Analysis of minor components in olive oil

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Abstract

Virgin olive oil is well known for its high content of phenolic substances that are thought to have health-promoting properties. These substances also contribute to the distinctive taste of the oil. In this study, tyrosol, vanillic acid, luteolin, and apigenin were identified and quantified by liquid chromatography mass spectrometry (LC-MS). In the seven samples analysed, tyrosol, the most abundant, was in the range of 1.4–29 mg/kg, vanillic acid was in the range of 0.67–4.0 mg/kg, luteolin was in the range of 0.22–7.0 mg/kg, and apigenin was in the range of 0.68–1.6 mg/kg. It was also shown that in olive oil, squalene can be analysed by using a refractive index detector. In the samples analysed, squalene occurred in the range of 3.9–9.6 g/l.

Introduction

The world production of olive oil is ca. 2.5 million tons, with Spain, Italy, Greece, and Maghrebian countries major producers. Abundance of oleic acid, a monounsaturated fatty acid, is the feature that sets olive oil apart from other vegetable oils. In particular, oleic acid ranges from 56% to 84% of total fatty acids, while linoleic acid, the major essential fatty acid and the most abundant polyunsaturated acid in our diet, is present in concentrations between 3% and 21% [1]. In addition to triglycerides and free fatty acids, olive oil contains a variety of nonsaponifiable compounds that add up to 1–2% of the oil and are important for its stability and unique flavour and taste.

It has been postulated that the components in olive oil in the Mediterranean diet, a diet which is largely vegetarian in nature, can contribute to the lower incidence of coronary heart disease and prostate and colon cancers. The Mediterranean diet includes the consumption of large amounts of olive oil, which contains high amounts of phenolic substances. The major phenolic compounds in olive oil are oleuropein, hydroxytyrosol (2-(3,4-dihydroxyphenyl)ethanol) and tyrosol (2-(4-hydroxyphenyl)ethanol). The phenolic compounds present in olive oil are strong antioxidants and radical scavengers. Typically, hydroxytyrosol is a superior antioxidant and radical scavenger to oleuropein and tyrosol [2].

It was shown by Owen et al. [3] that the health-promoting properties are a result of the unique profile of the phenolic fraction, along with high intakes of squalene and the monounsaturated fatty acid, oleic acid.

Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,20-tetracosahexane) is a terpenoid hydrocarbon occurring in high concentrations (60–75%) in the unsaponifiable fraction of olive oil.

The main focus of this study are the minor components such as polyphenols and squalene. In this study, the phenols [tyrosol, vanillin (4-hydroxy-3-methoxybenzaldehyde), vanillic acid (4-hydroxy-3-methoxybenzoic acid), p-coumaric acid (3-(4-hydroxyphenyl)-2-propenoic acid), ferulic acid (3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid), luteolin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one), apigenin (5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one), oleuropein] as well as squalene were analysed. The analytes found in the samples are shown in Fig. 1.

Section snippets

Materials and methods

The olives were grown in Greece and harvested in 2001. The oil was pressed in a small-scale laboratory press.

The polyphenols and other chemicals were bought from Merck (Darmstadt, Germany). Squalene was purchased from Sigma (St. Louis, MO, USA). Methanol, glacial acetic acid, acetonitrile, and acetone were of LiChrosolv quality from Merck. Water was prepared with Simplicity 185 (Millipore, Molsheim, France).

Sample preparation

One hundred milligrams of the olive oil was diluted with the eluent to 2 ml, centrifuged, and used for high-performance liquid chromatography (HPLC) analysis.

HPLC analysis

Twenty-five microliters of the diluted oil was injected onto a reversed-phase column. The HPLC equipment used was an HP 1100 (Agilent, Waldbronn, Germany) equipped with an isocratic pump and a refractive index detector. For injection, a manual injector (Rheodyne) was used. The analytes were separated on a LiChrospher 100 (5 μm, 250×4 mm;

Sample preparation

Five hundred milligrams of the olive oil was extracted with 500 μl of methanol in 2-ml Eppendorf reaction tubes. After vigorous shaking, the vials were centrifuged at 13,000 rpm for 5 min. The upper methanolic phase was used for HPLC analysis.

HPLC analysis

Five microliters of the methanolic extract was injected onto a reversed-phase column. The HPLC equipment used was an HP 1100 (Agilent) equipped with a quaternary pump and a diode array (HP 1100) and mass selective detector (MSD; Agilent). For injection an

Results and discussion

The analysis of phenolic substances by using mass selective detection after liquid chromatographic separation showed that from the tested substances, only tyrosol, vanillic acid, luteolin, and apigenin were found. The other substances (p-coumaric acid, ferulic acid, and oleuropein) normally found in olive oil could not be identified in the samples investigated samples [5]. Elenolic acids and its derivatives were also not found. Hydroxytyrosol, which is a commonly found substance in olive oil,

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