P1750

Sigma-Aldrich provided high purity grade nitric acid (Suprapure 65% HNO₃), calcium disodium ethylene-diaminetetraacetate hydrate (EDTA-Ca-Na₂), pepsin from porcine stomach mucosa (P-7000), pancreatin from porcine pancreas (P-1750), and bile extract porcine (B-6831) (St Louis, MO, USA). The National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA, provided stock solutions of Se (SRM3149), CRM SRM1566b (oyster tissue), and Rh (Rh SRM3144). In addition, this study made use of CRM from the National Metrology Institute of Japan (NMIJ7402-a, codfish tissue). Merck provided potassium hydroxide (KOH, 105033), sodium bicarbonate (NaHCO₃), and dialysis membrane (molecular weight cut-off 12–14 KDa, 13 mm internal diameter) (KGaA, Darmstadt, Germany). Medicell Membranes Ltd. provided flat dialysis membranes (MWCO 12–14 kDa, 15.9 mm wide) (Greenwich, London). In the study, deionized water from Milli-Q^® water (Millipore Sigma, Burlington, MA, USA) was employed.

A Hibiscus beverage and an Hibiscus commercial drink as the control beverage were subjected to a static in vitro gastrointestinal digestion model according to Blancas-Benitez, et al. [37 (link)] to evaluate the bioaacessibility of phenolic compounds (PC). Gastric digestion was simulated by adding pepsin (P-7000, Sigma-Aldrich, 0.2 mL of a 300 mg/mL solution in 0.2 M HCl-KCl buffer, pH 1.5, 40 °C, 2 h). Pancreatin (P-1750, Sigma-Aldrich, 3 mL of a 5 mg/mL solution in 0.1 M phosphate buffer, pH 7.5, 37 °C, 2 h) was added in order to simulate the intestinal digestion. After the intestinal digestion, this fraction was used to evaluated the BC released. This method differs from other methods such as the INFOGEST protocol [38 (link)], where a dialysis bag is used for the simulation of the passive diffusivity of metabolites in the small intestine; in this study, sample dialysis bags were not used because of the type of sample. The samples from this stage were centrifuged (Hermle Z 323 K; Wehingen, Germany) (3500× g, 15 min, 4 °C), and the supernatant (digested extract) was considered as soluble indigestible fraction. The residue was considered as indigestible-fraction. Both fractions were used to identify the BC by HPLC-DAD-ESI-MS described in the section below.

The in vitro gastrointestinal digestion was based on the protocol as detailed by Minekus et al. [24 (link)]. Simulated salivary fluid (SSF), simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared following the scheme previously reported [24 (link)]. In particular, the method (scaled up for 500 µL of liquid sample) included an oral phase composed of SSF at pH 7.0, with salivary α-amylase (75 U/mL; from human saliva Type IX-A, Sigma-Aldrich Co. Milan, Italy). The oral step was run at 37 °C for 2 min. Then, the oral bolus samples were mixed (ratio 1:1) with the SGF at pH 3.0 containing porcine pepsin (2000 U/mL; P7000; Sigma-Aldrich Co. Milan, Italy). The gastric phase was carried out for 120 min at 37 °C. Gastric chyme was mixed (1:1) with the SIF at pH 7.0 containing pancreatin (100 U/mL; P1750; Sigma-Aldrich Co. Milan, Italy) and bile salts (10 mM; B8631; Sigma-Aldrich Co. Milan, Italy). The intestinal phase was carried out for 120 min at 37 °C. During the in vitro digestion, appropriate amounts of HCl (1 M) and NaOH (1 M) were added for pH adjustment. At selected time points (i.e., gastric and pancreatic end-phases), corresponding digestion sample tubes for each EVOO sample were cooled on ice to stop the reaction. The experiment was performed in triplicate (n = 3) and all the in vitro digestion steps were carried out in amber bottles in the dark.