Abstract

Background and aims: Leukocyte infiltration, up-regulation of proinflammatory cytokines and severe oxidative stress caused by increased amounts of reactive oxygen species are characteristics of inflammatory bowel disease. The catechin (2R,3R)-2-(3,4,5-Trihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol-3-(3,4,5-trihydroxybenzoate), named epigallocatechin-3-gallate, EGCG, has been demonstrated to exert anti-inflammatory and antioxidative properties, reducing reactive oxygen species in the inflamed tissues. The aim of this study was to evaluate the therapeutic effects of EGCG in a murine model of colitis induced by oral administration of dextran sodium sulfate.

Methods: Mice received a daily oral administration of 6.9 mg/kg body weight EGCG or Piper nigrum (L.) alkaloid (2E,4E)-5-(1,3-benzodioxol-5-yl)-1-piperidin-1-ylpenta-2,4-dien-1-one, named piperine (2.9 mg/kg body weight) or the combination of the both — piperine was used in this combination to enhance the bioavailability of EGCG.

Results:In vivo data revealed the combination of EGCG and piperine to significantly reduce the loss of body weight, improve the clinical course and increase overall survival in comparison to untreated groups. The attenuated colitis was associated with less histological damages to the colon and reduction of tissue concentrations of malondialdehyde, the final product of lipid peroxidation. Neutrophils accumulation indicator myeloperoxidase was found to be reduced in colon tissue, while antioxidant enzymes like superoxide dismutase and glutathione peroxidase showed an increased activity. In vitro, the treatment with EGCG plus piperine enhanced the expression of SOD as well as GPO and also reduced the production of proinflammatory cytokines.

Conclusion: These data support the concept of anti-inflammatory properties of EGCG being generally beneficial in the DSS-model of colitis, an effect that may be mediated by its strong antioxidative potential.

Introduction

A combination of various etiological factors of inflammatory bowel disease (IBD) eventually leads to mucosal breakdown and ulcerations in active states of IBD. 1 The inflammation process includes a massive infiltration of polymorpho- and mononuclear phagocytic leukocytes producing large amounts of proinflammatory mediators, one of them being reactive oxygen species (ROS). Superoxide anion, hydrogen peroxide, and hypochlorous acid consist of highly reactive molecules as a result of the presence of unpaired electrons. 2,3 Due to dietary factors, the intestinal microflora, the large exposure to the outside environment and the interactions between cells of the immune system, the bowel is a major site of oxidant entry and production. 4

Recent studies have examined catechin (−)-epigallocatechin-3-gallate (EGCG),5,6 which represents up to 30% of the dry weight of green tea leaves. 7 Catechins have been shown to display antioxidant and anti-inflammatory properties in vitro. In animal models, 8,9 they have been shown to be more effective antioxidants compared to vitamins E and C. 10 This antioxidative and radical scavenging activity as shown in vitro and in vivo11,12 can be attributed to the presence of the phenolic hydroxyl groups on the B- and D-rings of the catechin molecule. 13 Recent studies applied EGCG intraperitoneally, using doses up to 50 mg/kg bodyweight. In the present study, we aimed at further investigating the antioxidant potential of EGCG combining it with a second dietary component, 1-piperoylpiperidin (piperine), an alkaloid from Piper nigrum (L.) (Piperaceae family). In order to correspond to applicable forms of intake even for humans, we administered EGCG intragastrically. After intragastric application, EGCG shows a low bioavailability and significant biotransformation leading to a reduction of the effective compound. 14 Extensive metabolism leads to the formation of glucuronidated, sulfated and methylated conjugates. 15 Uptake into enterocytes is thought to be dependant on membranous saturable monocarboxylate transporters (MCT), while cellular EGCG is being actively effluxed across the apical membrane by multidrug resistance-related protein (Mrp) 1 and 2 and to some extent by P-glycoprotein, a fact that may limit absorption of EGCG from the gut and its availability to the plasma. 16,17 The absolute bioavailability of EGCG in mice models is reported to be as much as 26.5%. 18

Therefore, it seemed necessary to increase the low bioavailability of EGCG after intragastrical application by coadministration of piperine effecting reduced small intestinal glucuronidation by 40 to 60% and increasing intestinal tissue and systemic concentrations of free EGCG. 19

Being continually exposed to ROS, the body has developed several endogenous antioxidant defense mechanisms: enzymes like superoxide dismutases (SOD), gluthathione peroxidases (GPO) and low molecular weight antioxidants such as vitamin C or vitamin E. 20 A deficiency in GPO genes for example leads to symptoms and pathology of IBD in mice. 21

However, the very imbalance between the increased production of ROS and the decreased detoxification by antioxidants initiates inflammatory cascades by upregulating different genes involved in the inflammatory response. 22,23 Furthermore, large amounts of ROS result in the damage of cellular proteins, 24 lipids, 25 cytoskeleton, 26 even DNA and ultimately, disruption of gastrointestinal barrier integrity with an increased gut permeability. 27,28 This appears to be a major pathogenic mechanism in IBD, 29 as chemiluminescence assays of samples from the colonic mucosa of patients with ulcerative colitis (UC) as well as animal models of UC show increased levels of ROS in active disease. 30,31

EGCG further interferes with other steps of the inflammatory process, e.g. it inhibits the secretion of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-8 through the attenuation of extracellular signal-regulated kinases (ERK) and NF-κB in human mast cell lines (HMC-1). 32 Furthermore, EGCG influences a number of signaling pathways, including activator protein 1 (AP-1) or the synthesis of eicosanoids and prostaglandin E2 (PGE2). 33 EGCG also shows anticarcinogenic effects in epidemiological and animal studies; administration of green tea, green tea extract or EGCG reduced tumor formation and growth and showed antiangiogenic and antimutagenic properties. 3436

The objective of the present study was to address whether EGCG exerts protection on DSS-induced chronic colitis in the mouse regarding its antioxidant and anti-inflammatory properties and whether the cotreatment with piperine increased the efficacy of EGCG.

Methods

Chemicals

EGCG (99.7% pure) was obtained from AXXORA Deutschland GmbH (Lörrach, Germany). Piperine (98% pure) was purchased from Fluka/Sigma-Aldrich (Steinheim, Germany). All other chemicals were of the highest grade commercially available.

Mice

All procedures using animals were reviewed and approved by the local animal subjects committee, University of Muenster (permit 8.87-50.10.36.08.128).

Female C57BL/6 mice (approx. 8–10 weeks of age, 18–20 g), were purchased from Charles River Laboratories (Sulzfeld, Germany) and were allowed to acclimate for 1 week prior to the start of experiments.

Five mice were housed per cage and maintained in air-conditioned quarters with a room temperature of 20 ± 2 °C, controlled relative humidity of 50 ± 10% and an alternating 12 hour light/dark-cycle. Mice were fed with standard laboratory diet and water or DSS-water and were allowed to eat and drink ad libitum.

Induction of chronic colitis

Murine chronic colitis was induced by weekly administration of 2 or 3% (w/v) solution of dextran sodium sulfate, DSS (MP Biomedicals, Illkirch, France) in turns with normal water for a period of approx. 60 days. This strategy leads to a reliable and reproducible chronic colitis. After this time, the animals were dispatched by an overdose of anesthesia. The colon was removed, opened and split longitudinally with the intestines of different mice being used for various analyses. In a separate series of experiments survival was examined.

Treatment

Treatment of different groups consisted of daily oral gavage of 200 μl of a 1.5 mM solution of EGCG and a 1 mM solution of piperine — resulting in 6.9 mg/kg bodyweight EGCG and 2.9 mg/kg bodyweight piperine for mice with a bodyweight of 18–20 g. The first group called “Control” received a gavage with only water, the second group called “Piperine” received only piperine, and the third group called “EGCG + Piperine” received the combination of EGCG plus piperine. Another group received only “EGCG”, resulting in no significant effects. For reference purposes, two groups treated with EGCG and EGCG plus piperine did not receive DSS, resulting in no colonic inflammation.

Histological colitis score

The colon was removed, opened and embedded in OCT compound (Tissue-Tek, Sukura Fine Tek Europe, Zoeterwoude, NL) and kept frozen at − 80 °C until further use. Sections of 5 mm thickness were stained with haematoxylin and eosin and then scored using a histological colitis score. 37

Each quarter of the colon was graded by three blinded investigators with a range from 0 to 3 as to amount of inflammation (acute and chronic), depth of inflammation and with a range from 0 to 4 as to the amount of crypt damage or regeneration. These changes were also quantified as to the percentage involvement within the quarters: (1) 1–25%; (2) 26–50%; (3) 51–75%; and (4) 76–100%. Each quarter was scored for each feature separately; points were multiplied by the factor of the involvement of the epithelium in a range from 0 to 12 for inflammation and for extent, and in a range from 0 to 16 for regeneration and for crypt damage. The average of the four quarters was representative for the whole colon.

Cell culture

Cell culture reagents were obtained from Cambrex and Lonza (Verviers, Belgium). Human HT-29 cells were maintained in RPMI medium, supplemented with 10 mM Hepes, 1 mM sodium pyruvate, 4.5 g/l glucose, 1.5 g/l sodium bicarbonate, 10% fetal bovine serum (FBS) and 1% penicillin (100U/ml)/streptomycin (100 μg/ml).

Cells were maintained in a water-saturated atmosphere of 95% O2 and 5% CO2 at 37 °C. For the experiments, cells were seeded into 6-well plates and used after 4 to 5 days of preculture. EGCG and piperine were dissolved in DMSO and added to the culture in different concentrations and combinations 6 h prior to the stimulation with bacterial lipopolysaccaride (LPS, 100 ng/ml). After 16 h of stimulation, the culture supernatants were analyzed by ELISA.

Enzyme-linked immunosorbent assay

The amount of IL-8 in HT-29 culture supernatants was assayed with enzyme immunoassay kits (BD Biosciences, Heidelberg, Germany) according to the manufacturer's instructions. The optical density at 450 nm was read using a microtiter plate photometer (Dynatech MR 4000, Dynatech, Ashford, UK).

MDA assay

Colorimetric reaction of thiobarbituric acid (TBA) with MDA, a secondary product of lipid peroxidation was caused by ROS. Analysis of lipid peroxidation damage in the mice colon was performed with the Thiobarbituric Acid Reaction described by Ohkawa. 38 The reaction mixture contained colon tissue, 50 μl 8.1% SDS, 200 μl 20% acetic acid solution of pH 3.5 and 1.3% TBA. It was heated at 95 °C for 60 min. After cooling to room temperature, 1 ml of the mixture of n-butanol and pyridine (15:1, v/v) was added and the mixture was shaken vigorously. After centrifugation at 4000 rpm for 10 min, the absorbance of the upper organic layer was measured at 532 nm (Genesys 10 Bio, Thermo Fisher Scientific Inc., Waltham, MA).

Immunohistochemistry

Colons were snap-frozen in Tissue-Tek (Sukura Fine Tek Europe, Zoeterwoude, NL). Cryostat sections of 5 μm were picked up on slides, air-dried, fixated for 15 min in pure acetone at − 20 °C and blocked with blocking solution (TNB, PerkinElmer, Zaventem, Belgium). A three-step staining was used, incubating the slides with solutions of rabbit anti-Gluthathione Peroxidase 1 (dilution 1:200)/rabbit anti-SOD1 (dilution 1:1000) (Abcam, Cambridge, UK), secondly biotinylated goat anti-rabbit Igs (1:100) (Becton Dickinson, Erembodegem, Belgium) and then Streptavidin Alexa Flour 546 conjugate (1:100) (Molecular Probes, Luiden, NL). In between each staining, the slides were washed three times for 2 min with PBS. Finally, the slides were counterstained with 4′,6-Diamidine-2′-phenylindoldihydrochloride (DAPI) (Fluka/Sigma-Aldrich, Steinheim, Germany). Positively stained cells were counted in at least 5–10 representative areas per section and they were expressed semiquantitatively.

MPO activity

The quantity of neutrophil infiltration in the inflamed colon caused by DSS-colitis can be characterized by MPO activity. 39 Tissue samples from distal colon were snap-frozen in liquid nitrogen, then homogenized and afterwards resuspended in 500 μL of 100 mM NaCl, 20 mM Tris (pH 7.5), and 0.1% Triton X-100 (Fluka/Sigma-Aldrich, Steinheim, Germany). After centrifugating the homogenate at 12.000 rpm for 20 min, the supernatant was removed for MPO assay. Ten microliters of the supernatant were added to 200 μl of 50 mM phosphate buffer (pH 6.0) containing 0.4 mg/mL o-phenylenediamine (Sigma Chemical Co., St. Louis, MO) and 10 μL of H2O2. After 20 min of incubation, the reaction was stopped with 50 μL of 0.4 mM H2SO4. Absorbance was measured at 490 nm (Genesys 10 Bio, Thermo Fisher Scientific Inc., Waltham, MA).

Immunoblotting

Cells of human HT-29 cells were seeded into plates and used after 6 to 8 days of preculture. EGCG and piperine were added in different concentrations and combinations 6 h prior to the stimulation with bacterial lipopolysaccaride (LPS, 100 ng/ml). After 16 h of stimulation, cells were removed from the plates, lysed in lysis buffer 20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X (all Sigma-Aldrich, Steinheim, Germany) and sonicated for 15 s. Total protein concentration was determined by Bradford assay (BioRad, Hercules, California, USA). 20 mg of total cellular protein were size separated on 4–20% tris glycin gel, blotted onto nitrocellulose membrane (Amersham Pharmacia Biotech, Illinois, USA), and blocked with blocking buffer (phosphate buffered saline (PBS) containing 10% (w/v) non-fat dry milk and 1% (w/v) bovine serum albumin (BSA)). Blots were incubated with polyclonal rabbit anti-SOD1 (dilution 1:2000) (Abcam, Cambridge, UK) overnight at 4 °C. Immunodetection was carried out using biotinylated goat anti-rabbit Igs (1:10,000) (Becton Dickinson, Erembodegem, Belgium) for 1 h at room temperature, followed by streptavidin horseradish peroxidase and enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Piscataway, USA).

Statistics

Results are expressed as mean ± standard deviation. 40 Student's t test or analysis of variance (ANOVA) along with Tukey's HSD test was used to compare results, with a P-value less than 0.05 considered to be significant. Survival of animals was evaluated by log rank test. Individual experiments were performed in quadruplicate (or triplicate for survival).

Results

DSS colitis is ameliorated by treatment with EGCG plus piperine

The average body weight of the mice was 20.3 ± 2.5 g at the beginning of the experiment. After starting DSS treatment mice receiving the combination of EGCG plus piperine exhibited a weight gain of 20% (relative weight 1.2 ± 0.1) (Fig. 1A), whereas DSS-treated control mice progressively lost weight. Particularly after increasing the DSS concentration to 3%, a significant difference was observed in the untreated control group (relative weight 0.9 ± 0.2). Group “Piperine” also exhibited a weight loss of −5% compared to the initial weight (relative weight 0.9 ± 0.1), whereas group “EGCG” showed a weight loss of – 1% (relative weight 0.9 ± 0.0). Body weight differences were statistically significant from day 45 until the end of the experiment. Post hoc tests (Tukey's test) showed group “EGCG + Piperine” to differ significantly from the three other groups treated with DSS. Reference groups of mice treated with only EGCG or EGCG plus piperine receiving no DSS exhibited a relative weight gain comparable to water-treated animals (data not shown). The data presented are representative of 4 individual experiments including 5 mice per group showing comparable results.

Reduced lethality in EGCG plus piperine treated mice

To further investigate the beneficial effect of EGCG, we conducted a survival analysis in our experiments. In one representative experiment, two mice of group “Control” and one mouse of group “Piperine” died in the second half of one experiment (Fig. 1B). The overall survival of mice from group “EGCG + Piperine” was not affected by the DSS-treatment. Pooling data from all experiments conducted with the same test conditions (n = 3 individual experiments including 5 mice in each group), evaluation of lethality with log rank tests showed the risk of an earlier death to be significantly higher in group “Control”.

Improved histological outcome of EGCG plus piperine treated mice

Histological analysis of colonic tissue of groups “Control” and “Piperine” revealed a strong epithelial disintegration with ulcerations, immune cell infiltrates, edema and wide areas of epithelial denudation (Fig. 2). Colonic tissue of group “EGCG” also showed epithelial disintegration and crypt shortening, though to a milder extent. In contrast, colonic tissue of mice from group “EGCG + Piperine” showed significantly less signs of epithelial damage with less ulcerations, mild inflammatory infiltrate and edema. To specify histological analysis, we applied a histology score of the colonic mucosa with respect to the categories severeness of inflammation, extent of inflammation, crypt damage and the percentile involvement. Groups “Control” and “Piperine” exhibited strong signs of inflammation with dense infiltrate of leucocytes in the mucosa and submucosa, loss of epithelium and crypt shortening in wide areas of the tissue leading to an assessment of 10.8 ± 3.2 points and 9.1 ± 2.0 points, group EGCG of 8.0 ± 2.0 points. By comparison, group “EGCG + Piperine” exhibited significantly less alterations of the microscopic architecture, with infrequent areas of mild inflammatory infiltrate and integrated epithelium resulting in a histological colitis score of 5.3 ± 1.3 (p ≤ 0.05 vs. treatment with water and vs. treatment with piperine). A score of 0 points was found for both reference groups treated with EGCG or EGCG plus piperine receiving no DSS, indicating no colonic inflammation. Concerning the bowel length as a macroscopic indication of the grade of inflammation, groups “Control”, “Piperine” or “EGCG” showed a reduced bowel length with comparison to group “EGCG ± Piperine”. A significant difference could only be found between groups “Control” and “EGCG ± Piperine” (Fig. 3).

Reduction of MPO activity upon treatment with EGCG plus piperine

The quantity of neutrophil infiltration in the inflamed colon caused by DSS-colitis can be characterized by MPO activity (Fig. 4A). MPO, a lysosomal peroxidase enzyme most abundantly present in neutrophil granulocytes, plays an important role in processing apoptotic material in the site of inflammation. Group “EGCG + Piperine” exhibited a significantly reduced MPO activity with comparison to groups “Control” or “Piperine”. MPO activity was also elevated in group “EGCG”.

Protective role of EGCG plus piperine on lipid peroxidation

To characterize the damaging process caused by ROS in the course of chronic colitis, we determined MDA concentrations in the colonic tissue (Fig. 4B). MDA, a secondary product of lipid peroxidation, originates from a reaction of reactive oxygen species with fatty acids of cell membranes and is toxic to other cell lipids and proteins and to the DNA. In groups “Control”, “Piperine” and “EGCG” the concentration of MDA in the colonic tissue was significantly higher than in the group “EGCG + Piperine”.

Further immunohistochemical analysis of colonic tissue revealed a strongly decreased expression of protecting enzymatic antioxidants GPO and SOD in control groups (Fig. 5). By comparison, coadministration of EGCG plus piperine could efficiently reverse the reduction of GPO/SOD expression in vivo.

EGCG-dependent SOD expression in epithelial cells was further examined by immunoblot in HT-29 line cells with different combinations and concentrations of EGCG and piperine. As shown in Fig. 6, a treatment with a combination of 15 mM EGCG plus 10 mM piperine or particularly a combination of 1,5 mM EGCG plus 1 mM piperine resulted in a significantly raised expression of SOD after stimulation with LPS whereas expression could not be raised by a treatment with 15 mM EGCG or 10 mM piperine alone.

EGCG plus piperine effects IL-8 secretion in HT-29 cells

To investigate the effect of EGCG plus piperine on inflammatory cytokine production, HT29 cells were treated with piperine and EGCG in vitro (Fig. 7). After 6 h of preincubation, cells were stimulated with LPS (100 ng/ml). Indeed, pretreatment with EGCG alone induced a non-significant (p = 0.87) reduction of IL-8 in HT-29 cells (253.1 ± 102.3 pg/ml). Pretreatment with piperine alone also did not significantly inhibit IL-8 (218.5 ± 82.1 pg/ml) production. However, the treatment with a combination of EGCG (15 mM) plus piperine (10 mM) led to a significant and exponential reduction of IL-8 in HT-29 cells (3.1 ± 4.3 pg/ml, p = 0.001).

Treatment with significantly lower doses of the combination of EGCG (1,5 mM) plus piperine (1 mM) also led to a significantly (p < 0.001) reduced production of IL-8 in HT-29 cells (33.2 ± 36.4 pg/ml, p = 0.006).

Discussion

The aim of this study was to evaluate the effects of EGCG with respect to inflammatory bowel disease, especially focussing on ROS as one of the assumed effector mechanisms of inflammation. In the colonic mucosa of patients with UC increased quantities of ROS are found to have supporting and noxious effects in the process of inflammation. 4144 Eventually, ROS lead to the severe situation of oxidative stress, which is potentially one of the fundamental reasons for tissue damage and ulcerations and consequently needs to be a target of therapeutic strategies. 45

EGCG, a catechin of the green tea plant Camellia sinensis (L.) Kuntze, shows strong antioxidative potential10 and was found to interfere with several steps of the inflammatory cascade in vitro and in vivo.46,9 In our experiments we coadministered the black pepper alkaloid piperine with EGCG, augmenting the bioavailability of EGCG. Indeed, cell culture experiments with HT-29 human colon cancer cells showed piperine to significantly lower intracellular levels of EGCG-3"-glucuronide, a fact that may result in higher amounts of free EGCG. 19

In comparison with other methods applying EGCG intraperitoneally, the intragastral application of EGCG in combination with piperine shows comparable results with lower concentrations being used and being closer to a real application in humans. Although the optimal dose needs to be determined e.g. as pharmacokinetic data are currently not available our data show that appropriate amounts of ECGC can be delivered by regular food intake as e.g. 3 cups of tea contain about 500 mg of green tea.

Furthermore, this study aimed at elucidating the mechanisms through which EGCG reduces colonic inflammation. Physiologically, increased amounts of enzymatic antioxidants are produced in the course of benign oxidative stress. 47,48 In contrast, lower antioxidant defense in the organ tissues is the result of severe enduring oxidative stress. 49 Our findings of lower enzymatic antioxidant levels in the colon of control groups indicate the DSS-model to induce severe oxidative stress. The ability of practically all ROS to inactivate one or several antioxidant enzymes is the probable reason for this imbalance in the system of antioxidants;50 resected mucosa and mucosal biopsies from IBD patients with active disease support these findings. 51

In the course of IBD inflammation, the failure to eliminate ROS leads to lipid peroxidation, affecting the cell membrane and modifying its permeability and selectivity and the activity of transmembrane transporters, receptors and enzymes. 52 In the present study we found the final product of lipid peroxidation, MDA, to be elevated in colonic tissue of untreated controls. These data are consistent with previous findings of increased levels of MDA in mucosal biopsies from IBD patients. 53

High levels of peroxides attract neutrophils chemotactically and have an influence on cytokine production. 54 This attraction might be one of the reasons for neutrophils to accumulate in the colon, which was indicated by the elevated MPO activity in the groups treated only with water or piperine. The reduction of ROS and peroxides might be partly responsible for the lowered numbers of neutrophils in groups treated with EGCG plus piperine.

Along with the increased antioxidant capacity in the colonic tissue of mice treated with EGCG plus piperine we found the antioxidant enzymes SOD and GPO to be increased in immunohistochemical analysis. These findings could be confirmed by in vitro data with HT-29 cells constitutively expressing SOD. Treatment with EGCG plus piperine could raise their expression and consecutively improve the antioxidant capacity in cell culture.

However, in this context the question remains whether the effects on our molecular endpoints like activity of antioxidant enzymes are downstream events of the modulation of ROS balance in colonic cells and tissues or caused by direct interaction of EGCG with various molecular targets like SOD, independent of its antioxidant properties. 55

Effects diminishing the inflammatory process could also be found in our experiments with epithelial cells, where the combined treatment with EGCG plus piperine significantly and strongly reduced the secretion of IL-8. Even treating the cells with only EGCG led to a modest reduction of IL-8, thus confirming earlier data. 32 From other studies it is known that treatment with only EGCG inhibits inflammatory transduction pathways like NF-κB, AP 1, IL-6 and TNF-α in vitro.5658

Again, the combination of EGCG plus piperine leads to a relevant overall reduction of IL-8, while EGCG alone did not show comparable effects. These results highly correlate with our in vivo data, where significant clinical effects were only achieved in groups treated with EGCG plus piperine. This may be due to the potential of piperine to reduce intracellular glucuronidated EGCG. The higher amount of free EGCG might potentiate its ability to reduce cellular inflammatory reactions.19

Even if the exact anti-inflammatory properties are not completely understood down to the molecular level, EGCG can reduce colitis especially when administered together with piperine. It is important to stress that in the present study none of the controls treated with piperine alone displayed significant anti-inflammatory effects, although piperine has already shown a certain therapeutic benefit. 59 Therefore, it cannot completely be excluded that piperine is the active drug being potentiated by EGCG, but all our experiments indicate that the main anti-inflammatory effect can be attributed to EGCG. In addition, our results were confirmed by recent studies, 5,6 employing higher concentrations of EGCG (10 mg/kg, 50 mg/kg body weight i.p. respectively), but without concomitant piperine.

As the consumption of green tea as well as of black pepper (world production 277.000 tons in 2007) is quite high, a coexposure to EGCG and piperine will presumably occur in many diets. This might be a reason for a variety of beneficial health effects and indicate the need for further studies to define the therapeutical potential of EGCG in human IBD. 60,61

Conference presentation: Poster presentation: Deutsche Gesellschaft für Verdauungs- und Stoffwechselkrankheiten (DGVS), September 2007, Bochum, Germany and United European Gastroenterology Week (UEGW), October 2008, Vienna, Austria Talk: Deutsche Arbeitsgemeinschaft Chronisch entzündliche Darmerkrankungen (DACED), June 2008, Mainz, Germany and Gesellschaft für Gastroenterologie in Westfalen e.V. (GGW), November 2008, Herne, Germany.

Acknowledgment

This manuscript was supported by a grant of the Deutsche Morbus Crohn/Colitis ulcerosa Vereinigung (DCCV). Financial Disclosure: There exist no financial arrangements related to the research or assistance with manuscript preparation. We thank S. Dufentester and E. Weber for expert technical support. Thanks to Stefan Brückner for medical informatics support. Specific contribution of the authors: Markus Brückner: design of the study, acquisition, analysis and interpretation of data, drafting the article, final approval of the version to be submitted, Sabine Westphal: conception and design of the study, acquisition, analysis and interpretation of data, critical revision of the article for important intellectual content, final approval of the version to be submitted, Wolfram Domschke: critical revision of the article for important intellectual content, final approval of the version to be submitted, Torsten Kucharzik: conception and design of the study, analysis and interpretation of data, final approval of the version to be submitted and Andreas Lügering: conception and design of the study, analysis and interpretation of data, critical revision of the article for important intellectual content, final approval of the version to be submitted.

  • EGCG

    Epigallocatechin-3-gallate

  • DSS

    dextran sodium sulfate

  • ROS

    reactive oxygen species

  • SOD

    superoxide dismutase

  • CAT

    catalase

  • GPO

    gluthathione peroxidase

  • ERK

    extracellular signal-regulated kinase

  • MPO

    myeloperoxidase

  • MDA

    malondialdehyde

  • TBA

    thiobarbituric acid

References

1
Andres
P.G.
Friedman
L.S.
Epidemiology and the natural course of inflammatory bowel disease
Gastroenterol Clin North Am
 
28
1999
255
281
vii
2
Kruidenier
L.
Verspaget
H.W.
Review article: oxidative stress as a pathogenic factor in inflammatory bowel disease—radicals or ridiculous?
Aliment Pharmacol Ther
 
16
2002
1997
2015
3
Rezaie
A.
Parker
R.D.
Abdollahi
M.
Oxidative stress and pathogenesis of inflammatory bowel disease: an epiphenomenon or the cause?
Dig Dis Sci
 
52
2007
2015
2021
4
Kaplan
M.
Mutlu
E.A.
Benson
M.
Fields
J.Z.
Banan
A.
Keshavarzian
A.
Use of herbal preparations in the treatment of oxidant-mediated inflammatory disorders
Complement Ther Med
 
15
2007
207
216
5
Abboud
P.A.
Hake
P.W.
Burroughs
T.J.
Odoms
K.
O'Connor
M.
Mangeshkar
P.
Wong
H.R.
Zingarelli
B.
Therapeutic effect of epigallocatechin-3-gallate in a mouse model of colitis
Eur J Pharmacol
 
579
2008
411
417
6
Mazzon
E.
Muia
C.
Paola
R.D.
Genovese
T.
Menegazzi
M.
De Sarro
A.
Suzuki
H.
Cuzzocrea
S.
Green tea polyphenol extract attenuates colon injury induced by experimental colitis
Free Radic Res
 
39
2005
1017
1025
7
Graham
H.N.
Green tea composition, consumption, and polyphenol chemistry
Prev Med
 
21
1992
334
350
8
Ahn
S.C.
Kim
G.Y.
Kim
J.H.
Baik
S.W.
Han
M.K.
Lee
H.J.
Moon
D.O.
Lee
C.M.
Kang
J.H.
Kim
B.H.
Oh
Y.H.
Park
Y.M.
Epigallocatechin-3-gallate, constituent of green tea, suppresses the LPS-induced phenotypic and functional maturation of murine dendritic cells through inhibition of mitogen-activated protein kinases and NF-kappaB
Biochem Biophys Res Commun
 
313
2004
148
155
9
Varilek
G.W.
Yang
F.
Lee
E.Y.
deVilliers
W.J.
Zhong
J.
Oz
H.S.
Westberry
K.F.
McClain
C.J.
Green tea polyphenol extract attenuates inflammation in interleukin-2-deficient mice, a model of autoimmunity
J Nutr
 
131
2001
2034
2039
10
Rice-Evans
C.A.
Miller
N.J.
Bolwell
P.G.
Bramley
P.M.
Pridham
J.B.
The relative antioxidant activities of plant-derived polyphenolic flavonoids
Free Radic Res
 
22
1995
375
383
11
Higdon
J.V.
Frei
B.
Tea catechins and polyphenols: health effects, metabolism, and antioxidant functions
Crit Rev Food Sci Nutr
 
43
2003
89
143
12
Schwedhelm
E.
Maas
R.
Troost
R.
Boger
R.H.
Clinical pharmacokinetics of antioxidants and their impact on systemic oxidative stress
Clin Pharmacokinet
 
42
2003
437
459
13
Nanjo
F.
Goto
K.
Seto
R.
Suzuki
M.
Sakai
M.
Hara
Y.
Scavenging effects of tea catechins and their derivatives on 1,1-diphenyl-2-picrylhydrazyl radical
Free Radic Biol Med
 
21
1996
895
902
14
Feng
W.Y.
Metabolism of green tea catechins: an overview
Curr Drug Metab
 
7
2006
755
809
15
Vaidyanathan
J.B.
Walle
T.
Glucuronidation and sulfation of the tea flavonoid (−)-epicatechin by the human and rat enzymes
Drug Metab Dispos
 
30
2002
897
903
16
Vaidyanathan
J.B.
Walle
T.
Cellular uptake and efflux of the tea flavonoid (−)epicatechin-3-gallate in the human intestinal cell line caco-2
J Pharmacol Exp Ther
 
307
2003
745
752
17
Hong
J.
Lambert
J.D.
Lee
S.H.
Sinko
P.J.
Yang
C.S.
Involvement of multidrug resistance-associated proteins in regulating cellular levels of (−)-epigallocatechin-3-gallate and its methyl metabolites
Biochem Biophys Res Commun
 
310
2003
222
227
18
Lambert
J.D.
Lee
M.J.
Lu
H.
Meng
X.
Hong
J.J.
Seril
D.N.
Sturgill
M.G.
Yang
C.S
Epigallocatechin-3-gallate is absorbed but extensively glucuronidated following oral administration to mice
J Nutr
 
133
2003
4172
4177
19
Lambert
J.D.
Hong
J.
Kim
D.H.
Mishin
V.M.
Yang
C.S.
Piperine enhances the bioavailability of the tea polyphenol (−)-epigallocatechin-3-gallate in mice
J Nutr
 
134
2004
1948
1952
20
Sies
H.
Strategies of antioxidant defense
Eur J Biochem
 
215
1993
213
219
21
Esworthy
R.S.
Aranda
R.
Martin
M.G.
Doroshow
J.H.
Binder
S.W.
Chu
F.F.
Mice with combined disruption of Gpx1 and Gpx2 genes have colitis
Am J Physiol Gastrointest Liver Physiol
 
281
2001
G848
G855
22
Grisham
M.B.
MacDermott
R.P.
Deitch
E.A.
Oxidant defense mechanisms in the human colon
Inflammation
 
14
1990
669
680
23
Barnes
P.J.
Karin
M.
Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases
N Engl J Med
 
336
1997
1066
1071
24
Rao
R.
Baker
R.D.
Baker
S.S.
Inhibition of oxidant-induced barrier disruption and protein tyrosine phosphorylation in caco-2 cell monolayers by epidermal growth factor
Biochem Pharmacol
 
57
1999
685
695
25
Gutteridge
J.M.
Lipid peroxidation and antioxidants as biomarkers of tissue damage
Clin Chem
 
41
1995
1819
1828
26
Banan
A.
Choudhary
S.
Zhang
Y.
Fields
J.Z.
Keshavarzian
A.
Oxidant-induced intestinal barrier disruption and its prevention by growth factors in a human colonic cell line: role of the microtubule cytoskeleton
Free Radic Biol Med
 
28
2000
727
738
27
Yakes
F.M.
Van Houten
B.
Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress
Proc Natl Acad Sci USA
 
94
1997
514
519
28
Rao
R.K.
Baker
R.D.
Baker
S.S.
Gupta
A.
Holycross
M.
Oxidant-induced disruption of intestinal epithelial barrier function: role of protein tyrosine phosphorylation
Am J Physiol
 
273
1997
G812
G823
29
Pravda
J.
Radical induction theory of ulcerative colitis
World J Gastroenterol
 
11
2005
2371
2384
30
Simmonds
N.J.
Allen
R.E.
Stevens
T.R.
Van Someren
R.N.
Blake
D.R.
Rampton
D.S.
Chemiluminescence assay of mucosal reactive oxygen metabolites in inflammatory bowel disease
Gastroenterology
 
103
1992
186
196
31
Grisham
M.B.
Volkmer
C.
Tso
P.
Yamada
T.
Metabolism of trinitrobenzene sulfonic acid by the rat colon produces reactive oxygen species
Gastroenterology
 
101
1991
540
547
32
Shin
H.Y.
Kim
S.H.
Jeong
H.J.
Kim
S.Y.
Shin
T.Y.
Um
J.Y.
Hong
S.H.
Kim
H.M.
Epigallocatechin-3-gallate inhibits secretion of TNF-alpha, IL-6 and IL-8 through the attenuation of ERK and NF-kappaB in HMC-1 cells
Int Arch Allergy Immunol
 
142
2007
335
344
33
Porath
D.
Riegger
C.
Drewe
J.
Schwager
J.
Epigallocatechin-3-gallate impairs chemokine production in human colon epithelial cell lines
J Pharmacol Exp Ther
 
315
2005
1172
1180
34
Fujiki
H.
Suganuma
M.
Okabe
S.
Sueoka
N.
Komori
A.
Sueoka
E.
Kozu
T.
Tada
Y.
Suga
K.
Imai
K.
Nakachi
K.
Cancer inhibition by green tea
Mutat Res
 
402
1998
307
310
35
Crespy
V.
Williamson
G.
A review of the health effects of green tea catechins in in vivo animal models
J Nutr
 
134
2004
3431S
3440S
36
Wang
Z.Y.
Cheng
S.J.
Zhou
Z.C.
Athar
M.
Khan
W.A.
Bickers
D.R.
Mukhtar
H.
Antimutagenic activity of green tea polyphenols
Mutat Res
 
223
1989
273
285
37
Dieleman
L.A.
Palmen
M.J.
Akol
H.
Bloemena
E.
Pena
A.S.
Meuwissen
S.G.
Van Rees
E.P.
Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines
Clin Exp Immunol
 
114
1998
385
391
38
Ohkawa
H.
Ohishi
N.
Yagi
K.
Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction
Anal Biochem
 
95
1979
351
358
39
Mullane
K.M.
Kraemer
R.
Smith
B.
Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium
J Pharmacol Methods
 
14
1985
157
167
40
Wiseman
S.A.
Balentine
D.A.
Frei
B.
Antioxidants in tea
Crit Rev Food Sci Nutr
 
37
1997
705
718
41
Keshavarzian
A.
Sedghi
S.
Kanofsky
J.
List
T.
Robinson
C.
Ibrahim
C.
Winship
D.
Excessive production of reactive oxygen metabolites by inflamed colon: analysis by chemiluminescence probe
Gastroenterology
 
103
1992
177
185
42
Monte
M.
Davel
L.E.
Sacerdote de Lustig
E.
Hydrogen peroxide is involved in lymphocyte activation mechanisms to induce angiogenesis
Eur J Cancer
 
33
1997
676
682
43
Kruidenier
L.
Kuiper
I.
Lamers
C.B.
Verspaget
H.W.
Intestinal oxidative damage in inflammatory bowel disease: semi-quantification, localization, and association with mucosal antioxidants
J Pathol
 
201
2003
28
36
44
Cao
W.
Vrees
M.D.
Kirber
M.T.
Fiocchi
C.
Pricolo
V.E.
Hydrogen peroxide contributes to motor dysfunction in ulcerative colitis
Am J Physiol Gastrointest Liver Physiol
 
286
2004
G833
G843
45
Riedle
B.
Kerjaschki
D.
Reactive oxygen species cause direct damage of Engelbreth–Holm–Swarm matrix
Am J Pathol
 
151
1997
215
231
46
Lin
Y.L.
Lin
J.K.
(−)-Epigallocatechin-3-gallate blocks the induction of nitric oxide synthase by down-regulating lipopolysaccharide-induced activity of transcription factor nuclear factor-kappaB
Mol Pharmacol
 
52
1997
465
472
47
Marklund
S.L.
Extracellular superoxide dismutase and other superoxide dismutase isoenzymes in tissues from nine mammalian species
Biochem J
 
222
1984
649
655
48
Halliwell
B.
Whiteman
M.
Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?
Br J Pharmacol
 
142
2004
231
255
49
MacMillan-Crow
L.A.
Crow
J.P.
Kerby
J.D.
Beckman
J.S.
Thompson
J.A.
Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts
Proc Natl Acad Sci USA
 
93
1996
11853
11858
50
Pigeolet
E.
Corbisier
P.
Houbion
A.
Lambert
D.
Michiels
C.
Raes
M.
Zachary
M.D.
Remacle
J.
Glutathione peroxidase, superoxide dismutase, and catalase inactivation by peroxides and oxygen derived free radicals
Mech Ageing Dev
 
51
1990
283
297
51
Mulder
T.P.
Verspaget
H.W.
Janssens
A.R.
de Bruin
P.A.
Pena
A.S.
Lamers
C.B.
Decrease in two intestinal copper/zinc containing proteins with antioxidant function in inflammatory bowel disease
Gut
 
32
1991
1146
1150
52
Ohyashiki
T.
Ohtsuka
T.
Mohri
T.
A change in the lipid fluidity of the porcine intestinal brush-border membranes by lipid peroxidation. Studies using pyrene and fluorescent stearic acid derivatives
Biochim Biophys Acta
 
861
1986
311
318
53
Chiarpotto
E.
Scavazza
A.
Leonarduzzi
G.
Camandola
S.
Biasi
F.
Teggia
P.M.
Garavoglia
M.
Robecchi
A.
Roncari
A.
Poli
G.
Oxidative damage and transforming growth factor beta 1 expression in pretumoral and tumoral lesions of human intestine
Free Radic Biol Med
 
22
1997
889
894
54
Curzio
M.
Esterbauer
H.
Poli
G.
Biasi
F.
Cecchini
G.
Di Mauro
C.
Cappello
N.
Dianzani
M.U.
Possible role of aldehydic lipid peroxidation products as chemoattractants
Int J Tissue React
 
9
1987
295
306
55
Na
H.K.
Kim
E.H.
Jung
J.H.
Lee
H.H.
Hyun
J.W.
Surh
Y.J.
(−)-epigallocatechin gallate induces Nrf2-mediated antioxidant enzyme expression via activation of PI3K and ERK in human mammary epithelial cells
Arch Biochem Biophys
 
476
2008
171
177
56
Na
H.K.
Surh
Y.J.
Intracellular signaling network as a prime chemopreventive target of (−)-epigallocatechin gallate
Mol Nutr Food Res
 
50
2006
152
159
57
Ahmed
S.
Marotte
H.
Kwan
K.
Ruth
J.H.
Campbell
P.L.
Rabquer
B.J.
Pakozdi
A.
Koch
A.E.
Epigallocatechin-3-gallate inhibits IL-6 synthesis and suppresses transsignaling by enhancing soluble gp130 production
Proc Natl Acad Sci USA
 
105
2008
14692
14697
58
Luo
D.
Min
W.
Lin
X.F.
Wu
D.
Xu
Y.
Miao
X.
Effect of epigallocatechingallate on ultraviolet B-induced photo-damage in keratinocyte cell line
Am J Chin Med
 
34
2006
911
922
59
Bae
G.S.
Kim
M.S.
Jung
W.S.
Seo
S.W.
Yun
S.W.
Kim
S.G.
Park
R.K.
Kim
E.C.
Song
H.J.
Park
S.J.
Inhibition of lipopolysaccharide-induced inflammatory responses by piperine
Eur J Pharmacol
 
642
2010
154
162
60
Sano
J.
Inami
S.
Seimiya
K.
Ohba
T.
Sakai
S.
Takano
T.
Mizuno
K.
Effects of green tea intake on the development of coronary artery disease
Circ J
 
68
2004
665
670
61
Su
L.J.
Arab
L.
Tea consumption and the reduced risk of colon cancer — results from a national prospective cohort study
Public Health Nutr
 
5
2002
419
425

Figures

Figure 1

A. Changes in the relative weight over 60-day period of alternating 2% (w/v) DSS treatment (arrows). Daily administration of H2O, piperine and the combination of EGCG & piperine started 1 week prior to the first DSS treatment. Asterisks/pounds depict significant differences in body weight (p < 0.05) between groups “Control”/“Piperine” and “EGCG + Piperine”. Data are means ± SD; n = 5 per group at the beginning of a single experiment. This experiment was repeated 4 times with comparable results. Overall weight loss between different treatments was significantly different calculated by including all experiments performed under the same settings (p = 0.001, ANOVA). B. Kaplan–Meier estimate of survival of groups “Control”, “Piperine” and “EGCG + Piperine” after 2% (w/v) DSS-administration (arrows). Regarding all comparable trials with the same test conditions, evaluation with a log rank test showed a significantly increased risk of earlier death in the control group receiving water only (p < 0.05).

Figure 1

A. Changes in the relative weight over 60-day period of alternating 2% (w/v) DSS treatment (arrows). Daily administration of H2O, piperine and the combination of EGCG & piperine started 1 week prior to the first DSS treatment. Asterisks/pounds depict significant differences in body weight (p < 0.05) between groups “Control”/“Piperine” and “EGCG + Piperine”. Data are means ± SD; n = 5 per group at the beginning of a single experiment. This experiment was repeated 4 times with comparable results. Overall weight loss between different treatments was significantly different calculated by including all experiments performed under the same settings (p = 0.001, ANOVA). B. Kaplan–Meier estimate of survival of groups “Control”, “Piperine” and “EGCG + Piperine” after 2% (w/v) DSS-administration (arrows). Regarding all comparable trials with the same test conditions, evaluation with a log rank test showed a significantly increased risk of earlier death in the control group receiving water only (p < 0.05).

Figure 2

Histological analysis of colonic tissue of mice subjected to the induction of chronic DSS colitis. Mice receiving only H2O in group “Control” exhibited a severely destroyed crypt architecture (A). Similary, mice of group “Piperine” showed a dense immune cell infiltrate, transmural inflammation and ulcerations (B). In comparison, mice treated with “EGCG + Piperine” showed only very mild changes with reduced epithelial denudation, and less ulcerations (C; D: uninflamed control). Representative examples of each group are shown.

Figure 2

Histological analysis of colonic tissue of mice subjected to the induction of chronic DSS colitis. Mice receiving only H2O in group “Control” exhibited a severely destroyed crypt architecture (A). Similary, mice of group “Piperine” showed a dense immune cell infiltrate, transmural inflammation and ulcerations (B). In comparison, mice treated with “EGCG + Piperine” showed only very mild changes with reduced epithelial denudation, and less ulcerations (C; D: uninflamed control). Representative examples of each group are shown.

Figure 3

Effects of different treatment of groups “Control”, “Piperine”, “EGCG” and “EGCG ± Piperine” on changes in bowel length induced by DSS. Mice of the groups “Control” (5.4 ± 0.5 cm), “Piperine” (5.9 ± 0.4 cm) or “EGCG” (5.9 ± 0.5 cm) showed a reduced bowel length with comparison to group “EGCG ± Piperine” (6.3 ± 0.2 cm). A significant difference could only be found between groups “Control” and “EGCG ± Piperine” (* p ≤ 0.05 vs. treatment with water, one way ANOVA followed by Tukey post-hoc test). Healthy reference mice without DSS treated with EGCG or EGCG plus piperine exhibited a bowel length of 6.7 ± 0.2 cm and 6.8 ± 0.3 cm.

Figure 3

Effects of different treatment of groups “Control”, “Piperine”, “EGCG” and “EGCG ± Piperine” on changes in bowel length induced by DSS. Mice of the groups “Control” (5.4 ± 0.5 cm), “Piperine” (5.9 ± 0.4 cm) or “EGCG” (5.9 ± 0.5 cm) showed a reduced bowel length with comparison to group “EGCG ± Piperine” (6.3 ± 0.2 cm). A significant difference could only be found between groups “Control” and “EGCG ± Piperine” (* p ≤ 0.05 vs. treatment with water, one way ANOVA followed by Tukey post-hoc test). Healthy reference mice without DSS treated with EGCG or EGCG plus piperine exhibited a bowel length of 6.7 ± 0.2 cm and 6.8 ± 0.3 cm.

Figure 4

A. MPO activity in colonic tissue. MPO activity determined in colon samples showed a significantly higher MPO activity (* p ≤ 0.05 vs. treatment with water, # p ≤ 0.05 vs. treatment with piperine) in mice of groups “Control” (1027.4 ± 464.4 mU MPO/mg bowel tissue) and “Piperine” (1238.5 ± 259.5 mU MPO/mg bowel tissue) in comparison with animals of groups “EGCG ± Piperine” (441.2 ± 205.2 mU MPO/mg bowel tissue). Overall MPO activity between the different treatments was significantly different (p ≤ 0.05, one way ANOVA). MPO activity was also elevated in group “EGCG” (943.4 ± 448.8 mU MPO/mg bowel tissue). B. Effects of treatment of group “EGCG + Piperine” on the concentration of MDA in bowel tissue. In groups “Control” (9.4 ± 0.5 mM MDA/g), “Piperine” (9.2 ± 0.4 mM MDA/g) and “EGCG” (8.7 ± 1.3 mM MDA/g) the concentration of MDA in the colonic tissue was significantly higher than in the group “EGCG ± Piperine” (4.8 ± 1 mM MDA/g) (* p ≤ 0.05 vs. treatment with water, # p ≤ 0.05 vs. treatment with piperine). Concentration of MDA between different treatments was significantly different (p = 0.001, one way ANOVA).

Figure 4

A. MPO activity in colonic tissue. MPO activity determined in colon samples showed a significantly higher MPO activity (* p ≤ 0.05 vs. treatment with water, # p ≤ 0.05 vs. treatment with piperine) in mice of groups “Control” (1027.4 ± 464.4 mU MPO/mg bowel tissue) and “Piperine” (1238.5 ± 259.5 mU MPO/mg bowel tissue) in comparison with animals of groups “EGCG ± Piperine” (441.2 ± 205.2 mU MPO/mg bowel tissue). Overall MPO activity between the different treatments was significantly different (p ≤ 0.05, one way ANOVA). MPO activity was also elevated in group “EGCG” (943.4 ± 448.8 mU MPO/mg bowel tissue). B. Effects of treatment of group “EGCG + Piperine” on the concentration of MDA in bowel tissue. In groups “Control” (9.4 ± 0.5 mM MDA/g), “Piperine” (9.2 ± 0.4 mM MDA/g) and “EGCG” (8.7 ± 1.3 mM MDA/g) the concentration of MDA in the colonic tissue was significantly higher than in the group “EGCG ± Piperine” (4.8 ± 1 mM MDA/g) (* p ≤ 0.05 vs. treatment with water, # p ≤ 0.05 vs. treatment with piperine). Concentration of MDA between different treatments was significantly different (p = 0.001, one way ANOVA).

Figure 5

Immunohistochemical analysis of colonic tissue after induction of chronic DSS colitis. (A) shows staining of SOD, and (B) staining of GPO in colonic tissue of animals treated with H2O only. In both images there is only a weak signal to be seen, indicating low antioxidant defense. In contrast, colonic tissue staining of animals treated whith EGCG plus piperine showed significant expression of SOD (C) and GPO (D). Representative examples of each group are shown.

Figure 5

Immunohistochemical analysis of colonic tissue after induction of chronic DSS colitis. (A) shows staining of SOD, and (B) staining of GPO in colonic tissue of animals treated with H2O only. In both images there is only a weak signal to be seen, indicating low antioxidant defense. In contrast, colonic tissue staining of animals treated whith EGCG plus piperine showed significant expression of SOD (C) and GPO (D). Representative examples of each group are shown.

Figure 6

Immunoblot showing SOD being constitutively expressed by HT-29 as seen in group 1. The treatment with the combination of EGCG plus piperine in different concentrations (lane 4 and 5) induced a significant expression of SOD (15–20 kDa protein) after stimulation with LPS whereas single treatment with EGCG (lane 3) or piperine (lane 6) did not induce comparable effects.

Figure 6

Immunoblot showing SOD being constitutively expressed by HT-29 as seen in group 1. The treatment with the combination of EGCG plus piperine in different concentrations (lane 4 and 5) induced a significant expression of SOD (15–20 kDa protein) after stimulation with LPS whereas single treatment with EGCG (lane 3) or piperine (lane 6) did not induce comparable effects.

Figure 7

Production of IL-8 after stimulation with LPS (100 ng/ml) in HT-29 cells after different treatments with EGCG/piperine alone or the combination of EGCG and piperine. Cells treated with the combination (15 mM EGCG + 10 mM piperine) exhibit an exponentially reduced secretion of proinflammatory cytokines IL-8 (p = 0.001), whereas cells treated with EGCG or piperine alone did not show a significant reduction in comparison to untreated controls.

Figure 7

Production of IL-8 after stimulation with LPS (100 ng/ml) in HT-29 cells after different treatments with EGCG/piperine alone or the combination of EGCG and piperine. Cells treated with the combination (15 mM EGCG + 10 mM piperine) exhibit an exponentially reduced secretion of proinflammatory cytokines IL-8 (p = 0.001), whereas cells treated with EGCG or piperine alone did not show a significant reduction in comparison to untreated controls.

Author notes

1
Markus Brückner and Sabine Westphal contributed equally to this work.
2
(work originates from this institution).