Four pairs of diastereomers were successfully isolated and separated from the water extract of Engelhardia roxburghiana by high performance counter-current chromatography (HPCCC) using a two-step procedure. The diastereomers were initially separated by a two-phase solvent system composed of n -hexane– n -butanol–0.1% trifluoroacetic acid (1:2:3, v/v/v) and followed by the same solvent system using hydroxypropyl-β-cyclodextrin (HP-β-CD) as an additive. The chromatographic conditions, elution mode, and concentrations of the additive were refined. The two-step HPCCC isolation yielded 43.7 mg (2S, 3S)-astilbin, 27.6 mg (2R, 3R)-astilbin, 5.9 mg (2S, 3R)-astilbin, 4.8 mg (2R, 3S)-astilbin, 6.9 mg (2S, 3S)-engelitin, 3.1 mg (2R, 3R)-engelitin, 8.2 mg (2S, 3R)-engelitin, and 6.0 mg (2R, 3S)-engelitin from 384 mg crude extract in four runs with purities of 99.3%, 96.2%, 99.8%, 99.9%, 97.0%, 96.5%, 96.1%, and 96.8%, respectively. The present study revealed that HP-β-CD can be used as an additive in HPCCC to effectively improve the resolution of the diastereomers. The established HPCCC method may serve as an approach to obtain high purity diastereomers on a large scale.
Wang Jing,Liu Shu,Ma Bing,Chen Lina,Song Fengrui,Liu Zhiqiang,Liu Chun-ming
Xanthine oxidase (XOD) catalyzes the metabolism of hypoxanthine and xanthine to uric acid, the overproduction of which could cause hyperuricemia, a risk factor for gout. Inhibition of XOD is a major treatment for gout, and biflavonoids have been found to act as XOD-inhibitory compounds. In this study, ultrafiltration liquid chromatography with photodiode-array detection coupled to electrospray-ionization tandem mass spectrometry (UF-LC-PDA–ESI-MS) was used to screen and identify XOD inhibitors from S. tamariscina . High-performance counter-current chromatography (HPCCC) was used to separate and isolate the active constituents of these XOD inhibitors. Furthermore, ultrahigh-performance liquid chromatography (UPLC) and triple-quadrupole mass spectrometry (TQ-MS) was used to determine the XOD-inhibitory activity of the obtained XOD inhibitors, and enzyme kinetics was performed with Lineweaver–Burk (LB) plots using xanthine as the substrate. As a result, two compounds in S. tamariscina were screened as XOD inhibitors: 65.31 mg amentoflavone and 0.76 mg robustaflavone were isolated from approximately 2.5 g S. tamariscina by use of HPCCC. The purities of the two compounds obtained were over 98 % and 95 %, respectively, as determined by high-performance liquid chromatography (HPLC). Lineweaver–Burk plot analysis indicated that amentoflavone and robustaflavone were non-competitive inhibitors of XOD, and the IC
50 values of amentoflavone and robustaflavone for XOD inhibition were 16.26 μg mL −1 (30.22 μmol L −1 ) and 11.98 μg mL −1 (22.27 μmol L −1 ), respectively. The IC
50 value of allopurinol, used as the standard, was 7.49 μg mL −1 (46.23 μmol L −1 ). The results reveal that the method for systematic screening, identification, and isolation of bioactive components in S. tamariscina and for detecting their inhibitory activity using ultrafiltration LC–ESI-MS, HPCCC, and UPLC–TQ-MS is feasible and efficient, and could be expected to extend to screening and separation of other enzyme inhibitors.
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