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.
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. 34–36
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.
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.
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 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 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).
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).
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.
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).
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).
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).
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).
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. 41–44 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.56–58
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
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.
dextran sodium sulfate
reactive oxygen species
extracellular signal-regulated kinase