antioxidants Article Identification of Tyrosyl Oleate as a Novel Olive Oil Lipophenol with Proliferative and Antioxidant Properties in Human Keratinocytes Cinzia Benincasa 1 , Chiara La Torre 2,†, Alessia Fazio 2,†, Enzo Perri 1, Maria Cristina Caroleo 2,†, Pierluigi Plastina 2,*,† and Erika Cione 2,† 1 CREA Research Centre for Olive, Fruit and Citrus Crops, 87036 Arcavacata di Rende, CS, Italy; [email protected] (C.B.); [email protected] (E.P.) 2 Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, CS, Italy; [email protected] (C.L.T.); [email protected] (A.F.); [email protected] (M.C.C.); [email protected] (E.C.) * Correspondence: [email protected]; Tel.: +39-0984-493128 † Department of Excellence 2018–2022. Abstract: Lipophenols are an emerging subclass of phenolic compounds characterized by the pres- ence of a lipid moiety. Recently, hydroxytyrosyl oleate (HtyOle), a derivative of hydroxytyrosol, has been identified in olive oil and by-products. Furthermore, HtyOle possesses anti-inflammatory, antioxidant, and tissue regenerating properties. In this work, the potential occurrence of tyrosyl oleate (TyOle) in olive oil was investigated based on the hypothesis that its precursors tyrosol and Citation: Benincasa, C.; La Torre, C.; oleic acid, both present in relatively high amount can be coupled together. Moreover, TyOle effects Fazio, A.; Perri, E.; Caroleo, M.C.; have been investigated in human keratinocytes to verify its proliferative and antioxidant properties. Plastina, P.; Cione, E. Identification The quantitative determination of TyOle was carried out by the external standard method in liquid of Tyrosyl Oleate as a Novel Olive chromatography coupled with mass spectrometry (LC/MS), in negative mode using multiple reac- Oil Lipophenol with Proliferative tion monitoring (MRM). The proliferative properties of TyOle on immortalized human keratinocytes and Antioxidant Properties in Human (HaCat) were evaluated by 3-(4,5-dimethylthiasol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Keratinocytes. Antioxidants 2021, 10, assay. Morphological changes were observed by fluorescent staining with phalloidin (for F-actin) 1051. https://doi.org/10.3390/ or 4,6-diamidino-2-phenylindole (DAPI, for chromatin) dye. The antioxidant activity was assessed at antiox10071051 the level of production of mitochondrial reactive oxygen species (ROS) induced with UV exposure. TyOle was identified in all the oil samples investigated. Interestingly, TyOle concentration was higher Academic Editors: Pierluigi Stipa and Giovanna Mobbili in defective or low-quality oils than in extra virgin oils. The formation of TyOle likely occurs during the crushing and kneading processes and its concentration is related to the increase of rancidity Received: 1 June 2021 and of the concentration of free precursors. Herein we show that TyOle induced an increase in the Accepted: 25 June 2021 viability of HaCat cells and cytoskeletal remodeling. Published: 29 June 2021 Keywords: polyphenol; lipophenol; phenolipid; tyrosol; hydroxytyrosol; fatty acid ester; antioxidant; Publisher’s Note: MDPI stays neutral proliferation; reactive oxygen species; reactive oxygen species (ROS) with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Olive oil represents the main source of fat in the Mediterranean diet and represents a valuable food product from both nutritional and economic point of view. Its worldwide Copyright: © 2021 by the authors. consumption has increased in the last five years [1]. The nutritional value and the health Licensee MDPI, Basel, Switzerland. promoting effects of olive oil rely on its favorable nutrient composition, including oleic acid This article is an open access article (OA) as the most abundant fatty acid and fat-soluble vitamins and the presence of phenolic distributed under the terms and compounds [2–5]. The latter are recognized in contributing to the positive health effects re- conditions of the Creative Commons lated to the consumption of extra virgin olive oil (EVOO) [6,7]. Noteworthy, the Regulation Attribution (CC BY) license (https:// 432/2012 of the EU approved the claim “olive oil polyphenols contribute to the protection creativecommons.org/licenses/by/ of blood lipids from oxidative stress” based on the scientific opinion of the European 4.0/). Antioxidants 2021, 10, 1051. https://doi.org/10.3390/antiox10071051 https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, x FOR PEER REVIEW 2 of 13 protection of blood lipids from oxidative stress” based on the scientific opinion of the Eu- ropean Food Safety Authority (EFSA) that “a daily intake of 20 g of olive oil, which con- tains at least 5 mg of hydroxytyrosol and its derivatives (e.g., oleuropein and tyrosol) pro- vides the expected beneficial effects” [8,9]. Tyrosol (2-(4-hydroxyphenyl)ethanol, Ty, Fig- ure 1) and hydroxytyrosol (2-(3,4-dihydroxyphenyl)ethanol, Hty, Figure 1) are among the main phenolic compounds found in olive and olive oil. They occur in their free form as well as in the esterified forms, mainly as secoiridoid derivatives (ligstroside and oleuro- pein, respectively) [10,11] or acylated, as in the case of hydroxytyrosyl acetate [12]. Recently, hydroxytyrosyl oleate (HtyOle, Figure 1), a derivative of Hty characterized by a lipophilic nature due the presence of an oleic acid fatty chain, has been identifified and quantified in EVOO [13,14], as well as in the by-products of olive oil industry, pomace and olive mill waste waters (OMWWs) [15]. Moreover, HtyOle has been found to possess Antioxidants 2021, 10, 1051 anti-inflammatory, antioxidant, as well as tissue-regenerating properties [14,15]. The2 con- of 12 jugation of fatty acids with active cosmetic ingredients leads to the formation of fatty de- rivatives with enhanced skin penetration ability with respect to small hydrophilic com- Foodpounds, Safety thus Authority increasing (EFSA) their antimicrobial, that “a daily intakeantioxidant of 20, gor of anti olive-ageing oil, which activities contains [16–18]. at leastIn this 5 mgwork, of hydroxytyrosol we investigated and the its possibility derivatives that (e.g., another oleuropein lipophenol and tyrosol) (tyrosyl provides oleate, theTyOle, expected Figure beneficial 1) could effects”occur in [8olive,9]. Tyrosoloil as a (2-(4-hydroxyphenyl)ethanol,result of the reaction between Ty, tyrosol Figure and1) andoleic hydroxytyrosol acid, both occurring (2-(3,4-dihydroxyphenyl)ethanol, in relatively high amount. Moreover, Hty, Figure proliferative1) are among and the antioxi- main phenolicdant properties compounds of TyOle found have in been olive investigated and olive oil. in human They occur keratinocyte in their cell free line form to asexplore well asthe in possibility the esterified that forms, TyOle mainly could ashave secoiridoid tissue regenerating derivatives pr (ligstrosideoperties like and its oleuropein, congener respectively)HtyOle. [10,11] or acylated, as in the case of hydroxytyrosyl acetate [12]. Figure 1. Structure of tyrosol (Ty),(Ty), hydroxytyrosolhydroxytyrosol (Hty),(Hty), tyrosyltyrosyl oleateoleate (TyOle)(TyOle) and and hydroxytyrosyl hydroxytyro- oleatesyl oleate (HtyOle). (HtyOle). 2. MaterialsRecently, and hydroxytyrosyl Methods oleate (HtyOle, Figure1), a derivative of Hty characterized by2.1. aChemicals lipophilic and nature Reagents due the presence of an oleic acid fatty chain, has been identifi- fied and quantified in EVOO [13,14], as well as in the by-products of olive oil industry, pomaceTyrosol and, olive oleicmill acid waste (OA), waters and methyl (OMWWs) oleate, [15 t-].butanol, Moreover, methanol, HtyOle dimethyl has been sulfoxide found to possess(DMSO) anti-inflammatory, and formic acid (LC/MS antioxidant, grade) aswere well purchased as tissue-regenerating from Sigma-Aldrich properties (Milan, [14,15 It-]. ® Thealy). conjugation Novozym of435 fatty (immobilized acids with active Candida cosmetic antarctica ingredients Lipase leads B) towas the from formation Novozymes of fatty derivatives(Bagsvæ rd, withDenmark enhanced). n-Hexane skin penetration and acetone ability (analytical with respect grade) towere small supplied hydrophilic from Carlo com- pounds,Erba Reagenti thus increasing (Milan, Italy). their antimicrobial,Ultrapure water antioxidant, (resistivity or of anti-ageing 18.2 MΩ· activitiescm) was obtained[16–18]. Infrom this a work,Milli-Q we plus investigated system (Millipore, the possibility Bedford, that anotherMA, USA). lipophenol Dulbecco’s (tyrosyl modified oleate, Eagle’s TyOle, Figuremedium1) could(DMEM), occur fetal in olivebovine oil serum as a result (FBS), of L- theglutamine, reaction penicillin/streptomycin, between tyrosol and oleic and acid,paraformaldehyde both occurring (PFA) in relatively were supplied high amount. from Thermo Moreover, Fisher proliferative Scientific and(Waltham, antioxidant MA, propertiesUSA). of TyOle have been investigated in human keratinocyte cell line to explore the possibility that TyOle could have tissue regenerating properties like its congener HtyOle. 2.2. Instrumentations 2. MaterialsNMR analyses and Methods were performed at 25 °C on a Bruker AC 300 spectrometer at 300 MHz 2.1.and Chemicals75 MHz for and 1H Reagents and 13C NMR, respectively. CDCl3 and tetramethylsilane were used as the solventTyrosol, and oleic the acid internal (OA), standard, and methyl respectively. oleate, t-butanol, HPLC analyses methanol, were dimethyl