Monohydric Phenol Research Articles

Experimentation on solvent extraction of polyphenols from natural waste

The main commercial product of the cashew tree is the cashew apple and nut. Cashew nut is regarded as lost crop in the content of agricultural produce of India despite its industrial and export potentials. After separation of nut from the cashew fruit, the nut shell is disposed into the environment and this is acting as a waste. The nut shell containing a dark reddish brown viscous liquid is called cashew nut shell liquid (CNSL) and consists of 70% anacardic acid (polyphenol), 18% cardol, and 5% cardanol. This waste material is having twofold advantages: CNSL is a low-cost phenol, which is having many industrial applications and after extraction of CNSL from the shell, the shell is acting as a fuel for many boilers. Cashew nut shell liquid was extracted from cashew nut shell by indirect leaching process using soxhlet extraction equipment. Different solvents were used in the extraction of CNSL from the cashew nut shell (CNS) and a comparison was made between them. Among all the solvents acetone gives more amounts of CNSL and its properties rely with the industrial CNSL. The operating conditions for the extraction were 60 °C and 1 atm; in every 25 g of cashew nut shell used for the extraction, 8.75 g CNSL was obtained. The CNSL was further separated into cardol, cardanol, and anacardic acid (polyphenol) using an extractant, ammonium hydroxide, with the aid of mechanical shaker equipment. Subsequently, the polyphenol was further separated into dihydric phenols (resorcinol) and monohydric phenol (phenol).

Tyrosinase kinetics: failure of the auto-activation mechanism of monohydric phenol oxidation by rapid formation of a quinomethane intermediate

When 3,4-dihydroxybenzylcyanide (DBC) is oxidized by mushroom tyrosinase, the first visible product, identified as the corresponding quinomethane, exhibits an absorption maximum at 480 nm. Pulse-radiolysis experiments, in which the o-quinone is formed by disproportionation of semiquinone radicals generated by single-electron oxidation of DBC, showed that the quinomethane (A480 6440 M-1.cm-1) is formed through the intermediacy of the o-quinone with a rate constant at neutral pH of 7.5 s-1. The oxygen stoichiometry of the formation of the quinomethane by tyrosinase-catalysed oxidation of DBC was 0.5:1. On the basis of oxygen utilization rates the calculated Vmax was 4900 nmol.min-1 and the apparent Km was 374 microM. The corresponding monohydric phenol, 4-hydroxybenzylcyanide (HBC), was not oxidized by tyrosinase unless the enzyme was pre-exposed to DBC, the maximum acceleration of HBC oxidation being obtained by approximately equimolar addition of DBC. These results are consistent with tyrosinase auto-activation on the basis of the indirect formation of the dihydric phenol-activating cofactor. The rapid conversion of the o-quinone to the quinomethane prevents the formation of the catechol by reduction of the o-quinone product of monohydric phenol oxidation from occurring in the case of the compounds studied. In the absence of auto-activation, the kinetic parameters for HBC oxidation by tyrosinase were estimated as Vmax 70 nmol.min-1 and Km 309 microM. The quinomethane was found to decay with a rate constant of 2k 38 M-1.s-1, as determined both by pulse-radiolysis and tyrosinase experiments. The second-order kinetics indicate that a dimer is formed. In the presence of tyrosinase, but not in the pulse-radiolysis experiments, the quinomethane decay was accompanied by a steady-state oxygen uptake concurrently with the generation of a melanoid product measured by its A650, which is ascribed to the formation of an oligomer incorporating the oxidized dimer.

Tyrosinase autoactivation and the problem of the lag period.

Evidence is presented for the binding of the quinone oxidation product of the monohydric phenol substrate, 4-hydroxyanisole, to mushroom tyrosinase. Column chromatography and SDS-PAGE separation showed labelling of the enzyme when incubated with 14C ring-labelled 4-hydroxyanisole. It is proposed that covalent binding to the enzyme and other proteins is through reaction of accessible nucleophilic groups, including thiols and amino groups, with the anisylquinone. This reductive addition enables the indirect generation of the catecholic substrate, which acts as an electron donor for the bicupric active site of met-tyrosinase and explains the lag kinetics of tyrosinase oxidation of non-cyclizing substrates. The effects of diluting the enzyme or the addition of amino acids on the lag period was consistent with a mechanism involving indirect generation of the dihydric phenol, which acts as the met-enzyme-recruiting substrate.

Tyrosinase kinetics: failure of acceleration in oxidation of ring-blocked monohydric phenol substrate.

When 2,5,6-trimethyl-4-hydroxyanisole is used as substrate for mushroom tyrosinase the oxidation rate is slow and the kinetics do not exhibit an initial acceleration (lag period), in contrast to the kinetics of oxidation of the parent compound, 4-hydroxyanisole. This finding is interpreted as evidence that the acceleration of oxidation of 4-hydroxyanisole is indirectly contingent on a reductive nucleophile addition to the orthoquinone product of the monohydric phenol, which is prevented by ring methylation. Such a view is consistent with the proposal that the lag-phase characteristic of the kinetics of monohydric phenol oxidation by tyrosinase is due to the activation of previously inactive enzyme by electron donation from an orthodiphenol substrate formed from the orthoquinone oxidation product.

Evidence of the Indirect Formation of the Catecholic Intermediate Substrate Responsible for the Autoactivation Kinetics of Tyrosinase

Tyrosinase (EC 1.14.18.1) exhibits unusual kinetic properties in the oxidation of monohydric phenol substrates consisting of a lag period that increases with increasing substrate concentration. The cause of this is an autocatalytic process dependent on the generation of a dihydric phenol substrate, which acts as an activator of the enzyme. Experiments with N-substituted dihydric phenol substrates (N-methyldopamine, N-acetyldopamine) demonstrate that oxygen consumption is retarded in the N-acetyl substituted material due to a diminished rate of cyclization. The oxygen uptake exhibited a similar pattern when N-acetyltyramine was oxidized, and this was reflected by a prolongation of the lag period. N,N-Dipropyldopamine was oxidized with normal kinetics but with an oxygen stoichiometry of 0.5 mol of oxygen/mol of substrate. We show that this is the result of the formation of a stable indoliumolate product with oxidation-reduction properties that prevent the formation of dopaminochrome, thus blocking further stages in the tyrosinase-catalyzed oxidation. Evidence that the indoliumolate product is formed by cyclization of the ortho-quinone is presented by pulse radiolysis studies, which demonstrate the formation of the ortho-quinone (by disproportionation of the corresponding semiquinones), which cyclizes to give the indoliumolate. The rate constant for cyclization was shown to be 48 s-1 (at pH 6.0). Tyrosinase-catalyzed oxidation of the monohydric phenol analogue, N, N-dimethyltyramine, was shown to require the addition of a dihydric phenol. Oxygen utilization then exhibited a stoichiometry of 1.0, indicating that the reactions proceed only as far as the cyclization. The analogous stable cyclic indoliumolate product was shown to be formed, with UV absorption and NMR spectra closely similar to the indoliumolate derived from N,N-dipropyldopamine. This material was methylated by catechol O-methyltransferase but was unreactive to redox reagents. The formation of the cyclic product accounts for the indefinite lag when N,N-dimethyltyramine is used as the substrate for tyrosinase in the absence of a dihydric phenol cofactor.

EFFICACY OF THE DISINFECTANT 'DISFECT-S' AGAINST PATHOGENS OF THE SILKWORM, Bombyx mori

In-vitro efficacy tests of Disfect-S (Poly alkyl monohydric phenol) on the viability of infective propagules of silkworm disease at varying concentrations of 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 2.0% & 3.0% were carried out. Efficacy of 'Disfect- S as surface sterilant of silkworm eggs was tested at concentrations of 0.2%, 0.4%, 0.6%, 0.8% and 1.0% with treatment durations of 1 min, 2 min, 5 min, 10 min and 20 min. The viability and the infectivity of Nosema bombycis spores was inactivated at 0.4% with 5 min. of treatment duration and the infectivity of conidia of Beauveria bassiana was completely arrested at 0.8% with 5 min. duration whereas Nuclear polyhedra virus (BmNPV) were completely inactivated at 0.8% for 10 minutes treatment duration. Disfect-S was highly effective as surface sterilant of silkworm eggs at 0.4% with 2 min. duration treatment without any deleterious effect on hatching. The present study gives an higher scope for Disfect-S as potential commercial disinfectant in sericulture.

Oxidation of monohydric phenol substrates by tyrosinase: effect of dithiothreitol on kinetics

The effect of thiol compounds on the monophenolase activity of tyrosinase was investigated using 4-hydroxyanisole as the substrate and dithiothreitol (DTT) as the model thiol compound. We have demonstrated three actions of DTT on tyrosinase-catalysed reactions: (1) direct reduction of the copper at the active site of the enzyme; (2) generation of secondary, oxidizable species by adduct formation with the o-quinone reaction product, 4-MOB, which leads to an increase in the total oxygen utilization by the reaction system; and (3) reversible inhibition of the enzyme. We confirm our previous observation that, at approx. 10 mol of DTT/mol of enzyme, the lag phase associated with monohydric phenol oxidation by tyrosinase is abolished. We suggest that this is due to reduction of the copper at the active site of the enzyme by DTT, since (a) reduction of active-site copper in situ by DTT was demonstrated by [Cu(I)]2-carbon monoxide complex formation and (b) abolition of the lag at low DTT concentration occurs without effect on the maximum rate of reaction or on the total amount of oxygen utilized. At concentrations of DTT above that required to abolish the lag, we found that the initial velocity of the reaction increased with increasing DTT, with a concomitant increase in the total oxygen utilization. This is due to the formation of DTT-4-methoxy-o-benzoquinone (4-MOB) adducts which provide additional dihydric phenol substrate either directly or by reducing nascent 4-MOB. We present n.m.r. evidence for the formation of mono- and di-aromatic DTT adducts with 4-MOB, consistent with a suggested reoxidation scheme in the presence of tyrosinase. Inhibition of the enzyme at concentrations of DTT above 300 pmol/unit of enzyme was released on exhaustion of DTT by adduct formation with 4-MOB as it was generated.

In vitro kinetics of oxidation of monohydric phenol substrates by tyrosinase

In vitro kinetics of oxidation of monohydric phenol substrates by tyrosinase

Oxidation of monohydric phenol substrates by tyrosinase. An oximetric study.

The purity of commercially available mushroom tyrosinase was investigated by non-denaturing PAGE. Most of the protein in the preparation migrated as a single band under these conditions. This band contained both tyrosinase and dopa oxidase activity. No other activity of either classification was found in the preparation. Oxygen consumption by tyrosinase during oxidation of the monohydric phenol substrates tyrosine and 4-hydroxyanisole (4HA) was monitored by oximetry in order to determine the stoichiometry of the reactions. For complete oxidation, the molar ratio of oxygen: 4HA was 1:1. Under identical conditions, oxidation of tyrosine required 1.5 mol of oxygen/mol of tyrosine. The additional oxygen uptake during tyrosine oxidation is due to the internal cyclization of dopaquinone to form cyclodopa, which undergoes a redox reaction with dopaquinone to form dopachrome and dopa, which is then oxidized by the enzyme, leading to an additional 0.5 mol of oxygen/mol of original substrate. Oxygen consumption for complete oxidation of 200 nmol of 4HA was constant over a range of concentrations of tyrosinase of 33-330 units/ml of substrate. The maximum rate of reaction was directly proportional to the concentration of tyrosinase, whereas the length of the lag phase decreased non-linearly with increasing tyrosinase concentration. Activation of the enzyme by exposure to citrate was not seen, nor was the lag phase abolished by exposure of the enzyme to low pH. Michaelis-Menten analysis of tyrosinase in which the lag phase is abolished by pre-exposure of the enzyme to a low concentration of dithiothreitol gave Km values for tyrosine and 4HA of 153 and 20 microM respectively.

Studies on the kinetics of oxidation of 4-hydroxyanisole by tyrosinase

The potential use of 4-hydroxyanisole as a chemotherapeutic agent in the treatment of malignant melanoma led us to investigate the kinetics of oxidation of this tyrosine analogue by tyrosinase. We found that addition of amino acids accelerated the reaction, resulting in a reduction in length of the characteristic lag period of monohydric phenol oxidation. The lag period was abolished completely by an aliquot of exhausted 4-hydroxyanisole/tyrosinase reaction mixture and by very low concentrations of thiol-containing compounds. We conclude that the reaction-accelerating property of non-thiol amino acids is due to the reductive addition of the ortho-quinone reaction product to nucleophilic groups of the amino acids. The dihydric phenol product which results is capable of met-tyrosinase recruitment by electron donation to the cupric active site generating the cuprous form of the enzyme which binds oxygen and is able to oxidise monohydric phenols. Abolition of the lag period by an aliquot of exhausted reaction mixture is probably due to recruitment of the met-enzyme by catecholic oligomers of the quinone product. Thiol containing compounds are able to abolish the lag period due to the ability of these compounds to reduce met-tyrosinase directly.

Oxygen permeability and surface free energy properties of silicone-containing copolymers of methyl methacrylate

Novel organofunctional siloxanes from γ-methacryloxypropyltrimethoxysilane (MPTMS) were synthesized by transesterification reactions with high monohydric alcohols, branched alcohols, ether alcohols, phenol, and mandelic acid. The factors effecting the substitution ratios were determined. Copolymers of these siloxanes with methyl methacrylate (MMA) were prepared by bulk copolymerization in glass molds and their swelling, thermal, oxygen permeability, and surface free energy characteristics were investigated in order to determine the effect of the substituted group on the produced copolymer. The changes in these properties by the crosslinking of the copolymer with ethylene glycol dimethacrylate (EGDM) were also investigated. The copolymers containing branched, nonpolar, and lengthy pendant groups were found to be highly oxygen permeable possibly due to the introduction of larger free volume by these groups. The surface energy analysis showed that the main variation was found to be in γ values rather than γ values and only ether alcohol substitution increased γ values to a greater extent.

Studies on indoleacetic acid oxidation by liquid medium from crown gall tissue cells: The role of malic acid and related compounds

1. 1. Indoleacetic acid oxidation by liquid medium from crown gall tissue culture cells has been studied. The reaction has a pH optimum of 4.5 and requires Mn 2+ and a monohydric phenol. A short lag phase is routinely observed. The appearance of peroxidase and indoleacetic acid oxidising activity in the medium of a tissue culture was followed over a 3 week time course. One function of this enzyme may be to prevent the accumulation of excess inhibitory concentrations of indoleacetic acid. 2. 2. Gel filtration of the medium through Sephadex G-25 produced 3 peaks. The high molecular weight fraction contained peroxidase but had almost completely lost the ability to oxidise indoleacetic acid unless recombined with the first low molecular weight peak. Malic and other compounds of related structure also reactivated in the same way as the natural activator. Mn 2+ and a monohydric phenol were required in each case. Neither effect was due to inhibition of endogenous catalase. The second low molecular weight peak inhibited indoleacetic acid oxidation by extending the lag phase. 3. 3. Both substrate activation and inhibition were observed in the presence of low levels of activator but were overcome with increasing amounts of activator. There was a non-linear dependence of reaction rate on enzyme concentration which was not apparent in the presence of 3.6 M urea. 4. 4. H 2O 2 is not an obligatory intermediate although it greatly decreased the lag phase and enhanced the rate of oxidation. Mn 2+ was unnecessary for these effects. Competitive studies indicated that both malic acid and H 2O 2 may act on the same process in the reaction mechanism. 5. 5. A model is put forward, based on the hypothesis that the reaction is autocatalytic with a cyclic mechanism involving unstable intermediates, for the participation of malic acid and Mn 2+ as well as monohydric phenols and H 2O 2 in the enzymic oxidation of indoleacetic acid by oxygen. 6. 6. In addition to malic acid, a wide range of non-phenolic compounds was tested for activating ability. The structural characteristics of activators are discussed.

Specificity of monohydric phenol oxidations by meta cleavage pathways in Pseudomonas aeruginosa T1

The oxidation of several mono-hydric phenols by wild type and mutant strains of Pseudomonas aeruginosa T1 has been studied. The data suggest, that a non-specific enzyme sequence of the meta cleavage pathway is induced by all of these phenols, which can catalyze the oxidation of phenol and its analogues to pyruvate, a fatty acid and a carbonyl compound, according to the general scheme of Dagley et al. (1964). Mutants unable to grow on phenol (hydroxylase-negative), have been isolated, and they are also unable to grown on or oxidize the cresols and the xylenols. Revertants of these mutants regain the capacity to grow on all these phenols and are indistinguishable from the wild type. Induced-substrate relationships for the earlier enzymes of the pathway have been determined, e.g., phenol in addition to catechol and the methylcatechols is an inducer for catechol 2,3-oxygenase. Analysis of the enzymic content of cells grown in a variety of steadystate conditions shows (a) that the ratio of the specific activities of the “phenol” hydroxylase and catechol 2,3-oxygenase is constant for each of their analogous substrates; and (b) that induction and catabolite repression of catechol 2,3-oxygenase and the “muconic semialdehyde” hydrolyase are coordinate, but that control of the “phenol” hydroxylase is independent.

The conversion of phenolase of p-coumaric acid to caffeic acid with special reference to the role of ascorbic acid

Caffeic acid was isolated as a product of p-coumaric acid oxidation catalysed by phenolase. Time course of p-coumaric acid conversion was studied in the absence and presence of ascorbic acid, and caffeic acid accumulation was found to be correlated with p-coumaric acid disappearance in the ascorbic acid-containing system. In the above reaction, p-coumaric acid undergoes a coupled oxidation with ascorbic acid as follows: p-coumaric acid+ascorbic acid+O 2 → caffeic acid. The effect on the initial reaction stage (lag phase) of concentrations of enzyme, monohydric phenol and ascorbic acid was studied and favourable conditions for obtaining caffeic acid in quantity are discussed.

Active Mitochondria Do Not Produce Ethylene

Although numerous reports on ethylene evolution from subcellular preparations have appeared (2,3, 4,5,8,9,11, 18, 19,20), some of these works have been criticized by Burg and Burg (1) and by Meigh (17). and the question whether or not mitochondria produce ethylene remains unclear. It is known that ethylene can be evolved nonenzymically from methionine and its derivatives, mediated either by ICu2+-ascorbate-H202 (10) or by FMN-light (22,23). Enzymic systems of ethylene production were described recently by Mapson and Wardale (13), Ku et al. (7), and Meheriuk and Spencer (14, 15, 16). More recently, Yang (21) has demonstrated that ethylene can rapidly be formed aerobically from methional by an enzyme system consisting of horseradish peroxidase, Mn2+ and sulfite ions, and a monohydric phenol. We have been able to isolate very active, homogeneous, relatively uninjured mitochondria from various fruits (6). It is the purpose of this article to indicate that such active mitochondria do not directly produce ethylene; it appears more likely that ethylene is produced by a soluble enzyme. Tomato fruits (Lycopersicon esculentum Mill., cv. VC 243-20) were harvested at various stages of maturity from greenhouse-grown plants (12). The fruits were washed with distilled water and chilled (0-4°) from 1 to 4 hours before processing by the method of Ku et al. (6). The flesh of the fruit wall was carefully removed, avoiding the inclusion of any skin, seeds, or the jelly-like tissue around the seeds.

Carbocyclization of 2,3,4-tri-O-methyl-1,6-anhydroglucopyranose under the action of metallic sodium in pyridine medium

1. Metallic sodium in pyridine medium causes carbocyclization of 1,6-anhydrides ofd-hexapyranoses, analogously to sodium in liquid ammonia. 2. Under the action of sodium in pyridine medium on 2,3,4-tri-O-methyl-1,6-anhydroglucopyranose, a mixture of dihydric phenols is formed-guaiacol and resorcinol; no monohydric phenol is detected in the reaction products.

Oscillopolarographic detection and determination of dihydric phenols

A 1 M-potassium nitrate solution has been proposed as supporting electrolyte for the qualitative detection of dihydric phenols by an oscillopolarographic method. Sufficiently distant incisions on curves of the function dV dt = f(V) permit their determination by the method of comparative titration with an average error of ± 3–4% Monohydric phenol does not interfere in their qualitative detection. Phloroglucinol and pyrogallol interfere in the detection as well as determination. The method of calibration curves permits determination of individual dihydric phenols with an error of ± 5%.

Dihydric phenols obtained in the carbocyclization of 1,6-anhydrides of D-hexoses

1. 2,3,4-Tri-O-methyl-1,6-anhydro-D-mannopyranose reacts with sodium in liquid ammonia under the conditions that have been found to be optimum for trimethyllevoglucosan with formation of phenol in 15–17% yield. This is only one-third of the yield in the analogous reaction with 2,3,4-tri-O-methyl-1,6-anhydro-D-gluco-, -D-ido-, and -D-gulo-pyranoses and only one-half of the yield in the reaction with 2,3,4-tri-O-methyl-1,6-anhydro-D-galactopyranose. 2. In the reaction of 1,6-anhydrides of hexoses with sodium in liquid ammonia, apart from monohydric phenol, about 10% of a mixture of three dihydric phenols (resorcinol, pyrocatechol, and resorcinol monomethyl ether) is formed.