Inhibitory effect of black chokeberry fruit polyphenols on pancreatic lipase – Searching for most active inhibitors

Journal of Functional Foods 49 (2018) 196–204

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Inhibitory effect of black chokeberry fruit polyphenols on pancreatic lipase – Searching for most active inhibitors

T

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Dorota Sosnowskaa, , Anna Podsędeka, Małgorzata Redzyniaa, Alicja Z. Kucharskab a b

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 4/10, 90-924 Łódź, Poland Department of Fruit, Vegetable and Plant Nutraceutical Technology, Wroclaw University of Environmental and Life Sciences, Chełmońskiego 37, 51-630 Wrocław, Poland

A R T I C LE I N FO

A B S T R A C T

Keywords: Black chokeberry fruits Phenolics profile Pancreatic lipase Inhibitors UPLC-MS

In this study, the effects of a chokeberry fruit crude extract (CE), its ethyl acetate fractions (EAF7 & EAF2) and a polyphenol-rich fraction (PRF) on esterase and lipolytic activities of pancreatic lipase (PL) were investigated. Ethyl acetate fractions, rich in flavonols, phenolic acids and low molecular procyanidins, showed a week inhibitory activity against PL while the CE and PRF, which contained the highly polymerized procyanidins exhibited the highest anti-lipase activity. The kinetic study showed that the inhibitory mode of CE was noncompetitive while PRF behaved like the mixed type inhibitor. Cumulative effects were observed when orlistat was used with either CE or PRF. These results indicate that black chokeberry fruits are a source of PL inhibitors with proanthocyanidins as the most active compounds.

1. Introduction Black chokeberry (Aronia melanocarpa), native to eastern parts of North America, is also cultivated in Eastern and Northern Europe. Chokeberry juice and fruit extracts exhibit diverse potential health benefits in animal and human studies, including antihyperlipidemic, hypoglycemic, hepatoprotective, antiproliferative, anti-inflammatory, antimicrobial, and anti-obesity properties (Baum, Howard, Prior & Lee, 2016; Bhaswant, Shafie, Mathai, Mouatt & Brown, 2017; Jurikova et al., 2017; Qin & Anderson, 2012; Takahashi et al., 2015). The latter activity seems to be very important because of an increasing number of obese people and association of the obesity with many diseases, particularly diabetes and cardiovascular disease. In 2016, > 1.9 billion adults worldwide (18 years and older), were overweight and over 650 million were obese. In addition, 41 million children under the age of 5 were overweight or obese (WHO, 2017). Reports on effects of chokeberry fruits on weight loss in human intervention studies are limited. Recent study revealed that four week administration of 100 mL/day of a glucomannan-enriched chokeberry juice to a group of 20 postmenopausal women with abdominal obesity resulted in the decreased body mass index (BMI) and waist circumference (Kardum et al., 2014). More evidence on the anti-obesity properties of chokeberry components has been provided by animal studies. Studies in rodent models suggest that chokeberry juice (Bhaswant et al., 2017), black chokeberry juice

concentrate (Baum et al., 2016), black chokeberry extract (Qin & Anderson, 2012), and anthocyanin-rich extract from chokeberry fruits (Takahashi et al., 2015) protect against weight gain and visceral fat accumulation. The mechanism of phytochemicals action against obesity is complex and multidirectional (Fu, Jiang, Guo & Su, 2016). The effects of this action include: reduction of dietary-fat absorption, energy intake, adipogenesis and lipogenesis processes, suppression of appetite or increase of energy expenditure and lipolysis. One of the most promising approaches to reduce the energy intake through gastrointestinal mechanisms is inhibition of lipases, especially pancreatic lipase. Pancreatic lipase (EC 3.1.1.3; triacylglycerol acyl hydrolase) produced by the pancreatic acinar cells, splits triacylglycerols into absorbable monoacylglycerols and fatty acids, and is responsible for the hydrolysis of 50–70% of total dietary fats in the intestinal lumen (Birari & Bhutani, 2007). The results of our previous studies showed that a crude extract from black chokeberries inhibited porcine pancreatic lipase in different assay systems (Podsędek, Majewska, Redzynia, Sosnowska, & Koziołkiewicz, 2014; Sosnowska, Podsędek, Redzynia & Żyżelewicz, 2015). Among the plant derived pancreatic lipase inhibitors, phenolic compounds are recognized as most active (Birari & Bhutani, 2007). Black chokeberry fruits contain more polyphenols, mostly anthocyanins and proanthocyanidins, than other berries and fruits (Denev et al., 2014; Mikulic-Petkovsek, Schmitzer, Slatnar, Stampar & Veberic, 2012;

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Corresponding author. E-mail addresses: [email protected] (D. Sosnowska), [email protected] (A. Podsędek), [email protected] (M. Redzynia), [email protected] (A.Z. Kucharska). https://doi.org/10.1016/j.jff.2018.08.029 Received 6 June 2018; Received in revised form 1 August 2018; Accepted 18 August 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.

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residue (0.5 L) was purified by solid-phase extraction as described by George, Brat, Alter and Amiot (2005) using a 12-Port Vacuum Manifold system. A C-18 Sep-Pak cartridge (10 g capacity, Waters Corp., Milford, MA) was pre-treated with successive applications of 40 mL of methanol and 40 mL of water. The aqueous residue (2 mL) was passed through the column. Phenolic compounds were bound to the C-18 cartridge, while sugars and other polar compounds were removed with water (40 mL). Then phenolic compounds were eluted with methanol (40 mL). After the removal of methanol under reduced pressure (T < 40 °C), the solid residue was dissolved in water and lyophilized that yielded 7.12 g of phenolic-rich fraction (PRF). All the samples were stored at −20 °C before further analyses.

Szajdek & Borowska, 2008; Wu, Gu, Prior & McKay, 2004). As much as seven different anthocyanins were detected in this fruit with cyanidin 3galactoside as the major pigment (Wu et al., 2004). Proanthocyanins are either oligomers or polymers of monomeric flavan-3-ols. Building blocks of procyanidins from black chokeberry fruits are exclusively (+)-catechin and (−)-epicatechin. The degree of polymerization (DP) of the majority of them is > 10 (Wu et al., 2004). Based on these results we decided to compare the anti-lipase activity of different subgroups of phenolic compounds from black chokeberry fruits. Therefore, this study aimed to (i) fractionate black chokeberry fruit polyphenols, (ii) determine the pancreatic inhibitory activity of these compounds and type of inhibition, (iii) analyze possible interactions between the black chokeberry polyphenols and orlistat, (iv) identify and quantify phenolic compounds contained in the crude extract and its fractions.

2.3. Assay for pancreatic lipase activity Inhibitory activities of orlistat (a positive inhibitor control) and the analyzed samples were expressed as the IC50 values (half-maximal inhibitory concentration). The IC50 value was concluded from the graph of lipase inhibition (%) versus the concentration of CE, EAF2, EAF7, PRF or orlistat per 1 mL of reaction mixture under assay conditions. A stock solution of orlistat (0.1 mg/mL) was prepared in methanol. Method 1: The pancreatic lipase activity was assayed by measuring the fatty acids released from emulsified triolein according to the method described previously (Sosnowska et al., 2015). Directly before the analysis, 200 mg samples of CE, EAF7, EAF2, and PRF were dissolved in 1.8 mL of 20 mM Tris-base buffer (pH 7.4, containing 150 mM NaCl, and 1.3 mM CaCl2) and their pH were adjusted to pH 7.3–7.4 with 2 or 6 M NaOH, depending on the initial pH value of a sample. The volume of each sample was filled to 2 mL with the same buffer. Thus, 1 mL of stock solution of each sample was equivalent to100 mg of lyophilized extract or its fraction. Briefly, 0.25 mL of diluted stock solution of sample (1–35-fold) was mixed with 0.5 mL of the triolein emulsion (0.6 g of triolein, 25 mL of Tris-buffer and 0.4 g of bile acids) and pre-incubated at 37 °C for 5 min before adding lipase solution (0.063 mL). Blanks, devoid of enzymes (replaced by Tris-base buffer), were prepared for background correction. The control contained all the aforementioned components but without the tested samples. Finally, the reaction mixtures were incubated in a shaking bath (200 rpm) at 37 °C for 30 min. The reaction was terminated by adding 0.23 mL of HCl. Then, 3 mL of isooctane was added and vortexed for 0.5 min. The upper layer (2 mL) was collected, followed by the addition of 0.4 mL copper reagent (5% cooper acetate, pH 6.1 regulated by pyridine) according to Zhang, Xiao, Yang, Wang & Li (2014). After vortexing for 1 min, the upper layer was centrifuged at 10,000 rpm for 10 min and its absorbance (A) was measured at 720 nm against a reagent blank. All samples were assayed in triplicate. Method 2: Lipase activity was also measured using 4-methylumbelliferyl oleate (4-MUO) as substrate according to the procedure described previously (Podsędek et al., 2014). The pancreatic lipase assay was conducted in a 96-well plate. The diluted stock solution of sample (25 µL, prepared as described in method 1) was combined with 25 µL of enzyme solution, mixed and incubated at 37 °C for 5 min. Then, 50 µL of 0.1 mM 4-MUO solution was added. The samples were mixed immediately and incubated at 37 °C for 20 min. The amount of 4-methylumbelliferone released by lipase was measured with a microplate reader (SynergyTM2, BioTek Instruments Inc.) at an excitation wavelength of 360 nm and at an emission wavelength of 460 nm. The sample blank and the control blank (without substrate) were prepared analogously.

2. Materials and methods 2.1. Reagents Lipase (EC 3.1.1.3) from porcine pancreas type II, TRIS-base, orlistat, triolein, bile from bovine and ovine, dimethyl sulfoxide, 4-methylumbelliferyl oleate (4-MUO), ascorbic acid, phloroglucinol, acetic acid, trifluoric acid, formic acid, acetonitrile, quercetin (> 95%), (+)-catechin (> 99%), (−)-epicatechin (> 98%), naringin (> 95%) and gallic acid (> 98%) were obtained from Sigma Aldrich (Steinheim, Germany). Cyanidin 3-glucoside (> 96%), cyanidin 3-galactoside (> 97%), 5-O-caffeoylquinic acid (> 99%), p-coumaric acid (> 90%), protocatechuic acid (> 90%), quercetin 3-O-glucoside (> 99%), quercetin 3-O-galactoside (> 98%), quercetin 3-O-rutinoside (> 99%), isorhamnetin 3-O-glucoside (> 95%) were procured from Extrasynthese (Lyon, France). Procyanidins B2 (> 97%) and B3 (> 90%), 3-O-caffeoylquinic acid (> 98%), 4-O-caffeoylquinic acid (> 98%), were purchased from TransMIT (Gieβen, Germany), and procyanidin B1 (> 96%), from PhytoLab (Vestenbergsgreuth, Germany). Sodium carbonate, sodium chloride, sodium acetate, sodium hydroxide, hydrochloric acid, cooper acetate, pyridine, ethyl acetate, isooctane, n-butanol were received from Chempur (Piekary Śląskie, Poland). Calcium chloride and Folin-Ciocalteu reagent were from POCH (Gliwice, Poland), and n-hexane, acetone and methanol from POCH BASIC (Avantor Performance Materials Poland S.A., Gliwice, Poland). Formic acid and acetonitrile were HPLC grade, while all reagents and solvents from Chempur, POCH and POCH BASIC were used of analytical grade. Ultrapure water (SimplicityTM Water Purification System, Millipore, Marlborough, MA, USA) was used to prepare all solutions. 2.2. Preparation of samples Black chokeberry fruits were collected in the middle of September at a plantation near Łódź, in the central region of Poland. Phenolic compounds were extracted from 1 kg of the pre-ground chokeberry fruits at first with 7 L of acetone:water (70:30, v/v), and next with 5 L methanol supplemented with 0.1% (v/v) of trifluoric acid. Both these steps were conducted for 60 min at room temperature. The slurries were centrifuged at 5000 rpm for 10 min and the supernatants were combined, and concentrated at 40 °C (using a vacuum rotary evaporator RII, Büchi, Switzerland) to remove organic solvents. The concentrate was freezedried that yielded 211.4 g crude extract (CE). A part of CE (200 g) was suspended in water (0.9 L) and subjected to extraction with n-hexane (3 × 0.9 L) to remove lipophilic substances. Then the pH of the aqueous layer was adjusted to 7 with 0.1 M NaOH before extraction with ethyl acetate (3 × 0.9 L). The pH of the remaining aqueous layer was adjusted to 2 with 0.1 M HCl before the next extraction with ethyl acetate (3 × 0.9 L). The ethyl acetate fractions were concentrated in vacuum at 40 °C and lyophilized that yielded 0.59 g of ethyl acetate pH 7 fraction (EAF7) and 3.48 g of ethyl acetate pH 2 fraction (EAF2). The aqueous

2.4. Determination of inhibitory type To determine the kinetics of pancreatic lipase inhibition by CE and PRF, the changes of lipase activity caused by different concentrations of CE (13, 16 and 18 mg/mL of reaction mixture) and PRF (1.2, 1.4 and 1.6 mg/mL) were investigated at six concentrations of the triolein 197

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7

2

10

CE

16

4.0e-1

3.5e-1

AU

3.0e-1

2.5e-1

28

2.0e-1

29 30 27

1.5e-1

5

1.0e-1

5.0e-2

13

6

19 22

9 14 18

1

2.00

3.00

4.00

5.00

35

6.00

27

16

4.0e-1

32

24 23 226

7.00

28

8.00

9.00

Time 10.00

EAF7 29

26

3.5e-1

3.0e-1

14

2.5e-1

36 33

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10

2.0e-1

6

8

12 11

1.5e-1

1.0e-1

3

1

35

15 17

19

31

4

37 38

5.0e-2

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

Time 10.00

Fig.1. UPLC-DAD chromatograms (280 nm) of crude extract (CE) and phenolic fractions (EAF7, EAF2 and PRF) obtained from black chokeberry fruit. The peak number corresponds to the number in Table 1.

reagent as described in our previous work (Podsędek et al., 2014). The absorbance of reaction mixtures (sample, Folin-Ciocalteu reagent, sodium carbonate and water) was measured at 760 nm after 20 min incubation at ambient temperature. Total phenolics content was expressed as mg of gallic acid equivalents (GAE) per 1 g of extract or its fractions. The content of total proanthocyanidins was determined after acid depolymerization to the corresponding anthocyanidins as described by Rösch, Bergmann, Knorr & Kroh (2003). The content of proanthocyanidins was expressed as mg of cyanidin equivalents (CYE) per 1 g of extract or fraction.

substrate (6.2, 9.2, 14.4, 18.5, 24.6, and 30.8 mg/mL of reaction mixture). The bile acids concentrations were always 1.5-fold lower than those of triolein. The inhibition types of CE and PRF were determined on the basis of Lineweaver-Burk plots. 2.5. Combined effects of orlistat and either crude extract (CE) or polyphenol-rich fraction (PRF) Two concentrations of orlistat (0.1 and 0.2 µg/mL of reaction mixture) were combined with two CE (5.3 and 7.4 mg/mL of reaction mixture) or PFR concentrations (1.2 and 1.4 mg/mL of reaction mixture). The results were expressed as a percentage inhibition compared to the inhibitor-free control. The pancreatic lipase activity was measured by method with cooper reagent according to procedure described in Section 2.3 (Method 1).

2.7. Identification of phenolics by UPLC–MS Phenolic compounds were identified using an Acquity UPLC system coupled with a quadruple-time of flight (Q-TOF) MS instrument (Waters Corp., Milford, MA, USA) equipped with an electrospray ionization (ESI) source. Separation was achieved on an Acquity TMBEH C18 column (100 × 2.1 mm, 1.7 µm; Waters). The mobile phase was a mixture of 0.1% formic acid and acetonitrile. The UPLC separation

2.6. Quantification of phenolic compounds by spectrophotometric methods Total phenolics content was determined using Folin-Ciocalteu 198

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EAF2

7 2

3.0

AU

2.5

29 30

2.0

28

5 1.5

27

9 1.0

1 16 10 13315

1 5.0e-1

2.00

1.4e-1

3.00

4.00

7

2

18 19

25 2425

5.00

26

6.00

32 33 7.00

34 37 8.00

38 9.00

Time 10.00

PRF

10 16

1.2e-1

AU

1.0e-1

8.0e-2

15 6.0e-2

4.0e-2

9 5

1

13 14

18

20 22 229 32 19 21 24 26 28 30 27

2.0e-2

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

Time 10.00

Fig.1. (continued)

acid degradation of proanthocyanidins were separated using a HPLC system (Waters, Milford, MA) equipped with a Symmetry C18 (250 × 4.6 mm i.d, 5 µm) column (Waters), a gradient pump (1525), a fluorescence detector (2475), an auto-injector (2707) and a Breeze 2 system controller. The mobile phase was a binary gradient with 2.5% acetic acid and 80% acetonitrile with a flow rate of 1 mL/min (Sójka, Klimczak, Macierzyński & Kołodziejczyk, 2013). The volume of injected samples was 20 μL. The fluorescence detection was recorded at an excitation wavelength of 278 nm and an emission wavelength of 360 nm. The calibration curves, which were based on peak area, were established using (+)-catechin, (−)-epicatechin, procyanidins B1, B2 and B3, as well as (+)-catechin-phloroglucinol and (−)-epicatechin-phloroglucinol adducts standards (for the samples after the hydrolysis reaction). The mDP was calculated as the molar ratio of all the flavanol units (phloroglucinol adducts + terminal units) to (−)-epicatechin and (+)-catechin, which correspond to terminal units.

conditions and the Q-TOF-MS operating parameters were described in the previous work (Sosnowska et al., 2016). The data were recorded using the Mass-LynxTM V 4.1 software. Polyphenolic compounds were putatively identified based on their UV–Vis characteristic, and MS and MS2 properties using the data gathered in-house and from literature. 2.8. Quantification of phenolic compounds by HPLC Phenolic compounds were quantified using a Dionex HPLC system (Germering, Germany) equipped with a Cadenza Imtakt column C5-C18 (75 × 4.6 mm) with a guard column, an Ultimate 3000 diode array detector, a LPG-3400A pump, an EWPS-3000SI autosampler, a column thermostat TCC-3000SD and Chromeleonv. 6.8 software. The mobile phase was 4.5% formic acid in acetonitrile. The chromatographic separation conditions were described in the previous work (Sosnowska et al., 2016). Caffeoylquinic acid isomers were quantified as 5-O-caffeoylquinic acid, p-coumaric acid derivatives as p-coumaric acid, anthocyanins as cyanidin 3-O-glucoside, quercetin derivatives as quercetin 3-O-glucoside, isorhamnetin derivative as isorhamnetin 3-Oglucoside, and protocatechuic acid and depside 1 as protocatechuic acid, and eriodictyol 7-glucuronide as naringin. The results were expressed as mg of a compound per 1 g of extract or its fraction.

2.10. Statistical analysis The results were expressed as the means ± standard deviations of triplicate measurements. The data were analyzed by means of a oneway analysis of variance (ANOVA). A Duncan post hoc test was used to determine differences between the means with significance level p < 0.05.

2.9. Analysis of mDP of proanthocyanidins

3. Results and discussion

Acid catalysed degradation of polymeric proanthocyanidins in the excess of phloroglucinol was used to determine the mean degree of polymerization (mDP) of proanthocyanidins. The direct phloroglucinolysis of CE, EAF7, EAF2, and PRF (40–60 mg) was performed as described by Wojdyło, Oszmiański, and Bielicki (2013). The products of

The anti-obesity potential of black chokeberry fruits may be partially related to the inhibitory activity against pancreatic lipase (PL), bringing about the reduced dietary fats absorption from the intestinal 199

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Table 1 Tentative identification of phenolic compounds in chokeberry fruit crude extract and fractions. Peak

tR (min)

λmax (nm)

[MS-H]- [MS + H]+ (m/z)

MS/MS (m/z)

Identity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

1.68 2.07 2.27 2.67 2.86 3.08 3.17 3.39 3.45 3.59 3.75 3.87 3.96 4.00 4.17 4.24 4.41 4.45 5.22 5.27 5.42 5.50 5.54 5.88 6.14 6.21 6.42 6.55 6.65 6.66 7.06 7.23 7.30 7.54 7.56 7.69 7.93 9.96

261;294 254;325 242;278 242;278 311 281;312 245;325 242;278 245;325 515 242;278 242;278 515 242;278 266;299 516 242;278 328 516 258;353 257;353 277 323 257;355 245;324 257;355 257;354 257;354 257;354 284 281 270 281 242;324 255;355 266;299 255;355 255;373

153 353 577 289 337 329 353 577 353 449 577 577 449 289 319 419 865 353 419 625 625 565 367 595 367 609 463 609 463 463 433 403 433 515 623 333 623 301

109 191/179/135 289

Protocatechuic acid 3-O-caffeoylquinic acid Procyanidin dimera (+)-Catechin p-coumaroylquinic acida Vanillate hexosidea 5-O-caffeoylquinic acid Procyanidin dimer B2 4-O-caffeoylquinic acid Cyanidin 3-O-galactoside Procyanidina Procyanidina Cyanidin 3-O-glucoside (−)-epicatechin Depsidea Cyanidin 3-O-arabinosidea Procyanidin trimera Caffeoylquinic acida Cyanidin 3-O-xylosidea Quercetin 3-O-dihexosidea Quercetin 3-O-dihexosidea Sinapoylsiringinic acid hexosidea Caffeoylquinic acid methyl ester 1a Quercetin 3-O-vicianosidea Caffeoylquinic acid methyl ester 2a Quercetin 3-O-robinosidea Quercetin 3-O-galactoside Quercetin 3-O-rutinoside Quercetin 3-O-glucoside Eriodictyol 7-glucuronidea Naringenin hexoside 1a Sinapoylsiringinic acida Naringenin hexoside 2a Dicaffeoylquinic acida Isorhamnetin 3-O-rhamnosylhexoside 1a Jaboticabina Isorhamnetin 3-O-rhamnosylhexoside 2a Quercetin

a

191/163 167 191/179/135 289 191/179/135 287 289 289 287

+

+

301/165/137 287 577/289 191 287 301 301 403/223/179 161 301 179 301 301 301 301/257 287 271/255 223/179 271/255 353/191 315 301/165 315 151

+

+

Tentatively identified (Li et al., 2012; Slimestad et al., 2005; Wu et al., 2004).

phenols were 39.96%, 32.18%, 32.94%, 30.67% and 27.55%, respectively. The total phenolics contents of CE, EAF7, EAF2 and PRF, determined by Folin-Ciocalteu method, were found to be 62.34, 419.35, 132.31 and 323.79 (mg GAE/g extract or fraction), respectively (Table 3). Surprisingly, the sums of concentrations of phenolic compounds, which were quantified by HPLC, were lower and amounted to 31.56, 306.52, 125.84 and 74.80 mg/g of CE, EAF7, EAF2 and PRF, respectively. The above differences can be attributed to the incomplete quantification of all peaks that were visible in the HPLC chromatographs, and potential interfering impact of other compounds on the results of the Folin-Ciocalteu assay (Everette et al., 2010). The results of the UPLC-MS and HPLC assays of low molecular weight phenolics, showed that anthocyanins dominated in CE and PRF, while hydroxycinnamic acids (HCA) and flavonols were the dominating phenolics in EAF2 and EAF7, respectively (Table 3). Apart from simple phenols, black chokeberry fruits are known as a rich source of proanthocyanidins which have been identified as procyanidins (Taheri, Connolly, Brand & Bolling, 2013; Wangensteen et al., 2014; Wu et al., 2004). In this study, the highest amounts of total proanthocyanidins, quantified by the acidbutanol test, were found in PRF (116.37 mg CYE/g) and EAF7 (70.09 mg CYE/g) (Table 3). Proanthocyanidins are oligomeric and polymeric flavanols with different degrees of polymerization (DP). The mean size of procyanidin molecules (mDP), which was determined by the floroglucinol-catalysed cleavage reactions method, depended on a sample and ranged from 1.63 to 42.35, with the highest value for CE. Wu et al. (2004) reported that polymeric procyanidins with DP > 10 dominated in chokeberry fruits (82% of total procyanidins).

tract. Black chokeberry fruits are valued as a rich source of phenolic compounds, mainly anthocyanins, flavanols, procyanidins and flavonols which have been reported as PL inhibitors (Jurikova et al., 2017; Sergent, Vanderstraeten, Winand, Beguin & Schneider, 2012). This study aimed at determination of phenolic profiles and the inhibitory effect of crude extract (CE) and its fractions (EAF7, EAF2 and PRF) on the PL activity as well as identification of potential anti-lipase agents from chokeberry fruits. 3.1. Composition of phenolic compounds Phenolic profiles of CE, EAF2, EAF7 were analyzed by UPLC-MS method (Fig. 1). Phenolic compounds were identified based on their retention times, Uv–vis spectra (200 – 600 nm), comparison with standard reference compounds ((+)-catechin, (−)-epicatechin, procyanidins B2, protocatechuic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, quercetin and quercetin 3glucoside, galactoside and rutinoside, and cyanidin 3-glucoside and galactoside) and the literature data for the rest compounds (Li et al., 2012; Slimestad, Torskangerpoll, Nateland, Johannessen & Giske, 2005). A total of 38 phenolic compounds, including twelve phenolic acids, ten flavonols, four anthocyanins, three flavanones, one depside and seven flavanols were detected in the tested samples (Table 1). Quantitative evaluation of phenolic compounds contents were determined by HPLC-DAD analysis. Cyanidin 3-O-galactoside was the main phenolic constituent in CE and PRF, quercetin 3-O-robinoside with quercetin 3-O-galactoside dominated in EAF 7 while 3- and 5-Ocaffeoylquinic acid in EAF2 (Table 2). Their percentages among total 200

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Table 2 Concentration of phenolic compounds identified in crude extract (CE) and fractions (EAF7, EAF2, PRF). Tentative identification

Content (mg/g)

Protocatechuic acid 3-O-caffeoylquinic acidA Procyanidin dimerB (+)-Catechin p-coumaroylquinic acidC Vanillate hexosideD 5-O-caffeoylquinic acid Procyanidin dimer B2B 4-O-caffeoylquinic acidA Cyanidin 3-O-galactosideE ProcyanidinB ProcyanidinB Cyanidin 3-O-glucoside (−)-epicatechin DepsideF Cyanidin 3-O-arabinosideE Procyanidin trimerB Caffeoylquinic acidA Cyanidin 3-O-xylosideE Quercetin 3-O-dihexosideG Quercetin 3-O-dihexosideG Sinapoylsiringinic acid hexosideH Caffeoylquinic acid methyl ester 1A Quercetin 3-O-vicianosideG Caffeoylquinic acid methyl ester 2A Quercetin 3-O-robinoside + Quercetin 3-O-galactosideG Quercetin 3-O-rutinoside + Quercetin 3-O-glucosideG Eriodictyol 7-glucuronideI Naringenin hexoside 1I Sinapoylsiringinic acidH Naringenin hexoside 2I Dicaffeoylquinic acidA Isorhamnetin 3-O-rhamnosylhexoside 1 J JaboticabinI Isorhamnetin 3-O-rhamnosylhexoside 2 J QuercetinG Total

CE

EAF7

EAF2

PRF

0.07 ± 0.01 4.45 ± 0.02 0 0 0.02 ± 0.01 0 4.13 ± 0.03 0 0.13 ± 0.02 12.61 ± 0.09 0 0 0.57 ± 0.03 0 0.18 ± 0.03 5.36 ± 0.03 0 0 0.73 ± 0.04 0.23 ± 0.02 0.09 ± 0.02 0.23 ± 0.03 0.02 ± 0.01 0.38 ± 0.03 0 1.04 ± 0.02 0.44 ± 0.02 0.48 ± 0.03 0 0.30 ± 0.3 0 0.02 ± 0.01 0.02 ± 0.01 0.04 ± 0.01 0.02 ± 0.01 0 31.56 ± 0.17

2.72 ± 0.04 0 2.98 ± 0.06 5.93 ± 0.06 0 0.24 ± 0.03 0 10.22 ± 0.06 0 4.58 ± 0.03 12.45 ± 0.05 17.16 ± 0.06 0.39 ± 0.02 67.67 ± 0.20 0.93 ± 0.02 11.48 ± 0.03 5.95 ± 0.04 0 2.42 ± 0.01 0 0 0 0 0 0 100.98 ± 3.31 37.56 ± 0.71 0 4.17 ± 0.03 0 0.38 ± 0.01 0 1.42 ± 0.03 12.63 ± 0.06 0.25 ± 0.02 4.00 ± 0.05 306.52 ± 3.05

7.59 ± 0.06 38.60 ± 0.05 0 0 1.14 ± 0.02 0 34.67 ± 0.08 0 5.08 ± 0.04 1.05 ± 0.02 0 0 0.05 ± 0.01 0 0.41 ± 0.03 2.15 ± 0.02 0 1.11 ± 0.02 0.51 ± 0.02 0 0 0 0 0 0.51 ± 0.02 11.73 ± 0.10 6.04 ± 0.05 11.85 ± 0.05 0 2.33 ± 0.03 0 0.49 ± 0.03 0.06 ± 0.01 0 0.10 ± 0.01 0.37 ± 0.02 125.84 ± 0.25

0.25 ± 0.01 18.75 ± 0.04 0 0 0.06 ± 0.01 0 6.21 ± 0.03 0 1.20 ± 0.03 24.07 ± 0.05 0 0 1.13 ± 0.03 0 1.08 ± 0.02 9.47 ± 0.03 0 0 1.26 ± 0.02 1.34 ± 0.04 0.55 ± 0.03 1.26 ± 0.03 0 2.10 ± 0.03 0 1.40 ± 0.03 2.20 ± 0.03 0.14 ± 0.01 0 2.18 ± 0.04 0 0 0.09 ± 0.01 0 0.07 ± 0.01 0 74.80 ± 0.13

A: quantified as 5-O-caffeoylquinic acid equivalents; B: quantified as (−)-epicatechin equivalent; C: quantified as p-coumaric acid equivalent; D: quantified as vanillin equivalent; E: quantified as cyanidin 3-O-glucoside equivalents; F: quantified as protocatechuic acid equivalent; G: quantified as quercetin 3-O-glucoside equivalents; H: quantified as gallic acid equivalents; I: quantified as naringin equivalent; J: quantified as isorhamnetin 3-O-glucoside equivalents. Table 3 Phenolic profiles (mg/g) of crude extract (CE) and phenolic fractions (EAF7, EAF2 and PRF) obtained from black chokeberry fruit. Phenolic compounds 1

Total phenolics Proanthocyanidins2 mDP3 Anthocyanins4 HCA4 HBA4 Flavonols4 Flavanols4 Others4 Sum of phenolics4

CE

EAF7

EAF2

PRF

62.34 ± 1.87a 13.55 ± 0.57a 42.35 ± 0.24d 19.27 ± 0.05c 8.77 ± 0.02b 0.78 ± 0.06a 2.22 ± 0.10a 0a 0.52 ± 0.04b 31.56 ± 0.17a

419.35 ± 11.36d 70.09 ± 2.76b 2.64 ± 0.17b 18.87 ± 0.05b 0a 3.89 ± 0.06b 144.21 ± 2.68d 122.37 ± 0.35b 17.18 ± 0.04d 306.52 ± 3.05d

132.31 ± 13.88b 7.17 ± 0.34a 1.63 ± 0.20a 3.76 ± 0.03a 81.60 ± 0.15c 10.33 ± 0.09d 18.30 ± 0.14c 0a 11.85 ± 0.05c 125.84 ± 0.25c

323.79 ± 11.26c 116.37 ± 10.84c 19.40 ± 0.44c 35.93 ± 0.07d 26.22 ± 0.05d 4.77 ± 0.04c 7.74 ± 0.09b 0a 0.14 ± 0.01a 74.80 ± 0.13b

Values are means ± standard deviations; n = 3–6. Mean values with different letters within the same row are statistically different (p < 0.05). 1 determined by Folin-Ciocalteu method. 2 determined after acid depolymerization. 3 mDP mean degree of polymerization of the proanthocyanidins estimeted by phloroglucinolysis. 4 determined by HPLC method.

fluorimetric method using 4-methylumbelliferyl oleate (4MUO), and spectrophotometric method with triolein/bile acids emulsion as substrates. The first method enables to determine only the esterase activity of the lipase because 4MUO is a simple synthetic ester. The second method allows for the assay of the lipolytic activity of PL and the

3.2. Pancreatic lipase (PL) inhibitory activity To identify phenolic compounds that are responsible for the antilipase activity of black chokeberry fruits, we determined the effect of CE, EAF2, EAF7 and PRF on pancreatic lipase activity by the

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increased with an increase in their DP and was higher than the activity of monomer and dimer of flavan-3-ol as well as chlorogenic acid (Sugiyama et al., 2007). Also other authors suggested the relationship between proanthocyanidins content and the extent of lipase inhibition by grape seed (Moreno et al., 2003), apple (Sugiyama et al., 2007) and lingonberry (McDougall, Kulkarni & Stewart, 2008) extracts. According to Gironés-Vilaplana et al. (2014) the lipase inhibition by fruits was correlated strongly with total anthocyanins. Worsztynowicz, Napierała, Białas, Grajek, and Olkowicz (2014) reported that among the black chokeberry fruit anthocyanins, only cyanidin-3-glucoside showed the lipase inhibitory activity in the assay with p-nitrophenol palmitate as substrate. In this study, the content of the latter anthocyanin in CE and PRF was the lowest among the identified pigments (Table 2). Surprisingly, EAF7 exhibited the lowest inhibitory activity in both the assays despite the highest content of total phenolics and flavonols, and high levels of total proanthocyanidins and anthocyanins. Noteworthy, the mDP of proantocyanidins was 16- and 7-fold lower for EAF7 than for CE and PRF, respectively. This suggests the significance of higher flavanol oligomers and polymers in inhibiting PL activity by black chokeberry fruit extract. On the basis of quantity, caffeoylquinic acid methyl ester dominated in this fraction (Table 2). This suggests that methyl group bound to quinic acid was responsible for the lower inhibitory activity of caffeoylquinic acids which were contained in CE, PRF and EAF2. 3-, 4- and 5-caffeoylquinic acids, and 3,4-, 3,5- and 4,5dicaffeoylquinic acids were found to be competitive inhibitors of PL (Hu et al., 2015). Noteworthy, all the tested samples inhibited pancreatic lipase to a lesser extent than orlistat (a clinically-approved lipase inhibitor). The IC50 values for orlistat were 0.70 ± 0.03 µg/mL for 4-MUO assay and 0.34 ± 0.01 µg/mL for triolein emulsion assay. The weaker effect of CE and its fractions may be ascribed to the complexity of composition and multiple interactions not only between the phenolic compounds but also with non-phenolic components. Noteworthy, also the standards of different phenolics demonstrated the weaker anti-lipase activities than orlistat (Li, Yang, Gao, Zhang & Wu, 2011; Sergent et al., 2012). So far, only a few authors tried to elucidate the kinetics of pancreatic lipase inhibition by fruit ingredients. In this study, the modes of pancreatic lipase inhibition by the most active samples (PRF and CE) were determined on the basis of the double-reciprocal Lineweaver-Burk plots (Fig. 2). The PL inhibition by CE was noncompetitive, while in case of PRF, the intersection of lines in the first quadrant suggests a mixed type inhibition. Thus, presumably the mechanism of PL inhibition by CE and PRF is different than in case of orlistat, which forms the covalent bond with serine residue in the lipase’s active site and is a competitive inhibitor (Yun, 2010). For comparison, some flavonols were reported to be the noncompetitive lipase inhibitors (Li et al., 2011) while cocoa procyanidins were mixed inhibitors (Gu, Hurst, Stuart & Lambert, 2011). According to literature (Ballinger & Peikin, 2002), orlistat may cause serious side effects and therefore its combinations with certain food components may provide safer route to prevention and treatment of obesity. To the best of our knowledge, this is the first report of cumulative effects observed for the mixtures of orlistat and black chokeberry phenolics. Our results showed that the sums of values of percentage PL inhibition by CE, PRF and orlistat, when used separately, were nearly the same (the lack of statistically significant differences) as the values of percentage PL inhibition caused by the mixtures of orlistat with either CE or PRF (Fig. 3). This may suggest that black chokeberry fruit components do not compete with orlistat (the competitive inhibitor) for the position in the active site and bind to other sites of PL molecules. The observed cumulative effect of orlistat combinations with either CE or PRF suggests that such preparations may support the antiobesity action of a pharmaceutical such as orlistat.

Table 4 Inhibition of pancreatic lipase as IC50 values of chokeberry extract and fractions. Sample

4-Methylumbelliferyl oleate μg/ml

Triolein mg/ml

CE EAF7 EAF2 PRF

60.31 ± 0.29b 421.96 ± 17.33d 118.78 ± 2.20c 14.51 ± 0.29a

14.31 ± 0.04b – – 2.44 ± 0.05a

Values are mean ± SD, n = 3. Samples without data did not inhibit 50% of enzyme. Mean values with different letters within the same column are statistically different (p < 0.05).

A

Control 10.6 mg/ml

7

12.7 mg/ml

6

14.8 mg/ml

5

1/[V]

4 3 2 1

-0.10

0 0.00 -1

-0.05

0.05

0.10

0.15

0.20

1/[S] (ml/mg)

-2

B

Control 1.2 mg

12

1.4 mg

10

1.6 mg

8

1/[[V]

6 4 2

-0.10

-0.05

0 0.00 -2

0.05

0.10

0.15

0.20

1/[S] (ml/mg)

-4

Fig. 2. Lineweaver-Burk plots showing the impact of the control, CE (A) and PRF (B) on the pancreatic lipase activity.

reaction mixture composition mimics to some extent the physiological conditions (McClements & Li, 2010). In both the assays, PRF displayed the highest inhibitory effect on pancreatic lipase while the IC50 values obtained for CE were from 4 to 6 times higher than for PRF (Table 4). The lower anti-lipase activity of CE may be associated with the presence of non-phenolic substances, such as sugars, organic acids and proteins which were removed when PRF was isolated. Furthermore, the concentrations of proanthocyanidins, flavonols and phenolic acids in PRF were 9-fold, 3-fold and nearly twice higher than in CE, respectively (Table 3). The HPLC analysis showed that cyanidin-3-galactoside, cyanidin-3-arabinoside, 3-caffeoylquinic and 5-caffeoylquinic acids predominated quantitatively in these fractions (Table 2). Noteworthy, the mDP of proanthocyanidins from CE was twice higher compared to PRF (42.35 versus 19.40). The results of our previous study in which emulsions of different lipids were used as substrates, showed that the degree of lipase inhibition by chokeberry juices was correlated with the mDP of proanthocyanidins (Sosnowska et al., 2016). Similarly, the antilipase activity of apple procyanidins (from trimer to heptamer) 202

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A

a Inhibition of lipase [%]

80 70

a

a

a

a

a

a

a

Orlistat (0.1 g/ml) Orlistat (0.2 g/ml) CE (5.3 mg/ml) CE (7.4 mg/ml)

60

Orlistat and CE

50 40 30 20 10 0 1

2

1

2

1

2

B

a

Inhition of lipase [%]

70 60 50

a

a

1

2

a

a

1

2

1

2

a

a

a

Orlistat (0.1 g/ml) Orlistat (0.2 g/ml) PRF (1.2 mg/ml) PRF (1.4 mg/ml) Orlistat and PRF

40 30 20 10 0 1

2

1

2

Fig. 3. Percentage lipase inhibition by orlistat, CE (A), and PRF (B) in various combinations; 1 – inhibition of lipase by single inhibitors; 2 – inhibition of lipase by mixtures of inhibitors. Different letters denote significant differences (p < 0.05) between the sum of the percentage inhibition caused by individual inhibitors (1) and the inhibition caused by a mixture of these inhibitors (2).

Conflict of interest

4. Conclusion

The authors declare no conflict of interest.

The growing interest in application of natural dietary phytochemicals as potential antiobesity agents in human has been observed over the last decades. The high PL inhibitory activity of black chokeberry fruit extract (CE) is ascribed to phenolic compounds. This study was performed because of the lack of detailed reports on the influence of different black chokeberry constituents on pancreatic lipase activity. Fractions of CE were isolated either with ethyl acetate (EAF7 and EAF2) or on the Sep-Pak C18 column (PRF). The highest lipase inhibitory activity was exhibited by PRF which was rich in proanthocyanidins and anthocyanins. Proanthocyanidins, particularly those with high DP may exert local effects in the gastrointestinal tract as lipase inhibitors due to their poor bioavailability. Further studies are necessary to establish a simple procedure of black chokeberry procyanidins extraction and to purify the most active of these compounds.

Ethics statement This article does not contain any studies with human or animal subjects. References Ballinger, A., & Peikin, S. R. (2002). Orlistat: Its current ststus as an anti-obesity drug. European Journal of Pharmacology, 440, 109–117. Baum, J. I., Howard, L. R., Prior, R. L., & Lee, S.-O. (2016). Effect of Aronia melanocarpa (Black Chokeberry) supplementation on the development of obesity in mice fed a high-fat diet. Journal of Berry Research, 6, 203–212. Bhaswant, M., Shafie, S. R., Mathai, M. L., Mouatt, P., & Brown, L. (2017). Anthocyanins in chokeberry and purple maize attenuate diet-induced metabolic synrome in rats. Nutrition, 41, 24–31. Birari, R. B., & Bhutani, K. K. (2007). Pancreatic lipase inhibitors from natural sources: Unexplored potential. Drug Discovery Today, 12, 879–889. Denev, P., Kratchanova, M., Ciz, M., Lojek, A., Vasicek, O., Nedelcheva, P., ... Vojtek, L. (2014). Biological activities of selected polyphenol-rich fruits related to immunity and gastrointestinal health. Food Chemistry, 157, 37–44. Everette, J. D., Bryant, Q. M., Green, A. M., Abbey, Y. A., Wangila, G. W., & Walker, R. B. (2010). Thorough study of reactivity of various compound classes toward the FolinCiocalteu reagent. Journal of Agricultural and Food Chemistry, 58, 8139–8144. Fu, C., Jiang, Y., Guo, J., & Su, Z. (2016). Natural products with anti-obesity effects and different mechanisms of action. Journal of Agricultural and Food Chemistry, 64, 9571–9585. George, S., Brat, P., Alter, P., & Amiot, M. J. (2005). Rapid determination of polyphenols and vitamin C in plant-derived products. Journal of Agricultural and Food Chemistry,

Acknowledgments The work was funded by the National Science Centre, allocated on the basis of Decision No DEC-2011/01/B/NZ9/02046. The publication was supported by the Wrocław Centre of Biotechnology, under the program The Leading National Research Centre (KNOW – Poland) for the years 2014–2018.

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D. Sosnowska et al.

Slimestad, R., Torskangerpoll, K., Nateland, H. S., Johannessen, T., & Giske, N. H. (2005). Flavonoids from black chokeberries, Aronia melanocarpa. Journal of Food Composition and Analysis, 18, 61–68. Sójka, M., Klimczak, E., Macierzyński, J., & Kołodziejczyk, K. (2013). Nutrient and polyphenolic composition of industrial strawberry press cake. European Food Research and Technology, 237, 995–1007. Sosnowska, D., Podsędek, A., Kucharska, A. Z., Redzynia, M., Opęchowska, M., & Koziołkiewicz, M. (2016). Comparison of in vitro anti-lipase and antioxidant activities, and composition of commercial chokeberry juices. European Food Research and Technology, 242, 505–515. Sosnowska, D., Podsędek, A., Redzynia, M., & Żyżelewicz, D. (2015). Effects of fruit extracts on pancreatic lipase activity in lipid emulsions. Plant Foods for Human Nutrition, 70, 344–350. Sugiyama, H., Akazome, Y., Shoji, T., Yamaguchi, A., Yasue, M., Kanda, T., & Ohtake, Y. (2007). Oligomeric procyanidins in apple polyphenol are main active components for inhibition of pancreatic lipase and triglyceride absorption. Journal of Agricultural and Food Chemistry, 55, 4604–4609. Szajdek, A., & Borowska, E. J. (2008). Bioactive compounds and health-promoting properties of berry fruits: A review. Plant Foods for Human Nutrition, 63, 147–156. Taheri, R., Connolly, B. A., Brand, M. H., & Bolling, B. W. (2013). Underutillized chokeberry (Aronia melanocarpa, Aronia arbutifolia, Aronia prunifolia) accessions are rich sources of anthocyanins, flavonoids, hydroxycinnamic acids, and proanthocyanidins. Journal of Agricultural and Food Chemistry, 61, 8581–8588. Takahashi, A., Shimizu, H., Okazaki, Y., Sakaguchi, H., Taira, T., Suzuki, T., & Chiji, H. (2015). Anthocyanin-rich phytochemicals from aronia fruits inhibit visceral fat accumulation and hyperglycemia in high-fat diet-induced dietary obese rats. Journal of Oleo Science, 64, 1243–1250. Wangensteen, H., Bräunlich, M., Nikolic, V., Malterud, K. E., Slimestad, R., & Barsett, H. (2014). Anthocyanins, proanthocyanidins and total phenolics in four cultivars of aronia: Antioxidant and enzyme inhibitory effects. Journal of Functional Foods, 7, 746–752. WHO (2017). Obesity and overweight < http://www.who.int/news-rom/fact-sheets/ detail/obesity-and-overweight > Accessed on July 17, 2018. Wojdyło, A., Oszmiański, J., & Bielicki, P. (2013). Polyphenolic composition, antioxidant activity, and polyphenol oxidase (PPO) activity of quince (Cydonia oblonga Miller) varieties. Journal of Agricultural and Food Chemistry, 61, 2762–2772. Worsztynowicz, P., Napierała, M., Białas, W., Grajek, W., & Olkowicz, M. (2014). Pancreatic α–amylase and lipase inhibitory activity of polyphenolic compounds present in the extract of black chokeberry (Aronia melanocarpa L.). Process Biochemistry, 49, 1457–1463. Wu, X., Gu, L., Prior, R. L., & McKay, S. (2004). Characterization of anthocyanins and proanthocyanidins in some cultivars of Ribes, Aronia and Sambucus and their antioxidant capacity. Journal of Agricultural and Food Chemistry, 52, 7846–7856. Yun, J. W. (2010). Possible anti-obesity therapeutics from nature – A review. Phytochemistry, 71, 1625–1641. Zhang, J., Xiao, L., Yang, Y., Wang, Z., & Li, G. (2014). Lignin binding to pancreatic lipase and its influence on enzymatic activity. Food Chemistry, 149, 99–106.

53, 1370–1373. Gironés-Vilaplana, A., Baenas, N., Villaño, D., Speisky, H., Garcia-Viguera, C., & Moreno, A. A. (2014). Evaluation of Latin-American fruits rich in phytochemicals with biological effects. Journal of Functional Foods, 7, 599–608. Gu, Y., Hurst, W. J., Stuart, D. A., & Lambert, J. D. (2011). Inhibition of key digestive enzymes by cocoa extracts and procyanidins. Journal of Agricultural and Food Chemistry, 59, 530–5311. Hu, B., Cui, F., Yin, F., Zeng, X., Sun, Y., & Li, Y. (2015). Caffeoylquinic acids competitively inhibit pancreatic lipase through binding to the catalytic triad. International Journal of Biological Macromolecules, 80, 529–535. Jurikova, T., Mlcek, J., Skrovankova, S., Sumczynski, D., Sochor, J., Hlavacova, I., ... Orsavova, J. (2017). Fruits of black chokeberry Aronia melanocarpa in the prevention of chronic diseases. Molecules, 22, 944 23 pages, DOI: 10.3390/molecules22060944. Kardum, N., Petrović-Oggiano, G., Takic, M., Glibetić, N., Zec, M., Debeljak-Martacic, J., & Konić-Ristić, A. (2014). Effects of glucomannan-enriched, aronia juice-based supplement on cellular antioxidant enzymes and membrane lipid status in subjects with abdominal obesity. The Scientific World Journal, Article ID 869250, 7 pages, DOI: 10. 1155/2014/869250. Li, J., Deng, Y., Yuan, Ch., Pan, L., Chai, H., & Keller, W. J. (2012). Antioxidant and quinone reductase-inducing constituents of black chokeberry (Aronia melanocarpa) fruit. Journal of Agricultural and Food Chemistry, 60, 11551–11559. Li, Y.-Q., Yang, P., Gao, F., Zhang, Z.-W., & Wu, B. (2011). Probing the interaction between 3 flavonoids and pancreatic lipase by methods of fluorescence spectroscopy and enzymatic kinetics. European Food Research and Technology, 233, 63–69. McClements, D. J., & Li, Y. (2010). Review of in vitro digestion models for rapid screening of emulsion-based systems. Food & Function, 1, 32–59. McDougall, G. J., Kulkarni, N. N., & Stewart, D. (2008). Current developments on the inhibitory effects of berry polyphenols on digestive enzymes. BioFactors, 34, 73–80. Mikulic-Petkovsek, M., Schmitzer, V., Slatnar, A., Stampar, F., & Veberic, R. (2012). Composition of sugars, organic acids, and total phenolics in 25 wild or cultivated berry species. Journal of Food Science, 77, C1064–C1070. Moreno, D. A., Ilic, N., Poulev, A., Brasaemle, D. L., Fried, S. K., & Raskin, I. (2003). Inhibitory effects of grape seed extract on lipases. Nutrition, 19, 876–879. Podsędek, A., Majewska, I., Redzynia, M., Sosnowska, D., & Koziołkiewicz, M. (2014). In vitro inhibitory effect on digestive enzymes and antioxidant potential of commonly consumed fruits. Journal of Agricultural and Food Chemistry, 62, 4610–4617. Qin, B., & Anderson, R. A. (2012). An extract of chokeberry attenuates weight and modulates insulin, adipogenic and inflammatory signalling pathways in epididymal adipose tissue of rats fed a fructose-rich diet. British Journal of Nutrition, 108, 581–587. Rösch, D., Bergmann, M., Knorr, D., & Kroh, L. W. (2003). Structure-antioxidant efficiency relationships of phenolic compounds and their contribution to the antioxidant activity of sea buckthorn juice. Journal of Agricultural and Food Chemistry, 51, 4233–4239. Sergent, T., Vanderstraeten, J., Winand, J., Beguin, P., & Schneider, Y.-J. (2012). Phenolic compound and plant extracts as potential natural anti-obesity substances. Food Chemistry, 135, 68–73.

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