Preventive effects of a nutraceutical mixture of berberine, citrus and apple extracts on metabolic disturbances in Zucker fatty rats

Abstract

Background: The prevention of obesity represents a major health and socio-economic challenge. Nutraceuticals are regularly highlighted for their beneficial effects in preventing the metabolic disturbances associated with obesity. However, few studies have described the combined action of nutraceutical mixtures combining polyphenols with alkaloids.

Objective: The aim of this study was to evaluate the effects of long-term dietary supplementation with a mixture of Berberine, Citrus and Apple extracts (BCA) in the primary prevention of obesity and its metabolic and vascular complications in the obese Zucker rat, a spontaneous model of genetic obesity and insulin resistance.

Methods: Sixteen 8-week-old obese Zucker male rats were randomly divided into two groups: all rats received oral gavage daily either with water, untreated obese (U-ObZ) or BCA (BCA-ObZ) mixture for thirteen weeks. Morphological and metabolic parameters were measured along the study. Cumulative concentration-response curves to insulin, acetylcholine and phenylephrine were determined on isolated thoracic aorta. Colon permeability measurements were performed using the Ussing chamber technique. Fecal samples collected at the beginning and the end of the protocol were used as a template for amplification of the V3-V4 region of the 16S rDNA genes.

Results: BCA supplementation reduced weight gain (p<0.05) and food intake (p<0.05) in the BCA-ObZ group rats compared to the U-ObZ group rats. It also improved glucose tolerance (p<0.001) and decreased fasting insulin and Homeostasis model assessment index (p<0.05). Through ex vivo experiments, the BCA mixture enhanced significantly aortic insulin relaxation (p<0.01), reduced α1-adrenoceptor-mediated vasoconstriction (p<0.01), and decreased distal colon permeability. Moreover, short-chain fatty acid producers such as Bacteroides, Blautia, and Akkermansia were found to be increased by the BCA mixture supplementation.

Conclusion: The results showed that a 13-week-supplementation with BCA mixture prevented weight gain and improved glucose metabolism in obese Zucker rats. We also demonstrated that BCA supplementation improved vascular function, colonic barrier permeability and gut microbiota profile.

Fig 1. Effect of BCA mix on body weight (A), adiposity index (B), food intake (C) and water intake (D).

Data were expressed as mean ± SEM. A Mann-Whitney test or Two-way ANOVA test were used for statistical analysis. n = 8 for each group. * p<0.05; ** p<0.01 BCA-ObZ vs. U-ObZ. U-ObZ: Untreated Obese Zucker; BCA-ObZ: BCA-Treated Obese Zucker.

Fig 2. Effect of BCA blend on glucose and insulin metabolism.

Glucose metabolism was assessed by oral glucose tolerance test (OGTT), performed at the beginning and end of the protocol. After a glucose bolus, plasma glucose was measured at baseline (A) and at week 13 of the protocol (B). The area under the curve (AUC) of OGTT was calculated at baseline and at week 13of the protocol (C). Insulin metabolism was assessed by calculating the insulin resistance assessment model (HOMA-IR) (D). Data were expressed as mean ± SEM. A Mann-Whitney test or Unpaired t test were used for statistical analysis. n = 8 * p<0.05; *** p<0.001 BCA-ObZ vs. U-ObZ. U-ObZ: Untreated Obese Zucker; BCA-ObZ: BCA-Treated Obese Zucker.

Fig 3. Effect of BCA treatment on aortic vasoreactivity.

CCRCs were constructed to assess aortic contractile responses using phenylephrine (Phe, 10⁻⁹ to 3.10⁻⁵ M (A) and relaxation responses using acetylcholine (Ach, 10⁻⁹ to 3.10⁻⁵ M) (B), insulin (Ins, 10⁻⁹ to 10⁻⁵ M) (C) and insulin (Ins, 10⁻⁹ to 10⁻⁵ M) with or not incubated BH4 (10⁻⁵ M) and L-Arg (10⁻³ M) (D). Percentages of contraction and relaxation calculated are relative to maximal changes from pre-contraction produced by KCl and Phe, respectively. Data are expressed as mean ± SEM and determined using NLME for Phe and Ach and LME for Ins. n = 6–8 each group. **p<0.01 BCA-ObZ vs. U-ObZ. ## p<0.01 U-ObZ+BH4+Larg vs. U-ObZ. U-ObZ: Untreated Obese Zucker; BCA-ObZ: BCA-Treated Obese Zucker; BH4: Tetrahydrobiopterin; L-Arg: L-arginine; NLME Non-linear Mixed Effect; LME: Linear Mixed effect.

Fig 4. Effect of BCA mixture on carbachol-induced contraction in isolated segments of distal colon from control and BCA-treated groups.

The responses were obtained after pretreatment with sodium butyrate (A) or propionate (B). Data are expressed as mean ± SEM. LME was used for statistical analysis. n = 8 for each group. U-ObZ: Untreated Obese Zucker; BCA-ObZ: BCA-Treated Obese Zucker; LME: Linear Mixed effect.

Fig 5. Permeability to fluorescein sulfonic acid was measured using Ussing chambers in the proximal colon (A) and in the distal colon (B).

Data are expressed as mean ± SEM. The Mann-Whitney test was used for statistical analysis. n = 8 for each group. ns: Not significant; * p<0.05 BCA-ObZ vs. U-ObZ. U-ObZ: Untreated Obese Zucker; BCA-ObZ: BCA-Treated Obese Zucker.

Fig 6. The panels represent the alpha diversity measures where; A) presents the observed library (total number of AVS observed); B) presents Chao1 index is the richness estimators (estimate the total number of AVS present in a community); C) presents Simpson is microbial indexes of diversity.

Boxes span the first to third quartiles; the horizontal line inside the boxes represents the median. U-ObZ: Untreated Obese Zucker; BCA-ObZ: BCA-Treated Obese Zucker.

Fig 7. The phylum composition of microbiome after the 13^th week of experimentation.

U-ObZ: Untreated Obese Zucker; BCA-ObZ: BCA-Treated Obese Zucker.