Inhibitory Effect of Flavonoids on Lipoxygenase-Dependent Lipid

Chapter 17

Inhibitory Effect of Flavonoids on LipoxygenaseDependent Lipid Peroxidation of Human Plasma Lipoproteins 1

Downloaded by SUFFOLK UNIV on January 24, 2018 | http://pubs.acs.org Publication Date: September 24, 1998 | doi: 10.1021/bk-1998-0701.ch017

J. Terao , E. L. da Silva, H. Arai, J-H. Moon, and M. K. Piskula National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, 2-1-2 Kannondai, Tsukuba, Ibaraki 305, Japan

There is increasing evidence that oxidative modification of plasma low-density lipoprotein (LDL) leads to the formation of lipid-laden foam cells in atherosclerotic lesions. In recent years, a role of lipoxygenase in this event has attracted much attention, although the mechanism for the modification still remains uncertain. Thus, we evaluated the inhibitory effect of several dietary antioxidants, including flavanoid aglycone, and glycosides, on the lipoxygenasedependent lipid peroxidation of human plasma L D L . 15Lipoxygenases (15-LOX) from soybean and rabbit reticulocytes were used as the enzymes. L D L oxidation was monitored by the measurement of cholesteryl ester hydroperoxides (CE-OOH), using reverse-phase HPLC. (-)-Epicatechin, quercetin, and quercetin monoglucosides (quercetin-3-O-β-glucopyranoside, quercetin 4'-Oβ-glucopyranoside, quercetin-7-O-β-glucopyranoside) were found to inhibit the accumulation of CE-OOH, whereas no inhibition was observed with 5-fold α-tocopherol-enriched L D L . It is, therefore, implied that dietary flavonoids, such as catechins and quercetin, can exert a role in decreasing cardiovascular diseases by inhibiting lipoxygenase-induced L D L oxidation. It is generally accepted that oxidatively modified, low-density lipoprotein (LDL) plays an important role in the early stages of atherogenesis (1-3). Although the exact mechanism by which oxidized L D L is formed in vivo is still unknown, L D L can be modified oxidatively by 15-lipoxygenases (15-LOX) in in vitro systems (4-7). High levels of lipoperoxides were found in L D L , and incubated with fibroblasts which overexpress 15-LOX (8,9). Therefore, the 15-LOX reaction seems to participate in the initial process of L D L oxidation as shown if Fig. 1. Several clinical trial studies have indicated that dietary antioxidants help in preventing the progression of

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©1998 American Chemical Society

Shibamoto et al.; Functional Foods for Disease Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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coronary artery disease (review, see ref. 10). These studies are focusing on the introduction into the L D L particles of chain-breaking antioxidants, such as atocopherol and the carotenoids, which decrease the extent of L D L oxidation. The use of 15-LOX inhibitors from dietary sources might be an interesting mode of prevention of atherosclerosis if 15-LOX plays an essential part in oxidative modification of L D L in vivo. Here, we selected (-)-epicatechin, quercetin, and quercetin monoglucosides as typical dietary flavonoids (Figure 2), which are recognized as lipoxygenase inhibitors (11,12). Their inhibitory effects on 15-LOXdependent L D L oxidation was evaluated by measuring the accumulation of cholesteryl ester hydroperoxides (CE-OOH). The results suggest that on a molar basis, these flavonoids are stronger inhibitors than α-tocopherol in the 15-LOXinduced oxidative modification of L D L . Effect of Lipophilic Antioxidants on Soybean Lipoxygenase-Dependent Peroxidation of Phospholipid-Bile Salt Micelles We first investigated the inhibition of some typical antioxidants in the enzymatic lipid peroxidation of bile-salt micelles of phosphatidylcholine (PC), using soybean lipoxygenase (13). Soybean enzyme can oxidize micellar phospholipids directly in the presence of bile salt (14,15). The inhibition ratio of each antioxidant was calculated from the phosphatidylcholine hydroperoxide (PC-OOH) concentration after 60 min. incubation with, and without, antioxidants. The 50% inhibition concentrations (IC50) were obtained, as shown, in Table 1. The IC50 of quercetin (90 μΜ), is much lower than α-tocopherol (220 μΜ). This indicates that quercetin possesses a higher inhibitory effect than α-tocopherol in the 15-LOX-dependent lipid peroxidation of esterefied lipids, in biological systems. β-Carotene showed no inhibitory effect, even at the concentration of 1 m M . Effect of Antioxidants on Soybean Lipoxygenase-Dependent Peroxidation of Human LDL. Human L D L was isolated from fresh human plasma by discontinuous densitygradient ultracentrifugation, according to the method of Kleinveld (16). Its suspension in PBS buffer (0.2 mg protein/ml) was oxidized by the addition of 10,000 U/ml of soybean lipoxygenase (Typel-B, 206,000 units/mg protein from Sigma Chemical Co.). L D L oxidation was carried out at 20°C, and the cholesteryl ester hydroperoxides (CE-OOH) was measured by reverse phase HPLC as the index of L D L oxidation levels (Figure 3). There was a progressive increase in the CE-OOH level without a detectable lag phase. In addition, 1 μΜ ascorbic acid was an effective antioxidant only in the first 3 hours. The L D L fraction used in the experiment, contained endogenous α-tocopherol, at the level of 11.4 η mol/mg protein. We prepared α-tocopherol-enriched L D L by the addition of D/L-atocopherol to plasma. L D L particles were isolated after incubation for 3 hours. This L D L contained 5-fold enriched α-tocopherol (50.2 nmol/mg protein), however, no inhibitory effects were identified by the enrichment treatment. In contrast, (-)-



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Fig. 1 Structure of Flavonoids Used in This Study

Quercetin

(-)-Epicatechin

Flavone

OH

OH „OGIc

,OH OH

GIcO

Il H OH

OGIc

Ο

Q3G Fig. 2 Possible Pathway for Oxidative Modification of Lipoprotein (LDL) Leading to Athersclerosis.

OH

IjMJ OH

OH

Ο

Q4'G Human Low-Density

Table I. The 50% Inhibition Concentrations (IC50) of Quercetin and Quercetin Monoglucosides on Mammalian 15-LOX-Induced Oxidation of Cholesteryl Esters in Human LDL Antioxidants IC50 (μΜ) Quercetin 0.35 0.47 Q3G Q7G 0.47 1.2 Q4'G Human LDL (0.4 μΜ) was oxidized with 15-LOX (1 μΜ) at 20°C, for 3 hours in the presence or absence of quercetin or quercetin glucosides.


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epicatechin and quercetin were found to be strong L O X inhibitors. Thus, it is indicated that (-)-epicatechin and quercetin act as effective inhibitors when L D L was subject to the lipoxygenase reaction.


Effect of Antioxidants on Mammalian 15-LOX-Dependent Lipid Peroxidation of Human LDL. Mammalian 15-LOX has been purified from various tissues, and its molecular properties have been studied (17). Mammalian 15-LOX is capable of oxygenating esterified fatty acids, not only in micelles, but also in biomembranes and plasma lipoproteins. It is suggested to participated in the oxidative modification of L D L . We applied L O X from rabbit reticulocytess as mammalian 15-LOX, and its reaction with L D L was used as the in vitro model system for the estimation of the antioxidant activity of flavonoids in the LOX-induced oxidative modification of L D L (Figure 4). Similar to soybean L O X , CE-OOH accumulated linearly by the reaction of reticulocyte 15-LOX with human LDL, and (-)-epicatechin and quercetin, at 1 μΜ, and inhibited the accumulation effectively. α-Tocopherol-enriched L D L did not inhibit the CE-OOH accumulation, and ascorbic acid only inhibited the first stage of 3 hours, at the level of 1 μΜ. Thus, flavonoids are superior to α-tocopherol and ascorbic acid in the 15-LOX-dependent oxidation of L D L . However, flavone at the same concentration was completely ineffective. Indeed, flavone contains a flavonoid nucleus, but does not have any additional phenolic groups. It is therefore likely that the substitution of the flavone nucleus with hydroxyl groups is essential to the ability of flavonoids to inhibit the 15-LOX catalyzed L D L oxidation. Effect of Quercetin and Quercetin Monoglucosides on Mammalian 15-LOXDependent Lipid Peroxidation of Human LDL A number of flavonoids are commonly found in foods in the form of glycosides. For example, quercetin is mainly present in the forms of 3,4'-di-0-P-glucosides, and 4'Οβ-glucoside in onion (18). Glucosidase from human plasma, and intestinal bacteria, are likely to hydrolyze the glucose moiety, resulting in a flavonoid aglycone (19). However, a recent study has shown the presence of flavonoids as glycosides in human plasma (20). In order to know the difference between a flavonoid aglycone, and its glycoside, in the inhibition of 15-LOX-induced L D L oxidation, we selected quercetin and quercetin glucosides (quercetin-3-0-/?-glucopyranoside; Q3G, quercetin 4'-0-p-glucopyranoside; Q4G, and quercetin-7-0-P-glucopyranoside; Q7G). Table 1 shows the IC 50 (the 50% inhibition concentration) of these aglycones and glucosides. Interestingly, binding of the glucose group at the 3- and 7- positions influenced the inhibitory effect very little, indicating that hydroxyl groups at 3- and the 7- positions, at least partly, do not take an essential part in the inhibition mechanism. On the other hand, IC 50 obtained from Q4'G was lower than those obtained from the other glucosides and aglycone. The hydroxyl group at the Β ring seems to, to some extent, contribute to the inhibition of 15-LOX-induced oxidation.



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Incubation time (hr) Fig. 3

Effect of antioxidants on LDL oxidation indiced by soybean 15-LOX.

18

Incubation Time (hr) Fig. 4. Effect of antioxidants on L D L oxidation induced by rabbit reticulocyte 15-LOX.


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Conclusion In 15-LOX-induced L D L oxidation, flavonoids containing a polyhydroxyl group can act as strong inhibitors. They may, therefore, have a role in the prevention of atherosclerosis if 15-LOX participates in the process of this degenerative disease. Endogenous α-tocopherol in L D L may not work as an effective inhibitor of this process. Quercetin glucosides, as well as aglycone, can inhibit this LOX-induced oxidation. These flavonoids may be promising compounds for the prevention of atherosclerosis.


Literature Cited

J.

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Inhibitory Effect of Flavonoids on Lipoxygenase-Dependent Lipid

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