5α cholestane

All solvents and chemicals were of analytical grade or HPLC/GC grade and purchased from RCI Labscan (Bangkok, Thailand). The total γ-oryzanol (98.5%) standard was purchased from Tsuno Rice Fine Chemical Co., Ltd. (Wakayama, Japan). PC standards, including docosanol (C₂₂), tetracosanol (C₂₄), hexacosanol (C₂₆), octacosanol (C₂₈) and triacontanol (C₃₀), and phytosterol standards, including campesterol, stigmasterol, β-sitosterol, sitostanol, and 5α-cholestane, were purchased from Sigma-Aldrich (St. Louis, MO, USA). N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and pyrogallol were also purchased from Sigma-Aldrich (St. Louis, MO, USA).

All consumables were from VWR (Ismaning, Germany). Derivatization reagents trifluoroacetic anhydride (TFAA), 1-(trimethylsilyl)imidazole (TSIM), and N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) were from Macherey-Nagel (Düren, Germany). Deionized water was prepared with an in-house ion-exchanger. 1,4-Dioxane and methyl tert-butyl ether (MtBE) were distilled before use. β-Glucuronidase/sulfatase from Helix pomatia type HP-2, 5α-cholestane (≥97%), pregnenolone (>98%), pregnenolone sulfate sodium salt (>98%), and cholesteryl sulfate sodium salt (>99%) were purchased from Sigma-Aldrich (Schnelldorf, Germany). Dehydroepiandrosterone sulfate sodium salt (>99%) and 25-hydroxycholesteryl sulfate sodium salt (>99%) were from Avanti Polar Lipids (Alabaster, AL, USA). All other sterol sulfate sodium salts were from Steraloids (Newport, RI, USA). All other reagents and solvents were purchased in HPLC grade or in pro analysis quality from Sigma-Aldrich (Schnelldorf, Germany).

A stock solution of cholesterol (Lipids Cholesterol Rich from adult bovine serum; Sigma-Aldrich, St. Louis, MO, USA) at 10 mg/mL was used to prepare cholesterol assimilation assays and to prepare a 6-point calibration curve as described in Section 2.3.6 [37 (link)]. A stock solution of 5-α-cholestane (Sigma-Aldrich, St. Louis, MO, USA) at 2.5 mg/mL was used as an internal standard in the lipid extractions.

The 16 cultivars of rice (Oryza sativa L.) analyzed in the present study were categorized as non-pigmented (white), black, or red, in accordance with their pericarp color (Figure S3), as follows: WR: Purple check (PC), Dongjin (DJ), and Heugdaegu (HDG); BR: Heughyang (HH), Heugjinju (HJJ), Heugnam (HN), Josengheugchal (JSHC), Maligate Pirurutong (MP), Suwon 493 (SW 493), and Suwon 505 (SW 505); RR: Aengmi (AM), Goryeong 8 (GR8), Hongjinju (HoJJ), Hanyangjo (HYJ), Siga-Chata (SC), and Jagwangdo (JGD). The rice samples were obtained from the Agricultural Genetic Resources Center at the National Academy of Agricultural Science (Suwon, Korea). The seeds were harvested in 2016 and the rice samples were manually hulled. The samples were pulverized on the same day using a mortar and a pestle. The rice powder was stored in a refrigerator at −20 °C prior to analysis. Pyridine, N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), methoxyamine hydrochloride, ribitol, 5α-cholestane, and fatty acid methyl ester (FAME) mixture (C₈–C₂₄) were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). Peonidin-3-O-glucoside and cyanidin-3-O-glucoside were purchased from Extrasynthese (Genay, France). All chemicals and reagents used in the study were HPLC grade.

Pumpkin seeds with husks, harvested at the commercial maturity stage, were obtained from Powdery Health Industry Co., Ltd., Jinmen, China) and used as the raw material for PSO extraction. Standards of fatty acid esters, triacylglycerol; α-, β-, γ- and δ-tocopherols (purity > 95%); squalene; 5α-cholestane; campesterol; stigmasterol; β-sitosterol; Bis(trimethylsilyl)trifluoroacetamide (BSTFA) + Trimethylchlorosilane (TMCS); DPPH, 2,4,6-Tris (2-pyridyl)-s-triazine (TPTZ); ABTS; and fluorescein sodium salt were purchased from Sigma-Aldrich Chemical Co., Ltd. (Shanghai, China). The 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was obtained from TCI Development Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade and were purchased from Sinopharm Chemical Regent (Shanghai, China).