UHPLC-DAD-FLD and UHPLC-HRMS/MS based metabolic profiling and characterization of different Olea europaea organs of Koroneiki and Chetoui varieties § Thomas Michel a,b,1 , Ines Khlif c,1 , Periklis Kanakis a , Aikaterini Termentzi a , Noureddine Allouche c , Maria Halabalaki a , Alexios-Leandros Skaltsounis a, * a Laboratory of Pharmacognosy and Natural Products Chemistry, School of Pharmacy, University of Athens, Panepistimioupoli Zografou, 15771 Athens, Greece b Universite ´ Nice Sophia Antipolis, ICN, UMR CNRS 7272, Parc Valrose, 06108 Nice CEDEX 2, France c Laboratoire de Chimie des Substances Naturelles, Faculte ´ des Sciences de Sfax, BP 1171, 3000 Sfax, Tunisia 1. Introduction The olive tree (Olea europaea L., Oleaceae) is one of the most important fruit trees in Mediterranean basin while olive oil, which is obtained from the whole olive fruit by mechanical means, is consumed all over the world for centuries. Virgin and Extra Virgin Olive oil has been established amongst other edible oils for its superior nutritional and medicinal value (Ghanbari et al., 2012b; Soler-Rivas et al., 2000). Several studies have demonstrated that the consumption of olive oil, rich in phenolic compounds is correlated with decreased risk of cardiovascular disease, obesity, metabolic syndrome and type-2 diabetes (Lo ´ pez-Miranda et al., 2010). Similarly, olive leaf extracts have been associated with antioxidant, antimicrobial, antiproliferative, and antiviral proper- ties (Benavente-Garcı ´a et al., 2000; Bouaziz and Sayadi, 2005; Briante et al., 2002; Goulas et al., 2009; Micol et al., 2005; Pereira et al., 2007). These biological effects have been mainly attributed to certain phenolic compounds found in Olea genus such as phenylethanols, secoiridoids, flavonoids and lignans. For instance, secoiridoid derivatives exhibit a diverse range of biological properties (Obied et al., 2007; Obied et al., 2008) and oleuropein, the major secoiridoid of olive fruit and leaves, has been assessed for its antioxidant potential, anti-inflammatory (Caroline Puel et al., 2006), antimicrobial (Pereira et al., 2007) and antiviral activities (Micol et al., 2005). Recent reports demonstrat- ed an anti-amyloidogenic effect of oleuropein suggesting a possible protective role against Alzheimer’s disease (Kostomoiri et al., 2013). Structurally related to oleuropein is ligstroside which is characterized by a hydroxytyrosol unit rather than tyrosol. Ligstroside is less concentrated in olive organs compared to Phytochemistry Letters 11 (2015) 424–439 A R T I C L E I N F O Article history: Received 28 October 2014 Received in revised form 17 December 2014 Accepted 23 December 2014 Available online 29 January 2015 Keywords: Olea europaea Koroneiki Chetoui metabolic profiling secoiridoid UHPLC-HRMS mass spectrometry dereplication A B S T R A C T The olive tree (Olea europaea L., Oleaceae) is one of the most important fruit trees in Mediterranean basin and has been associated with numerous biological assets. These effects have been mainly attributed to certain phenolic compounds found in fruits, olive oil and by-products of olive oil production. However, other Olea organs such as stems, roots and drupe stones have received little attention leading to limited knowledge about their phytochemical content. Thus, the main goal of the current study was the investigation of the chemical composition of diverse organs from two O. europaea varieties (i.e. Koroneiki and Chetoui) using combinations of modern analytical techniques. A fast UHPLC-DAD-FLD method was developed and applied for the profiling of different extracts of O. europaea organs as well as for the quantification of oleuropein. In addition, a dereplication strategy was developed using an Orbitrap platform (UHPLC-ESI-HRMS/MS) aiming to further characterization of the contained secondary metabolites. In total, 86 molecules were identified including compounds described for the first time in O. europaea such as coumarins. Some compounds were found to be organ specific such as nuzhenide derivatives in stone, flavonoids in leaves and oleuropein which was mainly found in Olea roots, in both varieties. Overall, it is noticeable that except olive oil, diverse organs of olive tree might comprise an alternative and valuable source of biologically active compounds. ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. § This paper is part of a special issue of selected presentations delivered at the 9th International Symposium on Chromatography of Natural Products, 26–29 May 2014, Lublin, Poland. * Corresponding author. Tel.: +30 2107754032; fax: +30 2107754594. E-mail address: [email protected](A.-L. Skaltsounis). 1 Equal contribution. Contents lists available at ScienceDirect Phytochemistry Letters jo u rn al h om ep ag e: ww w.els evier.c o m/lo c ate/p hyt ol http://dx.doi.org/10.1016/j.phytol.2014.12.020 1874-3900/ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Transcript
Phytochemistry Letters 11 (2015) 424–439
UHPLC-DAD-FLD and UHPLC-HRMS/MS based metabolic profilingand characterization of different Olea europaea organs of Koroneikiand Chetoui varieties§
Thomas Michel a,b,1, Ines Khlif c,1, Periklis Kanakis a, Aikaterini Termentzi a,Noureddine Allouche c, Maria Halabalaki a, Alexios-Leandros Skaltsounis a,*a Laboratory of Pharmacognosy and Natural Products Chemistry, School of Pharmacy, University of Athens, Panepistimioupoli Zografou, 15771 Athens, Greeceb Universite Nice Sophia Antipolis, ICN, UMR CNRS 7272, Parc Valrose, 06108 Nice CEDEX 2, Francec Laboratoire de Chimie des Substances Naturelles, Faculte des Sciences de Sfax, BP 1171, 3000 Sfax, Tunisia
A R T I C L E I N F O
Article history:
Received 28 October 2014
Received in revised form 17 December 2014
Accepted 23 December 2014
Available online 29 January 2015
Keywords:
Olea europaea
Koroneiki
Chetoui
metabolic profiling
secoiridoid
UHPLC-HRMS
mass spectrometry
dereplication
A B S T R A C T
The olive tree (Olea europaea L., Oleaceae) is one of the most important fruit trees in Mediterranean basin
and has been associated with numerous biological assets. These effects have been mainly attributed to
certain phenolic compounds found in fruits, olive oil and by-products of olive oil production. However,
other Olea organs such as stems, roots and drupe stones have received little attention leading to limited
knowledge about their phytochemical content. Thus, the main goal of the current study was the
investigation of the chemical composition of diverse organs from two O. europaea varieties (i.e. Koroneiki
and Chetoui) using combinations of modern analytical techniques. A fast UHPLC-DAD-FLD method was
developed and applied for the profiling of different extracts of O. europaea organs as well as for the
quantification of oleuropein. In addition, a dereplication strategy was developed using an Orbitrap
platform (UHPLC-ESI-HRMS/MS) aiming to further characterization of the contained secondary
metabolites. In total, 86 molecules were identified including compounds described for the first time
in O. europaea such as coumarins. Some compounds were found to be organ specific such as nuzhenide
derivatives in stone, flavonoids in leaves and oleuropein which was mainly found in Olea roots, in both
varieties. Overall, it is noticeable that except olive oil, diverse organs of olive tree might comprise an
alternative and valuable source of biologically active compounds.
� 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Phytochemistry Letters
jo u rn al h om ep ag e: ww w.els evier .c o m/lo c ate /p hyt ol
1. Introduction
The olive tree (Olea europaea L., Oleaceae) is one of the mostimportant fruit trees in Mediterranean basin while olive oil, whichis obtained from the whole olive fruit by mechanical means, isconsumed all over the world for centuries. Virgin and Extra VirginOlive oil has been established amongst other edible oils for itssuperior nutritional and medicinal value (Ghanbari et al., 2012b;Soler-Rivas et al., 2000). Several studies have demonstrated thatthe consumption of olive oil, rich in phenolic compounds iscorrelated with decreased risk of cardiovascular disease, obesity,metabolic syndrome and type-2 diabetes (Lopez-Miranda et al.,
§ This paper is part of a special issue of selected presentations delivered at the 9th
International Symposium on Chromatography of Natural Products, 26–29 May
1874-3900/� 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rig
2010). Similarly, olive leaf extracts have been associated withantioxidant, antimicrobial, antiproliferative, and antiviral proper-ties (Benavente-Garcıa et al., 2000; Bouaziz and Sayadi, 2005;Briante et al., 2002; Goulas et al., 2009; Micol et al., 2005; Pereiraet al., 2007). These biological effects have been mainly attributedto certain phenolic compounds found in Olea genus such asphenylethanols, secoiridoids, flavonoids and lignans.
For instance, secoiridoid derivatives exhibit a diverse range ofbiological properties (Obied et al., 2007; Obied et al., 2008) andoleuropein, the major secoiridoid of olive fruit and leaves, hasbeen assessed for its antioxidant potential, anti-inflammatory(Caroline Puel et al., 2006), antimicrobial (Pereira et al., 2007) andantiviral activities (Micol et al., 2005). Recent reports demonstrat-ed an anti-amyloidogenic effect of oleuropein suggesting apossible protective role against Alzheimer’s disease (Kostomoiriet al., 2013). Structurally related to oleuropein is ligstroside whichis characterized by a hydroxytyrosol unit rather than tyrosol.Ligstroside is less concentrated in olive organs compared to
T. Michel et al. / Phytochemistry Letters 11 (2015) 424–439 425
oleuropein and less information is available concerning itsbiological profile.
Recently, oleacein and oleocanthal which are dialdehydicaglycons of oleuropein and ligstroside respectively have drawngreat attention. Oleocanthal possess anti-inflammatory activitiessimilar to ibuprofen via its ability to inhibit cyclooxygenaseenzymes (Beauchamp et al., 2005). Further investigations havedemonstrated that oleocanthal inhibits tau fibrillisation associatedwith neurodegenerative diseases such as Alzheimer’s disease (Liet al., 2009; Monti et al., 2012) while oleacein found possessing aprotective antioxidant effect on erythrocyte oxidative damage(Paiva-Martins et al., 2009). Both compounds exert antiprolifera-tive and chemopreventive effects against several types of cancers(Elnagar et al., 2011; Garcıa-Villalba et al., 2010; Khanal et al.,2011; Menendez et al., 2008). Others olive biophenols likehydroxytyrosol, verbascoside and rutin show antioxidant andchemopreventive properties (Obied et al., 2007).
Chemical composition of olive fruits and olive oil as well as by-product of olive oil production (e.g. waste water, leaves) has beenextensively studied while other Olea organs such as stems, rootsand drupe stones have received little attention leading to a limitedknowledge about their phytochemical content. Indeed, recentstudies related to the phytochemical investigation of olive leaveshave been referred (Fu et al., 2010; Quirantes-Pine et al., 2013;Taamalli et al., 2011), while only few reports are available for olivedrupe stone (Silva et al., 2010; Silva et al., 2006). According to theliterature the main components of olive roots and stems arehydroxytyrosol, tyrosol, oleuropein and ligstroside (Del Rıo et al.,2003; Ortega-Garcıa and Peragon, 2010; Perez-Bonilla et al., 2006)as well as verbascoside and flavonoids such as taxifolin, luteolinand apigenin derivatives (in stem) (Japon-Lujan and Luque deCastro, 2007; Lujan et al., 2009; Lujan et al., 2008). However, ratherlimited data are available concerning other or minor constituentswhile there are not comparative studies exploring this topic. Such astudy would contribute significantly to the localisation ofalternative sources of olive bioactives and the discovery of newmolecules. Finally, new insight into the diverse biochemicalpathways in the whole tree is gained contributing to betterunderstanding of the nutritional and medicinal value of olivetree products.
The determination of the chemical identity of knownmetabolites in crude extracts (dereplication) is generallyperformed using Liquid Chromatography/Mass Spectrometry(LC-MS)-based methods. Recent developments, such as UltraHigh Performance LC (UHPLC) and High Resolution MS (HRMS)facilitate significantly the detection of hundreds compoundssimultaneously, in an untargeted manner even if present in lowquantity (Michel et al., 2013). Until now, qualitative screening ofolive polyphenols or biophenols have been performed using HPLCcoupled to electrospray ionisation and quadrupole time-of-flightmass spectrometry (HPLC-ESI-QTOF-MS) (Fu et al., 2009a;Quirantes-Pine et al., 2013; Silva et al., 2010; Taamalli et al.,2011) or equipped with a triple quadrupole mass analyzer (Lujanet al., 2008). However, relative new technologies such as Orbitrapanalyser, have also become competitive tools for the characteri-sation of natural products in complex matrices. Orbitrap analyzerprovides high resolution, high mass accuracy (<3 ppm) androbustness. Furthermore, combination of the Orbitrap analyzerwith an external accumulation device such as a linear ion trapenables multiple levels of collision induced fragmentation (MSn)for the elucidation of molecule structure (Makarov and Scigelova,2010). Therefore, such hybrid apparatuses could be consideredas platforms of choice for the characterization of small moleculessince they increase considerably the confidence of identificationprocedure, the dereplication of known compounds as well asthe putative identification of unknowns. In previous studies, we
have clearly demonstrated the power of Orbitrap analyzer forquantification and identification of natural compounds (Kanakiset al., 2013; Tchoumtchoua et al., 2013).
The aim of this work was to study the chemical compositionof olive leaves, stems, roots and stone extracts using combinationof modern techniques. A fast UHPLC-DAD method was developedand applied for the profiling of the methanol and ethyl acetateextract of O. europaea as well as for the quantification ofoleuropein in different organs. Furthermore, a UHPLC-ESI-HRMS/MS method was developed using an Orbitrap instrumentfor the further qualitative characterization thereof. The identifi-cation was based on retention time (Rt), accurate m/z dataobtained by ESI(�)-HRMS, proposed elemental composition (EC),ring double bond equivalent (RDBeq) values, HRMS/MS informa-tion as well as chemotaxonomic data. For comparative purposestwo Olea varieties, Koroneiki from Greece and Chetoui fromTunisia, the most characteristic of both countries, were investi-gated.
2. Materials and methods
2.1. Chemicals and standards
Ungraded methanol, ethyl acetate and hexane for extractionand MS grade acetonitrile, water and formic acid for UHPLC–MSanalyses were purchased from SDS Carlo Erba (Val de Reuil,France). Ungraded solvents were distilled before extraction step.Oleuropein was isolated from a dichloromethane extract of oliveleaves by column chromatography using a CH2Cl2-MeOH solventsystem of increasing polarity. Pure oleuropein was eluted withCH2Cl2-MeOH (90:10,v/v). The verification of its structure and thedetermination of purity (97%) were performed by HPLC and NMR(Puel et al., 2004).
2.2. Plant material
Two different olive tree (Olea europaea) varieties were studied:‘‘Koroneiki’’ from Greece and ‘‘Chetoui’’ from Tunisia as the mostrepresentative ones of these countries. The two years old‘‘Koroneiki’’ olive tree used for study was bought in a Greek localmarket. Leaves, stems and roots were separated and then dried byfreeze-drying, for 3 days. The ‘‘Chetoui’’ olive tree was harvested on2012 and then dried in room temperature. Dried material wasground to a fine powder using a hammer mill. For drupes, flesh wasseparated from stone, and only stones (containing seed) from bothvarieties were analysed.
2.3. Extraction and sample preparation procedure
20 g of powdered material from all organs were used forextraction which was performed using Pressurised Liquid Extrac-tion (PLE) and specifically an ASE 300 instrument (Dionex,Sunnyvale, CA, USA). 100 mL steel cells were used while methanoland ethyl acetate were selected as extraction solvents. Theextraction conditions were the following: temperature of 70 8C,pressure of 100 bar, 2 extraction cycles of 10 min, preheating andheating period of 1 and 5 min respectively, flush volume of 100%and purge of 60 s. The obtained extracts were evaporated using arotary evaporator under vacuum (Buchi Labortechnik AG,Switzerland) to afford dried crude extracts which were stored at2 8C. Liquid–liquid extraction method was additionally applied toall extracts for defatting purposes. Specifically, both methanol andethyl acetate extracts (500 mg) were dissolved in MeOH (200 mL)and then partitioned with three equal volumes of hexane(3 � 200 mL). The methanol layer of both extracts was analysedby UHPLC-DAD and UHPLC-HRMS.
T. Michel et al. / Phytochemistry Letters 11 (2015) 424–439426
2.4. UHPLC-DAD-Fluorimetry conditions
The analysis was performed using an UHPLC Acquity system(Waters, Milford, MA, USA) to ensure a high resolving power and abaseline separation of compounds in a reasonable separation time.All runs were performed on an Acquity UHPLC BEH C18 column(50 mm � 2.1 mm I.D., 1.7 mm), at 50 8C, with a flow rate of0.700 mL/min. A guard column (5 mm � 2.1 mm, 1.7 mm) with thesame stationary phase was also used. The mobile phase consistedof water + 0.1% FA (solvent A) and ACN + 0.1% FA (solvent B) andwas used in multistep gradient mode. The gradient was operated asfollow: 2 to 15% B for 3 min, isocratic at 15% for 2 min, 15 to 25% Bfor 2.5 min, 25 to 100% B for 3.5 min and final isocratic for 1 min at100%. Column equilibration was performed for 2 min betweeneach injection. The sample manager was thermostated at 10 8C,and the injection volume was set at 0.5 mL. The standard AcquityPDA module was used for online UV detection in the 200–490 nmrange, with a resolution of 2.4 nm and a sampling rate of10 spectra/s. The fluorescence detector was set with lex at350 nm and lem at 397 nm. Crude extracts were dissolved in amixture of water-acetonitrile (1:1) and then injected at 10 mg/mL.
2.5. UHPLC-ESI(-)-HRMS/MS conditions
UHPLC-HRMS/MS analyses were performed using an AccelaHigh Speed LC System hyphenated to a hybrid LTQ Orbitrap XLdiscovery mass spectrometer (Thermo Scientific, Bremen,Germany) equipped with an electrospray ionisation (ESI) probe.Separation was carried out on an Ascentis Express Fused-CoreTM
C18 column (100 � 2.1 mm i.d., 2.7 mm, Supelco, PA, USA). Anoptimum UHPLC separation gradient was developed in order toefficiently resolve all compounds. The flow rate was set at 800 mL/min and the solvent system was (A) water 0.1% Formic Acid and (B)acetonitrile. The elution program was: 2% B for 2 min; 10% B in2.5 min; 20% B in 13.5 min; 95% B in 14 min and hold for 1 min.After return back to 5% B in 0.5 min, column equilibration wasperformed for 1.5 min at the end of the run. The injection volumewas 5 mL and samples were injected at 0.1 mg/mL in water-acetonitrile solution (1:1). The HRMS & HRMS/MS data wereacquired in negative mode over a m/z range of 100–1500 for allextracts expect for seed extract (m/z range of 100–2000). The MSprofile was recorded in full scan mode (scan time = 1 micro scansand maximum inject time = 500 ms) in the form of TIC (Total IonCurrent) chromatogram. The ESI conditions were as follow:capillary temperature 350 8C; capillary voltage �3 V; tube lens�43.36 V; ESI voltage 3.1 kV. Nitrogen was used as sheath gas(30 Au) and auxiliary gas (10 Au). For the HRMS/MS acquisitions,a data-dependent method including the detection (full scan) andfragmentation of the 3 most intense peaks per scan was used.The mass resolving power was 30,000 for both levels and thenormalised collision energy in the ion trap was set to 35.0%(q = 0.25) for the HRMS/MS experiments. The raw data wereacquired and processed with Xcalibur 2.0.7 software from ThermoScientific.
2.6. Oleuropein calibration curve
Oleuropein was quantified by UHPLC-DAD via an externalcalibration curve. The stock solution of oleuropein was firstprepared at 10 mg/mL in a mixture of water-acetonitrile (1:1) andkept at 4 8C. Afterwards, a series of working standard solutions ofoleuropein at nine calibration levels (concentrations varying from50 to 7500 mg/mL) were prepared by serial dilution of the stocksolution with the same solvent. The curve (y = 200.41x � 3514.7, R2
0.9998) was constructed using the external standard method byplotting the peak-area of each standard versus the concentration.
Each standard solution was injected (0.5 mL) in triplicate andaveraged.
3. Results and discussion
3.1. Metabolite profiling by UHPLC-DAD-Fluorometry
The first step in the current study was extraction of the plantmaterial and since it regards a comparative study to ensurereproducibility and increase detection range. Therefore, PLE waschosen as the extraction method and two different solvents(methanol and ethyl acetate) were used to cover a wide polaritywindow. PLE is an established technique for the extraction ofnatural products since it offers comparative advantages such asspeed, efficiency, reproducibility, low solvent consumption andclean-up extracts (Kaufmann and Christen, 2002). Moreover, twodifferent detectors, DAD and FLD were initially employed in series,hyphenated to UHPLC device (UHPLC-DAD-FLD) providing com-plementary UV information. Typical chromatograms of methanoland ethyl acetate of both varieties are given in Fig. 1. For example,considering the same extraction solvent it can be observed thatthe UHPLC-DAD profiles of both varieties are similar for stemextracts (Fig. 1A). The same observation can be made for root andseed extracts (data not shown). Only in the case of leaf extracts,some differences between Koroneiki and Chetoui varieties weredetected but mostly quantitative ones (data not shown). Compari-son based on extraction solvent and observed retentions showsas expected that hydrophilic compounds are more abundant inthe methanol than in the ethyl acetate extract. Furthermore theDAD data allowed the first filtering of compounds regarding theirchemical identity and the determination of general chemicalclasses of constituents. These data combined with the Rtinformation were later on integrated with HRMS data assistingthe dereplication procedure. Thus, compounds exhibiting UVspectra of phenylpropanoids, secoiridoids and flavonoids whichare the major hydrophilic analytes in olive tree could be detected.UHPLC-DAD profiles at 280 nm of leaves, stems and roots showeda predominant metabolite at 5.1 min with a typical UV spectrumof secoiridoid. This compound was identified as oleuropein byco-injection with a standard. Additionally, the FLD profiling(Fig. 1B) indicates the presence of fluorescent molecules thatcould correspond to flavonoids or other phenolic compounds (e.g.coumarin).
3.2. Dereplication of olive extract by UHPLC-HRMS/MS
Orbitrap is a high resolution mass analyser providing accuratemass measurements (routinely Dm < 2–3 ppm) and high massresolving power (30,000 and even higher in the low mass region forOrbitrap Discovery). Indeed, the Orbitrap analyser enables thedetection and prediction of elemental composition (EC) with highconfidence even if compounds are at low level in complexmixtures. Based on this advantageous feature a comprehensivedereplication strategy was applied in order to identify moleculesfrom the different organs of O. europaea varieties (Tchoumtchouaet al., 2013). Only methanol extracts were further investigateddue to the bigger diversity of minor hydrophilic compounds asseen at the UHPLC-DAD profile.
Briefly, all well resolved peaks in TIC were selected and possibleEC were calculated. For reducing the possible EC candidates, masstolerance was set at 3 ppm, or less than 5 ppm in case of fragments.C, H, O and Cl were selected for elemental composition calculationsand only consistent RDBeq values were considered. Additionallychromatographic, spectrometric features such as UV absorbanceand HRMS/MS data as well as bibliographic and chemotaxonomicinformation were employed for identification. Furthermore adduct
Fig. 1. UPLC profiles of methanole and ethyl acetate extracts of Olea europaea stems recorded with different detectors: (A) UV detector l = 280 nm and (B) fluorometry. MeOH:
Methanol, EtOAc: Ethyl Acetate, O: Oleuropein.
T. Michel et al. / Phytochemistry Letters 11 (2015) 424–439 427
ions [M + Cl]� and [M � H + HCOOH]� especially observed in caseof secoiridoids supported the confirmation of EC for the [M � H]�
ion. The possible ECs for each selected peak were searched bothin in-house (Kanakis et al., 2013) and public databases (PubChem,ChemSpider, Metlin) to report known natural products and/oreliminate non-referenced formulae. Furthermore, some com-pounds were unambiguously identified by comparing theirretention time and spectral data with those of reference standards.
The compounds detected in methanol extracts are summarisedin Table 1 (in leaf, stem and root) and Table S1 (in seed –supplementary material) including their Rt, EC, monoistopic massof the pseudomolecular ion (m/z), RDBeq values and their majorHRMS/MS fragments. A total of 133 secondary metabolites weredetected in the different O. europaea organs and varieties belongingto various chemical classes including phenylethanols, hydroxy-cinnamic acid ester derivatives, secoiridoids, flavonoids, coumar-ins, lignans and triterpenes. Among them, 86 were identified orputatively characterised based on the dereplication strategy while44 peaks remained unidentified. The chemical structures ofrepresentative ones are displayed in Fig. 2.
4. Secoiridoids
Secoiridoids were the main class of secondary metabolitesidentified in olive tree. Among them, oleuropein (Rt 16.35 min)was the major compound in leaves stems and roots extracts whilenuzhenide derivatives were the main compounds in stone extracts.
4.1. Oleuropein derivatives
Oleuropein (53) at Rt 16.35 min is already described as the mostrepresentative secondary metabolite of olive leaves (Fu et al., 2010;
Herrero et al., 2011; Quirantes-Pine et al., 2013). It was detected atm/z 539.1761 and it was confirmed by comparison to authenticstandard. Moreover, two oleuropein isomers were also detected atRt 17.7 (55) and 18.5 min (58) (Table 1). All share similarfragmentation pattern with m/z 377, 307 and 275 to be the mainproduct ions. As described in literature, compound 58 couldcorrespond to oleuroside which differs from oleuropein in theposition of a double bound in the elenolic acid moiety (Fu et al.,2010; Herrero et al., 2011; Obied et al., 2008). Numerousoleuropein derivatives previously described in olive tree (Obiedet al., 2008; Quirantes-Pine et al., 2013; Taamalli et al., 2011) havebeen detected in all extracts such as demethyloleuropein (42, m/z
Regarding methoxyoleuropein, two peaks (51 & 52) weredetected at m/z 569.1865 with the same molecular formula andthese isomers are characterised by a similar fragmentationpathway, as shown in HRMS/MS data. A fragmentation schemeas well as a brief discussion regarding its product ions arepresented in supplementary material (Figure 1S). The two isomerscould correspond to (200R)-200-methoxyoleuropein and (200S)-200-methoxyoleuropein found in Jasminum officinale leaves belongingto Oleaceae family (Tanahashi et al., 1999). Additionally, based onthe proposed fragmentation and the retention time, we were ableto identify methoxyoleuroside (57, m/z 569.1865) eluting at18.2 min just before oleuroside.
Table 1Secondary metabolites identified in leaf, stem and root samples of Koroneiki and Chetoui cultivars. Rt, [M�H]�, EC and RDBeq information is given together with main fragments at MS/MS level. Relative abundance (% precursor ion
intensity) of the metabolites identified in different samples is indicated by + (0–5%), ++ (5–10%), +++ (10–50%) and ++++ (50–100%).
117 ni 20 365.1599 C19H25O7 7.5 229.1076 (C11H17O5. 3.5) +
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T. Michel et al. / Phytochemistry Letters 11 (2015) 424–439434
Another compounds of interest is jaspolyoside (64, Rt 20.65, m/z
925.2784), already identified in olive drupes (Di Donna et al., 2007;Obied et al., 2008), and found in all organs under investigation. Inthe current study jaspolyoside shares typical fragmentation motifsof secoiridoids such as loss of CH3OH from the decarboxymethylgroup (m/z 893.2632, C41H49O22) and loss of C4H6O1 (Fig. 2S,supplementary material). Furthermore, the HRMS/MS spectrumshows strong similarities with that of oleuropein. For instance, ionsat m/z 539.1749 (C41H49O22), m/z 377.1223 (C19H21O8) and m/z
307.0813 (C15H15O7) are typical fragments of oleuropein. Anothercharacteristic fragment ion is at m/z 763.2467 (C36H43O18),obtained by neutral loss of 162 mass units (glucose) from theparent ion giving rise to two ions at m/z 745.2287 (C36H41O17) andm/z 693.2020 (C32H37O17).
4.2. Ligstroside derivatives
Ligstroside (61, Rt 19.4 min) another well-known compoundin Oleaceae family is present in all organs at m/z 523.1811(C25H31O12) and has been confirmed by comparison with ligstro-side standard. Furthermore, its EC and fragmentation pattern agreewith the previous reports (Kanakis et al., 2013). Indeed, the mainfragment at m/z 361.1277 is obtained by loss of a glucose residuefrom deprotonated molecular ion (m/z 523.1711). Other char-acteristics fragments can be observed such as the loss of C4H6O (m/
z 291.0865) from m/z 361.1277 and the subsequent loss of CH3OH(m/z 259.0969). Interestingly, another ion at m/z 909.3020 (66,C42H53O22) was observed which possibly corresponds to a newmolecule structurally closed to jaspolyoside. Indeed, the fragmen-tation pathway presents similar fragments with that of ligstrosideand jaspolyoside with the difference of one hydroxyl groupsuggesting that the missing hydroxyl group is from the pheny-lethanol moiety.
4.3. Nuzhenide derivatives
Nuzhenide derivatives were exclusively found in the stones ofboth cultivars (Fig. 3). Among them, the well known nuzhenide(76, Rt 14 min) and nuzhenide-11-methyl oleoside were foundas the major compounds in seed extracts at m/z 685.2334(C31H41O17) and at m/z 1071.3520 (C48H63O27), respectively.The detection of the later compound at three retention times [i.e.Rt 19.84 (81), 20.38 (82) and 20.82 (84) min] suggests thepresence of isomers. All isomers exhibit a similar fragmentationscheme. As it is illustrated in Fig. 3 the ion at m/z 909.2985(C42H53O22) comprise a basic fragment and arises from the lossof a glucosyl moiety (162 mass unit) which by a further lossof a C4H6O produces the fragment at m/z 839.2580 (C38H47O21).The ion at m/z 685.2304 (C31H41O17), corresponding to thenuzhenide part can be derived by the loss of the 11-methyloleoside from the pseudo-molecular ion or from ion at m/z
909.2985. Likewise, the ion at m/z 523.1809 (C25H31O12) couldresult from two pathways: the first through the loss of a 11-methyl oleoside unit from the pseudo-molecular ion and thesecond through the loss of a glucosyl unit from the ion at m/z
685.2304. In the first case, the ion can at m/z 523.1809 canfurther give rise to the ion at 453.1374 (C21H25O11) by loss of aC4H6O unit. Finally, fragments corresponding to the 11-methyloleoside at m/z 403.1232 (C17H23O11) and the 11-methyl oleosideless water at m/z 385.1123 (C17H21O10) could be explained bydifferent pathways, some of which are suggested in Fig. 5.
Furthermore, other abundant 11-methyl-oleoside derivativespreviously detected in seeds such as nuzhenide-di(11-methyloleoside) (83, 1457.4713, C65H85O37) and nuzhenide-tri(11-methyloleoside) (85, 1843.5997, C78H108O50) were found in substantialamount (Silva et al., 2010). HRMS/MS spectrum of these compounds
H3CO
HOO
OH
OH
OCH3OHOlivil
O
OO
OHHOHO
OH
O
HO
R2 COO CH3O
R1
R1:H, R2:OH, OleuropeinR1:OCH3, R2:OH, Me thox yoleuropeinR1:, R2:H, Ligstroside
HO
HO
OH
Hydroxytyrosol
O
OHO
O
COOH
O
OHHOHO
OH
Oleos ide
O
OHHO
HO
O
OH
OH
Tax ifolin
O
O
OH
OH
OH
OO
OHHOHO
OH
Luteolin-7-O-Glucoside
O
COOCH3
OO
OHHOHO
OH
HO
OOOOHOHO
OH
Nuzhenide
O
OOHO
HO
O
Oleace in
Fig. 2. Chemical structures of characteristic compounds in Olea europaea.
T. Michel et al. / Phytochemistry Letters 11 (2015) 424–439 435
were characterised by successive loss of 368 mass units correspond-ing to 11-methyl oleoside. It is worthwhile to note that the major ionof the HRMS/MS spectrum of nuzhenide-11-methyl oleosidederivatives 81, 82 and 84 (Table 1S, supplementary material) atm/z 771.2299 (C34H43O20) does not actually corresponds to a directfragmentation due to neutral loss or rearrangement mechanisms.Indeed, the loss of 300 mass units from the pseudo molecular ioncould not be explained by any fragmentation scheme. Therefore,we suggest that the ion at m/z 771.2299 could correspond to adimer of 11-methyl oleoside in which both units had previouslylost water (m/z 385.1123).
Finally, two compounds [Rt 12.65 (75) and 14.71 (77)] at m/z
715.2448 (C32H43O18, 11.5) were detected in stone extracts. Thesemolecules share a similar fragmentation pattern with nuzhenidewith a difference of 30 mass units suggesting an additional uniton the tyrosol part. The intense fragment ion at m/z 553.1909(C26H33O13, 10.5) is likely due to the loss of a glucose unit. Thus,and as we previously described for methoxyoleuropein, weassigned these two compounds as methoxynuzhenide. However,data are not sufficient to confirm the structure of these compoundsand differentiate them.
4.4. Oleoside type derivatives
As previously described in literature (Fu et al., 2010; Obiedet al., 2007), three peaks 31, 33 and 36 (Rt 2.56, 4.94 and 5.46 min,respectively) were found at nominal mass of 389. Compound 33was identified as loganin based on its EC while 31 and 36 showeda different fragmentation pattern. The first one, characterised bya fragment at m/z 227.0553 (C10H11O6) is attributed to a loss of aglucosyl moiety. Successively, this ion gives rise to product ions atm/z 183.0659 (C9H11O4) and m/z 165.0546 (C9H9O3) respectively,
derived by a neutral loss of CO2 and double neutral loss of CO2 andH2O and was identified as oleoside.
Compound 36 exhibited a major fragment at m/z 345.1181(C15H21O9) suggesting the presence of a carboxylic group. Theoccurrence of additional ions in lower intensity such as at m/z
209.0451(C10H9O5), m/z 165.0559 (C9H9O3) and m/z 121.0661(C8H9O) suggest the presence of secologanoside (Table 1). Frag-ments at m/z 209.0451 and m/z 165.0559 respectively correspondto the loss of C6H12O6 (confirming the presence of a glucose) andC8H14O8. This later fragment pattern has already been observed formethoxy-oleuropein. The latter ions at m/z 121.0661 might beattributed to the loss of CO2 from ion at m/z 165. Moreover, thisresult is consistent with previous work which has demonstratedthat secologanoside elutes after oleoside under reverse phaseconditions (Fu et al., 2010; Obied et al., 2007).
The two compounds observed at m/z 403.1238 (37 & 39,C17H23O11,6.5) could be attributed to the two known isomersof oleoside methyl ester: oleoside 7-methyl ester and oleoside 11-methyl ester. Moreover, several isomers of 7-b-1-D-Glucopyrano-syl-11-methyl oleoside [Rt 5.14 (34), 5.39 (35) and 10.68 (44)], abiosynthetic precursor of ligstroside and oleuropein (Damtoftet al., 1993; Gutierrez-Rosales et al., 2010; Obied et al., 2008), wereidentified in Olea roots (Table 1 & Table 1S). Other biosyntheticintermediates were detected such as loganic acid glucoside at m/z
537.1968 (C22H33O15, 38) and 7-deoxyloganic acid at m/z 359.1339(C16H23O9, 41).
4.5. Phenylethanol related compounds
Another group of compounds found in olive extracts could becharacterised as phenylethanol related compounds. Among them,the well-known hydroxytyrosol, 3 (Rt 1.4 min, m/z 153.0563,
Fig. 3. Proposed fragmentation pathway for nuzhenide-11-methyl oleoside obtained in negative mode by ESI-HRMS/MS.
T. Michel et al. / Phytochemistry Letters 11 (2015) 424–439436
C8H9O3) and its glucoside isomers, 2 (Rt 1.3 min, m/z
315.1088, C14H19O8) were identified by comparison to literaturedata and their HRMS data. Additionally, compound 4 eluted at11.09 min exhibiting a pseudo-molecular ion at m/z 477.1395(C23H25O11) and a typical HRMS/MS fragmentation was identifiedas calceolarioside, already detected in Fraxinus species (Oleaceae)(Eyles et al., 2007) but for the first time in Olea leaves (Fig. 4).Indeed, the HRMS/MS spectra gave a major ion at m/z 323.0763(C15H15O8) indicating the loss of the hydroxytyrosol moiety. Inaddition, the caffeic acid moiety is manifested by an ion at m/z
315.1062 (C14H19O8) and at m/z 161.0243 (C9H5O3) corresponding
Fig. 4. ESI(-)-HRMS/MS spectra of c
to the hydroxytyrosol glucoside part and caffeic acid ion under itsdehydrated form, respectively.
5. Hydroxycinnamic acid derivatives
Besides secoiridoids, other phenolic compounds such asverbascoside, a heterosidic ester of caffeic acid and hydroxytyrosol,were found in all organs of O. euroapaea. Two peaks, 29 and 30 (Rt
12.4 and 13.5 min, respectively) with a molecular ion at m/z
623.1846 (C29H35O15) and a similar fragmentation motif, couldboth correspond to verbascoside. In fact, as it is mentioned in
alceolarioside (m/z 477.1395).
Fig. 5. Oleuropein content in mg/g of dry extract. I, Methanol Koroneiki. II, Methanol
Chetoui. III, Ethylacetate Koroneiki. IV, Ethylacetate Chetoui.
T. Michel et al. / Phytochemistry Letters 11 (2015) 424–439 437
literature (Cardinali et al., 2012; Mulinacci et al., 2005), the formercan be attributed to verbascoside and the later to isoverbascoside.The latter is an isomer of verbascoside with the caffeoyl moietylinked to the C6 of the central glucose. For both compounds aneutral loss of 162 units is observed giving rise to m/z 461.1647 thatand can be attributed to the loss of a caffeoyl moiety. Otherverbascoside related compounds were found at m/z 639.1919(C29H35O16) and at m/z 653.2073 (C30H37O16). These ions sharethe same fragmentation pattern and were respectively identifiedas b-hydroxyverbascoside (27) and b-methoxylverbascoside, (8).Interestingly, at the HRMS/MS data of both molecules, the same ionat m/z 621.1796 (C29H33O15) was present and it is derived from theloss of water and methanol. The hydroxy derivative has alreadybeen described in olive residues (Cardinali et al., 2012; Mulinacciet al., 2005) while the methoxy derivative has only been found inFraxinus species (Sanz et al., 2012), constituting here its first reportin Olea tree. Finally, metabolite 56 with a quasi-molecular ion atm/z 535.1452 (Rt 18.06, C25H27O13) could correspond to comse-logoside (p-coumaroyl-6-secologanoside) based on the suggestedEC and its fragment at m/z 491.1543 (C24H27O11) resulting from aCO2 loss, which is in accordance with literature (Jerman et al.,2010; Obied et al., 2007) (Table 1).
6. Flavonoids
Several flavonoids were detected in Olea extracts and all ofthem were identified by comparing their ECs and fragmentationpathways with those reported in literature and databases (Bendiniet al., 2007; Ghanbari et al., 2012a; Herrero et al., 2011; Quirantes-Pine et al., 2013; Taamalli et al., 2011) (Table 1). Among them,luteolin and apigenin derivatives were the most abundant. Forinstance, three luteolin rutinoside isomers (13, 14 and 24) werereported at m/z 593.1289 (C30H25O13) and three luteolin glucosideisomers (12, 17 and 20) were detected at m/z 447.0926(C21H19O11). For all of them, the ESI-HRMS/MS spectra show acommon ion at m/z 285.0401 (C15H9O6) which can be explainedby the loss of the rutinose (308 mass units) and the glucosyl(162 mass units) moiety. Likewise, apigenin-7-O-rutinoside, 16(m/z 577.1555, C27H29O14) and apigenin-7-O-glucoside, 15 (m/z
431.0975, C21H19O10) exhibit the same loss giving rise to the ionat m/z 269.0444 (C15H9O5) corresponding to apigenin aglycon.It is worth noting, that both luteolin (22) and apigenin (25) werealso detected. Other flavonoids found in Olea extracts are taxifolin(9, m/z 303.0507, C15H11O7), diosmetin (26, m/z 299.0554,C16H11O6) and quercetin (21, m/z 301.0347, C15H9O7) as well astheir glycosidic derivatives (Table 1). For instance, two diosmetinglycosides were identified, diosmin, 19 (Rt 15.02 min, m/z
607.1534, C28H31O15) and diosmetin glucoside, 18 (Rt 13.9, m/z
461.1079, C22H21O11) showing the same product ion at m/z
299.0554 and 284.031, both characteristic of diosmetin.
7. Coumarins
In this study, the use of the Orbitrap technology allowed us todetect for the first time in O. europaea two hydroxyl-coumarins andspecifically aesculin, 5 (Rt 2.9 min, m/z 339.0650, C15H15O9) and itsaglycon, aesculetin 6 (Rt 4.1 min, m/z 177.0193, C9H5O4) (Table 1).They were identified based on EC and HRMS/MS information aswell as the use of databases (Table 1). Furthermore, chemotaxa-nomic study indicates that these coumarins have already beenidentified in the Oleaceae family, especially in the genus Fraxinus
(Eyles et al., 2007; Kostova and Iossifova, 2007) as well as in Olea
africana (Tsukamoto et al., 1985; Tsukamoto et al., 1984). Thefragmentation of aesculin in MS/MS level led to an ion at m/z
177.0193 (C9H5O4) corresponding to the loss of the glucosyl part,while the aglycon aesculetin gives rise to a product ion at m/z
133.0301 (C8H5O2) explained by the CO2 loss. These two ions havealready been described in the literature (Li et al., 2012).
8. Lignans and triterpenic acids
Lignans, another group of compounds found in olive tree,mainly characterise the stems and roots. For instance, lignansalready isolated from the bark of O. europaea such as olivil, 70(Rt 8.70 min, m/z 375.1441, C20H23O7), (+)-1-hydroxypinoresinol40-b-D-glucoside, 71 (Rt 13.15 min, m/z 535.1809, C26H31O12) and(+)-1-acetoxypinoresinol 40-b-D-glucoside, 72 (Rt 14.32 min, m/z577,1921, C28H33O13) were identified. Olivil was identified basedon its EC and its fragment ions at m/z 195.0667 (C10H11O4) and atm/z 179.0716 (C10H11O3) which are consistent with the openingand cleavage of the tetrahydrofuran ring, further loss of a methylgroup as well as a loss of a water molecule, as described by Sanzet al. in Fraxinus species (Sanz et al., 2012). The HRMS/MS spectra ofhydroxypinoresinol and acetoxypinoresinol glucosides as well ascycloolivil glucoside, 69 show the same loss of 162 mass unitsconfirming the presence of glucose unit linked to lignans aglycon(Table 1).
Additionally to lignans, two well-known triterpenic acids ofolive namely maslinic acid (73) and oleanolic acid (74) have beenidentified taking into account their EC and by comparison with in-house standards (Table 1).
8.1. Comparison between different organs and varieties
Using UHPLC-ESI-HRMS/MS to profile different Olea organs, wewere able to characterize phenylethanols, secoiridoids, flavonoids,coumarins, lignans and triterpenic acids (Table 1 & Table 1S). Somecompounds have already been described in literature such ashydroxytyrosol, oleuropein, ligstroside, 7-deoxyloganic acid,cycloolivil glucoside in wood (Ortega-Garcıa and Peragon, 2010;Perez-Bonilla et al., 2006), flavonoids in leaves and stems (Lujanet al., 2009; Lujan et al., 2008; Quirantes-Pine et al., 2013) ornuzhenide derivatives in stone (Silva et al., 2010; Silva et al., 2006).Beside the common secoiridoid and verbascoside derivatives, thetype of constituents detected in leaves, stems, roots and stonesvaried significantly. Leaf samples are mainly characterized by thepresence of numerous flavonoids including quercetin, luteolin,apigenin and diosmetin glycosides. Stem also contained someflavonoids such as taxifolin as well as luteolin and apigeninderivatives. Glucosides of lignans were only found in wood, whilemolecules such as verbascoside, isoverbascoside and loganic acidglucosides were abundant in stem and root extracts. Oleosiderelated compounds like 7-b-1-D-Glucopyranosyl-11-methyl
T. Michel et al. / Phytochemistry Letters 11 (2015) 424–439438
oleosideisomers, 7-deoxyloganic acid and p-coumaroyl-6-secolo-ganoside were mainly identified in root samples. Nuzhenidederivative were the predominant secoiridoid compounds in stoneextracts. Furthermore, a difference that could be observed betweenorgans is the oleuropein content ranging from 3.85 to 506.89 mg/gof dry extracts (Fig. 5). Root extract contains more oleuropein(222.72 to 506.89 mg/g) than stem (82.96 to 146.1814 mg/g), leaf(14.13 to 236.01 mg/g) and stone (3.85 to 23.67 mg/g), makingOlea root as a potential source of oleuropein.
Regarding the composition of the two varieties under study,UHPLC-DAD profiling and UHPLC-HRMS/MS dereplication showedthat there were not important differences. Indeed, most of thecompounds found in Koroneiki are also present in Chetoui extracts.Only minor differences especially related to qualitative alterna-tions can be observed. For instance methoxyoleuropein isomersare more abundant in Koroneiki than in Chetoui extracts or b-methoxylverbascoside which is only present in Koroneiki samples.Conversely, oleuroside is predominant in Chetoui leaves while isa minor compound in Koroneiki leaves.
Overall, in the context of this study 86 different olive constituentshave been tentatively identified implying the valuable potentialsof Orbitrap analyser mainly related to accuracy, robustness andgenerally structure elucidation power.
9. Conclusion
Despite the numerous previous studies dealing with thephenolic composition of olive tree, the present work providesnew insight in the phytochemical content of olive leave, stem, rootand stone. The dereplication strategy applied using an UHPLChyphenated with an Orbitrap mass analyzer has proved to be apowerful tool for characterization of several compounds in thetwo varieties under investigation, Koroneiki and Chetoui. Thus,86 molecules were identified including compounds described forthe first time in O. europaea such as coumarins. Furthermore, somemetabolites found to be organ specific. For instance, olive stonecould be an excellent source of nuzhenide and its derivatives, whileflavonoids were mainly found in olive leaves. All organs containoleuropein and based on UHPLC-DAD quantification oleuropeincontent is higher in olive roots. These findings provide a betterunderstanding of the bioactive compounds of less recognizedorgans of olive tree. It is worth noting that several compoundsremained unidentified revealing the chemical diversity of Olea.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.phytol.2014.12.020.
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