Larry C. Ford and Drs. Noel L. Owen, Chemistry and Biochemistry and Steven G. Wood, Chemistry and Biochemistry
With antimicrobial resistance on the rise, research is turning more and more to natural sources for medicinal products (the original source, ironically, for most known antimicrobials). Under the aegis of the Natural Product Center here on campus, I had contact with the daughter of a shaman from the Yaqui Indian Tribe. The Yaqui people use Euphorbia polycarpa, commonly known as sandspurge, to treat snakebites, scorpion stings, and yeast infections. The latter ethnobotanical usage being the most testable, I chose to pursue the bioactive compounds in sandspurge.
The plant samples were macerated and subjected to a methylene chloride/methanol extraction. The resultant product was rotovaped to dryness and extracted based on solvent polarity. Methanol (polar) and hexane (nonpolar) were chosen for their relative polarities and their extracts tested against Candida albicans (the primary causative agent of yeast infections) in a number of standardized bioassays. These bioassays included diffusion assays measuring ratezonal inhibition as well as a more sophisticated liquid suspension assay. The tests yielded no antifungal activity, even with repeated trials. The extracts were then tested against Staphylococcus aureus (Gram positive) and Pseudomonas aeruginosa (Gram negative), bacteria commonly used in antimicrobial assays. While there was no activity against the representative Gram negative organism, there was reasonable antibacterial activity with Staphylococcus. Further assays were also run with HeLa cells, testing extracts against this extensively studied cervical cancer cell line. Again, activity was found. In both the Staph and HeLa tests, the activity was located entirely in the nonpolar hexane extracts. To further separate to better isolate specific bioactive compounds, I used silica gel column chromatography on the samples; hexane and an increasing polarity gradient of acetone separated the sample into a large number of fractions (~160). Thin liquid chromatography (TLC) allowed similar fractions to be combined. These pooled fractions were again run in Staph and HeLa cell bioassays. One mildly polar fraction of the original hexane extract (10% acetone/90% hexane) expressed much activity against Staph, and TLC analysis narrowed this activity to approximately 6 to 8 compounds. Unfortunately, the data does not appear reproducible. Extensive studies will not be done on this fraction until further efforts confirm or reject this antibacterial activity as simply an anomaly.
The cancer assay, on the other hand, produced exciting data. A number of fractions expressed notable anticancer activity. One fraction in particular demonstrated significant HeLa cell death. This sample, on the very polar end of the nonpolar extract, was selected for further study. TLC of this fraction indicated that a fair number of compounds were involved, but was unable to provide any more specific data. Therefore, High Performance Liquid Chromatography (HPLC) was employed to further separate and indicate the makeup of these active materials. HPLC data enabled us to restrict the active compounds to a narrow range in the chosen fraction. We have successfully identified the most likely region for this anticancer activity!
Further work will isolate these particular compounds (whose approximate identity has been determined) and focus on structural elucidation.