Comparison of laxative effects of fermented soybeans (Cheonggukjang) containing toxins and biogenic amines against loperamide-induced constipation mouse model

Abstract

Background/objectives: Cheonggukjang is a traditional fermented soybean paste with significant health-promoting effects. On the other hand, there have been insufficient studies on the safety and efficacy of Cheonggukjang, which is produced using traditional methods containing toxins and biogenic amines (BAs). This study compared the laxative effect of Cheonggukjang, containing high or low levels of toxins and BAs (HTBC or LTBC) in a loperamide (Lop)-induced constipation mouse model.

Materials/methods: To induce constipation, Lop (5 mg/kg) was administered orally to ICR mice twice a day for 4 days, and the dose was increased to 8 mg/kg after a 3-day rest period. Cheonggukjang (500 mg/kg, HTBC, or LTBC respectively) was administered for four weeks before the Lop treatment.

Results: The number of stools, fecal weight, water contents, gastrointestinal transit, and histological alterations were recovered significantly in the HTBC or LTBC groups. HTBC and LTBC administration did not induce significant changes in body weight, dietary intake, and behavior. The opioid-receptor downstream signaling pathway in colon tissues was also evaluated. The c-Kit, stem cell kinase, and mitogen-activated protein kinases subfamilies, including extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinases, and p38, were all downregulated in the HTBC or LTBC-administered mice colon compared to the Lop group.

Conclusion: These results show that Cheonggukjang, containing high levels of toxins and BAs, have a similar laxative effect in a mouse model of Lop-induced constipation.

Keywords: Fermented foods; biogenic amines; constipation; laxatives; toxicity.

Fig. 1. Schematic protocol for animal experiments.

Lop, loperamide; PC, positive control; HTBC, high level of toxins and biogenic amines containing Cheonggkujang; LTBC, low level of toxins and biogenic amines containing Cheonggkujang.

Fig. 2. Relative abundance of microorganisms in (A) HTBC or (B) LTBC.

HTBC, high level of toxins and biogenic amines containing Cheonggkujang; LTBC, low level of toxins and biogenic amines containing Cheonggkujang.

Fig. 3. Effects of HTBC and LTBC administration on the fecal parameters in mice with Lop-induced constipation. (A) Representative fecal pellet images collected over 24 h at D10 after Lop administration. (B) Total fecal number. (C) Total water content of fecal pellets.

Results were expressed as mean ± SD (n = 10 mice per group). Lop, loperamide; N, normal control group; C, Lop control group; PC, positive control group; HTBC, high level of toxins and biogenic amines containing Cheonggkujang; LTBC, low level of toxins and biogenic amines containing Cheonggkujang. ^***P < 0.001 vs. N group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001, vs. C group.

Fig. 4. Effects of HTBC or LTBC administration on the intestinal transit ratio in constipated mice induced by Lop. (A) The representative image of charcoal transit in the intestine. The covered distances were measured after 60 min of administration with charcoal. The arrows indicate that the charcoal meal has migrated. (B) The transit ratio of charcoal was calculated using the total length of the intestine and the distance of the charcoal diet.

Results were expressed as mean ± SD (n = 10 mice per group). Lop, loperamide; N, normal control group; C, Lop control group; PC, positive control group; HTBC, high level of toxins and biogenic amines containing Cheonggkujang; LTBC, low level of toxins and biogenic amines containing Cheonggkujang. ^***P < 0.001 vs. N group; ^###P < 0.001, vs. C group.

Fig. 5. Histological structures and mucin secretion of the transverse colon in Lop-induced constipation mice after HTBC and LTBC administrations. (A) The mice were evaluated by hematoxylin and eosin (H&E), and alcian blue stain, and the observed sections were photographed at 100× and 400× magnification using a microscope. (B) Muscular and (C) Mucosa layer thickness and (D) Inflammation indices were calculated using the Image J image software.

Results were expressed as mean ± SD (n = 10 mice per group). Lop, loperamide; N, normal control group; C, Lop control group; PC, positive control group; HTBC, high level of toxins and biogenic amines containing Cheonggkujang; LTBC, low level of toxins and biogenic amines containing Cheonggkujang. ^***P < 0.001 vs. N group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, vs. C group.

Fig. 6. Effect of HTBC and LTBC administrations on the expression levels of Cajal markers in Lop-induced constipation mice. Relative mRNA levels of (A) c-Kit and (B) SCF were examined by quantitative PCR in colons of Lop-induced constipation mice. (C) The protein expression levels of the cajal markers (c-Kit and SCF) were analyzed by western blot analysis in the colons of Lop-induced constipation mice. The protein levels of (D) c-Kit and (E) SCF (relative density normalized to β-Actin) were measured using an imaging densitometer and followed by statistical analysis.

Results were expressed as mean ± SD (n = 10 mice per group). Differences between the groups were determined as statistical significance by one-way ANOVA followed by Duncan's multiple range test. Lop, loperamide; N, normal control group; C, Lop control group; PC, positive control group; HTBC, high level of toxins and biogenic amines containing Cheonggkujang; LTBC, low level of toxins and biogenic amines containing Cheonggkujang; ANOVA, analysis of variance. ^*P < 0.05, **P < 0.01, and ^***P < 0.001 vs. N group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. C group.

Fig. 7. Effect of HTBC and LTBC administrations on MAPK signaling in Lop-induced constipation mice. The protein levels of the phospho and total ERK1/2, JNK, and p38 were analyzed in the Lop-induced constipation mice. The relative protein levels were quantified using an image analyzer. The band density was normalized using each total protein.

The results were expressed as mean ± SD (n = 10 mice per group). The differences between the groups were determined as statistical significance by one-way ANOVA followed by Duncan's multiple range test. Lop, loperamide; N, normal control group; C, Lop control group; PC, positive control group; HTBC, high level of toxins and biogenic amines containing Cheonggkujang; LTBC, low level of toxins and biogenic amines containing Cheonggkujang; MAPK, mitogen-activated kinase; ERK1/2, extracellular signal-regulated kinase 1/2; JNK, c-Jun N-terminal kinase; ANOVA, analysis of variance. ^***P < 0.001 vs. N group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. C group.