To prepare the quercetin-rich supplementation capsules, yellow onion peels purchased from Nonghyup located in Changnyeong, were washed three times in tap water and extracted with a 60% aqueous ethanol solution (50℃, 3 h) in a extractor (1 kL, Hansung F&C Co., Ltd., Seoul, Korea). The extracts were filtered with a filter press (Hankook Industry Co. Ltd., Seoul, Korea), the filtrates were concentrated to 2.4° Brix in a vacuum concentrator (1 kL, Hansung F&C Co.), and then the concentrates were processed to powder with a freeze dryer (SFDTS-200 kg Samwon Industry Co., Ltd. Seoul, Korea). Finally, the powder was adjusted to contain 100 mg quercetin/g dextrin (Samyang Co., Seoul, Korea). Capsules containing of only dextrin were used for placebo group.

Total soluble flavonoid of the extract was determined with aluminium nitrate using quercetin as a standard [10]. A 0.1 g sample (OPE) was mixed with 40 mL of 90% ethanol and centrifuged at 3,000 rpm for 10 min. 100 mL of solution obtained by extracting the residue with 80 % ethanol was diluted 10 times. An aliquot of 0.5 ml was added to test tubes containing 0.1 mL of 10 % aluminium nitrate, 0.1 mL of 1 M potassium acetate, 1.5 mL of 80 % ethanol, and 2.8 mL water. The absorbance of the supernatant was measured at 415 nm after 40 min at room temperature. Total flavonoid concentration was calculated using quercetin as standard. Quercetin measurements were done using high-performance liquid chromatography (HPLC) determination as previously described [11]. In brief, the hydrolysis of all glycosides to quercetin aglycone, followed by HPLC determination from samples, was analyzed. A 0.1 g sample (OPE) was mixed with 40 mL of 60% aqueous ethanol and 5 mL of 6 N HCl. After refluxing at 95℃ for 2 hours, the hydrolyzed solution was filtered into a 100 mL flask with 60% aqueous ethanol. Approximately 10 mL of the solution filtered through a 0.45 µm filter before injection for HPLC analysis. Quercetin in OPE was quantified using a Hewlett-Packard 1100 series HPLC system (Hewlett-Packard, Palo Alto, CA, USA) with a ZORBAX C18 column (150 × 4.6 mm, 5 µm, XDB-C18; Hewlett-Packard). Elution was performed using a mobile phase made up of water-5% acetic acid-acetonitrile (40:30:30) at a flow rate of 1.0 mL/min. UV detector was measured at 370 nm, and the sample injection volume was 20 µL. Quantification was extrapolated from the pure quercetin (Sigma Chemical Co., St. Louis, MO) standard curve.

Twelve healthy normal-weight women, all university students, participated in a randomized, placebo-controlled, double-blinded crossover design. The inclusion criteria were healthy women in the age range of 20-25 years. All subjects were healthy, and those with documented type 2 diabetes mellitus, hypertension, thyroid disorders, malabsorption syndrome, or any type of coronary heart disease were excluded. Furthermore, subjects who were taking any medications known to influence the variables to be studied were excluded. The subjects were given written informed consent, and the institutional review board at Hannam University approved the study protocol (2008-05k). During the quercetin depletion period (day 7), all subjects were requested to restrict their consumption of quercetin to avoid any influence on the study results. These food ingredients included onions, apples, red wine, tea, biological and freshly pressed fruit juices, berries, grapes, cherries, raisins, parsley, broccoli, cabbage, beans, and tomatoes. Subsequently, the subject were randomized into two groups and administered either capsules of placebo or OPE per day capsules containing 100 mg quercetin and 128 mg other mixed flavonoids (composition unknown) in two 2-week study periods, separated by a 1-week washout peroid. The subjects were instructed to maintain their usual patterns of dietary intake during the study. Compliance was monitored through biweekly phone calls for capsule counts, and a nutritionist checked for changes in usual dietary patterns at the end of the study. Venous blood samples were collected by nurse from the forearm in EDTA-treated and plain tubes after a fasting period, both at baseline and at 2 weeks after the intervention.

Body mass index (BMI) was calculated as weight in kg divided by height in meters squared; waist circumference was also measured. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) was measured from the left arm in seated patients with an automatic blood pressure monitor (TM-2654, A & D, Tokyo, Japan) after a 20 min rest. Two measurements were taken at least 5 minutes apart, and the mean was used for analysis. Serum total cholesterol (TC), low-density lipoprotein (LDL-cholesterol), glutamate oxalacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), and high-density lipoprotein (HDL-cholesterol) were measured with commercially available kits (Choongwae, Seoul, Korea) using enzymatic methods. Serum triglyceride was analyzed using a total glycerol test kit (Roche, Basel, Switzerland). All measurements were performed on a BS-220 auto analyzer (FULL Auto chemistry Analyzer Mindray Bio-Medical Electronics Co. China). Fasting serum glucose (FSG) concentrations were measured by the glucose oxidase method using a Beckman glucose analyzer (Beckman Instruments, Irvine, CA, USA). Atherogenic index (AI) was calculated according to the following formula: (total cholesterol-HDL cholesterol)/HDL cholesterol.

Plasma obtained from heparinized blood samples was centifugated at 3,000 rpm for 15 min and the supernatant fraction was separated. Glutathione peroxidase activitiy (GSH-Px) was determined according to the method described by Beutler [12]. 10 µliters of erythrocytic hemolysate were added to 100 µL of 1 M Tris-Hcl-5 mM EDTA buffer (pH 8.0), 20 µL of 0.1 M glutathione, 100 µL of 10 U/ml glutathione reductase, and 100 µL of 2 mM NADPH, and filled with H₂O to a final volume of 1 mL. After incubating at 37℃ for 10 minutes, the reaction was initiated by the addition of 10 µL of t-butyl hydroperoxide, and the absorbance was measured at 340 nm. The reaction was run for 90 sec, and the loss of NADPH was monitored by the change in A₃₄₀ nm/min. Catalase (CAT) activity was measured according to the method developed by Aebi [13] 100 µL of erythrocytic hemolysate was dissolved in 50 mL of 50 mM phosphate buffer (pH 7), and 2 mL of the mixture was added to a cuvette. The reaction was initiated by the addition of 1 mL of 30 nM H₂O₂ at 20℃. The H₂O₂ decomposition rate was measured at 240 nm for 30 seconds using a spectrophotometer. Superoxide dismutase (SOD) activity was assayed in erythrocyte-suspension by the procedure of Marklund and Marklund [14]. To 500 µL of the hemolysate were added 3.5 mL of water, 1 mL of ethanol and 0.6 mL chloroform. After the centrifugation at 3,000 rpm for 10 min, various dilutions were prepared from the supernatant. 20 µL pyrogallol was added to each dilution after incubation at 37℃ for 10 min. The reaction was monitored spectrophotometrically at 320 nm for 2 min. The unit of the enzyme was defined as the amount which inhibits the autoxidation of pyrogallol by 50%. Plasma total radical trapping antioxidant potential (TRAP) was measured using a modification of the photometric method developed by Rice-Evans and Miller [15]. The method for measuring antioxidant activity is predicated on the antioxidant-induced inhibition of the absorbance of the radical cation of 2,2'-azinobis (3-ethylbenzothiazoline 6-sulfonate) (ABTS^•+). The ABTS^•+ radical cation is formed by the interaction of ABTS^•+ (150 µM) with the ferrylmyoglobin radical species, which is, in turn, generated by the activation of metmyoglobin (2.5 µM) by H₂O₂ (75 µM). Ten microliters of sample/buffer/Trolox-standard was added to tubes containing 400 µL of PBS buffer, 20 µL of metmyoglobin, and 400 µL of ABTS and mixed by vortexing. The reaction was initiated by the addition of 170 µL of H₂O₂. After 6 min of incubation, the absorbance was measured at 734 nm using a spectrophotometer. Values are expressed as trolox equivalent antioxidant capacity (TEAC) and defined as the millimolar concentration of the trolox antioxidant capacity of a calibration curve.

Plasma concentrations of retinol, carotenoids, lycopene, tocopherol and coenzyme Q₁₀ were determined simultaneously by RP-HPLC (Reversed Phase High Pressure Liquid Chromatography) according to the method of Jakob and Elmadfa [16]. Briefly, plasma proteins were precipitated with ethanol and lipids were extracted with n-hexane. After evaporation, dry residue was redissolved with 150 µL of methanol-dichloromethane (85:15, v/v) and mixed and then 100 µL of this solution was injected into a guard column (Merck LiChrospher 100 RP18 (10 µm), 250 × 4 mm). Samples were run at a flow rate of 1.0 ml/min. on a Dionex HPLC system (Summit™ HPLC, USA). Absorbance was monitored at 325 nm for retinol, at 295 for tocopherol, at 450 nm for carotenoids, lycopene and at 270 nm for coenzyme Q₁₀. Concentrations were calculated from areas under the curve using an external calibration curve. To measure plasma vitamin C, venous blood samples were centrifuged 15 min at 1,000 rpm and supernatant 500 µL was added to 2 mL of 0.75 mM metaphosphoric acid solutions. After vortex-mix, it was centrifuged again at 2,400 rpm for 10 min for protein precipitation. The supernatant 500 µL along with 400 µL dinitrophenylhydrazone-thiourea-copper sulphate reagent composed of 0.01 mM of 2,4-dinitrophenylhydrazine, 0.027 M of copper sulfate, and 0.66 M of thiourea allowed to stands for 3 h at 37℃. After cooling for 10 min at cold water, incubate for 30 min at room temperature with 2 mL of 12 M H₂SO₄ and then analyzed spectrophotometrically at 520 nm. Plasma vitamin C concentration was calculated from standard curve from ascorbic acid (0.014, 0.028, and 0.11 mM) and blank from that of metaphosphoric acid solution.

For DNA damage determination, the alkaline comet assay was conducted according to Singh et al. [17]. The isolated leukocytes duplicated from one subject were subjected to oxidative stress by suspension in PBS with 100 µM H₂O₂ for 5 min on ice. The leukocytes, were mixed with 75 µL of 0.7% low melting agarose (LMA), and added to the slides precoated with 0.5% agarose. The slide was then immersed in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, and 1% sodium laurylasarcosine, 1% Triton X-100 and 10% DMSO) for 1 hr at 4℃. The slides were next placed into an electrophoresis tank containing 300 mM NaOH and 10 mM Na₂EDTA (pH 13.0) for 40 min. For electrophoresis of the DNA, an electric current of 25 V/300 ± 3 mA was applied for 20 min at 4℃. The slides were washed three times with a neutralizing buffer (0.4 M Tris, pH 7.5) for 5 min at 4℃, and then treated with ethanol for another 5 min before staining with 50 µL of ethidium bromide (20 µg/mL). Measurements were made by image analysis (Komet 4.0; Kinetic Imaging, Liverpool, U.K.) and fluorescence microscope (DMLB; LEICA Wetzlar, Germany), determining tail length (50 cells from each of two replicate slides).

Data were analyzed using the SPSS 14.0 package for Windows. Values were expressed as mean ± SE unless stated otherwise. Statistical differences between groups and within groups were considered significant at P < 0.05 by Student's t test and paired t test. Categorical variables were analyzed by using the χ² test.

To assess the effects of onion peel extract supplementation on risk factors of atherosclerosis, 12 healthy normal-weight, female college students (mean BMI, 20.2 ± 0.5 kg/m²; mean percentage body fat, 28.6 ± 1.0%) participated in this study. No adverse effects of onion peel extract or placebo treatment were reported during the 2-week intervention period. On assessing body weight, percentage of body fat, and waist hip ratio, blood pressure, and the blood biochemical indices of liver function enzyme activities, no significant differences were found between the placebo and onion peel extract treatment (Tables 1 and 2).

After onion peel extract supplementation, the serum total cholesterol, LDL-cholesterol, and AI significantly reduced (P < 0.05), and these effects were not observed after placebo (dextrin) intake (Table 2 and Fig. 1).

Significantly increased erythrocyte SOD activity was found at the end of both treatment periods. Also, the plasma total antioxidant capacity and other markers of antioxidant status including erythrocyte enzyme activity, plasma lipid antioxidant vitamin (retinol, γ-tocopherol, α-tocopherol, lycopene, α-carotene, β-carotene, and coenzyme Q10) concentrations, and lipid peroxidation parameters did not significantly change after two weeks of supplementation with the placebo or onion peel extract (Tables 3 and 4). In addition, onion peel extract supplementation did not significantly change ex vivo H₂O₂-induced DNA damage, while tail length increased following placebo supplementation (P < 0.05, Table 5).