Abstract
Calcium (Ca2+) signal transduction pathways play important roles in the regulation of diverse biological processes in eukaryotes ranging from unicellular (e.g., yeasts) to complex multicellular (e.g., humans) organisms. Small-molecule inhibitors of Ca2+-signaling pathways in humans can be of great medical importance, as represented by the immunosuppressants FK506 and cyclosporine A. A high-throughput drug screening assay for inhibitors of Ca2+-signaling has been developed on the basis of the ability of test compounds to restore the severe growth defect of a Ca2+-sensitive zds1 null-mutant strain YNS17 of Saccharomyces cerevisiae in a medium containing a high concentration of calcium ions. A previous screening of Thai medicinal plants using this yeast-based assay indicated that the crude extract of Kaempferia parviflora Wall. Ex. Baker contains a potent inhibitory activity. The aim of this study was to isolate and characterize the pure compound(s) responsible for this inhibitory activity against Ca2+-mediated cell-cycle regulation in yeast. Dichloromethane and methanol extracts of K. parviflora rhizomes were subjected to bioassay-mediated chromatographic fractionation using this yeast [YNS17 (Δzds1) strain]-based assay to screen for and select positive fractions. From the dichloromethane extract, four known flavonoid compounds with significant inhibitory bioactivity were obtained: compounds 1 (5-hydroxy-3,7-dimethoxyflavone), 2 (5-hydroxy-7-methoxyflavone), 3 (5-hydroxy-3,7,4’-trimethoxyflavone) and 4 (5,7-dimethoxyflavone). The inhibitory activity of all four compounds was dose-dependent. Compound 1 exhibited the highest activity and with no observed cytotoxic activity against the yeast. The Ca2+ induced severe growth defect, abnormal budding morphology, and G2 cell-cycle delay of the Δzds1 yeast strain were all alleviated or abrogated by 200 μM compound 1. Therefore, we conclude that 5-hydroxy-3,7-dimethoxyflavone possesses a potent inhibitory activity against the Ca2+-mediated cell-cycle regulation.
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Acknowledgments
This work was supported by the National Research University Project (FW645A) and Chulalongkorn University Century Academic Development Project to TM. The authors would like to thank Dr. Tanapat Palaga for use of the flow cytometer facility, which was purchased under the Thai Government Stimulus Package 2 (TKK2555) under the Project for Establishment of a Comprehensive Center for Innovative Food, Health Products and Agriculture, and Ms. Naunpun Sangphech for technical assistance on flow cytometric analysis.
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Boonkerd, S., Yompakdee, C., Miyakawa, T. et al. A flavonoid, 5-hydroxy-3,7-dimethoxyflavone, from Kaempferia parviflora Wall. Ex. Baker as an inhibitor of Ca2+ signal-mediated cell-cycle regulation in yeast. Ann Microbiol 64, 1049–1054 (2014). https://doi.org/10.1007/s13213-013-0742-6
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DOI: https://doi.org/10.1007/s13213-013-0742-6