Human bitter taste receptors are activated by different classes of

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Food and Beverage Chemistry/Biochemistry

Human bitter taste receptors are activated by different classes of polyphenols Susana Soares, Mafalda Santos Silva, Ignacio García-Estévez, Peggy Gro#man, Natércia Fernandes Brás, Elsa Brandão, Nuno Mateus, Victor De Freitas, Maik Behrens, and Wolfgang Meyerhof J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03569 • Publication Date (Web): 28 Jul 2018 Downloaded from http://pubs.acs.org on July 31, 2018

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Journal of Agricultural and Food Chemistry

Human Bitter Taste Receptors are Activated by Different Classes of Polyphenols Susana Soares1, Mafalda Santos Silva1, Ignacio García-Estevez1,2, Peggy Groβman, Natércia Brás3, Elsa Brandão1, Nuno Mateus1, Victor de Freitas1, Maik Behrens4,5, Wolfgang Meyerhof4,6

1

REQUIMTE, LAQV, Department of Chemistry and Biochemistry, Faculty of Sciences, University of

Porto, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal 2

Grupo de Investigación en Polifenoles (GIP). Facultad de Farmacia, University of Salamanca,

E37007, Salamanca, Spain 3

REQUIMTE, UCIBIO, Department of Chemistry and Biochemistry, Faculty of Sciences, University of

Porto, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal 4

DIFE - German Institute of Human Nutrition, Department of Molecular Genetics, Arthur-Scheunert-

Allee 114-116, 14558 Potsdam Rehbrücke, Germany 5

Leibniz-Institute for Food Systems Biology at the Technical University of Munich, Lise-Meitner-

Strasse 34, 85354 Freising, Germany 6

Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University

Kirrbergerstrasse,

Bldg.

48,

66421

Homburg,

Germany

1 ACS Paragon Plus Environment


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1

ABSTRACT

2

Polyphenols may contribute directly to plant-based foodstuffs flavor, in particular to astringency

3

and bitterness.

4

In this work, the bitterness of a small library of polyphenols from different classes [procyanidin

5

dimers type B, ellagitannins (punicalagin, castalagin and vescalagin) and phenolic acid ethyl esters

6

(protocatechuic, ferulic and vanillic acid ethyl esters] was studied by a cell-based assay. The bitter

7

taste receptors (TAS2Rs) activated by these polyphenols and the half-maximum effective

8

concentrations (EC50) of each agonist-TAS2Rs pair was determined. Computational methodologies

9

were used to understand the polyphenol molecular region responsible for receptor activation and

10

to get insights into the type of bonds established in the agonist-TAS2Rs pairs.

11

The results show the combinatorial pattern of TAS2Rs activation. TAS2R5 seems to be the only

12

receptor exhibiting a bias towards the activation by condensed tannins, while TAS2R7 seems more

13

tuned for hydrolyzable (ellagi)tannins. Additionally, at the concentrations usually found for these

14

compounds in foodstuffs, they can actively contribute to bitter taste, especially ellagitannins.

15

Keywords: procyanidins, tannins, astringency, phenolic acid ethyl esters

16


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INTRODUCTION

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Polyphenols comprise a wide group of structurally diverse compounds that are generally known for

19

their important health-promoting activities.1 These compounds are secondary metabolites of plants

20

being widespread in numerous plant-based foodstuffs which are largely consumed worldwide,

21

including tea, coffee, red fruits, and wine.2, 3

22

Polyphenols are usually divided in three major groups,2 the non-flavonoids, including mainly small

23

molecules like phenolic acids (e.g. protocatechuic, ferulic and gallic acids) and stilbenes; the

24

flavonoids, including flavanols (e.g. procyanidins), (iso)flavones (e.g. genistein), anthocyanins (e.g.

25

malvidin-3-glucoside), flavonols (e.g. quercetin), flavanones (e.g. naringenin) and chalcones (e.g.

26

xanthohumol); and, tannins that comprise a group of polyphenols with the special ability to interact

27

with proteins.4 Tannins are further divided in two major groups, the condensed and the

28

hydrolyzable tannins.4, 5 The former are oligomers or polymers of (epi)catechin and other flavan-3-

29

ol units and are therefore a subclass of the flavonoids/flavanols. Within this group there is a

30

diversity of structures due to the different type of bonds between the subunits (C8-C4 or C6-C4, B-

31

type or A-type interflavan linkages), degree of polymerization and degree of galloylation.

32

Hydrolyzable tannins are further divided into gallo- (GTs) and ellagitannins (ETs). ETs are

33

structurally highly complex being characterized by one or more hexahydroxydiphenoyl (HHDP)

34

units esterified to a sugar core, usually glucose. This complex class of polyphenols is further

35

categorized according to structural characteristics into four major groups: monomeric ETs (e.g.

36

punicalagin), C-glycosidic ETs with an open-chain glycoside core (e.g. castalagin, vescalagin),

37

oligomers, and complex tannins with flavan-3-ols.6, 7 ETs have an enormous structural variability

38

because of the different linkages of HHDP groups with the glucose moiety and their strong

39

tendency to form dimeric and oligomeric derivatives.

40

Polyphenols are widespread in foodstuffs. Pomegranates are one of the main valuable sources of

41

ETs that can also be found in other fruits and nuts (e.g., strawberries, raspberries, blackberries,

42

cloudberries, muscadine grapes, almonds and walnuts).8 Tea, namely green tea Camellia sinensis, is 3 ACS Paragon Plus Environment


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mainly rich in catechins, namely epigallocatechin gallate (EGCG), while procyanidin dimers,

44

oligomers and higher polymers are common in many other food products such as red grapes and

45

red wine, peaches, apples and chocolate.3 These compounds may contribute directly to the flavor

46

of foodstuffs’ flavor such as astringency and bitter taste.9 This latter can be appreciated in some

47

food items, such as beer, coffee, dark chocolate and red wine, but in most cases bitterness in food

48

is unwanted and efforts are taken to reduce bitter taste.9 The key goal is to reduce bitterness of

49

foodstuffs rich in polyphenols while keeping the health-promoting properties of polyphenols. To

50

achieve this it is important to determine which of the polyphenols are actually bitter and which of

51

the bitter taste receptors (TAS2Rs) are responsible for their detection. Although some polyphenols

52

have been identified as bitter compounds, there exists a considerable degree of inconsistency

53

across the sensory analysis-based literature. In general, these works assess the bitterness of

54

fractions/mixtures of compounds, such as polymeric fractions of tannic acid and tannins.10

55

Regarding structure/bitterness, some reports demonstrated that larger molecules tend to be less

56

bitter than smaller molecules. Conversely, others found that (−)-epicatechin was more bitter than

57

the stereoisomer (+)-catechin and that both were more bitter than the procyanidin trimers,

58

catechin-(4−8)-catechin-(4−8)-catechin and catechin-(4−8)-catechin-(4−8)-epicatechin.11 Robichaud

59

and colleagues found that tannic acid, a commercially available mixture of PGG and other esters

60

both more and less esterified (not well defined compound), was more bitter than both (+)-catechin

61

and a grape seed extract, which is rich in polymeric procyanidins.10

62

To overcome the limitations and inconsistencies of sensory studies, few studies were published

63

that analyzed the bitterness of polyphenols by cell-based assays through the heterologous

64

expression of TAS2Rs.12-14 In contrast to some of these studies, which mostly relied on testing only

65

commercially available compounds, the present work gathered a library of polyphenols both

66

isolated from food/vegetable sources as well as acquired commercially available, representative

67

compounds of different important classes of polyphenols: procyanidin dimers type B, ETs

68

(punicalagin, castalagin and vescalagin) and phenolic acid ethyl esters (protocatechuic, ferulic and 4 ACS Paragon Plus Environment

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vanillic acid ethyl esters). The agonist activities of these polyphenols were determined and the half-

70

maximum effective concentrations (EC50) determined. Computational methodologies were used to

71

understand the molecular region of polyphenols responsible for the binding, and to get insights

72

into the type of bonds established in the agonist-receptor pairs.



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MATERIAL AND METHODS

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Reagents. All reagents used were of analytical grade. Toyopearl HW-40(s) gel was purchased from

75

Tosoh (Tokyo, Japan). Epigallocatechin gallate (EGCG) was acquired from Biopurify Phytochemicals

76

Ltd. (Chengu, Sichuan, China). (+)-Catechin, (−)-epicatechin, ferulic acid ethyl ester, protocatechuic

77

acid ethyl ester and vannillic acid ethyl ester were purchased from Sigma-Aldrich, sodium

78

borohydride and tartaric acid were purchased from Aldrich and taxifolin was purchased from

79

Extrasynthèse (Genay, France).

80

Procyanidin Dimers Extraction and Isolation. Grape seeds (Vitis vinifera) were extracted as

81

described previously yielding four fractions.15,

82

ethanol/water/chloroform solution (1:1:2, v/v/v). The hydroalcoholic phase was then extracted

83

with ethyl acetate, and the organic phase was evaporated using a rotary evaporator (30 ºC). The

84

resulting residue corresponding essentially to procyanidins was fractionated through a TSK

85

Toyopearl HW-40(s) gel column (100 mm x 10 mmi.d., with 0.8 mL.min-1 methanol or

86

methanol/acetic acid), yielding four fractions according to the method described in the literature.17

87

All fractions were mixed with deionized water, and the organic solvent was eliminated using a

88

rotary evaporator under reduced pressure at 30 °C and then freeze-dried. Fractions were analyzed

89

by HPLC and the procyanidin dimers B1, B2, B2 3’-O-gallate, B7 and procyanidin trimer C1 were

90

isolated by preparative HPLC (HPLC Dionex Ultimate 3000, Thermo Fisher Scientific) equipped with

91

a reverse-phase C18 column (PrepLC C18, Waters) (150 mm x 2.5 mm; eluent A 1% formic acid

92

aqueous, eluent B: 1% formic acid in acetonitrile; gradient 0 min: 10% B, 37 min: 14.5% B, 40 min:

93

20% B, 55 min: 35% B, 57 min: 90% B; 0.5 mL.min-1) from the respective fraction.

94

Procyanidin Dimers Synthesis. The synthesis of procyanidin dimers B3 (catechin-(4-8)-catechin)

95

and B6 (catechin-(4-6)-catechin) as well as procyanidin dimer B4 (catechin-(4-8)-epicatechin)

96

followed the procedure described in the literature.18-20 Briefly, a taxifolin and (epi)catechin mixture

97

(ratio 1:3) was dissolved in ethanol under argon atmosphere and treated with sodium borohydride

98

(in ethanol). Using CH3COOH/H2O 50% (v/v), the pH was adjusted to 4.5 and the mixture was kept

16

Briefly, grape seeds were extracted with


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99

under argon atmosphere for 30 min. The reaction mixture was extracted with ethyl acetate. After

100

evaporation of the solvent, water was added, and the mixture was passed through reversed-phase

101

C18 gel, washed again, and recovered with methanol. After methanol evaporation, this fraction

102

was separated through a TSK Toyopearl HW-40(s) gel column (300 mm × 10 mm i.d., 0.8 mL.min-1,

103

methanol as eluent) coupled to a UV−Vis detector. From this, several fractions were recovered,

104

concentrated and analyzed by LC-MS (Finnigan DECA XP PLUS). Spectroscopical data were in

105

accordance with the literature.19, 21

106

Ellagitannins (ETs) Extraction and Isolation. Castalagin, vescalagin and grandinin were obtained

107

from medium-toasted oak chips (Quercus petraea (Matt.) Liebl wood) as referred in the

108

literature.22 A Sephadex LH-20 column was used, and different fractions containing the major ETs

109

were eluted with methanol/acidified water. The composition of these fractions was determined by

110

HPLC−DAD-MS as well as the ETs purity after purification by semipreparative HPLC. Punicalagin was

111

isolated from pomegranate, as previously reported.23 Briefly, dried husk powder (1 g) was extracted

112

ultrasonically with 30 mL of 40% ethanol for 30 min twice. After ethanol evaporation, the extract

113

was lyophilized and analyzed by LC−MS. Punicalagin puriﬁcaXon was performed by semipreparaXve

114

HPLC and its purity was determined by LC−MS.

115

Cell Transfection and Expression of TAS2Rs in Heterologous Cells. Functional expression studies

116

were carried out as described before.13 Human embryonic kidney (HEK)-293T cells stably expressing

117

the chimeric G protein subunit Gα16gust44 were seeded into poly-D-lysine-coated (10 μg xmL−1)

118

96-well plates under regular cell culture conditions [Dulbecco’s modified Eagle medium (DMEM),

119

10% FCS, 1% penicillin/streptomycin; 37 °C, 5% CO2, 95% humidity]. After 24−26h, cells were

120

transfected transiently with 150 ng expression plasmids using 300ng of Lipofectamine2000

121

(Invitrogen) per well. Expression vector were based on pEAK10 (Edge BioSystems) or pcDNA5/FRT

122

(Invitrogen). In addition to the TAS2R coding sequences, the plasmids contained the coding

123

sequences of the first 45 amino acids of rat somatostatin receptor 3 for cell surface localization and



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the herpes simplex virus (HSV) glycoprotein D epitope for immunocytochemical detection of the

125

receptors. The TAS2R sequences are according to the literature.24

126

Calcium Imaging Analysis. Twenty-four to 26 h after transfection, cells were loaded with the

127

calcium-sensitive dye Fluo-4-acetoxymethylester (2.0 μM, Molecular Probes) in serum-free DMEM.

128

Probenecid (Sigma-Aldrich GmbH), an inhibitor of organic anion transport, was added at a

129

concentration of 2.5 mM, to minimize the loss of the calcium indicator dye from cells. One h after

130

loading, the wells were washed three times with C1 buffer using a cell washer (BioTek). Cells were

131

incubated in washing buffer in the dark for 30 min between the washing steps. Fluorescence

132

changes were recorded at 510 nm following excitation at 488 nm by a fluorometric imaging plate

133

reader (FLIPR, Molecular Devices) before and after application of the test compounds. A second

134

application of 100 nM somatostatin-14 (Bachem) activating the endogenous somatostatin receptor

135

type 2 was used to assess cell vitality. All experiments were performed at least in duplicates. Mock-

136

transfected cells (cells transfected with empty pcDNA5/FRT or pEAK10 vectors used as negative

137

control) were always measured in parallel on the same microtiter plates using the same compound

138

concentrations used to examine the cells expressing the various TAS2Rs.

139

Unspecific responses test and screening of TAS2Rs. All compounds were initially tested at different

140

concentrations for unspecific calcium responses in mock-transfected HEK293T Gα16gust44 cells.

141

Different concentrations of sixteen polyphenol compounds were tested in pilot experiments to

142

determine the highest possible concentrations to be used for the screening experiment:

143

procyanidin dimers B1, B2, B3, B6, B7 and punicalagin were used up to 67 μM; procyanidin B2 3-O-

144

gallate, EGCG, vescalagin, castalagin, grandinin and ferulic acid ethyl ester were used up to 100 μM;

145

procyanidin B4 and vanillic and protocatechuic acids (400) ethyl esters were used up to 133 μM and

146

procyanidin trimer C1 was used up to 150 μM. The maximal compound concentrations used for the

147

TAS2R screening and dose-response assays were always lower than those concentrations. On the

148

basis of these pilot experiments, to identify the TAS2Rs that are sensitive to the selected

149

polyphenols, the 25 TAS2Rs, TAS2R1, TAS2R3, TAS2R4, TAS2R5, TAS2R7, TAS2R8, TAS2R9, TAS2R10, 8 ACS Paragon Plus Environment

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TAS2R13, TAS2R14, TAS2R16, TAS2R38, TAS2R39, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R31,

151

TAS2R45, TAS2R46, TAS2R30, TAS2R19, TAS2R20, TAS2R50, and TAS2R60, were expressed

152

individually in HEK293T cells stably expressing the chimeric G protein Gα16gust44, as referred

153

previously.

154

Determination of Half-Maximal Effective Concentrations (EC50) and Statistical Analysis. Having

155

identified responsive TAS2Rs, their concentration-dependent activation was examined and half-

156

maximal effective concentration (EC50) values for their bitter agonists were established. To

157

calculate the concentration−response curves, the ﬂuorescence changes of mock-transfected cells

158

were subtracted from the corresponding values of receptor-expressing cells by means of the FLIPR

159

software (Molecular Devices, Munich, Germany). To compensate for differences in cell density,

160

signals were normalized to background fluorescence for each well. Signals were recorded in at least

161

duplicate wells and the data averaged. Signal amplitudes were then plotted versus log agonist

162

concentration. EC50 values were calculated using SigmaPlot (Systat Software Gmbh, Erkrath,

163

Germany) by nonlinear regression using the function: () = min +

(max − min ) 1 + ( )

164

where x is the test compound concentration and nH the Hill coefficient. Statistical significance of

165

the difference between the several calculated EC50 and of the signal amplitudes was evaluated by

166

one-way analysis of variance, followed by the Bonferroni test. Differences were considered to be

167

statistically significant when P < 0.05.

168

Computational studies. The molecular structures of the four tested TAS2Rs (TAS2R5, TAS2R7,

169

TAS2R14 and TAS2R39) were created with GPCRDB homology modeling pipeline.25 The β2

170

adrenergic receptor (PDB ID: 3SN6),26 the serotonin 2B receptor (PDB ID: 5TUD)27 and the mu-

171

opioid receptor (PDB ID: 5C1M)28 were used as templates for constructing TAS2R5, TAS2R7 (and

172

TAS2R14), and TAS2R39 models, respectively. These models were optimized at physiological pH

173

using the Amber 12.0 simulation package29 (parm99SB force field).30 An explicit solvation model



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with TIP3P water molecules was used, filling a rectangular box with a minimum distance of 12 Å

175

between the box faces and any atom of each system. The minimization was carried out in two

176

steps: 1) the geometry of the water molecules and counter-ions were optimized; and 2) the

177

geometry of all atoms was optimized.

178

The GaussView software31 was used to build the 3D structures of the six phenolic ligands

179

(procyanidin B2 3-O- gallate, castalagin, punicalagin, malvidin-3- glucoside, ferulic acid ethyl ester

180

and pentagalloylglucose (PGG)).

181

Protein:ligand docking calculations were performed with the AutoDock 4.2 software.32 The VsLab

182

plug-in was employed,33 integrated in the VMD 1.9.2 software34 for files preparation, visual

183

inspection and analysis. The grid box was centered on two conserved residues of human TAS2Rs,

184

and comprised 70x70x80 points with a 0.375 Å spacing. The Trp89 and Phe247 residues of TAS2R14

185

were chosen due to their relevance for the binding of some recently proposed agonists.35 The

186

equivalent residues, obtained by sequence alignment, were used for the other receptors: TAS2R5

187

(Trp85 and Tyr247), TAS2R7 (Trp89 and Tyr247) and TAS2R39 (Phe117 and Asn274). The

188

Lamarckian genetic algorithm (LGA) was employed with the following parameters: population size

189

of individuals: 150; maximum number of energy evaluations: 2.5x10 6 and maximum number of

190

generations: 27,000. For all the calculations, 50 docking rounds were performed with step sizes of

191

2.0 Å for translations, and with orientations and torsions step size of 5.0°.


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RESULTS AND DISCUSSION

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The bitterness of polyphenol compounds is a prominent subject of relevance for consumer’s food

194

acceptance and choices and lastly for people’s food phytochemical-based health protection. So, the

195

current study intended to determine if the library of the 16 polyphenols selected, some of which

196

already have been described as bitter compounds36, 37 by sensory analysis, were able to activate

197

human bitter taste receptors (TAS2Rs). After identification of the activated receptors, the dose-

198

response curves for each agonist-receptor pair were determined. Finally, molecular docking studies

199

(detailed results presented as Supplementary Information) were made to assess which molecular

200

groups were most likely involved in the activation of each receptor and which type of bonds were

201

involved.

202 203

Identification of the TAS2Rs activated by polyphenol compounds and structure/activation

204

relationship. By tracing cytosolic calcium levels (Figure 2), the activation of TAS2Rs by sixteen

205

polyphenol compounds commonly present in human diet, namely condensed tannins (procyanidin

206

dimers B1, B2, B3, B4, B6, B7, procyanidin dimer B2 3-O-gallate (procyanidin B2g), EGCG and

207

procyanidin trimer C1), ellagitannins (ETs) (vescalagin, castalagin, punicalagin, and grandinin) and

208

phenolic acid ethyl esters (ferulic acid, protocatechuic acid and vanillic acid) was studied. One or

209

more TAS2Rs were identified as responsive for individual compounds, except for procyanidin

210

dimers B2, B3, B6 and trimer C1. These four compounds did not activate any TAS2R, at least in the

211

concentration range used (up to 67 - 150 μM). Figure 2 presents the fluorescence changes upon

212

TAS2R activation by an agonist of each family of polyphenol compounds. Overall, seven TAS2Rs

213

(TAS2R4, TAS2R5, TAS2R7, TAS2R14, TAS2R39, TAS2R43, TAS2R30) were activated by twelve

214

polyphenol compounds (Table 1).

215

The agonist/receptor pairs display an interesting activation pattern. First, different compounds

216

activate the same receptor. For example, procyanidins B1, B4, B7, B2g, EGCG and punicalagin

217

activate TAS2R5, whereas vescalagin, castalagin, punicalagin and grandinin activate TAS2R7. Also, 11 ACS Paragon Plus Environment


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all phenolic acid ethyl esters activate TAS2R14. Second, different receptors are activated by the

219

same compound. This is evidenced by EGCG, which activates TAS2R4, TAS2R5, TAS2R39, TAS2R43

220

and TAS2R30, and for procyanidin B1, procyanidin B2g, punicalagin and protocatechuic acid that

221

activate two different TAS2Rs each. These results confirm well the combinatorial activation

222

patterns of TAS2Rs previously reported and already seen for other polyphenol compounds.13

223

It can be observed that most compounds activated only one receptor, while five compounds

224

activate more than one receptor, namely procyanidin dimer B1, procyanidin B2g, EGCG, punicalagin

225

and protocatechuic acid ethyl ester. Among these, EGCG remarkably activated five different

226

receptors.

227

Additionally, looking at the structural diversity of the compounds studied here, these data also

228

have implications for structure−acXvity relaXonships. They suggest that the catechol or galloyl

229

group (Figure 1) could be a critical feature (although not essential) for the interaction of polyphenol

230

compounds with TAS2R5 since all compounds have at least one of these groups. First, the

231

compounds that activated this receptor, procyanidin dimers B1, B4, B7 have two catechol groups,

232

while procyanidin B2g and EGCG have one galloyl group. Punicalagin has also a galloyl derived

233

moiety. This is in agreement with a previous study that identified (−)-epicatechin, procyanidin

234

trimer, and pentagalloylglucose (PGG) as agonist for this receptor,13 and these compounds also

235

contain at least one of these molecular groups. Somehow, the molecular arrangement of the

236

galloyl groups to form the HHDP or the NHTP moieties seem to compromise the activation of

237

TAS2R5. Castalagin and vescalagin do not activate this receptor while punicalagin, that has two

238

galloyl groups apart from HHDP moiety, activates it.

239

However, the galloyl group is not essential for TAS2R5 activation. Other compounds that activate

240

TAS2R5, such as the synthetic ligand 1,10-phenanthroline lack these groups.24 Notably, the above

241

polyphenols together with (-)-epicatechin, procyanidin trimer C2 and PGG are the first natural

242

bitter compounds found for TAS2R5. So far, this receptor responded only to the synthetic


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compound phenanthroline and may therefore be the only TAS2R that is activated (“specifically”) by

244

natural tannins.

245

Another common feature was that all the ETs studied (punicalagin, castalagin, vescalagin and

246

grandinin) were agonists for receptor TAS2R7, in addition to the procyanidin dimer B1. All the ETs

247

share a glucose moiety in their structure; contrasting to punicalagin, the other ETs, castalagin,

248

vescalagin and grandinin present an open-chain glycoside core. It is interesting that in a previous

249

study malvidin-3-glucoside, an anthocyanin bearing a glucose moiety, was identified as agonist for

250

TAS2R7. This compound was also included in the molecular docking studies and it was observed

251

that the glucose hydroxyl groups of malvidin-3-glucoside actively contribute to TAS2R7 interaction

252

forming hydrogen bonds (Figure 4).

253

In general, the glycoside moiety is unlikely to represent the only key structural feature for TAS2R7

254

activation since most of the identified agonists for this receptor, chloroquine, quinine, diphenidol,

255

caffeine and sodium cromoglycate, do not present a glucose moiety. The common structural

256

feature to all these compounds is the presence of at least two adjacent cyclopentane or

257

cyclohexane ring systems in their structure. In fact, the molecular docking studies for punicalagin

258

and castalagin showed that for these compounds the interaction with TAS2R7 is favored by

259

hydrogen bonds between the hydroxyl groups of the rings systems (Figure 4). In fact, punicalagin

260

and castalagin rings themselves seem not to be able to do π-π stacking with aromatic amino acid

261

residues due to the high molecular weight and low flexibility of these compounds observed in the

262

theoretical studies. So, at the end, there could be several key molecular elements for TAS2R7

263

activation.

264

TAS2R14 was activated by the three phenolic acids ethyl ester compounds. Although EGCG has

265

been previously identified as agonist for this receptor, here it did not activate TAS2R14.38 According

266

to various studies that identified a large variety of putative bitter compounds structurally very

267

divergent as agonists,

268

TAS2R14 agonists, except that they contain one or several aromatic rings and at least one

14, 39, 40

it appears to be no obvious common structural motif shared by all



Page 14 of 31

269

electronegative side chain, like the phenolic acid ethyl esters studied here. In fact, regarding

270

flavonoids the importance of aromatic ring systems for TAS2R14 activation has been already

271

reported.14

272

To explain this promiscuity (structural and size diversity) among TAS2Rs agonists, it has been

273

hypothesized that TAS2R14 recognizes only a small frequently occurring part of bitter molecules

274

(such as the molecular ring system) and that the size of the binding pocket should not be limiting

275

for larger molecules.39, 41 In fact, a docking study observed that the receptor TAS2R14 is able to

276

accommodate agonists with a wide range of sizes, indicating that agonist-receptor contact points

277

do not envelop the ligand tightly.35 In agreement with these ideas, the molecular docking study for

278

the pair TAS2R14-ferulic acid ethyl ester support the fact that the ring system is the most important

279

molecular region for the binding, by establishing hydrophobic interactions with the TAS2R14.

280

TAS2R39 has been already identified as sensitive to several different natural and synthetic

281

compounds belonging to the receptors with intermediate agonist spectra.24 Here it has been

282

activated by B2g and EGCG. Other polyphenols, such as flavonoids12, 14 as well as the hydrolyzable

283

tannin PGG,13 have been previously identified as agonist for TAS2R39. It has been suggested that

284

this receptor seems, somehow, to be a bitter receptor for dietary compounds since many agonists

285

are dietary compounds.42

286

The structural features for an (iso)flavonoid to activate TAS2R39 have been determined by 3D-

287

pharmacophore models to be composed of two (or three) hydrogen bond donor sites, one

288

hydrogen bond acceptor site, and two aromatic ring structures, of which one has to be

289

hydrophobic.14 This is in line with the molecular docking studies for the pairs receptor-agonist

290

TAS2R39-B2g and TAS2R39-PGG. In both cases a significant π-π stacking between the aromatic

291

rings of galloyl groups and the receptor residues as well as hydrogen bonds between the hydroxyl

292

groups of the galloyl groups and the receptor residues were observed (Figure 4).

293

Regarding the TAS2R43 and TAS2R30 they are both activated by one compound only, EGCG and

294

protocatechuic acid ethyl ester, respectively. These receptors are also known to have a quite broad 14 ACS Paragon Plus Environment

Page 15 of 31


295

agonist spectra including both natural and synthetic compounds, lacking clear common motifs that

296

could be responsible for a specific recognition.24

297

Half-maximal activation agonist concentrations (EC50) and efficacy for the activated TAS2Rs. To

298

investigate the activation of the seven bitter receptors identified above by the polyphenols in

299

greater detail, concentration−response funcXons were recorded and the half-maximal activation

300

(EC50) agonist concentrations established (Figure 3). The concentration−response curves followed

301

sigmoid functions. The calculated EC50 values for the agonist−receptor pairs are presented in Table

302

2. Unfortunately, for some agonist-receptor pairs (procyanidin dimers B4 and B7, EGCG-TAS2R4

303

and EGCG-TAS2R30) it was not possible to determine the EC50 because unspecific responses were

304

observed in the control condition (mock) for the concentrations necessary to obtain a dose-

305

response curve.

306

Altogether, the data demonstrate that the EC50 values for the different agonist-receptor pairs have

307

a 100-fold range in the micromolar range. Whereas the EC50 value for grandinin is 2.43 μM for

308

TAS2R7, the value for protocatechuic acid ethyl ester is 155.64 μM. In general, and surprisingly, for

309

compounds that activated different receptors, similar ranges of the EC50 values were observed. The

310

EC50 values for procyanidin B1 are statistically equal for the two receptors activated (TAS2R5 and

311

TAS2R7). A similar situation was observed for procyanidin B2g and for EGCG. However, for

312

punicalagin the EC50 values exhibit a 10-fold difference in magnitude between the two receptors

313

activated. Also, the receptors activated by protocatechuic acid ethyl ester have significantly

314

different EC50 values.

315

Comparing the different families of compounds, interestingly, the EC50 value for all ETs are the

316

lowest for all the studied compounds and in the same magnitude (from 2.43 to 7.26 μM). This

317

means that these ETs are of high potency (low EC50 values) to this receptor. Furthermore, they have

318

also a high efficacy of TAS2R7 activation. This means that these ETs may elicit a strong bitter taste

319

in food even if they occur at low concentrations.



Page 16 of 31

320

The three phenolic acid ethyl esters activated the same receptor, the TAS2R14, with EC50 values

321

around 60 μM or 150 μM. Among the studied esters, ferulic acid ethyl ester was the one with the

322

highest potency. Comparing with other agonists for this receptor, these compounds present low

323

potency: the EC50 of saikosaponin is reached at 4.9 μM43 and flufenamic acid has an EC50 of 10

324

nM.39 Protocatechuic acid ethyl ester also activates TAS2R30 but it shows a lower potency (82.39

325

μM).

326

Here, the procyanidins activated intensively only the receptor TAS2R39, in particular procyanidin

327

B2g and EGCG (EC50 reached at 9.11 μM and at 8.50 μM, respectively) when comparing to other

328

agonists.24 EGCG was found to be the most efficient one in TAS2R39 activation but it also showed

329

high efficiency on TAS2R43 activation (EC50 reached at 16.72 μM).

330

Regarding signal amplitudes, which are related to the efficacy of receptor activation, they differ

331

across receptors for the same polyphenol compound (Table 3). Both procyanidin dimer B1 and

332

punicalagin activated the TAS2R7 receptor with higher efficacy relative to TAS2R5. Conversely,

333

protocatechuic acid ethyl ester activated TAS2R14 and TAS2R30 with similar efficacy. In general,

334

hydrolyzable tannins were the agonists with the highest efficacy of activation for TAS2R7.

335

Condensed tannins have a higher efficacy of activation for TAS2R39 and TAS2R43 compared to

336

TAS2R5. The high efficacy of EGCG on TAS2R39 activation has been previously observed when in

337

comparison with other polyphenols,44 even though the previously reported EC50 concentration is

338

20- fold higher than the one determined here (181.6 μM).

339

Furthermore, the compounds that possess two or more of these groups have the lowest EC50

340

concentrations, 6.29 μM for procyanidin B2 3-O-gallate and 12.3 μM for EGCG, respectively. This is

341

also similar to our previous study that estimated an EC50 value of 8.50 μM for PGG, one of the

342

compounds with a larger number of galloyl groups (five) among the studied compounds. In fact,

343

the molecular docking studies of the interaction between TAS2R5 and procyanidin B2 3-O-gallate

344

showed a larger number of significant hydrogen bonds by hydroxyl groups of the galloyl moiety.


Page 17 of 31


345

Representativeness of the bitter compounds studied here. The compounds studied here occur in

346

foodstuffs in a wide range of concentrations (Table 4). While several works have focused on the

347

identification and quantification of several condensed tannins in different foods,45 regarding ETs,

348

their analyses and quantitation are particularly difficult because they are only partially solubilized

349

in the extraction solvent or remain covalently bound to cell walls and other macromolecules of the

350

fruit.46 So, there are only a few studies regarding their quantification in foods.

351

Regarding phenolic acid ethyl esters, besides being present in red wine,37 these compounds also

352

occur in vegetables, such as potato (peels). Protocatechuic acid ethyl ester occurs in the peanut

353

seed (skin).47

354

For most of the compounds studied here, the concentrations at which they occur in foodstuffs,

355

they also activate the TAS2Rs. So, most of these compounds contribute actively to bitter taste of

356

foodstuffs.

357

Additionally, most of these compounds have been already identified as bitter by sensory analysis

358

and their determined thresholds are summarized in Table 4. Taste threshold refers to the minimum

359

concentration of compound needed to detect its taste stimulus (sensory analysis) or needed to

360

detect TAS2Rs activation (in vitro assay). Although not directly comparable, threshold correspond

361

to the “beginning” of the dose-response curve while EC50 is in the middle of the curve. So, it is

362

expected EC50 to be higher than threshold. For procyanidin dimer B1, EGCG, vescalagin, castalagin

363

and grandinin, the EC50 determined here are much lower than bitter thresholds determined by

364

sensory analysis (Table 4). This discrepancy between sensory and in vitro assays has been already

365

reported.13 For example, higher threshold concentrations and EC50 values for bitter hop compounds

366

were found in a sensory test compared to the taste receptor assay, whereas the ranking in order of

367

intensity for the compounds was the same.48 For the compounds studied here, these differences

368

could be justified by the absence of saliva (salivary proteins) in the in vitro approach to study the

369

TAS2Rs activation. Most of these polyphenols are also perceived as astringent. Actually, sensory

370

analyses report that some of the compounds can be more astringent than bitter (e.g. for phenolic 17 ACS Paragon Plus Environment


Page 18 of 31

371

acid ethyl esters astringency threshold is significantly lower than bitterness).37 So, in vivo they can

372

firstly interact with salivary proteins and only thereafter (for higher concentrations) interact with

373

TAS2Rs. In fact, some experiments made with other astringent polyphenols (data not shown) have

374

shown that the presence of salivary proteins can reduce TAS2Rs activation. Moreover, for instance,

375

the interaction with food proteins (β-casein and several gelatins) have already been proven to

376

reduce the bitterness of EGCG.49

377

In summary, the results here show once again the combinatorial pattern of TAS2Rs activation.

378

TAS2R5 may be the only receptor that is to some degree “specifically” activated by natural tannins,

379

more precisely by condensed tannins while TAS2R7 seems more tuned for hydrolyzable

380

(ellagi)tannins. Additionally, the docking studies of these specific protein-polyphenol interactions

381

identified the same type of interactions (pi-stacking, H-bonds to side chains and the backbone

382

carbonyl) that were first identified many years ago in protein precipitation studies as the

383

fundamental basis for protein polyphenol interactions. In the end, at the concentrations in which

384

these compounds are present in foodstuffs they contribute actively to bitter taste, especially ETs.


Page 19 of 31


385

Abbreviations Used

386

TAS2Rs: Bitter taste receptors

387

B2g: Procyanidin dimer B2 3’-O-gallate

388

EGCG: epigallocatechin gallate

389

ETs: ellagitannins

390

PGG: pentagalloylglucose

391



Page 20 of 31

392

Acknowledgments

393

This research was supported by a research project grant (PTDC/AGR-TEC/6547/2014) with financial

394

support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership

395

Agreement PT2020 (UID/QUI/50006/2013 - POCI/01/0145/FEDER/007265). Susana Soares and Elsa

396

Brandão gratefully acknowledges the Post-Doctoral grant from FCT (SFRH/BPD/88866/2012) and

397

the phD grant from FCT (SFRH/BD/105295/2014), respectively.

398

The authors also thank to FEDER-Interreg España-Portugal Programme (Project ref.

399

0377_IBERPHENOL_6_E) and to Spanish MINECO (Project ref. AGL2017-84793-C2-1-R co-funded by

400

FEDER) for the financial support. Ignacio Garcia-Estévez also thanks to the University of Salamanca

401

for the post-doctoral contract.

402

403 404


Page 21 of 31


405

Figure captions

406

Figure 1. Structures of the polyphenols studied with the common nomenclature of the different

407

groups and ring moieties.

408

Figure 2. Fluorescence changes of Fluo4-AM-loaded HEK293T-Gα16gust44 cells expressing the

409

TAS2Rs indicated in the graphs following administration of 40.0 μM procyanidin B2 3-O-gallate,

410

50.0 μM punicalagin or 300 μM protocatechuic acid ethyl ester. Responses of mock transfected

411

cells (empty control plasmid) are indicated by the grey solid line.

412

Figure 3. Concentration−response curves for HEK293T-Gα16gust44 cells transfected with cDNA

413

coding for the indicated TAS2R following stimulation with the indicated test compound. Error bars

414

represent the standard deviation. Experimental data are the full points, curve fitting is represented

415

by the solid line and signal in the control condition (mock plasmid) is the dashed line.

416

Figure 4. 3D representation of the first ranked docking pose of polyphenol compounds with human

417

TAS2Rs: TAS2R5:procyanidin B2 3-O-gallate, TAS2R7:malvidin-3-glucoside, TAS2R7:punicalagin,

418

TAS2R7:castalagin, TAS2R14:ferulic acid ethyl ester, TAS2R39:procyanidin B2 3-O-gallate, and

419

TAS2R39:PGG. Polyphenol compounds are represented with sticks and colored by atom type; the

420

receptor is represented in cartoon and colored in gray, while the interacting residues are

421

represented in ball-and-stick and colored by atom type. Aromatic, non-aromatic saccharide, and

422

non-aromatic non-saccharide rings are colored in red, green and purple, respectively.



Page 22 of 31

Tables

ETHYL ESTERS

HYDROLYZABLE TANNINS

CONDENSED TANNINS

Table 1. Summary of the human bitter taste receptors (TAS2Rs) activated by the polyphenol compounds tested. Compounds were divided by families. “+” indicates activation while “-“ indicates a lack-off activation. Only the activated TAS2Rs are presented. EGCG: epigallocatechin gallate TAS2Rs Compound R4 R5 R7 R14 R39 R43 R30 Procyanidin B1

-

+

+

-

-

-

-

Procyanidin B2

-

-

-

-

-

-

-

Procyanidin B3

-

-

-

-

-

-

-

Procyanidin B4

-

+

-

-

-

-

-

Procyanidin B6

-

-

-

-

-

-

-

Procyanidin B7

-

+

-

-

-

-

-

Procyanidin C1

-

-

-

-

-

-

-

Procyanidin B2g

-

+

-

-

+

-

-

EGCG

+

+

-

-

+

+

+

Vescalagin

-

-

+

-

-

-

-

Castalagin

-

-

+

-

-

-

-

Grandinin

-

-

+

-

-

-

-

Punicalagin

-

+

+

-

-

-

-

Ferulic acid

-

-

-

+

-

-

-

Protocatechuic acid

-

-

-

+

-

-

+

Vannillic acid

-

-

-

+

-

-

-


Page 23 of 31


Table 2. EC50 values for the test compounds and respective receptors. Values with the same letter are not significantly different (P < 0.05).

Ethylic Hydrolyzable Condensed Esters Tannins Tannins

Compound Procyanidin B1 Procyanidin B2G EGCG

EC 50 / µM TAS2R5

TAS2R7 a

119.34±10.71 123.95±17.27 6.29±3.22

b

12.30±3.63

b

-

a

TAS2R14

TAS2R39

TAS2R43

TAS2R30

-

-

-

-

-

-

9.11±6.05

b

-

b

-

8.50±2.84 16.72±13.71

b

-

Vescalagin

-

7.26±1.57

b

-

-

-

-

Castalagin

-

4.44±1.43

b

-

-

-

-

3.95±2.49

b

-

-

-

-

b

-

-

-

-

-

-

-

-

-

Punicalagin

40.43±2.77

c

Grandinin

-

2.43±1.29

Ferulic acid Protocatechuic acid Vanillic acid

-

-

66.65±4.36

-

-

155.64±46.36

-

-

c, d

151.17±7.81

a, e

a, e

-

82.39±2.18

a, d

-

Table 3. Signal amplitudes (given as relative fluorescence changes of ΔF/F) for the tested compounds. Values with the same letter are not significantly different (P < 0.001).

Ethylic Esters

Hydrolyza Conde ble nsed Tannins Tannin

Compound

Signal Amplitudes

TAS2R5 0.088±0.004 Procyanidin B1 0.13±0.009 Procyanidin B2G 0.19±0.003 EGCG Vescalagin Castalagin 0.19±0.03 Punicalagin Grandinin Ferulic acid Protocatechuic acid Vanillic acid

TAS2R7

TAS2R14

TAS2R39

TAS2R43

TAS2R30

0.21±0.012 0.49±0.03 0.61±0.006 0.43±0.03 2.43±1.29 -

0.36±0.01 0.32±0.01 0.37±0.02

0.26±0.007 0.41±0.003 0.29±0.02 -

0.32±0.09 -

Table 4. Concentration of some compounds studied here in common food sources, half-maximum concentration (EC50) determined in this study and sensory threshold previously determined as well as the sensory methods used to their determination.



Page 24 of 31

Figures Procyanidin dimers (C4-C8)

B1: R1 = OH; R2 = H; R3 = H; R4 = OH B2: R1 = OH; R2 = H; R3 = OH; R4 = H B3: R1 = H; R2 = OH; R3 = H; R4 = OH B4: R1 = H; R2 = OH; R3 = OH; R4 = H B2g: R1 = O-Galloyl; R2, R3 = H; R4 = OH

Condensed tannins Procyanidin dimers (C4-C6)

Epigallocatechingallate (EGCG)

B5: R1 = OH; R2 = H; R3 = OH; R4 = H B6: R1 = H; R2 = OH; R3 = H; R4 = OH B7: R1 = OH; R2 = H; R3 = H; R4 = OH

Ellagitannins

Punicalagin

Castalagin: R1 = OH; R2 = H Vescalagin: R1 = H; R2 = OH

Grandinin

Phenolic acid ethyl esters

Ferulic acid ethyl ester

Vanillic acid ethyl ester: R1=OCH3 Protocatechuic acid ethyl ester: R1=OH


Page 25 of 31


Figure 1. Procyanidin B2 3-O-gallate TAS2R39

600

400

Fluor RLU

Fluor RLU

600

Punicalagin TAS2R7

200 0

400 200 0

0

100

200 300 Time [s]

0

100

200 300 Time [s]

400

Protocatechuic acid ethyl ester TAS2R30

600

Fluor RLU

400

400 200 0 0

100 MOCK

200 300 Time [s]

400

300 µM

Figure 2.



0.5

R39

0.4

0.3

0.3

∆ F/F

∆ F/F

0.5 0.4

0.2

R7

0.2 0.1

0.1

0.0

0.0 0.01 -0.1

Page 26 of 31

0.1

1

10

0.1

100

-0.1

[B2g] / µ M

0.5

1

10

100

[punicalagin] / µM

R47

0.4

∆ F/F

0.3 0.2 0.1 0.0 1 -0.1

10

100

1000

[Protocatechuic acid] / µ M

Figure 3.


Page 27 of 31


Figure

4.



Page 28 of 31

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Graphic for table of contents Procyanidin B2g TAS2R5

600 400 200 0 0

100

200 300 Time [s]

400

TAS2R7

Punicalagin TAS2R7

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100

200 300 Time [s]

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Human bitter taste receptors are activated by different classes of

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