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Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Yamagata, Japan and Course of the Science of * Bioresources, The United Graduate School of Agricultural Sciences, Iwate University, Iwate, Japan
2To whom correspondence should be addressed. E-mail: takenaka{at}tds1.tr.yamagata-u.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • rats • soy protein • paraquat • oxidative stress • isoflavone • saponin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and anticarcinogenic effects (2
), and to regulate polyunsaturated fatty acid metabolism (3
–5
). Because soy protein preparations contain nonprotein components such as soy isoflavones, saponins, phytic acid and dietary fibers, many attempts have been made to distinguish between the biological effects of soy protein and its associated nonprotein components. The amino acid composition of soy protein, specific soy peptides, soy isoflavones and soy saponins have been proposed as factors responsible for hypocholesterolemic effects of soy protein (6
).
In contrast to its hypocholesterolemic effects, the antioxidative activity of soy protein has been poorly investigated. Rats fed a 20% soy protein diet were found to have lower concentrations of plasma thiobarbituric acid-reactive substances (TBARS)3
than rats fed a 20% casein diet (7
). Soy protein intake has been reported to inhibit oxidative modification of LDL in vitro (8
), which was attributed to radical scavenging activity of genistein (9
). Furthermore, antioxidative and antiatherosclerotic effects of soy isoflavones without soy protein have been reported recently (10
). Although several soy isoflavones have been shown to have antioxidative activity, the antioxidative activity of soy protein itself has not been examined thoroughly.
Reactive oxygen species cause oxidative damage to biomolecules and thus result in various diseases. Considerable effort has been applied to identifying antioxidants in foods and the mechanisms behind their antioxidative activity in vivo. In the present study, we investigated whether soy protein isolates had antioxidative effects on paraquat-induced oxidative stress in rats. Paraquat (1,1'-dimethyl-4,4'-bipyridilium dichloride; PQ) was added to the experimental diets to induce oxidative stress. Paraquat is a widely used herbicide and causes severe injury to the lungs and other organs. It produces oxidative stress by redox cycling with a variety of cellular diaphorases and oxygen to produce superoxide radicals (11
). Among the nonprotein components of soy protein isolates, soy isoflavones and saponins have high potentials for antioxidative activity. Therefore, we also examined the effects of soy isoflavones and saponins and tried to distinguish between the effects of each factor.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Daidzin, genistin and daidzein were purchased from Fujicco (Kobe, Japan), and genistein was purchased from Wako Pure Chemical (Osaka, Japan). Soy protein isolate (SPI; Fujipro R) was kindly donated by Fuji Oil (Osaka, Japan). Other chemicals were of reagent grade and available commercially.
Determination of isoflavones and saponins in SPI.
SPI (500 mg) was extracted three times using 3 mL methanol at 68°C for 30 min and the volume was adjusted to 10 mL. Isoflavone (daidzin, genistin, daidzein and genistein) concentrations were determined by HPLC using the La Chrom system (Hitachi, Tokyo, Japan). Eluted compounds were detected by monitoring UV absorbance at 256 nm. Samples were loaded onto a Develosil ODS-HG-5 column (Nomura Chemical, Aichi, Japan) and eluted using a linear gradient of 0–100% solvent B (methanol) in solvent A (50 g/L methanol in 10 g/L acetic acid) for 60 min at a flow rate of 0.8 mL/min. Total saponin concentrations were determined as described previously using saponin from soybeans (Wako Pure Chemical) as the standard (12
).
Animals and diets.
The care and use of the rats followed the institutional guidelines of Yamagata University. Rats were housed individually in a stainless steel cage with a 12-h light:dark cycle (0600–1800 h) at a temperature of 22–24°C and relative humidity at 
55%. In Experiment 1, 4-wk-old male Wistar rats (Japan SLC, Hamamatsu, Japan) were used to investigate the antioxidative activity of dietary soy protein. They were allowed free access to water and a commercial diet for 3 d to allow acclimation to these conditions. The rats were then divided randomly into six groups. Rats in each experimental group were fed a control diet containing casein as a nitrogen source (CAS; n = 5), a control diet containing PQ (CAS + PQ; n = 6), a soy protein diet containing soy protein as a nitrogen source (SPI; n = 5), a soy protein diet containing PQ (SPI + PQ, n = 6), a control diet supplemented with isoflavonoids and saponins to the level in the SPI diet (CAS + IS, n = 5) and a CAS + IS diet containing PQ (CAS + IS + PQ; n = 6). The isoflavone and saponin concentrations of SPI and the compositions of the experimental diets are shown in Table 1
.
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In Experiment 2, we used 4-wk-old male Wistar rats (Japan SLC) to investigate the effects of soy protein on paraquat absorption. Rats were randomly divided into two groups and were given CAS or SPI diet from 1000 to 1800 h every day. At 1130 h each day, when more than half of the daily food intake had been consumed, 2.5 mg paraquat was administered orally as 12.5 g/L in 5 g/L sucrose solution. Feces and urine were collected for the last 2 d (between d 4 and 6 after initiation) of the experiments. Food and water were provided as described above but for 6 d only. On d 6, rats were anesthetized with Nembutal (Dainippon Pharmaceutical) 90 min after the paraquat solution was administered, and blood was collected by cardiac puncture. A section of the liver was excised and used for preparation of the subcellular fraction. The lung and other parts of the liver were quickly frozen in liquid N2 and stored at -80°C until use.
Hepatic enzymes, lipids and glutathione.
Liver cytosolic, mitochondrial and microsomal fractions were prepared as described by Del Boccio et al. (13
). Glutathione peroxidase (GSH-Px) activity (14
), Glutathione reductase (GSSG-R) activity (15
) and glutathione-S-transferase (GST) activity (16
) were measured as described previously with slight modification. Protein content was measured by the method of Lowry et al. (17
) using bovine serum albumin as a standard. Liver lipids were extracted and purified by the method of Folch et al. (18
). Quantitative determination of total and oxidized glutathiones was performed according to Tietze with some modification (19
).
Serum and liver TBARS concentrations.
The serum TBARS concentration was determined as described previously (20
) and expressed as µmol of malondialdehyde/L blood. The liver TBARS concentration was measured as described previously (21
). A 500-mg liver sample was homogenized in 10 volumes of 50 g/L trichloroacetic acid (TCA)/0.01 mol/L HCl, and centrifuged at 13,600 x g, for 15 min at 2°C. The supernatant was extracted by vigorously vortexing the sample five times with an equal volume of diethyl ether to remove TCA and then used for total glutathione analysis. For GSSG analysis, equal volumes of 0.04 mol/L N-ethyl-maleimide (NEM) were added to the samples used for total glutathione analysis and incubated for 1 h at 25°C. After five extractions with equal volumes of diethyl ether to remove NEM, samples were used for GSSG analysis. A 100 µL aliquot of the samples was added to 1.6 mL of 0.1 mol/L sodium phosphate/5 mmol/L EDTA (pH 7.5), 100 µL of 6 kU/L glutathione reductase, and 100 µL of 10 mmol/L 5,5'-dithiobis-(2-nitrobenzoic acid). After mixing well, 100 µL of 4 mmol/L NADPH were added, and the absorbance was measured at 412 nm.
Serum, fecal and urine PQ concentrations.
Extraction and analysis of paraquat were performed according to Tsunoda (22
). A 0.2 mL aliquot of 0.23 mol/L perchloric acid was added to 0.2 mL serum or 0.2 mL urine and six volumes of 0.23 mol/L perchloric acid was added to 50 mg feces. After overnight incubation at 4°C, samples were centrifuged at 1500 x g, for 10 min at 4°C. The pellet was resuspended in 0.23 mol/L perchloric acid, centrifuged again and the supernatants from each centrifugation were combined. After 0.5 volume of 1 mol/L NaOH was added to the supernatant and its pH adjusted to >13 with 1 mol/L NaOH, the solution was applied to a Sep-Pak C18 cartridge and washed three times with distilled water. Paraquat was eluted using 2 mL 0.1 mol/L HCl followed by 3 mL distilled water, and stored at -20°C until use.
Statistics.
Data are expressed as means ± SEM. The homogeneity of variance between treatment was verified by Bartlett’s test. In experiment 1, data were analyzed using two separate two-way ANOVA (IS x PQ and SPI x PQ vs. CAS). Differences between two groups in Experiment 2 were tested by student’s t test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The levels of daidzein, genistein, daidzin, genistin and saponins in SPI are shown in Table 1
. Isoflavones and saponins were almost entirely removed from SPI by the triple methanol extraction. Because the levels of isoflavones and saponins in the SPI were comparable to those described in previous reports (23
), these data were used to construct the experimental diets for the subsequent studies (CAS + IS and CAS + IS + PQ; as shown in Table 1
).
Food intake, body weight and tissue weight.
The food intake and the body weight of rats in the CAS + PQ group were lower after 7 and 9 d, respectively, than in the CAS group (data not shown). Isoflavone and saponin (IS) supplements did not alter the effect of PQ on food intake and body weight gain. On the other hand, the SPI diet mitigated the decline in body weight that was observed in response to PQ (Table 2
and Fig. 1
). Rats fed PQ-containing diets had lower relative liver weights. Relative lung weights were greater due to PQ in rats fed CAS and CAS + IS but not in those fed SPI. SPI tended to lessen (P = 0.076) the increase in lung relative weight induced by PQ whereas IS did not (Table 2
and Fig.1
). Intake of SPI without PQ reduced liver relative weight compared with the livers of rats fed the CAS or CAS + IS diets.
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Serum (mmol/L) and liver (nmol/g) TBARS concentrations were not affected by IS or SPI intake. Because liver lipid concentrations were lower in the CAS + PQ than in the CAS group (data not shown), the TBARS concentration normalized to lipid content was significantly higher in the CAS + PQ than in the CAS group. IS intake did not affect TBARS concentration in liver. Intake of SPI reduced liver lipid content (data not shown), which elevated the TBARS concentration normalized to lipid content in the liver. Liver TBARS (nmol/mg total lipid) was affected by the interaction of SPI intake and PQ treatment (P = 0.043). TBARS concentration in the lung, a target organ of paraquat, did not differ among the experimental groups.
GSH, GSSG, and total glutathione concentrations.
Liver GSSG concentration was higher in the CAS + PQ group than in the CAS group. PQ treatment and dietary SPI interacted to affect glutathione oxidation (GSH/GSSG ratio) (P = 0.035) (Table 3
).
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Lung GSH-Px activity was increased by IS intake by not by SPI intake (Table 3)
. Lung GSSG-R activity was increased by PQ, but hepatic GSSG-R was not affected by PQ. It was, however, increased by dietary IS (Table 3)
. Both IS and SPI intakes increased liver GST activity (Table 3)
.
Serum, urine and fecal PQ concentrations.
In Experiment 2, body weight loss in the CAS + PQ group was mitigated in the SPI + PQ group (Table 4
). Paraquat concentrations in serum and excretions did not differ between the groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–26
). Therefore, we conclude that SPI generally reduces paraquat-induced oxidative stress but soy isoflavones and saponins do not. Serum, liver and lung TBARS were measured as indicators of lipid peroxidation to estimate the level of oxidative stress. The hepatic TBARS concentration normalized to lipid concentrations was higher in paraquat-fed rats, which exhibited progressive lipid peroxidation in liver. TBARS levels were not affected by dietary soy isoflavones. On the other hand, SPI intake lowered triglyceride concentration in serum and liver (data not shown) and, as a result, elevated TBARS normalized to lipid content. The effect of SPI intake on paraquat-induced increase in hepatic TBARS/total lipid was significant (SPI x PQ, P = 0.043), indicating that SPI intake had prevented lipid peroxidation induced by paraquat.
SPI affected glutathione metabolism in rats. Paraquat intake elevated hepatic GSSG concentration in rats fed the casein diet, and these effects were attenuated in rats fed SPI (Table 3)
. This suggests that intake of SPI might increase the antioxidative capacity of the liver. The effects of soy isoflavones and SPI on the activity of glutathione metabolizing enzymes have also been reported by others (27
,28
). Appelt and Reicks reported that soy protein isolate and soy flour increased GST activity in rat livers (27
), as did soy isoflavone supplements (28
). Hepatic GST activity of rats fed paraquat was significantly increased by soy isoflavones and saponins, or SPI intake in the present study (CAS + IS + PQ, SPI + PQ compared with CAS + PQ). The radical-scavenging activities and the effect on the activity of glutathione-related enzymes of SPI may be factors that prevented GSH oxidation.
The antioxidative activity of soy protein itself can potentially be explained by its amino acid composition or the effects of soy peptides. L-Arginine, which reportedly has antioxidative activity (29
), is more abundant in soy protein than in casein. Suetsuna et al. (30
) investigated the antioxidative activity of soy protein hydrolysate and reported two soy peptides with strong antioxidative activity. Antioxidative activity of histidine-containing peptides from soybean protein has also been reported (31
). The in vivo antioxidative activities of soy peptides and soy amino acids against paraquat-induced oxidative stress are now under investigation in our laboratory.
In the present study, we also examined the effect of SPI on absorption of paraquat. In the experimental procedure, rats had access to the casein or SPI diet from 1000 to 1800 h and were trained to eat >50% of the daily food intake between 1000 and 1130 h. Therefore, paraquat, ingested orally at 1130 h, might be mixed with casein or SPI in the digestive tract of the rat. Because serum paraquat concentrations of the two groups did not differ, SPI did not affect the toxicity of paraquat by preventing its absorption from digestive organs or its entrance into the systemic circulation.
In conclusion, soy protein but not soy isoflavones and saponins tended to reduce paraquat-induced oxidative stress in rats, and the effects could not be explained by lower paraquat absorption from the digestive organs of SPI-fed rats. Therefore, the present study suggests that soy protein itself has antioxidative effects on paraquat-induced oxidative stress in rats, but this activity cannot be explained by the presence of isoflavones and saponins.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CAS, casein; GSH-Px, glutathione peroxidase; GSSG-R, glutathione reductase; GST, glutathione-S-transferase; IS, soy isoflavones and saponins; NEM, N-ethyl-maleimide; PQ, paraquat; SPI, soy protein isolate; TBARS, thiobarbituric acid-reactive substances; TCA, trichloroacetic acid.
Manuscript received 10 December 2001. Initial review completed 14 January 2002. Revision accepted 24 April 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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