Inhibitory Effects of North American Wild Rice on Monocyte Adhesion

Sep 25, 2017 - The present study examined the effects of wild rice on monocyte adhesion, inflammatory and fibrinolytic mediators in low-density lipopr...
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Inhibitory Effects of North American Wild Rice on Monocyte Adhesion and Inflammatory Modulators in LDL Receptor-Knockout Mice Mohammed H. Moghadasian, Ruozhi Zhao, Nora Ghazzawi, Khuong Le, Franklin B. Apea-Bah, Trust Beta, and Garry Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03216 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 26, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

Jf-2017-032162 R2

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Inhibitory Effects of North American Wild Rice on Monocyte Adhesion and

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Inflammatory Modulators in LDL Receptor-Knockout Mice

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Mohammed H. Moghadasian, Ruozhi Zhao, Nora Ghazawwi,

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Khuong Le, Franklin B. Apea-Bah, Trust Beta and Garry X. Shen*

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Departments of Food and Human Nutritional Sciences1, and Internal Medicine2,

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University of Manitoba

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Corresponding author:

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Garry X. Shen MD PhD

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Diabetes Research Group

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University of Manitoba

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835-715 McDermot Ave

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Winnipeg, MB R3E 3P4

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Canada

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Tel: 204-789-3816

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Fax: 204-789-3987

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Email: [email protected]

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Running title: Wild rice reduced vascular inflammation

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Abstract

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The present study examined the effects of wild rice on monocyte adhesion, inflammatory and

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fibrinolytic mediators in low-density lipoprotein receptor-knockout (LDLr-KO) mice. Male

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LDLr-KO mice received cholesterol (0.06%, w/w) supplemented diet with or without white rice

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or wild rice (60%, w/w) for 20 weeks. White rice significantly increased monocyte adhesion,

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abundances of monocyte chemoattractant protein-1, tissue necrosis factor-α, intracellular cell

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adhesion molecule-1, plasminogen activator inhibitor-1, urokinase plasminogen activator (uPA),

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and uPA receptor in aortae and hearts of LDLr-KO mice compared to control diet. Wild rice

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inhibited monocyte adhesion to aorta, atherosclerosis and the abundances of the inflammatory

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and fibrinolytic regulators in the cardiovascular tissue of LDLr-KO mice compared to white rice.

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White or wild rice did not significantly alter the levels of cholesterol, triglycerides, or antioxidant

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enzymes in plasma. The anti-atherosclerotic effect of wild rice may result from its inhibition on

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monocyte adhesion and inflammatory modulators in LDLr-KO mice.

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Journal of Agricultural and Food Chemistry

Introduction

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Cholesterol-rich atherosclerotic plaques in arterial wall are the underlying pathology of the major

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cardiovascular diseases in adults. 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A reductase

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inhibitors or statins are routinely prescribed to decrease total and low-density lipoprotein (LDL)

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cholesterol levels in blood and reduced cardiovascular events.1 However, atherosclerotic

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cardiovascular diseases still remain as a major cause of mortality and morbidity worldwide.2, 3

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Atherosclerotic coronary artery disease was detected in patients with normocholesterolemia.4

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Those facts suggest that atherosclerotic cardiovascular disease may result from additional

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mechanisms, and cholesterol lowering medication alone may not be sufficient to prevent

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atherosclerotic cardiovascular diseases. Accumulate line of evidence suggest that vascular

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inflammation plays a critical role in the initiation of atherosclerosis.5 Anti-inflammatory

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medications potentially improve cardiovascular outcome, but the long-term safety of those

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medications remains to be determined.6 Monocyte adhesion is a key event of vascular

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inflammation and atherosclerosis. Inflammatory biomarkers, such as tumor necrosis factor-α

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(TNFα) and monocyte chemotactic protein-1 (MCP-1), play a crucial role in development of

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atherosclerotic lesions.7, 8 In the other hand, antioxidant agents including dietary antioxidants

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may attenuate lipoprotein oxidation, foam cell formation and atherosclerotic lesion

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development.9 Investigation on the effect of natural products on vascular inflammation in

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atherosclerotic animal models may help to determine the mechanism of the healthy benefits of

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the functional foods.

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Wild rice is a genus of grasses (Zizania), but not a type of rice. Several varieties of wild rice

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grow in nature. They include Zizania palustric L., Zizania Latifolia, Zizania aquatica) or Zizania 3 ACS Paragon Plus Environment

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Texana.10 North American wild rice (Zizania palustric L., presented as wild rice in subsequent

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sections) was a traditional food of Indigenous people in North America. Wild rice has been

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considered as healthy food and is currently available in grocery stores in North America. Wild

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rice has chewy dark outer layer and soft inner grain after cooking, and has been used as a

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component of salad or steamed mixed grains. We have recently summarized available

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information on biological properties of wild rice including nutritional values and various species

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of wild rice elsewhere.11

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Previous studies demonstrated that Asian wild rice has cholesterol lowering and antioxidant

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effects in rats.12 Recent studies by our group demonstrated that wild rice reduced atherosclerosis,

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plasma cholesterol and lipoproteins in LDL receptor-knockout (LDLr-KO) mice. In female

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LDLr-KO mice, the reduction of total, LDL and very low-density lipoprotein (VLDL)

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cholesterol by wild rice were 40%, 42% and 75% respectively, compared to control diet. In male

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LDLr-KO mice, the reduction of total, LDL and VLDL cholesterol by wild rice were relatively

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lower (15%, 12% and 35%) compared to female counterparts. However, the reduction of

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atherosclerosis in male LDLr-KO mice by wild rice was 71% compared to 61% in female LDLr-

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KO mice.13 The findings suggest the anti-atherosclerotic effect of wild rice in LDLr-KO mice is

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not precisely parallel to its cholesterol lowering effects in LDLr-KO mice. The mechanism for

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the anti-atherogenic effect of wild rice in male LDLr-KO mice may not be the same as that in

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female LDlr-KO mice.

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The present study examined the effects of wild rice on monocyte adhesion and relevant

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inflammatory or fibrinolytic mediators in cardiovascular tissue in addition to atherosclerotic

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lesion, lipid profile and the activities of antioxidant enzymes in male LDLr-KO mice.

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Materials and Methods

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Animals

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LDLr-KO mice (male, 6 weeks of age, average body weight 19 ± 2.5 grams) were purchased

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from the Jackson Laboratory (Bar Harbor, ME). The animals were housed in a temperature

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controlled room and stablized with regular mouse chow and tap water for 1 week. The mice

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were divided into 3 groups (n=8/group) with similar mean plasma total cholesterol

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concentrations and body weights after the stabilization.14

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Experimental diets

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Control diet is consisted of a regular mouse diet 9F 5020 (LabDiet, St Louis, MO) supplemented

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with 0.06% (w/w) cholesterol (Sigma Aldrich, St Louis, MO). Asian white rice was obtained

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from commercially available source (Sen Tai You, Nanjing Feng Yuan Rice Industry Co. Ltd,

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China). Wild rice was purchased from Flin Flon, MB, Canada. Rice was mechanically powdered

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at the Canadian Centre for Agri-Food Research in Health and Medicine, Winnipeg, Canada.

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White rice and wild rice diets were prepared by the replacement of control diet with white or

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wild rice powder (60%, w/w), and then pelleted. The dosages of the rice used in experiments

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were based on our previous publication.14 Both rice-containing diets were supplemented with

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0.06 % of cholesterol (w/w) as described.14 Macronutrients and representing energy in white and

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wild rice are presented in Table 1. The levels of total phenolic contents and phenolic compounds

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in wild and white rice are presented in Table 2.

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Nutritional intervention and sample collection

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Mice in the control, white rice and wild rice diet had access to ad libitum diet and water. Food

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intakes and body weights were weighed at weekly basis. At the end of the nutritional

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intervention, the mice were fasted overnight and then euthanized using carbon dioxide followed

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by blood collection via cardiac puncture. Abdominal aortae were placed in cultured dishes with

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ice-cold Hank’s balance salt solution (HBSS).15 Aortic roots were fixed in 10% buffered

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formalin and sectioned for histological assessment for atherosclerosis as described.13 Hearts and

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ascending aortae were collected, weighed and stored at -80°C until analysis. This study protocol

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of the present study was approved by the Animal Care and Protocol Committee at the University

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of Manitoba (protocol #13-053/1).

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Monocyte adhesion assay

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After remove extravascular fat tissue, aortae were longitudinally opened and fixed on the bottom

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of 35-mm culture dish containing 2 mL of ice-cold HBSS with 27-gauge needles within 1 h after

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tissue harvesting. Mouse WEHI-274.1 monocytes were labeled with 3 µg/mL of TRITC

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(Molecular Probes, Burlington, ON) for 15 min. Unbound dye was removed from the cells

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through a brief centrifugation.13 Fluorescently labeled monocytes (1 x 105) were added to each

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dish containing aortic strip. Mouse monocytes were incubated with aortic strips at room

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temperature for 30 min on a rotating mixer. Un-adhered monocytes were removed using 2

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washes with HBSS. Adhered monocytes were fixed using 2% glutaraldehyde in HBSS.

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Monocytes adhered to intima of aorta were counted under fluorescent microscopy using 10X

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magnification. At least 5 fields were counted from each aortic strip and the averages of adhered

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monocytes to each field were used for data analysis as described.15

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Inflammatory mediators in cardiovascular tissue

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The inflammatory mediators in extracts of hearts and aortae were analysed using Western

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blotting analysis as described.16 Monoclonal or polyclonal antibodies against mouse, urokinase

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plasminogen activator (uPA), uPA receptor (uPAR), plasminogen activator inhibitor (PAI)-1,

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intracellular cell adhesion molecule (ICAM)-1 and tissue necrosis factor (TNF)-α were obtained

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from Santa Cruz (Sandoz Cruz, CA), monocyte chemoattractant protein (MCP)-1 antibody was

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from ProMab (Richmond, CA), and β-actin antibody was from Abcam (Cambridge, MA).

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Corresponding secondary antibody conjugated with horse radish peroxidase (Santa Cruz) and

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enhanced chemilluminence reagents (GE Healthcare, Buckinghamshire, UK) were applied to

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visualize targeted antigens on nitrocellulose membranes. The abundance of antigens was

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assessed using Chemi-Doc system and Quantity One software (BioRad, Hercules, CA), and

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normalized with the abundance of β-actin in corresponding samples.

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Analysis of blood lipid profile and glucose

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The levels of glucose in blood of the mice were measured using AlphaTRAK glucose monitoring

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system from Abbott. Plasma total cholesterol, LDL-cholesterol, very low density lipoprotein

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(VLDL)-cholesterol, high density lipoprotein (HDL)-cholesterol and triglycerides were

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measured using Sekisui Diagnostics SL reagents.14

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Plasma MCP-1 and TNFα

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The levels of MCP-1 and TNFα in plasma of the mice were analysed using enzyme linked

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immunoabsorbant assay (ELISA) kits (Thermo Fisher Scientific in Ottawa, Canada for mouse

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MCP-1 and BD Bioscience in San Diego, CA for mouse TNFα).

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Plasma antioxidant enzyme activities

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The activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase in

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plasma of the mice fed with different diets were measured using assay kits from Cayman

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Chemical Co. (Ann Arbor, MI) as previously described.17

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Analyses of total phenolic content and compounds in rice samples

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Total phenolic content of the rice samples was determined using the Folin-Ciocalteu method as

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described previously.18 Gallic acid was used for calibration of total phenolics contents and

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phenolic compounds and the results were expressed as miligram gallic acid equivalents in per

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kilogram of milled rice (mg GAE/kg) at dry weight basis. The soluble (free and esterified) and

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insoluble (cell wall-bound) phenolic compounds in the rice samples were extracted and analyzed

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using liquid chromatograph-mass spectrometric analysis as previously described.19 The phenolic

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compounds were identified by comparing their retention times, UV-visible and mass spectral

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characteristics to authentic standards. Calibration curves of the standards were used to quantify

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the constituent phenolic compounds. The peak area of p-hydroxybenzoic acid was extracted at

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254 nm, while that of all the other phenolic compounds were extracted at 280 nm.

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Statistical methods

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Data presented in the figures and tables were presented as means ± standard deviation (SD).

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Statistical differences among multiple groups were analyzed using the one-way variance assay

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(ANOVA) followed with Tukey-Kramer multiple comparison tests. Probabilities