Porcine pancreas lipase

Folin-Ciocalteu phenol reagent (FC), 2, 2-diphenyl-1-picrylhydrazyl (DPPH), porcine pancreatic α-amylase (EC 3.2.1.1), Saccharomyces cerevisiae α-glucosidase (EC 3.2.1.20), and porcine pancreas lipase (EC 3.1.1.3) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical reagent grade. Authentic phenolic standards including caffeic, p-coumaric, ferulic and gallic acids, quercetin and (+)-catechin were obtained from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade methanol, acetonitrile and other solvents and reagents were purchased from Merck & Co. Inc. (NJ. USA) Deionized water prepared by Millipore purification system (Millipore Corp. Marlborough, MA, USA) was used for preparation of solutions.

All of the reagents including α-glucosidase, dimethyl sulfoxide (DMSO), potassium hydroxide (KOH), phosphate buffer, p-nitrophenyl-glucopyranoside (p-NPG), acarbose, phosphoric acid (H₃PO₄), human salivary α-amylase, 2-chloro-4-nitrophenyl-maltotrioside (CNP-G3), tris-HCl, porcine pancreas lipase, p-nitrophenyl butyrate (p-NPB), methanol, orlistat, acetonitrile, deuterated methanol (MeOH-d₄), deuterated chloroform (CHCl₃-d), eriodictyol, naringenin, and quercetin were purchased from Sigma Aldrich. The solvents used for extraction (ethanol—EtOH), HPLC (acetonitrile—MeCN), and LC–MS (MeCN and water—H₂O) analyses were purchased from Merck. The ultra-pure water used for the HPLC analysis was from an in-house Milli-Q system.

Porcine pancreas lipase (PPL), Amano Lipase PS from Burkholderia cepacia (BCL) and Candida rugosa lipase (CRL) were purchased from Sigma. Lipase from Thermomyces lanuginosus immobilized on particle silica gel (TLIM), lipase from Rhizomucor miehei immobilized on anion exchange resin (RMIM), lipase B from Candida antarctica immobilized on a macroporous acrylic resin (Novozym 435) and papain were purchased from Novo Nordisk. Bovine serum albumin (BSA) was purchased from Shanghai Huixing. Other reagents were commercially available and were used without further purification.

Commercial chemicals were used without further purification. Immobilized Candida antarctica lipase B (Novozym 435) was kindly donated by Novozymes Denmark. Porcine pancreas lipase (EC 3.1.1.3), Thermomyces lanuginosus lipase (EC 3.1.1.3), Amano lipase from Aspergillus niger (EC 3.1.1.3), and trypsin from porcine pancreas (EC 3.4.21.4) were purchased from Sigma-Aldrich. Analytical TLC (thin-layer chromatography) was performed on Merck pre-coated [silica gel 60 F254 20 × 20 cm)] plates. Melting points were determined with a melting point Thermo Scientific 9100 apparatus and are uncorrected. IR spectra were taken with a Bomem FT-IR MB spectrometer. NMR spectra were recorded in CDCl₃ with 300 MHz Bruker DRX Avance spectrometers. Mass spectra were recorded with an Agilent Technologies (HP) 5975C mass spectrometer by electron ionization (EI) (20–70 eV).

Optical rotations were determined with a Perkin Elmer 341 polarimeter. IR spectra were recorded on an FT-IR spectrophotometer, Perkin Elmer, Spectrum BX FTIR System. ¹H and ¹³C NMR measurements were obtained on Agilent-400MHz, Agilent-500MHz, and Agilent-600MHz NMR spectrometers with SiMe₄ as the internal reference. High-resolution mass spectrometry (HRMS) was performed using a Q-TOF mass spectrometer in the positive-ion ESI mode. HPLC was performed with a Hitachi/Merck L-6270 apparatus equipped with a UV-VIS detector (L 6200) and a differential refractometer detector (RI-71). TLC was performed on Merck Kiesegel 60 F254, 0.2 mm thick. Silica gel (Merck) was used for column chromatography. Purification by means of HPLC was done with a Silica gel column (Hibar 60, 7 m, 1 cm wide, 25 cm long). Chemicals were provided by Fluka (Buchs, Switzerland) and Aldrich (Sigma-Aldrich, Darmstadt, Germany). All solvents used were freshly distilled. Baker’s yeast was obtained from a local shop. The following enzymes were used in this work: Candida rugosa Lipase (Sigma, Darmstadt, Germany, Type VII, 950 U/mg) and Porcine Pancreas Lipase (Sigma, Darmstadt, Germany, Type II) (Sigma-Aldrich, Darmstadt, Germany). Enantiomeric excesses were determined by means of HPLC analyses on a chiral column (Chiralcel OD, Daicel, Japan, 254 nm) with n-hexane/i-PrOH 95:5 as eluent, flow rate 0.8 mL/min.