JOURNAL OF MEDICINAL FOOD J Med Food 17 (12) 2014, 1–3 # Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2014.3191
SHORT COMMUNICATION
Agaricus bisporus Attenuates Dextran Sulfate Sodium-Induced Colitis Min Young Um, Jae Ho Park, So Young Gwon, Jiyun Ahn, Chang Hwa Jung, and Tae Youl Ha Metabolism and Nutrition Research Group, Korea Food Research Institute, Seongnam, South Korea. ABSTRACT Agaricus bisporus (white button mushroom, WBM) is widely consumed in most countries and is reported to have anti-inflammatory and antioxidant activities. However, little is known regarding its effects in dextran sulfate sodium (DSS)-induced colitis, which are related to dysfunction of intestinal immunity. The aim of the present study was to investigate the effects of WBMs in an animal model of DSS-induced colitis. Male, 4-week-old ICR mice (n = 10 per group) were fed a normal diet with or without 10% WBM for 4 weeks, and colitis was induced by 3% DSS in drinking water for 7 days. WBMs prevented DSS-induced shortening of colon length (P = .033) and diminished diarrhea (P = .049) and gross bleeding (P = .001), resulting in a decreased disease activity index. Results of histological analysis showed that WBMs suppressed mucosal damage. In addition, WBMs attenuated the DSS-induced increase in myeloperoxidase activity (P = .012) and upregulation of proinflammatory cytokine tumor necrosis factor-a (P = .020) in the colon segment. Taken together, these findings suggest a possible role for the WBM as an immunomodulator that can prevent and/or treat ulcerative colitis.
KEY WORDS: Agaricus bisporus colitis dextran sulfate sodium inflammation
determined according to the method of Muchuweti et al.5 The 1,1-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assays described by Hayes et al.6 were used to determine the antioxidant activity of WBMs. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Korea Food Research Institute. Four-week-old, male ICR mice were purchased from Central Laboratory Animal, Inc. (Seoul, South Korea). After 7 days of acclimatization, animals were randomly assigned to three groups (n = 10 per group): the control group (normal diet, plain drinking water), DSS group (normal diet, drinking water containing 3% DSS), and DSS + WBM group (diet containing 10% WBM powder, drinking water containing 3% DSS) and fed a normal diet or WBM-added diet for 4 weeks. Colitis was induced by drinking water containing 3% DSS throughout the last 7 days of the experimental period. The normal diet was based on the AIN-93G diet formula. The mushroom powder was added to the experimental diet at a level of 10% of diet (wt/wt) by substituting it for equal amounts of corn starch. The dosage of WBMs was chosen from the results of previous animal studies.7,8 After sacrificing the animals in a fasting state, the colons were excised and their lengths were measured. The disease activity index (DAI) was calculated by scoring weight loss, stool consistency, and rectal bleeding as described by Murthy et al.9 The formalin-fixed colon tissues were processed, embedded in paraffin, and used for hematoxylin and eosin (H&E) staining. Myeloperoxidase (MPO) activity was determined by using the method described by
U
lcerative colitis is a chronic inflammatory bowel disease (IBD) with symptoms of diarrhea and rectal bleeding.1 Although the etiology of IBD remains unclear, accumulating evidence indicates that a dysregulated immune response associated with genetic and environmental factors plays a role in the disease pathology. Hence, there is great interest in agents that can block the immune response and inflammation. Agaricus bisporus, commonly known as the white button mushroom (WBM), is one of the most important edible mushrooms. WBMs are a valuable source of polyphenols, vitamins, minerals, fiber, and polysaccharides, and also possess immunomodulator, antitumor, anti-inflammatory, and antioxidant properties.2,3 Recently, Gunawardena et al.4 reported that WBM consumption inhibits nitric oxide and tumor necrosis factor (TNF)-a production in lipopolysaccharidetreated murine macrophages. However, little is known about the effects of WBMs on dextran sulfate sodium (DSS)induced colitis. The aim of the present study was to determine whether the WBM can prevent experimental colitis in mice. Fresh WBMs supplied by Byueo-Gun (Chungnam, South Korea) were washed with distilled water and sliced into small pieces. After freeze-drying, the WBMs were ground to a powder and stored at - 20C. Total polyphenol content was Manuscript received 12 March 2014. Revision accepted 5 September 2014. Address correspondence to: Tae Youl Ha, PhD, Metabolism and Nutrition Research Group, Korea Food Research Institute, Seongnam 463-746, South Korea, E-mail: tyhap@ kfri.re.kr
1
2
UM ET AL. Table 1. Contents of Total Polyphenol and Total Flavonoids from White Button Mushrooms and Antioxidant Activity
Total polyphenol (mg catechin/100g dry weight)
Total flavonoids (mg naringin/100g dry weight)
ABTS radical scavenging activity (IC50, mg/mL)
DPPH radical scavenging activity (IC50, mg/mL)
1636.8 – 17.5
123.0 – 10.2
466.4 – 60.5
1543.1 – 25.6 Results are mean – SEM of three experiments.
Table 2. Food Intake, Body Weight, and Colon Length Control
DSS
DSS + WBM
Food intake (g/day) 4.11 – 0.11 3.98 – 0.10 3.99 – 0.15 Initial body 26.62 – 1.20 26.73 – 1.10 26.65 – 0.88 weight (g) Final body 39.50 – 3.08* 35.90 – 2.36 37.80 – 2.12 weight (g) Colon length (cm) 8.93 – 0.76*** 6.90 – 0.65 7.84 – 0.73*,
##
Results are expressed as mean – SEM (n = 10 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. *P < .05, ***P < .001; significant difference from the DSS group. ##P < .01; significant difference from the control group. DSS, dextran sulfate sodium; WBM, white button mushroom.
Bradley et al.10 IL-1b, IL-6, and TNF-a levels in colon segments were determined by enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN, USA). Statistical analyses were performed using SPSS 14.0 software (SPSS, Inc., Chicago, IL, USA). Results are expressed as mean – SEM, and group differences were considered
significant at P < .05 based on one-way analysis of variance followed by Tukey’s post hoc test. Total polyphenol and total flavonoid concentrations were determined to be 1543.1 – 25.6 and 1636.8 – 17.5 mg/100 g d.w., respectively. WBMs exhibited DPPH and ABTS radical scavenging activity with an IC50 value of 123.0 – 10.2 and 466.4 – 60.5 lg/mL (Table 1). In the present study, we found that WBMs suppressed colon damage and inflammation in a mouse model of DSSinduced colitis. As shown in Table 2 and Figure 1A, DSSinduced acute colitis is characterized by body weight loss and loose and bloody stool. WBMs attenuated these clinical symptoms, as demonstrated by the lower DAI score in mice given DSS + WBM compared with mice given DSS alone (P = .005). In addition, WBMs minimized the DSS-induced shortening of colon length (P = .033) and attenuated DSSinduced histological changes in the colon (mucosal erosion, crypt loss, and distortion) (Fig. 1B). To better understand the effect of the WBM on DSSinduced colitis, we evaluated the activity of MPO, which is a biomarker of inflammation.11 We found elevated MPO
FIG. 1. Effect of the white button mushroom (WBM) on the disease activity index score (A). Histological analysis showing the effects of WBMs on dextran sulfate sodium (DSS)-induced colonic damage (B). Representative hematoxylin and eosin-stained sections (magnification, 200 · ). Effects of WBMs on cytokine levels and MPO activity in mice with DSS-induced colitis (C). Results are expressed as mean – SEM. (n = 10 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. *P < .05, **P < .01, ***P < .001; significant difference from the DSS group. ##P < .01, ###P < .001; significant difference from the control group. Color images available online at www.liebertpub.com/jmf
EFFECT OF AGARICUS BISPORUS ON DSS-INDUCED COLITIS
activity in colon tissues of mice treated with DSS, but enzyme activity in the DSS + WBM group was reduced by 50% (P = .012), consistent with histology results (Fig. 1C). Decreased MPO activity indicates an anti-inflammatory effect because MPO activity in the colon is linearly related to neutrophil infiltration.12 Hyperactivation of immune cells is another important factor of IBD progression, producing high levels of proinflammatory cytokines such as TNF-a, IL-6, and interferon-c. Elevated levels of proinflammatory cytokines are observed in the inflamed gut of IBD patients and in animals with DSS-induced colitis.12,13 Thus, decreasing proinflammatory cytokine levels offer an effective approach to the prevention and treatment of IBD. Our results showed increased levels of TNF-a, IL-1b, and IL-6 in the colon tissues of mice with DSS-induced colitis. WBMs decreased TNF-a levels in colonic tissues (P = .02) and appeared to decrease levels of IL-1b and IL-6; however, the differences were not significant. Our results indicate that the WBM ameliorates DSS-induced colitis in mice by suppressing proinflammatory mediators in the colon. The totality of the evidence indicates that dietary antioxidants protect the mucosal damage induced by DSS.14 Given that WBMs contain antioxidants, including phenolic compounds, we suggested that the beneficial properties of the WBM could be due, in part, to its antioxidant activity. Overall, our results demonstrate that the WBM suppresses DSS-induced inflammation and attenuates colonic damage in mice, suggesting that WBMs may be useful in the treatment and/or prevention of colitis. Further studies are required to elucidate the molecular mechanisms underlying these therapeutic effects of WBMs.
ACKNOWLEDGMENT This work was supported by the National Platform Technology Project from the Ministry of Knowledge Economy. AUTHOR DISCLOSURE STATEMENT There are no existing conflicts of interest. REFERENCES 1. Karagozian R, Burakoff R: The role of mesalamine in the treatment of ulcerative colitis. Ther Clin Risk Manag 2007;3: 893–903.
3
2. Wang HX, Liu WK, Ng TB, Ooi VE, Chang ST: The immunomodulatory and antitumor activities of lectins from the mushroom Tricholoma mongolicum. Immunopharmacology 1996;31: 205–211. 3. Liu J, Jia L, Kan J, Jin CH: In vitro and in vivo antioxidant activity of ethanolic extract of white button mushroom (Agaricus bisporus). Food Chem Toxicol 2013;51:310–316. 4. Gunawardena D, Bennett L, Shanmugam K, et al.: Anti-inflammatory effects of five commercially available mushroom species determined in lipopolysaccharide and interferon-c activated murine macrophages. Food Chem 2013;148:92–96. 5. Muchuweti M, Ndhlala AR, Kasiamhuru A: Analysis of phenolic compounds including tannins, gallotannins and flavanols of Uapaca kirkiana fruit. Food Chem 2006; 94:415–419. 6. Hayes JE, Allen P, Brunton N, O’Grady MN, Kerry JP: Phenolic composition and in vitro antioxidant capacity of four commercial phytochemical products: Olive leaf extract (Olea europaea L.), lutein, sesamol and ellagic acid. Food Chem 2011;126:948–955. 7. Xu Y, Na L, Ren Z, et al.: Effect of dietary supplementation with white button mushrooms on host resistance to influenza infection and immune function in mice. Br J Nutr 2013;109:1052–1061. 8. Wu D, Pae M, Ren Z, et al.: Dietary supplementation with white button mushroom enhances natural killer cell activity in C57BL/ 6 mice. J Nutr 2007;137:1472–1477 9. Murthy SN, Cooper HS, Shim H, Shah RS, Ibrahim SA, Sedergran DJ: Treatment of dextran sulfate sodium-induced murine colitis by intracolonic cyclosporin. Dig Dis Sci 1993;38:1722– 1734. 10. Bradley PP, Christensen RD, Rothstein G: Cellular and extracellular myeloperoxidase in pyogenic inflammation. Blood 1982; 60:618–622. 11. Kumar G K, Dhamotharan R, Kulkarni NM, Honnegowda S, Murugesan S: Embelin ameliorates dextran sodium sulfate-induced colitis in mice. Int Immunopharmacol 2011;11:724–731. 12. Nakhai LA, Mohammadirad A, Yasa N, et al.: Benefits of zataria multiflora boiss in experimental model of mouse inflammatory bowel disease. Evid Based Complement Alternat Med 2007;4: 43–50. 13. Dieleman LA, Palmen MJ, Akol H, et al.: Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clin Exp Immunol 1998;114: 385–391. 14. Andu´jar I, Recio MC, Giner RM, et al.: Inhibition of ulcerative colitis in mice after oral administration of a polyphenol-enriched cocoa extract is mediated by the inhibition of STAT1 and STAT3 phosphorylation in colon cells. J Agric Food Chem 2011;59: 6474–6483.
SHORT COMMUNICATION
Agaricus bisporus Attenuates Dextran Sulfate Sodium-Induced Colitis Min Young Um, Jae Ho Park, So Young Gwon, Jiyun Ahn, Chang Hwa Jung, and Tae Youl Ha Metabolism and Nutrition Research Group, Korea Food Research Institute, Seongnam, South Korea. ABSTRACT Agaricus bisporus (white button mushroom, WBM) is widely consumed in most countries and is reported to have anti-inflammatory and antioxidant activities. However, little is known regarding its effects in dextran sulfate sodium (DSS)-induced colitis, which are related to dysfunction of intestinal immunity. The aim of the present study was to investigate the effects of WBMs in an animal model of DSS-induced colitis. Male, 4-week-old ICR mice (n = 10 per group) were fed a normal diet with or without 10% WBM for 4 weeks, and colitis was induced by 3% DSS in drinking water for 7 days. WBMs prevented DSS-induced shortening of colon length (P = .033) and diminished diarrhea (P = .049) and gross bleeding (P = .001), resulting in a decreased disease activity index. Results of histological analysis showed that WBMs suppressed mucosal damage. In addition, WBMs attenuated the DSS-induced increase in myeloperoxidase activity (P = .012) and upregulation of proinflammatory cytokine tumor necrosis factor-a (P = .020) in the colon segment. Taken together, these findings suggest a possible role for the WBM as an immunomodulator that can prevent and/or treat ulcerative colitis.
KEY WORDS: Agaricus bisporus colitis dextran sulfate sodium inflammation
determined according to the method of Muchuweti et al.5 The 1,1-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assays described by Hayes et al.6 were used to determine the antioxidant activity of WBMs. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Korea Food Research Institute. Four-week-old, male ICR mice were purchased from Central Laboratory Animal, Inc. (Seoul, South Korea). After 7 days of acclimatization, animals were randomly assigned to three groups (n = 10 per group): the control group (normal diet, plain drinking water), DSS group (normal diet, drinking water containing 3% DSS), and DSS + WBM group (diet containing 10% WBM powder, drinking water containing 3% DSS) and fed a normal diet or WBM-added diet for 4 weeks. Colitis was induced by drinking water containing 3% DSS throughout the last 7 days of the experimental period. The normal diet was based on the AIN-93G diet formula. The mushroom powder was added to the experimental diet at a level of 10% of diet (wt/wt) by substituting it for equal amounts of corn starch. The dosage of WBMs was chosen from the results of previous animal studies.7,8 After sacrificing the animals in a fasting state, the colons were excised and their lengths were measured. The disease activity index (DAI) was calculated by scoring weight loss, stool consistency, and rectal bleeding as described by Murthy et al.9 The formalin-fixed colon tissues were processed, embedded in paraffin, and used for hematoxylin and eosin (H&E) staining. Myeloperoxidase (MPO) activity was determined by using the method described by
U
lcerative colitis is a chronic inflammatory bowel disease (IBD) with symptoms of diarrhea and rectal bleeding.1 Although the etiology of IBD remains unclear, accumulating evidence indicates that a dysregulated immune response associated with genetic and environmental factors plays a role in the disease pathology. Hence, there is great interest in agents that can block the immune response and inflammation. Agaricus bisporus, commonly known as the white button mushroom (WBM), is one of the most important edible mushrooms. WBMs are a valuable source of polyphenols, vitamins, minerals, fiber, and polysaccharides, and also possess immunomodulator, antitumor, anti-inflammatory, and antioxidant properties.2,3 Recently, Gunawardena et al.4 reported that WBM consumption inhibits nitric oxide and tumor necrosis factor (TNF)-a production in lipopolysaccharidetreated murine macrophages. However, little is known about the effects of WBMs on dextran sulfate sodium (DSS)induced colitis. The aim of the present study was to determine whether the WBM can prevent experimental colitis in mice. Fresh WBMs supplied by Byueo-Gun (Chungnam, South Korea) were washed with distilled water and sliced into small pieces. After freeze-drying, the WBMs were ground to a powder and stored at - 20C. Total polyphenol content was Manuscript received 12 March 2014. Revision accepted 5 September 2014. Address correspondence to: Tae Youl Ha, PhD, Metabolism and Nutrition Research Group, Korea Food Research Institute, Seongnam 463-746, South Korea, E-mail: tyhap@ kfri.re.kr
1
2
UM ET AL. Table 1. Contents of Total Polyphenol and Total Flavonoids from White Button Mushrooms and Antioxidant Activity
Total polyphenol (mg catechin/100g dry weight)
Total flavonoids (mg naringin/100g dry weight)
ABTS radical scavenging activity (IC50, mg/mL)
DPPH radical scavenging activity (IC50, mg/mL)
1636.8 – 17.5
123.0 – 10.2
466.4 – 60.5
1543.1 – 25.6 Results are mean – SEM of three experiments.
Table 2. Food Intake, Body Weight, and Colon Length Control
DSS
DSS + WBM
Food intake (g/day) 4.11 – 0.11 3.98 – 0.10 3.99 – 0.15 Initial body 26.62 – 1.20 26.73 – 1.10 26.65 – 0.88 weight (g) Final body 39.50 – 3.08* 35.90 – 2.36 37.80 – 2.12 weight (g) Colon length (cm) 8.93 – 0.76*** 6.90 – 0.65 7.84 – 0.73*,
##
Results are expressed as mean – SEM (n = 10 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. *P < .05, ***P < .001; significant difference from the DSS group. ##P < .01; significant difference from the control group. DSS, dextran sulfate sodium; WBM, white button mushroom.
Bradley et al.10 IL-1b, IL-6, and TNF-a levels in colon segments were determined by enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN, USA). Statistical analyses were performed using SPSS 14.0 software (SPSS, Inc., Chicago, IL, USA). Results are expressed as mean – SEM, and group differences were considered
significant at P < .05 based on one-way analysis of variance followed by Tukey’s post hoc test. Total polyphenol and total flavonoid concentrations were determined to be 1543.1 – 25.6 and 1636.8 – 17.5 mg/100 g d.w., respectively. WBMs exhibited DPPH and ABTS radical scavenging activity with an IC50 value of 123.0 – 10.2 and 466.4 – 60.5 lg/mL (Table 1). In the present study, we found that WBMs suppressed colon damage and inflammation in a mouse model of DSSinduced colitis. As shown in Table 2 and Figure 1A, DSSinduced acute colitis is characterized by body weight loss and loose and bloody stool. WBMs attenuated these clinical symptoms, as demonstrated by the lower DAI score in mice given DSS + WBM compared with mice given DSS alone (P = .005). In addition, WBMs minimized the DSS-induced shortening of colon length (P = .033) and attenuated DSSinduced histological changes in the colon (mucosal erosion, crypt loss, and distortion) (Fig. 1B). To better understand the effect of the WBM on DSSinduced colitis, we evaluated the activity of MPO, which is a biomarker of inflammation.11 We found elevated MPO
FIG. 1. Effect of the white button mushroom (WBM) on the disease activity index score (A). Histological analysis showing the effects of WBMs on dextran sulfate sodium (DSS)-induced colonic damage (B). Representative hematoxylin and eosin-stained sections (magnification, 200 · ). Effects of WBMs on cytokine levels and MPO activity in mice with DSS-induced colitis (C). Results are expressed as mean – SEM. (n = 10 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. *P < .05, **P < .01, ***P < .001; significant difference from the DSS group. ##P < .01, ###P < .001; significant difference from the control group. Color images available online at www.liebertpub.com/jmf
EFFECT OF AGARICUS BISPORUS ON DSS-INDUCED COLITIS
activity in colon tissues of mice treated with DSS, but enzyme activity in the DSS + WBM group was reduced by 50% (P = .012), consistent with histology results (Fig. 1C). Decreased MPO activity indicates an anti-inflammatory effect because MPO activity in the colon is linearly related to neutrophil infiltration.12 Hyperactivation of immune cells is another important factor of IBD progression, producing high levels of proinflammatory cytokines such as TNF-a, IL-6, and interferon-c. Elevated levels of proinflammatory cytokines are observed in the inflamed gut of IBD patients and in animals with DSS-induced colitis.12,13 Thus, decreasing proinflammatory cytokine levels offer an effective approach to the prevention and treatment of IBD. Our results showed increased levels of TNF-a, IL-1b, and IL-6 in the colon tissues of mice with DSS-induced colitis. WBMs decreased TNF-a levels in colonic tissues (P = .02) and appeared to decrease levels of IL-1b and IL-6; however, the differences were not significant. Our results indicate that the WBM ameliorates DSS-induced colitis in mice by suppressing proinflammatory mediators in the colon. The totality of the evidence indicates that dietary antioxidants protect the mucosal damage induced by DSS.14 Given that WBMs contain antioxidants, including phenolic compounds, we suggested that the beneficial properties of the WBM could be due, in part, to its antioxidant activity. Overall, our results demonstrate that the WBM suppresses DSS-induced inflammation and attenuates colonic damage in mice, suggesting that WBMs may be useful in the treatment and/or prevention of colitis. Further studies are required to elucidate the molecular mechanisms underlying these therapeutic effects of WBMs.
ACKNOWLEDGMENT This work was supported by the National Platform Technology Project from the Ministry of Knowledge Economy. AUTHOR DISCLOSURE STATEMENT There are no existing conflicts of interest. REFERENCES 1. Karagozian R, Burakoff R: The role of mesalamine in the treatment of ulcerative colitis. Ther Clin Risk Manag 2007;3: 893–903.
3
2. Wang HX, Liu WK, Ng TB, Ooi VE, Chang ST: The immunomodulatory and antitumor activities of lectins from the mushroom Tricholoma mongolicum. Immunopharmacology 1996;31: 205–211. 3. Liu J, Jia L, Kan J, Jin CH: In vitro and in vivo antioxidant activity of ethanolic extract of white button mushroom (Agaricus bisporus). Food Chem Toxicol 2013;51:310–316. 4. Gunawardena D, Bennett L, Shanmugam K, et al.: Anti-inflammatory effects of five commercially available mushroom species determined in lipopolysaccharide and interferon-c activated murine macrophages. Food Chem 2013;148:92–96. 5. Muchuweti M, Ndhlala AR, Kasiamhuru A: Analysis of phenolic compounds including tannins, gallotannins and flavanols of Uapaca kirkiana fruit. Food Chem 2006; 94:415–419. 6. Hayes JE, Allen P, Brunton N, O’Grady MN, Kerry JP: Phenolic composition and in vitro antioxidant capacity of four commercial phytochemical products: Olive leaf extract (Olea europaea L.), lutein, sesamol and ellagic acid. Food Chem 2011;126:948–955. 7. Xu Y, Na L, Ren Z, et al.: Effect of dietary supplementation with white button mushrooms on host resistance to influenza infection and immune function in mice. Br J Nutr 2013;109:1052–1061. 8. Wu D, Pae M, Ren Z, et al.: Dietary supplementation with white button mushroom enhances natural killer cell activity in C57BL/ 6 mice. J Nutr 2007;137:1472–1477 9. Murthy SN, Cooper HS, Shim H, Shah RS, Ibrahim SA, Sedergran DJ: Treatment of dextran sulfate sodium-induced murine colitis by intracolonic cyclosporin. Dig Dis Sci 1993;38:1722– 1734. 10. Bradley PP, Christensen RD, Rothstein G: Cellular and extracellular myeloperoxidase in pyogenic inflammation. Blood 1982; 60:618–622. 11. Kumar G K, Dhamotharan R, Kulkarni NM, Honnegowda S, Murugesan S: Embelin ameliorates dextran sodium sulfate-induced colitis in mice. Int Immunopharmacol 2011;11:724–731. 12. Nakhai LA, Mohammadirad A, Yasa N, et al.: Benefits of zataria multiflora boiss in experimental model of mouse inflammatory bowel disease. Evid Based Complement Alternat Med 2007;4: 43–50. 13. Dieleman LA, Palmen MJ, Akol H, et al.: Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clin Exp Immunol 1998;114: 385–391. 14. Andu´jar I, Recio MC, Giner RM, et al.: Inhibition of ulcerative colitis in mice after oral administration of a polyphenol-enriched cocoa extract is mediated by the inhibition of STAT1 and STAT3 phosphorylation in colon cells. J Agric Food Chem 2011;59: 6474–6483.
