Spectrofluorometric assay of thiamine hydrochloride in injections and multivitamin preparations

A spectrofluorometric method is described for the determination of thiamine hydrochloride. The method is based on formation of a fluorescent product by oxidation of thiamine HCl with 2,3,5,6-tetrabromo-1,4-benzoquinone in aqueous acetonitrile solution. The reaction product is stable for at least 6 h and shows excitation and emission maxima around 355 and 420 nm, respectively. The method is highly selective for thiamine HCl in the presence of other B vitamins. Thiamine HCl can be determined at concentrations as low as 20-160 ng/mL of the final solutions. When the method was applied to the determination of thiamine HCl added to some commercially available multivitamin preparations, recoveries were 98.78-99.98%.     

Antioxidant capacity and other bioactivities of the freeze-dried Amazonian palm berry, Euterpe oleraceae mart. (acai)

The fruit of Euterpe oleraceae, commonly known as acai, has been demonstrated to exhibit significantly high antioxidant capacity in vitro, especially for superoxide and peroxyl scavenging, and, therefore, may have possible health benefits. In this study, the antioxidant capacities of freeze-dried acai fruit pulp/skin powder (OptiAcai) were evaluated by different assays with various free radical sources. It was found to have exceptional activity against superoxide in the superoxide scavenging (SOD) assay, the highest of any food reported to date against the peroxyl radical as measured by the oxygen radical absorbance capacity assay with fluorescein as the fluorescent probe (ORACFL), and mild activity against both the peroxynitrite and hydroxyl radical by the peroxynitrite averting capacity (NORAC) and hydroxyl radical averting capacity (HORAC) assays, respectively. The SOD of acai was 1614 units/g, an extremely high scavenging capacity for O2*-, by far the highest of any fruit or vegetable tested to date. Total phenolics were also tested as comparison. In the total antioxidant (TAO) assay, antioxidants in acai were differentiated into "slow-acting" and "fast-acting" components. An assay measuring inhibition of reactive oxygen species (ROS) formation in freshly purified human neutrophils showed that antioxidants in acai are able to enter human cells in a fully functional form and to perform an oxygen quenching function at very low doses. Furthermore, other bioactivities related to anti-inflammation and immune functions were also investigated. Acai was found to be a potential cyclooxygenase (COX)-1 and COX-2 inhibitor. It also showed a weak effect on lipopolysaccharide (LPS)-induced nitric oxide but no effect on either lymphocyte proliferation and phagocytic capacity.     

Coexpression of the superoxide dismutase and the catalase provides remarkable oxidative stress resistance in Lactobacillus rhamnosus

Lactic acid bacteria (LAB) are generally sensitive to oxidative stress caused by reactive oxygen species (ROS). Antioxidant enzymes, especially superoxide dismutase (SOD) and catalase (CAT), can protect against ROS by eliminating superoxide and H(2)O(2), respectively. Lactobacillus rhamnosus is a valuable probiotic starter culture but is deficient in both SOD and CAT, and is thus likely to suffer from oxidative stress in industrial fermentation. To confer high level of oxidative resistance on L. rhamnosus , the SOD gene sodA from Streptococcus thermophilus and CAT gene katA from L. sakei were coexpressed in L. rhamnosus AS 1.2466. The enzyme activities of SOD and CAT were 147.80 ± 1.08 U/mg protein and 2.53 μmol of H(2)O(2) /min/10(8) cfu, respectively, in the recombinant L. rhamnosus CS. After incubation with 10 mM H(2)O(2), the survival ratio of L. rhamnosus CS was 400-fold higher than that of L. rhamnosus CAT. In long-term aerated conditions, viable cells of L. rhamnosus CS remained ～10(6) cfu/mL after incubation for 7 days, while no living cells of the control were detected. These results showed that the cooperation between SOD and CAT could significantly enhance oxidative resistance in L. rhamnosus . To our best knowledge, this is the first report of two synergistic antioxidant genes being coexpressed in the same Lactobacilli.     

Scavenging capacity of berry crops on superoxide radicals, hydrogen peroxide, hydroxyl radicals, and singlet oxygen

The antioxidant activities against superoxide radicals (O(2)(*)(-)), hydrogen peroxide (H(2)O(2)), hydroxyl radicals (OH(*)), and singlet oxygen ('O(2)) was evaluated in fruit juice from different cultivars of thornless blackberries (Rubus sp.), blueberries (Vaccinium spp.), cranberries (Vaccinium macrocarpon Aiton), raspberries (Rubus idaeus L. and Rubus occidentalis L.), and strawberries (Fragaria x ananassa Duch.). Among the different cultivars, juice of 'Hull Thornless' blackberry, 'Earliglow' strawberry, 'Early Black' cranberry, 'Jewel' raspberry, and 'Elliot' blueberry had the highest antioxidant capacity against superoxide radicals (O(2)(*)(-)), hydrogen peroxide (H(2)O(2)), hydroxyl radicals (OH(*)), and singlet oxygen ('O(2)). In general, blackberries had the highest antioxidant capacity inhibition of O(2)(*)(-), H(2)O(2), and OH(*). Strawberry was second best in the antioxidant capacity assay for these same free radicals. With regard to 'O(2) scavenging activity, strawberry had the highest value, while blackberry was second. Cranberries had the lowest inhibition of H(2)O(2) activity. Meanwhile, blueberries had the lowest antioxidant capacity against OH(*) and 'O(2). There were interesting and marked differences among the different antioxidants in their abilities to scavenge different reactive oxygen species. beta-Carotene had by far the highest scavenging activity against 'O(2) but had absolutely no effect on H(2)O(2). Ascorbic acid was the best at inhibiting H(2)O(2) free radical activity. For OH(*), there was a wide range of scavenging capacities from a high of 15.3% with alpha-tocopherol to a low of 0.88% with ascorbic acid. Glutathione had higher O(2)(*)(-) scavenging capacity compared to the other antioxidants.     

Biochemical characterization of a cambialistic superoxide dismutase isozyme from diatom Thallassiosira weissflogii: cloning, expression, and enzyme stability

A cDNA clone of 1081 bp encoding a second putative superoxide dismutase (SOD) from diatom Thallassiosira weissflogii was cloned by the polymerase chain reaction technique. The cDNA encodes a protein of 286 amino acid residues. Alignment of the truncated SOD sequence containing 217 amino acid residues with Mn-SODs from Vibrio mimicus and Escherichia coli, as well as two Fe-SODs from E. coli and Photobacterium leiognathi, this SOD showed greater homology to Mn-SOD. The residues required to coordinate the manganese ion were conserved in all reported Mn-SOD. The recombinant SOD has a half life of deactivation of 14.7 min at 65 degrees C. Its thermal inactivation rate constant Kd was 3.21 x 10(-2) min(-1). The enzyme was stable in a broad pH range from 4 to 12. The presence of imidazole (up to 0.8 M) and sodium dodecylsulfate (up to 4%) had little effect on the enzyme's activity. The atomic absorption spectrometric assay showed the presence of 0.3 atom of iron/manganese (2:1) in each SOD subunit. Reconstituted activity suggested that diatom SOD was cambialistic Fe/Mn-SOD.     

Mechanism of the superoxide scavenging activity of neoandrographolide - a natural product from Andrographis paniculata Nees

It was hypothesized that neoandrographolide might scavenge free radicals by donating the allylic hydrogen of the unsaturated lactone ring. It was found that the stoichiometry of the reaction between neoandrographolide and superoxide radical generated from KO(2) in DMSO was 2 to 1. One major reaction product was isolated and determined to be a diacid formed by the opening of the lactone ring. It was concluded that the antiradical activity of neoandrographolide proceeded by hydrogen abstraction from carbon C-15. A reaction mechanism was proposed.     

Effects of metal chelator, sodium azide, and superoxide dismutase on the oxidative stability in riboflavin-photosensitized oil-in-water emulsion systems

The effects of riboflavin photosensitization on the oxidative stability of oil-in-water (O/W) emulsions were determined using lipid hydroperoxides and headspace volatile analyses. The influences of a metal chelator, sodium azide, and superoxide dismutase (SOD) on oxidation pathways were tested to gain a better understanding of the role of transition metals, singlet oxygen, and superoxide anion, respectively. Emulsions with riboflavin and visible light irradiation had significantly higher lipid hydroperoxides and volatiles (p < 0.05) as compared to samples without light irradiation or riboflavin. The addition of ethylenediammetetraacetic acid (EDTA) decreased the formation of lipid hydroperoxides, hexanal, 2-heptenal, and 1-octen-3-ol in a concentration-dependent manner. Sodium azide, a singlet oxygen physical quencher, only inhibited the formation of 2-heptenal and 1-octen-3-ol. Overall, photosensitized riboflavin participated in both type I and type II pathways in O/W emulsions, and these pathways enhance the prooxidant activity of metals through their ability to produce lipid hydroperoxides and superoxide anion.     

Antioxidant activities of pomegranate fruit extract and its anthocyanidins: delphinidin, cyanidin, and pelargonidin.

Antioxidant activities of freeze-dried preparations of a 70% acetone extract of pomegranate (Punica granatum L.) and its three major anthocyanidins (delphinidin, cyanidin, and pelargonidin) were evaluated. Free radical scavenging activities were examined using an ESR technique with spin trapping; DMPO for hydroxyl (.OH) and superoxide (O(2)(.-) ) radicals; and [(MGD)(2)Fe(2+)] for nitric oxide (NO). Inhibitory effects on lipid peroxidation were estimated by the levels of malonaldehyde and 4-hydroxyalkenals in rat brain homogenates. Pomegranate extract exhibited scavenging activity against.OH and O(2)(.-). Anthocyanidins inhibited a Fenton reagent.OH generating system possibly by chelating with ferrous ion. Anthocyanidins scavenged O(2)(.)- in a dose-dependent manner. The ID(50) values of delphinidin, cyanidin, and pelargonidin were 2.4, 22, and 456 microM, respectively. In contrast, anthocyanidins did not effectively scavenge NO. Anthocyanidins inhibited H(2)O(2)-induced lipid peroxidation in the rat brain homogenates. The ID(50) values of delphinidin, cyanidin, and pelargonidin for them were 0.7, 3.5, and 85 microM, respectively. These findings suggest that the above anthocyanidins contribute to the antioxidant activity of pomegranate fruits.     

丛枝菌根真菌Glomus mosseae 提高超氧化物歧化酶和过氧化氢酶活性和真盐生植物盐地碱蓬耐盐性

Arbuscular mycorrhizal (AM)-mediated plant physiological activities could contribute to plant salt tolerance. However, the biochemical mechanism by which AM fungi enhance salt tolerance of halophytic plants is unclear. A pot experiment was conducted to determine whether salt tolerance of the C 3 halophyte Suaeda salsa was enhanced by the AM fungus Glomus mosseae. When 60-day-old S. salsa seedlings were subjected to 400 mmol L-1 NaCl stress for 35 days, plant height, number of leaves and branches, shoot and root biomass, and root length of G. mosseae-colonized seedlings were significantly greater than those of the nonmycorrizal seedlings. Leaf superoxide dismutase (SOD) activity at all sampling times (weekly for 35 days after salt stress was initiated) and leaf catalase (CAT) activity at 2 and 3 weeks after salt stress was initiated were also significantly enhanced in G. mosseae-colonized S. salsa seedlings, while the content of leaf malondialdehyde (MDA), a product of membrane lipid peroxidation, was significantly reduced, indicating an alleviation of oxidative damage. The corresponding leaf isoenzymes of SOD (Fe-SOD, Cu/Zn-SOD1, and Cu/Zn-SOD2) and CAT (CAT1 and CAT2) were also significantly increased in the mycorrhizal seedlings after 14 days of 400 mmol L-1 NaCl stress. Our results suggested that G. mosseae increased salt tolerance by increasing SOD and CAT activities and forming SOD and CAT isoforms in S. salsa seedlings.     

Isolation and characterization of superoxide dismutase from wheat seedlings

Two major superoxide dismutases (SODs; SODs I and II) were found in the crude enzyme extract of wheat seedlings after heat treatment, ammonium sulfate fractionation, anionic exchange chromatography, and gel permeation chromatography. The purification fold for SODs I and II were 154 and 98, and the yields were 11 and 2.4%, respectively. SOD I was further characterized. It was found that SOD I from wheat seedlings is a homodimer, with a subunit molecular mass of 23 kDa. Isoelectric focusing electrophoresis (IEF) and zymogram staining results indicated that the isoelectric point of SOD I is 3.95. It belongs to the MnSOD category due to the fact that it was insensitive to KCN or hydrogen peroxide inhibitor. This MnSOD from wheat seedlings was found to be stable over pH 7-9, with an optimum pH of 8, but was sensitive to extreme pH, particularly to acidic pH. It was stable over a wide range of temperatures (5-50 degrees C). Thermal inactivation of wheat seedling MnSOD followed first-order reaction kinetics, and the temperature dependence of rate constants was in agreement with the Arrhenius equation. The activation energy for thermal inactivation of wheat seedling MnSOD in the temperature range of 50-70 degrees C was found to be 150 kJ/mol. HgCl2 and SDS at a concentration of 1.0 mM significantly inhibited enzyme activity. Chemical modification agents, including diethyl pyrocarbonate (2.5 mM) and Woodward's reagent K (50 mM), significantly inhibited the activity of wheat seedling SOD, implying that imidazole groups from histidine and carboxyl groups from aspartic acid and glutamic acid are probably located at or near the active site of the enzyme.     

Characterization of the dimer-monomer equilibrium of the papaya Copper/Zinc superoxide dismutase and its equilibrium shift by a single amino acid mutation.

The coding region of the copper/zinc superoxide dismutase (Cu/Zn SOD) cDNA from papaya fruit, Carica papaya L. cv. Tainong 2, was cloned into an expression vector, pET-20b(+). The Cu/Zn SOD was expressed in Escherichia coli and purified by His-tag technique. Two active forms of the enzyme (30% dimer and 70% monomer) in equilibrium were observed. The activity of the dimeric enzyme was higher than that of the monomeric form. The thermal inactivation rate constant K(d) values calculated for the dimer and monomer at 90 degrees C were -0.0203 and -0.0216 min(-1), and the half-lives for inactivation were 41.9 and 31.8 min, respectively. This indicated that the dimeric enzyme was more stable than its monomeric form. The dimerization of the enzyme was inhibited under acidic pH (below 3.0) or imidazole buffer (above 0.5 M), whereas it was not affected under alkaline pH (above 9.0). Both activity and forms of the enzyme were not affected by 1-4% SDS. Furthermore, the dimeric enzyme was much more resistant to proteolytic attack after 3 h of incubation at 37 degrees C with trypsin or chymotrypsin. In addition, mutation of the papaya Cu/Zn SOD at position 48 from Leu to Phe (L48F) affected the association of monomer, whereas a mutant with Lys substitution (L48K) at the same position tended to dissociate into monomeric form.     

以黄连素/羟丙基-β-环糊精为荧光探针检测金刚烷胺

为了建立一种金刚烷胺(AMD)检测的新方法,以盐酸黄连素/羟丙基-P-环糊精(BRH/HP-β-CD)为荧光探针,将HP-β-CD分别与BRH、AMD相互作用,测定AMD、BRH/HP-β-CD及两者结合的荧光强度变化规律,并验证分析该荧光分光光度法的适用性。结果表明,HP-β-CD与BRH生成1∶1的包合物并显著增强黄连素水溶液的荧光强度,当加入AMD后,BRH/HP-β-CD包合物的荧光强度逐渐减弱,据此成功建立一种以BRH/HP-β-CD为荧光探针的金刚烷胺检测新方法。当AMD溶液浓度在0.05~4.5 mg·L^-1范围内时与荧光猝灭值△F之间呈线性关系,相关系数为0.989 3,检测限(S/N=3)为0.03 mg·L^-1。溶液pH值和常见的药物赋形剂均不会对测量结果造成干扰,将本方法用于盐酸金刚烷胺药片的分析,其回收率在92%~101%范围内,相对标准偏差小于1%,说明该方法能够成功用于金刚烷胺的测定。本研究为以超分子包合物为荧光探针用于金刚烷胺类药物的检测提供了理论依据。     

Decreased superoxide anion production in cultured human promonocyte cells (THP-1) due to polyphenol mixtures from olive oil processing wastewaters

The purpose of this study was to examine whether human monocytic line THP-1 after differentiation into adherent macrophages, taken as a model of human macrophages implicated in atheroma, is able to produce lower quantities of O(2)(*)(-) either in the presence of polyphenol-rich olive oil wastewater (OWW) fractions or after OWW preincubation and withdrawal from the medium. In these respective conditions, the purpose was to examine the scavenging activity and the cell action of OWW toward O(2)(*)(-) production. It was clearly seen that OWW fractions lowered the O(2)(*)(-) production in both conditions, leading to the conclusion that they were able to scavenge O(2)(*)(-) and to depress O(2)(*)(-) production in the cell. Given the role of O(2)(*)(-) in LDL oxidation and oxidized LDL in atheroma, these results support an antiatherogenic role of OWW and its potential utilization as a food complement.     

Protective effects of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone to PC12 cells against cytotoxicity induced by hydrogen peroxide

Oxidative stress has been considered as a major cause of cellular injuries in various clinical abnormalities. One of the possible ways to prevent reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical therapies to augment the endogenous antioxidant defense capacity. The present study found that 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), a chalcone isolated from the buds of Cleistocalyx operculatus, possessed cytoprotective activity in PC12 cells treated with H(2)O(2). The results showed that DMC could effectively increase cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) reduction], decrease the cell apoptotic percentage [annexin V/propidium iodide (AV/PI) assay], prevent the membrane from damage [lactate dehydrogenase (LDH) release], scavenge ROS formation, reduce caspase-3 activity, and attenuate the decrease of mitochondrial membrane potential (MMP) in PC12 cells treated with H(2)O(2). Meanwhile, DMC increased the catalytic activity of superoxide dismutase (SOD) and the cellular amount of glutathione (GSH), decreased the cellular amount of malondialdehyde (MDA), and decreased the production of lipid peroxidation in PC12 cells treated with H(2)O(2).     

红壤水分条件对柑橘叶片质膜透性以及活性氧代谢和保护酶活性的影响

通过盆栽柑橘控制盆内红壤不同含水量处理,研究了不同红壤水分条件对柑橘叶片质膜透性以及活性氧代谢和保护酶活性的影响.结果表明,柑橘叶片相对电导率在红壤相对含水量75% 处理时最低,并与红壤相对含水量呈显著负相关(R2 = 0.8236*,n=50);柑橘叶片丙二醛(MDA)含量随土壤水分含量的减少而显著增加,并与红壤相对含水量呈显著负相关(R2 = 0.9051*,n=50).柑橘叶片超氧阴离子自由基(O2.-)生成速率与过氧化氢(H2O2)含量在不同红壤水分条件下具有相近的变化曲线,在红壤相对含水量60% 的处理下达最大值后,随红壤水分减少或增加均呈下降趋势;O2.-、H2O2并不是引起柑橘在红壤水分亏缺或盈余胁迫状况下氧化伤害的单一原因.随着红壤含水量的变化,柑橘叶片抗氧化系统处于动态的平衡中,柑橘叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)酶活性在红壤干旱条件下都保持了较高的活性,但过氧化氢酶(CAT)活性随土壤水分减少却显著降低.     

Antioxidant activity of glyceollins derived from soybean elicited with Aspergillus sojae

The extract of soybean exposed to biotic elicitors such as food-grade fungus is known to have antioxidant activity. Glyceollins were major bioactive compounds present in soybean elicited by fungi and shown to have antifungal and anticancer activities. The purpose of present study was to evaluate the antioxidant activities of glyceollins by measuring ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, singlet oxygen quenching, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, hydroxyl radical scavenging activity, and lipid peroxidation inhibition. In addition, the antioxidant potential of glyceollins were measured by a fluorescent probe, 2',7'-dichlorofluorescin diacetate (DCFDA), and dihydroethidium (DHE) in mouse hepatoma hepa1c1c7 cells in which they were insulted with H2O2 to generate reactive oxygen species (ROS). Glyceollins showed a strong reducing power and inhibited lipid peroxidation, with significant scavenging activities of radicals including singlet oxygen, superoxide anion, ABTS, and DPPH. We also found that glyceollins significantly suppressed H2O2-induced ROS production in hepa1c1c7 cells. Therefore, glyceollins deserve further study as natural antioxidants and nutraceuticals.     

Novel nitric oxide and superoxide generation inhibitors, persenone A and B, from avocado fruit

One known, (2R)-(12Z,15Z)-2-hydroxy-4-oxoheneicosa-12,15-dien+ ++-1-yl acetate (1), and two novel compounds, persenone A (2) and B (3), have been isolated from avocado fruit (Persea americana P. Mill), as inhibitors of superoxide (O(2)(-)) and nitric oxide (NO) generation in cell culture systems. They showed marked inhibitory activities toward NO generation induced by lipopolysaccharide in combination with interferon-gamma in mouse macrophage RAW 264.7 cells. Their inhibitory potencies of NO generation (1, IC(50) = 3.6; 2, IC(50) = 1.2; and 3, IC(50) = 3.5 microM) were comparable to or higher than that of a natural NO generation inhibitor, docosahexaenoic acid (DHA; IC(50) = 4.3 microM). Furthermore, compounds 1-3 and DHA markedly suppressed tumor promoter 12-O-tetradecanoylphorbol-13-acetate-induced O(2)(-) generation in differentiated human promyelocytic HL-60 cells (1, IC(50) = 33.7; 2, IC(50) = 1.4; 3, IC(50) = 1.8; and DHA, IC(50) = 10.3 microM). It is notable that they were found to be suppressors of both NO- and O(2)(-)-generating biochemical pathways but not to be radical scavengers. The results indicate that these compounds are unique antioxidants, preferentially suppressing radical generation, and thus may be promising as effective chemopreventive agent candidates in inflammation-associated carcinogenesis.     

Evaluation of the antioxidant and pro-oxidant effects of tea catechin oxypolymers

Tea catechin oxypolymers (TCOP) were prepared by oxidizing tea catechin (TC, the content of EGCG was >85%) with H2O2. Their antioxidant and pro-oxidant effects were tested using a deoxyribose assay, a photoreduction of NBT assay, a lipoxygenase assay, a POV assay, and animal tests. The scavenging effects of TCOP to both the hydroxyl radical and superoxide radical were stronger than that of TC, and also they had no pro-oxidant effect; the rate constant for reactions of TC and TCOP for hydroxyl radical were 1.0 x 10(10) and (1.4-2.8) x 10(10) M(-1) x S(-1), respectively. TCOP can inhibit lipid peroxidation and lipoxygenase effectively, and it also can activate red cell SOD and reduce the MDA content in serum of mice very significantly. These results suggested that the antioxidant activity of TCOP was not less than or even more notable than that of TC.     

镉对褶纹冠蚌抗氧化因子的影响

研究了不同浓度的镉（Cd2＋）对褶纹冠蚌血清和肝胰脏中超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-PX）的活力和谷胱甘肽（GSH）、过氧化氢（H2O2）、丙二醛（MDA）的含量的影响。结果表明,褶纹冠蚌在1mg.L-1Cd2＋处理后,肝胰脏SOD活性随着暴露时间的延长而升高,且被显著诱导;在2mg.L-1Cd2＋处理后,肝胰脏SOD活性暴露48h被显著地诱导,其后开始回落,但是依然被显著地诱导。褶纹冠蚌经不同浓度Cd2＋处理后,肝胰脏的GPX活性显著被诱导;血清中的GSH含量先显著上升,在48h时达到峰值后显著下降;血清和肝胰脏的H2O2均被诱导;肝胰脏中MDA含量明显升高。褶纹冠蚌肝胰脏中SOD活性和血清中GSH含量可以用来指示水生生态系统中的镉污染。     

Levels of active oxygen species are controlled by ascorbic acid and anthocyanin in Arabidopsis

Stabilization of the levels of active oxygen species (AOS) is important to the survival of organisms. To clarify the system controlling levels of AOS in plants, this study used an electron spin resonance (ESR) method to directly measure superoxide radical (O(2)(.-)) scavenging activities in the wild-type Arabidopsis thaliana (Col and Ler ecotypes), two anthocyanin mutants (tt3 and ttg1), and an ascorbic acid mutant (vtc1). Under ordinary growth conditions, Arabidopsis contained superoxide-scavenging activity (SOSA) of approximately 300-500 SOD units/g of fresh weight. The ESR pattern indicated that most (40-50%) of this activity was due to ascorbic acid. For the analysis of SOSA under conditions of oxidative stress, synthesis of AOS was induced by gamma-irradiation. The radical scavenging activity in irradiated plants increased approximately 10-fold following an associated increase in the accumulation of ascorbic acid and anthocyanin. The accumulation of ascorbic acid and anthocyanin was suppressed by treatment with an antioxidant before irradiation and was induced by treatment with a radical-generating reagent. The contributions of ascorbic acid and anthocyanin to the total superoxide radical scavenging activity differed among ecotypes. In the Ler ecotype, ascorbic acid accumulated at twice the level of that in the Col ecotype, and induction of anthocyanin was half that in Col. To confirm the activity of ascorbic acid and anthocyanin against AOS stress, the viability of the wild type and mutants (tt2, tt3,tt5, ttg1, and vtc1) was examined after gamma-irradiation. Only the plants in which ascorbic acid and anthocyanin were induced had the ability to grow and flower.