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 HealthGaubius Laboratory

The Netherlands

Leiden University Medical CenterDepartment of CardiologyThe Netherlands

• 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.

European Comunity Garlic and Health project

• 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.

• Garlic clinical trials and animal studies with well-characterized and standardized garlic material, with a consistent study design and dosage are poorly found in the last 15 years.

Garlic and health

Possible effects of garlic on lipid metabolism

CHOLESTEROL

SYNTHESIS

CONVERSION

CHOLESTEROL

UPTAKE

receptors

STORAGE

ESTERS

DIETARY

BILE ACIDS

LDL receptor

CHOLESTERYL

BILE

other

BILIARYCHOLESTEROL

SECRETION

LIVER

INTESTINE

CM

CM Remnants

VLDL

LDL

Possible effects of garlic on atherosclerosis

Lipid core

Media

Intima

Endothelium

LDL

O2

oxLDL

Thrombus

Collagen- degrading proteinases

Shear stress

Fibrous capMonocytes

Inflammation

Oxidation

Contradictory effects Contradictory effects attributedattributed to garlic to garlic

• 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.

• 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.

• 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.

• 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.

• 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.

• Lipid metabolism

- No effects of garlic powders and isolated compounds on plasma lipid levels and markers of cholesterol synthesis and absorption under hyperlipidemic conditions.

- 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.

Previous reported effects of garlic (LI111, Previous reported effects of garlic (LI111, Morado 200, Allicin and DADS) in APOE*3-Morado 200, Allicin and DADS) in APOE*3-

Leiden miceLeiden mice

Study the effect of 2 different doses of Printanor garlic powder

• under mild human-like conditions

• on inflammation related parameters

• on atherosclerosis development

by means of a hyperlipidemic and atherosclerosis-susceptible APOE*3-Leiden transgenic mice.

Effect of garlic on inflammation processes and atherosclerosis under

human-like conditions: Aim

• 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: Garlic powders

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: 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.

Effect of garlic on inflammation and atherosclerosis under human-like conditions:

Experimental design

*

Control0.5%Prin

ControlKyolic

Control5% Prin

Effect of garlic on body weight gain of APOE*3-Leiden mice

Weeks

Bo

dy

wei

gh

t g

ain

(g

)

P<0.01

02468

10

0 4 8 12 16 20 24 0 4 8 12 16 20 24 0 4 8 12 16 20 24 28

**

**

Effect of garlic on food intake and body temperature of APOE*3-Leiden mice

Food intake(g/mouse/day)

2.79 ± 0.08

2.87 ± 0.12

2.75 ± 0.16

2.88 ± 0.10

Control

0.5% Prin

5% Prin

Kyolic

Free fatty acids(mM)

0.73 ± 0.13

0.79 ± 0.11

0.60 ± 0.10

0.62 ± 0.18

*

*

Effect of garlic on fecal fatty acids excretion of APOE*3-Leiden mice

To

tal

fat

excr

etio

n

(µm

ol/

100g

mo

use

/day

)0

200

400

600

800

Control 0.5%Prin

5% Prin

Kyolic

*

P<0.05

0

1

2

3

4

5

To

tal

dry

fec

es

(g/

day

)

Control 0.5%Prin

5% Prin

Kyolic

*

Effect of garlic on liver function of APOE*3-Leiden mice

Pla

sma

AL

AT

lev

els

(U/L

)

Weeks

Control0.5%Prin

ControlKyolic

Control5% Prin

0

50

100

150

200

0 4 8 12 16 20 24 0 4 8 12 16 20 24 0 4 8 12 16 20 24 28

Effect of garlic on plasma cholesterol levels of APOE*3-Leiden mice

Weeks

Ch

ole

ster

ol

leve

ls (

mM

) Control0.5%Prin

ControlKyolic

Control5% Prin

0

5

10

15

20

0 4 8 12 16 20 24 0 4 8 12 16 20 24 0 4 8 12 16 20 24 28

Effect of garlic on plasma triglyceride levels of APOE*3-Leiden mice

Weeks

Tri

gly

ceri

des

lev

els

(mM

) Control0.5%Prin

ControlKyolic

Control5% Prin

0

1

2

3

4

0 4 8 12 16 20 24 0 4 8 12 16 20 24 0 4 8 12 16 20 24 28

Effect of garlic on lipoprotein profile of APOE*3-Leiden mice

Cholesterol Triglycerides

Fractions

0

1

2

0 8 16 24 0 8 16 24 0 8 16 24 0 8 16 24

Control 0.5% Prin 5% Prin Kyolic

Before treatment with garlic preparations

0

1

2

0 8 16 24 0 8 16 24 0 8 16 24 0 8 16 24

mM

After treatment with garlic preparations

Effect of garlic on blood pressure of APOE*3-Leiden mice

Blo

od

pre

ssu

re

(mm

Hg

)

0

40

80

120

160

Control 0.5%Prin

5% Prin

Kyolic

12 weeks 16 weeks

Control 0.5%Prin

5% Prin

Kyolic

Effect of garlic on serum SAA and ICAM levels of APOE*3-Leiden mice

0

5

10

15

20

0 4 8 12 16 20 24 0 4 8 12 16 20 24 0 4 8 12 16 20 24 28

Control0.5%Prin

Control5% Prin

ControlKyolic

SA

A (

µg

/ml)

ICA

M (

µg

/ml)

0

10

20

30

40

0 4 8 12 16 20 24 0 4 8 12 16 20 24 0 4 8 12 16 20 24 28

Effect of garlic on plasma vWF levels of APOE*3-Leiden mice

050

100150200250

0 4 8 12 16 20 24 0 4 8 12 16 20 24 0 4 8 12 16 20 24 28

vWF

(%

PP

rat

)

Control0.5%Prin

Control5% Prin

ControlKyolic

Effect of garlic on atherosclerosis development of APOE*3-Leiden mice

Lesion typing Lesion areaCholesterol exposure

0

100

200

300

400

500

0

20

40

60

80

100

Control 0.5%Prin

5% Prin

Kyolic Control 0.5%Prin

5% Prin

KyolicControl 0.5%Prin

5% Prin

Kyolicµ

m2 10

00

%mM

Type I-IIIType IV-V

0

50

100

150

200

250

Ch

ole

ster

ol

exp

osu

re

(mM

) R= 0.8136

Area (µm2 1000)

Correlation between cholesterol exposure and atherosclerosis lesion area after garlic

treatment of APOE*3-Leiden mice

Y= 0.8308x + 289.82

0

200

400

600

0 50 100 150 200

Dietary garlic treatment on APOE*3-Leiden mice results in:

• 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.

Conclusions

Human intervention study

Collaboration with CHDR-Leiden

Future perspectives

Ackowledgements

Sonia Espirito Santo

Erik OffermanRia van den Hoogen

Louis Havekes

Bart van VlijmenHans Princen

Wim van Duyvenvoorde