Lipoxygenase assay

Compounds 111 having structural features of the dual cyclooxygenase (COX)/5-lipooxygenase (5-LO) inhibitor tepoxalin and the 5-LO inhibitor ABT-761 were prepared. Many of these hybrid compounds are potent COX and 5-LO inhibitors two compounds (111, r =McO, R = R" = R = H, R = NH2, R = Me and r = MeO, R = R = Me, R" = R = H, R = Cl) inhibited eicosanoid biosynthesis in an ex vivo assay, but neither improved on the main deficiency of tepoxalin, duration of 5-LO inhibitory activity (99BMCL979). Compounds 111 inhibit the production of arachidonic acid products associated with 5-lipoxygenase and cyclooxygenase and are useful in the treatment of inflammatory disorders (99USP5925769). 

Lopez-Nicolas JM, Bru R, Sanchez-Ferrer A and Garcia-Carmona F. 1994. An octaethylene glycol mon-ododecyl ether-based mixed micellar assay for lipoxygenase acting at neutral pH. Anal Biochem 221(2) 410-415. 

Isolation, Purification, Characterization, and Assay of Antioxy-genic Enzymes Isolation and characterization of superoxide dis-mutase, 105, 88 superoxide dismutase assays, 105, 93 assays of glutathione peroxidase, 105, 114 catalase in vitro, 105, 121 assays of lipoxygenase, 105, 126. 

A spectrophotometric assay procedure was used to measure the lipoxygenase isoenzyme activities of fresh and stored soya containing bread improver pastes and powders (77). 

A new class of compounds is reported to have dual inhibitory properties. They have a y-sultam skeleton and show potent inhibitory effects towards both COX-2 and 5-lipoxygenase as well as production of IL-1 in in vitro assays. These compounds have also proved to be effective in several animal arthritic models without any ulcerogenic activity. Among these compounds S-2474 ((E)-(5)-(3,5-di-fe/t.-butyl-4-hydroxy-benzylidene)-2-ethyl-1,2-isothiazolidine-1,1-dioxide) was selected as an anti-arthritic drug candidate and is now under clinical investigations (Inagaki et al., 2000). 

The assays presented in this section deal with the measurement of enzyme activity, which is expected to be proportional to the amount of active enzyme present in a sample, food or otherwise, unit ci.i is an overview of the important considerations in performing activity assays unitc 1.2 illustrates how these considerations are applied to the assay of a representative food-relevant glycosyl hydrolase. Chapters C2, C3, and C4 present the first units on particular types of activity assays. In Chapter C2, two units present peptidase activity assays that use either synthetic substrates (UNITC2.1) or common, commercially available protein substrates (unit C2.2). unitC3.i presents three different assays for lipase activity. unitc4.i presents assays for diphenol oxidases, and unitc4.2 for lipoxygenase. 

Enzymatic assay of lipid hydroperoxides A selective assay has been described for lipid hydroperoxides, based on the cyclooxygenase reaction with a sensitivity in the range of 20-200 pmol (Marshall et al., 1985 Pendleton and Lands, 1987 Lands, 1988). The substrate (15-HPETE) is not, however, commercially available and must therefore be synthesized from purified arachidonic acid by the method of Graft (1982) in which soya bean lipoxygenase is used to catalyse the stereospecific oxygenation of arachidonic acid. 

Linoleic acid is probably the best substrate for assay. It is readily dispersed at alkaline pH, and at acid or neutral pH it is no more difficult to suspend than the methyl ester or the glyceride. Although some species of lipoxygenase show more favorable action with esters than others, we know of no instance where such enzymes do not also act well on linoleic acid. The poor water solubility of linoleic acid and its changing dissociation in the pH range of interest are a disadvantage and usually require emulsifiers. The sulfate ester of linoleyl alcohol, which does not possess this disadvantage, has been proposed by Allen (108) and appears to be a welcome improvement. 

The overall modest yields achieved in these syntheses have recently been markedly improved by the use of the solid-phase copolymer of 4-vinylpyridine (P4-VP) (112) in the formation of the starting di-ynic esters. For example, when a suspension of P4-VP polymer in dichloromethane was stirred with the acid chloride from (1011 and then the propargyl alcohol (114), the ester (1151 was obtained excellent yield. By heating in xylene, (1151 underwent intramolecular cyclization to yield justicidin E(1041 and taiwanin C(1051 as the major products in addition, the isomers helioxanthin (1081 and retrohelioxanthin (1161 could also be isolated (Scheme 22) (113). Increased interest in these four lactone products has resulted from an assay which indicates 5-lipoxygenase inhibitory activity (114). 

Inhibition of PG biosynthesis by open chain diarylheptanoids was first reported by Itokawa and his coworkers (26, 31). Their results were implemented by studies on additional compounds first by Flynn (88) and later by Kinchi et al. (85). The latter group extended the assay of inhibitor activity also to the 5-lipoxygenase enzyme system. Their results are summarized in Table 2. Among macrocyclic diarylheptanoids only for garuganins (88-95), isolated from Garuga pinnata was some antiinflammatory activity claimed, but no specific data were disclosed (57, 58). 

As an assay for antioxidant activity, we examined the inhibition of lipoxygenase activity and radical scavenging activity using the a, a-diphenyl-P-picrylhydrazyl (DPPH) decolorization test. Aqueous, ethanolic and methanolic extracts of microalgae were used in the assays. 

Enzyme Purification. Broccoli contained sufficient levels of peroxidase, lipase and cystine lyase to permit their isolation in the amounts needed. Only traces of lipoxygenase and catalase were present. Activities (units/g vegetable see assay methods below) were peroxidase, 220 lipase, 12 lyase, 0.26. Catalase was M) units/g in broccoli compared to 19 in English green peas lipoxygenase was 2 units/g in broccoli compared to 110 in English green peas. Peroxidase, lipase and cystine lyase were purified by... 

Phenylethanoid glycosides from Forsythia fruits were assayed for their inhibitory effect on 5-lipoxygenase from rat peritoneal cells [54], Forsythiaside (27), suspensaside (28), acteoside (1) and p-hydroxyacteoside (29) showed a high inhibitory effect with IC50 of 2.50 x 10 M, 7.97 x 10 M, 5..27 x 10 M and 19.3 x 10 M, respectively. 

Slddlql and Tappel (W) were among the first to demonstrate the presence of lipoxygenase in peanuts. Using crude extracts and assaying them at pH 7.0, they found peanuts to contain only 1% of the lipoxygenase activity found in soybeans. This low... 

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