Pepsin from porcine stomach mucosa

Liposome’s stability was evaluated in simulated gastric juices (SGJ) and bile solution (BS) by turbidity measurements in predetermined time intervals (0 h, 3 h, 6 h and 24 h) by measuring the turbidity according to [35 (link)] at 450 nm using a turbidity meter (HI 83414, Hanna Instruments, Bedforshire, UK) at 25 °C. The SGJ and BS were prepared according to Pak et al. [36 (link)]. Briefly, SGJ were prepared by suspending pepsin from porcine stomach mucosa (Sigma, Taufkirchen, Germany) in sterile saline (0.5% w/v) to a final concentration of 0.22% w/v, adjusting pH to 2.0 with HCl. BS was prepared by dissolving porcine bile extract (Sigma, Taufkirchen, Germany) in DI water to a final concentration of 0.33%.

Ethyl acetate and n-hexane were purchased from VWR International (Radnor, PA, USA). Methanol and dichloromethane were obtained from Fisher Scientific (Loughborough, UK) and dimethyl sulfoxide and urea were supplied by Panreac Quimica SAU (Castellar del Vallés, Barcelona, Spain). Deuterated chloroform for spectroscopic experiments was obtained from VWR Chemicals Prolabo^® (Leuven, Belgium). For the cytotoxicity bioassays, Dulbecco’s Modified Eagle’s Medium (DMEM) was supplied by Lonza (Verviers, Belgium), premixed phosphate buffer saline solution (PBS, 10×) was supplied by Roche (Steinheim, Germany), fetal bovine serum, penicillin/streptomycin, l-glutamine, sodium pyruvate, trypsin and minimum essential medium non-essential amino acids (MEM NEAA), RPMI 1640 medium were purchased from Gibco (Paisley, UK). α-Cyclodextrin (α-CD) was obtained from Tokyo Chemical Industry (TCI Europe, Zwijndrecht, Belgium). Water (type I) was obtained from an Ultramatic system from Wasserlab. Lipase from porcine pancreas (L3126; enzymatic activity of 100–500 units), mucin from porcine stomach type II (M2378), pancreatin from porcine pancreas (P7545; 8 × U.S.P. specifications) and pepsin from porcine stomach mucosa (P7000; enzymatic activity of 500 units/mg solid) were obtained from Sigma Aldrich (Steinheim, Germany).

Pepsin from porcine stomach mucosa, dialysis membrane (14 kDa MWCO), DPPH, and type I collagen standard solutions from calf skin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alcalase^® 2.4 L (a proteinase from Bacillus licheniformis) was donated by Novozymes (Mexico City, Mexico). Papain enzyme from Carica papaya (30,000 U/mg) and Amicon ultrafiltration tubes (3 kDa MWCO) were purchased from Merck Corporation (Burlington, MA, USA). The protein marker and bovine serum albumin standard (2 mg mL⁻¹) were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Solvents for amino acid analysis were HPLC-grade (T.J. Baker, Chemicals, PA, USA). All other chemicals used in this investigation were of analytical grade and used as received.

SGF and SIF were prepared based on Gildas et al. [20 (link)], with modification. SGF was prepared by adding 9 g·L⁻¹ of sodium chloride (Merck, Darmstadt, Germany) and 3 g·L⁻¹ pepsin from porcine stomach mucosa (Sigma Aldrich Co, St Louis, MO, USA), which was then adjusted to pH 1.2 with 1 M hydrochloride acid. The mixture was sterile filtered through a 0.45 μm nylon membrane. Meanwhile, SIF was prepared by adding 9 g·L⁻¹ of sodium chloride (Merck, Darmstadt, Germany), 10 g·L⁻¹ trypsine from porcine pancreas (Sigma Aldrich Chemie, Steinem, Switzerland), and 3 g·L⁻¹ of bile salts (Fluka, St. Louis, USA), with its pH adjusted to 6.8 with 1 M sodium hydroxide. Both fluids were sterile filtered through a 0.45 μm membrane. Washed beads with entrapped G4 were added to 5 mL of each fluids consequently and incubated for 2 h at 37°C. The bead strengths were determined after the incubation period.

At the red colour maturity stage, Natal plums were harvested from the gardens of Tshwane University of Technology in Pretoria, Gauteng, South Africa. Fruits were washed, deseeded, and stored at −80° after harvesting. Kernels of Marula nut were obtained from Phalaborwa, Limpopo province from the rural framers and crushed with a stone the nut from the shell. Pancreatin from porcine pancreas, pepsin from porcine stomach mucosa, phenolic standards were purchased from Sigma Aldrich, Johannesburg, South Africa.