Clc ods c 18 column

Ten heart samples per group were collected four days after feeding, homogenized with 200 μL 5% acetonitrile and 0.05% TFA as solvent, pH 2.0 (1:2 v/v), centrifuged for 15 min at 12,000×g and the supernatant was applied onto a Shimadzu CLC-ODS C18 column (15 mm × 22 cm) equilibrated with the same solvent, using a flow rate of 0.4 mL/min. After 10 min, an acetonitrile linear gradient (5–80%) was applied for 10 min, followed by 20 min of 80% acetonitrile with 0.05% TFA, pH 2.0, and heme and biliverdin peaks were identified as previously described [33 (link)]. The experiments were performed at room temperature.

HPLC was performed on a Shimadzu CLC-ODS C18 column (15 mm × 22 cm) using a Shimadzu LC-10AT device (Tokyo, Japan), equipped with a diode array detector (SPD-M10A). Chromatography analysis was performed using 5% acetonitrile with 0.05% trifluoroacetic acid (TFA) as solvent, at a flow rate of 0.4 mL/minutes. Before injection, dried samples were diluted in 10% acetonitrile with 0.05% TFA and centrifuged for 15 minutes at 12,000 × g. Ten minutes after sample injection a 40 minutes linear acetonitrile gradient (5–80%) was applied, followed by 20 minutes of 80% acetonitrile. Supernatants were dried under vacuum, and stored at −20 °C protected from light until use as described above. AeBV peak area data were taken for statistical analyses.

Powder material (1 kg) was cold macerated in 1:2 ratio with 80% aqueous methanolic solvent (2 L) for 14 days with 12 h periodic stirring. Finally, macerate was filtered through Whatman No. 1 filter paper and filtrate was concentrated using rotary evaporator under reduced pressure at 40–45 °C and extract yield percentage was calculated as follows:

Quantitative phytochemical analysis of extract was performed to estimate the primary and secondary metabolites [14 (link)]. In vitro antioxidant potential of extract was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and reducing power assay following methods described previously [15 (link)]. The identification of metabolites was carried out by the means of a high-performance liquid chromatography (HPLC) system LC-10A (Shimadzu, Nagoya, Japan) using Shim-Pack CLC-ODS C—18 column (25 cm × 4.6 mm, 5 µm) [16 ]. Mobile Phase contained solvent A (H₂O: Acetic acid—94:6, pH = 2.27) and B (100% acetonitrile). The isocratic elution of fractions was carried out at flow rate of 0.1 mL/min at 30 °C and detected by ultra-violet (UV)-visible detector at 280 nm wavelength.