Liver health from leaf to lab: eco-friendly extraction and multi-dimensional analysis of European herbs

The threat posed by antibiotic-resistant bacteria and the severe or patient-dependent side effects of existing drugs are promoting greater knowledge of herbal remedies, which have previously been used as alternatives. The metabolic profiles and hepatoprotective effects of five European medicinal plants (Agrimonia eupatoria, Angelica archangelica, Angelica sylvestris, Sambucus nigra, and Sambucus ebulus) were investigated. Conventional and novel extraction techniques (infusion, magnetic-assisted extraction, ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE)) were optimized using a design of experiment approach for five parameters to increase the total phenolic content and antioxidant activity. The most sustainable extraction method with extracts of high bioactivity was MAE with 40 mL g-1, 5 min, 55°C, 400 W, and aqueous ethanol mixtures between 20 and 90% ethanol in water. To separate the complex plant metabolomes, a comprehensive two-dimensional liquid chromatography method with two columns in the second dimension (multi-2D LC × LC), which increased the peak capacity by 91% from 222 to 424 and the orthogonality from 85% to 92%, thus achieving almost complete coverage of the two-dimensional space compared to the optimized conventional LC × LC methods. The multi-2D LC × LC method was coupled with high-resolution mass spectrometry to screen 32 phenolic compounds. In addition, the method was combined with ion mobility to improve the separation of isomers and achieve a multidimensional non-target approach, resulting in 103 analytes tentatively identified at level 2. Bayesian optimization was tested as a tool to simplify LC × LC method development with optimized gradients within 3 to 9 runs, but optimization in the second dimension remained a challenge. To correlate the antioxidant and hepatoprotective effects of plant extracts and their components, preparative LC was used to enrich and screen potential fractions, identifying both known and unknown compounds responsible for the antioxidant effects of the plants. Damaged human hepatoma cells were treated with effective plant extracts and fractions, with the flower and many leaf extracts leading to a concentration-dependent improvement in cell viability. For further investigations, compounds from highly effective fractions could be further isolated and enriched to unambiguously identify hepatoprotective compounds in the future.