Figure 5.

Isolation of an ABCC11-inhibitory ingredient by means of recycling preparative HPLC. (a) Recycling preparative HPLC chromatograms for the separation of fractions Fr.#11-5-1 and Fr.#11-5-2. The upper chromatogram was recorded with a refractive index detector, and the lower one was recorded with a diode array and multiple-wavelength detector at 254 nm. After separation under the recycling mode (0–120 min), the mode was changed; Fr.#11-5-1 (123–126 min) and Fr.#11-5-2 (160–176 min) were collected, and all the wastes were collected and further processed as Fr.#11-5-3. R, recycled peaks for Fr.#11-5-1; r, recycled peaks for Fr.#11-5-2. (b) ABCC11-inhibitory activities of each subfraction (20 ppm) in terms of ABCC11-mediated [1,2,6,7-³H(N)]-DHEA-S transport activity measured by the vesicle transport assay; 1% DMSO was used for the vehicle control. Data are expressed as % of vehicle and the mean ± SD; n = 3. **, p < 0.01 vs. control (Dunnett’s test). (c) Purity verification of the isolated ingredient in Fr.#11-5-2 by spectrometric analyses. Left: UV chromatograms recorded at 265 nm. Right: LC-quadrupole time-of-flight-MS (LC-Q-TOF-MS) base peak chromatograms, excluding peaks derived from the plasticizing materials and injected solvent. †, a specific peak in Fr.#11-5-2 with a retention time of 5.83 min. (d) Full scan mass spectrum obtained in the positive ion mode of this peak (indicated by † in c) at 5.83 min. The inset is the magnified view for ions at m/z 271.0616 and 293.0428, which corresponded to the [M + H]⁺ and [M + Na]⁺ of the target constituent, respectively.