Transcript Urushiol oil extraction from Toxicodendron radicans (Poison Ivy)

Urushiol oil extraction from Toxicodendron radicans (Poison Ivy) and its
effect on cell viability in mouse melanoma B16-F10 cancer cells.
Zachary Speights* and Dr. Jeffrey Thompson, Department of Biology, York College of Pennsylvania
Poison Ivy
http://www.medicinenet.com/poison_ivy_oak_and_sumac/article.htm
Filtration of Poison
Ivy leaves
Introduction
Methods
Skin cancer is the most common form of cancer in the
United States, where each year there are more newly
diagnosed cases of skin cancer than breast, prostrate,
lung, and colon cancer combined. Melanoma is the least
common form of skin cancer, accounting for only 1% of
skin cancer cases, but has the highest mortality rate.
(American Cancer Society).
Urushiol oil extraction
Melanoma treatment almost always requires surgical
removal therefore surgical scarring and medical bills are
troublesome. Catching it early is important to avoid the
cancer from metastasizing but developing a less invasive
treatment has yet to be created.
Poison Ivy is a plant native to the United States that
contains urushiol oil, the substance that causes the
notoriously itchy rash. Previous research has shown this
oil to have effective antitumor/anticancer properties in
some other cell lines. (Kim et al. 2013) While some
cancer lines like gastric cancer have been explored using
uruhsiol oil as a treatment, little has been shown on the
effect of this oil and melanoma skin cancer.
Therefore, it was hypothesized that urushiol oil would
induce cell death upon increasing concentrations of
urushiol oil, ultimately causing cell death in a skin cancer
using a skin poison.
Collected 15 grams
of Poison Ivy leaves
Cell Treatment
Collect and prepare
poison ivy for urushiol
oil extraction
Conclusions
Tissue culture B16-F10
cells
Soaked in 100 mL
of ethanol for 7
days in cold room
under Nitrogen
Prepare serial dilutions
0-3% (vol/vol)
Control : ethanol
Filtered through
cheese cloth
Qualitative results:
Microscope
Treat cells with
extract-24 hours
Rotary evaporated
off ethanol
Quantitative
results:
MTS/PMS assay
Figures/Statistics
When visualized under the microscope at only 4x and 10x
magnification, morphological changes occurred to the
cells that indicated cell death. Cells began to shrivel and
form small spheres. The solution was also cloudy with
debris which may be an indication of internal cell
components from cell lysis.
Ethanol treated cells showed no significant change to the
treatment, and cells appeared to be healthy and living,
which confirms that ethanol was not affecting cell death
(Figure 1A).
Washed with 10 mL
of ethanol
Cell death was most visually observable at a 3% dosage
of urushiol oil, where there was little evidence of living
cells in the culture, however data shows cell death began
to occur in notably effective amounts at a 2% dosage
(Figure 1B,2; Cha 2012).
Results
Urushiol oil induced cell death upon treating B16-F10
melanoma cells with increasing concentrations of
urushiol oil.
96 well plate setup
A
B
Objectives
Future Studies
(1): Isolate an urushiol oil extract from
common poison ivy.
(2): Investigate cell viability in a mouse
melanoma cell line B16-F10 qualitatively
and quantitatively after treatment with
increasing concentrations of an urushiol
oil extract.
40X Magnification: Ethanol
4X Magnification: Treatment
Figure 1: Microphotographs of B16-F10 mouse melanoma cells treated with (A) 3.0% ethanol or (B) 3.0%
urushiol oil. Cells were treated for 24 hours and examined for cell viability under the microscope.
Literature Cited
American Academy of Dermatology. Available from:
https://www.aad.org/media/stats/conditions/skin-. Accessed 2016 September 24.
American Cancer society. Available from:
http://www.cancer.org/acs/groups/content/@research/documents/document/acspc047079.pdf. Accessed 2016 September 23.
B16-F10 (ATTC-CRL 6475) Product sheet. ATTC. Accessed September 24,2016.
Bandarchi, B., Ma, L., Navab, R., Seth, A., and Rasty, G. (2010). From melanocyte to metastatic
malignant melanoma. Dermatology Research and Practice, 1-8.
CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay. (2012). Madison, WI:
Promega Corporation.
Cha, J.D. and Kim, J.Y. (2012). Essential oil from Cryptomeria japonica induces apoptosis in human oral
epidermoid carcinoma cells via mitochondrial stress and activation of caspases. Molecules, 17(4), 3890-3901.
Nobili, S., Lippi, D., Witort, E., Donnini, M., Bausi, L., Mini, E., and Capaccioli, S. (2009).
Natural compounds for cancer treatment and prevention. Pharmacological Research, 59(6)
365-378.
Overwijk, W. W., and Restifo, N. P. (2001). B16 as a mouse model for human melanoma.
Current Protocols in Immunology. 20(1), 1-4.
Skin Cancer Foundation. Available from: http://www.skincancer.org/skin cancerinformation/melanoma/melanomatreatments. Accessed 2016 September 26.
Worley, S. L., Vaughn, B. J., Terry, A. I., Gardiner, C. S., and Dekrey, G. K. (2015). Time- and
dose-dependent effects of ethanol on mouse embryonic stem cells. Reproductive
Toxicology, 57, 157-164.
Yang, C. F., Li, W., Elliot, R., and Iltchencho, N. (2014). U.S. Patent No. WO2014081988 A1.
Washington, DC: U.S. Patent and Trademark Office.
Figure 2: Mean absorbance values of mouse melanoma B16-F10 cancer cells treated
with urushiol oil or ethanol at different concentrations (n=2). Absorbances represent
cell viability after 24 hours in solution. The best fit line, the equation for the line, and
the correlation coefficient are from linear regression analysis. The best fit curve and
correlation coefficient are from a quadratic, polynomial analysis.
Rotary Evaporation
-Compare urushiol oil with another plant of the same family
-Prepare dilutions and monitor over a time period
-Compare to a non cancerous cell line
-Determine mechanism of cell death
Figure 3: Mean absorbance values of Mouse melanoma B16F10 cancer cells treated with urushiol oil and ethanol at
different concentrations (n=2). Absorbances represent cell
viability after 24 hours in solution. Means of urushiol oil
absorbances are significantly different than ethanol up to 2%
concentrations. Data was analyzed suing a two-way ANOVA.
Acknowledgements
I would like to thank Dr. Jeffrey Thompson for supporting my research throughout
the duration of my project and for being a great role model. I would also like to
thank him for his patience and guidance to fulfil my vision for the project.