Identification of a Phosphodiesterase-Inhibiting Fraction from Roasted

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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

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Journal of Agricultural and Food Chemistry

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Identification of a Phosphodiesterase Inhibiting Fraction from Roasted Coffee (Coffea

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arabica) through Activity-guided Fractionation

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Short title: Phosphodiesterase Inhibitors from Roasted Coffee

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Teresa Röhrig, David Liesenfeld, Elke Richling*

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

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Schroedinger-Straße 52, D-67663 Kaiserslautern, Germany

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

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[email protected]).

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Abstract

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

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

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

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

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

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

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

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

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

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

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

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

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Keywords

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Phosphodiesterase,

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fractionation, melanoidins

cyclic

adenosine

monophosphate,


coffee,

activity-guided

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Introduction

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Phosphodiesterases (PDE) hydrolyze the phosphodiester bond of the secondary

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

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guanosine

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

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

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

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

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

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

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

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

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

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

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

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

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2.

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

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

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concentrations up to 200 mg/kg in roasted coffee

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

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

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coffee

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

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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



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

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

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to coffee selection for fractionation.

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Materials and Methods

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Chemicals

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

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

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

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Sigma-Aldrich

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phenylmethylsulfonyl fluoride from Alexis Biochemicals (Enzo Life Sciences,

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

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

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

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

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

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Coffee Samples and Extracts

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

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

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

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

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

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

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

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

(Sigma-Aldrich,

St.

Louis,

MO);


leupeptin,

pepstatin

A,

13-14.

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

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

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

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

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

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concentration during PDE assay (1:3 dilution).

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Liquid-liquid extraction of coffee

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

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

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

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literature 11.

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Activity-Guided Fractionation

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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freeze-drying were too low to calculate gravimetrical ratios.

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cAMP-PDE Activity Assay

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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least three independent experiments. Cells were checked regularly for mycoplasma

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contamination. The mechanism of inhibition was determined according to Lineweaver

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

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HPLC Analysis of Caffeine and Chlorogenic acids

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

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according to

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but identified with reference substances.

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

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

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

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

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

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

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

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

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

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

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tentatively identified by means of their fragments and subsequent literature

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

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

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

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

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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



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

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

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

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

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

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

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Browning Index (BI)

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

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

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

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

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

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

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observed between water or DMSO solved samples.

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Ultrafiltration of Fraction F8

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

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

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

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

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

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dried under nitrogen flow.

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Statistical analysis

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

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

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

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

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

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

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significant. Asterisks reflect the level of significance: p

Identification of a Phosphodiesterase-Inhibiting Fraction from Roasted

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