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Comparative study of phytosterol derivatives in monovarietal olive oils. RAQUEL BEATRIZ GÓMEZ-COCA, Gabriel D. Fernandes, Chellah del Águila-Sánchez, María del Carmen Pérez-Camino, and Wenceslao Moreda J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf501340q • Publication Date (Web): 27 May 2014 Downloaded from http://pubs.acs.org on June 2, 2014
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Comparative study of phytosterol derivatives in monovarietal olive oils# Raquel B. Gómez-Coca *, Gabriel D. Fernandes, Chellah del Águila-Sánchez, María del Carmen Pérez-Camino, Wenceslao Moreda Instituto de la Grasa –CSIC-, Avda. Padre García Tejero 4, E-41012-Sevilla, Spain Postal address: Avda. Padre García Tejero 4, 41012 Sevilla - Spain E-mail address:
[email protected] (R. B. Gómez-Coca)
AUTHOR INFORMATION Corresponding author *Tel.: +34 954 611 550 (ext. 251). Fax: +34 954 616 790. E-mail:
[email protected] 1 ACS Paragon Plus Environment
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Abstract: Plant sterols and their derivatives are minor compounds that have been
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extensively studied in vegetable oils, mainly in olive oil, where they are closely related
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with its identity. The objective of this work is to determine the content of free and
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esterified steryl glucosides and their profiles in olive oil in relation to different
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geographical situation of olive orchards, cultivar, farming modality, and sampling time.
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The orchards under study were located in the outer ring of the sub-metropolitan area
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of Madrid (Spain), where olives from Cornicabra, Manzanilla Cacereña, Manzanilla
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Castellana, and Picual varieties were grown under traditional and organic modes, and
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harvested in four different samplings. Conclusions state that cultivar, farming mode,
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and light-exposure, do not have outstanding effects, whereas pedoclimate might affect
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the steryl glucoside presence in a substantial way. Further studies are being carried
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out presently in order to confirm such statement. Also glucoside derivative profiles are
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discussed, and reasons for differences with results in previous studies pointed out.
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Keywords: olive oil, Solid Phase Extraction (SPE), Steryl Glucoside (SG), Esterified
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Steryl Glucoside (ESG), pedoclimate
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INTRODUCTION
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Phytosterols, as well as their derivatives, are compounds of the unsaponifiable matter
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of vegetable oils, whose amount and profile are important parameters of oil identity. In
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case of sterol derivatives, SG consist of a glucose moiety bound at the C3 position of
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the sterol residue via an acetyl bond. If additionally a fatty acid is esterified at the C6
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position of the sugar, ESG are formed.
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The presence of free and esterified steryl glucosides in edible matrices has been
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widely studied, although their synthesis pathway and influencing factors are not clear
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yet.1 The health promoting effects of steryl glucosides have been unknown for a long
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time. Their use for the treatment of pathologies related to elevated cholesterol in blood
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was never compared to those of FS solutions2 or functional foods,3 and it was not until
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2009 that studies on this field demonstrated the potential health benefits of
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glucosylated phytosterols in both free and esterified (acylated) forms.4,5 Actually, SG
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could get reductions of around 37% in cholesterol absorption in the gut of human
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subjects, a value similar to that 30% obtained with SE.4 As a consequence, research
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on possible steryl glucoside sources became more relevant and groups started
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publishing detailed data on the content and composition of glucosylated sterols in
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edible plant matrices to facilitate comparisons of the potential contribution of different
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foods as sources of steryl glucosides when included in the diet.6 Those studies did not
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usually included olive oil, which justifies our studies on the presence of both SG and
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ESG in this matrix.
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One would expect the SG and ESG composition to be closely related to the free
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sterol pool, and therefore the contribution of both SG and ESG to be bound to the oil
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identity. The procedure for isolating steryl glucosides from olive oil had been
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developed earlier. Initially the method was applied to a small number of oil samples of
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different categories and origins,7 and then to a wider number also from different
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cultivars and treated with different refining processes,8 reaching the conclusion that
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the only SG that might be present was β-sitosteryl glucoside (BSSG), whose
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concentration in olive oil was never higher than 3 mg kg-1. Regarding ESG, mass
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spectrometry results confirmed that in the majority of the samples the only ESG
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present -at concentration sometimes above 8 mg kg-1- was esterified β-sitosteryl
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glucoside (EBSSG), possibly eluting just before small quantities of esterified ∆5-
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avenasteryl glucoside (EAvSG).
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In this case we have determined the composition and content of free and esterified
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steryl glucosides in a more systematic way, paying special attention to the contribution
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of each steryl glucoside to the total glucoside concentration. We have focused on
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monovarietal olive oils from different geographical situation, farming modality, and
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sampling time. Our intention has been not only to start determining the factors
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affecting the steryl glucoside presence, but also to contribute in a direct way to nutrient
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databases.
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MATERIALS AND METHODS
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Reagents. The internal standard ChSG, commercial mixtures of ESG and SG, and
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the analytical-grade chemical reagents used in the course of this research were the
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same as those utilized previously. 8
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Samples and standard solutions. Virgin olive oils were obtained from Cornicabra,
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Manzanilla Cacereña, Manzanilla Castellana, and Picual olive varieties. They were
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directly extracted in the laboratory of Doctor Pérez Jiménez group (IMIDRA) using an
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Abencor® system identical to that described earlier.8 Our blank-sample (chemically
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refined olive-pomace oil) was obtained directly from the producers.
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Stock solutions (100 µg mL-1) and samples were treated as described formerly,7,8 in
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short: solutions of ESG standard were prepared by dissolving the standard mixture in
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chloroform.
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chloroform:methanol (2:1, by volume) blend. Samples consisted of 2 g oil dissolved in
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4 mL chloroform, spiked with 100 µL of ChSG stock solution. The hydroxyl groups
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were derivatised (1h at 60 ºC) with 200 µL of a mixture of Py:HMDS:TMCS (9:3:1, by
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volume) before carrying out the GC analysis.
Stocks
solutions
of
SG
and
of
ChSG
were
made
with
a
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Olive orchards and olive oil extraction. The orchards were situated in Madrid, the
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centre part of Spain, near the Manzanares River (Mediterranean climate), where they
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could be kept under optimal cultivation conditions. Specifically they were located in
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eight municipalities in the southern outer ring of the sub-metropolitan area (Table 1).
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In these regions winters are cold due to the altitude (average of 667 m above sea
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level), being January the coldest month with temperatures between 3 and 10 ºC, and
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148 sunshine h. These are dry climate areas, actually the common annual
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precipitations are between 400 and 500 mm per year in municipalities A-E, and below
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400 mm per year in municipalities F-H, that concentrate from October to May, being
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October and December are the wettest months (56 mm).
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Fruits from four different cultivars -Cornicabra, Manzanilla Cacereña, Manzanilla
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Castellana, and Picual cv.- were obtained from irrigated traditional orchards (drip
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irrigation). Additionally, olives from Manzanilla Castellana and Cornicabra cultivars
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were also obtain from organic orchards (Table 1).
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The olive plantations were in classical frame with around 300 trees per hectare. Fruits were handpicked according to the sampling time shown in Table 1.
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To assure maximum oil quality, olives were kept in darkness at 4 ºC, and oil was
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extracted within 24 h after harvesting using an Abencor® system and a small-quantity
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mill simulating commercial oil-extraction procedures (MC2 Ingeniería de Sistemas,
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Sevilla, Spain): the olives were crushed with an Abencor® hammer mill equipped with
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a 4-mm sieve; 700 g paste were processed using the system’s malaxer and centrifuge
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during 1 min at 3500 rpm. Conditions: malaxation temperature, 27 ºC; malaxation time,
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20 min, plus 10 min after water addition; amount of distilled water added to the paste,
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300 mL; amount of talc added, 20 g. The mixtures were left to decant during 24 h
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before proceeding to oil separation and filtration. To facilitate the work comprehension
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the oil samples were coded according to the Table 1.
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Instrumentation. GC analyses of the sterol glucosides were carried out with an
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Agilent 6890N Gas Chromatograph (Agilent Technologies, Santa Clara, California)
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equipped with an Agilent 7683B Automatic Liquid Sampler and FID. Acquisition of data
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was done with the Agilent ChemStation for GC System program. The conditions for
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the GC assays were: TRB-5HT column (5% diphenyl-95% dimethylpolysiloxane; 30 m
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x 0.32 mm ID x 0.10 µm film; Teknokroma, Sant Cugat del Vallés, Barcelona, Spain),
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1.0 µL injection volume, hydrogen carrier gas at 3 mL min-1 and ECP cool on-column
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injection. The oven temperature program was: 80 ºC (1 min), 50 ºC min-1 to 320 ºC (12
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min), and 40 ºC min-1 to 360 ºC (19 min). The detector temperature was 360 ºC.
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Steryl glucoside analysis. The SPE procedure developed previously8 was utilized.
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The method consist of the conditioning of the cartridges (ExtraBond Si-1 g; Scharlab
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S.L., Barcelona, Spain) with 5 mL tert-butyl methyl ether, 5 mL hexane, 5 mL
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chloroform, and 10 mL of a newly prepared blend consisting of chloroform:methanol
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4:1, by volume. Samples are then quantitatively transferred using 1 mL chloroform to
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wash the walls of the containers. The loaded cartridges are washed with 10 mL
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hexane, 10 mL tert-butyl methyl ether, and 10 mL chloroform, applied one after the
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other. The steryl glucosides are eluted with 5 mL of the chloroform:methanol 4:1, by
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volume, solution. The procedure was carried out under negative pressure (1 mm.Hg)
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in a vacuum manifold. The eluted fractions are evaporated to dryness under nitrogen
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flux, derivatised and chromatographed using cool on-column injection.
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The quantitative evaluation of the SG was carried out using ChSG as internal
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standard, whereas we quantified the ESG using three-point calibration curves (one
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specific curve for each ESG) built by spiking the in-house blank sample with the
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commercial standard. This evaluation together with the purity, qualitative and
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quantitative composition of the commercial standards is comprehensively described in
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our former work.8
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All statistical analyses were run using the software GraphPad Prism© version 5.01
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for Windows. The samples were compared by an ANOVA, with statistical significance
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of p
