Kurtis S. Staples and Dr. Steven G. Wood, Chemistry and Biochemistry
Carica papaya, commonly known as the papaya plant, is found in large abundance throughout the tropical regions of the planet. The fruit of the plant has been extensively studied with papain, a protease, being the center of research. Preliminary studies showed that compounds found in the leaves of the plant possessed anti-cancer properties. As alternatives for cancer treatment are constantly being sought, plant extracts have risen to the forefront in this hunt.1 An understanding of the structure and mode of activity of the compounds in this plant will further knowledge of how to develop chemotherapeutic agents.
Dried, mulled leaf sample was obtained from the University of Cape Coast in Ghana. This plant material was subjected to complete extraction using solvents covering a range of polarity. The extract thus obtained was then separated further via silica gel column chromatography. The fractions obtained from the column were subjected to a cytotoxicity bioassay, using human cancer cells,2 which identified the fractions containing the bioactive compounds. The active fractions were further purified using analytical and semi-preparative High Performance Liquid Chromatography (HPLC) (Fig. 1&2). Four of the individual compounds obtained from the HPLC were responsible for the bioactivity. These compounds exhibited potent cytotoxic activity with an LC50 (concentration resulting in 50% lethality) in the range of 25 μg/mL.
From these isolation studies, only 1-2 mg of each compound was obtained. It is necessary to have a minimum of 5 mg of pure sample to be able to obtain basic Nuclear Magnetic Resonance (NMR) data. Mass spectrometry (MS) experiments, however, were obtained and the most active of the four compounds was found to have a formula weight of 437. As this corresponds to a rather large molecule, it becomes necessary to obtain even more than 5 mg of the pure sample for NMR experiments. The MS experiment also showed the compound to have two carboxylate groups.
The original plant sample was exhausted in the first extraction. A letter was then sent to Ghana requesting more plant material. 5 lbs of plant leaves were received only a short time ago (August 10th). The fresh sample is currently being extracted and purified in an effort to perform the structural elucidation of the bioactive compounds. While waiting for the new sample to arrive, other sources were used in the search for these bioactive compounds. Plant samples from Western Samoa and Southern Florida were obtained, but these showed little if any of the desired bioactivity.
In an effort to better understand the mode of activity of these compounds, a phase II enzyme induction bioassay was used to detect compounds which might prevent mutagenesis.3 In this bioassay, the extracts were applied to a rat cancer cell line and compared to the known phase II enzyme inducing compound, sulforaphane. Sulforaphane is found in high concentrations in broccoli and has been determined to help in the prevention of cancer by converting cancer causing agents into metabolic waste products for elimination from the body. This bioassay returned results showing that semi-pure fractions of the extracts of Carica papaya were active in the range of nanograms per milliliter, comparable to sulforaphane. Further testing is being performed at this very time to determine the bioactivity of the pure compounds.
In addition to the laboratory research which has been performed, several presentations of this work were delivered at research conferences. In March, a poster was displayed as a part of the Cancer awareness week. In April of this year, a presentation was given at the BYU Spring Research Conference. First prize was awarded for the presentation. Another presentation was given in Moscow Idaho at the American Chemical Society—Northwest Regional Meeting. Each of these experiences was very beneficial towards the progress of the research project.
References
- M.K. Parmar et al., Chemotherapy for ovarian cancer, The New England Journal of Medicine (1996) 1268-70.
- F. Denizon and R. Lang, Rapid calorimetric assayfor cell growth and survivalModifications to the tetrazolium dye procedure giving improved sensitivity and reliability, Journal of Immunological Methods (1985) 271-7.
- The aid of Dr. Byron Murray and Ken Carpenter of Brigham Young University is gratefully acknowledged.
Figures
Figure 1 – HPLC chromatogram of semipure ethyl acetate extract.
Figure 2 – Table of relative bioactivities of semi-pure ethyl acetate extract.