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J. Agric. Food Chem. 2000, 48, 6292−6297
Influence of Jam Processing on the Radical Scavenging Activity and Phenolic Content in Berries Yoshiaki Amakura,* Yukiko Umino, Sumiko Tsuji, and Yasuhide Tonogai National Institute of Health Sciences, Osaka Branch, 1-1-43, Hoenzaka, Chuo-ku, Osaka 540-0006, Japan
Six selected phenolic aglycons (caffeic and ellagic acids, kaempferol, quercetin, myricetin, and morin) in nine types of berries, and their changes as influenced by jam processing, have been evaluated using optimized HPLC with diode-array detection. The berry samples, fresh and after jam processing, were analyzed, and the total amounts of selected phenolics as aglycons were identified and determined by acid hydrolysis. Their contents in fresh and jam samples did not indicate appreciable changes; therefore, the influence of jam processing on these selected phenolics in berries was suggested to be small, and was mostly present in berries as several conjugated forms that were glycosylated, esterified, etc., in the samples. The total phenolic content of each sample was also determined by the Folin-Ciocalteu method. The three samples of each berry, namely fresh, jam, and acid hydrolysate of the berry, had similar total phenolic contents. On the other hand, the scavenging effect on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was measured, and acid hydrolysates showed stronger activity than that of the fresh and jam-processed samples for all of the berry types. Keywords: berry; phenolic; HPLC; jam; radical scavenger INTRODUCTION
Phenolics such as flavonoids and phenolic acids are important secondary plant metabolites and are widely distributed in many fruits, vegetables, teas, and beverages (Haslam, 1977, 1981; King and Young, 1999; Merken and Beecher, 2000; Okuda et al., 1995; Tanaka, 1999). Most of the phenolics in plants occur as glycosides or esters (Daniel et al., 1989; Henning, 1981; Herrmann, 1989; Rommel and Wrolstd, 1993; Wilson and Hagerman, 1990). Among them, typical low-molecular-weight phenolics in foods (namely caffeic and ellagic acids, quercetin, kaempferol, morin, and myricetin, Figure 1), have been reported to exert potential health-promoting effects as antioxidants, and as antitumor and anticarcinogenic agents (Hatano, 1995; Okuda et al., 1984, 1992; Rapisarda and Tomaino, 1999). Additionally, ellagic acid and morin are included in the List of Existing Food Additives as natural antioxidants in Japan [Notification No. 120 (April 16, 1996), Ministry of Health and Welfare, Japan]. Berries such as strawberry, blueberry, and raspberry are traditionally favorite desserts all over the world. They are also used as processed food materials for juice, jam, dried fruit, ice cream, etc., and therefore are quite prevalent in our lives. Berries contain many phenolic substances, and attention is being paid to healthpromoting foods that have phenolic bioactivities (Ha¨kkinen et al., 1998, 1999; Versari, 1998). The analysis of phenolics (flavonols, phenolic acids, etc.) in berries using HPLC has already been discussed in several reports (Amakura et al., 2000; Ha¨kkinen et al., 1998, 1999; Rommel and Wrolatad, 1993; Shahrzad * To whom correspondence should be addressed. Telephone: +81-6-6941-1533. Fax: +81-6-6942-0716. E-mail
[email protected].
Figure 1. Structures of selected phenolics.
et al., 1996). However, reports describing changes of the actual aglycon amounts between fresh and processed berries has been rarely found. As mentioned above, the berries, including their phenolics, have often been proposed as health-promoting materials. Therefore, in view of the importance of these substances to health and to food chemistry, the determination of changes in selected phenolic contents upon processing of berries is required and is deemed important.
10.1021/jf000849z CCC: $19.00 © 2000 American Chemical Society Published on Web 11/05/2000
Jam Processing Influence on Phenolics in Berries
In this paper, we studied the fluctuations of six selected phenolics and the total phenolic amounts in fresh berries and in jam as processed berries. Furthermore, the scavenging activity on the 1,1-diphenyl-2picrylhydrazyl (DPPH) radical was measured, and the relationship between the phenolic contents and the radical scavenging effect was estimated and discussed. MATERIALS AND METHODS Samples, Chemicals, and Reagents. The nine edible types of berries used for this study were imported and donated by the Kobe quarantine station in Japan. All fresh berries were frozen and stored for 2-3 months at -20 °C until processed and analyzed. Ellagic acid dihydrate, quercetin dihydrate, and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Kaempferol, morin, and caffeic acid were purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). Myricetin was obtained from Aldrich Chemical (Milwaukee, WI). The standards were dissolved in methanol or ethanol. Methanol and acetonitrile were of HPLC grade. The Sep-Pak Plus tC18 cartridge (900 mg) used for refinement was from Waters (Milford, MA). Apparatus. The HPLC analysis was performed on a Shimadzu class LC-VP HPLC system with class LC-VP software, a pump (LC-10Advp), an autosampler (SIL-10AD), and a diode-array detector (SPD-M10Avp)(Shimadzu, Kyoto, Japan). An L-column ODS (5 µm, 250 × 4.6 mm i.d., Chemicals Inspection and Institute, Japan) was used for the analysis. Analytical Conditions. The HPLC conditions were modified from the method of Ha¨kkinen (1998): flow-rate, 1.0 mL/ min; volume injected, 10 µL; temperature, 40 °C; detection, 360 nm. The mobile phase used two solvents. Solvent A was 5 mmol/L potassium dihydrogenphosphate solution (pH 2.5) and solvent B was acetonitrile. The elution profile was 0-5 min, 0-10% B in A (linear gradient); 5-20 min, 10-40% B in A (linear gradient); 20-35 min, 40% B in A (isocratic); followed by 5 min of equilibrating with 100% A. The UV spectrum was recorded between 200 and 400 nm. The retention time and UV spectrum of the peak compared to the standard were used to identify the compound. An ambiguous peak in agreement with the standard on HPLC chromatogram was distinguished by photodiode array detection. Quantitative determination was carried out using calibration graphs obtained from standard solutions diluted with methanol or ethanol in the concentration range of 0.1-100 µg/mL. Results were expressed as mg of each phenolic per 100 g of fresh berries. Sample Preparations. All of the raw materials used were the same type of fresh berries homogenized. Jam Processing Procedure. The mixture (total amount, 20 g), consisting of the raw material (10 g) with added granulated sugar (10 g), was mildly heated to concentrate to ca.16 g (80% of total amount) with slow stirring for 10-15 min. This mixture was then allowed to stand to cool to room temperature, and the prepared substance was used as the jam sample (5565 °Brix, 10 g equivalent of each fresh berry). Preparation of Test Solution. The raw material (10 g) (or the jam sample, 10 g equivalent of fresh berry) was homogenized in 80% methanol (50 mL), and then the homogenate was filtered in vacuo. The filtrate was evaporated to ca. 10 mL, and then 0.1 mol/L HCl solution (100 µL) was added. The extract was directly loaded onto the Sep-Pak Plus tC18 cartridge (previously conditioned with 10 mL of methanol), followed by 10 mL of distilled water, and washed with 10 mL of distilled water. The cartridge was eluted with 10 mL of methanol. The eluate was collected in a flask and then evaporated to dryness under reduced pressure below 40 °C using a rotary evaporator. Every extract sample was dissolved in 10 mL of methanol then filtered through a 0.5-µm filter to give the test solution. Preparation of Acid Hydrolysate. A test solution (5 mL) in 1 mol/L HCl (5 mL) was heated in a boiling-water bath for 2 h, and the reaction mixture was evaporated to ca. 5 mL. The
J. Agric. Food Chem., Vol. 48, No. 12, 2000 6293 concentrate was directly loaded onto the Sep-Pak Plus tC18 cartridge, washed with 10 mL of distilled water, and then eluted with 10 mL methanol. The eluate was evaporated to dryness and dissolved in 5 mL of methanol. Determination of Total Phenolics. The total phenolic contents in the samples were determined with Folin-Ciocalteu reagent according to the method of Julkunen-Tiitto (1985) using gallic acid as a standard. Results were expressed as mg of gallic acid equivalent per 100 g of fresh berries. DPPH Radical Scavenging Assay. The free-radical scavenging capacity of the samples was tested as bleaching of the stable DPPH. A solution (4 mL) of a test sample in MeOH was added to a solution (1 mL) of DPPH in MeOH (final concentration of DPPH, 2.0 × 10-4 mol/L). After the solution was mixed for 10 s, it was left to stand for 30 min, and the absorbance of the resulting solution at 520 nm was measured. All experiments were carried out in triplicate and repeated at least three times. The scavenging activity on the DPPH radical was expressed as EC50, the concentration of the test sample required to give a 50% reduction in the absorbance from that of 2.0 × 10-4 mol/L DPPH in MeOH (Hatano, 1997). The potency is graphically displayed as 1/EC50. RESULTS AND DISCUSSION
The contents of the selected phenolics in fresh berries, jam, and acid hydrolysates of the berries, which were determined by RP-HPLC, are given in Table 1. All of the results were obtained in the three tests. Figure 2 illustrates the chromatograms of standards and strawberry (fresh, jam, and acid hydrolysate), at 360 nm, as examples. Selected Phenolics in Fresh Berries. Most fresh berry samples contained quercetin and kaempferol as shown in Table 1. In contrast, morin was not detected in the tested berries. Ellagic acid was detected in strawberry, blackberry, blueberry, and raspberry samples. Myricetin was detected in black current, cowberry, bayberry, and red current samples, but was not detected in berries that contained ellagic acid. On the other hand, samples hydrolyzed by acid showed the selected phenolics in amounts of about 5-100 times those of fresh samples (Table 1). In particular, bayberry was found to have a high increment of aglycon content (0.15-15.93 mg/100 g), but strawberry (1.02-23.81 mg/100 g), black current (0.77-22.40 mg/100 g), raspberry (0.92-20.01 mg/100 g), and blackberry (7.11-49.30 mg/100 g) also showed high aglycon contents, quantitatively. Caffeic acid was not detected in any of the fresh berry samples, but it was detected in the blueberries hydrolyzed by acid (0.89 mg/100 g). For each aglycon, ellagic acid was detected at a high content in strawberry (1.01-19.75 mg/100 g), raspberry (0.82-17.91 mg/100 g), and blackberry (6.87-42.44 mg/ 100 g), and quercetin was detected in cranberry (0.7814.02 mg/100 g), blueberry (0.07-7.08 mg/100 g), black current (0.17-9.09 mg/100 g), bayberry (0.04-8.77 mg/ 100 g), and blackberry (0.22-6.21 mg/100 g). Myricetin was detected in bayberry (0.10-5.79 mg/100 g) and black current (0.57-11.12 mg/100 g). The total selected phenolic contents were comparatively high in strawberry (23.81 mg/100 g), black current (22.40 mg/100 g), blackberry (49.30 mg/100 g), and raspberry (20.01 mg/ 100 g). Effects of Jam Processing on Selected Phenolics Contents. The amounts of the selected flavonols and phenolic acids in the berry jams were determined (Table 1). The production rate from conjugated forms to aglycons by jam processing was calculated as shown in Table 2. Consequently, the rates of selected phenolics pro-
6294 J. Agric. Food Chem., Vol. 48, No. 12, 2000
Amakura et al.
Table 1. Effects of Jam Processing and Acid Hydrolysis on Contents of Phenolics in Fresh Berriesa contents (mg/100 g of fresh berry weight, means ( SD, n ) 3) caffeic acid bayberry fresh after acid hydrolysis after jam processing after acid hydrolysis of jam black current fresh after acid hydrolysis after jam processing after acid hydrolysis of jam blackberry fresh after acid hydrolysis after jam processing after acid hydrolysis of jam blueberry fresh after acid hydrolysis after jam processing after acid hydrolysis of jam cowberry fresh after acid hydrolysis after jam processing after acid hydrolysis of jam cranberry fresh after acid hydrolysis after jam processing after acid hydrolysis of jam raspberry fresh after acid hydrolysis after jam processing after acid hydrolysis of jam red current fresh after acid hydrolysis after jam processing after acid hydrolysis of jam strawberry fresh after acid hydrolysis after jam processing after acid hydrolysis of jam
ellagic acid
myricetin
quercetin
kaempferol
TSPb
TPc
nd nd nd nd
nd nd nd nd
0.10 ( 0.01 5.79 ( 0.04 0.22 ( 0.03 4.18 ( 0.48
0.04 ( 0.01 8.77 ( 0.37 0.11 ( 0.01 7.91 ( 0.19
0.01 ( 0.01 1.37 ( 0.04 0.01 ( 0.01 1.29 ( 0.04
0.15 15.93 0.34 13.38
36.87 ( 1.35 36.58 ( 0.84 31.49 ( 1.62 28.27 ( 0.98
nd nd nd nd
nd nd nd nd
0.57 ( 0.01 11.12 ( 0.18 0.73 ( 0.05 8.83 ( 0.95
0.17 ( 0.01 9.09 ( 0.22 0.48 ( 0.17 7.06 ( 1.10
0.03 ( 0.01 2.19 ( 0.01 0.06 ( 0.03 1.57 ( 0.15
0.77 22.40 1.27 16.46
61.90 ( 0.66 66.48 ( 1.85 60.47 ( 1.09 58.37 ( 1.89
nd nd nd nd nd 0.89 ( 0.05 nd 0.50 ( 0.06
6.87 ( 0.16 42.44 ( 0.42 15.78 ( 0.85 43.87 ( 0.94
nd nd nd nd
0.22 ( 0.01 6.21 ( 0.58 1.65 ( 0.13 5.90 ( 0.83
0.02 ( 0.01 0.65 ( 0.08 0.05 ( 0.03 0.62 ( 0.08
7.11 49.30 17.48 50.39
76.23 ( 1.76 79.23 ( 1.26 76.91 ( 1.29 68.91 ( 1.02
0.78 ( 0.14 0.85 ( 0.08 0.70 ( 0.02 0.75 ( 0.10
nd nd nd nd
0.07 ( 0.01 7.08 ( 0.33 0.45 ( 0.07 6.04 ( 0.80
nd 1.72 ( 0.04 0.04 ( 0.02 1.52 ( 0.08
0.85 10.54 1.19 8.81
33.50 ( 0.19 37.04 ( 0.33 30.24 ( 1.32 29.56 ( 1.02
0.12 ( 0.00 2.46 ( 0.08 0.93 ( 0.05 2.90 ( 0.41
0.02 ( 0.01 0.50 ( 0.25 0.11 ( 0.05 0.53 ( 0.03
1.31 6.85 2.84 7.70
78.43 ( 1.26 82.12 ( 1.54 85.35 ( 1.79 72.54 ( 2.01
nd nd nd nd
0.78 ( 0.05 14.02 ( 0.19 2.19 ( 0.09 10.58 ( 0.38
0.04 ( 0.02 0.64 ( 0.33 0.04 ( 0.02 0.51 ( 0.02
0.82 14.66 2.23 11.09
81.55 ( 0.65 85.70 ( 1.19 84.48 ( 1.19 80.18 ( 1.21
nd nd nd nd
0.10 ( 0.01 1.71 ( 0.08 0.06 ( 0.02 2.10 ( 0.07
nd 0.38 ( 0.02 nd 0.36 ( 0.07
0.92 20.01 1.29 17.55
32.28 ( 1.22 33.80 ( 0.39 31.02 ( 1.04 29.16 ( 1.05
0.34 ( 0.02 2.06 ( 0.02 0.40 ( 0.01 2.11 ( 0.07
0.03 ( 0.01 0.36 ( 0.03 0.30 ( 0.02 0.36 ( 0.05
0.44 2.79 0.77 2.88
74.48 ( 0.82 71.71 ( 0.77 72.17 ( 1.95 69.31 ( 1.82
0.01 ( 0.01 3.42 ( 0.17 0.61 ( 0.06 3.69 ( 0.18
nd 0.64 ( 0.32 0.01 ( 0.01 0.70 ( 0.06
1.02 23.81 2.00 20.69
36.50 ( 0.66 37.61 ( 1.76 32.50 ( 1.74 30.34 ( 1.82
nd nd nd nd
nd nd nd nd
nd nd nd nd
nd nd nd nd
nd nd nd nd nd nd nd nd nd nd nd nd
0.82 ( 0.10 17.91 ( 0.32 1.23 ( 0.03 15.09 ( 0.80 nd nd nd nd 1.01 ( 0.19 19.75 ( 0.23 1.38 ( 0.18 16.30 ( 0.76
1.17 ( 0.03 3.89 ( 0.43 1.80 ( 0.06 4.27 ( 0.27
0.07 ( 0.02 0.37 ( 0.03 0.07 ( 0.01 0.41 ( 0.03 nd nd nd nd
a nd, not detected (
