Preparation of Z. mays extract
Z. Mays was harvested in Gangwon-do, Korea, from June to August and authenticated by Dr. Yong-Hwan Jung, Jeju Biodiversity Research Institute, Jeju Techno Park, Korea, where a voucher specimen (Voucher No. JBRI 140924–01). The dried powders from the whole plant (150 g), flag leaf (150 g), husk (150 g), cob (150 g), kernel (150 g), silk (150 g), tassel (150 g), and stalk (150 g) of Z. Mays were extracted with 70 % ethanol for 24 h, and the extract was incrassated by a rotary evaporator for 3 h. To remove the ethanol from the extract, it was mixed with water and incrassated again. Subsequently, the extract was filtered using filter paper and frozen on a freezing tray for 48 h. Freeze-drying powder of whole plant (21.3 g), flag leaf (21.0 g), husk (31.2 g), cob (6.3 g), kernel (11.8 g), silk (27.9 g), tassel (7.9 g), and stalk (20.7 g) were dissolved in DMSO for the experiments.
Cell culture and reagents
Mouse macrophage cell line, RAW264.7 was obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). The cells were maintained in RPMI 1640 (HyClone, Logan, UT, USA), containing 10 % fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and 1 % penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA), at 37 °C, under 5 % CO2. NIH/3 T3 mouse fibroblast cell line was maintained in DMEM (HyClone, Logan, UT, USA), containing 10 % FBS and 1 % penicillin/streptomycin at 37 °C, under 5 % CO2. Lipopolysaccharides (LPS) and Griess reagent were obtained from Sigma Aldrich (St. Louis, MO, USA). Mouse IL-4 was purchased from eBioscience (San Diego, CA, USA). Inducible nitric oxide synthase (iNOS) antibody was purchased from Millipore Corporation (Beverly, MA, USA).
Cell viability assay
Cell viability was measured using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; USB Corp., Cleveland, OH, USA) assay. Cells were plated in triplicate wells of 24-well plates, and cultured for 24 h. The cells were then treated with samples for 24 h, under a serum-free condition. Then, MTT reagent (1 mg/ml) was added to each well, and the cells were incubated for 3 h. The medium was removed, and the cells were solubilized with dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA). The absorbance was measured by spectrophotometer at a wavelength of 570 nm.
Nitric oxide determination
The concentration of nitric oxide (NO) in the culture supernatants was determined as nitrite, a major stable product of NO. The cells were plated in triplicate wells of 24-well plates and incubated overnight. The cells were then treated with samples for 24 h. The cell culture supernatants were incubated with Griess reagent for 30 min. The absorbance was measured by a spectrometer at a wavelength of 540 nm and calculated against a sodium nitrite standard curve.
Western blotting
iNOS protein levels were measured by western blotting. The protein extracts were loaded on a NuPAGE Novex 10 % Bis-Tris Gel (Invitrogen, Carlsbad, CA, USA) and transferred to a nitrocellulose membrane. The membranes were blocked with 5 % bovine serum albumin (BSA, Sigma, St. Louis, MO, USA) for 1 h and then incubated with primary antibodies, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody and detected using chemiluminescent HRP substrate (SurModics, Eden Prairie, MN, USA).
Transient transfection and luciferase assay
RAW264.7 cells were transfected with the iNOS, and NF-kB luciferase reporters using SuperFect® Transfection Reagent (Qiagen, Hilden, Germany). After 24 h of incubation, the cells were incubated in the presence or absence of Z. mays husk extract (ZMHE) induced by LPS for 24 h. The cells were then harvested and lysed, and the supernatants were assayed for their luciferase activity using a Dual Luciferase Assay System (Promega, Madison, WI, USA), and an Infinite® 200 PRO luminometer (Tecan, AG, Männedorf, Switzerland).
Enzyme-linked immunosorbent assay (ELISA)
Eotaxin-1 concentrations were quantified in culture supernatants of NIH/3 T3 after treatment of ZMHE induced by IL-4 using a commercially available ELISA kit (eBioscience, USA). Cell culture supernatants were collected 24 h after treatment with ZMHE, and assayed for eotaxin-1. Soluble intercellular adhesion molecule-1 (sICAM-1) concentrations were quantified in culture supernatants of RAW264.7 after treatment of ZMHE induced by LPS using a commercially available ELISA kit (R&D systems, Inc., Minneapolis, MN, USA). Cell culture supernatants were collected 24 h after treatment with 50 ppm ZMHE, and assayed for sICAM-1. The standard curve was linearized and subjected to regression analysis. The eotaxin-1 and sICAM-1 concentrations were determined using a standard curve.
Determination of total phenolic contents
The content of total phenols was determined by spectrophotometer, using gallic acid as standard, according to the method described by the International Organization for Standardization (ISO) 14502–1. Briefly, an aliquot of the diluted extracts (1.0 ml) was transferred into a separate tubes containing a 5.0 ml of a 1/10 dilution of Folin-Ciocalteu’s reagent in water. Then, a sodium carbonate solution (4.9 ml, 7.5 % w/v) was added. The tubes were then allowed to stand at room temperature for 60 min and then measured absorbance against water at a wavelength of 765 nm. Total phenolic contents was expressed as gallic acid equivalents in mg/g extract. The concentration of polyphenols in extracts was derived from a standard curve of gallic acid.
Determination of total flavonoids
Samples (0.25 ml of the extracts) was added containing distilled water (1 ml) and then 5 % NaNO2 (0.075 ml), 10 % AlCl3 (0.075 ml), and 1 M NaOH (0.5 ml) were added sequentially at 0.5, and 6 min. Finally, the volume of the reacting solution was adjusted to 2.5 ml with double-distilled water. The absorbance of the solution was measured by spectrophotometers at a wavelength of 410 nm. Total flavonoids were expressed as quercetin equivalents in mg/g extract.
Statistical analysis
All data are expressed as means ± standard deviations. Statistical significance of the data was determined using a Student’s t-test. A P < 0.05 was considered to be significant.