Improved Bioactivity of the Natural Product 5-Lipoxygenase Inhibitor Hyperforin by Encapsulation into Polymeric Nanoparticles.

Hyperforin, a highly hydrophobic prenylated acylphloroglucinol from the medical plant St. John's Wort, possesses anti-inflammatory properties and suppresses the formation of proinflammatory leukotrienes by inhibiting the key enzyme 5-lipoxygenase (5-LO). Despite its strong effectiveness and the unique molecular mode of interference with 5-LO, the high lipophilicity of hyperforin hampers its efficacy in vivo and, thus, impairs its therapeutic value, especially because of poor water solubility and strong plasma (albumin) protein binding. To overcome these hurdles that actually apply to many other hydrophobic 5-LO inhibitors, we have encapsulated hyperforin into nanoparticles (NPs) consisting of acetalated dextran (AcDex) to avoid plasma protein binding and thus improve its cellular supply under physiologically relevant conditions. Encapsulated hyperforin potently suppressed 5-LO activity in human neutrophils, but it failed to interfere with 5-LO activity in a cell-free assay, as expected. In the presence of human serum albumin (HSA), hyperforin was unable to inhibit cellular 5-LO activity, seemingly because of strong albumin binding. However, when encapsulated into NPs, hyperforin caused strong inhibition of 5-LO activity in the presence of HSA. Together, encapsulation of the highly hydrophobic hyperforin as a representative of lipophilic 5-LO inhibitors into AcDex-based NPs allows for efficient inhibition of 5-LO activity in neutrophils in the presence of albumin because of effective uptake and circumvention of plasma protein binding.

Tagetnoic acid, a new lipoxygenase inhibitor peroxy fatty acid from Tagetes minuta growing in Saudi Arabia.

A new peroxy fatty acid, tagetnoic acid (5) [4-((3S,6S)-6-((3E,8E)-octadeca-3,8-dien-1-yl)-3,6-dihydro-1,2-dioxin-3-yl)butanoic acid] and four known metabolites: ecliptal (5-formyl-α-terthiophene) (1), 5-(4-hydroxybut-1-ynyl)-2,2'-bithiophene (2), 22,23-dihydrospinasterone (3), and stigmasterol (4) were separated from the n-hexane fraction of the aerial parts of Tagetes minuta L. (Asteraceae). Their chemical structures were verified using IR, UV, 2D and 1D NMR, and HRMS. Compounds 3-5 displayed potent lipoxygenase inhibitory potential with IC50s 2.26, 1.83, and 1.17 µM, respectively compared to indomethacin (IC50 0.89 µM). Moreover, molecular docking study revealed that the potent activity of 5 is due to H-bonding and hydrophobic interaction. The results of this study suggested that Tagetes minuta dietary consumption would be useful for the individuals at risk of acute and chronic inflammatory disorders.

Mechanism of Action and Interactions between Thyroid Peroxidase and Lipoxygenase Inhibitors Derived from Plant Sources.

This study focused on the effect of kaempferol, catechin, apigenin, sinapinic acid, and extracts from plants (i.e., parsley, cumin, mustard, green tea, and green coffee) on thyroid peroxidase (TPO) and lipoxygenase (LOX) activity, antiradical potential, as well as the result of interactions among them. Catechin, sinapinic acid, and kaempferol acted as a competitive TPO inhibitors, while apigenin demonstrated an uncompetitive mode of inhibitory action. Ethanol extracts from all plants acted as competitive TPO inhibitors, while, after in vitro digestion, TPO activation was found especially in the case of mustard (24%) and cumin (19.85%). Most importantly, TPO activators acted synergistically. The TPO effectors acted as LOX inhibitors. The most effective were potentially bioaccessible compounds from green tea and green coffee (IC50 = 29.73 mg DW/mL and 30.43 mg DW/mL, respectively). The highest free radical scavenging ability was determined for catechin and sinapinic acid (IC50 = 78.37 µg/mL and 84.33 µg/mL, respectively) and potentially bioaccessible compounds from mustard (0.42 mg DW/mL) and green coffee (0.87 mg DW/mL). Green coffee, green tea, cumin, and mustard contain potentially bioaccessible TPO activators that also act as effective LOX inhibitors, which indicate their potentially health-promoting effects for people suffering from Hashimoto's disease.

Relative Inhibitions of 5-Lipoxygenase and Myeloperoxidase and Free-Radical Scavenging Activities of Daidzein, Dihydrodaidzein, and Equol.

The effects of bioavailability and metabolic transformation on the biological activities of daidzein are relatively unknown. The effects of daidzein, dihydrodaidzein, and equol at physiologically relevant concentrations on the production of leukotriene B4 and F2-isoprostanes, and myeloperoxidase enzyme activity in freshly isolated human neutrophils were examined. Equol, at physiological concentrations, inhibited leukotriene B4 production (IC50-200 nmol/L) in human neutrophils significantly more than daidzein and dihydrodaidzein (IC50 values >1000 nmol/L). Daidzein, dihydrodaidzein, and equol did not affect the enzymatic hydrolysis of leukotriene A4 to leukotriene B4, suggesting that they exerted their inhibitory effects on the 5-lipoxygenase activity. Daidzein (IC50 = 600 nmol/L) protected against free radical peroxidation of arachidonic acid significantly more than did equol and dihydrodaidzein (IC50 values >1000 nmol/L). Equol also showed significantly greater inhibition of myeloperoxidase activity (IC50 = 450 nmol/L) when compared to daidzein and dihydrodaidzein. Equol accumulated within the human neutrophils at significantly higher concentrations than daidzein and dihydrodaidzein after incubation with the three compounds at physiologically relevant concentrations. Neutrophils were able to accumulate intracellular daidzein, dihydrodaidzein, and equol up to a concentration of ∼600 nmol/L. Our results provide in vitro evidence that the biological activities of daidzein are profoundly influenced by bioavailability and metabolic transformation.

A high throughput screen identifies potent and selective inhibitors to human epithelial 15-lipoxygenase-2.

Lipoxygenase (LOX) enzymes catalyze the hydroperoxidation of arachidonic acid and other polyunsaturated fatty acids to hydroxyeicosatetraenoic acids with varying positional specificity to yield important biological signaling molecules. Human epithelial 15-lipoxygenase-2 (15-LOX-2) is a highly specific LOX isozyme that is expressed in epithelial tissue and whose activity has been correlated with suppression of tumor growth in prostate and other epithelial derived cancers. Despite the potential utility of an inhibitor to probe the specific role of 15-LOX-2 in tumor progression, no such potent/specific 15-LOX-2 inhibitors have been reported to date. This study employs high throughput screening to identify two novel, specific 15-LOX-2 inhibitors. MLS000545091 is a mixed-type inhibitor of 15-LOX-2 with a Ki of 0.9+/-0.4 µM and has a 20-fold selectivity over 5-LOX, 12-LOX, 15-LOX-1, COX-1, and COX-2. MLS000536924 is a competitive inhibitor with a Ki of 2.5+/-0.5 µM and also possesses 20-fold selectivity toward 15-LOX-2 over the other oxygenases, listed above. Finally, neither compound possesses reductive activity towards the active-site ferrous ion.

The antioxidant effect of ß-caryophyllene protects rat liver from carbon tetrachloride-induced fibrosis by inhibiting hepatic stellate cell activation.

Plant-based whole foods provide thousands of bioactive metabolites to the human diet that reduce the risk of developing chronic diseases. ß-Caryophyllene (CAR) is a common constituent of the essential oil of numerous plants, vegetables, fruits and medicinal herbs, and has been used as a flavouring agent since the 1930 s. Here, we report the antioxidant activity of CAR, its protective effect on liver fibrosis and its inhibitory capacity on hepatic stellate cell (HSC) activation. CAR was tested for the inhibition of lipid peroxidation and as a free radical scavenger. CAR had higher inhibitory capacity on lipid peroxidation than probucol, α-humulene and α-tocopherol. Also, CAR showed high scavenging activities against hydroxyl radical and superoxide anion. The activity of 5-lipoxygenase, an enzyme that actively participates in fibrogenesis, was significantly inhibited by CAR. Carbon tetrachloride-treated rats received CAR at 2, 20 and 200 mg/kg. CAR significantly improved liver structure, and reduced fibrosis and the expression of Col1a1, Tgfb1 and Timp1 genes. Oxidative stress was used to establish a model of HSC activation with overproduction of extracellular matrix proteins. CAR (1 and 10 µm) increased cell viability and significantly reduced the expression of fibrotic marker genes. CAR, a sesquiterpene present in numerous plants and foods, is as a natural antioxidant that reduces carbon tetrachloride-mediated liver fibrosis and inhibits hepatic cell activation.

Antioxidant and anti-inflammatory effects of Scoparia dulcis L.

Different extracts were obtained from Scoparia dulcis L. (Scrophulariaceae) by successive extraction with hexane, chloroform, and methanol. These extracts exhibited significant antioxidant capacity in various antioxidant models mediated (xantine oxidase and lipoxygenase) or not mediated (2,2-diphenyl-picrylhydrazyl, ferric-reducing antioxidant power, ß-carotene bleaching, lipid peroxidation) by enzymes. The antioxidant activity of the extracts was related to their phytochemical composition in terms of polyphenol and carotenoid contents. The chloroform extract was richest in phytochemicals and had the highest antioxidant activity in the different antioxidant systems. All the extracts exhibited less than 50% inhibition on xanthine oxidase but more than 50% inhibition on lipid peroxidation and lipoxygenase. The extracts strongly inhibited lipid peroxidation mediated by lipoxygenase.

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.

Inhibition of potato lipoxygenase by linoleyl hydroxamic acid: kinetic and EPR spectral evidence for a two-step reaction.

The reaction mechanism of an electrophoretically pure potato tuber lipoxygenase (ptLOX) was studied by EPR spectroscopy. An EPR spectrum of the 'native' ptLOX recorded at 4.5+/-0.5 K showed signals of a high-spin (pseudo) axial Fe(3+) with a g-value of approx. 6.3+/-0.1 with a shoulder at g=5.9+/-0.1, and a rhombic Fe(3+) signal at g=4.35+/-0.05. When the enzyme was treated with a 2-fold molar excess of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE], a 3-fold increase in the integral intensity of the g=6.3 signal was observed, indicating that 25% of the native ptLOX iron was in ferrous state. The positional isomer 9(S)-HPODE caused similar spectral changes. Therefore the catalytic centre of ptLOX appears to accommodate both positional isomers of linoleic acid hydroperoxides in a manner that ensures proper alignment of their hydroperoxy groups with the iron centre of the enzyme. Treatment of the Fe(3+)-ptLOX form with a 3-fold molar excess of linoleyl hydroxamic acid (LHA) completely quenched the g=6.3 signal. Concurrently, a dramatic increase in the signal at g=4.35 was detected, which was attributed to a newly formed LHA-Fe(3+)-ptLOX complex. The spectral characteristics of the complex are similar to those of a 4-nitrocatechol-Fe(3+)-ptLOX complex. From these observations, we conclude that LHA did not reduce Fe(3+) to Fe(2+), but rather formed a LHA-Fe(3+)-ptLOX complex. Formation of such a complex may be responsible for the inhibitory activity of LHA, at least in the initial stages of enzyme inhibition. A prolonged 15 min incubation of the complex at 23+/-1 degrees C led to the partial quenching of the g=4.35 signal. The quenching is attributed to the reduction of Fe(3+)-ptLOX by LHA, with concomitant formation of its oxidation product(s). A kinetic scheme for the inhibition is proposed.

Characterization of 5-lipoxygenase inhibitors in biochemical and functional in vivo assays.

Several potent and selective inhibitors of 5-lipoxygenase (5-LO) have been recently developed with excellent activity in certain in vivo assays of leukotriene production. The efficacy of three such inhibitors that have been in clinical trials (zileuton, A-78773 and ZD2138) were evaluated in: 1) ex vivo whole blood assay, 2) dermal Arthus reaction, and 3) functional airway response. In addition, a model of eicosanoid production in rat lung was developed that provides a simple assay for evaluation of the biochemical efficacy of 5-LO inhibitors in the lung. Bronchoalveolar lavage of rat lung with calcium ionophore A23187 resulted in rapid and robust production of PGE2, 6-keto-PGF1 alpha, thromboxane (TxB2), and leukotriene B4 (LTB4). Supplementation of lavage fluid with archidonic acid markedly augmented production of all eicosanoids except LTB4. All three inhibitors were potent and selective blockers of LTB4 production in the ex vivo whole blood assay and in the dermal Arthus reaction. In contrast, higher doses of inhibitor were needed to block LTB4 production in the rat lung lavage model than were needed to block ex vivo whole blood LTB4 production when both end points were measured in the same animal. Similarly, zileuton and A-78733 were less effective in suppressing the functional airway response to antigen in sensitized guinea pigs, whereas both inhibitors were effective in suppressing LTB4 production in the ex vivo whole blood assay. These results demonstrate that different 5-LO inhibitors have markedly distinct efficacy for inhibition of leukotriene production, depending on the animal model.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of lipoxygenase activity in lentil protoplasts by monoclonal antibodies introduced into the cells via electroporation.

The isolation of lentil protoplasts and the transfer of anti-lipoxygenase monoclonal antibodies into plant protoplasts by electroporation is reported. The dependence of the efficiency of monoclonal antibody incorporation on the field strength is shown as well. The transferred immunoglobulins retained their functional and structural integrity and were able to inhibit the intracellular target enzyme, with a linear relationship between inhibition of lipoxygenase activity and amount of incorporated monoclonal antibody. Moreover, the inhibition of lipoxygenase activity was well correlated with the increase of protoplast viability.