RESEARCH DAY POSTER

Report
Effect of Aqueous and Methanol Extracts of Tradescantia zebrina
and fluminensis on Human Cells
Ashley Brauner, Michael Pickart, Ph,D., University of Wisconsin-Stout
Collaborating Students: Benjamin Leist, Danielle Moehring, Calli Walsh, Samantha Langsford, and Lexi Blise
Tradescantia zebrina and Tradescantia fluminensis have
medicinal properties. Extracts of these plants may have
anticancer characteristics. Assays were preformed that measured
doubling time and clonogenic survival of SCC-13y (squamous
cell carcinoma), HFF-1 (human foreskin fibroblasts), and A549
(lung adenocarcinoma) cells. Proliferation inhibition was
determined via growth curve analysis. Compared to the negative
control of sterile water, cancer cell proliferation was decreased
with the addition of T. zebrina treatments. This study confirms the
general inhibitory effects of T. zebrina and T. fluminensis extracts
on cancerous and non-cancerous cells, allowing further research
to be conducted involving different cell lines and methods of
extraction.
T. zebrina on HFF-1 Cells
T. zebrina on HFF-1
600000
700000
Negative Control
600000
B
500000
1:40 Tz Extract
500000
Total Cell Count
A
400000
Total Cell Count
400000
Expon. (Negative Control)
y = -7500x + 71500
300000
Linear (1:40 Tz Extract)
200000
300000
0 Hours
72 Hours
200000
y = 24119e1.0817x
100000
100000
0
24
48
72
0
Negative Control
Time (Hours)
C
T. zebrina on SCC-13y Cells
1:40 Tz Extract
T. zebrina on SCC-13y Cells
2,500,000
D
2,500,000
Negative Control
Total Cell Count
2,000,000
2,000,000
1:40 Tz Decoction
1,500,000
1,000,000
Expon. (Negative Control)
Total Cell Count
Abstract Summary
Preliminary Investigation:
Inhibition of Proliferation
Extract Treatment Procedure
1,500,000
0 Hours
94 Hours
1,000,000
y = 132445e0.7075x
500,000
Linear (1:40 Tz Decoction)
500,000
y = -33500x + 292500
0
0
48
72
0
94
T. fluminensis on SCC-13y Cells
1:40 Tz Decoction
E
Negative Control
T. fluminensis on SCC-13y Cells
1,600,000
F
1,400,000
1,400,000
Cell Count (1:40 Tf Decoction)
1,200,000
Figure 1. A) T. fluminensis plant
B) T. zebrina plant
Extraction Methods
Table 1. Comparison between methanol and aqueous extraction methods,
along with challenges faced during development and the solutions
implemented to solve the problems.
Benefits
Aqueous Extraction
Methanol Extraction
Simple Procedure
Clear Extract
Sterile
Definite Concentration
Preserves Molecules
Challenges
Indefinite Concentrations
Solutions
Clonogenic Survival Methods
1,200,000
Negative Control
1,000,000
1,000,000
Linear (Cell Count (1:40 Tf
Decoction))
800,000
y = 35783e0.0255x
600,000
Expon. (Negative Control)
400,000
y = 783.85x + 88875
Total Cell Count
Total Cell Count
Figure 2 (above). Flowchart of treatment procedure. Aqueous extracts of Tradescantia zebrina and
Tradescantia fluminensis were diluted and applied to SCC-13y cells. Future experiments allow for
the use of different concentrations of T. fluminensis and T. zebrina methanol extract applied to
different cell lines. Figure 3 (below). Clonogenic survival assays were performed using confluent
SCC-13y plates of a 2.5% aqueous treatment and a control of sterile water. Each confluent plate
was trypsinized and counted using a hemacytometer. Cells were seeded in triplicate for both the
experimental and control groups at 100 and 200 cell densities in p60 dishes in DMEM media
supplemented in 10% FBS and 1% P/S. Cells were allowed to colonize for one week and were
then fixed with 4% paraformaldehyde and treated with crystal violet stain. Stained cells were then
counted to determine clonogenic survival rate.
800,000
0 Hours
144 Hours
600,000
400,000
200,000
200,000
0
0
0
50
100
150
200
1:40 Tf Extract
Negative Control
Time (Hours)
Figure 4. A) Growth curve analysis of a 2.5% T. zebrina aqueous extract. ~62,500
HFF1 cells were seeded, treated with T. zebrina aqueous extract, and counted 24 and
48 hours post-seeding. B) Comparative analysis of 2.5% T. zebrina extracts on HFF1
cells as compared to the negative control of sterile water. C) Growth curve analysis of
2.5% T. zebrina extracts on SCC-13y cells. ~280,000 SCC-13y cells were plated in
p60 dishes and treated with 2.5% T. zebrina aqueous extracts using equal
concentrations of sterile water as a negative control. Cells were harvested and
counted at 24, 72, and 96 hours post-seeding D) Comparative analysis of 2.5% T.
zebrina extracts with the negative control of sterile water on SCC-13y cells 0 hours
post-seeding and 94 hours post-seeding. E) Growth curve analysis of 2.5% T.
fluminensis extracts on SCC-13y cells. ~31,250 SCC-13y cells were plated and
treated with 2.5% T. fluminensis aqueous extract using sterile water as a negative
control. Control and treatment plates were harvested and counted with a
hemacytometer after 48, 96, and 144 hours post-seeding in order to generate a
growth curve F) Comparative analysis of 2.5% T. fluminensis extracts with the
negative control of sterile water 0 hrs post-seeding and 144 hours post-seeding. Error
bars are not shown for all graphs due to a lack of triplicate testing.
Future Goals and Research
Complex Procedure
Murky Extract
Non-sterile
More Concentrated Extracts
Used Improved Methanol
Used 0.22-µm Filter
Extraction Method
Sterilizer
Used Centrifuge Evaporator
1. Clonogenic Survival Assay – to determine the survival
rate of cells after the removal of T. zebrina and
fluminensis extract treatment.
2. Zebrafish Toxicity Treatment – to determine if the
antiproliferative properties of the extract are due to
cytostatic activity within the cell or due to toxicity of the
extract.
3. Flow cytometry readings using fluorescence-activated
cell sorter of cells treated with T. zebrina or T.
fluminensis extract would generate a cell cycle index
that could reveal cytostatic activity within the cell.

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