Silica gel

A Bruker Avance III HD 400 spectrometer operating at 400 MHz was utilized to obtain ¹H and ¹³C NMR spectra. The chemical shifts of ¹H and ¹³C NMR are expressed in δ (ppm) values regarding the solvent peaks δ_H 7.26 and δ_C 77 ppm for CDCl₃, and also δ_H 7.19, 7.55, 8.71 and δ_C 123.5, 135.5, 149.2 ppm for C₅D₅N. The coupling constants are obtained in Hertz (Hz). Aluminum-backed plates pre-coated with silica gel F254 (20 × 20 cm; 200 µm; 60 Å (Merck^™, Darmstadt, Germany) were used for TLC analysis. For column chromatography, silica gel 60/230–400 µm mesh size (Whatman^™, Sanford, ME, USA) was utilized. C18- reversed phase octadecyl silica (ODS) gel (Fluka™, Buchs, Switzerland) and Sephadex^® LH-20 (Sigma Aldrich^®, Bremen, Germany) were also used for final purification.

All chemicals used were purchased pure from commercially available sources such as Sigma Aldrich, VWR, Fisher or other chemical vendors. ¹H NMR (300 MHz) spectra were recorded on a Bruker Biospin NMR spectrometer. Thin layer chromatography was performed using Whatman silica gel 60 Å plates with florescent indicator and visualized using a UV lamp (254 nm) or KMnO₄ stain. Flash chromatography was performed on Grace with GraceResolve Normal Phase disposable silica columns. High performance liquid chromatography (HPLC) was performed on a Gilson 322 HPLC pump with a Gilson UV/VIS-155 detector and a Phenomenex Gemini C18 column (10 µm, 250 mm × 10 mm). Liquid chromatography electrospray ionization mass spectroscopy (LC–MS/ESI–MS) were acquired on an Agilent LC/MSD-SL with an 1100 HPLC and G1956B mass spectrometer with a Phenomenex Gemini 5 μm C18 110 Å 50 × 3 mm column.

The extract with the lowest MIC according to the preliminary screening was chosen for further assays. The phytochemical composition was investigated by liquid-liquid partitioning in which the bioactive extract (1.0 g) was resuspended in water (w) and partitioned into a separatory funnel in hexane (h), dichloromethane (d), ethyl acetate (ac), and n-butanol (b). A volume of 100 mL of each solvent was used in this step. The solvents were evaporated to give the 11 FO h (0.087 g), 11 FO d (0.333 g), 11 FO ac (0.017 g), 11 FO b (0.092 g), and 11 FO w (0.318 g) fractions. All the fractions were subjected to phytochemical analysis and an antibacterial activity assay. A phytochemical screening was performed by thin layer chromatography in glass plates covered with silica gel (Whatman, Kent, UK). The extract and fractions were investigated for the presence of cumarins, tannins, flavonoids, anthraquinones, essential oils, saponins, alkaloids, and triterpenes/steroids using standards and specific reagents for each class of secondary metabolites [12 ].

Anhydrous tetrahydrofuran was obtained from mBraun solvent purification system (A2 alumina). Reactions were monitored by thin-layer chromatography (TLC) on silica gel plates (60 F₂₅₄) with a fluorescent indicator, and independently visualized with UV light. Preparatory thin-layer chromatography (TLC) was performed on glass plates (7.5 × 2.5 and 7.5 × 5.0 cm) pre-coated glass plates coated with 60 Å silica gel (Whatman). Separations of isonitrile intermediates were carried out using flash chromatography (silica gel grade: 200-400 mesh, 40-63 μm) at medium pressure (20 psi). NMR spectra were recorded at 400 MHz in CDCl₃ and chemical shift values (δ) are reported in ppm. ¹H NMR spectra are reported in parts per million (δ) relative to the residual (indicated) solvent peak. Data for ¹H NMR are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, brs = broad singlet, d = doublet, t = triplet, q = quartet, ddd = double double doublet, m = multiplet, cm = complex multiplet), integration, and coupling constants in Hz. ¹³C NMR spectra were obtained on 400 MHz spectrometers (100 MHz actual frequency) and are reported in parts per million (δ) relative to the residual (indicated) solvent peak. High-resolution mass spectrometry (HRMS) data were obtained on spectrometer with a quadrupole analyzer.

Ultraviolet (UV) spectra and infrared (IR) spectra (KBr) were evaluated on a Shimadzu UV2401PC spectrophotometer (Shimadzu, Kyoto, Japan) and Bio-Rad FTS-135 spectrometer (Hercules, CA, USA), respectively. NMR spectra were obtained on DRX-400 or Advance III-600 spectrometers (Bruker, Bremerhaven, Germany) with TMS as the internal standard. Optical rotations were recorded on a JASCO P-1020 polarimeter (Horiba, Tokyo, Japan). The high-resolution mass spectra A were conducted on a Shimadzu LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan). Silica gel (100–200, 200–300 mesh, Qingdao Meigao Chemical Company, Qingdao, China), Sephadex LH-20 (20–150 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden.) and semi-preparative RP-HPLC purification (LC-20AT Shimadzu liquid chromatography system with ChromCoreTMC18 semi-preparative column, 250 mm × 10 mm, 5 μm) were used. Thin-layer chromatography (TLC) was conducted and detected under UV light or by heating after spraying with 10% H₂SO₄. Optical density and fluorescence were measured by a Microplate Reader (Tecan Infnite Pro series M200). A Nikon ECLIPSE 80i was used to perform the fluorescence experiments.

FT-IR spectrometer (Affinity-1, Shimadzu) was used to measure IR spectra. Optical rotations were measured in a polarimeter (MCP 300, Anton Paar) at 25°C. U-2910 spectrometer (Hitachi) was used to record UV spectra. Advance 600 spectrometer (Bruker) was used to measure ¹H NMR (600 MHz) and ¹³C NMR (150 MHz). Esquire 3000 plus spectrometer (Bruker) was used to measure ESIMS spectra. A micro TOF-QII mass spectrometer (Bruker) was used to record HRESIMS data. Sephadex LH-20 gel (Amersham Pharmacia) and silica gel (100–200 mesh and 200–300 mesh; Qingdao Marine Chemicals) were used in column chromatography. Analytical and preparative HPLC was performed on a Shimadzu Prominence system. Circular Dichroism Spectrometer (V100) was used to measure CD spectra.

¹H-, ¹³C-NMR, DEPT and 2D-NMR spectra were recorded on a Bruker AC 500 NMR spectrometer with tetramethylsilane (TMS) as an internal standard. HR-ESI-MS data were measured on a Bruker microTOF-QII mass spectrometer. CD spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics, Surrey, UK). Optical rotation values were measured with a PerkineElmer 341 polarimeter. Column chromatography was performed on silica gel (200–300 mesh; Qingdao Marine Chemical Factory, Qingdao, China), YMC gel (ODS-A, 12 nm, S-50 µm) and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden), respectively. The silica gel GF₂₅₄ used for TLC were supplied by the Qingdao Marine Chemical Factory, Qingdao, China. All solvents used were of analytical grade (Tianjin Fuyu Chemical and Industry Factory, Tianjin, China). HPLC was carried on Hitachi L-2400 with YMC ODS column. Spots were detected on TLC under UV light or by heating after spraying with 5% H₂SO₄ in EtOH (v/v).