Silica gel f254

EA fraction of AE1 was subjected to thin layer chromatographic (TLC) analysis for partial separation of active metabolites. Dried extract was re-dissolved in EA at a concentration of 20 mg/ml and 10 μl of solution was spotted on TLC plates (MERCK Silica gel F254). EA and chloroform in a ratio of 1:1 was used as running solvent. Under UV light, retention factor (R_f) values for all the bands were calculated. The compounds related to each band were collected by scratching and dissolving in one ml of EA. EA portion was separated by centrifugation at 6000 rpm for 10 min and was evaporated to dryness. After dissolving the dried compounds in DMSO at 100 μg/ml concentration, antibacterial activities were determined by disc diffusion method [19 (link)]. Zones of inhibition were observed and diameters were measured.

A Perkin-Elmer-241 MC polarimeter was used to measure optical rotations. ECD spectra were recorded on a J-810 spectropolarimeter. One- and two-dimensional NMR spectra were recorded on a Bruker ARX 600 spectrometer. Mass spectra (ESI) were recorded with a Finnigan LCQ Deca mass spectrometer. A UHR-QTOF maxis 4G mass spectrometer (Bruker Daltonics) was used to record HRESIMS data. A Dionex UltiMate-3400SD system with a LPG-3400SD pump and a photodiode array detector (DAD 3000RS) as well as a separation column (Eurosphere-10 C₁₈, 125 × 4 mm, Knauer) were used for HPLC analysis. Detection wave lengths were set at 235, 254, 280, and 340 nm. Semi-preparative HPLC analysis was performed with a Merck Hitachi Chromaster HPLC system (UV detector L7400; pump L7100; column Eurosphere-100 C₁₈, 300 × 8 mm, Knauer; flow rate at 5 mL/min). Silica gel 60 M (0.04–0.063 mm, Macherey-Nagel) or Sephadex LH-20 were used for column chromatography. TLC plates precoated with silica gel F₂₅₄ (Merck) were used to monitor isolation fractions. Distilled and spectral grade solvents were used for column chromatography and spectroscopic measurements, respectively.

Poly (d,l-lactide-co-glycolide, acid terminated, lactide:glycolide 50:50), PLGA, MW 24–38 kDa, and PVA, poly(vinyl alcohol) (MW = 31–50 kDa, 87–89% hydrolyzed) were obtained from Sigma-Aldrich (Gillingham, UK). Oxoid™ phosphate buffered saline tablets were purchased from Thermo Fisher Scientific (Waltham, MA). Tween 80 was supplied from El Nasr Pharmaceutical Chemicals Co. (Cairo, Egypt). Span 80 was obtained from Oxford Lab Fine Chem LLP (Navghar, India). Oleic acid was manufactured and packed by Piochem for Laboratory Chemicals (October City, Egypt). Dimethylformamide (DMF), sodium lauryl sulfate (SLS), MeOH, CH₂Cl₂, ethyl acetate, and n-butanol were purchased from Al-Gomhoria Company (Cairo, Egypt). We employed silica gel F254 (Merck, Kenilworth, NJ, 70–230 mesh), and Sephadex LH-20 (Sigma-Aldrich Chemical Co., St. Louis, MO) for column chromatography. Cisplatin injection was obtained from Mylan Pharmaceuticals Co. (Bengaluru, India).

The reagents and indicator molecules were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Alfa Aesar (Haverhill, MA, USA) and used without further purification. Solvents were freshly distilled prior to use and were dried over anhydrous Na₂SO₄. ¹H and ¹³C-NMR spectra were recorded on a 600-MHz Varian VNMRS spectrometer (Varian, Inc., NMR Systems, Palo Alto, CA, USA, Varian is now part of Agilent Technologies) in DMSO-d₆ or D₂O solutions; δ is given in ppm relative to tetramethylsilane (TMS) as internal standard. ¹H and ¹³C-NMR signals were assigned on the basis of one- and two-dimensional homo- and heteronuclear experiments (COSY, TOCSY, HMBC and HSQC). Melting points were taken on a Stuart SMP-3 apparatus (Global Science NZ Ltd., Auckland, New Zealand). The high-resolution accurate masses were determined with a Dionex Ultimate 3000 UHPLC system hyphenated with an Orbitrap Q Exactive Focus Mass Spectrometer equipped with electrospray ionization (ESI) (Thermo Fischer Scientific, Waltham, MA, USA). Reaction progress was observed by thin-layer chromatography on commercial silica gel plates (Merck silica gel F254 on aluminum sheets, Darmstadt, Germany) using different mobile phases. For column chromatography, Kieselgel 60 (particle size 0.040–0.063 mm, ordered from VWR Chemicals, Radnor, PA, USA) was employed.

Total lipids (TLs) were extracted from the tissue by the Folch method [14 (link)]. Further, the TL from the liver (approx. 400 mg) were successively separated into neutral lipids (NLs) and PLs on a Sep-Pak^® silica cartridge (Waters, Dublin, Ireland) by elution with chloroform and methanol. The recovered lipids were checked by TLC (silica gel F254, Merck KGaA, Darmstadt, Germany), and developed in chloroform/methanol/H₂O (65:25:4, v/v/v) solvent and sprayed with Dittmer reagent [15 (link)] to indicate PLs. Lipid content in the tissues was gravimetrically analyzed and was calculated as its per tissue weight. Cholesterol content in the TL was enzymatically measured using a commercial kit (Wako Chemical Industries, Ltd., Osaka, Japan). The fatty acid methyl esters (FAMEs) were prepared from the TL (approximately 10 mg) using a previous described method [16 (link)]. The FAMEs were injected into the gas chromatograph Shimazu GC-2014 gas chromatography system (Shimazu Corporation, Kyoto, Japan) equipped with a flame ionization detector with a fused silica capillary column, Omegawax 320 (0.32 mm × 30 m, Supelco, Bellefonte, PA, USA). The temperatures of the column, detector and injection port were set at 200, 260, and 250 °C, respectively. The amount of each fatty acid was calculated by using the internal standard (methyl tricosanoate, 23:0).

Column chromatographic procedures were performed using silica gel 60 (Merck—Darmstadt, Germany) or Sephadex LH-20 (Sigma-Aldrich—St. Louis, MO, USA), whereas analytical TLC separations were conducted using silica gel F₂₅₄ (Merck—Darmstadt, Germany). HPLC analysis was performed using a Dionex Ultimate 3000 chromatograph with a UVD-DAD-170 V as the detector, using a Luna Phenomenex C18 column (particle and pore size of 5 μm and 120 Å)—10 × 250 mm to semipreparative and 4.6 × 250 mm to analytical modes. Analytical grade solvents and reagents were used for every chromatographic procedure (Labsynth Ltd.a, SP, Brazil). NMR spectra were recorded on a Varian INOVA 500 (Palo Alto, CA, USA) operating at 500 and 125 MHz for ¹H and ¹³C nuclei, respectively. Spectra were recorded using CDCl₃ (Aldrich, St. Louis, MO, USA) as solvent and TMS as internal standard. ESI-HRMS spectra were recorded on a MicroTOF QII Bruker Daltonics (Billerica, MA, USA) spectrometer using positive or negative ionization modes.