Department of Food Science and Technology (FST) Oregon State University Phone: (541)

FINAL REPORT WTFRC Project # PH-1-126 Title: Principal Investigators: Health Benefits of Apples Ronald E. Wrolstad, Ph.D. Department of Food Science and Technology (FST) Oregon State University Phone: (541) 737-3591 E-mail: ron.wrolstad@orst.edu Balz Frei, Ph.D. Linus Pauling Institute (LPI) Oregon State University Phone: (541) 737-578 E-mail: balz.frei@orst.edu Co-Principal Investigator: Cooperators: Silvina B. Lotito, Post-doctoral scientist, LPI Robert W. Durst, Sr. Research Assistant, FST Maria Widyasar, GRA, FST (Years 1 & 2) Alfio DiMauro, Post-doctoral scientist, FST Objectives: The objectives of this project are: i) to measure the concentrations of individual phenolics and polyphenolic classes (anthocyanin pigments, flavonols, flavan-3-ols, procyanidins, cinnamates, phloridzin derivatives, etc.) in peel and flesh of Red Delicious, Granny Smith, and Fuji fresh-market apples; ii) to determine the in vitro and in vivo antioxidant activities of apple polyphenolics (whole extracts and individual compounds) by the Oxygen Radical Absorbing Capacity (ORAC) and Ferric Reducing Antioxidant Potential (FRAP) assays; and to investigate the biologically relevant antioxidant activity of apple polyphenolics by determining their effects on human plasma antioxidant and lipid oxidation and other (patho)physiological processes related to atherosclerosis and heart disease. Significant findings: 1. We demonstrated that apple extracts and apple polyphenolics added to human plasma significantly protected endogenous antioxidants (urate and α-tocopherol [vitamin E]) from oxidation and inhibited lipid peroxidation. The protection of lipoprotein lipids from oxidation is considered relevant to the prevention of atherosclerosis and heart disease. 2. We observed that when healthy volunteers (3 men and 3 women) consumed 5 Red Delicious apples (flesh and skin, providing 1,825 mg total phenols), the antioxidants (ascorbate [vitamin C], urate, and α- tocopherol) and lipids in their plasma were not better protected from oxidation upon incubation of plasma with a free radical generator. Despite the high in vitro antioxidant capacity of the individual polyphenols and the large amounts of total apple phenols ingested in this short-term study, our results do not support a significant in vivo antioxidant effect of apple polyphenols. Interestingly, however, we found a significant increase in the total antioxidant capacity of plasma (FRAP) in these individuals after apple consumption, which was due to increased plasma levels of ascorbate and urate. Consumption of a fructose solution matching the fructose content and mass of the apples likewise led to an increase in plasma antioxidant capacity paralleled by an increase in urate levels. Our data suggest that the in vivo antioxidant effects of apple consumption may be explained, in large part, by the well-known metabolic effect of fructose on urate production. 1

Methods: Extraction, separation, identification and quantification of apple polyphenolics Red Delicious (RD), Granny Smith (GS) and Fuji apples (FJ) of known history were supplied by Stemilt Growers, WA. Extraction protocols followed the procedures developed in year 1 of this project. Apples were peeled and cored, and peels and flesh were cryogenically milled with liquid nitrogen. Acetone extraction and chloroform partition incorporating centrifugation were used for preparation of aqueous extracts. Extracts were stored at -7 C before antioxidant and total polyphenolic analysis. Analytical and semipreparative HPLC were the primary analytical tools for separating and identifying polyphenolics. HPLC separations utilized the improved methodology developed by Schieber et al. (21). Electro-Spray Mass Spectroscopy (ESMS) was also used for identifying individual compounds. Quantities of individual compounds were measured by compound class (flavonols, flavanols, anthocyanins, procyanidins, cinnamates, etc.) with HPLC via the external standard method (Spanos et al., 199; Wen et al., 1999). Total phenolics were measured by the Folin-Ciocalteau procedure. Experiments with apple polyphenols and apple extracts The antioxidant capacity of different applecontained polyphenolics and apple extracts were evaluated by FRAP and ORAC. Total phenol content was determined by Folin-Ciocalteau. Individual catechins and phloridzin were measured by HPLC with electrochemical detection. Samples for 1-MCP Study: Red Delicious, Granny Smith and Fuji apples were provided by Stemilt Growers, WA. One box of each variety was fumigated with 1 ppm of 1-MCP for 18 hr at 2 C at the USDA Tree Fruit Research Laboratory, Wenatchee. Treatments were done 2 weeks after harvest. Apples were stored at C, 88% RH. Samples were shipped to the OSU Food Science & Technology Department at storage intervals of, 3 and 6 months. Study design for in vitro antioxidant activity of apple polyphenolics in human plasma Apple extracts containing different amounts of polyphenols were added to human plasma. The antioxidant capacity of plasma was measured without or with prior addition of apple extracts or apple polyphenols, using FRAP and ORAC. Human plasma was also oxidized by incubation with 5 mm AAPH (37 C, 4 h), which thermally decomposes and generates water-soluble peroxyl radicals at a constant rate. RD apple extracts (7.1 or 14.3 µg/ml total phenols) were added to the plasma samples before incubation with AAPH. Samples were incubated in a shaking water bath at 37 C under air atmosphere. Aliquots were withdrawn at different time points for determination of ascorbate, urate, α-tocopherol and cholesterol ester hydroperoxides (CE-OOH), using HPLC techniques (Frei et al., 1988). Study design for in vivo antioxidant activity of apple consumption Participants (n=6) consumed five RD apples (average apple weight: 238.7±4.5 g; total apple mass consumed: 137±38 g) provided by Stemilt Growers, WA. The participants were instructed to abstain from flavonoid-containing foods and dietary supplements for 24 h prior to the study. Blood was collected before ( h) and 1, 2, 3, 4, and 6 h after eating the apples. To evaluate the resistance of plasma to ex vivo oxidation, plasma was incubated separately with 5 mm AAPH (37 C, 4 h), and ascorbate, urate, α-tocopherol and CE-OOH were measured, as well as FRAP and ORAC (after precipitation of plasma proteins). To estimate the contribution of ascorbate, plasma was treated with ascorbate oxidase (FRAP AO ). After a washout period of two months, the same subjects consumed plain bagels (263.1±.9 g) and water matching the glycemic index and total mass of the five apples, or fructose (63.9±2.9 g) dissolved in water matching the fructose content and mass of the five apples. Repeated-measures analysis of variance (ANOVA) was used to evaluate time-dependent changes in plasma parameters, and the Tukey-Kramer test for post-hoc analysis. 2

Results and discussion: Experiments with apple polyphenols and apple extracts Table 1 shows the antioxidant capacity (FRAP and ORAC) of apple polyphenols and apple extracts in vitro. Table 1. Ferric Reducing Antioxidant Potential (FRAP) and Oxygen Radical Absorbance Capacity (ORAC) of apple polyphenols. Group Compound FRAP (µm) Quercetin Flavonols Rutin Flavanols ( )-Epicatechin (+)-Catechin ORAC (µm) 3.6 ±.26 a 2.9 ±.11 a 1.48 ±.21 b,d,e 2.9 ±.19 a 1.95 ±.18 b 2.52 ± 1.73 ±.6 b,d.2 a,b,d 2.12 ±.14 b,c.18 ±.4 c Phloridzin 1.94 ±.34 c,d Dihydrochalcones Phloretin 1.28 ±.2 d,e 1.72 ±.2 c Hydroxycinnamates Chlorogenic acid 1.21 ±.26 e 1.55 ±.24 c Values are expressed as the trolox equivalent concentration/µm polyphenol. Data shown are the mean ± SEM of at least 3 independent determinations. Data in the same column that are not sharing any superscripts are significantly different (p<.5). In both assays, apple-contained flavonols and flavanols were more effective than dihydrochalcones and the hydroxycinnamate, chlorogenic acid (Table 1). Aqueous apple extracts prepared from the flesh and skin of RD apples had the highest total phenolic content (357±6 mg/l) and antioxidant capacity (2877±91 and 352±89 µm trolox equivalents for FRAP and ORAC, respectively). GS and FJ extracts exhibited 192±7 and 1595±9 µm trolox equivalents for FRAP, respectively, and 1635±22 and 1697±178 µm trolox equivalents for ORAC, respectively. Considering the concentration and contribution of the individual polyphenols (Table 1), it can be calculated that for RD extracts the identified polyphenols account for only 14-2% of the total antioxidant capacity of extracts, suggesting that other compounds (such as procyanidins) make a significant contribution. Influence of cultivar and storage on antioxidant properties and total phenolics - Figure 1 shows the influences of cultivar and post-harvest storage on total phenolics (Fig. 1A) and antioxidant properties (Fig. 1B). Total phenolics are much higher in the peel than the flesh. RD was significantly higher in total phenolics than GS and FJ apples. Total phenolics showed a positive correlation with ORAC values, r 2 =.85 for peel and.87 for flesh. Antioxidant properties as measured by FRAP showed similar trends to ORAC with r 2 for ORAC and FRAP being.82 for peels and.7 for flesh. Anthocyanin pigments are believed to be a major contributor to both total phenolics and antioxidant properties of apple peels. There was no significant change in total phenolics with storage, however antioxidant properties showed a decreasing trend. The antioxidant properties of the non-polar chloroform phase were much lower than those of the aqueous phase, with values for the non-polar extract being typically 6% of the "aqueous" extract. GS non-polar extracts had the highest values, presumably because of the chlorophyll content. 3

Total Phenolics Contents of Peels and Flesh Red Delicious Fuji Granny Smith ORAC Contents of The Peels and Flesh Red Delicious Fuji Granny Smith 7 4 6 35 5 3 mg/g GAE 4 3 2 1 Peel Flesh Peel Flesh Peel Flesh µmol T.E/g 25 2 15 1 5 Peel Flesh Peel Flesh Peel Flesh month 3 months 6 months month 3 months 6 months FIGURE 1. Influences of cultivar and post-harvest storage on total phenolics (panel A) and antioxidant properties (panel B). Effect of 1-MCP treatment on apple polyphenolics and antioxidant properties - Treatment with 1-MCP had no significant effect on total phenolics, total anthocyanins, or the antioxidant properties. Mean values for the three cultivars are summarized in Table 2. Table 2. Marginal means of treatment effects on edible portions of apples Treatment Control 1-MCP Total Phenolics, mg/g GAE 1.86 1.96 Total Anthocyanins, mg/1g 1.79 1.66 ORAC, µmol TE/g 8.86 8.48 FRAP, µmol TE/g 8.94 9.13 In vitro experiments with human plasma and apple extracts To assess whether physiologically relevant concentrations of apple polyphenols can add to the antioxidant capacity of plasma, increasing amounts of RD extracts equivalent to 3.6 to 35.7 µg/ml total apple phenols were added to fresh human plasma, and FRAP and ORAC were measured (Fig. 2). We also calculated the expected increases in plasma FRAP and ORAC based on the amounts of RD extracts added and their FRAP and ORAC values. A linear 7-52% increase in plasma FRAP was observed (p<.1) (Fig. 2). ORAC was measured before and after addition of apple extracts, and before and after precipitation of proteins with perchloric acid (PCA). There was a non-significant trend towards increased ORAC after the addition of apple extracts in the range of 3.6 to 35.7 µg/ml total apple phenols (p=.9), with a maximal increase of about 8%. Precipitation of proteins with PCA decreased plasma ORAC by more than 7% to 819±14 µmol/l trolox equivalents. Adding apple extracts slightly, but significantly (p<.5), increased ORAC of PCA-precipitated plasma, with a maximal increase of about 12% at 35.7 µg/ml total apple phenols (Fig. 2). 4

FRAP (µm trolox equivalents) 8 75 7 65 6 55 5 45 A ORAC (µm trolox equivalents) 32 B 3 28 26 24 12 1 8 4 5 1 15 2 25 3 35 Apple polyphenols (µg/ml) 6 5 1 15 2 25 3 35 Apple polyphenols (µg/ml) FIGURE 2. FRAP and ORAC of human plasma supplemented with RD apple extract. Human plasma was spiked with RD apple extracts, and the antioxidant capacity was measured as FRAP (A) or ORAC (B) (filled symbols). ORAC was measured in whole plasma (circles) or after precipitation of plasma proteins with perchloric acid (triangles). The expected values for plasma FRAP and ORAC are also shown, calculated from the values for RD apple extracts themselves and the amounts of apple extracts added (open symbols, dotted lines). Results are shown as mean ± SEM of n=4 (FRAP) or n=3 (ORAC) independent experiments. When plasma was exposed to a constant flux of aqueous peroxyl radicals, endogenous ascorbate (7.±1.3 µm) was oxidized within 45 min of incubation, while urate (375±4 µm) was oxidized after ascorbate with a half-life (t 1/2 ) of 136±15 min (n=3 independent experiments). Addition of RD extracts (7.1 or 14.3 µg/ml total phenols) did not protect ascorbate from oxidation, but increased t 1/2 of urate significantly (p<.5). Similarly, t 1/2 of plasma α-tocopherol (24.7±1.2 µm) increased significantly in the presence of apple extracts (p<.5). Lipid peroxidation started after ascorbate depletion, and addition of RD extracts increased the lag time preceding detectable lipid peroxidation from 36.3±3.7 to 5.9±2.7 (p<.5) and 7.4±4.2 min (p<.1). Results are summarized in Table 3. Table 3. AAPH-induced oxidation of human plasma: kinetic parameters in the absence and presence of apple extracts Apple Extract (µg/ml phenols) Ascorbate Urate α-tocopherol CE-OOH lag phase (min) CEOOH rate (µm/min) 11.6 ± 1.5 136 ± 15 141 ± 18 36.3 ± 3.7.46 ±.19 7.1 12.2 ± 1.9 192 ± 16 164 ± 8 5.9 ± 2.7.39 ±.8 14.3 11.5 ± 2.6 28 ± 23 188 ± 8 7.4 ± 4.1.38 ±.5 Human plasma was incubated with 5 mm AAPH, in the absence or in the presence of Red Delicious apple extracts. The kinetic parameters of ascorbate, urate and α-tocopherol oxidation, and CE-OOH production were determined. Values in the presence of apple extracts were statistically compared to nonsupplemented plasma by one-way ANOVA (= p<.1; = p<.5). Effects of apple consumption on human plasma oxidation Six healthy volunteers consumed 5 whole (flesh and skin) RD apples, similar to the ones used to prepare the apple extracts. The resistance of plasma 5

antioxidants and lipids to oxidation after apple consumption was evaluated by incubation of plasma with 5 mm AAPH (37ºC, 4 h). No significant variation over time was observed in any of the parameters evaluated (Table 4). AAPH-mediated oxidation of ascorbate and α-tocopherol were not affected by apple consumption, as indicated by non-significant changes in t 1/2 (p>.5, repeated-measures ANOVA). Urate t 1/2 and lag phase of CE-OOH production showed a trend toward an increase at 2 h after apple consumption (Table 4), but these changes were not statistically significant. These data do not support a significant in vivo antioxidant effect of apple polyphenols in plasma after apple consumption. Table 4. AAPH-induced oxidation of human plasma: kinetic parameters before and after apple consumption Time (h) Ascorbate Urate α-tocopherol CE-OOH lag phase (min) 8.1 ± 1.4 137 ± 11 134 ± 15 42.2 ± 3.7 1 8.7 ± 1.8 147 ± 16 134 ± 19 47.5 ± 6. 2 7.4 ± 1.3 17 ± 17 15 ± 12 58.9 ± 7.5 4 8.6 ±.9 152 ± 14 158 ± 15 45.5 ± 6.9 Human plasma was obtained before and after apple consumption, and then incubated with 5 mm AAPH (n=6). The kinetic parameters of ascorbate, urate and α-tocopherol oxidation, and CE-OOH production were determined. Values were statistically compared by repeated-measures ANOVA. No significant variation over time was detected for any of the analyzed parameters (p>.5). Effects of apple consumption on total antioxidant capacity of human plasma Significant increases in plasma FRAP and FRAP AO were found following apple consumption (repeated measures ANOVA, p<.5), with significant (transient) increases at 1 h in both FRAP (from 445±35 to 499±34 µmol/l trolox equivalents) and FRAP AO (from 363±34 to 425±49 µmol/l) (Fig. 3). Ascorbate significantly increased from 64±4 µmol/l at baseline to 77±7 µmol/l at 1 h, and urate from 271±39 to 367±43 µmol/l. Consumption of fructose likewise led to parallel increases in plasma antioxidant capacity and urate levels, but did not affect ascorbate concentrations (Fig. 3). One hour after fructose consumption, FRAP and FRAP AO increased significantly from 441±58 to 486±62 µmol/l and 354±55 to 393±6 µmol/l, respectively. Similarly, a significant variation of urate over time was observed after fructose intake (p<.5). Post-hoc analysis indicated that the changes in urate were significant at 1 and 2 hours after fructose consumption. In contrast, FRAP and urate decreased significantly after bagel consumption (control) (Fig. 3). Our results indicate that ascorbate and urate contribute >9 % to the measured changes in FRAP after apple consumption. Values of FRAP AO (and its changes) were significantly correlated with urate (and its changes) after apple consumption (p<.1). In agreement, there was a highly significant correlation between FRAP AO and urate levels after fructose consumption (p<.1, r=.94). A highly significant correlation was also found between the increases in urate levels after apple consumption and after administration of fructose (p<.1, r=.7). 6

Changes in FRAP (microm trolox) Changes in FRAP AO (microm trolox) 8 6 4 2-2 -4 8 6 4 2-2 -4 Apples Fructose Bagels 1 2 3 4 6 1 2 3 4 6 Changes in Urate (microm) Changes in Ascorbate (microm) 25 2 15 1 5-5 -1 12 1 8 6 4 2-2 -4 1 2 3 4 6 1 2 3 4 6 Time (h) Time (h) FIGURE 3. Changes in antioxidant capacity (FRAP and FRAP AO ) and antioxidants (ascorbate and urate) in human plasma after apple consumption. Changes were determined in human plasma before () and 1, 2, 3, 4, and 6 h after apple (black bars), bagel (dark grey bars) or fructose (light bars) consumption. Data are shown as changes (µmol/l trolox equivalents), and expressed as the mean ± SEM (n=6). Statistical analysis (repeated measures ANOVA) showed a significant increase of FRAP and FRAP AO with time after apple and fructose consumption, and a significant decrease after bagel consumption (p <.5) = significantly different from time h (post-hoc Tukey-Kramer test). The antioxidant capacity of plasma was also measured as ORAC, in samples previously precipitated with PCA (Fig. 4). Basal plasma ORAC increased significantly following apple consumption (p<.1, repeated-measures ANOVA). Post hoc analysis revealed that ORAC was significantly higher at 1, 2, 3 and 4 hours after apple consumption. ORAC also increased significantly following bagel or fructose consumption (p<.5, repeated-measures ANOVA) (Fig. 4). No statistical significance was reached in the comparison of the areas under the curve (AUC) for ORAC changes vs. time (-6 h) (repeated-measures ANOVA). 7

.4 ORAC (mm trolox).3.2.1. 1 2 3 4 5 6 Time (h) FIGURE 4. Changes in ORAC in human plasma after apple consumption. Changes were determined in human plasma before () and 1, 2, 3, 4, and 6 h after apple (filled circles), bagel (empty circles) or fructose (triangles) consumption. Data are shown as changes (mm trolox equivalents), and expressed as the mean ± SEM. Statistical analysis (repeated measures ANOVA) showed a significant increase of ORAC with time after apple, bagel and fructose consumption (p <.5). In summary, our results indicate that apples are a significant source of polyphenols, which exhibit high antioxidant capacity (FRAP and ORAC) measured not only in chemical systems (extracts) but also in physiologically relevant biological systems (protection of plasma components against oxidation). However, despite the high amount of polyphenols ingested, our results do not support a significant antioxidant function of apple polyphenols in plasma after apple consumption, likely due to the limited bioavailability and extensive metabolism of these compounds in vivo. Interestingly, the total antioxidant capacity of plasma (FRAP) did increase after apple consumption, not due to apple polyphenols, but mainly due to fructose-mediated increases of plasma urate. Urate is an endogenous antioxidant, present in high concentrations in plasma. Although the metabolic effect of fructose leading to urate production has been described (Maenpaa et al, 1968), increases of this plasma antioxidant after apple (or other fruit) consumption have not been well established. This is a novel and unexpected finding, and can provide an alternative or additional explanation for the health benefits of fruit intake. Further investigations are needed to establish the in vivo relevance and potential health benefits of increased plasma urate levels. 8

Budget: Project Title: Health Benefits of Apples PI: R.E. Wrolstad a & B. Frei b Organization: a Food Science & Technology, b Linus Pauling Institute, OSU Project duration; 2-23 Project total (4 yrs): $248, Year Year 1 (2) Year 2 (21) Year 3 (22) Year 4 (23) Total 5, 66, 66, 66, Breakdown: Salaries a 29,28 42,351 39,162 36,34 Benefits b 13,52 13,929 17,858 16,942 Supplies c 5,452 7,72 5,98 11,718 Maintenance HPLC 1, 1, Travel d 1, 1, 1, Publication costs e 1, 1, 1, a Bob Durst (Sr. Research Assistant, FST); Maria Widyasari (GRA, FST, Years 1 & 2); Deborah Hobbs (research Assistant, LPI); Silvina Lotito (Post-doc, LPI, Years 3 & 4); Alfio DiMauro (Post-Doc, FST, Year 4). b OPE (42-49%) c Extraction solvents; HPLC supplies; maintenance of printer; ESMS fees; human aortic endothelial cells, cell culture media, chemicals, plastic ware, etc. d Travel in years 1 & 2 applied to materials and supplies; travel in year 3 to IFT for presentation of research findings. e Publication costs in years 1 & 2 applied to materials and supplies. Presentations at Professional Meetings: Silvina B. Lotito and Balz Frei. Antioxidant capacity of apple polyphenols. Poster presentation at Oxygen Club of California, 22 World Congress, Oxidants and Antioxidants in Biology, Santa Barbara, CA. March 6-9, 22 Silvina B. Lotito and Balz Frei. Apple extracts protect against antioxidant depletion and lipid peroxidation in human plasma. Poster presentation, 9 th Annual Meeting of the Oxygen Society, San Antonio, TX. November 2-24, 22 Silvina B. Lotito and Balz Frei. Apple consumption increases the total antioxidant capacity of plasma in humans due to an increase in urate levels. Poster presentation, 9 th Annual Meeting of the Oxygen Society, San Antonio, TX. November 2-24, 22 M. A. Widyasari and R. E. Wrolstad. Antioxidant Activities in Apples. Poster presentation, Institute of Food Technologists (IFT) Annual Meeting, Anaheim, CA. June 15-19, 22 Original Research Publications: Maria A. Widyasari. 22. Apple polyphenolics and their antioxidant properties: Influence of cultivars, post-harvest storage, and 1-MCP treatment. M.S. thesis, Oregon State University. Silvina B. Lotito and Balz Frei. Relevance of apple polyphenols as antioxidants in human plasma: contrasting in vitro and in vivo effects. Submitted for publication in Free Radic. Biol. Med. Silvina B. Lotito and Balz Frei. Apple consumption increases the antioxidant capacity of plasma due to a fructose-mediated increase in urate levels. Submitted for publication in Am. J. Clin. Nutr. 9