Chemicals and materials
Shallots and onions (Northern Yellow) were obtained from a local grocery store. Apples (Red Delicious and Cortland varieties) were obtained from the Cornell Orchards (Cornell University, Ithaca, NY). Porcine pepsin, bile extract, pancreatin, lactase (beta-galactosidase, from Kluyveromyces lactis, activity of 3000 units/mL), quercetin, and quercetin-4'-glucoside were purchased from Sigma Chemical Company (St. Louis, MO). Quercetin-3-glucoside was purchased from Indofine Chemical Company, Inc (Hillsborough, NJ). Caco-2 cells were obtained from the American Type Culture Collection (Rockville, MD) and were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco Life Technologies, Grand Island, NY) supplemented with 5% fetal bovine serum (Gibco Life Technologies, NY), 10 mM HEPES, 50 units/mL penicillin, 50 μg/mL streptomycin, and 100 μg/mL gentamicin, and were maintained at 37°C in 5% CO2.
In vitro digestion
Two hundred grams of each food sample were chopped, blended for 5 min with 200 mL saline (140 mM NaCl, 5 mM KCl) using a Waring blender, and then homogenized using a Virtis 45 homogenizer. The total homogenates were aliquotted in 15 mL centrifuge tubes and stored at -20°C until use.
For the digestion treatment, 2 g aliquots of the food sample were placed in a centrifuge tube with an equal amount of saline. The pH was decreased to 2.0 by drop-wise addition of 1M HCl, and porcine pepsin was added to a final concentration of 1.3 mg/mL. The digestate was incubated in a shaking water bath at 37°C for 30 minutes. The pH of the digestate was then increased to 5.8 with the drop-wise addition of 1M NaHCO3. Porcine bile extract and pancreatin were added to a final concentration of 1.1 and 0.175 mg/mL, respectively. The pH was increased to 6.5 by drop-wise addition of 1M NaHCO3, and the samples were incubated for 1 hour in a water bath at 37°C. Following digestion the pH was decreased to 2 by addition of HCl and the digestates were stored at -80°C for further analysis.
To examine and optimize the effects of digestion time and pH on the recovery of compounds from the digestate, the above parameters were varied. To examine the effects of pepsin digestion time, the pepsin digestates were incubated for 0, 30, 60 or 90 minutes and then incubated with the intestinal digestion enzymes for 60 minutes. To examine the effects of intestinal digestion, the samples were incubated with pepsin for 30 minutes, then incubated with pancreatin and bile for 0, 30, 60, or 90 minutes.
The effects of 100 μM ascorbic acid and a nitrogen environment on quercetin and quercetin glucoside recovery from onion and apple digestates were examined. Following homogenization and prior to digestion, the food samples were mixed 1:1 with saline containing 200 μM ascorbic acid, leaving a final sample concentration of 100 μM ascorbic acid. During the digestion procedure described above, the samples were flushed constantly with nitrogen.
The effect of pH of either 6.5 or 7.0 during intestinal digestion on quercetin and quercetin-3-glucoside recovery was compared. The effect of the storage pH was examined by comparing digested samples having either a final pH of 2.0 or 6.5. The effect of storage pH on 20 μM pure quercetin and 20 μM quercetin-3-glucoside was examined by comparing recoveries from samples stored at pH = 2.0, 3.5, 5.0 or 7.0. All samples were stored overnight at -80°C. Prior to HPLC analysis samples were thawed and extracted 4 times with acidified ethyl acetate (pH 2.0), evaporated to dryness and reconstituted in 2 mL acidified methanol (pH 2.0).
Doses of lactase (0.5 units to 3000 units per gram sample) were applied to 1 gram shallot extract and incubated for 15 minutes at 37°C. The final pH was brought to 2.0 and the samples were stored at -80°C. The time kinetics were examined by incubating 1 gram shallot extract with 100 units of lactase for 0, 15, 30, 60, 90, 120, 240, and 720 minutes.
The effect of both lactase and digestion were examined by digesting the samples with pepsin and pancreatin as described above, then incubating the samples with 100 units of lactase for 30 minutes at 37°C. Samples were stored overnight at -80°C.
Prior to HPLC analysis, all digestate samples were thawed and extracted 4 times with acidified ethyl acetate (pH 2.0), evaporated to dryness and reconstituted in 2 mL methanol.
Uptake of quercetin-4'-glucoside and quercetin from shallot digestates by Caco-2 cells
Caco-2 cells were seeded at a density of 5 × 105 cells per well in a collagen coated 6-well, flat bottom plate and incubated at 37°C in a 5% CO2 environment. Caco-2 cells were used between passages #10–25, and the cells reached confluence approximately 5 days post seeding. Culture media was changed three times a week. On day 14 post seeding, the DMEM was removed and the cells were rinsed three times with phosphate buffered saline (PBS).
Shallot homogenates were digested as previously described and were placed directly on the 14 day old Caco-2 cells, or the samples were diluted 1:2 or 1:4 in HBSS (Hank's Balanced Salt Solution). Cells were also incubated with non-digested shallot homogenates for comparison. For each treatment, two wells of cells were used for each sample, and each treatment was repeated in triplicate.
To examine the effect of lactase on quercetin uptake from shallots, shallots were digested then incubated with lactase (50, 100, 300, and 1000 units/g shallot) for 20 minutes at 37°C. The digested shallot homogenates and the digested plus lactase treated shallot homogenates were diluted 1:2 in HBSS and placed on the cells. In all experiments, Caco-2 cells were incubated with treatment for 30 minutes at 37°C in 5% CO2. The shallot treatment and HBSS was removed and the cells were rinsed three times with 20% methanol in PBS. Cells were scraped in acidified methanol (pH = 2.0) and the wells were rinsed three times with methanol. The scraped cells were sonicated for 15 minutes, centrifuged at 1600 g for 5 minutes, and the methanol supernatant was collected. The cells were rinsed three more times with methanol, the supernatants were collected and the methanol extracts were evaporated to dryness under nitrogen and reconstituted in 400 μl acidified methanol for HPLC analysis.
Induction of lactase activity in Caco-2 cells
Caco-2 cells were seeded at a density of 5 × 105
cells per well in a 6-well flat bottom plate. The cells were cultured in DMEM spiked with different doses of either lactose (10, 50, 100, 500, and 1000 μM), or forskolin (1, 10, 50, 100, and 200 μM). Media was changed every two days, and cells were harvested and lactase activity was measured at 14 days post-seeding. Lactase activity of Caco-2 cells was measured using a method adapted from Dahlqvist [31
]. Cells were trypsinized, collected, centrifuged and resuspended in homogenization buffer (50 mM sodium phosphate; 1 mM EDTA; 10 mM dithiothreitol; protease inhibitor cocktail, Sigma Chemical Co., St. Louis, MO). Cells were homogenized 5 times for 30 seconds with 1 minute of cooling between bursts using a benchtop homogenizer. Homogenates were treated with 56 mM lactose and incubated at 37°C for 60 minutes. Glucose oxidase, peroxidase, and o-dianisidine were applied to the cell homogenates and the final colored products were measured at 420 nm using a spectrophotometer [31
]. The results were compared to a glucose standard curve to determine the amount of glucose released by lactase in the Caco-2 cell monolayer. Protein was determined from crude cell homogenates colorimetrically using the Lowry method with comparisons to a bovine serum albumin standard curve. Results are expressed as milliunits/mg of protein, and one unit is defined as the lactase activity that hydrolyzes 1 μmole of lactose per minute at 37°C.
Quercetin and quercetin-3-glucoside content of untreated homogenates, digestates, and Caco-2 cell extracts were determined using an RP-HPLC procedure with a Supelcosil LC-18-DB column (150 mm × 4.6 mm, and 3 μm pore size). Waters 515 HPLC pumps (Waters Corp., Milford, MA) and a Waters 2487 dual wavelength absorbance detector (Waters Corp., Milford, MA) set at 370 nm were used for all HPLC analysis. Quercetin, quercetin-3-glucoside, and quercetin-4'-glucoside were used as standards. For the analysis of quercetin, quercetin-3-glucoside, and quercetin-4'-glucoside in the apple peel extracts, shallot extracts, and digestate extracts, the solvent system used was (A) acidified water (pH 2.0; triflouroacetic acid) and (B) acetonitrile. The gradient method was the following: 0.0 min, flow rate = 1.4, (A) 90% and (B) 10%; 53 min, flow rate = 1.5, (A) 80% and (B) 20%; 58 min, flow rate = 1.7, (A) 65% and (B) 35%; 64 min, flow rate = 1.4, (A) 90% and (B) 10%. Twenty μL injections were made for each sample. Quercetin, quercetin-3-glucoside, and quercetin-4'-glucoside concentrations in the apple peel extracts, shallot extracts, and in the digestates were extrapolated from the pure quercetin and quercetin-3-glucoside standard curves.
All data were reported as means ± SD for three replicates of each treatment. An analysis of variance (ANOVA) was used to compare results between treatment groups, and pairwise multiple comparisons were performed using Fisher's LSD with an individual error rate of 0.05. The statistical analysis was completed using Minitab Release 12 software (State College, PA).