Document Type

Thesis

College

College of Pharmacy and Health Sciences

Department

Pharmaceutical and Administrative Sciences

Degree

MS in Pharmaceutical Sciences

Date Completed

Spring 2024

First Committee Member

Gilzad Kohan, Hamed

Second Committee Member

Kinney, Shannon

Abstract

Objective: To evaluate the toxicological effects of a phytoecdysteroid and their mammalian metabolites through in vitro experiments using the MCF-7 cell line. Turkesterone levels of 11 commercially available turkesterone supplements were measured using High Pressure Liquid Chromatography (HPLC). Methods: The phytoecdysteroid 20-hydroxyecdysone (20HE) underwent two chemical reactions to synthesize two mammalian metabolites; 14-deoxy-20-hydroxyecdysone (14DO) and poststerone. These reactions were accomplished by zinc-mediated reduction and Jones oxidation, respectively. The structure of the products was confirmed via nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS). The turkesterone supplements were investigated using a HPLC on a Kinetex 2.6 μm C18 column, and a Hitachi L-2000 chromatograph. The associated Hitachi software was used to control and process the results of the chromatograph. Ultraviolet (UV) detection was used to determine the presence and quantity of turkesterone. Toxicological analysis was performed by utilizing the sulforhodamine-B (SRB) assay to determine relative changes in cellular proliferation. Quantitative polymerase chain reaction (qPCR) was also employed to determine the changes in expression of estrogen response element-positive genes (BRCA1, BCL2, and CHAT). Results: Analysis of the NMR and LC-MS spectra confirmed that the synthesis of 14DO and poststerone was successful. After evaluating the peaks of the turkesterone standard samples, a calibration curve was built. The brand Vinatura has been shown to have the highest concentration (110 μg/mL) of turkesterone. No significant changes in cell proliferation were seen with any of the drug treatments. The qPCR results suggest that the metabolites of 20HE and the supplement can significantly decrease BRCA1 expression in MCF-7 cells as compared to 20HE. 20HE, poststerone, and the Vinatura supplement were able to inhibit BCL2 expression to the same capacity of estradiol, while 14DO was significantly more effective at inhibiting BCL2 expression compared to estradiol. Conclusions: These results can potentially help to determine proper safety parameters for people that use phytoecdysteroid supplements who either have or are at risk for breast cancer.

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