Analysis of Active Compounds Using Target Protein Cofilin?Cucurbitacins in Cytotoxic Plant Bryonia cretica

We examined a two-step target protein binding strategy that uses cofilin as the target protein to analyze the active constituents in Bryonia cretica. In the first step, we prepared the target protein, and used it to analyze the compounds binding to it in the second step. We used the methanolic extract of B. cretica as a library of possible active compounds. We conducted LC-MS analysis using information from our previous study. The peaks in the HPLC profile were identified as cucurbitacin D, isocucurbitacin D, and cucurbitacin I. As far as we know, there is no known study of the activity of isocucurbitacin D in this research field. Therefore, we examined the effects of isocucurbitacin D on cell proliferation and cofilin protein in human fibrosarcoma cell line HT1080 to confirm the effectiveness of this strategy. The cytotoxicity assay, the fibrous/globular actin ratio assay, and the immunoblotting analysis revealed that isocucurbitacin D showed a cytotoxic effect with disruption of target protein cofilin. The target protein binding strategy is a direct and straightforward method for finding new drug seeds from crude sources, such as natural plant extracts.