Crohn’s disease and ulcerative colitis are chronic idiopathic inflammatory bowel diseases (IBD) mediated by immune dysfunction. Despite advancement in humanized monoclonal antibodies and available targeted therapies, there is still no cure for IBD. Therefore, many IBD patients remain refractory to the existing therapies (Fiocchi, 2012). Furthermore, IBD predisposes patients to intestinal surgeries and colorectal malignancy. Inflamed colonic tissue in IBD patients (Grisham and Granger, 1988; Rezaie et al., 2007) and models (Oz et al., 2012a) are rich in neutrophils and activated macrophages and subsequent increased reactive oxygen (ROS) and nitrogen (NOS) species (Oz et al., 2012b). The excess generation of toxic radicals surpasses the intestinal antioxidant defensive ability, thus resulting in oxidative damage (Grisham and Granger, 1988; Oz et al., 2012a,b).

Sulfasalazine has been used as a mainstay of therapy in IBD for decades. Sulfasalazine is a prodrug composed of 5-aminosalicylic acid (5-ASA) and sulfapyridine linked by an azo bond that is poorly absorbed in the stomach and small intestine. The azo linkage is cleaved by the azoreductases released from terminal ileum and colonic anaerobic microbiota to form a pair of amines with the active moiety, 5-ASA (Scheline, 1973; Oz and Ebersole, 2008, review). Sulfasalazine acts as an antioxidant against generated ROS and NOS, with metal chelating effect which reduces oxidative burst. Sulfasalazine may protect against fibrosis by accelerating apoptosis in stellate cell (Oakley et al., 2005). In addition, sulfasalazine induces T lymphocyte apoptosis, inhibits inflammatory intermediates cyclooxygenase/lipoxygenase and nuclear factor kappa B (NF-kB) transcription pathway for pro-inflammatory cytokines, and activates peroxisome proliferator-activated receptor (Wahl et al., 1998; Cavallini et al., 2001; Liptay et al., 2002; Doering et al., 2004; Rousseaux et al., 2005).

However, sulfasalazine has a double edged sword effect by generating additional oxidative stress, which may result in hepatotoxicity (Uko et al., 2012) and ulcerogenic potential. Furthermore, sulfasalazine can provoke hypospermia, and male infertility (Linares et al., 2011), the underlying mechanisms are not fully understood (Katsanos et al., 2012). Sulfasalazine is shown to increase thiobarbituric acid-reactive substances (TBARS), and catalse activity while decrease superoxide dismutase and glutathione levels in hepatic, and kidney suggesting oxidative damage can be a mechanism for nephro-and hepatotoxicity and male infertility related to sulfasalazine treatment (Alonso et al., 2009; Linares et al., 2009).

Additionally, 5-ASA induces apoptosis of intestinal epithelia and inhibits regeneration of colitic mucosa (Reinacher-Schick et al., 2000; Brown et al., 2010). Some of these side effects (e.g., hepatotoxicity, and severe blood disorders) are due to the sulfapyridine portion of sulfasalazine. These patients require escalation of medical therapies and surgery. Therefore, safe and effective drugs are needed for this vulnerable population.

About 30–50% of IBD patients use some type of Complementary and Alternative Medicine (CAM) therapy (Opheim et al., 2012) in addition to their medications whether or not discussed with their primary care providers. However, the safety and efficacy of these compounds and interaction with other drugs in use have not been fully investigated. Therefore, the consequences can be potentially dangerous. The range of CAM therapies include: (i) hypnosis (Szigethy et al., 2011), (ii) acupuncture to decrease response to stress (Rawsthorne et al., 2012), (iii) megadoses of Vitamins and minerals, (iv) prebiotics (Oz and Ebersole, 2008; Oz et al., 2009) (v) probiotics (Mack, 2011), and (vi) Herbal Medicines (Geerling et al., 2000; Keefer et al., 2012). Amongst herbal therapy, tea and tea extracts have received a great deal of attention and are available over the counter (OTC). Tea (Camellia sinensis) is an evergreen shrub which has been used for about 4000 years and is the most consumed beverage after water (Mukhtar et al., 1992; Sharma et al., 2007). Tea contains several components including vitamins (B and C), minerals, and caffeine. Three types of tea are available depending on the processing technique. Black tea is produced by rolling and fermenting the leaves and consumed the most (78% consumption). Green tea is prepared from steamed and dried leaves (20% consumption). Oolong tea is an intermediate form when leaves are semi-fermented (2% consumption).

Green tea Polyphenols (GrTP) are antioxidants and we have previously shown them to have inhibitory effects on NF-kB in vitro in intestinal epithelial cells (Yang et al., 2001) and anti-inflammatory effects in IL-2 deficient mice and some aspects of dextran sodium sulfate (DSS) induced-colitis models (Varilek et al., 2001; Oz et al., 2005). GrTP are shown to have a variety of beneficial effects including anti colorectal cancer possibly through decreasing the serum levels of triglyceride (Shimizu et al., 2008) and promotion of apoptosis (Shirakami et al., 2008; Oz and Ebersole, 2010). In addition, GrTP blocks cyclooxygenase (Cox2) and BCL-2 activity to protect against acetaminophen hepatotoxicity (Oz and Chen, 2008; Oz et al., 2009), as well as LPS induced and carbon tetrahydrochloride hepatotoxicity (Chen et al., 2004). Polyphenols are broken down by the gut microbiota. Similarly, about 70–90% is excreted into feces and the rest recovered from urine (Griffiths and Smith, 1972). Polyphenols are the main component of green tea which have received extensive attention and contains four known catechins: (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epicatechin (EC). EGCG accounts for about 40% of the total polyphenols in tea.

We hypothesized that the alternative therapy with GrTP and EGCG protect against inflammatory responses in DSS induced ulcerative colitis and in the IL-10 deficient model of spontaneous enterocolitis (resembling Crohn’s disease) models in a dose dependent manner similar to the standard-of-care agent sulfasalazine.

The animal studies were approved and performed in accordance with the guidelines for the care and use of laboratory animals accredited by the American Association of Accreditation of Laboratory Animal Care (AAALAC) at Veterans Administration (VA) and Laboratory Animal Research Resource Facility at the University of Kentucky Medical Center in Lexington, KY, USA. Animals were divided into groups of nine mice each (three/cage) and study was repeated at least once. All experiments conform to the relevant regulatory standards.

BALB/C wildtype animals tolerated GrTP, EGCG, and sulfasalazine in their daily diets with no severe side effects. Weight loss is a hallmark of colitis and colitic animals lost 8% of their body weight. GrTP (−2.5%) and Sulfasalazine (−5%) partially improved the weight loss in colitic animals, while, Mid and Low doses of EGCG had no effect on preserving animals’ weight (−8%). In contrast, High dose EGCG consumption further accelerated weight loss (−12%) (Figure 1). Colitic animals developed anemia due to bloody diarrhea, manifested with pale mucosa and a significant reduction in hematocrit (Control 41.5 ± 1.5 vs. colitic 25.2 ± 1.7 p < 0.05). EGCG and GrTP partially improved anemia and the hematocrit value. In contrast, sulfasalazine treatment further triggered anemia and the reduction in the hematocrit value (21.5 ± 2 p < 0.01) (Table 1). EGCG and GrTP administered to naïve control animals had no negative effect on the colonic weight or length. Colonic length became shortened (35%) and colonic weight increased in colitic animals due to the accumulation of inflammatory cells and EGCG partially improved the length (9%) with no effect on weight compared to colitic animals (Table 1). IL-10 deficient animals tolerated Low and Mid doses of GrTP and showed significantly improved enterocolitic symptoms while, lost weight and became moribund on high dose and were terminated.

Despite advances in humanized monoclonal antibodies and available targeted therapies, there is no cure yet for IBD. Biologic therapies, such as monoclonal antibody treatment are prohibitively expensive and have potential adverse effects including infections with fungi, JC virus, and tuberculosis. Many IBD patients also remain refractory to the existing therapies. Furthermore, IBD predisposes patients to intestinal surgeries and colorectal malignancy. Sulfasalazine is a standard care for treatment and maintenance in IBD also has severe adverse effects including hepatotoxicity (Uko et al., 2012), ulcerogenic and male infertility potential (Linares et al., 2011), as well as blood disorders in patients (Katsanos et al., 2012). Therefore, many of these patients seek CAM for symptom relief and improved quality of life.

Polyphenols are one of the most used herbal therapies available and have anti-inflammatory effect due to their antioxidant effects, alteration in the cell signaling, and particularly inhibition of the nuclear factor NF-κB pathway. Using IBD as a model of inflammation, we explored anti-inflammatory effects of the principal CAM, namely, GrTP and its most abundant cathechin EGCG, compared to the standard care, sulfasalazine treatment in murine colitis models. The susceptibility of mice to DSS-induced colitis and, polyphenols in specific EGCG-mediated anti-inflammatory action in this model have much in common with the similar phenomena observed with sulfasalazine.

While, colitic animals develop bloody diarrhea and anemia as IBD cardinal signs, GrTP and EGCG therapy were effective in improving hematocrit values. In contrast, sulfasalazine treatment further aggravated anemia in animals conceivably due to its adverse hemolytic effects as reported in IBD patients (Stein and Hanauer, 2000). In this study, EGCG was calculated according to the total amount of constituent present in the GrTP and administered at different doses of High (1), Mid (1/2), and Low (1/4) in daily diet. The Low dose EGCG appeared to be safe and to have the most effect on reducing colonic pathological lesions, normalizing global antioxidants ratio, partially improving colonic length and weight, without causing weight loss. However, Low dose was least beneficial in reducing SAA or in inhibiting reduction in leptin levels. While, GrTP normalized antioxidants, partially protected animals against weight loss and elevated TNFα but was less effective against colonic pathology as compared to Low dose EGCG.

In mice [3H](−)-EGCG is absorbed easily from the digestive tract and distributed widely into various organs, and excreted in the urine (6.4–6.6%) or feces (37.7–33.1%) within 24 h (Suganuma et al., 1998). In this study colonic GSH significantly decreased in colitic animals and improved in a dose dependent manner by EGCG treatment (DSS vs. High, Mid doses <0.05). However, sulfasalazine had no significant effect on the intestinal GSH. Of interest, the ratio of hepatic reduced-to-oxidized glutathione, GSH/GSSG, in colitic animals was decreased to about 1/2 of the amount in normal controls representing the DSS-induced colitis as a global oxidative stress model. Sulfasalazine, GrTP, and Low dose treatment normalized the hepatic glutathione ratio and the reversal of this important aspect of colitis in these models. Interestingly, High dose EGCG treatment caused fourfold increases in the hepatic glutathione ratio, demonstrating exaggerated global antioxidant imbalance presumably predisposing animals to excess weight loss with no improvement in colonic pathology.

In addition, EGCG and GrTP have been reported to have antimicrobial effects and to disrupt bacterial growth (Steinmann et al., 2013). In this study, GrTP and Low dose EGCG may have exerted their anticolitic effects through a combination of antimicrobial properties mucosal immunity and gut cleansing as well as antioxidants and anti-inflammatory action through inhibition of NF-kB activation and further IKK activity.

Leptin, an endocrine cytokine is a 16 kDa protein encoded by the ob gene which plays a central role in the maintenance of body weight and energy balance (Gaetke et al., 2002a). Leptin is secreted by the white adipocytes (Gaetke et al., 2002b, 2003) to regulate food intake and metabolism, while its action is controlled centrally by the hypothalamus (Zhang et al., 1994). Leptin regulates energy metabolism by increasing energy expenditure and decreasing food intake and body weight. The serum leptin concentration is linearly related to fat mass in ad libitum fed mice (Schwartz et al., 1997) and humans (Tuzun et al., 2004). Serum leptin rapidly declines with food restriction and is elevated with feeding (Schwartz et al., 1997; Gaetke et al., 2002a,b, 2003; Tuzun et al., 2004). Leptin deficiency affects both the innate and acquired immune systems (Mackey-Lawrence and Petri, 2012) as children and mice with congenital leptin or leptin receptor deficiency are reported to be susceptible to infections. The serum level of leptin is dysregulated in obesity (Schwartz et al., 1997) and during the active state of IBD (Tuzun et al., 2004). Leptin may present a potential mediator of inappropriate satiety and lipid dystrophy as well as deregulated immune response to altered gut microbiota in IBD patients. In this study colitic animals had significantly lower leptin levels. These findings are in accordance with leptin deficiency observed in other inflammatory diseases, including alcoholic hepatitis in mouse model (Tan et al., 2012). Additionally, Leptin has been shown to mediate resistance to enteric infection Entamoeba through its direct actions on intestinal epithelium which requires leptin receptor signaling through both the STAT3 and SHP2/ERK pathways (Guo et al., 2011). In the current study, GrTP and sulfasalazine had no effect on leptin levels. However, EGCG further reduced blood concentration of Leptin and High dose mediated additional weight loss possibly by blocking the appetite and decreasing food intake in colitic animals.

Our results have shown that 50 mg/kg sulfasalazine was sufficient to exert its protective effect against DSS-induced colitis. In contrast others reported that sulfasalazine at 10–20 mg/kg orally or IP did not prevent weight loss nor reduced diarrhea or gross pathology score in Trinitrobenzenesulfonic acid (TNBS)-induced colitis (Radi et al., 2011). This could be attributed to the insufficient drug dose used in the TNBS model against IBD.

Overall, these compounds had limited protective effects on DSS-induced colitis since colitis in general involves several key players including gut mucosal innate immune response, macrophage activation, ROS generation, and inflammatory response with subsequent loss of epithelia integrity, and increased luminal Gram-negative microbiota. Previously we and others have shown that GrTP inhibited signaling pathways involved in inflammation, including NF-kB in intestinal cells (Yang et al., 2001) and in IBD models (Varilek et al., 2001; Oz et al., 2005) and activated protein-1, AP-1(Abboud et al., 2008), which are key elements in production of pro-inflammatory mediators. Recently a mixture of EGCG and piperine was reported to protect against lipid peroxidation, neutrophils accumulation in DSS-induced colitic mice, while superoxide dismutase and glutathione peroxidase showed an increased activity in treated animals (Bruckner et al., 2012). To our knowledge this is the first report indicating: (a) GrTP to preserve gut microstructure and to protect against enterocolitis in IL-10 deficient model; (b) High dose EGCG consumption to provoke exaggerated global antioxidants and excess weight loss, rendering ineffective against colitis; (c) GrTP and Low dose EGCG to protect against colitis similar to the standard-of-care sulfasalazine, and to improve anemia.

Green tea polyphenols and Low EGCG improved antioxidants pools, decreased inflammatory cytokines and attenuated the severity of colitis in a manner similar to that of sulfasalazine. Thus polyphenols may become an alternative/additive therapy for IBD therapy and future human clinical trials of the polyphenols in IBD patients are warranted.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.