The impact of mangiferin from Belamcanda chinensis on experimental colitis in rats

Inflammatory bowel disease (IBD) is an important and commonly encountered clinical problem. The incidence of IBD has considerably increased in recent years in both Western and Eastern societies, also in children. Almost 25% of new diagnoses of the disease are made in patients under 18 years of age. The most common forms of IBD are Crohn’s disease (CD) and ulcerative colitis (UC). IBD is characterized by a chronic, relapsing inflammatory condition of the gastrointestinal tract, with both overlapping and distinct pathological and clinical features. Symptoms range from mild to severe during relapses and may disappear during remissions, depending on the involved segment of the gastrointestinal tract. Initially, both CD and UC are characterized by low-grade fever, fatigue, bloody diarrhea accompanied by mucorrhea, abdominal pain with cramps, unintended weight loss with reduced appetite and anemia. Differences between Crohn’s disease and ulcerative colitis patients become more evident with progression of the disease, along with development of intestinal and extraintestinal complications (Sartor 2006; Vasovic et al. 2016).

The pathogenesis of IBD is multifactorial and remains not completely understood. It is presumed that a complex interaction between genetic, environmental or microbial factors may lead to enhanced immune response, involving increased migration, proliferation and activation of Th1, Th2 and Th17 cells and increased concentrations of pro-inflammatory cytokines, including tumor necrosis factor α (TNF-α) and interleukin 17 (IL-17), that determine the development and persistence of inflammation. Moreover, oxidative stress and a loss of mucosal epithelial barrier may contribute to the development of intestinal inflammation (Pavlick et al. 2002; Sartor 2006; Shen and Durum 2010).

Despite a significant progress in IBD therapy, clinicians still expect more effective and safer medicines. Presently, aminosalicylates are considered drugs of choice for the management of majority of cases, while glucocorticosteroids, immunosuppressants and biological agents are reserved for more severe forms of the disease (van Assche et al. 2010; Dignass et al. 2012). The pharmacological treatment is fraught with severe side effects of drugs. Those effects are particularly important in the context of chronic and relapsing nature of IBD and the necessity of long-term pharmacotherapy (Kenneth and McQuaid 2012). Thus, there is a need for identification of new, more efficient and safer therapies for IBD. The assessment of substances obtained from plants, traditionally used in various inflammatory conditions, is one of the approaches for development of future IBD treatments.

Mangiferin (2-C-β-d-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone) belongs to xanthonoids, a class of natural components referred to as polyphenols. Mangiferin is mainly isolated from leaves, bark and fruit peel of Mangifera indica (L.) and rhizomes of Belamcanda chinensis (L.) DC. (syn.: Iris domestica (L.) Goldbl. & Mabb.; English: blackberry lilly, leopard flower; Traditional Chinese Medicine: she gan). These rhizomes are used in East Asian phytotherapy systems as an antipyretic, antiphlogistic, analgesic and expectorant agent. Studies with mangiferin based on experimental in vitro and in vivo assays have demonstrated its antioxidant, anti-inflammatory, immunomodulatory, antimicrobial, analgesic and radioprotective effects (Matkowski et al. 2013; Wauthoz et al. 2007).

This study was designed to elucidate the impact of mangiferin on 2,4,6-trinitrobenzensulfonic (TNBS) acid-induced colitis in rats.

Detailed effects (including the level of significance) of mangiferin on body weight, colon mass index, macro- and microscopic damages, TNF-α and IL-17 and MDA levels and SOD activity in colon tissues are presented in the Fig. 2. Summing up briefly, TNBS caused significant loss of body weight, increased colon mass index, elicited macro- and microscopic damages in colon tissues, increased TNF-α, IL-17 and MDA concentrations and decreased SOD activity, in comparison to the control group. Administration of mangiferin at the highest dose (100 mg/kg) significantly protected from TNBS-induced changes, such as macro- and microscopic damage, TNF-α, IL-17, MDA concentration increase and increased SOD activity, but not from TNBS-induced body weight loss and increased colon mass index. Mangiferin administered at the medium dose (30 mg/kg) protected only from macro- and microscopic damage and MDA level increase. The lowest dose of mangiferin (10 mg/kg) did not reverse any of the TNBS-induced changes. Administration of mangiferin in rats non-treated with TNBS did not exert any changes in any of studied parameters in comparison to the control group. Macroscopic changes of TNBS-induced colitis involved bowel wall thickening, hyperemia and edema. Microscopic examination of colon samples showed an intense interruption of colon tissues with ulceration and inflammation involving not only the epithelial layer but also all intestinal layers in some of studied specimens. The ulceration was associated with transmural infiltration of lymphocytes, macrophages and neutrophils. The mucosal infiltration of inflammatory cells was considerably high and it was minor in the submucosa and muscular layer. Rats with TNBS-induced colitis also showed structural distortion of crypts, desquamated areas or loss of epithelium. The inflammatory process was also linked with goblet cell depletion leading to the decline of the mucus layer of the epithelium (Fig. 3, 4). In comparison with the TNBS group mangiferin pre-treatment (30 or 100 mg/kg) before the induction of colitis significantly attenuated the colonic lesions and resulted in reduced score of macro- and microscopic damage. A histological examination of colonic samples from rats pre-treated with mangiferin (30 or 100 mg/kg) demonstrated a pronounced recovery of tissues of the colon, with decreased extent and reduced intensity of ulceration (Fig. 3), decreased transmural infiltration of inflammatory cells (Fig. 4a), increased amount of goblet cells refilled with their mucin constituents and restored epithelial cell layer (Fig. 4b). Mangiferin at the lowest dose (10 mg/kg) had no protective effect against TNBS-induced changes. The action of mangiferin on TNBS-induced colonic damage was dose dependent. It was significantly higher at the dose of 100 mg/kg compared to 30 mg/kg. Administration of mangiferin to rats non-exposed to TNBS did not exert any pathological changes of all studied parameters. Correlation coefficients between the microscopically evaluated degree damage of colonic tissue and TNF-α, IL-17, MDA concentrations (r = 0.692; 0.561; 0.663, respectively) and the SOD activity (r = −0.804) were statistically significant at p < 0.001. There was no directly proportional correlation between the score of microscopic damage and the above-mentioned parameters, but it has been demonstrated that the degree of microscopic damage done to the colon decreased along with decreasing TNF-α, IL-17 and MDA concentrations and increasing SOD activity.

Experimental animal models of IBD are valuable tools widely used in preclinical studies. In our study the TNBS model of IBD had been chosen because it is well characterized and shares some biochemical and immunological features and symptoms with the disease in humans (Westbrook et al. 2010).

Some efforts have been recently made to identify and extract purely natural compounds, particularly plant-derived ones, that could restore the normal immune response altered in IBD and act as protective agents in various inflammatory disorders. Polyphenols, represented by mangiferin, are one of the most important groups of natural substances, demonstrating strong anti-inflammatory and antioxidative properties (Márquez et al. 2010). Mangiferin has been widely evaluated in various in vitro assays that suggested its protective effect against oxidative damage and development of inflammation in tissues (Matkowski et al. 2013; Wauthoz et al. 2007). Our study was aimed at determination of the impact of mangiferin extracted from Belamcanda chinensis rhizome on the model of experimental colitis induced by rectal TNBS administration.

Based on results of the present study mangiferin did not exert any toxic effect on colon tissues. In the group of rats receiving mangiferin, but non-exposed to TNBS (M10, M30 and M100), no significant changes of all studied parameters were observed in comparison to the control group (group K). Rats exposed to TNBS (group C) developed colitis leading to body weight loss, colon mass index increase as well as macro- and microscopic damage in intestinal tissues compared to the control group. The inflamed colon of rats with TNBS-induced colitis demonstrated mucosal hyperemia, edema, increased mural thickness and distortion of crypts. Additionally, TNBS-induced colitis involved epithelial layer damage, depletion of goblet cells with insufficient production of mucus and a transmural infiltration of inflammatory cells. The mechanism of TNBS-induced intestinal inflammation is likely associated with accumulation of inflammatory cells in the mucosa and impairment of the epithelial monolayer on the mucosal intestinal surface, reflected by an excessive production of pro-inflammatory cytokines and induction of oxidative damage. In the present study, the inflammatory changes in colon tissues caused by TNBS administration were associated with increased TNF-α, IL-17, MDA expression and decreased SOD activity compared to the control group.

Based on the macroscopic, histological and biochemical results obtained for mangiferin pretreated groups with subsequent experimental colitis (CM10, CM30 and CM100), it can be concluded that mangiferin alleviates the course of experimental IBD. Mangiferin at doses of 30 and 100 mg/kg, but not at the dose of 10 mg/kg, reduced the intensity of experimental colitis in rats. Mangiferin at the dose of 100 mg/kg reduced tissue damage caused by TNBS to a greater extent than at the dose of 30 mg/kg, indicating that the protective effect of mangiferin against colon tissue injury is dose dependent. There was no statistically significant difference in measured parameters between control animals and those receiving mangiferin at the highest dose (100 mg/kg) and TNBS. That suggests that mangiferin at the highest dose not only reduces, but also prevents the development of colitis. Additionally, a decrease of the microscopic damage score accompanied by decreased TNF-α, IL-17 and MDA concentrations and increased SOD activity demonstrates the existence of a significant correlation between the degree of severity of microscopic injury and the anti-inflammatory and antioxidant action of mangiferin. Our results demonstrated that all studied doses of mangiferin exerted the antioxidant activity, but the anti-inflammatory effect is evident only at the highest dose. Tissue damage was the least pronounced when mangiferin was used at the highest dose, indicating that the antioxidant activity is insufficient to alleviate the course of experimental inflammatory bowel disease and that the anti-inflammatory activity of mangiferin plays a pivotal role in the mechanism of protective action of the compound.

TNF-α and IL-17 play an important role both in the pathogenesis of IBD and in experimental TNBS-induced colitis. Both cytokines participate in the development of intestinal inflammation ensuring regulation of proliferation, maturation, migration of inflammatory cells and their subsequent activation, enhancement of maturation of dendritic cells and increasing expression of matrix metalloproteases, chemokines and other pro-inflammatory cytokines (Shen and Durum 2010; Strober and Fuss 2011). Thus, a decrease of infiltration and activation of lymphocytes and a subsequent decrease of production of pro-inflammatory cytokines should contribute to reduction of intensity of inflammatory response. That kind of action has been demonstrated in this study and it is in line with targets of modern biological pharmacotherapy. In this study a trend was observed for lower secretion of TNF-α and IL-17 from intestinal tissue in animals pre-treated with mangiferin before the TNBS administration, compared to animals receiving only TNBS. TNF-α and IL-17 concentrations were lower when mangiferin was used at the dose of 100 mg/kg. Mangiferin (30 and 100 mg/kg) decreased size of ulcerated and inflamed areas, restored the epithelial layer, augmented amount of goblet cells and acid glycoproteins (mainly sialomucins) and improved adherents and tight junctions leading to appropriate cover of mucus layer over the intestinal epithelium. Together, those results demonstrate that mangiferin improves integrity of the intestinal mucosal epithelial barrier. Restoration of the intestinal barrier, separation of the gut lumen from the immune system of lamina propria and reduced infiltration of granulocytes, macrophages and lymphocytes into the lamina propria indicate that mangiferin decreases the excessive and uncontrolled antigenic stimulation of non-specific and specific immune responses and inhibits development of inflammation in the intestinal wall.

It is known that overproduction of reactive oxygen species (ROS) in intestinal mucosal cells induces the immune response leading to the injury of intestinal epithelial cells, disruption of integrity of the intestinal barrier and initiation of the intestinal inflammation. One of the most important processes involving free radicals is lipid peroxidation associated with oversynthesis of MDA (Pavlick et al. 2002). Our study demonstrated that mangiferin at all studied doses decreased the MDA level in colon tissues that was significantly increased after the administration of TNBS. It suggests an important antioxidant activity of mangiferin. One of several elements of the cell defense system is superoxide dismutase—the enzyme that catalyzes the reaction of dismutation of the superoxide anion. In the present paper only mangiferin administered at the highest dose increased SOD activity in TNBS-induced inflamed colon tissue, which suggests that the enzyme may be a less sensitive predictor of oxidative stress compared to MDA.

The anti-inflammatory action of mangiferin demonstrated in this paper is in line with results reported by other authors. Márquez et al. (2010) demonstrated a protective effect of the Mangiferae indica extract (containing mangiferin) on colonic tissues in experimental colitis caused by dextran sulfate sodium. Tested extract decreased serum IL-6 and TNF-α concentrations and reduced TNF-α and TNF-α type 2 receptor expression in colonic tissues. Recently, some studies using pure mangiferin have been also published. However, all of them used the murine model of colitis. Jeong et al. (2014) demonstrated that in TNBS-induced intestinal inflammation in mice mangiferin decreased the degree of colon shortening and reduced macroscopic lesions. Furthermore, mangiferin diminished expression of TNF-α, IL-1β, IL-6, inhibited IL-1R-associated kinase phosphorylation and activation of the nuclear factor kB. Similarly, the murine TNBS model of colitis was used by Lim et al. (2016) who demonstrated that TNBS-induced increase in TNF-α and IL-17 levels were reduced by pre-treatment with mangiferin. Two more papers reported the murine colitis model but the disease was induced by a different irritating factor—dextran sodium sulfate (DSS) in drinking water. Dou et al. (2014) demonstrated mainly the anti-inflammatory action of mangiferin (TNF-α, NF-κB, iNOS), whereas Somani et al. (2016) focused mainly on oxidative stress parameters (CAT, GSH, SOD, MDA, MPO), but also revealed some anti-inflammatory effects of mangiferin.

To our best knowledge, our study is the first to demonstrate the protective effect of pure mangiferin in experimentally induced colitis in rats. Besides some basic parameters of inflammation and oxidative stress that had been also studied by other researchers, we broadened our study by a more detailed histopathological evaluation. Besides the standard hematoxylin-eosin staining, demonstrating some typical micropathological damage to the colon tissue, we used alcian blue and Giemsa staining. The first reveals the colonic mucus layer with goblet cells producing sialomucins, whereas Giemsa staining allows to assess the magnitude of inflammatory cell infiltration to the colonic tissue. Both these processes play a very important role in the pathogenesis and determination of the degree of damage in colitis. Based on these findings we may state that mangiferin increases the integrity of epithelial layer and significantly decreases colonic infiltration by inflammatory cells.

Restoration of integrity of the mucosal epithelial barrier, decreased production of pro-inflammatory cytokines, prevention of lipid peroxidation and increased activity of antioxidant enzymes could be considered as mechanisms involved in the beneficial effect exerted by mangiferin. Considering findings from our and other studies it can be assumed that mangiferin alleviates the course of experimental colitis in rats and mice. Explanation of detailed molecular mechanisms involved in the beneficial effect of mangiferin requires further investigations of, e.g., the impact of mangiferin on the expression of protective proteins (e.g., trefoil factor 3, mucins), transcription factors, caspases or mitogen-activated protein kinases engaged in the activation of the inflammatory cascade in IBD.

In conclusion, mangiferin alleviates the colonic inflammatory changes in TNBS-induced experimental colitis in rats. Pre-treatment with mangiferin at the doses of 30 and 100 mg/kg diminishes macro- and microscopic colonic damage and leads to reduced MDA concentration. Moreover, pre-treatment with mangiferin at the dose of 100 mg/kg decreases TNF-α and IL-17 concentrations and increases SOD activity. The antioxidant activity is evident after administration of mangiferin at all studied doses and the anti-inflammatory effect is observed only after administration of mangiferin at the highest dose (100 mg/g). In parallel, the least pronounced injury of colon tissue develops following administration of mangiferin at the highest dose. It may, therefore, be concluded that the crucial mechanism underlying the protective impact of mangiferin observed in this report is associated with the compound’s anti-inflammatory action and that its antioxidant action is of an accessory character.