sonia m.s. espirito santo, bart j.m. van vlijmen, rien buytenhek, wim van duyvenvoorde, erik h....
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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
Effect of garlic powders and garlic active compounds on lipid metabolism and atherosclerosis in APOE*3-Leiden
mice
Effect of garlic powders and garlic active compounds on lipid metabolism and 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.
Aim of the study: Effects of garlic on lipid Aim of the study: Effects of garlic on lipid metabolism and atherosclerosismetabolism and atherosclerosis
Lipid metabolism
CHOLESTEROL
SYNTHESIS
CONVERSION
CHOLESTEROL
UPTAKE
receptors
HMG-CoA reductase
STORAGE
ESTERS
DIETARY
BILE ACIDS
LDL receptor
7-hydroxylase
27-hydroxylase
CHOLESTERYL
BILE
other
BILIARYCHOLESTEROL
ACAT
SECRETION
LIVER
INTESTINE
CHYLOMICRONS
CHYLOMICRONREMNANTS
LDL
VLDL
Atherosclerosis
Lipid core
MediaIntima
Endothelium
LDL
O2
oxLDL
Thrombus
Collagen- degrading proteinases
Shear stress
Fibrous capMonocytes
Inflammation
Oxidation
Contradictory effects Contradictory effects attributedattributed to garlic to garlic
• Soluble garlic extracts inhibit cholesterol biosynthesis in primary rat hepatocytes and human HepG2 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 C57Black/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. McCrindle 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 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 miceAtherosclerosis in APOE*3-Leiden mice
Adherence of monocytes to the activated vessel wall
Monocytes
Atherosclerosis in APOE*3-Leiden miceAtherosclerosis in APOE*3-Leiden mice
foam cells
intima
media{
Media not affected
Aorta with regular fatty streak
Atherosclerosis in APOE*3-Leiden miceAtherosclerosis in APOE*3-Leiden mice
{
media
intima
fibrosischolesterol
cleftsnecrosis
Affected media
Severe plaque in aorta
Effect of garlic on lipid metabolism and Effect of garlic on lipid metabolism and atherosclerosis in APOE*3-Leiden mice: atherosclerosis in APOE*3-Leiden mice:
Selection of compoundsSelection of compounds
• Lichtwer LI111 garlic powder: Reference dry garlic powder.
• Morado de Cuenca 200 (Mor200): 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 C57Black/6 mice
(D. Abramovitz et al., Coronary Artery Disease, 1999, 10: 515-519)
Effect of garlic on lipid metabolism and Effect of garlic on lipid metabolism and atherosclerosis in APOE*3-Leiden mice: atherosclerosis in APOE*3-Leiden mice:
Experimental scheme
Effect of garlic on lipid metabolism and Effect of garlic on lipid metabolism and atherosclerosis in APOE*3-Leiden mice: atherosclerosis in APOE*3-Leiden mice:
Experimental scheme-2
Matching and start of the studyWeight and food intakeBlood sampling/ plasma isolationPlasma lipid levels Cholesterol lipoprotein profile Chol. synthesis/ Intestinal absorption
Platelet activation measurements
Marker for liver function Liver lipid composition
Atherosclerotic aorta lesions area
Inflammation markers
2 4
6
8 10 12
14
80
8080
80
pp
60
p60
60
80
80
80
pp
6565
65
pp p
80 65
p p p p
8065
65
65
65
6065
65
16
Regular diet Western diet Atherogenic diet
60
p
Blood pressure
Compound biostability
65
p p
80
p
5
65
p
15
Run in
18 20 22
65
25
p
65
65
65
80 65
p
65
0
Atherosclerotic aorta lesions typing 605
General parameters Inflammation and atherosclerosis related parameters
Lipid metabolism related parameters
80
Effect of garlic on food intake under 3 dietary conditions
Control 3.6 3.2 3.3
LI111 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
*
*
*
Food intake
Regular diet Western diet Atherogenic diet
g/ mouse/ day
*P< 0.05
Effect of garlic on body weight under 3 dietary conditions
*P< 0.05
Control 20.9 23.3 23.2
LI111 21.0 22.0 22.6
Mor 200 21.5 23.2 22.1
DADS 20.3 21.0 20.6
Allicin 20.5 22.6 23.4
Body weight
* *
Regular diet Western diet Atherogenic diet
g
Effect of garlic on liver function under 3 dietary conditions
tP=0.051
Regular diet Western diet Atherogenic diet
Control 33.4 30.3 175
LI111 33.2 34.4 104
Mor 200 32.9 30.3 94.1
DADS 29.9 41.8 203
Allicin 25.2 28.2 171
Plasma alanine amino transferase
t
t
U/L
Effect of garlic on plasma cholesterol levels under 3 dietary conditions
*P< 0.05
Plasma cholesterol levels
Regular diet Western diet Atherogenic diet
mM
Control 3.1 9.3 63
LI111 2.9 9.3 57
Mor 200 3.0 9.1 44 *
DADS 2.9 8.3 70
Allicin 3.5 * 9.2 73
Regular diet Western diet Atherogenic diet
Effect of garlic on cholesterol lipoprotein distribution under 3 dietary conditions
*P< 0.05Fraction number
Ch
ole
ste
rol
(mM
)
0
1
2
3
4
1 5 9 13 17 21
0
1
2
3
4
1 5 9 13 17 21
VLDL
0.0
0.1
0.2
0.3
0.4
1 5 9 13 17 21
0.0
0.1
0.2
0.3
0.4
1 5 9 13 17 21
0
4
8
12
1 5 9 13 17 21
0
4
8
12
1 5 9 13 17 21
Control LI111 Morado 200
LDL HDL
Control DADS Allicin
Effect of garlic on markers of cholesterol synthesis and intestinal absorption under
3 dietary conditions Regular diet
*P< 0.05
0.0
0.5
1.0
1.5
Control LI111 Mor 200 DADS Allicin
0
10
20
30
40
Control LI111 Mor 200 DADS Allicin
campesterolb-sitosterol
Con
cen
trat
ion
(µ
mol
ste
rol /
mm
ol c
hol
este
rol)
squalenelathosterollanosterol
Markers of cholesterol synthesis
Markers of intestinal absorption
Liver lipid composition
Free cholesterol Triglycerides
mg lipid/ g protein
Cholesteryl esters
Control 19.4 43.7 154
LI111 15.9 * 31.5 * 130 *
Mor 200 15.3 * 35.0 * 127 *
DADS 17.8 49.7 171 *
Allicin 17.3 37.8 146
Effect of garlic on liver lipid levels under fast atherogenic conditions
*P< 0.05
Conclusions of the study of the effects of the study of the effects of garlic of garlic on lipid levels and lipid
metabolism (1)
• Under more human-like hyperlipidemic conditions (8-12 mM):
– 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 mM):
– Morado 200 showed a plasma cholesterol-lowering effect in APOE*3-Leiden mice.
– LI111 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 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
SA
A (
µg
/ m
l)
Control LI111 Mor 200 DADS Allicin
0
50
100
150
200
250
vWF
(%
PP
)
Control LI111 Mor 200 DADS Allicin
0
2
4
6
8
**
p=0.001
p<0.001
*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.
Effect of garlic on plaque area of the aortic root under fast atherogenic conditions
*P< 0.05Control LI111 Mor 200
0
100
200
300
400
Pla
qu
e ar
ea o
f th
e ao
rtic
ro
ot
(µm
2 x
100
0)
p = 0.017
Control DADS Allicin
0
100
200
300
400
p = 0.011
Under fast atherogenic conditions:
• LI111 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.
• LI111 and DADS treated groups showed a larger plaque area.
Conclusions of the study of the effects of of the study of the effects of garlic on garlic on fast atherosclerosis development
and other atherosclerosis related processes (2)
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
Future mild atherosclerosis development study under more human-like conditions:
Experimental scheme
Future mild atherosclerosis development study under more human-like conditions:
Experimental scheme
-2
Matching and start of the studyWeight and food intakeBlood sampling/ plasma isolationPlasma lipid levels Cholesterol lipoprotein profile
Fibrinogen, t-PA and soluble ICAM
Marker for liver function Liver lipid composition
Moncytes adherence
Atherosclerotic aorta lesions typing
6 14
60
6060
60
p
55
60
60
55
60
60
60
p
6060
60
p
60 60 p
60
Western diet
60
p
SAA and vWf
Compound biostability
6060
p
5
Run in22
24
60 60
0
Atherosclerotic aorta lesions area 555
General parameters Inflammation and atherosclerosis developmentrelated parameters
Lipid metabolism related parameters
In weeks
5
60