O-diphenolic Compounds Research Articles

Unraveling Substrate Specificity and Catalytic Promiscuity of Aspergillus oryzae Catechol Oxidase.

Catechol oxidases and tyrosinases are coupled binuclear copper enzymes that oxidize various o-diphenolic compounds to corresponding o-quinones. Tyrosinases have an additional monooxygenation ability to hydroxylate monophenol to o-diphenol. It is still not clear what causes the difference in the catalytic activities. We solved a complex structure of Aspergillus oryzae catechol oxidase with resorcinol bound into the active site. Catalytic activity of A. oryzae catechol oxidase was studied, for the first time, by high-resolution FT-ICR mass spectrometry to shed light on the reaction mechanism. The enzyme was also found to catalyze monooxygenation of small phenolics, which provides a novel perspective for the discussion of differences in the catalytic activity between tyrosinases and catechol oxidases. According to the results, two binding modes for resorcinol are suggested and a reaction mechanism for coupled binuclear copper enzymes is discussed.

Melatonin in Apples and Juice: Inhibition of Browning and Microorganism Growth in Apple Juice.

Synthetic melatonin (N-acetyl-5-methoxytryptamine, MT) is popular in the US and Asian markets as a health supplement. Here, we identified a naturally occurring melatonin source in apple juice. Melatonin was present in all 18 apple cultivars tested. The highest melatonin level of the edible part of apple was detected in the apple peel. The melatonin content in ‘Fuji’ apple juice is comparable to the level of its flesh. Melatonin was consumed during the process of juicing due to its interaction with the oxidants. Melatonin addition significantly reduced the juice color change to brown (browning). The mechanism is that melatonin scavenges the free radicals, which was indicated by the ASBT analysis; therefore, inhibiting the conversion of o-diphenolic compounds into quinones. Most importantly, melatonin exhibited powerful anti-microorganism activity in juice. The exact mechanisms of this action are currently unknown. These effects of melatonin can preserve the quality and prolong the shelf life of apple juice. The results provide valuable information regarding commerciall apple juice processing and storage.

In-vitro assay and inactivation kinetics of polyphenol oxidase of some Nigerian banana and plantain cultivars

Activity of polyphenol oxidase (PPO) from the pulp of three banana cultivars grown in Nigeria, namely: plantain, light green-skinned banana and red-skinned banana at unripe and ripe states were investigated. The efficiency of extraction of crude PPO enzyme and in-vitro assay of its activity were determined. Polyethylene glycol detergent gave the highest extraction efficiency. Enzyme activity and rate of browning were found to be highest in plantain and least in light green-skinned banana. Enzymes from all the cultivars were specific towards O-diphenolic compounds and not to monophenolics. The optima pH of PPO of the cultivars tends towards neutrality (6.6-7.0). Enzyme activity was destroyed between 2 and 4 min at 80°C. Thermal inactivation kinetic parameters revealed plantain's PPO to be the most thermal stable. Sodium metabisulphite was the most effective chemical inhibitor of the PPO. The electrophoretic patterns of banana PPO showed heterogeneity with greater number of bands in ripe.

In-vitro assay and inactivation kinetics of polyphenol oxidase of some Nigerian banana and plantain cultivars

Activity of polyphenol oxidase (PPO) from the pulp of three banana cultivars grown in Nigeria, namely: plantain, light green-skinned banana and red-skinned banana at unripe and ripe states were investigated. The efficiency of extraction of crude PPO enzyme and in-vitro assay of its activity were determined. Polyethylene glycol detergent gave the highest extraction efficiency. Enzyme activity and rate of browning were found to be highest in plantain and least in light green-skinned banana. Enzymes from all the cultivars were specific towards O-diphenolic compounds and not to monophenolics. The optima pH of PPO of the cultivars tends towards neutrality (6.6-7.0). Enzyme activity was destroyed between 2 and 4 min at 80°C. Thermal inactivation kinetic parameters revealed plantain's PPO to be the most thermal stable. Sodium metabisulphite was the most effective chemical inhibitor of the PPO. The electrophoretic patterns of banana PPO showed heterogeneity with greater number of bands in ripe.

Composition of pigments and colour changes in green table olives related to processing type

Brownish colourations in Natural green table olives (non-treated with alkali) make this product less attractive to consumers than Spanish-style green table olives (treated with alkali), which develop a more appreciated bright golden-yellow colour. These colour differences were studied in relation to changes in the composition of chlorophyll and carotenoid pigments, as well as polyphenolic compounds and polyphenol oxidase enzyme (PPO) activity. Natural green olives showed a different chlorophyll profile than Spanish-style. However, all the chlorophyll pigments formed in both processing types were Mg-free derivatives (mostly pheophytins) with similar colourations, ranging from grey to green brownish. In the carotenoid fraction no appreciable differences were found between both processing types. The fruit’s brownish colour was mainly due to polymeric substances with a size of >1000 daltons and polyphenolic nature, resulting from an enzymatic oxidation by PPO of the o-diphenolic compounds present in the fresh fruits.

Detection of an O-methyltransferase synthesising acetosyringone in methyl jasmonate-treated tobacco cell-suspensions cultures

Acetosyringone (3′,5′-dimethoxy-4′-hydroxyacetophenone) is a well-known and very effective inducer of the virulence genes of Agrobacterium tumefaciens but the precise pathway of its biosynthesis in plants is still unknown. We have used two tobacco cell lines, cultured in suspension and exhibiting different patterns of accumulation of acetosyringone in their culture medium upon treatment with methyl jasmonate, to study different steps of acetosyringone biosynthesis. In the two cell lines studied, treatment with 100μM methyl jasmonate triggered a rapid and transient increase in acetovanillone synthase activity followed by a progressive increase in S-adenosyl-L-methionine: 5-hydroxyacetovanillone 5-O-methyltransferase activity which paralleled the rise in acetosyringone concentration in the culture medium. This O-methyltransferase displayed Michaelis–Menten kinetics with an apparent Km value of 18μM for 5-hydroxyacetovanillone and its activity was magnesium-independent. Its molecular mass was estimated by gel permeation on an FPLC column and was found to be of ca. 81kDa. 5-Hydroxyacetovanillone was the best substrate among the different o-diphenolic compounds tested as methyl acceptors in the O-methyltransferase assay. No formation of 5-hydroxyacetovanillone could be detected in vitro from 5-hydroxyferuloyl-CoA and NAD in the extracts used to measure acetovanillone synthase activity, indicating that 5-hydroxyacetovanillone is probably formed by direct hydroxylation of acetovanillone rather than by β-oxidation of 5-hydroxyferulic acid. Taken together our results strongly support the hypothesis that acetosyringone biosynthesis in tobacco proceeds from feruloyl-CoA via acetovanillone and 5-hydroxyacetovanillone.

A simple method for the determination of bioactive antioxidants in virgin olive oils

The importance of olive polyphenols as bioactive compounds has grown in recent years as a result of intensive research on their anticancer, antiatherosclerotic and antihypertensive activities. However, there is currently no official method for determining the content of polyphenols in olive oils because of the technical difficulties in their determination. Here a simple method for the analysis of extra virgin olive oil o-diphenols by visible spectrometry is proposed and compared with the traditional method of solid phase extraction followed by colorimetric determination using sodium molybdate or Folin-Ciocalteu reagent or by high-performance liquid chromatography (HPLC) analysis using UV detection. This new approach to determining total o-diphenolic compounds exploits the oxidation of o-diphenols to quinones in a basic medium. Preliminary results showed a better correlation between the total o-diphenol determination by HPLC and by the proposed method (R(2) = 0.9229) than between the total o-diphenol determination by HPLC and by the molybdate colorimetric method (R(2) = 0.8689). A good correlation was also observed between the total phenolic content determined by HPLC and by the proposed method (R(2) = 0.8196), but this correlation was a little lower than the one obtained between the HPLC method and the Folin-Ciocalteu method (R(2) = 0.8752). The proposed method involves very little sample manipulation, requires inexpensive reagents and can be performed in less than 40 min for several samples at the same time, using olive oil samples of only 1-2 g.

The effect of fly attack (<i>Bactrocera oleae</i>) on the quality and phenolic content of <i>Chemlal</i> olive oil

The attack on olives by the pest Bactrocera oleae has been studied to determine its influence on the olive oil quality (free acidity, peroxide value, UV extinction, sensorial quality), the total polyphenol and the individual phenolic compounds. The Algerian chemlal olive variety was used in this work. Quantitative and qualitative analyses of phenolic compounds were performed using the colorimetric method and the GC coupled with mass spectrometry. The results demonstrated that the attack modified the quality of olive oil and confirmed the existence of hydrolytic and mostly oxidative processess which strongly reduced the amount of O-diphenolic compounds. The extent of modification was much greater when the olives were attacked and harvested at an advanced stage of maturity. Early harvesting could be an effective way to limit the damage caused by B. oleae and to improve the quality of virgin olive oil.

Production of o-diphenols by immobilized mushroom tyrosinase

The o-diphenols 4- tert-butyl-catechol, 4-methyl-catechol, 4-methoxy-catechol, 3,4-dihydroxyphenylpropionic acid and 3,4-dihydroxyphenylacetic acid were produced from the corresponding monophenols (4- tert-butyl-phenol, 4-methyl-phenol, 4-methoxy-phenol, p-hydroxyphenylpropionic acid and p-hydroxyphenylacetic acid) using immobilized mushroom tyrosinase from Agaricus bisporus. In all cases the yield was R diphenol ≥ 88–96%, which, according to the literature, is the highest yield so far, obtained using tyrosinase. The reaction was carried out in 0.5 M borate buffer pH 9.0 which was used to minimize the diphenolase activity of tyrosinase by complexing the o-diphenols generated. Hydroxylamine and ascorbic acid were also present in the reaction medium, the former being used to reduce mettyrosinase to deoxytyrosinase, closing the catalytic cycle, and the latter to reduce the o-quinone produced to o-diphenol. Inactivation of the tyrosinase by ascorbic acid was also minimized due to the formation of an ascorbic acid–borate complex. Concentrations of the o-diphenolic compounds obtained at several reaction times were determined by gas chromatography–mass spectrometry (GC–MS) and UV–vis spectroscopy. The experimental results are discussed.

Catalytic wet peroxide photo-oxidation of phenolic olive oil mill wastewater contaminants: Part II. Degradation and detoxification of low-molecular mass phenolic compounds in model and real effluent

Wet hydrogen peroxide photodegradation, catalyzed by aluminium–iron pillared montmorillonite (Al–Fe)PILC, of a mixture of eight model phenolic compounds present in olive mill wastewater (OMW), has been studied. Important percentages of phenol abatement after the wet hydrogen peroxide photocatalytic oxidation (WHPCO) (86% and 70%) have been achieved after 24h of the o-diphenolic compounds caffeic acid and hydroxytyrosol, respectively. Monophenolic compounds tyrosol, p-hydroxyphenylacetic acid and p-coumaric acid were the most resistant towards the WHPCO. The apparent rate constant kphenolicmolecule decreased with increasing initial pollutant concentration of organic molecule when the other parameters remained unchanged. Almost similar results were obtained with real OMW obtained by ultrafiltration of crude OMW. The toxicity of model and real OMW against the bioluminescent bacteria Vibrio fisheri was significantly decreased by 74% and 68%, respectively after the WHPCO. The subsequent biological treatment using a methanogenic consortium removed the remaining phenolic compounds by more than 70% even with the most recalcitrant compounds.

Antioxidant effect of phenolic compounds, alpha-tocopherol, and other minor components in virgin olive oil.

The effect of acidity, squalene, hydroxytyrosol, aldehydic form of oleuropein aglycon, hydroxytyrosyl acetate, tyrosol, homovanillic acid, luteolin, apigenin, alpha-tocopherol, and the mixtures hydroxytyrosol/hydroxytyrosyl acetate, hydroxytyrosol/tyrosol, and hydroxytyrosol/alpha-tocopherol on the oxidative stability of an olive oil matrix was evaluated. A purified olive oil was spiked with several concentrations of these compounds and, then, subjected to an accelerated oxidation in a Rancimat apparatus at 100 degrees C. Acidity, squalene, homovanillic acid, and apigenin showed negligible effect. At the same millimolar concentrations, the different o-diphenolic compounds yielded similar and significant increases of the induction time, alpha-tocopherol a lesser increase, and tyrosol a scarce one. At low concentrations of o-diphenols and alpha-tocopherol, a linear relationship between induction time and concentration was found, but at high concentrations the induction time tended toward constant values. To explain this behavior, a kinetic model was applied. The effect of the mixtures hydroxytyrosol/hydroxytyrosyl acetate was similar to that of a single o-diphenol at millimolar concentration equal to the sum of millimolar concentrations of both compounds. Concentrations of tyrosol >0.3 mmol/kg increase the induction time by 3 h. The mixtures hydroxytyrosol/alpha-tocopherol showed opposite effects depending on the relative concentrations of both antioxidants; so, at hydroxytyrosol concentrations <0.2 mmol/kg, the addition of alpha-tocopherol increased the induction time, whereas at higher hydroxytyrosol concentrations, the alpha-tocopherol diminished the stability.

Determination of phenols, flavones, and lignans in virgin olive oils by solid-phase extraction and high-performance liquid chromatography with diode array ultraviolet detection.

A simple analytical method for the quantitative determination of phenols, flavones, and lignans in virgin olive oils was developed. The polar fraction was isolated from small amounts of oil sample (2.5 g) by solid-phase extraction (SPE) using diol-phase cartridges, and the extract was analyzed by reversed-phase HPLC coupled with diode array UV detection. Chromatographic separation of pinoresinol, cinnamic acid, and 1-acetoxypinoresinol was achieved. Repeatability (RSD < 6.5%), recovery (> 90%), and response factors for each identified component were determined. SPE on amino-phase cartridges was used for isolating acidic phenols and as an aid for phenol identification. For the first time, 2-(4-hydroxyphenyl)ethyl acetate was detected in olive oils. The aldehydic structure of the ligstroside aglycon was confirmed by NMR spectroscopy. The colorimetric determination of total o-diphenolic compounds by reaction with molybdate was consistent with their HPLC determination. Differences between results obtained by liquid-liquid extraction and SPE were not statistically significant.

Inhibition of peroxynitrite dependent DNA base modification and tyrosine nitration by the extra virgin olive oil-derived antioxidant hydroxytyrosol

Hydroxytyrosol is one of the o-diphenolic compounds in extra virgin olive oil and has been suggested to be a potent antioxidant. The superoxide radical (O 2 •−) and nitric oxide (NO ) can react very rapidly to form peroxynitrite (ONOO −), a reactive tissue damaging species thought to be involved in the pathology of several chronic diseases. Hydroxytyrosol was highly protective against the peroxynitrite-dependent nitration of tyrosine and DNA damage by peroxynitrite in vitro. Given that extra virgin olive oil is consumed daily by many humans, hydroxytyrosol derived from this diet could conceivably provide a defense against damage by oxidants in vivo. The biological activity of hydroxytyrosol in vivo will depend on its intake, uptake and access to cellular compartments.

Effect of pH on the oxidation pathway of dopamine and dopa

The formation of 6-hydroxylated o-diphenolic compounds and other intermediates during the oxidation of dopamine, dopa and 3[3,4-dihydroxyphenyl]propionic acid by NaIO 4 or mushroom tyrosinase was studied using high pressure liquid chromatography with electrochemical detection. The results suggest the following: (1) acidic conditions are necessary to achieve accumulation of both dopaquinone and dopaminequinone during dopa and dopamine oxidation by NaIO 4 and also seem essential in the o-quinone hydroxylation pathway; (2) accumulation of o-quinones probably results in the formation of semiquinone owing to the interaction between o-quinone species and o-diphenolic precursors; (3) quinone methide is probably the reactive intermediate involved in the formation of 6-hydroxylated o-diphenolic compounds; (4) rapid hydroxylation of dopaquinone is due to the effect of the side chain carboxy group by enhancing the release of the β proton, thereby facilitating the tautomerization of dopaquinone to quinone methide; (5) neutral buffer promotes both the cyclization pathway of dopaquinone and dopaminequinone to form their respective aminochromes and the structural rearrangement of these aminochromes to 5,6-dihydroxyindole.

Involvement of l-tyrosine and phenol oxidase in the tanning of Aedes aegypti eggs

l-Tyrosine was found to be the initial precursor involved in the tanning of the chorion of Aedes aegypti eggs. This amino acid became detectable at about 24 h post-blood feeding and significant amounts of this compound accumulated in the ovaries during subsequent egg development. However, no o-diphenolic compounds were synthesized during the development of eggs within the mosquito ovaries. Oviposition of eggs to the outside environment initiated eggshell tanning due to activation of phenol oxidase, which then catalyzed the production of l-DOPA from l-tyrosine and further oxidized l-DOPA and dopamine to their respectivi o-quinones. Hardening and blackening of the chorion was completed in about 4 h. DOPA decarboxylase (DDC) also seemed to be involved in chorion tanning because a high percentage of l-DOPA, produced by the action of phenol oxidase following its activation, was decarboxylated to dopamine in in vitro assays. In addition, the application of the DDC inhibitor, m-hydroxybenzylhydrazine, resulted in an increased time required for the complete tanning of the chorion.

Solid-state 13C-NMR and diphenol analyses of sclerotized cuticles from stored product Coleoptera

Sclerotized cuticles from eight species of stored product Coleoptera including the maize weevil, Sitophilus zeamais; rice weevil, S. oryzae; granary weevil, S. granarius; red flour beetle, Tribolium castaneum; confused flour beetle, T. confusum; sawtoothed grain beetle, Oryzaephilus surinamensis; lesser grain borer, Rhyzopertha dominica and flat grain beetle, Cryptolestes pusillus, were subjected to solid-state 13C-nuclear magnetic resonance and/or chemical analyses. Solid-state NMR was used to measure relative amounts of protein (30–38%), chitin (32–36%), diphenolic compounds (24–33%) and lipid (2–5%) in cuticles from the three weevil species and the confused flour beetle, as well as wild-type and black mutant strains of the red flour beetle. NMR difference spectroscopic data were consistent with melanin and β-alanine being more abundant in the black and wild-type strains, respectively. Cuticles were extracted in cold acid and the o-diphenolic compounds were quantitated by reversed phase HPLC with electrochemical detection. In all species, the major diphenols were 3,4-dihydroxyphenylacetic acid (6–58 μmol g −1) and 3,4-dihydroxyphenylethanol (5–48 μmol g −1). Dopamine, N- β-alanyldopamine, N- β-alanylnorepinephrine, N-acetylnorepinephrine, 3,4-dihydroxybenzaldehyde and 3,4-dihydroxyphenylalanine were detected at much lower concentrations. The diphenols extracted with cold acid accounted for less than 10% of the total estimated by solid-state NMR.

Oxovanadium(IV) adsorption by plant roots. ESR identification of mobile and immobilized species

The adsorption of oxovanadium(IV) by plants ( Allium Sativum L.) has been investigated by means of ESR spectroscopy. It has been found that most of adsorbed VO 2+ ions accumulate in the roots as soluble low molecular weight complexes of o-diphenolic compounds. However, with increasing concentration, immobilized oxovanadium(IV) ions, possibly associated with acid polysaccharide components, are also detected. In the presence of EDTA, only the soluble VO(EDTA) complex species are observed, and they are mobile enough to be extensively translocated to the tops.

Dosage des composes phenoliques dans les extraits vegetaux par oxydation cuivrique en milieu non aqueux

(The spectrophotometric determination of phenolic compounds in vegetable extracts by oxidation with copper(II) in nonaqueous media) Oxidation with copper(II) in nonaqueous medium is used for the o-diphenolic compounds chlorogenic acid, rutin, pyrocatechol and epicatechol. A spectrophotometric study of the o-diphenol and o-quinone forms leads to the establishment of two systems of equations for the determination of these compounds. This study complements earlier results. The validity of the method is discussed on the basis of results obtained with Passe-crassane pear extracts.

Oxygen activation. II. The effect of catecholamines

1. Adrenal hormones greatly accelerated the autoxidation of p-hydroquinone by activating the O 2 molecule through complex formation by charge transfer. 2. O 2 activation by o-diphenolic compounds was also observed by using p-phenylenediamine as substrate. This explains the abnormally high O 2 uptake by the diamine in the presence of melanoma extracts, DOPA or catechol (the Greenstein-Riley effect). 3. The kinetics of catechol autoxidation is consistent with the view that one molecule is oxidized while a second catechol molecule acts as activator. 4. It is suggested that one of the primary roles of catecholamines is to active O 2.

N-Acetyl-dopamine as Sclerotizing Agent of the Insect Cuticle

SCLEROTIZATION, that is, the hardening and darkening of the insect cuticle, is generally believed1–3 to be due to the incorporation of o-quinones, the latter arising from the corresponding o-diphenols through the action of phenoloxidases. Tracer investigations4 confirmed the view that tyrosine metabolites are incorporated into the cuticle. However, the nature of the metabolites has not been identified, though several o-diphenolic compounds have been isolated5,6 from insect sources and tentatively regarded as precursors of sclerotin.