Polyphenols within drinks and foods are under intense scrutiny because of
Polyphenols within drinks and foods are under intense scrutiny because of their potential beneficial results on individual wellness. baby food (2nd foods Turkey & Rice Dinner) and General Mills Cheerios? were purchased from a local grocery store. Porcine bile extract porcine pancreatin type II Kit porcine lipase and porcine pepsin were purchased from Sigma Chemical Organization (St. Louis MO USA). HPLC grade solvents and all other chemicals were from either Fisher Scientific Organization or Sigma Chemical Company and were the best grade available. 2.2 In vitro digestion EGCg and PGG were separately subjected to a two-stage digestion model mimicking the human digestive system (Garrett Failla & Sarama 1999 Green Murphy Schulz Watkins & Ferruzzi 2007 Walsh Zhang Vodovotz Schwartz & Failla 2003 Six conditions were tested for each compound: pH changes pH changes with digestive components pH changes with Gerber? baby food pH changes with Cheerios? pH changes with digestive components and Gerber? baby food and pH changes with digestive components and Cheerios?. The polyphenol was added to 4.4 mL of simulated belly fluid comprised of 0.9% saline 9.1 mM mandelic acid in 0.01 M HCl pH 2. The polyphenol levels in the simulated belly were 1.5 mg/mL (1.6 mM PGG 3.3 mM EGCg). These levels are similar to the average total phenolic content of brewed tea (Astill Birch Dacombe Humphrey & Martin 2001 PGG was also examined at 0.4 mg/mL (0.4 mM). An aliquot of 25 μL was immediately removed for HPLC analysis and was used to establish the starting amount of phenol for the reaction (100%). The remaining answer was bubbled with nitrogen gas PSI-6206 for 5 min before initiating digestion by adding 500 μL of 100 mM HCl or 40 μg/μL pepsin in 100 mM HCl bringing the pH to 1 1.8 ± 0.1. A second aliquot of 25 μL was removed for analysis (0 h pH 1.8) and the remaining answer was sealed and incubated while rotating at 37° C. After 1 h of incubation another 25 μL aliquot was removed for evaluation (1 h pH 1.8). To the rest of the option 2140 μL of 0.2 M NaHCO3 or of NaHCO3 containing porcine bile extract (2.4 μg/μL) porcine pancreatin (0.4 μg/μL) and type II porcine lipase (0.2 μg/μL) was added achieving your final pH of 7.0 ± 0.1. The answer was once again bubbled with nitrogen before closing the test and incubating while spinning at 37° C for 2 h. After incubation your final aliquot was taken out for HPLC evaluation (2 h pH 7.0). For the circumstances that included the meals sources the meals was suspended in the saline option before the addition from the polyphenol. The Gerber? Turkey & PSI-6206 Grain Dinner baby meals was diluted five-fold with drinking water and 150 μL from the suspension system was put into the reaction mix. Cheerios? had been surface to an excellent natural powder using a pestle and mortar and 6.5 ± 0.2 mg was added to the reaction combination. For EGCg additional experiments were performed in which the solutions were adjusted to pH 5.0 or pH 6.0 in the first step of the incubation. Samples were taken immediately PSI-6206 (0 h pH 5.0 or 0 h pH 6.0) and after 1 h of incubation at 37° C after bubbling with nitrogen (1 h pH 5.0 or 1 h pH 6.0). Digestive components or food were not added in these experiments. 2.3 HPLC analysis As each aliquot was removed for HPLC analysis it was mixed with an equal volume of 1% (w/v) sodium lauryl sulfate in water to ensure recovery of all polyphenols including sorbed and insoluble materials. Each sample was centrifugally filtered through a 0.22 μm cellulose acetate membrane (Costar Spin-X? Centrifuge Tube Filter Fisher Scientific) for 1 m at 7 200 × and was immediately analyzed by HPLC with injection into the acidic mobile phase of the HPLC within 5 min of collecting the sample. The amount of EGCg and PGG in each sample was analyzed by HPLC using an Agilent 1050 system (Santa Clara CA USA) equipped with a diode array detector and controlled with Agilent ChemStation software (A.09.03). The system was equipped with two tandem 5 μm Hypersil ODS2 C18 cartridge columns (30×2.1 mm) with a guard column of the same material (Grace Davison Deerfield IL USA). Separation was achieved with a gradient of 0.13% trifluroacetic acid (TFA) in H2O (A) and PSI-6206 0.1% TFA in acetonitrile (B) at 0.5 mL/min in a 24 min program as follows: starting at 5% B linear increase.