Normal phase silica gel

Based on previous studies, the acetone extract [33 (link)], AaAc (28 g), was absorbed on silica gel in a 2:1 ratio to be placed in a glass column (100 × 400 mm²) packed with normal-phase silica gel (200 g, 70–230 mesh, Merck, Kenilworth, NJ, USA). After elution with 1500 mL of hexane as the mobile phase, the solvent was concentrated on a rotary evaporator to yield the hexane fraction (F-Hex). Then, 1500 mL of ethyl acetate was added to the column, which was concentrated to obtain the ethyl acetate fraction (F-AcOEt), and the process was repeated with acetone and methanol as mobile phases to obtain the acetone (F-Ac) and methanolic (F-MeOH) fractions, respectively. These fractions were evaluated in a murine model of xylene-induced local edema [38 (link)].

The HA from the ECS-1255 strain showed the highest degree of NTA, and it was fractionated using open-column chromatography. A chromatographic column was packed up with 35 g of normal-phase silica gel (70–230 mesh, Merck). The extract (3.6 g) was adsorbed on 5 g of normal-phase silica and added to the column. Fractionation was performed by three eluent dichloromethane-methanol systems (CH₂Cl₂-MeOH: 90 : 10, 70 : 30, 50 : 50 v/v), and the column was washed with MeOH J.T. Baker® (≥99.8%). Four fractions of 250 mL were collected from each system (a total of 1 L). However, a precipitate was formed during the last collection from the 70 : 30 system, which was also considered as a fraction; thus, five fractions (F1–F5) were collected. Each eluted fraction was concentrated with the same parameters used for AE, then lyophilized, and stored in glass vials. TLC analysis of the F5 fraction dissolved in MeOH was performed using a butanol- (99.4%) acetic acid- (≥98%) distilled water system (3 : 5 : 2 v/v), and the spots were stained with α-naphthol and visualized by UV light.

All solvents and reagents were purchased from Sigma-Aldrich (Deisenhofen, Germany) and used without further purification. 4-Hydroxyacetophenone was purchased from Acros (Thermo Fisher Scientific Geel–Belgium). The monitoring of reaction was done by the utilization of pre-coated silica gel plates (60 F₂₅₄) and thin layer chromatography (TLC). The normal phase silica gel (Merck, 70–230 mesh) was used to perform column chromatography purification, while the Merck silica gel (230–400 mesh) was utilized to perform the vacuum liquid chromatography.
A controllable single-mode microwave reactor, CEM Discover Microwave™ designed for synthetic utilization, was used. The reactor consists of power and pressure controls in addition to a magnetic stirrer. The temperature is controlled by an IR sensor. Melting points were measured using Sanyo MPD350 apparatus (Gallenkamp, Osaka, Japan) with digital display, and they were not corrected. A Perkin Elmer ATR spectrophotometer (Waltham, MA, USA) was used to record the infrared (IR) spectra. A Bruker Advance 400 MHz spectrometer (Fällanden, Switzerland) was used to record ¹H NMR and ¹³C NMR spectra. NMR samples were separately measured in DMSO, CDCl₃, and MeOD at room temperature. Mass spectra were recorded on Finnigan MAT95XL (ThermoFisher Scientific, Bremen, Germany) using (EI), at 70 eV.