Assigning a Role to the Endogenous Phenolic Compounds on Adventitious Root Formation of Olive Stem Cuttings

The objective of this study was to role the effect of phenolic compounds on the rooting potential of leafy cuttings of the recalcitrant to root olive cultivar ‘Kalamata’ and the easy to root ‘Arbequina’. Subsamples of cuttings were taken for analysis before planting (day 0) in the mist system and during the early phases of rhizogenesis (EPR). ‘Kalamata’ presented higher initial (day 0) total phenols in summer and total o-diphenols in autumn and spring compared to ‘Arbequina’, while ‘Arbequina’ had significantly higher initial total flavonoids and flavanols in autumn. A positive correlation was found between initial total phenols and rooting ability of ‘Arbequina’. In ‘Kalamata’ a positive correlation was established between initial total o-diphenols and rooting percentage while total flavonoids were negatively correlated with rooting. Generally, total phenols, o-diphenols, flavanols and flavonoids did not differ between the two cultivars and their concentration reduced significantly 15 days after planting. Furthermore, ‘Arbequina’ presented higher initial tyrosol, chlorogenic acid, luteolin-7-glucoside, rutin, quercetin and luteolin in summer and autumn compared to ‘Kalamata’. The above phenolics were positively correlated with the rooting of ‘Arbequina’. Significant changes were observed in the concentration of the individual phenolics during the EPR, whereas chlorogenic acid, rutin, quercetin and luteolin concentration increased significantly from day 1 to 5. In conclusion, there is a clear relationship between the phenolic profile and adventitious rooting of the two olive cultivars and in fact chlorogenic acid and rutin can be proposed as olive rooting enhancers.

     

Inhibitory effects of 4-vinylbenzaldehyde and 4-vinylbenzoic acid on the activity of mushroom tyrosinase

Tyrosinase (EC 1.14.18.1) catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones which form brown or black pigments. Here, the inhibitory effects of 4-vinylbenzaldehyde and 4-vinylbenzoic acid on the activity of mushroom tyrosinase have been investigated. The results showed that both 4-vinylbenzaldehyde and 4-vinylbenzoic acid could inhibit both monophenolase activity and diphenolase activity of the enzyme. For the monophenolase activity, 4-vinylbenzoic acid could lengthen the lag time, but 4-vinylbenzaldehyde could not. Both 4-vinylbenzaldehyde and 4-vinylbenzoic acid decreased the steady-state activity, and the IC₅₀ values were estimated as 93?μM and 3.0?mM for monophenolase activity, respectively. For the diphenolase activity, the inhibitory capacity of 4-vinylbenzaldehyde was stronger than that of 4-vinylbenzoic acid, and the IC₅₀ values were estimated as 23?μM and 0.33?mM, respectively. Kinetic analyses showed that inhibition by both compounds was reversible and their mechanisms were mixed-II type; their inhibition constants were also determined and compared.     

Inhibitory effects of Cefazolin and Cefodizime on the activity of mushroom tyrosinase

Tyrosinase (EC 1.14.18.1) catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones that form brown or black pigments. In the present paper, the effects of Cefazolin and Cefodizime on the activity of mushroom tyrosniase have been studied. The results showed that the Cephalosporin antibacterial drugs (Cefazolin and Cefodizime) could inhibit both monophenolase activity and diphenolase activity of the enzyme. For the monophenolase activity, Both Cefazolin and Cefodizime could lengthen the lag time and decrease the steady-state activities, and the IC₅₀ values were estimated as 7.0 mM and 0.13 mM for monophenolase activity, respectively. For the diphenolase activity, the inhibitory capacity of Cefodizime was obviously stronger than that of Cefazolin, and the IC₅₀ values were estimated as 0.02 mM and 0.21 mM, respectively. Kinetic analyses showed that inhibition by both compounds was reversible and their mechanisms were competitive and mixed-type, respectively. Their inhibition constants were also determined and compared. The research may offer a lead for designing and synthesizing novel and effective tyrosinase inhibitors and also under the application field of Cephalosporins.     

Inactivation kinetics of mushroom tyrosinase in the dimethyl sulfoxide solution

Mushroom tyrosinase (EC 1.14.18.1) is a kind of copper-containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones and then forms brown or black pigments. In the present paper, the effects of dimethyl sulfoxide on the enzyme activity for the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) have been studied. The results show that low concentrations of dimethyl sulfoxide (DMSO) can lead to reversible inactivation of the enzyme, and the IC ₅₀ is estimated to be 2.45 M. Inactivation of the enzyme by DMSO is classified as mixed type. The kinetics of inactivation of mushroom tyrosinase at low concentrations of DMSO solution has been studied using the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The results show the free enzyme molecule is more fragile than the enzyme–substrate complex in the DMSO solution. It is suggested that the presence of the substrate offers marked protection of this enzyme against inactivation by DMSO.     

Antioxidation and Tyrosinase Inhibition of Polyphenolic Curcumin Analogs

A series of polyphenolic curcumin analogs were synthesized and their inhibitory effects on mushroom tyrosinase and the inhibition of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical formation were evaluated. The results indictated that the analogs possessing m-diphenols and o-diphenols exhibited more potent inhibitory activity on tyrosinase than reference compound rojic acid, and that the analogs with o-diphenols exhibited more potent inhibitory activity of DPPH free-radical formation than reference compound vitamin C. The inhibition kinetics, analyzed by Lineweaver–Burk plots, revealed that compounds B₂ and C₂ bearing o-diphenols were non-competitive inhibitors, while compounds B₁₁ and C₁₁ bearing m-diphenols were competitive inhibitors. In particular, representative compounds C₂ and B₁₁ showed no side effects at a dose of 2,000 mg/kg in a preliminary evaluation of acute toxicity in mice. These results suggest that such polyphenolic curcumin analogs might serve as lead compounds for further design of new potential tyrosinase inhibitors.     

Kinetic study of monophenol and o-diphenol binding to oxytyrosinase

The complex reaction mechanism of tyrosinase involves three enzymatic forms, two overlapping catalytic cycles and a dead-end complex. The deoxytyrosinase form binds oxygen with a high degree of affinity, μM. The mettyrosinase and oxytyrosinase forms bind monophenols and o-diphenols, although the former is inactive on monophenols. Analytical expressions for the catalytic and Michaelis constants of tyrosinase towards phenols and o-diphenols have been derived. Thus, the Michaelis constant of tyrosinase towards monophenols and o-diphenols are related with the catalytic constants for monophenols and o-diphenols , respectively, and with the binding rate constants of the oxytyrosinase form with these substrates (k₊₄ and k₊₆, respectively), by means of the expressions and . From these expressions, we calculate the values of the binding rate constant of oxytyrosinase to the substrates (monophenols and o-diphenols) for tyrosinases from different biological sources, and reveal that the o-diphenols bind more rapidly to oxytyrosinase than the monophenols. In addition, a new kinetic constant (the Michaelis constant for o-diphenol in the monophenolase activity), is derived and determined. Thus, it has been shown that tyrosinase has apparently higher affinity towards o-diphenols in its monophenolase than in its diphenolase activity.     

Dopachrome conversion activity in Aedes aegypti: Significance during melanotic encapsulation of parasites and cuticular tanning

Phenol oxidase (PO) and dopachrome conversion enzyme (DCE) were partially purified from Aedes aegypti larvae by ammonium sulfate fractionation. PO from A. aegypti functions in the hydroxylation of monophenols (e.g., tyrosine and tyramine) to their related o-diphenols, and the oxidation of o-diphenols (e.g., l-dopa, dopamine, N-acetyldopamine) to their respective o-quinones. Partially purified DCE showed high specificity toward dopachrome generated from dopa with the l-configuration. The combined effects of PO and DCE significantly accelerated melanization pathways when l-dopa was used as substrate. Significant DCE activity also was detected in hemolymph samples from adult, female A. aegypti, and undoubtedly plays a role in melanotic encapsulation reactions.     

Regulation of enzymic oxidation of indole-3-acetic acid by phenols: Structure-activity relationships

Mono- and diphenols were tested for their effects on the decarboxylation of [1-¹⁴C]IAA catalysed by purified horseradish peroxidase (EC 1.11.1.7) in the presence or absence of 2,4-dichlorophenol (DCP). The number of hydroxyl groups and their position relative to each other and the nature and position of other substituents on the aromatic ring were found to affect the activity. Although the effects were complex, the following generalizations may be made. (1) Monophenols produce activation when no other cofactor is present. p-Substituted monophenols are more active than o- or m-compounds. In the presence of DCP, the activity varies from slight activation to strong inhibition. (2) m-Diphenols also produce activation in the absence of other cofactors while o- and p-diphenols, with the exception of 3,4-dihydroxyacetophenone and 3,4-dihydroxypropiophenone, produce strong inhibition in the presence or absence of DCP. The o-diphenolsare degraded in the IAA-oxidizing enzyme system and thus produce only a temporary inhibition. (3) m-Diphenols and 3,4-dihydroxyacetophenone produce a sustained inhibition in the presence of DCP. (4) Substitution at position 2 significantly alters the activity of m-diphenols. (5) O-Methylation alters the activity of most o-diphenols. In the absence of DCP, o-methoxyphenols and certain other phenols such as 3,4-dihydroxyacetophenone and 2,6-dihydroxyacetophenone either promote or inhibit IAA oxidation depending on concentration.     

Effect of Polyphenol Oxidase on Deodorization

A mixture of purified polyphenol oxidases (PPO), or acetone powders prepared from fruits and vegetables, and polyphenolic compounds (PPs) totally eliminated a methylmercaptan odor. 2-Methyl-thiochlorogenic acid was isolated from the reaction mixture of methylmercaptan and chlorogenic acid with burdock acetone powder. Further, the formation of 5-methylthiochlorogenic acid and 2,5-bis(methylthio)-chlorogenic acid was suggested. These facts demonstrate that the o-quinone compounds formed from o-diphenols by PPO rapidly reacted with methylmercaptan. The oxidation reaction of PPs by using acetone powder containing PPO or peroxidase is considered to be more effective for removing bad smells from our mouths and from the environment.     

Phenoloxidases from larval cuticle of the sheep blowfly,Lucilia cuprina: Characterization,developmental changes,and inhibition by antiphenoloxidase antibodies

A tyrosinase, enzyme A (EC 1.10.3.1, o-diphenol: O₂ oxidoreductase), and a laccase, enzyme B (EC 1.10.3.2, p-diphenol: O₂ oxidoreductase), have been partially purified and characterized from larval cuticle of the sheep blowfly, Lucilia cuprina. Enzyme A is active toward a range of o-diphenols but not p-diphenols, is strongly inhibited by thiourea and phenylthiourea, has a pH optimum between 6.5 and 7.0, and yields a single, 60,000 molecular weight subunit following SDS gel electrophoresis. Enzyme B is active toward both o-diphenols and p-diphenols, is only slightly inhibited by phenylthiourea, has a pH optimum near 4.5, is highly thermostable, and has an apparent molecular weight of 90,000. Enzyme A appears to be activated from an inactive proenzyme in the cuticle and to be present throughout the wandering phase of the final larval instar, declining at pupariation. Enzyme B is present in active form, increases greatly in the cuticle just at the time of pupariation, and then decreases as sclerotization occurs. Antibodies against enzyme A have been raised in sheep and rabbits, and against enzyme B in rabbits, but diets containing antiphenoloxidase antibodies did not affect development or mortality of fly larvae.     

Red clover coumarate 3′-hydroxylase (CYP98A44) is capable of hydroxylating p-coumaroyl-shikimate but not p-coumaroyl-malate: implications for the biosynthesis of phaselic acid

Red clover (Trifolium pratense) leaves accumulate several μmol of phaselic acid [2-O-caffeoyl-l-malate] per gram fresh weight. Post-harvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases (PPO) prevents breakdown of forage protein during storage. Forages like alfalfa (Medicago sativa) lack both foliar PPO activity and o-diphenols. Consequently, breakdown of their protein upon harvest and storage results in economic losses and release of excess nitrogen into the environment. Understanding how red clover synthesizes o-diphenols such as phaselic acid will help in the development of forages utilizing this natural system of protein protection. We have proposed biosynthetic pathways in red clover for phaselic acid that involve a specific hydroxycinnamoyl-CoA:malate hydroxycinnamoyl transferase. It is unclear whether the transfer reaction to malate to form phaselic acid involves caffeic acid or p-coumaric acid and subsequent hydroxylation of the resulting p-coumaroyl-malate. The latter would require a coumarate 3′-hydroxylase (C3′H) capable of hydroxylating p-coumaroyl-malate, an activity not previously described. Here, a cytochrome P450 C3′H (CYP98A44) was identified and its gene cloned from red clover. CYP98A44 shares 96 and 79% amino acid identity with Medicago truncatula and Arabidopsis thaliana C3′H proteins that are capable of hydroxylating p-coumaroyl-shikimate and have been implicated in monolignol biosynthesis. CYP98A44 mRNA is expressed in stems and flowers and to a lesser extent in leaves. Immune serum raised against CYP98A44 recognizes a membrane-associated protein in red clover stems and leaves and cross-reacts with C3′H proteins from other species. CYP98A44 expressed in Saccharomyces cerevisiae is capable of hydroxylating p-coumaroyl-shikimate, but not p-coumaroyl-malate. This finding indicates that in red clover, phaselic acid is likely formed by transfer of a caffeoyl moiety to malic acid, although the existence of a second C3′H capable of hydroxylating p-coumaroyl-malate cannot be definitively ruled out.     

Optical Rotatory Dispersion of Amino Acid Methylthiohydantoin

In order to clarify the postprandial glucose suppression via α-glucosidase (AGH) inhibitory action by natural compounds, flavonoids were examined in this study. Among the flavonoids (luteolin, kaempferol, chrysin, and galangin), luteolin showed the potent maltase inhibitory activity with the IC₅₀ of 2.3 mM, while less inhibitions were observed against sucrase. In addition, the effects of maltase inhibition by flavonoids were observed in the descending order of potency of luteolin>kaempferol>chrysin>galangin. Apparently, the AGH inhibition power greatly increased with the replacement of hydroxyl groups at 3′ and 4′-position of the B-ring. However, the inhibitory power of luteolin was poorer than a therapeutic drug (acarbose: IC₅₀; 430 nM). As a result of a single oral administration of maltose or sucrose (2 g/kg) in SD rats, no significant change in blood glucose level with the doses of 100 and 200 mg/kg of luteolin was observed. These findings strongly suggested that luteolin given at less than 200 mg/kg did not possess the ability to suppress the glucose production from carbohydrates through the inhibition of AGH action in the gut.     

Foliar oxidases as mediators of the rapidly induced resistance of mountain birch against <Emphasis Type="Italic">Epirrita autumnata</Emphasis>

Induced resistance of the mountain birch against its main defoliator Epirrita autumnata is a well-characterized phenomenon. The causal mechanism for this induced deterioration, however, has not been unequivocally explained, and no individual compound or group of traditional defensive compounds has been shown to explain the phenomenon. Phenolic compounds are the main secondary metabolites in mountain birch leaves, and the biological activity of phenolics usually depends on their oxidation. In this study, we found that the activity of polyphenoloxidases (PPOs), enzymes that oxidize o-diphenols to o-diquinones, was induced in trees with introduced larvae, and bioassays showed that both growth and consumption rates of larvae were reduced in damaged trees. PPO activity was negatively associated with both larval growth and consumption rates in trees with bagged larvae, but not in control trees. Our results suggest that the oxidation of phenolics by PPOs may be a causal explanation for the rapidly induced resistance of mountain birch against E. autumnata. This finding also helps to explain why correlations between insect performance and phenolics (without measuring indices explaining their oxidation) may not produce consistent results.     

Effect of Phenolic Antioxidants on Lipoxygenase Reaction

The effect of conventional antioxidants on soybean lipoxygenase reaction was examined. Inhibitory activities of o-diphenols such as pyrocatechol, homocatechol, propyl gallate and NDGA were higher than those of m- and p diphenols. The mode of inhibition by NDGA, one of the most effective inhibitors among the phenolic antioxidants tested, conformed to a competitive type and not to an induction period type. Under certain conditions, NDGA could be an irreversible inactivator for the enzyme. The effect of NDGA on the enzyme reaction could not be completely explained by the coupled oxidation theory. The inactivation by NDGA were effectively prevented by either of adding catalase, of incubating under anaerobic condition or in low pH medium or of adding borate. These facts showed that the inactivation of lipoxygenase took place in consonance with the autoxidation of NDGA.     

Improvement of olive oil phenolics content by means of enzyme formulations: Effect of different enzyme activities and levels

The present work deals with the development of an enzymatic treatment aiming at producing high-quality olive oil with increased phenolics content and antioxidant activity. Three different enzyme formulations, specifically pectinase, hemicellulase and cellulase (A), pectinase and hemicellulase (B), and only pectinase (C), were used either at three different level of each or in ternary and binary mixtures at a constant level. All of them were added to the olive paste (Italian cultivar Coratina) at the beginning of the malaxation step. Results demonstrated that an increased enzyme level led to higher phenolics content in the oils, and such an effect was found to be enzyme dependent, being greater when using formulation A, followed by formulations B and C. Two significant correlations were obtained between total polyphenols and o-diphenols contents versus antiradical power (R² = 0.8743 and R² = 0.8635, respectively), which pointed out the effectiveness of the proposed enzymatic treatment to produce olive oils characterized by low susceptibility to oxidation. A synergistic effect between the different enzymatic activities contained in the single formulations was observed by combining enzymes A and B. The ternary mixture was selected as the most efficient enzymatic system ensuring the highest phenolics content increase (40 and 37% for total polyphenols and o-diphenols contents, respectively) compared to the other enzymes when used at the same level.     

Structure-activity relationship of polyphenols on inhibition of chemical mediator release from rat peritoneal exudate cells

Summary The effect of phenolic compounds in foodstuffs on histamine and leukotriene B₄ (LTB₄) release from rat peritoneal exudate cells and their antioxidative activity were examined to assess their antiallergenic activities. Among them, triphenols such as pyrogallol and gallic acid inhibited histamine release from the cells, but diphenols did not. On the other hand, o- and p-diphenols such as catechol and hydroquinone with strong antioxidative activity inhibited LTB₄ release as strongly as pyrogallol, but an m-derivative resorcinol with weak antioxidative activity did not. Though carboxylated compounds and their noncarboxylated counterparts were antioxidative, the former exerted a much weaker inhibitory effect on the LTB₄ release than the latter. In flavonols, only myricetin with a triphenolic B ring strongly inhibited histamine release, but all flavonols strongly suppressed LTB₄ release irrespective of the number of OH groups in the B ring. Among flavonoids with an o-diphenolic B ring, flavonol and flavone with a C₄-carbonyl group strongly inhibited LTB₄ release, whereas the activity of anthocyan without C₄-carbonyl was much weaker than the above compounds. These results suggest that triphenolic structure is essential for the inhibition of histamine release. On the other hand, antioxidative activity and membrane permeability of phenolic compounds seemed to be essential for the inhibition of LTB₄ release. In addition, the C₄-carbonyl group seemed to be important for strongly inhibiting LTB₄ release.     

Interference of carbidopa and other catechols with reactions catalyzed by peroxidases

Background

A number of compounds, including ascorbic acid, catecholamines, flavonoids, p-diphenols and hydrazine derivatives have been reported to interfere with peroxidase-based medical diagnostic tests (Trinder reaction) but the mechanisms of these effects have not been fully elucidated.

Luteolin,a flavone,does not suppress postprandial glucose absorption through an inhibition of alpha-glucosidase action

In order to clarify the postprandial glucose suppression via alpha-glucosidase (AGH) inhibitory action by natural compounds, flavonoids were examined in this study. Among the flavonoids (luteolin, kaempferol, chrysin, and galangin), luteolin showed the potent maltase inhibitory activity with the IC50 of 2.3 mM, while less inhibitions were observed against sucrase. In addition, the effects of maltase inhibition by flavonoids were observed in the descending order of potency of luteolin > kaempferol > chrysin > galangin. Apparently, the AGH inhibition power greatly increased with the replacement of hydroxyl groups at 3' and 4'-position of the B-ring. However, the inhibitory power of luteolin was poorer than a therapeutic drug (acarbose: IC50; 430 nM). As a result of a single oral administration of maltose or sucrose (2 g/kg) in SD rats, no significant change in blood glucose level with the doses of 100 and 200 mg/kg of luteolin was observed. These findings strongly suggested that luteolin given at less than 200 mg/kg did not possess the ability to suppress the glucose production from carbohydrates through the inhibition of AGH action in the gut.     

The Use of Salt Hydrates as Water buffers To Control Enzyme Activity in Organic Solvents

The conversion of o-diphenols to o-quinones was carried out in chloroform using a dry powder prepared from mushrooms as the catalyst. Several salt hydrates proved effective in supplying the small amount of water necessary for catalysis. The efficacy of the hydrates was related to their water activities and their use provided a convenient method for controlling water activity in nearly non-aqueous conditions.