# Natural Extract Combination Modulates Intestinal Barrier and Hepatic Cholesterol via the Gut–Liver Axis In Vitro

## TL;DR

A natural extract combination improves intestinal and liver cholesterol regulation in lab models, offering potential for managing cholesterol-related health issues.

## Contribution

A novel multi-botanical formulation synergistically modulates the gut–liver axis to regulate cholesterol metabolism.

## Key findings

- MIX improved intestinal barrier integrity and cell viability compared to single extracts.
- MIX reduced hepatic cholesterol synthesis and enhanced LDL receptor expression synergistically.
- MIX increased bile acid production and free cholesterol excretion, improving hepatic clearance.

## Abstract

Background/Objectives: The gut–liver axis plays a central role in cholesterol homeostasis, linking intestinal absorption, microbial metabolites, and hepatic lipid regulation. Dysregulation of this axis contributes to hypercholesterolemia and cardiometabolic risk, beyond classical cholesterol synthesis pathways. This study evaluated a novel multi-botanical formulation (MIX) that combines Gastrodia elata, Black Garlic, Primula veris, and Emblica officinalis (AMLA) to integrate modulation of cholesterol metabolism through intestinal and hepatic mechanisms. Methods: Individual extracts were chemically characterised for polyphenols, flavonoids, polysaccharides, S-allyl-L-cysteine (SAC), and tannins. Caco-2 cells were treated with varying doses to determine optimal concentrations and for viability, transepithelial electrical resistance, and permeability analysis. Supernatants post-intestinal passage were applied to HepG2 cells under high-glucose conditions to assess viability, oxidative stress, SRC/ERK-MAPK signalling, cholesterol synthesis (HMGR), LDL uptake, PCSK9–LDLR–SREBP-2 axis, and bile acid production. Results: MIX enhanced intestinal barrier integrity (TEER, tight junctions, permeability) and preserved cell viability compared with single extracts. In HepG2 cells, MIX demonstrated synergistic effects: it reduced HMGR expression by 83–90% relative to individual extracts, increased LDLR expression by 43–97%, suppressed PCSK9 by up to 92%, and lowered total cholesterol and LDL uptake more effectively than RYRF. MIX also amplified bile acid production and free cholesterol excretion, indicating improved hepatic clearance pathways. SRC and ERK-MAPK signalling were favourably modulated, supporting hepatocyte survival under metabolic stress. Conclusions: The multi-botanical formulation exerts complementary and synergistic effects on intestinal absorption and hepatic cholesterol regulation, integrating suppression of cholesterol synthesis, enhanced LDL clearance, and stimulated elimination via bile acids. These findings highlight the potential of the MIX formulation to modulate metabolically induced cholesterol dysregulation, supporting further in vivo and clinical investigation.