Fig 1.
RP-HPLC-DAD and MALDI-TOF mass spec analysis of date palm extract.
A. RP-HPLC-DAD chromatograms for DPE at 280 nm (red) and 320 nm (black); and B. Positive reflectron mode MALDI-TOF MS spectra showing a series of peaks at m/z 734 (Δ238amu), m/z 762 (Δ266amu) and m/z 784 (Δ288amu) that may correspond to long-chain ω–hydroxyfatty acids (C16, C18, C20) esterified to the trans- Feruloyloxy octadecanoic acid. AI: absolute intensities; AU: absorbance units.
Fig 2.
RP-HPLC-DAD and MALDI-TOF mass spec analysis of a PAC-enriched DPE fraction.
A. RP-HPLC-DAD chromatogram for the PAC-enriched fraction of DPE following the Sephadex LH-20 column (DPE-PAC) collected at different wavelengths (Blue: 280, Green: 320, Black: 370, and Red: 520 nm); and B. MALDI-TOF MS spectra of DPE-PAC in positive reflectron mode, showing a series of PACs ranging from trimers to octamers. The Insert is a spectrum showing the overlapping isotope pattern for a DPE-PAC pentamer with Na+ and K+ adducts. AI: absolute intensities; AU: absorbance units.
Fig 3.
Percentage of A- and B-type interflavan bonds present in DPE-PAC.
The percentage of A- and B-type bonds was calculated using matrix algebra for overlapping isotopic peaks after MALDI-TOF MS.
Fig 4.
Determination of the ability of DPE to transactivate the ligand binding domain of the nuclear receptor FXR in vitro.
CV-1 cells were co-transfected with a Gal4 luciferase reporter and a Gal4 DNA-binding domain construct or a chimera in which the Gal4 DNA-binding domain is fused to the ligand-binding domain of FXR. The cells were treated with a known receptor-specific agonist, 100 μM chenodeoxycholic acid (CD) or DPE (mg/L). Results are expressed as normalized luciferase activity relative to DMSO (set at 1) (mean ± SEM). Statistical differences are represented by letters. Bars with the same superscript letter are not significantly different from each other.
Fig 5.
Determination of the ability of DPE to interact with a range of nuclear receptor ligand binding domains in vitro.
CV-1 cells were co-transfected with a Gal4 luciferase reporter and a series of chimeras in which the Gal4 DNA-binding domain is fused to the indicated nuclear receptor ligand-binding domain. The cells were treated with a known receptor-specific agonist or DPE (mg/L). Results are expressed as normalized luciferase activity relative to DMSO (set at 1) (mean ± SEM). The ligands used were as follows: A. Mouse constitutive androstane receptor (mCAR): 250 nM 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TC); B. Human CAR: 10 μM CITCO (CIT); C. Estrogen receptor alpha (ERα): 1 μM Estradiol (E2); D. Glucocorticoid receptor (GR): 100 nM Dexamethasone (DEX); E. Liver x receptor alpha (LXRα): 10 μM 22-hydroxycholesterol (22-C); F. Peroxisome proliferator-activated receptor (PPARα): 1 μM Clofibrate (CL); G. PPARϒ: 1 μM Troglitazone (T); H. Mouse pregnane x receptor (mPXR): 10 μM pregnane 16α-carbonitrile (PCN); I. human PXR: 10 μM Rifampicin (RIF); J. Retinoic acid receptor alpha(RARα): 1 μM all-trans retinoic acid (allT); K. Retinoid x receptor (RXR): 1 μM 9-cis-retinoic acid (RA); L. Thyroid hormone receptor beta (TRβ): 1 μM thyroid hormone (T3); and M. Vitamin D receptor (VDR): 100 nM 1α,25-dihydroxyvitamin D3 (D3). Statistical differences are represented by letters. Bars with the same superscript letter are not significantly different from each other.
Fig 6.
Determination of the ability of DPE to transactivate a range of nuclear receptors in vitro.
CV-1 cells were co-transfected with a Gal4 luciferase reporter and a series of chimeras in which the Gal4 DNA-binding domain is fused to the indicated nuclear receptor: A. RAR-related orphan receptor alpha (RORα), B. RORβ, C. mouse small heterodimer partner (SHP), and D. human SHP. The cells were treated with either DMSO or DPE (mg/L). Results are expressed as normalized luciferase activity relative to DMSO (set at 1) (mean ± SEM). Statistical differences are represented by letters.
Fig 7.
DPE acts as a co-agonist ligand for human and mouse FXR.
CV-1 cells were co-transfected with a luciferase reporter construct plus expression vectors as indicated A. Mouse FXR and B. Human FXR, and treated with vehicle (dimethylsulfoxide, DMSO) (white bars), 20, 50 or 100 mg/L DPE (gray bars), 100 μM CDCA (black bars) or 100 μM CDCA plus DPE as indicated (hatched bars). Results are expressed as fold change relative to the control (DMSO), normalized to the β-gal internal control (mean ± SEM). DPE: date palm extract; CD: chenodeoxycholic acid. Statistical differences are represented by letters. Bars with the same superscript letter are not significantly different from each other.
Fig 8.
DPE induces coactivator recruitment to bile acid-bound FXR.
CV-1 cells were co-transfected with a luciferase reporter construct plus expression vectors as indicated and treated with vehicle (dimethylsulfoxide, DMSO) (white bars), 20, 50, or 100 mg/L DPE (gray bars), 100 μM CDCA (black bars) or 100 μM CDCA + DPE (mg/L) (hatched bars). Results are expressed as fold change relative to the control (DMSO), normalized to the β-gal internal control (mean ± SEM). Statistical differences are represented by letters. Bars with the same superscript letter are not significantly different from each other.
Fig 9.
DPE differentially regulates FXR-target gene expression in vitro in Caco-2 cells.
Caco-2 cells were treated for 12 hours with either a negative control (DMSO), varying doses of DPE (20, 50 or 100 mg/L), 100 μM CDCA, or a combination of CDCA + DPE, as indicated. Relative gene expression is shown for A. ASBT, B. IBABP, C. FGF19, D. OSTα, E. OSTβ, and F. FXR. (Negative control (DMSO), white bars; DPE (20, 50 or 100 mg/L) (gray bars); CDCA (100 μM) (black bars); or in combination (hatched bars). Statistical differences are represented by letters. Bars with the same superscript letter are not significantly different from each other.