Effect of ginseng extract on induced damage in neuronal cells
Students:
Sandra Haltmeier, Kantonsschule Romanshorn
Fiona Wiesner, Kantonsschule Zürcher Oberland, Wetzikon
Supervision:
Prof. Dr. Volker Enzmann and Prof. Dr. Hans Rudolf Widmer
Tutors:
Dr. Stefano Di Santo and Stephanie Lötscher
Date:
13.03.2016-19.03.2016
1 Abstract
Ginseng is often used as a cure in Traditional Chinese Medicine. We wanted to proof
the effect of Ginseng as a preventive substance against induced damage in neuronal
cells. We have used retinal (661W) and neuronal stem cell (C17.2) lines. We could
find out, that the right concentration of Ginseng can protect the cells from death. The
661W cells need a concentration of 500 μg/ml. The C17.2 cells need a concentration
of 10 μg/ml or 500 μg/ml.
2 Introduction
Our project was based on the potential positive
effect of ginseng extract on two different cell
lines, which were treated with hydrogen peroxide. The extract was obtained from the root of
ginseng Panax quinquefolius (American Ginseng). Ginseng is believed to have numerous
beneficial qualities and is often used as a tonic
or as nutritional supplement, especially in Traditional Chinese Medicine. The plant is found in
Korea, China and south Siberia.
By treating some of the cells solely with different concentrations ginseng extract and
others additionally with hydrogen peroxide, we could distinguish differences in the
viability and metabolic activity between the cells.
The concentration of ginseng extract used were as following:
Concentration 1: 10 μg/ml
Concentration 2: 100 μg/ml
Concentration 3: 500 μg/ml
Furthermore, we worked with a hydrogen peroxide concentration of 500 μMol/L.
2.1 Cell lines used in our project
To examine the effect of ginseng extract we used two different cell lines.
661W – immortalized mouse retinal cell line (Figure 1)
This cell line possesses the same characteristics as cone photoreceptor cells and is
often used in ocular research.
C17.2 – immortalized mouse stem-like cell line (Figure 2)
C17.2 cells are stem-like cells and are able to differentiate into neurons. They are
obtained from the cerebellum of a mouse.
Figure 1: Untreated 661W cells
Figure 2: Untreated C17.2 cells
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3 Methods
After preparing and counting the cells we treated all of them with different concentrations of ginseng extract. Furthermore, we added hydrogen peroxide (H 2O2), a substance that should cause cell death. With the help of PrestoBlue and immunohistochemical staining we could then analyse the viability.
3.1 PrestoBlue analysis
We added 50 µl of PrestoBlue a blue resazurin-based solution, to our cell lines in the
24 well-plates containing 500 µl culture medium. Then we left the cells for one hour
to incubate and develop colour. After that we measure the cell viability with the GloMax. The change of the colour from blue to pink indicated an increased amount of
living cells. We got values for the colour intensity and when we subtract the numbers
for the colour of the medium without cells (blank), we received the realative colour.
Now we only had to set the values of the concentrations 1-3 in relation to our control
cells.
3.2 Immunohistochemical analysis
Immunohistochemistry visualises different cellular components. Firstly an added primary antibody (rabbit anti-βIII tubulin for the C17.2 cells and rabbit anti-GαT2 for the
661W cells) binds to specific elements in the cell. After incubating the cells for 24
hours and washing the substance, the second antibody (Goat anti-rabbit Alexa 488
for both cells), which is fluorescent, is added to the cells. This antibody will now bind
to the first antibody. By adding a drop of DAPI, a blue counterstain for nuclei, we
were later able to identify the cells and to depict the individual components under the
microscope.
4 Results
To analyse our experiment, we used two different and independent types of methods.
4.1 PrestoBlue analysis
4.1.1 661W
As the diagram 1 shows, the cells treated with Ginseng concentration 3 and H2O2
survived around 20% more than the control cells treated with H2O2 only. The Ginseng
concentrations 2 and 3 can help to increase the cell division under physiological conditions.
2
cell viability (% rel. to untreated Control)
661W PrestoBlue
120
100
80
without
H2O2
60
with H2O2
40
20
0
Control
Conc.1
Conc.2
Conc.3
Ginseng Concentration
Diagram 1: Percentage of viable retinal cells compared to the untreated control.
cell viability (% rel. to untreated Control)
4.1.2 C17.2
As the diagram 2 shows, the cells treated with concentration 1 and 3 and H 2O2 survived better than the control cells treated with H2O2 only. The increase is about 1215%. The numbers of concentration 2 are probably inaccurate which happened while
we pipetted.
C17.2 PrestoBlue
120
100
80
without
H2O2
60
40
with H2O2
20
0
Control
Conc.1
Conc.2
Conc.3
Ginseng Concentration
Diagram 2: Percentage of viable neuronal cells compared to the untreated control.
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4.2 Immunohistochemical analysis
4.2.1 661W
With the help of the immunohistochemical analysis we could depict the high mortality
rate of the cells treated with H2O2 (Figure 3). When the cells are treated with Ginseng, more cells are still alive (Figure 4). This observation tells us that a certain concentration of Ginseng extract is protective for the cells against the H2O2-induced
damage.
Figure 3: 661W retinal cells treated
with H2O2.
Figure 4: 661W treated with H2O2
and Ginseng concentration 3.
4.2.2 C17.2
Unfortunately, we weren’t able to examine the C17.2 with the help of immunohistochemical staining, because almost no cellular components were visible.
5 Discussion
Overall our experiment was successful.
With help of the PrestoBlue analyse we found out, that Ginseng is beneficial. The
concentrations one and three have a protective effect for the cell line C17.2. Around
15% less cells die, when the cells are treated with Ginseng concentration one or
three. For the retinal cell line 661W only the Ginseng concentration three is protective. About 20% less cells died. A lower concentration can help to increase the cell
division, but does not protect from H2O2.
The immunohistochemistry displayed the number and the shape of the cells. The cell
line C17.2 did not react how we expected. This was because the neurones were not
fully differentiated. We couldn’t take pictures of these cells treated with Ginseng or
H2O2. The pictures of the cell line 661W show that the cells treated with Ginseng
concentration two and H2O2 have a higher viability rate than the control cells treated
with H2O2 only.
For the next experiment we would plan a bit more time, so we could let the cells grow
longer. We could work with more cells and make additional analysis.
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6 References
Picture of the Ginseng root
http://www.anabolicmen.com/ginseng-testosteroneand-nitric-oxide-booster/
Information 661W cell line
Expression of Cone-Photoreceptor-Specific Antigens in a Cell line derived from Retinal Tumors in
Transgenic Mice, provided by Muayyad R. AlUbaidi, 2010
Information C17.2 cell line
http://www.phe-culturecollections.org.uk
7 Acknowledgments
We want to thank Prof. Dr. Volker Enzmann, Dr. Stefano Di Santo and Prof. Dr. Hans
Rudolf Widmer for the organisation of our experiment, the hospitality and the informative talks. We really enjoyed it.
Then we want to thank Stefanie Lötscher for her enormous patience, her help and
her kindness.
Furthermore, we want to thank the Inselspital Bern (Neurochirugie and Ophthamologie) and the DKF for the hospitality and the financial support.
In the end we want to thank the organisation “Schweizer Jugend forscht” for the organisation of this special research study week.
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