Ezchrom elite software

HPLC analysis of the studied extract was performed using a LaChrom Elite system (VWR-Hitachi, Radnor, PA, USA) equipped with two L7100 pumps, a L7200 autosampler, a L2450 diode array detector (DAD) performing the wavelength scanning from 200 nm to 600 nm, and EZ Chrom Elite software (Agilent Technologies, Massy, France). AEVM (1 mg/mL) was chromatographed with a reversed phase Interchim^® UP3ODB C₁₈ column (150 × 4.6 mm, 5 μm particle size). Gradient elution was developed with a mobile phase composed of water containing 1% phosphoric acid (A) and MeCN (B). The gradient was set as follows: 0–10 min, 9% B; 10–25 min, 9–12% B; 25–40 min, 12–16% B; 40–45 min, 16% B; 45–50 min, 16–40% B; 50–55 min, 9% B. A flow rate of 1 mL/min and an injection volume of 20 μL were selected. The quantification of individual anthocyanins was achieved by constructing calibration curves with commercial anthocyanin constituents that were representative of the five different groups occurring in bilberry (delphinidin, cyanidin, petunidin, malvidin and peonidin 3-O-glucosides). Standard solutions in the concentration range of 5–100 μg/mL were injected, and the chromatograms were monitored at a wavelength of 530 nm. Five-point calibration curves were constructed, and all of them exhibited satisfying linearities (R² > 0.99). Results were expressed as mg of constituent per g of dry extract.

Within 15 min after blood collection, plasma was separated by centrifugation (15 min at 2000 RPM). Plasma total triglycerides (Total TG) and total cholesterol (Total Chol) were measured by enzymatic assay using commercially available kits (Roche Diagnostics GmbH, Mannheim and mti Diagnostic GmbH, Idstein, Germany). Cholesterol lipoprotein fractions in serum were determined as described^{79 (link)}. Briefly, sera from each individual mouse were separated by size exclusion chromatography using a Superose and PC 3.2/30 column (Pharmacia Biotech, Uppsala, Sweden). Reagent (Roche Diagnostic, Mannheim, Germany) was directly infused into the eluate online and the absorbance was measured. The concentration of the different lipoprotein fractions was calculated from the area under the curves of the elution profiles by using the EZChrom Elite software (Scientific Software; Agilent Technologies, Santa Clara, CA).