Original Paper

Analytical and Bioanalytical Chemistry

, Volume 397, Issue 2, pp 643-654

First online:

Qualitative screening of phenolic compounds in olive leaf extracts by hyphenated liquid chromatography and preliminary evaluation of cytotoxic activity against human breast cancer cells

  • Shaoping FuAffiliated withInstitute of Chemistry and Applications of Plant Resources, School of Biological & Food Engineering, Dalian Polytechnic UniversityDepartment of Analytical Chemistry, Faculty of Sciences, University of Granada
  • , David Arráez-RomanAffiliated withDepartment of Analytical Chemistry, Faculty of Sciences, University of Granada
  • , Antonio Segura-CarreteroAffiliated withDepartment of Analytical Chemistry, Faculty of Sciences, University of Granada Email author 
  • , Javier A. MenéndezAffiliated withGirona Biomedical Research Institute (IdIBGi), Catalan Institute of Oncology, Girona (ICO-Girona)
  • , María P. Menéndez-GutiérrezAffiliated withRegenerative Cardiology Department, National Center for Cardiovascular Research Foundation
  • , Vicente MicolAffiliated withInstitute of Molecular & Cell Biology, University Miguel Hernández
  • , Alberto Fernández-GutiérrezAffiliated withDepartment of Analytical Chemistry, Faculty of Sciences, University of Granada

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In this work, high-performance liquid chromatography (HPLC) coupled to electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and electrospray ion trap multiple-stage tandem mass spectrometry (ESI-IT-MS2) has been applied to screen phenolic compounds in olive leaf extracts. The use of a small particle size C18 column (1.8 μm) provided great resolution and made separation of a lot of isomers possible. The structural characterization was based on accurate mass data obtained by ESI-TOF-MS, and the nature of fragmentation ions were further confirmed by ESI-IT-MS2 when possible. In addition, we employed tetrazolium salt (MTT)-based assays to assess the effects of olive leaf extracts on the growth of human tumor-derived cells. Upon this approach, we achieved an accurate profile of olive leaf phenolics along with the identification of several important isomers of secoiridoids and flavonoids. This will allow a better understanding of the complete composition of olive-leaf-bioactive compounds as well as their involvement in Olea europaea L. biochemical pathways. Importantly, olive leaf extracts exhibited dose-dependent inhibitory effects on the metabolic status (cell viability) of three breast cancer models in vitro. Since the tumoricidal activity of the extracts should be mainly attributed to the identified olive leaf phenolics, these findings warrant further investigation at the structure–function molecular level to definitely establish the anticancer value of these phytochemicals.


Survival plots obtained by using the MTT assay for SKBr3, MCF-7 and JIMT-1 breast cancer cell lines treated with olive leaf extract at increasing concentrations for 72 h.


Olive leaf Phenolic compounds High-performance liquid chromatography Time-of-flight mass spectrometry Ion trap multiple-stage tandem mass spectrometry Breast cancer