7 hydroxyflavone

3-Hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone, 6-methoxyflavon, diadzein and resveratrol were obtained from Sigma-Aldrich, USA with a purity of >98%. Di-hydroxylated and polyhydroxylated flavones were obtained from Indofine Chemical Co. (Hillsborough, NJ, USA) with a purity of >98%. Lipopolysaccharide (LPS) from Escherichia coli was obtained from Sigma-Aldrich, USA. All other chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China) or Sigma Aldrich, USA unless otherwise specified. Stock solutions of all flavonoid compounds were prepared in DMSO. All of the experiments were independently repeated at least three times. NMR spectra were recorded with Bruker Avance-400 NMR spectrometer (Madison, WI, USA). Electrospray ionization mass spectroscopy (ESI-MS) analysis was carried out with a Thermo Fisher TSQ Quantum Max Triple Stage Quadrupole mass spectrometer (MA, USA).

Soluble recombinant human TRAIL (rhsTRAIL) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA). DMSO (dimethylsulfoxide), DMF (dimethylformamide) and Triton X-100 were obtained from Sigma Chemical Company (St. Louis, MO, USA). 5-Hydroxyflavone, 6-methoxyflavone, 6-Hydroxyflavone, 7-methoxyflavone and 7-Hydroxyflavone were purchased from Sigma Chemical Company. 6-Hydroxyflavone and 7-Hydroxyflavone were obtained by biotransformation of 6-methoxyflavone, 7-methoxyflavone, 6-Hydroxyflavone and 7-Hydroxyflavone. The flavones were dissolved in DMSO (≤50 mM) or in DMF (50 mM) to a final concentration of 0.1% (v/v) in culture media.

One week after arrival at the University of Maryland School of Medicine's animal facilities, male and female mice were assigned to one of six experimental groups for drug treatment (n = 8 animals per group): (i) vehicle (10% dimethyl sulfoxide in sterile water as done in Xiao et al)²⁷ (ii) Fluoxetine, (iii) arachidonyl serotonin (Arch‐5HT), (iv) biochanin‐A, (v) genistein, and (vi) 7‐hydroxyflavone. Fluoxetine (10 mg/kg; Sigma‐Aldrich, Cat No. #F132) was used as a standard antidepressant comparison for all groups as done in,²⁸ while arch‐5HT (5 mg/kg; Sigma‐Aldrich, Cat No. A7357) was used as a standard FAAH inhibitor as done in.²⁹, ³⁰ Three different doses were selected based on published studies and were tested for each of the isoflavonoid compounds, and the lowest, maximally effective dose (indicated in bold) was selected and analyzed: biochanin‐A (20 mg/kg, 30 mg/kg, 40 mg/kg; Sigma‐Aldrich, Cat No. #D2016)³¹ genistein (10 mg/kg, 20 mg/kg, 30 mg/kg based on³²; Sigma‐Aldrich, Cat No. #G6649), 7‐hydroxyflavone (10 mg/kg, 20 mg/kg, 30 mg/kg; Sigma‐Aldrich, Cat No. #H4530).³³ The effect was measured after chronic administration of drugs, that is, 14 days of compounds/drug treatment followed by forced swim test. The minimum, maximally effective dose based on immobility time in forced swim test was selected.