## 1 Introduction  
The progress of the COVID-19 pandemic since 2019 has highlighted the need for using medicinal plants and/or natural products as promising alternatives to prevent or treat diseases [1]. Plants generally contain many effective bioactive compounds, such as flavonoids and polyphenols [2; 3; 4; 5], essential oils and terpenoids [6; 7; 8], fatty acids [9; 10], and alkaloids [11; 12]. In addition, the presence of antioxidants [13; 14] as well as antiinflammatory [15; 16] and antimicrobial agents [17; 18], which have different therapeutic effects, have been reported in many studies. The analysis of bioactive substances in medicinal plants or natural products relies on the use of feasible and effective extraction methods. Optimizing the variables that affect the extraction process (solvent quantity, solvent/plant ratio, temperature, pressure, time, heating rate) is as important as the selection of the extraction method to be used [19]. Response surface methodology (RSM) is an experimental optimization strategy in which mathematical models and statistical analysis are combined to reduce the number of experiments and the use of solvents, and save time. Furthermore, RSM reveals the relationships between the experimental factors and responses [20]. In addition, this method generates functional theoretical equations for response estimates at any desired level within the working range of experimental variables. Although there are many extraction methods, the most commonly used are Soxhlet extraction, maceration, and percolation. These methods have important limitations,such as high solvent consumption, long processing times, low extraction efficiencies, and they are not environmentally friendly [21]. Subcritical water extraction (SWE) is an efficient, low-cost, and green alternative method, capable of overcoming these limitations as demonstrated in several cases [22; 23; 24; 25; 26]. In SWE, water is used as the extraction solvent at temperatures in the range of 100-374 \({}^{\circ}\)C and with enough pressure to maintain the liquid state of water [27]. Although studies using SWE for the extraction of bioactive components from plants have been reported [28; 29; 30], this is the first time that SWE has been applied for the extraction of bioactive substances from \(O\). _mutabilis_. The _Boraginaceae_ family, which ranks ninth among the families with the highest number of species in Turkey, includes 357 taxa, 34 genera, 325 species, 16 subspecies, and 16 varieties. _Onosma_ L., the largest genus of the _Boraginaceae_ family, is represented by a total of 150 species worldwide [31; 32]. In Turkey, _Onosma_ L. is represented by 97 species, four varieties, and one hybrid species, of which 50 species and one type are endemic [33; 34]. Many of the _Onosma_ species have therapeutic potential, and these plant species are widely used in traditional medicine [35]. Specifically, it is known that _Onosma_ species are used in the relief of bladder pain, the treatment of kidney infections, and wound and burn healing [36]. In addition, studies with _Onosma_ species have shown that phenolic compounds have anticancer, antiinflammatory, antimicrobial, antioxidant, and wound healing properties [37]. Results related to the extraction of \(O\). _mutabilis_ using alternative, environmentally friendly methods are completely missing from the published literature. Furthermore, given the tunable properties of subcritical water, the hypothesis is that it could potentially extract bioactive substances that conventional extraction methods may not extract. The rationale of this work was to investigate the extraction of bioactive substances from \(O\). _mutabilis_ under subcritical conditions, as an environmentally friendly extraction strategy, and compare its efficiency and identity of extracted substances to conventional extraction methods. Therefore, the specific objectives are the following: (a) to determine the total phenol content and chemical profiles of the extracts obtained by SWE of the aboveground and root parts of \(O\).