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
European Comunity Garlic and Health project
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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.
Garlic and health
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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.
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Garlic clinical trials and animal studies with wellcharacterized and standardized garlic material, with a
consistent study design and dosage are poorly found
in the last 15 years.
Possible effects of garlic on lipid
metabolism
LDL
LIVER
LDL receptor
UPTAKE
SYNTHESIS
other
receptors
CHOLESTEROL
STORAGE
CHOLESTERYL
ESTERS
VLDL
CONVERSION
SECRETION
BILIARY
CHOLESTEROL
BILE
ACIDS
BILE
CM
Remnants
INTESTINE
DIETARY
CHOLESTEROL
CM
Possible effects of garlic on
atherosclerosis
Shear stress
Intima
Inflammation
Oxidation
Monocytes
Media
Fibrous
cap
LDL
Thrombus
Endothelium
oxLDL
O2
Lipid core
Collagen- degrading
proteinases
Contradictory effects attributed to garlic
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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.
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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.
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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.
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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.
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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.
Previous reported effects of garlic (LI111,
Morado 200, Allicin and DADS) in APOE*3Leiden mice
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Lipid metabolism
- No effects of garlic powders and isolated compounds
on plasma lipid levels and markers of cholesterol
synthesis and absorption under hyperlipidemic
conditions.
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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.
Effect of garlic on inflammation
processes and atherosclerosis under
human-like conditions: Aim
Study the effect of 2 different doses of Printanor
garlic powder
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under mild human-like conditions
on inflammation related parameters
on atherosclerosis development
by means of a hyperlipidemic and atherosclerosissusceptible APOE*3-Leiden transgenic mice.
Effect of garlic on inflammation and
atherosclerosis under human-like
conditions: Garlic powders
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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:
Experimental design
16 female APOE*3-Leiden mice per group
0.2% cholesterol diet for 28 weeks
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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.
Body weight gain (g)
Effect of garlic on body weight gain of
APOE*3-Leiden mice
Control
0.5%Prin
10
8
6
4
2
0
Control
5% Prin
* *
*
*
0
4
8 12 16 20 24
0
4
8 12 16 20 24
Control
Kyolic
*
0
4
8 12 16 20 24 28
Weeks
P<0.01
Effect of garlic on food intake and body
temperature of APOE*3-Leiden mice
Food intake
(g/mouse/day)
Free fatty acids
(mM)
Control
2.79 ± 0.08
0.73 ± 0.13
0.5% Prin
2.87 ± 0.12
0.79 ± 0.11
5% Prin
2.75 ± 0.16
0.60 ± 0.10
Kyolic
2.88 ± 0.10
0.62 ± 0.18*
*
Total dry feces
(g/ day)
5
*
4
3
2
1
0
Control 0.5%
Prin
5% Kyolic
Prin
Total fat excretion
(µmol/100g mouse/day)
Effect of garlic on fecal fatty acids excretion
of APOE*3-Leiden mice
800
*
600
400
200
0
Control 0.5%
Prin
5% Kyolic
Prin
P<0.05
Plasma ALAT levels (U/L)
Effect of garlic on liver function of
APOE*3-Leiden mice
Control
0.5%Prin
Control
5% Prin
Control
Kyolic
200
150
100
50
0
0
4
8
12 16 20 24
0
4
8
12 16 20 24
Weeks
0
4
8
12 16 20 24 28
Cholesterol levels (mM)
Effect of garlic on plasma cholesterol
levels of APOE*3-Leiden mice
Control
0.5%Prin
Control
5% Prin
Control
Kyolic
20
15
10
5
0
0
4
8
12 16 20 24
0
4
8
12 16 20 24
Weeks
0
4
8
12 16 20 24 28
Triglycerides levels (mM)
Effect of garlic on plasma triglyceride levels
of APOE*3-Leiden mice
Control
0.5%Prin
Control
5% Prin
Control
Kyolic
4
3
2
1
0
0
4
8
12 16 20 24
0
4
8
12 16 20 24
Weeks
0
4
8
12 16 20 24 28
Effect of garlic on lipoprotein profile of
APOE*3-Leiden mice
Before treatment with garlic preparations
2
Control
0.5% Prin
5% Prin
Kyolic
1
mM
0
0
8
16
24 0
8
16 24 0
8
16
24 0
8
16
24
8 16 24 0
8 16 24 0
Triglycerides
Cholesterol
Fractions
8
16
24
After treatment with garlic preparations
2
1
0
0
8
16
24 0
Blood pressure
(mmHg)
Effect of garlic on blood pressure of
APOE*3-Leiden mice
160
12 weeks
16 weeks
120
80
40
0
Control 0.5%
Prin
5% Kyolic Control 0.5% 5% Kyolic
Prin
Prin Prin
SAA (µg/ml)
Effect of garlic on serum SAA and ICAM
levels of APOE*3-Leiden mice
Control
0.5%Prin
20
Control
5% Prin
Control
Kyolic
15
10
5
ICAM (µg/ml)
0
0
4
8 12 16 20 24
0
4
8 12 16 20 24
0
4
8 12 16 20 24 28
0
4
8 12 16 20 24
0
4
8 12 16 20 24
0
4
8 12 16 20 24 28
40
30
20
10
0
Effect of garlic on plasma vWF levels of
APOE*3-Leiden mice
vWF (% PP rat)
Control
0.5%Prin
250
200
150
100
50
0
0
4 8 12 16 20 24
Control
5% Prin
0
4 8 12 16 20 24
Control
Kyolic
0
4 8 12 16 20 24 28
Effect of garlic on atherosclerosis
development of APOE*3-Leiden mice
Lesion area
Type I-III
Type IV-V
250
100
Control 0.5% 5% Kyolic
Prin Prin
80
60
40
20
200
µm2 1000
500
400
300
200
100
0
Lesion typing
%
mM
Cholesterol
exposure
150
100
50
0
Control 0.5% 5% Kyolic
Prin Prin
0
Control 0.5% 5% Kyolic
Prin Prin
Cholesterol exposure
(mM)
Correlation between cholesterol exposure
and atherosclerosis lesion area after garlic
treatment of APOE*3-Leiden mice
600
Y= 0.8308x + 289.82
R= 0.8136
400
200
0
0
50
100 150
Area (µm2 1000)
200
Conclusions
Dietary garlic treatment on APOE*3-Leiden mice
results in:
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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.
Future perspectives
Human intervention study
Collaboration with CHDR-Leiden
Ackowledgements
Sonia Espirito Santo
Wim van Duyvenvoorde
Erik Offerman
Ria van den Hoogen
Louis Havekes
Hans Princen
Bart van Vlijmen