Xanthohumol

Xanthohumol purchased from Sigma-Aldrich (Darmstadt, Germany) with purity of >98% was recrystallized from acetonitrile. Nicotinamide, glutarimide, acetamide, caffeine, and all used solvents were of analytical grade and were purchased from Sigma-Aldrich. The compounds used as coformers are included in the list of compounds authorized for the safe use as additives to food products, pharmaceutical preparations, and dietary supplements (so-called GRAS list (“Generally Recognized as Safe”) created by the FDA-American Food and Drug Agency).
Synthesis of the cocrystals. Xanthohumol and coformers at the 1:1 molar ratio were dissolved in acetonitrile at 30 °C. The concentrated solution was kept for 2 days at room temperature to obtain yellow crystals (with a plate and prismatic shapes) using the slow evaporation solution growth technique. Then crystals were collected from the crystallization vessels.

ACS-grade methanol for extraction, LC-MS Optima^TMacetonitrile, and UHPLC-MS/MS Optima^TM grade solvents (water, acetonitrile, methanol) were from Fisher (Thermo Fisher Scientific, Nepean, ON, Canada). SupraPur^TM formic acid (98%–100%) and ACS grade isopropyl alcohol were from VWR (VWR International LLC, Mississauga, ON, Canada). Commercially available analytical standards were sourced from Extrasynthese (Genay, Cedex, FR) (quercetin-3-O-glucoside ≥ 99%; kaempferol-3-O-rutinoside ≥ 98%; quercetin-3-O-rhamnoside ≥ 98.5%) or Sigma-Aldrich (St. Louis, MO, USA) (quercetin-3-O-rutinoside ≥ 95%; kaempferol-3-O-glucoside ≥ 97%; xanthohumol ≥ 96%; quercetin-3-O-(6’’-O-malonyl)-glucoside ≥ 85%). International Calibration Extract 3 (ICE-3), an alpha & beta acid standardized mixture, was sourced from Labor Veritas (AG, Zurich, CZ). Deuterated solvent (DMSO-d₆) was from CIL (Cambridge Isotope Laboratories Inc., Tewksbury, MA, USA). Pyridine, o-tolyl isothiocyanate, L-cysteine methyl ester, D-glucose, L-glucose, D-galactose, L-galactose were purchased from Sigma-Aldrich.

Dulbecco’s Modified Eagle Medium (DMEM) high glucose and Eagle’s Minimum Essential Medium (EMEM) were obtained from Biosera (Nuaille, France). Xanthohumol (XH), isoXanthohumol (IXH), 6-prenylnaringenin (6-PN) 8-prenylnaringenin (8-PN), naringenin (NAR), oxaliplatin (OxPt), 5-fluorouracil (5-FU), irinotecan (IRT), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer, phosphate-buffered saline (PBS), 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), DL-dithiothreitol (DTT), and neutral red were purchased from Sigma–Aldrich (Prague, Czech Republic). Fetal bovine serum (FBS) and gentamicin sulfate were supplied by Invitrogen (Carlsbad, CA, USA), and bovine serum albumin (BSA) by Fluka (Prague, Czech Republic). All other chemicals were of HPLC or analytical grade (Sigma–Aldrich, Czech Republic). Stock solutions of prenylflavonoids and naringenin were prepared in dimethyl sulfoxide (DMSO) and stored at 4 °C in the dark.

Caffeic acid, p-Coumaric acid, naringinen, chlorogenic acid, quercetin, isoquercetin, umbelliferone, harmine, luteolin, caffeine, 2,4,4′-trihydroxy chalcone, naringinin-7-glucoside, pseudoephedrine, glycyrrhizin, epicatechin, vanillic acid, gallic acid, β-glycyrrhetenic acid, xanthohumol and amentoflavone were purchased from Sigma Aldrich (St. Louis, MO, USA). Bisabolene, α-ionone, (Z)-3-hexenyl acetate, allyl anthranilate, isoeugenol, α-pinene, cuminaldehyde, thymol, caryophyllene oxide, carvone, and methyl disulphide were provided from Bedoukian Research (Danbury, CT, USA). Cynarin, emodin, rutin, and sennoside A were purchased from Chromadex (Wesel, Germany). Khellin was a gift from Prof. Dieter Treutter (Tech Univ. of Munich, Germany). Sarcophine was isolated from Sarcophyton ehrenbergi soft coral whereas vitexin and chrysin were isolated from Passiflora edulis leaf in Dr. Farag laboratory, Cairo University (Supplementary Fig. 6).

An experiment on the receptor binding mode by the antagonist was carried out in a pentobarbital-induced sleep model. The normal control group (NOR) was treated with saline, and the Saaz–Saphir mixture (75:25) was administered at 150 mg/kg and xanthohumol (Sigma-Aldrich, St Louis, MO, USA) and humulone (Aobious, Gloucester, MA, USA) at 20 mg/kg. Using edible oil, 6 mg/kg of bicuculline (Sigma-Aldrich) and 4 mg/kg of picrotoxin (Sigma-Aldrich) were intraperitoneally injected (i.p.) 30 min before hop extract administration [31 ]. Sleep latency and sleep duration were measured under the same conditions as in the pentobarbital sleep induction experiment.