Identification of a Phosphodiesterase-Inhibiting Fraction from Roasted

Apr 26, 2017 - We chose activity-guided fractionation and an in vitro test system to identify the ..... extracts were tested in the cAMP−PDE activit...
0 downloads 0 Views 912KB Size
Subscriber access provided by SUNY DOWNSTATE

Article

Identification of a phosphodiesterase inhibiting fraction from roasted coffee (Coffea arabica) through activity-guided fractionation Teresa Röhrig, David Liesenfeld, and Elke Richling J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 26 Apr 2017 Downloaded from http://pubs.acs.org on April 27, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

Journal of Agricultural and Food Chemistry

1

Identification of a Phosphodiesterase Inhibiting Fraction from Roasted Coffee (Coffea

2

arabica) through Activity-guided Fractionation

3 4

Short title: Phosphodiesterase Inhibitors from Roasted Coffee

5 6

Teresa Röhrig, David Liesenfeld, Elke Richling*

7

Department of Food Chemistry and Toxicology, University of Kaiserslautern, Erwin-

8

Schroedinger-Straße 52, D-67663 Kaiserslautern, Germany

9

* Corresponding author (tel +49 631 205 4061, fax +49 631 205 3085, email

10

[email protected]).

1 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 34

11

Abstract

12

Recent reports that coffee can significantly inhibit cAMP phosphodiesterases (PDE) in

13

vitro, as well as in vivo, have added another beneficial effect of coffee consumption.

14

However, the PDE-inhibiting substances remain mostly unknown. We chose activity-

15

guided fractionation and an in vitro test system to identify the coffee components

16

responsible for PDE inhibition. This approach indicated that a fraction of melanoidins

17

reveals strong PDE inhibiting potential (IC50 = 130 ± 42 µg/mL). These melanoidins

18

were characterized as water soluble, low molecular weight melanoidins (< 3kDa) with

19

a nitrogen content of 4.2% and a lower carbohydrate content in contrast to other

20

melanoidins. Fractions containing known PDE inhibitors such as chlorogenic acids,

21

alkylpyrazines, or trigonelline as well as N-caffeoyl-tryptophan and N-p-coumaroyl-

22

tryptophan did not exert PDE inhibiting activity. We also observed that the known PDE

23

inhibitor caffeine does not contribute to the PDE-inhibiting effects of coffee.

24

Keywords

25

Phosphodiesterase,

26

fractionation, melanoidins

cyclic

adenosine

monophosphate,

2 ACS Paragon Plus Environment

coffee,

activity-guided

Page 3 of 34

Journal of Agricultural and Food Chemistry

27

Introduction

28

Phosphodiesterases (PDE) hydrolyze the phosphodiester bond of the secondary

29

messengers 3´,5´-cyclic adenosine monophosphate (cAMP) and 3´,5´-cyclic

30

guanosine

31

monophosphates, AMP and GMP. Thus, PDEs, in addition to adenylate and guanylate

32

cyclases, play a major role in regulating cAMP and cGMP dependent signaling

33

pathways. The inhibition of PDEs is a common approach taken in pharmacology, and

34

has been applied to the treatment of hypertension, inflammation, and chronic

35

obstructive pulmonary disease 1. It was recently reported that coffee consumption

36

significantly inhibits cAMP-dependent PDEs in vitro as well as in vivo 2-3. PDE inhibition

37

might be involved in the various beneficial physiological effects of coffee consumption

38

such as weight reduction 4, as well as anti-diabetic

39

Caffeine has been known to be a PDE inhibitor since the discovery of PDEs

40

though its major physiological mode of action is assumed to be adenosine receptor

41

antagonism. Chlorogenic acids at concentrations ranging from 35 up to 100 mg per

42

100 mL in arabica coffee beverages 10 have been identified as potential PDE inhibitors

43

2.

44

roasting exhibit PDE-inhibiting potential. Alkylpyrazines represent a newly discovered

45

group of PDE inhibitors with weak IC50 values (0.4 – 1.6 mM) 2. However, alkylpyrazine

46

concentrations up to 200 mg/kg in roasted coffee

47

significant PDE inhibitory effect in vivo. Another group of components that are typically

48

formed during roasting are melanoidins, which can comprise up to 35% of roasted

49

coffee

50

PDEs or the physiological effects of PDE inhibition. Currently, the major PDE inhibitors

51

of coffee remain unknown. Thus, we employed activity-guided fractionation to identify

monophosphate

(cGMP)

to

their

5-6

corresponding

nucleoside

and anti-thrombotic effects 9

7-8.

even

Latest research of our group was focused on whether components formed during

12.

11

are considered too low for a

However, melanoidins have not yet been linked to either the inhibition of

3 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 34

52

the PDE-inhibiting substances in roasted coffee. Furthermore, we investigated the

53

effects of roasting degree, caffeine content, and brewing method on PDE activity prior

54

to coffee selection for fractionation.

55

Materials and Methods

56

Chemicals

57

The chemicals and reagents used in this study were purchased in p.a. quality. Purity

58

of reference compounds had purities >98%. AMP, benzamidine, bovine serum

59

albumin, caffeine, 5-caffeoylquinic acid, cAMP, and formic acid were purchased from

60

Sigma-Aldrich

61

phenylmethylsulfonyl fluoride from Alexis Biochemicals (Enzo Life Sciences,

62

Farmingdale, NY); rolipram from Calbiochem (EMD Millipore, Billerica, MA); and

63

adenosine-3’5’-cyclic phosphate [2,8-3H], ammonium salt 9.25 MBq/mL from

64

Hartmann Analytic (Braunschweig, Germany). The LXFL529L cell line was kindly

65

provided by Prof. Fiebig (University of Freiburg, Germany). N-Caffeoyl-L-tryptophan

66

and N-p-coumaroyl-L-tryptophan were synthesized according to literature

67

Coffee Samples and Extracts

68

Coffee samples were either purchased from a local supermarket, from a local roasting

69

manufactory (“Kaffeerösterei”, Kaiserslautern, Germany), or provided by Tchibo GmbH

70

(Hamburg, Germany) as powder or beans. Beans were ground with a Moulinette

71

(Moulinex, France). The samples consisted of 100% Arabica coffee. In particular, a

72

medium roast coffee (Tchibo), an espresso roast (Tchibo), a raw and a roasted coffee

73

(“Yellow Bourbon”, Kaffeerösterei), and a roasted decaffeinated coffee (Tchibo) were

74

used in our experiments. The raw and respective roasted coffees were freeze-dried

75

after grinding. All coffees were extracted using 60 g ground coffee per liter of hot water

(Sigma-Aldrich,

St.

Louis,

MO);

4 ACS Paragon Plus Environment

leupeptin,

pepstatin

A,

13-14.

Page 5 of 34

Journal of Agricultural and Food Chemistry

76

(~95 °C) in either a French press (5 min), a common filter machine (~ 6 min), or a

77

beaker with subsequent paper filtering using a Büchner funnel (5 min). The obtained

78

beverages were immediately cooled to 4°C, freeze-dried, and homogenized. The

79

decaffeinated coffee extract was dissolved in a solution of 15% caffeine to add caffeine

80

to the extract before the cAMP-PDE activity assay; this resulted in a 5% final caffeine

81

concentration during PDE assay (1:3 dilution).

82

Liquid-liquid extraction of coffee

83

500 mL freshly brewed coffee K2 (medium roast, filter machine) was extracted with 4

84

x 200 mL petrol ether, the organic phases were concentrated in vacuum, and dried

85

under nitrogen flow. Alkylpyrazines in this fraction were determined according to

86

literature 11.

87

Activity-Guided Fractionation

88

For the activity-guided fractionation a second batch of coffee extract from K2 was

89

prepared and labeled K2*. The medium roast coffee extract K2* (medium roast, filter

90

machine) was reconstituted with H2O at a concentration of 2 g/L and ultra-centrifuged

91

(100,000 RCF, 25°C, 3 h). The resulting insoluble pellet (water insoluble fraction (FN),

92

4.3%) and supernatant (soluble fraction (FS), 95.7%) were freeze-dried separately.

93

The soluble fraction was fractionated further with an Agilent 1200 series preparative

94

HPLC system (Model G1361A) (Agilent, Santa Clara, CA). HPLC conditions: ReproSil

95

100 C18 (5 µm, 250 x 20 mm, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany);

96

solvent system: A - 0.1% formic acid, B - acetonitrile; gradient profile: 2-12% B over 5

97

min, 12-30% B over 15 min, 30-90% B over 1 min, isocratic 90% B for 3 min, 90-2%

98

over 1 min, isocratic 2% B for 5 min; flow rate: 10 mL/min; injection volume: 10 mL;

99

sample concentration: 200 µg/mL in water (FS) or 50% methanol (fraction F8); UV-

100

detection: 270 nm, 325 nm. Fractionation mode was time-dependent every 3 min, 5 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

101

beginning from the injection peak at 5.5 min (F0). The chromatogram can be observed

102

in Figure 1. Yields after freeze-drying were as follows: F0, 8.5%; F1, 24.3%; F2, 16.8%;

103

F3, 7.4%; F4, 13.9%; F5, 6.9%; F6, 4.5%; F7, 4.7%; F8, 3.2%. The yield for F0, which

104

was collected from 0 min to the injection peak, probably resulted from small amounts

105

of fraction F1 reaching F0 as a consequence of minimal shifts of injection peak

106

retention time due to manual injection while fraction collection settings were time

107

dependent based on run start time. Losses during fractionation and drying were 10%.

108

Fraction F4 was further fractionated under the same HPLC conditions into F4a and

109

F4b to separate caffeine. Fraction F8 (100 µg/mL) was further fractionated under the

110

same HPLC conditions every 60 seconds from 24 to 28 min (see Figure 2) resulting in

111

fractions F8.1, F8.2, F8.3, F8.4, and F8.5. The yields of fractions F8.1 - F8.5 after

112

freeze-drying were too low to calculate gravimetrical ratios.

113

cAMP-PDE Activity Assay

114

Phosphodiesterases were isolated from LXFL529L cell lysate using a RUN III buffer

115

(100 mM TRIS/HCl, pH 7.4, containing 20 mM MgCl2, 0.2 mM EDTA, 10 mM

116

benzamidine, 1 mM β-mercaptoethanol, and a protease inhibitor mix containing PMSF,

117

leupeptin, pepstatin A). LXFL529L cells express mostly cAMP specific PDE IV

118

isozymes. 106 cells per cell culture dish were cultivated for 48 h and then harvested

119

with 2 x 200 µL RUN III buffer. Following ultrasound lysis and centrifugation (12,000

120

RCF, 15 min, 4°C) the supernatant, which contains the cytosolic phosphodiesterases,

121

was obtained. The cytosolic fraction was further diluted with RUN III buffer in order to

122

adjust the PDE hydrolysis rate to 20-25% after 10 min incubation. The influences of

123

various extracts and constituents on cAMP-PDE activity were measured according to

124

Montoya et al. (2014)2. Samples were dissolved in either water (coffee extracts, F0 -

125

F5) or DMSO (F6 - F8) and subsequently diluted to a concentration of 3% DMSO. 6 ACS Paragon Plus Environment

Page 6 of 34

Page 7 of 34

Journal of Agricultural and Food Chemistry

126

Rolipram (10 µM), a selective PDE 4 inhibitor, served as the positive control.

127

Experiments were performed in triplicates and IC50 values were determined after at

128

least three independent experiments. Cells were checked regularly for mycoplasma

129

contamination. The mechanism of inhibition was determined according to Lineweaver

130

and Burk, Dixon, and Cornish-Bowden15-17.

131

HPLC Analysis of Caffeine and Chlorogenic acids

132

The caffeine and chlorogenic acids present in the coffee extracts were quantified

133

according to

134

but identified with reference substances.

135

HPLC Analysis and HPLC-MS/MS Identification of Fractions F6, F7, F8

136

The HPLC analyses of fractions F6, F7, F8, as well as 8.1, 8.2, 8.3, 8.4, and 8.5, were

137

performed with an Agilent 1200 series HPLC system (Model G1312B) equipped with a

138

degasser (G1379B), binary pump (G1312B), auto-sampler (G1317C), column oven

139

(G1316B), and DAD detector (G1315). Substances that could not be identified through

140

the initial HPLC analyses were identified with a Perkin Elmer 200 series HPLC-UV

141

(PerkinElmer, Waltham, MA) equipped with a degasser, two micro pumps, an auto-

142

sampler, and UV detector (785A) coupled to a PE Sciex API 2000 triple quad mass

143

spectrometer (SCIEX, Framingham, MA). First precursor ions were tentatively

144

identified in a full scan, then a product ion scan was performed. Constituents were

145

tentatively identified by means of their fragments and subsequent literature

146

comparison. HPLC conditions: Synergi 4 µm polar RP 80Å (250 x 4.6 mm,

147

Phenomenex); solvent system: A - 0.1% formic acid, B - acetonitrile; gradient profile:

148

isocratic 10% B over 9 min, 10-25% B over 1 min, isocratic 25% B over 9 min, 25-50%

149

B over 15 min, 50-98% B over 1 min, isocratic 98% B for 5 min, 98-10% B over 1 min,

150

isocratic 10% B for 5 min; flow rate: 0.8 mL/min; injection volume: 50 µL; sample

18.

3- and 4-caffeoylquinic acid were quantified as 5-caffeoylquinic acid

7 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

151

concentration: 1 mg/mL in 10% acetonitrile; UV-detection: 248 nm, 260 nm, 280 nm.

152

ESI-MS(/MS) conditions: positive ion mode; ion spray voltage: 4700 V; temperature

153

450°C; declustering potential: 50 V; focusing potential: 340 V; entrance potential: 10,5

154

V; collision cell entrance potential: 12-30 V; negative ion mode; ion spray voltage: -

155

4500 V; temperature 450°C; declustering potential: -50 V; focusing potential: -340 V;

156

entrance potential: -10,5 V; collision cell entrance potential: -10 to -20 V.

157

Browning Index (BI)

158

The Browning index (BI) of coffee samples and fractions was measured based on

159

absorbance at 420 nm. The coffee extracts, at a concentration of 5 mg/mL in water,

160

and the fractions, at concentrations of either 1 mg/mL or 500 µg/mL in 20% DMSO,

161

were filtered through a membrane (0.45 µm Nylon, Restek, Bad Homburg, Germany)

162

and absorbance of 200 µL samples in a 96-Well plate was measured in triplicates. The

163

absorbance of the solvent control was subtracted from all samples. No difference was

164

observed between water or DMSO solved samples.

165

Ultrafiltration of Fraction F8

166

A 500 µg/mL sample of F8 was dissolved in a 1 M NaCl solution in order to break

167

possible ionic bonds. The ultrafiltration cartridge (Vivaspin 6, Sartorius, Göttingen,

168

Germany, 3 kDa) was filled and then centrifuged (4,000 RCF, 25°C, 1 h). After two

169

subsequent washing steps (1 mL NaCl 1M, 4,000 RCF, 25°C, 1.5 h), the filtrate was

170

freeze-dried and the residue was dissolved in dried acetone. The supernatant was then

171

dried under nitrogen flow.

172

Statistical analysis

173

The results of the cAMP-PDE inhibition assays are expressed as a mean ± standard

174

deviation of at least three independent experiments. Statistical analyses were carried 8 ACS Paragon Plus Environment

Page 8 of 34

Page 9 of 34

Journal of Agricultural and Food Chemistry

175

out with Analysis Tool in MS Excel 2013 software (Microsoft, Redmond, WA) and

176

Origin 9.1G (OriginLab, Northampton, MA). A one-sided Fisher’s F-test with a 95%

177

confidence interval was used to test for the equality of variance. A one-sided student’s

178

t-test was used to determine whether the differences between samples were

179

significant. Asterisks reflect the level of significance: p