rolf gebhardt university of leipzig medical fakulty institute of biochemistry garlic & health:...
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Rolf Gebhardt
University of Leipzig
Medical Fakulty
Institute of Biochemistry
Garlic & Health: WP 5.2The interaction of Garlic with
vessel wall and cholesterol biosynthesis
Deliverables:
1. DH2: Short report on properties and effects of garlic flavonoids
2. DH6: Publication on the molecular interactionof garlic compounds on AMP-dependent kinasepathway
3. DH7: Influence of garlic constituents on MMPand TIMP gene expression
4. DH11: Interference of garlic organosulfur compoundsand flavonoids on signal transduction
5. DH12: Influence of different garlic feeding on markers for tissue remodeling - w P14
6. DH18: Effect of garlic compounds on severalparameters in the ApoE mouse (Leiden) - w P14
Cholesterol biosynthesis:
3. Differential Effects of Garlic Extracts on Cholesterol-Biosynthesis- Sulfur fertilization- Environmental influences
1. Influence of Garlic Organosulfur Compounds:- Mechanistic studies
4. Consequences and Conclusions
2. Influence of flavonoids on cholesterol synthesis
Cholesterol Biosynthesis ishighly regulated at various steps
among others HMGCoA reductaseis a key enzyme
Many important biomoleculesare derived from intermediatesof cholesterol biosynthesis
--> the biosynthetic pathway should neverbe completely inhibited
Schematic view of the probable mode of action ofseveral garlic-derived organosulfur compounds
Comparison of different garlic-derived organosulfur compoundsfor inhibition of cholesterol biosynthesis
Inhibition of HMGCoA reductase and cholesterol biosynthesis
Test Compound EC50 values
HMGCoA Cholesterolreductase biosynthesis
Allicin > 300 17
Diallyl disulfide > 250 65
Allyl mercaptan > 1500 450
S-allyl cysteine n.d. > 1500
Diallyl sulfide > 1000 735
Dipropyl disulfide > 1500 > 1000
Effect of fructose and DADS on AMP levels, AMP-dep. kinase and cholesterol biosynthesis
Condition AMP AMPK Cholesterol biosynthesis
(nmoles/mg) (%) (%)
Control 0.34 100 100
Fructose 5.78 523 54
Diallyl disulfide 0.50 157 92
Fructose + DADS 4.85 686 25
Fruct. + DADS + araA 4.56 194 93
Effect of hypoxia, KCl and DADS on AMP levels, AMP-dep. kinase and cholesterol biosynthesis
Condition AMP AMPK Cholesterol biosynthesis
(nmoles/mg) (%) (%)
Control 0.41 100 100
low oxygen 4.75 408 72
low oxygen + DADS 4.59 566 59
KCl 2.58 346 79
KCl + DADS 2.73 424 63
KCl + DADS + araA 2.46 170 89
Correlation between AMPK activity andinhibition of cholesterol biosynthesis
Correlation coefficient:
r = - 0.96
for all conditions: - absence of DADS (closed symbols)
- presence of DADS (open symbols)
Influence of fructose, i.e. high AMP on the sensitivity ofhepatocytes towards DADS
Lowering of the cellular energy charge
- increases the sensi- tivity towards DADS
5- to 10-fold
Deliverables:
1. DH2: Short report on properties and effects of garlic flavonoids
2. DH6: Publication on the molecular interactionof garlic compounds on AMP-dependent kinasepathway
3. DH7: Influence of garlic constituents on MMPand TIMP gene expression
4. DH11: Interference of garlic organosulfur compoundsand flavonoids on signal transduction
5. DH12: Influence of different garlic feeding on markers for tissue remodeling - w P14
6. DH18: Effect of garlic compounds on severalparameters in the ApoE mouse (Leiden) - w P14
Structure of Favonoids used
University of Leipzig
All tested flavonoids belong to the flavonol group
They differ only in the amount and/or position of one or twohydroxyl groups
In the case of Luteolin and Kaempferol, several glycosideswere also tested
University of Leipzig
Comparison of the effects of Kaempferol, Luteolinand their glycosides in rat hepatocytes
Kaempferol: sensitive stimulation of cholesterol biosynthesis
high cytotoxicity (responsible for decline)
Luteolin: inhibition of cholesterol biosynthesis (no significant stimulation)
low cytotoxicity
0.01 0.1 1 10 100
Concentration ( M)
0
20
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80
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120
140
160
Inc
orp
ora
tio
n o
f [
14C
]ac
eta
te (
%)
Kaempferol
K.-3O-Glucosid
0.01 0.1 1 10 100
Concentration ( g/ml)
0
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[14C
]Ac
eta
te i
nc
orp
ora
tio
n (
%)
*
*
*: P < 0.01*
LuteolinLuteolin-7O-Gluc.
University of Leipzig
Comparison of the effects of Myricetin, Kaempferoland Luteolin in HepG2 cells
Myricetin: stimulation of cholesterol biosynthesis
Kaempferol: no significant effect
Luteolin: inhibition of cholesterol biosynthesis
0.1 1 10 100 1000
Concentration ( g/ml)
0
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14C
-Ac
eta
te I
nc
orp
ora
tio
n (
%)
0.1 1 10 100 1000
Concentration ( g/ml)
0
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14C
-Ac
eta
te I
nc
orp
ora
tio
n (
%)
*
** *
*
Luteolin
Kaempferol
Deliverables:
1. DH2: Short report on properties and effects of garlic flavonoids
2. DH6: Publication on the molecular interactionof garlic compounds on AMP-dependent kinasepathway
3. DH7: Influence of garlic constituents on MMPand TIMP gene expression
4. DH11: Interference of garlic organosulfur compoundsand flavonoids on signal transduction
5. DH12: Influence of different garlic feeding on markers for tissue remodeling - w P14
6. DH18: Effect of garlic compounds on severalparameters in the ApoE mouse (Leiden) - w P14
Garlic Powder: reinvestigation
Garlic Powder: expected influence
Inhibition of cholesterol biosynthesis
0.01 0.1 1
Concentration (mg/ml)
0
20
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120
140
160
Ch
ole
ste
rol
Bio
syn
thes
is (
%)
Hepatoc.
HepG2
fresh Garlic 1
Garlic Powder: reinvestigation
UnexpectedResult !!Garlic Powder
(Low Quality Garlic):
Stimulation of cholesterol biosynthesis !
0.01 0.1 1
Concentration (mg/ml)
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Ch
ole
ste
rol
Bio
sy
nth
es
is (
%)
Hepatoc.
HepG2
Field Study on Garlic Quality:
1. Controlled planting of four defined garlic varietiesin two different european countries
2. Controlled variation of soil fertilisation
3. Standardized harvesting, drying and powderpreparation
4. HPLC characterisation of organosulfur contentby standardized techniques in two laboratories
Influence of fertilization on garlic properties
Proper fertilization may change the response of hepatic cellswith respect to cholesterol biosynthesis:
-50
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-10
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Infl
ue
nc
e o
n C
ho
l. B
ios
yn
the
sis
0 50 100 200
Hepatoc.
HepG2
Stimulation
Inhibition
Stimulation may turn into inhibition
Inhibiting properties may be reinforced
Influence of fertilization on garlic properties
Influence of high sulfur fertilization: French harvest 2001
0 100 200 400
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-40
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-10
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Infl
ue
nc
e o
n C
ho
l. B
ios
yn
the
sis
Hepatoc.
HepG2
Stimulation
Inhibition
Stimulation
Influence of fertilization on garlic properties
Influence of high sulfur fertilization: French harvest 2001
Too much sulfur may reduce potency again
0 100 200 400
-50
-40
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-20
-10
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Infl
ue
nc
e o
n C
ho
l. B
ios
yn
the
sis
Hepatoc.
HepG2
Stimulation
Inhibition
Stimulation
Environmental influence on garlic properties
Comparison of French and Spanish harvest 2001
Only stimulation is found with spanish garlic
Too much sulfur may reduce potency again
0 100 200 400 2000
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100
Sti
mu
lati
on
0 100 200 400
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Infl
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ho
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ios
yn
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sis
Hepatoc.
HepG2
Stimulation
Inhibition
Stimulation
Questions:
1. What compounds of garlic inhibit or stimulatehepatic cholesterol-biosynthesis?
2. What is the molecular mechanism of stimulation?
Properties of acetate uptake:
0 2 4 6 8 10
Acetate Concentration (mM)
0
100
200
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500
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700
800
rel.
vel
oci
ty
0 2 4 6 8 10
Acetate Concentration (mM)
0
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rel.
vel
oci
ty
Influence of garlic on acetate uptake:
0 0.5 1.0 1.5 2
Acetate Concentration (mM)
0
50
100
150
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250
300re
l. v
elo
city
Influence of garlic on acetate uptake:
0.01 0.1 1
Concentration (mg/ml)
0
40
80
120
160
200
240re
l. v
elo
cit
y (
%)
Hepatoc.
Questions on acetate transport:
1. What transport system is involved?(alkyl monocarboxylate carrier?)
3. What is the molecular mechanism of stimulation?
2. What compounds are responsible forstimulation?
Current extensions:
1. Studies on greenhouse garlic of Odile
2. Studies on garlic from the core collectionof Rina
100%
S- TS S++
Deliverables:
1. DH2: Short report on properties and effects of garlic flavonoids
2. DH6: Publication on the molecular interactionof garlic compounds on AMP-dependent kinasepathway
3. DH7: Influence of garlic constituents on MMPand TIMP gene expression
4. DH11: Interference of garlic organosulfur compoundsand flavonoids on signal transduction
5. DH12: Influence of different garlic feeding on markers for tissue remodeling - w P14
6. DH18: Effect of garlic compounds on severalparameters in the ApoE mouse (Leiden) - w P14
Central Role of AMP-dependent kinase (AMPK) as amediator of stress signals, e.g. low energy charge
Energy consuming pathways e.g. fatty acid andcholesterol synthesis are shut down
Conclusion:
1. DADS influences glucose production in diabetic rats, but not in normal rats
2. DADS acts only in synergy with insulin
Does DADS increase sensitivity of diabetic hepatocytes for insulin?
Question:
Deliverables:
1. DH2: Short report on properties and effects of garlic flavonoids
2. DH6: Publication on the molecular interactionof garlic compounds on AMP-dependent kinasepathway
3. DH7: Influence of garlic constituents on MMPand TIMP gene expression
4. DH11: Interference of garlic organosulfur compoundsand flavonoids on signal transduction
5. DH12: Influence of different garlic feeding on markers for tissue remodeling - w P14
6. DH18: Effect of garlic compounds on severalparameters in the ApoE mouse (Leiden) - w P14
Garlic & Health: WP5 Cholesterol and cytokines
University of Leipzig
Aim: Interaction of garlic constitutents with vessel wall
0,0 1 5 10 50 100 5000
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100
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140
DADS AM SAC
Ab
sorb
an
ce (
%)
Concentration (µM)
Inhibition of Matrix Metalloproteinase-2 Production by DADS, AM and SAC in HCA-endothelial cells
Deliverables:
1. DH2: Short report on properties and effects of garlic flavonoids
2. DH6: Publication on the molecular interactionof garlic compounds on AMP-dependent kinasepathway
3. DH7: Influence of garlic constituents on MMPand TIMP gene expression
4. DH11: Interference of garlic organosulfur compoundsand flavonoids on signal transduction
5. DH12: Influence of different garlic feeding on markers for tissue remodeling - w P14
6. DH18: Effect of garlic compounds on severalparameters in the ApoE mouse (Leiden) - w P14
cancelled