Spd m10a

The leaves of Graptopetalum paraguayense were ground and lyophilized into powder at −20°C and stored in a moisture buster at 25°C before extraction. First, 1.5 g of Graptopetalum paraguayense powder was vortexed with 10 ml of 100% methanol (MeOH) for 5 minutes and then centrifuged for 5 min. After removal of the supernatant, 10 mL of 30% DMSO was added to each pellet to resuspend them. The suspension was mixed by vortexing for 5 min, centrifuged twice for 5 min, and filtered using a 0.45-μm filter at room temperature. The 30% DMSO supernatant was either stored at −20°C as a 150-mg/ml stock solution (referred to as GP extracts) or fractionated into four fractions (F1–F4) by a Sephadex LH-20 column. Using the analysis of AURKA, AURKB, and FLJ10540 protein levels via Western blotting, active molecules were obtained in fraction 3, referred to as the HH-F3 fraction. The HH-F3 fraction was further analyzed by HPLC with a UV detector (Shimadzu SPD-M10A), a normal-phase HPLC column (Phenomenex Luna 5 μm Silica (2) 100 Å, 4.6 × 250 mm), and ¹H- and ¹³C-NMR spectra to identify the structure of the active molecules. HH-F3 was then subjected to dialysis against water using a dialysis membrane (MWCO 12–14,000) (Spectrum Laboratories, Rancho Dominguez, CA) to obtain active compounds.

For the RP-HPLC fingerprint analysis of individual phenolic compounds present in the tomato juice extract, a Shimadzu system (Shimadzu Corp., Kyoto, Japan) consisting of two LC-10AD pumps, an SCTL 10A system controller, an SPD-M 10 A photodiode array detector, and a prepacked LUNA C 18 column (4 × 259 mm, 5 μm, Phenomenex) was used. A flow rate of 1 mL/min, injection volume of 20 μL, a gradient elution of acetonitrile-water-acetic acid (5 : 93 : 2, v/v/v) (solvent A) and acetonitrile-water-acetic acid (40 : 58 : 2, v/v/v) (solvent B), and a 0-50 min solvent B from 0% to 100% were applied [18 (link)]. The tomato juice extract was dissolved using a water : methanol mixture (20 : 80, v/v) and filtered through a 0.45 μm filter (Chromafil Xtra PET-45/25, Macherey-Nagel). The separation of compounds was monitored at 260 and 320 nm. The identification was performed based on the retention times and the UV spectra of the standards and the samples.