Anti-inflammatory properties of fruit juices enriched with pine bark extract in an in vitro model of inflamed human intestinal epithelium: The effect of gastrointestinal digestion Carmen Frontela-Saseta ⇑ , Rubén López-Nicolás, Carlos A. González-Bermúdez, Carmen Martínez-Graciá, Gaspar Ros-Berruezo Department of Food Science and Nutrition, Faculty of Veterinary Sciences, Regional Campus of International Excellence Campus Mare Nostrum, University of Murcia, Spain article info Article history: Received 10 September 2012 Accepted 16 November 2012 Available online 5 December 2012 Keywords: Fruit juices Gut inflammation In vitro digestion Pine bark extract abstract Enrichment of fruit juices with pine bark extract (PBE) could be a strategy to compensate for phenolic losses during the gastrointestinal digestion. A coculture system with Caco-2 cells and RAW 264.7 macro- phages was established as an in vitro model of inflamed human intestinal epithelium for evaluating the anti-inflammatory capacity of fruit juices enriched with PBE (0.5 g L 1 ) before and after in vitro digestion. The digestion of both PBE-enriched pineapple and red fruit juice led to significant changes in most of the analysed phenolic compounds. The in vitro inflammatory state showed cell barrier dysfunction and over- production of IL-8, nitric oxide (NO) and reactive oxygen species (ROS). In the inflamed cells, incubation with nondigested samples reduced (P< 0.05) the production of IL-8 and NO compared with digested sam- ples. ROS production increased in the inflamed cells exposed to digested commercial red fruit juice (86.8 ± 1.3%) compared with fresh juice (77.4 ± 0.8%) and increased in the inflamed cells exposed to digested enriched red fruit juice (82.6 ± 1.6%) compared with the fresh enriched juice (55.8 ± 6%). The anti-inflammatory properties of PBE-enriched fruit juices decreased after digestion; further research on the bioavailability of the assayed compounds is needed to properly assess their usefulness for the treatment of gut inflammation. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Inflammation is a nonspecific response of mammalian tissues to a variety of hostile agents and helps to restore homeostasis at in- fected or damaged sites (Davies and Hagen, 1997; Fiocchi, 2003). When the regulation of inflammatory responses fails, it can be- come chronic and contribute to the perpetuation and progression of disease. At the intestinal level, the failure is the result of dysfunctional epithelial and immune responses against normal en- teric microbiota and alterations of the gut homeostasis in response to genes and/or the environment (Ferguson et al., 2007). This dys- function is mainly characterised by the infiltration of inflammatory cells into the intestinal mucosa, where the resulting overproduc- tion of reactive oxygen species (ROS) is accompanied by the expression of a wide spectrum of proinflammatory cytokines and other mediators of inflammation associated with immune dysregulation (Conner and Grisham, 1996). The levels of these mediators amplify the inflammatory response being destructive and contributing to clinical symptoms (Romier et al., 2008). Current anti-inflammatory treatments comprise different drugs, which are frequently associated with undesirable side effects (Waldner and Neurath, 2009). Consequently, there is a growing sci- entific rationale for the use of dietary factors with antioxidant activity and, thus, the potential ability to modify any of the above-mentioned steps to contribute to the prevention, or even the treatment, of inflammatory processes (Romier-Crouzet et al., 2009; Sergent et al., 2010). Due to its antioxidant capacity, pheno- lic compounds are thought to have the potential to reduce or delay the development of inflammation, as shown in intestinal models (Ruiz and Haller, 2006; Romier-Crouzet et al., 2009; Sergent et al., 2010). Most studies (in vitro and in vivo) to date that have investigated the effect of pure polyphenol compounds on intestinal inflammation have shown promising results (Paradkar et al., 2004; Kim et al., 2005; Romier-Crouzet et al., 2009; Sergent et al., 2010); however, evidence about the role of dietary phenolics is lacking. Polyphenols are produced in plants and are regularly ingested in the diet (Manach et al., 2004), with fruit juices offering an attractive source (Bermúdez-Soto and Tomas-Barberán, 2004; Lichtenthaler and Marx, 2005). However, for effective health bene- fits, the necessary volume of ingested juice could involve a high in- take of simple sugars and excess calories (Breithpaut, 2001). Thus, 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.11.024 ⇑ Corresponding author. Tel.: +34 868887985; fax: +34 868887167. E-mail address: [email protected](C. Frontela-Saseta). Food and Chemical Toxicology 53 (2013) 94–99 Contents lists available at SciVerse ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
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Food and Chemical Toxicology 53 (2013) 94–99
Contents lists available at SciVerse ScienceDirect
Anti-inflammatory properties of fruit juices enriched with pine bark extractin an in vitro model of inflamed human intestinal epithelium: The effectof gastrointestinal digestion
Carmen Frontela-Saseta ⇑, Rubén López-Nicolás, Carlos A. González-Bermúdez, Carmen Martínez-Graciá,Gaspar Ros-BerruezoDepartment of Food Science and Nutrition, Faculty of Veterinary Sciences, Regional Campus of International Excellence Campus Mare Nostrum, University of Murcia, Spain
a r t i c l e i n f o
Article history:Received 10 September 2012Accepted 16 November 2012Available online 5 December 2012
Enrichment of fruit juices with pine bark extract (PBE) could be a strategy to compensate for phenoliclosses during the gastrointestinal digestion. A coculture system with Caco-2 cells and RAW 264.7 macro-phages was established as an in vitro model of inflamed human intestinal epithelium for evaluating theanti-inflammatory capacity of fruit juices enriched with PBE (0.5 g L�1) before and after in vitro digestion.The digestion of both PBE-enriched pineapple and red fruit juice led to significant changes in most of theanalysed phenolic compounds. The in vitro inflammatory state showed cell barrier dysfunction and over-production of IL-8, nitric oxide (NO) and reactive oxygen species (ROS). In the inflamed cells, incubationwith nondigested samples reduced (P < 0.05) the production of IL-8 and NO compared with digested sam-ples. ROS production increased in the inflamed cells exposed to digested commercial red fruit juice(86.8 ± 1.3%) compared with fresh juice (77.4 ± 0.8%) and increased in the inflamed cells exposed todigested enriched red fruit juice (82.6 ± 1.6%) compared with the fresh enriched juice (55.8 ± 6%). Theanti-inflammatory properties of PBE-enriched fruit juices decreased after digestion; further researchon the bioavailability of the assayed compounds is needed to properly assess their usefulness for thetreatment of gut inflammation.
� 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Inflammation is a nonspecific response of mammalian tissues toa variety of hostile agents and helps to restore homeostasis at in-fected or damaged sites (Davies and Hagen, 1997; Fiocchi, 2003).When the regulation of inflammatory responses fails, it can be-come chronic and contribute to the perpetuation and progressionof disease. At the intestinal level, the failure is the result ofdysfunctional epithelial and immune responses against normal en-teric microbiota and alterations of the gut homeostasis in responseto genes and/or the environment (Ferguson et al., 2007). This dys-function is mainly characterised by the infiltration of inflammatorycells into the intestinal mucosa, where the resulting overproduc-tion of reactive oxygen species (ROS) is accompanied by theexpression of a wide spectrum of proinflammatory cytokines andother mediators of inflammation associated with immunedysregulation (Conner and Grisham, 1996). The levels of thesemediators amplify the inflammatory response being destructiveand contributing to clinical symptoms (Romier et al., 2008).
ll rights reserved.
+34 868887167.ta).
Current anti-inflammatory treatments comprise different drugs,which are frequently associated with undesirable side effects(Waldner and Neurath, 2009). Consequently, there is a growing sci-entific rationale for the use of dietary factors with antioxidantactivity and, thus, the potential ability to modify any of theabove-mentioned steps to contribute to the prevention, or eventhe treatment, of inflammatory processes (Romier-Crouzet et al.,2009; Sergent et al., 2010). Due to its antioxidant capacity, pheno-lic compounds are thought to have the potential to reduce or delaythe development of inflammation, as shown in intestinal models(Ruiz and Haller, 2006; Romier-Crouzet et al., 2009; Sergentet al., 2010). Most studies (in vitro and in vivo) to date that haveinvestigated the effect of pure polyphenol compounds on intestinalinflammation have shown promising results (Paradkar et al., 2004;Kim et al., 2005; Romier-Crouzet et al., 2009; Sergent et al., 2010);however, evidence about the role of dietary phenolics is lacking.Polyphenols are produced in plants and are regularly ingested inthe diet (Manach et al., 2004), with fruit juices offering anattractive source (Bermúdez-Soto and Tomas-Barberán, 2004;Lichtenthaler and Marx, 2005). However, for effective health bene-fits, the necessary volume of ingested juice could involve a high in-take of simple sugars and excess calories (Breithpaut, 2001). Thus,
C. Frontela-Saseta et al. / Food and Chemical Toxicology 53 (2013) 94–99 95
supplementation with extract containing phenolic componentsshould improve the nutritional quality of processed fruit bever-ages. Moreover, studies at intestinal level must consider that foodis subjected to a gastrointestinal digestion process, which could af-fect the antioxidant potential and other functional components;this process probably exerts some beneficial effects on the gut epi-thelium (Bode and Dong, 2004). Pine (Pinus pinaster Ait) bark ex-tract (PBE) has been demonstrated to have strong antioxidantproperties, which are mainly attributed to its phenolic constituents(Maimoona et al., 2010). It consists of a concentrate of water-soluble polyphenols, mainly procyanidins, phenolic acids, cinnamicacids and their glycosides and taxifolin. The objective of thisresearch was to evaluate whether fruit juices enriched with PBEbefore/after a simulated gastrointestinal digestion proceduremodulated induced inflammation markers at the intestinal level.We established an in vitro model of inflamed human intestinal epi-thelium based on a co-culture system with Caco-2 cells and murinemacrophage cells (RAW 264.7), which were stimulated with bacte-rial lipopolysaccharide (LPS) to imitate an in vivo intestinal inflam-matory process.
2. Materials and methods
2.1. Samples
Four different fruit juices were studied: 1- pineapple (Ananas comosus L.) juice;2- red fruit juice, containing mainly of water and a mixture of red grape (26%), cher-ry (2%), raspberry (1%), blackberry (0.6%) and blackcurrant (0.6%) concentratedjuices; 3- pineapple juice with PBE (0.5 g L�1); 4- red fruit juice with PBE(0.5 g L�1). The juices were subjected to in vitro gastrointestinal digestion. Threeunits from each fruit juice were analysed for their phenolic content. Fruit juiceswere prepared at the pilot plant of Hero España S.A. (Alcantarilla, Murcia, Spain).
2.2. Chemicals
Enzymes and bile salts were purchased from Sigma Chemical Co. (St. Louis, MO,USA): pepsin (porcine, catalogue no. P-7000), pancreatin (porcine, catalogue No. P-1750) and bile extract (porcine, catalogue No. B-8756). The pepsin solution was pre-pared by dissolving 1.6 g of pepsin in 10 mL of 0.1 mol/L HCl. The pancreatin-bileextract solution was prepared by dissolving 0.2 g of pancreatin and 1.25 g of bile ex-tract in 50 mL of 0.1 mol/L NaHCO3. Reference standards (taxifolin, ferulic acid, gal-lic acid, chlorogenic acid and caffeic acid), LPS (Escherichia coli serotype 0127:B8)and 20 ,70-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide and reagents for HPLC analysis(acetonitrile and formic acid) were supplied by Merck KGaA, (Darmstadt, Germany).Water was treated in a Milli-Q water purification system (TGI Pure Water Systems,USA). Dulbecco’s Modified Eagle Medium (DMEM) (3.7 g NaHCO3/L, 2 mM gluta-mine, 10% heat-inactivated foetal calf serum, 1% nonessential amino acids/L, 1%penicillin–streptomycin) was purchased from Gibco BRL Life Technologies (Paisley,Scotland).
2.3. In vitro gastrointestinal digestion
The procedure was adapted from the method of Boato et al. (2002). The methodbriefly consists of two sequential steps: fruit juices were initially digested by pep-sin/HCl (pH 2) for 1 h at 37 �C, followed by digestion with bile salts/pancreatin (pH6.5) for 2 h at 37 �C. Control samples were run in parallel and consisted of an equiv-alent volume of purified water subjected to the same in vitro digestion.
2.4. Identification and quantification of phenolic compounds
Free phenolic acids in fruit juices with or without PBE enrichment were deter-mined before and after in vitro gastrointestinal digestion. Immediately after diges-tion, aliquots of the digested juices were stabilised by acidification (pH 2.0), filtered(0.45 lm) and then injected onto an HPLC Lichrocart C18 column (150 � 3.9 mm,5 lm; Waters, Milford, MA, USA). The HPLC analysis was performed on a Merck Hit-achi liquid chromatograph (Darmstadt, Germany), which was equipped with an L-7100 pump, an L-7490 refraction index detector and an L-7350 column oven,according to the method described by Frontela et al., (2011). Gallic acid, ferulic acidand taxifolin were quantified at 280 nm and chlorogenic acid and caffeic acid at320 nm according to the retention times of their corresponding standards. All theprocesses were carried out in darkness.
2.5. Cell culture
The Caco-2 cells and the RAW 264.7 macrophage cells were obtained from theEuropean Collection of Cell Cultures (ECACC; number 86010202, Salisbury, UK). Forthe experiments, the Caco- 2 cells were seeded at 4 � 105 cells/well onto Transwellinsert plates (24 mm diameter, 0.4 lm pore size; Transwell, Costar Corp.), and al-lowed to differentiate on filters for 21 d. During this period, the cells were main-tained in DMEM at 37 �C in an incubator with 5% CO2, 95% air atmosphere and95% relative humidity. The medium was changed every 2 d. Fruit juices with orwithout PBE before/after in vitro gastrointestinal digestion were freshly preparedand filter sterilised (0.22 lm) prior to addition 2 h daily to the culture media (4%final concentration) for four consecutive days before the experiment to investigatethe response of Caco-2 cells. On the day of the experiment, after a 2-h incubationperiod of Caco-2 cells with the samples, the medium was replaced again with afresh serum-free medium. In order to exclude any possible effect on cells due tochanges is osmolarity, the latter was measured in the cell growth medium beforeand after the addition of juices using a Vapro vapour pressure osmometer model5520 (Wescor Claremont, Ontario, Canada). Control cells were treated with anequivalent mix of enzymes and salts. Untreated cells were also run in paralleland subjected to the same changes of medium. The RAW 264.7 cells were platedat a density of 1.4 � 105 cells/well in 6-well tissue culture plates, overnight, to en-sure complete adherence. Then, transwell inserts containing differentiated Caco-2cells were placed to the 6-well plates preloaded with RAW 264.7 cells, accordingto the method of Tanoue et al. (2008). Then, 1 lg/mL LPS was added to the basolat-eral side as inflammatory stimulus of RAW 264.7. After an additional incubation of3 h, the culture supernatant from the apical side was collected to measure the IL-8and NO levels. The Caco-2 cells were then collected for determination of intracellu-lar ROS production.
2.6. Measurement of IL-8
The concentration of IL-8 in the culture medium in the supernatant was assayedin the media with an enzyme-linked immunosorbent assay (ELISA) kit (Thermo Sci-entific, Rockford, IL, USA) according to the manufacturers instructions. IL-8 secretionwas quantified using a reference standard curve provided with the kit. Results wereexpressed in terms relative to the positive control (Caco-2 cells co-cultured withLPS-stimulated RAW 264.7 in the absence of polyphenolic extract). The experimentwas performed three times, with each individual treatment being run in triplicate.
2.7. Measurement of nitric oxide (NO) levels
NO, which was present in the culture medium as nitrite and nitrate, was as-sayed using the colorimetric NO assay (Thermo Scientific, Rockford, IL, USA) accord-ing to the manufacturer’s protocol. Total NO contributed by nitrate and nitrite wasmeasured as nitrite from a nitrite standard curve provided with the kit, after con-verting all nitrate to nitrite. Briefly, the nitrate present in the supernatant was enzy-matically transformed into nitrite by nitrate reductase, and the total nitriteconcentration was measured photometrically at 530 nm using the Griess reagent(solution of sulfanilamide in 2 M HCl and N-(1-napthyl) ethylenediamine dihydro-chloride in 2 M HCl). Results were expressed in relative terms to the positive control(Caco-2 cells co-cultured with LPS-stimulated RAW 264.7 in the absence of poly-phenolic extract). The experiments were performed three times, with each individ-ual treatment being run in triplicate.
The tight junction permeability of the Caco-2 monolayers was determined bytransepithelial electrical resistance (TEER) during the incubation period (3 h), usingsamples with the same concentration as described earlier. TEER is believed to becorrelated with the change in paracellular permeability of the cell monolayer(Hashimoto et al., 1997). TEER values were monitored every 30 min. The effect ofeach assayed sample on the cell monolayer was expressed as the relative TEER va-lue to the value at zero time. Background resistance was determined by measuringacross a filter without cells in Hank’s balanced salt solution. Monolayers with resis-tances <500 X.cm2 were discarded. The TEER value was measured using a Millicell-ERS instrument (Millipore, Bedford, USA).
2.9. Intracellular accumulation of reactive oxygen species (ROS)
The intracellular accumulation of ROS in the Caco-2 cells was measured usingthe oxidant-sensitive fluorescent probe, DCFH-DA. DCFH converted from DCFH-DA deacetylase within the cells was oxidised by a variety of intracellular ROS toDCF, a highly fluorescent compound. After incubation with the samples (3 h), themonolayers were washed twice with PBS. The cells were then harvested and stainedwith 12.5 lM of DCFH-DA for 20 min in darkness at room temperature. A FACSortflow cytometer (BD Biosciences, San Jose, CA, USA) was used to analyse the intracel-lular ROS production and measure the fluorescent intensities of DCFH-DA (kex = 488 nm and k em = 530 nm). Approximately 10,000 counts were made for eachsample.
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2.10. Statistical analysis
Results were expressed as mean ± standard deviation from three independentdeterminations of each sample. Statistical analyses of the data were performed withthe Student’s t-test to compare the values with an appropriate control. A probabilityvalue of P 6 0.05 was considered to denote a statistically significant difference.
3. Results
3.1. Effect of in vitro gastrointestinal digestion on the phenolic content
Table 1 shows the content of each phenolic compound in thesamples before and after in vitro digestion for each analysed fruitjuice. As can be seen, gallic acid was the major phenolic compoundfound in all the samples analysed before and after digestion, fol-lowed by taxifolin. Both enriched commercial juices (pineappleand red fruits) showed no significant change in chlorogenic acidand taxifolin levels after the in vitro digestion process, whereas gal-lic acid and caffeic acid in pineapple juice and ferulic acid in redfruit juice were significantly (P 6 0.05) affected by gastrointestinaldigestion.
3.2. Effect of fresh and digested juices on IL-8 and NO production in thein vitro model of intestinal inflammation
In Fig. 1, IL-8 secretion in the coculture system constructed withthe Caco-2 cells and the RAW 264.7 cells is shown. As can be seen,
Table 1HPLC-DAD analysis of phenolic compounds in fruit juices before and after in vitro gastroi(n = 3).
* Indicates, within each phenolic compound, significant (P < 0.05) difference between th
Contro
l -
Contro
l + PBE
IC-P
BE PJ RFIC
-PJ
IC-P
J+P
IL-8
sec
retio
n(%
pos
itive
con
trol)
0
20
40
60
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100
120
j j j j
a
c
e
Fig. 1. Effect of fresh and digested fruit juices (enriched or not with pine bark extract) onthe positive control (Caco-2 cells co-cultured with LPS-stimulated RAW 264.7 in the absto Caco-2 cells incubated with DMEM alone. Values are mean ± SD of three independeinflammatory stimuli; PJ, pineapple juice; RF, red fruits juice. a–j, Means with different s
when the Caco-2/RAW 264.7 cells were stimulated with 1 lg/mLLPS, the basolateral side showed an increase in IL-8 secretion afterexposure to the digested fruit juices (from 3.45% to 78% with re-spect to the positive control [cells exposed to inflammatory stimuliin the absence of polyphenolic extract]) compared with exposureto the fresh fruit juices (from 2.2% to 45.5% with respect to the po-sitive control). However, after exposure to the red fruit juices, low-er IL-8 production was observed compared with the pineapplejuice. This inflammatory response was also observed when NOwas measured, and the results were expressed as a percentage ofNO secretion relative to the positive control (Fig. 2). Although NOwas constitutively expressed in the unstimulated Caco-2 cells(negative control), its secretion increased significantly in the pres-ence of inflammatory stimuli. The Caco-2 cells produced a higheramount of NO when they were exposed to digested fruit juices(from 32.3% to 61.3%) than when exposed to fresh juices (from8.5% to 26.4%). It must be noted that, despite the higher amountof phenolic compounds in the fruit juices added with the PBE, thisdid not always coincide with a decrease in NO production in theCaco-2 cells compared with the cells exposed to the commercialfruit juices.
3.3. Tight junction permeability of the Caco-2 monolayer
The effect of fresh and digested fruit selected in the presentstudy was assessed for its anti-inflammatory properties by
ntestinal digestion. Values (mg/100 mL) are expressed as mean ± standard deviation
IL-8 secretion in Caco-2 cells. Results are expressed in relative terms with regard toence of polyphenolic extract) (100% IL-8 secretion). Negative control correspondednt experiments. Abbreviations: PBE, pine bark extract; IC, Caco-2 cells exposed touperscripts are statistically different (P < 0.05).
Contro
l -
Contro
l + PBE
IC-P
BE PJ RFIC
-PJ
IC-P
J+PBE
IC-R
F
IC-R
F+PBE
Digeste
d PJ
Digeste
d RF
Digeste
d IC-P
J
Digeste
d IC-P
J+PBE
Digeste
d IC-R
F
Digeste
d IC-R
F+PBE
Nitr
ic o
xide
(% p
ositi
ve c
ontro
l)
0
20
40
60
80
100
120
e
a
efe
ef
h hh
hgg
g
i
dc
b
g
Fig. 2. Effect of fresh and digested fruit juices (enriched or not with pine bark extract) on NO secretion in Caco-2 cells. Results are in relative terms with regard to the positivecontrol (Caco-2 cells co-cultured with LPS-stimulated RAW 264.7 in the absence of polyphenolic extract) (100% NO production). Negative control corresponded to Caco-2 cellsincubated with DMEM alone. Values are mean ± SD of three independent experiments. Abbreviations: PBE, pine bark extract; IC, Caco-2 cells exposed to inflammatorystimuli; PJ, pineapple juice; RF, red fruits juice. a–i, Means with different superscripts are statistically different (P < 0.05).
800800
700 Control - C t lControl + PBE
600 IC-PBE
m2 )
500PJ RF
m/c
m 500 RFIC-PJ
(ohm 400 IC-PJ+PBE
IC RF
ER ( IC-RF
IC-RF+PBE
TEE 300 Digested PJ
Di t d RF200
Digested RFDigested IC-PJ 200 gDigested IC-PJ+PBE
100Digested IC-RF Digested IC-RF+PBEDigested IC RF+PBE
0 20 40 60 80 100 120 140 160 180 2000
Time (min)
0 20 40 60 80 100 120 140 160 180 200
Time (min)
Fig. 3. Effect of different samples on TEER of Caco-2 monolayers. The results are expressed as Ohm (resistance) � cm2 (surface area of the monolayer) vs. Time (min).
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evaluating the integrity of the Caco-2 cell barrier through the mea-surement of TEER (Fig. 3). Inflamed cells (positive control) showedthe highest decrease in the TEER value during 3 h. After 180 min,all the assessed monolayers showed a decrease in TEER values withrespect to the negative control (nonexposed cells).
Upon apical exposure of the inflamed Caco-2 monolayers to boththe commercial and the digested pineapple juice, increased perme-ability (reduced TEER) was observed compared with the effectcaused by pineapple juice enriched with PBE; however, this fruitjuice, as well as the digested red fruit juice, did not prevent a de-crease in TEER values below 500 X.cm2 in the inflamed Caco-2 cells.
3.4. Intracellular accumulation of reactive oxygen species (ROS)
After exposure of the Caco-2 cells to the fresh or the digestedfruit juices (pineapple and red fruits), enriched or not with PBE(0.5 g L�1) for 3 h, the intracellular accumulation of ROS was
measured by FACS flow cytometry. As shown in Fig. 4, the oxida-tive activity was increased in inflamed cells exposed to digestedcommercial red fruit juice (86.8 ± 1.3%) compared with the samejuice before digestion (77.4 ± 0.8%) and in inflamed cells exposedto the digested enriched red fruit juice (82.6 ± 1.6%) compared withthe nondigested enriched juice (55.8 ± 6%). In contrast, pineapplejuice (enriched and not with PBE) and the aqueous solution ofPBE (0.5 g L�1) decreased ROS production compared with thepositive control, although the results were rather average.
4. Discussion
Gastrointestinal conditions can significantly alter both thechemical structure and the function of phenolic compounds andalso their bioavailability (Bermúdez-Soto et al., 2007). In this study,the effects of the in vitro gastrointestinal digestion process on the
Fig. 4. FACS analysis of ROS generation (%) using DCFH-DA in Caco-2 cells exposed to different stimuli. Results are expressed as percentage of the positive control (Caco-2cells co-cultured with LPS-stimulated RAW 264.7 in the absence of polyphenolic extract) (100% ROS production). Negative control corresponded to Caco-2 cells incubatedwith DMEM alone. Values are mean ± SD of three independent experiments. Abbreviations: PBE, pine bark extract; IC, Caco-2 cells exposed to inflammatory stimuli; PJ,pineapple juice; RF, red fruits juice. Data with no common superscript letter are significantly different (P < 0.05).
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anti-inflammatory properties of pineapple and red fruit juices en-riched with a procyanidin-rich extract (D’Andrea, 2010) have beenaddressed in view of the latter’s previously established positive ef-fects on human health. The consumption of phenolics in the diet isassociated with many beneficial effects in the gut, such as a de-crease in inflammation (Evans et al., 2006; Sergent et al., 2010).However, in a study of different plant extracts, Kahkonen et al.(1999) concluded that a high concentration of phenolics does notnecessarily correspond to a high antioxidant activity, which couldprovide an anti-inflammatory effect. One of the inflammatory re-sponses is the oxidative burst that occurs in diverse cells (mono-cytes, neutrophils, eosinophils and macrophages) (Miguel, 2010).Enterocytes secrete the proinflammatory cytokine IL-8, whichstimulates the migration of neutrophils from intravascular to lumi-nal sites (Nanthakumar et al., 2000). In a previous study, afterin vitro gastrointestinal digestion of the same nonenriched fruitjuices used in the present study, a decrease in total phenolic con-tent was observed being gallic acid and taxifolin the major pheno-lics compounds found in all samples analysed before and afterdigestion (Frontela et al., 2011). Moreover, the chromatographicanalysis of samples showed a significant increase in monomericphenolics in nonenriched commercial juices and in PBE-enrichedjuices. When PBE was added to the fruit juices and then digested,a decrease in IL-8 secretion in the exposed Caco-2 cells was ob-served compared with cells exposed to the nonenriched digestedjuices. Polyphenols have been shown to have anti-inflammatoryproperties (Yoon and Beak, 2005). Thus, they could be used as acomplementary approach to conventional existing therapeutic aids
(i.e., nonsteroidal anti-inflammatory drugs) in the management ofinflammatory bowel disease. There is a need to find new and safecompounds able to contribute to the prevention, or even the treat-ment, of inflammatory diseases. Phenolic compounds could offeran alternative natural source for the treatment of these diseases(Sergent et al., 2010). According to Gauliard et al. (2008), phenoliccompounds of fruit juices can significantly alter cytokine and anti-oxidant production. In the present study, we observed a similartrend in the NO production of the cells. When the Caco-2 cells weresimultaneously incubated with digested fruit juices (with or with-out PBE) and the inflammatory stimuli, we observed a significantincrease in the production of NO compared with the same fruitjuice before digestion. The addition of the PBE did not cause signif-icant differences in NO secretion when the Caco-2 cells were ex-posed to fresh juices. However, it must be noted that after LPSstimuli, when the cells were exposed to digested samples, we ob-served similar values of NO production to those obtained in thenoninflammed cells. NO is an important modulator of the mucosalinflammatory response (Chen and Kitts, 2011). Our results suggestthat in vitro gastrointestinal digestion reduces the anti-inflamma-tory effect of fruit juices (pineapple and red fruits) enriched ornot with PBE. However, the observed values of NO production werelower than those observed by other authors studying the inhibitionof inflammatory mediators by polyphenolic plant extracts in hu-man intestinal Caco-2 cells (Romier-Crouzet et al., 2009). Duringthe inflammatory process, the intestinal mucosa can show barrierdysfunction, which can be used as an important biomarker of IBD(Nishitani and Mizuno, 2010). The effect of the inflammatory
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stimuli selected on the integrity of the Caco-2 cell barrier was eval-uated through the measurement of TEER during exposure to freshand digested fruit juices. TEER measurement provides a sensitivemethod for evaluating membrane-perturbing toxicants (Naraiet al., 1997). However, during PBE exposure, the overproductionof both the proinflammatory markers IL-8 and NO by the Caco-2cells after stimulation of RAW 264.7 with LPS in our experimentsdid not result in a decrease of TEER. Thus, components of PBE seemto protect the permeability of intestinal cells. As described above,the digested fruit juices conferred a lower protective effect in theCaco-2 monolayers than in the fresh samples. Injury of the cellsof the mucous membranes may provoke the production of ROSvia the permeation of macrophages and neutrophils. In general,ROS are thought to promote inflammation through these processes(Naik and Dixit, 2011). In the present study, extracts shown to beeffective in reducing the secretion of inflammatory mediators (IL-8 and NO) attenuating barrier dysfunction and reducing perme-ability (measured as TEER); however, extracts do not seem to exerta significant effect on ROS production from inflamed enterocytes.In view of the antioxidant characteristics of phenolic compounds,a reduction in the production of ROS by cells after exposure to di-gested red fruits juice + PBE (18.63 mg/100 mL) could be expected.However, the nonenriched red fruits juice (17.63 mg/100 mL)showed the lowest ROS production. In this regard, it should be con-sidered that Kahkonen et al. (1999), studying different plant ex-tracts, concluded that antioxidant activity does not necessarilycorrespond to a high amount of phenolics. In another study of plantextracts, Javanmardi et al. (2003) concluded that phenolics are notthe only factor responsible for antioxidant capacity. Moreover, su-gar or ascorbic acid present in the extract as probably cause ofinterference in antioxidant capacity measures should be consid-ered (Singleton and Rossi, 1965). A previous study (Frontela-Sasetaet al., 2011) assessing the antioxidant capacity of all samples usedin the present study showed an increase in antioxidant activity inenriched fruit juices after in vitro gastrointestinal digestion. In con-clusion, PBE added to fruit juices attenuated the inflammatory re-sponse in intestinal cells in an in vitro model. However, there was areduction in this activity following the gastrointestinal digestionprocess. Additional studies are needed to further establish theavailability of compounds derived from the digestion of PBE-enriched fruit juices in the gut.
Conflict of Interest
The author declare that there are no conflicts of interest.
Acknowledgements
To Fundación Séneca (Agencia Regional de Ciencia y Tecnología,Región de Murcia), for the postdoctoral fellowship of C. Frontela(09292/PD/08). To CONSOLIDER FUN-C-FOOD ‘Nuevos ingredien-tes funcionales para mejorar la salud’. To Hero Spain S.A, for pro-viding the fruit juices analysed. To Horphag Research Ltd., forproviding pine bark extract powder.
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