Etoac

The following chemicals were used as received: acetone (Sigma-Aldrich, ≥99.0%), chloroform (CHCl₃: VWR Chemicals, 99%), d-chloroform (CDCl₃: Apollo, >99%), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU: Sigma-Aldrich, 98%), diethyl ether (Et₂O: Sigma-Aldrich, ≥99.8%), N,N dimethylformamide (DMF: Fisher Scientific, LR grade), 2,6-di-tert-butyl-4-methylphenol (BHT: Alfa Aesar, 99%), ethyl acetate (EtOAc: Fisher Scientific, LR grade), hexane (Hex: VWR Chemicals, 99%), magnesium sulfate (MgSO₄: anhydrous, Fisher Scientific, LR grade), 3-mercapto-1-propanol (Tokyo Chemical Industry Ltd. UK, 96%), 1,3-propanediol (Sigma-Aldrich, 98%), propiolic acid (Acros Organics, 98%), silica gel (SiO₂: Apollo Scientific, 40-63 micron), sodium chloride (NaCl: Fisher Scientific, > 99%), sodium hydrogen carbonate (NaHCO₃: Fisher Scientific, >99%), sulfuric acid (Fisher Scientific, >95%), triethylamine (Et₃N: Fisher Scientific, LR grade). 1,6-hexanedithiol (Sigma-Aldrich, ≥97%) was vacuum distilled prior to use and stored in Young’s tapped ampuoles under N₂. Poly(l-lactic acid) (PLLA) (Ingeo™ Biopolymer 3100HP) was ordered from NatureWorks.

For the extraction of polar phenols, rat serum samples underwent enzymatic hydrolysis prior to liquid-liquid extraction (LLE) with ethyl acetate (EtOAc), as reported by Vasilakopoulou et al. [31 (link)]. 100 μL of sodium acetate (CH₃COONa, 0.1 M, pH 5, Thermo Fisher Scientific, MA, USA ) were added to 100 μL of serum samples. Several 20-μL aliquots of β-glucuronidase/ sulfatase (2000/40 U) were injected into the serum samples; the samples were vortexed and incubated for 45 min in a heating bath that was set at 37 °C. To end the enzymatic hydrolysis, 100 μL of phosphoric acid (H₃PO₄, 4% v/v, Thermo Fisher Scientific, MA, USA) were added to the incubated samples. The samples then underwent further treatment with LLE () with the addition of 700 μL EtOAc (Thermo Fisher Scientific, Pittsburgh, PA, USA) to the serum samples. The samples were then vortexed, centrifuged for 5 min at 5000× g, and their supernatants were collected. The extraction process was carried out three times in total, with the supernatants being pooled, evaporated, and reconstituted in 100 μL methanol-water (MeOH-H₂O, 1:1, v/v, Thermo Fisher Scientific, Pittsburgh, PA, USA), which were analyzed with UHPLC–HRMS (Thermo Fisher Scientific, Bremen, Germany).

Two kilograms of the fresh aerial parts of the plant (equal to 50% of the weight of a
wildly-growing plant) were air-dried in the shade at room temperature, grounded, and
exhaustively extracted by cold maceration with aqueous methanol (70%). The extract was
evaporated under reduced pressure at 40°C to yield 80 g residue. The residue was suspended
in distilled water and successfully fractionated with n-hexane, CH2Cl2, EtOAc, and n-BuOH
(Thermo Fisher, USA) saturated with H₂O. Each extract was evaporated under
reduced pressure to yield 3, 7, 12 and 22 g residues, respectively.

Each strain was cultivated at 21 °C under alternating light and dark (12 h each) in 10 cm Petri dishes containing PDA culture medium. The culture medium was then extracted by maceration in ethyl acetate (EtOAc, Thermo fisher scientific, Waltham, MA, USA) for 24 h at room temperature under agitation. The organic phase was recovered by vacuum filtration and washed three times with Millipore Corporation (MilliQ) water (Elga LabWater, High Wycombe, UK). The organic and water phases were dried under reduced pressure with a rotary evaporator (Büchi, Flawil, Switzerland) to yield crude mixtures. Agar from uninoculated PDA plates were used as controls.