Luna 5 μm silica

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.

Astaxanthin isomers were analyzed using
normal-phase HPLC with a photodiode array detector (SPD-M20A; Shimadzu,
Kyoto, Japan) as previously described.^{18 (link)} Briefly, two silica gel columns connected in tandem (Luna 5 μm
Silica (2), 2 mm × 150 mm × 4.6 mm, 100 Å, Phenomenex
Inc., Torrance, CA) were used as the stationary phase, and a mixture
of hexane, ethyl acetate, and acetone (70:20:10, v/v/v) was used as
the mobile phase. The column temperature was adjusted to 40 °C,
and the flow rate was maintained at 1.2 mL/min. The quantification
of astaxanthin isomers was carried out by peak area integration at
470 nm. The peaks of astaxanthin E/Z-isomers in the chromatograms were identified by retention times
and spectral data (i.e., whole band shapes of the absorption spectra,
maximum absorption wavelengths, and relative intensities of the Z-peak to the absorption maximum peak of the isomer [Q-ratios]).^{12 (link),14 (link),18 (link),19 (link)} The total (or each) Z-isomer
ratio of astaxanthin was determined as follows