C18 reversed phase column

CGA was quantified by HPLC. A Merck Hitachi LaChrome equipment with a L7000 interface module, a L7200 autosampler, a L7490 RI detector, a UV detector, a L7350 column oven and a L7100 pump, was used associated with the D-7000 HSM software. A reversed-phase C18 column (5 μm, 250 × 4.6 mm, Waters) at RT was used with a flow rate of 0,9 mL/min. The mobile phase consisted of A: water/acetic acid (19:1) and B: methanol (0 min: 95%, 3 min: 85%, 13 min: 75%, 25 min: 70%, 27 min: 40%, 28 min: 0%, 30 min: 95% and 38 min: 95% of A). CGA was detected at 320 nm and eluted at 15 min retention time. CGA was quantified in Cc leaf phenolic-derived extracts from the linear equation (y = 122,049 × − 1,236,603 where x and y represent CGA concentration and peak area, respectively; r² = 0.999), prepared with the injection of CGA pure standard (Sigma, USA) in methanol/water (50:50) (10–800 μg/mL). Cc leaf phenolic-derived extracts were diluted in methanol/water (50:50) (10 mg/mL) in duplicate and filtered through cellulose acetate filter, 0.22 μm pore size, prior to injection. The data acquisition was carried out using the D-7000 HSM software (Hitachi, Japan).

Silibinin was identified in mice blood plasma using Liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS). The High Pressure Liquid Chromatography (HPLC) separation was performed at 25°C using a Waters^® reversed phase C18 column (100 mm × 2.1 mm dimension; 5 μm particle size). ESI-MS was carried out using Waters^® micromass^® Q-TOF Mass Spectrometer at Regional Sophisticated Instrumentation Centre (RSIC), Panjab University, Chandigarh, India. For HPLC, the mobile phase consisted of acetonitrile and 10 mM ammonium acetate (pH 5.45) in the ratio of 50:50 (v/v). The detection of Silibinin was carried out at 288 nm and the flow rate was 100 μl/min with a total run time of 10 minutes. Mass spectrometric (MS) analysis was performed in negative ion mode under the following conditions: capillary voltage 2960 V, sample cone voltage 30 V, extraction cone voltage 1.0 V and desolvation temperature 350°C. The mass spectra were recorded in the m/z range of 100–700.

Optical rotations were measured on a JASCO P-2000 instrument. The 1D and 2D NMR spectra were recorded on either a Bruker DPX 400 instrument with tetramethylsilane as an internal standard and MeOH-d₄ as a solvent. HR-ESI-MS experiments were conducted using a Thermo Fisher QE Focus spectrometer. The semi-preparative HPLC procedure was conducted on a Shimadzu LC-16D instrument with an RID-20A (reflective index detector) and a reversed-phase C₁₈ column (250 × 10 mm, 5 μm, Waters SunFire). Column chromatography was performed using silica gel (200–300 mesh, China), octadecyl silica (ODS) (50 μm, Merck, Germany).

Serum folate levels were determined five days after STZ treatment and then every four weeks, using an automated chemiluminescence system (Immulite 1000; Siemens, Berlin, Germany).
Tail vein blood glucose level was monitored by using a glucometer (Accu-Chek; Roche, Penzberg, Germany). Blood glucose test strips were used to measure the blood glucose concentration in mice.
Plasma levels of Hcy were analyzed using high-performance liquid chromatography (HPLC) with a Waters 700 HPLC Pump and a reversed-phase C18 column (5 µm bead size; 4.6 mm × 250 mm) (Waters, Milford, CT, USA). Briefly, the mobile phase consisted of 0.08 M acetate buffer and 5% (v/v) methanol adjusted to pH 4.0 by addition of concentrated acetic acid and then filtered through a 0.45 µm membrane filter. The isocratic elution was performed using a flow rate of 1.0 mL/min at 30 °C and a pressure of 100–110 kgf/cm² (1500–1800 psi). A fluorescence detector with excitation at 390 nm and emission at 470 nm was used to detect Hcy. Before analysis of Hcy, the system was calibrated with authentic DL-homocysteine standards in the range of 50 to 4000 ng. Plasma Hcy was quantified relative to the standard obtained from Sigma Chemical Co. (St. Louis, MO, USA).

HPLC analysis of ascorbic acid was used following a method modified from Asami et al. (41 (link)). Small pieces of frozen samples (1–5 g) were homogenized with 20 mL of 4.5% metaphosphoric acid (HO₃P) in ultra turrax (IKA Laroratechnik, Malaysia) during 4 min. The extract was centrifuged at 16,096 × g for 15 min at 4°C, and the supernatant was filtered using filter paper (Whatman no. 1). ascorbic acid content was then quantified by Shimadzu’s HPLC system on reversed-phase C18 column (150 mm × 4.6 mm diameters, Waters Corporation, USA) under the following conditions: injected volume 20 µL; oven temperature 40°C; solvent potassium phosphate (10 mM); flow rate 1.5 mL/min, and the detection was performed with an ultraviolet wavelength of 242 nm. Total ascorbic content was calculated from calibration curves by comparison with external standard (ascorbic acid, Sigma-Aldrich, MO, USA) (42 , 43 (link)).