The combination of Cassia obtusifolia L. and Foeniculum vulgare M. exhibits a laxative effect on loperamide-induced constipation of rats

Abstract

Chronic constipation is a functional gastrointestinal disease that is detrimental to the quality of patient life. Cassia obtusifolia L. (CO) and Foeniculum vulgare M. (FV) are commonly used as medicinal foods in many countries. We aimed to examine the laxative effect and their underlying mechanism of CO and FV mixture on loperamide (lop)-induced constipated rats. To determine the laxative effects of these compounds, Sprague-Dawley rats were divided into six groups: the control, lop-induced constipated (2mg/kg), and three doses (100, 300, and 500mg/kg) of CO and FV mixture-, and Bisacodyl (bis, 3.3mg/kg)-treated groups. The mixture of CO and FV and bis were orally administered once a day for 4 weeks. For induction of constipation, the lop were treated with a dose of 2 mg/kg twice a day on the 3rd week after treatments of CO and FV extracts and bis. The results were revealed that the CO and FV mixture has the laxative effects more than those in CO and FV-alone treatments on constipated rats by determining the stool parameters, including stool number and weight. Indeed, stool parameters, such as, stool number, weight, and water contents and colonic peristalsis from the intestinal transit length and ratio were dramatically improved by CO and FV mixture treatment. Histological study also revealed that CO and FV mixture enhanced the thicknesses of mucosa and muscular layers of the colon in constipated rats. For their underlying mechanism, the mRNAs and proteins expression of muscarinic acetylcholine receptors (mAchR) M2 and M3 and their downstream signaling were preserved by CO and FV mixture treatment in constipated rats. Therefore, this study suggests that treatment with CO and FV mixture has beneficial effects against constipation. We further suggest that CO and FV mixture may be utilized as an alternative therapeutic strategy for constipation.

Conflict of interest statement

Competing Interests: We have the following interests. Seung Hee Jang is employed by TEAZEN, Inc. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Fig 1. The laxative effects of CO and FV treatment on lop-induced constipated rats.

Stool number (A), stool weight (B) daily measured in each group. N = 10 in each group. Data are expressed as mean ± SEM. Significance was measured by performing a one-way ANOVA followed by Bonferroni’s post-hoc test. # P < 0.05 and ## P < 0.01 vs. control. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. Lop-treated group. Lop, loperamide-induced constipated group; CO300, CO 300mg/kg/day-treated group; FV300, FV 300mg/kg/day-treated group; CFM300, CO and FV mixture 300 mg/kg/day-treated group, respectively; Bis, bisacodyl-treated group.

Fig 2. Body weight (BW) changes and feeding behavior during experimental procedure.

BW (A) was measured once a week, and food intake (B) and water consumption (C) were daily measured in each group. N = 10 in each group. Data are expressed as mean ± SEM. Significance was measured by performing a one-way ANOVA followed by Bonferroni’s post-hoc test. ### P < 0.001 vs. control. ** P < 0.01 and *** P < 0.001 vs. Lop-treated group. Lop, loperamide-induced constipated group; CFM 100, 300, and 500, CO and FV mixture 100, 300, and 500 mg/kg/day-treated group, respectively; Bis, bisacodyl-treated group.

Fig 3. Effects of CO and FV mixture treatment on stool parameters of constipated rats.

Stool number (A), stool weight (B), and water contents (C) were daily measured in each group. N = 10 in each group. Data are expressed as mean ± SEM. Significance was measured by performing a one-way ANOVA followed by Bonferroni’s post-hoc test. # P < 0.05 and ## P < 0.01 vs. control. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. Lop-treated group. Lop, loperamide-induced constipated group; CFM 100, 300, and 500, CO and FV mixture 100, 300, and 500 mg/kg/day-treated group, respectively; Bis, bisacodyl-treated group.

Fig 4. Effects of CO and FV mixture treatment on intestine transit length and gastrointestinal transit ratio of constipated rats.

Intestine transit length (A) and gastrointestinal transit ratio (B) were observed in 30 min later after receiving barium sulfate. N = 10 in each group. Data are expressed as mean ± SEM. Significance was measured by performing a one-way ANOVA followed by Bonferroni’s post-hoc test. ## P < 0.01 and ### P < 0.001 vs. control. ** P < 0.01 and *** P < 0.001 vs. Lop-treated group. Lop, loperamide-induced constipated group; CFM 100, 300, and 500, CO and FV mixture 100, 300, and 500 mg/kg/day-treated group, respectively; Bis, bisacodyl-treated group.

Fig 5. Effects of CO and FV mixture treatment on histological properties of transverse colon in constipated rats.

H&E staining of transverse colon in lop-, CO and FV mixture-, and bis-treated groups (A-F). Scale bar, 100 μm. Mucosa layer (G) and muscular layer (H) thicknesses are presented as graphs. N = 5 in each group. Data are expressed as mean ± SEM. Significance was measured by performing a one-way ANOVA followed by Bonferroni’s post-hoc test. ##P < 0.01 vs. control. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. Lop-treated group. Lop, loperamide-induced constipated group; CFM 100, 300, and 500, CO and FV mixture 100, 300, and 500 mg/kg/day-treated group, respectively; Bis, bisacodyl-treated group.

Fig 6. Effects of CO and FV mixture treatment on mRNA expression of mAchR M2 and M3 in the transverse colon.

qRT-PCR analysis of mAchR M2 (A) and M3 (B) mRNA expression. N = 10 in each group. Data are expressed as mean ± SEM. Significance was measured by performing a one-way ANOVA followed by Bonferroni’s post-hoc test. ### P < 0.001 vs. control. ** P < 0.01, and *** P < 0.001 vs. lop-treated group. Lop, loperamide-induced constipated group; CFM 100, 300, and 500, CO and FV mixture 100, 300, and 500 mg/kg/day-treated group, respectively; Bis, bisacodyl-treated group.

Fig 7. Effects of CO and FV mixture treatment on protein expression of mAchR M2 and M3 and PKC and PI3K as their downstream signaling pathway-related proteins in the transverse colon.

N = 10 in each group. Western blot analysis of mAchR M2 and M3 (A-C) and phosphorylated and total of PKC and PI3K (D-F) protein expression. Data are expressed as mean ± SEM. Significance was measured by performing a one-way ANOVA followed by Bonferroni’s post-hoc test. ### P < 0.001 vs. control. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. Lop-treated group. Lop, loperamide-induced constipated group; CFM 100, 300, and 500, CO and FV mixture 100, 300, and 500 mg/kg/day-treated group, respectively; Bis, bisacodyl-treated group.