Comparison of antioxidant capacity and phytochemical properties of wild and cultivated red raspberries (Rubus idaeus L.)

Journal of Food Composition and Analysis 23 (2010) 540–544

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Original Article

Comparison of antioxidant capacity and phytochemical properties of wild and cultivated red raspberries (Rubus idaeus L.) ¨ zgen 1,* C¸etin C¸ekic¸ 1,*, Mustafa O Department of Horticulture, Faculty of Agriculture, University of Gaziosmanpas¸a, 60240 Tokat, Turkey

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 January 2009 Received in revised form 29 June 2009 Accepted 5 July 2009

We investigated some of the chemical properties and antioxidant capacities of 14 wild red raspberry accessions selected from northern Turkey. In addition, the cultivars Heritage and Tulameen were included in the study to determine the variation between wild and cultivated raspberries. Total phenolics (TP), total monomeric anthocyanins (TMA), soluble solids (TSS), individual organic acids and sugars in the fruit were examined. Antioxidant capacity of fruits was determined by both ferric reducing ability of plasma (FRAP) and trolox equivalent antioxidant capacity (TEAC) assays. The fruit color and weight were determined as well. The result of this study indicated that some of the wild accessions of red raspberries have higher antioxidant capacity and phytonutrient content than existing domesticated cultivars. Moreover, significant variability was found for antioxidant capacity, TP, TMA, organic acids and sugars of wild raspberries. Principle component analysis showed that the accessions were divided into three groups: A2, A9, A12, A14 formed the first group with high phytonutrient properties; the cultivars Heritage and Tulameen grouped together with high phytonutrients but low color values; and the rest of the accessions formed the final group. The antioxidant capacity among samples averaged 14.6 and 14.1 mmol TE/gfw using FRAP and TEAC methods, respectively. ß 2009 Elsevier Inc. All rights reserved.

Keywords: Red raspberry Rubus idaeus L. Cultivar difference Anthocyanin FRAP Organic acid Phenolic TEAC Biodiversity Food analysis Food composition

1. Introduction Several studies have demonstrated the correlation between consumption of fresh fruits and vegetables with the prevention, delay or onset of chronic degenerative diseases including cancer (Kaur and Kapoor, 2001; Steinmetz and Potter, 1996). As with other fruits, berries may have additional health benefits, as they are rich sources of micronutrients and phytochemicals such as anthocyanins, phenolics and ascorbic acids (Beattie et al., 2005). In vitro studies indicate that anthocyanins and other polyphenols in berries have a range of potential anti-cancer and heart disease properties including antioxidant, anti-inflammatory and cell regulatory effects (Juranic and Zizak, 2005; Zafra-Stone et al., 2007). Red raspberries (Rubus idaeus L.), in particular, are known to demonstrate strong antioxidant capacity, mainly as a result of their high levels of anthocyanins and other phenolic compounds (Kafkas et al., 2008; Ka¨hko¨nen et al., 2001). However, phenolic compounds and antioxidant capacities can be influenced, mainly

* Corresponding authors. Tel.: +90 356 2521616x2110; fax: +90 356 2521488. ¨ zgen). E-mail addresses: [email protected] (C¸. C¸ekic¸), [email protected] (M. O 1 Both the authors have equal contribution. 0889-1575/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2009.07.002

by genetics and environment (Anttonen and Karjalainen, 2005; ¨ zgen et al., 2008). Connor et al., 2005a,b; McGhie et al., 2002; O Furthermore, Connor et al. (2005a) indicated that having high bioactive compounds variability among the accessions is important to obtain superior cultivars, and selection programs are the important resources. As an important commercial fruit crop, red raspberries are widely grown in all temperate regions of the world and are consumed by local people. Many of the most important modern commercial red raspberry cultivars derive from hybrids or selections from the wild. Northern Turkey is one of the important germplasm centers of this species (Davis, 1982). They grow naturally in high elevations (above 1000 m) and require moist, rich soils. In some cases, the wild forms of plants may demonstrate different phytonutrient profiles and flavors from the cultivated ones. Several researchers have stated that most of the wild species preserve higher antioxidant capacity, richer flavor, and more attractive color, fragrance and aromatic compounds (Halvorsen ¨ zgen et al., 2007). et al., 2002; O In this study, we investigated the variation of antioxidant capacity of wild red raspberry accessions in comparison with representatives of the cultivated raspberries. We also aimed to identify wild red raspberry accessions with high antioxidant capacity for potential use in cultivar development.

C¸. C¸ekic¸, M. O¨zgen / Journal of Food Composition and Analysis 23 (2010) 540–544 Table 1 The altitude, latitude and longitude where the genotypes were collected. Genotypes A1 A2 A5 A8 A9 A10 A11 A12 A13 A14 A15 A17 A20 A22

Province Trabzon Trabzon Trabzon Trabzon Trabzon Rize Rize Giresun Ordu Ordu Ordu Giresun Giresun Trabzon

Altitude (m) 1505 1506 1732 1718 1123 1486 1584 1381 1495 1480 1730 1813 1681 1775

Latitude 408 408 408 408 408 408 408 408 408 408 408 408 408 408

0

50 510 390 390 360 560 560 340 410 400 390 410 410 510

2.4. Determination of total antioxidant activity

Longitude 00

99 0100 4100 4700 4700 4900 1400 7200 8100 5800 9300 3900 3300 3500

398 398 398 398 408 418 418 388 378 378 378 388 388 398

541

0

25 260 400 400 230 080 080 260 550 550 560 150 180 090

00

96 2700 9100 7300 7900 0300 2500 4100 4400 7600 3400 0600 7700 0800

2. Materials and methods 2.1. Plant material and extraction As result of a national bramble selection program in Turkey, 14 superior wild red raspberry (R. idaeus L.) accesions were selected from northern Turkey where they grow naturally in the mountain area between 1120 and 1810 m elevations (Table 1). Commercially grown Heritage and Tulameen red raspberry cultivars were also included in the experiment to compare and contrast antioxidant properties of wild and cultivated red raspberries. Three independent samples of fully mature fruits (each weighing about 200 g) were harvested by hand in three separate plastic containers and transferred to the laboratory for physical and phytochemical analysis. The fruit color was measured using a Minolta portable chromameter (Minolta, Model CR-400) which provided CIE L*, a* and b* values. Then, fruit samples were frozen and stored at 30 8C for chemical analysis. At the time of analysis, three replicates of fruit samples were thawed at room temperature and homogenized in a standard food blender; excess fruits (more than 50 individual fruits) were used to minimize naturally occurring fruit-to-fruit variation. Slurries were used to determine total soluble solid (TSS) content by refractometery (Atago, Pal-1). 2.2. Total phenolic (TP) determination For the extraction and determination of TP content, a single extraction procedure was designed to assay phenols (Singleton and Rossi, 1965). For each replicate, a 3 g aliquot of slurry was transferred to polypropylene tubes and extracted with 20 mL of extraction buffer containing acetone, water, and acetic acid (70:29.5:0.5, v/v/v) for 2 h. After filtration, acetone was removed by rotary evaporation, after which the concentrated samples were brought to a final volume of 20 mL with deionised water. Next, Folin–Ciocalteu’s phenol reagent and water were incubated for 8 min, followed by the addition of 7% sodium carbonate. After 2 h, the absorbance was measured by an automated UV–vis spectrophotometer at 750 nm. Gallic acid was used as standard. The results were expressed as mg gallic acid equivalent (GAE) in gram fresh weight (fw) basis. 2.3. Total monomeric anthocyanins (TMA) TMA were estimated by a pH differential method (Giusti and Wrolstad, 2005) using a UV–vis spectrophotometer (model T60U, PG Instruments). Absorbance was measured at 533 nm and 700 nm in buffers at pH 1.0 and 4.5 using A = (A533  A700)pH 1.0  (A533  A700)pH 4.5 with a molar extinction coefficient of 30,900. Results were expressed as mg of cyanidin-3-sophoroside equivalent in gram fw basis.

Total antioxidant activity was estimated by two standard procedures: ferric reducing ability of plasma (FRAP) and trolox equivalent antioxidant capacity (TEAC) assays. The FRAP assay (Benzie and Strain, 1996) was conducted using three aqueous stock solutions containing 0.1 mol/L acetate buffer (pH 3.6), 10 mmol/L TPTZ [2,4,6-tris(2-pyridyl)-1,3,5-triazine] acidified with concentrated hydrochloric acid, and 20 mmol/L ferric chloride. These solutions were prepared and stored in the dark under refrigeration. Stock solutions were combined (10:1:1, v/v/v) to form the FRAP reagent just prior to analysis. For each assay laboratory duplicate, 2.97 mL of FRAP reagent and 30 mL of sample extract were mixed. After 10 min, the absorbance of the reaction mixture was determined at 593 nm on a spectrophotometer. For the standard TEAC assay, ABTS was dissolved in acetate buffer and prepared with potassium persulfate as described in ¨ zgen et al. (2006). The mixture was Rice-Evans et al. (1995) and O diluted in an acidic medium of 20 mM sodium acetate buffer (pH 4.5) to an absorbance of 0.700  0.01 at 734 nm for longer stability ¨ zgen et al., 2006). For the spectrophotometric assay, 3 mL of the (O ABTS+ solution and 10 mL of fruit extract were mixed and incubated for 10 min and the absorbance was determined at 734 nm. Trolox was used as a standard for both assays. The results were expressed as mmol trolox equivalent (TE) in gram fw basis. 2.5. Extraction of individual sugars and organic acids For the extractions of individual sugars and organic acids, fruit slurries (5 g) were diluted with purified water or meta-phosphoric acid (2.5%) solution for individual sugar and organic acid analysis, respectively. The homogenate was centrifuged at 6000 rpm for 5 min. Supernatants were filtered through a 0.45-mm membrane filter (Iwaki Glass) before HPLC analysis, and the mobile phase solvents were degassed before use. All the samples and standards were injected three times each and mean values were used. The HPLC analyses were carried out using a PerkinElmer HPLC system with Totalchrom navigator 6.2.1 software, a pump and UV detector (PerkinElmer, Series-200) (Waltham, MA, USA). The method for separation and determination of organic acids was modified from Shui and Leong (2002). The separation was carried out on an SGE wakosil C18RS 5 mm column (250 mm  4.6 mm i.d.). Optimum efficiency of separation was obtained using pH 2.5 sulfuric acid solution (solvent A), and methanol (solvent B). Other parameters adopted were as follows: injection volume, 20 mL; column temperature, 30 8C; and detection wavelength, 215 nm. Analysis of sugars was performed using a refractive index (RI) detector (PerkinElmer) (Bartolome et al., 1995). The separation was carried out on an SGE SS Exsil amino column (250 mm  4.6 mm i.d.). The elution solvent used was 80% acetonitril and 20% deionised water. The column was operated at 30 8C with 0.9 mL/min flow rate. Sample injection volume was 20 mL. 2.6. Statistical analysis Data were analyzed using SAS procedures and software (SAS, 2006). Analysis of variance was constructed using the PROC GLM procedure. The means were separated using the least significant difference (LSD) method at 0.01 significance level. PC was performed using PRINCOMP procedure and the accessions were plotted on the first three PCs using G3G procedure. 3. Results and discussion Fruit pomological characteristics of selected red raspberries are presented in Table 2. On average, the fruit weight was 1.3 g. As

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Table 2 Several pomological characteristics of red raspberry accessions sampled from Turkey along with two cultivars. Accession and cultivar

Fruit weight (g)

Dry matter (%)

Brix (%)

L*

A1 A2 A5 A8 A9 A10 A11 A12 A13 A14 A15 A17 A20 A22 Heritage Tulameen

0.9 1.7 1.2 1.1 1.1 1.2 0.9 1.2 1.1 0.7 1.2 1.1 1.0 1.2 2.5 2.8

18.2 23.3 17.6 19.4 19.7 20.6 19.4 23.1 18.8 21.4 21.9 22.9 22.3 20.8 16.6 20.9

10.4 12.7 10.5 12.9 11.4 10.1 10.2 13.4 9.6 12.2 13.1 10.8 10.0 13.3 11.8 14.7

43.4 49.6 41.6 43.3 45.0 47.8 48.2 43.3 44.2 42.2 47.0 40.5 42.5 41.7 36.1 37.9

LSD0.01

0.43

d c cd cd d cd d cd d d cd d d cd b a

h a i f f e f a g d c a b e j e

0.68

gh d gh cd f hi hi b ij e bc g ij bc ef a

0.73

a* de a de de b–d ab ab de c–e de a–c ef de de g fg

3.37

35.8 40.2 40.3 42.8 41.5 38.0 34.5 36.4 32.2 41.3 40.9 38.2 40.2 40.7 34.4 33.4

b* c–e a–c a–c a ab a–d de b–e e ab a–c a–d a–c a–c de de

4.48

17.0 18.8 20.4 21.0 23.7 17.3 15.7 17.5 15.7 24.1 22.0 19.4 21.8 21.0 16.9 16.5

e–g c–g a–f a–e ab d–g f–g c–g g a a–c b–g a–d a–e e–g e–g

4.03

LSD0.01: Least significant differences at 0.01 level.

expected, the weight of cultivated berries was much larger than wild accessions. A2 had the biggest berries among the wild accessions, with 1.7 g compared to 2.5 and 2.8 g for Heritage and Tulameen, respectively. Fruit dry matter content ranged from 16.6 to 23.3%. The average TSS content of berries was 11.7%. In terms of fruit color, the variation in b* was higher than those of L* and a*. In the chromaticity coordinates, where high b* indicates toward yellow direction and high a* indicates toward red direction, wild accessions looked darker red and towards to yellowish color. On the other hand, the appearance of cultivated red raspberries was a blue-based lighter red color. The average a* and b* values were 38.2 and 19.3, respectively. L* values represent lightness and wild accessions had higher L* values. There were significant differences among the accessions for TP, TMA and antioxidant capacity (Table 3). TP ranged from 1486 (A20) to 3479 (A2), with an average of 2046 mg GAE/g fw. We compared TP values, and except for A2, the other accessions and cultivars Table 3 Variation of total phenolics (TP), total monomeric anthocyanin (TMA), antioxidant capacity (FRAP and TEAC) of red raspberry accessions sampled from Turkey along with two cultivars. Accession and cultivar

TPa

A1 A2 A5 A8 A9 A10 A11 A12 A13 A14 A15 A17 A20 A22 Heritage Tulameen

2040 3479 1933 2120 2447 1664 2125 2084 1947 2386 1684 1625 1486 1586 1795 2342

LSD0.01

TMAb de a d–f cd b gh cd cd de b f–h gh gh gh e–g bc

243.17

258.0 199.1 216.1 214.0 296.2 137.5 179.1 237.0 214.6 239.5 197.6 188.8 65.6 200.6 239.5 195.9

b de cd cd a f e bc cd bc de de g de bc de

27.41

FRAPc

TEACd

13.9 19.8 11.5 11.7 17.9 11.2 12.0 15.2 16.0 19.7 13.2 13.2 14.4 11.4 18.1 14.4

13.5 21.5 13.3 11.6 17.0 11.3 12.9 17.5 16.2 19.3 10.6 11.1 8.9 11.1 17.5 12.9

de a fg fg b g fg cd c a ef ef c–e fg ab c–e

1.69

d a de d–f c d–f d–f bc c b fg e–g g e–g bc d–f

2.11

LSD0.01: Least significant differences at 0.01 level. a TP contents were estimated by the Folin–Ciocalteu assay of Singleton and Rossi (1965). Values are expressed as mg GAE/g fw. b TMA were determined by the pH-differential method of Giusti and Wrolstad (2005). Values are expressed as mg cy-3-soph/g fw. c FRAP values were determined by the method of Benzie and Strain (1996). Values are expressed as mmol of TE/g fw. d ¨ zgen et al. (2006). Values are TEAC values were determined by the method of O expressed as mmol TE/g fw.

displayed similar results (Khanizadeh et al., 2009). The result for A2 seems to indicate a rich source of phenolics. The average TMA content was 204.9 mg cy-3-soph/g fw, and A9 had the highest anthocyanin content with 296.2 mg cy-3-soph/g fw. A2 displayed the highest antioxidant capacity for FRAP and TEAC methods, resulting in 19.8 and 21.5 mmol TE/g fw, respectively. Among all the raspberries tested, A20 had the lowest TP and TMA content with the lowest antioxidant capacity determined by TEAC. ¨ zgen et al., 2008) Previous studies (e.g. Connor et al., 2005a,b; O reported strong relationships among measures of TP content, anthocyanin levels, and antioxidant capacity of Rubus species. From our data we calculated high correlation coefficients (r) between the TP values of red raspberry samples and their respective antioxidant capacities. Correlation coefficients between TEAC and TP, TMA, FRAP was r = 0.74, 0.73 and 0.81, respectively. When PC analysis was considered, the first three PC explained 30, 25 and 16% of the variation, making a total of 71% (Table 4). Among the variables tested, the phytonutrient-related parameters (TP, TMA, FRAP and TEAC) were highly correlated with PC1. The highly correlated variables with PCs were color measurements (L*, a*, b*) and fruit weight and dry matter content. Interestingly, while dry weight was positively correlated with PC2, fruit weight was negatively correlated. The highest correlation with PC3 was calculated from TSS content. Based on the PC analysis, the genotypes tested can be divided into three groups, as follows: A2, A9, A12, A14 formed the first group with high antioxidant capacity, TP, TMA content and darker red color (Fig. 1). The cultivars Heritage and Tulameen grouped together and had high phytonutrient content, lighter red color but larger fruit size. Table 4 First three principle component (PC) scores of the variables used to evaluate raspberry accessions sampled from Turkey along with two cultivars. Variable

PC1

PC2

PC3

Fruit weight Dry weight Brix L a b Total phenolics Total anthocyanin FRAP TEAC

0.22 0.03 0.26 0.02 0.03 0.02 0.48 0.37 0.48 0.53

0.40 0.36 0.03 0.35 0.55 0.51 0.14 0.06 0.07 0.01

0.39 0.16 0.59 0.49 0.24 0.30 0.17 0.02 0.08 0.22

3.03 0.30

2.51 0.25

1.60 0.16

Eigenvalue Proportion

[(Fig._1)TD$IG]

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543

fruits, particularly berries. The result of our study indicates that some of the wild accessions of red raspberries have higher antioxidant capacity and phytonutrient content than existing domesticated cultivars. Moreover, significant variability was found for the antioxidant capacity, TP, TMA, organic acids and sugars of wild raspberries. These findings may help and guide fruit breeders to develop new cultivars with high antioxidant capacity using wild types as a resource to help meet recent consumer trends. Acknowledgements This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No. TOVAG107O209); we greatly acknowledge the financial support. We would also like to thank all the project personnel for guiding us and collecting these valuable materials. References Fig. 1. Plot of red raspberry accessions sampled from Turkey along with two cultivars on the first three principle components (PC).

Table 5 Variation of individual sugars and organic acids of red raspberry accessions and cultivars. Accession/ cultivar

A2 A9 A10 A12 A14 A17 A20 A22 Heritage Tulameen

Sugars g/kg fresh wt

Organic acids g/kg fresh wt

Fructose

Glucose

Sucrose

Citric

Malic

30.9 27.3 30.8 32.0 33.9 31.4 30.4 33.3 35.1 36.8

25.1 22.5 22.3 25.7 23.8 24.4 22.1 24.4 24.3 28.3

9.0 8.0 7.9 10.0 9.2 7.5 6.7 10.6 10.2 12.2

18.2 15.8 13.6 12.4 11.4 13.9 13.7 12.3 10.5 9.6

0.81 0.63 1.14 0.73 1.15 0.71 0.73 0.51 1.16 0.91

LSD0.01

e f e de bc e e cd b a

2.25

bc cd d b b–d b–d d b–d b–d a

3.60

c d d b c de e b b a

1.11

a b c d e c c d f g

0.77

d e b e ab e e f a c

0.10

LSD0.01: Least significant differences at 0.01 level.

The rest of the accessions were in group III. The phytonutrient properties of these accessions were lower than either of the previous groups. The amounts of fructose, glucose and sucrose in the raspberries are given in Table 5. Fructose and glucose were found to be the predominant sugars in all samples analyzed. The fructose, glucose and sucrose concentrations averaged 32.2, 24.3 and 9.1 g/kg fw, respectively. The organic acid distribution of red raspberry was dominated by citric acid (mean value, 13.1 g/kg fw) (Table 5). Small amounts of malic and ascorbic acids (data not shown) were also detected. The citric acid content in raspberry fruit is about 10–12 times higher than malic acid. Cultivated raspberries were found to have less acidic characteristics. When the wild and cultivated raspberries were compared with pomologically, the most apparent difference appeared to be in fruit weight and color. Cultivated red raspberries were relatively lighter red in color and with larger fruits. Based on our findings, some of the wild accessions with high antioxidant capacity and better pomological characteristics such as A2, A9, A12 and A14 may be used in future breeding programs. 4. Conclusions Consumer interest in the relationship between diet and health has increased the demand for consumption of antioxidant-rich

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