Article pubs.acs.org/JAFC
In Vivo Interactions between Procyanidins and Human Saliva Proteins: Effect of Repeated Exposures to Procyanidins Solution Elsa Brandaõ , Susana Soares, Nuno Mateus, and Victor de Freitas* Centro de Investigaçaõ em Química (CIQ), Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal ABSTRACT: The general accepted mechanism for astringency arises from the interaction between tannins and salivary proteins (SP) resulting in (in)soluble aggregates. By HPLC analysis, it was observed that repeated sips of procyanidins (PC) solution practically depleted aPRPs (∼14%) and statherin (∼2%), and significantly reduced the amount of gPRPs. On the other hand, bPRPs were not significantly affected. In the analysis performed after the last exposure to PC solution, it was seen a significant recovering of the chromatographic peaks corresponding especially to aPRPs (∼74%) and statherin (∼80%). In vitro interaction between SP and PC results in the decrease of the chromatographic peaks of aPRPs and statherin, suggesting that these proteins were involved in the formation of a significant quantity of insoluble complexes. In general, the results suggest that the different families of SP can be involved in different stages of the development of astringency sensation. KEYWORDS: astringency, complexes, procyanidins, proline-rich proteins, protein−procyanidins interaction, statherin
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INTRODUCTION Polyphenols are a well-known class of compounds extremely important because of their organoleptic properties in vegetal foodstuffs, color and flavor, and because of numerous and potent health-promoting benefits associated with them.1 The action of polyphenols as antioxidants may prevent several diseases associated with oxidative stress such as cardiovascular diseases, cancers, inflammation, and others.2−5 Tannins, a complex group of polyphenols, are the best known class of astringent compounds. Astringency is a sensory experience elicited by a diverse range of plant-derived foods and beverages, including red wine, tea, soymilk, coffee, fruits, and vegetables. This complex sensory attribute is described as a dryness, roughening, and puckering sensation felt in the mouth.6,7 The mechanism for astringency was first proposed by Bate-Smith8 and is believed to be due to the ability of tannins to bind and precipitate salivary proteins (SP). Although the formation of protein−tannin complexes is actually assumed to play an important role in the sensation of astringency,9−11 the exact mechanism of this process is not well understood. Other factors are known to contribute to astringency sensation including a change in saliva viscosity,12 interactions between tannins and oral epithelial cells or with taste receptors,13 and an increase in friction.14−16 The in vitro reactivity of polyphenols with proteins (as well as gelatin) has already been described. Several studies have been performed to mimic protein−polyphenol interactions that take place in the oral cavity to produce physical-chemical responses that can be correlated to the astringency inducing capacity of these compounds.17−20 The interaction between proteins and tannins can also be affected by several factors, in particular pH of the medium, percentage of ethanol, ionic strength, temperature, and presence of carbohydrates. The general accepted mechanism for protein−tannin interaction was proposed by Siebert et al.18 Regarding this mechanism, a protein is considered as having a fixed number of © XXXX American Chemical Society
sites to which a tannin can bind, and according to the ratio of protein or tannin used, different protein−tannin complexes are formed. Indeed, proteins and polyphenols combine to form soluble complexes, and these can grow to colloidal size at which point they scatter light and become larger, leading to sediment formation.21 As referred to previously, astringency is thought to result from the interaction between SP and tannins. SP are structurally very diverse, but the most relevant ones for sensorial analysis have been grouped into six structurally related major classes, histatins, basic PRPs (bPRPs), acidic PRPs (aPRPs), glycosylated PRPs (gPRPs), statherin, and cystatins.22 Several biological properties have been associated with these proteins ranging from maintenance of ionic calcium concentration (aPRPs and statherin)23,24 to protection of oral tissues against degradation by proteolytic activity (cystatins).25 The intensity of perceived astringency plays a key role in determining the acceptability of various food products.26 Astringency can be perceived as a negative attribute and is referred to as a reason for consumers rejecting some polyphenol-derived foods.27,28 However, individual differences on saliva protein profile should be taken into account, because they may influence the perception of astringency. For instance, Horne et al.29 pointed to the importance of individual saliva flow and composition variations influencing this oral sensation. It is known that astringency increases upon successive exposures to tannins. The buildup of astringency has been illustrated in studies in which trained volunteers repeatedly sip astringent solutions, at defined intervals, while continuously rating astringency. Noble and co-workers27 showed this carryReceived: June 7, 2014 Revised: September 2, 2014 Accepted: September 8, 2014
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dx.doi.org/10.1021/jf502721c | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Figure 1. Illustration of the experimental approach used to study in vivo interaction between SP and PC.
over effect by a sensory study, evaluating the time−intensity curves for astringency of two red wines. The central physiological mechanism underlying astringency caused by different foods remains a controversial subject.30 The aim of this study is to provide new insights on the phenomenon of astringency perception. To evaluate the interaction of the different SP families, a repeated astringency model was developed in which it is possible to understand how the different families of SP interact in an in vivo assay with condensed tannins (procyanidins, PC). In addition, in vitro assays were also performed to investigate which SP families are more susceptible to form insoluble or soluble complexes, and the size of these complexes was evaluated by DLS measurements.
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Table 1. Time Intervals (min) after the Last Exposure to PC Solution (ST1_5) and Respective Designation in ST2 interval (min)
designation
5 10 20 30 40 45 50 55 60
ST2_6 ST2_7 ST2_8 ST2_9 ST2_10 ST2_11 ST2_12 ST2_13 ST2_14
The AS sample was analyzed by HPLC before and after interaction with PC solution (2.5 g L−1). The control condition was a mixture of AS (150 μL) and 0.1 M acetate buffer (pH 5.0, 12% ethanol) (50 μL) (final volume 200 μL). For the experiments with PC, the necessary volume (50 μL) of PC solution (2.5 g L−1) was added to AS (150 μL) to obtain the final volume 200 μL. After shaking, the mixture reacted at room temperature (20 °C) for 2 min and was then centrifuged (8000g, 5 min). One-half of the supernatant was directly injected into the HPLC, and the other half was filtered (cellulose acetate 0.22 μm) and only then injected into the HPLC. The percentage of insoluble complexes was obtained by subtracting the area of the chromatographic peaks of each family obtained in the HPLC analysis of the nonfiltered solution from the respective area obtained in the control saliva (AS without tannin). The percentage of soluble complexes was calculated in the same way but by subtracting the data obtained in the HPLC analysis of the nonfiltered solution from the respective area obtained in the filtered solution. Salivary Flow Rate. Salivary flow rates were measured at 11 am according to the procedure described by Gaviao et al.34 During 5 min, subjects spat out the saliva passively accumulating in their mouth into a preweighed plastic container. The salivary flow rate was assessed by three repetitions and was expressed in mL min−1. Dynamic Light Scattering (DLS) Measurements. The size of procyanidin/salivary protein aggregates present in solution was measured by DLS, using the Zetasizer Nano ZS Malvern instrument. The same procedure described for protein−tannin interaction in this section was applied herein. Thus, to determine the size of the aggregates, one-half of the supernatant resulting from protein− procyanidins reaction was evaluated by DLS, and the other one-half was filtered (cellulose acetate 0.22 μm) and then also analyzed by DLS. This technique measures the time-dependent fluctuations in the intensity of scattered light that occur because particles undergo Brownian motion. The analysis of these intensity fluctuations enables determination of the diffusion coefficients of particles, which are converted into a size distribution. The sample solution was illuminated by a 633 nm laser, and the intensity of light scattered at an angle of 173 was measured by an avalanche photodiode. This analysis provides information concerning particle size (obtained by average size parameter) and polydispersity. In general, Z average size results were considered valid when the polydispersity of solutions was below 0.5. These experiments were performed in triplicate. HPLC Analysis. The method used in the HPLC analysis was adapted from the method described by Soares et al.11 Ninety microliters of each solution were injected on HPLC Lachrom system (Merck Hitachi, L-7100) equipped with a Vydac C8 column (Grace
MATERIALS AND METHODS
Reagents. All reagents used were of analytical grade or better. Acetonitrile (ACN) was purchased from Panreac Quimica; trifluoroacetic acid (TFA) and sodium acetate were purchased from Fluka Biochemica; commercial procyanidins (Vitisol) were purchased from Berkem, Plant Extraction; and ethanol was purchased from AGA, Á lcool e Géneros Alimentares, SA. In Vivo Interaction between Procyanidins and Salivary Proteins. Human saliva was collected from healthy and nonsmoking volunteers. The experiences were conducted with four subjects aged from 21 to 31 years. Each volunteer contributed to three independent samples (n = 12). All participants were instructed to avoid food and beverages for at least 1 h before the sessions started. The recording procedure was explained in detail to the subjects who provided written consent prior to participation. The study was conducted according to the Declaration of Helsinki and was approved by the Ethics Committee of Medical School of University of Porto (EK84032011). The PC solution (2.5 g L−1) was prepared, and human saliva was collected from different subjects after repeated sips of this solution. The experimental procedure was divided into two stages: Stage 1 (ST1) comprises saliva collected during approximately 15 min with repeated sips of the PC solution at 3 min intervals. It was asked of the subjects to hold 5 mL of this solution in their mouth for 5 s, spit it out, and 3 min later the saliva was collected. This procedure was repeated five times (ST1_1−ST1_5). On the other hand, stage 2 (ST2) comprises saliva collected at different intervals during 60 min (ST2_6−ST2_14) after the last exposure to PC solution (Figure 1). These intervals (min) and respective designation are indicated in Table 1. ST1_5 was used as reference, so for example ST2_6 refers to saliva collected 5 min after ST1_5, ST2_7 refers to saliva collected 10 min after ST1_5, and so on. All samples of saliva were mixed with 10% TFA (final concentration 0.1%), centrifuged 5 min at 8000g, and the supernatant (acidic saliva, AS) was analyzed by high performance liquid chromatography (HPLC). In Vitro Interaction between Salivary Proteins and Procyanidins. To reduce the variability connected to diurnal variations31,32 and the influence of circadian rhythms,33 a pool of saliva collected at 11 am on three different days was prepared. Saliva was collected from the same subjects that participated in the in vivo astringency analysis. The saliva pool was mixed with 10% TFA (final concentration 0.1%), centrifuged (8000g for 5 min), and supernatant (AS) was separated from the precipitate and used for the following experiments. B
dx.doi.org/10.1021/jf502721c | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Davison Discovery Sciences); the column dimensions are 150 × 2.1 mm, and the particle diameter is 5 μm; detection was carried out at 214 nm, using a UV−vis detector (L-7420). The HPLC solvents were 0.2% aqueous TFA (eluent A) and 0.2% TFA in ACN/water 80/20 (v/v) (eluent B). The gradient applied was linear from 10% to 40% (eluent B) in 75 min, at a flow rate of 0.30 mL min−1. After the program, the column was washed with 100% eluent B for 20 min to elute S-type cystatins and other late-eluting proteins. After being washed, the column was stabilized with the initial conditions.11,35 Data and Statistical Analysis. The mean values of chromatographic peaks and standard error of mean (SEM) were evaluated using analysis of variance (ANOVA), followed by the Bonferroni test. The mean values of salivary flow rates, the percentage of SP involved in the formation of (in)soluble complexes, and standard deviations were evaluated by statistical analysis using t test unparametric. Differences were considered to be statistically significant when P was at least
