Ug120

Brazilin was purified from C. sappan extract using a preparative LC-20 series HPLC system (Shimadzu Corp., Tokyo, Japan) fitted with a reverse-phase C₁₈ column (UG120, 5-μm particle sizes, 4.6 I.D. x 250 mm UG120, Shiseido, Japan) with monitoring of eluate absorbance at 280 nm. The eluent system consisted of an isocratic mode of 100% (v/v) methanol, running at a flow rate of 0.5 ml/min at a column temperature of 25°C. Fraction with retention times of about 7.669 were amalgamated from repeat HPLC runs.

Frozen apple peel samples (~150 mg) were homogenized to a fine powder using a mortar and pestle. 3% pyrogallol and 60% KOH were added to a certain amount of sample, saponified in a water bath at 70 °C, and then cooled. After adding a 1% NaCl solution and a hexane and ethyl acetate mixture, the samples were homogenized by vortexing. The supernatant was collected by centrifugation. Again, a hexane and ethyl acetate mixture solution was added to the extracted supernatant. The supernatant was re-extracted following centrifugation. This process was repeated until all the pigment was gone. The collected supernatant was entirely concentrated with nitrogen gas and then dissolved in ethanol and used as the sample for carotenoids analyzed using HPLC (Shiseido SP LC SP3202, Tokyo, Japan).
A 10 μL aliquot was injected into the HPLC system with column Shiseido UG 120. Carotenoids were separated using a mobile phase (Acetonitrile: Methanol: Dichloromethane) at a flow rate of 1 mL/min, and the column temperature was maintained at 40 °C. Carotenoids were detected at a wavelength of 450 nm, and the concentrations of carotenoids were determined as β-carotene equivalents per g of fresh tissue weight. β-carotene was identified in the extracts by comparing retention times and online spectral data with standard samples.

Immature pear fruit extract (1 g fresh weight equivalent) was dissolved in 50% methanol (1 mL) and filtered through a 0.45 μm Millipore membrane filter. The dissolved solution was subjected to Octadecyl-silica (ODS)-HPLC analysis. Arbutin and PCA were separated on an ODS column (UG120, 4.6 mm internal diameter (ID) × 250 mm, 5 μm, Shiseido, Tokyo, Japan). The mobile phase was composed of H₂O/acetic acid (98:2, v/v, eluent A) and MeOH/H₂O (60:40, v/v, eluent B). The gradient program used was as follows: started at 100% A and increased to 100% B linearly over 45 min. The column temperature was maintained at 40 °C and the flow rate was 1 mL/min. Arbutin and PCA were monitored at 280 and 254 nm, respectively, using a photodiode array detector (SPD-M20D; Shimadzu, Kyoto, Japan). Arbutin and PCA contents in each sample were quantified by the chromatographic peak area of external standards. The calibration curves were plotted in the concentration range of 0.01–10 μg.