# In Vitro Influence of a Chemically Characterized Hippophae rhamnoides L. Fruit Extract on Healthy and Constipated Human Gut Microbiota Functionality and Aquaporin-3 Expression

## TL;DR

This study explores how a sea buckthorn fruit extract affects gut health and water transport in intestinal cells, offering a potential non-drug solution for constipation.

## Contribution

The study presents a chemically characterized sea buckthorn extract and its effects on gut microbiota and AQP-3 expression in constipation.

## Key findings

- The extract upregulated AQP-3 expression without causing cell toxicity.
- Fermentation with constipated microbiota reduced antioxidant capacity and increased SCFA production.
- The extract shows potential as a non-pharmacological approach for managing functional constipation.

## Abstract

To identify the underlying mechanisms by which H. rhamnoides fruit extract exerts regulatory effects on intestinal function, we investigated its chemical composition using UHPLC Q-Orbitrap HRMS and evaluated its biological effects on Aquaporin-3 (AQP-3) expression via Western blot in the intestinal epithelial cell line (HT-29). Moreover, fecal microbiota from healthy and constipated adults was employed to mimic the in vitro fermentation of the digested extract and evaluate its effects on gut microbiota functionality. Antioxidant capacity (i.e., Total Phenolic Contents (TPC), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) assays) was assessed prior to and after simulated digestion and fermentation processes. Short-chain fatty acids (SCFAs) were quantified using UHPLC-RID of the fermented samples. In the extract, 23 compounds belonging to a variety of classes (mainly polyphenols) were tentatively identified. The extract significantly upregulated AQP-3 expression in the absence of cytotoxicity. After in vitro fermentation with gut microbiota isolated from constipated subjects, ABTS and FRAP values significantly decreased, as well as TPC, suggesting a greater consumption of antioxidant compounds, consistent with the increased production of radical compounds associated with constipation. Fermentation with intestinal microbiota with healthy and constipated gut microbiota resulted in an increase in SCFA. These results provide preliminary insights into a non-pharmacological strategy for functional constipation.