Evaluation of the Effects of Instant Cascara Beverage on the Brain-Gut Axis of Healthy Male and Female Rats

Abstract

Instant cascara (IC) is a sustainable beverage obtained from dried coffee cherry pulp, rich in nutrients and bioactive compounds. The present research aimed to determine the effects of IC on general health and brain-gut axis parameters of healthy female and male rats. Wistar rats were exposed to IC (10 mg/mL) in their drinking water for 3 weeks. Body weight and solid and liquid intakes were monitored as indicators of food safety. Gastrointestinal transit was radiographically evaluated one day (acute) and 3 weeks (chronic) after the start of IC exposure. Locomotor activity, anxiety, and anhedonia of the animals after 3 weeks of treatment was also studied. Overall, compared to water-exposed animals, IC significantly increased food intake in males (p < 0.0001) and liquid intake in females (p < 0.05) without changes in body weight in either case. IC did not significantly modify gastrointestinal motility parameters after its acute or repeated intake and did not cause any significant behavioral alterations in males or females (p > 0.05). In conclusion, repeated intake of IC at the studied concentration did not negatively affect brain-gut axis functions of healthy male and female rats. Anxiety behavior, diarrhea, constipation, abnormal weight modifications, or other typical effects of toxicity were not observed in animals treated with the new powdered beverage, suggesting its food safety under the studied conditions.

Keywords: behavior; brain-gut axis; dried coffee cherry pulp; gastrointestinal motility; instant cascara; radiographic methods; rat; sex.

Figure 1

Experimental protocol. All rats were exposed to water (control group) or instant cascara (IC) beverage for 3 weeks, independently of the cohort of animals to which they were allocated. During the 3 weeks, body weight and food and drink intakes were regularly monitored. During the third week, different behavior assays were performed to analyze anhedonia (splash test, cohort 1), exploratory behavior (hole board test, cohort 1), and anxiety (plus maze test, cohort 2). Two radiographic motility studies were performed: after one day of IC beverage exposure (acute study, cohort 2) and after 3 weeks of IC beverage exposure (chronic study, cohort 1). At the end of the third week, animals were sacrificed, and a macroscopic analysis of the gastrointestinal organs was performed (cohort 1). The star represents the vaginal cytological smear evaluations performed to determine the phase of the estrous cycle in females in the indicated studies.

Figure 2

Effect of instant cascara (IC) beverage on the evolution of body weight and on the solid and liquid intake of rats of both sexes. The evolution of body weight (A) and the overall mean solid (B) and liquid (C) intake by each experimental group are shown. These parameters were recorded in the four experimental groups, distributed according to sex and the administered beverage (IC or water): Males-Control, Males-IC, Females-Control, and Females-IC. Data represent mean ± SEM (standard error of the mean). N = 12 animals per group (cohort 1). Sex-dependent statistically significant changes: ## p < 0.01, ### p < 0.001, #### p < 0.0001 (Females-Control vs. Males-Control); $$ p < 0.01, $$$ p < 0.001, $$$$ p < 0.0001 (Females-IC vs. Males-IC). Beverage-dependent statistically significant changes: **** p < 0.0001 (Males-IC vs. Males-Control); + p < 0.05 (Females-IC vs. Females-Control). (A): Two-way ANOVA followed by Bonferroni’s post-hoc test. (B,C): One-way ANOVA followed by Bonferroni’s post-hoc test.

Figure 3

The effect of instant cascara (IC) beverage on gastrointestinal transit of male and female rats was evaluated radiographically after barium administration. Gastrointestinal motility was evaluated in four experimental groups, distributed according to sex and the administered beverage (IC or water): Males-Control, Males-IC, Females-Control, and Females-IC. The values obtained at each time point in the semiquantitative analysis for the stomach (A), small intestine (B), cecum (C), and colorectum (D) are depicted, as well as the number of fecal pellets within the colorectum (E). Representative X-rays obtained at T2, T4, T8, and T24 from control males (blue frame, upper panel) and control females (red frame, lower panel) are shown in (F). Data represent mean ± SEM (standard error of the mean). n = 12 animals per group. Sex-dependent statistically significant changes: # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 (Females-Control vs. Males-Control); $ p < 0.05, $$ p < 0.01 $$$ p < 0.001, $$$$ p < 0.0001 (Females-IC vs. Males-IC). Two-way ANOVA followed by Bonferroni’s post-hoc test.

Figure 4

The effect of instant cascara (IC) beverage on the morphometric and densitometric analysis of gastrointestinal organs 3 weeks after IC administration. Area (A,C,E) and density (B,D,F) of the stomach (A,B), cecum (C,D), and fecal pellets (E,F) were measured with ImageJ from the X-rays obtained at 0, 1, 2, 3, 4, 6, 8, and 24 h after barium administration in four experimental groups, distributed according to sex and the administered beverage (IC or water): Males-control, Males-IC, Females-control, and Females-IC. The maximum values for area and barium density were obtained at T0 for the stomach and after averaging the values obtained at T3–T8 for the cecum and at T4–T8 for the fecal pellets. The study was performed 3 weeks after IC or water exposure. Data represent mean ± SEM (standard error of the mean). n = 12 animals per group. Sex-dependent statistically significant changes: # p < 0.05, ## p < 0.01, ### p < 0.001, [Females-control vs. Males-control]; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 (Females-IC vs. Males-IC). Beverage-dependent statistically significant changes: * p < 0.05 (Males-IC vs. Males-Control). One-way ANOVA followed by Bonferroni’s post-hoc test.

Figure 5

Effect of instant cascara (IC) beverage on the number and wetness of feces of male and female rats. The number of fecal pellets (A,B) and the differences between wet and dry weight (Wetness, (C,D)) are represented for the acute (A,C) and chronic (B,D) studies. There were four experimental groups, distributed according to sex and the beverage administered (IC or water): Males-Control, Males-IC, Females-Control, and Females-IC. Data represent mean ± SEM (standard error of the mean). n = 9 (A,C) or 12 (B,D) animals per group. Sex-dependent statistically significant changes: # p < 0.05 (Females-Control vs. Males-Control); $$ p < 0.01 (Females-IC vs. Males-IC). Two-way ANOVA followed by Bonferroni’s post-hoc test.

Figure 6

Effect of instant cascara (IC) beverage on the index of stained feces of male and female rats after 1 day ((A), acute study) or 3 weeks ((B), chronic study) of IC exposure. There were four experimental groups, distributed according to sex and the beverage administered (IC or water): Males-Control, Males-IC, Females-Control, and Females-IC. Data represent mean ± SEM (standard error of the mean). n = 9 (cohort 2) or 12 (cohort 1) animals per group. Sex-dependent statistically significant changes: $$ p < 0.01 (Females-IC vs. Males-IC). Two-way ANOVA followed by Bonferroni’s post-hoc test.