Hispidin

The hispidin was first isolated from Polyporus hispidulus, and the production and structure elucidation came from the mycelial broth of Phellinus linteus [15 (link)]. It can also be synthesized [40 (link)]. In this study, hispidin (C₁₃H₁₀O₅, 98%, soluble in DMSO at 20 mg/mL), MTT, and DCFDA were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The Nrf2 (ab52352) antibody was purchased from Abcam (Cambridge, MA, USA), and all other antibodies were purchased from GeneTex Inc. (San Antonio, TX, USA).

Methanol and ethanol of analytical grade (Fisher Chemical, Loughborough, UK) were used as extraction solvents. Folin-Ciocalteu, gallic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2-diphenyl-1-picrylhydrazyl (DPPH), sodium carbonate, potassium persulfate, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), acetic acid, hydrochloric acid, ferric chloride, acetonitrile, and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louise, MO, USA). All the organic solvents were HPLC grade. Ultrapure water from a purified system (Isopad Isomantle, Gemini BV, Pr. Beatrixlaan 301, 7312 DG Apeldoorn, The Netherlands) was used. External standards (purity ≥ 99%) caffeic acid, catechin, chlorogenic acid, diosmetin, ferulic acid, glycitin, isorhamnetin, (-)-epicatechin, luteolin-7-O-glucoside, luteolin-4′-O-glucoside, myricetin, naringin, p-hydroxybenzoic acid, quercetin, and rutin were purchased from Extrasynthese (Lyon Nord, Genay Cedex, France). Hispidin (purity ≥ 98%) was purchased from Sigma-Aldrich.

Methanol, n-hexane, ethyl acetate, n-butanol, petroleum ether, isopropanol, chloroform, dichloromethane, diethyl ether and dimethyl sulfoxide (DMSO) were of reagent-grade and purchased from Merck (Darmstadt, Germany). The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from Fluka (Buchs, Switzerland). Acetonitrile (LC/MS-grade), butylated hydroxytoluene (BHT), potassium hexacyanoferrate (K3Fe(CN)6), ropidium iodide (PI), Folin–Ciocalteu reagent, trypan blue, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Oil Red O solution 0.5% in isopropanol, Nile red (BioReagent, suitable for fluorescence), 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), 2′,2′-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS^•+), hispidin and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and trypsin–EDTA solution were provided by Hyclone (Logan, UT, USA).
A series 1260 Infinity HPLC system equipped with a model G1379B degasser, model G1312B binary gradient pump, model G1329B autosampler, model G1316A column oven, model G1315D photodiode array detection (PDA) system and a triple quadruple mass spectrometer 6420A were purchased from Agilent Technologies (Santa Clara, CA, USA).