Antioxidant and anti-inflammatory potential of bergamot powder against LPS-induced endothelial activation and dysfunction Morena Gabriele1, Stefania Frassinetti1, Ramona Antonucci1, Eleonora Moccia1, Vincenzo Longo1, Laura Pucci1 1
National Research Council (CNR), Institute of Agricultural Biology and Biotechnology, Department of Bio-Agricultural Science, Italy
Inflammation and oxidative stress are key factors of vascular injury underlying many chronic pathologies such as diabetes, obesity and
Bergamot extract
cardiovascular disease.
Polyphenols
Flavonoids
Flavonols
Ascorbic acid
ORAC
DPPH
ABTS
(mg GAE/g dw)
(mg CE/g dw)
(mg QE/g dw)
(mg AAE/100g dw)
(µmol TE/g dw)
(EC50= µg/ml)
(mmol TE/g dw)
17.44±0.40
16.74±0.27
3.91±0.37
66.93±0.05
950±0.37
720±70
13.63±0.05
Increasing evidence suggests an inverse correlation between fruit and
A
vegetable consumption and the risk of many chronic diseases.
B 125
125
Besides, it has been demonstrated that a high polyphenol-rich foods intake is
CAA-unit
associated to a lower incidence of cardiovascular diseases. Citrus Bergamia Risso, commonly named bergamot, is a hybrid plant of sour lemon and orange, belonging to the Rutaceae family, which grows in a very small coastal strip in Southern Italy, in the Reggio Calabria province.
50
**
75
** 85% hemolysis inhibition
50 25
M 8
n i t e c r e o u m Q a g r Be
to investigate, in inflamed human microvascular endothelial cells (HMEC-1) the protective effect of the whole-bergamot fruit extract against
***
***
0
CNT (CAA =0)
C. Bergamia powder obtained from lyophilized pulp and peel of whole fruits;
l ) k M M H m n P / a g l A 0 0 B A 1 ( x x T o o l l N t C o Tro Tro m a g r e B
l m / g 0 0 2 t
Bergamot extract effects on cellular antioxidant activity (A) and AAPH-induced hemolysis (B) in ex vivo human erythrocyte. Quercetin or Trolox were used as reference standard. Assays were carried out in triplicate and results were expressed as mean values ± SD. One-way ANOVA with Bonferroni multiple comparison test. * significantly different from CNT (AAPH-treated cells, CAA=0).
lipopolysaccharides (LPS)-induced endothelial activation and dysfunction.
A
100
***
to characterize and to evaluate the antioxidant capacity of an extract of
B 2.0
2.0
1.0
0.5
0.0
## **
## *
3A
1.0
0.5
0.0
T N C
l m
PS
l m
T N C
/ / L g g n + 1 0 t . 5 o 0 t m S o a m g LP a r g r Be Be
l m
PS
2.0
1.5
l m
###
1.0
0.5
0.0
1.0
*
3 C 7000000 1.5
###
0.5
T N C
Quantitative Real-Time PCR analysis of IL-6 (A), ICAM-1 (B), ET-1 (C) and CHOP (D) gene expression in 6 hours LPSexposed HMEC-1, following 1 hour pre-treatment with or without 50 µg/ml bergamot extract. Experiments were carried out in triplicate and results were expressed as mean ± SD. One-way ANOVA with Bonferroni multiple comparison test. * significantly different from control (CNT); # significantly different from LPS 0.1 ng/ml.
***
6000000
3D
# #
5000000
1.0
###
4000000 3000000
0.5
2000000 1000000
0.00
l l l S S m m m P / P / L g g/ L g n + + 0 1 m t . 1 5 . 0 o 00 t5 m o S t m S a mo a g LLPP a r g m eer ga r B g e B Ber B
TT N N CC
ROS determination (DCFH-DA) in 6 hours LPS-exposed HMEC-1, following 1 hour pre-treatment with or without 50 µg/ml bergamot extract. One-way ANOVA with Bonferroni multiple comparison test. * significantly different from control (CNT); # significantly different from LPS 0.1 ng/ml
Bergamot powder showed high levels of bioactive compounds and in vitro antioxidant capacity, as well a better hemolysis inhibition and erythrocytes antioxidant activity.
0
LPS
Bergamot
Bergamot + LPS
LPS exposure induced a significant up-regulation of IL-6, ICAM-1, ET-1 and CHOP gene expression, which were completely reversed in bergamot pre-treated cells. Otherwise, bergamot pre-treatment induced a slight decrement of ICAM-1 up-regulation.
[NO] % vs CNT
- 30.7%
+ 44.2%
+15.3%
Conclusions This preliminary data suggest a preventive action of bergamot-whole fruit powder, which is able to protect HMEC-1 from LPS-induced endothelial activation and dysfunction. This protective effect is probably linked to the high content of bioactive compounds that exhibit radical scavenging and anti-inflammatory activities.
0
1
1
0
0
Results
LPS exposure caused a higher ROS production and a reduction in NO level compared to control cells, which was totally inhibited by bergamot pre-treatment.
1
0
8000000
l l S m m P / / L g g n + 1 0 t . 5 o 0 t m S o a P m g L a r rg Be e B
l l S m m P / / L g g n + 0 1 t . 5 o 0 t m o S a P m g L a r rg Be e B
1
l l S m m P / / L g g n + 1 0 t . 5 o 0 t m S o a m LP a rg e g r B e B
0.0
T N C
**
2
T N C
*** ###
##
0.0
ROS production ET-1 fold increase
#
CHOP fold increase
***
ROS
###
1.0
D 1.5
***
1.5
CELLULAR ESTERASE 0.5
/ / L g g n + 1 0 t . 5 o 0 t m S o a m g LP a r g r Be Be
C
3B ICAM-1 fold increase
###
1.5
CHOP fold increase
## **
***
IL-6 fold increase
***
1.5
ICAM-1 fold increase
IL-6 fold increase
75
25
Aims:
ET-1 fold increase
***
% hemolysis vs CNT
100
NO determination (colorimetric assay) in 6 hours LPS-exposed HMEC-1, following 1 hour pre-treatment with or without 50 µg/ml bergamot extract. Experiments were carried out in triplicate and results were expressed as percentage with respect to control.
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