Contribution of Human Oral Cells to Astringency by Binding Salivary

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Contribution of human oral cells to astringency by binding salivary proteins/tannins complexes Susana Soares, Raul Ferrer-Gallego, Elsa Brandão, Mafalda Silva, Nuno Mateus, and Victor De Freitas J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 18 Sep 2016 Downloaded from http://pubs.acs.org on September 18, 2016

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Contribution of human oral cells to astringency by binding salivary proteins/tannins

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complexes

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Susana Soares⁰*, Raúl Ferrer-Galego#, Elsa Brandão⁰, Mafalda Silva⁰, Nuno Mateus⁰, Victor

4

de Freitas⁰

5



6

Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal

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8

Farmacia, Universidad de Salamanca, Campus Miguel de Unamuno, E 37007 Salamanca,

9

Spain.

REQUIMTE\LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências da

Grupo de Investigación en Polifenoles. Unidad de Nutrición y Bromatología, Facultad de

10

#

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*Corresponding author

Parc Tecnològic del Vi – VITEC, Ctra Porrera, 43730 Falset, Spain

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ABSTRACT

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The most widely accepted mechanism to explain astringency is the interaction and

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precipitation of salivary proteins by food tannins, in particular proline-rich proteins.

16

However, other mechanisms have been arising to explain astringency, such as binding of

17

tannins to oral cells. In this work, an experimental method was adapted to study the

18

possible contribution of both salivary proteins and oral cells to astringency induced by

19

grape seed procyanidins fractions. Overall, in the absence of salivary proteins, the extent of

20

procyanidin complexation with oral cells increased with increasing procyanidin degree of

21

polymerization (mDP). Procyanidins fractions rich in monomers were the ones with the

22

lowest ability to bind to oral cells. In the presence of salivary proteins and for procyanidins

23

with mDP 2 the highest concentrations (1.5 and 2.0 mM) resulted in an increased binding

24

of procyanidins to oral cells. This was even more evident for fractions III and IV at 1.0 mM

25

and upper concentrations. Regarding the salivary proteins affected, it was possible to

26

observe a decrease of P-B peptide and aPRP proteins for fractions II and III. This decrease is

27

greater as the procyanidins mDP increases. In fact, for fraction IV it was observed an almost

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total depletion of all salivary proteins. This decrease is due to the formation of insoluble

29

complexes salivary proteins/procyanidins.

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Altogether, these data suggest that some procyanidins are able to bind to oral cells and

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that the salivary proteins interact with procyanidins forming salivary proteins/procyanidins

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complexes that are also able to link to oral cells. The procyanidins that remain unbound to

33

oral cells, are able to bind to salivary proteins forming a large network of salivary

34

proteins/procyanidin complexes. Overall, the results presented herein provide one more

35

step to understand food oral astringency onset.

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KEYWORDS: proline-rich proteins, procyanidins, red wine, oral cells, astringency 2 ACS Paragon Plus Environment

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INTRODUTION

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Astringency is an important organoleptic sensory attribute of foodstuffs rich in tannins. It is

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a tactile sensation usually described as dryness, puckering and tightening of the oral cavity

41

resulting from the ingestion of food or beverages rich in these compounds1, 2. In some

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foodstuffs, such as red wine, this sensation is desired in balanced levels being even an

43

important quality parameter. On the other hand, in other foodstuffs, astringency is not

44

desirable at all, such as in the case of fruits, juices and tea.

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As astringency influences the overall quality of red wine and of other fruit derived

46

beverages, the knowledge of the compounds structure/activity relationship on this sensory

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property as well as the mechanisms underlying astringency development are important

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aspects of winemaking and beverages industry. This allows winemakers to manage and

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control unbalanced levels of astringency.

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Along the years a significant amount of research has been done towards astringency

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understanding. Presently, three major mechanisms were pointed as possible origins of

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astringency sensation. In 1954, Bate-Smith

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interaction of tannins with salivary proteins (SP) in the mouth. This is the most widely

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accepted mechanism and is vastly supported by the literature and relies on the interaction

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and precipitation of SP by food tannins, in particular proline-rich proteins (PRPs)

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major SP are usually grouped into five structurally related major classes namely histatins,

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PRPs, statherin (stat), cystatins (cyst) and mucins. Regarding astringency, PRPs are one of

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the most important classes of SP and are usually divided in three families according to their

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acidic/basic characteristics: basic PRPs (bPRPs) have mainly basic residues, acidic PRPs

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(aPRPs) are similar to bPRPs but have the first 30 N-terminal residues composed mainly by

2

proposed that astringency results from the

3-7

. The

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aspartic and glutamic acid and glycosylated PRPs (gPRPs) are bPRPs that have

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carbohydrates in their structure 8, 9.

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Several authors have found a significant correlation between the precipitation of tannins

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by proteins, such as SP, and astringency 10-12. Presently, it is known that protein and tannin

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structures’ are both relevant for the interaction, as well as pH, ionic strength and the

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presence of other molecules in solution such as carbohydrates

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that the increasing mean degree of polymerization (mDP) and galloylation degree of

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proanthocyanidins increase astringency perception of those tannins

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influence of SP, it has been reported since many years ago that bPRP are the most reactive

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SP toward tannins

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statherin SP are also highly reactive toward food tannins 17, 18.

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Other authors suggest that astringency could be detected by increased activation of

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mechanoreceptors located within the mucosa, like for other primary tastes such as

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bitterness. Another hypothesis suggests that astringency could be related to interactions

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between tannins and oral epithelial cells19. However, astringency is such a complex

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sensation that is unlikely to arise from only one physical-chemical mechanism.

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So, in this work, procyanidins with different mDP were isolated from a grape seed extract

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and it was adapted an experimental method to study the possible contribution of both SP

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and oral cells to astringency induced by these procyanidins which are normally present in

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red wine and are also widely distributed in vegetable foodstuffs and beverages.

16

13, 14

. It has been reported

7, 15

. Regarding the

. However, recent works concluded that aPRP, P-B peptide and

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EXPERIMENTAL

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Grape seed fractions (GSF) isolation

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Condensed tannins were extracted from Vitis vinifera grape seed extract and fractionated

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according to the method described in the literature20. Briefly, this extract was fractionated

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through a TSK Toyopearl HW-40(s) gel column (100 mmx10 mmi.d., with 0.8 mL.min-1

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methanol as eluent), yielding four fractions of procyanidin with different molecular weight.

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The first 30 min of elution were rejected. The first (GSFI), second (GSFII) and third (GSFIII)

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fractions were obtained after elution with 99.8% (v/v) methanol during 15 min (12 mL),

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other 15 min (12 mL) and other 4 h (192 mL), respectively. The fourth fraction (FIV) was

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eluted with methanol/5% (v/v) acetic acid during the next 14 h (670 mL). All fractions were

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mixed with deionized water, and the organic solvent was eliminated using a rotary

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evaporator under reduced pressure at 30 ºC and then freeze-dried.

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GSF characterization

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The procyanidin composition of fractions was determined by direct analysis by ESI-MS

96

(Finnigan DECA XP PLUS) and subsequent analysis of the average full mass spectra. The

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mean degree of polymerization (mDP) was determined by acid-catalysis reaction in the

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presence of phloroglucinol as described in the literature followed by LC-MS (Finnigan DECA

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XP PLUS) and HPLC analysis20, 21.

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Saliva collection

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Saliva was collected as referred previously in the literature17. Briefly, collection time was

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standardized at 2 p.m. in order to reduce concentration variability connected to circadian

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rhythms of secretion. The saliva pool was mixed with 10% TFA (final concentration 0.1%) to

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precipitate several high molecular weight SP (such as α-amylases, mucins, carbonic 6 ACS Paragon Plus Environment

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anhydrase and lactoferrin) and to preserve sample protein composition, since TFA partially

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inhibits intrinsic protease activity. However, peptides and proteins like histatins, basic,

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acidic and glycosylated PRPs, statherin, cystatins are soluble in this acidic solution and may

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be directly analyzed by RP-HPLC, as described ahead. After the centrifugation (8000 g for 5

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min), the supernatant was dialyzed in a cellulose dialysis membrane (MWCO: 3.5 KDa) for

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24 hours at 4 ºC with stirring against deionized water. Water was changed periodically.

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After dialysis, saliva was centrifuged and the supernatant was freeze-dried. The lyophylized

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saliva was re-solubilized in the same volume of water to maintain total protein

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concentration. The study was conducted according to the Declaration of Helsinki and was

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approved by the Ethics Committee of Medical School of University of Porto (EK84032011).

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HPLC saliva analysis

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90 μL of saliva were injected on a HPLC Lachrom system (L-7100) (Merck Hitachi) equipped

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with a Vydac C8 column (Grace Davison Discovery Sciences), with 5 μm particle diameter

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(column dimensions 150 x 2.1 mm); detection was carried out at 214 nm, using a UV-Vis

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detector (L-7420). The HPLC solvents were 0.2% aqueous TFA (eluent A) and 0.2% TFA in

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ACN/water 80/20 (v/v) (eluent B). The gradient applied was linear from 10 to 40% (eluent

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B) in 45 min, at a flow rate of 0.60 mL.min-1. After this program the column was washed

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with 100% eluent B for 20 min in order to other late-eluting proteins. After washing, the

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column was stabilized with the initial conditions.

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Cell culture

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One epithelial-like cell line (HSC-3) derived from human oral squamous cell carcinoma was

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used in this study. The cells were grown under standard culture conditions of 5% CO2 at 37

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°C in a humidified incubator in DMEM, supplemented with 10% fetal bovine serum, 100 7 ACS Paragon Plus Environment

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U/ml of penicillin G, 100 μg/ml of streptomycin sulphate, and 0.25 μg/ml of amphotericin

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B. Cells were dissociated with trypsin enzyme.

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Interaction of grape seed fractions with oral cells

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HSC-3 cells were seeded into 96 well flat bottomed tissue culture plates at a density of 1 ×

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105 cells/well, and grown to confluence before use in an assay. The cell monolayers were

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washed twice with PBS, pH 7.6, to remove residual growth medium, and water (control

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condition; Figure 1, A to D lines) or saliva (lines E to H) were added, in triplicate, at 30

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μl/well. Stock of test solutions of each fractions (6, 4.5, 3.0, 1.5 and 0.3 mM) were

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prepared in water or ethanol concentration of 12%, and 15 μL of each stock were added (to

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attain the final concentrations between 0.1 and 2 mM) and left in contact with the

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monolayers for 15 min. Several control conditions were also tested, such as GSF and saliva

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without oral cells (Figure 1; A line) and GSF and water without oral cells (Figure 1; H line),

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to know if there was any unspecific binding to the plate.

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After the incubation period, the solutions were removed from the wells and the oral cells

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were washed twice with PBS in order to remove the eventual formed aggregates that were

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not bounded. Then DMACA assay was done to measure the procyanidin content of each

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well.

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The incubation solutions from wells assays involving the interaction of SP with oral cells

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and GSF (Figure 1, B, C and D lines) were recovered to be analyzed by RP-HPLC.

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DMACA assay to measure the procyanidin content

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The method used was similar to the one described previously19. Briefly, a 0.1% solution of

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DMACA was prepared in acidified methanol (0.75 M H2SO4). After the interaction of HCS-3

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cells with GSF in absence and presence of saliva, the 96 wells depleted from the incubation

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solutions (as referred before) were incubated with 50 μl of DMACA solution for 20 min at

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room temperature, and the absorbance of each well was determined at 640 nm in a

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μQuant microtitre plate reader.

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RESULTS AND DISCUSSION

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Astringency has been highly associated to the interaction between salivary proteins (SP)

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and food tannins forming (in)soluble complexes that could precipitate in the oral cavity.

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However, some points of view are appearing suggesting that astringency could result from

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the contribution of several mechanisms. In fact, one of these mechanisms alone is not able

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to explain all the sensations associated to astringency. However, it is difficult to study

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simultaneously the several mechanisms and there is not much information about the

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contribution of oral cells to astringency. A first study done by Payne and co-workers19

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showed that oral cells have the ability to bind tannins. Though, the authors do not consider

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the effect of SP in this interaction. In this work, it was intended to study the effect of SP in

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the ability of tannins to bind to oral cells and the influence of ethanol on this interaction.

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In order to simulate in vitro what happens in the oral cavity, saliva volume used taken into

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account the volume of saliva normally present in the mouth after ingestion of a sip of wine

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(around 20 mL of saliva)11. It was also considered the average oral surface (214 cm2) 22 and

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the surface of each well (0.31 cm2) to determine the saliva volume to put in each well (30

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μL). Besides, it seems that wine ingestion usually results in a ratio of 2:1 saliva:wine11. So, it

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was added a volume of 15 μL of GSF stock solutions to each well.

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The final range of GSF concentrations was chose in order to cover the reported red wine

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concentrations at Phenol Explorer Database23 according to procyanidins mDP. Fraction I

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(GSF I) was found to contain mainly catechins and gallic acid, but also a small quantity of

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procyanidin dimers (mDP 1.1). GSF II contains essentially catechins and procyanidin dimers

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and galloyl derivatives (mDP 1.4). GSF III contains mainly procyanidin dimers and trimers

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and their galloyl derivatives but also a small quantity of procyanidin tetramers (mDP 2).

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GSF IV contains mainly procyanidin trimers and tetramers, their galloyl derivatives and also

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procyanidin pentamers (mean DP 4).

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Interaction of GSF with oral cells

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Procyanidins isolated from a grape seed extract were incubated with oral cells in different

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concentrations and after incubation the solutions were removed. Then oral cells were

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washed with buffer to remove procyanidins that were not effectively bound to oral cells.

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The wash process is sufficiently gentle to ensure that there is no damage of cell monolayer

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but could remove any weakly, hydrophobically-bound procyanidins. DMACA was used to

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detect GSF bounded to HSC-3 oral cells in the absence and presence of ethanol (12%). As

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shown by the results of this first assay in Figure 2A, it was observed that the GSF

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procyanidins bind to cell monolayers in a dose-dependent manner at concentrations

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between 0.5 and 2.0 mM. Beside GSF concentration, it was also observed a different

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binding related to the mDP of GSF. It was observed an increase in binding with mDP

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increase. So, the highest binding to HSC-3 oral cells was observed for the most polymerized

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fractions (GSF III and IV).

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GSF I and II are the ones with lowest binding ability to oral cells. This is probably related to

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the high quantity of monomers in these fractions while the other ones have dimers and

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highly polymerized structures.

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The same trend was observed in the presence of 12% ethanol (Figure 2B) but in this case

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the interaction of GSF with HSC-3 was slightly higher for all GSF, especially for GSF III and

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IV. This small effect of ethanol has been previously observed for the interaction of an

199

extract of procyanidins to oral cells 19.

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Figure 3 presents the interaction of GSF with HCS-3 oral cells in the presence of SP in water

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or in 12% EtOH. From these results it is possible to observe that the influence of SP is not

202

the same for all fractions. In fact, SP do not affect the interaction of GSF I with oral cells.

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The interaction with oral cells is very small for this fraction both in absence or presence of

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SP. For GSF II it is possible to observe that for the highest concentrations (1.5 and 2.0 mM)

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in water (Figure 3A), the presence of SP resulted in an increased binding of GSF

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procyanidins to oral cells. This is even more evident for GSF III and IV. In these cases, for 1.0

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mM and upper concentrations it is clearer the increase of procyanidins binding to oral cells

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induced by SP.

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For the experiments in 12% EtOH the results for GSF II, III and IV are different from the

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ones in water. It was not observed a difference between procyanidins binding to oral cells

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in presence of SP. From the literature it is already known that ethanol influences the

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interaction of procyanidins with proteins, usually inhibiting the formation of hydrophobic

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bonds and therefore its interaction and complexation.

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Salivary proteins interaction with GSF in absence and presence of oral cells

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In order to understand the influence of oral cells on the astringency and what happens to

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the SP during the interaction with oral cells, saliva was analyzed by HPLC before the

217

interaction with both GSF and with oral cells. Figure 4A shows the saliva (control) HPLC

218

chromatogram. The HPLC chromatogram of saliva is roughly divided into six SP family

219

regions: the first region comprises mainly proteins that belong to the classes of bPRPs. The

220

bPRPs identified in this region include IB-8b, IB-8c, IB-9, IB-4 and P-J. The second region

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comprises mainly a gPRPs, the bPRP3. The next region corresponds entirely to aPRPs,

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namely PRP1 and PRP3, and the next two peaks have phosphorylated forms of statherin 12 ACS Paragon Plus Environment

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and peptide P-B, respectively. The last region comprises cystatin proteins. The HPLC profile

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of saliva with 12% of ethanol or after saliva interaction with oral cells (without GSF) does

225

not change (data not shown).

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Initially, this interaction was made in the absence of oral cells to know the amount of

227

interaction between SP and procyanidins and which proteins are more affected. The

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interaction with saliva was only studied for the GSF that presented differences in the

229

interaction with oral cells in presence of SP (GSF II, III and IV) (Figure 3A). Besides, it was

230

only studied for one GSF concentration (1.0 mM) because it was the first concentration for

231

which it was observed differences by the presence of SP in DMACA assay (Figure 3A). As an

232

example, the saliva chromatogram before and after interaction with GSF IV 1.0 mM is

233

displayed in Figure 4B.

234

The observed changes in the chromatographic peaks were then calculated as the

235

percentage decrease of SP (Table 1). From the saliva analysis it is possible to observe a

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decrease of certain SP peaks, in special P-B and aPRP proteins for GSF II and III. This

237

decrease is greater as the mDP increases. In fact, for GSF IV, it was observed an almost

238

total depletion of SP. These decreases are due to the formation of insoluble complexes

239

SP/procyanidins that are removed by centrifugation prior to analysis by HPLC. Procyanidins

240

from GSF II and III that did not reacted with SP are overlaid with bPRP region. This was

241

verified by injection of GSF in the same chromatographic conditions (data not shown).

242

Unfortunately, this overlapping does not allow to get any information about the effect of

243

GSF onto bPRPs family.

244

After this experiment, the saliva recovered from the interaction with GSF (II, III and IV 1.0

245

mM) (Figure 1, Lines E to G, Columns 3 and 10) in the presence of oral cells was also

246

analyzed by HPLC. Figure 5 presents, as an example, the saliva analysis after interaction 13 ACS Paragon Plus Environment

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with GSF III in absence and presence of oral cells. In this example, the SP and polyphenols

248

were completely depleted. The variations observed in the chromatographic peaks area for

249

each protein and GSF are summarized in the table presented in Figure 5. For GSF II the area

250

of SP peaks is practically the same both in the absence and presence of oral cells. This is in

251

agreement with the previous results (Figure 3A) because there was also no significant

252

difference in the amount of GSF detected in oral cells at this concentration. For the other

253

fractions (GSF III and IV) it was observed a completely depletion of SP when the interaction

254

occurred in presence of oral cells (table presented in Figure 5). These results could explain

255

the higher detection of procyanidin observed in DMACA assay for GSF 1.0 mM of these

256

fractions.

257

For the experiments done in presence of 12% ethanol the HPLC profile of saliva are similar

258

with or without oral cells (data not shown) which is in agreement with the results from

259

DMACA assay .

260

Overall, the results obtained from the DMACA assay and from the HPLC analysis of SP are in

261

agreement. It was observed that for 1.0 mM and upper concentrations GSF II, III and IV

262

bind to oral cells and this binding is increased by SP. Simultaneously, SP disappear from the

263

solution. Altogether, these data suggests that some GSF procyanidins are able to bind to

264

oral cells (OC-GSF) and that the SP interact with GSF procyanidins forming SP/procyanidins

265

complexes that are also able to bind to oral cells (GSF-SP-OC) (Figure 6). The procyanidins

266

that remained unbound to oral cells, are able to bind to SP forming a large network of

267

complexes SP/procyanidins [OC-(GSF-SP)n]. However, this mechanism of interaction only

268

seems important in the absence or low concentrations of ethanol. In the presence of

269

ethanol the interaction of procyanidins with oral cells seems to be independent of SP.

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Ultimately, it seems that oral astringency is a very complex sensation and that depending

271

on the food matrix it could in some cases result from the combination of these two

272

mechanisms (oral cells binding and SP precipitation) while in other cases it could arise

273

mainly from one mechanism. In this way, the results presented herein provide one more

274

step to understand food oral astringency onset.

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ABBREVIATIONS USED

276

ACN, acetonitrile

277

aPRPs, acidic proline-rich proteins

278

bPRPs, basic proline-rich proteins

279

cyst, cystatins

280

DMACA, , 4-(dimethylamino)cinnamaldehyde

281

DMEM, Dulbecco’s

282

gPRPs, glycosylated proline-rich proteins

283

GSF, grape seed fraction

284

HPLC, high pressure liquid chromatography

285

LC-MS, Liquid chromatrography- mass spectrum

286

mDP, mean degree of polymerization

287

OC, oral cells

288

PRPs, proline-rich proteins

289

SP, salivary proteins

290

stat, statherin

291

TFA, trifluoroacetic acid

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American Society of Testing and Materials: Philadelphia, PA, 1989; Vol. 15.07.

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Saliva and to Proline-Rich Proteins Derived from It. J. Agric. Food Chem. 2000, 48, 838-843.

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FIGURES CAPTIONS Figure 1. Scheme of a 96 well plate assay, indicating all the control conditions made. In each column is presented the final grape seed fraction (GSF) concentrations. For GSF III and IV the same plate scheme was used. Figure 2. Relative intensity of GSF (different mDP fractions) bounded to HSC-3 oral cells in absence (A) and presence of 12% ethanol (B). Figure 3. Relative intensity of GSF (different mDP fractions) bounded to HSC-3 oral cells in absence (solid line) and presence (dashed line) of SP in water (A) and in 12% ethanol (B). Figure 4. A. RP-HPLC profile detected at 214 nm of control saliva (30 μL of saliva + 15 μL water) before the interaction with GSF and with oral cell. Each region/peak is assigned to the major family of SP identified. B. RP-HPLC profile detected at 214 nm of control saliva (solid line) before the interaction and after 15 min of interaction with GSF IV (final concentration 1.0 mM) in water (dashed line). Figure 5. RP-HPLC profile detected at 214 nm of saliva after the interaction with GSF III (1.0 mM) in absence of oral cells (solid line), and in presence of oral cell (dashed line) in water. After the interaction, the solutions were centrifuged and the supernatant was analyzed by HPLC. Table presents HPLC peaks intensity (in % comparing to the control saliva) after interaction with different GSF fractions (FII, FIII and FIV) in absence and presence of HSC-3 oral cells. T.A., trace amounts. Figure 6. Schematic representation of the mechanism of astringency sensation involving oral cells, salivary proteins and tannins.

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TABLES Table 1. HPLC peaks intensity (in % comparing to the control saliva) after interaction with different GSF fractions. FII

FIII

FIV

gPRPs

100,00

47,12

6,68

aPRPs

75,61

35,38

8,69

statherin

64,39

43,04

7,84

P-B

56,58

33,07

11,67

100,00

82,65

7,49

cystatins

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FIGURES Figure 1. 1

2

3

4

5

6

7

2 mM 1.5 mM 1 mM 0.5 mM 0.1 mM

10

11

12

Water

Water + GSF II

Oral cells + Water + GSF

Oral cells + water

Oral cells + Water + GSF

B

D E F

9

Water + GSF I

A

C

8

0.1 mM 0.5 mM 1 mM 1.5 mM 2 mM

Oral cells + Saliva + GSF

G

Saliva + GSF I

H

Oral cells Oral cells + Saliva + GSF + water + saliva Saliva + water Saliva + GSF II

Figure 2. A: Water

B: 12% EtOH GSF:

0.4

Abs (λ λ 640 nm)

I 0.3

(m DP 1.1)

II

(m DP 1.4)

III

(m DP 2)

IV (m DP 4) 0.2

0.1

0.0 0.0

0.5

1.0

1.5

2.0

2.5

Abs (λ λ 640 nm)

0.4

0.3

0.2

0.1

0.0 0.0

0.5

1.0

1.5

2.0

2.5

GSF / mM

GSF / mM

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Journal of Agricultural and Food Chemistry

Figure 3. A: Water 0.4

0.12

Abs (λ λ 640 nm)

Abs (λ λ 640 nm)

0.10 0.08 0.06 0.04

0.3

0.2

0.1

0.02 0.00 0.0

0.5

1.0

1.5

2.0

0.0 0.0

2.5

0.5

1.0

1.5

2.0

2.5

|GSF|/mM

|GSF|/mM FI (control)

FI (+ saliva)

FIII (control)

FIII (+ saliva)

FII (control)

FII (+ saliva)

FIV (control)

FIV (+ saliva)

B: 12% EtOH 0.4

0.12 0.10

0.3 Abs

Abs

0.08 0.06

0.2

0.04

0.1 0.02 0.00 0.0

0.5

1.0

1.5

2.0

0.0 0.0

2.5

0.5

1.0

1.5

2.0

2.5

|GSF|/mM

|GSF|/mM FI (control)

FI (+ saliva)

FIII (control)

FIII (+ saliva)

FII (control)

FII (+ saliva)

FIV (control)

FIV (+ saliva)

Figure 4. A.

B. bPRP gPRP

aPRP

stat P-B

cyst

6

4

4

Abs/Au

Abs/Au

6

2

2

0

0 5

10

15

20

25 t (min)

30

35

40

45

10

15

20

25

30

35

40

45

t (min)

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Figure 5 6

FII Abs/Au

4

2

0 15 10

20

25

30

35

40

FIII

FIV

- cells

+ cells

- cells

+ cells

- cells

gPRPs

100,00

83,65

47,12

T.A.

6,68

+ cells T.A.

aPRPs

75,61

83,42

35,38

T.A.

8,69

T.A.

statherin

64,39

72,19

43,04

T.A.

7,84

T.A.

statherin

56,58

78,34

33,07

T.A.

11,67

T.A.

cystatins

102,69

93,80

82,65

T.A.

7,49

T.A.

45

t (min)

Figure 6

Saliva proteins (SP) forming saliva film

Oral cells (OC)

Cells of oral cavity and saliva film

Food tannins bind to oral cells

(OC-SP)

Salivary proteins bind to tannins onto oral cells (OC-GSF, GSF-SP and OC-GSF-SP)

Formation of an extensive network of salivary proteins/tannins complexes [OC-(GSF-SP)n]

Food polyphenols (grape seed fractions) Salivary proteins

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TOC Graphic

Saliva proteins (SP) forming saliva film

Oral cells (OC)

Cells of oral cavity and saliva film

Food tannins bind to oral cells

(OC-SP)

Salivary proteins bind to tannins onto oral cells (OC-GSF, GSF-SP and OC-GSF-SP)

Formation of an extensive network of salivary proteins/tannins complexes [OC-(GSF-SP)n]

Food polyphenols (grape seed fractions) Salivary proteins

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FUNDING SOURCES The authors thank the financial support by one postdoctoral fellowship (SFRH/BPD/88866/2012) and one phD fellowship (SFRH/BD/105295/2014) from FCT (Fundação para a Ciência e Tecnologia). The work has also financial support by FCT/MEC through national funds and co-financed by FEDER (UID/ QUI/50006/2013 - POCI/01/0145/FERDER/007265), under the Partnership Agreement PT2020 and also by project AGL2014-58486-C2-1-R.

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TOC graphic

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