Pancreatic lipase

The pancreatic lipase (Sigma-Aldrich, UK) inhibitory activity was determined as described by Bustanji et al. (2010 ) with modifications. A volume of 50 μL pancreatic lipase solution (1 mg/mL in 2.5 mM tris-hydrochloride buffer pH 7.4 with 0.125 mM sodium chloride) was pre-mixed with 100 μL sample solution (initial concentrations used ranged from 5000 to 156.25 μg/mL for crude extracts and 1000 to 31.25 μg/mL for fractions) and incubated at 37 °C for 15 min. Following pre-incubation, 100 μL p-nitrophenyl butyrate (PNPB) (25 mM) was added to the enzyme-sample mixture and the volume was made up to 300 μL using tris-hydrochloride buffer. The reaction mixture was incubated at 37 °C for 60 min and the amount of p-nitro-phenol released was measured at 405 nm. The % inhibition and IC₅₀ were calculated as previously described. The kinetics of inhibition was calculated by increasing the concentration of PNPB and kinetic parameters were calculated from Line-weaver Burke plots.

The solvents used in this study were obtained from VWR International s.r.l. (Milan, Italy). Gallic acid, caffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid, ellagic acid, quercetin, catechin, rutin, ascorbic acid, propyl gallate, butylated hydroxytoluene (BHT), β-carotene, linoleic acid, pancreatic lipase, Tween 20, sodium potassium tartrate, sodium chloride, sodium carbonate, Folin-Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ), o-dianisidine (DIAN) color reagent, peroxidase-glucose oxidase (PGO), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, (ABTS) solution, sodium acetate, β-carotene, linoleic acid, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Dulbecco’s Modified Eagle Medium (DMEM), dimethyl sulfoxide (DMSO), and Fetal Bovine Serum (FBS) were purchased from Sigma-Aldrich s.r.l. (Milan, Italy). l-Glutamine and penicillin/streptomycin were purchased from Gibco, Life Technologies (Waltham, MA, USA).

Ultrapure water (˂5 µg/L TOC) obtained by a water purification system (Mili-Q Merck Millipore, Chile) was used. For mass spectrometry analysis, HPLC-grade methanol and MS-grade formic acid were used, obtained from J.T. Baker (Phillipsburg, NJ, USA). Gallic acid, Folin Ciocalteu commercial reagent, 2,4,6-tris(2-pyridyl)-s-triazine, sodium carbonate, acetic acid, ferric chloride hexahydrate, sodium acetate, Trolox, hydrochloric acid, 2,2′-Azobis(2-amidinopropane) dihydrochloride, absolute ethanol, fluorescein solution, phosphate buffer, acetylcholinesterase enzyme (AChE), butyrylcholinesterase enzyme (BChE), galantamine, Tris-HCl buffer, Ellman’s reagent (DTNB), acetylcholine, butyrylcholine, magnesium chloride, sodium chloride, alpha-glucosidase, 4-nitrophenyl α-d-glucopyranoside, acarbose, orlistat, pancreatic lipase, 4-nitrophenyl-dodecanoate, dimethyl sulfoxide (DMSO) and HPLC standard with a purity greater than 95% (usnic acid and atranorin) were obtained from Sigma (Sigma, St. Louis, MO, USA).

Spanish cocoa butter was purchased from Sunin Co. (Yongin, Korea). Palm stearin and shea butter were obtained from Ottogi Co. (Anyang, Korea) and Korea Similac (Pochun, Korea), respectively. HPLC-grade n-hexane (>99.5%), acetone (>99.3%), acetonitrile (>99.9%), and dichloromethane (>99.9%) were procured from J.T. Baker (Phillipsburg, NJ, USA). Silica gel–coated glass plates (20 cm × 20 cm, 0.25 mm thick) for thin-layer chromatography (TLC) were purchased from Merck KGaA (Darmstadt, Germany). Pancreatic lipase was acquired from Sigma Chemical Co. (St. Louis, MO, USA). Triolein (OOO; ≥99%), 1,2-dioleoyl-3-palmitoyl-rac-glycerol (POO; ≥99%), POP (≥99%), and PPP (≥99%), used as TAG standards, were also purchased from Sigma Chemical Co. All other reagents used herein were HPLC-grade.

Silymarin powder (SLM, ≥30% silybin (silybin A + silybin B) was obtained from Sigma Aldrich (https://www.sigmaaldrich.com/catalog/DataSheetPage.do?brandKey=SIGMA&symbol=S0292). The other compounds identified were the flavonolignans silychristin, silydianin, isosilybin A and isosilybin B and the flavanonol taxifolin. The extract title was 54.3% according to our previous work [24 (link)]), d-α-Tocopherol polyethylene glycol 1000 succinate (TPGS), silibinin (≥98%, HPLC), fluorescein 5(6)-isothiocyanate (FITC, ≥90%, HPLC), pepsin, bile salts, pancreatic lipase, pancreatin, phosphate buffered saline BioPerformance Certified pH 7.4 (PBS), Tween^®80, lecithin (≥99%, TLC) lyophilized powder, cholesterol BioReagent (≥99%), 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), human serum albumin (≥96%, HSA) lyophilized powder and all analytical grade and HPLC grade solvents were obtained from Sigma Aldrich (Saint Louis, MO, USA) with the support of Sigma Aldrich Italia (Milan, Italy). Soluplus^® was a gift from BASF (Ludwigshafen, Germany) with the support of BASF Italia, BTC Chemical Distribution Unit (Cesano Maderno, Monza e Brianza, Italy). Distilled water was obtained from a Simplicity^®UV Water Purification System, Merck Millipore (Darmstadt, Germany).