Mulberry leaf powder prevents atherosclerosis in apolipoprotein E-deficient mice

Biochemical and Biophysical Research Communications 358 (2007) 751–756 www.elsevier.com/locate/ybbrc

Mulberry leaf powder prevents atherosclerosis in apolipoprotein E-deficient mice Akiko Harauma a,d,1, Toshinori Murayama a,*, Kazuyuki Ikeyama d, Hideto Sano a, Hidenori Arai b, Ryo Takano d, Toru Kita c, Saburo Hara d, Kaeko Kamei d, Masayuki Yokode a a

Department of Clinical Innovative Medicine, Translational Research Center, Kyoto University Hospital, 54 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan b Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, Japan c Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Japan d Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Japan Received 25 April 2007 Available online 7 May 2007

Abstract Mulberry is commonly used to feed silkworms. Here we examined whether a dietary intake of mulberry leaf (ML) could affect atherogenesis in vivo and in vitro. Apolipoprotein E-deficient mice were fed either normal chow (control group) or a diet containing 1% ML powder (ML group) from 6 weeks of age. The mice were sacrificed after 12 weeks. The susceptibility of plasma lipoprotein to oxidation was assessed using diene formation. A significant increase in the lag time of lipoprotein oxidation was detected in the ML group compared with the control group. Furthermore, the ML group showed a 40% reduction in atherosclerotic lesion size in the aortae compared with the control. We also examined the direct anti-oxidative activity of ML in vitro. Aqueous extract of ML had a strong scavenging effect on 1,1-diphenyl-2-picrylhydrazyl and inhibited lipoprotein oxidation. These results confirm that ML contains anti-oxidative substances that might help prevent atherosclerosis.  2007 Elsevier Inc. All rights reserved. Keywords: Anti-oxidant; Apolipoprotein E-deficient mice; Atherosclerosis; Low-density lipoprotein; Mulberry leaves

Coronary artery disease develops as a result of various risk factors, including increased plasma low-density lipoprotein (LDL)-cholesterol levels, as well as LDL modifications, such as oxidation and aggregation. Oxidized LDL (Ox-LDL) has been implicated in the development of atherosclerosis [1–3]. Several biological properties of OxLDL might promote the development of atherosclerotic lesions, including the stimulation of monocyte adhesion [4], enhanced cytotoxicity [5], the formation of foam cells [6,7], and altered expression of cytokines and growth fac*

Corresponding author. Fax: +81 75 751 4769. E-mail address: [email protected] (T. Murayama). 1 Present address: Healthcare Research Institute, Wakunaga Pharmaceutical Co., Ltd., Japan. 0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.04.170

tors [8,9]. Because Ox-LDL formation is accelerated by oxidative stress, a variety of anti-oxidants have been suggested to prevent atherogenesis. Indeed, the development of atherosclerotic lesions in hyperlipidemic rabbits and monkeys can be reduced by anti-oxidants such as probucol [10,11]. However, studies investigating the effects of dietary anti-oxidants, including vitamin E, s-carotene, vitamin C and polyphenol, on the susceptibility of LDL to oxidation and on atherosclerosis have been inconclusive [12]. In addition to these vitamins, several herbs have been reported to inhibit LDL oxidation, although it is uncertain whether the administration of a clinically relevant dose would be feasible due to their relatively poor yields [13,14]. Mulberry leaf (ML), which has a high yield and is commonly used as a silk worm diet and an alternative medicine in China and

752

A. Harauma et al. / Biochemical and Biophysical Research Communications 358 (2007) 751–756

Japan, has recently been reported to contain potent antioxidants [15]. Here we examined whether the dietary administration of ML could affect the oxidative modification of lipoproteins and retard atherogenesis in apolipoprotein E-deficient (apoE / ) mice. Materials and methods Mice. The apoE / 129ola · C57BL/6 hybrid mice were a generous gift from Dr. Edward M. Rubin of the University of California at Berkeley, USA [16]. The mice were mated with C57BL/6 mice to produce F1 hybrids. The F1 apoE+/ mice were then backcrossed to C57/Bl6 mice for five generations. Mice homogeneous for the apoE-null allele on a C57/Bl6 background were subsequently generated by brother–sister mating and the males were used in the subsequent experiments [17,18]. The mice were kept in a temperature-controlled facility with a 14-h light/10-h dark photoperiod, and free access to food and water. After being weaned at 4 weeks of age, they were fed a normal chow diet (CMF; 8.7% fat and 0.063% cholesterol; Oriental Yeast, Tokyo, Japan) until they reached 6 weeks of age, when the experiments were started. The animals were fasted overnight before the collection of blood from the tail vein into tubes containing ethylenediaminetetraacetic acid (EDTA; 10 mM/L) for the preparation of plasma. All mice were sacrificed at the end of the study by cervical dislocation and their hearts were used in the subsequent analyses. This project was approved by the Animal Care Committee of Kyoto University, Japan. Test diets. ML powder was prepared by drying and milling for 8 s in a hot-air mill (Drymeister; Hosokawa Micron, Osaka, Japan). At 6 weeks of age, the mice were randomly divided into two groups, the control group (n = 7) and the ML group (n = 7), and were started on the test diets. The control group mice were fed a ground CMF diet, whereas the ML group received a ground CMF diet supplemented with 1% (w/w) of ML powder. The animals remained on their respective test diets for a further 12 weeks. Plasma cholesterol determination. The animals were fasted overnight and plasma was obtained in order to measure the total cholesterol levels at 6, 10, 14, and 18 weeks of age. The plasma total cholesterol levels were determined using a colorimetric assay (Cholescolor Liquid Kit; Toyobo, Osaka, Japan). Isolation of mouse non-HDL lipoprotein. Serum was obtained through the low-speed centrifugation (3000 rpm; 4 C; 10 min) of fresh blood that was collected in the presence of EDTA (25 mM). Non-HDL lipoproteins (density <1.063 g/mL) were isolated from the mouse plasma using densitygradient ultracentrifugation in order to measure their susceptibility to oxidation. Briefly, 700 lL of plasma was combined with KBr to achieve a final density of 1.063 g/mL in each tube. The tubes were centrifuged at 100,000 rpm at 4 C for 24 h (TC-100 Ultracentrifuge, rotor type TLA 100.2; Beckman–Coulter, Fullerton, CA). The non-HDL fraction was obtained from the top 250 ll and was dialyzed against PBS (pH 7.4) for 48 h at 4 C to remove the KBr. Conjugated-diene formation of lipoproteins. The susceptibility of the lipoproteins to oxidation was assessed by determining the lag phase of conjugated-diene formation using a modified version of the method reported previously [19]. Aliquots of the lipoprotein sample from each animal (300 lL) were gently mixed with CuSO4 solution in order to achieve a final concentration of 5 lmol/L. The appearance of the conjugated dienes was measured by continuously monitoring the absorbance at 234 nm using a spectrophotometer (DU 530 Life Science UV/vis Spectrophotometer; Beckman–Coulter) [20–24]. Tissue preparation and histochemistry. At the time of sacrifice, the hearts and proximal aortae were removed from the mice, cleaned of pericardial fat under a dissecting microscope and fixed in 10% formalin. The hearts were then cut directly under and parallel to the aortic cusps, and the upper portions were imbedded in OCT Compound (Sakura Finetek USA, Torrance, CA) and frozen at 70 C. The samples were sequentially cut into a total of 45 cross-sections (thickness: 6 lm)

around the aortic sinus. Of these, every third section (that is, 15 samples per mouse) was subjected to staining with oil red-O (Sigma– Aldrich, St. Louis, MO) followed by counterstaining with Meyer’s hematoxylin solution (Wako Pure Chemical Industries, Osaka, Japan) [18,25]. Image analysis and quantification of atherosclerotic lesions. The size of the atherosclerotic lesion in each aortic section was evaluated on the basis of oil red-O-staining using Image-Pro Plus (Media Cybernetics, Silver Spring, MD). In order to estimate the severity of the lesions, we calculated the ‘fraction area’ of the oil red-O-stained area compared with the wholevessel area, including the lumen, intima, media and adventitia, as described previously [18,26]. For each animal, every third section (that is, 15 samples per mouse) was examined, and the mean fraction area was calculated and expressed as a percentage. Extraction of ML. Dried ML powder (250 g) was extracted twice with 75% methanol (1.5 L) at 45 C for 4 h. After the extract had been concentrated to 300 ml, the residue was extracted five times with hexane (500 ml) in order to remove the lipid and hydrophobic fractions. The residue, which was diluted with water (300 ml), was then extracted six times with 1-butanol (1.5 L) and the water layer was concentrated and stored at 4 C [27]. The water-soluble ML fraction (50 ml) was dialyzed for 72 h at 4 C against water (15 L). The diffusible fraction (molecular weight (MW): <3500) was evaporated and treated at 145 C for 60 min, and then dissolved in HCl (pH 2–3) or NaOH (pH 8–9) solution. Scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH). The scavenging effect of each substance on the DPPH radical was measured by monitoring the decrease in absorbance at 517 nm. Each ML fraction in 1 ml water was added to 0.5 ml of a 400-lM ethanol solution of DPPH. After aggressive mixing for 10 s, the solution was left to stand for 30 min and the absorbance at 517 nm was then measured [15]. Isolation of human LDL. LDL (density: 1.019–1.063 g/ml) was isolated with sequential ultracentrifugation from the plasma of healthy volunteer subjects [28]. Statistical analysis. All data are reported as the mean ± the standard error of the mean (SEM). Statistical differences were determined using the Mann–Whitney U test with SYSTAT software for the Macintosh (version 5.2; Systat Software, Point Richmond, CA). A probability (P) value <0.05 was considered to be statistically significant.

Results Effect of dietary ML on body weight and plasma cholesterol in apoE / mice We initially examined the effect of the ML diet on plasma cholesterol levels and body weight. For this purpose, a total of 14 apoE / mice were assigned to either the ML group (n = 7) or the control group (n = 7), following the experimental protocol described in Materials and methods (Fig. 1A). As shown in Fig. 1B, no significant differences in body weight or plasma cholesterol concentration were observed between the two groups during the experimental period. Susceptibility of mouse non-HDL lipoproteins to oxidative modification We next studied the oxidative modification of lipoproteins in mice fed the ML diet. The non-HDL was isolated from the two groups of mice at 18 weeks of age, as described in Materials and methods. The susceptibility of the lipoproteins to oxidation was determined on the basis of the duration of the lag phase and the rate of

A. Harauma et al. / Biochemical and Biophysical Research Communications 358 (2007) 751–756

conjugated-diene formation (Fig. 2). A significant elongation of the lag phase of oxidation was detected in the ML group compared with the control group (175 min

753

versus 65 min). This indicated that lipoprotein oxidation was inhibited in apoE / mice by the dietary administration of ML powder.

A Control

Normal Chow Diet (CMF)

A

n=7

Mulberry

B

CMF containing 1% (wt / wt) mulberry powder

n=7 Age 6

10

14

18

wks.

Control ML 500

B

C 18

body weight (g)

30 25

200

20 15 100

10

Aortic lesion area (%)

300

plasma cholesterol (mg/dl)

400 Control ML 35

P < .05

16 14 12 10 8 6 4

5

2 0

0 6

10

14

18

0 Mulberry

Control

wks of age /

Fig. 3. Representative photomicrographs of the aortic sinus from apoE / mice. The animals in the control (A) and ML (B) groups were sacrificed at 18 weeks of age, and the aortic roots were stained with oil red-O and hematoxylin. The atherosclerotic lesion was quantified as the fraction area of each group (C). The ML group showed 62% less atherosclerosis compared with the control group.

Fig. 1. (A) Experimental protocols. apoE mice were randomly allocated to the control or ML groups at 6 weeks of age. The animals were fasted overnight before blood was collected from the tail at 6, 10, and 14 weeks of age, and from the heart at 18 weeks of age. The mice were sacrificed at 18 weeks of age. (B) Body weight and plasma cholesterol levels in the control () and ML ( ) groups of apoE / mice. 0.04

Absorbance (234 nm)

0.03

Non-HDL (Control)

Non-HDL (Mulberry)

0.02 Lag Time Control 65 min Mulberry 175 min

0.01

0 0

55

105

155

205

255

305

355

405

455

515

565

-0.01

Time (min) Fig. 2. Oxidation of non-HDL from apoE / mice in the control ( ) and ML ( ) groups. Conjugated-diene formation during the incubation of lipoprotein and CuSO4 was measured at 234 nm.

754

A. Harauma et al. / Biochemical and Biophysical Research Communications 358 (2007) 751–756 7

A 5

unit (× 10 )

6 5 4 3 2 1 0

n

le

lub

o ls

no

tha

u 1-b

B

wa

ra ef

bl

olu

-s ter

on

ra ef

bl

sa

ly dia

on

cti

cti

tio

c fra

on

cti

ra ef

bl

sa

ly dia

un

0.14 0.12 Control

Absorbance (234 nm)

0.1 0.08 Lag Time 0.06

Control 135 min Mulberry 675 min

0.04 0.02 water-soluble fraction 0 30 55 80 10 5 13 0 15 5 18 0 20 5 23 0 25 5 28 0 30 5 33 0 35 5 38 0 40 5 43 0 45 5 48 0 51 5 54 0 56 5 59 0 61 5 64 0 66 5 69 0 71 5

0 -0.02 Time (min)

Fig. 4. (A) Scavenging effects of ML on the DPPH radical. Each fraction of ML was added to the ethanol fraction of DPPH. The reduction in the absorbance of the solution at 517 nm was measured. A unit corresponded to 0.1 absorbance. (B) Inhibitory effects of the water-soluble ML fraction on the oxidation of human LDL. Conjugated-diene formation was measured at 234 nm in the presence ( ) or absence (j) of water-soluble ML.

Analysis of atherosclerotic lesions We then determined the atherosclerotic lesion size in each group of mice. Our group, and Nakajima and colleagues, have previously reported that apoE / mice fed a normal chow diet develop fibroproliferative lesions by the age of 18 weeks [25,29]. Consistent with these previous studies, the control mice in the present experiment showed advanced atherosclerotic lesions in the aortic root, as determined by staining with oil red-O (Fig. 3A). By contrast, the mice in the ML group showed a marked reduction in aortic-lesion size (Fig. 3B). Quantitative analysis demonstrated that the atherosclerotic area in mice in the ML group was as little as 61% of that observed in the control group (P = 0.014; Fig. 3C).

greater scavenging activity for the DPPH radical than did the butanol-soluble fraction. Dialysis separation of the water-soluble fraction suggested that the MW of the main active substance was less than 3500. Furthermore, the antioxidant activity of the low-MW fraction was stable against heat treatment at 145 C for 60 min. This fraction was also stable under acidic conditions (pH 2–3 in HCl solution), whereas the activity was halved under alkaline conditions (pH 8–9 in NaOH solution) (Fig. 4A). Finally, we investigated the preventive effect of the aqueous fraction against the oxidative modification of lipoproteins. Incubating the aqueous ML extract and human LDL with copper revealed a significantly longer lag time of LDL oxidation (675 min) compared with that in the control (135 min). This demonstrated that the ML extract protected the polyunsaturated fatty acids against oxidation (Fig. 4B).

Activity of the water-soluble ML Discussion We next examined whether ML showed direct anti-oxidative activity in vitro. For this purpose, an aqueous extract of ML was prepared as described in Materials and methods. As shown in Fig. 4A, the aqueous fraction showed

In this study, apoE / mice were fed a diet containing 1% ML (w/w) from 6 to 18 weeks of age. Dietary supplementation with ML powder prevented atherogenesis, but

A. Harauma et al. / Biochemical and Biophysical Research Communications 358 (2007) 751–756

did not affect either body weight or plasma cholesterol concentration during the experimental period. In addition, lipoproteins from the mice in the ML diet group showed increased resistance to oxidative modification. It is widely accepted that oxidative stress is closely related to atherogenesis. Previous studies have shown that vitamin E protects LDL against oxidation [30–32]. However, although some reports have found that vitamin E retards the development of atherosclerotic lesions in hyperlipidemic animals [33,34], others have demonstrated no anti-atherogenic benefits of this vitamin [35,36]. The polyphenolic flavonoids constitute a large class of related compounds, which comprise two phenylbenzene (chromanol) rings linked through a pyran ring [27]. Different classes of flavonoids are present in various fruit, vegetables, and beverages, including tea and wine. Polyphenolic flavonoids might prevent coronary artery disease by inhibiting LDL oxidation [37–45], which is thought to play a key role in the pathogenesis of atherosclerosis. The anti-oxidant activities of flavonoids are related to their chemical structure [46]. In contrast to a previous report suggesting that ML showed anti-oxidative activity in its 1-butanol-soluble fraction [15,47], our DPPH study revealed that the aqueous fraction had far greater anti-oxidative potential than the lipid-soluble fraction. In fact, we found that the aqueous fraction dramatically inhibited the formation of conjugated diene in human LDL. We therefore conclude that dietary ML can inhibit atherosclerosis partly through its anti-oxidative activity in the aqueous fraction. Interestingly, our results suggest that the active substances are small molecules (MW: <3500) that are stable to heat and acid, but not to alkaline conditions. The stability at high temperatures clearly indicates that the main substances are separate from ascorbic acid. Although the precise details of the active substances in ML remain to be discovered, this old Chinese herbal medicine might offer a novel approach for the prevention of atherosclerosis. Acknowledgments This research was supported, in part, by Grants-in-Aid (20045982, 12671111, 14571093, and 13307034) and Center of Excellence Grants (12CE2006) from the Ministry of Education, Science, Sports, and Culture of Japan, by a research grant for health sciences from the Japanese Ministry of Health and Welfare, and by the Establishment of International COE for Integration of Transplantation Therapy and Regenerative Medicine (the COE program) of the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank Akiko Kato for her excellent technical assistance. References [1] P. Holvoet, A. Mertens, P. Verhamme, K. Bogaerts, G. Beyens, R. Verhaeghe, D. Collen, E. Muls, F. Van de Werf, Circulating oxidized

[2]

[3]

[4]

[5]

[6]

[7]

[8] [9]

[10]

[11]

[12] [13]

[14] [15]

[16]

[17]

[18]

755

LDL is a useful marker for identifying patients with coronary artery disease, Arterioscler. Thromb. Vasc. Biol. 21 (2001) 844–848. P. Holvoet, S.B. Kritchevsky, R.P. Tracy, A. Mertens, S.M. Rubin, J. Butler, B. Goodpaster, T.B. Harris, The metabolic syndrome, circulating oxidized LDL, and risk of myocardial infarction in wellfunctioning elderly people in the health, aging, and body composition cohort, Diabetes 53 (2004) 1068–1073. A. Mertens, P. Verhamme, J.K. Bielicki, M.C. Phillips, R. Quarck, W. Verreth, D. Stengel, E. Ninio, M. Navab, B. Mackness, M. Mackness, P. Holvoet, Increased low-density lipoprotein oxidation and impaired high-density lipoprotein antioxidant defense are associated with increased macrophage homing and atherosclerosis in dyslipidemic obese mice: LCAT gene transfer decreases atherosclerosis, Circulation 107 (2003) 1640–1646. S. Mine, T. Tabata, Y. Wada, T. Fujisaki, T. Iida, N. Noguchi, E. Niki, T. Kodama, Y. Tanaka, Oxidized low density lipoproteininduced LFA-1-dependent adhesion and transendothelial migration of monocytes via the protein kinase C pathway, Atherosclerosis 160 (2002) 281–288. S.M. Colles, J.M. Maxson, S.G. Carlson, G.M. Chisolm, Oxidized LDL-induced injury and apoptosis in atherosclerosis. Potential roles for oxysterols, Trends Cardiovasc. Med. 11 (2001) 131–138. M. Yokode, T. Kita, H. Arai, C. Kawai, S. Narumiya, M. Fujiwara, Cholesteryl ester accumulation in macrophages incubated with low density lipoprotein pretreated with cigarette smoke extract, Proc. Natl. Acad. Sci. USA 85 (1988) 2344–2348. G.M. Chisolm, D. Steinberg, The oxidative modification hypothesis of atherogenesis: an overview, Free Radic. Biol. Med. 28 (2000) 1815– 1826. G.M. Chisolm 3rd, Y. Chai, Regulation of cell growth by oxidized LDL, Free Radic. Biol. Med. 28 (2000) 1697–1707. G.Z. Fei, Y.H. Huang, J. Swedenborg, J. Frostegard, Oxidised LDL modulates immune-activation by an IL-12 dependent mechanism, Atherosclerosis 169 (2003) 77–85. T. Kita, Y. Nagano, M. Yokode, K. Ishii, N. Kume, A. Ooshima, H. Yoshida, C. Kawai, Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia, Proc. Natl. Acad. Sci. USA 84 (1987) 5928–5931. T.E. Carew, D.C. Schwenke, D. Steinberg, Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit, Proc. Natl. Acad. Sci. USA 84 (1987) 7725–7729. A. Munteanu, J.M. Zingg, A. Azzi, Anti-atherosclerotic effects of vitamin E—myth or reality? J. Cell Mol. Med. 8 (2004) 59–76. A. Gugliucci, T. Menini, Three different pathways for human LDL oxidation are inhibited in vitro by water extracts of the medicinal herb Achyrocline satureoides, Life Sci. 71 (2002) 693–705. D. Heber, Herbs and atherosclerosis, Curr. Atheroscler. Rep. 3 (2001) 93–96. K. Doi, T. Kojima, Y. Fujimoto, Mulberry leaf extract inhibits the oxidative modification of rabbit and human low density lipoprotein, Biol. Pharm. Bull. 23 (2000) 1066–1071. A.S. Plump, J.D. Smith, T. Hayek, K. Aalto-Setala, A. Walsh, J.G. Verstuyft, E.M. Rubin, J.L. Breslow, Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells, Cell 71 (1992) 343–353. T. Murayama, M. Yokode, H. Horiuchi, H. Yoshida, H. Sano, T. Kita, Overexpression of low density lipoprotein receptor eliminates apolipoprotein B100-containing lipoproteins from circulation and markedly prevents early atherogenesis in apolipoprotein E-deficient mice, Atherosclerosis 153 (2000) 295–302. H. Sano, T. Sudo, M. Yokode, T. Murayama, H. Kataoka, N. Takakura, S. Nishikawa, S.I. Nishikawa, T. Kita, Functional blockade of platelet-derived growth factor receptor-beta but not of receptor-alpha prevents vascular smooth muscle cell accumulation in

756

[19]

[20]

[21]

[22]

[23] [24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

A. Harauma et al. / Biochemical and Biophysical Research Communications 358 (2007) 751–756 fibrous cap lesions in apolipoprotein E-deficient mice, Circulation 103 (2001) 2955–2960. B.M. Winklhofer-Roob, H. Puhl, G. Khoschsorur, M.A. van’t Hof, H. Esterbauer, D.H. Shmerling, Enhanced resistance to oxidation of low density lipoproteins and decreased lipid peroxide formation during beta-carotene supplementation in cystic fibrosis, Free Radic. Biol. Med. 18 (1995) 849–859. J.H. van Ree, W.J. van den Broek, V.E. Dahlmans, P.H. Groot, M. Vidgeon-Hart, R.R. Frants, B. Wieringa, L.M. Havekes, M.H. Hofker, Diet-induced hypercholesterolemia and atherosclerosis in heterozygous apolipoprotein E-deficient mice, Atherosclerosis 111 (1994) 25–37. H. Esterbauer, G. Striegl, H. Puhl, M. Rotheneder, Continuous monitoring of in vitro oxidation of human low density lipoprotein, Free Radic. Res. Commun. 6 (1989) 67–75. B. Frei, J.M. Gaziano, Content of antioxidants, preformed lipid hydroperoxides, and cholesterol as predictors of the susceptibility of human LDL to metal ion-dependent and -independent oxidation, J. Lipid Res. 34 (1993) 2135–2145. S.P. Gieseg, H. Esterbauer, Low density lipoprotein is saturable by pro-oxidant copper, FEBS Lett 343 (1994) 188–194. E. Schnitzer, I. Pinchuk, A. Bor, M. Fainaru, A.M. Samuni, D. Lichtenberg, Lipid oxidation in unfractionated serum and plasma, Chem. Phys. Lipids 92 (1998) 151–170. T. Murayama, M. Yokode, H. Kataoka, T. Imabayashi, H. Yoshida, H. Sano, S. Nishikawa, T. Kita, Intraperitoneal administration of anti-c-fms monoclonal antibody prevents initial events of atherogenesis but does not reduce the size of advanced lesions in apolipoprotein E-deficient mice, Circulation 99 (1999) 1740–1746. A. Nicoletti, S. Kaveri, G. Caligiuri, J. Bariety, G.K. Hansson, Immunoglobulin treatment reduces atherosclerosis in apo E knockout mice, J. Clin. Invest. 102 (1998) 910–918. N. Asano, E. Tomioka, H. Kizu, K. Matsui, Sugars with nitrogen in the ring isolated from the leaves of Morus bombycis, Carbohydr. Res. 253 (1994) 235–245. J.L. Goldstein, T. Kita, M.S. Brown, Defective lipoprotein receptors and atherosclerosis. Lessons from an animal counterpart of familial hypercholesterolemia, N. Engl. J Med. 309 (1983) 288–296. Y. Nakashima, A.S. Plump, E.W. Raines, J.L. Breslow, R. Ross, ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree, Arterioscler. Thromb. 14 (1994) 133–140. H.M. Princen, W. van Duyvenvoorde, R. Buytenhek, A. van der Laarse, G. van Poppel, J.A. Gevers Leuven, V.W. van Hinsbergh, Supplementation with low doses of vitamin E protects LDL from lipid peroxidation in men and women, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 325–333. J. Kaikkonen, E. Porkkala-Sarataho, J.D. Morrow, L.J. Roberts 2nd, K. Nyyssonen, R. Salonen, T.P. Tuomainen, U. Ristonmaa, H.E. Poulsen, J.T. Salonen, Supplementation with vitamin E but not with vitamin C lowers lipid peroxidation in vivo in mildly hypercholesterolemic men, Free Radic. Res. 35 (2001) 967–978. C.J. Fuller, I. Jialal, Effects of antioxidants and fatty acids on lowdensity-lipoprotein oxidation, Am. J. Clin. Nutr. 60 (1994) 1010S– 1013S.

[33] R.A. Parker, T. Sabrah, M. Cap, B.T. Gill, Relation of vascular oxidative stress, alpha-tocopherol, and hypercholesterolemia to early atherosclerosis in hamsters, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 349–358. [34] J. Orbe, J.A. Rodriguez, A. Calvo, A. Grau, M.S. Belzunce, D. Martinez-Caro, J.A. Paramo, Vitamins C and E attenuate plasminogen activator inhibitor-1 (PAI-1) expression in a hypercholesterolemic porcine model of angioplasty, Cardiovasc. Res. 49 (2001) 484– 492. [35] J.W. Heinecke, Clinical trials of vitamin E in coronary artery disease: is it time to reconsider the low-density lipoprotein oxidation hypothesis? Curr. Atheroscler. Rep. 5 (2003) 83–87. [36] J.M. Upston, P.K. Witting, A.J. Brown, R. Stocker, J.F. Keaney Jr., Effect of vitamin E on aortic lipid oxidation and intimal proliferation after arterial injury in cholesterol-fed rabbits, Free Radic. Biol. Med. 31 (2001) 1245–1253. [37] M. Aviram, B. Fuhrman, Polyphenolic flavonoids inhibit macrophage-mediated oxidation of LDL and attenuate atherogenesis, Atherosclerosis 137 (Suppl) (1998) S45–S50. [38] B. Fuhrman, M. Aviram, Flavonoids protect LDL from oxidation and attenuate atherosclerosis, Curr. Opin. Lipidol. 12 (2001) 41–48. [39] P.A. Belinky, M. Aviram, B. Fuhrman, M. Rosenblat, J. Vaya, The antioxidative effects of the isoflavan glabridin on endogenous constituents of LDL during its oxidation, Atherosclerosis 137 (1998) 49–61. [40] H.S. Demrow, P.R. Slane, J.D. Folts, Administration of wine and grape juice inhibits in vivo platelet activity and thrombosis in stenosed canine coronary arteries, Circulation 91 (1995) 1182–1188. [41] J.V. Formica, W. Regelson, Review of the biology of Quercetin and related bioflavonoids, Food Chem. Toxicol. 33 (1995) 1061– 1080. [42] H. Goker, M. Tuncbilek, G. Leoncini, E. Buzzi, M. Mazzei, Y. Rolland, R. Ertan, Synthesis and inhibitory activities on platelet aggregation of some flavonoid analogues, Arzneimittelforschung 45 (1995) 150–155. [43] L. Lanningham-Foster, C. Chen, D.S. Chance, G. Loo, Grape extract inhibits lipid peroxidation of human low density lipoprotein, Biol. Pharm. Bull. 18 (1995) 1347–1351. [44] S. Miura, J. Watanabe, M. Sano, T. Tomita, T. Osawa, Y. Hara, I. Tomita, Effects of various natural antioxidants on the Cu(2+)mediated oxidative modification of low density lipoprotein, Biol. Pharm. Bull. 18 (1995) 1–4. [45] S.T. Sinatra, J. DeMarco, Free radicals, oxidative stress, oxidized low density lipoprotein (LDL), and the heart: antioxidants and other strategies to limit cardiovascular damage, Conn. Med. 59 (1995) 579– 588. [46] C.A. Rice-Evans, N.J. Miller, P.G. Bolwell, P.M. Bramley, J.B. Pridham, The relative antioxidant activities of plant-derived polyphenolic flavonoids, Free Radic. Res. 22 (1995) 375–383. [47] B. Enkhmaa, K. Shiwaku, T. Katsube, K. Kitajima, E. Anuurad, M. Yamasaki, Y. Yamane, Mulberry (Morus alba L.) leaves and their major flavonol quercetin 3-(6-malonylglucoside) attenuate atherosclerotic lesion development in LDL receptor-deficient mice, J. Nutr. 135 (2005) 729–734.