Ase 350 system

PLEx was executed in a Dionex ASE 350 system (Dionex, Sunnyvale, CA, USA) with the powder of PHF obtained as mentioned above. Aliquot of 5.0 g of powder of PHF was mixed with diatomaceous earth (1/1) and placed in a 34 mL stainless–steel cells. The extraction was performed via 3 consecutively applied steps with absolute solvents of increasing polarity, in order to get the maximum possible number and amount of secondary metabolites of various polarities and miscibilities, namely, acetone, ethanol, methanol and their aqueous mixtures with water (1:10, 3:10), and pure water. Extraction time was of 22 min; pressure 10.6 MPa; temperature 75 °C (for acetone, ethanol and methanol) and 135 °C (for water). Organic solvents were removed in a rotary vacuum evaporator at 38 °C, while the residual water was removed in a freeze drying unit. The extracts after solvent evaporation were placed under nitrogen flow for 20 min and stored in dark glass bottles at −80 °C until analyzed.

Wild garlic leaves were collected at Fruska Gora mountain, Serbia. Collected leaves were sorted, washed in water, were frozen at −20 °C, freeze-dried, sealed in bags, and stored in the dark at room temperature in a desiccator. Prior extraction plant material was ground in a kitchen blender (0.325 mm), and moisture content (6.12%) was determined. Wild garlic extract was prepared following the recommended optimal conditions investigated by Tomšik et al. (2017) with slight modification [13 (link)]. The extraction was performed in ASE 350 system Dionex Corporation (Sunnyvale, CA, USA). Powdered wild garlic samples were placed into the extraction cell together with diatomic earth, and extraction was performed with water as solvent at 180 °C/1500 psi for 10 min. The extract was stored at 4 °C until further analysis.

An ASE 350 system (Dionex, Sunnyvale, CA, USA) with 100 mL stainless steel vessels was used as the pressurized solvent extractor. Approximately 15 g of dried rambutan peel was placed in an extraction cell. The extraction cells were placed in a carousel and the samples were extracted using one static cycle with 60% flush volume and 180 s purge time. The ASE parameters were set as temperature in the range from 60 to 120 °C, with aqueous ethanol concentration 50 to 90 vol% and extraction time between 10 and 50 min. The obtained extracts were evaporated to dryness using a rotary evaporator (Buchi Rotavapor R-215 Postfach Flawil, Switzerland) under 45 °C and 72 mPa until free of solvent and then stored at ~ −18 °C for further analysis.

PLE was run according to the methodology used by Leyva-Jiménez et al. [14 (link)] with a pressurized liquid extractor (ASE™ 350 system, Dionex, Sunnyvale, CA, USA). Briefly, for each extraction, 5 g of sample were mixed with 10 g of sea sand and loaded onto 33 mL stainless-steel extraction cells. After extraction, the extracts obtained were ice-cold to achieve a temperature of 20–25 °C and centrifuged at 17,000× g for 15 min in a centrifuge (Sorvall ST 16 R, Thermo Scientific, Leicestershire, UK). The supernatants were dried under vacuum in a Savant^TM SpeedVac Concentrator SC250 EXP (Thermo analysis Scientific, Sunnyvale, CA, USA) and they were stored at −20 °C until further use. Prior to use, the dry extracts were reconstituted with the same extraction solvent mixture to a concentration of 10 mg/mL and filtered with single-use syringe filters (0.20 µm pore size).

The PLE conditions and process applied to the grape stem powder were based on a previous process developed by the research group, where the optimal extraction conditions for obtaining the highest antioxidant capacity and total phenolic content (TPC) values were determined [17 (link)]. Briefly, PLE extractions were carried out in an ASE 350 system from Dionex Corporation (Sunnyvale, CA, USA) equipped with a solvent controller unit. Extraction cells (11 mL of capacity) were filled with a mixture of 1 g of grape stem powder dispersed on 1 g of diatomaceous earth. Ethanol:water (30%) as a solvent, at 120 °C for 10 min, yielded HPE (high polymeric phenolic extract), whereas LPE (low polymeric phenolic extract) was obtained at 100% ethanol, 80 °C, and 6 min extraction time. Each extract was independently recovered in a glass vial, and ethanol was removed at 37 °C by vacuum rotary evaporation (IKA RV 10 control, IKA, Staufen, Germany). Next, the extracts were freeze-dried (Telstar Lyobeta 15 equipment; Telstar, Madrid, Spain) and stored at −20 °C in darkness until further analysis.

Sample extraction was carried out using a Dionex™ ASE™ 350 system equipped with 22-mL stainless steel extraction cells under the following conditions: 120 °C, 5 min static extraction, 3 extraction cycles, 100% flush volume, and 60 s nitrogen purge. To reduce the risk of contamination, high purity solvents were used. During the method development, different solvents and solvent combinations were tested. The solvent volume used for the extraction resulted in approximately 50 mL under the specified conditions. More information on the solvents can be found in the respective sections below. In addition, the extraction cells, sand, and GFFs were pre-cleaned using the PLE system with acetone under the following conditions: 100 °C, 1 min static extraction, 3 extraction cycles, 100% flush volume, and 60 s nitrogen purge.