J. Agric. Food Chem. 2005, 53, 5577−5582
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Radical Scavenging and Anti-Lipoperoxidative Activities of Smallanthus sonchifolius Leaf Extracts KATERˇ INA VALENTOVAÄ ,*,† FRANTISˇ EK Sˇ ERSˇ ENˇ ,‡
AND JITKA
ULRICHOVAÄ †
Palacky´ University, Faculty of Medicine, Institute of Medical Chemistry and Biochemistry, Hneˇvotı´nska´ 3, CZ-77515 Olomouc, Czech Republic, and Comenius University in Bratislava, Faculty of Natural Sciences, Institute of Chemistry, Mlynska´ dolina CH-2, SK-842 15 Bratislava, Slovakia
Radical scavenging and anti-lipoperoxidative effects of two organic fractions and two aqueous extracts from the leaves of a neglected Andean crop-yacon (Smallanthus sonchifolius Poepp. & Endl., Asteraceae) were determined using various in vitro models. The extracts’ total phenolic content was 10.7-24.6%. They exhibited DPPH (IC50 16.14-33.39 µg/mL) and HO• scavenging activities (4.496.51 mg/mL). The extracts did not scavenge phenylglyoxylic ketyl radicals, but they retarded their formation. In the xanthine/xanthine oxidase superoxide radical generating system, the extracts’ activities were 26.10-37.67 superoxide dismutase equivalents/mg. As one of the extracts displayed xanthine oxidase inhibitory activity, the effect of the extracts on a nonenzymatically generated superoxide was determined (IC50 7.36-21.01 µg/mL). The extracts inhibited t-butyl hydroperoxideinduced lipoperoxidation of microsomal and mitochondrial membranes (IC50 22.15-465.3 µg/mL). These results make yacon leaves a good candidate for use as a food supplement in the prevention of chronic diseases involving oxidative stress. KEYWORDS: Yacon; phenolic compounds; antioxidant activity; lipid peroxidation; protective effects
INTRODUCTION
Yacon (Smallanthus sonchifolius) is an Andean crop used for centuries by the native inhabitants of South America as a food and in traditional medicine. Yacon tubers, for the local population, are a regular dietary component, and the leaves are used for their hypoglycemic activity (1). The tubers contain β-oligofructans as the storage saccharides (2, 3), and these are being at present recommended as prebiotics for improving mineral absorption and other beneficial effects (4-6). Because of their low caloric content, the tubers are a convenient dietary supplement for risk groups of the population (e.g., those with metabolic syndrome). However, until the 1980s, with the exception of Peru and Japan, practically no attention was devoted to this plant (7), which can be agriculturally cultivated in the European and northern American climate. Phenolic compounds have been isolated from the tubers of yaconsmainly chlorogenic acid (8) and other caffeic acid derivatives (9, 10)sand from the leaves of related Smallanthus fruticosus (centaureidin and sacuranetin) (11). We have already reported on the presence of large amounts of phenolic compounds in extracts from yacon leaves and tubers, mainly chlorogenic, protocatechuic, ferulic, rosmarinic, gallic, gentisic, and caffeic acids and their derivatives (12-14). We have also provided evidence for the antioxidant activity of two extracts in relation to the content of phenolics. Moreover, * Corresponding author phone: +420585632309; fax: +420585632302; e-mail:
[email protected]. † Palacky ´ University. ‡ Comenius University in Bratislava.
we showed that these extracts exhibited cytoprotective effects against tert-butyl hydroperoxide induced oxidative damage to rat hepatocytes (14). More recently, we have presented the effects of four different extracts of yacon leaves on rat hepatocyte viability, on oxidative damage, and on glucose metabolism in these cells and their insulin-like effect on cytochrome P450 (CYP) isoformes 2B and 2E mRNA expression in the rat hepatoma cell line (Fao cells). Phenolic compounds were probably responsible for the observed activities of the extracts (15). This study was undertaken to elucidate the antioxidant activity of four different extracts from S. sonchifolius leaves in different in vitro models of various degrees of complexity. The main objective was to contribute to the documentation of a new food supplement designed for the prevention of chronic diseases involving oxidative stress and disorders of glucose metabolism. MATERIALS AND METHODS Animals. Male Wistar rats weighing 200-250 g were conditioned in standard boxes for 15 days before the experiments. They were fed a standard laboratory diet, provided with water ad libitum, and kept on a 12:12 h light-dark cycle. Biological Material. Yacon (S. sonchifolius Poepp. & Endl., Asteraceae, Heliantheae, Melampodinae) plants, originally purchased from Ecuador, were grown at the Potato Research Institute in Havlı´cˇku˚v Brod. Voucher specimens were deposited in our collection at the Institute of Medical Chemistry and Biochemistry, Olomouc, Czech Republic. The leaves were collected in October 2000 at harvest time of the tubers and dried at ambient temperature. Extraction Procedure. Dried yacon leaves (20 g) were extracted as follows:
10.1021/jf050403o CCC: $30.25 © 2005 American Chemical Society Published on Web 06/15/2005
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( i) Organic fraction 1 (OF1): using a Soxhlet extractor with MeOH (3 × 8 h) and chlorophyll removal with petroleum ether, the aqueous layer was then acidified (0.01 M H3PO4) and extracted by ethyl acetate. Extract yield after evaporation of the solvent was 0.26 g. (ii) Organic fraction 2 (OF2): the dried drug was extracted by cool percolation with methanol/water (3:7); the extract was then acidified (0.01 M H3PO4) and extracted by ethyl acetate. Extract yield after evaporation of the solvent was 0.25 g. (iii) Decoction (DEC): the material was refluxed in water (20 mL) for 20 min and then left to cool at room temperature. Extract yield after freeze-drying was 3.30 g. (iv) Tea infusion extract (INF): 1000 mL of boiling water was poured onto the leaves and then allowed to extract for 20 min while cooling. Extract yield after freeze-drying was 6.06 g. All extracts were dried until a constant weight was achieved. The extraction procedure was repeated 3 times for each sample, and the reproducibility of the procedure was controlled by the measurement of total phenolic content in the samples. Reagents. β-Nicotinamide adenine dinucleotide (NADH) sodium salt, 1,1-diphenyl-2-picrylhydrazyl (DPPH, 90%), dimethyl sulfoxide (DMSO) for cell cultures, nitro blue tetrazolium chloride (NBT), phenazine methosulfate (PMS), sodium dodecylsulfate (SDS), superoxide dismutase (SOD) E.C. 1.15.1.1 from bovine erythrocytes (30 000 U), t-butyl hydroperoxide (tBH, 70% in water), 2-thiobarbituric acid (TBA, 98%), trichloroacetic acid (TCA), tris(hydroxymethyl)aminomethane (Tris), xanthine (99%) and xanthine oxidase (XOD) E.C. 1.1.3.22 from buttermilk (5 U), N-t-butyl-R-phenylnitrone (PBN), 5,5dimethyl-1-pyrroline-N-oxide (DMPO), and 30% H2O2 were purchased from Sigma-Aldrich Ltd., Czech Republic. FeSO4‚7 H2O and other chemicals and solvents were of analytical grade from Pliva-Lachema, Czech Republic. Analyses. Phenolic Content Analysis. Total phenolics in all extracts were determined using the Folin-Ciocalteau reagent (16). A total of 25 µL of the tested fraction in distilled water was mixed with 500 µL of the reagent (previously diluted 10-fold with water) and maintained at room temperature for 5 min; 500 µL of sodium bicarbonate (75 g/L) was added to the mixture. After 90 min at 30 °C, the absorbance was measured at 725 nm. Results were expressed as gallic acid equivalents. DPPH ScaVenging. A total of 375 µL of a methanol solution of the tested sample (6.25, 12.5, 25, 50, 75, and 100 µg/mL) was mixed with 750 µL of a methanol DPPH solution (20 mg/L). After 30 min, the absorbance at 517 nm was measured, and IC50 values were obtained from the inhibition curves (17, 18). Hydroxyl Radical ScaVenging. The Fenton reaction of H2O2 with FeSO4 and spontaneous decomposition of hydrogen peroxide at 25 °C were used as sources of hydroxyl (HO•) radicals. The scavenging activity of the tested samples was determined using electron paramagnetic resonance (EPR) spectroscopy. Because HO• radicals are very unstable and cannot be detected readily by the continuous wave EPR spectrometer, they were captured by a spin trap of DMPO, and the EPR spectra of this spin adduct were subsequently recorded. The reaction solution contained 5 × 10-4 M FeSO4, 0.025 M DMPO, the tested substances (0.05-10 mg/mL), and 0.05 M H2O2 in a phosphate buffer (0.19 M Na2HPO4, pH ) 7.4). The EPR spectra of these solutions were registered 40 min after the addition of H2O2, and the IC50 values were obtained from the dependences of scavenging activities on substance concentration. Phenylglyoxylic Ketyl Radical ScaVenging. The spontaneous decomposition of D,L-2,3-diphenyltartaric acid (DPTA) in propane-2-ol at 25 °C was used as a source of phenylglyoxylic ketyl radicals. Preparation and decomposition of DPTA is described in refs 19 and 20. Phenylglyoxylic ketyl radicals rise during the first phase of this decomposition, and they are transformed into benzoyl radicals during the second phase. Since the lifetime of these radicals is relatively short, they were stabilized as spin adducts with the spin trap of PBN. The lifetime of such spin adducts is longer (some tens of minutes); therefore, they can be easily recorded by a continual wave EPR spectrometer. The EPR spectra of solutions containing 0.025 M DPTA, 0.05 M PBN (control), or the desired amounts of yacon extracts or caffeic acid in propan-2-ol were recorded.
Valentova´ et al. Electron Paramagnetic Resonance Spectroscopy. All the EPR spectra were registered by a continual wave EPR spectrometer ERS 230 (ZWG Berlin, Germany), which operates in X-band (∼9.3 GHz), at a modulation amplitude 0.1 mT and a microwave power 5 mW. Superoxide Radical ScaVenging ActiVitysEnzymatic Assay. Antiradical activity was determined spectrophotometrically by monitoring the effect of the tested substances on the reduction of NBT to the blue chromogen formazan by O2•-. Superoxide radicals were generated by the xanthine/XOD system as described previously (21). Briefly, 0.1 mL of aqueous SOD standard solutions (5, 10, 25, 50, and 100 U/mL) or a sample solution (1 mg/mL) were separately added to a 1.0 mL mixture of 0.4 mM xanthine and 0.24 mM NBT in 0.1 M phosphate buffer (pH 7.8) containing 0.1 mM EDTA. Organic fractions were previously dissolved in DMSO (final concentration 0.1%). A total of 1.0 mL of XOD (0.05 U/mL), dissolved in the same phosphate buffer, was added, and the resulting mixture was incubated at 37 °C for 20 min. The reaction was terminated by adding 1.0 mL of 69 mM SDS solution, and the absorbance was measured at 560 nm (22). The superoxide scavenging activity was calculated as SOD equivalents (U/ mg) from the SOD standard curve. Effect on XOD ActiVity. The effect of the organic fraction OF1 on XOD activity was evaluated by measuring the formation of uric acid from xanthine in a double-beam spectrophotometer at room temperature. A total of 480 µL of XOD (0.592 U/mL) in a phosphate buffer (0.1 M) was mixed with 10 µL of the tested fraction in DMSO (final concentration 10, 25, 50, 75, 100, 125, and 150 µg/mL). The reaction was started by the addition of 500 µL of xanthine (400 µm) in the phosphate buffer, and the absorbance was recorded at 295 nm for 2 min. Superoxide Radical ScaVenging ActiVitysNonenzymatic Assay. Superoxide radicals were generated by the NADH/PMS system following a described procedure (23). A total of 50 µL of the tested fraction (final concentration 0, 2.5, 5, 10, 12.5, 20, 40, and 60 µg/mL) was mixed with 40 µL of NADH (996 µm), 60 µL of NBT (250 µm), and 150 µL of PMS (5.4 µm). All the reagents were dissolved in a phosphate buffer (19 mM, pH 7.4). Organic fractions were previously dissolved in DMSO (final concentration 0.1%). The reaction mixture was incubated for 30 min at 37 °C, and the absorbance was measured at 560 nm. IC50 values were obtained from inhibition curves. Protection Against tBH-Induced Lipoperoxidation. Rat liver microsomes and mitochondria were prepared by fractional centrifugation (24, 25) of rat liver homogenate in 3 mM Tris buffer containing 250 mM sucrose and 0.1 mM EDTA (pH 7.4). The mitochondrial and microsomal fractions were characterized by their protein content (26). For inhibition of lipoperoxidation, a mixture containing 2 mg/mL mitochondrial or 1 mg/mL microsomal fraction, 5-120 µg/mL extracts or phenolic acids, and 1 mM tBH was incubated in a water bath at 37 °C for 60 min. The incubation was then terminated by adding 2 mL of a mixture of 26 mM TBA and 918 mM TCA; the samples were subsequently incubated at 90 °C for 30 min. After cooling, the samples were centrifuged (10 min, 1000g, 20 °C), and the absorbance of the supernatant was measured at 535 nm (27). IC50 values were obtained from inhibition curves. Statistics. Data were analyzed with a one-way ANOVA using the StatView Statistical Package (SAS Institute, Inc.). Differences were considered statistically significant when p was
