Comparison of phenolic compounds and antioxidant ...

journal of functional foods 14 (2015) 736–746

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Comparison of phenolic compounds and antioxidant potential between selected edible fruits and their leaves Mirosława Teleszko, Aneta Wojdyło * Department of Fruit and Vegetable Technology, Wrocław University of Environmental and Life Sciences, Chełmon´skiego 37, 51-630 Wrocław, Poland

A R T I C L E

I N F O

Article history:

A B S T R A C T The aim of this study was to determine and compare a polyphenolic profile and antioxi-

Received 23 November 2014

dant activity in leaves and fruits of 7 selected species: Malus domestica (2 cultivars), Cydonia

Received in revised form 20

oblonga (4 cv.), Chaenomeles japonica (3 cv.), Ribes nigrum (3 cv.), Aronia melanocarpa (1 cv.), Vaccinium

February 2015

macrocarpon (11 cv.) and Vaccinium myrtillus. Polyphenolic profile was determined by LC-MS

Accepted 24 February 2015

and UPLC-PDA-FL and antioxidant activity were analysed by ABTS (2,2′-azino-bis(3-

Available online

ethylbenzothiazoline-6-sulphonic acid)) and FRAP (ferric reducing ability of plasma) test. The results showed that the leaves contained significantly higher polyphenol compounds

Keywords:

than the fruits. The highest concentration of total polyphenols were characterized by quince

Leaves

leaves (major group: polymeric proanthocyanidins) followed by cranberry > apple > choke-

Fruit

berry > Japanese quince > bilberry > and blackcurrant leaves (major group: flavonols). Also

Polyphenolic compounds

the strongest antioxidant potential was represented for quince leaves: 116.49 and 65.25 mmol

Antioxidant activity

trolox equivalents (TE)/100 g of dry matter (dm) in ABTS and FRAP tests, which correlated with the content of polymerized proanthocyanidins and flavonols. © 2015 Elsevier Ltd. All rights reserved.

1.

Introduction

As some studies show, leaves of well-known crops and wild growing plants (such as blackcurrant, chokeberry and bilberry) are a valuable source of antioxidant substances, especially polyphenols (Skupien´ , Kostrzewa-Nowak, Oszmian´ ski, & Tarasiuk, 2008; Tabart et al., 2007; Witzell, Gref, & Näsholm, 2003). It has been proven that chokeberry leaves (Aronia melanocarpa) extracts inhibit lipid and protein peroxidation in the brain of rats treated with oxidative stress-inducing factors (Cuvorova, Davydov, Prozorovskii, & Shvets, 2005). Skupien´ et al. (2008) described antileukaemic activity of chokeberry leaves

against human promyelocytes of several cell lines (HL60, HL60/ VINC and HL60/DOC), while Maslov et al. (2002) in a rat study revealed their antidiabetic properties. Potential pharmacological properties are also exhibited by quince leaf extract (QLE). Khademi, Danesh, Mohammad Nejad, Ghorbani, and Soleimani Rad (2013) investigated the ability of QLE in preventing atherosclerosis progression and determined the lipid-lowering effect (on tested rabbits). The effect of the examined extract (dose: 50 mg/kg) on lipid profiles was assessed by measuring total cholesterol, triacylglycerol, low- and high-density lipoprotein (LDL, HDL) and liver enzyme levels in plasma. In this respect, the properties of QLE were similar to those of atorvastatin (a medication used in hypercholesterolaemia). A reduction of lipid profile, increase

* Corresponding author. Department of Fruit and Vegetable Technology, Wroclaw University of Environmental and Life Sciences, Chełmon´skiego 37, 51-630 Wroclaw, Poland. Tel.: +48 71 320 77 06; fax: +48 71 320 77 07. E-mail address: [email protected] (A. Wojdyło). http://dx.doi.org/10.1016/j.jff.2015.02.041 1756-4646/© 2015 Elsevier Ltd. All rights reserved.


of HDL cholesterol and decrease of liver enzyme levels were observed. Aslan, Orhan, Orhan, and Ergun (2010) examined antidiabetic properties of hydro-ethanolic extracts from quince leaves. They reported a significant reduction of glucose level in diabetic rats’ blood by oral administration of QLE (dose: 250 and 500 mg/kg; time: 0–3 h). The same effect was observed when an antidiabetic drug (tolbutamide) in a dose of 100 mg/kg was employed. High biological potential has also been observed in sea buckthorn leaves (Hippophae rhamnoides L.). It is related to their ability to accelerate tissue regeneration and radioprotective, antiinflammatory, immunomodulatory and adaptogenic activity (Chawla et al., 2007; Ganju et al., 2005; Geetha et al., 2005; Gupta, Kumar, Pal, Banerjee, & Sawhney, 2005; Saggu et al., 2007; Upadhyay, Kumar, Siddiqui, & Gupta, 2011). The knowledge about the active compounds of leaves, their cytotoxicity or potential pharmacological properties, is still too limited. The starting point in the study of plants’ healthpromoting activity is to know the composition of bioactives, including polyphenols. Because of the wide action spectrum of these compounds, they may be complementary, natural raw material in fruit processing, enriching the health and sensory values of products, e.g. juices or purees (Teleszko, 2011). However, the available literature data indicate that this issue has rarely been addressed. For example, in the research of Kolniak-Ostek, Oszmian´ski, and Wojdyło (2013), apple leaves (Malus domestica Borkh.) in the amount of 0.5, 1.0 and 5.0% were added to cloudy apple drink. It was found that the use of leaves had a positive impact on the content of polyphenolic compounds in the final product (including phenolic acids, dihydrochalcones and flavonols) and significantly increased its antioxidant capacity. In view of the above, the aim of our study was to determine the polyphenolic profile in leaves of chosen plants and to compare leaves and fruits of examined species in terms of antioxidant contents.

2.

Materials and methods

2.1.

Reagents and chemicals

Quercetin and kaempferol 3-O-glucoside, cyanidin 3-rutinoside, p-coumaric acid, (+)-catechin, (−)-epicatechin were purchased from Extrasynthese (Lyon Nord, France). Chlorogenic acid and neochlorogenic acid were supplied by TRANS MIT GmbH (Giessen, Germany). Acetic acid, phloroglucinol, ascorbic acid, acetonitrile and methanol were purchased from Sigma– Aldrich (Steinheim, Germany).

2.2.

Plant material

Leaves and ripe fruits of apples (Malus domestica Borkh.; cv.: ‘Szampion’, ‘Ozark Gold’) cultivated in Poland were obtained from Research Station for Cultivar Testing in Zybiszów, chokeberry (Aronia melanocarpa (Michx.) Elliott; cv.: ‘Galicjanka’) from Orchards Company Trzebnica, cranberry (Vaccinium macrocarpon L.; cv.: ‘Bain Favorite’, ‘Ben Lear’, ‘Bergman’, ‘Drewer’, ‘Early

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Richard’, ‘Hollister Red’, ‘Howes’, ‘McFarlin’, ‘Pilgrim’, ‘Stankiewicz’, ‘Stevens’) from Berry Crops Experimental Field of Warsaw University of Life Sciences in Błonie, blackcurrant (Ribes nigrum L.; cv.: ‘Titania’, Tiben’, Tisel’) and Japanese quince (Chaenomeles japonica L.; cv.: ‘De˛ bosz’, ‘Witaminnyj’, ‘Z˙óltogaraczyj’) from Institute of Horticulture in Skierniewice. Quince leaves and fruits (Cydonia oblonga Mill.; cv.: ‘Kaszczenko’, ‘Marija’, ‘Pózńa Rejmana’, ‘Ronda’) were obtained from the Garden of Medicinal Plants, at the Wrocław Medical University. Bilberry leaves and fruits (Vaccinium myrtillus L.) were obtained from Forest District Olesńica. The morphological parts of each plant species were collected at the same time, ie. when the fruits were ripe. Before harvesting, fruits and leaves were prepared (inspection, washing), frozen at −70 °C and then lyophilized in ALPHA 1–4 LSC freeze drier (Christ, Osterode, Germany). The parameters of the process are: vacuum: 0.960 mbar, temperature of the shelves: +26 °C, drying time: 20 h.

2.3. Plant extract preparation to polyphenols content analysis Extracts were prepared by spreading about 0.2 g of freezedried leaves or 0.5 g of fruits by 10 mL of mixture containing HPLC-grade methanol (30 mL/100 mL), ascorbic acid (2.0 g/100 mL) and acetic acid in an amount of 1.0 mL/100 mL of reagent. The extracts were sonicated for 15 min (Sonic 6D, Polsonic, Poland), left for 24 h at 4 °C without light and then sonicated again for 15 min. The samples were centrifuged (MPW-380R; MPW Med. Instruments, Poland) for 10 min at 4 °C and 20,000 rpm. The resulting extract was analysed. Determination of polyphenols by UPLC coupled to PDA and FL detector. The analysis of polyphenolic compounds (including proanthocyanidins) was carried out on a UPLC system Aquity (Waters, USA) consisting of a binary solvent manager, sample manager, photodiode array detector (PDA) and fluorescence detector (FL, model λe). Empower 3 software was used for chromatographic data gathering and integration of chromatograms. A UPLC analyses were performed on a BEH Shield C18 analytical column (2.1 mm × 5 mm; 1.7 µm). The flow rate was 0.45 mL/min. A partial loop injection mode with a needle overfill was set up, enabling 5 µL injection volumes when 10 µL injection loop was used. Acetonitrile was used as a strong wash solvent and 10% acetonitrile as a weak wash solvent. All incubations were done in triplicate.

2.4.

Analysis of polyphenols compounds

Analysis was previously described by Wojdyło, Oszmian´ski, and Bielicki (2013).The analytical column was kept at 30 °C by column oven, sample manager at 4 °C. The mobile phase used for the separation was composed of aqueous formic acid 4.5% (A) and acetonitrile (B) in gradient mode set as follows: initial conditions 1% B; from 0 to 5 min 75% B; from 5.0 to 6.5 min 100% B; from 6.5 to 7.5 min the composition was kept constantly at 100% B; from 7.5 to 8.5 min reconditioning the column to initial gradient (1% B). The runs were monitored at the following wavelengths: flavan-3-ols (FL) at 280 nm, phenolic acid (FA) at 320 nm, and flavonol glycosides (F) at 360 nm, and were measured over the wavelength range of 200–600 nm in steps of 2 nm.

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Retention times and spectra were compared with those of pure standards. Anthocyanin was expressed as cyanidin-3-Orutinoside, quercetin derivatives and kaempferol 3-O-galactoside was expressed as quercetin and kaempferol-3-O-glucoside (respectively).The results were expressed in mg/100 g of dry matter (dm) of leaves/fruits.

2.5. Analysis of proanthocyanidins by phloroglucinolysis method Phloroglucinolysis was previously performed and described by Kennedy, Hayasaka, Vidal, Waters, and Jones (2001). Portions of freeze-dried leaves (0.02 g) or fruits (0.05 g) were precisely measured into 2 mL Eppendorf vials, then 0.8 mL of the methanolic solution of phloroglucinol (75 g/L) and ascorbic acid (15 g/L) were added. After addition of 0.4 mL of methanolic HCl (0.3 mol/L), vials were closed and incubated for 30 min at 50 °C with vortexing all time by thermo shaker (TS-100, BIOSAN). The reaction was stopped by placing the vials in an ice bath and adding 0.6 mL of sodium acetate buffer (0.2 mol/L). Next the vials were centrifuged immediately at 20,000 × g for 10 min at 4 °C. The analytical column was kept at 15 °C by column oven, sample at 4 °C. Mobile phase generated from 2.5% acetic acid (A) and acetonitrile (B) was mixed directly in the instrument. Solvent A and B were used in the following gradient: initial, 2% B; 0.6–2.17 min, 3% B; 2.17–3.22 min, 10% B; 3.22–5.00 min, 15% B and from 5.00 to 6.00 min followed by washing and reconditioning of the column (1.50 min). The calibration curves which were based on peak area were established using (+)catechin, (−)-epicatechin, (+)-catechins and (−)-epicatechinphloroglucinol adducts standards. The average degree of polymerization was calculating the molar ratio of all the flavan3-ol units (phloroglucinol adducts + terminal units) to (−)epicatechin and (+)-catechin which correspond to terminal units. For analysis excitation wavelength of 278 nm and emission wavelength of 360 nm was used. All incubations were done in triplicate and expressed in mg/100 g of dm of leaves/fruits.

2.6. Identification of polyphenols by the liquid chromatography–mass spectrometry (LC-MS) method The method was previously described by Wojdyło, Teleszko, and Oszmian´ski (2014). Freeze-dried powdered fruit and leaves (ca. 0.5 g) was extracted twice with 15 mL of 80% acetone acidified with 1% acetic acid. The extracts were sonicated for 15 min, centrifuged at 19,000 rpm for 10 min at 4 °C and concentrated on a vacuum rotary evaporator to a volume of ca. 3 mL. The samples were applied to the Sep-Pak C18 cartridge (Waters, Millford, USA) containing 1 g of the carrier, washed with distilled water to remove the sugar and organic acid (extract = 0°Brix) and then collected into a vacuum flask with 15 mL 80% methanol acidified with 1% HCl. The methanol extract was evaporated to dryness. The dry residue was dissolved in 4 mL of 4.5% formic acid, centrifuged for 5 min at 15,000 rpm and then given to the analysis. Identification of polyphenols (flavan-3-ols, dihydrochalcones, phenolic acids, flavonols) was carried out using an ACQUITY Ultra Performance LC™ system (UPLC™) with binary solvent manager (Waters, Milford, USA) and a Micromass Q-Tof Micro mass spectrometer (Waters, Manchester, UK) equipped with an electrospray

ionization (ESI) source operating in negative mode. For instrument control data acquisition and processing MassLynxTM software (Version 4.1) was used. Separations of individual polyphenols were carried out using a UPLC BEH C18 column (1.7 mm, 2.1 mm × 50 mm; Waters, Milford, USA) at 30 °C. Samples (10 µL) were injected and elution completed in 12 min with a sequence of linear gradients and isocratic flow rates of 0.45 mL/min. The mobile phase was composed of solvent A (4.5 mL/100 mL formic acid. v/v) and solvent B (100 mL/100 mL of acetonitrile). The program began with isocratic elution with 99% A (0–1 min) and then a linear gradient was used for 12 min, lowering A to 0%; from 12.5 to 13.5 min, returned to the initial composition (99% A), and then held constant to re-equilibrate the column. Analysis was carried out using full scan data-dependent MS scanning from m/z 100 to 1000. The effluent was led directly to an electrospray source with a source block temperature of 130 °C, desolvation temperature of 350 °C, capillary voltage of 2.5 kV and cone voltage of 30 V. Nitrogen was used as a desolvation gas at flow rate of 300 L/h.

2.7.

ABTS+· radical scavenging spectrophotometric assay

The free radical scavenging activity was determined by ABTS radical cation decolorization assay described by Re et al. (1999). The ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) was dissolved in water to a 7 mmol concentration. The ABTS radical cation (ABTS+·) was produced by reacting ABTS stock solution with 2.45 mmol potassium persulfate (final concentration) and kept in the dark at room temperature (20 ± 2 °C) for 12–16 h before use. The radical was stable in this form for more than 2 days when stored in the dark at room temperature. Before analysis the ABTS+· solution was diluted with bidistilled water to an absorbance of 0.700 (±0.02) at 734 nm. Aliquots of 30 µL of sample extract were added supernatant to 3.0 mL of diluted ABTS+· solution (A734 nm = 0.700 ± 0.02) and the absorbance was read exactly 6 min after initial mixing. The results were corrected for dilution and expressed in mmol trolox equivalent (TE)/100 g of dm of leaves or fruits. All determinations were performed in triplicates using Shimadzu UV–vis 2401 PC spectrophotometer (Tokyo, Japan).

2.8.

Ferric ions reducing/antioxidant power assay

The total antioxidant power of extracts was determined using the ferric reducing ability of plasma (FRAP) assay by Benzie and Strain (1996). The FRAP reagent was prepared by mixing acetate buffer (300 µM, pH 3.6), a solution of 10 µM TPTZ in 40 mmol HCl, and 20 µmol FeCl3 at 10:1:1 (v/v/v). The FRAP reagent (3 mL) and diluted sample extracts (1 mL) were added to each well and mixed thoroughly. The absorbance was taken at 593 nm after 10 min. Standard curve was prepared using different concentrations of trolox. All solutions were used on the day of preparation. All determinations were performed in triplicates. The results were corrected for dilution and expressed in mmol TE/100 g of dm.

2.9.

Statistical analysis

Statistical analysis was made using Statistica 9.0 (StatSoft, Poland). Significant differences (p ≤ 0.05) between means were evaluated by one-way ANOVA and Duncan’s multiple range test.

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3.

Results

3.1.

Polyphenols content

Determination of polyphenolic compounds (identification and quantification) in the leaves and fruits of apple, quince, Japanese quince, chokeberry, blackcurrant, cranberry and bilberry was performed using liquid chromatography–mass spectrometry (LC-MS) and ultra-performance liquid chromatography (UPLC) systems. Different fragment ions were obtained when collision-induced dissociation (CID) was applied to molecular species [M-H]− for the characterization of flavonoids and phenolic acids. Table 1 shows the following information on peaks observed during LC-MS/MS analyses: peak labels, retention times (Rt; min), wavelengths of absorbance maxima (λmax), MS and MS/MS spectra data. To summarize, 31 compounds including 2 dihydrochalcones, 6 flavan-3-ols, 8 phenolic acids and 15 flavonols were identified in leaves and fruits. The results of chromatographic analysis coupled with a statistical test (Table 2) showed that the leaves were not only a valuable source of tested antioxidants, but most of all contained significantly more polyphenols than the fruit. The only exception was Japanese quince (10% more polyphenols in fruits than in the leaves).

The differences in the content of polyphenolic compounds between different morphological parts of plants resulted from the specific species. The total amount of polyphenols in apple tree leaves was over 7 times higher with respect to the fruit (9457.59 and 1311.77 mg/100 g dm, respectively; p < 0.05). The content of polyphenols in cranberry leaves (11,095.46 mg/100 g dm – mean value) was almost 6-fold higher than in berries (1883.32 mg/100 g dm; p < 0.05). Quince leaves contained 4-fold more compounds compared to the fruit (11,796.72 and 2609.50 mg/100 g dm; p < 0.05). In bilberry leaves 6884.44 mg of polyphenols were detected, while only 1624.79 mg/100 g dm was found in fruits (p < 0.05). A similar regularity was also observed in chokeberry and blackcurrant leaves. The differentiation of the various morphological parts of these shrubs in terms of polyphenol contents was statistically significant (p < 0.05), although not so clear quantitatively as in other plant species. Chokeberry leaves contained 7806.51 mg polyphenols/100 g dm (22% more than ripe fruit), while the leaves of blackcurrant contained 1378.36 mg/100 g dm (37% more than berries). Interesting information was provided by analysis of the significance of the correlation, which was performed to verify the relationships between polyphenol contents in leaves and fruit. A positive but low value of the coefficient r in relation to total

Table 1 – Polyphenols identification in fruits and leaves by LC-MS method. Polyphenols group

Compounds

Rt (min)

λmax (nm)

[M-H]− (m/z) and fragment ions in MS/MS [M-H]− (m/z)

Species

Flavan-3-ols

Procyanidin B3 (+) Catechin Procyanidin B1 (−) Epicatechin Procyanidin B2 Procyanidin C1 Phloretin-2′xylo-glucoside Phloridzin Neochlorogenic acid p-Coumaric-quinic acid Chlorogenic acid Caffeic acid Cryptochlorogenic acid Unknown chlorogenic acid isomer 5-O-feruloylquinic acid 3,5-O-dicaffeoyloquinic acid Quercetin-3-O-vicianoside Myricetin-3-O-rutinoside Myricetin-3-O-galactoside Myricetin-3-xylopiranoside Quercetin-3-O-robinobioside Quercetin-3-O-galactoside Quercetin-3-O-rutinoside Quercetin-3-O-glucoside Kaempferol-3-O-galactoside Kaempferol-3-O-rutinoside Quercetin-3-O-rhamnoside Kaempferol-3-O-glucoside Methoxyquercetin-3-O-galactoside Dimethoxymyricetin-hexoside Methoxyquercetin-pentoside

2.72 3.15 3.96 4.70 4.81 5.55 6.99 8.06 2.36 3.26 3.62 3.62 3.85 5.12 5.86 8.37 6.70 6.40 6.80 6.74 7.01 7.17 7.29 7.43 8.05 8.49 8.50 8.55 8.69 8.80 8.93

277 278 277 278 277 278 285 285 325 305 326 325 324 326 326 324 354 353 353 353 356 354 354 352 345 345 352 345 356 356 356

577†, 289 289, 245 577, 289 289, 245 577, 289 865, 577, 289 567, 273 435, 273 353, 191 337, 163 353, 191 353, 191 353, 191 353, 191 367, 191, 173 515, 353, 191 595, 301, 463 625, 316 479, 316 449, 317 609, 301, 463 463, 301 609, 301 463, 301 447, 285 593, 285 447, 301 447, 285 477, 315 507, 345 447, 315

Jq A, Q, Jq, Cr A, Q, Jq, Cr A, Q, Jq, Ch, Cr A, Q, Jq A, Q, Jq A A A, Q, Ch, Bc A, Bc A, Q, Jq, Ch Bb A, Q Q Q Q Ch Bc Bc, Bb Cr Ch A, Q, Jq, Ch, Cr, Bc, Bb A, Q, Jq, Ch, Bc A, Q, Ch, Bc Q, Bc Q, Bc A, Cr Q, Bc Cr Cr Cr

Dihydrochalcones Phenolic acids

Flavonols

A – apple, Q – quince, Jq – Japanese quince, Ch – chokeberry, Cr – cranberry, Bc – black currant, Bb – bilberry. † The m/z values of the predominant ions are given in bold type.

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Table 2 – Polyphenols content and profile in leaves and fruits of tested plant species [mg/100 g dm]. Species

Morphological part

PA

Malus domestica (apple) Cydonia oblonga (quince) Chaenomeles japonica (Japanese quince) Aronia melanocarpa (chokeberry) Vaccinium macrocarpon (cranberry) Ribes nigrum (blackcurrant) Vaccinium myrtillus (bilberry)

Fruits Leaves Fruits Leaves Fruits Leaves Fruits Leaves Fruits Leaves Fruits Leaves Fruits Leaves

933.55 804.26 2,178.45 6,462.04 2,884.74 2,255.77 3,816.36 4,128.82 961.29 4,718.21 541.92 429.95 525.63 2,474.75

FL j k h a e g d c i b l m ł f

214.31 160.56 82.45 471.86 4,595.22 1,777.90 326.55 26.06 451.87 2,776.09 21.06 194.79 38.08 924.94

h j k e a c g ł f b m i l d

DHC

FA

28.74 b 6331.16 a nd nd nd nd nd nd nd nd nd nd nd nd

75.02 257.86 273.31 3,894.00 96.18 1,342.05 669.03 1,803.74 116.07 236.25 34.92 52.94 242.01 2,513.23

F l g f a k d e c j i m ł h b

58.35 1,903.75 75.29 968.82 67.23 1,548.88 273.96 1,847.89 170.60 3,364.91 73.95 700.68 76.99 971.52

m b j f ł d h c i a l g l e

A

TP

1.81 nd nd nd nd nd 1265.48 nd nd nd 327.62 nd nd nd

1311.77 9457.59 2609.50 11,796.72 7,643.37 6,924.60 6,351.38 7,806.51 1,883.32 11,095.46 999.48 1,378.36 1,624.79 6,884.44

ł c i a e f h d j b m l k g

PA – lymeric proanthocyanidins, FL – mono-, di- and oligomeric flavan-3-ols, DHC – dihydrochalcones, FA – phenolic acids, F – flavonols, A – anthocyanins, TP – total polyphenols, nd – not detected. Values are given as means (n = 3). a–m Statistically homogenous groups (Duncan’s test. p ≤ 0.05).

polyphenols (r = 0.03), polymeric proanthocyanidins (r = 0.43), flavan-3-ols (r = 0.42), phenolic acids (r = 0.47), and flavonols content (r = 0.48) indicates the insignificance of the correlation (no correlation between the content of these compounds in different morphological parts). The highest concentration of polyphenols characterized quince leaves, followed by cranberry > apple > chokeberry > Japanese quince > bilberry > and blackcurrant (p < 0.05). The observed trend was not confirmed by the analysis of antioxidants in fruits, which indicated that polyphenols are in the highest concentration in Japanese quince > chokeberry > quince > cranberry > bilberry > and apples. Convergent results were obtained only in the case of blackcurrant. Their leaves and berries contained the least polyphenols among all tested crops and wild growing species (p < 0.05). The observed differentiation followed directly from the content of the various antioxidant groups. In the leaves, proanthocyanidin polymers, mono-, di- and oligomers of flavan3-ols, phenolic acids, and flavonols (and also dihydrochalcones in apple tree) have been identified, whereas there was no presence of anthocyanins (only in fruits). The major polyphenol group in quince, Japanese quince, chokeberry and cranberry leaves was polymerized proanthocyanidins. Their content varied from 2255.97 to 6462.04 mg/100 g dm (for quince and Japanese quince, respectively; p < 0.05), which was 33 and 55% of the total phenolics. Chokeberry and cranberry leaves were also characterized by a high proportion of flavonols (1847.89 and 3364.91 mg/100 g dm, respectively), which was significantly higher compared to the mono-, di- and oligomers of flavan-3-ols (26.06 and 2276.07 mg/100 g dm, respectively) and phenolic acids (1803.74 and 236.25 mg/100 g dm, respectively). The leaves of quince were rich in phenolic acids (3894.00 mg/100 g dm). In turn, Japanese quince leaves – as in the case of fruit – were a valuable source of flavan-3-ols with the structure of mono-, di- and oligomers (1770.90 mg/100 g dm). However, dihydrochalcones (6331.16 mg/100 g dm) and flavonols (1903.75 mg/100 g dm) were the major polyphenolic compounds found in apple tree leaves. Over 50% of the polyphenols contained in blackcurrant leaves were flavonol compounds; 31% were polymers of

proanthocyanidins, while the proportion of monomers, dimers and oligomers of flavan-3-ols and phenolic acids was 14 and 4%, respectively. In bilberry leaves there dominated phenolic acids (2513.23 mg/100 g dm) and polymeric proanthocyanidins (2474.75 mg/100 g dm), i.e. respectively 37 and 36% of total polyphenol content. Comparing the results of the chromatographic determination of individual groups of polyphenols, it was found that the plant leaves contained significantly more phenolic acids and flavonols than the fruit (p < 0.05). The phenolic acid content ranged from 52.94 mg/100 g dm (blackcurrant leaves) to 3894.00 mg/100 g dm (quince leaves). Thus, these values are approximately 2- to 14-fold higher in relation to the fruit (34.92 and 273.31 mg/100 g dm; p < 0.05). A similar regularity concerned flavonol distribution. It was clearly illustrated by the example of apple tree. The leaves contained about 30-fold more of these compounds (1903.75 mg/100 g dm) than fruit (58.35 mg/100 g dm). In the leaves of cranberry, characterized by the highest concentration of flavonols among all tested materials (3364.91 mg/100 g dm), the difference was 20-fold, in the case of Japanese quince 23-fold, in bilberry and quince 13-fold, in blackcurrant 9-fold, and 7-fold in the case of chokeberry leaves and fruits (Table 2). Similar results were obtained in the analysis of flavan-3ols content (mono-, di-, oligo- and polymers). In plants of the genus Vaccinium, i.e. cranberry (V. macrocarpon) and bilberry (V. myrtillus), but also in quince, the concentration of these compounds was significantly higher (p < 0.05) in leaves than in fruits. In the case of chokeberry, the relationship concerned only the content of polymerized proanthocyanidins. In contrast, apple and Japanese quince leaves contained 25 and 61% less mono-, di- and oligomeric flavan-3-ols and 14 and 22% less polymeric proanthocyanidins in comparison to the fruits. A significant (p < 0.05) effect on the abundance of leaves in the active compounds was observed not only for plant species but also cultivar (Table 3). In quince leaves significantly differentiated content of mono-, di- and oligomeric flavan-3-ols (from 372.94 to 648.33 mg/100 g dm in ‘Kaszczenko’

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Table 3 – The impact of plant cultivar on polyphenols content in leaves [mg/100 g dm]. Species

Cultivar

PA

Malus domestica (apple) Cydonia oblonga (quince)

Szampion Ozark Gold Marija Kaszczenko Pózńa Rejmana Ronda unknown (wild form) De˛bosz Witaminnyj Z˙ółtogaraczyj Bain Favorite Ben Lear Bergman Drewer Early Richard Hollister Red Howes Mc Farlin Pilgrim Stankiewicz Stevens Titania Tiben Tisel

914.42 694.09 4951.71 11214.69 4012.99 5668.76 1979.00 1739.15 2735.10 2569.83 4107.28 5400.45 4539.03 4146.45 5336.28 5414.01 4812.64 4826.90 4222.42 4754.61 4340.19 792.09 238.95 258.87

Chaenomeles japonica (Japanese quince)

Vaccinium macrocarpon (cranberry)

Ribes nigrum (blackcurrant)

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

163.57 157.55 486.15 372.94 648.33 380.01 1378.33 1479.89 3643.04 610.34 2146.00 3619.16 2984.71 2166.29 4108.68 2901.09 3919.93 1797.73 2404.70 2524.12 1964.56 200.87 179.01 204.65

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

DHC

FA

3676.43 b 8985.88 a nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

316.74 198.98 4577.30 4544.66 3440.68 3013.35 2738.57 874.56 858.37 896.69 429.77 116.37 230.73 381.87 181.27 147.47 449.00 180.09 140.17 194.01 148.06 33.69 60.08 65.06

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

2262.19 1545.32 650.13 1403.69 854.78 966.67 1027.26 1342.90 1982.92 1842.43 3891.08 3627.29 3100.14 2286.62 2487.41 4280.74 3250.32 3488.07 3374.85 3786.28 3441.18 673.31 636.44 792.28

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

7333.35 11581.82 10665.30 17535.99 8956.77 10028.79 7123.15 5436.50 9219.44 5919.29 10574.13 12763.27 10854.61 8981.22 12113.64 12743.31 12431.89 10292.79 10142.13 11259.02 9893.98 1699.96 1114.48 1320.86

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

PA – polymeric proanthocyanidins, FL – mono-, di- and oligomeric flavan-3-ols, DHC – dihydrochalcones, FA – phenolic acids, F – flavonols, A – anthocyanins, TP – total polyphenols, nd – not detected. Values are given as means (n = 3). a–k Statistically homogenous groups (Duncan’s test. p ≤ 0.05).

and ‘Pózńa Rejmana’ cv.), phenolic acids (from 3013.35 to 4577.30 mg/100 g dm in ‘Ronda’ and ‘Marija’ cv.) and flavonols (‘Marija’: 650.13 mg/100 g dm; ‘Kaszczenko’: 1403.69 mg/100 g dm) was also observed. Cranberry leaves contained from 8981.22 to 12,763.27 mg of total polyphenols per 100 g dm (‘Drewer’ and ‘Ben Lear’ cv.; p < 0.05). Only quince leaves were a richer source of these compounds. The major group of polyphenols in cranberry leaves was polymerized proanthocyanidins in a concentration between 4107.28 mg in ‘Bain Favorite’ cv. leaves and 5414.01 mg/100 g dm in ‘Hollister Red’ cv. (p < 0.05). The level of flavan-3-ols with a lower degree of molecule polymerization (mono-, di- and oligomers) was quantitatively more varied and ranged from 1797.73 to 4108.68 mg/100 g dm (‘McFarlin’ and ‘Early Richard’ cv., respectively; p < 0.05). Flavonol content ranged from 2286.62 mg (‘Drewer’ cv.) to 4280.74 mg/100 g dm (‘Hollister Red’ cv.), while in leaves of ‘Howes’ cv. the highest concentration of phenolic acids was determined (449. 00 mg/100 g dm). The differences in polyphenol contents were also observed in leaves of Japanese quince. ‘Witaminnyj’ cv. was primarily noteworthy in this respect. Its leaves contained significantly more polymeric proanthocyanidins (2735.10 mg/100 g dm), mono-, di- and oligomers of flavan-3-ols (3643.04 mg/100 g dm) and flavonols (9219.44 mg/100 g dm) than the leaves of ‘De˛bosz’, ‘Z˙ółtogaraczyj’ cv. or the wild growing form of Japanese quince. The polyphenolic profile was also determined in apple tree leaves. In this respect, ‘Szampion’ cv. was richer in polymeric proanthocyanidins (914.42 mg/100 g dm), phenolic acids (316.74 mg/100 g dm), and flavonols (2262.19 mg/100 g dm) than ‘Ozark Gold’ cv. Despite this, ‘Szampion’ leaves were

characterized by a lower content of total polyphenols (7333.35 mg/100 g dm). It was due to the varied level of dihydrochalcones in both cultivars (Table 3). The leaves of ‘Ozark Gold’ cv. contained 8985.88 mg of these compounds in 100 g dm, i.e. more than 2-fold higher compared to the ‘Szampion’ cv. (3676.43 mg/100 g dm). Another source of bioactive compounds was blackcurrant leaves. The chromatographic determination of polyphenols was carried out for three cultivars (‘Titania’, ‘Tiben’, ‘Tisel’ cv.). The results of UPLC analysis clearly indicated that the richest source of total polyphenolics (1699.96 mg/100 g dm) and polymeric proanthocyanidins (792.09 mg/100 g dm) was ‘Titania’ cv. leaves. The highest concentration of mono-, di- and oligomers of flavan-3-ols (204.65 mg/100 g dm), phenolic acids (65.06 mg/100 g dm), as well flavonols (792.28 mg/100 g dm), was observed in ‘Tisel’ cv. The fact is that the leaves of blackcurrant were not such a valuable source of polyphenols as other analysed raw materials. However, it should be noted that both cultivars contained more flavonols than the leaves of ‘Marija’ cv. quince (p < 0.05). Regardless of the cultivar, the leaves of blackcurrant were richer in mono-, di- and oligomeric flavonols than apple tree leaves. Moreover, in the case of ‘Titania’ cv., more polymeric proanthocyanidins were determined (compared to the ‘Ozark Gold’ cv.).

3.2.

Antioxidant activity

The leaves of crops and wild growing plants were characterized by higher antioxidant activity than the fruit (Table 4; p < 0.05). The only exception was for chokeberry leaves. Their

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Table 4 – Antioxidant activity in leaves and fruits [mM TE/100 g of dm]. Species

Morphological part

ABTS

FRAP

Malus domestica (apple)

Fruits Leaves Fruits Leaves Fruits Leaves Fruits Leaves Fruits Leaves Fruits Leaves Fruits Leaves

8.72 ± 3.94 ł 35.94 ± 3.35 g 7.85 ± 2.54 m 116.49 ± 0.00 a 32.88 ± 3.74 j 60.30 ± 14.85 d 52.31 ± 0.01 e 50.01 ± 0.00 f 14.61 ± 4.89 l 96.02 ± 15.58 b 22.47 ± 3.07 k 32.91 ± 0.00 i 35.34 ± 0.01 h 79.30 ± 0.00 c

3.44 ± 2.51 m 15.30 ± 6.68 j 5.43 ± 1.89 ł 65.25 ± 0.00 a 19.51 ± 7.22 h 40.09 ± 9.71 e 36.64 ± 0.01 f 40.55 ± 0.00 d 8.40 ± 3.51 l 43.17 ± 14.63 c 11.82 ± 1.82 k 19.16 ± 0.00 i 26.81 ± 0.02 g 59.58 ± 0.00 b

Cydonia oblonga (quince) Chaenomeles japonica (Japanese quince) Aronia melanocarpa (chokeberry) Vaccinium macrocarpon (cranberry) Ribes nigrum (blackcurrant) Vaccinium myrtillus (bilberry)

Values are given as means (n = 3). a–m Statistically homogenous groups (Duncan’s test. p ≤ 0.05).

TEAC value, estimated on the basis of ABTS analysis (50.01 mmol TE/100 g dm), was slightly, although statistically significantly, lower compared to the berries (52.31 mmol TE/100 g dm). The strongest antioxidant potential (in ABTS and the FRAP test as well) was shown by quince leaves (116.49 and 65.25 mmol TE/100 g dm). These values were 15- and 12-fold higher compared to the fruits (7.85 and 5.43 mmol TE/100 g dm, respectively). Considering the antioxidant activity of leaves according to cultivars (Table 5), significant differences were

Table 5 – The impact of plant cultivar on antioxidant activity of leaves [mM TE/100 g dm]. Species

Cultivar

ABTS

Malus domestica (apple) Cydonia oblonga (quince)

Szampion OzarkGold Marija Kaszczenko Pózńa Rejmana Ronda unknown De˛bosz Witaminnyj Z˙ółtogaraczyj Bain Favorite Ben Lear Bergman Drewer Earli Richard Hollister Red Howes Mc Farlin Pilgrim Stankiewicz Stevens Titania Tiben Tisel

33.57 ± 0.00 38.31 ± 0.31 111.96 ± 0.60 134.92 ± 1.00 84.72 ± 0.00

b a c a d

10.57 ± 0.04 20.02 ± 0.01 57.17 ± 0.01 81.39 ± 0.05 44.52 ± 0.20

b a c a d

134.76 ± 0.02 68.37 ± 0.16 44.98 ± 0.86 67.78 ± 1.68 60.06 ± 0.53 89.33 ± 1.99 114.96 ± 1.99 93.51 ± 1.51 88.37 ± 0.00 118.23 ± 1.50 109.55 ± 0.00 111.91 ± 1.96 89.77 ± 1.00 88.64 ± 0.97 68.59 ± 0.00 83.35 ± 0.25 31.24 ± 0.61 17.64 ± 0.20 49.86 ± 0.60

b a d b c e b d e a c c e e g f b c a

77.85 ± 0.00 46.44 ± 0.14 30.73 ± 0.07 46.57 ± 0.62 36.63 ± 0.12 48.19 ± 0.00 63.03 ± 0.43 45.61 ± 0.32 36.36 ± 0.12 49.69 ± 0.00 57.72 ± 0.58 55.75 ± 0.14 44.37 ± 1.15 39.64 ± 0.14 16.28 ± 0.14 18.18 ± 0.58 17.93 ± 0.20 10.11 ± 0.09 29.45 ± 0.28

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

Chaenomeles japonica (Japanese quince) Vaccinium macrocarpon (cranberry)

Ribes nigrum (blackcurrant)

FRAP

Values are given as means (n = 3). a–k Statistically homogenous groups (Duncan’s test. p ≤ 0.05).

observed (p < 0.05). The results of ABTS and FRAP analysis clearly showed that the highest values of TEAC were exhibited by leaves of ‘Kaszczenko’ cv. quince (134.92 and 81.39 mmol TE/100 g dm, respectively), followed by ‘Ronda’ cv. (134.76 and 77.85 mmol TE/100 g dm, respectively) > ‘Marija’ cv. (111.96 and 57.17 mmol TE/100 g dm, respectively) > and ‘Pózńa Rejmana’ cv. (84.72 and 44.52 mmol TE/100 g dm, respectively). The antioxidant potential of cranberry leaves was estimated in the range from 68.59 to 118.23 mmol TE/100 g dm in the ABTS+• method (‘Stankiewicz’ and ‘Early Richard’ cv., respectively). The values obtained in FRAP analysis were lower and varied from 16.28 to 63.03 mmol TE/100 g dm (‘Stankiewicz’ and ‘Ben Lear’ cv.). Significantly lower activity against ABTS+• and Fe3+ ions was characteristic for bilberry leaves (79.30 and 59.58 mmol TE/100 g dm). Importantly, the apple tree leaves, which contained significantly more polyphenols than bilberry leaves, had one of the lowest levels of antioxidant activity in relation to ABTS+• (from 33.57 in ‘Szampion’ cv. to 38.51 mmol TE/100 g dm in ‘Ozark Gold’ cv.; p < 0.05) and the lowest in relation to the Fe3+ ions (10.57–20.02 mmol TE/100 g dm). In the leaves of quince and chokeberry, significantly higher TEAC values were observed, i.e. 60.30 and 50.01 mmol TE/100 g dm (ABTS method) and 40.09 and 40.55 mmol TE/100 g dm (FRAP method). Blackcurrant leaves were characterized by similar antioxidant activity to apple tree leaves. The TEAC value estimated in the ABTS method was 32.91, while in the FRAP test it was 19.16 mmol TE/100 g dm. The leaves of ‘Tisel’ cv. had significantly higher antioxidant activity values than in the case of ‘Titania’ and ‘Tiben’ cv. (49.86 and 29.45 mmol TE/100 g dm). The polyphenol profile of leaves determined their antioxidant activity, which was confirmed by Pearson correlation analysis. A significant correlation between activity against ABTS +• /Fe 3+ ions and the content of polymerized proanthocyanidins (r = 0.87 and 0.78, respectively) and phenolic acids (r = 0.67 and 0.87) was found. In the case of the total polyphenol content, an analogical relationship was observed only in relation to the ABTS method (r = 0.70). The statistical analysis indicated that the correlation between ABTS/FRAP results and flavan-3-ols (r = 0.47 and 0.25) or flavonols content (r = 0.17


and 0.11) was insignificant (p > 0.05). However, it does not mean that these compounds did not participate in the formation of antioxidant potential. The conclusions about the analysis of correlation should be related not only to the whole tested material (fruit, leaves), but also to specific species. For example, quince and cranberry leaves varied greatly in this regard. In the case of quince leaves, a significant (p < 0.05) impact of flavonol content on antioxidant activity measured by ABTS+• and FRAP methods was noted (r = 0.57 and 0.72, respectively). It also differs from the previously described trends. A similar situation was found in cranberry leaves according to mono-, di- and oligomeric flavan-3-ols (r = 0.80 and 0.58).

4.

Discussion

There are not many studies comparing the antioxidant properties and polyphenol content in fruit and leaves, which makes the discussion of our results difficult. However, Wang and Lin (2000) described a similar relationship according to blackberry, raspberry and strawberry. In this research, the total polyphenol content varied from 91 to 338 mg/100 g of fresh fruits (916–2310 mg/100 g dm) and from 10.5 to 32.3 mg/g for fresh leaves (30.9–129.2 mg/g dm). The leaves of thornless blackberry (‘Chester Thornless’, ‘Hull Thornless’, and ‘Triple Crown’), black raspberry (‘Jewel’), red raspberry (‘Autumn Bliss’, ‘Canby’, ‘Sentry’, ‘Summit’), and strawberry (‘Allstar’) were also found to have high antioxidant capacities compared to their fruit tissues. As the literature data show, little attention has been paid to research on biologically active substances of leaves, which were the research material in this work. However, it does not mean that morphological parts other than the fruits are useless for medical, cosmetic or food purposes. The classic example of their application is the versatile Camellia sinensis species, commonly called ‘tea’. The leaves and buds of this evergreen shrub are commonly known, e.g. in pharmacology (Namita, Mukesh, & Vijay, 2012). Tea contains about 4000 bioactive compounds, one third of which are polyphenols (Sumpio, Cordova, Berke-Schlessel, Qin, & Chen, 2006). According to Khokhar and Magnusdottir (2002), tea leaves include from 6500 to 13,490 mg of polyphenols per 100 g of dm, wherein the content of catechins can be up to 8500 mg. Our study showed that these compounds are present at similar concentrations in the leaves of bilberry (6884.44 mg/100 g dm), Japanese quince (6924.60 mg/100 g dm), chokeberry (7806.51 mg/100 g dm), apple tree (9457.59 mg/100 g dm), cranberries (11,095.46 mg/100 g dm) and quince (11,796.72 mg/100 g dm). Quince leaf polyphenols were examined by Oliveira et al. (2007). The total amount of phenolic acids (e.g. 3-Ocaffeoylquinic, 4-O-caffeoylquinic, 5-O-caffeoylquinic acid) and flavonols (quercetin and kaempferol derivatives) ranged from 490 to 1650 mg/100 g dm. These values are lower than those observed in our research. According to Costa et al. (2009), quince leaves contained from 29,400 to 16,400 mg of polyphenols (calculated as 5-O-caffeoylquinic acid) in 100 g dm. Their results were confirmed by the chromatographic determination of these compounds in our study (‘Kaszczenko’ cv.; 17,535.99 mg/100 g dm).

743

The polyphenolic profile of M. domestica leaves was more diverse. The leaves of apple tree (like the fruits) contain dihydrochalcones. According to Hunter and Hull (1993), as well as Picinelli, Dapena, and Mangas (1995), phloridzin and its aglycone are the main components of M. domestica leaves. These compounds are likely to represent between 2 and 15% of dry matter and 80–90% of all polyphenols (Gosch, Halbwirth, & Stich, 2010). The other compounds are mono- and dimeric flavan-3ols (136.78 mg/100 g dm), phenolic acids (107.29 mg/100 g dm) and flavonols (967.05 mg/100 g dm) (Picinelli et al., 1995). In apple fruits, phloridzin represents only 2–6% of total phenolics, while flavanols and proanthocyanidins dominate, with more than 90% (Guyot, Marnet, Larba, Sanoner, & Drilleau, 1998; Tsao, Yang, Young, & Zhu, 2003; Vrhovsek, Rigo, Tonon, & Mattivi, 2004). These values are also consistent with those obtained in our study. The study by Lee et al. (2014) described the profile of phenolic acids and flavonols in mature chokeberry leaves. The contents of both groups of these antioxidants were similar – as we also found in the results of our research. The authors observed 1148.30 mg of caffeoylquinic acid derivatives and 1065.90 mg of flavonols (including quercetin, isorhamnetin, kaempferol and apigenin glycosides) per kilogram of fresh leaves. The food industry is focused on chokeberry fruit processing. The health benefits of A. melanocarpa have been widely reported in the literature and have contributed to its versatile use, not only in the fruit but also in meat processing and baking (Białek, Rutkowska, & Hallmann, 2012). Chokeberry leaves are not known as a component of functional products. However, Lee et al. (2014) suggested that due to the high content of antioxidants they could successfully replace the leaves of tea. Chokeberry leaves are characterized by a slightly acidic, sweet taste. In South Korea they are collected in the initial growth phase and used as a spring vegetable. Phytochemical studies indicate that blackcurrant leaves are also a valuable source of polyphenols. The content of these compounds can be up to 5-fold higher than in fruits (Tabart, Kevers, Pincemail, Defraigne, & Dommes, 2006). The composition of blackcurrant leaves was previously described by He et al. (2010), as well as Raudsepp, Kaldmäe, Kikas, Libek, and Püssa (2010). They identified mainly flavonols (including kaempferol galactoside and glucoside, quercetin galactoside, and isorhamnetin), and phenolic acids. Depending on the extraction conditions (pH, type of solvent), the total polyphenol content in blackcurrant leaves was determined in the range from 4000 to 20,000 mg/100 g dm (Tabart et al., 2007). The values obtained in our study were much lower. The observed differences are probably due to weather conditions in the harvest season. The synthesis of polyphenolic compounds in plants depends on the degree of sunlight exposure and water relations during the vegetation period. Tattini et al. (2004) found a significant effect of these factors on the antioxidant content in Ligustrum vulgare leaves. The tested material from the shaded side of a bush (light exposure 6%) contained 3-fold less polyphenols than leaves grown under full sunlight exposure (100%). Wang and Lin (2000) also suggested that polyphenol concentrations in tissues determine the degree of plant maturity. Valuable plant materials also include bilberry leaves, which are the main waste products from the fruit cleaning process. As previous studies showed (Fraisse, Carnat, & Lamaison, 1996; Jaakola, Määttä-Riihinen, Kärenlampi, & Hohtola, 2004), they

744


are an excellent source of antioxidants. According to Riihinen, Jaakola, Kärenlampi, and Hohtola (2008), in 100 g fresh weight of bilberry leaves there is close to 1600 mg of polyphenols. Jaakola, Koskimäki, Riihinen, Tolvanen, and Hohtola (2008) noted the content of these compounds as 66–110 mg (procyanidins), 496–787 mg (flavonols) and 1443–1856 mg (phenolic acids) per 100 g dm. Similar values were obtained in our study. However, the concentration of procyanidins in the leaves was higher than in other authors’ research. In nature there are many more plant species characterized by a high content of biologically active substances, apart from Japanese quince. Until now, researchers were interested only in the fruits of this valuable plant. There is no information about the prohealthy potential of Japanese quince leaves and their abundance of polyphenolic compounds, which was clearly shown in our study. The knowledge about biological properties of cranberry leaves is also insufficient. One of the few studies on this topic is that by Booth, Kruger, Hayes, and Clemens (2012) about the possibility of using aqueous extracts of cranberry leaves for the production of functional foods. The studies were focused primarily on toxicological analysis. The aim was to verify whether cranberry leaf extract is safe for consumers’ health. Appropriate criteria were used to establish cranberry leaf as a food-grade raw material for use in manufacturing an extract for use in foods. Analytical evaluation provides evidence that at appropriate limits of detection there are no naturally occurring toxins of concern. In the tested extract, compounds with health-promoting properties, including flavonol glycosides, were identified. A high concentration of these antioxidants was also determined in leaves of 11 cranberry cultivars in our study. Another essential bioactive component of V. macrocarpon leaves is phenolic acids, with a total content between 532.3 and 676.8 mg/100 g for ‘Howes’ and ‘Early Black’ cv., respectively (Neto & Dao, 2010). Similar values were determined in the present study in leaves of ‘Howes’ and ‘Favorite Bain’ varieties (449.00 and 429.77 mg/100 g). The antioxidant properties of leaves from plant species such as apple, quince, Japanese quince, blackcurrant or cranberry have been rarely described in the literature, which makes it difficult to discuss the results of our study. A review of the available scientific data indicate that for most of them (including Japanese quince, apple tree, cranberry, and chokeberry leaves), TEAC values have not been recorded. However, Martz, Jaakola, Julkunen-Tütto, and Stark (2010), Oliveira et al. (2012), and Tabart et al. (2007) evaluated the antioxidant capacity of quince, bilberry and blackcurrant leaves using the DPPH method. It should be noted that the antioxidant activity against ABTS+• of tested leaves is comparable to tea leaves. Rusaczonek, S´widerski, and Waszkiewicz-Robak (2010) estimated the TEAC value for tea in the range of 46.40 (red tea) to 177.20 mmol TE/100 g dm (green tea), which is similar to the results obtained in our research, i.e. 50.01–116.49 mmol TE/100 g dm (for chokeberry and quince leaves, respectively).

5.

Conclusions

As the present study showed, the leaves of examined plants (such as quince, apple, chokeberry or cranberry) are a valuable

source of polyphenolic substances.They contained from 1378.36 to 11,796.72 mg/100 g of polyphenols (mean values), i.e. significantly more than the fruit (from 999.48 to 7643.37 mg/100 g dm). The highest concentration of antioxidants was exhibited by quince leaves, followed by cranberry > apple > chokeberry > quince > bilberry > blackcurrant leaves. Flavan-3-ols (mono-, di-, oligo- and polymers of proanthocyanidins) were the major polyphenol group in the tested raw material, except for apple tree and blackcurrant leaves (dihydrochalcones and flavonols dominated, respectively). Plant polyphenols, including those identified in leaves, are known for their anticancer, anti-inflammatory, antibacterial or – as was proved in the present study – antioxidant properties. Therefore, they could be considered as complementary raw material in fruit processing, enriching products (e.g. drinks) in bioactive compounds in the future. In the future perspective, closer attention to this issue may significantly contribute to the development of prevention and treatment of civilization diseases, such as cardiovascular diseases and diabetes.

Acknowledgements This work was supported by the European Union under Project No. POIG 01.01.02-00-061/09, acronym “Bioactive food”.

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