Sonia M.S. Espirito Santo, Bart J.M. van Vlijmen, Rien Buytenhek, Wim van Duyvenvoorde, Erik H....

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


foam cells

intima

media{

Media not affected

Aorta with regular fatty streak


{

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)


Experimental scheme


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


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

* *


g

Effect of garlic on liver function under 3 dietary conditions

tP=0.051


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


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


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


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)


0

50

100

150

200

250

vWF

(%

PP

)


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

Sonia M.S. Espirito Santo, Bart J.M. van Vlijmen, Rien Buytenhek, Wim van Duyvenvoorde, Erik H....

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