Influence of shading net on polyphenol profile and radical scavenging activity in different varieties of black currant berries

Scientia Horticulturae 160 (2013) 20–28

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Influence of shading net on polyphenol profile and radical scavenging activity in different varieties of black currant berries a ˇ Katarina Savikin , Maja Mikuliˇc-Petkovˇsek b , Boban Djordjevic´ c , Gordana Zdunic´ a,∗ , a Teodora Jankovic´ , Dejan Djurovic´ c , Robert Veberiˇc b a

Institute for Medicinal Plants Research “Dr Josif Panˇci´c”, Tadeuˇsa Koˇs´cuˇska 1, 11000 Belgrade, Serbia University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, 1000 Ljubljana, Slovenia c University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia b

a r t i c l e

i n f o

Article history: Received 4 March 2013 Received in revised form 10 May 2013 Accepted 13 May 2013 Keywords: Black currants Anthocyanins Flavonols Radical scavenging activity HPLC/MS Central Serbia

a b s t r a c t Changes of environmental factors, created under influence of various shading nets, could significantly affect some pomological and organoleptic characteristics of plants grown in such conditions as well as biosynthesis of phenolic compounds. Five black currant cultivars Ben Sarek, Ben Nevis, Ben Lomond, ˇ canska crna were cultivated either in the shade of green polyethylene nets or exposed to Ometa, and Caˇ direct sunlight during two experimental seasons. All berries in the control treatment (without shading nets) contained higher amounts of total phenolics in both experimental seasons: 474–520 mg GAE/100 g in 2010, and in 2011 it ranged from 401 to 501 mg GAE/100 g. Similar but less expressed trend was noticed regarding anthocyanins content. Growing in shade generally led to a lower content of flavonoid, phenolic acid and anthocyanin compounds, determined by HPLC/MS. Despite the reduction of phenolic compounds content, all tested varieties expressed significant radical scavenging activity against DPPH radical, and differences in antiradical activity were statistically significant only between varieties. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The area of conventionally or organically grown black currants (Ribes nigrum L.) has rapidly increased in Europe during the last two decades (Häkkinen et al., 1999a). In the group of soft fruit, currant production is in the second place in the world, behind strawberries. The impact of environmental factors on the flavonoid content and composition of berries has been studied (Anttonen and Karjalainen, 2005; Lugasi et al., 2011). Usage of different types of shade nets and anti-hail nets in some fruits production is getting wider (Hoppula and Karhu, 2006; Jakopic et al., 2009; Chorti et al., 2010). Shading is a culture practice which is often used to alleviate the excessive sunlight exposure of overhead plants (Liu and Liu, 2012). Shading can also decrease water evaporation from plants and soil, and decrease the environmental temperature around the plants, especially in the summer. The benefits of the utilization of nets include extending the harvest season (early and late maturation), and improving the yield, product quality and the overall agro-economical performance of agricultural crops (Shahak et al., 2004a). Plants in such environment are exposed to different growing conditions than plants growing without nets, particularly with

∗ Corresponding author. Tel.: +381 113031653; fax: +381 113031655. ´ E-mail address: [email protected] (G. Zdunic). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.05.007

regard to the intensity of sunlight. Effect of light on plant development is complex, involving the combined effects of several photo receptor systems (Oren-Shamir et al., 2001). Photo-selective nets (shading nets or colored shading nets) have been used with various perennial crops to improve fruit yield and quality, particularly in high irradiance environments (Shahak et al., 2004b). The use of photo-selective nets has been shown to have positive effects on fruit yield (in relation to full sun conditions) in some varieties of Vaccinium corymbosum L., depending on the color and shade percentage used (Retamales et al., 2008; Lobos et al., 2009). Light is also one of the most extensively studied environmental factor in the phenolic metabolism (Macheix et al., 1990). The light spectrum and the appropriate amount of energy are required for initiate synthesis of phenols (Ubi, 2004). In general, solar radiation is known to stimulate the enzymes phenylalanine ammonium lyase (PAL) and chalcone synthase (CHS) and other branch-point enzymes of the phenyl propanoid pathway. PAL catalyses the transformation of phenylalanine to trans-cinnamic acid, which leads to the formation of complex phenolic compounds such as flavonoids, tannins and lignin (Dixon and Paiva, 1995). Ilic´ et al. (2012) showed that changes of light intensity created under influence of various shading nets during cultivatin of tomato significantly affected the biosynthesis of phenolic compounds in tomato. In the recent years, fruits rich in phenolic compounds become more and more important (Määttä et al., 2004; Kondakova et al., 2009). Phenolic compounds, particularly anthocyanins and

K. Sˇ avikin et al. / Scientia Horticulturae 160 (2013) 20–28

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flavonols, manifest different biological activities that favorably affect the health of human body (Mazza, 2007; Pereira et al., 2009). Berries are important source of phenolic compounds and among them black currants showed very strong biological activity, such as antioxidant, anti-inflammatory, antimicrobial and anti-cancer, due to the presence of large quantities of these compounds (Beattie et al., 2005; Tabart et al., 2006; Mazza, 2007). Contents of flavonols in berry fruits vary and in black currant berries it may constitute over 30% of total phenolic compounds. The main black currant flavonols are kaempferol, quercetin and myricetin (Häkkinen et al., 1999a). The dark color of black currant berries is the result of high levels of anthocyanins in the epider´ mal cells (Krisch et al., 2009; Oszmianski and Wojdyło, 2009). The portion of anthocyanins in total phenols of black currant varieties can range from 60 to 85% (Lugasi et al., 2011). Many studies have shown that significant antioxidant activity of berries and their products are based also on a very high content of ascorbic acid (Moyer et al., 2002). Ascorbic acid exhibits a number of biological activities. It has been shown that it affects reducing damage to the DNA chains, cell walls and the appearance of cataracts (Benvenuti et al., 2004). Due to the great commercial, nutritional and medicinal values of black currants and also the lack of the data about the effects of shading nets in currants cultivation, the aim of our study was to determine the influence of changed environmental conditions caused by use of green shading nets on the biosynthesis of phenolic compounds and radical scavenging activity of different black currant varieties over two successive seasons. Thus, the results could support our hypothesis that although berries are cultivated in shade they still remain a valuable source of bioactive compounds.

network had been set above the plants, environmental measurements were performed every day at 7, 12 and 17 h. Measurements included the measurement of air temperature and the surface temperature of berries and leaves, relative humidity and light intensity. The air temperature and relative humidity were measured at height of 2 m using automatic weather stations MeteosCompact, PESSL INSTRUMENTS GmbH, Austria set in the immediate vicinity of the planting. Surface temperature of leaves and berries were measured with infrared thermometer DT-8188, PCE – GmbH, Germany (measurement range −50 to 550 ◦ C, accuracy ±1.5%; resolution between 0.1–50 and +200 ◦ C), and light intensity was measured with lux meter PRO-LX 1108, Electricals Electronics Enterprises, India (ranges 0.1–400,000 lx, accuracy ±3%, resolution 0.01–100 lx).

2. Material and methods

Total anthocyanin content was investigated according to the procedure described in European Pharmacopoeia 6.0, with slight modifications. Briefly, fresh berries (50 g) were crushed extemporaneously. To about 10 g of crushed, accurately weighed berries, 95 ml of methanol were added and mechanically stirred for 30 min then filtered into a 100 ml volumetric flask. Filter was rinsed and filtrate was diluted to 100 ml with methanol. A 50-fold dilution of this solution in a 0.1% (v/v) solution of hydrochloric acid in methanol was prepared. The absorbance of the solution was measured at 528 nm, using a 0.1% (v/v) solution of hydrochloric acid in methanol as the compensation liquid. The percentage content of anthocyanins, expressed as cyanidin-3-glucoside chloride, was calculated from the expression: A × 5000/718 × m (A = absorbance at 528 nm; 718 = specific absorbance of cyanidin-3-glucoside chloride at 528 nm; m = mass of the substance to be examined in grams).

2.1. Experimental conditions Five black currant cultivars were analyzed: Ben Sarek (Scotland), Ben Nevis (Scotland), Ben Lomond (Scotland), Ometa (Switzerland), ˇ canska crna (Serbia). Experimental fields were conducted in and Caˇ – (Serbia), situated between 44◦ 30 and 44◦ 45 N latvillage Mislodin itude and 20◦ 00 and 20◦ 20 E longitude, altitude between 80 and – is located almost in the center of the northern warm 90 m. Mislodin temperate belt, with a milder climate than the typical Pannonia, continental. The average annual temperature in this area is ca. 11◦ , and during the year, the amount of rainfall is 640 l/m2 . The plantation was established on sandy loam soil, with an average aq. pH of 6.3. Planting was done with one-year nursery trees at a distance of 1.8 m between rows, and 0.8 m in the row, what resulted in ca. 6950 plants/ha. Two factorial experiments (cultivar × treatment) were set up in random field and samples were taken from five bushes. Current shrubs were grown either in the shade of green polyethylene nets which, according to the manufacturer’s specifications (Sigma Promet, Beˇcej) retain 30% of light (shade treatment) or without the use of shade nets exposed to direct sunlight (control treatment). Polyethylene network was constructed from UV stabilized materials (300Kly). Network was set up across the shrubs of the shade treatment at a height of 2 m in early June and then removed after harvest. Berries were hand-harvested in the fourth and fifth years of vegetation in June and July 2010–2011, depending on the commercial ripening time for each cultivar (90% colored fruit). Berries were collected from five bushes (300 g per bush) of each cultivar. All of five samples from each cultivar were consolidated, thus representing average sample for further analysis. After harvesting, the berries were stored at +5 ◦ C and analyzed within 24 h. The shading nets were placed from middle of May till middle of June depending of early stage of veraison for each cultivar. After the

2.2. Determination of total phenolic content The total concentration of phenols was estimated by FolinCiocalteu method with slight modifications (Waterman and Mole, 1994). The berries (10 g) were extracted with methanol for 30 min on the ultrasonic bath and then filtered through filter paper. Two hundred microliters of extracts were added to 1 ml of 1:10 diluted Folin-Ciocalteu reagent. After 4 min, 800 ␮l of sodium carbonate (75 g/l) were added. After 2 h of incubation at room temperature, the absorbance at 765 nm was measured. Gallic acid (0–100 mg/l) was used for calibration of a standard curve. The results were expressed as milligrams of gallic acid equivalents per 100 g of fresh weight (mg GAE/100 g FW). Triplicate measurements were taken and mean values were calculated. 2.3. Total anthocyanins content

2.4. HPLC/MS analysis Extraction of berries was performed as described by MikulicPetkovsek et al. (2012), with slight modification. Frozen berry samples were ground to a fine paste in a mortar chilled with liquid nitrogen and 5 g were extracted with 10 ml methanol containing 3% (v/v) formic acid and 1% (w/v) 2,6-di-tert-butyl-4-methylphenol (BHT) in a cooled ultrasonic bath for 1 h. BHT was added to the samples to prevent oxidation. After extraction, the berries extracts were centrifuged for 10 min at 10,000 rpm. Each supernatant was filtered through a Chromafil AO-20/25 polyamide filter produced by Macherey-Nagel and transferred to a vial prior to injection into the HPLC system. Phenolic compounds were analyzed on a Thermo Finnigan Surveyor HPLC system (Thermo Scientific, San Jose, USA) with a diode array detector at 280 nm (flavanols and hydroxycinnamic), 350 nm (flavonols) and 530 nm (anthocyanins). Spectra of the compounds were recorded between 200 and 600 nm. The column was a Gemini

K. Sˇ avikin et al. / Scientia Horticulturae 160 (2013) 20–28

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2010

60

10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0

40 7h

30

12 h

20

17 h 10 0 control

shading

air temperature

control

shading

leaf temperature

control

100 90 80 70 60 50 40 30 20 10 0

Light intensity (lx)

Temperature C

50

7h humidity control

shading

berry temperature

12 h

light intensity control

17 h humidity shading light intensity shading

2011 60

40 7h

30

12 h

20

17 h 10 0

Light intensity (lx)

Temperature C

50

100 90 80 70 60 50 40 30 20 10 0

10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 7h

control

shading

air temperature

control

shading

leaf temperature

control

shading

berry temperature

12 h

17 h

humidity control

humidity shading

light intensity control

light intensity shading

Fig. 1. Temperature of air, leaves and berries surface, relative humidity and light intensity during the first (2010) and the second (2011) experimental season.

C18 (150 mm × 4.6 mm 3 ␮m; Phenomenex, Torrance, USA) operated at 25 ◦ C. The elution solvents were aqueous 1% formic acid (A) and 100% acetonitrile (B). Samples were eluted according to the linear gradient: 0–5 min, 3–9% B; 5–15 min, 9–16% B; 15–45 min, 16–50% B; 45–50 min, 50% isocratic (Marks et al., 2007); and finally washing and reconditioning of the column. The injection amount was 20 ␮l and flow rate 1 ml min−1 . All phenolic compounds were identified using a mass spectrometer (Thermo Scientific, LCQ Deca XP MAX) with an electrospray ionization (ESI) operating in negative (all phenolic groups except for anthocyanins) and positive (for anthocyanins) ion mode. The analyses were carried out using full scan data-dependent MSn scanning from m/z 115 to 1500. The injection volume was 10 ␮l and the flow rate maintained at 1 ml min−1 . The capillary temperature was 250 ◦ C, the sheath gas and auxiliary gas were 20 and 8 units, respectively; and the source voltage was 4 kV for negative ionization and 0.1 kV for positive ionization. Spectral data were elaborated using the Excalibur software (Thermo Scientific). The identification of compounds was confirmed by comparing retention times and their spectra as well as by adding the standard solution to the sample and by fragmentation. Concentrations of phenolic compounds were calculated from peak areas of the sample and the corresponding standards and expressed in mg kg−1 fresh weight (FW) of berries. For compounds lacking standards, quantification was carried out using similar compounds as standards. Thus, quercetin 3-malonylglucoside was quantified in equivalents of quercetin 3-galactoside, kaempferol 3-galactoside and kaempferol 3-rutinoside in equivalents of kaempferol, all myricetin glycosides in equivalents of myricetin, caffeoylhexose in equivalents of caffeic acid, p-coumaroylhexose in equivalents of p-coumaric acid, all cyanidin glycosides, petunidin3-rutinoside and peonidin-3-rutinoside in equivalents of cyanidin 3-glucoside and delphinidin glycosides were quantified in equivalents of delphinidin.

2.5. DPPH radical scavenging activity The free radical scavenging activity of berries on the stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical was carried out according to the procedure described previously (Silva et al., 2005), with slight modifications. The antiradical capacity of each extract was evaluated using a dilutions series, in order to obtain a large spectrum of sample concentrations. The extracts (100 ␮l) were mixed with 1400 ␮l of 80 ␮M methanolic solution of DPPH. The antiradical capacity of each sample was evaluated using a dilutions series, in order to obtain a large spectrum of sample concentrations. Extracts (100 ␮l) were mixed with 1400 ␮l of 80 ␮M methanolic solution of DPPH. Absorbance at 517 nm was measured after 20 min. The percentage of inhibition was calculated using equation:

Inhibition =

A0 − Ai × 100, A0

where A0 is absorbance of the control and Ai is absorbance of the samples. IC50 values were estimated using a nonlinear regression algorithm. All test analyses were run in triplicate. Trolox was used as a positive control. 2.6. Statistical analysis All data were reported as mean ± standard error of triplicate determinations. Since all observed parameters analyzed by twoway analysis of variance (ANOVA) showed significant interactions between treatments (cultivar × shading), null hypothesis of equal means for each treatment separately has been analyzed using oneway ANOVA with post hoc Fisher’s least significant difference test (LSD) as a method for comparing treatment group means at P < 0.05 level. Critical values for pairwise comparisons of the means are declared in tables for each treatment. Radical scavenging activity

K. Sˇ avikin et al. / Scientia Horticulturae 160 (2013) 20–28

2.92 0.002 0.036 4.62 0.003 0.056 501.5 ± 5.4 0.29 ± 0.003 1.13 ± 0.07 378.4 ± 2.2 0.28 ± 0.001 1.14 ± 0.03 401.2 ± 5.6 0.16 ± 0.002 1.22 ± 0.07 376.6 ± 2.1 0.15 ± 0.001 1.31 ± 0.04 457.3 ± 3.6 0.21 ± 0.003 1.21 ± 0.02 Data are presented as mean ± SD (n = 3); RSA – radical scavenging activity. a Critical values for pairwise comparisons of the means at P < 0.05 level (LSD0.05 ).

453.2 ± 4.4 0.21 ± 0.003 1.10 ± 0.05 437.4 ± 6.2 0.20 ± 0.001 1.11 ± 0.04 336.0 ± 1.1 0.17 ± 0.001 1.25 ± 0.05 443.0 ± 2.5 0.21 ± 0.004 1.11 ± 0.04 2011 Total phenolics Total anthocyanins DPPH RSA

459.6 ± 1.2 0.29 ± 0.002 1.17 ± 0.03

3.03 0.0018 0.029 5.14 0.0027 0.051 520.1 ± 3.5 0.26 ± 0.002 0.92 ± 0.05 410.6 ± 3.6 0.27 ± 0.001 0.93 ± 0.04 474.2 ± 2.3 0.23 ± 0.002 1.10 ± 0.03 381.1 ± 1.8 0.17 ± 0.001 1.18 ± 0.04 504.8 ± 2.5 0.18 ± 0.002 0.81 ± 0.02 495.6 ± 4.8 0.19 ± 0.001 0.88 ± 0.01 482.5 ± 6.0 0.20 ± 0.002 0.88 ± 0.04 415.5 ± 3.7 0.19 ± 0.001 0.96 ± 0.01 478.8 ± 7.6 0.18 ± 0.001 0.95 ± 0.03

Shade Shade Control Shade

Control

Shade

Control

Shade

Control

Ometa Ben Sarek Ben Lomond Ben Nevis

Total phenolic content in black currant fruit varieties is shown in Table 1. In the first experimental season (2010) in berries cultivated without shading, the highest content of total phenolics was detected in variety Ometa (520.1 mg GAE/100 g), while variety Ben Sarek contained the lowest amount (474.2 mg GAE/100 g). All varieties in the shaded treatment contained lower amounts of total phenolics when compared to the fully exposed ones. In the second experimental season (2011), total phenolic content in the control berries ranged from 401.2 (Ben Sarek) to 501.5 mg GAE/100 g (Ometa). Like in year 2010, berries grown in the shade had lower total phenol content than control non-shaded berries. Significant influence of the network on total phenolic content was recorded in all varieties, and the highest decrease of total phenolic content for about 25% was noticed in varieties Ben Nevis and Ometa, and the lowest in Ben Lomond. The effect of shading on the phenolic content in berries was extensively studied especially in grape berries (Spayd et al., 2002; Downey et al., 2004; Cortell and Kennedy, 2006). It has been shown that the amount of phenolics was higher for over 50% in berries with greater light exposure. On the other hand, shading did not significantly affected total phenolics in strawberry fruit (Anttonen et al., 2006). The diversity of obtained results might be due to the difference of plant adaptability to shading. When plants are cultivated in such environmental conditions that differs from their native habitat, their ability to grow and develop will mainly depend on their capacity to acclimatize at the level of photosynthesis (Pastenes et al., 2003). Grapes prefers full sun and shading largely reduces their capacity for polyphenol synthesis, whereas black currants can grows in semi-shade or no shade sites and reduction of polyphenol contents in shaded treatment is less pronounced. Three main flavonols found in black currants, quercetin, myricetin and kaempferol, tend to occur naturally as glycosides (Häkkinen et al., 1999a; Borges et al., 2010). Sugar substitution on flavonols usually appears as the O-glycosides at the 3-position. Flavonoids in investigated five black currant cultivars were determined by HPLC/MS method, and the results are summarized in Table 2. In all varieties, quercetin glycosides were the predominant flavonols, followed by myricetin glycosides. Kaempferol derivatives were present in much smaller amounts. Quercetin 3-rutinoside, myricetin 3-rutinoside, myricetin 3-glucoside and quercetin 3glucoside were the major flavonols detected in both experimental seasons, and quercetin-3-rutinoside was the main compound in

ˇ canska crna Caˇ

3.2. Total phenolics and flavonol composition

Table 1 Total phenolic (mg GAE/100 g), total anthocyanin (%) and DPPH radical scavening activity (IC50 mg/ml) of black currant varieties in both experimental seasons (2010 and 2011).

Temperature of the air, leaf and berries, as well as light intensity and relative humidity were measured in both experimental seasons (Fig. 1). Although some difference in measured parameters were noticed they were not statistically significant, except for light intensity (P < 0.05). The highest temperature of leaves and berries in both experimental seasons were recorded in the third measurement point (17 h) in control as well as in the shading treatment, while relative humidity was the lowest at 12 h. Average light intensity was 35–65% higher in shrubs exposed to the direct sunlight.

Control

3.1. Environmental measurements

456.9 ± 4.7 0.14 ± 0.001 0.89 ± 0.03

Shade Cultivar

3. Results and discussion

2010 Total phenolics Total anthocyanins DPPH RSA

Critical valuesa

data were presented in original values in table, but statistical test was performed on its reciprocal data since IC50 value is inversely proportional to antiradical activity. Degree of linear dependence between metabolites was calculated by Pearson’s correlation (r), and significance of correlation was performed using SPSS 11.5.

23

24

Table 2 Content of flavonoids (mg/kg) in black currant varieties in both experimental seasons (2010 and 2011). ˇ canska crna Caˇ Shade

Ben Nevis Control

Shade

Ben Lomond Control

Shade

Ben Sarek Control

Shade

Critical valuesd

Ometa Control

Shade

Control

9.3 0.2 7.8 1.8 17.4 0.7 10.8 3.0 2.7 4.0 29.5

± ± ± ± ± ± ± ± ± ± ±

0.5 0.01 0.5 0.1 1.2 0.06 1.2 0.2 0.3 0.3 2.3

21.0 1.2 19.3 5.5 36.0 2.1 18.5 7.6 5.7 6.5 51.1

± ± ± ± ± ± ± ± ± ± ±

1.7 0.1 1.8 0.3 1.7 0.08 1.9 0.8 0.6 0.8 4.9

12.4 3.6 7.9 2.0 15.4 3.2 7.6 3.2 7.5 6.4 38.5

± ± ± ± ± ± ± ± ± ± ±

1.2 0.3 0.4 0.1 1.6 0.2 0.4 0.2 0.3 0.7 3.4

17.4 4.6 12.0 2.2 18.6 5.3 9.6 3.7 8.3 8.2 41.5

± ± ± ± ± ± ± ± ± ± ±

1.4 0.6 1.5 0.2 1.7 0.4 1.1 0.3 0.5 0.4 4.1

12.1 0.2 9.8 1.4 19.4 0.7 12.0 1.8 3.8 3.4 25.2

± ± ± ± ± ± ± ± ± ± ±

1.1 0.01 0.5 0.1 1.7 0.02 1.1 0.08 0.2 0.4 1.0

17.5 0.2 14.6 2.0 24.4 0.9 12.3 2.3 4.0 3.6 21.5

± ± ± ± ± ± ± ± ± ± ±

1.0 0.01 1.9 0.2 2.5 0.07 0.7 0.3 0.4 0.5 1.6

15.2 5.9 17.3 4.2 16.9 2.5 11.6 3.9 7.3 8.1 34.0

± ± ± ± ± ± ± ± ± ± ±

1.6 0.2 1.0 0.3 1.2 0.3 1.3 0.4 0.7 0.7 3.1

13.9 5.2 15.7 3.5 15.5 2.0 10.4 3.0 7.0 7.1 32.2

± ± ± ± ± ± ± ± ± ± ±

1.5 0.7 1.2 0.1 1.3 0.3 1.0 0.1 0.3 0.5 3.2

20.3 5.0 19.6 2.4 29.0 1.1 17.0 3.3 7.4 5.5 33.7

± ± ± ± ± ± ± ± ± ± ±

1.9 0.5 1.5 0.2 2.7 0.1 1.7 0.2 0.4 0.4 4.0

16.0 3.8 14.8 2.0 22.4 1.1 13.2 2.2 6.6 4.5 23.8

± ± ± ± ± ± ± ± ± ± ±

2011 Myricetin 3-rutinosidea Myricetin 3-galactosidea Myricetin 3-glucosidea Myricetin 3-malonylglucosidea Quercetin 3-rutinoside Quercetin 3-galactoside Quercetin 3-glucoside Quercetin 3-malonylglucosideb Kaempferol 3-rutinosidec Kaempferol 3-galactosidec Epicatechin

14.4 0.4 13.4 0.9 19.3 3.3 7.2 3.3 3.3 2.1 29.2

± ± ± ± ± ± ± ± ± ± ±

1.7 0.01 1.2 0.06 1.7 0.8 0.2 0.4 0.3 0.2 1.1

11.7 0.2 10.2 2.6 17.3 0.3 7.3 3.1 1.9 1.9 31.1

± ± ± ± ± ± ± ± ± ± ±

1.0 0.01 1.6 0.2 1.8 0.01 0.5 0.2 0.1 0.1 2.2

9.6 2.6 6.2 1.3 15.7 3.9 11.5 2.7 5.9 6.8 30.7

± ± ± ± ± ± ± ± ± ± ±

0.7 0.1 0.6 0.09 1.1 0.2 1.1 0.1 0.5 0.3 2.2

14.0 3.4 7.8 1.3 19.8 3.0 12.4 2.2 7.5 7.4 33.5

± ± ± ± ± ± ± ± ± ± ±

1.1 0.2 0.3 0.07 1.0 0.2 1.0 0.2 0.5 0.7 1.9

9.1 0.1 6.3 1.4 18.1 0.6 11.2 1.6 3.7 2.9 19.9

± ± ± ± ± ± ± ± ± ± ±

0.6 0.01 0.4 0.1 0.8 0.01 1.0 0.09 0.2 0.2 1.3

13.0 0.1 9.5 1.9 20.0 0.5 11.5 1.7 3.9 2.9 21.2

± ± ± ± ± ± ± ± ± ± ±

1.1 0.01 0.4 0.2 1.3 0.01 1.0 0.6 0.3 0.2 1.3

12.4 3.9 12.6 2.5 16.2 2.3 12.7 3.3 5.9 6.4 31.3

± ± ± ± ± ± ± ± ± ± ±

1.0 0.2 0.6 0.2 0.8 0.1 1.1 0.2 0.4 0.4 2.5

10.5 2.2 11.1 2.9 16.4 3.4 12.8 3.4 5.4 6.9 33.6

± ± ± ± ± ± ± ± ± ± ±

0.8 0.1 1.0 0.1 1.2 0.1 1.0 0.1 0.3 0.2 2.9

14.8 3.8 12.9 2.1 26.5 3.2 19.4 2.9 4.8 5.1 32.7

± ± ± ± ± ± ± ± ± ± ±

1.2 0.3 1.5 0.1 2.2 0.2 1.8 0.2 0.2 0.2 2.5

17.7 4.6 15.8 3.2 30.8 3.6 21.7 3.3 6.6 5.4 34.8

± ± ± ± ± ± ± ± ± ± ±

Data are presented as mean ± SD (n = 3). a Expressed as mg myricetin/kg. b Expressed as mg quercetin-3-galactoside/kg. c Expressed as mg kaempferol/kg. d Critical values for pairwise comparisons of the means at P < 0.05 level (LSD0.05 ).

Cultivar

Shade

1.0 0.2 1.1 0.3 2.5 0.2 0.7 0.3 0.4 0.5 2.6

3.08 1.01 3.16 0.89 5.05 0.88 2.31 1.36 1.13 0.88 6.50

1.95 0.63 2.00 0.54 3.19 0.56 1.46 0.85 0.72 0.55 4.11

1.5 0.4 1.3 0.1 2.2 0.2 1.9 0.1 0.2 0.3 2.3

3.82 0.84 3.09 0.56 4.69 1.08 2.30 0.79 1.14 0.77 4.35

2.41 0.53 1.96 0.36 2.97 0.68 1.45 0.50 0.72 0.49 2.75

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2010 Myricetin 3-rutinosidea Myricetin 3-galactosidea Myricetin 3-glucosidea Myricetin 3-malonylglucosidea Quercetin 3-rutinoside Quercetin 3-galactoside Quercetin 3-glucoside Quercetin 3-malonylglucosideb Kaempferol 3-rutinosidec Kaempferol 3-galactosidec Epicatechin

4.94 1.24 0.41 7.82 1.95 0.65 54.5 ± 1.6 12.2 ± 0.6 4.4 ± 0.2 36.9 ± 2.2 9.9 ± 0.5 4.1 ± 0.2 28.4 ± 2.1 9.2 ± 0.8 3.7 ± 0.2 34.7 ± 2.5 9.9 ± 0.7 3.4 ± 0.2 32.4 ± 2.7 8.1 ± 0.4 4.0 ± 0.2 Data are presented as mean ± SD (n = 3). a Expressed as mg chlorogenic acid/kg. b Expressed as mg p-coumaric acid/kg. c Critical values for pairwise comparisons of the means at P < 0.05 level (LSD0.05 ).

24.5 ± 2.7 7.7 ± 0.5 3.8 ± 0.2 46.9 ± 3.0 10.4 ± 0.3 5.5 ± 0.3 36.3 ± 2.1 9.4 ± 0.3 4.8 ± 0.3 52.3 ± 2.3 19.9 ± 1.2 4.4 ± 0.2 2011 Neochlorogenic acida p-Coumaroylhexoseb Caffeoylhexoseb

39.2 ± 2.8 14.8 ± 1.2 3.7 ± 0.4

6.74 1.82 0.44 10.65 2.88 0.69 42.9 ± 2.9 11.2 ± 0.8 3.9 ± 0.2 44.2 ± 2.8 13.2 ± 1.0 4.9 ± 0.3 36.1 ± 1.7 10.1 ± 0.7 3.6 ± 0.2 44.2 ± 1.5 11.4 ± 0.8 3.8 ± 0.2 53.7 ± 3.9 11.6 ± 1.0 4.7 ± 0.3 46.5 ± 4.0 9.6 ± 1.0 4.9 ± 0.4 46.3 ± 1.7 12.0 ± 0.5 6.5 ± 0.3 35.1 ± 3.1 9.2 ± 0.8 4.8 ± 0.2 69.3 ± 5.1 24.7 ± 1.5 6.4 ± 0.4 35.4 ± 2.7 12.1 ± 1.0 3.1 ± 0.2

Cultivar Control Shade Shade Control Shade

Control

Shade

Control

Shade

Control

Ometa Ben Sarek Ben Lomond Ben Nevis

2010 Neochlorogenic acida p-Coumaroylhexoseb Caffeoylhexoseb

Shade

25

ˇ canska crna Caˇ

Table 3 Content of phenolic acids (mg/kg) in black currant varieties in both experimental seasons (2010 and 2011).

all varieties. The only exception was variety ‘Ben Sarek’ (season 2010), where similar contents of quercetin 3-rutinoside and myricetin 3-glucoside was observed. This flavonols composition in varieties in our study is similar to the other black currant varieties where quercetin 3-glucoside, myricetin 3-rutinoside, myricetin 3glucoside and quercetin 3-rutinoside were also the most abundant compounds (Sandell et al., 2009; Borges et al., 2010; Milivojevic et al., 2012). Some differences in the concentration of flavonols could be observed, which might be explained by the influence of genotype on the content of flavonoid compounds. However, our results are in accordance with previous studies where quercetin and myricetin were the major aglycones and kaempferol was detected in lower amount (Häkkinen et al., 1998; Mikkonen et al., 2001; Tabart et al., 2011). In non-shading berries, in all varieties except Ometa, the amounts of all flavonol glycosides were greater in the first cultivated season (Table 2), and the flavonol pattern of the major glycosides remains the same during both experimental seasons. Shading nets influenced the concentrations of the analyzed flavonoid glycosides in studied berries and in the first experimental ˇ canska season lower contents of the major flavonols in varieties Caˇ crna, Ben Nevis and Ben Lomond were noticed. Variety Ometa contained higher level of flavonols in 2010 year, while in the second season their amount decreased. Ben Sarek was the only variety with increased amount of flavonols in both experimental seasons, thus indicating that genetic differences between the cultivars influenced the capacity of plant to acclimate to low light conditions, as it was also shown in strawberry myrtle varieties (Franck et al., 2007). It has been shown that shading also cause reduction of flavonol concentration in wine grapes (Spayd et al., 2002; Downey et al., 2004), but some authors reported increased level of flavonols in shaded fruit (Bergqvist et al., 2001). These differences imply that, beside the light, other microclimate parameters affect flavonoid accumulation. Bergqvist et al. (2001) indicate that clusters of berries which have been exposed to direct sunlight had higher skin temperature than clusters in the shade, which could also contributed to increased synthesis of total phenols in them. Phenolic acids found in black currants are usually derivatives of hydroxycinnamic and p-hydroxybenzoic acids (Häkkinen et al., 1998, 1999b). In all studied black currant varieties, neochlorogenic acid was the major phenolic acid, followed by p-coumaric and caffeic acid derivatives (Table 3). According to Määttä et al. (2003), p-coumaric acid was the main hydroxycinnamic acid, and derivatives of caffeic and ferulic acids were also detected. Among varieties ˇ canska crna and Ben in our study cultivated without shading, Caˇ Nevis contained the highest amounts of phenolic acids in the first experimental season. During the second season, the amounts of phenolic acids decreased in all varieties, except in variety Ometa where the content was 20% higher. Flavan-3-ol(−)epicatechin was ˇ canska crna (51.06 mg/kg) found in the highest amount in variety Caˇ in the first experimental season. In 2011 year, the content of this compound varied between varieties from 21.20 to 34.83 mg/kg, and these amounts were in accordance with the previous report (Tabart et al., 2011). Shading affected phenolic acid accumulation in a similar way as did on flavonoid content (Table 3). In both experimental seasons, the reduction of phenolic acid concentration was observed in varieties Ben Nevis and Ben Lomond in comparison with berries cultivated without shading. In 2011 compared to 2010, the content ˇ canska crna, whereas in of phenolic acids increased in variety Caˇ variety Ometa their amount decreased by 24% and 27%, respectively. Variety Ben Sarek was the only variety with increased amount of phenolic compounds in berries grown with shading in comparison with non-shading berries in both experimental seasons. Our results show a large variation in the phenolic acids accumulation among varieties grown in the shade. This indicates

Critical valuesc

K. Sˇ avikin et al. / Scientia Horticulturae 160 (2013) 20–28

26

Table 4 Content of anthocyanins (mg/kg) in black currant varieties in both experimental seasons (2010 and 2011). ˇ canska crna Caˇ Shade

2011 Delphinidin 3-glucosidea Delphinidin 3-rutinosidea Delphinidin 3-xylosidea Delphinidin3-(6-Coumaroyl)glucosidea Cyanidin3-glucoside Cyanidin 3-rutinosideb Cyanidin3-(6-coumaroyl)glucosideb Petunidin 3-rutinosideb Peonidin 3-glucosideb

115.0 222.5 1.9 5.7

Shade

Control ± ± ± ±

10.1 19.6 0.3 0.6

463.4 730.9 6.9 27.6

± ± ± ±

25.4 36.8 0.8 1.8

203.8 567.0 3.2 52.5

Ben Lomond Control

± ± ± ±

12.9 26.5 0.2 2.5

389.2 951.8 5.5 87.1

± ± ± ±

Shade 21.2 33.0 0.7 2.7

204.0 497.1 3.4 7.1

Ben Sarek Control

± ± ± ±

16.8 15.1 0.6 0.8

304.3 723.7 6.4 10.8

± ± ± ±

Shade 13.2 27.1 0.3 1.2

519.3 917.2 8.7 41.3

Control ± ± ± ±

31.7 37.9 0.9 2.3

Critical valuesc

Ometa

397.1 678.4 6.9 31.3

± ± ± ±

Shade 23.7 34.4 0.9 1.7

472.8 890.3 11.3 14.6

Control ± ± ± ±

26.8 35.1 1.6 1.6

350.6 650.4 8.2 9.1

± ± ± ±

Cultivar

Shade

21.2 27.1 0.7 0.7

89.10 171.8 2.29 15.10

56.40 108.2 1.44 9.55

27.80 97.00 3.12

17.60 61.30 1.97

7.46 6.04

4.72 3.82

17.2 32.9 6.6 2.0

83.00 159.1 2.07 12.80

52.50 100.6 1.31 8.10

47.8 ± 3.9 231.2 ± 16.6 2.2 ± 0.5

154.6 ± 13.8 579.8 ± 10.2 8.0 ± 0.6

51.0 ± 2.4 293.5 ± 13.4 11.6 ± 0.9

96.9 ± 3.8 487.3 ± 23.4 19.0 ± 1.4

44.4 ± 2.9 289.8 ± 15.7 1.4 ± 0.3

67.1 ± 2.7 412.8 ± 12.8 2.2 ± 0.4

121.9 ± 15.3 499.6 ± 22.3 8.2 ± 0.9

104.6 ± 12.5 402.9 ± 15.0 6.7 ± 0.8

152.4 ± 10.9 584.0 ± 26.7 4.1 ± 0.6

108.5 ± 8.7 446.5 ± 22.8 2.8 ± 0.3

3.5 ± 0.3 2.7 ± 0.4

18.9 ± 1.3 13.2 ± 0.8

21.9 ± 1.7 15.9 ± 1.1

37.1 ± 2.6 26.5 ± 1.5

7.9 ± 0.8 4.4 ± 0.6

13.9 ± 1.4 8.4 ± 0.7

47.1 ± 3.9 33.6 ± 1.4

37.3 ± 2.8 27.5 ± 1.7

28.1 ± 1.8 23.1 ± 1.1

20.2 ± 1.4 15.8 ± 1.2

291.5 550.0 4.1 16.5

± ± ± ±

14.2 13.0 1.2 1.7

112.7 ± 3.4 578.5 ± 28.5 7.0 ± 4.6 6.8 ± 2.3 2.2 ± 1.3

175.6 334.7 2.5 11.3

± ± ± ±

9.7 22.4 1.4 3.5

214.7 519.5 2.6 66.6

± ± ± ±

11.7 26.6 1.1 6.7

189.5 636.2 3.4 87.6

± ± ± ±

17.6 36.4 0.8 16.3

84.2 ± 24.1 356.0 ± 25.6 4.8 ± 1.5

43.3 ± 2.4 248.6 ± 21.5 13.2 ± 6.8

43.4 ± 4.7 294.1 ± 23.5 17.7 ± 3.1

4.0 ± 2.4 2.4 ± 0.7

17.9 ± 8.8 12.0 ± 5.4

21.9 ± 3.8 13.2 ± 2.8

Data are presented as mean ± SD (n = 3). a Expressed as mg delphinidin/kg. b Expressed as mg cyanidin-3-glucoside/kg. c Critical values for pairwise comparisons of the means at P < 0.05 level (LSD0.05 ).

125.7 348.1 1.0 11.7

± ± ± ±

12.3 28.6 1.2 1.0

23.1 ± 7.7 192.2 ± 11.2 2.4 ± 0.2 3.5 ± 2.2 1.6 ± 1.2

192.0 588.7 3.5 20.3

± ± ± ±

13.3 29.8 0.6 3.5

41.8 ± 6.1 323.4 ± 20.0 4.2 ± 0.9 9.3 ± 1.5 5.3 ± 1.2

273.9 487.4 4.0 38.7

± ± ± ±

22.7 39.2 0.6 7.3

219.6 411.0 3.6 29.4

± ± ± ±

17.8 20.6 0.9 7.6

298.5 724.5 3.8 22.1

± ± ± ±

13.1 28.2 1.4 5.9

434.6 877.0 8.3 24.3

± ± ± ±

49.2 ± 10.2 242.5 ± 20.8 7.3 ± 1.6

52.2 ± 8.0 234.8 ± 20.5 6.3 ± 1.6

101.2 ± 9.9 491.4 ± 32.0 6.3 ± 1.8

147.8 ± 7.3 585.3 ± 19.4 7.4 ± 3.1

28.00 108.9 2.85

17.70 68.80 1.80

22.3 ± 4.5 14.6 ± 2.5

19.1 ± 3.8 13.1 ± 2.7

14.1 ± 4.1 10.5 ± 3.2

24.5 ± 1.6 19.6 ± 1.2

5.71 4.41

6.31 2.79

K. Sˇ avikin et al. / Scientia Horticulturae 160 (2013) 20–28

2010 Delphinidin 3-glucosidea Delphinidin 3-rutinosidea Delphinidin 3-xylosidea Delphinidin 3-(6-coumaroyl)-glucosidea Cyanidin3-glucoside Cyanidin 3-rutinosideb Cyanidin 3-(6-coumaroyl)-glucosideb Petunidin 3-rutinosideb Peonidin 3-glucosideb

Ben Nevis

K. Sˇ avikin et al. / Scientia Horticulturae 160 (2013) 20–28

that genetic characteristics of cultivars and climatic variables such as light and temperature can influence phenolic concentration in black currants. 3.3. Total anthocyanins and anthocyanin composition The values of total anthocyanins in the fruits significantly varied depending on the variety and intensity of solar radiation (Table 1). In the first experimental season (2010), total anthocyanin accumuˇ canska crna and lation was higher in the light exposed varieties Caˇ Ben Sarek, while in other three varieties content of anthocyanins was almost the same in shaded and control fruit. The highest content of anthocyanins in both experimental seasons was noticed in variety Ometa. In the second experimental season (2011), there was no difference between exposed and shaded fruit in the amount of ˇ canska crna total anthocyanins, with the exception of varieties Caˇ and Ben Nevis, where content of anthocyanins was 27% and 15% lower in shaded fruit, respectively. Reduction of total anthocyanin content (9%) in strawberries cultivated in shade was also shown by Anttonen et al. (2006). Also, anthocyanin concentration in grapes decreased for more than 30% in shaded treatment (Cortell and Kennedy, 2006). The major anthocyanins in black currants are delphinidin and cyanidin glycosides (Milivojevic et al., 2012). The main components were delphinidin 3-rutinoside, delphinidin 3-glucoside, cyanidin 3-glucoside and cyanidin 3-rutinoside, representing majority of total anthocyanin content, and it is consistent with results of other authors (Slimestad and Solheim, 2002; Wu et al., 2004; Bordonaba and Terry, 2008). Compounds presented in lower concentrations were petunidin 3-rutinoside, peonidin 3-rutinoside, and p-coumaroyl derivatives of the glucosides of delphinidin and cyanidin. Delphinidin was the major aglycon in quantity in all examined cultivars, and anthocyanin 3-rutinosides were predominant glycosides. These results were in accordance with published data (Slimestad and Solheim, 2002; Bordonaba and Terry, 2008). The amounts of individual anthocyanins in control decreased in the second experimental season in all varieties, except in variety Ometa (Table 4). Growing in shade, generally led to a lower content of anthocyanins. Depending on the variety, the reduction of anthocyanins ranged up to 30%. When it comes to individual variety in 2010, the largest influence of application of shading net has been ˇ canska crna and Ben Nevis. In this year, in observed in varieties Caˇ ˇ canska crna all quantified anthocyanins had a lower convariety Caˇ tent in the berries grown in shade, and 8 of 9 analyzed compounds in variety Ben Nevis. In the second experimental season (2011), the largest influence of treatment on the content of anthocyanins was manifested in the variety Ometa. It is interesting to note that Ben Sarek is the only cultivar where the content of the majority of anthocyanins was higher in the fruits grown in shade than in direct light in both experimental seasons. These results imply that both genotype and production environment affected the studied parameters of black currants. Direct sunlight and high temperature may lead to the sunburns in the fruit and the leaves, which could influence anthocyanins synthesis (Anttonen and Karjalainen, 2005). 3.4. Radical scavenging activity Due to the high content of phenolic compounds black currants are characterized by significant antioxidant activity. In our study, all tested varieties expressed high value of radical scavenging activity against DPPH radical and significant differences among varieties were noticed (Table 1). Higher activity was recorded in 2010 due to the higher amounts of phenolics, and Ben Lomond was found to be the most active variety. Wu et al. (2004) found that the antioxidant capacity depends on the content of total phenols. Although

27

significant difference in radical scavenging activity among treatments (direct sunlight and shading nets) was recorded for 2010, in 2011 we have not obtained such significance. Such results could be explained by potential influence of other antioxidant compounds, such as ascorbic acid, which contribute to the antioxidant activity but are not measured in our samples. 4. Conclusion Successful cultivation of berries depends on the influence of a large number of environmental factors, especially light and temperature. A moderate change of environmental factors by using shading nets has resulted in the improvement of conditions for the cultivation of those very useful fruits. In our study, all blackcurrant varieties in the shaded treatment contained lower amounts of total phenolics when compared to the fully exposed ones. Similar but less expressed trend was noticed with anthocyanins content. Among varieties, Ben Sarek was the only one that contained higher amounts of flavonols, phenolic acids and anthocyanins when growing in shade than in direct sun exposure. However, variety Ometa was the most abundant in total phenolics, total anthocyanins and major individual compounds in both shade and non-shade berries. Taking into account that in some varieties cultivated in shade the amount of flavonols in berries increased we could conclude that, beside the light, other microclimate or genetic parameters may affect flavonoid accumulation. All tested varieties expressed significant radical scavenging activity against DPPH radical, but differences in antiradical activity were statistically significant only between varieties, while among direct sunlight and shading nets it was recorded only for the first experimental season. Although the results obtained in this study showed that shading caused the decrease in the contents of flavonoids and anthocyanins, berries grown in such conditions still represent a good source of these biologically active compounds. Acknowledgments The authors acknowledge their gratitude to the Ministry of Education, Science and Technology of Serbia for financial support, project number 46013 and Slovenian Research Agency, program Horticulture No. P4-0013-0481. References Anttonen, M.J., Karjalainen, R.O., 2005. Environmental and genetic variation of phenolic compounds in red raspberry. J. Food Compos. Anal. 18, 759–769. Anttonen, M.J., Hoppula, K.I., Nestby, R., Verheul, M.L.J., Karjalainen, R.O., 2006. Influence of fertilization, mulch color, early forcing, fruit order, planting date, shading, growing environment, and genotype on the contents of selected phenolics in strawberry (Fragaria × ananassa Duch.) fruits. J. Agric. Food Chem. 54, 2614–2620. Beattie, J., Crozier, A., Duthie, G.G., 2005. Potential health benefits of berries. Curr. Nutr. Food Sci. 1, 71–86. Benvenuti, S., Pellati, F., Melegari, M., Bertelli, D., 2004. Polyphenols, anthocyanins, ascorbic acid, and radical scavenging activity of Rubus, Ribes, and Aronia. J. Food Sci. 69 (3), 164–169. Bergqvist, J., Dokoozlian, N., Ebisuda, N., 2001. Sunlight exposure and temperature effects on berry growth and composition of Cabernet Sauvignon and Grenache in the Central San Joaquin valley of California. Am. J. Enol. Vitic. 52 (1), 1–7. Bordonaba, J.G., Terry, L., 2008. Biochemical profiling and chemometric analysis of seventeen UK-grown black currant cultivars. J. Agric. Food Chem. 56, 7422–7430. Borges, G., Degeneve, A., Mullen, W., Crozier, A., 2010. Identification of flavonoid and phenolic antioxidants in black currants, blueberries, raspberries, red currants, and cranberries. J. Agric. Food Chem. 58, 3901–3909. Chorti, E., Guidoni, S., Ferrandino, A., Novello, V., 2010. Effect of different cluster sunlight exposure levels on ripening and anthocyanin accumulation in Nebbiolo grapes. Am. J. Enol. Vitic. 61, 23–30. Cortell, J., Kennedy, J., 2006. Effect of shading on accumulation of flavonoid compounds in (Vitis vinifera L.) Pinot Noir fruit and extraction in a model system. J. Agric. Food Chem. 54, 8510–8520. Dixon, R.A., Paiva, N.L., 1995. Stress-induced phenylpropanoid metabolism. Plant Cell 7, 1085–1097.

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