Transcript poster - Plant Research International

Extracts from garlic dry powders modulate LPS-induced liberation of cytokines.
H.-P. Keiß1, V.M. Dirsch1, T. Hartung2, A.M. Vollmar1
1Department
2Biochemical
of Pharmacy, Center of Drug Research, Butenandtstr 5-13, 81377 Munich, Germany
Pharmacology, University of Konstanz, Universitaetsstr. 10, 78457 Konstanz, Germany
[email protected]
Background:
Methods:
Garlic (Allium sativum) has been proposed to have anti-atherosclerotic potential,
with sulphur compounds seemingly the pharmacologically active substances.
Increasing evidence is given that inflammatory processes, such as
NF-B activation, cytokine liberation or expression of adhesion molecules, are
pivotal in the pathophysiology of atherosclerosis.
Garlic extracts
Garlic dry powder from unfertilized or sulphur fertilized allium sativum L. cv.
Printanor was used. Garlic dry powders were extracted either with aqueous media or
DMSO.
Cytokine liberation assay
Heparinized human whole blood was exposed to respective garlic extracts and
stimulated with lipopolysacharide (LPS) 10 ng/ml. Cytokines in the cell free
supernatants were collected and subsequently quantified by sandwich ELISA.
NF-B luciferase reporter gene assay
HEK 293 cells were transfected with a plasmid containing a NF- B driven fireflyluciferase gene and a plasmid containing a HSV-TK driven renilla-luciferase gene.
Cells were stimulated with supernatants from the cytokine liberation assay and NF-B
promoter activity was measured with a Dual-Luciferase Reporter System.
Aim:
The aim of our study was to examine the effects of different garlic extracts on
cytokine production and to elucidate the impact of increasing amounts of sulphur
compounds on the biological activity of the extracts.
1. Liberation of pro-inflammatory cytokines
Aqueous extracts
DMSO extracts
Il-1
TNF-
Il-1
TNF-
Printanor
wo. LPS
*
*
**
5000
**
7500
**
wo. LPS
g/
m
l
l
0.
m
1
0.
01
m
g/
m
LP
S
l
m
m
m
g/
- 10 ng/ml LPS
+ 10 ng/ml LPS
unfertilized garlic
fertilized garlic
• Aqueous extracts liberate Il-1 and TNF-
• Aqueous garlic extracts reduce LPS-induced liberation of Il-1 and TNF-,
however not dose dependently.
• Fertilized garlic shows a weaker pro-inflammatory, but a stronger antiinflammatory potential than unfertilized garlic
1
01
____________________________
_________________________ __________________________
unfertilized garlic
fertilized garlic
g/
m
l
Co
g/
m
l
m
1
0.
01
0.
0.
S
LP
m
g/
m
l
g/
m
l
1
0.
01
0.
m
Co
m
+ 10 ng/ml LPS
*
0
m
g/
10
________________________________
- 10 ng/ml LPS
unfertilized garlic
fertilized garlic
*
5000
2500
m
g/
m
l
l
l
l
m
g/
m
m
1m
g/
0,
______________________________
+ 10 ng/ml LPS
1
l
LP
S
m
g/
m
1
0,
m
l
l
Co
m
1
10
l
m
g/
m
g/
_________________________ __________________________
10000
10000
0
0
10
0.
1
1
m
m
g/
m
m
l
l
m
l
LP
S
g/
m
10
0.
1
1
m
m
g/
g/
m
m
l
l
Co
0
Printanor
12500
15000
0.
5000
m
**
10000
g/
m
*
10000
15000
g/
20000
Il-1 [pg/ml Blood]
TNF [pg/ml Blood]
Il-1 [pg/ml Blood]
20000
30000
20000
Printanor
TNF [pg/ml Blood]
Printanor
40000
_____________________________
+ 10 ng/ml LPS
unfertilized garlic
fertilized garlic
• DMSO extracts do not liberate Il-1 and TNF-
• DMSO extracts reduce LPS-induced liberation of Il-1 and TNF-
• Fertilization has no influence on the inhibitory effect
3. NF-B activity assay
To investigate whether the modulation of cytokine levels in human whole
blood leads to a change in the transactivation activity of the transcription
factor NF- B we performed a NF- B-luciferase reporter gene assay.
Therefore cells were transfected as described under methods and
subsequently stimulated with the supernatants of human blood, treated
with DMSO garlic extracts and LPS or with LPS alone.
2. Liberation of anti-inflammatory cytokine
DMSO extracts
Il-10
Printanor
500
l
l
m
m
g/
g/
**
5.0
**
**
unfertilized garlic
fertilized garlic
m
2.5
1
• Aqueous garlic extracts liberate Il-10
• Aqueous extracts increase LPS-induced liberation of Il-10
• Low concentrations of DMSO garlic extracts increase LPS-induced
liberation of Il-10
• High concentrations of aqueos fertilized garlic extract show a strong
inhibitory effect on Il-10 liberation
l
m
g/
m
0,
1
m
0,
01
g/
m
g/
m
m
l
l
m
g/
m
l
0.0
LP
S
+ 10 ng/ml LPS
unfertilized garlic
fertilized garlic
0,
01
__________________________
- 10 ng/ml LPS
unfertilized garlic
fertilized garlic
0.
___________________________
0.
01
m
m
g/
m
1
m
LP
S
l
l
m
Co
g/
0.
+ 10 ng/ml LPS
01
_____________________________
- 10 ng/ml LPS
0.
1
m
g/
m
l
10
m
g/
m
l
l
m
1m
g/
0.
____________________________
LP
S
l
g/
m
l
10
m
g/
m
l
1
m
m
1m
g/
0.
Co
0
7.5
o
0
10.0
C
500
*
1000
x-fold luciferase activity
1000
NF- B activity assay
Printanor
1500
**
1500
Il-10 [pg/ml Blood]
Il-10 [pg/ml Blood]
2000
0,
1
Aqueous extracts
DMSO garlic extract + LPS
• Supernatants of blood cells treated with DMSO garlic extracts
reduce NF-B activity
• Fertilized garlic was more active than unfertilized garlic
* < 0.05 Anova with Bonferroni post-test
** < 0.01 Anova with Bonferroni post-test
Conclusion:
This study indicates, that garlic dry powder extracts are able to decrease LPS-induced liberation of pro-inflammatory cytokines Il-1 and TNF-. On the other hand, they can
increase the liberation of the anti-inflammatory cytokine Il-10. These effects are strongly dependent on the solvent used for extraction. Furthermore, it was shown that sulphur
fertilization may have a positive influence on the action mediated by garlic. In summary, DMSO garlic extracts modify cytokine release from human blood in a way possibly
resulting in a lower expression of NF- B regulated genes.
This work was supported by the EU : “Garlic and Health Project”, Id-Nr: QLK1-CT-1999-00498