- Email: [email protected]
Phytochemical content and antioxidant capacity of water-soluble isolates from peanuts (Arachis hypogaea L.)
Food Research International 39 (2006) 898–904 www.elsevier.com/locate/foodres
Phytochemical content and antioxidant capacity of water-soluble isolates from peanuts (Arachis hypogaea L.) Christopher E. Duncan a, Daniel W. Gorbet b, Stephen T. Talcott a
a,*
Department of Food Science and Human Nutrition, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, USA b University of Florida, North Florida Research and Education Center, Marianna, FL 32344, USA Received 29 November 2005; accepted 22 May 2006
Abstract Numerous polyphenolics have been identified in peanuts, but information relating to their storage stability and contributions to total antioxidant capacity are lacking. This study investigated contributors to the antioxidant capacity of six peanut cultivars by means of fractionation from reversed phase C18 cartridges. Five water-soluble peanut isolates were prepared and subsequently stored at 35 C and evaluated for individual and total polyphenolics, antioxidant capacity, total reducing equivalents, and total amino acids after 0, 2, 4 and 8 weeks. The relative contribution of each isolate to total antioxidant capacity for each cultivar was additive, with the C18 non-retained fraction containing 100% of the p-coumaric acid and 82% of the total amino acids. During storage relatively small changes were observed for antioxidant activity and total reducing equivalents among cultivars and isolates, indicating the lack of oxidative or condensation reactions among the isolated constituents. Results demonstrated the overall stability and diversity of antioxidant polyphenolics found in peanuts, information that advances the marketability of the crop. 2006 Elsevier Ltd. All rights reserved. Keywords: Peanuts; Antioxidant; Fractionation; Polyphenolics; Storage
1. Introduction Peanuts may be consumed raw, roasted, pureed, or in a variety of other processed forms, and constitute as a multimillion-dollar crop world wide (Yu, Ahmenda, & Goktepe, 2005) with numerous potential dietary benefits. Recently several peanut cultivars were developed with elevated concentrations of the monounsaturated fatty acid oleic acid, in relation to other highly oxidizable polyunsaturated fatty acids. The high oleic trait provides peanuts with potentially greater health benefits and serves to prolong shelf life characteristics (Pattee, Isleib, Moore, Gorbet, & Giesbrecht, 2002; Reed, Sims, Gorbet, & O’Keefe, 2002; Talcott, Duncan, Del Pozo-Insfran, & Gorbet, 2005). Additionally, peanuts were found to lack naturally occurring trans-fatty acids (Sanders, 2001), which have been adversely associated with
*
Corresponding author. Tel.: +1 352 392 1991; fax: +1 352 392 9467. E-mail address: [email protected]fl.edu (S.T. Talcott).
0963-9969/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2006.05.009
various diseases (Awad, Chan, Downie, & Fink, 2000; Feldman, 1999; Peanut-Institute, 2002). Numerous phytochemical compounds are present in peanuts with potential antioxidant capacity including polyphenolics (Talcott, Passeretti, Duncan, & Gorbet, 2005), tocopherols (Hashim, Koehler, & Eitenmiller, 1993), and proteins (Bland & Lax, 2000). Other than contributions from these compounds, mature peanut kernels likely possess few other compounds in significant quantities that would impact antioxidant capacity. Up to 15 polyphenolics have been identified in peanuts (Duke, 1992) whereas Seo and Morr (1985) identified six from defatted peanut meal where p-coumaric acid was the predominant compound, accounting for 40–68% of the total phenolics present. Fajardo, Waniska, Cuero, and Pettit (1995) reported stress-induced synthesis of free and bound polyphenolics in peanuts with p-coumaric and ferulic acid present in the highest concentrations. Alone, p-coumaric acid has been shown to possess significant radical scavenging activities (Rice-Evans, Miller, & Paganga, 1996; RiceEvans, Miller, & Paganga, 1997) but its contribution to total
C.E. Duncan et al. / Food Research International 39 (2006) 898–904
antioxidant capacity in peanuts has not been reported. Containing 25% protein by weight, Talcott, Duncan et al. (2005) reported changes in soluble proteins and amino acids following dry roasting that along with moisture loss and formation of roasting by-products may have contributed to increased antioxidant capacity of peanuts. Sathe, Hamaker, Sze-Tao, and Venkatachalam (2002) identified the major protein in Inca peanuts to be albumin and accounted for 31% of the total protein. Proteins or amino acids may act to physically trap free radicals or participate in Maillard browning reactions during roasting, resulting in the formation of new antioxidant compounds (Borrelli, Visconti, Mennella, Anese, & Fogliano, 2002; Ehling & Shibamoto, 2005). Yanagimoto, Lee, Ochi, and Shibamoto (2002) demonstrated that pyrazines formed during peanut roasting had no antioxidant activity, while other classes of Maillard derivatives, namely pyrroles and furans, exhibited minor antioxidant capacity. As demonstrated in a previous study, the antioxidant activity of intact peanuts increased during roasting, possibly from the formation of Maillard reaction derivatives (Talcott, Passeretti et al., 2005). The soluble polyphenolics in dry roasted peanuts possess a radical scavenging capacity equivalent to many fruits and vegetables, yet values vary greatly among commercial peanut cultivars and among harvest years. Little information is available on specific compounds or a class of compounds that actually contribute to total antioxidant capacity. Therefore the objective of this work was to investigate contributors to antioxidant capacity in five isolates from six peanut cultivars and to evaluate changes in these isolates during accelerated storage at 35 C. 2. Materials and methods 2.1. Materials and processing Shelled kernels from six runner-type peanut cultivars were obtained from the University of Florida Agricultural Research Center in Marianna, FL, USA in 2004 and were stored frozen ( 20 C) under a blanket of nitrogen until utilized. Samples included the cultivars ‘Carver’, ‘Georgia Green’, and ‘DP-1’ and the high oleic varieties (>80% oleic acid) ‘Andru II’, ‘ANorden’, and ‘Hull’ (Anderson & Gorbet, 2002). Peanut were removed from frozen storage, warmed to room temperature, and 500 g from each cultivar roasted at 170 ± 3 C for 10 min in a Pyrex forced air convection oven (Aroma AeroMatic Oven, San Diego, CA, USA). Peanuts were agitated every 3 min to insure uniform roasting and the temperature monitored using a digital thermocouple (Component Design, Portland, OR, USA). Peanuts were cooled for manual testa removal, and held at 20 C for 14 days until extraction and analysis. 2.2. Physiochemical analyses Roasted peanut kernels were homogenized in a kitchenscale food processor to the smallest obtainable particle size
899
without creating a puree. Kernels were defatted by homogenizing with hexane (100 g/300 mL) using a tissuemizer (PT-10, Brinkman Instruments, Westbury, NY, USA). The mixture was held at 4 C for 24 h prior to solvent removal by vacuum filtration through Whatman #4 filter paper. A portion of the dried, defatted peanut flour was then tissumized in 80% methanol to remove soluble constituents and the solvent removed by rotary evaporation at 45 C. Isolates were re-dissolved with the aid of a sonic water bath into 0.1 M citrate buffer at pH 6.1, the pH of the original peanuts. Aqueous peanut extracts were divided into three equal portions for subsequent fractionation using Waters reversed phase C18 Sep-Pak Vac 20cc cartridges (Waters Corp., Milford, MA, USA; Fig. 1). The first portion was retained without modification as a stock extract (isolate I). The second portion was loaded onto a C18 cartridge and the non-retained fraction (isolate II) collected, while retained compounds were eluted with 30 mL of methanol (isolate III). The third portion was loaded onto a second C18 cartridge and eluted first with 30 mL of ethyl acetate (isolate IV) followed by 30 mL of methanol (isolate V). Solvents were removed by rotary evaporation at <35 C and each brought to an equivalent volume with 0.1 M citrate buffer at pH 6.1. The five isolates were divided into three replicates, filled into 15 mL screw-cap test tubes, individually flushed with nitrogen, and stored in the dark for 0, 2, 4, and 8 weeks at 35 C. Following storage, samples were held frozen ( 20 C) until analysis, with measurements taken directly from each extract after filtering through a 0.45 lm filter. Total reducing equivalents, including contributions from proteins and phenolic amino acids, were measured using the Folin–Ciocalteu assay with data expressed in mg/kg gallic acid equivalents. Free amino acids were measured in accordance with Fiedman, Pang, and Smith (1984) with values expressed in mg/kg equivalents of tryptophan. Individual polyphenolics were separated and characterized by HPLC according to the conditions of Talcott, Brenes, and Howard (2000) using a Dionex HPLC system (Dionex,
Roasted & Defatted Peanut Kernel Isolate I Stock Extract C18 Sep Pak Isolate III Methanol
Isolate II Non-retained
C18 Sep Pak
Isolate IV Ethyl Acetate
Isolate V Methanol
Fig. 1. Solid phase extraction scheme used to fractionate water-soluble phytochemicals from roasted, defatted peanuts.
900
C.E. Duncan et al. / Food Research International 39 (2006) 898–904
Sunnyvale, CA, USA). Separation was conducted on a Waters Spherisorb ODS-2 (4.6 · 250 mm; Waters Corp., Milford, MA, USA) column with detection at 280 nm using a Dionex PDA-100 photodiode array detector scanning from 200 to 400 nm. Concentrations of individual polyphenolics were expressed in equivalents of p-coumaric acid. Antioxidant capacity was determined using the oxygen radical absorbance capacity assay as previously described using a microplate reader (Talcott, Percival, Pitter-Moore, & Celoria, 2003). Peanut isolates were diluted in pH 7 phosphate buffer prior to analysis and values background corrected for the pH 6.1 citrate buffer. Antioxidant capacity was expressed in lM Trolox equivalents per gram of roasted peanut. 2.3. Statistical analysis Data were analyzed as a 6 · 4 · 5 full factorial that included six peanut cultivars analyzed at four sampling times and five isolates. Data represent the mean and standard error of triplicate determinations. Multiple linear regression, Pearson correlations, and analysis of variance were conducted using JMP software Version 5 (SAS Institute, 2002), with mean separation performed by the LSD test (P < 0.05). Recoveries were calculated based on concentrations present in isolate I unless where otherwise stated. 3. Results and discussion 3.1. Peanut isolate I Previous measurements of antioxidant capacity in intact, dry roasted peanuts held under a blanket of nitrogen at 35 C observed up to a 50% decrease over 8 weeks of storage that was independent of cultivar, fatty acid composition, or lipid oxidation (Talcott, Duncan et al., 2005). A goal of the current investigation was to determine a potential cause for these decreases during storage through selective isolation of soluble peanut constituents. Isolate I represented the all of the water-soluble phytochemical constituents isolated from peanuts, yet during storage this isolate did not exhibit a decrease in antioxidant capacity as previously observed for intact peanuts. Likewise, no difference between normal and high oleic acid varieties was observed. Results potentially indicated a difference in oxidative conditions created by the aqueous peanut isolates. In an effort to fractionate the antioxidative compounds present in whole peanuts, numerous intrinsic characteristics of the peanut matrix were likely altered. Changes in water activity, surface area, phytochemical interactions, and the water solubility of oxidizable compounds may be responsible for the difference in storage behavior and susceptibility to oxidation over time. For the cultivars Carver, Andru II, and ANorden no significant changes in antioxidant capacity were observed over 8 weeks storage at 35 C, while Georgia Green, DP-
1, and Hull exhibited antioxidant increases of 27%, 26%, and 32%, respectively (Fig. 2). The varying changes among cultivars during storage may have reflected small compositional differences among the cultivars, secondary reactions with soluble proteins, or varying degrees of oxidation that occurred during storage. Also small increases in total reducing equivalents were observed in all cultivars primarily between the second and fourth week of storage (data not shown). Since the Folin–Ciocalteu assay has an inherent inclination to react with reducing compounds other than polyphenolics, changes in soluble phenolics due to storage was an indication of varying reactivities of the phytochemicals during storage. Among the cultivars evaluated, Carver exhibited the highest antioxidant capacity (19.1 lM Trolox equiv./g) and total reducing equivalent concentration (852 mg/kg) (Table 1). Of the six cultivars evaluated, four exhibited a positive correlation between antioxidant activity and total reducing equivalents (r = 0.85, 0.82, 0.79, and 0.78 for Carver, Andru II, Georgia Green, and DP-1, respectively), similar to correlations previously observed for intact peanuts stored at 35 C (Talcott et al., 2005). The cultivars ANorden and Hull had a lower correlation coefficient (r = 0.56 and 0.47, respectively) that was due to their low total reducing equivalent concentration in relation to their antioxidant capacity. Separation of individual polyphenolics by reversed phase HPLC analysis was additionally conducted on the isolates and revealed many previously unidentified polyphenolic compounds that were likely contributors to the antioxidant and total reducing capacity of peanuts (Fig. 3). Predominant compounds selected from isolate I were tentatively characterized by UV spectroscopic properties and monitored for changes prior to storage and after 8 weeks at 35 C. Among those selected compounds changes were only observed during storage for peak 5 (kmax = 279.2 nm) and peak 7 (kmax = 256.2 nm), two compounds that were previously identified in peanuts (Talcott, Passeretti et al., 2005b). These compounds were present in isolates I and IV and exhibited average losses of 75% and 71%, respectively, among the cultivars. The remaining compounds varied insignificantly with storage and included peak 1 (kmax = 291.5 nm), peak 2 (kmax = 290.2 nm), peak 3 (kmax = 271.7 nm), peak 4 (p-coumaric acid; kmax = 306.5 nm), and peak 6 (kmax = 304.3 nm). 3.2. Peanut isolates II and III Water-soluble antioxidant constituents present in six peanut cultivars were assessed along with four additional isolates that were obtained based on their affinity to reversed phase C18 and their solubility in ethyl acetate or methanol. Average recovery of antioxidant constituents from isolates II and III were 17 ± 4% and 58 ± 5%, respectively, among the cultivars with an additional 25% that was not accounted for in the antioxidant capacity assay. Recoveries varied somewhat among cultivars due to differences in
C.E. Duncan et al. / Food Research International 39 (2006) 898–904
μM Trolox equivalents/g
Carver
Andru II
20
20
15
15
10
10
5
5
0
0 0
2
4
6
8
0
2
μM Trolox Equivalents/g
4
6
8
6
8
6
8
DP-1
Georgia Green 20
20
15
15
10
10
5
5
0
0 0
2
4
6
8
0
2
ANorden
4
Hull
20
20
15
15
ORAC (μM TE/g)
μM Trolox Equivalents/g
901
10
5
10
5
0
0 0
2
4
6
8
Week
0
2
4
Week
Fig. 2. The antioxidant capacity (lM Trolox equiv./g of peanut) observed for six peanut cultivars following sequential fractionation (see Fig. 1) and subsequent storage at 35 C for 8 weeks. Bars represent the standard error of the mean (n = 3).
partition efficiency from the C18 cartridges, solvent affinities, and from potential interactions with non-polyphenolic constituents. However, average concentrations of total reducing equivalents demonstrated an additive effect for
isolates II and III (50 ± 3% and 54 ± 3%, respectively), which confirmed complete partitioning from the C18 cartridge. As found in isolate I, an increase in total reducing equivalents was also observed in isolates II and III during
902
C.E. Duncan et al. / Food Research International 39 (2006) 898–904
Table 1 Antioxidant capacity (lM Trolox equiv./g), total reducing equivalents (mg/kg gallic acid equivalents; Folin–Ciocalteu assay), and total amino acids (mg/kg tryptophan equivalents; ninhydrin-reactive substances) present in each of six peanut cultivars immediately following phytochemical isolations (see Fig. 1) Cultivar
Isolate
Antioxidant capacity
Total reducing equivalents
Total amino acids
Mean
Err
Mean
Err
Mean
Err
Carver
I II III IV V
19.1 3.81 10.3 6.12 6.13
2.0 0.5 0.9 1.9 0.8
852 393 538 252 299
24 14 24 8.9 3.7
2990 2540 602 239 466
84 110 29 30 7.8
Andru II
I II III IV V
13.8 1.87 6.60 2.95 3.90
0.6 0.2 1.3 0.9 0.1
736 339 461 198 221
23 15 2.1 3.4 3.3
3460 2670 773 305 553
96 210 23 44 21
Georgia Green
I II III IV V
12.5 2.79 9.29 2.59 4.37
1.4 0.4 0.2 0.7 0.2
768 382 395 132 233
13 29 9.0 15 3.0
4190 3690 503 169 377
240 290 7.8 19 8.3
DP-1
I II III IV V
9.84 3.31 7.41 1.34 3.24
2.3 0.9 0.2 0.1 1.4
793 444 348 104 180
26 51 3.1 15 3.4
7670 9060 663 298 504
61 990 17 120 40
ANorden
I II III IV V
11.9 0.59 6.63 1.79 6.40
0.8 0.0 0.5 0.3 0.6
700 265 365 78.2 185
6.7 32 3.4 41 1.9
4730 5100 614 83.4 491
84 260 15 11 27
Hull
I II III IV V
12.1 0.67 5.20 1.43 7.54
0.9 0.1 1.2 0.1 0.2
718 445 373 <50 238
12 3.9 4.4 0.4 3.5
6500 4350 510 46.5 458
240 270 12 15 38
Data represent the mean and standard error (Err) for each isolate (n = 3).
the second and fourth week of storage (data not shown), but did not correspond to changes in antioxidant capacity which was unaltered by the conditions of storage (Fig. 2). Isolate II contained 95% of the ninhydrin-reactive amino acids present among the four isolates and exhibited an 82 ± 5% overall recovery from isolate I. This fraction contained the lowest antioxidant activity among the isolates and suggested that peanuts, though plentiful in total protein, do not derive an appreciable portion of their antioxidant activity from soluble proteins or amino acids. The most prevalent antioxidant polyphenolic in peanuts was pcoumaric acid and following fractionation was only found in isolate II with few other identifiable compounds (Table 1, Fig. 3). Therefore, the majority of antioxidant capacity in isolate II was likely due to the presence of p-coumaric acid. Insignificant changes in p-coumaric acid concentration was observed after 8 weeks storage at 35 C and despite its predominance, its overall contribution to the total antioxidant capacity of dry roasted peanuts was found to be minimal. Among the isolates, contributions
to total antioxidant capacity were largely derived from compounds present in isolate III. This fraction was low in ninhydrin-reactive substances and contained 58% of the total antioxidant capacity. This fraction was devoid of p-coumaric acid, yet despite the presence of numerous compounds present in low concentrations no predominant polyphenolic compound was identified that related to antioxidant capacity. 3.3. Peanut isolates IV and V The polyphenolics present in isolate III were again partitioned from reversed phase C18 with ethyl acetate (isolate IV) followed by methanol (isolate V) to further elucidate those compounds responsible for the metal reduction and antioxidant capacity in peanuts. As found in the previous isolates, no changes in antioxidant capacity were observed during storage (Fig. 2) indicating their inherent stability. Recoveries of these isolates among the peanut cultivars in relation to isolate III were found to be mostly additive in
C.E. Duncan et al. / Food Research International 39 (2006) 898–904
1,100
903
mAU
4 750
Isolate I
1 6 2
500
3
5 7
250
min
-100
0 1,100
5
10
15
20
25
30
35
40
45
50
55
60
mAU
65
72
Isolate II
750
4 500
1 2
250
min
-100
0 1,100
5
10
15
20
25
30
35
40
45
50
55
60
mAU
65
72
Isolate III
750
500
3
5
6
1
7
250
min
-100
0
5
10
15
20
25
30
35
40
45
50
55
60
65
72
Fig. 3. Typical reverse phase HPLC chromatograph at 280 nm of isolates I, II, and III (see Fig. 1) for the cultivar ‘Carver’. Spectral characteristics for predominant compounds include peak 1 (kmax = 291.5 nm), peak 2 (kmax = 290.2 nm), peak 3 (kmax = 271.7 nm), peak 4 (p-coumaric acid; kmax = 306.5 nm), peak 5 (kmax = 279.2 nm), peak 6 (kmax = 304.3 nm), and peak 7 (kmax = 256.2 nm).
their antioxidant capacity (33 ± 6% and 71 ± 12%) and total reducing equivalents (29 ± 7% and 55 ± 2%) for isolates IV and V, respectively. This separation technique demonstrated that numerous polyphenolic compounds present in peanuts contributed to their relatively high antioxidant capacity. 3.4. Complimentary effects of isolates II and III Responses observed for isolates II and III that related to their combined antioxidant capacity and total reducing equivalents were different. With partition efficiency near 100% for compounds exhibiting reducing capacity, those same compounds did not exhibit the same additive effect towards antioxidant capacity. With high concentrations of ninhydrin-reactive compounds present in isolate II and
a high antioxidant capacity present in isolate III, their combined contribution seemed to indicate a complimentary effect that enhanced the antioxidant capacity of isolate I, accounting for 25% higher antioxidant capacity on average among the peanut cultivars. This effect was not observed between isolates IV and V, where only small amounts of ninhydrin-reactive compounds were present and the resultant antioxidant capacity was additive. Heinonen et al. (1998) also reported an increased antioxidant capacity when protein was present along with ferulic acid. Similarly Herraiz, Galisteo, and Chamorro (2003) described protein– phenolic complexes between tryptophan and naturally occurring phenolic aldehydes with significant antioxidant activity. This complimentary effect between ninhydrinreactive compounds and soluble polyphenolics could only be elucidated following sequential partitioning of the
904
C.E. Duncan et al. / Food Research International 39 (2006) 898–904
phytochemical constituents and may partially explain the relatively high antioxidant capacity for dry roasted peanuts. Extraction and sequential partitioning of water-soluble phytochemicals from six cultivars of dry roasted peanuts was conducted to determine those compounds responsible for the antioxidant capacity and storage stability. A significant portion of the antioxidant capacity of peanuts can be contributed to compounds other than p-coumaric acid and ninhydrin-reactive compounds, both found in high concentrations in the C18 non-retained fraction (isolate II). The majority of antioxidant compounds were found to be polyphenolics with a high affinity to reversed phase C18, but no predominant antioxidant compound was identified. These findings suggest a multitude of compounds exist in peanuts in relatively low concentrations contributing to an overall antioxidant capacity. Differences between antioxidant capacity and total reducing equivalents following partitioning seemed to indicate a complimentary effect between polyphenolics and ninhydrin-reactive compounds in peanuts that is partially responsible for their relatively high antioxidant capacity in relation to numerous fruits and vegetables. The isolations did not elucidate a cause for previously observed decreases in antioxidant capacity during storage of whole peanuts, but rather revealed the relative stability of peanut polyphenolics during storage and characterized those compounds responsible for their antioxidant capacity. References Anderson, P. C., & Gorbet, D. W. (2002). Influence of year and planting date on fatty acid chemistry of high-oleic acid and normal peanut genotypes. Journal of Agricultural and Food Chemistry, 50, 1298–1305. Awad, A. B., Chan, K. C., Downie, A. C., & Fink, C. S. (2000). Peanuts as a source of b-sitosterol, a sterol with anticancer properties. Nutrition and Cancer, 36, 238–241. Bland, J. M., & Lax, A. R. (2000). Isolation and characterization of a peanut maturity-associated protein. Journal of Agricultural and Food Chemistry, 48, 3275–3279. Borrelli, R. C., Visconti, A., Mennella, C., Anese, M., & Fogliano, V. (2002). Chemical characterization and antioxidant properties of coffee melanoidins. Journal of Agricultural and Food Chemistry, 50, 6527–6533. Duke, J. A. (1992). Handbook of phytochemical constituents of GRAS herbs and other economic plants. Boca Raton, FL: CRC Press. Ehling, S., & Shibamoto, T. (2005). Correlation of acrylamide generation in thermally processed model systems of asparagine and glucose with color formation, amounts of pyrazines formed, and antioxidative properties of extracts. Journal of Agricultural and Food Chemistry, 53, 4813–4819. Fajardo, J. E., Waniska, R. D., Cuero, R. G., & Pettit, R. E. (1995). Phenolic compounds in peanut seeds: enhanced elicitation by chitosan and effects on growth and aflatoxin B1 production by Aspergillus flavus. Food Biotechnology, 9, 59–78.
Feldman, E. B. (1999). Assorted monounsaturated fatty acids promote healthy hearts. American Journal of Clinical Nutrition, 70, 953–954. Fiedman, M., Pang, J., & Smith, G. A. (1984). Ninhydrin-reactive lysine in food proteins. Journal of Food Science, 49, 10–13. Hashim, I., Koehler, P. E., & Eitenmiller, R. R. (1993). Tocopherols in runner and Virginia peanut cultivars at various maturity stages. Journal of the American Oil Chemists’ Society, 70, 633–635. Heinonen, M., Rein, D., Satue´-Gracia, M. T., Huang, S.-W., German, J. B., & Frankel, E. N. (1998). Effect of protein on the antioxidant activity of phenolic compounds in a lecithin-liposome oxidation system. Journal of Agricultural and Food Chemistry, 46, 917–922. Herraiz, T., Galisteo, J., & Chamorro, C. (2003). L-tryptophan reacts with naturally occurring and food-occurring phenolic aldehydes to give phenolic tetrahydro-b-carboline alkaloids: activity as antioxidants and free radical scavengers. Journal of Agricultural and Food Chemistry, 51, 2168–2173. Pattee, H. E., Isleib, T. G., Moore, K. M., Gorbet, D. W., & Giesbrecht, F. G. (2002). Effect of high-oleic trait and paste storage variables on sensory attributes stability of roasted peanuts. Journal of Agricultural and Food Chemistry, 50, 7366–7370. Peanut-Institute. (2002). Antioxidants from food sources, like peanuts and peanut butter, may protect against Alzheimer disease. Press Release, June 26. Reed, K. A., Sims, C. A., Gorbet, D. W., & O’Keefe, S. F. (2002). Storage water activity affects flavor fade in high and normal oleic peanuts. Food Research International, 35, 769–774. Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structure– antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biological Medicine, 20, 933–956. Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1997). Antioxidant properties of phenolic compounds. Trends in Plant Science, 2(4), 152–159. Sanders, T. H. (2001). Non-detectable levels of trans-fatty acids in peanut butter. Journal of Agricultural and Food Chemistry, 49, 2349–2351. SAS Institute, Inc. (2002). LMP Software Version 5; Cary, NC. Sathe, K. S., Hamaker, B. R., Sze-Tao, K. W. C., & Venkatachalam, M. (2002). Isolation, purification, and biochemical characterization of a novel water-soluble protein from Inca peanut (Plukenetia volubilis L.). Journal of Agricultural and Food Chemistry, 50, 4906–4908. Seo, A., & Morr, C. V. (1985). Activated carbon and ion exchange treatments for removing phenolics and phytate from peanut protein products. Journal of Food Science, 50, 262–263. Talcott, S. T., Brenes, C. H., & Howard, L. R. (2000). Contribution of periderm material and blanching time to the quality of pasteurized peach puree. Journal of Agricultural and Food Chemistry, 48, 4590–4596. Talcott, S. T., Duncan, C. E., Del Pozo-Insfran, D., & Gorbet, D. W. (2005). Polyphenolic and antioxidant changes during storage of normal, mid, and high oleic acid peanuts. Food Chemistry, 89, 77–84. Talcott, S. T., Passeretti, S., Duncan, C. E., & Gorbet, D. W. (2005). Polyphenolic content and sensory properties of normal and high oleic acid peanuts. Food Chemistry, 90, 379–388. Talcott, S. T., Percival, S. S., Pitter-Moore, J., & Celoria, C. (2003). Phytochemical composition and antioxidant stability of fortified yellow passion fruit (Passiflora edulis). Journal of Agricultural and Food Chemistry, 51, 935–941. Yanagimoto, K., Lee, K., Ochi, H., & Shibamoto, T. (2002). Antioxidative activity of heterocyclic compounds found in coffee volatiles produced by the Maillard reaction. Journal of Agricultural and Food Chemistry, 50, 5480–5484. Yu, J., Ahmenda, M., & Goktepe, I. (2005). Effects of processing methods and extraction solvents on concentration and antioxidant activity of peanut skin phenolics. Food Chemistry, 90, 199–206.
Phytochemical content and antioxidant capacity of water-soluble isolates from peanuts (Arachis hypogaea L.) Christopher E. Duncan a, Daniel W. Gorbet b, Stephen T. Talcott a
a,*
Department of Food Science and Human Nutrition, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, USA b University of Florida, North Florida Research and Education Center, Marianna, FL 32344, USA Received 29 November 2005; accepted 22 May 2006
Abstract Numerous polyphenolics have been identified in peanuts, but information relating to their storage stability and contributions to total antioxidant capacity are lacking. This study investigated contributors to the antioxidant capacity of six peanut cultivars by means of fractionation from reversed phase C18 cartridges. Five water-soluble peanut isolates were prepared and subsequently stored at 35 C and evaluated for individual and total polyphenolics, antioxidant capacity, total reducing equivalents, and total amino acids after 0, 2, 4 and 8 weeks. The relative contribution of each isolate to total antioxidant capacity for each cultivar was additive, with the C18 non-retained fraction containing 100% of the p-coumaric acid and 82% of the total amino acids. During storage relatively small changes were observed for antioxidant activity and total reducing equivalents among cultivars and isolates, indicating the lack of oxidative or condensation reactions among the isolated constituents. Results demonstrated the overall stability and diversity of antioxidant polyphenolics found in peanuts, information that advances the marketability of the crop. 2006 Elsevier Ltd. All rights reserved. Keywords: Peanuts; Antioxidant; Fractionation; Polyphenolics; Storage
1. Introduction Peanuts may be consumed raw, roasted, pureed, or in a variety of other processed forms, and constitute as a multimillion-dollar crop world wide (Yu, Ahmenda, & Goktepe, 2005) with numerous potential dietary benefits. Recently several peanut cultivars were developed with elevated concentrations of the monounsaturated fatty acid oleic acid, in relation to other highly oxidizable polyunsaturated fatty acids. The high oleic trait provides peanuts with potentially greater health benefits and serves to prolong shelf life characteristics (Pattee, Isleib, Moore, Gorbet, & Giesbrecht, 2002; Reed, Sims, Gorbet, & O’Keefe, 2002; Talcott, Duncan, Del Pozo-Insfran, & Gorbet, 2005). Additionally, peanuts were found to lack naturally occurring trans-fatty acids (Sanders, 2001), which have been adversely associated with
*
Corresponding author. Tel.: +1 352 392 1991; fax: +1 352 392 9467. E-mail address: [email protected]fl.edu (S.T. Talcott).
0963-9969/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2006.05.009
various diseases (Awad, Chan, Downie, & Fink, 2000; Feldman, 1999; Peanut-Institute, 2002). Numerous phytochemical compounds are present in peanuts with potential antioxidant capacity including polyphenolics (Talcott, Passeretti, Duncan, & Gorbet, 2005), tocopherols (Hashim, Koehler, & Eitenmiller, 1993), and proteins (Bland & Lax, 2000). Other than contributions from these compounds, mature peanut kernels likely possess few other compounds in significant quantities that would impact antioxidant capacity. Up to 15 polyphenolics have been identified in peanuts (Duke, 1992) whereas Seo and Morr (1985) identified six from defatted peanut meal where p-coumaric acid was the predominant compound, accounting for 40–68% of the total phenolics present. Fajardo, Waniska, Cuero, and Pettit (1995) reported stress-induced synthesis of free and bound polyphenolics in peanuts with p-coumaric and ferulic acid present in the highest concentrations. Alone, p-coumaric acid has been shown to possess significant radical scavenging activities (Rice-Evans, Miller, & Paganga, 1996; RiceEvans, Miller, & Paganga, 1997) but its contribution to total
C.E. Duncan et al. / Food Research International 39 (2006) 898–904
antioxidant capacity in peanuts has not been reported. Containing 25% protein by weight, Talcott, Duncan et al. (2005) reported changes in soluble proteins and amino acids following dry roasting that along with moisture loss and formation of roasting by-products may have contributed to increased antioxidant capacity of peanuts. Sathe, Hamaker, Sze-Tao, and Venkatachalam (2002) identified the major protein in Inca peanuts to be albumin and accounted for 31% of the total protein. Proteins or amino acids may act to physically trap free radicals or participate in Maillard browning reactions during roasting, resulting in the formation of new antioxidant compounds (Borrelli, Visconti, Mennella, Anese, & Fogliano, 2002; Ehling & Shibamoto, 2005). Yanagimoto, Lee, Ochi, and Shibamoto (2002) demonstrated that pyrazines formed during peanut roasting had no antioxidant activity, while other classes of Maillard derivatives, namely pyrroles and furans, exhibited minor antioxidant capacity. As demonstrated in a previous study, the antioxidant activity of intact peanuts increased during roasting, possibly from the formation of Maillard reaction derivatives (Talcott, Passeretti et al., 2005). The soluble polyphenolics in dry roasted peanuts possess a radical scavenging capacity equivalent to many fruits and vegetables, yet values vary greatly among commercial peanut cultivars and among harvest years. Little information is available on specific compounds or a class of compounds that actually contribute to total antioxidant capacity. Therefore the objective of this work was to investigate contributors to antioxidant capacity in five isolates from six peanut cultivars and to evaluate changes in these isolates during accelerated storage at 35 C. 2. Materials and methods 2.1. Materials and processing Shelled kernels from six runner-type peanut cultivars were obtained from the University of Florida Agricultural Research Center in Marianna, FL, USA in 2004 and were stored frozen ( 20 C) under a blanket of nitrogen until utilized. Samples included the cultivars ‘Carver’, ‘Georgia Green’, and ‘DP-1’ and the high oleic varieties (>80% oleic acid) ‘Andru II’, ‘ANorden’, and ‘Hull’ (Anderson & Gorbet, 2002). Peanut were removed from frozen storage, warmed to room temperature, and 500 g from each cultivar roasted at 170 ± 3 C for 10 min in a Pyrex forced air convection oven (Aroma AeroMatic Oven, San Diego, CA, USA). Peanuts were agitated every 3 min to insure uniform roasting and the temperature monitored using a digital thermocouple (Component Design, Portland, OR, USA). Peanuts were cooled for manual testa removal, and held at 20 C for 14 days until extraction and analysis. 2.2. Physiochemical analyses Roasted peanut kernels were homogenized in a kitchenscale food processor to the smallest obtainable particle size
899
without creating a puree. Kernels were defatted by homogenizing with hexane (100 g/300 mL) using a tissuemizer (PT-10, Brinkman Instruments, Westbury, NY, USA). The mixture was held at 4 C for 24 h prior to solvent removal by vacuum filtration through Whatman #4 filter paper. A portion of the dried, defatted peanut flour was then tissumized in 80% methanol to remove soluble constituents and the solvent removed by rotary evaporation at 45 C. Isolates were re-dissolved with the aid of a sonic water bath into 0.1 M citrate buffer at pH 6.1, the pH of the original peanuts. Aqueous peanut extracts were divided into three equal portions for subsequent fractionation using Waters reversed phase C18 Sep-Pak Vac 20cc cartridges (Waters Corp., Milford, MA, USA; Fig. 1). The first portion was retained without modification as a stock extract (isolate I). The second portion was loaded onto a C18 cartridge and the non-retained fraction (isolate II) collected, while retained compounds were eluted with 30 mL of methanol (isolate III). The third portion was loaded onto a second C18 cartridge and eluted first with 30 mL of ethyl acetate (isolate IV) followed by 30 mL of methanol (isolate V). Solvents were removed by rotary evaporation at <35 C and each brought to an equivalent volume with 0.1 M citrate buffer at pH 6.1. The five isolates were divided into three replicates, filled into 15 mL screw-cap test tubes, individually flushed with nitrogen, and stored in the dark for 0, 2, 4, and 8 weeks at 35 C. Following storage, samples were held frozen ( 20 C) until analysis, with measurements taken directly from each extract after filtering through a 0.45 lm filter. Total reducing equivalents, including contributions from proteins and phenolic amino acids, were measured using the Folin–Ciocalteu assay with data expressed in mg/kg gallic acid equivalents. Free amino acids were measured in accordance with Fiedman, Pang, and Smith (1984) with values expressed in mg/kg equivalents of tryptophan. Individual polyphenolics were separated and characterized by HPLC according to the conditions of Talcott, Brenes, and Howard (2000) using a Dionex HPLC system (Dionex,
Roasted & Defatted Peanut Kernel Isolate I Stock Extract C18 Sep Pak Isolate III Methanol
Isolate II Non-retained
C18 Sep Pak
Isolate IV Ethyl Acetate
Isolate V Methanol
Fig. 1. Solid phase extraction scheme used to fractionate water-soluble phytochemicals from roasted, defatted peanuts.
900
C.E. Duncan et al. / Food Research International 39 (2006) 898–904
Sunnyvale, CA, USA). Separation was conducted on a Waters Spherisorb ODS-2 (4.6 · 250 mm; Waters Corp., Milford, MA, USA) column with detection at 280 nm using a Dionex PDA-100 photodiode array detector scanning from 200 to 400 nm. Concentrations of individual polyphenolics were expressed in equivalents of p-coumaric acid. Antioxidant capacity was determined using the oxygen radical absorbance capacity assay as previously described using a microplate reader (Talcott, Percival, Pitter-Moore, & Celoria, 2003). Peanut isolates were diluted in pH 7 phosphate buffer prior to analysis and values background corrected for the pH 6.1 citrate buffer. Antioxidant capacity was expressed in lM Trolox equivalents per gram of roasted peanut. 2.3. Statistical analysis Data were analyzed as a 6 · 4 · 5 full factorial that included six peanut cultivars analyzed at four sampling times and five isolates. Data represent the mean and standard error of triplicate determinations. Multiple linear regression, Pearson correlations, and analysis of variance were conducted using JMP software Version 5 (SAS Institute, 2002), with mean separation performed by the LSD test (P < 0.05). Recoveries were calculated based on concentrations present in isolate I unless where otherwise stated. 3. Results and discussion 3.1. Peanut isolate I Previous measurements of antioxidant capacity in intact, dry roasted peanuts held under a blanket of nitrogen at 35 C observed up to a 50% decrease over 8 weeks of storage that was independent of cultivar, fatty acid composition, or lipid oxidation (Talcott, Duncan et al., 2005). A goal of the current investigation was to determine a potential cause for these decreases during storage through selective isolation of soluble peanut constituents. Isolate I represented the all of the water-soluble phytochemical constituents isolated from peanuts, yet during storage this isolate did not exhibit a decrease in antioxidant capacity as previously observed for intact peanuts. Likewise, no difference between normal and high oleic acid varieties was observed. Results potentially indicated a difference in oxidative conditions created by the aqueous peanut isolates. In an effort to fractionate the antioxidative compounds present in whole peanuts, numerous intrinsic characteristics of the peanut matrix were likely altered. Changes in water activity, surface area, phytochemical interactions, and the water solubility of oxidizable compounds may be responsible for the difference in storage behavior and susceptibility to oxidation over time. For the cultivars Carver, Andru II, and ANorden no significant changes in antioxidant capacity were observed over 8 weeks storage at 35 C, while Georgia Green, DP-
1, and Hull exhibited antioxidant increases of 27%, 26%, and 32%, respectively (Fig. 2). The varying changes among cultivars during storage may have reflected small compositional differences among the cultivars, secondary reactions with soluble proteins, or varying degrees of oxidation that occurred during storage. Also small increases in total reducing equivalents were observed in all cultivars primarily between the second and fourth week of storage (data not shown). Since the Folin–Ciocalteu assay has an inherent inclination to react with reducing compounds other than polyphenolics, changes in soluble phenolics due to storage was an indication of varying reactivities of the phytochemicals during storage. Among the cultivars evaluated, Carver exhibited the highest antioxidant capacity (19.1 lM Trolox equiv./g) and total reducing equivalent concentration (852 mg/kg) (Table 1). Of the six cultivars evaluated, four exhibited a positive correlation between antioxidant activity and total reducing equivalents (r = 0.85, 0.82, 0.79, and 0.78 for Carver, Andru II, Georgia Green, and DP-1, respectively), similar to correlations previously observed for intact peanuts stored at 35 C (Talcott et al., 2005). The cultivars ANorden and Hull had a lower correlation coefficient (r = 0.56 and 0.47, respectively) that was due to their low total reducing equivalent concentration in relation to their antioxidant capacity. Separation of individual polyphenolics by reversed phase HPLC analysis was additionally conducted on the isolates and revealed many previously unidentified polyphenolic compounds that were likely contributors to the antioxidant and total reducing capacity of peanuts (Fig. 3). Predominant compounds selected from isolate I were tentatively characterized by UV spectroscopic properties and monitored for changes prior to storage and after 8 weeks at 35 C. Among those selected compounds changes were only observed during storage for peak 5 (kmax = 279.2 nm) and peak 7 (kmax = 256.2 nm), two compounds that were previously identified in peanuts (Talcott, Passeretti et al., 2005b). These compounds were present in isolates I and IV and exhibited average losses of 75% and 71%, respectively, among the cultivars. The remaining compounds varied insignificantly with storage and included peak 1 (kmax = 291.5 nm), peak 2 (kmax = 290.2 nm), peak 3 (kmax = 271.7 nm), peak 4 (p-coumaric acid; kmax = 306.5 nm), and peak 6 (kmax = 304.3 nm). 3.2. Peanut isolates II and III Water-soluble antioxidant constituents present in six peanut cultivars were assessed along with four additional isolates that were obtained based on their affinity to reversed phase C18 and their solubility in ethyl acetate or methanol. Average recovery of antioxidant constituents from isolates II and III were 17 ± 4% and 58 ± 5%, respectively, among the cultivars with an additional 25% that was not accounted for in the antioxidant capacity assay. Recoveries varied somewhat among cultivars due to differences in
C.E. Duncan et al. / Food Research International 39 (2006) 898–904
μM Trolox equivalents/g
Carver
Andru II
20
20
15
15
10
10
5
5
0
0 0
2
4
6
8
0
2
μM Trolox Equivalents/g
4
6
8
6
8
6
8
DP-1
Georgia Green 20
20
15
15
10
10
5
5
0
0 0
2
4
6
8
0
2
ANorden
4
Hull
20
20
15
15
ORAC (μM TE/g)
μM Trolox Equivalents/g
901
10
5
10
5
0
0 0
2
4
6
8
Week
0
2
4
Week
Fig. 2. The antioxidant capacity (lM Trolox equiv./g of peanut) observed for six peanut cultivars following sequential fractionation (see Fig. 1) and subsequent storage at 35 C for 8 weeks. Bars represent the standard error of the mean (n = 3).
partition efficiency from the C18 cartridges, solvent affinities, and from potential interactions with non-polyphenolic constituents. However, average concentrations of total reducing equivalents demonstrated an additive effect for
isolates II and III (50 ± 3% and 54 ± 3%, respectively), which confirmed complete partitioning from the C18 cartridge. As found in isolate I, an increase in total reducing equivalents was also observed in isolates II and III during
902
C.E. Duncan et al. / Food Research International 39 (2006) 898–904
Table 1 Antioxidant capacity (lM Trolox equiv./g), total reducing equivalents (mg/kg gallic acid equivalents; Folin–Ciocalteu assay), and total amino acids (mg/kg tryptophan equivalents; ninhydrin-reactive substances) present in each of six peanut cultivars immediately following phytochemical isolations (see Fig. 1) Cultivar
Isolate
Antioxidant capacity
Total reducing equivalents
Total amino acids
Mean
Err
Mean
Err
Mean
Err
Carver
I II III IV V
19.1 3.81 10.3 6.12 6.13
2.0 0.5 0.9 1.9 0.8
852 393 538 252 299
24 14 24 8.9 3.7
2990 2540 602 239 466
84 110 29 30 7.8
Andru II
I II III IV V
13.8 1.87 6.60 2.95 3.90
0.6 0.2 1.3 0.9 0.1
736 339 461 198 221
23 15 2.1 3.4 3.3
3460 2670 773 305 553
96 210 23 44 21
Georgia Green
I II III IV V
12.5 2.79 9.29 2.59 4.37
1.4 0.4 0.2 0.7 0.2
768 382 395 132 233
13 29 9.0 15 3.0
4190 3690 503 169 377
240 290 7.8 19 8.3
DP-1
I II III IV V
9.84 3.31 7.41 1.34 3.24
2.3 0.9 0.2 0.1 1.4
793 444 348 104 180
26 51 3.1 15 3.4
7670 9060 663 298 504
61 990 17 120 40
ANorden
I II III IV V
11.9 0.59 6.63 1.79 6.40
0.8 0.0 0.5 0.3 0.6
700 265 365 78.2 185
6.7 32 3.4 41 1.9
4730 5100 614 83.4 491
84 260 15 11 27
Hull
I II III IV V
12.1 0.67 5.20 1.43 7.54
0.9 0.1 1.2 0.1 0.2
718 445 373 <50 238
12 3.9 4.4 0.4 3.5
6500 4350 510 46.5 458
240 270 12 15 38
Data represent the mean and standard error (Err) for each isolate (n = 3).
the second and fourth week of storage (data not shown), but did not correspond to changes in antioxidant capacity which was unaltered by the conditions of storage (Fig. 2). Isolate II contained 95% of the ninhydrin-reactive amino acids present among the four isolates and exhibited an 82 ± 5% overall recovery from isolate I. This fraction contained the lowest antioxidant activity among the isolates and suggested that peanuts, though plentiful in total protein, do not derive an appreciable portion of their antioxidant activity from soluble proteins or amino acids. The most prevalent antioxidant polyphenolic in peanuts was pcoumaric acid and following fractionation was only found in isolate II with few other identifiable compounds (Table 1, Fig. 3). Therefore, the majority of antioxidant capacity in isolate II was likely due to the presence of p-coumaric acid. Insignificant changes in p-coumaric acid concentration was observed after 8 weeks storage at 35 C and despite its predominance, its overall contribution to the total antioxidant capacity of dry roasted peanuts was found to be minimal. Among the isolates, contributions
to total antioxidant capacity were largely derived from compounds present in isolate III. This fraction was low in ninhydrin-reactive substances and contained 58% of the total antioxidant capacity. This fraction was devoid of p-coumaric acid, yet despite the presence of numerous compounds present in low concentrations no predominant polyphenolic compound was identified that related to antioxidant capacity. 3.3. Peanut isolates IV and V The polyphenolics present in isolate III were again partitioned from reversed phase C18 with ethyl acetate (isolate IV) followed by methanol (isolate V) to further elucidate those compounds responsible for the metal reduction and antioxidant capacity in peanuts. As found in the previous isolates, no changes in antioxidant capacity were observed during storage (Fig. 2) indicating their inherent stability. Recoveries of these isolates among the peanut cultivars in relation to isolate III were found to be mostly additive in
C.E. Duncan et al. / Food Research International 39 (2006) 898–904
1,100
903
mAU
4 750
Isolate I
1 6 2
500
3
5 7
250
min
-100
0 1,100
5
10
15
20
25
30
35
40
45
50
55
60
mAU
65
72
Isolate II
750
4 500
1 2
250
min
-100
0 1,100
5
10
15
20
25
30
35
40
45
50
55
60
mAU
65
72
Isolate III
750
500
3
5
6
1
7
250
min
-100
0
5
10
15
20
25
30
35
40
45
50
55
60
65
72
Fig. 3. Typical reverse phase HPLC chromatograph at 280 nm of isolates I, II, and III (see Fig. 1) for the cultivar ‘Carver’. Spectral characteristics for predominant compounds include peak 1 (kmax = 291.5 nm), peak 2 (kmax = 290.2 nm), peak 3 (kmax = 271.7 nm), peak 4 (p-coumaric acid; kmax = 306.5 nm), peak 5 (kmax = 279.2 nm), peak 6 (kmax = 304.3 nm), and peak 7 (kmax = 256.2 nm).
their antioxidant capacity (33 ± 6% and 71 ± 12%) and total reducing equivalents (29 ± 7% and 55 ± 2%) for isolates IV and V, respectively. This separation technique demonstrated that numerous polyphenolic compounds present in peanuts contributed to their relatively high antioxidant capacity. 3.4. Complimentary effects of isolates II and III Responses observed for isolates II and III that related to their combined antioxidant capacity and total reducing equivalents were different. With partition efficiency near 100% for compounds exhibiting reducing capacity, those same compounds did not exhibit the same additive effect towards antioxidant capacity. With high concentrations of ninhydrin-reactive compounds present in isolate II and
a high antioxidant capacity present in isolate III, their combined contribution seemed to indicate a complimentary effect that enhanced the antioxidant capacity of isolate I, accounting for 25% higher antioxidant capacity on average among the peanut cultivars. This effect was not observed between isolates IV and V, where only small amounts of ninhydrin-reactive compounds were present and the resultant antioxidant capacity was additive. Heinonen et al. (1998) also reported an increased antioxidant capacity when protein was present along with ferulic acid. Similarly Herraiz, Galisteo, and Chamorro (2003) described protein– phenolic complexes between tryptophan and naturally occurring phenolic aldehydes with significant antioxidant activity. This complimentary effect between ninhydrinreactive compounds and soluble polyphenolics could only be elucidated following sequential partitioning of the
904
C.E. Duncan et al. / Food Research International 39 (2006) 898–904
phytochemical constituents and may partially explain the relatively high antioxidant capacity for dry roasted peanuts. Extraction and sequential partitioning of water-soluble phytochemicals from six cultivars of dry roasted peanuts was conducted to determine those compounds responsible for the antioxidant capacity and storage stability. A significant portion of the antioxidant capacity of peanuts can be contributed to compounds other than p-coumaric acid and ninhydrin-reactive compounds, both found in high concentrations in the C18 non-retained fraction (isolate II). The majority of antioxidant compounds were found to be polyphenolics with a high affinity to reversed phase C18, but no predominant antioxidant compound was identified. These findings suggest a multitude of compounds exist in peanuts in relatively low concentrations contributing to an overall antioxidant capacity. Differences between antioxidant capacity and total reducing equivalents following partitioning seemed to indicate a complimentary effect between polyphenolics and ninhydrin-reactive compounds in peanuts that is partially responsible for their relatively high antioxidant capacity in relation to numerous fruits and vegetables. The isolations did not elucidate a cause for previously observed decreases in antioxidant capacity during storage of whole peanuts, but rather revealed the relative stability of peanut polyphenolics during storage and characterized those compounds responsible for their antioxidant capacity. References Anderson, P. C., & Gorbet, D. W. (2002). Influence of year and planting date on fatty acid chemistry of high-oleic acid and normal peanut genotypes. Journal of Agricultural and Food Chemistry, 50, 1298–1305. Awad, A. B., Chan, K. C., Downie, A. C., & Fink, C. S. (2000). Peanuts as a source of b-sitosterol, a sterol with anticancer properties. Nutrition and Cancer, 36, 238–241. Bland, J. M., & Lax, A. R. (2000). Isolation and characterization of a peanut maturity-associated protein. Journal of Agricultural and Food Chemistry, 48, 3275–3279. Borrelli, R. C., Visconti, A., Mennella, C., Anese, M., & Fogliano, V. (2002). Chemical characterization and antioxidant properties of coffee melanoidins. Journal of Agricultural and Food Chemistry, 50, 6527–6533. Duke, J. A. (1992). Handbook of phytochemical constituents of GRAS herbs and other economic plants. Boca Raton, FL: CRC Press. Ehling, S., & Shibamoto, T. (2005). Correlation of acrylamide generation in thermally processed model systems of asparagine and glucose with color formation, amounts of pyrazines formed, and antioxidative properties of extracts. Journal of Agricultural and Food Chemistry, 53, 4813–4819. Fajardo, J. E., Waniska, R. D., Cuero, R. G., & Pettit, R. E. (1995). Phenolic compounds in peanut seeds: enhanced elicitation by chitosan and effects on growth and aflatoxin B1 production by Aspergillus flavus. Food Biotechnology, 9, 59–78.
Feldman, E. B. (1999). Assorted monounsaturated fatty acids promote healthy hearts. American Journal of Clinical Nutrition, 70, 953–954. Fiedman, M., Pang, J., & Smith, G. A. (1984). Ninhydrin-reactive lysine in food proteins. Journal of Food Science, 49, 10–13. Hashim, I., Koehler, P. E., & Eitenmiller, R. R. (1993). Tocopherols in runner and Virginia peanut cultivars at various maturity stages. Journal of the American Oil Chemists’ Society, 70, 633–635. Heinonen, M., Rein, D., Satue´-Gracia, M. T., Huang, S.-W., German, J. B., & Frankel, E. N. (1998). Effect of protein on the antioxidant activity of phenolic compounds in a lecithin-liposome oxidation system. Journal of Agricultural and Food Chemistry, 46, 917–922. Herraiz, T., Galisteo, J., & Chamorro, C. (2003). L-tryptophan reacts with naturally occurring and food-occurring phenolic aldehydes to give phenolic tetrahydro-b-carboline alkaloids: activity as antioxidants and free radical scavengers. Journal of Agricultural and Food Chemistry, 51, 2168–2173. Pattee, H. E., Isleib, T. G., Moore, K. M., Gorbet, D. W., & Giesbrecht, F. G. (2002). Effect of high-oleic trait and paste storage variables on sensory attributes stability of roasted peanuts. Journal of Agricultural and Food Chemistry, 50, 7366–7370. Peanut-Institute. (2002). Antioxidants from food sources, like peanuts and peanut butter, may protect against Alzheimer disease. Press Release, June 26. Reed, K. A., Sims, C. A., Gorbet, D. W., & O’Keefe, S. F. (2002). Storage water activity affects flavor fade in high and normal oleic peanuts. Food Research International, 35, 769–774. Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structure– antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biological Medicine, 20, 933–956. Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1997). Antioxidant properties of phenolic compounds. Trends in Plant Science, 2(4), 152–159. Sanders, T. H. (2001). Non-detectable levels of trans-fatty acids in peanut butter. Journal of Agricultural and Food Chemistry, 49, 2349–2351. SAS Institute, Inc. (2002). LMP Software Version 5; Cary, NC. Sathe, K. S., Hamaker, B. R., Sze-Tao, K. W. C., & Venkatachalam, M. (2002). Isolation, purification, and biochemical characterization of a novel water-soluble protein from Inca peanut (Plukenetia volubilis L.). Journal of Agricultural and Food Chemistry, 50, 4906–4908. Seo, A., & Morr, C. V. (1985). Activated carbon and ion exchange treatments for removing phenolics and phytate from peanut protein products. Journal of Food Science, 50, 262–263. Talcott, S. T., Brenes, C. H., & Howard, L. R. (2000). Contribution of periderm material and blanching time to the quality of pasteurized peach puree. Journal of Agricultural and Food Chemistry, 48, 4590–4596. Talcott, S. T., Duncan, C. E., Del Pozo-Insfran, D., & Gorbet, D. W. (2005). Polyphenolic and antioxidant changes during storage of normal, mid, and high oleic acid peanuts. Food Chemistry, 89, 77–84. Talcott, S. T., Passeretti, S., Duncan, C. E., & Gorbet, D. W. (2005). Polyphenolic content and sensory properties of normal and high oleic acid peanuts. Food Chemistry, 90, 379–388. Talcott, S. T., Percival, S. S., Pitter-Moore, J., & Celoria, C. (2003). Phytochemical composition and antioxidant stability of fortified yellow passion fruit (Passiflora edulis). Journal of Agricultural and Food Chemistry, 51, 935–941. Yanagimoto, K., Lee, K., Ochi, H., & Shibamoto, T. (2002). Antioxidative activity of heterocyclic compounds found in coffee volatiles produced by the Maillard reaction. Journal of Agricultural and Food Chemistry, 50, 5480–5484. Yu, J., Ahmenda, M., & Goktepe, I. (2005). Effects of processing methods and extraction solvents on concentration and antioxidant activity of peanut skin phenolics. Food Chemistry, 90, 199–206.