The present work examines leaching of LIPOXYGENASE-1 from soybean flour employing stirred tank vessel. The effect of operating parameters, impellers speed, operational period, temperature, pH and scale-up were considered. The acetic acid pH: 5.2 increased the leaching of LIPOXYGENASE- 1. The sensitivity of agitator speed and geometrical-scale-up on the enzyme leaching has been conducted. The effect of agitator speed on the geometrically-scaled- up reactor has shown that similar amount of enzyme is leached at lower speed. The result is in para with the conventional system. The importance of operational period from 5 to 50 min on the enzyme leaching was evaluated and higher enzyme leaching obtained within 10 min. of operation. The Sigmaplot-6 for statistical verification and developing a correlation for the enzyme leaching was used.

Objective(s): Allylbenzenes have been recently developed as inhibitors of LIPOXYGENASEs. They decrease peroxidation activity via mimicking 1, 4-unsaturated bonds of fatty acids by their allyl portion. We designed and synthesized new derivatives of allyl benzenes (6a-f) with isopropoxy and amide substituents at ortho and meta positions towards allyl group, respectively. The inhibitory potency of the synthetized allylbenzenes against soybean 15-LIPOXYGENASE (SLO) and subsequently structureactivity relationships was assessed. Materials and Methods: 3-allyl-4-isopropoxybenzenamine (5) as starting material was synthesized by coupling of 4-nitropheol with allyl bromide, performing Claisen rearrangement and finally reduction of the nitro moiety. Final products 6a-f were prepared via amidation of 5 with the desired acyl chloride. Results: Among the compounds, N-(3-allyl-4-isopropoxyphenyl)adamantan carboxamide (6f) potentially showed best inhibition (IC50 = 1. 35 μ M) while 6a with cyclopropyl carboxamide moiety was the weakest inhibitor and 6e with phenyl carboxamide moiety showed no effect. Energy minimized 3D structures of the compounds were docked into the active site pocket of SLO. For the aliphatic amides, docking results showed compatibility between inhibitory potency and average Ki of the cluster conformers, in which their allyl moiety oriented towards SLO iron core. For the aliphatic analogs, by enlargement of the amide moiety size the inhibitory potency was increased. Conclusion: Docking results showed that orientation of the amide and allyl moieties of the inhibitors in the active site pocket is the major factor in inhibitory potency variation. Based on the mentioned orientation, for cycloaliphatic amides, by enlargement of the amide moiety both inhibition potency and calculated binding energy increases.

Objective(s): 15-LIPOXYGENASEs are one of the iron-containing proteins capable of performing peroxidation of unsaturated fatty acids in animals and plants. The critical role of enzymes in the formation of inflammations, sensitivities, and some cancers has been demonstrated in mammals. The importance of enzymes has led to the development of mechanistic studies, product analysis, and synthesis of inhibitors. Materials and Methods: The inhibitory activity of all synthetic compounds against SLO (soybean 15-LIPOXYGENASE: L1; EC 1, 13, 11, 12) was determined using the peroxide formation method. In this method, the basis of evaluation of LIPOXYGENASE activity is measuring the concentration of fatty acid peroxide. All measurements were compared with 4-methyl-2-(4-methylpiperazinyl)pyrimido[4, 5-b] benzothiazine (4-MMPB) as one of the known LIPOXYGENASE inhibitors. The radical scavenging ability of all synthetic compounds using stable free radicals (DPPH: 2, 2-diphenyl-1-picrylhydrazyl) was measured for further investigation. Results: In this study, a series of esters from phenolic acids with terpenoid alcohols was synthesized and their inhibitory potency against soybean 15-LIPOXYGENASE and their free radical scavenging properties were determined. Among the synthetic compounds, adamantyl protocatetuate 2j and bornyl protocatetuate 2o showed the most potent inhibitory activity with IC50 values of 0. 95 and 0. 78 μ m, respectively. Conclusion: By changing the alcohol and acyl portions of stylosin, it was found that electronic properties play main role in LIPOXYGENASE inhibition potency in contrast with steric features. Insertion of more reductive phenolic moiety such as catechuate and gallate lead to more LIPOXYGENASE inhibition potency of the esters as observed in their radical scavenging activity.

Background: LIPOXYGENASE enzymes play an important role in various mechanisms of organisms. So far, many studies on human and other organisms LIPOXYGENASE activity have been conducted. In eukaryotes, this enzyme converts arachidonic acid to a variety of inflammatory mediators. For example, leukotrienes are products of this enzyme reaction. This inflammatory mediator plays an important role in the healing process. Recent studies have shown that the LIPOXYGENASE enzyme extracted from an amphibious (Ambystoma mexicanum) is more effective in the healing process in comparison with human LIPOXYGENASE. Like in the case of other enzymes, the first step for enzyme identification and characterization is to produce a large amount of purified enzyme, but the recombinant production of these proteins in bacterial expression system has not yet been reported. Therefore, in the present study we have cloned and expressed LIPOXYGENASE axolotls (LOXe) in Escherichia coli (E. coli) BL21.Methods: The sequence encoding LOXe was designed based on the amino acid sequence of the protein and then, codon optimized in order to ensure the maximum expression in E. coli. At the next step, the synthetic DNA encoding LOXe inserted into the pUC57 vector using appropriate restriction sites and then, subcloned in the pET21-a, an expression vector in order to high production of the protein in bacteria. Recombinant vector transformed to E. coli BL21 as an expression host and expression of 71kDa protein LOXe (623 amino acids) was induced in the presence of IPTG (Isopropyl-beta-D-thiogalactopyranoside).Findings: The cloning of LOXe was performed successfully and possibility of expression of this enzyme in E. coli was confirmed.Conclusion: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that LOXe protein over-expressed successfully in E. coli cytoplasm.

Background and objective: Umbelliprenin, the natural prenylated coumarin distributed in the plants of apiaceae family, has shown various biological activities, especially as a cancer chemo preventive agent. In the present study, umbelliprenin, was examined for in vitro antioxidant activity, in vitro inhibitory activity against LIPOXYGENASE, and in vivo anti-inflammatory activity.Methods: The applied tests were interaction with 1,1-diphenyl-2-picryl-hydrazyl (DPPH) stable free radical, inhibition of lipid per oxidation, inhibition of soybean LIPOXYGENASE and in vivo inhibition of the carrageenin-induced rat paw edema.Results: Umbelliprenin did not show any significant antioxidant activity but exhibited a remarkable and potent inhibition against soybean LIPOXYGENASE (IC50=0.0725 mM). This compound, in the in vivo anti-inflammatory test, could also inhibit the carrageenin induced paw edema significantly (39%).Conclusion: The observed inhibition of LIPOXYGENASE may be a plausible mechanism for the potent cancer chemo preventive activity of umbelliprenin and may pose this compound as a valuable agent for the treatment of inflammatory diseases.