Monitoring the Glutathione Redox Reaction in Living Human Cells by Combining Metabolic Labeling with Heteronuclear NMR

The glutathione (GSH) redox reaction is critical for defense against cellular reactive oxygen species (ROS). However, direct and real‐time monitoring of this reaction in living mammalian cells has been hindered by the lack of a facile method. Herein, we describe a new approach that exploits the GSH biosynthetic pathway and heteronuclear NMR. [U‐¹³C]‐labeled cysteine was incorporated into GSH in U87 glioblastoma cells, and the oxidation of GSH to GSSG by a ROS‐producing agent could be monitored in living cells. Further application of the approach to cells resistant to temozolomide (TMZ), an anti‐glioblastoma drug, suggested a possible new resistance mechanism involving neutralization of ROS. This result was corroborated by the observation of up‐regulation of glutathione peroxidase 3 (GPx3). This new approach could be easily applied to redox‐dependent signaling pathways and drug resistance involving ROS.     

Disposable Amperometric Sensors for Thiols with Special Reference to Glutathione

The antioxidant ‘reduced glutathione’ tripeptide is conventionally called glutathione (GSH). The oxidized form is a sulfur‐sulfur linked compound, known as glutathione disulfide (GSSG). Glutathione is an essential cofactor for antioxidant enzymes; it provides protection also for the mitochondria against endogenous oxygen radicals. The ratio of these two forms can act as a marker for oxidative stress. The majority of the methods available for estimation of both the forms of glutathione are based on colorimetric and electrochemical assays. In this study, electrochemical sensors were developed for the estimation of both GSH and GSSG. Two different types of transducers were used: i) screen‐printed three‐electrode disposable sensor (SPE) containing carbon working electrode, carbon counter electrode and silver/silver chloride reference electrode; ii) three‐electrode disposable system (CDE) consisting of three copper electrodes. 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB) was used as detector element for estimation of total reduced thiol content. The enzyme glutathione reductase along with a co‐enzyme reduced nicotinamide adenine dinucleotide phosphate was used to estimate GSSG. By combining the two methods GSH can also be estimated. The detector elements were immobilized on the working electrodes of the sensors by bulk polymerization of acrylamide. The responses were observed amperometrically. The detection limit for thiol (GSH) was less than 0.6 ppm when DTNB was used, whereas for GSSG it was less than 0.1 ppm.     

High-throughput determination of glutathione and reactive oxygen species in single cells based on fluorescence images in a microchannel

A novel high-throughput method is presented based on fluorescence images of cells in a microchannel for determination of glutathione (GSH) and reactive oxygen species (ROS) inside single cells. We first present a method to determine GSH and ROS separately, in which GSH in cells is derivatized by 2,3-naphthalenedicarboxaldehyde (NDA), and intracellular ROS is labeled using dihydrorhodamine 123. The cells with either fluorescent derivatized GSH or fluorescent labeled ROS are introduced into a microchannel and fluorescence images of every moving cell in the microchannel are taken continuously using a highly sensitive thermoelectrically cooled electron-multiplying CCD. The fluorescence intensities of the images correspond to the masses of GSH or ROS. An average detection rate of 80-120 cells/min is achieved. We then propose a method for simultaneously determining GSH and ROS, in which ROS is first labeled in the cells. The labeled cells are then introduced into the whole channel and allowed to immobilize onto the glass substrate. The fluorescence images of all the cells in the channel are taken. NDA is then introduced into the channel to derivatize the GSH in the immobilized cells, and fluorescence images of all cells are taken again. An average analysis rate of 20 cells/min is achieved. The masses of GSH and ROS in the single cells can be obtained from the fluorescence intensities of the images using their calibration curves. Since the cells are not lysed, there is no problem with adsorption of biological macromolecules and cellular debris on the channel wall, so that channel treatment, necessary in usual single-cell analysis techniques using CE and microchip electrophoresis, is no longer necessary. For single global cells, this method can also be used to determine the concentrations of ROS and GSH, which has not been reported previously. The concentrations of ROS and GSH in single global cells can be calculated from the determined masses and the cell volume (derived from the diameter of the round fluorescence image of the derivatized GSH). For gastric cancer cells, the concentrations of GSH and ROS are in the range 0.35x10(-3)-1.3x10(-3) mol/L and 0.77x10(-) (6)-1.5x10(-6) mol/L, respectively.     

Crayfish Procambarus clarkii retina and nervous system exhibit antioxidant circadian rhythms coupled with metabolic and luminous daily cycles

Based on previous work in which we proposed midgut as a putative peripheral oscillator responsible for circadian reduced glutathione (GSH) crayfish status, herein we investigated the retina and optic lobe-brain (OL-B) circadian GSH system and its ability to deal with reactive oxygen species (ROS) produced as a consequence of metabolic rhythms and light variations. We characterized daily and antioxidant circadian variations of the different parameters of the glutathione system, including GSH, oxidized glutathione (GSSG), glutathione reductase (GR) and glutathione peroxidase (GPx), as well as metabolic and lipoperoxidative circadian oscillations in retina and OL-B, determining internal and external GSH-system synchrony. The results demonstrate statistically significant bi- and unimodal daily and circadian rhythms in all GSH-cycle parameters, substrates and enzymes in OL-B and retina, as well as an apparent direct effect of light on these rhythms, especially in the retina. The luminous condition appears to stimulate the GSH system to antagonize ROS and lipid peroxidation (LPO) daily and circadian rhythms occurring in both structures, oscillating with higher LPO under dark conditions. We suggest that the difference in the effect of light on GSH rhythmic mechanisms of both structures for antagonizing ROS could be due to differences in glutathione-system coupling strength with the circadian clock.     

Hydrogen Peroxide and Glutathione Dual Redox‐Responsive Nanoparticles for Controlled DOX Release

Polymer nanoparticulate drug delivery systems that respond to reactive oxygen species (ROS) and glutathione (GSH) simultaneously at biologically relevant levels hold great promise to improve the therapeutic efficacy to cancer cells with reduced side effects of chemo drugs. Herein, a novel redox dual‐responsive amphiphilic block copolymer (ABP) that consists of a hydrophilic poly (ethylene oxide) block and a hydrophobic block bearing disulfide linked phenylboronic ester group as pendant is synthesized, and the DOX loaded nanoparticles (BSN‐DOX) based on ABPs with varied hydrophobic block length are fabricated for DOX delivery. The self‐immolative leaving reaction of phenylboronic ester triggered by extracellular ROS and the cleavage of disulfide linkages induced by intracellular GSH both lead to rapid DOX release from BSN‐DOX, resulting in an on‐demand DOX release. Moreover, BSN‐DOX show better tumor inhibition and lower side effects in vivo compared with free drug.     

Rapid determination of reduced and oxidized glutathione levels using a new thiol-masking reagent and the enzymatic recycling method: application to the rat liver and bile samples

A microtiter plate assay for quantitation of reduced (GSH) and oxidized (GSSG) glutathione in the rat liver tissue and bile is described. The assay is based on the established enzymatic recycling method and a new thiol-masking reagent, 1-methyl-4-vinyl-pyridinium trifluoromethane sulfonate (M4VP). Samples were first processed by homogenization with (liver) or addition of (bile) sulfosalicylic acid. The total glutathione and GSSG were then determined before and after rapid (≤2 min) and efficient (100%) masking of the GSH content of the samples with M4VP followed by the enzymatic recycling assay. The percentages of error and coefficient of variation of the assay were within the accepted guidelines, indicating the accuracy and precision of the assay in the range of 6.25–100 pmol GSH per microplate well and 2.17–140 pmol GSSG per well, with lower limit of quantitation of 6.25 and 2.17 pmol per well for GSH and GSSG, respectively. Furthermore, the recoveries of added GSH or GSSG from the liver and bile samples were accurate and precise. The assay was applied to measurement of GSH, GSSG, and GSH:GSSG ratio in the liver and serially collected bile samples in sham-operated and ischemic rat livers, demonstrating a depletion of glutathione and a decrease in the GSH:GSSG ratio as a result of ischemia. The developed assay is rapid, sensitive, accurate, and precise and is suitable for studies of the redox status of liver under physiologic and pathophysiologic conditions.     

Scanning electrochemical microscopy studies of glutathione-modified surfaces. An erasable and sensitive-to-reactive oxygen species surface

A surface sensitive to reactive oxygen species (ROS) was prepared by reduction of a diazonium salt on glassy carbon electrode followed by the chemical coupling of glutathione (GSH) playing the role of an antioxidant species. The presence of active GSH was characterized through spectroscopic studies and electrochemical analysis after labeling of the -SH group with ferrocene moieties. The specific reactivity of GSH vs ROS was evaluated with scanning electrochemical microscopy (SECM) using the reduction of O(2) to superoxide, O(2)(?-), near the GSH-modified surface. Approach curves show a considerable decrease of the blocking properties of the layer due to reaction of the immobilized GSH with O(2)(?-) and the passage of GSH to the glutathione disulfide (GSSG). The initial surface could be regenerated several times with no significant variations of its antioxidant capacity by simply using the biological system glutathione reductase (GR)/NADPH that reduces GSSG back to GSH. SECM imaging shows also the possibility of writing local and erasable micropatterns on the GSH surface by production of O(2)(?-) at the tip probe electrode.     

Simultaneous and ultrarapid determination of reactive oxygen species and reduced glutathione in apoptotic leukemia cells by microchip electrophoresis

A microchip electrophoresis method coupled with laser-induced fluorescence (LIF) detection was established for simultaneous determination of two kinds of intracellular signaling molecules (reactive oxygen species, ROS, and reduced glutathione, GSH) related to apoptosis and oxidative stress. As the probe dihydrorhodamine-123 (DHR-123) can be converted intracellularly by ROS to the fluorescent rhodamine-123 (Rh-123), and the probe naphthalene-2,3-dicarboxaldehyde (NDA) can react quickly with GSH to produce a fluorescent adduct, rapid determination of Rh-123 and GSH was achieved on a glass microchip within 27 s using a 20 mM borate buffer (pH 9.2). The established method was tested to measure the intracellular ROS and GSH levels in acute promyelocytic leukemia (APL)-derived NB4 cells. An elevation of intracellular ROS and depletion of GSH were observed in apoptotic NB4 cells induced by arsenic trioxide (As(2)O(3)) at low concentration (1-2 microM). Buthionine sulfoximine (BSO), in combination with As(2)O(3) enhanced the decrease of reduced GSH to a great extent. The combined treatment of As(2)O(3) and hydrogen peroxide (H(2)O(2)) led to an inverse relationship between the concentrations of ROS and GSH obtained, showing the proposed method can readily evaluate the generation of ROS, which occurs simultaneously with the consumption of the inherent antioxidant.     

Simultaneous determination of glutathione and reactive oxygen species in individual cells by microchip electrophoresis

A microchip electrophoresis method was developed for simultaneous determination of reactive oxygen species (ROS) and reduced glutathione (GSH) in the individual erythrocyte cell. In this method, cell sampling, single-cell loading, docking, lysing, and capillary electrophoretic separation with LIF detection were integrated on a microfluidic chip with crossed channels. ROS was labeled with dihydrorhodamine 123 in the intact cell, while GSH was on-chip labeled with 2,3-naphthalene-dicarboxaldehyde, which was included in the separation medium. On-chip electrical lysis, characterized by extremely fast disruption of the cellular membrane (<40 ms), was exploited to minimize enzymatic effects on analyte concentrations during the determination. The microfluidic network was optimized to prevent cell leaking from the sample reservoir (S) into separation during the separation phase. The structure of the S was modified to avoid blockage of its outlet by deposited cells. Detection limits of 0.5 and 6.9 amol for ROS and GSH, respectively, were achieved. The average cell throughput was 25 cells/h. The effectiveness of the method was demonstrated in the simultaneous determination of GSH and ROS in individual cells and the variations of cellular GSH and ROS contents in response to external stimuli.     

SERS纳米探针对电刺激过程中细胞内产生活性氧的检测

电刺激是用于细胞内紊乱电活动引起疾病的一类重要治疗方式. 在电刺激过程中是否会诱导细胞内活性氧(ROS)水平的改变, 以及常规抗氧化抑制药物与电刺激治疗同时运用带来的影响, 目前尚未有相关研究. 本文设计了一种具有较好生物相容性的金/银核壳纳米棒表面增强拉曼(SERS)活性探针, 用于电刺激过程中细胞内产生ROS的检测. 将该探针与细胞共孵育, 使其内化入细胞, 对细胞进行不同时间的电刺激, 利用拉曼光谱对SERS探针的信号进行检测. 实验结果表明, 随着电刺激时间的延长, SERS信号减弱, 说明细胞内产生ROS的量明显增加. 该传感机制是利用ROS能刻蚀金/银核壳纳米棒的银壳, 从而使其变薄引起SERS信号减弱. 抗坏血酸(AA)和谷胱甘肽(GSH)两种抗氧化抑制剂类药物与电刺激同时运用时, 可观察到它们会对电刺激过程产生的ROS有清除作用. 该研究发展了一类用于细胞内ROS检测的光谱方法, 也为异常的氧化应激和肿瘤治疗过程中的组合用药提供了建议.     

Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from 'total GSH'. The linear range and limit of detection for both analytes were 7.5 × 10(-7) to 1 × 10(-5) M, and 5 × 10(-7) M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL(-1)), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL(-1) glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL(-1) (p < 0.05), which was rationalised by considering measurements of H(2)O(2) and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL(-1) glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.     

Selenium methylselenocysteine protects human hepatoma HepG2 cells against oxidative stress induced by tert-butyl hydroperoxide

Selenium methylselenocysteine (Se-MeSeCys) is a common selenocompound in the diet with a tested chemopreventive effect. This study investigated the potential protective effect of Se-MeSeCys against a chemical oxidative stress induced by tert-butyl hydroperoxide (t-BOOH) on human hepatoma HepG2 cells. Speciation of selenium derivatives by liquid chromatography–inductively coupled plasma mass spectrometry depicts Se-MeSeCys as the only selenocompound in the cell culture. Cell viability (lactate dehydrogenase) and markers of oxidative status—concentration of reduced glutathione (GSH) and malondialdehyde (MDA), generation of reactive oxygen species (ROS) and activity of the antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR)—were evaluated. Pretreatment of cells with Se-MeSeCys for 20 h completely prevented the enhanced cell damage, MDA concentration and GR and GPx activity and the decreased GSH induced by t-BOOH but did not prevent increased ROS generation. The results show that treatment of HepG2 cells with concentrations of Se-MeSeCys in the nanomolar to micromolar range confers a significant protection against an oxidative insult.     

Cationic Vesicles Based on Amphiphilic Pillar[5]arene Capped with Ferrocenium: A Redox‐Responsive System for Drug/siRNA Co‐Delivery

A novel ferrocenium capped amphiphilic pillar[5]arene (FCAP) was synthesized and self‐assembled to cationic vesicles in aqueous solution. The cationic vesicles, displaying low cytotoxicity and significant redox‐responsive behavior due to the redox equilibrium between ferrocenium cations and ferrocenyl groups, allow building an ideal glutathione (GSH)‐responsive drug/siRNA co‐delivery system for rapid drug release and gene transfection in cancer cells in which higher GSH concentration exists. This is the first report of redox‐responsive vesicles assembled from pillararenes for drug/siRNA co‐delivery; besides enhancing the bioavailability of drugs for cancer cells and reducing the adverse side effects for normal cells, these systems can also overcome the drug resistance of cancer cells. This work presents a good example of rational design for an effective stimuli‐responsive drug/siRNA co‐delivery system.     

Decrease of glutathione and fatty acids during iron-induced lipid peroxidation inEhrlich ascites tumor cells

In order to investigate a possible relationship between the intensity of lipid peroxidation (LP) in tumor cells and their proliferative activity various methods to quantify LP are desirable. In this study the decrease in the contents of fatty acids and glutathione was measured by established methods inEhrlich ascites tumor (EAT) cellsin vitro, in which LP was stimulated by the addition of ferrous iron, either as free ion or as histidinate chelate.When EAT cells were incubated for 30 min at 37 °C in the presence of 5 mM FeSO₄ the following changes were observed in comparison to appropriate control cells: The content of reduced glutathione (GSH) and total glutathione (GSH+2 GSSG) decreased significantly by 24 and 30% respectively. The decrease of 4 unsaturated (C 18:1; C 18:2; C 20:4; C 22:6) and 2 saturated fatty acids (C 16:0; C 18:0) by about 15% on the average was statistically significant only for C 16:0 and C 20:4).More pronounced effects were observed with 5 mM Fe(II)-histidinate. GSH and GSH+2 GSSG decreased by 54% and 40%, resp. The decrease of fatty acids by about 40% on the average was significant for all of the 6 fatty acids tested. These results are in agreement with previous studies on LP in EAT cells showing Fe(II)-histidinate to be a more powerful promoter of LP compared with free ferrous ion. The observation, that the content not only of GSH but also of total glutathione was decreased in iron-treated tumor cells is in contradiction to the hypothesis that GSH may act as a mere redox mediator of LP under the conditions used and points to a consumption of GSH by several possible pathways. The finding of decreased levels of unsaturated as well as saturated fatty acids in the presence of Fe(II)-histidinate underlines the extraordinary potency of iron as an initiator and catalyst of LP.This work was supported by the Association for International Cancer Research, St. Andrews, U.K.     

Up-regulation of defense enzymes is responsible for low reactive oxygen species in malignant prostate cancer cells

Reactive oxygen species (ROS) are involved in a diversity of important phenomena in the process of tumor development. To investigate the alterations of oxidative stress and their related systems in tumor progression, a variety of components in the antioxidative stress defense system were examined in prostate cancer cell lines, PC3 and LNCaP. Cell surface molecules involved in metastasis were expressed highly in PC3 cells compared with LNCaP cells, and strong invasion ability was shown in PC3 cells only. ROS level in LNCaP cells was twice higher than that in PC3 cells, although nitric oxide (NO) level was similar between the two cell lines. The content of GSH increased up to about 2-fold in PC3 compared with LNCaP. Activities of glutathione reductase, thioredoxin reductase, and glutathione S-transferase except catalase are significantly higher in PC3 cells than in LNCaP cells. Furthermore, oxidative stress-inducing agents caused down-regulation of GSH and glutathione S-transferase much more significantly in LNCaP cells than in PC3 cells. These results imply that malignant tumor cells may maintain low ROS content by preserving relatively high anti-oxidative capacity, even in the presence of stressful agents.     

Dithiophosphate-Induced Redox Conversions of Reduced and Oxidized Glutathione

Phosphorus species are potent modulators of physicochemical and bioactive properties of peptide compounds. O,O-diorganyl dithiophoshoric acids (DTP) form bioactive salts with nitrogen-containing biomolecules; however, their potential as a peptide modifier is poorly known. We synthesized amphiphilic ammonium salts of O,O-dimenthyl DTP with glutathione, a vital tripeptide with antioxidant, protective and regulatory functions. DTP moiety imparted radical scavenging activity to oxidized glutathione (GSSG), modulated the activity of reduced glutathione (GSH) and profoundly improved adsorption and electrooxidation of both glutathione salts on graphene oxide modified electrode. According to NMR spectroscopy and GC–MS, the dithiophosphates persisted against immediate dissociation in an aqueous solution accompanied by hydrolysis of DTP moiety into phosphoric acid, menthol and hydrogen sulfide as well as in situ thiol-disulfide conversions in peptide moieties due to the oxidation of GSH and reduction of GSSG. The thiol content available in dissolved GSH dithiophosphate was more stable during air oxidation compared with free GSH. GSH and the dithiophosphates, unlike DTP, caused a thiol-dependent reduction of MTS tetrazolium salt. The results for the first time suggest O,O-dimenthyl DTP as a redox modifier for glutathione, which releases hydrogen sulfide and induces biorelevant redox conversions of thiol/disulfide groups.     

UV released factors induce antioxidant defense in A375 cells

UV light leads to release of different secretory factors from irradiated cells of which some of them have been characterized. We have reported earlier that cells exposed to the supernatant medium from irradiated cells were resistant to killing by some genotoxic agents. In this study, we present our finding that demonstrates DNA damage induced by UV or H(2)O(2) is lowered on prior exposure to the UV released factors (UVRF). Production of ROS in cells and lipid peroxidation was also lowered. It was found that treatment of unexposed cells with UVRF present in the supernatant medium altered the antioxidant defense activity in cells. Significant was the increase in catalase (CAT) and Cu-Zn superoxide dismutase (SOD) activity, whereas glutathione peroxidase (GPx) and reduced glutathione (GSH) levels remained unaffected. Cells exposed to UVRF prior to UV or H(2)O(2) treatment also experienced such upregulation; however, the remarkable increase in the GPx activity exhibited by these cells was not observed in cells exposed to H(2)O(2) or UV alone. It appears that exposure to UVRF tinkered with antioxidant defense in cells to facilitate its proliferation upon assault by an agent that can produce oxidative damage.     

Modelling the Inhibition of Selenoproteins by Small Molecules Using Cysteine and Selenocysteine Derivatives

Small molecule-based electrophilic compounds such as 1-chloro-2,4-dinitrobenzene (CDNB) and 1-chloro-4-nitrobenzene (CNB) are currently being used as inhibitors of cysteine- and selenocysteine-containing proteins. CDNB has been used extensively to determine the activity of glutathione S-transferase and to deplete glutathione (GSH) in mammalian cells. Also, CDNB has been shown to irreversibly inhibit thioredoxin reductase (TrxR), a selenoenzyme that catalyses the reduction of thioredoxin (Trx). Mammalian TrxR has a C-terminal active site motif, Gly-Cys-Sec-Gly, and both the cysteine and selenocysteine residues could be the targets of the electrophilic reagents. In this paper we report on the stability of a series of cysteine and selenocysteine derivatives that can be considered as models for the selenoenzyme–inhibitor complexes. We show that these derivatives react with H₂O₂ to generate the corresponding selenoxides, which undergo spontaneous elimination to produce dehydroalanine. In contrast, the cysteine derivatives are stable towards such elimination reactions. We also demonstrate, for the first time, that the arylselenium species eliminated from the selenocysteine derivatives exhibit significant redox activity by catalysing the reduction of H₂O₂ in the presence of GSH (GPx (glutathione peroxidase)-like activity), which suggests that such redox modulatory activity of selenium compounds may have a significant effect on the cellular redox state during the inhibition of selenoproteins.     

Mechanism of Chicoric Acid Electrochemical Oxidation and Identification of Oxidation Products by Liquid Chromatography and Mass Spectrometry

Electrochemical oxidation of chicoric acid (ChA) was investigated using cyclic voltammetry and chronoamperometry at a glassy carbon electrode. Chicoric acid generates single quasi‐reversible redox wave in cyclic voltammetry over a wide pH range, and an ECEC‐dimerization mechanism is proposed. Effect of glutathione (GSH) on the electrochemical oxidation of chicoric acid (ChA) was investigated in Britton−Robinson buffer solution. Ultra‐high performance liquid chromatography (UPLC) coupled with mass spectrometry (MS) was used to show that the naturally occurring chicoric acid (ChA) underwent an electrochemical oxidation in the presence of glutathione (GSH) to form mono‐, bi‐, tri‐, and four‐glutathione conjugates of chicoric acid and a mono‐glutathione conjugate of a chicoric acid dimer. The obtained results are useful for understanding and predicting the oxidative degradation pathway of chicoric acid.     

Chromanols from Sargassum siliquastrum and their antioxidant activity in HT 1080 cells

Six meroterpenoids (compounds 1-6) of chromene class, including three known compounds (1-3), were isolated from Sargassum siliquastrum. The structure of these compounds was established by extensive 2D-NMR experiments such as (1)H gradient double quantum filtered correlation spectroscopy (gDQCOSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), gradient heteronuclear multiple quantum coherence (gHMQC), and gradient heteronuclear multiple bond correlation (gHMBC), and by comparison with published spectral data. The antioxidant activity of these compounds was evaluated by various antioxidant tests, such as scavenging effects on generation of intracellular reactive oxygen species (ROS), increments of intracellular glutathione (GSH) level, and inhibitory effects on lipid peroxidation in human fibrosarcoma HT 1080 cells. Compounds (1-6) significantly decreased generation of intracellular ROS and inhibited lipid peroxidation while they increased levels of intracellular GSH at a concentration of 5 μg/ml.