Lipoxygenases (EC 1.13.11.12) are suggested to be involved in the lipid peroxidation [27Shibata D, Axelrod B. Plant lipoxygenases J Lipid Mediat Cell Signal 1995; 12: 213-28.
[http://dx.doi.org/10.1016/0929-7855(95)00020-Q] ]. Hence, lipoxygenase inhibitors should have broad applications [28Richard-Forget F, Gauillard F, Hugues M, Jean-Marc T, Boivin P, Nicolas J. Inhibition of horse bean and germinated barley lipoxygenases by some phenolic compounds J Food Sci 1995; 60: 1325-9.]. Soybean lipoxygenase-1 is known to catalyze the oxygenation of polyenoic fatty acids containing a (1Z,4Z)-pentadiene system such as linoleic acid and linolenic acid to their 1-hydro- peroxy-(2E,4Z)-pentadiene products. In plants, the primary dioxygenation product is 13(S)-hydroperoxy-9Z,11E-octadienoic acid (13-HPOD) [29Grechkin A. Recent developments in biochemistry of the plant lipoxygenase pathway Prog Lipid Res 1998; 37: 317-52.
[http://dx.doi.org/10.1016/S0163-7827(98)00014-9] ]. Hence, the enzyme assay was usually performed using a UV spectrophotometer to detect the increase at 234 nm associated with the (2E,4Z)-conjugated double bonds newly formed in the product but not the substrate. In many previous reports, the data were obtained at pH 9 since soybean lipoxygenase-1 had its optimum pH at 9.0 [30Axelrod B, Cheesbrough TM, Laakso S. Lipoxygenase from soybeans Methods Enzymol 1981; 71: 441-57.
[http://dx.doi.org/10.1016/0076-6879(81)71055-3] ], but the absorption at 234 nm suffered from unstable baseline activity of unknown origin attributable to the presence of alkyl gallates at pH 9.0. This pseudoactivity of the blank control had to be subtracted from activity of the enzyme assay, making precise measurements difficult. Hence, lipoxygenase activity was measured as described previously [31Ha TJ, Nihei K, Kubo I. Lipoxygenase inhibitory activity of octyl gallate J Agric Food Chem 2004; 52: 3177-81.
[http://dx.doi.org/10.1021/jf034925k] [PMID: 15137872] ] in Tris-HCl buffer (0.1 M, pH 8.0) by the increase in absorbance at 234 nm from 30 to 90 sec after addition of the enzyme, using linoleic acid (18 µM) as a substrate. The results are listed in Table 2.

Table 2

IC₅₀, K_I, Log P Values and Inhibition Type of Alkyl Gallate against Soybean Lipoxygenase-1

The same series of alkyl gallates (2-9) were found to inhibit the linoleic acid peroxidation catalyzed by soybean lipoxygenase-1 without being oxidized. The inhibitory activity was a parabolic function of their lipophilicity and maximized with alkyl chain length between C₁₂ and C₁₆. Tetradecanyl (C₁₄) gallate exhibited the most potent inhibition with an IC₅₀ of 0.06 µM, followed by dodecyl (C₁₂) gallate with an IC₅₀ of 0.07 µM (Fig. 3). Octyl gallate (C₈) was previously described to inhibit this enzymatically catalyzed oxidation with an IC₅₀ of 1.3 µM. The inhibition kinetics analyzed by Lineweaver-Burk plots indicates that octyl gallate is a competitive inhibitor and the inhibition constant (K_I) was obtained as 0.54 µM (Fig. 4) [31Ha TJ, Nihei K, Kubo I. Lipoxygenase inhibitory activity of octyl gallate J Agric Food Chem 2004; 52: 3177-81.
[http://dx.doi.org/10.1021/jf034925k] [PMID: 15137872] ]. The inhibition of soybean lipoxygenase-1 of longer alkyl gallates is reversible but in combination with their ability to disrupt the active site competitively and to interact with the hydrophobic portion surrounding near the active site. In the case of tetradecanyl gallate, the enzyme quickly and reversibly binds this gallate and then its tetradecanyl group undergoes a slow interaction with the hydrophobic domain (Fig. 5, panel A). Apparently, the alkyl chain length is significantly related to the soybean lipoxygenase-1 inhibitory activity.

Fig. (3) Effects of dodecyl gallate (closed circle: 9) and tetradecyl gallate (open triangle: 10) on the activity of soybean lipoxygenase-1 for the catalysis of linoleic acid at 25 °C.

Fig. (4) Time-dependent inhibition of soybean lipoxygenase-1 in the presence of tetradecyl gallate (10). (A) Conditions were as follows: 40 μM linoleic acid, 1.3 nM lipoxygenase-1, and concentrations of tetradecyl gallate for curves 0-8 were 0, 0.04, 0.08, 0.1, 0.2, 0.4, 0.6, 1.0, and 4.0 µM. The kobs values at each inhibitor concentrations were determined by fitting the data to eq 1. (B) Dependence of the values for kobs on the concentration of tetradecyl gallate. The kobs values, determined in panel A, were fitted to eq 2.

Fig. (5) Time-dependent inhibition of soybean lipoxygenase-1 in the presence of tetradecyl gallate (10). (A) Conditions were as follows: 40 μM linoleic acid, 1.3 nM lipoxygenase-1, and concentrations of tetradecyl gallate for curves 0-8 were 0, 0.04, 0.08, 0.1, 0.2, 0.4, 0.6, 1.0, and 4.0 µM. The kobs values at each inhibitor concentrations were determined by fitting the data to eq 1. (B) Dependence of the values for kobs on the concentration of tetradecyl gallate. The kobs values, determined in panel A, were fitted to eq 2.

To further investigate the inhibitory effect of tetradecyl gallate (10) on dioxygenase enzyme, we assayed soybean lipoxygenase-1 activity with the inhibitor. Soybean lipoxygenase-1 showed time-dependent inhibition in the presence of dodecyl gallate (Fig.5, panel A). Increasing tetradecyl gallate concentrations led to the decrease in both the initial velocity (v_i) and the steady-state rate (v_s) in the progress curve. The progress curves obtained using various concentrations of the inhibitors were fitted to eq 1 to determine v_i, v_s, and k_obs. The plot for k_obs versus [I] are shown in panel B in Fig. (5). That plot showed a hyperbolic dependence on the concentration of the tetradecyl gallate, so the inhibition of lipoxygenase-1 by tetradecyl gallate followed a mechanism as the one illustrated below. The kinetic parameters, k₅, k₆, and K_i^app were derived from the plots by fitting the results to eq 2. Thus, analysis of data according to eq 2 yielded the following values: k₅ = 9.1(10^-3 s^-1, k₆ = 1.5(10^-3 s^-1, K_i^app = 0.48 µM. The kinetic model can be written as:

where E, S, I, and P denote enzyme, substrate, inhibitor (alkyl gallate) and product (13-HPOD), respectively. ES, EI and E^*I are respective complexes. Because k₅ is greater than k₆ the enzyme first quickly and reversibly binds with tetradecyl gallate and then undergoes a slow interaction of tetradecyl group with the hydrophobic portion near the active site.

Dodecyl 3,4-dihydroxybenzoate (11) inhibited linoleic acid peroxidation catalyzed by soybean lipoxygenase-1, whereas both dodecyl 3,5-dihydroxybenzoate (12) and dodecyl 4-hydroxybenzoate (13) showed little inhibitory activity, indicating that certain head portions are needed to elicit the inhibitory activity. Thus, catechol moiety seems to be important to elicit potent soybean lipoxygenase-1 inhibitory activity as a hydrophilic head portion [28Richard-Forget F, Gauillard F, Hugues M, Jean-Marc T, Boivin P, Nicolas J. Inhibition of horse bean and germinated barley lipoxygenases by some phenolic compounds J Food Sci 1995; 60: 1325-9., 32Whitman S, Gezginci M, Timmermann B, Holman TR. Structure-activity relationship studies of nordihydroguaiaretic acid inhibitors toward soybean, 12-human, and 15-human lipoxygenase J Med Chem 2002; 45: 2659-61.
[http://dx.doi.org/10.1021/jm0201262] [PMID: 12036375] ]. In addition, menthyl gallate (14), bornyl gallate (15) and decahydro-2-naphthyl gallate (16) also inhibited linoleic acid peroxidation catalyzed by soybean lipoxygenase-1, indicating that the hydrophobic tail portion seems to be flexible. As mentioned above, alkyl gallates were found to possess equally potent radical scavenging activity on DPPH radical. Apparently, intermediate free radicals are formed during the catalytic cycle of lipoxygenases [33Ruddat VC, Whitman S, Holman TR, Bernasconi CF. Stopped-flow kinetics investigations of the activation of soybean lipoxygenase-1 and the influence of inhibitors on the allosteric site Biochemistry 2003; 42: 4172-8.
[http://dx.doi.org/10.1021/bi020698o] [PMID: 12680771] ], but they remain tightly bound at the active site, thus not being accessible for free radical scavengers. In summary, alkyl gallates appear to combine both lipoxygenase inhibitory activity and free radical scavenging property in one agent and thus constitute effective antioxidants.

The one-electron reduction products of O₂, superoxide anion (O₂·-), and subsequently hydrogen peroxide (H₂O₂) and hydroxyl radical (HO·) derived from H₂O₂ and O₂·-, actively participate in the initiation of lipid peroxidation. Several oxidative enzymes in the human body produce free radicals. For example, xanthine oxidase produces the O₂·- radical as a normal product [34Grunfeld S, Hamilton CA, Mesaros S, et al. Rple of superoxide in the depressed nitric oxide production by the endothelium of genetically hypertensive rats Hepertesion 1995; 26: 854-7.]. In the control, superoxide anion reduces yellow nitroblue tetrazolium to blue formazan, so the generation of superoxide anion by the enzyme can be detected by measuring of the absorbance at 560 nm of formazan produced. Among the alkyl gallates tested, gallic acid was the most effective in scavenging superoxide anion generated by xanthine oxidase with an IC₅₀ of 2.6 µM. The inhibition kinetics analyzed by Lineweaver-Burk plots shows that gallic acid is a mixed type inhibitor (Fig. 6) and the inhibition constants, K_I and K_IS were obtained as 1.5±0.2 and 2.9±0.3 µM. Xanthine oxidase is a molybdenum-containing enzyme and converts xanthine to uric acid. This enzyme-catalyzed reaction is known to proceed via transfer of an oxygen atom to xanthine from the molybdenum center. Hence, formation of uric acid was also measured. As a result, gallic acid did not inhibit xanthine oxidase up to 200 µM, indicating that its potent antioxidant activity comes from scavenging superoxide radicals or modification of xanthine oxidase. Xanthine oxidase catalyzed formation of superoxide anion or hydrogen peroxide and uric acid but the reduced oxidase only catalyzed formation of hydrogen peroxide and uric acid [35Hille R, Massey V. Studies on the oxidative half-reaction of xanthine oxidase J Biol Chem 1981; 256: 9090-5.
[PMID: 6894924] ]. As the inhibition activity of superoxide anion by gallic acid was stronger than the scavenging activity of superoxide anion generated by PMS-NADH, the inhibition activity by gallic acid should comes from excursively reductive (oxidative) modification of xanthine oxidase bound by gallic acid as following scheme [36Masuoka N, Nihei K, Kubo I. Xanthine oxidase inhibitory activity of alkyl gallates Mol Nutr Food Sci 2006; 50: 725-31.]. The kinetic model can be written as:

where E, S and I denote enzyme, substrate and inhibitor (alkyl gallate), respectively. ES, EI and EIS are respective complexes.

Fig. (6) Lineweaver−Burk plots of superoxide anion generation by xanthine oxidase in the presence of gallic acid (1) at 25 °C, pH 10.0. Concentrations of gallic acid (1) for curves 0-3 were 0, 0.5, 1.0, and 2.0 µM, respectively.

Reaction of uric acid formation by xanthine oxidase is known to proceed via transfer of an oxygen atom to xanthine from the molybdenum center. X-ray crystallographic analysis of bovine milk xanthine oxidase indicated that one can model the binding of bicyclic substrates, e.g. with Phe 1009 perpendicular to the six-membered ring of xanthine and Phe 914 stacking flat on top of the substrate’s five-membered ring, which would then be able to form a covalent bond with one of the molybdenum ligands [37Enroth C, Eger BT, Okamoto K, Nishino T, Pai EF. Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion Proc Nat Acad Sci 2000; 97: 10723-8.
[http://dx.doi.org/10.1073/pnas.97.20.10723] [PMID: 11005854] [PMCID: PMC27090] ]. As the present study indicated that alkyl gallates having long chains were predominantly competitive inhibitors for xanthine, we deduced that dodecyl gallate binds with Phe 1009 perpendicular to the pyrogallol moiety and Phe 914 stacking flat on top of the dodecyl group and the dodecyl chain tightly interacts with hydrophobic protein pocket to act as an anchor, which is necessary to inhibit uric acid formation, as illustrated in Fig.(7). To confirm this postulation, the effect of menthyl gallate (14) and bornyl gallate (15) on xanthine oxidase was examined. Neither compound inhibited uric acid formation, indicating that both can not bind to the xanthine binding site since the bulky cyclic monoterpene moieties, which are exposed on the other side of the molecules, cannot be well embraced by the hydrophobic protein pocket in xanthine binding site.

Fig. (7) Binding of dodecyl gallate to xanthine binding site in xanthine oxidase. Dodecyl gallate binds with Phe 1009 perpendicular to the pyrogallol moiety and Phe 914 stacking flat on top of the dodecyl group. Amino acid residues around the alkyl chain of dodecyl gallate indicate the hydrophobic protein pocket, and the alkyl chain interacts with them.

The inhibition of uric acid formation by alkyl gallates was further examined at different concentration of oxygen. Octyl gallate, decyl gallate and dodecyl gallate were also predominantly competitive inhibitors of oxygen. As these gallates bound to xanthine binding site of xanthine oxidase, it indicated that alkyl gallates could bind to another site, which may be oxygen binding site (or superoxide anion generation site), in xanthine oxidase.

To study the effect of gallic acid and alkyl gallates on superoxide anion generation, we examined the scavenging activity of superoxide anion by alkyl gallates since the activity was well known. Superoxide anion was non-enzymatically generated in a PMS-NADH system. The scavenging activity of alkyl gallates for superoxide anion decreased as the alkyl side chain length increased. Differences in superoxide anion scavenging activity among alkyl gallates may be explained by the fact that an alkyl gallate with a shorter alkyl chain is a smaller molecule and more water soluble which allows it to easily react with the superoxide anion. However an alkyl gallate with a long alkyl chain hardly reacts with superoxide anion at low concentration but rapidly reacts at concentrations higher than 100 µM. It may explain that at low concentration alkyl gallate with a longer chain is less water soluble to hardly react with superoxide anion but at high concentration the amphipathic gallate aggregates to form a micelle which dissolves the components of the PMS-NADH system and the gallate in the micelles easily scavenges superoxide anion generated from the system [38McGovern SL, Caselli E, Grigorieff N, Shoichet BK. A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening J Med Chem 2002; 45: 1712-22.
[http://dx.doi.org/10.1021/jm010533y] [PMID: 11931626] ].

Effects of gallic acid and alkyl gallates on superoxide anion generation were examined under limited concentration of gallates to exclude influence of their scavenging activities. All gallates inhibited superoxide anion generation (Table 3), and the inhibition did not depend on alkyl chain length of gallates. It suggested that inhibition of superoxide anion generation with gallic acid and alkyl gallates having a short alkyl chain did not correspond to that of xanthine oxidase reaction itself. To confirm stoichiometry of xanthine oxidase reaction, oxygen consumption and hydrogen peroxide formation of the reaction were examined. No inhibition of oxygen consumption with gallic acid was observed, and the inhibition with alkyl gallates having a long chain was consistent with that of uric acid formation. No inhibition of hydrogen peroxide formation with gallic acid was also indicated. These results confirmed that gallic acid and alkyl gallates having short alkyl chain inhibited superoxide anion generation without inhibition of xanthine oxidase reaction.

Table 3

Kinetic Parameters of Gallic Acid and Its Alkyl Esters for Superoxide Anion Generation by Xanthine Oxidasea)

Xanthine oxidase-catalyzed reaction of oxygen to superoxide anion is known to proceed at the FAD-binding domain [37Enroth C, Eger BT, Okamoto K, Nishino T, Pai EF. Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion Proc Nat Acad Sci 2000; 97: 10723-8.
[http://dx.doi.org/10.1073/pnas.97.20.10723] [PMID: 11005854] [PMCID: PMC27090] ]. The binding site in the FAD-binding domain is considerably hydrophilic, and structure of the site is similar to that of vanillyl alcohol oxidase [39Fraaiji MW, van den Heuvel RHH, van Berkel WJH, Mattevi A. Structural analysis of flavinylation in vanillyl-alcohol oxidase J Biol Chem 2000; 275: 38654-8.], of which the isoalloxazine ring binds to a wide range of phenolic substrates. Compounds with a galloyl moiety bound to the isoalloxazine ring of FADH₂ in squalene epoxidase [40Abe I, Seki T, Noguchi H. Potent and selective inhibition of squalene epoxidase by synthetic galloyl esters Biochem Biophys Res Commun 2000; 270: 137-40.
[http://dx.doi.org/10.1006/bbrc.2000.2399] [PMID: 10733917] ]. As octyl, decyl and dodecyl gallates were predominantly competitive inhibitors of oxygen for uric acid formation and gallic acid and alkyl gallates equally inhibited superoxide anion generation, we deduced that galloyl moiety of gallic acid and alkyl gallates bound superoxide generation site in FAD-binding domain of xanthine oxidase. Binding of gallic acid and alkyl gallates to FADH₂ in the enzyme can easily reduce the enzyme, xanthine oxidase, and cannot prevent the binding of the first oxygen molecule to the site since oxygen is so small and reactive. We propose that FADH₂ in the reduced oxidase smoothly reduces first oxygen molecule to hydrogen peroxide (A pathway) rather than reducing the second oxygen molecule to superoxide anion (B pathway) as shown in Fig. (8).

Fig. (8) roposed mechanism of hydrogen peroxide formation by xanthine oxidase in the presence of gallic acid or alkyl gallates. Binding of gallates to the isoalloxazine ring in FADH2 reduces the enzyme, xanthine oxidase. FADH2 in the enzyme reduces oxygen to the flavin hydroperoxide and the hydroperoxide is smoothly reduced to hydrogen peroxide (A pathway).