Meoh d4

Among the fractions obtained from the SiG60-CC (250-mL size), the most active fraction for PPLI activity was evaporated and analysed. Briefly, the evaporated sample was dissolved in an appropriate deuterated solvent (Chloroform-d or MeOH-d4, Merck) at a ratio of 5–20 mg of compound to 600 µL of deuterated solvent. It was then transferred to an NMR tube and shaken until completely dissolved. The NMR spectrum was recorded on a Jeol JNM-ECZ (JNM-ECZ500R, Tokyo, Japan) 500MHz operated at 500 MHz for ¹H-NMR nuclei in order to detect the functional groups using tetramethylsilane as the internal standard. The chemical shift in δ (ppm) was assigned with reference to the signal from the residual protons in the deuterated solvents, while the chemical shift and J coupling value were determined using the MestReNova version 12.0.3 software.

MeOH and water for HPLC were purchased from VWR (Milan, Italy). Acetonitrile, HCOOH, and water for LC-MS analysis were bought from Merck (Merck KGaA, Darmstadt, Germany) MeOH-d4 (99.95%), Folin–Ciocalteu, DPPH, polyvinylpolypyrrolidone (PVPP), 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid (Trolox), NaNO₂, AlCl₃, NaOH, diammonium 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), Trolox, ellagic acid, quercetin, vitamin C, 2,3,5-Triphenyltetrazolium chloride (TPTZ), HCl FeCl₃ × 6 H₂O, FeSO₄, LPS, prednisone, pyocyanin (PCN), 2′-7′-dichlorodihydrofluorescein diacetate (DCFH2-DA), and L-1-p-Tosylamino-2-phenylethyl chloromethyl ketone (TPCK) reagents were purchased by Sigma-Aldrich (Milan, Italy).
WST-1 reagent was purchased from Roche (Basel, Switzerland). QUANTI-Blue was bought from InvivoGen (San Diego, CA, USA), Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was purchased by Sigma-Aldrich (Milan, Italy).
The THP-1 human monocytic leukemia cell line was purchased from the European Collection of Cell Cultures (Salisbury, UK). THP-1-XBlue-MD2-CD14 cell line (Invivogen; San Diego, CA, USA) has been derived from THP-1. The cultured murine monocyte macrophage cell line J774.A1 was purchased from the American Type Tissue Culture Collection (Rockville, MD, USA).

Unless otherwise noted, all commercially available compounds were used without further purification. Water was purified with a Millipore system. Bovine hearth cardiolipin and 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) were purchased from Larodan Inc (Solna, Sweden); Ferrous ammonium sulfate Fe(NH₄)₂(SO₄)₂ × 6H₂O (Fe II AS, Carlo Erba, Milan, Italy) (Na₂S × 9H₂O Merck, Milan, Italy), H₂O₂ 30% and 2-mercaptoethanol (Merck, Milan, Italy); chloroform, methanol, diethyl ether and n-hexane (HPLC grade) were used as received from J. T. Baker, Phillipsburg, NJ, USA. MeOH-d₄ and DMSO-d₆ were purchased from Merck (Milan, Italy). NMR spectra were recorded at ambient temperature on a Varian 500 MHz spectrometer (Agilent, Cernusco sul Naviglio, Milan, Italy). Solvents are detailed for each spectrum. UV spectra were recorded at room temperature with a Cary300Bio spectrophotometer (Agilent, Cernusco sul Naviglio, Milan, Italy). Hydrodynamic diameters of the obtained liposomes were measured using the dynamic light scattering (DLS) technique (Malvern Instruments Series NanoZS with a detection angle of 173°, Malvern Instruments, Malvern, UK). All measurements were recorded at 25 °C.