Fractionation of Plant Bioactives from Black Carrots (Daucus carota

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Fractionation of plant bioactives from black carrots (Daucus carota subspecies sativus varietas atrorubens Alef.) by adsorptive membrane chromatography and analysis of their potential anti-diabetic activity Tuba Esatbeyoglu, Miriam Rodriguez-Werner, Anke Schlösser, Martin Liehr, Ignacio Ipharraguerre, Peter Winterhalter, and Gerald Rimbach J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02292 • Publication Date (Web): 30 Jun 2016 Downloaded from http://pubs.acs.org on July 1, 2016

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Journal of Agricultural and Food Chemistry

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Fractionation of plant bioactives from black carrots (Daucus carota subspecies

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sativus varietas atrorubens Alef.) by adsorptive membrane chromatography and

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analysis of their potential anti-diabetic activity

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Tuba Esatbeyoglu1*, Miriam Rodríguez-Werner2, Anke Schlösser1, Martin Liehr1, Ignacio

5

Ipharraguerre1, Peter Winterhalter2, Gerald Rimbach1

6 7

1

8

2

Institute of Human Nutrition and Food Science, University of Kiel, Germany Institute of Food Chemistry, Technische Universität Braunschweig, Germany

9 10 11 12

*Corresponding author:

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Dr. Tuba Esatbeyoglu

14

Institute of Human Nutrition and Food Science

15

University of Kiel

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Hermann-Rodewald-Str. 6

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24118 Kiel, Germany

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Tel: +49 431 880 2583, Fax: +49 431 880 2628

19

Email: [email protected]

20

1

ACS Paragon Plus Environment


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Abstract:

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Black and purple carrots have attracted interest as colored extracts for coloring food due

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to their high content of anthocyanins. This study aimed to investigate the polyphenol

24

composition of black carrots. Particularly, the identification and quantification of phenolic

25

compounds of the variety Deep Purple carrot (DPC), which presents a very dark color,

26

was performed by HPLC-PDA and HPLC-ESI-MSn analyses. The separation of

27

polyphenols from a DPC XAD-7 extract into an anthocyanin fraction (AF) and copigment

28

fraction (CF; primarily phenolic acids) was carried out by membrane chromatography.

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Furthermore, possible anti-diabetic effects of the DPC XAD-7 extract and its AF and CF

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were determined. DPC samples (XAD-7, CF and AF) inhibited α-amylase and

31

α-glucosidase in a dose-dependent manner. Moreover, DPC XAD-7 and chlorogenic

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acid, but not DPC CF and DPC AF, caused a moderate inhibition of intestinal glucose

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uptake in Caco-2 cells. However, DPC samples did not affect glucagon-like peptide-1

34

(GLP-1) secretion and dipeptidyl peptidase IV (DPP-4) activity.

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Overall, DPC exhibits an inhibitory effect on α-amylase and α-glucosidase activity and

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on cellular glucose uptake indicating a potential anti-diabetic properties.

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anthocyanins,

black

38

Key words:

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chromatography, anti-diabetic activity

carrots,

HPLC,

phenolic

acids,

membrane

40

2


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Introduction:

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Cultivated carrots (Daucus carota L.) are classified into two main groups: the Western or

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carotene carrots (Daucus carota ssp. sativus var. sativus) and the Eastern or

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anthocyanin carrots (Daucus carota ssp. sativus var. atrorubens Alef.).1 Carrots

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enriched with anthocyanins are also called black or purple carrots due to their black

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color, which originate from Turkey, Afghanistan, Egypt and India.2 Different black carrots

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varieties are known e.g. Deep Purple, Antonina, Purple Haze and Beta Sweet that can

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be differentiated by the flesh color: white/yellow (Antonina), orange (Purple Haze and

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Beta Sweet) or violet flesh (Deep Purple). According to literature, phenolic compounds

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of black carrots were mainly characterized by UV-Vis spectra, HPLC and mass

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spectrometry3–11

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spectroscopy.4,6,12,13 Black carrot derived anthocyanins are glycosylated and acylated

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with hydroxycinnamic acids. In particular, their main anthocyanins are based on cyanidin

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aglycones and as minor compounds peonidin and pelargonidin derivatives have been

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identified.3–6,14,15

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xylosyl(feruloylglucosyl)galactoside.10,11,16 Due to the high stability of acylated

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anthocyanins, the use of black carrots as sources of food colorants has significantly

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increased.17,18 The main phenolic acid in black carrots is chlorogenic acid (5-CQA).10

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There is increasing evidence for potential health benefits of fruit and vegetable derived

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plant bioactives including anthocyanins.19–22 Recently, it has been suggested that black

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carrots may affect glucose metabolism thereby mediating potential health benefits.23

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Diabetes mellitus type 2 is a metabolic disorder of the glucose metabolism.24

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Anthocyanins have been associated with potential antidiabetic properties. Xiao et al.

and

their

The

structure

was

predominant

elucidated

by

anthocyanin

1

H

and

is

13

C

NMR

cyanidin-3-

3



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(2015) assumed that anthocyanin enriched foods may delay glucose absorption by

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inhibition of the enzymes α-amylase and α-glucosidase.25 Moreover, purified

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anthocyanin supplementation prevented insulin resistance in diabetic patients.26

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Anthocyanins exhibit potent anti-inflammatory properties which may contribute to their

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anti-diabetic activity.27 Furthermore, an extract of carrot roots was found to stimulate

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insulin-dependent glucose uptake in adipocytes.28

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The aim of the present study was to investigate the phenolic composition of the black

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carrot variety “Deep Purple carrot (DPC)” by HPLC-PDA and HPLC-ESI-MSn analyses

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as well as the separation of polyphenols from a DPC XAD-7 extract into an anthocyanin

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fraction (AF) and copigment fraction (CF). The raw extract of DPC was concentrated

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onto an Amberlite XAD-7 column to eliminate salts, proteins and sugars and thus

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resulted into an enrichment of polyphenols (XAD-7 extract).15 Adsorptive membrane

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chromatography29 has been applied for the separation of the black carrot XAD-7 extract

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into an anthocyanin fraction (AF) and a copigment fraction (CF), for the first time.

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Recently, Gras et al. (2016) separated anthocyanins and non-anthocyanin polyphenols

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from black carrot by liquid-liquid extraction.10 Furthermore, the determination of possible

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anti-diabetic effects of the DPC XAD-7 extract and its AF and CF has been conducted.

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Therefore, different enzyme inhibitory assays e.g. α-amylase and α-glucosidase have

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been applied. The inhibition of the intestinal glucose uptake in Caco-2 cells has been

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investigated by a fluorometric method. Glucagon-like peptide 1 (GLP-1) secretion in

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GLUTag cells was measured using an ELISA kit. The DPP-4 inhibitor activity of the DPC

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samples was determined photometrically.

86 4


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Materials and Methods:

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Sample. Black carrots (Daucus carota ssp. sativus var. atrorubens Alef.) were cultivated

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and provided by the Faculty of Agricultural Sciences and Landscape Architecture of the

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University of Applied Sciences of Osnabrück (Lower Saxony, Germany).

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Chemicals. Doubly deionized water was prepared using a Nanopure® resin (Nanopure,

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Barnstead, United States). Methanol and acetonitrile, HPLC grade, were acquired from

93

Fisher Scientific (Loughborough, UK). Acetonitrile, LC-MS grade, was obtained from

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Honeywell Specialty (Seelze, Germany) and formic acid, LC-MS grade, was supplied by

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Fisher Scientific. Formic acid, analytical grade, sodium hydroxide, ≥99% and sodium

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chloride, ≥99% were ordered from Carl Roth (Karlsruhe, Germany). Acetic acid,

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analytical grade, Amberlite® XAD-7 HP, chlorogenic acid, hemihydrate ≥98%,

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α-glucosidase, α-amylase, 3,5-dinitrosalicylic acid, 98%, potassium sodium tartrate

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tetrahydrate, ReagentPlus®, ≥99%, Triton® X-100, phlorizin and forskolin were obtained

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from Sigma-Aldrich (Steinheim, Germany). Ethanol was distilled (industrial quality). 2-

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Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) was ordered

102

from

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glucopyranoside (pNPG) and GLP-1 sandwich ELISA from Merck (Darmstadt,

104

Germany). All cell culture reagents were purchased from PAN-Biotech GmbH

105

(Aidenbach, Germany).

106

Extraction of anthocyanins. Anthocyanins from 900 g of fresh DPC were extracted

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with methanol/acetic acid (19:1, v/v). After 8 h-extraction, the sample was filtered and

108

the solvent was evaporated. The extracts were freeze-dried. In order to concentrate and

109

obtain an enrichment of the anthocyanins, the raw acidic methanolic extract was

Cayman

Chemical

(Ann

Arbor,

Michigan,

USA)

and

p-nitrophenyl-α-D-

5



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dissolved in Nanopure® water and applied onto an Amberlite XAD-7 column. The column

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was washed with Nanopure® water and the anthocyanins were eluted with a mixture of

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methanol/acetic acid (19:1; v/v). The extract was concentrated in vacuo and freeze-

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dried. Consequently, about 2 g XAD-7 extract was obtained.

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Adsorptive membrane chromatography. The separation of anthocyanins and

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copigments was performed onto a membrane adsorber, Sartobind S IEX 150 mL, from

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Sartorius Stedim Biotech (Göttingen, Germany). This membrane absorber is a strong

117

acidic cation exchanger with sulfonic acid groups (R-CH2-SO3-) and contains stabilized

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cellulose with a macro-porous structure on its surface. Before separation, anthocyanins

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are converted into flavylium cations by acidification. Thus, these cations can be retarded

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on the membrane adsorber surface and separated from other compounds that are not

121

positively charged. About 2 g XAD-7 extract was dissolved in 1 L methanol/acetic acid

122

(19:1; v/v) and filtered using a filter paper (MN 615 ¼ Macherey-Nagel, Düren,

123

Germany) and applied for the separation. A Sartopore 2 300 filter capsule was

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connected between the adsorber and the peristaltic pump Tandem 1082 (Sartorius). The

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regeneration and equilibration of the membrane absorber was carried out with 2.5 L of

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1N NaOH, 2.5 L of 0.01N HCl and 1 L of methanol/acetic acid (19:1, v/v) by pumping

127

with a flow rate of 100 mL/min. Then the extract solution was loaded. The copigments

128

were eluted with 1 L of methanol/acetic acid (19:1, v/v) and were collected. After the

129

separation of copigments, the retarded anthocyanins were eluted with 1 L of 1:1 (v:v)

130

mixture of aqueous 1M NaCl solution and methanol. Subsequently, AF was acidified

131

with acetic acid with a final concentration of 1% in order to stabilize these compounds.

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Samples of 2 mL were collected every 200 mL in the loading, washing and elution steps, 6


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then dried under nitrogen and analyzed by HPLC-PDA. The solvents of CF and AF were

134

removed in vacuo and both fractions were freeze-dried.

135

AF contained NaCl. Consequently, a desalination of anthocyanin fraction was required.

136

AF was dissolved in Nanopure® water/acetic acid (99.5:0.5, v/v) and applied onto an

137

Amberlite XAD-7 HP column. Salts were eliminated with 3 L Nanopure® water/acetic

138

acid (99.5:0.5, v/v), then anthocyanins were eluted with methanol/acetic acid (19:1; v/v),

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concentrated in vacuo, dissolved in Nanopure® water and freeze-dried.

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HPLC-PDA analyses. HPLC-PDA separations were performed on a RP-18 Luna 5µ C-

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18(2), 100 Å, 250 x 4.6 mm column (Phenomenex, Aschaffenburg, Germany) with a

142

HPLC guard cartridge system of the same material. Gradient elution was carried out with

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solvent systems A (Nanopure® water/acetonitrile/formic acid, 87:3:10, v/v/v) and B

144

(Nanopure® water/acetonitrile/formic acid, 40:50:10, v/v/v) at a flow rate of 0.5 mL/min.

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The linear gradient was as follows: 0 min 6% B, 20 min 20% B, 35 min 40% B, 40 min

146

60% B, 45 min 90% B, 55 min 6% B, 60 min 6% B. Anthocyanins were detected at

147

λ 520 nm and copigments were identified at λ 280 nm. The quantification was carried out

148

with cyanidin-3-O-glucoside as standard for anthocyanins and chlorogenic acid (5-CQA)

149

for copigments. All samples (DPC XAD-7, DPC CF and DPC AF) were injected three

150

times.

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HPLC-ESI-MSn analyses. Mass spectral analyses were recorded under the following

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operating conditions: positive ion and alternating mode; capillary, -2500 V; capillary exit

153

offset, 70 V; end plate offset, -500 V; skimmer 1, 20 V; skimmer 2, 10 V; dry gas, N2,

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11 L/min; dry temperature, 325 °C; nebulizer, 60 psi; scan range, m/z 100-2500. The

155

samples were separated on a C18 (2) Luna column (Phenomenex, Germany), 150 x 7



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2.0 mm, 3 µ. As mobile phase (A) Nanopure® water/acetonitrile/formic acid (95:3:2,

157

v/v/v) and (B) Nanopure®water/acetonitrile/formic acid (48:50:2, v/v/v) were used.

158

Samples were dissolved in mobile phase A and analyzed. The HPLC gradient was used

159

as described in HPLC-PDA analyses with a flow rate of 0.2 mL/min.

160

α-Amylase inhibition. The α-amylase assay was conducted according to Phan et al.30

161

and Wagner et al.31. In brief, 50 µL of DPC extracts were mixed with 100 mmol/L

162

KH2PO4 and 50 µL of 0.5 U/mL α-glucosidase and incubated at 37 °C for 5 min.

163

Afterwards, 50 µL of 10 mmol/L p-nitrophenyl-α-D-glucopyranoside (pNPG) was added

164

and incubated at 37 °C for 20 min. The reaction was stopped with 2 mol/L Na2CO3. The

165

absorbance was measured at λ 405 nm (Tecan, Crailsheim, Germany). Acarbose was

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used as positive control. Three independent experiments were performed in triplicate.

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For data analysis the software QtiPlot version 0.9.8.3 was used.

168

α-Glucosidase inhibition. α-Glucosidase inhibition was measured according to Phan et

169

al.30 and Wagner et al.31. Fifty micro liters of DPC extracts were mixed with 50 µL of 1%

170

starch solution as well as 50 µL of 10 U/mL α-amylase and incubated at 20 °C for 3 min.

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Then, 50 µL of colorant reagent, composed of 44 mM 3,5-dinitrosalicylic acid and 1.1 M

172

potassium sodium tartrate tetrahydrate solution, and 50 µl α-amylase (as a control) were

173

added, mixed and incubated at 99 °C for 15 min. The samples were cooled down to

174

ambient temperature and water was added (450 µL). The absorbance was measured at

175

λ 540 nm (Tecan, Crailsheim, Germany). Acarbose was used as positive control. Three

176

independent experiments were performed in triplicate. For data analysis QtiPlot version

177

0.9.8.3 was used.

8


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Determination of cytotoxicity using LDH assay. Fourteen days differentiated human

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colon carcinoma epithelial CaCo-2/TC-7 cells (150,000 cells per well in a 24-well plate)

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were incubated with 100 µg/mL DPC XAD-7, DPC CF and DPC AF and chlorogenic acid

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(35.4 µg/mL) and phlorizin (43.6 µg/mL) for 24 h. DMSO (0.1%) was used as vehicle

182

control and 1% Triton® X-100 as negative control. Cytotoxicity was evaluated by the

183

LDH assay according to Esatbeyoglu et al.32

184

Enteroendocrine GLUTag cells (kindly provided by Dr. D. J. Drucker, University of

185

Toronto, Ontario, Canada) were seeded into poly-D-lysine coated multiwell plates at a

186

density of 7.5 x 104 cells per cm2. After 24 h of pre-cultivation, cells were incubated with

187

Krebs Ringer Buffer (KRB, 0.5 mM MgCl2*2H2O, 1.5 mM CaCl2*6H2O, 0.1% BSA) for

188

30 min and subsequently treated with test compounds (50 µg/mL or 17.7 µg/mL,

189

respectively) for 2 h. Following treatment, medium was replaced with culture medium

190

containing 50 µg/mL neutral red (Carl Roth, Karlsruhe, Germany) and incubated for 2 h.

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Subsequently, cells were washed once with phosphate buffered saline (PBS, Gibco via

192

Thermo Fisher Scientific, Darmstadt, Germany) and incubated with neutral red

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extraction buffer (50% ethanol, 49% double distilled water, 1% glacial acetic acid) for

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15 min on a shaking platform. The absorbance of neutral red dye was measured at

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λ 450 nm and viability of the compound-treated cells was calculated as percentage

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absorbance of the vehicle treated cells for each treatment.

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Glucose uptake. CaCo-2/TC-7 cells were cultured in Dulbecco’s modified Eagle

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medium (DMEM) containing 4.5 g/L glucose, 4 mmol/L L-glutamine, 1 mmol/L sodium

199

pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin and 20% (v/v) fetal calf serum

200

(FCS). Cells were grown in 5% CO2/95% air at 37 °C in a humidified atmosphere until 9



Page 10 of 31

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90-95% confluency. For subculturing, cells were detached with 0.05% trypsin and 0.02%

202

EDTA in Ca- and Mg-free PBS.

203

The determination of glucose uptake in CaCo2-cells was performed according to Wu et

204

al.33 with some modifications. CaCo-2 cells were seeded in 24-well plates at a density of

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1.5 x 105 cells per well and were differentiated for two weeks replacing medium every

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second day. Subsequently, cells were treated with 100 µg/mL DPC XAD-7, DPC CF and

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DPC AF extracts and 35.4 µg/mL chlorogenic acid and 43.6 µg/mL phlorizin (as positive

208

control; phlorizin is a known inhibitor of intestinal glucose absorption34) in serum-free low

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glucose medium (DMEM; 1.0 g/L glucose, 100 U/mL penicillin, 100 µg/mL streptomycin)

210

for 24 h. After the incubation, cells were washed two times in Krebs-Ringer buffer

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(114 mM NaCl, 4.7 mM KCl, 1.16 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 20 mM

212

NaHCO3, 20 mM HEPES) and incubated with test compounds and with/or without

213

2-NBDG (50 µM) in triplicate for 1 h in the incubator at 37 °C. The reaction was stopped

214

with ice-cold PBS, cells were washed three times and fluorescence signal was

215

determined using Tecan infinite F200 (Tecan, Grödig, Austria) at an excitation

216

wavelength of λ 485 nm and an emission wavelength of λ 535 nm.

217

Determination of GLP-1 in GLUTag cells. Murine enteroendocrine GLUTag were

218

maintained in DMEM containing 1.0 g/L glucose, 8 mmol/L L-glutamine, 1 mmol/L

219

sodium pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin and 10% (v/v) FCS.

220

GLUTag cells were grown in 5% CO2/95% air at 37 °C in a humidified atmosphere. For

221

sub-culturing, cells were detached with 0.05% trypsin and 0.02% EDTA.

222

Enteroendocrine GLUTag cells were seeded into poly-D-lysine coated multiwell plates at

223

a density of 75,000 cells per cm2. After 24 h of pre-cultivation, cells were incubated with 10


Page 11 of 31


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Krebs-Ringer Buffer (0.5 mM MgCl2*2H2O, 1.5 mM CaCl2*6H2O, 0.1% BSA) for 30 min

225

and subsequently treated with 50 µg/mL DPC XAD-7, DPC CF and DPC AF extracts

226

and chlorogenic acid, respectively, for 2 h. Supernatants were collected and analyzed by

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a GLP-1 sandwich ELISA according to the manufacturer’s instructions (Merck,

228

Darmstadt, Germany). GLP-1 was quantified using a standard curve. Forskolin

229

(1.23 µg/mL) was used as a positive control. DMSO was used as vehicle control.

230

Results represent means of two independent experiments of pools of three independent

231

wells measured in duplicate + SEM.

232

Quantification of DPP-4 activity. Screening of test compounds (50 µg/mL DPC XAD-7,

233

DPC CF extracts and chlorogenic acid) towards potential DPP-4 inhibitor activity was

234

determined with the DPP-4 Drug Discovery Kit (Enzo Life Sciences, Lörrach, Germany)

235

according to the manufacturer´s protocol. Enzyme activity was continuously determined

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over 60 min with one measurement per minute at λ 405 nm. The DPP-4 inhibitor P32/98

237

(2.60 µg/mL; Enzo Life Sciences, Lörrach, Germany) was used as a positive control and

238

DMSO was used as vehicle control. Results represent means of three independent

239

measurements + SEM.

240

Statistical analyses. Student's t-test was applied for statistical analyses (Excel, Version

241

10, Microsoft Corporation). Results are presented as mean + SEM. p < 0.05 were

242

considered statistically significant.

243 244

Results and discussion:

245

Identification of phenolic compounds.

11



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Figure 1 shows the isocontour plot HPLC-PDA chromatograms from λ 200 to 650 nm

247

(A) and the chromatograms at λ 280 and 520 nm (B) of the XAD-7 extract. The

248

occurrence of anthocyanins was confirmed, which have absorption maxima at λ 280 and

249

520 nm, while copigments absorb only between λ 200 and 400 nm. Based on previous

250

studies, anthocyanins of black carrots were characterized by means of HPLC-PDA,

251

HPLC-MSn and NMR analyses.6,7,15 Black carrot extracts contained acylated

252

anthocyanins,

253

information on molecular ions and fragment ions of anthocyanins (Table 1) and

254

copigments (Table 2). These results are consistent with current literature data.10–12,16

255

Black carrot anthocyanins were acylated with hydroxycinnamic acids (e.g. sinapic acid,

256

ferulic acid and p-coumaric acid), thus stabilize its structure under acid conditions.5 We

257

detected acylated and non-acylated anthocyanins which is in accordance to Kammerer

258

and coworkers.5 For the quantification of anthocyanins, an external standard calibration

259

curve of cyanidin-3-O-glucoside from 10.0 to 480 mg/L was used at λ 520 nm with a

260

linearity

261

xylosyl(feruloylglucosyl)galactoside (compound 4). This result was in accordance with

262

previous reports.10,11,16 The acylated anthocyanin concentration was about 90% of total

263

anthocyanins. We detected about 1.2 g/kg fresh weight (≙ 0.14–0.23 g/kg dry weight)

264

total anthocyanins. In the literature, anthocyanin concentrations from 45.4 mg/kg to

265

17.4 g/kg dry weight (12–19% dry matter content) have been reported.5 Beside cyanidin

266

derivatives, peonidin derivatives were detected in our black carrots samples which is in

267

accordance to literature data.7

of

especially

R2=

cyanidin-based

0.999.

The

anthocyanins.

predominant

LC-MS

anthocyanin

data

was

provided

cyanidin-3-

12


Page 13 of 31


268

Chlorogenic acids were quantified using an external standard calibration curve of 5-CQA

269

from 11.7 to 350 mg/L with a linearity of R2= 0.997. Previous studies indicate that 5-CQA

270

may be the principal copigment in black carrots.8,9,35 In the analyzed DPC XAD-7

271

extract, 5-CQA was also the most abundant copigment, while neochlorogenic acid (3-

272

CQA) and cryptochlorogenic acid (4-CQA) occured as minor compounds. p-Coumaric

273

acid derivatives with a [M-H]- ion at m/z 337, caffeic acid derivatives with [M–H]- ion at

274

m/z 515 and ferulic acid derivatives with a [M-H]- ion at m/z 367 were detected in the

275

XAD-7 extracts in very small quantities. Their occurrence in carrots was verified

276

previously.8,35

277

Adsorptive membrane chromatography.

278

Based on an ion exchange chromatographic method, the separation of anthocyanins

279

from copigments has been carried out by adsorptive membrane chromatography. The

280

XAD-7 extract was dissolved in methanol:acetic acid (19:1, v/v) and applied onto the

281

membrane. Anthocyanins were adsorbed on the membrane surface and copigments,

282

which are colorless or weak colored, were eluted. For the elution of anthocyanins, a

283

mixture of aqueous 1M NaCl and methanol (1:1, v/v) was used. The anthocyanin fraction

284

(AF) contained NaCl. Subsequently, the desalination of AF was performed on a XAD-7

285

column. After separation of anthocyanins and copigments, HPLC-PDA analyses of AF

286

and the copigment fraction (CF) were carried out. In Figure 2, the HPLC-PDA

287

chromatograms of DPC AF and DPC CF are shown. Copigments have absorption

288

maxima between λ 200 and 400 nm, while anthocyanins absorb at λ 280 and 520 nm.

289

The HPLC-chromatogram of the DPC CF at λ 520 nm confirmed that anthocyanins were

290

not present in this fraction (Figure 2B; right). The most abundant copigments in DPC 13



Page 14 of 31

291

were identified as caffeoylquinic acids. 5-CQA (peak 8) was the major copigment of CF

292

(Figure 2C; right, and Table 2). Overall, the separation by means of adsorptive

293

membrane chromatography was successful and has not been reported in the literature

294

as far as black carrot is concerned.

295

In vitro anti-diabetic effects

296

The inhibition of α-amylase and α-glucosidase, which are involved in the digestion of

297

carbohydrates, may partly decrease post-prandial blood glucose levels.36 All DPC

298

samples dose-dependently inhibited the enzymes α-amylase (Figure 3) and

299

α-glucosidase (Figure 4) in vitro. Acarbose was used as a positive control for both

300

enzymatic assays. DPC CF was the strongest α-amylase inhibitor, whereas DPC XAD-7

301

showed the strongest inhibition against α-glucosidase. Xiao and Högger (2015)

302

suggested that various polyphenols e.g. anthocyanins may delay glucose absorption via

303

inhibition of α-amylase and α-glucosidase.25 Also various chlorogenic acids (composed

304

of caffeic acid with quinic acid), such as 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA,

305

and 4,5-diCQA, inhibited α-amylase and α-glucosidase.13,37 The esterification of caffeic

306

acid with quinic acid reduces their ability to inhibit α-amylase and α-glucosidase.37

307

Therefore, caffeic acid is a stronger inhibitor of these two enzymes as compared to

308

chlorogenic acids.37 Moreover, it is known that that chlorogenic acids with two caffeic

309

acids are stronger α-glucosidase inhibitors.13 An inhibition of these enzymes by DPC

310

has not been described. Overall, our data suggest that DPC polyphenols may exhibit

311

potential anti-diabetic activity.

312

All DPC samples and chlorogenic acid did not impair cell viability up to a concentration

313

of 100 µg/mL or 35.4 µg/mL in CaCo-2 cells, respectively (data not shown). 14


Page 15 of 31


314

Furthermore, all test compounds did not affect cell viability at 50 µg/mL or 17.7 µg/mL in

315

GLUTag cells, respectively (data not shown).

316

The inhibition of glucose uptake in the intestine may be an important strategy to control

317

blood glucose levels.33 2-NBDG was used as a fluorescein in our Caco-2 cell

318

experiments for the determination of cellular glucose uptake in response to the DPC

319

treatments. Only DPC XAD-7 and the pure standard compound 5-CQA significantly

320

inhibited 2-NBDG uptake compared to untreated control cells (Figure 5A). Phlorizin was

321

used as a positive control. Recently, it was shown that polyacetylenes from carrots

322

(variety Bolero) affect glucose uptake in adipocytes and myotubes.28 Furthermore, it has

323

been suggested that 5-CQA, a DPC constituent, regulates glucose metabolism via

324

AMPK activation thereby exhibiting anti-diabetic activity.38

325

GLP-1 is an incretin which is secreted by enteroendocrine L-cells. It plays a key role in

326

controlling glucose homeostasis.39 GLP-1 induces glucose-dependent stimulation of

327

insulin secretion thereby lowering blood glucose. GLP-1 is rapidly degraded by DPP-4.

328

DPP-4 inhibitors are potential hypoglycomics.39 Under the conditions investigated all

329

DPC samples did not induce GLP-1 secretion (Figure 5B). Furthermore, we did not

330

observe a DPP-4 inhibitory activity of DPC samples (Figure 5C). In contrast to our

331

results it has been shown by Nagamine and co-workers40 that various CQAs from sweet

332

potato leaf extracts may enhance the secretion of GLP-1. Differences between our and

333

literature data may be related to differences in polyphenol composition and administered

334

test concentrations in the corresponding cell culture assays.

335

15



Page 16 of 31

336

Black carrots are characterized as “cyanidin-type” due to their high content of cyanidin

337

derivatives. The main anthocyanin was cyanidin-3-xylosyl(feruloylglucosyl)galactoside,

338

while 5-CQA was the main copigment in DPC. For the first time, the XAD-7 extract of

339

DPC was separated into two groups of compounds, namely anthocyanins and

340

copigments, by an adsorptive membrane chromatographic method. This applied method

341

enabled the isolation of anthocyanins and copigments on a large scale within 2 h. Future

342

studies are required for the separation and scale up of individual compounds thus their

343

chemical structures can be confirmed, especially minor compounds. We identified DPC

344

as inhibitors of α-amylase and α-glucosidase. The inhibition of these enzymes may

345

partly mediate the anti-diabetic effects of DPC. Moreover, our results suggest that DPC

346

XAD-7 exhibits an inhibitory effect on glucose uptake. The potential anti-diabetic activity

347

of DPC, as observed in our in vitro studies, needs to be validated in appropriate in vivo

348

models in the future. Furthermore, the bioavailability of carrot-derived plant bioactives

349

including anthocyanins should be determined in future human studies.

350 351

Acknowledgments:

352

GLUTag cells were kindly provided by Dr. D. J. Drucker, University of Toronto (Toronto,

353

Ontario, Canada). We are grateful to Olaf Melzer from the Faculty of Agricultural

354

Sciences and Landscape Architecture of the University of Applied Sciences (Osnabrück)

355

for supplying black carrots.

356

16


Page 17 of 31


357

References

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Algarra, M.; Fernandes, A.; Mateus, N.; de Freitas, V.; Esteves da Silva, J. C. G.;

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Casado, J. Anthocyanin profile and antioxidant capacity of black carrots (Daucus

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carota L. ssp. sativus var. atrorubens Alef.) from Cuevas Bajas, Spain. J. Food

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vitro): a comparative study. J. Basic Clin. Physiol. Pharmacol. 2015, 26, 165–170.

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484 485

(39) Drucker, D. J. Incretin action in the pancreas: Potential promise, possible perils, and pathological pitfalls. Diabetes 2013, 62, 3316–3323.

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H.; Tsuda, T. Dietary sweet potato (Ipomoea batatas L.) leaf extract attenuates

488

hyperglycaemia by enhancing the secretion of glucagon-like peptide-1 (GLP-1).

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490

22


Page 23 of 31

491


Figure Captions:

492

Figure 1. (A) Isocontour plot of HPLC-PDA chromatogram of the anthocyanin-enriched

493

XAD-7 extract of Deep Purple carrot (DPC) monitored from λ 200 to 650 nm. (B) HPLC-

494

PDA chromatogram of anthocyanin-enriched XAD-7 extract of DPC monitored at λ 520

495

and 280 nm. Peak identification is given in Table 1.

496

Figure 2. Isocontour plot of HPLC-PDA (A) from λ 220 to 650 nm and chromatograms of

497

the copigment fraction (right side; CF) and the anthocyanin fraction (left side; AF) of

498

DPC at λ 520 nm (B) and at λ 280 nm (C) after separation by adsorptive membrane

499

chromatography. Peak identification is given in Table 1 and Table 2.

500

Figure 3. Dose-dependent inhibition of α-amylase activity by DPC XAD-7, DPC CF and

501

DPC AF. Acarbose was used as a positive control. Data are expressed as mean + SEM

502

of three independent experiments performed in triplicate.

503

Figure 4. Dose-dependent inhibition of α-glucosidase activity by the DPC XAD-7, DPC

504

CF and DPC AF extracts. Acarbose was used as a positive control. Data are expressed

505

as mean + SEM of three independent experiments performed in triplicate.

506

Figure 5. (A) Effects of DPC samples and chlorogenic acid on glucose uptake in CaCo-

507

2 cells. CaCo-2 cells were treated with 100 µg/mL DPC XAD-7, DPC CF and DPC AF

508

and 35.4 µg/mL chlorogenic acid and 43.6 µg/mL phlorizin as positive control for 15 min

509

and then exposed to 50 µM NBDG. Glucose uptake was measured after washing

510

immediately at λ 485 nm (extinction) und λ 535 nm (emission). All values are expressed

511

as mean + SEM of three independent experiments in triplicate. ** and *** indicate

512

significant differences in treated cells compared to control cells (p < 0.01 and p < 0.001,

513

respectively, Student’s t-test). (B) Secreted GLP-1 concentration in enteroendocrine 23



Page 24 of 31

514

GLUTag cells after 2 h of incubation. Cells were treated with 50 µg/mL DPC XAD-7,

515

DPC CF and DPC AF extracts and 17.7 µg/mL chlorogenic acid. Forskolin (1.23 µg/mL)

516

was used as positive control. Bars represent means of two individual experiments

517

performed in duplicate measured in a pool of three independent wells + SEM. (C)

518

Relative amount of remaining DPP-4 enzyme activity after incubation with the DPC

519

XAD-7 (50 µg/mL), DPC CF (50 µg/mL) and chlorogenic acid (17.7 µg/mL). P32/98

520

(2.60 µg/mL) was used as a positive control for DPP-4 inhibition. Bars represent means

521

of three different measurements + SEM.

24


Page 25 of 31


Tables. Table 1. Anthocyanin composition of Deep Purple Carrot (DPC) and its XAD-7 extract (n= 3; mean ± SEM). Peak

Anthocyanin

[M]+

Fragments

(m/z)

(m/z)

Concentration (mg Cya-3-glc /100g DPC)*

Concentration (mg Cya-3-glc /g XAD-7)*

1

Cyanidin-3-xylosyl-glucosyl-galactoside

743

287

3.73 ± 0.06

12.5 ± 0.20

2

Cyanidin-3-xylosyl-galactoside

581

287

9.16 ± 0.18

30.7 ± 0.62

3

Cyanidin-3-xylosyl(sinapoylglucosyl)galactoside

949

287

7.12 ± 0.12

23.8 ± 0.40

4

Cyanidin-3-xylosyl(feruloylglucosyl)galactoside

919

287

82.2 ± 0.14

275 ± 0.47

5

Cyanidin-3-xylosyl(coumaroylglucosyl)galactoside

889

287

17.1 ±0.45

57.3 ± 1.52

301

2.04 ±0.29

6.82 ± 0.96

Peonidin-3-xylosyl-galactoside 595 6 *Calculated as cyanidin-3-O-glucoside (Cya-3-glc) equivalents (fresh weight) at λ 520 nm

Table 2. Phenolic acid composition of Deep Purple Carrot (DPC) and its XAD-7 extract (n= 3; mean ± SEM). Concentration (mg 5-CQA/100 DPC)*

Concentration (mg 5-CQA/g XAD-7)*

191, 179, 135

10.2 ± 0.03

34.1 ± 0.11

191, 179

60.0 ± 0.84

201 ± 2.80

9 4-CQA 353 173, 179, 191, 135 15.8 ± 0.05 *Calculated as chlorogenic acid (5-CQA) equivalents (fresh weight) at λ 324 nm

52.9 ± 0.16

-

Peak

Phenolic acid

[M-H] (m/z)

7

3-CQA

353

8

5-CQA

353

Fragments (m/z)

25



Page 26 of 31

Deep Purple XAD-7

Absorption at 280 nm (uAU)

B)


A)

4 8,0E5 6,0E5 4,0E5 3

2,0E5 1 0,0

0

10

1,0E6

2 20

5 6 30

40

50

60 Time (min)

30

40

50

60 Time (min)

8

8,0E5 6,0E5 4,0E5 9 2,0E5

7

0,0 0

10

20

Figure 1

26


Page 27 of 31


Deep Purple AF

Deep Purple CF


4 1,2E6 8,0E5 3 2 5 1 6

4,0E5 0,0

0

10

20

30

40

50

60 Time (min)

4

3,0E6 2,5E6 2,0E6 1,5E6 1,0E6 5,0E5 0,0

1 0

10

3 5 2 6 20

30

40

50

60 Time (min)

Absorptioon at 280 nm (uAU)

C)


B)


A)

3,0E6 2,4E6 1,8E6 1,2E6 6,0E5 0,0

0

10

20

30

40

50

60 Time (min)

8

3,0E6 2,4E6 1,8E6 1,2E6 6,0E5 0,0

7 0

10

9 20

30

40

50

60 Time (min)

Figure 2

27


IC50 = 0.36 g/L

140 120 100 80 60 40 20 0

α-Amylase inhibition [%]



0.5

1 2.5 Acarbose [g/L]

5

1

IC50 = 5.15 g/L

140 120 100 80 60 40 20 0 1

2.5

5 7.5 DPC CF [g/L]

10

50

IC50 = 7.97 g/L

140 120 100 80 60 40 20 0

7.5

2.5

5 7.5 10 DPC XAD-7 [g/L]

50

IC50 = 18.6 g/L

250



0.1

Page 28 of 31

200 150 100 50 0 1

2.5

5 7.5 DPC AF [g/L]

10

50

Figure 3

28



120 100 80 60 40 20 0

IC50 = 5.96 g/L α-Glucosidase inhibition [%]

α-Glucosidase inhibition [%]

Page 29 of 31

0.5

1 2.5 Acarbose [g/L]

5

7.5

1

IC50 = 7.44 g/L

120 100 80 60 40 20 0

2.5

5 7.5 10 DPC XAD-7 [g/L]

50

IC50 = 7.32 g/L

120



0.1

IC50 = 5.04 g/L

120 100 80 60 40 20 0

100 80 60 40 20 0

1

2.5

5 7.5 DPC CF [g/L]

10

50

1

2.5

5 7.5 DPC AF [g/L]

10

50

Figure 4

29


A)

Glucose uptake relative to control


B)

Page 30 of 31

1.20 1.00

***

**

** 0.80 0.60 0.40 0.20 0.00 Control

Phlorizin

Control

Forskolin

DPC XAD-7

DPC CF

DPC AF

Chlorogenic acid

GLP-1 relative to DMSO control

10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

C)

DPC XAD-7

DPC CF

DPC AF

Chlorogenic acid

% of remaining DPP-4 - activity

140 120 100 80 60 40 20 0 Control

Inhibitor P32/98

DPC XAD-7

DPC CF

Chlorogenic acid

Figure 5 30


Page 31 of 31


TOC graphic Deep Purple carrot Copigment fraction XAD-7 extract OH

Anthocyanins

OH

HO

O OH O O

O

H HO HO

HO

HO O

O

OH

OH

O

Membrane Chromatography

OH OH

O

OH

3 OCH

& Anthocyanin fraction

Chlorogenic acids

OH OH HO

O OH O

O HO O

OH

O

H HO HO

HO 3 OCH

Enzyme inhibitory assays: - α-Amylase - α-Glucosidase Dipeptidyl peptidase IV (DPP-4) activity

O

O

OH

OH OH

O

OH

Glucose uptake in CaCo-2 cells Glucagon-like peptide-1 (GLP-1) in GLUTag cells

31


Fractionation of Plant Bioactives from Black Carrots (Daucus carota

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