Contents lists available at ScienceDirect Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff Metabolic profiling and investigations on crude extract of Olea europaea L. leaves as a potential therapeutic agent against skin cancer Debabrata Majumder a , Mousumi Debnath b,1, ⁎ , K.V. Libin Kumar c , Priyatosh Nath a , Rahul Debnath a , Chaitali Sarkar a , G.B.K.S. Prasad d , Yogesh Kumar Verma e , Debasish Maiti a,1 a Immunology Microbiology Lab, Department of Human Physiology, Tripura University, Suryamaninagar, Agartala 799022, Tripura, India b Department of Biosciences, Manipal University Jaipur, Dehmi Kalan, Jaipur 303007, Rajasthan, India c TUV SUD South Asia, Bangalore 560058, India d School of Studies in Biochemistry, Jiwaji University, Gwalior 474011, India e Rajathan Olive Cultivation Limited, State Institute of Agricultural Management Campus, Agricultural Research Station, Durgapura, Jaipur 302018, Rajasthan, India ARTICLE INFO Keywords: Apoptosis ERK 1/2 Melanoma Olive Cytotoxicity Oleuropein ABSTRACT Olive (Olea europaea L.) leaf extract (OLE) have shown therapeutic activity against different types of cancer. However, it is less known that olive leaf extract has anti myeloma activity. The in vitro cytotoxic activity of OLE was evaluated against mouse metastatic melanoma B16F10 cell lines. The extracts were analyzed for their an- tioxidant activity by DPPH radical scavenging activity and the presence of bioactive compounds by liquid chromatography-electrospray ionization-tandem mass spectrometry respectively. The aqueous OLE induced apoptosis of B16F10 cells through ERK 1/2 and p53 mediated pathway. The metabolic profiling of OLE showed 23 metabolites notably oleuropein aglycone, apigenin, luteolin, tyrosol, oleuropein and hydroxytyrosol. Oleuropein present in OLE may be responsible for disrupting the activation of ERK1/2 receptor in melanoma B16F10 cell lines. These findings pave path for further investigations of the structure and function at the mo- lecular level to establish the anticancer activity of oleuropein in melanoma skin cancer. 1. Introduction Olives (Olea europaea L.) are growing in popularity for contributing well-being and good health. These days the markets dealing with nu- traceutical are flooded with products made from olives. It has been reported that aqueous extract of olive leaves has the potential to lower the oxidative stress and also to improve the glucose control in diabetic rats (Al-Attar & Fawziah, 2017). Another research group from Algeria also reported that olive leaf extract possesses hypocholesterolemic ac- tivity (Cheurfa et al., 2019). OliveNet™ database shows the presence of an exhaustive list of compounds from olive (Bonvino et al., 2018). The olive leaf extract contains a wide variety of alcohols, secoiridoids, phenolics, flavonoids (Palmeri et al., 2016). The phytochemical profile was also investigated by Kontogianni et al. (2013) with liquid chro- matography-ultraviolet-visible (UV–Vis) diode array coupled to elec- trospray ionization multistage mass spectrometry. Benavente-Garcıa, Castillo, Lorente, Ortuno, and Del Rio (2000) also reported the presence of phenolic compounds. Oleuropein, hydroxytyrosol, verbascoside, apigenin-7-glucoside and luteolin-7-glucoside are the most abundant in olive leaf extracts. In the Mediterranean region, Olive is a part of the traditional medicine (Gorzynik-Debicka et al., 2018) and as anti-inflammatory https://doi.org/10.1016/j.jff.2019.05.005 Received 3 November 2018; Received in revised form 21 April 2019; Accepted 5 May 2019 Abbreviations: MEM, Minimal essential medium; FBS, fetal bovine serum; MEM-NEAA, MEM- non-essential amino acid solution non-essential amino acids; GAPDH, Glyceraldehyde 3 phosphate dehydrogenase; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMSO, Dimethyl Sulphoxide; AO/EB, Acridine orange/Ethidium bromide; PBS, phosphate buffer saline; Tris EDTA, ethylene diamine tetra acetic acid; SDS-PAGE, Sodium dodecyl sulphate polyacrylamide gel electrophoresis; TBST, Tris-buffered saline with 0.2% Tween-20; HRP, horseradish peroxidase; ECL substrate solution, Clarity max Western ECL blotting substrate; DPPH, Diphenyl picrylhydrazyl; MRM, multiple reaction monitoring; LC-ESI MS/MS, liquid chromatography-electrospray ionization-tandem mass spectrometry; UPLC, ultra-performance liquid chromatography; EMV, Electron Multiplier Voltage; ESI MS, electron spray ionization mass spectrometry; TIC, total ion chroma- tograms; CID, collision induced dissociation; ANOVA, One-way analysis of variance; SPSS, Statistical package for the social sciences; EIC, extracted ion scan; ERK 1/2, extracellular signal regulated kinases 1 and 2 MAPK: mitogen activated protein kinase; GPER, G protein coupled estrogen receptor 1; OLE, olive leaf extract; ROCL, Rajasthan Olive corporation limited ⁎ Corresponding author. E-mail address: mousumi.debnath@jaipur.manipal.edu (M. Debnath). 1 Equal contribution. Journal of Functional Foods 58 (2019) 266–274 1756-4646/ © 2019 Published by Elsevier Ltd. T