The antioxidative effects of the isoflavan glabridin on endogenous constituents of LDL during its oxidation

Abstract

The effect of the consumption of glabridin, an isoflavan isolated from Glycyrrhiza glabra (licorice) root, on the susceptibility of low density lipoprotein (LDL) to oxidation was studied in atherosclerotic apolipoprotein E deficient (E° mice) and was compared with that of the known flavonoids, quercetin and catechin. Glabridin inhibitory activity on in vitro oxidation of human LDL was also investigated by determining the formation of lipid peroxides and oxysterols and the consumption of LDL-associated lipophilic antioxidants. Determination of the extent of LDL oxidation by measuring the formation of thiobabituric acid reactive substances (TBARS) after 2 h of LDL incubation with CuSO₄ (10 μM) or 2,2′-azobis (2-amidino-propane) dihydrochloride (AAPH) (5 mM), revealed that glabridin or quercetin consumption resulted in a 53 and 54% reduction in copper ion induced oxidation, respectively, and a 95 and 83% reduction in AAPH induced LDL oxidation, respectively. No inhibition was obtained with consumption of catechin. About 80% of glabridin was found to bind to the LDL human particle. In the in vitro oxidation of LDL induced by AAPH (5 mM), glabridin inhibited the formation of TBARS, lipid peroxides and cholesteryl linoleate hydroperoxide (CLOOH) at all the concentrations tested (5–60 μM), while in oxidation induced by copper ions (10 μM), glabridin exhibited a pro-oxidant activity at concentrations lower than 20 μM, and a clear antioxidant activity at concentrations greater than 20 μM. Glabridin (30 μM) inhibited the formation of cholest-5-ene-3,7-diol (7-hydroxycholesterol), cholest-5-ene-3-ol-7-one (7-ketocholesterol) and cholestan-5,6-epoxy-3-ol (5,6-epoxycholesterol) after 6 h of AAPH induced LDL oxidation, by 55, 80 and 40%, respectively, and after 6 h of copper ion induced LDL oxidation, by 73, 94 and 52%, respectively. Glabridin also inhibited the consumption of β-carotene and lycopene by 38 and 52%, respectively, after 0.5 h of LDL oxidation with AAPH, but failed to protect vitamin E. The in vivo and in vitro reduction of the susceptibility of LDL to oxidation obtained with glabridin, may be related to the absorption or binding of glabridin to the LDL particle and subsequent protection of LDL from oxidation by inhibiting the formation of lipid peroxides and oxysterols, and by protecting LDL associated carotenoids.

Introduction

Human low density lipoprotein (LDL) is the major cholesterol carrier in the blood stream. It is well established that the cholesterol deposits in the arteries stem primarily from plasma LDL and that increased levels of plasma LDL correlate with an increased risk for atherosclerosis. Various lines of research provide strong evidence that LDL becomes oxidized in vivo and that oxidized LDL is involved in the formation of the early atherosclerotic lesions 1, 2, 3, 4, 5, 6, 7, 8.

Oxidation of LDL involves lipid peroxidation, in which the LDL polyunsaturated fatty acids are rapidly converted into lipid peroxides and into aldehydic lipid peroxidation products [3]. Cholesterol is oxidized during LDL oxidation and high levels of the oxysterol derivatives are found in oxidized LDL 9, 10, 11. Several reports suggest that the oxidation of LDL starts after the depletion of its endogenous lipophilic antioxidants, such as vitamin E, β-carotene, and lycopene 12, 13, 14, 15, 16, 17. Protection of LDL from oxidation is thus an effective strategy to prevent or to retard the progression of atherosclerosis. Compounds that can prevent lipid peroxidation (antioxidants) may increase the resistance of LDL to peroxidation. Lipophilic antioxidants, such as probucol, vitamin E or carotenoids, were shown to protect LDL from oxidation in vitro and in vivo 18, 19, 20, 21, 22, 23, 24, 25, 26, 27.

Plants produce a variety of phenolic antioxidants. Among the various phenolic compounds, the flavonoids are perhaps the most important group [28]. Flavonoids are components of a wide variety of edible plants, fruits, vegetables and grains, and are an integral part of the human diet [29].

Mangiapane et al. [30] demonstrated the ability of catechin, a naturally occurring flavanol derivative, to inhibit LDL oxidation induced by copper ions or by arterial wall cells. De Whalley et al. [31] reported that certain flavone and flavonol derivatives found in the diet significantly inhibited LDL oxidation by macrophages. These flavonoids conserved the vitamin E content of LDL and delayed the onset of detectable lipid peroxidation. It was demonstrated that consumption of red wine with meals reduced the susceptibility of human plasma and LDL to lipid peroxidation [32]. Consumption by atherosclerotic apolipoprotein E-deficient (E° mice) of red wine or of its major flavonoids quercetin, and to a lesser extent catechin, led to attenuation in the development of the atherosclerotic lesion, and this effect was associated with reduced susceptibility of their LDL to oxidation [33]. Extract of the root of Glycyrrhiza glabra (licorice), a plant originating from Asia where it has been employed to treat several diseases 34, 35, successfully reduced the susceptibility of LDL to oxidation 36, 37. Dietary supplementation of E° mice and humans with licorice extract resulted in a substantial reduction in the susceptibility of their LDL to oxidation. An analysis of the mice aortic arch lesions after consumption of licorice, in comparison with mice treated with placebo, was performed. Light microscopy revealed histopathologic atherosclerotic lesion in the aortic arch of both groups of mice, although the incidents of the lesion was far greater in the placebo-treated mice [37]. Four isoflavans, isolated from licorice roots, were found to inhibit LDL oxidation. Glabridin, one of the isoflavans isolated, was the major flavonoid and antioxidant constituent [36]. These isoflavans are considerably more lipophilic than the flavone or flavanol subclasses which were more extensively investigated. The LDL of E° mice fed with glabridin was found to be more resistant to oxidation than LDL from mice fed with placebo [37]. These results indicate that glabridin may be responsible for the effect of licorice in vivo.

In view of the in vivo results obtained with licorice extract and its major constituent glabridin, it is desirable to continue a deeper investigation to determine the mechanism by which glabridin protects LDL from oxidation, affecting the endogenous LDL constituents, such as cholesterol and antioxidants. The aim of the present study was to analyze the effect of glabridin on the consumption of lipophilic endogenous antioxidants and on the formation of cholesteryl linoleate hydroperoxide and oxysterols, during the oxidation of LDL.