1. Isolating and Identifying Novel Phytoestrogens from Gaultheria Shallon 1 Stephanie Fryman, 1 Cameron Davis, 1 Dr. Paige Baugher, 2 Dr. John Harrelson 1 Department of Biology, 2 Department of Pharmacy | Pacific University | 2043 College Way | Forest Grove, OR 97116 | paige.baugher@pacificu.edu Abstract: Phytoestrogens are secondary plant metabolites produced by plants that are similar in structure estrogen. Human consumption of these compounds has been associated with many health benefits. This projects aims to investigate novel plant sources of phytoestrogens within the Pacific Northwest. In this study, we investigated the potential phytoestrogen content of Gaultheria shallon (salal), a plant native to the western coastal regions of the United States. Data from our lab show that estrogen receptor positive cells proliferate in response to treatment with salal extract. We also found that this increase in proliferation can be blocked by inhibition of the estrogen receptor. Taken together, these data suggest that salal contains one or more compounds that activate the estrogen receptor. To begin to isolate these compounds, we used high performance liquid chromatography (HPLC). Data from our lab show that there are at least 19 different possible isolated compounds that may be acting as phytoestrogens. In the future, we propose to test each of these compounds individually for estrogenic activity. Background: Plants play a vital role in life to all organisms on Earth. With around 300,000 known species, plants can serve many different roles as well as fighting diseases. Phytoestrogens are estrogen-like compounds found in some foods and plants (6). They are found in high amounts in soybeans, whole wheat, tofu and many other beans. Phytoestrogens can act like estrogens by interacting with the human estrogen receptor and mimicking estrogen activity (1,2). A phytoestrogen can look structurally very similar to an estrogen molecule or quite different, but it will still follow this same chemical pathway as the estrogen (6). Estrogen is a compound that plays a vital role in the female body of humans and other animals. Estrogen has many functions in the body including the development of breasts, the thickening of endometrium, and regulating the menstrual cycle (3). Menopause occurs when a woman naturally stops producing eggs and the body produces less estrogen (7). Menstruation becomes less frequent and eventually stops. However, it is important for females to continue to receive estrogen even after menopause in order to continue their daily lives comfortably and avoid medical complications. Instead of taking estrogen, which can increase the risk of breast cancer, phytoestrogens can provide a safer alternative to receive estrogen (6). Phytoestrogens can increase proliferation in estrogen receptor expressing cells and can modulate estrogen receptor signaling. In this project, we are studying phytoestrogens in the plant Gaultheria shallon or salal. Salal is an evergreen shrub that is native to the western coastal regions of the United States (5). This plant produces a berry and is used as a food source by several indigenous mammalian herbivores (5). Previous research gave us the idea that phytoestrogens exist in Salal. The Nitinaht Native Americans of British Columbia ate the leaves of Salal the night before their wedding in order to increase fertility, suggesting that there is estrogenic activity in these leaves (4). The objective of our research is to continue to test Salal extracts to look for phytoestrogen activity and to isolate different compounds in the plant in order to find the compound that may act as a phytoestrogen. Results and Conclusions : Data from our lab show that MCF-7 breast cancer cells over expressing the estrogen receptor proliferate in response to treatment with crude salal extract. We also show that after blocking the estrogen receptor with ICI, there was no significant difference in proliferation with varying amounts of salal. Finally, we show that treatment with salal extract can significantly increase ERE-mediated transcription. Taken together, these data suggest that crude salal extract contains one or more compounds with phytoestrogenic activity. To begin isolation of these compounds, we used HPLC. 19 different compounds were isolated from crude extract using HPLC at 210nm and 254nm wavelengths. The 254nm shows possible conjugation of molecules at a given retention time, which would most likely indicate the phytoestrogens within salal. If isolation of the different components of salal is complete then, we should be able to test each isolated component to identify what is mimicking estrogen and whether the compound is working alone or with another possible compound. The E-SCREEN and ERE-luciferase assays should elucidate the possible novel phytoestrogens which could then be characterized using mass-spectrometry. Acknowledgements: Murdock Life Sciences Grant PRISM Dr. John Hayes Dr. Paige Baugher Dr. John Harrelson Dr. Staci Halpern Dr. David Cordes Dr. Jeanine Chan Heather Ritenour Reya Lii’lii References: 1.Duffy, C., Perez, A. Partridge. 2007. Implications of phytoestrogen intake for breast cancer. CA J Clin 57, 260-277 2.Mense, SM., T.K. Hei, R.K Ganju, H.K. Bhat. 2008. Phytoestrogens and breast cancer prevention possible mechanisms of action. Environmental Health Perspectives. 116, 426-433 3.Nelson, Lawrence. "Menstruation and the menstrual cycle fact sheet." Women's Health. National Institutes of Health, 21 Oct 2009. Web. 8 Nov 2011.. 4.Turner, N.J., et al., Ethno botany of the Nitinaht Indians of Vancouver Island. 1983, Victoria,British Columbia: British Columbia Provincial Museum. 5.United States. USDA. Plants Profile for Gaultheria shallon. Web.. 6.Warren, Barbour, and Carol Devine. "Phytoestrogens and Breast Cancer." Breast Cancer and Environmental Risk Factors. Cornell University, 07 2001. Web. 8 Nov 2011.. 7.Zieve, David, and Susan Stork. "Menopause." Pub Med Health. National Institutes of Health, 11 09 2010. Web. 8 Nov 2011.. V 0.01 V 0.01 0.1 1 10 100 nM E2 ug/ml salal EtOAc extract Relative Light Units * + + Crude Extraction of salal Component Isolation HPLC Estrogenic activity assay E-SCREEN ERE-Luciferase Compound Identification LC-MS NMR Estrogenic activity assay E-SCREEN ERE-Luciferase Figure1. Fold increase of MCF-7 breast cancer cell proliferation when treated with Gaultheria shallon extract using ethyl acetate, dichloromethane and hexane. Cells were incubated for 48 hours in concentration ranging from 0.1 µg/mL to 1000 µg/mL with a control of 0 µg/mL. Data were analyzed using wilcoxon Rank Sum (*indicates p<0.05, n=16). Error bars represent +/- SEM. Figure 6. The methodology of research for the investigation of novel phytoestrogens in Gaultheria shallon. Current research is focused on the first round of ERE- Luciferase Assay and Component isolation. Figure 4. Structures of 17ß-estradiol and representative examples of four commonly recognized families of phytoestrogens. Figure 2. Estrogen Receptor antagonist decreases proliferation response to crude EtOac extract. MCF-7 cells were plated with 5% hormone and were treated with either the vehicle or increasing concentrations of the EtOAc extract. Some were then given 1 µM ICI. After 5 days cells, were fixed and read at 595nm absorbance. (*) indicates p<0.5 and indicate significance relative to the EtOAc extract alone. (+)represent p<0.05 and indicate significance relative to the vehicle. Error bars represent +/- SEM. Figure 3. ERE-Luciferase Assay. Relative light units of T47D-Bluc breast cancer cells when treated with varying concentrations EtOAc extract of salal and estrogen. Relative light units measured on luminometer, after transcription occurs within the cells.(*) indicates P<0.05. Error bars represents +/- SEM. Figure 5. The retention time of different components of Gaultheria shallon EtOAc extract, shown at 254nm and 210nm wavelengths. Each peak is thought to represent a different possible phytoestrogen. Each peak at 254nm was isolated due to the fact that they are most likely conjugated compounds similar in structure to estrogen. Other components were separated based on the 210nm wavelength and possible association with the compounds of note at 254nm.