Article pubs.acs.org/JAFC
Protection of Epigallocatechin Gallate against Degradation during in Vitro Digestion Using Apple Pomace as a Carrier Liangyu Wu,†,‡ Luz Sanguansri,‡ and Mary Ann Augustin*,‡ †
College of Horticulture, Fujian Agriculture and Forestry University, 15 Shangxiadian Road, Fuzhou, Fujian 350002, People’s Republic of China ‡ CSIRO Food and Nutrition Flagship, 671 Sneydes Road, Werribee, Victoria 3030, Australia ABSTRACT: Apple pomace, a byproduct of the apple juice processing industry, may be used as a matrix for carrying phytochemicals. High-pressure processing (600 MPa for 5 min) or heat treatment (121 °C for 5 min) of wet apple pomace can increase the shelf life of the pomace but may influence the carrier properties of the wet pomace for phytochemicals. We examined the effects of these processing treatments on the adsorption capacity of apple pomace for epigallocatechin gallate (EGCG) and the stability of EGCG in simulated gastrointestinal fluids in vitro. Both processing treatments reduced the adsorption capacity but protected EGCG against degradation in the simulated gastrointestinal fluids. The extent of EGCG degradation in simulated gastrointestinal fluids in vitro in the presence of apple pomace was not influenced by gastric and intestinal enzymes, suggesting that pH had the overriding influence on EGCG degradation. This study showed the potential of apple pomace as a carrier for EGCG in functional food applications. KEYWORDS: apple pomace, epigallocatechin gallate, EGCG, polyphenol, adsorption, digestion
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INTRODUCTION Epigallocatechin gallate (EGCG), a polyphenolic compound present in tea, possesses a diverse spectrum of biological activities, including antioxidant,1,2 antibacterial,3,4 and antiviral5,6 properties, making it attractive as a bioactive for incorporation into functional foods. For EGCG to exert its beneficial health effects, it must be able to reach its site of action in the body after ingestion. The bioaccessibility and bioavailability of EGCG have been examined. Most studies have suggested that the gastrointestinal conditions may adversely affect EGCG bioavailability. For example, when EGCG was administered at 2 mg kg−1 body mass of a rat, the peak EGCG concentration in rat plasma detected within 20 min after administration was only 100 ng mL−1.7 Despite the fact that the peak concentration (Cmax) of EGCG was linearly proportional to the amount administered, the bioavailability was only 1.6−13.9% in healthy adults,8 because of the degradation of EGCG in the high pH of the intestinal fluids.9 EGCG is quite unstable at pH ≥ 5,10,11 and strategies are required to stabilize EGCG against degradation during gastrointestinal tract transit. Pharmacokinetic studies in mice showed that a novel stabilizing solution of ascorbic acid and tris(2-carboxyethyl)phosphine hydrochloride provides protection to EGCG after ingestion.12 Depending upon whether EGCG was ingested as the neat compound or as constituents of beverages or foods, the rate of EGCG degradation was variable.13 Apple pomace, a byproduct of the apple juice processing industry, is a potential matrix for carrying phytochemical compounds because the apple pomace stabilizes these compounds against degradation.14 Apple pomace does not contain EGCG,14 but we have previously shown that EGCG binds to apple pomace, making it attractive as a potential carrier for EGCG delivery into functional foods.15 © 2014 American Chemical Society
For practical purposes, it is useful to process the apple pomace to increase its shelf life. While freezing may be used, the pomace needs to be kept in a frozen state prior to its use. Alternatively, processing treatments, such as high pressure or heat treatment, of fruit pomace are two methods that may be used to reduce enzymatic browning and microbial load as well as improve the stability of foods.16−18 Experiments in our laboratory have shown that high-pressure processing and heat treatment reduced enzymatic browning of the apple pomace during storage (data not shown). In this study, the absorption characteristics of EGCG onto high-pressure-processed or heattreated apple pomace were compared to that of EGCG adsorption onto fresh frozen wet apple pomace. In addition, the protection afforded to EGCG in simulated gastrointestinal fluids in vitro as a result of its binding to apple pomace was investigated.
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MATERIALS AND METHODS
Materials and Chemicals. Apple pomace (∼50% Granny Smith and ∼50% Pink Lady) was a gift from a local apple juice producer (Summer Snow Company, Officer, Victoria, Australia). The pomace was obtained just after crushing and stored overnight at 4 °C. The pH of the pomace was 4.2 ± 0.2. The processing of apple pomace was carried out in the pilot plant at CSIRO, Werribee, Victoria, Australia. On the next day, the pomace was milled using a colloid mill (CR-40, Prime Machinery Group, Inc., Lansdale, PA), and the milled pomace (wet pomace) was stored at −20 °C. Wet pomace was removed from frozen storage and subjected to (i) high-pressure processing (HPP) at 600 MPa for 5 min using QFP 35L-600 high-pressure equipment (Avure Technology, Middletown, OH) or (ii) heat treatment (HT) at Received: Revised: Accepted: Published: 12265
September 25, 2014 November 20, 2014 November 24, 2014 November 24, 2014 dx.doi.org/10.1021/jf504659n | J. Agric. Food Chem. 2014, 62, 12265−12270
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Figure 1. (A) Particle size distribution and (B) confocal laser images of wet, HPP, and HT apple pomace. where C0 and Ct (mg L−1) are the EGCG concentrations of the liquid phase initially and at time t, V (L) is the volume of the solution, and W (g) is the dry mass of adsorbent used. The experiment was carried out in duplicate. In Vitro Digestion Simulation. A total of 10 mL of EGCG aqueous solution (10 g L−1) was mixed with 40 g of apple pomace, and the mixture was blended with a mixer (BSB-380, Breville Pty Ltd., Botany, New South Wales, Australia) for 1 h. Then 1 g of mixture was taken for in vitro digestion simulation. For the control, 1 mL of 2 g L−1 EGCG solution was used. In vitro digestions were carried out in simulated gastric fluids with or without added enzymes according to a published method.19 Preparation of Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF). For the preparation of SGF, 2.0 g of sodium chloride was dissolved in 500 mL of deionized water. To this solution was added 7 mL of 36% (w/v) hydrochloric acid and 3.2 g of pepsin (800−2500 units/mg of protein), and the mixture was made up to a volume of 1000 mL using deionized water. The pH was adjusted to1.2 using 1.0 M hydrochloric acid. For the preparation of SIF, 13.6 g of potassium dihydrogen phosphate was dissolved in 800 mL of deionized water. To this solution was added 154 mL of 0.2 M sodium hydroxide and 2.5 g of 8× USP pancreatin. The mixture was made up to a volume of 1000 mL using deionized water and stirred at 4 °C overnight. The final pH was adjusted to 6.8 using a few drops of 1.0 M sodium hydroxide. Sequential Gastric and Intestinal Digestion in Vitro. For each treatment, 22 digestion tubes were prepared and subjected to in vitro digestion. For each tube, apple pomace (1.0 g) was mixed with 10 mL of SGF, vortexed, placed into a glass tube, and held at 37 °C in a shaking water bath (120 rpm for 2 h). Following gastric digestion, the pH was adjusted from pH 1.2 to 6.8 and 10 mL of SIF was added. The tube was then placed in a shaking water bath (120 rpm for 3 h) at 37 °C. Samples were removed at 30 min intervals during the digestion for analysis of remaining EGCG. For each time point, two samples were removed for analysis. Extraction and Preparation of Samples for HPLC Analysis. For samples containing apple pomace, the contents of the whole tubes containing EGCG−apple pomace sample were used for extraction of EGCG. For EGCG−apple pomace samples exposed to simulated
121 °C for 5 min using an autoclave (Centenary Series, Atherton Company, Victoria, Australia). The processed pomace was stored at 4 °C and used within 1 week of preparation. The moisture contents of the wet, HPP, and HT apple pomace, as determined using the MA-30 moisture meter (Sartorius, GER), were 76.8 ± 0.4, 76.6 ± 0.8, and 76.4 ± 0.5% (w/w), respectively. The pH of the apple pomace after HPP and HT was 4.2 ± 0.2. Unless otherwise stated, EGCG, porcine pancreatin, pepsin, and other chemicals were purchased from SigmaAldrich Chemical Co. (St. Louis, MO). High-performance liquid chromatography (HPLC)-grade acetonitrile was from Fisher Scientific Company (Waltham, MA). Particle Size Distribution. The particle size distribution of the apple pomace was measured using a laser diffraction analyzer (Mastersizer 2000, Malvern Instruments, Ltd., U.K.). The apple pomace samples were diluted with water until an obscuration reading ranging from 13 to 17%. A general purpose model (on the basis of Mie and Fraunhofer scattering) was used to fit the particle size data, using a refractive index of 1.533 in the calculation. Analyses were carried out in triplicate on the diluted samples in distilled water. Confocal Laser Microscopy. The fresh frozen wet, HPP, and HT apple pomace were assessed using a Leica SP5 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). The sample was stained with a drop of fluorescent Congo red dye (0.2%, w/v) to stain polysaccharides. Observation was conducted at room temperature (∼22 °C) using a HC PL APO 20× objective, and excitation was achieved with the use of a 488 nm argon laser. Adsorption Kinetic Studies. Samples of pomace (0.50 g equivalent dry weight; pH 4.2 ± 0.2) were added to 100 mL of EGCG solution (200 mg of EGCG L−1) in separate conical flasks. The conical flasks were sealed by parafilm (Pechiney Plastic Packaging, Inc., Chicago, IL) and placed in a shaking water bath at 120 rpm at 25 °C. Aliquots (1.0 mL) were taken at specific time intervals (5, 15, 30, 60, 120, 180, 300, and 480 min) and subjected to HPLC analysis. The amount of EGCG adsorbed at time t, qt (mg g−1), was calculated using the following equation: qt =
(C0 − Ct )V W 12266
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gastric fluid, the whole sample was mixed with 10 mL of ethanol and stirred for 3 h prior to centrifugation. Extraction of samples at time t = 0 min showed that the amount extracted was the same as that originally added into the sample, showing that the extraction protocol was adequate. For EGCG−apple pomace samples exposed to sequential gastric and simulated intestinal fluids, the whole sample was mixed with 20 mL of ethanol, the pH was then adjusted to ∼3.5 to stabilize the EGCG against degradation, and the mixture was stirred for 3 h prior to centrifugation. All ethanol extracts were centrifuged at 10 000 rpm and 4 °C for 10 min, and the supernatant was used for HPLC analysis. For the control, where only EGCG solutions were added into gastrointestinal fluids (without apple pomace), samples were centrifuged at 10 000 rpm and 4 °C for 10 min and then the supernatant was used for HPLC analysis. The amount recovered at t = 0 min was also the same as that originally added. HPLC Analysis. The concentration of EGCG in the supernatants was determined following the method that we previously used.15 The HPLC [Surveyor system with photodiode array (PDA) detector and mass spectrometry (MS) pump, Thermo Finningan, NJ] conditions were as follows: injection volume, 10 μL; VYDAC C18 monomeric 238EV52 column (250 × 2.1 mm inner diameter × 5 μm particle size); column temperature, 28 °C; mobile phase A, acetonitrile + 0.08% trifluoroacetic acid; mobile phase B, water + 0.1% trifluoroacetic acid; and linear gradient elution, from 2% A/98% B to 24% A/76% B within 45 min and then change to 2% A/98% B until 50 min, with a flow rate of 200 μL/min. The ultraviolet−visible (UV−vis) detector wavelength was set at 280 nm. For quantification of EGCG, peak areas were compared to an external standard solution of EGCG. When an extract from apple pomace alone was analysed, there was no EGCG peak detected at the expected retention time for EGCG, confirming the absence of EGCG in the original apple pomace used. Data Analysis. All experiments were carried out at least in duplicate, and the data were analyzed using Origin 8.0 (Origin Lab, Inc., Northampton, MA) for a one-way analysis of variation (ANOVA). Duncan’s multiple range test was used to determine whether significant difference (p < 0.05) existed between mean values.
shown in Figure 1B. Although there were differences in the particle size distribution obtained using particle sizing data from light scattering (Figure 1A), the differences between the treatments of the apple pomace were not that obvious (Figure 1B). Adsorption of EGCG onto Apple Pomace. The adsorption capacity of EGCG onto wet apple pomace was significantly higher than that for HPP or HT (p < 0.05) processed apple pomace at 25 °C (Figure 2). In the initial
Figure 2. Adsorption of EGCG onto wet (blue diamonds), HPP (red squares), and HT (green triangles) apple pomace in sequential contact time at 25 °C. Initial [EGCG] = 200 mg L−1.
stages, there were sufficient available adsorption sites on the surface of apple pomace, resulting in rapid adsorption of EGCG onto the pomace. There was subsequent plateauing with increasing time because of the saturation of available adsorption sites. The apple pomace consists of insoluble fiber, such as lignin and cellulose. Hence, there are a large number of hydroxyl groups in these polymers that could potentially associate with EGCG through hydrogen bonding. Various processing treatments, such as heating and HPP, may cause multiple structural changes at the molecular, microstructural, and macroscopic levels and change the characteristics of the dispersed phase of the processed material.22 All of these changes are likely to impact on the adsorption capacity of small molecules, such as bioactive molecules, and the kinetics of the adsorption. Gastric and Intestinal Digestion in Vitro. The total EGCG in EGCG−apple pomace samples and EGCG alone exposed to gastrointestinal fluids is shown in Table 1 and Figure 3. Digestion in the Presence of Enzymes. As there is no EGCG in apple pomace, any EGCG detected in the system will be due to EGCG carried by the apple pomace. Upon exposure to SGF, there was minimal degradation of EGCG ( wet apple pomace. The recovery of EGCG in EGCG−HPP and EGCG−HT apple pomace mixtures was significantly higher (p < 0.05) than that of control and that for EGCG−wet apple pomace at the end of intestinal digestion. This demonstrates the ability of apple pomace to protect EGCG against degradation at ∼ neutral pH. Although the measured adsorption of EGCG to apple pomace follows the order of wet > HPP > HT apple pomace under equilibrium conditions when apple pomace is mixed with EGCG (Figure 2), it is possible for the binding affinity to change with pH of the gastrointestinal fluids. This was not measured in our study. However, the observation that apple pomace protects EGCG against degradation in SIF suggests that the protection afforded to EGCG may in part be due to the binding of EGCG to the pomace. It is also likely that the differences in the microstructure of the pomace may also have had an influence on degradation of bound EGCG. It is conceivable that a physical barrier to contact with the external environment provided by the apple pomace may contribute to the stabilization of EGCG. Other studies have suggested that processing treatments on plant tissue (e.g., tomato and potato) that alter structure affect the bioaccessibility of bioactives.26 Effect of the Enzyme. To determine if intestinal enzymes contributed to the rate of degradation of EGCG in simulated gastrointestinal conditions, the EGCG degradation in the absence and presence of enzymes (pepsin and pancreatin) was compared. Figure 4 shows the comparison of the digestion system with or without enzyme. The effect of the enzyme was
significantly different (p < 0.05) when only EGCG was delivered as an aqueous solution during intestinal digestion (Figure 4A). In contrast, the presence of enzyme did not have a marked effect on the EGCG−pomace mixtures during gastrointestinal digestion (panels B−D of Figure 4). In the presence of apple pomace, there already exists an interaction between EGCG and the pomace. It is not understood why there was a significant effect of the enzyme in EGCG only solutions (Figure 4 A) but not when EGCG was delivered in the presence of pomace (panels B−D of Figure 4). However, the effect of the enzyme, although significant, is small in comparison to the effect of pH on EGCG solutions (Figure 4A). Overall, the results suggest that pH had the overriding effect on the rate of EGCG degradation. In the in vitro system examined, the EGCG−pomace mixture markedly decreased the extent of degradation of EGCG during intestinal digestion. The enzyme had an effect on the digestion of EGCG solution, but the main factor influencing the degradation of EGCG was the pH. The observations that apple pomace protects EGCG against degradation implies that apple pomace could be considered as a suitable carrier for EGCG and polyphenols, with the potential to enhance the stability and bioavailability of polyphenolic compounds. The use of apple pomace as a carrier for EGCG is an alternative to use of encapsulation of EGCG in liposomes or biopolymeric nanopartricles for protecting EGCG against degradation in the gastrointestinal tract. Comparative studies between the efficacy of apple pomace as a carrier for EGCG compared to the use of encapsulation systems would be of interest. Experiments in vivo with humans are needed to validate the effects of apple pomace 12268
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Notes
The authors declare no competing financial interest.
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Figure 4. Amount of EGCG digested with (blue diamonds) or without (red squares) enzyme during the gastrointestinal digestion simulation, with the asterisks indicating that there is a significant difference (p < 0.05) at the same time point: (A) EGCG alone, (B) EGCG−wet apple pomace, (C) EGCG−HPP apple pomace, (D) EGCG−HT apple pomace.
on the bioavailability of polyphenols and to determine whether this can be translated to the achievement of desirable physiological properties.
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +61-3-9731-3486. Fax: +613-9731-3201. E-mail:
[email protected]. Funding
This work was funded in part by the Natural Science Foundation of Fujian Province (2014J01082) and the Doctoral Program of Higher Education China (20123515120007) awarded to Liangyu Wu. 12269
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