Bioactive compounds and antimicrobial activity of black currant (Ribes nigrum L.) berries and leaves extract obtained by different soil management system

Scientia Horticulturae 222 (2017) 69–75

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Bioactive compounds and antimicrobial activity of black currant (Ribes nigrum L.) berries and leaves extract obtained by different soil management system

MARK

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Svetlana M. Paunovića, , Pavle Maškovićb, Mihailo Nikolićc, Rade Miletića a b c

Fruit Research Institute, Department for Technology of Fruit Growing, Kralja Petra I/9, 32000 Čačak, Serbia University of Kragujevac, Faculty of Agronomy, Department of Chemistry and Chemical Engineering, Čačak, Cara Dušana 34, 32000 Čačak, Serbia University of Belgrade, Faculty of Agriculture, Department of Fruit Science, Belgrade, Nemanjina 6, 11080 Belgrade, Serbia

A R T I C L E I N F O

A B S T R A C T

Keywords: Black currant Soil management system Polyphenolic compounds Antimicrobial activity

Polyphenols compounds have been found in berries and leaves of black currant (Ribes nigrum L.) are known as agents acting preventively and therapeutically on the human organism. The effect of three soil management system (bare fallow, sawdust mulch and black plastic mulch) on the content of total anthocyanins, anthocyanin glycoside (delphinidin 3-glucoside, delphinidin 3-rutinoside, cyanidin 3-glucoside and cyanidin 3-rutinoside), flavonols (quercetin, myricetin and kaempferol), flavan-3-ols (catechin and epicatechin) and antimicrobial activity of seven black currant cultivars (‘Ben Lomond’, ‘Ben Sarek’, ‘Titania’, ‘Čačanska Crna’, ‘Tisel’, ‘Tiben’ and ‘Tsema’) was investigated. Total anthocyanins content was determined using the single pH and pH differential method. High performance liquid chromatography (HPLC) was employed for the identification of the most abundant metabolites presented in berries and leaves extracts. Microbial properties of extracts were examined using eight selected indicator strains. Soil management systems and cultivars showed highly significant differences in the tested parameters. Berries showed a higher content in anthocyanins, flavonols and flavan-3-ols than leaves. Quercetin was the most abundant flavonol in berries and leaves, and epicatechin the most abundant flavan-3-ol. Comparative research on black currant cultivars and soil management systems suggests an important relationship between cultivars and soil management systems. Soil management systems had an effect on the contents anthocyanins and anthocyanin glycoside in extract leaves, but not in extract berries of black currant cultivars. On the other hand, soil management systems have a significant positive effect on the synthesis and accumulation of flavonols and flavan-3-ols in both berries and leaves. Furthermore, all extracts showed strong antimicrobial activity. These results suggest that berries and leaves of black currant cultivars which growing on different soil management systems may be used as a source of beneficial compounds in food and pharmaceutical industries.

1. Introduction Black currant (Ribes nigrum L.) is recognized as a good source of polyphenols especially anthocyanins, phenolic acid derivatives, flavonols as well as proanthocyanidins compared to other berries (e.g., strawberry and raspberry) (Karjalainen et al., 2009; Mattila et al., 2011). Anthocyanins are responsible for the rich red, purple, blue, and black colors of berries and currants (e.g. black currants, blueberries, raspberries, red currants, strawberries) and other fruits (blood orange, red apples, red and black grapes), but also some vegetables such as purple cabbage and aubergines (Mazza and Miniati, 1993). Anthocyanins are the major group of phenolics in black currants, accounting for

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Corresponding author. E-mail address: [email protected] (S.M. Paunović).

http://dx.doi.org/10.1016/j.scienta.2017.05.015 Received 20 March 2017; Received in revised form 2 May 2017; Accepted 3 May 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.

approximately 80% of total phenolics. The four main pigments delphinidin 3-O-glucoside, delphinidin 3-O-rutinoside, cyanidin-3-Oglucoside and cyanidin-3-O-rutinoside contribute up to 97% of the total anthocyanin in black currants (Mazza, 2000; Anttonen and Karjalainen, 2006). Slimestad and Solheim (2002) further studied the anthocyanin composition of black currants and reported fifteen anthocyanin structures presented in the extracts of black currant berries. Many studies reported the high concentrations of phenolic compounds in black currants; a number of investigations have evaluated the different polyphenolic fractions of the fruits and to a lesser extent other plant parts like buds and leaves (Mikkonen et al., 2001; Maatta et al., 2003; Tabart et al., 2006; Raudsepp et al., 2010; Oszmiański et al., 2011).

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Ljubljana, Slovenia) and dried at 60 °C. The obtained dry extract was stored in the glass bottles at 4 °C until analysis.

Generally, berries and leaves of black currants showed very strong biological activity, including inhibition of cell proliferation, in addition to antimutagenic, antimicrobial, anti-inflammatory, anti-cancer and antihypertensive properties (Declume, 1989; Wang and Mazza, 2002; Puupponen-Pimiä et al., 2005; Mazza, 2007; Tabart et al., 2012). Black currants can thrive under a wide range of soils. The most common soil management system in black currant plantings is continuous tillage, but soils are also mulched with different types of materials (sawdust, black plastic, bark, wood chips etc.). The mulches delayed the onset of heat transfer through the soil, minimized the diurnal temperature variation, lowered the maximum temperature reached and reduced the initial cooling rate and weed control (Sinkevičiene et al., 2009). The generally recognized benefits of such mulches include increased early and total yields, positively affecting chemical, functional and quality properties fruit (Larsson, 1997; Melgarejo et al., 2012). Insufficient knowledge regarding the application of different soil management systems in black currant plantings and insufficient knowledge of the biological potentials of black currant berries and leaves as natural sources of antioxidants and other compounds vital for human health necessitate detailed research of this fruit. Therefore, the objective of this study was to evaluate and compare the effect of different soil management systems in a black currant planting on chemical composition and biological activity of berries and leaves of the selected cultivars. Chemical profile was established using rapid spectrophotometric assays and HPLC analysis. On the other hand, antimicrobial activitiy of prepared extracts was assessed against the eight bacterial strains.

2.3. Determinations of total anthocyanins content Total anthocyanins content was determined using the single pH and pH differential method. An aliquot of 0.25 mL of the extracted sample was dissolved in 0.25 mol L−1 of KCl buffer at pH 1.0 and 0.4 mol L−1 of CH3COONa buffer at pH 4.5. After 15 min, the absorbance was measured at λ = 515 nm and λ = 700 nm. Results were expressed as milligrams of cyanidin-3-glucoside equivalents per 100 g of dry extract (mg C3G/100 g DW). 2.4. Determination of anthocyanin glycoside Anthocyanin glycoside content (delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-glucoside and cyanidin-3-rutinoside) was determined using a Perkin Elmer Series 400 high performance liquid chromatograph equipped with a Hewlett-Packard 1040A photodiode array detector. A mobile phase consisted of 10% HCOOH and MeCN. Injection volume of the sample was 20 μm. The column was Li Chrospher 100 Rp-18e (250 × 4.6 mm, 5 μm particle size). The absorbance was measured at 290, 350 and 520 nm. Results are expressed as milligrams per 100 g of dry extract (mg/100 g DW). 2.5. HPLC analysis Quantification of individuals was performed by reversed phase HPLC analysis, using the modified method of Mišan et al. (2011). HPLC analysis was performed using a liquid chromatograph (Agilent 1200 series, Santa Clara, CA, USA), equipped with a diode array detector (DAD), Chemstation Software (Agilent Technologies), a binary pump, an online vacuum degasser, an autosampler and a thermostatted column compartment, on an Agilent, Zorbax Eclipse Plus-C18, 1.8 μm, 600 bar, 2.1 × 50 mm column, at a flow-rate of 0.8 mL/min. Gradient elution was performed by varying the proportion of solvent A (methanol) to solvent B (1% formic acid in water (v/v)) as follows: initial 0–2 min, 100% B; 2–4 min, 100–98% B; 4–6 min, 98–95% B;6–7 min, 95–73% B; 7–10 min, 75–48% B; 10–12 min 48% B; 12–20 min, 48–40% B. The total running time and post-running time were 21 and 5 min, respectively. The column temperature was 30 °C. The injection volume for samples and standards was 5 μL using an autosampler. The spectra were acquired in the range 210–400 nm and chromatograms plotted at 280, 330 and 350 nm. Two solvents were used for the gradient elution of catechin and epicatechin: A-(H2O + 2%HCOOH) and B-(80%ACN + 2%HCOOH + H2O). The elution program used was as follows: from 0 to 10 min 0% B, from 10 to 28 min gradually increases 0–25% B, from 28 to 30 min 25% B, from 30 to 35 min gradually increases 25k50% B, from 35 to 40 min gradually increases 50–80% B, and finally for the last 5 min gradually decreases 80–0% B. The absorbance of this mixture was measured at 289 nm. All identifications of individual compounds were based on the retention times of the original standards where available, and spectral data. Results are expressed as milligrams per 100 g of dry extract (mg/100 g DW).

2. Materials and methods 2.1. Plant material Aerial parts of black currant (Ribes nigrumL.) were collected in Čačak, Western Serbia, during 2012–2016 (Republic of Serbia 43°54′ N latitude, 20°21′ E longitude, 242 m above sea level). Seven cultivars were used: ‘Ben Lomond’, ‘Ben Sarek’, ‘Čačanska Crna’, ‘Tsema’, ‘Titania’, ‘Tisel’ and ‘Tiben’. Three soil management systems were employed: treatment 1–bare fallow i.e. continuous tillage, treatment 2–sawdust mulch, and treatment 3–black plastic mulch. The experiment was laid out in a randomised block design (7 cultivars × 3 soil management systems × 3 replications × 5 bushes), giving a total of 315 black currant bushes. Berries and leaves were selected visually and were at the same stage development and from similar locations in the bushes. Fruits were sampled at full ripeness in June, while leaves sampled were collected in July, respectively, as during that period of the year these plant parts are fully developed. 2.2. Sample preparation Fruit samples (10.0 g) were extracted by 96% ethanol (100.0 mL) as a solvent. The extraction process was carried out using an ultrasonic bath (model B-220, Branson Instruments, Smith-Kline Co., Danbury, CT, USA) at room temperature for 1 hour. After filtration, 5 mL of the liquid extract was used for extraction yield determination. The solvent was removed by a rotary evaporator (Devarot, Elektromedicina, Ljubljana, Slovenia) under vacuum and was dried at 30 °C to constant weight. The dried extracts were stored in glass bottles at 4 °C to prevent oxidative damage until analysis. The analyses of the extracts were carried out immediately after extraction. The ultrasound-assisted extraction (UAE) of leaves samples was performed in ultrasonic water bath (B-220, Branson and Smith Kline Company, Danbury, CT, USA). Plant sample (10 g) was placed in a volumetric flask and 100 mL of distilled water were added. The mixture was sonicated for 60 min. Solvents, from the extract, were removed by evaporation using a rotary evaporator (Devarot, Elektromedicina,

2.6. Test microorganisms The antimicrobial activity of the plant extract was tested in vitro against the following Gram-positive bacteria: Staphylococcus aureus (American Type Culture Collection (ATCC) 12600), Micrococcus lysodeikticus (ATCC 4698) and Bacillus mycoides (ATCC 6462); and the following Gram-negative bacteria: Klebsiella pneumonia (ATCC 6462), Pseudomonas glycinea (Faculty of Biological Sciences, Serbia (FSB) 40) and Escherichia coli (ATCC 11775) and fungi Candida albicans (ATCC 10259), Fusarium oxysporum (FSB91), Penicillium canescens (FSB24), 70

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highest content of total anthocyanins in berries was determined in ‘Čačanska Crna’ (372.9 mg C3G g−1), while the lowest in ‘Ben Sarek’ (207.5 mg C3G/100 g). In contrast, the highest values for anthocyanins in leaves was obtained in ‘Tiben’ (2.09 mg C3G/100 g), and lowest in ‘Tsema’ (2.03 mg C3G/100 g). Compared to the present results, Tabart et al. (2011) and Vagiri et al. (2013) also reported a higher content of total anthocyanins in berries. Moyer et al. (2002), Lister et al. (2002), Karjalainen et al. (2009) and Oancea et al. (2011) reported similar content of total anthocyanins in berries confirmed in the present study. Black currant contains four major anthocyanins; the 3-glucosides and 3-rutinosides of cyanidin and delphinidin (Slimestad and Solheim, 2002; Kähkönen et al., 2003; Määttä-Riihinen et al., 2004b). In our study, cyaniding-3-rutinoside contentin extract berries and leaves was highest compared to other anthocyanin glycosides. Anthocyanin glycoside in berries extract was 18.8–46.5 times higher than in leaves extract. Content of anthocyanin glycosides varied significantly among cultivars. Cyanidin-3-glucoside and cyanidin-3-rutinoside in berries were the highest in ‘Čačanska Crna’ and‘Titania’, while the highest values for delphinidin-3-glucoside and delphinidin-3-rutinoside were obtained in ‘Tsema’ and ‘Titania’. The results of anthocyanin glycoside detected in the leaves indicated that cvs. ‘Ben Lomond’, ‘Ben Sarek’, ‘Čačanska Crna’ and ‘Titania’ expressed the highest average content of cyanidin-3-glucoside and cyanidin-3-rutinoside, while the highest content of delphinidin-3-glucoside and delphinidin-3-rutinoside were found in cvs. ‘Tisel’ and ‘Tiben’. In fruits of black currant cultivars by a group of researchers dominates the content of delphinidin-3-rutinoside compared to cyanidin-3-rutinoside (Wu et al., 2004; Szajdek and Borowska, 2008; Scalzoa et al., 2008), while another group detected the most cyanidin-3-rutinoside (Rubinskiene et al., 2006; Bordonaba and Terry, 2008; Oszmianski and Wojdylo, 2009). Cultivars with enhanced levels of anthocyanins are in demand from the processing sector due to high antioxidant activity and potential health benefits (Brennan and Graham, 2009; Lister et al., 2002). The present research is among the few studies that have examined and compared the effect of different soil management systems on the total anthocyanins and anthocyanin glycosides in berries and leaves of black currant cultivars. Soil management systems had not an effect on the contents tested parameters in extract berries. The difference between soil management systems was statistically significant in extract of leaves. The total anthocyanins, cyanidin 3-glucoside and delphinidin 3-rutinoside in bare fallow were relatively higher than the sawdust mulch and black plastic mulch. On the other hand, the highest cyanidin 3-rutinoside and delphinidin 3-glucoside were obtained in black currants under plastic mulch and lowest in sawdust mulch. Cultivars showed significant differences across soil management systems. Anthocyanins contents in berries under sawdust treatment was higher in ‘Ben Lomond’, ‘Ben Sarek’, ‘Titania’ and ‘Čačanska Crna’, whereas under black plastic mulch higher values were recorded in ‘Tsema’, ‘Tisel’ and ‘Tiben’ (Fig. 1). All cultivars in extract of leaves had the highest anthocyanins content under black plastic mulch treatment (Fig. 2). It is most likely that the higher soil temperature and higher moisture of the soil under black plastic mulch favors the synthesis of cyanidin-3-rutinosideand delphinidin-3-rutinoside, whereas the smaller temperature fluctuations and smaller soil moisture under bare fallow promoted the synthesis of total anthocyanins, cyanidin 3-glucoside and delphinidin 3-glucoside. Anthocyanin production in black currants is genetically predetermined but different treatments can affect the biosynthesis of anthocyanins. Chaudry et al. (2004) reported that mulching treatments had some positive impact on plant growth, but the intensity of the effect can be different with different treatments. Significant differences in total anthocyanin were found between conventional and organic grown black currant (Vagiri et al., 2013). Kazimierczak et al. (2008) presented that anthocyanins levels were higher by 35% and 40% respectively in organically grown fruits as compared to conventionally produced.

Aspergillus glaucus (FSB32), Alternaria alternata (FSB51), Penicillium verrucosum (FSB21), Aspergillus niger (FSB31), Trichoderma viride (FSB11) and Phialophora fastigiata (FSB81). Pure cultures were generated by subculturing four times on the same media for seven days. 2.7. Minimum inhibitory concentration (MIC) Minimum inhibitory concentrations (MIC) of the extracts against the test bacteria were determined by microdilution method in 96-multiwell microtiter plates (Satyajit et al., 2007). All tests were performed in Muller–Hinton broth (MHB) with the exception of the yeast, in which case Sabouraud dextrose broth was used. A volume of 100 μL extract solution (in methanol, 200 μL/mL) was transferred into the first row of the plate. Fifty microliters of Mueller Hinton or Sabouraud dextrose broth (supplemented with Tween 80 to a final concentration of 0.5% (v/v)) were added to the other wells. A volume of 50 μL from the first test wells was pipetted into the second well of each microtiter line and then 50 microliters of scalar dilution were transferred from the second to the twelfth well. Ten microliters of resazurin indicator solution (prepared by dissolution of a 270-mg tablet in 40 mL of sterile distilled water) and 30 μL of nutrient broth were added to each well. Finally, 10 μL of bacterial suspension (106 CFU/mL) and yeast spore suspension (3 × 104 CFU/mL) was added to each well. For each strain, the growth conditions and the sterility of the medium were checked. Standard antibiotic amracin was used to control the sensitivity of the tested bacteria, whereas nystatin was used as a control against the tested yeast. Plates were wrapped loosely with cling film to prevent dehydration and prepared in triplicate. The plates were placed in an incubator at 37 °C for 24 h for the bacteria and at 28 °C for 48 h for the yeast. Subsequently, color change was assessed visually. Any color change from purple to pink or colorless was recorded as positive. The lowest concentration at which color change occurred was taken as the MIC value. The average of three values was calculated, and the obtained value was taken as the MIC for the tested compound and standard drug. 2.8. Statistical analysis The experimental data obtained during the five-year period were subjected to statistical analysis using Fisher's two-factor analysis of variance—ANOVA. Significant differences between the mean values of the tested factors and the interaction means were determined by LSD test at P ≤ 0.01 and P ≤ 0.05 significance levels. The results are presented in tabular and figure form IC50 values were calculated by nonlinear regression analysis from the sigmoidal dose–response inhibition curve. 3. Results and discussion 3.1. Total anthocyanins and anthocyanin glycoside of extract Interestingly, anthocyanins are a major phenolic group in soft fruits (Määttä-Riihinen et al., 2004a, 2004b), and have been shown to possess high in vitro antioxidant activity compared to other edible crops (Halvorsen et al., 2002). Black currant is one of the plants with the highest levels of anthocyanins among fruits (Häkkinen et al., 1999; Benvenuti et al., 2004). In recent years, numerous studies have shown that anthocyanins display a wide range of biological activities, such as cardiovascular disease, cancer, cataracts and neurological disorders including Alzheimer’s (Laplaud et al., 1997; Andriambeloson et al., 1998; Parthasarathy et al., 2001). The total anthocyanins and anthocyanin glycosides in extract berries and leaves are presented in Tables 1 and 2. Cultivars showed differences among the investigated parameters. The total anthocyanins content varied from 207.5 to 372.9 mg C3G/ 100 g and from 2.03 to 2.09 mg C3G/100 g for berries and leaves extracts, respectively. Anthocyanins contents in berries extract were almost 141 times higher than anthocyanins in leaves extract. The 71

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Table 1 Total anthocyanins and anthocyanin glycosidein extracts berries of black currant. Cultivar/Treatment

Cultivar(A)

Treatment(B)

‘Ben Lomond’ ‘Ben Sarek’ ‘Tsema’ ‘Titania’ ‘Čačanska Crna’ ‘Tisel’ ‘Tiben’ bare fallow sawdust black plastic

ANOVA Cultivar (A) Treatment(B) A×B

Total anthocyanins mgC3G/100 g

Cyaniding-3-rutinoside mg/100 g

Cyaniding-3-glucoside mg/100 g

Delphinidin-3-rutinoside mg/100 g

Delphinidin-3-glucoside mg/100 g

283.6 ± 15.2 d 207.5 ± 8.89 f 283.3 ± 8.14 d 258.6 ± 6.82 e 372.9 ± 5.47 a 301.0 ± 10.2 c 317.5 ± 10.0 b 284.9 ± 7.70 291.5 ± 7.91 291.2 ± 7.84

45.1 43.2 44.9 40.6 48.7 45.4 44.7 44.5 44.7 44.8

20.8 22.0 21.6 22.8 21.1 22.4 20.8 21.7 21.7 21.5

2.53 2.72 2.76 2.59 2.65 2.46 2.58 2.61 2.61 2.61

3.70 3.56 3.78 3.96 3.75 3.71 3.70 3.72 3.78 3.71

** ns ns

** ns ns

± ± ± ± ± ± ± ± ± ±

0.75 1.11 0.76 1.58 0.68 0.64 0.74 0.67 0.65 0.64

bc d c e a b c

± ± ± ± ± ± ± ± ± ±

0.81 0.66 0.73 0.75 0.77 0.63 0.69 0.48 0.48 0.46

d b c a d ab d

** ns ns

± ± ± ± ± ± ± ± ± ±

0.26 0.29 0.31 0.28 0.28 0.27 0.29 0.19 0.18 0.18

de ab a cd bc e cd

** ns ns

± ± ± ± ± ± ± ± ± ±

0.29 0.25 0.29 0.36 0.28 0.30 0.30 0.19 0.20 0.19

b c b a b b b

** ns ns

Means followed by different letters within the cultivar, treatment and year columns are significantly different at P ≤ 0.01 and P ≤ 0.05 according to LSD test and ANOVA (F-test) results.

3.2. Flavonols and flavan-3-ols of extract Berries such as bilberries, blueberries and black currants are good sources of flavonols, especially quercetin and myricetin (MäättäRiihinen et al., 2004a; Määttä-Riihinen et al., 2004a, 2004b; Häkkinen et al., 1999), whereas the most abundant flavan-3-ols are catechin, epicatechin, epigallocatechin and their galloyl-substituted derivatives (Tsao, 2010). The common flavan-3-ols present in different parts of black currant plants are epigallocatechin, gallocatechin, catechin, epicatechin and epigallocatechin gallate (Tabart et al., 2011). Substantial amounts of proanthocyanidins are also found in black currants (Wu et al., 2004). In order to identify compounds in extract of berries and leaves, HPLC-DAD analysis was performed and obtained results are shown in Tables 3 and 4. In our study, flavonol and flavan-3-ols content in the berries extracts were higher in comparison to the content in leaf extract. Quercetin level in berries was higher in cv. ‘Tiben’, whereas myricetin level was higher in cvs. ‘Ben Lomond’, ‘Titania’ and ‘Čačanska Crna’. The amount of kampferol was very low in all cultivars. In the leaves, flavonols content not varied significantly among cultivars. Previous studies, confirmed that quercetin was the dominant flavonol in black currant as same as it was found in our study (Hakkinen et al., 1999; Mättää et al., 2003), while other authors reported myricetin as the main flavonol (Mikkonen et al., 2001; Jakobek et al., 2007). Tabart et al. (2006) reported that quercetin was dominant in all berries, leaves, and cultivars, myricetin varied widely among the cultivars, while kaempferol content was very low. The contents flavan-3-ols of the berries were significantly higher

Fig. 1. Relationship between soil management systems and cultivar on the total anthocyanins in the extract of berries.

than in leaves. The highest epicatechin content was found in ‘Čačanska Crna’, while the lowest was in ‘Tsema’. The amount of catechin was highest in cultivars ‘Ben Lomond’ on the other hand, the lowest content was observed in cultivars ‘Titania’. Studies on black currant leaves show a five-fold higher content of total phenols than in fruits or other black currant parts (Tabart et al., 2006). The phenolic profile of black currant leaves includes flavonoids such as kaempferol, quercetin, myricetin and phenolic acids (Raudsepp

Table 2 Total anthocyanins and anthocyanin glycoside in leaves extracts of black currant. Cultivar/Treatment

Cultivar(A)

Treatment(B)

ANOVA Cultivar (A) Treatment(B) A×B

‘Ben Lomond’ ‘Ben Sarek’ ‘Tsema’ ‘Titania’ ‘Čačanska Crna’ ‘Tisel’ ‘Tiben’ bare fallow sawdust black plastic

Total anthocyanins mgC3G/100 g

Cyaniding-3-rutinoside mg/100 g

Cyaniding-3-glucoside mg/100 g

Delphinidin-3-rutinoside mg/100 g

Delphinidin-3-glucoside mg/100 g

2.07 2.06 2.03 2.06 2.07 2.08 2.09 2.15 1.95 2.10

1.27 1.30 1.18 1.28 1.32 1.11 1.14 1.15 1.10 1.44

1.18 1.17 1.11 1.17 1.18 1.11 1.11 1.19 1.13 1.12

0.06 0.06 0.07 0.06 0.06 0.08 0.07 0.06 0.05 0.08

0.14 0.16 0.16 0.16 0.05 0.16 0.17 0.18 0.16 0.14

** ** ns

± ± ± ± ± ± ± ± ± ±

0.05 c 0.05 d 0.03 e 0.05 d 0.05 c 0.04 b 0.04 a 0.04 a 0.03 c 001 b

± ± ± ± ± ± ± ± ± ±

0.07 0.06 0.09 0.06 0.06 0.09 0.09 0.05 0.03 0.05

a a b a a c bc b c a

** ** ns

** ** ns

± ± ± ± ± ± ± ± ± ±

0.02 0.02 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01

a a b a a b b a b b

** ** ns

± ± ± ± ± ± ± ± ± ±

0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.003 0.003 0.001

c c b c c a b b c a

± ± ± ± ± ± ± ± ± ±

0.004 0.002 0.004 0.004 0.002 0.002 0.002 0.002 0.003 0.003

d b b b c b a a b c

** ** ns

Means followed by different letters within the cultivar, treatment and year columns are significantly different at P ≤ 0.01 and P ≤ 0.05 according to LSD test and ANOVA (F-test) results.

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3.3. Antimicrobial activity Antimicrobial secondary metabolites are produced by many plants, as a part of plant’s normal growth process as well as a response to pathogen attack. Many investigations confirm the great antimicrobial potential of plant extracts (Oliveira et al., 2008; Tekwu et al., 2012; Vieira et al., 2014).The results of the analysis of the antimicrobial activity obtained by the dilution method are given in Tables 5 and 6, while MICs were determined for eight selected indicator strains. The black currant berries showed antimicrobial activity with MIC values ranging from 38.2 to 170.1 μg/mL. The most sensitive was the Escherichia coli with the MIC from 38.2 μg/mL (‘Ben Sarek’) and 56.9 μg/mL (‘Tisel’), followed by the bacteria Aspergillus niger with MIC of 40.6 μg/mL (‘Ben Sarek’) and 88.5 μg/mL (‘Tiben’) and Candida albicans with the MIC of 49.5 μg/mL (‘Čačanska Crna’) and 87.7 μg/mL (‘Tiben’). Antimicrobial activity in leaves ranging from 123.0 to 389.2 μg/mL. Unlike berries, in leaves the most sensitive was the Aspergillus niger with the MIC from 94.0 μg/mL (‘Titania’) and 157.0 μg/mL (‘Čačanska Crna’), followed by the bacteria Candida albicans with MIC of 147.6 μg/mL (‘Tiben’) and 283.6 μg/mL (‘Ben Sarek’) and Proteus vulgaris with the MIC of 155.7 μg/mL (‘Tiben’) and 306.8 μg/mL (‘Titania’). The berries showed the highest antimicrobial activity to the all strains in black plastic mulch, however the lowest to the strain in bare fallow. On the other hand, the lowest antimicrobial activity in leaves showed in black plastic mulch, while the highest in bare fallow. Cavanagh et al. (2003) confirmed that several fresh berries, including raspberry and blackcurrant, inhibited the growth of wide range of human pathogenic bacteria, both Gram-negative and Gram-positive. According to the authors, raspberry and black currant juices display good antibacterial activity against various bacteria, such as Enterococcus, Escherichia, Mycobacterium, Salmonella and Staphylococcus species, whereas Mycobacteria phlei appeared to be susceptile to all the products. Polyphenols compounds and antimicrobial activity in berries and leaves of black currant may have important applications in the future as natural antimicrobial agents for health promoting products as well as for food industry. Also, the comparative research on seven black currant cultivars and three soil management systems suggests an important relationship between cultivars and soil management systems, which should be considered when establishing commercial black currant orchards.

Fig. 2. Relationship between soil management systems and cultivar on the total anthocyanins in the extract of leaves.

et al., 2010). The leaves could therefore be of interest for industrial applications in health and functional foods, especially if plants could be bred for high levels of phenolic compounds. In the leaves, the highest content of epicatechin was determined in ‘Ben Lomond’, ‘Ben Sarek’, ‘Titania’ and ‘Čačanska Crna’. In contrast, the highest values for catechin was obtained in ‘Ben Lomond’ and ‘Tsema’. Auger et al. (2004) reported that epicatechin and catechin is a very important group of compounds in the Mediterranean diet. However, according to the authors, figs do not belong to fruit rich in both constituents, in comparison to plums, apples or various kinds of berries. Tabart et al. (2011) presented that the flavan-3-ols content was significantly higher in the buds compared to the berries and leaves explants. As with cultivars, significant differences were also observed among the soil management system. The contents of flavonols in berries were highest in currants grown on black plastic mulch, whereas the highest values for flavan-3-ols were obtained when mulching with sawdust. In contrast, flavonols in leaves were highest under bare fallow; and the highest values for flavan-3-olswere reached plastic mulch. Asami et al. (2003), Anttonen and Karjalainen (2006) and Fan et al. (2012) shown that some cultivation managements, such as planting date and mulching systems can enhance strawberry fruit yield and phytochemical compounds. Pliakoni and Nanos (2010) obtained higher concentration of phenolic compounds of nectarines with mulching, whereas Coventry et al. (2003) reported a higher total phenolics, flavonols and anthocyanins using mulch in wine grapes. In contrast, Melgarejoa et al. (2012) reported that the antioxidant activity and polyphenols content in plum decreased with the used mulching plastic.

Acknowledgment This study is part of Project Ref. No. 31093 financially supported by

Table 3 Flavonols and flavan-3-ols in extracts berries of black currant. Cultivar/Treatment

Quercetin mg /100 g

‘Ben Lomond’ ‘Ben Sarek’ ‘Tsema’ ‘Titania’ ‘Čačanska Crna’ ‘Tisel’ ‘Tiben’ bare fallow sawdust black plastic ANOVA Cultivar (A) Treatment (B) A×B

11.1 10.9 10.2 10.3 10.7 10.3 11.2 10.6 10.5 10.9 ** ** ns

± ± ± ± ± ± ± ± ± ±

0.66 0.55 0.71 0.81 0.77 0.74 0.66 0.46 0.46 0.46

Myricetin mg /100 g a b d d c d a b b a

6.25 6.02 5.87 6.34 6.34 5.94 5.95 6.11 5.98 6.21

± ± ± ± ± ± ± ± ± ±

0.57 0.56 0.51 0.45 0.51 0.58 0.61 0.36 0.34 0.36

Kaempferol mg /100 g a b b a a b b ab b a

3.84 3.58 3.16 3.15 3.73 4.04 3.86 3.65 3.69 3.53

** ** ns

** ** ns

Means followed by different letters within the cultivar and treatment columns are significantly. Different at P ≤ 0.01 and P ≤ 0.05 according to LSD test and ANOVA (F-test) results.

73

± ± ± ± ± ± ± ± ± ±

0.36 0.31 0.43 0.29 0.37 0.34 0.36 0.24 0.24 0.22

Epicatechin mg /100 g a b b a a b b ab b a

85.6 96.0 83.4 90.6 95.9 94.7 84.4 90.1 90.7 89.5 ** ** ns

± ± ± ± ± ± ± ± ± ±

2.70 0.84 2.49 1.87 1.11 0.65 3.07 1.41 1.42 1.41

Catechin mg /100 g d a f c a b e b a c

74.5 68.1 75.1 63.6 70.6 73.3 68.9 70.5 70.9 70.4 ** ns ns

± ± ± ± ± ± ± ± ± ±

0.76 1.33 0.82 2.34 1.21 1.40 2.12 1.07 1.06 1.06

a e a f c b d b a b

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S.M. Paunović et al.

Table 4 Flavonols and flavan-3-ols in extracts leaves of black currant. Cultivar/Treatment

Quercetin mg/100 g

Myricetin mg/100 g

Kaempferol mg/100 g

Epicatechin mg/100 g

‘Ben Lomond’ ‘Ben Sarek’ ‘Tsema’ ‘Titania’ ‘Čačanska Crna’ ‘Tisel’ ‘Tiben’ bare fallow sawdust black plastic ANOVA Cultivar (A) Treatment (B) A×B

0.59 0.59 0.58 0.59 0.59 0.56 0.56 0.63 0.57 0.54

0.21 0.20 0.20 0.19 0.20 0.19 0.26 0.24 0.17 0.21

0.097 0.099 0.096 0.098 0.098 0.098 0.096 0.109 0.083 0.101

7.65 7.65 7.43 7.67 7.65 7.49 7.49 7.48 7.50 7.74

± ± ± ± ± ± ± ± ± ±

0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.01 a 0.01 b 0.01 c

ns ** ns

± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.01 0.01 0.01 0.06 0.03 a 0.01 b 0.01 ab

ns * ns

± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 a 0.01 c 0.01 b

ns ** ns

± ± ± ± ± ± ± ± ± ±

0.06 0.06 0.07 0.06 0.06 0.06 0.06 0.04 0.04 0.03

Catechin mg/100 g a a b a a b b b b a

6.47 6.48 6.06 6.44 6.48 6.45 6.46 6.41 6.33 6.47

** ** ns

± ± ± ± ± ± ± ± ± ±

0.03 0.03 0.05 0.04 0.03 0.09 0.09 0.04 0.05 0.01

b a f e a d c b c a

** ** ns

Means followed by different letters within the cultivar and treatment columns are significantly. Different at P ≤ 0.01 and P ≤ 0.05 according to LSD test and ANOVA (F-test) results. Table 5 The antimicrobial activity of berries extracts. Cultivar/ Treatment

Staphylococcus aureus

Klebsiella pneumoniae

Escherichia coli

Proteus vulgaris

Proteus mirabilis

Bacillus subtilis

Candida albicans

Aspergillus niger

‘Ben Lomond’ ‘Ben Sarek’ ‘Tsema’ ‘Titania’ ‘Čačanska Crna’ ‘Tisel’ ‘Tiben’ bare fallow sawdust black plastic A Amaricin. B Nystatin.

118.9 108.1 104.2 120.7 100.7 104.6 114.2 108.4 117.4 104.7 0.97 /

129.8 112.4 135.0 125.9 117.0 113.3 143.7 129.1 124.1 122.7 0.49 /

49.1 38.2 49.5 40.8 41.7 56.9 53.0 50.6 46.3 44.1 0.97 /

138.9 116.3 116.3 111.5 117.0 130.2 95.5 133.0 115.9 102.9 0.49 /

137.1 109.4 129.3 124.1 138.0 128.5 122.0 126.9 131.0 123.0 0.49 /

170.1 106.1 134.1 122.0 118.5 117.6 139.3 138.4 129.4 121.3 0.24 /

81.6 56.0 70.8 64.7 49.5 81.6 87.7 84.2 61.1 65.5 / /

49.9 40.6 50.3 54.9 59.5 72.5 88.5 63.5 66.5 48.4 / 0.97

Table 6 The antimicrobial activity of leaves extracts. Cultivar/ Treatment

Staphylococcus aureus

Klebsiella pneumoniae

Escherichia coli

Proteus vulgaris

Proteus mirabilis

Bacillus subtilis

Candida albicans

Aspergillus niger

‘Ben Lomond’ ‘Ben Sarek’ ‘Tsema’ ‘Titania’ ‘Čačanska Crna’ ‘Tisel’ ‘Tiben’ bare fallow sawdust black plastic A Amaricin. B Nystatin.

358.8 225.2 246.0 156.3 159.1 283.6 162.1 193.5 222.0 266.4 0.97 /

167.8 286.5 282.0 306.7 166.4 191.0 185.3 160.0 225.2 294.5 0.49 /

200.7 192.5 221.3 389.2 210.3 230.0 273.5 201.5 257.9 276.7 0.97 /

170.7 271.8 246.0 306.8 126.7 173.1 155.7 123.8 210.9 287.1 0.49 /

178.7 316.7 305.1 278.3 237.3 319.6 258.8 199.0 269.4 343.6 0.49 /

260.5 355.9 321.2 447.8 178.0 367.6 303.9 275.0 318.1 364.6 0.24 /

176.5 283.6 232.8 176.5 221.4 274.9 147.6 179.8 196.0 272.8 / /

95.5 123.0 128.8 94.0 157.0 131.7 125.9 99.2 117.5 150.1 / 0.97

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