Assessment of red onion on antioxidant activity in rat

Food and Chemical Toxicology 50 (2012) 3912–3919

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Assessment of red onion on antioxidant activity in rat Bora Lee a,b, Ji-Hye Jung a, Hyun-Sook Kim c,⇑ a

Division of Biological Science, College of Science, Sookmyung Women’s University, Seoul 140-742, Republic of Korea Central Research Institute, Dr. Chung’s Food Co. Ltd., 1-25, Songjung-dong, Heungduk-gu, Chungjoo-si, Choongbuk-do 361-782, Republic of Korea c College of Human Ecology, Sookmyung Women’s University, Seoul 140-742, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 10 April 2012 Accepted 2 August 2012 Available online 10 August 2012 Keywords: Red onion Antioxidant activity Malondialdehyde Rat

a b s t r a c t Oxidative stress related to the aging process can increase the risk of degenerative disease. Red onions contain antioxidative compounds. This study was designed to investigate the effect of dietary red onion peel and/or flesh on antioxidative activity in rats. Twenty Sprague–Dawley male rats (18 weeks old) were divided into four groups. Each group was raised for 4 weeks on a red onion free control diet (ND), red onion diet containing 5% red onion peel (RP), 5% red onion flesh (RF), or 5% red onion peel + flesh (RPF). The results demonstrated that serum SOD activity was significantly increased in the RP and RPF groups, whereas glutathione peroxidase (GPx) activity was significantly higher in the RF group than in the ND group. Catalase activity and ORAC activity in liver showed upward tendency in the RP, RF, and RPF groups although the differences were not statistically significant. Liver malondialdehyde levels in the RPF group were significantly lower than those in the ND group were. In conclusion, red onion may enhance antioxidant defense mechanism through the induction of plasma SOD and GPx activities and inhibited liver lipid peroxidation. Therefore, red onion may exert important protective effects against oxidative stress related diseases. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.

1. Introduction The average life span of people has continued to increase as income levels climb and medical technology develops. According to the data on senior citizens announced by the Ministry of Health and Welfare, the percentage of people aged 65 years and over was greater than 10%, and this percentage is expected to reach approximately 20.8% in 2026. Thus, Korea already has an aging society that is expected to become a super-aged society in 2026 (National statistical office, 2009). During the course of aging, (Gariballa, 2004) the mobility rate of every type of chronic disease increases, and these increases are mostly influenced by dietary and nutritive conditions. Accordingly, improving the quality of life based on better nutrition is of the utmost importance, (Ahmed and Haboubi, 2010; Yim and Lee, 2004) and changes should be implemented before diseases occur due to aging. Bodily functions deteriorate when aging begins. Consequently, tissue damage accumulates gradually, or the amount of normal tissues and materials is reduced through intrinsic and extrinsic metabolic processes (Lebel et al., In press). Thus, tissue and physiological dysfunction occurs, and oxidative stress increases ⇑ Corresponding author. Address: Sookmyung Women’s University, Sunhun Building 307, Chungpa-Dong 2-Ka, Yongsan-Ku, Seoul, Republic of Korea. Tel.: +82 10 3182 0100; fax: +82 2 707 0195. E-mail address: [email protected] (H.-S. Kim).

(Blackwell et al., 2004). When oxidative stress increases and antioxidant systems in the body are weakened, DNA, lipids, and proteins including enzymes in endothelial cells are damaged (Kondo et al., 2009). Reactive oxygen species (ROS) are free radicals containing oxygen atoms that are produced naturally in the process of normal oxygen metabolism; ROS play important roles in cell signaling. However, ROS levels increase greatly during stress exposure and can result in damage to cellular structure, which can lead to diseases such as chronic heart disease, diabetes, inflammatory disease, and cancer (Mandal et al., In press). Types of ROS include superoxide anions (O2), hydroxyl radicals (OH), and hydrogen peroxide (H2O2). Superoxide dismutase (SOD) converts O2 to H2O2, which is then converted to H2O and O2 by catalase and glutathione peroxidase (GPx) (Kohen and Nyska, 2002). The enzymes comprise an antioxidant system that protects the body from oxidative stress (Lee and Wei, 2007). Flavonoids, which are found in polyphenols, are present abundantly in vegetables and fruits (Beecher, 2003). Flavonoids exert antioxidative (Giovannini et al., 2006), anti-inflammatory (Shapiro et al., 2007), anti-allergic, antifungal (Friedman, 2007), anti-platelet, anti-thrombotic, and anticancer effects (Fresco et al., 2006; Li et al., 2007). Quercetin (3,30 ,40 ,5.7-pentahydroxyflavone) is the prototypical flavonoid, and it is found primarily in broccoli, fruit, and onion (Allium cepa), particularly in the glycoside form (Scalbert and Williamson, 2000). Onion is an important food because it supplies various activated phytomolecules such as phenolic acid, flavonoids,

0278-6915/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.08.004

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copaenes, thiosulfinate, organosulfur compounds (OSCs), and anthocyanin (Slimestad et al., 2007). The phytochemical composition of onions is believed to vary according to species and cultivation technique. Among the species of onions, the red onion is abundant in polyphenols, flavonoids, flavonol, and tannin (Gorinstein et al., 2010). Some researchers reported that red onions had quercetin levels that were 14-fold that of garlic and levels that were twofold that of white onions (Gorinstein et al., 2008). Additionally, onion peel contains flavonoid levels that are 48-fold of that in its flesh, and cell-based investigations found that the peel has a greater capacity for controlling lipid peroxidation than the flesh does (Jang and Im, 2009). In a study of the antioxidant effects of onion flesh and peel, rats that consumed the peel had higher levels of antioxidants and lower levels of lipid peroxide than rats that ate the flesh did (Park and Kim, 2005; Park et al., 2007). Taking these results together, research on the physiological effects of red onion on disease prevention and on the differences in the levels of antioxidants between onion peel and onion flesh is warranted. Although many studies on the quercetin levels of onion and on disease prevention in relation to quercetin content have been conducted, the number of studies on the changes in physiological function caused by red onion consumption is insufficient; in particular, studies on the byproducts in onion peel are rare. Additionally, studies on food that can possibly control various changes caused by aging are also necessary. Accordingly, this study examined the antioxidant effects of onion peel and flesh, either consumed alone or in combination, on oxidant material generated during the beginning stages of aging. 2. Materials and methods 2.1. Evaluation of the antioxidant activity of red onion To compare the antioxidant activity of red onion with that of other vegetables, garlic, white onions, and red onions were purchased. The radical-scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) was determined according to the method described by Blois (1958). A 50 lL sample was incubated with DPPH solution for 10 min at room temperature, and DPPH activity was measured by reading the absorbance at 518 nm using a spectrophotometer. Oxygen radical antioxidant capacity (ORAC) was measured using the Rat ORAC Activity Assay kit (STA-345, Cell Biolabs, Inc. Arjons Drive, USA). All experiments were repeated three times, and the average value is reported.

Table 1 Composition of experimental diets. Ingredient

Group Experimental diet Red onion free control

Red onion peel

Red onion flesh

Red onion peel + flesh

Casein Corn starch Dyetrose Sucrose Cellulose Soybean oil TBHQ Mineral mixa Vitamin mixb

140 465.692 155 100 50 40 0.008 35

133 442.4074 147.25 95 47.5 38 0.0076 33.25

133 442.4074 147.25 95 47.5 38 0.0076 33.25

133 442.4074 147.25 95 47.5 38 0.0076 33.25

10

9.5

9.5

9.5

L-Cystine

1.8

1.71

1.71

1.71

Choline bitartrate Red onion peel Red onion flesh Total(g)

2.5

2.375

2.375

2.375

0

50

–

25

0

–

50

25

1000

1000

1000

1000

a

Mineral mix (AIN-93 M) (g/kg mixture): calcium carbonate 357, potassium phosphate monhydrate 250, potassium citrate monohydrate 28, sodium chloride 74, potassium sulfate 46.6, magnesium oxide 24.3, ferric citrate 6.06, zinc carbonate 1.65, cupric carbonate 0.63, potassium iodate 0.01, sodium selenate 0.0103, chromium potassium sulfate dodecahydrate 0.275, lithium chloride 0.0174, boric acid 0.0815, sodium fluoride 0.0635, nickel carbonate hudroxide tetrahydrate 0.0318, ammonium vanadate 0.0066, sucrose finely powdered (209.496) to make 1,000 g. b Vitamin mix (AIN-93 M) (g/kg mixture): niacin 3, calcium pantothenate 1.6, pyridoxine hci 0.7, thiamin HCI 0.6, riboflavin 0.6, folic acid 0.2, biotin 0.02, vitamin B12 (0.1%) 2.5, vitamin E DL-alpha tocopheryl acetate (500 iu) 15, vitamin A palmitate (500,000 iu/g) 0.2, vitamin K1 phylloquinone 0.075, sucrose finely powdered (974.705) to mate 1,000 g.

2.4. Measurement of hematological index The measurements of blood cell secretion and white blood cell (WBC) counts were executed using blood samples of rats in each group. Blood cell secretion and WBC counts were measured using a Coulter Counter and a Hemavet 850 (CDC, USA). The plasma anemia index was measured using a Hemavet 850 (CDC, USA).

2.2. Experimental animals and diets Animals used in this research were 18-week-old male Sprague–Dawley (SD) rats weighing 565 ± 54 g. The rats were bought from G-Bio Lab. Animal Inc., and they were housed in the laboratory at 22 ± 2 °C, 40–60% humidity, and with a 12h light/dark cycle with free access to solid feed and water. The rats were allowed to acclimate to the laboratory for 1 week before experimentation. The animals (n = 5 per group) were assigned into four dietary groups: normal diet (ND), normal diet with 5% red onion peel powder (RP), normal diet with 5% red onion flesh powder (RF), and normal diet with 5% red onion peel + flesh powder (RPF) for 4 weeks. The diets were a modification of AIN-93G (Table 1). Food intake and body weight were measured daily and weekly respectively. Red onions were purchased from Muan-gun, Jeollanam-do (Republic of Korea). The onion sample was divided into three parts: red onion peel, red onion flesh, and red onion peel + flesh. Afterward, samples were freeze-dried (Kobe freeze, 50 HP, Japan/Hyundai machine, 1.5 t, Korea) and disrupted (kyungchang machine, 10 kw, Korea) in a powder. All samples were stored at 20 °C until analysis. After an overnight fast, blood was collected by heart puncture, and serum was obtained by centrifuging the blood at 3000 rpm for 10 min at 4 °C. The liver, kidneys, heart, and lungs were removed, rinsed with distilled water, and then weighed. The plasma and organ samples were stored at 70 °C until analysis. All animal procedures were performed in accordance with the guidelines issued by Sookmyung Women’s University for the care and use of laboratory animals.

2.5. Measurement of antioxidative activity enzyme (SOD, GPx, catalase) Plasma was collected to determine SOD and GPx activity, the liver was homogenized in phosphate buffer solution (PBS) containing 1 mM EDTA, and the supernatants were then used to measure catalase activity. SOD, GPx, and catalase activities were measured using an SOD Assay Kit-WST (S311-10, Dojindo Molecular Technologies, Inc. Rockville, USA), a Glutathione Peroxidase Activity kit (900-158, Assay Designs, Inc., Ann Arbor, USA), and a Catalase Fluorometric Detection Kit (907-027, Assay Designs, Inc.), respectively. All experiments were repeated 3 times, and the average values are presented.

2.6. Measurement of malondialdehyde levels Liver lipid peroxide were measured using the OxiSelect™ TBARS assay kit (STA330, Cell Biolabs, Inc., San Diego, USA); all experiments were repeated three times, and the average values are presented.

2.7. Statistical analysis 2.3. Measurement of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) Plasma total AST and ALT levels were measured using an ELISA reader at 505 nm. Serum AST and ALT levels were measured using a BCS kit (Anyang-si, Gyeonggi-do, Korea). Serum AST and ALT levels are expressed as IU/mL of serum.

Statistical analysis was performed using SAS System ver. 9.0, and analysis of variance (ANOVA) was used for comparisons among all groups. All data are expressed as means ± S.D. unless otherwise noted. A probability level of 5% was considered significant. Differences between two groups were analyzed by the t-test. ANOVA was performed after Duncan’s multiple comparison test.

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Fig. 1. Comparison of DPPH radical-scavenging activity of garlic, white onion, red onion peel, red onion flesh, and red onion peel + flesh. abcDifferent letters within a column indicate significant differences (p < 0.05) from each other at a = 0.05 as determined by Duncan’s multiple range test (a > b > c). Statistical significance of differences was evaluated by Student’s t-test (⁄⁄ < 0.01) as compared with garlic.

3. Experiment results 3.1. Comparison of the antioxidant activity of garlic, white onion, red onion peel, red onion flesh, and red onion peel + flesh The antioxidant activity of red onion, garlic, and white onion was compared. Red onions were divided into peel, flesh, and peel + flesh. The data for DPPH radical-scavenging activity and ORAC values are shown in Figs. 1 and 2. First, red onion peel exhibited the highest DPPH radical-scavenging activity. In particular, red onion peel (p = 0.0018) and red onion peel + flesh (p = 0.002) exhibited significantly higher DPPH radical-scavenging activity than garlic. Red onion peel exhibited approximately six and fivefold higher DPPH radical-scavenging

activity than garlic and white onion, respectively. The ORAC values of white onion, red onion peel, red onion flesh, and red onion peel + flesh were significantly higher than that of garlic. In particular, red onion peel exhibited a significantly higher ORAC value than garlic (p = 0.0212). 3.2. Body weight gain, food intake, and caloric intake The body weight gain, food intake, and caloric intake for the 4 groups 4 weeks after treatment are listed in Table 2. The initial body weights, final body weights, and body weight gains were not significantly different among the groups. Additionally, the average daily food intake and caloric intake were also not significantly different among the groups.

Fig. 2. Comparison of ORAC value of garlic, white onion, red onion peel, red onion flesh, and red onion peel + flesh. abDifferent letters within a column indicate significant differences (p < 0.05) from each other at a = 0.05 as determined by Duncan’s multiple range test (a > b). Statistical significance of differences was evaluated by Student’s t-test (⁄ < 0.05) as compared with garlic.

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B. Lee et al. / Food and Chemical Toxicology 50 (2012) 3912–3919 Table 2 Body weight, food intake, and caloric intake of rat fed experimental diets. Initial weight (g) Red Red Red Red a

onion onion onion onion

free control peel flesh peel + flesh

573.0 ± 51.8 562.8 ± 54.5 559.3 ± 58.0 562.8 ± 55.1

a

Final weight (g)

Weight gain (g)

Food intake (g/day)

Caloric intake (kcal/day)

647.9 ± 59.1 625.7 ± 48.5 633.4 ± 62.6 640.7 ± 47.6

122.6 ± 43.0 122.7 ± 23.5 133.3 ± 27.6 149.7 ± 32.9

38.2 ± 3.8 41.2 ± 4.7 40.3 ± 4.1 40.4 ± 4.1

146.4 ± 14.6 156.8 ± 17.9 153.6 ± 15.7 153.7 ± 15.7

Values are presented mean ± S.D.

Table 3 Organ (liver, heart, kidney, lung) weights of rat fed experimental diets.

Red Red Red Red a

onion onion onion onion

free control peel flesh peel + flesh

Liver weight (g)

Heart weight (g)

Kidney weight (g)

Lung weight (g)

26.8 ± 5.1a 28.9 ± 3.7 28.4 ± 6.1 29.3 ± 1.9

1.9 ± 0.1 2.0 ± 0.2 2.1 ± 0.2 1.8 ± 0.1

3.7 ± 0.3 4.0 ± 0.5 3.9 ± 0.2 4.0 ± 0.5

2.3 ± 0.3 2.6 ± 0.3 2.5 ± 0.3 2.3 ± 0.3

Values are presented mean ± S.D.

3.3. Organ (liver, heart, kidney, lung) weight The organ weights of each group are stated in Table 3. Liver and kidney weights in the RP, RF, and RPF groups were higher compared with the ND groups, but there were no significant differences among the groups. Heart weight was highest in the RF group and lowest in the RPF group, but there were no significant differences among the groups. Lung weight was also not significantly different among the groups. 3.4. Effect of red onion on plasma AST and ALT levels In this study, plasma AST and ALT levels in the four groups were measured after the groups had consumed the experimental diets for 4 weeks Fig. 3. The ND group had higher AST and ALT levels than the other groups, but the differences were not significant. 3.5. Hematological index WBC, RBC, platelet counts, and hemoglobin and hematocrit levels are listed in Table 4. WBC and platelet counts were not significantly different among the groups and ranged 2.9–20.9 K/lL and

685–1436 K/lL, respectively. In addition, RBC levels in all four groups ranged 4.60–9.19 M/lL, although the values were significantly lower in the RF (p = 0.044) and RPF groups (p = 0.0364) than in the ND group. The RF group (p = 0.003) had significantly lower hemoglobin levels than the ND group, although the levels for all four groups ranged 10.0–26.7 g/dL. The RPF group (p = 0.001) had significantly lower hematocrit levels than the ND group, and the range of values for all four groups was 34.0–53.0%. 3.6. Effect of red onion on antioxidant enzyme (SOD, GPx, catalase) activity Antioxidant enzyme activity in each group is shown in Figs. 4(A) and (B). The SOD and GPx activities were measured in plasma while catalase activity was determined in liver. The SOD activity for all three experimental groups was significantly higher in the RP group. Additionally, SOD activity was significantly higher in the RP and RF groups than in the ND group (p = 0.0164 and 0.0012, respectively). GPx activity was significantly lower in the RP group than in the ND group (p < 0.001), whereas it was significantly higher in the RF group than in the ND group (p < 0.001). Catalase activity tended to be higher in the experimental groups than

Fig. 3. Effect of red onion on plasma AST, and ALT levels of rat fed experimental diets.

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Table 4 WBC, RBC, and Platelet counts in whole blood and Hemoglobin and Hematocrit levels in plasma of rat fed experimental diets. WBC level (K/lL) Red Red Red Red A B C

onion onion onion onion

free control peel flesh peel + flesh

A

11.6 ± 2.5 11.7 ± 3.6 9.0 ± 0.9 8.5 ± 1.8

RBC level (M/lL) aB

8.8 ± 0.5 8.3 ± 0.3ab 7.4 ± 0.6c⁄⁄C 8.0 ± 0.5bc⁄

Platelet level (K/lL) 667.2 ± 63.9 733.4 ± 123.4 717.8 ± 98.3 586.6 ± 290.2

Hemoglobin (g/dL) a

14.9 ± 0.5 14.6 ± 0.5ab 13.5 ± 0.5bc⁄⁄ 13.4 ± 1.5c

Hematocrit (%) 38.6 ± 1.5a 39.5 ± 3.6a 34.8 ± 3.3b 33.8 ± 1.5b⁄⁄

Values are presented mean ± S.D. Different letters (a–c) within a column indicate significant differences (p < 0.05) from each other at a = 0.05 as determined by Duncan’s multiple range test (a > b > c). Statistical significance of differences was evaluated by Student’s t-test (⁄ < 0.05, ⁄⁄ < 0.01) as compared with ND group.

Fig. 4(A). Effect of red onion on SOD and GPx activity in plasma of rat fed experimental diets. abcDifferent letters within a column indicate significant differences (p < 0.05) from each other at a = 0.05 as determined by Duncan’s multiple range test (a > b > c). Statistical significance of differences was evaluated by Student‘s t-test (⁄ < 0.05, ⁄⁄ < 0.01, ⁄⁄⁄ < 0.001) as compared with Red onion free control diet group.

Fig. 4(B). Effect of red onion on catalase and ORAC activity in liver of rat fed experimental diets.

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Fig. 5. Effect of red onion on Malondialdehyde level in liver of rat fed experimental diets. Statistical significance of differences was evaluated by Student‘s t-test (⁄ < 0.05) as compared with Red onion free control diet group.

in the ND group, but the differences were not statistically significant. 3.7. Effect of red onion on ORAC The results of ORAC are shown in Fig. 4(B). ORAC values of rat liver were higher in the experimental groups than in the ND group, but the differences were not significant. 3.8. Effect of red onion on malondialdehyde levels The effects of red onion on malondialdehyde levels are presented in Fig. 5. The RPF group exhibited significantly lower malondialdehyde levels (p = 0.0229) than the ND group. 4. Discussion Flavonoids are potential antioxidants that can remove oxidant material, and its main sources are vegetables and fruit. Among them, onion is abundant in bioactive materials such as flavonol and OSCs (Ali et al., 2000). According to previous studies on the analysis of quercetin content according to the part of the onion, onion peel contained more quercetin than onion flesh (Wiczkowski et al., 2008; Gennaro et al., 2002). Thus, this study measured the radical-scavenging capability of DPPH and ORAC to compare the antioxidant effects of garlic, white onion, red onion peel, red onion flesh, and red onion peel + flesh. The radical-scavenging capability of DPPH is a simple, sensitive, and widely used technique to measure the antioxidant activity of materials extracted from various materials and plants (Erdemoglu et al., 2009). The ORAC assay is a widely used method to measure the radical-donating capability of materials and is a basic, reliable method for measuring the antioxidant capability of food (Mhatre et al., 2009). In our experiments, the radical-scavenging capability of DPPH from red onion peel and red onion peel + flesh was significantly higher than that of garlic. Even in previous studies, the radical-scavenging activity of red onion was significantly higher than that of garlic and white onion under all conditions, including lyophilization, blanching, boiling, and baking (Jang and Im, 2009;

Gorinstein et al., 2008). Specifically, in the case of lyophilization, previous research has indicated that the radical-scavenging capacity of DPPH from red onion is approximately 1.2- and 1.7-fold higher than that of garlic and of white onion, respectively (Gorinstein et al., 2008). In the result obtained by measuring ORAC values, the ORAC value of red onion peel was significantly higher than that of garlic. According to a previous study, the ORAC of onion was higher than that of apple, tomato, pear, and peach (Proteggente et al., 2002). In the result obtained by measuring the ORAC values of chard, spinach, broccoli, carrot, onion, and celery, the fact that onion showed a high ORAC value next to spinach, broccoli, and chard in the fresh condition, and the highest ORAC value out of six kinds of vegetables in the lyophilized condition was in near agreement with that of previous studies (Ninfali and Bacchiocca, 2003). Taking these results together, the antioxidant capability of red onion was considered excellent compared to those of foods. Thus, this study divided animals into four diets groups (ND, RP, RF, and RPF) and examined the antioxidant activities in the rat after 4 weeks of following the diets. According to previous studies that investigated the level required to produce toxicity in quercetin and onion, it was reported that direct intake of a quercetin component of less than 0.25% and food-type intake, i.e., onion, of less than 6.4%, did not increase weight, sugar, liver weight and kidney weight. In this regard, this study set the quercetin concentration at 5% and conducted the experiments (Azuma et al., 2010). AST and ALT enzymes are used as indices of liver damage. Elevated AST and ALT levels may indicate degeneration, necrosis, and destruction of the liver due to cellular damage. In addition, these enzymes may be used as indices to assess whether experimental materials and diet treatments create toxicity (Bhardwaj et al., 2010). As differences in plasma AST and ALT levels were not observed in the experimental groups, the consumption of 5% onion did not appear to induce liver toxicity in rats. WBCs are a primary component of blood and protect the body from invading pathogens; conversely, RBCs transport oxygen and carbon dioxide to and from the lungs and tissues and control the electrolyte balance and viscosity of blood. Platelets are solid components that are important for blood coagulation. Hemoglobin is an important hemo-protein contained in RBCs that has various physiological

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functions such as the storage, transportation, and decomposition of oxygen, transportation of electrons, and the catalysis of redox reactions (Wijayanti et al., 2004). Hemoglobin should be maintained at proper levels to maintain the proper oxygen balance in tissues. Hematocrit is an index of the volume of RBCs in the blood that is used as an anemia index in combination with hemoglobin. In this study, no significant differences were found in RBC and platelet counts among the four groups. RBC counts were significantly lower in the RF and RPF groups than in the ND group, but the values were within the normal range. The hemoglobin level of the RF group was significantly lower than that of the ND group, and the hematocrit level of the RPF group was significantly lower than that of the ND group; in both cases, the values were within the normal ranges. Previous research indicated that the consumption of onions or onion byproducts result in reduced RBC counts and lower hemoglobin levels due to oxidative damage in RBCs, hemolytic anemia, and the formation of Heinz bodies (Ostrowska et al., 2004; Yamamoto et al., 2005; Roldán-Marín et al., 2009). This phenomenon occurred because oxides such as H2O2 were formed when disulfides were formed; as a result, hemoglobin and glutathione S-transferase (GST) were present in intact RBCs in rats that consumed onions, and OSCs, a bioactive components in onion, might exhibit toxic effects in rats, but not in humans (Munday et al., 2003). In experiments with dogs as well as rats, although RBC counts and hemoglobin and hematocrit levels significantly decreased on the third day after consuming onion, their values gradually increased up until day (Ali et al., 2000; Tang et al., 2008). Taking these results together, the reduced RBC counts and hemoglobin and hematocrit levels were due to the formation of sulfur compounds at the time of onion consumption, which resulted in acute anemia. However, the RBC counts and hemoglobin and hematocrit levels in all four groups were within the normal ranges, and consuming red onion at a concentration of 5% was expected to cause hemolytic anemia. In addition, the levels of these indices obtained after consuming onion for 4 weeks decreased in this study, but these values were expected to increase to the normal range during long-term onion consumption. Oxidative stress is caused by ROS such as O2, H2O2, and OH, and it causes changes in physiological systems in the body (Umakoshi et al., 2011). ROS are converted to nontoxic substances by SOD, GPx, and catalase. SOD converts O2 to H2O2, which then detoxified to H2O by GPx and catalase (Bang et al., 2009). In this study, SOD activity was higher in all three experimental groups than in the ND group, and GPx activity was significantly higher in the RF group. In general, higher SOD activity in experimental groups might offer some protective effects against generation of free radicals, thus reducing the oxidative stress by enhancing the conversion of superoxide radicals to H2O2, followed by deactivation of H2O2 by GPx in RF group (Heistad et al., 2009; Shakirin et al., 2012). As shown in Fig. 4(A), the GPx activity decreased in RP group unexpectedly in this study. Eventhough it was not easy to explain the possible causes of this phenomenon, we explain that the tendency of increment of catalase and ORAC activity (Fig. 4(B)) and slight decrement of liver MDA level (Fig. 5) could not be necessary implication of increased oxidative stress with decreased GPx activity. Catalase activity tended to be higher in the experimental groups than in the ND group. In a previous study, injecting 7% onion for 5 weeks into diabetic rats significantly increased the activities of kidney GPx, glutathione reductase, and GST (Bang et al., 2009). Similarly, SOD and GPx activity were increased when onion juice was injected into rats with selenite-induced cataract (Javadzadeh et al., 2009). Catalase activity was found to be higher in the neonatal rat than in the adult (Hamby-Mason et al., 1997 Sep). In contrast, GPx was shown to undergo a 70% increase in activity in rat between birth and adulthood (Baud et al., 2004). In cancer study, p53-induced upregulation SOD and GPx, but not catalase, increases

oxidative stress and apoptosis (Hussain et al., 2004). Both GPx and catalase detoxify H2O2 into H2O, but it is considered that the mechanism of GPx is different from the mechanism of catalase. According to this paper, increased GPx was influenced by red onion flesh and GPx may be more influential when H2O2 is detoxified into H2O. ORAC is a method of measuring antioxidant capability, which protects against an attack by peroxyl radicals (Ninfali et al., 2002; Zou et al., 2011). In this study, ORAC tended to be higher in the experimental groups than in the ND group. In a previous study that investigated the effect of the skin of pomegranate, a fruit abundant in flavonoids similarly to onion, on the antioxidant activity of rats, plasma ORAC values increased significantly and malondialdehyde levels decreased significantly after the consumption of pomegranate skin. Malondialdehyde is the last product produced from lipid peroxidation created by ROS-induced damage to the cellular membrane, and malondialdehyde levels are an important index of lipid peroxidation. However, a previous report suggested that increased levels of the antioxidant glutathione can have a favorable effect on controlling the production of malondialdehyde (Karaoz et al., 2002). In this study, the malondialdehyde level of the RPF group was significantly lower than that of the ND group. This result was in line with previous research indicating that onion extract lowered malondialdehyde levels in the testes of rats with cadmium toxicity and that malondialdehyde levels decreased significantly in rats with aflatoxin toxicity after the simultaneous consumption of garlic, onions, and cabbage (Ola-Mudathir et al., 2008; Abdel-Wahhab and Aly, 2003). Moreover, red onion was demonstrated to exhibit significantly greater antioxidant capability than garlic and white onion. In addition, rats consuming red onion had elevated SOD and GPx activities, enhanced ORAC, and reduced malondialdehyde levels. However, RBC counts and hemoglobin and hematocrit levels decreased in rats consuming red onion because of the sulfur compounds present in onion. In summary, red onion improves the antioxidant capability of rats, suggesting its great value as an antioxidant food to control lipid peroxidation. Specifically, if we can develop an antioxidant food to prevent aging in the beginning stage of aging using red onion, this food is expected to prevent diseases caused by aging.

5. Summary We compared the antioxidant capabilities of garlic, white onion, and red onion. The radical-scavenging capability of red onion peel, and red onion peel + flesh was significantly higher than that of garlic, and the ORAC of red onion peel was significantly higher than that of garlic. Based on this result, we selected red onion to perform an in vivo experiment using rats. Rats were divided into four groups, and the experimental groups consumed red onion for 4 weeks. AST and ALT levels were similar in all four groups after the 4-weeks period. Regarding RBC counts, the RP and RPF groups exhibited lower RBC counts than the ND group. Hemoglobin levels were decreased in the RF group, and hematocrit levels were decreased in the RPF group, although the levels in both cases were within the normal ranges. SOD activity was significantly increased in all groups consuming red onion, and GPx activity was significantly decreased RP group compared to that in the control group; however, GPx activity was significantly higher in the groups consuming red onion flesh. Both catalase activity and ORAC tended to increase in the experimental groups compared to that in the control groups, and malondialdehyde levels were significantly lower in the RPF group than in the ND group. According to these results, the consumption of red onion improved antioxidant enzyme – plasma SOD and GPx – activity and decreased lipid peroxidation (liver MDA). Accordingly, we expect to develop an antioxidant food based on red onion.

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