interaction between ellagitannins and salivary proline-rich proteins

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Chemistry and Biology of Aroma and Taste

INTERACTION BETWEEN ELLAGITANNINS AND SALIVARY PROLINE-RICH PROTEINS Susana Soares, Elsa Brandão, Ignacio García-Estévez, Fátima Fonseca, Carlos Guerreiro, Frederico Ferreira da Silva, Nuno Mateus, Denis Deffieux, Stephane Quideau, and Victor De Freitas J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02574 • Publication Date (Web): 05 Aug 2019 Downloaded from pubs.acs.org on August 7, 2019

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Journal of Agricultural and Food Chemistry

INTERACTION BETWEEN ELLAGITANNINS AND SALIVARY PROLINE-RICH PROTEINS Susana Soares1,*, Elsa Brandão1, Ignacio García-Estevez1,2, Fátima Fonseca3,4, Carlos Guerreiro1, Frederico Ferreira-da-Silva3,4, Nuno Mateus1, Denis Deffieux5, Stéphane Quideau5,*, Victor de Freitas1,* 1REQUIMTE,

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

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

de Investigación en Polifenoles (GIP). Facultad de Farmacia, University of Salamanca, E37007,

Salamanca, Spain 3i3S

- Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal

4IBMC 5Univ.

– Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal

Bordeaux, ISM (CNRS-UMR 5255), 351 cours de la Libération, 33405 Talence Cedex, France

*[email protected], [email protected], [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT

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The first contact of tannins with the human body occurs in the mouth, where some of these tannins are

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known to interact with salivary proteins, in particular with proline-rich proteins (PRPs). These interactions

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are important at a sensory level, especially for astringency development, but could also affect the biological

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activities of the tannins. This study gathers information on the relative affinity of the interaction, complexes

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stoichiometry and tannin molecular epitopes of binding for the interactions between the families of PRPs

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(bPRPs, gPRPs and aPRPs) with three representative ellagitannins (castalagin, vescalagin and punicalagin).

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These interactions were studied by saturation-tranfer difference-NMR and microcalorimetry. The effect of

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the PRPs-ellagitannins interaction on their antioxidant ability was also assessed by ferric reduction

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antioxidant power (FRAP) assays. The results support a significant interaction between the studied tannins

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and PRPs with binding affinities in the micromolar range. Punicalagin was always the ellagitannin with

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higher affinity. aPRPs were the salivary PRPs with higher affinity. Moreover, it was observed that when

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ellagitannins are present in low concentrations (5 G% to 50 G%< as it occurs in food, the antioxidant ability

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of these tannins when complexed with salivary PRPs could be significantly impaired.

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Keywords: STD-NMR, ITC, astringency, antioxidant ability

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INTRODUCTION

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Polyphenolic compounds are plant metabolites that are essentially present in plant-based and derived

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foodstuffs’ 1. They are responsible for some organoleptic properties2, 3, such as color and taste, as well as for

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some health benefits of plant-based food products 4.

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Tannins constitute a wide group of polyphenols typically separated in two main families, the condensed and

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hydrolyzable tannins 1. The condensed tannins are oligomers or polymers of flavan-3-ol monomers. The

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hydrolyzable tannins are galloylated monosaccharide (mainly glucose) derivatives. The hydrolyzable tannins

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include a subclass of C-glucosidic ellagitannins, that have a central core of an open-chain glucose and a

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characteristic carbon-carbon linkage between the glucose carbon-1 and the carbon-2 of the O-2 galloyl unit of

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the 2,3,5-nonahydroxyterphenoyl unit (NHTP group) 5. Two representative C-glucosidic ellagitanins are

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castalagin and vescalagin, the first ones to be isolated from Castanea (chestnut) and Quercus (oak) species.

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The glucopyranosic ellagitannins are another subclass of hydrolysable tannins, such as punicalagin that occurs

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in pomegranates. All these ellagitannins may be present in (alcoholic) beverages, such as wine and fruit juices

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produced from red grapes or from pomegranate6, 7. Punicalagin can occur in beverages due to its presence in

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pomegranates while castalagin and vescalagin can occur only in wines that are aged in oak barrels, or that are

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macerated with oak slaves or chips due to their extraction from wood by the alcoholic solution

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when some commercial tannins from oak or chestnut are added to these beverages. The presence of these

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ellagitannins in beverages is important because they can affect their chemical profile, their evolution and at

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the end their organoleptic properties. This has been already observed for red wine. In fact, red wine aged in

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barrels from two different European oak species, i.e., Q. robur and Q. petraea, exhibits a greater ellagitannin

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content than others 13, 14, and presented different sensory properties 15. This association has been attributed to

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the different content in ellagitannins.

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Among the organoleptic properties of plant-based beverages, astringency is one of the most important.

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Astringency is defined as roughening, dryness and puckering sensations that occur in the oral cavity due to the

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intake of alumns, acids, metals or tannins. High astringency is frequently described as an undesirable attribute

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of products rich in tannins. However, in some largely consumed food products like tea and wines, an

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equilibrated level of astringency is required for their in-mouth quality. The importance of tannins in these

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beverages is related to a strong interaction with salivary proteins, hence leading to astringency

8-12,

or even

16, 17.

Some 3

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mechanisms have been suggested to explain the development of astringency. The most accepted one relies on

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the interaction/precipitation of proteins from saliva by tannins, such as proline-rich proteins (PRPs), mucins,

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histatins, among others. PRPs are described by a high number of proline residues (25-42%) and they are further

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divided into three classes: basic (bPRPs), glycosylated (gPRPs) and acidic (aPRPs)18. aPRPs have a strong

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acidic N-terminal region, rich in aspartic and glutamic acid residues and bearing also some phosphate groups

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throughout their structure, whereas its C-terminal region is similar to that of bPRPs19. Regarding gPRPs,

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reports on the characterization of the carbohydrates linked to these proteins are recent. These post-translational

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modifications have been identified to be N-acetyl hexosamine, galactosyl and glucosylgalactosyl

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carbohydrates

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homeostasis of calcium concentration (aPRPs and statherin), and also a role in lubricating the oral surfaces

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(gPRPs and statherin).

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Several works in the literature point that some hydrolyzable tannins can be perceived as more astringent than

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condensed tannins, in particular castalagin and vescalagin21-23. In fact, for hydrolyzable tannins, the determined

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astringent taste thresholds (1.1 G% for castalagin and vescalagin)

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condensed tannins, namely the abundant procyanidin dimers (240 G% for procyanidin B1; 190 G% for

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procyanidin B2 and 300 G% for procyanidin B3)

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astringent, few works focused on their interaction with salivary proteins, except for pentagalloylglucose

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(PGG), which is a gallotannin. To our knowledge, the interaction of salivary PRPs with ellagitannins has not

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been yet investigated. Besides these interactions could be related to sensory properties, they could also have a

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significant impact on some biological activities of tannins, such as i) impair the bioavailability of these tannins

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through the development of insoluble or soluble complexes with salivary proteins 25, ii) reduce the digestion

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of other nutrients by inhibiting the function of digestive enzymes

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capacity of tannins.

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The present work focuses on studying the molecular interaction of the different PRPs (bPRPs, aPRPs and

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gPRPs) with model ellagitannins that have been already reported as astringent, the oak-derived C-glucosidic

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castalagin and vescalagin, and one related to the astringency sensation of the whole-fruit but that lacks

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individual sensory data, the pomegranate-derived glucopyranosic, punicalagin. These interactions were

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characterized regarding their relative affinity, stoichiometry of the complexes and tannin molecular epitopes

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of binding (ligand hydrogens’ that are near the protein) to the different PRPs.

20.

These proteins have main functions in oral cavity related to calcium binding to enamel,

24.

21, 23

are very low in comparison to some

Despite hydrolyzable tannins are perceived as more

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and/or iii) compromise the antioxidant


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This characterization will allow to understand which family of PRPs could be more involved on the astringency

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perception and to discriminate which ellagitannin could contribute more to astringency. These interactions

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were characterized by saturation transfer difference-nuclear magnetic resonance (STD-NMR) and

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microcalorimetry (ITC). STD-NMR technique comprises to obtain a difference spectrum by subtracting a

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spectrum acquired by irradiation at a region that has only resonances of the protein (on-resonance spectrum)

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to a spectrum acquired without protein saturation (off-resonance spectrum)27. Through the nuclear Overhauser

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effect, in the subtracted spectrum it will only appear the signals of the ligand protons that received saturation

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transfer from the protein. In this way, STD-NMR technique is a valuable tool for recognition of the ligand

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moieties involved on the binding process as well as to determine the dissociation constant (KD). ITC technique

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measures directly the heat transfers (release or absorption) in the course of a biomolecular interaction process.

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Measuring these heat transfers during the interaction allows to determine the KD, thr stoichiometry (n), the

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enthalpy ;PL< and the entropy ;Q < providing a thermodynamic characterization of the molecular interaction.

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Finally, to understand if these interactions could impair one of the most significant biological properties related

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to tannins (the antioxidant properties), it was also estimated ellagitannins antioxidant ability by the ferric

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reduction antioxidant power (FRAP) assay when tannins interact with these PRPs.



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MATERIAL AND METHODS

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Reagents. Toyopearl HW-40(s) gel was acquired from Tosoh (Tokyo, Japan). Deuterium oxide, 2,4,6-

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tripyridyls-triazine (TPTZ), sodium acetate, Trolox and iron(III) chloride (FeCl3) were purchased from Sigma-

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Aldrich. All the other reagents were of analytical grade.

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Isolation/identification of proteins from human saliva. Saliva was isolated and processed as described

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elsewhere28. This saliva was dialyzed for 24 h (cellulose membrane, MWCT 3.5 kDa, SpectrumLabs, Rancho

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Dominguez, CA, USA) against water (100x higher volume than saliva sample) at 4 ºC with stirring. Saliva

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was centrifuged and the supernatant was freeze-dried. The lyophilized saliva was re-solubilized in a minimal

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volume of water, filtered (PET, 0.44 G < and injected into a semi-preparative HPLC to isolate the different

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families of PRPs and P-B peptide with the conditions reported previously.

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The several fractions of PRPs were freeze-dried and the proteins present were identified by ESI-MS by flow

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injection analysis into an LTQ Orbitrap XL mass spectrometer (Thermo Fischer Scientific, Bremen, Germany)

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controlled by LTQ Tune Plus 2.5.5 and Xcalibur 2.1.0 with the conditions reported previously. Spectra were

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processed by a deconvolution process using the charge ratio analysis method by MagTran 1.03 software.

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The study was approved by the Ethics Committee and was conducted according to the Declaration of Helsinki.

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Isolation of castalagin, vescalagin and punicalagin. Castalagin and vescalagin were obtained from oak chips

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(Quercus petraea (Matt.) Liebl wood) as described in the literature29, 30. An extract obtained from these sources

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was applied to a Sephadex LH-20 column, and several fractions were eluted with acidified water/methanol

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mixture. The composition of these fractions was determined by L42#W& & %

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major ellagitannins were used to purify them by semipreparative HPLC. Punicalagin was isolated from

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pomegranate, as previously reported 31. Briefly, dried husk powder (1 g) was extracted ultrasonically with 30

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mL of 40% ethanol for 30 min twice. After ethanol evaporation, the extract was lyophilized and analyzed by

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2#W% = Punicalagin purification was performed by semipreparative HPLC. The purity of all ellagitanins was

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determined by 2#W% =

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Saturation Transference Difference (STD)-NMR. Each protein sample was prepared at a final concentration

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of 3 G% in D2O and the ellagitannins (0.1 to 3.5 mM range) were gradually added to the protein samples. The

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ellagitannins were added as a lyophilized powder to the protein sample allowing for the same protein sample

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to be used for all the titrations for each ellagitannin experiences.

and the ones containing the


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STD-NMR experiences were acquired on a Bruker Avance III 600 HD spectrometer, operating at 600.13 MHz,

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using a 5 mm PATXI 1H/D-13C/15N. The STD-NMR analysis conditions were the same as reported

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previously.

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The average binding constants (KA) were calculated by the presented model and equations (figure 1) 27:

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Isothermal Titration Microcalorimetry. A V-P MicroCalorimeter at 298 K controlled by Origin VPViewer

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software was used to make the ITC experiments. Solutions of each PRP were prepared in water ranging from

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20 to 30 G%= The ellagitannins solutions (titrant) were prepared in the range from 1 to 10 mM, and both

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solutions were degassed before titration. 1.4 mL of PRPs solution was loaded on the sample cell and the

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injection syringe was loaded with ellagitannins solution. After reaching baseline stability, ellagitannin solution

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was injected (4 to 12 G25 M

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to ensure full mixing.

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Raw data acquired from a plot of heat flow vs injection number were fitted by the AFFINIMETER software

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(S4SD – AFFINImeter, Santiago de Compostela, Spain) to find the binding constants, stoichiometry, and the

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thermodynamic parameters for the salivary proteins-ellagitannins interactions studied, using the following

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model and equations (Figure 2):

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Ferric Reducing Antioxidant Power (FRAP). The FRAP method was settled to quantify the ferric reducing

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capacity of plasma at low pH 32. A blue color develops when the ferric (Fe3+)-tripyridyltriazine complex is

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reduced to the ferrous (Fe2+) complex. The following solutions were prepared: i) 1, 6 or 30 G2 of ellagitannin

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(200 G% dissolved in water) were added to 39, 34 and 10 G2 of water (final volume 40 G2

interaction between ellagitannins and salivary proline-rich proteins

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