1. Effect of garlic on inflammation processes and atherosclerosis under human-like conditions in APOE*3-Leiden mice Sonia M.S. Espirito Santo TNO Prevention and Health Gaubius Laboratory The Netherlands Leiden University Medical Center Department of Cardiology The Netherlands

2. Development of high quality garlic under standardized conditions. Develop of a chemically well-characterized garlic material. Define organo-sulfur compounds metabolic pathway in the clove and in vivo after ingestion. Study in vitro and in vivo the influence of this standardized and well-characterized garlic material on atherosclerosis and cancer for disease prevention. European Comunity Garlic and Health project

3. Garlic has an historical record for its use in medicine. Some studies have suggested that garlic significantly reduces plasma lipid levels, systolic and diastolic blood pressure and that shows a strong antibiotic activity. Kik C, Kahane R, Gebhardt R. Nutr Metab Cardiovasc Dis. 2001;11:57-65. Garlic clinical trials and animal studies with well- characterized and standardized garlic material, with a consistent study design and dosage are poorly found in the last 15 years. Garlic and health

4. Possible effects of garlic on lipid metabolism CHOLESTEROL SYNTHESIS CONVERSION CHOLESTEROL UPTAKE receptors STORAGE ESTERS DIETARY BILE ACIDS LDL receptor CHOLESTERYL BILE other BILIARY CHOLESTEROL SECRETION LIVER INTESTINE CM Remnants VLDL LDL

5. Possible effects of garlic on atherosclerosis Lipid core Media Intima Endothelium LDL O2O2 oxLDL Thrombus Collagen- degrading proteinases Shear stress Fibrous cap Monocytes Inflammation Oxidation

6. Contradictory effects attributed to garlic Old human trials on healthy patients treated with garlic oil reported a decrease in plasma lipid levels. Bordia A, et al. Atherosclerosis.1975;21:15-19. Bordia A, et al. Recent human trials on hypercholesterolemic patients showed no effects of garlic powder (Kwai) and garlic oil treatment on plasma lipid levels. Simmons LA, et al. Atherosclerosis.1994;113:219-225. Berthold, et al. JAMA. 1998;279:1900-02. McCrindle BW, et al. Arch Pediatr Adolesc Med. 1998;152:1089-94. Isaacsohn JL, et al. Arch Intern Med.1998;158:1189-94. Gardner CD, et al. Atherosclerosis. 2001;154:213-220. Ziaei S, et al. Eur J Obstetrics & Gynecology. 2001;99:201-6. Allicin showed no effects on plasma lipid levels and decreased aortic fatty streaks-like lesions in hypercholesterolemic C57Black/6 mice. D. Abramovitz et al., Coronary Artery Disease, 1999, 10: 515-519. Induced-atherosclerosis in rabbits fed garlic oil and aged garlic extract (Kyolic) showed a decrease of aortic fatty streaks lesions. Bordia A, et al. 1980:428-37. Mand JK, et al. Indian Heart J. 1985;37:183-88. Efendy JL, et al. Atherosclerosis. 1997:132:37-42. Campbell JH, et al. J Nutr. 2001;131:1006S-09S. Durak I, et al. Nutr Metab Cardiovasc Dis. 2002 ;12:141-7. Hypercholesterolemic pigs and rats fed garlic oil and garlic powder (Kwai) showed no effects on atherosclerosis development. Heinle H,et al. Drug Res. 1994;44:614-17. Lee WM, et al. Exper Mol Pathol. 1980:33:345-8.

7. Lipid metabolism - No effects of garlic powders and isolated compounds on plasma lipid levels and markers of cholesterol synthesis and absorption under hyperlipidemic conditions. - Morado 200 garlic powder showed an improved liver function, decreased plasma cholesterol levels and serum amyloid A levels under atherogenic conditions. Fast atherosclerosis development –No decreasing effect of garlic on atherosclerosis of the aortic root was observed for all treatments. Espirito Santo SMS, et al. Effect of garlic powders and garlic isolated compounds on lipid metabolism. 2002. Previous reported effects of garlic (LI111, Morado 200, Allicin and DADS) in APOE*3- Leiden mice

8. Study the effect of 2 different doses of Printanor garlic powder under mild human-like conditions on inflammation related parameters on atherosclerosis development by means of a hyperlipidemic and atherosclerosis- susceptible APOE*3-Leiden transgenic mice. Effect of garlic on inflammation processes and atherosclerosis under human-like conditions: Aim

9. Printanor FR 2001 (0.5% Prin): 0.5% of dry garlic powder with low sulphur content. Printanor FR 2001 (5% Prin): 5% of dry garlic powder with high sulphur content. Kyolic (positive control): 0.16% of dry garlic powder rich in S-allylcysteine reported to result in 64% reduction of fatty streaks in New Zealand white rabbits. Campbell JH, Efendy JL, et al. J.Nutrition. 2001;131:1006S-1009S. Effect of garlic on inflammation and atherosclerosis under human-like conditions: Garlic powders

10. 16 female APOE*3-Leiden mice per group 0.2% cholesterol diet for 28 weeks General parameters: Body weight, food intake, marker of liver function, plasma lipid levels, blood pressure, and fecal fatty acids. Inflammation related parameters: All body and vessel wall inflammation markers (Serum amyloid A, soluble ICAM, and von willebrand factor) and whole blood stimulation assay. Atherosclerosis related parameters: Aortic root lesions typing, and total lesion area. Effect of garlic on inflammation and atherosclerosis under human-like conditions: Experimental design

11. * Control 0.5%Prin Control Kyolic Control 5% Prin Effect of garlic on body weight gain of APOE*3-Leiden mice Weeks Body weight gain (g) P<0.01 0 2 4 6 8 10 04812162024 04812162024 0481216202428 * * * *

12. Effect of garlic on food intake and body temperature of APOE*3-Leiden mice Food intake (g/mouse/day) 2.79 ± 0.08 2.87 ± 0.12 2.75 ± 0.16 2.88 ± 0.10 Control 0.5% Prin 5% Prin Kyolic Freefattyacids (mM) 0.73 ± 0.13 0.79 ± 0.11 0.60 ± 0.10 0.62 ± 0.18 * *

13. Effect of garlic on fecal fatty acids excretion of APOE*3-Leiden mice Total fat excretion (µmol/100g mouse/day) 0 200 400 600 800 Control0.5% Prin 5% Prin Kyolic * P<0.05 0 1 2 3 4 5 Total dry feces (g/ day) Control0.5% Prin 5% Prin Kyolic *

14. Effect of garlic on liver function of APOE*3-Leiden mice Plasma ALAT levels (U/L) Weeks Control 0.5%Prin Control Kyolic Control 5% Prin 0 50 100 150 200 04812162024 04812162024 0481216202428

15. Effect of garlic on plasma cholesterol levels of APOE*3-Leiden mice Weeks Cholesterol levels (mM) Control 0.5%Prin Control Kyolic Control 5% Prin 0 5 10 15 20 04812162024 04812162024 0481216202428

16. Effect of garlic on plasma triglyceride levels of APOE*3-Leiden mice Weeks Triglycerides levels (mM) Control 0.5%Prin Control Kyolic Control 5% Prin 0 1 2 3 4 04812162024 04812162024 0481216202428

17. Effect of garlic on lipoprotein profile of APOE*3-Leiden mice Cholesterol Triglycerides Fractions

18. Effect of garlic on blood pressure of APOE*3-Leiden mice Blood pressure (mmHg) 0 40 80 120 160 Control0.5% Prin 5% Prin Kyolic 12 weeks16 weeks Control0.5% Prin 5% Prin Kyolic

19. Effect of garlic on serum SAA and ICAM levels of APOE*3-Leiden mice 0 5 10 15 20 04812162024 04812162024 0481216202428 Control 0.5%Prin Control 5% Prin Control Kyolic SAA (µg/ml) ICAM (µg/ml) 0 10 20 30 40 0481216202404812162024 0481216202428

20. Effect of garlic on plasma vWF levels of APOE*3-Leiden mice 0 50 100 150 200 250 04812162024 04812162024 0481216202428 vWF (% PP rat) Control 0.5%Prin Control 5% Prin Control Kyolic

21. Effect of garlic on atherosclerosis development of APOE*3-Leiden mice Lesion typingLesion area Cholesterol exposure 0 100 200 300 400 500 0 20 40 60 80 100 Control0.5% Prin 5% Prin KyolicControl0.5% Prin 5% Prin KyolicControl0.5% Prin 5% Prin Kyolic µm 2 1000 % mM Type I-III Type IV-V 0 50 100 150 200 250

22. Cholesterol exposure (mM) R= 0.8136 Area (µm 2 1000) Correlation between cholesterol exposure and atherosclerosis lesion area after garlic treatment of APOE*3-Leiden mice Y= 0.8308x + 289.82 0 200 400 600 050100150200

23. Dietary garlic treatment on APOE*3-Leiden mice results in: Decreased body weight gain (P=0.007) for the 5% Printanor treated group: – lower (P=0.032) plasma free fatty acids – higher (P=0.043) fecal fatty acids excretion No effects on liver function, plasma lipids and glucose levels, and on blood pressure. No effects on markers of all body and vessel wall inflammation and on atherosclerosis development. Conclusions

24. Human intervention study Collaboration with CHDR-Leiden Future perspectives

25. Ackowledgements Sonia Espirito Santo Erik Offerman Ria van den Hoogen Louis Havekes Bart van Vlijmen Hans Princen Wim van Duyvenvoorde