Evaluation of Apigenin and Piperine against DSS-induced colitis in mice using body weight, colon weight, colon length and disease activity index

Introduction

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that affects the inner lining of the colon and rectum, leading to persistent inflammation. Colitis more broadly refers to inflammation of the colon, marked by mucosal damage, loss of epithelial integrity, infiltration of immune cells, and increased intestinal permeability (Owusu et al., 2020) [1] . Experimentally induced colitis closely resembles human ulcerative colitis, presenting with diarrhoea, rectal bleeding, weight loss, colon shortening and tissue changes. Persistent inflammation of the colonic lining impairs intestinal function, sustaining chronic disease and relapse (Beiranvand and Dehghan, 2025) [2] . Ulcerative colitis (UC) is a major cause of persistent vomiting and diarrhoea in animals, particularly dogs, cats, and pigs. In growing pigs, diarrhoea resulting from colon inflammation poses a serious challenge for the swine industry, as it compromises animal welfare, slows growth, and increases feed conversion ratios (Panah et al., 2021) [3] . DSS, a sulfated polysaccharide, triggers colonic inflammation by directly damaging epithelial cells, disrupting tight junctions, and compromising the mucosal barrier (Naveen et al., 2023) [4] . Corticosteroids are widely used as standard anti-inflammatory treatments for ulcerative colitis because of their strong immunosuppressive effects. Dexamethasone, a synthetic glucocorticoid, helps by reducing inflammatory cytokine production, inhibiting immune cell activity, and easing mucosal inflammation. However, long-term corticosteroid use can lead to complications such as metabolic imbalances, weakened immunity, delayed wound healing, and greater infection risk. These drawbacks highlight the need for safer alternatives, especially those derived from natural sources (Radlovic et al., 2025) [5] . Apigenin is a naturally occurring flavonoid found in many fruits, vegetables, and medicinal plants. It has been widely studied for its anti-inflammatory, antioxidant, and protective effects. These benefits arise from its ability to suppress pro-inflammatory cytokines, block inflammatory signalling pathways, and reduce oxidative stress (Singh et al., 2025) [6] .

Piperine, the primary alkaloid in Piper nigrum, is recognized for its strong anti-inflammatory and antioxidant properties. It has been shown to suppress the release of inflammatory mediators, thereby contributing to its protective effects (Sree et al., 2025) [7] . Piperine also enhances the bioavailability of other compounds, thereby boosting their therapeutic effectiveness when coadministered (Han et al., 2023) [8] . Recent studies indicate that combining natural compounds can provide stronger protection against colitis by addressing multiple disease mechanisms, including epithelial barrier disruption, oxidative stress, and inflammatory cytokine production (Boobalan et al., 2015) [9] . Considering the importance of the DSS-induced colitis model, the challenges of corticosteroid therapy, and the promising pharmacological properties of apigenin and piperine, this study aims to evaluate their protective effects against dextran sodium sulfate (DSS)-induced colitis in mice.

Materials and Methods

Drugs

Dextran sodium sulfate was procured from BLD Pharma tech, India-(Cat. No.BD123894), Apigenin from Tokyo Chemical Industry, Japan-(Cat. No. A1514) and Piperine from Tokyo Chemical Industry, Japan-(Cat. No. P0460).

Experimental Animals

Thirty adult male BALB/c mice (25-30 g) were obtained from Jeeva Life Sciences and housed under standard laboratory conditions (22-24 °C temperature, 30-70% humidity, 12 h light/dark cycle). After one week of acclimatisation, animals were maintained on a standard pellet diet with ad libitum access to water. All procedures were approved by the Institutional Animal Ethics Committee (Approval No. 06/30/C.V.Sc, Hyd.IAEC). Mice were randomly divided into five groups (n = 6) and treated for 14 days. III DSS (2%) per orally from 1 st day to 14 th day + Apigenin (5 mg/kg b.wt per orally from 1 st day to 14 th day) + Piperine (12.5 mg/kg b.wt per orally from 1 st day to 14 th day) 6

IV DSS (2%) per orally from 1 st day to 14 th day + Apigenin (10 mg/kg b.wt per orally from 1 st day to 14 th day) + Piperine (25 mg/kg b.wt per orally from 1 st day to 14 th day) 6

V DSS (2%) per orally from 1 st day to 14 th day + Dexamethasone sodium (1 mg/kg b.wt from 1 st day to 14 th day i/p) 6

Body weights

The body weights (g) were recorded on day 0 and subsequently on 7 th and 14 th days of the experimental period using an electronic weighing balance (Shimadzu Pvt. Ltd.).

Disease Activity Index (DAI)

The Disease Activity Index (DAI) was applied to assess the severity of intestinal injury, with scoring performed according to the DSS-induced damage criteria outlined by Zhao et al. (2023) [10] . Assessment of DAI is based on the weight change, faecal trait and stool occult blood (Table 2).

Colon weight and length

At the end of the experiment, the experimental animals were anesthetized with 4% chloral hydrate. The colon was collected, measured the length and weighed.

Statistical analysis

Data were expressed as mean±SE (n = 6). Statistical analysis was performed using SPSS software (version 21) by one-way ANOVA, followed by Duncan's multiple range test for post hoc comparisons. A value of p<0.05 was considered statistically significant.

Results

Body weights

The body weights of mice in Group I were significantly (p<0.05) higher than those in all other groups throughout the experimental period. Group II showed a significant reduction in body weight compared to the control group on days 7 and 14. In contrast, Groups III and IV exhibited a significant (p<0.05) improvement in body weight when compared to Group II. Furthermore, no significant difference was observed between Groups IV and V, indicating comparable effects on body weight. The findings are outlined in Table 3 and graphically represented in Figure 1.

Disease activity index (DAI) of colon

The disease activity index (DAI) in Group II was significantly (p<0.05) higher compared to Group I. Treatment groups III and IV showed a significant (p<0.05) reduction in DAI in a dose-dependent manner when compared to Group II. However, a significant (p<0.05) difference was also observed between Groups IV and V, with Group V exhibiting a greater reduction in DAI (Table 4; Fig. 2).

A significant (p<0.05) decrease in colon length was observed in Group II compared to the control group. In contrast, Groups III and IV demonstrated a significant (p<0.05) restoration of colon length in a dose-dependent manner relative to Group II. Furthermore, Group V showed a significantly (p<0.05) higher colon length compared to Group IV (Table 5; Fig. 3).

Colon weight in Group II was significantly (p<0.05) reduced compared to Group I. In contrast, Groups III and IV showed a significant (p<0.05) increase in colon weight when compared to Group II. However, no significant difference was observed between Groups IV and V (Table 6; Fig. 4).

Discussion

Inflammatory bowel disease (IBD) comprises a group of chronic disorders marked by recurrent intestinal inflammation, affecting both humans and domestic animals such as dogs and cats (Pratscher et al., 2024) [11] . The DSSinduced colitis model is a well-established method for studying ulcerative colitis (UC) (Yang and Merlin, 2024) [12] . DSS causes damage to the colonic epithelium and disrupts barrier function, facilitating the entry of bacteria and inflammatory substances into the mucosa, which initiates the disease process (Eichele and Kharbanda, 2017) [13] . Management of UC largely targets the reduction of abdominal discomfort and control of immune-mediated inflammation (Sandborn, 2013) [14] . Apigenin, a dietary flavonoid abundant in plant-derived foods and beverages, possesses significant pharmacological activities (Siddiquee et al., 2025) [15] . Oral administration has been shown to alleviate DSS-induced colitis through protection of the intestinal barrier and improvement in colon structure (Hu et al., 2023) [16] . Piperine, the bioactive compound of black pepper and long pepper, possesses significant pharmacological and anti-inflammatory activities. It has been reported to attenuate colorectal inflammation and enhance barrier function in experimental colitis models, including DSS-induced colitis (Gupta et al., 2015; Hu et al., 2025) [17,18] . In this study, mice in disease control group II showed a significant reduction in body weight over 14 days compared to other groups, likely due to impaired gastrointestinal absorption and fluid loss during diarrhoea, consistent with previous reports (Qu et al., 2021; Kalyankumarraju et al., 2021; Zheng et al., 2021) [19,20,21] . In contrast, treatment groups III and IV exhibited marked weight gain relative to group II, indicating the protective effects of apigenin and piperine. This improvement may be attributed to epithelial repair, restoration of metabolic activity, increased goblet cell numbers, and enhanced cellular biosynthesis. These findings agree with earlier studies (Hu et [22,23,24,25] . A significant reduction in colon weight and length was observed in disease control group II compared to group I, likely due to acute inflammation and epithelial cell loss. This is supported by gross lesions showing severe congestion and mucosal erosions. These findings are consistent with earlier reports (Mao et [26,27,28] . In contrast to this, a marked improvement in colon weight and length was observed in treatment groups III and IV, supported by histological evidence of epithelial regeneration and normalization of villi structure. This protective effect is likely due to the anti-inflammatory actions of apigenin and piperine, consistent with reported findings (Fu et al., 2022; Hu et al., 2025) [29,18] . Disease severity assessed using the Disease Activity Index (DAI), based on weight loss, stool consistency, and faecal blood, was markedly elevated in group II compared to group I, reflecting mucosal injury, impaired absorption, and intestinal bleeding. These findings are consistent with previous reports (Tian et al., 2020; Lv et al., 2020; Niu et al., 2022) [31,32,33] . Treatment groups III and IV showed significant improvement, with reduced DAI scores, weight gain, and alleviation of diarrhoea, likely due to the antiinflammatory and mucosal-protective effects of apigenin and piperine, as supported by earlier studies (Hu et al., 2015; Ji et al., 2025) [22,25] . Group V demonstrated further improvement, with greater reduction in weight loss and bloody stools compared to group IV.

Conclusion

In conclusion, the present study revealed that DSS (2% per orally from 1 st day to 14 th day) effectively induced colitis, as evidenced by body weight loss, colon shortening, and elevated disease severity assessed by the Disease Activity Index (DAI), based on weight loss, stool consistency, and faecal blood. Treatment with apigenin and piperine synergistically ameliorated colitis, as demonstrated by restoration of colon weight, colon length, body weight, and a significant reduction in Disease Activity Index (DAI) scores.

Acknowledgement

AcknowledgementsThe authors are thankful to the College of Veterinary Science, P. V. Narsimha Rao Telangana Veterinary University (PVNRTVU), Rajendranagar, Hyderabad for providing the necessary facilities to carry out the research work.

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