THE EFFECT OF LICORICE CONSUMPTION BY MICE ON LDL OXIDATION, MACROPHAGE FOAM CELL FORMATION, AND ATHEROSCLEROSIS

VIII. THE EFFECT OF LICORICE CONSUMPTION BY MICE ON LDL OXIDATION, MACROPHAGE FOAM CELL FORMATION, AND ATHEROSCLEROSIS

Dietary supplementation of licorice (200 Ag/day/mouse)to apolipoprotein E– deficient (Ej)mice for 6 weeks resulted in an 80% reduction in the suscep-

F IGURE 4 The effect of licorice extract supplementation to hypercholesterol- emic patients on the susceptibility of their LDL to atherogenic modifications: oxidation (a), aggregation (b), or retention (c). LDL was isolated from hyper- cholesterolemic patients before, after 1 month of licorice extract supplemen- tation, and after an additional 1 month of placebo supplementation. (a) LDL

oxidation: LDL (100 mg of protein/L) was incubated with 5 Amol/L CuSO 4 for 3 hr at 25jC. The formation of conjugated dienes was kinetically monitored at 234 nm and the lag time was measured. (b) LDL aggregation: The extent of LDL aggregation induced by vortexing was kinetically monitored at 680 nm, and results are given after 60 sec of vortexing. (c) LDL CS binding ability: LDL (200 mg of lipoprotein protein/L) was incubated with chondroitin sulfate (CS, 100 mg/L) for 30 min at 37jC. LDL was then precipitated, and the LDL-asso- ciated glycoseaminoglycan (GAG) content was determined in the precipitate. Results are expressed as mean F SEM. *p < 0.01 (vs. baseline at study entry).

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tibility of their LDL to copper-ion-induced oxidation, in comparison to LDL isolated from placebo-treated mice (84).

Administration of purified glabridin to Ej mice in their drinking water was followed by analysis of its antioxidative effect against ex vivo LDL oxi- dation (84). GC-MS analysis of the LDL derived from Ej mice after con- sumption of glabridin revealed that glabridin was absorbed, and bound to the LDL particle. Whereas no glabridin could be detected in LDL from control mice, LDL from mice that consumed glabridin (20 Ag/day/mouse) contained about 2 nmol of glabridin/mg LDL protein. LDL derived from Ej mice after consumption of 20 Ag glabridin/day/mouse for 6 weeks was significantly more resistant to copper-ion-induced oxidation (by 22%)than LDL derived from placebo-treated mice. Administration of glabridin (25 Ag/

F IGURE 5 Effects of glabridin consumption by E 0 mice, on the size of their aortic arch atherosclerotic lesion area. Photomicrographs of a typical atherosclerotic lesion of the aortic arch following treatment with placebo (a) or glabridin (b). The sections were stained with alkaline toludine blue. All micrographs are at the same magnification. (c) The lesion area is expressed in square micrometers

F SD. *p < 0.01 vs. placebo.

Licorice Root Flavonoid Antioxidants 607

day/mouse)to Ej mice for 3 months also reduced (by 50%)an additional atherogenic modification of LDL, i.e., its susceptibility to aggregation induced by vortexing (86). Most important, inhibition of atherogenic mod- ifications of LDL (oxidation and aggregation)in Ej mice following glabridin consumption was associated with a substantial reduction in macrophage foam cell formation and in the development of the atherosclerotic lesion area (Fig. 5).

IX. SUMMARY The beneficial health effects attributed to the consumption of fruits and

vegetables are related, at least in part, to their antioxidant activity. Of special

F IGURE 6 Major pathways by which licorice flavonoids inhibit LDL cholesterol oxidation and atherosclerosis. Licorice-derived flavonoids affect LDL directly by interacting with the lipoprotein and inhibiting LDL oxidation. Licorice flavonoids can also protect LDL indirectly, by their accumulation in the arteries and pro- tection of arterial macrophages against oxidative stress. The latter effect is associated with inhibition of the formation of ‘‘oxidized macrophages’’ and reduction in the capacity of macrophages to oxidize LDL. In addition, licorice- derived glabridin preserves paraoxonase activity, thereby increasing the hy- drolysis of lipid peroxides in lipopoteins and in atherosclerotic lesion, leading to attenuation in the progression of atherosclerosis.

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interest is the inverse relationship between intake of dietary nutrients rich in polyphenols and cardiovascular diseases. This effect is attributed to the polyphenols’ capability to inhibit LDL oxidation, macrophage foam cell formation, and atherosclerosis. Our current view on the major pathways by which licorice flavonoids protect LDL against oxidative modifications, and thereby reduce macrophage foam cell formation and the development of advanced atherosclerosis, are summarized in Figure 6. Licorice-derived glabridin can protect LDL against cell-mediated oxidation via two major pathways, including a direct interaction with the lipoprotein and/or an indirect effect through accumulation in arterial macrophages. Licorice-de- rived glabridin was shown to reduce the capacity of macrophages to oxidize LDL, owing to its binding to LDL and inhibition of its oxidation [by scavenging reactive oxygen species (ROS)and reactive nitrogen species (RNS)], also owing to its accumulation in arterial macrophages, followed by inhibition of macrophage lipid peroxidation and the formation of lipid- peroxide-rich macrophages. Furthermore, licorice-derived glabridin was shown to preserve serum paraoxonase (PON1)activity, resulting in PON1- induced hydrolysis of lipid peroxides in oxidized lipoproteins and in athero- sclerotic lesion.

All these antioxidative and antiatherosclerotic effects of licorice-derived glabridin were demonstrated in vitro, as well as in vivo (in humans and in the atherosclerotic apolipoprotein E–deficient mice). We conclude that licorice is

a source of some potent nutrients, which can attenuate the development of atherosclerosis secondary to its antioxidatie properties against lipid perox- idation in cells and in lipoproteins (87–89).

REFERENCES 1. Schaffner T, Taylor K, Bartucci EJ, Fischer-Dzoga K, Beenson JH, Glagov S,

Wissler R. Arterial foam cells with distinctive immunomorphologic and histochemical features of macrophages. Am J Pathol 1980; 100:57–80. 2. Gerrity RG. The role of monocytes in atherogenesis. Am J Pathol 1981; 103: 181–190. 3. Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature 1990; 343:425–430. 4. Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol ho- meostasis. Science 1986; 232:34–47. 5. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cho- lesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 1989; 320:915–924. 6. Aviram M. Modified forms of low density lipoprotein and atherosclerosis. Atherosclerosis 1993; 98:1–9.

Licorice Root Flavonoid Antioxidants 609 7. Aviram M. Beyond cholesterol: modifications of lipoproteins and increased

atherogenicity. In: Neri Serneri GG, Gensini GF, Abbate R, Prisco D, eds. Atherosclerosis Inflammation and Thrombosis. Florence, Italy: Scientific Press, 1993:15–36. 8. Aviram M, Rosenblat, M. Oxidative stress in cardiovascular diseases: role of oxidized lipoproteins in macrophage foam cell formation and atherosclerosis. In: Fuchs J, Podda M, Packer L, eds. Redox Genome Interactions in Health and Disease. New York: Marcel Dekker, 2004:557–590. 9. Aviram M. Review of human studies on oxidative damage and antioxidant protection related to cardiovascular disease. Free Rad Res 2000; 33:S85–S97. 10. Aviram M. Macrophage foam cell formation during early atherogenesis is determined by the balance between pro-oxidants and antioxidants in arterial cells and blood lipoproteins. Antiox Redox Signal 1999; 1:585–594. 11. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999; 340:115– 126. 12. Williams KJ, Tabas I. The response-to-retention hypothesis of early athero- genesis. Arterioscler Thromb Vasc Biol 1995; 15:551–561. 13. Williams KJ, Tabas I. The response-to-retention hypothesis of atherogenesis reinforced. Curr Opin Lipidol 1998; 9:471–474. 14. Nordestgaard BG. The vascular endothelial barrier-selective retention of lipoproteins. Curr Opin Lipidol 1996; 7:269–273. 15. Schwenke DC, Carew TE. Initiation of atherosclerotic lesions in cholesterol-fed rabbits. I. Focal increases in arterial LDL concentrations precede development of fatty streak lesions. Arteriosclerosis 1989; 9:895–907. 16. Schwenke DC, Carew TE. Initiation of atherosclerotic lesions in cholesterol-fed rabbits II: selective retention of LDL vs. selective increase in LDL permeability in susceptible sites of arteries. Arteriosclerosis 1989; 9:908–918. 17. Carmena R, Ascaso JF, Camejo G, Varela G, Hurt-Camejo E, Ordovas JM, Bergstom M, Wallin B. Effect of olive and sunflower oils on low density lipo- protein level, composition, size, oxidation, and interaction with arterial proteo- glycans. Atherosclerosis 1996; 125:243–255. 18. Camejo G, Hurt-Camejo E, Olson U, Bonders G. Proteoglycans and lipopro- teins in atherosclerosis. Curr Opin Lipidol 1993; 4:385–391. 19. Ismail NA, Alavi MZ, Moore S. Lipoprotein-proteoglycan complexes from injured rabbit aortas accelerate lipoprotein uptake by arterial smooth muscle cells. Atherosclerosis 1994; 105:79–87. 20. Kostner GM, Bihari-Varga M. Is the atherogenicity of Lp(a)caused by its reactivity with proteoglycans? Eur Heart J 1990; 11(suppl E):184–189. 21. Vijayagopal P, Srinivasan SR, Radhakrishnamurthy B, Berenson GS. Lipo- protein-proteoglycan complexes from atherosclerotic lesions promote choles- teryl ester accumulation in human monocyte/macrophages. Arterioscler Thromb 1992; 12:237–249. 22. Hurt E, Bonders G, Camejo G. Interaction of LDL with human arterial pro- teoglycans stimulates its uptake by human monocyte-derived macrophages. J Lipid Res 1990; 31:443–454.

610 Aviram et al. 23. Aviram M, Maor I, Keidar S, Hayek T, Oiknine J, Bar-El Y, Adler Z, Kertzman

V, Milo S. Lesioned low density lipoprotein in atherosclerotic apolipoprotein E-deficient transgenic mice and in humans is oxidized and aggregated. Biochem Biophys Res Commun 1995; 216:501–513. 24. Pentikainen MO, Lethonen EM, Kovanen PT. Aggregation and fusion of modified low density lipoprotein. J Lipid Res 1996; 37:2638–2649. 25. Schissel SL, Tweedie-Hardman J, Rapp JH, Graham G, Williams KJ, Tabas I. Rabbit aorta and human atherosclerotic lesion hydrolyze the sphingomyelin of retained low-density lipoprotein: proposed role for arterial-wall sphingomyeli- nase in subendothelial retention and aggregation of atherogenic lipoproteins. J Clin Invest 1996; 98:1455–1464. 26. Zhang WY, Gaynor PM, Kruth HS. Aggregated low density lipoprotein induces and enters surface-connected compartments of human monocyte-macrophages: uptake occurs independently of the low density lipoprotein receptor. J Biol Chem 1997; 272:31700–31706. 27. Hoff HF, O’Neil Y. Lesion-derived low density lipoprotein and oxidized low density lipoprotein share a lability for aggregation, leading to enhanced macro- phage degradation. Arterioscler Thromb Vasc Biol 1991; 11:1209–1222. 28. Hoff HF, Whitaker TE, O’Neil Y. Oxidation of low density lipoprotein leads to particle aggregation and altered macrophage recognition. J Biol Chem 1992; 267:602–609. 29. Maor I, Hayek T, Coleman R, Aviram M. Plasma LDL oxidation leads to its aggregation in atherosclerotic apolipoprotein E–deficient mice. Arterioscler Thromb Vasc Biol 1997; 17:2995–3005. 30. Hurt-Camejo E, Camejo G, Rosengren B, Lopez F, Ahlstrom C, Fager G, Bondjers G. Effect of arterial proteoglycan and glycosaminoglycans on low density lipoprotein oxidation and its uptake by human macrophages and arterial smooth muscle cells. Arterioscler Thromb 1992; 12:569–583. 31. Jialal I, Devaraj S. The role of oxidized low density lipoprotein in atherogenesis. J Nutr 1996; 126:1053S–1057S. 32. Steinberg D. Low density lipoprotein oxidation and its pathobiological signifi- cance. J Biol Chem 1997; 272:20963–20966. 33. Berliner JA, Heinecke JW. The role of oxidized lipoproteins in atherosclerosis. Free Rad Biol Med 1996; 20:707–727. 34. Aviram M. Oxidative modification of low density lipoprotein and atheroscle- rosis. Isr J Med Sci 1995; 31:241–249. 35. Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in athero- genesis. J Clin Invest 1991; 88:1785–1792. 36. Aviram M. Interaction of oxidized low density lipoprotein with macrophages in atherosclerosis and the antiatherogenicity of antioxidants. Eur J Clin Chem Clin Biochem 1996; 34:599–608. 37. Kaplan M, Aviram M. Oxidized low density lipoprotein: atherogenic and pro- inflammatory characteristics during macrophage foam cell formation: an inhibi- tory role for nutritional antioxidants and serum paraoxonase. Clin Chem Lab Med 1999; 37:777–787.

Licorice Root Flavonoid Antioxidants 611 38. Parthasarathy S, Santanam N, Auge N. Oxidized low-density lipoprotein, a

two-faced janus in coronary artery disease? Biochem Pharmacol 1998; 56:279– 284. 39. Parthasarathy S, Rankin SM. The role of oxidized LDL in atherogenesis. Prog Lipid Res 1992; 31:127–143. 40. Aviram M. Antioxidants in restenosis and atherosclerosis. Curr Interven Cardiol Rep 1999; 1:66–78. 41. Herttuala SY. Is oxidzed low density lipoprotein present in vivo? Curr Opin Lipidol 1998; 9:337–344. 42. Aviram M. The contribution of the macrophage receptor for oxidized LDL to its cellular uptake. Biochem Biophys Res Commun 1991; 179:359–365. 43. Kim JA, Territo MC, Wayner E, Carlos TM, Parhami F, Smith CW, Haberland ME, Fogelman AM, Berliner JA. Partial characterization of leukocyte binding molecules on endothelial cells induced by minimally oxidized LDL. Arterioscler Thromb 1994; 14:427–433. 44. Khan NBV, Parthasarathy S, Alexander RW. Modified LDL and its constituents augment cytokine-activated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells. J Clin Invest 1995; 95:1262–1270. 45. Rangaswamy S, Penn MS, Saidel GM, Chisolm GM. Exogenous oxidized low density lipoprotein injures and alters the barrier function of endothelium in rats in vivo. Circ Res 1997; 80:37–44. 46. Penn MS, Chisolm GM. Oxidized lipoproteins, altered cell function and athero- sclerosis. Atherosclerosis 1994; 108:S21–S29. 47. Brown AJ, Leong SL, Dean RT, Jessup W. 7-Hydroxycholesterol and its prod- ucts in oxidized low density lipoprotein and human atherosclerotic plaque. J Lipid Res 1997; 38:1730–1745. 48. Lynch SM, Morrow JD, Roberts LJ II, Frei B. Formation of non-cyclooxy- genase-derived prostanoids (F2-isoprostanes)in plasma and low density lipo- protein exposed to oxidative stress in vitro. J Clin Invest 1994; 93:998–1004. 49. Slatter DA, Paul RG, Murray M, Bailey AJ. Reactions of lipid derived malon- dialdehyde with collagen. J Biol Chem 1999; 274:19661–19669. 50. Jessup W, Mander EL, Dean RT. The intracellular storage and turnover of apolipoprotein B of oxidized LDL in macrophages. Biochim Biophys Acta 1992; 1126:167–177. 51. Chisolm GM, Hazen ST, Fox PL, Catchard MK. The oxidation of lipoproteins by monocyte-macrophages. J Biol Chem 1999; 274:25959–25962. 52. Parthasarathy S, Printz DJ, Boyd D, Joy L, Steinberg D. Macrophage oxidation of low density lipoprotein generates a modified form recognized by the scavenger receptor. Arteriosclerosis 1986; 6:505–510. 53. Aviram M, Fuhrman B. LDL oxidation by arterial wall macrophages depends on the antioxidative status in the lipoprotein and in the cells: role of prooxidants vs. antioxidants. Mol Cell Biochem 1998; 188:149–159. 54. Aviram M, Rosenblat M. Macrophage mediated oxidation of extracellular low density lipoprotein requires an initial binding of the lipoprotein to its receptor. J Lipid Res 1994; 35:385–398.

612 Aviram et al. 55. Aviram M, Rosenblat M, Etzioni A, Levy R. Activation of NADPH oxidase

is required for macrophage-mediated oxidation of low density lipoprotein. Metabolism 1996; 45:1069–1079. 56. Herttuala YS, Rosenfeld ME, Parthasarathy S, Glass CK, Sigal E, Witztum JL, Steinberg D. Colocalization of 15-lipoxygenase mRNA and protein with epitopes of oxidized low density lipoprotein in macrophage-rich areas of atherosclerotic lesions. Proc Natl Acad Sci USA 1990; 87:6959–6963. 57. Aviram M, Kent UM, Hollenberg PF. Microsomal cytochrom P450 catalyze the oxidation of low density lipoprotein. Atherosclerosis 1999; 143:253–260. 58. Meister A, Anderson ME. Glutathione. Annu Rev Biochem 1983; 52:711–760. 59. Rosenblat M, Aviram M. Macrophage glutathione content and glutathione peroxidase activity are inversely related to cell-mediated oxidation of LDL. Free Rad Biol Med 1997; 24:305–313. 60. Fuhrman B, Oiknine J, Aviram M. Iron induces lipid peroxidation in cultured macrophages, increases their ability to oxidatively modify LDL and affect their secretory properties. Atherosclerosis 1994; 111:65–78. 61. Fuhrman B, Oiknine J, Keidar S, Kaplan M, Aviram M. 1997 Increased uptake of low density lipoprotein (LDL)by oxidized macrophages is the result of enhanced LDL receptor activity and of progressive LDL oxidation. Free Rad Biol Med 1994; 23:34–46. 62. Mackness MI, Mackness B, Durrington PN, Connelly PW, Hegele RA. Paraoxonases biochemistry, genetics and relationship to plasma lipoproteins. Curr Opin Lipidol 1996; 7:69–76. 63. La Du BN, Adkins S, Kuo CL, Lipsig D. Studies on human serum paraoxonase/ arylesterase. Chem Biol Interact 1993; 87:25–34. 64. Aviram M. Does paraoxonase play a role in susceptibility to cardiovascular disease? Mol Med 1999; 5:381–386. 65. Aviram M. Paraoxonase protects lipoproteins from oxidation and attenuates atherosclerosis. Ateroscleroza 2000; 4:3–7. 66. La Du BN, Aviram M, Billecke S, Navab M, Primo-Parmo S, Sorenson RC, Standiford TJ. On the physiological role(s)of the paraoxonases. Chem Biol Interact 1999; 119/120:379–388. 67. Mackness MI, Harty D, Bhatnagar D, Winocour PH, Arrol S, Ishola M, Durrington PN. Serum paraoxonase activity in familial hypercholesterolaemia and insulin-dependent diabetes mellitus. Atherosclerosis 1991; 86:193–197. 68. Abbott CA, Mackness MI, Kumar S, Boulton AJ, Durrington PN. Serum paraoxonase activity, concentration, and phenotype distribution in diabetes mellitus and its relationship to serum lipids and lipoproteins. Arterioscler Thromb Vasc Biol 1995; 15:1812–1818. 69. Garin MC, James RW, Dussoix P, Blanche H, Passa P, Froguel P, Ruiz J. Paraoxonase polymorphism Met-Leu54 is associated with modified serum con- centrations of the enzyme: a possible link between the paraoxonase gene and increased risk of cardiovascular disease in diabetes. J Clin Invest 1997; 99:62– 66. 70. Aviram M, Rosenblat M, Bisgaier CL, Newton RS, Primo-Parmo SL, La Du

Licorice Root Flavonoid Antioxidants 613 BN. Paraoxonase inhibits high density lipoprotein (HDL)oxidation and pre-

serves its functions: a possible peroxidative role for paraoxonase. J Clin Invest 1998; 101:1581–1590. 71. Aviram M, Billecke S, Sorenson R, Bisgaier C, Newton R, Rosenblat M, Erogul J, Hsu C, Dunlp C, La Du BN. Paraoxonase active site required for protection against LDL oxidation involves its free sulfhydryl group and is different from that required for its arylesterase/paraoxonase activities: selective action of hu- man paraoxonase allozymes Q and R. Arterioscler Thromb Vasc Biol 1998; 18:1617–1624. 72. Aviram M, Rosenblat M, Billecke S, Erogul J, Sorenson R, Bisgaier CL, Newton RS, La Du B. Human serum paraoxonase (PON 1)is inactivated by oxidized low density lipoprotein and preserved by antioxidants. Free Rad Biol Med. 1999; 26:892–904. 73. Fuhrman B, Aviram M. Anti-atherogenicity of nutritional antioxidants. IDrugs 2001; 4:82–92. 74. Hertog MG, Kromhout D, Aravanis C, Blackburn H, Buzina R, Fidanza F, Giampaoli S, Jansen A, Menotti A, Nedeljkovic S. Flavonoid intake and long- term risk of coronary heart disease and cancer in the seven countries study. Arch Intern Med 1995; 155:381–386. 75. Aviram M, Fuhrman, B. Effects of flavonoids on the oxidation of LDL and atherosclerosis. In: Rice-Evans C, Packer L, eds. Flavonoids in Health and Dis- ease. 2d ed. New York: Marcel Dekker, 2003:165–203. 76. Rice-Evans CA, Miller NJ, Bolwell PG, Bramley PM, Pridham JB. The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Rad Res 1995; 22:375–383. 77. Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relation- ships of flavonoids and phenolic acids. Free Rad Biol Med 1996; 20:933–956. 78. Van Acker SABE, Van-den Berg DJ, Tromp MNJL, Griffioen DH, van Bennekom WP, Van der Vijgh WJF, Bast A. Structural aspects of antioxidants activity of flavonoids. Free Rad Biol Med 1996; 20:331–342. 79. Haraguchi H, Ishikawa H, Mizutani K, Tamura Y, Kinoshita T. Antioxidative and superoxide scavenging activities of retrochalcones in Glycyrrhiza inflata. Bioorg Med Chem 1998; 6:339–347. 80. Vaya J, Belinky PA, Aviram M. Antioxidant constituents from licorice roots: isolation, structure elucidation and antioxidative capacity toward LDL oxidation. Free Rad Biol Med 1997; 23:302–313. 81. Belinky PA, Aviram M, Mahmood S, Vaya J. Structural aspects of the inhibitory effect of glabridin on LDL oxidation. Free Rad Biol Med 1998; 24:1419–1429. 82. Belinky PA, Aviram M, Fuhrman B, Rosenblat M, Vaya J. The antioxidative effects of the isoflavan glabridin on endogenous constituents of LDL during its oxidation. Atherosclerosis 1998; 137:49–61. 83. Rosenblat M, Belinky P, Vaya J, Levy R, Hayek T, Coleman R, Merchav S, Aviram M. Macrophage enrichment with the isoflavan glabridin inhibits NADPH oxidase-induced cell mediated oxidation of low density lipoprotein. J Biol Chem 1999; 274:13790–13799.

614 Aviram et al. 84. Fuhrman B, Buch S, Vaya J, Belinky PA, Coleman R, Hayek T, Aviram M.

Licorice extract and its major polyphenol glabridin protect low-density lipoprotein against lipid peroxidation: in vitro and ex vivo studies in humans and in atherosclerotic apolipoprotein E-deficient mice. Am J Clin Nutr 1997; 66:267–275. 85. Fuhrman B, Volkova N, Kaplan M, Presser D, Attias J, Hayek T, Aviram M. Antiatherosclerotic effects of licorice extract supplementation to hypercholester- olemic patients: increased resistance of their LDL to atherogenic modifications, reduced plasma lipids levels, and decreased systolic blood pressure. Nutrition 2002; 18:268–273. 86. Aviram M, Fuhrman B. Polyphenolic flavonoids inhibit macrophage-mediated oxidation of LDL and attenuate atherogenesis. Atherosclerosis 1998; 137 (suppl):45–50. 87. Fuhrman B, Aviram M. Flavonoids protect LDL from oxidation and attenuate atherosclerosis. Curr Opin Lipidol 2001; 12:41–48. 88. Fuhrman B, Aviram M. Polyphenols and flavonoids protect LDL against atherogenic modifications. 2d ed. Handbook of Antioxidants: Biochemical, Nutritional and Clinical Aspects. 2001: 303–336. 89. Vaya J, Aviram M. Nutritional antioxidants: mechanisms of action, analyses of activities and medical application. Curr Med Chem Imm Endocr Metab Agents 2001; 1:99–117.