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Antiinflammatory and antioxidant activities from the basolateral fraction of Caco-2 cells exposed to a rosmarinic acid enriched extract MARISOL VILLALVA, Laura Jaime, Estela Aguado, Juan Antonio Nieto, Guillermo J. Reglero, and Susana Santoyo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06008 • Publication Date (Web): 18 Jan 2018 Downloaded from http://pubs.acs.org on January 19, 2018
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Journal of Agricultural and Food Chemistry
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Antiinflammatory and antioxidant activities from the basolateral
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fraction of Caco-2 cells exposed to a rosmarinic acid enriched extract
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Marisol Villalvaa, Laura Jaimea, Estela Aguadoa, Juan Antonio Nietoa, Guillermo
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Regleroa,b, Susana Santoyoa *
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a
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UAM+CSIC). 28049 Madrid, Spain
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b
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Madrid, Spain
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*Corresponding author: Susana Santoyo
Institute of Food Science Research (CIAL). Universidad Autónoma de Madrid (CEI
Imdea-Food Institute, Universidad Autónoma de Madrid (CEI UAM+CSIC), 28049
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Institute of Food Science Research (CIAL)
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Universidad Autónoma de Madrid
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28049 Madrid, Spain
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Tel: +34 910017926
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Fax: +34 9100.17905
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E-mail:
[email protected] 16
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ABSTRACT
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The potential use of Origanum majorana L. as a source of bioavailable phenolic
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compounds, specifically rosmarinic acid (RA), has been evaluated. Phenolic
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bioavailability was tested using an in vitro digestion process followed by a Caco-2
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cellular model of intestinal absorption. The HPLC-PAD-MS/MS analysis showed the
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main components in the extract were 6-hydroxyluteolin-7-O-glucoside and rosmarinic
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acid, followed by luteolin-O-glucoside. After digestion process, the amount of total
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phenolic compounds (TPC) only decreased slightly, although a remarkable reduction in
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RA (near 50%) was detected. Bioavailable fraction contained 7.37±1.39 mg/L digested
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extract of RA with small quantities of lithospermic acid and diosmin and presented an
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important antioxidant activity (0.89±0.09 mmol Trolox/L digested extract). Besides, this
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bioavailable fraction produced a significant inhibition in TNF-α, IL-1β and IL-6
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secretion, using a human THP-1 macrophages model. Therefore, RA content in the
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basolateral compartment could play an important role in the antioxidant and anti-
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inflammatory activities found.
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Key words: anti-inflammatory activity, antioxidant activity, rosmarinic acid, in vitro
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digestion, Caco-2 cells.
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INTRODUCTION
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Origanum majorana L. (marjoram) is a culinary herb often used in foods. Its essential
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oil and extracts have been indicated to possess antioxidant, antimicrobial, anticancer
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and anti-inflammatory activities.1,2 These activities have been attributed to the presence
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of a high percentage of phenolic acids and flavonoids in marjoram leaves3 particularly,
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to rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid
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naturally occurring in marjoram.4,5 Antioxidant activity of RA has been generally
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admitted, since this compound may act as free radical scavenger.6,7 Related to RA anti-
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inflammatory effects, Jiang et al.8 showed that this compound down regulated the levels
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of TNF-α, IL-6 and high mobility box 1 protein in bacterial lipopolysaccharide (LPS)
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induced RAW264.7 cells, indicating that RA might inhibit activation of the nuclear
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factor-κB pathway by inhibiting IκB kinase activity. Accordingly, Zdarilová et al.9
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reported that RA inhibited LPS-induced up-regulation of Il-1β, IL-6, TNF-α and
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suppressed expression of iNOS in human gingival fibroblasts. Further, Lembo et al.10
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indicated that RA produced a significant reduction in IL-1β, IL-6, IL-8 and TNF-α gene
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expression in HaCat cells after UVB irradiation.
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In order to extrapolate results founded in vitro to in vivo situation, it is important to
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know the bioavailability of bioactive compounds. Therefore, the use of an in vitro
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digestion/Caco-2 cell culture model has been proposed by several authors as an
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economical and useful alternative to study the bioavailability of these compounds.11,12
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Thus, although the transepithelial transport of RA in intestinal Caco-2 cells monolayers
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has been studied,13 it is crucial to investigate the effect that plant matrix plays on its
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bioavailability, since plant material matrix may alter absorption and bioavailability of
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phytochemicals.14 For that matter, it would be very interesting to determine the anti-
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inflammatory and antioxidant effect of the bioavailable fractions of marjoram extracts 3
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enriched in RA, in order to corroborate the biological activities described for these
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extracts.
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Solid –liquid extraction (SLE) is the most traditional technology used to extract active
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compounds from plant matrix. It is widely known that higher temperatures enhance the
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solubility of the solute in the solvent and thus improve its recovery. Nevertheless, the
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SLE temperature is limited by solvent boiling and, in some cases, by the loss of volatile
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compounds. In this regard, pressurized liquid extraction (PLE) allows the use of
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solvents in a liquid state at higher temperatures. Furthermore, a compression effect is
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made on vegetal particles, which also contributes to improve extraction yield; moreover,
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lower amount of solvent is required, extraction is faster and volatiles loss is
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minimized.15 Thus, several studies proposed PLE extraction as an alternative to
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conventional solid/liquid extraction in order to obtain phenolic compounds from herbs
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and spices.16,17
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However, it is hard to obtain a highly concentrated extracts in phenolic compounds
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using only PLE, due to the complexity and the presence of impurities in crude extracts
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of herbal raw materials. Nowadays, the use of adsorption resins has been proposed as
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one of the most useful tools for selective enrichment of phenolic compounds from plant
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material, such as naringenin recovery from orange juice18 or anthocyanins from
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grapes.19
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For this purpose, some of the most commonly employed resins are XAD-2, XAD-7,
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XAD-16 and Oasis HLB, that have been successfully used for phenolic compounds
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enrichment from natural extracts.20,21 Among them, XAD-7 has been proposed for
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rosmarinic acid enrichment from Lavandula vera22 or Rabdosia serra.23
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The aim of this work was to study the anti-inflammatory and antioxidant properties of
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the bioavailable fraction of PLE extracts enriched in rosmarinic acid. In order to
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increase the quantity of RA in PLE extracts of marjoram an amberlite XAD-7HP resin
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was employed. The bioavailability of both, original and enriched extracts, was
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determined by using an in vitro digestion/Caco-2 cell culture model. Thus, the anti-
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inflammatory and antioxidant activity of the basolateral fraction was measured.
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MATERIALS AND METHODS
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Chemicals
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Ethanol of analytical grade and Folin-Ciocalteu’s reagent were obtained from Panreac
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(Madrid,
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(Trolox, 97%), 2,2-Diphenyl-1-picrylhydrazyl free radical (DPPH), gallic acid for
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titration (97,5%), and thiazolyl blue tetrazolium bromide (MTT) were purchased from
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Sigma Aldrich (Madrid, Spain). Formic acid (99%) was obtained from Acros Organics
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(Madrid, Spain) and acetonitrile HPLC grade from Macron Fine Chemicals (Madrid,
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Spain). Reference substances (analytical standard or HPLC purity ≥ 95%) for phenolic
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compounds identification such as cryptochlorogenic acid, neochlorogenic acid and
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rosmarinic acid were purchased from Sigma Aldrich (Madrid, Spain). Apigenin,
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apigenin 7-O-glururonide, caftaric acid, diosmin, lithospermic acid, luteolin, salvianolic
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acid and vicenin 2 were from Phytolab (Madrid, Spain). Ethyl gallate, apigenin 7-O-
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glucoside, caffeic acid, luteolin 7-O-glucoside, p-coumaric acid and protocatechuic acid
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were obtained from Extrasynthese S.A. (Genay, France). Finally, luteolin-7-O-
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glucuronide was from HWI Analytic GmbH (Rülzheim, Germany). The water used in
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this study was ultrapure type 1 (Millipore, Madrid, Spain).
Spain).
(±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
acid
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Marjoram samples and PLE extraction
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Marjoram sample consisted of dried leaves obtained from an herbalist shop (Murciana
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herboristería, Murcia, Spain). The sample was grinded in a knife mill (Grindomix GM
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200, Retsch, Llanera, Spain) and the particle size was determined by sieving the ground
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plant material to the appropriate size (
