Savant spd121p

Mouse brains were removed and placed in ice-cold HBSS without Ca²⁺/Mg²⁺. The whole tissue was dissociated in 800 μL of acid saline solution (ASS, 0.9% NaCl in 15 mM HCl) using the pipette tip. After the dissociation, 2 mL of methanol/1 mL of chloroform were added and the sample was vortexed for 20 seconds. Then, 1 mL of chloroform/1 mL of ASS were added, and the sample was vortexed, incubated for 10 minutes in ice and centrifuged at 200g for 5 minutes at 4°C. The upper phase was discharged and the lower phase was mixed with 2 mL of methanol/1.8 mL of ASS. The sample was vortexed, incubated in ice for 10 minutes and centrifuged at 200g for 5 minutes at 4°C. Finally, the lower chloroform phase was dried out at 40°C using a speed-vac concentrator (Savant SPD121P, Thermo Scientific).

Liquid-liquid extraction and saponification of the samples was carried out as previously described by Minguez-Mosquera & Hornero-Mendez [19 (link)]. Briefly, 0.5 g of freeze-dried tissue (peel or flesh) was homogenized in acetone-butylated hydroxytoluene - 0.1% using an UltraTurrax (Ika, Staufen, Germany) and centrifuged (2000 rpm, 10 min, 4 °C). Extraction steps were repeated until complete removal of colour in the sample. The internal standard used was β-Apo-8′-carotenal (Sigma, St Louis, MO, USA). The extracts were combined and treated with diethyl ether. A solution of NaCl (10%, w/v) was added to separate the phases. The lower phase was discarded and the remaining phase was washed with Na₂SO₄ (2%, w/v) to remove water residues. Fifty mL of a methanolic solution of KOH (20%, w/v) was added and left for 1 h in darkness. The organic phase was washed several times with deionized water until washings were neutral. It was then filtered through a bed of anhydrous Na₂SO₄ and evaporated until dry using a speed vacuum (Thermo Scientific Savant SPD121P). The pigments were collected with 1 mL of acetone: methanol (7:3, v/v) and stored at −20 °C until needed. To prevent isomerization and photodegradation of carotenoids, all procedures were carried out under pale light.

A total of 21 samples of Radix Astragali were collected from different places in Gansu, Sichuan, Shanxi and Neimenggu provinces of China (Table 1). Voucher specimens (HQ101-HQ121) were deposited at Northwest Genuine Medicinal Materials Planting Cooperative (Jingyuan, Gansu, China). Powdered material from each plant was macerated in ethyl acetate (Sigma-Aldrich, St. Louis, MO, USA) and sonicated for 30 min. Subsequently, the mixture was shaken for one hour and left overnight. The next day, the mixture was shaken for another 10 min and filtered. The filtrate was evaporated to dryness under reduced pressure at 35 °C using a Savant SPD121P speed vacuum concentrator (Thermo Scientific, Waltham, MA, USA).

The removal of the LC-MS incompatible SDC and remaining lipids was done with an acid precipitation; a method modified from Scheerlink et al, 2015 [77 (link)]. The samples were adjusted to 2% v/v trifluoroacetic acid (TFA), vortexed thoroughly, and incubated at room temperature for 5 min. Next, the samples were centrifuged at 21,130 x g for 10 min, and the supernatant was harvested and vacuum dried (Savant Spd 121P speed vac concentrator, Thermo Scientific, Waltham, Massachusetts, USA). The samples were re-hydrated with 147 μL 50 mM TEAB, and the acid precipitation was repeated to ensure optimal removal of SDC. The samples were then desalted with Pierce™ C18 spin tips (Thermo Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s instructions, and vacuum dried before storage.

Catechins were extracted from oil samples supplemented with GTE and quantified as previously described [34 (link)]. To extract catechins, 0.5 g of oil sample was mixed with 1 mL of 90% ethanol in a 1.5 mL Eppendorf tube, vortexed for 1 min and centrifuged for 5 min at 20,000× g. The ethanolic phase (supernatant) was transferred into a 2 mL Eppendorf tube, and a second extraction of the remaining oil phase was carried out. The supernatants from the two extractions were combined and the ethanol was evaporated under vacuum in a SpeedVac concentrator Savant SPD121P (ThermoFisher Scientific, Asheville, NC, USA). The residue was solubilized in 1 mL or 2 mL of 1% (v/v) formic acid in water depending on the expected concentration of catechins and filtered through a 0.45 µL syringe filter into an HPLC vial. The samples were then analyzed using the reverse-phase HPLC conditions described earlier. The results are expressed in µg/g of oil.

PLGA nanoparticles were prepared by double emulsion based on a previously described procedure [18 (link)]. Briefly, PLGA (PLGA20: 20 mg; PLGA60: 60 mg) was dissolved in 2 mL of ethyl acetate and sonicated for 15 s (70% amplitude) using an ultrasonic homogenizer (Branson Digital Sonicator, Saint Louis, MO, USA), resulting in a w/o emulsion. An equal volume of PVA solution (7% (w/v) in water) was added and sonicated for an additional 30 s (70% amplitude), resulting in a w/o/w double emulsion. The organic solvent was evaporated in a Savant SPD121P vacuum centrifuge (Thermo Fisher Scientific, Waltham, MA, USA) at 10,000 rpm for approximately 1 h at 40 °C. Fluorescent nanoparticles were prepared, adding rhodamine (0.5 mg·mL⁻¹, 1 mg) to the organic solution. To prepare peptide-loaded nanoparticles, 400 µg of PLP139-151 was added to the PLGA organic solution. The prepared nanoparticle suspensions were stored at 4 °C until further use.