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Identification of Rhus verniciflua Stokes compounds that exhibit free radical scavenging and anti-apoptotic properties
Biochimica et Biophysica Acta 1570 (2002) 181 – 191 www.bba-direct.com
Identification of Rhus verniciflua Stokes compounds that exhibit free radical scavenging and anti-apoptotic properties Jeong-Chae Lee a, Kye-Taek Lim b, Yong-Suk Jang a,* a
Division of Biological Sciences and The Research Center for Bioactive Materials, Chonbuk National University, Chonju 561-756, South Korea b Biodefensive Substances Group, Institute of Biotechnology, Chonnam National University, Kwangju 500-757, South Korea Received 20 September 2001; received in revised form 17 January 2002; accepted 4 February 2002
Abstract Rhus verniciflua Stokes (RVS) is a widely used herbal plant with various biological properties. Our previous study using cultured neuronal cells showed that an ethanol extract of RVS had strong antioxidant properties. In this study, we characterized the antioxidant activity of the RVS ethanol extract and identified the active compounds responsible for this activity. From the RVS ethanol extract, we derived three water-eluted fractions and another three fractions eluted by organic solvents, and determined that the water-eluted fractions are what protect against reactive oxygen species (ROS) generated by iron and enzymes. Water-eluted fraction F2 was the most efficient antioxidant. Moreover, DNA fragmentation and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining experiments revealed that F2 also protects against thymocyte apoptosis mediated by hydroxyl radicals. Finally, EI-MS, 1H-NMR, and 13C-NMR spectra signals confirmed that the fraction contained flavonoid derivatives, including fustin, quercetin, butein, and sulfuretin. These results suggest that the flavonoid derivatives in F2 are the compounds in the RVS ethanol extract that act as antioxidants. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Antioxidant; Apoptosis; Flavonoid; Reactive oxygen specie; Rhus verniciflua Stokes
1. Introduction Reactive oxygen species (ROS), such as superoxide, hydroxyl radicals, and hydrogen peroxide, are constantly generated in small amounts during normal aerobic metabolism in living organisms [1]. ROS generation can be prevented by a wide array of primary antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase [1,2]. However, when the balance between cellular antioxidant defenses and ROS generation is disrupted, a condition referred to as oxidative stress occurs. Persistent oxidative stress can damage critical biomolecules and thus alter biologic processes, including signal transduction and gene expression, causing mitogenesis, muta-
Abbreviations: RVS, Rhus verniciflua Stokes; ROS, reactive oxygen species; TBARS, thiobarbituric acid-reactive substances; X/XO, hypoxanthine/xanthine oxidase; G/GO, glucose/glucose oxidase; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling * Corresponding author. Tel.: +82-63-270-3343; fax: +82-63-270-4312. E-mail address: [email protected] (Y.-S. Jang).
genesis, and cell death. Prolonged oxidative stress can cause several biologic disorders, including neurodegenerative diseases, immune dysfunction, and cancer [3,4]. There is evidence that ROS induce apoptosis, from studies in which antioxidants have been used to inhibit apoptotic mediators that induce the intracellular production of ROS and their behavior. For example, apoptotic agents such as ionizing and ultraviolet radiation, doxorubicin, cisplatin, and etherlinked lipids also generate ROS [5 –8]. In addition, apoptosis can be induced when a cell becomes less effective at scavenging or detoxifying ROS. Therefore, alleviating oxidative stress can reduce oxidative damage to, and apoptosis of, cells. As preventive and therapeutic measures to deal with diseases caused by oxidative stress become more common, the opportunities for the biochemical and clinical application of natural antioxidants increase. Consequently, numerous compounds have been used to treat diseases by reducing oxidative stress: in particular, plant-derived antioxidants, such as a-tocopherol, ascorbic acid, and flavonoids [1,9,10]. In Korea, much attention has been paid to Rhus verniciflua Stokes (RVS), because RVS is a known antiox-
0304-4165/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 2 ) 0 0 1 9 6 - 4
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idant [11,12]. Our previous results also indicate that an ethanol extract from RVS protects against oxidative damage by scavenging ROS or by altering the oxidized forms of chemicals [13 –15]. To date, however, there are no data that clearly indicate the mechanisms by which the RVS ethanol extract scavenges radicals or acts as an antioxidant, nor have the active compounds of the extract been identified. In this study, we prepared highly purified fractions of the RVS ethanol extract using solvents to elute the extract, and evaluated each fraction’s ability to scavenge free radicals generated by biologically relevant systems in vitro. We also assessed the degree to which each fraction protects against radical-mediated apoptosis by using the mouse thymocyte assay, a well-characterized model system for studying apoptosis [16]. Finally, we determined which active compounds are putatively responsible for the antioxidant property by analyzing MS and NMR spectra.
2. Materials and methods 2.1. Chemicals, plastics, and mice Unless otherwise specified, all chemicals used in this study were purchased from the Sigma Chemical (St. Louis, MO), and all the plastics were Falcon Labware purchased from Becton-Dickinson (Franklin Lakes, NJ). Inbred BALB/c mice (4 –6 weeks old) were purchased from Damul Science (Yoosung, Korea). 2.2. Preparation of RVS samples We prepared the initial ethanol extract from RVS as described previously [13]. Five grams of the ethanol extract dissolved in distilled water were applied to the silica gel ˚ , 28 –200 mesh). Ordered elution column (4 28 cm, 22 A was performed using distilled water, 30% ethanol, absolute ethanol, and 5% acetic acid, which yielded four eluted fractions (E1, E2, E3, and E4). The samples were collected and lyophilized to 2.2, 0.5, 0.8, and 0.4 g (44%, 10%, 16%, and 8% of the initial amounts) for E1, E2, E3, and E4, respectively. Fraction E1 was eluted again with distilled ˚ , 28 – 200 water using a silica gel column (2.5 60 cm, 22 A mesh) to yield three water-eluted fractions (F1, F2, and F3). These fractions were lyophilized to 0.7, 0.5, and 0.4 g (14%, 10%, and 8% of the initial amounts) for F1, F2, and F3, respectively. 2.3. Cell culture A single population of thymocyte cells was prepared using RPMI 1640 medium supplemented with antibiotics and 10% FBS (HyClone, Logan, UT). One million cells were resuspended in either 2 ml or 100 Al media for spreading onto either 35-mm culture dishes or 96-well flat-bottomed plates, respectively. Before the samples were
treated, the cultures were switched to medium supplemented only with 0.5% FBS. 2.4. Assay for scavenging activity against radicals We measured the scavenging activity of those samples exhibited against radicals generated by three different sources, such as Fe3+ ions, Fe2+ ions, and enzymes. Initially, we measured the scavenging activity of those samples exhibited against radicals generated by Fe3+ ions using three different assays: deoxyribose, ammonium thiocyanate, and DNA nicking assays. The deoxyribose and ammonium thiocyanate assays were essentially conducted according to the methods of Halliwell et al. [17] and Takao et al. [18], respectively. The DNA nicking assay was performed using supercoiled pBR322 plasmid DNA prepared from DH5a using WizardR Plus SV Minipreps (Promega, Madison, WI). Briefly, plasmid DNA (0.5 Ag) was added to Fenton’s reagents (30 mM H2O2, 50 AM ascorbic acid, and 80 AM FeCl3) containing RVS samples of different concentrations, and the final volume of the mixture was brought up to 20 Al. The mixture was incubated for 30 min at 37 jC, and the DNA was analyzed on a 1% agarose gel followed by ethidium bromide staining. We measured the scavenging activity of those samples exhibited against radicals generated by Fe2+ ions by using mitochondria from mouse cerebral cortex to determine the degree to which Fe2+ -dependent lipid inhibits peroxidation formation of thiobarbituric acid-reactive substances (TBARS). Mitochondria were isolated by gradient centrifugation of mouse cerebral cortex homogenate. Briefly, mouse cerebral cortex was homogenized in 4 ml buffer (0.25 M sucrose, 0.5 mM EDTA, and 10 mM Tris – HCl, pH 7.4) containing 12% Percoll using Dounce homogenizer. The homogenate was then layered onto a previously made gradient with 3.5 ml of 26% Percoll over 3.5 ml of 40% percoll. After a 5-min centrifugation at 30,000 g, mitochondria-rich fractions were collected and then resuspended in PBS, and the protein content of the suspension was measured by the methods of Lowry et al. [19]. The Fenton reaction system was used to induce formation of 2-TBARS in the mitochondrial suspension. Briefly, 20 Ag/ml of each RVS sample and 100 Al mitochondrial suspension (3 mg protein/ml) were mixed and the volume was brought up to 400 Al with PBS. The mixture was incubated for 10 min at 37 jC, and lipid peroxidation was initiated by adding newly prepared reaction solution (10 Al 2-mM ascorbic acid and 10 Al 25-AM FeSO4). After a 30-min incubation at 37 jC, TBARS formation was detected by measuring the absorbance at 530 nm as described previously [20]. Finally, we measured the scavenging activity of those samples exhibited against radicals generated by enzymes using the hypoxanthine/xanthine oxidase (X/XO) assay following the method of Gotoh and Niki [21] with a slight modification. Briefly, samples of different concentrations were added to the reaction solution containing 100 Al 30-
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mM EDTA (pH 7.4), 10 Al 30-mM hypoxanthine in 50 mM NaOH, and 200 Al 1.42-mM nitroblue tetrazolium (NBT). After a 3-min reaction, 100 Al 0.5-U/ml xanthine oxidase was added to the mixture and the volume brought up to 3 ml with 50 mM phosphate buffer (pH 7.4). After a 20-min incubation at room temperature, absorbance was measured at 560 nm. 2.5. Measuring cell viability We used the glucose/glucose oxidase (G/GO) system to generate radicals as described previously [22] and measured cell viability using an MTT assay [23]. Briefly, hydroxyl radicals were generated by the G/GO system (27.75 mM Dglucose and 20 mU/ml glucose oxidase in RPMI 1640 medium). Thymocytes were placed in 96-well plates and exposed to the radicals in a Haber– Weiss reaction for 4 h in the presence of RVS samples. At various times, 10 Al MTT solution (5 mg/ml in PBS) was added to each well and the plates were incubated for 4 h more at 37 jC. Finally, 70 Al acidic isopropanol was added to each well and the absorbance was measured at 560 nm using the SpectraCountk ELISA reader (Packard Instrument, Downers Grove, IL). 2.6. Measuring DNA synthesis and cytotoxicity We measured DNA synthesis by prestimulating thymocytes with 5 Ag/ml Concanavalin A (Con A) for 24 h, and incubating them with samples of different concentrations for another 24 h. We added 1 ACi [methyl-3H]thymidine (Amersham Pharmacia Biotech, Buckinghamshire, UK) to each well 8 h before the last incubation. Finally, cells were collected with a cell harvester (Inotech, Switzerland), and the tritium contents were measured using a liquid scintillation counter (Packard Instrument). We measured cellular cytotoxicity induced by treatment with RVS samples using a conventional MTT assay as described above. Cytotoxicity was calculated as follows % cytotoxicity ¼ ½ðAcontrol Asample Þ=Acontrol 100: 2.7. Detection of apoptosis We detected the induction of thymocyte apoptosis using DNA fragmentation and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assays. Briefly, thymocytes were spread onto 35-mm culture dishes and incubated with RPMI 1640 medium containing 0.5% FBS, 5 mU/ml GO, and 27.75 mM glucose in the presence of RVS samples of different concentrations. For the DNA fragmentation assay, 2 106 thymocytes were incubated with 500 Al lysis buffer (1% NP-40, 1% SDS in 50 mM Tris – HCl; pH 8.0) for 1 h at 65 jC. The DNA was extracted and the degree of fragmentation was analyzed using 2% agarose gel electrophoresis followed by ethidium bromide staining.
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For the TUNEL assay [24], aliquots of thymocytes were collected from liquid culture, centrifuged, and fixed with 0.3% buffered formaldehyde (pH 7.5) for 1 h at 4 jC. Cells were washed with PBS, resuspended in 70% ice-cold ethanol, and kept at 20 jC for 1 h. The cells were rehydrated with PBS and incubated in TdT buffer containing 30 mM Tris – HCl (pH 7.2), 140 mM sodium cacodylate, 1 mM CoCl2, 0.05 mg/ml BSA, 0.1 mM DTT, 7.5 U TdT, and 0.4 nM FITC-labeled dUTP. After a 30-min incubation at 37 jC, cells were washed with PBS, cytocentrifuged at 500 rpm for 3 min, and observed under a fluorescence microscope (Carl Zeiss, Germany). 2.8. Purification and identification of active compounds We separated active compounds within the RVS samples using reverse-phase HPLC (model 2690 with a 996-photo˚ diode array detector [Waters] and ABondapakk C18 125 A 10 Am, 3.9 300-mm column) using a gradient of acetonitrile in distilled water. We determined the structure of each compound from 1H-NMR and 13C-NMR spectra obtained using a Varian Unity Plus NMR (operated at 300 and 75 MHz, and 400 and 100 MHz, respectively; Walnut Creek, CA) in CD3OD containing tetramethylsilane (TMS) as an internal standard. 2.9. Statistical analyses All data were expressed as the mean F standard error. A one-way ANOVA using SPSS ver. 10.0 software was used for multiple comparisons. A value of p < 0.05 was considered significant.
3. Results 3.1. Scavenging effects of RVS samples on the generation of Fe3+-dependent hydroxyl radicals We first measured the scavenging activity of RVS samples on hydroxyl radicals generated by Fe3+ ions by measuring the degradation of deoxyribose, an indicator of the formation of thiobarbituric acid-malonaldehyde (TBAMDA) adducts (Fig. 1). The addition of RVS samples prevented hydroxyl radicals from degrading deoxyribose in a dose-dependent manner (Fig. 1A). Of the samples tested, F1, F2, and E3 scavenged hydroxyl radicals more than the other samples. Using concentration – activity curves, we calculated the concentration of each RVS sample required to inhibit 50% of TBA-MDA adduct formation (IC50). The IC50 of F1, F2, and E3 was 1.11, 0.72, and 1.31 mg/ml, respectively. In contrast, even the highest E2 and E4 concentrations inhibited only about 20% of adduct formation. We also determined the effects of hydroxyl-radical scavenging by RVS samples by using an ammonium thiocyanate assay to measure linoleic acid oxidation. RVS
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To further confirm the scavenging effect of the RVS samples, we investigated whether the samples could reduce Fe 3+ -dependent DNA nicking (Fig. 2). When pBR322 plasmid DNA was dissolved in the nickingreaction mixture, within 30 min we observed time-dependent increases in the formation of single-stranded nicked DNA, Form II, and of a double-stranded nicked and linear DNA, Form III (Fig. 2A, lanes 2 – 4). However, RVS samples added to the nicking-reaction mixture decreased the formation of Forms II and III DNA (Figs. 2B, C). For example, adding 20 Ag F1, F2, or F3 increased Form I DNA
Fig. 1. Inhibitory effects of RVS samples on FeCl3-dependent generation of hydroxyl radicals. Hydroxyl radicals were generated by Fenton’s reaction from (A) deoxyribose and (B) ammonium thiocyanate assay systems; the scavenging of hydroxyl radicals by RVS samples is expressed as % inhibition, as described in Materials and Methods. Concentrations of tested RVS samples ranged from 0.01 to 3 mg/ml. Results represent the mean values of three separate experiments. .: F1; n: F2; x: F3; o: E2; 5: E3; w: E4 .
samples effectively inhibited Fe3+ -dependent linoleic acid oxidation in a dose-dependent manner (Fig. 1B). As shown in the deoxyribose assay, the inhibitory effects of water fractions and E3 were higher than those of other RVS samples.
Fig. 2. Inhibitory effects of RVS samples on DNA nicking caused by hydroxyl radicals. (A) The DNA nicking reaction was initiated by adding 0.5 Ag pBR322 plasmid DNA to Fenton reaction solution. The reaction mixture was incubated at 37 jC and then analyzed by 1% agarose gel electrophoresis followed by ethidium bromide staining. Lanes 1 through 4 show the results for mixtures incubated for 0, 30, 20, and 10 min, respectively. Lane M shows the E/HindIII DNA marker. (B) Water-eluted RVS fractions were added to the reaction mixture and incubated for 30 min at 37 jC. Lanes 2 through 4 show the results from reaction mixtures containing 20 Ag F1, F2, and F3, respectively. Lanes 1 and 4 show native plasmid DNA and the E/HindIII DNA marker, respectively. (C) RVS samples were added to the reaction mixture and incubated for 30 min at 37 jC. Lanes 2 through 4 show the results from reaction mixtures containing 20 Ag E2, E3, and E4, respectively. Lanes 1 and 4 show native plasmid DNA and the E/HindIII DNA marker, respectively.
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formation, but resulted in loss and reduction of Fe3+ mediated Form III and Form II DNA, respectively (Fig. 2B, lanes 2– 4). In contrast, formation of Fe3+ -mediated Form II DNA was only slightly reduced by the addition of E2 and E4 (Fig. 2C, lanes 2 – 4). These results suggest that RVS samples scavenge hydroxyl radicals (generated in a Fe3+ -dependent manner), and that scavenging activity varies among the different sample fractions as follows: F2 > F1>E3>F3HE2 = E4. 3.2. Scavenging effects of RVS samples on Fe2+-dependent radical production This assay was performed by determining the amount of TBARS formed by Fe2+ -dependent lipid peroxidation. The TBARS concentration in mitochondrial suspension increased during the reaction, but decreased when RVS samples were added (Fig. 3). For example, the addition of 20 Ag/ml F1, F2, F3, or E3 significantly reduced the TBARS concentration ( P < 0.01) compared with the control treatment, whereas adding E2 and E4 affected TBARS concentration very little. In addition, among the RVS samples tested, F2 was the most efficient inhibitor of TBARS formed by lipid peroxidation. These results suggest that RVS samples are involved in scavenging ROS or iron chelators.
Fig. 4. Inhibitory effects of RVS samples on NBT reduction. Inhibitory effects of RVS samples were tested by monitoring NBT reduction caused by superoxide anions using the X/XO system, as described in Materials and Methods. The concentrations of tested RVS samples ranged from 0.01 to 3 mg/ml. Results are expressed as the mean values of triplicates. .: F1; n: F2; x: F3; o: E2; 5: E3; w: E4.
3.3. Scavenging effects of RVS samples on superoxide anion We tested for the effects of scavenging by RVS samples on the superoxide anion by monitoring the reduction of NBT caused by superoxide anions that are produced by xanthine oxidase-mediated degradation of hypoxanthine (Fig. 4). The RVS samples inhibited NBT reduction very efficiently. F1 and F2 inhibited superoxide anion production by 78.2 F 5.4% and 81.8 F 5.3%, respectively, when 3 mg/ml was added to the reaction solution. The failure of the absorbance of the reaction solution containing only NBT to change when samples were added suggests that the RVS samples did not directly reduce NBT (data not shown). Therefore, according to their concentration-dependent activity, RVS samples exhibited scavenging efficiency against superoxide anions in the following order: F2>F1>F3>E3HE2 = E4. These results indicate that F1 and F2 are active scavengers of both hydroxyl radicals and superoxide anion.
Fig. 3. Inhibitory effects of RVS samples on lipid peroxidation in mitochondria of mouse cerebral cortex. Twenty micrograms of each sample and 100 Al mitochondria suspension (3 mg protein/ml) were mixed, diluted to 400 Al with PBS, and incubated at 37 jC for 10 min. Lipid peroxidation was induced by adding 10 Al 2-mM ascorbic acid and 10 Al 25-AM FeSO4. TBARS levels were measured according to the methods of Lowry et al. [19]. Each bar represents the mean F S.E. values of triplicates; the figure shows representative results from three separate experiments. **P < 0.01; control and experimental treatments differed significantly.
3.4. Protective effects of RVS samples against thymocyte injury by glucose oxidase-mediated radicals Using the Haber– Weiss reaction, we further investigated the antioxidant activity of RVS samples by measuring the degree of cell injury caused by hydroxyl radicals produced by the G/GO system (Fig. 5). The addition of RVS samples (especially F1, F2, or E3) increased thymocyte viability 51.6% more than G/GO exposure alone ( P < 0.05). Adding
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no symptoms of cytotoxic effects when treated with high concentrations of water-eluted fractions, whereas those treated with E3 showed signs of highly toxic effects, even at the lowest concentration. These results suggest that water-
Fig. 5. Protective effects of RVS samples on thymocyte death induced by hydroxyl radicals. Thymocytes were exposed to hydroxyl radicals (from the G/GO system, generated in Haber – Weiss reactions) for 4 h in the presence of 50-Ag/ml samples. Each bar represents the mean F S.E. values of triplicates; the figure shows representative results from three separate experiments. * P < 0.05; control and experimental treatments differed significantly.
50 Ag/ml F1, F2, and E3 increased thymocyte viability by 79.4 F 4.6%, 84.3 F 5.7%, and 75.6 F 6.2%, respectively. In contrast, other RVS samples had no antioxidant effect at the concentrations tested. These results agree with the previous observation that the water fractions contain active scavengers of hydroxyl radicals and superoxide anions, and that F2 is the fraction that is the most effective against both radicals. 3.5. Effects of RVS samples on DNA synthesis in, and cytotoxicity of, thymocytes We determined the effect (defined as percentage inhibition of DNA synthesis) of RVS samples on DNA synthesis using a tritium incorporation assay (Fig. 6A). RVS samples inhibited DNA synthesis in thymocytes prestimulated with 5 Ag/ml Con A in a dose-dependent manner. Of the samples tested, E3 inhibited tritium uptake by thymocytes the most. The IC50 of F1, F2, F3, and E3 was 63.4, 64.9, 103.5, and 17.9 Ag/ml, respectively. These results suggest that RVS samples with higher antioxidant activity also inhibit DNA synthesis in thymocytes more than other samples (although E2 is an exception). RVS samples are likely to inhibit thymocyte DNA synthesis via either cytostatic or cytotoxic activity. We first performed MTT cytotoxicity assays to determine how RVS samples inhibit synthesis of thymocyte DNA (Fig. 6B). Cultured thymocytes exhibited a loss of cell viability, but
Fig. 6. Effects of RVS samples on DNA synthesis in, and cytotoxicity of, thymocytes. (A) Thymocytes were prestimulated with 5 Ag/ml Con A for 24 h, treated with RVS samples (at the concentrations indicated) for 24 h under low-serum conditions, and incubated in the presence of 1 ACi/ml [methyl-3H]thymidine for the last 8 h. (B) Thymocytes treated as in (A) were incubated in the presence of MTT solution for the last 4 h. Each bar represents the mean F S.E. values of triplicates; the figure shows representative results from three separate experiments. .: F1; n: F2; x: F3; o: E2; 5: E3; w: E4.
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eluted fractions of RVS samples, especially F1 and F2, are strong antioxidants with cytostatic effects, whereas E3 affects thymocytes cytotoxically rather than cytostatically, although E3 contains active radical scavengers. The details of this unexpected effect of E3 will be reported elsewhere (manuscript in preparation). 3.6. Effect of RVS samples on thymocyte apoptosis caused by hydroxyl radicals To confirm that RVS samples have antioxidant properties, we tested the protective effects of F1 and F2, which showed strong antioxidant activity, but not cytotoxicity, on thymocyte apoptosis caused by hydroxyl radicals. These effects were tested using DNA fragmentation (Fig. 7) and TUNEL staining (Fig. 8) assays. As shown in Fig. 7, DNA fragmentation detected quantitatively by agarose gel electrophoresis showed that thymocytes exposed to G/GO for 2 h exhibited a marked increase in the laddering of 200-bp DNA fragments (lane 2), a well-known characteristic of apoptosis. DNA laddering was hardly observed, however, in samples to which 100 Ag/ml of F1 (lane 3) and F2 (lane 4) had been added. The anti-apoptotic activity of F1 and F2 was confirmed by characterizing nuclear changes in thymocytes using TUNEL analysis (Fig. 8). The majority of thymocytes stained negatively in the absence of G/GO (Fig. 8A). When incubated with G/GO for 2 h, however, a large body within each cell was labeled with FITC-dUTP (Fig. 8B). Fewer cells contained positive FITC-dUTP staining of thymocytes
Fig. 7. Analysis of DNA fragmentation using agarose gel electrophoresis. Thymocytes were exposed to medium containing 27.75 mM glucose and 5 mU/ml glucose oxidase (G/GO) for 120 min in the presence of F1 and F2. Genomic DNA was prepared as described in Materials and methods and analyzed by 2% agarose gel electrophoresis followed by ethidium bromide staining. Lanes show results from control sample (lane 1) and samples treated with G/GO alone (lane 2), G/GO and 100 Ag/ml F1 (lane 3), and G/ GO and 100 Ag/ml F2 (lane 4).
Fig. 8. TUNEL staining of thymocytes. Thymocytes grown under lowserum conditions were exposed to G/GO for 120 min in the presence of F2. After incubation, thymocytes were stained with FITC-conjugated dUTP as described in Materials and Methods. (A) Control thymocytes; (B) thymocytes treated with G/GO alone; and (C) thymocytes treated with G/ GO and 100 Ag/ml F2. Photographs were taken under a UV-light fluorescence microscope at 400 . Arrows indicate apoptotic cells as shown by positive FITC-dUTP staining.
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˚ 10 Am, 3.9 300 mm) eluted with a linear Fig. 9. HPLC chromatogram of F2. HPLC was performed with a reverse-phase column (ABondapakk C18 125 A gradient of acetonitrile in distilled water. Column temperature was 30 jC, injection volume 10 Al/time, and flow rate 1.0 ml/min; detection was performed at 254 nm with a 996 photodiode array detector (Waters).
when treated with 100 Ag/ml F2 (Fig. 8C) or F1 (data not shown). These results indicate that, at least under these conditions, F1 and F2 can reduce the apoptotic death of thymocytes. 3.7. Active compounds in F2 Since F2 appeared to exhibit the most efficient antioxidant activity, the fraction was further purified using HPLC to identify its chemical makeup. The fraction had five peaks (Fig. 9), chemically identified as flavonoid derivatives by comparing the EI-MS, 1H-NMR, and 13C-NMR spectra with published data (Fig. 10) [12,25 – 29]. Compound 2 was initially identified as fustin after comparison with published data [12]. The compound exhibited an MS molecular ion peak at m/z 288 (70%) that corresponded to C15H12O6, and its 1 H-NMR and 13C-NMR spectra (operated at 300 and 75.4 MHz, respectively) showed aromatic signal characteristics for 2-(3V,4V-dihydroxyphenyl)-2,3-dihydro-3,7-dihydroxy4H-1-benzopyran-4-1, i.e., 3,3V,4V,7-tetrahydroxyflavanone (fustin). Compound 3 was identified as quercetin, because its 1H-NMR (300 MHz) spectra showed a pentahydroxyflavone signal with d 7.69 (1H, d, J = 2.1 Hz, H-2V), 7.55 (1H, dd, J = 2.1, 8.5 Hz, H-6V), 6.90 (1H, d, J = 8.5 Hz, H-5V), 6.42 (1H, d, J = 2.1 Hz, H-8), 6.20 (1H, d, J = 2.1 Hz, H-6); its 13C-NMR and EI-MS spectra were also consistent with previous data [25,27 –29]. Compound 4 was identified as butein, a chalcone derivative, by comparing its spectrum with those in the literature [12,26]. Its 1H-NMR (400 MHz) spectrum indicated the presence of a trans-a,h-unsaturated ketone peak with d 7.10 (1H, d, J = 16.1 Hz) and 7.53 (1H, d, J = 16.1 Hz)
and an m,p-3 substitution benzene structure with d 7.01 (1H, d, J = 2.2 Hz, H-3V), 7.13 (1H, dd, J = 2.2, 8.6 Hz, H-5V), 7.72 (1H, d, J = 8.6 Hz, H-6V), 7.42 (1H, d, J = 2.1 Hz, H-2), 7.24 (1H, d, J = 8.4 Hz, H-5), and 7.45 (1H, dd, J = 2.1, 8.4 Hz, H-6). Analysis of its 13C-NMR (100 MHz) spectrum suggested the presence of UCMO at 190.02 ppm. The proposed structure of compound 4 was fully supported by its EI-MS fragmentation pattern, which showed a tropylium ion peak at m/z 272 (99%)
Fig. 10. Chemical structure of compounds identified from fraction F2.
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and an UOUCH peak at m/z 71 (8%) corresponding to C15H12O5. Finally, the 1H-NMR (400 MHz) spectra of compound 5 indicated a benzene ring at d 7.50 (1H, d, J = 8.3 Hz), 7.13 (1H, dd, J = 8.3, 2.0 Hz), 7.41 (1H, d, J = 2.0 Hz), 6.59 (1H, d, J = 8.3 Hz), 6.74 (1H, d, J = 8.3 Hz). Its 13C-NMR (100 MHz) spectra indicated a ketone peak at 184.49 ppm, and an oxygen-bound benzene carbon signal at 168.19, 149.36, 146.69, and 147.68 ppm. The EI-MS fragmentation pattern of compound 5 showed an —OH peak at m/z 270 (99%) and 253 (18%), a —C2H4 peak at m/z 242 (11%), a —C2H5CMO peak at m/z 213 (10%), and a C7H7 + peak at m/z 91 (22%). Thus, by comparing these data with published reports [12,26], we concluded that compound 5 was 2-[(3V,4V-dihydroxyphenyl)methylene]-6-hydroxy-3(2H)-bezofuranone, i.e., 3V,4V,6trihydroxyaurone (sulfuretin). Unfortunately, we were not able to determine the chemical structure of compound 1 from its NMR and EI-MS spectra. These results suggest that the major compounds contained within the F2 fraction, which exhibited the most efficient antioxidant activity, are flavonoid derivatives.
4. Discussion We first used an Fe3+ -dependent system to test the scavenging activity of RVS samples on radicals generated by irons. We used this system because, of all the reduced forms of dioxygen, hydroxyl radicals are the most highly reactive and are thought to be the ROS that initiates cell damage. Results from the deoxyribose and ammonium thiocyanate experiments show that RVS samples contain active scavengers of hydroxyl radicals; deoxyribose degradation and linoleic acid oxidation were almost completely prevented in the presence of RVS samples (Fig. 1). In particular, the F2 fraction exhibited the most effective scavenging of hydroxyl radicals. We used DNA nicking assays to confirm that RVS samples exhibit scavenging activity against hydroxyl radicals produced by Fe3+ -dependent systems. The results were similar to those obtained from the deoxyribose and ammonium thiocyanate experiments; RVS samples (except fractions E2 and E4) inhibit DNA nicking mediated by hydroxyl radicals (Fig. 2). Next, we analyzed the role of Fe2+ in metalmediated lipid peroxidation, because hydroxyl radicals form in the presence of Fe2+ in vivo under normal conditions [30,31]. We demonstrated that the oxidation of Fe2+ to Fe3+ is closely linked to the onset of peroxidation, and that lipid peroxidation induced by the ascorbic acid –Fe2+ system is effectively inhibited by RVS samples, especially the water fractions and E3 (Fig. 3). We also found that F2 was the most effective fraction at inhibiting lipid peroxidation among those tested. We assumed that species other than superoxide, hydrogen peroxide, and hydroxyl radicals are involved in initiating Fe2+ -dependent lipid peroxidation. This assumption is realistic because several investigators have proposed that oxidizing species of Fe2+ include some iron –oxygen
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complexes, such as ferryl ion (FeO+ or FeOH3+ ) [32], perferryl iron (Fe2+ – O2 or Fe3+ – O2 ) [33], and the Fe2+ – O2 Fe3+ complex [34]. Moreover, the report [35] that desferrioxamine (DFX), a specific chelator of iron, completely inhibited the Fe2+ -dependent TBARS production supports our hypothesis. Thus, RVS samples may protect against Fe2+ -dependent lipid peroxidation, not only by directly scavenging ROS, but also through metal ion chelation or by altering iron redox chemistry. Next, using the X/XO assay system, we tested whether RVS samples can scavenge superoxide anions. The superoxide anion is generated in a variety of biological systems by either auto-oxidation processes or by enzymes, and its concentration increases under conditions of oxygenative stress and related situations [1,5,21]. RVS samples, especially the water fractions, actively scavenged superoxide anions in experiments with iron-dependent assay systems (Fig. 4). Finally, the degree to which RVS samples protect against radical-induced cell injury was tested in thymocytes using G/GO system. RVS samples significantly prevented thymocyte from death caused by hydroxyl radicals ( P < 0.05). The addition of 50 Ag/ml F1, F2, or E3 increased thymocyte viability 1.54-, 1.63-, and 1.46-fold, respectively, compared with viability of cells treated with GO alone (Fig. 5). Collectively, these results suggest that the water fractions and E3 effectively scavenge ROS and protect cells from radical-mediated injury; F2 was the most effective of these fractions. Several active compounds, both synthesized and naturally occurring, exhibit antiproliferative activity against various cancer cell lines [36 – 38]. These findings and the observation that an ethanol extract of RVS exhibits an antitumor effect [12,14,39] led us to predict that RVS samples can regulate cell proliferation. Our finding that DNA synthesis in mitogen-stimulated thymocytes was inhibited by RVS samples in a dose-dependent manner confirmed the prediction (Fig. 6A). Interestingly, the fractions that are effective antioxidants also inhibited the growth of thymocytes without any mutagenic effect. The MTT assay (Fig. 6B) showed that the viability of cultured thymocytes treated with 100 Ag/ml RVS water fractions for 24 h was 80% greater than control thymocytes. This led us to hypothesize that the inhibitory effect of the water fractions on DNA synthesis is due to cytostatic rather than cytotoxic effects. In contrast, we assumed that inhibition of DNA synthesis by E3 is due to cytotoxic rather than cytostatic mechanisms, although this fraction inhibited DNA synthesis more than any other. This assumption was based on the observation that thymocyte viability decreased, rather than remaining the same, with treatment at different E 3 concentrations. Although the exact mechanisms by which RVS samples inhibit thymocyte DNA synthesis need to be clarified through additional experiments, we believe that the inhibition is related to the activity of signal-transducing molecules involved in the cell cycle. Certain antioxidants have
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similar inhibitory effects on PI3-kinase, protein kinase C, protein tyrosine kinase, and some transcriptional factors; such inhibition in turn arrests cell growth and induces cell death in several carcinoma cell lines [12,26,38,40]. Apoptosis is the process that physiologically regulates cell number in developing organs and organisms. In many types of cells, it can be initiated by oxidative stress [1,3,41]. One of the hallmarks of apoptosis is the production of characteristic DNA fragments when DNA is cleaved by Ca2+ -dependent endonucleases at the linkage sites between nucleosomes [3,5]. The characteristic fragments are multimers of 180-bp fragments that are resolved into a DNA ladder on agarose gels during electrophoresis. As shown in Fig. 7, thymocytes exposed to G/GO for 2 h yielded a clear DNA ladder (lane 2), whereas the addition of 100 Ag/ml F1 or F2 to the thymocyte culture virtually eliminated the ladder (lanes 3 and 4). This finding was emphasized by results of the TUNEL assay used to characterize changes in thymocyte nuclei (Fig. 8). Most thymocytes incubated in G/GO for 2 h stained positively with FITC-dUTP, which indicates the presence of apoptotic cells (Fig. 8B). However, the addition of 100 Ag/ml F2 decreased the number of apoptotic cells (Fig. 8C), suggesting that fractions F1 and F2 inhibit apoptotic cell death caused by oxidation. Overall, the experimental results revealed that fraction F2 exhibited the most effective antioxidant activity. Consequently, we analyzed the fraction to identify active compounds that are antioxidant candidates. Chromatograms obtained from HPLC analysis showed five major peaks. We identified ortho-phenol flavonoids as major compounds by analyzing EI-MS, 1H-NMR, and 13C-NMR spectra and comparing them with published data (Figs. 9 and 10) [12,25 – 29]. Flavonoids are commonly found in most plants, and are the pharmacologically active constituents in many herbal plant medicines. Many flavonoids exhibit several pharmacological properties, acting as vasodilatory, anticarcinogenic, anti-inflammatory, antibacterial, immunestimulating, anti-allergic, and antiviral agents [42 –48]. The compounds identified in fraction F2 in particular are known to induce glutathione S-transferase, increase cell resistance to oxidative stress, and detoxify mutagenic xenobiotics [49]. Moreover, flavonoids containing one or more aromatic hydroxyl groups can replace a-tocopherol as a chain-breaking antioxidant and restore reduced glutathione as a protective agent in liver microsomal membranes [50]. In addition, depending on the cell type and cell cycle phase, flavonoids (including quercetin) encourage antiproliferation or differentiation by modulating cellular IP3 concentrations [51]. Hence, data from chemical analyses clearly support our experimental finding that F2 is the most efficient fraction; these data also explain why this water fraction acts as an antioxidant at low concentrations, and why it inhibits DNA synthesis depending on its concentration. In summary, many biochemical and clinical studies suggest that natural and synthetic antioxidant compounds help treat diseases mediated by oxidative stress. The results
presented in this report also indicate that RVS samples, especially the water-eluted fractions, contain agents that scavenge ROS, whether they are generated enzymatically or not. These agents also regulate cell proliferation and prevent oxidation-mediated apoptotic death of thymocytes. Using physicochemical procedures, we determined that the active compounds in fraction F2 are flavonoids. Therefore, we suggest that water-eluted RVS fractions might efficiently modulate intracellular redox states to prevent some diseases that are induced by oxidative stress. Further mechanistic and clinical studies will focus on isolating the mechanisms underlying the biochemical and biomolecular roles of active RVS compounds. Acknowledgements This work was supported by a grant from the Korean Ministry of Science and Technology and from Chollabukdo Province in support of regional research and development. Dr. J.-C. Lee was supported by the post-doctoral program at Chonbuk National University. Part of this work was conducted at the Research Center for Bioactive Materials at Chonbuk National University. References [1] J.M. Mates, F.M. Sanchez-Jimenez, Role of reactive oxygen species in apoptosis: implications for cancer therapy, IJBCB 32 (2000) 157 – 170. [2] B.N. Ames, M.K. Shigenaga, in: B. Halliwell, O.I. Aruoma (Eds.), Oxidants Are a Major Contributor to Cancer and Aging, Ellis Horwood, Chichester, 1993, pp. 1 – 15. [3] A.F.G. Slater, S. Orrenius, in: R.G. Cutler, L. Packer, J. Bertram, A. Mori (Eds.), Oxidative Stress and Apoptosis, Birkhauser, Basel, 1995, pp. 21 – 26. [4] H. Wiseman, B. Halliwell, Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer, Biochem. J. 313 (1996) 17 – 29. [5] R. von Harsdorf, P.F. Li, R. Dietz, Signaling pathway in reactive oxygen species-induced cardiomyocyte apoptosis, Circulation 99 (1999) 2934 – 2941. [6] B. Halliwell, J.M. Gutteridge, Role of free radicals and catalytic metal ions in human disease: an overview, Methods Enzymol. 186 (1990) 1 – 85. [7] M.N. Benchekroun, P. Pourquier, B. Schott, J. Robert, Doxorubicininduced lipid peroxidation and glutathione peroxidase activity in tumor cell lines selected for resistance to doxorubicin, Eur. J. Biochem. 211 (1993) 141 – 146. [8] B.A. Wagner, G.R. Buettner, C.P. Burns, Increased generation of lipid-derived and ascorbate free radicals by L1210 cells exposed to the ether lipid edelfosine, Cancer Res. 53 (1993) 711 – 713. [9] C.K. Sen, L. Packer, Antioxidant and redox regulation of gene transcription, FASEB J. 10 (1996) 709 – 720. [10] E. Middleton Jr., C. Kandaswani, Effects of flavonoids on immune and inflammatory cell functions, Biochem. Pharmacol. 43 (1992) 1167 – 1179. [11] D.H. Hong, S.B. Han, C.W. Lee, S.H. Park, Y.J. Jeon, M.J. Kim, S.S. Kwak, H.M. Kim, Cytotoxicity of urushiols isolated from sap of Korean lacquer tree (Rhus verniciflua Stokes), Arch. Pharmacol. Res. 22 (1999) 638 – 641. [12] N.C. Jung, Biological activity of urushiol and flavonoids from Lac
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Identification of Rhus verniciflua Stokes compounds that exhibit free radical scavenging and anti-apoptotic properties Jeong-Chae Lee a, Kye-Taek Lim b, Yong-Suk Jang a,* a
Division of Biological Sciences and The Research Center for Bioactive Materials, Chonbuk National University, Chonju 561-756, South Korea b Biodefensive Substances Group, Institute of Biotechnology, Chonnam National University, Kwangju 500-757, South Korea Received 20 September 2001; received in revised form 17 January 2002; accepted 4 February 2002
Abstract Rhus verniciflua Stokes (RVS) is a widely used herbal plant with various biological properties. Our previous study using cultured neuronal cells showed that an ethanol extract of RVS had strong antioxidant properties. In this study, we characterized the antioxidant activity of the RVS ethanol extract and identified the active compounds responsible for this activity. From the RVS ethanol extract, we derived three water-eluted fractions and another three fractions eluted by organic solvents, and determined that the water-eluted fractions are what protect against reactive oxygen species (ROS) generated by iron and enzymes. Water-eluted fraction F2 was the most efficient antioxidant. Moreover, DNA fragmentation and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining experiments revealed that F2 also protects against thymocyte apoptosis mediated by hydroxyl radicals. Finally, EI-MS, 1H-NMR, and 13C-NMR spectra signals confirmed that the fraction contained flavonoid derivatives, including fustin, quercetin, butein, and sulfuretin. These results suggest that the flavonoid derivatives in F2 are the compounds in the RVS ethanol extract that act as antioxidants. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Antioxidant; Apoptosis; Flavonoid; Reactive oxygen specie; Rhus verniciflua Stokes
1. Introduction Reactive oxygen species (ROS), such as superoxide, hydroxyl radicals, and hydrogen peroxide, are constantly generated in small amounts during normal aerobic metabolism in living organisms [1]. ROS generation can be prevented by a wide array of primary antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase [1,2]. However, when the balance between cellular antioxidant defenses and ROS generation is disrupted, a condition referred to as oxidative stress occurs. Persistent oxidative stress can damage critical biomolecules and thus alter biologic processes, including signal transduction and gene expression, causing mitogenesis, muta-
Abbreviations: RVS, Rhus verniciflua Stokes; ROS, reactive oxygen species; TBARS, thiobarbituric acid-reactive substances; X/XO, hypoxanthine/xanthine oxidase; G/GO, glucose/glucose oxidase; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling * Corresponding author. Tel.: +82-63-270-3343; fax: +82-63-270-4312. E-mail address: [email protected] (Y.-S. Jang).
genesis, and cell death. Prolonged oxidative stress can cause several biologic disorders, including neurodegenerative diseases, immune dysfunction, and cancer [3,4]. There is evidence that ROS induce apoptosis, from studies in which antioxidants have been used to inhibit apoptotic mediators that induce the intracellular production of ROS and their behavior. For example, apoptotic agents such as ionizing and ultraviolet radiation, doxorubicin, cisplatin, and etherlinked lipids also generate ROS [5 –8]. In addition, apoptosis can be induced when a cell becomes less effective at scavenging or detoxifying ROS. Therefore, alleviating oxidative stress can reduce oxidative damage to, and apoptosis of, cells. As preventive and therapeutic measures to deal with diseases caused by oxidative stress become more common, the opportunities for the biochemical and clinical application of natural antioxidants increase. Consequently, numerous compounds have been used to treat diseases by reducing oxidative stress: in particular, plant-derived antioxidants, such as a-tocopherol, ascorbic acid, and flavonoids [1,9,10]. In Korea, much attention has been paid to Rhus verniciflua Stokes (RVS), because RVS is a known antiox-
0304-4165/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 2 ) 0 0 1 9 6 - 4
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idant [11,12]. Our previous results also indicate that an ethanol extract from RVS protects against oxidative damage by scavenging ROS or by altering the oxidized forms of chemicals [13 –15]. To date, however, there are no data that clearly indicate the mechanisms by which the RVS ethanol extract scavenges radicals or acts as an antioxidant, nor have the active compounds of the extract been identified. In this study, we prepared highly purified fractions of the RVS ethanol extract using solvents to elute the extract, and evaluated each fraction’s ability to scavenge free radicals generated by biologically relevant systems in vitro. We also assessed the degree to which each fraction protects against radical-mediated apoptosis by using the mouse thymocyte assay, a well-characterized model system for studying apoptosis [16]. Finally, we determined which active compounds are putatively responsible for the antioxidant property by analyzing MS and NMR spectra.
2. Materials and methods 2.1. Chemicals, plastics, and mice Unless otherwise specified, all chemicals used in this study were purchased from the Sigma Chemical (St. Louis, MO), and all the plastics were Falcon Labware purchased from Becton-Dickinson (Franklin Lakes, NJ). Inbred BALB/c mice (4 –6 weeks old) were purchased from Damul Science (Yoosung, Korea). 2.2. Preparation of RVS samples We prepared the initial ethanol extract from RVS as described previously [13]. Five grams of the ethanol extract dissolved in distilled water were applied to the silica gel ˚ , 28 –200 mesh). Ordered elution column (4 28 cm, 22 A was performed using distilled water, 30% ethanol, absolute ethanol, and 5% acetic acid, which yielded four eluted fractions (E1, E2, E3, and E4). The samples were collected and lyophilized to 2.2, 0.5, 0.8, and 0.4 g (44%, 10%, 16%, and 8% of the initial amounts) for E1, E2, E3, and E4, respectively. Fraction E1 was eluted again with distilled ˚ , 28 – 200 water using a silica gel column (2.5 60 cm, 22 A mesh) to yield three water-eluted fractions (F1, F2, and F3). These fractions were lyophilized to 0.7, 0.5, and 0.4 g (14%, 10%, and 8% of the initial amounts) for F1, F2, and F3, respectively. 2.3. Cell culture A single population of thymocyte cells was prepared using RPMI 1640 medium supplemented with antibiotics and 10% FBS (HyClone, Logan, UT). One million cells were resuspended in either 2 ml or 100 Al media for spreading onto either 35-mm culture dishes or 96-well flat-bottomed plates, respectively. Before the samples were
treated, the cultures were switched to medium supplemented only with 0.5% FBS. 2.4. Assay for scavenging activity against radicals We measured the scavenging activity of those samples exhibited against radicals generated by three different sources, such as Fe3+ ions, Fe2+ ions, and enzymes. Initially, we measured the scavenging activity of those samples exhibited against radicals generated by Fe3+ ions using three different assays: deoxyribose, ammonium thiocyanate, and DNA nicking assays. The deoxyribose and ammonium thiocyanate assays were essentially conducted according to the methods of Halliwell et al. [17] and Takao et al. [18], respectively. The DNA nicking assay was performed using supercoiled pBR322 plasmid DNA prepared from DH5a using WizardR Plus SV Minipreps (Promega, Madison, WI). Briefly, plasmid DNA (0.5 Ag) was added to Fenton’s reagents (30 mM H2O2, 50 AM ascorbic acid, and 80 AM FeCl3) containing RVS samples of different concentrations, and the final volume of the mixture was brought up to 20 Al. The mixture was incubated for 30 min at 37 jC, and the DNA was analyzed on a 1% agarose gel followed by ethidium bromide staining. We measured the scavenging activity of those samples exhibited against radicals generated by Fe2+ ions by using mitochondria from mouse cerebral cortex to determine the degree to which Fe2+ -dependent lipid inhibits peroxidation formation of thiobarbituric acid-reactive substances (TBARS). Mitochondria were isolated by gradient centrifugation of mouse cerebral cortex homogenate. Briefly, mouse cerebral cortex was homogenized in 4 ml buffer (0.25 M sucrose, 0.5 mM EDTA, and 10 mM Tris – HCl, pH 7.4) containing 12% Percoll using Dounce homogenizer. The homogenate was then layered onto a previously made gradient with 3.5 ml of 26% Percoll over 3.5 ml of 40% percoll. After a 5-min centrifugation at 30,000 g, mitochondria-rich fractions were collected and then resuspended in PBS, and the protein content of the suspension was measured by the methods of Lowry et al. [19]. The Fenton reaction system was used to induce formation of 2-TBARS in the mitochondrial suspension. Briefly, 20 Ag/ml of each RVS sample and 100 Al mitochondrial suspension (3 mg protein/ml) were mixed and the volume was brought up to 400 Al with PBS. The mixture was incubated for 10 min at 37 jC, and lipid peroxidation was initiated by adding newly prepared reaction solution (10 Al 2-mM ascorbic acid and 10 Al 25-AM FeSO4). After a 30-min incubation at 37 jC, TBARS formation was detected by measuring the absorbance at 530 nm as described previously [20]. Finally, we measured the scavenging activity of those samples exhibited against radicals generated by enzymes using the hypoxanthine/xanthine oxidase (X/XO) assay following the method of Gotoh and Niki [21] with a slight modification. Briefly, samples of different concentrations were added to the reaction solution containing 100 Al 30-
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mM EDTA (pH 7.4), 10 Al 30-mM hypoxanthine in 50 mM NaOH, and 200 Al 1.42-mM nitroblue tetrazolium (NBT). After a 3-min reaction, 100 Al 0.5-U/ml xanthine oxidase was added to the mixture and the volume brought up to 3 ml with 50 mM phosphate buffer (pH 7.4). After a 20-min incubation at room temperature, absorbance was measured at 560 nm. 2.5. Measuring cell viability We used the glucose/glucose oxidase (G/GO) system to generate radicals as described previously [22] and measured cell viability using an MTT assay [23]. Briefly, hydroxyl radicals were generated by the G/GO system (27.75 mM Dglucose and 20 mU/ml glucose oxidase in RPMI 1640 medium). Thymocytes were placed in 96-well plates and exposed to the radicals in a Haber– Weiss reaction for 4 h in the presence of RVS samples. At various times, 10 Al MTT solution (5 mg/ml in PBS) was added to each well and the plates were incubated for 4 h more at 37 jC. Finally, 70 Al acidic isopropanol was added to each well and the absorbance was measured at 560 nm using the SpectraCountk ELISA reader (Packard Instrument, Downers Grove, IL). 2.6. Measuring DNA synthesis and cytotoxicity We measured DNA synthesis by prestimulating thymocytes with 5 Ag/ml Concanavalin A (Con A) for 24 h, and incubating them with samples of different concentrations for another 24 h. We added 1 ACi [methyl-3H]thymidine (Amersham Pharmacia Biotech, Buckinghamshire, UK) to each well 8 h before the last incubation. Finally, cells were collected with a cell harvester (Inotech, Switzerland), and the tritium contents were measured using a liquid scintillation counter (Packard Instrument). We measured cellular cytotoxicity induced by treatment with RVS samples using a conventional MTT assay as described above. Cytotoxicity was calculated as follows % cytotoxicity ¼ ½ðAcontrol Asample Þ=Acontrol 100: 2.7. Detection of apoptosis We detected the induction of thymocyte apoptosis using DNA fragmentation and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assays. Briefly, thymocytes were spread onto 35-mm culture dishes and incubated with RPMI 1640 medium containing 0.5% FBS, 5 mU/ml GO, and 27.75 mM glucose in the presence of RVS samples of different concentrations. For the DNA fragmentation assay, 2 106 thymocytes were incubated with 500 Al lysis buffer (1% NP-40, 1% SDS in 50 mM Tris – HCl; pH 8.0) for 1 h at 65 jC. The DNA was extracted and the degree of fragmentation was analyzed using 2% agarose gel electrophoresis followed by ethidium bromide staining.
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For the TUNEL assay [24], aliquots of thymocytes were collected from liquid culture, centrifuged, and fixed with 0.3% buffered formaldehyde (pH 7.5) for 1 h at 4 jC. Cells were washed with PBS, resuspended in 70% ice-cold ethanol, and kept at 20 jC for 1 h. The cells were rehydrated with PBS and incubated in TdT buffer containing 30 mM Tris – HCl (pH 7.2), 140 mM sodium cacodylate, 1 mM CoCl2, 0.05 mg/ml BSA, 0.1 mM DTT, 7.5 U TdT, and 0.4 nM FITC-labeled dUTP. After a 30-min incubation at 37 jC, cells were washed with PBS, cytocentrifuged at 500 rpm for 3 min, and observed under a fluorescence microscope (Carl Zeiss, Germany). 2.8. Purification and identification of active compounds We separated active compounds within the RVS samples using reverse-phase HPLC (model 2690 with a 996-photo˚ diode array detector [Waters] and ABondapakk C18 125 A 10 Am, 3.9 300-mm column) using a gradient of acetonitrile in distilled water. We determined the structure of each compound from 1H-NMR and 13C-NMR spectra obtained using a Varian Unity Plus NMR (operated at 300 and 75 MHz, and 400 and 100 MHz, respectively; Walnut Creek, CA) in CD3OD containing tetramethylsilane (TMS) as an internal standard. 2.9. Statistical analyses All data were expressed as the mean F standard error. A one-way ANOVA using SPSS ver. 10.0 software was used for multiple comparisons. A value of p < 0.05 was considered significant.
3. Results 3.1. Scavenging effects of RVS samples on the generation of Fe3+-dependent hydroxyl radicals We first measured the scavenging activity of RVS samples on hydroxyl radicals generated by Fe3+ ions by measuring the degradation of deoxyribose, an indicator of the formation of thiobarbituric acid-malonaldehyde (TBAMDA) adducts (Fig. 1). The addition of RVS samples prevented hydroxyl radicals from degrading deoxyribose in a dose-dependent manner (Fig. 1A). Of the samples tested, F1, F2, and E3 scavenged hydroxyl radicals more than the other samples. Using concentration – activity curves, we calculated the concentration of each RVS sample required to inhibit 50% of TBA-MDA adduct formation (IC50). The IC50 of F1, F2, and E3 was 1.11, 0.72, and 1.31 mg/ml, respectively. In contrast, even the highest E2 and E4 concentrations inhibited only about 20% of adduct formation. We also determined the effects of hydroxyl-radical scavenging by RVS samples by using an ammonium thiocyanate assay to measure linoleic acid oxidation. RVS
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To further confirm the scavenging effect of the RVS samples, we investigated whether the samples could reduce Fe 3+ -dependent DNA nicking (Fig. 2). When pBR322 plasmid DNA was dissolved in the nickingreaction mixture, within 30 min we observed time-dependent increases in the formation of single-stranded nicked DNA, Form II, and of a double-stranded nicked and linear DNA, Form III (Fig. 2A, lanes 2 – 4). However, RVS samples added to the nicking-reaction mixture decreased the formation of Forms II and III DNA (Figs. 2B, C). For example, adding 20 Ag F1, F2, or F3 increased Form I DNA
Fig. 1. Inhibitory effects of RVS samples on FeCl3-dependent generation of hydroxyl radicals. Hydroxyl radicals were generated by Fenton’s reaction from (A) deoxyribose and (B) ammonium thiocyanate assay systems; the scavenging of hydroxyl radicals by RVS samples is expressed as % inhibition, as described in Materials and Methods. Concentrations of tested RVS samples ranged from 0.01 to 3 mg/ml. Results represent the mean values of three separate experiments. .: F1; n: F2; x: F3; o: E2; 5: E3; w: E4 .
samples effectively inhibited Fe3+ -dependent linoleic acid oxidation in a dose-dependent manner (Fig. 1B). As shown in the deoxyribose assay, the inhibitory effects of water fractions and E3 were higher than those of other RVS samples.
Fig. 2. Inhibitory effects of RVS samples on DNA nicking caused by hydroxyl radicals. (A) The DNA nicking reaction was initiated by adding 0.5 Ag pBR322 plasmid DNA to Fenton reaction solution. The reaction mixture was incubated at 37 jC and then analyzed by 1% agarose gel electrophoresis followed by ethidium bromide staining. Lanes 1 through 4 show the results for mixtures incubated for 0, 30, 20, and 10 min, respectively. Lane M shows the E/HindIII DNA marker. (B) Water-eluted RVS fractions were added to the reaction mixture and incubated for 30 min at 37 jC. Lanes 2 through 4 show the results from reaction mixtures containing 20 Ag F1, F2, and F3, respectively. Lanes 1 and 4 show native plasmid DNA and the E/HindIII DNA marker, respectively. (C) RVS samples were added to the reaction mixture and incubated for 30 min at 37 jC. Lanes 2 through 4 show the results from reaction mixtures containing 20 Ag E2, E3, and E4, respectively. Lanes 1 and 4 show native plasmid DNA and the E/HindIII DNA marker, respectively.
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formation, but resulted in loss and reduction of Fe3+ mediated Form III and Form II DNA, respectively (Fig. 2B, lanes 2– 4). In contrast, formation of Fe3+ -mediated Form II DNA was only slightly reduced by the addition of E2 and E4 (Fig. 2C, lanes 2 – 4). These results suggest that RVS samples scavenge hydroxyl radicals (generated in a Fe3+ -dependent manner), and that scavenging activity varies among the different sample fractions as follows: F2 > F1>E3>F3HE2 = E4. 3.2. Scavenging effects of RVS samples on Fe2+-dependent radical production This assay was performed by determining the amount of TBARS formed by Fe2+ -dependent lipid peroxidation. The TBARS concentration in mitochondrial suspension increased during the reaction, but decreased when RVS samples were added (Fig. 3). For example, the addition of 20 Ag/ml F1, F2, F3, or E3 significantly reduced the TBARS concentration ( P < 0.01) compared with the control treatment, whereas adding E2 and E4 affected TBARS concentration very little. In addition, among the RVS samples tested, F2 was the most efficient inhibitor of TBARS formed by lipid peroxidation. These results suggest that RVS samples are involved in scavenging ROS or iron chelators.
Fig. 4. Inhibitory effects of RVS samples on NBT reduction. Inhibitory effects of RVS samples were tested by monitoring NBT reduction caused by superoxide anions using the X/XO system, as described in Materials and Methods. The concentrations of tested RVS samples ranged from 0.01 to 3 mg/ml. Results are expressed as the mean values of triplicates. .: F1; n: F2; x: F3; o: E2; 5: E3; w: E4.
3.3. Scavenging effects of RVS samples on superoxide anion We tested for the effects of scavenging by RVS samples on the superoxide anion by monitoring the reduction of NBT caused by superoxide anions that are produced by xanthine oxidase-mediated degradation of hypoxanthine (Fig. 4). The RVS samples inhibited NBT reduction very efficiently. F1 and F2 inhibited superoxide anion production by 78.2 F 5.4% and 81.8 F 5.3%, respectively, when 3 mg/ml was added to the reaction solution. The failure of the absorbance of the reaction solution containing only NBT to change when samples were added suggests that the RVS samples did not directly reduce NBT (data not shown). Therefore, according to their concentration-dependent activity, RVS samples exhibited scavenging efficiency against superoxide anions in the following order: F2>F1>F3>E3HE2 = E4. These results indicate that F1 and F2 are active scavengers of both hydroxyl radicals and superoxide anion.
Fig. 3. Inhibitory effects of RVS samples on lipid peroxidation in mitochondria of mouse cerebral cortex. Twenty micrograms of each sample and 100 Al mitochondria suspension (3 mg protein/ml) were mixed, diluted to 400 Al with PBS, and incubated at 37 jC for 10 min. Lipid peroxidation was induced by adding 10 Al 2-mM ascorbic acid and 10 Al 25-AM FeSO4. TBARS levels were measured according to the methods of Lowry et al. [19]. Each bar represents the mean F S.E. values of triplicates; the figure shows representative results from three separate experiments. **P < 0.01; control and experimental treatments differed significantly.
3.4. Protective effects of RVS samples against thymocyte injury by glucose oxidase-mediated radicals Using the Haber– Weiss reaction, we further investigated the antioxidant activity of RVS samples by measuring the degree of cell injury caused by hydroxyl radicals produced by the G/GO system (Fig. 5). The addition of RVS samples (especially F1, F2, or E3) increased thymocyte viability 51.6% more than G/GO exposure alone ( P < 0.05). Adding
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no symptoms of cytotoxic effects when treated with high concentrations of water-eluted fractions, whereas those treated with E3 showed signs of highly toxic effects, even at the lowest concentration. These results suggest that water-
Fig. 5. Protective effects of RVS samples on thymocyte death induced by hydroxyl radicals. Thymocytes were exposed to hydroxyl radicals (from the G/GO system, generated in Haber – Weiss reactions) for 4 h in the presence of 50-Ag/ml samples. Each bar represents the mean F S.E. values of triplicates; the figure shows representative results from three separate experiments. * P < 0.05; control and experimental treatments differed significantly.
50 Ag/ml F1, F2, and E3 increased thymocyte viability by 79.4 F 4.6%, 84.3 F 5.7%, and 75.6 F 6.2%, respectively. In contrast, other RVS samples had no antioxidant effect at the concentrations tested. These results agree with the previous observation that the water fractions contain active scavengers of hydroxyl radicals and superoxide anions, and that F2 is the fraction that is the most effective against both radicals. 3.5. Effects of RVS samples on DNA synthesis in, and cytotoxicity of, thymocytes We determined the effect (defined as percentage inhibition of DNA synthesis) of RVS samples on DNA synthesis using a tritium incorporation assay (Fig. 6A). RVS samples inhibited DNA synthesis in thymocytes prestimulated with 5 Ag/ml Con A in a dose-dependent manner. Of the samples tested, E3 inhibited tritium uptake by thymocytes the most. The IC50 of F1, F2, F3, and E3 was 63.4, 64.9, 103.5, and 17.9 Ag/ml, respectively. These results suggest that RVS samples with higher antioxidant activity also inhibit DNA synthesis in thymocytes more than other samples (although E2 is an exception). RVS samples are likely to inhibit thymocyte DNA synthesis via either cytostatic or cytotoxic activity. We first performed MTT cytotoxicity assays to determine how RVS samples inhibit synthesis of thymocyte DNA (Fig. 6B). Cultured thymocytes exhibited a loss of cell viability, but
Fig. 6. Effects of RVS samples on DNA synthesis in, and cytotoxicity of, thymocytes. (A) Thymocytes were prestimulated with 5 Ag/ml Con A for 24 h, treated with RVS samples (at the concentrations indicated) for 24 h under low-serum conditions, and incubated in the presence of 1 ACi/ml [methyl-3H]thymidine for the last 8 h. (B) Thymocytes treated as in (A) were incubated in the presence of MTT solution for the last 4 h. Each bar represents the mean F S.E. values of triplicates; the figure shows representative results from three separate experiments. .: F1; n: F2; x: F3; o: E2; 5: E3; w: E4.
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eluted fractions of RVS samples, especially F1 and F2, are strong antioxidants with cytostatic effects, whereas E3 affects thymocytes cytotoxically rather than cytostatically, although E3 contains active radical scavengers. The details of this unexpected effect of E3 will be reported elsewhere (manuscript in preparation). 3.6. Effect of RVS samples on thymocyte apoptosis caused by hydroxyl radicals To confirm that RVS samples have antioxidant properties, we tested the protective effects of F1 and F2, which showed strong antioxidant activity, but not cytotoxicity, on thymocyte apoptosis caused by hydroxyl radicals. These effects were tested using DNA fragmentation (Fig. 7) and TUNEL staining (Fig. 8) assays. As shown in Fig. 7, DNA fragmentation detected quantitatively by agarose gel electrophoresis showed that thymocytes exposed to G/GO for 2 h exhibited a marked increase in the laddering of 200-bp DNA fragments (lane 2), a well-known characteristic of apoptosis. DNA laddering was hardly observed, however, in samples to which 100 Ag/ml of F1 (lane 3) and F2 (lane 4) had been added. The anti-apoptotic activity of F1 and F2 was confirmed by characterizing nuclear changes in thymocytes using TUNEL analysis (Fig. 8). The majority of thymocytes stained negatively in the absence of G/GO (Fig. 8A). When incubated with G/GO for 2 h, however, a large body within each cell was labeled with FITC-dUTP (Fig. 8B). Fewer cells contained positive FITC-dUTP staining of thymocytes
Fig. 7. Analysis of DNA fragmentation using agarose gel electrophoresis. Thymocytes were exposed to medium containing 27.75 mM glucose and 5 mU/ml glucose oxidase (G/GO) for 120 min in the presence of F1 and F2. Genomic DNA was prepared as described in Materials and methods and analyzed by 2% agarose gel electrophoresis followed by ethidium bromide staining. Lanes show results from control sample (lane 1) and samples treated with G/GO alone (lane 2), G/GO and 100 Ag/ml F1 (lane 3), and G/ GO and 100 Ag/ml F2 (lane 4).
Fig. 8. TUNEL staining of thymocytes. Thymocytes grown under lowserum conditions were exposed to G/GO for 120 min in the presence of F2. After incubation, thymocytes were stained with FITC-conjugated dUTP as described in Materials and Methods. (A) Control thymocytes; (B) thymocytes treated with G/GO alone; and (C) thymocytes treated with G/ GO and 100 Ag/ml F2. Photographs were taken under a UV-light fluorescence microscope at 400 . Arrows indicate apoptotic cells as shown by positive FITC-dUTP staining.
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˚ 10 Am, 3.9 300 mm) eluted with a linear Fig. 9. HPLC chromatogram of F2. HPLC was performed with a reverse-phase column (ABondapakk C18 125 A gradient of acetonitrile in distilled water. Column temperature was 30 jC, injection volume 10 Al/time, and flow rate 1.0 ml/min; detection was performed at 254 nm with a 996 photodiode array detector (Waters).
when treated with 100 Ag/ml F2 (Fig. 8C) or F1 (data not shown). These results indicate that, at least under these conditions, F1 and F2 can reduce the apoptotic death of thymocytes. 3.7. Active compounds in F2 Since F2 appeared to exhibit the most efficient antioxidant activity, the fraction was further purified using HPLC to identify its chemical makeup. The fraction had five peaks (Fig. 9), chemically identified as flavonoid derivatives by comparing the EI-MS, 1H-NMR, and 13C-NMR spectra with published data (Fig. 10) [12,25 – 29]. Compound 2 was initially identified as fustin after comparison with published data [12]. The compound exhibited an MS molecular ion peak at m/z 288 (70%) that corresponded to C15H12O6, and its 1 H-NMR and 13C-NMR spectra (operated at 300 and 75.4 MHz, respectively) showed aromatic signal characteristics for 2-(3V,4V-dihydroxyphenyl)-2,3-dihydro-3,7-dihydroxy4H-1-benzopyran-4-1, i.e., 3,3V,4V,7-tetrahydroxyflavanone (fustin). Compound 3 was identified as quercetin, because its 1H-NMR (300 MHz) spectra showed a pentahydroxyflavone signal with d 7.69 (1H, d, J = 2.1 Hz, H-2V), 7.55 (1H, dd, J = 2.1, 8.5 Hz, H-6V), 6.90 (1H, d, J = 8.5 Hz, H-5V), 6.42 (1H, d, J = 2.1 Hz, H-8), 6.20 (1H, d, J = 2.1 Hz, H-6); its 13C-NMR and EI-MS spectra were also consistent with previous data [25,27 –29]. Compound 4 was identified as butein, a chalcone derivative, by comparing its spectrum with those in the literature [12,26]. Its 1H-NMR (400 MHz) spectrum indicated the presence of a trans-a,h-unsaturated ketone peak with d 7.10 (1H, d, J = 16.1 Hz) and 7.53 (1H, d, J = 16.1 Hz)
and an m,p-3 substitution benzene structure with d 7.01 (1H, d, J = 2.2 Hz, H-3V), 7.13 (1H, dd, J = 2.2, 8.6 Hz, H-5V), 7.72 (1H, d, J = 8.6 Hz, H-6V), 7.42 (1H, d, J = 2.1 Hz, H-2), 7.24 (1H, d, J = 8.4 Hz, H-5), and 7.45 (1H, dd, J = 2.1, 8.4 Hz, H-6). Analysis of its 13C-NMR (100 MHz) spectrum suggested the presence of UCMO at 190.02 ppm. The proposed structure of compound 4 was fully supported by its EI-MS fragmentation pattern, which showed a tropylium ion peak at m/z 272 (99%)
Fig. 10. Chemical structure of compounds identified from fraction F2.
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and an UOUCH peak at m/z 71 (8%) corresponding to C15H12O5. Finally, the 1H-NMR (400 MHz) spectra of compound 5 indicated a benzene ring at d 7.50 (1H, d, J = 8.3 Hz), 7.13 (1H, dd, J = 8.3, 2.0 Hz), 7.41 (1H, d, J = 2.0 Hz), 6.59 (1H, d, J = 8.3 Hz), 6.74 (1H, d, J = 8.3 Hz). Its 13C-NMR (100 MHz) spectra indicated a ketone peak at 184.49 ppm, and an oxygen-bound benzene carbon signal at 168.19, 149.36, 146.69, and 147.68 ppm. The EI-MS fragmentation pattern of compound 5 showed an —OH peak at m/z 270 (99%) and 253 (18%), a —C2H4 peak at m/z 242 (11%), a —C2H5CMO peak at m/z 213 (10%), and a C7H7 + peak at m/z 91 (22%). Thus, by comparing these data with published reports [12,26], we concluded that compound 5 was 2-[(3V,4V-dihydroxyphenyl)methylene]-6-hydroxy-3(2H)-bezofuranone, i.e., 3V,4V,6trihydroxyaurone (sulfuretin). Unfortunately, we were not able to determine the chemical structure of compound 1 from its NMR and EI-MS spectra. These results suggest that the major compounds contained within the F2 fraction, which exhibited the most efficient antioxidant activity, are flavonoid derivatives.
4. Discussion We first used an Fe3+ -dependent system to test the scavenging activity of RVS samples on radicals generated by irons. We used this system because, of all the reduced forms of dioxygen, hydroxyl radicals are the most highly reactive and are thought to be the ROS that initiates cell damage. Results from the deoxyribose and ammonium thiocyanate experiments show that RVS samples contain active scavengers of hydroxyl radicals; deoxyribose degradation and linoleic acid oxidation were almost completely prevented in the presence of RVS samples (Fig. 1). In particular, the F2 fraction exhibited the most effective scavenging of hydroxyl radicals. We used DNA nicking assays to confirm that RVS samples exhibit scavenging activity against hydroxyl radicals produced by Fe3+ -dependent systems. The results were similar to those obtained from the deoxyribose and ammonium thiocyanate experiments; RVS samples (except fractions E2 and E4) inhibit DNA nicking mediated by hydroxyl radicals (Fig. 2). Next, we analyzed the role of Fe2+ in metalmediated lipid peroxidation, because hydroxyl radicals form in the presence of Fe2+ in vivo under normal conditions [30,31]. We demonstrated that the oxidation of Fe2+ to Fe3+ is closely linked to the onset of peroxidation, and that lipid peroxidation induced by the ascorbic acid –Fe2+ system is effectively inhibited by RVS samples, especially the water fractions and E3 (Fig. 3). We also found that F2 was the most effective fraction at inhibiting lipid peroxidation among those tested. We assumed that species other than superoxide, hydrogen peroxide, and hydroxyl radicals are involved in initiating Fe2+ -dependent lipid peroxidation. This assumption is realistic because several investigators have proposed that oxidizing species of Fe2+ include some iron –oxygen
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complexes, such as ferryl ion (FeO+ or FeOH3+ ) [32], perferryl iron (Fe2+ – O2 or Fe3+ – O2 ) [33], and the Fe2+ – O2 Fe3+ complex [34]. Moreover, the report [35] that desferrioxamine (DFX), a specific chelator of iron, completely inhibited the Fe2+ -dependent TBARS production supports our hypothesis. Thus, RVS samples may protect against Fe2+ -dependent lipid peroxidation, not only by directly scavenging ROS, but also through metal ion chelation or by altering iron redox chemistry. Next, using the X/XO assay system, we tested whether RVS samples can scavenge superoxide anions. The superoxide anion is generated in a variety of biological systems by either auto-oxidation processes or by enzymes, and its concentration increases under conditions of oxygenative stress and related situations [1,5,21]. RVS samples, especially the water fractions, actively scavenged superoxide anions in experiments with iron-dependent assay systems (Fig. 4). Finally, the degree to which RVS samples protect against radical-induced cell injury was tested in thymocytes using G/GO system. RVS samples significantly prevented thymocyte from death caused by hydroxyl radicals ( P < 0.05). The addition of 50 Ag/ml F1, F2, or E3 increased thymocyte viability 1.54-, 1.63-, and 1.46-fold, respectively, compared with viability of cells treated with GO alone (Fig. 5). Collectively, these results suggest that the water fractions and E3 effectively scavenge ROS and protect cells from radical-mediated injury; F2 was the most effective of these fractions. Several active compounds, both synthesized and naturally occurring, exhibit antiproliferative activity against various cancer cell lines [36 – 38]. These findings and the observation that an ethanol extract of RVS exhibits an antitumor effect [12,14,39] led us to predict that RVS samples can regulate cell proliferation. Our finding that DNA synthesis in mitogen-stimulated thymocytes was inhibited by RVS samples in a dose-dependent manner confirmed the prediction (Fig. 6A). Interestingly, the fractions that are effective antioxidants also inhibited the growth of thymocytes without any mutagenic effect. The MTT assay (Fig. 6B) showed that the viability of cultured thymocytes treated with 100 Ag/ml RVS water fractions for 24 h was 80% greater than control thymocytes. This led us to hypothesize that the inhibitory effect of the water fractions on DNA synthesis is due to cytostatic rather than cytotoxic effects. In contrast, we assumed that inhibition of DNA synthesis by E3 is due to cytotoxic rather than cytostatic mechanisms, although this fraction inhibited DNA synthesis more than any other. This assumption was based on the observation that thymocyte viability decreased, rather than remaining the same, with treatment at different E 3 concentrations. Although the exact mechanisms by which RVS samples inhibit thymocyte DNA synthesis need to be clarified through additional experiments, we believe that the inhibition is related to the activity of signal-transducing molecules involved in the cell cycle. Certain antioxidants have
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similar inhibitory effects on PI3-kinase, protein kinase C, protein tyrosine kinase, and some transcriptional factors; such inhibition in turn arrests cell growth and induces cell death in several carcinoma cell lines [12,26,38,40]. Apoptosis is the process that physiologically regulates cell number in developing organs and organisms. In many types of cells, it can be initiated by oxidative stress [1,3,41]. One of the hallmarks of apoptosis is the production of characteristic DNA fragments when DNA is cleaved by Ca2+ -dependent endonucleases at the linkage sites between nucleosomes [3,5]. The characteristic fragments are multimers of 180-bp fragments that are resolved into a DNA ladder on agarose gels during electrophoresis. As shown in Fig. 7, thymocytes exposed to G/GO for 2 h yielded a clear DNA ladder (lane 2), whereas the addition of 100 Ag/ml F1 or F2 to the thymocyte culture virtually eliminated the ladder (lanes 3 and 4). This finding was emphasized by results of the TUNEL assay used to characterize changes in thymocyte nuclei (Fig. 8). Most thymocytes incubated in G/GO for 2 h stained positively with FITC-dUTP, which indicates the presence of apoptotic cells (Fig. 8B). However, the addition of 100 Ag/ml F2 decreased the number of apoptotic cells (Fig. 8C), suggesting that fractions F1 and F2 inhibit apoptotic cell death caused by oxidation. Overall, the experimental results revealed that fraction F2 exhibited the most effective antioxidant activity. Consequently, we analyzed the fraction to identify active compounds that are antioxidant candidates. Chromatograms obtained from HPLC analysis showed five major peaks. We identified ortho-phenol flavonoids as major compounds by analyzing EI-MS, 1H-NMR, and 13C-NMR spectra and comparing them with published data (Figs. 9 and 10) [12,25 – 29]. Flavonoids are commonly found in most plants, and are the pharmacologically active constituents in many herbal plant medicines. Many flavonoids exhibit several pharmacological properties, acting as vasodilatory, anticarcinogenic, anti-inflammatory, antibacterial, immunestimulating, anti-allergic, and antiviral agents [42 –48]. The compounds identified in fraction F2 in particular are known to induce glutathione S-transferase, increase cell resistance to oxidative stress, and detoxify mutagenic xenobiotics [49]. Moreover, flavonoids containing one or more aromatic hydroxyl groups can replace a-tocopherol as a chain-breaking antioxidant and restore reduced glutathione as a protective agent in liver microsomal membranes [50]. In addition, depending on the cell type and cell cycle phase, flavonoids (including quercetin) encourage antiproliferation or differentiation by modulating cellular IP3 concentrations [51]. Hence, data from chemical analyses clearly support our experimental finding that F2 is the most efficient fraction; these data also explain why this water fraction acts as an antioxidant at low concentrations, and why it inhibits DNA synthesis depending on its concentration. In summary, many biochemical and clinical studies suggest that natural and synthetic antioxidant compounds help treat diseases mediated by oxidative stress. The results
presented in this report also indicate that RVS samples, especially the water-eluted fractions, contain agents that scavenge ROS, whether they are generated enzymatically or not. These agents also regulate cell proliferation and prevent oxidation-mediated apoptotic death of thymocytes. Using physicochemical procedures, we determined that the active compounds in fraction F2 are flavonoids. Therefore, we suggest that water-eluted RVS fractions might efficiently modulate intracellular redox states to prevent some diseases that are induced by oxidative stress. Further mechanistic and clinical studies will focus on isolating the mechanisms underlying the biochemical and biomolecular roles of active RVS compounds. Acknowledgements This work was supported by a grant from the Korean Ministry of Science and Technology and from Chollabukdo Province in support of regional research and development. Dr. J.-C. Lee was supported by the post-doctoral program at Chonbuk National University. Part of this work was conducted at the Research Center for Bioactive Materials at Chonbuk National University. References [1] J.M. Mates, F.M. Sanchez-Jimenez, Role of reactive oxygen species in apoptosis: implications for cancer therapy, IJBCB 32 (2000) 157 – 170. [2] B.N. Ames, M.K. Shigenaga, in: B. Halliwell, O.I. Aruoma (Eds.), Oxidants Are a Major Contributor to Cancer and Aging, Ellis Horwood, Chichester, 1993, pp. 1 – 15. [3] A.F.G. Slater, S. Orrenius, in: R.G. Cutler, L. Packer, J. Bertram, A. Mori (Eds.), Oxidative Stress and Apoptosis, Birkhauser, Basel, 1995, pp. 21 – 26. [4] H. Wiseman, B. Halliwell, Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer, Biochem. J. 313 (1996) 17 – 29. [5] R. von Harsdorf, P.F. Li, R. Dietz, Signaling pathway in reactive oxygen species-induced cardiomyocyte apoptosis, Circulation 99 (1999) 2934 – 2941. [6] B. Halliwell, J.M. Gutteridge, Role of free radicals and catalytic metal ions in human disease: an overview, Methods Enzymol. 186 (1990) 1 – 85. [7] M.N. Benchekroun, P. Pourquier, B. Schott, J. Robert, Doxorubicininduced lipid peroxidation and glutathione peroxidase activity in tumor cell lines selected for resistance to doxorubicin, Eur. J. Biochem. 211 (1993) 141 – 146. [8] B.A. Wagner, G.R. Buettner, C.P. Burns, Increased generation of lipid-derived and ascorbate free radicals by L1210 cells exposed to the ether lipid edelfosine, Cancer Res. 53 (1993) 711 – 713. [9] C.K. Sen, L. Packer, Antioxidant and redox regulation of gene transcription, FASEB J. 10 (1996) 709 – 720. [10] E. Middleton Jr., C. Kandaswani, Effects of flavonoids on immune and inflammatory cell functions, Biochem. Pharmacol. 43 (1992) 1167 – 1179. [11] D.H. Hong, S.B. Han, C.W. Lee, S.H. Park, Y.J. Jeon, M.J. Kim, S.S. Kwak, H.M. Kim, Cytotoxicity of urushiols isolated from sap of Korean lacquer tree (Rhus verniciflua Stokes), Arch. Pharmacol. Res. 22 (1999) 638 – 641. [12] N.C. Jung, Biological activity of urushiol and flavonoids from Lac
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