Is There Room for Improving the Nutraceutical Composition of Apple

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Is there room for improving the nutraceutical composition of apple? Brian Farneti, Domenico Masuero, Fabrizio Costa, Pierluigi Magnago, Mickael Malnoy, Guglielmo Costa, Urska Vrhovsek, and Fulvio Mattivi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00291 • Publication Date (Web): 27 Feb 2015 Downloaded from http://pubs.acs.org on March 4, 2015

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Journal of Agricultural and Food Chemistry

Is there room for improving the nutraceutical composition of apple?

Brian Farneti1, Domenico Masuero2, Fabrizio Costa3, Pierluigi Magnago3, Mickael Malnoy3, Guglielmo Costa1, Urska Vrhovsek2, Fulvio Mattivi2*

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Department of Agricultural Sciences, Bologna University, Via Fanin 46, 40127 Bologna, Italy

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Department of Food Quality and Nutrition, Research and Innovation Centre, Fondazione Edmund

Mach (FEM), via E. Mach 1, 38010 San Michele all'Adige, Italy 3

Department of Genomics and Biology of Fruit Crop, Research and Innovation Centre, Fondazione

Edmund Mach (FEM), Via Mach 1, 38010 San Michele all’Adige (Trento), Italy

*Corresponding Author Fulvio Mattivi Department of Food Quality and Nutrition, Research and Innovation Centre, Fondazione Edmund Mach (FEM), via E. Mach 1, 38010 San Michele all'Adige, Italy. Telephone: +39 0461 615 259 E-mail: [email protected]

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Abstract

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In this study we assessed the main bioactive compounds of a broad apple germplasm collection,

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composed by 247 accessions of wild (97) and domesticated (150) species. Among the stilbenes,

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trans- and cis-piceid were found to be ubiquitary components of both wild and cultivated apples.

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Apple was suggested to be the second dietary source of resveratrols. Results confirmed that the

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selection pressure of breeding and domestication did not affect uniformly all the phytochemicals

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contained in apple. For instance, organic acids (malic and ascorbic acid) and some phenolics

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(stilbenes, hydroxycinnamic acids, and dihydrochalcones) were significantly influenced by

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selection while some relevant flavonoids (flavonols and flavan-3-ols) and triterpenoids (ursolic,

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oleanoic, and betulinic acids) were not. This comprehensive screening will assist in the selection of

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Malus accessions with specific nutraceutical traits suitable to establish innovative breeding

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strategies or to patent new functional foods and beverages.

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Keywords

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Malus x domestica Borkh.; phenolics; triterpenoids; ascorbic acid; metabolic phylogenesis;

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domestication; piceid, resveratrol, dietary intake

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Introduction

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Providing higher nutritional content in fruits and vegetables at affordable prices is likely to increase

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their consumption, which would be good for producers, growers and the logistic chain as well as for

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consumers. A new diet-health paradigm is evolving which places more emphasis on the positive

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aspects of diet. We are in the middle of a revolution that is changing the concept of food and our

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way of eating.1 Fresh fruit and vegetables have now assumed the status of functional foods, which

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should be capable of providing additional physiological benefit, such as preventing or delaying

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onset of chronic diseases, as well as meeting basic nutritional requirements. 1-3 Numerous

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epidemiological studies have shown the inverse association between fruit and vegetable

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consumption and risk of many chronic diseases including cardiovascular diseases, cancer, diabetes,

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osteoporosis, Alzheimer and other degenerative diseases. 4-9

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The consumption of apple (Malus x domestica Borkh.) fruit has been inversely related with lung

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cancer incidence,10-12 cardiovascular disease and coronary mortality,13 symptoms of chronic

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obstructive pulmonary disease,14-16 risk of thrombotic stroke17 and type II diabetes.18 These health-

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promoting properties are thought to be the result mainly of the large amounts of phenolic,

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triterpenes, fiber and vitamin compounds. Several mechanisms of action have been elucidated,

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including reduction of blood lipids and blood pressure, antioxidant and anti-inflammatory

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properties, weight loss, positive effects on glucose homeostasis and vascular functions, and

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modulation of the human gut microbiota.19

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Phytochemical concentrations significantly differ between cultivars20-22 and vary according to the

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fruit maturation as well as growing condition especially light availability and fertilization.21-24

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Apples are a rich dietary source of phenolic compounds which are responsible for most of the

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antioxidant activities of the fruit.25 Six main polyphenolic groups, distributed in the peel, pulp and

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core, are found among Malus germplasm: hydroxycinnamic acids, flavan-3-ols, anthocyanins,

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flavonols and dihydrochalcones,24 plus the stilbenes (trans- and cis-piceid), which were recently 3 ACS Paragon Plus Environment


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found in ‘Golden Delicious’ apples.26 Amongst them, flavan-3-ols and in particular oligomeric

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proanthocyanidins turn out as the main antioxidant source.25 Dihydrochalcones, mainly phloridzin,

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represent only a small amount of total polyphenols (around 3% of the total content), although in

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some russetted apple cultivars the concentration of phloridzin can rise up to 10%.20 Phloridzin has

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been shown to be a strong inhibitor of lipid peroxidation and, moreover, to improve insulin

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sensitivity by lowering blood sugar.27 In fact, phloridzin’s principal pharmacological action is to

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produce renal glycosuria and block intestinal glucose absorption through inhibition of the sodium-

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glucose symporters.28,29

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In addition to phenolic compounds apple fruit contains considerable amount of lipophilic

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triterpenoids principally localized into the cuticular wax layer.20,30,31 The composition of the

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cuticular wax has been studied because its relevance in avoiding fruit damage caused by abiotic and

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biotic agents.31 However the wax composition has been recently correlated also with the antioxidant

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and anti-inflammatory proprieties.20,32 The main triterpenoids detected in apple cuticular wax are

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ursolic, oleanoic and betulinic acids. Ursolic acid is a ubiquitous triterpenoid in the plant kingdom

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that was long considered to be biologically inactive, whereas in recent years it has attracted

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considerable interest because of its pharmacological effects such as the inhibition proliferation of

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HepG2 human hepatoma cells, MCF-7 human breast cancer cells, and Caco-2 human colon cancer

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cells.32

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Only recently, breeding for phytochemicals in horticultural crops has been identified as an

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important goal in the development of new cultivated lines. The plant’s genotype is considered of

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primal importance in the determination of its phytochemical profile, often surpassing the impact of

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cultural practices such as irrigation or fertilization. Horticultural crops such as strawberry, apple,

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tomato, potato, cabbage, broccoli, lettuce, onion, cranberry and raspberry are currently the subject

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of breeding programs in which the phytochemical content was considered a key component.33 The

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effect of domestication and particularly of breeding on apple bioactive compounds content is still 4 ACS Paragon Plus Environment

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unresolved and matter of debate. The inconsistency of recent results20,22,34 has not yet elucidated

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the effective impact of breeding activities on the total phytochemical content in apple fruit. We

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suggest that this open question deserve further investigation, since the breeding of the future

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cultivars should have the roots in a solid knowledge of the metabolic phylogenesis, taking into

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account the composition and relationships among the apples of the past (wild species, old cultivars)

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and of the present (elite cultivars).

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In the present study a broad apple germplasm collection, composed by both wild and domesticated

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species, was analysed to assess the amount of main bioactive compounds of apple fruit, namely

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phenolics, triterpenes and ascorbic acid. The availability of this repertoire of information allowed

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the study of the largest catalogue of apple bioactive compounds to date presented to the scientific

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community. It allowed to discover that apple is a significant dietary source of resveratrols (trans-

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and cis-piceid). Moreover, the effect of domestication on the actual concentration of these

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compounds and their potential role in breeding programs are discussed.

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Materials and methods

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Plant material and fruit sampling.

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The metabolic profiling was performed on an apple fruit collection represented by 247 accessions,

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composed by both wild Malus species (97) and Malus x domestica cultivars (150) (supplementary

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table 1). Plant materials were planted in triplicates for each accession and grown at the experimental

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orchard of the Fondazione Edmund Mach in the Northern part of Italy (Province of Trento). Each

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plant was maintained following standard agronomical management for pruning, thinning and pest

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disease control.

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A minimum of five homogeneous apples, harvested at optimal ripening stage as defined by the

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Extension Service of the Fondazione Edmund Mach and by fruit colour comparison, were collected 5 ACS Paragon Plus Environment


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from each accession and maintained at room temperature (~20°C) overnight before sampling. The

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entire collection was assessed for bioactive compound profiling in the first year, while in the second

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year only a subset, composed by 23 commercially important accessions, was assessed in order to

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validate the procedure.

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The core of three fruits per sample was removed with an apple corer. Three slices (cortex + skin) of

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each fruit, taken from opposite sides, were cut in small pieces, grinded under liquid nitrogen, and

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stored in falcon tubes at – 80 °C until the analysis.

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Phenolic compounds analysis.

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Extraction and analysis of phenols were performed according to the UPLC-MS/MS method

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published by Vrhovsek et al.26 An aliquot of 2 g of powdered tissues were extracted in sealed glass

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vials using 4 mL of water/methanol/chloroform solution (20:40:40). After vortexing for 1 min, the

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samples were mixed using an orbital shaker for 15 min at room temperature, and further centrifuged

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at 1000g (4 °C) for 10 min, after which the upper phases, made up of aqueous methanol extract,

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were collected. Extraction was repeated by adding another 2.4 mL of water/methanol (1:2) to the

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pellet and chloroform fractions. After the final centrifugation, the upper phases from the two

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extractions were combined and brought to the volume of 10 mL and filtered with a 0.2 µm PTFE

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filter prior to liquid chromatography-mass spectrometry analysis. Ultra-performance liquid

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chromatography was performed employing a Waters Acquity UPLC system (Milford, MA, USA)

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coupled to a Waters Xevo TQMS (Milford, MA, USA) working in ESI ionisation mode. Separation

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of the phenolic compounds was achieved on a Waters Acquity HSS T3 column 1.8 µm, 100 mm ×

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2.1 mm (Milford, MA, USA), kept at 40 °C, with two solvents: A (water containing 0.1% formic

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acid) and B (acetonitrile containing 0.1% formic acid). The flow was 0.4 mL/min, and the gradient

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profile was 0 min, 5% B; from 0 to 3 min, linear gradient to 20% B; from 3 to 4.3 min, isocratic

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20% B; from 4.3 to 9 min, linear gradient to 45% B; from 9 to 11 min, linear gradient to 100% B; 6 ACS Paragon Plus Environment

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from 11 to 13 min, wash at 100% B; from 13.01 to 15 min, back to the initial conditions of 5% B.

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From both standard solutions and samples, kept at 6°C, a volume of 2 µL was injected, after which

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the needle was rinsed with 600 µL of weak wash solution (water/methanol, 90:10) and 200 µL of

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strong wash solution (methanol/water, 90:10). Detection was performed by multiple reaction

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monitoring (MRM) with a Waters Xevo TQMS (Milford, MA, USA) triple quadrupole (QqQ)

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equipped with an electrospray (ESI) source. Capillary voltage was 3.5 kV in positive mode and

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−2.5 kV in negative mode; the source was kept at 150 °C; desolvation temperature was 500 °C;

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cone gas flow, 50 L/h; and desolvation gas flow, 800 L/h. Unit resolution was applied to each

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quadrupole. Each phenolic compound was analysed under the optimised MRM conditions

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(precursor and product ions, quantifier and qualifiers, collision energies, and cone voltages)

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described in Vrhovsek et al., 2012.26 Data were processed by Waters MassLynx 4.1 and TargetLynx

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software.

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Triterpenoids analysis.

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The extraction was performed according to Andre et al.20 5 mL of ethyl acetate and 5 mL of hexane

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were added to an aliquot of 500 mg of powdered tissues, shacked for 1 hour and centrifuged at

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2000g for 10 minutes. The dried extract was dissolved in 1 mL of ethanol and filtrated.

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Quantification and separation of triterpenoids was carried out with a Waters HPLC system Alliance

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2695 Separations Module equipped with a mass spectrometer Micromass ZQ and Waters Empower

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Pro software. The separation was performed on Phenomenex Luna C18 column, 250 x 2 mm, 5

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um and with a mobile phase consisting of 1% formic acid in water (solvent A) and methanol

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(solvent B). The column was heated at 35 °C with a flow of 0.3 ml/min under the following

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conditions: linear gradient from 50 to 85% of B in 5 min, to 90 % B in 25 min. The column was

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washed with 100% B for 5 min and equilibrated with the initial conditions for other 5 min. The

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injection volume was 20 µl. The conditions of the mass spectrometer were: capillary voltage 3000 7 ACS Paragon Plus Environment


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V, cone voltage 25 V, extractor voltage 10 V, source temperature 125 °C, desolvation temperature

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500 °C, cone gas flow (N2) 30 l/h, desolvation gas flow (N2) 1000 L/h. The scan speed was 0.1 s

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in SIM mode with detection in negative mode at m/z 455.50. The retention time for the betulinic

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acid was 21.5 minutes, 22.7 minutes for the oleanolic acid and 23.2 minutes for the ursolic acid.

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Organic acids analysis.

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An aliquot of 5 g of the edible part of the frozen fruit was homogenized in 50 ml of a solution of

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metaphosphoric acid (0.6%) in water containing sodium meta-bisulfite (1 g/L). Analysis of organic

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acids were performed as described in Vrhovsek et al.35 The separation was carried out on a Waters

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2690 HPLC system equipped with Waters 2996 DAD (Waters, Milford, MA), and Empower

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Software (Waters, Milford, MA.) on a reversed-phase column Discovery, RP C18 (Supelco, 250 x 4

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mm, 5 µm). The solvent used was 0.07% phosphoric acid in water. The isocratic separation was

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carried out at 30 °C in 10 min. After each run, the column was washed 5 min with 15% acetonitrile

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in 0.07% phosphoric acid in water and afterward equilibrated for 7 min prior to each analysis. The

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flow rate was 0.6 mL/min, the injection volume was 20 uL, and the UV-vis signal was set at 243

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nm for ascorbic acid and 210 nm for malic acid.

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Statistical analysis.

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Multivariate statistical analysis was performed on log transformed data using R 3.0.2 internal

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statistical functions and the external packages “PCA”, “gplots”, “ape”, and “diversitree”.

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Visualization of significant metabolite correlations (p < 0.01; R > 0.50) was conducted by the

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generation of a Correlation Analysis Network with Cytoscape.

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Results and discussion

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Total bioactive compounds of apple species.


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Total content of bioactive compounds, mainly phenolics, triterpenoids and ascorbic acid,

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significantly differed among wild Malus species as well as a M. x domestica cultivars (Table 1). The overall content of phenols in M. x domestica ranged between 225 µg/g FW (cv

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Laimburg Gelber Edelaefer) and 3307 µg/g FW (cv Permein dorato) with the elite market cultivars

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(supplementary Table 2) in the range between 435 µg/g FW (cv Scilate) and 2450 µg/g FW (cv

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Renetta Canada). More variation was found between the wild Malus species, ranging between 353

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µg/g FW (M. x Dawsoniana) and 6513 µg/g FW (M. angustifolia). Distribution of each phenolic

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compounds in the groups of genotypes assessed in this research is reported in Table 1. In detail,

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wild Malus species were characterized by a higher average content of all phenolic compounds than

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the M. x domestica, for which the differences between old and elite cultivars were negligible.

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Remarkably, the dihydrochalcones (phloridzin) content assessed in wild Malus species (average of

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262 µg/g FW with a maximum of 1081 µg/g FW) was significantly higher when compared with the

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levels recorded on the M. x domestica species (average of 52 µg/g FW with a maximum of 310 µg/g

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FW).

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The total content of triterpenoids in M. x domestica accessions ranged between 6 µg/g FW

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(cv Spitzlederer) and 1121 µg/g FW (cv Napoleone), with the market elite cultivars between 233

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µg/g FW (cv Renetta Canada) and 785 µg/g FW (cv Mutsu). Comparable variation was also found

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between the wild Malus species that ranged between 58 µg/g FW (M. x Hartwigii) and 1384 µg/g

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FW (M. sylvestris) (supplementary Table 1). Likewise average content of each triterpenoids

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(namely ursolic, oleanoic and betulinic acids) did not relevantly diverge between M. x domestica

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and wild Malus accessions. The most abundant triterpenoid, as already reported by Andre et al.20

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and Frighetto et al.,31 was ursolic acid followed by its isomeric compound oleanoic and betulinic

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acid (average values respectively around 400, 60 and 10 µg/g FW).

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Differences in organic acids content between M. x domestica and wild Malus accessions were more noticeable when compared to the other investigated metabolites. In fact, most of the wild 9 ACS Paragon Plus Environment


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Malus species had a relevant higher average content of both ascorbic and malic acid (140 and 16319

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µg/g FW, respectively). Contrarily, no remarkable differences were discovered between old and

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elite M. x domestica cultivars (average value of ascorbic and malic acid around 80 µg/g FW and

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7000 µg/g FW, respectively).

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Correlation network analysis of the investigated metabolites (Figure 1) was mainly assessed

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to check the accuracy of the used dataset rather than to gain knowledge about the metabolic

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pathways of apple secondary metabolites. The network was created from a significant Pearson

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correlation data set (P

Is There Room for Improving the Nutraceutical Composition of Apple

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