Biochanin a

Synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (CHOL), stigmasterol (STIGM), 7-dehydrocholesterol (7DCHOL), desmosterol (DESM), and camposterol (CAMPO) were purchased from Avanti Polar Lipids (Avanti Polar Lipids, Inc., Alabaster, AL, USA). Amphotericin B (AmB), nystatin (NyS), ergosterol (ERG), phloretin, biochanin A, genistein, quercetin, NaCl, KCl, NaOH, KOH, HEPES, EDTA, dimethylsulfoxide (DMSO), triton X-100, calcein, sephadex G-50, ethanol, methanol, chloroform, and hexadecane were obtained from Sigma-Aldrich Company Ltd. (Gillingham, UK). Figure 1 presents the chemical structures of the phospholipids, sterols, antibiotics, and polyphenols used in this study.

Cell lysates were obtained from 3T3-L1 or HepG2 cells. Cells were scraped and lysed with M-PER lysis buffer. After centrifugation for 15 min at 16,000×g, the supernatant was obtained, and protein content was quantified using Bradford reagent. Before drug treatment, the samples were diluted to achieve a protein concentration of 1 mg/mL. Samples were treated with the Kaem or DMSO for 2 h at 25 °C and then incubated with pronase (5, 10, and 20 µg/mL) or distilled water for 10 min at 25 °C. After the reaction, SDS was added to the sample and the samples were heated at 100 °C. A portion of each sample was used for LC–MS/MS analysis. Sample preparation and proteome analysis were conducted as indicated in the previous publication⁶⁹ . For western blot analysis, VDAC1 or Na⁺K⁺ ATPase was used as an internal control. For the structure–activity-relationship (SAR) analysis, kaempferol (Sigma-Aldrich, 60010), Acacetin (Sigma-Aldrich, 00017), isosakuranetin (Sigma-Aldrich, PHL82569), Biochanin A (Sigma-Aldrich, D2016), (−)Epicatechin (Sigma-Aldrich, E4018), Genistein (Sigma-Aldrich, G6649) were used.

Genistein (product number; G6649), daidzein (product number; 16,587), biochanin A (product number; 92,142), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (product number; D9132), 2,2-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) (product number; A3219), sulfanilamide (product number; S9251), naphthylethylenediamine dihydrochloride (NED) (product number; N9125), 3-morpholinosydnonimine hydrochloride (SIN-1) (product number; M5793), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) (product number; 93,285), diethylene-triamine-pentaacetic acid (DTPA) (product number; D1133), Evans blue (product number; E2129), 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH) (product number; 440,914), trichloroacetic acid (TCA) (product number; T6399), 2-thiobarbituric acid (product number; T5500), low density from human plasma (product number; L8292), gallic acid (product number; G7384), ascorbic acid (product number; 1,043,003), potassium persulfate (product number; 216,224), ammonium acetate (product number; A1542), phosphate-buffered saline (PBS) (product number; P3619), and ferric chloride (product number; 451,649) were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). Other reagents used were analytical grade.

Biochanin A (purity >98%) and β-estradiol (E2; purity >98%) were purchased from Sigma-Aldrich (Saint Louis, MO, United States).
Seven days after the surgery, rats were divided into six groups (n = 10 per group): sham, OVX, OVX + BCA (5 mg/kg), OVX + BCA (20 mg/kg), OVX + BCA (60 mg/kg), and OVX + E2 (0.35 mg/kg). BCA and estradiol were dissolved in 10% Tween 80 and 10% ethanol and administered by gavage every morning for 12 weeks. The doses of BCA and E2 and the treatment duration were determined based on previous studies (Aguiar et al., 2006 (link); Biradar et al., 2014 (link); Alauddin et al., 2018 (link)) and our preliminary experiment.
The body weights were monitored once a week for 4 weeks during the experiment.

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.

We employed 5 compounds, previously described as activators of TAS2Rs and EDCs in the literature. Genistein ≥98%, Daidzein ≥98%, Biochanin A and caffeine purchased from Sigma Aldrich (St. Louis, MO, USA) and 3′,4′,7-Trihydroxyisoflavone (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were dissolved in dimethyl sulfoxide (DMSO), ethanol, acetone and H₂O, respectively, and diluted in the media containing Dulbecco’s Modified Eagle Medium (DMEM, 1X), containing fetal bovine serum (FBS) (10%), L-glutamine (1%), penicillin/streptomycin (1%), non-essential amino acids (1%) and Ultra serum (2%), not exceeding a final DMSO concentration of 0.1% (v/v) to avoid the toxic effect of the cells. For acetone and ethanol, the toxicity effect was measured separately.