Kinetic model for the antiradical activity of the isolated p-catechol group in flavanone type structures using the free stable radical 2,2-diphenyl-1-picrylhydrazyl as the antiradical probe

The time evolution of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH*) concentration in four solvents (methanol, ethanol, propanol, and acetonitrile) during its reduction by three flavanones containing an isolated p-catechol group (taxifolin, eriodyctiol, and fustin) as well as the time evolution of the mass spectra of the reaction mixture has been determined by spectrophotometry and liquid mass spectrometry, respectively. In alcoholic solvents the reduction curves consisted of an initial short but fast kinetics step followed by a longer slow kinetics step; in contrast, in acetonitrile the reduction curves completely lacked the slow kinetics step. From the results, a kinetic model for the reaction of reduction of the DPPH* by the isolated p-catechol group in flavanone type structures is proposed. According to this model, the p-catechol group rapidly transfers two hydrogen atoms to DPPH*, through a fast rate constant k1, yielding the corresponding o-quinone. Then, the intermediate o-quinone forms an adduct with the alcoholic solvent, through a slow rate constant k2, and regenerates the p-catechol group. The regenerated p-catechol group reduces additional DPPH* through a fast rate constant k3, yielding the corresponding o-quinone, which can form a new adduct with the solvent to regenerate the p-catechol group, and so on. From the kinetics model, two explicit kinetics equations have been derived that fit very well the experimental data points acquired from all assayed compounds in all of the experiments carried out, thus allowing an accurate determination of the corresponding rate and stoichiometric constants.     

Synergistic effects of thiols and amines on antiradical efficiency of protocatechuic acid

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of protocatechuic acid and its structural analogues (methyl protocatechuate, 3',4'-dihydroxyacetophenone, 3,4-dihydroxybenzaldehyde, and 3,4-dihydroxybenzonitrile) were examined in aprotic and protic solvents. In aprotic acetonitrile, all test compounds scavenged two radicals. In protic methanol, however, these compounds rapidly scavenged five radicals except for protocatechuic acid, which consumed only two radicals. The result indicated that higher radical scavenging activity in methanol than in acetonitrile was due to a nucleophilic addition of the methanol molecule on the oxidized quinones, which led to a regeneration of catechol structures. To investigate the importance of the nucleophilic addition on the quinones for the high radical scavenging activity, DPPH radical scavenging activity of protocatechuic acid and its analogues was examined in the presence of a variety of nucleophiles. The addition of a strong nucleophile such as a cysteine derivative significantly increased the radical scavenging equivalence. Furthermore, thiol adducts at C-2 and C-2,5 of protocatechuic acid and its analogues were isolated from the reaction mixtures. These results strongly suggest that the quinone of protocatechuic acid and its analogues undergo a nucleophilic attack at C-2 to yield 2-substituted-3,4-diols. Then, a regenerated catechol moiety of adducts scavenge two additional radicals by reoxidation into quinones, which undergo the second nucleophilic attack at the C-5. This mechanism demonstrates a possibility of synergistic effects of various nucleophiles on the radical scavenging ability of plant polyphenols containing a 3,4-dihydroxy substructure like protocatechuic acid and its analogues.     

Structure-DPPH* scavenging activity relationships: parallel study of catechol and guaiacol acid derivatives

The scavenging behavior of a series of catechol and guaiacol acid derivatives toward DPPH(*) was examined having as a starting point the order of activity derived on the basis of theoretically calculated BDE values. The studied compounds were protocatechuic, homoprotocatechuic, dihydrocaffeic, and caffeic acids and also vanillic, homovanillic, dihydroferulic, and ferulic acids. Catechol and guaiacol were used as reference compounds. Observations from the parallel study were made with regard to structural features (number and position of hydroxyl groups and the side-carbon chain characteristics) that regulated the behavior of the compounds experimentally. The exceptional DPPH(*) scavenging behavior observed for homoprotocatechuic acid in ethanol and for caffeic acid in acetonitrile could not be supported by the respective BDE values. Ferulic was the most active among guaiacolic acids, whereas dihydroferulic exhibited the highest stoichiometry. Ionizable carboxylic groups seem to affect considerably the relative order of activity as was also evidenced using the ORAC assay. Questions raised about the validity of widely accepted views on criteria for SARs are discussed with regard to literature findings.     

Oxidative dimers produced from protocatechuic and gallic esters in the DPPH radical scavenging reaction

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging reactions of protocatechuic and gallic acids, and their methyl esters, have been investigated by NMR. In acetone, methyl protocatechuate was gradually converted to a Diels-Alder adduct of two molecules of the intermediate quinone in the reaction with DPPH radical, whereas methyl gallate rapidly gave a symmetrical dimer via a putative quinone precursor. Both dimers are rather unstable and their structures have been deduced by in situ NMR measurements of the reaction mixtures. Gallic acid also gave a corresponding symmetrical dimer in the same reaction as methyl gallate, although protocatechuquinone produced from protocatechuic acid did not yield a Diels-Alder adduct, unlike its methyl ester. Interestingly, these dimer formations were not observed in methanol solution.     

Anthocyanin-based natural colorants: a new source of antiradical activity for foodstuff

The antiradical capacity (radical scavenger capacity, RSC) of anthocyanin-based fruit extracts prepared in the laboratory (black chokeberry, black-thorn, and strawberry) was studied by using the 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH(*)). To determine their RSC, the second-order rate constant (k(2)) for the oxidation of these extracts by DPPH(*) was calculated. The value of k(2) was compared to that used in the food industry as natural (alpha-tocopherol) or synthetic (butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA)) antioxidants, as well as for a commercial elderberry concentrate and a synthetic colorant (Ponceau 4R). The k(2) values ((mg/mL)(-)(1) s(-)(1)), in methanol at 25 degrees C, were 1.87, 0.7, 0.42, 0.2, 0.05, 0.03, and 0.008 for alpha-tocopherol, black chokeberry, BHA, black-thorn, BHT, strawberry, and elderberry, respectively. Ponceau 4R lacked RSC. Therefore, these natural colorants proved to be a combined source of color and RSC for food material.     

Kinetic study of flavonoid reactions with stable radicals

The antiradical activities of some flavonols (kaempferol, quercetin, robinetin, quercetagetin, and myricetin), flavones (apigenin, baicalein, and luteolin), flavanones (naringenin and dihydroquercetin), and flavanols [(+)-catechin and (-)-epicatechin] were determined by measuring the reaction kinetics with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and alpha,gamma-bisdiphenylene-beta-phenylallyl (BDPA) radicals. The reactions, which follow the mixed second-order rate law, were investigated under pseudo-first-order conditions by use of a large excess of flavonoids, and their stoichiometry was determined by spectrophotometric titration. The results confirm stoichiometric factors of 1, 2, and 3 for flavonoids with one, two, and three hydroxyl groups in the B-ring, respectively, excluding kaempferol, which, despite a single OH group in the B-ring, has a factor of 2, which is explained by the 3-OH group supporting the reaction with free radicals. Structure-activity considerations indicate for the present series of flavonoids the importance of multiple OH substitutions and conjugation. The logarithms of reaction rate constants with the OH, DPPH, and BDPA radicals correlate well with the reduction potential of the flavonoids.     

Antiradical properties of commercial cognacs assessed by the DPPH(.) test

Antiradical activities of some commercial cognacs were evaluated by the DPPH(*) test. Different mathematical models for the evaluation of the antiradical efficiency of the cognac samples were proposed and discussed. Nonflavonoid phenols were found to be the main substances responsible of the radical scavenging activity of cognacs. In particular the strongest correlations between antiradical activity measurements and cognac chemical characteristics was found for ellagitannins, high molecular weight polyphenols, which are extracted from the wood and solubilized in the spirit mainly during first year aging.     

Anti- and pro-oxidant water soluble activity of Cichorium genus vegetables and effect of thermal treatment

Both the pro- and antiradical water soluble activity, toward DPPH(*), ROO(*), OH(*) radicals found in seven diet vegetables belonging to the Cichorium genus, and the effects of boiling, freezing, and freeze-drying on such activities were investigated. The vegetables were three red cultivars of Cichorium intybus var. silvestre from three different areas of production, that is, chicory from Chioggia, Treviso, and Verona, C. intybus var. foliosum (Belgian chicory), C. endivia var. latifolium (escarole), C. endiviavar. crispum ("crispa"), and a hybrid vegetable obtained by the cross between C. intybus var. silvestre and C. endivia var. latifolium (chicory from Castelfranco). The juices obtained by simple centrifugation of vegetables operating at 2 or 25 degrees C and submitted to the thermal technological treatments were assessed for antiradical activity using the DPPH(*) assay, the linoleic acid-beta-carotene system, and the deoxyribose assay. In all three assays used, each vegetable juice was shown to possess antiradical activity; there was a significant level in the C. endivia and the Belgian chicories and higher levels in the red C. intybus vegetables and the hybrid vegetable. All juice behaviors in the linoleic acid-beta-carotene system indicate that they also contain a thermally unstable component, which in a cold medium promptly promoted and accelerated linoleic acid peroxidation, therefore masking the presence of any thermally stable antiperoxyl radical components. The presence of these components, which efficiently protect linoleic acid from peroxidation, can be singled out only after inactivation by heating, or separation by dialysis, of the pro-oxidant components. Dialysis fractions showed that the pro-oxidant component has MW > 50000 Da and that the juices contain a number of antioxidant components which contribute to their antiradical activity.     

Antioxidant and antiradical activities in extracts of hazelnut kernel (Corylus avellana L.) and hazelnut green leafy cover

Phenolic compounds in the aqueous systems were extracted, from hazelnut kernel (HK) and hazelnut green leafy cover (HGLC), with 80% (v/v) ethanol (HKe and HGLCe) or 80% (v/v) acetone (HKa and HGLCa). The extracts were examined for their phenolic and condensed tannin contents and phenolic acid profiles (free and esterified fractions) as well as antioxidant and antiradical activities by total antioxidant activity (TAA), antioxidant activity in a beta-carotene-linoleate model system, scavenging of DPPH (2,2-diphenyl-1-picrylhydrazyl) radical, and reducing power. Significant differences (p < 0.05) in the contents of total phenolics, condensed tannins, and TAA existed among the extracts that were examined. HGLCa extract had the highest content of total phenolics (201 mg of catechin equivalents/g of extract), condensed tannins (542 mg of catechin equivalents/g of extract), and TAA (1.29 mmol of Trolox equivalents/g of extract) followed by HGLCe, HKa, and HKe extracts, respectively. Five phenolic acids (gallic acid, caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid) were tentatively identified and quantified, among which gallic acid was the most abundant in both free and esterified forms. The order of antioxidant activity in a beta-carotene-linoleate model system, the scavenging effect on DPPH radical, and the reducing power in all extracts were in the following order: HGLCa > HGLCe > HKa > HKe. These results suggest that both 80% ethanol and acetone are capable of extracting phenolics, but 80% acetone was a more effective solvent for the extraction process. HGLC exhibited stronger antioxidant and antiradical activities than HK itself in both extracts and could potentially be considered as an inexpensive source of natural antioxidants.     

Melanoidins exert a weak antiradical activity in watery fluids

The Maillard reaction has a dramatic impact on the overall acceptance and nutritional quality of most of the foods consumed daily in European countries. Melanoidins are polymeric structures formed in the last stage of the Maillard reaction with nearly unknown effect on the human health. The antiradical activity of several melanoidins isolated from model systems and coffee has been studied. A novel antiradical efficiency concept has been applied to describe the antiradical activity in an aqueous medium by bleaching the radical cation N,N-dimethyl-p-phenylenediamine (DMPD(*)(+)). Melanoidins exerted a significantly lower antiradical activity than classical antioxidant compounds (tannic acid, ferrulic acid, caffeic acid, gallic acid, and Trolox) in an aqueous medium. Significant differences have been observed according to the type of amino acid used as reactant during the formation of the melanoidin structure and the antiradical efficiency exerted.     

Isolation of an in vitro and ex vivo antiradical melanoidin from roasted barley

The antiradical properties of water-soluble components of both natural and roasted barley were determined in vitro, by means of DPPH* assay and the linoleic acid-beta-carotene system, and ex vivo, in rat liver hepatocyte microsomes against lipid peroxidation induced by CCl4. The results show the occurrence in natural barley of weak antioxidant components. These are able to react against low reactive peroxyl radicals, but offer little protection against stable DPPH radicals deriving from peroxidation in microsomal lipids. Conversely, roasted barley yielded strong antioxidant components that are able to efficiently scavenge free radicals in any system used. The results show that the barley grain roasting process induces the formation of soluble Maillard reaction products with powerful antiradical activity. From roasted barley solution (barley coffee) was isolated a brown high molecular mass melanoidinic component, resistant to acidic hydrolysis, that is responsible for most of the barley coffee antioxidant activity in the biosystem.     

Effect of solvent, temperature, and solvent-to-solid ratio on the total phenolic content and antiradical activity of extracts from different components of grape pomace

Grape byproducts were subjected to an extraction process under various different experimental conditions (namely, solvent type, temperature, solvent-to-solid ratio, time contact, and raw material) in order to study the effect of these conditions on the yield of phenolic compounds and the corresponding antiradical activity of extracts. Although the order of decreasing capacity to extract soluble materials was ethanol > methanol > water, methanol was the most selective for extracting phenolic compounds. Temperature and solvent-to-solid ratio were found to have a critical role in extraction efficiency; values of 50 degrees C (between 25 and 50 degrees C) and 1:1 (between 1:1 and 5:1) maximized the antiradical activity of phenolic extracts. In addition, extracts from grape samples previously subjected to distillation reached higher antiradical values in comparison to those coming directly from pressing; in both cases, seed extracts showed better results than those of stem when ethanol or water was employed, whereas the opposite occurred in the case of methanol. These differences were attributed to the different phenolic compositions of the considered fractions.     

Phenolic acids and derivatives: studies on the relationship among structure, radical scavenging activity, and physicochemical parameters

The antiradical activity of caffeic acid (1), dihydrocaffeic acid (5), and their corresponding n-alkyl esters was evaluated by using the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH(*)) method. Dihydrocaffeic acid (5) was the most potent compound, having an antiradical effect higher than that of (+/-)-alpha-tocopherol, whereas caffeic acid (1) was less efficient. Esterification of the carboxyl group of dihydrocaffeic acid (5) had a dramatic effect on its antiradical potency, but similar effects were not observed for caffeic acid (1) derivatives. The n-alkyl esters of both phenolic series had similar potencies, and their antiradical activities were independent of the alkyl chain length. Dose-dependent scavenger effects were found in both series. Acid-base properties of the compounds, evaluated by using potentiometry and spectrophotometry, showed that the catechol moiety had pK(a2) and pK(a3) values of 9. 24-9.02 and 11.38-10.99 in the dihydrocaffeic series and 8.48-8.24 and 11.38-11.07 in the caffeic series, respectively. Antiradical activity and pK(a) values of the compounds were not related.     

Murta leaves (Ugni molinae Turcz) as a source of antioxidant polyphenols

Extracts from Murta leaves are used by Chilean natives for their benefits on health and cosmetic properties, which are mainly due to the presence of polyphenolic compounds. Extraction of such compounds is strongly influenced by several variables, the effects of which are studied in this work; the antioxidant power of the resulting extracts was measured by two different methods [2,2-diphenyl-1-picrylhydrazyl (DPPH) and thiobarbituric acid reactive substances (TBARS)]. On the whole, maximum values of polyphenolic yields and antiradical power (DPPH method) were attained at 50 degrees C (from 25 to 50 degrees C) and a solvent-to-solid ratio (v/w) of 15:1 (15:1-25:1). The solvents assayed were ethanol, methanol, and water. The highest polyphenolic yield values (2.6% expressed as gallic acid) were reached with methanol, whereas maximum EC50 was attained by the ethanol extract (0.121 mol gallic acid/mol DPPH). Contact time was shown to have only a slight influence in alcoholic extraction, while in water a remarkable effect of increasing contact times (30-90 min) was observed. Just water was the solvent that offered the best result when the antioxidant power was measured by the TBARS method. High-performance liquid chromatography-mass spectrometry analysis revealed the presence of polyphenols, basically flavonols and flavanols, sometimes glycosilated; myricetin and quercetin glycosides were detected in all extracts, whereas epicatechin was present in alcoholic extracts and gallic acid was only present in water.     

Antioxidant and antiradical activities of flavonoids.

The relationship between the structure of 42 flavonoids and their antioxidant and antiradical activities was elucidated by heat-induced oxidation in a beta-carotene and linoleic acid system and by the 1,1-diphenyl-2-picrylhydrazyl decoloration test. From seven structurally divergent groups of flavonoids, only flavonols with a free hydroxyl group at the C-3 position of the flavonoid skeleton showed high inhibitory activity to beta-carotene oxidation. Antiradical activity depended on the presence of a flavonol structure or free hydroxyl group at the C-4' position. The effect of the 4'-hydroxyl was strongly modified by other structural features, such as the presence of free hydroxyls at C-3 and/or C-3' and a C2-C3 double bond.     

Effect of foliar application of selenium on the antioxidant activity of aqueous and ethanolic extracts of selenium-enriched rice

Selenium fertilizer was foliar applied to determine the effects of antioxidant activity of selenium-enriched rice assessed by alpha,alpha-diphenyl-beta-picylhydrazyl (DPPH) radical scavenging and the ferric thiocyanate (FTC) method. Results showed that selenium concentration in rice was significantly enhanced dose dependently. Aqueous or ethanolic extracts of rice displayed significantly higher antioxidant activity against lipid peroxidation. The activities of aqueous extracts were significantly higher than those of ethanolic extracts and increased with the increasing selenium concentration in rice. The DPPH assay showed that the kinetic behaviors of aqueous extracts were complex and slow, while ethanolic extracts reacted quickly with DPPH radical. Aqueous extracts of rice exhibited higher antiradical efficiencies than ethanolic extracts, and rice (1.275 mg Se kg(-)(1)) presented the lowest EC(50) values of 533.46 +/- 0.58 microg mL(-)(1). As compared to rice extracts, all of the reference antioxidants showed more than 4-fold antiradical efficiencies than rice extracts. This radical scavenging activity was significantly correlated with selenium concentrations in rice (R = 0.862, p < 0.05), while ethanolic extracts were inversely correlated with selenium concentration in rice.     

Free radical scavenging capacity in the aging of selected red Spanish wines

Free radical scavenging capacity by the DPPH(*) method and main physicochemical properties, polyphenols content by HPLC, color by a tristimulus colorimeter, and UV-vis spectra in the aging of selected red Spanish wines, were studied. As the wines age, they become darker (lower lightness, L) and increase their hue angle (lower red color) as well as the ratio of absorbance at 420 nm to that at 520 nm. Main polyphenolics identified in the samples were tannic acid, oenin, and gallic acid. The antiradical efficiency of the samples increased during aging, which could be related to an increase in the tannic acid concentration shown by the following correlationship: EC(50) = 1/(0.18 + 0.0011[tannic acid](mg/L)) with a correlation coefficient of 0.744.     

Simple detection method of powerful antiradical compounds in the raw extract of plants and its application for the identification of antiradical plant constituents

A simple detection method for a powerful radical scavenging compound in a mixture containing a large variety of compounds, such as the raw extract of edible plants, was developed using 1,1-diphenyl-2-picrylhydrazyl (DPPH) as the radical reagent. The method was established on the basis of the features of the typical chain-breaking antioxidation reaction mechanism, which suggests that the radical scavenging antioxidant should be converted to other stable nonradical compounds during the reaction. This method requires only a simple HPLC instrument, and the disappearance or decrease in the peak intensity, which is induced by the addition of DPPH. This change is monitored by the HPLC to detect the powerful radical scavenger from the complex mixture. The method was applied to the detection and identification of the most powerful antiradical compound in the extracts of three antioxidatively active plant extracts (Psidium guajava, Citrus depressa, and Hypericum chinense). The radical scavenging efficiency of a newly identified compound from H. chinense was also compared with that of Trolox and catechin using the method.     

Kinetic solvent effects on phenolic antioxidants determined by spectrophotometric measurements

The effects of polar (acetonitrile and tert-butyl alcohol) and apolar (cyclohexane) solvents on the peroxyl-radical-trapping antioxidant activity of some flavonoids, catechol derivatives, hydroquinone, and monophenols have been studied. The inhibition rate constants k(inh) of the antioxidants have been determined by following the increase in absorbance at 234 nm of a dilute solution of linoleic acid at 50 degrees C containing small amounts of antioxidant and radical initiator. Despite the low concentration of linoleic acid, the peroxidation process has been confirmed to be a free radical chain reaction described by the classical kinetic laws for this process. However, in the evaluation of k(inh), a careful analysis of the peroxidation curve, absorbance versus time, must be done because the final oxidation products of phenols may absorb at 234 nm. Phenols with two ortho-hydroxyls are the most active antioxidants, with inhibition rate constants in the range of (3-15) x 10(5) M(-1) x s(-1) (in cyclohexane). Nevertheless, it has been observed that in tert-butyl alcohol (a strong hydrogen bond acceptor) the rate constants dramatically decline to values not detectable by the present kinetic method. In acetonitrile (a weaker hydrogen bond acceptor) instead, the phenols with two ortho-hydroxyls scavenge the peroxyl radicals with rate constants close to those in cyclohexane. From the kinetic solvent effect, the equilibrium constant of the first solvation step of hydroquinone with tert-butyl alcohol has been determined at 50 degrees C, K(1) = 2.5 +/- 0.5 M(-1).     

Stoichiometric and kinetic studies of phenolic antioxidants from Andean purple corn and red-fleshed sweetpotato

Stoichiometric and kinetic values of phenolics against DPPH (2,2-diphenyl-1-picrylhydrazyl) were determined for Andean purple corn (Zea mays L.) and red sweetpotato (Ipomoea batatas). Both crops had higher antioxidant capacity and antiradical kinetics than blueberries and higher or similar anthocyanin and phenolic contents. The second-order rate constant (k(2)) was 1.56, 1.12, 0.57, and 0.26 (mg antiradical/mL)(-1) s(-1) for red sweetpotato, Trolox, purple corn, and blueberry, respectively. On the molar basis of active hydroxyl groups, k(2)' showed the same order as for k(2). Corn cob and sweetpotato endodermis contributed the most in phenolic compounds and antioxidant capacity. Both crops studied can be considered as excellent novel sources of natural antioxidants for the functional food and dietary supplement markets.