Extraction of bioactive components in immortelle (Helichrysum arenarium L.) by supercritical fluid extraction system and investigation of some pharmaceutical properties

Helichrysum arenarium L., widely used in traditional herbal medicinal products, stands out particularly with its supportive effects in digestive system disorders. In the European Union monograph, it has been scientifically approved for use in the treatment of bloating and digestive complaints. To enhance the efficacy of such herbal products and to obtain extracts with a safe compound profile, the selectivity and sustainability of the extraction methods employed are of great importance. Conventional solvent-based extraction methods are successful especially in extracting hydrophilic compounds such as phenolics and flavonoids; however, solvent residues remaining in the solution and stability issues depending on the processing time may arise. In contrast, the supercritical fluid extraction (SFE) method has emerged as an effective, eco-friendly alternative that leaves no solvent residue and allows the selective extraction of lipophilic compounds. The different selectivities of both methods for various compound groups formed the basis of the formulation developed in this study, where apolar compounds obtained by SFE were combined with polyphenols extracted with alcohol-water, resulting in a standardized herbal preparation with balanced bioactive compound diversity and bioavailability. In this thesis study, bioactive compounds of Helichrysum arenarium L. were obtained by SFE for the first time, and in order to increase extraction efficiency, temperature, pressure, and time parameters were optimized using a central composite design. As a result of optimization, the extraction conditions of 250 bar, 60 °C, and 180 min were determined as the optimum SFE working parameters. Under these optimum conditions, both Helichrysum arenarium L. and, for comparison, the frequently studied Helichrysum italicum L. species were extracted. The extraction yields were determined as 2.19% and 3.14%, respectively. For comparison, the same species were also extracted using conventional solvent extraction, and the extraction yields obtained by this method were 12.58% and 16.11%, respectively. The obtained extracts were subjected to comprehensive compositional analyses, making this study a pioneering work in which the characteristics of different extraction methods were comparatively evaluated. HPLC-UV analyses revealed that the apigenin-7-O-glucoside content in the alcohol extract of Helichrysum arenarium L. was 9060.00 mg/kg, whereas in the SFE extract it was 7.84 mg/kg, indicating that hydrophilic flavonoids are more efficiently extracted with alcohol-based solvents. On the other hand, SFE was found to be effective in the extraction of lipophilic compounds, particularly yielding high amounts of tocopherols and volatile terpenoids. HPLC-FLD analyses showed that the total tocol content of SFE extracts was up to 10 times higher than that of alcohol extracts. GC-MS volatile compound analysis identified borneol, eugenol, and carvacrol in Helichrysum arenarium L. extract, while neryl acetate, gamma-curcumene, and beta-selinene were characterized in Helichrysum italicum L. extract. GC-FID fatty acid analysis revealed linoleic acid (C18:2) as the dominant fatty acid in both species. Helichrysum italicum L., in particular, stood out with its high PUFA content (63–72%). In antioxidant activity assays, alcohol extracts showed higher radical scavenging capacity; however, SFE extracts also exhibited significant antioxidant activity under certain conditions. Antimicrobial activity tests demonstrated that extracts from both species did not show a marked effect against Staphylococcus aureus and Escherichia coli. Only the alcohol extract of Helichrysum italicum L. exhibited low-level inhibition against Staphylococcus aureus. Based on the identified bioactive compounds, six different combination formulations containing both lipophilic and hydrophilic compounds were developed, aiming to achieve a more balanced and synergistic effect. In these formulations, extracts obtained from both Helichrysum arenarium L. and Helichrysum italicum L. by SFE and solvent extraction methods were used. The formulation content was supported with plant oil-based systems designed to enhance the solubility of extracts, as well as enriched with specific vitamins and functional additives. To determine the solubility of apigenin-7-O-glucoside, the active compound of Helichrysum arenarium L., in simulated intestinal fluid, dissolution analyses were performed. The highest solubility value (97%) was obtained in the formulation prepared with extracts from both SFE and solvent extraction of Helichrysum arenarium L. In conclusion, this study systematically optimized the SFE parameters of Helichrysum arenarium L. for the first time, and the compositional profile of the obtained extracts was determined in detail. By combining SFE-derived extracts rich in lipophilic compounds with alcohol-water extracts containing high flavonoid content, an innovative formulation was developed that can be utilized as a functional food supplement. This product was presented as a strong candidate for industrial-scale applications in terms of sustainability, solvent safety, and effective compound profile.