Water extracts obtained from cistus herb (Cistus incanus L.) and promegranate peel (Punica granatum L.) are rich source of natural polyphenols. They contain ...
INFLUENCE OF CISTUS AND POMEGRANATE EXTRACTS ON ROS GENERATION IN V79 CELLS 1Aleksandra
Ślęzak, 2Helena Moreira, 2Benita Wiatrak, 3Jan Oszmiański, 2Kazimierz Gąsiorowski
1Department
of Paedriatric Bone Marrow Transplantation, Oncology and Hematology, Wrocław Medical Uniwersity, Wrocław, Poland; 2Department
3Department
of Basic Medical Science, Wrocław Medical Uniwersity, Wrocław, Poland;
of Fruit, Vegetable and Cereals Technology, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
Water extracts obtained from cistus herb (Cistus incanus L.) and promegranate peel (Punica granatum L.) are rich source of natural polyphenols. They contain (among others): catechins, gallic and ellagic acids, proanthocyanidins, phytosteroids, myricetins, ellagitannins and punicalagins. Their unique chemical composition suggests high antioxidative potential.
The aim of study was to compare an impact of the water extracts obtained from cistus and pomegranate on ROS and on H2O2 content within V79 cells (Chinese hamster pulmonary fibroblasts) after short (1h) and long (48hrs) exposure time.
MitoPy oxidation method 10
1H
9
48H
8 7 [E /E 0 ]
6 5 4 3 2 1
0 0
5 1
1
7
0
5
0
0 ]
L
M
O
c is t u s e x t r a c t [ g /m l]
0
R
,2
,1
X O
0 2 H
[1
T
5
0
2
M ito P Y flu o r e s c e n c e in te n s it y
3.
Figure.3 Impact of cistus extract on mitochondrial H2O2 level in V79 cell line (MitoPy test), after 1- hour and 48-hours incubation.
In 1-hour MitoPy test cistus extract decreased mitochondrial ROS level at low concentration (by 20%) and strongly increased at high concentration (9-fold) (Figure 3). 48-hours test with cistus revealed considerably decreased mitochondrial ROS level at all concentration range. The results were compared to H2O2 and TROLOX.
6
1H 5
48H
4 3 2 1
0 1
0
5 7
0 1
5
5 0
0
p o m e g ra n a te e x tra c t [ g /m l]
[1
0
M
]
L O R
,2
X O
0 2 H T
,1
0
2
[E /E 0 ]
M ito P Y flu o r e s c e n c e in te n s it y
4.
Figure.4 Impact of pomegranate extract on mitochondrial H2O2 level in V79 cell line (MitoPy test), after 1- hour and 48-hours incubation.
Pomegranate extract did not cause significiant differences in H2O2 level in V79 cells between 1h and 48hrs of incubation time. In both cases pro-oxidant activity of the extract was noted. The control V79 cultures contained H2O2 and TROLOX, alone.
Pomegranate extract, in 1-hour DCF-DA test, revealed pro-oxidant activity (Figure 2). Intracellular ROS level increased in a concentration-dependent manner up to 4-fold growth at 100µg/ml caused. After 48-hrs incubation with low concentrations of extract decrease in ROS amount by 13-36% was noted. Whereas higher concentrations caused only slight 1,2-fold increase in ROS level. The results were compared to V79 cells cultured with TROLOX.
1.
10
1H
9
48H
8
[E /E 0 ]
7 6 5 4 3 2 1 0 T R O LO X [1 0 M ]
0 ,1
0 ,2 5
5
10
75
100
c is t u s e x t r a c t [ g /m l]
Figure.1 Evaluation of cistus activity on intracellular ROS level in V79 cell line (DCF-DA test), after 1- hour and 48-hours incubation.
6
2. 1H
5
48H
4 [E /E 0 ]
TROLOX (Vitamine E), a strong antioxidant was used as a positive control.
In 1-hour test cistus extract exhibited ROS scavenging activity at low concentrations and strong pro-oxidant effect at high concentrations: 7-fold increase for 75µg/ml (Figure 1). After 48-hours incubation with cistus, decrease in ROS level was noted at all concentration range. The results were compared to TROLOX.
D C F f lu o r e s c e n c e in t e n s it y
Intracellular ROS level was estimated with DCF-DA oxidation assay, and mitochondrial H2O2 content was assessed with the MitoPy oxidation method. Samples were analyzed with CyFlow® Space cytometer.
D C F f lu o r e s c e n c e in t e n s it y
DCF-DA oxidation assay
3 2 1 0 T R O LO X [1 0 M ]
0 ,1
0 ,2 5
5
10
75
100
p o m e g ra n a te e x tra c t [ g /m l]
Figure.2 Evaluation of pomegranate activity on intracellular ROS level in V79 cell line (DCF-DA test), after 1- hour and 48-hours incubation.
In conclusion: • The extracts exhibited antioxidant activity at low concentrations while a pro-oxidant effect at high concentrations. • The pro-oxidant effects were significantly decreased after prolonged incubation with the extracts. • This is probably the result of the activation of intracellular antioxidant mechanisms, which need a longer time to reveal their full expression.
2ND WROCLAW SCIENTIFIC MEETINGS
