown seaweeds

Potential antioxidative activities of enzymatic extracts from seven species of brown seaweeds were evaluated using four different reactive oxygen species (ROS) scavenging assays containing DPPH (1,1-diphenyl-2-pricrylhydrazyl) free radical, superoxide anion, hydroxyl radical and hydrogen peroxide scavenging assay. The brown seaweeds were enzymatically hydrolyzed to prepare water-soluble extracts by using five carbohydrate degrading enzymes (Viscozyme, Celluclast, AMG, Termamyl and Ultraflo) and five proteases (Protamex, Kojizyme, Neutrase, Flavourzyme and Alcalase) of commercial and inexpensive enzymes obtained from Novozyme Co. (Novozyme Nordisk, Bagsvaerd, Denmark). The enzymatic extracts exhibited more prominent effects in hydrogen peroxide scavenging activity (approximately 90%) compared to the other scavenging activities and the activity of enzymatic extracts was even higher than that of the commercial antioxidants. In particular, Ultraflo and Alcalase extracts of S. horneri were dose-dependent and thermally stable. Moreover the two enzymatic extracts strongly inhibited DNA damage (approximately 50%). Those extracts showed significantly (p<0.05) remarkable scavenging effects in DPPH free radical scavenging assay and the activity indicated a marked correlation with phenolic contents. From the results, enzymatic extracts of the brown seaweeds might be valuable antioxidative sources.     

A novel method of measuring hydroxyl radical-scavenging activity of antioxidants using γ-irradiation

A new method using ESR spin trapping was proposed for measuring the scavenging activity of antioxidants for the hydroxyl (OH) radical. (-)-Epigallocatechin gallate (EGCg) and 5,5-dimethyl-1-pyrrolline N-oxide (DMPO) were used as the antioxidant and spin trapping agent, respectively. The conventional method using a Fenton reaction had problems associated with the estimation of activity, because the antioxidant disturbs the system for generating OH radical by coordinating on Fe²⁺ and by consuming H₂O₂, besides scavenging the spin adduct (DMPO-OH). Intense γ-irradiation was therefore used to generate OH radicals, and the intensity decrease in DMPO-OH after irradiation was followed to obtain the rate constant for the scavenging of DMPO-OH by EGCg. The intensities were extrapolated to zero time to estimate the quantity of DMPO-OH formed during γ-irradiation. By using these values, the reaction rate constant between OH radical and EGCg was calculated as a ratio to that of DMPO. It was shown that this method is useful for comparing the OH radical-scavenging activity of various antioxidants.     

试论茶多酚清除生物自由基的高效性

从生物自由基出发,本文综述了茶多酚(TP)清除自由基的效能.茶多酚对多种自由基具有卓越的清除特性,并明显优于其它抗氧化剂,但茶多酚浓度、体系pH值、自由基类型、儿茶素组成与结构对其清除效能有较大影响.     

The effect of antioxidants on the response of the rabbit urinary bladder to in vitro ischemia/reperfusion

To evaluate the protective effects of two naturally occurring antioxidants, α-Lipoic acid and coenzyme Q10 on the response to in vitro ischemia of the rabbit urinary bladder. We measured free fatty acid (FFA) content, phospholipid (PL) content, malondialdehyde (MDA) levels, and phospholipase A(2) activity (PLA) of subcellular compartments. Twenty New Zealand White male rabbits were separated into four groups of five rabbits each. The in vitro whole bladders from Groups 1 and 2 received a 3 h incubation under normal oxygenated physiological conditions. The bladders were stimulated by field stimulation at 1 and 3 h. The bladders from groups 3 and 4 underwent 1 h incubation time under normal oxygenated physiological conditions. After 1 h, the bladders were stimulated with field stimulation. After a maximal pressure response was recorded, the stimulation was turned off and the bath medium changed to one equilibrated with 95% nitrogen, 5% oxygen without glucose (ischemic medium) and incubated for 1 h with field stimulations occurring at 5 min intervals during this time. At the end of this hour of ischemia with repetitive stimulation, the bath was changed to an oxygenated medium with glucose for a 1-h reperfusion period after which the stimulation was repeated. The rabbits from groups 2 and 4 received α-Lipoic acid (10 mg/kg/day) + Coenzyme Q10 (3 mg/kg/day) by gavage for 4 weeks before the experiment. At the end of the experimental period, each bladder was opened longitudinally, and the muscle and mucosa separated by blunt dissection, frozen under liquid nitrogen, and stored at -80°C for biochemical analyses. Each tissue was fractionated by differential centrifugation into nuclear, mitochondrial, synaptosomal, and cytosol (supernatant) components. PL, FFA, MDA, and PLA were analyzed using standard biochemical techniques. Post-ischemic contractility only returned to 30% of control of the untreated group. However, post-ischemic contractility of the treated group returned to approximately 70% of control. PL loss in the muscle mitochondria and synaptosomes was prevented by antioxidant treatment, while the mucosal layer showed a significant drop in PL with antioxidants treatment. Administration of CoQ + LA significantly decreased MDA levels in both control and ischemic tissues in both the muscle and mucosal bladder layers, especially substantial in the microsomal and mitochondrial components. Treatment had variable effects on PLA(2) activity. Treatment of bladder dysfunction with antioxidants daily can be beneficial in man to prevent or delay the onset of progressive loss of bladder function especially that due to ischemic damage to mitochondrial and microsomal lipids. CoQ10 + LA can provide similar protection of the bladder muscle and mucosa against lipid oxidative stress as they have been shown to protect against protein oxidative damage.     

Antioxidant activity in vitro of two aromatic compounds from Caesalpinia sappan L

Two antioxidant compounds were isolated from C. sappan L by multiple steps of column chromatography and thin layer chromatography in succession with superoxide scavenging assay as activity monitor. Structures of the two compounds were determined by spectroscopic methods as 1',4'-dihydro-spiro[benzofuran-3(2H),3'-[3H-2]benzopyran]-1',6',6',7'-tetrol (compound 1) and 3-[[4,5-dihydroxy-2(hydroxymethyl) phenyl]-methyl]-2,3-dihydro-3,6-benzofurandiol (compound 2). Characterization of antioxidant properties of these two compounds was done by determining the inhibitory effect on xanthine oxidase activity as well as scavenging effect on superoxide anion and hydroxyl radicals. Our results indicated that compounds 1 and 2 inhibited xanthine oxidase activity and scavenged superoxide anion and hydroxyl radicals. Compounds 1 and 2 possessed similar radical scavenging activities as ascorbic acid, and they were more effective than other well-known antioxidants such as alpha-tocopherol, beta-carotene, and BHT. As inhibitors of free radical formation, compounds 1 and 2 were more effective than all the other antioxidants tested. In conclusion, compounds 1 and 2 can be regarded as primary antioxidants with radical-scavenging and chain-breaking activities as well as secondary antioxidants with inhibitory effect on radical generation.