Sodium borate

Acetone (99.9%), glacial acetic acid (99.8%), acetonitrile (99.9%), absolute ethanol, methanol (LC-MS grade), HCl (37%), chloroform, and sodium hydroxide were obtained from PanReac (Barcelona, Spain). Folin–Ciocalteu reagent, ABTS^•+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), DPPH^• (1,1-diphenyl-2-picrylhydrazyl), gallic acid, quercetin, cyanidin-3 glucoside, β-carotene, aluminium chloride, potassium bromide, potassium persulfate, sodium bicarbonate, sodium borate, sodium sulphate, sodium sulphite, cetrimonium bromide, citric acid, malic acid, and PMP (1-phenyl-3-methyl-5-pyrazolone) were acquired from Merck (Madrid, Spain). Pure compounds for the identification of monosaccharides, enzymes and electrolytes for in vitro digestion were also purchased from Merck. Bacterial strains Lactobacillus casei CECT 475 and Lactococcus lactis subsp. lactis CECT 185, as well as their respective culture media, were obtained from the Spanish Type Culture Collection (CECT) and PanReac, respectively.

Hydrochloric acid, pyridine, benzene sulfonyl chloride (BSC) and trifluoroacetic acid (TFA) were purchased from Fisher Scientific (Atlanta, GA, USA). Angiotensin-I-converting enzyme (rabbit lung), captopril and Hippuryl-l-Histidyl-l-Leucine (HHL) were obtained from Sigma Chemical Co., (St Louis, MO, USA). Sodium chloride, boric acid and sodium borate were purchased from Merck Co. (Darmstadt, Hesse, Germany). Peptide sequences YLLLK, WAFS and GVQEGAGHYALL were identified in the papain-generated protein hydrolysate from palm kernel cake (PKC) after fractionation and then were synthesized (First Base Laboratories Sdn Bhd, Selangor, Malaysia) to be used in this study.

Twenty-four hours after siRNA transfection, 3 × 10⁶ cells were pulse-labeled with 33 µM BrdU (Sigma-Aldrich) for 20 min at 37 °C, washed twice with PBS and released in BrdU-free medium for various times. At each time point, cells were resuspended in 750 µl PBS and fixed with 2250 µl of pure ethanol (Carlo Erba) for 30 min on ice. Cells were then washed with 1 ml (1%) BSA (Sigma-Aldrich) in PBS, incubated with 1 ml 2 N HCl (Carlo Erba) for 25 min at room temperature to denature DNA when 1.5 ml 0.1 M Sodium Borate (Sigma-Aldrich) was added for 2 min at room temperature. Cells were washed twice with 1 ml of PBS-BSA (1%) and incubated 1 h at room temperature with 100 µl of pure anti-BrdU antibody (Becton Dickinson) diluted 1:50 in PBS-BSA (1%). Washed cells were then resuspended in 100 µl anti-mouse FITC diluted 1:50 in PBS-BSA (1%) and incubated 1 h at room temperature. Cells were washed, resuspended in 2.5 µg/ml propidium iodide (Sigma-Aldrich), 250 µg/ml RNase and incubated overnight at 4 °C. The analysis of BrdU positive cell and DNA content was performed using a FACSCalibur with 488 nm laser (Becton Dickinson) and Cell Quest software (30,000 cells were analyzed for each sample).

Glu (98%, lot no. 140690-200401) was purchased from National Standard Material Centre (Beijing, China); Homoserine (IS) (99%, lot no. 5005F23A) was purchased from Alfa Aesar Chemical Co. Ltd (Shanghai, China); Gln (99%, lot no. BCBH4247V), GABA (97%, lot no. BCBN4574V), o-phthalaldehyde (OPA), β-mercaptoethanol, disodium hydrogen phosphate, sodium borate and perchloric acid were purchased from Sigma Aldrich Chemie (Steinheim, Germany). HPLC-grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). Water was purified by means of a Milli-Q plus system from Millipore (Bedford, MA).

Methanol, DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), TPTZ (2,4,6-tri(2-pyridyl)-s-triazine), ascorbic acid, hyaluronic acid (human umbilical cord), sodium hydroxide, hyaluronidase (bovine testes), calcium chloride, p-dimethylaminobenzaldehyde (PDMAB), and sodium borate were purchased from Sigma-Aldrich (USA). COX (ovine) and LOX inhibitor screening assay kits (Cayman Chemical Company, MI, USA), 96-well cell culture plate (Corning Life Sciences, Lowell, MA, USA), PMA (Sigma-Aldrich, St. Luis, MO, USA), and histamine release assay kit (SPI-Bio, France) were used.

The functionality of the osteoclasts was analyzed by using two different resorption assays: Osteo-assay Plates (#3987, Corning, Corning, NY, USA) and ivory slices. The Osteo-assay Plates are coated with inorganic three-dimensional crystalline material (bone biomimetic synthetic surface). Ivory slices were cut with a low speed water-cooled diamond saw. The slices had a diameter of 7 mm and a thickness of 0.5 mm. Osteoclasts were generated from hPBMCs in monoulture and co-cultures with cementoblasts as described above. After 21 days of culture, the cells were lysed by 2.5% chlorinated soda solution for 5 min at room temperature and washed by distilled water. Ivory slices were stained with a solution of 1% toluidine blue (#89640, Sigma-Aldrich, St Louis, MO, USA) in 1% sodium borate (#S9640, Sigma-Aldrich, St Louis, MO, USA) for 4 min. Resorption pits were observed and quantified using photomicrographs from five randomly chosen fields by inverted phase-contrast microscopy for Osteo-assay Plates and stereomicroscopy Discovery V8 (Zeiss, Oberkochen, Germany) for the ivory slices.