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Comparative Study
. 2013 Aug 5;8(8):e71762.
doi: 10.1371/journal.pone.0071762. Print 2013.

Ellagitannins From Rubus Berries for the Control of Gastric Inflammation: In Vitro and in Vivo Studies

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Free PMC article
Comparative Study

Ellagitannins From Rubus Berries for the Control of Gastric Inflammation: In Vitro and in Vivo Studies

Enrico Sangiovanni et al. PLoS One. .
Free PMC article

Abstract

Ellagitannins have shown anti-inflammatory and anti-Helicobacter pylori properties; however, their anti-inflammatory activity at gastric level was not previously investigated. The aim of this research was to evaluate the effects of ellagitannins from Rubus berries on gastric inflammation. Ellagitannin enriched extracts (ETs) were prepared from Rubus fruticosus L. (blackberry) and Rubus idaeus L. (raspberry). The anti-inflammatory activity was tested on gastric cell line AGS stimulated by TNF-α and IL-1β for evaluating the effect on NF-kB driven transcription, nuclear translocation and IL-8 secretion. In vivo the protective effect of ellagitannins was evaluated in a rat model of ethanol-induced gastric lesions. Rats were treated orally for ten days with 20 mg/kg/day of ETs, and ethanol was given one hour before the sacrifice. Gastric mucosa was isolated and used for the determination of IL-8 release, NF-kB nuclear translocation, Trolox equivalents, superoxide dismutase and catalase activities. In vitro, ETs inhibited TNF-α induced NF-kB driven transcription (IC₅₀: 0.67-1.73 µg/mL) and reduced TNF-α-induced NF-kB nuclear translocation (57%-67% at 2 µg/mL). ETs inhibited IL-8 secretion induced by TNF-α and IL-1β at low concentrations (IC₅₀ range of 0.7-4 µg/mL). Sanguiin H-6 and lambertianin C, the major ETs present in the extracts, were found to be responsible, at least in part, for the effect of the mixtures. ETs of blackberry and raspberry decreased Ulcer Index by 88% and 75% respectively and protected from the ethanol induced oxidative stress in rats. CINC-1 (the rat homologue of IL-8) secretion in the gastric mucosa was reduced in the animals receiving blackberry and raspberry ETs. The effect of ETs on CINC-1 was associated to a decrease of NF-κB nuclear translocation in ETs treated animals. The results of the present study report for the first time the preventing effect of ETs in gastric inflammation and support for their use in dietary regimens against peptic ulcer.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Experimental protocol for the in vivo study.
Before the experiment, the animals were randomly divided in 5 groups (6 rats in each group) and treated intragastrically (i.g.) by gavage. The dose of ET enriched extracts was calculated on the basis of a daily consumption of 125 g of fresh fruit by a human healthy adult of 70 kg. The day before the induction of gastric lesions, rats were placed in individual metabolic cages and deprived of food, with free access to tap water for 20 h. The last administration of ETs extracts, quercetin or vehicle was given 120 min before ethanol treatment.
Figure 2
Figure 2. Composition of blackberry (ETblack) and raspberry (ETrasp) ellagitannins.
Berries ETs were extracted with acetone/water 70:30, isolated by Sephadex LH 20 column chromatography, precipitated with hexane and quantified by UPLC-PDA-MS. Ellagitannins were detected at 260 nm.
Figure 3
Figure 3. Effect of ETs from blackberry and raspberry on NF-κB driven transcription induced by TNFα and IL-1β.
AGS cells at confluence were placed in a medium deprived of FCS, and stimulated with 10 ng/ml TNF-α (panel a and b) or (panel c and d) for 6 hrs. ETs were tested at 1–10 µg/ml; individual compounds at 0.5–10 µM. After 6 hours cells were harvested and luciferase assays were performed using Britelite™ Plus reagent (PerkinElmer Inc. Massachusetts, USA) according to manufacturer's instructions. Inhibition by 5 µM parthenolide, used as reference inhibitor, was 72% on TNF-α induced NF-κB driven transcription, and 71% on IL-1β induced NF-κB driven transcription. Results are the mean ± sd of three experiments in triplicate. * p<0.05, ** p<0.01, ***p<0.001.
Figure 4
Figure 4. Effect of ETs from blackberry and raspberry on NF-κB nuclear translocation induced by TNFα and IL-1β.
AGS cells at confluence were placed in a medium deprived of FCS, and stimulated with 10 ng/ml TNF-α (panel a) or IL-1β (panel b) for 1 hr. AGS cells were plated at the concentration of 1.5 × 106 cells/ml in 60-mm plates. NF-κB nuclear translocation was assessed using the NF-κB (p65) transcription factor assay kit (Cayman) followed by spectroscopy (signal read 450 nm, 0.1 s). Inhibition by 5 µM parthenolide used as reference inhibitor was 37% on TNF-α induced NF-κB nuclear translocation, and 40% on IL-1β induced nuclear translocation. Results are the mean ± sd of three experiments in triplicate. * p<0.05, ** p<0.01, ***p<0.001.
Figure 5
Figure 5. Effect of ETs from blackberry and raspberry on IL-8 release induced by TNFα and IL-1β.
AGS cells at confluence were placed in a medium deprived of FCS, and stimulated with 10 ng/ml TNF-α (panel a and b) or IL-1β (panel c and d) for 6 hrs. IL-8 was quantified by using Interleukin-8 High Sensitivity Human ELISA Set (Immunotools, Germany). The amount of IL-8 in the samples was detected by spectroscopy (signal read 450 nm, 0.1 s) by the use of biotinylated and streptavidin-HRP conjugate antibodies, evaluating 3,5,3,5′-tetramethylbenzidine (TMB) substrate reaction. Inhibition by 5 µM parthenolide, used as reference inhibitor, was 70% for both TNF-α and IL-1β induced IL-8 secretion. Results are the mean ± sd of three experiments in triplicate. * p<0.05, ***p<0.001.
Figure 6
Figure 6. Effect of ETs from blackberry and raspberry on IL-8 release induced by EtOH and H2O2.
Cells were grown in 24-well plates for 48 hrs (30000 cells/well) and then incubated for 12 hrs in the presence of 500 µM H2O2 (panels a and b), or for 24 hrs in the presence of 2% ethanol (panels c and d). IL-8 was quantified by using Interleukin-8 High Sensitivity Human ELISA Set (Immunotools, Germany). The amount of IL-8 in the samples was detected by spectroscopy (signal read 450 nm, 0.1 s) by the use of biotinylated and streptavidin-HRP conjugate antibodies, evaluating 3,5,3,5′-tetramethylbenzidine (TMB) substrate reaction. 10 µM quercetin, used as reference inhibitor, completely inhibited EtOH and H2O2 induced IL-8 secretion. Results are the mean ± sd of three experiments in triplicate. * p<0.05, ** p<0.01, ***p<0.001.
Figure 7
Figure 7. Protective effect of ETs from blackberry and raspberry against ethanol induced gastric injury.
Wistar rats were randomly divided in 5 groups (6 rats in each group). Controls were treated daily with vehicle (10% polyethylene glycol 400; PEG 400) for 10 days. Ethanol group received the vehicle (10% PEG 400) daily for 10 days, and then 1 ml of ethanol. ETblack group received 20 mg/kg of blackberry ETs dissolved in 10% PEG 400 for 10 days, and then 1 ml of ethanol. ETrasp group recived 20 mg/kg of raspberry ETs dissolved in 10% PEG 400 for 10 days, and then 1 ml of ethanol. Quercetin group (positive control) received 100 mg/kg of quercetin dissolved in 10% PEG 400 for 10 days, and then 1 ml of ethanol. The last administration of ETs, quercetin or vehicle was given 120 min before ethanol. Treatment was performed intragastrically by gavage. Gastric damage was assessed in a blind manner by a scoring system based on the number and severity of the lesions: 0, no lesions; 1, lesions 1–2 mm; 2, lesions 2–3 mm; 3, lesions >3 mm. Ulcer Index was calculated as the total number of lesions multiplied by their respective severity score. Results are the mean ± se, n = 6. ***p<0.001.
Figure 8
Figure 8. Effect of ETs from blackberry and raspberry on biochemical parameters ex vivo.
Rat gastric mucosa was homogenized in buffer A [10 mM TRIS-HCl (pH 8), 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 1% Triton X-100] using Tissue Lyser II (Qiagen) for 2 minutes at the highest frequency 30/s. The homogenates were centrifuged at 12,000 g for 10 min at 4° C. The supernatants were collected and stored at −80°C until use. The antioxidant capacity of the gastric mucosa homogenates was assessed by Oxygen Radical Absorbance Capacity (ORAC) assay and Trolox (4–160 µM) was used as the reference compound. The ORAC values were calculated as area under the curve and expressed as micromole of Trolox equivalent (TE) per gram of gastric mucosa sample ( µmol TE/g of gastric mucosa sample). SOD activity was determined from percent inhibition of the rate of WST-1-formazan formation, a coloured product that absorbs light at 450 nm. The amount of SOD in the samples was calculated by correlating the inhibition percentage of WST-1-formazan formation with the logarithm of the SOD units in a standard calibration curve. SOD activity is expressed as U/mg of proteins. CAT activity was determined by Catalase Assay Kit, a method based on the reaction of the enzyme with methanol in the presence of an optimal concentration of H2O2. The formaldehyde produced is measured colorimetrically at 540 nm with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole as the chromogen using a microplate reader. CAT activity is expressed as units (U) of CAT per mg of proteins. One unit of CAT is defined as the amount of enzyme that causes the formation of 1.0 nmol of formaldehyde per minute at 25°C. Results are the mean ± se, n = 6. * p<0.05, ** p<0.01.
Figure 9
Figure 9. Effect of ETs from blackberry and raspberry on CINC-1 (IL-8 homologue) ex vivo.
CINC-1 release was evaluated using GRO/CINC-1 (rat) EIA kit which uses a polyclonal antibody to rat GRO/CINC-1 labelled with the enzyme horseradish peroxidase. After a short incubation (10 minutes) the enzyme reaction was followed by spectroscopy (signal read 450 nm, 0.1 s). The concentration of rat GRO/CINC-1 in the samples was determined by interpolation with a GRO/CINC-1 standard curve. The results (mean ± se, n = 6) are expressed as pg of CINC-1 per mL of sample. * p<0.05, ** p<0.01.

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Grant support

The present research was partially funded by “Research Center for Characterization and Safe Use of Natural Compounds-G. Galli”. No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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