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Inhibitory potential of red cabbage against digestive enzymes linked to obesity and type 2 diabetes Anna Podsedek, Iwona Majewska, and Alicja Kucharska J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02499 • Publication Date (Web): 28 Jul 2017 Downloaded from http://pubs.acs.org on July 29, 2017

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Journal of Agricultural and Food Chemistry

Inhibitory potential of red cabbage against digestive enzymes linked to obesity and type 2 diabetes Anna Podsędeka*, Iwona Majewskaa, Alicja Z. Kucharskab a

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences,

Lodz University of Technology, Stefanowskiego 4/10, 90-924 Łódź, Poland b

Department of Fruit, Vegetable and Plant Nutraceutical Technology, Wroclaw University of Environmental

and Life Sciences, Chełmońskiego 37, 51-630 Wrocław, Poland

*

Corresponding author: Anna Podsędek

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 4/10, 90-924 Łódź, Poland Fax number: 48 42 6366618 Phone: 48 42 6313435 E-mail: [email protected]

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ABSTRACT

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Assays of the inhibitory potential against enzymes involved in carbohydrate and lipid

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digestion (α-amylase, α-glucosidase and lipase) as well as the total contents of phenolics and

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anthocyanins, anthocyanin profile, and antioxidant capacity revealed significant differences (p

5

< 0.05) between five varieties of red cabbage. Among the varieties, the highest inhibitory

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activity against α-glucosidase (IC50 = 3.87±0.12 mg DW of cabbage/mL) and lipase

7

(IC50=1.57±0.06 mg DW/mL) was exhibited by Koda variety, which showed the highest

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antioxidant capacity in ABTS (TEAC = 141±4.71 µmol/g DW) and FRAP (TEAC = 125±

9

1.94 µmol/g DW) assays, and had the highest total phenolics level (19.6±0.48 mg/g DW). The

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highest total anthocyanin content (12.0±0.16 mg/g DW) and inhibitory activity against α-

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amylase (IC50 = 69.0±3.65 mg DW of cabbage/mL) was shown by the Kissendrup variety.

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The anthocyanin profiles of these two varieties were characterized by the highest percentages

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of diacylated cyanidin derivatives. There was no correlation between the contents of phenolic

14

compounds and lipase inhibitory activity, but inhibition of α-amylase was correlated with

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concentrations of monoacylated and diacylated anthocyanins, while inhibition of α-

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glucosidase increased with total phenolics and diacylated anthocyanins levels.

17 18

KEYWORDS: red cabbage, α-amylase, α-glucosidase, lipase, antioxidant capacity

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INTRODUCTION

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The increasing prevalence of obesity is accompanied by a growing prevalence of type 2

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diabetes and is associated in part with major worldwide changes in caloric intake and diet

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composition. One of strategies aiming at prevention and treatment of obesity and type 2 2

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diabetes relies on application of nutrient digestion and absorption inhibitors. Suppression of

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activity of pancreatic lipase, α-glucosidase and α-amylase is positively associated with the

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reduction of fat and sugar absorption from the gastrointestinal tract. Plant derived foods,

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especially fruits and vegetables, can be a dietary source of polyphenolic inhibitors of digestive

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enzymes involved in lipid and carbohydrate metabolism.1-3

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Numerous fruits have been screened in vitro as the source of pancreatic lipase, α-

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glucosidase and α-amylase inhibitors,2-7 while there are only a few reports on the occurrence

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of such inhibitors in vegetables.5,8-10 The results of these studies suggest the positive

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correlation between the inhibitory activity of vegetables and polyphenol and anthocyanin

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contents. It was found that an anthocyanin extract and a polyphenol extract from red cabbage

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inhibited α-glucosidase and lipase, respectively.5,8 Therefore, we decided to determine the

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inhibitory activity of five red cabbage varieties against digestive enzymes degrading dietary

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sugars and lipids.

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Red cabbage (Brassica oleracea var. capitata rubra) is a rich dietary source of phenolic

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compounds, especially structurally differentiated acylated anthocyanins.11-14 This widely

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cultivated in Europe, North America, China and Japan vegetable demonstrated the potential

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therapeutic effect in diabetic and obese rats.15,16 To the best of our knowledge there are no

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literature reports comparing the anti-obesity and anti-diabetic effects, antioxidant capacity

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and anthocyanin profiles of different red cabbage varieties. The objective of this study was to

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determine: inhibitory activities of five varieties of red cabbage against digestive enzymes such

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as: porcine pancreatic lipase type II, intestinal rat α-glucosidase and α-amylase from porcine

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pancreas type VI-B, antioxidant capacities (radical-scavenging capacity (ABTS assay) and

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ferric-reducing antioxidant power (FRAP assay), the contents of total phenols (using Folin-

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Ciocalteu reagent) and total anthocyanins (by pH-differential assay) as well as anthocyanins

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profiles (by UPLC-PDA-MS technique). 3

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MATERIALS AND METHODS

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Chemicals. Gallic acid, cyanidin-3-glucoside, rat intestinal α-glucosidase (EC 3.2.1.20), α-

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amylase (EC 3.2.11) from porcine pancreas type VI-B, lipase (EC 3.1.1.3) from porcine

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pancreas type II, 4-methylumbelliferyl α-D-glucopyranoside (4-MUG), 4-methylumbelliferyl

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oleate (4-MUO), TRIS-base, potassium persulfate, 2,2’-azinobis(3-ethyl-benzothiazoline-6-

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sulphonic

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tetramethychroman-2-carboxylic acid (Trolox), acarbose, orlistat and acetonitrile were

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obtained from Sigma. Cyanidin 3-glucoside was purchased from Extrasynthese (Genay,

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France). Methanol and sodium carbonate were purchased from Chempur. Folin-Ciocalteu

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reagent, potato starch, hydrochloric acid, iodine, potassium iodide, sodium chloride and

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calcium chloride of analytical grade were purchased from POCH (Gliwice, Poland). Ultra

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purity water was prepared in the laboratory using a SimplicityTM Water Purification System

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(Millipore, Marlborough, MA, USA).

acid)

(ABTS),

2,4,6-tris-2-pyridyl-s-triazine

(TPTZ),

6-hydroxy-2,5,7,8-

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Plant Material. Koda and Kissendrup red cabbage (Brassica oleracea var capitata rubra)

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varieties were purchased from a farm of the PlantiCo Horticulture Breeding and Seed

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Production (Golębiew, Poland) while varieties Haco, Kalibos and Langedijker were obtained

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from commercial gardens near Łódź (central region of Poland). The edible parts of raw red

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cabbage were chopped, lyophilised (Alpha 1-2 LDplus, Christ, Osterode, Germany), finely

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ground using a coffee grinder and applied for preparation of crude extracts.

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Extraction Procedure. Samples of lyophilised cabbage (2 g) were extracted twice with 50

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mL of 70% methanol (v/v) for 15 min at room temperature using a magnetic stirrer and the

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suspensions were centrifuged at 4000 rpm for 15 min.17 The two supernatants were combined

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and concentrated using a rotary evaporator (Büchi Labortechnik AG, Switzerland) at 40 °C to

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the volume of 20 mL. Thus, 1 mL of crude extract was equivalent to 0.1 g of lyophilised red

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cabbage. 4

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Lipase Inhibition Assay. The activity of lipase was determined in the absence (control)

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and presence of the inhibitor by measuring the release of 4-methylumbelliferone from 4-

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MUO, using the fluorimetric method, as described previously.6 Reaction mixtures containing

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4-MUO as substrate, the lipase from porcine pancreas type II, crude extract and Tris buffer

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(pH 7.4) were incubated at 37 °C for 20 min. The controls contained the buffer instead of the

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extract. Orlistat was used as positive control. The amount of 4-methylumbelliferone released

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by lipase was measured with a microplate reader (SynergyTM2, BioTek Instruments Inc.) at

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an excitation wavelength of 360 nm and at an emission wavelength of 460 nm. The

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percentage inhibition (%) was calculated according to the following formula: ℎ % = 100 ∗ 1 −

 − 

  − 

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Where FA, FC, FB and FD were the values of fluorescence of the sample, control, blank sample

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and blank control, respectively.

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α-Glucosidase Inhibition Assay. The inhibition of α-glucosidase activity was determined

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by measuring the amount of 4-methylumbelliferone hydrolyzed from a fluorogenic substrate

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(4-MUG). The α-glucosidase solution was prepared as described previously by Podsędek et

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al.6 Aliquots (20 µL) of the red cabbage extracts were combined with 40 µL of freshly

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prepared enzyme solution and 40 µL of 0.5 mM 4-MUG solution in the Tris buffer (pH 6.9).

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The mixtures were incubated at 37 °C for 20 min and the fluorescence (FA) (excitation at 390

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nm and emission at 410 nm) was measured using a SynergyTM2 (BioTek Instruments Inc.).

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Blank and α-glucosidase controls were also prepared. The α-glucosidase control (FC)

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contained the buffer (20 µL), substrate solution (40 µL) and enzyme (40 µL). Blank (FB)

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contained the cabbage extract (20 µL), buffer (20 µL) and enzyme (40 µL) while the blank to

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control (FD) contained the buffer and enzyme solution. Acarbose was used as positive control.

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The percentage α-glucosidase activity inhibition was calculated according to the following equation: ℎ % = 100 ∗ 1 −

 − 

  − 

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Where: FA,FB, FC and FD were the values of fluorescence of the sample, blank sample, control

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and blank control, respectively.

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α-Amylase Inhibition Assay. The inhibiting effect of the red cabbage crude extracts on α-

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amylase activity was assayed as previously described.6 Before this assay the red cabbage

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extracts were concentrated tenfold. The reaction mixture contained 1% potato starch gel (the

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substrate), cabbage extract, phosphate buffer (pH 6.9) and α-amylase from porcine pancreas

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type VI-B. Acarbose was used as positive control. After incubation at 37 °C for 10 min, the

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reaction was stopped by the addition of HCl. After that, I2 in KI was added and the absorbance

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was read at 600 nm (using a SynergyTM2, BioTek Instruments Inc.). The percentage inhibition

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of α-amylase activity was calculated as follows: ℎ % = 100 ∗ 1 −

 − 

  − 

108

Where: AA and AB were the values of absorbance of mixtures containing crude extract and

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starch with or without amylase, respectively. AC and AD were the values of absorbance of

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mixtures containing starch and amylase or only starch.

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IC50 Values for Enzyme Inhibition. Inhibitory activities of crude extracts of red cabbage

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against the lipase, α-glucosidase and α-amylase were expressed as IC50 values. IC50 is defined

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as the concentration of the dry weight (DW) of red cabbage per mL of reaction mixture under

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assay conditions that halves the enzyme activity. The IC50 values were obtained from the

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least-squares regression line of the plots presenting the logarithm of the sample concentration

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(log) vs the percentage enzyme inhibition (%). 6

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Antioxidant Capacity Assays. The ABTS•+ and FRAP assays were carried out according

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to Re et al.18 and Benzie & Strain.19 The procedures were the same as described elsewhere.6

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The antioxidant capacity was expressed as µmol of Trolox/g DW of red cabbage (TEAC).

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The linearity range for ABTS assay was determined as 2-17 µM Trolox (R2 = 0.9981), and for

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FRAP assy as 0.5-15 µM Trolox (R2 = 0.9986).

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Total Phenolics Assay. Total phenolics content (TPC) assay was based on reaction with

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the Folin-Ciocalteu reagent and absorbance measurements at 760 nm as described

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previously.6 TPC was expressed as mg of gallic acid equivalents (GAE)/g DW of red

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cabbage. The calibration curve ranged from 0.5-4 µg gallic acid/mL (R2 = 0.9981).

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Total Anthocyanins Assay. Total anthocyanin content (TAC) was determined by the pH-

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differential method according to Nicoue et al.20 The absorbance of diluted crude extracts from

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red cabbage varieties was measured at 530 and 700 nm. TAC was expressed as mg of

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cyanidin-3-glucoside equivalents (CGE)/g DW of red cabbage.

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Identification and Quantification of Individual Anthocyanins. Anthocyanins were

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identified using an Acquity UPLC system coupled with a quadruple-time of flight (Q-TOF)

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MS instrument (Waters Corp., Milford, MA, USA), equipped with an electrospray ionization

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(ESI) source. Anthocyanins were separated on an Acquity TMBEH C18 column (100 x 2.1

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mm, 1.7 µm; Waters) operating at 30 °C. The mobile phase was a mixture of 0.1% formic

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acid (A) and acetonitrile (B). The gradient program was as follows: initial conditions 99%

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(A), 12 min 65% (A), 12.5 min 100% (B), 13.5 min 99% (A). The flow rate was 0.42 mL/min

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and the injection volume was 5 µL. The major operating parameters for the Q-TOF-MS were

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set as follows: capillary voltage of 2.0 kV, cone voltage of 40 V, cone gas flow of 1 L/h,

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collision energy of 28-30 eV, source temperature of 100 °C, desolvation temperature of 250

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°C, argon - collision gas, desolvation gas (nitrogen) at a flow rate of 600 L/h, data acquisition 7

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range of m/z 100-2000 Da, and the positive ionization mode. The data were collected by

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Mass-LynxTM V 4.1 software. Anthocyanins were identified basing on the comparison of

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their UV-Vis spectra and MS/MS fragmentation spectra with the previously published data.11-

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13

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Anthocyanins were quantified according to Mizgier et al.12 using a Dionex HPLC system

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(Germering, Germany) equipped with an Ultimate 3000 diode array detector. The detector

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was coupled with a LPG-3400A pump, an EWPS-3000SI autosampler, a column thermostat

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TCC-3000SD and Chromeleonv. 6.8 software. A Cadenza Imtakt column C5-C18 (75 x 4.6

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mm) equipped with a guard column were used. The mobile phase was a mixture of solvent A

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(4.5% formic acid, v/v) and solvent B (acetonitrile). The elution system was as follows: 0–1

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min 5% B; 20 min 25% B; 21 min 100% B; 26 min 100% B; 27 min 5% B. The flow rate of

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the mobile phase was 1.0 mL/min and the injection volume was 20 µL. The column operated

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at 30 °C and the separated compounds were monitored at 520 nm. All samples were analysed

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in duplicate. The results were calculated as mg of cyanidin-3-glucoside equivalents (CGE)/g

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DW of red cabbage. The linearity range for this assay was determined as 10-75 µg/mL (R2 =

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0.9995).

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Statistical Analysis. Unless otherwise stated, data were expressed as the means ± standard

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deviations of triplicate measurements. The results were analyzed by means of a one-way

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analysis of variance (ANOVA). A Tukey’s post hoc test was used to determine differences

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between the means at the significance level p

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Kissendrup > Langedijker > Haco > Kalibos, regardless of the assay method. These data were

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consistent with the previous results and confirmed the conclusion that the antioxidant

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potential of red cabbage is variety-dependent.13,17,35 For comparison, Wiczkowski et al.13

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reported that among five red cabbage varieties, the highest antioxidant capacity against

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superoxide anion radical, ABTS●+ cation radical and peroxyl radical was exhibited by the

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Langedijker Polona variety, followed by the Langedijker Dauer 2, Kissendrup and Koda ones.

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A few authors reported that acylation of anthocyanins increased their antioxidant activity.

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This relationship was observed for delphinidin37, pelargonidin38 and for cyanidin32

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derivatives. According to the latter authors, the diacylated form of cyanidin-3-diglucoside-5-

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glucoside showed the stronger antioxidant activity against ABTS●+cation radical and peroxyl

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radical in comparison to the monoacylated and not acylated forms. Among the five red

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cabbage varieties tested in our study, the percentage of diacylated cyanidin-3-diglucoside-515

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glucoside among total anthocyanins was the highest in the var. Kissendrup (24.74%) and

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Koda (23.44%), which showed the highest antioxidant potential.

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Pearson Correlation Coefficient Analysis. The Pearson correlation analysis was

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conducted to assess the interplay between the content of phenolic substances and both enzyme

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inhibition potential and antioxidant capacity. The data presented in Table 4, suggest the

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moderate correlation (the values of Pearson coefficient range from -0.413 to -0.655) between

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the contents of total phenolics and total anthocyanins and the degree of both α-amylase and

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α-glucosidase inhibition, which in turn suggests the synergistic or antagonistic effect between

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structurally different phenolic compounds. Therefore, the inhibitory effects of red cabbage

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against the aforementioned digestive enzymes cannot be predicted based on the high phenolic

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and anthocyanin levels. Also our previous study6 showed no correlation between the total

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phenolics content in fruit extracts and inhibition of pancreatic lipase, α-glucosidase or α-

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amylase (r < -0.530). Moreover, some other compounds present in the crude extracts may

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influence the inhibitory effect of phenolics. The solvent used for extraction (70% methanol) in

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this study is nonselective and dissolves not only phenolic compounds but also large amounts

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of sugars, organic acids and proteins.

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The data of this study showed that the inhibitory activities of the red cabbage extracts

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toward α-amylase were correlated with mono- and diacylated anthocyanins (r > - 0.70). In

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case of α-glucosidase, the strongest correlation was observed between the IC50 values and the

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total diacylated anthocyanins. Sreerama et al. suggested that the inhibition of α-glucosidase

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might be caused mainly by the occurrence of anthocyanins in methanolic extracts of different

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beans.26 Gironés-Vilaplana et al. showed that the inhibition of α-glucosidase was correlated

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with both the total contents of anthocyanins and non-red polyphenols in Latin-American

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fruits.4 16

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The inhibition of pancreatic lipase was negatively correlated with the contents of

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anthocyanins, with an exception of diacylated anthocyanins. The lack of correlation between

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inhibition of lipase activity and total anthocyanin content was also reported for the black and

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red beans.26 However, the inhibition of lipase by fruits4 and anthocyanin-containing extracts

366

from fruits, vegetables, legumes and cereals5 was positively correlated with total anthocyanins

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contents.

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The data presented in Table 4 (the values of Pearson correlation coefficient of r ≥ 0.72)

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provide evidence of the highly positive correlation between the contents of total phenolics,

370

total anthocyanins, not acylated, monoacylated and diacylated pigments contents, and

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antioxidant capacities (ABTS scavenging and FRAP assays). The high correlation between

372

the results of ABTS scavenging and FRAP assays and the contents of total phenols is not

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surprising because the latter compounds were assayed using the Folin-Ciocialteu reagent,

374

which is also applied to determine the reducing capacity. Also other authors observed the

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positive correlation between the levels of total phenolics and total anthocyanins, and the

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antioxidant potential of plant derived food.4,9,13,17,25

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To summarize, the inhibitory activities against α-glucosidase, α-amylase and lipase as well

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as the high antioxidant activities of five red cabbage varieties suggest that this vegetable may

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be used to prevent and treat diabetes and obesity. Our results showed that the biological

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activities and levels of phenolic compounds were variety-dependent. These results may help

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to exploit the potential of red cabbage as a healthy diet component for obese and diabetic

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people as well as a source of nutraceuticals. However, further studies are necessary to

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determine the effects of red cabbage phenolics in more complex systems and in various food

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matrices, both in the in vitro and in vivo digestion processes.

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Notes

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The authors declare no competing financial interest. 17

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Table 1 The total phenolics (TPC), total anthocyanins (TAC) and dry matter of the red cabbage TPC

TAC

(mg GAE/g DW)

(mg CGE/g DW)

Dry matter of lyophilised cabbage (%)

Haco

12.1±0.47b

6.38±0.27b

89.7±0.18d

Kalibos

10.1±0.10a

4.65±0.05a

84.8±0.17a

Kissendrup

18.7±0.22d

12.0±0.16d

90.1±0.20d

Koda

19.6±0.48e

10.4±0.16c

89.0±0.07c

Langedijker

16.3±0.59c

10.8±0.12c

86.8±0.25b

Red cabbage varieties

Values are expressed as mean ± standard deviation (n =3). Mean ± standard deviation in the same column with different letters denote statistically significant difference at p < 0.05. GAE, gallic acid equivalents; CGE, cyanidin-3-glucoside equivalents; DW, dry weight.

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Table 2 Profiles of anthocyanins in five red cabbage varieties Peak

Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

cyanidin 3-diglucoside-5-glucoside cyanidin 3,5-diglucoside cyanidin 3-(sinapoyl)-diglucoside-5-glucoside cyanidin 3-(sinapoyl)-triglucoside-5-glucoside cyanidin 3-(p-coumaroyl)-triglucoside-5-glucoside cyanidin 3-(feruloyl)-triglucoside-5-glucoside cyanidin 3-(sinapoyl)-diglucoside-5-glucoside cyanidin 3-(sinapoyl)-triglucoside-5-glucoside cyanidin 3-(sinapoyl)-diglucoside-5-glucoside cyanidin 3-(p-coumaroyl)(sinapoyl)-triglucoside-5-glucoside cyanidin 3-(feruloyl)(sinapoyl)-triglucoside-5-glucoside cyanidin 3-(caffeoyl)-diglucoside-5-glucoside cyanidin 3-(sinapoyl)(sinapoyl)-triglucoside-5-glucoside cyanidin 3-(p-coumaroyl)-diglucoside-5-glucoside cyanidin 3-(feruloyl)-diglucoside-5-glucoside cyanidin 3-(sinapoyl)-diglucoside-5-glucoside cyanidin 3-(sinapoyl)-glucoside-5-glucoside

[MS+H]+ (m/z) 773 611 979 1141 1081 1111 979 1141 979 1287 1317 935 1347 919 949 979 817

18 19 20 21

cyanidin 3-(p-coumaroyl)(sinapoyl)-diglucoside-5-glucoside cyanidin 3-(p-hydroxyferuoyl)(sinapoyl)-triglucoside-5-glucoside cyanidin 3-(feruloyl)(sinapoyl)-diglucoside-5-glucoside cyanidin 3-(sinapoyl)(sinapoyl)-diglucoside-5-glucoside

1125 1171 1155 1185

Fragments (m/z) 611/449/287 449/287 817/449/287 979/449/287 919/449/287 949/787/449/287 817/449/287 979/817/449/287 817/449/287 1125/449/287 449/287 773/449/287 1023/773/449/287 757/449/287 787/449/287 817/449/287 655/449/287 963/449/287 1009/449/287 993/449/287 1025/449/287 total1

% of contribution in total anthocyanins content Haco Kalibos Kissendrup Koda 22.1 39.4 18.3 36.4 2.36 0.81 1.64 2.03 0.70 0.70 0.86 1.63 1.22 1.62 1.64 4.92 2.54 8.31 3.90 4.63

Langedijker 36.7 3.34 0.48 0.75 4.29

2.25

5.45

2.12

3.17

3.95

4.39 0.41 1.66 1.33 0.70 1.51 19.9

0.65 0.15 1.08 1.19 0.05 0.27 13.1

1.91 0.07 1.89 1.46 0.52 1.00 23.6

2.16 0.25 1.21 1.47 0.87 0.39 9.90

3.91 0.41 0.88 0.71 0.54 0.82 19.4

24.8

20.7

19.5

10.6

17.4

1.59

0

1.20

0

0

5.55 0.18 3.85 2.96 6.14±0.28b

1.88 0 2.05 2.59 4.64±0.06a

8.43 0 4.54 7.42 11.9±0.28d

5.47 0.08 6.38 8.44 10.1±0.21c

3.31 0.20 2.09 0.82 10.5±0.25c

Data represent mean values ± standard deviation (n=2). Means in line related to total anthocyanin content followed by different letters are significantly different (p < 0.05). 1 – total anthocyanin content was expressed as mg of cyanidin-3 glucoside equivalents per gram dry weight of red cabbage.

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Table 3 Inhibitory activities against pancreatic lipase, α-amylase, and α-glucosidase of five red cabbage varieties IC50 (mg DW/mL) Variety

pancreatic lipase

α-amylase

α-glucosidase

Haco

1.90±0.10b

87.1±0.33b

7.07±0.32d

Kalibos

1.79±0.11ab

119±7.49d

6.00±0.57c

Kissendrup

2.19±0.02c

69.0±3.65a

4.97±0.46b

Koda

1.57±0.06a

101±4.84c

3.87±0.12a

Langedijker

4.10±0.12d

107±3.13cd

7.06±0.17d

Values are expressed as mean ± standard deviation (n =3). Mean ± standard deviation in the same column with different letters denote statistically significant difference at p < 0.05. DW, dry weight of red cabbage.

Table 4 Pearson’s correlation coefficients (r) between total phenolics, total anthocyanins,nonmono- and diacylated anthocyanins and antioxidant capacities, and digestive enzymes inhibitory activities of five red cabbage varieties IC50 (mg DW of red cabbage/mL)

TEAC (µmol Trolox/g DW of red cabbage)

α-amylase

αpancreatic glucosidase lipase

ABTS

FRAP

total phenolics

-0.476

-0.655

0.129

0.990

0.991

total anthocyanins

-0,537

-0.413

0.393

0.971

0.969

nonacylated anthocyanins

0,253

-0.280

0.557

0.734

0.736

monoacylated anthocyanins -0,709

-0.105

0.487

0.760

0.754

diacylated anthocyanins

-0.726

-0.261

0.793

0.792

-0,782

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Range: 4e-1

21

9.37

14 3.0e-1

8.19

2.5e-1

1 3.59

AU

2.0e-1

18

1.5e-1

9.02

8

3+4

1.0e-1

20

6.41

10 11 13 12 7.41

7.04 7.26

4.82

5 6.11

5.0e-2

2

9

17 19 8.79

6.71

3.87

0.0 3.00

4.00

5.00

6.00

7.00

8.00

Time 10.00

9.00

Fig.1.

160 d

TEAC (µ µmol Trolox/g DW)

140

d c

d

d

120 100 80

c b

Haco b

a

Kalibos a

60

Kissendrup Koda

40

Langedijker 20 0 ABTS

FRAP Methods

Fig.2.

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List of figure captions Fig.1. UPLC-DAD chromatogram (520 nm) of anthocyanins from red cabbage var. Haco Fig.2. The dependence of TEAC value on the red cabbage variety and assay method

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