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Characterization of the xanthine oxidase inhibitory activity of alk(en)yl phenols and related compounds
Phytochemistry 155 (2018) 100–106
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Review
Characterization of the xanthine oxidase inhibitory activity of alk(en)yl phenols and related compounds
T
Noriyoshi Masuokaa,b,∗, Isao Kuboc a
CDW Life Science Lab, Okayama Research Park Incubation Center, 5303 Haga, Kita-ku, Okayama 701-1221, Japan Department of Life Science, Faculty of Science, Okayama University of Science, Japan c Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3114, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Xanthine oxidase inhibition Superoxide anion suppression Xanthine oxidase modification Superoxide anion scavenging Antioxidant property
The inhibitory activity of xanthine oxidase (XO) is a combination of uric acid formation inhibition and superoxide anion (O2−) generation suppression. The inhibition of uric acid formation by XO is useful for the screening of natural compounds that prevent gout, while the suppression of O2− generation is useful for treating oxidative stress. Many edible plants contain abundant phenolic compounds and alk(en)yl phenols, and some of these compounds display XO inhibitory activity. This review focuses on XO inhibitory activity since this activity is used to characterize natural products. Recently, it was demonstrated that the inhibitory activity could be characterized using assays for XO inhibition, the suppression of O2− generation, DPPH radical scavenging and O2− radical scavenging. The inhibitory activity was divided three reaction types. The first is XO inhibition, the second O2− generation suppression by modification of enzyme molecules and the third two forms of O2scavenging. It was demonstrated that these three activities are related to both the hydroxy group arrangement in the phenol portion and the alk(en)yl chains. This characterization is useful for pursuing XO inhibitors and antioxidants in natural compounds.
1. Introduction Xanthine oxidoreductase (XOR) exists in two forms, xanthine dehydrogenase (XDH) (EC 1.1.1.204) and XO (EC 1.1.3.22). The enzyme is a homo-dimer, and the subunit contains 1 molybdenum-pterin, 2 iron–sulfurs and 1 flavin adenine dinucleotide (FAD). The structure was analyzed using X-ray crystallography (Enroth et al., 2000; Pauff et al., 2009). XO is formed from XDH under oxidative conditions. The enzyme reaction catalyzes two kinds of reactions. One is an oxidation reaction of hypoxanthine to xanthine and xanthine to uric acid at the Mo-pterin site, while the other is a reduction of NAD+ to NADH at the FAD site of XDH, and that of O2 to O2− or hydrogen peroxide at the FAD site of XO. These reactions are coupled and proceed in a “ping-pong” manner (Bay, 1975). The electron flux moves between the Mo-pterin and FAD cofactors though two iron-sulfur clusters. As XOR precludes the salvage pathway of the purine nucleotides adenine, guanine and hypoxanthine, the enzyme has a rate-limiting function of generating irreversible uric acid and is highly regulated at the transcriptional and post-translational levels. Increased XOR activity induces hyperuricemia and then gout. The substrate specificity at the FAD site is broad, and the oxidant metabolites generated by XO induce have different consequences, ranging
∗
from cytotoxicity to inflammation. It is suggested that increased XO activity is related to asthma, phlogosis, endothelial activation, leukocyte activation, vascular tone regulation and various pathophysiological conditions (Fong et al., 1973; McCord, 1985; Setiawan et al., 2016; Battelli et al., 2016). XO inhibitors lower the serum uric acid content and protect against western diet-induced aortic stiffness and impaired vasorelaxation in mice (Lastra et al., 2017). Lowering the serum uric acid level may help ameliorate endothelial dysfunction and reduce myocardial function so as to prevent heart failure (Harzand et al., 2012). Therefore, as the inhibition of XO ameliorates hyperuricemia and induced oxidative stress, many researchers have shown an interest in testing the XO inhibitory activity in both natural compounds and synthetic products (Hatano et al., 1990; Toda et al., 1991; Kubo et al., 2002; Liu et al., 2009; Tuzun et al., 2017; Belkhiri et al., 2017). However, although XO inhibitory activity has been studied for a long time, the activity is still not fully understood. For example, the XO inhibitory activity of gallic acid (4a) and the n-alkyl ester, dodecyl gallate (4f), were examined. Dodecyl gallate inhibited both the uric acid formation and the O2− generation catalyzed by XO but gallic acid did not affect uric acid formation and suppressed O2−generation. This suggests that
Corresponding author. CDW Life Science Lab, Okayama Research Park Incubation Center, 5303 Haga, Kita-ku, Okayama 701-1221, Japan. E-mail address: [email protected] (N. Masuoka).
https://doi.org/10.1016/j.phytochem.2018.07.006 Received 22 February 2018; Received in revised form 11 July 2018; Accepted 16 July 2018 Available online 23 July 2018 0031-9422/ © 2018 Elsevier Ltd. All rights reserved.
Phytochemistry 155 (2018) 100–106
N. Masuoka, I. Kubo
inhibited both the uric acid and O2− production catalyzed by XO, while cardanol C15:3 (2a) and salicylic acid (1e) did not. The alkenyl chain and phenolic portion in anacardic acid are independently responsible for the inhibitory activity. The inhibition was sigmoidal, and the reaction rate in the presence of anacardic acid is expressed asv = V max [S]/[(Km + [S]) (1 + [I]n/KI)] [When KI = (I) n, I = IC50.] The IC50 and slope factor (n) for the inhibition of uric acid formation were respectively 162 ± 10 μM and 1.7 ± 0.5, and they also suppressed O2− generation (IC50 = 53.6 ± 5.1 μM, n = 4.3 ± 0.5). Although the enzyme reaction similarly generated uric acid and O2−, the O2− generation was more potently suppressed than the uric acid formation. It was finally deduced that the suppression was the sum of the XO inhibition and O2− scavenging activity, even though anacardic acid does not have DPPH scavenging activity (Masuoka et al., 2016). As the alkenyl chain in anacardic acid is related to the inhibitory activity, alkyl gallates and caffeates were chosen as models to investigate the relationship. Akly1gallates (4a-g) were synthesized and tested (Nihei et al., 2003; Masuoka et al., 2006). Gallic acid and alkyl gallates (4a-c), having short side chains (< C6), did not inhibit the uric acid formation catalyzed by XO (Table 1). In the case of the alkyl gallates, which have longer C6 chains, stronger inhibition was observed. Octyl gallate (4e) competitively inhibited uric acid formation, and the docking experiment with this compound indicated it bound to the xanthine binding site in XO. Caffeic acid and the alkyl esters (5a-h) were also examined (Masuoka et al., 2012b). Alkyl caffeates competitively inhibited the uric acid formation catalyzed by XO, and the inhibition by alkyl caffeates increased with increasing alkyl chain length as alkyl gallates. Compared to alkyl gallates, the inhibition of the alkyl caffeates was more potent than that of the alkyl gallates. From these results, long alkyl chains in these inhibitors assist the inhibitor binding to the xanthine-binding site, using the hydrophobic interaction inside of XO, as indicated by the alkyl gallates as well as a
Abbreviations O2DPPH PMS ROS XDH XO XOR
Superoxide anion 1, 1-Diphenyl-2-picryhydrazyl Phenazine methosulfate Reactive oxygen species Xanthine dehydrogenase Xanthine oxidase Xanthine oxidoreductase
the O2− suppression of gallic acid is due to the antioxidant property and the alkyl chain of dodecyl gallate plays an important role in its inhibitory activity (Kubo et al., 2002). The alkenyl phenols, anacardic acid C15:3 6-[8′(Z), 11′(Z), 14′-pentadecatrienyl] salicylic acid (1a) and cardanol C15:3 (2a) were also tested. Anacardic acid C15:3 sigmoidally inhibited uric acid formation and O2− generation, but cardanol C15:3 did not (Masuoka and Kubo, 2004; Kubo et al., 2006). It indicates that both the phenol portion and alkenyl chain in alkenyl phenols play important roles in the inhibitory activity. In this review, we focus on the XO inhibitory activity of the phenolic compounds and alk(en)yl phenols isolated from edible plants and characterize their activity. Moreover, the XO inhibitory activity of natural and synthesized compounds (see Fig. 1) is broken down into three activity groups. 2. Natural sources of phenolic compounds Edible plants abundantly contain various kinds of phenolic compounds and alk(en)yl phenols. Alk(en)yl phenols, e.g. anacardic acids (1a-d), cardanols (2a-d) and cardols (3a, b), are present in cashew nuts, cashew apples, ginkgo nuts and cereal grains (Kubo et al., 1986; Irie et al., 1996; Kozubek and Tyman, 1999). Antioxidant, enzyme inhibitory, antibacterial and antitumor activities have been reported (Kubo et al., 1994; Hamad and Mubofu, 2015). The synthesis of various biologically active compounds has been reported using these compounds as the staring materials. In terms of the phenolic compounds, gallic acid (4a) is a widespread natural product that is known to be a potent antioxidant present in natural plant materials such as blackberry bark, henna, tea and uva ursi, and is often a component of hydrolysable tannins in many plants. Caffeic acid (5a) is also found in plants, fruits and vegetables. These compounds are increasingly of interest because of their broad biological effects, including antioxidative, antimicrobial and antitumor activities (Kartal et al., 2003; Gulcin, 2006; Cardenas et al., 2006). These compounds attract more interest than synthetic antioxidants due to the fact that they cause less damage to the human body (Lin and Yen, 1999). However, although gallic acid and caffeic acid are efficient antioxidants, their lower solubility in non-polar media limits their application in fatty food, especially various edible oils (Kalogeropoulos et al., 2009). To increase the solubility in non-polar media, n-alkyl esters of these phenolic compounds were synthesized. In particular, n-propyl, n-octyl and n-dodecyl gallates (4c-g) are widely used as food additives to scavenge ROS (Van Der Meeren, 1987). nAlkyl caffeates (5b-h) are likely to be useful in oily foods as antioxidants (Garrido et al., 2012; Wang et al., 2014). 3. How do phenolic compounds inhibit the uric acid formation catalyzed by XO? A sufficient amount of oxygen for the XO reaction was present in the aqueous reaction mixture, and XO activity was detected as uric acid formation generated from xanthine. Inhibition of uric acid formation, which is an XO inhibition effect, by phenolic compounds was tested. XO reactions were carried out at pH 10 to stabilize one of the reaction products (O2−) (Masuoka and Kubo, 2004). Anacardic acid C15:3 (1a)
Fig. 1. Structures of anacardic acids (1), cardanols (2), cardols (3) and related compounds (4–10). C15:3, C15:2 and C15:1 (above right) are the alkenyl chains of the compounds (1–3). 101
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Table 1 Inhibition of the uric acid formation catalyzed by XO with gallic acid, caffeic acid and n-alkyl esters. Compound
Gallic acid (4a) Methyl gallate (4b) Propyl gallate (4c) Hexyl gallate (4d) Octyl gallate (4e) Decyl gallate (4f) Dodecyl gallate (4g) Caffeic acid (5a) Methyl caffeate (5b) Propyl caffeate (5c) Butyl caffeate (5d) Pentyl caffeate (5e) Hexyl caffeate (5f) Heptyl caffeate (5g) Decyl caffeate (5h) a b c
IC50a(μM)
> 200 > 200 > 200 > 200c 262 ± 45 97 ± 3 49 ± 13 > 200 143 ± 13 30.2 ± 2.1 26.5 ± 2.0 18.0 ± 0.9 19.0 ± 1.0 12.5 ± 0.5 10.0 ± 1.0
For xanthine-binding (μM)b
Inhibition type
KI
KIS
– – – – 30.5 ± 1.8 35.8 ± 11.8 19.0 ± 6.0 – 38.6 ± 0.5 7.8 ± 0.8 7.3 ± 0.5 4.3 ± 0.4 3.6 ± 0.6 3.5 ± 0.5 3.3 ± 0.5
– – – – 943 ± 272 127 ± 23 46.7 ± 14.0 – – – – – – – –
– – – – Competitive Mixed Mixed – Competitive Competitive Competitive Competitive Competitive Competitive Competitive
IC50 values were measured at 200 μM xanthine and expressed as the mean ± SD. XO reaction was carried out with 0–200 μM xanthine under atmosphere. Hexyl gallate at 200 μM inhibited 13 ± 2% of the control activity.
en-diol structures (Blois, 1958).
squalene epoxidase (Abe et al., 2000). In the case of anacardic acid, the inhibition was noncompetitive for xanthine and sigmoidally inhibited. It is deduced that this interaction between the alkenyl chain of anacardic acid and the hydrophobic pocket in XO induces the cooperative binding and fitting of anacardic acid to the xanthine-binding site in XO. As the inhibition activity was related to the hydroxy groups in the phenol portion, other alkyl phenol compounds (6–10) were tested (Masuoka et al., 2012b, 2015). Protocatechuates (6a-c) and 3,5-dihydroxybenzoates (8a-c) displayed hardly any XO inhibition activity. By comparison, 3,4-dihydroxyphenyl alkanoates (7a-c) and 3,5-dihydroxyphenyl alkanoates (9a-c) exhibited weak inhibition at 200 μM. Similarly, hexyl and nonyl 2,4-dihydrobenzoates (10a, b) displayed hardly any inhibition activity, while dodecyl 2,4-dihydrobenzoate (10c) exhibited non-competitive inhibition (KI=KIS=187 ± 17 μM). These results indicate that it is necessary for XO inhibitors to have alkyl chains and particular hydroxy group arrangements in the phenol portion, since the phenol portion binds to the xanthine binding site, as in the case of the flavonoids (Lin et al., 2002; Masuoka et al., 2012a). It is suggested that it is necessary for XO inhibitors to be electron-rich. This is because the inhibitors electrostatically interact with the electron-deficient property of the XO molecules generated from XDH under oxidative conditions.
Conjugated en-diol structures. Table 2 Kinetic parameters of the gallic acid, caffeic acid and n-alkyl esters used for the O2− generated by XOa and for the DPPH scavenging activity. Compound
Gallic acid (4a) Methyl gallate (4b) Propyl gallate (4c) Hexyl gallate (4d) Octyl gallate (4e) Decyl gallate (4f) Dodecyl gallate(4g) Caffeic acid (5a) Methyl caffeate (5b) Propyl caffeate (5c) Butyl caffeate (5d) Pentyl caffeate (5e) Hexyl caffeate (5f) Heptyl caffeate (5g) Decyl caffeate (5h) Protocatechuic acid (6a) Heptyl protocatechuate (6b) Octyl protocatechuate (6c)
4. How do phenolic compounds suppress the O2− generation catalyzed by XO? 4.1. The O2− suppression and DPPH scavenging activity by phenolic acid and their alkyl esters The O2− generation catalyzed by XO was detected using blue formazan formation from the reaction with nitroblue tetrazolium (Toda et al., 1991; Masuoka and Kubo, 2004). The O2− suppression and DPPH scavenging activity of gallic acid, caffeic acid and their alkyl esters is summarized in Table 2. Although the enzyme reaction similarly generated uric acid and O2−, gallic acid and alkyl gallates (4a-g) displayed potent suppression of the O2− generation catalyzed by XO compared to inhibition of the uric acid formation (Masuoka et al., 2006). The suppression was unaffected by the alkyl chain length of gallates, unlike the inhibition of uric acid formation. The inhibition kinetics were analyzed as a mixed type. Caffeic acid and alkyl caffeates (5a-h) also suppressed O2− generation and scavenged DPPH radical similarly to the gallates. The O2− suppression of caffeates was less than that of the gallates. The DPPH scavenging activity of gallic acid, alkyl gallates (4a-g), caffeic acid and alkyl caffeates (5a-h) was assigned to their conjugated
Suppression of O2- generation IC50 (μM)b
KI
KIS
DPPH scavenging activityc
2.6 ± 0.3 7.4 ± 0.5 6.4 ± 1.0 5.2 ± 0.2 4.5 ± 0.5 3.9 ± 1.1 3.6 ± 0.2 11.5 ± 0.5 12.0 ± 0.5 11.5 ± 0.5 11.0 ± 0.5 10.0 ± 1.0 10.8 ± 1.5 7.6 ± 0.4 16.4 ± 6.4d 22.0 ± 0.6
1.5 ± 0.2 5.7 ± 0.4 4.5 ± 0.5 3.8 ± 0.4 3.9 ± 0.2 2.5 ± 0.6 1.8 ± 0.2 8.4 ± 0.8 4.6 ± 0.4 3.6 ± 0.2 4.0 ± 0.3 3.0 ± 0.5 3.1 ± 0.2 2.4 ± 0.2 9.1 ± 1.1 12.8 ± 0.3
2.9 ± 0.3 8.0 ± 0.6 6.8 ± 0.7 5.3 ± 0.6 4.7 ± 0.2 4.8 ± 1.0 4.4 ± 0.2 12.6 ± 1.1 15.8 ± 1.2 15.8 ± 0.4 28.4 ± 1.6 29.4 ± 1.3 25.4 ± 1.4 20.2 ± 0.9 38.3 ± 3.5 30.7 ± 0.7
6.51 6.34 6.02 5.74 6.09 6.02 7.28 4.63 4.73 4.83 4.69 4.49 3.77 5.71 4.81 3.53
65.2 ± 2.0
36.5 ± 2.1
87.6 ± 0.6
5.90 ± 0.83
82.0 ± 0.2
99.4 ± 0.4
87.3 ± 3.1
6.82 ± 0.17
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.30 0.12 0.17 0.22 0.14 0.18 0.54 0.20 0.26 0.11 0.12 0.22 0.60 0.23 0.15 0.23
a XO reaction was carried out at 0–200 μM xanthine in the presence of 0.0–2.0 μM of gallic acid or alkyl gallates under atmosphere. Values are expressed as the mean ± SD obtained from more than three separate experiments. b IC50 values are shown for 200 μM xanthine. c The values are the scavenged DPPH molecules/one molecule of the test compound. d IC50 values were estimated from inhibitions under 10 μM decyl caffeate.
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To further study this matter, the suppression of the O2− generation catalyzed by XO by other phenolic alkyl esters having conjugated endiol structures was tested. Protocatechuic acid and the esters (6a-c) displayed a DPPH scavenging activity (approximately 6 molecules per molecule) and mixed-type suppression of the O2− generation, similar to the gallates. 3,4-Dihydroxyphenyl alkanoates (7a-c) exhibited a ratio of approximately 3 DPPH molecules/tested molecule and sigmoidal suppression of the O2− generation. These results suggested that suppression of the O2− generation by phenolic acid and these alkyl esters is associated with the DPPH scavenging activity.
antioxidant activity (Kamal-Eldin et al., 2000). The suppression of O2− generation catalyzed by XO and the DPPH activity indicated in Table 3. Anacardic acid C15:3 sigmoidally inhibited XO (uric acid formation) activity, but cardanol C15:3 and cardol C15:3 (3a) did not (Masuoka and Kubo, 2004; Kubo et al., 2006). As O2− suppression by anacardic acid is stronger than XO inhibition, the O2− suppression by anacardic acid contained another form of O2− suppression activity. Cardols C15:3 and C10:0 sigmoidally suppressed O2− generation, but cardol C5:0 did not (Masuoka et al., 2015). These results indicate that O2− suppression is associated with the alk(en)yl chain length. To further investigate, suppression of the O2− generation catalyzed by XO with other phenolic alkyl ester compounds having no DPPH scavenging activity (≤0.04 ± 0.02) was examined. 3,5Dihydroxybenzoic acid and alkyl 3,5-dihydroxybenzoates (8a-c) exhibited no suppression activity. 3,5-Dihydroxyphenyl heptanoate (9a) also exhibited no suppression activity, while 3,5-dihydroxyphenyl decanoate and 3,5-dihydroxyphenyl tridecanoate (9c) displayed sigmoidal suppression. Hexyl 2,4-dihydroxybenzoate (10a) did not display any suppression activity but nonyl 2,4-dihydroxybenzoate (10b) did display sigmoidal suppression. Electron-rich phenol alkyl esters having no DPPH activity exhibited sigmoidal O2− suppression activity. From these results on the alk(en)yl phenols and related compounds, we deduced that the O2− suppression was associated with long alk(en) yl chains and that the O2− suppression with alk(en)yl phenols is induced by either O2− scavenging activity or specific enzyme modification as the inhibition of the uric acid formation was not observed.
4.1.1. Comparison of the O2− scavenging activity using a PMS-NADH system To compare the suppression of the O2− generation catalyzed by XO, the O2− scavenging activity was investigated using a PMS-NADH system (Nishikimi et al., 1972). The O2− scavenging activity of gallic acid and alkyl gallates is indicated in Fig. 2, and the scavenging activity was carefully analyzed, as follows. These scavenging activities of the gallates consist of two components. One group, having no or short alkyl chains, exhibited hyperbolic scavenging curves, and it was deduced that O2− was scavenged by electrons, these are suggested by the oxidation of the conjugated en-diol structure (Kawabata et al., 2002). Another group having long alkyl chains exhibited weak sigmoidal activity, and it was finally deduced that these compounds scavenged O2− and the phenolic radicals induced oxidative degradation reactions. Although the scavenging activity by gallic acid displayed the most potent O2− scavenging activity in these gallates, the O2− scavenging activity was low compared to the suppression activity of the O2− generation catalyzed by XO. The O2− scavenging activity of caffeic acid and alkyl caffeates displays a similar pattern as the gallates. As this O2− scavenging activity was also low compared to the suppression of the O2− generation catalyzed by XO, the suppression of the O2− generation is not primarily associated with O2− scavenging activity, but with other mechanisms (Masuoka and Kubo, 2016).
4.2.1. Similarity to the O2− scavenging activity using a PMS-NADH system To further examine the antioxidant activity, the scavenging of O2− using a PMS-NADH system was tested in the presence of cardols (C15:3, C15:2, C15:0) (Table 4). As the suppression activity of cardol C15:3 using the XO system is quite similar to the scavenging activity of cardol C15:3, it is deduced that this sigmoidal activity is not due to specific enzyme modification, but rather is due to the O2− scavenging activity (Masuoka et al., 2016).
4.1.2. Proposed mechanism of the O2− suppression by these phenolic compounds: modification of XO molecules Hille & Massey reported that XO takes several different oxidation states and that fully reduced XO contains a total of six electrons in the reduced iron-sulfur center, molybdenum (IV) and FADH2. Oxidation of four or six electron-reduced XO to two-electron-reduced XO generates hydrogen peroxide, while that of two-electron-reduced XO only generates O2− (Hille and Massey, 1981). The DPPH scavenging activity of alkyl gallates and caffeates indicated that the stable radical was reduced with the oxidation of the conjugated en-diol structures in their phenol portion. From these findings, it was deduced that these gallate and caffeate derivatives as well as related compounds bind the FAD site to reduce the enzyme and the modification of XO induces the O2− suppression. This O2− suppression by reduction-modification of the enzyme was also supported in the presence of flavonoids with high DPPH activity (Masuoka et al., 2012a). Therefore, we propose that the suppression of the O2− generation catalyzed by XO was induced by a reduction of XO molecules. The phenolic compounds and alkyl esters having DPPH scavenging activities are shown as reducing agents [B] in Fig. 3, eventually suppressing the O2− generation catalyzed by XO.
4.2.2. O2− scavenging mechanism of alk(en)yl phenols Resorcinol compounds with a long hydrocarbon chain at the 5-position have been reported to act as antioxidants and protect polyunsaturated fatty acids against peroxidation. However, Kamal-Eldin et al. (2000) reported that cardol C15:0 has a low hydrogen donation capacity and peroxyl radical-scavenging effect. This scavenging activity
4.2. The O2− suppression and DPPH scavenging activity by natural alk(en) yl phenols and related compounds Anacardic acidC15:3; 6-[8′(Z), 11′(Z), 14′-pentadecatrienyl] salicylic acid (1a), cardanol C15:3 (2a), cardol C15:3; 5-[8′(Z), 11′(Z), 14′-pentadecatrienyl] resorcinol and related compounds (C10:0 and C5:0) (3a-d) were examined. These compounds had low DPPH scavenging activity (having no conjugated en-diol structure), but had considerable
Fig. 2. The suppression of the O2− generated by the PMS-NADH system. Gallic acid and alkyl derivatives; gallic acid (●), methyl gallate (○), propyl gallate (▼), hexyl gallate (▽), octyl gallate (■), decyl gallate (□) and dodecyl gallate (◆). 103
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Table 4 O2− Scavenging activity of alkyl phenols using PMS-NADH. IC50 (μM)a
Compound Anacardic acid C15:3 Anacardic acid C15:2 Anacardic acid C15:1 Anacardic acid C15:0 Salicylic acid (1f) Cardanol C15:3 (2a) Cardanol C15:2 (2b) Cardanol C15:1 (2c) Cardanol C15:0 (2d) Cardol C15:3 (3a) Cardol C15:2 (3b) Cardol C15:0 (3c) Resorcinol (3f) a
(1a) (1b) (1c) (1d)
98 ± 2 (n = 6.7 ± 0.9, sigmoidal) 95 ± 3 (n = 5.0 ± 1.8, sigmoidal) 72 ± 5 (n = 10.5 ± 2.2, sigmoidal) 41 ± 3 (n = 9.4 ± 1.5, sigmoidal) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (No inhibitiona) 116 ± 10(n = 8.7 ± 1.8, sigmoidal) 116 ± 8 (n = 8.1 ± 1.3, sigmoidal) 117 ± 4 (n = 6.4 ± 1.5, sigmoidal) > 200 (No inhibitiona)
Indicates inhibition at 200 μM.
phenol portion in the inhibitors binds to the xanthine binding site. So, XO inhibitors have particular hydroxy group arrangements in the phenol portion and long alk(en)yl chains. (2) Modification of the XO molecule by the reducing activity of phenolic compounds. The enzyme molecule is reduced by the oxidation of the phenolic compounds having conjugated en-diol structures. When the reduced enzyme molecule catalyzes the reaction of xanthine to uric acid, the enzyme is further reduced and does not generate O2−, but rather, hydrogen peroxide. This reduced-modification does not inhibit uric acid formation, and this enzyme-reducing ability of phenolic compounds is evaluated using DPPH radical scavenging activity. (3) Scavenging of O2− with phenolic compounds. The scavenging activity is due to the reaction of these compounds with the O2− generated by XO and consists of two reaction types. One (3a) is responsible for the conjugated en-diol structure that is detected by DPPH activity, while the other (3b) is associated with the phenolic portion and the amphiphilic property of alk(en)yl phenols. The scavenging activities were determined using the PMS-NADH system.
Fig. 3. Proposed mechanism of the O2− or hydrogen peroxide generation catalyzed by xanthine oxidase. [A]: Uric acid formation inhibitors, [B]: reducing agents of the enzyme (molecule).
of cardols may be explained as follows. Cardols are amphiphilic molecules and the content in aqueous medium rapidly increases at levels greater than the critical micelle concentration (Stasiuk and Kozubek, 2010). An increase in the hydroxy group in an aqueous medium induces hydrogen donation to O2−, while radical formation at the hydroxy oxygen initiates changes in the electron density that destabilizes the phenolic ring and this in turn subsequently leads to oxidation of the ring (Hladyszowki et al., 1998). The scavenging activity of cardols may be related to the weak DPPH scavenging activity (Table 3). This mechanism may also underlie the weak and sigmoidal O2− scavenging activity of the alkyl gallates and caffeates having long alkyl chains (Fig. 2). 5. Classifying the XO inhibitory activity of phenolic compounds and alk(en)yl phenols The XO inhibitory activity of phenolic compounds is divided into three activity groups.
Therefore, the activities of the gallic acid, caffeic acid and alkyl gallates having short chains were assigned to the (2) and (3a) groups, while the alkyl gallates having long chains and alkyl caffeates were assigned to (1), (2) and (3b). Anacardic acids exhibited (1) and (3b) activities, while the cardols exhibited (3b) activity.
(1) Inhibition of uric acid formation by phenolic compounds (XO inhibition). This activity is measured by the inhibition of uric acid formation. The alk(en)yl chain in inhibitors potentially assists the inhibition using the hydrophobic interaction with XO and the
Table 3 Suppression of the O2− generation catalyzed by xanthine oxidase with alkyl phenols having no DPPH scavenging activity. Compound
IC50 (μM)a
DPPH scavengingb
Anacardic acid C15:3 (1a) Salicylic acid (1e) Cardol C15:3 (3a) Cardol C10:0 (3d) Cardol C5:0 (3e) Resorcinol (3f) Cardanol C15:3 (2a) 3,5-Dihydroxybenzoic acid (8a) Hexyl 3,5-dihydroxybenzoate (8b) Dodecyl 3,5-dihydroxybenzoate (8c) 3,5-Dihydroxyphenylheptanoate (9a) 3,5-Dihydroxyphenyldecanoate (9b) 3,5-Dihydroxyphenyltridecanoate (9c) Hexyl 2,4-dihydroxybenzoate (10a) Nonyl 2,4-dihydroxybenzoate (10b)
51.3 ± 1.5 (n = 4.2 ± 0.5, sigmoidal) > 200 (No inhibitiona) 115 ± 10 (n = 5.2 ± 0.2, sigmoidal) 106 ± 8 (n = 5.1 ± 0.2, sigmoidal) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (2.5% inhibitiona) 121 ± 4 (n = 1.9 ± 0.4, sigmoidal) 75.3 ± 5.0 (n = 5.5 ± 1.8, sigmoidal) > 200 (16% inhibitiona) 118 ± 1 (n = 3.7 ± 0.1, sigmoidal)
0.02 0.01 0.49 0.50 0.50 0.02 0.02 0.04 0.02 0.01 0.01 0.02 0.01 0.01 0.01
a b
Indicated inhibition at 200 μM. The value indicates scavenged DPPH molecules/one molecule of the tested compound.
104
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01
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6. Conclusions and perspectives
Hatano, T., Yasuhara, T., Yoshihara, R., Agata, I., Noro, T., Okuda, T., 1990. Effects of interaction of tannins with co-existing substances. VII. Inhibitory effects of tannins and related polyphenols on xanthine oxidase. Chem. Pharm. Bull. (Tokyo) 38 (5), 1224–1229. Hille, R., Massey, V., 1981. Studies on the oxidative half-reaction of xanthine oxidase. J. Biol. Chem. 256, 9090–9095. Hladyszowki, J., Zubik, L., Kozubek, A., 1998. Quantum mechanical and experimental oxidation studies of pentadodecylresorcinol, olivetol and resorcinol. Free Radic. Res. 28 (4), 359–368. Irie, J., Murata, M., Homma, S., 1996. Glycerol-3-phosphate dehydrogenase inhibitors, anacardic acids, from Ginkgo biloba. Biosci. Biotechnol. Biochem. 60 (2), 204–243. Kalogeropoulos, N., Konteles, S.J., Troullidou, E., Mourtzinos, I., Karathanos, V.T., 2009. Chemical composition, antioxidant activity and antimicrobial properties of propolis extracts from Greece and Cyprus. Food Chem. 116, 452–461. Kamal-Eldin, A., Pouru, A., Eliasson, C., Aman, P., 2000. Alkylresorcinols as antioxidants: hydrogen donation and peroxyl radical-scavenging effects. J. Sci. Food Agric. 81, 353–356. Kartal, M., Yıldız, S., Kaya, S., Kurucu, S., Topc¸u, G., 2003. Antimicrobial activity of propolis samples from two different regions of anatolia. J. Ethnopharmacol. 86, 69–73. Kawabata, J., Okamoto, Y., Kodama, A., Makimoto, T., Kasai, T., 2002. Oxidative dimmers produced from protocatechuic and gallic esters in the DPPH radical scavenging reaction. J. Agric. Food Chem. 50, 5468–5471. Kozubek, A., Tyman, J.H.P., 1999. Resorcinolic lipids, the natural non-isoprenoid phenolic amphiphiles and their biological activity. Chem. Rev. 99, 1–25. Kubo, I., Komatsu, S., Ochi, M., 1986. Molluscicides from the cashew Anacardium occidentale and their large-scale isolation. J. Agric. Food Chem. 34, 970–973. https://doi. org/10.1021/jf00072a010. Kubo, I., Kinst-Hori, I., Yokokawa, Y., 1994. Tyrosinase inhibitors from Anacardium occidentale fruits. J. Nat. Prod. 57, 545–552. Kubo, I., Masuoka, N., Ha, T.J., Tsujimoto, K., 2006. Antioxidant activity of anacardic acids. Food Chem. 99, 555–562. Kubo, I., Masuoka, N., Xiao, P., Haraguchi, H., 2002. Antioxidant activity of dodecyl gallate. J. Agric. Food Chem. 50 (12), 3533–3539. Lastra, G., Manrique, C., Jia, G., Aroor, A.R., Hayden, M.R., Barron, B.J., Niles, B., Padilla, J., Sowers, J.R., 2017. Xanthine oxidase inhibition protects against Western dietinduced aortic stiffness and impaired vasorelaxation in female mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. https://doi.org/10.1152/ajpregu. 00483.2016. May 24. Lin, C.M., Chen, C.S., Chen, C.T., Liang, Y.C., Lin, J.K., 2002. Molecular modeling of flavonoids that inhibits xanthine oxidase. Biochem. Biophys. Res. Commun. 294 (1), 167–172. Lin, M., Yen, C., 1999. Antioxidative ability of lactic acid bacteria. J. Agric. Food Chem. 47, 1460–1466. Liu, X., Chen, R., Shang, Y., Jiao, B., Huang, C., 2009. Superoxide radicals scavenging and xanthine oxidase inhibitory activity of magnesium lithospermate B from Salvia miltiorrhiza. J. Enzyme Inhib. Med. Chem. 24 (3), 663–668. Masuoka, N., Kubo, I., 2004. Characterization of xanthine oxidase inhibition by anacardic acids. Biochim. Biophys. Acta 1688, 245–249. Masuoka, N., Nihei, K., Kubo, I., 2006. Xanthine oxidase inhibitory activity of alkyl gallates. Mol. Nutr. Food Res. 50, 725–731. Masuoka, N., Matsuda, M., Kubo, I., 2012a. Characterisation of the antioxidant activity of flavonoids. Food Chem. 131, 541–545. Masuoka, N., Nihei, K., Masuoka, T., Kuroda, K., Sasaki, K., Kubo, I., 2012b. The inhibition of uric acid formation catalyzed by xanthine oxidase, Properties of the alkyl caffeates and cardol. J. Food Res. 1 (3), 257–262. Masuoka, N., Nihei, K., Maeta, A., Yamagiwa, Y., Kubo, I., 2015. Inhibitory effects of cardols and related compounds on superoxide anion generation by xanthine oxidase. Food Chem. 166 (1), 270–274. Masuoka, N., Kubo, I., 2016. Suppression of superoxide anion generation catalyzed by xanthine oxidase with alkyl caffeates and the scavenging activity. Int. J. Food Sci. Nutr. 67 (3), 283–287. https://doi.org/10.3109/09637486.2016.1153609. Masuoka, N., Tamsampaoloet, K., Chavasiri, W., Kubo, I., 2016. Superoxide anion scavenging activity of alk(en)yl phenol compounds by using PMS-NADH system. Heliyon 2, e00169. McCord, J.M., 1985. Oxygen-derived free radicals in postischemic tissue injury. N. Engl. J. Med. 312, 159–163. Nihei, K., Nihei, A., Kubo, I., 2003. Rational design of antimicrobial agents: antifungal activity of alk(en)yl dihydroxybenzoates and dihydroxyphenyl alkanoates. Bioorg. Med. Chem. Lett 13, 3993–3996. https://doi.org/10.1016/j.bmcl.2003.08.057. Nishikimi, M., Rao, N.A., Yagi, K., 1972. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem. Biophys. Res. Commun. 46, 849–854. Pauff, J.M., CaO, H., Hille, R., 2009. Substrate orientation and catalysis at the molybdenum site in xanthine oxidase. J. Biol. Chem. 284 (13), 8760–8767. Setiawan, H., Nagaoka, K., Kubo, M., Fujikura, Y., Ogino, K., 2016. Involvement of xanthine oxidoreductase-related oxidative stress in a dermatophagoides farina-induced asthma model of NC/Nga mice. Acta Med. Okayama 70 (3), 175–182. Stasiuk, M., Kozubek, A., 2010. Biological activity of phenolic lipids. Cell. Mol. Life Sci. 67, 841–860. Toda, S., Kumura, M., Ohnishi, M., 1991. Effects of phenolcarboxylic acids on superoxide anion. Planta Med. 57, 8–10. Tuzun, B.S., Hajdu, Z., Orban-Gyapai, O., Zomborszki, Z.P., Jedlinszki, N., Forgo, P., Kivcak, B., Hohmann, J., 2017. Isolation of chemical constituents of Centaurea virgata Lam. And xanthine oxidase-inhibitory activity of the plant extract and compounds. Med. Chem. 13 (5), 498–502.
XO inhibition of uric acid formation indicated that the inhibitors competitively inhibited XO. The phenol portion of the inhibitors binds to the xanthine binding site, and the alk(en)yl side chain enhances the binding to XO molecules. In addition, suppression of the O2− generation catalyzed by XO was affected by enzyme-reducing modification and two types of O2− scavenging activities. The enzyme-reducing modification capacity of phenolic compounds was evaluated by DPPH radical scavenging activity, while O2− scavenging was measured using a PMS-NADH system. The antioxidant activity of phenolic compounds was deduced as follows. Gallic acid, caffeic acid and alkyl gallates having short chains lower solubility in non-polar media such as edible oils and are easily oxidized with O2−, resulting in an increase of hydrogen peroxide generation (Masuoka et al., 2016). It is suggested that these compounds do not have utility as antioxidants in oily foods. Alkyl gallates and alkyl caffeates having long side chains are synthetic amphiphilic compounds and display sigmoidal O2− scavenging activity. Though these compounds are used as food additives, they may become oxidized because of their conjugated en-diol structure (Van Der Meeren, 1987). Anacardic acids and cardols are natural amphiphilic compounds without any conjugated en-diol structure, so it is suggested that these compounds are likely to be applicable as antioxidants, even in oily or fatty foods. For the prevention of hyperuricemia and oxidative stress, anacardic acids were shown to inhibit uric formation but cardol and cardanol do not. The anacardic acids exhibit both XO inhibition and superoxide anion scavenging activities. Anacardic acid C15:0 may be expected to have potential for the treatment of hyperuricemia since it had the most potent O2− scavenging activity among the anacardic acids, as long as untoward side-effects are not observed. Further study is warranted to test these hypotheses. Acknowledgments We thank Professor Ken-ichi Nihei, Faculty of Agriculture, Utsunomiya University, Japan, for presents of alkyl-phenol related compounds. References Abe, I., Seki, T., Noguchi, H., 2000. Potent and selective inhibition of squalene epoxidase by synthetic galloyl esters. Biochem. Biophys. Res. Commun. 270, 137–140. Battelli, M.G., Polito, L., Bortolotti, M., Bolobnesi, A., 2016. Xanthine oxidoreductasederived reaction species: physiological and pathological effects. Oxid. Med. Cell Longev. 2016, 3527579. Bay, R.C., 1975. Molybdenum iron-sulfur flavin hydroxylases and related enzymes. In: In: Boyer, P.D. (Ed.), The Enzymes, vol. 12. Academic Press New York, pp. 299. Belkhiri, E., Baghiani, A., Zerroug, M.M., Arrar, L., 2017. Investigation of antihemolytic, xanthine oxidase inhibition, antioxidant and antimicrobial properties of Salvia verbenaca L. aerial part extracts. Afr. J. Tradit., Complementary Altern. Med. 14 (2), 273–281. Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature 181, 1199–1200. Cardenas, M., Marder, M., Blank, V.C., Roguin, L.P., 2006. Antitumor activity of some natural flavonoids and synthetic derivatives on various human and murine cancer cell lines. Bioorg. Med. Chem. 14, 2966–2971. Enroth, C., Eger, B.T., Okamoto, K., Nishino, T., Nishino, T., Pai, E.F., 2000. Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: structurebased mechanism of conversion. Proc. Natl. Acad. Sci. U.S.A. 97, 10723–10728. Fong, K.L., McCay, P.B., Poyer, J.L., Keele, B.B., Misr, H., 1973. Evidence that peroxidation of lysosomal membranes is initiated by hydroxyl free radicals produced during flavin enzyme activity. J. Biol. Chem. 248 (22), 7792–7797. Garrido, J., Gaspar, A., Garrido, E.M., Miri, R., Tavakkoli, M., Pourali, S., Saso, L., Borges, F., Firuzi, O., 2012. Alkyl esters of hydroxycinnamic acids with improved antioxidant activity and lipophilicity protect PC12 cells against oxidative stress. Biochimie 94, 961–967. Gulcin, I., 2006. Antioxidant activity of caffeic acid (3,4-dihydroxycinnamic acid). Toxicology 217, 213–220. Hamad, F.B., Mubofu, E.B., 2015. Potential biological applications of bio-based anacardic acids and their derivatives. Int. J. Mol. Sci. 16, 8569–8590. Harzand, A., Tamariz, L., Hare, J.M., 2012. Uric acid, heart failure survival, and the impact of xanthine oxidase inhibition. Congest. Heart Fail. 18, 179–182.
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Isao Kubo has worked on chemistry, insect physiology and ecology in Kenya, Japan and USA. Since 1986, he became a Professor of natural products chemistry in University of California and (2016 -) a Professor Emeritus. His research involves in all areas of natural product chemistry. Since 1977, he is a member of American Association for the Advancement of Science (AAAS) Fellow.
Van Der Meeren, H.L., 1987. Dodecyl gallate, permitted in food, is a strong sensitizer. Contact Dermatitis 16, 260–262. Wang, J., Gu, S.S., Pang, N., Wang, F.Q., Pang, F., Cui, H.S., Wu, X.Y., Wu, F.A., 2014. Alkyl caffeates improve the antioxidant activity, antitumor property and oxidation stability of edible oil. PLoS One 9 (4), e95909.
Noriyoshi Masuoka worked on biochemistry in Okayama University Graduate School of Medicine and Dentistry. Since 2005, he became a professor of biochemistry at Okayama University of Science. Now (2017 -), he is a chief researcher in CDW Life science laboratory in Okayama. He investigates the functions of oxidative stresses and antioxidants, especially acatalasemia (catalase deficiency) and xanthine oxidase inhibitory activity of natural products.
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Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Review
Characterization of the xanthine oxidase inhibitory activity of alk(en)yl phenols and related compounds
T
Noriyoshi Masuokaa,b,∗, Isao Kuboc a
CDW Life Science Lab, Okayama Research Park Incubation Center, 5303 Haga, Kita-ku, Okayama 701-1221, Japan Department of Life Science, Faculty of Science, Okayama University of Science, Japan c Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3114, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Xanthine oxidase inhibition Superoxide anion suppression Xanthine oxidase modification Superoxide anion scavenging Antioxidant property
The inhibitory activity of xanthine oxidase (XO) is a combination of uric acid formation inhibition and superoxide anion (O2−) generation suppression. The inhibition of uric acid formation by XO is useful for the screening of natural compounds that prevent gout, while the suppression of O2− generation is useful for treating oxidative stress. Many edible plants contain abundant phenolic compounds and alk(en)yl phenols, and some of these compounds display XO inhibitory activity. This review focuses on XO inhibitory activity since this activity is used to characterize natural products. Recently, it was demonstrated that the inhibitory activity could be characterized using assays for XO inhibition, the suppression of O2− generation, DPPH radical scavenging and O2− radical scavenging. The inhibitory activity was divided three reaction types. The first is XO inhibition, the second O2− generation suppression by modification of enzyme molecules and the third two forms of O2scavenging. It was demonstrated that these three activities are related to both the hydroxy group arrangement in the phenol portion and the alk(en)yl chains. This characterization is useful for pursuing XO inhibitors and antioxidants in natural compounds.
1. Introduction Xanthine oxidoreductase (XOR) exists in two forms, xanthine dehydrogenase (XDH) (EC 1.1.1.204) and XO (EC 1.1.3.22). The enzyme is a homo-dimer, and the subunit contains 1 molybdenum-pterin, 2 iron–sulfurs and 1 flavin adenine dinucleotide (FAD). The structure was analyzed using X-ray crystallography (Enroth et al., 2000; Pauff et al., 2009). XO is formed from XDH under oxidative conditions. The enzyme reaction catalyzes two kinds of reactions. One is an oxidation reaction of hypoxanthine to xanthine and xanthine to uric acid at the Mo-pterin site, while the other is a reduction of NAD+ to NADH at the FAD site of XDH, and that of O2 to O2− or hydrogen peroxide at the FAD site of XO. These reactions are coupled and proceed in a “ping-pong” manner (Bay, 1975). The electron flux moves between the Mo-pterin and FAD cofactors though two iron-sulfur clusters. As XOR precludes the salvage pathway of the purine nucleotides adenine, guanine and hypoxanthine, the enzyme has a rate-limiting function of generating irreversible uric acid and is highly regulated at the transcriptional and post-translational levels. Increased XOR activity induces hyperuricemia and then gout. The substrate specificity at the FAD site is broad, and the oxidant metabolites generated by XO induce have different consequences, ranging
∗
from cytotoxicity to inflammation. It is suggested that increased XO activity is related to asthma, phlogosis, endothelial activation, leukocyte activation, vascular tone regulation and various pathophysiological conditions (Fong et al., 1973; McCord, 1985; Setiawan et al., 2016; Battelli et al., 2016). XO inhibitors lower the serum uric acid content and protect against western diet-induced aortic stiffness and impaired vasorelaxation in mice (Lastra et al., 2017). Lowering the serum uric acid level may help ameliorate endothelial dysfunction and reduce myocardial function so as to prevent heart failure (Harzand et al., 2012). Therefore, as the inhibition of XO ameliorates hyperuricemia and induced oxidative stress, many researchers have shown an interest in testing the XO inhibitory activity in both natural compounds and synthetic products (Hatano et al., 1990; Toda et al., 1991; Kubo et al., 2002; Liu et al., 2009; Tuzun et al., 2017; Belkhiri et al., 2017). However, although XO inhibitory activity has been studied for a long time, the activity is still not fully understood. For example, the XO inhibitory activity of gallic acid (4a) and the n-alkyl ester, dodecyl gallate (4f), were examined. Dodecyl gallate inhibited both the uric acid formation and the O2− generation catalyzed by XO but gallic acid did not affect uric acid formation and suppressed O2−generation. This suggests that
Corresponding author. CDW Life Science Lab, Okayama Research Park Incubation Center, 5303 Haga, Kita-ku, Okayama 701-1221, Japan. E-mail address: [email protected] (N. Masuoka).
https://doi.org/10.1016/j.phytochem.2018.07.006 Received 22 February 2018; Received in revised form 11 July 2018; Accepted 16 July 2018 Available online 23 July 2018 0031-9422/ © 2018 Elsevier Ltd. All rights reserved.
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inhibited both the uric acid and O2− production catalyzed by XO, while cardanol C15:3 (2a) and salicylic acid (1e) did not. The alkenyl chain and phenolic portion in anacardic acid are independently responsible for the inhibitory activity. The inhibition was sigmoidal, and the reaction rate in the presence of anacardic acid is expressed asv = V max [S]/[(Km + [S]) (1 + [I]n/KI)] [When KI = (I) n, I = IC50.] The IC50 and slope factor (n) for the inhibition of uric acid formation were respectively 162 ± 10 μM and 1.7 ± 0.5, and they also suppressed O2− generation (IC50 = 53.6 ± 5.1 μM, n = 4.3 ± 0.5). Although the enzyme reaction similarly generated uric acid and O2−, the O2− generation was more potently suppressed than the uric acid formation. It was finally deduced that the suppression was the sum of the XO inhibition and O2− scavenging activity, even though anacardic acid does not have DPPH scavenging activity (Masuoka et al., 2016). As the alkenyl chain in anacardic acid is related to the inhibitory activity, alkyl gallates and caffeates were chosen as models to investigate the relationship. Akly1gallates (4a-g) were synthesized and tested (Nihei et al., 2003; Masuoka et al., 2006). Gallic acid and alkyl gallates (4a-c), having short side chains (< C6), did not inhibit the uric acid formation catalyzed by XO (Table 1). In the case of the alkyl gallates, which have longer C6 chains, stronger inhibition was observed. Octyl gallate (4e) competitively inhibited uric acid formation, and the docking experiment with this compound indicated it bound to the xanthine binding site in XO. Caffeic acid and the alkyl esters (5a-h) were also examined (Masuoka et al., 2012b). Alkyl caffeates competitively inhibited the uric acid formation catalyzed by XO, and the inhibition by alkyl caffeates increased with increasing alkyl chain length as alkyl gallates. Compared to alkyl gallates, the inhibition of the alkyl caffeates was more potent than that of the alkyl gallates. From these results, long alkyl chains in these inhibitors assist the inhibitor binding to the xanthine-binding site, using the hydrophobic interaction inside of XO, as indicated by the alkyl gallates as well as a
Abbreviations O2DPPH PMS ROS XDH XO XOR
Superoxide anion 1, 1-Diphenyl-2-picryhydrazyl Phenazine methosulfate Reactive oxygen species Xanthine dehydrogenase Xanthine oxidase Xanthine oxidoreductase
the O2− suppression of gallic acid is due to the antioxidant property and the alkyl chain of dodecyl gallate plays an important role in its inhibitory activity (Kubo et al., 2002). The alkenyl phenols, anacardic acid C15:3 6-[8′(Z), 11′(Z), 14′-pentadecatrienyl] salicylic acid (1a) and cardanol C15:3 (2a) were also tested. Anacardic acid C15:3 sigmoidally inhibited uric acid formation and O2− generation, but cardanol C15:3 did not (Masuoka and Kubo, 2004; Kubo et al., 2006). It indicates that both the phenol portion and alkenyl chain in alkenyl phenols play important roles in the inhibitory activity. In this review, we focus on the XO inhibitory activity of the phenolic compounds and alk(en)yl phenols isolated from edible plants and characterize their activity. Moreover, the XO inhibitory activity of natural and synthesized compounds (see Fig. 1) is broken down into three activity groups. 2. Natural sources of phenolic compounds Edible plants abundantly contain various kinds of phenolic compounds and alk(en)yl phenols. Alk(en)yl phenols, e.g. anacardic acids (1a-d), cardanols (2a-d) and cardols (3a, b), are present in cashew nuts, cashew apples, ginkgo nuts and cereal grains (Kubo et al., 1986; Irie et al., 1996; Kozubek and Tyman, 1999). Antioxidant, enzyme inhibitory, antibacterial and antitumor activities have been reported (Kubo et al., 1994; Hamad and Mubofu, 2015). The synthesis of various biologically active compounds has been reported using these compounds as the staring materials. In terms of the phenolic compounds, gallic acid (4a) is a widespread natural product that is known to be a potent antioxidant present in natural plant materials such as blackberry bark, henna, tea and uva ursi, and is often a component of hydrolysable tannins in many plants. Caffeic acid (5a) is also found in plants, fruits and vegetables. These compounds are increasingly of interest because of their broad biological effects, including antioxidative, antimicrobial and antitumor activities (Kartal et al., 2003; Gulcin, 2006; Cardenas et al., 2006). These compounds attract more interest than synthetic antioxidants due to the fact that they cause less damage to the human body (Lin and Yen, 1999). However, although gallic acid and caffeic acid are efficient antioxidants, their lower solubility in non-polar media limits their application in fatty food, especially various edible oils (Kalogeropoulos et al., 2009). To increase the solubility in non-polar media, n-alkyl esters of these phenolic compounds were synthesized. In particular, n-propyl, n-octyl and n-dodecyl gallates (4c-g) are widely used as food additives to scavenge ROS (Van Der Meeren, 1987). nAlkyl caffeates (5b-h) are likely to be useful in oily foods as antioxidants (Garrido et al., 2012; Wang et al., 2014). 3. How do phenolic compounds inhibit the uric acid formation catalyzed by XO? A sufficient amount of oxygen for the XO reaction was present in the aqueous reaction mixture, and XO activity was detected as uric acid formation generated from xanthine. Inhibition of uric acid formation, which is an XO inhibition effect, by phenolic compounds was tested. XO reactions were carried out at pH 10 to stabilize one of the reaction products (O2−) (Masuoka and Kubo, 2004). Anacardic acid C15:3 (1a)
Fig. 1. Structures of anacardic acids (1), cardanols (2), cardols (3) and related compounds (4–10). C15:3, C15:2 and C15:1 (above right) are the alkenyl chains of the compounds (1–3). 101
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Table 1 Inhibition of the uric acid formation catalyzed by XO with gallic acid, caffeic acid and n-alkyl esters. Compound
Gallic acid (4a) Methyl gallate (4b) Propyl gallate (4c) Hexyl gallate (4d) Octyl gallate (4e) Decyl gallate (4f) Dodecyl gallate (4g) Caffeic acid (5a) Methyl caffeate (5b) Propyl caffeate (5c) Butyl caffeate (5d) Pentyl caffeate (5e) Hexyl caffeate (5f) Heptyl caffeate (5g) Decyl caffeate (5h) a b c
IC50a(μM)
> 200 > 200 > 200 > 200c 262 ± 45 97 ± 3 49 ± 13 > 200 143 ± 13 30.2 ± 2.1 26.5 ± 2.0 18.0 ± 0.9 19.0 ± 1.0 12.5 ± 0.5 10.0 ± 1.0
For xanthine-binding (μM)b
Inhibition type
KI
KIS
– – – – 30.5 ± 1.8 35.8 ± 11.8 19.0 ± 6.0 – 38.6 ± 0.5 7.8 ± 0.8 7.3 ± 0.5 4.3 ± 0.4 3.6 ± 0.6 3.5 ± 0.5 3.3 ± 0.5
– – – – 943 ± 272 127 ± 23 46.7 ± 14.0 – – – – – – – –
– – – – Competitive Mixed Mixed – Competitive Competitive Competitive Competitive Competitive Competitive Competitive
IC50 values were measured at 200 μM xanthine and expressed as the mean ± SD. XO reaction was carried out with 0–200 μM xanthine under atmosphere. Hexyl gallate at 200 μM inhibited 13 ± 2% of the control activity.
en-diol structures (Blois, 1958).
squalene epoxidase (Abe et al., 2000). In the case of anacardic acid, the inhibition was noncompetitive for xanthine and sigmoidally inhibited. It is deduced that this interaction between the alkenyl chain of anacardic acid and the hydrophobic pocket in XO induces the cooperative binding and fitting of anacardic acid to the xanthine-binding site in XO. As the inhibition activity was related to the hydroxy groups in the phenol portion, other alkyl phenol compounds (6–10) were tested (Masuoka et al., 2012b, 2015). Protocatechuates (6a-c) and 3,5-dihydroxybenzoates (8a-c) displayed hardly any XO inhibition activity. By comparison, 3,4-dihydroxyphenyl alkanoates (7a-c) and 3,5-dihydroxyphenyl alkanoates (9a-c) exhibited weak inhibition at 200 μM. Similarly, hexyl and nonyl 2,4-dihydrobenzoates (10a, b) displayed hardly any inhibition activity, while dodecyl 2,4-dihydrobenzoate (10c) exhibited non-competitive inhibition (KI=KIS=187 ± 17 μM). These results indicate that it is necessary for XO inhibitors to have alkyl chains and particular hydroxy group arrangements in the phenol portion, since the phenol portion binds to the xanthine binding site, as in the case of the flavonoids (Lin et al., 2002; Masuoka et al., 2012a). It is suggested that it is necessary for XO inhibitors to be electron-rich. This is because the inhibitors electrostatically interact with the electron-deficient property of the XO molecules generated from XDH under oxidative conditions.
Conjugated en-diol structures. Table 2 Kinetic parameters of the gallic acid, caffeic acid and n-alkyl esters used for the O2− generated by XOa and for the DPPH scavenging activity. Compound
Gallic acid (4a) Methyl gallate (4b) Propyl gallate (4c) Hexyl gallate (4d) Octyl gallate (4e) Decyl gallate (4f) Dodecyl gallate(4g) Caffeic acid (5a) Methyl caffeate (5b) Propyl caffeate (5c) Butyl caffeate (5d) Pentyl caffeate (5e) Hexyl caffeate (5f) Heptyl caffeate (5g) Decyl caffeate (5h) Protocatechuic acid (6a) Heptyl protocatechuate (6b) Octyl protocatechuate (6c)
4. How do phenolic compounds suppress the O2− generation catalyzed by XO? 4.1. The O2− suppression and DPPH scavenging activity by phenolic acid and their alkyl esters The O2− generation catalyzed by XO was detected using blue formazan formation from the reaction with nitroblue tetrazolium (Toda et al., 1991; Masuoka and Kubo, 2004). The O2− suppression and DPPH scavenging activity of gallic acid, caffeic acid and their alkyl esters is summarized in Table 2. Although the enzyme reaction similarly generated uric acid and O2−, gallic acid and alkyl gallates (4a-g) displayed potent suppression of the O2− generation catalyzed by XO compared to inhibition of the uric acid formation (Masuoka et al., 2006). The suppression was unaffected by the alkyl chain length of gallates, unlike the inhibition of uric acid formation. The inhibition kinetics were analyzed as a mixed type. Caffeic acid and alkyl caffeates (5a-h) also suppressed O2− generation and scavenged DPPH radical similarly to the gallates. The O2− suppression of caffeates was less than that of the gallates. The DPPH scavenging activity of gallic acid, alkyl gallates (4a-g), caffeic acid and alkyl caffeates (5a-h) was assigned to their conjugated
Suppression of O2- generation IC50 (μM)b
KI
KIS
DPPH scavenging activityc
2.6 ± 0.3 7.4 ± 0.5 6.4 ± 1.0 5.2 ± 0.2 4.5 ± 0.5 3.9 ± 1.1 3.6 ± 0.2 11.5 ± 0.5 12.0 ± 0.5 11.5 ± 0.5 11.0 ± 0.5 10.0 ± 1.0 10.8 ± 1.5 7.6 ± 0.4 16.4 ± 6.4d 22.0 ± 0.6
1.5 ± 0.2 5.7 ± 0.4 4.5 ± 0.5 3.8 ± 0.4 3.9 ± 0.2 2.5 ± 0.6 1.8 ± 0.2 8.4 ± 0.8 4.6 ± 0.4 3.6 ± 0.2 4.0 ± 0.3 3.0 ± 0.5 3.1 ± 0.2 2.4 ± 0.2 9.1 ± 1.1 12.8 ± 0.3
2.9 ± 0.3 8.0 ± 0.6 6.8 ± 0.7 5.3 ± 0.6 4.7 ± 0.2 4.8 ± 1.0 4.4 ± 0.2 12.6 ± 1.1 15.8 ± 1.2 15.8 ± 0.4 28.4 ± 1.6 29.4 ± 1.3 25.4 ± 1.4 20.2 ± 0.9 38.3 ± 3.5 30.7 ± 0.7
6.51 6.34 6.02 5.74 6.09 6.02 7.28 4.63 4.73 4.83 4.69 4.49 3.77 5.71 4.81 3.53
65.2 ± 2.0
36.5 ± 2.1
87.6 ± 0.6
5.90 ± 0.83
82.0 ± 0.2
99.4 ± 0.4
87.3 ± 3.1
6.82 ± 0.17
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.30 0.12 0.17 0.22 0.14 0.18 0.54 0.20 0.26 0.11 0.12 0.22 0.60 0.23 0.15 0.23
a XO reaction was carried out at 0–200 μM xanthine in the presence of 0.0–2.0 μM of gallic acid or alkyl gallates under atmosphere. Values are expressed as the mean ± SD obtained from more than three separate experiments. b IC50 values are shown for 200 μM xanthine. c The values are the scavenged DPPH molecules/one molecule of the test compound. d IC50 values were estimated from inhibitions under 10 μM decyl caffeate.
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To further study this matter, the suppression of the O2− generation catalyzed by XO by other phenolic alkyl esters having conjugated endiol structures was tested. Protocatechuic acid and the esters (6a-c) displayed a DPPH scavenging activity (approximately 6 molecules per molecule) and mixed-type suppression of the O2− generation, similar to the gallates. 3,4-Dihydroxyphenyl alkanoates (7a-c) exhibited a ratio of approximately 3 DPPH molecules/tested molecule and sigmoidal suppression of the O2− generation. These results suggested that suppression of the O2− generation by phenolic acid and these alkyl esters is associated with the DPPH scavenging activity.
antioxidant activity (Kamal-Eldin et al., 2000). The suppression of O2− generation catalyzed by XO and the DPPH activity indicated in Table 3. Anacardic acid C15:3 sigmoidally inhibited XO (uric acid formation) activity, but cardanol C15:3 and cardol C15:3 (3a) did not (Masuoka and Kubo, 2004; Kubo et al., 2006). As O2− suppression by anacardic acid is stronger than XO inhibition, the O2− suppression by anacardic acid contained another form of O2− suppression activity. Cardols C15:3 and C10:0 sigmoidally suppressed O2− generation, but cardol C5:0 did not (Masuoka et al., 2015). These results indicate that O2− suppression is associated with the alk(en)yl chain length. To further investigate, suppression of the O2− generation catalyzed by XO with other phenolic alkyl ester compounds having no DPPH scavenging activity (≤0.04 ± 0.02) was examined. 3,5Dihydroxybenzoic acid and alkyl 3,5-dihydroxybenzoates (8a-c) exhibited no suppression activity. 3,5-Dihydroxyphenyl heptanoate (9a) also exhibited no suppression activity, while 3,5-dihydroxyphenyl decanoate and 3,5-dihydroxyphenyl tridecanoate (9c) displayed sigmoidal suppression. Hexyl 2,4-dihydroxybenzoate (10a) did not display any suppression activity but nonyl 2,4-dihydroxybenzoate (10b) did display sigmoidal suppression. Electron-rich phenol alkyl esters having no DPPH activity exhibited sigmoidal O2− suppression activity. From these results on the alk(en)yl phenols and related compounds, we deduced that the O2− suppression was associated with long alk(en) yl chains and that the O2− suppression with alk(en)yl phenols is induced by either O2− scavenging activity or specific enzyme modification as the inhibition of the uric acid formation was not observed.
4.1.1. Comparison of the O2− scavenging activity using a PMS-NADH system To compare the suppression of the O2− generation catalyzed by XO, the O2− scavenging activity was investigated using a PMS-NADH system (Nishikimi et al., 1972). The O2− scavenging activity of gallic acid and alkyl gallates is indicated in Fig. 2, and the scavenging activity was carefully analyzed, as follows. These scavenging activities of the gallates consist of two components. One group, having no or short alkyl chains, exhibited hyperbolic scavenging curves, and it was deduced that O2− was scavenged by electrons, these are suggested by the oxidation of the conjugated en-diol structure (Kawabata et al., 2002). Another group having long alkyl chains exhibited weak sigmoidal activity, and it was finally deduced that these compounds scavenged O2− and the phenolic radicals induced oxidative degradation reactions. Although the scavenging activity by gallic acid displayed the most potent O2− scavenging activity in these gallates, the O2− scavenging activity was low compared to the suppression activity of the O2− generation catalyzed by XO. The O2− scavenging activity of caffeic acid and alkyl caffeates displays a similar pattern as the gallates. As this O2− scavenging activity was also low compared to the suppression of the O2− generation catalyzed by XO, the suppression of the O2− generation is not primarily associated with O2− scavenging activity, but with other mechanisms (Masuoka and Kubo, 2016).
4.2.1. Similarity to the O2− scavenging activity using a PMS-NADH system To further examine the antioxidant activity, the scavenging of O2− using a PMS-NADH system was tested in the presence of cardols (C15:3, C15:2, C15:0) (Table 4). As the suppression activity of cardol C15:3 using the XO system is quite similar to the scavenging activity of cardol C15:3, it is deduced that this sigmoidal activity is not due to specific enzyme modification, but rather is due to the O2− scavenging activity (Masuoka et al., 2016).
4.1.2. Proposed mechanism of the O2− suppression by these phenolic compounds: modification of XO molecules Hille & Massey reported that XO takes several different oxidation states and that fully reduced XO contains a total of six electrons in the reduced iron-sulfur center, molybdenum (IV) and FADH2. Oxidation of four or six electron-reduced XO to two-electron-reduced XO generates hydrogen peroxide, while that of two-electron-reduced XO only generates O2− (Hille and Massey, 1981). The DPPH scavenging activity of alkyl gallates and caffeates indicated that the stable radical was reduced with the oxidation of the conjugated en-diol structures in their phenol portion. From these findings, it was deduced that these gallate and caffeate derivatives as well as related compounds bind the FAD site to reduce the enzyme and the modification of XO induces the O2− suppression. This O2− suppression by reduction-modification of the enzyme was also supported in the presence of flavonoids with high DPPH activity (Masuoka et al., 2012a). Therefore, we propose that the suppression of the O2− generation catalyzed by XO was induced by a reduction of XO molecules. The phenolic compounds and alkyl esters having DPPH scavenging activities are shown as reducing agents [B] in Fig. 3, eventually suppressing the O2− generation catalyzed by XO.
4.2.2. O2− scavenging mechanism of alk(en)yl phenols Resorcinol compounds with a long hydrocarbon chain at the 5-position have been reported to act as antioxidants and protect polyunsaturated fatty acids against peroxidation. However, Kamal-Eldin et al. (2000) reported that cardol C15:0 has a low hydrogen donation capacity and peroxyl radical-scavenging effect. This scavenging activity
4.2. The O2− suppression and DPPH scavenging activity by natural alk(en) yl phenols and related compounds Anacardic acidC15:3; 6-[8′(Z), 11′(Z), 14′-pentadecatrienyl] salicylic acid (1a), cardanol C15:3 (2a), cardol C15:3; 5-[8′(Z), 11′(Z), 14′-pentadecatrienyl] resorcinol and related compounds (C10:0 and C5:0) (3a-d) were examined. These compounds had low DPPH scavenging activity (having no conjugated en-diol structure), but had considerable
Fig. 2. The suppression of the O2− generated by the PMS-NADH system. Gallic acid and alkyl derivatives; gallic acid (●), methyl gallate (○), propyl gallate (▼), hexyl gallate (▽), octyl gallate (■), decyl gallate (□) and dodecyl gallate (◆). 103
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Table 4 O2− Scavenging activity of alkyl phenols using PMS-NADH. IC50 (μM)a
Compound Anacardic acid C15:3 Anacardic acid C15:2 Anacardic acid C15:1 Anacardic acid C15:0 Salicylic acid (1f) Cardanol C15:3 (2a) Cardanol C15:2 (2b) Cardanol C15:1 (2c) Cardanol C15:0 (2d) Cardol C15:3 (3a) Cardol C15:2 (3b) Cardol C15:0 (3c) Resorcinol (3f) a
(1a) (1b) (1c) (1d)
98 ± 2 (n = 6.7 ± 0.9, sigmoidal) 95 ± 3 (n = 5.0 ± 1.8, sigmoidal) 72 ± 5 (n = 10.5 ± 2.2, sigmoidal) 41 ± 3 (n = 9.4 ± 1.5, sigmoidal) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (No inhibitiona) 116 ± 10(n = 8.7 ± 1.8, sigmoidal) 116 ± 8 (n = 8.1 ± 1.3, sigmoidal) 117 ± 4 (n = 6.4 ± 1.5, sigmoidal) > 200 (No inhibitiona)
Indicates inhibition at 200 μM.
phenol portion in the inhibitors binds to the xanthine binding site. So, XO inhibitors have particular hydroxy group arrangements in the phenol portion and long alk(en)yl chains. (2) Modification of the XO molecule by the reducing activity of phenolic compounds. The enzyme molecule is reduced by the oxidation of the phenolic compounds having conjugated en-diol structures. When the reduced enzyme molecule catalyzes the reaction of xanthine to uric acid, the enzyme is further reduced and does not generate O2−, but rather, hydrogen peroxide. This reduced-modification does not inhibit uric acid formation, and this enzyme-reducing ability of phenolic compounds is evaluated using DPPH radical scavenging activity. (3) Scavenging of O2− with phenolic compounds. The scavenging activity is due to the reaction of these compounds with the O2− generated by XO and consists of two reaction types. One (3a) is responsible for the conjugated en-diol structure that is detected by DPPH activity, while the other (3b) is associated with the phenolic portion and the amphiphilic property of alk(en)yl phenols. The scavenging activities were determined using the PMS-NADH system.
Fig. 3. Proposed mechanism of the O2− or hydrogen peroxide generation catalyzed by xanthine oxidase. [A]: Uric acid formation inhibitors, [B]: reducing agents of the enzyme (molecule).
of cardols may be explained as follows. Cardols are amphiphilic molecules and the content in aqueous medium rapidly increases at levels greater than the critical micelle concentration (Stasiuk and Kozubek, 2010). An increase in the hydroxy group in an aqueous medium induces hydrogen donation to O2−, while radical formation at the hydroxy oxygen initiates changes in the electron density that destabilizes the phenolic ring and this in turn subsequently leads to oxidation of the ring (Hladyszowki et al., 1998). The scavenging activity of cardols may be related to the weak DPPH scavenging activity (Table 3). This mechanism may also underlie the weak and sigmoidal O2− scavenging activity of the alkyl gallates and caffeates having long alkyl chains (Fig. 2). 5. Classifying the XO inhibitory activity of phenolic compounds and alk(en)yl phenols The XO inhibitory activity of phenolic compounds is divided into three activity groups.
Therefore, the activities of the gallic acid, caffeic acid and alkyl gallates having short chains were assigned to the (2) and (3a) groups, while the alkyl gallates having long chains and alkyl caffeates were assigned to (1), (2) and (3b). Anacardic acids exhibited (1) and (3b) activities, while the cardols exhibited (3b) activity.
(1) Inhibition of uric acid formation by phenolic compounds (XO inhibition). This activity is measured by the inhibition of uric acid formation. The alk(en)yl chain in inhibitors potentially assists the inhibition using the hydrophobic interaction with XO and the
Table 3 Suppression of the O2− generation catalyzed by xanthine oxidase with alkyl phenols having no DPPH scavenging activity. Compound
IC50 (μM)a
DPPH scavengingb
Anacardic acid C15:3 (1a) Salicylic acid (1e) Cardol C15:3 (3a) Cardol C10:0 (3d) Cardol C5:0 (3e) Resorcinol (3f) Cardanol C15:3 (2a) 3,5-Dihydroxybenzoic acid (8a) Hexyl 3,5-dihydroxybenzoate (8b) Dodecyl 3,5-dihydroxybenzoate (8c) 3,5-Dihydroxyphenylheptanoate (9a) 3,5-Dihydroxyphenyldecanoate (9b) 3,5-Dihydroxyphenyltridecanoate (9c) Hexyl 2,4-dihydroxybenzoate (10a) Nonyl 2,4-dihydroxybenzoate (10b)
51.3 ± 1.5 (n = 4.2 ± 0.5, sigmoidal) > 200 (No inhibitiona) 115 ± 10 (n = 5.2 ± 0.2, sigmoidal) 106 ± 8 (n = 5.1 ± 0.2, sigmoidal) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (No inhibitiona) > 200 (2.5% inhibitiona) 121 ± 4 (n = 1.9 ± 0.4, sigmoidal) 75.3 ± 5.0 (n = 5.5 ± 1.8, sigmoidal) > 200 (16% inhibitiona) 118 ± 1 (n = 3.7 ± 0.1, sigmoidal)
0.02 0.01 0.49 0.50 0.50 0.02 0.02 0.04 0.02 0.01 0.01 0.02 0.01 0.01 0.01
a b
Indicated inhibition at 200 μM. The value indicates scavenged DPPH molecules/one molecule of the tested compound.
104
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01
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6. Conclusions and perspectives
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Tyrosinase inhibitors from Anacardium occidentale fruits. J. Nat. Prod. 57, 545–552. Kubo, I., Masuoka, N., Ha, T.J., Tsujimoto, K., 2006. Antioxidant activity of anacardic acids. Food Chem. 99, 555–562. Kubo, I., Masuoka, N., Xiao, P., Haraguchi, H., 2002. Antioxidant activity of dodecyl gallate. J. Agric. Food Chem. 50 (12), 3533–3539. Lastra, G., Manrique, C., Jia, G., Aroor, A.R., Hayden, M.R., Barron, B.J., Niles, B., Padilla, J., Sowers, J.R., 2017. Xanthine oxidase inhibition protects against Western dietinduced aortic stiffness and impaired vasorelaxation in female mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. https://doi.org/10.1152/ajpregu. 00483.2016. May 24. Lin, C.M., Chen, C.S., Chen, C.T., Liang, Y.C., Lin, J.K., 2002. Molecular modeling of flavonoids that inhibits xanthine oxidase. Biochem. Biophys. Res. Commun. 294 (1), 167–172. Lin, M., Yen, C., 1999. Antioxidative ability of lactic acid bacteria. J. Agric. Food Chem. 47, 1460–1466. Liu, X., Chen, R., Shang, Y., Jiao, B., Huang, C., 2009. Superoxide radicals scavenging and xanthine oxidase inhibitory activity of magnesium lithospermate B from Salvia miltiorrhiza. J. Enzyme Inhib. Med. Chem. 24 (3), 663–668. Masuoka, N., Kubo, I., 2004. Characterization of xanthine oxidase inhibition by anacardic acids. Biochim. Biophys. Acta 1688, 245–249. Masuoka, N., Nihei, K., Kubo, I., 2006. Xanthine oxidase inhibitory activity of alkyl gallates. Mol. Nutr. Food Res. 50, 725–731. Masuoka, N., Matsuda, M., Kubo, I., 2012a. Characterisation of the antioxidant activity of flavonoids. Food Chem. 131, 541–545. Masuoka, N., Nihei, K., Masuoka, T., Kuroda, K., Sasaki, K., Kubo, I., 2012b. The inhibition of uric acid formation catalyzed by xanthine oxidase, Properties of the alkyl caffeates and cardol. J. Food Res. 1 (3), 257–262. Masuoka, N., Nihei, K., Maeta, A., Yamagiwa, Y., Kubo, I., 2015. Inhibitory effects of cardols and related compounds on superoxide anion generation by xanthine oxidase. Food Chem. 166 (1), 270–274. Masuoka, N., Kubo, I., 2016. Suppression of superoxide anion generation catalyzed by xanthine oxidase with alkyl caffeates and the scavenging activity. Int. J. Food Sci. Nutr. 67 (3), 283–287. https://doi.org/10.3109/09637486.2016.1153609. Masuoka, N., Tamsampaoloet, K., Chavasiri, W., Kubo, I., 2016. Superoxide anion scavenging activity of alk(en)yl phenol compounds by using PMS-NADH system. Heliyon 2, e00169. McCord, J.M., 1985. Oxygen-derived free radicals in postischemic tissue injury. N. Engl. J. Med. 312, 159–163. Nihei, K., Nihei, A., Kubo, I., 2003. Rational design of antimicrobial agents: antifungal activity of alk(en)yl dihydroxybenzoates and dihydroxyphenyl alkanoates. Bioorg. Med. Chem. Lett 13, 3993–3996. https://doi.org/10.1016/j.bmcl.2003.08.057. Nishikimi, M., Rao, N.A., Yagi, K., 1972. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem. Biophys. Res. Commun. 46, 849–854. Pauff, J.M., CaO, H., Hille, R., 2009. Substrate orientation and catalysis at the molybdenum site in xanthine oxidase. J. Biol. Chem. 284 (13), 8760–8767. Setiawan, H., Nagaoka, K., Kubo, M., Fujikura, Y., Ogino, K., 2016. Involvement of xanthine oxidoreductase-related oxidative stress in a dermatophagoides farina-induced asthma model of NC/Nga mice. Acta Med. Okayama 70 (3), 175–182. Stasiuk, M., Kozubek, A., 2010. Biological activity of phenolic lipids. Cell. Mol. Life Sci. 67, 841–860. Toda, S., Kumura, M., Ohnishi, M., 1991. Effects of phenolcarboxylic acids on superoxide anion. Planta Med. 57, 8–10. Tuzun, B.S., Hajdu, Z., Orban-Gyapai, O., Zomborszki, Z.P., Jedlinszki, N., Forgo, P., Kivcak, B., Hohmann, J., 2017. Isolation of chemical constituents of Centaurea virgata Lam. 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XO inhibition of uric acid formation indicated that the inhibitors competitively inhibited XO. The phenol portion of the inhibitors binds to the xanthine binding site, and the alk(en)yl side chain enhances the binding to XO molecules. In addition, suppression of the O2− generation catalyzed by XO was affected by enzyme-reducing modification and two types of O2− scavenging activities. The enzyme-reducing modification capacity of phenolic compounds was evaluated by DPPH radical scavenging activity, while O2− scavenging was measured using a PMS-NADH system. The antioxidant activity of phenolic compounds was deduced as follows. Gallic acid, caffeic acid and alkyl gallates having short chains lower solubility in non-polar media such as edible oils and are easily oxidized with O2−, resulting in an increase of hydrogen peroxide generation (Masuoka et al., 2016). It is suggested that these compounds do not have utility as antioxidants in oily foods. Alkyl gallates and alkyl caffeates having long side chains are synthetic amphiphilic compounds and display sigmoidal O2− scavenging activity. Though these compounds are used as food additives, they may become oxidized because of their conjugated en-diol structure (Van Der Meeren, 1987). Anacardic acids and cardols are natural amphiphilic compounds without any conjugated en-diol structure, so it is suggested that these compounds are likely to be applicable as antioxidants, even in oily or fatty foods. For the prevention of hyperuricemia and oxidative stress, anacardic acids were shown to inhibit uric formation but cardol and cardanol do not. The anacardic acids exhibit both XO inhibition and superoxide anion scavenging activities. Anacardic acid C15:0 may be expected to have potential for the treatment of hyperuricemia since it had the most potent O2− scavenging activity among the anacardic acids, as long as untoward side-effects are not observed. Further study is warranted to test these hypotheses. Acknowledgments We thank Professor Ken-ichi Nihei, Faculty of Agriculture, Utsunomiya University, Japan, for presents of alkyl-phenol related compounds. References Abe, I., Seki, T., Noguchi, H., 2000. Potent and selective inhibition of squalene epoxidase by synthetic galloyl esters. Biochem. Biophys. Res. Commun. 270, 137–140. Battelli, M.G., Polito, L., Bortolotti, M., Bolobnesi, A., 2016. Xanthine oxidoreductasederived reaction species: physiological and pathological effects. Oxid. Med. Cell Longev. 2016, 3527579. Bay, R.C., 1975. Molybdenum iron-sulfur flavin hydroxylases and related enzymes. In: In: Boyer, P.D. (Ed.), The Enzymes, vol. 12. Academic Press New York, pp. 299. Belkhiri, E., Baghiani, A., Zerroug, M.M., Arrar, L., 2017. Investigation of antihemolytic, xanthine oxidase inhibition, antioxidant and antimicrobial properties of Salvia verbenaca L. aerial part extracts. Afr. J. Tradit., Complementary Altern. Med. 14 (2), 273–281. Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature 181, 1199–1200. Cardenas, M., Marder, M., Blank, V.C., Roguin, L.P., 2006. Antitumor activity of some natural flavonoids and synthetic derivatives on various human and murine cancer cell lines. Bioorg. Med. Chem. 14, 2966–2971. Enroth, C., Eger, B.T., Okamoto, K., Nishino, T., Nishino, T., Pai, E.F., 2000. Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: structurebased mechanism of conversion. Proc. Natl. Acad. Sci. U.S.A. 97, 10723–10728. Fong, K.L., McCay, P.B., Poyer, J.L., Keele, B.B., Misr, H., 1973. Evidence that peroxidation of lysosomal membranes is initiated by hydroxyl free radicals produced during flavin enzyme activity. J. Biol. Chem. 248 (22), 7792–7797. Garrido, J., Gaspar, A., Garrido, E.M., Miri, R., Tavakkoli, M., Pourali, S., Saso, L., Borges, F., Firuzi, O., 2012. Alkyl esters of hydroxycinnamic acids with improved antioxidant activity and lipophilicity protect PC12 cells against oxidative stress. Biochimie 94, 961–967. Gulcin, I., 2006. Antioxidant activity of caffeic acid (3,4-dihydroxycinnamic acid). Toxicology 217, 213–220. Hamad, F.B., Mubofu, E.B., 2015. Potential biological applications of bio-based anacardic acids and their derivatives. Int. J. Mol. Sci. 16, 8569–8590. Harzand, A., Tamariz, L., Hare, J.M., 2012. Uric acid, heart failure survival, and the impact of xanthine oxidase inhibition. Congest. Heart Fail. 18, 179–182.
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Isao Kubo has worked on chemistry, insect physiology and ecology in Kenya, Japan and USA. Since 1986, he became a Professor of natural products chemistry in University of California and (2016 -) a Professor Emeritus. His research involves in all areas of natural product chemistry. Since 1977, he is a member of American Association for the Advancement of Science (AAAS) Fellow.
Van Der Meeren, H.L., 1987. Dodecyl gallate, permitted in food, is a strong sensitizer. Contact Dermatitis 16, 260–262. Wang, J., Gu, S.S., Pang, N., Wang, F.Q., Pang, F., Cui, H.S., Wu, X.Y., Wu, F.A., 2014. Alkyl caffeates improve the antioxidant activity, antitumor property and oxidation stability of edible oil. PLoS One 9 (4), e95909.
Noriyoshi Masuoka worked on biochemistry in Okayama University Graduate School of Medicine and Dentistry. Since 2005, he became a professor of biochemistry at Okayama University of Science. Now (2017 -), he is a chief researcher in CDW Life science laboratory in Okayama. He investigates the functions of oxidative stresses and antioxidants, especially acatalasemia (catalase deficiency) and xanthine oxidase inhibitory activity of natural products.
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