# Hepatic Cholesterol Regulation Through Multi-Botanical Extract Targeting of the PCSK9–LDLr–SREBP-2 Axis in HepG2 Cells

## TL;DR

A combination of four plant extracts helps regulate cholesterol in liver cells by targeting specific proteins, offering a potential non-statin alternative for managing cholesterol.

## Contribution

The study introduces a synergistic multi-botanical formulation that modulates cholesterol metabolism through a novel mechanism distinct from statins.

## Key findings

- The multi-botanical combination enhanced cell viability and reduced oxidative stress more effectively than individual extracts.
- The formulation downregulated PCSK9 and inhibited SREBP-2 activation while upregulating LDLr, indicating a coordinated regulatory effect.
- Bile acid production and free cholesterol excretion were significantly increased, suggesting improved cholesterol clearance.

## Abstract

Background/Objectives: Botanical and nutraceutical approaches have increasingly been considered as alternatives or complements to conventional lipid-lowering therapies, particularly in individuals with mild-to-moderate dyslipidemia or statin intolerance. This study aimed to evaluate a multi-botanical formulation, combining black garlic, sesame, Gastrodia elata, and Primula veris extracts, for its effects on hepatic cholesterol regulation and the PCSK9–LDLr–SREBP-2 axis in vitro. Methods: Each extract was chemically characterised for its polysaccharide, polyphenol, flavonoid, and sesamin content. HepG2 cells were exposed to normal (5 mM) or high-glucose (30 mM) conditions to mimic metabolic stress. Dose–response studies identified optimal concentrations for cell viability. Hepatic safety was assessed via MTT and ROS assays, while cholesterol metabolism was evaluated by measuring HMG-CoA reductase levels, total cholesterol, LDL levels, bile acid production, free cholesterol levels, and the expression of PCSK9, LDLr, and SREBP-2 using ELISA and Western blot. Results: All individual extracts improved cell viability, reduced oxidative stress, and moderately modulated cholesterol metabolism. The multi-botanical combination exhibited synergistic effects, enhancing cell viability (+47.5% vs. untreated), suppressing ROS, reducing HMGR levels, and lowering total intracellular cholesterol more effectively than single extracts or the statin-like reference RYRF. Importantly, the combination strongly downregulated PCSK9 and inhibited SREBP-2 proteolytic activation while upregulating LDLr, indicating coordinated transcriptional and post-translational regulation. Bile acid production and free cholesterol excretion were also significantly increased, supporting improved cholesterol clearance. Conclusions: This four-botanical formulation effectively modulates hepatic cholesterol homeostasis via a multifactorial, synergistic mechanism distinct from statin-like agents. The results suggest its potential as a safe, non-statin strategy to support cardiometabolic health. Future studies are warranted to confirm long-term efficacy and clinical relevance.