IMD 3 Summer Fellowship Report
Project Title : Assessment of Estrogen Mimic Activity in Extracts of Botanicals Collected in a Caribbean Rainforest
Student : Elizabeth S. Nicholson, University of Georgia , Athens , Georgia
Mentor : Dr. James L Wittliff, Hormone Receptor Laboratory, Department of Biochemistry & Molecular Biology
This investigation was conducted as part of my Summer Fellowship in the Hormone Receptor Laboratory. My daily supervisor was Dr. Irina Smolenkova and my research project used a sophisticated radio-ligand titration array protocol with recombinant human estrogen receptor (rhER). Briefly, this approach permits the detection of compounds in extracts and solutions which mimic female sex hormone activity.
Plants commonly used to treat “women's problems” by the Carib Indians on the island of Dominica were used in these studies. Botanicals included: Cecropia peltata ( Moraceae ), Eleutherine bulbosa ( Iriaceae ), Momordica charantia ( Cucurbitaceace ), Picrasma excelsa ( Simaroubaceae ), Stachytarpheto jamaicensis ( Verbenaceae ) and Wedelia trilobata ( Asteraceae). The stems and leaves from these plants were extracted in either absolute ethanol or a phosphate buffer, 50 mM, pH 74 containing 1.5 mM EDTA, 10 mM sodium molybdate and 10% glycerol. The radio-ligand titration array was used to test estrogen mimicry of the medicinal botanicals in the protocol shown below and representative results are shown in the diagrams. Interestingly, the plant extracts exhibited estrogen mimicry either by enhancing or inhibiting estrogen receptor binding. These preliminary results suggest that the molecular basis of the medicinal use of certain of these plants is due to the presence of estrogen-like compounds.
Estrogens and their mimics are involved in female development, maintenance of bone density, prevention of cardiovascular diseases, cancer treatment and various other health related events. An estrogen mimic is a compound that competes with naturally occurring estrogens such as estradiol-17 b for the ligand-binding site on the estrogen receptor protein.
A radio-ligand titration assay was performed using estradiol and ethanol as controls. Estradiol and the candidate compound competed against each other for the estrogen receptor and results were calculated using the K d values and the IC 50 values. In this assay, each receptor preparation was titrated with 6 increasing concentrations of radiolabeled [ 3 H]estradiol-17ß (e.g., 0.1, 0.2, 0.4, 0.7, 1.2, 2.4 nM), or increasing ratios of plant extract to estradiol in the absence and presence of a 200-fold excess of DES. Following overnight incubation at 4 ° C, unbound ligand was removed with dextran-coated charcoal, and the amount of receptor-bound ligand was determined. Specific binding capacity, B max , expressed as fmol/mg of extract protein, and binding affinity, K d values, expressed in M, were determined with OneSite® software (Chagrin Falls, OH) or Graphpad Prism® (San Diego, CA) from the results of the ligand binding isotherms using transformation into a Scatchard plot. This program gave a summary table of the results, binding isotherm, Scatchard plot and Klotz plot for each receptor-ligand complex.
Dr. Andrei Smolenkov, Investigator in the Hormone Receptor Laboratory, prepared the yeast expression system with the recombinant ER a . It was determined that several botanical extracts exhibited estrogen mimicry by binding to both estrogen receptor a and b isoforms, using the radio-labeled titration array, which detects inhibition of radio-labeled estradiol- ab
binding.
I appreciate the support in part by a grant from Phi Beta Psi Research Foundation and a Summer Fellowship from the Institute for Molecular Diversity & Drug Design.
Figure 1, Representative Competition Results with the Reference Compound (Estrone): [ 3 H]ESTRADIOL-17b Binding Graphed as Remaining SBC (OneSite TM ) w/ rhER a
Figure 2, Competition of Botanical Extracts Using [ 3 H]Estradiol-17 b
Binding Graphed as Remaining SBC (OneSite TM ) w/ rhER a
