Choline chloride

For NADES preparation, chemicals choline chloride, 1,2-propanediol, DL-malic acid, malonic acid, D-sorbitol, urea (all Thermo Fisher Scientific, Waltham, MA, USA), citric acid monohydrate, glycerol, D(−)-fructose (all Carl Roth, Karlsruhe, Germany), D(+)-glucose (Merck, Darmstadt, Germany) and methylurea (Acros Organics, Geel, Belgium) were used. Methylurea had a specified purity of 97%, and all other reagents were at least 98% or higher. In HPLC mobile phases, ultrapure water prepared from deionized water with a Barnstead MicroPure system (Thermo Fisher Scientific, Waltham, MA, USA), HPLC-grade acetonitrile (VWR, Radnor, PA, USA) and formic acid (Honeywell, Charlotte, NC, USA) were used. The solvent for NMR analyses was chloroform-d₁ (VWR). Other solvents used in this study were partly denatured ethanol (96% v/v, AustrAlco, Spillern, Austria) methanol (≥99.9%, Carl Roth) and dichloromethane (≥99.5%, Carl Roth). Reference substance spilanthol was isolated in the course of this work with a purity of 97% as determined by NMR.

A simplified outline of treatments and cell culture is presented in Figure 1. The doses of choline treatment were selected based on basal concentrations (4 μM) of free choline in plasma of cows during the first week of lactation (21 (link)) and plasma concentrations achievable with abomasal choline infusion (22 (link)). Choline chloride (Sigma Aldrich Inc.) was used as a source of supplemental choline. The commercial media used was selected for its relatively low concentration of Choline chloride (0.5 mg/L; Media-199, Gibco, ThermoFisher Scientific). The basal media contained 10% FBS (Gibco™, ThermoFisher Scientific), for which choline concentration was not available. Considering only the contribution of M-199 media, the concentration of choline ion in the basal media was 3.2 μM. The choline treatments provided an additional 5 or 10 μM choline. Therefore, the final concentrations of choline, accounting for the choline present in the basal media, were 3.2 (CHO3), 8.2 (CHO8), and 13.2 (CHOL13) μM. Because Choline chloride was used to provide the supplemental choline in CHO8 and CHO13, these treatments also added 5 and 10 μM additional chloride. However, the additional chloride is a tiny fraction of the 125 mM concentration in M-199 media or the 107 mM concentration in bovine plasma (27 (link)), suggesting that the additional chloride was not impactful.

In this research, analytical grade chemicals for DESs synthesis namely choline chloride, lactic acid, 1,3-butanediol and oxalic acid were purchased from Alfa Aesar (Thermo Fisher Scientific). EFB is one of the oilseed crop residues found abundantly in Southeast Asia^{48 (link)}. It was supplied from a Palm Oil Mill, Thailand. Commercial cellulose, sodium chlorite, and acetic acid were acquired from Sigma-Aldrich.

The methanol and acetonitrile used were HPLC (high-performance liquid chromatography) grade and purchased from Fisher Scientific, as well as the potassium sodium tartrate tetrahydrate used for reducing sugar determination and D-glucose standard (Madrid, Spain). Choline chloride, betaine, 1,2-propanediol, and albumin standard were from Thermo Fisher Scientific (Madrid, Spain), and triethylene glycol and Trolox standard were supplied by Acros Organics (Antwerp, Belgium). The HPLC standards, trifluoroacetic acid, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma Aldrich (St. Louis, MO, USA)l; Folin-Ciocalteu reagent and ethanol were obtained from Scharlau (Barcelona, Spain); and 3,5-dinitrosalicylic acid was purchased from Alfa Aesar (Kandel, Germany). Mueller–Hinton Broth culture media and bacteriological agar for microbiological assays were supplied by Oxoid (Basingstoke, Hampshire, UK).

Dead leaves of red maple (Acer rubrum) were collected from the ground in Houghton, Michigan, in October 2021. The leaves were thoroughly washed with pure water, laid out to dry at room temperature, and then ground into powders (≤300 μm). Three other types of dead leaves, namely, those of the Cercis canadensis, Quercus rubra, and Acer platanoides, were collected and treated in the same manner. Choline chloride (≥98%), oxalic acid dihydrate (98%), calcium oxalate monohydrate (99%), hydrochloric acid (36% solution), nitric acid (68-70% solution), and hydrogen peroxide (30% solution) were purchased from Thermo Fisher Scientific. Sodium sulfate (≥99%), sodium sulfite (≥98%), sodium hydroxide (≥97%), sodium chlorite (80%), sulfuric acid (95-98%), acetic acid (≥99.7%), tetracycline (98.0-102.0%), methanol (≥ 99.9%), chloroform (≥99.8%), magnesium oxide (97%), chloroplatinic acid hexahydrate (≥37.50% Pt basis), zinc oxide (99.99%), titanium (IV) oxide (P25, ≥99.5%), tungsten (VI) oxide (<100 nm particle size), cerium (IV) oxide (99.95%), zirconium (IV) oxide (99%), gallium (III) oxide (≥99.99%), molybdenum (IV) sulfide (99%), and tungsten (IV) sulfide (99%) were acquired from Sigma Aldrich.

BSG or MD biomass were obtained from Alken-Maes brewery (Alken, Belgium) and immediately dried at 65 °C until the residual water content was approximately at or below 5 wt%. Malic acid, glycerol, and choline chloride, used to prepare NADES formulations, were obtained from Thermo Fischer Scientific (Waltham, MA, USA). For the HPLC analysis, ferulic acid (FER; Thermo Scientific, Waltham, MA, USA), caffeic acid (CAFF; Thermo Scientific, Waltham, MA, USA), p-coumaric acid (COUM; Thermo Scientific, Waltham, MA, USA), and syringaldehyde (SYR; TCI chemicals, Tokyo, Japan) were used as standards for prevalent phenolic compounds. LC-grade formic acid (LiChropur; Merck, Darmstadt, Germany) was used to acidify the samples for HPLC analysis. Analytical reagent grade acetone (AnalaR NORMAPUR) and analytical reagent grade methanol (HiPerSolv CHROMANORM for LC-MS) were obtained from VWR (Radnor, PA, USA). Ultrapure (UP) water (18 MΩ·cm) was obtained using an Arium Pro System (Sartorius, Göttingen, Germany). Fresh UP water was prepared every day to prevent impurities.

Choline chloride was purchased from Thermo Fisher (Waltham, MA, USA). Daidzein (>98% purity) and Genistein (>95% purity) were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). Commercial antibodies, including their sources, concentrations or dilutions used, vendors, and RRID numbers, are listed in Table 5. Bicinchoninic acid (BCA) reagents, horseradish peroxidase (HRP)-conjugated secondary antibody, superblock (TBS), and enzyme-linked immunosorbent assay (ELISA). MaxiSorp 96-well plates were purchased from Thermo Fisher Scientific (Bedford, MA, USA). Amplex red soluble fluorophore and 4-Methylumbelliferyl phosphate (4-MUP) were purchased from Life Technologies (Carlsbad, CA, USA). Alkaline Phosphatase Streptavidin and the Proton Biotin Protein Labeling Kit were purchased from Vector Laboratories Inc. (Newark, CA, USA). Total and Phospho-Akt/mTOR Pathway panels were purchased from MilliporeSigma (Bedford, MA, USA). The Luminex MAGPIX system was purchased from Luminex Corp. (Austin, TX, USA). The SpectraMax M5 microplate reader was purchased from Molecular Devices Corp. (Sunnyvale, CA, USA).