Search for bioactive components from Ephedra species against adult T-cell leukemia using electron ionization MS- and NMR-based metabolomics

Metabolic profiling techniques are often used to find possible correlations between the metabolic profile of a compound and its biological activity. Electron ionization (EI)-MS and NMR are powerful tools for such analyses because they allow the simultaneous detection of diverse groups of primary and secondary metabolites. Based on a metabolomics method, we developed EI-MS for studying medicinal plants [1]. EI-MS shows fragment ion peaks more clearly than other ionization methods. These fragment data can be used like a fingerprint to identify the medicinal plant. We also previously reported that EI-MS- and NMR-based metabolomic techniques and loading plot analysis allowed the identification of bioactive components in Sophora flavescens [1, 2]. In the present study, we used EI-MS- and NMR-based metabolomic techniques to identify components from Ephedra species that showed inhibitory effects on the proliferation of adult T-cell leukemia (ATL) cells. ATL is caused by human T-cell leukemia virus type I infection, and because it is resistant to conventional chemotherapy, the identification of appropriate therapeutic agents is much needed. ATL cells were treated with acetone or methanol extracts of Ephedra species for 96h, then cell viability was analyzed by the WST-8 assay kit. Several acetone extracts of Ephedra species showed an inhibitory effect on ATL cells (IC₅₀= 8.6˜100µM). However, not all methanol extracts showed an inhibitory effect (IC₅₀ > 100µM). Principal component analysis of extracts selected from the loading plot analysis showed that each acetone extract had characteristic signals. Furthermore, there was a good correlation between some characteristic ions and a negative logarithmic IC₅₀ value on the viability of the ATL assay. These results suggest that component pattern analysis of Ephedra species is useful for finding bioactive compounds. Ephedra species could be classified based on the results of metabolite profiling with EI-MS and NMR. As a next step, we are going to collect as many plants and measurement samples possible using this methodology.

Acknowledgements: We thank Michiyuki Maeda (Kyoto University, Kyoto, Japan) and Naomichi Arima (Kagoshima University, Kagoshima, Japan) for supplying the cell lines.

Keywords: Ephedra, ATL, metabolomics, EI-MS, NMR.

References:

[1] Suzuki R, Kai H, Uesawa Y, Matsuno K, Okada Y, Shirataki Y. Electron ionization mass spectrometry-based metabolomics studies of Sophora flavescens can identify the geographical origin of root samples. Nat Prod Comm 2016; 11: 73 – 75

[2] Suzuki R, Hasuike Y, Hirabayashi M, Fukuda T, Okada Y, Shirataki Y. Identification of a xanthine oxidase-inhibitory component from Sophora flavescens using NMR-based metabolomics. Nat Prod Comm 2013; 8: 1409 – 1412