Chromabond c18

The model mixture was mixed with deionized water to reduce the ethanol content to 10% (v/v) and percolated through a SPE cartridge after conditioning with acetonitrile and equilibrating with an ethanol/water 9:1 (v/v) solution. Cartridges tested for extraction of flavor compounds were as follows: Chromabond C18 (500 mg) and Chromabond hydrophilic-lipophilic balanced HLB (200 mg) (Macherey–Nagel, Düren, Germany), Supelclean LC−8 and LiChrolut EN (Merck, Darmstadt, Germany). Compounds of interest were eluted with dichloromethane (2 × 3 mL), which was evaporated to 500 µL under a nitrogen flow.

Amino acids were extracted 24 h after the solution treatment (BABA, L-Asp, L-Gln, BABA:Asp, and BABA:Gln). A 1 mL of extraction buffer consisting of 0.1 M HCl with the addition of 4.6 μg/mL 2-aminoadipic acid as an internal standard was added to 100 mg of leaf powder, mixed thoroughly, and incubated on ice for 5 min. Samples were centrifuged for 10 min at 4 °C and 15,000× g. A total of 500 μL of supernatant was diluted with 100 μL of methanol. Subsequently, solid-phase extraction was performed using Macherey-Nagel Chromabond^® C18, 100 mg of solid-phase per 5 mL colony. The colony was washed with 1 mL of methanol and then with 1 mL of equilibration buffer (20% MeOH in 0.1 M HCl). Samples (600 μL) were applied to the column, and the column was washed with 400 μL of equilibration buffer, resulting in a volume of 1 mL of extracted amino acids. Extracted amino acids were derivatised and analysed, as described previously [46 (link)].

For the preparation of the enriched fraction for the LC-HRMS screening, one stored deep frozen agar plate cultures of S. ampullosporum Damon strain KSH534 was crushed in small pieces and extracted with EtOH 96% (2 × 250 mL) in an ultrasonic bath at room temperature. The resulted yellow solution was evaporated in vacuo to dryness. The dried crude extract was redissolved in EtOH 96%/H₂O (1:2, v/v) to a concentration of 50 mg/mL. The resulting solution was separated on SPE cartridges Chromabond^® C18 (loading 200 mg/3 mL, particle size 45 µm; Macherey-Nagel, Düren, Germany), targeted peptaibols 1–3 were eluted with EtOH 96%. After evaporation to dryness in vacuo, the enriched fraction was redissolved in CH₃CN and submitted to LC-HRMS.

LCMS-grade methanol, acetonitrile, water, and formic acid were purchased from Fisher Scientific (Hampton, NH, USA), while reserpine (>99%, HPLC-grade) and fluoxetine hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). The standards, daidzein (≥98%), genistein (≥98%), biochanin A (≥98%), and formononetin (≥98%) were purchased from ChemFaces (Wuhan, China). Phosphate buffer saline (PBS) buffer (pH 7.4) was purchased from R&M Chemicals (Petaling Jaya, Malaysia). The solid-phase extraction (SPE) cartridge used was a silica-based Chromabond C18 polypropylene column (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany). Holding water for the zebrafish during acclimatization and for experimental procedures was sourced from Coway-filtered tap water treated with water conditioner (Tension gon, Mydilab, Pulau Pinang, Malaysia). The syringe and needle (Ultra-Fine II, 0.30 mm (30 G) × 8 mm (5/16″), 0.5 mL) used for intraperitoneal (IP) injection were obtained from Becton Dickinson, Franklin Lakes, NJ, USA. All tanks used were standard plastic tanks (3 L, 5 L, and 7 L) bought from 3B Aquatics, Bangi, Malaysia.

SPE cartridges (Chromabond C18, 500 mg, 3 mL) were from Macherey-Nagel (Düren, Germany). Amberlite XAD-2 column material was from Supelco (Bellafonte, Pennsylvania, USA). Sephadex LH-20 material was from Sigma.