Anti-ulcerative colitis effects of chemically characterized extracts from C alliandra haematocephala in acetic acid-induced ulcerative colitis

Abstract

Background: Ulcerative colitis is a chronic immune-mediated inflammatory bowel disease that involves inflammation and ulcers of the colon and rectum. To date, no definite cure for this disease is available. Objective: The objective of the current study was to assess the effect of Calliandra haematocephala on inflammatory mediators and oxidative stress markers for the exploration of its anti-ulcerative colitis activity in rat models of acetic acid-induced ulcerative colitis. Methods: Methanolic and n-hexane extracts of areal parts of the plant were prepared by cold extraction method. Phytochemical analysis of both extracts was performed by qualitative analysis, quantitative methods, and high-performance liquid chromatography (HPLC). Prednisone at 2 mg/kg dose and plant extracts at 250, 500, and 750 mg/kg doses were given to Wistar rats for 11 days, which were given acetic acid on 8th day through the trans-rectal route for the induction of ulcerative colitis. A comparison of treatment groups was done with a normal control group and a colitis control group. To evaluate the anti-ulcerative colitis activity of Calliandra haematocephala, different parameters such as colon macroscopic damage, ulcer index, oxidative stress markers, histopathological examination, and mRNA expression of pro and anti-inflammatory mediators were evaluated. mRNA expression analysis was carried out by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Results: The phytochemical evaluation revealed polyphenols, flavonoids, tannins, alkaloids, and sterols in both extracts of the plant. Results of the present study exhibited that both extracts attenuated the large bowel inflammation and prevented colon ulceration at all tested doses. Macroscopic damage and ulcer scoreswere significantly decreased by both extracts. Malondialdehyde (MDA) levels and nitrite/nitrate concentrations in colon tissues were returned to normal levels while superoxide dismutase (SOD) activity was significantly improved by all doses. Histopathological examination exhibited that both extracts prevented the inflammatory changes, cellular infiltration, and colon thickening. Gene expression analysis by RT-qPCR revealed the downregulation of pro-inflammatory markers such as tumor necrosis factor-alpha (TNF-α) and cyclooxygenase-2 (COX-2) whereas the anti-inflammatory cytokines including Interleukin-4 (IL-4) and Interleukin-10 (IL-10) were found to be upregulated in treated rats. Conclusion: It was concluded based on study outcomes that methanolic and n-hexane extracts of Calliandra haematocephala exhibited anti-ulcerative colitis activity through modulation of antioxidant defense mechanisms and the immune system. In this context, C. haematocephala can be considered as a potential therapeutic approach for cure of ulcerative colitis after bioassay-directed isolation of bioactive phytochemicals and clinical evaluation.

Keywords: Calliandra haematocephala; HPLC; anti-inflammatory cytokines; oxidative stress; pro-inflammatory markers; ulcerative colitis.

FIGURE 1

The figure shows graphical overview of study.

FIGURE 2

HPLC quantification of flavonoids and polyphenols in CHnHE. Figure shows the relative quantities of flavonoids (quercetin and rutin) and polyphenols (gallic acid, vanillic acid, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, sinapic acid) in CHnHE. Bars in blue color represent the retention times of all phytochemicals whereas red colored bars represent the corresponding concentrations of these identified compounds.

FIGURE 3

HPLC quantification of flavonoids and polyphenols in CHME. Figure shows the relative quantities of flavonoids (quercetin and rutin) and polyphenols (gallic acid, vanillic acid, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, sinapic acid) in CHME. Bars in blue color represent the retention times of all phytochemicals whereas red colored bars represent the corresponding concentrations of these identified compounds.

FIGURE 4

HPLC chromatogram of CHnHE. Peaks in chromatogram represent the concentrations (ppm) of flavonoid and polyphenolic compounds according to their retention times (minutes).

FIGURE 5

HPLC chromatogram of CHME. Peaks in chromatogram represent the concentrations (ppm) of flavonoid and polyphenolic compounds according to their retention times (minutes).

FIGURE 6

Effect of plant extracts on colon tissues. Figure shows the macroscopic observation of damage in the colon of acetic acid-induced ulcerative colitis after treatment with CHnHE and CHME extracts at 250, 500, and 750 mg/kg doses. Damaged area was used to calculate macroscopic ulcer score and ulcer index. Figure shows a progressive decrease in tissue damage by plant extracts and SCG with increase in dose in contrast to CCG that shows remarkable tissue damage. Here, CCG, NCG, and SCG represent “colitis control group,” “normal control group,” and “standard control group” respectively.

FIGURE 7

Effect of C. haematocephala extracts (CHnHE and CHME) on macroscopic ulcer score (A) and ulcer index (B) in acetic acid induced ulcerative colitis. Figure shows significant dose dependent decline in macroscopic ulcer score and ulcer index after treatment for consective 11 days with CHnHE and CHME (250, 500, and 750 mg/kg doses) standardized with prednisone (2 mg/kg) in comparison (p < 0.001) to CCG. One-way ANOVA followed by post hoc Tukey’s test was applied to analyze the significance of differences among all treatment and control groups. Here, “a,” “b,” and “c” indicate significant differences from CCG, NCG, and SCG respectively.

FIGURE 8

Effect of C. haematocephala extracts (CHnHE and CHME) on oxidative stress markers; SOD (A), MDA (B), and NO (C) in acetic acid induced ulcerative colitis. Figure shows significant dose dependent increase in SOD activity and decline in MDA and NO synthesis after treatment for consective 11 days with CHnHE and CHME (250, 500, and 750 mg/kg doses) standardized with prednisone (2 mg/kg) in comparison (p < 0.001) to CCG. One-way ANOVA followed by posthoc Tukey’s test was applied to analyze the significance of differences among all treatment and control groups. Here, “a,” “b,” and “c” indicate significant differences from CCG, NCG, and SCG respectively.

FIGURE 9

Effect of C. haematocephala extracts (CHnHE and CHME) on histopathological architecture of colon in acetic acid induced ulcerative colitis. Figure shows histopathological slides representing the cellular and molecular aspects of colon inflammatory damage. All treatment groups and NCG exhibited the intact goblet cells and prevented the infiltration of inflammatory cells and edema in colon tissues. Here, CCG, NCG, and SCG represent “colitis control group,” “normal control group,” and “standard control group” respectively.

FIGURE 10

Effect of C. haematocephala extracts (CHnHE and CHME) on histopathological scale in acetic acid induced ulcerative colitis. Histopathological scale was quantified by inflammatory changes using histopathological slide examination given in Figure 9. This figure shows dose dependent decrease in histopathological scale after treatment with CHnHE and CHME (250, 500, and 750 mg/kg doses) standardized with prednisone (2 mg/kg) in comparison (p < 0.001) to CCG. One-way ANOVA followed by post hoc Tukey’s test was appliedto analyze the significance of differences among groups. Here, “a”, “b”, and “c” indicate significant differences from CCG, NCG and SCG respectively.

FIGURE 11

Effect of C. haematocephala extracts (CHnHE and CHME) on mRNA expression of pro and anti-inflammatory mediators; TNF-α (A), COX-2 (B), IL-4 (C), and IL-10 (D) in acetic acid induced ulcerative colitis. mRNA expression of all these mediators was normalized using GAPDH as housekeeping gene. Figure shows significant downregulation of mRNA expression of TNF-α cytokine and COX-2 enzyme whereas upregulation of mRNA expression of IL-4 and IL-10 after treatment for consective 11 days with CHnHE and CHME (250, 500, and 750 mg/kg doses) standardized with prednisone (2 mg/kg) in comparison (p < 0.001) to CCG. One-way ANOVA followed by post hoc Tukey’s test was appliedto analyze the significance of differences among all treatment and control groups. Here, “a,” “b,” and “c” indicate significant differences from CCG, NCG, and SCG respectively.