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Antioxidant capacity of phenolic phytochemicals from various cultivars of plums
Food Chemistry 81 (2003) 321–326 www.elsevier.com/locate/foodchem
Antioxidant capacity of phenolic phytochemicals from various cultivars of plums Dae-Ok Kima, Seung Weon Jeongb, Chang Y. Leea,* a
Department of Food Science and Technology, Cornell University, Geneva, NY 14456, USA b Korea Food Research Institute, Bundang, Seongnam, Kyounggi 463-746, South Korea
Received 27 May 2002; received in revised form 9 September 2002; accepted 9 September 2002
Abstract Polyphenolic phytochemical extractions of six cultivars of plums (Beltsville Elite B70197, Cacak Best, French Damson, Long John, Stanley, Yugoslavian Elite T101) and Gala apples were performed using 80% aqueous methanol with ultrasound assistance and extracts were analyzed for total phenolics, flavonoids, and antioxidant capacity. The total phenolic contents of various cultivars of plums were in a range of 174 to 375 mg/100 g, expressed as gallic acid equivalents (GAE), on a fresh weight basis. Total flavonoid concentrations ranged from 118 to 237 mg catechin equivalents (CE)/100 g fresh weight. The concentrations of total phenolics and flavonoids in Gala apples were 118 1.4 mg GAE and 62.0 6.9 mg CE per 100 g fresh sample weight, respectively. The stable radical chromogen, ABTS, commonly employed for the antioxidant activity measurement, was used to evaluate antioxidant capacity of plums and apples. The total antioxidant capacities, expressed as vitamin C equivalent antioxidant capacity (VCEAC), of fresh plums ranged from 266 to 559 mg/100 g. The order of total antioxidant capacity among different plum cultivars was as follows: Beltsville Elite B70197 >Cacak Best5French Damson> Yugoslavian Elite T101> Long John >Stanley. The total antioxidant capacity of fresh Gala apple was 205 5.6 mg VCEAC/100 g. There was a good correlation between total phenolics or flavonoids contents and VCEAC at the high level of P< 0.001. Dietary polyphenolics from plums may supply substantial antioxidants, which may provide health-promoting advantages to the consumer. # 2003 Published by Elsevier Science Ltd. Keywords: Antioxidant capacity; Free radical; Phenolic phytochemicals; Plums; Vitamin C equivalent antioxidant capacity (VCEAC)
1. Introduction Phenolic phytochemicals are important aromatic secondary metabolites in plants, many of which are commonly substituted by sugar moieties such as glucose, arabinose, xylose, rhamnose and galactose. Significant amounts of phenolic compounds frequently occur in foods such as fruits and vegetables and are routinely consumed in our diet. They importantly attribute to the sensory qualities (colour, flavour, taste) of fresh fruits, vegetables and their products. In addition, many phenolic phytochemicals have antioxidative, anticarcinogenic, antimicrobial, antiallergic, antimutagenic and antiinflammatory activities (Cao & Cao, 1999; Eberhardt, Lee, & Liu, 2000; Ito et al., 1998; Kawaii, * Corresponding author. Tel.: +1-315-787-2271; fax: +1-315-7872284. E-mail address: [email protected] (C.Y. Lee).
Tomono, Katase, Ogawa, & Yano, 1999; Kim, Choi, & Chung, 2000). Some phytochemicals, including flavonoids in fruits and vegetables, consumed as part of our daily diet, may reduce the risk of cardiovascular disease (Cook & Samman, 1996). Epidemiological studies show a significant inverse relationship between dietary intake of fruits and vegetables and the risk of coronary heart disease (Knekt, Ja¨rvinen, Reunanen, & Maatela, 1996). The distribution and composition of phenolic phytochemicals are affected by maturity, cultivars, horticultural practices, geographic origin, growing season, postharvest storage conditions and processing procedures (Burda, Oleszek, & Lee, 1990; De Freitas & Glories, 1999; Donovan, Meyer, & Waterhouse, 1998; Kalt, Forney, Martin, & Prior, 1999; Kim, Koh, & Koh, 2001; Lee & Jaworski, 1987; Spanos & Wrolstad, 1990). Plums contain copious amounts of natural phenolic phytochemicals, such as flavonoids and phenolic acids,
0308-8146/03/$ - see front matter # 2003 Published by Elsevier Science Ltd. PII: S0308-8146(02)00423-5
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which may function as effective natural antioxidants in our daily diet. Wang, Cao, and Prior (1996) demonstrated that plums had 4.4 times higher total antioxidant capacities than apples, the latter being one of the most commonly consumed fruits in our diet. They reported the total antioxidant capacity of various fruits including plums, using Trolox equivalents, where Trolox is a vitamin E analogue but not a natural compound. Plums demonstrated very good scavenger activity against oxygen-derived free radicals such as hydroxyl and peroxyl radicals (Murcia, Jime´nez, & Martı´nez-Tome´, 2001). Various phenolic compositions, in five different varieties of yellow and red plums, were recently analyzed by HPLC-DAD-ESIMS, but no antioxidant activity was reported (Toma´s-Barbera´n et al., 2001). In another report, antioxidant activity of chlorogenic acid and its isomers, isolated from prune, was evaluated (Nakatani et al., 2000). The measurement of antioxidant activity of individual compounds may lead to a misleading conclusion due to frequently observed antagonistic or synergistic interactions of various components of foods. Various kinds of antioxidant components in plums may play important roles in the combinative or synergistic contribution to total antioxidant activity. Yet plums, having very high concentrations of phenolic phytochemicals, remain underutilized in the average American diet (Vinson, Su, Zubik & Bose, 2001) and under-researched worldwide. Purposes of this study were to determine the content of total phenolic phytochemicals and flavonoids and to evaluate total antioxidant activity in various cultivars of plums using vitamin C equivalent antioxidant capacity (VCEAC). A simple chromogen, blue-green 2,20 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical, was employed for determination of total antioxidant activity.
2.2. Chemicals Gallic acid, ABTS as diammonium salt, (+)-catechin and Folin & Ciocalteu’s phenol reagent were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2,20 Azobis(2-amidino-propane)dihydrochloride (AAPH) was obtained from Wako Chemicals USA, Inc. (Richmond, VA, USA). All other chemicals used were of analytical grade. 2.3. Extraction of phenolics The phenolics in powdered freeze-dried plums and apples were extracted by the ultrasound-assisted method (Kim & Lee, 2002). Phenolics of the fruits were extracted from 10 g ground freeze-dried samples using 100 ml of 80% aqueous methanol. The mixture of freeze-dried powder and 80% aqueous methanol was sonicated for 20 min with continual nitrogen gas purging. The mixture was filtered through Whatman #2 filter paper (Whatman International Limited, Kent, England) using a chilled Bu¨chner funnel and rinsing with 50 ml 100% methanol. Extraction of the residue was repeated using the same conditions. The two filtrates were combined and transferred into a 1 l evaporating flask with an additional 50 ml of 80% aqueous methanol. The solvent was evaporated using a rotary evaporator at 40 C. The remaining phenolic concentrate was first dissolved in 50 ml of 100% methanol and diluted to a final volume of 100 ml using distilled deionized water (ddH2O) obtained with a NANOpure water system (Barnstead, Dubuque, Iowa, USA). The mixture was centrifuged at refrigerated temperatures, using a Sorvall RC-5B refrigerated superspeed centrifuge (Du Pont Company, Biomedical Products Department, Wilmington, DE, USA) at 10 K with GSA rotor for 20 min and stored at 4 C until analyses were performed.
2. Materials and methods 2.4. Determination of total phenolics 2.1. Fruits Six cultivars of plums (Beltsville Elite B70197, Cacak Best, French Damson, Long John, Stanley, Yugoslavian Elite T101) were picked at commercial maturity during the 2000 harvest season at the New York State Agricultural Experiment Station orchard in Geneva, New York. The Gala apple cultivar, picked at commercial maturity during the 2001 harvest season at the same location, was used as a reference sample. Immediately upon arrival in the pilot plant after harvest, plums and apples were stored in a 2–5 C cold room. Plums were carefully cut in half and the pits removed. Apple slices were prepared by hand. Pitted plums and sliced apples were frozen and lyophilized. Freeze-dried samples were ground to powder and then stored at 20 C until analyzed.
The concentration of total phenolics was measured by the method described by Singleton and Rossi (1965) with some modification. Briefly, an aliquot (1 ml) of appropriately diluted extracts or standard solutions of gallic acid (20, 40, 60, 80 and 100 mg/l) was added to a 25 ml volumetric flask containing 9 ml of ddH2O. A reagent blank using ddH2O was prepared. One mililiter of Folin & Ciocalteu’s phenol reagent was added to the mixture and shaken. After 5 min, 10 ml of 7% Na2CO3 solution was added with mixing. The solution was then immediately diluted to volume (25 ml) with ddH2O and mixed thoroughly. After incubation for 90 min at 23 C, the absorbance versus prepared blank was read at 750 nm. Total phenolic contents of plums and apples were expressed as mg gallic acid equivalents
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(GAE)/100 g fresh sample. All samples were analyzed in five replications.
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3. Results 3.1. Determination of total phenolics
2.5. Determination of total flavonoids Total flavonoids were measured by a colorimetric assay developed by Zhishen, Mengcheng, and Jianming (1999). A 1 ml aliquot of appropriately diluted sample or standard solutions of catechin (20, 40, 60, 80 and 100 mg/l) was added to a 10 ml volumetric flask containing 4 ml ddH2O. At zero time, 0.3 ml 5% NaNO2 was added to the flask. After 5 min, 0.3 ml 10% AlCl3 was added. At 6 min, 2 ml 1 M NaOH was added to the mixture. Immediately, the reaction flask was diluted to volume with the addition of 2.4 ml of ddH2O and thoroughly mixed. Absorbance of the mixture, pink in colour, was determined at 510 nm versus prepared water blank. Total flavonoids of fruits were expressed on a fresh weight basis as mg/100 g catechin equivalents (CE). Samples were analyzed in five replications. 2.6. Vitamin C equivalent antioxidant capacity (VCEAC) assay using ABTS radical ABTS radical anions were used, according to the method of Kim, Lee, Lee, and Lee (2002). In brief, 1.0 mM AAPH, a radical initiator, was mixed with 2.5 mM ABTS in phosphate-buffered saline (pH 7.4; 100 mM potassium phosphate buffer containing 150 mM NaCl). The mixed solution was heated in a water bath at 68 C for 13 min. The resulting blue-green ABTS solution was adjusted to the absorbance of 0.650 0.020 at 734 nm with additional phosphate-buffered saline. Twenty microliters of sample was added to 980 ml of the ABTS radical solution. The mixture was incubated in a 37 C water bath under restricted light for 10 min. A control (20 ml 50% methanol and 980 ml of ABTS radical solution) was run with each series of samples. The decrease of absorbance at 734 nm was measured at an endpoint of 10 min. Total antioxidant capacity of plums and apples, as determined by scavenging blue-green ABTS radical anions, was expressed on a fresh weight basis as mg/100 g vitamin C equivalents (VCEAC). The radical stock solution was freshly prepared, daily. All tested samples were replicated six times.
Total phenolics of six plum cultivars and Gala apples are shown in Fig. 1. The total phenolic contents of the fresh plums per 100 g ranged from 174 1.5 mg GAE in Stanley to 375 3.8 mg GAE in French Damson. Phenolic concentrations of the other plum varieties were as follows: Beltsville Elite B70197 (332 3.1 mg GAE), Cacak Best (319 1.4 mg GAE), Yugoslavian Elite T101 (217 4.9 mg GAE) and Long John (199 2.5 mg GAE). The total phenolic content of 100 g of fresh Gala apples was 118 1.4 mg GAE. 3.2. Determination of total flavonoids Total flavonoid contents of six fresh cultivars of plums and Gala apples are shown in Fig. 1. Beltsville Elite B70197 exhibited the highest flavonoids content of 237 6.3 mg CE/100 g fresh sample. This was followed by French Damson (215 9.7 mg CE), Cacak Best (200 2.5 mg CE), Yugoslavian Elite T101 (146 6.0 mg CE), Long John (126.3 3.4 mg CE) and Stanley (118 2.6 mg CE). The content of total flavonoids in 100 g fresh Gala apples was at the level of 62.0 6.9 mg CE. 3.3. VCEAC assay using ABTS radical The total antioxidant capacities of the fresh plum cultivars and Gala apples, as determined by scavenging ABTS radical anions, are presented in Fig. 2. The total
2.7. Statistical analysis Results are presented as mean value standard deviation. Statistical analysis between experimental results was based on Student’s t test. Significant difference was statistically considered at the level of P < 0.001 or P < 0.05.
Fig. 1. Contents of total phenolics and flavonoids in various cultivars of plum and Gala apple. The data are displayed with meanstandard deviation (bars) of five replications. Total phenolic and flavonoid contents were significantly different at P< 0.001 and P< 0.05, respectively. GA, ST, LJ, YE, CB, BE and FD stand for Gala apple, Stanley, Long John, Yugoslavian Elite T101, Cacak Best, Beltsville Elite B70197 and French Damson, respectively.
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Fig. 2. Vitamin C equivalent antioxidant capacity (VCEAC) of various cultivars of plum and Gala apple using free blue-green ABTS radical anions. The data are displayed with meanstandard deviation (bars) of six replications. VCEACs showed significant difference except between CB and FD at the level of P<0.05. GA, ST, LJ, YE, FD, CB and BE stand for Gala apple, Stanley, Long John, Yugoslavian Elite T101, French Damson, Cacak Best and Beltsville Elite B70197, respectively.
antioxidant capacity of Gala apples was 205 5.6 mg VCEAC/100 g. Beltsville Elite B70197, Cacak Best, French Damson, Long John, Stanley and Yugoslavian Elite T101 showed total antioxidant capacities of 559 12.9, 535 9.2, 524 25.5, 288 16.2, 266 14.5 and 317 21.7 mg VCEAC/100 g, respectively.
4. Discussion The total phenolic content of 100 g fresh plums ranged from 174 to 375 mg GAE and for Gala apples, 118 1.4 mg GAE (Fig. 1). Based on the total phenolics, the six plum cultivars studied may be classified into two groups, one exhibiting relatively high levels of polyphenolic phytochemicals and the other, low levels. The cultivars having relatively high concentrations of phenolic phytochemicals are French Damson, Beltsville Elite B70197 and Cacak Best, whereas the cultivars with lower concentrations are Stanley, Long John and Yugoslavian Elite T101. The total phenolic content of French Damson was about 2.2-fold higher than that of Stanley. French Damson and Stanley plums showed about 3.2 and 1.5 times higher total phenolic level than Gala apples, respectively. Plums were previously shown to have a higher total phenolic content than apples (Proteggente et al., 2002). The averages of total phenolic content of plums and apples were significantly different at a level of P< 0.001. Total phenolic content of a mixture of red plums (Black Star, Ciruela Santa Rosa, Raviota, June Black), cultivars of which were different from ours, was reported to be at the level of 320 mg GAE/100 g (Proteggente et al., 2002). Another study reported that total phenols of black plums was 144 mg/100 g, expressed as
catechin equivalents (Karakaya, El, & Tas¸ , 2001). Total phenolic concentrations of various apple cultivars were reported to be in the range of 50.9 to 140 mg GAE/100 g (Lee & Smith, 2000). These variations are due to differences among cultivars, growing seasons, other agricultural practices and also variations in assays’ protocols. Beltsville Elite B70197 ranked second highest in total phenolics and had the highest concentration of total flavonoids among the six cultivars of plums tested, followed by French Damson, Cacak Best, Yugoslavian Elite T101, Long John and Stanley. Beltsville Elite B70197, having the highest content of total flavonoids and Stanley, the lowest content, displayed levels 3.8-fold and 1.9-fold higher than Gala apples, at 62.0 mg CE/ 100 g, respectively. The total flavonoid contents of the plum cultivars and Gala apples were significantly different at a significance level of P < 0.05. Total phenolic content and total flavonoid content of plums and apples showed a linear relationship with a positive correlation coefficient of r2=0.934 (data not shown). Reactive free radicals, such as superoxide anion (O 2 ), hydroxyl radical (OH), and peroxy radical (ROO), are extremely reactive and are known to be a biological product in reducing molecular oxygen (Williams & Jeffrey, 2000). Damage mediated by free radicals results in the disruption of membrane fluidity, protein denaturation, lipid peroxidation, oxidative DNA and alteration of platelet functions (Fridovich, 1978; Kinsella, Frankel, German, & Kanner, 1993), which has been generally considered to be linked with many chronic health problems such as cancers, inflammation, aging and atherosclerosis. An antioxidant, which can quench reactive free radicals, can prevent the oxidation of other molecules and may, therefore, have health-promoting effects in the prevention of degenerative diseases. The VCEAC procedure, a decolorization assay using free blue-green ABTS radicals, was shown to be a very useful tool for expeditiously measuring the antioxidant activity in individual chemical compounds or complex fruits extracts (Kim et al., 2002). This method expresses antioxidant capacity on the basis of vitamin C equivalent. Vitamin C is ubiquitously present in fruits and has been shown to have preventive effects against carcinogenesis (Lee, Lee, Kang, & Lee, 2002). The total antioxidant activity of plums and apples, in terms of VCEAC, ranked as follows: Beltsville Elite B70197> Cacak Best5French Damson > Yugoslavian Elite T101> Long John > Stanley > Gala apple (Fig. 2). Beltsville Elite B70197 showed 1.8 times higher VCEAC than Stanley with the lowest VCEAC among plum cultivars. However, the VCEAC of Stanley plums was 1.3-fold higher than that of Gala apples. All plum varieties in this study had higher VCEAC than Gala apple. At the significance level of P < 0.05, VCEACs, among various cultivars of plums and Gala apples, showed significant difference except between Cacak Best and
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French Damson. Good correlation (r2=0.938) between total phenolic content and VCEAC was observed with a high significance level (P < 0.001) (Fig. 3A). A similar relationship (r2=0.942) with high significance (P < 0.001) was also obtained between total flavonoid content and VCEAC (Fig. 3B). This positive and significant relationship between total phenolic content and total antioxidant activity, observed in this study was previously reported using other assays (Kalt et al., 1999; Proteggente et al., 2002; Velioglu, Mazza, Gao, & Oomah, 1998). The results of this study imply that dietary polyphenolic phytochemicals from plums may supply substantial antioxidants, which, in turn, may provide health-promoting effects to consumers. Since it has been suggested that a higher consumption of fruits and vegetables with high phytochemicals can inhibit, prevent or retard chronic diseases (Birt, Hendrich, & Wang, 2001; Cook & Samman, 1996; Singletary, 2000; Yang, Landau, Huang, & Newmark, 2001) and the plums in our study showed substantially more
Fig. 3. Relationship between total phenolics and VCEAC (A) and between total flavonoids and VCEAC (B).
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antioxidant activity than apples, an increased consumption of this fruit is therefore recommended in our diet.
Acknowledgements The authors thank Ms. Nancy Smith, research support specialist, for revising the text and providing technical support and Mr. Jay Freer for selecting and providing the cultivars of plums.
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Antioxidant capacity of phenolic phytochemicals from various cultivars of plums Dae-Ok Kima, Seung Weon Jeongb, Chang Y. Leea,* a
Department of Food Science and Technology, Cornell University, Geneva, NY 14456, USA b Korea Food Research Institute, Bundang, Seongnam, Kyounggi 463-746, South Korea
Received 27 May 2002; received in revised form 9 September 2002; accepted 9 September 2002
Abstract Polyphenolic phytochemical extractions of six cultivars of plums (Beltsville Elite B70197, Cacak Best, French Damson, Long John, Stanley, Yugoslavian Elite T101) and Gala apples were performed using 80% aqueous methanol with ultrasound assistance and extracts were analyzed for total phenolics, flavonoids, and antioxidant capacity. The total phenolic contents of various cultivars of plums were in a range of 174 to 375 mg/100 g, expressed as gallic acid equivalents (GAE), on a fresh weight basis. Total flavonoid concentrations ranged from 118 to 237 mg catechin equivalents (CE)/100 g fresh weight. The concentrations of total phenolics and flavonoids in Gala apples were 118 1.4 mg GAE and 62.0 6.9 mg CE per 100 g fresh sample weight, respectively. The stable radical chromogen, ABTS, commonly employed for the antioxidant activity measurement, was used to evaluate antioxidant capacity of plums and apples. The total antioxidant capacities, expressed as vitamin C equivalent antioxidant capacity (VCEAC), of fresh plums ranged from 266 to 559 mg/100 g. The order of total antioxidant capacity among different plum cultivars was as follows: Beltsville Elite B70197 >Cacak Best5French Damson> Yugoslavian Elite T101> Long John >Stanley. The total antioxidant capacity of fresh Gala apple was 205 5.6 mg VCEAC/100 g. There was a good correlation between total phenolics or flavonoids contents and VCEAC at the high level of P< 0.001. Dietary polyphenolics from plums may supply substantial antioxidants, which may provide health-promoting advantages to the consumer. # 2003 Published by Elsevier Science Ltd. Keywords: Antioxidant capacity; Free radical; Phenolic phytochemicals; Plums; Vitamin C equivalent antioxidant capacity (VCEAC)
1. Introduction Phenolic phytochemicals are important aromatic secondary metabolites in plants, many of which are commonly substituted by sugar moieties such as glucose, arabinose, xylose, rhamnose and galactose. Significant amounts of phenolic compounds frequently occur in foods such as fruits and vegetables and are routinely consumed in our diet. They importantly attribute to the sensory qualities (colour, flavour, taste) of fresh fruits, vegetables and their products. In addition, many phenolic phytochemicals have antioxidative, anticarcinogenic, antimicrobial, antiallergic, antimutagenic and antiinflammatory activities (Cao & Cao, 1999; Eberhardt, Lee, & Liu, 2000; Ito et al., 1998; Kawaii, * Corresponding author. Tel.: +1-315-787-2271; fax: +1-315-7872284. E-mail address: [email protected] (C.Y. Lee).
Tomono, Katase, Ogawa, & Yano, 1999; Kim, Choi, & Chung, 2000). Some phytochemicals, including flavonoids in fruits and vegetables, consumed as part of our daily diet, may reduce the risk of cardiovascular disease (Cook & Samman, 1996). Epidemiological studies show a significant inverse relationship between dietary intake of fruits and vegetables and the risk of coronary heart disease (Knekt, Ja¨rvinen, Reunanen, & Maatela, 1996). The distribution and composition of phenolic phytochemicals are affected by maturity, cultivars, horticultural practices, geographic origin, growing season, postharvest storage conditions and processing procedures (Burda, Oleszek, & Lee, 1990; De Freitas & Glories, 1999; Donovan, Meyer, & Waterhouse, 1998; Kalt, Forney, Martin, & Prior, 1999; Kim, Koh, & Koh, 2001; Lee & Jaworski, 1987; Spanos & Wrolstad, 1990). Plums contain copious amounts of natural phenolic phytochemicals, such as flavonoids and phenolic acids,
0308-8146/03/$ - see front matter # 2003 Published by Elsevier Science Ltd. PII: S0308-8146(02)00423-5
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which may function as effective natural antioxidants in our daily diet. Wang, Cao, and Prior (1996) demonstrated that plums had 4.4 times higher total antioxidant capacities than apples, the latter being one of the most commonly consumed fruits in our diet. They reported the total antioxidant capacity of various fruits including plums, using Trolox equivalents, where Trolox is a vitamin E analogue but not a natural compound. Plums demonstrated very good scavenger activity against oxygen-derived free radicals such as hydroxyl and peroxyl radicals (Murcia, Jime´nez, & Martı´nez-Tome´, 2001). Various phenolic compositions, in five different varieties of yellow and red plums, were recently analyzed by HPLC-DAD-ESIMS, but no antioxidant activity was reported (Toma´s-Barbera´n et al., 2001). In another report, antioxidant activity of chlorogenic acid and its isomers, isolated from prune, was evaluated (Nakatani et al., 2000). The measurement of antioxidant activity of individual compounds may lead to a misleading conclusion due to frequently observed antagonistic or synergistic interactions of various components of foods. Various kinds of antioxidant components in plums may play important roles in the combinative or synergistic contribution to total antioxidant activity. Yet plums, having very high concentrations of phenolic phytochemicals, remain underutilized in the average American diet (Vinson, Su, Zubik & Bose, 2001) and under-researched worldwide. Purposes of this study were to determine the content of total phenolic phytochemicals and flavonoids and to evaluate total antioxidant activity in various cultivars of plums using vitamin C equivalent antioxidant capacity (VCEAC). A simple chromogen, blue-green 2,20 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical, was employed for determination of total antioxidant activity.
2.2. Chemicals Gallic acid, ABTS as diammonium salt, (+)-catechin and Folin & Ciocalteu’s phenol reagent were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2,20 Azobis(2-amidino-propane)dihydrochloride (AAPH) was obtained from Wako Chemicals USA, Inc. (Richmond, VA, USA). All other chemicals used were of analytical grade. 2.3. Extraction of phenolics The phenolics in powdered freeze-dried plums and apples were extracted by the ultrasound-assisted method (Kim & Lee, 2002). Phenolics of the fruits were extracted from 10 g ground freeze-dried samples using 100 ml of 80% aqueous methanol. The mixture of freeze-dried powder and 80% aqueous methanol was sonicated for 20 min with continual nitrogen gas purging. The mixture was filtered through Whatman #2 filter paper (Whatman International Limited, Kent, England) using a chilled Bu¨chner funnel and rinsing with 50 ml 100% methanol. Extraction of the residue was repeated using the same conditions. The two filtrates were combined and transferred into a 1 l evaporating flask with an additional 50 ml of 80% aqueous methanol. The solvent was evaporated using a rotary evaporator at 40 C. The remaining phenolic concentrate was first dissolved in 50 ml of 100% methanol and diluted to a final volume of 100 ml using distilled deionized water (ddH2O) obtained with a NANOpure water system (Barnstead, Dubuque, Iowa, USA). The mixture was centrifuged at refrigerated temperatures, using a Sorvall RC-5B refrigerated superspeed centrifuge (Du Pont Company, Biomedical Products Department, Wilmington, DE, USA) at 10 K with GSA rotor for 20 min and stored at 4 C until analyses were performed.
2. Materials and methods 2.4. Determination of total phenolics 2.1. Fruits Six cultivars of plums (Beltsville Elite B70197, Cacak Best, French Damson, Long John, Stanley, Yugoslavian Elite T101) were picked at commercial maturity during the 2000 harvest season at the New York State Agricultural Experiment Station orchard in Geneva, New York. The Gala apple cultivar, picked at commercial maturity during the 2001 harvest season at the same location, was used as a reference sample. Immediately upon arrival in the pilot plant after harvest, plums and apples were stored in a 2–5 C cold room. Plums were carefully cut in half and the pits removed. Apple slices were prepared by hand. Pitted plums and sliced apples were frozen and lyophilized. Freeze-dried samples were ground to powder and then stored at 20 C until analyzed.
The concentration of total phenolics was measured by the method described by Singleton and Rossi (1965) with some modification. Briefly, an aliquot (1 ml) of appropriately diluted extracts or standard solutions of gallic acid (20, 40, 60, 80 and 100 mg/l) was added to a 25 ml volumetric flask containing 9 ml of ddH2O. A reagent blank using ddH2O was prepared. One mililiter of Folin & Ciocalteu’s phenol reagent was added to the mixture and shaken. After 5 min, 10 ml of 7% Na2CO3 solution was added with mixing. The solution was then immediately diluted to volume (25 ml) with ddH2O and mixed thoroughly. After incubation for 90 min at 23 C, the absorbance versus prepared blank was read at 750 nm. Total phenolic contents of plums and apples were expressed as mg gallic acid equivalents
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(GAE)/100 g fresh sample. All samples were analyzed in five replications.
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3. Results 3.1. Determination of total phenolics
2.5. Determination of total flavonoids Total flavonoids were measured by a colorimetric assay developed by Zhishen, Mengcheng, and Jianming (1999). A 1 ml aliquot of appropriately diluted sample or standard solutions of catechin (20, 40, 60, 80 and 100 mg/l) was added to a 10 ml volumetric flask containing 4 ml ddH2O. At zero time, 0.3 ml 5% NaNO2 was added to the flask. After 5 min, 0.3 ml 10% AlCl3 was added. At 6 min, 2 ml 1 M NaOH was added to the mixture. Immediately, the reaction flask was diluted to volume with the addition of 2.4 ml of ddH2O and thoroughly mixed. Absorbance of the mixture, pink in colour, was determined at 510 nm versus prepared water blank. Total flavonoids of fruits were expressed on a fresh weight basis as mg/100 g catechin equivalents (CE). Samples were analyzed in five replications. 2.6. Vitamin C equivalent antioxidant capacity (VCEAC) assay using ABTS radical ABTS radical anions were used, according to the method of Kim, Lee, Lee, and Lee (2002). In brief, 1.0 mM AAPH, a radical initiator, was mixed with 2.5 mM ABTS in phosphate-buffered saline (pH 7.4; 100 mM potassium phosphate buffer containing 150 mM NaCl). The mixed solution was heated in a water bath at 68 C for 13 min. The resulting blue-green ABTS solution was adjusted to the absorbance of 0.650 0.020 at 734 nm with additional phosphate-buffered saline. Twenty microliters of sample was added to 980 ml of the ABTS radical solution. The mixture was incubated in a 37 C water bath under restricted light for 10 min. A control (20 ml 50% methanol and 980 ml of ABTS radical solution) was run with each series of samples. The decrease of absorbance at 734 nm was measured at an endpoint of 10 min. Total antioxidant capacity of plums and apples, as determined by scavenging blue-green ABTS radical anions, was expressed on a fresh weight basis as mg/100 g vitamin C equivalents (VCEAC). The radical stock solution was freshly prepared, daily. All tested samples were replicated six times.
Total phenolics of six plum cultivars and Gala apples are shown in Fig. 1. The total phenolic contents of the fresh plums per 100 g ranged from 174 1.5 mg GAE in Stanley to 375 3.8 mg GAE in French Damson. Phenolic concentrations of the other plum varieties were as follows: Beltsville Elite B70197 (332 3.1 mg GAE), Cacak Best (319 1.4 mg GAE), Yugoslavian Elite T101 (217 4.9 mg GAE) and Long John (199 2.5 mg GAE). The total phenolic content of 100 g of fresh Gala apples was 118 1.4 mg GAE. 3.2. Determination of total flavonoids Total flavonoid contents of six fresh cultivars of plums and Gala apples are shown in Fig. 1. Beltsville Elite B70197 exhibited the highest flavonoids content of 237 6.3 mg CE/100 g fresh sample. This was followed by French Damson (215 9.7 mg CE), Cacak Best (200 2.5 mg CE), Yugoslavian Elite T101 (146 6.0 mg CE), Long John (126.3 3.4 mg CE) and Stanley (118 2.6 mg CE). The content of total flavonoids in 100 g fresh Gala apples was at the level of 62.0 6.9 mg CE. 3.3. VCEAC assay using ABTS radical The total antioxidant capacities of the fresh plum cultivars and Gala apples, as determined by scavenging ABTS radical anions, are presented in Fig. 2. The total
2.7. Statistical analysis Results are presented as mean value standard deviation. Statistical analysis between experimental results was based on Student’s t test. Significant difference was statistically considered at the level of P < 0.001 or P < 0.05.
Fig. 1. Contents of total phenolics and flavonoids in various cultivars of plum and Gala apple. The data are displayed with meanstandard deviation (bars) of five replications. Total phenolic and flavonoid contents were significantly different at P< 0.001 and P< 0.05, respectively. GA, ST, LJ, YE, CB, BE and FD stand for Gala apple, Stanley, Long John, Yugoslavian Elite T101, Cacak Best, Beltsville Elite B70197 and French Damson, respectively.
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Fig. 2. Vitamin C equivalent antioxidant capacity (VCEAC) of various cultivars of plum and Gala apple using free blue-green ABTS radical anions. The data are displayed with meanstandard deviation (bars) of six replications. VCEACs showed significant difference except between CB and FD at the level of P<0.05. GA, ST, LJ, YE, FD, CB and BE stand for Gala apple, Stanley, Long John, Yugoslavian Elite T101, French Damson, Cacak Best and Beltsville Elite B70197, respectively.
antioxidant capacity of Gala apples was 205 5.6 mg VCEAC/100 g. Beltsville Elite B70197, Cacak Best, French Damson, Long John, Stanley and Yugoslavian Elite T101 showed total antioxidant capacities of 559 12.9, 535 9.2, 524 25.5, 288 16.2, 266 14.5 and 317 21.7 mg VCEAC/100 g, respectively.
4. Discussion The total phenolic content of 100 g fresh plums ranged from 174 to 375 mg GAE and for Gala apples, 118 1.4 mg GAE (Fig. 1). Based on the total phenolics, the six plum cultivars studied may be classified into two groups, one exhibiting relatively high levels of polyphenolic phytochemicals and the other, low levels. The cultivars having relatively high concentrations of phenolic phytochemicals are French Damson, Beltsville Elite B70197 and Cacak Best, whereas the cultivars with lower concentrations are Stanley, Long John and Yugoslavian Elite T101. The total phenolic content of French Damson was about 2.2-fold higher than that of Stanley. French Damson and Stanley plums showed about 3.2 and 1.5 times higher total phenolic level than Gala apples, respectively. Plums were previously shown to have a higher total phenolic content than apples (Proteggente et al., 2002). The averages of total phenolic content of plums and apples were significantly different at a level of P< 0.001. Total phenolic content of a mixture of red plums (Black Star, Ciruela Santa Rosa, Raviota, June Black), cultivars of which were different from ours, was reported to be at the level of 320 mg GAE/100 g (Proteggente et al., 2002). Another study reported that total phenols of black plums was 144 mg/100 g, expressed as
catechin equivalents (Karakaya, El, & Tas¸ , 2001). Total phenolic concentrations of various apple cultivars were reported to be in the range of 50.9 to 140 mg GAE/100 g (Lee & Smith, 2000). These variations are due to differences among cultivars, growing seasons, other agricultural practices and also variations in assays’ protocols. Beltsville Elite B70197 ranked second highest in total phenolics and had the highest concentration of total flavonoids among the six cultivars of plums tested, followed by French Damson, Cacak Best, Yugoslavian Elite T101, Long John and Stanley. Beltsville Elite B70197, having the highest content of total flavonoids and Stanley, the lowest content, displayed levels 3.8-fold and 1.9-fold higher than Gala apples, at 62.0 mg CE/ 100 g, respectively. The total flavonoid contents of the plum cultivars and Gala apples were significantly different at a significance level of P < 0.05. Total phenolic content and total flavonoid content of plums and apples showed a linear relationship with a positive correlation coefficient of r2=0.934 (data not shown). Reactive free radicals, such as superoxide anion (O 2 ), hydroxyl radical (OH), and peroxy radical (ROO), are extremely reactive and are known to be a biological product in reducing molecular oxygen (Williams & Jeffrey, 2000). Damage mediated by free radicals results in the disruption of membrane fluidity, protein denaturation, lipid peroxidation, oxidative DNA and alteration of platelet functions (Fridovich, 1978; Kinsella, Frankel, German, & Kanner, 1993), which has been generally considered to be linked with many chronic health problems such as cancers, inflammation, aging and atherosclerosis. An antioxidant, which can quench reactive free radicals, can prevent the oxidation of other molecules and may, therefore, have health-promoting effects in the prevention of degenerative diseases. The VCEAC procedure, a decolorization assay using free blue-green ABTS radicals, was shown to be a very useful tool for expeditiously measuring the antioxidant activity in individual chemical compounds or complex fruits extracts (Kim et al., 2002). This method expresses antioxidant capacity on the basis of vitamin C equivalent. Vitamin C is ubiquitously present in fruits and has been shown to have preventive effects against carcinogenesis (Lee, Lee, Kang, & Lee, 2002). The total antioxidant activity of plums and apples, in terms of VCEAC, ranked as follows: Beltsville Elite B70197> Cacak Best5French Damson > Yugoslavian Elite T101> Long John > Stanley > Gala apple (Fig. 2). Beltsville Elite B70197 showed 1.8 times higher VCEAC than Stanley with the lowest VCEAC among plum cultivars. However, the VCEAC of Stanley plums was 1.3-fold higher than that of Gala apples. All plum varieties in this study had higher VCEAC than Gala apple. At the significance level of P < 0.05, VCEACs, among various cultivars of plums and Gala apples, showed significant difference except between Cacak Best and
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French Damson. Good correlation (r2=0.938) between total phenolic content and VCEAC was observed with a high significance level (P < 0.001) (Fig. 3A). A similar relationship (r2=0.942) with high significance (P < 0.001) was also obtained between total flavonoid content and VCEAC (Fig. 3B). This positive and significant relationship between total phenolic content and total antioxidant activity, observed in this study was previously reported using other assays (Kalt et al., 1999; Proteggente et al., 2002; Velioglu, Mazza, Gao, & Oomah, 1998). The results of this study imply that dietary polyphenolic phytochemicals from plums may supply substantial antioxidants, which, in turn, may provide health-promoting effects to consumers. Since it has been suggested that a higher consumption of fruits and vegetables with high phytochemicals can inhibit, prevent or retard chronic diseases (Birt, Hendrich, & Wang, 2001; Cook & Samman, 1996; Singletary, 2000; Yang, Landau, Huang, & Newmark, 2001) and the plums in our study showed substantially more
Fig. 3. Relationship between total phenolics and VCEAC (A) and between total flavonoids and VCEAC (B).
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antioxidant activity than apples, an increased consumption of this fruit is therefore recommended in our diet.
Acknowledgements The authors thank Ms. Nancy Smith, research support specialist, for revising the text and providing technical support and Mr. Jay Freer for selecting and providing the cultivars of plums.
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