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Flavonols in fresh and processed Brazilian fruits
Journal of Food Composition and Analysis 22 (2009) 263–268
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Original Article
Flavonols in fresh and processed Brazilian fruits Rosemary Hoffmann-Ribani, Lı´sia S. Huber, Delia B. Rodriguez-Amaya * Department of Food Science, Faculty of Food Engineering, University of Campinas – UNICAMP, P.O. Box 6121, 13083-862 Campinas, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 May 2008 Received in revised form 24 November 2008 Accepted 1 December 2008
Flavonols (myricetin, quercetin and kaempferol) and flavones (luteolin and apigenin) were determined in Brazilian fruits, using a previously optimized and validated HPLC method. The flavonoids investigated were not detected in three cultivars each of mango and papaya. Quercetin was found in all the other fruits, the mean values varying from 0.3 mg/100 g in orange cultivar Peˆra to 7.5 mg/100 g in apple cultivar Fuji. Kaempferol was encountered in strawberry (0.7–0.9 mg/100 g), acerola (0.9–1.2 mg/100 g), pitanga (0.4 mg/100 g) and cashew-apple (
Keywords: Food analysis Food composition Flavonols Flavones Fruits Cultivar differences Processed fruits Brazilian fruits
1. Introduction Epidemiological studies have shown a correlation between increased consumption of flavonoids and reduced risk of cardiovascular diseases (Hertog et al., 1993, 1997; Knekt et al., 1996; Yochum et al., 1999) and certain types of cancer (Knekt et al., 1997; De Stefani et al., 1999; Garcia-Closas et al., 1999; Yang et al., 2001), but the evidence is still considered inconclusive (Hollman, 2001; Kris-Etherton et al., 2002), as such association was not observed in other studies particularly with cancer (Hertog et al., 1995). A difficulty encountered in these investigations has been the estimation of the flavonoid intake because of the limited data on the flavonoid composition of foods (Hollman and Katan, 1999; Arts and Hollman, 2005). Thus, determining the individual flavonoid concentrations in foods is considered a priority (Neuhouser, 2004; Scalbert et al., 2005); these analytical data cannot be replaced by the antioxidant activity reported in numerous papers. The flavonoid composition of some fruits has been reported, but more data are needed. Marked compositional variability has been observed due to varietal or cultivar differences, season, climate, degree of ripeness, processing and storage of the fruits (Robards
* Corresponding author. Tel.: +55 19 3521 4013; fax: +55 19 3521 2153. E-mail address: [email protected] (D.B. Rodriguez-Amaya). 0889-1575/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2008.12.004
and Antolovich, 1997; Aherne and O’Brien, 2002), but analytical uncertainty appears to be also involved. Except the flavanols, flavonoids are usually found in plants as glycosides. Quantitative determination of individual glycosides is difficult because several glycosides can occur for each flavonoid, each has a characteristic spectrum different from that of the aglycone and standards are not commercially available for most of these compounds (Hertog et al., 1992b). Food flavonoids levels are thus generally determined as aglycones after hydrolysis of food extracts in order to reduce the complexity of the analysis. Conditions for hydrolysis should, however, be optimized so that the aglycones are completely released, without provoking their degradation. The most widely used procedure for simultaneous extraction and hydrolysis is that established by Hertog et al. (1992b), which involves refluxing for 2 h at 90 8C with 1.2 M HCl in 50% aqueous methanol. Ha¨kkinen et al. (1998), using a mixture of flavonol and phenolic acid standards, compared the hydrolysis conditions of Hertog et al. with other conditions, utilizing 0.6 M or 1.2 M HCl in 50% aqueous methanol for 16 h at 21 8C or 35 8C under an atmosphere of nitrogen. The best results were obtained with 1.2 M HCl at 90 8C for 2 h and 0.6 M HCl at 35 8C for 16 h. These two conditions were then applied to samples of blackcurrant and strawberry. The best results for quercetin and myricetin in blackcurrant were obtained with 1.2 M HCl at 90 8C for 2 h, but kaempferol was not detected. In strawberry, this condition was also more efficient for quercetin, but myricetin and kaempferol
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were not detected. The method of choice was hydrolysis at 35 8C for 16 h with 1.2 M HCl. More recently Nuutila et al. (2002) compared the methods of Hertog et al. and Ha¨kkinen et al., using individual standards or mixtures of standards (phenolic acids, flavonols, flavones and catechins). The best results for majority of the compounds were obtained with Hertog et al.’s condition. Because the findings presented above indicate that the optimum conditions vary with different food samples, before initiating flavonoid analyses of Brazilian fruits, the conditions for extraction/hydrolysis were optimized for each fruit of interest by central composite rotational design and response surface analysis (Ribani and Rodriguez-Amaya, 2008). The optimum conditions did vary for the different fruits, which would be expected, considering the variation in the nature of the food matrix and in the composition and glycosylation of the flavonoids. The objective of the present study was to determine three major flavonols (myricetin, quercetin and kaempferol) and two major flavones (luteolin and apigenin) in fresh and processed Brazilian fruits, using a method that was previously optimized and validated. Flavonols and flavones are the most widely encountered flavonoids in fruits and vegetables. Brazil is rich in tropical, subtropical and temperate fruits, but only apple, orange and strawberry (Arabbi et al., 2004; Cordenunsi et al., 2005) had been analyzed in terms of the flavonol and flavone concentrations, and considering the well known between-sample variation, the number of sample lots analyzed was very limited. Additionally, it is important at this point when databases are being put together to compare analytical data obtained in Brazil and other countries.
2. Materials and methods 2.1. Fresh fruits The following fruits were analyzed: acerola (Malpighia glabra L. or Malpighia punicifolia L.) cultivars Olivier and Longa Vida and garden lots of undefined variety; apple (Malus domestica Borkh) cultivars Fuji, Gala and Golden; cashew-apple (Anacardium occidentale L.) supermarket lots of undefined variety; fig (Ficus carica L.) purple type; guava (Psidium guajava L.) white and red cultivar Ogawa; jabuticaba (Myrciaria jaboticaba Berg) supermarket lots of undefined variety; mango (Mangifera indica L.) cultivars Haden, Palmer and Tommy Atkins; oranges (Citrus sinensis) cultivars Bahia, Selecta, Lima and Peˆra; papaya (Carica papaya L.) cultivars Formosa, Golden and Solo; pitanga (Eugenia uniflora L.) supermarket and garden lots of undefined varieties; strawberry (Fragaria ananassa Duch.) cultivars Oso Grande, Sweet Charlie and Kamarossa (Table 1). 2.2. Processed fruits The processed products were chosen on the basis of the flavonoid levels found in the fresh fruits. The following products were analyzed: 1 brand of concentrated juice (brand A) and 2 brands of frozen pulp (brands B and C) of acerola; 3 brands of ready-to-drink juice (brands D–F), 3 brands of concentrated juice (brands A, D and G) and 3 brands of frozen pulp (brands B, C and H) of cashew-apple;
Table 1 Flavonol content of Brazilian fruits. Fruit
na
Concentration (mg/100 g fresh weight)b Myricetin
Quercetin
Kaempferol
Acerola (Malpighia glabra L. or Malpighia punicifolia L.) Garden lots (undefined variety) Cultivar Longa Vida Cultivar Olivier
6 5 3
nd nd nd
3.5–9.2 (5.0 0.1) 2.3–5.4 (4.1 1.1) 5.0–5.5 (5.3 0.2)
0.8–1.6 (1.2 0.3) 0.6–1.2 (0.9 0.3) 0.8–1.1 (1.0 0.1)
Apple (Malus domestica Borkh) Cultivar Fuji (red, with skin) Cultivar Golden Delicious (green, with skin) Cultivar Gala (red, with skin)
5 5 5
nd nd nd
6.3–8.6 (7.5 1.1) 2.8–4.2 (3.7 0.6) 4.0–8.0 (5.6 1.6)
Cashew-apple (Anacardium occidentale L.) Supermarket lots (undefined variety)
5
1.3–2.8 (2.0 0.5)
1.0–1.9 (1.3 0.4)
5
nd
0.8–1.9 (1.3 0.4)
nd
7 5
nd nd
0.8–1.1 (1.0 0.1) 1.0–1.2 (1.2 0.1)
nd nd
Jabuticaba (Myrciaria jaboticaba Berg) Supermarket lots (undefined variety)
8
nd
0.8–1.3 (1.1 0.2)
nd
Orange (Citrus sinensis) Cultivar Peˆra Cultivar Bahia Cultivar Lima Cultivar Selecta
5 5 5 4
nd nd nd nd
0.2–0.4 0.3–0.5 0.3–0.4 0.2–0.4
(0.3 0.1) (0.4 0.1) (0.3 0.1) (0.3 0.1)
nd nd nd nd
Pitanga (Eugenia uniflora L.) Garden lots (undefined variety) Supermarket lots (undefined variety)
3 4
3.3–4.2 (3.7 0.4) 2.7–3.6 (3.1 0.4)
5.6–7.3 (6.2 0.9) 5.1–6.7 (5.5 1.0)
0.4 (0.4 0.0) 0.3–0.6 (0.4 0.1)
Strawberry (Fragaria ananassa Duch.) Cultivar Kamarossa Cultivar Oso Grande Cultivar Sweet Charlie
3 5 2
nd nd nd
0.7–0.9 (0.8 0.1) 0.9–1.4 (1.1 0.2) 0.7–1.0 (0.9 0.2)
0.6–0.8 (0.7 0.1) 0.6–1.3 (0.9 0.2) 0.6–1.0 (0.8 0.3)
Fig (Ficus carica L.) Purple type Guava (Psidium guajava L.) Cultivar Ogawa (red) White type (white)
LQ, limit of quantitation, myricetin = 0.26 mg/100 g; quercetin = 0.23 mg/100 g, kaempferol = 0.26 mg/100 g; nd, not detected; LOD, limit of detection; myricetin = 0.086 mg/ 100 g, quercetin = 0.073 mg/100 g, kaempferol = 0.086 mg/100 g. a n = number of composite samples analyzed in duplicates. b Range (mean standard deviation).
R. Hoffmann-Ribani et al. / Journal of Food Composition and Analysis 22 (2009) 263–268 Table 2 Flavonol content of Brazilian processed fruits. Product
Concentrationa Quercetin
Kaempferol
Acerola Concentrated juice Brand A nd
Myricetin
1.0–1.8 (1.4 0.3)
0.4–0.5 (0.4 0.1)
Frozen pulp Brand B nd Brand C nd
2.1–2.9 (2.5 0.4)a 1.8–2.3 (2.1 0.2)b
0.7–1.0 (0.9 0.1)a 0.4–0.5 (0.4 0.0)b
Cashew-apple Ready-to-drink juice Brand D tr Brand E tr-0.2 Brand F tr-0.2
tr tr-0.2 tr-0.2
nd nd nd
Concentrated juice Brand A 0.3–0.4 (0.3 0.1)a Brand D 0.2–0.4 (0.3 0.1)a Brand G 0.2–0.3 (0.3 0.0)a
0.2 (0.2 0.0) tr-0.2 tr-0.2
nd nd nd
Frozen pulp Brand B 0.6–0.8 (0.7 0.1)a Brand C 0.2–0.4 (0.3 0.1)b Brand H 0.3–0.9 (0.5 0.3)a,b
0.4–0.5 (0.5 0.0)a 0.2 (0.2 0.0)b 0.2–0.6 (0.3 0.2)a,b
nd nd nd
Pitanga Concentrated juice Brand A 0.7–1.5 (1.2 0.4)a Brand I 1.6–2.0 (1.7 0.2)a
1.6–2.2 (2.0 0.3)a 2.2–2.8 (2.5 0.3)a
tr-0.2 tr-0.2
Frozen pulp Brand C 1.0–2.0 (1.6 0.5)
2.0–3.0 (2.5 0.5)
tr-0.2 (0.2 0.0)
Means of the same product (different brands) in a column with different letters are significantly different (P < 0.05). Products of the same brand letter were manufactured by the same company. nd, not detected; tr, trace; LOD, limit of detection, myricetin = 0.086 mg/100 g, quercetin = 0.073 mg/100 g, kaempferol = 0.086 mg/100 g. a Range (mean standard deviation), in mg/100 mL for the juice and mg/100 g for the pulp, of five sample lots analyzed individually in duplicates.
2 brands of concentrated juice (brands A and I) and 1 brand of frozen pulp (brand C) of pitanga (Table 2). 2.3. Chemicals The standards of myricetin, quercetin, kaempferol, luteolin and apigenin were purchased from Sigma Chemicals Co. (St. Louis, USA). Water was purified with a Milli-Q water purification system (Millipore, Bedford, USA). Methanol (HPLC-grade) was purchased from Mallinckrodt Baker Inc. (Phillipsburg, USA). Reagent grade formic acid and ascorbic acid were from Merck (Frankfurt, Germany) and Labsynth Ltd. (Diadema, Brazil), respectively. Stock standard solutions were prepared by dissolving the flavonoids in HPLC grade methanol to a concentration of approximately 500 mg/mL, stored at 18 8C and protected from light during storage and when in use. These solutions were found to be stable for over 2 months at 18 8C. To match the standard and sample solutions, the standard working solution was prepared as follows: standard stock solutions were diluted in 1.5 mL Milli-Q water to which 0.14% ascorbic acid was added. One mL of 6 M HCl was added and the solution was made up to 5 mL with methanol. 2.4. Sampling and sample preparation Samples were analyzed immediately after collection. Two to seven sample lots of ripe fresh fruits were collected for each cultivar of each fruit at different times from July 2004 to January 2005, harvest time for most of the fruits in Brazil, and analyzed individually in duplicate. For each lot, samples were purchased
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from three supermarkets (1 kg or a minimum of 3 units for big fruits per supermarket) in Campinas, Sa˜o Paulo. To obtain the composite sample submitted to analysis, small fruits (acerola, jabuticaba, strawberry and pitanga) taken from the three sampling sites were mixed, quartered to reduce the size of the sample from approximately 3 kg to 800 g and after removal of inedible portions (mainly seeds), homogenized first in a food processor with 0.135% (w/w) ascorbic acid as antioxidant, then in a Polytron homogenizer (Polytron System MR2100, Kinematica-AG, Luzern, Switzerland) at 25,000 rpm during 3 min The bigger fruits from the three sampling sites were cut longitudinally into quarters, opposite sections from each fruit were combined, and after removal of inedible parts (mainly seeds and peel for mango, orange and papaya), sliced (1– 2 cm) and homogenized as described for the small fruits. Fifteen grams of the composite sample were taken for analysis. For the processed fruits, five sample lots were analyzed individually in duplicate for each brand of each product. Each lot consisted of 3 units taken at random from the big supermarket lot. To obtain the composite sample, the contents of the 3 packages were mixed and 15 g of pulp or 15 mL of juice were taken for analysis. 2.5. Flavonoid extraction and hydrolysis The simultaneous extraction and hydrolysis procedure was based on that of Hertog et al. (1992b). However, instead of refluxing at 90 8C with 1.2 M HCL in 50% aqueous methanol for 2 h, the hydrolysis conditions (HCl concentration and refluxing time) were previously optimized for each fruit (Ribani and RodriguezAmaya, 2008). To the 15 g homogenized composite sample, 25 mL of methanol and 10 mL of HCl were added. The mixture thus formed consisted of different final molar concentration of HCl in 50% aqueous methanol HCl (v/v), with 0.04% of antioxidant. The optimum extraction/hydrolysis conditions were: acerola, 0.4 M HCl for 90 min; apple, 0.5 M HCl for 70 min; cashew-apple, 0.2 M HCl for 110 min; fig, 0.5 M HCl for 160 min; guava, 0.6 M HCl for 55 min; jaboticaba, 0.6 M HCl for 45 min; oranges, 1.5 M HCl for 90 min; pitanga, 0.6 M HCl for 40 min; strawberry, 1.2 M for 120 min. After refluxing, the extracts were cooled under running water and passed through a 130 mesh sieve. The volume was made up to 50 mL with methanol and an aliquot of about 5 mL was filtered through a 0.45 mm polytetrafluoroethylene (PTFE) filter (Millipore Ltd., Bedford, USA) prior to HPLC analysis. 2.6. HPLC analysis This analysis was performed on a Waters system, equipped with a Rheodyne injection valve with a 5 mL fixed loop, a quaternary pump (Waters model 600) and a UV–Vis photodiode array detector (Waters model 996), controlled by a Millenium workstation (version 32). A Symmetry C-18 (2.1 mm 150 mm, 3.5 mm) Waters column was used, the mobile phase consisting of methanol and water, both acidified with 0.3% formic acid, and the flow rate was 0.2 mL per min. A multilinear gradient was applied from 20:80 to 48:52 in 6 min, this proportion being maintained until 29 min, and then changed to 28:72 in 2 min, this proportion being maintained until 40 min. Finally, the mobile phase was bought back to the initial proportion of 20:80 in 3 min, maintaining this proportion until 60 min for column reequilibration before subsequent injection. Analytes were monitored from 200 to 600 nm and detection at 370 nm was used for quantification, which was done by external standardization. The full HPLC method, including the optimized extraction/ hydrolysis, was previously validated (Ribani and RodriguezAmaya, 2008). The limits of detection (LOD), calculated as the
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minimal concentration corresponding to 3.3 (SD/S), SD being the standard deviation of the blank and S the slope of the standard curve, were 0.086, 0.073, 0.086, 0.103 and 0.271 mg/100 g, respectively, for myricetin, quercetin, kaempferol, luteolin and apigenin. The calculated values were confirmed with actual analytical values under the chromatographic conditions established. The accuracy of the method was verified in recovery tests, using acerola samples spiked with 1.0 mg/100 g of flavonol standards and 1.7 mg/100 g of flavone standards, the test being done seven times for each flavonoid. The mean recoveries were 87, 95, 95, 96, and 95%, respectively, for myricetin, quercetin, kaempferol, luteolin and apigenin. The coefficients of variation showed good repeatability. The linearity of the HPLC method was checked for flavonols and flavones in the 0.03–4.8 mg/100 mL range (0.06–2.3, 0.03–4.8, 0.018–3.7, 0.11–2.6, 0.16–3.8 mg/100 mL for myricetin, quercetin, kaempferol, luteolin and apigenin, respectively). Calibration was performed by injecting the standard working solution in triplicate at five different concentrations for each flavonoid, based on the expected flavonoid content ranges in the samples. All standard curves passed through the origin, were linear in the concentration ranges expected in the samples, with coefficients of determination ranging from 0.9989 (for luteolin) to 0.9999 (for quercetin).
3. Results and discussion 3.1. Flavonol levels in fresh fruits The five flavonoids investigated were not detected in the mango cultivars Haden, Palmer and Tommy Atkins and in the papaya cultivars Formosa, Golden and Solo. Franke et al. (2004) did detect myricetin, quercetin and kaempferol in Hawaiian mango Haden, but at levels below the quantification limits of 0.04, 0.02 and 0.01 mg/100 g, respectively. These authors also detected in Hawaiian papaya myricetin and kaempferol, but not quercetin, at levels below the quantification limits. In a commercial frozen mango concentrate, Schieber et al. (2000) identified five quercetin glycosides and one kaempferol glycoside, the predominant flavonol glycosides quercetin 3-galactoside, quercetin 3-glucoside and quercetin 3-arabinoside amounting to 2.2, 1.6 and 0.5 mg/ 100 g, respectively. Thus, flavonols may be present in the Brazilian mangoes but at concentrations below our detection limits. Quercetin was found in all the other fruits, the mean level ranging from 0.3 to 7.5 mg/100 g, the highest concentration being found in apple cultivar Fuji (Table 1). Kaempferol was encountered in measurable amounts in strawberry, acerola and pitanga, ranging on the average from 0.4 to 1.2 mg/100 g, being highest in acerola. Myricetin was detected only in cashew-apple and pitanga at 2.0– 3.7 mg/100 g. The flavones luteolin and apigenin were not detected in all the fruits analyzed. This is in line with the USDA summary stating that fruits do not contain flavones, except for small amounts in lemons and pummelo juice (USDA, 2003). Franke et al. (2004) did find 0.8, 1.4, 2.6, 1.4–1.5, and 0.9 mg/100 g of luteolin in blueberry, ruby red grapefruit, red seedless grapes, two cultivars of oranges and black plum, respectively. Quercetin was the main flavonol in acerola. Its average concentration ranged from 4.1 to 5.3 mg/100 g, being lowest in the cultivar Longa Vida and highest in the cultivar Olivier. The mean concentration of kaempferol varied from 0.9 to 1.2 mg/100 g, being also lowest in ‘‘Longa Vida’’ and highest in the garden lots. As is well known, apple is a good to rich source of quercetin, depending on the cultivar. In the present study, the concentration of this flavonol varied from a mean of 3.7 mg/100 g in the cultivar Golden Delicious to 7.5 mg/100 g in the cultivar Fuji.
Unlike the other fruits, cashew-apple had myricetin (2.0 mg/ 100 g) as the major flavonol followed by quercetin (1.3 mg/100 g). Kaempferol ranged from below the LQ to 0.3 mg/100 g. The cashew-apple lots analyzed in the present study were purchased at local supermarkets and were mixtures of 3 types, elongated yellow, elongated red and round red, which botanically are not considered established cultivars. Only quercetin was found in fig, guava and jabuticaba with similar concentration ranges. The purple fig had an average of 1.3 mg/100 g quercetin. The red guava ‘‘Ogawa’’ had slightly lower quercetin concentration (1.0 mg/100 g) compared to the white type (1.2 mg/kg). Jaboticaba had 1.1 mg/100 g quercetin. Miean and Mohamed (2001) reported myricetin and apigenin contents of 55.0 and 57.9 mg/100 g dry weight, respectively, in guava; quercetin, kaempferol and luteolin were not detected. Quercetin was also the only flavonol detected in the four cultivars of oranges analyzed but at much lower levels (about 0.3 mg/100 g of pulp). This is not surprising since the flavanones hesperidin and narirutin are the predominant flavonoids in this fruit (Arabbi et al., 2004; Franke et al., 2004). Among the fruits investigated in the present study, pitanga had the highest myricetin content (3.1 and 3.7 mg/100 g, respectively, for the supermarket and garden lots). It also presented the second highest quercetin concentration (5.5 and 6.2 mg/100 g, respectively, for the supermarket and garden lots). This fruit also had kaempferol but at levels (0.4 mg/100 g for the supermarket and garden lots) lower than those of acerola and strawberry. In strawberry, the lowest quercetin and kaempferol values were observed in the cultivar ‘‘Kamarossa’’ (0.8 and 0.7 mg/100 g, respectively) and the highest in the cultivar ‘‘Oso Grande’’ (1.1 and 0.9 mg/100 g, respectively). 3.2. Flavonol levels in processed fruits Table 2 shows the flavonol levels encountered in processed fruits. While there are some between-sample and between-brand variations, the most important finding is that the processed products had flavonol contents considerably lower than those obtained for the fresh fruits, especially the cashew-apple products. Taking the frozen pulp, which is the product closest to the fresh fruit, the cashew-apple product had myricetin content about three to six times less than the fresh fruit; the quercetin content was about three to eight times less. For pitanga, the myricetin, quercetin and kaempferol levels were two to three times less in the frozen pulp compared to the fresh fruit. For acerola, the quercetin content of the frozen pulp was about 50% of the fresh fruit; the kaempferol level was also about 50% of that of the fresh fruit in brand C, but almost the same for brand B. Because the frozen pulps were blanched, the lower levels could be due to lower flavonol contents in the fruits used as raw material, removal of the peel and degradation during blanching. Enzymatic oxidation could also be involved if the raw material after pulping was held for some time before blanching and/or blanching was not sufficient to inactivate the oxidative enzymes. Surprisingly, for all three fruits, the flavonol concentrations of the frozen pulp were higher than those of the concentrated juice. The concentrated juice was expected to have higher levels because of the removal of water during the process. The low values indicate considerable degradation during the heat treatment applied to concentrate the juice, much more drastic than the pasteurization of the frozen pulp. 3.3. Comparison with published data Of the different fruits analyzed in this study, only apple, orange, strawberry and fig can be compared with previous data (Table 3).
R. Hoffmann-Ribani et al. / Journal of Food Composition and Analysis 22 (2009) 263–268 Table 3 Comparison of quercetin and kaempferol levels in apple, strawberries, orange and fig, reported in different studies. Fruit
Reference
n/cultivar or varietya
Concentration range (mg/100 g fresh weight) Quercetin
Kaempferol
5 1 2 4 1–2 18 1
2.8–8.6 0.4–10.1 2.5–8.0 2.1–3.8 2.1–7.2 2.0 0.4 2.6–7.4
nd—<0.26 nd <0.1 0.0 <0.2 nd nd
0.8–1.9 0.9 1.2
nd 0.0
Apple 3 cultivars 3 cultivars 2 cultivars 4 cultivars 6 cultivars Not specified 8 cultivars
Present study Arabbi et al. (2004) Franke et al. (2004) Harnly et al. (2006) Hertog et al. (1992a) Justesen et al. (1998) Price et al. (1999)
Fig 1 variety 1 variety
Present study Harnly et al. (2006)
5 8
Orange (pulp) 4 cultivars 2 cultivars 2 cultivars
Present study Arabbi et al. (2004) Franke et al. (2004)
4–5 1 3
0.2–0.5 0.8–0.9 0.4–0.7
nd nd <0.1
Present study Cordenunsi et al. (2005) Franke et al. (2004) Ha¨kkinen et al. (1999) Ha¨kkinen and To¨rro¨nen (2000) Harnly et al. (2006) Hertog et al. (1992a) Justesen et al. (1998) Kosar et al. (2004)
2–5 1 1 1 1
0.7–1.4 3.9–6.8 0.9 0.7 0.3–0.5
0.6–1.4 1.3–2.1 0.6 0.5–0.8 0.2–0.9
1.9 1.0 0.8–1.0 0.6 0.5 0.9 0.2
0.0 0.7–1.6 0.5 0.3 nd
Strawberry 3 cultivars 3 cultivars 1 variety 2 cultivars 6 cultivars 1 variety 1 variety Not specified 3 cultivars
7 3 4 1
nd, not detected. a Number of sample lots analyzed per cultivar or variety.
The other fruits were analyzed quantitatively in the present work for the first time. But even for the four fruits compared, considering the between-sample and between-cultivar compositional variations shown in Table 1, Table 3 indicates that more analyses are needed to obtain representative cultivar-specific data. The quercetin ranges in apple obtained in the present study compare very well with those of Franke et al. (2004), Hertog et al. (1992a) and Price et al. (1999). Those of Harnly et al. (2006) and Justesen et al. (1998) are on the lower side. The range of Arabbi et al. (2004) is much wider. Our quercetin range for orange is slightly lower than that of Franke et al. (2004) and distinctly lower than that of Arabbi et al. (2004). Our results with strawberry also agree with most of those reported in earlier studies (Hertog et al., 1992a; Justesen et al., 1998; Ha¨kkinen et al., 1999; Franke et al., 2004; Kosar et al., 2004), slightly higher than those of Ha¨kkinen and To¨rro¨nen (2000) and slightly lower than those of Harnly et al. (2006) for quercetin. Cordenunsi et al. (2005) obtained much higher levels. For fig, the mean quercetin value obtained by Harnly et al. falls within our range. Surprisingly, our results agree more with those obtained in other countries than with those of Arabbi et al. (2004) and Cordenunsi et al. (2005), who also analyzed Brazilian fruits. Apparently, analytical variability is involved, aside from natural variation. Arabbi et al. and Cordenunsi et al. did not hydrolyze the sample so the flavonoids were quantified as glycosides. However, quercetin (except for rutin) and kaempferol aglycones were used as external standards. It can be claimed that the distinctly higher results obtained by Arabbi et al. and Cardenunsi et al. are due to degradation of the flavonols during hydrolysis in the present study, although our method was optimized to avoid this degradation. However, Price
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et al. (1999) quantified quercetin as glycosides using the respective six glycoside standards and their results resemble those of the present work (Table 3). The other authors cited in the table did hydrolysis. In any case, these observations indicate that more work has to be done with the analytical methodology, including the formulation of certified reference materials and interlaboratory collaborative studies, as already done with carotenoids. Among the Brazilian fruits analyzed, the best sources of flavonoids are pitanga and cashew-apple, which contain the three flavonols investigated, acerola, having quercetin and kaempferol, and apple with high levels of quercetin. Pitanga and acerola, which used to be only garden fruits, are now being produced and processed commercially. Cashew-apple is a by-product of the cashew nut industry. References Aherne, S.A., O’Brien, N.M., 2002. Dietary flavonols: chemistry, food content, and metabolism. Nutrition 18, 75–81. Arabbi, P.R., Genovese, M.I., Lajolo, F.M., 2004. Flavonoids in vegetable foods commonly consumed in Brazil and estimated ingestion by the Brazilian population. Journal of Agricultural and Food Chemistry 52, 1124–1131. Arts, I.C.W., Hollman, P.C.H., 2005. Polyphenols and disease risk in epidemiologic studies. American Journal of Clinical Nutrition 81, 317S–325S. Cordenunsi, B.R., Genovese, M.I., do Nascimento, J.R.O., Hassimotto, N.M.A., dos Santos, R.J., Lajolo, F.M., 2005. Effects of the temperature on the chemical composition and antioxidant activity of three strawberry cultivars. Food Chemistry 91, 113–121. De Stefani, E., Boffetta, P., Deneo-Pellegrini, H., Mendilaharsu, M., Carzoglio, J.C., Ronco, A., Olivera, L., 1999. Dietary antioxidants and lung cancer risk: a casecontrol study in Uruguay. Nutrition and Cancer 34, 100–110. Franke, A.A., Custer, L.J., Arakaki, C., Murphy, S.P., 2004. Vitamin C and flavonoid levels of fruits and vegetables consumed in Hawaii. Journal of Food Composition and Analysis 17, 1–35. Garcia-Closas, R., Gonzalez, C.A., Agudo, A., Riboli, E., 1999. Intake of specific carotenoids and flavonoids and the risk of gastric cancer in Spain. Cancer Causes and Control 10, 71–75. Ha¨kkinen, S.H., Ka¨renlampi, S.O., Heinonen, I.M., Mykka¨nen, H.M., To¨rro¨nen, A.R., 1998. HPLC method for screening of flavonoids and phenolic acids in berries. Journal of the Science of Food and Agriculture 77, 543–551. Ha¨kkinen, S.H., Ka¨renlampi, S.O., Heinonen, I.M., Mykka¨nen, H.M., To¨rro¨nen, A.R., 1999. Content of the flavonols quercetin, myricetin, and kaempferol in 25 edible berries. Journal of Agricultural and Food Chemistry 47, 2274–2279. Ha¨kkinen, S.H., To¨rro¨nen, A.R., 2000. Content of flavonols and selected phenolic acids in strawberries and Vaccinium species: influence of cultivar, cultivation site and technique. Food Research International 33, 517–524. Harnly, J.M., Doherty, R.F., Beecher, G.R., Holden, J.M., Haytowitz, D.B., Bhagwat, S., Gebhardt, S., 2006. Flavonoid content of U.S. fruits, vegetables, and nuts. Journal of Agricultural and Food Chemistry 54, 9966–9977. Hertog, M.G.L., Feskens, E.J.M., Hollmann, P.C.H., Katan, M.B., Kromhout, D., 1993. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet 342, 1007–1011. Hertog, M.G.L., Feskens, E.J.M., Kromhout, D., 1997. Antioxidant flavonols and coronary heart disease risk. Lancet 349, 699. Hertog, M.G.L., Hollman, P.C.H., Katan, M.B., 1992a. Content of potentially anticarcinogenic flavonoids of 28 vegetables and 9 fruits commonly consumed in the Netherlands. Journal of Agricultural and Food Chemistry 40, 2379–2383. Hertog, M.G.L., Hollman, P.C.H., Venema, D.P., 1992b. Optimization of a quantitative HPLC determination of potentially anticarcinogenic flavonoids in vegetables and fruits. Journal of Agricultural and Food Chemistry 40, 1591–1598. Hertog, M.G., Kromhout, D., Aravanis, C., Blackburn, H., Buzina, R., Fidanza, F., Giampaoli, S., Jansen, A., Menotti, A., Nedeljkovic, S., Pekkarinen, M., Simic, B.S., Toshima, H., Feskens, E.J.M., Hollman, P.C.H., Katan, M.B., 1995. Flavonoid intake and long-term risk of coronary heart disease and cancer in the Seven Countries Study. Archives of Internal Medicine 155, 381–386. Hollman, P.C.H., 2001. Evidence for health benefits of plant phenols: local or systemic effects? Journal of the Science of Food and Agriculture 81, 842–852. Hollman, P.C.H., Katan, M.B., 1999. Dietary flavonoids: intake, health effects and bioavailability. Food Chemistry and Toxicology 37, 937–942. Justesen, U., Knuthsen, P., Leth, T., 1998. Quantitative analysis of flavonols, flavones, and flavanones in fruits, vegetables and beverages by high-performance liquid chromatography with photo-diode array and mass spectrometric detection. Journal of Chromatography A 799, 101–110. Knekt, P., Ja¨rvinen, R., Seppa¨nen, R., Hellio¨vaara, M., Teppo, L., Pukkala, E., Aromaa, A., 1997. Dietary flavonoids and risk of lung cancer and other malignant neoplasms. American Journal of Epidemiology 146, 223–230. Knekt, P., Jarvinen, R., Reunanen, A., Maatela, J., 1996. Flavonoid intake and coronary mortality in Finland: a cohort study. British Medical Journal 312, 478–481. Kosar, M., Kafkas, E., Paydas, S., Baser, K.H.C., 2004. Phenolic composition of strawberry genotypes at different maturation stages. Journal of Agricultural and Food Chemistry 52, 1586–1589.
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Kris-Etherton, P.M., Hecker, K.D., Bonanome, A., Coval, S.M., Binkoski, A.E., Hilpert, K.F., Griel, A.E., Etherton, T.D., 2002. Bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer. The American Journal of Medicine 113, 71S–88S. Miean, K.H., Mohamed, S., 2001. Flavonoid (myricetin, quercetin, kaempferol, luteolin, and apigenin) content of edible tropical plants. Journal of Agricultural and Food Chemistry 49, 3106–3112. Neuhouser, M.L., 2004. Dietary flavonoids and cancer risk: evidence from human population studies. Nutrition and Cancer 50, 1–7. Nuutila, A.M., Kammiovirta, K., Oksman-Caldentey, K.M., 2002. Comparison of methods for the hydrolysis of flavonoids and phenolic acids from onion and spinach for HPLC analysis. Food Chemistry 76, 519–525. Price, K.R., Prosser, T., Richetin, A.M.F., Rhodes, M.J.C., 1999. A comparison of the flavonol content and composition in dessert, cooking and cider-making apples; distribution within the fruit and effect of juicing. Food Chemistry 66, 489– 494. Ribani, R.H., Rodriguez-Amaya, D.B., 2008. Otimizac¸a˜o de me´todo para determinac¸a˜o de flavono´is e flavonas em frutas por cromatografia lı´quida de alta
eficieˆncia utilizando delineamento estatı´stico e ana´lise de superfı´cie de resposta. Quı´mica Nova 31, 1378–1384. Robards, K., Antolovich, M., 1997. Analytical chemistry of fruit bioflavonoids. Analyst 122, 11R–34R. Scalbert, A., Manach, C., Morand, C., Re´me´sy, C., Jime´nez, L., 2005. Dietary polyphenols and the prevention of diseases. Critical Review of Food Science and Nutrition 45, 287–306. Schieber, A., Ullrich, W., Carle, R., 2000. Characterization of polyphenols in mango puree concentrate by HPLC with diode array and mass spectrometric detection. Innovative Food Science and Emerging Technology 1, 161–166. USDA, 2003. USDA’s flavonoid database: flavonoids in fruit. Retrieved April 2008. http://www.nal.usda.gov/fnic/foodcomp/Data/Other/ADA2003_FruitFlav.pdf. Yang, C.S., Landau, J.M., Huang, M., Newmark, H.L., 2001. Inhibition of carcinogenesis by dietary polyphenolic compounds. Annual Review of Nutrition 21, 381– 406. Yochum, L., Kushi, L.H., Meyer, K., Folson, A.R., 1999. Dietary flavonoid intake and risk of cardiovascular disease in postmenopausal women. American Journal of Epidemiology 149, 943–949.
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Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Original Article
Flavonols in fresh and processed Brazilian fruits Rosemary Hoffmann-Ribani, Lı´sia S. Huber, Delia B. Rodriguez-Amaya * Department of Food Science, Faculty of Food Engineering, University of Campinas – UNICAMP, P.O. Box 6121, 13083-862 Campinas, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 May 2008 Received in revised form 24 November 2008 Accepted 1 December 2008
Flavonols (myricetin, quercetin and kaempferol) and flavones (luteolin and apigenin) were determined in Brazilian fruits, using a previously optimized and validated HPLC method. The flavonoids investigated were not detected in three cultivars each of mango and papaya. Quercetin was found in all the other fruits, the mean values varying from 0.3 mg/100 g in orange cultivar Peˆra to 7.5 mg/100 g in apple cultivar Fuji. Kaempferol was encountered in strawberry (0.7–0.9 mg/100 g), acerola (0.9–1.2 mg/100 g), pitanga (0.4 mg/100 g) and cashew-apple (
Keywords: Food analysis Food composition Flavonols Flavones Fruits Cultivar differences Processed fruits Brazilian fruits
1. Introduction Epidemiological studies have shown a correlation between increased consumption of flavonoids and reduced risk of cardiovascular diseases (Hertog et al., 1993, 1997; Knekt et al., 1996; Yochum et al., 1999) and certain types of cancer (Knekt et al., 1997; De Stefani et al., 1999; Garcia-Closas et al., 1999; Yang et al., 2001), but the evidence is still considered inconclusive (Hollman, 2001; Kris-Etherton et al., 2002), as such association was not observed in other studies particularly with cancer (Hertog et al., 1995). A difficulty encountered in these investigations has been the estimation of the flavonoid intake because of the limited data on the flavonoid composition of foods (Hollman and Katan, 1999; Arts and Hollman, 2005). Thus, determining the individual flavonoid concentrations in foods is considered a priority (Neuhouser, 2004; Scalbert et al., 2005); these analytical data cannot be replaced by the antioxidant activity reported in numerous papers. The flavonoid composition of some fruits has been reported, but more data are needed. Marked compositional variability has been observed due to varietal or cultivar differences, season, climate, degree of ripeness, processing and storage of the fruits (Robards
* Corresponding author. Tel.: +55 19 3521 4013; fax: +55 19 3521 2153. E-mail address: [email protected] (D.B. Rodriguez-Amaya). 0889-1575/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2008.12.004
and Antolovich, 1997; Aherne and O’Brien, 2002), but analytical uncertainty appears to be also involved. Except the flavanols, flavonoids are usually found in plants as glycosides. Quantitative determination of individual glycosides is difficult because several glycosides can occur for each flavonoid, each has a characteristic spectrum different from that of the aglycone and standards are not commercially available for most of these compounds (Hertog et al., 1992b). Food flavonoids levels are thus generally determined as aglycones after hydrolysis of food extracts in order to reduce the complexity of the analysis. Conditions for hydrolysis should, however, be optimized so that the aglycones are completely released, without provoking their degradation. The most widely used procedure for simultaneous extraction and hydrolysis is that established by Hertog et al. (1992b), which involves refluxing for 2 h at 90 8C with 1.2 M HCl in 50% aqueous methanol. Ha¨kkinen et al. (1998), using a mixture of flavonol and phenolic acid standards, compared the hydrolysis conditions of Hertog et al. with other conditions, utilizing 0.6 M or 1.2 M HCl in 50% aqueous methanol for 16 h at 21 8C or 35 8C under an atmosphere of nitrogen. The best results were obtained with 1.2 M HCl at 90 8C for 2 h and 0.6 M HCl at 35 8C for 16 h. These two conditions were then applied to samples of blackcurrant and strawberry. The best results for quercetin and myricetin in blackcurrant were obtained with 1.2 M HCl at 90 8C for 2 h, but kaempferol was not detected. In strawberry, this condition was also more efficient for quercetin, but myricetin and kaempferol
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were not detected. The method of choice was hydrolysis at 35 8C for 16 h with 1.2 M HCl. More recently Nuutila et al. (2002) compared the methods of Hertog et al. and Ha¨kkinen et al., using individual standards or mixtures of standards (phenolic acids, flavonols, flavones and catechins). The best results for majority of the compounds were obtained with Hertog et al.’s condition. Because the findings presented above indicate that the optimum conditions vary with different food samples, before initiating flavonoid analyses of Brazilian fruits, the conditions for extraction/hydrolysis were optimized for each fruit of interest by central composite rotational design and response surface analysis (Ribani and Rodriguez-Amaya, 2008). The optimum conditions did vary for the different fruits, which would be expected, considering the variation in the nature of the food matrix and in the composition and glycosylation of the flavonoids. The objective of the present study was to determine three major flavonols (myricetin, quercetin and kaempferol) and two major flavones (luteolin and apigenin) in fresh and processed Brazilian fruits, using a method that was previously optimized and validated. Flavonols and flavones are the most widely encountered flavonoids in fruits and vegetables. Brazil is rich in tropical, subtropical and temperate fruits, but only apple, orange and strawberry (Arabbi et al., 2004; Cordenunsi et al., 2005) had been analyzed in terms of the flavonol and flavone concentrations, and considering the well known between-sample variation, the number of sample lots analyzed was very limited. Additionally, it is important at this point when databases are being put together to compare analytical data obtained in Brazil and other countries.
2. Materials and methods 2.1. Fresh fruits The following fruits were analyzed: acerola (Malpighia glabra L. or Malpighia punicifolia L.) cultivars Olivier and Longa Vida and garden lots of undefined variety; apple (Malus domestica Borkh) cultivars Fuji, Gala and Golden; cashew-apple (Anacardium occidentale L.) supermarket lots of undefined variety; fig (Ficus carica L.) purple type; guava (Psidium guajava L.) white and red cultivar Ogawa; jabuticaba (Myrciaria jaboticaba Berg) supermarket lots of undefined variety; mango (Mangifera indica L.) cultivars Haden, Palmer and Tommy Atkins; oranges (Citrus sinensis) cultivars Bahia, Selecta, Lima and Peˆra; papaya (Carica papaya L.) cultivars Formosa, Golden and Solo; pitanga (Eugenia uniflora L.) supermarket and garden lots of undefined varieties; strawberry (Fragaria ananassa Duch.) cultivars Oso Grande, Sweet Charlie and Kamarossa (Table 1). 2.2. Processed fruits The processed products were chosen on the basis of the flavonoid levels found in the fresh fruits. The following products were analyzed: 1 brand of concentrated juice (brand A) and 2 brands of frozen pulp (brands B and C) of acerola; 3 brands of ready-to-drink juice (brands D–F), 3 brands of concentrated juice (brands A, D and G) and 3 brands of frozen pulp (brands B, C and H) of cashew-apple;
Table 1 Flavonol content of Brazilian fruits. Fruit
na
Concentration (mg/100 g fresh weight)b Myricetin
Quercetin
Kaempferol
Acerola (Malpighia glabra L. or Malpighia punicifolia L.) Garden lots (undefined variety) Cultivar Longa Vida Cultivar Olivier
6 5 3
nd nd nd
3.5–9.2 (5.0 0.1) 2.3–5.4 (4.1 1.1) 5.0–5.5 (5.3 0.2)
0.8–1.6 (1.2 0.3) 0.6–1.2 (0.9 0.3) 0.8–1.1 (1.0 0.1)
Apple (Malus domestica Borkh) Cultivar Fuji (red, with skin) Cultivar Golden Delicious (green, with skin) Cultivar Gala (red, with skin)
5 5 5
nd nd nd
6.3–8.6 (7.5 1.1) 2.8–4.2 (3.7 0.6) 4.0–8.0 (5.6 1.6)
Cashew-apple (Anacardium occidentale L.) Supermarket lots (undefined variety)
5
1.3–2.8 (2.0 0.5)
1.0–1.9 (1.3 0.4)
5
nd
0.8–1.9 (1.3 0.4)
nd
7 5
nd nd
0.8–1.1 (1.0 0.1) 1.0–1.2 (1.2 0.1)
nd nd
Jabuticaba (Myrciaria jaboticaba Berg) Supermarket lots (undefined variety)
8
nd
0.8–1.3 (1.1 0.2)
nd
Orange (Citrus sinensis) Cultivar Peˆra Cultivar Bahia Cultivar Lima Cultivar Selecta
5 5 5 4
nd nd nd nd
0.2–0.4 0.3–0.5 0.3–0.4 0.2–0.4
(0.3 0.1) (0.4 0.1) (0.3 0.1) (0.3 0.1)
nd nd nd nd
Pitanga (Eugenia uniflora L.) Garden lots (undefined variety) Supermarket lots (undefined variety)
3 4
3.3–4.2 (3.7 0.4) 2.7–3.6 (3.1 0.4)
5.6–7.3 (6.2 0.9) 5.1–6.7 (5.5 1.0)
0.4 (0.4 0.0) 0.3–0.6 (0.4 0.1)
Strawberry (Fragaria ananassa Duch.) Cultivar Kamarossa Cultivar Oso Grande Cultivar Sweet Charlie
3 5 2
nd nd nd
0.7–0.9 (0.8 0.1) 0.9–1.4 (1.1 0.2) 0.7–1.0 (0.9 0.2)
0.6–0.8 (0.7 0.1) 0.6–1.3 (0.9 0.2) 0.6–1.0 (0.8 0.3)
Fig (Ficus carica L.) Purple type Guava (Psidium guajava L.) Cultivar Ogawa (red) White type (white)
LQ, limit of quantitation, myricetin = 0.26 mg/100 g; quercetin = 0.23 mg/100 g, kaempferol = 0.26 mg/100 g; nd, not detected; LOD, limit of detection; myricetin = 0.086 mg/ 100 g, quercetin = 0.073 mg/100 g, kaempferol = 0.086 mg/100 g. a n = number of composite samples analyzed in duplicates. b Range (mean standard deviation).
R. Hoffmann-Ribani et al. / Journal of Food Composition and Analysis 22 (2009) 263–268 Table 2 Flavonol content of Brazilian processed fruits. Product
Concentrationa Quercetin
Kaempferol
Acerola Concentrated juice Brand A nd
Myricetin
1.0–1.8 (1.4 0.3)
0.4–0.5 (0.4 0.1)
Frozen pulp Brand B nd Brand C nd
2.1–2.9 (2.5 0.4)a 1.8–2.3 (2.1 0.2)b
0.7–1.0 (0.9 0.1)a 0.4–0.5 (0.4 0.0)b
Cashew-apple Ready-to-drink juice Brand D tr Brand E tr-0.2 Brand F tr-0.2
tr tr-0.2 tr-0.2
nd nd nd
Concentrated juice Brand A 0.3–0.4 (0.3 0.1)a Brand D 0.2–0.4 (0.3 0.1)a Brand G 0.2–0.3 (0.3 0.0)a
0.2 (0.2 0.0) tr-0.2 tr-0.2
nd nd nd
Frozen pulp Brand B 0.6–0.8 (0.7 0.1)a Brand C 0.2–0.4 (0.3 0.1)b Brand H 0.3–0.9 (0.5 0.3)a,b
0.4–0.5 (0.5 0.0)a 0.2 (0.2 0.0)b 0.2–0.6 (0.3 0.2)a,b
nd nd nd
Pitanga Concentrated juice Brand A 0.7–1.5 (1.2 0.4)a Brand I 1.6–2.0 (1.7 0.2)a
1.6–2.2 (2.0 0.3)a 2.2–2.8 (2.5 0.3)a
tr-0.2 tr-0.2
Frozen pulp Brand C 1.0–2.0 (1.6 0.5)
2.0–3.0 (2.5 0.5)
tr-0.2 (0.2 0.0)
Means of the same product (different brands) in a column with different letters are significantly different (P < 0.05). Products of the same brand letter were manufactured by the same company. nd, not detected; tr, trace; LOD, limit of detection, myricetin = 0.086 mg/100 g, quercetin = 0.073 mg/100 g, kaempferol = 0.086 mg/100 g. a Range (mean standard deviation), in mg/100 mL for the juice and mg/100 g for the pulp, of five sample lots analyzed individually in duplicates.
2 brands of concentrated juice (brands A and I) and 1 brand of frozen pulp (brand C) of pitanga (Table 2). 2.3. Chemicals The standards of myricetin, quercetin, kaempferol, luteolin and apigenin were purchased from Sigma Chemicals Co. (St. Louis, USA). Water was purified with a Milli-Q water purification system (Millipore, Bedford, USA). Methanol (HPLC-grade) was purchased from Mallinckrodt Baker Inc. (Phillipsburg, USA). Reagent grade formic acid and ascorbic acid were from Merck (Frankfurt, Germany) and Labsynth Ltd. (Diadema, Brazil), respectively. Stock standard solutions were prepared by dissolving the flavonoids in HPLC grade methanol to a concentration of approximately 500 mg/mL, stored at 18 8C and protected from light during storage and when in use. These solutions were found to be stable for over 2 months at 18 8C. To match the standard and sample solutions, the standard working solution was prepared as follows: standard stock solutions were diluted in 1.5 mL Milli-Q water to which 0.14% ascorbic acid was added. One mL of 6 M HCl was added and the solution was made up to 5 mL with methanol. 2.4. Sampling and sample preparation Samples were analyzed immediately after collection. Two to seven sample lots of ripe fresh fruits were collected for each cultivar of each fruit at different times from July 2004 to January 2005, harvest time for most of the fruits in Brazil, and analyzed individually in duplicate. For each lot, samples were purchased
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from three supermarkets (1 kg or a minimum of 3 units for big fruits per supermarket) in Campinas, Sa˜o Paulo. To obtain the composite sample submitted to analysis, small fruits (acerola, jabuticaba, strawberry and pitanga) taken from the three sampling sites were mixed, quartered to reduce the size of the sample from approximately 3 kg to 800 g and after removal of inedible portions (mainly seeds), homogenized first in a food processor with 0.135% (w/w) ascorbic acid as antioxidant, then in a Polytron homogenizer (Polytron System MR2100, Kinematica-AG, Luzern, Switzerland) at 25,000 rpm during 3 min The bigger fruits from the three sampling sites were cut longitudinally into quarters, opposite sections from each fruit were combined, and after removal of inedible parts (mainly seeds and peel for mango, orange and papaya), sliced (1– 2 cm) and homogenized as described for the small fruits. Fifteen grams of the composite sample were taken for analysis. For the processed fruits, five sample lots were analyzed individually in duplicate for each brand of each product. Each lot consisted of 3 units taken at random from the big supermarket lot. To obtain the composite sample, the contents of the 3 packages were mixed and 15 g of pulp or 15 mL of juice were taken for analysis. 2.5. Flavonoid extraction and hydrolysis The simultaneous extraction and hydrolysis procedure was based on that of Hertog et al. (1992b). However, instead of refluxing at 90 8C with 1.2 M HCL in 50% aqueous methanol for 2 h, the hydrolysis conditions (HCl concentration and refluxing time) were previously optimized for each fruit (Ribani and RodriguezAmaya, 2008). To the 15 g homogenized composite sample, 25 mL of methanol and 10 mL of HCl were added. The mixture thus formed consisted of different final molar concentration of HCl in 50% aqueous methanol HCl (v/v), with 0.04% of antioxidant. The optimum extraction/hydrolysis conditions were: acerola, 0.4 M HCl for 90 min; apple, 0.5 M HCl for 70 min; cashew-apple, 0.2 M HCl for 110 min; fig, 0.5 M HCl for 160 min; guava, 0.6 M HCl for 55 min; jaboticaba, 0.6 M HCl for 45 min; oranges, 1.5 M HCl for 90 min; pitanga, 0.6 M HCl for 40 min; strawberry, 1.2 M for 120 min. After refluxing, the extracts were cooled under running water and passed through a 130 mesh sieve. The volume was made up to 50 mL with methanol and an aliquot of about 5 mL was filtered through a 0.45 mm polytetrafluoroethylene (PTFE) filter (Millipore Ltd., Bedford, USA) prior to HPLC analysis. 2.6. HPLC analysis This analysis was performed on a Waters system, equipped with a Rheodyne injection valve with a 5 mL fixed loop, a quaternary pump (Waters model 600) and a UV–Vis photodiode array detector (Waters model 996), controlled by a Millenium workstation (version 32). A Symmetry C-18 (2.1 mm 150 mm, 3.5 mm) Waters column was used, the mobile phase consisting of methanol and water, both acidified with 0.3% formic acid, and the flow rate was 0.2 mL per min. A multilinear gradient was applied from 20:80 to 48:52 in 6 min, this proportion being maintained until 29 min, and then changed to 28:72 in 2 min, this proportion being maintained until 40 min. Finally, the mobile phase was bought back to the initial proportion of 20:80 in 3 min, maintaining this proportion until 60 min for column reequilibration before subsequent injection. Analytes were monitored from 200 to 600 nm and detection at 370 nm was used for quantification, which was done by external standardization. The full HPLC method, including the optimized extraction/ hydrolysis, was previously validated (Ribani and RodriguezAmaya, 2008). The limits of detection (LOD), calculated as the
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minimal concentration corresponding to 3.3 (SD/S), SD being the standard deviation of the blank and S the slope of the standard curve, were 0.086, 0.073, 0.086, 0.103 and 0.271 mg/100 g, respectively, for myricetin, quercetin, kaempferol, luteolin and apigenin. The calculated values were confirmed with actual analytical values under the chromatographic conditions established. The accuracy of the method was verified in recovery tests, using acerola samples spiked with 1.0 mg/100 g of flavonol standards and 1.7 mg/100 g of flavone standards, the test being done seven times for each flavonoid. The mean recoveries were 87, 95, 95, 96, and 95%, respectively, for myricetin, quercetin, kaempferol, luteolin and apigenin. The coefficients of variation showed good repeatability. The linearity of the HPLC method was checked for flavonols and flavones in the 0.03–4.8 mg/100 mL range (0.06–2.3, 0.03–4.8, 0.018–3.7, 0.11–2.6, 0.16–3.8 mg/100 mL for myricetin, quercetin, kaempferol, luteolin and apigenin, respectively). Calibration was performed by injecting the standard working solution in triplicate at five different concentrations for each flavonoid, based on the expected flavonoid content ranges in the samples. All standard curves passed through the origin, were linear in the concentration ranges expected in the samples, with coefficients of determination ranging from 0.9989 (for luteolin) to 0.9999 (for quercetin).
3. Results and discussion 3.1. Flavonol levels in fresh fruits The five flavonoids investigated were not detected in the mango cultivars Haden, Palmer and Tommy Atkins and in the papaya cultivars Formosa, Golden and Solo. Franke et al. (2004) did detect myricetin, quercetin and kaempferol in Hawaiian mango Haden, but at levels below the quantification limits of 0.04, 0.02 and 0.01 mg/100 g, respectively. These authors also detected in Hawaiian papaya myricetin and kaempferol, but not quercetin, at levels below the quantification limits. In a commercial frozen mango concentrate, Schieber et al. (2000) identified five quercetin glycosides and one kaempferol glycoside, the predominant flavonol glycosides quercetin 3-galactoside, quercetin 3-glucoside and quercetin 3-arabinoside amounting to 2.2, 1.6 and 0.5 mg/ 100 g, respectively. Thus, flavonols may be present in the Brazilian mangoes but at concentrations below our detection limits. Quercetin was found in all the other fruits, the mean level ranging from 0.3 to 7.5 mg/100 g, the highest concentration being found in apple cultivar Fuji (Table 1). Kaempferol was encountered in measurable amounts in strawberry, acerola and pitanga, ranging on the average from 0.4 to 1.2 mg/100 g, being highest in acerola. Myricetin was detected only in cashew-apple and pitanga at 2.0– 3.7 mg/100 g. The flavones luteolin and apigenin were not detected in all the fruits analyzed. This is in line with the USDA summary stating that fruits do not contain flavones, except for small amounts in lemons and pummelo juice (USDA, 2003). Franke et al. (2004) did find 0.8, 1.4, 2.6, 1.4–1.5, and 0.9 mg/100 g of luteolin in blueberry, ruby red grapefruit, red seedless grapes, two cultivars of oranges and black plum, respectively. Quercetin was the main flavonol in acerola. Its average concentration ranged from 4.1 to 5.3 mg/100 g, being lowest in the cultivar Longa Vida and highest in the cultivar Olivier. The mean concentration of kaempferol varied from 0.9 to 1.2 mg/100 g, being also lowest in ‘‘Longa Vida’’ and highest in the garden lots. As is well known, apple is a good to rich source of quercetin, depending on the cultivar. In the present study, the concentration of this flavonol varied from a mean of 3.7 mg/100 g in the cultivar Golden Delicious to 7.5 mg/100 g in the cultivar Fuji.
Unlike the other fruits, cashew-apple had myricetin (2.0 mg/ 100 g) as the major flavonol followed by quercetin (1.3 mg/100 g). Kaempferol ranged from below the LQ to 0.3 mg/100 g. The cashew-apple lots analyzed in the present study were purchased at local supermarkets and were mixtures of 3 types, elongated yellow, elongated red and round red, which botanically are not considered established cultivars. Only quercetin was found in fig, guava and jabuticaba with similar concentration ranges. The purple fig had an average of 1.3 mg/100 g quercetin. The red guava ‘‘Ogawa’’ had slightly lower quercetin concentration (1.0 mg/100 g) compared to the white type (1.2 mg/kg). Jaboticaba had 1.1 mg/100 g quercetin. Miean and Mohamed (2001) reported myricetin and apigenin contents of 55.0 and 57.9 mg/100 g dry weight, respectively, in guava; quercetin, kaempferol and luteolin were not detected. Quercetin was also the only flavonol detected in the four cultivars of oranges analyzed but at much lower levels (about 0.3 mg/100 g of pulp). This is not surprising since the flavanones hesperidin and narirutin are the predominant flavonoids in this fruit (Arabbi et al., 2004; Franke et al., 2004). Among the fruits investigated in the present study, pitanga had the highest myricetin content (3.1 and 3.7 mg/100 g, respectively, for the supermarket and garden lots). It also presented the second highest quercetin concentration (5.5 and 6.2 mg/100 g, respectively, for the supermarket and garden lots). This fruit also had kaempferol but at levels (0.4 mg/100 g for the supermarket and garden lots) lower than those of acerola and strawberry. In strawberry, the lowest quercetin and kaempferol values were observed in the cultivar ‘‘Kamarossa’’ (0.8 and 0.7 mg/100 g, respectively) and the highest in the cultivar ‘‘Oso Grande’’ (1.1 and 0.9 mg/100 g, respectively). 3.2. Flavonol levels in processed fruits Table 2 shows the flavonol levels encountered in processed fruits. While there are some between-sample and between-brand variations, the most important finding is that the processed products had flavonol contents considerably lower than those obtained for the fresh fruits, especially the cashew-apple products. Taking the frozen pulp, which is the product closest to the fresh fruit, the cashew-apple product had myricetin content about three to six times less than the fresh fruit; the quercetin content was about three to eight times less. For pitanga, the myricetin, quercetin and kaempferol levels were two to three times less in the frozen pulp compared to the fresh fruit. For acerola, the quercetin content of the frozen pulp was about 50% of the fresh fruit; the kaempferol level was also about 50% of that of the fresh fruit in brand C, but almost the same for brand B. Because the frozen pulps were blanched, the lower levels could be due to lower flavonol contents in the fruits used as raw material, removal of the peel and degradation during blanching. Enzymatic oxidation could also be involved if the raw material after pulping was held for some time before blanching and/or blanching was not sufficient to inactivate the oxidative enzymes. Surprisingly, for all three fruits, the flavonol concentrations of the frozen pulp were higher than those of the concentrated juice. The concentrated juice was expected to have higher levels because of the removal of water during the process. The low values indicate considerable degradation during the heat treatment applied to concentrate the juice, much more drastic than the pasteurization of the frozen pulp. 3.3. Comparison with published data Of the different fruits analyzed in this study, only apple, orange, strawberry and fig can be compared with previous data (Table 3).
R. Hoffmann-Ribani et al. / Journal of Food Composition and Analysis 22 (2009) 263–268 Table 3 Comparison of quercetin and kaempferol levels in apple, strawberries, orange and fig, reported in different studies. Fruit
Reference
n/cultivar or varietya
Concentration range (mg/100 g fresh weight) Quercetin
Kaempferol
5 1 2 4 1–2 18 1
2.8–8.6 0.4–10.1 2.5–8.0 2.1–3.8 2.1–7.2 2.0 0.4 2.6–7.4
nd—<0.26 nd <0.1 0.0 <0.2 nd nd
0.8–1.9 0.9 1.2
nd 0.0
Apple 3 cultivars 3 cultivars 2 cultivars 4 cultivars 6 cultivars Not specified 8 cultivars
Present study Arabbi et al. (2004) Franke et al. (2004) Harnly et al. (2006) Hertog et al. (1992a) Justesen et al. (1998) Price et al. (1999)
Fig 1 variety 1 variety
Present study Harnly et al. (2006)
5 8
Orange (pulp) 4 cultivars 2 cultivars 2 cultivars
Present study Arabbi et al. (2004) Franke et al. (2004)
4–5 1 3
0.2–0.5 0.8–0.9 0.4–0.7
nd nd <0.1
Present study Cordenunsi et al. (2005) Franke et al. (2004) Ha¨kkinen et al. (1999) Ha¨kkinen and To¨rro¨nen (2000) Harnly et al. (2006) Hertog et al. (1992a) Justesen et al. (1998) Kosar et al. (2004)
2–5 1 1 1 1
0.7–1.4 3.9–6.8 0.9 0.7 0.3–0.5
0.6–1.4 1.3–2.1 0.6 0.5–0.8 0.2–0.9
1.9 1.0 0.8–1.0 0.6 0.5 0.9 0.2
0.0 0.7–1.6 0.5 0.3 nd
Strawberry 3 cultivars 3 cultivars 1 variety 2 cultivars 6 cultivars 1 variety 1 variety Not specified 3 cultivars
7 3 4 1
nd, not detected. a Number of sample lots analyzed per cultivar or variety.
The other fruits were analyzed quantitatively in the present work for the first time. But even for the four fruits compared, considering the between-sample and between-cultivar compositional variations shown in Table 1, Table 3 indicates that more analyses are needed to obtain representative cultivar-specific data. The quercetin ranges in apple obtained in the present study compare very well with those of Franke et al. (2004), Hertog et al. (1992a) and Price et al. (1999). Those of Harnly et al. (2006) and Justesen et al. (1998) are on the lower side. The range of Arabbi et al. (2004) is much wider. Our quercetin range for orange is slightly lower than that of Franke et al. (2004) and distinctly lower than that of Arabbi et al. (2004). Our results with strawberry also agree with most of those reported in earlier studies (Hertog et al., 1992a; Justesen et al., 1998; Ha¨kkinen et al., 1999; Franke et al., 2004; Kosar et al., 2004), slightly higher than those of Ha¨kkinen and To¨rro¨nen (2000) and slightly lower than those of Harnly et al. (2006) for quercetin. Cordenunsi et al. (2005) obtained much higher levels. For fig, the mean quercetin value obtained by Harnly et al. falls within our range. Surprisingly, our results agree more with those obtained in other countries than with those of Arabbi et al. (2004) and Cordenunsi et al. (2005), who also analyzed Brazilian fruits. Apparently, analytical variability is involved, aside from natural variation. Arabbi et al. and Cordenunsi et al. did not hydrolyze the sample so the flavonoids were quantified as glycosides. However, quercetin (except for rutin) and kaempferol aglycones were used as external standards. It can be claimed that the distinctly higher results obtained by Arabbi et al. and Cardenunsi et al. are due to degradation of the flavonols during hydrolysis in the present study, although our method was optimized to avoid this degradation. However, Price
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et al. (1999) quantified quercetin as glycosides using the respective six glycoside standards and their results resemble those of the present work (Table 3). The other authors cited in the table did hydrolysis. In any case, these observations indicate that more work has to be done with the analytical methodology, including the formulation of certified reference materials and interlaboratory collaborative studies, as already done with carotenoids. Among the Brazilian fruits analyzed, the best sources of flavonoids are pitanga and cashew-apple, which contain the three flavonols investigated, acerola, having quercetin and kaempferol, and apple with high levels of quercetin. Pitanga and acerola, which used to be only garden fruits, are now being produced and processed commercially. Cashew-apple is a by-product of the cashew nut industry. References Aherne, S.A., O’Brien, N.M., 2002. Dietary flavonols: chemistry, food content, and metabolism. Nutrition 18, 75–81. 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