Effect of isoquinoline alkaloids on soybean lipoxygenase activity in vitro

Novel isoquinoline alkaloids were evaluated for their effect on the kinetics of a soybean lipoxygenase type I using linoleic acid as substrate. Some of these alkaloids were found to increase the initial reaction velocity, this property seems related to phenolic groups present in the molecule. The effect of these compounds on the soybean lipoxygenase activity was compared to that of others products which are known to affect this reaction. A reaction mechanism is then proposed: it appeared, in this reaction a correlative structure-activity of phenolic compounds we tested.     

Formation of free radical metabolites in the reaction between soybean lipoxygenase and its inhibitors. An ESR study

Recent studies showed that soybean lipoxygenase inhibitors like phenidone and nordihydroguaiaretic acid (NDGA) reduce the catalytically active ferric lipoxygenase to its inactive ferrous form. Addition of 13(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid (13-HPOD) regenerated the active ferric form. In this paper, it is shown that in such a system the inhibitors are oxidized to free-radical metabolites. Incubation of soybean lipoxygenase and linoleic acid with p-aminophenol, catechol, hydroquinone, NDGA, or phenidone resulted in the formation of the one-electron oxidation products of these compounds. Free-radical formation depended upon the presence of the lipoxygenase and 13-HPOD. The free radicals were detected by ESR spectroscopy, and their structure was confirmed by analysis of the spectra, using a computer correlation technique. These data support the proposed mechanism for the inhibition of lipoxygenase by phenolic antioxidants.     

Inhibitory effect on soybean lipoxygenase and docking studies of some secondary metabolites, isolated from Origanum vulgare L. ssp. hirtum

In this study, five secondary metabolites (caffeic acid, rosmarinic acid, lithospermic acid B, 12-hydroxyjasmonic acid 12-O-beta-glucoside and p-menth-3-ene-1,2-diol 1-O-beta-glucopyranoside) isolated from the polar extracts of the plant Origanum vulgare L. ssp. hirtum, were tested in vitro for their ability to inhibit soybean lipoxygenase. Among the examined compounds, lithospermic acid B demonstrated the best inhibitory activity on soybean lipoxygenase with IC50 = 0.1 mM. Docking studies have been undertaken as an attempt for better understanding the interactions of these compounds within the active site of soybean lipoxygenase. The predicted binding energy values correlated well with the observed biological data.     

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.     

Inhibitory effect on soybean lipoxygenase and docking studies of some secondary metabolites,isolated from Origanum vulgare L. ssp. hirtum

In this study, five secondary metabolites (caffeic acid, rosmarinic acid, lithospermic acid B, 12-hydroxyjasmonic acid 12-O-β-glucoside and p-menth-3-ene-1,2-diol 1-O-β-glucopyranoside) isolated from the polar extracts of the plant Origanum vulgare L. ssp. hirtum, were tested in vitro for their ability to inhibit soybean lipoxygenase. Among the examined compounds, lithospermic acid B demonstrated the best inhibitory activity on soybean lipoxygenase with IC₅₀ = 0.1 mM. Docking studies have been undertaken as an attempt for better understanding the interactions of these compounds within the active site of soybean lipoxygenase. The predicted binding energy values correlated well with the observed biological data.     

Pheomelanin Production by the Lipoxygenase-Catalyzed Oxidation of 5-S-Cysteinyldopa and 5-S-Cysteinyldopamine

5-S-cysteinyl-dopa (cysdopa) and 5-S-cysteinyl-dopamine (cysdopamine) are oxidized in vitro by soybean lipoxygenase (LOX) in the presence of hydrogen peroxide giving rise to the corresponding pheomelanins. The reaction is activated by caffeic acid and other catechols, suggesting a cofactor role for these compounds. The activating effect is proportional to the concentration of the cofactor, with a saturation profile. The activation extent of the various cofactors is directly related to LOX affinity for the same compounds. The possible implications of the peroxidative action of LOX in Parkinson's disease and in aging are discussed.     

The specificity of lipoxygenase-catalyzed lipid peroxidation and the effects of radical-scavenging antioxidants

The oxidation of low density lipoprotein (LDL) by lipoxygenase has been implicated in the pathogenesis of atherosclerosis. It has been known that lipoxygenase-mediated lipid peroxidation proceeds in general via regio-, stereo- and enantio-specific mechanisms, but that it is sometimes accompanied by a share of random hydroperoxides as side reaction products. In this study we investigated the oxidation of various substrates (linoleic acid, methyl linoleate, phosphatidylcholine, isolated LDL, and human plasma) by the arachidonate 15-lipoxygenases from rabbit reticulocytes and soybeans aiming at elucidating the effects of substrate, lipoxygenase and reaction milieu on the contribution and mechanism of random oxidation and also the effect of antioxidant. The specific character of the rabbit 15-lipoxygenase reaction was confirmed under all conditions employed here. However, the specificity by soybean lipoxygenase was markedly dependent on the conditions. When phosphatidylcholine liposomes and LDL were oxygenated by soybean lipoxygenase, the product pattern was found to be exclusively regio-, stereo-, and enantio-random. When free linoleic acid was incorporated into PC liposomes and oxidized by soybean lipoxygenase, the free acid was specifically oxygenated, whereas esterified linoleate gave random oxidation products exclusively. Radical-scavenging antioxidants such as alpha-tocopherol, ascorbic acid and 2-carboxy-2,5,7,8-tetramethyl-6-chromanol selectively inhibited the random oxidation but did not influence specific product formation. It is assumed that the random reaction products originate from free radical intermediates, which have escaped the active site of the enzyme and thus may be accessible to radical scavengers. These data indicate that the specificity of lipoxygenase-catalyzed lipid oxidation and the inhibitory effects of antioxidants depend on the physico-chemical state of the substrate and type of lipoxygenase and that they may change completely depending on the conditions.     

Medicinal plants: Concentrators and superconcentrators of copper and its role in metabolism of these species

With the combination of the atomic absorption method and spectrophotometry, we conducted the testing of medicinal plants of Russian flora (approximately 200 species) on the content of copper (Cu). We revealed 36 species—concentrators and superconcentrators of this element. The capability of these species to accumulate Cu is compared with the synthesis of physiologically active compounds (PAC), among which alkaloids and phenolic compounds prevail. The stimulating influence of Cu on the formation and accumulation of alkaloids of main structural types—derivatives of chinolysidine, isochinoline, tropane, and indole—is established. The data about the role of Cu-containing enzymes in the metabolism of alkaloids, as well as of phenolic compounds, are reviewed on the example of flavonoids. The role of concentrated copper in the medicinal effect of medicinal plants and, thus, the appearing perspective to widen their application spectrum, especially in the cases when the orientation of the action of PAC and Cu are different, is discussed.     

Enzymatic mass determination of high-specific-radioactivity 3H- and 14C-labeled polyunsaturated fatty acids

The soybean lipoxygenase reaction has been applied to calculating the specific activities of 3H- and 14C-labeled polyunsaturated fatty acids of the omega 3 and omega 6 families. The extent of the soybean lipoxygenase reaction with polyunsaturated fatty acids was determined by measuring the increase in absorbance at 234 nm. A salient feature of this application of of the soybean lipoxygenase assay involves the use of a solution which contains highly purified [1-14C]arachidonic acid of known specific radioactivity as a convenient and versatile standard. Because the amount of arachidonic acid in this standard can be easily measured by liquid scintillation counting, the problems of accurately weighing liquid fatty acid standards are avoided.     

Is ATP a substrate for 15-lipoxygenase?

Lipoxygenases catalyze peroxidation of polyunsaturated fatty acids containing the 1-cis, 4-cis pentadiene structure. Linoleic (18:2), linolenic (18:3), and arachidonic (20:4) acids are the predominant substrates for this class of enzymes. Effects of 15-lipoxygenase on the hydrolysis of adenosine 5'-triphosphate were investigated in vitro using soybean lipoxygenase and adenosine 5'-[gamma-32P]triphosphate. The amount of inorganic phosphate released from adenosine 5'-triphosphate was dependent upon enzyme as well as substrate concentrations, pH, and the duration of incubation. The ATPase activity with a Vmax value of 3.3 mumol.mg protein-1.h-1 and a Km value of 5.9 mM was noted in the presence of different concentrations of ATP at pH = 7.4. Phenidone, a lipoxygenase inhibitor, had no effect on this reaction. These findings suggest that soybean lipoxygenase catalyzes the release of inorganic phosphate from ATP primarily via hydrolysis.     

Activity of soybean lipoxygenase isoforms against esterified fatty acids indicates functional specificity

In soybean (Glycine max L.) vegetative tissue at least five lipoxygenase isozymes are present. Four of these proteins have been localized to the paraveinal mesophyll, a layer of cells that is thought to function in assimilate partitioning. In order to determine the role of the lipoxygenase isozymes within the soybean plant, the leaf lipoxygenases were cloned into bacterial expression vectors and expressed in Escherichia coil. The recombinant lipoxygenases were then characterized as to substrate preference, pH profiles for the most common plant lipoxygenase substrates, linoleic acid, and alpha-linolenic acid, and the reaction products with the substrates linoleic acid, alpha-linolenic acid, arachidonic acid, gamma-linolenic acid, and the triacylglycerol trilinolein. All five enzymes were shown to be (13S)-lipoxygenases against linoleic acid. The results of these assays also indicate that two of these isozymes are highly active against esterified fatty acid groups, such as those found in triacylglycerols. Lipid analysis of leaves from plants subjected to sink limitation conditions indicates that the soybean leaf lipoxygenases are active in vivo against both free fatty acids and esterified lipids, and that the quantities of lipoxygenase products found in leaf tissue show a positive correlation with the level of lipoxygenase in the leaf. Implications for the putative role of these enzymes in the paraveinal mesophyll are discussed.     

Phenolic compounds with antioxidant activity from Anthemis tinctoria L. (Asteraceae)

From the aerial parts of Anthemis tinctoria L. subsp. tinctoria var. pallida DC. (Asteraceae), one new cyclitol glucoside, conduritol F-1-O-(6'-O-E-p-caffeoyl)-beta-D-glucopyranoside (1), has been isolated together with four flavonoids, nicotiflorin (2), isoquercitrin (3), rutin (4) and patulitrin (5). The structures of the isolated compounds were established by means of NMR, MS, and UV spectral analyses. Methanolic extract and pure isolated compounds were examined for their free radical, scavenging activity, using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free stable radical, and for their inhibitory activity toward soybean lipoxygenase, using linoleic acid as substrate. Compounds 1 and 5 showed a strong scavenging effect in the DPPH radical assay. In addition 5 also exhibited high inhibitory activity on soybean lipoxygenase.     

A novel synthesis of 3-aryl coumarins and evaluation of their antioxidant and lipoxygenase inhibitory activity

A series of coumarin analogues bearing a substituted phenyl ring on position 3 were synthesized via a novel methodology, through an intermolecular condensation reaction of 2-hydroxyacetophenones and 2-hydroxybenzaldehyde, with imidazolyl phenylacetic acid active intermediates. The in vitro antioxidant activity of the synthesized compounds was evaluated using two different antioxidant assays (radical scavenging ability of DPPH stable free radical and inhibition of lipid peroxidation induced by the thermal free radical AAPH). Moreover, the ability of the compounds to inhibit soybean lipoxygenase was determined as an indication of potential anti-inflammatory activity.     

A novel approach to study of action of water-insoluble inhibitors of enzymic reactions

The effect of water-insoluble compounds on enzyme catalytic properties was studied using a colloidal solution of water in organic solvent as reaction medium. In this microheterogeneous medium enzyme is entrapped into hydrated reversed micelles of a surfactant, the dimensions of the internal hole of the micelles being dependent on the ratio of water to surfactant. At sufficiently low values of this ratio the molecule of entrapped enzyme has limited mobility in the micelle. Because of this the interaction of the enzyme with water-insoluble compound which is added in assay solution and intercalated in the surface layer of the micelle may be manifested. The suggested method was used to study the inhibitory action of dihydroriboflavin esters on D-amino acid oxidase from pig kidney and soybean lipoxygenase. The reaction medium was hydrated reversed micelles of Aerosol OT in octane. The method of sedimentation in an analytical ultracentrifuge has shown the dihydroriboflavin esters to be completely included into reversed micelles.     

Components of Stem Barks of Winchia calophylla A. DC. and Their Bronchodilator Activities

The Dai medicinal plant Winchia calophylla A. DC. (Apocynaceae) has efficacy as an anticough and anti-asthmatic medication. In order to investigate its relative bioactive components, we studied the chemical constituents of this plant. Using repeated column chromatography, 28 compounds, including loganin, six phenolic compounds, 17 indole alkaloids, three pyridine alkaloids, and a quinoline alkaloid,were isolated from the stem barks of W. calophylla. Loganin, paeonol, N (4)-methyl akuammicine, and cantleyine exhibited a moderate relaxation effect on isolated smooth muscles of guinea-pig tracheal spirals and lung strips and may be the bioactive components responsible for the bronchodilation produced by W. calophylla.     

Antioxidant effects of dopamine and related compounds.

The antioxidant and free radical scavenging effects of dopamine, noradrenaline, tyramine, and tyrosine were investigated and compared with alpha-tocopherol. The antioxidant effect of dopamine and its related compounds on peroxidation of linoleic acid were in the order of dopamine > alpha-tocopherol = tyramine > tyrosine > noradrenaline as measured by the thiocyanate method. These amine compounds had reducing power, and a scavenging effect on reactive oxygen species, i.e., superoxide anion and hydroxyl radical. The results for reducing power and scavenging effect of these amine compounds had a similar trend as their inhibition of linoleic acid peroxidation. The antioxidant activity of these amine compounds in soybean oil was also evaluated by the Rancimat method. The induction time to reach 100 meq/kg peroxide value (POV) of soybean oil for dopamine, alpha-tocopherol, tyramine, tyrosine, noradrenaline, and control were 9.0, 8.2, 8.0, 6.4, 4.6, and 4.3 h, respectively. The antioxidant efficacy of amine compounds seems to be correlated with the numbers of hydroxy groups and their position on the phenolic ring.     

Components of Stem Barks of Winchia calophylla A. DC. and Their Bronchodilator Activities

The Dai medicinal plant Winchia calophylla A. DC. (Apocynaceae) has efficacy as an anticough and anti-asthmatic medication. In order to investigate its relative bioactive components, we studied the chemical constituents of this plant. Using repeated column chromatography, 28 compounds, including loganin, six phenolic compounds, 17 indole alkaloids, three pyridine alkaloids, and a quinoline alkaloid, were isolated from the stem barks of W. calophylla. Loganin, paeonol, N (4)-methyl akuammicine, and cantleyine exhibited a moderate relaxation effect on isolated smooth muscles of guinea-pig tracheal spirals and lung strips and may be the bioactive components responsible for the bronchodilation produced by W. calophylla.     

Mung bean lipoxygenase in the production of a C6-aldehyde. Natural green-note flavor generation via biotransformation

Mung bean was investigated as a novel source of lipoxygenase in the natural production of the green-note aroma compound hexanal. Lipoxygenase extracted from mung bean catalyzed the oxidative reaction of linoleic acid, after which the intermediate hydroperoxide compound was split via green bell pepper hydroperoxide lyase to produce hexanal. In comparison to soybean lipoxygenase, mung bean lipoxygenase was found to be a good substitute as it produced 15.4 mM (76% yield) hexanal while soybean gave 60% yield. The mung bean pH profile comprised a wide peak (optimum pH 6.5) representing lipoxygenase-2 and lipoxygenase-3 isozymes, whereas two narrower peaks representing lipoxygenase-1 and lipoxygenase-2/3 isozymes were observed for soybean (optimum pH 10). Extraction at pH 4.5 was preferred, at which specific lipoxygenase activity was also the highest.     

Appearance of new lipoxygenases in soybean cotyledons after germination and evidence for expression of a major new lipoxygenase gene

The appearance and subsequent disappearance of lipoxygenase activity at pH 6.8 in germinated cotyledons of soybean (Glycine max [L.]) was shown using a variant soybean cultivar (Kanto 101) that lacks the two lipoxygenase isozymes, L-2 and L-3, that are present in dry seeds of a normal soybean cultivar (Enrei). Three new lipoxygenases, designated lipoxygenase L-4, L-5, and L-6, were purified using anionic or cationic ion exchange chromatography. The major lipoxygenase in 5-day-old cotyledons of the variant soybean was lipoxygenase L-4. Lipoxygenases L-5 and L-6 preferentially produced 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid (13S-HPOD) as a reaction product of linoleic acid, whereas lipoxygenase L-4 produced both 13S-HPOD and 9(S)-hydroperoxy-10(E), 12(Z)-octadecadienoic acid. All three isozymes have pH optima of 6.5, no activity at pH 9.0, and preferred linolenic acid to linoleic acid as a substrate. Partial amino acid sequencing of lipoxygenase L-4 showed that this isozyme shares amino acid sequence homology with lipoxygenases L-1, L-2, and L-3 but is not identical to any of them. This indicates that a new lipoxygenase, L-4, is expressed in cotyledons.     

Effect of endocannabinoids on soybean lipoxygenase-1 activity

Endocannabinoids appear to be involved in a variety of physiological processes. Lipoxygenase activity has been known to be affected by unsaturated fatty acids or phenolic compounds. In this study, we examined whether endocannabinoids containing both N-acyl group and phenolic group can affect the activity of soybean lipoxygenase (LOX)-1, similar to mammalian 15-lipoxygenase in physicochemical properties. First, N-arachidonoyl dopamine and N-oleoyl dopamine were found to inhibit soybean LOX-1-catalyzed oxygenation of linoleic acid in a non-competitive manner with a K_i value of 3.7 μM and 6.2 μM, respectively. Meanwhile, other endocannabinoids failed to show a remarkable inhibition of soybean LOX-1. Separately, N-arachidonoyl dopamine and N-arachidonoyl serotonin were observed to inactivate soybean LOX-1 with K_in value of 27 μM and 24 μM, respectively, and k₃ value of 0.12 min⁻¹ and 0.35 min⁻¹, respectively. Furthermore, such an inactivation was enhanced by ascorbic acid, but suppressed by 13(S)-hydroperoxy-9,11-octadecadienoic acid. Taken together, it is proposed that endocannabinoids containing polyunsaturated acyl moiety and phenolic group may be efficient for the inhibition as well as inactivation of 15-lipoxygenase.