J. Chil. Chem. Soc., 48, N 3 (2003) ISSN 0717-9324

RED WINE ANTIOXIDANTS. EVALUATION OF THEIR HYDROPHOBICITY
AND BINDING EXTENT TO SALIVARY PROTEINS.

Marta Pizarro I. y Eduardo Lissi G.

Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile
.E-mail: elissi@lauca.usach.cl (Received: August 21, 2002 ­Accepted: May 13, 2003)

ABSTRACT

Absorption by the organism of the antioxidants present in beverages depends on their hydrophobicity and their capacity to avoid precipitation by the proline rich proteins present in saliva. In the present work, we show that the antioxidants present in red wine samples are compounds of high hydrophilicity that are only poorly adsorbed by the proteins present in saliva. The antioxidant capacity of red wine samples is decreased only in 13 % when extracted with n-octanol (1:1 , V:V) , irrespective of the pH of the wine sample. Similarly, only a small fraction of the antioxidants (12.2 ± 3.4 %) is precipitated when wine samples are shaken for a few minutes with non-stimulated human saliva (5:1, V:V). This last result implies that most of the polyphenols present in the red wine samples are able to surpass the barrier imposed by the saliva.

INTRODUCTION

Wines, and in particular red wine, possess high concentrations of phenolic and polyphenolic compounds that confer to this beverage a high antioxidant capacity.^1-4 The total amount of antioxidants present in a wine sample depends on the employed variety, and it is particularly high in most red wines produced in Chile.^1,4 It has been proposed that a moderate wine ingesta could improve the antioxidant/prooxidant balance in the organism, therefore decreasing the risk associated to pathological conditions in which oxidative stress is involved, either as promoting factor and/or as an additional factor that contributes to the organism deterioration.⁵ In agreement with this proposal, several epidemiological studies have shown an inverse correlation between moderate alcohol intake and coronary heart diseases.^6-8 This protective effect appears to be particularly high regarding a moderate red wine ingesta.⁹ The role of the red wine antioxidants in this protection is supported by the fact that it has been detected an increase in the total antioxidant capacity of human plasma associated to a moderate wine intake.^10-14 This increase implies that at least some of the antioxidants of the red wine can be significantly absorbed and that they, or their catabolites retaining antioxidant activities, have relatively long life expectancies in the organism.

In order to be incorporated into the plasma, the active compounds of wine must be able to reach the intestine and be absorbed by this organ. This last process is, at least partially, influenced by the hydrophobicity of the compounds. It is interesting then to have an estimation of the hydrophobicity of the antioxidants present in wine. Furthermore, it has been suggested that the main function of proline-rich proteins present in saliva is to bind ingested plant polyphenols, rendering insoluble complexes.^15-16 The formation of these complexes depends upon the characteristics of the salivary proteins, the pH and the properties (molecular weight, flexibility and hydrophobicity) of the polyphenols.¹⁷ In particular, the high influence of the hydrophobicity in the association process makes possible a direct relationship between the polyphenol hydrophobicity and its insolubilization by salivary proteins.¹⁸ Since several of the phenolic compounds present in red wine (such as the proantocyanidines) are potential substrates for protein binding,^19-24, it was considered relevant to evaluate which impact could have this type of complexation upon the total antioxidant capacity of the beverage. The only study of this type has been carried out employing bovine serum albumin as model protein.²¹ Taking into account that this is a molecule substantially different than the proline rich salivary proteins, we report results bearing on the hydrophobicity of red wine antioxidants and their capacity to interact with the components of human saliva.

MATERIALS AND METHODS

The red wine total antioxidant capacity was measured as previously described by Campos and Lissi.¹ Basically, the capacity of wines and/or they extracts to bleach 2,2'azino-bis(3-ethylbenzthiazoline-6-sulfonic) acid (ABTS) derived pre-formed radicals was evaluated. The extent of the reaction following the addition of a small aliquot of wine is directly proportional to the total content of phenolic compounds with antioxidant properties present in the beverage.²⁵

ABTS derived radicals were prepared by incubation of an ABTS sample (0.05 mM) with potassium persulfate (0.044 mM) during 60 minutes at 50 C. During this treatment, the characteristic blue color of the radical develops, and remains stable for several hours. After addition of the wine sample, the color is bleached proportionally to the size of the added aliquot. The bleaching process takes place readily and is almost complete 15 minutes after the wine addition. All the reported results were obtained at this fixed time. The decrease in absorbance, measured at 734 nm, is compared to that obtained employing a suitable concentration of Trolox. This is a hydrophylic simile of vitamin E usually employed as standard in antioxidant measurements. The total antioxidant capacity of the tested sample, TRAP, measured in Trolox microequivalents, is calculated as

where

f is a dilution factor equal to the quotient between the total volume of the solution and the wine aliquot;
D_wine is the decrease in absorbance, measured at 734 nm, elicited by the wine addition;
D_Trolox is the decrease in absorbance, measured at 734 nm, elicited by Trolox; and
[Trolox] is the added (micromolar) Trolox concentration.

In order to evaluated the hydrophobicity of the compounds with antioxidant capacity present in the red wine (Cabernet Souvignon), wine samples were extracted with n-heptane or 1-octanol. These extractions were carried out at several pHs in order to assess if possible changes in the protonation status of the compounds were able to influence their partition between water and the organic solvents. The wine pH was adjusted employing NaOH or HCl. Extractions were performed by the hand-shaking method employing 1 volume of wine and 1 volume of 1-octanol or 10 volumes of n-heptane. After phase separation, improved by gentle centrifugation, an aliquot of the aqueous phase was poured in an ABTS radical solution for TRAP evaluation. The value obtained was compared to that measured prior to the extraction procedure. Also the wine absorbance, measured at 518 nm, was evaluated prior and after the extraction procedure. This allows an estimation of the fraction of the compounds that absorb to this wavelength (mostly tannins) that are solubilized in the organic solvent.

In order to evaluate the capacity of salivary proteins to bind the polyphenols present in wine, 5 mL of red wine (Cabernet Sauvignon) were shaken with 1 mL of non-stimulated human saliva and centrifuged. Prior to centrifugation, trichloroacetic acid (1 %) was added to some samples in order to further precipitate the phenol-protein complexes. Control experiments were carried out employing buffer solutions instead of saliva. A fraction of the supernatant was added to an ABTS radical solution and the decrease in absorbance was compared with that of a wine sample that has been shaken with buffer. Extraction of the antioxidants present in saliva by the wine can be considered as negligible.²⁶

RESULTS AND DISCUSSION

Proanthocyanidins (tannins) are wine constituents with antioxidant capacity.^23,27,28 which strongly interact with proteins.^24,29-31 Tannins play an important role regarding wine quality, and their interaction with salivary proteins produces a drying and puckering sensation in the mouth called astringency.^21,24,32 The capacity of a tannin to interact with salivary proteins is strongly correlated with its molecular weight,³¹ only remaining in solution those compounds of relatively low molecular weight. The formed complexes are insoluble in conditions similar to those found in the stomach and the small intestine, suggesting that the interaction of a tannin with salivary proteins could determine its bioavailability.³³ In spite of this, there are not direct measurements of the fraction of red wine antioxidants that are able to surpass this first barrier imposed by saliva. To the best of our knowledge, the reported results employ model proteins,²¹ or are devoted to evaluate the characteristics of the protein and/or the polyphenol that favor complex formation.^31,33,34 We have then measured the capacity of saliva to reduce the total antioxidant capacity of red wine samples. This was assessed by shaking together for a few minutes 5 mL of red wine and 1 mL of non-stimulated saliva. This treatment reduces both the color of the red wine (compared to that observed in a similar mixture of wine and buffer) and its capacity to bleach the pre-formed ABTS radicals Table 1). In fact, while the absorbance of wine measured at 518 nm was reduced in (16.5 ± 6.0) percent ( n = 4), the capacity to bleach pre-formed radicals was reduced in (12.2 ± 3.4) percent ( n = 5). These values were not significantly modified by treating the samples with trichloroacetic acid, pointing to the formation of insoluble complexes of high stability between some of the polyphenols and salivary proteins. These results indicate that salivary proteins present only a moderate capacity to remove the polyphenols present in the beverage. However, it is interesting to note that the percentage of reduction measured by absorbance and TRAP are not significantly different. The absorbance of the sample at 518 nm is dominated by light absorption by tannins and antocyanins,³⁵ while TRAP values measure the total content of phenolic groups. This implies that colored compounds with antioxidant activity are selectively complexed or, more probably, that this type of compounds constitute the main class of antioxidants present in red wine samples.³⁶

Table 1. Decrease (%) in absorbance (measured at 518 nm) and in the red wine capacity to trap ABTS derived radicals (TRAP) after exposure to saliva or extraction with organic solvents.

Since the capacity of a polyphenols to interact with proteins is strongly conditioned by its hydrophobicity, and that this parameter is relevant regarding absorption at the intestine level, it was evaluated the hydrophobicity of the compounds contributing to the TRAP value of the sample.

This was assessed by measuring the partition of the TRAP value between water and organic solvents. In particular, partition was measured between water:n-heptane (1:10, V/V) and water/1-octanol (1:1 , V/V) mixtures. Employing several types of red wines (Merlot, Pinot and Cabernet Sauvignon) and a white wine (Sauvignon Blanc) over the pH range 2.0 to 7.0, it was observed that less than 10 % of the wine TRAP was removed by extraction with n-heptane. This result, included in Table 1, implies that, even when the phenol groups are totally protonated, the hydrophilicity of the antioxidants present in wine is large enough to avoid removal by a tenfold excess of n-heptane.

In order to test the hydrophobicity of the antioxidants in conditions more similar to those prevailing in biological systems, it was measured their distribution between water and 1-octanol. This compound is frequently employed as organic solvent in the evaluation of the capacity of a given compound to be incorporated to biological membranes.³⁷ A volume of red wine was extracted, without pH adjusting, with an equal volume of 1-octanol. This treatment only reduced the capacity of wine components to react with free radials in (13 ± 1) percent, confirming so that most of the antioxidants are highly hydrophylic compounds. This result is fully compatible with the report of Ghiselli et al.,³⁶ who show that the main contributors to the antioxidant potential of red wines are the anthocyanins, the most hydrophylic family of compounds present in this beverage.³⁵ Similarly, the absorbance of the red wine sample, measured at 518 nm, decreases only in 3 ± 1 percent ( n = 5) after 1-octanol extraction. The difference between this value and the decrease observed when the wine is treated with saliva ( 16.5 ± 6 %) would indicate that salivary proteins are able to extract even compounds (tannins) of high hydrophilicity that are not extracted by 1-octanol. However, it is interesting to note that a similar difference is not observed when the decrease in TRAP elicited by saliva and 1-octanol are compared. In this case, both values are very similar. This would indicate that the colored compounds extracted in excess by salivary proteins do not contribute significantly to the red wine TRAP or, more likely, that 1-octanol is able to extract antioxidants (such as flavonoids), that do not associate to salivary proteins.

In conclusion, the results obtained in the present work indicate that most of the antioxidants present in red wines are compounds of high hydrophilicity that are only partially removed by 1-octanol extraction or interaction with salivary proteins. The latter result constitute the first evaluation of the reduction in red wine TRAP as a consequence of its exposition to salivary proteins.

Acknowledgments: This work has been supported by DICYT (Universidad de Santiago de Chile) and the program "Bases Moleculares de las Enfermedades Crónicas" (Pontificia Universidad Católica de Chile).

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