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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]

1

ACS Paragon Plus Environment


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ABSTRACT

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

3

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

4

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

6

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

8

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

10

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

11

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

12

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

13

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

15

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

16

glucosidase increased with total phenolics and diacylated anthocyanins levels.

17 18

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

19

20

INTRODUCTION

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

22

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

23

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

25

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

26

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

27

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

28

enzymes involved in lipid and carbohydrate metabolism.1-3

29

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

30

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

31

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

32

correlation between the inhibitory activity of vegetables and polyphenol and anthocyanin

33

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

34

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

35

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

36

sugars and lipids.

37

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

38

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

39

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

40

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

41

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

42

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

44

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

45

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

46

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

47

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

48

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-

51

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

52

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

53

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

54

sulphonic

55

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

56

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

57

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

58

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

59

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

60

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

61

(Millipore, Marlborough, MA, USA).

acid)

(ABTS),

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

(TPTZ),

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

62

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

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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)

75

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

76

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

77

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

78

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

79

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

80

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

81

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

82

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

−

−

83

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

84

and blank control, respectively.

85

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

86

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

87

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

88

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

89

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

90

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

91

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

92

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

93

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

94

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

95

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

5



96 97

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

−

−

98

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

99

and blank control, respectively.

100

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

103

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

106

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

107

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

109

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

110

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

114

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

115

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

116

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


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

118

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

119

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

121

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

123

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

124

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

128

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)

132

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

133

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

134

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

135

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

136

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

137

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

138

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

140

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



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141

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

142

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

143

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

144

13

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

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

156

0.9995).

157

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

158

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

159

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

160

between the means at the significance level p

326

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

327

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

328

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

329

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

330

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

334

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

335

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

336

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

337

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



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338

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.

340

Pearson Correlation Coefficient Analysis. The Pearson correlation analysis was

341

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

342

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

343

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

344

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

345

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

346

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

347

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

348

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

349

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

350

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

351

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

352

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

353

of sugars, organic acids and proteins.

354

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

355

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

357

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

358

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

359

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

360

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

361

fruits.4 16


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

363

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

364

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

365

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

367

contents.

368

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

369

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

370

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

371

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

373

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

375

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

376

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

377

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

378

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

380

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

381

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

382

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

383

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

384

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

385

Notes

386

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.

24


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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

25



15+16 8.40

Page 26 of 28

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.

26


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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

27



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