Effect of garlic on lipid metabolism,
inflammation processes and atherosclerosis
under human-like conditions in APOE*3Leiden mice
Sonia M.S. Espirito Santo
TNO Prevention and Health
Gaubius Laboratory
The Netherlands
L University Medical Center
Department of Cardiology
The Netherlands
Garlic and health
•
•
Garlic has an history for its use in cardiovascular
medicine.
Some studies have suggested that garlic significantly
reduces plasma lipid levels, systolic and diastolic
blood pressure and that it shows a strong antibiotic
activity.
Kik C, Kahane R, Gebhardt R. Nutr Metab Cardiovasc Dis. 2001;11:57-65.
•
Clinical trials and animal studies with wellcharacterized and standardized garlic material, with a
consistent study design and dosage are scarcely
found in the last 15 years.
Possible effects of garlic on lipid
metabolism
LDL
SYNTHESIS
UPTAKE
CHOLESTEROL
VLDL
STORAGE
CHOLESTERYL
ESTERS
BILE
ACIDS
BILIARY
CHOLESTEROL
CMR
CM
DIETARY
CHOLESTEROL
Possible effects of garlic on
atherosclerosis
INFLAMMATION
SHEAR STRESS
Fibrous
cap
Monocytes
LDL
Media
Thrombus
Endothelium
oxLDL
O2
OXIDATION
Lipid core
Collagen- degrading
proteinases
Contradictory effects in garlic studies
•
Old human trials in hypercholesterolemic patients reported
a decrease in plasma lipid levels.
Jain et al, 1993. Silagy et al, 1994. Steiner et al, 1996. Bordia et al, 1997.
•
Recent human trials in hypercholesterolemic patients
showed no effects of garlic treatment on plasma lipids.
Simmons et al,1994. Neil et al, 1996. Breithaupt et al, 1997. Berthold et al, 1998. McCrindle et al, 1998. Isaacsohn et al, 1998. Gardner et al, 2001. Ziaei et al, 2001. Phelps et al,
1998. Satitvipawee et al, 2003.
•
Induced-atherosclerosis in rabbits kyolic showed a
decrease in aortic fatty streaks-like lesions.
Efendy JL, et al. Atherosclerosis. 1997:132:37-42. Campbell JH, et al. J Nutr. 2001;131:1006S-09S.
•
Allicin treatment in hypercholesterolemic C57Black/6 mice
showed no effects on plasma lipid levels and decreased
aortic fatty streak-like lesions.
D. Abramovitz et al., Coronary Artery Disease, 1999, 10: 515-519.
•
Hypercholesterolemic pigs and rats fed garlic oil and garlic
powder (Kwai) showed no effects on atherosclerosis.
Heinle H,et al. Drug Res. 1994;44:614-17. Lee WM, et al. Exper Mol Pathol. 1980:33:345-8.
Effect of garlic on lipid metabolism,
inflammation processes and atherosclerosis
under human-like conditions: Aim
Study the effect of different garlic powders (Kwai,
Morado and Printanor) and garlic sulfur-containing
compounds (Allicin and DADS)
•
•
•
on lipid metabolism
on inflammation related parameters
on atherosclerosis development
by means of a hyperlipidemic and atherosclerosissusceptible APOE*3-Leiden transgenic mice.
APOE*3-Leiden Transgenic Mouse Model for
Study the Effect on Hyperlipidemia and
Atherosclerosis
•
•
•
•
Transgenic mice carrying a mutant form of human APOE
gene.
Dysfunctional APOE results on chylomicron/ VLDL
clearance defect.
Highly responsive to feeding cholesterol rich diets.
Develops atherosclerosis varying from fatty streaks to
complicated lesions.
Distribution of Lipids among Lipoprotein
Fractions in APOE*3-Leiden Mice
cholesterol
wild-type
Regular diet
triglycerides
APOE*3-Leiden
Regular diet
2.5
Cholesterol diet
VLDL
(mmol/l)
2.0
1.5
VLDL
1.0
HDL
0.5
HDL
VLDL
0.0
15 25 35 45
15 25 35 45
Fraction Number
15 25 35 45
Atherosclerosis in APOE*3-Leiden Mice
Stage I (Type I lesion)
Monocytes
Adherence of monocytes to the activated vessel wall
Atherosclerosis in APOE*3-Leiden Mice
Stage II (Type II lesion)
Media not
affected
foam cells
intima
media
{
Aorta with Regular Fatty Streak
Atherosclerosis in APOE*3-Leiden Mice
Stage III (Type IV-V lesion)
cholesterol
fibrosis
clefts
intima
necrosis
{
media
Affected media
Severe Plaque in Aorta
First project: Effects of garlic on lipid
metabolism and fast atherosclerosis in
APOE*3-Leiden mice
•
•
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.
First project: Conclusions
•
Lipid metabolism
• No effects of Kwai, Morado, Allicin and DADS on plasma
lipid levels and markers of cholesterol synthesis and
absorption under hyperlipidemic conditions.
• Morado garlic powder showed an improved liver
function, decreased plasma cholesterol levels and
serum amyloid A levels under severe 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.
J.Nutr.
Second project: Effect of garlic powder
Printanor on inflammation and
atherosclerosis under human-like conditions
Study the effect of 2 different doses of Printanor
•
•
•
under mild human-like conditions
on inflammation related parameters
on atherosclerosis development
by means of a hyperlipidemic and atherosclerosissusceptible APOE*3-Leiden transgenic mice.
Second project: Effect of garlic powder
Printanor on inflammation and
atherosclerosis under human-like conditions
•
Printanor FR 2001: 0.5% of dry garlic powder
•
Printanor FR 2001: 5% of dry garlic powder
•
Kyolic ® (positive control): 0.16%
Previously reported to result in 64% reduction of fatty
streaks in New Zealand white rabbits.
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
•
•
•
General parameters: Body weight, food intake, marker of
liver function, plasma lipid levels, blood pressure, and
fecal fatty acids.
Inflammation related parameters: General 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, total lesion area, and monocytes adhesion.
Effect of garlic on body weight gain in
APOE*3-Leiden mice
Body weight
(g)
30
26
*
*
22
18
0
4
8
*
*
*
12
16
20
Weeks
*
Body weight gain
(g)
Control
0.5% Printanor
5% Printanor
Kyolic
10
8
6
4
2
*
*
*
16
20
*
0
24
28
0
4
8
12
24
28
Weeks
*P<0.01
Effect of garlic on food intake and plasma
free fatty acids in APOE*3-Leiden mice
Table 1. Effect of Printanor and Kyolic® on food intake, total dry feces production, total fatty acids excretion in
feces and total fatty acid excretion after 28 weeks of study under Western diet conditions.
Food intake
Total dry feces
production
Total fatty acid
excretion in feces
Total fatty acid
excretion
g/mouse/day
g feces/100 g mouse/
day
µmol/ g feces
µmol/ 100 g
mouse/day
Control
2.79
±
0.08
1.5
±
0.3
101
±
21
144
±
25
0.5% Printanor
2.87
±
0.12
1.7
±
0.4
120
±
30
194
±
40*
5% Printanor
2.75
±
0.16
1.9
±
0.3*
108
±
29
200
±
43*
0.16% Kyolic®
2.88
±
0.10
1.7
±
0.4
85
±
26
128
±
48
Data are presented as meanSD for 18 mice per group. *P<0.05 as compared to the control group.
*P<0.05
Effect of garlic on plasma lipids in
APOE*3-Leiden mice
Table 2. Effect of Printanor and Kyolic® on plasma cholesterol and triglycerides after 28 weeks of
study under Western diet conditions. Cholesterol and triglycerides exposure during the 28 weeks
of the study.
Cholesterol
Triglycerides
Cholesterol
exposure
Triglycerides
exposure
mmol/L
mmol/L
mmol/L . weeks
mmol/L . weeks
Control
11.4
±
2.5
1.0
±
0.3
346.3
±
40.5
40.4
±
8.9
0.5%
Printanor
12.7
±
2.5
1.0
±
0.3
359.8
±
47.0
39.7
±
6.8
5% Printanor
12.6
±
4.4
0.9
±
0.2
379.1
±
67.3
40.5
±
5.6
0.16% Kyolic®
10.3
±
2.1
0.9
±
0.3
321.3
±
32.2
37.4
±
7.9
Data are presented as meanSD for 18 mice per group. *P<0.05 as compared to the control group.
Effect of garlic on lipoprotein profile in
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 in APOE*3Leiden 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 in 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 in
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 whole blood TNF levels
in response to LPS in APOE*3-Leiden mice
TNF production levels (pg/ml)
Regular chow diet
High-cholesterol diet
Western diet
60
*
*
*
Control
0.5%Printanor
5% Printanor
Kyolic
*
40
*
30
40 *
20
20
10
**
*
*
50
100
LPS (ng/ml)
150
0
0
0
0
50
100
LPS (ng/ml)
150
*P<0.05
Effect of garlic on atherosclerosis
development in APOE*3-Leiden mice
500
400
300
200
100
0
Control 0.5% 5% Kyolic
Prin Prin
Lesion typing
Type I-III
Type IV-V
100
%
mM
Cholesterol
exposure
80
60
40
20
0
Control 0.5% 5% Kyolic
Prin Prin
Effect of garlic on atherosclerosis
development in APOE*3-Leiden mice
Monocytes adhesion
Lesion area
50
# monocytes
µm2 x 1000
250
200
150
100
50
0
Control 0.5% 5% Kyolic
Prin Prin
40
30
20
10
0
Control 0.5%
Prin
5% Kyolic
Prin
Summary (1)
•
•
•
Treatment with Printanor (both doses) demonstrated
reduced body weight, which coincided with increased
feces production and increased fecal fatty acids
excretion.
Printanor (both doses) and Kyolic® treatment did not
affect plasma lipids, serum amyloid A, serum soluble
intercellular adhesion molecule-1, plasma von
Willebrand factor, and lipopolysaccharide-induced
Tumor Necrosis Factor- production by blood-derived
monocytes.
As analyzed after 28 weeks of treatment, Printanor
(both doses) and Kyolic did not affect atherosclerotic
lesion type, area or composition.
Conclusion
Under conditions relevant to the human
situation, the well-characterized and
production-controlled garlic powders
and garlic sulfur-containing
compounds do not display
hypolipidemic, anti-inflammatory or
anti-atherosclerotic properties.