Ethyl acetate

The peptides PSELT (H-Phe-Gln-Ile-Cys-Val-Ser-Sec-Gly-Tyr-Arg-OH), [Ser46,49] PSELT [called inert PSELT (I-PSELT) as a control], and PSELT-dansyl were synthesized by Fmoc solid phase methodology on an automated peptide synthesizer (CEM, Saclay, France) as previously described [19 (link)]. KCl, NaCl, NaHCO₃, CaCl₂, MgSO₄, KH₂PO₄, NaH₂PO₄, Na₂HPO₄, mannitol, glucose, Na-pyruvate, β-nicotinamide adenine dinucleotide (NADH), reduced disodium salt hydrate, 2,4 dinitrophenylhydrazine (DNPH), 2-thiobarbituric acid (TBA), bovine serum albumin (BSA), butylated hydroxyanisole, diethyl ether, ethylenediaminetetraacetic acid (disodium salt), diethylenetriamine pentaacetic acid, pyrogallol, streptomycin sulfate, tween-20, hydrogen peroxide (H₂O₂), and urea were purchased from Sigma Aldrich (St. Louis, MO, USA). Tunicamycin (TM) was from Cayman Chemical (Ann Arbor, MI, USA), Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12), penicillin/streptomycin and fetal bovine serum (FBS) were provided by Thermo Fisher Scientific (Waltham, MA, USA). All the solutions were freshly prepared before starting the experiments. Absolute ethanol, ethyl acetate, hydrochloric acid, methanol, and trichloroacetic acid (TCA) were from Carlo Erba Reagents (Cornaredo, Milano, Italy).

DPPH (1,1-diphenyl-2-picryl-hydrazyl radical), Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), BHT (butylhydroxytoluene), TBA (2-thiobarbituric acid), TMP (1,1,3,3-tetra-methoxy-propane), DNPH (di-nitrophenol hydrazine), and BSA (bovine serum albumin) were purchased from Sigma-Aldrich (Steinheim, Germany).
Guanidine hydrochloride, TCA (trichloroacetic acid), acetonitrile, ethanol, ethyl acetate were purchased from Carlo Erba Reagents (Val de Reuil Cedex, France).

Fenugreek seed powder was received from Herbal Acharn’s Home Co., Ltd. (Bangkok, Thailand). Rutin trihydrate (>98% purity), tricine buffer, collagenase from clostridium histolyticum and FALGPA (N-[3(2-furyl) acryloyl]-Leu-Gly-Pro-Ala), direct red 80, picric acid, and dimethyl sulfoxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile, ethyl acetate, absolute ethanol, formic acid, sodium dehydrate phosphate, disodium hydrogen phosphate, hydrochloric acid, and sodium hydroxide were purchased from Carlo Erba (Emmendingen, Germany). Commercial grade ethanol was purchased from Italmar Co., Ltd. (Bangkok, Thailand). Cholesterol was purchased from Cosmeplus Co., Ltd. (Bangkok, Thailand). Propylene glycol and solubilizer mixture were purchased from S. Tong Chemicals Co., Ltd. (Nonthaburi, Thailand). Sorbitan oleate was purchased from Croda Co., Ltd. (Bangkok, Thailand). Phospholipid (Phospholipon 90G) was purchased from Cargill Siam Ltd. (Bangkok, Thailand). Tocopherol acetate was purchased from Namsiang Co., Ltd. (Bangkok, Thailand). The preservative was purchased from Forecus Co., Ltd. (Bangkok, Thailand). Paraformaldehyde was purchased from Himadia Laboratories (Mumbai, India) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was purchased from Calbiochem (Burlington, MA, USA).

Formic acid, ethyl acetate, ethanol, methanol, UPLC-grade acetonitrile were from Carlo Erba Reagents (Italy). Anhydrous sodium sulphate, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ammonium persulfate, Tris-HCl, EDTA, phosphate-buffered saline, SDS, β-mercaptoethanol, NaCl were from Sigma Aldrich.

Chemical standards of phenolic compounds (benzoic acid, carvacrol, catechin, chlorogenic acid, t-cinnamic acid, 8-cinnamoyl harpagide (harpagoside), o-coumaric acid, p-coumaric acid, 2,3-dimethoxybenzoic acid, epicatechin, t-ferulic acid, gallic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 3-hydroxy-4-methoxybenzaldehyde, naringin, naringenin, quercetin, rutin, sinapinic acid, syringic acid, vanillic acid (all purity > 98%)), β-cyclodextrin (≥97%), n-hexane (HPLC-grade), diethyl ether (≥99%), and chloroform (HPLC-grade) were purchased from Sigma-Aldrich (Milan, Italy).
Ethyl acetate (≥99%), acetonitrile (HPLC-grade), methanol (HPLC-grade), ethanol (HPLC-grade), acetic acid (≥99%) as well as d-(+)-glucose were obtained from Carlo Erba Reagents (Milan, Italy). Sodium chloride (≥99%) was obtained from Honeywell (Seelze, Germany). NADES (glycolic acid/betaine mixture) was newly synthesized and supplied by University of Perugia. It was chosen between differently structured novel DES and NADES mixtures for its suitable properties (low freezing point and low viscosity, absence of aromatic compounds in its composition, low cost and natural source of the molecules forming it). Ultra-pure water was obtained using a Millipore Milli-Q Plus water treatment system (18 MΩ cm at 23 °C, Millipore Bedford Corp., Bedford, MA, USA).