GC-QTOF-MS as valuable tool to evaluate the influence of cultivar and sample time on olive leaves triterpenic components
Graphical abstract
Introduction
Pentacyclic triterpenes belong to plant secondary metabolites. These compounds are constituted by 30 carbons that are grouped in five cycles of six carbons with different substituents (Guinda, Rada, Delgado, Gutiérrez-Adánez, & Castellano, 2010) and they are biosynthesized by the acetate/mevalonate cytosolic pathway which yields (3S)-2,3-oxidosqualene (OS) (Crozier, Clifford, & Ashihara, 2006). The main triterpenoids found in the plant kingdom are oleanolic acid, maslinic acid, ursolic acid, erythrodiol and uvaol. These compounds play an important role in plant defense, as proven by the production of triterpenic phytoalexins (Van der Heijden, Threlfall, Verpoorte, & Whitehead, 1989) or saponins (Papadopoulou, Melton, Leggett, Daniels, & Osbourn, 1999) in response to biotic and abiotic stress. Indeed, triterpenoids are present in the surface of the leaves as constituents of waxes being involved in different roles such as maintenance of leaves structure, provide water, permeability, plant−insect interactions, etc.… (Bauer, Schulte, & Thier, 2004; Mintz-Oron et al., 2008; Shan, Wilson, Phillips, Bartel, & Matsuda, 2008). Moreover, triterpenoids have some important properties that cause beneficial effects in human health and act in disease prevention. They have been studied for their important effects as antimicrobial and antiviral (García-Granados et al., 1999; Rivas, Osuna, Mascaró, & Nájera, 2000; Xu, Zeng, Wan, & Sim, 1996), and their antioxidant (Montilla et al., 2003; Wang, Xia, & Cui, 2006; Yang et al., 2007), anti-inflammatory (Márquez Martín, De la Puerta Vázquez, Fernández-Arche, & Ruiz-Gutiérrez, 2006), anti-fungal (Duarte Rocha, Braga de Oliveira, de Souza Filho, Lombardi, & Castro Braga, 2004), anti-diabetic (Castellano, Guinda, Delgado, Rada, & Cayuela, 2013), hepatoprotective (Gutiérrez-Rebolledo, Siordia-Reyes, Meckes-Fisher, & Jiménez-Arellanes, 2016), antiatherogenic (Kirmizis & Chatzidimitriou, 2009), gastroprotector (Sánchez et al., 2006) and hypolipidemic (Pérez Gutiérrez, 2017) activities. Furthermore, some triterpenoids have shown to possess antitumor activities against a large amount of tumor types (Reyes-Zurita, Rufino-Palomares, Lupiáñez, & Cascante, 2009; Yamai et al., 2009; Zhou et al., 2011).
The olive tree (Olea europaea L.) is one of the most ancient and important crop in the Mediterranean basin, having this area the 95% of the olive orchards of the world (Ghanbari, Anwar, Alkharfy, Gilani, & Saari, 2012). This habitat is determined by Mediterranean climate, which is characterised by long, hot, dry summers and mild, rainy winters. Nevertheless, olive tree can cope with the low availability of water in soil by means of a series of morphological, physiological and biochemical mechanisms acquired in response to lean periods of water in summer (Sofo, Manfreda, Fiorentino, Dichio, & Xiloyannis, 2008).
During olive oil processing, large quantities of by-products are produced. Just in the Andalusian region, Southern Spain, amounts as high as 277,063 tons of olive stones, 985,552 tons of olive cake, and 432,984 tons of olive leaves and twigs are generated (Callejo López, Parra Heras, & Manrique Gordillo, 2010).
Olive by-products possess large amounts of bioactive compounds and olive leaf has been the object of special attention due to the presence of different families of bioactive compounds, such as phenolic derivatives, alditols and pentacyclic triterpenes, which have interesting pharmacological properties (Erba & Icier, 2010; Guinda et al., 2015). Oleanolic acid is the main olive leaf triterpenoid representing the 3.0–3.5% leaves dry weight (d.w.), followed by significant concentrations of maslinic acid and minor levels of ursolic acid, erythrodiol, and uvaol (Guinda et al., 2015). Despite that fact, olive residues are often disposed as waste after incineration or milling and, then, scattering on the field or used as animal feed, giving cause to environmental and economic problems (Guinda et al., 2015). For this reason, the utilization of these by-products in the elaboration of new products could be profitable for olive groves. In fact, nowadays, there is special interest on the use of olive by-products extracts to improve the nutritional profile of food products or to produce nutraceutics (Nunes, Pimentel, Costa, Alves, & Oliveira, 2016).
Determination of triterpenoids compounds has usually been carried out by gas chromatography coupled with mass spectrometry (Guinda, Albi, Pérez-Camino, & Lanzón, 2004; Sánchez Ávila, Priego Capote, & Luque de Castro, 2007) or flame ionization (Guinda et al., 2010; Harman-Ware, Sykes, Gary, & Davis, 2016; Jäger, Trojan, Kopp, Laszczyk, & Scheffler, 2009). Because of the low volatility and high molecular weight of these compounds, a previous derivatization step is necessary before the gas chromatography analysis. Derivatization of triterpenes is usually performed by silylation, which is a cumbersome procedure and needs reaction times from 30 min to 3 h (Boskou et al., 2006; Guinda et al., 2010; Janicsák, Veres, Zoltán Kakasy, & Máthé, 2006; Jemmali, Chartier, Dufresne, & Elfakir, 2016; Modugno, Ribechini, & Colombini, 2006).
Despite the fact that the triterpenoid profile of olive leaves has been studied before, there are no studies that report the evolution of these triterpenoids during their growth and the olive ripening period under the Andalusian climate. Thus, this work has focused on the determination of triterpenes compounds from olive leaves of six different cultivars grown in the same experimental field under the same environmental and agronomic conditions at four dates (June, August, October and December) in order to corroborate if the sampling time corresponding to the olive oil production (that generates high olive leaves by-products) is also the best one in terms of triterpenes accumulation.
Section snippets
Chemicals and reagents
Ethanol, the reagent used for extracting triterpenoids compounds from olive leaf samples was purchased from Fisher Scientific UK (Bishop Meadow Road, Fisher Chemical), and n-hexane HPLC grade was purchased from LAB-SCAN (44 - 101 Gliwice, ul. Sowinskiego 11, Poland). Standard compounds such as oleanolic and maslinic acid were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Dihydrocholesterol from Sigma-Aldrich (Saint Louis, MO, USA) was used as internal standard. Standard dilutions were
Analytical parameters of the method
An analytical verification of the method was performed considering linearity, sensitivity, repeatability, and accuracy. Two calibrations curves were elaborated with the standards maslinic acid and oleanolic acid. Table 1 lists the analytical parameters of the two standards used containing lineal range, calibration curve, determination coefficients, relative standard deviation (RSD%), limit of detection (LOD), limit of quantification (LOQ), and accuracy.
Calibration curves were constructed using
Conclusions
A GC-QTOF-MS has been established for the determination of triterpenic compounds in olive leaves for the first time. Despite the high number of reports about the triterpenic composition of olive leaves, to our knowledge, this is the first study that evaluates the effect of six cultivars (the most widely cultivated in Spain) and four sampling times on these compounds. Major changes occurred between June and August, and afterwards, the concentration of triterpenes stabilized. Quantitative
Acknowledgement
Vito Verardo thanks the MINECO for his “Ramon y Cajal” contract (RYC-2015-18795).
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