Effects of alcoholic solvents on antiradical abilities of protocatechuic acid and its alkyl esters

DPPH radical scavenging reactions of protocatechuic acid and its methyl ester were investigated in various solvents. In alcoholic solvents, methyl protocatechuate rapidly scavenged more than four equivalents of the radical, whereas approximately two equivalents were consumed in aprotic solvents. Methyl, ethyl, butyl, isopropyl, and tert-butyl protocatechuates were examined for their DPPH radical scavenging abilities in methanol or ethanol. As a result, the radical scavenging equivalence of sterically bulky esters tended to decrease compared to that of methyl or ethyl ester. The ABTS radical scavenging ability of those esters in water also showed the same tendency. Since 2-methoxy derivatives were detected in the reaction mixture of methyl protocatechuate and DPPH radical in methanol, a nucleophilic attack of an alcoholic molecule on the o-quinone intermediate, which is sensitive to steric hindrance from alkyl groups of both esters and alcoholic solvents, must be crucial for total radical scavenging abilities.     

Quinone hemiacetal formation from protocatechuic acid during the DPPH radical scavenging reaction

Protocatechuic acid was rapidly converted to protocatechuquinone 3-methyl hemiacetal and protocatechuquinone during the reaction with DPPH radical in methanol. The structure of the acetal was determined by comparing the NMR data with those of an authentic compound prepared by (diacetoxy)iodobenzene oxidation of protocatechuic acid.     

Radical scavenging activity of dicaffeoyloxycyclohexanes: contribution of an intramolecular interaction of two caffeoyl residues

Six regio- and stereoisomers of dicaffeoyloxycyclohexanes and 2,4-di-O-caffeoyl-1,6-anhydro-beta-D-glucose were synthesized as model compounds of dicaffeoylquinic acids, and their radical scavenging activity was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt) radical scavenging tests. Both DPPH and ABTS radical scavenging reactions of these compounds consisted of two different steps. In the first step, catechol moieties of the caffeoyl residues were rapidly converted to o-quinone structures and no significant difference in the reactivity was observed among the tested compounds. In the second step, however, the rate of the reaction increased as the intramolecular distance of the two caffeoyl residues decreased. A novel intramolecular coupling product, which could scavenge additional radicals, was isolated from the reaction mixture of trans-1,2-dicaffeoyloxycyclohexane and DPPH radical. The result suggests that the second step of the radical scavenging reaction is arising from an intramolecular interaction between the two caffeoquinone residues to regenerate catechol structures, and that the closer their distance is, the more rapidly they react. The radical scavenging activity of natural dicaffeoylquinic acids in a biological aqueous system might also depend on the positions of caffeoyl ester groups.     

Multiple pathways of the reaction of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH·) with (+)-catechin: evidence for the formation of a covalent adduct between DPPH· and the oxidized form of the polyphenol

The pathways of the reaction of 2,2-diphenyl picrylhydrazyl radicals (DPPH·) with (+)-catechin were studied in alcoholic solvents. The reaction mixtures were analysed by using reversed-phase liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS). The intermediate o-quinone of catechin, yellow dimers, trimers and, interestingly, an adduct of the oxidized form of catechin with DPPH radicals were identified. The mass of this adduct was 681 Da, suggesting that one molecule of the DPPH radical complexes with the oxidized form of catechin. It is concluded that once the intermediate o-quinone is formed, the reaction proceeds in two pathways, either the o-quinone reacts with catechin to form a hydrophilic dimer (type B), which is further oxidized to hydrophobic dimers (type A) and consequently to oligomers of higher molecular weights; or the A-ring of the o-quinone is further oxidized by a DPPH radical and that this oxidized intermediate then reacts with another DPPH radical to form the observed adduct. The identification of the latter mechanism could explain the contradictory results reported in the literature for the reaction of polyphenols with DPPH radicals.     

Multiple pathways of the reaction of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH and the oxidized form of the polyphenol

The pathways of the reaction of 2,2-diphenyl picrylhydrazyl radicals (DPPH) with (+)-catechin were studied in alcoholic solvents. The reaction mixtures were analysed by using reversed-phase liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS). The intermediate o-quinone of catechin, yellow dimers, trimers and, interestingly, an adduct of the oxidized form of catechin with DPPH radicals were identified. The mass of this adduct was 681 Da, suggesting that one molecule of the DPPH radical complexes with the oxidized form of catechin. It is concluded that once the intermediate o-quinone is formed, the reaction proceeds in two pathways, either the o-quinone reacts with catechin to form a hydrophilic dimer (type B), which is further oxidized to hydrophobic dimers (type A) and consequently to oligomers of higher molecular weights; or the A-ring of the o-quinone is further oxidized by a DPPH radical and that this oxidized intermediate then reacts with another DPPH radical to form the observed adduct. The identification of the latter mechanism could explain the contradictory results reported in the literature for the reaction of polyphenols with DPPH radicals.     

A novel oxidative dimer from protocatechuic esters: contribution to the total radical scavenging ability of protocatechuic esters

A novel oxidative dimer was isolated as a major product from a reaction mixture of methyl protocatechuate and DPPH radical in methanol. Its unusual benzobicyclo[3.2.1]octane structure was elucidated by extensive spectral analysis. This result suggests that the regeneration of catechol structures by the nucleophilic addition of an alcohol molecule on o-quinones and subsequent dimerization is one of the key reactions in the high radical-scavenging activity of protocatechuic esters in an alcoholic solvent.     

Oxidative dimer produced from a 2,3,4-trihydroxybenzoic ester

The DPPH radical-scavenging abilities of the naturally occurring phenolic acid, 2,3,4-trihydroxybenzoic acid, and its methyl ester were evaluated. Both compounds in acetonitrile scavenged as many as four radicals compared to three or fewer radical consumption in acetone or ethanol. Only the ester showed relatively high ability in methanol. Oxidation with o-chloranil in acetonitrile resulted in methyl 2,3,4-trihydroxybenzoate giving a novel benzocoumarin-type dimer, its chemical structure being confirmed by spectroscopic evidence. The formation of this dimer might partly account for the higher radical-scavenging efficiency of the ester in acetonitrile or methanol.     

2-O-alpha-D-glucopyranosyl-L-ascorbic acid scavenges 1,1-diphenyl-2-picrylhydrazyl radicals via a covalent adduct formation

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging mechanism of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) was studied. We found two undefined products, named X and Y, in the reaction mixture of AA-2G and the DPPH radical under acidic conditions by HPLC analysis. The reaction mixture was further subjected to LC-MS analysis. X was found to be a covalent adduct of AA-2G and the DPPH radical. On the other hand, Y could not be identified, probably because it was a mixture. A time-course study of the radical-scavenging reaction revealed that one molecule of AA-2G scavenged one molecule of DPPH radical to generate an AA-2G radical, which readily reacted with another molecule of the DPPH radical to form a covalent adduct (X). Subsequently, this adduct slowly quenched a third molecule of the DPPH radical, resulting in reaction products (Y). Therefore, one molecule of AA-2G has only one oxidizable -OH group, but can scavenge three molecules of the DPPH radical. The radical-scavenging mechanism of AA-2G elucidated in this study should be useful in understanding the biological roles of AA-2G per se in the food and cosmetic fields.     

Reaction of methylated urates with 1,1-diphenyl-2-picrylhydrazyl

The kinetics of the reaction between the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and methylated urates was studied. Urates that had methyl groups on the 1,3,9, or on the 1 and 3 or 1 and 9 nitrogens reacted with DPPH 15 to 77% faster than uric acid. Urates substituted with methyl groups on the 7 nitrogen or on both the 3 and 9 nitrogens reacted with DPPH at rates that were less than 0.1 that of uric acid. 3,7,9-Trimethyluric acid and 1,3,7,9-tetramethyluric acid reacted with DPPH at barely detectable rates. DPPH reacted with uric acid, the monomethylated urates, and some of the dimethylated urates in a ratio of 2:1. DPPH reacted with other dimethylated and trimethylated urates in a ratio of 1:1. Semiempirical MNDO calculations indicate that the most stable radical of uric acid is formed by hydrogen abstraction from the 3, 7 or 9 position. The most stable species resulting from loss of a second hydrogen lack hydrogens at the 3 and 7 positions or the 7 and 9 positions. For maximum reactivity with DPPH, methylated uric acid derivatives must have a hydrogen at nitrogen 7 and one of the hydrogens at either the 3 or 9 position.     

Oxidation of hydroxycinnamic acid and hydroxycinnamyl alcohol derivatives by laccase and peroxidase. Interactions among p-hydroxyphenyl,guaiacyl and syringyl groups during the oxidation reactions

Fungal laccase oxidized derivatives of hydroxycinnamic acid. The rates decreased in the order sinapic acid > ferulic acid ≥p-coumaric acid. The laccase oxidized sinapyl alcohol faster than coniferyl alcohol. The rates of oxidation of the hydroxycinnamic acid derivatives by an isoenzyme of peroxidase from horseradish decreased in the order p-coumaric acid > ferulic acid ≥ sinapic acid. The peroxidase oxidized coniferyl alcohol much faster than sinapyl alcohol. The laccase and the peroxidase predominantly oxidized (a) ferulic acid in a reaction mixture that contained p-coumaric acid and ferulic acid, (b) sinapic acid in a mixture of p-coumaric acid plus sinapic acid, and (c) sinapic acid in a mixture of ferulic acid plus sinapic acid. In a reaction mixture that contained both coniferyl and sinapyl alcohols, both fungal laccase and horseradish peroxidase predominantly oxidized sinapyl alcohol. From these results, it is concluded (1) that the p-hydroxyphenyl radical can oxidize guaiacyl and syringyl groups and produce their radicals and (2) that the guaiacyl radical can oxidize the syringyl group under formation of its radical; and that (3) in both cases the reverse reactions are very slow.     

Lipid radicals: properties and detection by spin trapping

Unsaturated lipids are rapidly oxidized to toxic products such as lipid hydroperoxides, especially when transition metals such as iron or copper are present. In a Fenton-type reaction Fe2+ converts lipid hydroperoxides to the very short-lived lipid alkoxyl radicals. The reaction was started upon the addition of Fe2+ to an aqueous linoleic acid hydroperoxide (LOOH) emulsion and the spin trap in the absence of oxygen. Even when high concentrations of spin traps were added to the incubation mixture, only secondary radical adducts were detected, probably due to the rapid re-arrangement of the primary alkoxyl radicals. With the commercially available nitroso spin trap MNP we observed a slightly immobilized ESR spectrum with only one hydrogen splitting, indicating the trapping of a methinyl fragment of a lipid radical. With DMPO or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) adducts were detected with carbon-centered lipid radical, with acyl radical, and with the hydroxyl radical. We also synthesized lipophilic derivatives of the spin trap DEPMPO in order to detect lipid radical species generated in the lipid phase. With all spin traps studied a lipid-derived carbon-centered radical was obtained in the anaerobic incubation system Fe2+/LOOH indicating the trapping of a lipid radical, possibly generated as a secondary reaction product of the primary lipid alkoxyl radical formed. Under aerobic conditions an SOD-insensitive oxygen-centered radical adduct was formed with DEPMPO and its lipophilic derivatives. The observed ESR parameters were similar to those of alkoxyl radical adducts, which were independently synthesized in model experiments using Fe3+-catalyzed nucleophilic addition of methanol or t-butanol to the respective spin trap.     

2-O-α-D-Glucopyranosyl-L-ascorbic Acid Scavenges 1,1-Diphenyl-2-picrylhydrazyl Radicals via a Covalent Adduct Formation

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging mechanism of 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) was studied. We found two undefined products, named X and Y, in the reaction mixture of AA-2G and the DPPH radical under acidic conditions by HPLC analysis. The reaction mixture was further subjected to LC–MS analysis. X was found to be a covalent adduct of AA-2G and the DPPH radical. On the other hand, Y could not be identified, probably because it was a mixture. A time-course study of the radical-scavenging reaction revealed that one molecule of AA-2G scavenged one molecule of DPPH radical to generate an AA-2G radical, which readily reacted with another molecule of the DPPH radical to form a covalent adduct (X). Subsequently, this adduct slowly quenched a third molecule of the DPPH radical, resulting in reaction products (Y). Therefore, one molecule of AA-2G has only one oxidizable –OH group, but can scavenge three molecules of the DPPH radical. The radical-scavenging mechanism of AA-2G elucidated in this study should be useful in understanding the biological roles of AA-2G per se in the food and cosmetic fields.     

Fatty acids,part 21: ring opening reactions of synthetic and natural furanoid fatty esters

Methyl 2,5-disubstituted C₁₈ furanoid fatty ester (viz. methyl 9,12-epoxyoctadeca-9,11-dienoate) was readily converted to methyl 9,12-dioxostearate using mineral or maleic acid. Conversion of the naturally occurring 2,3,5-trisubstituted furanoid fatty ester (viz. methyl 10,13-epoxy-11-methyloctadeca-10,12-dienoate) to the corresponding methyl 10,13-dioxo-11-methylstearate was much slower in rate under similar reaction conditions. The case of separating the dioxo derivatives from a mixture of other common fatty esters was demonstrated and the cyclodehydration of the isolated dioxo derivatives to the parent furanoid ester was rapidly achieved using dilute BF₃-methanol complex.     

Transesterification of canola oil in mixed methanol/ethanol system and use of esters as lubricity additive

Transesterification of canola oil was carried out with methanol, ethanol, and various mixtures of methanol/ethanol, keeping the molar ratio of oil to alcohol 1:6 and using KOH as a catalyst. Mixtures of alcohol increased the rate of transesterification reaction and produced methyl as well as ethyl esters. The increased rate was result of better solubility of oil in reaction mixture due to better solvent properties of ethanol than methanol and equilibrium due to methanol. With 3:3 molar ratio of methanol to ethanol {MEE (3:3)} the amount of ethyl ester formed was 50% that of methyl ester. Properties (acid value, viscosity, density) of all esters including mixed esters were within the limits of ASTM standards. Lubricities of these esters are in the order: ethyl ester>methyl ethyl ester>methyl ester.     

Conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel by immobilized Candida antarctica lipase

Acid oil, which is a by-product in vegetable oil refining, mainly contains free fatty acids (FFAs) and acylglycerols, and is a candidate of materials for production of biodiesel fuel. A mixture (acid oil model) of refined FFAs and vegetable oil was recently reported to be converted to fatty acid methyl esters (FAMEs) at >98% conversion by a two-step reaction system comprising methyl esterification of FFAs and methanolysis of acylglycerols using immobilized Candida antarctica lipase. The two-step system was thus applied to conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel. Under similar conditions that were determined by using acid oil model, however, the lipase was unstable and was not durable for repeated use. The inactivation of the lipase was successfully avoided by addition of excess amounts of methanol (MeOH) in the first-step reaction, and by addition of vegetable oil and glycerol in the second-step reaction. Hence, the first-step reaction was conducted by shaking a mixture of 66 wt% acid oil (77.9 wt% FFAs, 10.8 wt% acylglycerols) and 34 wt% MeOH with 1 wt% immobilized lipase, to convert FFAs to their methyl esters. The second-step reaction was performed by shaking a mixture of 52.3 wt% dehydrated first-step product (79.7 wt% FAMEs, 9.7 wt% acylglycerols), 42.2 wt% rapeseed oil, and 5.5 wt% MeOH using 6 wt% immobilized lipase in the presence of additional 10 wt% glycerol, to convert acylglycerols to FAMEs. The resulting product was composed of 91.1 wt% FAMEs, 0.6 wt% FFAs, 0.8 wt% triacylglycerols, 2.3 wt% diacylglycerols, and 5.2 wt% other compounds. Even though each step of reaction was repeated every 24 h by transferring the immobilized lipase to the fresh substrate mixture, the composition was maintained for >100 cycles.     

Studies on the 1,1-diphenyl-2-picrylhydrazyl radical scavenging mechanism for a 2-pyrone compound

The radical scavenging mechanisms for the 2-pyrone compound, 4-hydroxy-3,6-dimethyl-2H-pyrane-2-one (1), and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (4) in several solvent systems were evaluated by the quantitative change in compounds detected at 270 nm and subsequent HPLC analyses. The HPLC profile for each condition suggested that the reaction proceeded by a different mechanism in each solvent system. In organic solvents (CHCl3, iso-propanol, and EtOH), 1-[4-(3,4-dihydro-3,6-dimethyl-2,4-dioxo-2H-pyran-3-yl) phenyl]-1-phenyl-2-picrylhydrazine (2) was produced as an adduct of the DPPH radical and 1. On the other hand, the reaction in a buffer solution (an acetate buffer at pH 5.5) gave several degradation products with 1[4-(2,3-dihydro-2,5-dimethyl-3-oxo-fur-2-yl) phenyl]-1-phenyl-2-picrylhydrazine (5), this being structurally elucidated by spectroscopic analyses. The decrease of the DPPH radical in each reaction system suggests that compound 1 could scavenge about 1.5-1.8 equivalents of the radical in organic solvents and about 3.5-3.9 in the buffer solution.     

Predicting enzyme behavior in nonconventional media: correlating nitrilase function with solvent properties

The insolubility of nitrile substrates in aqueous reaction mixture decreases the enzymatic reaction rate. We studied the interaction of fourteen water miscible organic solvents with immobilized nitrile hydrolyzing biocatalyst. Correlation of nitrilase function with physico-chemical properties of the solvents has allowed us to predict the enzyme behavior in such non-conventional media. Addition of organic solvent up to a critical concentration leads to an enhancement in reaction rate, however, any further increase beyond the critical concentration in the latter leads to the decrease in catalytic efficiency of the enzyme, probably due to protein denaturation. The solvent dielectric constant (epsilon) showed a linear correlation with the critical concentration of the solvent used and the extent of nitrile hydrolysis. Unlike alcohols, the reaction rate in case of aprotic solvents could be linearly correlated to solvent log P. Further, kinetic analysis confirmed that the affinity of the enzyme for its substrate (K (m)) was highly dependent upon the aprotic solvent used. Finally, the prospect of solvent engineering also permitted the control of enzyme enantioselectivity by regulating enantiomer traffic at the active site.     

Production of methanol from aromatic acids by Pseudomonas putida.

When grown at the expense of 3,4,5-trimethoxybenzoic acid, a strain of Pseudomonas putida oxidized this compound and also 3,5-dimethoxy-4-hydroxybenzoic (syringic) and 3,4-dihydroxy-5-methoxybenzoic (3-O-methylgallic) acids; but other hydroxy- or methoxy-benzoic acids were oxidized slowly or not at all. Radioactivity appeared exclusively in carbon dioxide when cells were incubated with [4-methoxyl-14C]trimethoxybenzoic acid, but was found mainly in methanol when[methoxyl-14C]3-O-methylgallic acid was metabolized. The identity of methanol was proved by analyzing the product from [methoxyl-13C]3-O-methylgallic acid by nuclear magnetic resonance spectroscopy and by isolating the labeled 3,5-dinitrobenzoic acid methyl ester, which was examined by mass spectrometry. These results, together with measurements of oxygen consumed in demethylations catalyzed by cell extracts, showed that two methoxyl groups of 3,4,5-trimethoxybenzoate and one of syringate were oxidized to give carbon dioxide and 3-O-methylgallate. This was then metabolized to pyruvate; the other product was presumed to be the 4-methyl ester of oxalacetic acid, for which cell extracts contained an inducible, specific esterase. P. putida did not metabolize the methanol released from this compound by hydrolysis. Support for the proposed reaction sequence was obtained by isolating mutants which, although able to convert 3,4,5-trimethoxybenzoic acid into 3-O-methylgallic acid, were unable to use either compound for growth.     

Steady-state kinetics of combined oxidation of hydroquinone and o-dianisidine by hydrogen peroxide in the presence of horseradish peroxidase

The steady-state kinetics of horseradish peroxidase-catalyzed oxidation of hydroquinone was studied. Hydroquinone was shown to be a rapidly oxidizable substrate of the peroxidase. Values of kcat and Km for this substrate were determined in the pH range 4-7. The oxidation of hydroquinone and o-dianisidine was distinguished when both were present in the reaction mixture. o-Dianisidine was not oxidized until hydroquinone was completely converted. The rate of hydroquinone oxidation by peroxidase in the presence of o-dianisidine was 3-10 times higher than the rate of its individual oxidation. The activator decreased the Km for hydroquinone oxidation.