Silica gel

1D and 2D NMR spectra were recorded in CD₃OD and CDCl₃ on Bruker DRX 400 instrument at 295 K. Chemical shifts are given in ppm (δ) and were referenced to the solvent signals at 3.31/49.0 and 7.24/77.0 ppm, respectively. COSY (COrrelation SpectroscopΥ), HSQC (Heteronuclear Single Quantum Correlation), HMBC (Heteronuclear Multiple Bond Correlation), and NOESY (Nuclear Overhauser Effect SpectroscopYy) (mixing time 950 ms) experiments were performed using standard Bruker microprograms. Column chromatography (CC): Sephadex LH-20 (Pharmacia) and Silica gel (Merck, Art. 9385, Darmstadt, Germany). Preparative–thin-layer chromatography (Prep-TLC) plates were pre-coated with Silica gel (Merck, Art. 5721, Darmstadt, Germany). Fractionation was always monitored by TLC Silica gel 60 F– 254 (Merck, Art. 5554, Darmstadt, Germany) with visualization under UV (254 and 366 nm) and spraying with vanillin-sulfuric acid reagent. All obtained extracts, fractions, and isolated compounds were evaporated to dryness in a vacuum under low temperature and then were put in activated desiccators with P₂O₅ until their weights had stabilized.

The commercial reagents and solvents used for the organic syntheses were used as acquired (reagent grade purity) or were purified according to literature procedures [27 ]. Column chromatography (silica gel, 35–70 mesh, Merck, Darmstadt, Germany) or 20 × 20 cm preparative thin-layer chromatography (TLC) plates (coated with silica gel, 60 mesh, Merck, Darmstadt, Germany) were used to purify the reactional mixtures. ¹H NMR and ¹³C NMR spectra were recorded on Bruker Avance 300 (300.13 MHz) and 500 (125.76 MHz) spectrometers, respectively (Bruker, Wissembourg, France). CDCl₃, CD₃OD, and DMSO-d₆ were used as solvents, and tetramethylsilane (TMS) as an internal reference; the chemical shifts are expressed in δ (ppm) and the coupling constants (J) in Hertz (Hz). ESI-MS(+) spectra were recorded using a Micromass Q-Tof-2 spectrometer (Thermo Fisher Scientific, Manchester, UK), with CHCl₃ or with a mixture of MeOH/formic acid (1%) as a solvent. High-resolution mass spectra (HRMS) were recorded on a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) using CHCl₃ as a solvent. The full characterization (NMR, MS) is given in Supplementary Material.

Optical rotations were measured in CHCl₃ or MeOH at 25 °C using a Perkin-Elmer 241 polarimeter. UV spectra were recorded on a GBC Cintra 5 spectrophotometer. NMR spectra of all isolated compounds were recorded at 25 °C on Unity Inova 500NB high-resolution spectrometer (Agilent Technologies, CA, USA) operating at 500 MHz for ¹H-NMR and 100 MHz for ¹³C-NMR, respectively. Spectra were measured in CDCl₃ and CD₃OD and referenced against residual non-deuterated solvents. HRESIMS were measured on an Agilent 6520 Time of Flight (TOF) MS instrument. Column chromatography was carried out under TLC monitoring using silica gel (40–63 µm, Merck), and Sephadex LH-20 (25–100 µm, Pharmacia). For vacuum-liquid chromatography (VLC), silica gel (40– 63 µm) (Merck) was used. TLC was performed on silica gel 60 F₂₅₄ or RP-18 F₂₅₄ (Merck). LiChrolut RP-18 (40–63 μm) 500 mg, 3 mL (Merck) solid phase extraction (SPE) cartridges were also used. Semi-preparative HPLC was conducted by means of a Varian 920 LH instrument fitted with an autosampler module with a 1000 µL loop. The peak purities were monitored using a dual-wavelength UV detector settled at 254 and 360 nm. The columns were a 250 × 10 mm Spherisorb silica, particle size 5 µm (Waters) and a 300 × 7.5 mm Polymeric Reversed Phase (PLRP-S 100 Å), particle size 8 µm (Varian).

F-Ac (1.2 g) was selected for chromatographic separation, given its higher activity. The fraction was absorbed on silica gel (10 g, 70–230 mesh, Merck, Kenilworth, NJ, USA), placed in a glass column (50 × 300 mm²) packed with silica gel (80 g), and eluted with a dichloromethane:methanol gradient system as the mobile phase with a 10% increase in polarity, collecting fractional volumes of 100 mL. In total, 30 fractions were obtained, which were then pooled according to similarity via thin-layer chromatography (TLC) into four fractions (F1, F2, F3, and F4).
Due to the low availability of xylene and the yield of fractions, a TPA-induced local edema assay was used to evaluate the anti-inflammatory activity of F1–F4. TPA is a phorbol ester, widely used to study local inflammation by activating PKC with leukocyte infiltration and promoting eicosanoid release [45 ].

The following instructions were used to obtain spectroscopic data: melting points, Yanagimoto micromelting point apparatus (Yanaco New Science Inc., Kyoto, Japan); optical rotations, JASCO DIP-1000 digital polarimeter (JASCO Co., Tokyo, Japan); IR spectra, PerkineElmer 1720X FTIR spectrophotometer (PerkineElmer Inc., Waltham, MA, USA); UV spectra, HITACHI U-2000 spectrometer (Hitachi High-Technologies Co., Tokyo, Japan) (acetonitrile as a solvent); ¹H and ¹³C NMR spectra, Agilent VNMRS 600 spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA) (CDCl₃ was used as the solvent and TMS as the internal standard); FABMS, JEOL JMS-7000 mass spectrometer (JEOL Ltd., Tokyo, Japan); CD spectra, JASCO J-820 spectrometer (JASCO Co.); and HPLC, JASCO PU-1586 (RI 1531) (JASCO Co.). The following experimental conditions were used for column chromatography: (silica gel, 70–230 mesh; Merck, Darmstadt, Germany); medium-pressure liquid chromatography (MPLC; silica gel, 230–400 mesh; Merck); and TLC (silica gel 60 F₂₅₄; Merck).

The 1D and 2D NMR spectra were obtained using a Bruker Avance DRX Spectrometer, and the chemical shifts were recorded as δ values (ppm). Mass spectra were recorded using a high-resolution ESI Mass spectrometer. Silica gel (Merck, 63–200 μm particle size) and RP-18 (Merck, 75 μm particle size) were used for column chromatography. TLC was performed using Merck silicagel 60 F254 and RP-18 F254 plates. Isolated compounds were visualized after spraying with aqueous 20% H₂SO₄ and heating for about 5 min. Analytical-grade acetonitrile and distilled HPLC-grade water were purchased from Fisher Scientific (Pittsburgh, PA, USA). Open column chromatography was conducted using Silica gel (Merck, Darmstadt, Germany) and Sephadex LH-20 (Pharmacia, Uppsala, Sweden).