Reduction kinetics of the antiradical probe 2,2-diphenyl-1-picrylhydrazyl in methanol and acetonitrile by the antiradical activity of protocatechuic acid and protocatechuic acid methyl ester

This work evaluates the reduction kinetics of the antiradical probe 2,2-diphenyl-1-picrylhydrazyl (DPPH (*)) in methanol and acetonitrile by the antiradical activity of protocatechuic acid (3,4-dihydroxybenzoic acid, 1) and protocatechuic acid methyl ester ( 2). The reduction kinetics of DPPH (*) in both solvents by the antiradical activity of the p-catechol group in 2 is regular, that is, coincide with the proposed standard kinetic model for the reduction kinetics of DPPH (*) by the antiradical activity of an isolated p-catechol group. Therefore, the antiradical activity of 2 experimentally exhibits two rate-two stoichiometric constants in acetonitrile and three rate--three stoichiometric constants in methanol. In contrast, the reduction kinetics of DPPH (*) in both solvents by the antiradical activity of the p-catechol group in 1 is perturbed, that is, deviate from the proposed standard kinetic model. The deviations arise from the presence of the reactive carboxylic acid function which, in methanol, induces an additional reversible side reaction and, in acetonitrile, turns an irreversible reaction reversible, thus modifying the otherwise regular reduction kinetics of DPPH (*) by the antiradical activity of the p-catechol group in 1. On the other hand, the approximated theoretical kinetic equation that applies for those p-catechol groups whose reduction kinetics is regular and that experimentally exhibit three rate--three stoichiometric constants has been derived and used for fitting.     

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.     

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 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).     

Polymeric procyanidins as radical-scavenging components in red-hulled rice

The extracts from white-, black-, and red-hulled rice were prepared by sequential extraction with six different polar solvents, and their radical-scavenging activities were measured by methods using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH*) and tert-butyl hydroperoxyl radical (t-BuOO*). The extracts prepared with highly polar solvents, methanol and deionized water, exhibited higher DPPH* and t-BuOO* scavenging activities in all three cultivars. In addition, the acetone extract from red-hulled rice exhibited a high DPPH* and t-BuOO* scavenging activity, while no such activity was detected for the acetone extracts from white- and black-hulled rice. The major components responsible for the radical scavenging in the acetone extract from red-hulled rice were identified as procyanidins by acidic hydrolysis, vanillin assay, and Sephadex LH-20 chromatography. GPC analysis of the acetylated procyanidins revealed that the average molecular weight is about 5000, in a range of about 500-18,000.     

Effects of extraction solvent mixtures on antioxidant activity evaluation and their extraction capacity and selectivity for free phenolic compounds in barley (Hordeum vulgare L.)

Four kinds of solvent extracts from three Chinese barley varieties (Ken-3, KA4B, and Gan-3) were used to examine the effects of extraction solvent mixtures on antioxidant activity evaluation and their extraction capacity and selectivity for free phenolic compounds in barley through free radical scavenging activity, reducing power and metal chelating activity, and individual and total phenolic contents. Results showed that extraction solvent mixtures had significant impacts on antioxidant activity estimation, as well as different extraction capacity and selectivity for free phenolic compounds in barley. The highest DPPH* and ABTS*+ scavenging activities and reducing power were found in 80% acetone extracts, whereas the strongest *OH scavenging activity, O2*- scavenging activity, and metal chelating activity were found in 80% ethanol, 80% methanol, and water extracts, respectively. Additionally, 80% acetone showed the highest extraction capacity for (+)-catechin and ferulic, caffeic, vanillic, and p-coumaric acids, 80% methanol for (-)-epicatechin and syringic acid, and water for protocatechuic and gallic acids. Furthermore, correlations analysis revealed that TPC, reducing power, DPPH* and ABTS*+ scavenging activities were well positively correlated with each other (p < 0.01). Thus, for routine screening of barley varieties with higher antioxidant activity, 80% acetone was recommended to extract free phenolic compounds from barley. DPPH* scavenging activity and ABTS*+ scavenging activity or reducing power could be used to assess barley antioxidant activity.     

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.     

Effects of solvent polarity and acidity on the extraction efficiency of isoflavones from soybeans (Glycine max)

Soy isoflavones have been correlated with beneficial health effects. The predominant chemical forms of isoflavones present may affect their biological activities. Choosing the solvent system that can accurately quantify the amounts of individual isoflavones present in these products is paramount. Our objectives were to compare frequently used solvent systems and to evaluate the effects of polarity and acidity on the recovery of isoflavones from soybeans. Isoflavones were extracted from pulverized Manokin soybeans using six solvent systems, which are the combinations of three polarity levels (83% acetonitrile, 80% methanol, and 58% acetonitrile) and two acidity levels (nonacidified and acidified). The pulverized soybean was stirred for 2 h in each solvent system before filtration and concentration using rotary evaporation. The extract was resuspended in 16% acetonitrile and analyzed by high-performance liquid chromatography. Recoveries of pure standards were evaluated with all solvent systems. Solvents with a higher polarity extracted a significantly higher amount of total isoflavones. For individual isoflavones, 58% acetonitrile (highest polarity) extracted either the highest amounts or no less than other solvents, while 83% acetonitrile (lowest polarity) extracted either the lowest amounts or no more than other solvents except for the aglycone form. Acidification significantly reduced the recovery of the malonylglucoside form and the total isoflavones. The recovery study revealed that acidification favored the chemical transformations of isoflavones during the extraction. Among the six solvent systems examined, 58% acetonitrile aqueous solution without acid was the best for extraction of isoflavones from soybeans.     

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.     

Grouping of soil bacteria by analysis of colony formation on agar plates

Summary The colony formation of soil bacteria was studied in relation to incubation time. The process was simulated by a colony-forming curve (CFC) which was a superposition of several component curves (cCFC) given theoretically by the first-order reaction (FOR) model. This model had been previously proposed to define the colony formation of cells of a single culture. Soil bacteria were divided into several groups by these cCFC, as different types of bacteria produced their own colonies. Bacteria belonging to a single group grew at a similar rate on the plating medium and each group was characterized by a different growth rate. Most copiotrophic bacteria were fast growers and most oligotrophic bacteria slow growers.     

Kinetic characterization of the oxidation of chlorogenic acid by polyphenol oxidase and peroxidase. Characteristics of the o-quinone

Chlorogenic acid is the major diphenol of many fruits, where it is oxidized enzymatically by polyphenol oxidase (PPO) or peroxidase (POD) to its o-quinone. In spectrophotometric studies of chlorogenic acid oxidation with a periodate ratio of [CGA]0/[IO4-]0 < 1 and [CGA]0/[IO4-]0 > 1, the o-quinone was characterized as follows: lambda(max) at 400 nm and epsilon = 2000 and 2200 M-1 cm-1 at pH 4.5 and 7.0, respectively. In studies of o-quinone generated by the oxidation of chlorogenic acid using a periodate at ratio of [CGA]0/[IO4-]0 > 1, a reaction with the remaining substrate was detected, showing rate constants of k = 2.73 +/- 0.17 M-1 s-1 and k' = 0.05 +/- 0.01 M-1 s-1 at the above pH values. A chronometric spectrophotometric method is proposed to kinetically characterize the action of the PPO or POD on the basis of measuring the time it takes for a given amount of ascorbic acid to be consumed in the reaction with the o-quinone. The kinetic constants of mushroom PPO and horseradish POD are determined.     

Comparison of Solubility of Corn Proteins in Propanol,Ethanol, and tert‐Butyl Alcohol Solutions on the Tortilla Process Samples

To find the best solvent of those reported and to study changes in protein aggregation during corn processing to obtain tortillas, extractability of corn proteins with three alcoholic solutions (70% ethanol, 50% propanol, and 60% tert‐butyl alcohol) was compared in corn, nixtamal, masa, and tortillas. Relative solubility was assessed through size‐exclusion chromatography, SDS‐PAGE, and insoluble polymeric protein determination using the Dumas procedure. Differences in the behavior of solvents in the samples indicate that different protein interactions are promoted during each of the processing steps. All the three alcoholic solutions can be used to study changes in corn proteins, but the best solvent was 50% propanol. Ethanol (70%) extracted the lowest amounts of corn proteins in tortilla process samples.     

Enzymatic method with polyphenol oxidase for the determination of cysteine and N-acetylcysteine

Thiols, such as cysteine and N-acetylcysteine, are included in many pharmaceutical products for their mucolytic properties. The method described here uses mushroom polyphenol oxidase (PPO) to determine two thiols and consists of measuring the lag period in the formation of the product generated as PPO acts on o-diphenol in the presence of a thiol. In the experimental conditions, o-quinone is formed enzymatically and then reacts stoichiometrically with the thiol, originating the corresponding thiol-diphenol adduct, which does not absorb visible light. Once the thiol has been used up, the o-quinone can be observed in the medium. It must be borne in mind that the inhibition of PPO is practically null at low concentrations of thiol, and the only effect observed is the formation of the thiol-diphenol adduct. In the following, an exact kinetic method capable of rapidly and accurately assaying thiols with PPO and o-diphenol is optimized and is shown to be a straightforward way of calculating thiol concentration. The method has been successfully applied to the determination of cysteine in model solutions and of N-acetylcysteine in pharmaceutical products.     

Quantitative kinetic analysis of hydrogen transfer reactions from dietary polyphenols to the DPPH radical

Diphenylpicrylhydrazyl (DPPH) is widely used for quickly assessing the ability of polyphenols to transfer labile H atoms to radicals, a likely mechanism of antioxidant protection. This popular test generally pays no attention to the kinetics of H atom transfer, which however could be even more important than the total H-atom-donating capacities (stoichiometry, EC50) typically evaluated. In the present work, a series of dietary polyphenols belonging to the most representative families (flavonols from onion, flavanol monomers and oligomers from barley, and caffeic acid and caffeoyl esters from artichoke and endive) are characterized not only by their total stoichiometries (n(tot)) but also by their rate constants of first H atom abstraction by DPPH (k(1)), deduced from the kinetic analysis of the decay of the DPPH visible band following addition of the antioxidant. The mildly reactive DPPH radical allows a good discrimation between polyphenols, as demonstrated by the relatively large ranges of k(1) (ca. 400-5000 M(-)(1) s(-)(1)) and n(tot) (ca. 1-5) values typically measured with antioxidants having a single polyphenolic nucleus. With antioxidants displaying more than one polyphenolic nucleus (procyanidin oligomers, dicaffeoyl esters), the kinetic analysis makes it possible to demonstrate significant differences in reactivity between the subunits (two distinct k(1) values whose ratio lies in the range 3-10) and nonadditive stoichiometries.     

NMR analytical approach to clarify the molecular mechanisms of the antioxidative and radical-scavenging activities of antioxidants in tea using 1,1-diphenyl-2-picrylhydrazyl

(+)-catechin, ethyl gallate, ascorbic acid, and alpha-tocopherol were reacted with 1,1-diphenyl-2-picrylhydrazyl (DPPH), and the reaction mixtures were subjected to 13C-nuclear magnetic resonance (NMR) analyses to clarify the molecular mechanisms of the antioxidative and radical-scavenging activities of each antioxidant. When ascorbic acid was reacted with DPPH, it was oxidized to dehydroascorbic acid by DPPH. When a mixture of ascorbic acid and (+)-catechin was reacted with DPPH, ascorbic acid scavenged DPPH radical faster than (+)-catechin. Ascorbic acid also scavenged DPPH radical faster than ethyl gallate and alpha-tocopherol. When (+)-catechin was reacted with DPPH, the B-ring of (+)-catechin changed to an o-quinone structure. However, it was reduced to (+)-catechin by ethyl gallate or alpha-tocopherol. alpha-Tocopherol and ethyl gallate had almost identical antioxidative activities. Therefore, the order of radical-scavenging ability (speed) suggested by our 13C NMR study was as follows: ascorbic acid > alpha-tocopherol = ethyl gallate > (+)-catechin.     

Electron spin resonance estimation of hydroxyl radical scavenging capacity for lipophilic antioxidants

Hydroxyl radical scavenging capacity estimation for lipophilic antioxidants is a challenge due to their poor solubility in aqueous radical generating and measuring systems. In this study, an electron spin resonance (ESR) method was developed and validated for its application in estimating the relative hydroxyl radical (HO*) scavenging capacity for lipophilic antioxidants under physiological pH using a Fenton Fe2+/H2O2 system for radical generation and acetonitrile as a solvent. The Fenton Fe2+/H2O2 system generates a constant flux of pure HO* under the assay conditions. The method was validated by linearity, precision, and reproducibility using selected known lipophilic antioxidants including alpha-tocopherol, lutein, beta-carotene, and BHT. The potential effects of commonly used water-miscible and water-immiscible organic solvents on the Fenton Fe2+/H2O2 HO* generating system as well as their possible interactions with the fluorescent and spectroscopic probes were also reported. In addition, the limitation of the ESR assay was described.     

Thermal degradation kinetics of bixin in an aqueous model system

The kinetics of the thermal degradation of the natural cis carotenoid bixin in a water/ethanol (8:2) solution was studied as a function of temperature (70-125 degrees C), using high-performance liquid chromatography. The curves for the decay of bixin and formation of products (e.g., di-cis and all-trans isomers and a C17 degradation compound) did not adjust well to a first-order rate law, but very good fits were obtained using a biexponential model. This mathematical modeling gave the rate constant values for the formation of the primary products from bixin, and the energy barrier for each step was obtained. The di-cis isomers were formed immediately (15 kcal/mol) together with the decay of bixin, followed by a slow consumption, indicating their role as reaction intermediates. In fact, the di-cis isomers could easily revert to bixin (Ea approximately 3 kcal/mol) or yield the primary C17 degradation product, with an energy barrier of 6.5 kcal/mol. In turn, 24 kcal/mol was necessary for the Bix --> all-trans step, explaining its slower formation.     

Convenient synthesis of hydroxytyrosol and its lipophilic derivatives from tyrosol or homovanillyl alcohol

Hydroxytyrosol, a naturally occurred o-phenolic compound exhibiting antioxidant properties, was synthesized by a three-step high-yielding procedure from natural and low-cost compounds such as tyrosol or homovanillyl alcohol. First, the efficient chemoselective protection of the alcoholic group of these compounds was performed by using dimethyl carbonate (DMC) as reagent/solvent; second, the oxidation with 2-iodoxybenzoic acid (IBX) or Dess-Martin periodinane reagent (DMP) and in situ reduction with sodium dithionite (Na2S2O4) allowed the preparation of carboxymethylated hydroxytyrosol; finally, by a mild hydrolytic step, hydroxytyrosol was obtained in high yield and purity, as confirmed by NMR spectra and HPLC profile. By using a similar methodology, lipophilic hydroxytyrosol derivatives, utilized as additives in pharmaceutical, food, and cosmetic preparations, were prepared. In fact, at first the chemoselective protection of the alcoholic group of tyrosol and homovanillyl alcohol was performed by using acyl chlorides without any catalyst to obtain the corresponding lipophilic derivatives, and then these compounds were converted in good yield and high purity into the hydroxytyrosol derivatives by oxidative/reductive pathway with IBX or DMP and Na2S2O4.     

COMPUTED EFFECTS OF ADSORPTION KINETICS ON PESTICIDE MOVEMENT IN SOILS

For a model at high water fluxes, the inclusion of first-order adsorption-desorption rate constants leads to slower leaching of pesticide and increased spread than predicted using instantaneous equilibrium. The initial condition of the substance near the soil surface is also found to be important. A model with simultaneous fast and slow adsorption-desorption equilibria describes and explains published experimental data comparatively well. Under a pattern of intermittent rainfall, the effect of adsorption-desorption kinetics on pesticide movement is comparatively small.     

石榴汁花色苷热稳定性及其降解动力学研究

为了对石榴汁花色苷热降解的动力学进行了了解,测定了不同温度对石榴汁花色苷含量、色差的影响.结果表明,石榴汁花色苷对热不稳定,其色品指数a*.(Hunter a*)值随加热时间和温度升高呈下降趋势,而色品指数b*(Hunterb*)值呈上升趋势:石榴汁花色苷降解符合动力学一级反应,其反应活化能E0为52.67 kJ/mol,反应常数K0为6.37×106,得出了石榴汁花色苷降解的预测模型.经验证,模型与实测值拟合较好,表明该模型是合理的.