Antioxidant Effects of Spinach (Spinacia oleracea L.) Supplementation in Hyperlipidemic Rats

Sample preparation

Spinach, in dried powder form, was obtained from the Spinach Cluster Agency (Jeonnam, Korea) after being cultivated in Shinan Island, Jeonnam, Korea. For preparation of the hot water extracts (WE), 5 g of spinach powder was put in a glass bottle with 100 mL of deionized water, extracted with an autoclave, and freeze dried (Ilshin, Yangju, Korea). The ethanol extracts (EE) were prepared as follows: 5 g of spinach powder was extracted with 100 mL of ethyl alcohol and concentrated by evaporator (N-1110S-W, EYELA, Tokyo, Japan). WE and EE were redissolved in dimethyl sulfoxide to a concentration of 50 mg/mL and aliquots were kept at 20°C until used.

Measurement of total phenolic contents

Total polyphenol content (TPC) was measured by the method of Park et al. (15). Briefly, WE was mixed with 2 mL of 1 N Folin-Ciocalteu reagent and incubated at room temperature. Then 2 mL of 10% Na₂CO₃ were added and the mixture was incubated at room temperature. The absorbance was measured at 690 nm with an enzyme-linked immunosorbent assay (ELISA) reader (Tecan, Grödig, Salzburg, Austria). TPC was expressed as gallic acid equivalents (GAE).

Oxygen radical absorbance capacity assay

The oxygen radical absorbance capacity (ORAC) assay was carried out on a FLUOstar OPTIMA fluorescence plate reader (BMG LABTECH Gmbh, Ortenberg, Germany) with fluorescent filters (excitation wavelength 485 nm, emission wavelength 535 nm) according to the method of Park et al. (15). The results were calculated based on the difference in the area under the fluorescence decay curves between the blank and each sample. ORAC ROO· was expressed as μmol of Trolox equivalents (TE).

Cellular antioxidant activity assay

HepG2 cells were seeded at a density of 5×10⁴ cells/mL on 96-well microplates. Twenty-four hours after seeding, the growth medium was removed, and the wells were washed with phosphate buffered saline (PBS). Triplicate wells were treated for 1 h with 100 μL of extracts dissolved in treatment medium. Wells were washed with 100 μL of PBS, and 80 μM of AAPH in 100 μL of PBS plus 40 μM DCFH-DA was applied to the cells. Following the addition of the AAPH solution, the 96-well microplate was placed into a FLUOstar OPTIMA fluorescence plate reader at 37.8°C. Emission at 535 nm was measured with excitation at 485 nm. Triplicate control and blank wells were included on each plate: control wells contained cells treated with DCFH-DA and an oxidant, AAPH; blank wells contained cells treated with PBS without AAPH.

DNA damage determination by alkaline comet assay

The alkaline comet assays were conducted by the modified method (16) described by Singh et al. (17). For the measurement of antigenotoxic activity of spinach in vitro, human leukocytes were incubated for 30 min at 37°C with varying concentrations of WE or EE dissolved in DMSO (1, 10, 50, and 100 μg/mL). Following incubaton, leukocytes were resuspended in PBS with 200 μM H₂O₂, incubated on ice for 5 min, and washed with PBS. The slides were then immersed in a lysis solution and incubated for 1 h at 4°C. Next, the slides were equilibrated in electrophoresis buffer for 40 min. For electrophoresis of the DNA, an electric current of 25 V/300±3 mA was applied for 20 min at 4°C. The slides were washed three times with a neutralizing buffer and treated with ethanol for another 5 minutes before staining with 20 μL of ethidium bromide (20 μg/mL). A fluorescence microscope (Leica DMLB, Leica, Solms, Germany) was used to image the slides and Komet 4.0 software (Kinetic Imaging Ltd., Merseyside, UK) was used to analyze each image and determine the percent fluorescence in the tail (tail intensity [TI]; 50 cells from each of two replicate slides).

Animals and diets

All aspects of the in vivo experiment were conducted according to the guidelines provided by the Ethical Committee for Experimental Animal Care of Kyungnam University. Eight-week-old male Sprague-Dawley rats (SD, n=24) were purchased from KOATEC, Inc. (Pyeongtaek, Korea) and housed individually in a room with a 12-h light/12-h dark cycle, a temperature of 22~24°C, and a relative humidity of 50±5%. The rats were allowed free access to water and fed a commercially prepared diet for a 1 wk adjustment period. The rats were then randomly divided into three groups of eight animals each and fed an AIN-93 based normal diet (ND), a high fat-cholesterol diet (HFCD), or a HFCD supplemented with 5% freeze- dried spinach powder (HFCD+S). The HFCD contained 18% casein, 50% corn starch, 10% sucrose, 6.5% cellulose, 3.5% mineral mixture (AIN-93G), 1% vitamin mixture (AIN-93G), 0.2% choline bitartarate, 0.3% DL-methionine, 0.5% cholesterol, and 9.5% fat. Animals were monitored daily for general health. At the end of the experimental period, the rats were anesthetized with iso-flurane and blood was collected from the abdominal artery into a heparinized sterile tube. The plasma fraction was obtained from the blood samples by centrifugation at 450 g for 30 min and stored at −80°C until required for further analysis. The experiment was approved by Animal Care Committee, Kyungnam University, Gyeongnam, Korea.

Blood and hepatic lipid profiles

Plasma lipid profiles (i.e., total cholesterol, high-density lipoprotein [HDL]-cholesterol, triglycerides) were measured using assay kits from Bioclinical Systems (Anyang, Korea) and a photometric autoanalyzer (CH-100 plus; SEAC, Calenzano, Italy). Plasma low-density lipoprotein (LDL) cholesterol concentrations were calculated using the formula developed by Friedewald et al. (18). The concentration of total lipids in liver samples was determined using the method of Folch et al. (19). Total cholesterol (TC) and triglyceride concentrations in liver samples were analyzed with the same enzymatic kits used in the plasma analyses.

Baseline conjugated dienes in LDLs

The concentration of conjugated dienes in plasma was determined according to the method of Park et al. (16). Briefly, plasma was added to 700 μL of heparin citrate buffer and incubated for 10 min at room temperature. After centrifugation at 1,000 g for 10 min, the pellet was resuspended in 100 μL of 0.1 M Na-phosphate buffer containing 0.9% NaCl (pH 7.4). Lipids were extracted from 100 μL of the LDL suspension with chloroform/methanol (2:1), dried under nitrogen, and redissolved in cyclohexane. A spectrophotometer (UV-1205, Shimadzu, Tokyo, Japan) was used to determine the absorbance of the redissolved sample at 234 nm.

Hepatic thiobarbituric acid reactive substance (TBARS)

The thiobarbituric acid reactive substances (TBARS) level in plasma was estimated by the method of Buege and Aust (20). Briefly, 0.2 mL of plasma was mixed with 0.3 mL of distilled water and added to 1 mL of a mixture containing 0.38% thiobarbituric acid, 15% trichloroacetic acid, and 0.25 N HCl. The resulting mixture was incubated for 20 min in a boiling water bath prior to centrifugation at 2,000 g for 10 min. The absorbance of the supernatant was measured at 540 nm using 1,1,3,3- tetramethoxypropane (TMP) as standard. Lipid peroxidation was expressed as TBARS in nmol/mL plasma.

Liver lipid peroxidation levels were measured by the method of Ohkawa et al. (21) with some modification. Liver homogenates (0.4 mL) in 1.15% KCl, 0.1 mL of 8.1% SDS, 0.75 mL of 20% acetic acid, and 0.75 mL of 0.8% TBA were added to a test tube. The sample was vortexed and heated in a 95°C oil bath (OHB-2000, Tokyo Rikakikai Co., Tokyo, Japan) for 1 h. After cooling for 10 min, butanol-pyridine 15:1 (v/v) was added. The sample was mixed thoroughly and centrifuged at 3,515 g for 15 min. The fluorescence of the upper layer was measured at 552 nm. The amount of MDA present in the sample was converted to TBARS values using a TMP standard curve. A Pierce BCA protein assay kit (Thermo Scientific, Aalst, Belgium) was used to quantify the protein concentration of each liver sample.

Erythrocytic catalase

Erythrocytic hemolysates were prepared by the dilution of erythrocytes to 1:500 with distilled H₂O. One hundred microliter of erythrocytic hemolysate was dissolved in 50 mM phosphate buffer 50 mL (pH 7), and 2 mL of the mixture was added to a cuvette. The reaction was initiated by the addition of 1 mL of H₂O₂ 30 nM at 20°C. The H₂O₂ decomposition rate was measured at 240 nm for 30 sec using a spectrophotometer.

Hepatic antioxidant enzymes

To determine liver superoxide dismutase activity, liver samples were homogenized with 0.1 mL of 65 mM phosphate buffer (pH 7.8), and then centrifuged at 10,000 g for 20 min. Copper, zinc-superoxide dismutase (Cu/Zn-SOD) activity was measured in the resulting supernatant (i.e., the cytosolic fraction). The remaining pellet (i.e., the mitochondrial fraction) was dissolved in 0.1% triton and used for the determination of manganese-superoxide dismutase (Mn-SOD) activity. For the Mn-SOD activity assay, 4 mmol of KCN solution was added to the assay mixture to inhibit Cu/Zn-SOD. For Mn-SOD and Cu/Zn-SOD analyses, the samples were pre-incubated with 75 mM Na-xanthine and 10 mM hydroxylamine hydrochloride at 37°C for 10 min. Then 0.1 units of xanthine oxidase was added and samples were incubated at 37°C for an additional 20 min. The reaction was stopped by the addition of 1% sulphanilamide and 0.02% ethylenediamine dihydrochloride. After standing at room temperature for 20 min, the absorbance of the final mixture was measured at 540 nm. One nitrate unit (NU) of SOD activity was defined as the amount of protein required for 50% inhibition.

Catalase (CAT) activity was measured according to the method of Carrillo et al. (22). Liver tissue was homogenized in Na-K phosphate buffer (pH 7.0). The homogenates were centrifuged at 600 g for 10 min to obtain the supernatant. After centrifuging at 10,000 g for 20 min, the supernatant was discarded and the pellet was suspended in 1× RBC buffer. After incubating on ice for 10 min, the suspension was centrifuged at 10,000 g for 20 min. The pellet was washed in Na-K phosphate buffer (pH 7.0) and centrifugation was repeated. After washing, the pellet was resuspended in Na-K phosphate buffer. For the assay, Na-K phosphate buffer and sample were mixed in a quartz cuvette (QS, Hellma GmbH & Co., Müllheim, Germany) and the reaction was started by the addition of 300 μL of 30 mM H₂O₂ solution. The H₂O₂ decomposition rate was measured at 240 nm for 40 sec using a spectrophotometer.

Glutathione peroxidase (GSH-Px) activity was measured according to the method of Bogdanska et al. (23). Liver tissue was homogenized with 1 mL of 250 mM potassium phosphate buffer (pH 7.0), and then centrifuged at 10,000 g for 20 min. Twenty-five μL of supernatant, containing cytosolic fraction, was incubated with 10 mM EDTA, 10 mM NaN₃, 10 mM GSH, 2 mM NADPH, and 1 unit glutathione reductase at room temperature for 5 min. The reaction was initiated by the addition of 25 μL of 2.5 mM H₂O₂. The H₂O₂ decomposition rate was measured at 340 nm for 70 sec using a spectrophotometer.

The protein concentration of each supernatant was determined by BCA protein assay. Samples containing equal amounts of protein (25 μg) were used for the enzyme activity assays.

DNA damage in rat leukocytes

Twenty μL of whole blood were suspended with 150 μL of 0.7% low melting agarose (LMA), and added to slides that had been pre-coated with agarose. The slides were then treated in PBS with 200 μM H₂O₂ for 5 min on ice and washed with PBS. The following steps were the same as done in the in vitro DNA damage determination by comet assay.

Statistical analysis

All measurements were analyzed using the SPSS package for Windows (Ver. 14.0; SPSS, Chicago, IL, USA). Mean values among concentrations/animal groups were compared using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range tests (P<0.05).

Total polyphenol contents, antioxidant, and antigenotoxic effects of spinach

The TPC of WE and EE were 1.5±0.0 mg GAE/g and 0.5±0.0 mg GAE/g, respectively. The ORAC values of WE and EE increased in a concentration dependent manner (Fig. 1A). At the highest tested concentration (50 μg/mL), WE (7.6±0.1 TE) and EE (7.2±0.1 TE) showed similar ability to protect a fluorescent reporter from oxidative degeneration by AAPH. The viability of spinach-extract-treated HepG2 cells was 70% at the highest concentration tested (50 μg/mL, data not shown). These extracts reduced AAPH-induced oxidative stress in HepG2 cells (Fig. 1B), with higher concentrations producing greater increases in CAA. Pretreatment of the human leukocytes with spinach extracts for 30 min significantly reduced H₂O₂-induced oxidative stress (Fig. 1C), with higher concentrations showing increased ability to inhibit DNA damage.

Effects of spinach on blood and hepatic lipid profiles

In the in vivo studies, no adverse reactions or clinical signs were observed in HFCD rats throughout the feeding period. As shown in Table 1, there was a marked increase in the levels of TC and LDL-C in the HFCD group compared with the ND group. Plasma HDL-C decreased significantly in the HFCD group compared to the ND group. Plasma TC, HDL-C, and LDL-C levels did not differ between HFCD and HFCD+S rats. Additionally, while hepatic total lipid, TC, and triglyceride levels were significantly higher in the HFCD rats than in the ND rats, these concentrations did not differ between HFCD and HFCD+S groups.

Effects of spinach on antioxidant status

Liver TBARS levels were 79% higher in rats in the HFCD group versus the ND group, as shown in Fig. 2. Liver TBARS levels in rats that received the HFCD+S were not different from those of rats that received the ND. There was no difference in plasma TBARS level among treatment groups.

Plasma conjugated diene concentrations were significantly higher in the HFCD group than in the ND group (Table 2). Plasma conjugated diene concentrations were decreased in the HFCD+S group compared to the HFCD group; however, this difference was not statistically significant.

The activity of Mn-SOD was significantly lower in the livers of rats in the HFCD group than in the livers of rats in the ND group, as shown in Table 2. Liver Mn-SOD activity was greater in the HFCD+S group than in the HFCD group; however, this difference was not statistically significant. There was no difference in plasma Cu/Zn-SOD activity among treatment groups.

Tail DNA percentage was greater in the HFCD group than in the ND group, while tail DNA percentage in the HFCD+S group was not different from that of the ND group (Fig. 3).