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Bioaccessibility of individual phenolic compounds in extra virgin argan oil after simulated gastrointestinal process
Accepted Manuscript Bioaccessibility of individual phenolic compounds in extra virgin argan oil after simulated gastrointestinal process Ascensión Rueda, Samuel Cantarero, Isabel Seiquer, Carmen Cabrera-Vique, Manuel Olalla PII:
S0023-6438(16)30590-4
DOI:
10.1016/j.lwt.2016.09.028
Reference:
YFSTL 5741
To appear in:
LWT - Food Science and Technology
Received Date: 3 February 2016 Revised Date:
12 September 2016
Accepted Date: 21 September 2016
Please cite this article as: Rueda, A., Cantarero, S., Seiquer, I., Cabrera-Vique, C., Olalla, M., Bioaccessibility of individual phenolic compounds in extra virgin argan oil after simulated gastrointestinal process, LWT - Food Science and Technology (2016), doi: 10.1016/j.lwt.2016.09.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Bioaccessibility of individual phenolic compounds in extra
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virgin argan oil after simulated gastrointestinal process
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Ascensión Ruedaa*, Samuel Cantarerob, Isabel Seiquerc, Carmen Cabrera-
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Viquea, Manuel Olallaa
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a
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de Granada, Granada, Spain
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b
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Universidad de Granada, Granada, Spain
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Departamento de Nutrición y Bromatología. Facultad de Farmacia, Universidad
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Centro de Instrumentación Científica. Campus Universitario de Fuentenueva.
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c
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Experimental del Zaidín (CSIC), Armilla, Granada, Spain
Departamento de Fisiología y Bioquímica de la Nutrición Animal, Estación
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*Corresponding author (phone number: +34-958240755; Fax: +34-958249577;
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E-mail: [email protected])
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ACCEPTED MANUSCRIPT ABSTRACT
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The bioaccessibility of individual phenolic compounds in extra virgin argan oil
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was evaluated using an in vitro digestion method for first time. The analyses
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were carried out by ultra-performance liquid chromatography-electrospray
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ionisation tandem mass spectrometry in multiple reaction monitoring (UPLC-
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ESI-MS/MS). Hydroxyphenylacetic acid (4245 µg/kg), ferulic acid (2478 µg/kg),
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vanillin (2429 µg/kg) and 3,4-dihydroxybenzoic acid (2247 µg/kg) were the
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mayor compounds identified in argan oil extracts. After the digestion process,
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values of bioaccessibility of the different compounds varied from a minimum of
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2% (hydroxyphenylacetic acid) to a maximum of 84% (p-coumaric acid), but
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some such as caffeic acid became detectable, as a result from digestive
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transformations. The results indicate that the phenolic compounds of the argan
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oil are strongly affected during the digestive process, and bioaccessibility
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should be taken into account when assessing polyphenol content of oils.
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Keywords
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In vitro digestion, individual polyphenols, argan oil, UPLC
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1. Introduction Virgin argan oil is a product obtained from roasted seeds of the fruit of
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the argan tree Argania spinosa L. Skeels, which is endemic to SW Morocco.
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This oil has been used by the Berber population for centuries due to its
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sensorial and health-giving properties (Ahansal, Ben Sassi, Martini, Vaughan-
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Martini, Walker & Boussaid, 2008). The presence of phenols and phytosterols in
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argan oil has been associated with a cholesterol-lowering activity (Guillaume &
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Charrouf 2011) and antidiabetic effects have been shown in rats (Bellahcen,
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Mekhfi, Ziyyat, Legssyer, Hakkou, Aziz & Bnouham, 2012). In oils, polar lipids
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contribute greatly to nutritional value due to specific anti-inflammatory activities
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(Nasopoulou et al., 2014), but among the bioactive compounds present in virgin
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argan oil, polyphenols play an important role, since their antioxidant activity is
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related to cardioprotective (Pandey & Rizvi, 2009) and anti-inflammatory effects
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(Rosillo, Alcaraz, Sánchez-Hidalgo, Fernández-Bolaños, Alarcón-de-la-Lastra
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& Ferrándiz, 2014).
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Polyphenols such as caffeic acid, oleuropein, vanillic acid, tyrosol, ferulic
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acid, syringic acid, p-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, catechol,
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resorcinol and 4-hydroxybenzyl have been identified in virgin argan oil (Charrouf
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& Guillaume, 2007; Charrouf & Guillaume 2008), but his bioaccessibility has not
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been evaluated yet. Gallic acid has reported bioactivities such as antineoplastic,
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bacteriostatic, antimelanogenic and antioxidant properties. This molecule
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showed anticancer properties in prostate carcinoma cells. p-Hydroxybenzoic
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acid has been reported to have antioxidant activities against free radicals,
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antimicrobial activities and antimutagenic properties. vanillic acid showed
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antisicking and anthelmintic activities, and is also able to suppress hepatic
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fibrosis in chronic liver injury. Syringic acid show antioxidant, antibacterial and
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hepatoprotective activities (Heleno, Martins, Queiroz & Ferreira, 2015). The concept of bioaccessibility refers to the fraction of a compound which
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is released from the food matrix in the gastrointestinal tract and becomes
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available for absorption (Heaney, 2001). The polyphenol’s beneficial effects can
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only be truly effective if they reach the relevant tissues and exert their action at
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a sufficient concentration to have a biological effect. The most widely used
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procedure for screening polyphenolic compound bioaccessibility is the in vitro
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static gastrointestinal method (Carbonell-Capella, Buniowska, Barba, Esteve &
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Frígola, 2014). One of the most important methods to determine the
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bioaccessibility was proposed by Miller to simulate human digestion and
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absorption of dietary iron in the study of phenolic compound release. Over time
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this method has been used by other authors with modifications and has been
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employed in the screening of multiple foods (Gil-Izquierdo, Zafrilla & Tomás-
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Barberán,
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Rodríguez-Roque, Rojas-Graü, Elez-Martínez & Martín-Belloso, 2013). Some
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authors found that gastric digestion increases polyphenolic concentration,
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whereas the duodenal fraction significantly lessens polyphenolic content in
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pomegranate juice, broccoli and gooseberry (Pérez-Vicente, Gil-Izquierdo &
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García-Viguera, 2002; Chiang, Kadouh & Zhou, 2013; Vallejo, Gil-Izquierdo,
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Pérez-Vicente & García-Viguera, 2004). The method used by Bermudez-Soto et
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al. (2007) found that during intestinal digestion a significant decrease in
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anthocyanins (43%) and flavonols (26%) was observed, whereas chlorogenic
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acid increased (24%) in chokeberry. Separation and characterization of
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phenolic compounds has been conducted in argan fruit pulp, in alimentary and
Pérez-Vicente,
Gil-Izquierdo
&
García-Viguera,
2002;
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cosmetic oil and in the press cake (Charrouf & Guillaime 2007; El Abbassi,
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Khalid, Zbakh & Ahmad, 2014), but the analysis of individual phenolic
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compounds after subjecting argan oil to an in vitro digestion process has not
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been done yet. Due to the great interest of phenolic compounds, great efforts have been
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made in the last years to provide a technique highly sensitive for their
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determination, and methodologies such as gas chromatography, capillary
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electrophoresis and high performance liquid chromatography (HPLC), among
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others, have been applied (Carrasco-Pancorbo et al., 2005; Ignat et al., 2011).
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The use of ultra-performance liquid chromatography (UPLC) decreases the time
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needed for analysis (<20 minutes), improves the resolution and provides great
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sensitivity, and this method coupled with a MS/MS technique, makes it easy to
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detect a large number of compounds in a short time (Kartsova & Alekseeva,
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2008).
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The aim of this study was to assess the bioaccessibility of individual
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phenolic compounds in argan oil after an in vitro digestion method simulating
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the
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chromatography-electrospray ionisation tandem mass spectrometry (UPLC-ESI-
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MS/MS) in multiple reaction monitoring (MRM) was used as an analytical
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technique. The high sensitivity of this method enabled us to obtain low detection
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limits.
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digestion
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2. Materials and methods
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2.1. Materials
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2.1.1. Oils samples 5
process.
Ultra-performance
liquid
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lower than 0.8 % oleic acid, Norme Marocaine, 2003), obtained from specialist
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stores in Spain, were included in the study. The samples were produced in
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Morocco and obtained by cold pressing. The oil samples were carefully handled
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to avoid contamination and correctly stored and maintained at a temperature of
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4 ºC until analysis.
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2.1.2. Reagents and standards
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All chemicals were analytical reagent grade or higher purity. Ultrapure
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water was obtained from a Milli-Q purification system (Millipore, Bedford, MA).
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Methanol and acetonitrile were supplied by Sigma-Aldrich (LC-MS Ultra
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Chromasolv® Sigma Chemical Co., St. Louis, MO). Sodium bicarbonate,
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sodium carbonate and hydrochloric acid (37%) were purchased from Merck
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(Merck, Darmstadt, Germany). The commercial phenolic compound standards
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used were oleuropein, p-coumaric acid, o-coumaric acid, 4-hydroxyphenylacetic
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acid, quercetin, vanillin, gallic acid, 3,4-dihydroxybenzoic acid, caffeic acid and
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syringic acid, all purchased from Sigma-Aldrich. Ferulic acid, tyrosol, luteolin
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and vanillic acid were purchased from Fluka Chemicals (Madrid, Spain). Stock
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standard solutions were prepared by dissolving each phenolic compound at a
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concentration of 10 mg/mL in methanol, and the resulting solution was then
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stored at -18 °C.
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2.2. Methods
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2.2.1. In vitro digestion
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The in vitro digestion procedure (Fig. 1) was performed according to the
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method described by Mesías et al. (2009), slightly modified. The method
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consists of two sequential steps; an initial pepsin/HCl digestion to simulate 6
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duodenal digestion. Four grams of oil samples were mixed with 9 mL of
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bidistilled deionised water and sonicated (Vibracell VCX 130, Sonics &
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Materials INC, Danbury, Connecticut, USA). The pH was adjusted to 2 using
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HCl 1N. Pepsin (Sigma-Aldrich, Milan, Italy) was added to a final concentration
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of 0.05 g of pepsin / g sample and the samples were incubated in a shaking
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water bath (Bunsen, Madrid, Spain) at 110 oscillations/min, at 37 °C for 2 hours.
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For intestinal digestion, the pH was raised to 6 with NaHCO3 (1 M) dropwise,
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and 2.5 mL of a mixture of 0.4 g of pancreatin + 0.25 g of bile salts (Sigma-
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Aldrich, Milan, Italy) were added. The final pH was adjusted to 7 with NaHCO3
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(1 M) and the samples were incubated in a shaking water bath (110 rpm; 37 °C)
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for 2 hours. After gastrointestinal digestion, the digestive enzymes were
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inactivated by heat treatment for 4 min at 100 ºC in a polyethylenglycol bath.
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The samples were then cooled by immersion in an ice bath and centrifuged at
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10.000 rpm (Sorvall RC 6 Plus centrifuge, Thermo Scientific, Madrid, Spain) for
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30 min at 4 °C. The supernatants were carefully separated to obtain the
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bioaccessible fraction, which was filtered using Vivaspin® 2 ml Centrifugal
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Concentrator to remove the potential presence of protein, and then freeze-dried.
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Lyophilized samples were dissolved in 3 ml of methanol / acetonitrile 50% (v / v)
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for chromatographic analysis. Throughout the process, the samples were
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protected from light, cooled with an ice bath and submitted to sonication before
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each step.
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2.2.3. Phenolic extract
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A liquid–liquid extraction adaptation technique (Montedoro, Servili,
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Baldioli & Miniati, 1992) was used to determine individual phenolic compounds.
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in an orbital shaker (3005 GFL®, Grossburgwedel, Germany) for 1 hour and
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centrifuged (Hettich zentrifugen Universal 320, Buckinghamshire, England) at
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8000 rpm for 15 minutes. The supernatant was removed and the process was
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repeated twice. Using a rotary evaporator (R-215, Buchi Switzerland), a
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gelatinous residue was obtained, which was dissolved in 5 ml of methanol and
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filtered through a 0.45 µm Millipore syringe filter.
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2.2.4. UPLC analysis of polyphenols
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Analyses of polyphenols were carried out in chemical extracts and in bioavailable
fractions
obtained
after
the
in
vitro
digestion
of
oils.
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Chromatographic analysis of the polyphenols was performed in the Scientific
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Instrumentation Centre of the University of Granada using an Acquity UPLC H-
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Class with MS detection (Waters, Barcelona, Spain) and separations were
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achieved using an Acquity UPLC HSS T3 column (100 mm x 2.1 mm, 1.8 µm
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particle size).
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Chromatographic separation was carried out using a gradient elution with
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water + 0.5% acetic acid as eluent A and acetonitrile +0.5% acetic acid as
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eluent B. The flow rate was 650 µL/min, the column was maintained at 45 ºC
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and the injection volume was 10 µL. Gradient conditions were as follows: initial
193
mobile phase, 99% (A), which was linearly decreased to 70% (A) within 10.0
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min and to 5% within 12.0 min. Finally, it was returned to 99% in 0.1 min and
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maintained for 2.9 min to equilibrate the column. Total run time was 15 min.
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Mass spectrometry analysis was carried out using a Waters XEVO TQ-S
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tandem quadrupole mass spectrometer. The instrument was operated using
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ESI ionisation in negative ion mode. Data acquisition was performed using
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MassLynx 4.1 software with the QuanLynx program (Waters). For MS/MS
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following: capillary voltage 2.00 kV and cone voltage 10 V; the source
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temperature was 100 °C and the desolvation temperature, 300 °C. The cone
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gas (nitrogen) and desolvation gas (also nitrogen) were set at flow rates of 150
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and 500 L/h, respectively.
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The conditions used were adapted from previous papers (Goh & Gledhill,
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2011), seeking to obtain high sensitivity and to reduce analysis time. The
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IntelliStart™ Software was used to develop MRM acquisition methods for the 14
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phenolic compounds targeted in this analysis. Standard solutions of 10 mg/L of
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each phenolic compound at a flow rate of 10 µL/min were infused at first and
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then combined with the mobile phase flow. The collision voltage was optimised,
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selecting the most sensitive transition for quantification. Some phenolic
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compounds presented initially close retention times, but optimization of the
213
methods allowed the subsequent identification due to their different transitions.
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Quantification of the samples was performed against a calibration curve for
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each of the polyphenol analytes in solution. A chromatogram of digested argan
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oil sample is shown in Fig. 2 according to the conditions established in the
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methodology.
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Table 1 shows the parameters optimised for quantification using MRM,
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the calibration curves obtained, the recovery percentages and the limits of
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detection and of quantification (LOD and LOQ, respectively) obtained for each
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compound. After optimising the transitions, calibration curves were constructed
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for each compound, using various concentration ranges, but always including at
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least 5 different concentration points. LOD was determined by using a signal-to-
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noise ratio of 3, and LOQ, by using a signal-to-noise ratio of 10.
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the detection limits ranged from 0.07 µg/kg for hydroxyphenylacetic acid to 0.26
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µg/kg for quercetin. The recovery (%) was calculated as the percentage from
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the known amount of standard added to a refined vegetable oil. Analyses were
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carried out in triplicate.
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3. Results and discussion
In this experimental study, we first determined the initial content of
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polyphenols. A total of 14 phenolic compounds were analysed, structures of
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which are depicted as supplementary material (S1). Quercetin and oleuropein
235
were not detected before or after the in vitro digestion process. 11 compounds
236
were detected and quantified in argan oil extracts (Fig. 3), being the major ones
237
hydroxyphenylacetic acid (4245 µg/kg), ferulic acid (2478 µg/kg), vanillin (2429
238
µg/kg) and 3,4-dihydroxybenzoic acid (2247 µg/kg). Other compounds that have
239
been quantified in argan oil extracts are vanillic acid (1190 µg/kg) and tyrosol
240
(2048 µg/kg); these polyphenols were identified as major components of argan
241
oil by Chaurrof & Guillaume (2007). Khallouki et al. (2003) measured the
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following quantities in commercial argan oil: vanillic acid 123 µg/kg; syringic acid
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68 µg/kg, tyrosol 52 µg/kg. These levels are lower than those obtained in our
244
study, although the concentration of ferulic acid was slightly higher (3470
245
µg/kg). Aidoud et al. (2014) also measured lower quantities than those found in
246
our study with respect to vanillic acid (67 µg/kg), syringic acid (37 µg/kg) and
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tyrosol (12 µg/kg), and higher levels of ferulic acid (3147 µg/kg).
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Some polyphenols have been identified in the leaves and the fruit pulp of
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the argan tree (Charrouf & Guillaume, 2008; Aidoud, Ammouche, Garrido &
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Rodriguez,
2014).
In
the
press
cake,
3,4-dihydroxybenzoic
acid,
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hydroxyphenylacetic acid and vanillin have been identified (Joguet & Maugard,
252
2013). The simulation of digestion is widely used in many fields of food and
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nutritional science, as human trials are often expensive, require considerable
255
resources and may be ethically questionable (Lin, Shi, Wang &Shen, 2008). In
256
vitro models of digestion provide a useful alternative to animal and human
257
models and the results supplied are fast and accurate (Coni et al., 2000).
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In our study, polyphenol concentrations generally decreased during the
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simulated in vitro gastrointestinal digestion (Fig. 3). The highest levels of
260
polyphenols found in the bioavailable fraction of argan oil were vanillic acid, p-
261
coumaric acid and o-coumaric acid. Polyphenols shown a considerable
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structural diversity which largely influences their bioaccessibility; it has been
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shown that phenols may undergo structural modifications during the digestion
264
process that may reduce their bioaccessible fraction, mainly due to changes of
265
pH and interactions with other compounds, which differ depending on their
266
chemical structure (Dinnella et al., 2007). However, some compounds became
267
detectable or their concentration increased slightly after the digestion process,
268
as caffeic acid. On accordance, previous assays concerning transformations of
269
phenolic compounds during in vitro digestion of foods have shown that caffeic
270
acid is detected after the digestion process, but not before (Tarko et al., 2009).
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Caffeic acid may derive from the hydrolysis of chlorogenic acid in the small
272
intestine (Sato et al., 2011), but early studies show that it also may results of the
273
conversion of p-coumaric acid (Sato, 1969). Thus, in argan oil digested, it may
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be hypothesized that caffeic acid could come from p-coumaric acid.
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as the percentage from the initial quantity measured in the chemical extracts),
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for the oils analysed. With the exception of luteolin, which was detected in the
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chemical extracts but not after the in vitro digestion, values of bioaccessibility
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varied largely, from 2% (hydroxyphenylacetic acid) to 84 % (p-coumaric acid). A
280
value of 100% was assigned to caffeic acid, as it was only detected in the
281
bioaccessible fraction. The largest decreases (expressed in percentage of
282
reduction from the initial quantity) were observed in the following compounds:
283
syringic
284
hydroxyphenylacetic acid 98%; vanillin 95% and ferulic acid 94%. These losses
285
were greater than those reported by Dinnella et al. (2007), who describe that
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bioaccessibility of olive oil phenols vary from 37 to 90%. Other studies have
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indicated that the process of digestion reduces phenolic content by 47-62%
288
(Minekus et al., 2014). It is possible that the pancreatin digestion liberates
289
compounds able to associate with phenolic compounds (Joguet & Maugard,
290
2013). During gastrointestinal digestion, polyphenols may either interact with
291
other food constituents (e.g., chelation of ions), be further degraded (such as
292
anthocyanins in the small intestine), or metabolised, such as by hydrolysis via,
293
e.g., deglycosylation or cleavage by esterases (Hura, Limb, Decker &
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McClements, 2011).
3,4-dihydroxybenzoic
acid
95%;
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tyrosol
91%;
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To our knowledge, there is a lack of information concerning concentration
296
of individual polyphenols after in vitro digestion in argan oil, and only
297
bioaccessibility of total phenol content and antioxidant activity has been recently
298
reported by our research group (Seiquer et al., 2015).
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The analysis of phenolic compounds after subjecting argan oil to an in
302
vitro digestion process was performed for the first time. In general, the
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polyphenol concentration in the bioaccessible fraction of the argan oil was lower
304
than in the initial extract of the oil. In the bioaccessible fraction, argan oil contain
305
quantities of 3,4-dihydroxybenzoic acid, tyrosol, vanillic acid, caffeic acid,
306
syringic acid, p-coumaric acid, o-coumaric acid and ferulic acid.
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Polyphenols are highly sensitive to the mild alkaline conditions in the
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small intestine and a great proportion of these compounds can be transformed
309
into other unknown and/or undetected structural forms with different chemical
310
properties and, consequently, different bioaccessibility, bioavailability and
311
biological activity. Intestinal transformations of polyphenols seem to occur
312
during the digestion process of argan oil, leading to the release of the
313
compounds from the food matrix. However, these compounds could be
314
hydrolysed depending on the intestinal conditions.
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Decreases or increases of phenolic compounds in the bioaccessible
316
fractions of oils may take place, as it has been observed in the present study.
317
The results obtained indicate that only a minor fraction of argan oil polyphenols
318
may be considered bioavailable for subsequent absorption by the intestinal
319
cells. In any case, to better understand the transformation of argan oil
320
polyphenols during the digestion process more extensive study will be required,
321
to determine the presence of new metabolites.
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Conflict of interest
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The Authors declare that they have no conflict of interest.
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Acknowledgements
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This work was supported by the research group AGR141 of Junta de Andalucía
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of University of Granada.
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ACCEPTED MANUSCRIPT REFERENCES
331
Ahansal, L., Ben Sassi, A., Martini, A., Vaughan-Martini, A., Walker G. &
332
Boussaid, A. (2008). Biodiversity of yeasts isolated from the indigenous
333
forest of Argan (Argania spinosa (L.) Skeels) in Morocco. World Journal of
334
Microbiology and Biotechnology, 24, 777–782.
RI PT
330
Aidoud, A., Ammouche, A., Garrido, M. & Rodriguez, A. B. (2014). Effect of
336
lycopene-enriched olive and argan oils upon lipid serum parameters in
337
Wistar rats. Journal of the Science of Food and Agriculture, 94, 2943-
338
2950.
M AN U
SC
335
Bellahcen, S., Mekhfi, H., Ziyyat, A., Legssyer, A., Hakkou, A., Aziz, M. &
340
Bnouham, M. (2012). Prevention of chemically induced diabetes mellitus
341
in experimental animals by virgin argan oil. Phytotherapy Research, 26,
342
180-185.
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339
Bermúdez-Soto, M. J., Tomás-Barberán, F. A. & García-Conesa, M. T. (2007).
344
Stability of polyphenols in chokeberry (Aronia melanocarpa) subjected to
345
in vitro gastric and pancreatic digestion. Food Chemistry, 102, 865–74.
347 348 349 350
351 352
Carbonell-Capella, J. M., Buniowska, M., Barba, F. J., Esteve, M. J. & Frígola,
AC C
346
EP
343
M. (2014). Analytical methods for determining bioavailability and bioaccessibility of bioactive compounds from fruits and vegetables: a review. Comprehensive Reviews in Food Science and Food Safety, 13, 155-171.
Carrasco-Pancorbo, A., Cerretani, L., Bendini, A., Segura-Carretero, A., Gallina-Toschi,
T.
&
Fernández-Gutierrez,
15
A
(2005).
Analytical
ACCEPTED MANUSCRIPT 353
determination of polyphenols in olive oils. Journal of Separation Science,
354
28, 837–858. Charrouf, Z. & Guillaume, D. (2008). Argan oil: Occurrence, composition and
356
impact on human health. European Journal of Lipid Science and
357
Technology, 110, 632–636.
358 359
RI PT
355
Charrouf, Z. & Guillaume, D. (2007). Phenols and polyphenols from Argania spinosa, American Journal of Food Technology, 2, 679–683.
Chiang, C. J., Kadouh, H. and Zhou, K. (2013). Phenolic compounds and
361
antioxidant properties of gooseberry as affected by in vitro digestion. LWT-
362
Food Science and Technology, 51, 417–422.
M AN U
SC
360
Coni, E., Di Benedetto, R., Di Pasquale, M., Masella, R., Modesti, D., Attei, R.,
364
et al. (2000). Protective effect of oleuropein, and olive oil biophenol, on low
365
density lipoprotein oxidizability in rabbits, Lipids, 35, 45–54.
366
Dinnella,
TE D
363
C.,
Minichino,
P.,
D’Andrea,
A.M.
&
Monteleone.
(2007).
Bioaccessibility and antioxidant activity stability of phenolic compounds
368
from extra-virgin olive oils during in vitro digestion. Journal of Agriculture
369
and Food Chemistry, 55, 8423-8429.
AC C
EP
367
370
El Abbassi, A., Khalid, N., Zbakh, H. & Ahmad, A. (2014) Physicochemical
371
characteristics, nutritional properties, and health benefits of argan oil: a
372
review. Critical Reviews in Food Science and Nutrition, 11, 1401-1414.
373
Gil-Izquierdo, A., Zafrilla, P. & Tomás-Barberán, F. A. (2002). An in vitro method
374
to simulate phenolic compound release from the food matrix in the
375
gastrointestinal tract. European Food Research and Technology, 214,
376
155–159. 16
ACCEPTED MANUSCRIPT 377
Goh, E. & Gledhill, A. (2011). Analysis of polyphenols in fruit juices using
378
ACQUITY UPLC H-Class with UV and MS detection Waters Pacific,
379
Singapore, Waters Corporation, Manchester, UK.
381
Guillaume, D. & Charrouf, Z. (2011). Argan Oil. Alternative Medicine Review, 16, Number 3.
RI PT
380
Heleno, S. A., Martins, A, Queiroz, M. J. & Ferreira, I. (2015). Bioactivity of
383
phenolic acids: metabolites versus parent compounds: a review. Food
384
Chemistry, 173, 501-513.
SC
382
Hura, S. J., Limb, B. O., Decker, E. A. & McClements, D. J. (2011). In vitro
386
human digestion models for food applications. Food Chemistry, 125, 1–12.
387
Ignat, I., Volf, I., & Popa, V. I. (2011). A critical review of methods for
388
characterisation of polyphenolic compounds in fruits and vegetables. Food
389
Chemistry, 126, 1821-1835.
TE D
M AN U
385
Joguet, N. & Maugard, T. (2013). Characterization and quantification of phenolic
391
compounds of Argania spinosa leaves by HPLC-PDA-ESI-MS analyses
392
and their antioxidant activity. Chemistry of Natural Compounds, 48, 1069–
393
1071.
AC C
EP
390
394
Kartsova, L. A. & Alekseeva, A. V. (2008). Chromatographic and electrophoretic
395
methods for determining polyphenol compounds. Journal of Analytical
396
Chemistry , 63, 1024–1033.
397
Khallouki, F., Younos, C., Soulimani, R., Oster, T. Charrouf, Z., Spiegelhalder,
398
B., et al. (2003). Consumption of argan oil (Morocco) with its unique profile
399
of fatty acids, tocopherols, squalene, sterols and phenolic compounds
17
ACCEPTED MANUSCRIPT 400
should confer valuable cancer chemopreventive effects. European Journal
401
of Cancer Prevention, 12, 67–75.
402 403
Lin, Y., Shi, R., Wang, X. & Shen, H. M. (2008). Luteolin, a flavonoid with potential for cancer prevention and therapy. Current Cancer Drug Targets, 8, 634–646.
405
Mesías, M., Seiquer, I. & Navarro, M. P. (2009). Influence of diets rich in
406
Maillard reaction products on calcium bioavailability. Assays in male
407
adolescents and in Caco-2 cells. Journal of Agricultural and Food
408
Chemistry, 57, 9532–9538.
M AN U
SC
RI PT
404
409
Minekus, M., Alminger, M., Alvito, P., Balance, S., Bohn, T., Bourlieu, C., et al.
410
(2014). A standardised static in vitro digestion method suitable for food -
411
an international consensus. Food & Function, 5, 1113–1124. Montedoro, G., Servili, M., Baldioli, M. & Miniati, E. (1992). Simple and
413
hydrolyzable phenolic compounds in virgin olive oil. Their extraction,
414
separation, and quantitative and semiquantitative evaluation by HPLC.
415
Journal of Agricultural and Food Chemistry, 40, 1571–1576.
417 418 419
420 421
EP
Nasopoulou, C., Karantonis, H. C., Detopoulou, M., Demopoulos, C. A., &
AC C
416
TE D
412
Zabetakis, I. (2014). Exploiting the anti-inflammatory properties of olive (Olea europaea) in the sustainable production of functional food and neutraceuticals. Phytochemistry Reviews 13, 445–458.
Norme Marocaine (2003). Service De Normalisation Industrielle Marocaine Huile d ‘argane. Spécifications. Rabat, NM 08.5.090.
18
ACCEPTED MANUSCRIPT 422
Pandey, K. B. & Rizvi, S. I. (2009).Plant polyphenols as dietary antioxidants in
423
human health and disease. Oxidative Medicine and Cellular Longevity, 2,
424
270–278. Pérez-Vicente, A., Gil-Izquierdo, A. & García-Viguera, C. (2002). In vitro
426
gastrointestinal digestion study of pomegranate juice phenolic compounds,
427
anthocyanins, and vitamin C. Journal of Agricultural and Food Chemistry,
428
50, 2308–2312.
RI PT
425
Rodríguez-Roque, M. J., Rojas-Graü, M. A., Elez-Martínez, P. & Martín-Belloso,
430
O. (2013). Soymilk phenolic compounds, isoflavones and antioxidant
431
activity as affected by in vitro gastrointestinal digestion. Food Chemistry,
432
136, 206–212.
M AN U
SC
429
Rosillo, M. Á., Alcaraz, M. J., Sánchez-Hidalgo, M., Fernández-Bolaños, J. G.,
434
Alarcón-de-la-Lastra, C. & Ferrándiz, M. L. (2014). Anti-inflammatory and
435
joint protective effects of extra-virgin olive-oil polyphenol extract in
436
experimental arthritis. The Journal of Nutritional Biochemistry, 25, 1275–
437
1281.
EP
TE D
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Sato, M. (1969). The conversion by phenolase of p-coumaric acid to caffeic acid
439
with special reference to the role of ascorbic acid. Phytochemistry, 8, 353-
440
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362.
441
Sato, Y., Itagaki, S., Kurokawa, T., Ogura, J., Kobayashi, M., Hirano, T.,
442
Sugawara, M. & Iseki, K. (2011). In vitro and in vivo antioxidant properties
443
of
444
Pharmaceutics 403, 136–138.
chlorogenic
acid
and
caffeic
19
acid.
International
Journal
of
ACCEPTED MANUSCRIPT 445
Seiquer, I., Rueda, A., Olalla, M & Cabrera-Vique, C. (2015). Assessing the
446
bioavailability of polyphenols and antioxidant properties of extra virgin
447
argan oils by simulated digestion and Caco-2 cell assays. Comparative
448
study with extra virgin olive oil. Food Chemistry, 188, 496-503. Tarko, T., Duda-Chodak, A.,
Sroka, P. Satora, P. & Michalik, J. (2009).
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Transformation of Phenolics in Alimentary Tract, Food Technology and
451
Biotechnology, 47, 456–463
Vallejo, F., Gil-Izquierdo, A., Pérez-Vicente, A. & García-Viguera, C. (2004). In
453
vitro gastrointestinal digestion study of broccoli inflorescence phenolic
454
compounds, glucosinolates, and vitamin C. Journal of Agricultural and
455
Food Chemistry, 52,135–138.
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Figure captions.
459
Figure 1. Scheme of the in vitro digestion process applied to the oil samples.
460
Figure 2. UPLC–MS/MS chromatogram obtained from the digested argan oil
463
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sample.
Figure 3. Content of phenolic compounds in extra virgin argan oil before (oil extract) and after (digested oil) the digestion process.
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Figure. 4. Bioaccessibility of different polyphenols in argan oil (calculated as the
465
percentage from the initial quantity in the chemical extracts). 1: Gallic acid;
466
2: 3,4-Dihydroxybenzoic acid; 3: Tyrosol; 4: Hydroxyphenylacetic acid; 5:
467
Vanillic acid; 6: Caffeic acid; 7: Syringic; 8: Vanillin; 9: p-coumaric; 10:
468
Ferulic acid; 11: o-coumaric; 12: Quercetin; 13: Oleuropein; 14: Luteolin.
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Table 1
RT (min)
Calibration curvesa
Collision
LOD
LOQ
Recovery
voltage
energy
(µg/kg)
(µg/kg)
(%)
(V)
(eV)
0.47
96.5
y = 510255x - 43456.9
Cone
Transition
1.82
22.0
168.93 - 125.03
0.14
3, 4-Dihydroxybenzoic acid
4.25
30.0
152.93 - 53.08
24.0
0.09
0.31
99.0
y = 11961.3x - 819.205
Tyrosol
4.18
30.0
136.91 - 106.15
18.0
0.09
0.31
97.8
y = 2156.5x - 157.84
Hydroxyphenylacetic acid
4.50
30.0
150.95 - 79.09
18.0
0.07
0.24
96.4
y = 149507x - 11517
Vanillic acid
4.65
26.0
166.95 - 91.03
18.0
0.10
0.34
99.2
y = 30266.7x - 2551.55
Caffeic acid
4.92
10.0
178.95 - 135.05
14.0
0.07
0.22
98.3
y = 792138x - 57835.7
Syringic acid
5.23
12.0
197.08 - 167.03
18.0
0.05
0.17
96.6
y = 105523x - 7423.28
Vanillin
7.42
18.0
150.89 - 92.06
20.0
0.07
0.25
97.7
y = 68054.1x - 5533.4
p-Coumaric acid
5.95
18.0
162.95 - 119.06
28.0
0.11
0.39
98.4
y = 31614.3x - 3039.84
Ferulic acid
7.15
8.0
193.03 - 106.04
22.0
0.08
0.26
98.0
y = 2830.29x - 317.702
o-Coumaric acid
5.92
28.0
162.95 - 93.06
24.0
0.11
0.39
97.0
y = 31681.8x - 3026.2
Oleuropein
9.05
Luteolin
10.09
Quercetin
10.07
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Gallic acid
a
14.0
SC
Phenolic compound
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Retention time (RT), MS/MS parameters of the selected phenolic compounds and analytical parameters for the UPLC method.
2.0
539.34 - 275.16
22.0
0.02
0.08
99.8
y = 4460.53x - 99.2215
74.0
285.08 - 133.01
38.0
0.09
0.31
98.5
y = 844.103x - 88.224
2.0
301.14 - 151.01
24.0
0.26
0.86
98.3
y = 30.596x - 7.474
Calibration ecuation y = ax + b; y = área, x = Compound concentration.
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Hydroxyphenylacetic acid
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Vanillin
Tyrosol
Vanillic acid
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Gallic acid
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Caffeic acid
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o-Coumaric acid
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Oleuropein
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Quercetin
Luteolin
Syringic acid
Ferulic acid
p-Coumaric acid
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ACCEPTED MANUSCRIPT Highlights The UPLC-ESI-MS/MS method of polyphenol analysis was applied.
2.
11 compounds were quantified in argan oil before and after digestion.
3.
The digestive process strongly affects the polyphenol content in argan oil.
4.
Only a minor fraction of oil polyphenols may be considered bioavailable.
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S0023-6438(16)30590-4
DOI:
10.1016/j.lwt.2016.09.028
Reference:
YFSTL 5741
To appear in:
LWT - Food Science and Technology
Received Date: 3 February 2016 Revised Date:
12 September 2016
Accepted Date: 21 September 2016
Please cite this article as: Rueda, A., Cantarero, S., Seiquer, I., Cabrera-Vique, C., Olalla, M., Bioaccessibility of individual phenolic compounds in extra virgin argan oil after simulated gastrointestinal process, LWT - Food Science and Technology (2016), doi: 10.1016/j.lwt.2016.09.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Page 1 of 21
1
Bioaccessibility of individual phenolic compounds in extra
2
virgin argan oil after simulated gastrointestinal process
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Ascensión Ruedaa*, Samuel Cantarerob, Isabel Seiquerc, Carmen Cabrera-
5
Viquea, Manuel Olallaa
6
a
7
de Granada, Granada, Spain
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b
9
Universidad de Granada, Granada, Spain
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Departamento de Nutrición y Bromatología. Facultad de Farmacia, Universidad
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Centro de Instrumentación Científica. Campus Universitario de Fuentenueva.
10
c
11
Experimental del Zaidín (CSIC), Armilla, Granada, Spain
Departamento de Fisiología y Bioquímica de la Nutrición Animal, Estación
12
*Corresponding author (phone number: +34-958240755; Fax: +34-958249577;
14
E-mail: [email protected])
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ACCEPTED MANUSCRIPT ABSTRACT
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The bioaccessibility of individual phenolic compounds in extra virgin argan oil
23
was evaluated using an in vitro digestion method for first time. The analyses
24
were carried out by ultra-performance liquid chromatography-electrospray
25
ionisation tandem mass spectrometry in multiple reaction monitoring (UPLC-
26
ESI-MS/MS). Hydroxyphenylacetic acid (4245 µg/kg), ferulic acid (2478 µg/kg),
27
vanillin (2429 µg/kg) and 3,4-dihydroxybenzoic acid (2247 µg/kg) were the
28
mayor compounds identified in argan oil extracts. After the digestion process,
29
values of bioaccessibility of the different compounds varied from a minimum of
30
2% (hydroxyphenylacetic acid) to a maximum of 84% (p-coumaric acid), but
31
some such as caffeic acid became detectable, as a result from digestive
32
transformations. The results indicate that the phenolic compounds of the argan
33
oil are strongly affected during the digestive process, and bioaccessibility
34
should be taken into account when assessing polyphenol content of oils.
35
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Keywords
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In vitro digestion, individual polyphenols, argan oil, UPLC
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1. Introduction Virgin argan oil is a product obtained from roasted seeds of the fruit of
51
the argan tree Argania spinosa L. Skeels, which is endemic to SW Morocco.
52
This oil has been used by the Berber population for centuries due to its
53
sensorial and health-giving properties (Ahansal, Ben Sassi, Martini, Vaughan-
54
Martini, Walker & Boussaid, 2008). The presence of phenols and phytosterols in
55
argan oil has been associated with a cholesterol-lowering activity (Guillaume &
56
Charrouf 2011) and antidiabetic effects have been shown in rats (Bellahcen,
57
Mekhfi, Ziyyat, Legssyer, Hakkou, Aziz & Bnouham, 2012). In oils, polar lipids
58
contribute greatly to nutritional value due to specific anti-inflammatory activities
59
(Nasopoulou et al., 2014), but among the bioactive compounds present in virgin
60
argan oil, polyphenols play an important role, since their antioxidant activity is
61
related to cardioprotective (Pandey & Rizvi, 2009) and anti-inflammatory effects
62
(Rosillo, Alcaraz, Sánchez-Hidalgo, Fernández-Bolaños, Alarcón-de-la-Lastra
63
& Ferrándiz, 2014).
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Polyphenols such as caffeic acid, oleuropein, vanillic acid, tyrosol, ferulic
65
acid, syringic acid, p-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, catechol,
66
resorcinol and 4-hydroxybenzyl have been identified in virgin argan oil (Charrouf
67
& Guillaume, 2007; Charrouf & Guillaume 2008), but his bioaccessibility has not
68
been evaluated yet. Gallic acid has reported bioactivities such as antineoplastic,
69
bacteriostatic, antimelanogenic and antioxidant properties. This molecule
70
showed anticancer properties in prostate carcinoma cells. p-Hydroxybenzoic
71
acid has been reported to have antioxidant activities against free radicals,
72
antimicrobial activities and antimutagenic properties. vanillic acid showed
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antisicking and anthelmintic activities, and is also able to suppress hepatic
74
fibrosis in chronic liver injury. Syringic acid show antioxidant, antibacterial and
75
hepatoprotective activities (Heleno, Martins, Queiroz & Ferreira, 2015). The concept of bioaccessibility refers to the fraction of a compound which
77
is released from the food matrix in the gastrointestinal tract and becomes
78
available for absorption (Heaney, 2001). The polyphenol’s beneficial effects can
79
only be truly effective if they reach the relevant tissues and exert their action at
80
a sufficient concentration to have a biological effect. The most widely used
81
procedure for screening polyphenolic compound bioaccessibility is the in vitro
82
static gastrointestinal method (Carbonell-Capella, Buniowska, Barba, Esteve &
83
Frígola, 2014). One of the most important methods to determine the
84
bioaccessibility was proposed by Miller to simulate human digestion and
85
absorption of dietary iron in the study of phenolic compound release. Over time
86
this method has been used by other authors with modifications and has been
87
employed in the screening of multiple foods (Gil-Izquierdo, Zafrilla & Tomás-
88
Barberán,
89
Rodríguez-Roque, Rojas-Graü, Elez-Martínez & Martín-Belloso, 2013). Some
90
authors found that gastric digestion increases polyphenolic concentration,
91
whereas the duodenal fraction significantly lessens polyphenolic content in
92
pomegranate juice, broccoli and gooseberry (Pérez-Vicente, Gil-Izquierdo &
93
García-Viguera, 2002; Chiang, Kadouh & Zhou, 2013; Vallejo, Gil-Izquierdo,
94
Pérez-Vicente & García-Viguera, 2004). The method used by Bermudez-Soto et
95
al. (2007) found that during intestinal digestion a significant decrease in
96
anthocyanins (43%) and flavonols (26%) was observed, whereas chlorogenic
97
acid increased (24%) in chokeberry. Separation and characterization of
98
phenolic compounds has been conducted in argan fruit pulp, in alimentary and
Pérez-Vicente,
Gil-Izquierdo
&
García-Viguera,
2002;
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cosmetic oil and in the press cake (Charrouf & Guillaime 2007; El Abbassi,
100
Khalid, Zbakh & Ahmad, 2014), but the analysis of individual phenolic
101
compounds after subjecting argan oil to an in vitro digestion process has not
102
been done yet. Due to the great interest of phenolic compounds, great efforts have been
104
made in the last years to provide a technique highly sensitive for their
105
determination, and methodologies such as gas chromatography, capillary
106
electrophoresis and high performance liquid chromatography (HPLC), among
107
others, have been applied (Carrasco-Pancorbo et al., 2005; Ignat et al., 2011).
108
The use of ultra-performance liquid chromatography (UPLC) decreases the time
109
needed for analysis (<20 minutes), improves the resolution and provides great
110
sensitivity, and this method coupled with a MS/MS technique, makes it easy to
111
detect a large number of compounds in a short time (Kartsova & Alekseeva,
112
2008).
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The aim of this study was to assess the bioaccessibility of individual
114
phenolic compounds in argan oil after an in vitro digestion method simulating
115
the
116
chromatography-electrospray ionisation tandem mass spectrometry (UPLC-ESI-
117
MS/MS) in multiple reaction monitoring (MRM) was used as an analytical
118
technique. The high sensitivity of this method enabled us to obtain low detection
119
limits.
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2. Materials and methods
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2.1. Materials
123
2.1.1. Oils samples 5
process.
Ultra-performance
liquid
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125
lower than 0.8 % oleic acid, Norme Marocaine, 2003), obtained from specialist
126
stores in Spain, were included in the study. The samples were produced in
127
Morocco and obtained by cold pressing. The oil samples were carefully handled
128
to avoid contamination and correctly stored and maintained at a temperature of
129
4 ºC until analysis.
130
2.1.2. Reagents and standards
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All chemicals were analytical reagent grade or higher purity. Ultrapure
132
water was obtained from a Milli-Q purification system (Millipore, Bedford, MA).
133
Methanol and acetonitrile were supplied by Sigma-Aldrich (LC-MS Ultra
134
Chromasolv® Sigma Chemical Co., St. Louis, MO). Sodium bicarbonate,
135
sodium carbonate and hydrochloric acid (37%) were purchased from Merck
136
(Merck, Darmstadt, Germany). The commercial phenolic compound standards
137
used were oleuropein, p-coumaric acid, o-coumaric acid, 4-hydroxyphenylacetic
138
acid, quercetin, vanillin, gallic acid, 3,4-dihydroxybenzoic acid, caffeic acid and
139
syringic acid, all purchased from Sigma-Aldrich. Ferulic acid, tyrosol, luteolin
140
and vanillic acid were purchased from Fluka Chemicals (Madrid, Spain). Stock
141
standard solutions were prepared by dissolving each phenolic compound at a
142
concentration of 10 mg/mL in methanol, and the resulting solution was then
143
stored at -18 °C.
144
2.2. Methods
145
2.2.1. In vitro digestion
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The in vitro digestion procedure (Fig. 1) was performed according to the
147
method described by Mesías et al. (2009), slightly modified. The method
148
consists of two sequential steps; an initial pepsin/HCl digestion to simulate 6
ACCEPTED MANUSCRIPT gastric conditions, followed by a digestion with bile salts/pancreatin to simulate
150
duodenal digestion. Four grams of oil samples were mixed with 9 mL of
151
bidistilled deionised water and sonicated (Vibracell VCX 130, Sonics &
152
Materials INC, Danbury, Connecticut, USA). The pH was adjusted to 2 using
153
HCl 1N. Pepsin (Sigma-Aldrich, Milan, Italy) was added to a final concentration
154
of 0.05 g of pepsin / g sample and the samples were incubated in a shaking
155
water bath (Bunsen, Madrid, Spain) at 110 oscillations/min, at 37 °C for 2 hours.
156
For intestinal digestion, the pH was raised to 6 with NaHCO3 (1 M) dropwise,
157
and 2.5 mL of a mixture of 0.4 g of pancreatin + 0.25 g of bile salts (Sigma-
158
Aldrich, Milan, Italy) were added. The final pH was adjusted to 7 with NaHCO3
159
(1 M) and the samples were incubated in a shaking water bath (110 rpm; 37 °C)
160
for 2 hours. After gastrointestinal digestion, the digestive enzymes were
161
inactivated by heat treatment for 4 min at 100 ºC in a polyethylenglycol bath.
162
The samples were then cooled by immersion in an ice bath and centrifuged at
163
10.000 rpm (Sorvall RC 6 Plus centrifuge, Thermo Scientific, Madrid, Spain) for
164
30 min at 4 °C. The supernatants were carefully separated to obtain the
165
bioaccessible fraction, which was filtered using Vivaspin® 2 ml Centrifugal
166
Concentrator to remove the potential presence of protein, and then freeze-dried.
167
Lyophilized samples were dissolved in 3 ml of methanol / acetonitrile 50% (v / v)
168
for chromatographic analysis. Throughout the process, the samples were
169
protected from light, cooled with an ice bath and submitted to sonication before
170
each step.
171
2.2.3. Phenolic extract
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172
A liquid–liquid extraction adaptation technique (Montedoro, Servili,
173
Baldioli & Miniati, 1992) was used to determine individual phenolic compounds.
7
ACCEPTED MANUSCRIPT 15 g of oil was mixed with 10 ml of methanol 80% v/v, the mixture was shaken
175
in an orbital shaker (3005 GFL®, Grossburgwedel, Germany) for 1 hour and
176
centrifuged (Hettich zentrifugen Universal 320, Buckinghamshire, England) at
177
8000 rpm for 15 minutes. The supernatant was removed and the process was
178
repeated twice. Using a rotary evaporator (R-215, Buchi Switzerland), a
179
gelatinous residue was obtained, which was dissolved in 5 ml of methanol and
180
filtered through a 0.45 µm Millipore syringe filter.
181
2.2.4. UPLC analysis of polyphenols
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Analyses of polyphenols were carried out in chemical extracts and in bioavailable
fractions
obtained
after
the
in
vitro
digestion
of
oils.
184
Chromatographic analysis of the polyphenols was performed in the Scientific
185
Instrumentation Centre of the University of Granada using an Acquity UPLC H-
186
Class with MS detection (Waters, Barcelona, Spain) and separations were
187
achieved using an Acquity UPLC HSS T3 column (100 mm x 2.1 mm, 1.8 µm
188
particle size).
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Chromatographic separation was carried out using a gradient elution with
190
water + 0.5% acetic acid as eluent A and acetonitrile +0.5% acetic acid as
191
eluent B. The flow rate was 650 µL/min, the column was maintained at 45 ºC
192
and the injection volume was 10 µL. Gradient conditions were as follows: initial
193
mobile phase, 99% (A), which was linearly decreased to 70% (A) within 10.0
194
min and to 5% within 12.0 min. Finally, it was returned to 99% in 0.1 min and
195
maintained for 2.9 min to equilibrate the column. Total run time was 15 min.
196
Mass spectrometry analysis was carried out using a Waters XEVO TQ-S
197
tandem quadrupole mass spectrometer. The instrument was operated using
198
ESI ionisation in negative ion mode. Data acquisition was performed using
199
MassLynx 4.1 software with the QuanLynx program (Waters). For MS/MS
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201
following: capillary voltage 2.00 kV and cone voltage 10 V; the source
202
temperature was 100 °C and the desolvation temperature, 300 °C. The cone
203
gas (nitrogen) and desolvation gas (also nitrogen) were set at flow rates of 150
204
and 500 L/h, respectively.
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The conditions used were adapted from previous papers (Goh & Gledhill,
206
2011), seeking to obtain high sensitivity and to reduce analysis time. The
207
IntelliStart™ Software was used to develop MRM acquisition methods for the 14
208
phenolic compounds targeted in this analysis. Standard solutions of 10 mg/L of
209
each phenolic compound at a flow rate of 10 µL/min were infused at first and
210
then combined with the mobile phase flow. The collision voltage was optimised,
211
selecting the most sensitive transition for quantification. Some phenolic
212
compounds presented initially close retention times, but optimization of the
213
methods allowed the subsequent identification due to their different transitions.
214
Quantification of the samples was performed against a calibration curve for
215
each of the polyphenol analytes in solution. A chromatogram of digested argan
216
oil sample is shown in Fig. 2 according to the conditions established in the
217
methodology.
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Table 1 shows the parameters optimised for quantification using MRM,
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219
the calibration curves obtained, the recovery percentages and the limits of
220
detection and of quantification (LOD and LOQ, respectively) obtained for each
221
compound. After optimising the transitions, calibration curves were constructed
222
for each compound, using various concentration ranges, but always including at
223
least 5 different concentration points. LOD was determined by using a signal-to-
224
noise ratio of 3, and LOQ, by using a signal-to-noise ratio of 10.
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ACCEPTED MANUSCRIPT The R2 values were greater than 0.9901 for all the compounds used, and
226
the detection limits ranged from 0.07 µg/kg for hydroxyphenylacetic acid to 0.26
227
µg/kg for quercetin. The recovery (%) was calculated as the percentage from
228
the known amount of standard added to a refined vegetable oil. Analyses were
229
carried out in triplicate.
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230 231
3. Results and discussion
In this experimental study, we first determined the initial content of
233
polyphenols. A total of 14 phenolic compounds were analysed, structures of
234
which are depicted as supplementary material (S1). Quercetin and oleuropein
235
were not detected before or after the in vitro digestion process. 11 compounds
236
were detected and quantified in argan oil extracts (Fig. 3), being the major ones
237
hydroxyphenylacetic acid (4245 µg/kg), ferulic acid (2478 µg/kg), vanillin (2429
238
µg/kg) and 3,4-dihydroxybenzoic acid (2247 µg/kg). Other compounds that have
239
been quantified in argan oil extracts are vanillic acid (1190 µg/kg) and tyrosol
240
(2048 µg/kg); these polyphenols were identified as major components of argan
241
oil by Chaurrof & Guillaume (2007). Khallouki et al. (2003) measured the
242
following quantities in commercial argan oil: vanillic acid 123 µg/kg; syringic acid
243
68 µg/kg, tyrosol 52 µg/kg. These levels are lower than those obtained in our
244
study, although the concentration of ferulic acid was slightly higher (3470
245
µg/kg). Aidoud et al. (2014) also measured lower quantities than those found in
246
our study with respect to vanillic acid (67 µg/kg), syringic acid (37 µg/kg) and
247
tyrosol (12 µg/kg), and higher levels of ferulic acid (3147 µg/kg).
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248
Some polyphenols have been identified in the leaves and the fruit pulp of
249
the argan tree (Charrouf & Guillaume, 2008; Aidoud, Ammouche, Garrido &
250
Rodriguez,
2014).
In
the
press
cake,
3,4-dihydroxybenzoic
acid,
410
ACCEPTED MANUSCRIPT 251
hydroxyphenylacetic acid and vanillin have been identified (Joguet & Maugard,
252
2013). The simulation of digestion is widely used in many fields of food and
254
nutritional science, as human trials are often expensive, require considerable
255
resources and may be ethically questionable (Lin, Shi, Wang &Shen, 2008). In
256
vitro models of digestion provide a useful alternative to animal and human
257
models and the results supplied are fast and accurate (Coni et al., 2000).
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In our study, polyphenol concentrations generally decreased during the
259
simulated in vitro gastrointestinal digestion (Fig. 3). The highest levels of
260
polyphenols found in the bioavailable fraction of argan oil were vanillic acid, p-
261
coumaric acid and o-coumaric acid. Polyphenols shown a considerable
262
structural diversity which largely influences their bioaccessibility; it has been
263
shown that phenols may undergo structural modifications during the digestion
264
process that may reduce their bioaccessible fraction, mainly due to changes of
265
pH and interactions with other compounds, which differ depending on their
266
chemical structure (Dinnella et al., 2007). However, some compounds became
267
detectable or their concentration increased slightly after the digestion process,
268
as caffeic acid. On accordance, previous assays concerning transformations of
269
phenolic compounds during in vitro digestion of foods have shown that caffeic
270
acid is detected after the digestion process, but not before (Tarko et al., 2009).
271
Caffeic acid may derive from the hydrolysis of chlorogenic acid in the small
272
intestine (Sato et al., 2011), but early studies show that it also may results of the
273
conversion of p-coumaric acid (Sato, 1969). Thus, in argan oil digested, it may
274
be hypothesized that caffeic acid could come from p-coumaric acid.
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ACCEPTED MANUSCRIPT Fig. 4 shows the bioaccessibility of the different polyphenols (calculated
276
as the percentage from the initial quantity measured in the chemical extracts),
277
for the oils analysed. With the exception of luteolin, which was detected in the
278
chemical extracts but not after the in vitro digestion, values of bioaccessibility
279
varied largely, from 2% (hydroxyphenylacetic acid) to 84 % (p-coumaric acid). A
280
value of 100% was assigned to caffeic acid, as it was only detected in the
281
bioaccessible fraction. The largest decreases (expressed in percentage of
282
reduction from the initial quantity) were observed in the following compounds:
283
syringic
284
hydroxyphenylacetic acid 98%; vanillin 95% and ferulic acid 94%. These losses
285
were greater than those reported by Dinnella et al. (2007), who describe that
286
bioaccessibility of olive oil phenols vary from 37 to 90%. Other studies have
287
indicated that the process of digestion reduces phenolic content by 47-62%
288
(Minekus et al., 2014). It is possible that the pancreatin digestion liberates
289
compounds able to associate with phenolic compounds (Joguet & Maugard,
290
2013). During gastrointestinal digestion, polyphenols may either interact with
291
other food constituents (e.g., chelation of ions), be further degraded (such as
292
anthocyanins in the small intestine), or metabolised, such as by hydrolysis via,
293
e.g., deglycosylation or cleavage by esterases (Hura, Limb, Decker &
294
McClements, 2011).
3,4-dihydroxybenzoic
acid
95%;
SC
93%;
tyrosol
91%;
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To our knowledge, there is a lack of information concerning concentration
296
of individual polyphenols after in vitro digestion in argan oil, and only
297
bioaccessibility of total phenol content and antioxidant activity has been recently
298
reported by our research group (Seiquer et al., 2015).
299
12
ACCEPTED MANUSCRIPT 4. Conclusions
301
The analysis of phenolic compounds after subjecting argan oil to an in
302
vitro digestion process was performed for the first time. In general, the
303
polyphenol concentration in the bioaccessible fraction of the argan oil was lower
304
than in the initial extract of the oil. In the bioaccessible fraction, argan oil contain
305
quantities of 3,4-dihydroxybenzoic acid, tyrosol, vanillic acid, caffeic acid,
306
syringic acid, p-coumaric acid, o-coumaric acid and ferulic acid.
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Polyphenols are highly sensitive to the mild alkaline conditions in the
308
small intestine and a great proportion of these compounds can be transformed
309
into other unknown and/or undetected structural forms with different chemical
310
properties and, consequently, different bioaccessibility, bioavailability and
311
biological activity. Intestinal transformations of polyphenols seem to occur
312
during the digestion process of argan oil, leading to the release of the
313
compounds from the food matrix. However, these compounds could be
314
hydrolysed depending on the intestinal conditions.
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Decreases or increases of phenolic compounds in the bioaccessible
316
fractions of oils may take place, as it has been observed in the present study.
317
The results obtained indicate that only a minor fraction of argan oil polyphenols
318
may be considered bioavailable for subsequent absorption by the intestinal
319
cells. In any case, to better understand the transformation of argan oil
320
polyphenols during the digestion process more extensive study will be required,
321
to determine the presence of new metabolites.
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Conflict of interest
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The Authors declare that they have no conflict of interest.
325
Acknowledgements
327
This work was supported by the research group AGR141 of Junta de Andalucía
328
of University of Granada.
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ACCEPTED MANUSCRIPT REFERENCES
331
Ahansal, L., Ben Sassi, A., Martini, A., Vaughan-Martini, A., Walker G. &
332
Boussaid, A. (2008). Biodiversity of yeasts isolated from the indigenous
333
forest of Argan (Argania spinosa (L.) Skeels) in Morocco. World Journal of
334
Microbiology and Biotechnology, 24, 777–782.
RI PT
330
Aidoud, A., Ammouche, A., Garrido, M. & Rodriguez, A. B. (2014). Effect of
336
lycopene-enriched olive and argan oils upon lipid serum parameters in
337
Wistar rats. Journal of the Science of Food and Agriculture, 94, 2943-
338
2950.
M AN U
SC
335
Bellahcen, S., Mekhfi, H., Ziyyat, A., Legssyer, A., Hakkou, A., Aziz, M. &
340
Bnouham, M. (2012). Prevention of chemically induced diabetes mellitus
341
in experimental animals by virgin argan oil. Phytotherapy Research, 26,
342
180-185.
TE D
339
Bermúdez-Soto, M. J., Tomás-Barberán, F. A. & García-Conesa, M. T. (2007).
344
Stability of polyphenols in chokeberry (Aronia melanocarpa) subjected to
345
in vitro gastric and pancreatic digestion. Food Chemistry, 102, 865–74.
347 348 349 350
351 352
Carbonell-Capella, J. M., Buniowska, M., Barba, F. J., Esteve, M. J. & Frígola,
AC C
346
EP
343
M. (2014). Analytical methods for determining bioavailability and bioaccessibility of bioactive compounds from fruits and vegetables: a review. Comprehensive Reviews in Food Science and Food Safety, 13, 155-171.
Carrasco-Pancorbo, A., Cerretani, L., Bendini, A., Segura-Carretero, A., Gallina-Toschi,
T.
&
Fernández-Gutierrez,
15
A
(2005).
Analytical
ACCEPTED MANUSCRIPT 353
determination of polyphenols in olive oils. Journal of Separation Science,
354
28, 837–858. Charrouf, Z. & Guillaume, D. (2008). Argan oil: Occurrence, composition and
356
impact on human health. European Journal of Lipid Science and
357
Technology, 110, 632–636.
358 359
RI PT
355
Charrouf, Z. & Guillaume, D. (2007). Phenols and polyphenols from Argania spinosa, American Journal of Food Technology, 2, 679–683.
Chiang, C. J., Kadouh, H. and Zhou, K. (2013). Phenolic compounds and
361
antioxidant properties of gooseberry as affected by in vitro digestion. LWT-
362
Food Science and Technology, 51, 417–422.
M AN U
SC
360
Coni, E., Di Benedetto, R., Di Pasquale, M., Masella, R., Modesti, D., Attei, R.,
364
et al. (2000). Protective effect of oleuropein, and olive oil biophenol, on low
365
density lipoprotein oxidizability in rabbits, Lipids, 35, 45–54.
366
Dinnella,
TE D
363
C.,
Minichino,
P.,
D’Andrea,
A.M.
&
Monteleone.
(2007).
Bioaccessibility and antioxidant activity stability of phenolic compounds
368
from extra-virgin olive oils during in vitro digestion. Journal of Agriculture
369
and Food Chemistry, 55, 8423-8429.
AC C
EP
367
370
El Abbassi, A., Khalid, N., Zbakh, H. & Ahmad, A. (2014) Physicochemical
371
characteristics, nutritional properties, and health benefits of argan oil: a
372
review. Critical Reviews in Food Science and Nutrition, 11, 1401-1414.
373
Gil-Izquierdo, A., Zafrilla, P. & Tomás-Barberán, F. A. (2002). An in vitro method
374
to simulate phenolic compound release from the food matrix in the
375
gastrointestinal tract. European Food Research and Technology, 214,
376
155–159. 16
ACCEPTED MANUSCRIPT 377
Goh, E. & Gledhill, A. (2011). Analysis of polyphenols in fruit juices using
378
ACQUITY UPLC H-Class with UV and MS detection Waters Pacific,
379
Singapore, Waters Corporation, Manchester, UK.
381
Guillaume, D. & Charrouf, Z. (2011). Argan Oil. Alternative Medicine Review, 16, Number 3.
RI PT
380
Heleno, S. A., Martins, A, Queiroz, M. J. & Ferreira, I. (2015). Bioactivity of
383
phenolic acids: metabolites versus parent compounds: a review. Food
384
Chemistry, 173, 501-513.
SC
382
Hura, S. J., Limb, B. O., Decker, E. A. & McClements, D. J. (2011). In vitro
386
human digestion models for food applications. Food Chemistry, 125, 1–12.
387
Ignat, I., Volf, I., & Popa, V. I. (2011). A critical review of methods for
388
characterisation of polyphenolic compounds in fruits and vegetables. Food
389
Chemistry, 126, 1821-1835.
TE D
M AN U
385
Joguet, N. & Maugard, T. (2013). Characterization and quantification of phenolic
391
compounds of Argania spinosa leaves by HPLC-PDA-ESI-MS analyses
392
and their antioxidant activity. Chemistry of Natural Compounds, 48, 1069–
393
1071.
AC C
EP
390
394
Kartsova, L. A. & Alekseeva, A. V. (2008). Chromatographic and electrophoretic
395
methods for determining polyphenol compounds. Journal of Analytical
396
Chemistry , 63, 1024–1033.
397
Khallouki, F., Younos, C., Soulimani, R., Oster, T. Charrouf, Z., Spiegelhalder,
398
B., et al. (2003). Consumption of argan oil (Morocco) with its unique profile
399
of fatty acids, tocopherols, squalene, sterols and phenolic compounds
17
ACCEPTED MANUSCRIPT 400
should confer valuable cancer chemopreventive effects. European Journal
401
of Cancer Prevention, 12, 67–75.
402 403
Lin, Y., Shi, R., Wang, X. & Shen, H. M. (2008). Luteolin, a flavonoid with potential for cancer prevention and therapy. Current Cancer Drug Targets, 8, 634–646.
405
Mesías, M., Seiquer, I. & Navarro, M. P. (2009). Influence of diets rich in
406
Maillard reaction products on calcium bioavailability. Assays in male
407
adolescents and in Caco-2 cells. Journal of Agricultural and Food
408
Chemistry, 57, 9532–9538.
M AN U
SC
RI PT
404
409
Minekus, M., Alminger, M., Alvito, P., Balance, S., Bohn, T., Bourlieu, C., et al.
410
(2014). A standardised static in vitro digestion method suitable for food -
411
an international consensus. Food & Function, 5, 1113–1124. Montedoro, G., Servili, M., Baldioli, M. & Miniati, E. (1992). Simple and
413
hydrolyzable phenolic compounds in virgin olive oil. Their extraction,
414
separation, and quantitative and semiquantitative evaluation by HPLC.
415
Journal of Agricultural and Food Chemistry, 40, 1571–1576.
417 418 419
420 421
EP
Nasopoulou, C., Karantonis, H. C., Detopoulou, M., Demopoulos, C. A., &
AC C
416
TE D
412
Zabetakis, I. (2014). Exploiting the anti-inflammatory properties of olive (Olea europaea) in the sustainable production of functional food and neutraceuticals. Phytochemistry Reviews 13, 445–458.
Norme Marocaine (2003). Service De Normalisation Industrielle Marocaine Huile d ‘argane. Spécifications. Rabat, NM 08.5.090.
18
ACCEPTED MANUSCRIPT 422
Pandey, K. B. & Rizvi, S. I. (2009).Plant polyphenols as dietary antioxidants in
423
human health and disease. Oxidative Medicine and Cellular Longevity, 2,
424
270–278. Pérez-Vicente, A., Gil-Izquierdo, A. & García-Viguera, C. (2002). In vitro
426
gastrointestinal digestion study of pomegranate juice phenolic compounds,
427
anthocyanins, and vitamin C. Journal of Agricultural and Food Chemistry,
428
50, 2308–2312.
RI PT
425
Rodríguez-Roque, M. J., Rojas-Graü, M. A., Elez-Martínez, P. & Martín-Belloso,
430
O. (2013). Soymilk phenolic compounds, isoflavones and antioxidant
431
activity as affected by in vitro gastrointestinal digestion. Food Chemistry,
432
136, 206–212.
M AN U
SC
429
Rosillo, M. Á., Alcaraz, M. J., Sánchez-Hidalgo, M., Fernández-Bolaños, J. G.,
434
Alarcón-de-la-Lastra, C. & Ferrándiz, M. L. (2014). Anti-inflammatory and
435
joint protective effects of extra-virgin olive-oil polyphenol extract in
436
experimental arthritis. The Journal of Nutritional Biochemistry, 25, 1275–
437
1281.
EP
TE D
433
Sato, M. (1969). The conversion by phenolase of p-coumaric acid to caffeic acid
439
with special reference to the role of ascorbic acid. Phytochemistry, 8, 353-
440
AC C
438
362.
441
Sato, Y., Itagaki, S., Kurokawa, T., Ogura, J., Kobayashi, M., Hirano, T.,
442
Sugawara, M. & Iseki, K. (2011). In vitro and in vivo antioxidant properties
443
of
444
Pharmaceutics 403, 136–138.
chlorogenic
acid
and
caffeic
19
acid.
International
Journal
of
ACCEPTED MANUSCRIPT 445
Seiquer, I., Rueda, A., Olalla, M & Cabrera-Vique, C. (2015). Assessing the
446
bioavailability of polyphenols and antioxidant properties of extra virgin
447
argan oils by simulated digestion and Caco-2 cell assays. Comparative
448
study with extra virgin olive oil. Food Chemistry, 188, 496-503. Tarko, T., Duda-Chodak, A.,
Sroka, P. Satora, P. & Michalik, J. (2009).
RI PT
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Transformation of Phenolics in Alimentary Tract, Food Technology and
451
Biotechnology, 47, 456–463
Vallejo, F., Gil-Izquierdo, A., Pérez-Vicente, A. & García-Viguera, C. (2004). In
453
vitro gastrointestinal digestion study of broccoli inflorescence phenolic
454
compounds, glucosinolates, and vitamin C. Journal of Agricultural and
455
Food Chemistry, 52,135–138.
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Figure captions.
459
Figure 1. Scheme of the in vitro digestion process applied to the oil samples.
460
Figure 2. UPLC–MS/MS chromatogram obtained from the digested argan oil
463
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sample.
Figure 3. Content of phenolic compounds in extra virgin argan oil before (oil extract) and after (digested oil) the digestion process.
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Figure. 4. Bioaccessibility of different polyphenols in argan oil (calculated as the
465
percentage from the initial quantity in the chemical extracts). 1: Gallic acid;
466
2: 3,4-Dihydroxybenzoic acid; 3: Tyrosol; 4: Hydroxyphenylacetic acid; 5:
467
Vanillic acid; 6: Caffeic acid; 7: Syringic; 8: Vanillin; 9: p-coumaric; 10:
468
Ferulic acid; 11: o-coumaric; 12: Quercetin; 13: Oleuropein; 14: Luteolin.
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Table 1
RT (min)
Calibration curvesa
Collision
LOD
LOQ
Recovery
voltage
energy
(µg/kg)
(µg/kg)
(%)
(V)
(eV)
0.47
96.5
y = 510255x - 43456.9
Cone
Transition
1.82
22.0
168.93 - 125.03
0.14
3, 4-Dihydroxybenzoic acid
4.25
30.0
152.93 - 53.08
24.0
0.09
0.31
99.0
y = 11961.3x - 819.205
Tyrosol
4.18
30.0
136.91 - 106.15
18.0
0.09
0.31
97.8
y = 2156.5x - 157.84
Hydroxyphenylacetic acid
4.50
30.0
150.95 - 79.09
18.0
0.07
0.24
96.4
y = 149507x - 11517
Vanillic acid
4.65
26.0
166.95 - 91.03
18.0
0.10
0.34
99.2
y = 30266.7x - 2551.55
Caffeic acid
4.92
10.0
178.95 - 135.05
14.0
0.07
0.22
98.3
y = 792138x - 57835.7
Syringic acid
5.23
12.0
197.08 - 167.03
18.0
0.05
0.17
96.6
y = 105523x - 7423.28
Vanillin
7.42
18.0
150.89 - 92.06
20.0
0.07
0.25
97.7
y = 68054.1x - 5533.4
p-Coumaric acid
5.95
18.0
162.95 - 119.06
28.0
0.11
0.39
98.4
y = 31614.3x - 3039.84
Ferulic acid
7.15
8.0
193.03 - 106.04
22.0
0.08
0.26
98.0
y = 2830.29x - 317.702
o-Coumaric acid
5.92
28.0
162.95 - 93.06
24.0
0.11
0.39
97.0
y = 31681.8x - 3026.2
Oleuropein
9.05
Luteolin
10.09
Quercetin
10.07
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Gallic acid
a
14.0
SC
Phenolic compound
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Retention time (RT), MS/MS parameters of the selected phenolic compounds and analytical parameters for the UPLC method.
2.0
539.34 - 275.16
22.0
0.02
0.08
99.8
y = 4460.53x - 99.2215
74.0
285.08 - 133.01
38.0
0.09
0.31
98.5
y = 844.103x - 88.224
2.0
301.14 - 151.01
24.0
0.26
0.86
98.3
y = 30.596x - 7.474
Calibration ecuation y = ax + b; y = área, x = Compound concentration.
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Hydroxyphenylacetic acid
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Vanillin
Tyrosol
Vanillic acid
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Gallic acid
SC
Caffeic acid
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o-Coumaric acid
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Oleuropein
AC C
Quercetin
Luteolin
Syringic acid
Ferulic acid
p-Coumaric acid
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ACCEPTED MANUSCRIPT Highlights The UPLC-ESI-MS/MS method of polyphenol analysis was applied.
2.
11 compounds were quantified in argan oil before and after digestion.
3.
The digestive process strongly affects the polyphenol content in argan oil.
4.
Only a minor fraction of oil polyphenols may be considered bioavailable.
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