Investigation of Natural LipidâPhenolic Interactions on Biological

Article pubs.acs.org/JAFC

Investigation of Natural Lipid−Phenolic Interactions on Biological Properties of Virgin Olive Oil Muhammad H. Alu’datt,*,†,⊥ Taha Rababah,† Khalil Ereifej,† Sana Gammoh,† Mohammad N. Alhamad,§ Nizar Mhaidat,‡ Stan Kubow,∥ Ayman Johargy,# and Ola J. Alnaiemi† †

Department of Nutrition and Food Technology, Faculty of Agriculture, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan ‡ Department of Clinical Pharmacy, Faculty of Pharmacy, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan § Faculty of Agriculture, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan ∥ School of Dietetics and Human Nutrition, McGill University, 21,111 Lakeshore, Ste. Anne de Bellevue, Quebec Canada H9X 3 V9 ⊥ Faculty of Nursing, Umm Al-Qura University, P.O. Box 14405, Makkah, 21955, Saudi Arabia # Department of Medical Microbiology, Faculty of Medicine, University of Umm Al-Qura, Makkah, Saudi Arabia ABSTRACT: There is limited knowledge regarding the impact of naturally occurring lipid−phenolic interactions on the biological properties of phenolics in virgin olive oil. Free and bound phenolics were isolated via sequential methanolic extraction at 30 and 60 °C, and were identified and quantified using reversed phase high performance liquid chromatography, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and gas chromatography. Decreased oleic acid concentrations and increased concentrations of palmitoleic acid, stearic, linoleic, and linolenic acids were observed in virgin olive oil after removal of free and bound lipid phenolic compounds. The presence of p-hydroxybenzoic acid and tyrosol bound to glycerides was determined via LC-MS/MS, which indicates natural lipid−phenolic interactions in virgin olive oil. Both free and lipid bound phenolic extracts exerted antiproliferative activities against the CRC1 and CRC5 colorectal cancer cell lines. The present work indicates that naturally occurring lipid−phenolic interactions can affect the biological properties of phenolics in virgin olive oil. KEYWORDS: phenolic content, lipid-phenolic, interaction, antioxidant, antihypertensive, antitumorigenic, virgin olive oil

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INTRODUCTION Plant phenolic compounds have been indicated to play an important role in human health due to their possible role in reducing the risk of cardiovascular diseases, diabetes, and certain types of cancer.1 The use of phenolic compounds as nutraceuticals toward disease prevention has been constrained in association with lipid-based media because of their low solubility in this nonpolar media.2 Phenolic lipids can be synthesized by lipase-catalyzed transesterification between phenolic acids and triacylglycerols to enhance the solubility of hydrophilic phenolic compounds in nonpolar media.3 Such structured lipids can provide the health promoting properties of both lipids and phenolic compounds.3 Lipase-catalyzed esterification of phenolic acids with fatty alcohols can be achieved in solvent organic media by formation of phenolic lipid ester complexes4 or solvent free media via esterification of phenolic acids from green coffee with fatty alcohols.5 There is increasing interest, however, regarding the functional health properties of phenolic lipids resulting from natural phenolic lipids interactions found in foods such as cashews6 and propolis.7 The fatty acids in olive oil contain between 14 and 24 carbons.8 The main fatty acids found in olive oil are oleic acid, linoleic acid, palmitic acid, stearic acid, and linolenic acid.9 Minor quantities of myrsitic, margaric, heptadecenoic, arachidic, behenic, and lignoceric acids are found in olive oil.9 Variations © XXXX American Chemical Society

in fatty acid content are related to several factors including cultivar, place of production, degree of maturation, method of extraction, climate, and harvesting time.10 Virgin olive oil contains more than 30 different types of phenolic compounds. Several classes of phenolic compounds with antioxidant properties found in virgin olive oil include phenolic acids, phenolic alcohols, hydroxy-isocromans, flavonoids, secoiridoids, and lignans. The main phenolic compounds present in virgin olive oil are catechol, rutin, verbascoside, elenolic acid, oleuropein, hydroxytyrosol, tyrosol, caffeic acid, p-coumaric acid, and vanillic acid.11−17 The types and amount of phenolic compounds in virgin olive oil vary based on the degree of maturity, processing, storage time, and climatic conditions.13,14 The presence of virgin olive oil as an essential component in the Mediterranean diet has been strongly related to a lower chronic disease risk, which has been attributed to its high monounsaturated fatty acid and phenolic content.18 The levels of bioavailability of intact phenolic compounds with other food components such as protein, minerals, carbohydrate, and fat are limited. In vitro studies have several limitations related to bioavailability and stability of intact phenolic compounds Received: September 21, 2014 Revised: November 11, 2014 Accepted: November 12, 2014

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dx.doi.org/10.1021/jf504557k | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

et al.21 with modifications. A 5 mg portion of β-carotene was dissolved in 50 mL of chloroform. The β-carotene/linoleic acid stock solution was prepared by mixing 3 mL of β-carotene solution with 50 μL of linoleic acid and 400 mg of Tween 20. The chloroform was evaporated under nitrogen gas followed by addition of 100 mL distilled water, and the resulting emulsion was shaken for 2 min. A 100 μL portion of the bound phenolic methanolic extract (BP-30 °C or BP-60 °C) was mixed with 3 mL of the emulsion. The absorbance was measured at time 0 and after incubation for 1 h at 50 °C at 470 nm. A 100 μL portion of solvent was used in the blank control sample. The percent antioxidant activity (AA%) was calculated as amount of β-carotene bleaching in sample to control according to the following equation:

including the limit of polyphenol bioconversion and absorption as compared to in vivo studies which are less stable in human gut due to environmental conditions and diversity of microbes.19 To our knowledge, there is no information regarding the nature or biological properties of naturally occurring phenolic lipids that could result from natural lipid− phenolic interactions in olive oil. The specific objectives of this research were the following: (i) to investigate the occurrence of natural lipid−phenolic interactions in virgin olive oil via evaluation of the total content of free and lipid-bound phenolic compounds and identification of individual lipid phenolic compounds in virgin olive oil; and (ii) test the biological properties of free and bound phenolic compounds in terms of antioxidant activity, α-amylase and ACE inhibitory activities, and antitumorigenic activity.

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AA% = 100*((DRc − DRs)/DRc) Here, AA% is the percent antioxidant activity. DRc is the degradation rate of the blank control sample [DRc = (ln(A/B)/60)]. DRs is the degradation rate of the sample DRs = (ln(A/B)/60). A is the absorbance at t = 0, and B is the absorbance at t = 60 min. Measurement Inhibitory activity for α-Amylase Enzyme. The activity of α-amylase was evaluated according to the method described by McCue et al.23 with modifications. Porcine pancreatic α-amylase (10080, Sigma Chemical Co.) was prepared by mixing 0.030 g of the enzyme in 100 mL of distilled water. A potato starch solution of 0.5% (w/v) was prepared by dissolving 0.125 g of starch in 25 mL of phosphate buffer (pH 6.9; 65 °C/20 min). A colorimetric reagent was prepared by mixing well 19.8 g of sodium hydroxide with 10.6 g of 3,5dinitrosalicyclic acid solution (DNS) in 1.416 L of distilled water and then adding this to a mixture of 3.06 g of sodium potassium tartarate, 7.6 g of phenol, and 8.3 g of sodium metabisulfite, and mixing by shaking for 15 min. A 100 μL portion of phenolic extract (BP-30 °C or BP-60 °C), 500 μL of phosphate buffer (pH 7.0), and 500 μL of αamylase solution were incubated at 25 °C for 10 min. The control sample consisted of 100 μL of solvent. After the preincubation, 500 μL of potato starch solution was added, and the mixture was incubated in a 25 °C water bath for 10 min. The reaction stopped by adding 1 mL of DNS. The mixture was then incubated in a boiling water bath for 5 min and then cooled to room temperature. The total volume of reaction mixture was diluted to 10 mL by adding distilled water. To determine the amount of liberated maltose the absorbance was measured at 540 nm via a spectrophotometer (Spectrophotometer-UV 1800, Biotech Engineering Management Co., Ltd., U.K.). The percent α-amylase inhibition was expressed as amount of liberated maltose in sample to control and calculated according to the followin equation:

MATERIALS AND METHODS

Plant Materials. Nepali olive sample was obtained from Rahoub Valley (Irbid, Jordan). Samples were harvested in the winter of 2010 at proper harvesting stages according to the International Olive Oil Council method (IOOC).20 Olives were subjected to milling at commercial industrial two-phase olive oil (Irbid, Jordan) to obtain the virgin olive oil. Olive oil samples were filled in plastic containers of 10 L. The virgin olive oil was graded into upper and lower layers of 10 L containers after storage of three months to remove sediments and then stored at −18 °C prior to analysis. Phenolic Compounds Extraction. Free Phenolic Compounds Extraction. Free phenolic compounds were extracted according to the method described by Alu’datt et al.21 with modifications. A 15 mL sample of two layers from virgin olive oil was extracted with 25 mL of methanol at 30 °C for 1 h in a shaking water bath. The supernatant oil samples were filtered using Whatman 3 filter paper, and the resulting extract was designated as free phenolic extract at 30 °C (FP-30 °C). The supernatant that remained after methanol extraction at 30 °C was subjected to methanol extraction with 25 mL at 60 °C for 1 h in a shaking water bath. After filtration with Whatman 3 filter paper, the extract was designated as free phenolic extract at 60 °C (FP-60 °C). The FP-30 °C and FP-60 °C extracts were stored at −18 °C for further analysis. Bound Phenolic Compounds Extraction. Bound phenolic compounds were extracted according to the method described by Alu’datt et al.21 with modifications. The residue remaining after extraction of free phenolic compounds at 30 and 60 °C as described above was hydrolyzed with dilute alkaline solution (25 mL, pH 12.0, 0.1 M NaOH) for 24 h at 30 °C in a shaking water bath, and then the alkaline solution was freeze-dried (LFD-5508 freeze-dryer, Korea). The liberated bound phenolic compounds from alkaline hydrolysis were subjected to methanol extraction (25 mL) at 30 °C for 1 h. The supernatant was filtered using filter papers (Whatman 3), and the resulting extract was designated as bound phenolic at 30 °C (BP-30 °C). Following extraction with 25 mL of methanol at 60 °C for 1 h, the extract that was generated was designated as bound phenolic at 60 °C (BP-60 °C). All extracts were stored at −18 °C for further analysis. Total Phenolic Compounds Content. Determination of the total phenol content in each phenolic extract was conducted by using the Folin−Ciocalteu method as previously described by Hoff and Singleton.22 Standard curves of gallic acid were prepared using a stock solution of 5 mg/mL. A 100 μL portion of the extract containing phenolic compounds was added to 8.4 mL of distilled water. A 0.5 mL portion of the Folin−Ciocalteu solution was added and mixed well in the test tube after 4 min, which was followed by addition of 1.0 mL of 5% sodium carbonate and vortex-mixed for several seconds. The absorbance was measured 1 h later at 725 nm using UV−vis spectrophotometry (UV 1800, Biotech Engineering Management Co., Ltd., U.K.). The total phenolic content was expressed as mg/L gallic acid equivalents (mg GAE/L). Determination of Antioxidant Activity. Antioxidant activity of phenolic extract was determined according to the method of Alu’datt

α‐amylase inhibition % = 100[(Ac − As)/Ac] Here, Ac is the absorbance of the control sample at 540 nm, and As is sample absorbance. Determination Inhibitory Activity of Angiotensin 1-Converting Enzyme (ACE). ACE inhibition was measured according to the method described by Cushman and Cheung24 with modifications. A 0.3% (w/ v) solution of hippuryl-L-histidyl-L-leucine (HHL) solution was dissolved in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) HCl buffer with 300 mM sodium chloride (pH 8.3 at 37 °C). The ACE enzyme solution was prepared by mixing of 0.33 U in 1 mL of distilled water. An aliquot of 200 μL of HHL was mixed with 100 μL of either the BP-30 °C or BP-60 °C phenolic extract. An aliquot of 50 μL of ACE solution was added, and the mixture was incubated at 37 °C for 15 min. The reaction was stopped by adding 0.25 mL of HCl, and then 2 mL of ethyl acetate was added to extract the liberated hippuric acid. A 1 mL portion of the ethyl acetate layer was separated by centrifugation (3000 rpm/3 min) and evaporated by using boiling water bath for 15 min, and then 3 mL of distilled water was added. The amount of liberated hippuric acid was quantified by measuring the absorbance at 228 nm (UV 1800, Biotech Engineering Management Co., Ltd., U.K.). A blank was prepared by adding of 200 μL of HHL and 50 μL of ACE in 100 μL of distilled water. The amount of hippuric acid liberated in the control samples was defined as 100% ACE activity. The ACE inhibition was expressed as the amount of liberated hippuric acid in the sample to the control and calculated using the following equation: B



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absorbance of 280 nm was selected to determine the contents of individual phenolic compounds. The eluted compounds of samples and standards were detected at 280 nm. Mixture of external standards (p-hydroxybenzoic acid, gallic acid, caffeic acid, ferulic acid, vanillic acid, tyrosol, hydroxytyrosol, p-coumaric acid, syringic acid, apigenin, hesperidin, luteolin, quercetin, rutin, sinapic acid, and oleuropein) was used to quantify the contents of individual phenolic compounds. Analysis of the Fatty Acids by Gas Chromatography (GC). Fatty acid analysis was carried out according to the method described by Ereifej et al.27 with modifications. Fatty acid profiles of virgin olive oil before and after removal of free and bound phenolic compounds were analyzed by GC-MS (Varian 450-GC/Varian 320-MS). Fatty acid methyl esters of virgin olive were prepared as follows: 0.1 g of olive oil was dissolved in 2 mL of heptane and mixed with a 0.2 mL solution of 2 M methanolic potassium hydroxide and then shaken vigorously for 30 s. The upper layer of the solution containing fatty acid methyl esters was separated, and 15 μL was injected in a split/split injector. An Agilent HP-INNOwax column (30 m × 0.25 mm i.d., 0.25 μm film thickness) was employed. The oven temperature program was as follows: column temperature was programmed from 50 °C (kept for 4 min) to 200 °C at 25 °C/min, 2 min at 200 °C, from 200 to 240 °C at 4 °C/min, 18 min at 240 °C. A constant helium carrier gas flow rate of 0.8 mL/min was used. A standard of fatty acid methyl esters was used to identify the content of fatty acids and was expressed as percent of total content of fatty acids. HPLC Separation of Acylglycerols. Acylglycerols for virgin olive oil before and after removal of free and bound phenolic compounds were analyzed by HPLC. Olive oil samples were dissolved in tetrahydrofuran (THF). HPLC separation was carried out by Agilent 1100 series instrument (Wilmington, DE) equipped with UV−vis diode-array detection operated at 215 nm using a Zorbax SB-C18 reverse phase column (pore size 5 μm, 250 mm × 4.6 mm i.d., Restek Co., Bellefonte, PA) operated at 60 °C. Elution was carried out at flow rate of 0.8 mL/min, with an injection sample volume of 10 μL. The elution of the injected sample was carried out by a gradient solvent system using distilled water as solvent A, and acetonitrile/methyl tetra-butyl ether mixture (9:1, v/v) as solvent B. Linear gradient started from 87% solvent B, and increased to 100% solvent B over 25 min. The initial conditions were then re-established over 4 min. The fractionation of individual peaks obtained from HPLC analysis of glycerols for virgin olive oil before and after removal of free and bound phenolic compounds involved elution from the column and collection of the fractions that were pooled and freeze-dried (LFD-5508 freeze-dryer, Korea). A 4 mL portion of distilled water were added to each fraction, which was divided into two parts and then freeze-dried. The first part was extracted with methanol, while the second part was hydrolyzed with dilute alkaline solution (2 mL, pH 12.0, 0.1 M NaOH) for 24 h at 30 °C water bath followed by freeze-drying and then extracted with methanol. All fractionated samples were stored at −18 °C for further analysis. Statistical Analysis. Statistical analysis was performed by applying the General Linear Model (GLM) of the SAS software package (Version 9.1 SAS 2002 Institute Inc., NC). The least significant difference (LSD) multiple-range test was used for means separation using an α level of 0.05.

ACE inhibition % = [(A 228 blank − A 228 sample)/A 228 blank)100]

Here, A is the absorbance. Measurement of Anti-Tumorigenic Activity Using Human Colorectal Cancer Cells. Cell Culture. Two different human colorectal cancer cells (CRC1 and CRC5) were supplied by Dr. Rick Throm, University of Newcastle, Australia. The cell lines were cultured as monolayer and attached in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Lonza, Verviers, Belgium), 1% penicillin-streptomycin (5000 U penicillin/mL and 5000 U streptomycin/mL) (Lonza, Verviers, Belgium), and amphotericin B (250 μg/mL) (Lonza, Verviers, Belgium). Cells were incubated in a humidified atmosphere containing 5% CO2, at 37 °C in CO2 incubator (Shell Lab). Cell Viability Assay. The four types of phenolic extracts derived from the virgin olive oil layer characterized by having the highest phenolic content were chosen for studying the antitumorigenic effects of phenolics in olive oil. The cytotoxic effect of different concentrations of phenolic extracts (10−200 μL) was determined by using 3-(4,5-dimethylthiazo-l-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the method described by Wu et al.25 Cells were seeded at 5000 cells/well onto flat-bottomed 96-well culture plates, and allowed to grow for 24 h before treatment. Cells were incubated with five different concentrations of phenolic extracts (0, 10, 50, 100, and 200 μL; 0.0, 1.4, 7.1, 14.2, and 28.4 μg for FP-30 °C; 0.0, 0.7, 3.5, 7.0, and 14.0 μg for FP-60 °C; 0.0, 0.6, 3.0, 6.0, and 12.0 μg for BP-30 °C; and 0.0, 0.68, 3.4, 6.8, and 13.6 μg for BP-60 °C). After 48 h of incubation, cells were incubated with 10 μL of MTT for 4 h from Vybrant MTT assay cell proliferation assay kit (Molecular Probes, Eugene, OR) according to manufacturer’s instruction. After MTT incubation, the supernatant was aspirated, and the insoluble formozan product was dissolved with dimethyl sulfoxide (80 μL DMSO). Cell proliferation was measured by measuring absorbance using a microplate reader (model 3550, Bio-Rad Laboratories, CA) at 540 nm. Identification and Quantification of Individual Phenolic Compounds by Using Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry (LC/ESI-MS/MS). A 10 mL portion of free and bound phenolic extracts that were characterized with the highest phenolic content was evaporated under a stream of nitrogen and then dissolved in 1 mL of methanol and stored at −18 °C until further analysis. Standard curves were prepared by using a stock standard solution prepared by dissolving 5 mg of each phenolic standard in 50 mL of methanol. Virgin olive extracts were analyzed by LC-MS/MS according to the method described by Obied et al.26 The analysis of phenolic compounds was carried out using Agilent 1100 chromatography system (Agilent 1100, Agilent Technologies, Wilmington, DE) equipped with a diode array UV detector. The samples were injected into a Thermo C18 reversed phase column (pore size 5 μm, 250 mm × 4.6 mm i.d., Thermo Fisher Scientific, San Jose, CA). The analysis was carried out by a gradient solvent system using aqueous formic acid (1%) as solvent A, and methanol/acetonitrile/ formic acid mixture (89.5/9.5/1 v/v/v) as solvent B. A seven-step linear gradient elution for total run time of 65 min was carried out using the solvent gradient as follows: 0−10 min 90−70% solvent A and 10−30% solvent B, 10−15 min isocratic, 15−25 min 60% solvent A and 40% solvent B, 25−40 min 50% solvent A and 50% solvent B, 40−50 min to 100% solvent B, 50−55 min 90% solvent A and 10% solvent B and 55−65 min isocratic. An injection volume of 15 μL at constant flow of 0.75 mL/min was used for each analysis. The entire flow from the HPLC was directed into the tandem mass spectrometer. Triple-quadrupole mass spectrometer (API 3200; MDS Sciex, Concord, ON, Canada) was used. The mass spectral data was acquired on negative ion mode, capillary voltage 4000 V, ion source atmospheric pressure chemical ionization (APCI), a cone voltage 70 V, collision energy 10 eV, drying temperature 350 °C, drying gas N2 with 4.0 L/min flow rate, nebulizer gas helium with 40 psi flow rate, software used for data processing was Analyst software version 3.5.1. Diode array UV detector was used to scan between 200 and 400 nm to evaluate contents of individual phenolic compounds. A maximum

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RESULTS AND DISCUSSION Free and Bound Phenolic Distribution. Table 1 shows the content of extracted free and bound phenolic compounds from the lower layer A and upper layer B. The total phenolic content of olive oil from the combined fractions was 567.83 mg/L for layer A and 506.64 mg/L for layer B. The total content of extracted free phenolics in virgin olive oil ranged from 330.8 to 353.53 mg/L while the total content of extracted bound phenolics ranged from 175.84 to 214.3 mg/L. The above results are comparable with the upper range phenolic concentrations noted in previous studies, which found that the total content of phenolic compounds in virgin olive oil ranged C



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Table 1. Total Phenolic Content (mg/L), Antioxidant Activity (%), and Inhibitory Activities (%) of α-Amylase and ACE of Free (FP) and Bound (BP) Phenolic Compounds at 30 and 60 °C from Lower (A) and Upper (B) Layers of Extra Virgin Olive Oila FP-30 °C content (mg/L)

FP-60 °C content (mg/L)

BP-30 °C content (mg/L)

BP-60 °C content (mg/L)

237.37a A 232.32a A 2.52 antioxidant activity for FP-30 °C (%)

116.16b A 98.48b A 3.99 antioxidant activity for FP-60 °C (%)

100.74c A 86.09c B 1.83 antioxidant activity for BP-30 °C (%)

113.56b A 89.75bc B 2.90 antioxidant activity for BP-60 °C (%)

2.72 3.11

58.1a A 55.0a A 0.54 α-amylase inhibitory activity of FP30 °C (%)

44.1b A 39.3c B 0.60 α-amylase inhibitory activity FP60 °C (%)

45.3b A 45.1b A 0.30 α-amylase inhibitory activity BP30 °C (%)

6.7c A 5.7d A 0.24 α-amylase inhibitory activity BP60 °C (%)

0.46 0.43

27.99c A 21.49c B 1.03 ACE inhibitory activity FP60 °C (%)

37.67a A 33.20a B 0.41 ACE inhibitory activity BP30 °C (%)

30.15c A 27.27b A 0.87 ACE inhibitory activity BP60 °C (%)

0.71 0.94

layer of oil

33.41b A 27.70b B 0.89 ACE inhibitory activity of FP30 °C (%)

A B SE

33.12b A 24.89b B 0.26

17.51d A 11.60c B 0.98

37.57a A 29.39a B 0.35

28.51c A 24.32b A 1.41

0.36 1.08

layer of oil A B SE layer of oil A B SE layer of oil A B SE

SE

SE

Figure 1. Distribution of free and bound phenolic compounds (percent of total phenolic content) at 30 and 60 °C.

compounds in olive cake ranged between 10% and 25%. In contrast, Alu’datt et al.17 have reported that the percent content of bound phenolic compounds from olive meal represented less than 10% of total phenolic content. These results suggest that the phenolic compounds are present in free and bound forms in virgin olive oil. Antioxidant Activity of Free and Bound Phenolics Extracts. Table 1 demonstrates the antioxidant activity of free and bound phenolic extracts of virgin olive oil. The antioxidant activities were the highest (55.0−58.1%) in layers B and A of FP-30 °C. The antioxidant activity of BP-30 °C in layer B was higher (45.1) as compared to antioxidant activities with layer B from FP-60 °C (39.3). The lowest antioxidant activities were found in BP-60 °C for both layers B and A with values in the range 5.7−6.7%. The above results indicate that the antioxidant activities of extracted free or bound phenolic compounds extracted at 30 °C are higher than those observed with the phenolics extracted at 60 °C. These latter results are inconsistent with previous findings showing a negative relationship between temperature of extraction and antioxidant activity.30 The above ranges of antioxidant activities are similar to the 13.2−40.2% antioxidant activities reported by Gorinstein et al.31 in Spanish olive oils. Conversely, Baiano et al.16 found that the antioxidant activities of Italian virgin olive oils ranged between 70.67% and 87.83%. A weak positive linear correlation (0.571) was observed between total phenolic content and antioxidant activity (Table 2) which is in agreement with previous work showing that phenolic content was positively correlated with the antioxidant activity of olive oil.16 Inhibitory Activity of α-Amylase of Free and Bound Phenolics Extracts. Table 1 shows the α-amylase inhibitory activity of free and bound phenolic extracts from virgin olive oil. The maximum inhibitory activities of α-amylase were observed for layers A and B with the BP-30 °C extract with a range of values between 33.20% and 37.67%. The lowest inhibitory activities of α-amylase for the two layers were seen with the FP60 °C extract with a range of values of 21.49−27.99%. The bound phenolic extracts of virgin olive oil generally had a better α-amylase inhibitory activity than the free phenolic extracts. Also, the free or bound phenolic extracts at 30 °C had higher inhibitory activity of α-amylase than free or bound phenolic extracts at 60 °C. Variations in inhibitory activities of α-amylase by the phenolic extracts may be due to differences in type, quantity, and polarity of phenolic compounds in extracts. These results are in agreement with the findings by Loizzo et al.32 who

SE

SE

a

Means are averages of two replicates. Row values with the same small letters and column values with the same capital letters were not significantly different (p < 0.05). SE: standard error.

from 0.04 to 900 mg/kg.28,29 The variations in phenolic content are likely related to varieties of cultivar, extraction method, storage, and package conditions.8 In terms of individual fractions, the highest content of phenolic compounds in layers A and B was observed in the FP-30 °C extract with values of 237.37 and 232.2 mg/L, respectively. The lowest phenolic values were observed in the BP-30 °C extract with values of 100.74 and 86.09 mg/L for layers A and B, respectively. The above findings are in agreement with Alu’datt et al.21 who found that the majority of free phenolics were extracted from olive cake in first step cycle. Figure 1 illustrates the percent content of phenolic compounds of the FP-30 °C, FP-60 °C, BP-30 °C, and BP60 °C extracts. The highest percent phenolic content was observed for the FP-30 °C extract, which had 41.80% and 45.86% for layers A and B, respectively. On the other hand, total phenolics in the FP-60 °C extract represented only 20.64% and 19.44%, respectively. These latter results were consistent with the finding of Alu’datt et al.21 who found that a majority of phenolic compounds were extracted in the first three cycles of extraction from olive cake. The percent extraction of free phenolic compounds by heat treatment at 60 °C was lower than the percent phenolics obtained from the extraction carried out at 30 °C (Figure 1). The percent phenolic content of BP-30 °C in layers A and B was 17.74% and 16.99%, respectively. Similarly, the percent content of BP60 °C in layers A and B was 20.00% and 17.71%, respectively. These results are in agreement with values obtained by Alu’datt et al.21 who found that the percent content of bound phenolic D



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Table 2. Correlation Coefficients (r) between Phenolic Content, α-Amylase Inhibitory Activity, Anticancer Activity (CRC1 and CRC5), Antioxidant Activity, and ACE Inhibitory Activity of Phenolic Extracts from Virgin Olive Oil antioxidant activity

ACE inhibitory activity

α-amylase inhibitory activity

anticancer (CRC1)

anticancer (CRC5)

0.571

0.209 0.117

0.080 0.241 0.943

0.206 0.115 −0.911 −0.882

−0.830 −0.495 −0.720 −0.585

content antioxidant activity ace inhibitory activity α-amylase inhibitory activity

reported that the phenolic extracts of virgin olive oil inhibit αamylase activity. Several studies have reported the affinity of phenolic compounds to inhibit amylases and ACE enzymes.30 Inhibitory Activity of Angiotensin 1-Converting Enzyme (ACE) of Free and Bound Phenolic Extracts. Table 1 shows the inhibitory activities of free and bound phenolic extracts from olive oil on ACE enzyme inhibition. All free and bound phenolic extracts of olive oil had relatively good ACE inhibitory activity against enzyme apart from the free phenolic extract at 60 °C. Shahidi and Naczk30 have reported that the ACE inhibitory activity of phenolic compounds depends upon the polarity and solubility of phenolic compounds. The highest inhibitory activity of ACE was observed with the bound phenolic extract in layer A at 30 °C with value of 37.57% whereas the lowest inhibitory activity (17.51%) was found in the free phenolic extract at 60 °C. Similarly, the highest ACE inhibitory activity in layer B was observed in bound phenolic extract at 30 °C with value of 29.39%, and the lowest inhibitory activity was found in free phenolic extract at 60 °C with value of 11.60%. Similar ACE inhibitory activities of phenolic extracts of extra virgin have been previously reported by Loizzo et al.32 On the other hand, a high correlation was observed between ACE inhibitory activity and α-amylase inhibitory activity (0.943). Proliferation Inhibition Activity of Free and Bound Phenolic Extracts. The antiproliferative activity of extracted free and bound phenolic compounds from virgin olive oil against colorectal cancer cells is shown in Table 3. For the

with previous observations that oleuropein and hydroxytyrosol can have antiproliferative effects against colon cancer cell lines (HT-29 and SW620).33,34 Likewise, Fini et al.35 have reported that extracted phenolics from virgin olive oil inhibited proliferation of RKO and HCT116 colorectal cancer cells lines. Results showed that the temperature does not have an effect on the anticancer properties. In contrast, CRC1 anticancer activity showed a strong negative correlation with ACE inhibitory and α-amylase inhibitory activities with values of −0.911 and −0.882, respectively. The CRC5 anticancer activity had a weak negative correlation with antioxidant (−0.495) and α-amylase inhibitory (−0.585) activities. The CRC5 antitumorigenic activity had a strong negative correlation with phenolic content (−0.830) and ACE inhibitory activity (−0.720). LC-MS/MS Analysis of Individual Free and Bound Phenolic Compounds. Table 4 shows the phenolic profile identified by LC-MS/MS in the free and bound phenolic extracts of virgin olive oil. The LC-MS/MS profile of FP-30 °C extract showed the presence of gallic acid, p-hydroxybenzoic acid, vanillic acid, tyrosol, hydroxytyrosol, hesperidin, and quercetin that were not present in FP-60 °C extract. The major predominant individual phenolic compounds in FP-30 °C extract were luteolin, apigenin, and hydroxytyrosol with values of 49.02%, 32.33%, and 8.02% of total phenolic content, respectively, whereas the major predominant individual phenolic compounds in FP-60 °C extract were apigenin, luteolin, and sinapic acid with values of 45.84%, 25.04%, and 23.65% of total phenolic content, respectively. The above results are consistent with previous findings that gallic, phydroxybenzoic, vanillic, p-coumaric, and sinapic acids are the major phenolic acids found in virgin olive oil.12 Reboredó Rodriguez et al.36 reported that the contents of apigenin, vanillic acid, and p-coumaric in extra virgin olive oil were present in minor amounts. Regueiro et al.37 reported that the liquid chromatography-linear trap quadrupole (LC-LTQ-Orbitrap) analysis had an extensive identification of the phenolic compounds in walnuts. The LC-MS/MS profile of BP-30 °C was similar to the profile of BP-60 °C except for the presence of hesperidin in BP-30 °C extract and p-coumaric acid in BP-60 °C extract. The major predominant individual phenolic compounds in BP-30 °C extract were apigenin, luteolin, and quercetin with values of 44.39%, 25.04%, and 10.32% of total phenolic content, respectively, whereas the major predominant individual phenolic compounds in BP-60 °C extract were apigenin, luteolin, and rutin with values of 34.00%, 33.86%, and 25.18% of total phenolic content, respectively. The above results thus indicate the presence of flavonoids (apigenin, luteolin, and rutin) in detectable amounts as both free and bound phenolic compounds, which agrees with previous work showing that apigenin and lutolein are the major flavonoids compounds identified in virgin olive oil.15 The presence of hesperidin, rutin, and quercetin in olives has been previously reported by Alu’datt et al.17 On the other hand, Tuck and

Table 3. IC50 Values (μL/mL) of MTT Cytotoxicity Assay against Colorectal Cancer Cell Lines of Free Phenolic Compounds (FP) at 30 and 60 °C and Bound (BP) Phenolic Compounds at 30 and 60 °C from Virgin Olive Oila IC 50 (μL/mL) type of extract

CRC1

CRC5

SE

FP-30 °C FP-60 °C BP-30 °C BP-60 °C SE

93.05a A 96.55a A 84.22ab A 57.06b A 7.13

140.37a A 86.52b A 109.47ab A 100.61ab A 12.74

16.3 4.99 7.38 8.96

a

Means are average of two replicates. Column values with the same small letters and row values with same capital letters were not significantly different (p < 0.05). SE: standard error.

colorectal cancer cell line CRC 1, the lowest IC50 value was found with bound phenolic extract at 60 °C, which was significantly (p < 0.05) lower than both free phenolic extracts. For the CRC 5 colorectal cancer cell line, the lowest value of IC50 was found with the free phenolic extract at 60 °C that was significantly (p < 0.05) different from the IC50 of the free phenolic extract at 30 °C. The above results suggest that both free and bound phenolics can exert antiproliferative effects against CRC1 and CRC5 cells. These findings are consistent E



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Table 4. Percent of Total Phenolic Content of Free Phenolic Compounds (FP) at 30 and 60 °C and Bound (BP) Phenolic Compounds at 30 and 60 °C from Extra Virgin Olive Oil Identified using (LC/MS/MS)a type of phenolic compds

retention time (min)

FP-30 °C extract (%)

FP-60 °C extract (%)

BP-30 °C extract (%)

BP-60 °C extract (%)

gallic acid hydroxytyrosol tyrosol p-hydroxybenzoic acid vanillic acid p-coumaric acid sinapic acid rutin hesperidin oleuropein quercetin luteolin apigenin SE

4.44 8.1 12.2 12.6 15.1 19.2 21.4 27.4 27.5 32.9 37.4 39.2 43.8

0.97efg 8.02c 1.53def 1.46ef 2.58d ND 0.91efg 1.77de 0.23g 0.51fg 0.69efg 49.02a 32.33b 0.36

ND ND ND ND ND ND 23.65b 3.07c ND 2.4c ND 25.04b 45.84a 0.62

ND ND ND ND ND ND ND 9.15c 1.72d 4.00d 10.32c 30.43b 44.39a 0.83

ND ND ND ND ND 4.19c ND 25.18b ND 1.39d 1.38d 33.86a 34.00a 0.44

a

Mean value of two replicates. Percentage of total phenolic content based on peak areas. Column values with the same letters were not significantly different (p < 0.05). SE: standard error. ND: not detected.

Hayball11 reported that the major phenolic compounds in virgin olive oil are oleuropein, hydroxytyrosol, and tyrosol. The variation in profile of phenolic compounds for olive oil may be due to many factors including degree of olive maturity,13 and filtration of virgin olive oil during extraction and processing.14 Storage appears to be a major factor as tyrosol and hydroxytyrosol were found at low concentrations after storage ́ et of olive oil for more than six months.16 Reboredo-Rodriguez al.38 reported that the contents of individual phenolic compounds were 19.3−66.8 mg/kg for hydroxytyrosol and tyrosol, 3.3−8.4 mg/kg for apigenin and luteolin, 2.9−5.8 mg/ kg for pinoresinol, and trace contents for vanillic acid and pcoumaric acid. Effect of Removal of Free and Bound Phenolics on Fatty Acids Content. Table 5 shows the fatty acid composition (g/100 g olive oil) for virgin olive oil before and after removal of free and bound phenolic compounds. The major predominant fatty acids in virgin olive oil in either the presence or absence of phenolic compounds were oleic acid

(83.026−83.859%), palmitic acid (7.502−8.008%), and linoleic acid (5.837−6.190%) whereas myristic, palmitoleic, arachidic, eicosenoic, and behenic acids were found in minor amounts. Olive oil after removal of free and bound phenolic compounds showed a significant decrease in oleic acid (C18:1) content while concentrations of palmitoleic, stearic, linoleic, and linolenic acids were increased. The decrease in oleic acid content after removal of free and bound phenolics is likely due to conjugation of oleic acid with phenolic compounds in monoand diacylglycerols. The alkaline hydrolysis may have released the conjugated complex of oleic acid with phenolic compounds into the extracts. The increased levels of palmitoleic, stearic, linoleic, and linolenic acids may be the result of the breakage of bound phenolic compounds with free fatty acids by alkaline hydrolysis leading to the release of free fatty acids following the methanolic extraction of phenolics. The above findings are suggestive that phenolic compounds can be bound to either oleic acid in mono- and diacyglycerols or to palmitoleic, stearic, linoleic, and linolenic acids as free fatty acids. Effect of Removal of Free and Bound Phenolic Compounds on RP-HPLC Analysis of Acylglycerols. The RP-HPLC chromatogram of virgin olive acylglycerols before removal of phenolic compounds showed the presence of four major fractions (F1, F2, F3, and F4) (Figure 2a). The chromatogram for olive oil before removal of phenolic compounds was similar to the profile of olive oil after removal of free phenolic compounds except the presence of F1, which was subdivided to three major peaks F1a, F1b, and F1c (Figure 2b). This latter result suggests that the removal of phenolic compounds in F1 may have affected the polarity of acylglycerols. The RP-HPLC chromatogram for olive oil after removal of free and bound phenolic compounds was similar to the chromatogram for olive oil before removal of those phenolics except that fraction F4 was subdivided into three major peaks after hydrolysis, which suggests that alkaline hydrolysis may have led to partial hydrolysis of acylglycerols in ́ fraction F4. Reboredo-Rodriguez et al.38 and Reboredo39 ́ Rodriguez et al. reported that the predominant fatty acids in olive oil were oleic and linoleic acid. LC-MS/MS Analysis of Extracted Phenolic Compounds from Fractionated Acylglycerols via RP-HPLC. Table 6 shows the percent content of free and bound phenolic

Table 5. Fatty Acid Composition (g/100 g Virgin Olive Oil) of Virgin Olive Oil, Virgin Olive Oil after Removal of Free Phenolic Compounds, Virgin Olive Oil after Removal of Free and Bound Phenolicsa

fatty acid

formula

olive oil (g/100 g)

olive oil after removal of free phenolics (g/100 g)

myristic palmitic palmitoleic stearic oleic linoleic linolenic arachidic eicosenoic behenicacid squalene

C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 C24:6

0.008a 7.502a 0.053b 1.402b 83.859a 6.053b 0.257b 0.191a 0.111a 0.053a 0.514a

0.006a 7.876a 0.235a 1.382b 83.717a 5.837c 0.245b 0.189a 0.109a 0.051a 0.356a

olive oil after removal of free and bound phenolics (g/100 g)

SE

0.006a 8.008a 0.230a 1.686a 83.026b 6.190a 0.292a 0.197a 0.111a 0.056a 0.201a

0.001 0.138 0.005 0.050 0.093 0.028 0.005 0.005 0.003 0.001 0.097

a

Means are average of two replicates. Row values with the same letters were not significantly different (p < 0.05). SE: standard error. F



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Figure 2. RP-HPLC separation for acylgycerols before and after removal of free and bound phenolic compounds from virgin olive oil: (A) olive oil before removal of phenolic compounds, (B) olive oil after removal of free phenolic compounds, (C) olive oil after removal of free and bound phenolic compounds.

Table 6. Percent of Total Phenolic Content of Individual Phenolic Compounds of Free (before Hydrolysis) and Bound (after Hydrolysis) Phenolic Compounds by LC-MS/MS of RP-HPLC Fractionated Acylglycerols from Olive Oil before and after Removal of Free and Bound Phenolic Compoundsa fraction 1 individual phenolic compd p-hydroxybenzoic acid tyrosol p-hydroxybenzoic acid tyrosol p-hydroxybenzoic acid tyrosol a

free

free and bound

fraction 2 free

free and bound

fraction 3 free

free and bound

Olive Oil before Removal of Phenolic Compounds, Percent of Total Phenolic Content 80.05 47.07 ND ND ND ND 19.95 52.93 ND ND ND ND Olive Oil after Removal of Free Phenolic Compounds, Percent of Total Phenolic Content ND 45.63 ND ND ND ND ND 54.37 ND ND ND ND Olive Oil after Removal of Free and Bound Phenolic Compounds, Percent of Total Phenolic Content ND ND ND ND ND ND ND ND ND ND ND ND

fraction 4 free

free and bound

ND ND

ND ND

ND ND

ND ND

ND ND

ND ND

Percent of total phenolic content. ND: not detected.

phenolic interaction in oilseeds such as soybean and flaxseed. It appears that phenolic−lipid interaction in virgin olive oil can occur via two pathways that include either interaction of tyrosol with palmitoleic, stearic, linoleic, or linolenic acids in the free form or via interaction of p-hydroxybenzoic acid and oleic acid in conjugated form that is located in mono- and diacylglycerols. Free and bound phenolic extracts from virgin olive oil showed antioxidant action, α-amylase, and ACE inhibitory activities as well as antiproliferative effects in two colorectal cancer cell lines. Free phenolic extracts at 30 °C had superior antioxidant activities as compared to bound phenolic extracts whereas bound phenolic extracts at 30 °C had greater αamylase and ACE inhibitory activities than the free phenolic extracts. The major predominant phenolic compounds in free and bound phenolic extracts from virgin olive oil were apigenin, luteolin, and rutin. The presence of natural lipid−phenolic

compounds in RP-HPLC fractionated acylglycerols from olive oil before and after removal of the phenolic compounds. The presence of p-hydroxybenzoic acid and tyrosol was detected in both the free and acylglycerol-bound form. After removal of the free phenolic compounds, fraction number 1 showed the presence of p-hydroxybenzoic acid and tyrosol in the bound form, which were not detected before alkaline hydrolysis. After removal of free and bound phenolic compounds, p-hydroxybenzoic acid and tyrosol were not detected. This suggests that p-hydroxybenzoic acid and tyrosol were bound to the glycerol fraction. Phenolic compounds were not detected in the other RP-HPLC fractionated glycerols from olive oil either before or after removal of free and bound phenolic compounds. The above study is the first to demonstrate natural lipid and phenolic interactions in virgin olive oil although Alu’datt et al.40 have previously suggested the presence of lipid−protein− G



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(14) Gómez -Caravaca, A. M.; Cerretani, L.; Bendini, A.; SeguraCarretero, A.; Fernandez-Gutierrez, A.; Lercker, G. Effect of filtration systems on the phenolic content in virgin olive oil by HPLC-DADMSD. Am. J. Food Technol. 2007, 2, 671−678. (15) Suárez, M.; Macià, A.; Romero, M. P.; Motilva, M. J. Improved liquid chromatography tandem mass spectrometry method for the determination of phenolic compounds in virgin olive oil. J. Chromatogr. A 2008, 1214, 90−99. (16) Baiano, A.; Gambacorta, G.; Terracone, C.; Previtali, M. A.; Lamacchia, C.; La Notte, E. Changes in phenolic content and antioxidant activity of Italian extra-virgin olive oils during storage. J. Food Sci. 2009, 74, 177−183. (17) Alu’datt, M. H.; Rababah, T.; Ereifej, K.; Alli, I. Distribution, antioxidant and characterisation of phenolic compounds in soybeans, flaxseed and olives. Food Chem. 2013, 139, 93−99. (18) Fortes, C.; Forastiere, F.; Farchi, S.; Mallone, S.; Trequattrinni, T.; Anatra, F.; Schmid, G.; Perucci, C. A. The protective effect of the Mediterranean diet on lung cancer. Nutr. Cancer 2003, 46, 30−37. (19) van Duynhoven, J. V.; Vaughan, E.; Jacobs, D.; Kemperman, R.; Velzen, E. V.; Gross, G.; Roger, L. C.; Possemiers, S.; Smilde, A. K.; Dore, J.; Westerhuis, J. A.; Wiele, T. V. D. Metabolic fate of polyphenols in the human superorganism. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 4531−4538. (20) International Olive Oil Council (IOOC). Olive Oil Quality Omprovement, Technical Handbook; Madrid, Spain, 1984; pp 25−45. (21) Alu’datt, M.; Alli, I.; Ereifej, K.; Alhamad, M.; Al-Tawaha, A.; Rababeh, T. Optimisation, characterisation and quantification of phenolic compounds in olive cake. Food Chem. 2010, 123, 117−122. (22) Hoff, J. E.; Singleton, K. I. A method for determination of tannins in foods by means of immobilized protein. J. Food Sci. 1977, 42, 1566−1569. (23) McCue, P.; Kwon, Y. I.; Shetty, K. Anti-amylase, antiglucosidase and anti-angiotensin I-converting enzyme potential of selected foods. Food Biotechnol. 2005, 29, 278−294. (24) Cushman, D. W.; Cheung, H. S. Spectrometric assay and properties of the angiotensin-converting-I-enzyme of rabbit lung. Biochem. Pharmacol. 1971, 20, 1637−1648. (25) Wu, J. J.; Zhang, X. D.; Gillespie, S.; Hersey, P. Selection for TRAIL resistance results in melanoma cells with high proliferative potential. FEBS Lett. 2005, 579, 1940−1944. (26) Obied, H.; Bedgood, D. R., Jr.; Prenzler, P.; Robards, K. Chemical screening of olive biophenol extracts by hyphenated liquid chromatography. Anal. Chim. Acta 2007, 603, 176−189. (27) Ereifej, K.; Alu’datt, M. H.; Alkahalidy, H. A.; Alli, I.; Rababah, T. Comparison and characterization of fat and protein composition for camel milk from eight Jordanian locations. Food Chem. 2011, 127, 282−289. (28) Baldioli, M.; Servili, M.; Perretti, G.; Montedoro, G. F. Antioxidant activity of tocopherols and phenolic compounds of virgin olive oil. J. Am. Oil Chem. Soc. 1996, 73 (11), 1589−1593. (29) Tripoli, E.; Giammanco, M.; Tabacchi, G.; Di Majo, D.; Giammanco, S.; LaGuardia, M. The phenolic compounds of olive oil: Structure, biological activity and beneficial effects on human health. Nutr. Res. Rev. 2005, 18, 98−112. (30) Shahidi, F.; Naczk, M. Phenolics in Food and Nutraceuticals; CRC Press: Boca Raton, FL, 2004; pp 1−558. (31) Gorinstein, S.; Martin-Belloso, O.; Katrich, E.; Lojek, A.; Cíz, M.; Gligelmo-Miguel, N.; Haruenkit, R.; Park, Y. S.; Jung, S. T.; Trakhtenberg, S. Comparison of the contents of the main biochemical compounds and the antioxidant activity of some Spanish olive oils as determined by four different radical scavenging tests. J. Nutr. Biochem. 2003, 14, 154−159. (32) Loizzo, M. R.; Di Lecce, G.; Boselli, E.; Menichini, F.; Frega, N. G. Inhibitory activity of phenolic compounds from extra virgin olive oils on the enzymes involved in diabetes, obesity and hypertension. J. Food Biochem. 2011, 35, 381−399. (33) Notarnicola, M.; Pisanti, S.; Tutino, V.; Bocale, D.; Rotelli, M. T.; Gentile, A.; Memeo, V.; Bifulco, M.; Perri, E.; Caruso, M. G. Effects of olive oil polyphenols on fatty acid synthase gene expression

interactions was indicated via the presence of p-hydroxybenzoic acid and tyrosol in the acylglycerol fractions of virgin olive oil. The biological effects demonstrated with the lipid-bound phenolics in the present work require in vivo verification since there is extensive biotransformation of polyphenols by gut microbiota to secondary metabolites. The present findings, however, present the possibility that naturally occurring lipid bound phenolics might affect the bioaccessibility and bioavailability of the phenolic compounds. The present study indicates natural phenolic lipid interactions are present in virgin olive oil that may generate biologically active natural phenolic lipids for use in pharmaceutical and nutraceutical applications.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], malodat@just. edu.jo, [email protected]. Phone: +962776579511. Fax: +9627201078. Funding

Authors acknowledge Jordan University of Science and Technology (JUST) for financial support. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Scalbert, A.; Manach, C.; Morand, C.; Remesy, C.; Jimenez, L. Dietary polyphenols and the prevention of diseases. Crit. Rev. Food Sci. Nutr. 2005, 45, 287−306. (2) Figueroa-Espinoza, M. C.; Villeneuve, P. Phenolic acids enzymatic lipophilization. J. Agric. Food Chem. 2005, 53, 2779−2787. (3) Sabally, K.; Karboune, S.; St-Louis, R.; Kermasha, S. Lipasecatalyzed synthesis of phenolic lipids from fish liver oil and dihydrocaffeic acid. Biocatal. Biotransform. 2007, 25, 211−218. (4) Lue, B. M.; Karboune, S.; Yebaoh, F. K.; Kermasha, S. Lipasecatalyzed esterification of cinnamic acid and oleyl alcohol in organic solvent media. J. Chem. Technol. Biotechnol. 2005, 80, 462−468. (5) Guyot, B.; Bosquette, B.; Pina, M.; Graille, J. Esterification of phenolic acids from green coffee with an immobilized lipase from Candida antarctica in solvent-free medium. Biotechnol. Lett. 1997, 19, 529−532. (6) Trox, J.; Vadivel, V.; Vetter, W.; Stuetz, W.; Scherbaum, V.; Gola, U.; Nohr, D.; Biesalski, H. K. Bioactive compounds in cashew nut (Anacardium occidentale L.) kernels: Effect of different shelling methods. J. Agric. Food Chem. 2010, 58, 5341−5346. (7) Khalil, M. L. Biological activity of bee propolis in health and disease. Asian Pac. J. Cancer Prev. 2006, 7, 22−31. (8) Boskou, D.; Tsimidou, M.; Blekas, G. Polar phenolic compounds. In Olive Oil: Chemistry and Technology II; Boskou, D., Ed.; AOCS Press: Champaign, IL, 2006; pp 73−90. (9) International Olive Council (IOC). COI/T.15/NC No 3/Rev. 6. Trade standard applying to olive oils and olive pomace oils. Madrid, 2011. (10) Hashempour, A.; Ghazvini, R. F.; Bakhshi, D.; Sanam, S. A. Fatty acids composition and pigments changing of virgin olive oil (Olea europea L.) in five cultivars grown in Iran. Aust. J. Crop Sci. 2010, 4, 258−260. (11) Tuck, K. L.; Hayball, P. J. Major phenolic compounds in olive oil: metabolism and health effects. J. Nutr. Biochem. 2002, 13, 636− 644. (12) Carrasco Pancorbo, A.; Cruces-Blanco, C.; Segura-Carretero, A.; Fernandez Gutierrez, A. Sensitive determination of phenolic acids in extra-virgin olive oil by capillary zone electrophoresis. J. Agric. Food Chem. 2004, 52, 6687−6693. (13) Artajo, L. S.; Romero, M. P.; Motilva, M. J. Transfer of phenolic compounds during olive oil extraction in relation of ripening stage of the fruit. J. Sci. Food Agric. 2006, 86, 518−527. H



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and activity in human colorectal cancer cells. Gene Nutr. 2011, 6, 63− 69. (34) Fabiani, R.; De Bartolomeo, A.; Rosignoli, P.; Servili, M.; Montedoro, G. F.; Morozzi, G. Cancer chemoprevention by hydroxytyrosol isolated from virgin olive oil through G1 cell cycle arrest and apoptosis. Eur. J. Cancer Prev. 2002, 11, 351−358. (35) Fini, L.; Hotchkiss, E.; Fogliano, V.; Graziani, G.; Romano, M.; De Vol, E. B.; Qin, H.; Selgrad, M.; Boland, C. R.; Ricciardiello, L. Chemo preventive properties of pinoresinol-rich olive oil involve a selective activation of the ATM-p53 cascade in colon cancer cell lines. Carcinogenesis 2008, 29, 139−146. (36) Reboredo-Rodríguez, P.; Rey-Salgueiro, L.; Regueiro, J.; González-Barreiro, C.; Cancho-Grande, B.; Simal-Gándara, J. Ultrasound-assisted emulsification−microextraction for the determination of phenolic compounds in olive oils. Food Chem. 2014, 150, 128−136. (37) Regueiro, J.; Sánchez-González, C.; Vallverdú-Queralt, A.; Simal-Gándara, J.; Lamuela-Raventós, R.; Izquierdo-Pulido, M. Comprehensive identification of walnut polyphenols by liquid chromatography coupled to linear ion trap−Orbitrap mass spectrometry. Food Chem. 2014, 152, 340−348. (38) Reboredo-Rodríguez, P.; González-Barreiro, C.; CanchoGrande, B.; Simal-Gándara, J. Quality of extra virgin olive oils produced in an emerging olive growing area in north-western Spain. Food Chem. 2014, 164, 418−426. (39) Reboredo-Rodríguez, P.; González-Barreiro, C.; CanchoGrande, B.; Simal-Gándara, J. Improvements in the malaxation process to enhance the aroma quality of extra virgin olive oils. Food Chem. 2014, 158, 534−545. (40) Alu’datt, M. H.; Rababah, T.; Ereifej, K.; Brewer, S.; Alli, I. Phenolic−protein interactions in oilseed protein isolates. Food Res. Int. 2013, 52, 178−184.

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