Chemometric Analysis for the Evaluation of Phenolic Patterns in Olive

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Chemometric analysis for the evaluation of phenolic patterns in olive leaves from six cultivars at different growth stages Nassima Talhaoui, Ana Maria Gómez-Caravaca, Cristina Roldán, Lorenzo Leon, Raul de la Rosa, Alberto Fernandez-Gutierrez, and Antonio Segura-Carretero J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 22 Jan 2015 Downloaded from http://pubs.acs.org on January 26, 2015

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Journal of Agricultural and Food Chemistry

Chemometric Analysis for the Evaluation of Phenolic Patterns in Olive Leaves from Six Cultivars at Different Growth Stages

Nassima Talhaoui

1,2

, Ana María Gómez-Caravaca

1,2,*

, Cristina Roldán2, Lorenzo León 3,

Raúl De la Rosa 3, Alberto Fernández- Gutiérrez 1,2, Antonio Segura-Carretero 1,2

1

Department of Analytical Chemistry, University of Granada. Avda. Fuentenueva s/n, 18071

Granada (Spain). 2

Research and Development of Functional Food Centre (CIDAF), PTS Granada, Avda. del

Conocimiento s/n., Edificio Bioregión, 18016 Granada, Spain. 3

IFAPA Centro Alameda del Obispo, Avda Menéndez Pidal, s/n, E-14004 Córdoba, Spain.

* Corresponding autor. Ana Mª Gómez-Caravaca, E-Mail: [email protected]; Tel.: +34-958-637206; Fax: +34-958-637083

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ACS Paragon Plus Environment


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Abstract

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Leaves from six important olive cultivars grown under the same agronomic conditions were

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collected at four different times from June to December and analyzed by high performance

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liquid chromatography-diode array detector-time of flight-mass spectrometry (HPLC-DAD-

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TOF-MS). 28 phenolic compounds were identified and quantified. No qualitative differences

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were detected among leaves. However, for all cultivars, total concentrations of phenolic

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compounds decreased from June to August, then increased from October on, and reached

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higher levels again in December. Principal Component Analysis provided a clear separation

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of the phenolic content in leaves for different sampling times and cultivars. Hence, the

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availability of phenolic compounds depends on both the season and the cultivar. June and

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December seem to be a good time to collect leaves as a source of phenolic compounds.

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December coincide with the harvest period of olives in the Andalusian region. Thus, in

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December olive leaves could be valorized efficiently as olive by-products.

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Keywords: Olive leaves, phenolic compounds, HPLC-DAD-TOF-MS, cultivar, sampling

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time.

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INTRODUCTION

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Secondary metabolites such as phenolic compounds play a key role in plants; defense

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mechanisms against herbivores and biotic infections 1,2, and also in adaptation to abiotic stress

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3

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plant tolerance to salinity 4, and a link has also been established between tolerance to

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oxidative stress induced by water deficit and a rise in the antioxidant concentration in

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photosynthetic plants 5,6.

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The level of phenolics in plants varies extensively; it is affected by many factors that

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influence phenolic stability, biosynthesis and degradation. These include genetic and

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physiological factors as well as environmental factors 7. Therefore, the effect of phenolic

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compounds in plants resistance depends upon their respective biological activities, which in

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turn can be determined by the particular physico-chemical environments to which the

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compounds are exposed (high and low temperature, drought, alkalinity, salinity, UV stress,

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bacteria, fungi, insects, etc) 8,9.

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The olive tree (Olea europaea L.) is one of the oldest and most characteristic crops in the

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Mediterranean basin, as 95% of the world’s surface dedicated to olives is concentrated in this

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area10. Olive trees are considered drought tolerant because trees can survive on shallow soils

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with little supplemental water beyond winter rainfall that is typical of the Mediterranean

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climate. This is possible because, as can be observed in several plants of the Mediterranean

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shrubland biome, the olive tree has developed a series of physiological mechanisms to tolerate

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drought stress and grow under adverse climatic conditions 11. The most relevant mechanisms

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are: the regulation of stomata closure and transpiration, the regulation of gas exchange,

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osmotic adjustment and regulation of the antioxidant system

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cultivars have shown high resistance to diseases such as Verticillium wilt (caused by

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Verticillium dahliae) and olive scab (caused by Fusicladium oleagineum). Various studies

. In fact, many studies strongly support the idea that polyphenols play a significant role in

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11,12

. In addition, some olive


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have related this pathogenic resistance to a multifactorial phenolic component (tyrosol and its

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derivatives, oleuropein and rutin) 13–15.

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Spain is one of the world’s leading producer, importer and exporter countries in terms of

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olives oil and fruit, with a production of 7,820,060 tons. As result of olive processing, a huge

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quantity of olive by-products are produced annually; just in the Andalusian region around

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277,063 tons of olive stones, 985,552 tons of olive cake and 432,984 tons of olive leaves and

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twigs are generated

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agricultural tradition of the country. Olive leaves are one of those by-products that are used in

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many areas as animal food

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antioxidant and bioactive compounds, olive leaves have strong potential to be used in

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pharmaceutical preparations and as a supplement in the functional food industry

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several studies have been made on the health-promoting potential of olive leaves due to the

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phenolic compounds they contain

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the understanding of the metabolism of some phenolic compounds in olive leaves, or the

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influence of factors such as water deficit, genetic factor or seasonal period 21–25.

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In a previous work 26, our group reported the phenolic composition of ‘Sikitita’ (‘Chiquitita’

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in USA) , a newly bred olive cultivar

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phenolic compounds and cultivars were considered with the main aim of providing further

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insights into the evolution of olive leaves phenolic compounds in different olive cultivars

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during their growth and the olive ripening period under the Andalusian climate. We also

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highlight the optimal sampling time to use olive leaves as a source of bioactive compounds.

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. The use of by-products of this crop has long been part of the

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or energetic biomasses

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. Furthermore, as a great source of

. Others studies have also been carried out focused on

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. In the present work an exhaustive number of

MATERIALS AND METHODS

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. In fact,

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Chemicals and reagents 4


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Methanol, the reagent used for extracting the phenolic compounds from the olive- leaves

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samples, was purchased from Panreac (Barcelona, Spain), and HPLC-grade acetonitrile was

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purchased from Labscan (Dublin, Ireland). The acetic acid used was of analytical grade

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(assayed at >99.5%) and was purchased from Fluka (Switzerland). Water was purified using a

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Milli-Q system (Millipore, Bedford, MA, USA). Standard compounds such as

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hydroxytyrosol, tyrosol, luteolin, and apigenin were purchased from Sigma-Aldrich (St.

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Louis, MO, USA), and oleuropein from Extrasynthèse (Lyon, France). The stock solutions

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containing these analytes were prepared in methanol. All chemicals were of analytical reagent

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grade and used as received. All the solutions were stored in a dark flask at -20 °C until use.

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Samples

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Olive leaves (Olea europaea L.) from cultivars ‘Arbequina’, ‘Arbosana’, ‘Changlot Real’,

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‘Koroneiki’, ‘Picual’ and ‘Sikitita’ were used in this study. These cultivars were selected as

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some of the most widely used in new orchards currently in Spain, highly productive, well

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adapted to modern olive growing techniques and initially originated in different areas:

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‘Arbequina’ and ‘Arbosana’ from Catalonia (Spain), ‘Changlot Real’ from Valencia (Spain),

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‘Picual’ from Andalusia (Spain), ‘Koroneiki’ (Greece), and “Sikitita”, a new Spanish cultivar

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from cross-breeding between ‘Arbequina’ and ‘Picual’. All cultivars were grown under the

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same agronomic and environmental conditions in the same olive orchards located at “IFAPA,

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Centro Alameda del Obispo” in Córdoba, Spain (37°51'36.5"N 4°47'53.7"W). Samples were

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processed at four times: mid-June (fruit-set), mid-August, mid-October and mid-December

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(fruit-ripening) in 2012. Adult leaves were collected from three individuals of each cultivar,

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in five years old trees of these cultivars planted at 7 x 5 m spacing and trained as single-trunk

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vase. Standard cultural practices were followed, with minimal pruning to allow early bearing

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and irrigation by in-line drips with 2000 m3/ha per year to avoid water stress of plants. All the 5



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leaves collected from the same tree were pooled in a unique sample, immediately transferred

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to the laboratory, and dried outdoors. Finally, samples were stored at -80 ºC until needed.

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Extraction of phenolic compounds from olive leaves

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Sample extraction was performed as described previously by Talhaoui et al.

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leaves (0.5 g) were crushed and extracted via Ultra-Turrax IKA® T18 basic using 30 mL of

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MeOH/H2O (80/20). After solvent evaporation, the extracts were reconstituted with 2 mL of

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MeOH/H2O (50/50). Three replicates of each sample were processed.

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. Briefly, dry

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Determination of phenolic compounds by HPLC-DAD-TOF-MS

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Phenolic compounds were separated by a Poroshell 120 EC-C18 analytical column (4.6 × 100

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mm, 2.7 µm) from Agilent Technologies, on an Agilent 1200 series Rapid Resolution Liquid

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Chromatograph (Agilent Technologies, CA, USA). The gradient eluent, at flow rate of 0.8

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mL/min, was achieved using the method previously described by Talhaoui et al.

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column temperature was maintained at 25 °C and the injection volume was 2.5 µL.

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The HPLC system with diode-array detection was coupled to a micrOTOF (Bruker Daltonics,

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Bremen, Germany), an orthogonal-accelerated TOF mass spectrometer, using an electrospray

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interface (model G1607A from Agilent Technologies, Palo Alto, CA, USA). The effluent

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from the HPLC column was split using a T-type phase separator before being introduced into

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the mass spectrometer (split ratio = 1:3). Analysis parameters were set using a negative-ion

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mode with spectra acquired over a mass range from m/z 50 to 1000. The optimum values of

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the ESI-MS parameters were: capillary voltage, +4.5 kV; drying gas temperature, 190 °C;

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drying gas flow, 9.0 L/min; and nebulizing gas pressure, 2 bars. The accurate mass data on the

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molecular ions was processed through the newest Data Analysis 4.0 software (Bruker

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Daltonics, Bremen, Germany), which provided a list of possible elemental formulae via the 6


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. The

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Smart Formula Editor. The Smart Formula Editor uses a CHNO algorithm, which provides

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standard functionalities such as minimum/maximum elemental range, electron configuration

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and ring-plus double-bond equivalents, as well as a sophisticated comparison of the

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theoretical with the measured isotope pattern (Sigma Value) for increased confidence in the

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suggested molecular formula. Peak areas of phenolic compounds were integrated using

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Bruker Compass Target Analysis 1.2 software for compound screening (Bruker Daltonics,

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Bremen, Germany). All phenolic compounds showed good levels for quantification in the

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various samples on each date of sampling. Five standard calibration graphs were prepared for

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quantification of the phenolic compounds in the olive leaves using five commercial standards

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(oleuropein, hydroxytyrosol, tyrosol, apigenin, and luteolin).

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Statistical analysis

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All assays were run in triplicate. Values of different results were expressed as the means mg/g

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olive leaves. Results were tested for statistical significance by one-way ANOVA. Significant

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statistical differences among treatments (p

Chemometric Analysis for the Evaluation of Phenolic Patterns in Olive

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