Md 2010 plus

NIH1 was dissolved in DMSO to obtain a stock solution 10 mM. Samples of this stock solution were diluted until 1 mM in cell-free complete culture medium and left incubating at 37 °C for up to three days. At selected time points (0, 24, 48, and 72 h) samples were collected, diluted at 0.1 mM with mobile phase ACN/H2O 40:60, and analyzed through HPLC reversed-phase conditions on a Phenomenex Jupiter C18 (150 × 4.6 mm I.D.) column, UV detection at λ = 302 nm and a flow rate of 1 ml/min. Analyses were performed on a liquid chromatograph model PU 2089 PLUS equipped with a 20 µl loop valve and linked to MD 2010 Plus UV detector (Jasco Europe, Lecco, Italy). Areas of NIH1 peak, identified by co-injection, were analyzed and their percentage reductions vs time were reported in the graph.

0.5 mL of LIP-1, LIP-2, or LIP-3 was measured into a 5.0 mL volumetric flask, diluted to 1.5 mL with HPLC-gradient water and adjusted to 5.0 mL with HPLC grade methanol. HPLC analysis was performed on a system of two Jasco PU-2080 pumps connected to a Jasco MD-2010 Plus photodiode-array detector, in isocratic mode by 70% aqueous methanol on a Zorbax XDB-C8 column (5 μm, 4.6 × 150 mm) at a flow rate of 1.5 mL/min at λ = 243 nm. Each measurement was performed in triplicates, as well as the calibration that was performed by injecting 0.2, 0.5, 1.0, 3.0, and 5.0 μg of compound 3.

Sample preparation and analysis were carried out as previously described (Ben-Simhon et al., 2011 (link)). In short, methanolic extracts of pomegranate peel tissue were analyzed using a LaChrom Merck Hitachi HPLC system coupled with a diode array detector with 3D feature (Multiwavelength Detector, Jasco MD-2010 Plus), interface (Jasco LC-Net II/ADC) and scientific software (EZChrom Elite Client/Server version 3.1.6 build 3.1.6.2433). Extract aliquots were applied to a LiChrospher 100 RP-18 column with guard column (LichroCART cartridge, Merck Millipore), and eluted with a binary mobile phase consisting of phosphoric acid (0.1% v/v, pH 2.4) and acetonitrile. Anthocyanin identification and quantification were achieved using authentic standards. For each sampling stage, extract of peel tissue pooled from four to eight flowers/fruits from three replicate trees of each pomegranate variety was analyzed.

Three-dimensional high-performance liquid chromatography (HPLC) analysis was performed using a JASCO series (JASCO Corporation, Tokyo, Japan) system consisting of a JASCO PU-2089 Plus pump and a JASCO MD-2010 Plus photodiode array detector. Chromatographic separation was performed using a YMC-Pack ODS-AL S-5 column (5 μm, 250 mm × 4.6 mm i.d., YMC Co., Ltd., Tokyo, Japan) controlled at 40 °C (Shimadzu CTO-20 AC, Shimadzu Corporation, Kyoto, Japan). The mobile phases consisted of acetonitrile (A) and 50 mM sodium dihydrogen phosphate monohydrate (B). Gradient elution was performed at a flow rate of 0.8 mL/min with the following parameters: 0–60 min A:B (10:90, v/v) to A:B (90:10, v/v), 60–75 min A:B (90:10, v/v), and 75–80 min A:B (10:90, v/v). The injection volume was 10 μL (0.2 mg/mL). Scan data were collected from 220 to 600 nm.

H. indicus fractions obtained by soxhlet extraction were subjected to RP-HPLC-DAD analysis to identify and quantify their main phytomarkers. The analysis was performed using a Jasco modular HPLC (model PU 2089, Tokyo, Japan) coupled to a diode array apparatus (MD 2010 Plus) linked to an injection valve with a 20 µL sampler loop (40 µL injection volume). The system was composed of a Kinetex XB C18 (5μm, 15 × 0.46 cm, Phenomenex, Aschaffenburg, Germany) column, with a flow rate of 0.7 mL/min, a mobile phase consisting of two solvent solutions, A (water/formic acid, 99.9:0.1) and B (methanol), combined in a gradient system characterised by a starting point (0 “starting from”) of 5% B, which rose to 30% in 50 min, followed by an isocratic phase until 65 min; percentage of B fell to 5% in 5 additional min and came back to the initial condition in 10 min. All solvents used were of chromatographic grade (Sigma Aldrich, St. Louis, MI, USA). The recorded chromatograms of the phytocomplexes were compared to those of pure standards in terms of UV absorption and retention time.

The 1-octanol/water partition coefficient P was determined using HPLC based on the OECD Guideline for Testing Chemicals. A COSMOSIL Packed Column 5C18-MS-II (5 μm, 150 mm × 4.6 mm id, Nacalai Tesque, Inc., Kyoto, Japan) was fitted on an HPLC instrument (Multiwavelength Detector, MD-2010 Plus. JASCO, Tokyo, Japan) equipped with a pump (PU-2080, JASCO) and an oven (SSC-2120, Senshu Scientific Co., Ltd., Tokyo, Japan). The injection volume was 20 µL; the mobile phase was methanol–water 75% (v/v); and the flow rate was 1.0 mL/min in all cases. Compounds were detected by measuring the UV absorption at 240 nm. The temperature of the column was kept at 40.0 (± 0.1) °C during the measurement. The measurement was performed in triplicate, and the mean value was calculated. The dead time t₀ was measured with thiourea as the unretained compound, and the capacity factor k was calculated using the equation logk = log (t_r − t₀/t₀), where t_r represents the retention time of the compound. A calibration graph was determined experimentally using reference compounds (4-methylphenol, 4-chlorophenol, 4-phenylphenol, diphenylether, fluoranthene, and dichlorodiphenyltrichloroethane) with known logP values. The LogP values of phosphine–boranes were calculated based on a calibration graph (LogP = 2.6269 logk_w + 3.059, R² = 0.9915).