Verbascose

Analytical standards of arabinose, arabitol, L-ascorbic acid, citric acid, fructose, glucose, galactose, (-)-calcium hydroxycitrate tribasic [(-)-HCA], (-)-hydroxycitric acid lactone [(-)-HCAL], muco-inositol, myo-inositol, maltose, maltotriose, maltotetraose, mannitol, pinitol, phenyl-β-glucoside (internal standard), raffinose, sucrose, and verbascose were obtained from Sigma Aldrich (St. Louis, MO, USA). Derivatization reagents including hydroxylamine chloride, anhydrous pyridine, hexamethyldisilazane (HMDS), and trifluoroacetic acid (TFA) were also acquired from Sigma Aldrich (St. Louis, MO, USA).

CV-1, a green monkey kidney cell line (ATCC CCL 70), and HaCaT, a spontaneously transformed human keratinocyte cell line37 (link), were obtained from the American Type Culture Collection and the Cell Lines Service (CLS 300493, Eppelheim, Germany), respectively. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 1.8 mM CaCl₂ supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humid atmosphere of 5% CO₂. NHEKs were obtained from Lonza (Walkersville, MD) and maintained serum free in the keratinocyte basal medium KBM Gold (Lonza) containing 0.3 mM calcium supplemented with KGM-SingleQuot (Lonza). NHEKs were used for experiments within passage 2. Cells at 60 to 70% confluency were treated with vehicle, RFOs, or TO901317. Isomaltotriose and raffinose-series oligosaccharide family members including stachyose, verbascose, and d-(+)-raffinose·5H₂O were purchased from Sigma-Aldrich (St Louis, MO). The synthetic LXR agonist T0901317 was purchased from Cayman Chemical (Ann Arbor, MI).

The concentration of soluble sugars and oligosaccharides in the raw and extruded flours was determined by high-performance liquid chromatography (HPLC), using a procedure described by Muzquiz et al. (1992) [46 (link)] and modified by Pedrosa et al. (2012) [27 (link)]. The extraction was carried out using a 50% v/v aqueous ethanol, and the residue obtained, after evaporation and dryness, was redissolved in 1 mL of double-deionized water and centrifuged for 10 min at 10,000 rpm. After that, the obtained samples were filtered through a 0.45 µm Millipore membrane, and 20 µL of each extract was injected into an HPLC system (Beckman System Gold Instrument, Los Angeles, CA, USA) equipped with a Spherisorb-5-NH2 column (250 × 4.6 mm i.d., Waters, Milford, MA, USA), equilibrated with acetonitrile/water 60:40 (v/v), and a refractive index detector. The flow rate was 1 mL/min and the samples were analyzed in duplicate. Calibration curves for all standard sugar solutions were prepared. Individual sugars were quantified by comparison with external standards of pure sucrose, maltose, raffinose, stachyose. and verbascose (Sigma, St. Louis, MO, USA); ciceritol and galactinol were purified and kindly supplied by Dr. A. I. Piotrowicz-Cieslak (Olsztyn-Kortowo, Poland).