Laxative effects of triple fermented barley extracts (FBe) on loperamide (LP)-induced constipation in rats

Constipation is a widespread functional gastrointestinal illness that affects 3–15% of general population and causes discomfort and negative impact on quality of life [1‐4]. It can also cause restlessness, vomiting, gut obstruction, perforation, and may be linked with fatal pulmonary embolism or aspiration [5]. Currently, 20–30% of people older than 60 years use more than one laxative per week [6]. Drugs containing sennoside or magnesium oxide have powerful laxative/purgative activity and are mainly prescribed for constipation related illnesses; however, these drugs also induce side effects such as severe diarrhea [2], and their frequent use can induce melanosis coli, a risk factor for colorectal neoplasm [7].

There has recently been a rise in attention towards the role of functional foods in maintaining well-being, resulting in an increased demand for functional foods produced from natural sources [8]. Natural products are gaining interest in the biopharmaceutical industry as well as inspiring the search for novel potential sources of bioactive metabolites [9, 10]. Medicinal plants, crude drug substances as well as several herbs have antioxidant properties [11]. Grain consumption has been enhanced due to their favorable effects with respect to lowering the risk of diabetes, cardiovascular diseases, ischemic stroke, metabolic syndromes, and gastrointestinal cancer [12‐14]. Grains contain minerals, vitamins, phytochemicals and functional dietary fibers that are favorable for human body [14, 15]. Recently, fermented herbs have also been proposed as a potential source of medicinal and pharmaceutical ingredient, particularly since fermentation is believed to enhance the bioactivity of natural herbs through probiotic effect and biotransformation [16‐23].

Globally, barley grain is used in the brewing industry as a non-toxic cereal grain [24]. Furthermore, it is also used as an ingredient in various foods, beverages and animal forage [24]. The phenolic compounds present in barley (Hordeum vulgare L.) have shown antioxidants effects in the promotion of health [14, 25, 26]. This includes anticancer [24] and probiotic gastroprotective effects [27]. The functionality and bioavailability of these phenolic compounds is increased by fermentation process [28, 29], particularly the antioxidative effect [14, 15]. Various fermented barley extracts (FBe) have shown a number of potent pharmacological effects, especially improved antioxidative [14], uric acid-lowering [30], anti-atopic dermatitis [31, 32], hepatoprotective [14, 15], and immunostimulatory effects [33], compared with non-fermented extracts.

Rats have typically been used as experimental animals to test the efficacy of various drugs. Dietary habits, chemical compounds such as morphine, and psychological stress have been considered as the causes of constipation [2, 20‐22]. Normal rats are also useful experimental animals with regard to detecting various digestive disorders [20, 34‐36]. It has been established that loperamide (LP) can induce delays in colonic transit due to its inhibition of stool frequency in mice and increase in colonic contractions, resulting in spastic constipation [37]. This drug has been shown to inhibit colonic peristalsis and intestinal water secretion [38, 39], ultimately, extending the fecal evacuation time and delaying the intestinal luminal transit [40]. Therefore, LP-induced constipation has been considered a suitable animal model of spastic constipation [21, 22, 41].

The laxative effects of triple fermented rice extract by saccharification, Saccharomyces, and Weissella in normal rats [21] and in loperamide-treated rats [22] have been reported. Previously, we reported the less toxic behavior of the triple fermented barley extract using saccharification [20‐23]. However, there are currently no systematic assessments of the laxative effects of FBe in rodent models. Therefore, this study was intended to test the potential enhanced laxative effects of FBe in rat models of LP-induced constipation, using methods established in our previous studies [21, 22].

Constipation can arise from a variety of sources, including dietary habits, chemical compounds such as morphine and psychological stress [2]. It increases with age and may necessitate long-term treatment with laxatives. In the present study, in order to evaluate the potential laxative effects of FBe, we examined changes in fecal parameters (i.e., weight, numbers, and water content), fecal mucous content, gastrointestinal transit ratio (motility), and colonic mucosa histology (i.e., mean colonic mucosa thickness under alcian blue stain, number of colonic mucous-producing cells, and mean mucous membrane thickness of fecal pellets in the colon lumen) in LP-induced rats, a suitable animal model of spastic constipation [21, 22, 41]. The laxative effects of FBe were compared with SP (5 mg/kg), a cathartic stimulant activated by colonic bacteria [42, 43], as the reference drug [21, 22, 46].

Spastic constipation was induced via oral treatment with LP (3 mg/kg) once a day for 6 consecutive days 1 h before test substance administration, in accordance with our previous studies [21, 22, 47, 48]. The dosages of FBe (100–300 mg/kg) were selected according to our previous studies of fermented rice extracts in LP-induced constipation rat models [21, 22]. The 5 mg/kg dose of SP was also selected according to the previous studies [21, 22, 44]. Fecal pellets were collected one day before the first dose of the test substance and starting from immediately following the fourth administration for a 24-h duration, in order to measure fecal parameters and select the appropriate animals. Charcoal transfer was conducted after the sixth administration of the test substances.

A continuous oral supply of LP (3 mg/kg) for 6 days showed significant decreases in fetal water content and fecal pellet number, intestinal charcoal transit ratio, surface mucous thickness of fecal pellets found in the colon lumen at sacrifice, number and thickness of mucous-producing goblet cells in the colonic mucosa, and the mean diameter and number of fecal pellets remaining in the colon lumen at sacrifice. These findings are consistent with the classic signs of LP-induced spastic constipation [21, 22, 41]. However, these LP-induced spastic constipation-related decreases in intestinal motility and fecal discharge, as well as the histopathological changes in fecal and colon pellets in the colon lumen were significantly and dose-dependently inhibited by additional continuous oral administration of FBe (100–300 mg/kg) for 6 days. These findings provide evidence for the laxative effect of FBe on LP-induced spastic constipation in rats, without causing excess diarrhea. Thus, FBe at 100 mg/kg doses may act as a potent functional food ingredient or laxative agent to treat spastic constipation with low toxicity [55]. Our results also showed that the laxative effects of FBe (300 mg/kg) were milder than those of SP (5 mg/kg). However, favorable increases in intestinal motility and charcoal transit ratio were demonstrated with FBe 300 mg/kg and 200 mg/kg than with SP (5 mg/kg). FBe 100 mg/kg also showed similar inhibitory effects on the LP-induced decreased intestinal motility as SP 5 mg/kg.

No LP-related changes in body weight and weight gains were observed compared to intact vehicle control, which was similar to the results of our previous studies [21, 22]. Additionally, no meaningful changes in body weight and weight gains were detected for three different dosages of FBe (100, 200, and 300 mg/kg) compared with LP. It should be noted that FBe did not induce severe diarrhea as a side effect since FBe showed milder and favorable laxative effects compared with (SP 5 mg/kg). Furthermore, FBe did not impact body weight and weight gains. Contrastingly, SP (5 mg/kg) induced significant decreases in body weight and weight gains compared with both LP and vehicle controls, possibly due to its long-established powerful purgative and laxative activity [42, 43]. All rats used in this study in the intact control, LP control, and all FBe-treated groups showed body weight increases that were within the range for normal age-matched rats [56, 57].

Marked decreases in fecal discharge are typically seen in constipation; specifically, the delay of fecal pellets in the large intestinal lumen can induce over-absorption of water and subsequently, the water content of the discharged pellets are significantly decreased. Therefore, fecal parameters such as number of fecal water content and fecal pellets discharged are valuable indices for determining the effects of various laxative agents [44, 48]. LP has been shown to induce noticeable decreases in fetal water content and fecal pellet number as indications of spastic constipation [21, 22]. The increased fecal water content and fecal pellet number discharged detected in rats treated with FBe (100–300 mg/kg) compared with LP control rats suggest that FBe has promising laxative properties on spastic constipation. Decreases in fecal surface mucous content and increases in remnant fecal pellet number in the colon lumen have previously been observed in constipation [20, 36, 46, 58] as well as treatment with LP [21, 22]. Our results showed decreases in remnant fecal pellet number in the colon lumen and increases in surface mucous content following treatment with FBe (100–300 mg/kg), providing support for the hypothesis that FBe has promising laxative effects at these doses. In this study, rats treated with SP (5 mg/kg) also showed significant increases in the fecal water content, number of fecal pellets discharged, fecal water content, and the surface mucous thickness of remnant pellets in the colon lumen.

LP has been shown to decrease gastrointestinal charcoal transit ratio, a marker of intestinal motility, consistent with signs of spastic constipation [21, 22]. These signs were also observed in the LP-treated control rats used in the present study. Therefore, significant and dose-dependent increases in gastrointestinal charcoal transit ratio in rats treated with FBe (100–300 mg/kg) compared to LP control provide indirect evidence that FBe has promising laxative effects against LP-induced spastic constipation. Significant increases in intestinal motility, as measured by charcoal transit ratio, were demonstrated with FBe 300 mg/kg and 200 mg/kg compared with SP (5 mg/kg), and rats treated with FBe 100 mg/kg exhibited similar inhibitory effects on the LP-induced decreases in intestinal motility compared with SP 5 mg/kg.

Reduction in mucous production in the colonic mucosa on histopathological assessments are directly related with constipation [58]; specifically, marked decreases in the thickness of the colonic mucosa layer and mucous-producing cells have been observed [20‐22, 36, 59]. Additionally, treatment with 3 mg/kg of LP has been associated with marked decreases in mucosa thickness and colonic mucous-producing cells [21, 22]. In the present study, compared with intact controls, significant decreases in the surface mucous thickness of remnant fecal pellets found in the colon lumen at sacrifice, the number of mucous-producing cells in the colonic mucosa, and the mean colonic mucosa thickness were detected in rats following 6 days of consecutive oral application of LP (3 mg/kg). However, compared with LP controls, co-treatment with SP (5 mg/kg) and FBe (100–300 mg/kg) was associated with a significant increase in the number of mucous-producing cells in the colonic mucosa and the surface mucous thickness of remnant fecal pellets in the colon lumen. The effects of FBe were found to be dose-dependent. In addition, the colonic mucosa thickness significantly increased in rats treated with SP (5 mg/kg) and FBe (100–300 mg/kg; dose-dependent) compared with that in vehicle control rats.

Total polyphenols, total flavonoids and dietary fiber content of FBe were 3.66, 0.31 and 20.20%, respectively (Table 1). According to the meta-analysis of Yang et al. [60], intake of dietary fiber can clearly increase stool frequency in patients with constipation. Thus, a possible mechanism with which FBe improved the constipation seems to be its dietary fiber. However, further research is needed to elucidate the cause of laxative effect of FBe. These findings suggest that FBe has favorable laxative effects against LP-induced spastic constipation and that oral treatment of SP (5 mg/kg) was more favorable than FBe (300 mg/kg).

By comparing key factors associated with the laxative effect on the LP-induced spastic constipation in rats, the present work revealed that oral application of 100–300 mg/kg of FBe exhibited promising laxative effects, mediated by increase in gastrointestinal motility. Therefore, FBe may act as a promising functional food ingredient or a laxative agent for the treatment of spastic constipation, with less toxicity observed with the 100 mg/kg dose. The overall laxative effects of FBe 300 mg/kg on LP-induced constipation in rats were milder than those of SP 5 mg/kg; however, there were more favorable increases in intestinal motility in rats treated with FBe 300 and 200 mg/kg than in those treated with SP 5 mg/kg. Furthermore, FBe 100 mg/kg showed similar inhibitory effects on LP-induced decrease in intestinal motility as SP 5 mg/kg.