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Antioxidant and anti-atherogenic properties of phenolic acid and flavonol fractions of fruits of ‘Amari’ and ‘Hallawi’ date (Phoenix dactylifera L.) varieties Hamutal Borochov-Neori, Sylvie Judeinstein, Amnon Greenberg, Nina Volkova, Mira Rosenblat, and Michael Aviram J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf506094r • Publication Date (Web): 13 Mar 2015 Downloaded from http://pubs.acs.org on March 21, 2015

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Journal of Agricultural and Food Chemistry

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Antioxidant and anti-atherogenic properties of phenolic acid and flavonol fractions of fruits of ‘Amari’ and ‘Hallawi’ date (Phoenix dactylifera L.) varieties. Hamutal Borochov-Neori*1, Sylvie Judeinstein1, Amnon Greenberg1, Nina Volkova2, Mira Rosenblat2 and Michael Aviram2 1

Southern Arava Research and Development, M. P. Hevel Eilot 88820, Israel.

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The Lipid Research Laboratory, Rappaport Faculty of Medicine and the Rappaport Family

Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Rambam Medical Center, Haifa 31096, Israel.

*Corresponding author, Tel: +972-8-6355747; Tel: +972-8-6355730. E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT Date (Phoenix dactylifera L.) fruit phenolic-acid or flavonol fractions were examined in vitro

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for antioxidant and anti-atherogenic properties. Two fractions of each subgroup were prepared

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from two date varieties, ‘Amari’ and ‘Hallawi’, by solid phase extraction on C18. The fractions

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were analyzed for phenolics’ composition by RP-HPLC, and tested for ferric-reducing

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antioxidant power, free radical scavenging capacity, inhibition of Cu2+-induced LDL-oxidation

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and enhancement of HDL-mediated cholesterol efflux from macrophages. All four fractions

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exhibited variable capacities to reduce ferric ions, scavenge radicals and inhibit LDL-oxidation.

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Flavonol fractions were considerably better inhibitors of LDL-oxidation compared to phenolic-

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acid fractions, with IC50 of 9-31 nmol GAE mL-1 compared to 85-116 nmol GAE mL-1,

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respectively. Only the flavonol fractions stimulated cholesterol removal from macrophages.

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Within each subgroup, the levels of all the activities varied with fraction composition. The

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results demonstrated strong structure-activity relationships for date phenolics, and identified date

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flavonols as potential anti-atherogenic bioactives.

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Keywords: anti-atherogenic; antioxidants; date fruit; flavonols; phenolic acids; Phoenix

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dactylifera L.


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INTRODUCTION

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The date (Phoenix dactylifera L.) fruit has been utilized since ancient days as an important

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staple food and in ethno-medicine by the habitants of the Arabian Peninsula, Middle East and

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North Africa1, 2. The traditional use of dates in folk medicine is probably due to the fruit

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immunomodulatory3, antibacterial4 and antifungal5 properties. Hydrophilic extracts of the fruits

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possess potent antioxidant and free radical scavenging capacities6-10 that were attributed mostly

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to the phenolic compounds present in dates; the latter include primarily hydroxybenzoic and

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hydroxycinnamic acids and flavonols 6-9, 11-13.

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Atherosclerosis, the leading cause of morbidity and mortality in modern lifestyle, is initiated

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by macrophage cholesterol accumulation and foam cell formation14, and further enhanced by

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oxidative stress15. Phenolic compounds, especially flavonoids, are highly effective natural

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nutritional antioxidants16 capable of scavenging free radicals, chelating transition metal ions and

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inhibiting LDL oxidation17. Increased dietary intake of phenolics rich foodstuff was shown by

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epidemiological studies to be linked with reduced morbidity and mortality from coronary artery

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disease18. We have demonstrated earlier the beneficial anti-atherogenic effects of consumption of

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polyphenolics rich fruits, such as pomegranate and marula19, 20.

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Our recent in vivo study on date fruits9 indicated that regular consumption of two date

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varieties (‘Medjool’ and ‘Hallawi’) by healthy human subjects was associated with decreased

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levels of triglycerides in the serum. After consumption of ‘Hallawi’, but not ‘Medjool’ dates, the

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serum basal oxidative status and susceptibility to oxidation also decreased significantly. Notably,

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the soluble phenolics composition in the fruits of the two varieties differed considerably9, 13. In a

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parallel examination of the phenolics composition and in vitro anti-atherogenic activities of

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extracts from nine diverse date varieties13, we have found large varietal differences in the



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composition of the two major phenolics subgroups prevailing in dates, phenolic acids and

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flavonols. Moreover, the varieties differed considerably in their potency to inhibit LDL-

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oxidation and enhance serum-mediated cholesterol efflux from macrophages13, two processes

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relevant to atherosclerosis development. The varietal differences in the biological effects in both

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the in vivo and in vitro studies may reflect the diverse bioactivities exerted by different phenolic

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compounds due to structure-function relationships21, 22. However, to date, the differential

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contribution of individual date fruit phenolic acids and flavonols to the overall antioxidant and

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anti-atherogenic properties of the fruit were not characterized, and the involvement of other fruit

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components, such as dietary fiber23 was not excluded.

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Identifying the anti-atherogenic bioactives in dates is necessary to establish the fruit health

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value. In the present study, we have examined the antioxidant and anti-atherogenic properties of

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date fruit phenolic acids and flavonols separately, as a first step toward the identification of

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potentially anti-atherogenic date phenolics. The study employed phenolics’ subgroup

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fractionation from fruits of two date varieties that highly differ in their phenolics’ profile,

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‘Amari’ and ‘Hallawi’13, in order to obtain fractions of diverse composition. The isolated

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fractions were analyzed for their composition, ferric-reducing power, free radical scavenging

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capacity, inhibition of copper ion-induced LDL oxidation, and enhancement of HDL-mediated

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cholesterol efflux from macrophages. Date fruit phenolics’ structure-activity relationships were

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examined by comparing the nature and magnitude of activities manifested by the two phenolics’

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subgroups, as well as different compositions within each subgroup.

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MATERIALS AND METHODS

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Chemicals. Caffeic acid, catechin, catechin gallate, chlorogenic acid, o-coumaric acid, p-

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coumaric acid, epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, ferulic


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acid, gallic acid, gallocatechin, gallocatechin gallate, hydrocaffeic acid, phenyl acetate,

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pyrogallol, protocatechuic acid, sinapic acid, syringic acid, vanillic acid, tannic acid, and Folin-

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Ciocalteau reagent were all purchased from Sigma-Aldrich Co., Israel. Ellagic acid, 2-

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hydroxybenzoic (salicylic) acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, kaempferol-3-

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glucoside, procyanidin B2 and quercetin-3-β-glucoside were acquired from Fluka, France. 2.2'-

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Azobis, 2-amidinopropane hydrochloride (AAPH) was from Wako, Japan. Acetonitrile HPLC

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grade was purchased from Merck, Germany, and phosphoric acid from Frutarum, Israel. [3H]-

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labeled cholesterol was acquired from Amersham Biosciences, Inc., USA. Dulbecco's Modified

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Eagel's Medium (DMEM) and fetal calf serum (FCS) were purchased from Biological Industries,

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Beit Haemek, Israel.

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Date fruit. Commercial ready-to-eat tree ripen semi-dry fruits (“tamr” stage) of two date

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varieties, ‘Amari’ and ‘Hallawi’, were obtained shortly after harvest from Hadiklaim, Israel Date

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Growers Cooperative Ltd., and stored at -20 °C until processing.

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Date crude phenolics’ extract preparation. Pitted ‘Amari’ or ‘Hallawi’ fruits were grinded

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with KENWOOD meat grinder MG600 (KENWOOD Ariete, Italy), and homogenized in cold

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70% acetone solution containing 0.5% acetic acid at a fruit to solvent ratio of 1:3 (w/v) using

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mortar and pestle. The homogenate was kept at 4 ºC overnight and shaken on ice for additional 2

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hrs. The procured slurry was centrifuged at 12,000 x g for 10 min at 4 °C (Sorvall Instruments

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RC5C), and the clear supernatant was collected and stored at -20 ºC in light-protected vessels.

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Date phenolics’ subgroups fractionation. The crude phenolics’ extracts were fractionated into

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phenolic acids and flavonols subgroups by solid-phase extraction (SPE) on C18 column,

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following Oszmianski et al.24, with some modification. Two extraction columns [BakerbondTM

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spe octadecyl (C18) disposable extraction columns, sorbent weight 1000 mg, column size 6 ml,



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J.T.Baker®] were preconditioned with 5 mL of methanol. One column was equilibrated to

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neutral pH with 10 mL of double distilled water (DDW), pH 7.0, and the other, to acidic pH with

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10 mL of DDW, pH 2.0. The crude acetone extracts were diluted 20-fold with DDW and

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adjusted to pH 7.0 with 1 N NaOH. Total of 10 mL of the diluted extract were loaded onto the

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pH 7.0 column. The effluent containing the ionized phenolic acids and other polar components

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was saved. The column was then washed with 10 mL of DDW pH 7.0, and the two effluents

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were combined.

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Phenolic acids’ isolation. The combined effluents were adjusted to pH 2.0 with 1 N HCl, and

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loaded onto the acidified column. The phenolic acids were then eluted with 3 mL of methanol

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that were sequentially collected in 0.8, 1.0 and 1.2 mL aliquots. The second aliquot of eluent was

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dried in a miVac duo concentrator (Genevac Limited, UK) at 45 °C and kept at -20 °C.

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Flavonols’ isolation. After the removal of the polar compounds, the pH 7.0 column was adjusted

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to pH 2.0 by passing 10 mL DDW at pH 2.0, and washed with 3 mL 16% acetonitrile at pH 2.0.

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The flavonols were then eluted with 3 mL of ethyl acetate that were sequentially collected in 0.8,

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1.0 and 1.2 mL aliquots. The second aliquot of eluent was dried as described before and kept at -

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20 °C.

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Total phenolics’ content. The dried isolated fractions were each dissolved in 1.0 mL of 50%

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ethanol. The resulting solutions were diluted 5- and 10-fold with DDW and assayed for total

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phenolics’ content with Folin-Ciocalteau 2N phenol reagent, following Singleton and Rossi25.

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Aliquots of 100 µL were added to 900 µL reaction solution consisting of 200 µL freshly

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prepared 10-fold diluted Folin-Ciocalteau reagent, 100 µL 20% Na2CO3 and 600 µL DDW.

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Calibration curves were constructed with gallic acid (0 - 0.8 µmol mL-1). The absorbance at 765


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nm was measured after 1 h incubation, and the results were expressed in terms of gallic acid

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equivalents (GAE). Each measurement was done in triplicate.

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RP-HPLC analysis of isolated phenolics’ fractions. Phenolics’ analysis by RP-HPLC was

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carried out as described earlier13. The dissolved isolated fractions were filtered through a 0.45

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µm PTFE filter, and analyzed with a LaChrom Merck Hitachi HPLC system, consisting of Pump

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L7100, Column oven L7350 and Mixer-degasser L-7614, coupled with a diode array detector

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with 3D feature (Multiwavelength Detector, Jasco MD-2010 Plus), interface (Jasco LC-Net II /

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ADC) and scientific software (EZChrom EliteTM Client/Server version 3.1.6 build 3.1.6.2433)

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that provides real time data acquisition and post run data manipulation and integration

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capabilities. Twenty µL aliquots were injected by a manual injector (Rheodyne, Rohnert Park,

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CA) and loaded onto a Purospher®Star RP-18 endcapped column (250 x 4 mm LichroCART®

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cartridge, 5 µm particle size) with endcapped Lichrospher®100 RP-18 guard column (4 x 4 mm

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LichroCART® cartridge, 5 µm particle size). The binary mobile phase consisted of 0.1%

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phosphoric acid, pH 2.4 (solution A), and acetonitrile (solution B), and was delivered at a flow

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rate of 1 mL min-1. Elution was carried out with the following stepwise gradient outline: 1 to 10

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min, 5 to 15% solution B; 10 to 30 min, 15 to 30% solution B; 30 to 40 min, 30 to 100% solution

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B. The column was then washed and equilibrated by 10-min post runs with 100% and 5% B,

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respectively. Oven temperature was set at 40 °C, and the pressure was 158 atm. Acetonitrile was

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HPLC grade (LiChrosolv Merck, Germany); Column-filtered water was further distilled by

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Corning Megapure System, MP-6A, and passed through a 0.20 µm Nylon membrane.

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Phosphoric acid was of analytical grade.



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Phenolics’ classification and tentative identification were performed by the system software on

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the basis of UV/Vis absorbance spectra and the retention times of the chromatograms peaks, and

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the system phenolics’ library constructed from authentic standards.

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Individual phenolic compounds were quantified from the corresponding chromatogram peak

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areas calculated by the software in three repetitive runs, and calibration curves constructed with

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authentic standards of caffeic, coumaric, ferulic and salicylic acid, and kaempferol-3-glucoside.

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A cutoff area of 100,000 mV min was applied to discern between significant and non-significant

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peaks. The concentrations of the various hydroxybenzoic acids, hydroxycinnamic acids and

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flavonols were expressed in terms of nmol equivalents of the corresponding standards mL-1.

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Ferric-reducing antioxidant power (FRAP). The dissolved isolated fractions were diluted 5-

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and 10-fold with DDW, and assayed for ferric-reducing ability by the colorimetric test originally

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developed to assess the ferric reducing antioxidant power of plasma26. Fifty µL were added to

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950 µL reaction solution freshly prepared by mixing 50 mL 300 mmol L-1 acetate buffer, 5 mL

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10 mmol L-1 2,4,6-tripyridyl-s-triazine (TPTZ) and 5 mL 20 mmol L-1 ferric chloride.

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Absorbance at 593 nm was measured after 4 min incubation in a 37 °C water bath. Vitamin C

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was used for the calibration curve (0- 0.6 µmol mL-1), and the results were expressed in terms of

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vitamin C equivalents (VCE). Each measurement was done in triplicate.

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Free radical scavenging capacity. The radical generating compound 1,1-diphenyl–2-picryl-

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hydrazyl (DPPH) was employed to assess the capacity of the isolated fractions to scavenge free

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radicals27. Fifty µL aliquots of the isolated fractions at a concentration of 0.5 mmol L-1 GAE in

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50% ethanol were mixed with 1 mL of 0.1 mmol L-1 DPPH in ethanol, and the optical density

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(OD) at 517 nm was monitored continuously for 5 min at room temperature. For the control, 50

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µL of 50% ethanol were added to the DPPH solution. The free radical scavenging capacity in


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terms of percentage loss of OD at 517 nm was calculated using the following equation = 1 −

517 × 100 517

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where the sample and control OD at 517 nm correspond to the measurements after 5 min

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incubation in the presence and absence of the isolated phenolic fraction, respectively.

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LDL and HDL preparation. Fresh plasma was derived from 3 healthy normolipidemic

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volunteers (Rambam Hospital Helsinki Committee number 30572-10 - RBM). These volunteers

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received Mediterranean diet, but they did not consume any additional antioxidants. The plasma

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samples were inactivated (30 min at 56 °C) and then pooled together. The LDL and HDL

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fractions were isolated from the pooled plasma sample by discontinuous density gradient

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ultracentrifugation28. The LDL was separated at d=1.063 g mL-1, and the HDL at d= 1.210 g mL-

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4 °C, and then sterilized by filtration (0.45 µm), kept under nitrogen in the dark at 4 °C, and used

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within 2 weeks. The LDL and HDL protein concentration was determined with the Folin phenol

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reagent29.

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Copper ion-induced LDL oxidation. Prior to oxidation, the LDL was dialyzed overnight

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against EDTA-free, phosphate buffered saline (PBS) solution, pH 7.4, at 4 °C. LDL (100 µg of

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protein mL-1) was pre-incubated for 30 min at room temperature with increasing volume

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concentrations (0-25 µL mL-1) of the isolated phenolics’ fractions. Then, 5 µmol L-1 of CuSO4

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was added and the tubes were incubated for 1.5 h at 37 °C. At the end of the incubation, the

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extent of LDL oxidation was determined by measuring the generated amount of thiobarbituric

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acid reactive substances (TBARS) and of lipid peroxides. The TBARS assay was performed at

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532 nm, using malondialdehyde (MDA) for the standard curve30. The lipid peroxides assay,

. Both lipoproteins were dialyzed against 150 mmol L-1 NaCl, 1 mmol L-1 Na2EDTA (pH 7.4) at



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which analyzes lipid peroxides formation by their capacity to convert iodide to iodine, was

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measured spectrophotometrically at 365 nm31. The measurements were repeated three times.

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HDL-mediated cholesterol efflux from macrophages. J774 A.1 murine macrophage cells

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were purchased from the American Tissue Culture Collection (ATCC, Rockville, MD). Cells

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were grown in DMEM containing 5% FCS. The macrophages (1 mL of 106 cells mL-1) were

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pre-incubated at 37 °C for 20 h with 10 to 170 µM GAE of the isolated phenolics fractions.

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Control cells were incubated with an equivalent volume of 50% ethanol. The cells were then

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washed and incubated with [3H]cholesterol (2 µCi mL-1) at 37 °C for 1 h, followed by cell

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wash in ice-cold PBS for 3 times, and further incubation in the absence (basal cholesterol

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efflux rate) or presence of HDL (100 µg protein mL-1) for 3 h at 37 °C. Cellular and medium

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[3H]-labels were quantified and HDL-mediated cholesterol efflux was calculated as the ratio

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of [3H]-label in the medium / ([3H]-label in the medium + [3H]-label in the cells)32. HDL-

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mediated cholesterol efflux data were corrected for the non-specific loss of cholesterol (basal

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cholesterol efflux rate). The experiments were repeated three times.

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Statistical Analysis. The results are reported as means ± their respective standard deviations

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(SD) of three measurements (n=3). Statistical analysis of the data was performed with IBM SPSS

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Statistics, version 17.0. Conformity of all data sets to normal distribution and homogeneity of

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variances was established by the Shapiro-Wilk’s and Levene's tests, respectively. One-way

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ANOVA and Tukey-Kramer post-hoc means comparison test were employed to verify

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significant differences (p < 0.05, 0.01, or 0.001) in comparison to the control or among fractions.

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Correlations between fraction capacities were tested with the correlation analysis tool of

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Microsoft Excel 2010, and reported in terms of the correlation coefficient, r.

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RESULTS AND DISCUSSION


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Composition of isolated date phenolics’ subgroup fractions To obtain variable compositions of phenolic acid and flavonol fractions we employed fruits of

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two date varieties, ‘Amari’ and ‘Hallawi’ that largely differ in their phenolics’ composition13.

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Crude phenolics extracts were prepared with acidified 70% acetone, which was found to be more

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efficient than 50% ethanol in recovering anti-atherogenic capacities from dates13.

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Fractionation of the crude extracts of ‘Amari’ and ‘Hallawi’ fruits, carried out as described

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under Materials and Methods, yielded two fractions of phenolic acids, PhA 1 and PhA 2, and two

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of flavonols, Fl 1 and Fl 2. Fractions PhA 1 and Fl 1 were derived from ‘Amari’, and PhA 2 and

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Fl 2 from ‘Hallawi’ fruits. The yields of the isolated fractions are presented in Table 1, in terms

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of µmol GAE per g fruit. The yield of ‘Amari’ derived fractions was considerably larger than

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that of the fractions isolated from ‘Hallawi’, by approximately 10- and 3.5-fold for phenolic

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acids and flavonols, respectively. This is consistent with the higher content of soluble phenolics

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present in ‘Amari’ compared to ‘Hallawi’ dates13. By and large, the recovery of the various

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phenolics through the fractionation process was low. Optimization of the process conditions, e.

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g., the weight ratio of phenolics to solid phase and the flow rate during phenolics loading onto-

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and elution from the column, is likely to improve the yield.

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The isolated phenolics’ fractions were analyzed by RP-HPLC to classify and tentatively

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identify, as well as quantify, the phenolic constituents. Figure 1 presents the concentrations of

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the major phenolic acids in PhA 1 and PhA 2, in order of ascending retention time (Rt).

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Altogether, eight prominent peaks of phenolic acids were detected in the two fractions. The

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peaks at Rt of 9.7, 10.1, 11.4, 11.7, and 12.5 min corresponded to caffeic acid derivatives, and

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their concentrations are expressed as nmol caffeic acid mL-1. The compounds associated with Rt

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14.9, 16.7 and 27.4 min were coumaric and ferulic acid, and a salicylic acid derivative,



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respectively, and their concentrations are expressed accordingly in nmol mL-1 equivalents of

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coumaric, ferulic and salicylic acid. Seven and five phenolic acids constituted PhA 1 and PhA 2,

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respectively. The major hydroxycinnamic acid in both fractions was ferulic acid, comprising

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approximately 55 and 40 mole % of the total phenolic acid concentration in PhA 1 and PhA 2,

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respectively. This is consistent with our earlier findings on ferulic acid being the most abundant

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hydroxycinnamic acid in the fruits of nine date varieties, including ‘Amari’ and ‘Hallawi’13. The

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other major phenolic acids in PhA 1 were derivatives of caffeic acid; most prominent were those

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detected at Rt of 11.4, 11.7 and 12.5 min, consisting 11, 17, and 9 mole % of the total phenolic

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acid concentration, respectively. On the other hand, in PhA 2 the most abundant phenolic acid

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was a salicylic acid derivative that accounted to approximately 51 mole % of the total phenolic

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acid concentration. Notably, this hydroxybenzoic acid was absent in PhA 1.Small amounts of

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caffeic acid derivatives at Rt of 11.4 and 11.7 min were also present in PhA 2. Both fractions

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contained comparable small amounts of coumaric acid, accounting to merely 3 - 4 mole % of the

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total phenolic acid concentration.

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In summary, the two isolated fractions of date phenolic acids contained ferulic acid as a major

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component and comparable small amounts of coumaric acid, but differed considerably in the

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composition of the complementary set of phenolic acids; that of PhA 1 consisted mostly of

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caffeic acid derivatives, whereas that of PhA 2 comprised mostly a salicylic acid derivative. In

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this respect, PhA 1 relates to date varieties such as ‘Amari’, ‘Deglet Noor’ and ‘Medjool’ that are

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rich in caffeic acid derivatives and poor in salicylic acid derivative, whereas PhA 2 relates to

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varieties such as ‘Deri’, ‘Hadrawi’, ‘Hallawi’ and ‘Zahidi’ that are relatively low in caffeic acid

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derivatives and have significant amounts of the salicylic acid derivative13.


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Figure 2 illustrates the concentrations of the major flavonols in Fl 1 and Fl 2, in order of

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ascending Rt. Altogether, seven prominent peaks of flavonols were evident in the two fractions.

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On the basis of the authentic standard library all seven flavonols were tentatively classified as

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kaempferol derivatives; their concentrations in the Figure are expressed in nmol mL-1

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kaempferol-3-glucoside equivalents. Six and two peaks constituted Fl 1 and Fl 2, respectively.

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Both fractions shared a principal component at Rt of 16.0 min, amounting to approximately 30

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mole % of the total flavonol concentration. The other flavonols in Fl 1, corresponding to Rt of

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17.0, 18.4, 18.9, 20.8 and 21.3 min, accounted for approximately 13, 11, 15, 20 and 11 mole % of

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the total flavonols, respectively. The other flavonol in Fl 2, at Rt of 19.8 min, was the most

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abundant in this fraction, and was absent in Fl 1.

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In summary, the two isolated fractions of date flavonols considerably differed in composition;

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in addition to one prominent flavonol shared by the two fractions, Fl 1 consisted of significant

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amounts of five other flavonols, whereas, Fl 2 contained a single unique flavonol as the major

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component.

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Antioxidant and anti-atherogenic properties of isolated date phenolics’ subgroup fractions

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Ferric ion reducing power. The ferric reducing activity of the isolated fractions relative to

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vitamin C is shown in Table 1 in terms of the mole ratio of VCE to GAE. All the fractions

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exhibited considerable capacities to reduce ferric ions, that on the average were 80 to 88% that of

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vitamin C. Under the FRAP assay conditions, the flavonol fractions were slightly, yet

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significantly, better reductants, exceeding the reducing power of the phenolic acid fractions by

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approximately 10%.

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Free radical scavenging capacity. The capacity of the isolated fractions to scavenge free

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radicals is presented in Table 1. All the fractions demonstrated ability to neutralize free radicals,



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however, with different potencies. At an equivalent concentration of phenolic hydroxyls (24

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µmol GAE L-1 in the final reaction mixture), and after 5 min exposure, the phenolic acid

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fractions exhibited significantly higher efficiencies compared to the flavonol fractions. PhA 2

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was significantly the most potent fraction, decreasing DPPH concentration by 22%; PhA

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1followed with a radical scavenging capacity of 14%. The two flavonol fractions had comparable

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lower potencies, measuring approximately 39 and 61% of the radical scavenging capacities

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exhibited by PhA 1 and PhA 2, respectively.

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The antioxidant and antiradical capacity of phenolic molecules relate to their molecular

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structure, most significantly, the number and position of hydroxyl groups, extent of double bond

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conjugation, and the nature and position of substituents 21, 22. Negative correlation was obtained

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between the FRAP and DPPH assays (r = -0.906). It should be noted, however, that the

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equivalent mole concentration of phenolic hydroxyls of the various fractions employed in our

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DPPH assay did not correspond to an equivalent mole concentration of phenolic compounds, due

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to the variable number of hydroxyls per molecule among the phenolics comprising the fractions.

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This may have influenced the order of radical scavenging potencies of the phenolics’ fractions as

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presented in Table 1, since the reaction rate is affected by reactant mole concentration.

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Date phenolics’ effect on LDL oxidation. LDL-oxidation is an important process in the

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initiation of atherosclerosis development. We therefore studied the effect of the isolated date

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phenolics’ fractions on the susceptibility of LDL to oxidation. The extent of copper ion-induced

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LDL oxidation was measured in the presence of increasing concentrations of the isolated

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fractions. All the fractions considerably and dose-dependently inhibited LDL oxidation, as

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determined by the TBARS and lipid peroxides assays (Figure 3). By both assays, the potency to

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protect LDL from oxidation significantly differed (p < 0.01) among the four fractions. Moreover,


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the data sets obtained by the two assays exhibited a strong positive correlation (r = 0.995),

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strengthening the validity of the assay. The flavonol fractions were significantly more potent than

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the phenolic acid fractions (p < 0.001), requiring considerably lower phenolics concentrations to

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obtain 50% inhibition (IC50). Fl 2 was the most efficient inhibitor of LDL oxidation, followed by

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Fl 1, with IC50 of 10 and 31 nmol GAE mL-1 by the TBARS assay, and 9 and 28 nmol GAE mL-1

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by the lipid peroxides assay, respectively. PhA 1 was the least effective inhibitor, whilst PhA 2

293

was somewhat better, as implied by their IC50 of 116 and 99 nmol GAE mL-1 by the TBARS

294

assay, and 113 and 85 nmol GAE mL-1 by the lipid peroxides assay, respectively.

295

The competence of phenolic compounds to inhibit copper ion-mediated LDL oxidation

296

depends on their abilities to chelate or oxidize copper ions, act as hydrogen-donating antioxidant,

297

delocalize electrons, and partition between the aqueous medium and the lipophilic micro-

298

environment within the LDL particle; all of which are governed by their molecular structure21, 22,

299

33-35

300

other flavonoids36. Flavonols are superior to simple phenolic acids in electron delocalization

301

capability due to the large number of conjugated double bonds. A quantum–mechanical

302

investigation of the quercetin radical has revealed extended electronic delocalization between

303

adjacent rings, as well as cross-conjugation occurring at the C ring37. Moreover, the amphipathic

304

nature of flavonols is more pronounced, especially that phenolic acids are completely ionized at

305

the physiological pH of 7.4. Consistent with all the above, our study demonstrated that the

306

isolated date flavonol fractions were better inhibitors of LDL oxidation by 3- to 10-fold over the

307

phenolic acid fractions.

. Flavonols were found to be especially effective inhibitors of LDL oxidation compared to

308

Date phenolics effect on HDL-mediated cholesterol efflux. Reverse cholesterol transport

309

(RCT) is a major mechanism for the attenuation of atherosclerosis development. Macrophage



Page 16 of 32

16

310

cholesterol efflux is the first and potentially most critical step in macrophage RCT. We therefore

311

explored the effect of the isolated date phenolic acid and flavonol fractions on cholesterol

312

removal from J774A.1 macrophage cells. The extent of HDL-mediated cholesterol efflux from

313

macrophages that were pre-incubated with the date phenolics’ fractions was compared to that

314

measured in control cells that were pre-treated with an equivalent volume of 50% ethanol. The

315

results are shown in Figure 4, in terms of % change over control cells in the extent of cholesterol

316

efflux per 10 nmole GAE added. The two subgroups of date phenolics demonstrated markedly

317

different effects on cholesterol removal from the macrophages. Pre-treatment of the cells with

318

PhA 1 slightly, yet significantly, inhibited the efflux by approximately 1%; PhA 2 had practically

319

no effect. On the other hand, pre-incubation with the flavonol fractions increased the magnitude

320

of cholesterol efflux from the cells, especially with Fl 2 that significantly enhanced cholesterol

321

removal by approximately 6%.

322

A major factor in phenolics effect on cellular processes is their mode of interaction with the

323

membrane bilayer, i. e., the partition of less polar compounds in the hydrophobic interior of the

324

membrane, and the formation of hydrogen bonds between phospholipid polar head groups and

325

the more hydrophilic phenolics at the membrane/water interface38. The relatively polar phenolic

326

acids (ionized at a slightly alkaline pH) may cause cell membrane depolarization; a greater

327

depolarization was reported to be induced by hydroxycinnamic acids than the corresponding

328

hydroxybenzoic acids39. PhA 1 consists of mostly hydroxycinnamates, whereas PhA 2

329

accommodates equal proportions of hydroxycinnamates and hydroxybenzoates. PhA 1 is

330

therefore likely to cause a more extensive membrane depolarization (i. e., increased membrane

331

ion permeability) than PhA 2. Such a disturbance in membrane integrity may interfere with the

332

macrophage cellular functions, leading eventually to the adverse effect of PhA 1on cholesterol


Page 17 of 32


17

333

efflux. The positive effect exhibited by the flavonol fractions on cholesterol removal from

334

macrophages, may pertain to their more profound amphipathic nature that enables protection of

335

the cell membrane from oxidation by free radicals generated both extra- and intracellularly. In

336

this context, it is noteworthy that Fl 1, the less potent enhancer of cholesterol efflux, had a higher

337

proportion of components with shorter Rt (≤ 18.9 min) compared to Fl 2, and is therefore likely

338

to consist of somewhat more polar flavonols. The date flavonols may interact with the

339

macrophage plasma membrane, and modify it in ways that consequently augment HDL binding

340

to- and cholesterol removal from the cells. Moreover, their interaction with the membrane and/or

341

internalization may initiate signaling pathways implicated in the expression of transporters

342

involved in serum-mediated cholesterol efflux from macrophage foam cells. Under a similar

343

experimental set up, pre-treatment of macrophages with 20 µM quercetin increased HDL binding

344

to- and cholesterol efflux from the cells by 27% and 50%, respectively, compared to control

345

cells40. Moreover, the cellular levels of mRNA for the ATP-binding cassette A1 (ABCA1)

346

transporter, a mediator of macrophage cholesterol efflux, and the nuclear factor PPARα that

347

induces ABCA1 expression in macrophages, increased by 42% and 77%, respectively40.

348

Taken together, our current study demonstrates that both phenolic acids and flavonols of date

349

fruits have activities that affect processes pertinent to atherosclerosis development. However, the

350

nature and potencies of the activities exhibited by the two phenolics’ subgroups varied

351

considerably. The date phenolic acids were efficient reductants and free radical scavengers,

352

whereas the flavonols were superior inhibitors of LDL oxidation, and, in addition, stimulated

353

cholesterol efflux from macrophages. Furthermore, different phenolics’ compositions of the

354

same subgroup, i. e., more subtle molecular structure differences, displayed variable levels of

355

activities, as is evident from the performance disparities comparing PhA 1 to PhA 2, and Fl 1 to



18

356

Fl 2. All in all, the results are consistent with the strong structure-function relationships

357

established for phenolic compounds21, 22, 33-36, and support our interpretation that the large

358

differences in the anti-atherogenic potency of phenolics' extracts from a diverse collection of

359

date varieties were inflicted by the substantial varietal divergence in phenolics composition13.

360

We have recently shown that regular consumption of ‘Medjool’ and ‘Hallawi’ dates by

361

healthy human subjects had beneficial anti-atherogenic effects9. The two date varieties differed

362

in both the range and magnitude of the favorable effects, and the fruit phenolics’ composition,

363

suggesting a possible role for phenolic compounds in the fruit health traits. Identifying the anti-

364

atherogenic bioactives in dates is necessary to establish the fruit health value, and utilize dates in

365

nutritional approaches directed at cardiovascular heath preservation. Our present study identifies

366

date flavonols as potentially efficient anti-atherogenic biochemicals. The in vitro biological

367

assays employed nano- to low micromolar concentrations of the isolated date flavonols, well in

368

the range of blood phenolics’ concentrations41. Thus, the present findings may be relevant to the

369

in vivo anti-atherogenic capacities of date fruits; the latter may comprise flavonols action in

370

concert and/or synergy with other fruit components, e. g. other phenolics and nutritional fibers.

371

We are currently exploring the identity and in vitro activities of flavonol constituents from a

372

diverse collection of date varieties. However, to establish causal relationships between date fruit

373

flavonol composition and anti-atherogenic benefits, in vivo studies with the purified flavonols are

374

needed.

375 376

ABBREVIATIONS USED

377

AAPH- 2.2'-azobis, 2-amidinopropane hydrochloride; ABCA1- ATP- binding cassette A1;

378

DDW- double distilled water; DMEM- Dulbecco's Modified Eagel's Medium; DPPH- 1,1-

379

diphenyl–2-picryl-hydrazyl; FCS- fetal calf serum; FRAP- Ferric-reducing antioxidant power;


Page 18 of 32

Page 19 of 32


19

380

FW- fresh weight; GAE- gallic acid equivalents; HDL- high density lipoprotein; LDL- low

381

density lipoprotein; MDA- malondialdehyde; OD- optical density; PBS- phosphate buffered

382

saline; PPARα- peroxisome proliferator-activated receptor alpha; RCT - reverse cholesterol

383

transport; RP-HPLC- reversed phase high performance liquid chromatography; SD- standard

384

deviation; TBARS- thiobarbituric acid reactive substances; VCE- vitamin C equivalents.

385

ACKNOWLEDGEMENT

386

Date fruit for this research were provided by HADIKLAIM, Israel Date Growers' Cooperative

387

Ltd.

388

REFERENCES

389

1. Barreveld, W. H. Date palm products. Food and Agriculture Organization of the United

390 391 392

Nations, Agricultural Services Bulletin No. 101, 1993, FAO, Rome, Italy. 2. Vayalil, P. K. Date Fruits (Phoenix dactylifera Linn): An Emerging Medicinal Food. Crit. Rev. Food Sci. Nutr. 2012, 52, 249-271.

393

3. Puri, A.; Sahai, R.; Singh, K. L.; Saxena, R. P.; Tanton, J. S.; Saxena, K. C. Immunostimulant

394

activity of dry fruits and plant materials used in Indian traditional medical system for

395

mothers after child birth and invalids. J. Ethnopharmacol. 2000, 71, 89-92.

396 397 398 399

4. Sallal, A.-K; Ashkenani, A. Effect of date extract on growth and spore germination of Bacillus subtilis. Microbios 1989, 59, 203-210. 5. Shraideh, Z. A.; Abu-El-Teen, K. H.; Sallal, A-K. Ultrastructural effects of date extract on Candida albicans. Mycopathologia 1998, 142, 119-123.

400

6. Vayalil, P. K. Antioxidant and antimutagenic properties of aqueous extract of date fruit

401

(Phoenix dactylifera L. Arecaceae). J. Agric. Food. Chem. 2002, 50, 610-617.



20

402

7. Mansouri, A.; Embarek, G.; Kokkalouc, E.; Kefalas, P. Phenolic profile and antioxidant

403

activity of the Algerian ripe date palm fruit (Phoenix dactylifera). Food Chem. 2005, 89,

404

411-420.

405

8. Al-Farsi, M.; Alasalvar, C.; Morris, A.; Baron, M.; Shahidi, F. Comparison of antioxidant

406

activity, anthocyanins, carotenoids, and phenolics of three native fresh and sun-dried date

407

(Phoenix dactylifera L.) varieties grown in Oman. J. Agric. Food Chem. 2005, 53, 7592-

408

7599.

409

9. Rock, W.; Rosenblat, M.; Borochov-Neori, H.; Volkova, N.; Judeinstein, S.; Elias, M.;

410

Aviram, M. Effects of date (Phoenix dactylifera L., Medjool or Hallawi Variety)

411

consumption by healthy subjects on serum glucose and lipid levels and on serum oxidative

412

status: a pilot study. J. Agric. Food Chem. 2009, 57, 8010-8017.

413

10. Amoros, A.; Pretel, M.T.; Almansa, M.S.; Botella; M.A.; Zapata, P.J.; Serrano, M.

414

Antioxidant and nutritional properties of date fruit from Elche Grove as affected by

415

maturation and phenotypic variability of date palm. Food Sci. Technol. Int. 2009, 15, 65-

416

72.

417

11. Regnault-Roger, C.; Hadidane, R.; Biard, J. F.; Boukef, K. High performance liquid and thin-

418

layer chromatographic determination of phenolic acids in palm (Phoenix dactilifera)

419

products. Food Chem. 1987, 25, 61-67.

420

12. Hong, Y. J.; Tomas-Barberan, F. A.; Kader, A. A.; Mitchell, A. E. The flavonoid glycosides

421

and procyanidin composition of Deglet Noor dates (Phoenix dactylifera). J. Agric. Food

422

Chem. 2006, 54, 2405 –2411.


Page 20 of 32

Page 21 of 32


21

423

13. Borochov-Neori, H.; Judeinstein, S.; Greenberg, A.; Volkova, N.; Rosenblat, M.; Aviram, M.

424

Date (Phoenix dactylifera L.) fruit soluble phenolics composition and anti–atherogenic

425

properties in nine Israeli varieties. J. Agric. Food Chem., 2013, 61, 4278–4286.

426

14. Lusis, A. J. Atherosclerosis. Nature. 2000, 404, 233-241.

427

15. Schulze, P. C.; Lee, R. T. Oxidative stress and atherosclerosis. Curr. Arterioscler. Rep. 2005,

428 429

7, 242-248. 16. Aviram, M.; Kaplan, M.; Rosenblat, M.; Fuhrman, B. Dietary antioxidants and paraoxonases

430

against LDL oxidation and atherosclerosis development. Handb. Exp. Pharmacol. 2005,

431

170, 263-300.

432 433 434

17. Geleijnse, J. M.; Hollman, P. Ch. Flavonoids and cardiovascular health: which compounds, what mechanisms? Am. J. Clin. Nutr. 2008, 88, 12-13. 18. Hertog, M.G.; Kromhout, D.; Aravanis, C.; Blackburn, H.; Buzina, R.; Fidanza, F.;

435

Giampaoli, S.; Jansen, A.; Menotti, A.; Nedeljkovic, S.; Pekkarinen, M.; Simic, B. S.;

436

Toshima, H.; Feskens, E. J. M.; Hollman, P. C. H.; Katan, M. B. Flavonoid Intake and

437

Long-term Risk of Coronary Heart Disease and Cancer in the Seven Countries Study.

438

Arch. Intern. Med., 1995, 155, 381-386.

439

19. Aviram, M.; Rosenblat, M.; Gaitini, D.; Nitecki, S.; Hoffman, A.; Dornfeld, L.; Volkova, N.;

440

Presser, D.; Attias, J.; Liker, H.; Hayek, T. Pomegranate juice consumption for 3 years by

441

patients with carotid artery stenosis reduces common carotid intima-media thickness, blood

442

pressure and LDL oxidation. Clin. Nutr. 2004, 23, 423-433.

443 444

20. Borochov-Neori, H.; Judeinstein, S.; Greenberg, A.; Fuhrman, B.; Attias, J.; Volkova, N.; Hayek, T.; Aviram, M. Phenolic antioxidants and antiatherogenic effects of Marula



22

445

(Sclerocarrya birrea Subsp. caffra) fruit juice in healthy humans. J. Agric. Food. Chem.

446

2008, 56, 9884-9891.

447 448 449

21. Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biol. Med. 1996, 20, 933-956. 22. Brown, J.E.; Khodr, H.; Hider, R.C.; Rice-Evans, C.A. Structural dependence of flavonoid

450

interactions with Cu2+ ions: implications for their antioxidant properties. Biochem. J.

451

1998, 330, 1173-1178.

452

23. Mrabet, A.; Rodríguez-Arcos, R.; Guillén-Bejarano, R.; Chaira, N.; Ferchichi, A.; Jiménez-

453

Araujo, A. Dietary Fiber from Tunisian Common Date Cultivars (Phoenix dactylifera L.):

454

Chemical Composition, Functional Properties, and Antioxidant Capacity. J. Agric. Food

455

Chem. 2012, 60, 3658−3664.

456 457 458 459 460 461 462 463 464

24. Oszmianski, J.; Ramos, T.; Bourzeix, M. Fractionation of phenolic compounds in red wine. Am. J. Enol. Vitic. 1988, 39, 259–262. 25. Singleton, V. L.; Rossi, J. A. Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. Am. J. Enol. Vitic. 1965, 16, 144-158. 26. Benzie, I.F.F.; Straino, J.J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Anal. Biochem. 1996, 239, 70-76. 27. Malterud, K.M.; Farbort, T.L.; Huse, A.C.E.; Sund, R. B. Antioxidant and radical scavenging effects of arthraquinones and anthorones. Pharmacol. 1993, 47, 77-85. 28. Aviram, M. Plasma lipoprotein separation by discontinuous density gradient

465

ultracentrifugation in hyperlipoproteinemic patients. Biochem. Med. 1983, 30, 111-118.

466

29. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the

467

Folin phenol reagent. J. Biol. Chem. 1951, 193, 265-275.


Page 22 of 32

Page 23 of 32


23

468 469 470

30. Buege, J. A.; Aust, S. D. Microsomal lipid peroxidation. Methods Enzymol. 1978, 52, 302310. 31. El-Saadani, M.; Esterbauer, N.; El-Sayed, M.; Goher, M.; Nassar, A.Y.; Jurgens, G.

471

Spectrophotometric assay for lipid peroxides in serum lipoproteins using commercially

472

available reagent. J. Lipid Res. 1989, 30, 627-30.

473

32. Gu, X.; Kozarsky, K.; Krieger, M. Scavenger receptor class B, type I-mediated [3H]

474

cholesterol efflux to high and low density lipoproteins is dependent on lipoprotein binding

475

to the receptor. J. Biol. Chem. 2000, 275, 29993-30001.

476

33. Cheng, J-C.; Dai, F.; Zhou, B.; Yang, L.; Liu, Z-L. Antioxidant activity of hydroxycinnamic

477

acid derivatives in human low density lipoprotein: Mechanism and structure–activity

478

relationship. Food Chem. 2007, 104, 132-139.

479

34. Laranjinha, J. Caffeic acid and related antioxidant compounds: Biochemical and cellular

480

effects. In Handbook of Antioxidants, 2nd edition revised and expanded; Cadenas, E.;

481

Packer, L. Eds; Marcel Dekker, Inc.; New York, NY, 2001; pp 279-302.

482

35. Fuhrman, B.; Aviram, M. Polyphenols and flavonoids protect LDL against atherogenic

483

modifications. In Handbook of Antioxidants, 2nd edition revised and expanded; Cadenas,

484

E.; Packer, L. Eds; Marcel Dekker, Inc.; New York, NY, 2001; pp 303-336.

485

36. Vaya, J.; Mahmood, S.; Goldblum, A.; Aviram, M.; Volkova, N.; Shaalan, A.; Musa, R.;

486

Tamir, S. Inhibition of LDL oxidation by flavonoids in relation to their structure and

487

calculated enthalpy. Phytochem. 2003, 62, 89-99.

488

37. Russo, N.; Toscano, M.; Uccella, N. Semiempirical molecular modeling into quercetin

489

reactive site: structural, conformation, and electronic features. J. Agric. Food Chem. 2000,

490

48, 3232–3237.



24

491 492 493 494 495

38. Hendrich, A.B. Flavonoid-membrane interactions: possible consequences for biological effects of some polyphenolic compounds. Acta. Pharmacol. Sin. 2006, 27, 27-40. 39. Glass, A.D.M.; Dunlop, J. Influence of phenolic acids on ion uptake. IV. Depolarization of membrane potentials. Plant Physiol. 1974, 54, 855-858. 40. Rosenblat, M.; Volkova, N.; Khatib, S.; Mahmood, S.; Vaya, J.; Aviram, M. Reduced

496

glutathione (GSH) increases quercetin stimulatory effects on HDL- or apoA1- mediated

497

cholesterol efflux from J774A.1 macrophages. Free Radic. Res., 2014, doi:

498

10.3109/10715762.2014.963574

499 500

41. Manach, C.; Scalbert, A.; Morand, C.; Rémésy, C.; Jiménez, L. Polyphenols: food sources and bioavailability. Am. J. Clin. Nutr. 2004, 79, 727-747.

501

FOOTNOTE: Financial support was obtained from the Date Palm Growers' Desk of Israel

502

Plants Production and Marketing Board, the Jewish National Fund (KKL) and the JCA

503

Charitable Foundation.


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504

FIGURE CAPTIONS

505

Figure 1: Composition of phenolic acid fractions isolated from date fruits. Fraction

506

composition was analyzed by RP-HPLC. Chromatogram peak concentrations in nmol mL-1 are

507

presented in order of ascending retention times. Results are the mean ± SD (n=3).

508

Figure 2: Composition of flavonol fractions isolated from date fruits. Fraction composition was

509

analyzed by RP-HPLC. Chromatogram peak concentrations in nmol mL-1 are presented in order

510

of ascending retention times. Results are the mean ± SD (n=3).

511

Figure 3: The effect of phenolic acid and flavonol fractions isolated from date fruits on copper

512

ion-induced LDL oxidation. LDL (100 µg protein mL-1) was incubated with increasing

513

concentrations (0-25 µL mL-1) of the isolated date phenolics’ fractions in the presence of 5 µmol

514

L-1 CuSO4. The extent of LDL oxidation was determined by the TBARS and lipid peroxides

515

assays. The phenolics concentrations needed to achieve 50% inhibition (IC50) are shown in terms

516

of nmol GAE mL-1.

517

Results are the mean ± SD (n=3). Bars labeled with different uppercase (TBARS assay) or

518

lowercase (lipid peroxide assay) letters are significantly different at p < 0.01; "***" indicates

519

significant differences at p < 0.001 comparing to PhA 1 and PhA 2 (Tukey-Kramer posthoc).

520

Figure 4: The effect of phenolic acid and flavonol fractions isolated from date fruits on HDL-

521

mediated cholesterol efflux from macrophages. J774A.1 macrophages (1 mL of 106 cells mL-1)

522

were pre-incubated at 37 °C for 20 h with 20 to 170 µM GAE of the isolated phenolics' fractions.

523

After cell wash the cells were labeled with [3H]-cholesterol and the extent of HDL (100 µg

524

protein mL-1)-mediated cholesterol efflux from the cells was determined. Phenolics' fraction

525

effect is presented in terms of % change over the control in cholesterol efflux per 10 nmol GAE

526

added.



26

527

Results are the mean ± SD (n=3). * Denotes significant difference at p < 0.05 (one-way

528

ANOVA) between fraction-treated- and control cells.


Page 26 of 32

Page 27 of 32


27

529

Table 1: Yield and Antioxidant Properties of Phenolic Acid and Flavonol Fractions Isolated from

530

‘Amari’ and ‘Hallawi’ Date Fruitsa

Ferric Reducing Date

Phenolics’

Fraction

Variety

Subgroup

Label

‘Amari’

Phenolic Acids

‘Hallawi’ ‘Amari’

Flavonols

‘Hallawi’

Yield Power

Free Radical Scavenging Capacity

(µmol GAE

(VCE/GAE,

per g fruit)

mole ratio)

PhA 1

4.27±0.62

0.81±0.02b

14±1b

PhA 2

0.38±0.09

0.80±0.01b

22±3a

Fl 1

1.37±0.15

0.88±0.01a

8±1c

Fl 2

0.43±0.11

0.87±0.03a

9±2c

(% OD 517 nm loss)

531

a

532

density at 517 nm.

533

Values are mean ± SD (n=3). Values within a column with different letters are significantly

534

different at a 95% confidence interval (Tukey-Kramer posthoc).

Abbreviations: GAE - gallic acid equivalents; VCE - vitamin C equivalents; OD 517 nm - optical



28

Phenolic Acid Concentration (nmol mL-1)

Figure 1

400

PhA 1 PhA 2

300 200 100 0 9.7

10.1 11.4 11.7 12.5 14.9 16.7 27.4 Retention Time (min)


Page 28 of 32

Page 29 of 32


29

Flavonol Concentration (nmol mL-1)

Figure 2

50 40

Fl 1 Fl 2

30 20 10 0 16.0

17.0

18.4 18.9 19.8 20.8 Retention Time (min)


21.3


Page 30 of 32

30

Figure 3

Inhibition of LDL Oxidation (IC50, nmol GAE mL-1)

120

D

TBARS Lipid Peroxides

d C

90

c

60 *** B

30

b

*** A

a

0 PhA 1

PhA 2


Fl 1

Fl 2

Page 31 of 32


31

Change in Cholesterol Efflux (% per 10 nmol GAE added)

Figure 4:

9.0 *

7.0 5.0 3.0 1.0 * -1.0 PhA 1

PhA 2


Fl 1

Fl 2


32

GRAPHIC FOR TABLE OF CONTENTS


Page 32 of 32

Date (Phoenix dactylifera L.) - ACS Publications - American Chemical

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