Original Contribution
Antioxidant Constituents from Licorice Roots: Isolation, Structure Elucidation and Antioxidative Capacity Toward LDL Oxidation

https://doi.org/10.1016/S0891-5849(97)00089-0Get rights and content

Abstract

The present study analyzed the antioxidative properties of natural compounds from the root of the plant Glycyrrhiza glabra (licorice) toward LDL oxidation. Seven constituents, with antioxidant capacity were isolated from Glycyrrhiza glabra. The isolated compounds were identified as the isoflavans Hispaglabridin A (1), Hispaglabridin B (4), Glabridin (3), and 4′-O-Methylglabridin (2), the two chalcones, isoprenylchalcone derivative (5) and Isoliquiritigenin (6), and the isoflavone, Formononetin (7). Among these compounds, Glabridin constituted the major amount in the crude extract (11.6%, w/w) as detected by high-performance liquid chromatography (HPLC) analysis. The antioxidative capacities of the isolated compounds (1–7) were tested against β-carotene destruction and LDL oxidation. The isoflavans (1–4) at a concentration of 50 μM inhibited β-carotene consumption, following 90 min of incubation at 50°C, similar to the inhibitory effect of the whole licorice crude extract (at 16 mg/l). The chalcones (5 and 6) exhibited moderate inhibition and the isoflavone 7 was almost inactive, whereas vitamin E (50 μM) completely inhibited β-carotene consumption. The inhibitory effect of the constituents 1–7, at a concentration of 30 μM on 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced LDL oxidation was determined by measuring the amount of the thiobarbituric acid reactive substances (TBARS) and the amount of lipid peroxides. While compounds 1–6 exhibited high inhibitory activity, compound 7 and vitamin E were not active. A dose-dependent inhibitory effect of Glabridin, on the formation of cholesteryl linoleate hydroperoxide (CLOOH), in an AAPH-induced LDL oxidation system was also shown. Glabridin, at 5 or 40–60 μM concentration, inhibited the CLOOH formation by 62% and 90%, respectively. These results suggest that constituents 1–6 are very potent antioxidants toward LDL oxidation with Glabridin being the most abundant and potent antioxidant. As LDL oxidation is a key event in the formation of the early atherosclerotic lesion, the use of these natural antioxidants may be proven beneficial to attenuate atherosclerosis.

Introduction

Oxidative damage to various tissues by free radicals have been implicated as the cause of diverse diseases.1, 2, 3 Several lines of evidence from both in vitro and in vivo studies suggest that oxidation of low-density lipoprotein (LDL), the major cholesterol-carrier in human plasma, critically contributes to human atherosclerosis.4, 5, 6, 7, 8, 9, 10

Oxidation of LDL involves lipid peroxidation, in which the polyunsaturated fatty acids in the LDL core and in the phospholipids, are rapidly converted into lipid hydroperoxides and into aldehydic lipid peroxidation products.[11] Oxidatively modified LDL is taken up at enhanced rate by macrophages, via the scavenger receptor, which, unlike the LDL receptor, is not regulated by the cellular cholesterol content, leading to the formation of lipid-laden macrophage foam cells.[12] Early atherosclerotic lesion is characterized by massive accumulation of foam cells.[13] Oxidized LDL is atherogenic, not only because of its contribution to foam cell formation but also as a result of its effects on the secretion of potent substances from arterial wall cells, and its cytotoxicity toward endothelial cells and smooth muscle cells.8, 14, 15, 16

Protection of LDL from oxidation should be an effective strategy to prevent or to slow the progression of atherosclerosis.11, 17, 18, 19 Compounds that can prevent lipid peroxidation (antioxidants) may increase the resistance of LDL to peroxidation. Contrary to the protective effect of antioxidants on lipid peroxidation, antioxidants might also accelerate oxidative damage to DNA, proteins, and carbohydrates.20, 21, 22, 23, 24, 25 Thus, utilization of antioxidants for therapeutical applications require examination of their effects upon different biological molecules.

Plants produce a variety of antioxidants against molecular damage from reactive oxygen species (ROS), and phenolics compose the major class of plant-derived antioxidants. Among the various phenolic compounds, the flavonoids are perhaps the most important group.[26] Flavonoids are components of a wide variety of edible plants, fruits, vegetables, and grains and are an integral part of the human diet.[27] They have the property to scavenge free radicals and to prevent lipid peroxidation.28, 29, 30 Catechin, a naturally occurring flavonoid,[31] as well as other flavonoids,32, 33 were shown to markedly inhibit LDL oxidation, in vitro.

Glycyrrhiza glabra is the licorice plant, and has a history of consumption for the past 6000 years.[34] The licorice roots have long been used as flavoring and sweating agents, as well as demulcents and expectorants in western countries. Furthermore, licorice root extracts have been used in Japan and China to treat allergic inflammation.35, 36

At present, glycyrrhizin and its aglycone, glycyrrhetinic acid, the main components in licorice root, are clinically used in treatments of hyperlipaemia, atherosclerosis, viral diseases, and allergic inflammation such as chronic hepatitis and atopic dermatitis,36, 37 while minor components of licorice, which are mostly flavonoids, have some biological action supplementing the efficacy of licorice.[38] Certain flavonoids isolated from licorice roots show effects on arachidonic acid metabolism,[39] as well as antiplatelet agents,[40] as antitumorigenic,[41] as antimicrobial,42, 43, 44 as antiviral,[45] as antiinflammatory[37] and also as antioxidants.38, 46 The root extracts of Glycyrrhiza glabra exhibited antioxidant activity and two antioxidant flavonoid compounds were isolated from it: Glabrene and Glabridin.[46]

In a previous research carried out in this group,[47] the antioxidative activity of licorice crude extract toward LDL oxidation was investigated in vitro and in vivo. It was shown that: (1) crude licorice extract completely inhibit LDL oxidation in a dose-dependent manner in vitro, and (2) daily consumption of 0.1 g (orally supplemented) of this extract by normolipidemic volunteers for 2 weeks, remarkably increased the resistance of their LDL to AAPH as well as to copper ions-induced lipid peroxidation.

In the present study, we examined the activity of seven compounds, isolated from the licorice root, in preventing β-carotene consumption and LDL oxidation.

Section snippets

Chemicals

The roots of Glycyrrhiza glabra used in this study were provided by Fertilizers & Chemicals Ltd. (Haifa, Israel). Beta-carotene, linoleic acid, vitamin E, tween 20, cholesteryl linoleate (CL), thiobarbituric acid, and trichloroacetic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Polysciences, Inc. (Warrington, PA, USA) supplied the 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH). Aluminum oxide 90 active, particle size 0.063–0.200 mm for column chromatography was from

Isolation and Identification of Antioxidant Compounds from Licorice Extract

Commercially available Glycyrrhiza glabra (licorice) roots were ground and extracted in acetone. The extract obtained was first separated into fractions using liquid-liquid extraction (Fig. 1) and then chromatographed repeatedly on silica gel, to isolate and purify potent antioxidants. Each fraction, in this separation and throughout the whole study was tested for its antioxidant activity using the β-carotene-linoleate model system. The structural elucidation of the isolated antioxidant

Discussion

The present study showed that licorice extract contained several potent antioxidant constituents as determined by their ability to inhibit β-carotene consumption and LDL oxidation. These constituents might be responsible for our previous findings that crude licorice extract can increase the resistance of LDL toward oxidation in in vitro and in vivo experiments.[47] The structure of these constituents (1–7) was determined as flavonoid derivatives and Glabridin (compound number 3) was found to be

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