Potassium dihydrogen phosphate

Grounded F. vesiculosus samples from July 2017 were purchased from Algaplus Lda (production site located at Ria de Aveiro coastal lagoon, Northern Portugal, 40°36′43″ N, 8°40′43″ W) an enterprise dedicated to the production of edible seaweeds in an integrated multi-trophic aquaculture (IMTA) system. HPLC grade acetone, ethanol, methanol, n-hexane, ethyl acetate, acetonitrile, dimethylsulfoxide, hydrochloric acid, glacial acetic acid, sodium chloride, sodium hydroxide, potassium hydroxide, sodium and potassium tartarate, tris-HCl, and starch were acquired from Fisher (Pittsburgh, PA, USA). The enzymes α-glucosidase from Saccharomyces cerevisiae (EC No. – 3.2.1.20), α-amylase from porcine pancreas (EC No. – 3.2.1.1) and lipase from porcine pancreas (EC No. – 3.1.1.3) together with 2,4-dimethoxybenzaldehyde (DMBA), 4-nitrophenyl α-D-glucopyranoside (PNPG), 4-nitrophenyl butyrate (PNPB) and formic acid were purchased from Sigma (St. Louis, MO, USA). Sodium di-hydrogen phosphate and potassium di-hydrogen phosphate were acquired from Panreac (Barcelona, Spain). Dinitrosalycilic acid and Acarbose were purchased from Acros Organics (Hampton, NH, USA), calcium chloride from ChemLab (Eernegem, Belgium) and orlistat from AlfaAesar (Ward Hill, MA, USA). All reagents were of analytical grade or of the highest available purity.

Angiotensin-converting enzyme (ACE, from human, 95%), hydrochloric acid (HCl, ACS reagent, 37%), trisodium citrate dihydrate (C₆H₅Na₃O₇·2H₂O, 99%), fluorescein, 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH), sodium carbonate (Na₂CO₃), 3,5-Dinitrosalicylic acid (DNS) color reagent, aluminium chloride (AlCl₃), sodium nitrite (NaNO2 ACS reagent, 97.0%), α- and β-glucosidase, α- and β-pNPG, and α-amylase were acquired from Sigma-Aldrich (Buchs, Switzerland). Potassium phosphate dibasic trihydrate (K₂HPO₄·3H₂O) was acquired from Merck (Buchs, Switzerland), ethanol (EtOH, absolute PA, 99.5%), potassium dihydrogen phosphate (KH₂PO₄, 99.5%), sodium chloride (NaCl, 99.8%), N-[3-(2-furyl)acryloyl]-Phe-Gly-Gly (FAPGG) and sodium hydroxide (NaOH) were acquired from Panreac (Barcelona, Spain). Folin-Ciocalteu solution, 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, 98%), and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) were acquired from Fluka (Munich, Germany). Acetonitrile (ACN) and methanol (MeOH) (both HPLC grade, 99.99%) from Thermo Fisher Scientific (Loughborough, UK), and PSA/C18/Mg₂SO₄ (25/25/150 mg, DisQuE) was purchased from Waters (Milford, MA, USA). The ultrapure water used on the assays was obtain using an Ultrapure water purification system (Milli-Q^® Direct 8 at 18 MW cm, 23 °C, Millipore Corporation, Burlington, MA, USA).

HPLC-grade Acetonitrile and water were purchased by Panreac (Madrid, Spain). Standards of kaempferol, myricetin, quercetin, rutin, caffeic, chlorogenic, and protocautechic acids of HPLC-grade were purchased by Sigma-Aldrich (Madrid, Spain). We also acquired CCCP from Sigma (C2759).
Tert-butylhydroperoxide (t-BOOH), glutathione reductase (GR), reduced (GSH) and oxidized (GSSG) glutathione, dichlorofluorescin (DCFH), o-phthaldialdehyde (OPT), nicotine adenine dinucleotide phosphate reduced salt (NADPH), 2,4-dinitrophenylhydrazine (DNPH), H₂O₂, 1,1,3,3-tetraethoxypropane (TEP), gentamicin, penicillin G, and streptomycin were purchased from Sigma Chemical Co. (Madrid, Spain), and rapamycin was purchased from Merck (number 553,210). Acetonitrile, methanol of HPLC grade, dimethyl sulfoxide (DMSO) of analytical grade, sodium hydroxide, sodium chloride, di-sodium hydrogen phosphate anhydrous, potassium di-hydrogen phosphate, as well as formic, hydrochloric, perchloric, and sulfuric acids were acquired from Panreac (Barcelona, Spain). Bradford reagent was from BioRad Laboratories S.A. DMEM culture media and fetal bovine serum (FBS) were from Cultek (Madrid, Spain). All other reagents were of analytical quality.

Nifedipine (>98%, NFD) was obtained from Kemprotec Limited (Carnforth, UK), while Eudragit L-100-55 (methacrylic acid-ethyl acrylate copolymer 1:1) was donated by Evonik Rohm GmbH (Essen, Germany). Cyclic olefin copolymer filaments (transparent, 1.75 mm diameter size) (COC) were obtained from Creamelt^® 3D Printing Materials (Rapperswil-Jona, Switzerland) and UV polymerizable resin (405 nm) was obtained from Anycubic^® (Shenzhen, China). Deoxycholic acid sodium salt (>98%) was obtained from Fluka Biochemica (Cleveland, OH, USA). Solvents such as methanol, ethanol, dimethylsulfoxide, and other chemicals such as sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, and polysorbate 80 (Tween 80) were all of ACS grade or above and were purchased from Panreac (Barcelona, Spain). Cellulose acetate dialysis membranes with a molecular weight cut-off of 3500 Da were purchased from Medicell Ltd. (London, UK). The Anycubic Photon Mono X (LCD-based SLA printer, 405 nm light source, 0.05 mm 3840 × 2400 XY resolution, 0.01 mm Z resolution, 192 × 120 × 245 mm build volume) and Anycubic Mega Zero (FDM printer, 0.1 mm layer resolution 0.125 mm XY resolution, 0.4 mm nozzle diameter) were obtained from Anycubic^® (Shenzhen, China).

Melatonin (M5250-5G) and dexamethasone (D1756-5G) were purchased from Sig-ma-Aldrich (Madrid, Spain). Poly(D,L-lactic-co-glycolic) (PLGA) acid 50:50 (D170500523; Resomer^® 503; 24,000–38,000 g/mol) was purchased from Evonik Industries (Essen, Ger-many). Sodium chloride was obtained from Merck (1.06404.1000, Merck KGaA, Darmstadt, Germany). Potassium di-hydrogen phosphate (131509.1211), di-sodium hydrogen phosphate 12-hydrate (131678.1211), polyvinyl alcohol 72,000 g/mol (A2255,0250), methanol (221091.1612), ethanol (221086.1612), acetonitrile (221881.1612) and dichloromethane stabilized with 20 ppm of amylene (361254.1612) were acquired from PanReac AppliChem (Barcelona, Spain). All solvents cited were HPLC grade. Water was purified from a Milli-Q^® filtration system (Millipore Corporation, Billerica, MA, USA). Acetone and all other chemicals were reagent grade and were used as received.

Liquid Sauton media were prepared by sequentially adding, with constant agitation, the following compounds: citric acid (Fluka Chemika, Steinheim, Germany) 2 g/L, potassium dihydrogen phosphate (Panreac, Barcelona, Spain) 0.5 g/L, ferric ammonium citrate (Sigma-Aldrich, St. Louis, USA) 0.05 g/L, magnesium sulphate heptahydrate (Fluka Chemika) 1 g/L, zinc sulphate (Panreac), and 2–4 g/L L-asparagine (Scharlau, Sentmenat, Spain) or L-glutamate (Panreac). Finally, glycerol (from 15 to 60 mL/L) was added (Panreac), and the pH was adjusted to 7.2–7.3. All media were sterilized by autoclaving for 15 min at 121 °C. Middlebrook 7H10 medium (Becton and Dickinson, Le Pont de Claix, France) supplemented with 10% oleic albumin dextrose catalase enrichment medium was prepared as described previously [7 (link)], and Middlebrook 7H9 medium (Becton and Dickinson, Le Pont de Claix, France) was supplemented with 10% albumin dextrose catalase enrichment medium.