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

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.


Introduction
Constipation is one of the most frequent functional gastrointestinal disorder [1,2]. The prevalence of chronic constipation is 3-15% of the global population, especially in the elderly over 65 years of age and women [3]. This disease is often caused by metabolic imbalance, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Animal study design and induction of constipation
The 6 weeks-aged (weight 150-200 g) male Sprague-Dawley rats (Samtako Biokorea, Daejeon, Korea) were used for all experiments. Animals were housed in cages maintained at 23 ± 2˚C with 50 ± 10% humidity on a 12 h light:dark schedule. The rats were divided into 6 groups: control group, lop-induced constipated group, lop-treated with administration of 100, 300, and 500 mg/kg CO and FV mixture group, and bisacodyl (bis)-treated group (n = 10 in each group). For CO and FV alone treatment, each Co and FV extract was daily administered by oral gavage with a dose of 300 mg/kg for 4 weeks. CO and FV mixture was also daily administered with a dose of 100, 300, and 500mg/kg for 4 weeks, respectively. Finally, for the bis-treated group as a positive control group, bis was orally administered once a day with a dose of 3.3mg/kg/day. For induction of constipation, the rats were orally administered 2mg/kg of lop in 0.9% sodium chloride twice a day for 1week on the 3 rd -week after starting the administration of CO, FV extracts and bis. For euthanasia, the rats were anesthetized by CO2 inhalation to minimize suffering.

Analysis of body weight, food intake, and water consumption
The body weight, food intake, and water consumption of rats in each group was daily measured during the experimental period. All measurements were performed in triplicate to ensure accuracy.

Measurement of stool parameters
The excreted fecal pellets were daily collected throughout the experiment. Fecal samples were then weighed and counted three times for the analysis of stool number and weight. For analysis of stool water content, feces samples were dried at 40˚C for 24 h and weighed. Stool water content was then determined by subtracting dried feces from wet feces.
(1.4g/ml; Dongin-dang pharmaceutical Co., Ltd., Siheung, Korea). At 1 h after treatment of barium sulfate, the rats were sacrificed and the small intestines in each group were rapidly dissected. Thereafter, the distance moved by the barium sulfate was measured for the analysis of intestine transit length. For the analysis of intestine transit ratio, it was calculated by the following formula: Intestine transit ratio (%) = distance moved by the barium sulfate (cm)/total intestine length (cm) X 100.

Histological analysis of the transverse colon
The rats were euthanized and the transverse colons in each group were rapidly removed and fixed with 10% neutral-buffered formalin. After fixation, the tissues were embedded in paraffin, and embedded tissues were cut into 5-μm thick sections. The sections were then stained with hematoxylin-eosin (H&E) and examined using light microscopy (Carl Zeiss, Jena, Germany). The thicknesses of mucosa and muscular layers in the transverse colon were measured by AnalySIS 2.3 software (Carl Zeiss).

Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from the frozen tissue of transverse colon by using Ribospin TM II kit (GeneAll biotechnology Co., LTD, Seoul, Korea). 1μg of total RNA in each group was applied to reverse transcription PCR by using ImProm II Reverse Transcriptase (Promega, Medison, USA) with oligo-dT priming. qRT-PCR was conducted using a TaKaRa Thermal Cycler Dice Real Time System Single TP 815 (Takara, Shiga, Japan) with SYBR Green (Takara) used to examine the mRNA expression of mAchR M2 and M3 in the colon. The primer sequences were as follows: mAchR M2 forward:

Statistical analysis
All data are reported as the mean ± SEM. Statistical significance was analyzed using one-way ANOVA with Bonferroni post-hoc test (Prism 5.0.3, GraphPad Software Inc., San Diego, USA). P <0.05 was considered statistically significant.

Effects of CO, FV, and combined treatment on lop-induced constipated rats
To test the laxative effects of CO, FV, and their combined treatments on lop-induced constipated rats, the fecal parameters, including stool numbers and weights were determined. As shown in Fig 1, stool numbers and weights were significantly decreased after treatment of lop, while 300 mg CO and FV-alone treated groups were shown to increase these fecal parameters compared with those in lop-treated group. Furthermore, combined administration of 300 mg CO and FV have the preventive effects against constipation more than the CO and FV-alone treated groups on constipated rats by lop. These results demonstrated that CO and FV, especially, combined treatment of CO and FV improved lop-induced constipation in rats through the enhancement of fecal parameters.

Effects of CO and FV combined treatment on body weight and feeding behavior of constipated rats
To investigate whether CO and FV mixture treatment could affect the body weight and feeding behavior of constipated rats, body weight, food intake, and water consumption were measured during the experimental period. As shown in Fig 1, Body weight was steadily increased in each group. However, no differences in body weight changes in all groups were observed after administrations of lop, Co and FV mixture, and bisacodyl (bis), as a positive control drug (Fig 2A). Food intake was significantly decreased in lop-induced constipated rats (26.8% decrease vs. control group), while all doses of CO and FV mixture treatment were shown to similar levels with that of control group ( Fig 2B). Finally, water consumption did not induce significant changes in all groups ( Fig 2C). Collectively, these results demonstrated that CO and FV mixture treatment did not induce any alteration of body weight, food intake, and water consumption.

Effects of CO and FV combined treatment on stool parameters of constipated rats
To determine the laxative effects of CO and FV combined treatment on constipated rats, we examined the stool numbers, weights, and water contents in lop-, CO and FV mixture-, and bis-treated groups, respectively. Stool number was significantly reduced by 33.2% after treatment of lop compared with those in control group. Otherwise, CO and FV mixture treatments increased the stool numbers compared with those in lop-treated group (34.9%, 44.5%, and 63.2% increases in 100, 300, and 500 mg of CO and FV mixture -treated groups vs. lop-treated group, respectively; Fig 3A). Stool weight was also decreased in lop-treated group compared with that in control group (20.6% decrease vs. control group). However, this level was restored in all doses of CO and FV mixture-treated groups compared with that in lop-treated group (34.8%, 41.1%, and 69.0% increases in CO and FV mixture-100, 300, and 500 mg-treated groups vs. lop-treated group, respectively; Fig 3B). Furthermore, water contents of stool were shown to similar levels in control group when CO and FV mixtures were treated in constipated rats, while lop treatment reduced the water contents of stool (34.2% decrease vs. control group; Fig 3C). Similar results were found for bis treatment, as a positive drug.

Effects of CO and FV combined treatment on intestine transit length and gastrointestinal transit ratio of constipated rats
As shown in Fig 4, the significant decreases in both intestine transit length and intestine transit ratio of barium sulfate were observed in lop-induced constipated rats compared with those in control group (11.3% and 13.4% decreases of intestine transit length and ratio in lop-treated group vs. control, respectively). Otherwise, these decreases were attenuated by CO and FV mixture treatment in lop-induced constipated rats, which were similar levels with those in control group.

Effects of CO and FV combined treatment on histological properties of transverse colon in constipated rats
To determine whether CO and FV mixture treatment could alleviate the histopathological alterations of the transverse colon in constipated rats, we determined the morphology of the transverse colon by H&E staining. The results showed that the thicknesses of mucosa and muscular layers of transverse colon in lop-induced constipated rats were decreased compared with those in control group (48.4% and 36.2% decreases of mucosa and muscular layer vs. control group, respectively). However, CO and FV mixture treatment showed that the thicknesses of mucosa and muscular layers were maintained, with the most effective in 500mg/kg CO and FV mixture-treated groups compared with those in lop-treated group (15.8%, 40.9%, and 116% increases of mucosa layer and 20.0%, 37.3%, and 91% increases of muscular layer in CO and FV mixture-100, 300, and 500 mg-treated groups vs. lop-treated group, respectively; Fig  5). Additionally, similar results were found for bis treatment. Therefore, these results indicate that Co and FV mixture treatment could maintain the histological structures of transverse colon in constipated rats.

Effects of CO and FV combined treatment on mAchRs and their downstream signaling pathway in constipated rats
To determine whether CO and FV mixture treatment could affect the regulations of gene expression related to the muscle contraction, we performed the quantitative RT-PCR (qRT-PCR) of two mAchRs (mAchR M2 and M3), which play a role for smooth muscle contraction, of the transverse colons in constipated rats. The mRNA expression levels of mAchR M2 and M3 were significantly reduced in lop-induced constipated rats compared with those in control group, but the reduced levels were substantially inhibited when treated CO and FV mixture and bis (Fig 6). In addition, these protein expression patterns were also decreased in lop-treated group, while the reduced mAchR M2 protein levels were gradually increased as concentration of CO and FV mixture was increased and mAchR M3 protein levels were significantly increased in all CO and FV mixture treated group compared with those in lop-treated group (Fig 7A, 7B and 7C). Moreover, phosphorylation of PKC and PI3K, as the mAchRs downstream signaling pathway, were significantly reduced by lop treatment. However, these reductions were dramatically prevented by CO and FV mixture treatment as similar values in control group (Fig 7A, 7D and 7E). Hence, these results indicate that the laxative effects of CO and FV mixture may act through the regulation of mAchR M2 and M3 and their downstream signaling pathway.

Discussion
Recently, herbal plants and natural products have been attracted much attention to develop the therapeutic drugs for the treatment of constipation due to their effectiveness and safety properties [28]. Accordingly, their laxative effects and underlying mechanisms on constipation have been intensively studied [29]. CO and FV have the beneficial effects against many diseases, including hepatoprotection [30,31], neuroprotection [32], and antioxidant [33] [34]. CO exhibited the preventive effect against hyperlipidemia by decreasing serum low-density lipoprotein and triglycerides [35]. Additionally, FV has the therapeutic effects against cardiovascular disorders by reducing blood pressure without affecting the heart functions [36]. The previous studies have also demonstrated that FV has the anti-cancer effects in breast (MCF-7) and liver (Hepg) cancer cell lines [37]. In the present study, we sought to determine the laxative effects of CO and FV mixture in lop-induced constipated rats.
In this study, we first demonstrated that cotreatment of CO and FV have preventive effects against the constipation on lop-treated rats. Indeed, lop-induced constipation is well established and widely used as a model of spastic constipation [38]. Lop suppresses the water secretion and peristalsis in the colon, which causes to delay stool evacuation time and intestine luminal transit [39]. Consequently, these states directly affect the reduction of feed and water intakes in lop-exposed rats. Therefore, food intake and water consumption are considerable factors for the evaluation of constipation [28]. In our study, the analysis of the food intake revealed that the treatment of lop significantly decreased the food intake in rats, although the water consumption did not change among groups. Otherwise, administration of CO and FV mixture increased the food intake in lop-induced constipated rats, which were similar levels in control group.
In constipation, the obstacle of water absorption causes to decrease fecal discharges and delay fecal pellet transit in the large intestine. These alterations of fecal properties have been used as indicators of constipation symptoms and as indices of therapeutic effects against constipation [40]. Therefore, improvements of discharged fecal parameters, including increases of stool numbers and soften stools, and intestinal transit time, is one of the important strategy for the constipation treatment [41,42]. Previous studies showed dramatically decreased the stoolrelated parameters, including stool numbers, weights and water contents in lop-treated rats [12,43]. Consistently, our data demonstrated that these parameters were significantly reduced Histological studies have demonstrated that lop treatment caused the significant alterations in the transverse colon such as decreases of both mucosa and muscular layers. Previous studies have reported that constipation was accompanied by the markedly decreases of the colonic mucosa and muscular layers [12,44]. Therefore, the preventive effects against these pathological states of colon were direct evidence of laxative effects of therapeutic drug candidates. As expected, treatment of CO and FV mixture dramatically increased mucosa and muscular layers of colon in constipated rats. mAchRs belongs to the Ach receptors that are expressed in many cells (such as, neurons, heart, and smooth muscle cells) in the body and their roles are involved in many cell functions, including medication of cholinergic transmission, immune responses, and regulation of cell growth [45]. mAchR M1, M2, and M3 are expressed in the colon and have crucial roles for the intestinal activities, particularly motility and secretion [46]. Previous studies were reported to the decreases of mAchRs and their downstream proteins, such as PKC and PI3K in constipation [13]. Regarding with this, we demonstrated that both mRNA and protein expression of mChR M2 and M3 were significantly decreased after lop treatment. Particularly, these mAchR M2 and M3 decreases were dramatically recovered by Co and FV mixture treatment. Furthermore, protein expression of PKC and PI3K as downstream signaling pathway was revealed that CO and FV mixture treatment dramatically prevent the decreases levels after lop administration. Taken together, we further confirmed that laxative effect of Co and FV cotreatment may mediate by activation of AchR M2 and M3-related signaling pathway in constipated rats.
In conclusion, our study suggests that CO and FV mixture treatment has the laxative effect by recovering stool-parameters, colonic morphology, and activation of mAchRs and their downstream signaling pathway in constipation. Furthermore, our study provides that CO and FV cotreatment could be considered as a therapeutic drug candidate for the prevention or treatment of constipation.