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. 2024 Apr 29;7(5):1270–1277. doi: 10.1021/acsptsci.3c00323

Mangiferin, A Naturally Occurring Glucosylxanthone, Induces Apoptosis in Caco-2 Cells In Vitro and Exerts Protective Effects on Acetic Acid-Induced Ulcerative Colitis in Mice through the Regulation of NLRP3

Gangappa Dharmapuri †, Anil Kumar Kotha †, Suresh K Kalangi ‡,*, Pallu Reddanna †,*
PMCID: PMC11091985  PMID: 38751614

Abstract

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Inflammatory bowel diseases (IBD), an inflammatory disease, include Crohn’s disease and ulcerative colitis. Dysregulated autoimmune response to gut dysbiosis is mainly involved in the pathogenesis of IBD and is triggered by various inciting environmental factors. With its rising prevalence in every continent, IBD has evolved into a global disease, which is on the rise, affecting people of all ages. There is a growing incidence of IBD in the elderly population, as evidenced by epidemiological data. IBD is characterized by an inflammatory process that requires a lifelong treatment. The main challenge in IBD management is the adverse side effects associated with almost all of the currently available drugs. Hence, there is a search for drugs with more efficacy and fewer side effects. Natural products with great structural diversity and ease of modification chemically are being explored, as they were shown to control IBD by safely suppressing pro-inflammatory pathways. The present study aims at understanding the role of mangiferin, a COX-2 inhibitor isolated from tubers of Pueraria tuberosa in the treatment of IBD and colon cancer, in vitro on the Caco-2 human colon cancer cell line and in vivo in the acetic acid-induced IBD mouse model. In the acetic acid-induced colitis model, it prevented the decrease in length of the colon, mucosal erosion, and cellular infiltration in a dose-dependent manner. The expression levels of various pro-inflammatory markers like COX-2, IL1β, TNF-α, INF-γ, IL-6, NLRP3, and caspase-1 were downregulated in an acetic acid-induced mouse model on treatment with mangiferin in a dose dependent manner. Mangiferin also showed anticancer effects on Caco-2 cells by increasing the expression of Fas ligand, Fas receptor, FADD, caspase-8, and caspase-3 proteins, whereas Bid and Bcl-2 proteins showed decreased expression. These data suggest that mangiferin, an inhibitor of COX, induces apoptosis in colon cancer cells in vitro and protects mice from acetic acid-induced colitis in vivo.

Keywords: inflammatory bowel disease (IBD), Pueraria tuberosa (PT), ulcerative colitis (UC), nucleotide-binding domain, leucine-rich repeat containing proteins (NLRP3)

The two most common categories of inflammatory bowel disease (IBD) are supported by chronic inflammation of the intestine. Inflammatory bowel disease (IBD) is influenced by a complex interplay of genetic and immunological elements, and the etiology is far from fully understood. Microbiological and environmental factors such as smoking, diet, medication, topographical, social circumstances, and stress play a major role in the pathogenesis of IBD. The presence of intestinal flora is responsible to cause gut inflammation in most animal models of IBD. IBD is the result of immune dysfunction in the intestinal lining, which further results in damaging the epithelial layer leading to exposure of the mucosal immune system with antigenic material of gut lumen.1 Under physiological conditions, the intestinal lining is in the state of controlled inflammation and is regulated by an equilibrium of proinflammatory and anti-inflammatory cytokines. In modulating inflammation, cytokines play a key role and become a target of IBD therapy. An increase in the synthesis of nonspecific inflammatory mediators such as cytokines, chemokines, eicosanoids, and reactive oxygen species results in the infiltration of leukocytes to the sites of inflammation, enhancing tissue destruction and ultimately leading to clinical manifestation of disease. TNF-α and IL-6 play a key role in the inflammation of intestinal mucosa in animal models of IBD.2 In chronic cases of IBD, the risk of colorectal,3 melanoma, and nonmelanoma skin cancer (NMSC) may increase.4

All stages of tumorigenesis are affected by inflammation. Activation of the caspase-1 inflammasome leads to acute and chronic inflammation. A diverse population of immune cells, such as macrophages, neutrophils, dendritic cells, natural killer (NK) cells, and lymphocytes, triggers inflammation-associated tumor development. Inflammasome, a cytoplasmic complex composed of multiple proteins that serves as a molecular framework for initiating the activation of the cysteine protease caspase-1 orchestrates inflammation in immune cells. Upon activation of a specific nucleotide-binding domain, leucine-rich repeat-containing proteins such asAIM2-like receptors, or pyrin, inflammasome complexes are assembled.5 Among all inflammasomes, NOD-like receptor (NLRP) family pyrin domain (PYD)- containing 1 (NLRP1), NLRP3, NLR family CARD domain-containing 4 (NLRC4), and pyrin absent in melanoma 2 (AIM2) inflammasomes are most widely explored. inflammasomes are generally get initiated as soon as after the sensing of bacterial toxins, secretion system components, nucleic acids, pathogenic crystals, or any altered cellular components.6 NLRP1, NLRP3, NLRC4, NLRP6, and AIM2 influence cancer pathogenesis by regulating innate and adaptive immune responses, cellular death, proliferation, and/or the gut microbiota. Initiation of the inflammasome and IL18 signaling cascade predominantly confer protection in colitis-associated colorectal cancer.5

IBD is one of the major health issue affecting people worldwide with at least one in thousand persons in western countries7 and Indians topping the Southeast Asian countries (SEA).8 Present day therapy for IBD involves agents that suppress the immune system, leading to an increased risk for developing infections and anti-inflammatory drugs, which are accompanied by side effects. Hence there is a search for safer and more effective drugs and in this connection, there is a growing interest toward the natural products.9 Also, more and more patients globally are looking for complementary and alternative medicine (CAM). For the past 20–30 years natural products including medicinal herbs provided approximately half of the drugs developed for clinical use.10 Many plants sourced phenols such as flavonoids, coumarins, quinones, tannins, phenolic acids exert potential beneficial effects.11 Efficacy of these natural compounds depend on their ability to decrease the levels of inflammatory mediators and inflammatory cytokines.10 Several medicinal plants were shown to suppress inflammation and thus offering protection against many inflammatory diseases, including IBD. Many natural bioactive compounds have been used and some have proven promising in the treatment of IBD.12 Mangiferin a natural glycoside with its good permeability and bioavailability have been shown to reduce inflammation yet the exact mechanism is uncertain.13−19 In the present study, we have evaluated the effect of mangiferin, an inhibitor of cyclooxygenease-2 isolated from tubers of Pueraria tuberosa on acetic acid induced colitis model of mice in vivo and on human colon cancer cell line Caco-2 in vitro.

2.0. Materials and methods

2.1. Chemicals and Reagents

MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), Trypsin-EDTA, Tris, Ethylene diamine tetra acetic acid (EDTA), diethyl dithio-carbamate (DDC), Tween-20, Glycerol, Phenol, carrageenan, and ammonium sulfate were procured from Sigma Chemical Co. (St. Louis, MO). Celecoxib was a generous gift from Aurobindo Pharma (Hyderabad, India). The Mangiferin (100 mg/mL) stock solutions were prepared in dimethyl sulfoxide and further dilutions were made in PBS for in vitro and in vivo treatment. Polyclonal antibodies against COX-2, IL-1β, TNF-α, INF-γ, and IL-6 were purchased from Santa Cruz Biotechnology (California, U.S.A). iNOS antibody was procured from Thermo Fisher Scientific. All other chemicals and solvents were of analytical grade and were procured from authorized companies.

2.2. Extraction and Purification of Mangiferin

Pueraria tuberosa tubers were shade-dried for a week, and the powdered tubers were extracted with n-hexane/ethyl acetate/methanol at room temperature. These extracts were then filtered and evaporated under reduced pressure to obtain the respective extracts. The methanolic extract was chromatographed through the silica gel column using n-hexane and n-hexane-ethyl acetate in the stepwise gradient. The n-hexane-ethyl acetate (2:8) eluates, on concentration gave a yellow colored solid. This was further purified over a silica gel column using n-hexane-ethyl acetate step gradient to yield pure mangiferin. The purified mangiferin inhibited COX-1 activity by 79.4% and COX-2 activity by 45.9% (unpublished data).

2.3. Acute colitis Model

Male Blab/c mice weighing about 20–25 g were obtained from the National Institute of Nutrition, India. Animals were kept in stainless steel cages in a room with a 12 h light/dark exposure cycle. Commercially available diet and lab sterilized water were provided ad libitum throughout the course of experimentation. After 10 days of adaptation, the total number of 30 mice were divided in to 5 different groups (n = 6 mice/group): The control mice (group 1) received 2 mL of saline alone. Colitis induced mice (group 2–6) received 2 mL of 4% (v/v) acetic acid by rectal administration with a soft 6-Fr pediatric catheter, inserted into anus up to a length of 4 cm. The colitis induced mice (group 3–6) after 7 days of treatment were treated by oral administration of 5 mg/kg bw (group 3), 10 mg/kg bw (group 4), 20 mg/kg bw (group 5) mangiferin, and 20 mg/kg bw celecoxib (group 6) for 7 days. After 7 days of treatment, the colon was excised, opened longitudinally, and rinsed with normal saline solution. Tissue samples were taken for histopathologic examination and biochemical studies. The mice studied in this experiment was handled as per the guidelines for the care and use of Laboratory animals published by NIH (National Institute of Health). The experimental protocol was approved by Institutional Animal Ethics Committee (IAEC), University of Hyderabad, India and the Registration number allotted was 151/GO/ReBi/S/99/CPSCEA.

2.4. Visualization of typical colon length

The changes in the length of colon were visualized in the mice of control and treatment groups, and the data were analyzed.

2.5. Histology of colon tissue

Colon tissues taken from control and experimental animals were initially rinsed in PBS and fixed in Bouin’s fixative (70% saturated picric acid, 25% formaldehyde, and 5% glacial acetic acid) overnight. This was followed by the washing of tissues thoroughly with distilled water. Subsequently the tissues underwent dehydration by sequential immersion in 70%, 80%, and 90% alcohol and finally in absolute alcohol for 10 min each. After dehydration, the tissues were processed in alcohol and benzene (3:1 for 10 min, 1:1 for 10 min, and benzene and paraffin (1:1) for 10 min) to embed them in paraffin wax. The tissues were then placed in molten paraffin for 2–3 h to facilitate infiltration, after which they were allowed to harden. Thin sections (10 μm) were taken on Leitz microtome and mounted on poly lysine-coated slides. Sections were deparaffinized by incubating in xylene for 10 min, sequential rehydration in 90, 80, and 70% alcohol for 10 min each. The tissue sections were observed under light microscope at 400× magnification, and photographs were taken.

2.6. SDS PAGE and Western Blotting

Colon tissue was homogenized in 100 mM Tris-HCl (pH 8.0) buffer containing 0.3 M mannitol, 1 mM EGTA, 1 mM EDTA, 4 mM K2HPO4, 1 mM DTT, 1 mM Sodium orthovanadate, 0.1% SDS, 2 mM PMSF and 40 μL/ml of complete protease inhibitor solution. The homogenate was centrifuged for 30 min at 10,000 rpm at 4 °C and the resultant supernatant was used for SDS-PAGE and Western blot analysis. Protein content in the supernatant was quantified using the Bradford method (Bradford, 1976). SDS-PAGE and Western blot analyses for the detection of COX- 2, IL1β, TNF-α, INF-γ, NLRP3, Caspase-1, IL-6 and β-actin in the colon tissue homogenates were performed.

2.7. Estimation of Histamine levels

Colonic tissue (0.5 g) was homogenized in 1 mL of 0.4 N perchloric acid. An aliquot of the supernatant fluid was transferred to a 25 mL glass-stoppered tube containing 0.5 mL of 5 N NaOH, 1.5 g of solid NaCl and 10 mL n-butanol. The tubes were shaken to extract histamine from the butanol layer. After centrifugation, the aqueous phase was removed by aspiration. The histamine formed was measured on a spectrophotometer, with an excitation wavelength of 360 nm and fluorescence wavelength of 450 nm.12,20 Celecoxib was used as a positive control.

2.8. Cell Culture

The Human colon cancer cell line, Caco-2 was obtained from NCCS (Pune, India), were grown, and maintained in RPMI media supplemented with 10% heat-inactivated FBS, 100 IU/ml penicillin, and 100 μg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 °C. The cells were sub cultured twice each week and seeded at a density of approximately 2 × 105 cells/ml fresh culture medium with 10% FBS. For LPS and mangiferin treatments, either LPS (1 μg/mL) or mangiferin (10, 20, 30 μM respectively) were added into the medium.

2.9. Cell Viability assay

Caco-2 cells were seeded in 96 well plates at a density of 5× 104 cells/well and exposed to varying concentrations of mangiferin (10 nM to 100 μM) for 48–72 h. Subsequently the cell viability was determined colorimetrically by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay where the cells were incubated with 20 μL of MTT (5 mg/mL) at 37 °C for 4 h. After dissolving the crystals in DMSO the plates were read in a microtiter plate reader at 570 nm. The impact of mangiferin on growth inhibition was evaluated by calculating the percentage of cell viability; cells treated with the PBS alone were considered as control. Each concentration was tested in three independent experiments each conducted with four replicates. Percent growth in the treated samples was calculated with relation, to the control. The IC50 was determined from cell survival plots.

2.10. Preparation of whole-cell extracts and immunoblot analysis

Caco-2 cells were incubated with35 μM mangiferin for different time periods (3h, 6h, 12, 24), and the cells were lysed as per the methods described previously.21 For the preparation of whole-cell extract, cells were rinsed with PBS and suspended in a lysis buffer [20 mM Tris, 1 mM EDTA, 150 mM NaCl, 1% NP40 (Nonidet P40), 0.5% sodium deoxycholate, 1 mM 2-glycerophosphate, 1 mM Na3VO4 (sodium orthovanadate), 1 mM PMSF, 10 μg/mL leupeptin and 20 μg/mL aprotinin]. The mixtures were shaken at 4 °C for 30 min and then centrifuged (10 000 g) for 10 min, and the supernatants were collected as whole-cell extracts. The protein content was quantified by the Bradford method.22 An equal amount of total cell lysate was separated by SDS/8–12%-(w/v)-PAGE along with protein molecular mass standards and then transferred on to nitrocellulose membranes. Membranes were stained with 0.5% Ponceau S in 1% acetic acid to check the transfer. The membranes were blocked with 5% (w/v) nonfat dried skimmed milk powder and then incubated with the primary antibodies in 10 mL of antibody-diluted buffer (1 × Trisbuffered saline and 0.05% Tween 20 with 5% milk) with gentle shaking at 4 °C for 8–12 h and then incubated with peroxidase-conjugated secondary antibodies. The blots were probed with β-actin antibodies to confirm equal protein loading. The expression levels of Bcl-2 were decreased, and caspase −3 was increased in mangiferin treated cells.

3. Results and Discussion

3.1. Effect of Mangiferin on mice colitis model In Vivo

3.1.1. Mangiferin restores colon length in acetic acid induced colitis mice

Shortening of colon length, an important characteristic feature of colitis, was observed in mice rectally administered with 2 mL of 4% (v/v) acetic acid. The length of the colon was shortened in acetic acid treated mice from 5.8 to 4.3 cm when compared to control. However, when the mice were treated with different dosages (5 mg/kg body weight (bw), 10 mg/kg bw, 20 mg/kg bw) of mangiferin/day, the colon length was restored to normal (Figure 1).

Figure 1.

Figure 1

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Visualization of typical colon length: Mice were induced with 2 mL of 4% (v/v) acetic acid and then with mangiferin daily through oral gavage for 7 days control mice received only saline.; 1. Control (2 mL of saline); 2. Two ml of 4% (v/v) acetic acid induced mice; 3. Five mg/kg mangiferin treated and 2 mL of 4% (v/v) acetic acid induced mice; 4. Ten mg/kg mangiferin and 2 mL of 4% (v/v) acetic acid induced mice; 5. Twenty mg/kg mangiferin and 2 mL of 4% (v/v) acetic acid induced mice.

3.1.2. Mangiferin restores body weight of mice with colitis

Mice were checked for body weight after the induction of colitis. On acetic acid treatment, the body weights of mice were found to be decreased by 15–20% compared to their normal weight. This decrease in body weights were restored to normal on treatment with different doses of mangiferin. The body weight of mice treated with 5 mg/mL mangiferin was gained by 5%, body weight was gained by 12% on treatment with 10 mg/mL mangiferin, and 20 mg/mL mangiferin treated mice were equal to control mice. Celecoxib treated mice could also restore their body weight to normal (Figure 2)

Figure 2.

Figure 2

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Mangiferin restores body weight: Body weight of mice was checked after treatment with mangiferin.

3.1.3. Effect of mangiferin on colon histopathological changes induced by acetic acid

After the induction of colitis with acetic acid, histopathological studies were carried out. The control group (a) showed distinct colon tissue with smooth intact mucosa and no erosion of tissue or inflammatory cell infiltration (Figure 3). In the colitis model group (b) mucosal erosion, granulation tissue hyperplasia, glandular enlargement was observed (indicated by black arrows). In the mangiferin treated groups ((c, d, and e)), mucosal erosion and cellular infiltration was decreased dose dependently (Figure 3).

Figure 3.

Figure 3

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Mangiferin reduces inflammation in the colon tissue: After induction of colitis with acetic acid, mice were treated with different doses of mangiferin. Colon tissues were used to identify the effects of mangiferin on ulcerative colitis in vivo. a) Control group b) Colitis induced group with 2 mL of 4% (v/v) acetic acid c) 2 mL of 4% (v/v) acetic acid + mangiferin (5 mg/kg body weight), d) 2 mL of 4% (v/v) acetic acid + mangiferin10 mg/kg body weight, (e) 2 mL of 4% (v/v) acetic acid + mangiferin 20 mg/kg body weight,.

3.1.4. Effect of mangiferin on the expression of pro-inflammatory proteins in colon tissue of mouse model of colitis

Further studies on protein expression were carried out to check whether mangiferin has any inhibitory effect on the expression of pro-inflammatory proteins, COX-2, IL-1β TNF-α, INF-γ, NLRP3, Caspase-1, and IL-6. From the results presented in Figure 4, we have noticed that pro-inflammatory proteins were significantly upregulated in response to acetic acid treatment. Mangiferin treatment to the mice with colitis potently reduced pro-inflammatory protein levels. This down regulation of pro-inflammatory proteins indicates strong anti-inflammatory effects of mangiferin in vivo on colitis induced mice.

Figure 4.

Figure 4

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Effect of Mangiferin on the expression of COX-2, IL-1β, TNF-α, INF-γ and IL-6 NLRP3 proteins in the colon tissue of control and experimental mice: Western blot analysis showing the expression of COX-2, IL-1β, TNF-α, INF-γ, NLRP3, Caspase-1, and IL-6 proteins in the colon tissues obtained from mice treated with (lane 1) saline, (lane 2) 2 mL of 4% (v/v) acetic acid, (lane 3) 2 mL of 4% (v/v) acetic acid + mangiferin (5 mg/kg body weight), (lane 4) 2 mL of 4% (v/v) acetic acid + mangiferin 10 mg/kg body weight, (lane 5) 2 mL of 4% (v/v) acetic acid + mangiferin 20 mg/kg body weight, (lane 6) 2 mL of 4% (v/v) acetic acid + celecoxib 20 mg/kg body weight. Densitometric analysis of COX-2, IL-1β, and TNF-α, INF-γ, IL-6, NLRP3 levels from colon tissues of mice subjected to treatments as indicated. β-actin was used as loading control. The values represent the mean ± SEM for three independent experiments.

3.1.5. Effect of Mangiferin on histamine levels of colon tissue

Levels of histamine were studied in the colitis mice model. Histamine levels were drastically increased in acetic acid induced colitis mice, and these levels were decreased on treatment with mangiferin. The control mice showed less than 0.5 ng/mL of histamine. In acetic acid induced mice, histamine levels increased to 1.5 ng/mL. On treatment with magiferin the histamine levels decreased dose dependently (Figure 5).

Figure 5.

Figure 5

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Estimation of Histamine content: The levels of histamine were determined in the homogenized tissue samples. Celecoxib was used as positive control. (1) saline, (2) 2 mL of 4% (v/v) acetic acid, (3) 2 mL of 4% (v/v) acetic acid + mangiferin (5 mg/kg body weight), (4) 2 mL of 4% (v/v) acetic acid + mangiferin10 mg/kg body weight, (5) 2 mL of 4% (v/v) acetic acid + mangiferin 20 mg/kg body weight, (6) 2 mL of 4% (v/v) acetic acid + celecoxib 20 mg/kg body weight

3.2. Effect of Mangiferin on Colon cancer cell line in vitro

3.2.1. Effect of mangiferin on Caco-2 cell cytotoxicity

In chronic cases of IBD there is a risk of colon cancer. In-vitro studies were taken up to study the role of mangiferin in colon cancer cells. Caco-2 cells were treated with mangiferin at different concentrations, and cell viability was measured via MTT assay. Different concentrations of mangiferin ranging from 0.01 to 100 μM were used to determine cell viability. Mangiferin, showed growth inhibition on Caco-2 cells with an IC50 of 40 μM (Figure 6).

Figure 6.

Figure 6

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Mangiferin exerts antiproliferative effect in Caco-2 Cells. Caco-2 cells were treated with various concentrations of mangiferin and the cell viability was assessed using the MTT assay. The values presented as the means + SEM for three independent experiments.

3.2.2. Effect of Mangiferin on the expression of FasL, Fas receptor, FADD, Bid, caspase-8, Bcl-2 and caspase-3 proteins

Caco-2 cells on treatment with mangiferin for different time points showed an increase in the expression of FasL, Fas receptor, FADD, caspase-8 and caspase-3 proteins, whereas Bid showed time dependent decreased expression. The expression of Bcl-2 is associated with mitochondrial membrane integrity and plays a crucial role in the regulation of apoptosis. A time dependent decrease in Bcl-2 protein levels was observed on treatment with mangiferin for different time points (Figure 7).

Figure 7.

Figure 7

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Western blot analysis showing the expression of various proteins on treatment with mangiferin: Caco-2 cells treated with mangiferin (35 μM conc) at various time points samples were centrifuged (2000 rev./min for 2 min). A 100–200 μg sample of protein was loaded on to the gel and Western-blot analysis was carried out using the corresponding antibodies.

Inflammatory bowel disease, which includes both Crohn’s disease and Ulcerative Colitis, is caused by chronic inflammation of the colon. The currently available drugs for inflammation bowel disease are either not effective enough or associated with side effects. In this scenario, there is a need for the discovery of drugs with better efficacy and safety. Natural compounds have been used for different ailments. Extracts of many plants are known to be used in the treatment of IBD but their exact mechanism of action is still unknown. Mangiferin is known to act as anti-inflammatory, antioxidant and antidiabetic by targeting MAP kinases and apoptotic pathways.23−26 In this study we exploited acetic acid induced colitis model to check the efficacy of mangiferin on IBD. Several studies have described the acetic acid induced colitis as a model that is consistent and straightforward to investigate IBD there by establishing an ideal environment for collecting and analyzing colon tissue.27,28

In this study, 2 mL of 4% (v/v) acetic acid was given to mice intra rectally to induce IBD. Reduction in the length of colon a characteristic feature of IBD was clearly seen in animals induced with acetic acid. The reduction in body weight, which was observed in acetic acid induced mice, was restored on treatment with different doses of mangiferin. Infiltration of neutrophils were observed in histopathological sections of colon tissue of mice induced with acetic acid. Expression of pro-inflammatory proteins like COX-2, TNF-α, IL-1β, and IL-6, revealed the symptoms of IBD on treatment with acetic acid. Dysregulated activation of NLRs (nucleotide binding domain, leucine rich repeat containing receptors) leads to inflammation and are responsible for several metabolic disorders, autoimmune diseases, and inflammatory disorders.28 “Inflammasome” formation is the result of NLRs activation, and this results in the activation of procaspase-1 to caspase1 and this finally results in conversion inactive form of several substrates such as pro IL-1β and IL-18 into their active forms. NLRP3 is the most studied inflammasome, and this contributes to several inflammatory diseases. Studies have demonstrated that mice lacking NLRP3 exhibit increased susceptibility to colitis and colitis-associated colorectal cancer induced by the DNA damaging agent azoxymethane (AOM) and chemical colitogenic dextran sulfate sodium.29 alternative study has proposed that mice deficient in NLRP3 exhibit increased resistance toward DSS induced colitis compared with wild-type mice (ref), whereas another investigation has reported a comparable tumor burden between wild-type mice and mice deficient in NLRP3, treated with AOM and DSS.29 It is important to note that mice lacking ASC and caspase-1 are also vulnerable to DSS-induced colitis and colitis-associated colorectal cancer, providing substantial evidence to supporting a protective role of inflammasomes in an inflammatory model of colorectal cancer.5 Mangiferin reduces the expression levels of NLRP3 and caspase 1 when treated dose dependently. In this study, mangiferin treatment was effective in restoring colon length, reducing infiltration of neutrophils, and decreasing the expression of pro-inflammatory proteins. Celecoxib was used as a standard anti-inflammatory drug for comparison, and the mangiferin effect was comparable to celecoxib. Markers of allergy are normally seen in IBD patients. Histamine a major contributor of allergic reaction was estimated in this study, and it was found that levels of histamine were increased on induction of colitis, whereas mice treated with mangiferin showed decreased histamine levels dose dependently. These studies thus demonstrate the antiallergic effects of mangiferin.

Ulcerative colitis leads to colon cancer in several IBD patients as evidenced by many studies. 10–15% of deaths in IBD are linked to colon cancer.30 In this scenario, the effect of mangiferin was tested on the colon cancer cell line Caco-2 in vitro. Mangiferin inhibited the growth of Caco-2 cells with an IC50 value of 40 μM. The expression levels of antiapoptotic and pro-apoptotic proteins like Bcl-2, FasL, Fas receptor, FADD, Bid, caspase-8 and caspase-3 was studied by Western blot analysis. In mangiferin treated cells at different time points, the expression of antiapoptotic proteins was decreased. FasL, Fas receptor, FADD, Bid, caspase-8 and caspase-3, which are key players of apoptosis, showed time dependent activation in Caco-2 cells.

4. Conclusions

Mangiferin, an anti-inflammatory compound isolated from Pueraria tuberosa, ameliorated the acetic acid induced ulcerative colitis in mice in terms of restoration of colon length and body weight in a dose dependent manner. Mangiferin also decreased the expression of inflammatory proteins like COX-2, IL-1β TNF-α, INF-γ, NLRP3, Caspase-1, and IL-6, which were elevated in colitis induced mice. Mangiferin also showed anticancer effect on Caco-2 cells in vitro with an IC50 value of 40 μM, which are mediated by enhanced expression of pro-apoptotic proteins while decreasing the expression of antiapoptotic proteins (Figure 8).

Figure 8.

Figure 8

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Schematic representation on the effects of mangiferin on Acetic acid-induced ulcerative colitis in mice and on Caco-2 cells in vitro.

Acknowledgments

This study was supported by DST-DRL funded project VI/D&P/560/2016-17/TDT (G-1) and BSR Faculty Fellowship from the University Grants Commission, (UGC), Govt of India to PR (Award Letter No. F. 18-1/2011 (BSR). DG acknowledges UGC for the award of Dr.D.S.Kothari Postdoctoral Fellowship, Government of India for providing postdoctoral fellowship (UGC-DSKPDF) (No.F.4-2/2006 (BSR) BL/17-18/0307). KAK acknowledges the support of the Council of Scientific and Industrial Research (CSIR) for Senior Research Associate fellowship (No (13(8956-A)/2017 Pool).

Author Present Address

$ Molecular Microbiology and Immunology Division, CSIR-Central Drug Research Institute, Sector-10, Janaki Puram Extension, Lucknow 226031, India

The authors declare no competing financial interest.

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