Effect of garlic powders and garlic active compounds

Effect of garlic powders and garlic active compounds on lipid metabolism and atherosclerosis in APOE*3 -Leiden mice Sonia M. S. Espirito Santo, Bart J. M. van Vlijmen, Rien Buytenhek, Wim van Duyvenvoorde, Erik H. Offerman, Louis M. Havekes and Hans M. G. Princen Gaubius Laboratory, TNO-PG, Leiden, the Netherlands EU project

Aim of the study: Effects of garlic on lipid metabolism and atherosclerosis • Effects on lipid levels and lipid metabolism under more human-like conditions. – Lipid levels, cholesterol synthesis and cholesterol absorption. • Effects on fast atherosclerosis development and other processes that might be related with atherosclerosis. – Inflammation, blood pressure, platelet activation and atherosclerosis analysis.

Lipid metabolism LDL LIVER LDL receptor SYNTHESIS UPTAKE HMG-Co. A reductase other receptors CHOLESTEROL ACAT 7 -hydroxylase 27 -hydroxylase VLDL STORAGE CHOLESTERYL ESTERS CONVERSION SECRETION BILIARY CHOLESTEROL BILE ACIDS BILE CHYLOMICRON REMNANTS INTESTINE DIETARY CHOLESTEROL CHYLOMICRONS

Atherosclerosis Shear stress Intima Inflammation Media Oxidation Fibrous cap Monocytes LDL Thrombus Endothelium ox. LDL O 2 Lipid core Collagen- degrading proteinases

Contradictory effects attributed to garlic • Soluble garlic extracts inhibit cholesterol biosynthesis in primary rat hepatocytes and human Hep. G 2 cells. R. Gebhardt, Lipids, 1993, 28(7): 613 -9. • Lichtwer garlic powder reduces plasma lipid levels and blood pressure in hypercholesterolemic rats. M Ali et al. , Prostaglandins Leukot Essent Fatty Acids, 2000, 62(4): 253 -9. • Allicin showed no effects on plasma lipid levels and decreased aorta atherosclerotic lesions in C 57 Black/6 mice fed a high fat/ cholesterol diet. D. Abramovitz et al. , Coronary Artery Disease, 1999, 10: 515 -519. • Garlic powder showed no effects on plasma lipid levels in patients with moderated hypercholesterolemia. B. W. Mc. Crindle et al, Arch Pediatr Adolesc Med, 1998, 152(11): 1089 -94 and C. D. Gardner et al, Atherosclerosis, 2001, 154(1): 213 -2. • Garlic oil showed no effects on plasma lipid levels, cholesterol synthesis and cholesterol absorption in patients with moderated hypercholesterolemia. H. K. Berthold et al, JAMA, 1998, 17(23): 1900 -2.

Aim of the study: Effects of garlic on lipid levels and lipid metabolism (1) Study the effect of 2 garlic powders and 2 garlic active compounds on plasma lipid levels, cholesterol synthesis and intestinal absorption under more human-like conditions using hyperlipidemic and atherosclerosis-susceptible APOE*3 -Leiden transgenic mice.

APOE*3 -Leiden transgenic mouse as a model for hyperlipidemia and atherosclerosis studies • Transgenic mice carrying a mutant form of human APOE*3 gene. • Dominant dysfunctional APOE*3 -Leiden results in defect in chylomicron/ VLDL clearance and more human-like lipoprotein profile. • Highly responsive to feeding cholesterol-rich diets. • Develops atherosclerosis varying from fatty streaks to more human-like complicated lesions.

Atherosclerosis in APOE*3 -Leiden mice Monocytes Adherence of monocytes to the activated vessel wall

Atherosclerosis in APOE*3 -Leiden mice Media not affected foam cells intima media { Aorta with regular fatty streak

Atherosclerosis in APOE*3 -Leiden mice cholesterol fibrosis clefts intima necrosis { media Affected media Severe plaque in aorta

Effect of garlic on lipid metabolism and atherosclerosis in APOE*3 -Leiden mice: Selection of compounds • Lichtwer LI 111 garlic powder: Reference dry garlic powder. • Morado de Cuenca 200 (Mor 200): Garlic powder rich in organic sulfur-containing compounds. • Diallyl disulfide (DADS): Isolated garlic compound rich in sulfur. • Allicin: Reference isolated garlic compound reported to result in 43% reduction of fatty streaks in C 57 Black/6 mice (D. Abramovitz et al. , Coronary Artery Disease, 1999, 10: 515 -519)

Effect of garlic on lipid metabolism and atherosclerosis in APOE*3 -Leiden mice: Experimental scheme -2 0 Run in Matching and start of the study Weight and food intake Blood sampling/ plasma isolation Plasma lipid levels Cholesterol lipoprotein profile Chol. synthesis/ Intestinal absorption Marker for liver function Liver lipid composition Blood pressure Platelet activation measurements Inflammation markers 80 80 p p p Regular diet 6 Western diet 2 4 8 80 80 80 p p p 15 65 65 65 10 12 65 65 65 p p p 80 65 14 Atherogenic diet 16 65 65 65 18 20 22 65 65 65 p p p Atherosclerotic aorta lesions typing Atherosclerotic aorta lesions area Compound biostability p General parameters Lipid metabolism related parameters p p Inflammation and atherosclerosis related parameters 60 60 p 60 65 5 5 p 25 60 60 60 p

Effect of garlic on food intake under 3 dietary conditions Food intake Regular diet Western diet Atherogenic diet g/ mouse/ day Control 3. 6 3. 2 3. 3 LI 111 3. 3 3. 0 2. 6 * Mor 200 3. 4 3. 0 2. 8 * DADS 3. 0 2. 6 2. 4 * Allicin 3. 6 3. 3 3. 0 *P< 0. 05

Effect of garlic on body weight under 3 dietary conditions Body weight Regular diet Western diet Atherogenic diet g Control 20. 9 23. 3 23. 2 LI 111 21. 0 22. 6 Mor 200 21. 5 23. 2 22. 1 DADS 20. 3 21. 0 * 20. 6 * Allicin 20. 5 22. 6 23. 4 *P< 0. 05

Effect of garlic on liver function under 3 dietary conditions Plasma alanine amino transferase Regular diet Western diet Atherogenic diet U/L Control 33. 4 30. 3 175 LI 111 33. 2 34. 4 104 t Mor 200 32. 9 30. 3 94. 1 t DADS 29. 9 41. 8 203 Allicin 25. 2 28. 2 171 t. P=0. 051

Effect of garlic on plasma cholesterol levels under 3 dietary conditions Plasma cholesterol levels Regular diet Western diet Atherogenic diet m. M Control 3. 1 9. 3 63 LI 111 2. 9 9. 3 57 Mor 200 3. 0 9. 1 44 * DADS 2. 9 8. 3 70 Allicin 3. 5 * 9. 2 73 *P< 0. 05

Effect of garlic on cholesterol lipoprotein distribution under 3 dietary conditions Regular diet Western diet 0. 4 4 0. 3 3 VLDL Atherogenic diet Control LI 111 Morado 200 12 LDL HDL Cholesterol (m. M) 8 0. 2 2 0. 1 1 0. 0 4 0 1 5 9 13 17 21 1 0. 4 4 0. 3 3 0. 2 2 0. 1 1 0. 0 0 1 5 9 13 17 21 0 5 9 13 17 21 1 5 9 12 13 17 21 Control DADS Allicin 8 4 0 1 5 9 13 17 Fraction number 21 1 5 9 13 17 21 *P< 0. 05

Effect of garlic on markers of cholesterol synthesis and intestinal absorption under 3 dietary conditions Concentration (µmol sterol / mmol cholesterol) Regular diet squalene lathosterol lanosterol 1. 5 1. 0 Markers of cholesterol synthesis 0. 5 0. 0 Control LI 111 Mor 200 DADS Allicin 40 campesterol b-sitosterol 30 20 Markers of intestinal absorption 10 0 Control LI 111 Mor 200 DADS Allicin *P< 0. 05

Effect of garlic on liver lipid levels under fast atherogenic conditions Liver lipid composition Free cholesterol Triglycerides Cholesteryl esters mg lipid/ g protein Control 19. 4 43. 7 154 LI 111 15. 9 * 31. 5 * 130 * Mor 200 15. 3 * 35. 0 * 127 * DADS 17. 8 49. 7 171 * Allicin 17. 3 37. 8 146 *P< 0. 05

Conclusions of the study of the effects of garlic on lipid levels and lipid metabolism (1) • Under more human-like hyperlipidemic conditions (8 -12 m. M): – No beneficial effects of garlic powders and garlic isolated compounds were observed on lipid levels, cholesterol synthesis and intestinal absorption in APOE*3 -Leiden mice. • Under fast atherosclerosis conditions (45 -70 m. M): – Morado 200 showed a plasma cholesterol-lowering effect in APOE*3 -Leiden mice. – LI 111 and Morado 200 had a cholesterol and triglyceride liver storage lowering effect in APOE*3 -Leiden mice.

Aim of the study: Effects of garlic on fast atherosclerosis development and other atherosclerosis related processes (2) Study the effect of 2 garlic powders and 2 garlic active compounds on inflammation processes, blood pressure, platelet activation and fast atherosclerosis development using hyperlipidemic and atherosclerosis-susceptible APOE*3 -Leiden transgenic mice.

Effect of garlic on Serum Amyloid A and von Willebrand factor concentrations under fast atherogenic conditions 8 250 p=0. 001 p<0. 001 200 * * 4 2 v. WF (%PP) SAA (µg/ ml) 6 150 100 50 0 0 Control LI 111 Mor 200 DADS Allicin *P< 0. 05

Effect of garlic on blood pressure and platelet activation under fast atherogenic conditions • No effects of garlic powders and garlic isolated compounds were observed on the percentage of activated platelets in blood under all dietary treatments. • No influence of garlic powders and garlic isolated compounds were found on blood pressure under regular and Western dietary conditions by cuff-tail method.

Plaque area of the aortic root (µm 2 x 1000) Effect of garlic on plaque area of the aortic root under fast atherogenic conditions 400 p = 0. 017 p = 0. 011 300 200 100 0 0 Control LI 111 Mor 200 Control DADS Allicin *P< 0. 05

Conclusions of the study of the effects of garlic on fast atherosclerosis development and other atherosclerosis related processes (2) Under fast atherogenic conditions: • LI 111 and Morado 200 showed an improved liver function. • Morado 200 had a lowering effect on plasma cholesterol levels. • Morado 200 and Allicin showed a lowering effect on serum amyloid A concentration, suggesting a decreased liver specific inflammation. • LI 111 and DADS treated groups showed a larger plaque area.

Future mild atherosclerosis development study under more human-like conditions Study the effect of 2 garlic powders of the same strain with a high and a low content of sulfur-containing compounds and allicin as a positive control on inflammation related parameters and on mild human-like atherosclerosis development conditions by means of a hyperlipidemic and atherosclerosis -susceptible APOE*3 -Leiden transgenic mice.

Future mild atherosclerosis development study under more human-like conditions: Experimental scheme In weeks -2 0 Run in Matching and start of the study Weight and food intake Blood sampling/ plasma isolation Plasma lipid levels Cholesterol lipoprotein profile Marker for liver function Liver lipid composition SAA and v. Wf Fibrinogen, t-PA and soluble ICAM Atherosclerotic aorta lesions typing 60 60 p 60 Atherosclerotic aorta lesions area Moncytes adherence Compound biostability p General parameters Lipid metabolism related parameters 24 Western diet 6 14 60 60 60 p 60 60 60 22 60 60 60 p 60 5 55 55 55 p Inflammation and atherosclerosis development related parameters