# Sustainable Extraction of Hedera helix Bioactive Compounds via Synergy of Natural Deep Eutectic Solvent and Ultrasound: Process Optimization, Mechanistic Insights and Anti-Colon Cancer Activity

## TL;DR

This study explores a sustainable method to extract bioactive compounds from Hedera helix using natural solvents and ultrasound, showing effectiveness and potential for industrial use.

## Contribution

The study introduces a novel ultrasound-assisted extraction method using a natural deep eutectic solvent for efficient and sustainable extraction of Hedera helix compounds.

## Key findings

- A ternary natural deep eutectic solvent formulation achieved the highest extraction efficiency for eight target compounds from Hedera helix leaves.
- Optimized ultrasound extraction conditions included 45 minutes of ultrasonic time, a solid/liquid ratio of 1:54 g/mL, and a temperature of 42 °C.
- The NADES-UAE method showed strong anti-colon cancer activity against HT-29 cells in vitro.

## Abstract

Hedera helix (HE) contains diverse bioactive constituents, including triterpenoid saponins, flavonoids, and phenolic acids, which exhibit various pharmacological activities. In this study, ultrasound-assisted extraction (UAE) combined with natural deep eutectic solvent (NADES) was employed to enhance the extraction efficiency and elucidate the underlying mechanisms. Among the tested formulations, a ternary system composed of malonic acid (Mal), N,N′-dimethylurea (DMU), and 1,4-butanediol (1,4-BDO) achieved the highest efficiency for extracting eight target compounds from the HE leaves. In addition, the key interactions among NADES components were confirmed by Fourier-transform infrared (FT-IR) spectroscopy, providing valuable insights into the extraction mechanism. The UAE process was systematically optimized through single-factor experiments. Subsequently, response surface methodology (RSM) identified the optimal conditions as ultrasonic time of 45 min, solid/liquid ratio of 1:54 g/mL, and ultrasonic temperature of 42 °C. Scanning electron microscopy (SEM) elucidated the microstructural alterations in plant cell walls induced by NADES-UAE, alongside the enhanced penetration and disruption mechanisms. In vitro bioactivity revealed that the NADES-extracted HE exerted strong inhibitory effect on HT-29 colon cancer cells. Overall, these findings demonstrate the high effectiveness and sustainability of NADES-UAE for extracting HE bioactive compounds and provide valuable implications for the industrial-scale production of plant-based functional products.