Synthesized T-X was donated by Prof. Sung Hoon Kim (Kunkuk University, South Korea). 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline 6-sulfonic acid ammonium salt) (ABTS), potassium persulfate, sodium phosphate buffer, potassium ferricyanide, trichloroacetic acid (TCA), ferric chloride, carmine, and loperamide hydrochloride were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). All reagents used were of analytical grade.

The DPPH assay was performed with some modifications according to the method of Brand-Williams et al.¹⁷. T-X (100 μL at 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 mg/mL) was added to 200 μL of 0.2 mM DPPH solution dissolved in methanol. The samples were incubated for 30 min at room temperature in the dark, and then absorbances at 517 nm were measured using a microplate reader (Multiskan FC 357, Thermo Scientific, China).

The ABTS assay was performed with some modifications according to the method of Re et al.¹⁸. Briefly, ABTS radical cations were prepared by mixing 7.4 mM ABTS solution and 2.6 mM potassium persulfate solution in equal quantities and allowing the mixture to react for 24 h at room temperature in the dark before use. The ABTS+ solution was diluted with deionized water to obtain an absorbance at 734 nm. T-X (50 μL at 3.125, 6.25, 12.5, 25, and 50 mg/mL) was added to 950 μL of diluted ABTS+ solution. The samples were incubated for 30 min at room temperature in the dark, and then absorbances were measured at 734 nm using a microplate reader.

The reducing power assay was used with some modifications according to the method of Oyaizu¹⁹. T-X (100 μL at 3.125, 6.25, 12.5, 25, and 50 mg/mL) was added to 0.2 M sodium phosphate buffer (pH 6.6) and 10% potassium ferricyanide were mixed and added in an amount equal to that of the sample, and the mixture was allowed to react for 20 min at 50°C. The resulting solution was mixed with 100 μL of 10% trichloroacetic acid and centrifuged at 1,000 × g for 10 min. A 200-μL sample of the supernatant was mixed with 40 μL of 0.1% ferric chloride, and then absorbances were measured at 700 nm using a microplate reader.

Twenty-four male Sprague-Dawley rats (5 weeks old) were purchased from Samtacho (Osan, Korea) and individually housed in a room with temperature (23 ± 2°C) and humidity (55 ± 10%) controlled under 12 h light/dark cycle. All rats consumed a standard irradiated chow diet (Purina Mills, Seoungnam, Korea) and water ad libitum to stabilize their metabolic condition for 1 week. The animal procedures were conducted in accordance with the Guidelines for Care and Use of Laboratory Animals and were approved by the Animal Ethnics Commit-tee of Chonnam National University, Yeosu, Korea (approval number: CNU IACUC-YS-2016-7).

After 1 week of adaptation, the rats were randomly divided into four treatment groups (n = 6 each): normal untreated rats (NOR group), loperamide-treated control rats (CON group), loperamide and T-X (15 mg/kg)-treated rats (T-X group), and loperamide and commercial Dulcolax S (5.5 mg/kg)-treated rats (DS group). All rats were provided AIN-76A basal diets and water ad libitum. Constipation was induced in the rats by oral administration of 1 mL loperamide (4 mg/kg) suspended in 0.9% sodium chloride twice per day, at 9 am and 6 pm, for 14 days. The control group was administered normal saline only. During the experimental periods, T-X (15 mg/kg) was orally administered to rats at 10 am. A comparison group was treated with commercial Dulcolax S (5.5 mg/kg) as the standard drug. We measured daily food intake, water intake, and body weight gain of all the rats, and treatment continued for 14 days. The feed efficiency ratio (FER) in the experimental period was calculated by dividing the dietary intake quantities by body weight gain.

The number and weight of fecal pellets from individual rats were determined daily at 9 am during the experimental period. We dried fecal pellets for 24 h at 70°C and determined the water content using the following equation: Fecal water content (%) = [(fecal wet weight - fecal dry weight)/fecal wet weight] × 100.

Blood was collected from the heart using a syringe under ether anesthesia. The serum was separated by centrifugation at 2,000 × g for 20 min and stored at -80°C. Organs, namely liver, heart, and kidneys, were removed, weighed, frozen in liquid nitrogen, and stored at -80°C until the biochemical analysis was conducted. Serum total protein, albumin, aspartate aminotransferase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatinine concentrations were determined using an automatic biochemical analyzer (Hitachi 747; Hitachi Co., Tokyo, Japan).

Liver tissue was homogenized in a glass-Teflon homogenizer with 50 mM phosphate buffer (pH 7.4) to obtain 1:9 (w/v) whole homogenate that was then centrifuged at 25,000 rpm for 20 min at 4°C. The supernatant was used to assess hepatic antioxidant enzyme activities. Total GSH content and superoxide dismutase (SOD) and catalase (CAT) activities were determined using a commercially available kit supplied by Biovision (CA, USA) according to the methods suggested by the manufacturer. Hepatic glutathione S-transferase (GST) activity was determined using 1-chloro-2,4-dinitrobenzene (CDNB) in the presence of 0.1 mM GSH following the method of Habig et al.²⁰. The formation of dinitrophenyl thioether in response to GST was monitored for 3 min at 37°C using a spectrophotometer at 340 nm. Glutathione reductase (GR) was measured using the method of Pinto et al. [21]. Briefly, the supernatant was mixed with 5 mM NADPH and 1 M glutathione disulfide (GSSG), and the formation of NADP+ was monitored with a spectrophotometer at 340 nm. For glutathione peroxidase (GPx) activity, the supernatant was mixed with 100 mM GSH, 1 mM EDTA, 5 mM NADPH, and 1 unit of GR in 0.1 M phosphate buffer (pH 7.0), and then incubated for 3 min. 10 mM cumene hydroperoxide was added to the reaction mixture, and the oxidation of NADPH into NADP+ was determined using a spectrophotometer at 340 nm.

The GIT ratio was determined using the method of Niwa et al. with some modifications²². On the 15th day of the experimental period, 1 mL carmine (3 g suspended in 50 mL of 0.5% carboxymethylcellulose) was orally administered to the rats as a marker. After 30 min, the animals were sacrificed, and then small intestines were quickly removed. We determined the distance over which carmine had travelled and the total length of the small intestine to calculate the GIT ratio. The GIT ratio was expressed as the percentage of distance travelled by carmine relative to the total length of the small intestine.

The concentrations of GAS, MTL, SS, and CGRP in serum were evaluated using commercially available ELISA kits.

All data are expressed as the mean ± SD. Statistical analyses were performed using the IBM SPSS Statistics version 20.0 (Chicago, IL, USA). Multiple groups were compared by one-way analysis of variance, followed by Tukey-Kramer multiple range testing to determine significant differences in all parameters. Values were considered statistically significant at P < 0.05.

As shown in Fig. 1, we investigated the in vitro antioxidant activities of T-X using DPPH, ABTS, and reducing power assays. The IC₅₀ value, the concentration inhibiting 50% of free radical production efficacy of T-X against DPPH radicals, was 355.4 mg/mL. In addition, T-X exhibited stronger scavenging activity (25.1 mg/mL) against ABTS radicals than the other treatments, and absorbances in the reducing power assay tended to increase as the concentration of T-X increased. These results showed that T-X effectively scavenged various reactive radicals in a concentration-dependent manner.

The effects of T-X on body weight, food intake, and FER in rats are presented in Table 1. Final body weight, body weight gain, and food intake were significantly higher in the NOR group than in the other groups. Food intake tended to be decreased in the CON group compared to that in the T-X and DS groups. In addition, there was no significant difference in FER among the groups.

In the present study, Dulcolax S was used as the standard drug to treat constipated rats. As shown in Table 2, organ weights, namely weights of whole livers, kidneys, and hearts, were not affected by treatment with T-X or Dulcolax S, which indicated that T-X had no side effects in vivo.

The effects of T-X on the number, wet weight, and water content of fecal pellets from loperamide-induced constipated rats are shown in Fig. 2. To confirm that constipation was induced by loperamide, we investigated fecal parameters. In the CON group, the number of fecal pellets was significantly lower than that in the NOR group (17.4 counts/day in CON vs. 22.0 count/day in NOR), which indicated that constipation was establishment in response to loperamide. After constipation was induced, the number, wet weight, and water content of fecal pellets in the T-X and DS groups markedly increased relative to those in the CON group. In particular, T-X treatment increased the water content of fecal pellets above normal (18.4% in T-X vs. 17.1% in NOR).

To examine the effect of T-X on the regulation of biochemical parameters in loperamide-induced constipated rats, serum AST and ALT levels were determined to assess hepatic function and BUN levels were determined to assess renal function. As shown in Table 3, serum protein, albumin, BUN, and creatinine levels were not influenced by T-X treatment. In addition, indexes of liver cell injury, such as AST and ALT levels, were not significantly different among the groups.

Figure 3 shows the hepatic GSH content and antioxidant enzyme activities of rats with constipation induced by loperamide. In the present study, there were no significant differences in GSH content and GST activities among the groups. GR activity tended to increase in the T-X and DS groups, respectively, compared to that in the CON group, although there was no significant difference among the groups. In the CON group, antioxidant enzyme activities such as GSH-Px, GR, SOD, and CAT were markedly decreased compared to those in the NOR group, whereas GSH-Px, SOD, and CAT activities were significantly elevated in both the T-X and DS groups when compared with the CON group.

In the present study, we investigated the effects of T-X on gastrointestinal motility, as shown in Fig. 4. Indexes of gastrointestinal motility such as transit distance and GIT ratio markedly decreased in the loperamide-induced constipated rats. In contrast, treatment with T-X or Dulcolax S markedly increased the transit distance and GIT ratio in loperamide-induced constipated rats relative to those in the NOR group. These results indicated that T-X ameliorated constipation.

We also evaluated the effects of T-X on serum gastrointestinal hormones, namely GAS, MTL, SS, and CGRP. As shown in Fig. 5, serum GAS and MTL concentrations in the CON group were significantly lower than those in the NOR group. However, colonic motility indexes such as serum GAS and MTL concentrations were markedly increased in the T-X and DS groups relative to those in the CON group. On the other hand, serum SS concentrations in the CON group were significantly higher than those in the NOR, T-X, and DS groups. Serum CGRP concentration was markedly higher in the CON group than in the NOR and DS groups; however, T-X did not change serum CGRP concentrations in loperamide-induced constipated rats.