Inhibitory Effects of North American Wild Rice on Monocyte Adhesion

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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

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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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

Inhibitory Effects of North American Wild Rice on Monocyte Adhesion

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