IC50and
1. Introduction
Lipoxygenases (EC 1.13.11.12) are non-heme iron-containing enzymes that catalyze the site-specific oxygenation of polyunsaturated fatty acids to produce hydroperoxides. Lipoxygenases are suggested to be involved in the early event of atherosclerosis by inducing plasma low-density lipoprotein (LDL) oxidation [1, 2]. Lipoxygenase inhibitors have also been suggested to be potential cancer chemopreventives [3,4]. On the other hand, lipid peroxidation is well known as one of the major factors in deterioration during the storage and processing of foods, because it can lead to the development of unpleasant rancid or off flavors as well as potentially toxic end products [5]. Hence, lipoxygenase inhibitors should have broad applications [6].
Lipoxygenase inhibitors act in different ways; by chelating the iron of the active site of the enzyme [7, 8] and/or by reducing the ferric form of the enzyme to an inactive ferrous form [9, 10] (Fig. 1). Resorcinol exhibits no effect on this enzyme, indicating that the pentadeca(en)yl tail portion seemed an essential element in eliciting the inhibitory activity. The available information now demonstrates that lipoxygenase inhibitors can be designed when appropriate head portions are selected. 5-Pentadeca(en)yl salicylic acids, commonly known as anacardic acids, were previously reported to inhibit the linoleic acid
2. Results
To begin with, ethylenediaminetetraacetic acid (EDTA,
A homologous series of alkyl (C1-C20) protocatechuates (3,4-dihydroxybenzoates) and their related derivatives were previously synthesized by one-step esterification utilizing DCC (
The synthesized alkyl protocatechuates (Fig. 2)were tested for their effects on the soybean lipoxygenase-1 catalyzed oxidation of linoleic acid (
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39.4 | Competitive | 19.7 |
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2.3 | Competitive | 0.98 |
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0.55 | Competitive | 0.23 |
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0.25 | – | – |
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0.15 | – | – |
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0.09 | – | – |
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0.06 | Slow-binding | – |
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0.05 | – | – |
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0.31 | – | – |
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0.42 | – | – |
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0.32 | – | – |
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0.1 | Slow-binding | – |
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0.09 | – | – |
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>100 | – | – |
The inhibitory activity of soybean lipoxygenase-1 was measured by two methods for comparison, because this enzyme seems to be sensitive to assay conditions. In the current study, linoleic acid (
Subsequently, the inhibition kinetics of soybean lipoxygenase-1 by octyl protocatechuate (
To investigate the inhibitory effect of dodecyl protocatechuate (
The plot for
Thus, analysis of data yielded the following values:
where E, S, I, and P denote enzyme, substrate, inhibitor (dodecyl protocatechuate) (
Subsequently, the progress curves of 13-HPOD (
Soybean lipoxygenase-1 was used if dodecyl protocatechuate (
Some lipoxygenases reduce the ferric form of the enzyme to an inactive ferrous form [9,10]. This enzyme reduction mechanism is directly measurable by electron spin resonance(ESR)and fluorescence and also is indirectly observable when the inhibitor-dependent decrease in 13-HPOD (
Since the head portion of alkyl protocatechuates is the same, the data are interpreted to mean that changes in the hydrophobic tail portions correlate and are responsible for this specific activity. As far as protocatechuic acid (
Based on the head and tail concept, octylgallate(3,4,5-trihydroxybenzoate) (
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Anacardic acid (C15:0) Dodecyl 3,4,5-trihydroxybenzoate Dodecyl 3,5-dihydroxybenzoate Dodecyl 3,4-dihydroxy-5-methoxybenzoate Dodecyl 3,4-dihydroxycinnamate Dodecyl 4-hydroxybenzoate Dodecyl 3-hydroxybenzoate |
14.3 0.07 60.3 10.5 3.3 200 200 |
The data obtained so far indicates that alkyl 3,4-dihydroxybenzoates can be expected as superior as lipoxygenase inhibitors similar to the alkyl 3,4,5-trihydroxybenzoates. It should be noted, however, that alkyl 3,4,5-trihydroxybenzoates were oxidized in part in basic condition. The lipoxygenase assay was performed using a UV spectrophotometer to detect the increase at 234 nm associated with the (2
The data obtained so far indicates that alkyl 3,4-dihydroxybenzoates can be expected as superior as lipoxygenase inhibitors similar to the alkyl 3,4,5-trihydroxybenzoates. It should be noted, however, that alkyl 3,4,5-trihydroxybenzoates were oxidized in part in basic condition. The lipoxygenase assay was performed using a UV spectrophotometer to detect the increase at 234 nm associated with the (2
In contrast to their DPPH scavenging activity, the lipoxygenase inhibitory activity of alkyl protocatechuates was found to correlate with their alkyl chain lengths and require a certain head portion. This indicates that lipoxygenase inhibitors can be designed when appropriate head portions are selected. For example, dodecyl 3,5-dihydroxybenzoate (
3. Discussion
Alkyl protocatechuate antioxidants may act in a variety of ways including direct quenching of reactive oxygen species, inhibition of enzymes involved in the production of the reactive oxygen species, and chelation of low valent metal ions such as Fe2+, Fe3+ or Cu2+. In connection with this, both pyrogallol (
Alkyl protocatechuates act as both lipoxygenase inhibitors and scavengers. Safety is a primary consideration for antioxidants in food products. After ingestion, the alkyl protocatechuates are likely hydrolyzed, at least in part, to protocatechuic acid and the corresponding alcohols that are common in edible plants. For example, pecan nuts contain protocatechuic acid as a one of predominant phenolic acids [28]. If alkyl protocatechuates can reach the sites where antioxidants are needed, a more lipophilic alkyl side chain may partition into lipophilic membranes of cells and organelles, where it presumably exerts its antioxidant activity, similar to the phytyl chain in tocopherols and tocotrienols [29]. The site of antioxidant location is known to be important, however, it is not clear if alkyl protocatechuates can reach, without being metabolized, the sites where antioxidants are needed for protection from oxidative damages. It should be noted, however, that the role of alkyl protocatechuates in the human body is unknown when orally ingested. It is not clear if alkyl protocatechuates are absorbed into the system through the intestinal tract and delivered to the places where lipoxygenase inhibitors are needed without being metabolized.
4. Materials and methods
Acknowledgements
The work was presented in part at the Symposium of Diet and the Prevention of Gender Related Cancers in Division of Agricultural and Food Chemistry for the 222nd ACS National Meeting in Chicago, Il.
Abbreviations
LDL, low-density lipoprotein; SAR, structure and activity relationships; NDGA, nordihydroguaiuretic acid; DPPH, 1,1-diphenyl-2-picrylhydrazyl; EDTA, ethylenediaminetetraacetic acid; IC50, the half maximal inhibitory concentration; DCC,
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