Abstract

1. Ursolic Acid Concentration in Sage Extract Cytotoxicity of Sage Extract compounds to an Immortalized Colon Cancer Line • Nathan Etsitty1, Zhong Ye2, Zhiyi Qiang2, Benjamin Herman3 and Dr. Suzanne Hendrich2 • Northern Arizona University • Department of Food Science & Human Nutrition, Iowa State University, Ames, IA • Native American Outreach Program Student Mentor, Iowa StateUniversity, Ames, IA, 50010 Results The determination of UA in Sage extract by HPLC The equation for a standard curve was established by the concentration range of 1, 10, 20, 50, and 100 uM of pure ursolic acid: y = 0.0008x + 1.0919 with R2 of 0.9991. Figure 2 indicates the standard curve is linear in the concentration range from 1-100 uM which means we can calculate the concentration of UA in sage extract based on the standard curve (peak area to the concentration) in the range from 1-100uM. Were the results to have been nonlinear, this would not have been possible. Based upon the peak area at the concentration level of pure UA and the peak area of in sage extract, the concentration of UA in sage extract could be determined using the equation: Y=aX+b Y where X is the peak area, Y represents the concentration of UA, a represents the slope, and b represents rise of the solution . Figure 1 shows that UA in 10 mg/ml sage extract is at a concentration of 400 umol/L (uM). The diluted UA standard and sage extract to 3, 30, 60, 150, 300 uM with DMSO also are shown since the 100 uL of those solutions were added to 200 uL working media in 96 well plate for cytotoxicity measurement, making the final concentrations 1, 10, 20, 50, and 100 uM. Sage A (0.5 mg/mL) was selected for this study because of the higher concentration of UA (0.4 nmol/20uL=20uM) than that in B (0.2 nmol/20 uL= 10 uM). 20 uM was not enough for 100 uM in cytotoxicity measurement, so a higher concentration of sage extract (10 mg/mL) and the UA was tested by diluting a 400 uM solution to 3, 30, 60, 150, and 300 uM (Qiang, 2008). The measurement of cytotoxicity of UA and Sage extract by MTS assay The OD (optical density) average values for UA, Sage, and DMSO were seperated by the different levels of concentration of 1, 10, 20, 50, ad 100 uM. Table 1 shows what concentration levels were placed in each well. Table 2 shows the OD values along with their average for each concentration level along with the standard deviation. It was recorded that lower levels (1, 10, and 20 uM) of concentrations showed no levels of toxicity while the higher concentration levels (50 and 100 uM) were toxic to cells (P>0.05). Cytotoxicity was determined by calculating the P-value (P>0.05) which indicates no toxic levels, and a P-value (P<0.05) that show toxic levels within the compounds compared with the OD values of the control. Methods & Materials HPLC ~Materials: Sage extract (0.5 mg/mL of two sage extracts A & B (#C80202) with 75.0% of ursolic acid) was purchased from Sabinsa. Ursolic acid (M.W.=456.68 g/mol) was purchased from Sigma. A reverse phase analytical Ymcpack-ODS C18 column (250 mm* 4.6 mm * 5um) was run at room temperature using small vials (2ml) were filled with (1) acetic acid (0.1%) – mobile phase A, (2) acetonitrile – mobile phase B. ~Methods: 1. Standard protocol for preparing compounds for HPLC analysis with different concentrations levels as listed: *1uM, 10uM, 20uM, 50uM, and 100uM of ursolic acid and sage extract (0.5 mg/mL) with two replicates Each compound (UA & Sage) was loaded into small vials (2ml) with different concentration levels and analyzed by HPLC. 2. Procedure for running HPLC: UA and Sage concentrations was determined by HPLC as outlined by Hu et al. (2002). Where that study used soyasaponins, we used UA and sage extract. Results were analyzed as described by Hu et al. (2002). Results were calculated according to the following equations: Concentration of ursolic acid in sage extract (nmol/L) = Conc. (y) *R. Conc. (y) is the concentration of the target compound. This was calculated by establishing a standard curve with x of the ratio of PA (peak area) of pure ursolic acid and y of the concentration of the standard. R is the dilution times (Qiang et al., 2008). Caco-2 cell model ~Materials: Working Media made of: FBS-Fetal Bovine Serum, HEPES buffer, Beta-glutamine, and an anitbiotic was purchased from Gibco. 6 plate transwells and flask(s) (75 cm2), an incubator, a safety hood, 75% ethanol for sterilization, and Red-Evac (which is an instrument used to remove media) were employed to manage the cell culture. ~Methods: 1. Procedures for Observing and Maintaining Cells: Media was replaced every 2-3 days to maintain a good environment for growing cells. Before old media was changed and replaced with new media, percentage of cell growth was monitored using a microscope until it reached 100%. Cells utilized for this study originated from a passage of cells which utilized generations 24 to 26. These Caco-2 cells were prepared as described by Hu et al. (2004). Cytotoxicity ~Materials: 48 well plates, centrifuge tubes, and DMSO (controls) were purchased from Sigma, UA & Sage, Red-Evac, MTS, respectively. ~Methods: 1. Protocol for passage of cells into a 48 well plate: Procedures for transferring cells to a 48 well plate were reported by HU et al. (2004). In this study, UA and sage along with a control were analyzed rather than soyasaponins and the other compounds reported by Hu et al. (2004). Both studies share the same technique but measured the toxicity of different compounds. 2. Procedure for testing the 48 well plates for cytotoxicity: When the cells that is in a 48 well plate reach 50% cell growth or above, they are then treated using the cytotoxicity experiment. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was conducted with UA along with Sage Extract to measure the toxic and non-toxic concentration ranges for them. Different concentration levels (1uM, 10uM, 20uM, 50uM, 100uM) of each compound were used along with a control known as DMSO (Dimethyl Sulfoxide). Each compound was placed in a 48 well plate for each concentration of compounds and then placed in the presence of metabolically active cells. Then 200 uL solution in each 48 well was transferred to 96 well plate measured by DU Series 700 UV/Vis Scanning Spectrophotometer (Beckmancoulter, USA) to test the OD (optical density value) (Ye, 2008). The average and Standard Devaition (SD) were calculated. Abstract Ursolic acid (UA, also known as pentacyclic triterpene) is a compound that comes from the extracts of a plant called sage (salvia officinalis). UA compound has been tested in many studies and has proven to be effective for anti-cancer and anti-tumor growth along with its use for anti-inflammatory purposes. 20 umol/L of UA was found within 0.5 mg/mL sage extract (0.5mg/mL) by HPLC (High Performance/Pressure Liquid Chromatography). The Caco-2 cell model is used to simulate the human intestine and its functions, allowing observations and measurements of cytotoxicity and bioavailability of the compound and extract without the use of live subjects. Using the Caco-2 cell model, we tested various concentrations of UA and sage extract to determine cytotoxicity. At low concentrations (i.e., 1uM, 10uM, and 20uM) showed no toxic levels compared with control (P>0.05), while 50 uM and 100 uM were toxic (P<0.05). In conclusion, UA and sage extract at low concentrations are not toxic to cells, making these concentrations appropriate for use to test the bioavailability of UA and compounds in the sage extract by the Caco-2 cells. Introduction UA (ursolic acid) is a compound that can be found in many plants like sage, and has been found to be very useful for anti-cancer, anti-tumor, and anti-inflammatory purposes. Sage is an herb that is widely used and contains many polyphenols. Sage extract is an effective antioxidant but a particular constituent of the extract called ursolic acid is known to slow the process of cell growth in cancer cells. The focus of this work is to determine the concentration of UA in sage extract and to establish guidelines outlining the concentrations at which UA and sage extract are toxic to colon cancer cells using the Caco-2 cell model. This will allow researchers to begin work toward determining concentrations of UA and sage extract that might be useful for colon cancer treatments. In order to begin testing the concentrations of UA and sage extract which cause cytotoxicity, the concentrations of these compounds in solution must be determined via HPLC (High Performance Liquid Chromatography), which separates, identifies, purifies, and quantifies each compound that’s being tested (Qiang, 2008). We used the HPLC to determine the concentration of UA within sage extract. After the compounds are separated and read by the HPLC, they are ready to be analyzed for cytotoxicity to the Caco-2 cell model (Ye, 2008). For this procedure cells are created, grown, and maintained for the use in testing how various compounds affect the cells. The cells that are being used in this procedure are an immortalized colon cancer cell line which may be used rather than live subjects to model the response of colon cancer cells to various treatments. The experiment was done so the determination of UA and Sage by HPLC can be analyzed and the cytotoxicity by Caco-2 cells can be reached to further the study of absorbability within the two compounds. Within this research, the procedures of HPLC and cytotoxicity by Caco-2 cells were achieved. Compared to other studies, the cytotoxicity of different compounds used showed different reactions and some weren’t focused on the absorbability part that this project wants to achieve. The absorbability of UA and Sage in the human intestine has not been achieved, so a method known as Caco-2 cells will be used to identify its absorbability rate and reaction. Table 1. The info for each well in the 96 plate is as follows (cytotoxicity) *Note: RA- Rosmarinic acid; UA- Ursolic acid; P1- Prunella 1; P2- Prunella; Sage- Sage extract; D- DMSO Table 2. OD (optical density) values *Note: Bold numbers are the OD values of UA, Sage, and DMSO Conclusion In conclusion, we determined 20 uM of UA was present in sage extracts which allowed us to focus on concentrations around that level for conducting cytotoxcity analyses. Within the cytotoxicity, it was evident that lower levels of concentration proved to be non toxic allowing those levels of use to be recommended for further investigations. Furthermore, with the toxic levels that were identified, the absorbability levels of Caco-2 cells can be tested at these concentrations to ensure that cells remain viable throughout the testing procedures. Fig. 1 0.44 nmol of ursolic acid standard Fig. 2: The standard curve of ursolic acid Acknowledgements I would like to thank everyone who helped me successfully complete my research experiment this summer which includes the Department of Food Science & Human Nutrition, GWC, NSF, and the Agronomy Department. Most of all, I’d like to thank my family again because without their support to strive for success, this project would of never happened for me.