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The objectives of the study include optimizing acetogenin extraction and identifying acetogenin-rich fractions, determining of the level of acetogenin bioavailability by using in vitro systems, and examining a possibility of neuroprotection by pawpaw antioxidants.
Non-Technical Summary: Acetogenins a group of compounds, exhibiting a wide range of bioactivities including anticancer property. However, the low content found in plant materials may be hampering the wider availability and therefore the use in clinical research. In addition, acetogenins may be bioavailable, the level of uptake or metabolism is unknown. While acetogenins are toxic to various cell types, antioxidant components in pawpaw fruit may reduce the toxicity to nerve cells, thereby giving neuroprotection. The purpose of the study is to optimize acetogenin extraction, to identify bioactive fractions, to determine the level of uptake and metabolism of acetogenins by cells with the use of cell culture based assays, and to investigate the effect of pawpaw antioxidant components, in relation to changes in cell antioxidant levels and apoptosis caused by acetogenins. For the Objective 1, extraction of acetogenins will be examined with different pawpaw plant parts including the stem bark, fruit pulp, peels and seeds. Combinations of different organic solvents will be used to optimize the extraction efficiency. Extracts will be fractioned and will be tested for bioactivity with cultures of human cell lines such as A-549 (human lung carcinoma), MCF-7 (human breast carcinoma), and HT-29 (human colon adenocarcinoma). For the Objective 2, pawpaw pulp will be subjected to in vitro digestion. Caco 2 cells will be exposed to digested pawpaw pulp to investigate uptake and metabolism of acetogenins. For the Objective 3, cultures of PC12, a dopaminergic rat pheochromocytoma cell line, will be subjected to pawpaw extracts at different concentrations. Interaction between proapoptic acetogenins and other bioactive pawpaw components will be monitored with assays including thiobarbituric acid reactive substances (TBARS) assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) test, flow cytometric analysis and Western blotting in regard to cell viability, antioxidant level, and apoptosis.