The use of cyclodextrins as solubility enhancers in the ORAC method may cause interference in the measurement of antioxidant activity.

The ability of cyclodextrins to enhance the water solubility of lipophilic compounds is used to modify the water-based Oxygen Radical Absorbance Capacity (ORAC) method to measure antioxidant activity in vitro. However, the use of these solubility enhancers may alter fluorescent readings, which has led to contradictory results being described in the literature. The low specificity of these oligosaccharides and their controlled release effect can result in cyclodextrins forming inclusion complexes with other reagents in the assay, changing the kinetics. In this study, the cause of cyclodextrins' interference in the ORAC method is evaluated through a physicochemical and computational approach. Cyclodextrins showed a clear increase in the fluorescent signal both in the presence and absence of the antioxidant oxyresveratrol, the precise effect being dependent on the type and concentration of cyclodextrin. Although the glucidic nature of cyclodextrins could play a minimal role in this effect, it seems that the main cause was the encapsulation of other substrates in the reaction, fluorescein and AAPH.

Cyclodextrins and antioxidants.

In recent years, the growth of the functional foods industry has increased research into new compounds with high added value for use in the fortification of traditional products. One of the most promising functional food groups is those enriched in antioxidant compounds of a lipophilic nature. In spite of the numerous advantages reported for such antioxidant molecules, they may also have disadvantages that impede their use in functional foods, although these problems may well avoided by the use of encapsulant agents such as cyclodextrins. This explains the recent increase in the number of research papers dealing with the complexation of different guest molecules possesing important antioxidant properties using natural and modified cyclodextrins. This paper presents a review of the most recent studies on the complexes formed between several important types of antioxidant compounds and cyclodextrins, focusing on the contradictory data reported in the literature concerning to the antioxidant activity of the host/guest molecule complexes, the different complexation constants reported for identical complexes, the bioavailability of the antioxidant compound in the presence of cyclodextrins and recommendation concerning the use of natural or modified cyclodextrins. Moreover, the use of cyclodextrins as antibrowning agents to prevent enzymatic browning in different foods is revised. Finally, we look at studies which suggest that cyclodextrins act as ''secondary antioxidants," enhancing the ability of traditional antioxidants to prevent enzymatic browning.

Development of a reversed phase high performance liquid chromatography method based on the use of cyclodextrins as mobile phase additives to determine pterostilbene in blueberries.

In this work, a reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the determination of pterostilbene in food samples. The novel method is based on the addition of cyclodextrins (CDs) to the mobile phase where the complexation of pterostilbene by CDs is carried out. In order to select the most suitable conditions for the RP-HPLC method, the effect of several physico-chemical parameters on the complexation of pterostilbene by CDs was studied. Our results show that the addition of 12 mM HP-ß-CD to a 50:50 (v/v) methanol:water mobile phase at 25°C and pH 7.0 significantly improves the main analytical parameters. In addition, it was seen that pterostilbene forms a 1:1 complex with HP-ß-CD, showing an apparent complexation constant of 251±13 M(-1). Finally, in order to study the validity of the proposed method, blueberries were analyzed and the concentration of pterostilbene has been determined.

Kinetic mechanism and product characterization of the enzymatic peroxidation of pterostilbene as model of the detoxification process of stilbene-type phytoalexins.

The enzymatic peroxidation of pterostilbene, a strong antifungal belonging to the stilbene family, by peroxidase (POX), is reported for the first time as a model of phytoalexin detoxification carried out by the enzymatic pool of pathogens. Kinetic characterization of the pterostilbene oxidation reaction pointed to an optimum pH of 7.0, at which value the thermal stability of POX was studied. Moreover, the data showed that pterostilbene inhibits POX activity at high concentrations of substrate. Several kinetic parameters, including V(max), K(m) and K(I), were calculated and values of 0.16ΔAbs min(-1), 14.61 µM, and 31.41 µM were reported. To understand the possible physiological role of this reaction in the phytoalexin detoxification process, the products of pterostilbene oxidation were identified using HPLC-MS and a radical-radical coupling reaction mechanism was proposed. Three main products with a high molecular weight and pronounced hydrophobicity were identified: pterostilbene cis dehydromer, pterostilbene trans dehydromer and pterostilbene open dimer. The dimeric structures of these molecules indicate that the pterostilbene oxidation reaction took place at the 4'-OH position of the hydroxystilbenic moieties and the three above mentioned dimeric products were found, due to the ability of electron-delocalized radicals to couple at various sites. Finally, the capacity of cyclodextrins (CDs) as starch model molecules in plants to complex both the substrate and the products of the oxidation reaction was evaluated. The inhibition process of POX activity was modified at high pterostilbene concentrations due to sequestering of the substrate reaction and to the different affinity of the reaction products for CDs.

Use of reversed phase high pressure liquid chromatography for the physicochemical and thermodynamic characterization of oxyresveratrol/beta-cyclodextrin complexes.

Knowledge of the complexation process of oxyresveratrol with beta-cyclodextrin (beta-CD) under different physicochemical conditions is essential if this potent antioxidant compound is to be used successfully in both food and pharmaceutical industries as ingredient of functional foods or nutraceuticals, despite its poor stability and bioavailability. In this paper, the complexation of oxyresveratrol with natural CDs was investigated for first time using RP-HPLC and mobile phases to which alpha-, beta-, and gamma-CD were added. Among natural CDs, the interaction of oxyresveratrol with beta-CD was more efficient than with alpha- and gamma-CD. The decrease in the retention times with increasing concentrations of beta-CD (0-4 mM) showed that the formation constants (KF) of the oxyresveratrol/beta-CD complexes were strongly dependent on both the water-methanol proportion and the temperature of the mobile phase employed. However, oxyresveratrol formed complexes with beta-CD with a 1:1 stoichiometry in all the physicochemical conditions tested. Moreover, to obtain information about the mechanism of the oxyresveratrol affinity for beta-CD, the thermodynamic parameters DeltaG degrees, DeltaH degrees and DeltaS degrees were obtained. Finally, to gain information on the effect of the structure of different compounds belonging to the stilbenoids family on the KF values, the complexation of other molecules, resveratrol, pterostilbene and pinosylvin, was studied and compared with the results obtained for the oxyresveratrol/beta-CD complexes.

Complexation of pinosylvin, an analogue of resveratrol with high antifungal and antimicrobial activity, by different types of cyclodextrins.

The complexation of pinosylvin, a potent antimicrobial and antifungal stilbenoid, by cyclodextrins (CDs) is described for first time in this work. Steady-state fluorescence was used to demonstrate that natural (alpha-, beta-, and gamma-CD) and modified (HP-beta-CD, methyl-beta-CD, and ethyl-beta-CD) CDs are able to complex pinosylvin following a 1:1 stoichiometry. However, substantial differences in the strength of the complexation exist between the CDs tested. Although among natural CDs the interaction of pinosylvin with beta-CD was more efficient than with alpha- and gamma-CD, the results show that the complexation constants (K(F)) were higher for all of the modified CDs than for natural CDs, the highest K(F) being that determined for HP-beta-CD-pinosylvin complexes (12112 +/- 761 M(-1)). Moreover, deprotonation of the hydroxyl group of pinosylvin led to a sharp fall in the K(F) values with respect to those observed for the complexes formed between the protonated structure of this stilbenoid and the CDs. Moreover, a pK(a) value is reported for the first time for pinosylvin. Furthermore, when the temperature of the system was increased, a significant drop was observed in the complexation constant values. From these K(F) values and to throw light on the mechanism of pinosylvin affinity for HP-beta-CD, three thermodynamic parameters, DeltaH degrees , DeltaS degrees , and DeltaG degrees , were calculated. The results show that the complexation of pinosylvin by HP-beta-CD is a spontaneous and exothermic process with negative values for entropy changes. Finally, to gain information on the effect of the structure of different compounds belonging to the stilbenoid family on the K(F) values, the complexation of other molecules such as (E)-resveratrol and pterostilbene was studied and compared with the results obtained for the HP-beta-CD-pinosylvin complexes.

Physicochemical study of the complexation of pterostilbene by natural and modified cyclodextrins.

In this paper, the interaction between pterostilbene and cyclodextrins (CDs) is described for the first time using steady-state fluorescence. It was seen that pterostilbene forms a 1:1 complex with all of the natural (alpha-, beta-, and gamma-CDs) and modified (HP-beta-CD, methyl-beta-CD, and ethyl-beta-CD) CDs tested. Among natural CDs, the interaction of pterostilbene with beta-CD was the most efficient. However, all of the modified CDs showed higher complexation constants (K(F)) than beta-CD. The highest K(F) was found for HP-beta-CD (17520 +/- 981 M(-1)), in which its value showed a strong dependence upon pH in the region where the pterostilbene begins the deprotonation of its hydroxyl group. Moreover, the values of K(F) decreased as the system temperature increased. To obtain information on the mechanism of pterostilbene affinity for CD, the thermodynamic parameters of the complexation (DeltaH degrees , DeltaS degrees , and DeltaG degrees ) were studied. Finally, a comparison of the K(F) values obtained for three types of stilbenes revealed that both the stoichiometry and the K(F) values of the complex are dependent upon the structure of the guest molecule. While the trans-resveratrol-HP-beta-CD and pterostilbene-HP-beta-CD complexes showed a 1:1 stoichiometry with a higher K(F) value for the trans-resveratrol-HP-beta-CD complexes, trans-stilbene showed a 1:2 stoichiometry.