Influence of solvents on the antioxidant property of flavonoids Enrico Finotti 1 , Danila Di Majo 2 1 Introduction The flavonoids are a chemical group present in plants as by- products. They represent a large class of coloured chemical constituents naturally occurring in fruits, nuts, seeds, leaves, and flowers. There is a multiplicity of flavonoids and various hydroxylation, methoxylation, and glycosylation patterns. In nature, the flavonoids do not occur in a free state but as glyco- sides, the third position being the most commonly glycosylated site [1–3]. They have been considered more as vitamins [4] than potential carcinogenic compounds [5], but many experi- mental results and epidemiological studies give considerable evidence that flavonoids may have a role in preventing health hazards due to oxidative stress [1, 6, 7]. In citrus fruits, three different classes represent the flavonoids group: the flavones, the flavanols, and the glycosilated flavanols. The interest in these classes of compounds is due to their pharmacological activity as radical scavengers [7]. We focused our attention on these flavonoids because of the fact that they are not only solu- ble in a water-alcohol mixture, but also have high solubility in fats and oils [8]. This means that the compounds present differ- ent chemical forms related to the environment in which they are solved [9, 10]. This feature could make these compounds an ideal tool for the study of their reactivity when they are exposed to oxidative stress by hydrophilic radical sources or by lipophilic radical sources. The aim of this study is to evalu- ate the antioxidant capacity of flavones, flavanols, and glycosi- lated flavanols, when they are solved in a hydrophilic environ- ment (water) in the presence of an hydrophilic radical source, such as the 2,29-azo-bis(2-amidinopropane) (ABAP), and in a lipophilic environment (toluene) in the presence of a lipophilic radical source, such as 2,2-azo-bis(2,4-dimethylvaleronitrile) (AMVN). Their antioxidant values have been estimated by cro- cin bleaching, because it is the more suitable method since it is able to detect the antioxidant capacity in both lipophilic and hydrophilic environments. 2 Materials and methods 2.1 Crocin bleaching inhibition The crocin bleaching inhibition method was used to deter- mine the antioxidant capacity of the glycosylated flavanols: naringin, neohesperidin, neoericitrin, hesperidin, narirutin didymin, and the related flavones (purchased from Extrasyn- these, Genay, France) such as naringenin, hesperetin eriodic- tyol and isosakuratenin, at 0.01 mm each in both lipophilic (toluene) and hydrophilic (water) environments. This method is based on the crocin bleaching as a result of its oxidation by a source of radicals, AMVN (Waco Chemicals, Richmond, CA, USA) in lipophilic environment and ABAP (Waco Chemi- cals) in the hydrophilic environment [11, 12]. 2.2 Competition kinetic assay Saffron from Sigma Chemical Co. (St. Louis, MO, USA) was pretreated by diethyl ether (Merck, Darmstadt, Germany) in order to remove traces of lipids and other contaminants, and after the crocin was extracted twice from the saffron by metha- nol (Merck). Crocin was measured in methanol at 443 nm and calculated using the absorption coefficient (e = 1.33 6 10 5 mol –1 cm –1 at 443 nm). The crocin bleaching inhibition method was used to determine the antioxidant capacity of the single compounds. This method is based on the crocin bleaching as a result of its oxidation by a source of radicals, AMVN in a lipo- philic environment and ABAP in the hydrophilic environment. This reaction can be monitored by recording the correspondent (bleaching rate, Vo) decrease in absorbance at 443 nm, for 10 min, When an antioxidant or pseudo-antioxidant compound is added, it reacts with the free radicals and, as a consequence, the crocin bleaching rate is reduced (V a ). The reaction follaved the competitive reaction equation: V 0 =V a ¼ 1 þ K a =K c ½Pseudo-antioxidant=½Crocin where K a and K c are the respective absolute second order con- stants. From the [pseudo-antioxidant]/[crocin] vs. V 0 /V a linear regression plot, the slope K a /K c can be calculated. Its value indicates the relative capacity (antioxidant capacity) of differ- ent molecules to interact with the ROO9 radicals. Reactions were carried out at 40 8C. After AMVN or ABAP were added to the reaction solution (final volume was 1 mL in toluene for a lipophilic environment and water for a hydrophilic environ- ment), the bleaching rate of crocin was recorded after 10 min. Blanks without crocin were run to rule out spectral interfer- ences between the compound and crocin. Each kinetic analysis was compared to a kinetic crocin bleach containing only AMVN or ABAP (V 0 ) and used for the calculations according to the competitive reaction equation. 186 Nahrung/Food 47 (2003) No. 3, pp. 186 – 187 i 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0027-769X/2003/0306-0186$17.50+.50/0 In polyphenols redox systems, the solvent plays a fundamental role in the chemical behaviour of these compounds. Antioxidants can react in different ways with the prooxidant molecules. We have found differ- ences in the antioxidant capacity of flavonoids such as naringin, neo- hesperidin, neoericitrin, hesperidin, narirutin didymin and the related flavones naringenin, hesperetin eriodictyol and isosakuratenin, when they are in the presence of radicals and solved in water or in an alcohol mixture. Correspondence: Dr. Enrico Finotti, Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione, Via Ardeatina No. 546, I-00178, Roma, Italy E-mail: e.finotti@agora.it Fax: +39-06-5031592 1 Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione, Roma, Italy 2 Scuola di Specializzazione in Scienza dell’Alimentazione, Universita ` degli Studi di Palermo, Italy Abbreviations: ABAP, 2,29-azo-bis(2-amidinopropane; AMVN, 2,29- azo-bis(2,4-dimethylvaleronitrile) Keywords: Antioxidant properties / Flavonoids / Polyphenols /