Octacosanol

All standards of fatty acid methyl esters were purchased from Sigma (St. Louis, MO, USA). Methanol, n-hexane, petroleum ether, chloroform, potassium iodide, Folin–Ciocalteu reagent, sodium carbonate, starch, concentrated sulphuric, nitric and hydrochloric acid (37%) were purchased from Kemika (Zagreb, Croatia). Anhydrous sodium sulphate and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) were purchased from Sigma (St. Louis, MO, USA).
Sodium and potassium hydroxides in the form of pellets, chloroform and analytical standards (dihydrocholesterol, campesterol, stigmasterol, β-sitosterol, eicosanol, docosanol, tricosanol, tetracosanol, pentacosanol, hexacosanol, heptacosanol, octacosanol, squalene and (±)-α-tocopherol) were purchased from Sigma (St. Louis, MO, USA). Ethanol and a silylating mixture according to Sweeley were purchased from Fluka (Buchs, Switzerland).
Hydroxytyrosol, tyrosol, oleuropein, lutein and apigenin were purchased from Extrasynthese (Genay, France). Caffeic acid and gallic acid were purchased from Sigma (St. Louis, MO, USA). Standard stock solutions of each compound were 1 mg/mL in Methanol.
All mineral standards were purchased from Merck (Darmstadt, Germany) and diluted to the appropriate obtained concentration.
All standards stored at +4 °C in amber vials and in a dark place until analysis.

E. pela wax—semifinished, first grade—was purchased from the Emeishan Institute of Insect Wax in Sichuan Province, southwestern China.
Sodium hydroxide, hydrochloric acid, chloroform, tetrahydrofuran, and lithium aluminum hydride (analytical grade) were supplied by Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China) A total of four PC standards, tetracosanol, hexacosanol, octacosanol, and triacontanol, were obtained from Sigma-Aldrich (Burlington, MA, USA) with a purity of >98%. Scopolamine hydrobromide was acquired from APExBIO (Houston, TX, USA) and dissolved in sterile normal saline at a final concen-tration of 4 mg/kg. Donepezil was purchased from the American MCE Company (Kowloon, Hongkong) and dissolved in 0.9% sterile normal saline at a final concentra-tion of 3 mg/kg. Sodium carboxymethyl cellulose (CMC-Na) was purchased from Bei-jing Soleibao Technology Co., Ltd. (Beijing, China). Assay kits for the determination of acetylcholine (Ach), acetylcholinesterase (AChE), superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) were purchased from Nanjing Jiancheng Institute of Biological Engineering (Nanjing, China). Normal saline (0.9%) was obtained from Hebei Tiancheng Pharmaceutical Co. Ltd. (Cangzhou, China).

All animal husbandry and animal experiments complied with regulations of the University of Tsukuba for animal experiments and were approved by the Animal Experiment Committee of the University of Tsukuba. Seven-week-old C57BL/6 mice were obtained from CLEA Japan (Tokyo, Japan). Mice were housed in colony cages under a 12 h light/12 h dark cycle, with unlimited access to food and water. At 10 weeks of age, mice were randomly divided into the following four groups: chow group (fed on standard chow with vehicle treatment), HFD group (fed with vehicle treatment), the HFD + octacosanol group (60 mg/kg/day; Sigma-Aldrich, Tokyo, Japan), and HFD + policosanol group (60 mg/kg/day; NOF, Tokyo, Japan). octacosanol and policosanol were dissolved in 10 mg/ml Acacia-gum water. octacosanol, policosanol, or the same volume of vehicle was administered via oral gavage once a day for four weeks. At the end of four weeks, all mice were sacrificed in the early light phase in a non-fasting state. HFD was obtained from Oriental Yeast (Tokyo, Japan) and the composition of HFD is shown in Supplementary Table S1. The policosanol extracted from rice bran oil was obtained from NOF (Tokyo, Japan). The composition of aliphatic alcohols in policosanol is C22; 1%, C24; 9%, C26; 9%, C28; 18%, C30; 21%, C32; 9%, C34; 16%, C36; 14%, and others; 3%.